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This is to certify that the

dissertation entitled

Serotonergic regulation of growth hormone secretion
in cattle.

presented by
Roy Patrick Radcliff

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Animal Science

 

 

   

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MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 c:/CIRC/DateDue.p65-p 15

 

SEROTONERGIC REGULATION OF GROWTH HORMONE SECRETION IN
CATTLE

By
Roy Patrick Radcliff

A DISSERTATION
Submitted to
Michigan State University
In partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Animal Science

2001

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ABSTRACT

SEROTONERGIC REGULATION OF GROWTH HORMONE SECRETION IN
CATTLE

By

Roy Patrick Radcliff

Growth hormone is required for postnatal growth and galactopoiesis in
ruminants. Secretion of growth hormone is controlled by hypothalamic
hormones, which in turn are controlled by neurotransmitters, one of which is
serotonin. Activation of serotonin receptors with quipazine induces secretion of
growth hormone in cattle. However, the mechanism of quipazine-induced
secretion of growth hormone is not known. My objective was to investigate the
mechanism of quipazine-induced secretion of growth hormone in cattle. I
hypothesized that thyrotropin releasing-homone (T RH) mediates serotonin-
induced secretion of growth hormone in cattle.

Using an in vitro perifusion culture system, | determined that quipazine did
not induce secretion of growth hormone directly from anterior pituitary cells, nor
were posterior pituitary cells mediating the effects of quipazine on secretion of
growth hormone. Therefore, the effects of quipazine on secretion of growth
hormone in vivo are not directly on the pituitary gland. Wl‘lh the perifusion culture
system, I also determined that quipazine did not induce secretion of growth
hormone releasing-hormone (GHRH) or somatostatin (SS) from bovine
hypothalamic slices. These data indicate that quipazine-induced secretion of

growth hormone in vivo is not mediated by GHRH or SS.

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I used a meal-fed steer model to investigate the possibility of two other
hypothalamic peptides as mediators of quipazine-induced secretion of growth
hormone. Quipazine induced secretion of growth hormone to similar magnitudes
before versus after feeding. Pituitary adenylate cyclase-activating polypeptide
(PACAP) induced secretion of growth hormone to greater magnitude before
versus after feeding, indicating that PACAP probably does not mediate
quipazine-induced secretion of growth hormone. Thyrotropin releasing-hormone
induced secretion of growth hormone to similar magnitudes before versus after
feeding, indicating that TRH might mediate quipazine induced secretion of growth
honnone.

I injected meal-fed steers with 3 or 6 pg of 3,3’,5-triiodo-L-thyronine (T3)
daily to increase negative feedback on the thyrotropic axis and down regulate
TRH receptor expression on the pituitary gland and/or TRH synthesis and
secretion from the hypothalamus. Injections of T3 decreased endogenous
secretion of 3,3’,5, 5’-tetraiodo-L-thyronine without affecting basal secretion of
growth hormone. Injections of T3 did not affect GHRH- or TRH-induced secretion
of growth hormone. These data indicate that injections of T3 did not affect
releasable amounts of growth hormone or TRH receptor expression on the
pituitary gland. However, daily injections of T3 inhibited but did not completely
block quipazine-induced secretion of growth hormone.

In conclusion, TRH at least partially mediates serotonin-induced secretion

of growth hormone in cattle.

This dissertation is dedicated to
James Edward “Uncle Ed” Norman.
A man who has always been, and will continue to be, an inspiration to me.

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ACKNOWLEDGEMENTS

I would like to express my deepest and most sincere gratitude to my major
professor, Dr. H. Allen Tucker. His patient guidance and strong commitment to
graduate education is something I hope to pass on to the next generations of
scientists. l was extremely fortunate to have such a wise and outstanding mentor
throughout my entire graduate career. From him, I not only learned the lessons
of science that will help me achieve my career goals, but also some lessons in
life that will enable me to achieve my life goals. Thank you.

I’d also like to thank all the members of my guidance committee: Dr
Donald Jump, Dr. Keith Lookingland, Dr. Dale Romsos, and Dr. Mike
VanderHaar. Each member of my committee has taught me something either in
the classroom or through discussions of my research and results.

I would also like to thank all the past and present members of the Animal
Reproduction Laboratory. In particular Dr. Roy Fogwell, Dr. Christopher
McMahon and Larry Chapin, l have enjoyed the times we have spent together in
work and play and it has been it has been a honor to get to know you and your
families.

I would also like to thank Sheldon’s Packing and Bellingar Packing for
their generosity in providing tissue for my in vitro work as well as their patience
with me when I disrupted their routines in the slaughter houses.

I would me amiss if I did not thank all the graduate students and friends in
the Department of Animal Science. Many have helped me with experiments

throughout my graduate carreer. In particular Mr. Kent Tjardes, Mr. Mike Peters,

 

Mr. i m F
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Mr. Tom Pilbeam and Dr. Ted Ferris. Our weekday and weekend excursions
were very beneficial as stress management tools.

Finally, I would like to thank my family, especially my parents, Jack and
Joan Radcliff. If every child had the love and support that I have had in life, I am

sure the world would be a better place.

vi

 

 

 

 

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TABLE OF CONTENTS

LIST OF TABLES ................................................................................................. x
LIST OF FIGURES .............................................................................................. xi
INTRODUCTION .................................................................................................. 1
REVIEW OF LITERATURE .................................................................................. 4
l Characterization of Growth Hormone ....................................................... 4
II Hypothalamic Regulation of Growth Hormone Secretion ........................ 5
A. Effects of GHRH and SS on Growth Hormone Synthesis and
Secretion ......................................................................................... 5
8. Discovery, Characterization, and Localization of TRH ..................... 8
C. Characterization and Signal Transduction Mechanism of TRH
Receptors ...................................................................................... 1 1
D. Discovery, Characterization, and Localization of PACAP .............. 13
E. Characterization and Signal Transduction Mechanism of PACAP
Receptors ...................................................................................... 1 5
III Serotonergic Receptor Regulation of Growth Hormone Secretion ....... 17
A. Characterization of Serotonergic Innervation of the Hypothalamus
...................................................................................................... 17
B. Characterization, Localization and Signal Transduction Mechanism
of Serotonin Receptors .................................................................. 19

C. Serotonergic Receptor Regulation of Growth Hormone Secretion 23

CHAPTER 1: Serotonin Induced Secretion of Growth Hormone ................. 26
INTRODUCTION ...................................................................................... 27
MATERIALS AND METHODS ................................................................. 28

Perifusion of Bovine Anterior and Posterior Pituitary Cells ................. 28
Experimental Protocol ......................................................................... 31
Statistical Analysis .............................................................................. 32
RESULTS ................................................................................................. 33
Effect of Quipazine on Secretion of Growth Hormone from Dispersed
Pituitary Cells ................................................................................ 33
DISCUSSION ........................................................................................... 35

vii

CHAPTER 2: Serotonin Induced Secretion of GHRH and SS ....................... 38

INTRODUCTION ...................................................................................... 39
MATERIALS AND METHODS ................................................................. 4O
Perifusion of Bovine Hypothalamic Slices ........................................... 40
Experimental Protocols ....................................................................... 42
Statistical Analysis .............................................................................. 43
RESULTS ................................................................................................. 44
Effect of Quipazine on Secretion of GHRH and SS from bovine
Hypothalamic Slices ...................................................................... 44
DISCUSSION ........................................................................................... 47
CHAPTER 3: Role of TRH and PACAP in Serotonergic Regulation of
Growth Hormone Secretion ............................................................................. 49
INTRODUCTION ...................................................................................... 50
MATERIALS AND METHODS ................................................................. 53
Management of Animals ..................................................................... 53
Blood Sample Collection and Processing ........................................... 54
Experimental Protocols ....................................................................... 54
Statistical Analysis .............................................................................. 57
RESULTS ................................................................................................. 58
Effect of Quipazine Before and After Feeding on Secretion of Growth
Hormone (Experiment 1) ............................................................... 58
Effect of Various Doses of TRH on Secretion of Growth Hormone
(Experiment 2) ............................................................................... 60
Effect of TRH Before or After Feeding on Secretion of Growth
Hormone (Experiment 3) ............................................................... 60
Effect of Various Doses of PACAP on Secretion of Growth Hormone
(Experiment 4) ............................................................................... 64
Effect of PACAP Before and After Feeding on Secretion of Growth
Hormone (Experiment 5) ............................................................... 68
DISCUSSION ........................................................................................... 68
CHAPTER 4: Serotonin-Induced Secretion of Growth Hormone in
Hyperthyroid Meal-fed Steers .......................................................................... 76
INTRODUCTION ...................................................................................... 77
MATERIALS AND METHODS ................................................................. 80
Management of Animals ..................................................................... 80
Blood Sample Collection and Processing ........................................... 80
Experimental Protocols ....................................................................... 81
Statistical Analysis .............................................................................. 84
RESULTS ................................................................................................. 85
Effects of T3 on basal, GHRH-, TRH- and Quipazine-Induced Secretion
of Growth Hormone and Total T4 ................................................... 85

viii

 

DISC
SUMMARY
IMPUCATI

BIBLIOGRI

 

 

DISCUSSION ......................................................................................... 100

SUMMARY AND CONCLUSIONS ................................................................... 107
IMPLICATIONS ................................................................................................ 1 13
BIBLIOGRAPHY .............................................................................................. 1 16

 

Table 1.

 

LIST OF TABLES

Table 1. Treatment assignments for Experiment 1. ....................................... 33

 

Figure 1

Figure 2.

 

LIST OF FIGURES

Figure 1. Proposed model for direct action of serotonin on the bovine

Figure 2.

pituitary gland. Serotonergic neurons (oval) project axons to the
pituitary stalk with terminals juxtaposed to portal vessels which
transport it to the pituitary gland. Serotonin acts at the posterior
pituitary gland (PP) to induce secretion of growth hormone (GH) from
the anterior pituitary gland (AP). ........................................................ 29

Effect of quipazine on secretion of growth hormone from
dispersed bovine pituitary cells. TOP PANEL; Data presented are
least squares mean concentrations (:l: pooled standard error of mean;
SEM) of growth hormone in medium effluent from dispersed anterior
pituitary gland cells perifused with vehicle medium (AP-vehicle; n=6)
or quipazine (AP-Quipazine; n=6), and the combination of anterior and
posterior pituitary gland cells perifused with vehicle medium (AP/PP-
vehicle; n=6) or quipazine (AP/PP-Quipazine; n=6). Treatments were
imposed from 61 to 80 min (indicated by box on X axis). All chambers
were exposed to 60 mM KCI from 181 to 200 min (indicated by box on
X axis) to test somatotrope viability. BOTTOM PANEL; Statistical
analysis was performed on least squares mean AUC (:I: pooled SEM)
for growth hormone calculated from 60 to 120 min and adjusted for
pre-treatment concentrations using concentration of growth hormone
at 60 min as a covariate. Least squares means were not different from
each other. ........................................................................................ 34

Figure 3 Proposed model for GHRH and SS mediating serotonin-induced

secretion of growth hormone in cattle. Serotonergic neurons (oval)
project axons to hypothalamus that terminate on GHRH and SS
neurons (ovals). Terminals of GHRH and SS neurons are juxtaposed
to portal vessels which transport them to the anterior pituitary gland
(AP) where they stimulate and inhibit secretion of growth hormone,
respectively. ...................................................................................... 41

xi

Figure 4

Figure 5.

Figure 4.

Figure 5.

Effect of quipazine on secretion of GHRH from bovine
hypothalamic slices. TOP PANEL; Data presented are least
squares mean concentrations (:l: pooled standard error of mean; SEM)
of GHRH in medium effluent from bovine hypothalamic slices
perifused with control medium (control; n=8), 10"5 M quipazine (n=8),
10.3 M quipazine (n=8), or 10"° M quipazine (n=8). Treatments were
imposed from 61 to 80 min (indicated by the box on the X axis) and
slices were exposed to 60 mM KCI from 181 to 200 min (indicated by
the box on the X axis) to test tissue viability. BOTTOM PANEL;
Statistical analysis was performed on least squares mean AUC (:I:
pooled SEM) for GHRH calculated from 61 to 120 min and adjusted
for pre-treatment concentrations using the concentration of GHRH at
60 min as a covariate. Quipazine at 10"° M induced a greater AUC
than the AUC of controls (P = 0.02) ................................................... 45

Effect of quipazine on secretion of SS from bovine hypothalamic
slices. TOP PANEL; Data presented are least squares mean
concentrations (1: pooled standard error of mean; SEM) of SS in
medium effluent from bovine hypothalamic slices perifused with
vehicle medium (control; n=8), 1043 M quipazine (n=8), 10"3 M
quipazine (n=8), or 10’10 M quipazine (n=8). Treatments were
imposed from 61 to 80 min (indicated by the box on the X axis) and
slices were exposed to 60 mM KCI from 181 to 200 min (indicated by
the box on the X axis) to test tissue viability. BOTTOM PANEL;
Statistical analysis was performed on least squares mean AUC (:l:
pooled SEM) for SS calculated from 61 to 120 min and adjusted for
pre-treatment concentrations using the concentration of SS at 60 min
as a covariate. Least squares means were not different from controls.
.......................................................................................................... 46

Figure 6. Proposed model for TRH and PACAP mediating serotonin-

induced secretion of growth hormone in cattle. Serotonergic
neurons (oval) project axons to hypothalamus that terminate on TRH
and PACAP neurons (ovals). Terminals of TRH and PACAP neurons
are juxtaposed to portal vessels which transport them to the anterior
pituitary gland (AP) where they stimulate secretion of growth
hormone. ........................................................................................... 52

xii

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Figure 7

 

Figure 7. Effect of quipazine on secretion of GH in meal-fed steers. TOP
PANEL; Data presented are mean concentrations (1 standard error of
mean; SEM) of growth hormone in serum of steers injected with 0.2
mg serotonin receptor agonist (quipazine)lkg BW either 1 h before
(n=8) or 1 h after (n=8) feeding. Arrows indicate times of injection.
BOTTOM PANEL; Statistical analysis was performed on least
squares mean AUC (:l: pooled SEM) calculated over 60 min from time
of injection and adjusted by covariance using concentrations of growth
hormone in serum immediately before injection of quipazine. Least
squares mean AUC before versus after feeding was not different (P =
0.7). ................................................................................................... 59

Figure 8. Effect of quipazine on peak concentrations of GH in meal-fed
steers. Data presented are least squares mean peak concentration (:I:
pooled standard error of mean) of growth hormone in serum of steers
injected with 0.2 mg serotonin receptor agonist (quipazine)lkg BW
either 1 h before (n=8) or 1 h after (n=8) feeding. Peak concentration
of growth hormone following injection of quipazine was not different
before versus after feeding (P = 0.55). .............................................. 61

Figure 9. Effect of various doses of TRH on secretion of GH in meal-fed
steers. TOP PANEL; Data presented are mean concentrations (i
standard error of mean, SEM) of growth hormone in serum of steers
injected with saline (control; n=6) or TRH (0.1, 0.3, 1, 3 or 10 uglkg
BW; n=6 per dose). Arrow indicates time of injection. BOTTOM
PANEL; Statistical analysis was performed on least squares mean
AUC (i pooled SEM;) calculated over 60 min from time of injection
and adjusted by covariance using concentrations of growth hormone
in serum immediately before injection. Least squares means with *
differ from control (P < 0.02). ............................................................. 62

Figure 10. Effect of various doses of TRH on peak concentrations of GH in
meal-fed steers. Data presented are least squares mean peak
concentrations (i pooled standard error of mean) of growth hormone
in serum of steers after injection of saline (control; n=6) or TRH (0.1,
0.3, 1, 3 or 10 jig/kg BW; n=6 per dose). Least squares mean with *
differs from control (P < 0.01). ........................................................... 63

xiii

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Figure 11. Effect of TRH on secretion of GH before and after feeding in
meal-fed steers. TOP PANEL; Data presented are mean
concentrations (:l: standard error of mean; SEM) of growth hormone in
serum of steers injected with 0.3 pg TRH/kg BW either 1 h before
(n=16) or 1 h alter (n=16) feeding. Arrows depict times of injection.
BOTTOM PANEL; Statistical analysis was performed on least
squares means AUC transformed by natural logarithm (i pooled
SEM;GH LN-AUC;) calculated over 60 min from time of injections and
adjusted by covariance using concentrations of growth hormone in
serum immediately before injection of TRH. Least squares mean LN-
AUC before versus after feeding was not different (P = 0.22). .......... 65

Figure 12. Effect of TRH on peak concentrations of GH before and after
feeding in meal-fed steers. Data presented are least squares mean
peak concentrations (1 pooled standard error of mean) of growth
hormone in serum (transformed by natural logarithm) of steers
injected with TRH (0.3 jig/kg BW) either 1 h before (n=16) or 1 h after
(n=16) feeding. LN-Peak concentration of growth hormone following
injection of TRH before versus after feeding was not different (P =
0.85). ................................................................................................. 66

Figure 13. Effect of various doses of PACAP on secretion of GH in meal-
fed steers. TOP PANEL; Data presented are least squares mean
AUC (i pooled standard error of mean; SEM) transformed by natural
logarithm (LN-GH AUC). BOTTOM PANEL; least squares mean
peak concentrations (i pooled SEM) of growth hormone in serum
(transformed by natural logarithm; LN-Peak) of steers injected with
saline (control; n=10) or PACAP (0.1 (n=5), 0.3 (n=5), 1 (n=10), 3
(n=10) or 10 (n=10) jig/kg BW) calculated over 40 min from time of
injection and adjusted by covariance using concentrations of growth
hormone in serum immediately before injection. Least squares
means with * differ from control (P < 0.01), 1' differs from control (P <
0.1). ................................................................................................... 67

Figure 14. Effect of PACAP on secretion of GH before and after feeding in
meal-fed steers. TOP PANEL; Data presented are mean
concentrations (.+_ standard error of mean; SEM) of growth hormone in
serum of steers injected with 3 pg PACAP/kg BW either 1 h before
(n=8) or 1 h after (n=8) feeding. Arrows indicate times of injection.
BOTTOM PANEL; Statistical analysis was performed on least
squares mean AUC (:l: pooled SEM) calculated over 60 min from time
of injection and adjusted by covariance using concentrations of growth
hormone in serum immediately before injection of PACAP. Least
squares mean AUC before feeding differs from AUC after feeding (P =
0.05). ................................................................................................. 69

xiv

1““‘1‘

Figure 15

Figure 16.

 

Figure 15 Effect of PACAP on peak concentrations of GH before and after
feeding in meal fed steers. Data presented are least squares mean
peak concentration (:I: pooled standard error of mean) of growth
hormone in serum of steers injected with PACAP (3 jig/kg BW) either
1 h before (n=8) or 1 h after (n=8) feeding. Peak concentration of
growth hormone following injection of PACAP was greater before
versus after feeding (P = 0.002) ........................................................ 70

Figure 16. Model of negative feedback of 3,3’,5-triiodo-L-thyronine (T3) on
thyrotropin releasing-hormone (TRH) neurons in the
hypothalamus and the anterior pituitary gland. Injecting
exogenous T3 (bold arrow) will increase negative feedback on TRH
neurons and (or) the anterior pituitary gland thereby reducing thyroid
stimulating hormone (TSH) secretion from the anterior pituitary gland
and 3, 3’,5, 5’-tetraiodo-L—thyronine (T 4) secretion from the thyroid
gland as well as the ability of serotbnin to induce secretion of growth
hormone from the anterior pituitary gland. ......................................... 79

Figure 17. Effect of daily injections of T3 on basal secretion of GH in meal-
fed steers. Data presented are least squares mean AUC (:I: pooled
standard error of mean) of growth hormone of corn oil-injected
(control; n=4) and T3-treated (3 jig/kg BW; n=4) meal-fed steers. The
AUCs were calculated from 0800 to 1400 h on days -1, 5 and 10
relative to initiation of injections, and transformed by natural logarithm
(GH LN-AUC). Injection of T3 did not affect least square mean GH
LN-AUC on days -1, 5 or 10 (P > 0.16) ............................................. 87

Figure 18. Effect of daily injections of T3 on concentrations of total T4 in
meal-fed steers. Data presented are least squares mean
concentrations (:l: pooled standard error of mean) of total T4 in serum
of corn oil-injected and T3-treated (3 jig/kg BW) meal-fed steers on
days -1, 5 and 10 relative to initiation of injections. Compared with
corn oil-injected steers, daily injection of T3 reduced concentration of
total T4 in serum on d 5 and 10. Points with * differ P = 0.001. Points
with ** differ P = 0.0001. .................................................................... 88

Figure 19. Effect of daily injections of T3 on GHRH-induced secretion of GH
in meal-fed steers. TOP PANEL; Data presented are mean
concentrations (i standard error of mean; SEM) of growth hormone in
serum of corn oil-injected and T3-treated meal-fed steers after
intravenous injection of sterile water (H20) or GHRH dissolved in H20
(0.2 jug/kg BW) at 0900 h. Arrow indicates time of injection.

