Distribution of NOS isoforms in a porcine endotoxin shock model

Abstract

Sepsis is a major cause of morbidity and mortality. NO, an endogenous vasodilator, has been associated with the hypotension, catecholamine hyporesponsiveness, and myocardial depression of septic shock. Although iNOS is thought to be responsible for the hypotension and loss of vascular tone occurring several hours after endotoxin administration, little is known on the effects of constitutive eNOS on LPS-induced organ dysfunction. This study assessed the distribution of eNOS and iNOS in various vascular beds in conscious pigs challenged with LPS. Cardiac and regional hemodynamic parameters were recorded over 8 h in the presence and absence of aminoguanidine, a rather selective inhibitor of iNOS activity, and N-methyl-L-arginine, a nonspecific NOS inhibitor. Our data show that LPS-induced cardiac depression was associated with coronary, renal, and mesenteric vasoconstrictions and a hepatic vasodilatation. LPS also induced increases in eNOS in the heart and lungs, whereas iNOS was mostly detected in the liver. Nitrotyrosine formation was mainly detected in the lungs, with traces in the kidney, liver, and gut. Accordingly, our results suggest that the early decrease in blood pressure and cardiac depression are likely due to activated eNOS, whereas both isoforms are involved in the hepatic vasodilation. In contrast, carotid, coronary, mesenteric, and renal vasoconstrictions were significant at 5 and/ or 6 h after LPS infusion, suggesting that NO is not the primary mediator, facilitating and/or unmasking the release of vasoconstrictor mediators. Consequently, developing newer tissue- or isoform-specific NOS inhibitors can lead to novel therapeutic agents in septic shock.

Fig. 1

Hemodynamic changes recorded for 8 h on MAP, CO, SVR, PAP, HR, and dP/dt in conscious pigs subjected to LPS infused at 200 μg kg⁻¹ over 60 min *P < 0.05 vs. B. B indicates baseline.

Fig. 2

Hemodynamic changes recorded for 8 h on Car BF, Cor BF, Mes BF, Ren BF, Hep BF, Car VR, Cor VR, Mes VR, Ren VR, and Hep VR in conscious pigs subjected to LPS infused at 200 μg kg⁻¹ over 60 min *P < 0.05 vs. B. B indicates baseline; Car BF, carotid blood flow; Car VR, carotid resistance; Cor BF, coronary blood flow; Cor VR, coronary vascular resistance; Hep BF, hepatic blood flow; Hep VR, hepatic vascular resistance; Mes BF, mesenteric blood flow; Mes VR; mesenteric vascular resistance; Ren BF, renal blood flow; Ren VR, renal vascular resistance.

Fig. 3

Immunoblot analysis of lung tissue treated with LPS with and without AG, an inducible NOS inhibitor, using a monoclonal mouse antinitrotyrosine.

Fig. 4

iNOS activity recorded at 8 h in various tissues, for example, liver, kidney, lung, heart, spleen, and large and small intestine in pigs challenged with LPS at 200 μg kg⁻¹ over 60 min.

Fig. 5

eNOS recorded by immunofluorescence staining in the heart and lung in control pigs (without LPS) and treated with LPS at 200 μg kg⁻¹ over 60 min Green indicates smooth muscle actin (vasculature); magenta, eNOS; white, eNOS and smooth muscle actin (vasculature).

Fig. 6

iNOS recorded by immunofluorescence staining in the liver in control pigs (without LPS) and treated with LPS at 200 μg kg⁻¹ over 60 min Blue indicates 4′,6-diamidino-2-phenylindole (nuclei); green, smooth muscle actin (vasculature); red, iNOS.

Fig. 7

Effects of AG infused at 1 mg kg⁻¹ min⁻¹ on MAP, CO, SVR, PAP, HR, and dP/dt in eight conscious pigs treated with LPS at 200 μg kg⁻¹ over 60 min *P < 0.05 vs. LPS; ^†P < 0.05 LPS vs. B. B indicates baseline.

Fig. 8

Effects of AG infused at 1 mg kg⁻¹ min⁻¹ on Car BF, Cor BF, Mes BF, Ren BF, Hep BF, Car VR, Cor VR, Mes VR, Ren VR, and Hep VR in eight conscious pigs treated with LPS at 200 μg kg⁻¹ over 60 min *P < 0.05 vs. LPS;^†P < 0.05 LPS vs. B. B indicates baseline; Car BF, carotid blood flow; Car VR, carotid resistance; Cor BF, coronary blood flow; Cor VR, coronary vascular resistance; Hep BF, hepatic blood flow; Hep VR, hepatic vascular resistance; Mes BF, mesenteric blood flow; Mes VR; mesenteric vascular resistance; Ren BF, renal blood flow; Ren VR, renal vascular resistance.

Fig. 9

iNOS activity recorded in various tissues, for example, liver, kidney, lung, heart, spleen, and large and small intestine in pigs challenged with LPS at 200 μg kg⁻¹ over 60 min in the presence and in the absence of AG.

Fig. 10

Effects of L-NMA infused at 300 μg kg⁻¹ min⁻¹ on MAP, CO, SVR, PAP, HR, and dP/dt in seven conscious pigs treated with LPS at 200 μg kg⁻¹ over 60 min *P < 0.05 vs. LPS; ^†P < 0.05 LPS vs. B. B indicates baseline.

Fig. 11

Effects of L-NMA infused at 300 μg kg⁻¹ min⁻¹ on Car BF, Cor BF, Mes BF, Ren BF, Hep BF, Car VR, Cor VR, Mes VR, Ren VR, and Hep VR in seven conscious pigs treated with LPS at 200 μg kg⁻¹ over 60 min *P < 0.05 vs. LPS; ^†P < 0.05 LPS vs. B. B indicates baseline; Car BF, carotid blood flow; Car VR, carotid resistance; Cor BF, coronary blood flow; Cor VR, coronary vascular resistance; Hep BF, hepatic blood flow; Hep VR, hepatic vascular resistance; Mes BF, mesenteric blood flow; Mes VR; mesenteric vascular resistance; Ren BF, renal blood flow; Ren VR, renal vascular resistance.