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Published as doi: 10.1096/fj.05-5049fje.
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(The FASEB Journal. 2006;20:1492-1494.)
© 2006 FASEB

Tyrosine kinase inhibitors and ATP modulate the conversion of smooth muscle L-type Ca2+ channels toward a second open state

Shinsuke Nakayama*,1, Yasushi Ito{dagger}, Shinji Sato{dagger}, Atsushi Kamijo*, Hong-Nian Liu* and Shunichi Kajioka*,2

Department of
* Cell Physiology and

{dagger} Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

1Correspondence: Department of Cell Physiology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: h44673a{at}nucc.cc.nagoya-u.ac.jp

ABSTRACT

Properties of smooth and cardiac L-type Ca2+ channels differ prominently in several physiological aspects, including sympathetic modulation. To assess the possible underlying mechanisms, we applied the whole cell patch-clamp technique to guinea pig detrusor smooth muscle cells, in which only L-type Ca2+ channel currents are observed in practice. During depolarization to large positive potentials, the conformation of the majority of L-type Ca2+ channels is converted from the normal (O1) to a second open state (O2), which undergoes little inactivation during depolarization. Extracellular application of genistein, a known tyrosine kinase inhibitor, significantly attenuated the voltage-dependent conversion of Ca2+ channels to O2, accompanied by reduction of availability, whereas genistin, an inactive analog, had little effect. In the absence of ATP in the patch pipette, intracellular application of either genistein or tyrphostin-47 suppressed the conversion to O2. Computer calculation revealed that the acceleration of the O1 to an inactivated state qualitatively reconstructs the unique effects of PTK inhibitors antagonized by ATP. We concluded that under normal conditions smooth muscle L-type Ca2+ channels are already modulated by tyrosine-kinase and ATP-related mechanism(s) and thereby easily achieve the second conversion, which yields voltage-dependent modulation of L-type Ca2+ current analogous to that in cardiac myocytes during ß-adrenoceptor stimulation.—Nakayama, S., Ito, Y., Sato, S., Kamijo, A., Liu, H.-N., Kajioka, S. Tyrosine kinase inhibitors and ATP modulate the conversion of smooth muscle L-type Ca2+ channels toward a second open state.


Key Words: persistent Ca2+ channel current • genistein • genistin

PROPERTIES OF SMOOTH and cardiac L-type Ca2+ channels differ prominently in several physiological aspects, although these Ca2+ channels are splice products with 95% homology (1) . It is well known that cardiac muscle L-type Ca2+ channels play a central role in the inotropic action of sympathetic nerves through stimulation of ß-adrenoceptors and initiation of the cyclic AMP cascade, resulting in phosphorylation of the channel protein (2 , 3) . On the other hand, sympathetic stimulation relaxes most smooth muscles, including detrusor, via activation of ß-adrenoceptors (4) , and smooth muscle L-type Ca2+ channels are not sufficiently enhanced to alter physiological function (especially in terms of cyclic AMP cascade (5) ). It is of interest to address what mechanisms produce this contrast in Ca2+ channel regulation.

It is also known that ß-adrenoceptor stimulation alters the gating properties of cardiac L-type Ca2+ channels (6 , 7) . Recently, lines of evidence have shown that ß-adrenoreceptor-mediated potentiation of cardiac L-type Ca2+ channel current is accompanied by U-shaped inactivation (8 9 10) , i.e., reduction of the degree of inactivation at positive conditioning potentials. In smooth muscle L-type Ca2+ channels, we have previously reported similar U-shaped inactivation produced by a second, long channel open state (O2) (11) . This long open state (O2) is the result of a transition from the normal open state (O1) following depolarizations to more positive potentials than required for the closed (C) to O1 transition (12) . It is considered that the presence of multiple open states can allow variation in the routes of Ca2+ influx, from action potential generation to persistent Ca2+ influx (13 , 14) .

There is now an accumulating body of evidence that the activity of smooth muscle L-type Ca2+ channels can be modulated through mechanisms related to the tyrosine kinase cascade (15 16 17 18 19 20) . In cardiac muscle, it has recently been reported that ß-adrenoreceptor stimulation can also activate the tyrosine kinase cascade (21) . In the present study, we therefore aimed to address the hypothesis that under normal conditions, smooth muscle L-type Ca2+ channels are already modulated by factors related to the PTK cascade such that they are conditioned to undergo the transition to the O2 state, thus resulting in functional diversity in L-type Ca2+ channels. We examined the effects of PTK inhibitors on voltage-sensitive Ca2+ channel current in guinea pig detrusor smooth muscle cells. We chose this model because L-type Ca2+ channels are the main Ca2+ channels expressed in this smooth muscle, and the majority of these L-type Ca2+ channels undergo the O2 state conversion during a conditioning step of high positivity.

MATERIALS AND METHODS

Preparation of cells
Guinea pigs of ~3 wk after birth were killed by cervical dislocation and exsanguination after anesthetizing them with diethyl ether. The animals were treated in accordance with the Animal Experimental Guides of Nagoya University Graduate School of Medicine. The urinary bladder was quickly dissected. After removal of epithelium, detrusor muscle was cut into small pieces, which were then incubated with a Mg2+-free, Ca2+-free solution containing collagenase (1.5 mg/ml, No 034–10533; Wako Chemical, Osaka, Japan), trypsin inhibitors (2 mg/ml, type I-S; Sigma, St. Louis, MO, USA) and Pronase (1.5 mg/ml; Fulka, Buchs, Switzerland) for 12–15 min at 37°C. After rinsing with a Mg2+-free, Ca2+-free solution containing no enzymes, smooth muscle cells were dissociated by triturating with a fire-blunted glass pipette. Some of the cell suspension was stored in a low-Mg2+ (~0.05 mM), low-Ca2+ (~0.1 mM) solution at 5°C before use, and used for up to 6 h.

Current recording
The whole cell membrane currents were recorded using a voltage-clamp amplifier (Axopatch 200A: Axon Instruments, Foster City, CA, USA) through an AD/DA converter (TL-1, Axon Instruments). The measurements were carried out at room temperature (21–26°C). A cut-off frequency of 5 kHz was applied to reduce the noise. The resistance of the patch pipette was in the region of 3–6 M{Omega}, when a Cs+-rich pipette solution was used. Single smooth muscle cells of 15–70 pF membrane capacity were used for the electrical recordings. The capacitive current was electrically compensated. The series resistance (6–13 M{Omega} on rupture of the patch membrane) was partially compensated (by 40–60%). The time to clamped voltage was set to be less than 0.2 ms. The voltage error was normally less than 7 mV, when the time constant of the tail current was analyzed. Unless otherwise stated, the voltage of the cell membrane was clamped at –60 mV (holding potential).

