Extracellular Calcium-Sensing Receptor Is Critical in Hypoxic Pulmonary Vasoconstriction

Abstract

Aims: The initiation of hypoxic pulmonary vasoconstriction (HPV) involves an increase in cytosolic calcium ([Ca²⁺]_i) in pulmonary artery (PA) smooth muscle cells (PASMCs). Both the processes depend on extracellular Ca²⁺. Extracellular Ca²⁺ can be sensed by extracellular calcium-sensing receptor (CaSR). This study aims at determining whether CaSR is pivotal in the initiation of HPV. Results: Experiments were performed in cultured PASMCs, isolated PAs, and rats including CaSR knockdown preparations. Both hypoxia and H₂O₂ equivalent to the level achieved by hypoxia increased [Ca²⁺]_i in an extracellular Ca²⁺-dependent manner in PASMCs, and this was inhibited by CaSR knockdown or its negative allosteric modulator, Calhex231. Hypoxia-increased H₂O₂ generation was diminished by mitochondria depletion. Mitochondria depletion abolished hypoxia-induced [Ca²⁺]_i increase (HICI), which was reversed by H₂O₂ repletion. CaSR knockdown or Calhex231, however, prevented the reversible effect of H₂O₂. HICI was abolished by catalase-polyethylene glycol (PEG-Catalase), not superoxide dismutase-polyethylene glycol (PEG-SOD) pretreatment, attenuated by ryanodine receptor3-knockdown or inhibition of store-operated Ca²⁺ entry. HPV in vitro and in vivo was inhibited by Calhex231 and by CaSR knockdown. Innovation: A novel mechanism underlying HPV is revealed by the role of CaSR in orchestrating reactive oxygen species and [Ca²⁺]_i signaling. Conclusions: The activation of mitochondrial H₂O₂-sensitized CaSR by extracellular Ca²⁺ mediates HICI in PASMCs and, thus, initiates HPV. Antioxid. Redox Signal. 17, 471–484.

Introduction

H ypoxic pulmonary vasoconstriction (HPV) matches ventilation with perfusion and also leads to pulmonary hypertension. The initiation of HPV involves an increase in cytosolic calcium ([Ca²⁺]_i) in pulmonary artery smooth muscle cells (PASMCs) (20, 33, 37, 38, 43). However, the signaling pathways for hypoxia-induced [Ca²⁺]_i increase (HICI) remains highly controversial (20, 33, 37, 38, 43).

Many previous studies have shown consistently that HICI and HPV were largely dependent on extracellular Ca²⁺ (9, 10, 13, 19, 25, 29, 34, 35, 42). Apparently, the extracellular Ca²⁺ dependence can be interpreted as the Ca²⁺ source for Ca²⁺ influx from extracellular space stimulated by hypoxia. Extracellular Ca²⁺ entry may be activated by hypoxia-altered level of reactive oxygen species (ROS) (1, 18, 26, 39, 41). The suggested Ca²⁺ entry pathways by hypoxia include store- (4, 23, 29, 35, 42) and voltage-operated Ca²⁺ influx (1, 20, 43). Meanwhile, Ca²⁺ release channel(s) especially ryanodine receptors (RyRs) have also been broadly documented to be involved in HICI and HPV (8, 12, 15, 32, 34, 48).

Innovation

Hypoxic pulmonary vasoconstriction (HPV) matches lung ventilation with blood perfusion and also leads to pulmonary hypertension. The mechanism underlying HPV remains largely unknown. The Ca²⁺ sensitivity of calcium-sensing receptor (CaSR) in pulmonary artery smooth muscle cells is enhanced by mitochondria-derived reactive oxygen species (ROS) during hypoxia. The subsequent activation of sensitized CaSR by extracellular calcium then triggers intracellular calcium release mainly from ryanodine receptor and extracellular calcium influx primarily from store-operated calcium entry pathway. HPV in vitro and in vivo is inhibited by Calhex231 and by CaSR knockdown. The pivotal role of CaSR in orchestrating ROS and cytosolic calcium ([Ca²⁺]_i) signaling is revealed as a new mechanism for the triggering of HPV, and the CaSR is also suggested as a novel target for the treatment of HPV-related diseases.

Extracellular Ca²⁺ is also the ligand for extracellular calcium-sensing receptor (CaSR) (14). The dependence of HICI and HPV on extracellular Ca²⁺ may not be understood solely as Ca²⁺ influx secondary to the depletion of intracellular Ca²⁺ stores by hypoxia. Substantial progress in this controversial point warrants a novel attempt to elucidate how these different Ca²⁺ pathways orchestrate to induce [Ca²⁺]_i elevation during hypoxia. As a matter of fact, however, it has never been studied whether extracellular Ca²⁺ plays any role(s) in addition to the source of Ca²⁺ influx from extracellular space in HICI and HPV.

CaSR is a G-protein-coupled membrane receptor that senses extracellular Ca²⁺ concentration and conveys this information to intracellular space through multiple signal pathways including [Ca²⁺]_i and extracellular regulated protein kinase (14). CaSR protein is expressed in vascular smooth muscle cells (6, 21, 45). The activation of CaSR in vascular smooth muscle cells increases [Ca²⁺]_i and induces a vasoconstriction of the gerbilline spiral modiolar artery (45). Additionally, functional CaSR appears sensitive to redox status (2, 47).

