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(Circulation. 1998;98:1400-1406.)
© 1998 American Heart Association, Inc.

Clinical Investigation and Reports

Dysfunctional Voltage-Gated K⁺ Channels in Pulmonary Artery Smooth Muscle Cells of Patients With Primary Pulmonary Hypertension

Jason Xiao-Jian Yuan, MD, PhD; Ann M. Aldinger, BS; Magdalena Juhaszova, PhD; Jian Wang, MD; John V. Conte, Jr, MD; Sean P. Gaine, MD; Jonathan B. Orens, MD; ; Lewis J. Rubin, MD

From the Departments of Medicine (J.X.-J.Y., A.M.A., J.W., S.P.G., J.B.O., L.J.R.), Physiology (J.X.-J.Y., M.J., L.J.R.), and Surgery (J.V.C.), University of Maryland School of Medicine, Baltimore, Md.

Correspondence to Lewis J. Rubin, MD, University of Maryland School of Medicine, 10 S Pine St, Suite 800, Baltimore, MD 21201. E-mail lrubin{at}umaryland.edu

	Abstract

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Background—Primary pulmonary hypertension (PPH) is a rare disease of unknown cause. Although PPH and secondary pulmonary hypertension (SPH) share many clinical and pathological characteristics, their origins may be disparate. In pulmonary artery smooth muscle cells (PASMCs), the activity of voltage-gated K⁺ (K_V) channels governs membrane potential (E_m) and regulates cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt). A rise in [Ca²⁺]_cyt is a trigger of vasoconstriction and a stimulus of smooth muscle proliferation.

Methods and Results—Fluorescence microscopy and patch clamp techniques were used to measure [Ca²⁺]_cyt, E_m, and K_V currents in PASMCs. Mean pulmonary arterial pressures were comparable (46±4 and 53±4 mm Hg; P=0.30) in SPH and PPH patients. However, PPH-PASMCs had a higher resting [Ca²⁺]_cyt than cells from patients with SPH and nonpulmonary hypertension disease. Consistently, PPH-PASMCs had a more depolarized E_m than SPH-PASMCs. Furthermore, K_V currents were significantly diminished in PPH-PASMCs. Because of the dysfunctional K_V channels, the response of [Ca²⁺]_cyt to the K_V channel blocker 4-aminopyridine was significantly attenuated in PPH-PASMCs, whereas the response to 60 mmol/L K⁺ was comparable to that in SPH-PASMCs.

Conclusions—These results indicate that K_V channel function in PPH-PASMCs is inhibited compared with SPH-PASMCs. The resulting membrane depolarization and increase in [Ca²⁺]_cyt lead to pulmonary vasoconstriction and PASMC proliferation. Our data suggest that defects in PASMC K_V channels in PPH patients may be a unique mechanism involved in initiating and maintaining pulmonary vasoconstriction and appear to play a role in the pathogenesis of PPH.

Key Words: potassium • electrophysiology • pulmonary heart disease

	Introduction

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Primary pulmonary hypertension (PPH) is a rare disease with an incidence of 1 to 2 per million per year that is characterized hemodynamically by elevated pulmonary vascular resistance, leading to progressive right heart failure and death. The predominant pathophysiological and pathological features include vasoconstriction, vascular remodeling (medial and intimal proliferation), vascular injury (induced by shear stress, ischemia, and inflammation), and in situ thrombosis.^{1 2 3} Impaired endothelium-dependent pulmonary vasorelaxation, increased endothelin-1 synthesis, and imbalanced secretion of vasoactive prostanoids have been implicated in the development of both PPH and secondary pulmonary hypertension (SPH).^{4 5 6 7} However, several lines of evidence indicate that intrinsic abnormalities of pulmonary vascular smooth muscle are present in PPH and may be important in its pathogenesis. Medial hypertrophy, suggesting a stimulus for vasoconstriction, is the earliest and most consistent pathological finding of PPH,^{1 2 8 9} and smooth muscle stretch induced by vasoconstriction is a promoter of smooth muscle cell hypertrophy and hyperplasia.¹⁰ Pulmonary arteries from patients with PPH tend to have more smooth muscle hypertrophy compared with vessels from patients with SPH.¹¹ Additionally, vascular rings from PPH patients are more sensitive to vasoconstrictors than rings from normal subjects,¹² and patients with PPH tend to be more responsive to acute vasodilator than patients with SPH.^{2 8} Approximately 26% of PPH patients exhibit significant and sustained reductions in pulmonary artery pressure and vascular resistance in response to Ca²⁺ channel blockers,¹³ suggesting that elevated cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary arterial smooth muscle cells (PASMCs) is involved, at least in part, in the development and maintenance of PPH.

