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Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway.

Randriamboavonjy V, Schrader J, Busse R, Fleming I - J. Exp. Med. (2004)

Bottom Line: Insulin failed to relax endothelium-intact rings of porcine coronary artery.Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner.The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

View Article: PubMed Central - PubMed

Affiliation: Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.Goethe-Universität, D-60590 Frankfurt am Main, Germany.

ABSTRACT
Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

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Insulin-stimulated and NO-dependent release of ATP and adenosine from washed human and murine platelets. (a) Effect of L-NA (300 μmol/L, 30 min), the guanylyl cyclase inhibitor, NS 2028 (10 μmol/L, 30 min), and the G kinase inhibitor, KT 5823 (1 μmol/L, 30 min), on the insulin (1 μmol/L, 10 min)-induced release of ATP from washed human platelets. (b) Effect of L-NA (300 μmol/L, 30 min) on the release of adenosine from washed human platelets incubated with either solvent (Sol) or insulin (1 μmol/L, 10 min). In some experiments, adenosine deaminase (50 U/ml) was added to the supernatant before stimulation. In each case, the ability of the supernatant to relax precontracted rings of porcine coronary artery was assessed, and the results were divided into two categories, i.e., the relaxation induced by the supernatant of insulin-stimulated platelets was >60% (responders) or relaxation was <20% (non responders). (c) Effect of insulin (1 μmol/L, 10 min) and NO (DETA NONOate, 1 μmol/L, 10 min) on the release of ATP from platelets derived from either wild-type (eNOS+/+) or eNOS−/− mice. Experiments were performed in the presence of solvent (Sol), L-NA, or Rp-8CPT-cGMPs (10 μmol/L). The results shown represent the mean ± SEM of data obtained in 6–10 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 versus the response obtained using the supernatant from insulin-stimulated platelets.
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fig4: Insulin-stimulated and NO-dependent release of ATP and adenosine from washed human and murine platelets. (a) Effect of L-NA (300 μmol/L, 30 min), the guanylyl cyclase inhibitor, NS 2028 (10 μmol/L, 30 min), and the G kinase inhibitor, KT 5823 (1 μmol/L, 30 min), on the insulin (1 μmol/L, 10 min)-induced release of ATP from washed human platelets. (b) Effect of L-NA (300 μmol/L, 30 min) on the release of adenosine from washed human platelets incubated with either solvent (Sol) or insulin (1 μmol/L, 10 min). In some experiments, adenosine deaminase (50 U/ml) was added to the supernatant before stimulation. In each case, the ability of the supernatant to relax precontracted rings of porcine coronary artery was assessed, and the results were divided into two categories, i.e., the relaxation induced by the supernatant of insulin-stimulated platelets was >60% (responders) or relaxation was <20% (non responders). (c) Effect of insulin (1 μmol/L, 10 min) and NO (DETA NONOate, 1 μmol/L, 10 min) on the release of ATP from platelets derived from either wild-type (eNOS+/+) or eNOS−/− mice. Experiments were performed in the presence of solvent (Sol), L-NA, or Rp-8CPT-cGMPs (10 μmol/L). The results shown represent the mean ± SEM of data obtained in 6–10 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 versus the response obtained using the supernatant from insulin-stimulated platelets.

Mentions: In experiments in which insulin elicited the generation of a platelet-derived relaxing factor, there was a significant, insulin-induced release of ATP from platelets (Fig. 4 a). However, ATP levels were not increased by insulin in samples (non responders) which failed to relax the arterial rings. The insulin-induced release of ATP was attenuated by L-NA and by NS2028 (10 μmol/L) and the KT 5823 (1 μmol/L). On the other hand, both the NO donor DETA-NONOate (1 μmol/L) and the cyclic GMP analogue–G kinase activator Sp-cGMPS (10 μmol/L) stimulated the release of ATP from platelets. ATP concentrations were 574.9 ± 21.25 versus 2,006.3 ± 540.7 and 1,394.0 ± 207.7 nmol/L ATP in the supernatant of solvent-stimulated platelets versus DETA-NONOate– and Sp-cGMPS–stimulated platelets (P < 0.01, n = 5–6). The levels of ATP released from these platelets did not elicit aggregation and were markedly lower than the levels detected in the supernatant from thrombin-stimulated platelets. The ATP concentration was 574.0 ± 21.3 nmol/L ATP in the supernatant of solvent-stimulated platelets versus 4,567.0 ± 794.0 nmol/L ATP in the supernatant from thrombin-stimulated platelets (P < 0.01, n = 6). Similar results were obtained when adenosine levels in the supernatant were assayed (Fig. 4 b). Moreover, adenosine levels in the supernatant were attenuated in the presence of adenosine deaminase (Fig. 4 b).


Insulin induces the release of vasodilator compounds from platelets by a nitric oxide-G kinase-VAMP-3-dependent pathway.