This study aimed at investigating the mechanism underlying extracellular Ca²⁺ dependence of HICI and its associations with RyR (8, 15, 34, 48), store-operated Ca²⁺ influx (SOC) (4, 23, 29, 35, 42), and HPV. It was found that functional CaSR, which can be sensitized by hypoxia-induced ROS, serves as a bridge between extracellular Ca²⁺ and [Ca²⁺]_i elevation and, therefore, is pivotal in HPV.

Results

Expression of a functional CaSR: sensing extracellular Ca²⁺

Immunocytostaining showed the major localization of CaSR in the cytosol in permeabilized PASMCs and the membrane localization of the mature functional protein in nonpermeabilized PASMCs (Fig. 1A). Western blot on cell lysate simultaneously isolated from cytoplasm and membrane revealed a 170 kDa band, presumably a prematured form (24, 50) and the other 55–72 kDa one, respectively (Fig. 1B).

FIG. 1.

Expression of a functional calcium-sensing receptor (CaSR) in pulmonary artery smooth muscle cells (PASMCs). (A) Indirect immunocytochemical staining of PASMCs in culture for CaSR (red) and nucleus (blue) in permeabilized (upper left) and nonpermeabilized preparations (upper right) as well as in the presence of a specific blocking peptide (lower left, nonpermeabilized preparations) and in a negative control without incubation of anti-CaSR antibody (lower right, nonpermeabilized preparations; n=3). (B) Western blot for CaSR in the absence (left) and presence (right) of a specific blocking peptide in cell lysate prepared from cytoplasm and membrane (n=3). (C) Enhanced phosphorylation of ERK1,2 in PASMCs in response to a 3 min stimulation of 4.0, 6.0, and 7.5 mM [Ca²⁺]_o, respectively (n=3, *p<0.05). (D) CaSR functional evaluation by cytosolic calcium ([Ca²⁺]_i) monitoring in PASMCs in response to elevated [Ca²⁺]_o (n=23). (E) Inhibition of [Ca²⁺]_i response by 1 μM Calhex231 (n=21). (F) Dose-dependent inhibition of [Ca²⁺]_i response by Calhex231 (*p<0.05).

To determine whether the CaSR protein is functional, PASMCs were exposed to increased extracellular Ca²⁺ concentration ([Ca²⁺]_o) (5). A 3-min [Ca²⁺]_o elevation from 2.0 to 6.0 and 7.5 mM enhanced phosphorylation of ERK1,2 (p44/p42; Fig. 1C), an indication of CaSR activation (14) and increased [Ca²⁺]_i (n=23, p<0.05 for each, Fig. 1D, F). (1S,2S,1′R)-N1-(4-chlorobenzoyl)-N2-[1-(1-naphthyl)ethyl]-1,2-diaminocyclo-hexane (Calhex231), the negative allosteric modulator of CaSR, dose-dependently inhibited [Ca²⁺]_o elevation-stimulated [Ca²⁺]_i increase (p<0.05, Fig. 1E, F). Thus, PASMCs can sense [Ca²⁺]_o through CaSR-mediated [Ca²⁺]_i signaling.

HICI: extracellular Ca²⁺ dependence, Calhex231 inhibition, and CaSR dependence

Hypoxia, by a quick superfusion of PASMCs with hypoxic medium for 3 min, increased [Ca²⁺]_i (n=51, p<0.01, Fig. 2A, C). The HICI was completely abolished or significantly inhibited by the removal of extracellular Ca²⁺ (n=43, Fig. 2B, C), the presence of Calhex231 (n=8, Fig. 2D, E), or CaSR knockdown (p<0.01 vs. blank or nonspecific shRNA respectively, Fig. 3C–F). Of note, CaSR knockdown abolished/inhibited [Ca²⁺]_i response to 6.0 and 7.5 mM [Ca²⁺]_o, whereas it does not affect the store Ca²⁺ contents in PASMCs (Supplementary Figs. S1 and S2; Supplementary Data are available online at www.liebertonline.com/ars). Thus, HICI depends on extracellular Ca²⁺-associated CaSR activation or a permissive role of CaSR.

FIG. 2.

Extracellular Ca²⁺ dependence of hypoxia-induced [Ca²⁺]_i increase and inhibition by Calhex231. (A) Representative curve of [Ca²⁺]_i responses to three repetitive episodes of hypoxia (Hy1, Hy2, and Hy3) each for 3 min in the presence of 2.0 mM extracellular Ca²⁺ (n=51). (B) Representative curve of [Ca²⁺]_i responses to repetitive episodes of hypoxia in the presence (Hy1 and Hy3) or absence (Hy2) of 2.0 mM extracellular Ca²⁺ (n=43). (C) Averaged [Ca²⁺]_i levels at baseline and hypoxic episodes (*p<0.05). (D) Representative curve of [Ca²⁺]_i responses to repetitive episodes of hypoxia in the absence (Hy1 and Hy3) and presence (Hy2) of 1 μM Calhex231 (n=8). (E) Dose-dependent inhibition of [Ca²⁺]_i peak response to hypoxia by Calhex231 (four parameter logistic regression, p<0.05, n=8 for each dose).

FIG. 3.

