Progesterone depression of norepinephrine-stimulated cAMP accumulation in hypothalamic slices

Molecular Brain Research, 5 (1989) 109-119 Elsevier

109

BRM 70119

Progesterone depression of norepinephrine-stimulated cAMP accumulation in hypothalamic slices Nicolas Petitti and Anne M. Etgen Departments of Psychiatry and Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 (U.S.A.)

(Accepted 11 October 1988) Key words: Norepinephrine; Progesterone; cAMP; Adrenergic receptor; Preoptic area; Hypothalamus

The present experiments examined the effects of progesterone on adrenergic receptor coupling to adenylate cyclase in hypothalamic and preoptic area slices by monitoring norepinephrine (NE)-stimulated increases in cAMP accumulation. Progesterone treatment of estrogen-primed rats decreased NE-induced slice cAMP accumulation. The reduced cAMP response was estrogen-dependent since it was not demonstrable in slices from rats exposed to progesterone without prior estrogen priming. Neither generalized increases in phosphodiesterase activity nor decreases in the catalytic activity of adenylate cyclase could account for the reduced ability of NE to stimulate cAMP accumulation in hypothalamic slices. Moreover, the cAMP response to two other activators of adenylate cyclase, adenosine and vasoactive intestinal peptide, was not decreased in slices from rats treated with estrogen plus progesterone. Selective adrenergic agonists and antagonists were employed to determine which adrenergic receptors mediate cAMP accumulation in progesterone-exposed slices. Slice cAMP levels were elevated by the fl receptor agonist isoproterenol but not by ctI (phenylephrine) or ct2 (clonidine) agonists. However, clonidine potentiated the effect of isoproterenol on slice cAMP formation whereas phenylephrine did not. Likewise, NE-stimulated cAMP accumulation was completely antagonized only by a combination of both fl (propranolol) and a 2 (yohimbine) antagonists. The data suggest that in slices from estrogen plus progesterone-treated rats, a 2 receptors contribute significantly to NE stimulation of cAMP accumulation. The overall depression of the cAMP response to NE in progesterone-exposed slices may involve a decrease of a 1receptor facilitation of cAMP synthesis.

INTRODUCTION

ceptors may also be involved in N E stimulation of c A M P accumulation 11,4°.

N e u r o t r a n s m i t t e r s and n e u r o m o d u l a t o r s can regulate neuronal functions by initiating a cellular cascade involving cyclic adenosine 3 ' : 5 ' - m o n o p h o s phate ( c A M P ) , a second messenger associated with the regulation of protein p h o s p h o r y l a t i o n and subsequent cellular response 19. N o r e p i n e p h r i n e (NE) is known to initiate such a cascade. N E increases formation of c A M P in rat brain slices through the activation of adenylate cyclase, an event involving both a and fl adrenergic r e c e p t o r stimulation s. This action is unique in brain slices in that a adrenergic agonists augment the c A M P accumulation induced by the activation of fl adrenergic receptors s,l°,ll. T h e a receptor potentiation of the c A M P response seems to be m e d i a t e d primarily by (it1 receptors, although a 2 re-

Ovarian steroid h o r m o n e s can m o d u l a t e the synthesis, release and turnover of N E and its receptors in the central nervous system 1'2,6,21,26,32,51-53. W e recently r e p o r t e d that N E induction of c A M P accumulation in hypothalamus and preoptic area ( P O A ) slices fluctuates in association with changes in circulating ovarian steroids 14. O u r data indicate further that a I receptor stimulation potentiates fl receptor induction of c A M P accumulation in hypothalamic and P O A slices 15. These findings are consistent with the results of other work on hypothalamic 7, hippocampal 16, and cortical slices 39,40. In addition, we found that estrogen t r e a t m e n t of ovariectomized female rats simultaneously enhances a 1 and depresses fl receptor function 15 as m e a s u r e d by the actions of spe-

Correspondence: N. Petitti, Department of Psychiatry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, N Y 10461, U.S.A.

