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(Hypertension. 1998;31:1125-1129.)
© 1998 American Heart Association, Inc.

Scientific Contributions

Single L-Type Calcium Channels in Smooth Muscle Cells From Resistance Arteries of Spontaneously Hypertensive Rats

Yusuke Ohya; Takuya Tsuchihashi; Shuntaro Kagiyama; Isao Abe; ; Masatoshi Fujishima

From the Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan.

Correspondence to Yusuke Ohya, MD, PhD, Second Department of Internal Medicine, Kyushu University, Faculty of Medicine, Maidashi 3–1-1, Higashi-ku, Fukuoka 812–82, Japan. E-mail ohya{at}intmed2.med.kyushu-u.ac.jp

	Abstract

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Abstract—The amplitude of the whole-cell L-type Ca²⁺ channel current recorded from vascular smooth muscle cells is reportedly greater in spontaneously hypertensive rats (SHR) than in Wistar-Kyoto rats (WKY). However, no study has examined properties of single Ca²⁺ channels in arterial cells from these strains. To further test the hypothesis that activation of L-type Ca²⁺ channels in arterial smooth muscle cells would be enhanced in SHR, we recorded single Ca²⁺ channel currents in resistance mesenteric artery cells from SHR and WKY (8 to 9 weeks of age) using a cell-attached patch clamp technique. With 50 mmol/L Ba²⁺ in the recording pipette, the depolarizing pulse from a holding potential of -40 mV evoked the single L-type Ca²⁺ channel current. Opening of the single channels was more frequent in cells from SHR than from WKY. Single-channel conductance (20 pS) and open time (1 ms at 0 mV) did not differ in the two strains. The results suggest that an increased amplitude of the whole-cell current can be attributed to the enhanced opening of single Ca²⁺ channels in the arterial smooth muscle cells from SHR compared with WKY.

Key Words: muscle, smooth, vascular • electrophysiology • calcium channels • vascular resistance • hypertension, genetic

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Changes in the properties of ion channels in vascular smooth muscle cells from hypertensive animals have been studied by use of the whole-cell patch clamp technique. The amplitude of whole-cell L-type Ca²⁺ channel currents is increased in resistance mesenteric arteries of young SHR, in cerebral arteries of adult stroke-prone SHR, and in azygous vein of neonatal SHR compared with age-matched WKY.^{1 2 3} A greater amplitude of the whole-cell Ca²⁺ channel current would be related with the increase in Ca²⁺ influx into the cell, which may contribute to alterations in the function of vascular smooth muscle cells. On the other hand, the number of radiolabeled dihydropyridine (PN200-110) bindings to the aorta of SHR did not differ from that of WKY,^{4 5} suggesting that the density of Ca²⁺ channels in the membrane is not increased in SHR. If this observation could be generalized to other vascular tissues, an alteration other than that in the density of channels would be responsible for the increased whole-cell amplitude of the Ca²⁺ channel current in SHR. We therefore hypothesized that the activation of single Ca²⁺ channel is enhanced in arterial cells from SHR compared with those from WKY. However, the alteration of single Ca²⁺ channels in SHR has not yet been clarified. Single-channel recording by use of the patch clamp technique can evaluate directly whether the channel properties are altered, but whole-cell recording is not adequate for that purpose. The present study used the single-channel recording by means of the cell-attached patch clamp technique to evaluate the basic characteristics of single Ca²⁺ channels in arteries from SHR and WKY.

	Methods

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Animals
Experiments were performed on 8- to 9-week-old SHR and WKY that had been obtained from the Disease Model Cooperative Research Association (Kyoto, Japan; SHR/IZM and WKY/IZM)⁶ at 4 weeks of age and maintained thereafter at the Institute of Experimental Animals at Kyushu University. The study protocol was approved by the Committee on Ethics of Animal Experimentation in Faculty of Medicine, Kyushu University. Systolic blood pressure was measured by the tail-cuff method. Systolic blood pressure of 8- to 9-week-old SHR (183±6 mm Hg, n=8) was significantly higher than that of age-matched WKY (136±4 mm Hg, n=8; P<.05).

