INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

PULMONARY ARTERY VASOCONSTRICTION and medial hypertrophy are cardinal manifestations of hypoxic pulmonary hypertension. Although significant progress has been made in understanding the signaling events that link chronic hypoxia to alterations in vascular tone and structure, important unresolved issues remain. Recent evidence suggests that the peptidergic vasoconstrictor endothelin (ET)-1 is an important mediator of the vascular changes associated with hypoxic pulmonary hypertension. Both steady-state mRNA for the preproET-1 transcript as well as lung levels of the mature peptide are elevated in hypoxic pulmonary hypertension (7). In addition, circulating levels of ET-1 are elevated in a variety of animal models of pulmonary hypertension, as well as humans with both primary and secondary pulmonary hypertension (1, 34). Importantly, pharmacological inhibition of ET-1-receptor activation ameliorates the development of pulmonary hypertension in chronically hypoxic rats, suggesting that the increased levels of ET-1 may be etiologic in the development of hypoxic pulmonary hypertension (4-6).

However, the mechanisms through which ET-1 affects pulmonary artery smooth muscle cell (PASMC) activation both in the normotensive and the hypoxic-hypertensive circulation have not been fully characterized. At least two mechanisms of action of ET-1 in PASMC exist, 1) inhibition of voltage-gated K⁺ (K_v) channels and 2) inositol 1,4,5-trisphosphate-mediated Ca²⁺ release from intracellular stores (15). An important unresolved controversy is the relative contribution of ET_A and ET_B receptors to these downstream signaling events in the hypertensive PASMC. Previous investigators found that in normotensive resistance PASMC inhibition of K_v channels was mediated by ET_B-receptor activation (22). The membrane depolarization resulting from K_v-channel inhibition primed the cells for subsequent hypoxia-induced Ca²⁺ influx (29). In contrast to the effect on K_v channels, release of intracellular Ca²⁺ stores was mediated by activation of both ET_A and ET_B receptors (10). The relevance of these observations to the hypertensive pulmonary circulation is uncertain.

Although both K_v-channel inhibition and release of intracellular Ca²⁺ stores could be important in the genesis of hypoxic pulmonary hypertension, data suggest that ET-receptor coupling to downstream signaling events may be altered in the hypertensive pulmonary circulation. McCulloch et al. (16) found that in the normotensive PASMC, ET-1 potency was increased after pharmacological inhibition of ET_A receptors, suggesting a predominant inhibitory role for the ET_A receptor. In contrast, in hypertensive PASMC, activation of the ET_A receptor mediated pulmonary vasoconstriction, suggesting a switch from smooth muscle cell inhibition to activation via the ET_A receptor (16). These studies were limited to isolated pulmonary artery rings and did not elucidate the cellular mechanisms of action of ET-1.

On the basis of this information, we hypothesized that in hypoxic-hypertensive pulmonary circulation a switch in receptor coupling between ET-1 and either K_v channels or release of intracellular Ca²⁺ stores occurs, resulting in enhanced ET_A-mediated activation of the PASMC. To evaluate this hypothesis we used whole cell patch clamp to measure the effect of ET-1 on macroscopic K_v current in freshly dispersed PASMC isolated from resistance pulmonary arteries from normoxic and chronically hypoxic rats. We also measured the effects on cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) using the Ca²⁺-sensitive fluorophore indo 1. The relative contribution of ET_A and ET_B receptors was assessed using the receptor-selective blockers BQ-123 and BQ-788. Our findings are consistent with the hypothesis that there is unique coupling of ET receptors to K_v channels and that this coupling is altered in hypoxic pulmonary hypertension. The net result of these alterations is enhanced ET_A-mediated inhibition of K_v channels, which, in combination with increased ET-1 peptide, may contribute to membrane depolarization and subsequent activation of voltage-gated Ca²⁺ channels.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Hypoxic rats. Male Sprague-Dawley rats (200-300 g) were placed in a hypobaric chamber for 3-4 wk. Air was exchanged and pressure was monitored continuously with a mercury altitude gauge (17,000 ft above sea level altitude; PO₂ = ~76 Torr). We took the rats out of the chamber for 10 min three times per week to clean the cages and replenish food. Chronically hypoxic rats were studied after 3-4 wk of hypobaric hypoxia. Control rats were housed at Denver's altitude with otherwise identical care.