0169-328X/89/$03.50 ~ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

110 cific receptor agonists and antagonists on cAMP accumulation. Our earlier study 14 also found that subsequent exposure of estrogen-primed rats to progesterone (P) partially or completely suppresses NE enhancement of cAMP accumulation. The purpose of the present studies was to examine the mechanism by which P alters NE-induced accumulation of cAMP in hypothalamic and POA slices. Various NE receptor agonists and antagonists were used to distinguish the NE receptor subtypes that mediate cAMP accumulation. Other experiments used phosphodiesterase inhibitors, a direct activator of adenylate cyclase, and additional agonists of cAMP accumulation to determine the contribution of other components of the adenylate cyclase/cAMP generating system. MATERIALS AND METHODS

Tissue slice preparation Experiments were performed using sexually mature female Sprague-Dawley rats (Taconic Farms) weighing 150-175 g. Animals were housed under a 14 h light-10 h dark cycle in a temperature-controlled environment (25 °C) with ad libitum food and water. All animals were ovariohysterectomized (OVX) bilaterally under Metofane anesthesia 4 - 7 days prior to use. In all cases, estrogen treatment consisted of two subcutaneous (s.c.) injections of 2 /~g of estradiol benzoate (EB) given 24 and 48 h before sacrifice. The P treatment consisted of an injection of 500/tg of P given s.c. 3.5 h before sacrifice. EB and P were dissolved in peanut oil and injected in a volume of 0.1 ml. Animals were sacrificed by decapitation, and the brain quickly removed and placed into ice-cold artificial cerebrospinal fluid 55. The entire hypothalamus and POA were removed as described by Hatton 2°, and slices (each 350ktm thick) were cut on a Mcllwain tissue chopper beginning approximately 2 mm anterior to the optic chiasm and ending 1 mm anterior to the mammillary bodies. In all experiments, brain slices were prepared between 10.30 and 11.00 h to eliminate potential diurnal variation in cAMP 27 and adrenoreceptor content 25'28. Based on anatomical landmarks observed in comparable slices from fixed tissue, slices of POA and middle hypothalamus (MH) were obtained as described earlier ~5. Each slice was maintained at 34-35 °C with shaking (80

oscillations/min) in an individual tissue culture well containing 300/~1 of artificial cerebrosPinal fluid in an O2/CO 2 (95/5) saturated environment. The incubation conditions were identical to those used in our previous work with rat hypothalamic slices 14'15. Slices were left undisturbed for 75 min to allow nucleotide levels to stabilize 13"17.

Drug treatment Following the equilibration period, the POA and MH slices from one animal were exposed to the same experimental treatment (see individual experiments). Drugs were added directly to the incubation wells as concentrated solutions in 3/A of appropriate vehicle: distilled water for adenosine, vasoactive intestinal peptide (VIP), NE agonists and antagonists; 0.01 N HC1 for NE; 1% ethanol for forskolin. Non-drug control slices received equal volumes of vehicle at the same time. Incubation time with NE, forskolin, and NE agonists was always 20 min. NE antagonists were added 5 min prior to NE. Phosphodiesterase inhibitors 3-isobutyl-l-methylxanthine (IBMX) and I>4(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO20-1724) were added at the beginning of the equilibration period in a volume of 3/~1 of absolute ethanol. Experiments were terminated by transferring slices rapidly to 400/al of ice cold 5% trichloroacetic acid (TCA). Determination of cAMP content The slices were disrupted by sonication, and the supernatant (containing cAMP) and pellet (containing tissue protein) separated by centrifugation. The TCA pellet was dissolved in 2 N N a O H for later determination of protein content 29'31. The supernatant was acidified with 1 N HC1, and TCA was removed with 4 volumes of washed ether. The resulting aqueous extracts were concentrated by lyophilization and analyzed for cAMP content using a modified Gilman protein binding assay 5 as reported previously 13. Data were converted to pmol of cAMP per mg tissue protein. For all experiments except those with adenosine and VIP, values for the 4 POA or 3 MH slices were averaged to give a single value for each brain region for each rat. In experiments involving adenosine and VIP, values from individual POA or MH slices were used.

111

Phosphodiesterase assay Slices obtained from E B - and EB + P-primed rats were incubated under the conditions described above for 95 min. The slices were then disrupted by sonication in Tris buffer, and the supernatant and protein pellet s e p a r a t e d by centrifugation. The supernatant was analyzed for calcium-independent and calcium + c a l m o d u l i n - d e p e n d e n t p h o s p h o d i e s t e r a s e activity using the p r o c e d u r e described by Wolff and Brostrom 54.

