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Role of Prostanoids in Glucose- and
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and Oxygen Consumption

presented by

Robert Wesley Coatney

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° Wm:

ROLE OF PROSTANOIDS IN
GLUCOSE- AND OLEATE-INDUCED
CHANGES IN INTESTINAL BLOOD FLOW

AND OXYGEN CONSUMPTION

By

Robert Wesley Coatney

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Physiology

1993

ABSTRACT
ROLE OF PROSTANOIDS IN GLUCOSE- AND OLEATE-INDUCED
CHANGES IN INTESTINAL BLOOD FLOW AND OXYGEN CONSUMPTION
By

Robert Wesley Coatney

Intestinal blood flow increases after a meal and the stimuli for this hyperemia are the
digested products of food. Several studies indicate that endogenous prostanoids, and
speciﬁcally TxAQ, may play a role in the regulation of intestinal blood flow and oxygen
uptake during nutrient absorption. Most of these studies utilized a balanced food that
contained carbohydrates, fats, and proteins. Therefore it is unknown if prostanoids play
a role in the regulation of the increases in blood ﬂow and oxygen uptake induced by the
individual nutrients. Glucose and oleate are absorbed by different processes and evoke
different vascular responses, and different types or quantities of prostanoids may play a
role in the regulation of the responses to these individual nutrients. The ﬁrst objective of
this study was to determine if prostanoids play a role in the regulation of glucose— and
oleate-induced increases in blood ﬂow and oxygen uptake. This was studied by
determining the effect of cyclooxygenase inhibition on the increases in blood ﬂow and
oxygen uptake induced by glucose and oleate, and if and which prostanoids are released
in response to luminal placement of glucose and oleate. Additionally, the effect of SQ
29548, a TxA2 l endoperoxide receptor blocker, on the glucose- and oleate-induced
increases in blood ﬂow and oxygen uptake was also determined.

Both glucose and oleate increased blood ﬂow and oxygen uptake when placed into

the lumen. Cyclooxygenase inhibition enhanced the increases in blood ﬂow and oxygen
uptake induced by both glucose and oleate. Cyclooxygenase inhibition also decreased
resting blood flow, but did not affect resting oxygen uptake. Even though a decrease in
blood ﬂow, i.e. an increase in initial resistance, was shown to inﬂuence the magnitude of
the glucose- and oleate-induced vascular responses, the enhancement of the glucose— and
oleate-induced hyperemias by cyclooxygenase inhibition was in addition to the decrease
in resting blood flow. Oleate induced a greater increase in blood flow than did glucose
before and after cyclooxygenase inhibition. Further, the level of enhancement of the
oleate-induced increase in blood flow by cyclooxygenase inhibition was greater than that
of glucose. The increases in oxygen uptake induced by glucose and oleate were similar
before and after cyclooxygenase inhibition. Additionally, cyclooxygenase inhibition
enhanced the glucose- and oleate-induced increases in oxygen uptake the same degree.

Luminal placement of glucose and oleate increased the venous concentrations and
releases of P652, TxA2, P612, and PGFZG. Oleate induced a greater release of PGE2, TxAz,
and PGF2m than did glucose.

The TxA2 / endoperoxide receptor blocker, SQ 29548, did not affect resting jejunal
blood ﬂow or oxygen uptake. SQ 29548 enhanced the glucose-induced increase in oxygen
uptake, but did not affect the glucose-induced hyperemia. TxA2 receptor blockade
enhanced the increases in both blood flow and oxygen uptake induced by oleate.

Several in vivo studies have suggested that prostanoids might limit the food-
induced increase in oxygen uptake by limiting the increase in tissue oxidative metabolism.
However, in in vivo conditions changes in intestinal oxygen uptake could be inﬂuenced

by changes in tissue oxidative metabolism as well as vascular factors. Therefore, the

second objective this study was to determine the effects of endogenous prostanoids on
jejunal mucosal oxygen consumption separate from their vascular actions. Therefore, the
effects of 1) cyclooxygenase inhibitors (mefenamate and indomethacin), 2) substrate
loading with arachidonate, 3) U-44069, a TxAJendoperoxide analogue, and 4) SQ 29548,
a TxAzlendoperoxide receptor blocker on the rate of jejunal mucosal oxygen consumption
(Q07) were determined in vitro in the absence and presence of nutrients, glucose or
oleate, using constant volume manometry.

The jejunal mucosa consumed oxygen in vitro in absence (basal Q02) and
presence of nutrients. Cyclooxygenase inhibition, and arachidonate individually and in
combination decreased the basal mucosal Q02. Hence, prostanoids might enhance basal
QO2 whereas arachidonate per se or non-cyclooxygenase products of arachidonate
decrease basal Q02 in vitro. Glucose, at a concentration of 5 mM, maximally increased
Q02. Cyclooxygenase inhibition enhanced the glucose-induced increase in mucosal QO ,
whereas substrate loading with arachidonate attenuated the glucose-induced increase in
Q02. This affect of arachidonate on the glucose-induced increase in Q02 was abolished
by cyclooxygenase inhibition, suggesting that it was due to prostanoids. Neither the TxA2
analogue, U-44069, nor the TxA2 I endoperoxide receptor blocker affected basal mucosal
Q02. The TxA2 analogue decreased the glucose-induced increase in Q02, whereas the
TxA2 I endoperoxide receptor blocker enhanced the glucose-induced increase in mucosal
Q02. Hence, endogenous prostanoids, and in particular TxAz, might act to limit the
increase in mucosal QO2 induced by glucose.

Oleate alone did not affect mucosal Q02 in vitro. Oleate plus bile or taurocholate

signiﬁcantly decreased mucosal Q02 at concentrations greater than 2.0 mM. Oleate at a

concentration of 5 mM plus taurocholate produced the maximum decrease in Q02. This
effect of oleate plus taurocholate is opposite of its effect in viva. Cyclooxygenase
inhibition and arachidonate individually and in combination decreased basal Q02 as well
as Q02 in the presence of oleate plus taurocholate. However, it appears that these
treatments did not affect the decrease in Q02 induced by oleate plus taurocholate. Thus
it seems that prostanoids do not affect the oleate-induced decrease in mucosal Q02 in
vitra. The reason for the difference in the effect of oleate on Q02 in vitra, and the oxygen

uptake in viva is unknown.

This work is dedicated to my wife, Anna,
and our sons, Kevin and Garrett,
with love and devotion.

vi

ACKNOWLEDGEMENTS

First and foremost, I would like to acknowledge the advice, encouragement,
support, and scientiﬁc expertise of my mentor, Dr. C.C. Chou. Thank you Dr. Chou for
your support and guidance, as well as the opportunity to do this research.

I also want to express my gratitude to the members of my guidance committee,
Dr. Robert Pittman, Dr. Ed Robinson, Dr. William Smith, and Dr. John Stick for their
support and input during my doctoral training.

I am sincerely grateful and indebted to Dr. Adamu Alemayehu for his intellectual

input, advice, encouragement, and friendship.

vii

TABLE OF CONTENTS

Page
LIST OF TABLES ......................................................................................................... ix
LIST OF FIGURES ........................................................................................................ xi
INTRODUCTION ........................................................................................................... 1
LITERATURE REVIEW ................................................................................................ 4
MATERIALS AND METHODS ................................................................................... 34
Effect of Prostanoids on Glucose- and Oleate-Induced Changes in Blood
Flow and Oxygen Uptake in viva ...................................................................... 34
Effect of Prostanoids on Mucosal Oxygen Consumption in vitra ................... 44
RESULTS ....................................................................................................................... 62
Effect of Prostanoids on Glucose- and Oleate-Induced Changes in Blood
Flow and Oxygen Uptake in viva ...................................................................... 62
Effect of Prostanoids on Mucosal Oxygen Consumption in vitra ................... 89
DISCUSSION ................................................................................................................. 107
Effect of Prostanoids on Glucose- and Oleate-Induced Changes in Blood
Flow and Oxygen Uptake in viva ...................................................................... 107
Effect of Prostanoids on Mucosal Oxygen Consumption-in vitra ................... 119
SUMMARY AND CONCLUSION ............................................................................... 138
LIST OF REFERENCES ............................................................................................... 143

viii

Table

10.

11.

12.

13.

14.

LIST OF TABLES

Paar:
Treatment chemicals used in Series I - XII in vitra experiments ..................... 50
The effect of taurocholate on jejunal mucosal QO2 ........................................... 54
The effect of oleate plus taurocholate on mucosal QO2 .................................... 56
The effect of taurocholate on the 5.0 mM oleate—induced change in
mucosal QO2 ....................................................................................................... 59
Effect of mefenamate on glucose-induced changes in blood flow,
resistance, arteriovenous oxygen content difference, and oxygen uptake ........ 63
Effect of mefenamate on oleate-induced changes in blood ﬂow,
resistance, arteriovenous oxygen content difference, and oxygen uptake ........ 66
Effect of angiotensin II on glucose-induced changes in blood flow,
resistance, arteriovenous oxygen content difference, and oxygen uptake ........ 77
Effect of angiotensin II on oleate-induced changes in blood flow,
resistance, arteriovenous oxygen content difference, and oxygen uptake ........ 78
The Effect of Cyclooxygenase Inhibitors on Jejunal Mucosal QO2 ................. 90
The Effect of Arachidonate on Jejunal Mucosal QO2 ....................................... 91
The Effects of Mefenamate and Arachidonate, alone and combined,
on Mucosal QO2 .................................................................................................. 93
The Effect of U-44069 on Jejunal Mucosal QO2 .............................................. 95
The Effect of SQ 29548 on Jejunal Mucosal QO2 ............................................ 96

The effect of mefenamate on oleate plus taurocholate-induced changes in
mucosal QO2 ....................................................................................................... 100
ix

15.

16.

The effect of arachidonate on oleate plus taurocholate-induced changes in
mucosal QO2 ....................................................................................................... 103

The effect of SQ 29548 on oleate plus taurocholate-induced changes in
mucosal QO2 ....................................................................................................... 106

LIST OF FIGURES

Page

Possible mechanisms and pathways involved in the regulation of intestinal
blood flow during nutrient absorption ............................................................. 13

Intestinal preparation ........................................................................................ 36

The effect of a bolus of arachidonate (300 ug/kg, administered into the
pulmonary artery) on aortic pressure before and 1 hour after
administration of mefenamate (lOmg/kg i.v.) ................................................. 40

Relationship between oxygen consumed and incubation time when the

media contained only 0 mM glucose (open circle) or 5 mM glucose

(black diamond). The slope of the relationship represents the mucosal

oxygen consumption rate in nl-min"-mg dry wt“. * P < 0.05 relative to

the slope for 0 mM glucose. Standard error bars are included but are

smaller than the symbols .................................................................................. 46

Effect of glucose on mucosal Q02. * P < 0.05 relative to 0 mM glucose.
# P < 0.05 relative to preceding lower glucose concentration ....................... 47

Effect of 10% gallbladder bile on mucosal Q02 in the absence and
presence of 5 mM glucose. (n= 8 determinations! 4 dogs) * P < 0.05
relative to no treatment (T x). # P < 0.05 relative to 5 mM glucose ............ 48

Effect of TxAQ / endoperoxide receptor blocker, SQ 29548, on the

decrease in QO2 induced by U-44069, a TxA2 receptor analogue, in the
presence of 5 mM glucose. (n= 6 determinations/ 3 dogs) * P < 0.05

relative to 5 mM glucose. # P < 0.05 relative to U-44069 ......................... 52

Mucosal Q02 in the absence and presence of increasing concentrations of

taurocholate [TC]. * P < 0.05 relative to basal value ([TC] 0). # P < 0.05
relative to preceding taurocholate concentration ............................................. 55

xi

 

10.

11.

12.

13.

14.

15.

The effect of oleate ([OA]) in the absence and presence of taurocholate

([TC] 0.1 mM) or 10% bile. "‘ P < 0.05 relative to basal value

([TC] 0, [0A] 0). # P < 0.05 relative to the preceding oleate

concentrations. The effect of 5.0 mM oleate in the absence and presence

of 10% bile were performed in a separate series of experiments and are
included for comparison ................................................................................... 57

Correlation between changes in jejunal blood ﬂow and oxygen uptake
(V02) induced by glucose plus bile. Open circles represent responses
before mefenamate. Triangles represent responses after mefenamate ........... 65

Correlation between changes in jejunal blood flow and oxygen uptake
(V0,) induced by oleate plus bile. Open circles represent responses
before mefenamate. Triangles represent responses after mefenamate ........... 68

Comparison of the glucose- and oleate-induced percent change from
control for blood flow and oxygen uptake before and after mefenamate.
* P < 0.05. # P < 0.05 relative to the respective value before
mefenamate. @ P < 0.05 relative to the respective value for glucose ......... 69

Relationship between initial resistance and the change in resistance

induced by glucose plus bile. Open circles and dashed line represent the
response before mefenamate. Triangles and solid line represent the

response after mefenamate. Slopes of the regression lines are signiﬁcantly
different, * P < 0.05 ........................................................................................ 72

Relationship between initial resistance and the change in resistance

induced by oleate plus bile. Open circles and dashed line represent the
response before mefenamate. Triangles and solid line represent the

response after mefenamate. Slopes of the regression lines are signiﬁcantly
different, * P <0.05 ......................................................................................... 73

Relationship between initial resistance and the change in resistance

induced by glucose plus bile before and during angiotensin H infusion.

Open circles represent the response before angiotensin H and the closed
circles represent the response during angiotensin II infusion. Slope of the
regression line for the angiotensin II series (dashed line) is signiﬁcantly
different from that for after mefenamate (solid line) * P < 0.05.

Mefenamate data are reproduced from Figure 13 ........................................... 75

xii

16.

17.

18.

19.

20.

21.

22.

23.

Relationship between initial resistance and the change in resistance

induced by oleate plus bile before and during angiotensin II infusion.

Open circles represent the response before angiotensin II and the closed
circles represent the response during angiotensin H infusion. Slope of the
regression line for the angiotensin 11 series (dashed line) is significantly
different from that for after mefenamate (solid line) * P < 0.05.

Mefenamate data are reproduced from Figure 14 ........................................... 76

Arterial and jejunal venous plasma concentrations of 6-keto-PGFla, Tsz,
POE), and PGFZG (pg-ml") during luminal placement of normal

saline (NS), glucose (GL), and oleate (OA). * P < 0.05 relative to the
venous concentration obtained during the corresponding NS ........................ 81

Jejunal releases of 6-keto-PGFm, Tsz, PGEz, and PGFm (ng-min"-100g“)
during luminal placement of normal saline (NS), glucose (GL), and oleate
(OA). * P < 0.05 relative to the value obtained during the corresponding
NS; # P < 0.05 relative to the corresponding GL value ............................... 83

Jejunal blood flow and oxygen uptake during luminal placement of

normal saline or glucose before and after intraarterial administration of

SQ 29548. * P < 0.05 relative to normal saline value. # P < 0.05

relative to the glucose value before SQ 29548 ............................................... 86

Jejunal blood flow and oxygen uptake during luminal placement of

normal saline or oleate before and after intraarterial administration of SQ
29548. * P < 0.05 relative to normal saline value. # P < 0.05 relative to

the glucose value before SQ 29548 ................................................................. 88

Effect of mefenamate [Mef], arachidonate [AA], and mefenamate plus
arachidonate in combination on the mucosal Q02 in the absence and presence
of 5 mM glucose [Glue]. * P < 0.05 relative to the basal value

([Gluc] 0, [Met] 0, [AA] 0). # P < 0.05 relative to mefenamate

([Gluc] 0, [Met] 40, [AA] 0), or arachidonate

([Gluc] 0, [Mef] 0, [AA] 100) values. @ P < 0.05 relative to 5 mM

glucose value ([Gluc] 5, [Mef] 0, [AA] 0). § P < 0.05 relative to
arachidonate value in 5 mM glucose ([Gluc] 5, [Met] 0, [AA] 100) ............ 95

Effect of SQ 29548, a TxA2 / endoperoxide receptor blocker in mucosal
Q02 in the absence and presence of 5 mM glucose. * P < 0.05 relative to
corresponding value without SQ 29548 .......................................................... 97

Effect of mefenamate ([Mef]) on mucosal Q02 in the absence and

presence of taurocholate ([TC]) and taurocholate plus oleate ([OA]).

* P < 0.05 relative to basal value ([TC] 0, [DA] 0). # P < 0.05 relative

to corresponding value without mefenamate ([Mef] 0) .................................. 101

xiii

24.

25.

26.

Effect of arachidonate on mucosal Q02 in the absence and presence of
taurocholate ([TC]) and taurocholate plus oleate ([OA]). Also shown is

the effect of arachidonate plus mefenamate in combination in the

presence of taurocholate plus oleate. * P < 0.05 relative to basal value

([TC] 0, [0A] 0, [AA] 0). # P < 0.05 relative to corresponding value

with out arachidonate ([AA] 0). @ P < 0.05 relative to

([TC] 0.1, [0A] 0.5, [AA] 100) ....................................................................... 104

Theoretical model for the effect of prostanoids on the basal mucosal
Q02 in vitro. The cells shown represent intestinal epithelial cells from
the crypt region. R, - receptor; (+) - enhances .............................................. 128

Theoretical model for the effect of prostanoids on the glucose-induced

increase in mucosal Q02 in vitro. The cells shown represent intestinal
epithelial cells from the villus region. R, - receptor; (-) - decreases;

? - action or pathway unknown or uncertain .................................................. 130

xiv

INTRODUCTION

Intestinal blood ﬂow increases after a meal (18,23,42,44), and the stimuli for this
hyperemia are the digested products of food (24,42). The mechanisms involved in the
initiation and regulation of this hyperemia are multifactorial and include oxidative
metabolism, enteric nerves and local reﬂexes, gastrointestinal hormones and peptides, and
local tissue chemicals such as histamine and prostanoids (18,23,42). Additionally different
nutrients may inﬂuence intestinal blood ﬂow via different mechanisms or combinations
of mechanisms (26,42). Systematic study of various constituents of food revealed that
glucose and oleate are the main nutrients that individually stimulate an intestinal
hyperemia (24). These two nutrients inﬂuence intestinal blood ﬂow differently (26).

Recent studies have shown that endogenous prostanoids may play a role in the
regulation of intestinal blood ﬂow and oxygen uptake during nutrient absorption
(20,41,67). The food-induced increase in blood ﬂow and oxygen uptake is accompanied
by an increase in the release of prostanoids by the jejunum (20). Cyclooxygenase
inhibition enhances the food- and oleate-induced increase in blood ﬂow and oxygen
uptake (41,77). Arachidonate attenuates the food-induced increase in blood flow and
oxygen uptake (67). Most of these studies investigated the role of prostanoids in the
regulation of the nutrient-induced hyperemia utilizing a balanced food that contained

equal parts by weight of carbohydrate, fat, and protein. Therefore it is unknown if

2

prostanoids play a role in the regulation of the increases in blood ﬂow and oxygen uptake
induced by individual nutrients. Inasmuch as glucose and oleate are absorbed by different
processes and evoke different vascular responses, different types or quantities of
prostanoids may play a role in the regulation of the responses to these individual
nutrients. The ﬁrst objective of this study was to investigate if prostanoids play a role in
the regulation of glucose- and oleate-induced increases in blood ﬂow and oxygen uptake.
This was studied by determining l) the effect of cyclooxygenase inhibition on the
increases in blood ﬂow and oxygen uptake induced by glucose and oleate, and 2) if and
which prostanoids are released in response to luminal placement of glucose and oleate.

Cyclooxygenase inhibition enhances and arachidonate attenuates the food-induced
increase in blood ﬂow and oxygen uptake in parallel (41,67). The food-induced increases
in blood flow and oxygen uptake before and after cyclooxygenase inhibition or
arachidonate are linearly and positively correlated (41,67). Since intestinal oxygen uptake
is independent of blood ﬂow throughout the physiological range of blood flow (48,67),
the enhancement of the food-induced increase oxygen uptake is most likely due to the
enhancement of oxidative metabolism rather than the enhancement of blood ﬂow.
However, in in viva conditions changes in intestinal oxygen uptake could be inﬂuenced
by changes in tissue oxidative metabolism as well as vascular factors. (48,59) Since these
studies were performed utilizing a naturally perfused segment of intestine in viva, the
effects of endogenous prostanoids on intestinal oxygen uptake cannot be completely
separated from their vascular actions. The second objective this study was to determine
the effects of endogenous prostanoids on jejunal mucosal oxygen consumption separate

from their vascular actions. Therefore, the effects of l) prostanoids synthesis inhibition

3

by cyclooxygenase inhibitors (mefenamate and indomethacin), 2) substrate loading with
arachidonate, 3) U-44069, a TxA2/endoperoxide analogue, and 4) SQ 29548, a TxA2/
endoperoxide receptor blocker on jejunal mucosal oxygen consumption were determined
in vitra in the absence and presence of nutrients, glucose or oleate, using constant volume

manometry.

LITERATURE REVIEW

Postprandial Intestinal Hyperemia

In pioneering studies nearly sixty years ago, Herrick et al. (51) and Essex et al.
(32) showed that blood ﬂows through the cranial mesenteric, femoral, carotid and
coronary arteries increase shortly after the ingestion of a milk, egg, and glucose meal in
conscious dogs. Since that time the increase in blood ﬂow to the splanchnic organs after
a meal (postprandial hyperemia) has been the subject of much study. The postprandial
hyperemia is considered a "functional hyperemia", which occurs to meet the metabolic
demands of the digestive organs during digestion and assimilation of food following
feeding.
Anatomical Considerations

The splanchnic vascular bed is perfused by the celiac, superior mesenteric, and
inferior mesenteric arteries. Blood ﬂow to the stomach is supplied primarily by the celiac
artery, while the superior mesenteric artery supplies primarily the small intestine, and the
inferior mesenteric artery supplies the colon (23). Total splanchnic blood ﬂow averages
approximately 25% of the cardiac output, with the small intestine receiving 10-15%, the
colon receiving 3-5%, and the organs perfused by the celiac artery receiving the
remainder (15,18). The resting small intestinal blood ﬂow ranges from 30 to 100

m1/min/100 grams of tissue (18,23). Blood ﬂow to different layers of the small intestine

5

is not distributed equally. Under resting conditions, the mucosal layer receives 75-80%
of the blood perfusing the whole bowel wall, while the submucosa receives approximately
5%, and the muscularis receives 20-25% (18,48). Within the mucosal layer the villi
receive approximately 60% of the mucosal ﬂow, while the crypts receive the remainder
(18). The relative distribution of the blood ﬂow to the different layers within the bowel
wall corresponds to the metabolic activities of the respective tissues. At rest the oxygen
consumption of the muscle layer is approximately one—quarter of that of the mucosa (17).
The intestinal mucosa has a high capillary density, and these capillaries form an extensive
network beneath the mucosal epithelium in both the villus and the crypt (18,48). These
features of the mucosal capillaries facilitate ﬂuid transport between the epithelial cell and
blood, and serve to promote intestinal absorption and secretion. At rest, approximately 20-
30% of the intestinal capillaries are perfused with blood, leaving a large reserve to
accommodate the needs imposed by absorption and secretion (18).
Overall Cardiovascular Resmnse to a Meal

The cardiovascular response to feeding consists of two distinct phases: a brief
anticipation/ingestion phase is followed by a longer digestive/absorptive phase (15). The
anticipation/ingestion phase is characterized as a generalized systemic cardiovascular
response. When conscious dogs see, smell, and ingest food, the cardiac output, heart rate,
and aortic pressure increase. Coronary vascular resistance decreases, while renal and
mesenteric resistances increase, and vascular resistance to skeletal muscle either increases
and/or decreases (105,106). These cardiovascular responses are brief. Within 5 - 30
minutes after ingestion all the above variables, except the mesenteric vascular resistance,

return to pre-feeding values. These cardiovascular responses that occur during the

6

anticipation/ingestion phase appear to be the result of activation of the sympathetic
nervous system, as they are blocked by adrenergic blocking agents (105).
The digestive/absorptive phase begins as the anticipation/ingestion phase is waning

(23). It is characterized by an increase in blood ﬂow that is conﬁned to the organs
involved in digestion and assimilation. Blood ﬂow to the stomach increases when food
enters the stomach (22,54). The gastric hyperemia is transient, and is not due to
distention, but is due to presence of nutrients in the lumen. As this gastric hyperemia
subsides, blood ﬂow to the small intestine and pancreas starts to increase, reaching its
peak 30 - 90 minutes after feeding and lasting for several hours (22,99,105). In conscious
dogs the average increase in cranial mesenteric blood flow after feeding ranges from 28
to 132% above resting values, and pancreatic blood ﬂow doubles (23). The increase in
ﬂow to the digestive organs is partly compensated by a decreased ﬂow to inactive skeletal
muscle. However, inasmuch as cerebral and cardiac blood ﬂows are unchanged during
digestion (44,106), and renal blood ﬂow increases after ingestion of a protein-rich meal
(48), the increased ﬂow in digestive organs must come from an increase in cardiac output
and/or a decreased ﬂow in some organs (23).
Localization of the Postprandial Hmremia

Even though the increase in blood flow after a meal is conﬁned primarily to the
organs of the digestive system that are actively engaged in digestion and absorption, the
increase in blood ﬂow is not simultaneously and uniformly distributed to all sections of
the gastrointestinal tract, nor to all tissue layers of the bowel wall. In conscious dogs,
blood flow to the stomach, duodenum, jejunum and pancreas were increased at 30 and

90 minutes after oral ingestion of a meal, but the ﬂow to distal ileum did not increase

7
until 90 minutes after feeding (44). Explanations for the delay in the increase in ileal

blood flow are that chyme did not enter the ileum until after 30 minutes but before 90
minutes, and that the hyperemia is localized to those segments exposed to chyme.

The distribution of blood ﬂow to the gastrointestinal tract after feeding has been
systematically characterized by Chou et al. (22) in anesthetized dogs. Introduction of
predigested food directly into the stomach promptly increased blood ﬂow through the
celiac artery for about 30 minutes, but ﬂow in the superior mesenteric artery (SMA) did
not increase until 30 minutes after the introduction. The increased SMA ﬂow lasted as
long as the chyme was present in the lumen of the small intestine. Infusion of predigested
food into the duodenal lumen, on the other hand, increased SMA ﬂow within 5 minutes,
but did not alter celiac artery blood ﬂow. The increased SMA ﬂow lasted as long as the
infusion was continued. It thus appears that following a meal there is an initial increase
in celiac ﬂow with no change in SMA flow, but as the chyme empties from the stomach
and enters the small intestine, the celiac ﬂow returns toward control, while SMA ﬂow
rises. This same study (22) provided further evidence that blood ﬂow increases primarily
to the gut segment that is exposed to chyme. Intraduodenal infusion of predigested food
increased blood ﬂow through the SMA, but did not alter the ﬂow to an isolated jejunal
segment which was not exposed to chyme. Conversely, when predigested food was placed
into the lumen of the isolated jejunal segment, blood ﬂow to the segment increased, but
SMA ﬂow was not altered. Subsequent experiments utilizing two adjacent isolated jejunal
segments further show that the increased blood ﬂow was due to exposure of the mucosa
to nutrients. Placement of predigested food into the lumen of one segment increased ﬂow

only to the segment containing food, but did not alter ﬂow to the other segment that

8

contained an equal volume of normal saline (22).

