Contribution of elevated intracellular calcium to pulmonary arterial myocyte alkalinization during chronic hypoxia

Abstract

In the lung, exposure to chronic hypoxia (CH) causes pulmonary hypertension, a debilitating disease. Development of this condition arises from increased muscularity and contraction of pulmonary vessels, associated with increases in pulmonary arterial smooth muscle cell (PASMC) intracellular pH (pHi) and Ca(2+) concentration ([Ca(2+)]i). In this study, we explored the interaction between pHi and [Ca(2+)]i in PASMCs from rats exposed to normoxia or CH (3 weeks, 10% O2). PASMC pHi and [Ca(2+)]i were measured with fluorescent microscopy and the dyes BCECF and Fura-2. Both pHi and [Ca(2+)]i levels were elevated in PASMCs from hypoxic rats. Exposure to KCl increased [Ca(2+)]i and pHi to a similar extent in normoxic and hypoxic PASMCs. Conversely, removal of extracellular Ca(2+) or blockade of Ca(2+) entry with NiCl2 or SKF 96365 decreased [Ca(2+)]i and pHi only in hypoxic cells. Neither increasing pHi with NH4Cl nor decreasing pHi by removal of bicarbonate impacted PASMC [Ca(2+)]i. We also examined the roles of Na(+)/Ca(2+) exchange (NCX) and Na(+)/H(+) exchange (NHE) in mediating the elevated basal [Ca(2+)]i and Ca(2+)-dependent changes in PASMC pHi. Bepridil, dichlorobenzamil, and KB-R7943, which are NCX inhibitors, decreased resting [Ca(2+)]i and pHi only in hypoxic PASMCs and blocked the changes in pHi induced by altering [Ca(2+)]i. Exposure to ethyl isopropyl amiloride, an NHE inhibitor, decreased resting pHi and prevented changes in pHi due to changing [Ca(2+)]i. Our findings indicate that, during CH, the elevation in basal [Ca(2+)]i may contribute to the alkaline shift in pHi in PASMCs, likely via mechanisms involving reverse-mode NCX and NHE.

Keywords: Na+/Ca2+exchange; Na+/H+ exchange; hypoxic pulmonary hypertension.

Figure 1

Bar graphs showing mean ± SEM values for resting intracellular Ca²⁺ concentration ([Ca²⁺]_i; A) and basal intracellular pH (pH_i; B) in pulmonary arterial smooth muscle cells isolated from rats exposed to normoxia ($n=204$ for [Ca²⁺]_i and $n=550$ for pH_i) and chronic hypoxia ($n=455$ for [Ca²⁺]_i and $n=934$ for pH_i). Asterisk indicates a significant difference from the normoxic value ($P<0.05$).

Figure 2

A, Effect of exposure to KCl (80 mM; $n=97$ for normoxic and $n=138$ for hypoxic); removal of extracellular Ca²⁺ (Ca²⁺-free; $n=83$ for normoxic and $n=69$ for hypoxic); treatment with NiCl₂ (500 nM; $n=72$ for normoxic and $n=79$ for hypoxic) or treatment with SKF96365 (SKF; 10 μM; $n=79$ for normoxic and $n=47$ for hypoxic) on intracellular Ca²⁺ ([Ca²⁺]_i) in rat pulmonary arterial smooth muscle cells (PASMCs). B, Change in intracellular pH (pH_i) induced in PASMCs from normoxic and chronically hypoxic rats by exposure to KCl ($n=42$ for normoxic and $n=37$ for hypoxic); removal of extracellular Ca²⁺ ($n=92$ for normoxic and $n=34$ for hypoxic); treatment with NiCl₂ ($n=89$ for normoxic and $n=42$ for hypoxic) or treatment with SKF ($n=55$ for normoxic and $n=66$ for hypoxic). Data are expressed as mean ± SEM change (Δ) in [Ca²⁺]_i or pH_i. Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values.

Figure 3

A, Effect of removal of extracellular bicarbonate (HEPES; $n=28$ cells for normoxic and $n=32$ cells for hypoxic) and exposure to 3 mM ($n=88$ for normoxic and $n=77$ for hypoxic) or 10 mM ($n=25$ for normoxic and $n=32$ for hypoxic) ammonium chloride (NH₄Cl) on intracellular pH (pH_i) in pulmonary arterial smooth muscle cells (PASMCs) from normoxic and chronically hypoxic rats. B, Changes in intracellular Ca²⁺ ([Ca²⁺]_i) induced by exposure of PASMCs to HEPES-buffered extracellular solution ($n=85$ for normoxic and $n=48$ for hypoxic) or NH₄Cl (3 mM: $n=69$ for normoxic and $n=82$ for hypoxic; 10 mM: $n=100$ for normoxic and $n=48$ for hypoxic). Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values.

