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Am J Physiol Heart Circ Physiol 277: H1732-H1744, 1999;
0363-6135/99 $5.00
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Vol. 277, Issue 5, H1732-H1744, November 1999

Ca2+-dependent Clminus channels in mouse and rabbit aortic smooth muscle cells: regulation by intracellular Ca2+ and NO

Yoji Hirakawa, Marion Gericke, Richard A. Cohen, and Victoria M. Bolotina

Vascular Biology Unit, Whitaker Cardiovascular Institute, Boston Medical Center, Boston, Massachusetts 02118


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ca2+-dependent Cl- (Cl-Ca) channels and their regulation by intracellular Ca2+ concentration ([Ca2+]i) and nitric oxide (NO) were characterized in mouse and rabbit aortic smooth muscle cells (SMC) using patch clamp and fura 2 imaging. Single channels (1.8 pS) and whole cell Cl-Ca currents were activated by caffeine-induced Ca2+ release. Single Cl-Ca channels were also activated by >= 200 nM Ca2+ in inside-out membrane patches and remained active for >5 min in <= 1 µM Ca2+ but showed rapid rundown in 2 mM Ca2+. Authentic NO or S-nitroso-N-acetylpenicillamine (SNAP) did not affect their activation or rundown in inside-out patches. In the whole cell, SNAP (100 µM) and 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (50 µM) did not affect Cl-Ca current, but at a higher concentration SNAP (1 mM) induced a sustained [Ca2+]i rise, accompanied by a dramatic decrease in caffeine-induced Ca2+ release and Cl-Ca current. These results indicate that 1) mouse and rabbit aortic SMC possess 1.8-pS Cl-Ca channels that are activated by Ca2+ release from the stores, 2) both activation and rundown of single Cl-Ca channels depend on [Ca2+]i, and 3) NO does not affect Cl-Ca channels directly or via cGMP but can inhibit their activation indirectly by decreasing Ca2+ release from the stores.

single chloride channel; rundown; noise analysis; caffeine-induced calcium release; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CALCIUM (Ca2+)-dependent chloride (Cl-Ca) currents can be activated by Ca2+ sparks (39) and by global Ca2+ release from intracellular stores triggered by a variety of contractile agonists in vascular smooth muscle cells (SMC) (for review see Refs. 5, 22). Under physiological conditions, activation of Cl-Ca channels produces inward current and membrane depolarization that can activate L-type Ca2+ channels, Ca2+ influx, and contraction of vascular SMC (2, 20, 26). Inactivation or inhibition of Cl-Ca current could cause membrane repolarization and inhibition of Ca2+ influx, and this mechanism has been proposed to mediate endothelium-dependent hyperpolarization and relaxation of SMC (9, 24, 32). However, it is still unclear whether nitric oxide (NO), the main endothelium-derived hyperpolarizing and relaxing factor (8, 30), can affect Cl-Ca channels.

Aortic SMC are widely used in studies of the mechanisms of endothelium-dependent vascular relaxation, but Cl-Ca channels have not been studied in freshly dispersed aortic SMC. Thus, before addressing the questions about the effects of NO on Cl-Ca channels, we first characterized single Cl-Ca channels and whole cell Cl-Ca currents in a novel preparation of SMC from mouse aorta. To the best of our knowledge, this is the first study of ion channels performed in freshly dispersed mouse aortic SMC. This preparation is a valuable tool for electrophysiological studies of smooth muscle in a growing variety of genetically modified mouse models. Because NO-induced relaxation has been extensively studied in rabbit aorta, we also characterized Cl-Ca channels and their regulation by Ca2+ release from intracellular stores in SMC freshly dispersed from rabbit aorta.

Activation of whole cell Cl-Ca current in a variety of SMC has been shown to strictly depend on intracellular Ca2+ concentration ([Ca2+]i) rise caused by contractile agonists (for review, see Refs. 5, 22). The mechanism of inactivation of the whole cell Cl-Ca current or rundown of single Cl-Ca channels is not that clear. Ca2+ regulation of Cl-Ca channels has been studied mostly at the level of whole cell Cl-Ca currents because of extremely small conductance and rapid rundown of single Cl-Ca channels in excised membrane patches. Some authors suggested that Cl-Ca current strictly follows [Ca2+]i (25), whereas others observed that Cl-Ca current decreased faster than [Ca2+]i after the release of Ca2+ from the stores (34). It was proposed that Ca2+/calmodulin-dependent protein kinase can induce inactivation of Cl-Ca currents in equine tracheal SMC by uncoupling channel activity from [Ca2+]i (34). However, it is unclear whether this process could be responsible for rundown or inactivation of single Cl-Ca channels. In the present study, we provide a detailed description of activation and rundown of single Cl-Ca channels in excised membrane patches. We believe that our finding of the dependence of the rundown of single Cl-Ca channels on intracellular Ca2+ will open new possibilities for their prolonged recording in excised membrane patches, which was a nonresolvable problem for many years.

Many different targets for NO have been found in SMC, but it is unknown whether single Cl-Ca channels can be affected by NO directly, similar to Ca2+-dependent K+ (K+Ca) channels (1, 4, 28). Also, one could propose the existence of some indirect regulation of Cl-Ca channels by NO through its effects on Ca2+ stores, because Ca2+ released from the stores activates Cl-Ca channels. NO has been shown to affect ryanodine receptors in caffeine-sensitive stores of cardiac and skeletal myocytes (23, 29, 35, 36), although it is unclear how this might affect Cl-Ca channels. In the present study, we address these questions and show that NO does not inhibit single Cl-Ca channels in excised membrane patches but an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), can suppress caffeine-induced activation of Cl-Ca currents by decreasing Ca2+ release from the stores. Preliminary results have been published in abstract form (14).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparations

Mouse aortic SMC. Freshly isolated mouse aortic SMC (mSMC) were obtained by a modified method using papain digestion (6) as follows. C57BL/6 mice (15-20 g) were anesthetized by inhalation of diethyl ether and killed by cervical dislocation. The thoracic aorta was rapidly removed, cleaned of connective tissues, cut into small pieces, and rinsed in dissociation medium (DM) containing (in mM) 110 NaCl, 5 KCl, 10 NaHCO3, 0.5 NaH2PO4, 0.5 KH2PO4, 2 MgCl2, 10 taurine, 10 HEPES, and 11 glucose and 0.02% bovine serum albumin (pH 7.2). Pieces of aorta were incubated in DM with papain (40 U/ml; Fluka, Buchs, Switzerland) and dithiothreitol (DTT, 2 mM; Sigma, St. Louis, MO) for 15 min at 37°C with constant stirring with a small magnetic stirrer (Fisher, Pittsburgh, PA). After incubation, they were rinsed twice in fresh DM and then gently triturated with a heat-polished Pasteur pipette. Isolated cells were stored at 4°C until use for up to 4 h. A small drop of the cell suspension was placed in a 35-mm polystyrene tissue culture dish (Corning) or a 0.15-mm glass-bottom chamber (Bioptechs, Butler, PA), and SMC were allowed to adhere to the bottom before they were washed with normal bath solution.

