AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 276: H1827-H1838, 1999;
0363-6135/99 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kawakubo, T.
Right arrow Articles by Sokabe, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kawakubo, T.
Right arrow Articles by Sokabe, M.
Vol. 276, Issue 6, H1827-H1838, June 1999

Characterization of a newly found stretch-activated KCa,ATP channel in cultured chick ventricular myocytes

Takashi Kawakubo1, Keiji Naruse2, Tatsuaki Matsubara1, Nigishi Hotta1, and Masahiro Sokabe2

Departments of 1 Internal Medicine III and 2 Physiology II, Nagoya University School of Medicine, Nagoya 466-8550, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

With the use of the patch-clamp technique, five kinds of stretch-activated (SA) ion channels were identified on the basis of their single-channel conductances and ion selectivities in cultured chick ventricular myocytes. Because a high-conductance K+-selective channel predominated among these channels, we concentrated on characterizing its properties mostly using excised inside-out patches. With 145 mM KCl solution in the pipette and the bath, the channel had a conductance of 199.8 ± 8.2 pS (n = 22). The ion selectivities among K+, Na+, Ca2+, and Cl- as estimated from their permeability ratios were PNa/PK = 0.03, PCa/PK = 0.025, and PCl/PK = 0.026. The probability of the channel being open (Po) increased with the Ca2+ concentration in the bath ([Ca2+]b; dissociation constant Kd = 0.51 µM at +30 mV) and membrane potential (voltage at half-maximal Po = 39.4 mV at 0.35 µM [Ca2+]b). The channel was blocked by gadolinium, tetraethylammonium, and charybdotoxin from the extracellular surface and, consequently, was identified as a Ca2+-activated K+ (KCa) channel type. The channel was also reversibly activated by ATP applied to the intracellular surface (Kd = 0.74 mM at 0.10 µM [Ca2+]b at +30 mV). From these data taken together, we concluded that the channel is a new type of KCa channel that could be designated as an "SA KCa,ATP channel." To our knowledge, this is the first report of KCa channel in heart cells.

patch clamp; stretch-activated channel; calcium-activated potassium channel; adenosine 5'-triphosphate-activated channel


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IT HAS BEEN DEMONSTRATED that several types of Ca2+-activated K+ (KCa) channels are present in various muscular and nonmuscular preparations (16, 25). Among these channels, a large-conductance type (big KCa channel) similar to that first reported by Marty (16) is the most popular and extensively studied type. Big KCa channels from a variety of tissues share common properties of conductance, ion selectivity, voltage dependency, and Ca2+ dependency (4, 16, 25). Because of their abundance and large conductance, KCa channels are supposed to participate in repolarizing the membrane potential after depolarization. Recently, it has been suggested that KCa channels in smooth muscle may serve as a negative-feedback pathway by responding to fluctuations in membrane potential and intracellular Ca2+ concentration, and in this way they may serve to regulate the level of vascular tone (5).

Recently, special types of KCa channels that are activated by some factors in addition to membrane potential and intracellular Ca2+ concentration have been reported. Robertson et al. (22) showed that a KCa channel in pulmonary arterial smooth muscle cells isolated from rats can be activated by Mg2+-ATP, and therefore they called it a KCa,ATP channel. The existence of KCa channels modulated by membrane stretch has also been reported in the apical membrane of cultured medullary thick ascending limb cells (28), in the apical membrane of rat and rabbit cortical collecting tubules (21), in G292 osteoblastic-like cells (7), and in embryonic rat neuroepithelial cells (17). Curiously enough, there has been no report of the KCa channel in cardiac myocyte despite such a ubiquitous distribution of KCa channels in a variety of cell types.

In the past decade, a new type of ion channel called stretch-activated (SA) channel, which is supposed to be activated by membrane tension (26), has emerged. SA channels are also carried by a variety of cell types (18). In cardiac myocytes some types of K+ channels have been reported to be stretch activated (23, 29). It is believed that this type of channel would be activated when cells are mechanically deformed. This aspect of SA channels is particularly intriguing in heart cells because these cells are always subjected to periodic stretch and sometimes receive mechanical overload that induces arrhythmias or hypertrophy.

Originally, we started our study to further characterize the SA channels in heart cells using a preparation similar to that used by Ruknudin et al. (23). We were able to identify five different types of SA channels with conductances ranging from 25 to 200 pS. Because the channel with the largest conductance (200 pS) was most frequently observed, virtually in most patches, we decided to characterize this channel and found that it is a big KCa channel. In this report we present our detailed analyses of the biophysical and pharmacological properties of the newly found KCa channel in cultured chick heart cells. The channel turned out to be modulated by both membrane stretch and intracellular ATP, which is a completely new aspect in regard to the big KCa channels. Furthermore, this is the first report of the existence of a big KCa channel in heart cells.


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

Cell culture. Ventricles were dissected from 10- to 12-day-old White Leghorn embryos under sterile conditions. Cell suspensions were prepared by exposing the ventricles to nominally Ca2+-Mg2+-free saline containing 2 mg/ml collagenase (Sigma Chemical) for 10 min at 37°C. Cells were grown in Dulbecco's modified Eagle's medium (GIBCO BRL) supplemented with heat-inactivated horse serum (10% vol/vol), chick embryo extract (2% vol/vol), penicillin, and streptomycin. The cultures were maintained in an atmosphere of 95% air-5% CO2 at 37°C and 95% relative humidity. Cells from 3 to 14 days of culture were used for experiments because the patches from younger cells were more fragile, preventing us from making stable recordings.

