Increase in Functional Ca²⁺ Channels in Cerebral Smooth Muscle With Renal Hypertension

From the Departments of Neurosurgery and Physiology, University of Maryland School of Medicine, Baltimore, Md.

	Abstract

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Abstract—The hypothesis that availability of functional Ca²⁺ channels in vascular smooth muscle is augmented in hypertension was tested in basilar artery cells from Wistar rats exhibiting stable systolic blood pressure (BP_sys) for 2 to 11 weeks after partial renal artery ligation (Goldblatt 2-kidney 1-clip [2K1C] model). Cells were freshly isolated and patch-clamped using a nystatin–perforated patch method. BP_sys ranged from 110 to 280 mm Hg and correlated with normalized kidney mass. Macroscopic current-voltage curves were fit to a Boltzmann function to obtain maximum conductance (g_max), steepness and midpoint potential for the voltage dependence of activation (k and E_1/2, respectively), and extrapolated reversal potential for the chord conductance (E_rev). Linear regression of normalized conductance (ng_max=g_max/cell capacitance) versus BP_sys for 103 cells indicated a strong relationship, with a slope of 0.0019 nS · pF^-1 · mm Hg^-1 (P<0.0001). Similar analysis of data from 35 other cells exposed to 500 nmol/L Bay K 8644 gave a slope of 0.0041 nS · pF^-1 · mm Hg^-1 (P=0.001). Voltage-dependent parameters, k, E_1/2, and E_rev, were not significantly related to BP_sys. Single-channel measurements in cell-attached patches revealed that the number of channels in 32 patches was significantly related to BP_sys (P=0.0024) but that slope conductance, open dwell times at 0 mV, and distribution between 2 open states were not. Finally, in a subgroup of 61 cells from animals made hypertensive (180 mm Hg<BP_sys<200 mm Hg) for 1/2 to 6 weeks, we found that elevation of ng_max depended on duration of hypertension (P=0.003), with no elevation at 1/2 week. We conclude that in the 2K1C model, availability of functional Ca²⁺ channels increases with BP_sys with no change in channel properties and that measurable BP_sys elevation occurs before the increase in functional channels.

Key Words: hypertension • Goldblatt model • two-kidney one-clip model • Ca²⁺ channel • smooth muscle cell

	Introduction

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In several types of cells, including platelets, red blood cells, and smooth muscle cells of hypertensive humans and other vertebrates, [Ca²⁺]_i is elevated.¹ In vascular smooth muscle, [Ca²⁺]_i is critical for development and maintenance of force and is one of the principal determinants of vascular tone.² Given that increased peripheral vascular resistance and augmented contractile responses to vasoconstrictors are hallmarks of hypertension, it is evident that altered Ca²⁺ handling in vascular smooth muscle should be considered an important factor in hypertension.^{1 3 4}

Several lines of evidence suggest that augmented Ca²⁺ influx through L-type Ca²⁺ channels in vascular smooth muscle contributes to hypertension. There is increased basal Ca²⁺ influx and augmented Ca²⁺ influx after agonist activation of vascular smooth muscle in hypertension.^{5 6} Organic Ca²⁺ channel blockers are more effective in reducing BP, peripheral resistance, and vasoconstrictor responses in genetic^{7 8 9} and acquired^{10 11} hypertensive subjects, and there is increased sensitivity to the Ca²⁺ channel agonist Bay K 8644 in genetic^{12 13 14 15} and acquired^{16 17 18} hypertension. Also, direct measurements of Ca²⁺ channel currents have given evidence of larger currents in genetic hypertension, suggesting an increase in functional Ca²⁺ channel activity.^{19 20 21 22 23}

Ca²⁺ channel currents have been studied in vascular smooth muscle cells from 2 genetic strains, SHR and SP-SHR, with cells from normotensive genetically related WKY rats used as controls. The earliest reports were on primary cultures of azygos vein from neonatal SHR¹⁹ and, later, from SP-SHR,²⁰ with larger macroscopic currents having been found in both studies. Since arterial tone is more important for BP control and freshly isolated cells may be more representative of in vivo conditions than cultured cells, it was important that comparable experiments be carried out in freshly isolated arterial cells. Larger currents were subsequently reported in freshly isolated mesenteric artery cells from 4- to 5-week-old²¹ and from 20-week-old SHR,²² although the latter finding was in disagreement with the study of Ohya et al²¹ involving cells from 16- to 18-week-old animals. Recently, larger currents were also reported in freshly isolated cerebral arterial cells from adult SP-SHR.²³

The significance of larger Ca²⁺ currents in genetic models of hypertension is uncertain. First, this finding has not been generalized to other forms of hypertension. Second, it is not known whether this finding is a primary manifestation of the hypertensive phenotype or whether it is acquired secondary to hypertension. Elucidation of these 2 points would come from study of a nongenetic model, in which development of hypertension and augmentation of Ca²⁺ channel density could be correlated. Third, the molecular basis for larger currents is unclear, ie, whether they represent (1) an alteration in channel properties, (2) an alteration in channel availability due to second-messenger mechanisms, or (3) upregulation of channel expression, with more channel protein molecules inserted into the cell membrane. Complete characterization of channel properties requires single-channel recordings, which have thus far not been reported in cells from hypertensive animals. Also, all reports to date have made use of conventional whole cell recordings in which second-messenger modulation of channel availability may be unduly altered. This last difficulty is best avoided by use of a perforated-patch method in which cytoplasmic disturbance is minimized.