Rabbit aortic SMC. Male New Zealand White rabbits (2-2.5 kg) were exsanguinated after injection of pentobarbital sodium (30 mg/kg) and heparin (150 U/kg). A segment of thoracic aorta was rapidly removed, cleaned of connective tissues, cut into small pieces, and rinsed in DM. Pieces of aorta were incubated in DM with papain (50 U/ml) and DTT (2 mM) for 30 min at 37°C in a shaking water bath. After incubation, they were rinsed twice in fresh DM and then gently triturated with a heat-polished Pasteur pipette. Isolated cells were stored at 4°C until use for up to 4 h.

Electrophysiological Studies

Single-channel recording. Single-channel currents were recorded using the standard patch-clamp technique (12) in cell-attached or inside-out membrane patches. Pipettes were pulled from borosilicate glass capillaries with a filament (1B150F; WPI, Sarasota, FL) on a horizontal puller (model P-87; Setter Instrument, Novato, CA) and heat polished with a microforge (model MF-9; Narishige, Tokyo, Japan). To improve signal-to-noise ratio during single-channel recording, pipettes with tip resistance of 20-25 MOmega were coated with Sylgard (Dow Corning, Midland, MI). The currents were recorded with a low-noise patch-clamp amplifier (Axopatch 200B; Axon Instruments, Foster City, CA). Data were filtered at 1 kHz and stored on digital audio tapes (DT-120RA; SONY, Tokyo, Japan) using a digital tape recorder (DTR-1203; Bio-Logic, Claix, France) for later analysis. Representative current traces were additionally filtered at 200-500 Hz for better visual resolution on the figures. pClamp6 software (Axon Instruments) was used for data acquisition and analysis. Probability of the channels being open (NPo, where N = no. of channels in the patch) was analyzed and plotted over time to illustrate the time course of channel activity in representative experiments. The original traces of single-channel currents are shown at different times of the experiment as defined in Figs. 1, 2, 4, and 11.

Whole cell recording. Whole cell currents were recorded using an amphotericin B-perforated patch-clamp technique unless otherwise indicated. Amphotericin B (300 µg/ml) was included in the pipette solution, sonicated for 2-3 min, and then kept in the dark and used within 2 h. Heat-polished glass pipettes (1B150; WPI) with tip resistance of 1-3 MOmega were used. The very tip of the pipette was filled with amphotericin B-free pipette solution, and then the pipette was backfilled with amphotericin B-containing solution. Electrical connection with series resistance of 20-30 MOmega was achieved within 5 min after making gigaohm seal contact. With this technique, whole cell currents could be recorded without changing cytosolic Ca2+ concentration because the pores formed by amphotericin B are not permeable for Ca2+ and intracellular messenger molecules (21). The currents were recorded with Axopatch 200B, filtered at 1-2 kHz, and stored on a computer hard disk for later analysis using pClamp6 software. The cell capacitance and the series resistance were compensated. Leakage currents were not subtracted in any current traces. Whole cell currents were evoked by ramp depolarizations (from -100 to +50 mV for 150 or 750 ms, every 1 or 2 s) from a holding potential of -30 or -60 mV (as specified in the legends to Figs. 5, 7, 8, and 10). All experiments were performed at room temperature (20-21°C).

Noise Analysis of Whole Cell Current

Nonstationary noise analysis was done using standard methods (13) as described earlier (37). Whole cell Cl-Ca current was recorded at constant holding potentials (-30, -60, and/or -100 mV) during application of caffeine (n = 8). Data were filtered at 2 kHz and sampled at 10 kHz. Mean current (I) and current variance (sigma 2) were determined during 25- to 50-ms intervals. During the sampling intervals, I changes by <10%. Analysis of the dependence of sigma 2 on I was performed using Microcal Origin (Microcal Software, Northampton, MA). The single-channel current (i) was estimated for each experiment by fitting the equation sigma 2(I) = Ii - I2/N + B, where sigma 2(I) is the variance, N is the number of channels, and B is a free parameter reflecting the background noise. Single-channel conductance was estimated from the slope of the single-channel current-voltage relationship.

Intracellular Ca2+ Measurement

Freshly dispersed individual SMC were placed in a chamber with a 0.15-mm glass bottom (Bioptechs) and incubated with fura 2-AM (5 µM) for 30 min at room temperature. After being washed with regular bath solution (in a shaking water bath at 37°C for 15 min), the cells were transferred to the stage of an inverted microscope (Olympus IX70, Tokyo, Japan) equipped with a ×40 fluorescence objective (Olympus Uapo/340, NA 0.9).

Fluorescence was measured at room temperature (20-21°C) using a dual-excitation fluorescence imaging system from IonOptics (Milton, MA). Cells were illuminated at 340 and 380 nm, and the emitted light was collected at 510 nm by an intensified charge-coupled device camera. [Ca2+]i was calculated from the measured ratio of 340-nm to 380-nm signals (R) using the formula (10) [Ca2+]i = Kd · beta  · (R - Rmin)/(Rmax - R), where Rmin, Rmax, and beta  were determined from in vitro calibration and a dissociation constant (Kd) of 145 nM for fura 2 was used. After each experiment, cells were permeabilized with ionomycin (5 µM) and fura 2 was quenched with Mn2+ (10-20 mM). The resulting image was used as a background image and was subtracted from the experimental traces. IonWizard software (IonOptics) was used for data acquisition and analysis. For simultaneous measurement of fluorescence and whole cell currents, IonWizard was synchronized with pClamp6.

Solutions

The normal bath solution contained (in mM) 130 NaCl, 10 tetraethylammonium chloride (TEA), 2 CaCl2, 2.8 KCl, 2 MgCl2, 10 HEPES, and 5.5 glucose. The pH was adjusted to 7.4 with NaOH. In some experiments, Na+ or Cl- was replaced by equimolar N-methyl-D-glucamine (NMDG) or glutamate, respectively. For Ca2+-free solution, Ca2+ was not added and the solution contained 2 mM EGTA to buffer the residual Ca2+. For low-Ca2+ (100, 200, and 400 nM and 1 µM) solutions, 1 mM EGTA was included together with 390, 550, 720 and 870 µM Ca2+, respectively. The normal pipette solution for perforated patch recordings contained (in mM) 100 cesium aspartate, 40 CsCl, 3 MgCl2, 0.1 EGTA, and 10 HEPES. The pH was adjusted to 7.2 with CsOH. For conventional whole cell recordings, MgCl2 was replaced by equimolar MgATP. For single-channel recordings, the normal pipette solution contained (in mM) 140 NaCl, 2 CaCl2, and 10 HEPES (pH 7.2). As indicated in some experiments, cation currents were eliminated by substituting all membrane-permeant cations with impermeant NMDG, and the pipette solution contained (in mM) 140 NMDG and 10 HEPES (pH 7.2 with HCl).

