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(Hypertension. 1997;30:442.)
© 1997 American Heart Association, Inc.

Articles

Calcium Sensitivity and Agonist-Induced Calcium Sensitization in Small Arteries of Young and Adult Spontaneously Hypertensive Rats

Linda M. Shaw; Jacqueline Ohanian; Anthony M. Heagerty

From the Department of Medicine, Manchester Royal Infirmary, Manchester, UK.

Correspondence to Dr J. Ohanian, Department of Medicine, Manchester Royal Infirmary, Oxford Rd, Manchester, M13 9WL UK. E-mail johanian{at}fs1.cmht.nwest.nhs.uk

	Abstract

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Abstract The sensitivity of the myofilaments to Ca²⁺ is increased during agonist-induced contraction of vascular smooth muscle. Given the important contribution of vascular tone to the elevation of peripheral resistance observed in genetic hypertension, we have investigated whether alterations in myofilament Ca²⁺ sensitivity occur in small arteries from spontaneously hypertensive rats (SHR) and age-matched Wistar-Kyoto (WKY) controls during the developmental and established phases of hypertension. Segments of mesenteric, renal, and femoral artery with an average lumen diameter <300 µm from 5- or 20-week-old rats were mounted in a wire myograph. Morphological measurements were made and the vessels permeabilized with Staphylococcus aureus -toxin. Dose-response curves to increasing concentrations of Ca²⁺ were obtained and the ability of 100 nmol/L endothelin-1 (ET-1) or 10 µmol/L norepinephrine (NE) in the presence of 10 µmol/L GTP to enhance tension in response to low Ca²⁺ (pCa6.7) was determined. Systolic, diastolic, and mean blood pressures were higher in SHR than in WKY at 5 and 20 weeks. The media thickness:lumen diameter ratio was increased in mesenteric and femoral arteries from SHR compared with WKY at 5 and 20 weeks. There was no difference in media thickness:lumen diameter ratio in renal arteries or between 5- and 20-week animals in any vascular bed. The pCa curves were not different in mesenteric, renal, or femoral arteries from hypertensive compared with normotensive rats or between age groups, except in femoral arteries at 20 weeks, which exhibited a greater sensitivity to Ca²⁺ in SHR. Tension developed in response to maximal Ca²⁺ (pCa5.0) was greater in permeabilized mesenteric arteries from SHR compared with WKY at 20 weeks of age only; media stress was again similar in both strains but increased in older animals compared with younger animals in mesenteric arteries from WKY. The submaximal contraction induced by pCa6.7 was greater in femoral and renal than mesenteric arteries. GTP (10 µmol/L) augmented the tension developed to pCa6.7 in mesenteric arteries at 5 and 20 weeks and in renal arteries at 20 weeks. Addition of 100 nmol/L ET-1 or 10 µmol/L NE in the continued presence of GTP markedly increased tension in mesenteric arteries at 5 and 20 weeks. In renal arteries, 10 µmol/L NE enhanced Ca²⁺ sensitivity in the presence of GTP in SHR at 5 and 20 weeks and WKY at 5 weeks. In femoral arteries, there was a tendency for ET-1 and NE to increase Ca²⁺ sensitivity, but this increase was significant in WKY at 20 weeks (ET-1) and SHR at 5 weeks (NE) only. We have demonstrated that the sensitivity of the myofilaments to Ca²⁺ and ET-1– or NE-induced Ca²⁺ sensitization is not different in permeabilized small mesenteric, renal, or femoral arteries from SHR compared with WKY controls. Only in SHR mesenteric arteries at 20 weeks of age was there evidence of increased active tension in response to maximal Ca²⁺, despite structural differences, consistent with increased muscle mass in femoral arteries from SHR. We conclude that it is unlikely that a ubiquitous abnormality of the sensitivity of the contractile apparatus to Ca²⁺ or agonist-induced Ca²⁺ sensitization in vascular smooth muscle underlies the elevated total peripheral resistance associated with hypertension.