The vascular response of the different layers of the bowel wall to feeding is also
not uniform. In conscious dogs the increase in blood ﬂow to the gastric body following
oral feeding is conﬁned primarily to the mucosal layer (11,44). In conscious as well as
anesthetized dogs, the food-induced or glucose-induced increase in small intestinal blood
ﬂow is due primarily to an increase in mucosal-submucosal blood ﬂow, without
signiﬁcantly altering blood ﬂow to the muscle layer (22,44,74,111). However, blood ﬂow
to the muscle layer may increase, if the chyme distends the gut wall and induces rhythmic
contractions (17).

These Studies indicate that the increase in blood ﬂow to the digestive organs after
a meal is primarily localized to those organs and tissues actively engaged in digestive
functions such as absorption and secretion. In particular, small intestinal blood ﬂow
increases only in those segments which contain chyme, and the increased ﬂow is
primarily localized to the mucosal-submucosa] layer.

Luminal Stimuli Responsible for the Postprandial Intestinal Hmremia

Studies in conscious animals have shown that the chemical nature of a meal
inﬂuences the level of postprandial intestinal hyperemia (23). For example, in conscious
primates, a high-fat, high-protein meal produces a more profound and sustained
mesenteric hyperemia than does a carbohydrate meal, and a fruit meal produces an
intermediate response (107). Anesthetized dogs also show a similar response (89). A high-
fat diet (45% fat, 18% protein, 29% carbohydrate), a high-protein diet (8% fat, 64%
protein, 22% carbohydrate), and a high-carbohydrate diet (8% fat, 18% protein, 68%

carbohydrate) produced 31%, 24%, and 18% increases in jejunal blood ﬂow, respectively

9

(89). Chou er al. (24) conducted the ﬁrst systematic study to determine the role played
by the various constituents of chyme in induction of intestinal hyperemia utilizing in situ
loops of canine small intestine. They ﬁrst compared the vascular actions of major
constituents of chyme, i.e., digested and undigested foodstuffs, hydrolytic enzymes and
bile. A commercially available canned dog food was homogenized and separated into two
portions. One portion was retained to be used in the experiments as "undigested food",
whereas the other portion was incubated and mixed with a pancreatic enzyme preparation
for 5 hours to permit digestion (predigested food). Jejunal blood ﬂow was promptly and
signiﬁcantly increased by intraluminal placement of predigested food, but was not
increased by luminal placement of undigested food, the pancreatic enzyme preparation,
or bile. Additionally, when the liquid and solid phases of the predigested food
preparation were separated by centrifugation, and each then placed into the lumen, only
the liquid phase increased jejunal blood ﬂow. Therefore, the stimuli responsible for the
intestinal hyperemia are the hydrolytic products of food digestion contained in the liquid
phase (24).

Chou er al. (24) and Kvietys et al. (58) investigated further which products of food
digestion inﬂuence intestinal blood ﬂow, studying the effects of glucose, oleate,
monoolein, and amino acids on jejunal blood ﬂow. These studies showed that at
concentrations which mimic postprandial luminal concentrations, only glucose (150 mM)
signiﬁcantly increased blood flow when placed in the lumen. Other studies have also
shown that glucose increases intestinal blood flow (26,71,90,9l). The endproducts of fat
digestion i.e., the long-chain fatty acid oleate and the monoglyceride monoolein, had no

effect on intestinal blood ﬂow when placed in the lumen in aqueous solution. However,

10
when these lipids were solubilized by gallbladder bile (26,58) or sodium taurocholate

(24), or the pH of the oleate solution was increased (26), and then placed into the lumen,
intestinal blood flow increased markedly, and the level of hyperemia was signiﬁcantly
greater than that produced by glucose solution. The triglyceride triolein had no effect on
intestinal blood ﬂow, either with or without the addition of bile (58).

Individual amino acids, and di- and tri-peptides are the major products of protein
digestion. At physiological concentrations, few if any of the common dietary amino acids
affect intestinal blood ﬂow when placed into the lumen. Chou et al. (24) and Kvietys et
al. (58) have shown that the luminal placement of a solution containing 16 amino acids,
individually or in combination, at concentrations found postprandially, had no effect on
jejunal blood ﬂow. However, a solution containing these amino acids at concentrations
10 times greater than the postprandial luminal concentrations, did increase intestinal blood
ﬂow (24). Jejunal blood ﬂow was also increased by intraluminal placement of 28 mM
glutamic acid, 20 mM aspartic acid (24) or 300 mM glycine (103). The concentrations
used in these studies, however, exceed the concentrations found in the intestinal lumen
after a meal, and their effect may be pharmacological (42,48). In contrast to the apparent
lack of physiological effect by amino acids, a predigested high protein test diet has been
shown to increase intestinal blood flow when placed in the lumen, and the magnitude of
the hyperemia induced by this diet was similar to that induced by a predigested high
carbohydrate test diet (89). The products of protein digestion responsible for the
hyperemia are unknown. It is well known that many gastrointestinal peptides are
vasodilators in the small intestine (19,25).

Bile is an important constituent of chyme. The postprandial concentration of bile

l 1
in the upper small intestinal lumen ranges from 10 to 33% (48,58). Chou er al. (24) have

shown that placement of a solution containing 33% gallbladder bile into the jejunal lumen
did not affect local blood ﬂow. However, addition of this bile solution to the predigested
food signiﬁcantly enhanced the hyperemic effect of the predigested food (24).
Furthermore, Kvietys et al. (58) have shown that adding bile to the following nutrient
solutions Signiﬁcantly alters the vasoactivity of the nutrients. Thus, 10% bile renders
oleate capable of increasing intestinal blood ﬂow and signiﬁcantly enhances the glucose-
induced increase in blood flow. At a higher concentration (33%), bile renders amino acids
and a short chain fatty acid, caproic acid, vasoactive. The action of bile to render long
chain fatty acids capable of increasing intestinal blood flow is related to its role in micelle
formation and solubilization of the fatty acids (26,58). The means by which bile enhances
the glucose-induced hyperemia is unknown, but it is not due to enhancement of glucose
absorption (26,90). The mechanism by which bile renders amino acids and caproic acid
capable of increasing jejunal blood ﬂow is also unknown. Although bile or the bile salt
taurocholate in the jejunal lumen do not alter local blood flow, they increase local ileal
blood ﬂow when placed into the ileal lumen (24,60,90). This bile-induced ileal hyperemia
is likely related to the absorption of bile salts in the ileum.

In conclusion, the luminal stimuli responsible for postprandial intestinal hyperemia
are the digestive products of fats, carbohydrates, and proteins. Of these three major
nutrients, the micellar lipids (on a weight basis) induce the greatest hyperemia, followed
by glucose. Bile plays an important role in the hyperemia by potentiating the food- or
glucose-induced hyperemia, and by rendering fatty acid and amino acids capable of

inducing the hyperemia. Most of the studies investigating the mechanisms of postprandial

12

intestinal hyperemia, therefore, have utilized oleic acid, glucose, and bile as test nutrients.
Mechanisms of the Pgstprandial Intestinal Hmremia

Intestinal blood ﬂow increases primarily in the mucosal layer of those sections of
the small intestine that are exposed to nutrients. This suggests that postprandial intestinal
hyperemia is a locally mediated vascular response to nutrients in the lumen. Although the
sympathetic nervous system mediates the cardiovascular responses during the
anticipation/ingestion phase, it does not inﬂuence the mesenteric vasodilation during
digestion and absorption. Similarly, the parasympathetic nervous system does not mediate
the postprandial intestinal hyperemia, as vagotomy and denervation of the intestinal
segment do not signiﬁcantly alter food—, glucose- or oleate-induced increase in intestinal
blood ﬂow (23,34,48,7l). Therefore the major mechanisms involved in the initiation and
maintenance of the postprandial intestinal hyperemia are local and intrinsic to the
intestine.

The possible pathways and mechanisms involved in regulation of intestinal blood
ﬂow after a meal are summarized in Figure 1. From this ﬁgure it should be evident that
the mechanisms involved in the regulation are complex, with many factors potentially
playing a role as well as interacting with one another. Nutrients could act directly on the
intestinal vasculature once absorbed, or indirectly by means of metabolic, local neural or
paracrine regulatory mechanisms. There is some evidence in support of each of these
mechanisms of action. Additionally different nutrients may inﬂuence intestinal blood ﬂow
via different mechanisms or combinations of mechanisms (23,42). The mechanisms which
have been implicated as playing a role in the regulation of the intestinal hyperemia that

will be reviewed include 1) direct effeet of the constituents of chyme, 2) the enteric

13

 

[DIGESTED PRODUCTS OF FOOD IN our LUMEiT]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

       

 

 

 

 

 

 

 

 

 

 

 

 

MUCOSAL
MECHANO—. CHEMO TISSUGﬁ ﬁggn'ga'ég
RECEPTORS PEPTIDES
PROSTAN l
ENTERIC NERVOUS 0 DS
SYSTEM (ENS)
I j \

i Y !
MOTILITY ABSORPTION SECRETION
TISSUE METABOLISM

’ P
o, \
METABOLITES
TRANSMURAL ADENOSINE ABSORBED
PRESSURE CHEMICALS
A
BLOOD FLOW
’ CAPILLARY EXCHANGE T
Figure 1. Possible mechanisms and pathways involved in the regulation of

intestinal blood ﬂow during nutrient absorption.

14

nervous system and its reflexes, 3) gastrointestinal hormones and peptides, 4) local tissue
autacoids, and 5) local tissue metabolism.

Some constituents of chyme are vasodilators and therefore can increase local blood
ﬂow once they are absorbed from the gut lumen. Unlike glucose and amino acids, which
do not alter blood flow when infused intra—arterially (21), intra-arterial infusion of a
micellar solution of oleic acid, caproic acid, or taurocholate increases jejunal blood ﬂow
(26). Similarly, intra-arterial infusion of various bile salts also increases ileal blood ﬂow
(60). Carbon dioxide and hydrogen ions are also vasodilators in the small intestine (23).
These vasodilator luminal contents, after their absorption into the mucosal interstitium,
may increase local blood ﬂow by acting directly on vascular smooth muscle or indirectly
via other regulatory mechanisms shown in Figure 1.

The enteric nervous system and its receptors play an important role in regulation
of intestinal functions. The receptors can sense the composition of the intestinal contents
(chemoreceptors, osmoreceptors) and mechanical stimuli (mechanoreceptors) to initiate
reﬂexes, which in turn alter intestinal functions, such as transmucosal transport and motor
activity (18). Whether or not the enteric nervous system plays a signiﬁcant role in
postprandial regulation of intestinal blood ﬂow is controversial. Several studies suggest
that local nerves do not play an important role in the regulation of the nutrient-induced
jejunal hyperemia. Infusion of hexamethonium (ganglionic blocker), methysergide
(serotonin antagonist), tetrodotoxin (Na+ channel blocker), as well as extrinsic denervation
of the jejunal segment, all failed to alter the increases in jejunal blood ﬂow and oxygen
uptake induced by intraluminal glucose or oleate (71). Atropine not only failed to alter

the oleate-induced increases in jejunal blood ﬂow and oxygen uptake (63,72), it actually

15

enhanced a food-induced increase in jejunal blood ﬂow (72). Thus, the food-induced
hyperemia is not mediated by a vasodilator effect of the cholinergic muscarinic
mechanism.

The non-cholinergic, non-adrenergic nerves of the enteric nervous system,
however, may play a role in the regulation. Mechanical stimulation of the mucosal surface
that mimics the movement of chyme in the gut lumen increases local blood ﬂow (17,23).
Similarly, the jejunal hyperemia induced by intraluminal placement of hypertonic glucose
as well as the hyperemia induced by isotonic glucose or oleate was blocked by mucosal
application of a local anesthetic, dibucaine (21,71). However, dibucaine also inhibited the
glucose- or oleate-induced increase in jejunal oxygen uptake in vivo, and inhibited
glucose absorption and mucosal oxygen consumption in vitro (71). The inhibitory action
of dibucaine on the hyperemia, therefore, may be in part due secondarily to its inhibition
of glucose transport and tissue oxygen consumption (71).

Recent studies have shown that capsaicin-sensitive afferent nerves (C-ﬁbers) may
play a role in regulation of gastrointestinal blood ﬂow (19,79). The vasodilatory action
of these nerves are mediated by neuropeptides such as cholecystokinin (CCK), substance
P (SP), and vasoactive intestinal polypeptide (VIP) (19). Rozsa and Jacobson (79) have
shown that the oleate-bile-induced jejunal hyperemia was prevented by treating the jejunal
lumen with lidocaine or capsaicin, and that the hyperemia was absent in rats treated with
capsaicin during their neonatal life. They also showed that VIP antiserum attenuated this
hyperemia, but SP or CCK antiserum, as well as hexamethonium, atropine, or reserpine,
failed to affect the hyperemia (79). Gallavan et al. (40) further Showed that VIP is

released during the jejunal hyperemia induced by intraluminal placement of an oleate-bile

16

solution. These studies suggest that capsaicin-sensitive sensory afferent nerves and VIP
may participate in the jejunal hyperemia produced by oleate.

Although the role of the gastrointestinal (GI) hormones and peptides in regulation
of digestive functions is well established, the precise physiological role of these peptides
in regulating the postprandial hyperemia is still not clearly deﬁned. Most of these peptides
are vasoactive and could act as endocrine, paracrine, or neurocrine mediators in regulating
intestinal blood flow (19). Gastrin, CCK, secretin, gastric inhibitory polypeptide (GIP),
SP, VIP, neurotensin (NT), calcitonin gene-related peptide, glucagon, and enkephalins are
vasodilators in the small intestine, whereas somatostatin and peptide YY are
vasoconstrictors (19,25). Early studies have implicated the involvement of circulating GI
hormones in the vascular response to a meal (25,48). Gastrin, CCK, and secretin are
released into the systemic circulation after a meal and are known to affect GI functions
associated with digestive processes (25,48). However, before a circulating GI hormone
is implicated in the postprandial hyperemia, it must be shown that the hormone can
produce vasodilation at a postprandial blood concentration. Comparisons of the
postprandial blood concentrations of these peptides and their minimal concentrations
required to produce vasoactivity in recent reviews (19,25) and studies (40,46,70) have
indicated that the postprandial blood concentrations of CCK, secretin, gastrin, GIP, and
NT are insufﬁcient to affect intestinal blood ﬂow. It thus appears that these peptides as
well as others do not mediate the postprandial hyperemia as circulating hormones.
However, the local tissue concentrations of these peptides must be much higher than those
in the circulating blood, particularly in the vicinity of their releases (19). Some peptides,

therefore, could contribute in regulation of postprandial intestinal blood flow as paracrines

17

or neurocrines, if their local tissue concentrations are high enough to produce their
vascular actions. The candidate peptides are CCK, VIP, SP, and NT ( 19,48). For example,
the oleate-induced intestinal hyperemia is accompanied by a sustained increase in VIP
release (40) and a transient increase in CCK and NT releases into the local venous blood
(40,46). The hyperemia induced by oleate was blocked by VIP antiserum (79). The
hyperemia induced by mechanical stimulation of the mucosa of the small intestine is
accompanied by an increase in VIP release (19), and the role of CCK may vary with
species and the type of meals, e.g., fats vs carbohydrates (19).

The small intestine releases a variety of local tissue chemicals (autacoids) that
have been implicated as paracrine mediators of the postprandial intestinal hyperemia.
These include serotonin, bradykinin, histamine, and the prostanoids. Serotonin at low
concentrations and bradykinin are vasodilators in the intestine, and both signiﬁcantly
affect intestinal motility (17). Furthermore, both autacoids are released into the portal vein
under various physiological conditions, such as feeding, intraduodenal instillation of
glucose, and mechanical mucosal stimulation (34,48). These two autacoids, therefore, may
contribute to the postprandial intestinal hyperemia, but their physiological importance has
not been clearly deﬁned.

Histamine increases intestinal blood ﬂow and oxygen uptake, and these effects are
mediated by H1 and H2 receptors (27). This study further showed that tripelennamine, an
H1 receptor blocker, signiﬁcantly attenuated the increase in jejunal blood ﬂow induced
by luminal placement of predigested food, and abolished the corresponding increase in
oxygen uptake. Metiamide, an H2 receptor blocker, on the other hand, failed to alter the

food-induced increases in both (27). Hence, endogenous histamine may mediate, via Hl

18

receptors, the food-induced increases in intestinal blood ﬂow and oxygen uptake.

Prostanoids are the cyclooxygenase products of arachidonic acid, and are
vasoactive in the intestine (20,36,45). The presence of nutrients in the lumen stimulates
their production and release in canine jejunum (20). Their role in regulation of intestinal
blood flow is complicated, because the products include both vasodilators and
vasoconstrictors (45). Of the four most commonly produced prostanoids in the intestine,
prostaglandin (PG)I2 is a more potent vasodilator than PGEz, whereas TxA2 is a more
potent vasoconstrictor than PGFu, on weight basis (20). This same study further showed
that these four prostanoids are released into the jejunal venous blood under resting
conditions, and that the food-induced increases in blood ﬂow and oxygen uptake are
accompanied by an increase in their releases (20). The relative magnitude of the food-
induced increases in their releases, however, is not equal. This suggests that prostanoids
could either enhance or inhibit the food-induced hyperemia, depending on the amount of
vasodilator and vasoconstrictor prostanoids released when the nutrient is in the gut lumen.
However, the several studies indicate that the endogenous prostanoids as a whole act to
inhibit the intestinal hyperemia induced by a meal containing equal weights of
carbohydrate, protein, and fat in dogs. The food-induced increases in blood ﬂow and
oxygen uptake are enhanced by inhibition of prostanoid synthesis with cyclooxygenase
inhibitors (mefenamate or indomethacin) (41), but are attenuated by substrate loading with
prostanoid precurser (arachidonate) (67).

The next step was to identify which prostanoid vasoconstrictor plays a signiﬁcant
role in limiting the hyperemia. TxA2 is a much more potent vasoconstrictor than PGFZ,

and quantitatively venous TxA2 concentration during nutrient absorption is twofold greater

19

than that of PGFZ, (20). Furthermore, TxA2 produces a greater vasoconstriction in the
mucosa (the site of nutrient absorption) than in the muscularis (3), and it is the major
prostanoid produced by the mucosa (65). The role of TxA2 in the regulation of the food-
induced increase in blood ﬂow and oxygen uptake has been assessed utilizing its analog
(II-44069), its receptor blocker (SQ 29548), and TxA2 synthase inhibitors (irnidazole and
U—63557A) (2,68,69). These studies showed that endogenous TxA2 limits the food-induced
increase in intestinal oxygen uptake and capillary ﬁltration coefﬁcient, but does not affect
the increase in blood ﬂow.

As in any organ, intestinal blood ﬂow regulation is closely linked to the metabolic
status of the intestine. Intestinal absorption, secretion, and rhythmic contractions are
accompanied by an increase in intestinal oxygen uptake (17,18,48). The neurohumoral and
local tissue chemicals discussed above can not only act directly on the vasculature to alter
local blood ﬂow; they can also inﬂuence the intestinal functions and metabolism, which
in turn alters local blood ﬂow. Almost all of these factors that enhance or inhibit
postprandial intestinal hyperemia also produce parallel changes in oxygen uptake (18). For
example, the enhancement of the food-induced hyperemia by indomethacin or
mefenamate, and the attenuation of the hyperemia by histamine H1 receptor blocker or
arachidonate, are accompanied by simultaneous enhancement and attenuation of the food-
induced increase in oxygen uptake, respectively (20,27,41,67). Also, the increases in blood
ﬂow and oxygen uptake before and after mefenamate or arachidonate are linearly and
positively correlated (41,67). Inasmuch as changes in intestinal oxygen uptake are blood
ﬂow independent over the physiological range (30—140 ml/min/ 100 g) (67), the action of

these chemicals on food-induced increase in intestinal oxygen uptake is most likely due

20

to their action on tissue oxidative metabolism, rather than to their action on blood ﬂow.
This is further supported by the ﬁnding that cyclooxygenase inhibition increases
carbohydrate absorption and metabolism while enhancing the food-induced hyperemia
(43). Therefore, there is strong possibility that these enhancers or inhibitors can act
indirectly via intestinal functions and oxidative metabolism to affect local blood flow.
Of all the possible mechanisms involved in initiating and maintaining the
postprandial intestinal hyperemia, tissue oxidative metabolism has been proposed to be
the most important (18,42). It has been estimated that two-thirds of the hyperemia can be
attributed to tissue metabolism (18,48). The primary function of the intestinal circulation
is to deliver oxygen to the functioning tissue, and the arteriolar and precapillary sphincter
tone are closely linked to the metabolic status of the intestinal tissue. The metabolic
theory of blood flow regulation predicts that an increase in tissue oxygen demand, as a
result of increased tissue metabolism, will result in a decrease in tissue oxygen tension
and an increase in the concentration of vasodilator metabolites in the interstitial ﬂuid (42).
Both events will relax the arterioles and precapillary sphincters, leading to an increase in
local blood ﬂow and the number of capillaries being perfused. Oxygen delivery to tissues
is increased and the capillary to cell diffusion distance is decreased, both of which serve
to maintain tissue oxygen tension at a level that does not limit the rate of aerobic
metabolism (42). Thus, the increased tissue oxygen demands can be met by increasing
blood flow, capillary density, and tissue oxygen extraction (18,48). The intestinal
hyperemia occurring during glucose absorption is a good example. This hyperemia is
characterized by increases in intestinal oxygen uptake (12 - 25%), the capillary

permeability-surface area (18 - 36%), and oxygen extraction (0 - 15%), with the

21
corresponding increase in blood flow (7 - 19%) (26,71,74,88,90,9l).

Sit et al. (90) have investigated the role of glucose absorption and metabolism in
glucose-induced hyperemia. They compared the effects of glucose (absorbed and
metabolized), 3-O—methylglucose (absorbed but not metabolized) and 2-deoxyglucose
(neither absorbed nor metabolized). Luminal placement of glucose-bile solution increased
both blood flow (19%) and oxygen uptake (17%), whereas 3-O-methylglucose-bile
slightly increased blood ﬂow (7%), but did not signiﬁcantly increase oxygen uptake. The
glucose-induced hyperemia was signiﬁcantly greater than that induced by 3-0-
methylglucose. The nonabsorbable and non-metabolized analogue 2-deoxyglucose failed
to increase both blood flow and oxygen uptake. Thus, both absorption and oxidative
metabolism play signiﬁcant roles in glucose-induced jejunal hyperemia.

The mediators proposed to link tissue metabolism and intestinal blood ﬂow are
tissue oxygen tension (P0,) and vasodilator metabolites. Bohlen (8,9) has shown that
tissue P02 in the villus tip decreases during glucose absorption in the rat intestine. This
glucose-induced decrease in tissue P02 is concomitant with, and inversely correlated with,
the increase in blood ﬂow. This suggests that the glucose-induced hyperemia is in part
mediated by a decrease in tissue P0,.

The possible vasodilator metabolites that have been proposed to be involved in the
regulation of postprandial intestinal hyperemia are adenosine, K*, H‘, and tissue
osmolality (18,42,48). Adenosine is a vasodilator in the intestinal mucosa, and the food-
induced increases in blood ﬂow and oxygen uptake are accompanied by an increase in
adenosine release into the local venous blood and lymph (81,82,83,84). Furthermore, the

food-induced jejunal hyperemia is inhibited by adenosine antagonists (aminophylline, 8-

22

phenyltheophylline, adenosine deaminase), but is enhanced by dipyridamole (a chemical
which ampliﬁes adenosine accumulation in the interstitial ﬂuid) (81,84). Therefore,
adenosine may play a role in postprandial intestinal hyperemia. Potassium and hydrogen
ions are vasodilators, but their importance in regulation of postprandial intestinal
hyperemia has not been systemically investigated ( 18,48). An increase in plasma
osmolarity increases intestinal blood ﬂow. However, there is conﬂicting evidence
regarding the extent to which changes in tissue osmolarity inﬂuence postprandial intestinal
blood ﬂow (42,48). Tissue hyperosmolarity may play a role in contributing the
hyperemia in rats (10), but its importance in other species is still unclear. For example,
a recent study showed that luminal placement of a glucose-electrolyte solution does not
signiﬁcantly alter mucosal tissue osmolarity in the canine jejunum and proximal ileum
(31).

In summary, the intestinal vascular response to the presence of nutrients in the
lumen is mediated by a variety of regulatory pathways which vary with the nutrient
(Figure 1). The mechanisms involved are local and intrinsic to the intestine. Presence of
nutrients in the lumen initiates nutrient absorption and stimulates the enteric nervous
system and releases of local GI peptides and autacoids. These neurohumoral factors can
alter local blood ﬂow directly by acting on vascular smooth muscle, or indirectly by
altering intestinal functions and tissue metabolism. The possible mediators linking
metabolism and blood ﬂow are tissue oxygen tension and adenosine. It is likely that the
postprandial intestinal hyperemia results from a complex interplay of many factors, most

of which enhance, while a few, such as prostanoids, inhibit the hyperemia.

23
Prostanoids

Prostanoids are the products of arachidonic acid metabolism formed via the
cyclooxygenase pathway. Prostanoids are synthesized by virtually all mammalian tissues
(96). Prostanoid formation involves three phases: 1) mobilization of arachidonic acid from
membrane glycerophospholipids via the actions of lipases, 2) conversion of the liberated
arachidonate to prostaglandin (PG) G2 and then to prostaglandin endoperoxide, PGHZ, by
PGH synthase, and 3) speciﬁc isomerization or reduction of PGH2 to the major
biologically active prostanoids, PGEZ, PGFZ, PGDZ, prostacyclin or PGIZ, and thromboxane
(Tx) A2 (94.95.96). Prostanoids are not stored in cells (95), rather they are synthesized
in response to various stimuli. Therefore cells must rapidly mobilize arachidonate for
conversion to prostanoids (95). The mobilization of arachidonate is an important control
point in the regulation of prostanoid synthesis and is considered to be the rate limiting
step (96). The exact biochemical details of arachidonate mobilization on unresolved (96),
but it is known that arachidonate can be mobilized via at least two pathways (95,96,97).
The first pathway involves the action of phospholipase A2 to cleave arachidonate from the
sn-2 position of phosphatidylcholine or phosphatidylethanolamine. The second pathway
involves the sequential action of phospholipase C, and a diglyceride lipase on
phosphatidylinositol. The mobilization of arachidonate by phospholipase A2 is currently
considered to be quantitatively the most important pathway (95). However, in some cell
types, such as platelets, the phospholipase C pathway is responsible for a large portion
of the arachidonate mobilization in response to certain stimuli (95,96).