In the present study, our principal goal was to test the hypothesis that augmented density of functional Ca²⁺ channels occurs also in a nongenetic form of hypertension. We chose the 2K1C Goldblatt model for study because the pathophysiology involving angiotensin has been well studied in this model.²⁴ We studied freshly isolated basilar artery cells because channel properties and effects of second messengers on channel availability have been well characterized in these cells.^{25 26 27} Two additional hypotheses that use of this model allowed us to address were (1) whether biophysical properties of Ca²⁺ channels are altered with hypertension and (2) whether BP elevation precedes or follows the increase in channel density. Briefly, we found that the density of functional Ca²⁺ channels in basilar artery cells correlated strongly with BP in the 2K1C model, that no change in macroscopic or single-channel properties could account for this effect, and that measurable BP elevation occurred before the increase in functional Ca²⁺ channels.

	Materials and Methods

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Goldblatt Animals
We used a 2K1C Goldblatt model to induce hypertension.^{28 29} Female Wistar rats 4 to 6 weeks of age were used. Surgery for renal artery clipping was always performed on the left, with animals anesthetized to a surgical level with ketamine (60 mg/kg IP) plus xylazine (7.5 mg/kg IP). After surgery, animals were given free access to food and water and were observed for development of hypertension. All surgical protocols and postoperative surgical management were in accord with National Institutes of Health guidelines for animal use. At weekly intervals after surgery and at the time of tissue harvest, BP was measured using a rat tail-cuff plethysmograph (Harvard Instruments). Data on BP are given as peak BP_sys at the time of tissue harvest.

Rats that underwent renal artery clipping were maintained for 4 to 11 weeks after surgery and had kidney mass measured when they were killed for study. The efficacy of renal artery clipping in producing hypertension varied between animals, with some animals remaining normotensive and others developing various levels of hypertension. Generally, in animals that would exhibit elevated pressures, this was observed within 2 to 3 weeks after surgery. Overall, values of BP_sys ranged between 110 and 280 mm Hg. Sixty animals were killed 2 to 11 weeks after exhibiting a new stable level of BP or stable normotension after surgery. Of the 60 animals, 29 were used for cell preparations for the experiments reported in the present study, and 21 were used for a different experiment. For these 50 animals, a plot of BP_sys versus normalized kidney mass (mass of operated kidney divided by mass of unoperated kidney) indicated an inverted "V-shaped" relationship (Figure 1): BP_sys was inversely related to normalized kidney mass down to a value of 0.6, beyond which, as the operated kidney decreased in mass, BP_sys tended to normalize. The normalized kidney mass in 16 other unoperated rats of comparable age was 0.97±0.03 (mean±SD). Ten other animals maintained for 4 to 9 weeks after surgery were not used for study because of a discrepancy between BP_sys and normalized kidney mass. An additional 3 animals were operated and killed 1/2 week after development of hypertension and were used for the experiments reported in Figure 7.

View larger version (20K): [in this window] [in a new window]   Figure 1. BPsys correlates with normalized kidney mass in the 2K1C model. The normalized kidney mass was obtained by dividing the mass of the operated (left) kidney by the mass of the unoperated kidney. The data are from 29 animals used for the experiments reported here () and 21 animals used for other experiments (), all of which exhibited stable blood pressure >2 weeks before they were killed for study. Linear regression of the data with normalized kidney mass of 0.6 indicated an intercept of 434 mm Hg and a slope of -304 mm Hg; corresponding values for the data with normalized kidney mass of 0.6 were 125 mm Hg and 232 mm Hg, respectively (solid lines).

View larger version (17K): [in this window] [in a new window]   Figure 7. Relation between Ca2+ channel density and duration of hypertension. Bar graph showing mean ngmax for cells from animals with 180 mm Hg<BPsys<200 mm Hg for the times indicated along the abscissa. Data plotted at 2 to 3 weeks were pooled from 4 cells harvested at 2 weeks and 23 cells harvested at 3 weeks; data plotted at 5 to 6 weeks were pooled from 15 cells harvested at 5 weeks and 5 cells harvested at 6 weeks. One-way ANOVA indicated a significant difference between groups (P=0.003). A Student-Newman-Keuls multiple comparison test indicated that the mean at 1/2 week was significantly different from the value at 2 to 3 weeks (P<0.05) and from the value at 5 to 6 weeks (P<0.001). Error bars represent SE; the number of cells studied is indicated near each bar.