Figure 4

Role of Na⁺/H⁺ exchange in mediating Ca²⁺-dependent changes in intracellular pH (pH_i). Effect of ethyl isopropyl amiloride (EIPA; 10 μM) on basal pH_i (A) and intracellular Ca²⁺ ([Ca²⁺]_i; B) in pulmonary arterial smooth muscle cells (PASMCs) from normoxic ($n=40$ for pH_i and $n=42$ for [Ca²⁺]_i) or chronically hypoxic ($n=49$ for pH_i and $n=100$ for [Ca²⁺]_i) rats. Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values. C, EIPA prevented the change in pH_i induced by changing [Ca²⁺]_i via perfusion with KCl (80 mM; $n=42$ for untreated and $n=47$ for EIPA), Ca²⁺-free solution ($n=92$ for untreated and $n=35$ for EIPA) or NiCl₂ (500 nM; $n=89$ for untreated and $n=31$ for EIPA) in PASMCs from chronically hypoxic rats. Data are presented as mean ± SEM. Two asterisks indicate significant difference between values in the presence and absence of EIPA.

Figure 5

Role of Na⁺/Ca²⁺ exchange in mediating Ca²⁺-dependent changes in intracellular pH (pH_i). All data are presented as mean ± SEM. A, Effect of bepridil (BPD; $n=119$ for normoxic and $n=72$ for hypoxic), dichlorobenzamil (DCB; $n=86$ for normoxic and $n=68$ for hypoxic), and KB-R7943 (KBR; $n=97$ for normoxic and $n=127$ for hypoxic) on basal intracellular Ca²⁺ ([Ca²⁺]_i) in pulmonary arterial smooth muscle cells (PASMCs) from normoxic and chronically hypoxic rats. B, Effect of BPD ($n=90$ for normoxic and $n=39$ for hypoxic), DCB ($n=70$ for normoxic and $n=70$ for hypoxic), and KBR ($n=89$ for normoxic and $n=83$ for hypoxic) on basal pH_i in PASMCs. A and B, asterisk indicates significant difference from baseline; two asterisks indicate significant difference between normoxic and hypoxic values. C, The effect of pretreating cells from chronically hypoxic rats with BPD, DCB, or KBR on the change in pH_i induced by changing [Ca²⁺]_i with 80 mM KCl ($n=37$, 63, 111, and 37 for control, BPD, DCB, and KBR, respectively), Ca²⁺-free extracellular solution ($n=34$, 74, 103, and 35 for control, BPD, DCB, and KBR, respectively), or 500 nM NiCl₂ ($n=42$, 89, 98, and 56 for control, BPD, DCB, and KBR, respectively). Asterisk indicates significant difference from baseline; two asterisks indicate significant difference between values in the absence (control hypoxic) and presence of Na⁺/Ca²⁺ exchange inhibitor.

Figure 6

Schematic illustrating proposed mechanism by which changes in intracellular Ca²⁺ ([Ca²⁺]_i) influence intracellular pH (pH_i) in pulmonary arterial smooth muscle cells (PASMCs) during chronic hypoxia (CH). CH has been shown to upregulate the expression of canonical transient receptor potential (TRPC) proteins, leading to increased Ca²⁺ influx through nonselective cation channels (NSCC) and elevated intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because these channels can also conduct Na⁺, activation may also contribute to depolarization. Depolarization during CH leads to Ca²⁺ influx through reverse-mode Na⁺/Ca²⁺ exchange (NCX), which, along with upregulation of Na⁺/H⁺ exchanger isoform 1 (NHE1) expression, leads to increased activation of Na⁺/H⁺ exchange and elevated intracellular pH (pH_i). Both alkaline pH_i and elevated [Ca²⁺]_i have been associated with PASMC proliferation and contraction. Inhibitors are shown in red. Dashed gray line represents hypothesized pathway requiring additional confirmation. DCB: dichlorobenzamil; KBR: KB-R7943; SKF: SKF96365; BPD: bepridil; EIPA: ethylisoproplyamiloride.