Norepinephrine Regulation of Growth Hormone Release from Goldfish Pituitary Cells. I. Involvement of...

Journal of Neuroendocrinology, 2000, Vol. 12, 311–322

Norepinephrine Regulation of Growth Hormone Release from GoldfishPituitary Cells. I. Involvement of a2 Adrenoreceptor and Interactionswith Dopamine and Salmon Gonadotropin-Releasing Hormone

E. K. Y. Lee,* V. C. C. Chan,* J. P. Chang,† W. K. Yunker† and A. O. L. Wong**Department of Zoology, University of Hong Kong, Hong Kong.†Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada.

Key words: growth hormone, a2 adrenoreceptor, norepinephrine, epinephrine, dopamine, gonadotropin-releasing hormone, goldfish pituitarycells.

Abstract

Adrenergic regulation of growth hormone (GH) release in the goldfish was examined in vitro usingdispersed goldfish pituitary cells under column perifusion. Norepinephrine and epinephrinesuppressed basal GH release from goldfish pituitary cells in a reversible and dose-dependentmanner. At high doses, a transient rebound of GH release was observed after termination ofnorepinephrine and epinephrine treatment. In this study, the dose-dependence of adrenergicinhibition on basal GH release was mimicked by the a2 agonists clonidine and UK14304. Basal GHsecretion, however, was not affected by the b agonist isoproterenol and a1 agonist methoxamine. Inaddition, the inhibitory actions of norepinephrine and clonidine on basal GH release were blockedby the a2 antagonists yohimbine and RX821002. The b antagonist propranolol and a1 antagonistsprasozin and benoxathian were not effective in this respect. Salmon gonadotropin-releasinghormone (sGnRH) and dopamine, two known GH-releasing factors in fish, stimulated GH releasefrom goldfish pituitary cells and their GH-releasing actions were inhibited by simultaneous treatmentwith norepinephrine. Furthermore, the GH rebound after norepinephrine treatment was significantlyenhanced by prior exposure to sGnRH and this effect was not observed with dopamine treatment.These results, taken together, suggest that in the goldfish adrenergic input at the pituitary levelinhibit basal GH release through activation of a2 adrenoreceptors. This a2 inhibitory influence mayinteract with dopaminergic and GnRH input to regulate GH secretion from goldfish pituitary cells.The ‘post-inhibition’ GH rebound after NE treatment and its sensitivity to sGnRH potentiation mayalso represent a novel mechanism for GH regulation in fish.

In mammals, growth hormone (GH) secretion is stimulated exert their regulatory actions. In the goldfish, dopamine (3),gonadotropin (GTH)-releasing hormone (GnRH 4), neuro-by GH-releasing hormone (GHRH) and inhibited by somato-

statin. The release of these two GH regulators into the portal peptide Y (5), GHRH (6), bombesin (7), thyrotropin-releasing hormone (8) and cholecystokinin (9) are known toblood is in turn modulated by neurotransmitters and neuro-

peptides within the central nervous system (CNS; for a recent stimulate GH release directly at the pituitary level. Whereassomatostatin (10), norepinephrine (11); and serotonin (12)review, see (1)). Unlike mammals, teleost fish do not have a

hypophyseal portal blood system and the median eminence function as inhibitors of basal GH secretion. Among the GHregulators examined in the goldfish, catecholamines are par-is functionally incorporated into the anterior pituitary (2).

In this case, pituitary cells are directly innervated by the ticularly interesting in terms of their regulatory role inpituitary functions. Dopaminergic neurones have been identi-hypothalamus and neuroendocrine factors from the CNS can

be delivered to the vicinity of individual pituitary cells to fied in the preoptic nuclei of the goldfish hypothalamus and

A part of the present study has been presented in the 10th Annual Meeting of the Society for the Study of Endocrinology, Metabolism andReproduction, Hong Kong (1995) and 11th Asia-Oceania Congress of Endocrinology, Seoul, South Korea (1998).

Correspondence to: Dr Anderson O. L. Wong, Department of Zoology, The University of Hong Kong, Pokfulam Road, Hong Kong (e-mail:[email protected]).

© 2000 Blackwell Science Ltd

312 GH release and a2 adrenoreceptors

these neurones innervate the anterior pituitary through a Materials and methodspreoptico-hypophyseal pathway (13). In the same animal Animalsmodel, adrenergic neurones are located in the isthmal teg-

Goldfish (Carassius auratus) of the common or comet variety, with bodymentum, periventricular nuclei and basal hypothalamus. weight ranging from 25 g to 40 g, were purchased from local pet stores andProjection of these adrenergic neurones into the infundibulum maintained in 200 l aquaria at 18–20 °C under a photoperiod of 12 L512D

cycle for at least 2 weeks before experiments. The fish were fed to satiationalong a preoptico-infundibular pathway has been reporteddaily with commercial fish feed and were killed for pituitary cell preparation(14). Besides, adrenergic fibers from the isthmal tegmentum,by spinosectomy after treatment with 0.05% MS222 according to the animal

the equivalent of the locus ceruleus in mammals, have been care regulations at the University of Hong Kong. Since the goldfish used inproposed to be the source of adrenergic input to the pituitary the present study were gonadally regressed and exhibited no sexual dimorph-

ism in external morphology, both male and female fish were used in thein fish (2). In other studies, however, a humoral origin ofpreparation of pituitary cells.pituitary adrenergic input has been suggested as norepi-

nephrine is not present at significant levels in the goldfish Reagents and test substancespituitary (15). In teleosts, norepinephrine and epinephrine Norepinephrine, epinephrine, dopamine, bovine serum albumin (BSA) and