Key Words: rats, inbred SHR • myofilament calcium sensitivity • hypertension • small arteries

	Introduction

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Intracellular free Ca²⁺ concentration is a major determinant of smooth muscle tone, and the level of this ion is tightly regulated by a number of Ca²⁺-mobilizing and Ca²⁺-homeostatic mechanisms.¹ During agonist-induced contraction, intracellular Ca²⁺ levels rise through two mechanisms: release from intracellular stores and influx across the plasma membrane.² There is evidence that NE-stimulated Ca²⁺ influx is considerably higher in SHR than in WKY, which may contribute to the enhanced sensitivity to this agonist in mesenteric arteries.¹ However, in addition to increasing intracellular Ca²⁺, vasoconstrictor hormones sensitize the contractile myofilaments to Ca²⁺, and an abnormality in this mechanism could lead to enhanced vasoconstriction. Agonist-induced Ca²⁺ sensitization appears to be a G protein–mediated effect³ and involves downstream effectors such as myosin light chain phosphatase,⁴ protein kinase C,⁵ and tyrosine kinases.^{6 7} Recently Kanagy and Webb⁸ demonstrated that carotid artery strips from hypertensive rats had a greater responsiveness than strips from normotensive rats to mastoparan, a peptide that activates G proteins directly, suggesting that there may be altered G protein–mediated signaling in hypertension. Furthermore, Satoh et al⁹ reported augmented 5-hydroxytryptamine– and GTPS-induced Ca²⁺ sensitization in coronary arteries from SHRSP, again implicating altered intracellular signaling pathways. Also, it is possible that myofilament Ca²⁺ sensitivity is greater in arterial smooth muscle from hypertensive animals, although the evidence is conflicting. Increased Ca²⁺ sensitivity has been demonstrated in saponin-skinned aortic and portal vein tissue from SHR compared with WKY,¹⁰ and Bian and Bukoski¹¹ showed that after Ca²⁺ depletion, mesenteric arteries from SHR developed greater force to increasing concentrations of Ca²⁺ in the presence of 10 µmol/L NE than vessels from normotensive strains. In contrast, no alteration in the sensitivity of myofilaments to Ca²⁺ was detected in skinned rat tail artery rings¹² or coronary arteries⁹ from the SHRSP compared with vessels from normotensive control animals. A possible explanation for these conflicting reports may be the diversity of vascular beds, size of vessel, and agonists studied.

Contraction mediated directly by Ca²⁺ and agonist-induced Ca²⁺ sensitization may be investigated in small arteries permeabilized with -toxin. In this preparation, small pores are formed in the plasma membrane that allow the levels of ions such as Ca²⁺ to be controlled but leave receptors coupled to their signaling systems.^{3 13} Using this preparation, we have investigated whether the sensitivity of the myofilaments to Ca²⁺ is altered in the early and established phases of hypertension in small arteries from the mesenteric, renal, and femoral vascular beds in hypertensive compared with normotensive rats. We have examined also whether there is altered NE- or ET-1–induced Ca²⁺ sensitization in these vessels at two stages of the hypertensive process and whether structural changes of the arterial wall influence these responses.

	Methods

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Animals
For this study, male SHR and their control strain WKY were used. They were obtained from Charles River Ltd (Margate, Kent, UK). All procedures performed were carried out by trained personnel in accordance with institutional guidelines and the UK Animals (Scientific Procedures) Act of 1986.

Blood Pressure Measurement
Direct blood pressures were measured as previously described.¹⁴ Briefly, at 5 or 20 weeks of age, femoral artery cannulas were implanted under anesthesia (0.26 mg fentanyl citrate, 8.25 mg fluanisone, and 4.1 mg midazolam per kilogram body weight), and the proximal tip of the catheter was advanced into the abdominal aorta to permit direct measurement of aortic blood pressure. The distal region of the catheter was exteriorized between the scapulae, flushed with saline containing 100 U/mL heparin, and blocked with a stainless steel spigot. Analgesia was provided by 0.3 mg buprenorphine per kilogram body weight. Twenty-four hours after catheter implantation, unrestrained, conscious blood pressure recordings were made with a Viggo-Spectramed TXX-R pressure transducer connected to a chart recorder. Mean aortic blood pressure was calculated as diastolic blood pressure plus one third pulse pressure.