In guinea-pig detrusor, it has been reported that both T- and L-type Ca2+ channels contribute to electrically stimulated contractions (22) . It has been also reported that T-type Ca2+ channels are activated at membrane potentials close to the holding potential applied in the present study, suggesting that T-type Ca2+ channels are already inactivated in our experiments. Furthermore, dihydropyridine Ca2+ channel antagonists can nearly completely abolish voltage-operated inward Ca2+ current. These facts indicate that we deal with only L-type Ca2+ channels practically.

A paired pulse protocol was used to estimate the population of L-type Ca2+ channels, which can be converted from the normal to a second open state (11 , 12 , 23 , 24) . Test steps to 0 mV (100 ms) with and without preconditioning depolarization (+80 mV, 4 s) were alternately applied with appropriate intervals. We have previously shown that the Cs+-rich, 2 mM EGTA-containing pipette solution, which blocks outward K+ channel currents and Ca2+-activated channel currents during test and repolarization steps, enables us to analyze voltage-dependent modulation of Ca2+ channel kinetics. Furthermore, the preconditioning procedure at +80 mV can rule out the contribution of Ca2+-dependent inactivation on L-type Ca2+ channels, because of a very small driving force for Ca2+ ions at +80 mV.

Solutions and drugs
The "normal" solution had the following composition (mM): 125 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgCl2, 11.8 glucose, and 11.8 HEPES (N-2-hydroxyethylpiperadine-N-2-ethanesulphonic acid); pH was adjusted to 7.4 with Tris base. Modification of the extracellular medium for cell preparation was made by iso-osmotical substitution with NaCl, and no chelating agent was added. The composition of the pipette solution was (mM): 14.1 CsCl, 1.4 MgCl2, 7 TEA (tetraethyl ammonium, 2 EGTA (ethylene glycol-bis-(ß-aminoethylether) N, N, N', N'-tetraacetic acid), 1 ATP, 0.1 GTP (guanosine 5'-triphosphate), 10 HEPES/Tris (pH 7.2). In some experiments, ATP and GTP were simultaneously removed from the pipette solution in order to potentiate the effect of intracellular applications of tyrosine kinase inhibitors.

The following chemicals were used in the present study: genistein, genistin, EGTA (free acid), and sodium orthovanadate from Sigma (St. Louis, MO, USA); ATP (disodium salt) and GTP (trisodium salt) from Seikagaku Corporation (Tokyo, Japan); tyrphostin-47 and PP2 from BIOMOL (Plymouth Meeting, PA, USA). Stock solutions of genistein, genistin, and tyrphostin-47 were prepared by dissolving these drugs in DMSO (dimethyl sulfoxide). The drugs were diluted just before use.

Statistics and data analysis
The numerical data were expressed as means ± SD. Differences between means were evaluated by Student’s t-tests or repeated-measures ANOVA; when a significant difference was permitted by ANOVA in a category of groups, differences between a control group and other groups were evaluated by Dunnett’s test. P < 0.05 was normally taken as a statistically significant difference. Single and double asterisks indicate P < 0.05 and P < 0.01, respectively.

The deactivation time constant of tail current was estimated by fitting the discrete data points iteratively with a single exponential function (11 , 12) .

Simulation of Ca2+ channel current and U-shaped inactivation property was performed using Mathcad 2000 (MathSoft, Cambridge, MA, USA). Numerical solutions of simultaneous differential equations were obtained using a Runge-Kutta procedure. We applied a kinetic scheme in which one closed and two open states (C-O1-O2) are sequentially linked and the C and O1 states have their corresponding inactivated states (I0 and I1) (23) .

RESULTS

Distinct effects of genistein on conditioned and unconditioned Ca2+ channel currents
In enzymatically isolated detrusor smooth muscle cells, Ca 2+ channel currents were evoked by test steps (0 mV, 100 ms) with and without a preconditioning step (+80 mV, 4 s). We have previously shown that relatively large depolarizations convert the channel conformation from the normal (O1) to a second open state (O2) in which Ca2+ channels inactivate only very slowly or not at all during depolarization, and deactivate slowly on repolarization (12 , 25) . The U-shape of the steady state inactivation curves seen with preconditioning steps (4–5 s) of large positivity is considered to reflect the O2 state (11 , 23) . In the present study, we therefore designed the paired pulse protocol to examine mechanisms and factors affecting development of O2.

Genistein, a soybean isoflavone, is known to selectively inhibit protein tyrosine kinase (PTK) (26) . First, we examined the effects of this drug on Ca2+ channel currents evoked by test steps with and without a preconditioning depolarization. The patch pipette contained CsCl and TEA to suppress K+ channel current, and ATP to slow the rundown of (L-type) Ca2+ channel current. Figure 1 shows an example of such experiments. In Figure 1A , conditioned (red) and unconditioned (blue) currents are shown superimposed. Under superfusion with normal extracellular solution (a = control), the peak amplitude of the conditioned inward current (Ic) recorded during the test step was larger than that of unconditioned inward current (Iu). Application of 10 µM genistein in the extracellular medium gradually reduced both Ic and Iu. The suppression was more pronounced in Ic; thereby, the Ic/Iu ratio decreased. The current traces (b) and (c) were obtained 4.5 and 10.5 min, respectively, after application of genistein. Ic and Iu were 53.2 and 79.4%, respectively, after 4.5 min (b), and 28.9 and 50.6% of the control Ic and Iu values, after 10.5 min (c). Ic/Iu thus decreased from 1.46 (a) to 0.98 (b), and to 0.83 (c). Slowly deactivating tail currents were observed after preconditioning depolarization. The decay time constant of the slow-tail current ({tau}tail) in (a) was 11.2 ms. During the subsequent continuous application of genistein, {tau}tail varied between 8 and 12 ms, irrespective of the change in amplitude of the test and tail currents. Similar results were obtained from other isolated detrusor smooth muscle cells examined. Because Ca2+-dependent inactivation was negligible during the conditioning depolarization at +80 mV (12) , it is suggested that mechanisms other than this inactivation process caused the pronounced suppression of Ic.


