Specificity of the β2-adrenergic receptor stimulating cyclic AMP accumulation in the intermediate lobe of rat pituitary gland

European Journal of Pharmacology, 81 (1982) 411-420

411

Elsevier Biomedical Press

S P E C I F I C I T Y O F T H E I~Iz-ADRENERGIC R E C E P T O R S T I M U L A T I N G CYCLIC A M P A C C U M U L A T I O N IN T H E I N T E R M E D I A T E L O B E OF RAT P I T U I T A R Y G L A N D HELENE MEUNIER * and FERNAND LABRIE ** MRC Group in Molecular Endocrinology, Le Centre Hospitalier de l'Universitk Laval, Quebec, GIV 4G2, Canada

Received 23 February 1982, accepted 7 April 1982

H. MEUNIER and F. LABRIE, Specificity of the fl2-adrenergic receptor stimulating cyclic A M P accumulation in the intermediate lobe of rat pituitary gland, European J. Pharmacol. 81 (1982) 411-420. Changes of cyclic AMP levels were used to assess the specificity of the fl-adrenergic receptor in primary cultures of cells prepared from the intermediate lobe of rat pituitary gland. During a 4 min incubation, fl-adrenergic agonists led to a 4 to 6 fold stimulation of cyclic AMP concentration with the following order of potency (K D values): zinterol (0.75 nM)> hydroxybenzylisoproterenol (1.0 nM)> (-)-isoproterenol (4.6 n M ) > soterenol > (7.7 n M ) > ( - ) epinephrine (10 nM)>OPC 2009 (procaterol, 11 nM)>>(-)-norepinephrine (300 nM). The potent antagonists cyanopindolol, (-)-propranolol and hydroxybenzylpindolol reversed the stimulatory effect of (-)-isoproterenol at K D values of 0.4-0.6 nM. Other fl-adrenergic antagonists had the following order of potency: pindolol = (-)-alprenolol = timolol (0.9-1.0 nM) >>metoprolol (100 nM)> dichloroisoproterenol (300 nM)> butoxamine (1100 nM). The fl I-selec" tive antagonist practolol had a low potency at 7000 nM. The stereoselectivity of the receptor is indicated by the 400 to 70 fold higher potency of the (-)-isomers of isoproterenol, epinephrine and propranolol as compared to their (+)-stereoisomers. The data show that the fl-adrenergic receptor in the intermediate lobe of the rat pituitary gland is mainly of the fl2-subtype. Study of this pure population of postsynaptic fl-adrenergic receptors where binding could be correlated with other parameters of cellular activity (cyclic AMP formation and a-MSH secretion) should yield useful information about the less accessible adrenergic systems of the brain. Cyclic AMP

Intermediatelobe

a-MSH

Dopaminergic fl2-Adrenergicpituitary

1. I n t r o d u c t i o n

The intermediate lobe of the rat pituitary gland is made up of a homogeneous population of cells (Howe, 1973) specialized in the secretion of peptides derived from proopiocortin, a - M S H being the major secretory product (Lissitzky et al., 1978; Dub6 et al., 1978). The rate of secretion of these peptides is stimulated by fl-adrenergic agents and inhibited by dopaminergic substances (Hadley et al., 1975; Bower et al., 1974; Tilders et al., 1975;

* Fellow of the Conseil de la Recherche en Sant6 du Qurbec. ** MRC Career Investigator. To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

Munemura et al., 1980b; C6t6 et al., 1981). Since the adenylate cyclase system has been well demonstrated to play a mediatory role in controlling the activity of pars intermedia cells (C6t+ et al., 1981), we have used changes of cyclic A M P levels induced by a large series of adrenergic agonists and antagonists to study in detail the specificity of the B-adrenergic receptor. The present data show that the pars intermedia fl-adrenergic receptor is of the fl2-subtype. Since the properties of the subtypes of the fl-adrenergic receptor appear to be constant between tissues, it is hoped that the knowledge gained with this relatively simple model will yield useful information about the less accessible fl-adrenergic systems of the brain.

412 2. Materials and methods

2.1. Materials Dopamine, (--)- and (+)-isoproterenol, ( - ) and (+)-epinephrine, ( - ) - and (+)-norepinephrine, (-)-alprenolol, yohimbine, serotonin and acetylcholine were purchased from Sigma. The following compounds were gifts: haloperidol (McNeil), phentolamine (CIBA), prazocin (Pfizer), phenylephrine (Sterling-Wintrop), cyanopindolol, hydroxybenzylpindolol and pindolol (Sandoz), timolol (Frosst), dichloroisoproterenol (Lilly), ( - )and (+)-propranolol (Ayerst), metoprolol (Astra), butoxamine (Burroughs-Wellcome), practolol (ICI and Ayerst), zinterol and ( - ) - s o t e r e n o l (Mead Johnson) and hydroxybenzylisoproterenol (Phillips-Duphar). Dulbecco's modified Eagle's Medium (DMEM) and sera were obtained from Grand Island Biologicals Co. Dextran-coated charcoaladsorbed sera were prepared by overnight incubation of the sera at 4°C with 1% charcoal (Norit A) and 0.1% Dextran T 70 obtained from Fisher and Pharmacia, respectively, followed by a second incubation at room temperature for 2 h. Sera were then inactivated by heating at 56°C for 30 rain. 2.2. Preparation of pars interrnedia cells Adult female Sprague-Dawley rats (Charles River CD Strain obtained from Canadian Breeding Farms, St. Constant, Quebec), usually 100, at random stages of the estrous cycle and weighing 150-200g, were used for the preparation of primary cultures of pars intermedia cells. The rats were decapitated rapidly and the neurointermediate (NI) lobe, containing both the posterior and the intermediate lobes, was then separated from the anterior lobe and placed in Hepes buffer (NaC1 137 raM; KCI 5 mM; N a 2 H P O 4 0.7 mM; n-2-hydroxyethylpiperazine ethanesulfonic acid (Hepes) pH 7.2 25 mM; glucose 10 mM; CaC12 360/~M). NI lobes were then rinsed several times with sterile Hepes buffer and transferred with a Pasteur pipette to a 16X 125 mm sterile and siliconized glass culture tube. NI lobes were then allowed to settle and the supernatant was discarded.

Enzymatic digestion was performed at 37°C in a Dubnoff shaking incubator after addition of 10 ml of 0.1% hyaluronidase (Sigma), 0.35% collagenase (Boehringer) and 3% bovine serum albumin (BSA, Schwartz-Mann) in Hepes buffer. After one hour, pars intermedia cells were further dissociated by gentle aspiration of NI lobes with a Pasteur pipette (once or twice at 20 min intervals) during an additional hour of incubation. After this two-hour incubation, the supernatant containing dissociated cells was transferred to a new siliconized tube containing 5 ml of D M E M and 10% fetal calf serum (dextran-coated charcoal-adsorbed and heat-inactivated), 1% non-essential amino acids, 50 units/ml of penicillin, 50 /~g/ml of streptomycin and 2% BSA. Dispersed cells were collected by centrifugation at 100 × g for 10 min and resuspended in DMEM containing serum and BSA. Meanwhile, the remaining non-dissociated fragments of the NI lobes were subjected to a second enzymatic digestion in 7ml of 0.12% Viokase in Hepes buffer for 30 min. After decantation, the supernatant containing the freshly dissociated cells was combined with the previous fraction of dissociated cells and centrifuged. The pellet was resuspended in 7 ml of Viokase (0.25%) for a final 5 min digestion which was stopped by adding 5 ml of D M E M / s e r u m / B S A . The cells were again collected by centrifugation, washed once through 4% BSA in D M E M and three times with D M E M / serum/BSA. The pooled cells were then resuspended in D M E M containing 10% charcoal-treated fetal calf serum, 1% non essential amino acids, 50 units/ml of penicillin and 50 /~g/ml of streptomycin, pH 7.25. The cells were then distributed in Linbro multi-well tissue culture plates in 1 ml aliquots containing 100000 cells. The plates were placed in a water-jacketed incubator at 37°C under a water-saturated atmosphere of 95% air, 5% CO 2 and used 5 days later. The final yield was usually 2 X l0 s cells/intermediate lobe. 2.3. Incubation procedure After 5 days in culture, the cells were washed once with D M E M before incubation with D M E M containing 5 mM Hepes in the absence of sera for

