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(Hypertension. 1995;25:110-116.)
© 1995 American Heart Association, Inc.

Articles

Myofilament Calcium Sensitivity of Normotensive and Hypertensive Resistance Arteries

Ka Bian; Richard D. Bukoski

From the Hypertension and Vascular Research Laboratories, Department of Internal Medicine, University of Texas Medical Branch, Galveston Island.

Correspondence to Richard Bukoski, PhD, Hypertension and Vascular Research Laboratories, J-65, University of Texas Medical Branch, Galveston Island, TX 77550.

	Abstract

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Abstract We measured intracellular Ca²⁺ and isometric force simultaneously in endothelium-denuded mesenteric resistance arteries of 12- to 15-week-old male spontaneously hypertensive rats (SHR), Wistar-Kyoto (WKY) rats, and Wistar rats. Basal Ca²⁺ did not differ among vessels of these strains (SHR, 86.6±4.5 nmol/L; WKY, 78.5±4.7 nmol/L; Wistar, 83.1±3.9 nmol/L). Myofilament Ca²⁺ sensitivity was determined by measuring the intracellular Ca²⁺ and force responses to cumulative addition of extracellular Ca²⁺ (0.025 to 2.5 mmol/L) in the presence of 100 mmol/L K⁺ or 10 µmol/L norepinephrine after depletion of releasable intracellular Ca²⁺ stores. With 100 mmol/L K⁺, no between-strain differences in active stress, intracellular Ca²⁺, or myofilament Ca²⁺ sensitivity were observed. With 10 µmol/L norepinephrine, the active stress response of SHR vessels to 0.025 and 0.05 mmol/L Ca²⁺ was increased compared with both normotensive strains. The intracellular Ca²⁺ response was not different in vessels of SHR and WKY rats but was depressed in Wistar vessels. Myofilament Ca²⁺ sensitivity of SHR was elevated compared with both WKY and Wistar rats (P<.05) (ED₂₅ for SHR, 74.4±5.1 nmol/L; WKY, 89.8±5.5 nmol/L; Wistar, 86.9±3.4 nmol/L). No strain differences in intracellular Ca²⁺ or active stress responses of SHR and WKY vessels were detected during cumulative addition of norepinephrine with constant extracellular Ca²⁺ (1.5 mmol/L). These results indicate that no hypertension-associated defect in vascular Ca²⁺ handling exists in mesenteric arteries of the SHR. Moreover, although resistance arteries of SHR exhibit enhanced myofilament Ca²⁺ sensitivity compared with two normotensive strains after depletion of intracellular Ca²⁺ and activation with 10 µmol/L norepinephrine, this difference is not observed under the more physiological manipulation of cumulative addition of norepinephrine in the presence of constant Ca²⁺. We conclude that enhanced myofilament Ca²⁺ sensitivity is unlikely to contribute to the hypertension of SHR.

Key Words: hypertension, genetic • muscle, smooth, vascular • fura 2 • calcium • vascular resistance

	Introduction

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It is recognized that Ca²⁺ plays a major role in triggering vascular smooth muscle force generation and that the level of free intracellular Ca²⁺ within the smooth muscle cell is tightly regulated by means of sequestration and extrusion transport processes.^{1 2 3 4} Over the past two decades, experimental data from a variety of sources have supported the hypothesis that altered vascular smooth muscle cell Ca²⁺ handling plays a causal role in the heightened vascular reactivity observed in hypertension of genetic origin. For example, it has been demonstrated that arteries of spontaneously hypertensive rats (SHR) have a heightened reactivity to divalent cations^{5 6} and an increased sensitivity to extracellular Ca²⁺.^{7 8 9} It has also been demonstrated that ATP-dependent Ca²⁺ uptake is depressed in isolated subcellular fractions prepared from SHR arteries.^{10 11 12} More recently, with the introduction of fluorescent Ca²⁺ indicators such as fura 2 and quin 2, it has been demonstrated that free intracellular Ca²⁺ is elevated in SHR cultured aortic smooth muscle cells.^{13 14 15} There is also evidence that Ca²⁺ handling is abnormal in platelets of hypertensive humans and animals.^{16 17 18 19 20 21 22} These data have been used to support the postulate that disturbed vascular smooth muscle cell Ca²⁺ handling contributes to hypertension since platelets are viewed by many as a functional model of the vascular smooth muscle cell.