Figure 1
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  		Figure 1. Effects of genistein on whole-cell Ca2+ channel currents evoked with (red) and without (blue) preconditioning depolarization. A test step of 0 mV (100 ms) was repeated at 45-s intervals except the interval before the last two test steps. A preconditioning step (+80 mV, 4 s) was applied before every other application of the test step. A) Pairs of whole cell membrane currents (with and subsequently without preconditioning depolarization) are normalized by cell capacitance and shown superimposed. Only the last 25 ms of the conditioning step is shown. Dotted lines indicate zero current levels. The left calibration bar (10 pA/pF) is for (a), and the right one (5 pA/pF) is for (b) and (c). After observing a control pair of membrane currents (a), 10 µM genistein was applied in the extracellular medium. The current traces (b) and (c) were recorded during continuous application of genistein. The acquisition times of (a–c) are indicated by horizontal bars in B. The upper graph in B shows changes in the amplitude of the peak inward current during test step. Red squares and blue circles are for the peak amplitudes of inward currents evoked with (Ic) and without preconditioning depolarization (Iu), respectively. The ratio of (Ic/Iu) in the lower graph was estimated by pairing inward currents evoked by preconditioned test step and subsequent unconditioned test step. The symbol (triangle) for the ratio is plotted in the middle time of each pair. Note the reversal of the Ic/Iu ratio. C) Correlation of Ic/Iu (x axis) and Iu (y axis) is plotted. Each blue circle represents one of 25 individual detrusor cells used for the paired pulse protocol in normal solution (the initial pair of test inward currents after rupture of the cell membrane were used to estimate each set of x-y values). Solid and open squares (orange) represent individual cells in the presence of 10 µM (n=5) and 50 µM genistein (n=3), respectively. Recordings were made 5–12 min after exposure to the drug. D) Inactivation curve in the presence of 10 µM genistein. Inactivation property was examined over a wide voltage range (-100 to +80 mV) ~12 min after application of genistein. The test potential was 0 mV, and the duration of the conditioning step was 4 s. The mean amplitude of the test inward current normalized by that preconditioned at –60 mV (I/I-60, green circles) was plotted against the conditioning potential (n=4). The red square represents (I+80/I-60 = Ic/Iu) before application of genistein. Vertical bars, SD.

Figure 1C shows a correlation plot of the current density of test inward current (Iu = y axis) and the Ic/Iu ratio (x axis). In the 25 individual detrusor cells examined using the paired pulse protocol under normal conditions, the average of the current density Iu was –8.58 ± 4.63 pA/pF, and the Ic/Iu ratio was 1.39 ± 0.32. In the presence of genistein, these two parameters were smaller (Table 1) .


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  		Table 1. The current density (Iu) was and the Ic/Iu ratio in normal solution and in the presence of extracellular genistein

In the presence of 10 µM genistein (~12 min after the application), the inactivation property was examined over a wide voltage range (n=4). The test potential and duration of conditioning step were 0 mV and 4s, respectively. To minimize effects of the preceding voltage sequence, the conditioning potentials were applied in the following order: –60, +80, –80, –100, +60, –40, +40, +20, –20, 0 mV (11 , 23) . As shown in Fig. 1D , the inactivation curve was still clearly U-shaped, with a maximal degree of inactivation at 0 mV [0.038 ± 0.018 of the test current conditioned at –60 mV (= I-60)]. The recovery of the test current amplitude at +80 mV (I+80) reached 0.75 ± 0.13 of I–60 (n=4) in the presence of genistein. This value was, however, significantly smaller than the Ic/Iu ratio (1.41±0.2) measured before application; (I+80/I–60 = Ic/Iu). Also, curve fitting of the data from –100 to 0 mV with a sigmoid function provided a half-inactivation potential (Ei0.5) of –43.6 mV, suggesting a hyperpolarizing shift compared to those previously obtained in normal solution (Ei0.5: –31.6 mV) (11) (See also online supplemental material.).

Effects of genistin, an inactive analog of genistein
Genistin is a glycosylated analog of genistein, which has no effect on PTK (16 , 27 , 28) . Figure 2 shows the effect of genistin on voltage-dependent Ca2+ channel currents. The same, paired pulse protocol shown in Fig. 1 was used. A control pair of conditioned and unconditioned membrane currents was observed (a); 10 µM genistin was then applied to the extracellular medium. During the application of genistin, Ic and Iu slowly decreased by similar degrees, and the Ic/Iu ratio remained at around the control concentration. The current traces (b) and (c) were obtained ~4.5 and ~15 min after application of genistein, respectively. Ic and Iu were 90.1 and 94.4%, respectively, of the control in (b), and 73.3 and 74.8% of the control in (c). The Ic/Iu ratio of this cell was 1.38 in control (a), 1.32 in (b) and 1.35 in (c). In the presence of 10 µM genistin, the paired pulse protocol was applied in five other cells. The average of the current density Iu was –6.77 ± 4.01 pA/pF, and the Ic/Iu ratio was 1.49 ± 0.15 (6–15 min after genistin application, n=5). Taken together, the results with genistein and genistin suggest that PTK-related mechanisms play important roles in the basal availability of Ca2+ channels, especially for the conversion to the O2 state.


Figure 2
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  		Figure 2. Effects of genistin, an inactive analog of genistein. The paired pulse protocol shown in Fig. 1 was used. Test steps (0 mV, 100 ms) with and without preconditioning depolarization (+80 mV, 4 s) were alternately applied as shown in B. A) Pairs of conditioned and unconditioned current traces: Conditioned ones are indicated by arrowheads. Only the last 25 ms of the conditioning step is shown. Dotted lines indicate zero current levels. The left calibration bar (10 pA/pF) is for (a), and the right one (5 pA/pF) is for (b) and (c). After observing a control pair of membrane currents (a), 10 µM genistin was applied in the extracellular medium. The current traces (b) and (c) were recorded during continuous application of this drug. The acquisition times of (a–c) are indicated by horizontal bars in B. In B, the upper graph shows the time courses of changes in Ic (squares) and Iu (circles), while the lower graph is for the Ic/Iu ratio (triangles). The symbol for the ratio is plotted in the middle time of each pair.

Intracellular application of genistein and tyrphostin-47
Genistein was next applied through the patch pipette. The patch pipette also contained 1 mM ATP (as it did during experiments examining the effects of extracellular applications). The inclusion of 10 µM genistein in the patch pipette did not have much effect on Ca2+ channel currents evoked by the paired pulse protocol (n=4). However, because it is considered that greater concentrations are required when applied through a patch, 30 µM genistein was included for the next series of experiments. (ATP was also included.) Figure 3 A and B show an example of such experiments. The current traces in A(a) show the initial pair of the L-type Ca2+ channel currents with and without a preconditioning depolarization step (+80 mV, 4 s). These current traces were obtained ~2 min after rupture of the cell membrane with a patch pipette containing 30 µM genistein (and 1 mM ATP). Subsequent traces (b), (c), and (d) were 3rd, 8th, and 14th pairs respectively and were recorded at times indicated in B. As shown in B, both Ic and Iu temporarily increased in (b) (to 107.8 and 115.8% of the initial values, respectively) and subsequently slowly decreased by similar degrees (to 97.1 and 105.2% (c); to 55.8 and 55.8% of the initial values, respectively (d))(top), while the Ic/Iu ratio remained around the initial level (1.44 in (a); 1.34 in (b); 1.33 in (c); 1.44 in (d))(bottom). Similar recordings were obtained from three other cells. Although the recordings were made at different times after the rupture, the maximal Iu for all cells was 111.1 ± 7.9% of the initial amplitude (n=4). Genistein is known to inhibit PTK by competing with ATP (26) . Because of slow dialysis through the patch, in order to be effective, intracellular application of genistein may require reduction of intracellular ATP concentration. In the next series of experiments, we therefore examined the effect of intracellular application of PTK inhibitors with the patch pipette containing no ATP.


