Capacitative Ca2 Entry in Pulmonary Artery Smooth Muscle Cells

By inhibiting SR Ca2+ uptake, SERCA inhibitors cause contraction of pulmonary artery smooth muscle (15, 27, 32). Some of the effects of CPA on rat pulmonary artery smooth muscle are illustrated in Figure 1.

Figure 1. Store-depletion by SERCA inhibitors activates contraction, divalent cation influx and a Ni2+-sensitive cation current. A: Contraction of intact pulmonary artery induced by CPA. B: In isolated myocytes from the same artery CPA induces a transient rise in [Ca2+]j when applied in Ca2+-free solution, due to CaJ+ release, but a sustained rise follows when Ca2* is readmitted. C: The ability of CPA to accelerate quenching of fura-2 fluorescence by Mn2+ confirms that the sustained rise in [Ca2*]i is due to CCE. D: Currents flowing through store-operated channels activated by CPA in physiological solution are small in amplitude, noisy, and abolished by low concentrations of Ni2+ (Modified from Ref. 27).

Figure 1. Store-depletion by SERCA inhibitors activates contraction, divalent cation influx and a Ni2+-sensitive cation current. A: Contraction of intact pulmonary artery induced by CPA. B: In isolated myocytes from the same artery CPA induces a transient rise in [Ca2+]j when applied in Ca2+-free solution, due to CaJ+ release, but a sustained rise follows when Ca2* is readmitted. C: The ability of CPA to accelerate quenching of fura-2 fluorescence by Mn2+ confirms that the sustained rise in [Ca2*]i is due to CCE. D: Currents flowing through store-operated channels activated by CPA in physiological solution are small in amplitude, noisy, and abolished by low concentrations of Ni2+ (Modified from Ref. 27).

Contraction results from a rise in [Ca2+]j brought about through several mechanisms. As in other cell types, the SERCA inhibitors prevent the SR from buffering Ca2+as it enters the cells, resulting in the accumulation of Ca2+inthe cytosol. This can occur even in the absence of stimulated Ca2+ entry and is enhanced by the unopposed leak of Ca2+from the SR, which eventually depletes the Ca2+ store. Invariably, however, SERCA inhibitors have been found to produce a sustained contraction of pulmonary arterial smooth muscle that is dependent upon the presence of extracellular Ca2+, implying aneedforCa2+ entry (15, 27, 32). At least some of the Ca2+ may enter through voltage-operated Ca2+ channels, because nifedipine reduces the contractile response to SERCA inhibitors (27). These channels may be activated following the initial rise in [Ca2+]j caused by SERCA inhibition, which would bring about the activation of Ca2+-dependent CI' channels and consequently cause depolarization of the cell membrane. Other studies found no effect of L-type Ca2+ channel blockers on the contractile response (15), and the drugs were ineffective at blocking the rise in [Ca2+], produced by SERCA inhibitors in pulmonary artery myocytes (7, 27,48). Thus, at least part ofthe contraction is mediated by Ca2+ entry through apathway that is independent of voltage-gated Ca2+ channels. We found in rat pulmonary artery that nifedipine inhibited the CPA-induced contraction if applied within the first 30 min, but its effect then disappeared (15). This suggests that the initial effect of SERCA inhibitors is to release SR Ca2+, activate CI" channels and stimulate voltage-dependent Ca2+ entry. But, as the stores become depleted, CCE emerges as the predominant pathway by which Ca2+ entry sustains contraction. This is further supported by the finding that both the sustained contraction and the Ca2+ influx pathway activated by CPA in rat pulmonary artery were 50% blocked by the cation channel blockers SKF965, Ni2+, and Cd2+, all at concentrations causing similar inhibition of a store-operated current in isolated myocytes (27).