Dispersed PASMC preparation. Single PASMC were obtained with the modified method of Archer et al. (2). Briefly, normoxic and hypoxic rats were anesthetized with intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Lungs were quickly removed from chest en bloc and transferred into a beaker containing cold (4°C) physiological salt solution (PSS). The PSS was composed of (in mM) 135 NaCl, 5.5 KCl, 2 CaCl₂, 1 MgCl₂, 10 HEPES, and 10 glucose; pH was adjusted to 7.4 with 5 M NaOH. Distal third and fourth divisions of the intrapulmonary arteries were carefully isolated, cleaned of adventitia, and opened longitudinally. After 15- to 20-min recovery in cold nominally Ca²⁺-free PSS, the arteries were preincubated for 15 min in cold Ca²⁺-free PSS digestion solution containing (in mg/ml) 1 papain, 0.75 dithiothreitol, 1 BSA, and 1 trypsin inhibitor (for fluorescence studies 20 µM CaCl₂ was added), after which the digestion solution was heated to 37°C for 18 min. After digestion the tissues were thoroughly washed, and single PASMC were dispersed by gentle trituration with a fire-polished glass pipette. Drops of cell suspension were placed onto the perfusion chamber for electrophysiological or microfluorometric studies. All cells were studied within 60 min of dissociation.

Immunohistochemistry. Immunohistochemical studies were performed to identify cells expressing smooth muscle -actin using methods previously described (21). Briefly, serial Formalin-fixed, paraffin-embedded sections (6 µm) were placed on poly-L-lysine-coated slides, deparaffinized in three changes of xylene, and rehydrated in 100, 95, and 75% ethyl alcohol. The dilution that yielded optimal specific staining was determined in pilot experiments. Mouse anti-rat smooth muscle cell -actin primary antibody (1:100 dilution; Atlantic Antibody, Stillwater, MN) was used. Control experiments were performed using rabbit serum. Biotinylated anti-rabbit immunoglobulin (1:200 dilution; Vector Laboratories, Burlingame, CA) was applied for 30 min, followed by a 30-min incubation in a Vectastain avidin-biotin-alkaline phosphatase reagent. The substrate 3-amino-9-ethyl-carbazole yielded a red reaction product, and sections were counterstained in methyl green.

Measurements of membrane potential and I_K(v). Gently dispersed single PASMC were placed in the perfusion chamber on the stage of an Olympus CK-2 inverted microscope (Tokyo, Japan) for ~15 min to allow adherence to the bottom of the chamber and then were continuously superfused with PSS. To eliminate the effect of Ca²⁺ influx on I_K(v), during current recording we changed the superfusate to Ca²⁺-free PSS. Patch-clamp pipettes (2-4 M) were pulled from borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) using Flaming/Brown pipette puller model P-80 (Sutter Instrument, Novato, CA) and fire polished. The internal solution contained (in mM) 125 KCl, 5 Na₂ATP, 0.5 NaGTP, 5 MgCl₂, 10 HEPES, and 10 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), pH 7.2 adjusted with KOH.

