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thesis entitled
Prostaglandin Metabolism In
Papillary Collecting Tubule
Cells From Rabbit Kidney
presented by

Frank Charles Grenier

has been accepted towards fulfillment
of the requirements for

 

degree in _Bj.o.ch.em_Ls t ry

Doctor of Philosophy

Major professor

Date nag/5431703) /5 /r./ 5/

0-7639

 

 

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2 9 1998

 

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charge from circulation records

 

 

 

PROSTAGLANDIN METABOLISM IN
PAPILLARY COLLECTING TUBULE
CELLS FROM RABBIT KIDNEY

By

Frank Charles Grenier

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1981

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ABSTRACT

PROSTAGLANDIN METABOLISM IN
PAPILLARY COLLECTING TUBULE
CELLS FROM RABBIT KIDNEY

By

Frank Charles Grenier

Homogeneous (>97%) populations of renal papillary collecting tubule
(RPCT) cells were isolated from the rabbit kidney. RPCT cells were
characterized as being derived from the collecting tubule on the basis
of anatomical source, size, enzyme histochemistry, and cyclooxygenase
antigenicity; in addition RPCT cells synthesized 3',5'-cyclic AMP (CAMP)
in response to arginine vasopressin, formed hemicysts when grown to

confluency and adhered with morphological asymmetry to Millipore

filters.

Homogenates of RPCT cells when incubated with [3HJ-arachidonic

acid formed 6-keto-PGF1G, PGFZQ, PGEz and PGDZ. At

arachidonic acid concentrations below 2 E! the major product formed was
6-keto-PGF1a; at higher concentrations PGEZ was the major
radioactive prostaglandin formed.

A series of hormones were tested for their influence on the release
of immunoreactive prostaglandins (iPG) by intact RPCT cells grown in
monolayer culture; the major product (ca. 75%) under both bassal and
stimulated conditions was iPGEZ. At very low concentrations (1
10-10.!) bradykinin, lysyl-bradykinin and methionyl-lysylbrady-
kinin all caused 3-5 fold increases in iPGEZ formation.

Significantly, neither arginine vasopressin (AVP) (10'7_M) nor
desamino-AVP (10-7.!) caused prostaglandin release by RPCT cells.
These results indicate that kinins can act directly on the collecting
tubule to elicit PGE2 formation; furthermore, the effect of kinins may
be natriuretic since PGEZ has been shown by others to inhibit Na+
resorption by the medullary collecting tubule.

RPCT cells synthesized cAMP in response to AVP, parathyroid hormone
and glucagon but not to adrenergic agents. In addition, exogenous
PGEZ and P612, increased intracellular cAMP concentrations in RPCT
cells.

The effects of PGE2 and P612 on AVP-induced cAMP production by
RPCT cells were investigated. At 10‘7 M AVP and 10'5 M PGEZ
or P612, the increase in both total and intracellular cAMP levels was
less than that expected for an additive response of AVP plus
prostaglandin. This suggests that prostaglandins can partially block

AVP-induced cAMP accumulation in the collecting tubule.

TO KEARSTIE

ii

 

ACKNOWLEDGEMENTS

I would like to thank Dr. "Wild Bill" Smith for his advice,
encouragement and financial support. His kindness was only superceded
by his generosity and good looks.

I would also like to thank all the members of the laboratory and
secretarial staff for many hours of enjoyment in and out of the
laboratory.

I would like to express my gratitude to Thomas Rollins for doing
the electron microscopy reported in this thesis.

A special thanks goes out to Arlyn Garcia-Perez for being social
director, and to Rick Huslig and Dave DeWitt both of whom began this
journey with me. whether chasing sheep through petrified muck during
December or floating through "ozone trips", we've shared innunerable
indignities.

And most importantly I would like to thank my wife Kearstie. She

stood by her man.

Page

LIST OF TABLES ....................................................... v

LIST OF FIGURES ...................................... . .............. vi

INTRODUCTION ......................................................... 1
CHAPTER

I. LITERATURE REVIEW.... ........................................ 3

Prostaglandin Metabolism ..................................... 3

Kidney Function .............................................. 8

Localization of Prostaglandin Synthesis ..................... 11

Prostaglandin Function in the Kidney ........................ 13

Prostaglandins and Water Resorption..... .................... 14

The Kallikrein-Kinin system and Prostaglandins .............. 16

II. PROSTAGLANDIN SYNTHESIS BY RPCT CELLS ISOLATED BY

TRYPSIN DISSOCIATION . ................ . ..................... 20

Materials and Methods ....................................... 21

Results and Discussion ...................................... 27

III. KINlN-INDUCED PROSTAGLANDIN SYNTHESIS BY RPCT CELLS

IN CULTURE.... ...... .................... ..... . .............. 49

Materials and Methods ...... . ............. .. ............... ..50

Results ..................................................... 61

Discussion .................................................. 89

IV. INTERRELATIONSHIPS AMONG BRADYKININ, PGEZ, VASOPRESSIN

AND CAMP IN RPCT CELLS. O O O O O O I O O O O O ....... O O O O O O O O O O O ....... 94

Materials and Methods ....................................... 95

Results .............. . ...................................... 99

Discussion.. .................. . ............................ 112

SUMMARY DISCUSSION ........ . ...... .... .............................. 118

BIBLIOGRAPHY ....................................................... 122

TABLE OF CONTENTS

iv

LIST OF TABLES

Table Page
I Prostaglandin products formed from arachidonic
acid by cell p0pulations isolated from rabbit
renal papillae ......................... . ................... 41
II Characterization of 6-keto-PGF1G (Rf values) ................ 42
III Cross-reactivities of prostaglandin anti-sera with
various prostaglandins......................... ............. 54

IV Formation of 3',5'-cyclic AMP by RPCT cells in
response to hormonal effectors..................... ......... 71

V Synthesis of iPGEz by RPCT cells in response to
hormonal effectors ................... ...... ................. 79

VI Characterization of prostaglandins formed by RPCT
cells in response to hormonal effectors ..................... 81

Figure Page
1 Overview of prostaglandin metabolism ........................ ....5
2 Schematic representation of the sites of cyclooxygenase

in the kidney.... ......... ... ........ ........... ............... IO
3 Trypsin-isolated RPCT cells in suspension. ..... .. .............. 29
4 Fluorescent photomicrographs of RPCT cells treated with

and without anti-cyclooxygenase serum............ .............. 32
5 [14CJ-glucose oxidation by isolated RPCT cells ................. 34
6 [14CJ-leucine incorporation by isolated RPCT cells ............. 36
7 A radiochromatogram of the products formed upon incubation

of [3 H]- arachidonic acid ................ . ...................... 4O
8 Time course for the biosynthesis of various prostaglandin

products by a RPCT cell homogenate ................. . ........... 45
9 Prostaglandin biosynthesis by RPCT cell homogenates

at different initial concentrations of

[3H]-arachidonic acid ......................................... 48
10 Phase photomicrograph of RPCT cells grown eight days

in monolayer culture ......... ................ .................. 64
11 Electron micrographs of (A) intact collecting tubule

from rabbit, (B) isolated RPCT cells grown on

Millipore filters, and (C) junctional complex of

RPCT cell grown on Millipore filter...... ....... . .............. 66
12 Hemicysts present in culture of RPCT cells....... .............. 69
13 Release of cAMP into media by RPCT cells incubated

with AVP, oxytocin and PTH....... ...... .......... .............. 73
14 Time course of intracellular and extracellular cAMP

by RPCT cells in response to 10'7 M AVP ......... .............. 75
15 RPCT cell growth in monolayer culture ..... ....... .............. 78

LIST OF FIGURES

vi

Figure

16

17
18

19

20

21

22

23

Time course of iPGEz synthesis by RPCT cells in
monolayer culture... ...... . ........ .. ...... . ..... . .........

Dependence of iPGEZ synthesis on kinin concentration .......

Dependence of intracellular cAMP formation on
prostaglandin concentration.................... ........... .

Time course for intracellular cAMP formation caused by PGE2
and bradykinin................................... ..........

Effect of stirring on intracellular cAMP formation caused
by PGEZ and bradykinin ........ ..... ......... ...............

Time course of the effect of PGE2 on intracellular cAMP
formation caused by AVP ....................................

Effect of AVP, PGEZ and bradykinin on intracellular cAMP
synthesis by RPCT cells..................... ...............

Model illustrating the effects of prostaglandins,
bradykinin, cAMP and AVP in RPCT cells .....................

....85
....87

...101

...103

...106

...109

...111

...114

INTRODUCTION

Various experimental approaches have been used to investigate how
prostaglandins are involved with kidney processes including 1) whole
animal clearance methods 2) perfusion of isolated kidneys, 3)
thicropuncture and stopped-flow techniques and 4) techniques using renal
:slices and renal homogenates (38,57). A drawback of all these methods,

f1owever, is that in each case many different kidney cells may influence
aarry measurements made using these systems. It is virtually impossible
t:c> interpret such data on the individual cell level. Nevertheless,
these methods have been useful in defining both the prostaglandin
b‘i osynthetic capacity of the kidney and the hormones which cause
prostaglandin biosynthesis by the kidney (57).

Frustrated by the relative indirectness of these methods, Burg
5232 ad, developed a technique for microperfusing individual segments of
kridney tubules (42). The tremendous advantage of this technique was its
liack of complexity. Only one tubular cell type was being studied and
therefore the interpretation of the data was greatly simplified. Using
perfused collecting tubules, for example, Grantham gt _a_l_. were able to
(demonstrate that PGE1 inhibits arginine vaSOpressin (AVP)-stimulated
vvater resorption in the collecting tubule (61). This information was
lJnattainable prior to Burg's advancement.

The major limitation of Burg's method, however, is the difficulty

‘in making biochemical measurements with isolated tubule segments.

1

2

Dissection of tubule segments is laborious and yields segments only 1-2
mm in length (42). It would require several hundred tubule segments
(106 cells) to make many biochemical measurements (e.g. PGEZ and
3',5'-cyclic AMP radioimmunoassays) that could be made easily with one
partially confluent 24-well culture dish. Renal medullary interstitial
cells, for instance, have been used in culture to make a series of
biochemical measurements pertaining to prostaglandin metabolism (1). As
with tubule segments though, the data obtained with cultured cells can
also be cell specific.

Our interest in investigating prostaglandin metabolism in isolated
collecting tubule cells was based on two observations. The first was
that immunohistochemical localization of the prostaglandin-forming
enyzme, cyclooxygenase, demonstrated that the collecting tubule cell was
the only tubular cell type that stained for the enzyme (52). The second
was Grantham's observation that PGE1 inhibits water resorption in
kidney (61). Taken together, these data suggested that prostaglandins
synthesized by the collecting tubule may be closely involved with kidney
function. In order to test our hypothesis biochemically, we chose to
isolate a population of collecting tubule cells from the rabbit kidney

and to study prostaglandin metabolism in these cells.

LITERATURE REVIEW

Prostaglandin Metabolism. The pathways for the biosynthesis of

 

various prostaglandins are illustrated in Fig. 1. The first step in
this "arachidonate cascade" is the liberation of arachidonic acid from
an esterified precursor in reSponse to an exogenous stimulus. The
precursor is most commonly a phospholipid. Triglycerides, even in
triglyceride-rich cells such as renal medullary interstitial cells, are
apparently not a primary source of arachidonic acid (1).

The release of arachidonic acid from phospholipids may occur via
two different mechanisms. The first pathway involves the
straightforward action of a phospholipase A2 on a
2-arachidonyl-phosphoglyceride to yield free arachidonic acid. Bills et
al. have shown that platelets contain a phospholipase A2 activity
which will preferentially catalyze the release of fatty acids from
phOSphatidylcholine containing arachidonic acid at the 2-position (2,3).
A second pathway for arachidonate release also exists in platelets. A
cytosolic phospholipase C specific for phosphatidylinositol catalyzes
the cleavage of phosphatidylinositol, yielding diglyceride and inositol
phosphate (4,5,6) and a diglyceride lipase then catalyzes the release of
arachidonic acid from the diglyceride. This phospholipase C-
diglyceride lipase pathway provides for the specific release of
arachidonic acid because approximately 90% of the phosphatidylinositol

in platelets contains arachidonic acid esterified at the 2-position

Figure 1. Overview of prostaglandin biosynthesis.

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(7,8). It is not known whether one or both of these pathways are
usually operational in cells other than platelets. Hong et_al. have
recently shown that BALB/3T3 cells contain a phospholipase A2 activity
which is activated by bradykinin, thrombin and the calcium ionophore,
A23187; these effectors caused arachidonic acid release via a
phospholipase A2 activity and not by activation of a phospholipase C
(9).

Free arachidonic acid released from phospholipids is converted to
the endoperoxide, PGGZ, by a cyclooxygenase which is part of the PGH
synthase. The reaction mechanism involves the abstraction of the
Lyhydrogen atom from carbon 13 of arachidonic acid, the addition of one
molecule of oxygen at carbon 11 and, after a series of intramolecular
rearrangements, the addition of a second molecule of oxygen at carbon 15
(10,11,12,13,14). PGGZ is converted to another endoperoxide, PGHZ,
by the peroxidase function of the PGH synthase which converts the
15-hydroperoxy prostaglandin to a 15-hydroxy prostaglandin. The
half-lives of PGHZ and PGGZ in aqueous solutions at pH 7.5 are
approximately 5 min (15).

PGH synthase (also called cyclooxygenase) was the first of the
prostaglandin biosynthetic enzymes to be extensively characterized
(16,17). It has been purified from sheep and bovine vesicular glands
and is a dimer with a subunit molecular weight of 70,000 daltons (17).
The cyclooxygenase activity of the enzyme requires an Fe2+ or
Mn2+ containing porphyrin whereas the peroxidase activity is
functional only with an Fe2+ containing porphyrin (18,19). The
enzyme is located on the cytoplasmic side of the endoplasmic reticulum

(20).

7

PGHZ is normally converted to the biologically active
prostaglandins, PGDZ, PGEZ, PGan, P612 (prostacyclin) and
TxAz (thromboxane AZ). All these prostaglandins with the possible
exception of P602 are synthesized by the kidney. Enzymatic
conversions to PGDZ and PGE2 are catalyzed by PGH-PGD and PGH-PGE
isomerases (21,22,23,24) and conversions to PGIZ and TxAz by P612
and TxAz synthases (25,26). Presently, it is unknown if PGan can
be formed enzymatically directly from PGHZ; a 9-keto-PGE2-reductase
can convert PGEZ to PGFga in some cells and this enzyme is present
in the kidney (27). PGDZ, PGEZ and PGan can also be formed at
significant rates non-enzymatically from PGHz. 0f the above enzymes
only PGDZ isomerase has been purified to homogeneity. The spleen
enzyme is cytosolic, has a molecular weight of 30,000 daltons and
requires glutathione (23). The brain enzyme is also cytosolic, but does
not require glutathione and has a molecular weight of 85,000 (24).

The catabolism of biologically active prostaglandins to inactive
prostaglandins can occur either enzymatically or non-enzymatically.
TxAz and PGIZ are rapidly converted non-enzymatically to Tsz and
6-keto-PGF1a (28,29). The half-lives of TxAz and PGIZ in
aqueous solution at pH 7.5 are approximately 30 sec and 5 min,
respectively (30,31). PGIZ, PGDZ, PGEZ and PGan can be
enzymatically inactivated by 15-hydroxy-prostaglandin dehydrogenase,
which convert active 15-hydroxy-prostaglandins to inactive
15-keto-prostaglandins (32). 15-keto-prostaglandins can be further
modified by 15—keto-prostaglandin A13 reductase (which reduces the
13,14 double bond) and 9-hydroxy-prostaglandin reductase (33,34). All

of these catabolic enzymes are found in the mammalian kidney, but only

8

the NAD+-dependent 15-hydroxy prostaglandin dehydrogenase has been
studied in much detail. This enzyme is a soluble protein and is found
principally in the kidney cortex in both the nephron and the vasculature
(32,33,36). Outside the kidney this enzyme is found in large amounts in
the lung and active prostaglandins escaping the kidney via the
circulation can be catabolized there. The 9-keto-PGE2 reductase may
also be considered catabolic in the kidney because it converts active
PGEZ to PGFZQ which is essentially inactive in the kidney.

In the kidney, another type of functional inactivation may occur by
sequestering biologically active prostaglandins in the lumen of the
nephron. PGEZ, for example, is a potent natriuretic substance at the
serosal surface of the collecting tubule, but is inactive at the lumenal
surface (37). Prostaglandins are secreted and reabsorbed along much of
the nephron, but have been shown to enter the nephron primarily at the

level of the loop of Henle.

Kidney Function. The mammalian kidney has two primary functions.

 

They are: a) to remove waste products, particularly those from protein
metabolism, from the blood and b) to help regulate fluid and electrolyte
balance in the body (39). The nephron is the excretory tubule which is
responsible for the exchange of water and solutes from the glomerular
filtrate and the passage of this fluid onto the collecting tubule at the
distal end of the nephron (Fig. 2). From the collecting tubules, the
filtrate is passed to the ureter and is evacuated as urine. Epithelial
cells of the renal tubule change the composition of the urine through a

combination of secretion and resorption.

Figure 2. Schematic re resentation of the functional unit of the
kidney and t e sites of prostaglandin synthesis in the
kidney (shaded segments).

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11

The principal function of the collecting tubule is to accept hypo-
or iso-osmotic filtrate from the distal convoluted tubule and to
concentrate it under stimulation by the anti-diuretic hormone,
arginine-vasopressin (AVP) (40). AVP is synthesized in the hypothalamus
and is stored in granules in the posterior pituitary (41). Release of
AVP into the vasculature is brought about by changes in the osmolality
of the blood. Highly sensitive osmoreceptors in the hypothalamic nuclei
can respond to as little as a 2% change in the osmotic pressure of the
blood. The manipulation of the concentration of urine occurs by
changing the Na+ and water permeability of the collecting tubule
lunenal membrane. Urine can be concentrated by passive removal of water
down a concentration gradient between the relatively hypoosmotic urine
and the hyperosmotic interstitium (40).

The binding of vasopressin to kidney plasma membranes has been
examined in detail by Jard (43). The plasma membrane from pig kidney
contains a lysine-vasopressin sensitive adenylate cyclase activity which
is activated by vasopressin (half-maximal activation at 5 x 10"9 M)
(43,44). Collecting tubule segments also have an adenylate cyclase
activity which stimulated by arginine-vasopressin (45). The link
between cAMP and increased permeability of the collecting tubule lumenal
membrane is not presently understood, but probably involves a
phosphorylation-dephosphorylation cycle(s) which alter membrane

permeability (46).

Localization of Prostaglandin Synthesis in the Kidngy. Lee et a1.

were the first to report that the renal medulla contained

12

prostaglandin-like material (47). Crowshaw later showed that
prostaglandin biosynthesis was greater in the renal medulla than the
renal cortex (48). Using sucrose density gradient centrifugation to
fractionate kidney homogenates, Anggard £3.21- showed biosynthesis to be
located primarily in the microsomal fractional of renal medullary cells
(49). Because prostaglandins generally act near their sites of
synthesis, defining the specific cellular sites of synthesis within the
kidney can provide insights into prostaglandin function (50). Nutgeren
.EE.El- showed that medullary interstitial cells and medullary collecting
tubule cells could synthesize prostaglandins (51). The histochemical
assay used, however, involved staining for arachidonic acid-dependent
peroxidase activity with 3,3'-diaminobenzidine and was both indirect and
nonspecific.

Using a rabbit antibody raised against the rate-limiting enzyme of
prostaglandin synthesis, cyclooxygenase, Smith et a1. localized
prostaglandin biosynthesis by immunohistochemistry (Fig. 2) (52). The
enzyme was found in medullary and cortical collecting tubule cells,
medullary interstitial cells and endothelial cells lining all arteries
and arterioles in the rabbit, rat, sheep, cow and guinea pig kidneys.
The enzyme was also detected in the parietal layer cells of Bowman's
capsule in the rabbit and the mesangial cells in ovine and bovine
glomeruli. In the hydronephrotic kidney, the enzyme was found in the
thin limb of Henle cells (53). While this immunohistochemical technique
does not eliminate the possibility that cyclooxygenase is present in
cells other than those listed above, it does suggest that if the enzyme

is present in other cells it probably exists at low concentrations.

13

Cyclooxygenase, for instance, is known to be present in vascular smooth
muscle cells (54) which were not detected by this technique.

The particular prostaglandins synthesized by a given cell type can
not be determined from the cyclooxygenase localization. One can use
either of two approaches to determine which cells form which
prostaglandins. The first is to isolate individual cell types from the
kidney and to characterize the prostaglandins synthesized by these
cells. The second is to raise antibodies against the enzymes that
catalyze the formation of the active prostaglandins and to localize
these enzymes immunohistochemically. Historically, the obstacle to this
second approach has been that only the PGD2 isomerase has been
purified to homogeneity and thus only this enzyme would be suitable for
raising antibodies using traditional immunological techniques.
Recently, however, Dewitt §£.21- have been able to raise monoclonal
antibodies against both PGIz synthase and TxAz synthase using
hybridoma technology even though only impure enzyme preparations of
these synthases were available (55). In the kidney, PGIZ synthase was
found in both vascular endothelial cells and smooth muscle cells and in
a specialized type of interstitial cell found in the medullary
interstitium (55). As with the localization of cyclooxygenase, this
technique may not be sensitive enough to localize all the PGIZ
synthase containing cells in the kidney. TxAZ synthase localization

has just been begun.

Prostaglandin Function in the Kidney. Arterial blood entering the
kidney contains prostaglandins at concentrations substantially below

those known to cause most physiological effects in the kidney (e.g. < 50

14

pH for PGEZ (56)). It is likely, therefore, that the effects of
prostaglandins in the kidney are caused entirely by prostaglandins
synthesized intrarenally. Prostaglandins synthesized in the kidney are
known to be involved in controlling renal blood flow, the
renin-angiotensin system, natriuresis, hypertension and water resorption
(47, 58). Thus, prostaglandins synthesized in glomerular arterioles can
affect renin release in juxtaglomerular cells (59,60), prostaglandins
formed in the renal vasculature can attenuate decreases in renal blood
flow caused by vasoconstrictors (57) and prostaglandins formed in the
collecting tubule and possibly the renal medullary interstitial cells
can regulate Na+ and water transport in the collecting tubule
(37,52,61). The particular prostaglandin which causes each of these
effects has not been determined conclusively.

The molecular basis by which prostaglandins exert their effects
have been carefully evaluated in only a few systems, none of which are
in the kidney. Inhibition of platelet aggregation by PGIZ, PGE1 and
PGDZ involves the interaction of these prostaglandins with specific
receptors which ultimately affect adenylate cyclase activity in the
platelet (62,63,64). PGEZ may also function independently of a
cAMP-mediated mechanism in w1-38 fibroblasts and S49 lymphoma cells in
which amino acid and Mg2+ transport are affected, respectively
(65,66). PGFZa exerts its effect on the corpus luteum independently

of a cAMP-mediated mechanism as well (67).

Prostaglandins and Water Resorption. Evidence for an antagonism

 

between AVP-stimulated water resorption and prostaglandins has come from

several sources. In hypophysectomized dogs pretreated with the

15

cyclooxygenase inhibitor, indomethacin, AVP caused a greater increase in
urine osmolality than in untreated animals (68,69,70). Indomethacin
pretreatment also potentiated AVP-stimulated cAMP synthesis suggesting
that prostaglandins may block water resorption by inhibiting cAMP
synthesis (70). Using perfused segments of rabbit cortical collecting
tubules, Grantham et al. showed that AVP-stimulated water resorption is
inhibited by 50% using 10-95 PGEI (61). The phosphodiesterase
inhibitor, theophylline, also caused water resorption, but PGEl
potentiated this effect. PGEl alone caused a small increase in water
resorption. These results suggested that PGEl, like AVP, can cause
cAMP synthesis in cortical collecting tubules which then leads to water
resorption. Grantham postulated, however, that PGE1 and AVP also
compete for a common receptor which is linked to adenylate cyclase and
that PGEl inhibits AVP-stimulated water resorption when both are
present simultaneously.

Direct evidence for an inhibition of cAMP synthesis by
prostaglandins, however, has not been clear cut. Edelman found that
10'7M_PGE1 inhibited AVP-stimulated adenylate cyclase activity in
crude homogenates of hamster papillae (71). Both Dousa and Torikai
found, however, that 10'5M_PGE2 had no effect on AVP-stimulated
adenylate cyclase in isolated rat medullary collecting tubules (72,73).
Using rat papillary collecting tubules, Dousa also found no inhibition
of adenylate cyclase by PGEZ, but did observe an inhibition of total
AVP-stimulated cAMP formation by 10'5M_PGE2 when intact tubules
were assayed (74). No inhibition was observed at concentrations below
IO'QM PGEZ. Neither PGE1 nor PGEZ stimulate cAMP synthesis at

concentrations below 10'7M_in any renal test system.

16

The data on the interaction between AVP and PGE are conflicting.
In the study by Grantham on isolated cortical collecting tubules, PGE1
inhibits water resorption at 10-9 M which is one order of magnitude
below the lowest concentration of PGE1 or PGEZ shown to inhibit cAMP
formation in any other system. The potentiation of
theophylline-stimulated water resorption by 10'9 M PGEl also
suggests that PGEl can independently stimulate cAMP synthesis in the
isolated tubule at a concentration two orders of magnitude below that
reported in other systems. While there is little doubt that PGEZ and
PGE1 can inhibit AVP-stimulated water resorption in the collecting
tubule, there is still some question as to the concentrations at which
PGEZ is effective physiologically and whether a cAMP-independent
mechanism of PGE2 inhibition may also be important. Moreover, the
importance of PGE1 is doubtful because PGE1 is probably not formed
by the kidney in XIXQ-

The simplest model reported to explain the relationship between AVP
and PGE2 suggests that AVP simultaneously stimulates cAMP and PGE2
synthesis in the collecting tubule. PGEZ then serves as a feedback
inhibitor of water resorption. The possible role of PGE2 alone on
water resorption is not known. The importance of other prostaglandins
synthesized by the kidney, PGan, TxAz, PGDZ and PGIZ on water

resorption also remains to be investigated.

The Kallikrein-Kinin System and Prostaglandins. Kallikrein is a
proteolytic enzyme which is widely distributed in body tissues and
fluids, including the blood (41). Lysyl-bradykinin (kallidin) is formed

intrarenally from kininogen by the action of kallikrein (75). The

17

source of kininogen for renal kinin synthesis, however, is presently
unknown (75,76). Lysyl-bradykinin can be converted to bradykinin in the
kidney by an aminopeptidase (76). Kininases are the enzymes which
render these kinins biologically inactive. The most abundant renal
kininase is probably the peptidyl dipeptidase, kininase II (77).
Inhibition of kininase II with specific inhibitors results in
substantially higher intrarenal levels of kinins suggesting that
kininases are of major importance in regulating renal kinin levels

(78).

Renal kallikrein is significantly different from plasma kallikrein
and urinary kallikrein is generally believed to be a reflection of
intrarenal kallikrein synthesis and not due to filtration of plasma
kallikrein (78). Carretero et_al. have shown that the inner cortex of
the kidney contains the highest levels of kallikrein and ward ££.91-
have shown that kallikrein is associated with the plasma membrane
fraction (79,80). Stopped-flow techniques have been used to localize
kallikrein secretion to the level of the distal tubule (81). Release of
kallikrein into the tubular fluid is well documented (82), but it is not
known if release into the vasculature and interstitium also occurs. Due
to the distal location of kallikrein secretion it has been proposed that
kallikrein exerts its tubular effects primarily at the distal and
collecting tubules (83).

Kallikrein and thus kinins are known to be related to sodium and
water excretion by the kidney (84). For example, Mills gt 31. have
reported that sodium excretion and kallikrein excretion are closely
correlated in rabbits and rats (85). Decreased kallikrein excretion

(occurs in essential hypertension suggesting that retention of sodium

18

during hypertension may be partially regulated by kallikrein (86).
Aldosterone, an adrenal cortical hormone which stimulates sodium
resorption by the kidney, also causes kallikrein excretion (87).
Kallikrein may thus serve to oppose the anti-natriuretic effect of

aldosterone i vivo.

 

Consistent with the effect of kallikrein on sodium excretion,
bradykinin infusion caused natriuresis in both the perfused dog kidney
and the anesthesized dog (88,89,105). McGiff et a1. were the first to
show that intrarenal arterial infusion of bradykinin also caused the
release of a PGE-like compound in the dog kidney (90). It has since
been demonstrated that PGan and PGIZ are also released from the
bradykinin-stimulated dog kidney (91,92). Sites within the kidney which
are known to synthesize prostaglandins in response to bradykinin are the
renal vasculature, the medullary interstitial cells and the papillary
collecting tubule (1,93,94,95).

