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(Hypertension. 1997;29:1329-1336.)
© 1997 American Heart Association, Inc.

Articles

Effects of Age on Ca²⁺ Currents in Small Mesenteric Artery Myocytes From Wistar-Kyoto and Spontaneously Hypertensive Rats

Irina M. Lozinskaya; ; Robert H. Cox

From the Bockus Research Institute, The Graduate Hospital, and Department of Physiology, University of Pennsylvania, Philadelphia.

Correspondence to Robert H. Cox, PhD, Bockus Research Institute, The Graduate Hospital, One Graduate Plaza, Philadelphia, PA 19146. E-mail rcox{at}mail.med.upenn.edu

	Abstract

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Abstract The purpose of this study was to test the hypothesis that differences in voltage-gated Ca²⁺ channels increase with age during the development of sustained hypertension in the spontaneously hypertensive rat (SHR). Using patch-clamp methods, we measured whole-cell Ca²⁺ currents in freshly isolated myocytes from small mesenteric arteries of juvenile (5 to 7 weeks), young (10 to 12 weeks), and mature (19 to 23 weeks) Wistar-Kyoto rats (WKY) and SHR. Indirect tail artery systolic pressure increased progressively with age in SHR (from 125±5 to 159±5 to 192±5 mm Hg) but only in the younger WKY (from 107±6 to 130±4 to 136±4 mm Hg). Peak Ca²⁺ current density (current per cell capacitance) was larger in SHR than WKY myocytes at all ages (at 6 weeks, 3.5±0.4 versus 2.3±0.2 pA/pF; at 12 weeks, 3.8±0.2 versus 3.1±0.2; at 20 weeks, 4.9±0.4 versus 3.3±0.4). Cell capacitance (surface area) was significantly smaller in 12-week-old SHR than WKY (25.2±1.1 versus 31.8±1.6 pF), but no differences were found in the 6- or 20-week-old groups. There were significant differences in Ca²⁺ current with strain, age, and voltage but no significant age-strain interactions. The ratio of peak Ca²⁺ current for SHR to that of WKY declined linearly with voltage at all ages, suggesting differences in the voltage dependence of Ca²⁺ current activation. The voltage dependence of Ca²⁺ current was shifted to the left in SHR compared with WKY at all ages. Activation curves were shifted significantly in the negative voltage direction only in 20-week-old SHR myocytes. We have found differences with age in Ca²⁺ current density and its voltage dependence in SHR compared with WKY during the phase of development in which blood pressure becomes established in the SHR. The net effect of these differences predicts a larger Ca²⁺ current in SHR at voltages in the physiological range of membrane potential.

Key Words: calcium channels • age • patch-clamp techniques • rats, inbred SHR

	Introduction

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It is generally accepted that established hypertension is characterized by an increased peripheral resistance.¹ While an augmented geometric resistance contributes to this increase,² augmented vascular responsiveness to a wide variety of agonists is also a major contributor.³ It has been demonstrated that augmented contractile responses to agonists occur in intact segments³ as well as in single cells⁴ of hypertensive arterial smooth muscle. Furthermore, differences in Ca²⁺ handling (influx and efflux) as well as in free cytoplasmic Ca²⁺ levels under basal conditions and in response to agonist activation have been reported in hypertension.^{5 6 7} These and other studies suggest the existence of alterations in excitation-contraction coupling in hypertensive smooth muscle and focus on altered Ca²⁺ regulation.³ An understanding of the cellular mechanisms responsible for these observations remains incomplete.