Figure 3
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  		Figure 3. Effects of genistein application via the patch pipette containing 1 mM ATP. Note the antagonistic action of ATP to PTK inhibition. A) Paired whole cell currents: Conditioned currents are indicated by arrow heads. Dotted lines indicate zero current levels. Test steps (0 mV, 100 ms) with and without preconditioning depolarization (+80 mV, 4 s) were alternately applied as shown in B. The patch pipette contained 30 µM genistein and 1 mM ATP. Initial pair recorded ~2 min after rupture of the cell membrane is shown (a). (Before starting to record the series of paired membrane currents, capacitive currents were carefully compensated by adjusting the parameters in the amplifier, and the amplitude of unconditioned current was checked.) b–d) were subsequently recorded during the intracellular application of genistein. The acquisition times of (a–d) are indicated by horizontal bars in B. In B, squares, circles, and triangles indicate changes in Ic, Iu, and the Ic/Iu ratio caused by intracellular application of genistein with ATP in the pipette. The symbol for the ratio is plotted in the middle time of each pair.

Figure 4 shows paired current traces and the time courses of changes in Ic (red), Iu (blue) and Ic/Iu ratio (green). The current traces (a–d) in A were recorded at times indicated in B. As shown in the figure, removal of ATP yielded dramatic changes in the effects of intracellular application of genistein. In contrast to the results shown in Fig. 3 , Ic and Iu decreased faster to 23.3 and 32.4% after ~18 min (c), and to 8.2 and 13.5%, respectively, of the initial values after ~27 min (d). The suppression of Ic was more pronounced. As a result, the Ic/Iu ratio was reversed [from 1.25 (a) to 0.90 (c) and to 0.76 (d)], mimicking the features of Ca2+ current suppression observed during extracellular (bath) application of genistein (Fig. 1) . Similar recordings were obtained from 7 out of the 10 other cells examined.


Figure 4
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  		Figure 4. Effects of genistein application via the patch pipette containing no ATP. A) Paired whole-cell currents. Dotted lines indicate zero current levels. The same paired pulse protocol shown in Fig. 3 was applied. B) The time courses of changes in Ic (squares), Iu (circles), and the Ic/Iu ratio (triangles). The patch pipette contained 30 µM genistein, but no ATP. Conditioned and unconditioned experiments are shown in red and blue, respectively. The Ic/Iu ratio is shown in green. The left calibration bar for (a) and (b); right for (c) and (d).

To confirm whether PTK-related mechanisms underlie the genistein-induced suppression of Ca2+ channel current, we next examined the effect of tyrphostin-47, another PTK inhibitor with broadband spectrum (29 , 30) . Instead of genistein, 30 µM tyrphostin-47 was included in the ATP-free pipette solution, and the same paired pulse protocol was applied. As shown in Fig. 5 , intracellular application of tyrphostin-47 produced essentially the same inhibitory effects on Ca2+ channel current. Both Ic and Iu were gradually decreased during the whole-cell recording, but the suppression of Ic was more pronounced. The Ic/Iu ratio progressively decreased to 0.35. Similar results were obtained from three other cells.


Figure 5
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  		Figure 5. Effects of tyrphostin-47 application via the patch pipette containing no ATP. Instead of genistein, 30 µM tyrphostin-47 was included in an ATP-free pipette solution. Other explanations are the same as Fig. 3 and 4 .

Figure 6 summarizes characteristic features of Ca2+ channel current seen during intracellular application of genistein and Tyrphostin-47. When ATP was included in the pipette, genistein had little effect on the Ic/Iu ratio. In contrast, in the absence of ATP, the Ic/Iu ratio decreased progressively as Iu decreased during application of either genistein or tyrphostin-47 in the pipette. Also, in the absence of ATP, genistein and tyrphostin-47 progressively enhanced the rate of inactivation in Iu, as Iu decreased. On the other hand, the rate of inactivation in Ic was not significantly altered (data not shown, P > 0.05 for both genistein and tyrphostin-47). The decay time constant of the slow-tail current ({tau}slow-tail) induced by the conditioning depolarization was not affected by these PTK inhibitors even in the absence of ATP.


Figure 6
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  		Figure 6. Graphs summarizing ATP-dependent suppression of Ca2+ channel current caused by PTK inhibitors. Statistics were made by grouping the pairs of Ic and Iu for the initial (1.0) and with the amplitude of Iu closest to 0.75, 0.50, and 0.25 of the initial value. Effects of tyrosine kinase inhibitors were analyzed by correlating the changes in Ic/Iu (A), the inactivation ratio of Iu at the end of the test current (0 mV, 100 ms) (B), and the decay time constant of slow-tail current induced by conditioning depolarization ({tau}slow-tail) (C) with the reduction of Iu. Single and double asterisks indicate P < 0.05 and P < 0.01, respectively, (vs. the group of 1.0 Iu) in Dunnett’s test.

Intracellular dialysis of an ATP-free solution in the absence of PTK inhibitors
ATP is involved in numerous kinase activities, including PTK. The paired pulse protocol was, thus, applied in the detrusor cells dialyzed with a pipette solution containing neither ATP nor PTK inhibitors. Figure 7 A shows an example of changes in the test currents with (upper trace) and without preconditioning depolarization at +80 mV (4 s)(lower trace). Both currents gradually decreased during the dialysis. As shown in B, the Ic/Iu ratio and the rate of inactivation in Iu (Iu (100 ms)/Iu(peak)) decreased, as Iu decreased, while {tau}slow-tail of the tail current after the conditioning depolarization hardly changed (n=8). The decrease in Ic/Iu ratio was, however, significantly smaller than that in the presence of PTK inhibitors in the pipette.