In contrast with these reports, our laboratory has found that basal levels of intracellular Ca²⁺ are not elevated in intact mesenteric resistance arteries of SHR compared with those of Wistar-Kyoto (WKY) rats²³ and that the force and intracellular Ca²⁺ responses of resistance arteries to maximal activation with 100 mmol/L K⁺ and 10 µmol/L norepinephrine are similar in vessels of the two strains.²⁴ Given the fact that our previous results do not support the widely held hypothesis that vascular Ca²⁺ metabolism is deranged in vascular smooth muscle of genetically hypertensive animals, we have performed additional experiments to test two key hypotheses. One states that intracellular Ca²⁺ mobilization is heightened in mesenteric resistance arteries of SHR compared with vessels of normotensive WKY and Wistar rats. The second states that myofilament Ca²⁺ sensitivity is greater in vessels of SHR compared with those of the normotensive strains.

	Methods

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Animals
All procedures were approved by the institutional Animal Care and Use Committee of the University of Texas Medical Branch. Male SHR and normotensive WKY and Wistar rats were purchased from Charles River (Wilmington, Mass) at 10 to 12 weeks of age and used between the ages of 12 and 15 weeks. They were housed in colony rooms maintained at a constant temperature and humidity and with fixed dark/light cycles and given free access to Purina rat chow and tap water. Several days after rats arrived, systolic blood pressure was determined with the indirect pneumatic tail-cuff technique. On the day of the experiment, the animals were placed in an atmosphere of CO₂ until they stopped breathing; then the thorax was opened and the heart punctured. Branch II and III resistance arteries were dissected from the mesenteric bed and denuded of endothelium by thrusting a human hair in and out of the lumen of the vessel segment as described by Osol et al²⁵ and previously used in our laboratory^{26 27 28} ; arteries then were mounted in a single-channel wire myograph (Living Systems Instruments) as previously described.²⁹ Resting diameter and wall thickness were determined for each vessel using a x40 water immersion objective (Zeiss Instruments) and filar micrometer eyepiece, and the vessels were allowed to equilibrate at 37°C for 30 minutes. The vessel segments were stretched to their optimal length for force development by construction of a length-tension curve using 100 mmol/L K⁺ as agonist.³⁰ The media thickness at the final length was calculated from values of the diameter of the segment, its axial length, and the media volume, which was assumed to remain constant. The functional status of the endothelium was assessed by examining the relaxation response to acetylcholine (0.1 µmol/L) after precontraction with norepinephrine. The absence of a relaxation response was taken as evidence of a functionally disrupted endothelium. Force values were normalized to cross-sectional area of the vessel segment and expressed as active stress (millinewtons per millimeter squared) or to the maximal response to a given agonist and expressed as percent of maximal tension.

Simultaneous Measurement of Force and Ca²⁺
Intracellular Ca²⁺ and stress development were determined as described.³¹ A modification that was introduced was the use of a bicarbonate-buffered salt solution (PSS) of the following composition (mmol/L): NaCl 130, KCl 4.7, MgSO₄7H₂O 1.17, glucose 5, CaCl₂ 1.50, and NaHCO₃ 15, at pH 7.4, which was gassed with 95% air/5% CO₂ in place of the nongassed HEPES-buffered solution that was used in the original report.³¹ In addition, the illumination system was converted from a transmission fluorescence to an epifluorescence arrangement, which increased the signal-to-noise ratio from 0.2 to 2.0. The myograph was placed on the stage of an inverted microscope interfaced with a dual excitation wavelength fluorometer (SPEX-AR/CM) by means of a liquid light guide. Fluorescent light was focused on the vessels by means of a x20 fluor objective (Nikon). Emitted light was filtered with a 510-nm barrier filter (Nikon BA-510) and detected with a photomultiplier tube. Data were stored and analyzed on a personal computer.

