INTRODUCTION

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ACUTE HYPOXIC VASOCONSTRICTION is intrinsic to the pulmonary circulation and can be demonstrated in isolated lungs (21), resistance pulmonary arteries (PAs) (15) and single, isolated smooth muscle cells (SMCs) from resistance PAs (19). The mechanism of hypoxic pulmonary vasoconstriction (HPV) remains to be fully elucidated. Although neurohumoral factors such as endothelin, nitric oxide (NO), leukotrienes, and prostanoids are important modulators of HPV (8), the initiation of the response is intrinsic to the PA SMC and may involve the inhibition of one or more K⁺ channels (3, 29, 46). The K⁺ channel(s) involved in HPV appear to belong to the family of 4-aminopyridine (4-AP)-sensitive, voltage-dependent K⁺ channels (Kvs) (2, 3, 46). Recent studies suggest that resistance PA SMC express several Kv channels, including Kv1.5, Kv2.1, Kv9.3, Kv1.2, and Kv1.4 (6, 27, 45) and a large-conductance, calcium-sensitive K⁺ channel (BKCa) (45). The mechanism by which changes in oxygen are sensed and K⁺ channel activity is modified has not been identified. However, it has been proposed that it may involve a change in the redox status of the cytosol (7, 37). Certainly, there is substantial evidence that K⁺ channels can be redox modulated. Reducing agents inhibit PA whole cell and single-channel K⁺ currents and depolarize cells in a similar manner to hypoxia (17, 30, 31, 44), whereas oxidizing agents enhance K⁺ currents and hyperpolarize cells (17, 30, 31). In addition, rotenone, which blocks complex I in the electron transport chain (ETC) and as such would be expected to modify the cytosolic redox status in a similar manner to hypoxia, inhibits Kv channels and causes pulmonary vasoconstriction (2, 5). We have previously reported that acute hypoxia causes a decrease in activated oxygen species (AOS) and an increase in reduced glutathione (GSH) (1, 2), an effect shared by rotenone (2). The "redox hypothesis" suggests two components to the mechanism of HPV: a sensor (the cytosolic redox component) and an effector (the K⁺ channel). This mechanism remains controversial because recombinant K⁺ channels have also been shown to be inhibited by hypoxia (13, 25, 27).

Chronic hypoxia (CH) results in both structural and functional changes in the PA including a decreased pressor response to subsequent acute hypoxic challenges (24). Although it is known that some of the reduced pressor response can be attributed to an increase in NO in CH (14, 20), this effect cannot completely account for the loss of HPV or the fact that responses to other vasoconstrictors are maintained or enhanced [possibly because of an increase in smooth muscle mass associated with vascular remodeling in CH (9, 24)]. Chronic inhibition of Kv channels may result in the membrane depolarization found in PA SMC taken from CH (35), because these channels determine the resting membrane potential of PA SMC (3, 35, 43). This depolarization could then initiate downregulation of Kv channels (18). The effect of CH on the redox status of the lung has yet to be established. The present study was undertaken to determine whether the decreased HPV found in CH is due to loss of hypoxia-sensitive K⁺ channels (the effector), functional inhibition of the channels due to an impaired oxygen sensor (as evidenced by increased glutathione or decreased radicals), or both.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Chronic hypoxia. Adult, male Sprague Dawley rats (300-350 g) were used for all experiments. Control animals were maintained in room air. Two groups of CH rats were used. One group was gradually acclimatized, in a hypobaric chamber, to a change in altitude over 5 days and then maintained at a simulated altitude of 22,000 feet (0.45 atmospheres) for at least 3 wk (n = 6). A second group of rats was exposed to a simulated altitude of 22,000 feet for 2 days after 6 h acclimatization at 13,000 feet (n = 11). The 2-day time point was selected because medial hypertrophy of the PA media has not occurred, simplifying hemodynamic interpretations, whereas at 3 wk remodeling and hypertension are well established. Some animals in each group were also studied after return to room air for 2 days. All studies were begun within 1 h of removal from the hypobaric chamber. CH pulmonary hypertension was confirmed in anesthetized (pentobarbital sodium; 50 mg/kg ip), ventilated, open-chest rats, by measuring mean PA pressure by direct puncture of the PA with a 25-gauge needle connected to a transducer (Radnoti Instruments). Cardiac output was measured by using a Transonic Systems flowmeter, placed around the aorta. Right ventricular hypertrophy was assessed by using the ratio right ventricle to left ventricle + septum (RV/LV+S).