The mobilization of arachidonate and subsequent prostanoids synthesis can be

elicited by a large number of stimuli. The stimuli have been categorized as physiological,

24

physical, or pharmacological (96). Physiological stimuli include hormones, such as
histamine, and bradykinin, and proteases, such as thrombin (96). These "physiological"
stimuli are thought to act via cell surface receptors to cause the activation of
phospholipases and the selective mobilization of arachidonate (96). An example of a
physical stimulus which causes prostanoids synthesis is the shear forces acting on
endothelial cells which stimulate PGI2 production. Pharmacological stimuli include the
Ca++ inophore A23187, and phorbol esters. These agents are thought to non-Speciﬁcally
increase in the activity of phospholipases and thus mobilize arachidonate (96).

After arachidonate is mobilized, it is converted to an endoperoxide, PGH2, by PGH
synthase (95). PGH synthase exhibits both the cyclooxygenase activity to convert
arachidonate to PGGZ, and the peroxide activity to convert PGG2 to PGH2 (96). These
activities are contained on the same protein. Current theory suggests that PGH synthase
within the cell does not require "activation" but is essentially in an active state awaiting
the mobilization of arachidonate (96). Non-steroidal anti-inﬂammatory drugs, such as
mefenamate and indomethacin, inhibit prostanoid synthesis by interacting with the
cyclooxygenase site of PGH synthase and competing with arachidonate for binding
(95,96). Hence, the terminology cyclooxygenase inhibitors.

Endoperoxide PGH2 is then converted to the biologically active prostanoids. In
general this conversion is considered to be cell speciﬁc, with a given cell forming
predominantly one type of the major prostanoids in relative abundance (95,97). The
conversion of PGH2 to PGEZ, PGFm, PGIZ, and TxA2 is generally considered to be
catalyzed by PGE synthase, PGF synthase, PGI synthase, and TxA synthase, respectively.

Prostanoids are local hormones which act near the site of their production (95),

25

as autocrine and paracrine mediators. In general, prostanoids are thought to act via cell
surface receptors (96,97). There is pharmacological evidence for a distinct receptor or
set of receptors for each major prostanoid (Smith, Gardiner). For example, three
pharmacologically distinct PGEz receptors, EPl, EP2, and EP3, have been described
(47,97). There is also some evidence for at least two subtypes of TxAzlendoperoxide
receptors (47). Phannacologically distinct receptors for PGI2 and PGFZ, also exist (47).
These prostanoid receptors appear to be coupled to guanine nucleotide regulatory proteins
for signal transduction (95,97). The general intracellular responses to prostanoids include
an increase or decrease in CAMP concentration, or an increase in intracellular Ca++
concentration (95,97). For example, there is considerable evidence that in the renal tubule
PGE, can act via an EP2 receptor to increase CAMP formation, an EP3 receptor to
decrease CAMP formation, or an EPl receptor to mobilize Ca++ (97). These responses are
the same as the changes in second messengers which occur during signal transduction by
guanine nucleotide protein linked receptors (95). Therefore it appears that prostanoids act
via cell surface receptors that are linked to guanine nucleotide regulatory proteins which
cause changes in second messengers such as CAMP, IP_,, and Ca”.
Prostanoids in the Gastrointestinal Tract

There has been considerable interest in the physiological and pharmacological
effects of prostanoids in the intestinal tract (110). Prostanoids are produced throughout
the intestinal tract. A multitude of studies have shown that prostanoids or their analogues
are capable of inﬂuencing intestinal functions including, motility, secretion, absorption,
and blood ﬂow. Numerous other studies which utilized cyclooxygenase inhibitors,

substrate loading with arachidonate, and TxA2 synthase inhibitors and receptor blockers

26

provide evidence that endogenous prostanoids may play a physiological role in the
regulation of these intestinal functions.

Prostanoid synthesis occurs throughout the small intestine in the mucosal as well
as the muscular layers of the gut wall LeDuc and Needleman (65) have Shown that
microsomal preparations of canine jejunal mucosa produce TxA2 (15%), PGI2 (14%),
PGE, (10%), PGF” (5%) and PGD2 (4%) (with 51% non-speciﬁc hydroxy-fatty acids).
These authors suggested that the cyclooxygenase activity of the intestinal mucosa was
relatively low because PGH2 enhanced prostanoids production more than did arachidonate.
Other studies also indicate that the intestinal mucosa is capable of synthesizing these same
prostanoids, but the proﬁle of the prostanoids synthesized varies between the studies
(l,5,7,64,108). The muscular layer produces predominantly PGI2 and lesser amounts of
PGE2 and PGF” (65,80).

Chou et al. (20) has shown that PGI2 (44%) > PGE, (32%) > TxA2 (21%) > PGFm
(2.5%) were produced by an isolated jejunal segment and released in to the venous blood
draining the segment at rest. These prostanoids represent those produced and released by
the whole gut wall rather than any individual tissue layer, but are similar to the in vitro
studies (l,5,7,65,108) in that TxA” PGEZ, and PGI2 are the prostanoids commonly
produced.

Several stimuli have been shown to increase intestinal prostanoid production in
vitro and in viva. Kallidin, a kinin, increases PGEz production by ileal mucosa in vitro
(109). Lawson and Powell (64) have shown that bradykinin Stimulates the synthesis of
PGE, and 6-keto-PGFm (metabolite of PGIZ) by ileal mucosa in vitro. Two studies

indicate that prostanoids are produced in response to a meal. Dupont etal. (29) showed

27

that PGE2 and PGF” concentrations increase in the intestinal tract of miniature swine
after a liquid meal. These authors concluded that feeding stimulated prostanoid release.
More recently, Chou et al. (20) showed that intraluminal placement of a digested food
mixture that contained equal parts by weight of fats, carbohydrate, and protein
signiﬁcantly increased the venous concentrations and release of PGE2, 6-keto-PGFm
(metabolite of PGIZ), Tsz (metabolite of TxAz), and PGF” from isolated jejunal segment
in viva. The release of PGI2 and TxA2 increased twofold, PGE2 increased fourfold, and
PGFZ, increased eightfold (20). These studies indicate that the small intestine is capable
of synthesizing and releasing prostanoids at rest and in response to stimuli including
nutrients in the lumen.

One of the earliest observed effects of prostanoids was their ability to alter
intestinal smooth muscle tone and contractions (110). There are numerous reports
describing the effects of prostanoids on intestinal motility, however the effects reported
by early studies show considerable variability that seems to depend on the type of
prostanoid, the dose and route administered, and the species studied (100). Yet from these
studies, in general PGE, and PGI2 relax Circular intestinal smooth muscle, while PGF2m
stimulates contraction of the circular smooth muscle (100). Further systematic study
indicates that the effect of prostanoids on intestinal motility may depend on the pattern
of motility at the time of prostanoid administration (100). In this study, PGE, and PGI2
administered intravenously or via local intraarterial infusion decreased the frequency of
the migrating myoelectrical complexes in fasted dogs, and decreased spiking activity in
fed dogs. This suggests that PGE, and PGI2 inhibit intestinal motility in both the fasted

and fed state. PGFZ. administered intravenously or via local intraarterial infusion

28

interrupted the migrating myoelectrical complex and induced spiking activity similar to
a fed pattern in fasted dogs, but had no effect on motility of fed dogs (100). This suggests
that PGFZ, increases spiking activity in fasted but not fed dogs. The same study further
showed that cyclooxygenase inhibition with indomethacin induced spike activity in fasted
dogs, but had no effect on motility in fed dogs (100), suggesting that prostanoids may
play a role in regulating intestinal motility. Tollstrom et al. (101) reported similar ﬁndings
that PGEz decreased motility, where as PGFZ, increased phasic contractions in fasted
humans.

Prostanoids, especially PGEz, are potent secretogues (110). Exogenous PGE2 and
PGF”, increase intestinal secretion both in vitro and in viva (14,110). Buhkave and Rask-
Madsen (14) showed that physiological concentrations of PGE; (10“- 10 8M) inhibits Na+2
absorption and enhances ﬂuid secretion. Indomethacin enhanced these responses to PGEz
as well as increases Na+ and net ﬂuid absorption. Similar effects of PGE2 have been
reported in viva. Brunsson et al. (13) reported the PGE2 intraluminally or intraarterially,
or arachidonate intraluminally increased net ﬂuid secretion in the rat jejunum. These
authors suggest that the mechanism by which PGE2 enhances net secretion may be dose-
dependent since at low concentrations (approximately 10 9- 10'8M) PGE2 increases ﬂuid
secretion via a neurogenic mechanism that was blocked by intravenous hexamethonium
or intraluminal lidocaine, where as at higher concentrations (10 8- 10‘7M) PGE2 increases
secretion by increasing tissue CAMP levels (13). The ﬁnding that low concentrations of
PGE2 may increase secretion via a non-CAMP mechanism is supported by the in vitro
study of Buhkave and Rask-Madsen (14). PGFq or its analogue 16,16-dimethyl PGE2

administered into the superior mesenteric artery (2pg/kg/hr) signiﬁcantly increased net

29

ﬂuid secretion by the canine jejunum and ileum in viva (56). Conversely, indomethacin
enhances water absorption (56). These studies suggest that PGE2 may play a role in
regulating intestinal secretion. The effects of other prostanoids on intestinal ion transport
and secretion are less well studied. PGF” has been reported to either have no effect on
ion transport and net ﬂuid movement (14), or increase net secretion (75). Prostacyclin,
PGIz, has been reported to attenuate the increase in secretion induced by PGE2 (110).
More recently, TxA2 has been shown to decrease Na+ absorption by the intestinal mucosa
in vitro (93). These studies suggest that not only can exogenous prostanoids affect
intestinal ion transport and secretion but that endogenous prostanoids may play a
physiological role in the regulation of intestinal ion and ﬂuid transport.

There is evidence that prostanoids also inﬂuence nutrient absorption by the
intestine. Inasmuch as some prostanoids have been shown to inhibit Na+ absorption, they
might also inhibit glucose absorption; a transport process that is dependent on a Na“
gradient. Coupar and McColl (28) have reported that PGE, and PGF,“ inhibit glucose
absorption by the rat jejunum. Additionally, local intraarterial infusion of PGE2 or PGFm
decreased glucose absorption by isolated jejunal segments independent of the affects of
these prostanoids on blood ﬂow (6). Grass er al. (50) have shown that PGE, analogues,
enprostil and RS-86505-007, inhibit glucose absorption by jejunal mucosa in vitro. These
authors suggest that the inhibitory effect of these PGE, analogues may be due to an
enhanced "barrier" property of the intestinal mucosa possibly by increased mucus
secretion. Enprostil has also been reported to decrease glucose absorption and post-meal
serum glucose levels in humans (86). Gallavan and Chou (43) have shown that

cyclooxygenase inhibition by mefenamate enhances glucose absorption and carbohydrate

30

metabolism while a digested food mixture was in the lumen. These studies suggest that
prostanoids can inhibit glucose absorption, and that endogenous prostanoids may play a
role in regulating glucose absorption and metabolism. The effects of prostanoids on the
absorption of other nutrients is unexplored.

Prostanoids may also play a role in the regulation of intestinal blood ﬂow under
resting and postprandial conditions. However, their role in the regulation of intestinal
blood ﬂow is complicated. Different prostanoids have different vascular actions in the
mesenteric circulation as PGI2 is a more potent vasodilator than PGEZ, and TxA2 is a
more potent vasoconstrictor than PGF20L (20,45). In addition to their vascular actions,
prostanoids might also affect intestinal functions and oxidative metabolism and thereby
indirectly inﬂuence blood ﬂow. At rest the jejunum produces and releases prostanoids into
the venous blood (20). Cyclooxygenase inhibitors decrease resting intestinal blood ﬂow
(41,45), suggesting that vasodilator prostanoids may regulate intestinal blood ﬂow under
resting conditions.

Endogenous prostanoids may also play a role in the regulation of the postprandial
increase in blood ﬂow and oxygen uptake. The food-induced increases in jejunal blood
ﬂow and oxygen uptake are accompanied by an increase in the release of prostanoids
(20). Inhibiting prostanoid synthesis with cyclooxygenase inhibitors, mefenamate or
indomethacin, enhances the food-induced increase in blood ﬂow and oxygen uptake (41).
Substrate loading with arachidonate, the precursor of prostanoids, attenuates the food-
induced increase in blood ﬂow and oxygen uptake (67). Additionally, mefenamate blocked
the arachidonate-induced attenuation of the food-induced increase in blood ﬂow and

oxygen uptake (20). The effect of arachidonate was therefore most likely due to

31

prostanoids (20). These studies indicate that endogenous prostanoids as a whole act to
limit the food-induced increase in blood ﬂow and oxygen uptake.

From these aforementioned studies it was proposed that the effect of endogenous
prostanoids may be due, in part, to their actions on intestinal functions and tissue
oxidative metabolism (20,41,67). This thesis is supported by the following evidence.
Cyclooxygenase inhibition enhances whereas arachidonate attenuates the food-induced
increase in blood ﬂow and oxygen uptake in a parallel manner (41,67). Further, the
increase in blood ﬂow and oxygen uptake before and after mefenamate or arachidonate
are linearly and positively correlated (41,67). Since intestinal oxygen uptake is largely
independent of blood ﬂow even when food is in the lumen (67,48), the enhancement by
cyclooxygenase inhibition and attenuation by arachidonate of the food-induced increase
in oxygen uptake is most likely due to the enhancement or attenuation of intestinal tissue
oxidative metabolism. Cyclooxygenase inhibition has also been shown to increase
carbohydrate absorption and metabolism while enhancing the food-induced hyperemia
(43). Additionally, PGE, and PGF” have been shown to inhibit glucose absorption (6,28).
As described above, prostanoids have been shown to alter other intestinal functions as
well. Hence, it seems likely that endogenous prostanoids might limit intestinal functions
and oxidative metabolism, and thereby indirectly limit the food-induced hyperemia.

These studies suggest that a vasoconstrictor prostanoid might play a role in
limiting the food-induced hyperemia. Thromboxane A2 is a more potent vasoconstrictor
than PGF” (20,45), and the venous concentration of TxA2 is greater than that of PGF,“
during nutrient absorption (20). TxA2 is produced by the intestinal mucosa (65), and is

a more potent vasoconstrictor in the mucosa (the primary site of the nutrient-induced

32

hyperemia) than in the muscularis (3). Hence, the role of TxA2 in the regulation of the
food-induced hyperemia was assessed. TxA2 synthase inhibitors as well as a TxA2 /
endoperoxide receptor blocker enhance the food-induced increase in oxygen uptake and
capillary ﬁltration coefﬁcient (capillary exchange density), but do not affect the increase
in blood ﬂow (2,68,69). Thus endogenous TxA2 limits the food-induced increase in
oxygen uptake and capillary exchange density, but does not affect blood ﬂow. From these
Studies it was proposed that TxA2 might limit the food-induced increase in oxygen uptake
by limiting the increase in intestinal oxidative metabolism (2).

These studies indicate that endogenous prostanoids, and in particular TxA2 aCt to
limit the food-induced increases in blood ﬂow and oxygen uptake. Most of the
aforementioned studies investigated the role of prostanoids utilizing a food mixture that
contained equal parts by weight of fats, carbohydrates, and proteins. Therefore it is
unknown if prostanoids play a role in the regulation of the increases in blood ﬂow and
oxygen uptake induced by individual nutrients. Inasmuch as glucose and oleate are
absorbed by different processes and evoke different vascular responses (26), different
types or quantities of prostanoids may play a role in the regulation of the responses to
these nutrients. Therefore the ﬁrst objective of this study is to determine if prostanoids
play a role in the regulation of glucose- and oleate-induced increases in blood ﬂow and
oxygen uptake.

In the above studies it was proposed that prostanoids and TxA2 limit the food-
induced increase in oxygen uptake by limiting the food-induced increase in intestinal
oxidative metabolism. However, in in viva conditions, changes in intestinal oxygen uptake

could be inﬂuenced by changes in tissue oxidative metabolism as well as vascular factors

33

(48,59). Inasmuch as the aforementioned studies were performed using naturally perfused
segments of the intestine in viva, the effects of the prostanoids on intestinal oxygen
uptake cannot be completely separated from their vascular actions. The second objective
of the present study was to determine the effect of the prostanoids, particularly TxAz, on
oxygen consumption of the jejunal mucosa in vitro, independently of their vascular action,

utilizing a constant volume manometry.

MATERIALS AND METHODS

Effect of Prostanoids on Glucose- and Oleate-Induced Changes in Blood Flow and
Oxygen Uptake in viva.
Surgical Preparation

All experiments were conducted on mongrel dogs (15-30 kg) of either sex which were
deprived of food for 24 hours. The animals were anesthetized with pentobarbital sodium
(30 mg/kg i.v.) and supplemented as needed to maintain a surgical plane of anesthesia.
All animals were ventilated with a positive-pressure respirator (Harvard Apparatus, S.
Natick, MA) that was adjusted to achieve normal arterial blood pH, 02 tension, and CO2
tension before each experiment. Systemic arterial pressure was continuously monitored
through a cannula in a femoral artery.

In all experiments a midline incision was made and a segment of jejunum (20 - 40
g, approximately 30 cm aboral to the ligament of Treitz) perfused by a single artery and
drained by a single vein was exteriorized. Before cannulation of the intestinal blood
vessels, heparin sodium (500 U/kg) was administered intravenously. In all experiments,
the single vein draining the segment was cannulated for measurement of venous outflow
by timed collection with a graduated cylinder and stop watch. Arteriovenous oxygen
content difference (Cao2 - Cvoz) was determined continuously by perfusing femoral

arterial blood and a portion of the venous outﬂow blood (6 ml/min) through separate

34

35

cuvettes of an arteriovenous oxygen content difference analyzer (A-Vox Systems, San
Antonio, TX) (87) with a Gilson pump (Minipuls 2, Gilson Medical Electronics ,
Middleton, WI). The venous outﬂow and outﬂows from the cuvettes were directed to a
reservoir. The blood from the reservoir was pumped back to the animal via a femoral vein
at a rate equal to the total outﬂows. A rubber tube, for the placement and withdrawal of
solutions, was placed into the lumen of the segment and both ends of the segment were
tied and cut away from the adjacent jejunum to exclude collateral ﬂow (24). Luminal
pressure was recorded via the rubber tube connected to a pressure transducer (Stratharn
P23Gb) during the presence of a solution in the lumen. The segment was covered with
a plastic sheet to prevent desiccation, and kept at 37° C with a heat lamp and a
therrnoregulator (Yellow Springs Instruments, Model 63RC, Yellow Springs, OH). Figure
2 diagrammatically shows the experimental preparation. The weight of the segment was
obtained at the end of each experiment. Oxygen uptake (V01) was calculated as the
product of blood ﬂow and (Cao2 - Cvoz). Blood ﬂow and oxygen uptake were expressed
as ml-min"o100g". Resistance was calculated as the quotient of mean arterial pressure and
blood ﬂow and expressed as mmHg-ml“-min“-100g".

Solutions: Solutions containing 200 mM glucose or 40 mM oleate were prepared to
be approximately isotonic in distilled water or normal saline and included 10%
gallbladder bile obtained from the animal prior to each experiment. The nutrient solutions
and normal saline for intraluminal placement were kept at 37° C during the experiment.
These concentrations of glucose and oleate are within the ranges previously reported to
be found in the upper small intestinal lumen postprandially (12,53). Mefenamate was

prepared by adding the appropriate quantity of mefenarnic acid (Sigma Chemical, St.

36

  
  

 

 

Normal
' : Salme
or
Nutrient
Systemic
Arterial
Pressure
I
F oral
Femoral 32in
Artery

 

AVOx

 

(C302 ' CV02)

 

Figure 2. Intestinal preparation.

37
Louis, M0) to normal saline and then adding 1.0 M NaOH until the drug went into

solution. SQ 29548, a TxAz/endoperoxide receptor blocker, and U—44069, a
TxAzlendoperoxide analogue, (Cayman Chemical CO., Ann Arbor, M1) were prepared in
normal saline.
Exmrimental Protocol

The objectives of the ﬁrst two series of experiments were to determine the effect of
cyclooxygenase inhibition by mefenamate on glucose- and oleate-induced increases in
jejunal blood ﬂow and oxygen uptake, and to determine the jejunal production of PGE2,
6-keto-PGFw (metabolite of PGIZ), PGF”, and TxB2 (metabolite of TxAz) when the lumen
contained normal saline or glucose, and normal saline or oleate. The effect of intraluminal
placement of glucose (series I, n=7) or oleate (series II, n=7) on jejunal blood ﬂow and
oxygen uptake was determined before and one hour after administration of mefenamate.
The same experimental protocol was used in both series. Ten to ﬁfteen milliliters of
normal saline was placed into the lumen for 15 minutes. This procedure was repeated
until blood ﬂow and (Cao2 - Cvoz) reached a steady state. Then either glucose or oleate
was placed into the lumen for 15 minutes and blood ﬂow and (Cao2 - Cvoz) measured.
The nutrient solution was then withdrawn and replaced with normal saline for 15 minutes.
Mefenamate (10 mg/kg, slowly i.v.) was then administered. One hour after administering
mefenamate, the effect of the same nutrient was again determined as described above. The
dose of mefenamate used in this study has been shown to effectively block prostanoid
synthesis (20,37,69). In this series of experiments, the efﬁcacy of mefenamate in blocking
prostanoid synthesis was tested in two dogs by bolus injection of arachidonate (Sigma

Chemical, St. Louis, MO) (300 pg/kg) through a catheter placed into the pulmonary artery

38

via a femoral vein. A large bolus of arachidonate injected into the pulmonary circulation
produces large circulating levels of the vasodilator PGI2 (30). The arachidonate injection
produced a decrease in aortic pressure, which was blocked 1 hour after mefenamate
administration (Figure 3). Data obtained during the last 3 minutes of the 15 minute
normal saline or nutrient placement period were used for analysis because the blood ﬂow
and (Cao2 - Cvoz) at that time were at a steady state (20,24). The Change in blood ﬂow
due to glucose or oleate was determined as the difference in the steady state blood ﬂow
during luminal placement of normal saline and the respective nutrient

For the determination of prostanoid concentrations, blood samples were collected in
plastic test tubes containing the cyclooxygenase inhibitor mefenamate (5 jig/ml blood).
The samples were taken simultaneously from a femoral artery and the venous outﬂow
from the jejunal segment 11 and 15 minutes after the luminal placement of normal saline
or glucose (series I), and normal saline or oleate (series II). The venous outﬂow was
measured at the time of sampling. Blood samples were centrifuged immediately for 10
minutes. The plasma was removed and stored at -20°C until radioirnmunoassay.

Radioimmunoassay was performed as previously described (20) using a standard
technique. Brieﬂy, Tsz, PGEz, PGFm, and 6-keto-PGFm antisera (Advanced Magnetics,
Cambridge, MA) were appropriately diluted in phosphate- buffered saline [0.1 N
phosphate buffer, 0.9% NaCl, 0.5% gelatin, (PBSG)]. Aliquots (100 pl) of the appropriate
antisera were mixed with 100 pl aliquots of either Standard (Advanced Magnetics,
Cambridge, MA) or plasma sample. The standard solutions were made by serial dilution
of the prostanoid standard with prostanoid-free canine plasma, which was prepared by

stripping the plasma collected from mefenamate treated dogs with charcoal. The standard

39

Figure 3. The effect of a bolus of arachidonate (300 rig/kg, administered into the
pulmonary artery) on aortic pressure before and 1 hour after
administration of mefenamate (lOmg/kg i.v.).

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41

solutions and plasma samples were then mixed with the appropriate radioactive tracer
prostanoid (New England Nuclear, Boston,MA), 6000 cpm in 100 III PBSG, so that the
total reaction volume was 300 III. After incubation (0.5-1 hour at ambient temperature,
then 16-24 hours at 4°C), the protein-bound material was isolated by adding 1.0 ml of
dextran-coated charcoal suspension (5 mg/ml charcoal, 0.5 mg/ml dextran in PBSG). The
tubes were vortexed and the centrifuged for 12 minutes at 1500 rpm and 0°C. The
supernatant of each tube was decanted into a vial containing 15 ml scintillation cocktail
(Safety-Solve, Research Products International, Mt. Prospect, IL), and the radioactivity
was determined in a beta scintillation counter (model A300C, Packard, Downers Grove,
IL). Duplicate values were averaged and compared with the standard curve performed
with each assay. The antisera cross-reactivity was as follows: 6-keto-PGFml with Tsz,
PGDz, PGAQ, PGA,, PGBZ, PGBl <0.01%, PGF,“ (7.8%), 6-1ceto-PGEl (6.8%), PGEl
(0.7%), and PGE, (0.6%); TxB2 with 6-keto-PGFm, PGF”, PGF”, PGE” PGEz, PGDZ,
PGAz, PGA,, PGB,, and PGE2 < 0.1%; PGE, with Tsz, 6-keto-PGFm, PGF”, 6-keto-
PGEl, PGBZ, PGBI, PGD2 < 1.0%, PGF” (1.3%), PGEl (50%), PGA2 (6.0%), and PGAl
(3.0%); and PGFm with 6-keto-PGE1, PGBZ, PGBI, PGDz, PGAz, PGA, < 0.5%, 6-keto-
PGFml (1.1%), PGF,“ (100%), PGEl (1.1%), and PGE2 (0.3%).

In the next four series of experiments (series III- VI), the surgical preparation was the
same as described above with one addition. The single artery supplying the segment was
cannulated, and a perfusion circuit established between a femoral artery and the single
artery. The circuit was designed to allow the jejunal segment to be naturally perfused by
aortic blood and for the local, intraarterial infusion of treatment chemicals (3,67).