Tissue loading with and calibration of fura 2 was performed as follows. Basal fluorescence of the vessel at 340, 360, and 380 nm was determined, followed by addition of 3 µmol/L fura 2-AM (Molecular Probes) dissolved in dimethyl sulfoxide and pluronic F-12. After incubation for 1 hour at 37°C, the vessel was washed five times with fresh PSS, and vessel fluorescence at 360 nm was redetermined to assess the extent of loading with fura 2. After the experimental protocol (see below), Ca²⁺-sensitive fura 2 in the vessel segments was calibrated by the sequential addition of 20 mmol/L ionomycin to determine R'_max, 20 mmol/L EGTA (buffered to pH 7.4 with Tris-HCl) to determine R'_min, and 10 mmol/L MnCl₂ in Ca²⁺-free PSS to quench fura 2 fluorescence. For each vessel, baseline fluorescence was subtracted from all data points, and the Ca²⁺ concentration at a given time point was estimated using the experimentally derived parameters R'_max, R'_min, and ß' as previously described,³² where R'_max and R'_min are the ratios of fluorescence (340/380 nm) in the presence of saturating Ca²⁺ (R'_max) and in the absence of Ca²⁺ (<15 nmol/L, R'_min), and ß' is the ratio of fluorescence at 380 nm in the absence and presence of Ca²⁺.

The dissociation constant used was empirically determined in a separate series of experiments. For each determination, vessel segments were dissected from the mesentery of an individual rat and pooled. The pooled segments, with a wet weight of approximately 3 mg, were loaded with fura 2 as described above and then washed and placed in a solution of the following composition: 100 mmol/L KCl, 15 mmol/L NaCl, 50 mmol/L Tris-HCl at pH 7.2, and 1 µg/mL digitonin. The vessels were then pelleted, and the supernatant was transferred to a stainless steel chamber with a volume of 250 µL that was fitted with a glass coverslip and maintained at 37°C. The fluorescence of the captured dye (excitation, 340 and 380 nm; emission, 510 nm) in response to varying levels of free ionized Ca²⁺ was then determined in an EGTA (10 mmol/L) clamped solution. The free Ca²⁺ in this solution was verified using a Ca²⁺-specific electrode. K_d values were then calculated from Hill plots of log(F-F_min/F_max-F) versus log[Ca²⁺].³³ The estimated values were not significantly different from one another (SHR, 235±18 nmol/L; WKY, 228±34 nmol/L; Wistar, 230±11 nmol/L; n=7-8; P=NS). We therefore ruled out the possibility that differential hydrolysis of fura 2-AM by vascular tissue of the three strains resulted in a species of the dye with an altered affinity for Ca²⁺.

Two different approaches for assessment of the Ca²⁺-force relation were taken. The first assessed the Ca²⁺-force relation after depletion of releasable stores of Ca²⁺. Baseline fluorescence was determined to provide an estimate of basal free intracellular Ca²⁺, after which the vessels were contracted once with a mixture of 100 mmol/L K⁺ and 10 µmol/L norepinephrine. Intracellular Ca²⁺ stores were then depleted by repeatedly inducing contraction with 10 µmol/L norepinephrine in nominally Ca²⁺-free medium until no contractile responses were observed. The segments were then exposed to either 100 mmol/L K⁺ plus 1 µmol/L phentolamine or 10 µmol/L norepinephrine. Ca²⁺ was then cumulatively added back to the bath, and the intracellular Ca²⁺ and force responses were recorded. ED₂₅ and ED₅₀ values for Ca²⁺ were determined by plotting, for each level of extracellular Ca²⁺ that was examined, percent maximal force response versus the level of intracellular Ca²⁺ achieved. The second method for assessing the Ca²⁺-force relation consisted of determining the intracellular Ca²⁺ and force responses to the cumulative addition of norepinephrine in the presence of constant, 1.5 mmol/L, extracellular Ca²⁺.