Preparation of Single Cells
Single smooth muscle cells were obtained from the resistance mesenteric arterial branch (diameter <300 µm) by collagenase treatment as previously reported.^{2 7} In brief, rats were anesthetized with ether and then decapitated. The small mesenteric arteries were dissected, and connective tissue was carefully removed. The arteries were rinsed and incubated for about 15 minutes at 36°C in a Ca²⁺-free solution (in mmol/L: 145 NaCl, 6 KCl, 10 glucose, 10 HEPES, pH 7.3 titrated with NaOH). The tissue was then incubated for about 45 to 50 minutes at 36°C in the Ca²⁺-free solution containing 0.3% collagenase (Wako Chemical). The digested tissue was resuspended in Ca²⁺-free solution without collagenase, cut into small pieces with scissors, and gently agitated with a glass pipette to disperse single cells. Cells were stored at 6°C to 8°C in Ca²⁺-free solution containing 1 mmol/L MgCl₂ and 0.2% BSA (Sigma Chemical Co) until use. Cells were used for current recording within 4 hours after cell preparation.

Electrical Recording
Conventional whole-cell and cell-attached single-channel recordings were made with a patch pipette through a voltage-clamp amplifier (Axopatch 1-D, Axon Instruments) according to the method of Hamill et al.⁸ Conditions and procedures were basically the same as those we had previously described.^{2 7 9} We recorded single-channel currents without the presence of organic Ca²⁺ channel agonists such as Bay K 8644, since this agent greatly modifies channel properties.^{10 11}

The recording pipette was made from Pyrex glass tubing (Narishige) that had a resistance of 4 to 5 m with the recording solutions. Currents were recorded at room temperature (22°C to 24°C). Membrane currents were low-pass filtered at 2 kHz, digitized at a sampling frequency of 5 to 10 kHz, and stored in a personal computer system for subsequent analysis. Traces were finally presented after the currents had been low-pass filtered at 1 kHz. For the recording of membrane currents and data analysis, pClamp (Axon Instruments) was used on the PC-AT compatible computer. Single-cell capacitance was determined with a cancellation network in the patch amplifier.⁸ Capacitive and leak currents were eliminated by P/4 protocol in the whole-cell recording and by subtraction using traces with no channel openings in the single-channel recording. Liquid junction potential was not corrected.

For the recording of the whole-cell Ca²⁺ currents, the bath solution contained (in mmol/L) BaCl₂ 50, TrisCl 75, glucose 10, and HEPES 10 at pH 7.3 titrated with TrisOH. The pipette solution contained (in mmol/L) Cs aspartate 120, CsCl 30, EGTA 10, ATP Na₂ 3, MgCl₂ 3, and HEPES 10 at pH 7.3 titrated with CsOH.

For the recording of the single Ca²⁺ channel currents, the pipette solution contained (in mmol/L) 50 BaCl₂, 75 TrisCl, 10 glucose, and 10 HEPES at pH 7.3 titrated with TrisOH. The bath contained high-K⁺ solution to depolarize the cell membrane to 0 mV, which consisted of (in mmol/L) 150 KCl, 1 MgCl₂, 10 EGTA, and 10 HEPES at pH 7.3 titrated with KOH.

Statistical Methods
Data are expressed as mean±SEM. Statistical significance was determined by an unpaired t test or one-way ANOVA. A value of P<.05 was considered as statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
With use of the whole-cell configuration, whole-cell L-type Ca²⁺ channel currents were recorded (Fig 1). A holding potential of -40 mV was used to minimize the T-type Ca²⁺ channel currents. Mesenteric arterial cells from SHR of this age (8 to 9 weeks of age) showed a significantly greater amplitude than did those from the age-matched WKY (P<.05). The current density at 0 mV, the same potential as used in the single-channel recording, was -6.6±0.5 pA/pF in SHR (n=20) and -4.3±0.5 pA/pF in WKY (n=24) (P<.05) (Table).

