Neuropeptide FF2 receptor distribution in the human brain. An immunohistochemical study.

Human neuropeptide FF2 (hFF2) receptor has been postulated to mediate central autonomic regulation by virtue of its ability to bind with high affinity to many amidated neuropeptides. In the present immunohistochemical study, we identified hFF2 positive neurons in the forebrain and medulla oblongata of individuals, who died suddenly of mechanical trauma or hypothermia. Morphologically, these neurons demonstrated features identified with both projection neurons and interneurons. In the forebrain, the highest density of hFF2 expressing neurons was observed in the anterior amygdaloid area and dorsomedial hypothalamic nucleus, especially in its caudal part. A lesser density of hFF2 neurons was identified in the ventromedial hypothalamic nucleus, lateral and posterior hypothalamic areas whereas few cells were visualized in the paraventricular hypothalamic nucleus, perifornical nucleus, horizontal limb of the diagonal band, ventral division of the bed nucleus of the stria terminalis, nucleus basalis of Meynert and ventral tegmental area. In the medulla, significant numbers of hFF2 neurons were observed in the dorsal motor nucleus of vagus and to a lesser extent in the area of catecholaminergic cell groups, A1/C1. These data provide first immunohistochemical evidence of hFF2 localization in the human brain, which is consistent with that reported for tissue distribution of FF2 mRNA and FF2 binding sites within the brain of a variety of mammalian species. The distribution of hFF2 may help in identifying the role of amidated neuropeptides in the human brain within the context of central autonomic and neuroendocrine regulation.

Influence of dehydration on the expression of neuropeptide Y Y1 receptors in hypothalamic magnocellular neurons.

Regulation of vasopressin (VP) and oxytocin (OT) secretion involves integration of neural signals from hypothalamic osmoreceptors, ascending catecholaminergic and peptidergic cell groups in the brain stem, and local and autoregulatory afferents. Neuropeptide Y (NPY) is one factor that stimulates the release of VP and OT from the supraoptic (SON) and paraventricular nuclei of the hypothalamus via activation of Y1 receptors (Y1R). The current studies were designed to assess the regulation and distribution of NPY Y1R expression in the SON of male rats that were either given 2% NaCl drinking water (24-72 h) or water deprived (48 h). Subjecting male rats to these conditions resulted in significant increases in both the number of cells expressing Y1R immunoreactivity (ir) and the amount of Y1R protein per cell within the SON. Y1R immunoreactivity was increased in the magnocellular but not medial parvocellular paraventricular nuclei, and Y1R mRNA levels were increased in the SON of salt-loaded rats. Subpopulations of both VP and OT cells in the hypothalamus express Y1R immunoreactivity and a greater percentage of VP-ir cells express Y1R after salt loading. To control for potential effects of dehydration-induced anorexia, a group of euhydrate animals was pair fed with animals consuming 2% NaCl. No detectable change in Y1R expression was observed in the SON of pair-fed animals, even though body weights were significantly lower than controls. These data demonstrate that NPY Y1R gene and protein expression are increased in the SON of salt-loaded and water-deprived animals and provide a mechanism whereby NPY can support VP/OT release during prolonged challenges to fluid homeostasis.

Structure and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 26RFa in vertebrates.

A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.

Targeted disruption of GPR7, the endogenous receptor for neuropeptides B and W, leads to metabolic defects and adult-onset obesity.

Gold-thioglucose (GTG) induces lesions in the ventromedial nucleus of the hypothalamus, resulting in hyperphagia and obesity. To identify genes involved in the hypothalamic regulation of energy homeostasis, we used a screen for genes that are dysregulated in GTG-induced obese mice. We found that GPR7, the endogenous G protein-coupled receptor for the recently identified ligands neuropeptide B and neuropeptide W, was down-regulated in hypothalamus after GTG treatment. Here we show that male GPR7-/- mice develop an adult-onset obese phenotype that progressively worsens with age and was greatly exacerbated when animals are fed a high-fat diet. GPR7-/- male mice were hyperphagic and had decreased energy expenditure and locomotor activity. Plasma levels of glucose, leptin, and insulin were also elevated in these mice. GPR7-/- male mice had decreased hypothalamic neuropeptide Y RNA levels and increased proopiomelanocortin RNA levels, a set of effects opposite to those evident in ob/ob mice. Furthermore, ob/ob GPR7-/- and Ay/a GPR7-/- double mutant male mice had an increased body weight compared with normal ob/ob or Ay/a male mice, suggesting that the obesity of GPR7-/- mice is independent of leptin and melanocortin signaling. Female mice did not show any significant weight increase or associated metabolic defects. These data suggest a potential role for GPR7 and its endogenous ligands, neuropeptide B and neuropeptide W, in regulating energy homeostasis independent of leptin and melanocortin signaling in a sexually dimorphic manner.

