AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets

Ingrid Fleming; Christian Schulz; Birgit Fichtlscherer; Bruce E. Kemp; Beate Fisslthaler; Rudi Busse

doi:10.1160/TH03-04-0228

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2003; 90(05): 863-871
DOI: 10.1160/TH03-04-0228

Platelets and Blood Cells

Schattauer GmbH

AMP-activated protein kinase (AMPK) regulates the insulin-induced activation of the nitric oxide synthase in human platelets

Ingrid Fleming

¹Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany

,

Christian Schulz

¹Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany

,

Birgit Fichtlscherer

¹Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany

,

Bruce E. Kemp

²St.Vincent’s Institute of Medical Research, Fitzroy, Victoria, Australia

,

Beate Fisslthaler

¹Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany

,

Rudi Busse

¹Institut für Kardiovaskuläre Physiologie, Klinikum der J.W. Goethe-Universität, Frankfurt am Main, Germany

› Author Affiliations

Abstract

Summary

Little is known about the signaling cascades that eventually regulate the activity of the endothelial nitric oxide synthase (eNOS) in platelets. Here, we investigated the effects of insulin on the phosphorylation and activation of eNOS in washed human platelets and in endothelial cells.

Insulin activated the protein kinase Akt in cultured endothelial cells and increased the phosphorylation of eNOS on Ser¹¹⁷⁷but failed to increase endothelial cyclic GMP levels or to elicit the relaxation of endothelium-intact porcine coronary arteries. In platelets, insulin also elicited the activation of Akt as well as the phosphorylation of eNOS and initiated NO production which was associated with increased cyclic GMP levels and the inhibition of thrombin-induced aggregation. The insulin-induced inhibition of aggregation was accompanied by a decreased Ca²⁺response to thrombin and was also prevented by N^ωnitro-L- arginine. In platelets, but not in endothelial cells, insulin induced the activation of the AMP-activated protein kinase (AMPK), a metabolic stress-sensing kinase which was sensitive to the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin and the AMPK inhibitor iodotubercidin. Moreover, the insulin-mediated inhibition of thrombin-induced aggregation was prevented by iodotubercidin. Insulin-independent activation of the AMPK using 5-aminoimidazole-4-carboxamide ribonucleoside, increased platelet eNOS phosphorylation, increased cyclic GMP levels and attenuated platelet aggregation.

These results highlight the differences in the signal transduction cascade activated by insulin in endothelial cells and platelets, and demonstrate that insulin stimulates the formation of NO in human platelets, in the absence of an increase in Ca²⁺, by activating PI3-K and AMPK which phosphorylates eNOS on Ser¹¹⁷⁷.

Keywords

Aggregation - 5-aminoimidazole-4-carboxamide ribonucleoside - iodotubercidin - endothelium - calcium

Full Text

References

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