EFFECTS OF CHRONIC AND ACUTE y-HYDROXYBUTYRATE ADMINISTRATION ON MASS AND THE RELEASE OF GROWTH HORMONE AND CORTICOSTERONE

A Thesis

Presented to

the Faculty of the College of

Science and Technology

Morehead State University

In Partial Fulfillment

of the Requirements for the Degree

Master of Science in Biology

by

Eric C. Goshorn

October, 1998 /-t?p-1\'/ I"\s 4 ,he-a ·1 ~ ~lC\ .9~ G ls> 1 lo .JL.

Accepted by the faculty of the College of Science and Technology,

Morehead State University, in partial fulfillment of the requirements for the

Master of Science degree.

Master's Committee:

/2..-lo -

ii EFFECTS OF CHRONIC AND ACUTE y-HYDROXYBUTYRATE ADMINISTRATION ON MASS AND THE RELEASE OF GROWTH HORMONE AND CORTICOSTERONE

Eric Christopher Goshorn, M.S. Morehead State University, 1998

Director of Thesis·

Gamma-hydroxybutyrate (GHB) is a naturally occurring compound found both in neural (Roth and Suhr 1970) and extraneural tissue (Nelson et al. 1981 ). While the endogenous function of peripheral GHB has yet to be elucidated, experimentation has found that this compound induces anesthesia, and the release of growth hormone and prolactin from the anterior pituitary (Oyama and Takiguchi 1970; Takahara et al. 1977). This study was undertaken to determine the effect of chronic GHB injections on the weight gain of rats during early postnatal development. Acute injections were also administered to rats not previously treated with exogenous GHB to

iii ascertain the mechanism of growth hormone release and to determine GHB's effect on corticosterone release.

At the age of 3 days, both male and female rats from the chronic group were injected intraperitoneally with 100 mg/kg, 500 mg/kg, or 0 mg/kg of

GHB. These injections were then performed twice a week for 38 days, with the weight being recorded before each injection. Nine days following the end of this injection period, those rats that had received 500 mg/kg of GHB, were once again injected with of 500 mg/kg of GHB. The plasma was then assayed for growth hormone and corticosterone levels. Rats in the acute group were injected with 500 mg/kg GHB alone, or in combination with the following: 1) chlorpromazine, a dopamine. antagonist, 2) cyproheptadine, a serotonin , or 3) pyridoxine, a system stimulator. The plasma growth hormone and corticosterone were then determined using ELISA or RIA, respectively.

Results indicate that chronic GHB administration does not lead to significant weight gain for male or female rats. Furthermore, chronic administration of GHB leads to a reduction of GHB-induced growth hormone release in male rats, possibly due to a receptor down-regulation. In contrast to the males, na"ive female rats had no rise in plasma growth hormone following GHB injection. Both chlorpromazine and cyproheptadine caused a reversal of the GHB-induced rise in plasma growth hormone levels, indicating possible action through both the dopaminergic and systems.

iv Pyridoxine leads to a decrease in GHB-induced growth hormone, leaving some question as to the role of the dopaminergic system in GHB's activity.

Acute injections of exogenous GHB produced elevated plasma corticosterone levels in males and females that were not responsive to the dopamine and serotonin antagonists.

Accepted by: Chair

V Acknowledgments

Like my time at Morehead State University, the list of people I would like to thank is long and quite extensive. I would like to thank all of the faculty and staff in the Department of Biological and Environmental Sciences. In particular, those individuals associated with the biology and chemistry departments. I would like to thank the members of my committee, Dr. David

Magrane, Dr. David Saxon, and Dr. Craig Tuerk. And I would like to thank the department for funding for the project.

I would also like to thank Michael Spencer for lab assistance, and Dr.

Brian Reeder for advice on statistical analysis.

Lastly, I would like to thank my family and friends for their constant encouragement and support. A special thanks goes to my wife, Rachel

Leanne Short, who assisted me in what she described as the endless literature search.

vi Table of Contents

I. Introduction...... 1

Metabolism...... 2 Distribution...... 4 Receptor...... 6 Relationship to GABA...... 7 Effects...... 7 Possible Mechanisms...... 10 Research Objectives...... 13 Rationale for Using Antagonists and .. 13 II. Methodology...... 15 Ar:iimals...... 15 ...... 15 Chronic Experiment.,...... 15 Acute Experiment...... 16 Growth Hormone Assay...... 18 Corticosterone Assay...... 20 Statistical Analysis ...... :...... 21

Ill. Results...... 22 Chronic Experiment...... 22 Acute Experiment...... 28 Growth Hormone Assay...... 28 Corticosterone Assay...... 31 IV. Discussion...... 35

· V. References...... 41

vii List of Figures

Figure Page

1. Chemical structure of y-hydroxybutyric acid...... 1

2. Metabolic pathway for y-hydroxybutyrate...... 3

3. Overview of experimental design...... 17

4. The effect of varying concentrations of y-hydroxybutyrate on the weight gain of chronically treated male rats...... 23

5. Standardized weights of male rats injected with varying concentrations of y-hydroxybutyrate...... 24

6. The effect of varying concentrations of GHB on the weight gain of . chronically treated female rats...... 25

7. Standardized weights of female rats injected with varying concentrations of y-hydroxybutyrate...... 26

8. Plasma growth hormone levels of male rats in response to acute injections of y-hydroxybutyrate, chlorpromazine, cyproheptadine or pyndox1ne...... 29

9. Plasma growth hormone levels of female rats in response to acute injections of y-hydroxybutyrate, chlorpromazine, cyprbheptadine or pyndox1ne ...... 30

10. Plasma corticosterone levels of male rats in response to acute injections of y-hydroxybutyrate, chlorpromazine, cyproheptadine or pyndoxine ...... 32

11. Plasma corticosterone levels of female rats in response to acute injections of y-hydroxybutyrate, chlorpromazine, cyproheptadine or pyndox1ne ...... 33

viii List of Tables

Table Page

1. Distribution of GHB in neural and peripheral tissues of adult rats...... 5

2. The effect of y-hydroxybutyrate on the organ development of chronically treated male rats...... 27

3. The effect of y-hydroxybutyrate on the organ development of chro11ically treated female rats...... 27

ix Introduction

Gamma-hydroxybutyrate (GHB), shown in Figure 1, is a 4-carbon compound containing a hydroxyl and carboxylic acid moiety, on primary carbons 4 and 1 respectively. It was originally used experimentally to determine if there existed a metabolic or physiological role for butyric acid

(Laborit 1964). The addition of the hydroxyl group was hoped to delay~­ oxidation, so these roles could be more firmly established (Laborit 1964). It was known that this molecule is structurally similar to y-aminobutyric acid

(GABA), and it was thought that perhaps GHB might be able to cross the blood-brain barrier (GABA cannot), and function as a GABA precursor facilitating its synthesis in the brain (Laborit 1964; Feigenbaum and Howard

1996).

