Effects of hypoxia and mitochondrial inhibition on the capacitative calcium entry in rabbit pulmonary arterial smooth muscle cells
Introduction
Endoplasmic reticulum (ER) is the main intracellular Ca2+ store that releases Ca2+ in response to appropriate stimuli such as hormones or neurotransmitters. In electrically nonexcitable cells, a rise in [Ca2+]c can occur through the release of Ca2+ from 1,4,5-trisphosphate-sensitive ER Ca2+ stores [1]. This release leads to the decrease of ER Ca2+ content and, as a consequence, the activation of extracellular Ca2+ entry through store-operated Ca2+ channels (SOCs) or Ca2+ release-activated Ca2+ channels (CRAC), a process dubbed “capacitative Ca2+ entry (CCE)” by Putney [2]. This highly Ca2+-selective CCE pathway reported in many kinds of cells has been suggested to play many important physiological functions in various kinds of cells [3], [4].
Since it has been reported that CCE could play important roles in maintaining pulmonary vascular tone [5], [6], [7] and cell proliferation [8], attention has been recently given to the CCE in pulmonary arterial smooth muscle cells (PASMCs). It should be noted that the pulmonary artery is somewhat different from the other systemic arteries. To optimize the ventilation-perfusion ratio in the lung, the pulmonary artery contracts rather than relaxes in response to hypoxia, which is completely opposite to the response in systemic arteries [9]. This is called hypoxic pulmonary vasoconstriction (HPV). HPV can occur in isolated pulmonary arteries, thus the oxygen sensor and subsequent constrictor mechanism(s) must be located in either the vascular smooth muscle [10] or the endothelium [11]. When the isolated pulmonary artery is exposed to hypoxia for a long time, it shows biphasic responses. After an initial transient constriction (phase 1), a slowly developing and sustained constriction (phase 2) occurs [5], [12], [13]. Phase 1 develops independently of the endothelium and is considered to be intrinsic to the smooth muscle cells and to result from membrane depolarization and Ca2+ influx via voltage-dependent Ca2+ channels [14]. It has been suggested that the full development of phase 2 requires vasoconstrictors from intact endothelial cells [11], [15]. However, recent evidence suggests that, it is endothelium independent and intrinsic to smooth muscle without the need for endothelial factors [6], [13].
After the discovery of K+ channels that are suppressed by hypoxia in pulmonary arterial smooth muscle cells (PASMCs) [9], [10], HPV has been hypothesized to be a series of cellular events. The suppression of K+ currents results in the depolarization of membrane potential, which leads to increase in [Ca2+]c due to the activation of voltage-dependent Ca2+ channels [10], [16]. In addition to the hypothesis supporting the role of K+ channels, there is an increasing body of evidence regarding the roles of Ca2+ release from intracellular Ca2+ stores in HPV [5], [13], [17], [18], [19]. Therefore, these data suggest that, the Ca2+ release upon hypoxia and subsequent activation of CCE might be very important not only in the maintenance of pulmonary arterial tone but also in the underlying mechanisms of the HPV. However, to date, only a few reports have appeared on the role of CCE in HPV. Robertson and colleagues [5] reported that, in isolated rat pulmonary artery, a considerable portion of the biphasic contraction of HPV appears to be contributed by Ca2+ influx through CCE pathways. Meanwhile, Dipp and colleagues [13] suggested that in isolated rabbit pulmonary arteries hypoxia releases intracellular Ca2+ from ryanodine-sensitive stores by a mechanism intrinsic to PASMCs without need for Ca2+ influx across the plasma membrane or an endothelial factor.
Our previous report using cultured rabbit PASMCs suggested that, hypoxia-induced increase in [Ca2+]c may be ascribed to both Ca2+ release from SR and the subsequent activation of nifedipine-insensitive CCE [19]. During the processes mitochondria appear to modulate hypoxia-induced Ca2+ release from SR which potentially implicates the mitochondria in the induction of HPV. To further examine this possibility, we have investigated the CCE pathway in cultured rabbit PASMCs and the effects of hypoxia and mitochondrial inhibition on the CCE. We demonstrate here that both hypoxia and mitochondrial inhibitors lead to net cellular gain of Ca2+ during the CCE transient. This could be important for understanding of the mechanisms of HPV.
Section snippets
Culture of pulmonary arterial smooth muscle cells (PASMCs)
The reason and the limitation of the use of cultured PASMCs as well as the procedures for smooth muscle cell culture were previously described [19]. In brief, New Zealand white rabbits were anesthetized, their lungs were excised, placed in PBS solution, and transferred into the sterile petri dishes in clean bench. Under the stereomicroscope, the fifth to seventh branches of the pulmonary arteries were dissected from the right lobes of the lung. Isolated pulmonary arteries were moved to Ca2+ and
Results
Capacitative Ca2+ entry (CCE) was triggered by the depletion of intracellular Ca2+ stores with cyclopiazonic acid (CPA, 10 μM), which reversibly inhibits sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA). In the absence of extracellular Ca2+, CPA generated a transient rise in [Ca2+]c, which came back into the resting level after a few minutes. After that, the addition of 2 or 10 mM Ca2+ to the bathing solution led to a further increase in [Ca2+]c through CCE pathways (Fig. 1A, B). When the cells
Discussion
In the present study, we demonstrated the presence of CCE in the rabbit PASMCs. Although Ca2+ entry through CCE channels has been characterized in many systemic vascular smooth muscle cells [22], [23], [24], there are relatively a few reports in PASMCs. The presence of CCE has been reported on the primary cultured PASMCs of the dog [6] and human [8], and isolated arteries in rat [5], [7]. Therefore, our data make a very useful addition to the growing body of literature that suggests the
Acknowledgements
We thank Min-Jung Kim for building the protocol of cell culture. This study was supported by grants from the Korea Science and Engineering Foundation (97-0403-1301-5) and Basic Medical Research Fund from Ministry of Education (Korea) in 1997.
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