Prostanoid synthesis inhibition by mefenamate increased resting vascular resistance

42

of the jejunal segment, which has been shown to alter the jejunal vascular response to
intraluminal placement of digested food (41). Therefore, in series III and IV, angiotensin
II was infused intraarterially via the perfusion circuit to reduce resting blood ﬂow of the
segment to approximately the same extent as seen after prostanoid synthesis inhibition
with mefenamate. The protocol was the same as that for series I and II. The effect of
intraluminal placement of glucose (series III, n=7) or oleate (series IV, n=7) on jejunal
blood ﬂow and oxygen uptake was determined before and during angiotensin H infusion
(0.65 ngmin’1-100g"). These series of experiments served as controls for the effect of
increased resting vascular resistance on the jejunal responses to glucose and oleate.
The objective of the next two series of experiments was to determine the effect of SQ
29548, a TxA2/endoperoxide receptor blocker, on the glucose- and oleate-induced changes
in blood ﬂow and oxygen uptake. SQ 29548 has been shown to be a highly speciﬁc
thromboxane A2 receptor antagonist (73,98). The protocol was the same as that used in
the previous series of experiments. The effect of intraluminal placement of glucose (series
V, n=7) or oleate (series VI, n=7) on jejunal blood ﬂow and oxygen uptake was
determined before and after administration of SQ 29548. After determining the effect of
glucose or oleate on jejunal blood ﬂow and (Cao2 - Cvoz), SQ 29548 (2 pg) was
administered intraarterially into the perfusion circuit. Glucose or oleate was then placed
into the lumen for 15 minutes and the jejunal responses again determined. This dose of
SQ 29548 was previously shown to be effective in blocking the vasoconstriction induced
by U-44069, a TxAQ/endoperoxide analogue (2,3). In these series of experiments, the
efficacy of SQ 29548 was tested by administering a bolus of U-44069 (0.1 pg)

intraarterially before and after the administration of SQ 29548. Before SQ 29548, U-

43
44069 decreased jejunal blood ﬂow 44i8%, where as after SQ 29548, U-44069 did not

signiﬁcantly affect blood flow.

Statistical Analysis: All values are expressed as the mean .t SE. The data were
analyzed using a Student’s t test modiﬁed for comparison of paired sample means, or a
random block ANOVA employing Neuman-Student-Keuls post hoc test for comparison

of the means. Statistical signiﬁcance was set at p<0.05.

44

Effect of Prostanoids on Mucosal Oxygen Consumption in vitro.
Measurement of Mucosal Oxygem Consumption in vitro.

Jejunal mucosal oxygen consumption was measured using constant volume
manometry (Warburg Apparatus, Fisher Scientiﬁc, Pittsburgh, PA). Krebs-Ringers
phosphate buffer (KRPB) solution that contained one-half the normal calcium
concentration was the media used in all experiments (66). Segments of jejunum were
harvested from mongrel dogs anesthetized with pentobarbital sodium (30 mg/kg), and
placed in oxygenated ice-cold KRPB. Blood was removed from the segment by perfusing
KRPB via the local artery. The segment was cut open along the mesenteric border, and
intact mucosal sheets were obtained by gently separating the mucosa from the underlying
gut wall using a microscope slide (66,85). Small pieces of mucosa, 1x2 mm, were
obtained by cutting ﬁrst thin strips of mucosa and then small pieces from the strips. These
small pieces were transferred to fresh oxygenated cold KRPB. Numerous small pieces
were blotted on ﬁlter paper, then weighed to the nearest 0.1 gram, and placed in Warburg
ﬂasks. Each ﬂask contained a total volume of 3 ml incubation media which included
oxygenated KRPB with or without nutrient and with or without the treatment chemical
to be tested. Each flask which contained a treatment chemical was paired with a control
ﬂask containing no treatment chemical.

For absorption of C0,, the center well of the ﬂask contained 0.2 ml of 10% KOH and
ﬂuted ﬁlter paper. The ﬂasks were attached to manometers, ﬂushed with 100% oxygen,
placed in a water bath (37°C), and shaken at 90 cycles per min. After a 10-15 min
equilibration period, manometer pressure readings were obtained every 10 minutes for 1

hour. Additional mucosal tissue samples were weighed, oven dried, and re-weighed to

45

obtain the percent dry weight, which was used to calculate the dry weight of the
experimental samples. The amount of oxygen consumed, in microliters 02 per milligram
dry weight, was calculated as described by Umbreit et al. (102). Figure 4 shows an
example of the relationship between the oxygen consumed and incubation time. The
regression lines were constructed using the method of least squares, and homogeneity of
the regressions in each run was tested by F test. The slope of the regression line is the

oxygen consumption rate, Q02, expressed in nl-min"omg dry weight".

Exgrimental Protocol for Determining the Effect of Prostanoids on Glucose-induced

 

Change in Mucosal (X), in vitro.

In a preliminary series of experiments (n= 8 mucosal tissues from 4 dogs), mucosal
Q02 was determined when the incubation media contained 0, 2.5, 5, 10, 25, 50, and 100
mM glucose. The mucosal Q02 were 11532, 14715, 15818, 15617, 16016, 15716, and
15717 nl-min'1-mg dry weight", respectively (Figure 5). This indicated that 5 mM glucose
was the minimum concentration that maximally increased the mucosal Q02.
Postprandially the lumen contains nutrients as well as bile. Additionally, glucose plus bile
was placed into the lumen to increase blood ﬂow and oxygen uptake in the preceding as
well as numerous previous in viva studies (26,90,91) and enhances the glucose-induced
increase in blood ﬂow in viva (58). Since it was desired to mimic the postprandial and
in viva conditions in these in vitro studies, the effect of 10% gallbladder bile on mucosal
Q02 in the absence and presence of glucose was determined. As shown in Figure 6, 10%
bile signiﬁcantly decreased basal Q02 as well as the glucose-induced increase in Q02.

Since bile signiﬁcantly decreased Q02 it was not used in subsequent studies. Therefore,

46

12-0‘ o 0 mM Glucose (n-56)
O 5 mM Glucose (fl-70)

10.0- y=0.173x an ”.3".
r=0.99 »

Oxygen Consumed
(pl / mg dry tissue)

 

 

 

O -1 O 20 30 4O 50 60
Time (minutes)
Figure 4. Relationship between oxygen consumed and incubation time when the

media contained only 0 mM glucose (open circle) or 5 mM glucose
(black diamond). The slope of the relationship represents the mucosal
oxygen consumption rate in nl-min"omg dry wt". "‘ P < 0.05 relative to
the slope for 0 mM glucose. Standard error bars are included but are
smaller than the symbols.

47

 

 

 

€175T , r .
E150" 7 I 67
21:: & % §é
5°: % N 4
5:2: 6 N %

 

 

 

 

 

 

 

 

 

 

 

 

O 2.5 5 10 25 50
GLUCOSE CONCENTRATION (mM)

Figure 5. Effect of glucose on mucosal Q02. * P < 0.05 relative to 0 mM
glucose. # P < 0.05 relative to preceding lower glucose concentration.

48

200
1 80
1 60
1 4O
1 20
1 00

80

60

002 (nl/min/mg)

20

 

No Tx 10% Bile 5 mM Bile +
Glucose Glucose

Figure 6. Effect of 10% gallbladder bile on mucosal Q02 in the absence and
presence of 5 mM glucose. (n= 8 determinations/ 4 dogs) * P < 0.05
relative to no treatment (T x). # P < 0.05 relative to 5 mM glucose.

49

in the subsequent series of experiments, the media contained either 0 mM or 5 mM
glucose with or without treatment Table 1 summarizes the treatment chemicals, the
number of determinations, and the number of dogs used in each of the 12 series of
experiments. In each individual experiment, each ﬂask that contained a treatment chemical
was paired with a control ﬂask containing no treatment chemical and utilized the mucosal
tissue from the same jejunal segment. Furthermore, the mucosal Q02 for all
determinations were determined in duplicate.

The objective of Series I to IV experiments was to determine if endogenous
prostanoids affect the basal (0 mM glucose) and a glucose (5 mM glucose)-induced
increase in jejunal mucosal Q02. Series I and II determined the effect of cyclooxygenase
inhibition, and Series III and IV determined the effect of substrate loading with
arachidonate, the precursor of prostanoids. The objective of Series V and VI was to
determine if the effect of arachidonate on the mucosal QO2 was due to prostanoids. Thus
effects of mefenamate (40 nM) alone, arachidonate (100 nM) alone, and the combination
of mefenamate (40 M) and arachidonate (100 nM) were determined.

The objective of Series VII to X was to determine if TxA2 affects the basal and
glucose-induced increase in jejunal mucosal Q02. A TxMendopemxide analogue, U-
44069, and a TxAQ/endoperoxide receptor blocker, SQ 29548, were utilized for this
purpose. These chemicals, U-44069 and SQ 29548, have been shown to be a potent
thromboxane A2 agonist, and highly speciﬁc TxA2 receptor antagonist, respectively
(52,73,98). The concentrations of U-44069 (0.28, 2.8, 28.0 nM) utilized were
approximately 0.1, 1, and 10 times the concentration of Tsz (metabolite of TxA2) found

in the venous blood from a jejunal segment containing predigested food in its lumen (20).

50

Table 1. Treatment chemicals used in Series I-XII in vitro experiments.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

SERIES Glucose Treatment
Concentration
0 mM 5 mM nldogs

Ia - x Mefenamate (40 M) 1214
lb x Indomethacin (10 M) 4/3
IIa x Mefenamate (40 nM) 18/7
IIb x Indomethacin (10 W) 6/3
III x Arachidonate (1, 10, 100, 1000 M) 8/3
IV x Arachidonate (1, 10, 100, 1000 M) 9/4
Va x Mefenamate (40 nM) 12/6
Vb x Arachidonate (100 pM) 12/6
Vc x Arachidonate (100 nM) & Mefenamate (40 M) 1216
VIa x Mefenamate (40 M) 9/4
VIb x Arachidonate (100 M) 9/4
We x Arachidonate (100 nM) & Mefenamate (40 W) 9/4
VII x U-44069 (0.28, 2.8, 28.0 nM) 8/4
VIII x U-44069 (0.28, 2.8, 28.0 nM) 16/9
IX x SQ-29548 (10 nM) 10/5
X x SQ-29548 (10 nM) 12/6
XI x Arachidonate (100 nM) & SQ—29548 (10 nM) 10/5
XII x Arachidonate (100 nM) & SQ-29548 (10 M) 9/4

 

 

 

 

 

 

 

Each treatment was paired with a control determination, the media of which did not contain
any treatment chemical. n = number of determinations/number of dogs used.

51
The concentration of SQ 29548 (10 nM) used was chosen based on a preliminary study.

This study showed that SQ 29548 at this concentration completely blocked the inhibitory
action of 280 nM U-44069 on mucosal Q02 in the presence of 5 mM glucose as shown
in Figure 7.

The objective of Series XI and XII was to determine if the effect of arachidonate on
the mucosal QO2 was due to the action of TxMendopemxide. Therefore, the effect of a
combination of SQ 29548 and arachidonate on mucosal QO2 was determined. The Series
XII experiments were performed in conjunction with Series VI experiments utilizing

mucosal tissue from the same jejunal segments.

Exmrimental Protocol for Determining_t_he Effect of Prostanoids on Oleate-induced
Change in Mucosal 00, in vitro.

The objective of Series XIII ta XV was to determine the effect of prostanoids on
oleate-induced changes in mucosal Q02 in vitro. Prior to these series of experiments, ﬁve
preliminary studies were performed to determine the concentration of oleate that would
be used. The ﬁrst preliminary study determined the effect of sonicated oleate alone on
mucosal Q02. The oleate solution was prepared by adding the appropriate quantity of
oleate to KRPB and sonicating the solution for 1 minute. The effect of 0, 0.1 and 5.0 mM
sonicated oleate was determined (n= 4 determinations). Mucosal QO2 was 118:4, 116:3,
and 11413 nl-min"-mg", respectively. Hence, sonicated oleate did not alter mucosal Q02.
Studies performed in viva have shown that luminal placement of oleate did not alter
jejunal blood ﬂow, but that luminal placement of oleate plus bile or a bile salt,

taurocholate, increased jejunal blood ﬂow (26,58). This suggests that oleate may also need

Figure 7.

002 (nl/ min/ mg)

52

200
1 80
1 60
1 4O
1 20
1 OO
80
60
4O
20

 

5 mM U-HOGQ 50-29548 1015M

GLUCOSE 280 nM [1-44039 250 nM

Effect of TxA2 / endoperoxide receptor blocker, SQ 29548, on the
decrease in QO2 induced by U-44069, a TxA2 receptor analogue, in the
presence of 5 mM glucose. (n= 6 determinations/ 3 dogs) * P < 0.05
relative to 5 mM glucose. # P < 0.05 relative to U-44069.

53

to be combined with bile or a bile salt in order to inﬂuence mucosal Q02 in vitro. Since
it was previously shown that 10% gallbladder bile signiﬁcantly decreased QO2 (Figure 6,
page 48), the effect of taurocholate on mucosal QO2 was determined in the second
preliminary study. Mucosal Q02 was determined when the media contained 0, 0.01, 0.1,
0.5, 1.0, 2.0, and 10.0 mM taurocholate. The results are summarized in Table 2.
Taurocholate did not alter Q02 at concentrations of 0.01, and 0.1 mM. At concentrations
of 0.5, 1.0, 2.0, and 10.0 mM taurocholate signiﬁcantly decreased Q02 in a dose
dependent fashion (Figure 8). The maximum concentration of taurocholate that did not
alter Q02 was 0.1 mM. Therefore this concentration of taurocholate was used in the
subsequent studies.

The effect of oleate plus taurocholate was determined in the third preliminary study.
Mucosal Q02 was determined when the media contained no treatment (basal), 0.1 mM
taurocholate, or oleate at concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 mM plus 0.1
mM taurocholate. The results are summarized in Table 3. Oleate, at concentrations of 0.1
and 0.5 mM, plus taurocholate did not alter Q02, whereas 1.0 mM oleate plus
taurocholate tended to decrease Q02, and 2.0, 5.0, and 10.0 mM oleate plus taurocholate
signiﬁcantly decreased mucosal QO2 (Figure 9). The minimum concentration of oleate
which maximally decreased mucosal QO2 was 5.0 mM.

Since oleate plus bile increased oxygen uptake in viva, it was unexpected that oleate
plus taurocholate would decrease Q02 in vitro. Therefore, additional studies were
performed to further characterize this effect. The fourth preliminary study determined the
mucosal QO2 when the media contained no treatment, 5.0 mM sonicated oleate, or 5.0

mM oleate plus 10% bile (n= 7 determinations] 3 dogs). Sonicated oleate did not alter

54

Table 2. The effect of taurocholate on jejunal mucosal Q02.

 

 

Media

Contents QO2 Change % Change n
No Treatment 121 i 3 14/6
TC 0.01 mM 117 i 3 -3 j; 2 -2.6 i: 1.6% 6/3
TC 0.1mM 122 _-l_-_ 3 2 i 3 2.1 i: 2.1% 10/4
TC 0.5 mM 112 .‘t 2*# -10 1 2 -9.1 i 2.2% 4/2
TC 1.0 mM 107 j; 3* -13 i 3 -ll.2 i 3.2% 10/4
TC 2.0 mM 103 -_l-_ 2* -15 i 5 -l3.8 i 4.6% 6/3
TC 10.0 mM 77 i 2*# -44 i 5 -36.4 i 4.7% 4/2

 

QO2 values and changes are means 3; SE and are in n1 02-min"-mg dry wt". TC =
Taurocholate. * P<0.05 relative to No Treatment. # P < 0.05 relative to preceding lower
TC concentration. n = number of determinations/number of dogs.

55

 

 

 

 

 

 

 

160 a
1 40 -
’6‘»
E 120 - ‘
E 100 -
é so .
5 60 a
N O
O 40 - ’0‘
0 a
2°‘ I“:
O . .6
[TC] 0 0.01 0.1 0.5 1.0 2.0 10.0 mM
Figure 8. Mucosal Q02 in the absence and presence of increasing concentrations

of taurocholate [TC]. * P < 0.05 relative to basal value ([TC] 0).
# P < 0.05 relative to preceding taurocholate concentration.

56

Table 3. The effect of oleate plus taurocholate on mucosal Q02.

 

 

Media

Contents QO2 Change % Change 11
No Treatment 114 i 6 7/3
TC 0.1 mM 118 _-_+_ 5 4i 2 3.5 i 2.0% 7/3
TC + 0A 0.1 mM 118 i. 5 3 i 3 2.6 :t 1.5% 7/3
TC + 0A 0.5 mM 116 i 6 2 i: 2 1.7 i 2.1% 7/3
TC + 0A 1.0 mM 112 -_l-_ 6 -2 :1 -1.7 :1; 3.4% 7/3
TC + 0A 2.0 mM 108 i 8"‘# -13 i 2 -10.9 i 2.4% 4/3
TC + 0A 5.0 mM 94 i 6*# -21 _4_-_ 5 -18.4 i 3.8% 7/3
TC + OA 10.0 mM 88 i 8* -26 i 5 -22.8 i: 4.2% 3l3

 

Q02 values and changes are means i SE and are in n1 02-min"-mg dry wt".
TC = Taurocholate (0.1 mM). OA = Oleate. * P < 0.05 relative to No Treatment.
# P < 0.05 relative to preceding TC + OA concentration. n = number of
determinations/number of dogs.

57

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

g 120: T T\ / T T *# I
>1oo- \ / \ T *# ,..
-- \ / A
g § g §¢
5, 50- § % \ /
s .0- § % S?
. V v
0 s /A .\ / -
[0A] 0 O 0.1 0.5 1.0 2.0 5.0 10.0 5.0 5.0 mM
[TC] 0 | ———————— 0.1mM -------- | o Bile

([TC] 0.1 mM) or 10% bile. * P < 0.05 relative to basal value ([TC] 0,
[0A] 0). # P < 0.05 relative to the preceding oleate concentrations. The
effect of 5.0 mM oleate in the absence and presence of 10% bile were
performed in a separate series of experiments and are included for
comparison.

58
Q02, whereas 5.0 mM oleate plus 10% bile signiﬁcantly decreased mucosal Q02. The

results of this study are shown in Figure 9 combined with those of the previous study for
comparison. The decrease in QO2 induced by 5.0 mM oleate plus bile was not
signiﬁcantly different from that of 5.0 mM oleate plus 0.1 mM taurocholate. The ﬁfth
preliminary study determined if the concentration of taurocholate used affects the decrease
in QO2 induced by 5.0 mM oleate, i.e. does a greater concentration of taurocholate alter
the effect of 5.0 mM oleate. The mucosal QO2 was determined when the media contained
no treatment (basal); 5.0 mM sonicated oleate; 0.1, 0.5 or 1.0 mM taurocholate; and 5.0
mM oleate plus 0.1, 0.5, or 1.0 mM taurocholate. The results are summarized in Table
4. As in the previous study, sonicated oleate did not alter Q02. Likewise, 0.1 mM
taurocholate did not affect Q02, but 0.5 and 1.0 mM taurocholate decreased Q02. Oleate
(5.0 mM) plus 0.1, 0.5 and 1.0 mM taurocholate decreased Q02. Even though 0.5 and 1.0
mM taurocholate decreased Q02, the oleate-induced decrease in Q02 was similar in the
presence of all three taurocholate concentrations. Hence, the effect of 5.0 mM oleate plus
taurocholate was not inﬂuenced by these concentrations of taurocholate.

Based on these preliminary studies 5.0 mM oleate plus 0.1 mM taurocholate, which
was the minimal concentration that maximally decreased Q02, was selected for use in the
subsequent studies. Further evidence from these studies which substantiate the selection
of this combination is that the change in QO2 induced by this combination was similar
to that induced by 5.0 mM oleate plus bile as well as 5.0 mM oleate plus greater
concentrations of taurocholate. Additionally, 5 mM oleate is within the lower end of the
range of fatty acids found in the upper small intestinal lumen postprandially (12,53).

Since it was unexpected that oleate plus taurocholate would decrease mucosal Q02, the

59

Table 4. The effect of taurocholate on the 5.0 mM oleate-induced change in mucosal Q02.

 

 

Media

Contents Q02 Change % Change n
No Treatment (Basal) 130 1; 3 4/3
5 mM Oleate 126 i 4 4 i 2 2.5 i 2.1% 3/3
TC 0.1 mM 130 i 3 4/3
TC 0.1 mM + 0A 105 i 2*# -25 .i: 4 -19.7 i: 2.1% 4/3
TC 0.5 mM 119 i 2* 4/3
TC 0.5 mM + OA 99 i 5*# -20 i 3 -16.2 _+_- 2.3% 4/3
TC 1.0 mM 112 i 3* 4/3
TC 1.0 mM + 0A 96 i: 3*# -16 i: 3 -14.3 i 3.4% 4/3

 

QO2 values and changes are means i SE and are in n1 Ozomin"-mg dry wt".
TC = Taurocholate. 0A = Oleate (5.0 mM). * P < 0.05 relative to No Treatment.
# P < 0.05 relative to the corresponding TC concentration.

60

subsequent studies were also performed in the presence of 0.5 mM oleate plus 0.1 mM
taurocholate, a combination which did not affect Q02.

In the subsequent series of experiments the media contained KRPB only (no treatment
or basal), 0.1 mM taurocholate, 0.5 mM oleate plus 0.1 mM taurocholate, or 5.0 mM
oleate plus 0.1 mM taurocholate with and without treatment chemical. In each individual
experiment, the mucosal Q02 with and without treatment chemical was determined using
the mucosal tissue from the same jejunal segment.

The objective of Series XIII and XIV was to determine if endogenous prostanoids
effect the oleate-induced decrease in mucosal Q02. Series XIII determined the effect of
cyclooxygenase inhibition with mefenamate on mucosal Q02, and Series XIV determined
the effect of substrate loading with arachidonate on mucosal Q02. Additionally in Series
XIV, the effect of mefenamate on the arachidonate-induced changes in QO2 was
determined to investigate if the changes were due to prostanoids. The objective of Series
XV was to determine if endogenous TxA2 I endoperoxide effects the oleate-induced
changes in mucosal Q02. The constituents of the media were as follows.

In Series XIII mucosal QO2 was determined when the media contained no treatment
(basal), 0.1 mM taurocholate, 0.5 mM oleate plus 0.1 mM taurocholate, and 5.0 mM
oleate plus 0.1 mM taurocholate without and with mefenamate (40 M).

In Series XIV mucosal QO2 was determined when the media contained no treatment
(basal), 0.1 mM taurocholate, 0.5 mM oleate plus 0.1 mM taurocholate, and 5.0 mM
oleate plus 0.1 mM taurocholate without and with arachidonate (100 nM). The effect of
the combination of arachidonate and mefenamate was also determined in this series.

In Series XV mucosal QO2 was determined when the media contained no treatment

61
(basal), 0.1 mM taurocholate, 0.5 mM oleate plus 0.1 mM taurocholate, and 5.0 mM

oleate plus 0.1 mM taurocholate without and with SQ 29548 (10 pM).

Chemicals: Mefenamic acid, sodium arachidonate, indomethacin, and oleic acid were
obtained from Sigma Chemical (St. Louis, MO). U-44069 and SQ 29548 were obtained
from Cayman Chemical Co (Ann Arbor, MI). Soduim taurocholate was obtained from
ICN Biomedicals (Irvine, CA).

Statistical Analysis: The mucosal QO2 values in nl-min"-mg dry weight", are
expressed as the mean :1: SE. The effects of treatments on the mucosal Q02 were
analde and compared utilizing ANOVA and random block ANOVA, and a Student-

Newman-Kuel’s post hoc test. Statistical signiﬁcance was set at P<0.05.

RESULTS

Effect of Prostanoids on Glucose- and Oleate-Induced Changes in Blood Flow and
Oxygen Uptake in viva.

Systemic arterial pressure (130 i 6 mmHg) was not signiﬁcantly altered by luminal
placement of normal saline, glucose, or oleate, nor by intraarterial administration of
angiotensin II, SQ 29548, or U-44069. The administration of mefenamate increased aortic
pressure to 144 i 3 mmHg. However, the data presented here were obtained under
conditions of steady state aortic pressure.

In series I the responses to luminal placement of glucose were determined before and
one hour after intravenous administration of mefenamate. The effect of mefenamate on
the glucose-induced increases in jejunal blood ﬂow and oxygen uptake (V 02) are
summarized in Table 5. Luminal placement of glucose signiﬁcantly increased blood ﬂow
both before and after mefenamate administration. However, the magnitude of the glucose-
induced increase in blood ﬂow was signiﬁcantly greater after mefenamate. This is true
whether the data are expressed as the absolute change in blood ﬂow (10.6 _-g-_ 4.2 before
vs. 24.9 i 2.7 ml-min"o100g" after mefenamate) or as the percent change from control
(17.4 1; 2.5% before vs. 67.1 i 6.4% after mefenamate). Thus, mefenamate enhanced the
magnitude of the glucose-induced increase in jejunal blood ﬂow.

Luminal placement of glucose signiﬁcantly increased jejunal oxygen uptake both

62

Table 5.

63

Effect of mefenamate on glucose-induced changes in blood ﬂow,

resistance, arteriovenous oxygen content difference, and oxygen uptake.

 

 

 

 

Lumen Content Difference Percent

NS Glucose Change
Before Mefenm
Blood Flow 60116.2 70717.8 10.612.4* 17.412.5%*
Resistance 2.351029 2.001024 -0.351007* -14.612.1%*
(Cao2 - Cvo,) 4.261053 4.231049 00310.15 -2.314.4%
Oxygen Uptake 2.371010 2.801015 0.4310.09* 18.012.4%*
After Mefenamate
Blood Flow 37.413.2# 62315.7 24.912.7*# 67.116.4%*#
Resistance 4.1 110.50# 2.471027 -1.6410.25*# -39.411.7%*#
(Cao2 - Cvoz) 6.9010.46# 5.211076 -l.6710.38*# -22.914.1%*#
Oxygen Uptake 2.491019 3.1710.20# 0.6810.13*# 26.514.2%*#

 

Values are mean 1 SE. Blood ﬂow and oxygen uptake values are expressed in
m1.min"o100g", (Cao2 - Cvoz) in ml 02 / 100 ml, and resistance in
mmHg-ml"-min"-100g". Difference equals glucose value minus normal saline (N S)

value. * P < 0.05.

# P < 0.05 relative to value before mefenamate.

64

before and after mefenamate administration. The administration of mefenamate did not
signiﬁcantly alter oxygen uptake when normal saline was in the lumen. However,
mefenamate signiﬁcantly enhanced the glucose-induced increase in oxygen uptake from
0.43 1 0.09 (18.0 1 2.4%) before to 0.68 1 0.13 ml-min"-100g" (26.5 1 4.2%) after
mefenamate administration. Thus, mefenamate also enhanced the glucose-induced increase
in jejunal oxygen uptake. As shown in Figure 10, the glucose-induced changes in blood
ﬂow are signiﬁcantly correlated with the changes in oxygen uptake both before and after
mefenamate administration.

In series II the responses to luminal placement of oleate were determined before and
one hour after intravenous administration of mefenamate. The effect of mefenamate on
the oleate-induced increases in jejunal blood ﬂow and oxygen uptake are summarized in
Table 6. Luminal placement of oleate signiﬁcantly increased blood ﬂow both before and
after mefenamate administration. However, the magnitude of the oleate-induced increase
in blood ﬂow was signiﬁcantly greater after mefenamate. This is true whether the data
are expressed as the absolute change in blood ﬂow (28.0 1 3.8 before vs. 53.7 1 6.8
m1~min"-100g" after mefenamate) or as the percent change from control (43.9 1 4.5%
before vs. 186.6 1 18.3% after mefenamate). Thus, mefenamate enhanced the magnitude
of the oleate-induced increase in jejunal blood ﬂow.

Luminal placement of oleate signiﬁcantly increased jejunal oxygen uptake both before
and after mefenamate administration. The administration of mefenamate did not alter
oxygen uptake when normal saline was in the lumen. However, mefenamate signiﬁcantly
enhanced the oleate-induced increase in oxygen uptake from 0.35 1 0.12 (15.9 1 5.3%)

before to 0.73 1 0.24 ml-min"-100g" (27.2 1 5.3%) after mefenamate administration.

65

80 -
0 Before Mefenamate
701 A After Mefenamate

60 -
50‘ y-19.2x + 7.1
40 . r=0.60 (p(0.05)

30-
20-
10‘

o I I I I I ﬂ
0.00 0.25 0.50 0.75 1.00 1.25 1.50

Change in V02 (ml/min/100g)

 

 

Change in Blood Flow (ml/min/100g)

 

Figure 10. Correlation between changes in jejunal blood ﬂow and oxygen uptake
(V02) induced by glucose plus bile. Open circles represent responses
before mefenamate. Triangles represent responses after mefenamate.

66

Table 6. Effect of mefenamate on oleate-induced changes in blood ﬂow,
resistance, arteriovenous oxygen content difference, and oxygen uptake.

 

 

 

Men Content Difference Percent

NS Oleate Change
Before Mefenamate
Blood Flow 65.7125 93.7130 28013.8“ 43.914.5%*
Resistance 2.031009 1.381005 -0.6410.09* -31.714.6%*
(Cao2 - Cvoz) 3.501017 2.811012 -0.6910.13* -19.013.2%*
Oxygen Uptake 2.281011 2.631012 0.351012“ 15.915.3%*
After Mefenamate
Blood Flow 30.913.5# 84.517.5 53.716.8*# 186.6118.3%*#
Resistance 4.871034# 1.751016 -3.121041*# -62.614.6%*#
(Cao2 - Cvoz) 7.531041# 3.521033 -4.0110.30*# -53.618.1%*#
Oxygen Uptake 2.241013 2.9710.l9# 0.731024*# 27.215.3%*#

 

Values are mean 1 SE. Blood ﬂow and oxygen uptake values are expressed in
mlomin"-100g", (Cao2 - Cvoz) in ml 02 / 100 m1, and resistance in
mmHg-ml".min"-100g". Difference equals oleate value minus normal saline (NS) value.

* P < 0.05. # P < 0.05 relative to value before mefenamate.

67

Thus, mefenamate also enhanced the oleate-induced increase in jejunal oxygen uptake.
There is a signiﬁcant correlation between the oleate-induced changes in oxygen uptake
and blood ﬂow both before and after mefenamate, as shown in Figure 11.

Figure 12 shows and compares the glucose- and oleate-induced percent change from
control for blood ﬂow and oxygen uptake before and after mefenamate administration.
The magnitude of the hyperemia induced by oleate was signiﬁcantly greater than that
induced by glucose both before and after mefenamate administration. This is true whether
compared as the absolute change in blood ﬂow (10614.2 ml-min"-100g" for glucose vs
28013.8 ml.min"-100g" for oleate before mefenamate, and 24912.7 ml-min"0100g" for
glucose vs 53.7168 ml-min"- 100g" for oleate after mefenamate) or as the percent change
from control (17.4125 % for glucose vs. 43.9145 % for oleate before mefenamate, and
67.1164 % for glucose vs 186.6118.3 % for oleate after mefenamate). Mefenamate
administration enhanced both the glucose- and oleate-induced increases in blood ﬂow. To
compare the enhancement, by mefenamate, of the glucose- and oleate-induced hyperemias,
the magnitude of enhancement of the glucose-induced increase in blood ﬂow (calculated
as the glucose-induced change in blood ﬂow after mefenamate minus the glucose-induced
change in blood ﬂow before mefenamate) was compared to the magnitude of enhancement
of the oleate-induced increase in blood ﬂow. The magnitude of enhancement of the
oleate-induced increase in blood ﬂow was signiﬁcantly greater than the enhancement of
the glucose-induced hyperemia (25.6 1 6.8 for oleate vs 14.4 1 4.1 m1-min"-100g" for
glucose). Therefore it seems that mefenamate enhanced the oleate-induced increase in
blood ﬂow more than it enhanced the glucose-induced increase in blood ﬂow. The

magnitude of the increase in jejunal oxygen uptake induced by glucose and oleate were

68

    

’3 80 -

8 0 Before Mefenamate

S 70- A After Mefenamate

'é 60d

> A

\E’ 50. ‘ 340.5X ‘1' 19.3
. 3 r=0.82 (p(0.01)

r: 40‘

'0

8 30-

E

.5 2° ‘

8.

r: 10- 0

E

0 0 ﬂ

 

 

0.00 0.25 0:50 0.1/5 1.2m 1325 1.50
Change in V02 (ml/min/ 1009)

Figure 11. Correlation between changes in jejunal blood ﬂow and oxygen uptake
(V02) induced by oleate plus bile. Open circles represent responses
before mefenamate. Triangles represent responses after mefenamate.

69

220

20° D Before Mefenamate
150 B After Mefenamate

160
140
120
100

% Change in Blood Flow

 

B 8 3 3

95 Change in Oxygen Uptake
3

 

GLUCOSE OLEATE

Figure 12. Comparison of the glucose- and oleate-induced percent change from
control for blood ﬂow and oxygen uptake before and after mefenamate.
* P < 0.05. # P < 0.05 relative to the respective value before
mefenamate. @ P < 0.05 relative to the respective value for glucose.

 

70

similar before and after mefenamate administration (0.43 1 0.09 (18.0 1 2.4%) for glucose
vs 0.35 1 0.12 (15.9 1 5.3%) for oleate before mefenamate, and 0.68 1 0.13 mlcmin’
l~100g" (26.5 1 4.2%) for glucose vs. 0.73 10.24 ml-min"-100g" (27.2 1 5.3%) for oleate
after mefenamate). Furthermore, mefenamate enhanced the glucose- and oleate-induced
increase in oxygen uptake by the same magnitude (0.35101 m1-min"-100g" for glucose
vs 03810.1 mlomin'l-IOOg" for oleate).

In series I and series II, the administration of mefenamate produced a decrease in
resting blood ﬂow and a corresponding increase in vascular resistance. In series I resting
blood ﬂow decreased from 60.1 1 6.2 to 37.4 1 3.2 ml.min"-100g" and resistance
increased from 2.35 1 0.29 to 4.11 1 0.50 mmHg-ml-min"-100g" (Table 5). In series II
resting blood ﬂow decreased from 65.7 1 2.5 to 30.9 1 3.5 ml-min"-100g" and resistance
increased from 2.03 1 0.19 to 4.87 1 0.34 mmHg-ml-min"-100g" (Table 6).

Myers and Honig (70) have shown that initial resistance plays a signiﬁcant role in
determining the magnitude of the vascular response to a stimulus. Moreover, Gallavan and
Chou (41) have shown that initial resistance does play a role in determining the jejunal
vascular response to food. For example, there is a signiﬁcant correlation between initial
resistance and the food-induced change in vascular resistance such that the greater the
initial resistance the greater the food-induced change in resistance. Thus it is possible that
the enhancement of the hyperemias induced by glucose and oleate in series I and series
II may have been due to the changes in initial resistance, i.e. the decrease in resting blood
ﬂow. This possibility was examined by analyzing the relationship between initial
resistance and the change in resistance produced by glucose and oleate. For the purpose

of analysis, the data from series I and series II were each divided into two groups; initial

71

resistance and the change in resistance before mefenamate, and the initial resistance and
the change in resistance after mefenamate. The data were analyzed assuming that the
regression line passed through the origin (41). The relationship between initial resistance
the glucose-induced change in resistance before and after mefenamate are shown in Figure
13 and that for oleate is shown in Figure 14. In all cases there was a signiﬁcant
correlation between initial resistance and the magnitude of the change in resistance
produced by glucose and oleate (P<0.05). Hence, the greater the initial resistance, the
greater the magnitude of the vascular response produced by glucose and oleate. However,
for both glucose and oleate the slope of the regression line after treatment with
mefenamate was signiﬁcantly different from that before mefenamate. That is, for a given
initial resistance, both glucose and oleate elicit a signiﬁcantly greater decrease in
resistance after cyclooxygenase inhibition with mefenamate.

To further test this relationship the jejunal vascular responses to glucose and oleate
were examined before and during the intraarterial infusion of angiotensin II in series III
and series IV, respectively. The infusion of angiotensin H signiﬁcantly increased jejunal
vascular resistance from 2.51 1 0.14 to 4.48 1 0.21 mmHg-ml'min"-100g" in series III
and from 2.38 1 0.23 to 4.90 1 0.24 mmHg-ml-min"-100g" in series IV. In addition, the
magnitude of the glucose-induced decrease in resistance increased from -0.42 1 0.12
before to -1.10 1 0.10 mmHg-ml-min"-100g" during the infusion of angiotensin H. The
magnitude of the oleate-induced decrease in resistance also increased from -0.73 1 0.21
before to -1.96 1 0.14 mmHg-ml-min'lo100g" during the infusion of angiotensin 11. As in

the previous two series, there was a signiﬁcant correlation between initial resistance and

the decrease in resistance produced by glucose (Figure 15) and oleate (Figure 16). The

Change in Resistance

72

Initial Resistance
(mmHg/ml/min/l 009)

 

 

-1.
3
o
2
2
E
>
E
or —3~
:1:
E
E
V
-4-1-
-5--
Figure 13.

 

O Glucose before Mefenamate
A Glucose after Mefenamate

Relationship between initial resistance and the change in resistance
induced by glucose plus bile. Open circles and dashed line represent the
response before mefenamate. Triangles and solid line represent the
response after mefenamate. Slopes of the regression lines are
signiﬁcantly different, * P < 0.05.

Change in Resistance

 

0
0
-1.
A
U.
o
9.
2
E
5
E
E -3-
3:
E
5;
.4.
-5--
Figure 14.

    

73

Initial Resistance
(mmHg/mI/min/100g)
1 2 3 4 5 e 7

o Oleate before Mefenamate
O Oleate after Mefenamate

Relationship between initial resistance and the change in resistance
induced by oleate plus bile. Open circles and dashed line represent the
response before mefenamate. Triangles and solid line represent the
response after mefenamate. Slopes of the regression lines are
signiﬁcantly different, * P <0.05.

74

slope of the regression line for the relationship before angiotensin H was not different
from that for the relationship during angiotensin H infusion for both glucose and oleate,
so a single regression line was constructed for each. However, the slope of the regression
line for the angiotensin H series was signiﬁcantly different from that after mefenamate
administration for both glucose (Figure 14) and oleate (Figure 15) from series I and II.
Thus, although initial resistance does play a role in determining the jejunal vascular
response to glucose and oleate, the enhancement of both the glucose- and oleate-induced
hyperemias by mefenamate is, in part, in addition to and independent from the increase
in initial resistance.

In series III and series IV the jejunal blood ﬂow and metabolic responses to glucose
and oleate, respectively, were also determined before and during angiotensin II infusion.
The glucose-induced increase in blood ﬂow and oxygen uptake before angiotensin H
infusion were 11.8 1 3.5 and 0.26 1 0.05 ml-min"°100g", respectively (T able 7). During
angiotensin II infusion, resting blood ﬂow decreased, but oxygen uptake was not
signiﬁcantly altered. During angiotensin H infusion, the glucose-induced increase in blood
ﬂow (10.8 1 3.2 m1~min"o100g"), and oxygen uptake (0.20 1 0.09 ml~min"-100g") were
not different from the values before angiotensin H infusion.

Table 8 summarizes the changes in blood ﬂow and oxygen uptake induced by oleate
before and during angiotensin H infusion. As in series III, during angiotensin H infusion
resting blood ﬂow decreased, but oxygen uptake was not signiﬁcantly altered. Further, the
oleate-induced increase in blood ﬂow (25.1 1 7.8 mlomin"-100g"), and oxygen uptake
(0.28 1 0.05 ml-min"-100g") before angiotensin H infusion were not different from the

oleate-induced increase in blood ﬂow (18.4 1 6.8 ml-min'lo100g"), and oxygen uptake

Change in Resistance

-1.-

(mmHg/ml/min/1 OOg)

 

-5J-

Figure 15.

  
 

75

Initial Resistance
(mmHg/ ml/ min/ 1 009)
1 2 :5 4 5 e 7
I I w
1 o

‘

 
     

°\

r=0.94

o Glucose before Angiotensin
O Glucose during Angiotensin

Relationship between initial resistance and the change in resistance
induced by glucose plus bile before and during angiotensin H infusion.
Open circles represent the response before angiotensin H and the closed
circles represent the response during angiotensin H infusion. Slope of
the regression line for the angiotensin H series (dashed line) is
signiﬁcantly different from that for after mefenamate (solid line)

* P < 0.05. Mefenamate data are reproduced from Figure 13.

Change in Resistance

76

Initial Resistance

 

 

  

(mmHg/ ml/ min/ 1 009)
0 1 2 3 4 5 6 7 8
O ‘ l ' ' 1 F 1 ﬁ'
-1 --
’3
8 e
E _2 __ \ y=-0.38x
g \ \r=0.85
E e
\ \ \
0| -3 -- \
E O Oleate before Angiotensin
f, e Oleate during Angiatensin
-4..
-5J-
Figure 16. Relationship between initial resistance and the change in resistance

induced by oleate plus bile before and during angiotensin H infusion.
Open circles represent the response before angiotensin H and the closed
circles represent the response during angiotensin H infusion. Slope of
the regression line for the angiotensin H series (dashed line) is
signiﬁcantly different from that for after mefenamate (solid line)

* P < 0.05. Mefenamate data are reproduced from Figure 14.

77

Table 7. Effect of angiotensin H on glucose-induced changes in blood ﬂow,
resistance, arteriovenous oxygen content difference, and oxygen uptake.

 

 

 

wen Content Difference Percent
NS Glucose Change
Before Angiotensin H
Blood Flow 46.1127 57.9155 1 1.813.5* 21.817. 1 %*
Resistance 2.511014 2.101016 -0.421011* -17.014.2%*
(Cao2 - Cvoz) 5.271037 4.901052 03710.30 -7.8515.7%
Oxygen Uptake 2.381009 2.64101 1 0.2610.05* 11.112.1%*

During Angiotensin H Infusion

Blood Flow 26.311 .711 37.113341 l0813.2* 40.917.3%*#
Resistance 4.481021# 3.381025# 4.101010% -24.912.9%*#
(Caoz - Cvoz) 8.3111.05# 7.4610.83# 0861067 -11.0618.7%
Oxygen Uptake 2.241029 2.441025 0.2010091 9.012.4%*

 

Values are mean 1 SE; n=7. Blood ﬂow and oxygen uptake values are expressed in
ml-min"-100g", (Cao2 - Cvoz) in ml 02 / 100 ml, and resistance in
mmHgoml"-min"-100g". Difference equals glucose value minus normal saline (NS)
value. * P < 0.05. # P < 0.05 relative to value before angiotensin H.

78

Table 8. Effect of angiotensin H on oleate-induced changes in blood ﬂow,
resistance, arteriovenous oxygen content difference, and oxygen uptake.

 

 

 

 

Linnen Content Difference Percent

NS Oleate Change
Before Angiotensin H
Blood Flow 50513.1 75.6182 25.117.8* 51.5117.7%*
Resistance 2.381023 1.661018 -0.731021* -28.416.3%*
(Cao2 - Cvoz) 4.641028 3.641022 -l.0010.39* -19.816.4%*
Oxygen Uptake 2.321008 2.601012 02810.05* 12.711.9%*
During Angioteniin H Infu_si1)g
Blood Flow 25.611.3# 44.2118.2# 18.416.8* 70.317.7%*#
Resistance 4.9010.24# 2.9410.27# -1.9610.14*# -40.513.1%*#
(Cao2 - Cvoz) 8.431033# 5.9010.52# -2.5311.17*# -30.015.7%*#
Oxygen Uptake 2.211008 2.471012 0.261005" 11.411.7%*

 

Values are mean 1 SE; n=7. Blood ﬂow and oxygen uptake values are expressed in
ml-min"o100g", (Cao2 - Cvo,) in ml O2 [100 m1, and resistance in
mmHg-ml'l-min"o100g". Difference equals oleate value minus normal saline (NS) value.

* P < 0.05. # P < 0.05 relative to value before angiotensin II.

79

(0.26 1 0.05 ml-min"-100g") during angiotensin H infusion. Hence, in contrast to
mefenamate, angiotensin II infusion did not signiﬁcantly alter the increase in jejunal blood
ﬂow and oxygen uptake induced by glucose or oleate.

The second objective of series I and II was to determine the production of PGE” 6-
keto-PGFm, Tsz, and PGF,“ when the lumen contained normal saline or glucose, and
normal saline or oleate. Figure 17 shows the arterial and jejunal venous concentrations
of the four prostanoids when the lumen contained normal saline or glucose, and normal
saline or oleate. Each value is the average of the prostanoid concentration in the samples
obtained at the 11th and 15th minute after luminal placement of one of the solutions. The
venous concentration of each prostanoid was always higher than the corresponding arterial
concentration in all conditions, indicating that all four prostanoids were produced and
released from the jejunum whether the luminal contents were normal saline, glucose or
oleate. Arterial concentrations of all four prostanoids did not change signiﬁcantly when
the luminal contents were changed, but venous concentrations changed when the luminal
contents were changed to glucose or oleate. Compared with the corresponding value when
normal saline was in the lumen, glucose tended to increase the venous concentrations of
6-keto-PGF1a and PGF”, and signiﬁcantly increased venous PGE2 and TxB2
concentrations. Oleate also tended to increase the venous concentration of 6-keto-PGFm,
and signiﬁcantly increased venous PGE” Tsz, and PGF2m concentrations. Although the
venous PGE” Tsz, and PGF2m concentrations when oleate was in the lumen tended to
be higher than those when glucose was in the lumen, the differences were not statistically
different.

Figure 18 compares the rate of individual prostanoid releases when the lumen

80

contained normal saline or glucose, and normal saline or oleate. The rate of prostanoid
release was calculated as the product of blood ﬂow and the arteriovenous prostanoid
concentration difference. In series I, when normal saline was in the lumen PGEZ, PGI2
(6-keto-PGFml precursor), TxAz, (TxB2 precursor), and PGF,“ were released from the
jejunum. The relative magnitude of the release (in ng-ml"-min"-100g") was PGE2 (28 1
6) > PGI2 (12 1 2) > TxA2 (9 1 2) > PGF,“ (4 1 1). When the relative magnitude of the
release was expressed as the percent fraction of the sum of all four prostanoid releases,
vasodilators, PGFq (52%) and PGI2 (22%) comprised 75% of the total release when
normal saline was in the lumen. When glucose was placed into the lumen, the release of
all four prostanoids increased signiﬁcantly. PGEZ, TxA, and PGF2m releases doubled,
while PGI2 release increased nearly ﬁfty percent The relative magnitude of the release
(in ng-ml"-min"dOOg") was PGE2 (57 1 16) > PGI2 (17 1 3) = TxA2 (l8 1 4) > PGF2m
(7 1 2). The total release of prostanoids doubled. However, vasodilators prostanoids, PGE:
and PGIZ, still comprised 74% of the total release.

In series II, when normal saline was in the lumen PGE), PGIZ, TxAz, and PGF,“ were
released from the jejunum. The relative magnitude of the release (in ng-ml"-min"-100g")
was PGE2 (32 1 8) > PGI2 (l4 1 3) > TxA2 (7 1 2) > PGF,“ (4 1 1). Vasodilator
prostanoids, PGEz and PGIZ, comprised 79% of the total prostanoid release. These values
are not different from those in series I when normal saline was in the lumen. When oleate
was placed into the lumen, the release of all four prostanoids increased signiﬁcantly.
PGE, and PGF” releases increased threefold, TxA2 release increased fourfold, and the
PG, release increased by more than sixty percent. The relative magnitude of the release

(in ng-ml".min"-100g") was PGE2 (136 1 30) > TxA, (35 1 9) > PGI2 (23 1 4) > PGF2m

 

Figure 17.

81

Arterial and jejunal venous plasma concentrations of 6-keto-PGFm,
Tsz, PGEQ, and PGan (pg-m1") during luminal placement of normal
saline (NS), glucose (GL), and oleate (0A). * P < 0.05 relative to the
venous concentration obtained during the corresponding NS.

82

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83

Figure 18. Jejunal releases of 6-keto-PGFm, Tsz, PGEI, and PGF,“
(ng~min"400g") during luminal placement of normal saline (NS),
' glucose (GL), and oleate (OA). * P < 0.05 relative to the value
obtained during the corresponding NS; # P < 0.05 relative to the
corresponding GL value.

84

NS

 

l-"-1
NS

 

 

 

 

 

 

 

 

 

assesses" E§8§ﬁ33°
(600 L/Ugm/bu) canopy 335‘, (500 L/ugtu/ﬁu) ”aqua ”2:19:11

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85

(19 1 6). The total release of prostanoids increased nearly threefold. However,
vasodilators prostanoids, PGE, and PGIZ, still comprised 75% of the total release.

The releases of PGE2, TxA2 and PGF,“ when oleate was in the lumen were
signiﬁcantly greater than those when glucose was in the lumen. The release of PGI2 when
oleate was in the lumen tended to be higher than that when glucose was in the lumen, but
was not statistically different

The next series of experiments were performed to determine the if endogenous TxA2
plays a role in the regulation of the changes in blood ﬂow and oxygen uptake induced by
glucose (series V), and oleate (series VI). Figure 19 shows the jejunal blood ﬂow and
oxygen uptake during luminal placement of normal saline or glucose before and after
intraarterial administration of SQ 29548. Luminal placement of glucose signiﬁcantly
increase jejunal blood ﬂow before and after administration of SQ 29548. Administration
of SQ 29548 did not alter blood ﬂow when normal saline was in the lumen (53.5 1 3.7
before and 53.1 1 3.2 ml-min"-100g" after SQ 29548). The magnitude of the glucose-
induced increase in blood ﬂow before SQ 29548 was not different from that after SQ
29548. This true whether the data are expressed as the absolute change in blood ﬂow
(12.2 1 1.7 before and 13.9 1 2.1 ml-min"o100g" after SQ 29548), or as the percent
change from control (24.8 1 3.8% before and 26.0 1 3.7% after SQ 29548).

The administration of SQ 29548 did not alter jejunal oxygen uptake when normal
saline was in the lumen (2.21 1 0.18 before and 2.19 1 0.17 after SQ 29548). Luminal
placement of glucose increased jejunal oxygen uptake before and after SQ 29548
administration. However, the magnitude of the glucose-induced increase in oxygen uptake

after administration of SQ 29548 (0.44 1 0.08 mlomin'l-100g'l or 19.6 1 3.4%) was

 

JEJUNAL BLOOD FLOW
(ml/mln/l 009)

JEJUNAL OXYGEN UPTAKE
(ml/mln/l 009)

Figure 19.

86

D Normal Saline
100- - Glucose

 

 

 

 

 

 

 

 

 

E glormol Saline
3.50 . ucoee

3.00- *#
2.50-
2.00-
1.50-
1.00-
0.50-

 

 

 

 

 

 

 

 

 

o.oa
Before After

30 29548 80 29548

Jejunal blood ﬂow and oxygen uptake during luminal placement of
normal saline or glucose before and after intraarterial administration of '
SQ 29548. * P < 0.05 relative to normal saline value. # P < 0.05
relative to the glucose value before SQ 29548.

87
signiﬁcantly greater than that before SQ 29548 (0.26 1 0.03 ml-min"-100g" or 12.2 1

1.4%). Hence, SQ 29548 enhanced the increase in jejunal oxygen uptake induced by
glucose, but did not affect the glucose-induced hyperemia.

Figure 20 shows the jejunal blood ﬂow and oxygen uptake during luminal placement
of normal saline or oleate before and after administration of SQ 29548. Luminal
placement of oleate signiﬁcantly increased blood ﬂow before and after SQ 29548.
Administration of SQ 29548 did not affect resting jejunal blood ﬂow (56.6 1 1.7 before
and 56.8 1 3.9 ml-min"°100g" after SQ 29548). However, the magnitude of the oleate-
induced increase in blood ﬂow was signiﬁcantly greater after administration of SQ 29548.
This is true whether the data are expressed as the absolute change in blood ﬂow (32.7 1
4.3 before vs 45.6 1 4.8 mlomin"o100g" after SQ 29548), or the percent change from
control (57.1 1 8.3% before vs 82.5 1 12.5%).

In series VI, the administration of SQ 29548 did not alter jejunal oxygen uptake when
normal saline was in the lumen (2.40 1 0.21 before and 2.47 1 0.22 after SQ 29548).
Luminal placement of oleate increased jejunal oxygen uptake before and after SQ 29548
administration. However, the magnitude of the oleate-induced increase in oxygen uptake
after administration of SQ 29548 (0.34 10.03 mlomin"~100g" or 14.9 1 1.4%) was greater
than that before SQ 29548 (0.22 1 0.03 ml-min"-100g" or 9.7 1 1.2%). Hence, SQ 29548

enhanced both the increase in jejunal blood ﬂow and oxygen uptake induced by oleate.

88

1: Normal Saline
m Oleate *#

1oo *

JEJUNAL BLOOD FLOW
(ml/min/iOOg)

8388

 

O

:1 Normal Saline

 

3.50 m Oleate
2
E
3 ’3
8 E
g E
a
a
7
Before After
80 29548 50 29548

Figure 20. Jejunal blood ﬂow and oxygen uptake during luminal placement of
normal saline or oleate before and after intraarterial administration of
SQ 29548. * P < 0.05 relative to normal saline value. # P < 005'
relative to the glucose value before SQ 29548.

89
Effect of Prostanoids on Mucosal Oxygen Consumption in vitro.

Effect of Prostanoids on the Glucose-induced Change in Mucosal m in vitro.