Data Analysis
Results were analyzed for between-strain differences using either the Student's t test when single values between two strains were compared or ANOVA with a repeated-measures design and linear contrast analysis for comparisons between pairs of means (SYSTAT). A value of P.05 was assumed to indicate a significant difference.

	Results

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The animals used in this study were age-matched at 86.4±1.0 days for SHR, 83.9±1.57 days for WKY rats, and 83.2±1.84 days for Wistar rats. The systolic blood pressure of SHR was significantly greater than that of both WKY and Wistar rats, and the blood pressure of Wistar rats was also greater than that of WKY rats (Table 1). When set at the optimal length for active stress development, no between-strain differences were detected in the lumen diameter, media thickness, or wall-to-lumen ratio of vessels of SHR compared with those from the normotensive strains (Table 1). However, media thickness and the wall-lumen ratio were significantly greater in Wistar compared with WKY vessels (Table 1).

View this table: [in this window] [in a new window]   Table 1. Chief Vital Statistics

Fura 2 was calibrated using an in situ method for each vessel studied. No between-strain differences were detected for R'_max and ß', whereas the R'_min value was slightly but significantly elevated in vessels of SHR compared with those of WKY and Wistar rats (Table 2). We also performed a separate series of experiments to empirically determine the K_d value for Ca²⁺ of fura 2 that had been hydrolyzed by and subsequently extracted from vessels of each strain (Table 2). The basal level of free intracellular Ca²⁺ was estimated using these derived calibration constants, and no between-strain differences were detected (Table 2).

View this table: [in this window] [in a new window]   Table 2. Fura 2 Constants

The Ca²⁺-force relation was assessed by monitoring changes in intracellular Ca²⁺ and force during the cumulative addition of extracellular Ca²⁺ to Ca²⁺-depleted vessels in the presence of either 100 mmol/L K⁺ (plus phentolamine) or 10 µmol/L norepinephrine. Fig 1, left, shows a representative trace of the force and intracellular Ca²⁺ responses of a normotensive vessel in the presence of 10 µmol/L norepinephrine. Two aspects of the responses should be noted. One is that there is a dose-dependent increase in force and intracellular Ca²⁺ in response to extracellular Ca²⁺ up to 0.8 mmol/L. The second is that higher levels of extracellular Ca²⁺ induce relaxation of the segments and a fall in intracellular Ca²⁺. This relaxation was observed in vessels from all three strains when activated with 10 µmol/L norepinephrine but not with K⁺.

View larger version (11K): [in this window] [in a new window]   Figure 1. Typical recordings show time course of changes in intracellular Ca2+ and stress of mesenteric resistance arteries. Left, Response to cumulative addition of Ca2+ in the presence of 10 µmol/L norepinephrine after Ca2+ depletion; values at bottom indicate extracellular Ca2+ concentration (mmol/L). Right, Response to cumulative addition of norepinephrine in the presence of constant extracellular Ca2+ (1.50 mmol/L); values at the bottom indicate the -log of molar concentration of norepinephrine.

A maneuver used for assessment of the myofilament Ca²⁺ sensitivity was depletion of releasable intracellular Ca²⁺ pools by repeatedly (two to three times) inducing contraction with 10 µmol/L norepinephrine in the presence of nominally Ca²⁺-free PSS. This procedure resulted in a fall in the basal level of intracellular Ca²⁺ (Table 3). As noted above, basal Ca²⁺ in the subset of vessels that underwent Ca²⁺ depletion was similar among the three strains (Table 3). Depletion of releasable Ca²⁺ pools resulted in a significant fall in intracellular Ca²⁺ in vessels of all three strains, although the magnitude of the fall was significantly greater in SHR and Wistar rats compared with WKY rats. It thus appears that basal Ca²⁺ and the change in Ca²⁺ wrought by Ca²⁺ depletion are unrelated to blood pressure of the animal.