Preparation of NO Solution

A standard 1-liter intravenous solution bag was filled with distilled water (750 ml) that had been bubbled with nitrogen gas to remove oxygen. Approximately 30 meq of Bio-Rad analytical grade anion exchange resin were mixed in the water before the bag was filled. The resin retains any nitrites or nitrates that may be formed by NO reacting with oxygen. The contents of the bag were bubbled with nitrogen gas for another 30 min. Any dead space nitrogen gas was expelled from the bag before it was filled to capacity with NO gas. The contents were then mixed thoroughly, and the bag was placed in a refrigerator at 4°C. The concentration of NO in the solution equilibrated to give a 3.1 ± 0.6 mM (n = 5) saturated solution at least for 1 wk as measured by a chemiluminescent NO analyzer (Sievers NOA model 270, Boulder, CO). At the time of use, subsequent dilutions were made from this stock by simply drawing off the solution from the bag with a syringe. Serial dilutions were prepared in 100 × 16-mm Vacutainer blood collection tubes filled with 9 ml of bath solution that was deoxygenated by bubbling for 1 h with nitrogen gas on ice.

Drugs

Caffeine, ionomycin, niflumic acid, amphotericin B, DTT, NMDG, TEA, SNAP, 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT-cGMP), MgATP, R-24571, calyculin A, and bovine serum albumin (fatty acid, endotoxin free) were purchased from Sigma. Acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) and fura 2-AM were from Molecular Probes (Eugene, OR). Papain was from Fluka. SNAP, papain, DTT, bovine albumin, and amphotericin B were prepared from powder at the time of each experiment. Other drugs were prepared as a stock solution and were diluted to the final concentration with the bath solution at the time of the experiment.

Statistics

Data are shown as means ± SE, with n indicating the number of experiments. Statistical significance between groups was assessed using Student's paired or unpaired t-test. Values of P < 0.05 were considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Single Cl-Ca Channels in Mouse and Rat Aortic SMC

General characteristics. Single Cl--channel currents were recorded and compared in aortic SMC freshly isolated from mouse (mSMC) and rabbit (rSMC). Figure 1 shows a typical recording of inward single-channel currents and analysis of NPo in cell-attached and inside-out configurations of the same membrane patch from mSMC. There were virtually no channel openings in resting mSMC (Fig. 1A), but caffeine (10 mM) applied to the cell activated small inward currents (Fig. 1B; n = 6). The channel activation was always transient and lasted <= 30 s even in the continuous presence of caffeine. Excising an inside-out membrane patch into 2 mM Ca2+-containing bath solution activated the same channel (Fig. 1C; n = 7). In rSMC we found similar channels that were activated either by caffeine in cell-attached membrane patches (Fig. 2A; n = 11) or by excising inside-out membrane patch into 2 mM Ca2+ (Fig. 2B; n = 19). In both preparations of mSMC and rSMC, the single-channel currents (Fig. 3, A and B) in inside-out membrane patches under symmetrical Cl- conditions had a linear current-voltage (I-V) relationship with a reversal potential of 0 mV and an identical single-channel conductance of 1.8 ± 0.2 pS.


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  		Fig. 1.   Single Ca2+-dependent Cl- (Cl-Ca)-channel currents in mouse aortic smooth muscle cells (mSMC). Representative experiment with single-channel currents recorded in cell-attached membrane patch in control, after application of caffeine (10 mM), and after inside-out patch was excised into 2 mM Ca2+. Probability of channels being open (NPo) is shown during time course of experiment (top). Left traces at bottom are original traces of single-channel currents during different periods of experiment shown at top: control (A), during application of caffeine (B), after inside-out membrane patch was excised (C). Right traces show higher time resolution of single-channel currents at corresponding time points labeled with asterisks on left traces. Single-channel currents are recorded at -100 mV applied to membrane patch. Inward currents are shown as downward deflections; zero current level is shown by arrows. Pipette contains only N-methyl-D-glucamine (NMDG, 140 mM) and HEPES (10 mM), and membrane patch is excised into regular bath solution containing 2 mM Ca2+.



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  		Fig. 2.   Single Cl-Ca-channel currents in rabbit aortic SMC (rSMC). Typical traces of single-channel currents recorded at -100 mV applied to membrane. Membrane patch configuration and main components of pipette and bath solutions are shown in insets. Inward currents are shown as downward deflections; zero current is shown by arrow at end of each trace. A: activation of single-channel currents in cell-attached membrane patch during application of caffeine (10 mM) to whole cell. B: single-channel currents before and after inside-out membrane patch was excised into 2 mM Ca2+-containing bath solution. Moment of excising patch is shown by vertical bar interrupting time course of recording for ~2 s to eliminate mechanical artifacts. Bottom inset shows distinct levels of single-channel currents at higher time resolution. Tetraethylammonium chloride (TEA, 10 mM) and Ni2+ (5 mM) are present in pipette. C: single-channel currents recorded before and after an inside-out membrane patch was excised in 2 mM Ca2+. Inward currents recorded with NMDG (140 mM) in pipette disappear after reduction of Cl- concentration in bath solution from 149 to 9 mM (at time indicated). D: single-channel current recording before and after inside-out membrane patch was excised into Ca2+ (2 mM)-containing bath solution. Niflumic acid (Niflu A, 100 µM) and TEA (10 mM) were present in pipette solution.



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  		Fig. 3.   Current-voltage (I-V) relationship of single Cl-Ca-channel current in mSMC (A) and rSMC (B) in inside-out membrane patches obtained from mSMC and rSMC under symmetrical Cl- conditions. Each point represents mean ± SE from 3-29 different experiments (no. of experiments shown for each point). Single-channel conductance gamma  is calculated from slope of I-V relationship.

Substitution of all permeable cations with impermeable NMDG in the pipette did not affect the amplitude or appearance of inward single-channel currents in mSMC (Fig. 1; n = 63) or rSMC (Fig. 2C; n = 106). Inward currents disappeared when the concentration of Cl- in the bath solution was reduced from 149 to 9 mM by substitution with glutamate (Fig. 2C; n = 3). Ni2+ (5 mM; n = 10) or TEA (10 mM; n = 9) did not affect single-channel currents (Fig. 2B), but the currents were absent when niflumic acid (100 µM), a Cl--channel inhibitor, was included in the pipette solution (Fig. 2D; n = 9). These data are consistent with the presence of single Cl- channels of 1.8-pS conductance in aortic SMC from mouse and rabbit.

Ca2+-dependent activation. Ca2+ dependence of single Cl- channels was studied in excised inside-out membrane patches by exposing the intracellular surface of the membrane to different concentrations of Ca2+. Channel activation did not occur when patches were excised from rSMC in Ca2+-free (n = 41) or 100 nM Ca2+-containing (n = 29) bath solution (Fig. 4A). The channels could be activated only when the patch was excised in [Ca2+]i >= 200 nM (Fig. 4, B-D; n = 53 for mSMC, and n = 97 for rSMC). The initial activity of the channels (immediately after the patch was excised) in 200 nM [Ca2+]i was lower than in >= 400 nM [Ca2+]i (Fig. 4, B-D).


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  		Fig. 4.   Ca2+-dependent activation and rundown of single Cl-Ca channels in inside-out membrane patches. NPo and original traces of single-channel currents during different periods of experiment are shown before and after inside-out membrane patches from rSMC were excised into bath with different concentrations of Ca2+ [100 and 400 nM (A), 200 and 400 nM (B), 1 µM (C), and 2 mM (D)]. Pipette solution contained 140 mM NMDG. Inward currents (shown as downward deflections) were recorded at -100 mV applied to membrane; zero current level is shown by a dotted line.