The importance of prostaglandins in mediating bradykinin-induced
natriuresis has not been clear cut. In the perfused dog kidney,
indomethacin did not inhibit bradykinin-induced natriuresis (96) while
in the anesthesized dog, the prostaglandin synthesis inhibitor,
meclofenamate, inhibited bradykinin-induced natriuresis (88). This
discrepancy is typical of much of the data collected on the effect of
prostaglandins on sodium transport in the kidney.

Large intravenous doses of PGE2 have been shown to be natriuretic
in both man and animals (97,98,99). In other work, cyclooxygenase
inhibitors were found to increase, decrease or have no effect on sodium
excretion in the conscious dog (100,101,102,103). In studies with

nephron suspensions, renal slices and the perfused rabbit nephron, PGE

19

and PGF did not effect sodium transport (56,104). It was suggested
therefore that prostaglandins caused natriuresis by causing changes in
renal hemodynamics and not by direct tubular effects. Stokes and Kokko
have found, however, that peritubular PGEZ caused a natriuresis in
isolated perfused cortical and medullary collecting tubules (37).
Overall, while kinins can cause natriuresis and prostaglandin synthesis
in the kidney, the significance of renal prostaglandin synthesis in
relation to sodium excretion by the kidney remains unresolved.

Another function of bradykinin in the kidneythat is apparently
mediated, at least in part, by prostaglandins is bradykinin-induced
diuresis. On intrarenal arterial infusion bradykinin caused diuresis in
the dog and indomethacin pretreatment inhibited this diuresis (96).
Moreover, bradykinin-induced diuresis could not be overcome by the
simultaneous infusion of AVP (105,106). These data have been
interpreted as meaning that bradykinin-induced prostaglandin synthesis
may enhance diuresis by causing an antagonism of AVP-stimulated water

resorption.

CHAPTER II

ISOLATION OF RENAL PAPILLARY
COLLECTING TUBULE (RPCT) CELLS:
CHARACTERIZATION OF THE PROSTAGLANDIN
PRODUCTS FORMED BY CELL HOMOGENATES

Prostaglandins are formed by 4-5 different cells types in the
kidney: medullary interstitial cells, endothelial cells of arteries and
arterioles, glomerular epithelial and mesangial cells and both cortical
and medullary collecting tubule cells. Homogenous populations of both
renal medullary interstitial cells and vascular endothelial cells have
been prepared and studies with these isolated cells have provided
important insights into prostaglandin metabolism by these cells.
Parallel studies with collecting tubules could lead to a further
understanding of the function of prostaglandins in the kidney. In
chapter I, a method is described for isolating collecting tubule cells
and the characterization of the prostaglandin products fonmed by

homogenates of these cells.

20

MATERIALS AND METHODS

Materials. Samples of PGDZ, PGEZ, PGFZQ, Tsz,
6-keto-PGFla and Flurbiprofen (dl-2-(fluoro-4-biphenylyl) propionic
acid) were generous gifts of Drs. John Pike and Udo Axen of the Upjohn
Company, Kalamazoo, Michigan. Unlabeled arachidonic acid was obtained
from Nu-Chek Prep, Inc., Elysian, Minnesota and
[5,6,8,9,11,12,14,15-3H]-arachidonic acid (60-100 Ci/mmol),
.L-[4,5-3H(N)]-leucine (40-60 Ci/mmol) and Df[14C(U)]-glucose
(150-250 mCi/mmol) were purchased from New England Nuclear Corporation
Boston, Massachusetts. Trypsin (1/250), Dulbecco's modified Eagle
media, fetal calf serum, antibiotic-antimycotic (100x) and glutamine
were purchased from Grand Island Biological Company. Fluorescein iso-
thiocyanate (FITC)-conjugated goat anti-rabbit lgG was obtained from
Miles Laboratories, Inc. Rabbit anti-cyclooxygenase serum and preimmune
serum were prepared as described previously (52). Silica gel G was pur-
chased from Merck. Nitroblue tetrazolium, NADH, bovine hemoglobin and
EDTA were purchased from Sigma Chemical Company. Type CLS collagenase
was from Worthington Biochemical Corporation. All other chemicals were

reagent grade and were obtained from comnon comnercial sources.

Isolation of renal_papillary collecting tubule (RPCT) cells. New
Zealand white rabbits (2-4 kg) were sacrificed by intravenous injections

of lethal doses of Nembutal (0.5%) and the kidneys removed and placed in

21

22

phosphate-buffered saline (PBS). The papillae (0.7 g) were dissected
from each kidney, transferred to Krebs-Ringer buffer (less Ca2+ and
MgZ+ salts), pH 7.5, and minced finely into sections of 0.3-0.5

cm3 with a razor blade at room temperature. The minced tissue was
placed in 3 ml of Krebs-Ringer buffer, pH 7.5, containing 0.5 mg of
trypsin per ml. The tissue was drawn up and down a disposable pipet (3
mm bore) 15-20 times per min for 5 min and filtered through a stainless
steel mesh (0.25 mm? pore size). The initial filtrate normally
contained 5-8 x 106 cells of which 95-99% were small non- collecting
tubule cells. The remaining residue was removed from the filter and
resuspended in 3 ml Krebs-Ringer buffer and again drawn up and down a
disposable pipet 15-20 times per min (1 mm bore) until the tissue was
completely dispersed (5 min). The trypsinized tissue was again filtered
and the filtrate (containing 1-2 x 106 cells of which up to 40-80%

were non-collecting tubule cells) diluted 1:2 with H20 and allowed to
stand for 4-5 min. Reducing the osmolity caused the lysis of all small
cells. Krebs-Ringer buffer (3-4 ml) was then added to the suspension
and the collecting tubule cells were collected by centrifugation at 500
x g for 2 min. The cell pellet was washed twice by resuspension and
centrifugation in Krebs-Ringer buffer, pH 7.5. The final cell pellet
was dispersed in Krebs-Ringer or another buffer and counted with a
hemacytometer following staining with either trypan blue or erythrosine
red at a final concentration of 0.04% (w/v) (107). The entire procedure
normally required 90 min to complete. The overall purity of the cells

isolated was judged to be greater than 97%.

23

L§HJ-leucine metabolism. Suspensions of RPCT cells were divided
into 5 vials so that each contained 1-4 x 105 cells in 1 ml of
Krebs-Ringer buffer, pH 7.5, containing a 1:50 dilution of
antibiotic-antimycotic. Controls included 1 ml of buffer without cells
and 1 ml of the cell suspension to which cycloheximide (1 ug/ml) was
added. [3HJ-Leucine (2-4 uCi) was added to each vial, and the cells
were incubated at 37°C under a 10% C02 atmosphere for different time
intervals. To st0p further metabolism of [3H]-leucine, 100 umoles of
unlabeled leucine and 1 mg of bovine serum albumin were added to each
vial followed immediately by 5 ml of ice cold 10% trichloroacetic acid.
Each vial was left at 4°C for 40 minutes and centrifuged to pellet the
protein. The supernatant was removed and the pellet washed with 30 ml
of ice-cold 10% trichloroacetic acid on a Whatman GF/C glass fiber
filter. The filter was dried and counted in 6 ml of Bray's

scintillation fluid (108).

[14CJ-glucose metabolism. RPCT cells were isolated using a

 

Ca2+ MgZ+, HCO3' and glucose-free Krebs-Ringer buffer, pH

7.5. The final cell pellet was resuspended in Krebs-Ringer buffer
lacking only HCO3 and glucose. The cells were divided into 5 vials
each containing 1-4 x 105 cells in 1 ml of buffer containing a 1:50
dilution of antibiotic-antimycotic. Two controls were performed, one
without cells; the other, in which cells were incubated in the presence
of NaN3 (0.02%). [14CJ-Glucose (5 uCi) was added to each vial and

the vials were sealed and incubated at 37°C. Glucose oxidation was
measured as 14C02 trapped as H14C03' on KOH-soaked filter

paper which was placed in a plastic ladle above the media. After

24

different incubation times, 0.5 ml of 10% trichloroacetic acid was added
to each vial and one hr later the filter paper was removed and counted

in 6 ml of Bray's scintillation fluid.

NADH-diaphorase and anti1gyclooxygenase immunohistochemical
staining. Staining was performed on cells adhered to coverslips. A few
dr0ps of a RPCT cell suspension (5 x 105 cells per ml of Dulbecco's
modified Eagle media containing 10% fetal calf serum, a 1:50 dilution of
antibiotic-antimycotic and Z‘mM glutamine) were placed on each
coverslip, and the samples incubated at 37°C under a 10% C02
atmosphere for 6-8 hr at which time approximately 40% of the cells had
adhered. Coverslips with cells attached were washed with PBS, frozen in
isopentane (-60°C) and then dried in a desiccator under water aspiration
for 1 hr. The adhered RPCT cells were stained for NADH-diaphorase
activity according to the procedure of Farber et al. (109).
Immunohistochemical staining for the prostaglandin-forming cycooxygenase
was also performed on cells quick frozen, as above, except that
coverslips to which cells were adhered were dipped into
chloroform/methanol (2/1, v/v at -10°C for 4-5 seconds and immediately
lyophilized. After drying for 1 hr the adhered RPCT cells were stained
for cyclooxygenase antigenicity as described previously (52,110).
Fluorescence and bright-field microscopy was performed using a Leitz
Orthoplan microscope. Photomicrographs were obtained with an Orthomat

Camera using Kodak Trix Pan film (ASA 400).

Prostaglandin synthesis and characterization. RPCT cells or the

 

small cells obtained in the first filtrate were suspended in 2 ml of 0.1

25

M_tris-chloride, pH 7.5 containing lemM phenol. Ovine hemoglobin
(0.15mg) was added to each sample of cells. The cells were homogenized
in a glass pestle homogenizer at 4°C for 1-1 1/2 minutes and then added
to 1ml of buffer containing [3H]-arachidonate diluted to varying
specific activities with unlabeled acid. A sample to which Flurbiprofen
(IO'QM) was added served as a control. The vials were incubated

with shaking for 15 min at 37°C and the reactions terminated by adding
21 ml of chloroform/methanol (1/1, v/v). The protein was removed by
centrifugation and 9 ml of chloroform and 4.5 ml of 0.05 M HCl were
added to the supernatant. The chloroform phase was removed and dried
under a stream of N2. The residues were dissolved in 100 pl of
chloroform/methanol (1/1, v/v) and applied to thin layers of silica gel
G (0.3 mm). Chromatography was performed in 2-butanone/isopropyl
ether/methylene chloride/benzene/HOAc (40/40/5/5/1)-solvent system A-for
1 1/2 hr and standards visualized with I2 vapor. Regions containing

the PGan/6-keto-PGF1a, PGEZ, P002, and Tsz standards

were scraped into scintillation vials and counted, or radioscanning was
performed on a Berthold Varian Aerograph Radioscanner. Individual

prostaglandin derivatives were further characterized as follows.

PGEgand PGDZ. Radioactivity chromatographing with PGEZ and

 

PGDZ in solvent system A was eluted from the silica gel with
chloroform/methanol (1/1, v/v) and rechromatographed both in
hexane/ethyl acetate/HOAc (40/40/1)-solvent system B-and
chloroform/methanol/H0Ac/H20 (90/9/1/0.65)-solvent system C on thin
layers of silica gel G. In addition, aliquots of radioactive material

from both the PGEZ and PGDZ regions of chromatograms developed in

26

solvent system A were reduced by treatment with 5 mg of NaBH4 in 0.5
ml of methanol containing 10 mg each of carrier PGEZ and PGDZ.

After 40 min at room temperature, 6.5 ml of chloroform/methanol (1/1,
v/v), 3 ml of chloroform and 1.5 ml of 0.05 N_HCl were added to each
reaction vial. The lipid layer was removed, dried under N2,
redissolved in 100 pl of chloroform/methanol (1/1) and chromatographed

in solvent system B.

PGFZ and 6-keto-PGFle. Material chromatographing in the
combined PGan/6-keto-PGF1a region of thin layer plates
developed in solvent system A was eluted and rechromatographed in
solvent systems 8 and C to resolve these two derivatives. The
PGan/G-keto-PGFla region from chromatography in solvent system
A was also treated with NaBH4, as described above, except that 10 ug
of PGE2, PGan, and 6—keto-PGF1Q were used as carriers in each
reaction vial. Another aqliquot of the PGan/6-keto-PGF10
region from chromatography in solvent system A was methylated by
treating with ethereal diazomethane for 15 minutes at room temperature.
The ether was removed under a stream of N2 and the residue dissolved
in 100 ul of chloroform/methanol (1/1) and chromatographed in solvent

system C.

Tsz. Radioactivity migrating with Tsz during chromatography

in solvent system A was rechromatographed in solvent systems B and C.

RESULTS AND DISCUSSION

Isolation and identification of collecting tubule cells. Rabbit
renal papillae contain four major cell types. Three of these types, the
medullary interstitial cells, the vascular endothelial cells and the
epithelial cells comprising the thin segment of Henle's loop are small
(diameter < 6.50) and thus can be distinguished by microscopic

examination from the fourth cell type, the collecting tubule epithelial
cell (RPCT), which has a diameter of > 10 u- Dissociation of papillae
by mincing and subsequent treatment with trypsin yielded mixtures of
both large RPCT cells and contaminating small cell types. All small
cells disappeared when the isotonic preparative media was diluted 1:2
vri th water and incubated for 3 min. Apparently, the small cells were
preferentially lysed by this treatment while the number and morphology
of the large cells was unaffected.

The size and shape of the large cells (Fig. 3) were similar to
collecting tubule cells observed in histological sections of renal
Papillae (111). Two or three RPCT cells were often seen adhered along
their long axis forming a slight arc. Strings of RPCT cells 10-15 cells
T n length were also observed.

Both the RPCT cells in histological sections and the isolated large
CeTls stained positively for NADH-diaphorase activity. None of the
Small cells isolated were prominently stained for this enzyme. These

observations are consistent with the distribution of NADH-diaphorase in

27

28

Figure 3. Phase contrast photomicrograph of trypsin-isolated RPCT
cells in suspension. Magnification, x250.

29

 

3D

the renal medulla of the rabbit and serve to further confirm the
identity of the isolated large cells as RPCT cells (109).

Isolated RPCT cells were subjected to immunohistofluorescence
staining with rabbit anti-cyclooxygenase serum and rabbit pre-immune
serum, respectively (Fig. 4). The RPCT cells stained much more
intensely with the immune serum in agreement with staining seen in

tissue sections (110,112).

