Takaya J, Kimura M, Lasker N, Aviv A
Hypertension Research Center, University of Medicine & Dentistry of New Jersey, Newark 07103-2714.
Biochem J. 1991 Sep 1;278 ( Pt 2)(Pt 2):411-5. doi: 10.1042/bj2780411.
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.
研究了Ca2+在调节佛波醇12,13 - 二丁酸酯（PdBu）与完整人血小板结合中的作用。胞质游离Ca2+浓度[（Ca2+]i）的改变，而非细胞外Ca2+，对佛波酯的结合参数有显著影响。Ca(2+)耗尽的血小板显示最大结合容量（Bmax）显著下降，平衡解离常数（Kd）增加，希尔系数（h）降低，提示存在对Ca(2+)敏感和不敏感的PdBu结合位点群体。在1 mM - Ca2+缓冲液中，凝血酶（0.1 NIH单位/ml）和离子霉素（0.5 microM）使[Ca2+]i升高至约300 - 500 nM，同时Kd显著下降，但对Bmax无明显影响。在Ca(2+)耗尽的血小板中未观察到凝血酶对PdBu结合有影响。H7对蛋白激酶C（PKC）的抑制与凝血酶诱发的更大的[Ca2+]i瞬变及Kd下降相关。星形孢菌素也降低了PdBu结合的Kd。我们提出PKC抑制剂对Kd的这种作用也是[Ca2+]i依赖性的。在完整血小板中的这些观察结果表明，激动剂或非激动剂诱导的[Ca2+]i升高的主要作用是增加PKC对PdBu以及可能对内源性二酰甘油的亲和力。然而，[Ca2+]i本身的升高并不会增加PdBu结合的Bmax。