View larger version (22K): [in this window] [in a new window]   Figure 1. Whole-cell L-type Ca2+ channel currents recorded in cells from SHR and WKY. Left, Current-voltage relationship of WKY (n=20) and SHR (n=20). Current amplitudes at various command potentials were normalized by cell capacitance and plotted. Data are shown as mean±SEM. *P<.05 vs WKY. Liquid junction potential was not corrected. Right, Traces of Ca2+ channel currents evoked by command potentials (-20 to 40 mV in a 10-mV step) in WKY (cell capacitance of 17 pF) and SHR (17 pF). Dotted line indicates zero current level. Arrowhead indicates the beginning of voltage step; holding potential was -40 mV.

View this table: [in this window] [in a new window]   Table 1. Comparison of Characteristics of L-Type Ca2+ Channels in WKY and SHR

Single L-type Ca²⁺ channel currents were recorded by use of the cell-attached configuration. Depolarizing command steps were applied from a holding potential of -40 mV every 2 s. Openings of the Ca²⁺ channels were brief, and the amplitude was 1 pA at a command potential of 0 mV. Application of 1 µmol/L nifedipine to the bath solution abolished the channel opening (data not shown).

The incidence of at least one channel opening per tested patch did not significantly differ between SHR and WKY (SHR, 16 of 24 patches, 67%; WKY, 14 of 27 patches, 52%). Fig 2A shows 12 consecutive recordings obtained in single cells from SHR and WKY. The opening of the channels was more frequent in SHR than in WKY. To clarify this difference, we evaluated time-dependent changes in the channel activity (Fig 2B and 2C). As an indicator of channel activity, values of NPo per depolarization were plotted against time, where N is the number of channels available for opening in the patch membrane and Po is the probability of the channels being open: NPo=(total duration of channel opening during the command potential)/(duration of the command pulse). Bars in Fig 2B and 2C correspond to the traces in Fig 2A. NPo fluctuated in both SHR and WKY, but a higher NPo appeared frequently in SHR. In addition, the appearance of a blank trace that showed no opening of the channel was more frequent in WKY (27% of the total trace) than in SHR (10%) in this figure. The cumulative activity (cumulative NPo) during 64 consecutive traces was then compared between SHR and WKY (Fig 2D). The cumulative NPo of SHR reached a higher level than did that of WKY. The mean value of cumulative NPo was higher in SHR (5.2±0.4; n=12; P<.05) than in WKY (3.0±0.3; n=12) (Table). In addition, the appearance of the blank sweep was less frequent in SHR (14±2%; n=12) than in WKY (19±2%; n=12) (Table). These observations suggest that channel activity is higher in SHR than in WKY.

View larger version (37K): [in this window] [in a new window]   Figure 2. A, Typical recordings showing single L-type Ca2+ channel currents, recorded from cells of SHR and WKY. Twelve consecutive traces are shown for each strain. Command potential of 0 mV was applied every 2 s from a holding potential of -40 mV. Horizontal lines in traces represent the close level. Arrows indicate beginning of command pulse. B and C, Time-dependent changes in channel activity for WKY (B) and SHR (C) during 64 consecutive stimulations. As an indicator of channel activity, the values of NPo per depolarization are plotted against time; NPo=(total duration of channel opening during the command potential)/(duration of the command pulse). Bars in this figure correspond to the traces in A. D, Cumulative NPo was obtained from the data in B and C.

The distribution of the amplitude for unitary currents at 0 mV is shown in Fig 3. Distribution of the amplitude did not differ in SHR and WKY. Single-channel conductance was then obtained from the current-voltage relationship. Single-channel conductance obtained from pooled data was nearly the same in SHR (20±1 pS, n=3 to 5) and WKY (19±1 pS, n=3 to 5).

