Mokczdar and Cellular ~~docrin~o~, 54 (1987) 203-211 Elsevier Scientific Publishers Ireland, Ltd.

MCE 01760

a-Adrenergic stimulation of growth hormone release in anterior pituitary reaggregate cell cultures

Philippe Maertens and Carl Denef ~orufo~ of Cell Ph~acoZo~, b’niuersity of Letmen, School of Medicine, Leuuen,

(Received 22 April 1987; accepted 27 July 1987)

203

perifused rat

Key wora!r: a-Adrenergic receptor; Epinephrine; Growth hormone; Anterior pituitary

It has been previously shown that P-adrenergic agonists stimulate growth hormone (GH) release from perifused rat anterior pituitary cell aggregates cultured in serum-free defined medium for 5-6 days. The present data show that the natural /3-agonist epinephrine (EPI) stimulates GH release through an additional ru-adrenergic mecha~sm. The involvement of this mechanism was suggested by the following findings. The intrinsic activity of EPI, at concentrations 1 10 nM, was significantly higher than that of isoproterenol (ISO) in stimulating GH release. Phenylephrine (PHE), a specific agonist of a,-adrenergic receptors, provoked a significant rise of GH release. The effect was concentration dependent between 100 nM and 10 FM. The stimulatory effect of EPI and PHE was lowered, respectively blocked, by low concentrations of the potent qadrenergic antagonist prazosin. The EPI-induced GH release could be totally blocked only by ad~stration of both LY- and &receptor antagonists. Under the same conditions and using concentrations up to 1 PM, the qagonist clonidine had no or only a slight stimulatory effect; dopamine had no effect. Administration of both PHE and IS0 resulted in a more than additive stimulation of GH release. The effectiveness of PHE but not clonidine, together with the high potency of the q-blocker prazosin suggest that the a-adrenergic receptor is predominantly of the q-subtype. When tested on days 1, 3, 6 and 8 in culture, both ar- and P-adrenergic responses appeared to be higher in the presence of the glucocorticoid dexamethasone compared to the_responses in the absence of dexametha- sone. In the latter case, both adrenergic responses tended to decrease during culture. PHE also stimulated GH release in perifused freshly removed intact pituitaries or freshly dispersed pituitary cells, suggesting that the observed a-adrenergic effect was not a property developing in aggregates under culture conditions.

Introduction secretion. cu,-Adrenergic agents stimulate adreno- corticotropic hormone (ACTH) (Vale and Rivier,

There is considerable experimental evidence 1977; Giguere et al., 1981) and P-endorphin supporting the involvement of a-adrenergic mech- (Raymond et al., 1981) as well as thyrotropin anisms in the control of the pituitary hormone release (abuse et al., 1983; Peters et al., 1983)

from monolayer cultures of rat anterior pituitary

Address for correspondence: Philippe Maertens, Labora- cells. cy,-Adrenergic receptors have also been found

t&y of Cell Pharmacology, University of Leuven, School of in homogenates of porcine neurointermediate lobe Medicine, Campus Gasthuisberg, B-3000 Leuven, Belgium. pituitary tissue (Battaglia et al., 1983). In vivo

0303-7207/87/$03.50 a 1987 Elsevier Scientific publishers Ireland, Ltd.

204

experiments have demonstrated that the cY,-adren- ergic system is involved in inhibiting prolactin (PRL) release (Lien et al., 1986) while &-adren- ergic agents stimulate PRL release in perifused pituita~ cell aggregates (Denef and Baes, 1982). With regard to GH secretion, many reports in the literature indicate that catecholamines not only influence but are a prerequisite for normal GH secretion in rats (for reviews, see Weiner and Ganong, 1978; Krulich, 1979; Martin, 1980). It has generally been assumed that they exert their influence on pituitary GH secretion via hypo- thalamic factors. Although al,-adrenoceptor ago- nists seem to have a dual effect on GH release in man (Struthers et al., 1986), the current view is that or,-agonists, such as clonidine, stimulate GH release in rats via postsynaptic cu,-receptors by way of changes in growth hormone-releasing fac- tor (GRF) release (Eden et al., 1981; Eriksson et al., 1982; M&i et al., 1984). Activation of central adrenoceptors of the tY,-subtype, on the other hand, leads to i~bition of GH secretion in rats (Krulich et al., 1982; Cella et al., 1987).