Metabolic characterization of RPCT cells. The isolated RPCT cells

 

excluded both trypan blue and erythrosine red dyes. A maximum of 106
RPCT cells were obtained per g of papillae by our method although the
average yield was approximately half of that value. Figs. 5 and 6
illustrate the metabolism of [14C]-glucose and [3HJ-leucine,
respectively, by isolated cells. RPCT cells both oxidized
[14CJ-glucose to 14C02 and incorporated [3HJ-leucine into
trichloroacetic acid-precipitable material in a time-dependent fashion
for about 2 h. Cells preincubated for 30 min with NaN3 (0.02%) showed
a 75-90% reduction in [14C]-glucose oxidation. Incorporation of
[3H]-leucine into tricholoracetic acid-precipitable radioactivity was
inhibited at least 80% when cells were preincubated with cycloheximide
(1 ug/ml) for 30 min. In contrast, streptomycin, a prokaryotic protein
synthesis inhibitor, at a concentration of 100 ug/ml did not block
[3HJ-leucine incorporation.

Isolated RPCT cells adhered to both glass and plastic petri dishes.
Approximately 40% of the isolated cells became attached within a few
hours when incubated in Dulbecco's modified Eagle media supplemented

with 10% fetal calf serum, 2.9M glutamine and antibiotic-antimycotic.

Figure 4.

31

Fluorescence photomicrographs of isolated RPCT cells
treated with (A) anti- -c clooxygenase or (B) rabbit
perimmune serumthen FIT labe ed goat anti- rabbit IgG.

he rounding of the cells as compared to those in Fig.
3 was caused by chloroform/methanol fixation prior to
staining. Magnification, x 250.

32

 

33

Figure 5. [14CJ-glucose oxidation by isolated RPCT cells. _
RPET cells were incubated for the indifiated times with
[ CJ-glucose] and the formation of [ C]-C02
measured as described in the text.

34

 

 

I J
M N 'l
(91199.,01 x Z/cpt xde)
wsnoavuw asoonio-o”

 

TlME(hr)

35

Figure 6. [3HJ-leucine incorporation into trichloroacetic acid
precipitable-products by isolated RPCT cells. RPCT
cells were incubated for the indicated times with
[ H]-leucine and the formation of trichloroacetic acid

precipitable-radioactivity measured as described in the
ext.

36

 

 

 

 

l
N a Q
Fl
($1163,01x5/,_01x undo)
wsnoauaw amonzi'I-I-te

TIME (hr)

37

The attached cells could be removed from these surfaces by treatment
with trypsin (0.05% in PBS for 5 min) and subcultured on other petri
dishes where they retained distinctive RPCT cell morphology and
continued to exclude vital dyes. Subcultured RPCT cells when grown in a
10% C02 atmosphere still excluded vital dyes after 10-14 days during
which time only the culture media was exchanged. No increase in cell
number occurred during this time.

Attempts were also made to isolate RPCT cells following tissue
dissociation in which EDTA (0.001-0.01%) was substituted for trypsin.
EDTA treatment of minced papillae did provide relatively high yields of
RPCT cells (6-10 x 106 cells per g papillae). The EDTA-isolated cells
also had the same capacity as trypsin-isolated cells both to oxidize
glucose and to convert exogenous leucine into protein. However, the
EDTA-isolated cells were permeable to vital dyes, were lysed rapidly by
treatment with trypsin and were unable to repair their permeability

defect during a 48 hr incubation in culture media.

"Small" cell isolation. Relatively large numbers of small cells

 

were obtained (5-8 x 106 cells per g of papillae) and were
contaminated with only 1-5% CT cells. The small cells were Spherical
and approximately 6p in diameter. None of these cells stained with
vital dyes or for NADH-diaphorase activity. I made no attempt to
distinguish between vascular endothelial cells, medullary interstitial

cells and the epithelial cells of the thin loop of Henle.

Characterization of arachidonic acid metabolites formed by RPCT

cells. Fig. 7 shows a radiochromatogram of the products synthesized

38

from [3H]-arachidonic acid by CT cell homogenates. The major products
synthesized by homogenates of both CT cells and small cells
chromatographed with authentic PGDZ, PGEZ and PGFZQ standards as
expected (49,113). The identity of radioactivity chromatographing with
P602 and PGE2 in solvent system A was verified by chromatography in
solvent systems B and C and by reduction with NaBH4 and chromatography
in solvent system B. The two peaks of radioactivity seen in the

PGan region of the thin-layer plate (Fig. 7) were seldom resolved
clearly. However, as described below, further examination indicated
that 50% of the radioactivity migrating with PGFZa was actually
[3HJ-6-keto-PGF10 while only 30% was found to be due to

[3HJ-PGF2a. Tsz was not synthesized in significant quantities

((3%). When cell homogenates were incubated with [3HJ-arachidonic

acid in the presence of Fluriprofen (10'4M) only unreacted arachi-
donate was recovered indicating that all products were derived from the
activity of the prostaglandin-forming cyclooxygenase and not a lipoxy-
genase. Thus, the small amount of radioactivity chromatographing in the
monohydroxy acid region of the thin-layer plate (Fig. 7) is likely HHT
and not a 20 carbon product (114). Data derived from incubations with
five different RPCT homogenates are summarized in Table 1.

Material with Rf = 0.17-0.19 in solvent system A was eluted from
the silica gel G with chloroform/methanol (1/1, v/v). The presence of
both radioactive 6-keto-PGF1Q and PGan was indicated by
comparing the chromatographic properties of the eluted material with
those of various other prostaglandin derivatives in solvent systems 8
and C (Table 2). Treatment of the radioactive material with ethereal

diazomethane converted 40% of the radioactivity into a product which

Figure 7.

39

A radiochromatogram of the products formed upon
incubation of [ HJ-arachidonic acid (100 EU) with a
RPCT cell homogenate. Products were separated by
chromatography in solvent 5 stem A. Radioscanning was
performed as described in t e text. AA-arachidon1c
acid; RC-ricinoleic acid.

40

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42

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43

also chromatographed with the methyl ester prepared from
6-keto-PGF1G. NaBH4 treatment of the radioactive metabolite and
authentic 6-keto-PGF1G failed to alter the chromatographic
properties of either compound in agreement with the finding of
Pace-Asciak (115), although PGDZ and PGE2 were reduced to PGF
isomers by NaBH4 (>90%) under these conditions.

We performed two control experiments which verified that
6-keto-PGF1a formation was actually being catalyzed by the
biosynthetic machinery originating from the intact RPCT cells and not by
enzymes derived from cellular debris which might possibly have remained
after hypotonic lysis of small papillary cells. In the first
experiment, we examined the supernatant obtained from the final wash of
RPCT cells, but found no prostaglandin biosynthetic activity. In the
second experiment, we compared 6-keto-PGFla synthesis by homogenates
of two different preparations of RPCT cells: one in which trypsin (0.5
mg/ml) was included and the other in which trypsin was excluded during
the hypotonic lysis step. We reasoned that trypsin would inactivate any
prostaglandin biosynthetic enzymes not sequestered within the intact
plasma membrane. We found no differences in the amounts of the
different radioactive products formed per cell by homogenates of RPCT
cells which had been isolated by each of these two procedures.

Fig. 8 shows a time course for the production of various
prostaglandin derivatives when isolated RPCT cell homogenates were
incubated with 2.5 EM_arachidonic acid. PGEZ and 6-keto-PGF1a
were synthesized at initial rates of 150 pmol/min/IO6 cells. The

reactions were complete within 10 min and prior to the complete

 

Figure 8.

44

Time course for the biosynthesis of various
prostaglandin pr ducts by a RPCT cell homogenate
incubated with [ HJ—arachidonic acid (2.5 uM).
Reactions were performed for the indicated-times and
the products analyzed as described in the text.

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46

utilization of substrate, apparently reflecting the self-catalyzed
destruction of the cyclooxygenase (116).

Fig. 9 compares the amounts of different prostaglandins synthesized
at different initial concentrations of arachidonate. At concentrations
of arachidonic acid of less than 2 EM, 6-keto-PGF1a was the major
product formed while at higher arachidonate concentrations, PGE2 was
the major product. Vane and coworkers (117) and Sun et al. (118) have
made similar observations of the effect of substrate concentrations on
the prostaglandin product distribution in other tissues. Apparently,
the Km of the PGIZ synthase is much lower than that of the isomerase
catalyzing PGEZ formation. We suSpect that the fall in overall
6-keto-PGF1Q production noted at high concentrations of arachidonic
acid (e.g. 100 EU) may have resulted from the presence of small but
inhibitory levels of hydroperoxide contaminants (119) in the substrate
solution.

Although PGIZ is known to be formed by the renal cortex
(120,121), apparently by renal vascular endothelial cells (122), the
renal medulla has previously been reported to form only the classical
prostaglandin derivatives (1,49,113,123,124). Thus, it was somewhat
surprising to find that isolated collecting tubule cells as well as
populations enriched in medullary interstitial cells have the capacity
to produce significant quantities of PGIZ. Since this work was
reported, Dunn (124) and Silberbauer (125) have also reported that

PGIZ can be produced by the renal medulla.

47

Figure 9. Prostaglandin biosynthesis by RPCT cell homogenates at
different initial concentrations of [3HJ-arachidonic
acid. Incubations were performed for 15 min and the
radioactive products measured as described in the text.

48

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CHAPTER III

KININ—INDUCED PROSTAGLANDIN SYNTHESIS
BY RENAL PAPILLARY COLLECTING TUBULE
CELLS IN CULTURE

In Chapter II, I presented a method for isolating RPCT cells
following dissociation with trypsin and defined the prostaglandin
biosynthetic capacity of these cells. In this chapter, I describe an
improved isolation procedure for RPCT cells and the further
characterization of these cells in terms of their morphology, enzyme
histochemistry, responsiveness to honnones and growth in tissue culture.
I also present the results of experiments designed to determine which
prostaglandins are released by RPCT cells in culture and what hormonal

factors stimulate the release of these prostaglandins.

49

MATERIALS AND METHODS

Materials. Trypsin (1/250), Dulbecco's modified Eagle media (DMEM),
antibiotic-antimycotic (100x) and fetal bovine serum were all purchased
from the Grand Island Biological Company. Collagenase (CLS II) was
obtained from Worthington Biochemicals, Inc. Bradykinin triacetate,
lysyl-bradykinin, bovine serum albumin, histamine, thrombin, glucagon,
norepinephrine, iSOproterenol, epinephrine, calcitonin, cholera toxin,
arginine vasopressin (AVP), angiotensin II, NADPH, NADH, NADP, NAD and
3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma Chemical
Company. Flufenamic acid was from Aldrich Chemical Company. The
Ca++ ion0phore A23187 was obtained from Cal-Biochem. [3H]PG02
(120 Ci/mmole), [3H]arachidonic acid (130 Ci/mmole), [3H]PGE2 (130
Ci/mmole), [3HJPGE2a (120 Ci/mmole), [3H]TxB2 (150 Ci/mmole),
[3H]6-keto-PGF1Q (100 Ci/mmole), [3H]Lfleucine (60 Ci/mmole),
[3H]3',5'-cyclic AMP (31 Ci/mmole) and radioimmunoassay supplies for
the 3',5'-cyclic AMP assay were all purchased from New England Nuclear
Corp. Antisera against PGEg and PGFZQ were obtained from Miles
Laboratories, Inc. Antisera against Tsz and 6-keto-PGF1G were
purchased from Immunalysis, Inc., Milton, MA. Prostaglandin standards
were generously supplied by Dr. John Pike of the Upjohn Company.
[3H]PGH2 was prepared essentially as described by Hamberg and
Samuelsson (125). Dr. Roderick Walker of the University of Illinois,

Chicago, graciously donated samples of desamino—arginine vaSOpressin

50

51

(dD-AVP) (126). Methionyl-lysyl-bradykinin was purchased from Chemical
Dynamic Corp., South Plainfield, N.J. Parathonnone (1-34 bovine
parathyroid hormone (PTH), 6000 I.U./mg) was from Beckman Instruments.
Glutaraldehyde, Araldite 502, Epon 812, 0504, 2,4,5-tri(dimethylamino-
ethyl)-phenol and propylene oxide were from Electron Microscopy
Sciences. Uranyl acetate was from Polysciences, Inc., Warrington, PA.
MF cement, HAMK-OZ4-12 filters and calf-skin collagen were obtained from
Millipore Corp. All other chemicals were reagent grade or better and

were purchased from common chemical sources.

Cell isolation. Collecting tubule cells were isolated under

 

sterile conditions from renal papillae using a modification of the
procedure described in Chapter II. The papillae were considered to be
those portions of the medulla containing no thick ascending limbs.
Papillae (0.5-0.8 g) were carefully dissected from rabbit kidneys,
minced in a petri dish with a sterile razor blade and transferred to a
plastic culture tube containing 4 ml of 0.1% collagenase in Krebs buffer
(composition, l".flfli 118 NaCl, 25 NaHC03, 14 glucose, 4.7 KCl, 2.5
CaClz, 1.8 M9304 and 1.8 KH2P04), pH 7.3. The minced tissue was
incubated for l.5-2.5 hours at 37° under a 5% C02 atmosphere. The
tissue was then gently agitated by drawing it up and down in a large
bore 10 ml pipet 4-5 times. The cell suspension was filtered through a
Gelman stainless steel mesh (Cat. No. 4320-1) and the filtrate diluted
with 2 volumes of distilled H20 to burst cells other than collecting
tubule cells. The collecting tubule cells were pelleted by
centrifugation on a tabletop clinical centrifuge. The cell pellet was

resuspended in 10% bovine serum albumin in phosphate buffered saline

52

(PBS; composition in mm: 151 NaCl, 45 KH2P04 and 2.5 NaOH), pH 7.2

and centrifuged again to pellet the cells. This latter step removed
considerable amounts of debris from broken cells. The final cell pellet
was resuspended in DMEM containing 10% fetal bovine serum and
antibiotic-antimycotic (1/100) and an aliquot taken for counting. When
using 24-well replicate culture dishes (16 mm, well diameter), cells
were plated to a density of 6-9 x 104 cells per well and grown at 37°

under a 10% C02 atmosphere.

Incubation of cells with effectors. Treatment of RPCT cells with

 

effectors was done in triplicate using 24-well culture dishes. Cells
were first rinsed free of media with Krebs buffer, pH 7.3, and then 0.6
ml of buffer containing an effector was added to the cells. The cells
were incubated for the desired lengths of time at 37°C under a 5% C02
atmosphere. Typically, 0.02- 0.10 ml of buffer was removed from each
well for radioimmunoassays. The remaining buffer was removed and
discarded and 0.20 ml of 0.1% sodium dodecyl sulfate (SDS) added to each
well. The sample was incubated at 37°C for 15 min and the solubilized
protein assayed by the Lowry procedure (127) using bovine serum albumin
as a standard.