View larger version (24K): [in this window] [in a new window]   Figure 3. A and B, Histograms showing amplitude of single L-type Ca2+ channels in SHR (A) and WKY (B) at a membrane potential of 0 mV, fitted with a single gaussian function. Mean amplitudes of WKY (from 119 events) and SHR (from 190 events) were both 1.0 pA. C, Single-channel conductance was obtained from the current-voltage relationship. Unitary current amplitude was plotted against membrane potential. Data are mean±SEM from three to six experiments in both SHR and WKY. Solid lines are drawn by fitting data to the linear relationship with a slope conductance of 21 pS in SHR and 20 pS in WKY.

Fig 4 shows histograms of open time in SHR and WKY. Open time of 5 ms or longer appeared in 3% of the total events in WKY and in 5% in SHR and was not included in this graph. Distribution of open time did not differ between SHR (=1.1±0.1 ms, n=8) and WKY (=1.0±0.1 ms, n=8) (Table). Because 95% of the openings lasted for up to 5 ms in the both strains, openings of L-type Ca²⁺ channels in arteries from the two strains revealed predominantly the "mode 1" behavior.¹²

View larger version (15K): [in this window] [in a new window]   Figure 4. Histograms showing open time of single L-type Ca2+ channels in SHR and WKY obtained at 0 mV. Data sets for WKY (288 events) and SHR (395 events) were fitted to a single exponential with time constants of 1 ms. Not shown is open time of 5 ms or longer that appeared in 3% of the total events in WKY and in 5% in SHR.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrated that the opening of the L-type Ca²⁺ channels was increased in arterial smooth muscle cells from SHR compared with WKY. However, the single-channel conductance and open time did not differ between SHR and WKY. Thus, an increased opening of the single channels would contribute greatly to the increased amplitude of the whole-cell current.

The unitary inward current recorded in the present study was considered to be L-type Ca²⁺ channel currents from the following findings: (1) single-channel conductance and open time were basically the same as those of the L-type Ca²⁺ channel in other arterial tissues studied^10,11,13; (2) the holding potential was -40 mV, which inactivated the T-type Ca²⁺ channels as well as Na⁺ channels¹⁰; and (3) the channel opening disappeared with the application of nifedipine, suggesting that the channel is sensitive to dihydropyridines.^{10 11}

Whole-cell amplitude (I) consisted of several parameters, such as the amplitude of the single-channel current (i), the total number of channels in cell membrane (N_T), the fraction of channels that is available for opening (P_F; availability), and the open probability of each channel (Po), while P_F and Po were affected by time and voltage: I=i N_T P_F Po.^{14 15} The availability (P_F) describes slow gating between "modes," ie, the transition between the available and unavailable states (typically in order of seconds, to 10 s).^{12 14 15 16} The open probability (Po) describes fast gating, ie, how the available channel moves between the closed, open, and inactivated states during depolarization (typically in order of milliseconds, to 10 ms). In the present study, the amplitude of single channels did not significantly differ between SHR and WKY. The most evident alteration in SHR compared with WKY was a higher channel activity (NPo), where NPo corresponds to N_T P_F Po. Because it is difficult to determine precisely whether the patch membrane contains one or multiple channels, we could not separate N and Po (or N_T, P_F, and Po) as in the previous studies for vascular Ca²⁺ channels.^{9 10} However, if the channel number (N_T) is not increased in arterial tissues in SHR compared with WKY, as suggested by studies that examined dihydropyridine bindings in the aorta,^{4 5} the increased opening of the channel (NPo) would be attributable to the increased availability (P_F) and/or open probability (Po) of the channels.

Slow kinetics (change in availability, P_F) can be evaluated with the application of consecutive sweeps; runs of nonblank and blank sweeps change slowly.^{14 16} Distribution of blank sweep is used to characterize the slow kinetics; the low percentage of blank sweep corresponds to the high availability.¹⁴ In the present study, single channels stayed in the state of low NPo (mostly in mode 1) or in the blank sweep (mode 0) in both rat strains. The presence of mode 0 (blank) sweep was less frequent in SHR than in WKY. Thus, the availability (P_F) is likely to be higher in SHR than in WKY.