Single-channel recording and analysis. We used the patch-clamp technique for high-resolution detection of single-channel currents. Patch electrodes were fabricated from 100-lambda glass capillaries (Drummond Scientific) in two stages on a vertical electrode puller (PP-83, Narishige) and were heat polished on a microforge (MF-83, Narishige). Tips of freshly pulled pipettes were filled by being placed in a 1-ml sample vial containing a filtered solution for 3-5 min, and then the shanks were backfilled. Pipettes were mounted on a micromanipulator and connected to the probe of a patch-clamp amplifier (model 3900, Dagan Instruments). Mechanical stretch in the patch was made by applying negative pressure in the pipette with a pneumatic transducer tester (DPM-1B, BIO-Tek Instruments) connected to a pipette holder. Most recordings were done with excised inside-out patches, although cell-attached patches were used in a limited case. All experiments were conducted at room temperature (23 ± 2°C). Currents were stored on VHS videotapes through a PCM recorder (VR-10B, Instrutech). The stored raw data were replayed through a four-pole Bessel filter and analyzed using software (PAT vers. 6.02) written by Dr. John Dempster (University of Strathclyde, Glasgow, UK). Channel-opening events were detected by using the 50% amplitude-threshold criterion from segments of records of 10-200 s in length. Single-channel current amplitude was measured from the peak-to-peak distance on the amplitude histogram or directly from chart records. The probability of the channel being open (Po) was calculated from amplitude histograms as the ratio of open peak area to total area. In patches containing N number of channels with low Po, in which there were very few overlapped openings, Po was determined from the equation (26) Po (%) = (1 - P1/Nc) × 100, where Pc is the probability of the channel not being open in the record. In most kinetic analyses, i.e., determination of mean open and closed times, we used the data from the patches containing a single channel. In inside-out patches, membrane potential was defined as the negative value of the pipette potential. Upward current deflections in Figs. 1-5, 7, and 8 represent cation movement from the cytoplasmic side to the external side of the membrane and vice versa for anions.

Solutions. The standard external solution (in the pipette) facing the extracellular surface of the patch contained 145 mM KCl, 1 mM CaCl2, 10 mM HEPES, and 10 mM glucose adjusted to pH 7.40 with KOH. The internal solution (in the bath) facing the cytoplasmic surface of the patch was the same as that in the pipette except that the concentration of CaCl2 was below 1 mM. When the Ca2+ concentration was below 10 µM, it was adjusted with EGTA on the basis of calculations made using the program EqCal (Biosoft) according to the stability constants from Owen (20). Most experiments were done with excised inside-out patches and with these solutions, unless otherwise noted.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SA channels in cultured myocytes. Figure 1 shows typical examples of five different types of SA single-channel currents recorded from separate inside-out patches. As shown, negative pressures in the pipette caused Po to increase without apparent adaptation in every case. We first categorized the channels on the basis of their conductances as determined from current-voltage (I-V) relations under two different ionic conditions: 1) 145 mM KCl in the pipette and 145 mM KCl in the bath, or 2) 145 mM NaCl in the pipette and 145 mM KCl in the bath. With 145 mM KCl saline in the pipette and the bath, we identified four types of SA channels with conductances of ~25 pS (Fig. 1, A and B), 50 pS (Fig. 1C), 100 pS (Fig. 1D), and 200 pS (Fig. 1E). The 25-pS type was further separated into two subtypes, a K+-selective channel (Fig. 1B), with a permeability ratio of Na+ to K+ (PNa/PK) of 0.10, and a nonselective cation channel (Fig. 1A), with a PNa/PK of 1.0. The 50-, 100-, and 200-pS channels were K+ selective (PNa/PK = 0.08, 0.05, and 0.03, respectively). These results are roughly in line with the earlier report of Ruknudin et al. (23). Because the activity of the 200-pS channel predominated in most patches observed, we focused on this type and characterized its biophysical and pharmacological properties. Interestingly, as shown by the following results, this channel was voltage dependent and strongly activated by Ca2+ from the cytoplasmic side of the membrane, which suggests that it is a big KCa channel.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 1.   Typical records of single-channel currents of 5 different types of stretch-activated (SA) channels. Identification of SA channels was made through pressure sensitivity of channels. A: 25-pS SA channel (nonselective) at -80 mV. -C, closed level of channel. B: 25-pS SA channel (K+ selective) at +120 mV. C: 50-pS SA channel (K+ selective) at +80 mV. D: 100-pS SA channel (K+ selective) at +40 mV. E: 200-pS SA channel (K+ selective) at +30 mV. All traces were obtained from excised inside-out patches with symmetrical 145 mM KCl solutions except for trace in A, where pipette contained 145 mM NaCl and bath contained 145 mM KCl. Arrows at baselines indicate application time of suction.

Channel density of 200-pS channel. Channel frequencies in 100 patches were 4 channels for nonselective 25-pS type, 22 channels for K+-selective 25-pS type, 21 channels for 50-pS type, 39 channels for 100-pS type, and 245 channels for 200-pS type. The frequencies for 25-pS (nonselective), 25-pS (K+ selective), 50-pS, and 100-pS channel types were somewhat similar to those reported by Ruknudin et al. (23); however, our frequency for 200-pS channel was much higher than that reported by Ruknudin et al. The reason for this difference may be that they used cell-attached patches with intracellular surfaces exposed to a lower Ca2+ concentration than that (0.35-1.0 µM) used for our inside-out patches. The estimated intracellular Ca2+ concentration in our preparation was 0.1-0.35 µM (see Fig. 7D). The membrane area of the patch could be estimated to be 2-5 µm2, based on the geometric measurements obtained using high-power videomicroscopy (26). Therefore, the channel densities of the 200-pS channel seemed to be in the range of 0.5-1.2 channels/µm2.

Ion selectivity. The ion selectivity of the 200-pS channel was estimated from I-V curves for inside-out patches by exposing the cytoplasmic surface of the membrane to 145 mM KCl, 72.5 mM KCl, 145 mM NaCl, or 72.5 mM CaCl2 solutions. Figure 2A shows single-channel activities of a 200-pS channel in an excised inside-out patch at the indicated membrane potentials. Figure 2B shows I-V relationships under these various ionic conditions. With symmetrical 145 mM KCl solutions (n = 22), a nearly linear I-V relationship with a reversal potential near zero and a mean slope conductance of 199.8 pS was obtained. In inside-out patches with 145 mM NaCl in the bath (n = 8), there was a positive shift in the reversal potential and the I-V curve became sublinear and was well fitted by the Goldman-Hodgkin-Katz current equation (Fig. 2B). The permeability ratio PNa/PK was calculated using the equation PNa/PK = ([K+]o/[Na+]i) exp(-ErevF/RT), where Erev is the reversal potential, F is Faraday's constant, R is the gas constant, T is absolute temperature (°K), [Na+]i is the bath (cytoplasmic surface) concentration of Na+, and [K+]o is the pipette (extracellular surface) concentration of K+. The calculated PNa/PK was 0.033 (n = 6), a value consistent with those for KCa,ATP channels from other preparations.