View this table: [in this window] [in a new window]   Table 3. Effect of Extracellular Ca2+ Depletion on Intracellular Ca2+ Concentration

When the intracellular Ca²⁺ and force responses to the cumulative addition of Ca²⁺ in the presence of 100 mmol/L K⁺ plus 1 µmol/L phentolamine were examined, no strain differences in the active stress response (Fig 2, top) were detected. Because there was a trend for a difference at higher concentrations of extracellular Ca²⁺, we carried out a power analysis to determine the fail-safe N value for rejecting the null hypothesis. The analysis (power=0.80, level=0.05) indicated that a total of 33 separate observations in each group would be needed to demonstrate a significant difference. Moreover, no difference in the amount of Ca²⁺ mobilized was detected (Fig 2, bottom). The plots of percent tension versus the steady-state concentration of intracellular Ca²⁺ that was achieved were not different between vessels of the two strains (Fig 3). As predicted from Fig 3, there was no strain difference in myofilament Ca²⁺ sensitivity (ED₂₅, 111±6.9 versus 117±8.2 nmol/L, SHR versus WKY; ED₅₀, 144±10.2 versus 140±10.5 nmol/L, SHR versus WKY; n=10 and 11, respectively; P=NS).

View larger version (14K): [in this window] [in a new window]   Figure 2. Line graphs show active stress response (top) and intracellular Ca2+ response (bottom) of spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) vessels to cumulative addition of extracellular Ca2+ in the presence of 100 mmol/L K+ plus 1 µmol/L phentolamine after Ca2+ depletion. Values are mean±SEM; n=10 and 11, respectively. No between-strain differences were detected.

View larger version (12K): [in this window] [in a new window]   Figure 3. Line graph shows intracellular Ca2+-force relation of spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat resistance arteries determined by cumulative addition of extracellular Ca2+ in the presence of 100 mmol/L K+ plus 1 µmol/L phentolamine as shown in Fig 2. The ordinate indicates the percentage of the response to 2.5 mmol/L Ca2+. Values are mean±SEM of 10 to 11 observations for the first nine data points and 7 to 9 observations for the remainder. No between-strain differences were detected.

When the Ca²⁺ and force responses to cumulative addition of Ca²⁺ in the presence of 10 µmol/L norepinephrine were determined, there was a significant increase in the magnitude of active stress developed in response to the low doses of Ca²⁺ (0.025 to 0.1 mmol/L) in resistance arteries of SHR compared with vessels of WKY and Wistar rats (Fig 4, top). No difference in the intracellular Ca²⁺ response to addition of extracellular Ca²⁺ through 0.8 mmol/L was detected between SHR and WKY vessels, whereas intracellular Ca²⁺ was significantly attenuated in Wistar vessels in response to low levels of extracellular Ca²⁺ (Fig 4, bottom). The intracellular Ca²⁺ concentration achieved in response to 1.6 and 2.5 mmol/L extracellular Ca²⁺ was depressed in WKY compared with SHR vessels. Plots of percent tension versus the absolute level of free intracellular Ca²⁺ shown in Fig 5 indicate that myofilament Ca²⁺ sensitivity, as reflected by calculated ED₂₅ values, is increased in resistance arteries of SHR (P<.05) compared with those of both normotensive strains. ED₂₅ values for Ca²⁺ were 74.4±5.1 nmol/L for SHR (n=15), 89.8±5.5 nmol/L for WKY rats (n=12), and 86.9±3.4 nmol/L for Wistar rats (n=24). Moreover, ED₅₀ values of SHR were significantly different from those of Wistar (P<.05) but not WKY rats (SHR, 97.3±6.2 nmol/L; WKY, 106±5.6 nmol/L; Wistar, 111.8±3.9 nmol/L).