The increase in cytoplasmic free Ca²⁺ that activates vascular smooth muscle occurs as a result of both intracellular Ca²⁺ release and extracellular Ca²⁺ influx.⁸ Force development depends primarily on Ca²⁺ release from intracellular stores with subsequent myosin light chain phosphorylation.⁹ However, in the absence of Ca²⁺ influx, the steady, tonic component of smooth muscle contraction (ie, force maintenance) cannot be sustained.¹⁰ Also, the tonic level of contraction has been shown not to depend on the degree to which the intracellular Ca²⁺ stores are filled before activation.¹¹ Voltage-gated Ca²⁺ channels have been shown to provide a steady-state Ca²⁺ influx pathway for agonist-mediated (eg, norepinephrine) contractions.¹² These results suggest that Ca²⁺ influx is the major factor determining force maintenance in smooth muscle, along with a contribution from Ca²⁺-independent mechanisms.⁹

Previous studies have documented differences in voltage-dependent Ca²⁺ channels in genetic models of hypertension, the spontaneously hypertensive rat (SHR and stroke-prone SHR), compared with its control counterpart, the Wistar-Kyoto rat (WKY).^{13 14 15 16 17} Most of these studies were performed with high concentrations (10 to 50 mmol/L) of Ba²⁺ or Ca²⁺ as the charge carrier to improve resolution of the small Ca²⁺ currents in rat vascular myocytes. However, the results of these studies are somewhat conflicting. It has generally been shown that Ca²⁺ current or Ba²⁺ current is larger in myocytes from SHR or stroke-prone SHR than in their normotensive counterparts^{13 14 15 16 17} and that the differences between the strains is correlated with differences in blood pressure,¹⁶ but this is not a universal finding. Ohya et al¹⁴ compared Ca²⁺ currents in WKY and SHR from young (6-week-old) and mature (18-week-old) animals and found significant differences in Ca²⁺ currents only in the young animals.

In contrast to the results of Ohya et al,¹⁴ we recently reported larger Ca²⁺ currents in myocytes isolated from small mesenteric arteries of 20-week-old SHR than in those from WKY¹⁸ measured with 2 mmol/L extracellular Ca²⁺. These differences in Ca²⁺ currents between WKY and SHR may be a part of the cause of the blood pressure differences between the strains or may represent the response to elevated blood pressure. If the former is correct, it might be expected that such differences in Ca²⁺ currents would be present in younger animals. However, if the latter is correct, it could be expected that differences in the Ca²⁺ currents might be smaller or absent in younger animals. Accordingly, we performed experiments in 5- to 7- and 10- to 12-week-old animals, in which hypertension is not fully developed in the SHR, and compared Ca²⁺ currents with those of 20-week-old animals, in whom blood pressure had stabilized at steady levels.

	Methods

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Tissue and Solutions
Myocytes were enzymatically isolated from mesenteric artery branches of juvenile (5 to 7 weeks), young (10 to 12 weeks), and mature (19 to 23 weeks) male WKY and SHR with methods we have previously described.¹⁸ Briefly, the mesenteric artery and attached branches were removed from the animals after euthanasia and cleaned of surrounding fat and matrix. Individual branches were removed, cut open longitudinally, and incubated in a Ca²⁺-free solution at 37°C for about 40 minutes. The branches were then cut into small pieces, and collagenase (200 to 250 U/mL) and elastase (20 to 25 U/mL) were added to the incubation solution for about 25 to 35 minutes at 37°C. The tissue and enzyme solution were separated by filtration through 210-µm mesh, washed with 1 mL enzyme-free buffer, and triturated gently with a Pasteur pipette. Released cells were separated from the remaining tissue by filtration through 210-µm mesh. An aliquot of cells was placed in a chamber mounted on the stage of an inverted microscope (Nikon Diaphot), and cells were allowed to adhere to the glass bottom of the chamber. After initiation of perfusion with Ca²⁺-free buffer to remove debris and loosely attached cells from the chamber, perfusion (at about 1 mL/min) was switched to 10 mL of a solution containing 0.2 mmol/L Ca²⁺ and then to 10 mL of one containing 2 mmol/L Ca²⁺. With this method, chamber Ca²⁺ concentration increased in an approximate ramplike manner from 0 to 0.2 mmol/L and then from 0.2 to 2 mmol/L. We found this procedure to maintain cells in a relaxed, elongated state and result in improved Ca²⁺ tolerance of the myocytes. All procedures were performed in accordance with institutional guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee.