Isolated perfused lung model. Rats were anesthetized and ventilated with room air as previously described (14). After a thoracotomy and heparinization, the PA pressure was measured with a dual-lumen cannula, allowing simultaneous perfusion and pressure monitoring. Heart and lungs were then removed en bloc and suspended in a humidified chamber at 38°C. Lungs were ventilated with normoxic gas [20% O₂-5% CO₂/balance N₂; PO₂ = 172 ± 0.9 mmHg (n = 8)] or hypoxic gas [2.5% 0₂-5% CO₂/balance N₂; PO₂ = 44 ± 2 mmHg (n = 8)] with a positive end-expiratory pressure (2.5 cmH₂0) and perfused at 0.04 ml · min¹ · gm rat wt¹ with a Krebs solution containing 4% albumin. Pressure changes under these constant-flow conditions are the result of altered resistance. Meclofenamate (17 µM) and N^G-nitro-L-arginine methyl ester (L-NAME; 50 µM) were included to inhibit prostaglandin and NO synthesis, respectively. To determine lung reactivity, all lungs were subjected to a protocol consisting of 10 min normoxia, a bolus injection of angiotensin II (AII; 0.15 µg) into the afferent line, followed after 8 min by a 6-min hypoxic challenge. The responses to rotenone (10 µM), 4-AP (Kv channel blocker; 5 mM), tetraethylammonium (nonspecific K⁺ channel blocker; 10 mM), and KCl (20 mM) were tested in both control and CH rats with the agent being tested given after the hypoxic challenge. The pH was maintained between 7.36-7.40.

Chemiluminescence. Measurement of AOS from lungs of normoxic and CH rats was performed simultaneously with the measurement of PA pressure, using luminol-enhanced chemiluminescence as previously described (1, 4). This technique, although not selective for a specific radical species, has been shown to measure the superoxide dismutase-sensitive production of radicals and peroxides from the surface of the lung (4). Whole lung chemiluminescence does not permit localization of the source of AOS to specific lung structures (i.e., PA vs. parenchyma). Briefly, isolated lungs were suspended and perfused in a black box, in the dark, within 2 mm of a foil-shielded Lucite rod. Luminescence was conveyed to a red-sensitive, cooled photomultiplier tube (RCA C31034A) powered to 1,760 V. The signal was sent to a Princeton discriminator (EG & G, Princeton Applied Research) and digitally displayed on a photon counter. Chemiluminescence was recorded continuously and expressed in counts per 0.1 s. Although this technique for measurement of superoxide has been recently challenged (10), it has also been extensively validated under the conditions used (26, 36).

Electrophysiology. To specifically investigate changes in K⁺ current and resting membrane potential caused by CH, the conventional, whole cell patch-clamp technique was used (11). Rats were anesthetized, as above, and the heart and lungs were removed en bloc. Resistance PAs (~200 µm diameter, 4th or 5th division) were dissected daily and placed in "Ca²⁺-free" Hanks' solution for 30 min at 4°C. The Hanks' solution contained (in mM) 140 NaCl; 4.2 KCl; 1.2 KH₂PO₄; 0.5 MgCl₂; 10 HEPES; 0.1 EGTA (pH 7.4). Arteries were then transferred to a papain solution containing 1 mg/ml papain, 0.75 mg/ml bovine albumin, and 0.85 mg/ml dithiothreitol and digested at 4°C for 30 min and then at 37°C for 10 min. Arteries were washed thoroughly with Hanks' solution without EGTA ("low-Ca²⁺") for at least 15 min and then maintained on ice in Hanks' supplemented with 1 mg/ml glucose. Gentle trituration produced a suspension of single cells, which were pipetted into a perfusion chamber on the stage of an inverted microscope. After a brief period to allow partial adherence to the bottom of the recording chamber, cells were perfused with a normoxic solution of composition (in mM): 115 NaCl; 25 NaHCO₃; 4.2 KCl; 1.2 KH₂PO₄; 1.5 CaCl₂; 10 HEPES (pH 7.4, PO₂ = 120 mmHg by bubbling with 20% O₂-3.5% CO₂-balance N₂). Electrode resistances ranged from 3-5 m after fire polishing and when filled with a solution of composition (in mM): 140 KCl; 1.0 MgCl₂; 10 HEPES; 0.1 EGTA (pH 7.2). Hypoxic solutions (PO₂ = 40 mmHg) were bubbled with 3.5% CO₂-balance N₂. All drugs were applied to the cells dissolved in the extracellular perfusate via gravity perfusion at a rate of 2 ml/min. Estimation of cell capacitance (in pF) was made from whole cell capacitance compensation, with current density calculated by dividing each whole-cell amplitude by cell capacitance (pA/pF). Cells were voltage clamped at a holding potential of 70 mV, and currents were evoked by +20 mV steps to more positive potentials by using test pulses of 200-ms duration at a rate of 0.1 Hz. Currents were filtered at 1 kHz and sampled at 2 or 4 kHz. For membrane potential recordings, cells were held at their resting membrane potential in current-clamp mode. All data were recorded and analyzed using pClamp 6.03 software (Axon Instruments, Foster City, CA). All experiments were performed at 22°C.

