High glucose rapidly activates the nitric oxide/cyclic nucleotide pathway in human platelets via an osmotic mechanism

Summary

The aim was to evaluate whether high glucose influences the nitric oxide (NO)/cyclic nucleotide pathway in human platelets via osmotic stress and to clarify the role of protein kinase C (PKC) in this phenomenon. The study was carried out on 33 healthy lean male volunteers, aged 28.3±1.3 years. NO synthesis was detected as L-citrulline production after L-arginine incubation in platelets incubated for 6 min with 22.0 mM D-glucose and isoosmolar concentrations of mannitol, L-glucose and fructose. To evaluate the influence of PKC, experiments with D-glucose and mannitol were repeated in the presence of the PKC-beta selective inhibitor LY379196, and NO synthesis was detected after a 6-min incubation with phorbol 12-mirystate 13-acetate (PMA), a non-selective PKC activator. Platelet content of guano-sine-3’, 5’-cyclic monophosphate (cGMP) and adenosine-3’, 5’-cyclic monophosphate (cAMP) was measured by radio-immunoassay in platelets incubated with iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose. NO-dependence of cyclic nucleotide enhancements was evaluated by inhibiting NO synthase and guanylate cyclase. Platelet aggregation to ADP and collagen was evaluated in Platelet-Rich Plasma (PRP) in the presence of a 6-min incubation with D-glucose and mannitol, both without and with LY379196 and the guany-late cyclase inhibitor (H-[1, 2, 4]Oxadiazolo [4, 3-a]quinoxaline-1-one)(ODQ). Iso-osmolar concentrations of D-glucose, mannitol, L-glucose and fructose, and PMA increased NO production (p=0.0001). Effects of D-glucose and mannitol were blunted by LY379196. D-glucose and mannitol enhanced platelet cGMP and cAMP (p=0.0001) with a mechanism blunted by NO synthase and guanylate-cyclase inhibition, but did not modify platelet aggregation. In conclusion, glucose activates the NO/cyclic nucleotide pathway in human platelets with an osmotic mechanism mediated by PKC-beta.

Keywords

Glucose - platelets - nitric oxide - osmotic stress - protein kinase C

• References

• 1 Loscalzo J. Nitric oxide insufficiency, platelet activation and arterial thrombosis. Circ Res 2001; 88: 756-62.
• 2 Trovati M, Anfossi G, Massucco P. et al. Insulin stimulates nitric oxide synthesis in human platelets and, through nitric oxide, increases platelet concentrations of both guanosine-3’,5’-cyclic monophosphate and adenosine-3’,5’-cyclic monophosphate. Diabetes 1997; 46: 742-9.
• 3 Way KJ, Katai N, King GL. Protein Kinase C and the development of diabetes vascular complications. Diabetic Medicine 2001; 18: 945-9.
• 4 Idris I, Gray S, Donnelly R. Protein kinase C activation: isoenzyme-specific effects on metabolism and cardiovascular complications in diabetes. Diabetologia 2001; 44: 659-73.
• 5 Kroll MH, Sullivan R. Mechanisms of platelet activation. In Thrombosis and Hemorrhage. Loscalzo J and Schafer AI editors, Williams & Wilkins; Baltimore, USA: 1998: 261-91.
• 6 Assert R, Scherk G, Bumbure A. et al. Regulation of protein kinase C by short term hyperglycaemia in human platelets in vivo and in vitro. Diabetologia 2001; 44: 188-95.
• 7 Anfossi G, Massucco P, Mularoni E. et al. Organic nitrates and compounds that increase intraplatelet cyclic guanosine monophosphate (cGMP) levels enhance the anti-aggregating effects of the stable prostacyclin analogue Iloprost. Prostaglandins Leukot Essent Fatty Acids 1993; 49: 839-45.
• 8 Maurice DH, Haslam RJ. Molecular basis of the synergistic inhibition of platelet function by nitrovasodilators and activators of adenylate cyclase:inhibition of cyclic AMP breakdown by cyclic GMP. Mol Pharmacol 1990; 37: 671-81.
• 9 Ling S, Little P, Williams I MR. et al. High glucose abolishes the antiproliferative effect of 17β -estradiol in human vascular smooth muscle cells. Am J Physiol Endocrinol Metab 2002; 282: E746-E751.
• 10 Fleming I, Busse R. Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 2003; 284: R1-R12.
• 11 Elzagallaai A, Rosé SD, Trifarò JM. Platelet secretion induced by phorbol esters stimulation is mediated through phosphorylation of MARCKS: a MARCKSderived peptide blocks MARCKS phosphorylation and serotonin release without affecting pleckstrin phosphorylation. Blood 2000; 95: 894-902.
• 12 Palmer RMJ, Ferrige AG, Moncada S. A novel citrulline- forming enzyme implicated in the formation of nitric oxide by vascular endothelial cells. Biochem Biophys Res Commun 1989; 158: 348-52.
• 13 Queen LR, Xu B, Horinouchi K. et al. β 2 -Adrenoceptors activate nitric oxide synthase in human platelets. Circ.Res 2000; 87: 39-44.
• 14 Born GVR. Aggregation of blood platelets by adenosine diphosphate and its reversal. Nature 1962; 194: 927-9.
• 15 Weiss HJ, Rogers J. Thrombocytopenia due to abnormalities in platelet release reaction. Blood 1972; 39: 2-8.
• 16 Trovati M, Mularoni EM, Burzacca S. et al. Impaired insulin-induced platelet antiaggregating effect in obesity and in obese NIDDM patients. Diabetes 1995; 44: 1318-22.
• 17 Schwarzer E, Turrini F, Ulliers D. et al. Impairment of macrophage function after ingestion of Plasmodium falciparum-infected erythocytes or isolated malarial pigment J Exp Med. 1992; 176: 1033-41.
• 18 Trovati M, Anfossi G. Insulin, insulin resistance and platelet function: similarities with insulin effects on cultured vascular smooth muscle cells. Diabetologia 1998; 41: 609-22.
• 19 Shaffler A, Arndt H, Scholmerich J. et al. Amelioration of hyperglycemic and hyperosmotic induced vascular dysfunction by in vivo inhibition of protein kinase C and p38MAP kinase pathway in the rat mesenteric microcirculation. Eur J Clin Invest 2000; 30: 586-93.
• 20 Park CW, Kim JH, Lee JH. et al. High glucose-induced intercellular adhesion molecule-1 (ICAM-1) expression through an osmotic effect in rat mesangial cells is PKC-NF-Kappa B-dependent. Diabetologia 2000; 43: 1544-53.
• 21 Miyata Y, Asano Y, Muto S. Hyperosmotic mannitol activates basolateral NHE in proximal tubule from P-glycoprotein null mice. Am J Physiol (Renal Physiol) 2002; 202: F718-F729.
• 22 Hirata K, Kuroda R, Sakoda T. et al. Inhibition of endothelial nitric oxide synthase activity by protein kinase C. Hypertension 1995; 25: 180-5.
• 23 Huang Q, Yuan Y. Interaction of PKC and NOS in signal transduction of microvascular hyperpermeability. Am J Physiol (Heart Circ Physiol) 1997; 273: H2442-H2451.
• 24 Berck BC, Corson MA, Peterson TE. et al. Protein kinases are mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow. J Biomech 1995; 28: 1439-50.
• 25 Wedgwood S, Bekker JM, Black SM. Shear stress regulation of endothelial NOS in fetal pulmonary arterial endothelial cells involves PKC. Am J Physiol (Lung Cell Mol Physiol) 2001; 281: L490-8.
• 26 Hortelano S, Genaro AM, Bosca L. Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide. J Biol Chem 1992; 267: 24937-40.
• 27 Mckenna TM, Li S, Tao S. PKC mediates LPS- and phorbol-induced cardiac cell nitric oxide synthase activity and hypocontractility. Am J Physiol (Heart Circ Physiol 38) 1995; 269: H1891-H1898.
• 28 Scott-Burden TE, Elizondo E, Ge T. et al. Simultaneous activation of adenyl cyclase and protein kinase induce production of nitric oxide by vascular smooth muscle cells. Mol Pharmacol 1994; 46: 274-82.
• 29 Rosado JA, Sage SO. Protein kinase C activates non-capacitative calcium entry in human platelets. J Physiol 2000; 529: 159-69.
• 30 Gresele P, Guglielmini G, De Angelis M. et al. Acute, short-term hyperglycemia enhances shearstress induced platelet activation in patients with Type II diabetes mellitus. J Am Coll Cardiol 2003; 41: 1013-20.
• 31 Keating FK, Sobel BE, Schneider DJ. Effects of increased concentrations of glucose on platelet reactivity in healthy subjects and in patients with and without diabetes mellitus. Am J Cardiol 2003; 92: 1362-5.
• 32 Chlopicki S, Olszanecki R, Janiszewski M. et al. Functional role of NADPH oxidase in activation of platelets. Antioxid Redox Signal 2004; 6: 691-8.
• 33 Vasquez-Vivar J, Kalyanararaman B, Martasek P. et al. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U.S.A. 1998; 95: 9220-5.
• 34 Katusic ZS. Vascular endothelial dysfunction: does tetrahydrobiopterin play a role?. Am J Physiol Heart Circ Physiol 2001; 281: H981-H986.
• 35 Gryglewski RJ, Palmer RM, Moncada S. Superoxide anion is involved in the breakdown of endotheliumderived vascular relaxing factor. Nature 1986; 320: 454-6.
• 36 Trovati M, Anfossi G. Mechanisms involved in platelet hyperactivation and platelet-endothelium interrelationships in diabetes mellitus. Curr Diab Rep 2002; 2: 316-22.
• 37 Vinik I A, Erbas T, Sun Park T. et al. Platelet dysfunction in Type 2 diabetes. Diabetes Care 2001; 24: 1476-85.