CaSR dependence of hypoxia-induced [Ca²⁺]_i increase. (A) Western blot showing shRNA-mediated CaSR knockdown. (B) Averaged densitometry in control and CaSR shRNA-treated PASMCs (*p<0.05, n=3 for each). (C–E) Representative curve of [Ca²⁺]_i responses to hypoxia (Hy) in blank control (n=11, C), control shRNA (n=14, D) and CaSR specific shRNA-treated PASMCs (n=9, E) in the presence of 2.0 mM extracellular Ca²⁺. (F) Averaged [Ca²⁺]_i peaks in response to hypoxia (*p<0.05).

Ca²⁺ sensitivity of CaSR: enhanced by ROS

Hypoxia, as indicated by a quick decline in PO₂ from 154±4 to 19±1 mmHg (n=4), stimulated an abrupt increase in ROS (n=22, Fig. 4Ai), estimated (30) to a level equivalent to 15.6 μM extracellular H₂O₂ (n=21–26 for each dose, Fig. 4Aii). The hypoxia-stimulated ROS generation in PASMCs was confirmed using a redox-sensitive green fluorescent protein (GFP)-RoGFP (Fig. 4Aiii) (40). Hypoxia-stimulated ROS generation is not affected by Calhex231 (Supplementary Fig. S3). About 15.6 μM H₂O₂, not a lower concentration of 13.0 μM (Supplementary Fig. S4), which did not induce any [Ca²⁺]_i transient in the absence of extracellular Ca²⁺, triggered a [Ca²⁺]_i transient in the presence of 2 mM [Ca²⁺]_o (n=5, Fig. 4Bi, Biii). These results are consistent with previous studies showing that H₂O₂ increased [Ca²⁺]_i in smooth muscle cells (18, 46), and the [Ca²⁺]_i increase by low μM H₂O₂ depended on extracellular Ca²⁺ (46). 1 μM Calhex231 and CaSR-knockdown significantly inhibited 15.6 μM H₂O₂-induced [Ca²⁺]_i transient (n=13, Fig. 4Bii, Biii, C). [Ca²⁺]_o elevation-induced [Ca²⁺]_i signal in PASMCs is enhanced by the concomitant presence of 15.6 μM H₂O₂ or under hypoxia (Supplementary Fig. S5). Thus, the Ca²⁺ sensitivity of CaSR in inducing a [Ca²⁺]_i transient can be enhanced by ROS at the level achieved by hypoxia in PASMCs.

FIG. 4.

Enhancement of Ca²⁺ sensitivity of CaSR by reactive oxygen species (ROS). (A) Hypoxia-triggered ROS generation in PASMCs was estimated by DCFDA. Representative curves of DCFDA fluorescence in PASMCs in response to hypoxia (n=22, Ai) and to a series of extracellular application of H₂O₂ (n=21–26, Aii). Averaged time-course alterations of ratios of mito-RoGFP fluorescence intensity from excitation at 405 and 488 nm and emission at 515 nm from PASMCs on exposure to hypoxia, 15.6 (μM H₂O₂, or normoxic control (n=3 for each, Aiii). (B) Representative curve of [Ca²⁺]_i in PASMCs in response to 15.6 μM H₂O₂ in the absence and then presence of 2.0 mM [Ca²⁺]_o (n=5, Bi); representative curve of [Ca²⁺]_i in PASMCs in response to 15.6 μM H₂O₂ in the absence and then co-presence of 2.0 mM [Ca²⁺]_o and 1 μM Calhex231 (n=13, Bii); averaged [Ca²⁺]_i baseline and peak responses to 15.6 μM H₂O₂ (*p<0.05, Biii). (C) CaSR-mediated [Ca²⁺]_i response to 15.6 μM H₂O₂. Representative curves of [Ca²⁺]_i in response to 15.6 μM H₂O₂ in the presence of 2.0 mM [Ca²⁺]_o in control PASMCs (n=12, Ci), PASMCs treated with nonspecific- (n=8, Cii) and CaSR-specific shRNA (n=7, Ciii). Averaged [Ca²⁺]_i baseline and peak responses to 15.6 μM H₂O₂ (*p<0.05, Civ).

CaSR-dependent [Ca²⁺]_i increase by hypoxia: role of mitochondria-derived H₂O₂

Mitochondria depletion (7, 39) using ethidium bromide almost completely diminished ROS generation by hypoxia (n=27, p<0.01 vs. control with or without pyruvate (Py) and uridine (Ur), Fig. 5A). Hypoxia triggered similar [Ca²⁺]_i increase in control PASMCs cultured in the presence (n=8, p<0.01, Fig. 5Bi, Bv) and absence of Py and Ur (Fig. 2A). Mitochondria depletion completely abolished HICI, and this was reversed by exposure of PASMCs to 15.6 μM H₂O₂ (Fig. 5Bii, Bv). The exposure of H₂O₂-reversed [Ca²⁺]_i increase during hypoxia in mitochondria-depleted PASMCs was prevented by 1 μM Calhex231 (n=15, p<0.01 vs. co-exposure of H₂O₂ in the absence of Calhex231, Fig. 5Biii, Bv) and by CaSR knockdown (Fig. 5Biv, Bv), suggesting that mitochondria-derived ROS sensitizes CaSR and thereafter triggers HICI.

FIG. 5.