Small Artery Structure
On the day of study, rats were stunned by a blow to the head and killed by cervical dislocation, in accordance with Institutional and American Veterinary Medical Association guidelines on euthanasia.¹⁵ Femoral small arteries (third-order branch vessels originating distal to the femoral artery were used in 20-week rats, but due to size limitations, second-order vessels were used in 5-week rats), mesenteric small arteries (third-order branches of the superior mesenteric artery), and renal small arteries (first-order branches of the main renal artery) were dissected out and cleaned of extraneous fat and connective tissue. Segments (approximately 2 mm long) were mounted as ring preparations in an isometric myograph¹⁶ and warmed to 37°C.

The myograph was designed with a small Perspex window situated directly below the mounting heads so that when it is mounted on the stage of a light microscope (Leitz, UK Ltd), the artery wall can be imaged by using a water-immersion objective (x40, Zeiss Ltd). A calibrated micrometer eyepiece (x8, Zeiss Ltd) was used to measure the thickness of the media. Media CSA (equivalent to media volume per unit length) was calculated from the media thickness and internal circumference. The resting tension–internal circumference relation was determined for each segment of artery as described previously.¹⁶ From this value, the internal circumference (L₁₀₀) that the artery would have if relaxed and under a transmural pressure of 100 mm Hg (13.3 kPa) was calculated. The corresponding internal diameter (l₁₀₀) was calculated as L₁₀₀/. Each artery was then set to the normalized effective internal diameter (l₀), where l₀=0.9 l₁₀₀.¹⁶ Assuming constant media volume, the media thickness could be calculated for l₀.

Permeabilization
After the vessels were incubated in relaxing solution for 15 minutes, they were permeabilized according to the method of Kitazawa et al.³ Briefly, a 10-µL droplet of pCa6.7 solution containing 1250 units of Staphylococcus aureus -toxin and 10 µmol/L A23187, to deplete the sarcoplasmic reticulum of Ca²⁺, was placed onto each vessel segment. After tension development had reached a plateau (a period of 15 to 20 minutes), the vessels were equilibrated in relaxing solution and bubbled with 100% O₂. All experiments were carried out at room temperature.

After permeabilization, each artery was stimulated by a series of solutions of increasing Ca²⁺ concentration. A pCa curve was obtained by normalizing the tension for each submaximal Ca²⁺ contraction with respect to maximal tension and plotting as a function of pCa. The contractile response to the maximum Ca²⁺ concentration was calculated as active tension (mN/mm), defined as force response divided by twice vessel segment length and active media stress (mN/mm²), defined as the active tension divided by media thickness at l₀, which has been described previously.¹⁷

The Ca²⁺-sensitizing effects of (1) 10 µmol/L GTP, (2) 10 µmol/L GTP+100 nmol/L ET-1, and (3) 10 µmol/L GTP+10 µmol/L NE at pCa6.7 were determined. After each addition, the contraction elicited by that particular compound was allowed to reach a plateau before the next was added. Initial dose-response curves showed that 100 nmol/L ET-1 and 10 µmol/L NE produced the maximum contractile response to these agonists.

Solutions
Vessels were dissected out and held in the myograph in physiological saline solution of the following composition (in mmol/L): NaCl 119, KCl 4.7, CaCl₂ 2.5, NaHCO₃ 25, MgSO₄ 1.17, KH₂PO₄ 1.18, K₂EDTA 0.026, and glucose 5.5, which was bubbled with 95% O₂/5% CO₂ to give a pH of 7.4 at 37°C. The composition of solutions involved in permeabilization were as follows. Relaxing solution consisted of (in mmol/L) PIPES 30, sodium creatine phosphate 10, Na₂ATP 5.16, magnesium methane sulfonate 7.31, potassium methane sulfonate 74.1, K₂EGTA 1; pH was adjusted to 7.1 with KOH. In the -toxin or activating solutions, 10 mmol/L EGTA was used, and a specified amount of calcium methane sulfonate was added to give the desired concentration of free Ca²⁺ ions.^{3 18}

Drugs
Staphylococcus aureus -toxin was obtained from GIBCO-BRL. A23187, GTP, NE, and ET-1 were obtained from Sigma Chemical Company. A23187 was dissolved in DMSO (final concentration <1%), and GTP, NE, and ET-1 were dissolved in distilled water.