A rise in [Ca²⁺]_cyt in PASMCs is a major trigger for vasoconstriction and an important stimulus for smooth muscle hypertrophy.^{14 15 16} Membrane potential (E_m) controls the function of sarcolemmal voltage-gated Ca²⁺ channels and is thus a key determinant of [Ca²⁺]_cyt and pulmonary vascular tone.¹⁴ The resting E_m in PASMCs is regulated by the K⁺ currents through voltage-gated K⁺ (K_V) channels.¹⁷ Inhibition of K_V channels results in cell depolarization, thereby increasing [Ca²⁺]_cyt and causing pulmonary vasoconstriction.^{17 18 19 20} Accordingly, we hypothesized that attenuated K⁺ channel function, resulting in membrane depolarization and an increase in [Ca²⁺]_cyt, may play a role in the pathogenesis of PPH. Using patch clamp techniques and quantitative fluorescence microscopy, we compared K_V channel activity, resting E_m, and [Ca²⁺]_cyt regulation in PASMCs obtained from patients with PPH and SPH.

	Methods

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Subjects
The clinical and hemodynamic characteristics of the 21 subjects from whom lung tissue was obtained are shown in the Table. The diagnosis of PPH was established clinically in 5 patients on the basis of the criteria used in the National Institutes of Health Registry on PPH¹ and confirmed histopathologically. Ten subjects had pulmonary hypertension resulting from known causes (SPH) (Table). Two patients undergoing lobectomy for bronchogenic carcinoma, who had no evidence of pulmonary hypertension by physical examination, ECG, echocardiogram, or pathological examination of resected lung tissue, and 4 patients with obstructive disease, who had normal pulmonary arterial pressures, were the sources of tissue for normotensive control experiments (nonpulmonary hypertension [NPH]).

View this table: [in this window] [in a new window]   Table 1. Demographic, Clinical, and Hemodynamic Characteristics of Study Population

Preparation and Culture of PASMCs
We used primary cultured PASMCs in this study. Lung tissue, removed from patients in the operating room, was immediately placed in cold (4°C) saline and taken to the laboratory for dissection. Muscular pulmonary arteries were incubated in Hanks' balanced salt solution (20 minutes) containing 2 mg/mL collagenase (Worthington Biochemical). The adventitia was stripped, and endothelium was removed. The remaining smooth muscle was digested with 2.0 mg/mL collagenase, 0.5 mg/mL elastase, and 1 mg/mL bovine albumin (Sigma Chemical Co) at 37°C to make a cell suspension of PASMCs. The single PASMC was resuspended, plated onto 25-mm coverslips, and incubated in a humidified atmosphere of 5% CO₂ in air at 37°C in 10% fetal bovine serum DMEM for 1 week.

Immunofluorescence Labeling
The PASMCs were stained with the membrane-permeant nucleic acid stain, 4', 6'-diamidino-2-phenylinodole (DAPI, 5 µmol/L), and the blue fluorescence emitted at 461 nm was used to estimate total cell numbers in the cultures. A specific monoclonal antibody raised against smooth muscle -actin was used to evaluate cellular purity of cultures, and a secondary antibody conjugated with indocarbocyanine (Cy3) was used to display the fluorescent image (emitted at 570 nm). The cell images were processed by a MetaFluor/MetaMorph Imaging System (University Imagine); the Cy3 fluorescence was colored red and DAPI fluorescence was colored green to display images with red-green overlay.

