Alpha-2 Adrenergic Receptor Functional Coupling to G Proteins in Rat Brain During Postnatal Development H. KEVIN HAPPE, DAVID B. BYLUND, and L. CHARLES MURRIN

During postnatal development, alpha-2 adrenergic receptors (A2AR) change in both density and distribution. In forebrain, receptor density increases about 4-fold over neonatal levels, reaching adult levels before postnatal day (P) 28, whereas in hindbrain, including cerebellum, there is a decrease in overall receptor density. We examined the coupling of A2AR to G proteins using agonist-stimulated [S]GTPgS binding as a functional assay. In forebrain the A2AR agonist-stimulated [S]GTPgS binding increases rapidly after P7, reaching its highest levels at P21 and then declining slightly to adult levels. This binding increases more slowly than receptor number, suggesting that the appearance of G proteins, rather than the A2AR, determines the developmental appearance of functional A2AR-G protein interactions in forebrain. Basal [S]GTPgS binding and [S]GTPgS binding stimulated by other neurotransmitter receptor systems (GABA-B, mu opiate, and muscarinic) increase with a time course similar to A2AR-stimulated [S]GTPgS binding. In contrast, in hindbrain, A2AR-stimulated [S]GTPgS binding decreases during postnatal development in parallel with the decrease in A2AR levels, whereas [S]GTPgS binding stimulated by other neurotransmitter receptor systems increases in parallel with basal [S]GTPgS binding. Functional receptor-G protein coupling in hindbrain appears to be dependent on the developmental appearance of G proteins for most neurotransmitter systems. However, for A2AR the decrease in receptor density is the overriding factor. These studies 1) demonstrate the functional measurement of A2AR-G protein coupling in native tissue for the first time, 2) demonstrate that A2AR are coupled to G proteins throughout postnatal development, and 3) describe developmental increases and decreases in functional A2AR in brain. Alpha-2 adrenergic receptors (A2AR) are one of the three major classes of receptors for norepinephrine and epinephrine (Bylund, 1988; Bylund et al., 1994). They are widely distributed in the body and play important roles in a variety of physiological and pathological processes, including regulation of blood pressure, nociception, locomotion, and processing of stressful stimuli (Ruffolo et al., 1993, 1995). A2AR have been used to ameliorate withdrawal symptoms from opiates and alcohol, as anesthetic adjuvants in surgery, and may be of some benefit in treating cognitive deficits in the elderly (Ruffolo et al., 1995). These actions and uses point to the importance of understanding the role of A2AR in central nervous system (CNS) function. A2AR are members of the G protein coupled receptor superfamily and appear to interact primarily with Gi/o proteins (Chabre et al., 1994; Limbird et al., 1995). When A2AR are stimulated, GDP is released from the Gi/o protein complex, allowing GTP to bind in its place. This leads to the dissociation of the a from the bg subunits of the heterotrimeric G protein complex and the subsequent regulation of signal transduction systems within the cell. This activation of G proteins is one of the functional consequences of stimulation of A2AR and, as such, provides a measure of the functional activity of these receptors in tissue. Relatively little is known about the development of central A2AR in general, and even less is known about their function during the developmental period. We examined A2AR functional activity during development using the [S]GTPgS binding assay (Hilf et al., 1989; Sim et al., 1995). This approach has been used in the study of several receptors, including A2AR expressed in cultured cells (Tian et al., 1994; Gillison et al., 1997; Wise et al., 1997). We report here the functional linkage of A2AR to G proteins at birth and the subsequent alterations in the magnitude of this coupling during the first postnatal month. Increases in A2AR-agonist induced GTP-binding in forebrain generally parallel the increase in A2AR levels, but there are interesting discrepancies from this. In cerebellum and brainstem, the highest levels of Received for publication June 30, 1998. 1 This work was supported by National Institutes of Health Grant NS 38201. Part of this work has been reported previously in abstract form (Happe et al., 1998). ABBREVIATIONS: A2AR, alpha-2 adrenergic receptors; CNS, central nervous system; carbachol, carbamyl choline; P, postnatal day. 0022-3565/99/2883-1134$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 288, No. 3 Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 288:1134–1142, 1999 1134 at A PE T Jornals on M ay 9, 2017 jpet.asjournals.org D ow nladed from both receptors and functional receptor-G protein coupling are present at birth and decline in parallel to relatively low levels postnatally. Experimental Procedures Materials. [S]GTPgS (1000–1500 Ci/mmol) was obtained from New England Research Products (Boston, MA) and [H]RX 821002 (53 Ci/mmol) from Amersham Life Science (Arlington Heights, IL). Atropine sulfate, carbamyl choline (carbachol), epinephrine bitartrate, dithiothreitol, GTPgS, and baclofen were purchased from Sigma Chemical Company (St. Louis, MO). UK 14,304 was a gift from Allergan Pharmaceuticals (Irvine, CA). Rauwolscine HCl and RX 821002 HCl were purchased from Research Biochemicals Inc. (Natick, MA). Methadone was purchased from Mallinckrodt (St. Louis, MO) and GDP was purchased from United States Biochemical Corp. (Cleveland, OH). All other chemicals were research grade. Animals. Adult female Sprague-Dawley rats, 185 to 250 g, (SASCO, Kingston, NY) were housed three to four per cage and fed ad libitum. Rat pups were bred in our colony. Litters were culled to nine pups and monitored for normal growth by body weight (Happe and Murrin, 1990). Brains were collected at P0 (day of birth), P7, P14, P21, and P28. All animal use procedures were in strict accordance with The National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the local Animal