The Journal of Neuroscience, October 1992, 12(10): 3889-3876 Estradiol Selectively Regulates a,,-Noradrenergic Receptors in the Hypothalamus and Preoptic Area Nicolas Petitti, George B. Karkanias, and Anne M. Etgen Departments of Psychiatry and Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461 We previously demonstrated that estradiol administered in of CAMP synthesis is correlated with a modest (20-30%) ele- viva elevates the number of a,-adrenoceptors in preoptic vation in the total number of specific 3H-prazosin binding sites area (POA) and hypothalamic membranes from ovariecto- in hypothalamic and POA membranes(Etgen and Karkanias, mized female rats and potentiates a, receptor augmentation 1990). Interestingly, progesterone(P) abolishes(Y, receptor po- of ,&adrenoceptor-stimulated CAMP formation in slices from tentiation of CAMP synthesis in slicesfrom E,-primed female these brain regions. Present studies examined (1) if estradiol rats (Petitti and Etgen, 1989, 1990, 1992) without affecting the selectively regulates any cY,-adrenoceptor subtype, and (2) number or affinity of 3H-prazosin binding sites(Etgen and Kar- which ,x, receptor subtype mediates the augmentation of kanias, 1990). CAMP synthesis. Hypothalamic and POA membranes from Norepinephrine (NE) receptors have long been divided into estradiol-treated rats, when compared to ovariectomized rats, p, o(,, and c+ subtypes. However, it is now clear that thesethree had modestly (3040%) but significantly elevated numbers classesof adrenergic receptors each can be subdivided further of 3H-prazosin ((Y,) binding sites. Estradiol affected neither basedupon both pharmacologicaland molecular biological cri- the number of a, receptor sites in frontal cortex nor the teria (for recent reviews, seeLomasney et al., 1991; Szabadi and affinity of 3H-prazosin binding in any brain region examined. Bradshaw, 1991). The /3 receptorscan be subdivided into 0, and Results of binding studies conducted in the presence of p2 receptor subtypes (Minneman and Molinoff, 1980), the 01, chlorethylclonidine, a selective, irreversible inactivator of the receptors can be subdivided into alA and ollB(and perhaps a,,-) (Y,~ receptor subtype, indicated that the estrogen-dependent receptor subtypes (McGrath, 1982; Morrow and Creese, 1986; increase in total a, binding sites in POA and hypothalamic Minneman et al., 1988; Terman et al., 1990; Harrison et al., membranes was attributable to a selective, five- to sixfold 199 l), and the LYEreceptors can be subdivided into at least aZA, increase in (Y,~ receptor number. Progesterone had no mea- Q, and 01~~receptor subtypes(Bylund, 1988; Bylund et al., 1988; surable effects on a, receptor binding. Blockade of alB re- Harrison et al., 1991). Morrow and Creese (1986) originally ceptors with chlorethylclonidine eliminated phenylephrine reported that two classesof (Y, receptor binding sites in rat ce- augmentation of isoproterenol-stimulated CAMP formation rebral cortex could be differentiated by their affinities for the in slices, whereas the a,A antagonist 5methyl-urapadil did competitive antagonistsWB4 101 and phentolamine, and des- not. This suggests that the a,B receptor subtype potentiates ignated them a,* and CY,~.Han et al. (1987a,b) subsequently CAMP formation. Thus, the increased (Y, receptor augmen- demonstrated that thesebinding sitescould be distinguished by tation of CAMP formation seen in slices from estradiol-treat- their different sensitivities to inactivation by the site-directed ed rats is correlated with increased (Y,~ receptor number. alkylating agent chlorethylclonidine (CEC). They demonstrated that the o(,~site was relatively CEC insensitive whereasthe (Y,~ In some tissues,including brain slices, activation of a,-adren- site was sensitive to irreversible inactivation by CEC. ergic receptors potentiates agonist- and forskolin-stimulated The identity of the (Y, receptor subtype mediating CAMP po- CAMP accumulation (Perkins and Moore, 1973; Daly et al., tentiation remains unresolved. For example, Johnson and 1980; Duman et al., 1985; Sugdenet al., 1985; Etgen and Petitti, Minneman (1986, 1987) suggestthat the a), receptor subtype 1987; Petitti and Etgen, 1990, 1991). Ovarian steroidsmodulate that augmentsCAMP accumulation is similar to the 01,receptor a,-adrenoceptor augmentation of CAMP formation in brain ar- that activates inositol phospholipid hydrolysis, most likely the easthat regulate female reproductive function. When compared (Y,~subtype, whereasRobinson and Kendall (1989) suggestthat to hypothalamic and preoptic area (POA) slices from ovariec- it is not. Recent studies employing both CEC and antagonists tomized (OVX) rats, slices from estradiol (E,)-treated females with high selectivity for the o(,* subtype