The possibility that catecholamines can also regulate GH secretion by acting directly on the pituitary through specific receptor mechanisms, becomes more and more apparent. Using whole rat anterior pituitary glands (MacLeod, 1969; Bir- ge et al., 1970) and bovine pituitary slices in- cubated in vitro (Shofield, 1967) no significant effect of the catecholamines epinephrine (EPI), norepinephrine (NE) and phenylephrine (PHE) on GH release could be found, probably due to lack of dynamic detection systems. However, in perifu- sion experiments of dispersed cells (Perkins et al., 1983,1985) it has been demonstrated that EPI and l-isoproterenol (ISO) could directly stimulate GH release in vitro through &-adrenergic receptors. We have confirms these findings in reaggregated pituitary cells in culture (Swennen et al., 1985; Baes and Denef, 1987) and demonstrated that the magnitude of the &effect was strongly enhanced by intercellular communication. Although at least part of this potentiation was exerted at the level of EPI interaction with P-receptors, the p~ticipation of a-receptors was not fully ruled out. The present study provides evidence that GH can also be released from rat anterior pituitary cell aggregates by a direct effect of EPI on ol-adrenergic receptors

and that this effect was not an artefact or adapta- tion to culture conditions.

Materials and methods

Animals Three-month-old male Wistar rats (300 g) were

used in most experiments. They were housed un- der a 14 h light, 10 h dark schedule, in a constant temperature (22-23OC) and hu~~ty (60-70~) environment, and received a standard diet (Hope Farms) and water ad libitum. The animals were killed by decapitation.

Reaggregaie ceil cultures The anterior pituitaries were removed and dis-

persed after trypsin and EDTA treatment into single cells as described previously (Denef et al., 1978). 1.5-2 X lo6 cells were obtained per anterior pituitary. The viability was greater than 95%, as judged by trypan blue exclusion. The cells were suspended and seeded at a density of 1 x lo6 cells/2 ml culture medium in a 35 mm untreated Petri dish. The dishes were subjected to coniinu- ous gyratory shaking on an Ilfors HT shaker (Bottmingen, Switzerland) in a humidified CO,& incubator (1.2% CO,) at 37 *C to allow reaggrega- tion (Baes and Denef, 1987). The aggregates were cultured in a serum-free defined medium (Baes and Denef, 1987) supplemented with 50 pM tri- iodothyronine (Ta) (Serva, Heidelberg, F.R.G.) and 80 nM dexamethasone (Serva), unless otherwise stated. The latter hormone was added as it strongly potentiates GH responses and promotes intercellu- lar communication with GH cells (Baes and De- nef, 1987). After 2 days in culture, aggregates from six dishes were collected and transferred in 6 ml new culture medium to 50 mm Petri dishes.

Perifwion of pituitary cell aggregates After 5 or 6 days of culture, the dynamics of

GH release from the aggregates (and in some experiments from intact pituitaries as a whole) was studied in a perifusion system as previously described (Denef et al., 1984). In the experiments, where freshly dispersed cells were perifused, the cells, mixed with 1.6 ml pre-swollen Bio-Gel P-2 beads (Bio-Rad, Richmond, CA, U.S.A.), which

205

had been washed extensively with perifusion medium, were loaded on the same perifusion chambers. The perifusion medium consisted of Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY), supplemented with 1 g/l NaHCO,, 15 mM Hepes and 15 mM TES buffer, adjusted to pH 7.5. Eight perifusion preparations could be run simultaneously at a flow rate of 0.125 ml/mm. After a 3 h equilibration period, the aggregates (from 1.5 X lo6 cells) were exposed to 20 min pulses of the drugs. The eluate was collected as 4 n-tin fractions in polystyrene tubes containing 100 ~12% bovine serum albumin (BSA) (Serva) in saline and stored at - 20 o C until assay.

Hormone assays Rat GH was measured by RIA with reagents

kindly supplied by Dr. A.F. Parlow through the National Hormone and Pituitary Program, NIADDK, Baltimore, MD, U.S.A. Anti-rat GH serum 5 and GH-RP 2 standard were used. All assays were run in duplicate. Reliability of the assay was shown previously (Baes and Denef, 1987).

