Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

doi:10.1016/S0024-3205(02)01497-2

Life Sciences

Volume 70, Issue 19, 29 March 2002, Pages 2321-2333

https://doi.org/10.1016/S0024-3205(02)01497-2 Get rights and content

Abstract

We have investigated the effects of hypoxia on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (P_O2<50 mmHg) external solution increased [Ca²⁺]_i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca²⁺]_i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca²⁺, hypoxic stimulation elicited a transient [Ca²⁺]_i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca²⁺ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m–chlorophenyl–hydrazone (CCCP) enhanced the [Ca²⁺]_i rise in response to hypoxia. A short application of caffeine gave a transient [Ca²⁺]_i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca²⁺]_i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca²⁺]_i rise, nifedipine did not decrease [Ca²⁺]_i. These data suggest that the [Ca²⁺]_i increase in response to hypoxia may be ascribed to both Ca²⁺ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca²⁺ entry. Mitochondria appear to modulate hypoxia induced Ca²⁺ release from the SR.

Introduction

Hypoxic pulmonary vasoconstriction (HPV) is very important in the pulmonary circulation, whereby the degree of vasoconstriction to hypoxia optimizes the ventilation-perfusion ratio. The hypoxia-induced vasoconstriction in one part of the lung diverts blood away from the poorly ventilated part into oxygen-rich areas [1]. Since HPV has been observed in isolated pulmonary arteries, the oxygen sensor and subsequent constrictor mechanism(s) is believed to be present within either the vascular smooth muscle [2] or the endothelium [3].

The discovery of K⁺ channels which are reversibly suppressed by hypoxia in pulmonary arterial smooth muscle cells (PASMCs) has given rise to the possibility of a central role of smooth muscle in HPV [2], [4]. If the suppression of voltage dependent K⁺ channels [2] or the decreased open probability of O₂-sensitive K⁺ channels [4] results in depolarization of the membrane, the intracellular Ca²⁺ ([Ca²⁺]_i) might increase by the activation of voltage-dependent Ca²⁺ channels [1], [5]. In contrast to the above hypothesis, which is based on the triggering role of hypoxia-related K⁺ channels, it has been recently reported that direct activation of intracellular Ca²⁺ release from sarcoplasmic reticulum (SR) by hypoxia might play an initial triggering role in HPV [6], [7], [8], [9]. Gelband and Gelband [7] proposed that the initial event in HPV might be the release of Ca²⁺ from intracellular Ca²⁺ stores and the consequent inhibition of voltage-dependent K⁺ channels by increased [Ca²⁺]_i could keep the membrane potential depolarized. In addition, it has been reported that when caffeine- and ryanodine-sensitive intracellular Ca²⁺ stores are depleted in canine pulmonary artery, the hypoxic contraction of the vessel is significantly reduced [6]. All these data raise the possibility that the SR Ca²⁺ pool plays an important role in HPV. However, it is still not clear whether Ca²⁺ release from intracellular Ca²⁺ stores contributes to the development or maintenance of HPV as well as the detailed characteristics of the Ca²⁺ release mechanisms from the SR by hypoxia.

In the present study, we have studied the effects of hypoxia on [Ca²⁺]_i in freshly isolated and cultured rabbit PASMCs loaded with fura-2. When the cells were exposed to a glucose-free and hypoxic (Po2<50 mmHg) solution, PASMCs showed a steady rise in [Ca²⁺]_i, of which the initial transient component comes from the intracellular Ca²⁺ stores. The continuous Ca²⁺ influx was not due to the opening of L-type Ca²⁺ channels, but probably to the activation of store-operated channels. We also observed that mitochondrial metabolic inhibitors, CCCP or rotenone, potentiate the hypoxia- or caffeine-induced [Ca²⁺]_i increase.

Section snippets

Preparation of smooth muscle cells

New Zealand white rabbits weighing 1.5–2 kg were anesthetized with pentobarbital sodium (30 mg/kg). The whole heart with the lung was rapidly excised, placed in PBS solution, and transferred into the sterile petri dishes in clean bench. The lung was removed from the heart and rinsed several times with fresh PBS solution. Under a stereomicroscope, the fifth to seventh branches of the pulmonary arteries were dissected free and cleaned of adventitia from the right lobes of the lung. Isolated

Results

The first objective of the study was to test whether [Ca²⁺]_i in PASMCs increases in response to hypoxia, and whether such a response is specific to the PASMCs. To do this, we compared [Ca²⁺]_i using fura-2 in pulmonary and coronary arterial smooth muscle cells of the rabbit. As shown in Fig. 1, hypoxic stimulation increased [Ca²⁺]_i in freshly isolated PASMCs (Fig. 1A), but not in coronary SMCs (Fig. 1B). However, the hypoxia induced [Ca²⁺]_i responses in freshly isolated PASMC were small and

