Distribution of secretin receptors in the rat central nervous system: an in situ hybridization study.

Secretin shows a wide distribution in the brain. Functional significance of central secretin is stressed since it has been associated with autism and schizophrenia. The presence of the secretin receptor was previously demonstrated in the brain by different methods. Neurons in the cerebellum, hypothalamic paraventricular and supraoptic nuclei, and in the vascular organ of lamina terminalis were shown to express secretin receptor mRNA by using in situ hybridization with digoxigenin-labeled probe. In this work, we used a very sensitive radioactive in situ hybridization technique and systematically mapped the expression of secretin receptor mRNA in the brain. The densest labeling was observed in the nucleus of solitary tract and in the laterodorsal thalamic nucleus, where decreasing number of receptors was seen in the vascular organ of lamina terminalis, and the lateral habenular complex, and then in the supraoptic nucleus. Only a few scattered labeled cells were observed in the median frontal gyrus, entorhinal cortex, hypothalamic paraventricular nucleus, perifornical region, lateral hypothalamic area, head of the caudate nucleus, spinal trigeminal nucleus, and cerebellum. Secretin receptor mRNA showed a far wider distribution than was known before, suggesting a more significant functional relevance than thought earlier.

Intranasal application of secretin, similarly to intracerebroventricular administration, influences the motor behavior of mice probably through specific receptors.

Secretin and its receptors show wide distribution in the central nervous system. It was demonstrated previously that intravenous (i.v.) and intracerebroventricular (i.c.v.) application of secretin influenced the behavior of rat, mouse, and human. In our previous experiment, we used a special animal model, Japanese waltzing mice (JWM). These animals run around without stopping (the ambulation distance is very limited) and they do not bother with their environment. The i.c.v. secretin attenuated this hyperactive repetitive movement. In the present work, the effect of i.c.v. and intranasal (i.n.) application of secretin was compared. We have also looked for the presence of secretin receptors in the brain structures related to motor functions. Two micrograms of i.c.v. secretin improved the horizontal movement of JWM, enhancing the ambulation distance. It was nearly threefold higher in treated than in control animals. The i.n. application of secretin to the left nostril once or twice a day or once for 3 days more effectively enhanced the ambulation distance than i.c.v. administration. When secretin was given twice a day for 3 days it had no effect. Secretin did not improve the explorative behavior (the rearing), of JWM. With the use of in situ hybridization, we have found very dense secretin receptor labeling in the cerebellum. In the primary motor cortex and in the striatum, only a few labeled cells were seen. It was supposed that secretin exerted its effect through specific receptors, mainly present in the cerebellum.

[Structure and function of incretin receptor].

G protein-coupled receptor (GPCR) is integral membrane protein with seven alfa-helices and most diverse families of protein in mammals. It is located on cell membrane and activated by binding neurotransmit proteins and hormones. It has critical role of functional regulation in central nerve system and peripheral organs. Recently, many orphan GPCRs have been identified from the data of genomic sequence in human genomic project. GIP receptor and GLP-1 receptor belong to glucagon receptor subfamily of class B and are widely expressed in many organs. GIP receptor is expressed in intestine, adipose tissue, brain, adrenal gland, and bone, while GLP-1 receptor is expressed in intestine, CNS, lung, kidney and heart. GIP and GLP-1 have not only pancreatic effect, such as potentiation of insulin secretion but also many extrapancreatic effects.

Expression of receptors for luteinizing hormone, gastric-inhibitory polypeptide, and vasopressin in normal adrenal glands and cortisol-secreting adrenocortical tumors in dogs.

