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Am J Physiol Lung Cell Mol Physiol 292: L1002-L1012, 2007. First published January 5, 2007; doi:10.1152/ajplung.00161.2006
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Inhibition of hypoxia-induced calcium responses in pulmonary arterial smooth muscle by acetazolamide is independent of carbonic anhydrase inhibition

Larissa A. Shimoda,1 Trevor Luke,1 J. T. Sylvester,1 Hui-Wen Shih,2 Ahamindra Jain,2 and Erik R. Swenson3

1Division of Pulmonary and Critical Care Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland; 2Department of Chemistry, University of California at Berkeley, Berkeley, California; and 3Division of Pulmonary and Critical Care Medicine, University of Washington School of Medicine, St. Louis, Missouri

Submitted 25 April 2006 ; accepted in final form 13 December 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxic pulmonary vasoconstriction (HPV) occurs with ascent to high altitude and can contribute to development of high altitude pulmonary edema (HAPE). Vascular smooth muscle contains carbonic anhydrase (CA), and acetazolamide (AZ), a CA inhibitor, blunts HPV and might be useful in the prevention of HAPE. The mechanism by which AZ impairs HPV is uncertain. Originally developed as a diuretic, AZ also has direct effects on systemic vascular smooth muscle, including modulation of pH and membrane potential; however, the effect of AZ on pulmonary arterial smooth muscle cells (PASMCs) is unknown. Since HPV requires Ca2+ influx into PASMCs and can be modulated by pH, we hypothesized that AZ alters hypoxia-induced changes in PASMC intracellular pH (pHi) or Ca2+ concentration ([Ca2+]i). Using fluorescent microscopy, we tested the effect of AZ as well as two other potent CA inhibitors, benzolamide and ethoxzolamide, which exhibit low and high membrane permeability, respectively, on hypoxia-induced responses in PASMCs. Hypoxia caused a significant increase in [Ca2+]i but no change in pHi. All three CA inhibitors slightly decreased basal pHi, but only AZ caused a concentration-dependent decrease in the [Ca2+]i response to hypoxia. AZ had no effect on the KCl-induced increase in [Ca2+]i or membrane potential. N-methyl-AZ, a synthesized compound lacking the unsubstituted sulfonamide group required for CA inhibition, had no effect on pHi but inhibited hypoxia-induced Ca2+ responses. These results suggest that AZ attenuates HPV by selectively inhibiting hypoxia-induced Ca2+ responses via a mechanism independent of CA inhibition, changes in pHi, or membrane potential.

pulmonary vascular smooth muscle; intracellular Ca2+

ALVEOLAR HYPOXIA, AS OCCURS with ascent to high altitude or chronic lung diseases, results in hypoxic pulmonary vasoconstriction (HPV). Exaggerated HPV causes high altitude pulmonary edema (2) and may complicate the course of patients with chronic cardiopulmonary disease. Despite extensive study, the exact cellular mechanisms by which hypoxia induces HPV remain poorly understood. HPV is generally believed to require Ca2+ influx into pulmonary arterial smooth muscle cells (PASMCs), as HPV is attenuated by removal of extracellular Ca2+ and by antagonists of voltage-gated Ca2+ channels (25, 27, 34, 40–42, 48). HPV can also be modulated by changes in intracellular pH (pHi) (33) and extracellular pH (21).

When taken prior to ascent, acetazolamide (AZ), a carbonic anhydrase (CA) inhibitor, can prevent the development of acute mountain sickness (44) through its effects on renal bicarbonate reabsorption and chemoreceptor activity to increase ventilation and arterial oxygenation. Work in intact animals and isolated perfused lung preparations has demonstrated that AZ also attenuates the magnitude of HPV and slows the onset of the response (7, 8, 15). The mechanisms by which this occurs remain uncertain. As a respiratory stimulant, AZ raises alveolar PO2 and by this mechanism might blunt HPV; however, a direct effect independent of ventilation-induced changes in alveolar PO2 is also evident since AZ inhibits HPV when ventilation, alveolar PO2, and PCO2 are carefully controlled or held constant (7, 8, 15). Vascular smooth muscle contains CA (3), and AZ causes vasodilation in systemic blood vessels (5, 28, 29). Mechanisms involved may include modulation of K+ channels, membrane potential (Em), Ca2+ signaling, or pHi (28, 29). Furthermore, in various cell types, CA inhibitors block Ca2+ channels (13, 17, 26, 55), inhibit a Cl–-dependent ATPase (5), and activate Ca2+-activated K+ channels (29, 46, 47). The effect of AZ and/or CA inhibition on PASMC function is unknown.