Materials EB and P were purchased from Steraloids (Wilton, NH). Metofane was obtained from P i t m a n - M o o r e (Atlanta, G A ) . Forskolin was purchased from Calbiochem-Behring (LaJolla, C A ) and VIP from Bachem (Torrance, C A ) . Prazosin was d o n a t e d by Pfizer ( G r o t o n , CT). I B M X , adenosine, N E and all o t h e r N E receptor agonists and antagonists were purchased from Sigma (St. Louis, M O ) . RO-20-1724 was o b t a i n e d from Roche Pharmaceutical (Nutley, N J).

Statistics Significant differences between means were determined using analysis of variance. Planned post hoc comparisons were made using a D u n n e t t (for comparison with controls) or a N e w m a n - K e u l s multiple range test. Differences were considered statistically significant if P < 0.05. RESULTS

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NE CONCENTRATION, pM Fig. 1. Concentration-dependent elevation of cAMP content by NE in POA (open symbols) and MH (closed symbols) slices from rats given EB (2 pg, 24 and 48 h before sacrifice) with (solid lines) or without (dashed lines) 500/~g of P 3.5 h before sacrifice. Data are expressed as the fold increase in cAMP content relative to control slices incubated without added NE and represent the mean (+ S.E.M.) of 4-7 independent replications. Data for EB alone are from ref. 15.

than did M H slices. In slices from EB + P-treated animals, the maximal c A M P response to 100 # M N E was a 6-fold increase in the P O A and a 2- to 3-fold increase in the MH. In contrast, in slices from EBtreated animals, the maximal c A M P response to 100 /aM N E was a 12-fold increase in the P O A and a 5fold increase in the MH. Fig. 2 shows that depression of the c A M P response to N E is d e p e n d e n t on prior estrogen exposure. P O A and M H slices from O V X controls and from O V X animals injected with 500 #g of P 3.5 h before sacrifice showed similar levels of N E - i n d u c e d c A M P accumulation. A t 100 ktM N E , P O A slices showed nearly a 10-fold increase, and M H slices exhibited a 5- to 6fold increase in c A M P content.

Effects of P on NE-stimulated cAMP In our previous study TM,P t r e a t m e n t of E B - p r i m e d rats attenuated the c A M P response to 10/~M N E in hypothalamic and P O A slices. This reduction in c A M P response was selective for EB + P-treated rats in that it was not seen in O V X rats given P alone. A n initial experiment was p e r f o r m e d to d e t e r m i n e if reduced accumulation of c A M P would be observed over a N E concentration range. Fig. 1 compares the c A M P response of P O A and M H slices from animals treated with EB or with E B + P to increasing concentrations of NE. U n d e r both conditions, N E (5-100 p M ) p r o d u c e d a c o n c e n t r a t i o n - d e p e n d e n t increase in c A M P accumulation. As r e p o r t e d previously 14,15, P O A slices showed a larger c A M P response to N E

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NE CONCENTRATION, MM Fig. 2. Concentration-dependent elevation of cAMP content by NE in POA (open symbols) and MH (closed symbols) slices from OVX control females (solid lines) or OVX females given 500pg of P 3.5 h before sacrifice (dashed lines). Each point represents the mean of 3 or 4 independent replications.

112 TABLE I

Effects ofR0-20-1724 and forskolin on cAMP accumulation in POA and M H slices from EB- and EB + P-primed rats Values represent the m e a n of 3 - 6 independent replications. There were significant main effects of both h o r m o n e and drug treatment for both brain regions: P O A h o r m o n e , FI,26 = 6.39, P < 0.02; P O A drug treatment, F4,26 = 22.0, P < 0.00001; M H hormone, F1,23 = 10.4, P < 0.005; M H drug treatment, F4,23 = 21.9, P < 0.00001.

Treatment

EB

EB + P

POA Control NE (100/aM) RO-20-1724 (0.7 m M ) RO-20-1724 (0.7 m M ) + NE (100/tM) Forskolin (100/~M)

4.88 50.1 76.0 247 242

MH + + + + +

0.50 5.71 20.5 11.6 81.5

7.31 49.4 76.8 386 150

POA + + + + +

1.08 15.1 25.5 55.4 39.1

5.71 27.4 52.8 99.3 278

MH + + + + +

0.53 1.77" 1.39 1.53" 34.9

6.69 28.7 92.6 184 156

+ + + + +

0.62 11.3" 7.35 12.1" 23.7

* Significantly less than same treatment with EB alone (P < 0.05), N e w m a n - K e u l s .