When measuring the doubling time of cells, day 1 represented 18
hour old cells. On subsequent days, cells from 3 different wells were
removed by treatment with PBS containing 0.03% EDTA, pH 7.3. The cells
in one aliquot were counted with a hemacytometer, and the other aliquot
was assayed for protein after the addition of 0.5 ml of 0.5% 505. Media
in all wells was changed daily. The effects of AVP and bradykinin on

3',5'-cyclic AMP and prostaglandin formation, respectively, were

53

determined prior to the treatment with PBS containing 0.03% EDTA, pH
7.3.

 

Prostaglandin radioimmunoassays. Immunoreactive prostaglandins,
19052, iPGan, iTsz and 6-keto-PGF10 were measured with ‘
anti-PGEZ, anti- PGan, anti-Tsz and anti-6-keto-PGF10
sera, respectively; iPGDz was measured using anti-Tsz sera with a
cross-reactivity against PGDZ of 2.5%. All the prostaglandin
radioimmunoassays employed a single antibody to bind the prostaglandins
and dextran T-70-coated charcoal (25 mg dextran, 250 mg charcoal, in
25 ml of PBS, pH 7.2 to precipitate any unbound prostaglandins). The
sensitivities of the various assays were 10-500 pg for PGEZ and
PGan, 20-1,000 pg for 6-keto-PGFla and Tsz and SOD-10,000 pg
for PGDZ when using the anti-Tsz sera. The cross-reactivities of
the anti- sera with different prostaglandins are shown in Table 3.

The large amounts of iPGE2 relative to other prostaglandins
synthesized by RPCT cells allowed us to measure iPGEZ directly using
aliquots of incubation media without chromatographic separation from
other products. The very low cross-reactivity of anti-PGFZQ sera
with other prostaglandins also permitted measurements of iPGan
directly without isolating PGF“. Because of the small amounts of
6-keto~PGFla formed and the cross reaction of PGE2 with
anti-6-keto-PGF10 serum, it was necessary to purify 6-keto-

PGFla prior to assay. To measure iPGDg, PGDZ was first purified
by thin-layer chromatography in two solvent systems and then assayed
using anti- Tsz serum. All the effectors used were checked to

detenmine if they had an independent effect on the radioimmunoassays.

54

TABLE 3. Cross-reactivities of prostaglandin anti-sera with various

 

 

 

prostaglandins.
a% Cross-reactivity
Sera PGEZ TXBZ PGan 6-keto-PGF1a PGDz
anti-PGEZ 100 (0.1 1.6 (0.1 1.5
anti-TXBZ 0.26 100 0.07 0.05 2.5
anti—PGFZQ <0 01 <0.01 100 <0.01 <0.01
anti-6-ket0-PGF1G 2.1 (0.1 4.5 100 (0.3

 

a% Cross- reactivity was calculated after determining the amount
of a prostaglandin derivative which would cause 50% displacement of the
bound, radiolabeled parent hapten from the antibody (e. g. [3 HJPGEZ
from anti- -PGE2) relative to the amount of unlabeled parent antigen
(e.g. PGEZ) needed to cause the same displacement.

55

When necessary, extraction of the incubation media and
chromatographic separation of prostaglandins was perfonned as follows.
Prostaglandins were extracted from 1 ml of Krebs buffer, pH 7.3, with
8 ml of chloroform/methanol (2/1; v/v) and 1.5 ml of 0.05 M_HCl. The
lipid layer was removed and the solvent evaporated under a stream of
N2 gas. The residue was dissolved in 0.10 ml of chloroform/methanol
(1/1; v/v) and spotted on a silica gel G thin layer plate (Analtech).
The prostaglandins were routinely resolved by chromatography in a
solvent system containing isopropyl ether/Z-butanone/benzene/methylene
chloride/acetic acid (40/40/5/5/1; v/v/v/v/v). Regions of the sample
with the same chromatographic mobilities as PGDZ and 6-keto- PGFla
standards were scraped from the plate and eluted from the silica gel
with 5 ml of chloroform/methanol (1/1; v/v) and the solvent evaporated
as above. Material extracted from the PGDZ region of the first plate
was spotted on a second silica gel G thin layer plate and subjected to
chromatography in hexane/ethyl acetate/acetic acid (40/40/1; v/v/v) and
the PGDZ region scraped and eluted as above. Both 6-keto-PGF10
and PGDZ were redissolved in ethanol/PBS, pH 7.2 (1/4, v/v) and
assayed. The recoveries of P602 and 6-keto-PGF16 throughout these
procedures were monitored using [3HJPG02 and

[3H]-6-keto-PGFla, respectively, and averaged 65-70%.

3',5'-cyclic AMP radioimmunoassay. To quantitate extracellular

 

3',5'-cyclic AMP, samples were routinely assayed without purification of
3',5'-cyclic AMP by removing aliquots from incubation mixtures
containing various effectors with RPCT cells. All effectors used were

tested for potential interference with the radioimmunoassay.

56

To verify that it was 3',5'-cyclic AMP which was actually being
measured, some samples were treated with phosphodiesterase: 0.05 ml of
bovine phosphodiesterase (3 mg/ml) in 0.05 M tris-chloride, pH 7.7, was
added to 0.1 ml aliquots removed from incubation mixtures in which
3',5'-cyclic AMP was generated and the sample incubated for 45 minutes
at 37°. After destruction of phosphodiesterase, 3',5'-cyclic AMP levels
were determined by radioimmunoassay and found to be reduced by more than
90%. Furthermore, in samples spiked with 5 x 105 dpm of [3H]
3',5'-cyclic AMP and then treated with phosphodiesterase and extracted
and chromatographed according to the procedures of Goldberg et_gl.
(128), greater than 85% of the original tritium label was converted to
products (ca. 85% 5'-AMP) with chromatographic mobilities different than
3',5'- cyclic AMP.

Measurements of intracellular 3',5'-cyclic AMP were performed as
follows. At the appr0priate times after addition of the effectors, the
incubation buffer was aspirated. The cells were washed quickly with 1
ml of ice-cold PBS, pH 7.2, and then 350 ul of ice-cold 1% perchloric
acid was added to each well. [3H]3',5'-Cyclic AMP (3000 dpm) was
added to each well and the samples incubated at 4° for 2.5 hr. The
perchloric acid solution was then neutralized with 3 M_KOH and
centrifuged at 2000 x g for 10 min to remove particulate material.
Supernatants derived from each sample were applied to columns of Dowex
AG-1X8 (0.5 x 4.0 cm) equilibrated with 0.1 N formic acid; 3',5'-cyclic
AMP was eluted with 5 ml of 2 N formic acid. The eluant was lyophilized
and the residue redissolved in 250 pl of H20. An aliquot of this
sample was used to determine the recovery of [3H]3',5'-cyclic AMP

(typically 60-70%), and the remainder used for radioimmunoassays.

 

57

Statistical Analyses. Experiments involved using 24-well culture

 

dishes in which a minimun of 3 or 4 replicates and thus 6 or 8

treatments were analyzed for any effects. A completely random analysis

 

of variance was used to test for differences between samples means at
p<0.05 (129). Dunnett's test was used for comparing differences between
effector means with the control mean (129). Error bars on the figures

are 1 SEM.

Electron microscopy. Samples examined by electron microscopy

 

included renal papillary tissue and RPCT cells grown in monolayers
attached to either culture dishes or Millipore filters (130). Primary
fixation of all samples was performed for 24 hr at 4° in 2%
glutaraldehyde in 0.1 M_sodium cacodylate, pH 7.4. In the case of
papillary tissue, cubes (ca. 1 mm3) were cut from fresh tissue and
immersed in fixative. Cultured RPCT cells were overlayed with the
fixative and the fixation performed in culture dishes. To grow RPCT
cells on millipore filters, one edge of a HAMK-OZ4—12 Millipore filter
was glued to the bottom of a culture dish using MF cement, the entire
dish seeded with RPCT cells and the cells incubated on the filter for
3-4 days in culture media. Following fixation in glutaraldehyde all
samples were washed 4-6 hr with frequent changes of 0.1 M sodiun
cacodylate, pH 7.4. The samples were then postfixed in 1% 0504
overnight at 4°. The samples were washed and then dehydrated by
sequential exposure to 50%, 70%, 80%, 90% and 100% (3x) solutions of
ethanol. After the third treatment with 100% ethanol, RPCT cells were
removed from culture dishes by scraping with a rubber policeman,

transferred to 4 dram screw t0p vials and collected by centrifugation at

58

500 x g for 2 min. To these cell pellets or tissue blocks or the entire
millipore filter was added propylene oxide, which was, after a brief
incubation period, then mixed with an equivalent volume of Epon-araldite
resin (Epon 812:Araldite 502:d0decenyl succinic anhydride; 25/20/60;
v/v/v), and the samples agitated for 4 hr at 24°. Extra resin was then
added to provide a ratio of resin to propylyene oxide of 2, and the
mixture agitated overnight. The samples were then collected by ‘
centrifugation and resuspended in Epon-Araldite resin to which had been
added 0.024 volumes of 2,4,6-tri—(dimethylaminomethyl)phenol. The
samples were placed in Beem capsules and incubated for 72 hr at 60°.

The capsules were sectioned and the sections examined and photographed
using a Phillips Model 201 transmission electron microscope. The
sections were counterstained with 2% uranyl acetate in H20. Kodak

EM4463 film was used for photography.

Enzyme Histochemistry. Histochemical staining for succinate

 

dehydrogenase, NADH diaphorase and a-glycerophosphate dehydrogenase
(109,131) was performed on quick-frozen RPCT cells cultured on glass
cover slips and compared to staining of sections (10 um) of rabbit
kidney cut on Ames Lab-Tek cryotome. Light microscopy was performed on
a Leitz Orth0plan microscope equipped with an Orthomat camera.

Photomicrosc0py was performed using Kodak TriX Pan Film (ASA 28).

Measurements of Potential Differences. Lexan polycarbonate tubing

 

(5/8" x 3/4") was cut into 1/2" cylinders and the surfaces of the
cylinders sanded smooth. The cylinders were boiled in Alconox for 1

hour, rinsed with distilled H20 and allowed to dry. A thin film of

59

silicone cement was put on one surface of the cylinder. A polycarbonate
Nucleopore filter (0.8 um; Cat. No. 111109) was then attached to this
edge and the cement allowed to dry overnight. Three dabs of silicone
cement were put on the filter edges of the cylinder in order to fonn a
tripod so that a space was present below the filter when the unit rested
on a flat surface. Approximately 2 mm of clearance was satisfactory.

The filter unit was washed with 75% ethanol and then 0.08 ml of a
collagen solution (1/1 mixture of 8 mg/ml calf-skin collagen and 0.1%
acetic acid) was spread uniformly across the inner surface of the
filter. The collagen solution was allowed to dry overnight. The
collagen was fixed by exposing the entire unit to vapors of concentrated
NH4OH (30%) for 1 hr. The collagen-coated filter units were stored in
petri dishes at 37°C under a humid 5% C02 atmosphere. Prior to
seeding the filters with cells, the filter unit was rinsed with DMEM
containing 10% fetal bovine serum. Cells were seeded at a density of
1-2 x 106 cells/cm2 of filter, in order to insure confluency. The
unit was then placed in a 100 mm2 petri dish containing 12 ml of media
and incubated at 37° under a 5% C02 atmosphere.

Measurements of potential differences were made in either DMEM
containing 10% fetal bovine serun or in 20 EU tris-chloride, pH 7.2
containing 1 EM MgClz, 1 EH CaClz and 140 EU NaCl. Buffer was added
to the inside and outside of the filter unit such that the fluid levels
were equal and thus no hydrostatic pressure was present. The inside and
outside solutions were connected to two solutions of 3‘M KCl by 12 inch
salt bridges. The salt bridges were made by drawing a hot mixture of 3
.M KCl and 3% bacterial grade agar into polyethylene tubing (PE 240) and

allowing the agar to solidify. The bridges were stored immersed in 3‘M

 

60

KCl. The two 3 M_KCl solutions were connected to a Corning model 10 pH
meter with two Fischer calomel electrodes (Cat. No. 13-639-52). A
potential of zero was determined by placing both agar bridges in either
the inside or outside solutions. A potential difference was measured by
moving one bridge back to the other solution.

Madin-Darby canine kidney (MDCK) cells used as positive controls
for potential difference measurements were obtained from the American
Type Culture Collection and were grown in DMEM containing 10% fetal

bovine serum at 37° under a 5% C02 atmosphere.

RESULTS

Histological Characterization of Isolated Papillary Collecting

Tubule (RPCT) Cells. We previously isolated RPCT cells from rabbit

 

kidney by a procedure which involved mincing of the papillae, agitation
of the tissue with 0.05% trypsin and then hypotonic lysis of the
dispersed cells; an average of 106 RPCT cells were obtained per g of
papillae. We have since found that we can increase the cell yield 10-
fold by substituting a milder 1.5-2.5 hr incubation with collagenase
(0.1%) at 37° for the trypsin-agitation step of the earlier procedure.
Approximately 60% of the isolated cells adhered to plastic petri dishes.
When grown to confluency (8-10 days) and examined by light microscopy,
RPCT cells were polygonal (Fig. 10), and resembled cells in confluent
cultures of Madin-Darby canine kidney (MDCK) cells, a transfonned
epithelial cell line apparently derived from the canine distal tubule
(130,132). Cells having the highly elongated appearance of smooth
muscle cells were seen occasionally in some RPCT cell cultures but never
represented more than 0.5% of the total cells in one day cultures.

Fig. 11A-C are electron micrographs comparing the morphological

properties of collecting tubules lg situ (Fig. 11A) with RPCT cells

 

grown in monolayers on Millipore filters (Fig. 118 and 11C). The
isolated RPCT cells had intact plasma membranes and the mitochondrial
and nuclear membranes were well-preserved (Fig. 118). There was little

vacuolization induced by the isolation procedure. Lipid droplets were

61

62

apparent in approximately 20% of both the isolated cells and the intact
tubules. RPCT cells which were grown on Millipore filters resembled

the parent cells in having relatively few microvilli but those present
studded the apical surface (Fig. 118) suggesting polarity in adhesion
and growth. This same pattern of adherence has also been observed with
MDCK cells (130). Cell junctions were consistently present between the
apical and basolateral surfaces of RPCT cells grown on Millipore filters
(Fig. 11C).