Ca2+-dependent rundown. After inside-out membrane patches were excised in 2 mM Ca2+, activation of single Cl-Ca channels was followed by rapid rundown, which was dependent on the Ca2+ concentration at the intracellular surface of the membrane patches. In 2 mM Ca2+, the channel activity completely disappeared within 1-2 min after the patches were excised [Fig. 4D; 72 ± 14 s (n = 12) for rSMC and 63 ± 12 s (n = 4) for mSMC]. Rundown was significantly slower at lower [Ca2+]i. Indeed, as shown in Fig. 4C, the channel could be active for >10 min when patches were excised in 1 µM Ca2+ and the maximum time of the single-channel recording was primarily determined by the lifetime of the membrane patches and varied from 5 to 11 min (n = 14). Interestingly, when the patches were excised from rSMC and exposed to Ca2+-free solution (with 2 mM EGTA added) for 3 min, subsequent application of Ca2+ (1 µM) failed to activate Cl-Ca channels (n = 4), indicating that along with Ca2+-dependent rundown, there is also some Ca2+-independent rundown of single Cl-Ca channels.

Excising the membrane patch from rSMC in 2 mM Ca2+ in the presence of ATP (3 mM) did not affect activation of Cl-Ca channels and did not change the time required for their rundown [71 ± 14 s (n = 9) compared with control 72 ± 14 s (n = 12); P > 0.1]. Similar activation and rundown of Cl-Ca channels were also observed in the combined presence of ATP and the calmodulin inhibitor R-24571 (3 µM). The time required for rundown under these conditions was 58 ± 20 s (n = 4, P > 0.1). A high concentration of the protein phosphatase 1 and 2A inhibitor calyculin A (100 µM) in the bath neither prevented nor accelerated the rundown of Cl-Ca channels. When patches were excised into 1-2 µM Ca2+ in the presence of calyculin, the channels remained active for >5 min (n = 7), which was similar to the duration observed under control conditions.

Whole Cell Ca2+-Dependent Cl- Current

General characteristics. There was no difference in the basal characteristics and the regulation of Cl-Ca currents between mSMC and rSMC, although the cell capacitance of fully relaxed mSMC (10.5 ± 2.1 pF; n = 74) was about one-half that of rSMC (20.3 ± 3.8 pF; n = 95). Under conditions in which K+ channels were inhibited by Cs+ (140 mM) in the pipette, bath application of caffeine (10 mM) transiently activated a whole cell current in both mSMC (n = 13) and rSMC (n = 63). Figure 5A shows a typical whole cell current in rSMC recorded at -30 mV with voltage ramps applied every 1 s. The caffeine-induced current showed some outward rectification and had a reversal potential of -22.4 ± 2.3 mV (n = 29), which is close to the calculated equilibrium potential for Cl- under our experimental conditions (ECl = -30 mV). When Cl- concentration of the bath solution was reduced from 149 to 49 mM by equimolar substitution with glutamate, the reversal potential of the caffeine-induced current shifted in the positive direction by 18.7 ± 5.1 mV (n = 3), consistent with Cl- selectivity of the current. The caffeine-induced current was insensitive to TEA (10 mM, Fig. 5, B and D; n = 35), Ni2+ (5 mM, Fig. 5, B and D; n = 9), and nifedipine (5 µM, Figs. 5D and 8C; n = 6) but was completely inhibited by niflumic acid (100 µM, Fig. 5, C and D; n = 8). On the basis of all these data, the caffeine-induced whole cell current was identified as Cl- current. Importantly, bath application of niflumic acid (100 µM, n = 5) did not affect the basal "leakage" current (when TEA was present in the bath and Cs+ was in the pipette solution to prevent K+ currents), indicating the absence of detectable whole cell Cl- current under basal conditions. There was no difference in Cl- current density between mSMC and rSMC. At -60 mV, inward current densities were -10.2 ± 0.9 (n = 12) and -10.1 ± 0.9 (n = 18) pA/pF, respectively.


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  		Fig. 5.   Main characteristics of whole cell Cl-Ca current induced by caffeine. A-C: changes in whole cell current are shown during application of caffeine (10 mM) to rSMC. Ramp depolarizations (from -100 to +50 mV, 750 ms) are applied every 1 s. Holding potential is -30 mV. I-V relationships are shown on right during corresponding ramps before and during caffeine application. Ni2+ (5 mM) and TEA (10 mM) are present in bath solution in B, niflumic acid (100 µM) and TEA (10 mM) are present in C. D: summary data showing density of peak Cl-Ca current activated by caffeine in control and in presence of TEA (10 mM), Ni2+ (5 mM) + TEA (10 mM), nifedipine (5 µM) + TEA (10 mM), and niflumic acid (niflu. a.; 100 µM) + TEA (10 mM). Inward currents at -80 mV are shown by downward bars, and outward currents at +50 mV are shown by upward bars. Numbers show how many experiments are summarized for each treatment. * P < 0.05 were considered to be significant.

Noise analysis. To characterize the single channels that underlie the whole cell current activated by caffeine, we performed noise analysis of the whole cell current. Figure 6A shows an example of the whole cell current recorded in rSMC at -60 mV during application of caffeine. Figure 6B shows an example of the distribution of current variance during the rising phase of the current at -60 mV fitted with the equation sigma 2(I) = iI - I2/N + B, where I is a mean whole cell current and i is an estimated single-channel current. Figure 6C summarizes the dependence of estimated single-channel current on membrane potential in different rSMC (n = 8). Single-channel conductance (obtained from the slope of the I-V plot) was estimated to be gamma  = 2.0 pS, which was similar to the conductance of the single Cl-Ca channels recorded in inside-out membrane patches (Fig. 3, A and B). The reversal potential of the estimated single-channel current was around -25 mV, which is very close to the reversal potential of the whole cell Cl-Ca currents. These data are consistent with single Cl-Ca channels of ~2 pS being responsible for caffeine-induced whole cell currents in rSMC.


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  		Fig. 6.   Noise analysis of whole cell Cl-Ca current activated by caffeine. A: an example of whole cell Cl-Ca current recorded at -60 mV during application of caffeine (10 mM) in rSMC. B: representative plot of current variance (sigma 2) vs. mean current at -60 mV in same rSMC. Each point represents current variance during sequential 25-ms intervals recorded during rising phase of caffeine-induced Cl-Ca current. C: I-V relationship of estimated single Cl-Ca-channel current obtained from a polynomial fit on B. Each point represents mean ± SE from 3-8 different experiments (shown by no. under each point). Single-channel gamma  is calculated from slope of I-V relationship.