Cell Preparation
Single smooth muscle cells were isolated from basilar arteries using methods similar to those previously used in this laboratory.^{25 30} The basic isolation solution (PS1) contained (mmol/L) NaCl 116.3, KCl 5.4, NaH₂PO₄ 10.4, MgSO₄ 0.83, glucose 5.5, and NaHCO₃ 26.2, equilibrated with 95% O₂/5% CO₂. Animals were killed by intraperitoneal injection of an overdose of sodium pentobarbital (120 mg/kg) and then underwent transcardiac perfusion with 150 mL PS1 along with PAP (18 µg/mL) at a perfusion pressure of 100 cm H₂O. After harvesting the brain, the basilar artery was dissected in PS1 plus CaCl₂ (0.02 mmol/L) and PAP (18 µg/mL). Enzymatic digestion was carried out at 37°C in PS1 plus CaCl₂ (0.02 mmol/L) and PAP (12 µg/mL) (PS2) along with (mg/mL) collagenase 2, elastase 0.4, DNase 0.2, soybean trypsin inhibitor 1, and fatty acid–free BSA 1 for 75 to 95 minutes, followed by PS1 plus PAP (12 µg/mL) without added Ca²⁺ (PS3) plus (mg/mL) protease 0.4, soybean trypsin inhibitor 0.4, and BSA 0.4 for 2 to 5 minutes. After enzymatic treatment, the artery was transferred to PS3 and triturated to release single myocytes. Cells were stored at 4°C in a modified KB solution that contained (mmol/L) KCl 85, K₂HPO₄ 30, MgSO₄ 5, sodium pyruvate 5, taurine 20, creatine 5, and ATP · Na₂ 2, plus fatty acid–free albumin (1 mg/mL) (pH 7.2).³¹ Patch-clamp experiments were generally carried out within 2 to 10 hours of cell harvest.

Solutions
For whole-cell recordings, we used a perforated-patch technique.³² The pipette solution contained (mmol/L) CsCl 130, MgCl₂ 8, and HEPES 10 (pH 7.2) as the base solution. Nystatin was solubilized in dimethyl sulfoxide, vortexed, sonicated, and then diluted into the final pipette solution. Working solution was made daily before each experiment by adding 33 µL of nystatin stock into 10 mL of the above base solution to yield a final nystatin concentration of 165 µg/mL and dimethyl sulfoxide concentration of 3.3 µL/mL. The bath solution contained (mmol/L) TEA 125, 4-aminopyridine 5, MgCl₂ 1, BaCl₂ 10, HEPES 10, and glucose 12.5, pH 7.3 with HCl. For single-channel experiments, the pipette solution contained (mmol/L) BaCl₂ 40, TEA 100, 4-aminopyridine 5, HEPES 10, and glucose 12.5, pH 7.2 with TEA · OH plus Bay K 8644 (200 nmol/L) to increase the open-channel probability.²⁵ The bath solution contained (mmol/L) KCl 145, TEA 10, EGTA 5, HEPES 10, and glucose 12.5, pH 7.3 with KOH. Enzymes used for cell isolation and other chemicals and reagents were obtained from Sigma Chemical Co or Fisher Scientific.

Voltage-Clamp Experiments
Macroscopic currents were recorded using a nystatin-perforated whole-cell technique.³² Pipettes with tip resistances of 1 to 3 M that were made from borosilicate glass (Kimax) were used. Cells with seal resistances of <2 G generally were discarded. After the capacitative transient in response to small test pulses had stabilized, cell capacitance was estimated from the dial settings for capacitative compensation on the voltage-clamp amplifier. In preliminary experiments, we verified the accuracy of this method using calculations based on the integral of the uncompensated capacitative current. Membrane currents were measured during 200-ms pulses from an HP of -60 mV or during ramp pulses (-60 to +60 mV, 0.45 mV/ms) from an HP of -60 mV. In both cases, test pulses were applied at 20-s intervals. Leakage currents were estimated from small depolarizing or hyperpolarizing pulses and, when measurable, were subtracted after appropriate scaling. Single-channel currents were recorded by a cell-attached patch technique using 1- to 3-M pipettes coated with Sylgard 184 (Dow Corning) and heat-polished. Membrane currents were amplified (Axopatch 200A, Axon Instruments, Inc) and sampled on-line at 2 to 5 kHz by a microcomputer equipped with either a Labmaster DMA (Scientific Solutions) and running pCLAMP software (version 6.0.2, Axon Instruments, Inc) or a CED1401 D-A converter (Cambridge Electronic Design Limited) running CED Patch and Clamp Software (version 6.0). All experiments were performed at room temperature, 22°C to 25°C.

Data Analysis
After access to the cytoplasm had been obtained, membrane currents were monitored either with 200-ms step pulses to 0 mV or with ramp pulses given at 20-s intervals. As previously found,^{26 27} the inward current gradually grew larger during the first several minutes of dialysis, a phenomenon referred to as run-up. For the analyses reported here, we took the largest current observed after completion of run-up, when the current had stabilized to its maximum value.

Open-channel dwell times were computed for openings of individual channels, with exclusion of simultaneous openings due to multiple channels. For open dwell time analysis, data were filtered at 500 Hz (-3 dB), allowing us to resolve events >360 µs in duration. Because of this, we ignored transitions <500 µs.