HEPES were purchased from Sigma (St Louis, MO, USA). sGnRH wassecretion from chromaffin cells of head kidneys and walls ofobtained from Peninsula Laboratories (Belmont, CA, USA). Agonistscardinal veins are the major source of catecholamines foundand antagonists for adrenoreceptors, including phenylephrine, clonidine,in systemic circulation, especially during the acute phase of UK14304, isoproterenol, methoxamine, phentolamine, propranolol, prazosin,

stress responses (for review, see (16)). yohimbine, benoxathian, and RX821002, were obtained from ResearchBiochemicals Inc. (Wayland, MA, USA). Medium M199 and horse serumDopamine, the precursor of norepinephrine and epineph-for cell culture were purchased from Gibco BRL Life Technologies (Grandrine, is an important GH-releasing factor in the goldfish,Island, NY, USA). Dopamine, norepinephrine, epinephrine and adrenergicespecially in fish undergoing gonadal regression (17). This analogs were stored under argon gas at −20 °C to prevent oxidation of drugs.

biogenic amine stimulates GH release both in vivo (17) and sGnRH was dissolved in 0.1% acetic acid to give a stock solution at 1 mconcentration and stored frozen at −80 °C. Phenylephrine, isoproterenol,in vitro (18) through pituitary D1 receptors coupled topropranolol, clonidine, methoxamine, phentolamine, benoxathian, RX821002,cAMP- (19) and Ca2+-dependent pathways (20). In teleosts,norepinephrine, epinephrine and dopamine were dissolved directly in deion-such as the goldfish and other carp species, dopamine also ized water to give a 10-m stock solution, which were then diluted to

functions as a GTH-release inhibitor. Suppression of basal appropriate concentrations using perifusion medium. Preparation of mediumwith UK14304, prazosin and yohimbine was similar to that of other adrenergicGTH release and blocking of the GTH-releasing effect ofdrugs except that they were first dissolved in dimethyl sulfoxide (DMSO)GnRH are mediated through pituitary D2 receptors (21). Inbefore subsequent dilution. The final concentrations of DMSO were alwaysour previous studies, we have shown that norepinephrineless than 0.1% and DMSO up to 0.1% was previously shown to have no

stimulates GTH release in goldfish both in vivo (22) and in effect on basal GH release from goldfish pituitary cells (22). In this study,vitro (23). The GTH-releasing action of NE can be exerted test substances were freshly prepared 15 min before the initiation of drug

treatment to minimise the potential problem of drug oxidation.directly at the pituitary cell level via a1 adrenoreceptors (24).In in-vivo experiments, intraperitoneal injection of norepi- Preparation of goldfish pituitary cellsnephrine induces a dose-dependent inhibition of serum GH Dispersed goldfish pituitary cells were prepared as described by Chang et al.levels in the goldfish. However, i.c.v. injection of norepi- (25) with minor modifications. Briefly, pituitaries were excised from goldfish

and washed three times in washing medium (Medium M199 with Hanks’nephrine is not effective in this respect (11). These resultssalts, 25 m HEPES, 26 m NaHCO3, 100 U/ml penicillin, 100 mg/ml strepto-have prompted us to speculate that, in the goldfish, the sitemycin, 250 ng/ml fungizone, and 0.3% BSA; pH 7.2) to remove blood clots

of norepinephrine action to inhibit GH secretion is not within and other contaminating tissues. After washing, pituitaries were diced intothe CNS, but is located outside the blood–brain-barrier. fragments of #0.6 mm in thickness using a McILwain tissue chopper (Mickle

Laboratory Engineering, Gomshall, UK) and exposed to trypsinSimilar to other vertebrates, the pituitaries of teleosts are(25 mg/10 ml, GibcoBRL) for 35 min at 28 °C. After incubation, trypsinlocated outside the blood–brain-barrier and could be a pos-digestion was terminated by adding trypsin inhibitor (25 mg/10 ml, Sigma).

sible site of action for this adrenergic inhibition. Although Pituitary fragments were rinsed in DNase II (0.1 mg/10 ml, Sigma) followedthe receptor specificity of norepinephrine inhibition on basal by a two-step washing with Ca2+ -free medium (Medium M199 with Hanks’

salts without CaCl2, supplemented with 25 m HEPES, 26 m NaHCO3,GH release has not been examined in our previous studies,100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml fungizone, and 0.3%it is possible that adrenergic input to the pituitary canBSA; pH 7.2). Following these washing steps, pituitary fragments were

differentially regulate body growth and reproduction in the dispersed in Ca2+-free medium with DNase II (0.1 mg/10 ml ) by gentlegoldfish by modulating GH and GTH release, respectively. suction and extrusion using a BioRad DPTP transfer pipet (BioRad

Laboratories, Richmond, CA). Pituitary cells were harvested by filtrationIn this study, using an in-vitro column perifusion system,through a sterile nylon mesh (#30 mm pore size) followed by centrifugationwe tested the hypothesis that norepinephrine, by actingat 200×g for 10 min at 4 °C. After resuspending the cell pellet in 5 ml Ca2+-directly at the pituitary cell level through specific adrenorecep- free medium, total cell yield and percentage viability were estimated by cell

tors, inhibits both basal and stimulated GH secretion in the counting in the presence of trypan blue. The average cell yield was about0.5–0.7 million cells per pituitary with a mean viability of 95.4±0.7% (n=16).goldfish. Dose-dependence of norepinephrine and epinephrine

treatment on basal GH release from goldfish pituitary cells Column perifusion of goldfish pituitary cellswas examined. The receptor specificity of adrenergic inhibi- After cell counting, pituitary cells were cultured in plating medium (Mediumtion on basal GH release was characterized using agonists M199 with Earle’s salts, 25 m HEPES, 26 m NaHCO3, 100 U/ml penicillin,

100 mg/ml streptomycin, and 250 ng/ml fungizone; pH 7.2) with preswollenand antagonists specific for the a1, a2 and b adrenoreceptorCytodex beads (Sigma) for 2 h at 28 °C under 5% CO2 and saturated humidity.subtypes. Given that sGnRH and dopamine are two majorAfter the completion of cell attachment on Cytodex beads (>90%), horseGH-releasing factors in the goldfish, interactions of these two serum was introduced to cell culture to give a final concentration of 1% (v/v).

GH regulators with norepinephrine in regulating GH release Following a further incubation of 15–18 h, Cytodex beads with pituitary cellswere loaded into 0.5 ml microcolumns (#2.5 million cells/column;from goldfish pituitary cells were also investigated.

© 2000 Blackwell Science Ltd, Journal of Neuroendocrinology, 12, 311–322

GH release and a2 adrenoreceptors 313

ACUSYST-S’ cell perifusion system, CELLEX Biosciences Inc., Minneapolis, Effects of a1, a2, and b adrenergic agonists on GH releaseMN, USA) and perifused at a flow rate of 15 ml/h with prewarmed (28 °C)perifusion medium (Medium M199 with Hanks’ salts, 25 m HEPES, 26 m Increasing doses of the a1 agonists phenylephrine andNaHCO3, 0.1% BSA, 100 U/ml penicillin, 100 mg/ml streptomycin, and methoxamine, a2 agonists clonidine and UK14304 and b250 ng/ml fungizone; pH 7.2). Pituitary cells were routinely perifused for agonist isoproterenol were administered as 10-min pulses to3–4 h before the initiation of sample collection. After this initial perifusion

goldfish pituitary cells under column perifusion (Fig. 2).period, basal GH release from goldfish pituitary cells was relatively stable inthe absence of stimulation. Test substances, including dopamine, sGnRH, Representative results at 1 m dose of these adrenergic agon-norepinephrine, epinephrine and adrenergic analogues, were introduced into ists are shown in Fig. 2 and the respective dose–responseperifusion columns from a drug reservoir through a three-way stopcock. The curves (including those of norepinephrine and epinephrine)total dead volume of the perifusion system was estimated to be 1.32±0.04 ml

are summarized in Fig. 2. Similar to norepinephrine and(n=6) and thus it would take at least 5 min before pituitary cells exposed toepinephrine, the a2 agonists clonidine (0.1 n–10 m) anddrugs applied from the drug reservoir. Perifusate from individual columns

was collected in 5-min fractions and stored frozen at −20 °C. GH contents UK14304 (10 n–1 m) induced a dose-dependent suppres-in these samples were later assayed using a radioimmunoassay previously sion on basal GH release. The ED50 of the inhibitory actionvalidated for goldfish GH (10). of clonidine was estimated to be 287.2±18.5 n. The maximalData transformation and statistics inhibitory response and minimal effective dose of clonidine

were estimated to be −437.7±29.4% basal and 0.1 n,GH data (in ng/ml ) from individual columns were expressed as a percentageof the mean GH contents in the first six fractions collected at the beginning respectively. The higher maximal inhibitory response of cloni-of perifusion experiment prior to drug treatment (referred to as ‘% basal’). dine (P<0.05) compared to those of norepinephrineThis transformation was done to allow pooling of GH data from separate

(−167.3±15.8% basal ) and epinephrine (−242.4±94.9%columns without distorting the profile of GH release during the course ofbasal ) might be due to the fact that clonidine is moreperifusion (22). GH responses were quantified by calculating the net change

of GH release after a particular drug treatment (i.e. a net change of area hydrophobic than catecholamines and therefore takes aunder the curve). Dose–response curves were analysed using the GraphPad longer time to wash out from perifusion columns. (Since onlyPrism computer program (GraphPad Software, San Diego, CA) to estimate three doses of UK14304 were tested in these experiments, thethe ED50 values and maximal GH responses. Minimal effective dose was

ED50 of UK14304 was not determined). Unlike the a2defined as the lowest dose of the drug tested that induced a significant changein GH release with respect to basal levels (i.e. a net GH response that is agonists, the a1 agonist methoxamine (10 n–1 m) and bsignificantly different from zero). GH data were subjected to statistical agonist isoproterenol (0.1 n–10 m) were not effective inanalysis using Student’s t-test or followed by Fisher’s least significant altering basal GH secretion (Fig. 2). The a1 agonist pheny-difference (LSD) test. Differences were considered significance at P<0.05.

lephrine did not affect basal GH release at low doses(0.1 n–0.1 m) but a small inhibitory effect was observed at

Results high doses(1 m and 10 m). The minimal effective dose ofphenylephrine tested was 1 m and the ED50 was estimated