Regulation of Cl-Ca currents by Ca2+ release from intracellular stores. Simultaneous recording of [Ca2+]i and whole cell currents in rSMC and mSMC showed that Cl- current is transiently activated simultaneously with caffeine-induced [Ca2+]i rise (Fig. 7A). Figure 7B shows the time course of the current (measured at -30 mV) and [Ca2+]i (from Fig. 7A), both normalized to their peak amplitude and plotted on the same graph. The rise in [Ca2+]i and activation of Cl- current peaked in ~1-2 s, but the decline of Cl-Ca current was significantly faster than that of [Ca2+]i. The declining phase of Cl-Ca current in mSMC had a duration of tau 1/2 = 2.6 ± 0.8 s compared with 5.9 ± 2.7 s for that of [Ca2+]i (n = 13, P < 0.001). Bath application of the Ca2+ ionophore ionomycin (1-10 µM) activated the same Cl- current as that activated by caffeine in mSMC (n = 3) and rSMC (n = 5). When the current had already been activated by ionomycin, caffeine caused no additional effect (n = 3). The same Cl- current was also activated by dialysis of mSMC (n = 3) and rSMC (n = 2) with 1-2 µM Ca2+ applied through the pipette in regular whole cell configuration.


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  		Fig. 7.   Simultaneous recording of [Ca2+]i and whole cell Cl-Ca current. A: typical result of simultaneous recording of intracellular Ca2+ concentration ([Ca2+]i, top trace) and whole cell Cl-Ca current (bottom trace) during application of caffeine (10 mM). Whole cell current was recorded at a holding potential of -30 mV with ramp depolarizations (-100 mV to +60 mV for 150 ms) applied every 1 s. B: simultaneous [Ca2+]i and current transients (from traces in A) normalized to their maximal amplitude.

Caffeine applied to the cells with intervals >= 5 min repeatedly induced a transient [Ca2+]i rise (Fig. 8) and activation of Cl-Ca current in the presence of extracellular Ca2+ (Fig. 8A and Fig. 9). In the absence of extracellular Ca2+, caffeine was still able to increase [Ca2+]i and activate Cl-Ca current, but its effect gradually disappeared after a few applications (Fig. 8B). Activation of Cl- current by caffeine was abolished when the rise in [Ca2+]i was prevented by pretreatment of rSMC (n = 25) with a membrane-permeant Ca2+ chelator, BAPTA-AM (20 µM for 20 min, Fig. 8D).


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  		Fig. 8.   Repetitive activation of whole cell Cl-Ca currents. Typical recordings of whole cell Cl-Ca currents in rSMC during repetitive application of caffeine (10 mM) in control (A), in absence of extracellular Ca2+ (B), in presence of 5 µM nifedipine (C), and in rSMC pretreated with BAPTA-AM (20 µM) for 20 min (D). Current was recorded at holding potential -30 mV with ramp depolarizations (from -100 to +50 mV) applied every 1 or 2 s.



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  		Fig. 9.   Effect of SNAP and 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (CPT-cGMP) on caffeine-induced whole cell Cl-Ca current and peak [Ca2+]i rise. Percent change in amplitude of peak Cl-Ca current (measured at -60 mV) is shown in mSMC during repeated applications (with 4- to 5-min intervals) of caffeine (10 mM) in untreated cells (open bars) and in cells treated (solid bars) with SNAP (100 µM, A), CPT-cGMP (50 µM, B), and SNAP (1 mM, C) for 1, 5, and 10 min. In each experiment, percent change in caffeine-induced Cl-Ca current was estimated by normalizing currents activated by consecutive applications of caffeine to initial current, which is shown as 100%. In similar experiments, changes in peak [Ca2+]i rise after consecutive applications of caffeine were determined before and during mSMC treatment with SNAP (hatched bars in A and C). Nos. above bars show nos. of experiments summarized.

Although caffeine-induced activation of Cl-Ca currents did not require influx of extracellular Ca2+ (as seen in Fig. 8B), the activity of L-type Ca2+ channels appeared to be important for repeated activation of Cl-Ca currents. Indeed, caffeine-induced Cl-Ca current gradually disappeared when nifedipine (5 µM) was present in Ca2+-containing bath solution (Fig. 8C; n = 5), similar to what occurred in Ca2+-free solution. Washout of nifedipine resulted in partial recovery of caffeine-induced current. Treatment of rSMC with BAY K 8644 (1 µM) for 2-3 min before the next application of caffeine increased the amplitude of caffeine-induced Cl-Ca current (n = 3; not shown). These results indicate that Cl-Ca currents in aortic SMC are directly activated by Ca2+ release from intracellular stores and may be indirectly regulated by the state of filling of the stores.

Effect of NO on Caffeine-Induced Ca2+ Release and Ca2+-Activated Cl- Channels

Inhibition of whole cell Cl-Ca currents could provide a mechanism of NO-induced hyperpolarization and relaxation of vascular SMC. To determine whether NO inhibits Cl-Ca currents, we applied it directly to intact cells and excised membrane patches.

Treatment of intact mSMC with the NO donor SNAP (100 µM) or with a membrane-permeant analog of cGMP, CPT-cGMP (50 µM), did not affect whole cell Cl-Ca current. Figure 9A summarizes the data showing that there is no significant difference in the peak Cl-Ca current amplitude (at -60 mV) evoked by three consecutive applications of caffeine (with 4- to 5-min intervals to allow refilling of caffeine-sensitive stores) in the absence and presence of 100 µM SNAP. Figure 9B summarizes the changes in Cl-Ca currents in similar experiments in the absence and presence of CPT-cGMP.

Interestingly, in similar experiments, a higher concentration of SNAP (1 mM) did not significantly affect Cl-Ca current within 1 min after its application but significantly inhibited caffeine-induced Cl-Ca current after 5 min of treatment (Fig. 9C). After 5-min pretreatment of rSMC with 1 mM SNAP, caffeine also failed to activate single Cl-Ca channels in cell-attached patches (n = 5; not shown), although subsequent excision of inside-out membrane patches into Ca2+-containing solution produced normal activation of Cl-Ca channels despite the continued presence of SNAP (n = 11).

To test the possible role of intracellular Ca2+ in mediating the effects of a high concentration of SNAP on Cl-Ca channels, the effect of SNAP on caffeine-induced Ca2+ release was recorded separately or simultaneously with Cl-Ca currents. SNAP (100 µM) did not significantly affect the caffeine-induced transient rise in [Ca2+]i even when applied for 5 min (Fig. 9A). In contrast, 1 mM SNAP caused a slowly developing, sustained rise in basal [Ca2+]i and progressive disappearance of the caffeine-induced Ca2+ release (Fig. 10, top). This effect was accompanied by disappearance of caffeine-induced Cl-Ca currents measured simultaneously with [Ca2+]i (Fig. 10). Importantly, SNAP did not affect the basal whole cell current, and the SNAP-induced sustained [Ca2+]i rise did not activate Cl-Ca current in the absence of caffeine. The correlation between the time-dependent effects of SNAP (1 mM) on caffeine-induced Ca2+ release and Cl-Ca current (Figs. 9C and 10) points to the possibility that downregulation of Cl-Ca currents occurs secondary to the effect of SNAP on Ca2+ release from intracellular stores. It is important to point out that there was some difference in the degree of inhibition of Cl-Ca current and Ca2+ release (Figs. 9 and 10), which could be caused either by additional direct effect of NO on Cl-Ca channels or by the partial Ca2+-dependent inactivation of Cl-Ca channels (34) as a result of the major increase in basal [Ca2+]i observed in the presence of 1 mM SNAP.


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  		Fig. 10.   Effect of SNAP on [Ca2+]i and whole cell Cl-Ca current. A representative experiment with simultaneous recording of [Ca2+]i and whole cell Cl-Ca current in mSMC during repeated application of caffeine (10 mM) under control conditions and after addition of SNAP (1 mM). Top: changes in [Ca2+]i over course of whole experiment. Bottom: simultaneous recording of [Ca2+]i and Cl-Ca current is shown during corresponding periods. Current was recorded at -30 mV with ramp depolarizations (from -100 to +60 mV, 150 ms) applied every 1 s.

To determine whether NO can inhibit single Cl-Ca channels directly, we excised inside-out membrane patches from mSMC in the absence or presence of authentic NO (1 µM). Figure 11 shows that NO did not prevent normal activation of single Cl-Ca channels when inside-out membrane patches were excised into 400 nM Ca2+ (n = 12). Excising the membrane patch in the presence of SNAP (1 mM) also did not affect activation of Cl-Ca channels (n = 5). NO also did not affect the time required for the rundown of Cl-Ca channels. In 1 µM Ca2+, complete rundown occurred after 393 ± 74 s (n = 9) in the presence and after 405 ± 66 s (n = 6) in the absence of NO (P > 0.5). These results indicate that there is no direct effect of NO on activation or rundown of single Cl-Ca channels.


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  		Fig. 11.   NO does not affect single Cl-Ca channels in excised membrane patches. Representative experiments showing NPo for Cl-Ca channels before and after inside-out membrane patches were excised in 400 nM Ca2+ under control conditions (A) and in presence of NO (1 µM) applied just before patch was excised (B). Original traces of single-channel currents at -100 mV applied to membrane are shown at bottom of each panel at specified time. Inward currents are shown as downward deflections; zero current level is shown by dotted line. Note that there are >2 channels in each membrane patch, resulting in several levels of single-channel currents. Pipette contained 140 mM NMDG.

It is also important to mention that NO (1 µM) applied to intact mSMC was never observed to activate single Cl-Ca channels in cell-attached membrane patches (Fig. 11; n = 29). When applied to excised membrane patches, NO never activated single Cl-Ca channels after they had run down (n = 5).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Single Cl-Ca Channels in Aortic SMC

A small (1.8 pS) channel was activated in cell-attached membrane patches of SMC from mouse and rabbit aorta by extracellular application of caffeine or by excising inside-out membrane patches into Ca2+-containing solution. The Ca2+ dependence and Cl- selectivity of this channel were evident from 1) strict dependence of channel activation on [Ca2+]i, 2) insensitivity of inward currents to the complete substitution of cations with NMDG in the pipette, 3) sensitivity to substitution of Cl- with glutamate, and 4) inhibition with niflumic acid, a Cl--channel blocker. Noise analysis of the caffeine-induced whole cell current showed an estimated single-channel conductance (2.0 pS) similar to that of single Cl-Ca channels (1.8 pS) recorded in excised patches. This result strongly suggests that these 1.8-pS channels indeed are responsible for the whole cell Cl-Ca current in SMC from mouse and rabbit aorta. Single Cl-Ca channels with similar conductance were reported in the A7r5 fetal rat aortic SMC line (1.8 pS) (31) and in human mesenteric artery (2.8 pS) (19), but those studies were limited because of the fast rundown of Cl-Ca channels in excised membrane patches.

Whole Cell Cl-Ca Current in Aortic SMC

Increasing [Ca2+]i with caffeine or ionomycin, or by simple dialysis of SMC with 1-2 µM Ca2+ in the patch pipette, activated a whole cell Cl-Ca current that was similar to those described in other SMC (for review, see Ref. 22). The Ca2+ dependence and Cl--selective conductance of this current were evident from 1) simultaneous activation of the current with the rise in [Ca2+]i, 2) absence of current activation in the presence of BAPTA, 3) the reversal potential approximating ECl, 4) inhibition by niflumic acid but not TEA or Ni2+, and 5) sensitivity to variation in Cl- concentration. The rise in [Ca2+]i also activated K+Ca current in mSMC and rSMC, but its contribution was excluded in our experiments with 140 mM Cs+ present in the pipette and 10 mM TEA added to the bath solution. There are some reports showing that caffeine can activate Ca2+-independent nonselective cation currents in nonvascular SMC (11). However, these currents were not present in mSMC and rSMC, because Ni2+, an inhibitor of nonselective cation channels, did not affect caffeine-induced whole cell current.

Ca2+-Dependent Activation and Inactivation of Cl-Ca Channels

Activation of Cl-Ca channels was strictly Ca2+ dependent, because 1) single channels were activated by excising membrane patch only into Ca2+-containing bath solution with a threshold of [Ca2+]i between 100 and 200 nM; 2) simultaneous recording of [Ca2+]i and whole cell current showed a strong correlation between the rise in [Ca2+]i and activation of Cl-Ca current; 3) chelation of intracellular free Ca2+ with BAPTA prevented activation of Cl-Ca current; and 4) intracellular dialysis with 1-2 µM Ca2+ solution activated Cl-Ca current. Our data showed that threshold [Ca2+]i for activation of single channels was between 100 and 200 nM, consistent with the estimates obtained previously from simultaneous recording of [Ca2+]i and whole cell current in other SMC (25, 33). Because of the relatively high [Ca2+]i threshold necessary to activate the channel, it is apparent why there was no basal Cl-Ca channel activity and no corresponding whole cell currents observed in resting SMC that had a basal [Ca2+]i <100 nM.

The mechanisms of Cl-Ca channel inactivation and/or rundown appeared to be more complicated. The faster rate of decline of the Cl-Ca current compared with that of [Ca2+]i after caffeine-induced Ca2+ release supports the possibility of Ca2+-dependent inactivation of Cl-Ca channels recently proposed by Wang and Kotlikoff (34). In excised membrane patches, we found that rundown of single Cl-Ca channels strongly depends on [Ca2+]i, because increasing Ca2+ concentration significantly accelerated the disappearance of channel activity. Our results with both single channels and whole cell current are consistent with strong Ca2+ dependence of the inactivation and/or rundown of Cl-Ca channels, although there were no effects of R-24571 (inhibitor of Ca2+/calmodulin) or calyculin A (inhibitor of protein phosphatases 1 and 2A) on the Ca2+-dependent rundown. Moreover, some Ca2+-independent rundown was also observed that occurred after the channel was excised from the cell even in the absence of Ca2+. At the level of single channels, it was not possible to distinguish the rundown caused by inactivation from that induced by excising the membrane patch. Importantly, our results indicate that one can significantly decrease the rundown of single Cl-Ca channels in inside-out membrane patches by reducing Ca2+ concentration applied to the intracellular surface of the membrane, which might prove helpful for longer recording of single Cl-Ca channels.

NO Does Not Affect Single Cl-Ca Channels, But Whole Cell Cl-Ca Current Can Be Indirectly Inhibited by a High Concentration of SNAP That Affects Intracellular Ca2+

Our data indicate that NO does not inhibit Cl-Ca channels in aortic SMC directly. Indeed, activation of single Cl-Ca channels by a physiological concentration of Ca2+ (400 nM), which could be attained during the application of agonists, was not affected by authentic NO (1 µM) or SNAP (<= 1 mM) in inside-out membrane patches. Application of NO (1 µM) to the inside-out membrane patch also did not affect rundown of Cl-Ca channels. These findings are consistent with the absence of a direct effect of NO on Cl-Ca channels in excised membrane patches. This is in contrast to K+Ca channels, which we found to be affected by NO under similar experimental condition (4).