Figure 4 (page 46) shows the relationship between the amount of oxygen consumed
by the jejunal mucosa and the incubation time when the incubation media contained only
0 or 5 mM glucose in all 12 series of experiments. The mucosal oxygen consumption
rates (Q02) were 116 and 173 nl-min"omg dry wt" when the media contained 0 and 5 mM
glucose, respectively. Thus, addition of 5 mM glucose to the media signiﬁcantly increased
mucosal QO2 by 48 %.

Table 9 shows the effects of cyclooxygenase inhibitors on mucosal Q02 in the
absence and presence of 5 mM glucose. In the absence of glucose both mefenamate and
indomethacin signiﬁcantly inhibited the Q02 by 10511.3 and 9.210.4%, respectively. The
magnitude of the decrease in QO2 induced by these two cyclooxygenase inhibitors was
not signiﬁcantly different Inclusion of 5 mM glucose in the media markedly increased
the Q02 from 11812 to 16716, and 11913 to 15112 nl-min".mg dry wt", respectively.
This glucose-induced increase in mucosal QO2 was further enhanced by both mefenamate
(8.910.1%) and indomethacin (l0610.8%). Mefenamate and indomethacin enhanced the
Q02 by the same magnitude. Thus, cyclooxygenase inhibition decreased the basal jejunal
mucosal Q02, but it enhanced the glucose-induced increase in Q02.

The effect of arachidonate (AA) on jejunal mucosal QO2 is shown in Table 10. Over
the concentration range 1 to 1000 1.1M, arachidonate dose-dependently decreased Q02 in
the absence and presence of 5 mM glucose. Thus arachidonate inhibited the basal QO2
as well as the glucose-induced increase in Q02. In order to determine if the arachidonate-

induced inhibition of mucosal QO2 was due to arachidonate or its cyclooxygenase

 

90

Table 9. The Effect of Cyclooxygenase Inhibitors on Jejunal Mucosal Q02.

 

 

Series Media Contents QO2 Changes % Change

Ia 0 mM Glucose 118 1 2

0 mM Glucose + Mef. 106 1 2* -12 1 2 -105 1 1.3%
lb 0 mM Glucose 119 1 3

0 mM Glucose + Indo. 108 1 2* -11 1 1 -9.2 1 0.4%
Ha 5 mM Glucose 167 1 6

5 mM Glucose + Mef. 182 1 7* +15 1 l +8.9 1 0.1%
Hb 5 mM Glucose 151 1 2

5 mM Glucose + Indo. 168 1 3* +16 1 1 +106 1 0.8%

 

QO2 values and changes are means 1 SE and are in n1 02-min"-mg dry wt".
Mef. = Mefenamate (40 pM). Indo.= Indomethacin (10 M). * P < 0.05 relative to
corresponding glucose alone. See Table 1 for number of experiments.

91

Table 10. The Effect of Arachidonate on Jejunal Mucosal Q02.

 

 

Series Media Contents QO2 Changes % Change
HI 0 mM Glucose 118 1 l
0 mM Glucose + AA 1 1.1M 113 1 2* -5 1 2 -4.2 1 2.0%
0 mM Glucose + AA 10 M 112 1 1* -6 1 1 -4.9 1 1.0%
0 mM Glucose + AA 100 M 106 11*@ -12 1 2 -9.9 11.0%
0 mM Glucose + AA 1000 1.1M 99 1 2*@ -l9 1 2 -16.11 2.0%
IV 5 mM Glucose 192 1 7
5 mM Glucose + AA 1 M 172 1 3* -21 1 6 ~10.7 1 2.7%
5 mM Glucose + AA 10 M 170 1 6* -23 1 4 -11.9 1 3.1%
5 mM Glucose + AA 100 M 1611 5*@ -301 5 -15.6 1 2.1%
5 mM Glucose + AA 1000 M 151 1 4*@ -40 1 4 -20.7 1 1.4%

 

 

QO2 values and changes are means 1 SE and are in n1 02-min"-mg dry wt".
AA = Arachidonate. * P < 0.05 relative to corresponding 0 mM or 5 mM Glucose alone.
@ P < 0.05 relative to preceding lower AA concentration.

92

metabolites, prostanoids, the effects of mefenamate and arachidonate individually and in
combination were determined in the absence and presence of 5 mM glucose in the same
series of experiments. The results are summarized in Table 11 and depicted graphically
in Figure 21. In the absence of glucose, mefenamate and arachidonate individually
decreased mucosal QO2 9.411.l% and l0411.1%, respectively. The combination of
mefenamate (40 nM) and arachidonate (100 1.1M) signiﬁcantly decreased mucosal QO2
by 17.511.3%. This decrease in QO2 was signiﬁcantly greater than that induced by either
mefenamate or arachidonate individually. In the .presence of 5 mM glucose, mefenamate
individually increased QO2 6.511.7%, whereas arachidonate individually decreased QO2
11.211.2%. The mucosal QO2 (162 1 6 nl-min'l-mg dry wt") when the media contained
the combination of mefenamate and arachidonate in 5 mM glucose was not signiﬁcantly
different from the Q02 values when the media contained 5 mM glucose without treatment
(160 1 6 nl-min‘l-mg dry wt"), and mefenamate individually in 5 mM glucose (172 1 6
n1.min"-mg dry wt"). However, this QO2 was signiﬁcantly greater than that when the
media contained arachidonate individually in 5mM glucose (142 1 5 nl-min"-mg dry wt").
Thus, mefenamate abolished the arachidonate-induced decrease in Q02 in the presence
of glucose.

Table 12 shows the effect of U-44069, a TxA2 / endoperoxide analogue, on jejunal
mucosal Q02. In the absence of glucose, U-44069 did not signiﬁcantly affect mucosal
Q02. However, in the presence of 5 mM glucose U-44069 signiﬁcantly decreased the Q02
in a dose dependent manner. The effect of SQ 29548, a TxA2 / endoperoxide receptor
blocker, is shown in Table 13. In the absence of glucose, SQ 29548 did not signiﬁcantly

affect mucosal Q02. However, in the presence of 5 mM glucose, it signiﬁcantly increased

 

93

Table 11. The Effects of Mefenamate and Arachidonate, alone and combined,
on Mucosal Q02.

 

 

Series Media Contents QO2 Change % Change
V 0 mM Glucose 114 1 2
0 mM Glucose + Mef. 103 1 2* -11 1 1 -9.4 1 1.1%
0 mM Glucose + AA 102 1 2* -l2 1 1 -10.4 11.1%
0 mM Glucose + Mef. + AA 94 1 2*@ -20 1 2 -175 1 1.3%
VI 5 mM Glucose 160 1 6
5 mM Glucose + Mef. 170 1 6* +10 1 3 +6.5 1 1.7%
5 mM Glucose + AA 142 1 5* -18 1 2 -11.2 11.2%
5 mM Glucose + Mef. + AA 162 1 6# +2 1 2 +1.11 1.8%

 

QO2 values and changes are means 1 SE and are in n1 Ozomin"-mg dry wt".
Mef. = Mefenamate (40 11M); AA = Arachidonate (100 11M); Mef. + AA = both
Mefenamate (40 1.1M) and Arachidonate (100 M). * P < 0.05 relative to respective
0 or 5 mM Glucose alone. # P < 0.05 relative to 5 mM Glucose + AA value. @ P < 0.05
relative to both 0 mM Glucose + AA and 0 mM Glucose + Mef. values.

94

Q02 (nl/min/m
'5
O

 

 

 

 

 

 

 

 

 

o ,
[Glucll ----- 0 ----- l | ————— 5 ————— |
[Mef] 0 4o 0 40 o 40 o 40 p.11
[AA] 0 o 100 100 o o 100 100 MM

Figure 21. Effect of mefenamate [Mef], arachidonate [AA], and mefenamate plus
arachidonate in combination on the mucosal Q02 in the absence and
presence of 5 mM glucose [Gluc]. * P < 0.05 relative to the basal
value ([Gluc] 0, [Mef] 0, [AA] 0). # P < 0.05 relative to mefenamate
([Gluc] 0, [Met] 40, [AA] 0), or arachidonate ([Gluc] 0, [Met] 0, [AA]
100) values. @ P < 0.05 relative to 5 mM glucose value ([Gluc] 5,
[Met] 0, [AA] 0). § P < 0.05 relative to arachidonate value in 5 mM
glucose ([Gluc] 5, [Mef] 0, [AA] 100).

Table 12. The Effect of U-44069 on Jejunal Mucosal Q02.

95

 

 

Series Media Contents QO2 Changes % Change
VH 0 mM Glucose 119 1 2
0 mM Glucose + U 0.28 nM 118 1 2 -11 2 ~06 1 1.7%
0 mM Glucose + U 2.8 nM 115 1 2 -3 1 2 -2.8 1 1.6%
0 mM Glucose + U 28.0 nM 116 1 2 -3 1 3 -2.1 1 3.0%
VIH 5 mM Glucose 177 1 6
5 mM Glucose + U 0.28 nM 168 1 6* -9 1 2 -5.2 1 1.2%
5 mM Glucose + U 2.8 nM 164 1 6* -13 1 2 -7.6 1 1.0%
5 mM Glucose + U 28.0 nM 152 1 7*# -23 1 5 -12.4 1 2.5%

 

QO2 values and changes are means 1 SE and are in n1 02-min"omg dry wt".
U = U-44069. * P < 0.05 relative to 5 mM Glucose. # P < 0.05 relative to preceding

U-44069 concentration.

96

Table 13. The Effect of SQ 29548 on Jejunal Mucosal Q02.

 

 

 

Series Media Contents QO2 Change % Change

D( 0 mM Glucose 114 1 2

0 mM Glucose + SQ 111 1 2 -3 1 1 -2.8 1 1.2%

XI 0 mM Glucose + AA 102 1 2* -12 11 -10.4 11.1%

0 mM Glucose + AA + SQ 100 1 2* -14 11 -12.111.0%
X 5 mM Glucose 166 1 7

5 mM Glucose + SQ 181 1 7* +14 1 3 +8.4 1 1.5%
VI 5 mM Glucose 160 1 6

5 mM Glucose + AA 142 1 5* -18 1 2 -11.2 11.2%

XH 5 mM Glucose + AA + SQ 149 1 5*# -11 1 1 -6.5 1 1.3%

 

QO2 values and changes are means 1 SE and are in n1 02-min"omg dry wt".
SQ = SQ 29548 (10 11M); AA = Arachidonate (100 M). * P < 0.05 relative to
corresponding 0 or 5 mM Glucose value. # P < 0.05 relative to 5 mM Glucose + AA.
The series XH experiments were performed in conjunction with series VI using the
same mucosal tissue from the same jejunal segments.

97

20 ' ’:’:’:’:’:‘

 

 

 

 

 

 

 

 

 

[Glue] 1
[80] O

 

Figure 22. Effect of SQ 29548, a TxA2 / endoperoxide receptor blocker in mucosal
Q02 in the absence and presence of 5 mM glucose. * P < 0.05 relative
to corresponding value without SQ 29548.

98
the Q02 by 8.411.5%, from 166 1 7 to 181 1 7 nlvmin"-mg dry wt" (Figure 22). Thus,

the TxA2 analogue inhibited, whereas the TxA2 receptor blocker enhanced the glucose-
induced increase in mucosal Q02.

Table 13 also shows the effect of SQ 29548 on the arachidonate—induced decrease in
mucosal Q02. In the absence of glucose, arachidonate individually or in combination with
SQ 29548 signiﬁcantly decreased the Q02. The magnitude of the decreases in QO2 under
these two conditions, i.e., -10.411.1% vs -12.111.0% were not signiﬁcantly different.
Thus SQ 29548 did not affect the arachidonate-induced decrease in Q02 in the absence
of glucose. In the presence of 5 mM glucose, arachidonate individually and in
combination with SQ 29548 signiﬁcantly decreased the mucosal Q02. However, the
decrease in QO2 induced by arachidonate individually (-11.211.2%) was signiﬁcantly
greater than that produced by arachidonate in combination with SQ 29548 (-6.511.3%).
Thus, TxA2 receptor blocker, SQ 29548, did not signiﬁcantly affect the arachidonate-
induced inhibition of the basal mucosal Q02, but it attenuated the arachidonate—induced

inhibition of the glucose-induced increase in mucosal Q02.

Effect of Prostanoids on t_he Olgate-induced Change in Mucosal m, in vitro.

As shown in the preliminary studies, 0.1 mM taurocholate did not alter Q02 in series
XHI-XV experiments. In all three series 0.5 mM oleate plus taurocholate did not affect
mucosal Q02, whereas 5.0 mM oleate plus taurocholate signiﬁcantly decreased QO2 an
average of 1715 nl-min"-mg'l (14.613.2%).

The effect of cyclooxygenase inhibition by mefenamate on the Q02 in the absence and

99

presence of taurocholate, and oleate plus taurocholate is summarized in Table 14 and
depicted in Figure 23. Mefenamate decreased the basal QO2 by 12.312.2%. Mefenamate
also decreased Q02 in the presence of 0.1 mM taurocholate (7.712.3%), and 0.5 mM
oleate plus taurocholate (7.512.2%). Oleate (5.0 mM) plus taurocholate decreased mucosal
QO2 by 13.413.1% (from 11013 to 9513 nl-min"-mg dry wt"). In the presence of 5.0 mM
oleate plus taurocholate, mefenamate further decreased mucosal QO2 by 11.612.7% (from
9513 to 8315 nl-min"~mg dry wt") for a total decrease in QO2 of 24.514.1% from the
basal value (11013 to 8315 nl-min"~mg dry wt"). The magnitude of the decrease in QO2
produced by mefenamate was similar in all conditions. The magnitude of the decrease in
QO2 induced by the combination of 5.0 mM oleate plus taurocholate and mefenamate
(24.514.1%) was not different from the sum of the decreases in Q02 induced by
mefenamate individually (12.312.2%) and 5.0 mM oleate plus taurocholate individually
(13.413.1%), suggesting that the effects might be additive. Further, the 5.0 mM oleate
plus taurocholate-induced decrease in QO2 when the media did not contain mefenamate
(13.413.l%) was not different from the 5.0 mM oleate plus taurocholate-induced decrease
on QO2 when the media contained mefenamate (l4.412.6%, from 9715 to 8315 n1~min'
lomg dry wt"). Thus mefenamate decreased basal QO2 as well as Q02 in the presence of
taurocholate and oleate plus taurocholate. However, it appears that it did not alter the
decrease in QO2 induced by 5.0 mM oleate plus taurocholate.

The effect of arachidonate on mucosal Q02 in the absence and presence of
taurocholate and oleate plus taurocholate is summarized in Table 15. Figure 24 also shows
these results graphically. Arachidonate decreased basal QO2 by 8.111.0%. It also

decreased Q02 in the presence of taurocholate (9.712.3%), and 0.5 mM oleate plus

 

 

100

 

 

Table 14. The effect of mefenamate on oleate plus taurocholate-induced changes in
mucosal Q02.
Series Media Contents QO2 Change % Change 11
XIH No Treatment (Basal) 110 1 3 9/5
Mefenamate (Mef.) 97 1 5*# -12 1 2 -12.3 1 2.2% 8/5
TC 0.1 mM 107 1 4 8/5
TC + Mef. 99 1 5*# -8 1 3 -7.7 1 2.3% 8/5
TC + 0A 0.5 mM 107 1 3 8/5
TC + GA 0.5 mM + Mef. 100 1 5*# -8 1 2 ~75 1 2.2% 8/5
TC + 0A 5.0 mM 95 1 3* 7/5
TC + 0A 5.0 mM + Mef. 83 1 5*# -111 4 -11.6 1 2.7% 7/5

 

QO2 values and changes are means 1 SE and are in n1 02-min"-mg dry wt".
TC = Taurocholate (0.1 mM). OA = Oleate. * P < 0.05 relative to No Treatment
(Basal) value. # P < 0.05 relative to corresponding value without mefenamate.

101

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

140«
120- -
3100 T *1? ]12t2 1’\ *#]Gt3 T ..
\ I \ / _ , v...) *#
/ s x ‘ 1,-
g g § ¢ g ; :
5 so % x % \ . .
N 40- % § / \ i
a a V s
A § é § 1 -
[Tc] |--o--| |—-o.1--| |--o.1--| |--o.1--| mM
13:1, "2: °;.;' ".'°:;' "3°51: "3°13" :3:

Figure 23. Effect of mefenamate ([Mef]) on mucosal Q02 in the absence and
presence of taurocholate ([TC]) and taurocholate plus oleate ([OA]).
* P < 0.05 relative to basal value ([TC] 0, [0A] 0). # P < 0.05 relative
to corresponding value without mefenamate ([Mef] 0).

102
0.1 mM taurocholate (9.811.7%). Oleate (5.0 mM) plus taurocholate decreased QO2 by

12.913.2% (from 13113 to 11414 nl-min"-mg dry wt"). In the presence of 5.0 mM oleate
plus taurocholate, arachidonate further decreased mucosal QO2 by 5.811.7% (from 11414
to 10815 nl-min"-mg dry wt") for a total decrease in QO2 of 17.112.2% from the basal
value (13113 to 10815 nl-min".mg dry wt"). The magnitude of the decrease in QO2
produced by arachidonate was similar in all conditions. The magnitude of the decrease
in QO2 induced by the combination of 5.0 mM oleate plus taurocholate and arachidonate
(17.112.2%) was not different from the sum of the decreases in QO2 induced by
arachidonate individually (8.111.0%) and 5.0 mM oleate plus taurocholate individually
(12.913.3%), suggesting that the effects might be additive. Further, the 5.0 mM oleate
plus taurocholate-induced decrease in QO2 when to media did not contain arachidonate
(12.913.3%) was not different from the 5.0 mM oleate plus taurocholate-induced decrease
on QO2 when the media contained arachidonate (10.712.2%, from 12113 to 10815 nl-min'
l-mg dry wt"). Thus, even though arachidonate decreased basal QO2 as well as Q02 in the
presence of 0.1 mM taurocholate, 0.5 mM oleate plus taurocholate, and 5.0 mM oleate
plus taurocholate, it seems that it did not alter the decrease in QO2 induced by 5.0 mM
oleate plus taurocholate.

In an attempt to determine if the effect of arachidonate on QO2 was due to
cyclooxygenase products, the effect of the combination of mefenamate and arachidonate
on Q0, was determined in the presence of 0.5 and 5.0 mM oleate plus taurocholate
(Table 15). The combination of mefenamate and arachidonate decreased mucosal Q02 in
both conditions. In the presence of 0.5 mM oleate plus taurocholate mefenamate and

arachidonate in combination decreased QO2 by 16.615.%. This decrease in QO2 was

103

 

 

Table 15. The effect of arachidonate on oleate plus taurocholate-induced changes in
mucosal Q02.
Series Media Contents ' QO2 Change % Change It
XIV No Treatment (Basal) 131 1 3 10/5
Arachidonate 100 1.1M (AA) 121 1 3* ~10 1 2 ~81 1 1.0% 10/5
TC 0.1 mM 133 1 2 10/5
TC + AA 120 1 3* ~11 1 3 ~97 1 2.3% 10/5
TC + 0A 0.5 mM 1311 4 4/2
TC + 0A 0.5 mM + AA 120 1 3*# ~12 1 3 ~98 1 1.7% 4/2
TC + 0A 0.5 mM + AA + Mef. 110 1 6*#@ ~21 1 5 ~16.6 1 5.2% 4/2
TC + 0A 5.0 mM 114 1 4* 6/3
TC + GA 5.0 mM + AA 108 1 5*# -6 1 3 -5.8 1 1.7% 6/3
TC + 0A 5.0 mM + AA + Mef. 105 1 5*# ~8 1 3 ~7.1 1 2.0% 6/3

 

QO2 values and changes are means 1 SE and are in n1 Ozomin"-mg dry wt".
TC = taurocholate (0.1 mM). OA = oleate. AA = arachidonate (100 pM). Mef.
mefenamate, (40 11M). * P < 0.05 relative to No Treatment (Basal) value. # P < 0.05

relative to corresponding value without AA. @ P < 0.05 relative to

TC + GA 0.5 mM + AA.

104

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1012 111:3 12:3
140 - “*3
FI- *# *# 1:1; *# o
1 20 ~ $7 \ *#
A \ + *1
“E" 100 - é \ '1'
e / \
-- so 1 / \
E / §
:5 60- é \
8. 4o - ; i
O / \
20 - / \
O 6
[TC] |—-o--| |- 0.1 -| |-—-—o.1--—-| |—---o.1--—-| mM
[0A] |—-0—-| |--0--| I 0.5 i |—-'-- 5.0 ----| mM
[AA] 0 100 0 100 0 100 100 0 100 100 pM
[Mef] 4o 40 [1M
Figure 24. Effect of arachidonate on mucosal Q02 in the absence and presence of

taurocholate ([TC]) and taurocholate plus oleate ([OA]). Also shown is
the effect of arachidonate plus mefenamate in combination in the
presence of taurocholate plus oleate. * P < 0.05 relative to basal value
([TC] 0, [0A] 0, [AA] 0). # P < 0.05 relative to corresponding value
with out arachidonate ([AA] 0). @ P < 0.05 relative to ([T C] 0.1, [CA]
0.5, [AA] 100).

105
signiﬁcantly greater than that induced by arachidonate individually (9.811.7%). or

mefenamate individually (9.1126 %, data from series XIH). Oleate (5 mM) plus
taurocholate decreased QO2 by 13.1122%. In the presence of 5.0 mM oleate plus
taurocholate mefenamate and arachidonate in combination decreased QO2 an additional
7.1120%. This decrease in QO2 tended to be slightly greater than that induced by
arachidonate in 5.0 mM oleate plus taurocholate (5.211.7%) but was not statistically
signiﬁcant Hence mefenamate appears to have either not altered or added to the affect
of arachidonate on Q02 in the presence of 0.5 and 5.0 mM oleate plus taurocholate.
Mefenamate did not abolish or even attenuate the effect of arachidonate.

The effect of the TxA2 / endoperoxide blocker, SQ 29548, on mucosal Q02 in the
absence and presence of taurocholate and oleate plus taurocholate are summarized in
Table 16. SQ 29548 did not alter the basal QO2 nor did it affect Q02 in the presence of
0.1 mM taurocholate, or 0.5 mM oleate plus taurocholate. Even though 5.0 mM oleate
plus taurocholate decreased Q02, SQ 29548 did not alter this oleate-induced decrease in

Q02. Hence, SQ 29548 did not alter mucosal Q02 in any of these conditions.

 

106

 

 

Table 16. The effect of SQ 29548 on oleate plus taurocholate-induced changes in
mucosal Q02.
Series Media Contents Q02 Change % Change 11
XVI No Treatment (Basal) 117 1 3 8/4
SQ 29548 10 M 113 1 4 -3 1 2 ~33 1 1.6% 8/4
TC 0.1 mM 119 1 2 8/4
TC + SQ 116 14 -3 1 2 ~33 1 2.1% 8/4
TC + CA 0.5 mM 115 1 2 8/4
TC + 0A 0.5 mM + SQ 113 1 2 ~2 1 2 ~2111.9% 8/4
TC + 0A 5.0 mM 93 1 2* 7/4
TC + 0A 5.0 mM + SQ 93 1 3* -1 1 2 ~07 1 2.6% 7/4

 

QO2 values and changes are means 1 SE and are in n1 02-min"-mg dry wt".
TC = Taurocholate (0.1 mM). OA = Oleate. * P < 0.05 relative to No Treatment
(Basal) value.

DISCUSSION

Effect of Prostanoids on Glucose- and Oleate~Induced Changes in Blood Flow and
Oxygen Uptake in viva.

Intestinal blood ﬂow increases after a meal (18,23,4244), and the stimuli for this
hyperemia are the digested products of food (24,42). The mechanisms involved in the
initiation and regulation of this hyperemia are multifactorial and include oxidative
metabolism, enteric nerves and local reﬂexes, gastrointestinal hormones and peptides, and
local tissue chemicals such as histamine and prostanoids (18,23,4248). Additionally
different nutrients may inﬂuence intestinal blood ﬂow via different mechanisms or
combinations of mechanisms (26,42). Systematic study of various constituents of food
revealed that glucose and oleate are the main nutrients that individually stimulate an
intestinal hyperemia (24). These two nutrients inﬂuence intestinal blood ﬂow differently
(26).

Recent studies have shown that endogenous prostanoids may play a role in the
regulation of intestinal blood flow and oxygen uptake during nutrient absorption
(20,41,67). The food-induced increase in blood ﬂow and oxygen uptake is accompanied
by an increase in the release of prostanoids by the jejunum (20). Cyclooxygenase
inhibition enhances the food~ and oleate-induced increase in blood ﬂow and oxygen

uptake (41,77). Arachidonate attenuates the food-induced increase in blood ﬂow and

107

108

oxygen uptake (67). Most of these studies investigated the role of prostanoids in the
regulation of the nutrient-induced hyperemia utilizing a balanced food that contained
equal parts by weight of carbohydrate, fat, and protein. One study in exception
investigated the effect of cyclooxygenase inhibitors on glucose- and oleate-induced
increases in a calculated blood ﬂow (77), but did not determine the effects on changes in
oxygen uptake nor determine if prostanoids are released in response to the individual
nutrients. Therefore it is unknown if prostanoids play a role in the regulation of the
increases in blood ﬂow and oxygen uptake induced by individual nutrients. Inasmuch as
glucose and oleate are absorbed by different processes and evoke different vascular
responses, different types or quantities of prostanoids may play a role in the regulation
of the responses to these individual nutrients. The objective of this study was to
investigate if prostanoids play a role in the regulation of glucose- and oleate-induced
increases in blood ﬂow and oxygen uptake. This was studied by determining l) the effect
of cyclooxygenase inhibition on the increases in blood ﬂow and oxygen uptake induced
by glucose and oleate, and 2) if and which prostanoids are released in response to luminal
placement of glucose and oleate.