Electrophysiological Methods
Membrane currents were recorded using the whole-cell, patch-clamp configuration¹⁹ at room temperature (about 23° to 25°C). Micropipettes (2 to 3 M resistance) were made from capillary tubing (WPI Kwik-fil) with a programmable puller (Sutter Instruments, model P-80/PC) and fire polished. Series resistance and capacitance compensation were adjusted maximally using a patch-clamp amplifier with a 100-M head stage (Dagan, model 8900). Experimental protocols were controlled using a computer (Dell 466/L) and PCLAMP software (Axon Instruments). Current signals were converted from analog to digital form at a sampling rate of 10 kHz using a Labmaster A/D board (Axon Instruments, version 5.5.1) and stored in a computer for subsequent analysis. Multiple responses to hyperpolarizing (20-mV) voltage-clamp steps (n=5) were obtained for each protocol, averaged, and used to provide capacitance and leak compensation of the raw data. Experimental current records were analyzed using PCLAMP software.

Procedures
Cell break-in was accomplished by gentle suction at a holding potential of -60 mV. Membrane potential was stepped at 15-second intervals from -60 to 0 mV for 3 to 5 minutes during cell dialysis with the pipette solution until the current stabilized. Current-voltage relations were then determined from a holding potential of -90 mV. Voltage-clamp steps 75 milliseconds long were applied from -60 to +40 mV in 10-mV increments every 15 seconds. Peak values of Ca²⁺ currents were determined at each voltage using the CLAMPFIT module of PCLAMP. Activation curves were obtained from values of Ca²⁺ currents extrapolated from the exponential phase of the Ca²⁺ current inactivation back to the time of the initiation of the voltage-clamp step.²⁰ From the current-voltage curves, the apparent reversal potential was determined, and conductance values were calculated.²⁰ Conductance values were normalized by dividing by the maximal value to determine the activation curves.

Solutions and Chemicals
The tissue incubation buffer had the following composition (mmol/L): NaCl 140, KCl 5, MgCl₂ 1, HEPES 10, and glucose 10, with pH 7.4 (titrated with NaOH) and osmolality of 296±2 mOsm/L. The solution used to fill the patch pipettes had the following composition (mmol/L): CsCl 100, TEA-Cl 20, NaCl 5, MgATP 5, HEPES 10, and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) 10 at pH 7.2 (titrated with CsOH) and had an osmolality of 306±3 mOsm/L. CLS3 collagenase was obtained from Worthington Biochemical and porcine pancreas elastase from ICN. Bay K8644 and nifedipine were purchased from Research Biochemicals International, and all other chemicals were purchased from Sigma Chemical Co.

Statistical Analysis
Statistical comparisons of membrane currents were performed by ANOVA for unpaired data using the StatView (ABACUS Concepts) application on a Macintosh computer (PowerMac 7100). Statistical comparisons of peak currents were performed by a three-way ANOVA with two between factors (strain and age) and one within factor (voltage). The values were adjusted for the number of mean comparisons, and probability values less than .05 were considered to be significant. Average values are given as mean±SEM for individual cells studied in the various groups for each procedure.

	Results

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Systolic pressure was significantly higher in SHR than WKY at all three ages. In SHR, systolic pressure increased progressively with age (125±5 to 159±5 to 192±5 mm Hg), whereas in WKY, it increased significantly only between the two younger age groups (107±6 to 130±4 to 136±4 mm Hg). Also, systolic pressure did not show any significant changes with age from 18 to 23 weeks in SHR or WKY. Compared with WKY, SHR had a larger ratio of heart weight to body weight, with the difference between the two groups increasing with age (at 6 weeks, 3.7±0.1 versus 4.1±0.1 mg/g; at 12 weeks, 3.0±0.1 versus 3.6±0.1; at 20 weeks, 2.6±0.1 versus 3.8±0.2). Such findings have been routinely reported in this animal model.

From a holding potential of -90 mV, inward currents recorded under the conditions of these experiments activated at about -40 mV, exhibited a maximum near 0 mV, and had an apparent reversal potential between +50 and +60 mV (Fig 1). Inspection of current traces revealed only minor apparent qualitative differences among myocytes from the various groups (strain and age) with regard to these properties.