Mitochondria-derived H₂O₂ in CaSR-mediated [Ca²⁺]_i increase by hypoxia. (A) Representative curves of DCFDA fluorescence in response to hypoxia in control PASMCs in the absence (n=19) or presence of pyruvate (Py) and uridine (Ur; n=10) and in mitochondria-depleted PASMCs (n=26, Ai). Averaged DCFDA fluorescence intensity (*p<0.01 vs. control PASMCs in the presence or absence of Py and Ur, Aii). (B) Representative curve of [Ca²⁺]_i responses to two repetitive episodes of hypoxia (Hy1 and Hy2) in PASMCs cultured with Py and Ur (n=8, Bi), to hypoxia in the absence (Hy1) and presence of 15.6 μM H₂O₂ (Hy2) in mtDNA-depleted PASMCs (n=13, Bii), to hypoxia in the absence (Hy1) and co-presence of 15.6 μM H₂O₂ and 1 μM Calhex231 (Hy2) in mtDNA-depleted PASMCs (n=15, Biii), to hypoxia in the absence (Hy1) and presence of 15.6 μM H₂O₂ (Hy2) in mtDNA-depleted PASMCs with CaSR knockdown (n=13, Biv). Averaged [Ca²⁺]_i peak responses to hypoxia (*p<0.05, Bv). (C) Representative curve of [Ca²⁺]_i responses to hypoxia in the PEG-SOD (n=16, Ci) and PEG-catalase (n=15, Cii)-pretreated PASMCs. PASMCs were pretreated overnight with 1000 U/ml PEG-SOD or 5000 U/ml PEG-catalase. Averaged [Ca²⁺]_i baseline and peak responses to hypoxia (*p<0.05, Ciii). Extracellular Ca²⁺ is 2.0 mM for all.

PEG-Catalase, not PEG-SOD loaded into PASMCs (7), abolished HICI (Fig. 5Ci–Ciii), clarifying the specific role of intracellular H₂O₂ in HICI.

HICI: RyR and SOC dependence

To determine the downstream target of CaSR activation by hypoxia, we manipulated the intracellular Ca²⁺ release and SOC pathways. The inhibition of RyR by 100 μM ryanodine, but not the inhibition of IP₃ receptor by 20 μM XeC, significantly attenuated HICI (p<0.05 vs. control, Fig. 6Ai, Aiv for ryanodine; p=nonsignificance vs. control, Fig. 6Aii, Aiv for XeC). These are consistent with previous studies using either genetically manipulated PASMCs (48) or pharmacological inhibitors (8, 11, 15, 34). Being accordant with other investigators (4, 23, 35), SOC inhibitor, 50 μM SKF96365 significantly inhibited HICI (p<0.05 vs. control, Fig. 6Aiii, Aiv). Similarly, Ryanodine and SKF96365, not XeC, inhibited H₂O₂-induced [Ca²⁺]_i increase (Fig. 6B, C).

FIG. 6.

Inhibition of hypoxia- and H₂O₂-induced [Ca²⁺]_i increase by manipulations of intracellular Ca²⁺ release and extracellular Ca²⁺ influx. (A) Representative curve of [Ca²⁺]_i responses to hypoxia alone (Hy1) and the second episode of hypoxia (Hy2) in the presence of ryanodine (n=10, Ai), XeC (n=17, Aii) or SKF96365 (n=8, Aiii). Averaged [Ca²⁺]_i peak responses to the second episode of hypoxia (*p<0.05, Aiv). (B) Representative curve of [Ca²⁺]_i responses to 15.6 μM H₂O₂ in the absence and the subsequent presence of ryanodine (n=15, Bi) or XeC (n=17, Bii). Averaged [Ca²⁺]_i peak responses to H₂O₂ (*p<0.05, Biii). (C) Representative curve of [Ca²⁺]_i responses to 15.6 μM H₂O₂ in the presence of verapamil (n=12, Ci) or SKF96365 (n=17, Cii). Averaged [Ca²⁺]_i peak responses to 15.6 μM H₂O₂ (*p<0.05, Ciii). (D) Western blotting and densimetric analysis of STIM1 (Di, left) and RyR3 (Di, right) level in control and shRNA-treated PASMCs (n=3 for each, *p<0.01, Di). Representative curve of [Ca²⁺]_i responses to hypoxia and 15.6 μM H₂O₂ in control (n=6, Dii), STIM1 knockdown (n=9, Diii), or RyR3 knockdown PASMCs (n=24, Div). Averaged [Ca²⁺]_i peak responses to hypoxia or H₂O₂ (*p<0.05, Dv). Extracellular Ca²⁺ is 2.0 mM for all.

RyR3 (48) and STIM1 knockdown block or profoundly inhibited hypoxia- and H₂O₂-induced [Ca²⁺]_i increase, respectively (Fig. 6D), revealing that RyR3 and STIM1 can be the major molecular targets of CaSR activation or sensitization upon hypoxia and H₂O₂ stimulation. However, this study does not exclude the involvement of voltage-sensitive pathways (1, 25, 43), as verapamil also inhibited H₂O₂-induced [Ca²⁺]_i elevation (n=12, p<0.05 vs. control, Fig. 6C).