View larger version (17K): [in this window] [in a new window]   Figure 4. Line graphs show active stress response (top) and intracellular Ca2+ response of spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY) rat, and Wistar rat mesenteric resistance arteries to cumulative addition of extracellular Ca2+ in the presence of 10 µmol/L norepinephrine after Ca2+ depletion. Values are mean±SEM; n=15 for SHR, 12 for WKY, and 24 for Wistar. Top, *Significantly greater response of SHR than WKY and Wistar at P<.05; sunburst, greater response of SHR than Wistar at P<.05. Bottom, *Significant difference between SHR and WKY at P<.05; sunburst, significant difference between SHR and WKY vs Wistar at P<.05.

View larger version (17K): [in this window] [in a new window]   Figure 5. Line graph shows intracellular Ca2+-force relation of spontaneously hypertensive rat (SHR), Wistar-Kyoto (WKY) rat, and Wistar rat resistance arteries determined by cumulative addition of extracellular Ca2+ in the presence of 10 µmol/L norepinephrine as shown in Fig 4. Values reflect mean±SEM of 12 to 24 observations. *Significant difference between SHR vs WKY and Wistar at P<.05.

Because we noted the differential force response of SHR and normotensive vessels to extracellular Ca²⁺ in the presence of 10 µmol/L norepinephrine, we also assessed the active stress and intracellular Ca²⁺ responses of SHR and WKY vessels to cumulative addition of norepinephrine in constant extracellular Ca²⁺. Fig 1, right, illustrates the active stress intracellular Ca²⁺ and force responses of a vessel isolated from a Wistar rat to cumulative addition of norepinephrine. Two aspects of this response should be noted. One is the graded increase in intracellular Ca²⁺ in response to the cumulative doses of norepinephrine; the second is the fact that at lower norepinephrine concentrations, there is no spike or overshoot in the intracellular Ca²⁺ response, whereas at 3 µmol/L norepinephrine and higher, the intracellular Ca²⁺ response is characterized by a peak followed by a plateau response to a lower but steady-state level.

Fig 6, top, illustrates the active stress response to cumulative addition of norepinephrine. No differences between SHR and WKY rats were detected. Moreover, no differences in steady-state concentrations of intracellular Ca²⁺ were detected between the strains (Fig 6, bottom). The Ca²⁺-force relations were similar between the two strains (Fig 7), as evidenced by similar ED₅₀ values for Ca²⁺ (188±13 versus 171±12 mmol/L, SHR versus WKY; n=10 and 11, respectively; P=NS).

View larger version (12K): [in this window] [in a new window]   Figure 6. Line graphs show active stress response (top) and intracellular Ca2+ response (bottom) of spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat vessels to cumulative addition of norepinephrine in the presence of 1.5 mmol/L extracellular Ca2+. Values are mean±SEM; n=10 and 11, respectively.

View larger version (12K): [in this window] [in a new window]   Figure 7. Line graph shows intracellular Ca2+-force relation of spontaneously hypertensive rat (SHR) and Wistar-Kyoto (WKY) rat resistance arteries. Values are steady-state levels obtained after cumulative addition of norepinephrine at the concentrations indicated in Fig 6. Values are mean±SEM; n=10 and 11, respectively.

	Discussion

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We have examined the Ca²⁺-force relation in mesenteric arteries of SHR, WKY rats, and Wistar rats in an effort to test the hypothesis that intracellular Ca²⁺ mobilization is heightened in mesenteric resistance arteries of SHR compared with vessels of normotensive WKY and Wistar rats and to examine the postulate that myofilament Ca²⁺ sensitivity is greater in vessels of SHR compared with those of the normotensive strains. The results in general do not support these hypotheses and have important implications with regard to the broader hypothesis that altered vascular cell Ca²⁺ handling plays a causal role in the heightened reactivity of vascular smooth muscle observed in hypertension of genetic origin.