The method of maximum simultaneous openings was used to determine the number of channels in a patch of membrane.³³ This method is accurate when the probability of channel opening is relatively high. In the present study, we estimated this variable at 0 mV with Bay K 8644 in the pipette, conditions under which the open probability for the L-type channel is near maximal.²⁵

Data were fit to Equations 1, and 2 in the text, to the gaussian distributions in Figure 4, and to the logistic equation in Figure 5 using the iterative nonlinear least-squares method of Marquardt-Levenberg (NFIT 1.1, Island Products, or Origin 4.1, Microcal). Scatterplots shown in Figures 1, 3, 4A to 4D, 6A, and 6C were fit to a linear regression equation (Origin 4.1, Microcal). For the group comparisons of Figure 7, we used a 1-way ANOVA with the Student-Newman-Keuls method for pairwise multiple comparison. All data are given as mean±SE.

View larger version (37K): [in this window] [in a new window]   Figure 4. Cell capacitance and voltage dependence of Ca2+ current are not related to BPsys. Cell capacitance and I-V curves were measured in 103 cells studied without Bay K 8644. I-V curves were fit to Equation 1 to obtain k and E1/2, which are the steepness and midpoint potential of activation, and Erev, the extrapolated reversal potential for the chord conductance. Scatter plots (A to D) and corresponding frequency histograms (E to H) are shown for cell capacitance (A and E), the value of k (B and F), the value of E1/2 (C and G), and the value of Erev (D and H). For the scatterplots (A to D), all abscissas correspond to the one shown in panel D. Superimposed linear regressions are shown; none of the slopes for any of the 4 variables was significantly different from zero. For the frequency histograms (E to H), the bin width was set as SD/5.5; superimposed gaussian fits are also shown.

View larger version (19K): [in this window] [in a new window]   Figure 5. Response of Ca2+ channel current to Bay K 8644. A, Original records obtained during 200-ms ramp pulses (-60 to +50 mV, 0.45 mV/ms) in a cell (968011AK, 17.2 pF) from a rat with BPsys of 150 mm Hg before (record a) and after (record b) the addition of 500 nmol/L Bay K 8644. B, Concentration-response relationship for effect of Bay K 8644 on Ca2+ channel conductance. Data are from 15 basilar artery cells from normotensive unoperated Wistar rats. Values are mean±SE with 3 cells at each concentration. The curve represents a fit to the following logistic equation: g'=(g''-1)/[1+(c1/2/c)n]+1, where g' is the relative conductance (obtained by dividing gmax after drug by gmax before drug, with values of gmax derived from a fit to Equation 1), g'' is the maximum value of g', c is the concentration of drug, c1/2 is the concentration at which half the maximum effect is observed, and n is the Hill coefficient; nonlinear least squares fit gave values of g''=2.04, c1/2=38 nmol/L, and n=3. C, Original records obtained during 200-ms ramp pulses in a cell (964301AO, 14.4 pF) from a rat with BPsys of 210 mm Hg for 4 weeks before (record a) and after (record b) addition of 500 nmol/L Bay K 8644.

View larger version (25K): [in this window] [in a new window]   Figure 3. Functional Ca2+ channel density is related to BPsys. For 103 cells studied without () and 35 cells studied with () 500 nmol/L Bay K 8644 in the bath solution, I-V curves were fit to Equation 1 to obtain gmax, which was divided by cell capacitance to obtain ngmax. Values of ngmax are plotted against BPsys. Linear regression gave intercepts of -0.063 and -0.18 nS/pF and slopes of 0.0019 and 0.0041 nS · pF-1 · mm Hg-1 for the data without and with Bay K 8644, respectively (solid lines). The 95% confidence intervals for the regression lines are also shown (dotted lines). The slope of both regression lines was significantly different from zero (P<0.0001 and P=0.001 for data without and with Bay K 8644, respectively).

	Results

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Macroscopic Ca²⁺ Channel Density
When basilar artery cells were obtained from animals displaying stable BP for 2 to 11 weeks after surgery, either normal or elevated (110<BP_sys<280 mm Hg), the magnitude of the macroscopic Ca²⁺ channel current correlated with BP_sys. Records from 2 representative cells from animals with BP_sys values of 130 and 240 mm Hg, obtained during 200-ms step depolarizations to 0 mV from an HP of -60 mV, are shown in Figure 2B (records a and b, respectively). Values of peak current measured at various potentials in the same cells were plotted against membrane potential to obtain the steady-state I-V relationship. These plots showed that at all potentials, peak current was larger in the cell from the animal with higher BP_sys (Figure 2C). In other cells, we measured the Ca²⁺ channel current during ramp pulses (-60 to +60 mV, 0.45 mV/ms; HP=-60 mV) to obtain a pseudo–steady-state I-V relationship. This technique allowed rapid estimation of the I-V curve with minimal effect of rundown. Results obtained using ramp depolarizations on 2 representative cells from animals with BP_sys values of 140 and 200 mm Hg (Fig 2D, records a and b) were in accord with those observed with step depolarizations (Fig 2C, open circle and open square): the current was larger at all potentials when recorded in a cell from a hypertensive animal; the maximum current was observed at 0 to +15 mV; when larger, the maximum current tended to occur at slightly more negative potentials; and the current became outward at +50 to +70 mV.

