Cytosolic calcium regulates phorbol diester binding affinity in intact phagocytes.
The mobilization of internally sequestered stores of Ca2+ and activation of protein kinase C appear to be involved in neutrophil activation. We have examined the inter-relationship of these two pathways by investigating the effects of modulating Ca2+ activity on the binding of [3H]phorbol 12,13-dibutyrate (PDBU) to protein kinase C in intact phagocytes. Differentiated HL-60 cells were equilibrated with [3H]PDBU prior to stimulation with various agents known to alter Ca2+ homeostasis in cells. Agents that elevated cytosolic Ca2+, such as f-Met-Leu-Phe and A23187, up-regulated radioligand binding by increasing the affinity of the PDBU/protein kinase C interaction. These effects were time- and agonist concentration-dependent and temperature-sensitive. The kinetics of the up-regulation of binding by f-Met-Leu-Phe coincided with the kinetics of Ca2+ mobilization (by quin2 fluorescence measurements). The putative intracellular Ca2+ antagonist 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate alone down-regulated [3H]PDBU binding and inhibited the up-regulation of ligand binding by f-Met-Leu-Phe and A23187. Low concentrations of La3+ (0.1-10 microM) also inhibited up-regulation of radioligand binding to f-Met-Leu-Phe and A23187, whereas higher concentrations (0.1-1 mM) alone increased [3H] PDBU binding and supported further up-regulation of ligand binding by the Ca2+-mobilizing agents. These data suggest a role for Ca2+ in the regulation of phorbol diester binding to protein kinase C in intact cells.
Dougherty
R W
RW
Niedel
J E
JE
D000634:Aminoquinolines / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010587:Phagocytosis; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D013997:Time Factors
D000634:Aminoquinolines; D010703:Phorbol Esters; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; C035739:Quin2; D002118:Calcium