Under normal or normoxic conditions, CaSR activation (by elevating [Ca²⁺]_o to 6.0 mM) -induced [Ca²⁺]_i signal in PASMCs was inhibited by XeC (Fig. 7A–D), indicating that CaSR activation-induced [Ca²⁺]_i signal is mediated by IP₃ receptor, the well-established downstream target of CaSR (14). However, the downstream target turns to be RyR under hypoxic condition in PASMCs (Fig. 6). To reveal the discrepancy, the role of protein kinase A (PKA) pathway was explored. XeC did (Fig. 7F, H), whereas ryanodine failed to (Fig. 7G, H), inhibit HICI in the presence of H-89, a PKA inhibitor, indicating that IP₃ receptor mediates HICI, while PKA signaling pathway is blocked. On the contrary, ryanodine did, whereas XeC failed to, inhibit HICI in the absence of H-89 (Fig. 7I–L), implying that rynodine receptor mediates HICI, while PKA signaling pathway is not blocked.

FIG. 7.

The effect of protein kinase A (PKA) inhibition on CaSR-mediated [Ca²⁺]_i signal in PASMCs. (A) Representative tracing of [Ca²⁺]_i in PASMCs in response to repetitive elevation of [Ca²⁺]_o from 2.0 to 6.0 mM (n=11). (B) Effect of 20 μM XeC on [Ca²⁺]_o elevation-induced [Ca²⁺]_i response (n=11). (C). Effect of 100 (μM ryanodine (Ry) on [Ca²⁺]_o elevation-induced [Ca²⁺]_i response (n=10). (D) Averaged [Ca²⁺]_i responses in A–C (*p<0.05). (E) Representative tracing of [Ca²⁺]_i in PASMCs in response to hypoxia in the absence and then presence of 10 (μM H-89, a PKA inhibitor (n=6). (F) Effect of XeC on hypoxia-induced [Ca²⁺]_i response in the presence of 10 (μM H-89 (n=8). (G) Effect of ryanodine on hypoxia-induced [Ca²⁺]_i response in the presence of 10 (μM H-89 (n=10). (H) Averaged [Ca²⁺]_i responses in E–G (*p<0.05). (I) Representative tracing of [Ca²⁺]_i in PASMCs in response to repetitive episodes of hypoxia (n=51). (J) Effect of XeC on hypoxia-induced [Ca²⁺]_i response (n=17). (K) Effect of ryanodine on hypoxia-induced [Ca²⁺]_i response (n=10). (L) Averaged [Ca²⁺]_i responses in I–K (*p<0.05). For comparison, some results in Figures 2A and 6A were included here as I–L.

HPV: role of CaSR in vitro and in vivo

Phenylephrine (PE)-preconstricted PA rings showed no response to acetylcholine confirming the removal of endothelium; however, they contracted when each hypoxic challenge was applied to the vessels (Fig. 8Ai). Calhex231 dose-dependently inhibited the hypoxia-induced constriction of the vessel (four parameter logistic regression, p<0.05, Fig. 8Aiv). 15.6 μM H₂O₂ contracted rings in a way similar to hypoxia (Fig. 8Bi) that was also inhibited by 1 μM Calhex231 (n=5, p<0.01, Fig. 8Bii, Biii), suggesting the mechanistic role of H₂O₂ in sensing CaSR and the subsequent constriction of PA by hypoxia. Furthermore, the hypoxia-induced constriction of PA isolated from CaSR knockdown rats was profoundly inhibited as compared with control (n=3, p<0.01, Fig. 8C).

FIG. 8.

Inhibition of hypoxic pulmonary vasoconstriction (HPV) and H₂O₂-induced pulmonary artery constriction in vitro by Calhex231 and CaSR knockdown. (A) Isometric tension of endothelium-removed pulmonary artery (PA) rings in response to acetylcholine (Ach) and two episodes of hypoxia (Hy1 and Hy2) sequentially each after preconstriction by phenylephrine (PE) and to a final exposure of 80 mM K⁺ (KCl-physiological salt solution (KPSS), an equimolar substitution of K⁺ for Na⁺; n=3, Ai). Isometric tension of endothelium-removed PA in response to Ach and two episodes of hypoxia in the absence (Hy1) and presence of 1 μM Calhex231 (Hy2, n=3, Aii). Averaged responses to PE, Ach, and hypoxia as well as the effect of 1 μM Calhex231 (normalized to tension induced by KPSS, *p<0.01, n=3, Aiii). Dose-dependent inhibition of HPV in vitro by Calhex231 (four parameter logistic regression, p<0.05, Aiv). (B) Isometric tension of endothelium-removed PA in response to Ach, hypoxia, and 15.6 μM H₂O₂ in the absence (n=6, Bi) and presence of 1 μM Calhex231 (n=5, Bii). Averaged responses to PE, Ach, hypoxia, and H₂O₂ as well as the effect of 1 μM Calhex231 (*p<0.01, Biii). (C) Western blotting and densimetric analysis of CaSR level in endothelium-removed PA isolated from rats treated with shRNA (n=4, *p<0.01, Ci). Isometric tension in response to Ach, hypoxia in endothelium-removed PA isolated from control (n=5, Cii), and CaSR shRNA-treated rats (n=5, Ciii). Averaged responses to PE, Ach, and hypoxia (*p<0.01, Civ). The maximum constriction response to KPSS was used to normalize the tension for each experiment.