Interactions with phosphodiesterase These studies determined whether P treatment of EB-primed animals influences phosphodiesterase activity in POA and/or MH slices. Preliminary results with IBMX suggested that P might increase phosphodiesterase activity in slices from EB-primed rats (unpublished observations). Since IBMX interacts with adenosine receptors, additional experiments were conducted with a second phosphodiesterase inhibitor known not to interact with adenosine receptors 3°. Slices were incubated with RO-20-1724 (0.7 mM) beginning at the time of slice preparation. Basal levels of cAMP were elevated 10-15 fold by RO-20-1724 in slices from EB- and EB + P-primed animals. The addition of 100 pM NE produced a further increase in slice concentrations of cAMP (Table I) that is more robust in EB- than in EB + P-primed slices. The percent changes were approximately the same response seen in EB- and in EB + P-primed slices exposed to 100pM NE in the absence of RO-20-1724 (Fig. 1). Direct measurements of phosphodiesterase activity in slices from EB- and EB + P-treated rats were also performed (Table II). There was no significant difference in either calcium-independent or calcium + calmodulin-dependent phosphodiesterase activity in slices from animals under the two hormonal treatments.

Effects of forskolin These studies determined whether P treatment of EB-primed rats directly influences adenylate cyclase activity in POA and/or MH slices. Forskolin, a potent activator of adenylate cyclase in most types of animal

cells44, was used for this purpose. Slices from both brain regions showed concentration-dependent increases in cAMP levels when incubated with forskolin (Fig. 3). Maximal increases in cAMP content of POA slices were reached at 100/~M forskolin (30fold) and in MH slices at 200 ~M forskolin (15-fold). As reported previously, forskolin-stimulated cAMP accumulation was more robust in POA than in MH slices. Moreover, the cAMP response to forskolin was at least as great in slices from EB + P-treated rats as in slices from EB-treated rats (Table I).

Effects of VIP and adenosine The following experiments were performed to determine whether the reduction in cAMP response following P treatment of EB-primed rats was selective for the NE system. Fig. 4 compares the cAMP re-

T A B L E II

Phosphodiesterase activity in slices from EB- and EB + Ptreated rats Values reported are pmol cyclic A M P hydrolyzed/mg protein/min (~ + S.E.M.).

Treatment

Phosphodiesterase activity POA

MH

EB Calcium-independent Calcium + calmodulin

876+302 (n = 4) 1223+357 (n = 4)

420+48 (n = 3) 708+53 (n = 3)

EB + P Calcium-independent Calcium + calmodulin

606+129 (n = 4) 966+161 (n = 4)

3 5 5 + 1 6 ( n = 3) 606+42 (n = 3)

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Forskolin, ,uM Fig. 3. Concentration-dependent elevation of cAMP content by forskolin in POA (solid lines) and MH (dashed lines) slices from EB + P-treated rats. Each value represents the mean (+ S.E.M.) of 3 or 4 independent replications. sponse of P O A and M H slices from E B - and EB + Pt r e a t e d rats to V I P and adenosine. These c o m p o u n d s have previously b e e n identified as potentiating c A M P levels in several brain regions 9,13,37,44'45. Both V I P and adenosine p r o d u c e d significant elevations in

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c A M P content of both P O A and M H slices from E B and EB + P - t r e a t e d animals. P t r e a t m e n t of E B p r i m e d rats did not decrease VIP- or adenosine-stimulated c A M P accumulation in P O A or M H . Threeway analysis of variance ( h o r m o n e x brain region x drug t r e a t m e n t ) showed significant main effects of h o r m o n e ( P < 0.03), brain region ( P < 0.001) and drug t r e a t m e n t ( P < 0.00001). T h e r e was also a significant interaction between h o r m o n e and drug treatm e n t (P < 0.02) and between brain region and drug t r e a t m e n t (P < 0.002). The significant interactions were attributable to the fact that V I P stimulated much greater c A M P accumulation in M H slices derived from EB + P-treated rats than in M H slices from E B - p r i m e d rats ( N e w m a n - K e u l s , P < 0.01).