Hypercortisolism caused by an adrenocortical tumor (AT) results from adrenocorticotropic hormone (ACTH)-independent hypersecretion of glucocorticoids. Studies in humans demonstrate that steroidogenesis in ATs may be stimulated by ectopic or overexpressed eutopic G protein-coupled receptors. We report on a screening of 23 surgically removed, cortisol-secreting ATs for the expression of receptors for luteinizing hormone (LH), gastric-inhibitory polypeptide (GIP), and vasopressin (V(1a), V(1b), and V(2)). Normal adrenal glands served as control tissues. Abundance of mRNA for these receptors was quantified using quantitative polymerase chain reaction (QPCR), and the presence and localization of these receptors were determined by immunohistochemistry. In both normal adrenal glands and ATs, mRNA encoding for all receptors was present, although the expression abundance of the V(1b) receptor was very low. The mRNA expression abundance for GIP and V(2) receptors in ATs were significantly lower (0.03 and 0.01, respectively) than in normal adrenal glands. The zona fasciculata of normal adrenal glands stained immunonegative for the GIP receptor. In contrast, islands of GIP receptor-immunopositive cells were detected in about half of the ATs. The zona fasciculata of both normal adrenal glands and AT tissue were immunopositive for LH receptor; in ATs in a homogenous or heterogenous pattern. In normal adrenal glands, no immunolabeling for V(1b)R and V(2) receptor was present, but in ATs, V(2) receptor-immunopositive cells were detected. In conclusion, QPCR analysis did not reveal overexpression of LH, GIP, V(1a), V(1b), or V(2) receptors in the ATs. However, the ectopic expression of GIP and V(2) receptor proteins in tumorous zona fasciculata tissue may play a role in the pathogenesis of canine cortisol-secreting ATs.

Regulation of aldosterone secretion by several aberrant receptors including for glucose-dependent insulinotropic peptide in a patient with an aldosteronoma.

CONTEXT: Primary adrenal Cushing's syndrome can result from the aberrant adrenal expression of several hormone receptors; this mechanism has not been explored in detail in aldosterone-producing tumors. OBJECTIVE: The objective of the study was to evaluate a 56-yr-old male patient with an aldosteronoma for the regulation of aldosterone secretion by aberrant hormone receptors. RESULTS: Renin-independent stimulation of aldosterone secretion was observed in vivo after a mixed meal, oral glucose, or administration of glucose-dependent insulinotropic peptide (GIP), vasopressin, and tegaserod. The mixed meal-mediated stimulation of aldosterone was not present in five other cases of aldosteronoma. A smaller response of aldosterone after GIP infusion was observed in a normal subject. Aldosterone secretion was stimulated by GIP in primary cultures of this patient's aldosteronoma. Increased expression of GIP receptor was found in this aldosteronoma by real-time RT-PCR and immunohistochemistry. The GIP receptor protein was also found at lower levels in zona glomerulosa cells of the normal adjacent adrenal gland. Increased expression of serotonin 4 and ACTH receptors was also present in this aldosteronoma. CONCLUSIONS: This case report provides new evidence of the implication of aberrant hormone receptors in the regulation of this aldosteronoma and suggests that further detailed studies of the role of aberrant hormone receptors in this frequent pathology should be undertaken.

Molecular identification and characterization of the dog motilin receptor.

Motilin, a 22-amino acid peptide hormone secreted by endocrine cells of the intestinal mucosa, plays an important role in the regulation of gastrointestinal motility. The actions of motilin agonists have been extensively investigated in dogs due to physiological similarities between the dog and human alimentary tracts. The amino acid sequence of the dog motilin receptor, however, was previously unknown. We have cloned a cDNA from dog stomach corresponding to the motilin receptor. The deduced protein shared 71% and 72% sequence identity with the human and rabbit motilin receptors, respectively. Expression of the dog motilin receptor in CHO cells promoted the typical cellular responses to the agonists, motilin and erythromycin. The rank order of potency determined for these agonists was similar to that found for the human motilin receptor, with motilin being more potent than erythromycin. Immunohistochemistry of the dog stomach revealed that the motilin receptor was localized in neuronal cell bodies and fibers. This is the first study detailing the cloning, expression, and functional characterization of the dog motilin receptor. Determination of the full sequence and functional properties of the dog motilin receptor will provide useful information enabling us to interpret previous and future studies of motilin agonists in dogs.

Food-dependent Cushing's syndrome: from molecular characterization to therapeutical results.

