In PGF2α (10 μM) contracted coronary arterial segments with endothelium lowering of O2 resulted in a transient increase in tension followed by relaxation which was most pronounced at 1% O2 (Figure ). In segments without endothelium, the concentration-response curve for lowering O2 was rightward shifted (Figure , Figure ), but the relaxation induced by 1% O2 was similar in segments with (73.2 ± 7.2%, n = 5) and without endothelium (75.0 ± 3.1%, n = 4). In a pressure myograph, 1% O2 also increased diameter in segments with endothelium (19.4 ± 2.7%, n = 14) to a similar degree as in segments without endothelium (20.8 ± 2.6%, n = 14).
Figure 2 Effect of the endothelin receptor antagonist SB217242 on hypoxic vasodilation. (A) Effect of the endothelin receptor antagonist SB217242 (10 μM) on concentration-response curves for oxygen (O2) lowering in coronary arterial segments with and without (more ...)
The endothelin receptor antagonist SB217242 significantly increased hypoxic relaxation regardless of the presence or absence of endothelium (Figure ). Addition of SB217242 to the organ bath resulted in less than 1% reduction in tone indicating limited ET-1 mediated basal vasoconstrictor tone in the coronary arteries. Tissue ET-1 was measured in arteries both with and without endothelium exposed to 1 hour of hypoxia compared to normoxic controls and no statistical differences in ET-1 concentration were found (Figure ). ET-1 added to the organ bath in control experiments could also be detected. Concentration response curves were constructed for ET-1, and we found ET-1 reversed vasodilation induced by 1% O2 in arteries with and without endothelium. The sensitivity towards ET-1 was increased at 1% O2 compared to 95% O2 both in arteries with and without endothelium (Figure ).
Concentration-responses for exogenously added NO were rightward shifted in coronary arterial segments contracted to the same level with endothelin compared to PGF2α (Figure ). In PGF2α and ET-1 contracted arterial segments, an inhibitor of guanylyl cyclase, ODQ (3 μM) inhibited the response to exogenously added NO markedly (Figure ), but failed to change the response to O2 lowering (Figure ). Coronary arterial relaxation induced by O2 lowering from 95 to 1% O2 was reduced after inhibition of nitric oxide synthase with L-NOARG 300 μM (Figure ).
Figure 3 Role of the nitric oxide (NO)-cyclic GMP pathway in hypoxic vasodilation. (A) Concentration-response curves for NO in the absence and the presence of an inhibitor of soluble guanylyl cyclase, ODQ (3 × 10-6 M) in arteries contracted with endothelin (more ...)
Basal NO release was evaluated by the addition of a scavenger of NO, oxyhaemoglobin (10 μM) and basal release of NO was calculated as a reduction in electrode current as previously reported [19
]. The procedure was performed during both normoxic and hypoxic conditions. There was no significant difference in basal release of NO at 21% O2
(64.2 ± 16 nM, n = 6) versus 1% O2
(64.3 ± 14 nM, n = 5). In arteries contracted to the same level by adding additional PGF2α
(Figure ), the NO concentration was markedly enhanced at 1% O2
compared to 21% O2
(Figure ). Dialysate concentrations of the NO synthase inhibitor ADMA from arteries investigated in normal HEPES were at the lower detection limit (<0.06 μM) and showed no tendency to increase during hypoxia. We found that following the addition of ADMA 10 μM to the organ bath, significant amounts of ADMA could be recovered in the dialysate and this was independent of oxygenation (21% O2
: 1.9 ± 0.3 μM; 1% O2
: 2.0 ± 0.3 μM, n = 6).
Figure 4 Effect of acute hypoxia on nitric oxide release. Effect of acute hypoxia (1% O2) on (A) constriction to PGF2α and (B) the release of NO. Results are means ± SEM of 5 experiments. Differences were evaluated with a paired t test: *P < (more ...)
A non-specific potassium channel blocker, TEA significantly inhibited relaxation induced by O2 lowering (Figure ). Concentration-responses for exogenously added NO were significantly inhibited in the presence of TEA (Figure ). A subanalysis showed that the presence of ODQ (3 μM) together with TEA gave an additional inhibition of NO induced relaxation as compared to TEA alone at 10-5 and 3*10-5 M NO.
Figure 5 Effect of the potassium channel blocker TEA on hypoxic vasodilation. (A) Concentration-response curves for O2 lowering in the absence and the presence of an inhibitor of soluble guanylyl cyclase, ODQ (3 × 10-6 M) and the absence or presence of (more ...)
In arteries with endothelium, the transient contractions disappeared and a leftward shift in concentration-response curves for O2 lowering was observed in segments treated with the superoxide scavenger, tiron, while tiron had no effect in segments without endothelium (Figure ). In arterial segments with endothelium, an inhibitor of superoxide formation, apocynin leftward shifted concentration-response curves for O2 lowering, but this was not the case for arteries without endothelium (Figure ). The vasodilation to 1% O2 was unaltered in the presence of either tiron or apocynin. Additional tests of the potential role of H2O2 showed that the cell permeable enzyme PEG catalase 300 u/ml failed to change hypoxic relaxation (Figure ) while H2O2 -induced relaxation was inhibited (Figure ). A test for the potential role of superoxide showed that PEG-SOD 70 u/ml failed to change the curves for O2 lowering (Figure ).
Figure 6 Effect of the free radical scavenger tiron and the putative NADPH oxidase inhibitor apocynin on hypoxic vasodilation. Effect of (A) the free radical scavenger tiron (10 μM) and (B) the putative NADPH oxidase inhibitor, apocynin (10 μM) (more ...)
Figure 7 Effect of PEG-catalase and PEG-SOD on hypoxic vasodilation. (A) Effect of PEG-catalase (100 u/ml) on concentration-response curves for oxygen lowering and (B) Effect of PEG catalase (100 u/ml) on concentration-response curves for hydrogen peroxide. (C) (more ...)
The mitochondrial inhibitors rotenone (1 μM) and antimycin A (1 μM) both significantly inhibited vasodilatation to O2 lowering (Figure ). The combination of the two inhibitors did not have an additional effect compared to either of the inhibitors alone (Figure ). The mitochondrial inhibitors did not change NO relaxation (Figure ).
Figure 8 Effect of Rotenone and Antimycin A on hypoxic vasodilation. Effect of rotenone (1 μM), antimycin A (1 μM), and rotenone (1 μM)+ antimycin A (1 μM) on concentration-response curves for (A) oxygen lowering and (B) Concentration-response (more ...)