View larger version (17K): [in this window] [in a new window]   Figure 2. The Ca2+ channel current in basilar artery cells is larger with hypertension. A, Voltage-clamp protocol representing a 200-ms depolarizing step from an HP of -60 to 0 mV. B, Superimposed records of current obtained during step depolarizations to 0 mV in a cell (a; 95N30C3, 15.4 pF) from a rat with BPsys of 130 mm Hg, and in a cell (b; 963291AF, 15.2 pF) from another rat with BPsys of 240 mm Hg that had been hypertensive for 6.5 weeks. C, I-V curves showing peak inward current as a function of potential for the same cells as in panel B from rats with BPsys of 130 mm Hg () and 240 mm Hg (). D, Superimposed pseudo–steady-state current voltage curves recorded during ramp depolarizations (-40 to +50 mV, 0.45 mV/ms) in a cell (a; 965171AB, 16.5 pF) from a rat with BPsys of 140 mm Hg, and in a cell (b; 967241AD, 15.5 pF) from a rat with BPsys of 200 mm Hg that had been hypertensive for 6 weeks. The abscissa in panel C corresponds to the one in panel D. Linear leak and capacitative currents were subtracted.

To quantify the relationship between magnitude of the current and BP_sys, I-V data from individual cells between -40 mV and +40 mV were fit to the Boltzmann function:

(1)

where I is current, E is membrane potential, g_max is the maximum conductance at positive potentials, k and E_1/2 are the steepness and midpoint potential of activation, respectively, and E_rev is the extrapolated reversal potential for the chord conductance. Ca²⁺ channel current density was then calculated as ng_max, obtained by dividing g_max by cell capacitance. Quantifying the magnitude of the current in this way was thought to be preferable to use of a single measure, such as the peak of the I-V curve, because it is based on multiple measurements and is independent of voltage.

I-V data obtained with either step (22 cells) or ramp (90 cells) depolarizations were collected from 112 cells isolated from 21 rats displaying stable BP for 2 to 11 weeks after surgery. Good voltage clamp was obtained in 103 of the 112 cells studied. A scatterplot of individual values of ng_max versus BP_sys for the 103 cells with good voltage clamp is shown (Figure 3, ). Linear regression gave a slope of 0.0019 nS · pF^-1 · mm Hg^-1, which was significantly different from zero (P<0.0001). Excluded from this analysis were data from 9 cells that exhibited poor voltage clamp, defined as an I-V curve with a fitted value of k<1.96 SD or a value of E_1/2 outside of 1.96 SD (see Figure 4).

Cell capacitance was analyzed separately to examine the contribution of this factor to the increase in channel density with BP_sys. Values of capacitance for the 103 cells were not significantly related to BP_sys (regression slope, -0.019 pF/mm Hg; P>0.05; Figure 4A) and were found to be normally distributed (Figure 4E). This analysis indicated that larger currents observed with elevated BP were likely due to the greater number of functional channels and not a larger cell size.

Macroscopic Properties of Ca²⁺ Channels
We evaluated certain properties of the Ca²⁺ channel to ascertain whether they were affected by BP. The above analysis using Equation 1 yielded information on the voltage dependence of activation of the Ca²⁺ channels. We evaluated values of k and E_1/2 in the 103 cells used for Figure 3. Linear regression indicated that neither parameter was significantly related to BP_sys. For k, the slope was -0.0037 mV/mm Hg (P>0.05, Figure 4B), and for E_1/2, the slope was -0.042 mV/mm Hg (P>0.05, Figure 4G). Both parameters were normally distributed (Figure 4, panels F and G, respectively). This analysis indicated that larger currents observed with elevated BP were not associated with any significant change in the voltage-dependent properties of the channel.

Also available for analysis from Equation 1 was E_rev. This variable, representing the extrapolated reversal potential for the chord conductance, underestimates the true reversal potential of the macroscopic current, which approaches the voltage axis asymptotically. Nevertheless, in the present context, E_rev is sensitive to the presence of other currents, especially outward current through K⁺ or other cationic channels. We thus evaluated values of E_rev for the 103 cells. Values of E_rev were not significantly related to BP_sys, with a regression slope of -0.0032 mV/mm Hg (P>0.05, Figure 4D), and were normally distributed (Figure 4H). This suggested no systematic error in our analysis due to the appearance of a new or larger non–Ca²⁺ channel current.

