A study on the cellular and molecular mechanisms of peptide receptor signal transduction processes has been initiated by taking two approaches: The first approach is to analyze electrophysiological responses at membrane and molecular levels in the selected peptide receptor-bearing cells: AR42-J cells for cholecystokinin (CCK) receptors and swiss 3T3 cells for bombesin receptors. The second approach is to study the peptide receptor signal transduction process in detail using the Xenopus oocyte surrogate system, after injecting mRNA's extracted from the above cells. Whole cell voltage-clamp and patch-clamp techniques were applied to those cultured cells. These cells do not have profound voltage-sensitive K-channels. Application of bombesin (1 micromolar, 20 microliters) to 3T3 cells produced a conductance increase, and a strong activation of Ca-mediated K-current. The major source of increase intracellular Ca concentration appears to be intracellular. A two electrode voltage clamp technique was applied for analyzing the Xenopus oocyte response to mRNA injection. Size fractionated mRNAs by the sucrose gradient technique were further assayed in order to prepare cDNA-libraries. Both CCK and bombesin receptors induced by mRNA injection produced inward current (C1-Flux) at the membrane holding potential of -60 mV, with several seconds to minutes long latencies consisting of two components, an initial peak transient current flow followed by secondary sustained slow current flow. These responses do not require an intracellular release of Ca ions. In order to compare these responses with central (neuronal) CCK-receptor functions, we also injected mRNA extracted from the rat brain cortex, which was successfully translated by the oocyte system into functional receptors.