Figure 1. Chemical structure of y-hydroxybutyric acid.

GHB was found capable of rapidly crossing the blood-brain barrier

(Laborit 1964; Cash 1996; Feigenbaum and Howard 1996), and was

1 discovered to be a natural metabolite, found in both neural (Roth and Suhr

1970) and extraneural tissues (Nelson et al. 1981 ). GHB was at one time

marketed as a health aid to persons with insomnia, and to bodybuilders as an

inducer of growth hormone release (Chin et al. 1992; Sanguineti et al. 1997).

Within 3 months of sales, it was banned in California and Florida due to acute

poisoning (Chin et al. 1992). Ingestion of the is reported to give a "high"

or disoriented effect, and it is easily synthesized illegally by dissolving sodium

hydroxide in near boiling gamma-butyrol lactone (Sanguineti et al. 1997).

Metabolism of GHB

GHB is widely considered to be primarily an endogenous metabolite of

GABA, and a pathway using this precursor for GHB formation is presented in

Figure 2. In the brain, GABA is converted into succinic semialdehyde (SSA)

by the GABA-transaminase (GABA-T). The SSA can then either be

oxidized and enter the citric acid cycle (Maitre 1996), or it can be reduced to

GHB by the action of a cytosolic enzyme, SSA reductase (SSR2) (Rumigny et

al. 1980). GHB has also been shown to be formed from 1,4-butanediol via the action of an dehydrogenase in neural and extraneural tissues.

There are two known to oxidize GHB back to succinic semialdehyde, GHB dehydrogenase (GHB-D) and GHB-oxoacid transhydrogenase (GHB-T) (Nelson and Kaufman 1994). The former is a cytosolic enzyme which catalyzes an NADP+ -dependent reaction that

2 (2)0 (3)(Y°

(8)

NADP NADPH

NADPH NADP.

NAO

~ADH 0 0 5 ( > II II H~QH·

Figure 2. Metabolic pathway for GHB (Redrawn from Vayer et al. 1987). Keys to intermediates are listed by number and enzymes are designated by letters. 1) gamma-aminobutyric acid, 2) 1,4-butanediol, 3) gamma-butyrolactone, 4) succinic semialdehyde , 5) GHB, 6) succinic acid. A) GABA-transaminase, 8) alcohol dehydrogenase, C) peripheral lactonase, D) SSA reductase, E) GHB-dehydrogenase, GHB-transhydrogenase, F) Succinate dehydrogenase

3 oxidizes GHB and reduces 0-glucuronate to L-gulonate (Kaufman and Nelson

1981). The latter is a mitochondrial enzyme that oxidizes GHB and

reduces an oxoacid such as a-ketoglutarate without the NADP+ requirement

(Kaufman et al. 1988).

In the brain, GHB-D is the predominant degradative enzyme for GHB

during the fetal and early neonatal period, while GHB-T takes over this role in

the later post-natal period. However in peripheral tissue, both enzymes have

active parts throughout development (Nelson and Kaufman 1994).

SSA can then be converted to succinate by SSA dehydrogenase, from which it can enter the citric acid cycle and be metabolized for energy

(Kaufman and Nelson 1987).

Distribution of GHB

As previously indicated, GHB is found in both neural and extraneural locations (Table 1). In the brain, GHB levels vary by region (Snead and

Morley 1981; Maitre 1997), and the highest concentrations are found in the midbrain and in the caudate regions.

From a developmental standpoint, the highest levels of neural GHB are found in neonates (400% higher than in adults) and decline significantly through the postnatal period. GABA, succinic semialdehyde dehydrogenase

(SSDH), and GABA-aminotransferase are at their lowest levels during the

4 prenatal period and increase through postnatal development (Snead and

Morley 1981 ). In the neonatal brain, the activity of the catabolic enzyme for

GHB is higher than the activity of the anabolic enzyme (GABA-T) required for its synthesis from GABA, indicating that GABA is not the primary source of

GHB (Snead and Morley 1981).

GHB exists in various peripheral tissues, with concentrations being greatest in brown fat, kidney, heart and muscle (see Table 1.). The high levels of GHB found in these organs suggested that it must participate in some way in their metabolism (Nelson et al. 1981). GABA is likely not the precursor of GHB in extraneural tissue, as it is found in only trace amounts.

Table 1. Distribution of GHB in neural and peripheral tissues of adult rats (Taken from Snead and Morley 1981; Maitre 1997).

Neural GHB (nmols/g) Peripheral GHB (nmols/g) Whole brain 2.29 Heart 12.4 Frontal cortex 0.4 Kidney 28.4 Temporal cortex 5.75 Liver 1.42 Hippocampus 1.2 Lungs 1.54 Striatum 1.8 Muscle 10.2 Substantia nigra 4.6 Brown fat 37.4 Caudate 11 .4 Pons-medulla 4.68 Midbrain 15.0 Cerebellum 8.48

5 The GHB Receptor

Studies indicate that there are two classes of GHB binding sites, one with high and the other with low affinity binding (Snead and Liu 1984). In the

CNS, GHB binding sites seem to exist only on neurons (Maitre 1997). The receptor has not been sequenced, although it is known to be composed of less than 10% protein, and the remaining structure is mainly composed of lipid material (Cash 1996). Binding capacities have been shown to increase

2 with Mg •, although it is not an absolute requirement (Cash 1996). GHB membrane binding sites are absent in peripheral tissue (Snead and Liu 1984;

Cash 1996).