Randriamboavonjy V, Schrader J, Busse R, Fleming I - J. Exp. Med. (2004)

Insulin-stimulated and NO-dependent release of ATP and adenosine from washed human and murine platelets. (a) Effect of L-NA (300 μmol/L, 30 min), the guanylyl cyclase inhibitor, NS 2028 (10 μmol/L, 30 min), and the G kinase inhibitor, KT 5823 (1 μmol/L, 30 min), on the insulin (1 μmol/L, 10 min)-induced release of ATP from washed human platelets. (b) Effect of L-NA (300 μmol/L, 30 min) on the release of adenosine from washed human platelets incubated with either solvent (Sol) or insulin (1 μmol/L, 10 min). In some experiments, adenosine deaminase (50 U/ml) was added to the supernatant before stimulation. In each case, the ability of the supernatant to relax precontracted rings of porcine coronary artery was assessed, and the results were divided into two categories, i.e., the relaxation induced by the supernatant of insulin-stimulated platelets was >60% (responders) or relaxation was <20% (non responders). (c) Effect of insulin (1 μmol/L, 10 min) and NO (DETA NONOate, 1 μmol/L, 10 min) on the release of ATP from platelets derived from either wild-type (eNOS+/+) or eNOS−/− mice. Experiments were performed in the presence of solvent (Sol), L-NA, or Rp-8CPT-cGMPs (10 μmol/L). The results shown represent the mean ± SEM of data obtained in 6–10 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 versus the response obtained using the supernatant from insulin-stimulated platelets.
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fig4: Insulin-stimulated and NO-dependent release of ATP and adenosine from washed human and murine platelets. (a) Effect of L-NA (300 μmol/L, 30 min), the guanylyl cyclase inhibitor, NS 2028 (10 μmol/L, 30 min), and the G kinase inhibitor, KT 5823 (1 μmol/L, 30 min), on the insulin (1 μmol/L, 10 min)-induced release of ATP from washed human platelets. (b) Effect of L-NA (300 μmol/L, 30 min) on the release of adenosine from washed human platelets incubated with either solvent (Sol) or insulin (1 μmol/L, 10 min). In some experiments, adenosine deaminase (50 U/ml) was added to the supernatant before stimulation. In each case, the ability of the supernatant to relax precontracted rings of porcine coronary artery was assessed, and the results were divided into two categories, i.e., the relaxation induced by the supernatant of insulin-stimulated platelets was >60% (responders) or relaxation was <20% (non responders). (c) Effect of insulin (1 μmol/L, 10 min) and NO (DETA NONOate, 1 μmol/L, 10 min) on the release of ATP from platelets derived from either wild-type (eNOS+/+) or eNOS−/− mice. Experiments were performed in the presence of solvent (Sol), L-NA, or Rp-8CPT-cGMPs (10 μmol/L). The results shown represent the mean ± SEM of data obtained in 6–10 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001 versus the response obtained using the supernatant from insulin-stimulated platelets.
Mentions: In experiments in which insulin elicited the generation of a platelet-derived relaxing factor, there was a significant, insulin-induced release of ATP from platelets (Fig. 4 a). However, ATP levels were not increased by insulin in samples (non responders) which failed to relax the arterial rings. The insulin-induced release of ATP was attenuated by L-NA and by NS2028 (10 μmol/L) and the KT 5823 (1 μmol/L). On the other hand, both the NO donor DETA-NONOate (1 μmol/L) and the cyclic GMP analogue–G kinase activator Sp-cGMPS (10 μmol/L) stimulated the release of ATP from platelets. ATP concentrations were 574.9 ± 21.25 versus 2,006.3 ± 540.7 and 1,394.0 ± 207.7 nmol/L ATP in the supernatant of solvent-stimulated platelets versus DETA-NONOate– and Sp-cGMPS–stimulated platelets (P < 0.01, n = 5–6). The levels of ATP released from these platelets did not elicit aggregation and were markedly lower than the levels detected in the supernatant from thrombin-stimulated platelets. The ATP concentration was 574.0 ± 21.3 nmol/L ATP in the supernatant of solvent-stimulated platelets versus 4,567.0 ± 794.0 nmol/L ATP in the supernatant from thrombin-stimulated platelets (P < 0.01, n = 6). Similar results were obtained when adenosine levels in the supernatant were assayed (Fig. 4 b). Moreover, adenosine levels in the supernatant were attenuated in the presence of adenosine deaminase (Fig. 4 b).

Bottom Line: Insulin failed to relax endothelium-intact rings of porcine coronary artery.Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner.The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

View Article: PubMed Central - PubMed

Affiliation: Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.Goethe-Universität, D-60590 Frankfurt am Main, Germany.

ABSTRACT
Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.

Show MeSH
Related in: MedlinePlus