Date of Award
Doctor of Philosophy (PhD)
Edwin E. Daniel
Galanin, a 29 amino acid polypeptide, is widely distributed in enteric nerves of the gastrointestinal tract (GIT) in mammals. Previous functional studies demonstrated that galanin may function as neurotransmitter and neuromodulator in GIT. In canine small intestine, galanin induces TTX-insensitive inhibition of small intestinal circular muscle motility in vivo and in vitro, suggesting a direct smooth muscle action of galanin (Fox et al, 1986). Intraarterial infusion of galanin inhibits vasoactive intestinal polypeptide release from isolated perfused canine small intestinal circular muscle, indicating a neuronal action of galanin (Fox et al, 1988). We used ¹²⁵I-porcine galanin as a ligand to study the galanin receptors in the circular muscle and deep muscular plexus from the canine small intestine. The separation, purification and characterization of nerve and muscle membranes were carried out using the technique developed in our laboratory by Ahmad et al (1988). Specific binding sites for galanin were found in both nerve and smooth muscle membranes. The galanin receptor on synaptosomes showed some similar characteristics to that on smooth muscle membranes: (1) The equilibrium binding analysis showed a high affinity and high capacity binding sites in nerve (Kd = 1.1 nM, Bmax = 244 fmol/mg) and in muscle (Kd = 0.58 nM, Bmax = 389 fmol/mg); (2) The specific binding of ¹²⁵I-galanin was inhibited by galanin or N-terminal galanin fragments (galanin 1-16, 1-15, 1-11), but not inhibited by C-terminal fragment galanin 15-29, suggesting a crucial role of the N-terminal region of the galanin molecule in receptor recognition. Computer analysis suggested a two-site model of galanin receptor; (3) The receptor-bound ¹²⁵I-galanin was only partially dissociated by addition of excess (1 μM) unlabelled galanin. However, in the presence of a GTP analog, GTPγS, the dissociation of bound ¹²⁵I-galanin was accelerated and completed, implicating involvement of G proteins in binding of galanin to a two-site receptor. This was supported by the observation that GTPγS abolished the high affinity galanin binding site, leaving only a low affinity binding site in competition studies; (4) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cross-linked galanin-receptor complexes from synaptosomes and smooth muscle membranes revealed a similar radioactive band at an approximate molecular mass of 50,000 dalton. Although, the neuronal and muscular galanin receptors have the similar properties, studies on characterizing the G proteins involved in these two groups of receptors using bacterial toxins indicated that galanin-receptor interaction in nerves involves a pertussis toxin-sensitive G protein, while the receptor in smooth muscle plasma membranes is coupled to a cholera toxin-sensitive, pertussis toxin-insensitive G protein. We conclude that, in canine small intestine, galanin may act as a neurotransmitter and/or neuromodulator by interacting with a specific receptor subtype coupled by distinct G proteins on smooth muscle membrane as well as synaptosomes.
Chen, Cora Ke, "Galanin receptors in canine small intestine" (1993). Open Access Dissertations and Theses. Paper 3871.