Temporal variation in endotoxin-induced vascular hyporeactivity in a rat mesenteric artery organ culture model

1 Endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is well described in rodent aorta, but has not been investigated in smaller vessels in vitro. 2 Segments of rat superior mesenteric artery were incubated in culture medium with or without foetal bovine serum (10%) for 6, 20 or 46h in the presence or absence of bacterial lipopolysaccharide (LPS; 1-100 mug ml(-1)). 3 Contractions to PE were measured with or without nitric oxide synthase (NOS) inhibitors: L-NAME (300 muM), aminoguanidine (AMG; 400 muM) 1400W (10 muM) and GW273629 (10 muM): the guanylyl cyclase inhibitor, ODQ (3 muM); the COX-2 inhibitor, NS-398 (10 muM) Contractile responses to the thromboxane A, mimetic, U46619 were also assessed. 4 In the presence of serum, LPS induced hyporeactivity at all time points. In its absence, hyporeactivity only occurred at 6 and 20 h. 5 L-NAME and AMG fully reversed hyporeactivity at 6 h, whereas they were only partially effective at 20 h and not at all at 46 h. In contrast partial reversal of peak contraction was observed with 1400W (62% at 46 h), GW273629 (57% at 46 h) and ODQ (75% at 46 h). COX-2 inhibition produced no reversal. 6 In contrast to PE, contractions to U46619 were substantially less affected by LPS. 7 We describe a well-characterized reproducible model of LPS-induced hyporeactivity, which is largely mediated by the NO-cyclic CMP-dependent pathway. Importantly, long-term (2-day) production of NO via iNOS is demonstrated. Moreover, conventional doses of L-NAME and AMG became increasingly ineffective over time. Thus, the choice of inhibitor merits careful consideration in long-term models.