Hemicysts commonly observed in cultures of MDCK cells (132) were
seen in cultures of RPCT cells which had been grown to confluence
without media changes (Fig. 12); addition of ouabain (10"5 M) to the
media prevented formation of the hemicysts. We observed electrical
potential differences of 0.5-1.0 mV (apical surface negative) in each of
five separate experiments with cultures of MDCK cells seeded on
collagen-coated Nucleopore filters. However, we detected a potential
difference (0.4 mV, apical surface negative) with only one of five
different cultures of RPCT cells. There are two possible explanations
for our inability to achieve consistent results in measuring potential
differences with RPCT cells. First, the quantities of RPCT cells
required to cover the relatively large Nucleopore discs used for
potential difference measurements could only be obtained after 2-3 cell
passages during which time increasing contamination (> 10%) of
nonpolygonal fibroblast-like cells had occurred; and second, unlike MDCK
cells, RPCT cells do not fonn a stable confluent monolayer, but instead
begin to detach and die upon reaching confluency (see below, Figure

15).

63

Figure 10. Phase photomigraph of RPCT cells grown eight days in
monolayer culture. Magnification, x 250. Nucleus (N).

64

 

65

Figure 11. Electron micrographs of (A) intact collecting tubule
from rabbit renal papillae (magnification, X4,500),
(B) isolated RPCT cells grown in Millipore filters
(magnification, X12,250) and (C) junctional complex of
RPCT cells grown on Millipore filter (magnification
X20,000). Nucleus (N), mitochondria (M), basement
membrane (BM), oil dr0plets (0), rough endOplasmic
reticulum (RER), basolateral surface (8), apical
surface (AS), Millipore filter (MF), desmosome (D),
tight junction (TJ), and gap junction (Gd).

66

\

 

67

 

68

Figure 12. Hemicysts present in culture of RPCT cells grown to
confluence without changing the growth medium.
Magnification, X80.

69

 

70

The RPCT cells gave intense and uniformly positive staining for
both NADH diaphorase and a-glycerophosphate dehydrogenase activities but
did not stain for succinate dehydrogenase activity. This pattern of
staining distinguishes collecting tubules from other epithelial cells of

the papillae and inner medulla (133).

Biochemical Characteristics of RPCT Cells. We had previously
identified isolated RPCT cells in suspension as being derived from the
collecting tubules on the basis of tissue source, cell diameter and
immunohistochemical pr0perties. To determine if monolayer cultures of
RPCT cells retained differentiated biochemical characteristics expected
for collecting tubules, we measured the ability of one day old RPCT
cells to synthesize 3',5'-cyclic AMP in response to a variety of
effectors (Table 4). Agents capable of increasing 3',5'-cAMP levels at
relatively high concentrations (:10'7 M) were AVP, dD-AVP, lysine
vaSOpressin, cholera toxin, oxytocin, glucagon and, unexpectedly,
parathyroid hormone; in contrast, adrenergic agents, calcitonin and
histamine were ineffective. Fig. 13 compares the concentration
dependence of the AVP-, parathyroid hormone- and oxytocin-induced
increases in extracellular 3',5'-cyclic AMP. As anticipated, the most
active agent on a molar basis was AVP. The concentration dependence
observed with AVP is similar to that seen with intact tubules dissected
from the outer medulla (134).

Fig. 14 shows the time course of the AVP-induced appearance of both
intracellular and extracellular 3',5'-cyclic AMP. There was an
approximately linear increase in extracellular 3',5'-cyclic AMP during

the 60 min incubation period whereas intracellular 3',5'-cyclic AMP

71

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72

Figure 13. Release of cAMP into media by RPCT cells incubated with
different concentrations of AVP (O——————O), oxytocin
(A——————A) and PTH (I——————0). Incubations were
performed at 37° for 60 min with IBMX (10-4 M) and
the indicated concentrations of effectors. ‘—
Radioimmunoassays for extracellular cAMP were performed
as described in the text.

73

 

l
10'9 10‘7

l
Io—Il

[EFFECTOR] (Ml

 

 

 

1
I043

 

 

 

1 1 1
o o o
3 8 9

(ugatmd "as bit/mu: og/salouu) dtNVO

74

Figure 14. Time course of the formation of intrace11u1ar
(A—————A) and extrace11u1ar (I——————l) cAMP by RPCT
ce11$ in response to 10'7 M AVP. Incubations were
performed for the indicated times in the presence of

IBMX (10'4 fl) and radioimmunoassays for CAMP were
performed as described in the text.

75

 

 
   
 

intracellular

20

 

 

4
3

O
O
200

00

(upload nao owl/salon”) dWV°

TIME (min)

76

levels peaked during the first 5 min with AVP and then gradually

decreased.

Growth of RPCT Cells in Monolayer Culture. After a lag period of

 

3-4 days there was a four-fold increase in both cell number and cellular
protein in culture dishes seeded with freshly isolated RPCT cells (Fig.
15). After l0 days the cells reached confluence and both cell protein
and cell number began decreasing. The AVP—responsiveness characteristic
of differentiated collecting tubules and noted with one day old RPCT
cells was retained by RPCT cells maintained in tissue culture for up to
l0 days; 3-4 fold increases in 3',5'-cyclic AMP were elicited with

lO'7 M_AVP. Furthermore, ten-day-old RPCT cells were similar in

size and appearance to the original cells and stained positively for
NADH diaphorase. In addition, the rate of iPGEZ synthesis was

increased to the same extent (3-4 fold) by treatment with bradykinin
(10'9 M) in cultures of both one- and ten-day-old RPCT cells. Thus,
several differentiated characteristics of collecting tubule cells were
retained when RPCT cells were grown almost to confluency in primary

monolayer culture.

Prostaglandin Biosynthesis by RPCT cells in Response to Various
Effectors. Table 5 summarizes the results of a series of experiments in
which the influence of a number of effectors on the synthesis of iPGEz
by RPCT cells was determined. Agents which consistently increased
iPGE2 synthesis by RPCT cells after brief (10 min) incubation times
included the kinin derivatives, the Ca++ ionophore A23187, cholera

toxin, and thrombin. No significant response was elicited by

77

Figure 15. RPCT cell growth in monolayer culture. 24-well cluster
dishes were seeded with approximately 30,000 CT cells
per well. Protein content and cell number were
determined at the indicated times as described in the
text.

78

—v——v— (b'rl) ueumd neg

0 O
8 st 8 N 9.

 

 

1 l I I l

 

 

 

 

.._. (2-01 X) Jaqwnu ueo

Time in Culture (days)

TABLE 5.
effectors.

79

Synthesis of iPGEz by RPCT cells in response to hormonal

 

aiPGE
Limoles/ug cel

7 protein)

 

 

Effector Concentration Incubation Time (min)
(5) 10 so
None 4513.5 8519.2
IBMX 10-4 4413.9 8218.9
bradykinin 10-7 184115.1* 334126.1*
lysyl-bradykinin 10-7 196112.8* 305123.7*
methionyl-lysyl-bradykinin 10-7 190120.7* 320127.6*
AVP 10-7 5917.6 102110.2
dDAVP 10-7 4915.2 11511o.7
cholera toxin 10-7 125115.1* 237123.2*
norepinephrine 10-5 5716.8 154116.2*
epinephrine 10-5 6714.2 165115.7*
isoproterenol 10-5 6313.2 9016.9
thrombin 2.5 u/ml 108110.9* 154113.3*
A23187 10-7 14719.2* 430136.7*
histamine 10-5 5513.4 ND
angiotensin II 10'7 7518.6 140110.3*

 

aValues represent means 1 SEM; those marked with an asterisk (*)
indicate a significant change from control values (p<0.01).

measured as described in the text.

iPGEz was

80

parathyroid hormone (10'7 M), calcitonin (10‘7 M), histamine,

AVP, dD-AVP, lysine vasopressin (10'7 M), oxytocin (10’7 M) or
isoproterenol even after 60 min incubation periods at relatively high
concentrations of each of these agents. Angiotensin II, epinephrine and
norepinephrine each increased iPGE formation up to 2-fold but only after
a 60 min incubation.

Homogenates of RPCT cells synthesize various prostaglandins in
different proportions from arachidonic acid depending on the initial
fatty acid substrate concentration. Therefore, we characterized the
prostaglandins formed by intact RPCT cells in response to several
effectors to determine if any of these agents caused the preferential
formation of a prostaglandin derivative other then PGE. We found that
the types of prostaglandins formed by RPCT cells under basal conditions
and in response to bradykinin, AVP, dD-AVP, A23187, angiotensin II,
epinephrine and norepinephrine were very similar in all cases (Table 6).
The major product was of the E series (70-75%). Smaller amounts of
iPGan (ca. 15%), iPGDz (5-10%) and i-6-keto-PGF1a (5%) were
also found. No iTsz was detected under any conditions. These
results indicate that there was no differential effect by any agent
tested on the synthesis of any particular class of prostaglandin.

We performed two sets of experiments to determine if significant
catabolism of PGE formed by RPCT cells could occur during the incubation
of these cells with effectors. In the first experiment, [3H]PGE2
was added in Krebs buffer, pH 7.3, to intact RPCT cells at a
concentration (10'7 M) designed to mimic conditions which occur
following synthesis of iPGEz by cells in response to bradykinin

(10'8 M). Following 3 hr incubations at 37° without effectors or

81

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.w m1m<h

82

with AVP (10-7 M), bradykinin (10-8 M), angiotensin II (10-7
.M) or epinephrine (10'5.fl): prostaglandins were extracted and
separated by thin-layer chromatography and the mobility of the
radioactivity with respect to prostaglandin standards determined. In
all cases, greater than 90% of the original radioactivity was recovered
and of this material more than 90% cochromatographed with PGEZ. In a
second experiment, homogenates of RPCT cells (107) were prepared in
1 ml of 0.1 M_tris-chloride, pH 7.4, containing 10‘4 M_NADH,
10-4 M NADPH, 10-4 M NAD, 10-4 M NADP, 10-4 M fl ufenamic
acid and 10"7 M [3HJPGE2, and the sample was incubated for 3 hr
at 37°. Following this incubation, less than 5% of the added
radioactivity cochromatographed with products other than PGEZ. Under
the same conditions using guinea pig lung homogenates (1 mg protein/ml)
more than 60% of the added [3H]PGE2 was converted to less polar
products. These results indicate that no significant catabolism of
prostaglandins occurs in RPCT cells during our experiments and thus, the
results obtained in assaying the parent prostaglandins accurately
reflect prostaglandin formation by RPCT cell incubates.

PGEZ, P602 and PGan all can be formed from the
prostaglandin endoperoxide, PGHZ, nonenzymically in varying ratios
depending on the experimental conditions (125). Therefore, we tested
RPCT cells for PGH-PGE isomerase activity, the enzyme which catalyzes
the formation of PGE2 from PGHZ. [3H]PGH2 (30 MM) was incubated
with homogenates of both freshly isolated and nine day old RPCT cells
(0.6 mg protein/ml) in 0.1 M_tris-chloride, pH 7.4, containing 5 MM
reduced glutathione. The rate of formation of [3H]PGE2 was 2
pmoles/min/ug of cell protein with both freshly isolated and nine day

 

83

old cells. No synthesis of [3HJPGE2 above that seen with no-enzyme
controls was observed with cell homogenates which had been boiled (100°
for 2 min). The specific activity of PGH-PGE isomerase in RPCT cells is
100 times the specific rate of PGE2 release observed with
bradykinin-treated cells (Fig. 15). Our results establish that PGH-PGE
isomerase activity is present in RPCT cells and suggest that PGEZ
released by intact cells is actually synthesized through an

enzyme-catalyzed isomerization of PGHZ.

iPGE Biosynthesis by RPCT Cells in Response to Bradykinin. The

 

kinin derivatives were the only effectors of potential physiological
importance that caused a rapid increase in the synthesis of
prostaglandins by RPCT cells (Table 5). Therefore, we examined the
effects of the kinins in more detail. Increased synthesis of iPGEz by
RPCT cells could be detected within 1 min of exposing the cells to
bradykinin (Fig. 16). By 3 min a maximal 4-6 fold increase relative to
the control had occurred. Bradykinin, lysyl—bradykinin and
methionyl-lysyl-bradykinin exhibited similar dose-response curves with
half-maximal responses occurring at concentrations of approximately
10"11 M (Fig. 17). Thus, RPCT cells are extremely sensitive to
induction of prostaglandin formation by kinins. By comparison,
half-maximal responses to bradykinin in Swiss mouse 3T3 cells occur at
approximately 10'8 M bradykinin (135). At concentrations of
10'8 M, the responses of RPCT cells to bradykinin, lysyl-bradykinin
and methionyl-lysyl-bradykinin were not additive.

The release of arachidonic acid and PGE2 from 3T3 cells induced

by bradykinin is reportedly blocked by inhibitors of RNA and protein

84

Figure 16. Time course of the iPGEZ synthesis by RPCT cells in
monolayer culture. Incubations were as described in the

text8with (I___———l) and without (L——————A) bradykinin
(10- n)-

 

85

 

 

 

 

 

 

L

l I
O O
in <-

|60 '-
IZO '-

(ugewd neo 611 /se|oui,i) 239d!

TIME (min)

86

Figure 17. Dependence of iPGEz synthesis on kinin concentration.
Incubations were performed for 10 min at 37° with the
indicated concentrations of bradykinin (0__———43),
lysyl-bradykinin (A———————A) and
methionyl-lysyl-bradykinin (l——————O) and iPGE2
analyzed as described in the text.

 

87

 

l
l0‘7

 

 

I
IO-S

l
'O-I I

[KININ] (M)

 

 

 

 

l l l
o o . o
a «a n

I20 )-

| l
S .53

(Uieimd use fin/inOI/seiown 239d!

88

synthesis (136). However, we found that even when RPCT cells were
preincubated for 60 min with cycloheximide (5 ug/ml), subsequent
formation of iPGEz in response to bradykinin was unaffected for up to
90 min; under these conditions, incorporation of [3H]leucine into
trichloroacetic acid-precipitable material decreased from 10,600 cpm in
the absence of inhibitor to 700 cpm in the presence of cycloheximide.
Thus, unlike 3T3 cells, RPCT cells do not contain a protein, undergoing
rapid turnover, which is involved in the release of fatty acid

precursors of prostaglandins.

DISCUSSION

The collecting tubule appears to be the only region of the renal
tubule where prostaglandins are synthesized in healthy kidneys
(51,52,123). We have segregated renal papillary collecting tubule cells
from other prostaglandin- forming cells of the papillae so that we could
determine what factors influence prostaglandin metabolism in the
collecting tubule.