Effects o f adrenergic receptor agonists and antagonists Table III depicts the effects of several adrenergic agonists and antagonists on c A M P accumulation in P O A and M H slices from E B + P - t r e a t e d rats. A n a l ysis of variance revealed significant main effects of drug t r e a t m e n t ( F = 23.0, df = 7,44; P < 0.0001). Post hoc analysis (Dunnett) indicated that neither the a I agonist phenylephrine (10 p M ) , the/3 agonist isoproterenol (10 /~M) nor the combination of

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Fig. 4. Effects of other cAMP agonists on cAMP content of POA (A) and MH (B) slices from female rats primed with EB only (2/xg, 24 and 48 h before sacrifice; open bars) or with EB plus 500/2g of P 3.5 h before sacrifice (hatched bars). Equilibrated slices were exposed for 20 min to vehicle (CON), 100 /zM adenosine (ADEN) or 10 ktM VIP prior to cAMP determination. Each value represents the mean (+ S.E.M.) of 5-10 slices. * Significantly greater than VIP response in slices from rats receiving only EB (P < 0.01, Newman-Keuls).

Control NE (100/~M) Isoproterenol (10/xM) Phenylephrine (10ktM) Isoproterenol + phenylephrine (10/~M each) NE (100/xM) + prazosin (10k~M) NE (100/~M) + propranolol (10/2M) NE (100/~M) + yohimbine (50/xM)

6.59 + 41.9 + 8.24 + 3.11 +

MH 0.67 3.50* 1.59 0.26

6.03 + 19.8 + 11.4 + 6.31 +

0.79 3.12" 0.41 0.33

8.81 + 2.61

9.58 + 5.22

27.8 + 4.76**

15.0 + 2.61

9.34 + 1.48

10.7 + 1.81

8.93 + 1.79

10.2 + 4.54

* Significantly greater than control (P < 0.01, Dunnett), ** Significantly greater than control (P < 0.01, Dunnett) but less than NE alone (P < 0.01, Newman-Keuls).

114 phenylephrine and isoproterenol increased cAMP accumulation significantly when compared to control values. In contrast, NE (100/aM) significantly elevated cAMP levels approximately 6-fold in POA (P < 0.01) and approximately 3-fold in MH (P < 0.05). In contrast to the small effect of adrenergic agonists, there were significant effects of adrenergic antagonists to block cAMP accumulation induced by 100 ,uM NE. The cAMP response of POA slices to 100/aM NE was reduced 40% by the selective c~1 antagonist prazosin (10/aM). The cAMP level in the presence of prazosin plus NE was significantly greater than in controls (Dunnett, P < 0.01) but less than in slices exposed to NE alone (Newman-Keuls, P < 0.01). Both the fl antagonist propranolol (10/aM) and the a 2 antagonist yohimbine (50/aM) reduced NEstimulated cAMP to levels equal to those of control slices. MH slice cAMP accumulation produced by

100/aM NE was not significantly inhibited by prazosin, propranolol or yohimbine. The small and somewhat variable cAMP response to 100/aM NE in MH slices made it difficult to draw conclusions regarding the effects of antagonists on NE-induced cAMP accumulation. In order to clarify both the small and somewhat variable cAMP response to adrenergic agonists and the ineffectiveness of adrenergic antagonists in MH slices, additional experiments included 1 mM IBMX in the incubation to increase the overall slice cAMP content. Fig. 5 shows the cAMP response to NE agonists of POA and MH slices from EB + P-treated rats incubated with 1 mM IBMX. Two-way analysis of variance (brain region x drug treatment) revealed significant main effects of drug treatment only (F = 35.7; df = 6,46; P < 0.00001). Neither phenylephrine (aa) nor clonidine (a2) alone affected cAMP

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Fig. 5. Effects of adrenergic receptor agonists on cAMP content of POA (A) and MH (B) slices from EB + P-treated rats incubated in the presence of 1 mM 1BMX throughout the experiment. Equilibrated slices were incubated an additional 20 rain with vehicle (CON), 100¢tM NE (NE), or 10~uM agonists alone or in combination. ISO, isoproterenol; PHE, phenylephrine; CLO, clonidine. Each value represents the mean (+ S.E.M.) of 3-6 independent replications. * Significantly greater than control (P < 0.01, Dunnett) but less than NE (P < 0.01, Newman-Keuls). ** Significantly greater than ISO or CLO alone (P < 0.05, Newman-Keuls).