GSH assay. To assess whether hypoxia induces a more reduced state, levels of GSH were measured from control lungs during normoxia and acute hypoxia (6 min) and also from CH lungs (2 days and 3 wk). GSH levels were measured according to the method of Hissin and Hilf (12), using the fluorescent reagent o-phthalaldehyde, which reacts specifically with GSH at pH 8. Lungs were isolated as described above, perfused with a Krebs solution, and flash frozen in liquid nitrogen. A 250-mg sample of lung tissue from either control or CH rats was homogenized in 0.1 M sodium phosphate-0.005 M EDTA buffer with 1 ml 25% H₂PO₃ as a protein precipitant and then centrifuged at 10,000 g for 30 min. For determination of GSH levels, 4.5 ml EDTA buffer (pH 8) was added to 0.5 ml of the supernatant. A 100-µl sample of the resulting suspension was then mixed with 1.8 ml of phosphate-EDTA buffer and 100 µg o-phthalaldehyde and, after 15 min mixing at room temperature, read in a spectrofluorometer at a fluorescence of 420 nm with activation at 350 nm. Readings from tissue homogenates were compared with standard linear curves produced by the same methods using known concentrations of GSH.

Immunoblotting. Levels of protein expression of two candidate oxygen-responsive Kv channels, Kv1.5, and Kv2.1, and the BKCa channel were determined in PA homogenates from normoxic (n = 3) and 3-wk CH rats (n = 3). Resistance (200-500 µm diameter) PAs were dissected as described above and homogenized in buffer containing (in mM) 100 Tris (pH 7.5); 100 NaCl, 2 EDTA, 1 Na₃VO₄, 50 NaF, 0.1 N-tosyl-L-phenylalanine chloromethyl ketone; 1 phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 2 dithiothreitol (Sigma-Aldrich, St. Louis, MO). Samples were then cleared of debris by centrifugation at 600 g for 10 min. The cleared samples were boiled in the presence of 0.625 M Tris (pH 6.8), 2% SDS, 50 mM dithiothreitol, glycerol, and bromphenol blue. A 505-mg sample of protein was loaded into each lane of a 7.5% discontinuous SDS-PAGE gel. After electrophoresis, proteins were electroblotted onto nitrocellulose membrane (Amersham, Little Chalfont, UK) and blocked with buffer containing 10 mM Tris (pH 7.5), 100 mM NaCl, 0.1% Tween-20 (TBS-T) and 5% milk (Blotto) for 1.5 h. Membranes were then incubated with indicated primary antibodies (Alomone, Jerusalem, Israel) in TBS-T containing 3% BSA for 4 h, washed for 1 h, incubated with the secondary antibodies (Pierce, Rockford, IL) in Blotto for 1 h, and subsequently washed in TBS-T for another hour. Results were visualized via enhanced chemiluminescence (Amersham), and data were recorded on BioMax-MR film (Kodak, Rochester, NY). The specificity of the antibody for the intended antigen was confirmed in competition experiments in which incubation with an excess of the relevant antigen neutralized the antibody (data not shown). Kv and KCa channel expression reflects differences in channel levels because protein loading (as indicated by the Ponceau stain) was quite uniform. The immunoblots were done on pooled proteins from PAs taken from normoxic and CH rats (n = 3 rats for each group).

Statistics. Data are presented as means ± SE. Current-voltage curves and isolated lung data were analyzed by using factorial and repeated-measures ANOVAs, and membrane potential data were analyzed using the unpaired Student's t-test. A value of P < 0.05 was considered statistically significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hemodynamics. Pulmonary hypertension developed gradually over increasing length of exposure to CH. Table 1 shows the differences in PA pressure, cardiac output (CO), and total pulmonary resistance (mean PA pressure/mean CO) for rats that had been exposed to CH for 2 days or 3 wk vs. control. PA pressures were elevated and CO reduced, even after only 2 days exposure to hypoxia.