Figure 7
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  		Figure 7. Changes in Ca2+ channel current in ATP-free control. The same, paired pulse protocol used in Figs. 4 and 5 was applied in the detrusor cells dialyzed with a pipette solution containing neither ATP nor PTK inhibitors. A) paired whole cell currents. The current traces (a–d) represent, respectively, the initial pair and the pairs with the amplitude of Iu closest to 0.75, 0.5, and 0.25 of the initial value. (The traces are 1st, 4th, 6th, and 8th pairs.) B) Graphs summarizing ATP-free control. See Fig. 6 for abbreviations. Single and double asterisks indicate significant differences of P < 0.05 and P < 0.01, respectively, vs. the group of 1.0 Iu (Dunnett’s test). Single and double sharps indicate significant differences of P < 0.05 and P < 0.01, respectively, vs. corresponding data obtained with 30 µM genistein shown in Fig. 6 (unpaired Student’s t test).

Intracellular application of PP2
PP2 is known to inhibit the tyrosine kinase activity of src in a noncompetitive manner against ATP (31) . Lastly, we examined the effect of PP2 (30 µM) application via the patch pipette containing ATP. The paired pulse protocol was again applied. Figure 8 A shows an example of such experiments. The initial pair (a) was obtained ~2 min after the rupture the membrane. The amplitude of the both Ic and Iu increased significantly for initial ~10 min [(b) = 4th pair: maximal amplitude], then slowly decreased [(c) = 12th, (d) = 24th pair]. It is noted that even in the presence of ATP, intracellular application of PP2 significantly reduced the Ic/Iu ratio, accompanied by acceleration of Iu inactivation. Essentially, similar results were obtained in three other cells. The graphs in B summarize the effects of PP2 on the Ic/Iu ratio, Iu inactivation rate and {tau}slow-tail.


Figure 8
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  		Figure 8. Effects of PP2 application via the patch pipette containing ATP. The same, paired pulse protocol used in Fig. 7 was applied. A) Paired whole cell currents. The current traces (a–d) represent the initial pair and the pairs with the amplitude of Iu closest to 0.75 and 0.5, respectively, of the initial value. (The traces are 1st, 4th, 12th, and 24th pairs.) The patch pipette contained 30 µM PP2 and 1 mM ATP. B) Graphs summarizing the effects of PP2 in the presence of ATP. Statistical analysis was performed by grouping the pairs of Ic and Iu with the maximal (1.0) (i.e., (b) in A) and with the amplitude of Iu closest to 0.75 and 0.50 of the maximal Iu. (Iu-max), because PP2 largely increased Iu for the initial ~10 min. Single and double asterisks indicate significant differences of P < 0.05 and P < 0.01, respectively, vs. the group of 1.0 Iu-max (Dunnett’s test).

DISCUSSION

The present patch-clamp experiments have demonstrated significant suppression of L-type Ca2+ channel current in detrusor smooth muscle by a soy-bean isoflavone, genistein. The result is consistent with previous reports on PTK inhibitors in vascular and intestinal smooth muscles (16 , 17 , 19) . In addition, we have observed that the genistein-induced suppression of L-type Ca2+ current is much more pronounced after preconditioning depolarization. This unique effect of genistein is ascribed to tyrosine kinase-related mechanisms or factors for the following reasons. First, application of tyrphostin-47, another tyrosine kinase inhibitor, reproduced this voltage-dependent inhibitory effect (Fig. 5) , but genistin, an inactive analog of genistein did not (Fig. 2) . Secondly, irrespective of extracellular or intracellular application (32) , PTK inhibitors caused similar changes in the Ic/Iu ratio (e.g., Fig. 1 and Fig. 4 ), but intracellular application of PTK inhibitors required removal of ATP from the pipette solution. This antagonistic effect of ATP on PTK inhibition is a feature of genistein and is structurally similar PTK inhibitors (26) .

During conditioning depolarization at +80 mV, Ca2+-dependent inactivation of L-type Ca2+ channels is considered to be negligible, because at this membrane potential, the driving force of Ca2+ entry is very much attenuated, and the patch pipette contains a sufficiently high concentration of Ca2+ chelating agent to ensure a small Ca2+ entry (11) . It is deduced that PTK inhibitors act on mechanisms other than Ca2+-dependent inactivation to produce the more pronounced suppression of Ic.

Previously, by use of a minimum kinetic model, we have shown that "voltage-dependent" U-shaped inactivation and slow deactivation properties are closely linked to each other and are both attributed to a second open state (O2), the conformational change of which requires relatively more positive membrane potentials than those causing the transition from the closed (C) to normal open state (O1) (23) . The experiments in the present study, comparing conditioned and unconditioned test currents, were designed in order to elucidate the effect of PTK-related mechanisms on the O2 state. Using the minimum kinetic model (Fig. 9 A), we have calculated the percentage of available Ca2+ channels and plotted it against the conditioning potential. The duration of the conditioning step was 4 s (Fig. 9B ). In the kinetic scheme, only C and O1 have their corresponding inactivated states, I0 and I1, respectively. The set of the rate constants used in the computer simulation is listed in Table 2 (33 34 35) . The availability of Ca2+ channels reconstructed for normal conditions (circles) is clearly U-shaped with a minimum (5.8%) at 0 mV, and with nearly full recovery at +80 mV (90.2%), mimicking inactivation curves previously reported in guinea-pig detrusor cells (11 , 36) . [Because the degree of activation at + 20 to +80 mV is larger than that at 0 mV (the test potential), the amplitude of test current is proportionally increased by conditioning steps of positive potentials.]


Figure 9
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  		Figure 9. Computer modeling of inactivation curves and test currents recorded under normal conditions and during application of PTK inhibitors in smooth muscle Ca2+ channels. A minimum kinetic scheme was used in the present study (Aa); one closed (C) and two open states (O1 and O2) are sequentially linked. Only the C and O1 states have their corresponding inactivated states, I0 and I1 (i.e., Ca2+ channels in O2 is not inactivated). The k(s) represent the rate constants of transition (listed in Table 2 ). Note that Ca2+-dependent inactivation was not involved in this model. Ab) A possible kinetic scheme brought about by PTK inhibitors, where the transition from O1 to I1 (the rate constant, k14*) is accelerated, and Ca2+ channels do not practically undergo the second voltage-dependent conversion toward O2. Inversely, computer simulations based on the kinetic schemes Ab and Aa mimic cardiac currents recorded in control and on ß-adrenoceptor stimulation, respectively. In B, percentage of available Ca2+ channels (C+O1+O2) at the end of conditioning step (4 s) was plotted against the conditioning potential. Circles represent available channels under normal conditions, whereas squares and triangles represent those in case of x5 and x15 k14, respectively. C) Computer calculation for Ca2+ channel current evoked by a step depolarization to 0 mV. Normal condition (a); x5 (b); x15 k14 (c). As the k14 rate increased, the recovery phase of availability (at positive conditioning potentials) was attenuated, and simultaneously, inactivation of Ca2+ channel current evoked by a step depolarization was accelerated.


