Immunoreactive beta-endorphin in a subpopulation of mouse spleen macrophages.

Using radioimmunoassay and immunofluorescence with antibodies to beta-endorphin (beta EP) and ACTH, we have shown that a subpopulation of mouse spleen cells, expressing Mac-1, a marker of macrophage differentiation, contains immunoreactive (ir)-beta EP, ir-ACTH, and smaller amounts of presumptive higher molecular weight forms of both. Neither nonadherent spleen cells, nor adherent or nonadherent cells from peripheral blood, contained detectable levels of these peptides. These findings suggest that beta EP and ACTH may be synthesized in a subpopulation of spleen macrophages, and are consistent with the possibility that these or related peptides may modulate lymphocyte function in the specific microenvironment of the spleen.

N-acetyl endorphin in rat spermatogonia and primary spermatocytes.

In previous reports modest levels of beta-endorphin have been found by radioimmunoassay in rat testis, and localized by immunofluorescence to the interstitial cells. We have confirmed these previous reports and extended them by showing that the majority of testicular endorphins are acetylated forms, N-acetyl gamma-endorphin, N-acetyl alpha-endorphin, and N-acetyl beta-endorphin1-27. In addition, N-acetylated endorphins are not found in interstitial cells, but are confined to spermatogonia and primary spermatocytes.

Pro-opiomelanocortin messenger ribonucleic acid and posttranslational processing of beta endorphin in spleen macrophages.

We have previously demonstrated low levels of immunoreactive (ir)-beta-endorphin (beta-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an approximately 3.5,000-molecular weight (mol wt) species, putatively beta-EP, an approximately 11.5,000-mol-wt species, putatively beta-lipotropin, and a higher molecular weight species (putative beta-EP precursor, pro-opiomelanocortin (POMC). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is beta-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-beta-EP1-16 (alpha-endorphin), N-acetyl-beta-EP1-17 (gamma-endorphin), N-acetyl-beta-EP1-27, and N-acetyl-beta-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species beta-EP1-31.

Attenuated stress response to acute lipopolysaccharide challenge and ethanol administration in vasopressin V1b receptor knockout mice.

The arginine vasopressin (Avp) 1b receptor (Avpr1b) present on anterior pituitary corticotrophs is involved in the stimulation of adrenocorticotrophic hormone (ACTH) secretion, especially during times of stress. Corticotrophin-releasing hormone (CRH) is considered the major ACTH secretagogue during acute stress whereas Avp appears to be the more dominant mediator of the hypothalamic-pituitary-adrenal (HPA) axis response during chronic stress situations. To investigate the role of the Avpr1b in the HPA axis response to acute stress, we measured ACTH and corticosterone (CORT) plasma levels in Avpr1b knockout (KO) mice and wild-type controls in response to bacterial lipopolysaccharide (LPS) challenge and ethanol (EtOH) administration. Mice deficient in Avpr1b had markedly compromised plasma ACTH and CORT responses to acute (30 min) LPS, but normal ACTH and CORT response to more extended exposure (4 h) to the immune system activator. The plasma ACTH and CORT levels stimulated by intoxicating, sedative doses of EtOH (3.2 and 4 g/kg) were significantly decreased in the Avpr1b KO mice compared to wild-type littermates. Significantly higher EtOH-induced plasma ACTH and CORT secretion was measured in female than in male Avpr1b wild-type mice. There were no differences in the blood alcohol levels following acute EtOH administration in Avpr1b KO or wild-type mice of either gender. Our results clearly suggest that Avpr1b plays a significant role in the HPA axis response to acute immune stress and EtOH intoxication.

Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues.

The human APJ receptor is a G protein-coupled receptor which functions as an efficient alternative co-receptor for a number of human immunodeficiency virus type 1 and simian immunodeficiency virus strains. We have cloned the rat APJ receptor, which we term B78/apj, and have mapped the mRNA distribution of both the receptor and its natural ligand apelin in rat tissues. Northern blot analysis showed a similar pattern of expression for B78/apj and apelin mRNAs with hybridising transcripts seen in the lung, heart, skeletal muscle, kidney, brain and liver. In situ hybridisation histochemistry studies revealed intense B78/apj gene expression in the parenchyma of the lung, a sub-population of glomeruli in the kidney, the corpora lutea of the ovary and isolated cells of the anterior lobe of the pituitary. B78/apj mRNA had a striking and unique distribution within the central nervous system (CNS) where receptor expression was found in cells within the meninges around the brain, in the posterior magnocellular and medial parvocellular areas of the hypothalamic paraventricular nucleus and in the supraoptic nucleus. This hypothalamic distribution offers a possible specific role of this receptor in mediating neuroendocrine responses in the CNS.