Response to Bay K 8644
We also evaluated the response to the Ca²⁺ channel agonist Bay K 8644, the application of which should reveal the maximum number of available functional channels. Figure 5 shows pseudo–steady-state I-V curves from a representative cell from an animal with BP_sys of 150 mm Hg, obtained during ramp depolarizations before (record a) and after (record b) the addition of 500 nmol/L Bay K 8644. Addition of the drug caused the current to increase 2-fold in magnitude and to activate more steeply and at more negative potentials.²⁵ For these experiments to be meaningful, it was important to ensure that the optimal concentration of drug was used to reveal the maximum number of channels. To determine the concentration-response relationship for Bay K 8644, we used basilar artery smooth muscle cells from unoperated normotensive Wistar rats. Fifteen cells were studied after a single application of drug. We measured the effect of drug by fitting the pseudo–steady-state I-V curves to Equation 1 and dividing g_max after drug administration by g_max before drug administration. We found that a half-maximum effect occurred at 38 nmol/L and that 500 nmol/L of the drug would be sufficient to ensure a maximum response (Figure 5B). Figure 5 also shows pseudo–steady-state I-V curves from a representative cell from an animal with a BP_sys of 210 mm Hg, obtained during ramp depolarizations before (record a) and after (record b) the addition of 500 nmol/L Bay K 8644. The findings in this cell were comparable to those observed in cells from normotensive animals, with a somewhat greater than 2-fold increase in current, a steep turning on of the current, and a shift to the left of the I-V curve.

Data with Bay K 8644 were collected from 35 cells isolated from 11 rats manifesting stable BP for 2 to 11 weeks after surgery. As previously, pseudo–steady-state I-V curves from these cells were fit to Equation 1, and Ca²⁺ channel density was estimated as ng_max. A scatterplot of individual values of ng_max versus BP_sys for the 35 cells is shown (Figure 3, ). Linear regression gave a slope of 0.0041 nS · pF^-1 · mm Hg^-1, which was significantly different from zero (P=0.001). Notably, 500 nmol/L Bay K 8644 produced the same magnitude of effect at all BPs, ie, an approximate doubling of ng_max, as indicated by the doubling of the regression slope from 0.0019 to 0.0041 nS · pF^-1 · mm Hg^-1. These data suggested no appreciable change in response to Bay K 8644 with elevated BP_sys, and they corroborated the positive relationship between channel density and BP_sys observed without drug.

Single-Channel Properties of Ca²⁺ Channel
Two other properties, single-channel conductance and open dwell time kinetics, were evaluated because a change in either could account for an increase in macroscopic Ca²⁺ channel current. To simplify these analyses, we compared pooled data obtained from normotensive animals with pooled data obtained from hypertensive animals. Figure 6A shows values (mean±SE) of open-channel current at different potentials for 6 patches from rats with BP_sys of 110 to 140 mm Hg (open circles) and for 23 patches from rats with BP_sys of 170 to 240 mm Hg (open squares). Linear regression indicated a slope conductance of 20.2 pS that was not affected by BP.

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of blood pressure on single-channel properties. A, Slope conductance measured in cells from normotensive and hypertensive animals. Values represent mean±SE for 6 patches from rats with BPsys of 110 to 140 mm Hg () and 23 patches from rats with BPsys of 170 to 240 mm Hg (). Linear regression of pooled data indicated a slope conductance of 20.2 pS. B, Open-channel dwell times at 0 mV measured in cells from normotensive and hypertensive animals. Values represent summed data on 1640 channel openings from 9 patches from 3 rats with BPsys of 120 to 130 mm Hg () and 1979 channel openings from 13 patches from 7 rats with BPsys of 170 to 250 mm Hg (). All data were obtained with 200 nmol/L Bay K 8644 in the pipette. Both sets of data were fit to Equation 2 with identical time constants (1=0.47 ms, 2=4.3 ms), with the fraction of short openings being 84% and 88% and the scaling factor being 1270 and 1360 for the normotensive and hypertensive data, respectively (lines). C, Plot of maximum number of superimposed openings during 200-ms pulse to 0 mV measured in 32 cell-attached patches from rats with various levels of BPsys. All data were obtained with 200 nmol/L Bay K 8644 in the pipette. Linear regression of data indicated an intercept of -0.19 openings and a slope of 0.031 openings/mm Hg; the slope was significantly different from zero (P=0.0024).

Open-channel dwell times of >0.5 ms were analyzed for individual nonsuperimposed openings at 0 mV. To minimize bias, a similar number of openings was analyzed in both groups, 1640 for the normotensive group and 1979 for the hypertensive group. Figure 6B shows values summed from 9 patches from 3 rats with BP_sys of 120 to 130 mm Hg (open circles) and from 13 patches from 7 rats with BP_sys of 170 to 250 mm Hg (open squares). The 2 data sets were fit to the following equation:

(2)

where t is time, N is the number of events, S is a scaling factor, and f and (1-f) are the fractions of openings having time constants of ₁ and ₂, respectively. Both sets of data were fit with identical time constants, ₁=0.47 ms and ₂=4.3 ms, and with f=0.84 and f=0.88 for the normotensive and hypertensive data, respectively (lines). The scaling factor, S, reflecting only the total number of events being analyzed, was fit with values of 1270 and 1360 for the normotensive and hypertensive data, respectively. This analysis suggested that the open state of the channel, as characterized by the time constants of opening and the fraction of time spent in each state, was not greatly affected by BP.