Measurement of [Ca²⁺]_cyt
The [Ca²⁺]_cyt in single PASMC was measured by use of fura-2 and quantitative fluorescence microscopy.¹⁷ The fura-2–loaded (3 µmol/L for 30 minutes) cells on coverslips were superfused with the bath solution for 30 minutes at 35°C. Fura-2 fluorescence (510-nm light emission excited by 380- and 340-nm illumination) from PASMCs and background fluorescence were measured with an Olympus IMT2 microscope equipped for epifluorescence microscopy. The fluorescence signals emitted from the cells were collected (at 2 Hz) with a photomultiplier tube and stored for later analysis. When Ca²⁺ is measured with 2 excitation wavelengths for fura-2, [Ca²⁺]_cyt is related to the ratio of measured 510-nm fluorescence signals elicited at 380 and 340 nm.

Measurements of K⁺ Currents and E_m
Whole-cell and single-channel K⁺ currents (I_K) were recorded with an Axopatch-1D amplifier and pClamp software (Axon Instruments) by use of patch clamp techniques (Figure 1A through 1C).¹⁷ Patch pipettes (2 to 4 M) were fire polished on a microforge. The currents were filtered at 1 to 2 kHz (-3 dB) and digitized at 4 to 6 kHz.

View larger version (35K): [in this window] [in a new window]   Figure 1. Three recording configurations for patch clamp measurement of single-channel and whole-cell IK in PASMCs. A, While electrode is being pressed against cell membrane, slight suction applied to pipette interior results in formation of a gigaohm seal (giga seal) between electrode and cell membrane. Single-channel IK can be recorded in electrically isolated membrane patch (called cell-attached patch). B, After disruption of patch membrane (by applying brief and strong suction of pipette interior), IK from whole cell can be recorded (called whole-cell recording). C, After establishment of whole-cell recording configuration, withdrawal (pull) of electrode from cell excises membrane patch that inner side of membrane faces pipette interior and outer side of membrane faces bath solution (called outside-out patch). Single-channel IK in cell-attached patch (A, right), whole-cell IK (B, right), and single-channel IK in outside-out patch (C, right) are amplified by patch clamp amplifier (Patch Clamp), digitized with data acquisition system (Data Acqui), displayed on computer (PC-486), and then analyzed with pClamp software. Current levels when channels are closed (C) are indicated by horizontal arrows. Upper reflection represents outward current.

Whole-Cell I_K Recording
Step-pulse protocols and data acquisition were performed by a TL-1 digital interface (Axon Instruments) coupled to a computer (Figure 1B). Series resistance and whole-cell capacitance were compensated for by adjustment of the internal circuitry of the amplifier. Leakage and capacitance currents were subtracted by use of the P/4 protocol in pClamp software. The normal bath (extracellular) solution contained (mmol/L) NaCl 141, KCl 4.7, MgCl₂ 1.2, CaCl₂ 1.8, glucose 10, and HEPES 10, pH 7.4, with 1 mol/L NaOH. The patch pipette (intracellular) solution contained (mmol/L) KCl 125, ATP 5, EGTA 10, MgCl₂ 4, and HEPES 10, pH 7.2.

Single-Channel I_K Measurement
For cell-attached recording (Figure 1A), the extracellular solution was the same as that described for whole-cell current recording. The patch pipette (extracellular) solution contained (mmol/L) KCl 135, HEPES 10, MgCl₂ 1.2, glucose 10, and EGTA 10, pH 7.4. The extracellular and intracellular solutions for measuring currents in outside-out patches (Figure 1C) were the same as those for whole-cell current recording.

Membrane Potential Recording
E_m in single PASMC was measured in current-clamp configuration when the cell was held at no current (I=0). The extracellular and intracellular solutions were the same as those for whole-cell current recording.

Chemicals
4-Aminopyridine (4-AP, Sigma) and tetraethylammonium (TEA, Chemika Fluka) were directly dissolved in the bath superfusate on the day of use. Charybdotoxin (ChTX, Acurate) and cyclopiazonic acid (CPA, Sigma) were dissolved in water and DMSO, respectively, to make stock solutions of 100 µmol/L and 20 mmol/L; aliquots of the stock solutions were diluted 1:2000 to 1:4000 to make final concentrations of 25 nmol/L and 10 µmol/L. Similar dilution of DMSO alone into the bath solution was used as control and had no effect on K⁺ currents, E_m, and [Ca²⁺]_cyt.

Statistical Analysis
Data are expressed as mean±SE. Statistical analysis was performed by use of the unpaired Student's t test or ANOVA. Differences were considered significant when P<0.05.

	Results

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Mean pulmonary arterial pressures in SPH (46±4 mm Hg, n=9) and PPH (53±4 mm Hg, n=5) patients were comparable (P=0.30) and were significantly higher than in NPH patients (19±1 mm Hg, n=5, P<0.001). Other hemodynamic parameters were comparable in SPH and PPH (Table).

Virtually all cells stained by the nucleic acid dye DAPI cross-reacted with the smooth muscle -actin antibody (Figure 2), indicating that the cultured cells were smooth muscle cells without contamination by fibroblasts and endothelial cells. Additionally, there was no apparent morphological difference between SPH- and PPH-PASMCs (Figure 2).

View larger version (66K): [in this window] [in a new window]   Figure 2. Primary cultured PASMCs isolated from patients with SPH and PPH. Cells were stained with specific antibody against smooth muscle -actin (red fluorescence) and the nucleic acid dye DAPI (green fluorescence). Scale line is 10 µm.

Resting E_m and [Ca²⁺]_cyt in SPH- and PPH-PASMCs
Resting [Ca²⁺]_cyt in SPH-PASMCs was not significantly different from that in NPH-PASMCs. However, the resting [Ca²⁺]_cyt in PPH-PASMCs was significantly higher than in NPH- and SPH-PASMCs (P<0.05) (Figure 3). The comparable resting [Ca²⁺]_cyt in NPH- and SPH-PASMCs and the significantly different [Ca²⁺]_cyt between SPH- and PPH-PASMCs support the hypothesis that PPH-PASMCs may have a unique defect that does not exist in SPH-PASMCs, despite comparable pulmonary arterial pressures in SPH and PPH patients.

View larger version (31K): [in this window] [in a new window]   Figure 3. Summarized data showing [Ca2+]cyt in PASMCs from patients with NPH (open bar), SPH (hatched bar), and PPH (solid bar). Inset, Averaged resting membrane potential (Em) in PASMCs from SPH (hatched bar) and PPH (solid bar) patients. Data are expressed as mean±SE with the number of cells tested in parentheses. *P<0.05, ***P<0.001 vs SPH-PASMCs.

In smooth muscle cells, prolonged membrane depolarization causes sustained elevation of [Ca²⁺]_cyt.¹⁸ Consistent with the higher resting [Ca²⁺]_cyt, the resting E_m in PPH-PASMCs was significantly more depolarized than in SPH-PASMCs (Figure 3, inset). E_m is determined primarily by K⁺ permeability through sarcolemmal K⁺ channels. Whether the more depolarized E_m in PPH-PASMCs is due to inhibited K⁺ channels was then examined by comparing K⁺ currents between SPH- and PPH-PASMCs.

Reduced Whole-Cell K_V Currents in PPH-PASMCs
At least 3 types of K⁺ currents have been described in PASMCs²⁰ : (1) K_V currents [I_K(V)], (2) Ca²⁺-activated K⁺ (K_Ca) currents, and (3) ATP-sensitive K⁺ (K_ATP) currents. Whereas K_Ca and K_ATP currents were minimized with pipette (intracellular) solutions containing 10 mmol/L EGTA and 5 mmol/L ATP, the whole-cell I_K(V) was isolated in SPH-PASMCs (Figure 4A, left). Neither the K_Ca channel blockers TEA (1 mmol/L) and ChTX (25 nmol/L) nor the K_ATP channel blocker glibenclamide (10 µmol/L) affected I_K(V). Similar to rat PASMCs, the I_K(V) in SPH-PASMCs appeared to consist of a transient current and a steady-state current. In PPH-PASMCs, the amplitudes of the currents, measured at the beginning [10 to 50 ms for the transient I_K(V)] and end [250 to 290 ms for the steady-state I_K(V)] of the test pulses (300 ms) (Figure 4A, right), were significantly diminished compared with SPH-PASMCs (Figure 4B). Because the size of the SPH- and PPH-PASMCs appeared similar (Figure 2), the reduced I_K(V) was unlikely to be due to size-related differences in cell capacitance.

