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(Circulation Research. 1995;77:370-378.)
© 1995 American Heart Association, Inc.

Articles

Voltage-Gated K⁺ Currents Regulate Resting Membrane Potential and [Ca²⁺]_i in Pulmonary Arterial Myocytes

Xiao-Jian Yuan

From the Department of Medicine, Division of Pulmonary and Critical Care Medicine, and the Department of Physiology, University of Maryland School of Medicine, Baltimore.

Correspondence to X.-Jian Yuan, MD, PhD, Pulmonary Division, UMAB Medical School, 10 S Pine St, Suite 800, Baltimore, MD 21201.

	Abstract

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Abstract The membrane potential (E_m) of pulmonary arterial smooth muscle cells (PASMCs) regulates pulmonary arterial tone by controlling voltage-gated Ca²⁺ channel activity, which is a major contributor to [Ca²⁺]_i. The resting membrane is mainly permeable to K⁺; thus, the resting E_m is controlled by K⁺ permeability through sarcolemmal K⁺ channels. At least three K⁺ currents, voltage-gated K⁺ (K_V) currents, Ca²⁺-activated K⁺ (K_Ca) currents, and ATP-sensitive (K_ATP) currents, have been identified in PASMCs. In this study, both patch-clamp and quantitative fluorescent microscopy techniques were used to determine which kind(s) of K⁺ channels (K_V, K_Ca, and/or K_ATP) is responsible for controlling E_m and [Ca²⁺]_i under resting conditions in rat PASMCs. When the bath solution contained 1.8 mmol/L Ca²⁺ and the pipette solution included 0.1 mmol/L EGTA, depolarizations (-40 to +80 mV) elicited both K_Ca and K_V currents. Removal of extracellular Ca²⁺ and increase of intracellular EGTA concentration (to 10 mmol/L) eliminated the Ca²⁺ influx–dependent K_Ca current. 4-Aminopyridine (4-AP, 5 to 10 mmol/L) but not charybdotoxin (ChTX, 10 to 20 nmol/L) significantly reduced K_V current under these conditions. In current-clamp experiments, 4-AP decreased E_m (depolarization) and induced Ca²⁺-dependent action potentials; this depolarization increased [Ca²⁺]_i in intact PASMCs. Neither ChTX nor the specific blocker of K_ATP channels, glibenclamide (2 to 10 µmol/L), caused membrane depolarization and the increase in [Ca²⁺]_i. However, pretreatment of PASMCs with ChTX enhanced the 4-AP–induced increase in [Ca²⁺]_i. These results suggest that the 4-AP–sensitive K_V currents that are active in the resting state are the major contributors to regulation of E_m and thus [Ca²⁺]_i in rat PASMCs.

Key Words: voltage-gated K⁺ channels • Ca²⁺-activated K⁺ channels • ATP-sensitive K⁺ channels • membrane potential • intracellular Ca²⁺

	Introduction

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Elevation of [Ca²⁺]_i plays a primary role in triggering contraction in vascular smooth muscle cells, including PASMCs. Both Ca²⁺ influx through sarcolemmal Ca²⁺ channels and Ca²⁺ release from intracellular Ca²⁺ stores (eg, sarcoplasmic reticulum) contribute to the rise in [Ca²⁺]_i. By controlling Ca²⁺ influx via voltage-gated Ca²⁺ channels, E_m in vascular smooth muscle cells is an important regulator of vascular tone.^{1 2} The Ca²⁺ current through voltage-gated Ca²⁺ channels is proportional to the amount of time a Ca²⁺ channel spends in the open state, which is strongly regulated by E_m. It has been proposed that the voltage dependence of Ca²⁺ channels underlies the E_m dependence of vascular tone, with 3-mV depolarization increasing Ca²⁺ influx as much as twofold.^{1 2} In vascular smooth muscle cells, E_m is controlled by K⁺ permeability through sarcolemmal K⁺ channels. Because of high membrane input resistance of 2.6 to 17 G in resting vascular smooth muscle cells,^{3 4 5} a very small change in outward K⁺ currents should result in a marked change in E_m.

At least four types of K⁺ channel current have been identified in vascular smooth muscle cells^{2 6 7} : (1) I_K(V), which is composed of transient (A-type)^{6 8 9} and steady state (delayed rectifier) components,^{10 11 12} (2) I_K(I),¹³ (3) I_K(Ca),¹⁴ and (4) I_K(ATP).^{3 15} E_m appears to be controlled by an equilibrium between the outward currents provided by the K⁺ channels and the inward currents provided by Ca^{2+16 17} and Cl^- channels.^{18 19} However, it has been proposed that under resting physiological conditions, K⁺ permeability through K_V channels is responsible for determining the resting E_m^{16 20 21 22 23 24 25} and depolarization-dependent repolarization,^{1 2} whereas K_ATP and K_Ca channels contribute to the hyperpolarization induced by various exogenous and endogenous vasodilators¹⁵ and the repolarization following increased [Ca²⁺]_i,²⁶ respectively. In vascular smooth muscle cells, the K_ATP and K_Ca channels, as targets of endogenous vasodilators and negative feedback regulators of vascular tone, link E_m to the state of cellular metabolism and [Ca²⁺]_i homeostasis.

Regulation of PA tone and contraction depends largely on resting E_m and [Ca²⁺]_i in PASMCs. Membrane ionic channels are major contributors in controlling E_m and, subsequently, [Ca²⁺]_i. Thus, the primary goal of the present study was to use the selective blockers of respective K⁺ channels to identify those K⁺ channels that are responsible for regulating E_m and, more importantly, [Ca²⁺]_i under resting physiological conditions. The data provide evidence that 4-AP–sensitive I_K(V) is the major K⁺ current controlling resting E_m and [Ca²⁺]_i in the primary cultures of rat PASMCs.