Data Calculation and Statistical Analysis
Each contraction was measured from the baseline and calculated as tension and media stress. All results were expressed as mean±SEM. Individual group sizes are indicated in the text, tables, and figure legends. Repeated measures ANOVA was used to determine differences between SHR and WKY pCa dose-response curves. ANOVA plus the least significant difference test was used to assess differences in blood pressure and in mesenteric and renal arteries. Because femoral arteries from the two age groups were taken from different locations, differences between strains and pCa₅₀ values (the pCa required to elicit half-maximal tension) were calculated using Student’s t test. All other comparisons were by Student’s t test. P<.05 was considered statistically significant.

	Results

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Blood Pressure
Systolic blood pressure, diastolic blood pressure, and mean blood pressure were greater at 5 and 20 weeks in SHR than in WKY (P<.05, Table 1). There was an increase in blood pressure in both strains of rat with age (P<.05, Table 1).

View this table: [in this window] [in a new window]   Table 1. Systolic, Diastolic, and Mean Blood Pressures for 5- and 20-Week-Old SHR and WKY

Morphological Measurements
Values of lumen diameter, media thickness, media CSA, and media thickness:lumen diameter are shown in Table 2 and Fig 1. Mesenteric and femoral arteries from SHR clearly showed structural differences compared with WKY controls at both 5 and 20 weeks of age. The only difference detected in the renal arteries from SHR was an increase in media thickness at 20 weeks of age. Within-strain age-related differences in morphology of mesenteric and renal vessels were observed also (Table 2, Fig 1). These comparisons could not be made for femoral arteries, as they were different order vessels at 5 and 20 weeks.

View this table: [in this window] [in a new window]   Table 2. Morphological Characteristics of Small Arteries From Three Vascular Beds for 5- and 20-Week-Old SHR and WKY

View larger version (20K): [in this window] [in a new window]   Figure 1. Relationship between tension developed to Ca2+ (pCa5.0) and media thickness to lumen diameter ratio in permeabilized small arteries from SHR and WKY. Segments of mesenteric, renal, and femoral artery from 5- and 20-week-old WKY and SHR were mounted as ring preparations in a wire myograph, media thickness and lumen diameter measured, the vessels permeabilized with -toxin, and the tension developed to maximal (pCa5.0) Ca2+ determined as described in "Methods." Media thickness to lumen diameter ratios were calculated and plotted against tension. Data from 5-week animals are in the upper panel and 20-week animals in the lower panel; , , , WKY; , •, , SHR. Data are mean±SEM from a minimum of 5 and maximum of 16 separate experiments; numbers of animals in individual experiments are shown in parentheses. Statistical comparison is by Student’s t test for differences between strains in femoral arteries and by ANOVA plus the least significant difference test for differences in mesenteric and renal arteries. *P<.05 vs WKY for media:lumen ratio. +P<.05 vs WKY for pCa5.0.

Ca²⁺-Induced Contraction
There was no significant difference in the active tension or media stress developed in response to pCa5.0 (maximal Ca²⁺) between SHR and WKY controls in any of the vessels studied, except for mesenteric arteries from 20-week-old SHR, which developed approximately 25% greater tension than those from 20-week-old WKY (Table 3, Fig 1). Fig 1 clearly shows that the greater media:lumen ratio in femoral and mesenteric arteries from the hypertensive animals did not result in enhanced active tension development to pCa5.0 compared with vessels from age-matched normotensive animals. However, age-dependent changes were observed, such that 20-week animals developed approximately 60% greater tension to pCa5.0 than 5-week animals in mesenteric arteries (Table 3). Media stress was increased also in older animals compared with younger animals in mesenteric arteries, but only in the normotensive animals (Table 3). There was no difference in maximum tension or media stress in response to pCa5.0 between 5- and 20-week animals in renal arteries (Table 3).