Based on these considerations, we hypothesized that AZ blunts HPV by CA inhibition via modulation of pHi, Em, or intracellular Ca2+ concentration ([Ca2+]i) in PASMCs. To test this hypothesis, we used fluorescent microscopy and the Ca2+-, Em-, and pH-sensitive dyes, fura-2, DiBAC4(3), and 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), respectively, to determine the effect of hypoxia and other agonists on PASMC Ca2+, Em, and pHi and the ability of AZ to modulate these responses. To determine whether the effect of AZ is attributable to CA inhibition, we tested another powerful sulfonamide CA inhibitor, ethoxzolamide (EZ) (24), and N-methyl-AZ (N-Meth-AZ), in which one of the amine hydrogens of the sulfonamide moiety (SO2NH2) responsible for CA inhibition is replaced with a methyl group (SO2NHCH3) to prevent binding to CA (23, 35). To determine whether AZ inhibits a membrane-bound CA with its activity oriented to the extracellular space or an intracellular cytosolic isozyme, we used benzolamide (BZ), a potent hydrophilic cell-impermeant sulfonamide CA inhibitor (24). A portion of these results has been described previously (43).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Isolation of PASMCs. All procedures and protocols in this study were approved by the Johns Hopkins University Animal Care and Use Committee. The method for obtaining single PASMCs has been described previously (39). Intrapulmonary arteries (200–600 µm outer diameter) were isolated from male Wistar rats (250–350 g) and cleaned of connective tissue. After removal of the endothelial cells, the arteries were allowed to recover for 30 min in cold (4°C) HBSS containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and 10 glucose, with pH adjusted to 7.2 with 5 M NaOH, followed by 20 min in reduced-Ca2+ (20 µM CaCl2) HBSS at room temperature. The tissue was enzymatically digested in reduced-Ca2+ HBSS containing collagenase (type I; 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM) at 37°C for 20 min. Following digestion, single smooth muscle cells were dispersed by gentle trituration with a wide-bore transfer pipette in Ca2+-free HBSS, and the cell suspension was placed on 25-mm glass cover slips. PASMCs were transiently cultured in Ham's F-12 media supplemented with 0.5% fetal bovine serum and 1% penicillin/streptomycin for 24–48 h before study.

Measurement of [Ca2+]i. The methods for measurement of [Ca2+]i have been described previously (51). PASMCs were placed in a laminar flow cell chamber and perfused with modified Krebs bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.1 glucose, and 1.2 KH2PO4. [Ca2+]i was measured in cells incubated with 5 µM fura-2 AM for 60 min at 37°C, then washed with physiological salt solution for 15 min at 37°C to remove extracellular dye and allow complete deesterification of cytosolic dye. Ratiometric measurement of fluorescence from the dye was performed on a workstation (Intracellular Imaging, Cincinnati, OH) consisting of a Nikon TSE 100 Ellipse inverted microscope with epifluorescence attachments. The light beam from a xenon arc lamp was filtered at 340 and 380 nm and focused onto the PASMCS under examination via a x20 fluorescence objective (Super Fluor 20, Nikon). Light emitted from the cell at 510 nm was returned through the objective and detected by a cooled charge-coupled device (CCD) imaging camera. An electronic shutter (Sutter Instruments) was used to minimize photobleaching of dye. Protocols were executed and data collected online with InCyte software (Intracellular Imaging). [Ca2+]i was estimated from in vitro calibration solutions.

