Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes.

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.

Genomic organization and functional characterization of the mouse GalR1 galanin receptor.

Galanin mediates diverse physiological functions in digestive, endocrine, and central nervous systems through G-protein-coupled receptors. Two galanin receptors have been cloned but the gene structures are unknown. We report genomic and cDNA cloning of the mouse GalR1 galanin receptor and demonstrate that the coding sequence is uniquely divided into three exons encoding the N-terminal portion through the fifth transmebrane domain, the third intracellular loop, and the sixth transmembrane domain through the C-terminus. Functional analysis of the encoded cDNA revealed active ligand binding and intracellular signaling. The expression is detected in brain, spinal cord, heart and skeletal muscle.

Hypocholesterolemic activity of a novel inhibitor of cholesterol absorption, SCH 48461.

The amount of cholesterol that circulates in the plasma as lipoproteins can be affected by the balance of cholesterol metabolism within and between the intestines and liver. In the present report, we describe a novel hypocholesterolemic agent and document its pharmacological effects in animal models of hypercholesterolemia. The oral administration of (3R,4S)-1,4-bis-(4-methoxyphenyl)-3-(3-phenylpropyl)-2-azetidinone (SCH 48461) reduced plasma cholesterol concentrations in cholesterol-fed hamsters, rats and rhesus monkeys with ED50s of 1, 2 and 0.2 mg/kg per day, respectively, SCH 48461 was also highly effective in reducing hepatic cholesteryl ester accumulation in cholesterol-fed hamsters and rats after 7 days of treatment. In one 3 week study, rhesus monkeys were fed a 0.25% cholesterol/22% saturated fat diet with or without SCH 48461. At the end of the 3 week period the control group's VLDL + LDL-cholesterol increased to 180 Mg/dl from a baseline of approximately 65 mg/dl while plasma apolipoprotein B levels had doubled. Animals treated daily with 1 mg/kg SCH 48461 maintained their baseline levels of VLDL + LDL-cholesterol, HDL-cholesterol, and plasma apolipoproteins B and A-I. After 3 weeks the diets of the two groups were switched. Within 1 week SCH 48461 (1 mg/kg per day) rapidly reversed the elevated VLDL + LDL-cholesterol levels of the previous control group to near baseline values. SCH 48461 exerted its hypocholesterolemic effect through the inhibition of cholesterol absorption. A dose of 10 mg/kg per day inhibited cholesterol absorption in cholesterol-fed hamsters by 68% while a similar reduction was achieved in chow-fed monkeys with 3 mg/kg per day. This latter dose inhibited cholesterol absorption in cholesterol-fed monkeys by 95%. Treatment of cholesterol-fed monkeys with 10 mg/kg per day SCH 48461 significantly increased fecal neutral sterol excretion (52 vs. 32 mg/kg) but had no effect on acidic sterol excretion. Using a 2-h absorption model in cholesterol-fed hamsters, SCH 48461 caused a 46% inhibition of unesterified [14C]cholesterol accumulation in the intestinal wall and a 90% inhibition of cholesteryl ester formation at a dose of 10 mg/kg. Similar data were observed when the plasma radioactivity was assessed, indicating inhibition of both free (61%) and esterified (85%) cholesterol appearance. In contrast, CI-976, a potent acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, did not affect the uptake of free cholesterol into the intestines while inhibiting cholesterol esterification (98% inhibition).(ABSTRACT TRUNCATED AT 400 WORDS)