	Materials and Methods

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Cell Preparation and Culture
Rat primary cultured (3 to 7 days) PASMCs were used for the present study. The methods used for isolation and culture of PASMCs are described elsewhere.⁵ Briefly, the right and left branches of the rat main PA, with some intrapulmonary arterial branches, were aseptically removed from Sprague-Dawley rats (125 to 250 g) and placed in 1 mL Hanks' balanced salt solution (Sigma Chemical Co) containing 1.5 mg collagenase (Worthington Biochemical Co) for 20 minutes at 37°C. The adventitia was then dissected free of the vessel, and the endothelium was carefully rubbed off with forceps. Single PASMCs were obtained by digesting the resultant PA smooth muscle tissue with 1.5 mg/mL collagenase, 0.5 mg/mL elastase (Sigma), and 1 mg/mL bovine serum albumin (Sigma). Cells were incubated in a humidified atmosphere of 5% CO₂ in air at 37°C for 3 to 7 days after being plated onto coverslips and fed twice weekly with 10% FBSCM (Irvine Scientific). Twelve to 24 hours before experiments, the bovine serum concentration in FBSCM was decreased to 0.3% in order to stop cell proliferation.

Recording of Membrane Currents and E_m in Single PASMCs
The patch-clamp technique was used to measure membrane currents and E_m under voltage-clamp or current-clamp (current=0) mode, respectively, in the whole-cell configuration.^{5 27} Pipettes were pulled from borosilicate glass capillaries (VWR Scientific) with a vertical pipette puller (PP-83, Narishige) and fire polished with a Narishige microforge. The pipettes used for recording whole-cell currents and resting E_m had resistances of 2 to 4 M when filled with the regular pipette solution (134 mmol/L KCl). Coverslips containing the cells were placed in the recording chamber (volume, 0.75 mL) and superfused with external (bath) solution at a rate of 1.3 mL/min. Voltage-clamp command potentials were generated by using an Axopatch-1D patch-clamp amplifier (Axon Instruments) under the control of PCLAMP software. The interpulse interval in the voltage-clamp experiments was 10 to 25 seconds. Data acquisition was carried out with a digital interface (TL-1 DMA, Axon Instruments) coupled to an IBM-compatible computer. Membrane current and voltage were monitored on a storage oscilloscope (Tektronix). Whole-cell currents were filtered at 2 kHz (-3 dB), digitized at 1 to 2 kHz, and stored on hard disk of the computer for off-line analysis. Series resistance and whole-cell capacitance were compensated (60% to 70%) by adjusting the internal circuitry of the patch-clamp amplifier. The leakage currents were subtracted by using P/4 protocol in PCLAMP software. All electrophysiological experiments were performed at room temperature (22°C to 24°C).

Measurement of [Ca²⁺]_i in Single PASMCs
The [Ca²⁺]_i in PASMCs was measured by using the fluorescent dye fura 2 and a quantitative fluorescent microscopy system (Photoscan M-series, Photon Technology International).²⁸ The cells grown on 25-mm coverslips were incubated in 3 mL 10% FBSCM containing 5 µmol/L of the acetoxymethyl ester form of fura 2 (fura 2-AM) for 30 minutes at room temperature under an atmosphere of 5% CO₂/95% air. The fura 2–loaded cells on coverslips were then superfused (at a rate of 2.2 mL/min) with the standard bath solution (PSS) for 30 minutes at 32°C. This allows sufficient time to wash away any extracellular fura 2-AM and for intracellular esterase to cleave cytosolic fura 2-AM into the active fura 2.

A single cell of interest was identified and illuminated with light from a 75-W xenon lamp. Filtered emitted light at wavelengths of <550 nm was passed to a rotating chopper disk, allowing light to pass alternately through two 10-nm bandpass filters centered at 340 and 380 nm (Omega Optical). Excitation light was reflected by a dichroic mirror and focused onto the cell being studied with a x40 Nikon UV-Fluor objective. Emitted light was low pass–filtered (510 nm) and passed through an aperture positioned over the cell. Fura 2 fluorescence (510-nm light emission excited by 340- and 380-nm illumination) from this area of the cell and background fluorescence were measured by using a photomultiplier tube via an Olympus IMT2 microscope equipped for epifluorescence microscopy. The fluorescence signals were collected continuously and stored in a 386 IBM-compatible computer (Everex Computer System) for later analysis.

[Ca²⁺]_i was calculated from the ratio (R) of measured 510-nm fluorescence signals (F) elicited at 340 nm and 380 nm, according to the following equations:

F_{340, cell} and F_{380, cell} indicate the measured fluorescence from the cell when illuminated at 340 nm and 380 nm, respectively; F_{340, bkg} and F_{380, bkg}, the background fluorescence signals, respectively; K_d (225 nmol/L), the dissociation constant of the Ca²⁺:fura 2 complex; S_f2, 340 fluorescence of the Ca²⁺-free fura 2; S_b2, 380 fluorescence of the Ca²⁺-saturated fura 2; and R_min and R_max, the ratio (F₃₄₀/F₃₈₀) of the fluorescence signals of fura 2 that is free of Ca²⁺ and the ratio of the fluorescence signals of the Ca²⁺:fura 2 complex at the highest [Ca²⁺], respectively. R_max and R_min were determined by using methods described elsewhere.^{29 30}