30

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Fig. 6. Effects of adrenergic receptor antagonists on cAMP content of POA (A) and MH (B) slices from EB + P-treated rats incubated in the presence of 1 mM IBMX throughout the experiment. Equilibrated slices were incubated an additional 20 min with vehicle (CON), 100¢tM NE alone (NE), or 100/~M NE plus 10/~M prazosin (PRZ), 10/~M propranolol (PRO) or 50/~M yohimbine (YOH) alone or in combination. Antagonists were added 5 rain before NE. Each value represents the mean (+ S.E.M.) of 3-8 independent replications. * Significantly less than NE (P < 0.01, Newman-Keuls). ** Significantly less than NE (P < 0.05, Newman-Keuls).

115 levels in either POA or MH slices. In contrast, the/~ agonist isoproterenol significantly elevated cAMP levels in POA and MH slices when compared to nondrug controls (Dunnett, P < 0.01), but the elevation was less than that produced by 100 #M NE alone (Newman-Keuls, P < 0.01). Phenylephrine did not enhance the cAMP response to isoproterenol. Instead clonidine synergized with isoproterenol to produce a greater increase in cAMP in both POA and MH than either drug alone (Newman-Keuls, P < 0.01); however, this increase in cAMP accumulation was still significantly less than that evoked by 100/~M NE (Newman-Keuls, P < 0.01). Fig. 6 shows the effects of several adrenergic antagonists on the cAMP response to 100 p M NE in P O A and MH slices incubated with 1 mM IBMX. Two-way analysis of variance revealed significant main effects of drug treatment only (F = 29.0; df = 6,50; P < 0.00001). NE-stimulated cAMP accumulation was reduced 15-20% by the a 1 antagonist prazosin in P O A slices (Newman-Keuls, P < 0.05). In contrast, prazosin did not significantly reduce cAMP levels in the MH slices (Newman-Keuls, P > 0.10). The cAMP response to 100 #M NE in both P O A and MH slices was partially reduced by propranolol (Newman-Keuls, P < 0.01) and by yohimbine (Newman-Keuls, P < 0.01). Neither propranolol nor yohimbine reduced cAMP to control levels (Dunnett, P < 0.01). The combination of proprano1ol and yohimbine produced the most potent inhibition of cAMP accumulation; in both POA and M H slices, cAMP levels were reduced to those of control slices. The effect of combined drug treatment on the cAMP response to NE was significantly greater than that of either p~'opranolol (Newman-Keuls, P < 0.01) or yohimbine (Newman-Keuls, P < 0.01) alone. In contrast, the combination of propranolol and prazosin did not inhibit NE-induced cAMP accumulation further than propranolol alone (NewmanKeuls, P > 0.10). DISCUSSION In agreement with our earlier work 14'15, the present data demonstrate that ovarian steroids modify NE responsiveness in brain slices. The results suggest that P treatment of EB-primed rats decreases the capacity of NE to induce cAMP accumulation in P O A