pHi measurements. pHi within PASMCs was monitored using the cell-permeant pH-sensitive dye BCECF-AM. After incubation with BCECF-AM for 60 min at 37°C under an atmosphere of 21% O2-5% CO2, cells were washed with Krebs solution for 15 min at 37°C to remove extracellular dye and allow complete deesterification of cytosolic dye. Cells were excited with light at 490 and 440 nm, and light emitted from the cells was detected at 530 nm. The ratio of 490-to-440-nm emission was calculated and converted into pH values by performing a calibration curve after each experiment. PASMCs were subjected to a high K+ buffer containing 10 µM nigericin, which allowed the cell to adopt the pH of the high K+ buffer. Two high K+/nigericin buffers were used to set pH to 6.5 or 7.5. pHi was estimated from in situ calibration after each experiment. Cells were perfused with a solution containing (in mM) 105 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 20 HEPES-Tris, and 0.01 nigericin to allow pHi to equilibrate to external pH (32). A two-point calibration was created from fluorescence measured as pHi was adjusted with KOH from 6.5 to 7.5. Intracellular H+ ion concentration ([H+]i) was determined from pHi using the formula pHi = –log([H+]i).

Em measurements. Changes in Em were measured using the fluorescence dye DiBAC4(3), which was excited at 490 nm and detected at 510 nm. Cells were loaded by continuous perfusion with Krebs solution containing 500 nM DiBAC4(3) for at least 15 min before beginning measurements to insure stable uptake of dye. Baseline DiBAC4(3) fluorescence was monitored for 5 min with control Krebs, followed by 10 min with Krebs in the presence or absence of AZ (100 µM). At the end of this period, cells were challenged with KCl (60 mM) or endothelin-1 (ET-1; 10–8 M). Since DiBAC4(3) fluorescence is detected at a single wavelength, any change in the concentration of the dye in the perfusion medium (i.e., differing concentrations between perfusion reservoirs) could result in a change in fluorescence that is not due to a change in Em. To minimize the possibility of this type of error, all experiments were performed by perfusing from a single reservoir into which AZ, KCl, or ET-1 were dissolved directly.

CA inhibitors. AZ and EZ were obtained from Sigma Scientific. BZ was a gift from Dr. Thomas H. Maren (University of Florida, Department of Pharmacology). The synthesis and purification of N-Meth-AZ followed that described by Maren (23). In N-Meth-AZ, one of the amine hydrogens of the sulfonamide moiety (SO2NH2) responsible for CA inhibition by AZ is replaced with a methyl group (SO2NHCH3) to prevent binding to CA, but the rest of the molecule remains unaltered in terms of its general size, aromatic ring structure, and charge characteristics. Figure 1 shows the chemical structures of the four sulfonamides, molecular weight, inhibition constant against CA, lipid/water solubilities, and ionization constants (pKa).


Figure 1
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  		Fig. 1. Structure and properties of the carbonic anhydrase (CA)-inhibiting and -noninhibiting sulfonamides. MW, molecular weight; KI, dissociation constant for inibitor binding; CA II, carbonic anhydrase II.

 
Statistical analysis. All data are presented as means ± SE. Statistical comparisons were performed using Student's t-test (paired or unpaired) as appropriate. Differences were considered to be significant when P < 0.05.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of hypoxia and AZ on pHi. To test whether the mechanism by which AZ inhibited HPV in isolated lungs involved modulation of hypoxia-induced changes in PASMC pHi, pHi was measured during control conditions and after changing to hypoxic solution in the absence and presence of 100 µM AZ (Fig. 2). AZ alone caused a small but statistically significant decrease in resting pHi (Table 1). Exposure to 4% O2 had no significant effect on PASMC pHi, with pHi measured at 7.23 ± 0.02 during normoxia and 7.24 ± 0.03 after 15 min of hypoxia (n = 85 cells in 5 experiments). Similarly, hypoxia had no effect on pHi in PASMCs pretreated with AZ (7.22 ± 0.03 to 7.24 ± 0.03; n = 55 cells in 4 experiments).


Figure 2
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  		Fig. 2. Composite traces demonstrating the effect of hypoxia on intracellular pH (pHi) in the absence and presence of acetazolamide (AZ; 100 µM). Whereas AZ caused a small decrease in pHi, hypoxia had no effect on pHi in either control cells (n = 85 cells in 5 experiments) or cells treated with AZ (n = 55 cells in 4 experiments).