Statistical analysis Where appropriate, two-way analysis of vari-

ance was used to determine the statistical signifi- cance of differences. Data are presented as the mean + SEM, except when the SEM overlaps with the symbol used (where only the symbol is shown); the number of independent experiments for each response, performed on different days, is denoted by ‘n’.

Chemicals and solutions I-Epinephrine, I-isoproterenol, phenylephrine

and dopamine (all from Sigma Chemical Co., St. Louis, MO) were always freshly dissolved in 1% ascorbic acid in water, to prevent catecholamine oxidation, and added as a lOO-fold concentrated solution to the perifusion medium. Clonidine (a gift from Dr. J. Leysen, Janssen Pharmaceutics, Beerse, Belgium) was dissolved in 1% ascorbic acid in a 1 mM concentration, aliquoted and stored at - 20” C. At the time of use it was further diluted and finally used as lOO-fold con- centrated solution. df-Propranolol (Imperial Chemical Industries, Gent, Belgium) was freshly

dissolved and diluted in 0.9% NaCl. Prazosin hy- drochloride (a gift from Dr. J.E. Leysen, Janssen Pharmaceutics) was dissolved and diluted in ethanol. Both antagonists were added as a lOOO- fold concentrated solution to the medium.

Results

Stimulation of GH release by EPI, Comparison with IS0

We previously reported that EPI was less potent than the P-adrenergic agonist IS0 in stimulating GH release via a &-mechanism, the approximate EC,, being 1.2 X lo-’ M and 2 X 10e8 M respec- tively (Swennen et al., 1985; Baes and Denef, 1987). Fig. 1 shows that, at a concentration of 10 nM, IS0 is indeed more potent in stimulating GH release than EPI. GH release was stimulated by a factor 5.9 * 0.4 and 3.9 * 0.25 respectively (in- tegrated area under the response curve divided by average basal release). From a concentration of 100 nM, EPI stimulated GH release by a factor of 14.2 f 2.2, which was significantly higher than the stimulation by IS0 (~6.3 * 0.5) suggesting EPI acts via a second mechanism.

2700 T % 2LOO 5 = 2100 DC 5 1800

1wnn ISO 0 2 1500 [Pi .

2 1200

6 900

s 600

300 100

0 20 40 60 80

TIME lmlnl

Fig. 1. Effect of IS0 (10 and 100 nM) and EPI (10 and 100

nM) on GH release from perifused anterior pituitary cell

aggregates from adult male rats. The different concentrations

were tested simultaneously, each in a separate perifusion prep-

aration. Values represent mean secretion rate/4 mink SEIvf

(n = 4) in percent of baseline secretion during 20 min exposure

(indicated by bars) to the agonists IS0 (0) and EPI (0). Left

panel: 10 nM IS0 and EPI; right panel: 100 nM IS0 and EPI.

F-test: IS0 10 nM versus EPI 10 nM: P < 0.05; IS0 100 nM

versus EPI 100 nM: P < 0.001.

206

Effect of a- and /3-antagonists on EPI-stimulated GH release

In order to study whether the stimulation of GH release by EPI was partially mediated by an a-adrenergic mechanism, the aggregates were chal- lenged with EPI together with a- and /3-adreno- ceptor antagonists (Fig. 2). In three independent experiments, integrated hormone release during EPI (100 r&I) exposure fell from 1882 rf: 405% to 666 + 83% of basal release (64% inhibition) when given together with 10 nM prazosin, a potent a,-adrenergic antagonist (F-test: P < 0.001). 1 PM dl-propranolol, a P-adrenergic antagonist, caused a similar inhibition of GH secretion; hormone release fell to 531 f 50% of basal release (72% inhibition; P < 0.001). The EPI-induced GH re- lease could be totally blocked by simultaneous administration of the a- and P-antagonist.

Effect of PHE on GH release Fig. 3A shows that 1 PM PHE, a specific agonist

of a,-adrenergic receptors, strongly stimulated GH

4000

3600 i

= 3200- : 2 2800 -

o 2400- ,\" w 2000- m

4 1600-

0 CONTROL

A t PRAZOSIN 1OnM

l . PROPRANOLOL lutl 0 t PRAZOSIN

I PROPRANOLOL

;r 5

1200

800

400

100

0 10 20 30 40 50 60 70 80

TIME (mrd

Fig. 2. Effect of the o-antagonist prazosin (10 nM) and the

&urtagonist propranolol (1 PM) on EPI-stimulated GH re-

lease from perifused anterior pituitary cell aggregates of adult

male rats. The antagonists or vehicles were present throughout the entire perifusion. Values are the meanf SEM of three

independent experiments. Bar indicates the time of exposure to

EPI.

