Endotoxin-induced vascular hyporeactivity to phenylephrine (PE) is well described in rodent aorta, but has not been investigated in smaller vessels in vitro. Segments of rat superior mesenteric artery were incubated in culture medium with or without foetal bovine serum (10%) for 6, 20 or 46 h in the presence or absence of bacterial lipopolysaccharide (LPS; 1 – 100 μg ml−1). Contractions to PE were measured with or without nitric oxide synthase (NOS) inhibitors: L-NAME (300 μM), aminoguanidine (AMG; 400 μM) 1400W (10 μM) and {"type":"entrez-nucleotide","attrs":{"text":"GW273629","term_id":"282549412","term_text":"GW273629"}}GW273629 (10 μM); the guanylyl cyclase inhibitor, ODQ (3 μM); the COX-2 inhibitor, NS-398 (10 μM). Contractile responses to the thromboxane A2 mimetic, U46619 were also assessed. In the presence of serum, LPS induced hyporeactivity at all time points. In its absence, hyporeactivity only occurred at 6 and 20 h. 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), {"type":"entrez-nucleotide","attrs":{"text":"GW273629","term_id":"282549412","term_text":"GW273629"}}GW273629 (57% at 46 h) and ODQ (75% at 46 h). COX-2 inhibition produced no reversal. In contrast to PE, contractions to U46619 were substantially less affected by LPS. We describe a well-characterized reproducible model of LPS-induced hyporeactivity, which is largely mediated by the NO-cyclic GMP-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. Keywords: Vascular hyporeactivity, endotoxin, nitric oxide, phenylephrine, organ culture, NOS inhibitors, guanylyl cyclase inhibition, endothelium, thromboxane A2 Introduction Sepsis is the leading cause of morbidity and mortality in critically ill patients with a mortality rate exceeding 50% (Deitch, 1998). Gram-negative bacterial infections are responsible for approximately 50% of cases of septic shock (Thiemermann, 1997). This condition is characterized by an exaggerated host systemic inflammatory response usually leading to a high cardiac output, low systemic vascular resistance circulation with a low blood pressure (Parillo, 1993). In such patients, therapy with an α1 adrenergic agonist is often required to reverse the hypotension. However, hypotension may be resistant to high doses of vasopressor agents, a state known as vascular hyporeactivity. Administration of endotoxin (lipopolysaccharide; LPS), a cell wall component ubiquitous to Gram-negative bacteria, to animals and human volunteers has been used extensively to mimic Gram-negative sepsis (Thiemermann, 1997; Deitch, 1998). In many models, endotoxin-induced vascular hyporeactivity is associated with enhanced formation of nitric oxide (NO) within the blood vessel, involving activation of mainly inducible (iNOS) but also constitutive (eNOS) isoforms of NO synthase (NOS) (Julou-Schaeffer et al., 1990; Thiemermann, 1994). Once formed, NO can activate soluble guanylyl cyclase resulting in vascular smooth muscle relaxation through the formation of guanosine 3′-5′ cyclic-monophosphate (cyclic GMP). Consistent with this, inhibition of NOS or soluble guanylyl cyclase has been shown to reverse vascular hyporeactivity in vivo and in vitro, either partially (Yen et al., 1995; Mitolo-Chieppa et al., 1996; Wu et al., 1998) or completely (Julou-Schaeffer et al., 1990; Hall et al., 1996; Scott et al., 1996). Endotoxin is known to induce the expression of other enzymes, including cyclo-oxygenase (COX-2) (Bishop-Bailey et al., 1997) suggesting that additional mechanisms are likely to contribute to endotoxin-induced changes in vascular reactivity. For example, increased prostanoid production and enhanced potassium channel activity have been reported to contribute to the development of hypotension and vascular hyporeactivity (Wu et al., 1995b; Fatehi-Hassanabad et al., 1996; Clapp & Tinker, 1998). The majority of in vitro investigations into mechanisms of vascular hyporeactivity have used aortic tissues from rodents (Julou-Schaeffer et al., 1990; Wu et al., 1995a; Scott et al., 1996, Hall et al., 1996). However, this is a large conduit vessel that only makes a small contribution to systemic vascular resistance. In contrast, inducing hyporeactivity in smaller rodent blood vessels has proved very difficult. Using vessels harvested from endotoxaemic animals (ex-vivo) or vessels superfused with endotoxin, Mitchell et al. (1993) and Glembot et al. (1995) were unable to demonstrate diminished responses to vasoconstrictor agents. However, hyporeactivity could be demonstrated in small mesenteric and femoral vessels ex vivo, but only if the bathing medium contained L-arginine, the precursor for NO synthesis. (Schneider et al., 1992; 1994; Mitolo-Chieppa et al., 1996). The ex vivo and in vitro models described to date differ in the degree of hyporeactivity obtained and in the response to blockade of NO synthesis. These differences may relate to the species or origin of blood vessel, the endotoxin model under investigation, the dose and duration of incubation with LPS, or the type of NOS inhibitor used. We sought to develop a reproducible in vitro model of hyporeactivity in a smaller artery and chose to study a mesenteric vessel since the mesenteric circulation is an important contributor to vascular tone, receiving 30 – 40% of the total cardiac output. Using an organ culture method, we investigated the effect of LPS on vascular reactivity to the α1 agonist, phenylephrine (PE) and the thromboxane A2 mimetic, U46619 in rat superior mesenteric artery. We sought to characterize the response to PE in terms of the dose and duration of LPS incubation, the effect of serum and the role of the endothelium. The contribution of the NO pathway was also assessed using a variety of NOS inhibitors. Preliminary results have been presented in abstract form (O'Brien et al., 1999; 2000a, 2000b).