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Septic shock is the most common cause of death in intensive care units. Although the underlying pathogenetic mechanisms are complex and not completely understood, enhanced formation of nitric oxide (NO) contributes importantly tocirculatory shock pathogenesis. Endogenous NO is formed from L-arginine by NO synthase (NOS). NO has been implicated in a number of physiological and pathological processes including immune and inflammatory responses and regulation of blood pressure. Several isoforms of NOS has been recognized. The constitutive form(cNOS) is believed to be present in neruons, endothelial cells, and platelets and its activity is Ca2+-dependent. The inducible form (iNOS) is found in macrophages, glial cells, hepatocytes and vascular smooth muscle cells andits activity is Ca2+-independent. While low level of NO is produced by cNOS, the iNOS can be stimulated by LPS and a number of cytokines to produce large amounts of NO which, with regards to circulation, can contribute to circulatory failure through several loci such as hypotension, vascular hyporeactivity to vasocons
trictor agents, myocardial dysfunction and maldistribution in organ blood flow . Although the activityof iNOS is Ca2+-independent, recent evidence has suggested an intimate relationship between intracellular calcium concentration ( [Ca2+]i )and the induction of iNOS protein. Using both in vivo and in vitro paradigms and agents known to affect [Ca2+]i, the present project sought to examine the effects of [Ca2+]i manipulation on NO production and blood pressure regulation. To provide a relatively homogeneous cells population, vascular smooth muscle cells (VSMCs) were cultured from the aorta of Sprague-Dawley rats. The standard procedure to induce iNOS in vascular smooth muscle cells (VSMCs) by stimulation with LPS and INF( was employed. Possible modulatroy effects of changing [Ca2+]i with dantrolene ( an inhibitor of Ca2+ release from sarcoplasmic reticulum) and the calcium channel blocker nefedipine (1 to 100 (M) were studied. NO level was indirectly monitored by measuring the level of its stable metabolite nitrite in the culture medium. Cell viability was monitored by the MTT assay. For in vivo experiments, rats were anesthetized by pentobarbital (50 mg /Kg i. p.) and cannulated for direct measurements of cardiovascular parameters through the carotid artery and drug administration through the jugular vein. In addition, in vitro experiments on tension recording were carried out using isolated rings from the descending aorta of rats treated with dantrolene and LPS. Results indicated that in VSMCs (1) Stimulation by LPS for48 hr increased the production of NO. That induction of iNOS accounted for this effect was revealed by immunocytochemical staining with iNOS antibody and the reduction of LPS-elicited NO production by aminoguanidine ( an iNOS inhibitor). (2) While interferon-( (IFN-() alone had no effect on NO production, it significantly potentiated the effect of LPS on NO production. (3) Both nefedipine (1 to 100 (M) and dantrolene (1 to 100 (M) dose-dependently suppressed the production of NO induced by LPS plus IFN-(. and (4) Low dose of dantrolene (1 (M) potentiated the inhibitory effect of nefedipine on NO production and iNOS protein induction. Results from in vivo experiments also indicated systemic administration of LPS (10 mg/ Kg, i.v.) caused a prolonged hypotension and tachycardia. Dantrolene (1 mg/Kg, i.v.) attenuated the LPS-induced late hypotension but not tachycardia. Tension recording of isolated aortic rings indicated an impaired contrac tile responses in rats treated with LPS. However, dantrolene did not result in a significant improvement on this impairment by LPS. In conclusion, results from VSMCs cultures and in vivo experiments suggested a casual relationship bet ween [Ca2+]i and the induction of iNOS protein. Our results also suggest that combination use of low dose of dantrolene and nifedipine may have clinical potential in protection against hypotension during sepsis shock.
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