Discussion

In the present study, the hypoxic responses of coronary and pulmonary artery cells were compared. Hypoxic stimulation raised [Ca²⁺]_i in freshly isolated and cultured pulmonary SMCs, but it had no effect on the coronary arterial SMCs. These contrasting effects of hypoxic stimulation on [Ca²⁺]_i are in good agreement with the well-known fact that pulmonary arteries constrict in response to hypoxia, while systemic arteries respond in the opposite way [1], [2], [14], [15]. Therefore, it seems likely

Acknowledgements

We thank Dr. DW Hilgemann for correction of the English and helpful discussions as well as Bae YM for helping us to measure O₂ tension. This study was supported by grants from the Ministry of Science and Technology (1998) and the Korea Science and Engineering Foundation (97-0403-1301-5).

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These results, together with previous reports that relaxation of rabbit pulmonary arteries is usually seen during hypoxic stimulation (Marriott and Marshall, 1990; MacLean et al., 1993; Vadula et al., 1993), suggest that such a mechanism may not play an important role in hypoxic increase in [Ca2+]i. As described above, a number of reports have shown that hypoxic contraction in isolated lungs, PAs and PASMCs are relatively insensitive to multiple CaV channel blockers (Demiryurek et al., 1993; Jabr et al., 1997; Robertson et al., 2000; Shimoda et al., 2000; Sham et al., 2000; Dipp and Evans, 2001; Kang et al., 2002). Whether hypoxia in fact inhibits Ca2+-activated K+ (KCa) channels remains unclear, since during hypoxia this channel has been shown to be inhibited (Park et al., 1995; Peng et al., 1997; Vandier et al., 1998), activated (Archer et al., 1996; Cornfield et al., 1996; Nossaman et al., 1997), and unaffected (Post et al., 1995).
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ROS generation in mitochondria is, however, also quite complex, and it has been proposed in competing converse hypotheses that it is either a hypoxia‐induced decrease in ROS generation by the mitochondria, or a hypoxia‐induced increase in mitochondrial ROS generation that mediates pulmonary hypoxic vasoconstriction (Moudgil et al., 2005; Waypa et al., 2001, 2002; Waypa and Schumacker, 2002, 2008). Experimental data support each of these competing hypotheses; additional data also suggest an alternative calcium‐related mechanism whereby mitochondria regulate hypoxic pulmonary vasoconstriction (Kang et al., 2002, 2003). Of three well‐described heme oxygenases (HO‐1, ‐2, and ‐3), the expression of only one (HO‐1) has been definitively shown to be inducible (HO‐1 is inducible by hypoxia, and potentially by increased oxidative stress or shear stress) (Paffett and Walker, 2007; Shibahara et al., 2007).
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Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

Abstract

Introduction

Section snippets

Preparation of smooth muscle cells

Results

Discussion

Acknowledgements

J Biol Chem

Jpn J Pharmacol

Cell Calcium

Mechanisms of acute hypoxic and hyperoxic changes in pulmonary vascular reactivity

Direct role of potassium channel inhibition in hypoxic pulmonary vasoconstriction

Am J Physiol

The role of the endothelium in hypoxic pulmonary vasoconstriction

Exp Physiol

Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O2-sensing potassium current

Br J Pharmacol

Hypoxic and metabolic regulation of voltage-gated K+ channels in rat pulmonary artery smooth muscle cells

Exp Physiol

Prominent role of intracellular Ca2+ release in hypoxic vasoconstriction of canine pulmonary artery

Br J Pharmacol

Ca2+ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels

Circulation

Hypoxia enhances agonist-induced pulmonary arterial contraction by increasing calcium sequestration

Am J Physiol

Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat

J Physiol

Tharpsigargin stimulates Ca2+ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

Am J Physiol

Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells

Am J Physiol

Contrasting effects of hypoxia on tension in rat pulmonary and mesenteric arteries

Am J Physiol

Characterization of hypoxia-induced [Ca²⁺]_i rise in rabbit pulmonary arterial smooth muscle cells

Regulation of the resting potential of rabbit pulmonary artery myocytes by a low threshold, O₂-sensing potassium current

Hypoxic and metabolic regulation of voltage-gated K⁺ channels in rat pulmonary artery smooth muscle cells

Prominent role of intracellular Ca²⁺ release in hypoxic vasoconstriction of canine pulmonary artery

Ca²⁺ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels

Tharpsigargin stimulates Ca²⁺ entry in vascular smooth muscle cells: nicardipine-sensitive and -insensitive pathways

Capacitative Ca²⁺ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells