Selective Block of Phase 1 of Hypoxic Pulmonary Vasoconstriction in Isolated Arteries

Many studies have shown that phase 1 of HPV may be inhibited by Ca2+ channel blockers (11, 32,42,58), or by depletion of ryanodine-sensitive SRCa2+ stores (12,13, 23, 29, 58). More recently, it has been proposed that inhibition of SR Ca2+ATPase activity (12, 52) and subsequent activation of Ca2+entry via a store-refilling current may underpin phase 1 of HPV (52).

Our investigations proved to be consistent with all of the above except for a role for Ca2+ entry via a store-refilling current. As mentioned previously, we found that depletion of ryanodine-sensitive SR Ca2+ stores abolished both phase 1 and the maintained plateau constriction in pulmonary arteries without endothelium (12, 13). In contrast, however, other pharmacological manoeuvres had discrete effects on phase 1 of HPV.

We found phase 1, but not phase 2 of HPV to be abolished by pre-incubation of isolated pulmonary arteries with nifedipine, a Ca2+ channel antagonist, in the absence of extracellular Ca2+ (Fig. 2Ca) (Dipp and Evans, unpublished data). Thus, nifedipine is most likely blocking SR Ca2+ release by hypoxia, as predicted by previous investigations on systemic artery smooth muscle (57). Whatever the mechanism, however, nifedipine is clearly without effect on maintained smooth muscle constriction by hypoxia, and is therefore unable to block maintained smooth muscle SR Ca2+ release by hypoxia.

Figure. 2. Pharmacological separation of 3 components of HPV in isolated pulmonary arteries. Black indicates component 1, grey indicates component 2, and white indicates component 3. A: Constriction by hypoxia of an artery ring with (a and b) or without © and d) endothelium in the presence of cyclopiazonic acid (a and c) or 8-bromo-cADPR (b and d). B: Constriction by hypoxia of an artery ring without endothelium in the absence of extracellular Ca2+ and in the presence of Lai+ (a) or in the presence of 8-bromo-cADPR and K+-induced pre-constriction (b). C: Constriction by hypoxia of an artery ring with endothelium in the absence of extracellular Ca2+ and in the presence of nifedipine (a) or in the presence of 8-bromo-cADPR and K+-induced pre-constriction (b).

Figure. 2. Pharmacological separation of 3 components of HPV in isolated pulmonary arteries. Black indicates component 1, grey indicates component 2, and white indicates component 3. A: Constriction by hypoxia of an artery ring with (a and b) or without © and d) endothelium in the presence of cyclopiazonic acid (a and c) or 8-bromo-cADPR (b and d). B: Constriction by hypoxia of an artery ring without endothelium in the absence of extracellular Ca2+ and in the presence of Lai+ (a) or in the presence of 8-bromo-cADPR and K+-induced pre-constriction (b). C: Constriction by hypoxia of an artery ring with endothelium in the absence of extracellular Ca2+ and in the presence of nifedipine (a) or in the presence of 8-bromo-cADPR and K+-induced pre-constriction (b).

Consistent with the findings of Robertson et al. (52), we also found that preincubation of pulmonary arteries with an SR Ca2+ ATPase antagonist, cyclopiazonic acid, abolished phase 1 of HPV, and without effect on maintained smooth muscle constriction by hypoxia (Fig. 2Aa and Ac) (12). In contrast to Robertson et al. (52), however, we find no evidence for the involvement of capacitative Ca2+ entry during phase 1, because removal of extracellular Ca2+ under the conditions of our experiments had no effect on HPV in arteries without endothelium (Fig. 1C) (13). Moreover, we found that lanthanum, which has been used as a selective blocker of the store-refilling current (52), also blocked phase

1 of HPV after removal of extracellular Ca2+, raising the possibility that lanthanum is also capable of inhibiting a component of SR Ca2+ release at relatively low concentrations (Fig. 2Ba) (Dipp and Evans, unpublished data). Despite these facts it is important to note that store refilling by some route likely occurs during prolonged exposure to hypoxia (60), will certainly be required to maintain SR Ca2+release, and may be modulated by hypoxia (31).

Contrary to our findings, Robertson et al. (52) found no evidence of a ryanodine- and caffeine-sensitive component of constriction. We have no concrete explanation for these differences, but they may result from the use of pre-constriction, pentobarbitone anaesthesia, and/or the "physiological" parameters within which HPV was induced. One or a combination of these factors may also explain the varied dependence of phase 1 of HPV on extracellular Ca2+ under different experimental conditions.

Strikingly, we also found cyclopiazonic acid to be without effect on phase

2 of HPV in arteries without endothelium (Fig. 2Ac) (12), even though Ca2+ release from ryanodine-sensitive SR stores in the smooth muscle underpins both phases of HPV. Thus, it is possible that phase 1 may be mediated by inhibition of SR Ca2+ ATPase activity by a fall in ATP supply (36, 59), and a consequent increase in net Ca2+ efflux from the SR via ryanodine receptors (RyRs). To achieve this whilst allowing for a second phase of maintained SR Ca2+ release, however, one would require the presence of at least two spatially segregated intracellular Ca2+ stores each served by a discrete SR Ca2+ ATPase (SERCA) subtype, one being sensitive to cyclopiazonic acid whilst the other is not (7). Alternatively, other mechanisms sensitive to cyclopiazonic acid may be involved (see below).

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