Although the most direct support for CCE involvement in the contraction of pulmonary arteries derives from studies with SERCA inhibitors, evidence is emerging that CCE also contributes to the contraction brought about by receptor activation. Thus following store depletion in Ca2+-free medium by continuous exposure to the a-adrenoceptor agonist, phenylephrine, the restoration of Ca2+in the presence of the a-adrenoceptor antagonist, phentolamine, produced a contraction that was presumed to reflect CCE (24). The interpretation of this result is, however, critically dependent on the receptor-mediated events being abolished by phentolamine, because CCE and receptor-operated Ca2+ entry are not easily distinguished on pharmacological grounds. Contraction resistant to calcium antagonists has also been shown to result when extracellular Ca2+ is returned to vessels exposed briefly to agonist in Ca2+-free medium to deplete the stores (15). Providing sufficient time was allowed for the receptor-activated events to cease, the contraction probably resulted from Ca2+ entry that was stimulated as a consequence of store depletion. Although CCE has been linked to pulmonary artery constriction, contractile responses to SERCA inhibitors are only a fraction of the amplitude of those generated by receptor agonists (27). Moreover, in the presence of physiological levels of Ca2+, receptor-operated and voltage-gated influx may be sufficient to prevent store depletion and CCE activation during agonist-induced contraction (16, 21). Thus CCE is probably a relatively minor source of contractile Ca2+ under physiological conditions in vivo.

In support of this, we recently found that although low concentrations of La3+

had little effect on CPA-induced contraction, La3+ was a potent inhibitor of agonist-induced contraction (unpublished results). In cerebral arteries, CCE was found to mediate a substantial rise in smooth muscle [Ca2+]j without evoking contraction, suggesting that [Ca2+]j rose within a localized cell compartment that was inaccessible to the contractile proteins (9). It is likely therefore that CCE

serves other functions within the cell. Replenishment of Ca stores is likely to be its major role, but CCE has also been linked to the proliferation of PASMCs in culture (13, 37). Thus CCE could be important in the regulation of cell growth and the vascular remodeling that occurs in pulmonary hypertensive disease.

A direct pathway linking the extracellular medium to refilling of the Ca2+ store was initially suggested for vascular muscle in 1981 (4). Direct evidence for a store depletion-activated Ca2+ influx pathway in pulmonary artery smooth muscle appeared only in the last few years (7, 13, 27, 32,48). Using fluorescent [Ca2+]i indicators, these studies identified an increase in [Ca2+]j brought about by store depletion, which required the presence of extracellular Ca2+, was insensitive to calcium antagonists, inhibited by blockers ofcation channels and inhibited by membrane depolarization. However, while thapsigargin and CPA activated this pathway in myocytes from rat and human pulmonary artery (7, 13, 27), they were an insufficient stimulus in the dog (48). The reason appears to be that dog pulmonary artery myocytes have separate IP3-sensitive andryanodine/caffeine-sensitive Ca2+ stores, both of which must be depleted in order to activate CCE.

4. Ca2+ Stores and Capacitative Ca2+ Entry in Hypoxic Pulmonary Vasoconstriction

Several strands of evidence support a key role for SR Ca2+ release in mediating the contractile response of pulmonary artery smooth muscle to hypoxia. Ca2+ store depletion with caffeine and/or ryanodine has been reported to blunt the hypoxia-induced rise in [Ca2+]i in freshly isolated or cultured smooth muscle cells (33, 40) and to reduce the contraction of intact arteries to varying degrees (21, 22, 32). Other studies found that this treatment abolished hypoxic vasoconstriction (6, 11, 41). This led to the hypothesis that Ca2+ release via ryanodine receptor-channels is an early event in the cascade activated by hypoxia. The mechanism by which the cell senses a fall in 02 tension and triggers the release of Ca2+ is still the subject of much debate. An attractive hypothesis invokes the diffusible messenger cyclic adenosine diphosphate-ribose (cADPR), which mobilizes Ca2+ from ryanodine-sensitive stores (8). It is based on the premise that a change in cellular redox state brought about by hypoxia increases the level of P-NADH, which inhibits cADPR hydrolase to promote cADPR formation from P-NAD+. Inhibition of HPV by the cADPR antagonist, 8-bromo-cADPR, lends strong support for such a mechanism (8).