View larger version (34K):
[in this window]
[in a new window]
  		Fig. 2.   Voltage-dependent behavior of single-channel currents of 200-pS K+ channel. A: single-channel activities in an inside-out patch in symmetrical 145 mM KCl solutions at indicated membrane potentials. B: current-voltage (I-V) relationships for 200-pS SA channels from inside-out patches with symmetrical 145 mM KCl (), 145 mM KCl in pipette and 145 mM NaCl in bath (), 145 mM KCl in pipette and 72.5 mM KCl in bath (), and 72.5 mM CaCl2 in pipette and 145 mM KCl in bath (open circle ). Data points represent averages from 8, 7, 6, and 5 patches, respectively. Curves are drawn according to Goldman-Hodgkin-Katz current equation. V, membrane potential.

Similarly, PCa/PK was calculated using the equation PCa/PK = [K+]i exp(-ErevF/RT)[1 + exp(-ErevF/RT)]/4[Ca2+]o, where [K+]i is the bath concentration of K+ and [Ca2+]o is the pipette concentration of Ca2+. With 72.5 mM CaCl2 in the pipette and 145 mM KCl in the bath, PCa/PK was 0.025 (n = 7). PCl/PK was calculated as 0.026 from the measurement of the reversal potential with 72.5 mM KCl in the bath and 145 mM KCl in the pipette (n = 7). This value is comparable to that of a KCa channel from rat brain synaptosomal membranes as reported by Nomura et al. (19).

Effects of K+-channel blockers. In general there are two types of channel blockers, fast and slow blockers. Fast blockers induce a graded decrease of the single-channel currents, whereas slow blockers make "all-or-none" types of gaps in the open-channel currents, which sometimes are hard to discriminate from channel closing. To avoid this difficulty, we used a high Ca2+ concentration (1 mM) in the bath to keep the channel open for most of the time over a wide potential range. First, we tested the effect of charybdotoxin (CTX), a specific blocker for big KCa channels (2), on the 200-pS channel using the backfill procedure (3), in which 145 mM KCl solution containing 20 nM CTX was backfilled. Figure 3A shows time-dependent changes in the channel activities at +30 mV, which reflect gradual diffusion of the drug to the patch membrane. The Po of the 200-pS channel was significantly decreased 5 min after the start of the backfill and almost completely disappeared after an additional 15 min.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 3.   Pharmacological properties of 200-pS SA channel. A: blockade of a 200-pS SA channel by charybdotoxin (CTX). Blockade occurred without significant reduction in current amplitude. Traces were obtained at indicated times after start of backfilling of pipette. Pipette tip was first filled with 145 mM KCl and then backfilled with same solution containing 20 nM CTX; bath contained 145 mM KCl and 1 mM CaCl2. Membrane potential was held at +30 mV under a suction of -10 mmHg in pipette. B: blockade of a 200-pS SA channel by 20 µM gadolinium (Gd) using backfill procedure. Method and conditions were same as in A. Blockade occurred without significant reduction in current amplitude. C: time courses of open probability (Po) of control group (n =16), CTX group (n =9), and Gd group (n =10). Time was measured from start of backfill of solutions. Membrane potentials were held at +30 mV in all groups. D: blockade of a 200-pS SA channel by 1 mM tetraethylammonium (TEA). TEA reduced current amplitude in a dose-dependent manner. Conditions were same as in A except that pipette was backfilled with 145 mM KCl containing 1 mM TEA.

The effect of 20 µM gadolinium (Gd3+), a potent blocker of SA channels, was also examined using the same method. As shown in Fig. 3B, a slow blockade of the 200-pS channel became visible at 5 min, and complete blockade was apparent 15 min after the backfill. Although the kinetics of this Gd3+ blockade were not systematically investigated in the present study, its qualitative features seemed to be similar to the Gd3+ blockade of the endogenous SA cation channels in Xenopus oocytes (31). Figure 3C shows time courses of Po from the control, CTX, and Gd3+ groups. Using the backfill procedure, we tested the effect of extracellular Mg2+ (3 mM) and found that the Po of the channel was not changed (control 68.3% vs. Mg2+ 66.1%, n = 3). We also tested the effect of intracellular Mg2+ and again observed no significant change in Po (70.5% vs. 68.1%, n = 3).

Fast blocking of the 200-pS channel by externally applied tetraethylammonium (TEA) using the backfill procedure is shown in Fig. 3D. In contrast to the all-or-none type of blockade described above, TEA decreased the mean single-channel current in a dose-dependent manner and increased the open-channel current noise of the 200-pS channel. This type of "flickery block" is characteristic of a blocking process with relatively fast kinetics. The single-channel conductance was 151 pS with 0.1 mM TEA, 126 pS with 0.3 mM TEA, and 62 pS with 0.8 mM TEA backfilled in the pipette, thus yielding an estimated dissociation constant (Kd) of ~0.4 mM. These data are in accordance with the TEA-induced reduction of the single-channel conductance in a variety of big KCa channels (hereafter, we refer to KCa channels by name).

Calcium and voltage dependencies. The effect of intracellular Ca2+ on the channel activity was studied by exposing the cytoplasmic side of the patch to a range of various Ca2+ concentrations ([Ca2+]b). Figure 4A demonstrates Ca2+ activation of the 200-pS channel in an inside-out patch with various [Ca2+]b at a membrane potential of +30 mV in a symmetrical 145 mM KCl solution. Although Po was extremely low (0.1%) at 0.1 µM [Ca2+]b, Po increased with the increase in [Ca2+]b. Ca2+ dependency of the open and closed times was estimated from the dwell-time histograms. The histograms of open-time distribution were well fitted by single-exponential functions, whereas those of closed times were fitted by double-exponential functions (Fig. 4B). Ca2+ dependency of Po and the mean open and closed times are summarized in Fig. 4C, in which the mean long closed time decreased and the mean open time increased with the increase in [Ca2+]b, whereas short closed time showed little change. This type of [Ca2+]b dependency, with a decrease in long closed time and an increase in open time with an increase in [Ca2+]b, was also seen in other preparations (25).