413

4 min. All substances were tested in the presence of 10 -4 M ascorbic acid. Incubations were stopped upon addition of 1 ml of 0.1 M acetic acid which lyses the cells and releases intracellular cyclic AMP.

2.4. Cycfic AMP and a-MSH radioimmunoassays

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2.5. Calculations Radioimmunoassay data were analyzed with a desk-top calculator using a program based on Model II of Rodbard and Lewald (1970). Statistical significance was determined according to the multiple-range test of Kramer (1956). Dose-response curves and 50% effective doses (EDs0) were calculated using an iterative non-linear least squares regression, as described (Rodbard, 1974). All data are presented as the means - S . E . M . of triplicate determinations, except when the S.E.M. overlaps with the symbol used (where only the symbol is shown).

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had no effect up to 10/~M (data not shown). As shown in fig. 3, (-)-isoproterenol, ( - ) - e p i nephrine and (-)-norepinephrine led to a maxi-

Results 1.2-

As illustrated in fig. 1, 100 nM ( - ) - i s o p r o terenol led to rapid stimulation of cyclic AMP accumulation in pars intermedia cells, a 4 fold increase being measured as soon as 30 s after addition of the fl-adrenergic agonist with a progressive return to basal levels at 7 min. That the effect observed was due to activation of the fladrenergic receptor is indicated by the complete reversal of the stimulatory effect of l0 nM ( - ) isoproterenol by the fl-adrenergic antagonist ( - ) propranolol while the et-adrenergic antagonist phentolamine had no effect (fig. 2). The specific eq- and aE-adrenergic antagonists prazocin and yohimbine as well as serotonin and acetylcholine,

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mal 5, 3 and 2.5 fold stimulation of cyclic AMP accumulation at EDs0 values of 4.6, 30 and 3000 nM, respectively. The stereoselectivity of the /3adrenergic receptor is clearly indicated by the find-

ing that the (+)-stereoisomers of isoproterenol and epinephrine were respectively 400 and 70 times less potent than the (-)-isomers (table 1). ( + ) Norepinephrine was inactive up to the highest concentration studied (10 ~ M). The specificity of the fl-adrenergic receptor was further characterized with a series of/3-adrenergic agonists. Two potent compounds, namely zinterol (fl2-selective) (Temple et al., 1976) and hydroxybenzylisoproterenol (HBI, a non-selective agonist) stimulated cyclic AMP accumulation at lower EDs0 values than (-)-isoproterenol, namely 0.75 and 1.0 nM (fig. 4, table 1). While HBI is a full agonist, zinterol led to a stimulation which reached 80% of the levels measured in the presence of a maximal concentration of (-)-isoproterenol. Two other/32selective agonists~ OPC 2009 (procaterol) (Saitoh et al., 1978; Himori and Taira, 1977) and soterenol (Dungan et al., 1968; Minneman et al., 1979b) acted as partial agonists at ED50 values of 11 and 7.7 nM, respectively (fig. 5, table 1). fl-Adrenergic antagonists were next used to further study the specificity of the cyclic AMP response. Fig. 6 and table 1 show the effect of a series of highly potent antagonists: cyanopindolol (0.4 nM) >/(-)-propranolol (0.5 nM) I> hydroxybenzylpindolol (0.6 nM)>pindolol (0.9 n M ) =