Galanin-like peptide is target for regulation by orexin in the rat hypothalamus.

Galanin-like peptide (GALP) is a newly discovered 60 amino acid peptide from the porcine hypothalamus. GALP has been shown to be expressed predominantly in the arcuate nucleus (ARC) of the rat hypothalamus, a region considered to be one of the most important feeding-regulating centers in the brain. GALP-containing neurons in the ARC express leptin receptors, but relationships between GALP and other feeding-regulating neurons have not yet been fully elucidated. Given that Orexin (OX)-containing neurons make synaptic inputs to the ARC, we thus examined the relationship between GALP and OX in the ARC by use of a dual immunostaining technique. OX-immunoreactive fibers appeared to be closely apposed to GALP-immunoreactive cell bodies and their processes. We also examined whether the OX receptor, OX(1)-R was expressed in the GALP-containing neurons. Immunoreactivity for both OX(1)-R and GALP was detectable in 9.6 % neurons (range 4.2-14.6%) in the ARC. These findings strongly suggest that GALP may participate in the regulation of feeding behavior under the influence of leptin and OX.

Orexin receptor-1 (OX-R1) immunoreactivity in chemically identified neurons of the hypothalamus: focus on orexin targets involved in control of food and water intake.

The neuropeptides orexin-A and orexin-B are produced in neurons of the lateral hypothalamic area and have been implicated to be involved in the regulation of food/water intake and sleep-wake control. The orexins act at two different G-protein-coupled orexin receptors (OX-R1 and OX-R2) that are derived from separate genes and expressed differentially throughout the central nervous system. In the present study, we have used a polyclonal antipeptide antiserum to analyse in detail the distribution of OX-R1-immunoreactive neurons in the rat hypothalamus. In order to identify the chemical mediators of orexin action in the hypothalamus, the OX-R1-containing neurons were characterized with regard to the content of peptides shown previously to affect ingestive and drinking behaviour. Neurons containing OX-R1 immunoreactivity were widely distributed in the hypothalamus with cell bodies located in the suprachiasmatic, periventricular, paraventricular (both magno- and parvocellular division), supraoptic, arcuate, ventromedial, dorsomedial and tuberomammillary nuclei and the lateral hypothalamic area. In magnocellular neurons of the paraventricular and supraoptic nuclei, OX-R1 immunoreactivity was seen in both vasopressin- and oxytocin-containing neurons. OX-R1 immunoreactivity was demonstrated in vasopressin and vasoactive intestinal polypeptide (VIP) neurons of the suprachiasmatic nucleus, in somatostatin neurons of the periventricular nucleus and in corticotropin-releasing hormone (CRH) neurons of the parvocellular paraventricular nucleus. In the arcuate nucleus, OX-R1 immunoreactivity was present in neuropeptide Y (NPY) and agouti-related peptide (AGRP) neurons of the ventromedial part as well as in proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) neurons of the ventrolateral division. In the lateral hypothalamic area, OX-R1 immunoreactivity was demonstrated in melanin-concentrating hormone (MCH)- and orexin-containing neurons. In the hypothalamic tuberomammillary nucleus, OX-R1-immunoreactivity was shown in many histamine-containing neurons. The results support the idea that orexins have important actions on hypothalamic neurons that control food intake and fluid balance, but also that orexins may regulate other neuroendocrine systems.

Orexin-A, an hypothalamic peptide with analgesic properties.