concluded that the exhibit enhancedo(, receptor augmentation of CAMP formation receptors mediating CAMP potentiation and phosphoinositol (Petitti and Etgen, 1990). This changein LY,receptor facilitation hydrolysis in brain slices fit neither the current (Y,* nor the (Y,~ classification (Minneman and Atkinson, 1991). Therefore, the purposesof the present studies were (1) to evaluate the possi- Received Feb. 5, 1992; revised Apr. 7, 1992; accepted May 5, 1992. bility that the small E,-induced increase in total 01,receptor This work was supported by DHHS Grants MH41414 and RSDA MH00636 to A.M.E., by BRSG Grant RR05397, and by the Department of Psychiatry, binding in hypothalamus and POA is attributable to selective Albert Einstein Colleee of Medicine. alterations in a specific cu,receptor subtype, and (2) to identify Correspondence sh&ld be addressed to Dr. A. M. E&en, Department of Psy- chiatry, F113, Albert Einstein College of Medicine, 1300 Morris Park Avenue, which oc,-adrenoceptorsubtype mediates the augmentation of Bronx, NY 1046 1. CAMP formation in hypothalamic and POA slices.The selective Copyright 0 1992 Society for Neuroscience 0270-6474/92/123869-08$05.00/O aYIAreceptor antagonist 5-methyl-urapadil(5-MU; Gross et al., 3870 Petitti et al. * Estradiol increases a,.-Adrenergic Receptors 1988), WB4101, an antagonist with higher affinity for the allA tion was preincubated for 10 min at 37°C with vehicle or with 10 PM than for the LY,~subtype, and CEC, an irreversible, a,,-selective CEC. Reactions were stopped by addition of 6 ml of ice-cold Na-HEPES buffer and centrifugation for 10 min at 20,000 x g. The supematant alkylating agent, were used to distinguish the (Y, receptor sub- was discarded and the pellet resuspended in 6 ml Tris-MgCl, buffer. types, Specific ‘H-prazosin binding after CEC inactivation reflects the (Y,~- Some of these findings have appeared in preliminary form adrenergic receptor population. The LY,~receptor population was deter- (Etgen et al., 1992). mined by subtracting the binding of 3H-prazosin after CEC inactivation from total specific ‘H-prazosin binding. Determinationof sliceCAMP accumulation.Each POA and MH slice Materials and Methods was maintained at 34-35°C in a shaking water bath (80 oscillations/ min) in an individual tissue culture well containing 300 ~1 of a modified Animals and hormonetreatments. Female Sprague-Dawley rats ob- Yamamoto’s medium (Yamamoto, 1972) in an O,/CO, (95:5)-saturated tained from Taconic Farm (Germantown, NY) and weighing 150-l 75 environment. The incubation conditions were identical to those used gm were ovariectomized bilaterally under Metofane anesthesia 4-7 d in our previous work except that NaHCO, was replaced by 10 mM prior to use. Estrogen treatment consisted of two subcutaneous injec- HEPES in the Yamamoto’s medium. Slices were preincubated for 75 tions of 2 pg of E, benzoate (EB) given 24 and 48 hr before death. P min prior to the addition of the NE agonists. CEC and 5-MU were treatment consisted of an injection of 500 fig of P given subcutaneously added directly to the incubation wells 30 min and 10 min, respectively, 3.5 hr before sacrifice. EB and P were dissolved in peanut oil and injected prior to phenylephrine (PHE) and isoproterenol (ISO). At the end of in a volume of 0.1 ml. the 20 min incubation period with the NE agonists, the slices were Tissuepreparation. Animals were killed by decapitation, and their transferred rapidly to 400 ~1 of ice cold 5% (w/v) trichloroacetic acid. brains were rapidly removed and placed on ice. The entire hypothalamus The slices were disrupted by sonication, and the supematant (containing and POA were removed, and 350 pm slices were cut on a McIlwain CAMP) and pellet (containing tissue protein) were separated by cen- tissue chopper beginning approximately 2 mm anterior to the optic trifugation. The pellet was dissolved in 2.0 M NaOH for determination chiasm and ending 1 mm anterior to the mammillary bodies. Based on of protein content. The supernatant was acidified with 1 .O M HCl, and anatomical landmarks observed in comparable slices from fixed tissue, trichloroacetic acid was removed with 4 vol of washed ether. The re- four slices of POA and three slices of middle hypothalamus (MH) were sulting aqueous extracts were concentrated by lyophilization and ana- obtained as described earlier (Etgen and Petitti, 1986, 1987) and used lyzed for CAMP content using a modified Gilman protein binding assay for CAMP assays and for preparation of membranes for some radioli- (Brostrom and Kon, 1974). gand binding assays. The MH slices include the arcuate nucleus, the Chemicals. EB and P were purchased from Steraloids, Inc. (Wilton, ventromedial nucleus, the dorsomedial nucleus, and much of the lateral NH). Metofane