Reagents and Solutions
The standard extracellular PSS used for recording I_K and E_m and for measuring [Ca²⁺]_i contained (mmol/L): NaCl 141, KCl 4.7, CaCl₂ 1.8, MgCl₂ 1.2, HEPES 10, and glucose 10, buffered to pH 7.4 with 5 mol/L NaOH. In Ca²⁺-free PSS, the CaCl₂ was replaced by MgCl₂, and 0.5 to 1.0 mmol/L EGTA was added. The perfusion speeds are 1.3 and 2.2 mL/min in the patch-clamp setup and the quantitative fluorescent microscopy system. That is, the perfusion speed used for measuring [Ca²⁺]_i is 1.69 (2.2/1.3) times faster than that used for measuring E_m. The internal (pipette) solution used for recording I_K and E_m consisted of the following (mmol/L): KCl 125, MgCl₂ 4, HEPES 10, EGTA 10, and Na₂ATP 5, buffered to pH 7.2 with 1 mol/L KOH. Usually, 90 µL KOH (1 mol/L) was needed to buffer the 10-mL internal pipette solution (original pH, 4.85) to pH 7.2. Thus, actual [K⁺]_i was slightly increased from 125 to 134 mmol/L. This increase in [K⁺]_i would slightly shift the K⁺ equilibrium potential by 2 mV (the calculated K⁺ equilibrium potential was shifted from -84.0 to -85.8 mV). In some experiments, the EGTA concentration in the Ca²⁺-free pipette solution was reduced from 10 to 0.1 mmol/L in order to diminish intracellular Ca²⁺ chelation.

4-AP (Aldrich Chemical Co) was dissolved directly into PSS on the day of use. The solution containing 4-AP was buffered to pH 7.4 by using HCl before each experiment. ChTX (Accurate Chemical & Scientific Co) was dissolved in DMSO (Sigma) and diluted to a final concentration of 10 to 20 nmol/L in the bath solution. Glibenclamide (Sigma) was dissolved in DMSO to make a stock solution of 100 mmol/L; an aliquot of the stock solution was diluted 1:50 000 to 1:10 000 into PSS to make a final concentration of 2 and 10 µmol/L, respectively. Similar dilutions of DMSO alone into PSS were used as controls and had no effect on K⁺ currents, E_m, or [Ca²⁺]_i.

Statistics
Data are expressed as mean±SEM. Statistical analysis was performed by using paired or unpaired Student's t test or ANOVA, as appropriate. Differences were considered to be significant at P<.05.

	Results

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Whole-Cell Currents in PASMCs
When the cells were superfused with 1.8 mmol/L Ca²⁺–containing PSS and dialyzed with the pipette solutions containing 0.1 mmol/L EGTA, several components of whole-cell currents were recorded by depolarizing cells from a holding potential of -70 mV to a series of test potentials ranging from -40 to +80 mV (Fig 1). There were two components of I_K as well as I_Ca and I_Cl(Ca). I_Ca and I_Cl(Ca) were eliminated by removal of extracellular Ca²⁺ (Fig 1, center); I_Cl(Ca) was inhibited by the Cl^- channel blocker^{18 19} niflumic acid (10 µmol/L) (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of removal of extracellular Ca2+ on whole-cell currents recorded in a PASMC dialyzed with the pipette solution containing 0.1 mmol/L EGTA and 5 mmol/L ATP. Representative families of superimposed currents, elicited by depolarizing cells with a series of test potentials between -40 and +80 mV from a holding potential of -70 mV, were recorded before, during, and after removal of extracellular Ca2+ from 1.8 mmol/L Ca2+–containing PSS. Leakage and capacitance currents were subtracted. Corresponding I-V curves were also shown under current records. ICa (), IK (), and ICl () denote inward Ca2+ current, outward K+ current, and inward Ca2+-activated Cl- current, respectively.

Two components of I_K, I_K(V) and I_K(Ca), could be differentiated by the dependence on extracellular Ca²⁺ (Fig 1). The Ca²⁺ influx–dependent I_K(Ca) component was evidenced by the hump in an N-shaped I-V relation (I-V curve) between 0 and +60 mV,³¹ which could only be obtained when 1.8 mmol/L Ca²⁺ was present in the bath solution (compare Figs 1 and 2). Removal of extracellular Ca²⁺ and increase of intracellular EGTA concentration (from 0.1 to 10 mmol/L) virtually eliminated the Ca²⁺-dependent component, I_K(Ca),³² of the total I_K (Figs 1 and 2). This suggests that the K_Ca (but not K_V) channel activity depends greatly on availability of free [Ca²⁺]_i in the resting PASMCs.

View larger version (14K): [in this window] [in a new window]   Figure 2. Whole-cell IK(V) (A) and corresponding I-V curve (B) recorded in a PASMC superfused with Ca2+-free PSS containing 0.5 mmol/L EGTA and dialyzed with Ca2+-free pipette solution containing 10 mmol/L EGTA and 5 mmol/L ATP. Currents were elicited by depolarizing the cell to test potentials between -60 and +80 mV in a 20-mV increment from a holding potential of -70 mV. One-component (A, upper inset) and two-component (A, lower inset) exponential fits (smooth lines on top of current records) of inactivation of IK(V), elicited by a 900-millisecond voltage step to +80 mV, are shown. Time constants for fast and slow inactivating components of IK(V) were 30 and 434 milliseconds, respectively; these values were based on two-component exponential fit. The equation used for the two-component fit is as follows: IK=A0+A1e(-t/1)+A2e(-t/2), where A1 (1063 pA) and A2 (555 pA) represent current amplitudes for two inactivation components at time (t)=0, 1 and 2 represent respective inactivation time constants, and A0 (1218 pA) corresponds to amplitude of steady state current.

