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.

Autoradiographic quantitation and anatomical mapping of GTP sensitive-galanin receptors in the guinea pig central nervous system.

Galanin is a 29-amino acid peptide widely distributed in the mammalian central nervous system. Galanin receptors in the guinea pig brain were visualized using [125I]galanin by in vitro receptor quantitative autoradiography. Scatchard analysis of [125I]galanin binding to slide-mounted sections revealed saturable binding to a single class of high affinity receptors with a KD of approximately 1 nM. Specific [125I]galanin binding sites were detected in a large number of brain areas (concentration range: from non detectable to 99.32 fmol/mg of tissular proteins). The anatomical mapping revealed high densities essentially in the telencephalon (e.g. lateral septal nuclei, amygdala, hippocampal dentate gyrus) and the diencephalon (e.g. the anterodorsal and medial habenular thalamic nuclei, the paraventricular, dorsomedian and median mammillary hypothalamic nuclei, the posterior lobe of the pituitary). Addition of Mg2+ and GTP increased binding in some areas such as the zona incerta, the median eminence and the arcuate nucleus, and decreased it in other areas such as the amygdala, the hippocampus and the mammillary nuclei. This regional heterogeneity in the effect of Mg2+ and GTP can be interpreted as: (1) different rates of galanin receptor occupancy by endogenous peptide; (2) a differential coupling of GTP binding proteins to galanin receptors in the brain structures; and (3) a different nature of receptors. At any rate, this study provides evidence for a specific GTP-sensitive galanin receptor in guinea pig brain with an extensive distribution suggesting various physiological implications. Comparison with studies performed in several mammals shows that the overall distribution of galanin receptors is well preserved among species. These data suggest that galanin may possess similar functional properties in the different species tested so far. Nevertheless, very distinct differences were found in some areas like the cortex, the hippocampus and the pituitary.

The age-related increase in galanin binding sites in the rat brain correlates with behavioral impairment.

The regional distribution of [125I]galanin specific binding sites was determined in young (three- to four-month-old), 14-15-month-old and aged (26-27-month-old) male Sprague-Dawley rats, previously tested for their performances in the Morris water-maze task, using the radioautographic method on brain sections. A significant increase in specific binding was observed in piriform and entorhinal cortex, ventral subiculum, and dorsal dentate gyrus in the aged rats, whereas no significant changes were observed in dorsal subiculum, amygdala, septal area and various subcortical structures. The area-specific regional increase in specific binding density in aged rats was significantly correlated with the impairment of the behavioral performance in the Morris water-maze task. The change in [125I]galanin specific binding was a result of an increase in the number of galanin binding sites, but not of an increase in affinity.

[125I]galanin binding sites in the rat frontal lobe are guanine nucleotide-sensitive and display a low regional index of occupancy.

As the neuropeptide galanin is possibly involved in spatial learning, we investigated both the precise location and the binding features of the receptors within the rat frontal cortex using quantitative autoradiography. Galanin receptors predominated in layers I and V of medial and lateral frontal cortex with a low regional index of occupancy by endogenous galanin. These receptors might be of functional relevance since the guanylnucleotide Gpp(NH)p inhibited [125I]galanin specific binding in each labeled region in the frontal lobe. Nevertheless, the striking areal difference of expression of this effect within the medial frontal cortex suggests that [125I]galanin binding sites might be coupled to one or more types of G-proteins related to the functionally distinct cortical subareas.

Age-related increase in galanin-binding sites in the rat brain: correlation with behavioral impairment.

The regional distribution of 125I-galanin specific binding sites was determined by radioautography on brain sections in young (3- to 4-month-old) and aged (26- to 27-month-old) male Sprague-Dawley rats, previously tested for their performances in the Morris water maze task. In aged rats, a significant increase in specific binding was observed in piriform, perirhinal and entorhinal cortex, the CA1 field of the ventral hippocampus, ventral subiculum, and dorsal dentate gyrus, whereas no significant change was observed in the ventral dentate gyrus, the dorsal subiculum, the CA3 field of the hippocampus, the amygdala or the septal area. The area-specific regional increase in specific binding density in aged rats was significantly correlated with the impairment of their behavioral performances in the Morris water maze task. The change in 125I-galanin specific binding in the aged rats was a result of an increase in the number of galanin-binding sites, without change in affinity.

Regional stimulatory and inhibitory effects of guanine nucleotides on [125I]galanin binding in rat brain: relationship with the rate of occupancy of galanin receptors by endogenous galanin.