Effects of hypoxia, rotenone, and K+ channel blockers on chemiluminescence. Unenhanced light emission was not significantly different between groups; however, basal levels of luminol-enhanced chemiluminescence were significantly attenuated in CH (Table 1). Acute hypoxic ventilation produced rapid, sustained decreases in the chemiluminescence signal in normoxic rats, which returned to basal levels on return to normoxia (Fig. 1A). The absolute decrease in chemiluminescence that occurred with acute hypoxia was reduced after both short- and long-term CH (Table 1 and Fig. 1B). Returning short-term CH rats to normoxic air for 2 days recovered the chemiluminescence signal (Fig. 1C). Like hypoxia, the ETC complex I blocker rotenone also produced sustained decreases in chemiluminescence in control lungs (Fig. 1A) that were almost completely abolished after long-term CH (Fig. 1B) but returned in rats allowed to recover for 2 days (Fig. 1C). Meclofenamate, L-NAME, and AII had no effects on chemiluminescence in either control or CH lungs (data not shown for L-NAME and meclofenamate).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Representative traces from isolated, perfused lungs showing simultaneous recording of chemiluminescence (CL) and pulmonary arterial pressure (Ppa). A: response in control lung to angiotension II (AII), hypoxia, and rotenone (10 µM). B: response in 3-wk chronic hypoxia (CH) lung to AII, hypoxia, and rotenone. C: response in 3-wk CH returned to normoxia for 2 days to AII, hypoxia, and rotenone. Bar indicates period of acute hypoxic challenge. Rotenone added at arrow. D: means ± SE changes () in Ppa in control and CH rat lungs to acute hypoxia and rotenone (10 µM). * P < 0.05, different from control.

Effects of hypoxia, rotenone, and K+ channel blockers on PA pressure. Hypoxia, rotenone, AII, 4-AP (5 mM), and KCl (20 mM) all produced significant vasoconstriction in control lungs whereas tetraethylammonium (10 mM) had no effect on baseline pressure. After 2-day and 3-wk CH, the hypoxic- and rotenone-induced vasoconstriction were almost completely abolished (Fig. 1D), whereas responses to AII, 4-AP, and KCl were maintained (or augmented; Fig. 2).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Means ± SE Ppa caused by AII (0.15 µg), hypoxia, 4-aminopyridine (4-AP; 5 mM), and KCl (20 mM). Responses recorded from control, short-term CH (2-day CH) and long-term CH rats (3-wk CH). *Significantly (P < 0.05) different from control.

Changes in GSH levels in acute and chronic hypoxia. Further evidence of a reduced cytosol in CH was found from studies of GSH levels. Acute hypoxic exposure (6 min) of normoxic lungs from control rats caused a significant increase in GSH from control levels. Baseline GSH levels were elevated in lungs from both 2-day and 3-wk CH rats to values similar to those seen during acute hypoxia (Fig. 3). In CH rat lungs, an acute hypoxic challenge had no additional effect on GSH levels, suggesting the lungs were already maximally reduced (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Means ± SE levels of reduced glutathione (GSH) recorded from whole lung homogenates taken from normoxic rats (n = 6), normoxic rats exposed to a 6-min hypoxic challenge (n = 4), rats exposed to 2 days hypoxia (n = 3), rats exposed to 3-wk hypoxia (n = 6), and rats returned to normoxia for 2 days after 2 days hypoxia (n = 4). *Significantly (P < 0.05) different from normoxia.