Importantly, our data also suggest that NO does not affect Cl-Ca channels indirectly via cGMP or other second messengers in intact aortic SMC. Indeed, the membrane-permeant analog of cGMP (CPT-cGMP, 50 µM) did not affect whole cell Cl-Ca currents evoked by caffeine. These data are inconsistent with the possibility of cGMP-dependent inhibition of Cl-Ca channels recently proposed in SMC from opossum esophagus (38).

Although NO in vascular SMC does not inhibit the activity of Cl-Ca channels directly or through cGMP, it is attractive to speculate that it could still suppress activation of Cl-Ca currents indirectly by affecting the basal level of [Ca2+]i or Ca2+ release from intracellular stores. Indeed, progressive disappearance of caffeine-induced Cl-Ca current followed the disappearance of caffeine-induced Ca2+ release in SMC treated with a high concentration of SNAP (1 mM). The effect of SNAP starts right after application, with a progressive rise in basal [Ca2+]i. The time-dependent reduction in caffeine-induced Ca2+ release can explain the reduction in corresponding Cl-Ca currents. On the other hand, the rise in basal [Ca2+]i can cause some Ca2+-dependent inactivation of Cl-Ca channels described by Wang and Kotlikoff (34). This additional inactivation could be the reason why Cl-Ca current was inhibited more than corresponding Ca2+ release.

The effect of NO on Ca2+ release from caffeine-sensitive stores has been extensively studied in cardiac and skeletal muscle during the last few years, but the results are still controversial. Some authors found NO-induced activation of ryanodine (and caffeine)-sensitive ion channels that release Ca2+ from the stores (29, 35), whereas others showed that NO can inhibit these channels and suppress Ca2+ release from the stores (18, 23, 36). Our experiments were not designed to resolve this controversy, but theoretically could be explained by NO-induced activation of Ca2+-release channels in SMC that causes a gradual, time-dependent depletion of the stores and a rise in basal [Ca2+]i. It is important to emphasize, however, that very high concentrations of NO donors were necessary to affect caffeine-sensitive Ca2+ release channels, Ca2+ stores, and, in our experiments, activation of Cl-Ca currents, which raises a question as to the physiological significance of such effects of NO donors. Other mechanisms can affect the filling state of caffeine-sensitive stores and secondary activation of Cl-Ca channels in SMC. For example, L-type Ca2+ channels in airway smooth muscle provide an important pathway by which caffeine-sensitive stores are refilled (16, 17). Although Ca2+ influx itself is not necessary for Cl-Ca-channel activation in aortic SMC, we found that Ca2+ influx through L-type Ca2+ channels is very important for repetitive activation of Cl-Ca currents with caffeine. The progressive disappearance of caffeine-induced Ca2+ release and Cl-Ca currents during application of SNAP (1 mM) could partially result from the inhibition of L-type Ca2+ channels by NO. Indeed, inhibition of whole cell L-type Ca2+ current by NO donors has been reported in SMC (3, 7, 15, 25) and theoretically can indirectly affect activation of Cl-Ca channels. Thus our studies demonstrated that NO does not affect Cl-Ca channels directly or via cGMP, but Cl-Ca current in SMC could be suppressed indirectly via different effects of NO on intracellular free and stored Ca2+.


    ACKNOWLEDGEMENTS

The authors thank Dr. S. Sims and Dr. S. Zakharov for helpful comments and Dr. T. Adachi for measurement of NO concentration.


    FOOTNOTES

This study was supported by the National Heart, Lung, and Blood Institute (Grants HL-54150-02, HL-31607-14, HL-55993-03), the American Heart Association (Grant 9417730), and the Naito Foundation Subsidy for Inter-Institute Research.

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

Address for reprint requests and other correspondence: V. M. Bolotina, Vascular Biology Unit, Boston Medical Center, R 408, 88 E. Newton St., Boston, MA 02118-2393 (E-mail: vbolotina{at}med-med1.bu.edu).

Received 17 December 1998; accepted in final form 28 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abderrahmane, A., D. Salvail, M. Dumoulin, J. Garon, A. Cadieux, and E. Rousseau. Direct activation of KCa channel in airway smooth muscle by nitric oxide: involvement of a nitrosylation mechanism? Am. J. Respir. Cell Mol. Biol. 18: 1-13, 1998[Abstract/Free Full Text].

2.   Amedee, T., and W. A. Large. Microelectrode study on the ionic mechanisms which contribute to the noradrenaline-induced depolarization in isolated cells of the rabbit portal vein. Br. J. Pharmacol. 97: 1331-1337, 1989[Medline].

3.   Blatter, L. A., and W. G. Wier. Nitric oxide decreases [Ca2+]i in vascular smooth muscle cells by inhibition of the calcium current. Cell Calcium 15: 122-131, 1995.

4.   Bolotina, V. M., S. Najibi, J. J. Palacino, P. J. Pagano, and R. A. Cohen. Nitric oxide directly activates calcium-dependent potassium channels in vascular smooth muscle. Nature 368: 850-853, 1994[Medline].

5.   Carl, A., H. K. Lee, and K. M. Sanders. Regulation of ion channels in smooth muscles by calcium. Am. J. Physiol. 271 (Cell Physiol. 40): C9-C34, 1996[Abstract/Free Full Text].

6.   Clapp, L. H., and A. M. Gurney. Outward currents in rabbit pulmonary artery cells dissociated with a new technique. Exp. Physiol. 76: 677-693, 1991[Abstract].

7.   Clapp, L. H., and A. M. Gurney. Modulation of calcium movements by nitroprusside in isolated vacular smooth muscle cells. Pflügers Arch. 418: 462-470, 1991[Medline].

8.   Cohen, R. A., F. Plane, S. Najibi, I. Huk, T. Malinski, and C. J. Garland. Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery. Proc. Natl. Acad. Sci. USA 94: 4193-4198, 1997[Abstract/Free Full Text].

9.   Droogmans, G., G. Callewaert, I. Declerck, and R. Casteels. ATP-induced Ca2+ release and Cl- current in cultured smooth muscle cells from pig aorta. J. Physiol. (Lond.) 440: 623-634, 1991[Abstract/Free Full Text].

10.   Grynkiewicz, G., M. Poenie, and R. Y. Tsien. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3340-3450, 1985.

11.   Guerrero, A., F. S. Fay, and J. J. Singer. Caffeine activates a Ca2+-permeable, nonselective cation channel in smooth muscle cells. J. Gen. Physiol. 104: 375-394, 1994[Abstract/Free Full Text].

12.   Hamill, O. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch clamp technique for high resolution current recording from cells and cell free membrane. Pflügers Arch. 391: 85-100, 1981[Medline].

13.   Heinemann, S. H., and F. Conti. Nonstationary noise analysis and application to patch clamp recordings. Methods Enzymol. 207: 131-149, 1992[Medline].