 

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  		Table 1. Effect of carbonic anhydrase inhibitors

 
Effect of hypoxia and AZ on [Ca2+]i. Perfusing cells with solution equilibrated with 4% O2, 5% CO2 produced a rapid, reversible increase in [Ca2+]i (Fig. 3), from 176.2 ± 10.2 nM to a maximum of 258.5 ± 13.9 nM ({Delta}[Ca2+]i = 81.9 ± 9.5 nM; n = 115 cells in 5 experiments). The maximum change in [Ca2+]i in response to hypoxia occurred within 7 min of beginning perfusion with hypoxic solution. On return to normoxia, [Ca2+]i rapidly returned to normoxic levels. Application of AZ (10–100 µM) had no effect on basal [Ca2+]i in PASMCs (Table 1). AZ caused a concentration-dependent decrease in the hypoxia-induced rise in [Ca2+]i, with a significant reduction in the peak change in [Ca2+]i induced by hypoxia at both 30 and 100 µM AZ (Fig. 3). From the concentration-inhibition plot, the IC50 was estimated to be 50 µM.


Figure 3
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  		Fig. 3. A: composite traces illustrating the effect of hypoxia on [Ca2+]i in the absence (n = 115 cells in 5 experiments) and presence of AZ (10–100 µM). In the absence of AZ, hypoxia caused a rapid, reversible increase in [Ca2+]i. AZ caused a dose-dependent inhibition of the hypoxia-induced Ca2+ response with almost complete blockade at 100 µM (n = 73 cells in 5 experiments). B: bar graphs representing effect of AZ on the peak change in [Ca2+]i induced by hypoxia (n = 38 cells in 3 experiments for 10 µM; n = 60 cells in 4 experiments for 30 µM). C: concentration response plot demonstrating IC50 of AZ. *Significant difference from control value (P < 0.05).

 
Effect of AZ on KCl-induced Ca2+ responses. To examine whether the effect of AZ on hypoxia-induced Ca2+ responses was due to a nonspecific inhibition of Ca2+ signaling, we determined the effect of AZ on the response to KCl. Exposure to KCl (60 mM) caused a significant increase in [Ca2+]i (153.2 ± 14.5 to 276.6 ± 19.8 nM; n = 40 cells in 4 experiments), reaching a mean peak change in [Ca2+]i of 123.4 ± 20.6 nM (Fig. 4). Following pretreatment with 100 µM AZ, the mean change in [Ca2+]i in response to KCl was not altered, reaching a mean of 134.7 ± 18.2 nM (from 157.8 ± 11.9 to 292.6 ± 21.2 nM; n = 43 cells in 4 experiments).


Figure 4
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  		Fig. 4. A: composite traces illustrating the effect of AZ (100 µM) on KCl-induced increases in [Ca2+]i. In the absence of AZ, KCl (60 mM) induced a rapid increase in [Ca2+]i (n = 40 cells in 4 experiments). Addition of AZ had no effect on the KCl-induced increase in [Ca2+]i (n = 43 cells in 4 experiments). B: bar graph representing mean change in [Ca2+]i induced by KCl in the absence and presence of AZ.

 
Effect of EZ and BZ on Ca2+ responses. As with AZ, neither EZ nor BZ altered basal [Ca2+]i (Table 1). However, in contrast to the effects of AZ, EZ (Fig. 5) and BZ (Fig. 6) had no significant effect on the hypoxia-induced increase in [Ca2+]i at any of the concentrations tested (10–100 µM). As with AZ, both EZ and BZ caused a significant decrease in resting pHi (Table 1). These results suggest that CA inhibition in PASMCs results in intracellular acidosis but that this is not related to the inhibition of the hypoxia-mediated rise in [Ca2+]i by AZ.