View larger version (36K): [in this window] [in a new window]   Figure 1. Currents recorded from representative mesenteric artery myocytes from 6-, 12-, and 20-week-old Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Currents were recorded in 10-mV steps at -60 to +40 mV from a holding potential of -90 mV, with external [Ca2+] of 2 mmol/L. Currents are separated in three consecutive test potential ranges in the three columns, with a -60-mV current included in all sets as a baseline reference. Voltage protocols used for recording the currents are shown at the top and refer to each column. Only currents along the bottom row are labeled, but the same relative positions of the current traces apply to all six rows for those test voltages. Cell capacitances were as follows: 6 weeks, WKY=19.4 pF and SHR=19.5 pF; 12 weeks, WKY=23.8 pF and SHR=22.4 pF; 20 weeks, WKY=37.3 pF and SHR=27.0 pF. Vertical and horizontal calibration bars shown at the lower left represent 100 pA and 15 milliseconds, respectively.

These inward currents were found to be dihydropyridine sensitive, with 10 nmol/L Bay K8644 increasing the current (53±10%, n=17) and 100 nmol/L nifedipine inhibiting the current completely (Fig 2). The currents were sensitive to some inorganic divalent cations, with 0.1 mmol/L Cd²⁺ also inhibiting the current completely. However, 0.1 mmol/L Ni²⁺ (26±6%, n=4) or amiloride (21±7%, n=6) only partially inhibited the inward current (Fig 2). These characteristics of the inward currents were not significantly different between WKY and SHR. On the basis of these characteristics, as well as on those described below, the inward currents were identified as L-type Ca²⁺ currents (I_Ca).

View larger version (30K): [in this window] [in a new window]   Figure 2. Pharmacology of inward currents in mesenteric artery myocytes from Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Voltage protocols used for the various compounds are shown at the top and apply to each column. Test voltages listed as 1, 2, and 3 represent -40, -30, and -10 mV for A and -30, -20, and 0 mV for B. Baseline current recorded at -60 mV is also shown in A and B. Test voltage for C, D, and E was 0 mV. A, Inward currents recorded before (top) and during (bottom) perfusion with 10 nmol/L Bay K8644 (12-week-old WKY, 37.3 pF). B, Inward currents recorded before and during perfusion with 0.1 mmol/L Ni2+ (12-week-old WKY, 33.4 pF). C, Inward currents recorded before and during perfusion with 100 nmol/L nifedipine (20-week-old SHR, 26.8 pF). D, Inward currents recorded before and during perfusion with 0.1 mmol/L Cd2+ (6-week-old SHR, 33.7 pF). E, Inward currents recorded before and during perfusion with 0.1 mmol/L amiloride (12-week-old SHR, 29.9 pF). Vertical calibration bar represents 150 pA for A and 100 pA for B through E; horizontal bar represents 20 milliseconds for all.

Comparison of maximum values of I_Ca recorded in this study revealed differences between the strains in juvenile and mature but not young animals (at 6 weeks, 46±4 versus 71±8 pA; at 12 weeks, 101±8 versus 99±6; at 20 weeks, 112±7 versus 163±15). To compensate for the effects of differences in cell size on I_Ca, capacitance values were determined for each cell and used to normalize I_Ca. This approach implicitly assumes that factors other than cell surface area that contribute to cell capacitance (eg, membrane thickness, cell shape, and dielectric constant) are not different between age or strain. Average cell capacitance values are summarized in the Table. Cell capacitance values were significantly smaller in 12-week-old SHR than WKY but were not different between 6- and 20-week-old animals. Cell capacitance increased progressively and significantly from 6 to 20 weeks in SHR but only from 6 to 12 weeks in WKY. When I_Ca was normalized to cell surface area, it was found that significant strain [F(1,124)=34.55, P<.00001] and age [F(2,124)=19.8, P<.0001] differences were present, but there was no strain-age interaction [F(2,124)=1.35, P=.24] (Table).