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4.   Ca2+ dependency of 200-pS SA channel. A: single-channel current traces of 200-pS channel in an inside-out patch at various Ca2+ concentrations in bath ([Ca2+]b). Po of channel in an inside-out patch increased as [Ca2+]b increased. Membrane potential was held at +30 mV. Pipette and bath contained 145 mM KCl. B: dwell-time histograms of open (OT), long closed (lCT), and short closed times (sCT) for patch in A at 0.35 µM [Ca2+]b. OT histogram could be fitted by a single-exponential function, and lCT and sCT histograms could be fitted by a double-exponential function with time constants indicated. C: plot of Po vs. [Ca2+]b. Data () were taken from patch in A. Dotted line was drawn according to a Boltzmann-type function. [Ca2+]b dependence of OT, lCT, and sCT are also indicated. In response to a change in [Ca2+]b, lCT decreased from 514.24 ms at 0.1 µM [Ca2+]b to 1.91 ms at 0.8 µM [Ca2+]b, whereas OT increases from 2.12 to 11.83 ms.

Figure 5A shows representative recordings of the 200-pS channel currents at various membrane potentials in a symmetrical 145 mM KCl solution with a 0.35 µM [Ca2+]b. As the membrane potential was made more positive, the mean open time of the channel became longer. In Fig. 5A, a few brief channel openings are visible at -20 mV, whereas at higher voltages Po increases accordingly, and even second channel openings become apparent at +50 mV. An example of dwell-time histograms at -20 and +50 mV are shown in Fig. 5B. The voltage dependence of Po is demonstrated in Fig. 5C, in which the data points are well fitted by a Boltzmann function of the form
<IT>P</IT><SUB>o</SUB> = 1/{1 + exp[−<IT>K</IT> (<IT>V</IT> − <IT>V</IT><SUB>0</SUB>)]} (1)
where V is a given membrane potential, V0 is the voltage at half-maximal Po, and K is the logarithmic potential sensitivity indicating an e-fold increase in membrane potential. For this particular data set, V0 was 39.2 mV and K-1 was 14.7. Figure 5C shows the voltage dependence of the mean open and closed times, where the mean open time increases and the mean long closed time decreases with voltage, whereas the mean short closed time remains unchanged. A similar tendency was reported in other preparations (6, 7, 25).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5.   Voltage dependency of 200-pS SA channel. A: single-channel current traces in an inside-out patch at indicated membrane potentials. Pipette and bath contained 145 mM KCl and 0.35 µM [Ca2+]b. B: dwell-time histograms of OT and lCT at 2 different membrane potentials for patch in A. OT histograms were fitted by a single-exponential function, and lCT histograms were fitted by a double-exponential function, with time constants indicated. Dotted lines drawn in lCT histograms were fitted to time region longer than 2 ms. C: plot of Po vs. membrane potential. Data () were taken from patch in A. Dotted line was drawn according to Boltzmann function: Po = 1/{1 + exp[-K(V - V0)]}, with V0 = 39.2 mV and K-1 = 14.7, where V is a given membrane potential, V0 is voltage at half-maximal Po, and K is logarithmic potential sensitivity. Voltage dependence of mean OT, mean lCT, and mean sCT are also indicated. As membrane potential increases, lCT decreases from 133.52 ms at -20 mV to 35.66 ms at +30 mV and then decreases further to 10.89 ms at +50 mV, whereas OT increases from 1.98 ms at -20 mV to 3.56 ms at +30 mV and 9.90 ms at +50 mV.

Figure 6 shows Po as a function of membrane potential at various [Ca2+]b from 45 separate inside-out patches. Po increased with depolarization at all [Ca2+]b tested, and the data set for each [Ca2+]b was reasonably fitted by Eq. 1. The value for K-1 was 18.07 ± 0.72 (n = 5).


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 6.   Voltage dependence of Po at different [Ca2+]b. Dotted lines were drawn according to Boltzmann equation (see Eq. 1 in text). Data points represent means ± SE.

The theoretical activation curves in Fig. 6 give Po values at membrane potentials of -20, -10, 0, +10, +20, and +30 mV with various Ca2+ concentrations, from which we could obtain Hill plots. Hill coefficients were 4.34 ± 0.17 mV (n = 6).

From the above results [i.e., high unitary conductance (200 pS) and sensitivity to Ca2+, voltage, TEA, and CTX], we concluded that the 200-pS SA channel was a Ca2+-activated K+ channel (KCa channel).

Pressure dependency. We next investigated SA properties of the KCa channel. In both inside-out and cell-attached patches in cultured chick cardiac cells, the activity of the KCa channel was increased by the application of negative pressure in the patch pipette. Figure 7A shows typical current traces of responses to the pressure in the pipette. Typically, membrane stretch by negative pressure was applied in a stepwise fashion with a 10-mmHg increment, and the response of the channel activity lagged behind the pressure change by only a few seconds. Positive pressure in the pipette also activated the KCa channel in a way similar to negative pressure within 20 mmHg. However, positive pressure as low as 20 mmHg frequently deteriorated or sometimes broke the seal in 10-15 s, preventing us from obtaining good results.


View larger version (26K):
[in this window]
[in a new window]
  		Fig. 7.   Pressure dependence of Ca2+-activated K+ (KCa) channel. A: single-channel activity of KCa channel at different pressures. Negative pressures in pipette progressively increased channel activity. Values at left indicate pressure. Pipette contained 145 mM KCl and 1 mM CaCl2, and bath contained 145 mM KCl and 0.35 µM CaCl2. Membrane potential was held at +30 mV. B: dwell-time histograms of OT and lCT at 0 and 50 mmHg for patch in A. lCT decreased from 331.53 ms at 0 mmHg to 52.09 ms at 50 mmHg, whereas OT increased from 4.22 to 10.89 ms. C: plots of pressure dependency of Po, mean OT, mean sCT, and mean lCT for patch in A. D: pressure dependency of Po at 2 different [Ca2+]b from inside-out patches, Po with no Ca2+ on either side of membranes from inside-out patches, and Po from cell-attached patches. Data points represent means ± SE calculated from 11 experiments with 0.1 µM [Ca2+]b, 12 experiments with 0.35 µM [Ca2+]b, 4 experiments with no Ca2+ on either side of membranes, and 8 experiments from cell-attached patches.