OBJECTIVE: Cortisol secretion in ACTH-independent macronodular adrenal hyperplasia (AIMAH) may be regulated by the aberrant expression of several G-protein-coupled receptors. Bilateral adrenalectomy is the treatment of choice in most cases. We searched for aberrant receptor expression in a patient with AIMAH and evaluated the response to medical and surgical treatment. PATIENT: A 35-year-old woman with amenorrhea, hirsutism, and hypertension presented ACTH-independent cortisol secretion with high androgen levels. Abdominal computed tomography showed bilateral adrenal macronodules (4.5 cm right and 1.0 cm left). Scintigraphy with I(131)-norcholesterol showed bilateral uptake, prevalent on the right side. Several in vivo stimulation tests were assessed before and after treatment and in vitro studies were performed after unilateral adrenalectomy. RESULTS: Plasma cortisol increased after a standard meal test (60%) and oral glucose loading (147%), and the response was blunted by pretreatment with 100 microg s.c. octreotide. The therapy with long-acting release octreotide (octreotide-LAR) showed an improvement in urinary free cortisol (UFC) levels. Unilateral adrenalectomy was performed and histopathology revealed macronodular AIMAH. Cortisol and androgens increased after perifusion of tumoral tissue with glucose-dependent insulinotropic polypeptide (GIP), and GIP and LH-receptor overexpression was found in both the adrenal nodules and the adjacent cortex. After surgery, UFC and androgen levels normalized followed by clinical improvement. CONCLUSIONS: GIP and LH-receptor expression may coexist in AIMAH, influencing the functional and morphological phenotype. Aberrant hormone receptor expression enables specific pharmacological treatment, but long-term studies are needed to evaluate its real efficacy. Unilateral adrenalectomy may be a safe initial option, particularly for asymmetric bilateral adrenal enlargements.

Secretin receptor oligomers form intracellularly during maturation through receptor core domains.

Oligomerization of numerous G protein-coupled receptors has been documented, including the prototypic family B secretin receptor. The clinical significance of oligomerization of this receptor became clear with the recent observation that a misspliced form present in pancreatic cancer could associate with the wild-type receptor and act as a dominant negative inhibitor of its normal growth inhibitory function. Our goal was to explore the molecular mechanism of this interaction using bioluminescence (BRET) and fluorescence (FRET) resonance energy transfer and fluorescence microscopy with a variety of receptor constructs tagged with luciferase or cyan or yellow fluorescent proteins. BRET signals comparable to those obtained from cells coexpressing differentially tagged wild-type receptors were observed for similarly tagged secretin receptors in which all or part of the amino-terminal domain was deleted. As expected, neither of these constructs bound secretin, and only the partially truncated construct sorted to the plasma membrane. Receptors lacking the majority of the carboxyl-terminal domain, including that important for phosphorylation-mediated desensitization, also produced BRET signals above background. These findings suggested that the receptor's membrane-spanning core is responsible for secretin receptor oligomerization. Interestingly, alanine substitutions for a -GxxxG- helix interaction motif in transmembrane segment 7 created nonfunctional receptors that were capable of forming oligomers. Furthermore, treatment of receptor-expressing cells with brefeldin A did not eliminate the BRET signals, and morphologic FRET experiments confirmed the expected subcellular localizations of receptor oligomers. We conclude that secretin receptor oligomerization occurs through -GxxxG- motif-independent interactions of transmembrane segments during the maturation of nascent molecules.

Gastrointestinal growth factors and neoplasia.

Gastrointestinal (GI) hormones are chemical messengers that have been recognized for over a century as regulatory factors for normal physiologic functions in the GI tract and pancreas, including absorption, secretion, motility, and digestion. These hormones traditionally act in a true endocrine fashion with release from a distant site to regulate physiologic functions of specific target organs. In general, GI hormones bind to their G-protein-coupled receptors (GPCRs) to produce their endocrine effects. In addition to effects on physiologic functions of the GI tract and pancreas, selected GI hormones can act in an endocrine, paracrine, and/or autocrine fashion to stimulate the proliferation of normal and neoplastic GI tissues as well as non-GI tissues. This review will focus on effects of GI hormones on neoplastic tissues concentrating on the hormones that have been best characterized for these effects.

