This study on the cellular and molecular mechanisms of peptide receptor signal transduction processes has used two approaches. The first approach is to analyze the electrophysiological responses to application of neuropeptides in selected peptide receptor-bearing cells: Swiss 3T3 cells for bombesin (BN) receptors and AR42J cells for cholecystokinin (CCK) receptors. The second approach is to study the peptide receptor signal transduction process in detail using the Xenopus oocyte surrogate system, after injection of mRNAs extracted from the above cells. Whole cell voltage-clamp and techniques are applied to those cultured cells. The Swiss 3T3 cells do not have profound voltage sensitive K-channels, but some cells possess a transient type of Ca-current (lCa(t) channels. Application of bombesin to 3T3 cells induces a K-conductance increase by activating Ca-mediated K-current (IK(Ca)) flow. AR42J cells possess voltage-sensitive IK, INa, ICa(t) and ICa(I), and Ca-sensitive ICI(Ca). Application of CCK to AR42J cells induces a conductance increase to Cl ions, whereas Na and K ions do not show significant effects. In both cell lines, ligand binding with receptors triggers a rise in intracellular Ca ion concentration by releasing from intracellular sources. The two-electrode voltage clamp technique has been applied to assay the functionally transcribed CCK and bombesin receptors in the Xenopus oocytes, after injection of their respective mRNAs. Size-fractionated mRNAs obtained by the sucrose gradient technique are further assayed in order to clone and sequence these receptor cDNAs and genes. Pharmacological analysis of BN receptors expressed by injecting mRNAs of various tissues indicates that there are subtypes in brain and esophageal tissues.