Although single-channel properties were not altered, the number of channels in a patch was found to be correlated with BP_sys (Figure 6C). The method of maximum simultaneous openings was used to determine the number of channels in a patch of membrane.³³ We assessed the maximum number of superimposed openings in 32 patches studied during 200-ms step pulses (n=30) from HP of -60 to 0 mV. For all of these experiments, pipettes with similar resistances of 1 to 3 M containing 200 nmol/L Bay K 8644 were used, and care was taken to not exert undue negative pressure during seal formation that would cause incorporation of a larger membrane area into the pipette. At all values of BP_sys, we found patches that exhibited low numbers of channels (Figure 6C). However, as BP_sys increased, the probability of finding a greater number of channels increased, and only at high levels of BP_sys did we observe large numbers of channels. Overall, the number of channels was related to BP_sys; linear regression gave a slope of 0.031 openings/mm Hg, which was significantly different from zero (P=0.0024). This finding of an increase in number of channels per patch with higher BP_sys corroborated our other findings of an increase in macroscopic Ca²⁺ channel current with no change in channel properties.

Hypertension Precedes Increase in Current
We sought to ascertain the temporal relationship between the increase in density of functional Ca²⁺ channels and elevation in BP. First, we prepared a new group of animals with renal artery ligation and followed them closely with tail-cuff plethysmography after surgery. Of the group, we identified 3 animals at 1/2 week after development of hypertension (180 mm Hg<BP_sys<200 mm Hg), and from them, we obtained 14 basilar cells for evaluation of Ca²⁺ channel density. Second, we performed a post hoc analysis of the data presented in Figure 3 and identified 47 cells from animals with 180 mm Hg<BP_sys<200 mm Hg that could be reanalyzed according to duration of hypertension. Of these, 27 cells had been harvested at 2 to 3 weeks, and 20 cells had been harvested at 5 to 6 weeks. The 3 groups of cells exhibited significantly different values of ng_max (Figure 7; by ANOVA, P=0.003). Cells from animals with hypertension for 1/2 week had values of ng_max not significantly different from values found in normotensive animals (Figure 3, open squares; BP_sys=140±6 mm Hg; by t test, P>0.05) but significantly less than those found in animals hypertensive to the same extent for 2 to 3 weeks (P<0.05) or for 5 to 6 weeks (P<0.001) This analysis suggested that short-term hypertension was less likely to be associated with increased channel density than longer-term hypertension.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The principal new finding of the present study is that the density of functional Ca²⁺ channels in basilar artery smooth muscle cells correlates strongly with BP_sys in the 2K1C model. This is the first demonstration of altered density of functional Ca²⁺ channels in vascular smooth muscle in a nongenetic acquired form of hypertension. To optimize our measurements, we applied 2 methods not previously used for quantitative assessment of Ca²⁺ channel density in vascular smooth muscle: (1) we used a perforated-patch technique to minimize disruption of cytoplasmic second-messenger systems, and (2) we estimated values of normalized conductance from measurements at multiple potentials by fitting to a Boltzmann function (Equation 1) rather than relying on a single measurement, typically the peak of the I-V curve. Although we observed variability in normalized conductance between cells, both from the same animal and from different animals, our finding of a significant correlation between normalized conductance and BP_sys was robust and became stronger when channels were maximally activated by Bay K 8644. With the addition of Bay K 8644, the slope of the relationship between channel density and BP_sys doubled from 0.0019 to 0.0041 nS · pF^-1 · mm Hg^-1. These findings, made over a broad range of BP, extend observations made in genetic models of hypertension in which currents from 2 groups, control and hypertensive, were compared.^{19 20 21 22 23} Also, our findings are consistent with reports that in various forms of hypertension, increased sensitivity to nifedipine is related to the degree of hypertension.¹⁰

The channel whose density correlated with hypertension was the L-type Ca²⁺ channel, in agreement with findings from genetically hypertensive animals.^{19 20 21 22 23} For our experiments, we used Cs⁺ in the pipette to minimize contamination from K⁺ currents, and we used Ba²⁺ instead of Ca²⁺ as the charge carrier to improve the signal-to-noise ratio and to obviate Ca²⁺-induced inactivation of Ca²⁺ channels. Under the conditions of the present study, the macroscopic current that we recorded can reliably be said to be due exclusively to L-type Ca²⁺ channels.²⁶ In confirmation of this, we found that the relation between channel density and BP_sys steepened when measured in the presence of a dihydropyridine specific for L-type channels. Also, single-channel measurements indicated a slope conductance of 20 pS, the value characteristic of the L-type Ca²⁺ channel recorded under these conditions.²⁵ In contrast to other studies that have also reported on alterations in T-type current with hypertension,^{19 23 34 35} we did not examine this point specifically in our experiments because basilar artery cells,²⁶ like certain other types of arterial smooth muscle,²² show no evidence of T-type channels.