Measurement of [Ca²⁺]_i. [Ca²⁺]_i was estimated using the Ca²⁺-sensitive fluorophore indo 1 as previously described (26). Briefly, after single PASMC dispersion in PSS containing 5 µM indo 1-AM (Molecular Probes, Eugene, OR), drops of cell suspension were placed onto a glass coverslip and incubated for 30 min at room temperature in the dark, and then the glass coverslip (indo 1-AM-loaded PASMC attached) was washed with PSS and incubated for another 30 min for indo 1-AM deesterification. The glass coverslip was inserted into a perfusion chamber on the stage of an inverted Nikon Diaphot microscope (Tokyo, Japan) equipped with a ×100 fluorescence objective lens. An ultraviolet light beam from a 75-W xenon arc lamp was filtered at 365 nm to excite indo 1. Emitted fluorescence signal was directed to a dichroic mirror. Transmitted light and reflected light were filtered at 480 and 405 nm, respectively, and measured by two separate photometers (PTI, Princeton, NJ). The emission ratio of 405 to 480 nm was calculated as an index of [Ca²⁺]_i and stored in an Macintosh PowerPC computer (Apple, Cupertino, CA) using a 12-bit MacLab/8 interface (Analog Digital Instruments, Milford, MA). For calculating [Ca²⁺ ]_i the following formula was used: [Ca²⁺]_i = K_d(R  R_min)/(R_max  R), where K_d is the apparent dissociation constant of the Ca²⁺/indo 1 complex and is taken as 250 nM (8); R_max is the fluorescence ratio (R) for the Ca²⁺-saturated dye; R_min is R for the Ca²⁺-unbound dye; and  is the ratio of the 480-nm fluorescence obtained for the Ca²⁺-free and Ca²⁺-bound forms of the dye. Values for R_max and R_min were determined in vivo in PASMC after permeabilization with 25 µM ionomycin and exposure to either 10 mM Ca²⁺ or 10 mM EGTA (0 mM Ca²⁺) solutions, respectively (31). In the experiments measuring the effect of Ca²⁺ influx on the Ca²⁺ transient, a Ca²⁺-free superfusate was used. All experiments were conducted at 32°C.

Chemicals. BQ-788 (gift from Banyu Pharmaceutical, Tsukuba, Japan) and indo 1-AM were dissolved in DMSO and diluted before use. BQ-123 (Banyu Pharmaceutical), ET-1 (Peptides International, Louisville, KY), iberiotoxin (Alomone Labs), and other chemicals obtained from Sigma (St. Louis, MO) were dissolved in deionized water. For both BQ-123 and BQ-788 a concentration of 5 µM was used, on the basis of the reported selectivity of these compounds for ET_A and ET_B receptors, respectively (11, 12).

Statistical analysis. All data are expressed as means ± SE, and n refers to the number of cells tested. Statistical significance was determined with Student's t-test, and a P value < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell isolation and myosin staining. Myosin staining of sections of intrapulmonary artery revealed two morphologically distinct populations of medial smooth muscle cells. The majority of cells were cuboidal in shape and stained only weakly for -actin. Cells in the inner medial layer were spindle shaped and arranged circumferentially and exhibited intense -actin staining. Enzymatic dispersion produced a mixture of cuboidal cells and elongated, spindle-shaped cells. The latter cells, which appeared grossly similar to smooth muscle cells in the subintimal layer, also stained strongly positive for -actin and were used for all subsequent electrophysiological studies (Fig. 1).

View larger version (32K): [in this window] [in a new window]   Fig. 1.   A: smooth muscle -actin staining of section of intrapulmonary artery (original magnification ×20). Endothelium was separated from media in this section and is not visible. Cells in inner media demonstrated strong -actin staining indicated by red color (black arrow). Majority of cells in outer media showed weak -actin staining (red arrow). B: -actin staining of enzyme-dissociated cells (original magnification ×60). Spindle-shaped cell of type used for electrophysiological studies is indicated by arrow. C: fluorescence image of same field as in B, confirming that spindle-shaped cell highly expresses smooth muscle -actin.

Passive electrical properties of PASMC. Using current-clamp technique and a ramp protocol, we measured RMP, whole cell input resistance, and capacitance in both normotensive (NT) and hypertensive (HT) PASMC. We found that the RMP was depolarized in HT PASMC [NT: 48.7 ± 1.5 mV (n = 35); HT: 43.2 ± 1.5 mV (n = 28), P < 0.01]. In contrast, whole cell capacitance and input resistance were the same in the PASMC from both NT and HT rats [capacitance: 12.7 ± 0.7 pF (n = 39) vs. 13.7 ± 0.6 pF (n = 31); input resistance: 4.7 ± 0.5 G (n = 39) vs. 4.0 ± 0.6 G (n = 31); P = not significant (NS)].