A large body of experimental evidence demonstrates that resting levels of free intracellular platelet Ca²⁺ are elevated in platelets of hypertensive humans and SHR.^{16 17 18 19 20 21 22} As noted above, these results have been interpreted to indicate that free intracellular Ca²⁺ is also elevated in vascular smooth muscle. However, the results of the present study indicate that basal intracellular Ca²⁺ is not elevated in mesenteric resistance arteries of SHR compared with those of either WKY or Wistar rats and thus are inconsistent with the premise that basal Ca²⁺ is elevated in vascular smooth muscle of the SHR compared with normotensive strains.

The results obtained with vessels of SHR and WKY rats and now Wistar rats confirm and extend our earlier reports that intracellular Ca²⁺ does not differ between primary and first-passage cultures of mesenteric artery myocytes of SHR and WKY rats²³ and intact vessel segments of these strains.²⁴ These results do contrast with reports that basal and stimulated Ca²⁺ levels are elevated in cultured aortic myocytes^{13 14 15} and in intact aortic rings of SHR compared with WKY rats.³⁴ It is not known why intracellular Ca²⁺ may be elevated in aorta of SHR and not in the mesenteric bed. Aside from potential methodological differences, it is important to remember that the aorta is a conduit vessel that does not significantly contribute to peripheral resistance. Thus, the data obtained in the present study using resistance arteries may be more directly relevant to questions regarding the potential contribution of altered vascular Ca²⁺ handling to the SHR model of hypertension. In addition to basal Ca²⁺, we have also found that Ca²⁺ mobilization in response to graded increases in norepinephrine was not different in the resistance arteries of SHR and WKY rats (Fig 4, bottom). This indicates that alterations in agonist-induced Ca²⁺ mobilization are not associated with elevated blood pressure in this model.

The results summarized in Table 3 are of interest because they show that depletion of the releasable Ca²⁺ pool results in a decrease in the resting level of free intracellular Ca²⁺ in all of the vessels studied. It is possible that the magnitude of the fall in intracellular Ca²⁺ induced by Ca²⁺ depletion reflects the size of the releasable pool of Ca²⁺; ie, a greater decrease in Ca²⁺ could reflect a larger releasable pool. Although the magnitude of the fall in Ca²⁺ induced by Ca²⁺ depletion is greater in SHR than WKY rats, it was not different between SHR and Wistar rats, indicating that the size of the releasable pool of Ca²⁺ is unrelated to blood pressure.

Although these data indicate a dissociation between Ca²⁺ handling and blood pressure, this interpretation must be accepted with caution. For instance, all of the measurements depend on accurate calibration of the hydrolyzed fura 2. As noted in "Methods," we used an in situ approach to determine calibration constants for fura 2 on a vessel-by-vessel basis and used a novel approach to rescue hydrolyzed fura 2 from vessels of each strain and empirically determine the dissociation constant (K_d) of fura 2 for Ca²⁺. We have therefore ruled out the possibility that differential hydrolysis of fura 2-AM by vascular smooth muscle of the three strains results in a species of fura 2 with altered affinities for Ca²⁺. The possibility does remain, however, that differences in the intracellular milieu of vascular myocytes of the three strains could contribute to altered fluorescence properties that remain unidentified. Thus, short of making simultaneous measurements of intracellular Ca²⁺ using the fluorescent indicator and a Ca²⁺-selective electrode as recently described by Jensen and colleagues,³⁵ we believe that we have made a concerted effort to be accurate in our estimation of Ca²⁺ concentration.