There are mixed reports on the effects of SERCA inhibitors on HPV. Thapsigargin and CPA inhibited the hypoxic contraction in rabbit pulmonary artery (41) and abolished it in the rat (11). In stark contrast, CPA and thapsigargin potentiated the contractile response to hypoxia in dog pulmonary artery (21). These apparently conflicting results may be reconciled by considering species differences in the functional properties of the SR Ca2+ stores. In canine myocytes, the caffeine- and ryanodine-sensitive store is structurally and functionally distinct from the IP3-sensitive store and SERCA inhibitors deplete only the latter (21, 48). Thus if hypoxia only affects the ryanodine-sensitive store, SERCA inhibitors would not be expected to affect the response. The potentiation of HPV seen in dog vessels may be explained if some of the released by hypoxia is normally accumulated via SERCA into the store. In contrast, the caffeine/ryanodine- and stores display substantial functional overlap in rodent myocytes (27). So SERCA inhibitors (as well as caffeine and ryanodine) could deplete Ca2+ from both stores in these cells and consequently inhibit the Ca2+-releasing effect of hypoxia.

Although Ca2+ release is important for HPV, it is not yet clear if the SR is the major source of contractile Ca2+. This is because HPV and the associated rise in [Ca2+]j are greatly diminished when Ca2+ is removed from the extracellular medium (6, 11, 22, 33,41), implying that Ca2+ influx also contributes. It is still possible that hypoxia initially stimulates Ca2+ influx, which binds to the ryanodine receptor and triggers Ca2+-induced Ca2+ release. Alternatively, hypoxia-induced Ca2+release could trigger an increase in [Ca2+]j by stimulating Ca2+ entry pathways. In support of this, nisoldipine reduced the contractile response to caffeine in canine pulmonary artery (21). Organic calcium antagonists have been widely found to blunt HPV and the associated rise in [Ca2+]:, both in isolated pulmonary arteries (22, 32, 33) and intact perfused lungs (25, 36). Interestingly, the potentiated contractile response to hypoxia observed in canine pulmonary artery in the presence of CPA was much less sensitive to nisoldipine than the control response to hypoxia in the absence of CPA (21). Since removing extracellular Ca2+ blocked this effect, hypoxic vasoconstriction was likely due to the recruitment of voltage-independent Ca2+ entry. This pathway resembled CCE in being blocked by SKF96365. Another recent study showed that voltage-independent Ca2+ entry contributes to HPV in rat pulmonary artery (32). The underlying pathway was permeable to Mn2+ and blocked by SKF96365 and low concentrations of La3+, all of which are characteristic markers ofnon-selective cation channels. Although it has only recently attracted serious interest, evidence for a voltage-independent Ca2+ entry pathway in HPV has in fact been available for some time. In 1993, a Ca2+ entry pathway insensitive to nifedipine and verapamil was shown to underlie a sustained rise in [Ca2+]i induced by hypoxia in cultured pulmonary arterial myocytes (33).

Although the voltage-independent Ca2+ entry pathway has been proposed to reflect CCE, this has yet to be confirmed. Its properties are equally compatible with cation channels mediating receptor-operated Ca2+ entry. Thus we found in rat pulmonary artery that La3+, which was shown to inhibit HPV, is a poor inhibitor of CCE but a potent blocker of agonist-induced Ca2+ entry. Since agonist-induced pre-tone is required in most experiments to achieve a clear response to hypoxia, the use of currently available CCE inhibitors to investigate their role is problematic. The fact that pre-tone is needed implies that there is synergy between the pathways activated by agonist and hypoxia. Thus, by affecting this synergy, a drug interfering with receptor-mediated events could give the impression of inhibiting HPV without actually altering the events mediating HPV. More selective approaches for interfering with CCE will be needed to enable its contribution to HPV to be clearly elucidated.

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