View this table: [in this window] [in a new window]   Table 3. Tension and Media Stress Induced by Maximal Calcium (pCa5.0) in Permeabilized Small Arteries

Comparison Between Vascular Beds
The tension developed to maximal Ca²⁺ (pCa5.0) was greater in femoral arteries than mesenteric arteries from both hypertensive and normotensive rats at 5 and 20 weeks of age and from renal arteries at 5 weeks only (Table 3). These data correlate with the markedly greater media:lumen ratio of femoral arteries compared with mesenteric and renal vessels (Fig 1).

Ca²⁺ Sensitivity
There was a greater sensitivity to Ca²⁺ in femoral arteries from SHR than WKY at 20 weeks: pCa₅₀ values were 6.35 and 6.07 for SHR and WKY, respectively; P<.05 (Fig 2).

View larger version (13K): [in this window] [in a new window]   Figure 2. Ca2+ tension relationship in -toxin–permeabilized femoral small arteries from 20-week SHR () and WKY (). Responses were normalized to tension achieved at pCa5.0. Values are mean±SEM; n=12.

There was no difference in Ca²⁺ sensitivity of tension development at either 5 or 20 weeks in the remaining vessels studied between SHR and WKY controls. No age-dependent changes in Ca²⁺ sensitivity were detected in any of the vessels studied.

Agonist-Induced Ca²⁺ Sensitization
In the second stage of this study, we investigated whether NE and ET-1 in the presence of GTP were able to increase the sensitivity of the myofilaments to Ca²⁺ and whether these responses were altered in hypertension. An illustration of the protocol used and the sensitization response induced by 100 nmol/L ET-1 in a mesenteric artery from a 5-week WKY is shown in Fig 3.

View larger version (6K): [in this window] [in a new window]   Figure 3. Agonist-induced Ca2+ sensitization in an -toxin–permeabilized small artery. A segment of mesenteric artery from a 5-week WKY was mounted in a wire myograph and permeabilized with -toxin as described in "Methods." The tension developed to pCa6.7 and the effects of addition of GTP (10 µmol/L) and ET-1 (100 nmol/L) were recorded.

Mesenteric Arteries
GTP (10 µmol/L) slightly enhanced the submaximal contraction induced by pCa6.7 (Fig 4). The addition of 100 nmol/L ET-1, in the continued presence of GTP, markedly augmented tension at constant low Ca²⁺. A similar increase in tension in the presence of GTP and low constant Ca²⁺ was observed with 10 µmol/L NE. There was no difference in the Ca²⁺ sensitization induced by ET-1 and NE between vessels from SHR and WKY controls at either 5 or 20 weeks of age.

View larger version (28K): [in this window] [in a new window]   Figure 4. Ca2+-sensitization responses induced by ET-1 and NE in the presence of low Ca2+ and GTP. -Toxin–permeabilized mesenteric arteries were stimulated with pCa6.7, GTP (10 µmol/L), GTP+ET-1 (100 nmol/L), and GTP+NE (10 µmol/L) as described in "Methods." Top, Tension responses from 5-week-old animals; bottom, 20-week-old animals. Open bars represent WKY controls; hatched bars, SHR. Data are mean±SEM; n=6. *P<.05, agonist vs pCa6.7 WKY; +P<.05, agonist vs pCa6.7 SHR, by Student’s paired t test.

Renal Arteries
GTP (10 µmol/L) augmented the tension developed to submaximal Ca²⁺ (pCa6.7) in arteries from both SHR and WKY at 20 weeks of age only (Fig 5). ET-1 did not induce a further increase in tension compared with GTP alone in either strain or at either age. In contrast, NE in the presence of GTP increased tension at constant low Ca²⁺ in renal arteries from SHR at 5 and 20 weeks and from WKY at 5 weeks.