Effect of chronic hypoxia on I_K(v). I_K(v) was studied under conditions that eliminated the other predominant outward currents: ATP-dependent and Ca²⁺-dependent K⁺ currents [I_K(Ca)]. To eliminate contamination from these currents, 5 mM ATP (to inactivate ATP-dependent K⁺ channels) and 10 mM BAPTA (to chelate cytosolic free Ca²⁺) were included in the pipette solution. In addition, Ca²⁺ was omitted in the bath solution to minimize the possibility that Ca²⁺ influx would activate I_K(Ca). Membrane potential was held at 80 mV, and a family of depolarizing pulses was applied from 60 to +60 mV in 10-mV increments. Figure 2A shows a representative example of outward currents from a NT PASMC under these conditions. The current was evoked at a membrane potential more negative than 30 mV after a short period of delay and reached a steady state with a slow inactivation. Application of 100 nM iberiotoxin (a specific Ca²⁺-dependent K⁺ channel blocker) reduced K⁺ current (I_K) by 2.3 ± 1.4% (3 cells from 3 rats), indicating that the I_K(Ca) contribution to the total I_K was negligible.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Voltage-gated K+ current [IK(v)] from normotensive (NT) and hypertensive (HT) pulmonary artery smooth muscle cells (PASMC). Family of representative IK(v) records by depolarizing cells from 60 to +60 mV in 10-mV increments for 400 ms from NT (A) and HT (B) PASMC. C: current-voltage (I-V) curve generated from NT () and HT () PASMC. I-V curve shows that there was no change in IK(v) threshold. D: maximum IK(v) density at +60 mV from both NT (filled bar) and HT (open bar) PASMC. IK(v) in HT PASMC was significantly reduced (* P < 0.01).

Effect of ET-1 on I_K(v) in NT and HT PASMC. To determine whether ET-1 affected I_K(v) under our experimental conditions, we applied ET-1 (10¹²-10⁸ M) before measurement of outward current elicited by depolarization to +60 mV. Concentration-response tests in NT PASMC showed multiple concentration-dependent effects. At a very low concentration (10¹² M) ET-1 increased I_K(v) by 10% in two of five cells, had no effect in one cell, and decreased I_K(v) by 5% in two cells. At higher concentrations suppression of I_K(v) became the predominant effect. At the highest concentration used (10⁸ M) all cells responded with a decrease in I_K(v). Representative examples are shown in Fig. 3, A and B. The effect of ET-1 was only partially reversible (<20%). Similarly, in HT PASMC, the I_K(v) inhibition was also concentration dependent, with multiple effects at lower concentrations. At higher concentrations (10⁹ M and 10⁸ M), all cells showed a I_K(v) decrease. Despite a tendency for some cells to show a small increase in I_K(v) at lower concentrations, the magnitude of the increase was significantly less than the magnitude of the inhibition in I_K(v). Thus, when group means of the effect of ET-1 on I_K(v) were calculated, the net effect was a decrease in I_K(v) at all but the lowest concentration tested. There was a concentration-dependent decrease in I_K(v) in both NT and HT PASMC. The inhibition was maximal at an ET-1 concentration of 10¹⁰ M in normotensive PASMC (Fig. 3C). Although the magnitude of the inhibition in I_K(v) tended to be greater at higher concentrations of ET-1 in HT versus NT PASMC, this trend did not achieve statistical significance.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of endothelin (ET)-1 on IK(v). A: representative example of augmentation of IK(v) that occurred in some PASMC exposed to a low concentration of ET-1 (1012 M). B: representative example of inhibition of IK(v) that occurred in all cells exposed to a high concentration of ET-1 (108 M). C: concentration-response curve shows that ET-1 effect was concentration-dependent with a maximum seen at a concentration of 1010 M in NT cells () and 109 M in HT cells ().

8

View larger version (6K): [in this window] [in a new window]   Fig. 4.   Representative example of effect of combined application of 4-aminopyridine (4-AP) and ET-1. After application of 4-AP (5 mM) IK(v) was reduced significantly. Subsequent application of ET-1 (108 M) did not induce an additional decrease in IK(v).