The voltage-gated Ca²⁺-independent I_K, I_K(V), could then be isolated by using the Ca²⁺-free bath solution containing 0.5 to 1 mmol/L EGTA and the Ca²⁺-free pipette solution containing 10 mmol/L EGTA and 5 mmol/L ATP.³² During a maintained depolarization (eg, +80 mV) under these conditions, I_K(V) rose to an early peak and then inactivated when the cell was superfused with Ca²⁺-free PSS (0.5 mmol/L EGTA present) and dialyzed with 10 mmol/L EGTA Ca²⁺-free pipette solution (Fig 2). Two exponentials were required to best fit the decay of I_K(V) (Fig 2): a fast component with a time constant of 30 milliseconds and a slow component with a time constant of 434 milliseconds at +80 mV in this cell. This suggests that I_K(V) is generated by K⁺ efflux through either of two types of K_V channel, eg, A-type^{6 8 9} and delayed rectifier^{11 12 32} K⁺ channels, or one type of K_V channel that has multiple states.³²

Effects of ChTX and 4-AP on I_K(V) in PASMCs
When cells were dialyzed with Ca²⁺-free pipette solution (plus 10 mmol/L EGTA) and superfused with Ca²⁺-free PSS containing 0.5 mmol/L EGTA, ChTX (10 nmol/L) negligibly affected I_K elicited by test potentials from -40 to +80 mV (Fig 3A and 3C). The ChTX-insensitive I_K, however, was significantly decreased by 10 mmol/L 4-AP (Fig 3B and 3D); this indeed indicated that this current is the voltage-dependent Ca²⁺-insensitive I_K, termed I_K(V).^{22 32} Averaged data shown in Fig 3C and 3D demonstrated that ChTX (10 nmol/L) negligibly affected I_K(V), which was elicited by depolarizing cells from -70 to +60 mV, whereas bath application of 10 mmol/L 4-AP significantly diminished the steady state I_K(V), which was elicited by voltage steps to +60 mV (by 48%, Fig 3C) and to -40 mV (by 85%; Fig 3D, crosshatched bars). It is noteworthy that 4-AP inhibited the transient I_K(V) to a greater extent than the steady state I_K(V). These data are consistent with the observations by other investigators.^{9 10 32}

View larger version (18K): [in this window] [in a new window]   Figure 3. Effects of ChTX and 4-AP on voltage-gated K+ current (IK) in PASMCs. A, Whole-cell currents, elicited by depolarizing the cells to a series of 300-millisecond command steps ranging from -40 to +80 mV from a holding potential of -70 mV, were recorded before and during application of 10 nmol/L ChTX in a PASMC that was superfused with Ca2+-free PSS and dialyzed with 10 mmol/L EGTA– and 5 mmol/L ATP–containing pipette solution. B, Current records were obtained by using the same protocol as in panel A before and after bath application of 10 mmol/L 4-AP. Difference currents (subtraction, treatment-sensitive currents) were obtained by subtracting currents recorded during application of ChTX (A) or 4-AP (B) from currents recorded under control conditions. Leakage and capacitance currents were subtracted. C, Summary of effects of ChTX (10 nmol/L) and 4-AP (10 mmol/L) on the steady state IK (measured at 285 to 295 milliseconds of the 300-millisecond test pulse, n=5) elicited by depolarizing the cells to +60 mV from a holding potential of -70 mV. D, Composite data on inhibitory effects of 10 mmol/L 4-AP on the transient (TR, measured at 5 to 50 milliseconds) and the steady state (SS) IK elicited by depolarizing the cells (n=6) to -40 mV from a holding potential of -70 mV. Data are mean±SEM. **P<.01, *P<.05 vs control.

Effects of ChTX, Glibenclamide, and 4-AP on E_m in PASMCs
In rat primary cultured PASMCs, the average resting E_m values that were measured by using current-clamp technique were -41±1 mV (n=27) and -39±1 mV (n=21), respectively, in the presence and absence of 1.8 mmol/L external Ca²⁺. Extracellular application of either ChTX (10 nmol/L, Fig 4A and 4B) or glibenclamide (10 µmol/L, Fig 4C) had no effect on E_m. 4-AP (5 to 10 mmol/L), however, significantly and reversibly depolarized PASMCs and elicited extracellular Ca²⁺-dependent action potentials when the cells were superfused with 1.8 mmol/L Ca²⁺–containing PSS (Fig 4D and 4E).

View larger version (18K): [in this window] [in a new window]   Figure 4. Effects of ChTX, glibenclamide (Gli), and 4-AP on resting Em in the presence and absence of extracellular Ca2+ in PASMCs. The average Em at the resting state in the PASMCs tested in the present study is -40 mV. Bath application of either 10 nmol/L ChTX (A and B) or 10 µmol/L Gli (C) negligibly affected Em in the cells superfused with 1.8 mmol/L Ca2+–containing or Ca2+-free PSS. Bath administration of 5 mmol/L (D) and 10 mmol/L (E) 4-AP resulted in the long-duration spikes (which are composed of two components: a transient [action potential] component and a steady plateau component) in PASMCs bathed in 1.8 mmol/L Ca2+–containing PSS; removal of extracellular Ca2+ eliminated 4-AP–induced action potentials but had no effect on the steady state depolarization (F). Perfusion speed of the patch-clamp setup by which Em was measured was 1.3 mL/min.