Comparative distribution of vasopressin V1b and oxytocin receptor messenger ribonucleic acids in brain.

The comparative distributions of the vasopressin V1b receptor (V1bR) and the oxytocin receptor (OTR) messenger RNAs (mRNAs) are described in male rat brain using in situ hybridization histochemistry. V1bR transcripts were present in forebrain and hypothalamus and were less abundant in mid- and hindbrain regions, similar to the gradient observed with OTR transcripts. Microscopic analyses indicated that V1bR expressing cells typically demonstrated the morphology of neurons and confirmed V1bR gene expression in regions including the olfactory bulb, supraoptic, suprachiasmatic, and dorsomedial hypothalamic nuclei, piriform and entorhinal cortices, hippocampus, substantia nigra, and dorsal motor nucleus of the vagus. Most regions that expressed V1bR mRNA also expressed OTR mRNA, although OTR gene expression was much more extensive than that of the V1bR. V1bR and OTR mRNA distributions were distinct from each other and from that of the V1a receptor mRNA in brain. A few brain regions express only V1bR transcripts such as the dorsomedial hypothalamic nucleus and the external plexiform layer of the olfactory bulb. Other brain regions, such as the fields of Ammon's horn, the suprachiasmatic nucleus, the substantia nigra pars compacta, and the piriform cortex express mRNAs that encode all three receptor subtypes (V1a, V1b, and OTR), whereas brain areas including the red nucleus and supraoptic nucleus express V1bR and OTR transcripts only. These data suggest functional specialization of the V1b, OTR and V1a receptors in brain.

The diurnal rhythm in vasopressin V1a receptor expression in the suprachiasmatic nucleus is not dependent on vasopressin.

The mammalian suprachiasmatic nucleus is the site of the circadian rhythm generator. The degrees of expression there of several neuropeptides, including vasopressin and vasoactive intestinal polypeptide, follow a diurnal rhythm. The vasopressin V1a receptor, whose activation results in phosphoinositol hydrolysis and mobilization of intracellular calcium, is expressed in this nucleus. This study used double simultaneous hybridization histochemistry to show that V1a receptor transcripts are present in both vasopressin and vasoactive intestinal polypeptide neurons and that their levels follow a diurnal rhythm. Furthermore, the expression of the V1a receptor is 12 h out of phase from that of vasopressin. However, the receptor's diurnal rhythm is still maintained in the vasopressin-deficient Brattleboro rat, indicating that the V1a receptor's rhythm is independent of any vasopressin feedback.

Induction of oxytocin receptor gene expression in rabbit amnion cells.

Oxytocin (OT)-stimulated PGE2 release by rabbit amnion is enhanced by the up-regulation of oxytocin receptors (OTR), which increase about 200-fold at the end of pregnancy. As recent studies have shown that PGs are essential for parturition, the rise in amnion OTR and associated PGE2 synthesis are probably essential for labor initiation. The present work was directed toward understanding the mechanisms of OTR up-regulation. Levels of agents that stimulate adenylyl cyclase activity and cortisol are increased in amniotic fluid at the end of pregnancy. Addition of either forskolin or cortisol to cultured amnion cells caused an increase in OTR ligand-binding sites and steady state OTR messenger RNA (mRNA) levels. Forskolin treatment elevated OTR mRNA levels rapidly, but transiently, whereas cortisol's effects were slower and sustained. Actinomycin or cycloheximide, added 3 h after forskolin, led to a sustained elevation in OTR mRNA levels, suggesting that forskolin increases the activities of OTR mRNA-destabilizing factors along with increasing OTR mRNA concentration. Cortisol did not appear to affect OTR mRNA stability. Measurement of OTR mRNA transcription rates showed that forskolin's effects were maximal within 1 h of treatment. In contrast, cortisol-induced transcription was not apparent until 8 h. The effects of forskolin and cortisol on OTR gene transcription were synergistic. Thus, the increase in OTR mRNA levels occurring after either forskolin or cortisol treatments is the result of induction of OTR gene expression, but the effects of the two agents appear to occur at separate sites.

Immunoreactive arginine vasopressin in the rat thymus.