and MH slices. Maximal concentrations of NE (100 #M) produce approximately 12-fold (POA) and 5fold (MH) increases in cAMP content of slices from OVX 15 or EB-treated females. In contrast, when slices are derived from EB-primed rats given 500 #g of P 3.5 h prior to sacrifice, the maximal elevation in cAMP levels in response to 100 # M NE is 6-fold in the POA and 2- to 3-fold in the MH (Fig. 1). The Pinduced reduction in NE-stimulated cAMP accumulation is an estrogen dependent process since it is not demonstrated in slices from OVX rats given P without prior exposure to estrogen (Fig. 2). The depressed capacity of slices to accumulate cAMP in response to NE may arise from either decreased adenylate cyclase activity or increased cAMP hydrolysis. It is unlikely that the observed changes are due to direct inhibition of adenylate cyclase. The cAMP response to forskolin, a direct activator of adenylate cyclase 46, was as great in slices from EB + P-treated rats as in slices from EB-treated rats (Table I). We also analyzed the possibility that P increases the rate of cAMP hydrolysis by phosphodiesterases. Evidence from several sources does not support this notion. First, in the presence of the phosphodiesterase inhibitor RO-20-1724, NE stimulation of slice cAMP accumulation was still depressed. Second, comparison of both calcium-independent and calcium + calmodulin-dependent phosphodiesterase of P O A and MH slices from EB- and EB + P-treated animals revealed no differences in enzyme activity. Third, forskolin-stimulated cAMP accumulation was not attenuated in slices from EB + P-treated rats when compared to EB-treated rats in either the POA or MH. Thus a large body of evidence supports the conclusion that P inhibition of NE-stimulated cAMP accumulation is not attributable solely to increased phosphodiesterase activity. It is notable that Thomas et al. 49 found that the reduction in adenylate cyclase activity in rabbit myometrium following P treatment of EB-primed animals was due to adenosine inhibition of adenylate cyclase. However, this is probably not the situation in P O A or MH slices. First, adenosine increased rather than decreased cAMP accumulation in brain slices from both EB- and EB + P-treated rats. Second, the adenosine antagonist theophylline (200 or 500 #M) does not reverse the depressant effect of P on NE-activated cAMP accumulation (unpublished observa-

116 tions). Thus in P O A and M H slices, the predominant action of adenosine is to stimulate adenylate cyclase. Third, the adenosine elevation of c A M P was similar in slices from EB- and EB + P-treated rats. Therefore, it is unlikely that adenosine inhibition of adenylate cyclase is the mechanism by which P attenuates the c A M P response to N E in M H or P O A slices. Present data also indicate that the reduction of c A M P accumulation in EB + P-treated rats is specific for NE. Elevations of slice c A M P concentrations by VIP and adenosine, both activators of brain adenylate cyclase 9'13'37'41'44, were not reduced in EB + Ptreated rats when compared to EB-treated rats. In MH slices (Fig. 4B), the VIP-induced c A M P level in EB + P rats was actually greater than in EB-primed rats. We cannot rule out the possibility that P may also reduce the stimulatory ability of other agonists of adenylate cyclase. However, the data indicate that P does not cause an overall depressant effect on receptor-activated c A M P production. In agreement with other work in brain slices 7' 16.39,40.48, our results demonstrate that the response to NE in P O A and M H slices is mediated by interactions with both a and fl receptors. However, due to the small and somewhat variable c A M P response of slices from EB + P-treated animals to the adrenergic agents, it was difficult to identify the receptor subtypes mediating the c A M P response in the absence of a phosphodiesterase inhibitor. In the presence of IBMX, a clearer elucidation of adrenergic agonist and antagonist effects was attained. Stimulation of fl receptors with isoproterenol elevated c A M P levels in slices from both brain regions, whereas neither of the a agonists (phenylephrine or clonidine) increased cAMP when given alone. This is not surprising, since a adrenergic systems do not directly activate cAMP production but rather facilitate or potentiate the activation by other agonists 8'1°. Surprisingly, the response to isoproterenol was augmented by the addition of the a 2 agonist clonidine. Thus the data support the hypothesis that in EB + P-treated rats, a 2 receptors synergize with fl receptor activation to induce c A M P accumulation in P O A and M H slices. Results obtained with adrenergic antagonists provide further support for this hypothesis. The combination of fl and a 2 receptor antagonists produced the most potent inhibition of NE-stimulated c A M P accumulation in both P O A and M H slices. Antagonism of