View this table: [in this window] [in a new window]   Table 1. Summary of Ca2+ Current Parameters

Fig 3 shows a comparison of average values of peak Ca²⁺ current density for myocytes from WKY and SHR as a function of voltage at the three ages. The number of observations per data point in Figs 3 through 6 are given in the first line of the Table. The peak I_Ca was the maximum (inward) current recorded at each test voltage step. There were significant differences in the voltage dependence of peak I_Ca with regard to strain [F(1,124)=28.8, P<.0001] and age [F(2,124)=16.82, P<.0001]. In addition, there was a significant difference in I_Ca values with regard to voltage and strain [F(4,496)=20.87, P<.0001] and voltage and age [F(8,496)=16.69, P<.001]. In general, peak Ca²⁺ current densities were larger in SHR than WKY myocytes over the entire voltage range tested at all three ages. The maximum values of peak I_Ca are summarized in the Table and were significantly larger at all three ages in SHR than WKY. In addition, there were significant strain-by-voltage and age-by-voltage interactions among the I_Ca data.

View larger version (22K): [in this window] [in a new window]   Figure 3. Comparison of average current density–voltage data for peak Ca2+ currents from myocytes of 6-week-old (A), 12-week-old (B), and 20-week-old (C) Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Traces at the top show families of currents recorded with 75-millisecond-long voltage clamp steps from -60 to +40 mV in 10-mV steps from a holding potential of -90 mV. indicates WKY currents; , SHR currents. Vertical and horizontal calibration bars represent 150 pA and 20 milliseconds, respectively. Peak current was determined at each test voltage, divided by cell capacitance, and then averaged. Symbols are identified in the figure and represent mean values; vertical bars are ±1 SEM.

View larger version (9K): [in this window] [in a new window]   Figure 4. Voltage dependence of the ratio of the average value of peak ICa for 6-week-old (A), 12-week-old (B), and 20-week-old (C) spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). Symbols represent values determined by dividing average ICa values for SHR (ISHR) by those for WKY (IWKY) at each voltage. These current ratios were best fit with a linear regression curve shown in the panels. The slopes of the regression curves were 1.57 (r=.86) for the 6-week-old group, 1.26 (r=.75) for the 12-week-old group, and 1.46 (r=.94) for the 20-week-old group.

View larger version (13K): [in this window] [in a new window]   Figure 5. Voltage dependence of normalized peak Ca2+ current for 6-week-old (A), 12-week-old (B), and 20-week-old (C) Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). For each cell, peak ICa values were determined at each voltage, normalized by dividing by the maximum ICa value for the cell, and then averaged at each voltage. Symbols are identified in the figure and represent mean values; vertical bars are ±1 SEM.

View larger version (13K): [in this window] [in a new window]   Figure 6. Voltage dependence of ICa activation for 6-week-old (A), 12-week-old (B), and 20-week-old (C) Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). For determination of activation, conductance (g) values were determined with ICa values extrapolated to the time of initiation of the voltage-clamp step, the estimated reversal potential (Erev), and the following equation—g=I/(Vm-Erev)—and then normalized by the maximum conductance. Symbols are identified in the figure and represent mean values; vertical bars are ±1 SEM, g/gmax is the ratio of conductance to maximum value; Vm is voltage.

Average values of peak I_Ca for the SHR group were divided by similar values for WKY myocytes at the various voltages for the three age groups (Fig 4). The ratio I_SHR/I_WKY decreased linearly with voltage. These data were transformed for statistical analysis by determining I_SHR/[I_Ca(WKY)] and [I_Ca(SHR)]/I_WKY ratios and were analyzed separately. In this transform, I_X is the average value for the group (X=WKY or SHR) and I_Ca(X) are values for individual cells. For both of these transforms, there was a significant voltage dependence of I_Ca ratio [F(2,124)=5.16, P=.007] but no significant age effect. These results suggest a difference in the voltage dependence of I_Ca between WKY and SHR at all ages.