Effect of BQ-123 and BQ-788 on ET-1-mediated inhibition of I_K(v). We tested the relative contributions of the ET_A and ET_B receptors to ET-1-mediated inhibition of I_K(v) in PASMC using the ET_A-receptor-selective inhibitor BQ-123 and the ET_B-receptor-selective inhibitor BQ-788. As Fig. 5 demonstrates, in NT PASMC ET_A-receptor blockade had no effect on ET-1-mediated I_K(v) inhibition, whereas ET_B-receptor blockade significantly reduced the effect. The combination of ET_A- and ET_B-receptor blockade produced an effect in excess of either inhibitor alone. In contrast, in HT PASMC ET_A-receptor blockade was more effective than ET_B-receptor blockade. Again, the combination of ET_A- and ET_B-receptor blockade completely eliminated the response.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effect of BQ-123 and BQ-788 on ET-1-mediated inhibition of IK(v). BQ-123 (5 µM) did not affect ET-1 inhibition of IK(v) in NT PASMC (filled bar) but reduced ET-1 inhibition by 37% in HT PASMC (open bar). BQ-788 (5 µM) reduced ET-1 inhibition by 48% in NT PASMC and 59% in HT PASMC, respectively. Combined application of BQ-123 and BQ-788 almost eliminated ET-1 inhibition in both NT and HT PASMC. * P < 0.05 from ET-1 alone; P < 0.05 from normotensive cells.

Effect of ET-1 on [Ca²⁺]_i in NT and HT PASMC. We measured the effect of ET-1 on [Ca²⁺]_i in NT and HT PASMC using indo 1 fluorescence. Resting [Ca²⁺]_i did not differ between NT and HT PASMC [81 ± 11 nM (19 cells from 6 rats) in NT PASMC; 88 ± 8 nM (17 cells from 6 rats) in HT PASMC; P = NS]. We tested the [Ca²⁺]_i response to a range of concentrations of ET-1. At concentrations of 10¹²-10¹⁰ M no response was detected. At a concentration of 10⁹ M 14% cells (1 of 7 cells) responded, whereas at 10⁸ M, the percentage of cells responding increased to 82% (9 of 11 cells). The response occurred within 60 s of addition of 10⁸ M ET-1 and was characterized by an initial large Ca²⁺ transient followed by a train of smaller transients. The peak response was greater in HT PASMC [NT: 730 ± 100 nM (n = 14 cells from 8 rats) vs. HT: 1,238 ± 253 nM (n = 10 cells from 5 rats), P < 0.05], and the pattern of the response tended to be more irregular in HT cells. Representative examples of responses to ET-1 are shown in Fig. 6. We repeated measurements of [Ca²⁺]_i in PASMC in the absence of extracellular Ca²⁺ (0 Ca²⁺). The magnitude of the initial transient was not reduced by removal of extracellular Ca²⁺ (1,277 ± 298; n = 9 cells from 3 rats). However, the duration of the transient was shorter (control: 115 ± 17 s; 0 Ca²⁺: 57 ± 9 s; P < 0.05), and the prolonged train of transients was eliminated in the absence of extracellular Ca²⁺ (Fig. 6C).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Representative examples of effect of ET-1 on cytosolic free Ca2+. A: ET-1 at 108 M induced Ca2+ transients within 60 s with a peak followed by a train of smaller transients in both NT (A) and HT (B) PASMC in presence of 2 mM extracellular Ca2+. Removal of extracellular Ca2+ did not attenuate magnitude of the initial peak but curtailed duration of train of Ca2+ transients from 115 to 57 s (P < 0.05; C). ET-1 was added at 0 time point. R405/480, 405- to 480-nm fluorescence ratio.