Based on simple morphological and histochemical criteria, these
primary RPCT cell cultures were comprised of at least 97% collecting
tubule epithelia. The properties which identify these cells as being
derived from the collecting tubule are as follows: (a) the cells are
isolated from papillae carefully dissected from the outer medulla; (b)
RPCT cells uniformly stain positively for NADH diaphorase and
a-glycerophosphate dehydrogenase activities and cyclooxygenase
antigenicity and negatively for succinate dehydrogenase activity;
papillary collecting tubule cells exhibit this unique pattern of
staining but cells of the thin limb and descending limb of Henle's loOp,
medullary interstitial cells, capillaries and venules do not (52,131);
(c) the average diameter of the isolated cells measured on cell
suspensions both prior to adherence and after trypsinization of adherent
cells is 12-15 um -- as expected for collecting tubules, which are
approximately twice the diameter of any other papillary cell; (d) RPCT

cells contain AVP-responsive adenylate cyclase activity and the

89

9Q

half-maximal concentration of AVP necessary for activation (l0‘10

_M) is similar to that reported by others for isolated collecting tubule
segments both with respect to AVP-dependent adenylate cyclase respon-
siveness (135) and to water permeability (61); RPCT cells also release
3',5‘-cyclic AMP in response to glucagon but not adrenergic agents as
expected for collecting tubules (137,138); and (e) morphological charac-
teristics such as the size of the nuclei, numbers of lipid droplets,
relative paucity of mitochondria, asymnetric growth on millipore filters
and formation of hemicysts are properties expected of collecting tubule
epithelial cells. The only characteristic of RPCT cells which was not
anticipated from previous studies of collecting tubules was the
increased production by the isolated cells of 3',5'-cyclic AMP in
response to parathyroid hormone. Although parathyroid hormone does not
activate adenylate cyclase activity in the cortical or inner medullary
collecting tubule (139), our results raise the possibility that the
papillary collecting tubule is responsive to this hormone.

A number of properties of MDCK cells, a transformed epithelial cell
line derived from the canine distal tubule, have been described
previously (130,132). RPCT cells are similar to MDCK cells
morphologically, in the formation of hemicysts and in their responses to
most effectors of adenylate cyclase activity (132). The most notable
differences are that parathyroid hormone increases 3',5'-cyclic AMP
synthesis by RPCT but not MDCK cells and that oxytocin and AVP are
equipotent in their actions on MDCK but not on RPCT cell adenylate
cyclases (132).

We have demonstrated that RPCT cells can be grown and maintained in

monolayer culture for at least l0 days with no major changes in

91

differentiated cell characteristics such as morphology, histochemical
pr0perties, AVP-dependent 3',5'-cyclic AMP production and
bradykinin-dependent prostaglandin formation. Thus, RPCT cells, when
grown in primary monolayer culture, provide a rational model for
studying the biochemical events associated with water and inorganic ion
transport which appear to be the principal functions of the collecting
tubule (57,140). However, as with any primary cell culture system, one
cannot eliminate the possibility that certain properties of the parent
cells are modified or lost during isolation and culture.

The results of our studies on prostaglandin formation by RPCT cells
suggest that kinins can act directly on the medullary collecting tubule
to cause PGEZ biosynthesis. The concentration at which each of the
major renal kinins -- bradykinin, lysyl-bradykinin and
methionyl-lysyl-bradykinin (141) -- cause half-maximal stimulation of
prostaglandin synthesis in RPCT cells (10'11 M) is four orders of
magnitude lower than that required for prostaglandin synthesis in other
cell culture systems (135) and is less than one tenth of the
concentration needed for half-maximal contraction of uterine smooth
muscle (142). Thus, RPCT cells are extraordinarily responsive to kinin
derivatives. Since the concentrations of kinins in urine (141) are
greater than necessary to cause maximal iPGE by RPCT cells, we suspect
that kinins may act at the blood surface of the collecting tubule (75)
to induce prostaglandin formation. Kinin- induced prostaglandin
synthesis by renal collecting tubules may cause a natriuresis (133)
since PGEZ does inhibit both active Na+ resorption by medullary
collecting tubule segments (37) and passive Na+ resorption by the

anatomically adjacent medullary thick ascending limb (143).

92

Epinephrine, norepinephrine and angiotensin II increased
prostaglandin formation by RPCT cells, but only by 2-fold, at most, and
only after extended treatment times at relatively high concentrations.
We speculate, therefore, that these agents are not major physiological
effectors of prostaglandin synthesis by collecting tubules. Both
sympathetic nerve stimulation and angiotensin II do increase
prostaglandin synthesis in intact kidneys, and we suspect that these
agents act principally on the renal vasculature (144) and/or glomerular
epithelial and mesangial cells (145).

Although AVP does induce prostaglandin formation in the renal
medulla (45,146) and the anuran bladder (147), neither AVP nor the
nonpressor analog dD-AVP (126) had any effect on the rate of synthesis
of prostaglandins by RPCT cells during a 60 min incubation period; yet,
under these same experimental conditions both AVP and dD-AVP do cause a
3-4 fold increase in the production of 3',5'-cyclic AMP. These results
suggest that AVP when acting as an antidiuretic agent has no short term,
acute influence on prostaglandin synthesis by medullary collecting
tubules.

The results of our product characterizations indicate that PGEZ
is the major prostaglandin released by medullary collecting tubules and
that PGEZ is synthesized enzymatically. The lack of PGE2 catabolism
by RPCT cells suggests that this prostaglandin must be inactivated at
other cellular sites either in the kidney or, after entering the venous
drainage, by the lung. P012 is formed at significant rates by RPCT
cell homogenates, especially at low concentrations of arachidonic acid.
However, the lack of appreciable 6-keto-PGF1G release in these

current studies suggests that either (a) P012 synthase activity

93

is lost during acclimation of RPCT cells to monolayer culture conditions
or (b) that P012 synthase activity is simply not expressed by intact

cells.

CHAPTER IV

INTERRELATIONSHIPS AMONG BRADYKININ,
PGEZ, VASOPRESSIN AND CAMP IN RENAL
PAPILLARY COLLECTING TUBULE CELLS

In the previous chapter I described studies on prostaglandin
metabolism by RPCT cells in culture. PGEZ was found to be the major
prostaglandin product and PGE2 synthesis was shown to be induced by
low concentrations of renal kinins, but not by AVP. In this chapter, I
describe the results of experiments directed toward understanding the

effects of prostaglandins alone and in combination with AVP on cAMP

metabolism by RPCT cells.

94

 

 

MATERIALS AND METHODS

Materials. Trypsin (1/250), Dulbecco's modified Eagle media
(DMEM), antibiotic-antimycotic (100X) and fetal bovine serum were all
purchased from Grand Island Biological Company. Collagenase (CLS II)
was obtained from Worthington Biochemicals, Inc. Bradykinin triacetate,
bovine serum albumin, cholera toxin, arginine vasopressin (AVP) and
3-isobutyl-1 methylxanthine (IBMX) were purchased from Sigma Chemical
Company. [3H]arachidonic acid (130 Ci/mmole), [3H]PGE2 (130
Ci/mmole), [3H]3',5'-cyclic AMP (31 Ci/mmole) and radioimmunoassay
supplies for the 3',5'-cyclic AMP assay were all purchased from New
England Nuclear Corp. Antiserun against PGEZ was obtained from Miles
Laboratories, Inc. Prostaglandin standards, PGE2, P002, PGFZQ
and P012, were either donated by or purchased from the Upjohn Company.
All other chemicals were reagent grade or better and were purchased from

common chemical sources.

Cell isolation. Renal papillary collecting tubule (RPCT) cells

 

were isolated from rabbit papillae as described in Chapter III. The
final cell pellet obtained was resuspended in DMEM containing 10% fetal
bovine serum and antibiotic-antimycotic (1/60) and an aliquot taken for
counting. Typically the RPCT cells were seeded in 24-well cluster
culture dishes (16 mm, well diameter) at a density of 6-9 x 104 cells

per well and grown at 37°C under a 10% C02 atmosphere.

95

 

96

Incubation of cells with effectors. Treatment of RPCT cells with
effectors was done in triplicate or quadruplicate using 24-well culture
dishes. Cells were first rinsed free of media with Krebs buffer
(composition, in MM: 118 NaCl, 25 NaHCO3, 14 glucose, 4.7 KCl, 2.5
CaClz, 1.8 M9504 and 1.8 KH2P04), pH 7.3 with or without IBMX
depending on the experiment being done. Effectors were then added to
the cells in Krebs buffer. In experiments in which intracellular cyclic
AMP was measured, 0.3 ml of Krebs buffer (1 effectors) was added to each
well for the desired time intervals. For iPGEz assays, 0.3-1.0 ml
Krebs buffer 1 effectors was added to each well and 0.03-0.10 ml was
removed for radioimmunoassay at the desired time intervals. Cell
protein was assayed by the Lowry procedure following the solubilization
of cell protein by adding 0.20 ml of 0.1% sodium dodecyl sulfate to the
wells and incubation at 37°C for 10 min. Bovine serum albunin was used

as the protein standard.

iPGEZ radioimmunoassay. Immunoreactive PGEZ, iPGEz, was

 

measured using anti-PGEZ antibody with an assay yielding a sensitivity
range of 10-500 pg for PGEZ. The large amount of iPGEz synthesized

by RPCT cells relative to other prostaglandins allowed us to measure
iPGEz directly without chromatographic separation from the other
prostaglandins present. The iPGEz radioimmunoassay employed a single
antibody to bind iPGE2 and dextran T-70 coated charcoal (25 mg

dextran, 250 mg charcoal in 25 ml phosphate buffered saline, pH 7.2) to
precipitate any unbound iPGEz. All the effectors used were checked to

detenmine if they had an independent effect on the radioimmunoassay.

97

3',5'-cyclic AMP radioimmunoassay. Measurements of intracellular
cAMP were performed as follows. At the appropriate times after addition
of the effectors, the incubation media was aspirated. 0.35 ml of
ice-cold 6% trichloracetic acid (TCA) was added to each well and the
entire culture dish frozen in a -70°C freezer. After 15 min the dish
was thawed, [3H]3',5'-cyclic AMP (5000 dpm) added to each well and the
samples incubated at 4°C for 2 h. The TCA solution was then removed
from the wells and extracted 4 times with 5 ml portions of
water-saturated diethyl ether. The residual ether was removed by
placing the samples in a 65°C water bath for 15 min and then the samples
were lyophilized. The lyophilized residue was redissolved in 0.15-0.50
ml 0.05 M_sodium acetate, pH 6.2. An aliquot of this sample was used to
determine [3HJCAMP recovery (typically 80-90%) and another aliquot
used for radioimmunoassay. 1251 cpm from the radioimmunoassay were
counted on a Beckman gamma counter in order to exclude interfering

[3HJ-cAMP cpm.

Stirring_experiments. To make measurements on cells that had been

 

stirred several modifications were necessary. 1 ml of RPCT cells in
DMEM containing 10% fetal bovine serum were seeded at a density of 6-9 x
104 cells per 35 mm culture dish. The culture dishes were tilted at

an angle such that the RPCT cells only adhered to approximately 35-40%
of the surface area of the dish. Prior to treatment of the RPCT cells
with effectors the cells were rinsed free of media as described above.
Effectors were then added to the cells in 1 ml of Krebs buffer and the
buffer mixed vigorously by one of two methods. In the first method the

buffer was pipetted up and down continually for the entire time interval

 

98

of the experiment. In the second method a small stir bar (15 mm x 1.5
mm) was used to stir the buffer. Extreme care must be taken to regulate
the speed of stirring to ensure the stir bar remains on the portion of
the culture dish not containing cells. Any cells that contact the stir
bar are immediately removed from the surface of the culture dish. After
mixing by either method, the Krebs buffer was removed and 1 ml of 6% TCA
was added to the dish. The measurement of cAMP was then done as
described above except that one additional extraction with diethyl ether

was done.

Statistical analyses. Experiments involved using 24-well culture

 

dishes in which 3 or 4 replicates and thus 8 or 6 treatments were
analyzed for any effects. A completely random analysis of variance was
used to test for differences between sample means at p < 0.05 (129).
Dunnett's test was used for comparing differences between the effector

means and the control mean.

RESULTS

The Effect of Prostaglandins on Intracellular cAMP Concentrations

 

in RPCT Cells. Fig. 18 illustrates the dose response curves for the

 

effects of PGIZ, PGEZ, P002 and PGan on intracellular cAMP

levels in RPCT cells incubated in the presence of 10'4 M IBMX. With
all effectors tested, the maximal increase in intracellular cAMP
concentrations occurred at 10 min. In the absence of phosphodiesterase
inhibitors no changes in cAMP levels were observed. P012 and PGE2
caused 3.5- and 2-fold increases in cAMP, respectively. Half-maximal
increases in cAMP concentrations occurred at approximately 5 x 10"6

_M for both P012 and PGE2. Neither P002 nor PGFZa altered

intracellular cAMP concentrations. The effects of PGIZ and PGE2 at
10'5 M were additive, suggesting that two distinct receptor sites

exist in RPCT cells. Pretreatment of RPCT cells with aspirin (1 mM for
1.5 h) did not change the relative increase in intracellular cAMP
synthesis observed with either PGE2 or P012; however, with aspirin

the levels of cAMP were depressed approximately 30% in both control and
treated cells presumeably due to suppression of endogenous prostaglandin

synthesis.

The Effect of Bradykinin on Intracellular cAMP Levels in RPCT

 

Cells. Fig. 19 illustrates the time courses for the increases in

intracellular cAMP levels caused by the additions of PGE2 (10'5 M

99

100

Figure 18. Dependence of intracellular cAMP formation on
prostaglandin concentration. Incubations were
performed for 10 min at 37°C and cAMP assayed as
described in the text.

101

 

 

 

Io'

 

 

.5
I

| '6
[EFFECTOFQ (M)

 

Io"?

 

l

 

I
,O
0
¢

I l
O O
.9- 0
("mead nos I'm/nit" ovseiow»
dWVO HV‘In‘I'IBOVL-ILNI

102

Figure 19. Time course for PGEZ (10‘5 M and 10-9 M) and
bradykinin (10-7 M) stimulated intracellular cAMP
formation by RPCT cells. cAMP was assayed as
described in the text.

 

103

 

 

 

i
’2»
LIJ
E
l ,1
g .2
is: '9
\ 1i“
? 0.
g \
N
8
0.