The hypothalamic peptide orexin-A and the orexin-1 receptor are localized in areas of the brain and spinal cord associated with nociceptive processing. In the present study, localization was confirmed in the spinal cord and demonstrated in the dorsal root ganglion for both orexin-A and the orexin-1 receptor. The link with nociception was extended when orexin-A was shown to be analgesic when given i.v. but not s.c. in mouse and rat models of nociception and hyperalgesia. The efficacy of orexin-A was similar to that of morphine in the 50 degrees C hotplate test and the carrageenan-induced thermal hyperalgesia test. However, involvement of the opiate system in these effects was ruled out as they were blocked by the orexin-1 receptor antagonist SB-334867 but not naloxone. Orexin-1 receptor antagonists had no effect in acute nociceptive tests but under particular inflammatory conditions were pro-hyperalgesic, suggesting a tonic inhibitory orexin drive in these circumstances. These data demonstrate that the orexinergic system has a potential role in the modulation of nociceptive transmission.

Galanin-R1 and -R2 receptor mRNA expression during the development of rat brain suggests differential subtype involvement in synaptic transmission and plasticity.

The present study employed 35S-labelled oligonucleotides and in situ hybridization to examine the distribution in the developing rat brain of mRNA encoding two galanin receptor subtypes, i.e. Gal-R1 and Gal-R2. Gal-R1 and/or Gal-R2 mRNA was detected at embryonic day (E) 20 and from postnatal day (P) 0-70. Gal-R1 mRNA was highly expressed in olfactory regions, ventral hippocampal CA fields, dorsomedial thalamic areas and many hypothalamic nuclei at all ages studied. In adult brain, Gal-R2 mRNA was most abundant in the dentate gyrus, anterior and posterior hypothalamus, raphe and spinal trigeminal nuclei, and in the dorsal motor nucleus of the vagus. At P0-P7, Gal-R2 mRNA was more widely distributed and abundant than at other ages, with highest levels of expression detected throughout the neocortex and thalamus. Thus, Gal-R2 transcripts had a more restricted distribution than Gal-R1 and were differentially abundant at different ages, while the distribution and relative abundance of Gal-R1 mRNA did not alter substantially during postnatal development. In general, Gal-R1 and -R2 mRNAs were localized in regions previously shown to contain [125I]-galanin binding sites and galanin-positive terminals in adult brain. Galanin-immunostaining was assessed in postnatal brain to determine whether peptide innervation correlated with observed transient receptor expression, but was not particularly enriched in Gal-R2 mRNA-positive areas of P4 or P7 brain. These results, together with earlier findings [e.g. Burazin, T. C. D. & Gundlach, A. L. (1998) J. Neurochem., 71, 879-882], suggest that Gal-R1 receptors have a broad role in normal synaptic transmission, while Gal-R2 receptors, in addition to a similar role in particular pathways, may be involved in processes prominent during the establishment and maturation of synaptic connections in developing brain and during neural damage and repair in the mature nervous system.

Development of a fluorescent ligand-binding assay using the AcroWell filter plate.

One of the most powerful tools for receptor research and drug discovery is the use of receptor-ligand affinity screening of combinatorial libraries. Early work involved the use of radioactive ligands to identify a binding event; however, there are numerous limitations involved in the use of radioactivity for high throughput screening. These limitations have led to the creation of highly sensitive, nonradioactive alternatives to investigate receptor-ligand interactions. Pall Gelman Laboratory has introduced the AcroWell, a patented low-fluorescent-background membrane and sealing process together with a filter plate design that is compatible with robotic systems. Taken together, these allow the AcroWell 96-well filter plate to detect trace quantities of lanthanide-labeled ligands for cell-, bead-, or membrane-based assays using time-resolved fluorescence. Using europium-labeled galanin, we have demonstrated that saturation binding experiments can be performed with low-background fluorescence and signal-to-noise ratios that rival traditional radioisotopic techniques while maintaining biological integrity of the receptor-ligand interaction. In addition, the ability to discriminate between active and inactive compounds in a mock galanin screen is demonstrated with low well-to-well variability, allowing reliable determination of positive hits even for low-affinity interactions.