The effect of membrane stretch on the open-close kinetics was analyzed on the basis of open- and closed-time distributions. Dwell-time histograms of those times at 0 and 50 mmHg of negative pressure are shown in Fig. 7B. For this channel, a double-exponential function was required to fit the closed-time distribution, and a single-exponential function was enough to fit the open-time distribution, suggesting that there are at least two closed states (C1 and C2) and one open state (O1). Such a property is similar to the results reported in other types of SA channels (9, 12). With this particular patch, the mean long closed time decreased and the mean open time increased with membrane stretch (Fig. 7C). The change in open time was much smaller than that in long closed time, suggesting that suction in the pipette increased Po primarily by decreasing the mean long closed time of the channel. This is also similar to the findings in the previously mentioned reports (9, 12). Five additional patches showed basically a similar tendency. With the assumption of a simple linear kinetics model (C1 left-right-arrow  C2 left-right-arrow  O1), only the transition from C1 to C2 is stretch dependent.

We also examined the effect of membrane stretch on the KCa channel in cell-attached patches. Membrane stretch activated the KCa channels in a similar manner, although there was a high degree of variability in their responses. Figure 7D summarizes the data from excised inside-out patches with 0.1 (n = 11) or 0.35 µM (n = 12) [Ca2+]b, from excised inside-out patches with no Ca2+ in the pipette or bath (n = 4), and from cell-attached patches (n = 8). From these results, the intracellular Ca2+ concentration in our preparations could be estimated between 0.1 and 0.35 µM, a reasonable range for the resting heart cell. In the apical membrane of cultured medullary thick ascending limb cells, the activation of KCa channels by membrane stretch was reported to be attributed to some indirect processes, such as Ca2+ influx mediated by SA cation channels or volume-induced Ca2+ releases from cytoplasmic stores (9, 28). However, we observed stretch-dependent channel activation with 0 mM CaCl2 and 4 mM EGTA in the pipette and bath (Fig. 7D). To rule out the possibility of Ca2+ crossing from the patch pipette into a nonstirred layer at the cytoplasmic surface of the membrane patch, we performed a set of cell-attached experiments with 0 mM CaCl2 and 4 mM EGTA in the patch and bath solutions. Under these conditions, we still observed an increase of Po by negative pressure in the pipette (data not shown).

ATP dependency. Because Robertson et al. (22) have shown that KCa channels in pulmonary arterial smooth muscle cells isolated from rats can be activated by Mg2+-ATP, we also tested the effect of ATP on our KCa channel in inside-out patches. Aliquots of concentrated ATP solution were successively added to the bath to give various final concentrations at the cytoplasmic side ([ATP]b). Figure 8A shows typical current traces demonstrating progressive activation of the KCa channel by this dose protocol. ATP dependency of Po and the mean open and closed times are shown in Fig. 8B. Po was extremely low without ATP but increased steeply with an increase of ATP. The mean long closed time decreased and the mean open time increased with an increase of ATP. The changes in long closed time and open time are consistent with the changes in Po. Similar to the effect of membrane stretch, the primary effect of ATP on the gating kinetics seems to be a reduction of the mean long closed time. The observed sensitivity to ATP suggested that the channel should be designated as a KCa,ATP channel. Figure 8C shows ATP dependency of Po from eight separate patches. In the absence of Ca2+ in the pipette and bath, 1 mM ATP applied to the cytosolic surface of inside-out patches also increased the Po of the KCa,ATP channel from 0.051 to 42.2%. Albarwani et al. (1) have reported that the KCa,ATP channel in pulmonary arterial smooth muscle cells from the rat was maximally activated with 1 mM Mg-ATP. The KCa,ATP channel in this study required a little more ATP to be maximally activated. The Hill coefficient calculated from a range of 0.5 to 1.75 mM [ATP]b in this study was ~5.0, whereas that in the study of Albarwani et al. between 10 µM and 1 mM [ATP]b was ~1.2. Our KCa,ATP channel is more sensitive to changes in [ATP]b.


View larger version (31K):
[in this window]
[in a new window]
  		Fig. 8.   Effect of ATP on single-channel activity of KCa channel. A: records of KCa-channel openings at several concentrations of intracellular ATP. Pipette contained 145 mM KCl and 1 mM CaCl2, and bath contained 145 mM KCl and 0.1 µM CaCl2. Membrane potential was maintained at +30 mV. B: plot of Po vs. ATP. Data () were taken from patch in A. Dotted line was drawn according to a Boltzmann-type function. ATP dependency of OT, lCT, and sCT are also indicated. C: ATP dependency of Po. Data points represent means ± SE calculated from 8 inside-out patches. Dotted line represents a fit of data points to a Boltzmann-type function. KCa channels reached half-maximal Po at a concentration of 0.78 mM. D: voltage dependence of Po at different bath concentrations of ATP ([ATP]b; in mM) from a single inside-out patch. At a given [ATP]b, voltage causes a marked increase in Po, as observed in effect of [Ca2+]b on voltage dependency (see Fig. 6).

We also examined interaction between ATP and voltage (membrane potential) in the activation of the KCa,ATP channel. The results from a single KCa,ATP channel are shown in Fig. 8D. Increases in [ATP]b shift the curve to left, which is similar to the effect of [Ca2+]b on Po. Two other separate patches gave similar results.

Effects of other nucleotides. We examined the effect of other nucleotides on the KCa,ATP channel in inside-out patches. Concentrated nucleotide solutions were directly added to the bath, as was done with Ca2+ and ATP, and negative pressure (-20 mmHg) was applied in the pipette. ADP and AMP activated the KCa,ATP channels but with lesser effects than ATP. Table 1 summarizes the effects of various agents on the KCa,ATP channel.