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  		Table 2. The rate constants (ms–1)

A characteristic feature seen during application of PTK inhibitors is the accelerated inactivation of the test current (without conditioning depolarization), while Ca2+ entry during the test depolarization step was clearly reduced (Table 2) . The kinetic scheme in Fig. 9A , without incorporating Ca2+-dependent inactivation, has been proposed to account for "voltage-dependent" U-shaped inactivation (23) . Therefore, we suggest that modification of this kinetic scheme may account for this paradoxical effect. The rate constant k14 for the O1 to I1 transition (asterisk in Fig. 9Ab ) could be responsible for the accelerated inactivation with PTK inhibitors. Use of 5 times the rate constant k14 decreased the availability of Ca2+ channels at +80 mV to 64.7% (squares in Fig. 9B ). Use of 15 times the rate constants k14 further decreased it to 37.7% (triangles in Fig. 9B ). These changes in the availability of the Ca2+ channels were accompanied by progressive increases in the inactivation rate of the test current (C: step depolarization to 0 mV). Furthermore, it was possible to use the set of the rate constants in Table 2 to reconstruct slow-tail currents evoked after conditioning depolarization. Also, the decay time constant ({tau}=~10 ms) was little affected by increasing k14 up to 15 times (simulation not shown). Overall, acceleration of the O1 to I1 transition appears to qualitatively account for the effects of PTK inhibitors on Ca2+ channel current kinetics.

The test current in Fig. 9Cc , mimicking a late phase of the PTK effect (e.g., (d) in Figs. 5 and 7 ), has been simulated with a rate constant of 15 times k14. Using this rate constant, the calculated Ic(+80)/Iu(–60) ratio decrease to ~55% (vs. normal). This degree of suppression may not be sufficient to reconstruct the observed changes, taking the difference in degree of activation between the test (0 mV) and conditioning steps (+80 mV). (For example, in Fig. 5B (d), the Ic(+80)/Iu(-60) ratio decreased to 42% of that in (a)) This discrepancy might be explained by additional attenuation (or blockade) of the O1 to O2 transition (k12 in Fig. 9Ab ), and/or the acceleration of the O2 to I2 transition, if the I2 state exists.

In both native and cloned L-type smooth muscle Ca2+ channels, slow deactivating and U-shaped inactivation properties are preserved even in the presence of H-7 (extracellular medium) and ATP{gamma}S (included in the pipette) (23 , 24) . These facts have implied that the transition between O1 and O2 is not due to chemical modulation (phosphorylation/dephosphorylation) of channel protein, but due to a physical process (i.e., a voltage-dependent transition). The modulation of Ca2+ channel current by genistein and tyrphostin-47 seen in the present study is indicative of involvement of PTK-related mechanism(s), but it is also consistent with the previous notion of a voltage-dependent transition. Treatments with PTK inhibitors gradually decreased the Ic, Iu, and Ic/Iu ratio. However, alternate applications of test steps, with and without preconditioning depolarization, revealed that slow-tail currents were evoked only after preconditioning depolarization.

With respect to the effects of ATP, it may be noteworthy that intracellular application of genistein temporally potentiated L-type Ca2+ channel current in guinea pig detrusor cells, especially when ATP was included in the pipette (Fig. 3A and 3B). This effect might be due to another mechanism relating to PTK. In cardiac myocytes, biphasic modulation of L-type Ca2+ channel current (temporal suppression followed by potentiation) has been reported. This subsequent potentiation might correspond to the effect of intracellular application of genistein in the presence of ATP (37) . Conversely, removal of ATP from the patch pipette accelerated the reduction in Ca2+ channel current even in the absence PTK inhibitors (Fig. 7) . This is probably mainly due to the run-down phenomenon in the absence of ATP (38 , 39) . The decrease in Ic/Iu ratio was significantly smaller without PTK inhibitors. Besides prevention of run-down, the existence of ATP itself might have some supporting effect on the conversion of the Ca2+ channel conformation toward O2 state. Alternatively, it could be explained by suppression of a wide range of phosphorylation processes, including PTK. Furthermore, ATP is known to be essential for many cellular functions maintaining the intracellular environment, such as Ca2+ pumps. These points require further investigation.

It is well known that ß-adrenoreceptor stimulation potentiates L-type Ca2+ channel current in cardiac myocytes presumably via cyclic AMP-dependent phosphorylation of the channel protein (6 , 7) . Furthermore, recent studies have suggested that cardiac L-type Ca2+ channels additionally acquire U-shaped inactivation on ß-adrenoreceptor stimulation (8 9 10) . Together with these data, the present results using PTK inhibitors provide us with a unique insight into the regulatory mechanism on smooth muscle Ca2+ channel activity. Namely, it is speculated that under normal conditions smooth muscle L-type Ca2+ channels are already set in a ‘stimulated’ channel mode mimicking cardiac Ca2+ channels after ß-adrenoreceptor stimulation, and are not further potentiated by sympathetic signals. It is noteworthy that L-type Ca2+ channels in some smooth muscles show incomplete conversion to noninactivating state during depolarizations of high positivity (40) . These smooth muscle Ca2+ channels might undergo further potentiation via similar mechanisms operating in cardiac myocytes.

The O2 state of smooth muscle L-type Ca2+ channels may not be conferred by phosphorylation of the channel protein itself, but by association of PTK and/or related signaling molecule(s) with the Ca2+ channel protein, probably with support of ATP (37) . This notion is consistent with the fact that a broad phosphotyrosine phosphatase inhibitor, orthovanadate (1 mM) (41) did not prevent the PTK-inhibitor-induced suppression of smooth muscle Ca2+ channel current (evoked by either conditioned or unconditioned test step; unpublished observation). For example, association of such molecules with the C-terminus of the Ca2+ channel protein might play a latch role in converting the channel conformation toward the inactivated state. It has been shown that, pp60c-src, a nonreceptor PTK, which is abundant in smooth muscle, can potentiate L-type Ca2+ channel current (15) , and that PTK is indeed associated with the {alpha}1 subunit of smooth muscle L-type Ca2+ channels (17) . This association may occur via auxiliary subunits (e.g., ß3-subunit (42 43 44) ).