View larger version (40K): [in this window] [in a new window]   Figure 4. Comparison of whole-cell IK(V) in PASMCs from patients with SPH and PPH. A, Families of currents elicited by depolarizing cells to test potentials ranging from -40 to +80 mV (holding potential, -70 mV). B, Summarized amplitudes of currents, elicited by test potentials of +20, +40, +60, and +80 mV, were measured at 10 to 50 ms [for transient IK(V)] and 250 to 290 ms [for steady-state IK(V)] while duration of test pulse was 300 ms. Data are expressed as mean±SE with number of cells in parentheses. *P<0.05, ***P<0.001 vs SPH-PASMCs.

Comparison of Single-Channel I_K in SPH- and PPH-PASMCs
In cell-attached membrane patches of SPH-PASMCs, a large-amplitude K_Ca current and a small-amplitude I_K(V) (slope conductance, 44 to 65 pS; n=8) were elicited by depolarization to +90 mV (Figure 5A). The calculated slope conductances of K_Ca currents are 217±8 pS (n=17) and 215±7 pS (n=9) in SPH- and PPH-PASMCs, respectively. In 44 SPH-PASMC membrane patches tested, the K_Ca current was evident in 40 patches (91%), and I_K(V) was apparent in 32 patches (73%) (Figure 5B). In contrast, in 16 PPH-PASMC membrane patches tested, K_Ca current was observed in all patches (100%), but I_K(V) was detectable in only 2 patches (12%), suggesting that the small-conductance I_K(V) is diminished in PPH-PASMCs.

View larger version (31K): [in this window] [in a new window]   Figure 5. Single-channel IK in cell-attached patches of PASMCs from patients with SPH and PPH. A, Representative IK(V) (small amplitude) and KCa current [IK(Ca), large amplitude] elicited by sustained +90-mV test pulse (left) in SPH-PASMC. The horizontal arrows denote current level when channels are closed. Vertical and horizontal bars denote 5 pA and 50 ms, respectively. Current-voltage relationship curves for IK(V) () and IK(Ca) () are shown on right. B, Percentage of patches from SPH- (hatched bars) and PPH- (solid bars) PASMCs in which IK(Ca) and IK(V) were present. Number of cells tested is shown in parentheses.

Diminished Response of [Ca²⁺]_cyt to 4-AP in PPH-PASMCs
In excised outside-out patches, 5 mmol/L 4-AP (Figure 6A) had no effect on the large-conductance K_Ca current, whereas 1 mmol/L TEA (Figure 6B) significantly inhibited the K_Ca current (the steady-state open probability was decreased from 0.65 to 0.23). These results suggest that 4-AP predominantly blocks K_V channels, whereas low doses of TEA selectively block K_Ca channels.²⁰

View larger version (43K): [in this window] [in a new window]   Figure 6. Effects of 4-AP and TEA on single-channel KCa current [lqsb]IK(Ca)] and [Ca2+]cyt in PASMCs from SPH patients. A, IK(Ca) in outside-out patch before, during, and after application of 5 mmol/L 4-AP (top) and corresponding amplitude histograms (bottom). Vertical and horizontal bars denote 5 pA and 50 ms, respectively. B, IK(Ca) before, during, and after application of 1 mmol/L TEA (top) and corresponding amplitude histograms (bottom). C, [Ca2+]cyt measured in SPH-PASMCs when 4-AP (a) or TEA (b) was superfused. Solid bars (c) denote [Ca2+]cyt (mean±SE, n=11) before (Cont) and during application of TEA.

Application of 1 mmol/L TEA (Figure 6C) or 25 nmol/L ChTX did not affect [Ca²⁺]_cyt (by 3±1 nmol/L, n=10, and 1±1 nmol/L, n=17, respectively) in SPH-PASMCs. The K_ATP channel blocker glibenclamide (10 µmol/L) also had no effect on [Ca²⁺]_cyt (by 2±1 nmol/L, n=17). These results suggest that K_Ca and K_ATP channels may be relatively inactive under resting conditions because of low [Ca²⁺]_cyt (50 to 100 nmol/L) and a high concentration of intracellular ATP (1 to 3 mmol/L).²⁰

The K_V channel blocker 4-AP, however, reversibly increased [Ca²⁺]_cyt in PASMCs from NPH and SPH patients (Figure 7A). This effect was apparently caused by membrane depolarization induced by reduction of I_K(V), because a similar effect could be induced by 60 mmol/L K⁺ (which shifts the K⁺ equilibrium potential to -21 mV) (Figure 7B). In contrast, the 4-AP–induced increase in [Ca²⁺]_cyt was significantly attenuated in PPH-PASMCs (Figure 7A and 7C). The effect of 60 mmol/L K⁺ on [Ca²⁺]_cyt was similar in cells from SPH and PPH patients (Figure 7B and 7D).