Changes in K+ channel activity and expression after CH. There was a significant change in cell capacitance after 3-wk CH (7.8 ± 0.5 pF control, n = 16 vs. 12.6 ± 1.5 pF CH, n = 17, P < 0.01), indicating an increase in cell size. With this change in cell size accounted for, CH caused a significant decrease in K⁺ channel density (Fig. 4A). However, in both control and CH, the majority of the current was inhibited by 4-AP [2 mM; 84 ± 3% inhibition in control (n = 3); 73 ± 4% inhibition after 3-wk CH (n = 3)]. Resting membrane potentials recorded from SMC from 3-wk CH rats were significantly depolarized compared with controls (55 ± 1.7 mV in control n = 7, and 40 ± 0.8mV in CH, n = 16, P < 0.001). In CH cells, 4-AP (2 mM) caused a further depolarization of 15.1 ± 2 mV (n = 5, P < 0.002). Sensitivity of the K⁺ channel currents to hypoxia was tested in both normoxic and long-term CH rats. Hypoxia (40 mmHg) caused partial suppression of currents recorded from normoxic rats (n = 6) that was reversible on return to normoxia (Fig. 4B). In contrast, there was little effect of acute hypoxia on K⁺ currents recorded from PA SMCs from CH rats (n = 6, Fig. 4C). Consistent with the reduction in current density, the expression of Kv1.5 and Kv2.1 channel protein was significantly decreased in CH rats whereas levels of BKCa channel protein remained unchanged (Fig. 5).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   A: average current density-voltage plot showing current (I) densities (in pA/pF) recorded from single smooth muscle cells from control () rat pulmonary artery smooth muscle cells (PA SMC) and 3-wk CH PA SMC (). Each point is plotted as means ± SE. **Significantly (P < 0.01) different from control. B: average current density-voltage plot from control PA SMC during normoxia () and 4-min hypoxia (). *P < 0.05, different from control. Inset: representative traces from a PA SMC in control, hypoxia, and after return to normoxia (recovery) to show the reversibility of hypoxic inhibition. Vertical scale bar, 1,000 pA; horizontal scale bar, 50 ms. C: average current density-voltage plot from 3-wk CH PA SMC during normoxia () and 4-min hypoxia ().

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Immunoblots of protein levels of Kv1.5, Kv2.1, and BKCa channels from pulmonary arteries from control (C) or 3-wk (H) rats. Lower panel indicates Ponceau stain.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The development of pulmonary hypertension on exposure to CH is well-known (22, 24, 32). Our hemodynamic data confirm these observations and indicate that pulmonary hypertension develops quickly, with increases in PA pressure and decreases in CO after only 2 days (Table 1). HPV is intrinsic to the pulmonary circulation and is an important means of optimizing lung ventilation with perfusion. In our model, it would appear that HPV is not the mechanism that sustains pulmonary hypertension, because hypertension develops even though the hypoxic response of the lung is lost. McMurtry et al. (22) originally reported that several weeks of exposure to CH reduced the ability of the lung to constrict to these acute hypoxic challenges. They later showed that this blunting effect could be demonstrated after as little as 40 h exposure to hypoxia (23). Our data confirm these findings, with attenuation of HPV after only 2 days CH. It has been proposed that the mechanism by which this oxygen sensitivity is decreased might be associated with the increased production of NO that occurs in CH (14, 16, 33). It is likely that this increase accounts for some of the decreased contractile response to acute hypoxia because HPV in CH rats can be partially reversed by inhibition of NO synthase (34). However, the studies presented here were all done in the presence of the NO synthase inhibitor L-NAME, and HPV was still found to be diminished, suggesting that an increase in NO production is not the primary mechanism involved. Furthermore, the loss of HPV is selective, shared only with the loss of the rotenone-induced constriction. Kv channels still control membrane potential in CH and initiate vasoconstriction when inhibited (Fig. 2).

There is evidence to suggest that HPV may occur, at least in part, via inhibition of several 4-AP-sensitive Kv channels in PA SMCs (2, 3, 46). This may occur via a direct inhibitory effect of hypoxia on the channel or may be secondary to changes in cytosolic redox and/or levels of AOS. Although the ultimate effector for hypoxia is the K⁺ channel, the identity of the oxygen sensor that closes the K⁺ channel has been debated, and indeed there may be more than one. Candidates for the oxygen sensor include NADH or NADPH oxidase, a superoxide radical-generating complex in SMC plasma membranes, which is closely related to the neutrophil NADPH oxidase (42). If NADH or NADPH oxidase were the sensor, inhibition of these enzymes should elicit K⁺ channel inhibition and vasoconstriction in a similar manner to hypoxia. Although pharmacological inhibitors of NADPH oxidase, such as diphenyleneiodonium, do inhibit K⁺ channels, they in fact cause relaxation and inhibit HPV (40). This vasodilator effect results from the fact that diphenyleneiodonium, a nonspecific probe, also inhibits the Ca²⁺ channel. Recent data from Weissmann et al. (41) indicate that a different NADPH oxidase inhibitor, 4-(2-aminoethyl)benzene-sulfonyl fluoride, transiently constricts the normoxic isolated rabbit lung and suppresses its response to hypoxia. In the absence of additional data on the potential nonselectivity of 4-(2-aminoethyl)benzene-sulfonyl fluoride, this might indicate a role for NADPH oxidase as an oxygen sensor in the lung. These conflicts illustrate the problems associated with the use of current pharmacological tools to determine the roles of these oxidases in oxygen sensing. Recently, using isolated, perfused mouse lungs from NADPH oxidase-deficient "knock-out" mice, it was shown that hypoxic K⁺ channel inhibition in PA SMC and HPV is unaltered despite the absence of NADPH oxidase activity and AOS production, suggesting that the oxygen sensor is not the complete NADPH oxidase enzyme (5). These data suggest that the radicals measured by chemiluminescence are not themselves the sensor, because HPV is preserved in mice that do not make AOS. Interestingly, rotenone-induced constriction is also preserved in these mice.

It has also been suggested that the oxygen sensor is the mitochondrial ETC (2). Acute hypoxia rapidly shifts the cytosolic redox status of the cell by increasing the accumulation of reducing equivalents. Inhibitors of the proximal ETC, such as rotenone (complex I) and antimycin (complex III), also shift cytosolic redox status and cause vasoconstriction in a manner analogous to hypoxia (2). Because the effector for hypoxia, in this model, is ultimately the closure of Kv channels, it would be expected that agents that modify cellular redox status would also modify K⁺ channel activity. Indeed, oxidizing agents reverse HPV (38, 39) and increase K⁺ channel activity in PA SMC (31). Conversely, the electron shuttlers duroquinone and coenzyme Q inhibit K⁺ channels in PA SMC, depolarize membrane potential, and cause vasoconstriction (31), as do rotenone, antimycin (2), GSH, NADH, and NADPH (30). In agreement with a change in cytosolic redox status, our results show that acute hypoxia rapidly increases levels of GSH within 6 min, reflecting a more reduced cytosol (Fig. 3). In CH, baseline levels of GSH are increased to levels similar to those achieved during acute hypoxic challenges, suggesting that the redox status of the CH lung is already more reduced than that of normoxic controls. Acute hypoxia has no further effect on GSH levels under chronically hypoxic conditions, suggesting that the reduction induced by CH overwhelms any further reduction that can be achieved by acute hypoxia. Thus the ability of the cell to sense a change in oxygen is lost and there is no inhibition of K⁺ current (Fig. 4).

Long-term CH has been previously reported to reduce whole-cell K⁺ channel current and cause downregulation of Kv1.5 channel message in PA SMC (45). Because long-term CH is associated with vascular remodeling including smooth muscle hypertrophy, calculations of current density (whole-cell K⁺ current divided by cell capacitance) allow a more accurate indication of changes in K⁺ channel activity, because changes in cell size are accounted for. CH decreased current density significantly from control. Although K⁺ current density was reduced, vasoconstrictor responses to 4-AP were unchanged in CH (Fig. 2). In contrast, vasoconstriction to hypoxia and rotenone was almost completely abolished. These data, along with membrane potential recordings indicating that the membrane can still be depolarized by 4-AP, show that Kv channels in the SMC membrane remain functional after CH. In addition, a similar percentage of the total current was 4-AP sensitive after CH. The attenuated current density may be due to the inhibition of the currents by the initial increase in GSH (30) resulting in subsequent membrane depolarization. Prolonged depolarization downregulates expression of Kv1.5 channels in GH3 pituitary cells within hours (18). Because CH results in prolonged membrane depolarization, this mechanism may account for the observed decrease in Kv1.5 channel expression. It has not been determined whether expression of Kv2.1 is similarly regulated by changes in membrane potential. Several Kv channels and combinations of Kv channels have been shown to be oxygen sensitive, including Kv2.1 (13, 25, 27, 28). It could therefore be argued that CH specifically downregulates the hypoxia-sensitive channel, which could also account for the observed loss of HPV and the lack of inhibitory effect of hypoxia on whole-cell K⁺ channel activity (Fig. 4). However, it should be remembered that the increase in cytosolic GSH occurs within minutes, before any changes in K⁺ channel protein levels. In addition, there is parallel impairment of both rotenone and hypoxic changes in AOS and pulmonary constriction. These observations would make it more likely that it is the chronic reduction of the oxygen sensor that results in loss of HPV.

In conclusion, data from whole lung, patch-clamp, and immunoblot studies suggest that the attenuation of HPV after CH is due to a loss of the inhibition of K⁺ channel activity by acute hypoxia. This is likely to be due to an inability of acute hypoxia to further modify the cytosolic redox status beyond the already reduced environment caused by CH. It is unclear whether the change in expression of Kv1.5 and Kv2.1 also play a role in the loss of HPV, but it is likely that chronic membrane depolarization associated with CH is responsible for the decrease in channel protein.