The voltage dependence of I_Ca in WKY and SHR was compared more directly by normalizing values of peak I_Ca by the maximum I_Ca for each cell before averaging (Fig 5). At all three ages, peak I_Ca values were generally larger in myocytes from SHR than WKY at voltages between -40 and -10 mV, which also suggests a hyperpolarizing shift in the voltage dependence of I_Ca activation. To quantify the differences in voltage dependence of normalized I_Ca, we used the following approach. The voltage value at which I/I_max=0.5 [I/I_max(0.5)] on the rising phase (ie, the negative voltage region) of the current-voltage curve was determined by a least-squares fit of the currents around that value (number of points 4) for each cell by a third-order polynomial. Voltage values at I/I_max(0.5) were then averaged for WKY and SHR myocytes. The voltage values at I/I_max(0.5) (Table) were significantly different in 20-week-old but not in 6- or 12-week-old SHR compared with WKY. Age had a significant effect on voltage at I/I_max(0.5) for both groups between 6 and 12 or 6 and 20 weeks of age.

The voltage dependence of the peak I_Ca ratio primarily reflects differences in the voltage dependence of I_Ca activation. Accordingly, the voltage dependence of activation was determined from peak I_Ca data (Fig 6). Differences in voltage at one-half maximum activation (V_0.5) for I_Ca activation curves were found between mature (20-week-old) WKY and SHR, but the differences between strains at the other two ages were not statistically significant. There was a significant age effect on the V_0.5 of I_Ca activation, with a significant increase from 6 to 12 weeks in both strains. In all comparisons, k values (slope of the activation-voltage curve at V_0.5) were not significantly different between strain and age (Table).

The relationship between average values of I_Ca and systolic pressure in WKY and SHR at the three ages studied is summarized in Fig 7. The straight line in the figure represents a linear regression fit to all the data points by least-squares analysis. This analysis shows that a strong correlation (r²=.916) exists between blood pressure and Ca²⁺ current density.

View larger version (16K): [in this window] [in a new window]   Figure 7. Correlation between maximum Ca2+ current density and tail-artery systolic pressure for Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) at 6, 12, and 20 weeks of age. The straight line was fit by linear regression analysis to all the data.

	Discussion

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In this study, we found differences in whole-cell Ca²⁺ currents in myocytes from small mesenteric arteries of SHR at 6, 12, and 20 weeks compared with myocytes from WKY. These include differences in Ca²⁺ current density and its voltage dependence. Peak Ca²⁺ current density was larger in myocytes from young SHR than those from WKY at all voltages between -40 and +20 mV. Furthermore, there were significant age-related changes in I_Ca for both SHR and WKY.

The larger currents recorded in the SHR were not the result of differences in cell size, as the currents were normalized to cell capacitance. This would be expected to minimize the effects of differences in cell surface area between cells in the various groups. This suggests that the differences in Ca²⁺ current density are the result of a larger number of channels or larger open probability of the channels. There is some evidence in the literature of a larger number of dihydropyridine binding sites in SHR arteries than WKY arteries,²¹ which could be the basis for the larger I_Ca density in SHR. However, this conclusion has recently been challenged by Hermsmeyer et al,²² who showed a smaller number of dihydropyridine binding sites in stroke-prone SHR using a novel fluorescent dihydropyridine probe.

The peak I_SHR/I_WKY ratio decreased with voltage in a linear manner in animals at all three ages. Despite the fact that the value for the slope of the voltage-dependence curve was smaller in the 12-week-old group, there was no significant effect of age on this relationship. If the voltage dependence of I_Ca in WKY and SHR was identical (ie, a scaled difference), the I_SHR/I_WKY ratio would be constant (equal to the scaling factor) and voltage independent. These results suggest that the voltage dependence of I_Ca differs between WKY and SHR myocytes. This was confirmed in two ways. First, voltage values where normalized I_Ca (I/I_max) was equal to 0.5 were found to be more negative for SHR than WKY myocytes. Second, comparison of V_0.5 for the calculated activation curves showed that these values were generally more negative in SHR. This difference in voltage dependence of I_Ca should contribute to the relatively larger I_Ca in SHR myocytes at physiological values of membrane voltage.²³

Previous studies of Ca²⁺ currents in vascular myocytes in hypertension have suggested the presence of two Ca²⁺ channel types, L and T, especially at younger ages.^{13 14 15 16 17} However, in our previous study in myocytes from 20-week-old animals,¹⁸ we found evidence for only one current component, an L-type. Likewise, in the present study, we found no evidence for the presence of T-type Ca²⁺ currents at 6 or 12 weeks in either WKY or SHR.