Characterization of hypothalamic neurons expressing a neuropeptide receptor, GALR2, using combined in situ hybridization-immunohistochemistry.

Our laboratory is interested in characterizing the neurotransmitter and hormonal phenotype of neurons in the rat hypothalamus expressing novel neuropeptide receptors of the neuropeptide Y and galanin families. In this review, we describe a technique combining nonradioactive in situ hybridization to detect mRNA and fluorescence immunohistochemistry to detect protein antigens. We examined paraffin sections of rat hypothalamus using confocal microscopy to determine whether mRNA for the galanin receptor, GALR2, was colocalized at the cellular level of resolution with somatostatin or tyrosine hydroxylase immunoreactivity. We found that many neurons in the hypothalamus expressed both GALR2 mRNA and either somatostatin or tyrosine hydroxylase immunoreactivity. The simultaneous detection of mRNA and protein immunoreactivity in individual neurons using the confocal microscope for visualization is an excellent tool for the analysis of newly characterized genes in the central nervous system.

Expression of the galanin receptor subtype Gal-R2 mRNA in the rat hypothalamus.

The distribution of galanin receptor subtype 2 (Gal-R2) mRNA-expressing cells was examined by in situ hybridization in the rat hypothalamus using a full-length rat 35S-riboprobe. Gal-R2 receptor mRNA-expressing cells were found at moderate to high levels of expression in most nuclei and regions of hypothalamus. The labeling was observed within well-defined anatomical nuclei: preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, dorsomedial, mammillary nuclei. The supraoptic and ventromedial nuclei were almost devoid of labeling. Some scattered labeled cells were also observed in the pituitary. This distribution of Gal-R2 mRNA-expressing cells corresponds well with that of galanin binding sites studies. As compared to the distribution of the galanin receptor subtype 1 (Gal-R1), our results indicate that the Gal-R2 type is differentially distributed, although a significant overlap exists in some regions such the preoptic area, arcuate and dorsomedial nuclei. The functional implications of these results are discussed in light of the role of galanin receptors plays in neuroendocrine regulation and feeding behavior.

Expression of the novel galanin receptor subtype GALR2 in the adult rat CNS: distinct distribution from GALR1.

Recent molecular cloning studies by our laboratory and others have identified the existence of a novel rat galanin receptor subtype, GALR2. In the present study, we examined the regional and cellular distribution of GALR2 mRNA in the rat central nervous system (CNS) by in situ hybridization. For comparative purposes, adjacent sections were probed for GALR1 mRNA expression. Our findings indicate that dorsal root ganglia express by far the highest levels of GALR2 mRNA in the rat CNS. Hybridization signal is mainly concentrated over small and intermediate primary sensory neurons. In spinal cord, the large alpha motoneurons of the ventral horn are moderately labeled and several small, but less intensely labeled, cells are scattered throughout the gray matter. In brain sections, the highest levels of GALR2 mRNA are detected in granule cells of the dentate gyrus, in the mammillary nuclei, and in the cerebellar cortex. Moderate levels of GALR2 mRNA are observed in the olfactory bulb, olfactory tubercle, piriform and retrospinal cortices, hypothalamus (namely the preoptic area, arcuate nucleus, and dorsal hypothalamic area), substantia nigra pars compacta, and sensory trigeminal nucleus. Moderate to weak hybridization signal is also present in several other hypothalamic nuclei, specific layers of the neocortex, periaqueductal gray, and several nuclei within the pons and medulla, including locus coeruleus, lateral parabrachial, motor trigeminal, pontine reticular, hypoglossal, vestibular complex, ambiguus, and facial and lateral reticular nuclei. This novel pattern of GALR2 distribution within the rat CNS differs considerably from that of GALR1, suggesting that specific physiologic effects of galanin may be ascribed to the GALR2 galanin receptor subtype.

The lymnaea cardioexcitatory peptide (LyCEP) receptor: a G-protein-coupled receptor for a novel member of the RFamide neuropeptide family.