A study by Snead (1992) on GHB-induced seizures found that rats injected with pertussis toxin (PTX) were significantly protected from the seizure associated effects of GHB. This suggested that GHB receptors were associated with a G protein, as PTX is widely known to be an inhibitor of G proteins through ADP-ribosylation (Voet and Voet 1995). Later analyses found that in the presence of GTP, but not ATP, the binding affinity of GHB for its brain membrane receptor is decreased significantly. This gives evidence that the GHB receptor acts through a G protein, because in the presence of

GTP, the receptor-G protein complex dissociates to a point where a state of low affinity receptor binding predominates (Ratomponirina et al. 1995). In addition, ADP-ribosylation of the G proteins by PTX prevented them from coupling to the GHB receptor.

6 Some researchers have suggested that perhaps GHB is not the primary ligand for this receptor, and that the ligand is a molecule yet to be elucidated, because some psychotrophic substances that bear no structural semblance to GHB have been found to bind to the receptor, with similar affinity as GHB (Cash 1996).

Relationship to GABA

It is widely held that GHB is a GABA receptor . This concept is largely due to the original assumption that GHB is primarily derived from

GABA. However, there is substantial evidence which suggests that GHB is not exclusively derived from GABA endogenously (Feigenbaum and Howard

1996). A study by Snead et al. (1982) found that inhibiting the formation of

GABA has no significant effect on GHB levels in the rat brain. GHB has only a slight effect on GABAnergic mechanisms, and even this may be indirect

(Feigenbaum and Howard 1996). Of the two subtypes of GABA receptors,

GHB is unable to serve as an agonist to the GABAA receptor (Mathivet et al.

1997), but it is a weak agonist of the GABA8 receptor (Mathivet et al. 1997;

Nissbrandt and Engberg 1996; Snead 1996).

Physiological Effects of GHB

Early research on cats, and later humans, indicated that administration of GHB caused depression of the central nervous system

(Roth and Gairma 1966). However, initial researchers were uncertain if the

7 depressive effects were caused by GHB or its precursor, y-butyrolactone

(GBL), both of which had been used in the experiments. It was soon

determined that the physiological effects of exogenously administered GBL

was attributed to the conversion into GHB by lactonase which is present in

the blood serum (Roth and Gairman 1966).

When the amount of GHB in the brain reaches the critical level of 70

mg/kg, the animals lose their righting reflex and a state of anesthesia ensues

(Giarman and Roth 1964). The LD50 in rats was shown to be 1.7 g/kg with

respiratory depression as the cause of death (Laborit 1964).

During periods of hypoxia, GHB has been demonstrated to increase

the survival time in the animals studied (Atru et al. 1980), and this action is thought to be caused by depressing the cerebral metabolic rate. Under

extreme anoxic conditions, the most favorable reaction for an animal is the reduction of succinic semialdehyde into y-hydroxybutyric acid, leading to the formation of NAD+ reserves, which are in short supply during hypoxia and can be used to eliminate excess lactate. During exercise, GHB can also be converted into succinic acid, which can enter the Krebs cycle upon conversion to succinate (Kleimenova et al. 1979). In addition, GHB causes a slight drop in body temperature leading to hypothermia, and appears to protect against convulsions (Laborit 1964).

8 GHB has also been used experimentally to control alcohol dependence since GHB and share some chemical and pharmacological properties. Both are short carbon chains containing hydroxyl groups, and both have anesthetic and motor impairing effects. Columbo et al. (1994) demonstrated that GHB has a suppressing effect on voluntary alcohol intake, and ultimately reduces the amount of ethanol consumption by alcohol dependent test animals. Fadda et al. (1989) has found that GHB was able to antagonize ethanol withdrawal syndrome in rats. It was demonstrated that a significant protection was provided at lower than anesthetic doses. In addition, chronic ethanol intake confers tolerance to the and anaesthetic effect of GHB.

Takahara et al. (1977) evaluated the role that GHB played in regulating the secretion of pituitary hormones. They found that GHB caused a significant rise in the plasma levels of prolactin (PRL) and growth hormone

(GH) (Oyama and Takiguchi 1970; Takahara et al. 1977). Takahara et al.

(1977) suggested the release of prolactin might be associated with a possible decrease in the release of dopamine from the hypothalamus (see next section), as dopamine is also known as prolactin inhibiting factor (PIF)

(Porterfield 1997). GHB might induce serotonin release from its nerve terminals stimulating the release of prolactin and GH (Takahara et al. 1977).

9 It has not yet been determined what long term effects GHB will have on body mass (Chin et al. 1992).

Possible Mechanisms of Actions of GHB on Hormone Release

While the metabolic and physiological roles of GHB have undergone extensive research, many of the mechanisms of its action are not well understood (Chin et al. 1992).

GHB and opium induce similar effects, so researchers hypothesized that possibly GHB has some action through the opiate system, possibly as an opiate agonist (Feigenbaum and Simantov 1996). However, studies have shown that , an opiate receptor antagonist, could not block the GH releasing effect of GHB (Gerra et al. 1995). In addition, there was only negligible binding of GHB to theµ, 6 and K subtypes of the opiate receptor at any concentration of GHB (Feigenbaum and Simantov 1996). This suggests that GHB does not work directly through the opiate system.

A large body of evidence indicates that GHB might have a strong influence on the dopaminergic system by serving as a relatively specific inhibitor of dopamine release (Feigenbaum and Howard 1996a). It is known that dopamine and its agonists increase the hypothalamic secretion of GHRH

(Brambilla et al. 1997). GHB is capable of significantly inhibiting impulse flow of dopamine through central dopamine neurons, presumably by action on their storage granular membranes (Feigenbaum and Howard 1996a; Menon

10 et al. 1974). This inhibition of release resulted in an increase in central dopamine (DA) levels (Roth and Suhr 1970). GHB has also been found to inhibit rather than enhance dopamine releasing agents, and enhance drugs that block dopamine release (Feigenbaum and Howard 1996a). A lengthy inhibition of DA release might disinhibit DA autoreceptors resulting in an increased expression and activity of tyrosine hydroxylase (the rate-limiting enzyme in DA biosynthesis), explaining the increase in DA synthesis that has been observed (Roth and Suhr 1970; Feigenbaum and Howard 1996a).