Adrenergic regulation of GH release from goldfish pituitary to be >1 m. The maximal inhibitory response could not becells deduced for phenylephrine due to a lack of fitting with the

GraphPad Prism program. In these experiments, the GHIncreasing doses (0.1 n–1 m) of norepinephrine (Fig. 1)response to the highest dose (10 m) of phenylephrine testedand epinephrine (Fig. 1), given as 10-min pulses, suppressed(−62.6±18.4% basal ) was much smaller (P<0.05) than thatbasal GH release from goldfish pituitary cells in a dose-of the corresponding dose of clonidine (−425±25.6% basal ),dependent manner with ED50 values of 18.5±1.4 andnorepinephrine (−162.2±17.4% basal ) and epinephrine15.5±4.6 n, respectively. In this study, individual doses of(−225.0±22.3% basal ). Unlike norepinephrine and epineph-norepinephrine and epinephrine were tested in naive pituitaryrine, termination of drug treatment using micromolar dosescells without prior drug treatment in separate perifusionof clonidine, UK14304, and phenylephrine did not trigger acolumns. The maximal inhibitory responses on GH releasesignificant level of ‘post-inhibition’ GH rebound.induced by norepinephrine and epinephrine were similar

(−167.3±15.8% basal for NE and −242.4±94.9% basal foreprinephrine, P>0.05). The peak inhibition on GH release Effects of a1, a2, and b adrenergic antagonists onwas observed 15–20 min after the initiation of norepinephrine norepinephrine inhibition of GH releaseor epinephrine treatment. Norepinephrine or epinephrineinhibition on basal GH release in general would recover The receptor specificity of norepinephrine inhibition on basal

GH release was further characterized in perifused goldfishwithin 30–40 min after drug treatment. At high doses ofnorepinephrine (100 n, 1 m, and 10 m) and epinephrine pituitary cells using adrenergic antagonists specific for differ-

ent subtypes of adrenoreceptors. In these experiments, the(1 m), GH release following the recovery phase increased tolevels significantly higher than that of the pretreatment basal. inhibitory effect of norepinephrine (10-min pulse, 0.5 m) was

tested during a 2-h continuous perifusion of either the generalThe peak height of this GH rebound after norepinephrine(100 n, 1 m, and 10 m) and epinephrine treatment (1 m) a antagonist phentolamine (5 m) or b antagonist propranolol

(5 m, Fig. 3). Norepinephrine consistently suppressed basalwas 117.7±6.9, 127.3±10.9, 128.5±8.6, and 122.5±6.8%basal, respectively (vs 100.0±2.5% basal of the pretreatment GH release and this inhibitory action was blocked by simul-

taneous treatment with phentolamine but not propranolol.basal, P<0.05). Since the rate of basal GH release fromgoldfish pituitary cells is known to be stable over a period of To further elucidate the possible involvement of a1 and a2

adrenoreceptors in norepinephrine action, norepinephrine4–5 h in this perifusion system (26), it is unlikely that theobserved GH rebound is the result of an ‘up-drifting’ of basal (0.5 m, Fig. 3) and the a2 agonist clonidine (0.5 m, Fig. 4)

were given during the continuous perifusion of the a1 antag-GH secretion.



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F. 1. Effects of norepinephrine (NE) and (–)epinephrine (EP) on basal GH release from perifused goldfish pituitary cells. Increasing doses(0.1 n–10 m) of NE () and EP () were given as 10-min pulses as indicated by vertical bars. GH data, expressed as Mean±SEM (n=5–7), werepooled from at least five independent experiments and the mean basal GH level was 45.4±3.6 ng GH/ml. GH responses were quantified as the netchange in GH release after a pulse of drug treatment (i.e. a net change of area under the curve) and the dose–response curves for NE and EPinhibition on basal GH release are presented in Fig. 2. In this study, separate columns were used for individual doses of NE and EP tested.

onist prazosin (5 m) or a2 antagonist yohimbine (5 m). The treatment as a similar GH response was not observed afterclonidine perifusion.inhibitory actions of norepinephrine and clonidine on basal

GH release were abolished in the presence of yohimbine, andprazosin was without effects in this respect. Using a similar Effects of norepinephrine on sGnRH- and dopamine-experimental design, the inhibitory actions of norepinephrine stimulated GH release(0.5 m, Fig. 3) and clonidine (0.5 m, Fig. 4) were furtherexamined in the presence of the a1 antagonist benoxathian To test for the possible interactions between norepinephrine

and other GH-regulators in goldfish, sGnRH (100 n) and(5 m) and a2 antagonist RX821002 (5 m). RX821002, sim-ilar to yohimbine, was effective in blocking the inhibitory dopamine (1 m), two known GH-releasing factors in fish,

were introduced as 5-min pulses during a 1.5-h continuousactions of norepinephrine and clonidine on GH release. Theinhibitory actions of norepinephrine and clonidine, however, perifusion of norepinephrine (10 m). Norepinephrine treat-

ment suppressed basal GH release as well as the GH-releasingwere not affected by simultaneous treatment with benoxath-ian. In these studies, the a2 antagonists yohimbine and actions of sGnRH (Fig. 5) and dopamine (Fig. 5). In the

case of sGnRH studies, the GH rebound after NE treatmentRX821002 not only blocked the inhibitory action of norepi-nephrine on basal GH release but also abolished the ‘post- (with a peak height of 265.4±49.3% basal ) was significantly

greater than that observed in the preceding norepinephrineinhibition’ GH rebound after norepinephrine treatment.Again, this GH rebound was specific to norepinephrine dose–response studies (Fig. 1; 128.5±8.6% basal at 10 m



