title: Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium.
abstract: The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.
authors.affiliation: Department of Pharmacological Sciences, Genentech, Inc., South San Francisco, California 94080.
authors.forename: A
authors.initials: A
authors.lastname: Perianin
authors.forename: R
authors.initials: R
authors.lastname: Synderman
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D001572:Benzofurans; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D004987:Ethers / Q000494:pharmacology; D016257:Fura-2; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D037342:Pertussis Toxin; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D000470:Alkaloids; D001572:Benzofurans; D004075:Diglycerides; D004987:Ethers; D010566:Virulence Factors, Bordetella; C044295:1,2-dioctanoylglycerol; D015759:Ionomycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D016257:Fura-2