BOTTOM PANEL; Statistical analysis was performed on least
squares mean AUCs (i pooled SEM) calculated from 0900 to 1000 h.
Bars with different letters differ P < 0.001. ......................................... 90

 

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Figure 25. Effect of daily injections of T3 on quipazine-induced secretion of
GH In meal-fed steers. TOP PANEL; Data presented are mean
concentrations (i standard error of mean; SEM) of growth hormone in
serum of corn oil-injected and T3-treated meal-fed steers after
intravenous injection of 0.9% saline or a serotonin receptor agonist
(quipazine) dissolved in 0.9% saline (0.2 mg/kg BW) at 0900 h. Arrow
indicates time of injection. BOTTOM PANEL; Statistical analysis was
performed on least squares mean AUCs (:t pooled SEM) calculated
from 0900 to 1000 h. Compared with injection of saline, injection of
quipazine increased GH AUC in corn oil-injected but not T3-treated
steers. Bars with different letters differ P < 0.05. .............................. 98

Figure 26. Effect of daily injections of T3 on quipazine-induced peak
concentrations of GH in meal-fed steers. Data presented are least
squares mean peak concentrations (i pooled standard error of mean)
of growth hormone transformed by natural logarithm (LN-Peak GH) in
serum of corn oil-injected and T3-treated meal-fed steers after
intravenous injection of sterile saline or serotonin receptor agonist
(quipazine) dissolved in saline (0.3 jig/kg BW) at 0900 h. Bars with
different letters differ P < 0.01. .......................................................... 99

 

Figure 27. Effect of daily injections of T3 on quipazine-induced secretion of
T4 in meal-fed steers. Data presented are least squares mean
concentrations (i pooled standard error of mean) of total T4 in serum
of corn oil-injected and T3-treated meal-fed steers immediately before
(0900 h ) and 4 h after (1300 h) intravenous injection of sterile 0.9%
saline or a serotonin receptor agonist (quipazine) dissolved in 0.9%
saline. Injection of saline did not affect concentrations of total T4 in
serum of corn oil-injected or T3-treated steers. Injection of quipazine
did not affect concentrations of total T4 in serum of corn oil-injected or
T3-treated steers. ............................................................................. 101

Figure 28. Proposed model for serotonergic regulation of growth hormone
secretion in cattle. Ovals with connected lines represent serotonin
and thyrotropin releasing hormone (TRH) neurons. The cylinder
represents portal vessels between the hypothalamus and the anterior
pituitary gland, which is represented by the large circle. The model
depicts the neuronal pathway in which serotonin may induce secretion
of growth hormone. Serotonergic neurons stimulate TRH neurons. In
turn, TRH is secreted into portal vessels which transport it to the
anterior pituitary gland where it stimulates secretion of growth
hormone. ......................................................................................... 112

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INTRODUCTION

Animal agriculture provides nourishment to mankind through production of
meat, milk and eggs. The ultimate goal of research in animal agriculture is to
improve the production and production efficiency of animal agriculture. Through
such research, many products have been developed that benefit all aspects of
animal agriculture. For example, crops that are more nutritious have been
developed to feed both man and animals. Development of products such as
Lutalyse‘” (prostaglandin f2“) for estrus synchronization, Synovex S®
(progesterone and estradiol benzoate implants) to improve growth performance

in feedlot steers and Posilac‘” (recombinant bovine somatotropin) to improve milk

production in dairy cattle all provide options to animal industry that can be used
to improve production. Understanding the endogenous regulators of animal
growth and development has aided scientists in developing the products and
strategies that are used today to improve animal agriculture.

In cattle, growth hormone is the primary regulator of postnatal growth and
milk production (Bauman et al., 1982; Bauman, 1992). Injecting cattle with
exogenous growth hormone increases protein and decreases adipose deposition
in the carcass (Eisemann et al., 1989; Binelli et al., 1995; Radcliff et al., 1996).
Exogenous growth hormone also increases mammary development in heifers
(Radcliff et al., 1996) and milk production in cows (Bauman, 1992). Thus, growth

hormone is a valuable tool for improving production of meat and milk.

 

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Currently, exogenous growth hormone is only approved for use in lactating
dairy cattle and requires a labor force to inject the cows every 2 weeks. Even if
growth hormone was approved for use in growing animals the requirement for
labor and pharmaceutical supplies would be cost prohibitive.

An alternative to administering exogenous growth hormone, would be to
increase endogenous secretion of growth hormone. Basic research investigating
the regulation of endogenous growth hormone secretion would allow
development of other cost effective products or strategies to ultimately improve
animal growth and development. The focus of this dissertation is the regulation
of growth hormone secretion in cattle.

Secretion of growth hormone from the anterior pituitary gland seems to be
controlled mainly by growth hormone-releasing hormone (GHRH), which
stimulates, and somatostatin (SS), which inhibits secretion of growth hormone
(Frohmian et al., 1992). However, other hypothalamic peptides such as
thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating
polypeptide (PACAP) may also play a role in regulating secretion of growth
hormone in cattle (Vines et al., 1976; Vines, 1976; Vines et al., 1977; Hashizume
et al., 1994). Since GHRH, SS, TRH and PACAP are all peptides, injection or
implantation are the only routes of administration that will maintain their biological
activity. Therefore, administration of GHRH, SS, TRH or PACAP would generate
similar costs in labor and pharmaceuticals as administration of growth hormone.

However, the endogenous secretion of these peptides is ultimately controlled by

neurotransn
less labor in

Hypo
control sears
1997b). An:
controlling 55
al., 1996). I—
growth horn.-

The c
Mild) serot:
Oblet‘tlves we
Induces Secra
serotonin reg
fissUSE; and 3

 

 

neurotransmitters that are not peptides, and these compounds may offer another
less labor intensive means of manipulation.

Hypothalamic neurotransmitters such as norepinephrine and dopamine
control secretion of GHRH and SS in cattle (West at al., 1997a; West at al.,
1997b). Another neurotransmitter, serotonin, has also been implicated in
controlling secretion of growth hormone in cattle (Gaynor et al., 1995; Gaynor et
al., 1996). However, the mechanisms by which serotonin induces secretion of
growth hormone is not known.

The overall goal of this dissertation was to investigate the mechanisms by
which serotonin induces secretion of growth hormone in cattle. Specific
objectives were to determine: 1) if activation of serotonin receptors directly
induces secretion of growth hormone from bovine pituitary cells; 2) if activation of
serotonin receptors affects secretion of GHRH and SS from bovine hypothalamic
tissue; and 3) the roles of TRH and PACAP in mediating serotonin receptor

agonist-induced secretion of growth hormone in cattle.

-13

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REVIEW OF LITERATURE

l Characterization of Growth Hormone
Growth hormone is a 191 amino acid protein hormone that is synthesized
and stored in specific cells in the anterior pituitary gland called somatotropes.

Somatotropes make up approximately 50% of the cells found in the anterior

- n

pituitary gland (T homer et al., 1998). Growth hormone plays a role in stimulation
of growth as well as in a number of metabolic processes, such as hepatic IGF-I
production and stimulation of hormone sensitive lipase in adipose tissue (T homer
et al., 1998). The size and amino acid sequence of growth hormone varies
between species as well as within species. The majority of bovine growth
hormone is synthesized as a 207 amino acid precursor and proteolytically
cleaved to form the 191 amino acid protein (Woychik et al., 1982). Most variation
within a species is due to alternative splicing of the mRNA. The gene coding for
bovine growth hormone is approximately 1800 base pairs long. It is composed of
5 exons and 4 introns (Woychik et al., 1982). Variant forms of growth hormone
and their biological activity have been reported in cattle (Hampson and Rottman,
1987; Krivi et al., 1989). However, the 191 amino acid form is the most abundant
and most biologically active (Krivi et al., 1989).

Growth hormone is stored in secretory granules inside somatotropes and
released in a pulsatile pattern into peripheral circulation by exocytosis (McCann
and Ojeda, 1992). It is well established that secretion of growth hormone from

the anterior pituitary gland is controlled by two hypothalamic neuropeptides.

These neurc

of growth hC

 

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A. Effects 0

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These neuropeptides are GHRH which induces, and SS which inhibits secretion

of growth hormone (T horner et al., 1998).

ll Hypothalamic Regulation of Growth Hormone Secretion
A. Effects of GHRH and SS on Growth Hormone Synthesis and Secretion
lmmunohistochemical experiments in cattle revealed a dense population a:
of GHRH neuronal cell bodies located mainly in the arcuate nucleus of the i
hypothalamus with a smaller population located lateral and dorsal to the arcuate
nucleus within and around the ventromedial nuclei (Leshin et al., 1994). The
distribution of GHRH neurons in cattle is similar to that described in rats
(Merchenthaler et al., 1984; Bloch et al., 1983). Terminals of GHRH neurons are
located in the external zone of the median eminence (Leshin et al., 1994)
juxtaposed to portal vessels. Norepinephrine induces release of GHRH in cattle
(West at al., 1997a) which is transported via hypophysial portal blood to the
anterior pituitary gland where it binds to specific receptors on somatotropes
(Seifert et al., 1985; Velicelebi et al., 1986). Binding of GHRH to it’s receptor
activates the adenylate cyclase-cAMP-protein kinase A pathway via the
stimulatory Gs protein (Mayo et al., 1995; Wong et al., 1995). Activation of this
pathway accomplishes two things: it induces release of growth hormone from
somatotropes by increasing intracellular free calcium (Holl et al., 1988) and
induces transcription of growth hormone mRNA by phosphorylating c-AMP
response element binding protein, which binds to DNA and induces transcription

of specific genes (Barinaga et al., 1983).

Cell .

eminence a'i

   
  
 

of rats. shee
al.,1995). Ir:-
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1997C). DOp
GHRH. SS is
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bovine artistic

 

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Cell bodies of SS neurons that terminate near portal vessels of the median
eminence are located mainly in the periventricular nucleus of the hypothalamus
of rats, sheep and cattle (Frohman et al., 1992; Leshin et al., 1994; Willoughby et
al., 1995). In cattle, SS fibers and varicosities have also been reported in the
arcuate nucleus (Leshin et al., 1994) indicating that SS may play a role in
regulating secretion of GHRH. In fact, experiments with perifused bovine
hypothalamic slices show that SS inhibits secretion of GHRH (West at al.,
1997c). Dopamine induces release of SS in cattle (West et al., 1997b) and like
GHRH, SS is transported to the anterior pituitary gland via portal vessels.
Somatostatin inhibits GHRH-induced secretion of growth hormone in cultured
bovine anterior pituitary cells in a dose dependent manner, but had no effect on
basal secretion of growth hormone (Padmanabhan et al., 1987). The mRNA for
five different SS receptors (SSTR1 — SSTR5) have been identified in rodent
hypothalamus and anterior pituitary gland (Reisine et al., 1995; Beaudet and
Tannenbaum, 1995). However, SSTR2 is thought to be the major receptor
responsible for suppression of growth hormone secretion because it has greatest
expression in the anterior pituitary gland of all the SS receptor subtypes. The
ability of SSTR2 to bind specific SS analogs is positively correlated with a
particular analog’s ability to inhibit secretion of growth hormone (Reisine and
Bell, 1995). Somatostatin inhibits activation of adenylate cyclase via G; or Go
proteins thereby reducing cAMP and blocking the ability of GHRH to induce

synthesis and secretion of growth hormone (Frohman et al., 1992).

 

Grov
including ca.
et al., 1988
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Growth hormone is secreted in a pulsatile manner in all animals studied,
including cattle (Vasilatos and Wangsness, 1981; Wheaton et al., 1986; Kraicer
et al., 1988). Because GHRH stimulates and SS inhibits secretion of growth
hormone it is reasonable to speculate that release of growth hormone is
dependant on the interaction of GHRH and SS. In fact, one hypothesis is that
secretion of GHRH and SS are 180° out of phase (T annenbaum and Ling, 1984).
A pulse of growth hormone occurs when GHRH in portal blood is high and SS is
low. Conversely, circulating concentrations of growth hormone are low when SS
is high and GHRH is low in portal blood. There is one experiment conducted in
rats that lends support to this hypothesis. However, concentrations of GHRH
and SS were quantified in portal blood in one group of rats and concentrations of
growth hormone in serum in another group (Plotsky and Vale, 1985). Thus,
actually aligning pulses of growth hormone with peaks and troughs of GHRH and
SS in the same group of animals was not done. Indeed, there is evidence that
the regulation of growth hormone secretion is more complex, and this is
described below.

In sheep, only 70% of growth hormone pulses occur coincident with or
immediately following a pulse of GHRH. Furthermore, pulses of SS are not out of
phase with GHRH pulses and do not correlate with troughs in growth hormone
secretion (Frohman et al., 1990; Thomas et al., 1991). Normally, individual
patterns of growth hormone secretion are random among a group of animals
(Carlsson and Jansson, 1990; Wheaton et al., 1986). In calves, growth hormone

secretion can be synchronized among a group of animals by restricting free

access to fe
at. 1993; G
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meal-fed m0
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access to feed for a fixed 2-h period each day (Moseley et al., 1988; Gaynor et
al., 1993; Gaynor et al., 1995). In this meal-fed model, endogenous growth
hormone secretion is pulsatile and relatively high shortly before feeding,
decreases during feeding, and remains low for 2 to 3 h after feeding. Thus, the
meal-fed model allows prediction as to when growth hormone will be high or low
in a group of calves with reasonable accuracy. Using this model, McMahon and
coworkers (2000) compared the activity of GHRH and SS neurons around the
time of feeding. As concentrations of growth hormone decreased from shortly
before to shortly after feeding, activity of GHRH neurons in the arcuate nucleus
also decreased. However, activity of SS neurons in the periventricular nucleus
also decreased during this period. These data suggest that the post-feeding
suppression of growth hormone is not due to an increase in SS secretion into
portal blood.

Another hypothalamic peptide, TRH, can also induce secretion of growth
hormone from cultured bovine anterior pituitary cells (Smith and Convey, 1975).
In fact, when activity of TRH neurons was examined in meal-fed calves,
neuronal activity decreased as concentrations of growth hormone decreased
from before to after feeding (Radcliff et al., 1999). These data suggested that
hypothalamic regulation of growth hormone may involve more than just GHRH
and SS.

B. Discovery, Characterization, and Localization of TRH
It has been recognized since the early 1950’s that the hypothalamus

exerts an important influence on regulation of the pituitary-thyroid axis. However,

iwas not ur
and it’s Cheri
Burgus et at
human. res;
TRH peptide
human prep'
TRH is cons»;
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HOifelt, 1980
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it was not until 1969 that TRH was isolated from ovine and porcine hypothalami
and it’s chemical structure determined (pyroGlu-His-Pro-NHZ) (Boler et al., 1969;
Burgus et al., 1969). PreproTRH is 255 and 242 amino acids long in the rat and
human, respectively (Lechan et al., 1986; Yamada et al., 1990). In the rat, five
TRH peptides are proteolytically cleaved from the prepro-peptide, while the
human prepro-peptide yields six TRH peptides. Regardless, the structure of
TRH is conserved across species. Because TRH is a tripeptide, it was very
easily synthesized. The early availability of synthetic TRH produced an initial
rush of information on various aspects of it’s localization and endocrinology.

The highest concentrations of TRH in the rat hypothalamus are in the
median eminence (Jackson and Reichlin, 1974). Within the median eminence,
however, TRH is only present in nerve fibers and terminals (Johansson and
Hokfelt, 1980; Lechan and Jackson, 1982). There are no TRH neuronal cell
bodies found in the median eminence. Neuronal cell bodies that stain positive for
TRH are scattered throughout the hypothalamus and central nervous system
(Lechan and Jackson, 1982; Hisano et al., 1986; Fliers et al., 1994). However,
the majority of immunoreactive TRH neuronal cell bodies projecting to the
median eminence are located in the parvocellular region of the paraventricular
nucleus (Lechan and Jackson, 1982; Hisano et al., 1986; Fliers et al., 1994).
Indeed, retrograde tracing of neurons with wheat germ agglutinin injected into the
median eminence of rats revealed the paraventricular nucleus as the only area
that had neurons that stained positive for both wheat germ agglutinin and pro-

TRH (Kawano et al., 1991). Experiments utilizing ablation and deafferentation

suggest tha‘
around the f
area. then c
Pahovns,lé
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suggest that TRH neurons located in the paraventricular nucleus project laterally
around the fomix before projecting medially through the lateral retrochiasmatic
area, then caudally to terminate in the median eminence (Palkovits et al., 1982;
Palkovits, 1984). Terminals in the median eminence are juxtaposed to portal
vessels, which transport TRH to the anterior pituitary gland. In vitro experiments
using synaptosomes isolated from sheep and rat hypothalami demonstrated that
dopamine induces release of TRH (Bennett et al., 1975; Schaeffer et al., 1977).
Histamine and serotonin have also been shown to induce release of TRH from
cultured rat medial basal hypothalamus (Charli et al., 1978; Chen and Ramirez,
1981).

Thyrotropin-releasing hormone induces secretion of thyroid-stimulating
hormone and prolactin from the anterior pituitary gland of humans (Bowers et al.,
1971; Jacobs et al., 1973), rats (Mueller et al., 1973; Blake, 1974), sheep
(Debeljuk et al., 1973; Fell et al., 1973), goats (McMurtry and Malven, 1974) and
cattle (Scheme, 1972; Convey et al., 1973; Vines et al., 1976). In addition, TRH
induces secretion of growth hormone in pre- and post-pubertal cattle (Vines et
al., 1976; Vines et al., 1977), pregnant cattle (Vines et al., 1977) and lactating
cattle (Convey et al., 1973; Tucker et al., 1975; Boume et al., 1977). Infusion of
TRH for 6-h increased secretion of growth hormone for the entire infusion period
(Tucker et al., 1975). Even though concentrations of growth hormone declined
slightly during the infusion period, growth hormone was still three times greater
than saline-infused controls at the end of the infusion period, and administration

of a second heterologous stimulus further increased concentrations of growth

10

hc

at.

hormone. In fact, when TRH and GHRH are simultaneously administered to
cattle, there is a synergistic increase in secretion of growth hormone (Hodate et
al., 1985). These data indicate that GHRH and TRH may induce the secretion of
growth hormone via different mechanisms, and combined with the fact that TRH
induces secretion of growth hormone during various physiological states in cattle
suggests that TRH may play a role in the normal hypothalamic regulation of
growth hormone secretion in cattle.
C. Characterization and Signal Transduction Mechanism of TRH Receptors
The first evidence that TRH binds to a specific receptor was that tritium
labeled TRH would competitively bind to plasma membranes isolated from both
rat thyrotropic tumor cells (Grant et al., 1972) and bovine anterior pituitary cells
(Labrie et al., 1972). Receptors for TRH on intact cells were first identified on a
clonal rat pituitary cell line that secretes growth hormone and prolactin (GH3)
using tritium labeled TRH (Gourdji et al., 1973; Hinkle and Tashjian, 1973). Since
then, TRH receptor binding has been demonstrated in the anterior pituitary gland,
retina and throughout the central nervous system in rodents, ruminants,
carnivores and fowl (Burt and Taylor, 1982; Taylor and Burt, 1982; Sharif et al.,
1991). A recent report describes the presence of TRH receptor mRNA in
somatotropes from the rat (Konaka et al., 1997). Using in situ hybridization,
Konaka and coworkers found that 61% of the anterior pituitary cells stained
positive for TRH receptor mRNA. Staining for growth hormone and prolactin in
these same cells revealed that 62% and 31% of the cells that stained positive for

TRH receptor mRNA were somatotropes and lactotropes, respectively. The

11

presence of TRH receptors on somatotropes and lactotropes may explain why
TRH induces release of both growth hormone and prolactin in cattle (Vines et al.,
1976; Vines, 1976; Vines et al., 1977).

The receptor for TRH has been cloned from mouse (Straub et al., 1990),
rat (de la Pena et al., 1992; Seller et al., 1993) and human tissue (Duthie et al.,
1993). Comparison of the receptors from the rat, mouse and human show very
high homology except for the COOH terminal tail (Duthie et al., 1993). The
cloned receptors from mouse, rat and human tissues are all functional, and the
differences in the COOH end have not produced any observable differences in
function. The human gene encoding the TRH receptor has three exons and two
introns with upstream promoter sequences for the glucocorticoid response
element, activator protein-1 sites, cyclic-AMP response elements and the
pituitary specific Pit-1 factor, indicating that TRH receptor expression is regulated
by many different signals (Matre et al., 1999). The TRH receptor has seven
transmembrane elements (Sellar et al., 1993) coupled to a G-protein (Gq or G11).

Binding of TRH to the receptor causes activation of phospholipase C81
and hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-
trisphosphate and diacylglycerol (Aragay et al., 1992; Hsieh and Martin, 1992;
Svoboda et al., 1996). Inositol 1,4,5-trisphosphate induces release of
intracellular calcium stores, which depolarize the cell and open voltage-gated
calcium channels. The increase in intracellular calcium (from intracellular and
extracellular sources) induces secretion of thyroid-stimulating hormone, prolactin

or growth hormone depending on the cell type (Albert and Tashjian, 1984).