0 20 40 60 80 -0 -7 -6 -5

TIME lmml cone of agonst (log Ml

Fig. 3. A: Effect of PHE (1 PM) on GH release from perifused

anterior pituitary cell aggregates of adult male rats. Bar in-

dicates the time of exposure to PHE. Values are the meanf

SEM of nine independent experiments. B: Concentration-ef-

fect relationship of PHE and clonidine on GH release from

perifused anterior pituitary cell aggregates from adult male

rats. Values represent the meanf SEM (n = 4) secretion/4

min, expressed as percent of baseline secretion. The concentra-

tions were tested simultaneously, each in a different perifusion

preparation. The values under the stimulation curve are 113 f

6%, 167 f 13%, 491 f 58% and 1084* 198% of basal release for

10 nM, 100 nM, 1 PM and 10 PM PHE respectively and

106k2.3’5 (n = 4) and 124* 12% of basal release (n = 3) for

10 nM and 1 CM clonidine respectively.

release (431 f 28% above basal). This effect was obtained in nine independent experiments done with different cell preparations. During the 20 min pulse, the secretion rate rapidly reached its maxi- mum and then rapidly declined even before PHE

A T soat B P

1200-

900-

600-

300-

1OOr

I I CONTROL

I 1 I I I

0 20 40 60 80 -8 -1 -6 -5 TIME (mm1 cone of PHE llog MI

Fig. 4. A: Antagonizing effect of 10 nM prazosin on

PHE-stimulated GH release from perifused anterior pituitary

cell aggregates from adult male rats. MeanskSEM from four

independent experiments. The values under the stimulation

curve are 434 f 38% and 122 f 4% of basal release for prazosin-

and vehicle-treated perifusion preparations respectively. F-test: P < 0.001. E: Dose-dependent blockade of PHE-stimulated

GH release by 10 nM prazosin (single experiment). Prazosin

and vehicle were present throughout the whole perifusion.

207

2000

z 1750 d

g 1500

J 1250

G 1000

z m 750

b 500

' 250 100

. PHE 1un It. IS0 1onn . PHE . IS0 expected o PHE t IS0 observed

0 10 20 30 40 50 60 70 80

TIME (mln)

Fig. 5. Secretion pattern of anterior pituitary cell aggregates from adult male rats subjected to simultaneous perifusion with

PHE and ISO. Bar indicates time of exposure (n = 3).

was omitted from the perifusion medium. The effect of PHE was concentration dependent be- tween 10 nM and 10 PM (Fig. 3B). Simultaneous perifusion with low concentrations of prazosin blocked the stimulatory effect of PHE on GH release, even when 10 PM concentrations of the a-agonist were used (Fig. 4). There was no or only a slight effect of the q-agonist clonidine on GH release. No effect was found with dopamine either alone or in combination with PHE (data not shown).

PHE

-0 ’ ’ I ’ ’ ’ ’ ’ 0123456709 123456709

TIME (days I” culture1

Fig. 6. Effects of 1 PM PHE (left panel) and 100 nM IS0 (right panel) on different days in culture (two independent experiments). Values represent mean secretion rate values ex- pressed in percent of baseline secretion during 20 min exposure to the agonist. Full line: cells cultured in medium containing

Fig. 7. Perifusion of freshly dispersed anterior pituitary cells (left panel; stimulation factor: xl.34 of the area under the stimulation curve) and an intact anterior pituitary (right panel; stimulation factor: ~1.34) of adult male rats (representative experiment out of three respectively two similar experiments). Perifusion of the dispersed cells and the intact pituitaries was done in the same experiment. The dispersed cells were loaded on a B&Gel P-2 column as described in the Methods section and perifused under similar conditions as the aggregates or intact pituitaries. Bars indicate time of exposure to 10 pM

80 nM DEX; broken line: without DEX. PHE.

