The activity of constitutive nitric oxide synthase is increased by the pathway cAMP/cAMP-activated protein kinase in human platelets. New insights into the antiaggregating effects of cAMP-elevating agents.
Human platelets synthesize nitric oxide (NO) through an endothelial-type NO synthase (ecNOS) activated also by substances enhancing 3',5'-cyclic adenosine monophosphate (cAMP) concentrations, such as catecholamines, beta-adrenoceptor agonists and adenosine. To verify whether cAMP directly activates ecNOS through the cAMP-dependent protein kinase A (PKA), we evaluated (i) the influence of 8-Br-cAMP, adenosine and forskolin on ecNOS activity and phosphorylation at Ser(1177) and (ii) the effect of PKA inhibition on ecNOS activity. Platelets from 10 healthy male volunteers were used for aggregation studies and measurement of NOS activity (conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline) following exposure to 8-Br-cAMP, adenosine and forskolin, both in the absence and in the presence of the PKA inhibitor Rp-cAMPS (100 micromol/l). The phosphorylation of the PKA substrate vasodilator-stimulated phosphoprotein (VASP) at Ser(157) and Ser(239) and of ecNOS at Ser(1177) was evaluated by Western blot. NOS activity (pmol L-citrulline/10(8) platelets) increased from 0.090+/-0.002 to 0.148+/-0.013 with 500 micromol/l 8-Br-cAMP (p<0.0001), to 0.140+/-0.008 with 30 micromol/l adenosine (p<0.0001) and to 0.140+/-0.009 with 10 micromol/l forskolin (p<0.0001). Rp-cAMPS decreased baseline NOS activity from 0.093+/-0.001 to 0.075+/-0.006 (p<0.02) and prevented the stimulation by 8-Br-cAMP, adenosine and forskolin. Platelet exposure to 8-Br-cAMP and forskolin, beside the phosphorylation of the specific PKA substrate VASP, markedly increased the expression of ecNOS protein phosphorylated at Ser(1177). The study shows that NOS activity of human platelets is increased by the cAMP/PKA pathway which is involved in NO synthesis induced by adenosine, forskolin and potentially by every antiaggregating substance enhancing intraplatelet cAMP via receptor-dependent and -independent mechanisms.
Russo
Isabella
I
Diabetes Unit, Department of Clinical and Biological Sciences of the Turin University, Ospedale San Luigi Gonzaga Hospital, Regione Gonzole, 10, Orbassano 10043, Turin, Italy.
Doronzo
Gabriella
G
Mattiello
Luigi
L
De Salve
Alessandro
A
Trovati
Mariella
M
Anfossi
Giovanni
G
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000241:Adenosine / Q000494:pharmacology; D000328:Adult; D001792:Blood Platelets / Q000201:enzymology*; D015815:Cell Adhesion Molecules / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008297:Male; D008840:Microfilament Proteins; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D015854:Up-Regulation; D000098662:Vasodilator-Stimulated Phosphoprotein
D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D008840:Microfilament Proteins; D010750:Phosphoproteins; D010975:Platelet Aggregation Inhibitors; D000098662:Vasodilator-Stimulated Phosphoprotein; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases; D000241:Adenosine