12

Diacylglycerol also serves as a second messenger. Diacylglycerol activates
protein kinase C, which in turn phosphorylates many intracellular proteins (Kahn
et al., 1998) that regulate a number of cellular functions including ion pumps, that
either repolarize or maintain depolarization of secretory cells as well as
transcription and translation of protein hormones.
D. Discovery, Characterization, and Localization of PACAP

Pituitary adenylate cyclase-activating polypeptide is another hypothalamic
peptide that induces secretion of growth hormone from cultured bovine anterior
pituitary cells (Hashizume et al., 1994). Pituitary adenylate cyclase-activating
polypeptide is a 38 amino acid peptide that was originally isolated from ovine
hypothalamic tissue (Miyata et al., 1989). The N-terminal portion, amino acids 1
- 28, of the peptide has 68% homology with porcine vasoactive intestinal peptide
(VIP). The C-terminal portion, amino acids 29 - 38, has no homology with any
known peptide. The name comes from the peptide’s ability to increase cyclic-
AMP in rat pituitary cells. There are two isoforrns of PACAP. The more abundant
form is the 38 amino acid peptide (PACAP-38); the second is a truncated form
which is made up of the 27 N-terminal amino acids (PACAP-27) (Miyata et al.,
1990). lmmunohistochemical experiments with ovine hypothalamic tissue have
shown that PACAP neuronal cell bodies are located in the paraventricular and
supraoptic nuclei. Stained fibers were located throughout the hypothalamus with
dense staining in the median eminence juxtaposed to portal vessels (Koves et
al., 1990). As determined by radioimmunoassay, the greatest concentration of

PACAP-38 was in the hypothalamus. However, PACAP-38 was also measured

13

in extracts from other regions of the brain and several peripheral organs. The
shorter PACAP-27 was also measured in rat hypothalamic tissue but was found
in much lesser amounts (Arimura et al., 1991). In fact, PACAP-38 was the major
form measured in hypophysial portal blood, which contained significantly greater
amounts of PACAP-38 than peripheral blood (Dow et al., 1994). Thus, the
greatest source of PACAP is the hypothalamus.

In vitro, PACAP-38 induces secretion of growth hormone from bovine
(Hashizume et al., 1994), porcine (Martinez-Fuentes et al., 1998b; Martinez-
Fuentes et al., 1998a) and rat anterior pituitary cells (Velkeniers et al., 1994).
Hashizume and coworkers (1994) also demonstrated that the stimulatory effects
of PACAP-38 on growth hormone secretion from bovine anterior pituitary cells
was blunted when treated with SS and PACAP simultaneously. Pituitary
adenylate cyclase-activating polypeptide also increased growth hormone mRNA
accumulation in porcine (Martinez-Fuentes et al., 1998b) and rat anterior pituitary
cells in culture (Velkeniers et al., 1994). Experiments testing the effects of
PACAP in vivo are limited. Exogenous PACAP induces secretion of growth
hormone in male and lactating female rats (Rawlings and Hezareh, 1996). In
sheep, however, infusion of 1 nmollmin of PACAP into the carotid artery for 10
min failedto induce secretion of growth hormone (Sawangjaroen and Curlewis,
1994). The inability of PACAP to induce secretion of growth hormone in ewes
may be due to the small dose that was infused, or alternatively PACAP may not

play a role in regulating secretion of growth hormone in sheep.

14

E. Characterization and Signal Transduction Mechanism of PACAP
Receptors

Specific binding of PACAP was first demonstrated with 125l labeled
PACAP-27 in bovine brain membranes (Ohtaki et al., 1990). Unlabeled PACAP-
27 and PACAP-38 displaced ‘25l-PACAP-27 while VIP did not. After purification,
the receptor showed a higher affinity for PACAP-38 than PACAP-27 (Ohtaki et
al., 1993). To date, three distinct PACAPNIP receptors (PVR1, PVR2 and
PVR3) have been cloned. All three receptors have seven transmembrane
domains and are linked to G-proteins. The three PACAPNIP receptors can be
divided into two types.

Type I receptors bind PACAP-38 and PACAP-27 with greater affinity than
VIP. The PVR1 is a type I receptor that hasbeen cloned from rat (Hosoya et al.,
1993; Pisegna and Wank, 1993; Spengler et al., 1993), human (Ogi et al., 1993),
and bovine (Miyamoto et al., 1994) tissue, and activates both adenylate cyclase
and phospholipase C pathways. In the rat, six splice variants of PVR1 have
been cloned, a short form called PVR1s and five variants with inserts in the
putative third intracellular l00p of the receptor (Spengler et al., 1993; Joumot et
al., 1995). There are two distinct 28 amino acid inserts (PVR1hip and
PVR1hop1), a 27 amino acid insert that is identical to hop1 except that it lacks a
serine residue (PVR1hop2), and two combination inserts (PVR1hiphop1 and
PVR1hiphop2). All six variants activate adenylate cyclase and phospholipase C
except PVR1hip, which only activates adenylate cyclase (Rawlings and Hezareh,

1996).

15

The PVR2 and PVR3 receptors are Type II receptors which bind PACAP-
38, PACAP-27 and VIP with approximately equal affinity (Rawlings, 1994). The
PVR2 receptor has been cloned from rat (lshihara et al., 1992) and human tissue
(Sreedharan et al., 1993; Couvineau et al., 1994; Gagnon et al., 1994). It
probably corresponds to the classic VIP receptor. In cells transfected with the
PVR2, both PACAP and VIP activate adenylate cyclase with equal potency
(lshihara et al., 1992; Sreedharan et al., 1993; Couvineau et al., 1994; Gagnon et
al., 1994; Ciccarelli et al., 1994). The PVR3 receptor has been cloned from rat
(Usdin et al., 1994; Lutz et al., 1993), mouse (Inagaki et al., 1994) and human
tissue (Svoboda et al., 1994; Adamou et al., 1995). The PVR3 activates
adenylate cyclase but not phospholipase C (Rawlings and Hezareh, 1996).

All three PVR receptors are found in the anterior pituitary gland. The
PVR1 is the most abundant, PVR2 expression is lowest and PVR3 expression is
intermediate (Rawlings and Hezareh, 1996). In culture, 89% of rat anterior
pituitary cells that stained positive for growth hormone also bound biotinylated
PACAP-38 (Vigh et al., 1993). Addition of a membrane permeable cAMP
antagonist blocked the PACAP-induced influx of extracellular calcium (Canny et
al., 1992; Rawlings et al., 1993). In addition, PACAP-38 failed to mobilize inositol
1,4,5-trisphosphate sensitive calcium stores (Rawlings and Hezareh, 1996)
indicating that the phospholipase C pathway was not activated. These data
suggest that PACAP receptors on somatotropes are Type II receptors (PVR2
and(or) PVR3) because they activate the adenylate cyclase- cAMP second

messenger pathway, not the phospholipase C pathway. If the actions of PACAP-

16

38

Cr

III 3

Al

38 on somatotropes were mediated by Type I receptors then the phospholipase

C pathway should have also been activated.

III Serotonergic Receptor Regulation of Growth Hormone Secretion
A. Characterization of Serotonergic Innervation of the Hypothalamus

Serotonin belongs to the biogenic amine family of neurotransmitters that
are synthesized from amino acid precursors. Serotonin is also referred to as 5-
hydroxytryptamine (5-HT). Serotonin is derived from the amino acid tryptophan
by two enzymatic reactions. The first reaction, which is the rate limiting reaction,
is catalyzed by tryptophan hydroxylase which puts a hydroxyl group in the 5
position on the indole ring of tryptophan to make 5-hydroxytryptophan. The
second reaction, catalyzed by the enzyme 5-hydroxytryptophan decarboxylase,
produces serotonin.

The serotonergic system in the brain originates from the midline of the
brain stem and projects rostrally throughout the cortex, hippocampus and
olfactory bulb, and caudally to the spinal cord. Serotonin has been implicated in
sleep, aggression, sexual activity, appetite, learning, memory, depression,
obsessive compulsive disorder, anorexia, bulimia, sudden infant death
syndrome, Alzheimer's disease and schizophrenia. It is the single largest brain
neurotransmitteisystem known and can be characterized as a “giant” neuronal
system (Azmitia and Whitaker-Azmitia, 1991). Several experiments using

autoradiography have demonstrated the dense serotonergic innervation of the

hypothalamus (Conrad et al., 1974; Bobillier et al., 1976; Bobillier et al., 1979;

17

Azmitia and Segal, 1978). The serotonergic fibers within the hypothalamus
originate from cell bodies in the raphe nuclei with their axons projecting to the
hypothalamus through the medial forebrain bundle, ventricular tract, and arcuate
tract (Azmitia and Segal, 1978). Additionally, cell bodies that accumulate labeled
serotonin (Beaudet and Descarries, 1979) and stain positive for serotonin
(Frankfurt et al., 1981) have been identified within the dorsomedial nucleus of the
hypothalamus. However, their function has yet to be identified. Early retrograde
tracing experiments using either the fluorescent dye true blue or fluorescent
microspheres inserted into the paraventricular nucleus revealed labeled cell
bodies in the dorsal raphe and median raphe nuclei as well as the medial
Iemniscus (Sawchenko et al., 1983; Petrov et al., 1992). In contrast, anterograde
tracing experiments from the dorsal and medial raphe nuclei revealed very few or
no labeled terminals in the paraventricular nucleus (Azmitia and Segal, 1978;
Moore et al., 1978; Vertes and Martin, 1988; Vertes, 1991; Vertes et al., 1999).

A more recent report by Larsen and coworkers (1996) combined retrograde and
anterograde tracings to identify the origin of serotonergic terminals in the
paraventricular nucleus. Injection of the tracer, cholera toxin subunit B, into the
medial portion of the paraventricular nucleus or into the third ventricle resulted in
labeled serotonergic neurons in the dorsal and medial raphe nuclei. In addition,
injection of the anterograde tracer lectin PHA—L into either the dorsal or medial
raphe nuclei resulted in labeled terminals in close proximity to the ependymal
lining of the third ventricle with very few fibers and terminals labeled in the

paraventricular nucleus. These data indicate that earlier retrograde tracing

18

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inne

I'IUt

V

in:

PAC

a: _

330

the p

St
‘3 10 Si

A
Vega
V...

(Du

sil.

.HW

experiments that attempted to identify neurons projecting to the paraventricular
nucleus may have actually traced neurons projecting to the ependymal lining and
was the result of the tracer leaking into the supraependymal layer. However,
injection of cholera toxin subunit B into the lateral portion of the paraventricular
nucleus resulted in labeling of neurons in raphe magnus and no labeling in the
dorsal and medial raphe nuclei. Additionally, injection of lectin PHA-L into the
raphe magnus resulted in anterograde labeling of a dense network of fibers and
terminals throughout the paraventricular nucleus (Larsen et al., 1996). Thus, it
appears that different nuclei in the hypothalamus receive serotonergic input from
specific raphe nuclei. Regardless of the exact origin, the dense serotonergic
innervation of the hypothalamus could influence the secretion of several
hypothalamic hormones such as corticotrophin releasing-hormone, TRH,
PACAP, GHRH and gonadotropin releasing-hormone. Specificity of serotonin’s
action may be a function of it’s origin as well as it’s receptor subtype present on
the postsynaptic cell.
B. Characterization, Localization and Signal Transduction Mechanism of
Serotonin Receptors

There are 14 known serotonin receptor subtypes which can be classified
into seven separate subfamilies (5-HT1 — 5-HT7). Unless noted in this review
each receptor has it’s own gene. Due to the vast number of serotonin receptors,
only those that are located in the hypothalamus are discussed in detail. In the 5-
HT1 family, there are five subtypes (5-HT1A, 5-HT13,5-HT1D, 5-HT1E and 5-HT1F)

all of which have seven transmembrane domains and inhibit adenylate cyclase

19

via Gp

hasha

hunen
anygde
Uthis
uteri
in6293

(levy er

via G-proteins (Barnes and Sharp, 1999). Of the five 5-HT1 subtypes only 5-HT1E
has been reported in the hypothalamus.

Currently, there are no reports of radioligand binding specifically to 5—HT1E
receptors. However, in the human and monkey, 5-HT15 mRNA has been isolated
from entorhinal cortex and the caudate putamen with lesser amounts found in the
amygdala and hypothalamus (Bruinvels et al., 1994b; Bruinvels et al., 1994a).
Little is known about the physiological role of the 5-HT1E receptor. However,
when it is expressed in either murine Ltk-cells or human embryonic kidney cell
line 293, treatment with serotonin inhibits forskolin-stimulated adenylate cyclase
(Levy et al., 1992; McAllister et al., 1992), indicating that activation of 5-HT1E
receptors inhibits the adenylate cyclase second messenger system.

The 5-HT2 receptor subfamily is made up of three receptor subtypes (5-
HTZA, 5-HT33 and 5-HTzc). All three receptors have seven transmembrane
domains coupled to phospholipase C, and they mobilize intracellular calcium
(Barnes and Sharp, 1999). Of the 5-HT3 receptors, 5-HT23 is the only one
located in the hypothalamus. However, I have also included some discussion on
5-HT2c because of it’s abundance in the choroid plexis which lines the third
ventricle.

The 5-HT23 receptor is restricted to a few locations. In the rat
immunoreactivity appears only in the cerebellum, lateral septum, and dorsal
hypothalamus (Duxon et al., 1997a). There are few data available on the
physiological role of 5-HT23 receptors. One report indicates that a 5-HT23 agonist
has anxiolytic effects (Duxon et al., 1997b).

20

 

(I)

5’.’

(Pi

Autoradiographic experiments have produced a detailed map of the 5-
HTzc receptor distribution in the brain. Firstly, very large amounts of the 5-HT3c
receptor are located in the choroid plexis. Binding sites are also widely
distributed throughout the cortex, limbic system, and basal ganglia (Barnes and
Sharp, 1999). Several behavioral and physiological responses have been
attributed to 5-HTzc receptor activation. These include hypolocomotion,
hypophagia, anxiety, penile erections and hypertherrnia (as reviewed by Koek et
al., 1992). Recent reports indicate that 5-HT2c receptors may inhibit secretion of
noradrenaline and dopamine (Millan et al., 1998; Di Matteo et al., 1998).
Administration of cyproheptadine (5-HT2 receptor antagonist) to steers inhibits
secretion of growth hormone (Gaynor et al., 1995)

The third serotonin receptor subfamily (5-HT3) contains only one receptor
subtype (5-HT3A). However 3 splice variant (5-HT3AS) has been reported (Hope
et al., 1993). The 5-HT3 receptor subfamily differs from the other serotonin
receptors in that it is a ligand gated ion channel with homology to other ligand
gated ion channels such as nicotinic, GABAA and glycine receptors (Barnes and
Sharp, 1999). However, it is not present in the hypothalamus.

The fourth serotonin receptor family (5-HT4) contains four receptor
subtypes (5-HT4(3), 5-HT4ib), 5-HT4(c), and 5-HT4(3)). Unlike the previously
discussed subfamilies, 5-HT4 receptors all appear to come from a single gene
and are all splice variants with identical amino acid sequences up to Leu358
(Blondel et al., 1998). All four splice variants have seven transmembrane

domains and activate adenylate cyclase (Gerald et al., 1995; Claeysen et al.,

21

.‘EM‘L—u

1996; Van den Wyngaert et al., 1997), but none of the four variants have been
identified in the hypothalamus.

Very little is understood about the fifth subfamily of serotonin receptors (5-
hts). To date there is no direct evidence for the existence of a native 5-ht5
receptor; therefore, it is still denoted with lower case letters. Initially the 5-ht5
receptor was identified by screening mouse cDNA libraries and was named 5-
mg. (Plassat et al., 1992). Later the 5-ht53 receptor was identified by the same
group (Matthes et al., 1993). The 5-htsA and 5-ht53 receptors have since been
identified from rat (Erlander et al., 1993; Wisden et al., 1993) and the 5-htsA from
human (Rees et al., 1994) cDNA libraries.

The 5-ht3 receptor was initially identified from it’s cDNA sequence by two
different groups (Monsma et al., 1993; Ruat et al., 1993a) and thus far is the only
serotonin receptor in the 5-ht3 subfamily. Both Northern blot analysis and in situ
hybridization revealed that 5-ht3 mRNA is mostly confined to the central nervous
system (Monsma et al., 1993; Ruat et al., 1993a). The 5-ht3 receptor has seven
transmembrane regions and experiments using artificial expression systems
indicate that it activates adenylate cyclase (Monsma et al., 1993; Ruat et al.,
1993a; Boess et al., 1997). With the recent discovery of specific 5—ht5 receptor
ligands (Sleight et al., 1998) and demonstration of their ability to bind in the rat
and porcine striatum (Boess et al., 1998), the functional roles of the 5-ht5
receptor may soon be identified.

The last subfamily of serotonin receptors (5-HT7) are encoded by a single

gene but splice variants produce four receptors (5-HT7(3), 5-HT7(3), 5-HT7(3), and

22

AH v

 

5-HT7M) (Heidmann et al., 1997; Jasper et al., 1997). The 5—HT7 receptor has
seven transmembrane domains and activates adenylate cyclase in both artificial
expression systems and native tissues (Plassat et al., 1993; Ruat et al., 1993b;
Heidmann et al., 1997; Heidmann et al., 1998). The 5-HT7 receptor is expressed
at relatively high amounts in the thalamus, hypothalamus and hippocampus (T 0

et al., 1995; Stowe and Barnes, 1998). However, large tissue-specific

"I

expression of the different splice variants is not apparent. Artificial expression of

the 5-HT7 receptor has demonstrated it’s ability to activate adenylate cyclase and .;-.
increase intracellular calcium concentrations without inducing any phospholipase

C activity (Baker et al., 1998), indicating that 5-HT7 may play a role in neuronal

activation.

The large number of different serotonin receptors discovered in the past
decade in addition to the specific distribution of each of the receptor subtypes,
lends support to the many functions that serotonin is believed to have in the
central nervous system. Due to the complexity of the serotonergic system, it will
be several more years before we understand all the physiological actions of
serotonin.

C. Serotonergic Receptor Regulation of Growth Hormone Secretion

It is well established that the major control of growth hormone secretion is
by two hypothalamic peptides, GHRH and SS. However, there is considerable
evidence that GHRH and SS are not the only regulators of growth hormone
secretion. Serotonin is one of several substances from the hypothalamus that

may play a role in regulation of growth hormone secretion. However, reports

23

concerning the role of serotonin are conflicting, and the mechanism of action has
yet to be delineated. For example, the serotonin receptor agonist, quipazine,
which activates several different serotonin receptors, has been reported to inhibit
secretion of growth hormone in chickens (Hall et al., 1983). In contrast, the same
serotonin receptor agonist stimulates secretion of growth hormone in cattle.
Additionally, the 5-HT2/5-HTzc receptor antagonist cyproheptadine inhibits
secretion of growth hormone in cattle (Sartin et al., 1987; Gaynor et al., 1995;
Gaynor et al., 1996). But, when bovine anterior pituitary cells in culture were
treated with quipazine, there was no effect on secretion of growth hormone
(Gaynor et al., 1996). In sheep, the 5-HT1A receptor agonist buspirone inhibited
secretion of growth hormone when administered either intravenously or
intracerebroventricularly while cyproheptadine administration via either route had
no effect (Spencer et al., 1991). In the rat, the nonselective serotonin receptor
agonists quipazine and MK212 (6-chloro-2(piperazinyl)-pyrazine) and serotonin
itself stimulate secretion of growth hormone (Vijayan et al., 1978; Arnold and
Femstrom, 1981; Willoughby et al., 1982; Willoughby et al., 1987; Chapman et
al., 1993). However, when cultured rat anterior pituitary cells were treated with
serotonin there was no effect on growth hormone secretion (Balsa et al., 1998).
One investigator measured the amounts of serotonin and it’s metabolite, 5-
hydroxyindoleacetic acid (5-HIAA), in four different rat models that are commonly
used to investigate growth hormone secretion. The ratio of 5-HIAA to serotonin
(5-HIAA15-HT) can be used as an index of neuronal activity. In all four rat

models, serotonergic neuronal activity was positively correlated with

24

concentrations of growth hormone in serum (Smythe et al., 1982). These data
suggest that the effects of serotonin on growth hormone secretion are species
specific and mediated in the hypothalamus, not in the anterior pituitary gland.
There have been reports that in the rat, serotonin's action on growth
hormone secretion is mediated via GHRH (Murakami et al., 1986), TRH (Chen

and Ramirez, 1981), and PACAP (Yamauchi et al., 1996), all of which are

"1

possible mediators. But research to support any one of these hypothalamic

”nu—m.—
0 . -

hormones as the mediator of serotonin-induced growth hormone secretion in
cattle is lacking. It has also been reported that there is an interaction of
serotonergic and az-adrenergic systems with regards to growth hormone
secretion. In rats, an intact serotonergic system had to be present in the

hypothalamus for az-adrenergic receptor agonists to stimulate growth hormone
secretion (Conway et al., 1990). Conversely, in cattle pre-treatment with an a;-
adrenergic receptor antagonist suppressed serotonin receptor agonist-induced
secretion of growth hormone (Gaynor et al., 1996). One thing is clear from all of
these data, we are not sure how serotonin induces secretion of growth hormone

in vivo in cattle or rats.