A novel, long-acting agonist of glucose-dependent insulinotropic polypeptide suitable for once-daily administration in type 2 diabetes.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone with a potentially therapeutic role in type 2 diabetes. Rapid degradation by dipeptidylpeptidase IV has prompted the development of enzyme-resistant N-terminally modified analogs, but renal clearance still limits in vivo bioactivity. In this study, we report long-term antidiabetic effects of a novel, N-terminally protected, fatty acid-derivatized analog of GIP, N-AcGIP(LysPAL(37)), in obese diabetic (ob/ob) mice. Once-daily injections of N-AcGIP(LysPAL(37)) over a 14-day period significantly decreased plasma glucose, glycated hemoglobin, and improved glucose tolerance compared with ob/ob mice treated with saline or native GIP. Plasma insulin and pancreatic insulin content were significantly increased by N-AcGIP(LysPAL(37)). This was accompanied by a significant enhancement in the insulin response to glucose together with a notable improvement of insulin sensitivity. No evidence was found for GIP receptor desensitization and the metabolic effects of N-AcGIP(LysPAL(37)) were independent of any change in feeding or body weight. Similar daily injections of native GIP did not affect any of the parameters measured. These data demonstrate the ability of once-daily injections of N-terminally modified, fatty acid-derivatized analogs of GIP, such as N-AcGIP(LysPAL(37)), to improve diabetes control and to offer a new class of agents for the treatment of type 2 diabetes.

Glucose-dependent insulinotropic peptide stimulates thymidine incorporation in endothelial cells: role of endothelin-1.

We have previously characterized the receptor for glucose-dependent insulinotropic polypeptide (GIPR) in vascular endothelial cells (EC). Different EC types were found to contain distinct GIPR splice variants. To determine whether activation of the GIPR splice variants resulted in different cellular responses, we examined GIP effects on human umbilical vein endothelial cells (HUVEC), which contain two GIPR splice variants, and compared them with a spontaneously transformed human umbilical vein EC line, ECV 304, which contains four GIPR splice variants. GIP dose-dependently stimulated HUVEC and ECV 304 proliferation as measured by [3H]thymidine incorporation. GIP increased endothelin-1 (ET-1) secretion from HUVEC but not from ECV 304. Use of the endothelin B receptor blocker BQ-788 resulted in an inhibition of [3H]thymidine incorporation in HUVEC but not in ECV 304. These findings suggest that, although GIP increases [3H]thymidine incorporation in both HUVEC and ECV 304, this proliferative response is mediated by ET-1 only in HUVEC. These differences in cellular response to GIP may be related to differences in activation of GIPR splice variants.

In vitro autoradiographic localization of (125)i-secretin receptor binding sites in rat brain.

Although the existence of the receptor for secretin in the brain was suggested, the localization of secretin receptor and the neuronal function of secretin have not been clarified yet. In the present study, the localization of secretin receptor was investigated in the rat brain by using an in vitro autoradiography technique. Frozen section autoradiography with (125)I-secretin showed intense binding in the nucleus of solitary tract, laterodorsal thalamic nucleus, and accumbens nucleus; moderate binding in the hippocampus, caudate/putamen, cerebellum, cingulate and orbital cortices. Scatchard plot analysis gave the Kd value of 125 pM with Bmax of 134 fmol/mg tissue in the hippocampus. The binding specificity was confirmed with secretin and its analogs, VIP, PACAP, and glucagon. These results indicate the secretin receptor system might have some neural functions in the brain, which could give the basis for therapeutic use of secretin in autistic children.

Food-dependent androgen and cortisol secretion by a gastric inhibitory polypeptide-receptor expressive adrenocortical adenoma leading to hirsutism and subclinical Cushing's syndrome: in vivo and in vitro studies.

Aberrant gastric inhibitory polypeptide (GIP) receptor expression in bilaterally hyperplastic adrenals or unilateral adrenal adenomas is a rare form of adrenal hyperfunction. So far, only few cases have been described. In all these cases, cortisol was the predominant steroid released in a food-dependent manner, leading to the development of non-ACTH-dependent Cushing's syndrome. In the present study, we describe a novel case of a GIP receptor-expressive adrenocortical adenomatous nodule, detected incidentally by computed tomography scanning in a 41-yr-old lady with hirsutism but no clinical signs of Cushing's syndrome, on physical examination. Hormonal investigations in morning fasting samples showed slightly elevated androgen levels, low-normal baseline cortisol, normal suppression of cortisol after dexamethasone administration, and ACTH levels that were not suppressed and did stimulate after CRH administration. The elevated urinary free cortisol excretion, in conjunction with an atypical cortisol diurnal rhythm, raised the possibility of an aberrant stimulation of cortisol production by the adrenal tumor. Further studies demonstrated food-dependent secretion of cortisol, which was abolished by prior octreotide administration. Notably, substantial amounts of adrenal androgens were also secreted after food consumption. Removal of the tumor resulted in undetectable cortisol and androgen levels that did not respond to food consumption. Histological examination of the excised tumor revealed an adrenocortical adenomatous nodule originating from the inner zona reticularis, consisting mainly of compact cells. A steroidogenic secretory pattern, indicating the concomitant release of adrenal androgens and cortisol, was also observed in vitro from tumor cells cultured in the presence of GIP. The in vitro secretory response to GIP was higher for the adrenal androgen DHEA, compared with cortisol. The expression of the GIP receptor in tumor cells, but not in the adjacent normal adrenal, was demonstrated by RT-PCR), using specific oligonucleotide probes for this receptor. In summary, we describe a patient with a GIP-expressive cortisol and androgen oversecreting adrenocortical nodule with the unusual presentation of hirsutism and not the typical clinical signs of Cushing's syndrome. It is of note that food intake in this patient provoked a substantial increase in both adrenal androgen and cortisol levels that, together with the histological appearance of this nodule, was compatible with a zona reticularis-derived tumor. Thus, aberrant expression of the GIP receptor does not exclusively involve cells of a zona fasciculata phenotype, as previously reported, but may also occur in other types of differentiated adrenocortical cells.

Motilin and motilin receptors: characterization and functional significance.

In order to get more insight into the mechanism of action of the gastrointestinal peptide, motilin, and its role in human physiology, we aimed at characterizing motilin and motilin receptors. Motilin. Sequence analysis of the motilin precursor from several species indicated that the N- and C-terminal regions of the motilin precursor have evolved at different rates. Sequence analysis of the motilin precursor in brain tissue of rabbit and man and motilin radioimmunoassay on tissue extracts, proved that motilin is a brain-gut peptide. Plasma motilin levels are increased in patients with ulcerative colitis or Crohn's disease. A weak correlation between the motilin genotype and the susceptibility to inflammatory bowel disease was demonstrated. Motilin receptors. Motilin receptors are expressed early postnatally and can be regulated by changes in its plasma level. The pharmacophore of motilin consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. In vivo and in vitro studies showed that in the rabbit and human antrum, smooth muscle and neuronal motilin receptors exist which have different characteristics. In the rabbit duodenum motilin's action depends upon the influx of extra- and intracellular Ca2+. Nevertheless, in primary smooth muscle cultures, Ca2+ influx through L-type Ca2+ channels is the major transduction mechanism. The existence of central motilin receptors was demonstrated by autoradiography. Receptor binding studies allowed the identification of two binding sites. In contrast to antral smooth muscle cells, the response to motilin in the human TE671 medulloblastoma cell line, expressing the motilin receptor, relies on intracellular IP3-sensitive Ca2+ stores. The antibiotic erythromycin-A (EM-A) binds to the motilin receptor and induces contractions with the same regional and species specificity as motilin. This interaction was supported by the discovery of motilin antagonists. Structure activity studies led to the development of more powerful erythromycin derivatives, which lack antibiotic properties and which are now in clinical trial for treatment of hypomotility disorders. Conclusion and perspectives. The physiological role of motilin and its receptors in the brain requires further investigation. Erythromycin and its derivatives act as motilin agonists with clinically useful prokinetic potential. The motilin receptor has recently been cloned and has substantial structural homology with the growth hormone secretagogue receptor. This may not only lead to the further characterization of motilin receptor subtypes and aid the development of safe and selective motilin receptor agonists and antagonists, useful for the treatment of GI disorders, but may also give a new dimension to the role of motilin in human physiology.

GIP-dependent adrenal Cushing's syndrome with incomplete suppression of ACTH.

ACTH-independent Cushing's syndrome may be due to the development of ectopic hormone receptors in adrenal tissue. Thus, in food-dependent Cushing's syndrome the adrenals aberrantly express receptors for gastric inhibitory polypeptide (GIP). We present the case of a 60-year-old woman with food-dependent Cushing's syndrome whose cortisol levels increased after stimulation with CRH. In this patient with Cushing's syndrome the finding of low basal plasma cortisol levels in the late night and early morning as well as a paradoxical rise of plasma cortisol during a 7-h infusion with dexamethasone (carried out without any restriction in food intake), suggested that cortisol production was stimulated at times of food intake. Hourly measurements of plasma cortisol for 48 h revealed prominent meal-related peaks. A plasma cortisol response, elicited by oral glucose administration, could be prevented by octreotide. Plasma ACTH was low or undetectable. CRH administration was followed by a ACTH response from 3 to 16 ng/l and a plasma cortisol response from 230 to 680 nmol/l. Octreotide treatment for nearly five months induced a partial clinical and biochemical remission. Total bilateral adrenalectomy was performed. The left adrenal was grossly enlarged (7 x 5.5 x 4 cm) and the right adrenal was slightly enlarged (6 x 4 x 1.8 cm). Microscopy revealed bilateral nodular hyperplasia. Cell suspensions of adrenal tissue from the patient did respond in a dose-dependent fashion to stimulation with GIP and were very sensitive to stimulation with synthetic ACTH1-24. However, CRH had no significant effect on cortisol production in vitro. Using RT-PCR amplification and cDNA hybridization, GIP receptor was found to be overexpressed in the left and right adrenal tissues from this patient as compared to adrenal tissues from a normal individual or from non GIP-dependent adrenal Cushing's syndrome. There was no evidence of presence of adrenal CRH receptors. Thus, in this patient with food-dependent Cushing's syndrome, the CRH-induced plasma ACTH and cortisol response is probably mediated by an incomplete suppression of the HPA axis as a result of the intermittent food-dependent nature of Cushing's syndrome.

Identification of binding domains of the growth hormone-releasing hormone receptor by analysis of mutant and chimeric receptor proteins.

The hypothalamic peptide GH-releasing hormone (GHRH) stimulates the release of GH from the pituitary through binding and activation of the GHRH receptor, which belongs to the family of G protein-coupled receptors. The objective of this study was to identify regions of the receptor critical for interaction with the ligand by expressing and analyzing truncated and chimeric epitope-tagged GHRH receptors. Two truncated receptors, GHRHdeltaN, in which part of the N-terminal domain between the putative signal sequence and the first transmembrane domain was deleted, and GHRHdeltaC, which was truncated downstream of the first intracellular loop, were generated. Both the receptors were deficient in ligand binding, indicating that neither the N-terminal extracellular domain (N terminus) nor the membrane-spanning domains with the associated extracellular loops (C terminus) are alone sufficient for interaction with GHRH. In subsequent studies, chimeric proteins between the receptors for GHRH and vasoactive intestinal peptide (VIP) or secretin were generated, using the predicted start of the first transmembrane domain as the junction for the exchange of the N terminus between receptors. The chimeras having the N terminus of the GHRH receptor and the C terminus of either the VIP or secretin receptor (GNVC and GNSC) did not bind GHRH or activate adenylate cyclase after GHRH treatment. The reciprocal chimeras having the N terminus of either the VIP or secretin receptors and the C terminus of the GHRH receptor (VNGC and SNGC) bound GHRH and stimulated cAMP accumulation after GHRH treatment. These results suggest that although the N-terminal extracellular domain is essential for ligand binding, the transmembrane domains and associated extracellular loop regions of the GHRH receptor provide critical information necessary for specific interaction with GHRH.

Galanin/GMAP- and NPY-like immunoreactivities in locus coeruleus and noradrenergic nerve terminals in the hippocampal formation and cortex with notes on the galanin-R1 and -R2 receptors.