Removal of extracellular Ca²⁺ had no effect on the 4-AP–induced steady state depolarization that primarily results from a decreased K_V conductance but completely abolished the 4-AP–induced action potentials (Fig 4F) that are presumably due to an increased Ca²⁺ conductance. Composite data shown in Fig 5 indicate that neither 10 nmol/L ChTX (n=9) nor 10 µmol/L glibenclamide (n=13) affected E_m (a change of 1.0±0.7 or 1.2±0.9 mV, respectively), whereas 10 mmol/L 4-AP significantly depolarized the cells by 16±1 mV (n=11) and 13±2 mV (n=12) (steady state depolarization), respectively, in the absence and presence of extracellular Ca²⁺ (Fig 5).

View larger version (28K): [in this window] [in a new window]   Figure 5. Composite data from a number of experiments showing the changes in resting Em induced by ChTX (10 nmol/L), glibenclamide (Gli, 10 µmol/L), and 4-AP (10 mmol/L) in the absence or presence of extracellular Ca2+ (1.8 mmol/L) in PSS. The crosshatched bars represent the steady state change of Em; the solid bar denotes the transient depolarization induced by 4-AP. It is noteworthy that 4-AP did not elicit any action potentials in the absence of extracellular Ca2+. Inset shows the average resting Em in the presence (PSS, n=25) or absence (0 Ca2+, n=20) of extracellular Ca2+. Data are mean±SEM, with numbers of cells indicated under bars.

In the presence of extracellular Ca²⁺, the 4-AP–induced steady state depolarization, in addition to decreased K_V conductance, may also be attributable to a sustained Ca²⁺ influx³³ and Ca²⁺-activated Cl^- efflux.^{18 19 34} The peak of the 4-AP–induced transient depolarization (action potential) was also averaged in Fig 5 (37±4 mV, n=12). Compared with the 13 mV of 4-AP–elicited steady state depolarization, this transient peak was presumably due to the increased Ca²⁺ influx through voltage-gated Ca²⁺ channels that resulted from membrane depolarization via the decreased K⁺ efflux through K_V channels.

In the presence of 15 nmol/L ChTX and absence of extracellular Ca²⁺, 4-AP (0.3 to 10 mmol/L) depolarized PASMCs in a dose-dependent manner (Fig 6). Under conditions in which PASMCs were dialyzed with solutions containing 10 mmol/L EGTA and superfused with Ca²⁺-free PSS, bath application of 0.3, 1, 3, and 10 mmol/L 4-AP decreased resting E_m (depolarization) by 1.9±0.6, 3.2±0.5, 7.5±1.1, and 15.8±1.0 mV, respectively (Fig 6A and 6B). Furthermore, 4-AP also inhibited I_K(V) in a dose-dependent manner (Fig 6A, inset).

View larger version (13K): [in this window] [in a new window]   Figure 6. Graded effects of 4-AP on resting Em in the presence of 15 nmol/L ChTX in PASMCs superfused with Ca2+-free PSS. A, Em was -46 mV before application of 4-AP and gradually depolarized to -43, -38, and -29 mV after application of 1, 3, and 10 mmol/L 4-AP, respectively, in the presence of 15 nmol/L ChTX. In the inset, single whole-cell K+ current traces elicited by a repeated voltage step to +80 mV from a holding potential of -70 mV were obtained before (), during ( and ), and after (dotted line) bath applications of 3 mmol/L () and 10 mmol/L () 4-AP. Vertical and horizontal bars denote 150 pA and 100 milliseconds, respectively. B, Averaged changes of Em in PASMCs during bath applications of 0.3 (n=3), 1 (n=13), 3 (n=10), and 10 (n=14) mmol/L 4-AP, respectively. Data are expressed as mean±SEM. ***P<.001, **P<.01 vs control. Perfusion speed of the patch-clamp setup by which Em was measured was 1.3 mL/min.

In the cells dialyzed with 10 mmol/L EGTA and 5 mmol/L ATP, inability of ChTX and glibenclamide to depolarize PASMCs can be attributable to deactivated K_Ca and K_ATP channels.^{3 26 32} Actually, this is exactly what the data (Figs 4 through 6) showed. The same reagents (4-AP, ChTX, and glibenclamide) were then used to determine, in intact (nondialyzed) cells, which K⁺ channels (K_V, K_Ca, and/or K_ATP channels) contribute to regulating [Ca²⁺]_i in PASMCs (see below).

Effects of ChTX, Glibenclamide, and 4-AP on [Ca²⁺]_i in PASMCs
Consistent with the effects on E_m, only 4-AP (10 mmol/L), but neither ChTX (20 nmol/L) nor glibenclamide (10 µmol/L), increased [Ca²⁺]_i under resting conditions in intact (nondialyzed) PASMCs (Fig 7). The 4-AP–induced increase in [Ca²⁺]_i featured an initial relatively rapid rise, which after reaching the maximal level, declined gradually to an elevated [Ca²⁺]_i plateau (Fig 7A and 7D). Bath application of 5 µmol/L verapamil, a blocker of voltage-gated Ca²⁺ channels, significantly inhibited the 4-AP–induced increase in [Ca²⁺]_i (Fig 8A and 8C), whereas removal of extracellular Ca²⁺ abolished the response of [Ca²⁺]_i to 4-AP (Fig 8B and 8C). These results indicate that the 4-AP–induced increase in [Ca²⁺]_i is due to Ca²⁺ influx through voltage-gated Ca²⁺ channels, which are opened by the membrane depolarization resulting from decreased K_V channel activity.