25

CHAPTER 1

Serotonin Induced Secretion of Growth Hormone

26

INTRODUCTION

Serotonin has been implicated in the regulation of growth hormone
secretion. However, the effects of serotonin seem to be species specific.
Serotonin inhibits secretion of growth hormone in chickens and sheep (Hall et al.,
1983; Spencer et al., 1991) but stimulates secretion of growth hormone in cattle
and rats (Sartin et al., 1987; Gaynor et al., 1995; Gaynor et al., 1996; Smythe et
al., 1975; Vijayan et al., 1978; Willoughby et al., 1982; Willoughby et al., 1987).
The exact mechanisms by which serotonin exerts it’s effects are not known.
Serotonin does not induce secretion of growth hormone directly from cultured
bovine or rat anterior pituitary cells (Gaynor et al., 1996; Balsa et al., 1998).
However, Balsa and coworkers (1998) reported that serotonin induced secretion
of growth hormone from dispersed rat anterior pituitary cells co-cultured with
posterior pituitary cells in medium supplemented with corticosterone. Even
though the increase in secretion of growth hormone was significant, the
magnitude of response was quite small (approximately 3.9 versus 4.5 ng per
plate for control and treated cells respectively, when incubated for 180 min). It is
therefore possible that serotonin acts directly on the posterior pituitary gland to
induce secretion of growth hormone from the anterior pituitary gland. In fact,
Balsa and coworkers speculated that a melanocyte-stimulating hormone (aMSH)
from the posterior pituitary cells was mediating the effects of serotonin on
secretion of growth hormone from anterior pituitary cells. However, they could

not confirm aMSH as the mediator. Thus, Balsa and coworkers were unable to

27

explain how serotonin caused secretion of growth hormone from anterior pituitary
cells co-cultured with posterior pituitary cells.

Gaynor and coworkers (1996) tested the effects of quipazine on secretion
of growth hormone from bovine anterior pituitary cells. However, the anterior
pituitary cells were not co-cultured with posterior pituitary cells. Therefore, I
proposed the mechanism of action depicted in Figure 1 and hypothesized that
activation of serotonergic receptors would induce secretion of growth hormone
from bovine anterior pituitary cells co-cultured with posterior pituitary cells. My
objective was to determine if quipazine, which induces secretion of growth
hormone in cattle (Sartin et al., 1987; Gaynor et al., 1996), would induce release
of growth hormone from bovine anterior pituitary cells co-cultured with posterior

pituitary cells.

MATERIALS AND METHODS

Perifusion of Bovine Anterior and Posterior Pituitary Cells

Two bovine pituitary glands were collected at a local abattoir, placed in ice
cold calcium, magnesium free-Hank’s balanced salt solution (pH 7.4, CMF-
HBSS; Life Technologies Inc., Grand Island, NY) that had been sterilized by
filtration with a 0.22 pm Sterivex®-GS filter (Millipore®, Bedford, MA) and
transported back to the laboratory. Working inside a biological safety hood,
using aseptic technique, each pituitary gland was dissected to separate anterior

pituitary tissue from posterior pituitary tissue. In separate vials, anterior and

28

Raphe Nuclei w

Pituitary Stalk
Portal Vessel O Serotonfl)

 

 

+

 

Pituitary +(—l PP

Blood Vessel ) GH (7W

milk, meat

Figure 1. Proposed model for direct action of serotonin on the bovine
pituitary gland. Serotonergic neurons (oval) project axons to the pituitary stalk
with terminals juxtaposed to portal vessels which transport it to the pituitary
gland. Serotonin acts at the posterior pituitary gland (PP) to induce secretion of
growth hormone (GH) from the anterior pituitary gland (AP).

 

 

29

posterior pituitary tissues were individually enzymatically dispersed using
techniques described previously for anterior pituitary tissue (Padmanabhan et al.,
1987). Briefly, tissue was cut into approximately 1 mm cubes. The cubes were
then placed in CMF-HBSS containing collagenase (3 mg/ml) and incubated in a
shaking water bath at 37° C for 45 min. Cells were triturated after 15, 30 and 45
min of incubation. Collagenase solution and cells were then poured through

sterile gauze into 50 ml conical tubes and centrifuged to pellet cells. Supernatant

rial-JACAM r. .o-L 1.. B!

was removed, and cells resuspended and rinsed with CMF-HBSS three times.

Harvested anterior and posterior pituitary cells from each pituitary gland
were suspended in separate vials with Dulbecco’s modified Eagle’s medium
(DMEM; Life Technologies Inc.) with low glucose (1,000 mg/L) containing 1%
newborn calf serum (Life Technologies Inc.), 25 mM N-2-hydroxyethylpiperazine-
N’-2 ethanesulfonic acid (HEPES; Sigma Chemical Co., St. Louis, MO), penicillin
(10 U/ml; Life Technologies Inc.), streptomycin (10 ug/ml; Life Technologies Inc.)
and fungizone (0.25 uglL; Life Technologies Inc.). Medium was also
supplemented with 1045 M hydrocortisone (H4001; Sigma Chemical Co. St. Louis,
MO), a concentration similar to concentration of corticosterone used by Balsa
and coworkers (1998). Concentration of cells was 10‘3 cells/ml. After dispersion
and resuspension, cell viability of both anterior and posterior pituitary cells was
greater than 90% as determined by trypan blue exclusion.

The perifusion system of Gaynor and coworkers (1996) was modified for
use in this experiment. Briefly, barrels of 3-ml syringes (Becton-Dickinson and

Co.) were used as perifusion chambers. Rubber stoppers were placed on the

30

tips of syringes and a layer of sterile glass wool was inserted. Syringes were
packed by gravity to a volume of 0.5 ml with sterile Cytodex l beads, which had
been previously swollen in 0.9% sodium chloride solution. Cells from each
pituitary gland were dispensed into chambers as a mixture of anterior (1 ml) and
posterior (0.25 ml) pituitary cells or just anterior pituitary cells (1 ml). Chambers
were then filled to 3 ml with DMEM and placed in a humidified atmosphere of
95% 02-5% 002 at 37°C for 24 h to allow cells to adhere to the Cytodex l beads.
On the following day chambers were connected to peristaltic pumps, and
the cells were perifused at 37°C with DMEM gassed with 95% 075% C02 at a
flow rate of 0.15 mein for 180 min. After this initial flushing period, chambers
were ready for experimental protocols. Experimental treatments were dissolved

in medium and sterilized through a 0.22 pm filter into separate vials that were

connected to the peristaltic pump via a three-way stopcock. The stopcock
allowed for easy switching from medium to medium containing treatments and
back to medium. During experiments, 3-ml fractions of medium effluent were
collected into 12 X 75 mm polypropylene tubes inside the incubator. Fractions
were stored at -20°C until assayed. Radioimmunoassay was used to quantify
concentrations of growth hormone in medium effluent as described previously
(Gaynor et al., 1995).
Experimental Protocol

Six chambers containing 106 anterior pituitary cells only (three chambers
per pituitary gland), and six chambers containing a mixture of anterior (106 cells)

and posterior (0.25 X 10'5 cells) pituitary cells (three chambers per pituitary gland)

31

E?

tn

1A4
VJ

".5,
“WV

were assigned to one of two treatments in the first replicate (Table 1) and
perifused with DMEM for 180 min. Medium effluent (3 ml) was collected at 20-
min intervals, and all samples were assayed for growth hormone. Pre-treatment
samples were collected at 20, 40 and 60 min. Treatments were either control
medium or 10'6 M quipazine dissolved in medium. This dose of quipazine was
the middle dose used by Gaynor and coworkers (1996) and was similar to the
dose of serotonin used by Balsa and coworkers (1998). Cells were perifused
with treatments from 61 to 80 min. Post-treatment samples were collected from
81 to 180 min. Cells were then perifused with medium for 120 min to allow them
to recover from treatments imposed in the first replicate. Using the same cells,
treatments were reversed and the experiment replicated. At the end of the
second replicate, cells were perifused with DMEM containing 60 mM potassium
chloride (KCI) from 181 to 200 min, then medium from 201 to 240 min. In viable
secretory cells, KCI causes depolarization and an influx of extracellular calcium
which induces secretion. These samples were assayed for growth hormone to
determine somatotrope viability. Data from the first and second replicate were
pooled and aligned by time of treatment for statistical analysis.
Statistical Analysis

Areas under the response curves (AUC) of growth hormone were
calculated from 61 to 120 min using the trapezoidal rule and were subjected to
analysis of variance using Mixed Models procedures of Statistical Analysis
System (SAS;SAS, 1999). To account for differences in basal release between

chambers, pre-treatment growth hormone values at 60 min were used as a

32

 

Ef

A
'5.1
VI I.

covariate in the statistical analysis. Cow, replicate, treatment and covariate were
included in the model. Potassium chloride-stimulated release was calculated as

the average concentration of growth hormone in medium samples collected from
200 to 240 min. Comparison between average baseline concentrations (average

concentration from 20 to 60 min) and KCI-stimulated concentrations were

conducted using Student’s paired t-test (Gill, 1978).

Table 1. Treatment assignments for Experiment 1.

 

 

 

Chamber Cow Replicate 1 Replicate 2
1 (AP3 cells only) 1 Control Quipazine
2 (AP cells only) 2 Control Quipazine
3 (AP cells only) 1 Control Quipazine
4 (AP cells only) 2 Quipazine Control
5 (AP cells only) 1 Quipazine Control
6 (AP cells only) 2 Quipazine Control
7 (AP and PP” cells) 1 Control Quipazine
8 (AP and PP cells) 2 Control Quipazine
9 (AP and PP cells) 1 Control Quipazine
10 (AP and PP cells) 2 Quipazine Control
11 (AP and PP cells) 1 Quipazine Control
12 (AP and PP cells) 2 Quipazine Control

3 Anterior pituitary gland.

b Posterior pituitary gland.

RESULTS

Effect of Quipazine on Secretion of Growth Hormone from Dispersed

Pituitary Cells

Profiles of concentrations of growth hormone in medium effluent are

shown in Figure 2 (top panel). Perifusion of anterior pituitary cells with medium

containing 10'6 M quipazine did not alter AUC for growth hormone when

33

‘.‘(

 

 

 

     

160 -
+ AP-Vehicle
A —-— AP-Quipazine
2 12° . —+— AP/PP-Vehicle
3 —~— AP/PP-Quipazine
o
0
o
I so -
a)
c
v
I 4o-
0
o . Em! . FEEL j
0 40 so 120 160 200 240
Minutes
3000 -
E 2500 -
E 2000 -
E 1500 -
O
or
C 1000 -
v
(:9: 500 -
o .

 

   

AP- AP- APIPP- APIPP-
Vehlcle Quipazine Vehicle Qulpazlne

Figure 2. Effect of quipazine on secretion of growth hormone from
dispersed bovine pituitary cells. TOP PANEL; Data presented are least
squares mean concentrations (i pooled standard error of mean; SEM) of growth
hormone in medium effluent from dispersed anterior pituitary gland cells
perifused with vehicle medium (AP-vehicle; n=6) or quipazine (AP-Quipazine;
n=6), and the combination of anterior and posterior pituitary gland cells perifused
with vehicle medium (AP/PP-vehicle; n=6) or quipazine (AP/PP-Quipazine; n=6).
Treatments were imposed from 61 to 80 min (indicated by box on X axis). All
chambers were exposed to 60 mM KCI from 181 to 200 min (indicated by box on
X axis) to test somatotrope viability. BOTTOM PANEL; Statistical analysis was
performed on least squares mean AUC (i pooled SEM) for growth hormone
calculated from 60 to 120 min and adjusted for pre-treatment concentrations
using concentration of growth homone at 60 min as a covariate. Least squares
means were not different from each other.

34

 

in A?
‘ Vll

compared with perifusion of anterior pituitary cells with medium alone (P = 0.99;
Figure 2, bottom panel). Similarly, perifusion of a combination of anterior and
posterior pituitary cells with medium containing 10‘6 M quipazine did not alter
AUC for growth hormone compared with anterior and posterior pituitary cells
perifused with medium alone (P = 0.64; Figure 2, bottom panel). Perifusion with
medium containing 60 mM KCI increased secretion of growth hormone from all

chambers, indicating that somatotropes were viable at the end of the experiment.

DISCUSSION

In the current experiment, activation of serotonergic receptors did not alter
secretion of growth hormone from either anterior pituitary cells alone or in co-
culture with posterior pituitary cells. Our laboratory showed previously that the
serotonin receptor agonist quipazine had no effect on secretion of growth
hormone from dispersed bovine anterior pituitary gland cells (Gaynor et al.,
1996). However, Balsa and coworkers (1998) hypothesized that posterior
pituitary cells (melanotropes) mediated the effects of serotonin on secretion of
growth hormone in the rat, and reported that serotonin increased secretion of
growth hormone from rat anterior pituitary gland cells co-cultured with posterior
pituitary gland cells in medium containing corticosterone.

Secretion of growth hormone is calcium-dependent. Glucocorticoids
modulate calcium channel subunit mRNA expression as well as calcium currents

in clonal GHa cells (Fomina et al., 1993; Fomina et al., 1996). However, Balsa

35

 

SL

and coworkers reported the addition of corticosterone to media had no effect on
serotonin-induced secretion of growth hormone from rat anterior pituitary cells
alone. Similar results were obtained in the current experiment with bovine
anterior pituitary cells.

Balsa and coworkers (1998) reported that for serotonin to induce secretion
of growth hormone, rat anterior pituitary cells had to be co-cultured with posterior
pituitary cells in medium supplemented with corticosterone, Therefore,
suggesting that something from the posterior pituitary cells in combination with
corticosterone was mediating the effects of serotonin on secretion of growth
hormone. Balsa and co-workers initially speculated that aMSH may be a
mediator. However, they did not observe an increase in aMSH secretion from
posterior pituitary cells cultured in medium containing corticosterone and treated
with serotonin. This led Balsa and coworkers to speculate that something that
has yet to be identified was secreted from the posterior pituitary cells in response
to serotonin, in combination with corticosterone, ultimately inducing the secretion
of growth hormone from anterior pituitary cells.

I tested the effects of a nonspecific serotonin receptor agonist, quipazine,
on secretion of growth hormone from bovine anterior pituitary cells alone and
anterior pituitary cells co-cultured with posterior pituitary cells with added
glucocorticoids. My results contrast those of Balsa and coworkers (1998) in that
the serotonin receptor agonist did not induce secretion of growth hormone from
anterior pituitary cells co-cultured with posterior pituitary cells in medium

containing hydrocortisone. One possibility for the differing results is that the

36

mechanism of serotonin-induced secretion of growth hormone is different in
cattle than in rats. Another possibility is that Balsa and coworkers conducted
their experiment in a static culture system with cells that had been in culture for
72 h. | used a perifusion system to reduce the possibility of negative feedback
on the secretory cells and more closely mimic an in vivo environment for the
cells. I also used cells that had been in culture for only 24 h. It is also possible
that by the time I conducted the experiment the posterior pituitary cells could
have died. I tested the viability of the somatotropes with KCL at the end of the
experiment, but I did not assay the KCL samples for aMSH, to confirm that the
posterior pituitary cells were still viable.

The data from this experiment support the conclusion of Gaynor and
coworkers (1996) in that the effects of serotonin receptor agonist on secretion of
growth hormone in cattle are not directly on the anterior pituitary gland. In
addition, these data demonstrate that in cattle the effects of quipazine on
secretion of growth hormone are not mediated by posterior pituitary cells.
Therefore, I rejected the model proposed in Figure 1. These data combined with
those of Gaynor and co-workers led me to hypothesize that the effects may be

mediated via hypothalamic hormones.

37

CHAPTER 2

Serotonin Induced Secretion of GHRH and SS

38

 

 

 

INTRODUCTION

In Chapter 1, I concluded that serotonin does not induce secretion of
growth hormone directly from the pituitary gland in cattle, and speculated that
hypothalamic hormones could be mediating serotonin’s effects. The central
serotonergic system is the largest brain system known with projections
throughout the neural axis (Azmitia and Whitaker-Azmitia, 1991). The
hypothalamus, which controls most anterior pituitary gland functions, contains
large numbers of serotonergic terminals and fibers (Conrad et al., 1974; Bobillier
et al., 1976; Bobillier et al., 1979; Azmitia and Segal, 1978). Several serotonin
receptor subtypes have been identified in the hypothalamus (5-HT1E, 5-HT23, 5-
HTzc and 5-HT7) and have been reviewed by Barnes and Sharp (1999).
Therefore, it is possible that hypothalamic hormones mediate serotonin’s effects
on secretion of growth hormone. However, exactly which hypothalamic
hormone(s) mediate serotonin-induced secretion of growth hormone is not
known.

Secretion of growth hormone is controlled mainly by GHRH which induces
and SS which inhibits secretion (Thomer et al., 1998). Therefore, it would seem
reasonable to speculate that the effects of serotonin on secretion of growth
hormone could be mediated by GHRH and(or) SS. In fact, in the rat, GHRH has
been implicated as a mediator of serotonin-induced secretion of growth hormone
(Murakami et al., 1986). However, other hypothalamic peptides (T RH and

PACAP) also have been implicated in serotonin-induced secretion of growth

39

hormone in the rat (Chen and Ramirez, 1981; Yamauchi et al., 1996). I
hypothesized that GHRH and SS mediate serotonin-induced secretion of growth
hormone in cattle. A proposed model of this hypothesis is depicted in Figure 3.
My objective was to determine if activation of serotonin receptors would affect

release of GHRH or SS from cultured bovine hypothalamic slices.

MATERIALS AND METHODS

Perifusion of Bovine Hypothalamic Slices

Brain tissue was obtained from cattle killed at a local abattoir within 10 min
of death. Tissue was processed and cultured as described in a previous report
(West at al., 1997a) with slight modification. Briefly, brains with attached, intact
pituitary stalks were removed from the cranial cavity. A block of tissue containing
the thalamus, hypothalamus and pituitary stalk was dissected from each brain
with a scalpel. Coagulated blood and blood vessels were carefully removed with
scissors and forceps from the pituitary stalk and dorsal surface of the block of
tissue. The block was then bisected along the sagittal midline through the
thalamus, third ventricle and pituitary stalk. Using a custom made jig, one 1-mm
thick parasagittal slice was cut from the medial surface of each
hemihypothalamus. All extrahypothalamic tissue was removed, and each
hypothalamic slice was rinsed in ice-cold oxygenated CMF-HBSS. Each
hypothalamic slice was then placed in a 5 ml perifusion chamber and filled with

oxygenated, ice-cold minimal essential medium-0t (pH 7.4, MEM-a; Life

Technologies Inc.) supplemented with 25 mM HEPES, 0.1% bovine serum

40

Raphe Nuclei

Hypothalamus ®
Pituitary Stalk

Portal VesselO SS GHRH

K J

GH OW

milk, meat

 

Anterior Pituitary

 

 

Blood Vessel

 

Figure 3 Proposed model for GHRH and SS mediating serotonin-induced
secretion of growth hormone in cattle. Serotonergic neurons (oval) project
axons to hypothalamus that terminate on GHRH and SS neurons (ovals).
Terminals of GHRH and SS neurons are juxtaposed to portal vessels which
transport them to the anterior pituitary gland (AP) where they stimulate and inhibit
secretion of growth hormone, respectively.

41

albumin (A3859; Sigma Chemical Co.), penicillin (10 Ulml), streptomycin (10
ug/ml), ascorbic acid (1 glL; Merck and Co. Inc., Rathway, NJ), bacitracin (25
mglL; Sigma Chemical Co.), diprotin A (10 mglL; Peninsula Laboratories,
Belmont, CA) and soybean trypsin inhibitor (150 mg/L; Sigma Chemical Co.) that
had been sterilized by filtration. Chambers were then placed in a Kool MateTM
Series Thermoelectric Cooler and Warmer (Igloo Products Corp., Houston, TX)
that was maintained at approximately 4° C and transported back to the
laboratory.

Chambers containing hypothalamic slices were randomly assigned to
treatments and connected to peristaltic pumps in an incubator at 37°C. Slices
were perifused with oxygenated MEM-ot at a rate of 0.15 mein for 120 min to
establish stable basal secretion of GHRH and SS. After this initial period, slices
were used for experimental protocols. Treatments were dissolved in medium
and filtered into separate vials as described in Chapter 1: Perifusion of Bovine
Anterior and Posterior Pituitary Cells. During experiments, medium effluent
was collected in 3-ml fractions inside the incubator at 37° C. Fractions were
promptly removed from the incubator and stored at -20°C until assayed for

GHRH and SS as described by West and coworkers (1997a).

Experimental Protocols
This experiment was conducted across two consecutive weeks and two
trips to the local abattoir. Each week, sagittal hypothalamic slices (1 mm thick)

were perifused with MEMa at a flow rate of 0.15 ml per min. Medium effluent

42

was collected at 20-min intervals for 180 min. Pre-treatment samples were
collected at 20, 40 and 60 min. Treatments included control medium, or
quipazine at either 10‘ M, 10'8 M, or 10"° M dissolved in medium. Treatments
were given from 61 to 80 min. Slices were then perifused with medium from 81
to 180 min. The treatments were then re-randomized and slices perifused with
medium for 120 min to allow the tissue to recover. Beginning at 300 min the
experiment was then repeated. At the end of the second replicate each week,
slices were perifused with medium containing 60 mM KCI from 181 to 200 min, to
depolarize the secretory cells and determine tissue viability, then medium for an
additional 40 min. Growth hormone-releasing hormone and SS were measured
in all samples. Data from the first and second replicates each week were pooled
and aligned by time of treatment. Data from both weeks were also pooled (n = 3

and 5 per treatment for weeks one and two respectively).