The 10 min episodes of hypoxia successively triggered typical HPV in rats (n=4, Fig. 9Ai), which was dose-dependently inhibited by Calhex231 (four-parameter logistic regression, p<0.05, Fig. 9Aiv). HPV was found to be typical in control rats treated with nonspecific shRNA (n=3, Fig. 9Bi), whereas significantly inhibited in CaSR knockdown rats (n=4, p<0.01 vs. control, Fig. 9Bii, Biii). CaSR knockdown did not affect PE-induced pulmonary vascular constriction in rats (not shown) or plasma Ca²⁺ level (Supplementary Fig. S6).

FIG. 9.

Inhibition of HPV in vivo by Calhex231 and CaSR knockdown. (A) Pressure of pulmonary artery (Ppa) in response to successive episodes of hypoxia in the absence (n=3, Ai) and presence of 1 μM Calhex231 (n=3, Aii). Averaged increase in pulmonary vascular resistance (ΔPVR *p<0.01, Aiii). Dose-dependent inhibition of HPV in vivo by Calhex231 (four parameter logistic regression, p<0.05, Aiv). (B) Ppa in response to successive episodes of hypoxia in control (n=3, Bi) and CaSR shRNA-treated rats (n=4, Bii). Averaged alterations of ΔPVR (*p<0.01, Biii). The pulmonary vascular resistance (PVR) was calculated by Ppa-left ventricular end-diastolic pressure (LVEDP)/cardiac output (CO; mmHg·min·L⁻¹) and used to determine HPV as ΔPVR=PVR_10% _O₂−PVR_20% _O₂/PVR_20% _O2 (%).

Discussion

The basal function of CaSR in PASMCs was first evaluated by elevating the extracellular Ca²⁺ to supraphysiological levels, the direct stimulus of CaSR (14) (Fig. 1). In the subsequent experiments revealing the actual role of CaSR in HPV, the extracellular Ca²⁺ was maintained at a physiological level.

Hypoxia induces an [Ca²⁺]_i increase (HICI) in PASMCs that depends on extracellular Ca²⁺ (Fig. 2). These results are broadly consistent with most of the previous observations well documented in literature including those on isolated or cultured PASMCs (9, 25, 34, 35), isolated pulmonary arteries (PAs) (13, 19, 29), and perfused lung or lung slices (10, 42). In the absence of extracellular Ca²⁺, however, hypoxia was reported to cause a small and transient increase followed by a sustained decrease in [Ca²⁺]_i below baseline in canine PASMCs (22, 23), a small increase in rat PASMCs (25) or transient(s) in 34% of rat PASMCs examined (27). A transient contraction of rat PA rings by hypoxia in Ca²⁺-free medium was also noted (12). This discrepancy can be due to differences in experimental preparations (3, 32) and heterogeneity of [Ca²⁺]_i response to hypoxia in PASMCs (27). Extracellular Ca²⁺ was shown to differentially affect hypoxic vasoconstriction in different species (32), and hypoxia also differentially affected [Ca²⁺]_i in PASMCs isolated from different regions of PA tree (3). Additional and potential reasons can be the spontaneous Ca²⁺ release in the absence of extracellular Ca²⁺ reported in a variety of cell types including vascular smooth muscle cells (31, 44) and endogenous peroxidase activity. Generally, the extracellular Ca²⁺ dependence of HICI was understood as the importance of Ca²⁺ influx in all previous studies. The present study, however, showed further that both Calhex231 and CaSR knockdown inhibited HICI in PASMCs (Figs. 2 and 3). This provides, to the best of our knowledge, the first clue of the CaSR involvement in HICI and HPV. These novel findings further imply that the extracellular Ca²⁺ dependence of HICI cannot be simply or merely interpreted as the resource of Ca²⁺ influx as presumed. An additional role of extracellular Ca²⁺, the ligand of CaSR can be an initial trigger for the activation of CaSR during hypoxia and the subsequent [Ca²⁺]_i elevation and constriction.

The actual role of ROS in HPV remains controversial, and even both increased and decreased ROS by hypoxia were documented as recently reviewed (33, 37, 38). The present study is consistent with those showing increased ROS by hypoxia (10, 26, 39 –41). The oxidation of mitochondria-localized RoGFP observed here also suggests the origin of ROS, and this is generally consistent with the previous study showing increased oxidation of RoGFP in the intermembrane space but decreased oxidation of RoGFP in the mitochondrial matrix (40). H₂O₂ at the level achieved by hypoxia increased [Ca²⁺]_i in the presence of extracellular Ca²⁺, which was diminished by the removal of extracellular Ca²⁺, the presence of Calhex231, or CaSR knockdown (Fig. 4). These results suggest that ROS elevated by hypoxia cannot directly stimulate intracellular Ca²⁺ release by itself and that H₂O₂ induces [Ca²⁺]_i elevation through CaSR in PASMCs. It also implies that the Ca²⁺ sensitivity of CaSR should be enhanced sufficiently by the H₂O₂ level or oxidized status achieved by hypoxia for mediating [Ca²⁺]_i elevation in the presence of constant extracellular Ca²⁺ level (without elevating [Ca²⁺]_o as the stimulus of CaSR). To better describe this process mechanistically, the present study adopts the expression of “sensing/sensitization,” a concept usually used to indicate an altered activity of ion channel under oxidized/reduced status underlying HPV (33, 43) Thus, H₂O₂ at the level achieved by hypoxia induces [Ca²⁺]_i signaling in PASMCs through sensitizing CaSR. The mechanism underlying how hypoxia/ROS sensitizes CaSR remains unclear. One speculation can be the regulation of CaSR dimerization by disulfide bridge(s), although it is not an absolute requirement (2, 47).

