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Endothelium-mediated effects of prostaglandins and nitric oxide on

Mette Stæhr

Summary

Prostaglandins are derived from cyclooxygenase (COX) metabolism of arachidonic acid. They serve as important physiologic modulators of vascular tone as well as salt and water homeostasis in the kidney. The kidney, and especially the inner medulla, is capable of synthesizing all types of prostaglandins, but also vascular endothelial cells can produce various prostaglandins. Two isoforms of the COX enzymes are known; COX-1, which is constitutively expressed and COX-2 which is constitutively expressed in some cell types and can be induced in others by factors such as high salt, bacterial endotoxins, and cytokines. Another important regulator of vascular tone is nitric oxide (NO) synthesized by three different NOsynthases (NOS); the neuronal (nNOS), the endothelial (eNOS), and the inducible (iNOS). Several factors suggest a linkage between the two pathways. In this project we especially focus on the effect of prostaglandin E2 (PGE2) on eNOS activity.

 Part 1: We hypothesized that a serial stimulation of vascular cyclooxygenase-2 with subsequent activation of endothelial nitric oxide synthase (eNOS) is responsible for the decrease in blood pressure, cardiac performance and vascular reactivity in endotoxemia caused by lipopolysaccharide (LPS). The hypothesis was tested in catheterized, conscious, freely moving wild type mice and mice with targeted deletion of COX-2 and eNOS (C57BL/6J background) that were given an intravenous LPS bolus (2 mg/kg, 055:B5). In vitro studies were performed on murine aorta rings. LPS caused a concomitant decrease in mean arterial blood pressure (MAP) and heart rate (HR) that was significant after three hours and sustained through the experiment (eight hours). The LPS-induced changes in MAP and HR were not different from control in COX-2-/- and eNOS-/- mice. A prostacyclin receptor (IP) antagonist (BR5064), blocked the hypotensive effect of an IP agonist (beraprost), but did not attenuate the LPSinduced decrease in MAP and HR. LPS decreased eNOS and nNOS mRNA abundances in several organs while iNOS mRNA was enhanced. In aortic rings, LPS suppressed α1-adrenoceptor-mediated vascular tone. Inhibition of COX-2 activity (NS 398), disruption of COX-2, endothelium removal or eNOS deletion (eNOS-/-) did not improve vascular reactivity after LPS while the NO synthase blockers 1400W and L-NAME prevented loss of tone. Thus, our studies suggest that COX-2 and eNOS activity are not necessary for LPS-induced decreases in blood pressure, heart rate and vascular reactivity whereas inducible NOS activity appears crucial. Combined, COX-2 and eNOS are not obvious therapeutic targets for cardiovascular rescue during gram-negative endotoxemic shock. 

Part 2: During bacterial-endotoxemia endothelial damage and vascular smooth muscle dysfunction are observed. Large amounts of NO are released by iNOS and eNOS leading to vasodilatation and hypotension. Protein phosphatase 2B (calcineurin) activates eNOS by dephosphorylation and induces iNOS in macrophages, vascular smooth muscle cells and neutrophil granulocytes. Based on this we hypothesized that the calcineurin inhibitor, Cyclosporin A (CsA) counteracts the lipopolysaccharide (LPS)-induced suppression of vasoreactivity. The hypothesis was tested in catheterized, conscious, freely moving wild type mice (C57BL/6J) that were given an intravenous bolus of CsA (20 or 40 mg/kg) followed by continuous infusion of CsA (20 or 40 mg/kg/day). CsA infusion continued throughout the experiment. After one day the mice were given an LPS bolus (2 mg/kg, 055:B5) and blood pressure and heart rate were followed for additionally Abstract 2 one day. In vitro: Mouse aortic rings were incubated with LPS (50ìg/mL, E. Coli 055:B5) and CsA (10- 5M, 10-6M or 10-7M) for 18h, at 37°C and the effect of phenylephrine was recorded with a myograph. LPS caused a concomitant decrease in mean arterial blood pressure (MAP). CsA infusion increased blood pressure significantly but did not counteract the LPS induced fall in blood pressure. LPS exposure reduced contractility in aortic rings from wild type mice significantly. CsA 10-6 M and 10-7M had no significant effect on vasoreactivity after LPS incubation but vasoreactivity were significantly different after incubation with LPS and CsA 10-5M compared to rings incubated with LPS alone. We conclude that the calcineurin inhibitor, Cyclosporin A, can improve vasoreactivity significantly after LPS incubation compared to vessels incubated with LPS alone, whereas infusion of CsA (20 or 40 mg/kg) does not counteract the LPS induced fall in blood pressure. 

Part 3: Elevated dietary salt intake increases renal medullary COX-2 and PGE2-synthase activity and cGMP excretion. It was hypothesized that COX-2 activity attenuates blood pressure increase by stimulation of NO synthesis by eNOS in systemic vasculature and kidney medulla during a high NaCl intake. COX- 2-/- and +/+ mice were given a diet with 0.004% (LS) or 4% (HS) NaCl for 18 days during continuous blood pressure recordings by indwelling catheters. Food, water intake and diuresis were determined in metabolic cages. Urine osmolality and electrolyte excretion were determined. Organs and blood were removed for mRNA/protein analysis and measurement of plasma renin concentration. Food intake was not significantly different between groups whereas water intake, diuresis, Na+, Nitrite, and cGMP excretion was significantly elevated with high salt diet. Plasma renin concentration was elevated by LS compared to HS in both strains but the increase was attenuated in COX-2-/- vs. COX- 2+/+. There was a significant dependence of blood pressure on salt intake and genotype: COX-2-/- exhibited higher blood pressure than COX-2+/+, particularly at night, both on HS and LS intake, and the largest blood pressure increase observed was at HS in COX-2-/- at night (107.2±0.6 mmHg (day) vs. 117.6±1.0 mmHg (night)). Also COX-2+/+ littermates displayed an increase in blood pressure on HS vs. LS (93.5±0.9 mmHg (LS) vs. 104.4±1.2 mmHg (HS)). There was no change in the expression of eNOS mRNA in kidney medulla by salt intake in either COX genotypes, whereas nNOS was significantly increased in kidney medulla in COX-2 -/- compared to COX-2 +/+. In conclusion; C57BL/6J exhibit salt intake-dependent blood pressure and COX-2 appears to be crucial for normal blood pressure homeostasis. During LS, COX-2 stimulates renin secretion. COX-2 activity attenuates circadian blood pressure variation during high salt intake.