It was found that the magnitude of the vasodilatation induced by severe hypoxia was unaffected by the addition of L-NA and indomethacin, demonstrating that it was not due to an involvement of the endothelial factors nitric oxide (NO) and/or prostaglandins. & Collins, 1994 (rabbit coronary)). However, in some studies, the responses have been found to be independent of the endothelium (Graser & Rubanyi, 1992 (canine coronary); Aalkjaer & Lombard, 1995 (rat cerebral, mesenteric)). Again, the responses observed may depend on the type and size of vessel under study and on the species. As indicated above, experimental conditions and protocols may also influence the results obtained. To date, only one study has investigated the role of the endothelium in the contractile responses of isolated rat mesenteric vessels during hypoxia. Aalkjaer & Lombard (1995) demonstrated that after > 30 min of severe hypoxia, mesenteric arteries exhibited a reduced contractile response to adenosine vasopressin (AVP), which was unaffected by removal of the endothelium. This study clearly demonstrates that established hypoxia reduces contractile responses to subsequently applied AVP by an endothelium-independent mechanism(s) but it cannot be assumed that the vasodilator response of precontracted tissues to hypoxia is also independent of the endothelium; indeed in other tissues, such as the canine coronary artery, hypoxic relaxations of Proflavine depolarized tissues were prevented by inhibitors of endothelial prostaglandin synthesis (Graser & Rubanyi, 1992). The present study therefore initially investigated the role of the endothelial vasodilator substances nitric oxide (NO) and prostaglandins in the vasodilator responses of isolated rat mesenteric arteries to severe hypoxia. Hypoxia may be expected to increase the production Proflavine of lactic acid due to stimulation of anaerobic glycolysis. It has previously been suggested that, during hypoxia, lactic acid may accumulate within Proflavine the cells reducing intracellular pH (pHi) and indeed, addition of metabolic inhibitors has been found to decrease pHi in a number of isolated smooth muscle preparations (Wray, 1990; Smith 1996). As alterations in pHi are well known to alter vascular contractility (Austin & Wray, 19931998) it has been postulated that the change in tension associated with hypoxia may be a result of a change in pHi and indeed in a preliminary study, one of us has previously found that metabolic inhibition with cyanide reduced both the tone and pHi of depolarized mesenteric arteries. It is unclear, however, whether the acidifications observed with metabolic inhibition are responsible for the vasodilatation (Smith 1996). The effect of hypoxia (i.e. a decrease in 1996). Vessels were precontracted with high-K+ solution, in the continued presence of Rp-cAMPS, and the effects on hypoxic vasodilatation examined and compared with control responses to hypoxia obtained in the same system. Due to financial constraints, these experiments were performed without continual perfusion and therefore pHi was not measured. We have previously shown, however, that the magnitude of the vasodilator responses observed to both hypoxia and metabolic inhibition in this experimental set-up are similar to those observed when vessels are constantly perfused. The effects of Rp-cAMPS on vasodilatory responses of depolarized tissues to Proflavine isoprenaline (0.1 M) were also examined. Measurement of pHi After mounting and equilibration, the vessel was placed on the stage of a Leica DM IRB inverted microscope where it was incubated with 5-10 M of the acetoxymethylester form of the pH-sensitive dye carboxy-SNARF (Molecular Probes) for 2-3 h. After washing, vessels were excited PR52B at 340 nm and emissions collected at 570 nm and > 600 nm via photomultiplier tubes. The ratio of these emissions was calibrated in terms of absolute pH units using either the K+-H+ ionophore nigericin or, more usually, by an method using the free acid form of SNARF. We have previously shown that there is a good agreement between these methods in vascular cells (Austin & Wray, 19931996). All results offered are indicated as means s.e.m. with representing the number of experiments. All reactions were expressed Proflavine as changes in active wall pressure (mN mm?1) from resting levels and normalized while a percentage of the response to 60 mM KCl or 1 M U46619 while appropriate. Time programs of changes were identified from simultaneous recordings of the guidelines with zero time being the addition of the experimental manoeuvre. Variations between groups were compared by ANOVA and Student’s test (combined or unpaired) and by the Student-Newman-Keuls test for multiple comparisons. RESULTS Resting guidelines The vessels used in this study experienced a mean diameter of 288 6 m (= 30). The = 4). Changing the gassing combination from 95 % air flow-5 % CO2 to 95 % N2-5 % CO2 resulted in a decrease in = 4) (Fig. 1example of the effect of hypoxia within the tone of an isolated.