TABLE 1 Activity of fl-adrenergic agents on c A M P levels in rat pars intermedia cells. K D values for agonistic activity were taken directly as the concentration of the agent giving 50% of maximal stimulation (EDs0 value) of c A M P levels. For assessing antagonistic activity, c o m p o u n d s were tested in the presence of 10 n M (-)-isoproterenol and apparent dissociation constants were calculated according to the equation K D = IC50/(1 + S / K ) (Cheng and Prusoff, 1973). In this equation, S is the concentration of (-)-isoproterenol (10 nM), K is the EDs0 value of (-)-isoproterenol for stimulation of cyclic A M P levels (4.6 nM) and IC50 is the concentration of the agent giving 50% reversal of the stimulatory effect of (-)-isoproterenol. For ( - ) - e p i n e p h r i n e and ( )-norepinephrine, 10 m M haloperidol w a s also present. Antagonists

EDs0 (nM)

Antagonists

K D (nM)

Zinterol Hyd roxybenzylisoproterenol ( -- )-Isoproterenol ( -- )-Soterenol ( -- )-Epinephrine OPC 2009 ( )-Norepinephrine ( + )-Isoproterenol ( + )-Epinephrine ( + )-Norepinephrine

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Cyanopindolol ( - )-Propranolol Pindolol ( - )-Alprenolol Timolol ( + )-Propranolol Metoprolol Dichloroisoproterenol Butoxamine Practolol

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0.00 Fig. 4. Effects of increasing concentrations of hydroxybenzylisoproterenol (O) (-)-isoproterenol (O) and zinterol ( 0 ) on cAMP accumulation in pars intermedia cells.

(--)-alprenolol (0.9 n M ) ~ t i m o l o l (1.0 nM). Metoprolol (100 nM), a fairly selective i~antagonist (Petrack and Czernik, 1973), was 200 times less potent than the non-selective t-adrenz O < .J

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Fig, 5. Effect of increasing concentrations of OPC 2009 (procaterol) (O) and soterenol ( O ) on cAMP accumulation in pars intermedia cells. Results are expressed as percent of 100 nM ( - )-isoproterenol-induced cAMP levels (2.7 pmol/105 cells).

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ANTAGONIST (LOG M) Fig. 6. Reversal of 10 nM (-)-isoproterenol-induced stimulation of cAMP levels by increasing concentrations of cyanopindolol (O), hydroxybenzylpindolol (O), ( -)-propranolol ( ~ ) , pindolol ( , ) and metoprolol ( A ) in pars intermedia cells.

ergic antagonist (-)-propranolol. Dichloroisoproterenol completely reversed the stimulation induced by (-)-isoproterenol at a K D value of 300 nM (table 1). The two selective fl-adrenergic antagonists butoxamine (t2) (Levy, 1965) and practolol (Ell) (Levy and Wilkenfeld, 1969) were active at K D values of 1100 and 7000 nM, respectively. The stereoselectivity of the response is further illustrated by the 80 fold greater potency of the ( - ) - as compared to the (+)-isomer of propranolol to reverse the stimulatory action of ( - ) isoproterenol on cyclic AMP accumulation. Since pars intermedia cells contain not only fl-adrenergic but also dopaminergic receptors, we used the long-term cultured cells to study the interaction of catecholamines with these two receptors. We thus also determined the experimental conditions which should permit the interaction with each receptor to be studied independently. The dopaminergic antagonist haloperidol was used first to prevent the inhibition of cyclic AMP induced by activation of the dopamine receptor. Fig. 7 shows that ( - )-isoproterenol had no affinity for the dopaminergic receptor up to 1/tM, its

416 2.5-

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Fig. 7. Effect of increasing concentrations of (--)-isoproterenol ( © ,Q), ( - )-epinephrine (~,~IP) and ( --)-norepinephrine (A , A ) on cAMP accumulation in the presence (filled symbols) or absence (open symbols) of 10 nM haloperidol in pars intermedia cells. Haloperidol was added 25 rain before the adrenergic agents to prevent binding to the dopaminergic receptor.