Figure 5
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  		Fig. 5. Top: composite traces illustrating the effect of ethoxzolamide (EZ; 10–100 µM), a CA inhibitor with high membrane permeability, on hypoxia-induced increases in [Ca2+]i. Bottom: bar graph representing average peak change in [Ca2+]i in response to hypoxia in the absence (n = 115 cells) and presence of EZ (n = 60 cells in 5 experiments for 10 µM; n = 68 cells in 4 experiments for 30 µM; n = 88 cells in 7 experiments for 100 µM). EZ had no effect on the peak change in [Ca2+]i induced by hypoxia at any of the concentrations tested.

 

Figure 6
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  		Fig. 6. Top: composite traces illustrating the effect of benzolamide (BZ; 10–100 µM), a CA inhibitor with poor membrane permeability, on hypoxia-induced increases in [Ca2+]i. Bottom: bar graph representing average peak change in [Ca2+]i in response to hypoxia in the absence (n = 115 cells) and presence of BZ (n = 65 cells in 5 experiments for 10 µM; n = 125 cells in 8 experiments for 30 µM; n = 50 cells in 4 experiments for 100 µM). BZ had no effect on the peak change in [Ca2+]i induced by hypoxia at any of the concentrations tested.

 
Effect of N-Meth-AZ on hypoxia-induced Ca2+ responses. To further test the hypothesis that AZ inhibited [Ca2+]i response to hypoxia via a mechanism unrelated to CA inhibition, we determined the effects of an AZ-derivative compound in which the sulfonamide group responsible for CA inhibition was substituted with a methyl group (N-Meth-AZ). The purity of the compound was assessed by high-resolution fast atom bombardment mass spectrometry, which showed only one species to be present in the recrystallized sample. In contrast to AZ, BZ, and EZ, N-Meth-AZ (100 µM) had minimal effect on basal pHi (7.13 ± 0.02 to 7.12 ± 0.01; n = 81 cells in 5 experiments; Fig. 7). Pretreatment with N-Meth-AZ for 10 min caused a very small but statistically significant increase in basal [Ca2+]i (193.4 ± 11.0 to 200.9 ± 11.3 nM; n = 63 cells in 4 experiments). The increase in [Ca2+]i induced by hypoxia in control PASMCs was markedly reduced in PASMCs treated with N-Meth-AZ, from 141.1 ± 32.2 nM (n = 78 cells in 4 experiments) to 33.2 ± 17.4 nM (n = 63 cells in 3 experiments) (Fig. 8A). To test whether the inhibitory effect of N-Meth-AZ on hypoxia-induced Ca2+ responses was due to generalized cell toxicity, we also tested the effect of N-Meth-AZ on KCl-induced Ca2+ responses. The mean increase in [Ca2+]i in response to KCl (60 mM) was similar in the presence (n = 73 cells in 4 experiments) and absence (n = 83 cells in 4 experiments) of 100 µM N-Meth-AZ (Fig. 8B).


Figure 7
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  		Fig. 7. Composite traces demonstrating the effect of 100 µM AZ (top) and N-methyl-AZ (N-Meth-AZ; bottom) on pHi in pulmonary arterial smooth muscle cells (PASMCs). AZ caused a small decrease in pHi (n = 55 cells in 4 experiments), whereas N-Meth-AZ (n = 81 cells in 5 experiments) had no significant effect on pHi.

 

Figure 8
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  		Fig. 8. Composite traces (left) and bar graphs (right) demonstrating the effect of hypoxia (4% O2) (A) and 60 mM KCl (B) on [Ca2+]i in the absence and presence of 100 µM N-Meth-AZ. Hypoxia caused a significant increase in [Ca2+]i in control cells (n = 78 cells in 4 experiments) but had no effect on [Ca2+]i in the presence of N-Meth-AZ (n = 63 cells in 3 experiments). In contrast, [Ca2+]i increased to a similar extent in response to KCl in the absence (n = 83 cells in 4 experiments) and presence (n = 73 cells in 4 experiments) of N-Meth-AZ. *Significant difference from control value (P < 0.05).

