CONCENTRATION AND REGULATION OF CYCLIC-NUCLEOTIDES, CYCLIC-NUCLEOTIDE-DEPENDENT PROTEIN-KINASES AND ONE OF THEIR MAJOR SUBSTRATES IN HUMAN PLATELETS - ESTIMATING THE RATE OF CAMP-REGULATED AND CGMP-REGULATED PROTEIN-PHOSPHORYLATION IN INTACT-CELLS

被引:146
作者
EIGENTHALER, M [1 ]
NOLTE, C [1 ]
HALBRUGGE, M [1 ]
WALTER, U [1 ]
机构
[1] UNIV WURZBURG, MED KLIN, KLIN BIOCHEM LAB, JOSEF SCHNEIDER STR 2, W-8700 WURZBURG, GERMANY
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1992年 / 205卷 / 02期
关键词
D O I
10.1111/j.1432-1033.1992.tb16803.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Vasodilators capable of elevating cAMP or cGMP inhibit the activation of human platelets and stimulate the phosphorylation of a 46-kDa protein (vasodilator-stimulated phosphoprotein, VASP) mediated by cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). The availability of purified proteins and specific antisera against VASP, PKG and the catalytic subunit of PKA enabled us to measure and estimate the concentration of these regulatory proteins in intact human platelets. In addition, the rate of PKA- and PKG-mediated VASP phosphorylation in intact human platelets was estimated. For these calculations, a homogeneous population of human platelets and a homogeneous intracellular distribution of proteins and second messengers was assumed. Unstimulated washed human platelets contain 4.4-mu-M cAMP and 3.1-mu-M catalytic subunit of PKA, which is equivalent to 6.2-mu-M cAMP-binding sites due to PKA. Unstimulated washed human platelets also contain 0.4-mu-M cGMP and 7.3-mu-M PKG monomer, equivalent to 14.6-mu-M cGMP-binding sites due to the PKG. The intracellular concentration of VASP in platelets was estimated to be 25-mu-M. Treatment of washed human platelets with 10-mu-M (or 10 nM) prostaglandin E1 (PGE1) elevated the intracellular cAMP concentration to 27-mu-M (10-mu-M with 10 nM PGE1) within 30 s, accompanied by a rapid, up to 55% (35%), conversion of VASP from the dephosphorylated form (46-kDa protein) to the phosphorylated form (50-kDa protein). Treatment of washed human platelets with 100-mu-M (or 1-mu-M) sodium nitroprusside elevated the platelet cGMP level to 4-mu-M (0.9-mu-M with 1-mu-M sodium nitroprusside) within 2 min, accompanied by a less-rapid VASP phosphorylation of 45% (27% with 1-mu-M sodium nitroprusside). PGE1 and sodium nitroprusside had no significant effect on human platelet cGMP or cAMP levels, respectively. The results suggest for human platelets that relatively small increases in cAMP levels are required for activation of most of PKA, whereas even several-fold increases in platelet cGMP levels are capable of stimulating only a small fraction of total PKG. This interpretation was also supported by phosphorylation experiments with purified VASP, PKG and catalytic subunit of PKA. The results also support the hypothesis that in human platelets both cAMP/PKA- and cGMP/PKG-regulated VASP phosphorylation are components of an efficient and sensitive signal-transduction pathway, most likely involved in the inhibition of platelet activation. In particular, the high concentration of the converter enzymes PKA and PKG suggest that rapidity in reaching steady-state levels of phosphorylation in response to cAMP- or cGMP-elevating agents is of considerable importance for the regulation of human platelets.
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收藏
页码:471 / 481
页数:11
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