Agonist-mediated Ca2+ release in permeabilized UMR-106-01 cells. Transport properties and generation of inositol 1,4,5-trisphosphate.
Permeabilized and intact UMR-106-01 cells attached to culture plates or coverslips were used to evaluate compartmentalized generation and the effective concentration of inositol 1,4,5-trisphosphate (In-1,4,5-P3) during agonist-mediated Ca2+ release. In permeabilized cells, Ca2+ release had the following characteristics. In-1,4,5-P3 released approximately 65% of the Ca2+ incorporated into intracellular stores. Prostaglandin F2 alpha (PGF2 alpha), endothelin, or GTP(gamma S) alone released a small amount or no Ca2+. However, the agonists together with GTP(gamma S) were as effective as In-1,4,5-P3 in releasing Ca2+. Both agonist- and In-1,4,5-P3-mediated Ca2+ release required the presence of permeable ion. Agonists, like In-1,4,5-P3, stimulated 45Ca uptake from low Ca2+ medium devoid of permeable ions into Ca2(+)-loaded intracellular stores. The permeabilized cell system was then used to evaluate compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation. Mass measurement shows that in intact resting cells In-1,4,5-P3 concentration was 1.4 microM and was reduced to 0.05 microM following permeabilization. Stimulation with agonists increases In-1,4,5-P3 concentration from 0.05 to 0.34 microM. Ca2+ release by this concentration of In-1,4,5-P3 evenly distributed in the cytosol can account for only part of the agonist-mediated Ca2+ release. However, the effects of saturating In-1,4,5-P3 concentration and agonists were blocked by the specific inhibitor heparin. Measurement of heparin dependency of In-1,4,5-P3-mediated Ca2+ release was used to calculate an affinity for In-1,4,5-P3 of 0.39 microM. Similar measurements with agonists show that In-1,4,5-P3 concentration at the site of Ca2+ release during agonist stimulation is 11.2 microM. Hence, the total increase in In-1,4,5-P3 is reflected in considerably higher localized concentrations. This is interpreted to suggest compartmentalized generation and action of In-1,4,5-P3 during agonist stimulation.
Department of Physiology, University of Texas Southwestern Medical Center, Dallas 75235.
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Khademazad
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Muallem
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002463:Cell Membrane Permeability; D015232:Dinoprostone / Q000494:pharmacology; D016232:Endothelins; D004730:Endothelium, Vascular; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology*; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D007700:Kinetics; D012516:Osteosarcoma; D010455:Peptides / Q000494:pharmacology*; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism*
D016232:Endothelins; D010455:Peptides; D013873:Thionucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015544:Inositol 1,4,5-Trisphosphate; D006160:Guanosine Triphosphate; D006493:Heparin; D015232:Dinoprostone; D002118:Calcium