Mystixin-7 and mystixin-11 increase cytosolic free Ca2+ and inositol trisphosphates in human A-431 cells.
Mystixin-7 and mystixin-11, small peptides structurally related to corticotropin-releasing factor (CRF), have been shown to attenuate vascular leakage in injured skin. The goal of this study was to characterize changes in cytosolic Ca2+ concentration ([Ca2+]i) in human epidermoid A-431 cells treated with these two peptides and to investigate the mechanisms by which these changes occurred. The resting [Ca2+]i in A-431 cells at 37 degrees C was 76 +/- 2 nM (n = 373). When cells were treated with either peptide, [Ca2+]i increased immediately. The increase depended on the peptide concentration, with a median effective concentration of 299 +/- 9 pM for mystixin-7 and 2.23 +/- 0.04 pM for mystixin-11. The increases also depended on extracellular Ca2+ and were blocked by Cd2+, Co2+, verapamil, and nifedipine. alpha-Helical CRF-(9-41), a synthetic CRF receptor antagonist, and pertussis toxin also blocked the increase in [Ca2+]i induced by the two peptides. Taken together, these results suggest that mystixin-7 and mystixin-11 interact with CRF receptors to activate pertussis-sensitive G proteins coupled to L-type Ca2+ channels that allow an uptake of extracellular Ca2+. Because U-73122, an inhibitor of 1,4,5-inositol trisphosphate production, partially inhibited the increase in [Ca2+]i, we measured inositol trisphosphates in cells stimulated by the two peptides. Both increased inositol trisphosphate levels within 1 min. The increase was inhibited by the removal of extracellular Ca2+ or treatment with U-73122. The results suggest that the Ca2+ influx stimulated by mystixin-7 and mystixin-11 induces an increase in inositol trisphosphates, resulting in a mobilization of Ca2+ from 1,4,5-inositol trisphosphate-sensitive Ca2+ pools.
Kiang
J G
JG
Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA.
D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects*; D002460:Cell Line; D003346:Corticotropin-Releasing Hormone / Q000494:pharmacology*; D003600:Cytosol / Q000187:drug effects; D004848:Epithelium / Q000187:drug effects; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology*
D015220:Calcium Channels; D010455:Peptides; D015544:Inositol 1,4,5-Trisphosphate; D003346:Corticotropin-Releasing Hormone; D002118:Calcium