title: 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.
authors.affiliation: Hypertension Research Center, University of Medicine & Dentistry of New Jersey, Newark 07103-2714.
authors.forename: J
authors.initials: J
authors.lastname: Takaya
authors.forename: M
authors.initials: M
authors.lastname: Kimura
authors.forename: N
authors.initials: N
authors.lastname: Lasker
authors.forename: A
authors.initials: A
authors.lastname: Aviv
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D013917:Thrombin / Q000494:pharmacology
chemical_list: D015240:Phorbol 12,13-Dibutyrate; D013917:Thrombin; D002118:Calcium