We assessed a number of channel properties to determine whether they were altered with hypertension. On the basis of macroscopic current recordings, we found that the voltage dependence of activation (E_1/2 and k of Equation 1) and the extrapolated reversal potential of the chord conductance (E_rev of Equation 1) were not significantly affected by BP. Also, in the first single-channel recordings on cells from hypertensive animals, we found that open-channel conductance, open dwell times (₁ and ₂ of Equation 2), and the distribution between open-channel states (f of Equation 2) were not appreciably altered by BP. Our single-channel recordings, however, corroborated our macroscopic current recordings by showing greater numbers of channels in patches from hypertensive animals. Our negative finding on voltage dependence of activation agrees with reports of little or no shift in freshly isolated mesenteric artery cells^{21 22} but differs from reports on cultured azygos vein cells in which a significant shift in voltage dependence was observed.^{19 20}

The second major finding of the present study is that the increase in functional Ca²⁺ channels in basilar artery smooth muscle cells followed the development of hypertension; ie, it appeared to be a secondary manifestation of hypertension. In our experiments with acquired hypertension, this question was relatively easily addressed in a subgroup of animals that was followed closely with tail-cuff plethysmography after surgery, was killed soon after establishment of hypertension, and had basilar artery cells assessed for Ca²⁺ channel density. By contrast, studies on genetic models of hypertension have led to equivocal or contradictory conclusions regarding this question. On the basis of his finding that smooth muscle cells from neonate azygos vein not exposed to elevated BPs showed larger currents, Hermsmeyer³⁶ considered that increased Ca²⁺ channel availability might be causally responsible for hypertension rather than secondarily involved. A subsequent study by Ohya et al²¹ confirmed that arterial smooth muscle cells from young animals showed greater channel density but found that this difference was later lost when animals matured. Although the basilar artery likely contributes little to systemic resistance, the present study nevertheless provides no evidence that increased channel availability must be in place for elevated systemic pressures to be manifested.

A finding of augmented channel density can be due to 1 or a combination of 3 things: altered channel properties, altered channel availability due to a second-messenger mechanism, or altered channel expression and insertion into the cell membrane. In the present study, we showed that channel properties, including voltage dependence, single-channel conductance, and open dwell time characteristics, were not significantly altered and thus could not account for the increase in channel density with hypertension. Some authors have attributed quantitative differences in macroscopic currents in genetic hypertension to increased channel expression.²³ An increase in density of functional channels, however, does not necessarily signify more channel protein molecules per unit membrane area. Absent any significant change in channel properties, altered availability can also be due to altered second-messenger function. For example, an increase in channel availability with hypertension could be due to impaired endothelial function, as has been documented in SHR, 2K1C, and other models of hypertension.^{37 38 39 40 41 42} In the basilar artery, hypertension-induced endothelial dysfunction would result in loss of NO and, thus, larger Ca²⁺ currents, because NO decreases the availability of Ca²⁺ channels via a cGMP-mediated mechanism that does not alter channel properties.²⁷ In our preparation, we cannot exclude the possibility that channel expression was increased along with a concomitant increase in channel insertion into the cell membrane. However, because our experimental methods were specifically designed to minimize second-messenger alterations in the smooth muscle cells during measurement of their Ca²⁺ channel density, altered availability due to an endothelium-dependent second-messenger mechanism also cannot be ruled out. Additional work will be required to distinguish between these 2 mechanisms.

In conclusion, we have shown in the 2K1C model of acquired hypertension that the density of functional Ca²⁺ channel in basilar artery cells correlated strongly with BP, that no change in macroscopic or single-channel property could account for this observation, and that measurable BP elevation occurred before the increase in functional Ca²⁺ channels. Our finding of an increase in Ca²⁺ channel density secondary to hypertension may have important implications for understanding development of hypertension-related vasculopathy in the cerebral circulation. One of the best-documented effects of hypertension on the cerebral circulation is the shift in the autoregulatory curve, with some evidence suggesting that the shift may be related to augmented Ca²⁺ channel activity. Also, an increase in functional Ca²⁺ channels could underlie the vulnerability of smooth muscle cells in the cerebral circulation to the hypertension-associated vasculopathic condition called arteriolosclerosis, which predisposes to hypertensive hemorrhage.⁴³

	Selected Abbreviations and Acronyms

 

2K1C = two-kidney one-clip BP = blood pressure BPsys = systolic BP E1/2 = midpoint potential for voltage dependence of activation Erev = reversal potential gmax = maximum conductance HP = holding potential I-V = current-voltage k = steepness for voltage dependence of activation ngmax = normalized gmax PAP = papaverine HCl SHR = spontaneously hypertensive rat(s) SP-SHR = stroke-prone SHR TEA = tetraethylammonium chloride WKY = Wistar-Kyoto

	Acknowledgments

 
This study was supported by grants from the National Heart, Lung, and Blood Institute (HL-42646) and the American Heart Association (AHA), with funds contributed in part by the AHA, Maryland Affiliate, Inc. We thank Lioudmila Melnitchenko for her expert technical assistance and Dr Ashiwel Undie for help with statistical analysis.

	Footnotes

 
Reprint requests to Dr J. Marc Simard, Department of Neurosurgery, University of Maryland School of Medicine, 22 South Greene St, Baltimore, MD 21201.

Received July 14, 1997; accepted April 2, 1998.

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