Effect of BQ-123 and BQ-788 on ET-1-mediated increase in [Ca²⁺]_i. We tested the effect of ET_A- and ET_B-receptor blockade on the initial Ca²⁺ transient by pretreating cells with either BQ-123 or BQ-788 before exposure to 10⁸ M ET-1. In both NT and HT PASMC, both BQ-123 and BQ-788 reduced the percentage of cells responding to ET-1 and attenuated the magnitude of the Ca²⁺ transient. In NT PASMC ET_A blockade reduced the percentage of cells responding to ET-1 from 74 (14 of 19 cells) to 33 (3 of 9 cells)%, whereas ET_B blockade reduced the percentage to 8% (1 of 12 cells). The magnitude of the transient in cells that responded was reduced from 730 ± 100 nM (n = 14 of 19 cells) to 210 ± 45 (n = 3 of 9 cells) and 234 (n = 1 of 12 cells) nM, respectively. In HT PASMC ET_A blockade reduced the percentage of responding cells from 71 (10 of 14 cells) to 17 (1 of 6 cells)%, whereas ET_B blockade eliminated the response in all cells tested (0 of 8 cells). Because of the small number of responding cells, a comparison of transient magnitude could not be made.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ET-1-mediated pulmonary artery vasoconstriction is a critical event in the genesis of hypoxic pulmonary hypertension. Our studies addressed mechanisms through which ET-1 activates the pulmonary artery myocyte, comparing cells from normotensive rats to those isolated from chronically hypoxic rats. We chose to study only cells from distal third and fourth branch pulmonary arteries because prior investigators have demonstrated diversity of ion channel activity along the pulmonary arterial tree (17). Cells isolated from these "distal" pulmonary arteries have been reported to have predominantly K_v channels and a robust intrinsic hypoxic response, suggesting that they more accurately model hypoxic vasoconstriction in the intact lung (30). Even within these small pulmonary arteries there are multiple "phenotypes" of smooth muscle cells. Consistent with a prior report of Hislop and Reid (9), on histological sections we found there were two morphologies of cells in the media. In the inner media PASMC were elongated and arranged circumferentially and strongly expressed smooth muscle -actin. In the remainder of the media, cells were more cuboidal and generally stained only weakly for -actin. Freshly dispersed cells similarly displayed multiple morphologies. We found that elongated cells always displayed strongly positive -actin immunoreactivity. In contrast, rounded or cuboidal cells variably expressed -actin. Therefore, we restricted electrophysiological studies to the elongated cells, which likely represent a vasoconstrictor phenotype.

Passive electrical properties did not differ between NT and HT PASMC. This suggests that gross differences in cell surface area and membrane permeability did not exist. However, membrane potential was more depolarized in hypertensive cells. This is consistent with several prior reports using both patch clamp and microelectrodes in isolated cells and intact resistance arteries (14, 25). One notable exception is a report by Suzuki and Twarog (27), who found hyperpolarization in resistance pulmonary arteries from chronically hypoxic rats. However, it is not certain whether the endothelium was intact in that preparation, and subsequent studies of isolated cells have failed to confirm that observation. The patch-clamp technique is not an ideal method of measuring membrane potential, because dialysis of intracellular contents with the pipette solution may alter channel and pump activity. However, because all cells were studied under identical conditions, the difference between NT and HT PASMC was probably qualitatively accurate. Thus the observed depolarization would likely place HT PASMC closer to the activation threshold for voltage-gated Ca²⁺ channels and thus render them more susceptible to activation by concurrent depolarizing stimuli such as hypoxia or circulating vasoconstrictors.

We focused the remainder of our studies on the effect of ET-1. First, we tested whether ET-1 inhibited K_v channels. Interestingly, at very low concentrations ET-1 had variable, albeit modest, effects. At a concentration of 1 pM, approximately equal proportions of cells showed increased, decreased, or unaffected I_K(v). This suggests that there may be subtle diversity of electrophysiological behavior even within the morphologically homogeneous population of cells chosen for study. At higher concentrations, ET-1 inhibited I_K(v) in most cells. Inhibition of I_K(v) by ET-1 is consistent with prior reports in NT PASMC (23, 24). HT PASMC displayed similar behavior, although at concentrations <10⁸ M the percentage of cells in which I_K(v) was suppressed by ET-1 was greater in HT than NT PASMC. I_K(v) was partially inhibited by 4-AP, and ET-1 had no additional inhibitory effect in the presence of 4-AP. The nature of 4-AP-resistant K_v channels is uncertain. Yuan et al. (32, 33) demonstrated that 4-AP has a similar inhibitory effect on oxygen-regulated K⁺ channels in PASMC. Although not conclusive proof, this suggests that ET-1 and hypoxia may act thorough a similar class of ion channel.