As shown in Fig. 8 , even in the presence of ATP, intracellular application of PP2 (a src inhibitor noncompetitive against ATP) (31) decreased the Ic/Iu ratio, accompanied by the acceleration of Ic inactivation. These changes were essentially similar to the effects of intracellular application of either genistein or Tyrphostin-47 in the absence of ATP, but occurred only very slowly. Also, intracellular application of PP2 caused temporal potentiation of Ca2+ channel current, much more prominent than that caused by genistein in the presence of ATP. These results support the involvement of PTK- (especially src-) relating mechanisms in the second voltage-dependent conversion of L-type Ca2+ channels and also suggest the presence of multiple sites for PTK actions.

In conclusion, the present patch-clamp measurements of pairing conditioned and unconditioned Ca2+ channel currents have revealed that PTK- and ATP-related mechanism(s) confer the noninactivating O2 state to smooth muscle L-type Ca2+ channels, in addition to increasing their availability. It is also speculated that this mechanism may provide an important link of channel kinetics between smooth and cardiac Ca2+ channels, i.e., even under normal conditions, smooth muscle Ca2+ channels are set in a channel mode, similar to cardiac L-type Ca2+ channels on ß-adrenoreceptor stimulation. Furthermore, the present study opens the possibility that Ca2+ influx through the noninactivating O2 state might play an important role in cell growth and cell-matrix interaction, because PTK-related mechanisms or factors underlie this open state. To elucidate the detailed control and functional diversity of L-type Ca2+ channels, it seems to merit further investigation on PTK-related modulation of the noninactivating O2 state, including the roles of ß- and other auxiliary subunits.

ACKNOWLEDGMENTS

The authors are grateful to Professors Hamid I. Akbarali (University of Oklahoma), Alison F. Brading (Oxford University), Masaki Kameyama (Kagoshima University), Drs. Masahiro Aoyama (Nagoya University) and Lorraine M. Smith (Edinburgh University) for stimulating discussion and improving the manuscript. This work was partly supported by Grants-in-Aid for scientific research from the Japan Society for the Promotion of Science.

FOOTNOTES

2 Present address: Oita University School of Medicine, Oita 879-5593, Japan.

Received for publication November 18, 2005. Accepted for publication February 27, 2006.