A novel G-protein-coupled receptor (GRL106) resembling neuropeptide Y and tachykinin receptors was cloned from the mollusc Lymnaea stagnalis. Application of a peptide extract from the Lymnaea brain to Xenopus oocytes expressing GRL106 activated a calcium-dependent chloride channel. Using this response as a bioassay, we purified the ligand for GRL106, Lymnaea cardioexcitatory peptide (LyCEP), an RFamide-type decapeptide (TPHWRPQGRF-NH2) displaying significant similarity to the Achatina cardioexcitatory peptide (ACEP-1) as well as to the recently identified family of mammalian prolactin-releasing peptides. In the Lymnaea brain, the cells that produce egg-laying hormone are the predominant site of GRL106 gene expression and appear to be innervated by LyCEP-containing fibers. Indeed, LyCEP application transiently hyperpolarizes isolated egg-laying hormone cells. In the Lymnaea pericardium, LyCEP-containing fibers end blindly at the pericardial lumen, and the heart is stimulated by LyCEP in vitro. These data confirm that LyCEP is an RFamide ligand for GRL106.

Molecular cloning and gene expression of growth hormone-releasing peptide receptor in rat tissues.

We cloned a fragment of the rat GH-releasing peptide (GHRP) receptor homologue and examined the tissue distribution of GHRP receptor mRNA in rats. Sequence analysis showed that the open reading frame is well conserved between rat and human with 96% identity in a 364-amino acid overlap. By reverse transcription-polymerase chain reaction we detected GHRP receptor mRNAs in the rat brain including the hypothalamus, anterior pituitary, and renal pelvis in twenty-eight tissues tested. Microdissection revealed that GHRP receptor mRNAs were localized predominantly in the arcuate nucleus and ventromedial hypothalamus.

Galanin receptors in the hippocampus and entorhinal cortex of aged Fischer 344 male rats.

Galanin (GAL) has been proposed to be an inhibitory modulator of cholinergic memory pathways because it acts within the hippocampus to inhibit the release and antagonize the postsynaptic actions of acetylcholine. Here we have used: 1) slice binding and quantitative autoradiography to assess the density and occupancy of GAL receptors; and 2) in situ hybridization histochemistry to assess expression of the GALR1 receptor subtype in the ventral hippocampus of 3-month-old and 21-month-old Fischer 344 male rats. We detected a small but significant (p < or = 0.0003) age-related reduction in 125I-GAL binding-site density in the ventral hippocampus and entorhinal cortex under standard binding conditions. Post-hoc analysis indicated that this reduction with age persisted in the CA1 radiatum and entorhinal cortex following GTP-induced desaturation to unmask pre-existent GAL receptors occupied by endogenous ligand. It was not associated with a significant change in peak GALR1 gene expression in the hippocampus. Because a portion of GAL receptors in this region have been postulated to function as presynaptic auto-receptors on cholinergic fiber terminals, the reduction in GAL binding sites with age may be a consequence of age-related alterations in GAL receptor expression by basal forebrain cholinergic neurons which project to the ventral hippocampus.

Somatotroph function in the neonatal pig.

The objective of this study was to evaluate developmental changes in somatotroph function and related gene expression in neonatal pigs. Male piglets were sacrificed at 1, 7, 14, 21, 28, 35, and 42 d of age (8/age group) for the collection of tissue and blood. Serum concentrations of GH were determined. Quantitations of mRNA were performed for pituitary Pit-1, GH, and GHRH receptor. Cultures of pituitary cells from each pig were stimulated with 0, 0.1, 1, or 10 nM GHRH; 2 mM 8-Br-cAMP; or 100 nM phorbol myristate acetate. Elevated serum concentrations of GH were observed at 1 d of age, followed by a pronounced decrease to basal levels thereafter (P < 0.0001). A mild transient increase in circulating GH occurred at Day 28. In vitro GH secretion was significantly stimulated by secretagogue treatments (P < 0.0001). Age-related declines in in vitro GH secretion were observed regardless of if the cells were stimulated by GHRH or by secretagogues that bypass the GHRH receptor (P < 0.001). Similarly, cellular GH content varied with age (P = 0.01). Levels of pituitary GH mRNA (P = 0.01) and GHRH receptor mRNA (P = 0.0002) decreased with age. The quantity of GHRH receptor mRNA was correlated with GH mRNA levels (r = 0.55, P = 0.02), serum GH concentrations (r = 0.55, P = 0.02), and in vitro GH secretion (r = 0.66, P = 0.001). Pituitary Pit-1 mRNA levels at 7 and 14 d of age were significantly elevated relative to all other sampling times (P = 0.0002). Levels of Pit-1 and GH mRNAs were significantly correlated (r = 0.64, P = 0.003). These results demonstrate a strong developmental regulation of somatotrophic function and related gene expression during the early neonatal period of the pig. Age-related decreases in secretory function may be mediated by concurrent mechanisms relating to the expression of the GHRH receptor and of GH.

Autofeedback suppression of growth hormone (GH) secretion in transgenic mice expressing a human GH reporter targeted by tyrosine hydroxylase 5'-flanking sequences to the hypothalamus.

Transgenic mice expressing a tyrosine hydroxylase-human (h) GH fusion gene in the hypothalamus exhibit a dwarf phenotype. The GH feedback mechanism(s) underlying the growth retardation in these animals was investigated by assessing peptide and messenger RNA (mRNA) levels of the hormones of the hypothalamic-GH-IGF-I axis. Pituitary GH content, hypothalamic GH-releasing hormone (GHRH) and somatostatin (SRIH) content, and serum IGF-I levels were measured by RIA. mRNA levels of hypothalamic GHRH and SRIH and of pituitary GH and the GHRH receptor were measured by Northern blot hybridization. Transgenic mice of both sexes and their wild-type littermates were studied at 2-4 months of age. The pituitary GH content was markedly reduced by 85% in male and by 87% in female transgenic mice compared to that in wild-type controls (P < 0.01 for both). The pituitary GH mRNA content was also decreased by 73% (P = 0.002) in transgenic male mice. Circulating IGF-I levels were significantly reduced by 66% and 68% in male and female transgenic mice, respectively (P = 0.001). The hypothalamic GHRH content was significantly reduced by 19% and 33% (P < 0.05) in male and female transgenic mice, respectively. No significant difference was detected, however, in the hypothalamic SRIH content between wild-type and transgenic mice. Hypothalamic GHRH mRNA levels were significantly decreased by 35% (P = 0.002) in transgenic male mice compared to those in wild-type littermates. In contrast, SRIH mRNA was not significantly changed. An even greater reduction (61%; P = 0.003) was observed in pituitary GHRH receptor mRNA in transgenic mice. These data indicate that the GH deficiency and dwarf phenotype of the tyrosine hydroxylase-hGH transgenic mouse can be attributed primarily to impaired hypothalamic GHRH production. The mechanism of GH feedback inhibition appears to involve direct suppression of GHRH gene expression by locally produced hGH in the hypothalamus.

Differential gene expression of growth hormone (GH)-releasing hormone (GRH) and GRH receptor in various rat tissues.

Growth hormone (GH)-releasing hormone (GRH) acts on specific receptors in the anterior pituitary to stimulate the synthesis and release of GH. Recent reports suggest that GRH is also synthesized in extrahypothalamic tissues. To evaluate the potential roles of extrahypothalamic GRH, we studied the gene expression of GRH and GRH receptors in various rat tissues by reverse transcribed (RT)-polymerase chain reaction (PCR). Total RNA was extracted from twenty-three rat organs and RT-PCR was performed with GRH and GRH receptor primers. Highly-sensitive RT-PCR-Southern blotting showed that GRH and GRH receptor mRNA coexist in the widespread tissues (14 of 25 tissues). GRH mRNA was relatively abundant in the cerebral cortex, brain stem, testis, and placenta, while GRH receptor mRNA was abundant in renal medulla and renal pelvis. Northern blot hybridization using poly A+ RNA indicated that the transcript of GRH receptor gene found in the renal medulla was similar to the longer transcript (about 4 Kb) of pituitary GRH receptor in the size. These results suggest that GRH plays a potential role not only in the neuroendocrine axis, but also in the autocrine and paracrine systems in extrahypothalamic tissues.