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F. 2. Effects of the a1, a2 and b adrenergic agonists on basal GH release from perifused goldfish pituitary cells. Increasing doses of the a1 agonistsmethoxamine (10 n–1 m) and phenylephrine (0.1 n–10 m), a2 agonists clonidine (0.1 n–10 m) and UK14304 (10 n–1 m) and b agonistisoproterenol (0.1 n–10 m) were given as 10-min pulses to test their effect on basal GH secretion. Representative results of the profiles of GHrelease in response to these adrenergic agonists given at 1 m dose (as indicated by vertical bars) are presented in (). GH responses were quantifiedas the net change in GH release after a pulse of drug treatment (i.e. a net change of area under the curve) and the corresponding dose–responsecurves of these adrenergic agonists (including those of NE and EP) are summarized in (). GH data, expressed as Mean±SEM (n=4–5), werepooled from at least four independent experiments and the mean basal GH level was 49.8±5.5 ng GH/ml. Drug treatments giving a similar magnitudeof GH responses are grouped together by the same underscore (P>0.05, anova followed by Fisher’s LSD test).

norepinephrine, P<0.05). A similar potentiation of the GH (Fig. 6), defined as a net change of area under the curveover the pretreatment basal, was increased in a dose-rebound, however, was not observed in the corresponding

studies with dopamine. In this case, the peak height of GH dependent manner with respect to the concentration ofsGnRH tested. The peak height of GH rebound for therebound with dopamine treatment was only 123.3±16.5%

basal (P>0.05, when compared to the GH rebound after control and treatment groups exposed to 1 n, 10 n, and1000 n sGnRH was 128.1±6.8, 120.9±6.7, 202.3±19.4,10 m NE in Fig. 1). In a parallel study (Fig. 6), increasing

concentrations (1 n–100 n) of sGnRH were given as 5-min and 262.9±52.2% basal, respectively.pulses during the 1.5 h continuous perifusion of norepi-nephrine (10 m). Among these treatment groups, basal GH Discussionrelease was inhibited to the same extent after norepinephrineperifusion when compared to the control group without Adrenergic regulation of GH release has been extensively

studied in mammals, but similar studies in lower vertebrates,sGnRH treatment (Fig. 6). In the presence of norepi-nephrine, the GH responses to increasing doses of sGnRH especially in fish, are lacking. In mammals, especially in the

rat, norepinephrine stimulates GH release through a2 adreno-were small but dose-dependent (Fig. 6). The magnitude of‘post-inhibition’ GH rebound after norepinephrine perifusion receptors in the hypothalamus by inducing GHRH release




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F. 4. Effects of the a1 and a2 adrenergic antagonists on clonidine inhibition of basal GH release from perifused goldfish pituitary cells. Clonidine(Clon, 0.5 m) was given as 10-min pulses (as indicated by black bars) during the 2-h continuous perifusion of () the a1 antagonist prazosin (Prazo,5 m) or a2 antagonist yohimbine (Yohim, 5 m), and () the a1 antagonist benoxathian (Benox, 5 m) or a2 antagonist RX821002 (RX821, 5 m)(as indicated by white bars). In this study, benoxathian and RX821002 were dissolved directly in perifusion medium and therefore medium M199was used as the solvent control. Prazosin and yohimbine, however, were first dissolved in DMSO before subsequent dilution with perifusion mediumand thus DMSO (0.1%) was used as the solvent control. GH data, expressed as Mean±SEM (n=4), were pooled from four independent experimentsand the mean basal GH level was 44.7±5.3 ng GH/ml. The profiles of GH release are presented on the left and the quantified GH responses areshown on the right. In these experiments, GH responses were quantified as the net change in GH release after a pulse of drug treatment (i.e. a netchange of area under the curve). Drug treatments giving similar magnitude of GH responses are denoted by the same letter (P>0.05, anova followedby Fisher’s LSD test) and GH responses that are significantly different from basal GH release (P<0.05, Student’s t-test) are marked by asterisks.

F. 3. Effects of the a1, a2 and b adrenergic antagonists on norepinephrine (NE) inhibition of basal GH release from perifused goldfish pituitarycells. NE (0.5 m) was given as 10-min pulses (as indicated by black bars) during the 2-h continuous perifusion of () the general a antagonistphentolamine (Phentol, 5 m) or b antagonist propranolol (Propran, 5 m), () the b1 antagonist prazosin (Prazo, 5 m) or a2 antagonist yohimbine(Yohim, 5 m), and () the a1 antagonist benoxathian (Benox, 5 m) or a2 antagonist RX821002 (RX821, 5 m) (as indicated by white bars). In thisstudy, phentolamine, propranolol, benoxathian and RX821002 were dissolved directly in perifusion medium and therefore medium M199 was usedas the solvent control. Prazosin and yohimbine, however, were first dissolved in DMSO before subsequent dilution and thus DMSO (0.1%) was usedas the solvent control. GH data, expressed as Mean±SEM (n=4), were pooled from four independent experiments and the mean basal GH levelwas 46.6±6.3 ng GH/ml. The profiles of GH release are presented on the left and the quantified GH responses are shown on the right. In theseexperiments, GH responses were quantified as the net change in GH release after a pulse of drug treatment (i.e. a net change of area under thecurve). Drug treatments giving a similar magnitude of GH responses are denoted by the same letter (P>0.05, anova followed by Fisher’s LSD test)and GH responses that are significantly different from basal GH release (P<0.05, Student’s t-test) are marked by asterisks.