 
Effect of AZ on Em. AZ has recently been proposed to alter the activation of Ca2+-activated K+ channels, causing hyperpolarization (29, 46, 47). Since depolarization is believed to contribute to the hypoxia-induced increase in [Ca2+]i, AZ-induced hyperpolarization could prevent activation of voltage-gated Ca2+ channels and an increase in [Ca2+]i in response to hypoxia. To test this possibility, we used the Em-sensitive fluorescent dye DiBAC4(3). The ability of the dye to measure changes in Em was first verified by measuring the response to 1) the K+ channel opener, pinacidil, which causes hyperpolarization; 2) KCl, which causes depolarization by reducing the gradient for K+ efflux; and 3) ET-1, which causes depolarization via inhibition of K+ channels (Fig. 9). Baseline fluorescence was relatively stable over time, decreasing by 4.11 ± 0.4% over 10 min (n = 90 cells in 3 experiments). In response to pinacidil (100 µM), fluorescence decreased 19.76 ± 3.3% (n = 58 cells in 3 experiments), whereas fluorescence increased 61.58 ± 12.8% in response to 60 mM KCl (n = 109 cells in 4 experiments) and 75.6 ± 13.4% in response to ET-1 (n = 82 cells in 5 experiments). Application of AZ (100 µM) had no significant effect on basal Em (–2.86 ± 4.5%; n = 178 cells in 8 experiments) and did not alter the increase in fluorescence induced by either KCl (53.13 ± 14.8%; n = 90 cells in 3 experiments) or ET-1 (66.0 ± 10.4%; n = 87 cells in 5 experiments).


Figure 9
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  		Fig. 9. A: composite traces demonstrating effect of KCl (60 mM) and AZ (100 µM) on DiBAC4(3) (490 nm) fluorescence. B: bar graph showing mean change in fluorescence in untreated cells (control; n = 90 cells in 3 experiments) and cells exposed to the hyperpolarizing agent, pinacidil (Pin; 100 µM, n = 58 cells in 3 experiments) or the depolarizing agents, KCl (n = 109 cells in 4 experiments) or endothelin-1 (ET-1; 10 nM, n = 82 cells in 5 experiments). C: bar graphs showing mean change in fluorescence in basal fluorescence (n = 178 cells in 8 experiments) and in response to KCl (n = 90 cells in 3 experiments) and ET-1 (88 cells in 5 experiments) in the presence of 100 µM AZ.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we found that inhibition of CA with AZ, BZ, or EZ caused a small but significant acid shift in PASMC pHi, consistent with a mild acidosis arising from loss of CA-mediated facilitated CO2 diffusion (44). Challenge with moderate hypoxia caused a significant increase in PASMC [Ca2+]i that was prevented by AZ but not BZ or EZ. The lack of effect of BZ and EZ on the hypoxic response was not due to ineffective concentrations of these two more powerful CA inhibitors, as both reduced basal pHi to a similar or greater extent than did AZ. Moreover, N-Meth-AZ, which has no CA-inhibiting action (23) and had no effect on pHi, also prevented the hypoxia-induced increase in [Ca2+]i. The inhibitory action of AZ and N-Meth-AZ on Ca2+ signaling was specific for hypoxia, as no effect on KCl-induced Ca2+ responses was observed. Finally, AZ had no effect on either resting Em or agonist-induced depolarization. These results suggest that AZ prevents a rise in [Ca2+]i in response to hypoxia in PASMCs by a mechanism that does not involve intracellular acidification or a change in Em and is independent of CA inhibition.

CA inhibitors were originally developed as diuretics with primary action on the kidney to interfere in acid-base related ion transport events. Although their use as diuretics has been supplanted by more potent alternatives, they are presently used to treat glaucoma, metabolic alkalosis, epilepsy, and acute mountain sickness, conditions in which alteration in systemic acid-base status or CA-dependent ion transport can be beneficial. CA is expressed in most cells, and there have been 14 different isozymes identified. This rich diversity of expression has led some to search for nonclassical (i.e., non-acid-base) functions of the enzyme. Swenson et al. (45) showed that CA inhibition impairs ventilation-perfusion matching in the lung, a process that is thought to result from both O2- and CO2-dependent changes in bronchial and vascular smooth muscle tone. To directly test whether CA inhibition alters HPV, a major mechanism that maintains ventillation perfusion (VA/Q) matching, Deem et al. (7) found that AZ blunts and slows HPV in the isolated perfused rabbit lung. Iturriaga et al.(16) found similar effects on another hypoxia-sensitive response: the neural output of the peripheral receptors of the carotid body, which contain CA, as does vascular smooth muscle (3).

