From this study, we predicted that the human calcitonin gene-related peptide (hCGRP) fragment hCGRP-(8-37) would be a selective antagonist for CGRP receptors but an agonist for calcitonin (CT) receptors. In rat liver plasma membrane, where CGRP receptors predominate and CT appears to act through these receptors, hCGRP-(8-37) dose dependently displaced 125I-[Tyr0]rat CGRP binding. However, hCGRP-(8-37) had no effect on adenylate cyclase activity in liver plasma membrane. Furthermore, hCGRP-(8-37) inhibited adenylate cyclase activation induced not only by hCGRP but also by hCT. On the other hand, in LLC-PK1 cells, where calcitonin receptors are abundant and CGRP appears to act via these receptors, the bindings of 125I-[Tyr0]rat CGRP and 125I-hCT were both inhibited by hCGRP-(8-37). In contrast to liver membranes, interaction of hCGRP-(8-37) with these receptors led to stimulation of adenosine 3',5'-cyclic monophosphate (cAMP) production in LLC-PK1 cells, and moreover, this fragment did not inhibit the increased production of cAMP induced not only by hCT but also by hCGRP. Thus hCGRP-(8-37) appears to be a useful tool for determining whether the action of CGRP as well as that of CT is mediated via specific CGRP receptors or CT receptors.
Calcitonin gene-related peptide (CGRP) is present in the stomach, and exogenous CGRP stimulates gastric somatostatin release. A study was undertaken to elucidate the functional linkage between CGRP and somatostatin in the stomach. Newborn Wistar rats were made CGRP deficient by intraperitoneal injection of capsaicin 2 days after birth, and then 2.5 mo later, release of CGRP and somatostatin was examined by vascular perfusion of the isolated stomach. In CGRP-deficient rats, neither the content nor basal secretion of gastric somatostatin differed from that in normal rats, and although none of several secretagogues induced CGRP secretion, the somatostatin response to glucagon was well preserved, indicating the presence of normally functioning D cells. On the other hand, arterial infusion of capsaicin significantly increased the release of not only CGRP but also somatostatin from the stomach of normal rats. In CGRP-deficient rats, however, capsaicin produced no corresponding effect. Finally, human CGRP-(8-37), a CGRP-receptor antagonist, completely inhibited the increase of gastric somatostatin induced by both rat alpha-CGRP and capsaicin infusion in normal rats. Thus the capsaicin-induced increase of somatostatin release appears to be mediated by CGRP in the stomach.
The role of protein kinase C in muscarinic agonist-induced insulin release from rat insulinoma cells was investigated. The dose-dependent stimulation of insulin secretion by carbamylcholine (carbachol) was associated with dose-dependent increase in the release of 3H-inositolphosphates from prelabeled rat insulinoma cell line (RINr) cells. After preincubation with 32P-orthophosphates, carbachol also evoked a rapid decrease in 32P-labeling of phosphatidylinositol-4,5-bisphophate with concomitant increase in 32P-labeling of phosphatidic acid. Furthermore, carbachol significantly increased membrane-associated protein kinase C activity with a simultaneous decrease of its activity in cytosol. Although phorbol-12,13-dibutyrate (PDBu), a protein kinase C activator, also stimulated insulin release, insulin secretion induced by concomitant administration of carbachol and PDBu was clearly less than the level expected on the basis of an additive action. Moreover, PDBu significantly inhibited inositolphospholipid turnover stimulated by carbachol. Finally, PDBu inhibited the binding of 3H-scopolamine binding revealed that PDBu decreased the number of muscarinic receptors without altering its affinity. These findings suggest that activation of protein kinase C not only mediates muscarinic stimulation of insulin secretion from RINr cells but also operates a negative feedback mechanism in a signal transduction system, at least in part, via down-regulation of muscarinic receptors.
To evaluate the functional relationship between the liver calcitonin gene-related peptide (CGRP) receptor and guanine nucleotide-binding proteins, we investigated the effects of nucleotides not only on adenylate cyclase activation by CGRP, but also on 125I-[Tyr0]rat CGRP binding to rat liver plasma membranes. In the presence of GTP, rat CGRP stimulated adenylate cyclase activity in a dose-dependent manner in rat liver plasma membranes, and this effect was reduced in the absence of GTP. Salmon calcitonin also enhanced adenylate cyclase activation in the presence of GTP, but only in higher concentrations. On the other hand, guanine nucleotides not only decreased 125I-[Tyr0]rat CGRP binding to rat liver plasma membranes, but also accelerated the dissociation of label binding, and the removal of Mg2+ from incubation medium attenuated this inhibitory action of GTP on 125I-[Tyr0]rat CGRP binding to membranes. Scatchard analysis of the data revealed that the reduction of 125I-[Tyr0]rat CGRP binding by GTP was due to the decrease in binding affinity without a significant change in binding capacity. These findings lead us to conclude that binding of CGRP to its receptors activates adenylate cyclase in rat liver plasma membranes via a guanine nucleotide-dependent process, suggesting the involvement of guanine nucleotide-binding stimulatory protein in the action of CGRP.
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