Research by Volpi et al. (1997) revealed that in patients with

Parkinson's disease, GHB was still able to cause GH secretion whereas

GABA was not. This suggests that GHB is not working through a

GABAnergic pathway as many earlier researchers had assumed, and indicates it is acting, in this case, through non-dopamine mediation, such as serotonergic neurotransmission or through a pathway.

It has been demonstrated that alpha-2 receptor-stimulated GH release is mediated by serotonergic neurotransmission (Conway et al. 1990; Smythe et al. 1975). The ability of the serotonin receptor antagonist, metergoline, to decrease GHB-induced GH release indicates GHB action may be through the serotonergic system (Gerra et al. 1995). Research has shown that GHB caused no marked changes in norepinephrine levels (Roth and Suhr 1970), but does increase serotonin concentration (Walmeler and Fehr 1978).

Hedner and Lundborg (1983) found that GHB does not change the

11 endogenous brain concentration of 5-hydroxytryptamine (5-HT, serotonin), however it does cause an increase in the rate of 5-HT synthesis and degradation (Hedner and Lundborg 1983). Additional research by Vescovi and Coiro (1997) found that alcoholics showed a down regulation in the serotonin receptor subtype 5-HT1 D. In this instance, GHB is unable to cause the release of GH.

GH is thought to act as an adrenal secretagogue, and cause decreased cortisol clearance and metabolism, or possibly a decrease in serum cortisol binding globulin leading to increased serum free cortisol in humans with elevated GH (Weaver et al. 1994). Previous research on human subjects have found that plasma cortisol rose significantly following acute

GHB administration (Oyama and Takiguchi 1970). In rats, corticosterone serves as the primary glucocorticoid instead of cortisol (Griffin and Ojeda

1996). The effect of GHB on plasma corticosterone levels has not been analyzed experimentally in rats.

The ability of GHB to increase GH levels may be a model to indirectly evaluate the hypothalamic action of this drug (Gerra et al. 1995). The secretion of GH is under the control of the CNS, through action of two specific hypothalmic neuropeptides released from hypophyseal-portal system, GH­ releasing hormone (GHRH), and somatostatin (SS). The actions of GHRH on the somatotrophs of the anterior pituitary are stimulatory while the effects of

SS are inhibitory (Argente et al. 1996; Conway et al. 1990; Muller et al. 1995).

12 Research obiectives

The primary goals of this research were to examine the action of GHB on GH release in rats, as well as to determine if there was an effect on plasma corticosterone levels. There were seven major questions asked in this research. During the early postnatal period will exogenous GHB 1) increase the body mass of rats in a dose dependent fashion, 2) alter basal GH levels, and 3) result in alterations in the development of tissues such as the adrenals and gonads? In adult rats, 4) what pathway does GHB use to cause the release of GH, 5) what effect will GHB have on the release of corticosterone, 6) would chronic administration of GHB reduce GHB-induced effects, and 7) what response variations exist between male and female rats?

It is hypothesized in this study, that GHB is able to cause the release of GH by increasing modular action of a neurotransmitter on the release of

GHRH or SS. It is also hypothesized that this release of GH through chronic injections of GHB will result in a dose-dependent increase in weight gain in both male and female rats. And finally, it is hypothesized that GHB administration will result in an increase in plasma corticosterone, similar to that seen for cortisol in humans.

Rationale for Using Neurotransmitter Antagonists and Agonists

As indicated in the previous sections, GHB has an effect on the levels of serotonin and dopamine found in the brain, and both of these

13 have been shown to influence the release of GH from the

anterior pituitary. In order to elucidate if the ~HS-induced GH release is due

to either of these transmitters, the following drugs were used in conjunction

with GHB: Chlorpromazine, cyproheptadine and pyridoxine.

Cyproheptadine is a serotonin receptor antagonist (Conway et al.

1990; Smythe et al. 1975), capable of completely blocking the ability of 5HT

to elevate serum GH levels in rats at a: dosage of 10 mg/kg (Smythe et al.

1975). If this drug reverses the effect of GHB, it may be assumed 5HT plays

a role in GHB mediation.

Chlorpromazine is a dopamine receptor antagonist (Menon et al. 1974;

Porterfield 1977; Sato et al. 1995) capable of reversing the effects of

dopamine. If administration of this drug hinders the ability of exogenous GHB to elevate serum GH levels, this would indicate dopamine plays a key role in

GHB mediation.

Pyridoxine is a drug which helps facilitate the decarboxylation of L­ dopa to dopamine by acting as a coenzyme of Dopa carboxylase. In this manner, pyridoxine by elevating dopamine levels (Delitala et al. 1976; Reiter and Root 1978), causes a significant rise in GH levels within 60 minutes of administration, while simultaneously decreasing prolactin levels (Delitala et al.

1976). If this drug is capable of enhancing GHB action, it would strongly suggest dopaminergic involvement.

14 Methodology

Animals

Pregnant rats were purchased from Harlan Sprague Dawley, and

placed in standard plastic cages with wood chip bedding, and housed in the

animal care facility in Lappin Hall. The ambient temperature was maintained

at approximately 22 °C, with a 12-hour lighVdark cycle. Both food and water were freely available throughout the experiment. Twenty-four days after birth, the juvenile rats were weaned and separated into groups of three.

Drugs

Gamma-hydroxybutyrate (sodium salt), pyridoxine, chlorpromazine, and cyproheptadine were purchased from the Sigma Chemical Co.

Cyproheptadine was dissolved in 50% (v/v)·ethanol, and the other three drugs were dissolved in physiological saline (0.9% sodium chloride w/v). All drugs were at room temperature when administered.