                              
View this table:
[in this window]
[in a new window]
  		Table 1.   Effect of other chemicals on KCa,ATP channel


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we have revealed the presence of five distinct types of SA channels in cultured chick ventricular myocytes by using the patch-clamp technique. Among them, a K+ channel with the largest conductance predominated and was identified as a KCa channel on the basis of several characteristics, including high K+ selectivity, a large unitary conductance, voltage and Ca2+ dependencies, and sensitivities to TEA and CTX. The KCa channels in this study were also activated by application of ATP to the intracellular surface of inside-out patches. The channel could therefore be identified as a stretch-, Ca2+-, and ATP-activated K+ (SA KCa,ATP) channel.

Calcium and voltage dependencies. The gating of the KCa channels in chick cardiac myocytes was sensitive to changes in [Ca2+]b between 0.1 and 1.0 µM (Fig. 4). A KCa channel with a similar sensitivity to [Ca2+]b (<0.1 µM) was reported in smooth muscle cells (25), whereas channels with higher sensitivity (activation at <0.01 µM) in pituitary cells (30) and lower sensitivity (1-10 µM activation range) in cultured rat muscle cells (4) have been reported. The Hill coefficient of our KCa channel in cardiac myocytes was between 4.0 and 5.0. For other preparations the values have been determined to be 2.87 in cultured rat skeletal muscle (4), 2.82 in rat pulmonary arterial smooth muscle cells (1), and 1.63 in rat brain synaptosomal cells (19). Most of the values from mammalian skeletal muscle have been reported to be between 1.5 and 3.0.

A comparison of the voltage sensitivity of the present KCa channels with those in various preparations may be made by using the inverse slope of the plot of the natural logarithm of the ratio [Po/(1 - Po)] versus membrane potential. Our channel had an inverse slope of 14.8-18.5 mV, a value determined over a range of membrane potential between -80 and +80 mV and [Ca2+]b between 0.25 and 0.70 µM. For other preparations the values for the inverse slope have been determined to be 9 mV in clonal anterior pituitary cells (30), 15-16 mV in cultured rat skeletal muscle (4), 11.3 mV for smooth muscle cells of the guinea pig taenia coli (11), and 15-20 mV for canine colonic myocytes (6). Thus the inverse slope of most KCa channels consistently lies between 10 and 20 mV, suggesting that the voltage-sensing mechanism of KCa channels, including ours, seems to be well conserved. The Ca2+ and voltage dependencies of our KCa channel lie in the range of those of previously reported KCa channels.

Activation by membrane stretch. An interesting aspect of the KCa channel in cardiac myocytes described in this study is its mechanosensitive property, a direct activation by membrane stretch. Stretch activation of some KCa channels, however, has been ascribed to other mechanisms rather than to stretch itself, e.g., increases of intracellular Ca2+ concentration by Ca2+ influx through a SA cation channel that permits an influx of Ca2+ or volume-induced release of Ca2+ from cytoplasmic stores (21). Although we cannot completely preclude such mechanisms in our channel, we have observed stretch activation of the KCa channels under conditions designed to eliminate changes in Ca2+ concentration in the pipette and bath solutions. In cell-attached patches with 0 mM Ca2+ in the patch pipette and the bath, membrane stretch by suction also increased the Po of the KCa channel in a manner similar to that in excised patches, suggesting that the stretch activation of our KCa channel does not require any intracellular messengers. These results strongly suggest that the channel is activated directly by membrane stretch.

Ruknudin et al. (23) have reported that one of five SA channels found in their study had a large conductance (190 pS), a value similar to ours (23). However, our channel is much more permeable to K+ over Na+ than their channel, and their channel had no clear voltage dependence. At present, without other data to compare, we cannot completely conclude whether our channel is different from theirs.

Activation mechanism by ATP. In the present study, we found that the KCa channel is activated by physiological levels of ATP from the cytoplasmic side of the membrane. It is possible that ATP activation of the KCa channel involves protein phosphorylation that requires ATP. Activation of KCa channels by cAMP-dependent phosphorylation has been reported in a variety of cells (8, 14, 24). We tested the involvement of such a cAMP-dependent protein phosphorylation by using dibutyryl cAMP, a membrane-permeable cAMP analog, which had no significant effect when applied to the inside-out patch (see Table 1). We also tested the effect of cAMP on the channel in the intact cell to check whether there is a cAMP-dependent intracellular mechanism that could modulate the channel activity. However, even in cell-attached patches, extracellular application of dibutyryl cAMP up to 1 mM had no effect on the channel activation. It is unlikely that activation of the KCa channel involves cAMP-dependent protein phosphorylation. What is puzzling is that adenosine 5'-O-(3-thiotriphosphate) (ATPgamma S) had no effect on the KCa channel (see Table 1). At present we do not know whether the channel discriminates the difference between ATP and ATPgamma S or whether ATP hydrolysis is involved in the ATP activation. However, the latter mechanism seems to be unlikely because Mg2+ required for ATP hydrolysis had no effect on the ATP activation.

On the other hand, it is known that muscarinic K+ channels in atrial myocytes of guinea pig hearts are directly activated by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgamma S) in the absence of agonists such as acetylcholine (11). We applied GTPgamma S (1 mM) to the cytoplasmic surface of inside-out patches, but little activation of the KCa channels was detected. Thus G protein-mediated signal transduction systems may not play a major role in the activation of our KCa channel.

Recently, Lemos and Takeda (15) have reported that application of 10 µM ATP to the external solution containing a normal concentration of Ca2+ caused a large increase in KCa-channel activity in cell-attached patches. This is due to the ATP-induced increase in Ca2+ concentration from both Ca2+ release from internal stores and influx from the extracellular solution. In the present study, external solutions that contained ATP did not activate KCa channels in cell-attached patches (data not shown), and when ATP was in the bath, there was no difference in Po with or without Ca2+ in the pipette in excised inside-out patches (data not shown). Therefore, it is not likely that the activation of our KCa,ATP channel by ATP involves an ATP-induced increase in intracellular Ca2+ concentration or [Ca2+]b (15). These results strongly suggest that the ATP-activation of our KCa channel is mediated by a direct interaction of ATP with the channel.

It is known that, under physiological conditions, the intracellular ATP concentration is a few millimoles per liter. However, we observed in cell-attached patches that the activity of the KCa,ATP channels was extremely low, whereas in inside-out patches, 1 mM ATP at the cytoplasmic surface caused a significant activation. It is possible that in cell-attached patches some kind of cytosolic factors may suppress the ATP-induced activation of the KCa,ATP channel.