The mitochondria-derived H₂O₂ was validated to sensitize CaSR and trigger HICI using mitochondria depletion strategy (Fig. 5). Ryanodine at the concentration inhibiting RyR or SOC inhibitor, but not IP₃ receptor inhibitor, significantly attenuated HICI (Fig. 6) that confirms previous reports (4, 8, 11, 15, 23, 34, 35, 48). An additional finding from the present study is that ryanodine/RyR3 knockdown and SOC inhibition/STIM1 knockdown similarly attenuated 15.6 μM HICI in PASMCs (Fig. 6), suggesting that RyR3 and STIM1 are likely the key components or downstream targets underlying CaSR activation or sensitization-triggered [Ca²⁺]_i signaling during hypoxia. In other words, the pivotal role of sensitized CaSR is to arrange or direct the mitochondria-derived H₂O₂, extracellular Ca²⁺, the intracellular Ca²⁺ release pathway of RyR3, and the STIM1-participated extracellular Ca²⁺ influx pathway of SOC to orchestrate the [Ca²⁺]_i elevation during hypoxia in PASMCs. However, this study does not exclude the role of voltage-sensitive channels and myofilament Ca²⁺ sensitivity in HICI and HPV (1, 25, 28, 43). The inhibitory effect of ryanodine on HICI was swapped to XeC by the presence of a PKA inhibitor (Fig. 7), indicating that the switch of downstream target of CaSR activation from IP₃ receptor under normal/normoxic condition (14) to RyR under hypoxia is possibly related with PKA.

HPV is typically biphasic, an initial and transient one followed by a sustained constriction (4, 11, 16, 28, 29). The critical role of CaSR revealed in the current study appears applicable mainly to the initial phase and may also be involved in the sustained constriction (Supplementary Fig. S7).

Calhex231 and CaSR knockdown inhibited hypoxia-induced constriction of PA (Fig. 8). This is consistent with that CaSR activation in vascular smooth muscle cells can induce [Ca²⁺]_i elevation (17) and cause constriction of the gerbilline spiral modiolar artery (45) and that hypoxia- and peroxide-induced increase in [Ca²⁺]_i and contraction are intimately related in PASMCs (28, 29). Calhex231 also inhibited H₂O₂-induced PA constriction (Fig. 8), functionally confirming the signaling role of H₂O₂ in sensing CaSR, which, in turn, triggers PA constriction during hypoxia. The dose-dependent inhibition of HPV by Calhex231 was noted in rats, and the critical role of CaSR was further validated by the attenuated HPV in CaSR knockdown rats (Fig. 9). The effect of Calhex231 revealed in the current study appears to be specific, as it does not affect PE, high K⁺, or angiotensionII-induced PA constriction (Supplementary Figs. S8 and S9). Taken together, these results provide functional evidence for the pivotal role of CaSR-mediated Ca²⁺ signaling in HPV, as illustrated in the mechanistic flow chart next (Fig. 10).

FIG. 10.

The illustration of CaSR-mediated Ca²⁺ signaling in HPV.

The blockage of hypoxia-triggered Ca²⁺ signaling in PASMCs by loading PEG-catalase and the essential role of CaSR in hypoxia-triggered Ca²⁺ signaling and HPV (Figs. 2 –4, 5C, and 6 –9) may warrant future studies to determine whether CaSR activity can be modulated by endogenous peroxidase and whether CaSR manipulation can potentially serve as a therapeutic target for HPV-related diseases such as hypoxic pulmonary hypertension and edema.

Materials and Methods

Cell culture

All experiments involving Sprague-Dawley rats were approved by the Institutional Animal Care and Use Committee.

Rat PASMCs were cultured from the third branches of intralobar artery explants and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (7, 36). The PASMCs at ∼70% confluence in 1–2 passages were employed in experiments. To deplete mitochondrial DNA, PASMCs were treated with 110 μg/ml sodium Py, 50 μg/ml Ur, and 200 ng/ml ethidium bromide as fully described earlier (7). PASMCs were isolated from at least three rats for each kind of experiment.

[Ca²⁺]_i measurement

[Ca²⁺]_i measurements were performed in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline (HBS) containing (mM): NaCl 135, KCl 5.0, CaCl₂ 2.0, MgSO₄ 1.2, d-glucose 10, and HEPES 5 (pH 7.40) or without Ca²⁺ but with 1 mM ethylene glycol tetraacetic acid (EGTA) (0 [Ca²⁺]_o) using Fura-2 as earlier (7, 49). For hypoxic stimulation, the HBS under normoxic condition in the bath was quickly switched to hypoxic HBS through a sealed perfusion system. The hypoxic HBS was made ready before experiments by equilibration with and continuous bubbling of 100% N₂.