Combination of a- and P-agonists mimics the EPI effect

As shown in Fig. 5, the effect of 1 PM PHE perifused together with 10 nM IS0 was higher than the sum of the effect of 1 PM PHE (408 + 29% of basal) and of 10 nM IS0 (562 &- 88% of basal) alone. The difference between the expected (870 f 65% of basal) and the observed value (1251 rf 212% of basal) was highly significant (F-test: P < 0.01).

a-Stimulation, a characteristic developing in aggre- gates during culture conditions?

To explore the possibility that cu-adrenergic stimulation of GH secretion in reaggregate cell cultures was not induced by the culture condi- tions, we examined the cu-adrenergic response of freshly dispersed cells and intact pituitaries, as well as the influence of time of residence in cul- ture with and without the glucocorticoid dexa- methasone (DEX). Fig. 6 shows that when tested on days 1, 3, 6 and 8 in culture, both (Y- and /3-adrenergic responses appeared to be higher in the presence of DEX compared to the responses in the absence of DEX. The effects on GH release did not tend to increase in culture neither in the presence nor in the absence of DEX. On the

100

1

sKlgle cells 300

I

Intact pctultary E 80 e * 240

0 20 40 60 80 0 20 40 60 80 TIME lmm)

208

contrary, in the absence of DEX, both adrenergic responses tended to decrease. As can be seen in Fig. 7, PHE clearly stimulated GH release in freshly removed intact pituitaries as well as in freshly dispersed cells.

Discussion

The present results demonstrate that EPI stimulates GH release not only through P-adreno- ceptors but in addition through an a-adrenergic mechanism. The involvement of both these recep-

tors was indicated by the findings that (1) from concentrations of 100 nM, EPI became more ef- fective than IS0 in stimulating GH release, which is not consistent with the definition of P-adreno-

ceptor-mediated stimulation; (2) the EPI effect could only partially be blocked by propranolol; (3) the EPI effect on GH release was diminished

by prazosin, a highly selective a-antagonist (U’Prichard et al., 1978); (4) the effect of EPI could totally be blocked by simultaneous applica- tion of the (Y- and P-blocker; (5) PHE, an (Y-

adrenergic agonist, stimulated GH release and potentiated the stimulatory effect of the P-agonist

ISO. Although the aim of this study was not to give

a rigorous characterization of the a-receptor sub-

type involved, the observed a-effect appeared to be cu,-mediated. PHE, a specific agonist of (pi- adrenergic receptors, provoked a clear-cut, con- centration-dependent rise of GH release (Fig. 3), while clonidine, a specific a,-agonist, had almost no effect. The small agonistic activity of 1 PM clonidine on GH release can possibly be explained by its low affinity for a,-receptors, along with its usual preferential affinity for a,-receptors (And& et al., 1976). Furthermore, prazosin, highly effec- tive as a,-antagonist, its potency for cu,-receptors being 3-4 orders of magnitude higher than for cr,-receptors (U’Prichard et al., 1978), reduced the effect of EPI and blocked that of PHE. Moreover, prazosin was effective at low nanomolar con- centrations. A concentration of 10 nM virtually completely blocked the stimulatory effect of 0.1, 1 and 10 PM PHE on GH release. We did not use methoxamine as agonist, as in rabbit uterus the drug binds with equal affinity to CY~- and cY,-recep- tors (Hoffman et al., 1980). Neither did we use the

a-antagonist phentolamine as it does not dis- criminate between the two receptor subtypes (Hoffman et al., 1979) and as it has been sug- gested to have also some /3-agonistic activity (Gould and Reddy, 1976).

During exposure to PHE, GH secretion rate immediately reached its maximum and then rapidly returned to near baseline levels, even be- fore omission of PHE from the perifusion medium. The brevity of this response offers an explanation for the negative results obtained by others in different, more static in vitro systems. Indeed the lack of an in vitro effect of a-agonists on GH release may be due to a rapid desensitization of

this adrenoceptor subtype. Rapid desensitization of P-adrenoceptors mediating GH release has al- ready been described (Perkins et al., 1983, 1985; Baes and Denef, 1987).

