Shinya Sakita

ACUTE VIGOROUS EXERCISE ATTENUATES SENSITIVITY OF PLATELETS TO NITRIC OXIDE

We tested whether inhibition of platelet activation by nitric oxide (NO) might be altered by strenuous exercise. Sixteen healthy male non-smokers, aged 20-26 years, underwent treadmill testing. All subjects reached Bruce stage IV without chest pain or abnormal ST-T wave changes. Platelet aggregation by Borns method and cyclic GMP accumulation in the washed platelets were determined before and immediately after exercise. Dose-response curves for platelet aggregation by collagen were constructed both in the absence and presence of 2 microM SIN-1, an NO donor, to quantify the antiaggregation effects of NO. After exercise, platelet aggregation by collagen was modestly enhanced and inhibition of platelet aggregation by SIN-1 was significantly attenuated by exercise. This attention was accompanied by a blunted cyclic GMP response of the platelets to the NO donor. We conclude that impaired sensitivity of the platelets to NO, in addition to the enhancement of platelet aggregation, may partially explain the epidemiological evidence that acute strenuous exercise precipitates coronary events.

Acute vigorous exercise primes enhanced NO release in human platelets

Activation of platelets by acute vigorous exercise has been demonstrated by various parameters, including an increase in agonist-induced platelet [Ca2+]i levels. However, direct evidence is lacking regarding how acute exercise affects platelet-derived NO. Twenty-three healthy male non-smokers (21-59 years) underwent a symptom-limited treadmill exercise test. Washed platelets were prepared from blood samples obtained before and immediately after exercise. All subjects completed at least Bruce stage 2 and were each negative for ischemia. With a low dose (2 microg/ml) of collagen, NO release from washed platelets, detected by the NO-selective microelectrode, was significantly increased after exercise (pmols/10(8) platelets, before: 0.64+/-0.11, after: 1.03+/-0.18; P<0.005) without changes in aggregation ability. This enhanced NO release was accompanied by increased platelet [Ca2+]i levels (before: 232+/-25, after: 296+/-37; P<0.01). With a high dose (5 or 10 microg/ml) of collagen, NO release and aggregation were both modestly, but significantly, enhanced after exercise. The exercise-induced enhancement of platelet NO release in response to collagen was also suggested by increase in platelet cyclic guanosine monophosphate accumulation and augmenting effect of N(G)-monomethyl-L-arginine on platelet aggregation. In summary, acute strenuous exercise primes enhanced NO release and may play a protective role against exercise-induced activation of platelets in normal subjects.

Ibudilast modulates platelet-endothelium interaction mainly through cyclic GMP-dependent mechanism.

3-Isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast) has been widely used in Japanese clinics for its antiasthmatic and antithrombotic effects. We investigated the mechanisms involved in the antiplatelet effects of the agent, specifically focusing on platelet-endothelium interaction. Ibudilast inhibits both phosphodiesterase (PDE) 3 and 5, the two major PDE isoforms of human platelets, with an IC50 of 31 and 2.2 microM, respectively. Cyclic guanosine monophosphate (GMP) accumulation in washed human platelets exposed to ibudilast alone increased significantly only at high concentrations of the agent (100 microM), whereas > or = 1 microM ibudilast enhanced cyclic GMP levels in the platelets cocultured with bovine aorta endothelial cells (ECs). In contrast, ibudilast enhanced cyclic AMP accumulation only at 100 microM, either with or without ECs. The synergistic effect of ibudilast and EC on cyclic nucleotide accumulation also was demonstrated by the inhibitory capability of the drug and the cells on platelet aggregation. The synergism between ibudilast and aspirin-pretreated ECs was more pronounced than that between ibudilast and N(omega)-nitro-L-arginine (L-NNA)-pretreated ECs. Ibudilast affected neither ATP diphosphohydrolase activity nor NO release from EC up to a concentration of 10 microM. We conclude that ibudilast exhibits antiplatelet properties mainly by inhibiting PDE5 to potentiate antiplatelet function of endothelium-derived NO.

Effect of Nifedipine on Cyclic Gmp Turnover in Cultured Coronary Smooth Muscle Cells

We investigated the effects of nifedipine on cyclic GMP turnover and the pertinent enzyme activities in cultured coronary smooth muscle cells (SMC). Nifedipine at high concentrations slightly decreased basal soluble guanylate cyclase activity and inhibited the action of sodium nitroprusside (SNP) but had no effect on the particulate form of the enzyme. In contrast, nifedipine inhibited cyclic GMP hydrolysis by directly inhibiting the partially purified calmodulin-stimulated isoform of phosphodiesterase (type I PDE) with IC50 of 4.2 microM. Nifedipine > or = 1.0 microM enhanced cyclic GMP accumulation in response to 1.0 microM SNP, although nifedipine alone exerted no influence on cyclic GMP levels. Enhancement of cyclic GMP accumulation by nifedipine in response to SNP was not affected by BAY K 8644, a calcium channel agonist. These properties may be shared by other dihydropyridines since nicardipine and nisoldipine also inhibited type I PDE with similar IC50. However, some other structurally unrelated calcium channel blockers, diltiazem and verapamil, had little effect on cyclic nucleotide hydrolysis or on cyclic GMP accumulation in response to SNP. Nifedipine may synergistically enhance cyclic GMP accumulation in response to nitric oxide (NO)-releasing agents by directly inhibiting type I PDE in coronary SMC. Such effects of nifedipine may partly contribute to coronary vasodilation and prevention of coronary spasm in patients with ischemic heart disease.

Impaired NO release from bovine aortic endothelial cells exposed to activated platelets

We have previously shown that aggregated human platelets elicited a decrease in intracellular adenosine triphosphate (ATP), enhanced adenosine egress and damage to mitochondria in bovine aortic endothelial cells (ECs). To test whether such metabolic and ultrastructural changes could be associated with functional impairment of ECs, we investigated the effects of activated platelets on nitric oxide (NO) and prostacyclin release, and on the antiaggregation property of ECs. Pretreatment of ECs with aggregated platelets transiently stimulated basal NO release while prolonged (> or = 30 min) exposure dose-dependently inhibited NO release, both basal and in response to ATP or serotonin, with NO synthase activity being attenuated in these cells. Supplementary L-arginine (L-A) restored NO release completely. Prostacyclin release was also stimulated transiently but not affected by prolonged pretreatment. The antiaggregation property of ECs was attenuated by pretreatment with activated platelets but restored with L-A supplement. Although the effects of activated platelets and 0.5 mM acetylsalicylic acid (ASA) to attenuate the antiaggregation property of ECs were additive, activated platelets had no effect on ECs treated with 0.2 mM N omega-nitro-L-arginine (L-NA), suggesting a common mechanism. We conclude that prolonged exposure to aggregated platelets may affect the antiaggregation property of ECs by directly inhibiting NO synthesis, which may be normalized by L-A supplementation.

Co-culture of Platelets with Activated J774.1 Cells: Effects on Platelet Aggregation and Cyclic Nucleotides

Recent in situ hybridization studies revealed the presence of inducible nitric oxide synthase (iN0S)-positive cells in deendothelialized lesion following balloon injury in animal models. Since iNOS-positive cells existed particularly at the luminal surface of the lesion,’ we investigated the interaction between platelets and iNOS-positive cells, focusing on platelet aggregation and cyclic nucleotides in the platelets directly exposed to the activated cells. This was made possible by using impedance aggregometry for measuring platelet aggregation and guanineor adenine-prelabeling method for determining platelet cyclic nucleotides. We used murine macrophage cell line 5774.1 cells as a model for iNOS-positive cells since induction of iNOS has been well documented in 5774.1 cells activated by lipopolysaccharide (LPS) and/or cytokines.2