Physiological role of SA KCa,ATP channels. In this study we used only embryonic heart cells; therefore, it may be too early to discuss the physiological role of the KCa,ATP channel. Taking this limitation into account, we offer some speculation on the possible functions of this channel in the heart.

Intracellular Ca2+ concentration in heart cells under physiological conditions is roughly 0.10 µM in the diastolic phase and 1.0 µM in the systolic phase. The corresponding membrane potentials are roughly -80 and +30 mV. Intracellular ATP concentration in heart cells is suggested to be 4-5 mM in both phases. With a 0.10 µM [Ca2+]b at -80 mV, Po of the KCa,ATP channel in this study proved to be quite low (Fig. 6), and ATP is not likely to increase Po at this potential (Fig. 8). On the other hand, with a 1.0 µM [Ca2+]b at +30 mV, corresponding to the systolic phase, Po increased to the maximal level even with no ATP (Fig. 6). Thus ATP may not affect the KCa,ATP channel under normal conditions.

Under pathological conditions, e.g., during ischemia, intracellular Ca2+ concentration is suggested to be 0.8-1.0 µM in the diastolic phase and 1.3-1.5 µM in the systolic phase. Intracellular ATP concentration during ischemia decreases to 25-30% of its normal level, namely, 1.0-1.3 mM, in both phases. With a 0.8 µM [Ca2+]b at -80 mV, Po of the channel seems to be >30% (Fig. 6). A 1 mM [ATP]b does not seem to affect the Po at this potential (Fig. 8D). The channel may be fully activated in the systolic phase during ischemia, as under normal conditions. Thus, under pathological conditions in both phases, the channel would remain at a highly activated level, contributing to hyperpolarization of the membrane potential and reduction of the excitability. The effect of ATP on the channel under both conditions is not clear, because [Ca2+]b seems to dominate in controlling the voltage dependence of the channel. ATP may contribute to a more subtle regulation of the KCa,ATP-channel activity. On the other hand, membrane stretch seems to increase the Po of the channel (Fig. 7D) and to accelerate the hyperpolarizing process.

Albarwani et al. (1) have suggested that KCa,ATP channels in pulmonary arterial smooth muscle cells isolated from the rat are involved in controlling pulmonary vascular tone under hypoxic conditions. After hypoxia, the sensitivity of KCa,ATP channels to intracellular Ca2+ concentration, and therefore to voltage, is reduced as a consequence of reduced intracellular ATP concentration. The activity of these channels is therefore reduced, exerting a depolarization, which would potentially favor Ca2+ influx through voltage-gated Ca2+ channels, ultimately increasing pulmonary vascular tone. This may also mean that activation of KCa,ATP channels causes dilation of pulmonary arteries. Taguchi et al. (27) have suggested that dilation of cerebral arteries in response to increases in the intracellular concentration of cAMP is mediated by activation of KCa channels. Brayden and Nelson (5) also indicated that activation of KCa channels by any means could lead to vasodilation of myogenic arteries. Thus one important role of KCa channels might be to exert vasodilation. Kume et al. (13) have suggested that beta -adrenergic stimulation of KCa channels may cause relaxation of tone in airway smooth muscle, namely, bronchodilation by cAMP-dependent and membrane-delimited pathways. Likewise, KCa,ATP channels in the heart may be involved in dilation or relaxation of the heart. However, it remains to be determined whether the KCa,ATP channel is expressed in matured heart cells and how the KCa,ATP channel is involved in the heart under physiological and pathological conditions.


    ACKNOWLEDGEMENTS

This work was partly supported by Grants-in-Aid 09044283 and 09257220 for scientific research (to M. Sokabe) from the Ministry of Education, Science and Culture of Japan, a grant (to M. Sokabe) from the Japan Space Forum, and a grant (to M. Sokabe) from CREST.


    FOOTNOTES

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: M. Sokabe, Dept. of Physiology II, Nagoya University School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan (E-mail: msokabe{at}med.nagoya-u.ac.jp).

Received 29 June 1998; accepted in final form 14 January 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albarwani, S., B. E. Robertson, P. C. G. Nye, and R. Z. Kozlowski. Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat. Pflügers Arch. 428: 446-454, 1994[Medline].

2.   Anderson, C. S., R. Mackinnon, C. Smith, and C. Miller. Charybdotoxin block of single Ca2+-activated K+ channels: effects of channel gating, voltage, and ionic strength. J. Gen. Physiol. 91: 317-333, 1988[Abstract/Free Full Text].

3.   Auerbach, A. Single-channel dose-response studies in single, cell-attached patches. Biophys. J. 60: 660-670, 1991[Abstract/Free Full Text].

4.   Barrett, J. N., K. L. Magleby, and B. S. Pallota. Properties of single calcium-activated potassium channels in cultured rat muscle. J. Physiol. (Lond.) 331: 211-230, 1982[Abstract/Free Full Text].

5.   Brayden, J. E., and M. T. Nelson. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 256: 532-535, 1992[Abstract/Free Full Text].

6.   Carl, A., and K. M. Sanders. Ca2+-activated K+ channels of canine colonic myocytes. Am. J. Physiol. 257 (Cell Physiol. 26): C470-C478, 1989[Abstract/Free Full Text].

7.   Davidson, R. M. Membrane stretch activates a high-conductance K+ channel in G292 osteoblastic-like cells. J. Membr. Biol. 131: 81-92, 1993[Medline].

8.   Ewald, D. A., A. Wiliams, and I. B. Levitan. Modulation of Ca2+-dependent K+-channel activity by protein phosphorylation. Nature 315: 503-506, 1985[Medline].

9.   Filipovic, D., and H. Sackin. A calcium-permeable stretch-activated cation channel in renal proximal tubule. Am. J. Physiol. 260 (Renal Fluid Electrolyte Physiol. 29): F119-F129, 1991[Abstract/Free Full Text].

10.   Hu, S. L., Y. Yamamoto, and C. Y. Kao. The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli. J. Gen. Physiol. 94: 833-847, 1988[Abstract/Free Full Text].