Immunoreactive arginine vasopressin (ir-AVP), coeluting with authentic nonapeptide on reverse phase HPLC, is present in the thymus of Sprague-Dawley, Long-Evans, and homozygous (di/di) Brattleboro rats, and BALB/c mice. By immunohistochemistry, ir-AVP positive cells are sparse, and do not appear to be lymphocytes. Adrenalectomy and dexamethasone administration to intact rats produces an identical response in terms of thymic ir-AVP, with a rise after 1-2 days followed by a fall to levels below baseline after 8 days. The rise 2 days after adrenalectomy, and the fall 8 days later, were both prevented by administration of aldosterone, but not by corticosterone or dexamethasone to adrenalectomized animals. The role(s) of thymic ir-AVP, and the physiological significance of its mineralocorticoid dependence, remain to be established.

Immunohistochemical localization of the vasopressin V1b receptor in the rat brain and pituitary gland: anatomical support for its involvement in the central effects of vasopressin.

Biological effects of vasopressin (VP) are mediated by four different receptors, two of which (the V1a and the oxytocin receptors) have been well characterized in the rodent brain, suggesting that these are the main receptors responsible for the central effects of VP. However, transcripts of the V1b VP receptor (V1bR) have been detected throughout the rat brain by RT-PCR and in situ hybridization, indicating that the V1bR adds to the population of central VP receptors. Because there are no specific ligands for the V1bR, the receptor protein itself has been difficult to visualize. In the present study, the distribution of the V1bR protein was investigated in the rat forebrain, midbrain, hindbrain, and cerebellum by immunohistochemistry using an antiserum raised against a synthetic fragment of the carboxylterminal of the rat V1bR protein. Immunohistochemistry revealed the presence of the V1bR in pituitary corticotrophs as expected. In naive, untreated rats, fiber networks containing V1bR-immunoreactivity were mainly concentrated in the hypothalamus, amygdala, cerebellum, and particularly in those areas with a leaky blood brain barrier or close to the circumventricular organs (medial habenula, subfornical organ, organum vasculosum laminae terminalis, median eminence, and nuclei lining to the third and fourth ventricles). A strikingly dense network was present in the external zone of the median eminence. Colchicine treatment was required to reveal the localization of V1bR-immunoreactive cell bodies. V1bR-containing cell bodies and associated protrusions were mainly located in the hippocampus, caudate putamen, cortex, thalamus, olfactory bulb, and cerebellum. These results demonstrate the widespread distribution of the V1bR protein in the rat brain over multiple, functionally distinct neuronal systems. These data suggest that the V1bR mediates different physiological functions of VP in the brain.

Estrogen increases renal oxytocin receptor gene expression.

Estrogens have been implicated in the sodium and fluid imbalances associated with the menstrual cycle and late pregnancy. An estrogen-dependent role for renal oxytocin receptors in fluid homeostasis is suggested by the present findings which demonstrate that estradiol benzoate treatment increases the expression of the oxytocin receptor messenger ribonucleic acid and 125I-OTA binding to oxytocin receptors in the renal cortex and medullary collecting ducts of ovariectomized female rats. Moreover, estradiol induced high levels of oxytocin receptor expression in outer stripe proximal tubules of ovariectomized female and adrenalectomized male rats. Proximal tubule induction was inhibited in a dose-dependent manner by the antiestrogen tamoxifen, but cortical expression of oxytocin receptors in macula densa cells was unaffected by tamoxifen. These data demonstrate cell-specific regulation of oxytocin receptor expression in macula densa and proximal tubule cells, and suggest a important role for these receptors in mediating estrogen-induced alterations in renal fluid dynamics by possibly affecting glomerular filtration and water and solute reabsorption during high estrogen states.

Hypothalamic-pituitary corticotroph function after shunting of magnocellular vasopressin and oxytocin to the hypophyseal portal circulation.