Statistical Analysis

Areas under the response curve for GHRH and SS were calculated for the
interval between 61 and 120 min using the trapezoidal rule and subjected to
analysis of variance using Mixed Models procedures of SAS (SAS, 1999). To
account for differences in pre-treatment concentrations, concentrations of GHRH
and $8 at 60 min were used as covariates in their respective statistical analysis.
Week of experiment, replicate, treatment and covariate were included in the
model. Potassium chloride-stimulated release was calculated as the average

concentration of GHRH or SS in the three samples following KCI treatment.

43

Comparisons between average baseline concentrations (average concentration
from 20 to 60 min), and KCI-stimulated concentrations were conducted using

Student’s paired t-test (Gill, 1978).
RESULTS

Effect of Quipazine on Secretion of GHRH and SS from bovine
Hypothalamic Slices

Profiles of concentrations of GHRH and SS in medium effluent are
presented in Figures 4 (top panel) and 5 (top panel) respectively. Compared with
hypothalamic slices perifused with medium alone, perifusion with medium
containing 10"° M of quipazine, increased AUC for GHRH (P = 0.02; Figure 4
bottom panel). However, higher doses of the quipazine (10'6 and 10'“ M) did not
increase AUC for GHRH.

Perifusion of hypothalamic slices with medium containing quipazine did
not affect AUC for SS compared with hypothalamic slices perifused with medium
alone (Figure 5; bottom panel). All slices responded to perifusion with medium
containing 60 mM KCI by releasing both GHRH and SS, indicating that the tissue

was viable at the end of the experiment.

45° ' -0— Control
400 « —-— 10‘” M Quipazine
—‘— 10" M Quipazine
-0— 10‘6 M Quipazine

   

350 -
300 -
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GHRH (pg/ml)

 

 

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12000 -

9000 -

6000 -

GHRH ( pg 0 ml'1 min)

3000 -

A .s.

Control 10“°M 10°M 10 M
Quipazine Quipazine Quipazine

 

 

Figure 4. Effect of quipazine on secretion of GHRH from bovine
hypothalamic slices. TOP PANEL; Data presented are least squares mean
concentrations (:l: pooled standard error of mean; SEM) of GHRH in medium
effluent from bovine hypothalamic slices perifused with control medium (control;
n=8), 106 M quipazine (n=8), 10‘8 M quipazine (n=8), or 10° M quipazine (n=8).
Treatments were imposed from 61 to 80 min (indicated by the box on the X axis)
and slices were exposed to 60 mM KCI from 181 to 200 min (indicated by the box
on the X axis) to test tissue viability. BOTTOM PANEL; Statistical analysis was
performed on least squares mean AUC (+ pooled SEM) for GHRH calculated
from 61 to 120 min and adjusted for pre-treatment concentrations using the
concentration of GHRH at 60 min as a covariate. Quipazine at 10'10 M induced a
greater AUC than the AUC of controls (P= 0. 02).

45

 
 
  
   
  

 

 

         
   

  
   
  
 
 
 
 
  

   
    
   

  
 
  
 

      

  
  
  
   
 

      
   

       

      
   
   

     
     
  

      
      
     
  
 

  
   
 

 

       
  

   
       

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Figure 5. Effect of quipazine on secretion of SS from bovine hypothalamic
slices. TOP PANEL; Data presented are least squares mean concentrations (i
pooled standard error of mean; SEM) of SS in medium effluent from bovine
hypothalamic slices erifused with vehicle medium (control; n=8), 1043 M
quipazine (n=8), 10 M quipazine (n=8), or 10'10 M quipazine (n=8). Treatments
were imposed from 61 to 80 min (indicated by the box on the X axis) and slices
were exposed to 60 mM KCI from 181 to 200 min (indicated by the box on the X
axis) to test tissue viability. BOTTOM PANEL; Statistical analysis was
performed on least squares mean AUC (: pooled SEM) for SS calculated from
61 to 120 min and adjusted for pre-treatment concentrations using the
concentration of SS at 60 min as a covariate. Least squares means were not
different from controls.

46

DISCUSSION

Hypothalamic control of growth hormone secretion is mediated mainly by
GHRH and SS (T homer et al., 1998). In this experiment, I tested the hypothesis
that GHRH and (or) $8 mediate the effects of serotonin on growth hormone
secretion in cattle. The serotonin receptor agonist, quipazine, at the lowest dose
tested (10'10 M), significantly increased AUC for GHRH compared with controls,
indicating an increase in GHRH release. However, higher doses of quipazine
had no effect on AUC for GHRH compared with controls. Thus, the response in
secretion of GHRH was not a typical dose response. In addition, all slices,
including controls, demonstrated a slight increase in GHRH and SS secretion
during the time that treatments were imposed (Figures 4 and 5). In the current
experiment, activation of serotonin receptors with 10'10 M quipazine increased
AUC 18% over that of controls. This increase is very small compared to other
receptor agonists that induce secretion of GHRH.

In past experiments using a similar culture system, the az-adrenergic
receptor agonists clonidine and guanabenz each increased secretion of GHRH in
a dose responsive manner. The minimal dose of each agonist required to
increase AUC of GHRH was 10’5 M. Additionally, clonidine and guanabenz at
the highest dose tested (10" M) each increased AUC of GH 2-fold compared with
controls (West et al., 1997a). Therefore, it is possible that the small increase in
GHRH secretion induced by 10"° M quipazine in the current experiment was

merely an artifact of the culture system.

47

The increase in GHRH secretion in vitro contrast with the effects of
quipazine and GHRH on secretion of growth hormone in vivo. For example, a
previous report using a meal-fed steer model (feed available for 2 h each day)
shows that injection of quipazine either 1 h before feeding or 1 h after feeding
induced large increases in concentrations of growth hormone in serum (Gaynor
et al., 1995). Furthermore, the increases were of similar magnitude before
versus after feeding. In contrast, injection of GHRH induces a much greater
increase in concentrations of growth hormone in serum before compared with
after meal-feeding of steers (Moseley et al., 1988; McMahon et al., 2000). If
GHRH is the mediator of serotonin-induced secretion of growth hormone, then
GHRH-induced secretion of growth hormone would be expected to be of a similar
magnitude before and after meal—feeding. The serotonin receptor agonist that I
used in the current experiments was biologically active because the same lot of
material induced secretion of growth hormone in vivo (Chapter 3).

In conclusion, serotonin receptor agonist-induced secretion of growth
hormone in cattle is not mediated via GHRH or SS from the hypothalamus.

Therefore, I rejected the model proposed in Figure 3.

48

CHAPTER 3

Role of TRH and PACAP in Serotonergic Regulation of Growth Hormone

Secretion

49

INTRODUCTION

In Chapters 1 and 2, I concluded that serotonin receptor agonist-induced
secretion of growth hormone in vivo was neither a direct effect of the agonist on
the pituitary gland nor mediated via change in secretion of GHRH or SS from the
hypothalamus. However, two other hypothalamic neuropeptides have been
implicated in serotonin-induced secretion of growth hormone. Chen and Ramirez
(1981) reported that serotonin induced secretion of TRH from superfused rat
hemi-hypothalami in a dose responsive manner. However, in cattle, hemi-
hypothalami are too large to culture as one piece . To overcome this problem,
slices of bovine hypothalamic tissue (600 to 1000 pm thick) have been cultured
and medium effluent assayed for GHRH and SS. However, TRH neuronal cell
bodies are located in the paraventricular nucleus with axons projecting laterally
around the fomix then dorsally through the retrochiasmatic area to the median
eminence. Due to the course of TRH axons from the paraventricular nucleus to
the median eminence, it is impossible to cut a slice thin enough to maintain
tissue viability while maintaining axonal integrity. Therefore, experiments similar
to Chen and Ramirez (1981) could not be conducted with bovine hypothalami.
However, in this chapter, I describe in vivo experiments designed to test the
possibility that TRH mediates serotonin-induced secretion of growth hormone.

A second hypothalamic neuropeptide that may mediate serotonin-induced
secretion of growth hormone is PACAP. Pituitary adenylate cyclase-activating

polypeptide increases secretion of growth hormone from cultured bovine anterior

50

pituitary cells (Hashizume et al., 1994). Evidence for PACAP’s role in
serotonergic regulation of growth hormone comes from a report by Yamauchi
and coworkers (1996). In rats, intravenous injection of PACAP induced a dose
responsive increase in concentrations of growth hormone in plasma. The
PACAP-induced increase in growth hormone secretion was blocked with a
truncated form of PACAP called (PACAP (6-38)), which is a PACAP receptor
antagonist (Robberecht et al., 1992). Prior injection with PACAP (6-38) also
blocked 5-hydroxytryptophan- (serotonin precursor) induced growth hormone
secretion in these rats. Thus, blockade of PACAP receptors inhibited serotonin-
induced secretion of growth hormone indicating that PACAP could be mediating
serotonin-induced secretion of growth hormone.

To determine if either TRH or PACAP were possible mediators of
serotonin-induced secretion of growth hormone in cattle (Figure 6), I used the
meal-fed steer model. In this model, growth hormone secretion is synchronized
among a group of steers by allowing them ad libitum access to feed for only 2 h
each day. Growth hormone-releasing hormone-induced secretion of growth
hormone is suppressed 2 h alter feeding compared with 2 h before feeding
(Moseley et al., 1988; McMahon et al., 2000). In contrast, Gaynor and coworkers
(1996) reported that injection of a serotonin receptor agonist either 1 h before or
1 h after feeding induced quantitatively similar increases (AUC and peak
responses) in concentrations of growth hormone in serum. Therefore, if either
TRH or PACAP is the mediator of serotonergic regulation of growth hormone,

then their administration shortly before feeding would be expected to increase

51

 

Serotoni

 

 

  

 

 

Raphe Nuclei
? ?
Hypothalamus PACAP ®
Pituitary Stalk
Portal VesselO PACAP TRH O
+ -I-
Anterior Pituitary
Blood Vessel I GH Tm

milk, meat

Figure 6. Proposed model for TRH and PACAP mediating serotonin-
induced secretion of growth hormone in cattle. Serotonergic neurons (oval)
project axons to hypothalamus that terminate on TRH and PACAP neurons
(ovals). Terminals of TRH and PACAP neurons are juxtaposed to portal vessels
which transport them to the anterior pituitary gland (AP) where they stimulate
secretion of growth hormone.

52

concentrations of growth hormone in serum to a magnitude similar to their
administration after feeding. l hypothesized that injection of TRH or PACAP
before and after feeding would induce secretion of growth hormone to similar
magnitudes. Thus, my objectives were to determine: 1) if activation of serotonin
receptors with a nonspecific agonist would induce similar increases in growth
hormone secretion before and after feeding to confirm the work of Gaynor and
coworkers (1996), 2) a dose of TRH that would maximally stimulate secretion of
growth hormone before feeding, 3) if the dose of TRH determined in objective 2
would induce secretion of growth hormone to similar magnitudes before and after
feeding, 4) a dose of PACAP that would maximally induce secretion of growth
hormone before feeding and 5) if the dose of PACAP from objective 4 would
stimulate growth hormone secretion to similar magnitudes before and after

feeding.

MATERIALS AND METHODS

Management of Animals

Holstein steer calves were housed in rooms maintained at 20 1: 1° C with
18 h of light and 6 h of dark each day. Steers were fed a pelleted diet balanced
to meet nutrient requirements (NRC, 1989) that contained 18% crude protein and
19.6% acid detergent fiber (Land O’Lakes, Indianapolis, IN). Beginning at 8 to 10
weeks of age, steers were adapted to meal-feeding over a 14—d period by

gradually decreasing the amount of time feed was offered from 24 h to 2 h each

53

day. Thereafter, steers were allowed free access to feed from 1000 to 1200 h
each day. Water was available at all times. Steers were maintained on the
meal-feeding regimen for at least 14 d before an experiment was conducted.
Steers were weighed once a week for calculating drug doses.
Blood Sample Collection and Processing

Steers were fitted with indwelling jugular catheters (18 Ga Micro-
renethane®; Braintree Scientific Inc., Braintree, MA) at least 18 h before the
experiment began. A sterile solution of 3.5% sodium citrate was used to
maintain catheter patency. Blood samples (8 ml) were collected with 10 ml
syringes (Becton Dickenson & Co., Rutherford, NJ) and catheters immediately
flushed with 3.5% sodium citrate. Blood was transferred to 16 by 100 mm glass
tubes and stored at room temperature for 6 h and then at 4° C for 18 h. Blood
samples were then centrifuged at 1500 X g for 25 min and serum was harvested
and stored at —20° C until assayed. Concentrations of growth hormone in serum
were determined by radioimmunoassay as described previously (Gaynor et al.,
1995).
Experimental Protocols

Experiment 1 was designed to test the effect of quipazine before and after
feeding on concentrations of growth hormone in serum. Meal-fed Holstein steers

(21 :l: 0.6 weeks old, 131 :l: 8 kg BW) were randomly assigned to receive 0.2 mg

quipazine/kg BW (Gaynor et al., 1995) intravenously 1 h before feeding (0900 h;
n=4) or 1 h after feeding (1300 h; n=4) in a crossover design. Blood samples
were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915, 0920, 0930, 0940

and 1000 h from steers injected with quipazine at 0900 h. Blood samples were
collected at 1200, 1220, 1240, 1300, 1305, 1310, 1315, 1320, 1330, 1340 and
1400 h from steers injected with quipazine at 1300 h. The following day
treatments were reversed between the two groups of steers. Each steer served
as its own control by comparing pre-treatment with post-treatment concentrations
of growth hormone in serum.

Experiment 2 was a TRH dose-response experiment to determine the
minimal dose of TRH that would induce maximal secretion of growth hormone.
Six meal-fed Holstein steers (13.2 i 0.5 weeks old, 100 i 5 kg BW) were used in
a 6 X 6 Latin Square design, balanced for residuals, to test the effects of six
doses of TRH (0, 0.1, 0.3, 1, 3 and 10 pg TRH/kg BW) on secretion of growth
hormone on six different days. Each steer received each treatment one time and
treatment days were 48 h apart. On each treatment day, TRH (Sigma Chemical
00., St. Louis, MO) was dissolved in 0.9% saline solution, and each steer
received it’s assigned dose of TRH intravenously at 0900 h (before feeding).
Blood samples were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915,
0920, 0930, 0940 and 1000 h.

Experiment 3 was conducted to test the effect of TRH injection before and
after feeding on concentrations of growth hormone on serum. Dose of TRH used
was 0.3 uglkg BW as determined in the second experiment. This third
experiment was conducted in two replicates with eight Holstein steers per

replicate. In replicate one, steers averaged 20 i 0.5 weeks of age and 156 i 8

kg BW. In replicate two, steers averaged 15 i 0.6 weeks of age and 100 i 6 kg

55

BW. Each replicate was a crossover design with steers randomly assigned to
receive an intravenous injection of TRH either 1 h before feeding (0900 h) or 1 h
after feeding (1300 h) on the first treatment day. Times of TRH administration
were reversed the following day. Blood samples were collected at 0800, 0820,
0840, 0900, 0905, 0910, 0915, 0920, 0930, 0940 and 1000 h from steers injected
with TRH at 0900 h, and 1200, 1220, 1240, 1300, 1305, 1310, 1315, 1320, 1330,
1340 and 1400 h from steers injected with TRH at 1300 h.

Experiment 4 was a PACAP-38 dose-response experiment conducted in
two replicates to determine the minimal dose of PACAP-38 that would maximally
stimulate secretion of growth hormone. Each replicate used five Holstein steers
in a 5 X 5 Latin Square design to test the effects of five doses of PACAP-38
(Synpep Corp., Dublin, CA) on secretion of growth hormone. The first replicate

tested doses of 0, 0.1, 0.3, 1, and 3 pg PACAP-38/kg BW. Steers in replicate
one averaged 14 i 0.6 weeks of age and 96 i 3 kg BW. Because the highest
dose of PACAP-38 (3 pg/kg BW) was the only dose that increased
concentrations of growth hormone in serum, l repeated the experiment with
doses of 0, 1, 3, 10 and 10 pg PACAP-38Ikg BW. The 10 pg PACAP-38mg BW
dose was used twice to complete the 5 X 5 Latin Square which provided an n of
10. Steers in replicate two averaged 17 :l: 0.5 weeks of age and 111 i 4 kg BW.
In both replicates, treatment days were 48 h apart and on each treatment day
PACAP-38 was dissolved in 0.9% saline solution and respective treatments were

injected intravenously at 0900 h. Blood samples were collected at 0800, 0820,

56

0840, 0900, 0905, 0910, 0915, 0920, 0930, 0940 and 1000 h. Data from
replicates 1 and 2 were pooled for final statistical analysis.

Experiment 5 was conducted to determine the effect of PACAP-38
administered 1 h before and 1 h after feeding on secretion of growth hormone.
Eight Holstein steers (20.5 :t 1 week of age and 145 :t 9 kg BW) were used in a
crossover design where they were randomly assigned to receive an injection of
PACAP-38 (3 pg/kg BW, determined in Experiment 4) either before or after
feeding on the first day of sampling. Treatments were reversed on the second
day of treatment. Blood samples were collected at 0800, 0820, 0840, 0900,
0905, 0910, 0915, 0920, 0930, 0940 and 1000 h from steers injected with
PACAP at 0900 h, and 1200, 1220, 1240, 1300, 1305, 1310, 1315, 1320, 1330,
1340 and 1400 h from steers injected with PACAP-38 at 1300 h.

Statistical Analysis

In Experiments 1, 2 and 3, AUCs of growth hormone were calculated for
60 min following treatment using the trapezoidal rule. In Experiment 4 and 5,
AUCs were calculated for 40 min following treatment because growth hormone
secretion had returned to basal levels. In all five experiments, the highest
concentration of growth hormone in serum during the 60 min period following
treatment was determined as the peak concentration of growth hormone. Both
AUC and peak concentrations were subjected to analysis of variance using
Mixed Models procedures of SAS (SAS, 1999). In Experiments 3 and 4, data for
AUCs and peak concentrations of growth hormone were transformed by natural

logarithm (LN) to achieve homogeneous variance and normality. To account for

57

differences in pre-treatment concentrations of growth hormone in serum in all
experiments, concentrations of growth hormone in serum immediately before
treatment were used as covariates in the statistical analysis. The statistical
model for Experiments 1 and 2 included the covariate, animal, treatment and
sampling day. In Experiment 3, the model included the covariate, animal,
replicate, treatment, sampling day, and interactions of experiment by sampling
day and experiment by treatment. The statistical model for Experiment 4
included the covariate, replicate, animal within replicate, day within replicate and
treatment. The statistical model for Experiment 5 included the covariate, animal
and treatment. Sampling day was not significant (P > 0.25); therefore, it was
removed from the statistical analysis to add another degree of freedom to the

error term.

RESULTS

Effect of Quipazine Before and After Feeding on Secretion of Growth
Hormone (Experiment 1)

All serum samples from this experiment were assayed for growth hormone in a
single assay with an intra-assay coefficient of variation of 11.6%. Basal secretion
of growth hormone was greater before feeding than after. Injection of quipazine
increased concentrations of growth hormone in serum both before and after

feeding (P < 0.05; Figure 7). Furthermore, injection of quipazine 1 h after feeding

58

  
 

   

30 -
Quipazine

I

Quipazine

  

Feeding

20-

  

GH (nglml)

101

 

 

o I I I I I

0800 0900 1000 1200 1300 1400
Time (h)

1400
1200
1000
800
600
400
200

 

 

GH AUC (ng - ml'1 min)

Before After
Feeding

Figure 7. Effect of quipazine on secretion of GH in meal-fed steers. TOP
PANEL; Data presented are mean concentrations (1: standard error of mean;
SEM) of growth hormone in serum of steers injected with 0.2 mg serotonin
receptor agonist (quipazine)lkg BW either 1 h before (n=8) or 1 h after (n=8)
feeding. Arrows indicate times of injection. BOTTOM PANEL; Statistical
analysis was performed on least squares mean AUC (i pooled SEM) calculated
over 60 min from time of injection and adjusted by covariance using
concentrations of growth hormone in serum immediately before injection of
quipazine. Least squares mean AUC before versus after feeding was not
different (P = 0.7).

59

induced a similar growth hormone AUC increment as injection of quipazine 1 h
before feeding (P = 0.71; Figure 7). Injection of quipazine before and after
feeding induced similar increments in peak concentrations of growth hormone
(Figure 8).

Effect of Various Doses of TRH on Secretion of Growth Hormone
(Experiment 2)

In this experiment, all serum samples were assayed for growth hormone in
a single assay with an intra-assay coefficient of variation of 15.8%. Compared
with controls, injection of TRH at 0.1 and 10 pg/kg BW did not significantly
increase AUC of growth hormone, while TRH at 0.3, 1 and 3 pg/kg BW
significantly increased AUC of growth hormone (Figure 9). Compared with
controls, 0.3 pg TRH/kg BW was the only dose that significantly increased least
squares mean peak concentrations of growth hormone in serum (Figure 10).

