title: Corticotropin-releasing factor-like peptides increase cytosolic [Ca2+] in human epidermoid A-431 cells.
abstract: This study investigated whether sauvagine and urotensin I change [Ca2+]i in human epidermoid A-431 cells and whether these changes are correlated with their anti-edema properties in vivo. A-431 cells were used because they possess the corticotropin-releasing factor (CRF) receptor 2. Treatment with either sauvagine or urotensin I led to an immediate increase in [Ca2+]i, the magnitude of which depended on the concentration of the drug. Sauvagine was more effective than urotensin I, with a median effective concentration (EC50) of 1.4 +/- 0.2 fM, compared to an EC50 of 66 +/- 7 fM for urotensin I. Both were more effective at stimulating increases in [Ca2+]i than CRF (EC50 of 6.8 +/- 0.1 pM). There was a correlation between the EC50 for increasing [Ca2+]i and the median effective dose (ED50) for inhibiting edema induced by heating rat paw (r = 0.99). Removal of extracellular Ca2+ or incubation with La3+ eliminated the increase in [Ca2+]i stimulated by either peptide. Pretreatment with a CRF receptor antagonist reduced the increase in [Ca2+]i by these peptides. This occurred in an antagonist concentration-dependent manner, with median inhibitory concentrations (IC50) of 1.99 +/- 0.04 nM and 0.85 +/- 0.04 nM, respectively. Both pertussis toxin (an inhibitor of G proteins) and U-73122 (an inhibitor for inositol trisphosphate (InsP3) production) partially inhibited the increases. InsP3 was measured to determine whether these peptides mobilized Ca2+ from an InsP3-sensitive store. Both sauvagine and urotensin I increased InsP3. The InsP3 increases were inhibited by U-73 122 and CRF antagonist, but not by removal of external Ca2+. Both peptides elevated protein tyrosine phosphorylation. In summary, these peptides increase [Ca2+]i as a result of Ca2+ influx via CRF receptor-operated Ca2+ channels coupled to pertussis toxin-sensitive G proteins and a Ca2+ mobilization from InsP3-sensitive Ca2+ pools. Their in vivo effectiveness at inhibiting edema is related to their respective capacities to stimulate elevations of [Ca2+]i, supporting a role for intracellular Ca2+ in this process.
authors.affiliation: Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. Dr._Juliann_Kiang@WRSMTP-ccmail.army.mil
authors.forename: J G
authors.initials: JG
authors.lastname: Kiang
mesh_terms: D029845:Amphibian Proteins; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D006801:Humans; D007293:Inosine Triphosphate / Q000378:metabolism; D010446:Peptide Fragments / Q000494:pharmacology; D036361:Peptide Hormones; D010455:Peptides / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D018019:Receptors, Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism; D014579:Urotensins / Q000494:pharmacology*
chemical_list: D029845:Amphibian Proteins; C101531:CRF receptor type 2; D002121:Calcium Channel Blockers; D010446:Peptide Fragments; D036361:Peptide Hormones; D010455:Peptides; D018019:Receptors, Corticotropin-Releasing Hormone; D014579:Urotensins; D007293:Inosine Triphosphate; D014443:Tyrosine; C025727:sauvagine; D003346:Corticotropin-Releasing Hormone; C029676:urotensin I; C045089:corticotropin releasing hormone (9-41); D002118:Calcium