Nonetheless, it remains possible that factors outside the realm of calibration may have affected the results. For example, it is possible that microcompartmentalization of Ca²⁺ pools exists within the cell and that these compartments cannot be distinguished with our signal-averaging approach. In addition, it is possible that influx of the cation into the cell is elevated in SHR but that sequestration and extrusion processes effectively remove it, having no net effect on cytosolic Ca²⁺ but resulting in greater turnover. Although such a phenomenon has been described in platelets,³⁶ the effect of altered Ca²⁺ turnover within the cell on stability of the Ca²⁺-calmodulin complex and its ability to activate myosin light chain kinase and initiate force generation are not understood.

Another important aspect of this report is the result of our assessment of myofilament Ca²⁺ sensitivity. We found no differences in myofilament Ca²⁺ sensitivity of SHR and WKY vessels during activation with 100 mmol/L K⁺. In contrast, we found that the ED₂₅ value for Ca²⁺ was greater in SHR vessels than in segments from both WKY and Wistar rats. These results indicate that myofilament Ca²⁺ sensitivity is enhanced in mesenteric resistance arteries of SHR compared with normotensive vessels. This observation agrees with the recent report of Soloviev and Bershtein,⁸ who examined Ca²⁺ sensitivity in saponin-skinned aorta of SHR and WKY rats, and earlier reports from Mulvany and Nyborg,⁹ which showed that the force response to extracellular Ca²⁺ is enhanced in SHR compared with WKY rats. It should be noted, however, that in contrast with the present report, neither of these studies included measurements of free intracellular Ca²⁺.

A concern that we had with our measurements was the possibility that the maneuver used to deplete releasable Ca²⁺ was nonphysiological and could complicate interpretation of the results. For example, it has been shown in white cells that Ca²⁺ depletion enhances flux of extracellular Ca²⁺ into the cell by causing the release of a soluble messenger.³⁷ To assess this further, we determined myofilament Ca²⁺ sensitivity of SHR and WKY vessels during cumulative addition of norepinephrine in the presence of a constant level of extracellular Ca²⁺ (1.50 mmol/L) and found no difference in myofilament Ca²⁺ sensitivity. We have concluded that the difference in myofilament Ca²⁺ sensitivity of SHR versus WKY and Wistar vessels observed during cumulative addition of extracellular Ca²⁺ is real but requires nonphysiological manipulation to be detectable. Its contribution to the physiological state is therefore questionable.

A final point to address is the finding that there was a large difference in the apparent myofilament Ca²⁺ sensitivity depending on which measurement approach was used. When the Ca²⁺-force relation was determined during cumulative addition of extracellular Ca²⁺ to the norepinephrine-activated preparation, myofilament Ca²⁺ sensitivity was in the range of 70 to 80 nmol/L, whereas when it was assessed during cumulative addition of norepinephrine in the presence of constant extracellular Ca²⁺, the value was on the order of 180 nmol/L. We suspect that the lower ED₅₀ values obtained with the first method are the result of constant exposure to the high level of norepinephrine, which could be expected to fully activate G protein–linked pathways that enhance myofilament Ca²⁺ sensitivity,^{38 39} whereas the higher value is the result of combined graded increases in activation of the coupling pathways and intracellular Ca²⁺. The precise mechanisms involved warrant further investigation.

In conclusion, the results of the present studies have important implications for the proposed role of altered cellular Ca²⁺ handling metabolism as a causal factor in genetic hypertension. If Ca²⁺ is not elevated in vascular smooth muscle of the mesenteric bed yet resistance in this bed is elevated in the intact animal, one is led to conclude that disturbed vascular Ca²⁺ metabolism does not play a role in maintaining elevated peripheral resistance. Our data therefore support the growing sentiment that in genetic hypertension, structural changes, which are the result of vascular hypertrophy or remodeling, may play a larger role in maintaining elevated peripheral resistance than do changes in vascular reactivity.⁴⁰

	Acknowledgments

 
This work was supported by a grant from the National Institutes of Health (HL-41816), Bethesda, Md. The authors would like to thank Wilma Frye for her assistance in preparing this manuscript.

Received January 31, 1994; first decision March 9, 1994; accepted September 8, 1994.

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