My objective was to determine a minimal dose of TRH that would
maximally stimulate secretion of growth hormone. Therefore, 0.3 pg TRH/kg BW
was chosen to be used in Experiment 3. -
Effect of TRH Before or After Feeding on Secretion of Growth Hormone
(Experiment 3)

In this experiment, serum samples were assayed for growth hormone in a
two separate assays with intra-assay coefficients of variation of 7.1% and 11.2%
and an inter-assay coefficient of variation of 7.1%. As in Experiment 1, basal
secretion of growth hormone was greater before feeding than after. Injection of

TRH increased concentrations of growth hormone in serum both before and after

60

Peak GH (nglml)

 

 

Before After
Feeding

Figure 8. Effect of quipazine on peak concentrations of GH in meal-fed
steers. Data presented are least squares mean peak concentration (1 pooled
standard error of mean) of growth hormone in serum of steers injected with 0.2
mg serotonin receptor agonist (quipazine)lkg BW either 1 h before (n=8) or 1 h
after (n=8) feeding. Peak concentration of growth hormone following injection of
quipazine was not different before versus alter feeding (P = 0.55).

61

60q

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GH (nglml)

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+ 0.3 ug TRH
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steers. TOP PANEL; Data presented are mean concentrations (3: standard error
of mean, SEM) of growth hormone in serum of steers injected with saline
(control; n=6) or TRH (0.1, 0.3, 1, 3 or 10 pg/kg BW; n=6 per dose). Arrow
indicates time of injection. BOTTOM PANEL; Statistical analysis was performed
on least squares mean AUC (i pooled SEM;) calculated over 60 min from time of
injection and adjusted by covariance using concentrations of growth hormone in
serum immediately before injection. Least squares means with * differ from
control (P < 0.02).

62

Peak GH (ngIML)

 

 

Control 0.1 0.3 1 3 10
TRH (uglkg BW)

Figure 10. Effect of various doses of TRH on peak concentrations of GH in
meal-fed steers. Data presented are least squares mean peak concentrations (:t
pooled standard error of mean) of growth hormone in serum of steers after
injection of saline (control; n=6) or TRH (0.1, 0.3, 1, 3 or 10 pg/kg BW; n=6 per
dose). Least squares mean with * differs from control (P < 0.01).

63

 

feeding (P < 0.05; Figure 11), and the magnitude and pattern of the increase was
similar at both times. Injection of TRH after feeding induced a similar increase in
growth hormone AUC as injection of TRH before feeding (P = 0.22; Figure 11).
Injection of TRH increased least squares mean peak concentrations of
growth hormone to similar magnitudes before versus after feeding (Figure 12).

Effect of Various Doses of PACAP on Secretion of Growth Hormone

3“!

(Experiment 4)

In this experiment, serum samples were assayed in two separate assays

M:

with intra-assay coefficients of variation of 11.9% and 9.0% and an inter-assay
coefficient of variation of 13.7%. When adjusted for pre-treatment differences in
concentrations of growth hormone in serum, injection of PACAP-38 at 0.1, 0.3, 1
and 10 pg/kg BW did not significantly increase growth hormone AUC compared
with controls. However, injection of PACAP-38 at 3 pg/kg BW tended to increase
growth hormone AUC (P = 0.09; Figure 13). When adjusted for differences in
pre-treatment concentrations of growth hormone in serum, peak concentrations
of growth hormone after injection of 0.1, 0.3 and 1 pg PACAP-38kg BW were not
different from control (Figure 13). However, compared with controls, injection of
3 or 10 pg PACAP-38lkg BW increased mean peak concentrations of growth
hormone in serum. Since my objective was to determine a minimal dose of
PACAP-38 to maximally stimulate secretion of growth hormone, 3 pg PACAP-

38/kg BW was used in the subsequent experiment.

64

TR I TRH

 

 

  

 

 

3.0-. I I
g | |
20 - a)
a: .5'
5 3|
I
(D 10 :_ I2
« l
0 - z I
0800 0900 1000 1200 1300 1400
Time (h)
7 .
E 6-
85 s-
5‘ E 4 -
5 .
cl 3 '
I l:
o . 2-
Z
a 1-
o _ . it: M... .v.. .1 .. H...
Before After
Feeding

Figure 11. Effect of TRH on secretion of GH before and after feeding in
meal-fed steers. TOP PANEL; Data presented are mean concentrations (1
standard error of mean; SEM) of growth hormone in serum of steers injected with
0.3 pg TRH/kg BW either 1 h before (n=16) or 1 h after (n=16) feeding. Arrows
depict times of injection. BOTTOM PANEL; Statistical analysis was performed
on least squares means AUC transformed by natural logarithm (i pooled
SEM;GH LN-AUC;) calculated over 60 min from time of injections and adjusted
by covariance using concentrations of growth hormone in serum immediately
before injection of TRH. Least squares mean LN-AUC before versus after
feeding was not different (P = 0.22).

65

 

w

LN-PEAK GH
(LN nglml)
N

1%

     

and.“ 1 .

Before After
Feeding

 

Figure 12. Effect of TRH on peak concentrations of GH before and after
feeding in meal-fed steers. Data presented are least squares mean peak
concentrations (1 pooled standard error of mean) of growth hormone in serum
(transformed by natural logarithm) of steers injected with TRH (0.3 pg/kg BW)
either 1 h before (n=16) or 1 h after (n=16) feeding. LN-Peak concentration of
growth hormone following injection of TRH before versus after feeding was not
different (P = 0.85).

66

  

GH LN-AUC
(LN-ng - ml'1 min)

   

 

 

Control 0.1 0.3 1 3 10
PACAP (uglkg BW)

3.
(:5:
xé :-
so:
we
‘tz
2:1 1-
.l

 

 

Control 0.1 0.3 1 3 10
PACAP (uglkg BW)

Figure 13. Effect of various doses of PACAP on secretion of GH in meal-
fed steers. TOP PANEL; Data presented are least squares mean AUC (i
pooled standard error of mean; SEM) transformed by natural logarithm (LN-GH
AUC). BOTTOM PANEL; least squares mean peak concentrations (:I: pooled
SEM) of growth hormone in serum (transformed by natural logarithm; LN-Peak)
of steers injected with saline (control; n=10) or PACAP (0.1 (n=5), 0.3 (n=5), 1
(n=10), 3 (n=10) or 10 (n=10) pg/kg BW) calculated over 40 min from time of
injection and adjusted by covariance using concentrations of growth hormone in
serum immediately before injection. Least squares means with * differ from
control (P < 0.01), 1' differs from control (P < 0.1).

67

 

Effect of PACAP Before and After Feeding on Secretion of Growth
Hormone (Experiment 5)

All serum samples from this experiment were assayed for growth hormone in a
single assay with an intra-assay coefficient of variation of 11.6%.Two steers that
were supposed to receive an injection of PACAP-38 1 h after feeding were
removed from the data set because a vial of PACAP-38 was accidentally broken.
Therefore, the number of steers that received PACAP-38 after feeding was 6. As
reported in Experiments 1 and 3, basal secretion of growth hormone was greater
before feeding than after. Injection of PACAP-38 increased concentrations of
growth hormone in serum both before and after feeding (P < 0.05). However,
injection of PACAP-38 1 h before feeding induced greater AUC of growth
hormone than injection of PACAP-38 1 h after feeding (P = 0.05; Figure 14).
Injection of PACAP 1 h before feeding induced a greater least squares mean
peak concentration of growth hormone than injection 1 h after feeding (Figure

15).
DISCUSSION

Basal secretion of growth hormone was greater before feeding than after
in Experiments 1, 3 and 5 which confirms the work of Moseley and coworkers
(1988) and Gaynor and coworkers (1996) showing that feeding suppresses
secretion of growth hormone. In Experiment 1 of this chapter I repeated the work

of Gaynor and coworkers (1996). My results confirm their findings, that

68

25. PACAP

 

 

   

 

   

20 - I l
E I l PACAP
. m
‘°’ ‘5 l=l
Si ‘6
d)
I 10 " 0
(D ILL l
5 :.
S.
0 . , , . .
0800 0900 1000 1200 1300 1400
Time (h)
*
,E 500 -
E
1; 400 -
E
‘ 300 -
O)
C
V 200 -
0
D
< 100 -
I ;s
w o _ ans—em .1 4a., .so- ..a;~Le\-I.L
Before After
Feeding

Figure 14. Effect of PACAP on secretion of GH before and after feeding in
meal-fed steers. TOP PANEL; Data presented are mean concentrations (i
standard error of mean; SEM) of growth hormone in serum of steers injected with
3 pg PACAP/kg BW either 1 h before (n=8) or 1 h after (n=8) feeding. Arrows
indicate times of injection. BOTTOM PANEL; Statistical analysis was performed
on least squares mean AUC (d: pooled SEM) calculated over 60 min from time of
injection and adjusted by covariance using concentrations of growth hormone in
serum immediately before injection of PACAP. Least squares mean AUC before
feeding differs from AUC after feeding (P = 0.05).

69

 

25-

 

 

 

20 -
:c A .
0 E 15
x \
“ 2’
‘1’ v 10 -

5 ..

0

Before After
Feeding

Figure 15 Effect of PACAP on peak concentrations of GH before and after
feeding in meal fed steers. Data presented are least squares mean peak
concentration (3: pooled standard error of mean) of growth hormone in serum of
steers injected with PACAP (3 pg/kg BW) either 1 h before (n=8) or 1 h after
(n=8) feeding. Peak concentration of growth hormone following injection of
PACAP was greater before versus after feeding (P = 0.002)

70

 

administration of a nonselective serotonin receptor agonist stimulates secretion
of growth hormone to similar magnitudes before and after feeding in meal-fed
steers. These data suggest that the pituitary gland does not become refractory
after feeding to serotonin-induced secretion of growth hormone. Since quipazine
does not directly affect secretion of growth hormone from cultured anterior
pituitary cells, it was prudent to investigate the roles of other known hypothalamic
neuropeptides as possible mediators of serotonin-induced growth hormone
secretion.

Previous experiments showed that GHRH-induced secretion of growth
hormone is much greater before versus after feeding (Moseley et al., 1988;
McMahon et al., 2000). In contrast, quipazine induces similar increases in
secretion of growth hormone before and after feeding. Furthermore, I showed
that quipazine does not affect secretion of GHRH from cultured hypothalamic
slices (Chapter 2). Collectively, these two experiments suggested it is unlikely
that GHRH is the mediator of serotonin-induced secretion of growth hormone.
Therefore, some other hypothalamic neuropeptide must mediate serotonin’s
effects on secretion of growth hormone. In this chapter, I tested the likelihood of
two other hypothalamic peptides as mediators of serotonin-induced secretion of
growth hormone, namely TRH and PACAP.

Earlier work in our laboratory determined that the minimal dose of TRH
required to maximally stimulate secretion of prolactin from the anterior pituitary
gland of cattle was between 50 and 100 pg per heifer (Vines, 1976). However,

Vines (1976) also reported that maximal secretion of growth hormone was not

71

 

achieved even with the highest dose of TRH tested. Therefore, it was necessary
for me to conduct a dose-response experiment to determine a minimal dose of
TRH that would maximally stimulate secretion of growth hormone. In Experiment
2, I determined that 0.3 pg TRH/kg BW was the minimal dose of TRH to
maximally increase both peak concentrations of growth hormone and AUC
calculated over a 60-min period after injection of TRH. This dose of TRH is
similar to the dose of TRH required to maximally stimulate secretion of prolactin
determined by Vines (1976). In fact, when Vines’ doses are converted to a per
kg BW basis using the median weight of the heifers in his experiment, the 50 pg
dose used converts to approximately 0.36 pg TRH/kg BW. The highest dose of
TRH that Vines administered was approximately 0.73 pg/kg BW. In the current
experiment there were three doses (1, 3 and 10 pg/kg BW) larger than the 0.3
pig/kg BW. Compared with controls, 0.3 pg TRH/kg BW was the only dose that
increased peak concentrations of growth hormone, although 0.3, 1 and 3 pg
TRH/kg BW increased AUC of growth hormone.

An interesting observation from this experiment is that the doses of TRH
greater than 0.3 pg/kg BW increased release of growth hormone for a longer time
Period. In fact concentrations of growth hormone in serum after administration of
TRH at doses of 1, 3 and 10 pg/kg BW were still greater than controls 60 min
after injection. One explanation for this is that TRH receptors are internalized in

reeponse to agonist binding (Dmlota et al., 1998b). In another report, TRH at 5 X
1 0‘10 M did not induce receptor lntemalization while TRH at 5 X 10'9 induced

massive receptor internalization. lntemalization occurred within 3 to 5 min and

72

if .._—: ‘r-..I:—?‘

was maintained for 30 min (Drrnota et al., 1998a). In the current experiment it
appears that the higher doses induced a prolonged release of growth hormone.
Perhaps the higher doses induced receptor lntemalization and desensitization. If
circulating concentrations of TRH were still high when the receptors were
recycled, they could be reactivated and prolong secretion of GH. l speculate that

if I had collected blood samples for an additional hour, AUC of growth hormone

"; '11

x-» A
L- ’ ‘ ..

would have been similar in steers receiving TRH at doses from 0.3 to 10 pglkg

It’-

BW. Therefore, I concluded that 0.3 pglkg BW was an optimal dose in that it was
the minimal dose that would maximally stimulate secretion of growth hormone.
Thus, I decided to use the 0.3 pglkg BW dose in subsequent experiments.

Based on the following information I hypothesized that some hypothalamic
peptide other than GHRH or SS mediates quipazine’s actions on secretion of
growth hormone. Activation of serotonin receptors induces similar increases in
growth hormone secretion before versus after feeding, and this effect is not
exerted directly on the anterior pituitary gland, Clonidine, an a2-adrenergic
receptor agonist induces secretion of GHRH (West et al., 1997a). Both GHRH
and clonidine induce greater secretion of growth hormone before versus after
feeding (Moseley et al., 1988; Gaynor et al., 1993; McMahon et al., 2000). If
TRH is the mediator of serotonin receptor agonist-induced secretion of growth
hormone, then TRH-induced secretion of growth hormone would be expected to

be of similar magnitude before and after feeding. In Experiment 3 of this chapter,
TRH was administered to meal-fed steers before and after feeding. Indeed,

ir1jection of TRH before and after feeding induced similar peak concentrations of

73

growth hormone and AUC. These data suggested that TRH could be the
mediator of serotonin-induced growth hormone secretion in cattle.

Another neuropeptide that could mediate serotonin-induced secretion of
growth hormone is PACAP. Treating bovine anterior pituitary cells with PACAP
increased secretion of growth hormone (Hashizume et al., 1994). However,

when sheep were treated with PACAP, there was no effect on concentration of

. rue-ta

growth hormone in serum (Sawangjaroen and Curlewis, 1994). The effects of
PACAP on secretion of growth hormone in cattle have not been investigated. In 2.
Experiment 4 of this chapter, PACAP was administered at six different doses.
When the data were adjusted for differences in pre-treatment concentrations of
growth hormone, the 3 pglkg BW dose was the only dose that tended to
increase growth hormone AUC (P < 0.1). However, both the 3 and 10 pglkg BW
dose increased peak concentrations of growth hormone (P < 0.01). Thus, I
concluded that PACAP does increase concentrations of growth hormone in
serum of cattle. The discrepancy between my data in cattle and the results of
Sawangjaroen and coworkers (1994) in sheep may be explained by close and
(or) injection protocol. Sawangjaroen infused a total of 10 nM PACAP over a 10-
rnin period into the carotid artery. I administered doses that ranged from 2.2 nM
to 220 nM for a 100 kg BW steer as a bolus injection into the jugular vein. The
two highest doses (66 and 220 nM for a 100 kg steer) were the only doses that
increased growth hormone secretion.
Injection of PACAP induced a greater AUC of GH before feeding than

a1‘I:er. Because quipazine induces similar increments in secretion of growth

74

hormone before and after feeding, and PACAP does not, I suggest that PACAP
is not the mediator of serotonin-induced secretion of growth hormone. These
results are explainable. Feeding also suppresses GHRH-induced secretion of
growth hormone. Both GHRH and PACAP activate adenylate cyclase and
increase intracellular cAMP (Mayo et al., 1995; Wong et al., 1995; Miyata et al.,

1989). If the post-feeding suppression of growth hormone secretion is due to

"‘3

inhibition of adenylate cyclase then it is reasonable that PACAP-induced

[E ‘7 "_—"'-'3"““.

secretion of growth hormone would also be suppressed after feeding. In
contrast, TRH uses a phospholipase C second messenger system (Aragay et al.,
1992; Hsieh and Martin, 1992; Svoboda et al., 1996), and it induces release of
similar amounts of growth hormone before and after feeding. In addition,
serotonin induces secretion of TRH from rat hypothalamic tissue in culture (Chen
and Ramirez, 1981).

.In conclusion, these results support the hypothesis that TRH mediates
serotonin-induced secretion of growth hormone and further suggest that PACAP
is not a mediator of serotonin-induced secretion of growth hormone. Therefore, I
rejected the portion of the model proposed in Figure 6 depicting serotonergic
neurons stimulating the secretion of PACAP which in turn stimulates secretion of

growth hormone from the anterior pituitary gland.

75

CHAPTER 4

Serotonin-Induced Secretion of Growth Hormone in Hyperthyroid Meal-fed

Steers

76

INTRODUCTION

Thus far, evidence described in this dissertation suggests that TRH may
mediate serotonin-induced secretion of growth hormone in cattle. However,
there has been no direct test of this hypothesis. One way to test this hypothesis

would be to use a TRH receptor antagonist to block TRH receptors on the

. *‘fi-l

anterior pituitary gland, then challenge with a serotonin receptor agonist and

measure subsequent increments in secretion of growth hormone.
Benzodiazepines have been shown to inhibit TRH-induced secretion of thyroid
stimulating hormone (TSH) and growth hormone from perifused rat pituitary
glands (Roussel et al., 1986). In prior experiments not described in this thesis, I
attempted to block TRH receptors in meal-fed steers with a benzodiazepine
called chlordiazepoxide. In vitro, chlordiazepoxide is a competitive TRH receptor
antagonist (Simasko and Horita, 1984; Drummond, 1985; Joels and Drummond,
1989). In vivo, chlordiazepoxide has anxiolytic and sedative effects
(Baldessarini, 1993). During my attempts to block TRH receptors in meal-fed
steers, chlordiazepoxide was more potent as a sedative than as a TRH receptor
antagonist. Therefore, chlordiazepoxide was not a suitable TRH receptor
antagonists to test my hypothesis.
Another way to block TRH effects on the anterior pituitary gland would be
to use negative feedback of 3, 3’, 5-triiodo-L-thyronine (T 3) to down regulate TRH
r eceptors on the anterior pituitary gland. In addition, T3 feeds back negatively on

the hypothalamus.

77

In the anterior pituitary gland and on GH3 cells, T3 inhibits expression of
TRH receptors (Lean et al., 1977; Perrone and Hinkle, 1978). In pituitary tumor
cell lines, T3 increases the rate of growth hormone mRNA accumulation
(Samuels and Shapiro, 1976; Seo et al., 1977; Martial et al., 1977). However, in
GH3 cells, the concentration of T3 required to induce a half maximal increase in
growth hormone mRNA synthesis is 45-fold greater than the concentration
required to induce a half maximal loss of TRH receptors (Hinkle et al., 1979).
Therefore, it should be possible to down regulate TRH receptors without affecting
synthesis of growth hormone.

Within the hypothalamus, T3 inhibits synthesis of TRH mRNA in neurons in
the paraventricular nucleus. These neurons control the secretion of TSH from
the anterior pituitary gland. This effect is selective, because T3 has no effect on
TRH mRNA synthesis in TRH neurons in other regions of the brain (Segerson et
al., 1987; Koller et al., 1987; Dyess et al., 1988).

Hormonal hyperthyroidism has been induced in cattle with daily injections
of T3 dissolved in corn oil (Thrift et al., 1999). Daily injections of T3 increased
concentrations of T3 and decreased concentrations of 3, 3’, 5, 5’-tetraiodo-L-
thyronine (thyroxin; T4) in serum to below the detection limit of the assay. Since
T4 is the predominant form released from the thyroid gland, these data indicate

that T3 injections effectively shut down the thyroid axis.

If daily injections of T3 can effectively shut down the thyroid axis, negative

~.._r::':?'.‘t'll

*-
_‘_am

13:.

feedback must be exerted at the level of the hypothalamus and (or) the pituitary

Q'and, or even at the level of the thyroid gland (Figure 16). Negative feedback at

78

Hypothalamus O'?’

TRH
- Anterior
/ Pituita

T3

l

T4 TSH

 

 

     
   
  

 

Growth Hormone

 

Thyroid +
Gland

Figure 16. Model of negative feedback of 3,3’,5-triiodo-L-thyronine (T3) on
thyrotropin releasing-homone (TRH) neurons in the hypothalamus and the
anterior pituitary gland. Injecting exogenous T3 (bold arrow) will increase
negative feedback on TRH neurons and (or) the anterior pituitary gland thereby
reducing thyroid stimulating hormone (TSH) secretion from the anterior pituitary
gland and 3, 3’, 5, 5’-tetraiodo-L-thyronine (T 4) secretion from the thyroid gland as
well as the ability of serotonin to induce secretion of growth hormone from the

anterior pituitary gland.