By using immunofluorescence methodology, extensive galanin (GAL) and GAL message-associated peptide (GMAP)-positive terminal networks were observed in the hippocampal formation. The majority of the GAL/GMAP fibers were dopamine beta-hydroxylase- (DBH) positive, that is, they were noradrenergic. This finding was established with GAL/GMAP-DBH double-staining and with 6-hydroxy-dopamine treatment, which totally abolished all fibers in which GAL/GMAP and DBH coexisted. Also, reserpine treatment caused a marked depletion of GAL. No evidence for GAL/GMAP coexistence with 5-hydroxytryptamine was obtained. In the ventral hippocampus, GAL/GMAP-, DBH-negative fibers were seen in the stratum oriens, the anterior stratum radiatum, along the granule cell layer and in the strata oriens and alveus. In the locus coeruleus (LC), around 80% of the GMAP-positive neurons contained neuropeptide tyrosine (NPY), and about 40% of the NPY-positive neurons expressed GMAP. GAL-R1 receptor mRNA was expressed in Barrington's nucleus (close to the LC), but was not detected in the hippocampal formation/dorsal cortical areas. GAL-R2 receptor mRNA was found in the granule cell layer in the dentate gyrus. The present results show that most, but not all, immunohistochemically detectable GAL/GMAP in the hippocampal formation/dorsal cortex is present in noradrenergic nerve terminals originating in the LC, which has a robust GAL/GMAP synthesis. The functional role of GAL may be related to noradrenaline, possibly by a presynaptic action. However, the presence of GAL in other systems and of GAL-R2 receptor mRNA in granule cells also indicates other targets.

Evidence for the existence of galanin receptors on cultured astrocytes of rat CNS: colocalization with cholinergic receptors.

The cellular localization of binding sites for [125I]galanin was studied in explant cultures of rat neocortex, cerebellum, locus coeruleus and spinal cord by means by of autoradiography. Binding sites for the peptide were observed on a great number of astrocytes in all CNS regions studied. In addition to astrocytes, many neurones were intensely labelled by [125I]galanin. Binding of [125I]galanin (10(-8) M) to both astrocytes and neurones was markedly reduced or inhibited by the unlabelled peptide at high concentration (10(-6) M), suggesting 'specific' binding of the radioligand. Evidence for the colocalization of galanin and cholinergic receptors on astrocytes was provided by combined autoradiographic and immunohistochemical studies. Many astrocytes were labelled by [125I]galanin and immunostained with antibodies to either muscarinic or nicotinic receptors. Electrophysiological studies revealed that addition of galanin (10(-9) to 10(-7) M) to the bathing fluid caused a dose-dependent hyperpolarization of the majority of astrocytes studied. When galanin (10(-8) M) and the cholinergic agonists muscarine and nicotine (10(-6) M) were tested on the same astrocyte, all three compounds induced a hyperpolarization, suggesting a colocalization of functional galanin and cholinergic receptors on the glial membrane.

Ultrastructural localization of galanin and galanin receptors in the guinea pig median eminence.

The aim of this work was to compare the localization of galanin and galanin receptors in the guinea pig median eminence at the light and electron microscopic level. Concerning galanin the highest labeling was shown in the external part of the median eminence. At the ultrastructural level, galanin immunoreactivity was observed only in nerve terminals containing granular vesicles of approximately 120 nm in diameter. Light microscopic autoradiographs of semithin sections exhibited a moderate labeling in the external part of the median eminence. Galanin receptors were labeled in vitro on semithin sections (2 microm) using the highly specific radioligand [125I]galanin. Ultrastructural data showed that most of galanin binding sites overlaid membrane appositions between nerve terminals and also between nerve terminal and tanycyte. By considering the percentages in the distribution of the binding it appeared that galanin receptors were located on some nerve ending membranes. Our observations were not really in favor of a presence of receptors in tanycytes. The presence of galanin nerve endings in the external part suggests that like in the rat the peptide may have a direct hypophysiotrophic role. In contrast, the occurrence of numerous binding sites gives additional arguments in favor of a local action (paracrine and/or autocrine) of galanin occurring via galanin receptors located essentially on the pericapillary nerve terminals in the guinea pig median eminence.