stimulatory effect not being affected by haloperidol. However, when the interaction of (-)-epinephrine and (-)-norepinephrine with the dopamine receptor was prevented by 10 nM haloperidol, the EDs0 values for their actions were decreased from 30 to 10 and 3000 to 300 nM, respectively, while their maximal responses were increased. Interaction of dopaminergic agents with the/3-adrenergic receptor can be prevented by the addition of 100 nM (-)-propranolol. That changes of a-MSH secretion are related to changes of cyclic AMP levels is well illustrated in fig. 8 which shows that the stimulation of cyclic AMP accumulation by (-)-isoproterenol was accompanied by an increased a-MSH release while the dopaminergic agonist CB-154 (2-bromo-aergocryptine) inhibited both basal and isoproterenol-stimulated cyclic AMP levels and c~-MSH release. 4. Discussion

Analysis of the brain catecholamine receptors coupled to the adenylate cyclase system has proven

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417

difficult due to the heterogeneity of the cell population and to the indirect actions which can affect cyclic AMP accumulation (Horn and Phillipson, 1976; Skolnick and Daly, 1976). Moreover, in brain slices and in vesicular preparations, epinephrine and norepinephrine can modulate cyclic AMP accumulation by acting on both a- and fl-adrenergic receptors (Horn and Philipson, 1976). As shown by immunocytological and biochemical data, the intermediate lobe of the pituitary gland represents a homogeneous population of cells secreting a-MSH and other peptides derived from proopiocortin (Lissitzky et al., 1978; Dub6 et al., 1978; Pelletier et al., 1977). Since this tissue contains fl-adrenergic and dopaminergic receptors, it should offer an ideal system to study the correlation between cellular activity (such as a-MSH secretion) and biochemical events (such as receptor levels, adenylate cyclase activity and cyclic AMP accumulation) involved in the action of these two receptors. In fact, the activity of each receptor can be studied independently by using an appropriate concentration of a specific antagonist of the other receptor. In addition, this system offers the possibility of studying in detail the interaction between two types of receptors having opposite effects on cellular activity. The present system also offers a pure population of postsynaptic receptors, as opposed to the presence of both pre- and postsynaptic receptors in preparations in brain tissue. There are also advantages in using intact cells to analyze the activity of receptors. As an example, studies in rat pinealocytes have clearly demonstrated that there are distinct differences in the changes in cyclic AMP accumulation induced by fl-adrenergic agonists, as compared with the changes obtained previously when pineal homogenates were used (Auerbach et al., 1981). In order to avoid the possible deleterious effects of enzymes used for cell dispersion, pars intermedia cells were used 5 days after plating. The full recovery of the fl-adrenergic receptor after 5 days in culture could possibly explain our finding of stimulation of cyclic AMP levels by epinephrine and norepinephrine alone while no effect was observed previously (Munemura et al., 1980a). The same explanation probably applies to the higher potency of (-)-isoproterenol measured in the pre-

sent study (EDs0 value = 4.6 nM) as compared to that in a previous study ( ~ 30 nM), using acutely dispersed cells (Munemura et al., 1980b). The time course of fl-adrenergic stimulation of cyclic AMP levels in pars intermedia cells was similar to that observed in wild-type S-49 lymphoma cells in the absence of a phosphodiesterase inhibitor (Erdos and Maguire, 1980). The possibility that elevated cyclic AMP levels are due to increased adenylate cyclase activity rather than to decreased phosphodiesterase action is supported by the finding that the stimulatory effect of the fl-adrenergic agonists is seen in the presence of inhibitors of cyclic nucleotide phosphodiesterase activity such as theophylline and isobutylmethylxanthine (Munemura et al., 1980b) as well by the direct stimulatory effect of fl-adrenergic agonists on adenylate cyclase activity in pars intermedia homogenate (C6t6 et al., 1980; C6t6 et al., 1981). Such findings validate the use of changes in cyclic AMP levels as parameter of fl-adrenergic activity. The order of potency (-)-isoproterenol > ( - ) epinephrine >>(-)-norepinephrine suggests the f12 nature (Lands et al., 1967a,b) of the adrenergic receptor in pars intermedia cells. Although no drug so far available shows absolute specificity for the fll and fl2-subtypes of the adrenergic receptors, the specificity of the pars intermedia fl-adrenergic receptor is best defined by the potency of a large series of selective fl-adrenergic agonists and antagonists to modulate cyclic AMP levels. As illustrated in fig. 9, there was a close correlation (r--0.933, P<0.01) between the ability of compounds to influence adenylate cyclase activity in a typical fl2-adrenergic tissue, namely rat lung (Minneman et al., 1979a) and the effect on cyclic AMP levels in pars intermedia cells. On the other hand, there was a complete lack of correlation (r -- 0.396, P < 0.01) with adenylate cyclase data obtained with rat heart, a typical//1-tissue. The fl2-nature of the adrenergic receptor in pars intermedia cells is also in agreement with the specificity of the fl2-receptor in rat cerebellum (Dolphin et al., 1979) as well as in other fl2-systems (Burges and Blackburn, 1972; Lefkowitz, 1975; Lacombe et al., 1976). The fl2-nature of the pars intermedia adrenergic receptor is also well supported by the low activity of practolol (7000