11.   Ito, H., T. Sugimoto, I. Kobayashi, K. Takahashi, T. Katada, M. Ui, and Y. Kurachi. On the mechanism of basal and agonist-induced activation of G protein-gated muscarnic K+ channel in arterial myocytes of guinea pig heart. J. Gen. Physiol. 98: 517-533, 1991[Abstract/Free Full Text].

12.   Kim, Y., E. R. Dirksen, and M. J. Sanderson. Stretch-activated channels in airway epithelial cells. Am. J. Physiol. 265 (Cell Physiol. 34): C1306-C1318, 1993[Abstract/Free Full Text].

13.   Kume, H., I. P. Hall, R. J. Washabau, K. Takagi, and M. I. Kotlikoff. beta -Adrenergic agonists regulate KCa channels in airway smooth muscle by cAMP-dependent and -independent mechanisms. J. Clin. Invest. 93: 371-379, 1994.

14.   Kume, H., A. Takai, H. Tokuno, and T. Tomita. Regulation of Ca2+-dependent K+-channel activity in tracheal myocytes by phosphorylation. Nature 341: 152-154, 1989[Medline].

15.   Lemos, V. S., and K. Takeda. Neuropeptide Y2-type receptor-mediated activation of large-conductance Ca2+-sensitive K+ channels in a human neuroblastoma cell line. Pflügers Arch. 430: 534-540, 1995[Medline].

16.   Marty, A. Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 291: 497-500, 1981[Medline].

17.   Mienville, J.-M., J. L. Barker, and G. D. Lange. Mechanosensitive properties of BK channels from embryonic rat neuroepithelium. J. Membr. Biol. 153: 211-216, 1996[Medline].

18.   Morris, C. E. Mechanosensitive ion channels. J. Membr. Biol. 113: 93-107, 1990[Medline].

19.   Nomura, K., K. Naruse, K. Watanabe, and M. Sokabe. Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers. J. Membr. Biol. 115: 241-251, 1990[Medline].

20.   Owen, J. D. The determination of the stability constant for calcium-EGTA. Biochim. Biophys. Acta 451: 321-325, 1976[Medline].

21.   Pächa, J., G. Frindt, H. Sackin, and L. G. Palmer. Apical maxi K channels in intercalated cells of CCT. Am. J. Physiol. 261 (Renal Fluid Electrolyte Physiol. 30): F696-F705, 1991[Abstract/Free Full Text].

22.   Robertson, B. E., P. Corry, P. C. G. Nye, and R. Z. Kozlowski. Ca2+- and Mg-ATP activated potassium channels from rat pulmonary artery. Pflügers Arch. 421: 94-96, 1992[Medline].

23.   Ruknudin, A., F. Sachs, and J. O. Bustamante. Stretch-activated ion channels in tissue-cultured chick heart. Am. J. Physiol. 264 (Heart Circ. Physiol. 33): H960-H972, 1993[Abstract/Free Full Text].

24.   Sadoshima, J., N. Akaike, H. Kanaide, and M. Nakamura. Cyclic AMP modulates Ca-activated K channel in cultured smooth muscle cells of rat aortas. Am. J. Physiol. 255 (Heart Circ. Physiol. 24): H754-H759, 1988[Abstract/Free Full Text].

25.   Singer, J. J., and J. V. Walsh, Jr. Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique. Pflügers Arch. 408: 98-111, 1987[Medline].

26.   Sokabe, M., and F. Sachs. Quantitative video microscopy of patch clamped membranes: stress, strain, capacitance, and stretch channel activation. Biophys. J. 59: 722-728, 1991[Abstract/Free Full Text].

27.   Taguchi, H., D. D. Heistad, T. Kitazono, and F. M. Faraci. Dilatation of cerebral arterioles in response to activation of adenylate cyclase is dependent on activation of Ca2+-dependent K+ channels. Circ. Res. 76: 1057-1062, 1995[Abstract/Free Full Text].

28.   Taniguchi, J., and W. B. Guggino. Membrane stretch: a physiological stimulator of Ca2+-activated K+ channel in thick ascending limb. Am. J. Physiol. 257 (Renal Fluid Electrolyte Physiol. 26): F347-F352, 1989[Abstract/Free Full Text].

29.   Van Wagoner, D. R. Mechanosensitive gating of atrial ATP-sensitive potassium channels. Circ. Res. 42: 973-983, 1993.

30.   Wong, B. S., H. Lecar, and M. Adler. Single calcium-dependent potassium channels in clonal anterior pituitary cells. Biophys. J. 39: 313-317, 1982[Abstract/Free Full Text].

31.   Yang, X. C., and F. Sachs. Block of stretch-activated ion channels in Xenopus oocytes by gadolinium and calcium ions. Science 243: 1068-1071, 1989[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 276(6):H1827-H1838
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
C.-R. Kong, N. Bursac, and L. Tung
Mechanoelectrical excitation by fluid jets in monolayers of cultured cardiac myocytes
J Appl Physiol, June 1, 2005; 98(6): 2328 - 2336.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
J. Wang, S. Morishima, and Y. Okada
IK channels are involved in the regulatory volume decrease in human epithelial cells
Am J Physiol Cell Physiol, January 1, 2003; 284(1): C77 - C84.
[Abstract] [Full Text] [PDF]

Home page
 J. Thorac. Cardiovasc. Surg.Home page
A. C. Nicolosi, G. West, J. G. Markley, B. Logan, and G. N. Olinger
Gadolinium attenuates regional stunning in the canine heart in vivo
J. Thorac. Cardiovasc. Surg., July 1, 2002; 124(1): 57 - 62.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
B. B. Lerman, E. D. Engelstein, and D. Burkhoff
Mechanoelectrical Feedback: Role of {beta}-Adrenergic Receptor Activation in Mediating Load-Dependent Shortening of Ventricular Action Potential and Refractoriness
Circulation, July 24, 2001; 104(4): 486 - 490.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
A. C. Nicolosi, C. S. Kwok, S. J. Contney, G. N. Olinger, and Z. J. Bosnjak
Gadolinium prevents stretch-mediated contractile dysfunction in isolated papillary muscles
Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1122 - H1128.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow