Na+/H+ exchange activity induced by thrombin is not inhibited by protein kinase inhibitors, staurosporine, K-252a, H-7 and sphingosine, in human platelets.
In human platelets stimulated with thrombin (40 mU/ml), Na+/H+ exchange activity [the ethylisopropylamiloride (EIPA)-sensitive increase of cytoplasmic pH (pHc)] and protein kinase C (PKC) activity [phosphorylation of 47 kDa protein (P47), a substrate for PKC] were determined in the presence of protein kinase inhibitors, staurosporine (0.05-1 microM), K-252a (0.5-10 microM), H-7 (100 microM) and sphingosine (20-40 microM). Staurosporine and K-252a completely blocked PKC activity. H-7 and sphingosine reduced the P47 phosphorylation to 64% and 35%, respectively. On the contrary, the thrombin-induced pHc increase was not inhibited by staurosporine, K-252a or H-7. Sphingosine elevated the resting pHc by 0.26-0.42 independently of the Na+/H+ exchanger and inhibited the thrombin-induced pHc increase. However, after the resting pHc elevated by sphingosine had been reduced to the initial level by adding sodium propionate, the thrombin-induced pHc increase was observed again. These results suggested that sphingosine inhibited the thrombin-induced pHc increase by elevating the resting pHc. Thus, we concluded that the Na+/H+ exchanger was activated by thrombin through a pathway independent of PKC.
Japanese Red Cross Central Blood Center, Tokyo.
A
A
Ogawa
Y
Y
Ishikawa
S
S
Sasakawa
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000470:Alkaloids / Q000494:pharmacology*; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology*; D002352:Carrier Proteins / Q000097:blood*; D006801:Humans; D006859:Hydrogen / Q000097:blood; D026121:Indole Alkaloids; D007546:Isoquinolines / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D010879:Piperazines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012964:Sodium / Q000097:blood; D017923:Sodium-Hydrogen Exchangers; D013110:Sphingosine / Q000494:pharmacology*; D019311:Staurosporine; D013917:Thrombin / Q000494:pharmacology*
D000470:Alkaloids; D002227:Carbazoles; D002352:Carrier Proteins; D026121:Indole Alkaloids; D007546:Isoquinolines; D010879:Piperazines; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; D006859:Hydrogen; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; C049985:staurosporine aglycone; D012964:Sodium; D011493:Protein Kinase C; D013917:Thrombin; D019311:Staurosporine; D013110:Sphingosine; C039614:ethylisopropylamiloride