Turner and Kozlowski (29) reported studies of the effect of ET-1 on hypoxia-induced depolarization of PASMC. They found that there was synergy between the two in that subthreshold concentrations of ET-1 primed the cells to depolarize in response to hypoxia. They concluded that the central role for ET-1 in hypoxic vasoconstriction in vivo may be based on this electrophysiological synergy. Our findings that HT PASMC were depolarized relative to NT cells, in combination with similar susceptibility to ET-1-mediated K_v inhibition, suggests that the hypertensive pulmonary artery myocyte may be "primed" to respond to hypoxia through the dual mechanisms of intrinsic depolarization and enhanced pulmonary ET-1 production.

All known biological effects of ET-1 are mediated via ET receptors. Two receptor subtypes, ET_A and ET_B, have been described on pulmonary artery myocytes. We therefore tested the role of each in coupling ET-1 to inhibition of I_K(v). In NT PASMC we found that the ET_A-receptor inhibitor BQ-123 did not alter the ET-1 effect. In contrast, the ET_B-receptor inhibitor BQ-788 reduced the effect by ~50%. This is qualitatively similar to a prior report by Salter and Kozlowski (22), who found that in NT PASMC, ET-1 inhibited I_K(v) via the ET_B receptor. However, in our studies, the combination of BQ-788 and BQ-123 was more effective than BQ-788 alone, completely eliminating the effect of ET-1. Although the explanation for this finding is uncertain, it suggests that there is some form of synergy between ET_A and ET_B receptors such that coupling of ET_A receptors to K_v channels can only occur in the presence of ET_B activation. Similar ET receptor cross talk was found in a study by Mickley et al. (18) of ET-1 vasoconstriction in intact rat mesenteric arterioles, suggesting that this may be a generalized phenomenon in the vasculature.

Effects of ET-receptor inhibition differed in HT PASMC. Although BQ-123 alone had no effect on NT PASMC, it reduced ET-1-mediated I_K(v) inhibition by 63% in HT cells. Again, the combination of BQ-123 and BQ-788 completely eliminated the response. Thus it appears that activation of the ET_A receptor alone becomes sufficient to reduce I_K(v) in HT PASMC. I_K(v) inhibition was not caused by release of intracellular Ca²⁺ stores, because the use of 10 mM BAPTA in the internal pipette solution, in combination with low extracellular Ca²⁺, would have prevented calcium ions from reaching the K_v channel. The increased efficacy of ET_A-receptor activation is consistent with a prior report from McCulloch et al. (16); they found that chronic hypoxia potentiated ET_A-receptor-mediated vasoconstriction in rat intrapulmonary arteries. It is also consistent with the finding of Li et al. (13) that there was increased ET_A, but not ET_B, receptor expression in hypoxic rat lung.

We wondered whether the pattern of receptor coupling to K_v channels was similar to another important mechanism of smooth muscle cell activation, release of intracellular Ca²⁺ stores. Release of intracellular Ca²⁺ was measured using the Ca²⁺-sensitive fluorophore indo 1. We confirmed that the initial large Ca²⁺ transient was caused by release of intracellular stores by testing the effect of removal of extracellular Ca²⁺. Under the latter condition the peak increase in [Ca²⁺]_i was not reduced from that observed with 2 mM extracellular Ca²⁺, although the duration was attenuated and subsequent regenerative spikes in [Ca²⁺]_i were eliminated. These results are similar to multiple prior reports and validate the use of initial peak height as an index of release of intracellular Ca²⁺ stores.

We found that, in contrast to the effect on K_v channels, either BQ-123 or BQ-788 alone was sufficient to inhibit the Ca²⁺ transient in either NT or HT PASMC. Thus it appears that in PASMC, simultaneous activation of both ET_A and ET_B receptors is required for this signal transduction pathway. This finding differs somewhat from that reported by Hyvelin et al. (10). In that earlier study, BQ-123 completely abolished the Ca²⁺ transient in main pulmonary artery myocytes and in 21% of intrapulmonary myocytes, whereas BQ-788 had no effect on main pulmonary myocytes but inhibited the transient in 70% of intrapulmonary myocytes. The principal difference between our result and theirs was that we found BQ-123 to have significantly more efficacy in intrapulmonary myocytes. Whether this discrepancy represents a strain difference (Sprague-Dawley vs. Wistar rats) or was caused by other methodological differences (such as restriction of our studies to a morphologically distinct subset of PASMC) is uncertain.

We found that the requirement for simultaneous activation of both subtypes of ET receptor to stimulate the [Ca²⁺]_i transient did not differ between NT and HT cells. Therefore, our data suggest that coupling of ET receptors to intracellular Ca²⁺ stores utilizes a different signal transduction pathway than does coupling to K_v channels. Another line of evidence supporting that hypothesis was analysis of the threshold response. The threshold for inhibition of K_v channels by ET-1 was 1-10 pM. In contrast, release of intracellular Ca²⁺ stores required 1-10 nM ET-1. Thus it appears that a more sensitive signaling pathway is utilized for coupling of ET receptors to K_v channels in PASMC.

These findings may help clarify the signaling mechanisms utilized in the hypertensive pulmonary circulation, and they could have relevance to prior observations in the intact pulmonary circulation. We previously reported (19) that in the chronically hypoxic rat lung, inhibition of nitric oxide synthase by N^G-nitro-L-arginine (L-NNA) unmasked vasoconstrictor tone. In the isolated, PSS-perfused chronically hypoxic rat lung L-NNA vasoconstriction could be eliminated by removal of extracellular Ca²⁺ or by simultaneous blockade of low- and high-threshold voltage-gated Ca²⁺ channels, consistent with PASMC membrane depolarization (20). L-NNA vasoconstriction was also reduced ~60% by ET_A-receptor blockade and eliminated by simultaneous inhibition of both ET_A and ET_B receptors, suggesting that ET-1 was stimulating the membrane depolarization (19). That pattern of inhibition, partial reduction with BQ-123 and complete inhibition with the combination of BQ-123 and BQ-788, is nearly identical to the pattern we found for ET-1-mediated inhibition of I_K(v) in hypertensive PASMC. We cannot rule out the possibility that multiple mechanisms contribute to ET-1-mediated membrane depolarization in the hypertensive pulmonary circulation in vivo. In addition to inhibition of I_K(v), ET-1 may also activate Cl conductance through release of intracellular Ca²⁺ stores (3). However, our results suggest the latter event cannot be the sole mechanism of depolarization, because it would be prevented by either ET_A- or ET_B-receptor blockade.

Our findings may also help to explain another paradox. Multiple investigators have found that ET_A-receptor blockade reduces pulmonary artery pressure in chronically hypoxic rats by 60-100% (4-6). Inhibition of L-type Ca²⁺ channels has a similar effect, again suggesting that the pulmonary vasoconstriction stimulated by ET-1 in chronically hypoxic rats is mediated by membrane depolarization and activation of voltage-gated Ca²⁺ channels. In vivo, the circulating concentration of ET-1 in chronically hypoxic animals and humans is in the range of 3-5 nM (1, 7). Our studies suggest that at that concentration maximum inhibition of PASMC K_v channels would be seen. In the normotensive pulmonary circulation ET_A blockade alone would not be sufficient to prevent ET-1-mediated inhibition of I_K(v). However, our data are consistent with the hypothesis that the high degree of efficacy of ET_A-receptor blockade in vivo could be explained by the switch in coupling between ET-1 and K_v channels from ET_B to ET_A receptors.

In conclusion, we found that ET-1 inhibited K_v channels in PASMC from both normoxic and chronically hypoxic rats. Although we did not directly measure the change in membrane potential caused by inhibition of I_K(v), the dependence of PASMC on I_K(v) to set resting membrane potential suggests that ET-1 could cause membrane depolarization through this mechanism. Chronic hypoxia resulted in a switch in receptor coupling, eliminating the absolute requirement for ET_B activation seen in normoxic PASMC, although the maximum effect was seen when both ET_A and ET_B receptors were inhibited. Therefore, our findings are consistent with the hypothesis that at least a portion of the activity of ET-1 in the hypoxic pulmonary circulation is inhibition of K_v channels mediated by activation of ET_A and, to a lesser extent, ET_B receptors. This effect, exerted on a background of increased ET-1 production and constitutively depolarized PASMC, would prime the PASMC for further hypoxic inhibition of K_v channels resulting in membrane depolarization, activation of voltage-gated Ca²⁺ channels, and, ultimately, pulmonary artery vasoconstriction.