Protein kinase C stimulates dense tubular Ca2+ uptake in the intact human platelet by increasing the Vm of the Ca(2+)-ATPase pump: stimulation by phorbol ester, inhibition by calphostin C.
The effects of protein kinase C (PKC) on Ca2+ transport were investigated in human intact platelets. The indicator quin2 was used to measure the free cytoplasmic Ca2+ concentration ([Ca2+]cyt) and to search for possible PKC effects on the Ca(2+)-ATPase extrusion pump located in the plasma membrane. The Ca2+ indicator chlorotetracycline (CTC) was used to study PKC effects on the dense tubular Ca(2+)-ATPase uptake pump. The activity of PKC was stimulated by phorbol 12-myristate 13-acetate (PMA) and was inhibited with calphostin C. Neither PKC activation nor inhibition had any effect on [Ca2+]cyt or the Ca2+ extrusion pump. Substantial activation of the dense tubular pump was observed with PMA. In resting platelets bathed in 2 mM external Ca2+ giving [Ca2+]cyt = 102-106 nM, activation of PKC by PMA (100 nM) increases the rate and extent of dense tubular Ca2+ uptake to 1.62 +/- 0.35 and 1.25 +/- 0.3 times control value (respectively). The Vm of the dense tubular pump was measured by using ionomycin to manipulate [Ca2+]cyt. It is shown that PMA increases the Vm by a factor of 1.7 +/- 0.4 but has no effect on the Km value (= 180 nM). An unexpected finding was that PKC activity supports a portion of the basal activity of the dense tubular Ca2+ pump in resting platelets. Preincubation with the inhibitor calphostin C (100 nM) decreases the rate and extent of dense tubular Ca2+ uptake in resting platelets by 38 +/- 5% and 29 +/- 21% (respectively). This is due to a 28 +/- 9% decrease in the Vm of the dense tubular pump. This suggests that there is a low level of stimulation of dense tubular Ca2+ pump mediated by PKC in resting platelets.
Tao
J
J
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, FL.
Johansson
J S
JS
Haynes
D H
DH
D001792:Blood Platelets / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000187:drug effects / Q000502:physiology*; D003864:Depression, Chemical; D005470:Fluorometry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009281:Naphthalenes; D011083:Polycyclic Compounds / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D013268:Stimulation, Chemical; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D009281:Naphthalenes; D011083:Polycyclic Compounds; D011493:Protein Kinase C; D000252:Calcium-Transporting ATPases; C058819:calphostin C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium