Translocation of protein kinase C in human polymorphonuclear neutrophils. Regulation by cytosolic Ca2(+)-independent and Ca2(+)-dependent mechanisms.

[3H]Phorbol dibutyrate [( 3H]PDB) rapidly and reversibly binds to human polymorphonuclear neutrophils (PMN). Ca2+/diacylglycerol/phospholipid-dependent protein kinase C appeared to be the receptor for this binding because: a diacylglycerol, dioctanoylglycerol, competed with [3H]PDB for PMN binding sites; a blocker of protein kinase C-phospholipid interactions, sphinganine, inhibited PMN binding of [3H]PDB; and changes in cytosolic Ca2+ apparently regulated PMN binding of the label. Relevant to the last point, disrupted PMN contained 9 X 10(5) phorbol diester receptors/cell, whereas intact PMN had only 1.6 X 10(5) such receptors that were accessed by the ligand. This number fell to 1.0 X 10(5) in Ca2(+)-depleted PMN and rose to 2.5 X 10(5) in cells stimulated with the Ca2+ ionophore, ionomycin. This ionomycin effect lasted for greater than 16 min, correlated temporally with changes in cytosolic Ca2+, did not occur in Ca2(+)-depleted PMN, and was blocked by sphinganine. A second ionophore, A23187, likewise induced Ca2(+)-dependent rises in [3H]PDB binding. These results fit the standard model, wherein rises in cytosolic Ca2+ cause protein kinase C to translocate from cytosol to plasmalemma and thereby become more available to [3H]PDB. In contrast, two humoral agonists, N-formyl-Met-Leu-Phe (fMLP) and leukotriene (LT)B4, had actions that did not fit this model. They stimulated PMN to increase the availability of PDB binding sites by a sphinganine-sensitive mechanism, but their actions differed from those of ionophores. They induced biphasic (t = 15 and 60 s) increases in [3H]PDB binding while eliciting monophasic (t = 15 s), short-lived (t less than 1 min) rises in cytosolic Ca2+. In Ca2(+)-depleted PMN, moreover, fMLP and LTB4 stimulated slow (t greater than or equal to 30 s), monophasic, prominent rises in [3H]PDB binding and binding site number without appreciably altering cytosolic Ca2+. We suggest, therefore, that fMLP and LTB4 translocate protein kinase C using two sequential mechanisms. The first involves Ca2+ transients and thus produces abrupt (t = 15 s), rapidly reversing responses. The second mechanism uses an unrelated signal to effect a more slowly evolving (t = 60 s) movement of protein kinase C to plasmalemma. Hence, the standard model does not explain all instances of protein kinase C translocation, and a cytosolic Ca2(+)-independent signal contributes to the regulation of protein kinase C as well as those responses elicited by the effector enzyme.