;/\/

 

 

l
o
9

200 -

(ulelon "so fill/salon”)

dWV° HV'lflTlEOVHlNI

60

4O

20

TIME (min)

 

104

or 10-9 M) or bradykinin (10'7 M). In all cases the peak

intracellular cAMP concentration occured 10 min after the addition of
effectors. The effect of bradykinin on cAMP accumulation was completely
inhibited by pretreatment of the RPCT cells with aspirin (1 mM per 1.5
h). In the absence of aspirin, the concentration of iPGEZ present in
the media following a 10 min incubation with bradykinin was typically
10'9 M_or two orders of magnitude below the lowest concentration

of exogenous PGEZ or P012 that would stimulate cAMP synthesis.

Moreover, a ten-fold dilution of the media surrounding the cells (so the
extracellular concentration of PGE2 was 10'10 M in response to
bradykinin) did not affect the ability of bradykinin to increase
intracellular cAMP levels two fold.

To investigate the basis for the apparent concentration
differential between the effects on cAMP of prostaglandins synthesized
endogenously and prostaglandins added exogenously, we performed the
following experiment. RPCT cells were first seeded on culture dishes
lying at an angle so that the cells adhered to only one side of each
dish. This allowed the media to be mixed with a stir bar situated on
the opposite side of the dish without disrupting the cells mechanically.
Different dishes containing RPCT cells were then incubated with the
desired effector (PGEZ (10'5 M) or bradykinin (lo-7 M)) in the
presence and absence of stirring. As shown in Fig. 20, stirring
eliminated the increase in cAMP concentrations caused by bradykinin but
not by exogenous PGEZ. These results imply that in the presence of
bradykinin and in the absence of stirring, prostaglandins fonned by RPCT
cells can accumulate at locally high concentrations in extracellular

spaces near the cell membranes and that by increasing the rate of

105

Figure 20. Effect of stirring on intracellular cAMP formation
caused by PGEZ (lo-5 M) or bradykinin (10-7
.M). cAMP was assayed after incubation for 10 min at
25°C as described in the text.

106

 

 

 

(Daniel
(W bOIDlB

(WLQDMS

(pants)

(ngllz39d

 

. (ngll239d

(mulls)
‘IOELLNOO

'lOtLLNOO

 

l I

l l
0 0
§ 9.
(Ulalmd “93 bd/Ulw ovsaiow»
dWVO uv‘ln'rlaovaml

 

107

movement of newly formed prostaglandins away from the cell surface by
stirring, the effects of endogenous prostaglandins on cAMP accumulation

are abolished.

Effect of PGE2 on AVP Stimulated Intracellular cAMP Accumulation.

 

Fig. 21 illustrates the time courses for the effects of PGE2

(10'5 M), AVP (10-9 M) and AVP plus PGE on the accumulation of

cAMP in RPCT cells. At 5, 10 and 20 min the amounts of intracellular
cAMP observed in response to AVP or AVP plus PGEZ are not

significantly different. Since PGEZ alone causes a 2-fold increase in
intracellular cAMP concentrations, the results indicate that the effects
of AVP and PGE2 are not additive. Results similar to those shown

in Fig. 4 were also obtained when 10"7 M AVP was substituted for

10'9 M AVP and when bradykinin (10‘7 M) was substituted for

PGE2 (lo-5 M).

Fig. 22 illustrates the effect of AVP, PGEZ and bradykinin on
intracellular cAMP levels in RPCT cells 10 min after the addition of the
effectors. With either AVP plus PGEZ or AVP plus bradykinin, the
observed levels of intracellular cAMP are significantly lower (40-50%)
than the levels expected based on the calculated additivity of the cAMP
responses due to PGEZ or bradykinin plus AVP. At concentrations of
PGE2 less than 10'7 M, no effect on AVP stimulated cAMP synthesis
could be detected. The inhibition of AVP stimulated cAMP synthesis by
bradykinin was eliminated by pretreatment of RPCT cells with aspirin (1
mM for 1.5 h). P012 also inhibits AVP-stimulated cAMP synthesis by

RPCT cells in a manner similar to that observed with PGEZ.

 

 

108

Figure 21. Time course of the effect of 10"5 M PGEZ on
intracellular cAMP synthesis stimuTated by AVP (10"9
.M). cAMP was assayed as described in the text.

 

1505 msE
oe

 

109

 

i. 1 01w 1

Ergmuoas .

1298124/

12%_1~moe+1zoo_1o><\ .

  

 

00m

 

 

(uleimd "as brl/saiouu)

dWV3 WTIBOVHLNI

110

Figure 22. Effect of AVP (10-7 M), Poi-:2 (10-5 M), and
bradykinin (10"7 M) on intracellular cAMP
synthesis by RPCT cells. Incubations were performed
for 10 min at 37°C and cAMP assayed as described in

the text.

'omed
»d in

111

 

 

lJ

alt-

Cl> I l l
p, 8
(almond llao btvulw ovsalouu)
dwva uv1n'l‘laovaml NI slow-lo

 

M09)
)3 MN

>18+dAV
dAV

>18

calm)
+dAV

339a + dAv
dAV
339d

“ICELLNOO

DISCUSSION

A model illustrating the interrelationships of prostaglandins,
bradykinin, cAMP and AVP in RPCT cells is illustrated in Fig. 23.
Bradykinin, but not AVP, caused iPGEZ biosynthesis by RPCT cells.
Receptors for PGEZ on RPCT cells are envisioned to be present both
exogenously and on the serosal surface of RPCT cells. Stokes observed
that only PGEZ added to the serosal surface of cortical collecting
tubules caused natriuresis; lumenal PGEZ had no effect (37). In
addition, our data suggests that PGEZ synthesized due to bradykinin
exits the cell and acts exogenously. While no known functions of
prostaglandins synthesized due to bradykinin currently exist in RPCT
cells, two potential functions are to mediate bradykinin—induced
natriuresis and diuresis.

The reported natriuretic effect of bradykinin in the kidney is
probably due to an effect on renal hemodynamics and an effect on tubular
sodium transport (37,56,104). Stokes and Kokko have shown that
exogenous PGEZ inhibits sodium backflux, but not sodium efflux in the
perfused rabbit cortical collecting tubule (37). Bradykinin-stimulated
PGE2 formation may cause a similar effect in the papillary collecting
tubule. The means by which PGEZ causes natriuresis in the cortical
collecting tubule, however, is unknown. cAMP synthesized due to PGE2

by RPCT cells may be involved in this process. PGEZ caused

112

 

 

113

Figure 23. Model illustrating the effects of prostaglandins,
bradykinin (BK), cAMP and AVP in RPCT cells.

114

URINE BLOOD
\ COLLECTING weuua CELL

GOMPARTMENT all

 

ANTIDIURESIS <——-— cAW<

COMPARTMENT 1"2

 

Pol-i2 <——zo:4 <———— BK

 

PGEZ % 9PGEz

NATRlLRESlS 7 <CAMPM Q _
(HC

 

 

 

115

natriuresis in the cortical collecting tubule at the same concentration
at which PGE2 caused cAMP synthesis by RPCT cells.

Bradykinin has also been shown to cause diuresis in the perfused
dog kidney (89). Inhibition of prostaglandin synthesis with
indomethacin abolishes bradykinin-induced diuresis (96) implying
prostaglandins mediate bradykinin-induced diuresis somewhere in the
kidney and as a possible corollary that prostaglandins synthesized due
to bradykinin may inhibit anti-diuresis caused by AVP. In fact when
bradykinin and AVP are simultaneously infused in the dog,
bradykinin-induced diuresis is not inhibited (105,106). One role of
PGE2 synthesized due to bradykinin by RPCT cells thus may be to
inhibit AVP—stimulated cAMP synthesis and thereby inhibit water
resorption and enhance diuresis.

The interaction between AVP and prostaglandins in collecting
tubules remains confused. Our data suggests that PGEZ may cause a
decrease in AVP-stimulated cAMP biosynthesis. However, our data also
indicates that AVP and AVP plus PGEZ cause an equivalent amount of
total intracellular cAMP biosynthesis by RPCT cells. In order for water
resorption to be inhibited by PGEZ, cAMP biosynthesis must ostensibly
decrease.

One speculative explanation for how this could occur would be that
two compartments of cAMP biosynthesis existed in RPCT cells. In a two
compartment model total intracellular cAMP could be unchanged, but cAMP
synthesis could decrease in compartment 1 (which controls water
resorption) while increasing in compartment 2 in the presence of AVP
plus PGEZ. The possibility that PGEZ could inhibit AVP-stimulated

water resorption independently of an inhibition of cAMP synthesis is

116

unlikely in lieu of the work of Grantham t al. Using perfused rabbit

cortical collecting tubules, Grantham §£._l- showed that PGEl

inhibited AVP-stimulated water resorption, but not cAMP-stimulated water
resorption (61). This data indicated that PGEl inhibited water
resorption by inhibiting cAMP biosynthesis. Neither Grantham nor Dorisa
found any evidence suggesting PGEl or PGEZ, respectively, could
stimulate phosphodiesterase in the collecting tubule (61,74).

There remains, however, several discrepancies between our data and
Grantham's data. Grantham observed that PGE1 inhibited AVP-stimulated
water resorption at 10"9 M. We did not observe an inhibition of AVP
induced cAMP biosynthesis below 10-6 M PGEZ. Additionally,

Grantham used PGEl and not PGEZ and he used cortical collecting

tubules and not papillary collecting tubules. Either of these
differences could be responsible for the difference in the sensitivities
in these two systems. The isolation and culturing of RPCT cells may
have caused a loss of sensitivity by these cells.

In order to resolve if the non-additive response of AVP plus PGEZ
is due to the inhibition of AVP-induced cAMP biosynthesis several
experiments could be done. First, one could measure AVP-stimulated
adenylate cyclase activity in plasma membrane fractions from RPCT cells
and determine if PGEZ or PGEl can inhibit this activity directly.
Secondly, if one could find an effector which could cause an additive
effect with AVP on cAMP biosynthesis in RPCT cells, the non-additive
effect of AVP plus PGEZ would appear to be specific. And lastly, one
could try to find PGEZ analogues which do not affect cAMP biosynthesis

alone, but which do inhibit AVP—stimulated cAMP biosynthesis.

117

Additionally, PGEl should be tested to determine if it affects RPCT

cells at a lower concentration than PGEZ.

 

SUMMARY DISCUSSION

A number of important points have been presented in the previous

chapters. A brief synopsis of these observations is presented below.

Isolation of RPCT cells. A convenient procedure for isolating

 

homogeneous populations (97%) of RPCT cells was developed. The cells
obtained were shown to be derived from the collecting tubule by a series
of morphological, histochemical and biochemical tests. RPCT cells
isolated by this new procedure provide a reasonable model in which to
study prostaglandin metabolism in collecting tubule cells. In addition,
the cell yields obtained (107 cells/9 Papillae) allow one to make
biochemical measurements with RPCT cells which cannot be made with

isolated tubule segments.

RPCT cells biosynthesize cAMP in response to AVP and parathyroid
hormone. The cyclic nucleotide theory of AVP-induced water resorption
predicts that AVP-stimulated cAMP synthesis precedes water resorption.
RPCT cells, as expected, synthesized cAMP in response to AVP. The
half-maximal response to AVP occured at 10'10 M which was
consistent with previously reported values for AVP-induced cAMP

synthesis and water resorption in collecting tubules.

118

119

Parathyroid hormone was also shown to elevate extracellular cAMP
levels which has not been previously reported in papillary collecting

tubules.

RPCT cells can synthesize 6-keto-PGFlg. The capacity of RPCT

 

cells to synthesize 6-keto-PGFla was the first indication that the
kidney medulla can synthesize PGIZ. RPCT cells in culture, however,
synthesized predominantly PGEZ (70-75%) under conditions of basal and

stimulated prostaglandin biosynthesis.

Bradykinin, but not AVE, caused iPGEZbiosynthesis by RPCT cells.

 

The current model of collecting tubule function predicts that AVP would
cause prostaglandin biosynthesis in collecting tubule cells. AVP,
however, did not induce prostaglandin biosynthesis by RPCT cells. The
only important physiological stimulator of prostaglandin biosynthesis
found was bradykinin. Bradykinin caused half-maximal increase in
prostaglandin biosynthesis at 10‘11 M which was three orders of
magnitude below the values reported to cause prostaglandin synthesis by
both 3T3 cells and vascular endothelial cells. Because PGE2 causes
natriuresis in the isolated cortical collecting tubule and bradykinin
causes natriuresis in the kidney, one could speculate that bradykinin
induced PGEZ biosynthesis may cause natriuresis in the papillary

collecting tubule.

PGEZ and PGIz stimulated cAMP biosynthesis by RPCT cells.
PGEZ and P012, but not PGDZ and PGan, caused cAMP

biosynthesis by RPCT cells. The responses to PGE2 and PGIZ were

120

additive suggesting that two separate receptors exist for PGEZ and

P012. PGEZ and P012 may also have distinct functions in RPCT

cells.

Prostaglandins cause cAMP biosynthesis through exogenous cell
receptors. Bradykinin induced prostaglandin biosynthesis caused cAMP
biosynthesis by RPCT cells. These prostaglandins can exit the cell and
cause cAMP biosynthesis by interaction with exogenous cell receptors.
The maintenance of the extracellular concentrations of PGE2 (10'5-
10'6) necessary to cause cAMP biosynthesis is probably achieved by
sequestering endogenously synthesized prostaglandins is an unstirred

layer around the RPCT cells.

PGE;_inhibition of AVP-stimulated cAMP biosynthesis. The

 

observed biosynthesis of cAMP by RPCT in reSponse to AVP plus PGE2 is
approximately 40% less than the calculated amount of biosynthesis
expected based on the assumption that the responses to AVP and PGE2
along should be additive. This non-additive response, however, did not
occur at a concentration below 10'6.fl PGEZ. 10"6 M PGEZ is

three orders of magnitude greater than level of PGE1 which inhibited
AVP-stimulated water resporption in the cortical collecting tubule.
This discrepancy in sensitivity in these systems remains to be

determined.

 

 

COPYRIGHT ACKNOWLEDGEMENTS

I would like to thank Prostaglandins (Gero-X, Inc.) and The
American Journal of Physiology for their permission to reproduce
segments of the publications by Frank C. Grenier and William L. Smith
(1978) Prostaglandins 16(5), 759-772 and Frank C. Grenier, Thomas E.
Rollins and William L. Smith (1981) Am. J. Physiol. 24; F1360-F1370.

121

 

10.
11.

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