View larger version (33K): [in this window] [in a new window]   Figure 7. Effects of 4-AP and 60 mmol/L K+ (60 K+) on [Ca2+]cyt in PASMCs from patients with NPH, SPH, and PPH. A and B, Representative records of [Ca2+]cyt in response to 4-AP (5 mmol/L, A) and 60 K+ (B) in SPH- (left) and PPH- (right) PASMCs. C and D, Summarized data (mean±SE with number of cells tested in parentheses) showing the 4-AP– (C) and 60 K+- (D) induced peak rises in [Ca2+]cyt in PASMCs from NPH (open bars), SPH (crosshatched bars), and PPH (solid bars) patients. *** P<0.001 vs NPH- and SPH-PASMCs.

	Discussion

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The absence of a suitable animal model of PPH and its rarity have hampered progress in clarifying the pathogenesis of PPH. By obtaining pulmonary vascular tissue from patients with both SPH and PPH, we were able to compare and contrast at a cellular level the mechanisms underlying this disease. Our results demonstrate that compared with SPH-PASMCs, PPH-PASMCs have (1) a higher resting [Ca²⁺]_cyt and a more depolarized resting E_m, (2) an inhibited I_K(V), and (3) a diminished response of [Ca²⁺]_cyt to the K_V channel blocker 4-AP. These observations indicate that the K_V channels are dysfunctional PPH-PASMCs. The resultant membrane depolarization and increased [Ca²⁺]_cyt may play a pivotal role in vasoconstriction and possibly vascular proliferation, which are important components of the pathogenesis of PPH.

Dysfunctional PASMC K_V Channels in the Pathogenesis of PPH
In PASMCs, I_K(V) is composed of the rapidly inactivating I_K(V) [transient I_K(V)] and the slowly inactivating or noninactivating I_K(V) [steady-state I_K(V)].^{17 19 20} The transient I_K(V), which resembles A-type K⁺ current, is involved mainly in regulating the duration of action potential, whereas the steady-state I_K(V), which resembles slowly inactivating or noninactivating delayed rectifier K⁺ current, plays an important role in governing resting E_m. In PASMCs, inhibition of I_K(V) raised [Ca²⁺]_cyt by depolarizing the cell membrane and increased pulmonary arterial pressure,^{17 18 19 20} whereas inhibition of K_Ca currents and K_ATP currents had no effects on resting E_m, [Ca²⁺]_cyt, or pulmonary vascular tone.^{17 19} These results suggest that in PASMCs, I_K(V) is a major determinant of E_m and [Ca²⁺]_cyt at rest¹⁷ and is an important contributor to the maintenance of basal pulmonary vascular tone.¹⁹

Weir et al²¹ recently demonstrated that the anorexic agents aminorex and fenfluramine inhibited the 4-AP–sensitive I_K, caused membrane depolarization, and increased pulmonary arterial pressure. Because appetite suppressant use has been implicated in the development of PPH in some patients who take these drugs,²² it is possible that a predisposition to the development of PPH based on the function of PASMC K_V channels may exist in susceptible individuals.

Endogenous K_V channels turn over very rapidly; the half-lives of the channel mRNA and protein are 0.5 and 4 hours, respectively.²³ The short half-life of the K_V channels suggests that the cells undergo rapid exchange of channel mRNAs. Compared with SPH-PASMCs, the mRNA level of K_V1.5 (a delayed rectifier K_V channel subunit) is significantly attenuated in PPH-PASMCs.²⁴ The decreased K_v1.5 mRNA expression would reduce the number of the functional K_V channels and decrease K_V current availability. Thus, 1 mechanism involved in the attenuated I_K(V) in PPH-PASMCs is inhibited gene transcription and/or reduced mRNA stability of K_V channels.