The possibility that our cultured cells would have undergone major deviations from normal, such as up-regulation of adrenoceptors in the ab-

sence of catecholamines or adaptation to culture environment can reasonably be excluded. No con- sistent rise in responsiveness during the culture period was found in the presence of DEX whereas a decrease of the (Y- and P-response was seen in the absence of DEX. An additional important finding in this respect was that PHE was able to stimulate GH release in intact pituitaries and

freshly dispersed cells (Fig. 7) further supporting the conclusion that the a-adrenergic response was

not a characteristic acquired during culture. Perkins et al. (1983, 1985) did not find an effect

of the a-blocker phentolamine on ISO-induced

GH secretion of dispersed rat anterior pituitary cells. They did not mention its influence on the EPI effect nor was prazosin used in their experi-

ments. However, looking at some of their data (Fig. 1 in Perkins et al., 1985) 100 nM EPI caused a remarkably more sustained GH release than 100 nM ISO, which is consistent with our findings. Although this difference was quite obvious, no comments were given by the authors.

The present observations raise the question concerning the potential physiological role of an a-adrenergic mechanism in the control of GH secretion. That such response may occur is con- sistent with the finding of LY- and /3-adrenergic binding sites in rat pituitary (Peters et al., 1983;

209

Petrovic et al., 1983; De Sousa, 1985; Perkins et al., 1985). The findings of relatively high amounts of EPI in the median eminence (Palkovits et al., 1980) indicate a central source of EPI secretion into the portal circulation. Moreover, Johnston et al. (1983) found that the concentration of EPI in plasma from the transected pituitary stalk of ana~thet~d rats was 60-908 higher than the EPI concentration in peripheral venous plasma providing evidence in favour of a direct action of centrally derived EPI upon anterior pituitary hormonal release. The concentration of EPI in portal blood is within the range required for a direct effect on GH release. Nevertheless, some earlier studies disagree with the latter findings (in-Jonath~ et al., 1977; Gudelsky and Porter, 1979; Reymond and Porter, $982). Apparent dis- crepancies may be due in part to differences in sex and anaesthesia. Consistent as well as conflicting with our results are al-effects observed in vivo. Whereas Ruth et al. (1976) found a small and short-lasting release of GH, induced by very large doses of PHE, injected ~~avenously into rats under urethane anaesthesia, KLrulich et al. (1982) reported an lu,-adrenergic inhibition of GH release after intraventricular administration of (Ye- agonists. Similar conflicting in vitro versus in viva results exist as far as ACTH secretion is con- cerned. The net effect of a-adrenergic agonists adjusters in vivo in the dog is i~bition of ACTH secretion (Ganong, 19’77) whereas the same agonists provoke stimulation of ACTH release from the pituitary in vitro (Gig&e et al., 1981). Besides species differences these discrepancies could be explained by an effect at the level of the central nervous system overriding the effect at the pituitary level, at least under the test conditions used. Whether there are physiolo~cal conditions in which the pituitary site of action predo~nat~ remains to be studied.

During stress peripheral levels of circulating EPI in the rat can exceed 10 nM (Kvetnansky et al., 1978). Gibbs (1985) reported a 3-fold increase in EPI concentrations in portal blood as compared to basal levels in heat-stressed adrenalectomized rats. These data indicate that EPI may be involved in the regulation of GH release during stress. However, there are obvious species differences in the effect of stress on GH secretion: GH levels

secondary to stress are elevated in man, sheep and cow but are suppressed in the rat, mouse, dog or pig (Martin, 1976). It seems therefore unlikely that EPI is involved in GH release during stress. How- ever, there exist some data that show a stress-in- duced increase of GH secretion in the rat under certain conditions. Removal of the preoptic region of the telencephalon in the rat not only blocked but also reversed the normal stress-induced decre- ment of GH release (Rice and CritchIow, 1976; Miod~s~ews~ and C&.&low, 1982). Stress-in- duced inhibition of GH secretion appears to de- pend on increased activity of somatostatin neu- rons located in the periventricular nucleus of the preoptic region. In addition, antiserum to soma- tostatin produces a blockade ‘of the GH response to stress in the rat (Arimura et al., 1976; Terry et al., 1976). Under conditions of stress, somatosta- tin secretion may normally dominate and thus override the direct stimulatory effect of circulating or portal blood EPI on somatotrophs. However, it is possible that the direct ar-adrenergic stimulation of somatotrophs fulfills a compensator mecha- nism or that the direct stimulatory effect plays a role in habituation to stress. Clearly, it is fair to say that the exact physiological role of EPI-stimu- lated GH release is not yet clear.