Surgical pituitary stalk compression (PSC) causes neural lobe denervation and development of ectopic magnocellular terminals in the external zone of the median eminence, resulting in shunting of magnocellular vasopressin (VP) and oxytocin (OT) to the pituitary portal circulation. To determine the effects of PSC on hypothalamic and pituitary function, VP and CRH receptors and POMC messenger RNA (mRNA) in the pituitary, and CRH, VP, and OT mRNA levels in the PVN were examined in 7-day PSC and sham-operated rats. Immunohistochemical staining revealed marked accumulation of immunoreactive VP in the swollen pituitary stalk and the external zone of the distal median eminence of PSC rats. Plasma ACTH and corticosterone levels were significantly increased by PSC, an effect that was attenuated by minipump infusion of desamino-[D-Arg8]-vasopressin (DDAVP) given to correct the diabetes insipidus. [3H]VP binding to anterior pituitary membranes from PSC rats was reduced by 50% compared to that in sham-operated controls, but VP V1b receptor mRNA levels measured by in situ hybridization were unchanged. Pituitary CRH receptors measured by binding autoradiography and CRH receptor mRNA levels did not change after PSC. POMC mRNA was unchanged in the anterior pituitary lobe, but markedly increased in the intermediate lobe of PSC rats, with or without DDAVP infusion. In the hypothalamic supraoptic and paraventricular nuclei, PSC resulted in reduction of VP mRNA (-83%) and OT mRNA (-38%) levels, probably reflecting retrograde degeneration of magnocellular neurons. This decrease was more pronounced for VP mRNA than for OT mRNA CRH mRNA levels in parvicellular cells of the paraventricular nuclei of PSC rats were reduced by 55%. Correction of the diabetes insipidus by DDAVP treatment further decreased hypothalamic VP mRNA levels, but restored CRH mRNA to control levels. The data show that continuous exposure of the pituitary to high VP levels from ectopic magnocellular fibers in the median eminence results in VP, but not CRH, receptor loss. Pituitary VP receptor down-regulation and decreased hypothalamic CRH expression are likely to contribute to the reduced corticotroph responsiveness to VP reported in this experimental model.

Cellular localization of vasopressin V1a receptor messenger ribonucleic acid in adult male rat brain, pineal, and brain vasculature.

Vasopressin V1a receptor (V1aR) transcripts were localized in brain, pineal, and superficial brain vascular tissues of adult male rats using hybridization histochemistry and an [35S]riboprobe complementary to the messenger ribonucleic acid (mRNA) encoding the fifth to the midseventh transmembrane regions of the receptor. V1aR mRNA was extensively distributed throughout brain and was expressed in 1) superficial cells of the granule cell layers of the main olfactory bulb, hippocampal dentate gyrus, and cerebellum; 2) numerous anatomically distinct brain nuclei; 3) isolated cells dispersed throughout the central nervous system; 4) cells of the choroid plexus, occasional blood vessels in the olfactory bulb and interpeduncular nucleus, and extraparenchymal intracranial vasculature; and 5) some white matter structures. Numerous cells expressing V1aR transcripts were found in forebrain structures, including primary olfactory (piriform) cortex, the anterior and posterior olfactory nuclei; dorsal, intermediate, and ventral lateral septal nuclei; the septo-fimbrial nucleus and accumbens nucleus; and numerous hypothalamic regions with the most intense hypothalamic labeling in the arcuate, stigmoid, suprachiasmatic, and periventricular nuclei and the lateral hypothalamic area. Cells expressing V1aR transcripts were ubiquitous throughout the midbrain, pontine, and medullary regions. A lower intensity signal was found in cells of the parvocellular paraventricular and anteroventral nucleus of the thalamus, circumventricular organs including the pineal, and the subfornical organ. V1aR transcripts were not generally detected in parenchymal vasculature, but could be found over large blood vessels in the interpeduncular nucleus and medial olfactory bulb; transcripts were commonly detected in perivascular brain cells. V1aR mRNA was abundantly expressed by choroid plexus, endothelial cells of midline blood vessels between the main olfactory bulbs, and superficial vascular tissue on all brain surfaces. These data confirm the presence of the vascular/hepatic-type V1aR gene in brain tissue and document an extensive expression. The distribution of V1aR mRNA suggests that there are at least two types of vasopressin-responsive cells in brain: one type exemplified by lateral septal ara neurons innervated by classical axodendritic/somatic synaptic vasopressinergic terminals and a second, perivascular/vascular type that would facilitate humoral vasopressinergic signaling in the brain.

Expression of vasopressin V1a and V2 receptor messenger ribonucleic acid in the liver and kidney of embryonic, developing, and adult rats.