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F. 5. Effects of norepinephrine (NE) on the GH-releasing actions of salmon gonadotropin-releasing hormone (sGnRH) and dopamine in perifusedgoldfish pituitary cells. sGnRH (100 n, ) and dopamine (1 m, ) were given as 5-min pulses (as indicated by black bars) during the 1.5-hcontinuous perifusion of NE (10 m) (as indicated by white bars). GH data, expressed as Mean±SEM (n=3–5), were pooled results from at leastthree independent experiments. Given that NE was dissolved directly in perifusion medium, the culture medium M199 was used as the solvent control.The profiles of GH release are presented on the left whereas the quantified GH responses are shown on the right. GH responses were quantified asthe net change in GH release after a pulse of drug treatment (i.e. a net change of area under the curve). Treatments giving a similar magnitude ofGH responses are denoted by the same letter (P>0.05, anova followed by Fisher’s LSD test). GH responses that are significantly different from basalGH release (P<0.05, Student’s t-test) are marked by asterisks. The mean basal GH level of these perifusion experiments was 40.1±5.2 ng GH/ml.

from the arcuate nucleus (ARC; (27)) and inhibiting somatos- (30) and a2 adrenoreceptors have been identified in the nerveterminals of serotonin neurones (31). These findings raise thetatin secretion from the periventricular nucleus (28, 29). In

the same animal model, a2 adrenergic stimulation of GHRH possibility that GHRH release induced by b2 adrenergicstimulation is indirect and mediated through serotonin inter-release requires an intact serotoninergic system in the ARC



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F. 6. Effect of salmon gonadotropin-releasing hormone (sGnRH ) on the ‘post-inhibition’ GH rebound after norepinephrine (NE) treatment inperifused goldfish pituitary cells. Increasing doses (1–100 n) of sGnRH was given as 5-min pulses (as indicated by black bars) during the 1.5-hcontinuous perifusion of NE (10 m) (as indicated by white bars). In this study, a continuous perifusion of NE without sGnRH treatment was usedas the control group. GH data, expressed as Mean±SEM (n=3–4), were pooled from at least three independent experiments. The profiles of GHrelease are presented in the top panel (). Average basal GH release after NE inhibition (), GH responses to increasing doses of sGnRH (), andthe magnitude of ‘post-inhibition’ GH rebound after NE treatment ( ) are shown in the lower panels. Basal GH release after NE inhibition wasdefined as the mean of GH contents in the five fractions collected 20 min after initiation of NE perifusion. GH responses to sGnRH were quantifiedas the net change in GH release after sGnRH pulses (i.e. a net change of area under the curve). ‘Post-inhibition’ GH rebound was quantified bycalculating the sum of net GH release above the pretreatment basal in the 10 fractions following termination of NE perifusion. Treatment groupsgiving a similar magnitude of GH responses are denoted by the same letter (P>0.05, anova followed by Fisher’s LSD test). GH responses that aresignificantly different from basal GH release (P<0.05, Student’s t-test) are marked by asterisks. The mean basal GH release in these perifusionexperiments was 37.9±4.1 ng GH/ml.

neurones. In some studies (32) but not others (33), a small norepinephrine action is not within the CNS. In this study,norepinephrine and epinephrine inhibited basal GH releaseincrease in GH release from rat pituitary cells through

activation of pituitary b adrenoreceptors has been reported, from perifused goldfish pituitary cells with ED50 values of18.5±1.4 and 15.5±4.6 n, respectively. ED50 values in thebut the functional significance of such a small GH response

still remains elusive. In the goldfish, unlike mammals, adre- nanomolar dose range suggest that in the goldfish adrenergicsystem can exert a potent inhibitory effect on basal GHnergic influence on basal GH release is inhibitory rather than

stimulatory. In our previous studies, serum GH levels in the release at the pituitary cell level. Although the source ofadrenergic input to the goldfish pituitary has not been clearlygoldfish could be attenuated in a dose-dependent manner by

intraperitoneal injection of norepinephrine (11). This inhibit- defined, the possibility of a humoral origin can not beexcluded. In teleosts, norepinephrine and epinephrine secre-ory action, however, was not observed when norepinephrine

was injected directly into the brain, indicating that the site of tion from chromaffin cells in head kidneys and walls of large



veins (e.g. cardinal veins) in general represent stress signals worth mentioning that in the same animal model norepi-nephrine stimulates GTH release both in vivo (22) and infrom the peripheral tissues (16). Adrenergic inhibition of

basal GH release may be a part of the stress responses in vitro (23) through activation of pituitary a1 adrenoreceptors(24). Since norepinephrine has been shown to stimulatefish, especially during exposure to environmental stressors. A

temporary suspension of body growth may be adaptive in GnRH release from goldfish hypothalamic slices under cul-ture conditions (38), a part of the GTH response induced byconserving metabolic energy to provide for the increased

energy demands during stress situations. NE treatment in vivo may be indirect through its centralaction on GnRH neurones. Nevertheless, our present findingsIn this study, the inhibitory actions of norepinephrine and