418 4

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nM), a selective ill-antagonist first described by Dunlop and Shanks (12). In analogy with previous findings in a fiE-specific tissue, namely rat lung (Minneman et al., 1979a), zinterol and soterenol behaved as partial agonists on cyclic A M P accumulation in pars intermedia cells. A similar effect was observed with OPC 2009. This action is probably related to their fladrenergic antagonistic activity expressed at high concentrations in both heart and lung rat tissue (Minneman et al., 1979a). ( - ) - E p i n e p h r i n e and ( - ) - n o r e p i n e p h r i n e , which are two valuable drugs for the identification of flE-adrenoreceptors (Lands et al., 1967a) were found to be respectively 3.5 and 45 times less potent than the non-selective ( - ) - i s o proterenol in a fiE-rich tissue (Minneman et al., 1979a), namely rat lung. In pars intermedia cells, (--)-epinephrine and ( - ) - n o r e p i n e p h r i n e , tested in the presence of 10 nM haloperidol to prevent negative interference from the dopaminergic receptor, were 2 and 60 times less potent than ( - ) isoproterenol thus doubling the selectivity of these drugs. Since the lung contains 86% of flE-adren-

ergic receptors (Minneman et al., 1979b), it appears from the present data that the proportion of fiE-Subtype receptors is higher in the pars intermedia cells. It is possible, at least on the basis of the techniques available, that the pars intermedia cells contain a small proportion of ill-receptors. In fact, most tissues examined so far contain a mixture of fl~- and fiE-receptor subtypes. The relative proportion of the two receptor subtypes ranged from 85% fl~ in rat heart to 98% fl2 in rat cerebellum (Minneman et al., 1979b). Although more than two subtypes of fl-adrenergic receptors may exist (Boissier et al., 1971), it appears likely that various proportions of ill- and fiE-SUbtypes leading to the same physiological response (Carlsson et al., 1972) may exist in different tissues and explain the organspecific pharmacological responses. The coexistence of the two receptor subtypes could be due to their presence in the same cell type or to the cellular heterogeneity of the tissues examined. Stimulation of a - M S H secretion in rat pars intermedia cells can thus be added to the other fiE-specific adrenergic responses, namely bronchodilatation, vasodilatation, i n h i b i t i o n ' o f uterine contraction (Lands et al., 1967a,b), glycogenolysis in skeletal muscle and liver, as well as relaxation of smooth muscle (for review see Minneman et al., 1981). The present findings demonstrate that the pure population of pars intermedia cells responds to a predominantly f/E-Subtype of adrenergic control apparently free of a-adrenoreceptors. The possibility is thus offered of measuring receptor binding, cyclic A M P accumulation and a specific cellular response, namely a - M S H secretion in the same pure cell population. This system should be of great help for detailed studies of the mechanisms controlling the activity of flE-adrenoreceptors.

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