The mechanism by which AZ prevents HPV in vivo may be multifactorial. The respiratory stimulation generated by an induction of metabolic acidosis leads to increased ventilation and thus enhanced alveolar PO2 at any given inspired PO2 (44) and thus a reduction of the primary stimulus to HPV. However, the prevention of HPV by AZ in isolated perfused lung preparations and awake animals, where ventilation and/or alveolar PO2 are carefully controlled (7, 8, 15), indicate that AZ can reduce HPV via mechanisms other than elevation of alveolar PO2.

In this study, we show that AZ acts directly on the vascular smooth muscle to inhibit [Ca2+]i responses to hypoxia and that these effects do not depend on CA inhibition or intracellular acidification. In our cells, all three CA inhibitors (AZ, BZ, and EZ) resulted in a small but significant acid shift in basal pHi. This is consistent with CA inhibition-induced acidosis described in corneal endothelium (4), nonpigmented ciliary epithelium (52), lactotrophs (11), neuronal cells (19, 20), and rat heart, brain, liver, and spleen tissue (36), and rules out the possibility that, at least in the case of highly diffusible EZ, there was a lack of intracellular penetration of the drug. Our findings suggest that CA actively participates in regulation of basal pHi in PASMCs under normal conditions.

That AZ decreased pHi in PASMCs presented a possible mechanism for preventing HPV. Intracellular pH can modulate a number of cell functions, including PASMC contraction (9, 18). While we observed no change in pHi when oxygen concentration was reduced, consistent with reports in systemic vascular smooth muscle (10, 12, 37), previous studies in feline PASMCs demonstrated that moderate hypoxia caused an increase in pHi that was associated with smooth muscle cell contraction (22). Moreover, HPV in isolated perfused rat lungs was enhanced when pHi was increased by addition of weak bases, whereas decreasing pHi by addition of weak acids or by inhibition of Na+/H+ exchange blunted HPV (33). If alkalinization of PASMCs during hypoxia contributes to HPV, a decrease in pHi, as seen with CA inhibitors, could have adverse effects on HPV and provide a mechanism for AZ-induced antagonism of HPV. This hypothesis is opposed, however, by results from experiments we performed to assess the effect of CA inhibition on PASMC Ca2+ signaling during hypoxia. In PASMCs, hypoxia caused a significant, rapid, and reversible increase in [Ca2+]i. This increase in [Ca2+]i in response to hypoxia had previously been shown to require Ca2+ influx (1, 6, 50) and was required for generation of HPV (25, 27, 34, 40–42, 48). We found that AZ inhibited the rise in [Ca2+]i induced by hypoxia, suggesting an ability to modulate Ca2+ signaling. The effect of AZ was concentration dependent with a calculated IC50 of 50 µM. This concentration corresponds to the concentrations of AZ that inhibited HPV in isolated lungs and intact animals (7, 8). In contrast, neither BZ nor EZ was able to alter the hypoxia-induced change in [Ca2+]i, yet caused similar, if not greater, acidification of PASMCs.

We further investigated the possibility that the effects of AZ on HPV and hypoxia-induced Ca2+ responses were independent of CA inhibition using N-Meth-AZ. As expected from data demonstrating a loss of CA inhibitory action in vitro (23), substitution of one of the amines in the sulfonamide group with a methyl group resulted in the inability of the compound to induce acidification. However, N-Meth-AZ reduced the increase in [Ca2+]i in response to hypoxia to a similar extent as the same concentration of AZ. Based on our data, we can therefore rule out inhibition of CA and consequent acidification as the mechanism by which AZ prevents hypoxia-induced Ca2+ mobilization.