Chronic Experiment

The chronic experiment is summarized on the left side of the protocol flow chart in Figure 3. On the third postnatal day, the rat pups were sexed and weighed. The male and females were divided into three groups, with each group containing 5 males and 4 females. The first group received 100

15 mg/kg of GHB, the second received 500 mg/kg of GHB, and the last group

received of 0.9 % saline, which was used as a placebo. These amounts of

GHB are well within the range of previous studies (Atru et al. 1980, Colombo et al. 1994, Fadda et al. 1989). All injections were performed intraperitoneally using a 1 cc syringe with a 27 gauge needle, with a total volume of 0.1 cc. The pups were weighed and injected twice a week at approximately 5:00 p.m., for ' a period of 38 days.

At the end of the 38 days, the rats were euthanized using a small animal guillotine, and blood was collected in heparinized 15 ml test tubes.

The pituitary, left kidney, left adrenal, and left testis/ovary were removed from each animal, cleaned of excess fatty tissue, and weighed.

Acute experiment

The protocol for the acute experiment is shown in the right side of the flowchart in Figure 3. Male and female rats, 7 weeks of age, were divided into 5 groups and weighed. The first group received an injection of physiological saline only (4 males, 4 females). Group 2 was injected with

GHB, 500 mg/kg (5 males, 4 females). Group 3 was injected with chlorpromazine, 42.6 mg/kg (5 males, 3 females}. Group 4 was injected with pyridoxine, 20 mg/kg (4 males, 3 females). The last group, group 5, was injected with cyproheptadine, 10 mg/kg (5 males, 4 females). Fifteen minutes following the first injection, groups 3-5 received an injection of GHB,

16 I Rats-3 days post-natal I

•• • ' Saline 100 rrg'kg 500 rrg'kg (Qmtrol) GHB GHB

For 38 days, rats were Injected and weighed 1EXP~1 twice a week •• •• I 9 day pause in injections I

,, ••

Rats in these two groups were 1~~1 sacrificed by guillotine, and weights of pituitary, left adrenal, left kidney, and left testis'ovary were detemined. ~ta!

Control• I Pyridoxine• Ollorprorrazine• 11 O;proheptaclne• I 1,s mnutes I + • ' 500 I Vehide I 500 500 500 500 mg/kg rrg'kg rrg'kg rrg'kg rrg'kg GHB GHB GHB GHB GHB

I 70 minute interval I

Growth hormone assay (ELISA) Corticosterone assay (RIA)

Figure 3. Overview of experimental design.

17 500 mg/kg. The total volume of each injection was 0.2 cc. The initial injections were performed at approximately 1 :00 p.m. After a period of 70 minutes following the GHB injection, the animals were euthanized using a small animal guillotine.

The time period between removing the rat from its cage and sacrificing the animal was kept short as possible to prevent a raise in corticosterone levels. Blood collected from each rat upon sacrifice was centrifuged to separate the plasma. The plasma was then frozen at -20°C until it could be analyzed.

To determine the effect of chronic exposure of exogenous GHB on growth hormone release, 5 male and 4 female rats, from the chronic experiment also received an injection of GHB, 500 mg/kg/0.2cc. These rats had not received GHB for 9 days to determine if there was a long-lasting effect. The rats were sacrificed and plasma analyzed in the same manner as the rats from the acute experiment.

Growth Hormone Assay

Determination of rat plasma growth hormone (rGH) was made using an enzyme immunoassay (EIA) system (Amersham International). Plasma samples were diluted 1 :5 in assay buffer [0.025M phosphate buffer pH 7.5 containing 0.1 % sodium azide.] Standards were prepared using serial

18 dilutions (1.6, 4.1, 10.24, 25.6, 64, 160 ng/ml) from a rat growth hormone

(rGH) stock solution of 400 ng/ml. Fifty microliters of the plasma samples and rGH standards were pipetted into individual wells of a microtitre plate, coated with anti-goat lgG. An equal volume of antiserum (goat anti-rGH serum) was then added to each well, and allowed to incubate at room temperature for 3 hours. After the incubation period, 50 µI of rGH conjugate was pipetted into each well and incubated at room temperature for 30 minutes. The wells were then aspirated with a pipette and washed with wash buffer (0.01 M phosphate buffered saline pH 7.4 containing 0.2% Tween). A 100 µI aliquot of Amdex was then added to each well and allowed to incubate at room temperature for

30 minutes. Amdex is an amplification reagent that contains a straight chain dextran backbone covalently bound to hundreds of horseradish peroxidase molecules (Amersham 1996). The wells were again aspirated and washed.

For the color forming reaction, an 100 µI aliquot of TMB substrate (3,3' ,5,5'­ tetramethylbenzidine/hydrogen peroxide solution, in 20% dimethylformamide) was added to each well, and allowed to react at room temperature for a period of 30 minutes. The reaction was ended with the addition of 100 µI of

1 M H2S04. The microtitre plate was then analyzed for optical density (OD) using a MR 600 microplate reader (Dynatech Laboratories, inc.) at 450 nm.

All samples and standards were run in duplicate.

19 The rGH level of each sample was determined using a standard curve generated from the optical densities of the known rGH concentrations using

Microsoft Excel.

Corticosterone Assay

Determination of plasma corticosterone was made using a radioimmunoassay (RIA) kit (ICN Biomedicals, Inc.). Plasma samples were pipetted into glass tubes and diluted 1 :500 in steroid diluent. The tubes were then incubated at 98 °C in an lsotemp water bath (Fisher Scientific) for 10 minutes. With the exception of the blank, 100µ1 of anti-corticosterone was pipetted to the assay tubes. A 100µ1 volume of corticosterone-3H tracer

(-10, 000 cpm/1 00µ1) was then pipetted to all of the reaction tubes, and the tubes were mixed and incubated at 4°C for 16 hours.

Following the incubation, 200µ1 of charcoal dextran solution (4°C) was added and the tubes were mixed and incubated for 20 minutes. The tubes were then centrifuged at 2500 rpm for 15 minutes at 4°C, using a Marathon

21 K/BR centrifuge (Fisher Scientific). The supernatant was then decanted into scintillation vials. A volume of 16 ml of scintillation fluid was then added to each vial and counts per minute (cpm) were recorded using a Beckman scintillation counter (LS 5801 Series).

20 All samples and standards were run in duplicate. The corticosterone levels of each sample was determined using a standard curve generated from the cpm of known corticosterone concentrations using Microsoft Excel.

Statistical Analysis

Analysis of variance (ANOVA) was used to determine if a significant difference existed between groups. In the chronic study, all groups were compared to the group receiving the placebo, while in the acute experiment groups were compared against those receiving 500 mg/kg of GHB. The

ANOVA and graphical data were performed using Microsoft Excel. In all graphs, points represent the mean ± the standard error value for each group indicated.

21 Results

The administration of 500 mg/kg of GHB in both the chronic and acute

experiments was observed to produce an anesthetic effect immediately

following the injections. This sedative state lasted for approximately two

hours.

Chronic Experiment

The effect of chronic GHB injections on male and female rats are shown in Figures 4 and 6 respectively. To eliminate the possibility that

discrepancies in the starting weights of the animals had some impact on the study, each day the animals were weighed, the weights were standardized by simply dividing by the starting weight of each animal. This information is shown in Figures 5 and 7.

For both the males and females, analysis of variance revealed no significant difference between the groups injected with GHB, both 100 mg/kg and 500 mg/kg, and the control group (F<5.3, p>0.05). However, while they may have not been significant, different trends do seem to exist between the males and females. For the males, the highest weight gain was obs_erved in the group receiving 100 mg/kg GHB, and the lowest weight gain was observed in the group receiving 500 mg/kg GHB (Day 38, F=0.6727,

P=0.4358), with the difference growing closer to significant over time.

22 1 8 0 160 1 40

ti) -E 1 20 ....ns C, 100 -.r::. 80 -C, "Gi ;: 60 40 I\) w 20 ,:, ~ 0 0 1 0 20 30 40 Time (Days)

E._1 00 mg / kg • 5 0 0 mg /kg ... C on tro I

Figure 4. The effect of varying concentrations of chronically administered y-hydroxybutyrate on the weight gain of male rats. 20 1 8 1 6 .r:. -0, 'cii 1 4 ~ 1 2 'O Cl,) .!'.::! 1 0 'O "-ca 8 - 'O C ca 6 U)- 4 I\) +:>- 2 0 0 1 0 20 30 40 Time ( D ays)

F 1 00 mg / kg • 5 0 0 mg /kg • Co ntro!]

Figure 5. Standardized weights of male rats chronically injected with varying concentrations of y­ hydroxybutyrate. 140

1 2 0

1 0 0 'in E ...(Q 80 ~ .r:. -Cl 60 ·a;

I\) 3: 0, 40

20

0 0 1 0 20 30 40 Time (Days)

• 1 0 0 m g / kg • 500 mg / kg ....,,. Control

Figure 6. The effect of varying concentrations of chronically administered y-hydroxybutyrate on the weight gain of female rats. 1 8 1 6

L: 1 4 -C) ·; ;: 1 2 'O Q) 1 0 .!::! 'O... 8 I\) cu 0) 'O C 6 cu en- 4 2 0 0 1 0 20 30 40 Time (Days)

• 1 0 0 m g /kg • 5 0 0 mg / kg .. Con trol ]

Figure 7. Standardized weights of female rats chronically injected with varying concentrations of -y­ hydroxybutyrate. For the females, injection of both 100 mg/kg and 500 mg/kg led to a

slight increase in body weight over the group receiving the placebo.

When the weights of the organs were analyzed, it was found that for

the males, there was no significant difference between the groups receiving

GHB, either 100 mg/kg or 500 mg/kg, and those receiving saline (F<5.1174,

.P>0.05). The mean weights for each group is shown in Table 2.

Table 2. The effect of chronically administered y-hydroxybutyrate on the organ development of male rats. All weights are expressed as grams of tissue per kilogram of total animal mass.

Pituitary Left Kidney Left Adrenal Left Testis Control 0.0334 4.4546 0.1102 5.6247 100 mg/kg 0.0343 4.3123 0.1012 5.4714 500 mg/kg 0.0337 4.4093 0.0999 5.7095

For the females, there was also no significant difference in organ weights between the groups. The groups receiving 100 mg/kg GHB showed

, a considerable discrepancy in the weights of the left kidney and left adrenal

(F=3.8157, P=0.0935; F=1.0451, P=0.3407 respectively) between the groups.

The mean values for these groups are listed in Table 3.

Table 3. The effect of chronically administered y-hydroxybutyrate on the organ development of female rats. All weights are expressed as grams of tissue per kilogram of total animal mass.

Pituitary Left Kidney Left Adrenal Left Ovary Control 0.0382 4.3301 0.1499 0.3149 100 mg/kg 0.0384 4.4137 0.1343 0.3704 500 mg/kg 0.0404 4.3599 0.1361 0.3670

27 Acute Experiment

Growth Hormone Assa;

As shown in Figure 8, administration of GHB in males led to a non­ significant increase of plasma GH to 260 ng/ml from a mean of 176 ng/ml in the controls. The male rats receiving the dopamine receptor antagonist chlorpromazine had their plasma GH return to the basal level of 175 ng/ml

(P=0.5195: F=0.4539), and those male rats receiving the serotonin receptor antagonist, cyproheptadine, had the plasma GH levels fall to 145 ng/ml (P=

0.3192; F= 1.128). The rats receiving the Dopa carboxylase coenzyme, pyridoxine, had their plasma GH fall to 32 ng/ml (P=0.0983; F=3.636).

For male rats that had received chronic injections of GHB throughout their post natal development, administration of 500 mg/kg of GHB did not produce an increase in plasma GH, while an elevation of plasma GH did occur in the male na'ive rats receiving the first injection (500mg/kg) of GHB

(P=0.1166; F=3.094).

As indicated in Figure 9, GHB administration to female rats did not cause the increase in plasma GH as seen for the males. Instead GH levels fell from 176 ng/ml in the control to 115 ng/ml (P=0.2421, F=1.684).

Compared to the controls, non-significant reductions in plasma GH levels in female rats were observed following acute injections of chlorpromazine [95 ng/ml (P=0.5010, F=0.5254)], cyproheptadine [83 ng/ml (P=0.2394,

F=1.706)], and pyridoxine [78 ng/ml (P=0.2189, F=1.975)].

28 .....__E 400 g, 350 ~ 300 - S 250 200 - Ea.. :co 150 .c 100 ~ := 50 I\) co ~ 0 C, Vehicle Chronic GHB Pyrid . Chlor. Cypro.

Figure 8. Plasma growth hormone levels of male rats in response to 500 mg/kg of y-hydroxybutyrate alone or with various neurotransmitter influencing compounds. The abbreviations pyrid., chlor. and cypro. are for the drugs pyridoxine, chlorpromazine and cyproheptadine respectively. The group indicated in the graph as chronic, had received chronic injections of 500 mg/kg of GHB for a period of 38 days. -----

...... E 250 C) ._.C 200 C1) C 0 150 E ~ 0 100 :c .c i 50 0 ~ (,J 0 0 C, Vehicle Chronic GHB Pyrid . Chier. Cypro.

Figure 9. Plasma growth hormone levels of female rats in response to 500 mg/kg of y-hydroxybutyrate alone or with various neurotransmitter influencing compounds. The abbreviations pyrid., chlor. and cypro. are for the drugs pyridoxine, chlorpromazine and cyproheptadine respectively. The group indicated in the graph as chronic, had received chronic injections of 500 mg/kg of GHB for a period of 38 days. For the female rats that had received chronic injections of GHB, the acute administration of the drug caused virtually no change in plasma GH levels compared to that of the control group, and had a P-value of 0.1397 and

F-value of 2.896 compared to the na"ive group receiving GHB.

Corticosterone Assay

As shown in Figure 10, administration of GHB to male rats, caused a tremendous increase in plasma corticosterone levels, when compared to saline-injected controls. This increase from 19 ng/ml to 431 ng/ml was significant (P=0.0009, F=37.215). This effect was not abated by any of the drugs given in combination, and in fact all reported levels are higher, although not significantly. The group receiving chlorpromazine had a mean plasma corticosterone level of 464 ng/ml, while the group receiving cyproheptadine had a concentration of 580 ng/ml (P=0.0869, F=3.959). The final group receiving pyridoxine had a plasma corticosterone level of 512 ng/ml. All groups were significantly elevated from the controls, but there were no significant differences between the treatment groups.

For the male rats that had received chronic injections of GHB, the acute administration of the drug (500 mg/kg) resulted in the same increase in plasma corticosterone, 483 ng/ml, as seen in the na"ive rats that received the

500 mg/kg dosage (see Figure 10).

31 W700 - r c, 600 * * * * * E. 500 Q) C 400 0 ~ ...a> 300 w I\) 200 ~(.) t 100 8 0 Vehicle Chronic GHB Pyrid. Cypro.

Figure 10. Plasma corticosterone levels of male rats in response to 500 mg/kg y-hydroxybutyrate and various drugs. (* Significantly different from controls). The abbreviations pyrid., chlor. and cypro. are for the drugs pyridoxine, chlorpromazine and cyproheptadine respectively. The group indicated in the graph as chronic, had received chronic injections of 500 mg/kg of GHB for a period of 38 days. ..-.. 1000 -E ""en * * * ._..C 800 a, C 600 0 ~ ..,a, 400 "'0 ..,(.) 200 ~ w 0 w (..) 0 Vehicle Chronic GHB Pyrid . Chlor. Cypro.

Figure 11 . Plasma corticosterone levels of female rats in response to 500 mg/kg -y-hydroxybutyrate and various drugs. (* Significantly different from controls). The abbreviations pyrid., chlor. and cypro. are for the drugs pyridoxine, chlorpromazine and cyproheptadine respectively. The group indicated in the graph as chronic, had received chronic injections of 500 mg/kg of GHB for a period of 38 days. As illustrated in Figure 11, the administration of 500 mg/kg of GHB to female rats caused a significant rise in plasma corticosterone as seen in the male rats, which had received the 500 mg/kg dosage (P=0.0004, F=50.762).

The addition of the serotonin and dopamine receptor antagonists and the coenzyme for Dopa carboxylase did not alter the effect of GHB. The female groups receiving chlorpromazine, cyproheptadine, and pyridoxine had mean plasma corticosterone concentrations of 555, 776, and 670 ng/ml respectively.

Unlike the males however, the female rats that had received chronic injections of GHB, had a significantly reduced effect on GHB induced corticosterone release, 103 ng/ml (P=0.0153, F=11.268). This was an 80% reduction.

34 Discussion

Previous research has indicated that GHB leads to the release of GH

(Oyama and Takiguchi 1970; Takahara et al. 1977), a hormone that has a definite growth promoting function (Griffith and Ojeda 1996). As indicated in

Figure 5, chronic injections of y-hydroxybutyrate did not result in ,any significant increases in the masses of developing male rats. In fact, the group receiving the highest dosage, 500mg/kg, had the smallest amount of total body mass. Failure to measure a significant increase in mass may be due to several factors. GH is known to be released endogenously in a pulsatile manner (Jaffe et al. 1998). Chronic injections of GHB, as were performed in this research, may lead to an inability of the drug to stimulate GH release, therefore consecutive injections could not result in additional growth.

Evidence for this is shown in Figure 8, in which male rats that had previously been injected chronically (indicated on the bar graph as chronic) with GHB for a period of 38 days failed to exhibit a rise of plasma GH in response to a acute injection of 500 mg/kg of GHB. This effect may be caused by the large dose of GHB leading to a down regulation of GHB receptors, or perhaps by a down regulation of an intermediate's receptors in the pathway, such as dopamine or serotonin. Recent research by Ratomponirina et al. (1998) found that chronic peripheral administration of 500 mg/kg of GHB rapidly

35 down-regulates GHB receptors in rats. This effect is long lasting or possibly even permanent, since the rats in the group chronically injected with 500 mg/kg of GHB had not been injected for a period of 9 days before the acute portion of the experiment. It is of interest to note that the male rats achieving the highest amount of weight gain were those receiving 100 mg/kg. While not statistically significant, weight gain was higher in this group than the control.

This suggests that perhaps at this lower dosage, chronic injections do not lead to receptor down regulation.

For the chronically treated females (Figure 7), administration of both the 100 and 500 mg/ml concentrations of GHB resulted in slight increases in mass over time, 6.6 and 3.9 % respectively. This is interesting since acute administration of GHB in female rats did not result in elevated growth hormone levels (see Figure 9).

In an effort to elucidate the mechanism of action by which GHB causes the release of GH, the effects of chlorpromazine and cyproheptadine were determined. In both male and female rats, each of these drugs caused a non­ significant decrease in the GH release induced by GHB (Figures 8 and 9).

The chlorpromazine led to 33% and 17% reduction of

GHB induced GH release in males and females respectively, whereas, the serotonin antagonist cyproheptadine resulted in 44% and 28% reductions in males and females respectively. Such reductions in GH release caused by these antagonists suggest both dopamine and serotonin are involved in the

36 mechanism involved in y-hydroxybutyrate's GH releasing activity, with serotonin having a more prominent role as in the case of normal basal secretion (Conway et al. 1990).

Pyridoxine (Vitamin B6) is metabolized in vivo to pyridoxal-5'­ phosphate which can function as a for dopa decarboxylase which converts L-dopa to dopamine (Delitala et al. 1976; Geraldet al. 1982). The effect of 20 mg/kg of this cofactor on GHB induced GH release is intriguing.

In males, the drug caused a 88% reduction in GH release, and a 32%

' reduction in females. This result is quite unexpected since pyridoxine produces the release of growth hormone in normal human subjects (Delitala et al. 1976). A possible explanation is that pyridoxine has a more rapid effect on G H release than does G HB, and by the time the serum was assayed, the hormone may have been cleared from the plasma. However, in humans the maximum GH peak occurs more rapidly as a result of GHB injection than by pyridoxine injection (Delitala et al. 1976; Takahara et al. 1977). In rats, growth hormone has a plasma half life of 5-6 minutes (Badger et al. 1991; Goya et al.

1987) so if pyridoxine does cause a rapid plasma GH surge, it may be cleared before the analysis took place. These results coincided with past research that has found GHB inhibits rather than enhances dopamine releasing agents

(Feigenbaum and Howard 1996a). If this is the case, the reduction in GH secretion caused by chlorpromazine may just be due to action on the normal

37 pathways for basal GH release, as opposed to action on the GHBnergic system.

Further research should be devoted to determining possible action by the and cholinergic systems on the GHB pathway. Evidence suggests that, in normal conditions, the catecholaminergic and cholinergic systems stimulates GH secretion by releasing GHIH and inhibiting SS, and the release of adrenaline regulates its pulsatile secretion by action on its alpha and beta receptors (Argents et al. 1996; Muller et al. 1995).

The discrepancies between the male and female responses to GHB is quite intriguing, and is not easily explained, since previous research on this subject has been performed almost exclusively on males. One explanation is the sexual dimorphism in the regulation of GH secretion that exists between normal male and female rats (Jaffe et al. 1998). Males tend to have large infrequent pulses, predominately during sleep, whereas females have smaller more uniform pulses throughout the day (Jaffe et al. 1998; Muller et al. 1995).

This difference is thought to be caused primarily by the more prominent role of hypothalamic somatostatin (SS) in males than in females (Jaffe et al.

1998).

GHB was found in this research to have the ability to increase the concentration of plasma corticosterone in both male and female subjects by a factor of 10-20 fold (Figures 10 and 11 ). The mechanism for this release is not known. However, it is probably not due to the release and consequent

38 action of growth hormone as described by Weaver et al. (1994), since the

females without the increased GH levels also had this elevated corticosterone

release. GHB may again be acting in the hypothalamus, causing the release

of corticotropin-releasing hormone (CRH) which could in turn release

adrenocorticotropin hormone (ACTH) from the anterior pituitary, and

ultimately cause the release of corticosterone from the adrenal gland. CRH secretion from the hypothalamus has been found to be stimulated in response to-norepinephrine, acetylcholine, serotonin, and dopamine (Calogero et al.

1988; Calogero et al. 1989a; Calogero et al. 1989b). Caloogero et al. 1988 also suggests that CRH secretion is weakly stimulated by epinephrine and dopamine. However, when used in combination with GHB, the dopamine and serotonin receptor antagonists, chlorpromazine and cyproheptadine, were unable to prevent the GHB induced corticosterone release, indicating these two particular neurotransmitters appear not to be involved in GHB's corticosterone releasing process.

Another possible mechanism to explain the elevation of corticosterone is that GHB could be prompting the release of ACTH by acting directly on corticotrophs in the anterior pituitary. Finally, GHB may be acting directly on the cells of the adrenal cortex causing the release of corticosterone. These possible modes of stimulation were not studied in this research.

For the male rats, chronic injection of GHB had no effect on sensitivity for animals to release corticosterone (Figure 10). For the females however,

39 chronic administration of GHB decreased the drug's ability to release corticosterone by 80% (see Figure 11 ). A study by Ogilvie and Rivier (1997) found that female rats secrete more ACTH and corticosterone than male rats in response to the same amount of ethanol. This effect appears to be · dependent on the sex hormone estradiol (Nowak et al. 1995; Ogilvie and

Rivier 1997). If this increased ACTH release also holds true for GHB, then perhaps chronic injections of the drug would result in a more rapid ACTH receptor down regulation in female rats than males.

Conclusion

The primary goals of this research were to examine the action of GHB on GH and corticosterone release in both chronic and acute experiments.

While the receptor antagonists for the neurotransmitters serotonin and dopamine were able to reduce GHB effects, results indicate that GHB did not significantly stimulate GH secretion in either sets of experiments at the doses of 100 mg/kg and 500 mg/kg used in this research. Consequently, there was no dose dependent weight increase caused by this drug. However, injections of GHB did cause significant increases in plasma corticosterone.

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47