SMOOTH-MUSCLE CELLS FROM GUINEA-PIG STOMACH POSSESS HIGH-AFFINITY GALANIN RECEPTORS THAT MEDIATE RELAXATION

Galanin-like immunoactivity occurs in nerves and plexi in muscle layer s throughout gastrointestinal tract including the stomach. Galanin can affect gastric emptying and contraction or relaxation of gastric musc le in different species. The aim of this study was to investigate the direct effect of galanin on dispersed gastric smooth muscle cells and to characterize any galanin receptors that mediated any effect. Disper sed gastric smooth muscle cells were prepared from guinea pig stomach by collagenase digestion. Porcine galanin (p-galanin; 1 mu M) did not stimulate contraction when present alone; however, p-galanin (1 mu M) inhibited carbachol-induced contraction with a half-maximal effect at 7 nM. p-Galanin (1 mu M) increased cellular adenosine 3',5'-cyclic mon ophosphate (cAMP) content by 10 s and caused a maximal increase of 80% over basal. I-125-galanin (porcine) bound to dispersed cells in a tim e- and temperature-dependent manner. Binding was saturable, reversible , and specific. Binding of I-125-galanin was inhibited almost equally by porcine and rat galanin (K-i = 6-8 nM) but was not inhibited by the galanin-associated peptide [preprogalanin-(108-123)]. The fragment ga lanin-(1-16) was equally potent to rat galanin; however, the fragment galanin-(9-29) was 56-fold less potent (K-i = 370 nM). Computer analys is demonstrated there were two binding sites for p-galanin on gastric smooth muscle cells, a high-affinity site (K-d = 2.6 nM) with low capa city (B-max = 175 fmol/mg protein) and a low-affinity site (Kd = 150 n M) with large capacity (B-max = 3,611 fmol/mg protein). I-125-galanin binding to gastric smooth muscle cell membrane was inhibited by 5'-gua nylyl imidodiphosphate [Gpp(NH)p] in a dose-dependent manner(IC50 = 75 nM). Computer analysis demonstrated that with Gpp(NH)p the high-affin ity binding site was lost. These results demonstrate that gastric smoo th muscle cells possess high-affinity galanin receptors, occupation of which causes cAMP generation and muscle relaxation. The NH2-terminus of galanin determines the receptor affinity that is regulated by a gua nine nucleotide-binding protein.