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Phorbol 12,13-dibutyrate binding to intact human platelets. The role of cytosolic free Ca2+.

ABSTRACT: The role of Ca2+ was examined in regulating the binding of phorbol 12,13-dibutyrate (PdBu) to intact human platelets. Alterations in the cytosolic free Ca2+ concn. [( Ca2+]i), but not extracellular Ca2+, substantially influenced the binding parameters of the phorbol ester. Ca(2+)-depleted platelets demonstrated a significant decline in the maximal binding capacity (Bmax), an increase in equilibrium dissociation constant (Kd) and a decrease in the Hill coefficient (h), suggesting the presence of Ca(2+)-sensitive and Ca(2+)-insensitive populations of PdBu-binding sites. In 1 mM-Ca2+ buffer, thrombin (0.1 NIH unit/ml) and ionomycin (0.5 microM) evoked a rise in [Ca2+]i to approx. 300-500 nM, associated with a significant decline in Kd, but without an apparent effect on Bmax. No effect of thrombin was observed on PdBu binding in Ca(2+)-depleted platelets. Inhibition of protein kinase C (PKC) by H7 was associated with a greater thrombin-evoked [Ca2+]i transient and a decline in Kd. Staurosporine also decreased the Kd for PdBu binding. We propose that this effect of the PKC inhibitors on the Kd was also [Ca2+]i-dependent. These observations in intact platelets indicate that the primary role of agonist- or non-agonist-induced rise in [Ca2+]i is to increase the affinity of PKC for PdBu and, presumably, endogenous diacylglycerol. However, in itself a rise in [Ca2+]i does not increase the Bmax, for PdBu binding.

SUBMITTER: Takaya J

PROVIDER: S-EPMC1151358 | biostudies-other | 1991 Sep

REPOSITORIES: biostudies-other

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Project description:Staurosporine, a potent protein kinase C (PKC) inhibitor, was studied for its effects on the binding of phorbol 12,13-dibutyrate (PDBu) to human polymorphonuclear leucocytes (PMNs). Treatment of PMNs with staurosporine concentrations in the range 50 nM-2 microM at 37 degrees C (but not at 4 degrees C) and with 1 nM [3H]PDBu at both temperatures enhanced specific PDBu binding to PMNs by approx. 10-600% relative to control values. The potentiation was rapid (detectable within 1 min) and reached a plateau after 10 min of cell treatment. Scatchard analysis of the binding showed that staurosporine increased the total number of PDBu-binding sites (Bmax) from (178 +/- 9) x 10(3) (control) to (324 +/- 15) x 10(3) sites per PMN and lowered the apparent dissociation constant (Kd) from 9.6 +/- 0.8 (control) to 3.3 +/- 0.3 nM. In Ca(2+)-depleted cells, staurosporine induced similar changes in Kd values, whereas the Bmax. increased by 60%. Treatment of PMNs with 500 nM staurosporine enhanced PDBu binding in the particulate fraction by 120 +/- 7% and decreased PDBu binding in the soluble fraction by 69 +/- 4%, whereas PKC histone-phosphorylating activity of both fractions was almost completely inhibited. Incubation of staurosporine-pretreated particulate fractions with soluble fractions enriched the particulate fraction in PDBu-binding sites at the expense of the soluble fraction, without altering the binding affinity of PDBu for either fraction. Stimulation of PMNs with chemotactic N-formyl peptides induced a transient increase in PDBu binding. This effect was potentiated by approx. 52% by staurosporine. These results show that, in addition to its interference with PKC protein-phosphorylating activity, staurosporine enhances both PDBu-binding capacity and affinity to PMNs, through a mechanism involving Ca(2+)-dependent and -independent processes. Alterations of PDBu binding to soluble and particulate fractions suggest that the enhanced binding capacity in intact PMNs may be due to translocation of PDBu receptors, presumably PKC units. This phenomenon may affect PKC-dependent cellular responses mediated by physiological stimulation.

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Phorbol 12,13-dibutyrate binding to intact human platelets. The role of cytosolic free Ca2+.

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