Intracellular ROS measurement

The detection of intracellular ROS using H₂DCFDA and its calibration exploiting the DCFDA oxidation speed versus extracellular application of a series of H₂O₂ were previously described in detail (7, 30, 49). The mitochondrial targeting RoGFP was generated from pAcGFP1-Mito vector (Clontech) by incorporating four mutations (C48S, Q80R, S147C, and Q204C) in GFP coding region (40) using a QuikChange Multi Site-directed mutagenesis kit (Stratagene), and the mutations were confirmed by sequencing. After transfection with Lipofectamine 2000, RoGFP images in PASMCs were obtained under a confocol microscope (OLYMPUS IX71) with excitation of 405 or 488 nm and emission at 515±10 nm corresponding to the oxidized and reduced status, respectively. The ratios of fluorescence intensity from excitation at 405 and 488 nm were analyzed using FLUOVIEW V.5.0 software to quantify the extent of oxidative status.

Delivery of shRNA

Effective shRNA specifically against CaSR, STIM1, RyR3, and nonspecific control (Origene) were transfected into PASMCs using Lipofectamine 2000 as previously described (7). The shRNA constructs simultaneously encode GFP for identification of transfected PASMCs individually. The oligo-DNA encoding the control and the effective shRNA targeting CaSR were subcloned into the Lenti-X shRNA expression vector (Clontech), respectively, confirmed by sequencing and packaged into lentiviral particles using Lenti-X HTX packaging system and 293T cells (Clonetech). 72 h before hemodynamic experiments, 1 ml lentivirus (∼10⁶ TU/ml) was intravenously injected into each rat.

Immunochemical staining and western blot

To determine the expression of CaSR in PASMCs and endothelium-removed PAs, immunostaining and western blot were conducted as previously described in detail (7, 50) using an anti-CaSR antibody (Pierce Biotechnology).

Measurement of force

The endothelium-removed rings of intralobar PA were mounted in a wire myograph immersed in individual water-jacketed chambers containing physiological salt solution (118 NaCl, 24 NaHCO₃, 1 MgSO₄, 0.44 NaH₂PO₄, 4 KCl, 5.5 glucose, and 1.8 CaCl₂ mM, and pH 7.35–7.45), bubbled continuously with 95% O₂-5% CO₂ at 37°C, and stretched to a predetermined optimal passive tension of 500 mg. Force displacement was recorded using a PowerLab/4SP data acquisition system (ML118, AD Instruments). Each ring was initially contracted by 0.1 μM PE and subsequently challenged by 0.01 μM acetylcholine to confirm its contractility and endothelium nonintegrity, respectively. Since a small pretone by agonist can facilitate the hypoxic response in rat intralobar PA (16, 28), the rings were contracted by 0.1 μM PE 5 min before and during the hypoxic challenge. In some experiments, oxygen tension in the myograph chamber was simultaneously monitored via an isolated dissolved oxygen meter and electrode (ISO2, World Precision Instrument). Hypoxia was induced to a stable PO₂ level of 33 mmHg in PSS close to the rings within ∼1.7 min by continuously bubbling with 95% N₂-5% CO₂.

Hemodynamic measurements

Rats were anesthetized with urethane (1.2 g/kg ip), and a catheter was inserted in the trachea and connected to a ventilator supplying room air or a 10% O₂-90% N₂ gas mixture. To continuously monitor pressure of pulmonary artery, a PE-50 tubing with a curved tip was passed from the right jugular vein into the main PA. The catheterization was also performed for the measurement of left ventricular end-diastolic pressure, and cardiac output (CO) was measured using the thermodilution method with the CO pod (ML313C, AD Instruments) and the PowerLab/4SP data acquisition system. A 200 μl of room temperature 0.9% NaCl solution was injected into the left jugular vein catheter with its tip near the right atrium, and changes in blood temperature were detected by a T-type ultra-fast thermocouple probe (MLT1402, Physitemp) positioned in the aortic arch from the right carotid artery.

Statistical analysis

Data are reported as means±standard error. The Student's t-test and one-way analysis of variance analysis were made for two or multiple group comparisons, respectively. A difference was considered significant at p<0.05.

Footnotes

Acknowledgments

The research was supported by 973 program-2012CB518201, NSFC grants (81170048, 30971162, 30670926 and 30770940), and a grant from key laboratory of high altitude medicine of MOE.

Author Disclosure Statement

No competing financial interests exist.

Abbreviations Used

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Supplementary Material

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Extracellular Calcium-Sensing Receptor Is Critical in Hypoxic Pulmonary Vasoconstriction

Abstract

Introduction

Innovation

Results

Expression of a functional CaSR: sensing extracellular Ca2+

HICI: extracellular Ca2+ dependence, Calhex231 inhibition, and CaSR dependence

Ca2+ sensitivity of CaSR: enhanced by ROS

CaSR-dependent [Ca2+]i increase by hypoxia: role of mitochondria-derived H2O2

HICI: RyR and SOC dependence

HPV: role of CaSR in vitro and in vivo

Discussion

Materials and Methods

Cell culture

[Ca2+]i measurement

Intracellular ROS measurement

Delivery of shRNA

Immunochemical staining and western blot

Measurement of force

Hemodynamic measurements

Statistical analysis

Footnotes

Acknowledgments

Author Disclosure Statement

Abbreviations Used

References

Supplementary Material

Expression of a functional CaSR: sensing extracellular Ca²⁺

HICI: extracellular Ca²⁺ dependence, Calhex231 inhibition, and CaSR dependence

Ca²⁺ sensitivity of CaSR: enhanced by ROS

CaSR-dependent [Ca²⁺]_i increase by hypoxia: role of mitochondria-derived H₂O₂

[Ca²⁺]_i measurement