Galanin has been shown to stimulate feeding or modulate neuroendocrine secretions when administered centrally. In the present work, using quantitative autoradiography, we documented the existence of [125I]galanin specific binding sites in several hypothalamic nuclei expected to mediate these effects. In standard binding conditions, [125I]galanin specific binding can be visualized in the hypothalamic ventromedial nucleus, stria terminalis, piriform cortex, central amygdaloid nucleus and medial amygdaloid nucleus, while it is almost undetectable in most neuroendocrine or autonomic hypothalamic areas. We hypothesized that high endogenous galanin levels in these regions might mask galanin receptors. We first showed that a high ionic strength/acid wash of brain slices is effective in removing more than 80% of specifically prebound [125I]galanin in all tested regions. After such treatments, specific binding sites could be revealed in the hypothalamus namely in the parvocellular paraventricular nucleus, periventricular nucleus, arcuate nucleus and median eminence. In contrast, regions already labeled in standard conditions exhibited a slight decrease in [125I]galanin binding. Thus, regions were ranked from low to high rate of occupancy of galanin receptors by endogenous galanin, the rate of occupancy of galanin receptors being maximal in median eminence (greater than 90%). We thus studied the regional effect of guanine nucleotides on [125I]galanin specific binding. A high concentration (100 microM) of guanyl 5'-yl imidodiphosphate, a nonhydrolyzable analog of GTP directly added to the incubation medium, inhibited [125I]galanin binding in all telencephalic regions. On the same sections and only in regions of high index of galanin receptor occupancy (arcuate nucleus, median eminence, dorsomedial nucleus, paraventricular nucleus, and periventricular hypothalamic nucleus), guanyl 5'-yl imidodiphosphate paradoxically enhanced [125I]galanin binding. The effects of acid preincubation and guanyl 5'-yl imidodiphosphate incubation on [125I]galanin binding were strongly correlated in these hypothalamic areas (r = 0.97). In all regions, guanyl 5'-yl imidodiphosphate increased the rate of dissociation of [125I]galanin. In competition studies, guanyl 5'-yl imidodiphosphate decreased the IC50 s of unlabeled galanin which were homogenized around 4 nM in most telencephalic and hypothalamic regions. Thus, the guanyl 5'-yl imidodiphosphate-induced stimulation of [125I]galanin specific binding measured in the neuroendocrine and autonomic hypothalamus is linked to an increase in receptor capacity and not to a rise in receptor affinity. Both inhibitory and stimulatory guanyl 5'-yl imidodiphosphate effects observed in [125I]galanin equilibrium binding studies were dose-dependent and guanine nucleotide-specific with guanyl 5'-yl imidodiphosphate more potent than GTP or GDP.(ABSTRACT TRUNCATED AT 400 WORDS)

Structural requirements for galanin interaction with receptors from pancreatic beta cells and from brain tissue of the rat.

The binding activity of several galanin fragments and analogs was measured on specific receptors present in rat brain and the rat pancreatic beta cell line Rin m 5F. In both tissues it was observed that: 1) galanin(3-29), galanin(10-29) and [Ile2]-galanin were ineffective for inhibiting [125I] galanin binding and 2) active peptides had the following rank order of potency: galanin(1-29) greater than [Ac-Trp2]-galanin(2-29) greater than galanin(2-29) greater than galanin(1-15) greater than [Phe2]-galanin greater than [Tyr2]-galanin. It was concluded that the N-terminal portion of galanin is very important for interaction with central or peripheral receptors. The aromatic amino acid in position 2 (Trp in native galanin) plays a crucial role.

Characterization of galanin receptors in the insulin-secreting cell line Rin m 5F: evidence for coupling with a pertussis toxin-sensitive guanosine triphosphate regulatory protein.

The present work characterizes galanin receptors in the insulin-secreting pancreatic beta-cell line Rin m 5F and documents their regulation by guanine nucleotides. Binding of [125I]galanin to cell membranes was found to be temperature dependent, rapid, saturable, reversible, and highly peptide specific. Optimal steady state conditions were achieved after a 60-min incubation at 15 C. The concentration dependence of galanin binding determined by adding increasing concentrations of [125I]galanin indicated that galanin receptors were saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of receptors, with a Kd of 0.3 nM and a binding capacity of 82 fmol/mg protein. Guanyl 5'-yl imidodiphosphate dramatically enhanced the dissociation of bound [125I]galanin. Some guanine nucleotides inhibited [125I]galanin binding to membranes with the following order of potency: guanyl 5'-yl imidodiphosphate greater than GTP = GDP. Other nucleotides had no effect. The effect of the guanine nucleotides was Mg2+ dependent, but Na+ independent, although Mg2+ ions alone (5 mM) slightly enhanced [125I]galanin binding, and Na+ ions alone (100 mM) induced a 60% decrease in the binding. Finally, overnight treatment of Rin m 5F cells with pertussis toxin (0.4 microgram/ml) dramatically reduced [125I]galanin binding to cell membranes. This was related to a 4-fold decrease in receptor affinity, with no change in binding capacity. In conclusion, for the first time evidence of the existence of galanin receptors on functional pancreatic beta-cells is presented. Also, other findings support the fact that galanin receptors are functionally associated with a pertussis toxin-sensitive GTP-binding protein mediating guanine nucleotide control of galanin binding.

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase.

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.