a 1 receptors caused little (POA) or no reduction (MH) in NE activation of c A M P accumulation and did not potentiate the inhibition produced by fl antagonism. Therefore, the antagonist data also suggest that activation of fl and a2 receptors mediates NE enhancement of c A M P accumulation in EB + P-treated P O A and MH slices with little or no participation of a I receptors. It is also possible that a 2 receptors have direct facilitatory actions since propranolol does not completely inhibit the cAMP response to N E (Fig. 5). The situation in slices from EB + P-treated rats stands in marked contrast to that in slices from O V X or EB-treated female rats reported previously 15. In those experiments, both M H and P O A slices from O V X and EB-exposed animals showed an ctI enhancement of cAMP accumulation in that phenylephrine augmented the c A M P response to isoproterenol. Estrogen treatment also caused a relative increase in a 1 activity and a concomitant decrease in fl activity. The c A M P response to NE was blocked more effectively by the a 1 antagonist prazosin than by the fl antagonist propranolol, and the c A M P response to isoproterenol alone was reduced 15. However, it must be noted that the previous studies were conducted in the absence of IBMX. Therefore, we cannot rule out the possibility that the attenuation of the ct1 response is attributable to the presence of I B M X in the current experiments. While the augmentation of fl receptor stimulation of adenylate cyclase by a I sites has been shown in numerous studies 7"8'15'16'22'39'40'43'48, the current data demonstrate that a 2 receptors may also participate in cyclase activation. This conclusion is consistent with recent reports which demonstrate that a2 receptors may participate in NE-stimulated c A M P accumulation in cortical slices I1'12'4°. Moreover, our work suggests that the relative contribution of a~ and a2 sites may vary as a function of hormonal state. Indirect effects of P and NE-stimulated c A M P accumulation must also be considered since it has been suggested that P facilitates NE release 23'24'34'36. Thus endogenous NE release in the P O A and M H of animals given a regimen of EB + P may be greater than in animals given EB alone. This could lead to desensitization, a phenomenon which has been demonstrated in the fl and a 1 adrenergic receptor systems 33'42'46'47. However, it is also possible that P

117 could decrease N E receptors i n d e p e n d e n t of changes in N E release. The desensitization of a l receptors could also lead to a n o t h e r biochemical consequence. Activation of a~ receptors in many tissues, including the rat brain, stimulates the turnover of phosphoinositides, leading to the activation of protein kinase C 3'4. A d d i t i o n a l l y , activation of protein kinase C can augment c A M P accumulation 18"22"35"38"5°. Thus, protein kinase C activation by a I receptors could be involved in the stimulatory effects of N E on c A M P production in O V X and E B - p r i m e d rats. In EB + P-treated rats, the a I component appears to be attenuated. If a l - m e d i a t e d protein kinase C activation is also reduced, a decrease in measurable N E - s t i m u l a t e d slice c A M P accumulation might well be predicted. In summary, present results support the conclusion that P t r e a t m e n t of E B - p r i m e d rats decreases the capacity of N E to induce c A M P accumulation in P O A and M H slices. The reduction appears to be estrogen d e p e n d e n t since it is not d e m o n s t r a t e d in slices without prior exposure to EB. Neither an increase in phosphodiesterase activity nor a decrease in adenylate cyclase activity a p p e a r to cause the r e d u c e d abiliREFERENCES 1 Advis, J.P., McCann, S.M. and Negro-Vilar, A., Evidence that catecholaminergic and peptidergic (luteinizing hormone-releasing hormone) neurons in suprachiasmatic-medial preoptic, medial basal hypothalamus and median eminence are involved in estrogen-negative feedback, Endocrinology, 107 (1980) 892-901. 2 Barraclough, C.A. and Wise, P.M., The role of catecholamines in the regulation of pituitary luteinizing hormone and follicle-stimulating hormone secretion, Endocr. Rev., 3 (1982) 91-119. 3 Berridge, M.J., Inositol triphosphate and diacylglycerol as second messengers, Biochem. J., 220 (1984) 345-360. 4 Berridge, M.J. and Irvine, R.F., Inositol triphosphate, a novel second messenger in cellular signal transduction, Nature (Lond.), 312 (1984) 315-321. 5 Brostrom, C.O. and Kon, C., An improved protein binding assay for cyclic AMP, Anal. Biochem., 58 (1974) 459-468. 6 Crowley, W.R., Effects of ovarian hormones on norepinephrine and dopamine turnover in individual hypothalamic and extrahypothalamic nuclei, Neuroendocrinology, 34 (1982) 381-386. 7 Daly, J.W., Padgett, W., Creveling, C.R., Cantacuzene, D. and Kirk, K.L., Cyclic AMP generating systems: regional differences in activation by adrenergic receptors in rat brain, J. Neurosci., 1 (1981) 49-59. 8 Daly, J.W., Padgett, W., Nimitkitpaisan, Y., Creveling, C.R., Cantacuzene, D. and Kirk, K.L., Fluoronorepinephrine: specific agonists for the activation of alpha and beta

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