Expression of store-operated Ca2+ entry and transient receptor potential canonical and vanilloid-related proteins in rat distal pulmonary venous smooth muscle

Abstract

Chronic hypoxia causes remodeling and alters contractile responses in both pulmonary arteries and pulmonary veins. Although pulmonary arteries have been studied extensively in these disorders, the mechanisms by which pulmonary veins respond to hypoxia and whether these responses contribute to chronic hypoxic pulmonary hypertension remain poorly understood. In pulmonary arterial smooth muscle, we have previously demonstrated that influx of Ca(2+) through store-operated calcium channels (SOCC) thought to be composed of transient receptor potential (TRP) proteins is likely to play an important role in development of chronic hypoxic pulmonary hypertension. To determine whether this mechanism could also be operative in pulmonary venous smooth muscle, we measured intracellular Ca(2+) concentration ([Ca(2+)](i)) by fura-2 fluorescence microscopy in primary cultures of pulmonary venous smooth muscle cells (PVSMC) isolated from rat distal pulmonary veins. In cells perfused with Ca(2+)-free media containing cyclopiazonic acid (10 μM) and nifedipine (5 μM) to deplete sarcoplasmic reticulum Ca(2+) stores and block voltage-dependent Ca(2+) channels, restoration of extracellular Ca(2+) (2.5 mM) caused marked increases in [Ca(2+)](i), whereas MnCl(2) (200 μM) quenched fura-2 fluorescence, indicating store-operated Ca(2+) entry (SOCE). SKF-96365 and NiCl(2), antagonists of SOCC, blocked SOCE at concentrations that did not alter Ca(2+) responses to 60 mM KCl. Of the seven known canonical TRP (TRPC1-7) and six vanilloid-related TRP channels (TRPV1-6), real-time PCR revealed mRNA expression of TRPC1 > TRPC6 > TRPC4 > TRPC2 ≈ TRPC5 > TRPC3, TRPV2 > TRPV4 > TRPV1 in distal PVSMC, and TRPC1 > TRPC6 > TRPC3 > TRPC4 ≈ TRPC5, TRPV2 ≈ TRPV4 > TRPV1 in rat distal pulmonary vein (PV) smooth muscle. Western blotting confirmed protein expression of TRPC1, TRPC6, TRPV2, and TRPV4 in both PVSMC and PV. Our results suggest that SOCE through Ca(2+) channels composed of TRP proteins may contribute to Ca(2+) signaling in rat distal PV smooth muscle.

Fig. 1.

Representative phase-contrast microscopy images (objective ×20) of rat distal pulmonary venous smooth muscle cells (PVSMC) in 2–3 days of culture (A) and 4–6 days of culture (B). C: α-actin fluorescent immunostaining in red, with nuclei counterstained in green in PVSMC cultured for 4–6 days (objective ×40). D: representative traces of time course of [Ca²⁺]_i responses to KCl (60 mM) in rat distal PVSMC.

Fig. 2.

A: time course of [Ca²⁺]_i change (Δ[Ca²⁺]_i) before and after restoration of extracellular [Ca²⁺] to 2.5 mM in distal PVSMC perfused with Ca²⁺-free Krebs ringer bicarbonate (KRB) solution containing 10 μM CPA, 1 mM EGTA, and 5 μM nifedipine (Nifed+CPA; n = 8 experiments in 239 cells) and in control cells, which did not receive CPA but were otherwise treated similarly (Nifed; n = 6 experiments in 168 cells). B: average peak change in [Ca²⁺]_i during restoration of extracellular [Ca²⁺] between 15 and 30 min in cells shown in A. *P < 0.0001 vs. cells absent of CPA.

Fig. 3.

A: time course of fura-2 fluorescence at 360 nm normalized to values at time 0 before and after administration of MnCl₂ (200 μM) to distal PVSMC perfused with Ca²⁺-free KRB solution containing 10 μM CPA, 1 mM EGTA, and 5 μM nifedipine (+CPA; n = 7 experiments in 177 cells) and in control cells, which did not receive CPA but were otherwise treated similarly (−CPA; n = 5 experiments in 137 cells). B: average decrease in fura-2 fluorescence at 10 min after administration of MnCl₂ to cells shown in A. *P < 0.0001 vs. cells absent of CPA.

Fig. 4.

Time course effects of different doses of SKF-96365 (A) and NiCl₂ (B) on [Ca²⁺]_i change (Δ[Ca²⁺]_i) before and after restoration of extracellular [Ca²⁺] to 2.5 mM in distal PVSMC perfused with Ca²⁺-free KRB solution containing 10 μM CPA, 1 mM EGTA, and 5 μM nifedipine. Average peak change in Δ[Ca²⁺]_i before (C, between 0 and 15 min) and after (D, between 15 and 30 min) restoration of extracellular [Ca²⁺] in cells exposed to SKF-96365 (1, 10, and 50 μM; C), NiCl₂ (50, 200, and 500 μM; D), or vehicle control. *Significant difference from respective control (P < 0.05). **P < 0.05 vs. value at 1 μM SKF-96365 or 50 μM NiCl₂. ***P < 0.05 vs. value at 10 μM SKF-96365 or 200 μM NiCl₂.

Fig. 5.

A: effect of 50 μM SKF-96365 and 500 μM NiCl₂ on time course of fura-2 fluorescence at 360 nm normalized to values at time 0 before and after administration of MnCl₂ (200 μM) to distal PVSMC perfused with Ca²⁺-free KRB solution containing 10 μM CPA, 1 mM EGTA, and 5 μM nifedipine. B: average change in fura-2 fluorescence at 360 nm in cells exposed to SKF-96365 (n = 5 experiments in 120 cells), NiCl₂ (n = 5 experiments in 118 cells), or vehicle control (n = 5 experiments in 117 cells) at 10 min after administration of MnCl₂. *Significant difference from control (P < 0.0001).

Fig. 6.

Effect of nifedipine (5 μM) (A; n = 5 experiments in 145 cells), SKF-96365 (50 μM) (B; n = 6 experiments in 165 cells), and NiCl₂ (500 μM) (C; n = 6 experiments in 170 cells) on [Ca²⁺]_i responses to KCl (60 mM) in rat distal PVSMC. *P < 0.0001 vs. control.

Fig. 7.

Expression of TRPC mRNA normalized to β-actin in rat proximal and distal PVSMC (top: n = 5) and PV (bottom: n = 5) as determined by real-time quantitative PCR. *P < 0.05 vs. respective proximal cells or tissue.

Fig. 8.

Expression of TRPV mRNA normalized to β-actin in rat proximal and distal PVSMC (top: n = 5) and PV (bottom: n = 5) as determined by real-time quantitative PCR.

Fig. 9.

Expression of TRPC and TRPV proteins in rat proximal and distal PVSMC and PV as determined by Western blotting using rat brain as positive control. Data show representative blots for TRPC1, TRPC4, TRPC6, TRPV2, TRPV4, and β-actin in rat distal PVSMC (top: n = 5) and PV (bottom: n = 3). *P < 0.05 vs. respective proximal cells or tissue.