 

 

79

the level of the hypothalamus would decrease TRH synthesis and release, which
may be induced by afferent neurons such as serotonin. Negative feedback at the
pituitary gland could cause a decrease in TSH synthesis and (or) TRH receptor
expression. If daily injections of T3 decrease either TRH synthesis or TRH
receptor expression and TRH is the mediator of serotonin-induced secretion of

growth hormone, then the increase in growth hormone secretion induced by a

serotonin receptor agonist should be suppressed in steers chronically treated

 

‘1 Al 54-74
t.,.-

with T3. I hypothesized that daily injections of T3 would inhibit TRH- and
quipazine-induced secretion of growth hormone. Therefore, my objectives were
to determine if daily injections of T3 affected: 1) basal secretion of growth
hormone in meal-fed steers, 2) releasable amounts of growth hormone from the
anterior pituitary gland by measuring GHRH-induced secretion of growth
hormone, 3) TRH-induced secretion of growth hormone and 4) quipazine-

induced secretion of growth hormone.

MATERIALS AND METHODS

Management of Animals

Holstein steer calves were adapted to meal-feeding and housed as
described in Chapter 3.
Blood Sample Collection and Processing

Blood samples were collected, processed and assayed as described in

Chapter 3. In addition, selected blood samples were also assayed for total T4

80

using a commercially available kit (Diagnostic Products Corp., Los Angeles, CA)
validated for bovine serum (Williams et al., 1987).
Experimental Protocols

Experiments in this chapter were designed to test the effects of daily T3
(Sigma Chemical Co., St Louis, MO) injections on basal, GHRH-, TRH- and
quipazine-induced secretion of growth hormone. In the first experiment Holstein
steers (138.5 1 6 kg BW: 20 i 0.5 weeks of age) were injected subcutaneously
each day, 2 h before meal-feeding (1000 to 1200 h) with T3 (3 pglkg BW; n=4)
dissolved in corn oil to induce negative feedback on the hypothalamic-pituitary-
thyroid axis thereby decreasing TRH receptor expression and(or) TRH synthesis
in the hypothalamus. Controls (n=4) received corn oil. Fresh T3 was initially
dissolved in 100% ethanol to a concentration of 5 mglml then diluted with corn oil
to a final concentration of 500 pglml every third day. Ethanol was also added to
corn oil, at the same ratio as that used to dissolve T3. Injection volumes were
adjusted for changes in BW once per week to maintain a constant dose of T3.
Two days before commencement of T3 treatment (day 0), all steers were fitted
with indwelling jugular catheters. On days -1, +5 and +10 relative to initiation of
T3 treatment, blood samples were collected at 20-min intervals from 0800 h to
1400 h to be assayed for growth hormone to determine if T3 treatment affected
basal secretion of growth hormone in meal-fed steers. Samples collected at
0900 h were also assayed for total T4 concentrations to determine secretory

activity of the thyroid gland as an index of the effectiveness of T3 treatment.

81

4.—

After 20 d of T3 treatment, meal-fed steers were again fitted with indwelling
jugular catheters. On day +21, steers (within thyroid status; i.e. within corn oil
and T3—treated groups) were randomly assigned to receive an intravenous
injection of either GHRH (0.2 pglkg BW) dissolved in sterile water or sterile water
at 0900 h in a crossover design. The following day treatments were reversed.
Blood samples were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915,

0920, 0930, 0940, 1000 and 1300 h on each treatment day.

Ila-1L ”I’m 3...:

On day +24 of T3 treatment, meal-fed steers (within thyroid status) were
randomly assigned to receive an intravenous injection of either TRH (0.3 pglkg
BW) dissolved in sterile 0.9% saline solution or sterile 0.9% saline solution at
0900 h in a crossover design. The following day treatments were reversed.
Blood samples were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915,
0920, 0930, 0940, 1000 and 1300 h on each treatment day.

On day +27 of T3 treatment, meal-fed steers (within thyroid status) were
randomly assigned to receive an intravenous injection of either quipazine (0.2
mg/kg BW) dissolved in sterile 0.9% saline solution or sterile 0.9% saline solution
0900 h in a crossover design. The following day treatments were reversed.
Blood samples were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915,
0920, 0930, 0940, 1000 and 1300 h on each treatment day.

Sera harvested from blood samples from 0800 to 1000 h on days +21,
+22, +24, +25, +27 and +28 were assayed for growth hormone. Sera from blood
samples taken at 0900 and 1300 h on days +21, +22, +24, +25, +27 and +28

were assayed for total T4.

82

The previous experiment was repeated with another group of meal-fed
steers. However, in this experiment the dose of T3 was doubled, and the order in
which the growth hormone secretagogues were administered was rearranged to
control for any carry over effects. Holstein steers (108.8 i 4 kg BW: 16.5 i 0.4
weeks of age) were injected subcutaneously each day with either corn oil (n=4)
or T3 (6 pglkg BW; n=4) dissolved in corn oil. Fresh T3 and vehicle were

prepared every third day as in the first experiment.

.4

E33531!
._ - -

{ifl—:7

After 22 d of T3 treatment, meal-fed steers were fitted with indwelling
jugular catheters. On day 23, steers (within thyroid status) were randomly
assigned to receive an intravenous injection of either quipazine (0.2 mglkg BW)
dissolved in sterile 0.9% saline solution or sterile 0.9% saline solution at 0900 h
in a crossover design. The following day treatments were reversed. Blood
samples were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915, 0920,
0930, 0940, 1000, 1100, 1200, 1300, 1400 and 1500 h on each treatment day.

On day 26 of T3 treatment, meal-fed steers (within thyroid status) were
randomly assigned to receive an intravenous injection of either GHRH (0.2 pglkg
BW) dissolved in sterile water or sterile water at 0900 h in a crossover design.
The following day treatments were reversed. Blood samples were collected at
0800, 0820, 0840, 0900, 0905, 0910, 0915, 0920, 0930, 0940, 1000, 1100, 1200.
1300, 1400 and 1500 h on each treatment day.

On day 29 of T3 treatment, meal-fed steers (within thyroid status) were

randomly assigned to receive an intravenous injection of either TRH (0.3 pglkg

BW) dissolved in sterile 0.9% saline solution or sterile 0.9% saline solution at

83

0900 h in a crossover design. The following day treatments were reversed.
Blood samples were collected at 0800, 0820, 0840, 0900, 0905, 0910, 0915,
0920, 0930, 0940, 1000, 1100, 1200, 1300, 1400 and 1500 h on each treatment
day.

Sera harvested from all blood samples collected from 0800 to 1000 h were

assayed for growth hormone. Sera harvested from blood samples collected at

.31

0800, 0900, 1000, 1100, 1200, 1300, 1400 and 1500 h were assayed for total T4.

Statistical Analysis

To determine the effects of T3 treatment on basal secretion of growth
hormone, total AUC of growth hormone secretion was calculated from 0800 to
1400 h for corn oil- and T3-treated steers on days -1, 5 and 10 relative to
initiation of T3 treatment in the first experiment (day 0). Total AUC and
concentrations of total T4 were subjected to analysis of variance using Mixed
Models procedures of SAS (SAS, 1999). The statistical model included steer
nested within T3 treatment, T3 treatment, day and T3 treatment by day
interaction. Total AUC of growth hormone and total T4 concentrations of control
and T3-treated steers were compared on each day.

Secretion of growth hormone in response to growth hormone
secretagogues (GHRH, TRH and quipazine) was estimated by calculating total
AUC of growth hormone from 0900 h (time of injection) to 1000 h using the
trapezoidal rule. Secretion of growth hormone (AUC) in response to an injection

of GHRH, TRH or a quipazine in the steers injected with 3 pg T3lkg BW (first

experiment) was compared to the responses in growth hormone secretion to an

injection of GHRH, TRH or a quipazine in the steers injected with 6 pg T3/kg BW
(second experiment) using a students t-test (Gill, 1978). The dose of T3 (3 vs. 6
pglkg BW) did not affect secretion of growth hormone in response to GHRH (P =
0.83), TRH (P = 0.61) or quipazine (P = 0.86). Therefore, data were pooled and
subsequent analyses were conducted to compare T3-treated steers (regardless
of dose) with steers injected with corn oil.

Total AUC of growth hormone was calculated from 0900 to 1000 h for
GHRH, TRH and quipazine challenges and subjected to analysis of variance
using Mixed Models procedures of SAS (SAS, 1999). The statistical model
included concentration of growth hormone at 0900 as a covariate when
significant (P < 0.25), steer nested within T3 treatment, experiment, T3 treatment,
challenge, T3 treatment by challenge interaction and day when significant (P <
0.25). Data for basal secretion of growth hormone and peak concentrations of
growth hormone from the TRH challenge and quipazine were transformed by

natural logarithm to achieve homogeneous variance before statistical analysis.

RESULTS

Effects of T3 on basal, GHRH-, TRH- and Quipazine-Induced Secretion of
Growth Hormone and Total T4

In the first experiment in which half of the steers were injected with 3 pg

T3/kg BW, serum samples from days —1, +5 and +10 were assayed for growth

hormone in a single assay with an intra-assay coefficient of variation of 14.6%.

85

Total AUC of growth hormone from 0800 to 1400 h for T3-treated steers was not
different from corn oil-injected steers on days —1, +5 or +10 of T3 treatment
(Figure 17).

In contrast, by day +5, T3-treated steers had significantly lower
concentrations of total T4 in serum compared with com oil-injected steers (Figure

18). On day +10, concentrations of total T4 in serum of T3-treated steers were

- . “a
a.”
1

below the detection limit of the assay (1 pg/dl).

Serum samples from the GHRH challenges (from first and second
experiments) were assayed for growth hormone in two separate assays with
intra-assay coefficients of variation of 11.9% and 10.0% and an inter-assay
coefficient of variation of 11.7%. Serum samples from the TRH challenges (from
first and second experiments) were assayed for growth hormone in two separate
assays with intra-assay coefficients of variation of 6.1% and 8.1% and an inter-
assay coefficient of variation of 12.1%. Serum samples from quipazine
challenges (from first and second experiments) were assayed for growth
hormone in two separate assays with intra-assay coefficients of variation of 9.5%
and 8.6% and an intra-assay coefficient of variation of 8.8%. All serum samples
assayed for total T4 (first and second experiments) were assayed in two assays
with intra-assay coefficients of variation of 0.4% and 4.5% and an inter-assay
coefficient of variation of 6.5%.

The profile of concentrations of growth hormone in serum (pooled data

from first and second experiments) of corn oil-treated steers injected with sterile

86

‘ - Corn 0“

GH LN-AUC
(LN-ng .mr1 min)
0 A N u h (It a: N on

 

 

Figure 17. Effect of daily injections of T3 on basal secretion of GH in meal-
fed steers. Data presented are least squares mean AUC (i pooled standard
error of mean) of growth hormone of corn oil-injected (control; n=4) and T3-
treated (3 pglkg BW; n=4) meal-fed steers. The AUCs were calculated from 0800
to 1400 h on days —1, 5 and 10 relative to initiation of injections, and transformed
by natural logarithm (GH LN-AUC). Injection of T3 did not affect least square
mean GH LN-AUC on days -1, 5 or 10 (P > 0.16).

87

 

-O— Corn oil

6 ' * 5 T3 **

 

Total T4 (pgldl)

 

 

 

Day

Figure 18. Effect of daily injections of T3 on concentrations of total T4 in
meal-fed steers. Data presented are least squares mean concentrations (i
pooled standard error of mean) of total T4 in serum of corn oil-injected and T3-
treated (3 pglkg BW) meal-fed steers on days -1, 5 and 10 relative to initiation of
injections. Compared with com oil-injected steers, daily injection of T3 reduced
concentration of total T4 in serum on d 5 and 10. Points with * differ P = 0.001.
Points with ** differ P = 0.0001.

88

water was similar to that of T3-treated steers injected with sterile water (Figure
19; top panel). In addition, the profile of concentrations of growth hormone in
serum of corn oil-treated steers injected with GHRH was similar to that of T3-
treated steers injected with GHRH. The treatment (GHRH or sterile water) by
thyroid status (T 3- or corn oil-treated) interaction was not significant (P = 0.55).

In com oil-treated steers, compared with injection of sterile water, injection of

...11

GHRH markedly increased AUC of growth hormone (Figure 19; bottom panel).
Likewise, in T3-treated steers, compared with injection of sterile water, injection of
GHRH markedly increased AUC of growth hormone. Compared with corn oil-
treated steers injected with GHRH, injection of T3-treated steers with GHRH
induced secretion of growth hormone to a similar magnitude as indicated by both
AUC (Figure 19, bottom panel) and mean peak concentrations of growth
hormone (Figure 20).

Similar to results shown in Figure 15, compared with injection of corn oil,
chronic T3 markedly reduced concentrations of total T4 in serum (Figure 21). In
corn oil-treated steers, injection of sterile water did not affect concentrations of
total T4 in serum at 1300 h compared with 0900 h. Similarly, in T3-treated steers,
injection of sterile water did not affect concentrations of total T4 in serum at 1300
h compared with 0900 h. In corn oil-treated steers, injection of GHRH did not
affect concentrations of total T4 in serum at 1300 h compared with 0900 h.
Additionally, in T3-treated steers, injection of GHRH did not affect concentrations

of total T4 in serum at 1300 h compared with 0900 h.

89

6° ' + Corn oil/H20
—O— Corn oil/GHRH

   

40

30

GH (nglml)

20

 

 

2000 -

1500 '

1000 '

500 -

 

 

GH AUC (ng - mr1 min)

Corn oil Corn oil T3 T3
"20 GHRH H2O GHRH

Figure 19. Effect of daily injections of T3 on GHRH-induced secretion of GH
in meal-fed steers. TOP PANEL; Data presented are mean concentrations (:l:
standard error of mean; SEM) of growth hormone in serum of corn oil-injected
and T3-treated meal-fed steers after intravenous injection of sterile water (H20)
or GHRH dissolved in H20 (0.2 pglkg BW) at 0900 h. Arrow indicates time of
injection. BOTTOM PANEL; Statistical analysis was performed on least
squares mean AUCs (i pooled SEM) calculated from 0900 to 1000 h. Bars with
different letters differ P < 0.001.

90

Peak GH (nglml)

 

 

Corn oil Corn oil T3 T3
H20 GHRH H20 GHRH

Figure 20 Effect of daily injections of T3 on GHRH-induced peak
concentrations of GH in meal-fed steers. Data presented are least squares
mean peak concentrations (1 pooled standard error of mean) of growth hormone
in serum of com oil-injected and T3-treated meal-fed steers after intravenous
injection of sterile water (H20) or GHRH dissolved in H20 (0.2 pglkg BW) at
0900 h. Bars with different letters differ P < 0.001.

91

_ r‘ '.a‘.‘e_l

AJ
'- A.—

A

 

lid

 

— T4@0900
— T4@1300

 
  
 

‘1

it: J 311.5

._.;._' 3....”

. :7
2‘ a 4444

Total T4 (pgldl)

~—' (~-

. . - ,‘ I “if...
.,,._ .3.._
g. . r‘
'. 77". 3. it
.‘.- _
"... I
. .“ . . , '4
r “0" _ ‘5.
., .- r- r, J
t ."z‘ . r. ‘ i . J: '3
.7 .~ E
. “(L ‘

,0
~<

. - :1
“A“

 

 

o meaii .
Corn oil Corn oil T3 T3

H20 GHRH H20 GHRH

Figure 21. Effect of daily injections of T3 on GHRH-induced secretion of T4
in meal-fed steers. Data presented are least squares mean concentration (:t
pooled standard error of mean) of total T4 in serum of corn oil-injected and T3-
treated meal-fed steers immediately before (0900 h ) and 4 h after (1300 h)
intravenous injection of sterile water (H20) or GHRH dissolved in H20. Injection
of H20 did not affect concentrations of total T4 in serum of corn oil-injected or T3-
treated steers. Injection of GHRH did not affect concentrations of total T4 in
serum of corn oil-injected or T3-treated steers.

92

The profile of concentrations of growth hormone in serum (pooled data) of
corn oil-treated steers injected with saline was similar to that of T3-treated steers
injected with saline (Figure 22; top panel). Likewise, the profile of concentrations
of growth hormone in serum of corn oil-treated steers injected with TRH was
similar to that of T3-treated steers injected with TRH. The treatment (TRH or

saline) by thyroid status (T 3- or corn oil-treated) interaction was not significant (P

“I

= 0.34). However, the main effect of thyroid status tended to affect secretion of

 

‘*‘_-f
l. .’ ‘-
I

growth hormone in response to injection of TRH (P = 0.1). In corn oil-injected
steers, compared with injection of saline, injection of TRH increased AUC (Figure
22; bottom panel) as well as mean peak concentrations of growth hormone
(Figure 23). In contrast, in T3-treated steers, compared with injection of saline,
injection of TRH did not increase AUC of growth hormone (Figure 22, bottom
panel). However, with respect to mean peak concentrations of growth hormone
in T3-treated steers, injection of TRH increased mean peak concentrations to 2.9
i 0.2 LN-ng/ml compared with 2.0 i 0.2 LN-ng/ml in saline injected steers (Figure
23). Compared with corn oil-treated steers injected with TRH, injection of T3-
treated steers with TRH increased secretion of growth hormone to similar
magnitudes as indicated by both AUC (Figure 22;bottom panel) and mean peak
concentrations of growth hormone (Figure 23).

Concentrations of total T4 in serum were greater in corn oil-treated steers
than in serum of T3-treated steers. In com oil-treated steers, injection of saline
did not affect concentrations of total T4 in serum at 1300 h compared with 0900 h

(Figure 24). Similarly, in T3-treated steers, injection of saline did not affect

93

30 - + Corn oil/Saline
—O— Corn oil/T RH
+ T3/Saline

—o— T3/TRH

    
 
 

N
0

GH (nglml)

a
O

 

 

 

 

0 . . .
0800 0900 1000

Time (h)

 

 

Corn oil Corn oil T3 T3
Saline TRH saline TRH

Figure 22. Effect of daily injections of T3 on TRH-induced secretion of GH
in meal-fed steers. TOP PANEL; Data presented are mean concentrations (i
standard error of mean; SEM) of growth hormone in serum of com oil-injected
and T3-treated meal-fed steers after intravenous injection of 0.9% saline or TRH
dissolved in 0.9% saline (0.3 pglkg BW) at 0900 h. Arrow indicates time of
injection. BOTTOM PANEL; Statistical analysis was performed on least
squares mean AUCs (i pooled SEM) calculated from 0900 to 1000 h. Bars with
different letters differ P < 0.01.

a:
3%.
0‘?
n.-z
z..l
JV

 

 

Corn oil Corn oil T3 T3
Saline TRH saline TRH

Figure 23. Effect of daily injections of T3 on TRH-induced peak
concentrations of GH in meal-fed steers. Data presented are least squares
mean peak concentrations (i pooled standard error of mean) of growth hormone
transformed by natural logarithm (LN-Peak GH) in serum of corn oil-injected and
T3-treated meal-fed steers after intravenous injection of sterile saline or TRH
dissolved in saline (0.3 pglkg BW) at 0900 h. Bars with different letters differ P <
0.01.

95

— T4@0900
° — T,@1300

L

 

l
to. .. ng-l

iliF'l-‘um

 

Corn oil Corn oil T3
Saline TRH Saline TRH

 

Total T4 (pgldl)
O -\ N b) h 0| Q N on

. .; . .' a
n ‘ 343'? 1“. I.

q " 0 ~' r:-
o .- t.»

Figure 24. Effect of daily injections of T3 on TRH-induced secretion of T4 in
meal-fed steers. Data presented are least squares mean concentrations (i
pooled standard error of mean) of total T4 in serum of corn oil-injected and T3-
treated meal-fed steers immediately before (0900 h ) and 4 h after (1300 h)
intravenous injection of sterile 0.9% saline or TRH dissolved in 0.9% saline.
Injection of saline did not affect concentrations of total T4 in serum of corn oil-
injected or T3-treated steers. Injection of TRH increased concentrations of total
T4 in serum of both corn oil-injected and T3-treated steers at 1300 h. Within
treatment, bars with different letters differ P < 0.01.

96

 

concentrations of total T4 in serum at 1300 h compared with 0900 h. As
expected, in com oil-treated steers, injection of TRH increased concentrations of
total T3 in serum at 1300 h compared with 0900 h. In addition, in T3-treated
steers, injection of TRH also increased concentrations of total T4 in serum at
1300 h compared with 0900 h.

The profile of concentrations of growth hormone in serum (pooled data) of
corn oil-treated steers injected with saline was similar to that of T3-treated steers
injected with saline (Figure 25; top panel). The profile of concentrations of
growth hormone in serum of com oil-treated steers injected with quipazine had a
pattern similar to that of T3-treated steers injected with quipazine. The treatment
(quipazine or saline) by thyroid status (T 3- or corn oil-treated) interaction was
significant (P = 0.07). In com oil-treated steers, compared with injection of
saline, injection of quipazine increased secretion of growth hormone as indicated
by both AUC (Figure 25; bottom panel) and mean peak concentrations of growth
hormone (Figure 26). In contrast, in T3-treated steers, compared with injection of
saline, injection of the quipazine did not increase AUC of growth hormone (Figure
25; bottom panel). However, with respect to mean peak concentrations of growth
hormone in T3-treated steers, injection of quipazine increased mean peak
concentrations to 2.9 i 0.2 LN-ng/ml compared with 2.0 i- 0.2 LN-nglml in saline
injected steers (Figure 26). Compared with corn oil-treated steers injected with
quipazine, injection of T3-treated steers with quipazine increased both AUC
(Figure 25; bottom panel) and mean peak concentrations of growth hormone to

similar magnitudes (Figure 26).

97

rT'—-.---7*~1:g
_ P

40 ' —e— Corn oil/Saline
—0— Corn oil/Quipazine
+ T3/Saline

3° ' —D— T3/Quipazine

    

20-

GH (nglml)

10-

 

 

14UU '

1200 .

1000 -
800 -
600 -
400 -
200

 

 

GH AUC (mg . ml-1 min)

0

Corn oil Corn all T, T3
Saline Quipazine Sallne Quipazine

Figure 25. Effect of daily injections of T3 on quipazine-induced secretion of
GH in meal-fed steers. TOP PANEL; Data presented are mean concentrations
(i standard error of mean; SEM) of growth hormone in serum of corn oil-injected
and T3-treated meal-fed steers after intravenous injection of 0.9% saline or a
serotonin receptor agonist (quipazine) dissolved in 0.9% saline (0.2 mglkg BW)
at 0900 h. Arrow indicates time of injection. BOTTOM PANEL; Statistical
analysis was performed on least squares mean AUCs (:t pooled SEM) calculated
from 0900 to 1000 h. Compared with injection of saline, injection of quipazine
increased GH AUC in corn oil-injected but not T3-treated steers. Bars with
different letters differ P < 0.05.

98

 

 

LN-Peak GH
(LN-nglml)

 

 

Corn oil Corn oil T3 T3
Saline Quipazine Saline Quipazine

Figure 26. Effect of daily injections of T; on quipazine-induced peak
concentrations of GH in meal-fed steers. Data presented are least squares
mean peak concentrations (1: pooled standard error of mean) of growth hormone
transformed by natural logarithm (LN-Peak GH) in serum of com oil-injected and
T3-treated meal-fed steers after intravenous injection of sterile saline or serotonin
receptor agonist (quipazine) dissolved in saline (0.3 pglkg BW) at 0900 h. Bars
with different letters differ P < 0.01.

99

 

 

Concentrations of total T4 in serum of corn oil-treated steers were greater
than in serum of T3-treated steers. In corn oil-treated steers, injection of saline
did not affect concentrations of total T4 in serum at 1300 h compared with 0900 h
(Figure 27). Likewise, in T3-treated steers, injection of saline did not affect
concentrations of total T4 in serum at 1300 h compared with 0900 h. In corn oil-
treated steers, injection of quipazine did not affect concentrations of total T4 in
serum at 1300 h compared with 0900 h. Similarly, in Tg-treated steers, injection
of quipazine did not affect concentrations of total T4 in serum at 1300 h

compared with 0900 h.

DISCUSSION

Injection of the serotonin receptor agonist, quipazine, induces secretion of
growth hormone in cattle (Gaynor et al., 1996). However, the mechanism by
which the serotonin receptor agonist induces secretion of growth hormone is not
known. An earlier experiment by Gaynor and coworkers (1996) and in vitro
experiments described in Chapter 1 of this dissertation suggests that the effects
of the serotonin receptor agonist are not directly on the pituitary gland. In other
experiments utilizing in vitro and in vivo approaches, l have collected data that
suggest that the effects of serotonin on secretion of growth hormone are not
mediated by SS, GHRH or PACAP. However, the experiments that I conducted
in Chapter 3 of this thesis suggest that TRH could be mediating serotonin-

induced secretion of growth hormone.

100

’1'. ..._._........ “‘1:
F IL
41.

 

— T4@0900
— T4@1300

 
  
   

Corn oil Corn oil T3 T3
Saline Quipazine Saline Quipazine

 

Figure 27. Effect of daily injections of T; on quipazine-induced secretion of
T4 in meal-fed steers. Data presented are least squares mean concentrations
(i pooled standard error of mean) of total T4 in serum of corn oil-injected and T3-
treated meal-fed steers immediately before (0900 h ) and 4 h after (1300 h)
intravenous injection of sterile 0.9% saline or a serotonin receptor agonist
(quipazine) dissolved in 0.9% saline. Injection of saline did not affect
concentrations of total T4 in serum of corn oil-injected or Tag-treated steers.
Injection of quipazine did not affect concentrations of total T4 in serum of corn oil-
injected or T3-treated steers.

101

 

 

 

Therefore, my hypothesis in Chapter 4 was that TRH mediates serotonin
receptor agonist-induced secretion of growth hormone. To test this hypothesis, I
had to block the effects of TRH on secretion of growth hormone. To do this, I
used negative feedback of T3 on the hypothalamus and pituitary to decrease
TRH synthesis and secretion and(or) TRH receptor expression in the anterior
pituitary gland. Previous experiments demonstrated that T3 feeds back at the
level of the hypothalamus to down regulate TRH synthesis (Segerson et al.,
1987; Koller et al., 1987; Dyess et al., 1988) as well as at the level of the anterior
pituitary gland to down regulate TRH receptors (Lean et al., 1977; Perrone and
Hinkle, 1978). A recent report (Thrift et al., 1999) showed that injecting heifers
with 2 mg T3/d induced negative feedback on the thyroid axis and decreased
serum concentrations of T4 to. below detection levels of their assay. In this
chapter, I also decreased concentrations of T4 in serum of cattle to below
detectable levels by injecting 3 pg T3/kg BW daily.

The hormone T3 also increases synthesis of growth hormone mRNA
(Samuels and Shapiro, 1976; Seo et al., 1977; Martial et al., 1977). However, at
least in GH3 cells, the concentration of T3 required to increase growth hormone
mRNA is 45-fold greater than the concentration required to down regulate TRH
receptors (Hinkle et al., 1979). Another report demonstrated that
hyperthyroidism in cattle induced by injecting T4 increased concentrations of T3 in
plasma by 65% but did not affect basal secretion of growth hormone (Hannon
and Trenkle, 1991 ). In my experiment I chose to inject T3 so that I could measure

total T4 in serum as an indicator of thyroid gland secretory activity. In my

102

Iii-’5 TTrZT’

.Di';
|.|

 

experiment, injecting T3 down regulated the thyroid axis in meal-fed steers
without affecting basal secretion of growth hormone throughout the 28 and 30
days of injecting T3, in the first and second experiments respectively.

Even though daily injections of T3 did not affect basal secretion of growth
hormone, it remained to be determined if daily injections of T3 affected release of
growth hormone in the bovine anterior pituitary gland. To my knowledge no one
has tested the effects of hyperthyroidism on GHRH-induced secretion of growth
hormone in cattle. However, in the rat, hyperthyroidism does not affect GHRH-
induced secretion of growth hormone (Dieguez et al., 1986; Root et al., 1986).
Data from this chapter indicate that hyperthyroidism does not affect GHRH-
induced secretion of growth hormone in cattle.

Similarly, T3-induced hyperthyroidism did not affect TRH-induced secretion
of growth hormone in meal-fed steers. However, the AUC of growth hormone
following injection of TRH was numerically less in T3-treated steers. These data
contrast results reported in rats (Chihara et al., 1976). Because basal, GHRH-,
and TRH-induced secretion of growth hormone in meal-fed steers were not
affected by injections of T3, it appears that the anterior pituitary gland’s ability to
synthesize and secrete growth hormone is not affected. The failure of T3-
treatment to decrease TRH-induced secretion of growth hormone suggests that
the TRH receptors on the anterior pituitary were not down regulated by T3-

treatrnent. It is possible that injections of T3 at doses greater than 6 pglkg BW

would in fact down regulate TRH receptor expression and inhibit TRH-induced

secretion of growth hormone.

103

 

There are several reports suggesting that serotonin plays a role in
regulating secretion of growth hormone. In the rat, concentrations of growth
hormone in serum are highly correlated with serotonin turnover (ratio of
metabolite, 5-hydroxyindoleacetic acid to serotonin) in the brain (Smythe et al.,
1982). Similar results have been observed in cattle (Gaynor and coworkers,
unpublished). Previous experiments in this dissertation and those of Gaynor et
al. (1996) demonstrated that the effects of a serotonin receptor agonist on
secretion of growth hormone are mediated via hypothalamic hormones. Chen
and Ramirez (1981) reported that serotonin induced secretion of TRH from
perifused rat hemihypothalami. In this chapter, I demonstrated that inhibition of
the hypothalamic-pituitary-thyroid axis with negative feedback inhibits but does
not completely block serotonin receptor agonist-induced secretion of growth
hormone in cattle. However, inhibition of the hypothalamic-pituitary-thyroid axis
with negative feedback did not inhibit TRH-induced secretion of GH. One
interpretation of these data is that injections of T3 inhibited TRH mRNA
transcription and translation within the hypothalamus but not expression of TRH
receptors on the anterior pituitary gland. These data suggested that TRH may be
one of several mediators of serotonin-induced secretion of growth hormone in
cattle.

It is possible that some other hypothalamic peptide that was not examined
in this dissertation may play a role, in concert with TRH, in mediating serotonin-
induced secretion of growth hormone. Support for this hypothesis comes from a

report by Gaynor and coworkers (1996) in which meal-fed steers were pretreated

104

Uv". n*e;uwr-

 

with either yohimbine or idazoxan (oz-adrenergic receptor antagonists) and then
injected with quipazine (serotonin receptor agonist). Pretreatment with these or;-
adrenergic receptor antagonists partially blocked serotonin receptor agonist—
induced secretion of growth hormone. These data indicate that serotonergic and
aradrenergic neuronal systems work in concert to regulate secretion of growth
honnone.

In this chapter, the T4 data obtained after injection of the serotonin
receptor agonist do not support the hypothesis that TRH mediates serotonin
receptor agonist-induced secretion of growth hormone. However they do not
negate it either. If TRH mediates serotonin receptor agonist-induced secretion of
growth hormone, then the serotonin receptor agonist should also increase
secretion of TSH and ultimately increase serum concentrations of total T4. In this
chapter, both vehicle-injected controls and T3-treated steers responded to TRH
with an increase in serum concentrations of total T4. However, injection of
quipazine did not increase serum concentrations of total T4 in either corn oil-
injected controls or T3-treated steers. One explanation for these results may be
that when we determined the dose of serotonin receptor agonist, we determined
a dose optimal for inducing secretion of growth hormone (Gaynor et al., 1996)
not TSH or T4 secretion. In the rat approximately 61% of the cells that contain
TRH receptor mRNA are somatotropes (Konaka et al., 1997). If serotonin
receptor agonist-induced secretion of growth hormone is mediated by TRH,
perhaps the dose required to induce TSH secretion and ultimately T4 secretion is

different from the dose required to induce secretion of growth hormone.

105

gift—.- n‘wm
.- 4

 

In conclusion, these results demonstrate that daily injections of T3
negatively feed back on the thyroid axis and reduces serum concentrations of
total T4. I also demonstrated that daily injections of T3 reduces, but doesn’t
completely abolish quipazine-induced secretion of growth hormone. These data
support the hypothesis that TRH mediates, in part, serotonin receptor agonist-

induced secretion of growth hormone in cattle.

106

 

SUMMARY AND CONCLUSIONS

My overall goal in this dissertation was to understand the mechanisms by
which serotonin induces secretion of growth hormone in cattle. Specifically, my
objectives were to: 1) determine if a serotonin receptor agonist (quipazine) acts

directly on the anterior pituitary gland to induce secretion of growth hormone in

cattle and; 2) determine the role of SS, GHRH, TRH and PACAP in mediating the

effects of serotonin receptor agonist-induced secretion of growth hormone in
cattle. To address my objectives I used both in vitro and in vivo approaches.

In a previous experiment, Gaynor and coworkers (1996) reported that
quipazine did not induce secretion of growth hormone from dispersed bovine
anterior pituitary cells in a perifusion culture system. In contrast, a more recent
report by Balsa and coworkers (1998) demonstrated that serotonin induced
secretion of growth hormone from rat anterior pituitary cells co-cultured with
posterior pituitary cells in a static system with medium supplemented with
corticosterone. The requirement for corticosterone can be explained because
glucocorticoids are required for expression of voltage gated calcium channels
(Fomina et al., 1993; Fomina et al., 1996), However, Balsa and coworkers were
unable to explain the requirement for posterior pituitary cells for serotonin
induced-secretion of growth hormone, and speculated that there was an
unknown factor from posterior pituitary cells that mediated the effects of

serotonin on secretion of growth hormone.

107

 

In chapter 1 I reported that quipazine does not induce secretion of growth
hormone from bovine anterior pituitary cells co-cultured with posterior pituitary
cells in medium supplemented with cortisol. Results presented in Chapter 1 also
showed that the serotonin receptor agonist did not directly induce secretion of
growth hormone from perifused dispersed anterior pituitary cells. Therefore, my
data support the conclusion of Gaynor et al (1996) that serotonin does not
directly induce secretion of growth hormone from the anterior pituitary gland in
cattle. In addition I demonstrated that the posterior pituitary gland does not
mediate the effects of serotonin on the secretion of growth hormone.

Collectively, the data supported the idea that the effects of serotonin on secretion
of growth hormone are mediated by hypothalamic hormones.

Murakami and coworkers (1986) reported that serotonin induced-secretion
of growth hormone in rats was inhibited by passive immunization against rat
GHRH, indicating that GHRH mediates serotonin-induced secretion of growth
hormone in rats. In Chapter 1, I used a perifusion culture system to test the
effects of quipazine on secretion of GHRH and SS from bovine hypothalamic
tissue. Activation of serotonin receptors did not increase secretion of GHRH or
decrease secretion of SS from perifused bovine hypothalamic slices. Therefore,
I concluded that the effects of serotonin on growth hormone in cattle were not
mediated by either GHRH or SS in cattle. This conclusion is supported by in vivo
work from Moseley and coworkers (1988) and Gaynor and coworkers (1996).
Both of these experiments utilized meal-fed steers. Moseley and coworkers

reported that GHRH-induced secretion of growth hormone is greater before than

108

ll $n—ammfi
>‘

a
i

 

after feeding, while Gaynor and coworkers reported that serotonin receptor
agonist-induced secretion of growth hormone is similar before versus after
feeding. If GHRH was mediating the effects of serotonin receptor agonists, then
GHRH-induced secretion of growth hormone would be expected to be similar
before and after feeding.

Previous reports have implicated both PACAP (Yamauchi et al., 1996) and
TRH (Chen and Ramirez, 1981) as possible mediators of serotonin-induced
secretion of growth hormone in rats. Yamauchi et al (1996) reported that prior
treatment of rats with a PACAP receptor antagonist suppressed serotonin-
induced secretion of growth hormone. Chen and coworkers (1981) perifused rat
hemihypothalami. treated them with medium containing serotonin and measured
TRH in the medium effluent. Chen and coworkers reported that serotonin
increased secretion of TRH from perifused hemihypothalami. In Chapter 2, I
determined the minimal doses of TRH and PACAP required to maximally
stimulate secretion of growth hormone in meal-fed steers. I demonstrated that
activation of serotonin receptors induced secretion of growth hormone before
feeding when concentrations of growth hormone in serum are normally high, as
well as after feeding when concentrations of growth hormone in serum are low,
and that the magnitude of response in growth hormone secretion was similar
before and after feeding. If either PACAP or TRH is mediating the effects of
serotonin then they too would be expected to induce similar amounts of growth
hormone secretion before and after feeding. Using the doses of TRH and

PACAP determined in Chapter 2, I tested the effects of TRH and PACAP before

109

 

 

and after feeding. Injection of PACAP before feeding induced greater secretion of
growth hormone than injection of PACAP after feeding. Therefore, I concluded
that PACAP probably does not mediate serotonin-induced secretion of growth
hormone in cattle. In contrast, injection of TRH before feeding induced secretion
of similar amounts of growth hormone as injection of TRH after feeding. Thus,
TRH could be mediating serotonin receptor agonist-induced secretion of growth
honnone.

In Chapter 4, I developed and utilized a T3 negative feedback paradigm to
down regulate TRH synthesis and(or) TRH receptor expression. Previous
reports indicate that T3 inhibits expression of TRH receptors in the pituitary gland
(Lean et al., 1977; Perrone and Hinkle, 1978) as well as TRH synthesis in the
paraventricular nucleus of the hypothalamus (Segerson et al., 1987; Koller et al.,
1987; Dyess et al., 1988). When meal-fed steers were injected daily with T3,
endogenous secretion of T4 was inhibited indicating that this paradigm was
effective in down regulating the thyrotropic axis. However, daily injections of T3
did not affect TRH-induced secretion of growth hormone, which, indicates that
TRH-receptors on the anterior pituitary gland were not down-regulated. In
contrast, when steers were injected with quipazine, growth hormone secretion
was inhibited in T3-treated steers compared with control steers that received corn
oil. These data indicate that down regulation of TRH synthesis inhibits serotonin-
induced secretion of growth hormone. However, T3-treatment did not completely

abolish serotonin receptor agonist-induced secretion of growth hormone,

110

1. 4

 

indicating that TRH may not be the sole mediator of serotonin-induced secretion
of growth hormone.

In conclusion, the serotonin receptor agonist does not act directly on the
pituitary gland to induce secretion of growth hormone. Additionally, the data
reported in this dissertation suggests that serotonin-induced secretion of growth
hormone in cattle is mediated by TRH, while at the same time supporting the
conclusion that the effects of the serotonin receptor agonist on secretion of
growth hormone are not mediated by SS, GHRH or PACAP. I propose the model
in Figure 28 as the mechanism of serotonin induced secretion of growth hormone
in cattle. Specifically, serotonin neurons activate TRH neurons and induce the
secretion of TRH into portal vessels. Portal vessels then transport TRH to the

anterior pituitary where it induces the secretion of growth hormone.

111

im: in. MIR
.‘

 

Raphe Nuclei Serotonin

Paraventricular

Nucleus TRH

Ponal
Vessel . TRH .

 
 
 

  

Anterior
Pituitary

    

Growth Hormone

Figure 28. Proposed model for serotonergic regulation of growth hormone
secretion in cattle. Ovals with connected lines represent serotonin and
thyrotropin releasing hormone (TRH) neurons. The cylinder represents portal
vessels between the hypothalamus and the anterior pituitary gland, which is
represented by the large circle. The model depicts the neuronal pathway in
which serotonin may induce secretion of growth hormone. Serotonergic neurons
stimulate TRH neurons. In turn, TRH is secreted into portal vessels which
transport it to the anterior pituitary gland where it stimulates secretion of growth
honnone.

112

 

IMPLICATIONS

The data presented in this dissertation lends support to the hypothesis
that TRH is a mediator of serotonin-induced secretion of growth hormone in
cattle. However, further investigation is required to determine if other
hypothalamic peptides are involved in the serotonergic regulation of growth
hormone secretion, and whether or not this would be an appropriate
neuroendocrine system to target in order to increase endogenous secretion of
growth hormone.

Future research to determine if serotonin receptors are present on TRH
neurons and exactly which serotonin receptor is involved would aid in identifying
compounds to specifically increase secretion of growth hormone. Ultimately, we
may be able to modulate serotonin receptors and increase growth and lactation
with orally active compounds similar to serotonin reuptake inhibitors that are
currently used in humans.

Since hyperthyroidism did not completely inhibit serotonin-induced
secretion of growth hormone, it is possible that other hypothalamic peptides are
involved in serotonergic regulation of growth hormone secretion in cattle. Two
possible peptides are galanin and ghrelin (endogenous growth hormone
releasing peptide-6 receptor agonist). Using specific receptor antagonists to
block the action of these hormones at the anterior pituitary, then challenging the
steers with a serotonin receptor agonist and measuring growth hormone would

determine if either of these hormones were involved in serotonergic regulation of

113

growth hormone secretion. Alternatively, development of a portal vessel
cannulation technique in cattle would allow scientists to measure the
hypothalamic hormones in portal blood and determine if injection of a serotonin
receptor agonist increases secretion of any of these hypothalamic hormones.
Granted, this technique has already been developed in sheep, but prior research

shows that activation of serotonin receptors does not increase secretion of

El

growth hormone in sheep. Therefore, sheep would not be an acceptable model.

'

The fore-mentioned research would further our goal of understanding
neuroendocrine regulation of growth hormone secretion. However, investigating
the mechanism of feeding-induced suppression of growth hormone secretion in
cattle would also improve our understanding of regulation of growth hormone
secretion in cattle. Specific questions to answer might be what is the mechanism
by which somatotropes become refractory to GHRH after feeding and does
feeding reduce the neuroendocrine signals that stimulate secretion of growth
hormone. Answering these questions may ultimately lead to methods to improve

growth and lactation by increasing endogenous secretion of growth hormone.

114

 

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