REFERENCES

  1. Hofmann, F., Biel, M., Flockerzi, V. (1994) Molecular basis for Ca2+ channel diversity. Annu. Rev. Physiol. 17,399-418[CrossRef]
  2. Kameyama, M., Hofmann, F., Trautwein, W. (1985) On the mechanism of ß-adrenergic regulation of the Ca channel in the guinea-pig heart. Pflügers Arch. 405,285-293[CrossRef][Medline]
  3. Cavalie, A., Allen, T. J., Trautwein, W. (1991) Role of the GTP-binding protein Gs in the beta-adrenergic modulation of cardiac Ca channels. Pflügers Arch. 419,433-443[CrossRef][Medline]
  4. Bülbring, E., Ohashi, H., Tomita, T. (1981) Adrenergic mechanisms. Bülbring, E. Brading, A. F. Jones, A. W. Tomita, T. eds. Smooth Muscle: An Assessment of Current Knowledge ,219-248 Edward Arnold London.
  5. Beech, D. J., McHugh, D. (1996) Regulation of opening of voltage-gated Ca channels in smooth muscle cells. Bolton, T. B. Tomita, T. eds. Smooth Muscle Excitation ,39-54 Academic Press London.
  6. Yue, D. T., Herzig, S., Marban, E. (1990) ß-adrenergic stimulation of calcium channels occurs by potentiation of high-activity gating modes. Proc. Natl. Acad. Sci. U. S. A. 87,753-757[Abstract/Free Full Text]
  7. Ochi, R. (1993) Single-channel mechanism of ß-adrenergic enhancement of cardiac L-type calcium current. Jpn J Physiol. 43,571-589[CrossRef][Medline]
  8. Mitarai, S., Kaibara, M., Yano, K., Taniyama, K. (2000) Two distinct inactivation processes related to phosphorylation in cardiac L-type Ca2+ channel currents. Am. J. Physiol. 279,C603-C610
  9. Findlay, I. (2002) ß-Adrenergic stimulation modulates Ca2+- and voltage-dependent inactivation of L-type Ca2+ channel currents in guinea-pig ventricular myocytes. J. Physiol. 541,741-751[Abstract/Free Full Text]
  10. Findlay, I. (2003) Physiological modulation of inactivation in L-type Ca2+ channels. J. Physiol. 554,275-283[CrossRef][Medline]
  11. Nakayama, S., Brading, A. F. (1993) Inactivation of the voltage-dependent Ca2+ channel current in smooth muscle cells isolated from the guinea-pig detrusor. J. Physiol. 471,107-127[Abstract/Free Full Text]
  12. Nakayama, S., Brading, A. F. (1993) Evidence for multiple open states of the Ca2+ channels in smooth muscle cells isolated from the guinea-pig detrusor. J. Physiol. 471,87-105[Abstract/Free Full Text]
  13. Nakayama, S., Smith, L. M., Tomita, T., Brading, A. F. (1996) Multiple open states of calcium channels and their possible kinetic schemes. Bolton, T. B. Tomita, T. eds. Smooth Muscle Excitation ,13-25 Academic Press London.
  14. Fry, C. H., Wu, C., Sui, G. P. (1998) Electrophysiological properties of the bladder. Int. Urogynecol. J. 9,291-198
  15. Wijetunge, S., Hughes, A. D. (1995) pp60c-src increases voltage-operated calcium channel currents in vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 217,1039-1044[CrossRef][Medline]
  16. Liu, H., Li, K., Sperelakis, N. (1997) Tyrosine kinase inhibitor, genistein, inhibits macroscopic L-type calcium current in rat portal vein smooth muscle cells. Can. J. Physiol. Pharmacol. 75,1058-1062[CrossRef][Medline]
  17. Hu, X. Q., Singh, N., Mukhopadhyay, D., Akbarali, H. I. (1998) Modulation of voltage-dependent Ca2+ channels in rabbit colonic smooth muscle cells by c-Src and focal adhesion kinase. J. Biol. Chem. 273,5337-5342[Abstract/Free Full Text]
  18. Seki, T., Yokoshiki, H., Sunagawa, M., Nakamura, M., Sperelakis, N. (1999) Angiotensin II stimulation of Ca2+-channel current in vascular smooth muscle cells is inhibited by lavendustin-A and LY-294002. Pflügers Arch. 437,317-323[CrossRef][Medline]
  19. Wijetunge, S., Lymn, J. S., Hughes, A. D. (2000) Effects of protein tyrosine kinase inhibitors on voltage-operated calcium channel currents in vascular smooth muscle cells and pp60c-src kinase activity. Br. J. Pharmacol. 129,1347-1354[CrossRef][Medline]
  20. Jin, X., Morsy, N., Shoeb, F., Zavzavadjian, J., Akbarali, H. I. (2002) Coupling of M2 muscarinic receptor to L-Type Ca2+ channel via c-src kinase in rabbit colonic circular smooth muscle. Gastroenterol. 123,827-834[CrossRef]
  21. Schäfer, M., Frischkopf, K., Taimor, G., Piper, H. M., Schlüter, K.-D. (2000) Hypertrophic effect of selective ß1-adrenoceptor stimulation on ventricular cardiomyocytes from adult rat. Am. J. Physiol. 279,C495-C503
  22. Chow, K. Y., Wu, C., Sui, G. P., Fry, C. H. (2003) Role of the T-type Ca2+ current on the contractile performance of guinea pig detrusor smooth muscle. Neurourol. Urodyn. 22,77-82[CrossRef][Medline]
  23. Aoyama, M., Murakami, M., Iwashita, T., Ito, Y., Yamaki, K., Nakayama, S. (2003) Slow deactivation and U-shaped inactivation properties in cloned Cav1.2b channels in CHO cells. Biophys. J. 84,709-724[Medline]
  24. Smith, L. M., Kajioka, S., Brading, A. F., Nakayama, S. (1999) Effects of phosphorylation-related drugs on slow Ca2+ tail current in guinea-pig detrusor cells. Eur. J. Pharmacol. 370,187-193[CrossRef][Medline]
  25. Nakayama, S., Klugbauer, N., Kabeya, Y., Smith, L. M., Hofmann, F., Kuzuya, M. (2000) {alpha}1-Subunit of smooth muscle Ca2+ channel preserves multiple open states induced by depolarization. J. Physiol. 526,47-56[Abstract/Free Full Text]
  26. Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., Fukami, Y. (1987) Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262,5592-5595[Abstract/Free Full Text]
  27. Flatman, P. W., Adragna, N. C., Lauf, P. K. (1996) Role of protein kinases in regulating sheep erythrocyte K-Cl cotransport. Am. J. Physiol. 271,C255-C263[Medline]
  28. Ogura, T., Shuba, L. M., McDonald, T. F. (1999) L-type Ca2+ channel current in guinea pig ventricular myocytes treated with modulators of tyrosine phosphorylation. Am. J. Physiol. 276,H1724-H1733[Medline]
  29. Sears, C. L., Firoozmand, F., Mellander, A., Chambers, F. G., Eromar, I. G., Bot, A. G., Scholte, B., De Jonge, H. R., Donowitz, M. (1995) Genistein and tyrphostin 47 stimulate CFTR-mediated Cl– secretion in T84 cell monolayers. Am. J. Physiol. 269,G874-G882[Medline]
  30. Tsang, F., Choo, H. H., Dawe, G. S., Wong, W. S. (2002) Inhibitors of the tyrosine kinase signaling cascade attenuated thrombin-induced guinea pig airway smooth muscle cell proliferation. Biochem. Biophys. Res. Commun. 293,72-78[CrossRef][Medline]
  31. Karni, R., Mizrachi, S., Reiss-Sklan, E., Gazit, A., Livnah, O., Levitzki, A. (2003) The pp60c-Src inhibitor PP1 is non-competitive against ATP. FEBS Lett. 537,47-52[CrossRef][Medline]
  32. Belevych, A. E., Warrier, S., Harvey, R. D. (2002) Genistein inhibits cardiac L-Type Ca2+ channel activity by a tyrosine kinase-independent mechanism. Mol. Pharmacol. 62,554-565[Abstract/Free Full Text]
  33. Nakayama, S., Brading, A. F. (1995) Interaction of Ca2+ agonist and depolarization on Ca2+ channel current in guinea pig detrusor cells. J. Gen. Physiol. 106,1211-1224[Abstract/Free Full Text]
  34. Marks, T., Jones, S. W. (1992) Calcium currents in the A7r5 smooth muscle-derived cell line. An allosteric model for Ca channel activation and dihydropyridine agonist action. J. Gen. Physiol. 99,367-390[Abstract/Free Full Text]
  35. Rothberg, R. S., Magleby, K. L. (2001) Testing for detailed balance (microscopic reversibility) in ion channel gating. Biophys. J. 80,3025-3026[Medline]
  36. Nakayama, S., Brading, A. F. (1995) Possible contribution of long open state to non-inactivating Ca current in detrusor cells. Am. J. Physiol. 269,C48-C54[Medline]
  37. Wang, Y. G., Lipsius, S. L. (1998) Genistein elicits biphasic effects on L-type Ca2+ current in feline atrial myocytes. Am. J. Physiol. 275,H204-H212[Medline]
  38. Yazawa, K., Kameyama, A., Yasui, K., Li, J.-M., Kameyama, M. (1997) ATP regulates cardiac Ca2+ channel activity via a mechanism independent of protein phosphorylation. Pflügers Arch. 433,557-562[CrossRef][Medline]
  39. Yamaoka, K., Kameyama, M. (2003) Regulation of L-type Ca2+ channels in the heart: overview of recent advances. Mol. Cell Biochem. 253,3-13[CrossRef][Medline]
  40. Imaizumi, Y., Muraki, K., Watanabe, M. (1991) Measurement of noninactivating Ca current in smooth muscle cells. Methods Neurosci. 4,44-60
  41. Du, X. L., Gao, Z., Lau, C. P., Chiu, S. W., Tse, H. F., Baumgarten, C. M., Li, G. R. (2004) Differential effects of tyrosine kinase inhibitors on volume-sensitive chloride current in human atrial myocytes: Evidence for dual regulation by Src and EGFR kinases. J. Gen. Physiol. 123,427-439[Abstract/Free Full Text]
  42. Murakami, M., Yamamura, H., Suzuki, T., Kang, M.-G., Ohya, S., Murakami, A., Miyoshi, I., Sasano, H., Muraki, K., Hano, T., et al (2003) Modified cardiovascular L-type channels in mice lacking the voltage-dependent Ca2+ channel ß subunit. J. Biol. Chem. 278,43261-43267[Abstract/Free Full Text]
  43. Murakami, M., Aoyama, M., Suzuki, T., Sasano, H., Nakayama, S., Iijima, T. (2003) A new splice variant isoform of the ß2 subunit of the voltage-dependent calcium channel and its gene structure. Mol. Cell Biochem. 254,217-225[CrossRef][Medline]
  44. Klöckner, U., Itagaki, K., Bodi, I., Schwartz, A. (1992) ß-subunit expression is required for cAMP-dependent increase of cloned cardiac and vascular calcium channel currents. Pflügers Arch. 420,413-415[CrossRef][Medline]




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