epinephrine on GH release from goldfish pituitary cells were suggest that in the goldfish adrenergic input at the pituitarycell level may differentially regulate GTH and GH releasemimicked by the a2 agonists clonidine and UK14304 but not

by the a1 agonist methoxamine nor the b agonist isoproter- through activation of a1 and a2 adrenoreceptors, respectively.In our perifusion experiments, a rebound of GH releaseenol. Furthermore, the inhibition on basal GH release

induced by norepinephrine or the a2 agonist clonidine was was observed after termination of high doses of norepi-nephrine and epinephrine treatment. Interestingly, in thoseblocked by the a antagonist phentolamine and the a2 antagon-

ists yohimbine and RX821002. The b antagonist propranolol studies testing the interactions of norepinephrine with sGnRHand dopamine, a significant enhancement of this GH reboundand the a1 antagonists prazosin and benoxathian were not

effective in this regard. These results, as a whole, indicate was observed after norepinephrine perifusion with a previousexposure to sGnRH. The potentiating effect induced bythat adrenergic inhibition of basal GH release from goldfish

pituitary cells is mediated via receptors resembling mamma- sGnRH was dose-dependent. Apparently, the effect wasspecific to sGnRH as a prior exposure to dopamine did notlian a2 adrenoreceptors. The rank order of potency based on

the comparison of ED50s of adrenergic agonists tested (i.e. further increase the GH rebound. In a separate study, norepi-nephrine also inhibited basal GH release and theNE# EP�clonidine>phenylephrine&isoproterenol ) is con-

sistent with the pharmacological profile of a2 adrenoreceptors GH-releasing action of PACAP, which is a newly identifiedGH-releasing factor in the goldfish (39). Again, similar toreported in mammals (34). In these in-vitro experiments, it is

noted that a small but significant inhibition of basal GH dopamine, the GH rebound after norepinephrine perifusionwas not enhanced by prior treatment with PACAP (data notrelease was observed at high doses of the a1 agonist pheny-

lephrine. Given that the minimal effective dose of this a1 shown). In mammals, like the rat, a rebound of GH releaseafter somatostatin inhibition has been reported in mixedagonist was at 1 m level with ED50 estimated to be >1 m,

it is likely that the effect of phenylephrine on basal GH populations of pituitary cells (40) and purified somatotrophs(41). This ‘post-somatostatin’ GH rebound can be increasedrelease was due to a cross-reactivity with a2 adrenoreceptors.

To our knowledge, the present study represents the first in a dose-dependent manner in rat somatotrophs by applyingincreasing doses of GHRH during somatostatin perifusionreport in fish to document adrenergic inhibition of GH release

at the pituitary cell level through activation of a2 (41). These observations have been attributed to somatostatininhibition of GH exocytosis without a concurrent suppressionadrenoreceptors.

In the goldfish, GH release is regulated by a number of of GH synthesis. Given that episodic GH release in mammalsis due to a 180° out-of-phase secretion of GHRH andneuropeptides and neurotransmitters (for review, see (35)).

Among these regulators, GnRHs (including sGnRH and somatostatin from the hypothalamus (for review, see (42)),it is possible that the ‘post-somatostatin’ GH rebound maychicken GnRH-II ) and dopamine are the major physiological

GH-releasing factors, both in vivo (17, 35) and in vitro potentiate the GH-releasing effect of the subsequent GHRHpulses. This may account for the in-vivo potentiation of(3, 4). Specific binding sites for GnRHs (35) and dopamine

(18) have been identified on goldfish somatotrophs. The somatostatin pretreatment on GHRH-stimulated GH releasereported in some studies (43). In the goldfish, the pusatilityGH-releasing effect of dopamine is additive to that of sGnRH

(20) through activation of pituitary D1 receptors (3). In of GH release has not been previous examined. Although thepresent study indicates that removal of adrenergic inhibitionaddition, long-term treatments with sGnRH (35) and dopa-

mine analogues (17) are effective in stimulating body growth may serve as a stimulatory signal on GH release from goldfishpituitary cells and this stimulatory action can be selectivelyin the goldfish. In this study, to further examine the role of

adrenergic input in regulating GH release from goldfish potentiated by sGnRH, the physiological relevance of thisGH rebound is unclear and future investigations are war-pituitary cells, the possible interactions of norepinephrine

with the GH-releasing effects of sGnRH and dopamine were ranted. At present, it is also not clear regarding the mechan-isms mediating this ‘post-inhibition’ GH rebound. However,tested. In this case, norepinephrine suppressed basal GH

release and blocked the GH responses induced by sGnRH it is tempting to speculate that norepinephrine may inhibitGH exocytosis but not its synthesis in a manner similar toand dopamine, respectively. These results suggest that, in the

goldfish, adrenergic input to the pituitary may serve as a that of somatostatin. In this case, the continual synthesis ofGH in the presence of norepinephrine would lead to annegative regulator of both basal and stimulated GH release,

presumably through activation of pituitary a2 adrenorecep- increase in the total number of GH secretory vesicles, whichwould then be released upon the removal of norepinephrinetors. The involvement of a2 adrenoreceptors in norepi-

nephrine inhibition of stimulated GH release is supported by inhibition. We are currently testing this hypothesis by examin-ing total cellular GH contents and steady-state GH mRNAthe observations that the GH-releasing actions of GnRHs

and dopamine D1 agonists can be attenuated by clonidine in levels in goldfish pituitary cells with norepinephrine treat-ment. In this study, it is also noted that a GH rebound wasgoldfish pituitary cells under static incubation (37). It is



immunoreactivity in the goldfish pituitary: regulation of pituitary hor-not observed after perifusion with the a2 agonists clonidinemone secretion by CCK-like peptides in vitro. Gen Comp Endocrinoland UK14304. The cause for such a discrepancy with the1993; 92: 88–103.

corresponding results of norepinephrine experiments is still 10 Marchant TA, Fraser RA, Andrews PC, Peter RE. The influence ofunknown. mammalian and teleost somatostatins on the secretion of growth hormone

from goldfish (Carassius auratus L.) pituitary fragments in vitro. RegulIn summary, the present study provides evidence that inPept 1987; 17: 41–52.goldfish norepinephrine and epinephrine inhibit basal GH

11 Chang JP, Marchant TA, Cook AF, Nahorniak CS, Peter RE. Influencesrelease by acting directly at the pituitary cell level. Adrenergic of catecholamines on growth hormone release in female goldfish,inhibition on basal GH release from goldfish pituitary cells Carassius auratus. Neuroendocrinology 1985; 40: 463–470.

12 Wong AOL, Murphy CK, Chang JP, Neumann CM, Lo A, Peter RE.is mediated through receptors resembling mammalian a2Direct actions of serotonin on gonadotropin-II and growth hormoneadrenoreceptors. This adrenergic inhibition not only affectsrelease from goldfish pituitary cells: interactions with gonadotropin-basal GH release but also attenuates the GH responses toreleasing hormone and dopamine and further evaluation of seotonin

sGnRH and dopamine. Since sGnRH and dopamine are two receptor specificity. Fish Physiol Biochem 1998; 19: 23–34.major physiological GH-releasing factors in fish, the present 13 Kah O, Dulka JG, Dubourg P, Thibault J, Peter RE. Neuroanatomical

substrate for the inhibition of gonadotropin secretion in goldfish: exist-findings may suggest that in the goldfish the a2 inhibitoryence of a dopaminergic preoptico-hypophyseal pathway. Neuroendo-influence at the pituitary level is an integral component ofcrinology 1987; 45: 451–458.the neuroendocrine control of GH secretion. In this study, 14 Hornby PJ, Piekut DT. Distribution of catecholamine-synthesizing

the identification of a GH rebound after the removal of enzymes in goldfish brains: presumptive dopamine and norepinephrineadrenergic inhibition is particularly interesting. This ‘postin- neuronal organization. Brain Behav Evol 1990; 35: 49–64.

15 Sloley BD, Trudeau VL, Peter RE. Dopamine catabolism in goldfishhibition’ GH rebound which can be modulated selectively by(Carassius auratus) brain and pituitary: lack of influence of catecholestrog-sGnRH may represent a novel mechanism to regulate GHens on dopamine catabolism and gonadotropin secretion. J Exp Zoolrelease in fish. 1992; 263: 398–405.

16 Reid SG, Bernier NJ, Perry SF. The adrenergic stress response in fish:control of catecholamine storage and release. Comp Biochem Physiol (C)

Acknowledgements 1998; 120: 1–27.17 Wong AOL, Chang JP, Peter RE. In vitro and in vivo evidence that

The present study was supported by RGC (HK) and CRCG grants (HKU ) dopamine exerts growth hormone-releasing activity in goldfish.to A.O.L.W. and NSERC grants (Canada) to J.P.C., respectively. Financial Endocrinol Metab 1993; 27: E925–E932.supports from the Industry Department (HK) and Department of Zoology 18 Wong AOL, Chang JP, Peter RE. Characterization of D1 receptors(HKU ) in the forms of a Biotechnology Internship (to V.C.C.C.) and a mediating dopamine-stimulated growth hormone release from pituitaryPostgraduate Studentship (to E.K.Y.L.) are also acknowledged. Special cells of the goldfish, Carassius auratus. Endocrinology 1993; 133: 577–584.thanks are given to Dr R. E. Peter (University of Alberta, Canada) for the 19 Wong AOL, Van Der Kraak G, Chang JP. Cyclic 3∞,5∞-adenosinesupply of GH standard and antiserum for GH RIA. We are also indebted to monophosphate mediates dopamine D1-stimulated growth hormoneour colleagues in HKU, including D .K. O. Chan, K. L. Yu, S. M. Chan and release from goldfish pituitary cells. Neuroendocrinology 1994; 60:F. C. Leung, for their support throughout the project. 410–417.

20 Wong AOL, Van Goor F, Jobin RM, Neumann CM, Chang JP.Interactions of cyclic adenosine 3∞,5∞-monophosphate, protein kinase C,Accepted 4 October 1999and calcium in dopamine- and gonadotropin-releasing hormone-stimulated growth hormone release in the goldfish. Endocrinology 1994;135: 1593–1604.References

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22 Chang JP, Peter RE. Influence of norepinephrine and a-adrenergic2 Kah O, Anglade I, Lepretre E, Dubourg P, De Monbrison D. Themechanisms on gonadotropin secretion in female goldfish, Carassiusreproductive brain in fish. Fish Physiol Biochem 1993; 11: 85–98.auratus. Gen Comp Endocrinol 1984; 55: 89–95.3 Wong AOL, Chang JP, Peter RE. Dopamine stimulates growth hormone

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