Another ~nt~guing consideration is that the same final effect - stimulation of GH release - of a single catecholamine, is mediated by two different receptor systems. In certain tissues, cy- and @receptors mediate opposite effects whereas in other tissues, stimulation of (Y- and &adreno- ceptors can lead to the same biological response. The latter is for example the case in rat liver, where 1y- as well as /3-adrenergic stimulation result in increased glycogenolysis and gluconeogenesis (Exton and Harper, 1975). Simpson et al. (1986) cultured heart muscle cells from the neonatal rat in serum-free medium and found that contractile activity required both cyl- and /3,-adrenergic recep- tor stimulation. The synergistic activity of (Y- and /3-adrenergic agonists on GH release in our experi- ments could be seen as an amplification of the signal, a phenom~on often observed when agonists with different mechanisms of signal transduction cooperate. It is now welf established that &- and &-adrenergic receptors both activate adenylate cyclase and stimulate generation of

210

adenosine-3’,5’-monophosphate (CAMP) and that signal transduction by a,-adrenergic receptors may occur through a variety of other effector systems including Ca2+ mobilization, phosphatidy~nositol hydrolysis, e~~~rnent of Nat or K” flux and arachidonic acid release (Exton, 1985). Interest- ingly, there might also exist distinct subtypes of a,-adrenergic receptors linked to different signal transduction systems as well as an ‘undifferenti- ated’ class of adrenergic receptors which can be stimulated only by ‘duai’ agonists like EPI, as suggested by O’Brien and Rall (1987). It was beyond the aim of our study to demonstrate that a,-adrenergic agonists induce an elevation in cyto- solic Ca2+ in anterior pituitary cells. In previous studies we demonstrated /I-adrenergic stimulation of CAMP accumulation in rat anterior pituitary cell cultures (Swennen et al., 1985).

It has been proposed that there is a balance between OL- and P-receptors which is under control of hormones such as glucocorticoids and thyroid hormone (Guellaen et al., 1982; Bilezikian and Loeb, 1983; De Souza, 1987). Modulation by glu- cocorticoids was also found in the present system, but both 01- and P-adrenergic effects on GH re- lease appeared to be similarly affected by gluco- corticoids.

Evidence has already been provided that GH release stimulated by EPI is modulated by facila- tory interactions of somatotrophs with other cells, the latter being most likely lactotrophs (Baes and Denef, 1987). This facilitation entirely depends on the presence of a glucocorticoid (DEX). Our re- sults indicate that glucocorticoids also enhance the q-adrenergic effects of EPI. In addition, in ungo- ing experiments using perifused aggregates of pituitary cells separated by gradient sedimentation at unit gravity followed by Percoll isopycnic sedi- mentation, we found that the poorest a-response was seen in aggregates composed of a highly (88%) purified population of large somatotrophs, sup- porting the hypothesis of an involvement of inter- cellular communications in the a-adrenergic stimulation of GH secretion as well. Which cell type is involved in potentiating the a-adrenergic effect remains to be studied, although cortico- trophs and/or thyrotrophs are good candidates as they bear q-receptors in rat pituitary (Gig&e et al., 1981; Peters et al., 1983). Previously we showed

evidence that P-receptors are located on lactotrophs (Denef and Baes, 1982; Baes and De- nef, 1987).

In conclusion, the present data provide experi- mental evidence for a possible functional role of EPI as a growth hormone releaser through a direct action on pituitary (Y- as well as /&adrenergic receptors. The possibility that intercellular signal- ing may be involved in a-stimulation of GH re- lease, as has been previously shown for /3-stirnula- tion (Baes and Denef, 1987) remains a promising area for future research.

Acknowledgements

The authors thank A. De Wolf and M.J. Vanderheyden for the excellent technical assis- tance, M. Bareau for typing the manuscript and Dr. A.F. Parlow and the National Hormone and Pituitary Program for the generous gifts of RIA material.

The present work has been supported by grants from the ‘Belgian Programme for the Re- inforcement of the Scientific Potential in the New Technologies - PREST’ and from the ‘Gecon- certeerde Onderzoeksakties’.

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