The ontogenic expression of mRNAs encoding the V1a and V2 vasopressin receptors (V1aR and V2R) was visualized in liver and kidney of embryonic, developing, and adult rats using in situ hybridization histochemistry. Transcripts were detected at 16 and 19 days gestational age in kidney, with V1aR mRNA predominating in the developing cortex and V2R in the medulla. V1aR transcripts in 1-day-old kidneys were in vascular elements, in cells of developing medullary collecting ducts, and over mesangial cells of deep glomeruli, consistent with a role for the V1aR in regulating cellular growth. Expression of V1aR mRNA in the adult was found mainly in medullary vascular elements, arcuate and interlobular arteries, short segments of the cortical distal tubule, and transitional epithelium and smooth muscle of the pelvic wall and ureter. V2R mRNA, at 16 and 19 days gestational age, was in cells of developing medullary and cortical collecting ducts and, after birth, in cells of differentiating thick limbs of the loops of Henle, papillary surface epithelium, overlying macula densa, and short distal nephron segments. This distribution is in accord with the known role of V2 receptors in regulating water excretion. In contrast to kidney, liver did not express V2R mRNA and expressed V1aR transcripts only after birth. V1aR mRNA labeling was over cells bordering central veins on day 1 and surrounding central veins by day 5. A gradient was maximal on postnatal day 21, with V1aR mRNA most abundant in hepatocytes surrounding central veins and virtually absent in periportal hepatocytes. By day 60, most hepatocytes expressed V1aR transcripts, and the gradient was reduced. The ontogenic expression and receptor mRNA gradient are consistent with a role for hepatic V1a receptors in glucoregulation. These experiments confirm the presence of both V1a and V2 receptors in kidney and show that vasopressin receptor mRNA expression is developmentally regulated and tissue specific.

Distribution of V1a and V2 vasopressin receptor messenger ribonucleic acids in rat liver, kidney, pituitary and brain.

The hepatic, vascular-type (V1aR) and the renal, antidiuretic-type (V2R) vasopressin receptor cDNAs were recently cloned from rat liver and kidney libraries, respectively. DNA fragments containing the region encoding the putative 5/6 transmembrane loops of these receptors were subcloned, separately, into RNA polymerase promoter-containing vectors from which 35S-labeled sense and antisense riboprobes were synthesized. In situ hybridization histochemistry showed high levels of V1aR transcripts in the liver and the renal medulla among the vascular bundles. Sparser labeling was found in the renal cortex, but there were no grains over the glomeruli. V1aR mRNA was detected in many brain areas, including the hippocampal formation, central amygdala, dorsolateral septum, lateral hypothalamus, suprachiasmatic, ventromedial, dorsomedial, and arcuate nuclei of the hypothalamus, nucleus of the solitary tract, cerebellum, spinal nucleus of the trigeminal tract, reticular formation, inferior olivary nucleus, and choroid plexus. Rare labeled cells were seen along the periphery of the posterior pituitary. V2R transcripts were not detected in the liver or brain, but were present in high amounts in the inner and outer renal medulla, primarily associated with collecting ducts. Sparser labeling was found in the renal cortex, and no grains were seen over the glomeruli. These data confirm the expression of the V1a vasopressin receptor in liver and brain and demonstrate that kidney expresses mRNAs encoding V1a and V2 vasopressin receptors.

Ovarian immunoreactive beta-endorphin and estrous cycle in the rat.

Adult female Sprague-Dawley rats were killed at different stages of a 4-day estrous cycle, and ovaries and anterior pituitaries examined for content of immunoreactive beta-endorphin by RIA and for localization by indirect immunofluorescence. Two anti-beta-endorphin antisera, both recognizing different antigenic determinants of human-beta-endorphin, showed intense immunofluorescence staining of cells localized predominantly in ovarian corpora lutea. At proestrus, both large and small luteal cells stained positively but only the large luteal cells were immunofluorescence positive at other stages of the estrous cycle. In addition, less intense staining of granulosa cells was occasionally observed in secondary and antral follicles; scattered cells in the interstitium were also weakly positive. In contrast, cells of primordial and primary follicles, and those of theca tissue were consistently negative. Ovarian levels of immunoreactive beta-endorphin were found to be lowest at estrus (2.1 +/- 0.18 ng/g; n = 8, mean +/- SE) and significantly raised in stepwise manner over metestrus and diestrus to a peak (approximately 4 X estrous levels) at proestrus; in contrast, immunoreactive beta-endorphin content of anterior pituitaries remained unaltered during the same period. Sephadex G-50 gel chromatography of ovarian extracts revealed three distinct peaks of immunoreactive beta-endorphin, a minor peak in the void volume, and two major peaks of unequal size eluting at mol wt approximately 11.5K and approximately 3.5K. The major species of low molecular weight immunoreactive beta-endorphin on reverse phase HPLC was beta-endorphin1-31. We conclude from the findings that, in adult rat ovaries, luteal, granulosa, and interstitial cells are responsible for the production of immunoreactive beta-endorphin and that this production, being related to the estrous cycle, is presumably under the direct or indirect influence of gonadotropins.