In series I of this study luminal placement of glucose increased blood ﬂow and
oxygen uptake before and after cyclooxygenase inhibition. Inhibiting prostanoid synthesis
with mefenamate doubled the increase in blood ﬂow and nearly doubled the increase in
oxygen uptake induced by glucose. This indicates that endogenous prostanoids may act
to limit the glucose-induced increases in blood ﬂow and oxygen uptake. The increases
in blood ﬂow and oxygen uptake induced by glucose before mefenamate in this study are

similar to those previously reported (26,74,9091). Sit er al. (91) has shown that the

 

109

glucose-induced hyperemia is, at least in part, related to both glucose absorption and an
increase in oxidative metabolism. Additionally, Gallavan and Chou (43) have shown that
cyclooxygenase inhibition enhances glucose absorption and aerobic carbohydrate
metabolism while enhancing the food-induced hyperemia. Hence cyclooxygenase
inhibition would be expected to enhance the glucose-induced increase in blood ﬂow and
oxygen uptake, as was shown in the present study. However, it has been reported that
cyclooxygenase inhibitors did not affect the glucose-induced increase in blood ﬂow (77).
In this previous study the blood ﬂow value was calculated based on submucosal arteriolar
diameter and red blood cell velocity measured by videomicroscopy in rats (77), whereas
in the present study blood ﬂow was measured by timed collection of the venous outﬂow
from the jejunal segment The reason for the difference in these studies is unclear, but
may be related to the difference in the techniques or species used in these studies.

As shown in Table 5, the enhancement of the glucose-induced hyperemia by
cyclooxygenase inhibition is accompanied by an enhancement of the glucose-induced
increase in oxygen uptake. Furthermore, the glucose-induced changes in blood ﬂow are
signiﬁcantly correlated with the changes in oxygen uptake both before and after
mefenamate (Figure 10). Since intestinal oxygen uptake is largely independent of blood
ﬂow over the physiological range of blood ﬂow of 30 to 140 m1-min"-100g" (48,67), the
enhancement of the glucose-induced increase in oxygen uptake by cyclooxygenase
inhibition is most likely due to the enhancement of tissue oxidative metabolism rather
than the enhancement of blood ﬂow. This is supported by the ﬁndings that
cyclooxygenase inhibition enhances glucose absorption and carbohydrate metabolism (43).

Furthermore PGE2 and PGF” have been shown to inhibit glucose absorption (6,28).

 

110

Hence, endogenous prostanoids might act to limit the glucose-induced increase in oxygen
uptake by limiting oxidative metabolism, and thereby indirectly limit the increase in blood
ﬂow.

In series I] luminal placement of oleate increased blood ﬂow and oxygen uptake
before and after cyclooxygenase inhibition. Inhibiting prostanoid synthesis with
mefenamate enhanced the increases in blood ﬂow and oxygen uptake induced by oleate.
This suggests that endogenous prostanoids may also act to limit the oleate-induced
increases in blood ﬂow and oxygen uptake. The increases in blood ﬂow and oxygen
uptake induced by oleate before mefenamate in this study are similar to those previously
reported (2040.46.58.71). Cyclooxygenase inhibitors have previously been shown to '
enhance the increase in blood ﬂow induced by oleate in rats (77). The present study
supports this ﬁnding and further shows that cyclooxygenase inhibition also enhances the
increase in oxygen uptake induced by oleate.

As shown in Table 6. the enhancement of the oleate-induced hyperemia by
cyclooxygenase inhibition is accompanied by an enhancement of the oleate-induced
increase in oxygen uptake. Furthermore, the oleate-induced changes in blood ﬂow are
signiﬁcantly correlated with the changes in oxygen uptake both before and after
mefenamate (Figure 11). As was the case for glucose. the enhancement of the oleate~
induced increase in oxygen uptake by cyclooxygenase inhibition is most likely due to the
enhancement of tissue oxidative metabolism rather than the enhancement of blood ﬂow.
Hence, endogenous prostanoids may also act to limit the oleate-induced increase in
oxygen uptake by limiting oxidative metabolism. and thereby indirectly limit the increase

in blood ﬂow. In contrast to glucose, there is no further evidence regarding the effect of

lll

cyclooxygenase inhibitors on lipid absorption or metabolism.

In both series I and II cyclooxygenase inhibition signiﬁcantly decreased resting
blood ﬂow. Cyclooxygenase inhibitors have previously been shown to decrease resting
intestinal blood ﬂow (30.41.44). Myers and Honig (70) have shown that initial resistance
plays a signiﬁcant role in determining the magnitude of the vascular response to a given
stimuli. Granger and Norris (49) have also shown that the sensitivity of resistance vessels
to increases in oxygen demand during digestion is enhanced when control arteriovenous
oxygen content difference is high or the initial ﬂow level is low. Both of these conditions
existed after cyclooxygenase inhibition in both series I and H (Table 5 and 6). Moreover,
Gallavan and Chou (41) have shown that initial resistance. i.e. resting blood ﬂow, plays
a signiﬁcant role in determining the magnitude of the food-induced vascular response
such that the greater the initial resistance the greater the food-induced decrease in
resistance. Therefore the relationship between initial resistance and the change in
resistance induced by glucose and oleate was examined utilizing the same approach as
described by Gallavan and Chou (41). As shown in Figures 13 and 14. it is evident that
this relationship also holds true for both glucose and oleate, i.e. the greater the initial
resistance, the greater the decrease in resistance induced by glucose or oleate.

Based on this relationship it could be argued that the enhancement of the glucose-
and oleate-induced hyperemias by cyclooxygenase inhibition might be due to the decrease
in resting blood ﬂow. However. for both glucose and oleate the slope of the regression
line for the relationship before cyclooxygenase inhibition was signiﬁcantly different from
that after cyclooxygenase inhibition (Figures 13 and 14). In series III and IV this

relationship was further tested by determining the glucose~ and oleate-induced vascular

112

responses before and during angiotensin H infusion. Angiotensin H infusion increased
initial resistance (decreased resting blood ﬂow) to a level similar to that after
cyclooxygenase inhibition, but did not signiﬁcantly affect the changes in blood ﬂow and
oxygen uptake induced by glucose or oleate (Tables 7 and 8). In these series there was
also a signiﬁcant correlation between initial resistance and the change in resistance
induced by both glucose (Figure 15) and oleate (Figure 16). The slopes of the regression
lines in the angiotensin H series were similar to those before mefenamate for both glucose
and oleate. However, for both glucose and oleate, the slope of the regression line for the
angiotensin H series was signiﬁcantly different from that for after cyclooxygenase
inhibition. Therefore, although initial resistance does inﬂuence the glucose- and oleate-
induced vascular responses, the enhancement of the glucose~ and oleate-induced
hyperemias by cyclooxygenase inhibition is, at least in part. in addition to and
independent from the decrease in resting blood ﬂow. Thus, for a given initial resistance
the decrease in resistance induced by glucose or oleate is greater after cyclooxygenase
inhibition.

Cyclooxygenase inhibitors have previously been shown to enhance the increases
in blood ﬂow and oxygen uptake induced by luminal placement of a balanced food
mixture that contained equal parts of carbohydrate, fat. and protein (41). The present
study shows that cyclooxygenase inhibition also enhances the increase in blood ﬂow and
oxygen uptake induced by both glucose and oleate. Inasmuch as cyclooxygenase
inhibition enhances the glucose- and oleate-induced increases in blood ﬂow and oxygen
uptake. the enhancement of the balanced food-induced increases in blood ﬂow and oxygen

uptake by cyclooxygenase inhibitors in the previous study could have been due to the

113

enhancement of carbohydrate (glucose)- or fat (oleate)~induced increases in blood ﬂow
and oxygen uptake either individually or in combination. In the present study the glucose-
and oleate-induced increases in blood ﬂow were correlated with the increases in oxygen
uptake before and after cyclooxygenase inhibition. A similar correlation has been shown
to exist between the changes in blood ﬂow and oxygen uptake induced by food before
and after cyclooxygenase inhibition as well as substrate loading with arachidonate (41,67).
These previous studies also proposed that endogenous prostanoids may act to limit the
food-induced increase in oxygen uptake by limiting oxidative metabolism and that they
may thereby indirectly limit the increase in blood ﬂow.

Comparatively, in the present study the increase in blood ﬂow induced by oleate
was greater than that induced by glucose both before and after cyclooxygenase inhibition.
Cyclooxygenase inhibition enhanced both the glucose- and oleate-induced increase in
blood ﬂow. However. the level of enhancement of the oleate-induced hyperemia was
greater than that of glucose. The increase in oxygen uptake induced by glucose and oleate
were similar before and after cyclooxygenase inhibition. Further, cyclooxygenase
inhibition enhanced the glucose~ and oleate-induced increase in oxygen uptake the same
degree. Inasmuch as cyclooxygenase inhibition enhanced the oleate-induced increase in
blood ﬂow more than that of glucose. it might be expected that more prostanoids,
possibly vasoconstrictors, are produced in response to oleate which cause a greater
limitation of the oleate induced increase in blood ﬂow.

The second objective of this study was to determine which prostanoids are
produced and released by the jejunum during luminal placement of glucose and oleate.

For this purpose the jejunal release of PGFq. PGI2 (as metabolite 6-keto-PGFM), TxA2 (as

114

metabolite Tsz), and PGF,“ was determined when the lumen contained normal saline or
glucose and normal saline or oleate. These four prostanoids are commonly found in the
gastrointestinal tract ( 1.4,5.7.20.65,80) and have been shown to be released by the
jejunum under resting and postprandial conditions (20).

As shown in Figures 17 and 18 the jejunum produces and releases PGEZ, PGI .
TxAz. and lesser amounts of PGF,“ when normal saline is in the lumen. i.e. the resting
condition. This ﬁnding is in agreement with other studies (20). The relative magnitude
of release in series I was PGE, (52%) > PGI2 (23%) > TxA2 (17%) > PGF2m (7%), and
in series 11 PGE2 (56%) > PGI2 (25%) > TxA2 (12%) > PGF20L (7%). The releases of
prostanoids in these two series was similar. In both series. vasodilator prostanoids. PGE2
and PGIz, comprise the majority, 75% in series I and 79% in series 11, of the prostanoids
released. Chou er al. (20) has previously shown that the canine jejunum produces and
releases these four prostanoids at rest under the similar experimental conditions as those
in the present study. The relative magnitude of prostanoids released in the present study
is similar to that in the previous study with the following exception. In both series of the
present study slightly less PGI2 and slightly more PGE, were released than in the previous
study. Consequently in the present study the relative magnitude of PGE, release was
greater than that of PGIT In contrast, in the previous study the PGI2 release was greater
than that of PGE2 (20). The reason for this difference is unclear as the studies were
performed under similar conditions using the same species. as well as the same techniques
to determine prostanoid concentrations. Even though the relative magnitude of the release
of PGE2 and PGI2 differed in these studies, vasodilator prostanoids comprised

approximately 75% of the release in both the previous and present studies. Ahlquist er al.

115
(1) found that canine small intestinal mucosa produces PGE, > PGF,“ = PGI2 > TxAz.

LeDuc er al. (65) has shown that the canine jejunal mucosa produces TxA2 = PGI2 >
PGE; > PGF”, while PGI2 was the major prostanoid produced in the muscularis tissue.
The prostanoid releases determined in the present study are from the whole gut wall. yet
they are comparable to the releases in these studies.

Placing glucose in the lumen doubled the release of PGE), TxAQ, and PGFm, and
nearly doubled the release of PGIz. Luminal placement of oleate increased the release of
TxA2 fourfold. PGE2 and PGF,“ threefold. and PGI2 nearly twofold. This suggests that
glucose and oleate stimulate the synthesis and release of these four prostanoids.
Furthermore. oleate stimulated the release of a greater amount of prostanoids, in particular
TxAz, than did glucose. The relative magnitude of prostanoids released in response to
either glucose or oleate was similar to the relative magnitude of the prostanoids released
under resting conditions, i.e. PGE; > PGI2 > TxA, > PGFm. This suggests that glucose
and oleate primarily increased the quantity of prostanoids released with little alteration
of the relative magnitude of prostanoids released. The exception to this is that oleate
tended to stimulate the release of more TxAz. These prostanoid releases are from the
whole gut wall and do not indicate the sites or tissues which are responsible for the
synthesis and release of these prostanoids. The ﬁnding that oleate stimulates the release
of a greater amount of prostanoids than glucose supports the ﬁnding that inhibiting
prostanoid synthesis enhances the increase in blood ﬂow induced by oleate more than that
induced by glucose.

Chou et al. (20) have shown that luminal placement of a balanced food increased

the release of PGE, fourfold, PGF,“ eightfold, and nearly doubled the release of TxA2 and

116

PGIz. Comparing the prostanoid releases in the present study to the prostanoid releases
in response to food in the previous study (20), it appears that a similar amount of TxAz,
but slightly lesser amounts of PGE2, PGIz, and PGF,“ were released in response to
glucose. Additionally, it appears that more TxA2 and PGE2. a similar amount of PGF“,
and slightly less PGI2 were released in response to oleate compared to food. The relative
magnitude of prostanoids released in response to food in the previous study was PGE, >
PGI2 > TxA2 > PGF”. which is similar to the relative magnitude of prostanoids released
in response to glucose and oleate in the present study.

In summary cyclooxygenase inhibition enhances the increases in blood ﬂow and
oxygen uptake induced by both glucose and oleate. Additionally. cyclooxygenase
inhibition enhances the oleate-induced increase in blood ﬂow more than that induced by
glucose, but enhances the g1ucose- and oleate-induced increases in oxygen uptake by the
same magnitude. This suggests that endogenous prostanoids act to limit the increases in
blood ﬂow and oxygen uptake induced by both glucose and oleate, but limit the oleate~
induced increase in blood ﬂow more than that of glucose. Additionally, both glucose and
oleate stimulate the release of prostanoids, but oleate stimulates a greater release of
prostanoids, and in particular TxAz, than does glucose. These effects of cyclooxygenase
inhibition on the individual nutrient-induced increase in blood ﬂow and oxygen uptake
are similar to its effect on the food-induced increase in blood ﬂow and oxygen uptake.

In series I and II cyclooxygenase inhibition enhanced the glucose- and oleate-
induced increases in blood ﬂow and oxygen uptake. This suggests that vasoconstrictor
prostanoids might play a role in the regulation of the increases in blood ﬂow and oxygen

uptake induced by both glucose and oleate. Thromboxane A2 is a more potent

117

vasoconstrictor than PGF,“ (20,45). Nutrient absorption and the nutrient-induced
hyperemia are localized to the mucosal layer (23.44.48), and TxA2 is synthesized
primarily in the jejunal mucosa (65). and is a more potent vasoconstrictor in the mucosa
than in the muscularis (3). Previous studies have shown that TxA2 can act to limit the
increases in oxygen uptake and capillary exchange density induced by a balanced food
without affecting the food-induced hyperemia (2,68,69). Additionally. both glucose and
oleate stimulated an increase in the release of TxA2 in series I and II above. Hence, it
seems likely that TxA2 might also inﬂuence blood ﬂow and oxygen uptake induced by
glucose and oleate. Therefore the objective of series V and VI was to determine if TxA2
plays a role in the regulation of the increases in blood ﬂow and oxygen uptake induced
by glucose and oleate. This was investigated using a TxA2 / endoperoxide receptor
blocker. SQ 29548, which has previously been used in similar studies (2.3).

In the present study (series V and VI) SQ 29548 did not affect resting blood ﬂow
or oxygen uptake. This ﬁnding is in agreement with previous studies using SQ 29548
(2.3), as well as thromboxane synthase inhibitors (imidazole and U63557A) (68,69). These
ﬁndings indicate that endogenous TxA2 does not play a signiﬁcant role in regulating
jejunal blood ﬂow or oxygen uptake under resting conditions.

Thromboxane A2 / endoperoxide receptor blockade with SQ 29548 signiﬁcantly
enhanced the increase in oxygen uptake induced by glucose, but did not affect the
glucose-induced increase in blood ﬂow. This suggests that endogenous TxA2 might act
to limit the glucose-induced increase in oxygen uptake without altering the glucose-
induced hyperemia. Previous studies have shown that the glucose-induced increase in

oxygen uptake can be met by an increase in oxygen extraction (88.90). an increase in

118

blood ﬂow (26). or a combination of an increase in blood ﬂow and oxygen extraction
(26). Since the glucose-induced increase in blood ﬂow was not affected by SQ 29548, the
enhancement of the glucose-induced increase in oxygen uptake in the present study was
most likely met by an increase in oxygen extraction. SQ 29548 as well as TxA2 synthase
inhibitors also enhance in food-induced increase in oxygen uptake without affecting the
food-induced hyperemia (2.68). These studies suggest that the food-induced increase in
oxygen uptake was also met by an increase in oxygen extraction (2.68). The TxA2
synthase inhibitors and receptor blocker also enhance the food-induced increase in
capillary ﬁltration coefﬁcient (an index of capillary exchange density) without altering the
food-induced hyperemia (2,69). In the previous studies it was proposed that the effect of
TxA2 might be related to its action on intestinal metabolism. i.e. TxA2 might limit the
food-induced increase in tissue oxidative metabolism and thereby indirectly limit the
increase in capillary exchange density (2). It seems possible that TxA2 might also act to
limit the glucose-induced increase in oxygen uptake and oxygen extraction by limiting
tissue oxidative metabolism.

Thromboxane A2 / endoperoxide receptor blockade with SQ 29548 enhanced the
increases in blood ﬂow as well as oxygen uptake induced by oleate. This suggests that
TxA2 might act to limit the oleate-induced increases in blood ﬂow and oxygen uptake.
A previous study has shown that the increase in oxygen uptake induced by oleate is met
primarily by an increase in blood ﬂow as oleate did not alter the PS product (an index
of capillary exchange density) and either decreases or does not alter oxygen extraction as
indexed by arteriovenous oxygen content difference (26). Since the oleate-induced

increase in oxygen uptake is met primarily by an increase in blood ﬂow, the enhancement

119

of the oleate-induced increase in blood ﬂow by SQ 29548 may be due to the enhancement
of the oleate-induced increase in oxygen uptake. Hence, TxA2 might limit the oleate-
induced increase in oxygen uptake thereby limit the increase in blood ﬂow. However, a
direct vascular effect of TxA2 could also contribute to the limitation of the oleate-induced
hyperemia.

In summary in series V and VI SQ 29548 did not alter resting jejunal blood ﬂow
and oxygen uptake. Thus suggesting that TxA2 does not play a role in the regulation of
resting blood ﬂow and oxygen uptake. TxA2 receptor blockade with SQ 29548 enhanced
the glucose-induced increase in oxygen uptake without affecting the glucose-induced
hyperemia. It also enhanced the increases in blood ﬂow and oxygen uptake induced by
oleate. This suggests that endogenous TxA2 might act to limit the glucose-induced
increase in oxygen uptake as well as limit the oleate-induced increases in blood ﬂow and

oxygen uptake.

Effect of Prostanoids on Mucosal Oxygen Consumption in vitro.

In the in vivo studies above, cyclooxygenase inhibition enhanced the glucose~ and
oleate-induced increases in blood ﬂow and oxygen uptake. Additionally the increase in
blood ﬂow and oxygen uptake induced by glucose and oleate were linearly and positively
correlated before and after cyclooxygenase inhibition. Since intestinal oxygen uptake is
independent of blood ﬂow over the physiological range of blood ﬂow, the enhancement
of the glucose~ and oleate-induced increase oxygen uptake was most likely due to the
enhancement of oxidative metabolism rather than the enhancement of blood ﬂow.

However, in in vivo conditions changes in intestinal oxygen uptake could be inﬂuenced

120

by changes in tissue oxidative metabolism as well as vascular factors (48,59). Since these
studies were performed utilizing a naturally perfused segment of intestine in viva, the
effects of endogenous prostanoids on intestinal oxygen uptake cannot be completely
separated from their vascular actions. The aim of these in vitro studies was to determine
the effect of endogenous prostanoids on jejunal mucosal oxygen consumption in the
absence and presence of nutrients, glucose and oleate, separate from their vascular actions.
Therefore. the effects of 1) prostanoid synthesis inhibition by cyclooxygenase inhibitors
(mefenamate and indomethacin), 2) substrate loading with arachidonate, 3) U-44069, a
TxA2/endoperoxide analogue, and 4) SQ 29548. a TxAzlendoperoxide receptor blocker on
jejunal mucosal oxygen consumption were determined in vitro using constant volume
manometry.

Effect of Prostanoids on the Glucose-induced Increase in Mucosal 009 in vitro.

This part of present study determined the effects of cyclooxygenase inhibition.
substrate loading with arachidonate, a TxA2 analogue, and a TxA2 receptor blocker on
basal (no glucose) Q02, and a glucose-induced (5 mM) increase in Q02 in vitro. The
basal and glucose-induced mucosal QO2 values reported in this study are higher than
those previously reported for canine jejunal mucosa (66,70), but are slightly lower than
those reported for piglet jejunal mucosa (78) and rat ileal mucosa (38). Including 5 mM
glucose in the media increased the mucosal QO2 by 48%. The magnitude of this glucose~
induced increase in mucosal QO2 is similar to previously reported glucose-induced
increases in mucosal Q02 in vitro (66.70.78). Additionally. 5 mM glucose was minimum
concentration of glucose that maximally increased Q02. It was recently reported that 5

mM glucose also maximally increases Q02 in piglet mucosa (78).

121

The effect of inhibiting prostanoid synthesis on mucosal QO2 was determined
using cyclooxygenase inhibitors. mefenamate and indomethacin. Both cyclooxygenase
inhibitors decreased the basal Q02. This suggests that cyclooxygenase products,
prostanoids, might act to enhance the basal mucosal Q02 in vitro. In contrast. previous
studies (20.41) as well as the in viva studies above have shown that cyclooxygenase
inhibitors do not alter resting jejunal oxygen uptake in viva. suggesting that endogenous
prostanoids do not inﬂuence the resting jejunal oxygen uptake.

Inhibiting prostanoid synthesis enhanced the increase in QO2 induced by glucose
as shown in Tables 9 and 11. This suggests that endogenous prostanoids limit the
glucose-induced increase in mucosal Q02. This is consistent with the previously described
ﬁnding that cyclooxygenase inhibition enhances the glucose-induced increase in oxygen
uptake in viva. Additionally, it supports the thesis that the enhancement of the glucose-
induced increase in oxygen uptake by cyclooxygenase inhibition in vivo is due to the
enhancement of tissue oxidative metabolism and not an increase in blood ﬂow.
Cyclooxygenase inhibition has also been shown to enhance carbohydrate metabolism and
glucose absorption in vivo (43), which further supports these ﬁndings.

In an attempt to determine the effect of increasing prostanoid synthesis on mucosal
Q02 in the absence and presence on glucose, the effect of substrate loading with
arachidonate. the precursor of prostanoids. was determined. Arachidonate decreased the
basal Q02. This suggests that prostanoids might act to limit the basal Q02 in vitro. In
contrast it has previously been shown that arachidonate plus normal saline in the jejunal
lumen did not alter resting jejunal oxygen uptake in viva (20,67). Since cyclooxygenase

inhibition decreased basal Q02, suggesting that prostanoids might enhance the basal Q02,

122

it would be expected that substrate loading with arachidonate might enhance basal Q02.
Therefore, the next objective was to determine if the arachidonate-induced decrease in
basal QO2 was due prostanoids, the cyclooxygenase products of arachidonate. For this
purpose the effect of a cyclooxygenase inhibitor and arachidonate in combination on QO2
was determined in the absence of glucose. Mefenamate and arachidonate in combination
decreased the basal mucosal QO2 more than either the cyclooxygenase inhibitor or
arachidonate individually. Hence, cyclooxygenase inhibition appears to have added to.
rather than abolished the arachidonate-induced decrease in basal Q02. This suggests that
the arachidonate-induced decrease in basal QO2 was not due to cyclooxygenase products.
Rather, the decrease in the basal QO2 induced by arachidonate might have been due to
the affect of arachidonate per se or a non-cyclooxygenase product of arachidonate.
Arachidonate also attenuated the increase in QO2 induced by glucose. This affect
of arachidonate is consistent with that previously described where the addition of
arachidonate to a food mixture attenuates the food-induced increase in jejunal oxygen
uptake in viva (20.67). Additionally, since cyclooxygenase inhibition enhances the
glucose-induced increase in Q02. suggesting that prostanoids limit the glucose-induced
increase in Q02. increasing prostanoid synthesis by substrate loading with arachidonate
would be expected to attenuate the increase in QO2 induced by glucose. However, in light
of the ﬁnding that the arachidonate-induced decrease in basal QO2 was not due to
prostanoids. the next objective was to determine if the effect of arachidonate on the
glucose-induced increase in QO2 was due to cyclooxygenase products, prostanoids. or
arachidonate per se. Therefore the effect of mefenamate and arachidonate in combination

on mucosal Q02 in the presence of 5 mM glucose was determined. Cyclooxygenase

123

inhibition abolished the arachidonate-induced attenuation of the increase in QO2 induced
by glucose. This suggests that the attenuation of the glucose-induced increase in QO2 by
arachidonate was most likely due to its cyclooxygenase products, prostanoids. This further
suggests that prostanoids act to limit the glucose-induced increase in mucosal Q02.
Cyclooxygenase inhibition has also been shown to also abolished the arachidonate-induced
attenuation of the food-induced increase in oxygen uptake in viva (20).

In this study, as described above, cyclooxygenase inhibitors and arachidonate
individually and in combination decreased the basal Q02 in vitro. This suggests that
prostanoids might act to enhance the basal mucosal QO2 whereas arachidonate or possibly
non-cyclooxygenase metabolites of arachidonate might act to decrease the basal Q02 in
vitro. These ﬁndings differ from those of previous in vivo studies. The in vivo studies
showed that cyclooxygenase inhibitors and arachidonate did not alter resting jejunal
oxygen uptake (20,41,67). These studies suggests that prostanoids do not alter resting
jejunal oxygen uptake in vivo. The reason for this disparity between the in vitro ﬁndings
in this study and in viva ﬁndings of previous studies is unknown. The in vitro condition
in the absence of glucose might not fully mimic the environment of the jejunal mucosa
under resting conditions in viva. Also the oxygen uptake in the in vivo studies represents
the oxygen uptake of the whole gut wall and might not completely reﬂect the mucosal
oxygen consumption.

In the present study cyclooxygenase inhibitors enhanced. whereas substrate loading
with arachidonate attenuated the glucose-induced increase in mucosal Q02. Additionally.
the attenuation of the glucose-induced increase in QO2 by arachidonate was abolished by

mefenamate. and thus likely to be due to prostanoids. Therefore, endogenous prostanoids

124

limit the glucose-induced increase in Q02 in vitro. As described above, these ﬁndings are
consistent with those of previous in vivo studies (20,41,67). It is also in agreement with
the ﬁndings in the present study that prostanoids limit the glucose-induced increase in
jejunal oxygen uptake in viva. It was proposed above that endogenous prostanoids might
act to limit the glucose-induced increase in oxygen uptake in viva by limiting the increase
in tissue oxidative metabolism. These in vitro ﬁndings support this thesis as they indicate
that prostanoids limit the glucose-induced increase in mucosal oxygen consumption
independent of their vascular actions. In previous studies it was proposed that endogenous
prostanoids might act to limit the food-induced increase in oxygen uptake by limiting the
increase in tissue metabolism rather than blood ﬂow (20.41.67). The present study also
supports this thesis as it indicates that endogenous prostanoids can act to limit a nutrient-
induced increase in mucosal QO2 independent of their vascular actions. The role of
prostanoids in the regulation of the glucose- and nutrient-induced hyperemias, therefore.
may be to limit the glucose- or nutrient-induced increase in oxygen uptake and thereby
indirectly limit the hyperemia.