[Ca²⁺]_cyt, Pulmonary Vasoconstriction, and Vascular Remodeling
In vascular smooth muscle cells, [Ca²⁺]_cyt can be increased by Ca²⁺ influx through Ca²⁺ channels and Ca²⁺ release from intracellular stores.^{14 15 16} Because of the voltage dependence of the sarcolemmal Ca²⁺ channels, membrane depolarization is an important cause of elevated [Ca²⁺]_cyt in PASMCs.¹⁴ The voltage window for sustained elevation of [Ca²⁺]_cyt through smooth muscle voltage-gated Ca²⁺ channels ranges from -40 to -15 mV.¹⁸ Therefore, the more depolarized PASMCs in PPH patients result in an increased Ca²⁺ influx and elevated [Ca²⁺]_cyt.

The ratio of cytosolic free [Ca²⁺] ([Ca²⁺]_cyt) to the intracellularly stored [Ca²⁺] in the sarcoplasmic reticulum ([Ca²⁺]_SR) is about 1:10 000 to 50 000. The resting [Ca²⁺]_cyt in PPH-PASMCs is 23% higher than in SPH-PASMCs, which would be expected to result in a significant increase in [Ca²⁺]_SR. Agonist-induced vasoconstriction is triggered by an initial release of Ca²⁺ from sarcoplasmic reticulum. The higher [Ca²⁺]_SR may thus be responsible for the augmented agonist-mediated pulmonary vasoconstriction in PPH patients.¹²

A rise in cytosolic [Ca²⁺], in addition to triggering cell contraction, can rapidly (within 50 to 300 ms) increase nuclear [Ca²⁺] and promote cell proliferation by moving quiescent cells into the cell cycle and by propelling the proliferating cells through mitosis.^{15 16 25} Thus, increased [Ca²⁺]_cyt may also play a pivotal role in the hypertrophy of small pulmonary arteries and muscularization of pulmonary arterioles, which are characteristic of PPH.¹¹

Possible Origin of PPH: Involvement of Dysfunctional K_V Channels
A variety of endothelium-derived vasoactive substances, such as nitric oxide, endothelium-derived hyperpolarizing factor (epoxides), prostacyclin, and endothelin,^{26 27 28 29} exert their effects in part through alteration of ion channel function in PASMCs. Thus, an impairment of endothelium-dependent pulmonary relaxation, an imbalance in the ratio of vasoconstrictors and vasodilators, and a dysfunctional K_V channel in PASMCs may contribute to the development or progression of PPH. We postulate that the early stages of PPH are characterized by pulmonary vasoconstriction resulting from dysfunctional K_V channels (Figure 8), which lead to E_m depolarization and an increase in [Ca²⁺]_cyt in PASMCs. Subsequently, impaired endothelium-dependent vasodilation and elevated [Ca²⁺]_cyt potentiate vasoconstriction and eventually lead to vascular remodeling. Altered secretion of endothelium-derived constricting and relaxing factors further contributes to vasoconstriction and vascular wall thickening. Ultimately, dynamic vasoconstriction is replaced by extensive vascular remodeling.

View larger version (27K): [in this window] [in a new window]   Figure 8. Proposed cellular mechanisms responsible for development of PPH. Process appears to be initiated by abnormal gene transcription and expression of KV channels. Resultant reduction of KV currents [IK(V)] causes membrane depolarization and opens voltage-gated Ca2+ channels. Increased Ca2+ influx through sarcolemmal Ca2+ channels and Ca2+-induced Ca2+ release from intracellular Ca2+ stores (mainly sarcoplasmic reticulum [SR]) raise [Ca2+]cyt, which triggers pulmonary vasoconstriction. Rise in [Ca2+]cyt would also increase nuclear Ca2+ concentration ([Ca2+]n) and stimulate cell proliferation, which causes pulmonary vascular remodeling. Endothelium-derived relaxing factors (EDRF) may participate in regulating Em and [Ca2+]cyt through activation of K+ (KCa and KV) channels and/or inhibition of voltage-gated Ca2+ channels in PASMCs. (+) indicates increase (or enhance); (-), decrease (or inhibit).

	Acknowledgments

 
This work was supported by grants from the PPH Cure Foundation, the PPH Research Foundation, and the National Institutes of Health (HL-54043 and HL-02659). Dr Yuan is an established investigator of the American Heart Association.

Received March 4, 1998; revision received May 20, 1998; accepted June 3, 1998.

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