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

J. Takaya, M. Kimura, N. Lasker, Abraham Aviv

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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. Ca2+-depleted platelets demonstrated a significant decline in the maximal binding capacity (B(max)), an increase in equilibrium dissociation constant (K(d)) and a decrease in the Hill coefficient (h), suggesting the presence of Ca2+-sensitive and Ca2+-insensitive populations of PdBu-binding sites. In 1 mM-Ca2+ buffer, thrombin (0.1 NIH unit/ml) and ionomycin (0.5 μM) evoked a rise in [Ca2+](i) to approx. 300-500 nM, associated with a significant decline in K(d), but without an apparent effect on B(max). No effect of thrombin was observed on PdBu binding in Ca2+-depleted platelets. Inhibition of protein kinase C (PKC) by H7 was associated with a greater thrombin-evoked [Ca2+](i) transient and a decline in K(d). Staurosporine also decreased the K(d) for PdBu binding. We propose that this effect of the PKC inhibitors on the K(d) 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 B(max) for PdBu binding.

Original languageEnglish (US)
Pages (from-to)411-415
Number of pages5
JournalBiochemical Journal
Volume278
Issue number2
DOIs
StatePublished - Jan 1 1991

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All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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