Organic Ca²⁺ channel blockers have proven to be effective therapeutic agents for the treatment of hypertension.²⁴ A part of their action includes the reduction of peripheral resistance.²⁵ These agents have also been shown to inhibit contractile responses in isolated arterial smooth muscle²⁶ and reduce Ca²⁺ influx.⁷ The effects of this diverse group of agents have been shown to be greater in hypertensive than normotensive subjects both in humans^{27 28} and in animal models.^{21 24 25 29} These results suggest that voltage-gated Ca²⁺ channels contribute to the determination of peripheral resistance and that differences in their contribution to excitation-contraction coupling processes exist between normal and hypertensive subjects.

This conclusion is supported by observations involving the effects of activating the L-type Ca²⁺ channels with the dihydropyridine agonist Bay K8644 on resting tone in arterial segments from normotensive and hypertensive animals. It has been consistently shown that Bay K8644 alone produces a larger contractile response in arteries from several different hypertensive animal models, including the SHR, compared with their normotensive controls that depends on influx through L-type Ca²⁺ channels.^{30 31 32 33} Hernandez et al³³ suggested that one or more of the following explanations could account for the differences: (1) Bay K8644 releases a contracting factor from endothelial cells; (2) the resting membrane potential is depolarized in hypertensive smooth muscle cells, resulting in a larger effect of Bay K8644 on Ca²⁺ influx through L-type channels; (3) the L-type channels are altered in such a manner that their activation by Bay K8644 produces a larger contractile effect.

The results of our experiments provide evidence in support of the third possibility above. Maximum values of I_Ca are larger in SHR than WKY, and their voltage dependence is shifted in the negative direction, which would favor a higher open probability of Ca²⁺ channels at a given value of membrane potential and a greater effect of Bay K8644 on open probability.²³ There is also evidence for a depolarized membrane potential in SHR arteries³⁴ that could contribute to the larger contractile response of Ca²⁺ L-type channel activation. Nelson et al^{12 23} have shown that the open probability of L-type channels at physiological values of resting membrane potential is sufficient to allow this pathway to supply activator Ca²⁺ for contraction. Thus, the results of the present study provide support for a role of Ca²⁺ channels in the altered contractile function of hypertensive arterial smooth muscle, but they do not exclude an effect of the other factors as suggested by Hernandez et al.³³

What is the relationship between these differences in Ca²⁺ current density and blood pressure in these two rat strains? The data summarized in Fig 7 show that a strong correlation (r²=.916) exists between blood pressure and Ca²⁺ current density. Such a correlation does not indicate that a causal relation exists between these two functions. However, as indicated above, if the strain differences in Ca²⁺ currents were a response to elevated blood pressure, differences in the youngest animals (approximately 6 weeks) would be expected to be much smaller than those in the oldest. However, this was not the case. Although absolute values of I_Ca were smaller in juvenile animals, relative differences between the two strains (ie, I_SHR/I_WKY) were not age dependent. This suggests that the differences in I_Ca between WKY and SHR either contribute to the differences in blood pressure between the strains (are causal) or are completely unrelated. The results of these experiments do not allow us to determine which of these alternatives is correct.

In conclusion, the results of this study have documented a larger Ca²⁺ current density in SHR aged 6 to 20 weeks, when arterial pressure is rapidly developing along with differences in the voltage dependence of activation. These differences predict a larger contribution of voltage-gated Ca²⁺ channels to Ca²⁺ influx in the SHR. These differences may contribute to the larger contractile responses to agonists previously reported³ and to the greater effectiveness of organic Ca²⁺ channel blockers in hypertensive subjects.²⁴

	Acknowledgments

 
This work was supported in part by research grant HL-28476 from the US Public Health Service.

Received June 25, 1996; first decision July 25, 1996; accepted December 4, 1996.

	References

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