Recent studies have shown that endogenous TxA2 may play a role in limiting the
food-induced increases in jejunal oxygen uptake and capillary exchange capacity without
inﬂuencing the food-induced increase in blood ﬂow (2.68.69). It seems that the action of
TxA2 might be to limit intestinal oxidative metabolism and oxygen uptake (2).
Additionally, it was shown the present study that TxA2 also limits the increase in oxygen
uptake induced by glucose in viva. without altering the glucose-induced hyperemia.
Hence, TxA2 might limit the glucose-induced increase in intestinal oxidative metabolism.

Therefore, the next objective of the present study was to determine if TxA2 affects jejunal

125

mucosal Q02 in the absence and presence of glucose. Neither the TxA2 I endoperoxide
analogue. U~44069. nor the TxA2 / endoperoxide receptor blocker. SQ 29548 affected the
basal mucosal Q02. This suggests that endogenous TxA2 / endoperoxide does not play a
role in regulating the basal mucosal Q02 in vitro. Consistent with this ﬁnding it has been
shown that SQ 29548 and thromboxane synthetase inhibitors (imidazole and U63557A)
do not affect resting jejunal oxygen uptake in viva (2,68).

The TxA2 analogue, U~44069, decreased the glucose-induced increase in mucosal
Q02 in a dose dependent manner. The concentrations of U~44069 which attenuated the
glucose-induced increase in QO2 were approximately 0.1, 1, and 10 times the Tsz (a
stable metabolite of TxAz) concentration in venous blood from a jejunal segment that
contained food in the lumen (20). Additionally. these concentrations are within the range
of Tsz released in response to luminal placement of glucose as shown in this study.
Hence. TxA2 / endoperoxide, at concentrations released during nutrient absorption, as well
as glucose absorption, can act to limit a glucose-induced increase in the mucosal QO2
independent of its vasoconstrictor action.

Blocking the TxA2 / endoperoxide receptors with SQ 29548 enhanced the glucose-
induced increase in the mucosal Q02. This suggests that endogenous. mucosally
synthesized TxA2 / endoperoxide can act to limit the glucose-induced increase in mucosal
Q02 in vitro. It has previously been shown that SQ 29548, as well as TxA2 synthetase
inhibitors, enhance the food-induced increase in jejunal oxygen uptake and capillary
exchange capacity without affecting the food-induced increase in blood ﬂow in viva
(2,68,69). In the discussion of these studies. it was proposed that the action of TxA2 may

be related to its affect on intestinal oxidative metabolism. The results of the present study

126

support this thesis as it shows that TxA2 limits the glucose-induced increase in mucosal
QO2 independent of its vascular actions. Additionally, these ﬁndings suggest that the
enhancement of the glucose-induced increase in oxygen uptake in vivo by SQ 29548 in
the present study might be due to the enhancement of mucosal oxygen consumption.

To determine if the arachidonate-induced decrease in the basal and glucose-
induced increase in mucosal QO2 was due to TxA2 / endoperoxide. the effects of TxA2
/ end0peroxide receptor blockade with SQ 29548 on the arachidonate-induced decrease
in the mucosal QO2 was determined in the absence and presence of glucose. SQ 29548
did not alter the arachidonate-induced decrease in the basal mucosal Q02. This suggest
that TxA2 / endoperoxide does not play a role in the arachidonate-induced decrease in
basal Q02. SQ 29548 attenuated the arachidonate-induced decrease in the increase in the
mucosal QO2 induced by glucose. Thus, TxA2 / endoperoxide was, in part. responsible for
the arachidonate-induced decrease of the glucose-induced increase in the mucosal Q02.
Consistent with these ﬁndings, SQ 29548 has also been shown to reverse the
arachidonate-induced attenuation of the increase in jejunal oxygen uptake induced by food
in vivo (20).

The present studies suggest that endogenous prostanoids. other than TxAz. enhance
the basal Q02. This enhancement of the basal QO2 is most likely due to an increase in
oxidative metabolism to meet an increase in energy expenditure and demand as a result
of an increase in intestinal functions. In the absence of nutrients. a function which might
be increased is the secretion of electrolytes, water. and possibly mucus. The secretion of
electrolytes and water is thought to occur primarily from the cells in the crypt regions of

the small intestine. With regard to prostanoids, PGE, is a more potent secretagogue than

127
PGF,“ (14). PGI2 has little affect on intestinal secretion (110). PGE, is produced

predominantly in the crypt and subepithelial regions of the mucosa (92). Further, the
epithelium of the crypt region has been reported to be more responsive to PGEz than the
villus epithelium. i.e. PGE, induces a greater increase in cAMP in the crypt cells (92). An
increase in cAMP, as a second messenger, has been associated with an increase in
electrolyte as well as chloride secretion (13,110). Based on these considerations a
theoretical model can be conceptualized to account for the effect of prostanoids on basal
QO2 as diagrammed in Figure 25. Under basal conditions in vitro, i.e. in the absence of
exogenous nutrients, the cells of the mucosa produce and release prostanoids. In
particular, cells in the crypt and subepithelial regions release predominantly PGEZ. This
PGE2 could then interact with receptors on epithelial cells in this region to cause an
increase in the second messenger, CAMP, and subsequently increasing the secretion of
electrolytes and water. This secretion is considered an "active" process that requires
energy, in the form of ATP, to provide and maintain the Na+ gradient within the secreting
cells via the Na” + ATPase pump. The increase in energy expenditure to maintain the
Na+ gradient during secretion would most likely be met by an increase in oxidative
metabolism and, hence. oxygen consumption. Prostanoids released in other regions of the
mucosa as well as prostanoids other than PGEz could also enhance mucosal activities.
other than nutrient absorption, and thereby also result in an increase in oxidative
metabolism. Which prostanoids might be involved as well as which mucosal activities
might be affected is open to speculation. Endogenous prostanoids could also act to
directly increase basal oxidative metabolism, without ﬁrst increasing a mucosal function.

However, the mechanism by which such an action would occur is unknown.

128

Arachidonic acid

\

 

p65,. Pal,
TxAz. PGF...
. PGE... PGF... Pol,

 

Na‘ :, ~———-——Na

 

Na"
20"

 

Ci‘efflux .‘

' e -— 0
H20 4":- ' H2

 

Figure 25. Theoretical model for the effect of prostanoids on the basal mucosal
Q02 in vitro. The cells shown represent intestinal epithelial cells from
the crypt region. R, ~ receptor; (+) ~ enhances.

129

In the present study glucose increased mucosal Q02. Endogenous prostanoids, and
speciﬁcally TxAz. appear to limit this glucose-induced increase in Q02. The increase in
QO2 induced by glucose was most likely due to an increase in glucose transport and
carbohydrate metabolism (43,66). Glucose transport involves a glucose-Na“ cotransporter
which requires a Na” gradient that is maintained by the Na*/l(+ ATPase pump. An
increase in glucose-Na+ transport would increase the activity of the Na“/I(+ ATPase pump
in order to maintain the Na+ gradient and thereby increasing ATP utilization. The increase
in ATP utilized would most likely be supplied by an increase in oxidative metabolism and
hence oxygen consumption. Glucose transport is considered to occur in epithelial cells
present primarily on the outer two-thirds of the villi. Microsomes from villus epithelial
cells have been shown to produce PGEz, TxAz, PGF”, and PGI2 (1.5.92). Based on these
considerations the following theoretical model can be conceptualized to account for the
actions of prostanoids on the glucose-induced increase in QO2 (Figure 26). In the presence
of glucose. the cells of the mucosa produce and release prostanoids. In the villus regions,
prostanoids, and speciﬁcally TxA2. are released and interact with their cell surface
receptors. This would increase or possibly decrease the levels of second messengers in
the cells. although it is not known which speciﬁc second messenger systems would be
affected. Changes in second messenger levels could act to decrease the activity of the
Na"/K+ ATPase pump or to decrease the activity of the Na+ ~ glucose cotransporter. The
result of either of these events would decrease glucose transport as well as decrease the
ATP utilized, and thereby, decrease oxidative metabolism. The prostanoids could also act
to directly decrease carbohydrate metabolism (43), however, the mechanism by which

such an action might occur is unknown.

130

Arachidonic acid

\

PGE, Pal,
Ter. PGF...

 

 

 

 

 

Na’ ' H Glucose

Glucose

 

Figure 26. Theoretical model for the effect of prostanoids on the glucose-induced
increase in mucosal Q02 in vitro. The cells shown represent intestinal
epithelial cells from the villus region. R, - receptor; (~) ~ decreases;

? ~ action or pathway unknown or uncertain.

131

Although the precise mechanism by which endogenous prostanoids and speciﬁcally
TxA2 limit the glucose-induced increase in mucosal Q02 in vitro is unknown. several
studies support the thesis that prostanoids inﬂuence glucose absorption and carbohydrate
metabolism. Cyclooxygenase inhibition has been shown to enhance jejunal glucose
absorption and carbohydrate metabolism in vivo (43). Other studies have shown that PGE2
and PGF,“ inhibit glucose absorption in viva (6,28). Likewise, PGE2 analogues have been
shown to decrease glucose transport by the intestinal mucosa in vitro (50) and decrease
post-meal serum glucose levels in humans (86). Hence it seems likely that prostanoids
might act to limit glucose transport and carbohydrate metabolism and thereby limit
mucosal oxygen consumption.

In summary. cyclooxygenase inhibitors and arachidonate individually and in
combination decrease the basal mucosal Q02. This suggests that prostanoids may enhance
basal QO2 whereas arachidonate per se or its non-cyclooxygenase metabolites might
decrease basal mucosal Q02 in vitro. Neither the TxA2 / endOperoxide analogue nor the
receptor blocker affected basal Q02. Hence. TxA2 does play a role in regulating the basal
Q02 in vitro. Cyclooxygenase inhibitors enhance. whereas arachidonate attenuates the
increase in QO2 induced by glucose. Further, cyclooxygenase inhibition abolished the
arachidonate-induced attenuation of the increase in QO2 induced by glucose. The TxA2
/ endoperoxide analogue decreased whereas the receptor blocker enhanced Q02 in the
presence of glucose. This indicates that endogenous mucosal prostanoids, and in particular
TxAz. can act to limit the glucose-induced increase in mucosal QO2 independent of their
vascular actions. These prostanoids may play a role in the regulation of glucose- as well

as nutrient-induced increases in blood ﬂow and oxygen uptake by limiting mucosal

132

oxygen consumption and thereby indirectly limiting the increase in blood ﬂow or capillary

exchange density.

Effect of Prostﬂigs on Lhe Ole_ate~induced Change in MucosalQO, in vitro.

The aim of this part of the study was to determine the effects of prostanoids and
TxA2 on an oleate-induced increase in mucosal Q02 in vitro. However, oleate either
decreased or did not alter mucosal OQz.

Luminal placement of oleate plus bile or a bile salt has been shown above as well
as in previous studies to signiﬁcantly increase jejunal oxygen uptake in viva (26.58.70).
Hence, it was originally hypothesized that oleate would also increase mucosal Q02 in
vitro. The ﬁrst goal of this study was to determine the concentration of oleate that would
be used in series XIII ~ XV experiments which were aimed at determining the effect of
prostanoids on oleate-induced changes in Q02. Five preliminary studies were performed
in an attempt to determine this concentration. Since gallbladder bile was shown to
signiﬁcantly decrease mucosal QO2 (Figure 6) and glucose alone increased Q02, the effect
of sonicated oleate alone was determined in the ﬁrst preliminary study. In this study
sonicated oleate did not alter jejunal mucosal Q02. Likewise, sonicated oleate alone does
not alter jejunal blood ﬂow and oxygen uptake in vivo (26,58). However, oleate
emulsiﬁed with gallbladder bile or a bile salt. taurocholate, has been shown to increase
jejunal blood ﬂow and oxygen uptake in viva (26.58). This suggested that oleate might
also need to be combined with bile or taurocholate, a bile salt in order to affect mucosal
Q02 in vitro. Since bile decreases QO2 (Figure 6), the effect of taurocholate on mucosal

QO2 was determined in the second preliminary study. This study showed that taurocholate

133

does not alter QO2 at concentrations less than 0.1 mM. but decreases QO2 at
concentrations of 0.5 mM and greater. Similarly. Faust et al. (35) has also shown that 1.0
and 2.0 mM taurocholate signiﬁcantly decreases, but 0.1 mM taurocholate does alter
mucosal oxygen consumption in vitro. Since 0.1 mM taurocholate was the maximum
concentration which did not alter QO2 it was used in the subsequent studies.

The effect of oleate plus taurocholate on mucosal QO2 was then determined in the
third preliminary study. Oleate at concentrations of 0.1 and 0.5 mM plus taurocholate did
not alter mucosal Q02. whereas 1.0 mM oleate plus taurocholate tended to decrease, and
2.0, 5.0, and 10.0 mM oleate plus taurocholate signiﬁcantly decreased mucosal Q02. The
decrease in Q02 in the presence of 5.0 mM oleate plus taurocholate was greater than that
in 2.0 mM oleate plus taurocholate. but not different from that in presence of 10.0 mM
oleate plus taurocholate. Hence. 5.0 mM oleate plus taurocholate was the minimum
concentration which maximally decreased Q02.

Since it was unexpected that oleate would decrease mucosal QO2 the fourth and
ﬁfth preliminary studies were performed to further conﬁrm this effect. To investigate if
the effect of oleate plus bile was different from that of oleate plus taurocholate the effect
of 5.0 mM oleate plus 10% gallbladder bile on mucosal QO2 was determined and then
compared to the effect of 5.0 mM oleate plus taurocholate. As shown in Figure 9. 5.0
mM oleate plus bile also decreased mucosal Q02. The effect of 5.0 mM oleate plus bile
was similar to that of 5.0 mM oleate plus taurocholate. The ﬁfth study was performed to
determine if the concentration of taurocholate affects the oleate-induced response. Even
though 0.5 and 1.0 mM taurocholate decreased Q02 and 0.1 mM taurocholate did not alter

Q02, the magnitude of the decrease in QO2 induced by 5.0 mM oleate was not affected

134

by the concentration of taurocholate used. Hence. it appears that taurocholate or bile is
required for oleate to affect mucosal Q02. Additionally, it seems that bile as well as
various concentrations of taurocholate are similarly able to render oleate capable of
affecting Q02. This action of taurocholate or bile may be related to the emulsifying of the
oleate.

These studies show that 0.5 mM oleate plus taurocholate does not affect Q02. but
that 5.0 mM oleate plus taurocholate signiﬁcantly decreases mucosal Q02 in vitro. In
contrast, several studies (26.58.70). including the in vivo studies above, have shown that
oleate plus bile increases jejunal oxygen uptake in vivo. The reason why oleate plus
taurocholate does not also increase Q02 in vitro is unknown. However, the fact that the
in vitro studies were performed using jejunal mucosa separated from the other intestinal
layers. whereas the in viva studies were performed using whole jejunal segments might
suggest that the mechanism by which oleate plus bile increases jejunal oxygen
consumption might not be conﬁned entirely to the mucosal layer.

Separating the mucosa from the underlying layers could have disrupted the neural
connections. Recent studies have suggested that primary afferent nerves (C ﬁbers) in the
jejunum, in addition to their sensory functions. may play a role in the regulation of the
oleate-induced increase in blood ﬂow in viva (79). Capsaicin can deplete these C-ﬁbers
of their neurotransmitters, and has also been shown to inhibit the oleate-induced
hyperemia (79). Additionally, local anesthetic agents, lidocaine and dibucaine, have been
shown to attenuate or inhibit the oleate-induced increase in blood ﬂow (71,79) and
oxygen uptake (71), further suggesting that a neurally mediated mechanism may play a

role in regulating the oleate-induced responses. Vasoactive intestinal peptide (VIP) has

135

been proposed to be the neuropeptide released in response to oleate plus bile (19.40.79).
VIP has been reported to increase ileal oxygen uptake (19). If the oleate-induced increase
in blood and oxygen uptake is indeed mediated by a neural mechanism as suggested by
these studies, then removing the mucosal layer from the underlying intestinal layers might
have disrupted the neural pathways and thereby ablated the mechanism by which oleate
plus bile induced an increase in jejunal oxygen consumption.

The mechanism by which oleate plus taurocholate decreases QO2 at the higher
concentrations used in the present study is also unknown. Oleate, at concentrations greater
than 4 mM, emulsiﬁed by taurocholate has been shown to cause some disruption of a
monolayer of cultured intestinal epithelial cells after 1 hour of exposure (62). The cause
of this disruption is unknown. however it was not due to lipid peroxidation of cell
membranes by the oleate (62).

Even though the effect of oleate plus taurocholate on Q02 in vitro was different
from its effect on oxygen uptake in vivo. the effect of prostanoids on the oleate-induced
changes in mucosal QO2 was still studied. The effect of cyclooxygenase inhibition with
mefenamate. substrate loading with arachidonate, and the TxA2 / endoperoxide receptor
blocker. SQ 29548, on mucosal QO2 was determined in the absence and presence of 0.1
mM taurocholate, 0.5 mM oleate plus taurocholate, and 5.0 mM oleate plus taurocholate.
In all series XIII ~ XV experiments 0.1 mM taurocholate and 0.5 mM oleate plus
taurocholate did not alter mucosal QO2 whereas 5.0 mM oleate plus taurocholate
decreased QO2 by an average of l4.613.2%.

Mefenamate, a cyclooxygenase inhibitor, and arachidonate decreased basal Q02.

These effects of mefenamate and arachidonate on basal OQ2 in these series are the same

136

as those previously described and discussed (pages 121-122) for the in vitro glucose
studies, and will not be discussed again here. Both cyclooxygenase inhibition and
arachidonate also decreased Q02 in the presence of 0.1 mM taurocholate, 0.5 mM oleate
plus taurocholate, and 5.0 mM oleate plus taurocholate. Cyclooxygenase inhibition
decreased Q02, including basal Q02, the same magnitude in all conditions. Likewise
arachidonate decreased Q02. including basal. the same magnitude in all conditions.
Additionally the decrease in QO2 induced by 5.0 mM oleate plus taurocholate before
mefenamate was similar to that after mefenamate. The same was true for the 5.0 mM
oleate plus taurocholate-induced decrease in QO2 before and after arachidonate. Hence,
it appears that cyclooxygenase inhibition and arachidonate decreased the basal QO2 but
did not inﬂuence the oleate-induced decrease in Q02. Therefore, prostanoids apparently
do not play a role in the regulation of the oleate-induced decrease in mucosal Q02 in
vitro.

As shown in Table 16, the TxA2 / endoperoxide receptor blocker did not effect
basal QO2 nor Q02 in the presence of 0.1 mM taurocholate, 0.5 mM oleate plus
taurocholate, or 5.0 mM oleate plus taurocholate. Hence. TxA2 / endoperoxide does not
play a role in the regulation of the basal or oleate-induced decrease in mucosal Q02.
Since the TxA2 / endoperoxide receptor blocker did not affect Q02 in these conditions the
effect of the TxA2 analogue. U—44069. was not determined.

In summary. oleate. at concentrations greater than 5.0 mM. plus taurocholate decreased
mucosal Q02. This effect of oleate is opposite the effect of oleate plus bile in viva.
Cyclooxygenase inhibition and arachidonate decreased basal Q02, but do not signiﬁcantly

alter the decrease in QO2 induced by 5.0 mM oleate plus taurocholate. Additionally, TxA2

137

receptor blockade did not affect in oleate-induced decrease in Q02. Thus it appears that

prostanoids, and TxAz, do not regulate the decrease in QO2 induced by oleate in vitro.

 

SUMMARY and CONCLUSION

Effect of Prostanoids on Glucose- and Oleate-Induced Changes in Blood Flow and

Oxygen Uptake in viva.

The objective of this study was to determine if prostanoids play a role in the
regulation of glucose~ and oleate-induced increases in blood ﬂow and oxygen uptake. This
was studied by determining the effect cyclooxygenase inhibition on the increases in blood
ﬂow and oxygen uptake induced by glucose and oleate, and determining if and which
prostanoids are released in response to luminal placement of glucose and oleate.
Additionally, the effect of a TxA2 / endoperoxide receptor blocker. SQ 29548, on the
glucose~ and oleate-induced increase in blood ﬂow and oxygen uptake was also
determined. The ﬁndings are as follows:

1. Glucose and oleate increased blood ﬂow and oxygen uptake when place into the
lumen. Cyclooxygenase inhibition enhanced the increases in blood and oxygen uptake
induced by both glucose and oleate.

2. Cyclooxygenase inhibition decreased resting blood ﬂow, but did not affect resting
oxygen uptake. Even though a decrease in blood ﬂow, i.e. an increase in initial
resistance. was shown to inﬂuence the magnitude of the glucose~ and oleate-induced
vascular responses, the enhancement of the glucose- and oleate-induced hyperemias

by cyclooxygenase inhibition was in addition to the decrease in resting blood ﬂow.

138

139

3. Oleate induced a greater increase in blood ﬂow than did glucose before and after
cyclooxygenase inhibition. Further. the level of enhancement of the oleate-
induced increase in blood ﬂow by cyclooxygenase inhibition was greater than the
enhancement of the glucose-induced increase in blood ﬂow.

4. The increases in oxygen uptake induced by glucose and oleate were similar before and
after cyclooxygenase inhibition. Cyclooxygenase inhibition enhanced both the glucose-
and oleate-induced increases in oxygen uptake.

5. The glucose- and oleate-induced increases in blood ﬂow are correlated with the
increases in oxygen uptake before and after cyclooxygenase inhibition.

6. Luminal placement of glucose and oleate increased the venous concentrations and
releases of PGF... TxAz, PGIZ. and PGF”. Oleate induced a greater release of PGE2,
TxA” and PGF,“ than did glucose.

7. The TxA2 / endoperoxide receptor blocker. SQ 29548, did not affect resting jejunal
blood ﬂow or oxygen uptake. SQ 29548 enhanced the glucose-induced increase in
oxygen uptake, but did not affect the glucose-induced hyperemia. TxA2 receptor
blockade enhanced the increases in both blood ﬂow and oxygen uptake induced by
oleate.

These ﬁndings suggest that endogenous prostanoids limit the glucose- and oleate-
induced increases in blood ﬂow and oxygen uptake. Additionally, it appears that
prostanoids limit the oleate-induced hyperemia more than that of glucose. Further study
suggests that endogenous TxA2 limits the increase in oxygen uptake induced by glucose.
but does not affect the glucose-induced hyperemia. Endogenous TxA2 appears to limit the

increases in both blood ﬂow and oxygen uptake induced by oleate.

140

Effect of Prostanoids on Mucosal Oxygen Consumption in vitro.

The objective of following the in vitro studies was to determine the effects of
endogenous prostanoids on jejunal mucosal oxygen consumption separate from their
vascular actions in the absence and presence of nutrients. Therefore, the effects of l)
prostanoids synthesis inhibition by cyclooxygenase inhibitors (mefenamate and
indomethacin), 2) substrate loading with arachidonate. 3) U~44069, a TxA2/endoperoxide
analogue, and 4) SQ 29548, a TxA2/endoperoxide receptor blocker on jejunal mucosal
oxygen consumption were determined in vitro in the absence (basal) and presence of

glucose or oleate.

Effect of Prosta_ngigs on t_he GluLcose-induced Increase in Mucosal 001 in vitro.

This part of present study determined the effects of cyclooxygenase inhibitors,
substrate loading with arachidonate, a TxA2 analogue, and a TxA2 receptor blocker on
basal (no glucose) Q02, and a glucose-induced (5 mM) increase in Q02 in vitro.

1. Cyclooxygenase inhibitors. and arachidonate individually as well as in combination
decrease basal mucosal Q02 in vitro. This suggests that prostanoids might enhance
basal QO2 whereas arachidonate per se or non-cyclooxygenase products of
arachidonate decrease basal Q02 in vitro.

2. Glucose signiﬁcantly increased mucosal Q02. The minimum concentration of glucose
which maximally increased QO2 was 5 mM.

3. Cyclooxygenase inhibition enhanced the glucose-induced increase in mucosal Q02,
whereas substrate loading with arachidonate attenuated the glucose-induced increase

in Q02. This affect of arachidonate on the glucose-induced increase in QO2 was

141

abolished by cyclooxygenase inhibition. suggesting that it was due to cyclooxygenase
products of arachidonate. prostanoids. These ﬁndings indicate that prostanoids limit
the glucose-induced increase in Q02 in vitro.

Neither the TxA2 analogue. U~44069. nor the TxA2 / endoperoxide receptor blocker
affected basal mucosal Q02.

The TxA2 analogue decreased the glucose-induced increase in Q02, whereas the TxA2
/ endoperoxide receptor blocker enhanced the glucose-induced increase in mucosal
Q02. This suggests that endogenous TxA2 might act to limit the increase in mucosal
QO2 induced by glucose.

These ﬁndings indicate that prostanoids, and TxAz. can act to limit the glucose~

induced increase in mucosal Q02 independent of their vascular actions.

Effect of Prostanoids on the Oleate-induced Chan e in Mucosal in vitro.

This objective of this part of present study was determined the effects of

cyclooxygenase inhibitors, substrate loading with arachidonate, and a TxA2 receptor

blocker on basal and the oleate-induced change in Q02 in vitro.

1.

2.

Oleate alone did not affect mucosal Q02 in vitro. Oleate plus bile or taurocholate
signiﬁcantly decreased mucosal QO2 at concentrations greater than 1.0 mM. Oleate
at a concentration of 5.0 mM plus taurocholate produced the maximum decrease in
Q02. This effect of oleate plus taurocholate in vitro is opposite of its effect on jejunal
oxygen uptake in viva.

Cyclooxygenase inhibitor and arachidonate individually and in combination decreased

basal QO2 as well as Q02 in the presence of taurocholate. and both concentrations of

142

oleate plus taurocholate. It appears that these treatments did not affect the decrease
in QO2 induced by 5.0 mM oleate plus taurocholate.
3. The TxA2/ endoperoxide receptor blocker. SQ 29548, did not affect the mucosal Q02
in the absence or presence of oleate plus taurocholate.
Thus it seems that prostanoids do not affect the oleate-induced decrease in mucosal
Q02 in vitro. The reason for the difference in the effect of oleate on Q02 in vitro. and the

oxygen uptake in vivo is unknown.

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