14.   Hirakawa, Y., M. Gericke, R. A. Cohen, and V. M. Bolotina. Calcium-dependent chloride channels in vascular smooth muscle cells from mouse and rabbit aorta: regulation by calcium and nitric oxide (Abstract). Biophys. J. 74: A98, 1998.

15.   Hu, H., N. Chiamvimonvat, T. Yamagishi, and E. Marban. Direct inhibition of expressed cardiac L-type Ca2+ channels by S-nitrosothiol nitric oxide donors. Circ. Res. 81: 742-752, 1997[Abstract/Free Full Text].

16.   Janssen, L. J., and S. M. Sims. Emptying and refilling of Ca2+ store in tracheal myocytes as indicated by ACh-evoked currents and contraction. Am. J. Physiol. 265 (Cell Physiol. 34): C877-C886, 1993[Abstract/Free Full Text].

17.   Janssen, L. J., and S. M. Sims. Ca2+-dependent Cl- current in canine tracheal smooth muscle cells. Am. J. Physiol. 269 (Cell Physiol. 38): C163-C169, 1995[Abstract/Free Full Text].

18.   Kannan, M. S., Y. S. Prakash, D. E. Johnson, and G. C. Sieck. Nitric oxide inhibits calcium release from sarcoplasmic reticulum of porcine tracheal smooth muscle cells. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1-L7, 1997[Abstract/Free Full Text].

19.   Klockner, U. Intracellular calcium ions activate a low-conductance chloride channel in smooth-muscle cells isolated from human mesenteric artery. Pflügers Arch. 424: 231-237, 1993[Medline].

20.   Klockner, U., and G. Isenberg. Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through Ca-activated chloride current. Pflügers Arch. 418: 168-175, 1991[Medline].

21.   Korn, S. J., and R. Horn. Influence of sodium-calcium exchange on calcium current rundown and the duration of calcium-dependent chloride currents in pituitary cells, studied with whole cell and perforated patch. J. Gen. Physiol. 94: 789-812, 1989[Abstract/Free Full Text].

22.   Large, W. A., and Q. Wang. Characteristics and physiological role of the Ca2+-activated Cl- conductance in smooth muscle. Am. J. Physiol. 271 (Cell Physiol. 40): C435-C454, 1996[Abstract/Free Full Text].

23.   Meszaros, L. G., I. Minarovic, and A. Zahradnikova. Inhibition of the skeletal muscle ryanodine receptor calcium release channel by nitric oxide. FEBS Lett. 380: 49-52, 1996[Medline].

24.   Nelson, M. T., M. A. Conway, H. J. Knot, and J. E. Brayden. Chloride channel blockers inhibit myogenic tone in rat cerebral arteries. J. Physiol. (Lond.) 502: 259-264, 1997[Abstract/Free Full Text].

25.   Pacaud, P., G. Loirand, G. Gregoire, C. Mironneau, and J. Mironneau. Calcium-dependence of the calcium-activated chloride current in smooth muscle cells of rat portal vein. Pflügers Arch. 421: 125-130, 1992[Medline].

26.   Pacaud, P., G. Loirand, C. Mironneau, and J. Mironneau. Noradrenaline activates a calcium-activated chloride conductance and increases the voltage-dependent calcium current in cultured single cells of rat portal vein. Br. J. Pharmacol. 97: 139-146, 1989[Medline].

28.   Shin, J. H., S. Chung, E. J. Park, D.-Y. Uhm, and C. K. Suh. Nitric oxide directly activates calcium-activated potassium channels from rat brain reconstituted into planar lipid bilayer. FEBS Lett. 415: 299-302, 1997[Medline].

29.   Stoyanovsky, D., T. Murphy, P. R. Anno, Y. M. Kim, and G. Salama. Nitric oxide activates skeletal and cardiac ryanodine receptors. Cell Calcium 21: 19-29, 1997[Medline].

30.   Tare, M., H. C. Parkington, H. A. Coleman, T. O. Neild, and G. J. Dusting. Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium. Nature 346: 69-71, 1990[Medline].

31.   Van Renterghem, C., and M. Lazdunski. Endothelin and vasopressin activate low conductance chloride channels in aortic smooth muscle cells. Pflügers Arch. 425: 156-163, 1993[Medline].

32.   Von der Weid, P. Y., V. N. Serebryakov, F. Orallo, C. Bergmann, V. A. Snetkov, and K. Takeda. Effects of ATP on cultured smooth muscle cells from mouse aorta. Br. J. Pharmacol. 108: 638-645, 1993[Medline].

33.   Wang, Y. X., and M. I. Kotlikoff. Muscarinic signaling pathway for calcium release and calcium-activated chloride current in smooth muscle. Am. J. Physiol. 273 (Cell Physiol. 42): C509-C519, 1997[Abstract/Free Full Text].

34.   Wang, Y. X., and M. I. Kotlikoff. Inactivation of calcium-activated chloride channels in smooth muscle by calcium/calmodulin-dependent protein kinase. Proc. Natl. Acad. Sci. USA 94: 14918-14923, 1997[Abstract/Free Full Text].

35.   Xu, L., J. P. Eu, G. Meissner, and J. S. Stamler. Activation of the cardiac calcium release channel (ryanodine receptor) by poly-S-nitrosylation. Science 279: 234-237, 1998[Abstract/Free Full Text].

36.   Zahradnikova, A., I. Minarovic, C. Venema, and L. G. Meszaros. Inactivation of the cardiac ryanodine receptor calcium release channel by nitric oxide. Cell Calcium 22: 447-453, 1997[Medline].

37.   Zakharov, S. I., D. A. Mongayt, R. A. Cohen, and V. M. Bolotina. Monovalent cation and L-type Ca2+ channels are responsible for calcium paradox-like phenomenon in rabbit aortic smooth muscle cells. J. Physiol. (Lond.) 514: 71-81, 1999[Abstract/Free Full Text].

38.   Zhang, Y., F. Vogalis, and R. K. Goyal. Nitric oxide suppresses a Ca2+-stimulated Cl- current in smooth muscle cells of opossum esophagus. Am. J. Physiol. 274 (Gastrointest. Liver Physiol. 37): G886-G890, 1998[Abstract/Free Full Text].

39.   ZhuGe, R., S. M. Sims, R. A. Tuft, K. E. Fogarty, and J. V. Walsh, Jr. Ca2+ sparks activate K+ and Cl- channels, resulting in spontaneous transient currents in guinea-pig tracheal myocytes. J. Physiol. (Lond.) 513: 711-718, 1998[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 277(5):H1732-H1744
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[Abstract] [Full Text] [PDF]

Home page
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Physiol Rev, July 1, 2005; 85(3): 1061 - 1092.
[Abstract] [Full Text] [PDF]

Home page
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Am J Physiol Renal Physiol, August 1, 2003; 285(2): F326 - F335.
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Ca2+-activated Cl- channels in corpus cavernosum smooth muscle: a novel mechanism for control of penile erection
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Home page
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Calcium Influx Factor Directly Activates Store-operated Cation Channels in Vascular Smooth Muscle Cells
J. Biol. Chem., August 18, 2000; 275(34): 26158 - 26163.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
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Properties of a Native Cation Channel Activated by Ca2+ Store Depletion in Vascular Smooth Muscle Cells
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