View larger version (18K): [in this window] [in a new window]   Figure 7. Representative traces (A through C) illustrating effects of 4-AP (10 mmol/L, A), ChTX (20 nmol/L, B), and glibenclamide (Gli, 10 µmol/L; C) on [Ca2+]i in unstimulated (resting) cultured rat PASMCs. The cells were superfused with 1.8 mmol/L Ca2+–containing PSS. Broken lines in each record represent [Ca2+]i values measured before application of 4-AP (solid bar), ChTX (crosshatched bar), and Gli (open bar), respectively. Composite data (D through F) show the responses of [Ca2+]i to 4-AP (10 mmol/L, n=12; D), ChTX (20 nmol/L, n=6; E), and Gli (10 µmol/L, n=8; F). Solid bars denote peak increase in [Ca2+]i induced by 4-AP (D through F). Data are mean±SEM. ***P<.001, *P<.05 vs control. Perfusion speed of the quantitative fluorescent microscopy system by which [Ca2+]i was measured was 2.2 mL/min.

View larger version (22K): [in this window] [in a new window]   Figure 8. Effects of verapamil and removal of extracellular Ca2+ on 4-AP–induced elevation in [Ca2+]i in PASMCs superfused with PSS. Representative traces illustrate that bath application of 5 µmol/L verapamil reduced the response of [Ca2+]i to 4-AP by 40% (A), whereas removal of extracellular Ca2+ (for 2 minutes with 0.5 mmol/L EGTA present) abolished the 4-AP–induced rise in [Ca2+]i (B). C, The values of [Ca2+]i were obtained before (control), during (4-AP), and after (washout) bath applications of 10 mmol/L 4-AP when cells were superfused with PSS (open bars, n=6), 5 µmol/L verapamil–containing PSS (crosshatched bars, n=6), and Ca2+-free PSS (with 0.5 mmol/L EGTA present) (solid bars, n=17). 4-AP slightly, but not significantly (P=.12), decreased resting [Ca2+]i in PASMCs superfused with Ca2+-free PSS. Data are mean±SEM. ***P<.001, *P<.05 vs control; #P<.01 vs PSS in the presence of 4-AP. Perfusion speed of the quantitative fluorescent microscopy system by which [Ca2+]i was measured was 2.2 mL/min.

The observation that the 4-AP–induced increase in [Ca²⁺]_i was not maintained at the maximal level suggests that a repolarization mechanism (eg, activation of K_Ca channels) and Ca²⁺ extrusion and sequestration processes become active after the membrane depolarization and/or the rise in [Ca²⁺]_i. Indeed, administration of 20 nmol/L ChTX in the presence of 4-AP delayed the decline of 4-AP–induced elevation of [Ca²⁺]_i (Fig 9). This suggests that the membrane repolarization process induced by activation of K_Ca channels due to a rise in [Ca²⁺]_i at least partially contributes to the negative-feedback regulation of depolarization-induced increase in [Ca²⁺]_i in PASMCs.

View larger version (19K): [in this window] [in a new window]   Figure 9. ChTX enhances 4-AP–induced elevation of [Ca2+]i in PASMCs. A, Extracellular application of 20 nmol/L ChTX negligibly affected [Ca2+]i, whereas 10 mmol/L 4-AP significantly increased [Ca2+]i. Although ChTX alone had no effect on [Ca2+]i, bath application of 20 nmol/L ChTX in the presence of 10 mmol/L 4-AP attenuated the decline phase of elevated [Ca2+]i. Broken line denotes the monoexponential fitting of the [Ca2+]i decay curve in the absence of ChTX. Perfusion speed of the quantitative fluorescent microscopy system by which [Ca2+]i was measured was 2.2 mL/min. B, Averaged data obtained from eight experiments illustrate that the decay phase of the 4-AP–induced increase in [Ca2+]i was significantly blunted by application of ChTX (P<.05 vs control, ANOVA). Data are mean±SEM.

Time Courses of 4-AP–Induced Membrane Depolarization and Increase in [Ca²⁺]_i
The time courses t_md, t_AP, and t_Ca were determined as follows: in each experiment, t represents the time from the introduction of 4-AP into the tissue chamber to the beginning of the change in the measured parameter. However, in the present study, E_m and [Ca²⁺]_i were measured by using two different experimental setups. The perfusion speeds are 1.3 and 2.2 mL/min (see "Materials and Methods"), respectively, in the patch-clamp setup (for measuring E_m) and the quantitative fluorescent microscopy system (for measuring [Ca²⁺]_i). Thus, the perfusion rate is 1.69 times faster in the fluorescent microscopy system than in the patch-clamp setup. Therefore, all individual t_Ca values were multiplied by 1.69 to correct for the faster perfusion rate. The rate of 4-AP–induced action potentials (t_AP) in the presence of extracellular Ca²⁺ is 69±9 seconds (n=27), and the time to initiate membrane depolarization (t_md) in the absence of extracellular Ca²⁺ is 24±2 seconds (n=19). Moreover, the perfusion-corrected time to increase [Ca²⁺]_i (t_Ca) is 85±5 seconds (n=57); thus, t_md<t_AP<t_Ca. These results suggest that 4-AP–induced membrane depolarization and action potentials precede the 4-AP–induced increase in [Ca²⁺]_i.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
PASMCs Have Voltage-Gated, Ca²⁺-Activated, and ATP-Sensitive K⁺ Channels
The primary cultured PASMCs used in the present study possess similar physiological, pharmacological, and biochemical properties compared with the freshly dissociated smooth muscle cells and isolated PA rings.^{35 36} In this cell preparation, a transient (A-type) K_V current, a steady state (delayed rectifier) K_V current,^{5 37} a K_Ca current (Reference 5⁵ and the present study), and an L-type voltage-gated Ca²⁺ current³⁸ have been described. The electrophysiological and pharmacological properties of these channels are also comparable to those identified in freshly dissociated PASMCs.^{4 10 11 12 16 25 39} In the isolated PA rings from which PASMCs were dissociated, cromakalim, the opener of K_ATP channels, significantly blocks 20 mmol/L K⁺–induced or hypoxia-induced contraction.⁴⁰ This result indicates that K_ATP channels, described in many types of vascular smooth muscle cells,^{2 7} are also present in rat PA smooth muscle.³

ChTX and glibenclamide block K_Ca and K_ATP channels with the concentrations for half block (K_i) of 1 to 10 nmol/L^{1 26 41} and 20 to 100 nmol/L,^{41 42} respectively. Although each drug has been shown to block some other types of K⁺ channel as well, ChTX and glibenclamide appear to be fairly selective inhibitors of K_Ca and K_ATP channels, respectively, in vascular smooth muscle cells.^{2 7 41} 4-AP, demonstrated to be without effect on K_Ca¹¹ and K_ATP² channels, blocks K_V channels with widely differing affinities; K_i ranges from 10 µmol/L to 10 mmol/L.^{10 11 22 41 43}

Voltage-Gated K⁺ Channels Play a Major Role in the Regulation of E_m in PASMCs
The results from the present study indicate that (1) neither the K_Ca channel blocker ChTX nor the K_ATP channel blocker glibenclamide affects E_m and [Ca²⁺]_i under resting conditions, in spite of their respective blockade effects on I_K(Ca) and I_K(ATP); (2) the K_V channel blocker 4-AP not only depolarizes but also increases [Ca²⁺]_i in PASMCs via its inhibitory effect on I_K(V); and (3) ChTX, albeit without effect on resting [Ca²⁺]_i, enhances the 4-AP–induced rise in [Ca²⁺]_i in PASMCs. These data suggest that I_K(V) is the major K⁺ current that is active under resting conditions and contributes to the regulation of resting E_m and thus [Ca²⁺]_i in PASMCs, whereas I_K(Ca), activated by increased [Ca²⁺]_i and depolarization, is responsible for membrane repolarization as a negative-feedback pathway in regulating E_m and Ca²⁺ influx–induced increase in [Ca²⁺]_i.^{2 7 16 26 32}

It has been proposed that (1) increasing intracellular ATP concentration from 0 to 1 mmol/L significantly depolarizes rabbit PASMCs,³ (2) hypoxia-induced pulmonary vasoconstriction is inhibited by the K_ATP channel opener cromakalim,^{40 44} and (3) anoxia-induced pulmonary vasodilation is inhibited by the K_ATP channel blocker glibenclamide.⁴⁴ The physiological role of I_K(ATP) in regulating E_m and [Ca²⁺]_i under resting conditions in PASMCs is not certain because of the very low open probability of K_ATP channels at physiological ATP concentrations.⁴² Hyperpolarization caused by activation of K_ATP channels, however, has been demonstrated to be the mechanism responsible for vascular relaxation induced by a number of endogenous vasodilators.^{2 3 7 42}

Role of Ca²⁺-Activated and ATP-Sensitive K⁺ Channels in Regulating E_m and [Ca²⁺]_i
The activity of K_Ca channels increases steeply with both depolarization and increasing [Ca²⁺], and the [Ca²⁺] needed to produce half-maximal activation of I_K(Ca) (at 0 mV) is 0.5 to 2 µmol/L^{2 6 7 14 45 46} in various smooth muscle cells, whereas the K_Ca channels in rat PASMCs show channel openings only with >300 nmol/L [Ca²⁺] present on the cytoplasmic side.⁴⁷ Thus, K_Ca channels in PASMCs are virtually inactive under resting conditions where E_m and [Ca²⁺]_i are -40 mV and 0.1 µmol/L, respectively. When the cells are depolarized and [Ca²⁺]_i is increased, however, activation of K_Ca channels tends to repolarize the cells, close voltage-gated Ca²⁺ channels, and reduce vascular tone.^{2 7 16 17 26}

The concentration of ATP for half-maximal inhibition of I_K(ATP) is 20 to 140 µmol/L, whereas 1 to 3 mmol/L ATP completely blocks K_ATP channels.^{3 15 42 48} Internal ATP concentration is usually in the millimolar range⁴² ; thus, K_ATP channels in muscle cells would normally be held closed by ATP in the resting state. However, because of the high membrane input resistance in vascular smooth muscle cells in the range of 2.6 to 17 G,^{1 2 3 4 5} the opening of even a few K_ATP channels by various endogenous vasodilators and metabolic inhibitors would have substantial effects on E_m and vascular tone.^{2 7 42}

Resting E_m measured in the primary cultures of PASMCs used in the present study (E_m, -40 mV) is somewhat more depolarized than that (E_m, -55 mV) reported in freshly dissociated PASMCs.^{3 4} This discrepancy may result from the higher concentration of ATP (5 mmol/L)³ and Mg²⁺ (4 mmol/L)²⁵ in the pipette solutions used in the present study. In rabbit PASMCs, Clapp and Gurney³ reported that increasing intracellular ATP concentrations from 0 to 3 mmol/L significantly shifted E_m from -70 to -53 mV and virtually blocked the glibenclamide-induced depolarization (from 15 to 1 mV). Furthermore, in canine renal arterial smooth muscle cells, increasing intracellular Mg²⁺ from 0 to 10 mmol/L caused an 20-mV change of resting E_m (from -52 to -33 mV) and significantly reduced the 4-AP–induced depolarizations (from 21 to 6 mV).²⁵ These observations imply that ATP- and Mg²⁺-induced inhibitions of I_K(ATP) and I_K(V), respectively, may contribute to the relatively more depolarized E_m measured in the present study.