Single-channel conductance of L-type Ca²⁺ channels was about 20 pS in both SHR and WKY. We used 50 mmol/L Ba²⁺ to record the single-channel current. According to the study by Gollasch et al,¹⁷ the relationship between the slope conductance () and Ba²⁺ concentration ([Ba²⁺]_o) can be fitted with the Hill equation: =_max/[1+(k_0.5/[Ba²⁺]_o)ⁿ] with an apparent dissociation constant (k_0.5) equal to 35.27 mmol/L, a power factor (n) equal to 0.38, and a maximum conductance (_max) equal to 42.2 pS. From the equation, the estimated slope conductance with 50 mmol/L Ba²⁺ is about 22 pS, which is not different from our data.

In the present study without Ca²⁺ channel agonists, about 95% of single-channel openings lasted up to 5 ms; channel openings revealed mode 1 behavior. Thus, the distribution of open time (<5 ms) was fitted to one exponential with a time constant of about 1 ms in both SHR and WKY. The appearance of long-lasting openings in the present study was less frequent than that in bovine pial arteries¹⁴ but was almost the same as that in guinea pig basilar arteries.¹⁸ The reason for the discrepancy among studies is unknown, but it might be due to the difference in tissues or to the recording conditions, such as stimulus frequency, holding potential level, and temperature.

A mechanism for the altered activation of L-type Ca²⁺ channels in SHR has not been clarified in the present study; however, several possible mechanisms could be suggested. First, the regulation of channel activity might be altered. It has been reported that the regulation of the activation of L-type Ca²⁺ channels involves phosphorylation by protein kinase C^{19 20} and by cyclic AMP–dependent kinase,^{18 21} some ATP-related mechanism,⁶ and GTP-binding protein–dependent mechanism.^{22 23} Because the activities of protein kinase C^{24 25} and GTP-binding protein^{26 27} in vascular smooth muscle cells are reported to be altered in SHR, some of these intracellular mechanisms may explain the alteration. Another possible mechanism is that an altered phenotype of L-type Ca²⁺ channels that shows a high availability for opening may distribute with higher density in membrane of SHR arteries than of WKY arteries.

We have previously reported that the amplitude of L-type Ca²⁺ channel current was increased in SHR at 4 to 5 weeks of age compared with age-matched WKY, while the differences disappeared in rats at 16 weeks of age and older.² Our hypothesis was that vascular injury that developed during hypertension or maturation might affect the activity of Ca²⁺ channels. However, two recent studies showed that the amplitude of L-type Ca²⁺ channels in mesenteric arteries from SHR at 18 weeks of age²⁸ or cerebral arteries from stroke-prone SHR at 17 weeks of age and older³ remained increased compared with that in age-matched WKY. The reason for the discrepancy is unknown at present; however, possible explanations could be suggested. First, the genetic heterogeneity of SHR or WKY might explain the discrepancy; it was shown that varieties of genetic heterogeneity exist in and among substrains of SHR or WKY.⁶ To clarify this possibility, studies are ongoing to evaluate the age-dependent change in amplitude of Ca²⁺ channel current using SHR/IZM and WKY/IZM; the genetic heterogeneity of IZM substrains are reported to be small.⁶ Another possible explanation is that differences in the recording conditions would contribute to the discrepancy, as suggested by others.²⁸

In conclusion, we have shown that the opening of single L-type Ca²⁺ channels in mesenteric artery cells from SHR is increased compared with those from WKY. The amplitude and open time of the single-channel current did not differ between SHR and WKY. These findings suggest that the enhanced amplitude of the whole-cell current is attributable to the increased opening of single channels. The mechanism for this increased activity remains to be determined.

	Selected Abbreviations and Acronyms

 

NPo = channel activity NT = total number of channels in cell membrane PF = availability Po = open probability SHR = spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This study was supported by grants from the Ministry of Education, Science, and Culture, Japan (Nos. 06770497 and 07670788).

Received June 16, 1997; first decision July 22, 1997; accepted December 9, 1997.

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