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

Abstract

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 (>90%). We thus studied the regional effect of guanine nucleotides on [ 125I]galanin specific binding. A high concentration (100 μM) 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 IC 50s 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. In conclusion: (1) Taking advantage of the low rate of dissociation of galanin from its binding sites in autoradiographic conditions, we validated a method of high ionic strength/acid preincubation in autoradiography, allowing estimation of the regional rate of occupancy of galanin receptors. (2) This allowed us to demonstrate that high levels of the endogenous peptide interfere with the visualization of galanin receptors in most neuroendocrine and autonomie hypothalamic nuclei. (3) Based on classical features of sensitivity of agonist binding to guanine nucleotides, galanin receptors seem coupled to GTP binding proteins in all brain regions studied, but there is a regional heterogeneity of [ 125I]galanin binding site sensitivity to guanylnucleotides. Such differences between hypothalamic and telencephalic areas are discussed either as the interference of bound endogenous galanin on brain sections and/or as a differential coupling of GTP binding proteins to galanin receptors among brain structures.