The 4-AP–sensitive transient (measured at 5 to 50 milliseconds) and steady state I_K(V) (measured at 285 to 295 milliseconds of the 300-millisecond test pulse), at a test potential of -40 mV, were 26±11 and 14±4 pA (Fig 3D), respectively. This indicates that K_V channels are active at resting E_m in PASMCs. 4-AP–induced blockade of K_V channels not only depends on voltage and time but also depends on the state of channels per se.⁴⁹ The resting state blockade of 4-AP on K_V channels occurs at negative voltage, when the channels are mostly at their resting state.⁴⁹ Blockade of the 4-AP–sensitive K_V channels at such a resting state would, therefore, cause membrane depolarization and subsequently open voltage-gated Ca²⁺ channels, promote Ca²⁺ influx, and ultimately increase [Ca²⁺]_i. This is indeed what was observed in the present study.

When the PASMCs were bathed in PSS containing 4-AP, the membrane depolarization (Fig 4D and 4E) was prolonged by (1) inhibition of K_V channels by 4-AP^{10 11 25} ; (2) Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels, which are inactivated incompletely at a voltage range between -40 and -20 mV³³ ; (3) inhibition of K_Ca channels by chelating [Ca²⁺]_i with 10 mmol/L EGTA, as decreasing intracellular EGTA from 10 mmol/L to 0.1 mmol/L significantly shortens the depolarization duration²⁴ ; (4) inhibition of K_ATP channels by the 5 mmol/L ATP and 1.2 mmol/L MgCl₂ present in the pipette solution,^{1 2 3 7} ; and (5) Cl^- efflux through Ca²⁺-activated Cl^- channels.^{18 19 34} Thus, the inhibition of repolarization that results from activation of K_Ca, K_V, and K_ATP channels, as well as the inward currents due to Ca²⁺ influx and Cl^- efflux, may account for the prolongation of Ca²⁺-dependent spikes induced by 4-AP (Figs 4D, 4E, and 5).

Conclusion
In conclusion, sarcolemmal K_V channels in PASMCs are active under resting conditions and are responsible for maintaining resting E_m^{4 16 20 21 22 23 25 32} and thereby helping to regulate Ca²⁺ conductance and [Ca²⁺]_i.^{1 2 33} Accordingly, K_V channel activity plays a fundamental role in regulating PA tone under physiological conditions. Nevertheless, K_Ca and K_ATP channels in PASMC membranes respond to changes of intracellular Ca²⁺ and ATP, respectively.² Consequently, K_Ca channel activity appears to play an important role in controlling E_m and, subsequently, [Ca²⁺]_i^{11 26} by controlling repolarization as a negative-feedback regulator in PASMCs, whereas K_ATP channel activity regulates E_m and [Ca²⁺]_i in response to alteration of the cellular metabolic state.² Furthermore, K_Ca and K_ATP channels may also be targets of various exogenous and endogenous vasoactive substances. Development and utilization of pharmacological activators of K⁺ (K_V, K_Ca, and K_ATP) channels, especially K_V channels, in PASMCs may greatly benefit the treatment of pulmonary hypertension, which is a major contributor to many cardiopulmonary diseases.

	Selected Abbreviations and Acronyms

 

4-AP = 4-aminopyridine ChTX = charybdotoxin DMSO = dimethyl sulfoxide Em = membrane potential FBSCM = fetal bovine serum culture medium I-V = current-voltage ICa = inward Ca2+ current ICl(Ca) = Ca2+-dependent Cl- current IK = whole-cell K+ current IK(ATP) = ATP-sensitive K+ current IK(Ca) = Ca2+-activated K+ current IK(I) = inwardly rectifying K+ current IK(V) = voltage-gated K+ current KATP channel = ATP-sensitive K+ channel KCa channel = Ca2+-activated K+ channel Kir channel = inwardly rectifying K+ channel KV channel = voltage-gated K+ channel PA = pulmonary artery PASMCs = PA smooth muscle cells PSS = physiological salt solution tAP = time course of the action potential tCa = time course of the increase in [Ca2+]i tmd = time course of 4-AP–induced membrane depolarization

	Acknowledgments

 
This study was supported by a Grant-in-Aid from the American Heart Association, Maryland Affiliate, Inc; a Special Research Initiative Support grant from the University of Maryland School of Medicine; and National Institutes of Health grant HL-54043. Dr Yuan is a Parker B. Francis Fellow in Pulmonary Research and a recipient of the Giles F. Filley Memorial Award from the American Physiological Society. Dr Yuan thanks A. Aldinger, R.T. Bright, and E.M. Santiago for their technical assistance and is also grateful to Drs M.P. Blaustein, M.L. Tod, L.J. Rubin, and M.T. Nelson for their review of the manuscript.

Received November 17, 1994; accepted April 25, 1995.

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				S. Bonnet, E. Dumas-de-La-Roque, H. Begueret, R. Marthan, M. Fayon, P. Dos Santos, J.-P. Savineau, and E.-E. Baulieu Dehydroepiandrosterone (DHEA) prevents and reverses chronic hypoxic pulmonary hypertension PNAS, August 5, 2003; 100(16): 9488 - 9493. [Abstract] [Full Text] [PDF]

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				Y. Yu, M. Sweeney, S. Zhang, O. Platoshyn, J. Landsberg, A. Rothman, and J. X.-J. Yuan PDGF stimulates pulmonary vascular smooth muscle cell proliferation by upregulating TRPC6 expression Am J Physiol Cell Physiol, February 1, 2003; 284(2): C316 - C330. [Abstract] [Full Text] [PDF]

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