Pharmacological characterization and region-specific expression in brain of the beta 2- and beta 3-subunits of the rat GABAA receptor.

The cDNA for a third beta-subunit of the rat GABAA receptor has been cloned using another beta-subunit, which we had previously cloned [(1989) FEBS Lett. 246, 145-148], as a probe. The approximately 8-kb cDNA for this beta-subunit (termed beta 2) encodes a protein of 474 amino acid residues that shares approximately 80% sequence identity with the rat and bovine beta 1- and beta 3-subunits. Coexpression of the cloned beta-subunit cDNA with the alpha 1-subunit cDNA of the rat GABAA receptor in Xenopus oocytes produced a functional receptor and Cl- channel with pharmacological characteristics of a GABAA receptor. In contrast to interchanging alpha-subunits [(1988) Nature 335, 76-79], exchange of beta 2- or beta 3-subunits in an alpha 1/beta receptor complex did not markedly alter the pharmacological properties of expressed receptors. In situ hybridization histochemistry with synthetic subunit-specific oligo-deoxynucleotide probes revealed a region-specific expression of alpha 1-, beta 2- and beta 3-subunit mRNAs in the rat central nervous system. These observations provide an additional molecular basis for the functional heterogeneity in the GABAA receptor complex.

Multiple GABAA receptor alpha subunit mRNAs revealed by developmental and regional expression in rat, chicken and human brain.

GABAA receptor alpha subunit transcripts were detected by Northern analysis of rat, chicken and human brain mRNA using a series of 32P-labelled antisense RNA probes derived from human alpha 1 subunit cDNAs. These alpha subunit mRNAs differ in their distribution among various brain regions in the rat and at least one species is detected primarily in fetal brain. GABAA receptor alpha 1 subunit probes encoding the putative extracellular domain detect at least five alpha subunit transcripts in rat brain, whereas probes encoding the putative intracellular domain detect only two mRNAs. These data suggest the presence in brain of multiple GABAA receptor alpha subunits having homologous extracellular domains and whose expression is regionally and developmentally regulated. These alpha subunit transcripts may encode proteins that comprise GABAA isoreceptors differing in their pharmacological and physiological properties.

Cloning and expression of a novel rat GABAA receptor.

Two full-length cDNA clones encoding alpha- and beta-subunits of a GABAA receptor have been isolated from a rat cerebral cortex cDNA library. The mature alpha-subunit protein consists of 428 amino acids with a calculated Mr of 48,680. This protein is highly homologous (approximately 99% amino acid identity) with the bovine brain alpha 1-subunit receptor [(1988) Nature 335, 76-79]. The mature rat beta-subunit receptor is a 448 amino acid polypeptide and shares approximately 80% amino acid identity with the previously characterized bovine GABAA receptor beta-subunit [(1987) Nature 328, 221-227]. Co-expression of the cloned DNA in Xenopus oocytes produces a functional receptor and ion channel with pharmacological characteristics of a GABAA receptor. GABAA alpha- and beta-subunit mRNA is detectable in the cortex, cerebellum and hippocampus.

Localization of cannabinoid receptor mRNA in rat brain.

Cannabinoid receptor mRNA was localized in adult rat brain by 35S-tailed oligonucleotide probes and in situ hybridization histochemistry. Labelling is described as uniform or non-uniform depending on the relative intensities of individual cells expressing cannabinoid receptor mRNA within a given region or nucleus. Uniform labelling was found in the hypothalamus, thalamus, basal ganglia, cerebellum and brainstem. Non-uniform labelling that resulted from the presence of cells displaying two easily distinguishable intensities of hybridization signals was observed in several regions and nuclei in the forebrain (cerebral cortex, hippocampus, amygdala, certain olfactory structures). Olfactory-associated structures, basal ganglia, hippocampus, and cerebellar cortex displayed the heaviest amounts of labelling. Many regions that displayed cannabinoid receptor mRNA could reasonably be identified as sources for cannabinoid receptors on the basis of well documented hodologic data. Other sites that were also clearly labelled could not be assigned as logical sources of cannabinoid receptors. The localization of cannabinoid receptor mRNA indicates that sensory, motor, cognitive, limbic, and autonomic systems should all be influenced by the activation of this receptor by either exogenous cannabimimetics, including marijuana, or the yet unknown endogenous "cannabinoid" ligand.