Increased nitric oxide synthesis in uraemic platelets is dependent on L-arginine transport via system y(+)L.
Bleeding tendency in uraemic patients seems to be related to alterations in the activity of the L-arginine-nitric oxide (NO) signalling pathway in platelets. We have reported previously that L-arginine influx into human platelets is mediated by the high-affinity cationic amino acid transport system y(+)L. In the present study we examined the dependency of nitric oxide synthase (NOS) activity on L-arginine transport in platelets isolated from healthy controls and uraemic patients on haemodialysis. We investigated basal and ADP-stimulated NOS activity, as reflected by the conversion of L-[(3)H]arginine to L-[(3)H]citrulline, in platelets obtained from healthy controls and uraemic patients on haemodialysis. To determine whether NOS activity depended on L-arginine transport, we analysed the effects of competitive inhibitors of L-arginine transport via system y(+)L on NOS activity. Basal NOS activity was increased from 0.21+/-0.06 to 0.7+/-0.2 pmol/10(8) platelets ( n=9, P<0.05) in uraemic patients. Stimulation by ADP (10 micro M) significantly increased NOS activity (inhibitable by L-NAME) in control platelets (252%) but failed to increase further the elevated NOS activity in uraemic platelets. Homocysteine and L-leucine, competitive inhibitors of system y(+)L, markedly inhibited NOS activity in uraemic platelets. These observations indicate that platelets from uraemic patients on haemodialysis generate more NO than control platelets and that entry of L-arginine via system y(+)L is most likely rate-limiting for platelet NO production in chronic renal failure.
Brunini
T M C
TM
Departamento de Farmacologia e Psicobiologia, Instituto de Biologia, Av. 28 de Setembro 87, CEP 20551-030, Rio de Janeiro, Brazil. cribeiro@physiol.ox.ac.uk
Yaqoob
M M
MM
Novaes Malagris
L E
LE
Ellory
J C
JC
Mann
G E
GE
Mendes Ribeiro
A C
AC
D000244:Adenosine Diphosphate / Q000494:pharmacology; D027241:Amino Acid Transport System y+L / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport; D001792:Blood Platelets / Q000378:metabolism*; D016022:Case-Control Studies; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D006710:Homocysteine / Q000494:pharmacology; D006801:Humans; D007930:Leucine / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis* / Q000097:blood*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D006435:Renal Dialysis; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D014511:Uremia / Q000097:blood* / Q000628:therapy
D027241:Amino Acid Transport System y+L; D004791:Enzyme Inhibitors; D013439:Sulfhydryl Reagents; D006710:Homocysteine; D009569:Nitric Oxide; D000244:Adenosine Diphosphate; D001120:Arginine; D019001:Nitric Oxide Synthase; D007930:Leucine; D005033:Ethylmaleimide; D019331:NG-Nitroarginine Methyl Ester