AZ has been proposed to cause membrane hyperpolarization (5, 28, 29, 47), which could act to decrease Ca2+ influx through voltage-gated Ca2+ channels. We found that AZ had no effect on resting Em in our cells, results consistent with findings in rat kidney (14). The lack of a measurable effect of AZ on Em was not due to an inability to detect hyperpolarization in our system, as a decrease in cell fluorescence was readily observed in response to pinacidil, a K+ channel activator. One limitation of this study was that we were unable to consistently demonstrate a change in Em in response to hypoxia with DiBAC4(3) (data not shown) and thus could not determine directly the effect of AZ on hypoxia-induced depolarization. It is possible that the rate of change in PO2 in our system was sufficiently slow to prevent observation of an Em response. Another possibility is that our cells do not exhibit hypoxia-induced depolarization, and/or the magnitude of change is below the threshold for detection. This would be consistent with data from several investigators who were able to elicit hypoxia-induced depolarization in isolated PASMCs only when cells were first depolarized with other agents (49) or severe PO2 or chemical hypoxia were used (30, 53, 54). Although significant depolarization was observed in canine PASMCs (31), the reproducibility of the response was unclear since only a single representative trace was presented. Other indirect evidence that would point away from AZ-induced hyperpolarization as the mechanism for repressed Ca2+ mobilization during hypoxia would be that EZ was as effective as AZ in opening KCa channels (46) but had no effect on the hypoxia-induced rise in [Ca2+]i in our cells. It is also unlikely that AZ acts to repress depolarization by preventing inhibition of K+ channels since AZ did not alter depolarization in response to ET-1 or KCl (Fig. 9), which increase Em via inhibition of voltage-gated K+ channels (39) and by reducing the gradient for K+ efflux, respectively.

While the results from experiments examining the effect of AZ on Em appear to rule out hyperpolarization-driven reduction in Ca2+ influx as a mechanism of inhibited Ca2+ mobilization in response to hypoxia, inhibitors of CA have also been shown to directly block a range of voltage-dependent Ca2+ channels (13, 17, 26, 55), some of which have been shown to participate in HPV (25, 40–42, 48). Thus we also tested the ability of AZ and N-Meth-AZ to inhibit Ca2+ mobilization in response to KCl, which depends on Ca2+ influx through voltage-gated Ca2+ channels (38, 51). In PASMCs, the KCl-induced increase in [Ca2+]i was similar in magnitude to that induced by hypoxia but was unaffected by pretreatment with AZ or N-Meth-AZ. These results indicate that AZ had no effect on the activation of voltage-gated Ca2+ channels and suggest that the effect of AZ on PASMC Ca2+-signaling during hypoxia was not due to toxicity or a nonspecific inability to mobilize intracellular Ca2+. The differential inhibitory effect of AZ on the increases in [Ca2+]i in response to hypoxia and KCl might also point to the intriguing implication that hypoxia causes Ca2+ influx through pathways other than L-type Ca2+ channels, as recently suggested (51).

In summary, we found that AZ blocks the rise in PASMC [Ca2+]i that occurs in response to hypoxia by a mechanism independent of CA inhibition and its generation of a mild intracellular acidosis. AZ does not appear to exert its inhibitory effect on the hypoxia-mediated increase in [Ca2+] in PASMCs by either changes in Em or by blockade of voltage-sensitive calcium channels. The molecular target or pathway that is altered by AZ, but not other sulfonamide CA inhibitors, and is responsible for HPV inhibition remains to be discovered. The use of compounds such as N-Meth-AZ, which are similar in structure to AZ but lack its CA-inhibiting activity (and attendant side effects), may prove beneficial in elucidating the action of AZ in blunting HPV and provide new directions to explore regarding therapeutic strategies.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by funds from the National Heart, Lung, and Blood Institute to L. A. Shimoda (HL-67919) and E. R. Swenson (HL-24163) and by unrestricted grants from the University of California, Berkeley to A. Jain.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. A. Shimoda, Div. of Pulmonary and Critical Care Medicine, Johns Hopkins Univ., 5501 Hopkins Bayview Circle, JHAAC 4A.52, Baltimore, MD 21224 (e-mail: shimodal{at}welch.jhu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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