View larger version (35K): [in this window] [in a new window]   Figure 5. Ca2+-sensitization responses induced by ET-1 and NE in the presence of low Ca2+ and GTP. -Toxin–permeabilized renal arteries were stimulated with pCa6.7, GTP (10 µmol/L), GTP+ET-1 (100 nmol/L), and GTP+NE (10 µmol/L) as described in "Methods." Top, Tension responses from 5-week-old animals; bottom, 20-week-old animals. Open bars represent WKY controls; hatched bars, SHR. Data are mean±SEM; n=4. *P<.05, agonist vs pCa6.7 WKY; +P<.05, agonist vs pCa6.7 SHR, by Student’s paired t test.

Femoral Arteries
GTP (10 µmol/L) did not augment the tension developed to submaximal Ca²⁺ (pCa6.7) significantly (Fig 6). There was a tendency for tension to increase in the presence of ET-1 and GTP, although this increase was significant in arteries from WKY at 20 weeks of age only. Similarly increased Ca²⁺ sensitivity in response to NE was slight, and significant changes were observed in vessels from SHR at 5 weeks of age only.

View larger version (33K): [in this window] [in a new window]   Figure 6. Ca2+-sensitization responses induced by ET-1 and NE in the presence of low Ca2+ and GTP. -Toxin–permeabilized femoral arteries were stimulated with pCa6.7, GTP (10 µmol/L), GTP+ET-1 (100 nmol/L), and GTP+NE (10 µmol/L) as described in "Methods." Top, Tension responses from 5-week-old animals; bottom, 20-week-old animals. Open bars represent WKY controls; hatched bars, SHR. Data are mean±SEM; n=4. *P<.05, agonist vs pCa6.7 WKY; +P<.05, agonist vs pCa6.7 SHR, by Student’s paired t test.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
To investigate whether the sensitivity of the myofilaments to Ca²⁺ was altered in small arteries in hypertension, we studied -toxin–permeabilized vessels in vitro taken from the mesenteric, renal, and femoral vascular beds from SHR and WKY controls during the onset and established phases of hypertension. At 5 weeks of age, blood pressure was elevated in SHR compared with age-matched WKY controls, and structural differences were already present in the mesenteric and femoral arteries. Furthermore, the structural modifications that occurred varied between the individual beds, enabling us to investigate the contractile response to a directly acting stimulus, ie, Ca²⁺, in conjunction with four different structural adaptations: (1) remodeling, which involved reduced lumen diameter and increased media:lumen ratio, with no accompanying change in media CSA, in mesenteric arteries at 5 weeks in SHR; (2) growth, which was demonstrated by increased wall thickness and no change in lumen diameter in femoral arteries at 5 and 20 weeks in SHR; (3) remodeling and growth in mesenteric arteries at 20 weeks in SHR (remodeling and growth indices were calculated as in Reference 19¹⁹ ; data not shown); and (4) no significant media:lumen ratio difference in renal arteries at 5 and 20 weeks of age in SHR compared with WKY. These structural alterations are in good agreement with previous studies in mesenteric^{14 17 20 21} and femoral^{22 23} arteries, confirming that the contributions of remodeling and growth to the structural modifications that occur in the vascular wall of small arteries in SHR vary according to the vascular bed studied.¹⁹ We did not detect any difference in either the maximum contractile response or sensitivity to Ca²⁺ in any of the arteries studied at 5 weeks of age between normotensive and hypertensive animals, despite elevated blood pressure and increased media:lumen ratio in femoral and mesenteric arteries. These data demonstrate that increased myofilament sensitivity to Ca²⁺ is unlikely to be involved in the developing stage of hypertension in this animal model. Later, when hypertension was established, there was an increase in the tension developed to maximum Ca²⁺ in mesenteric arteries from SHR. However, this effect was lost when the structural differences were accounted for, suggesting that the increased tension was due to increased media thickness. A similar response has been demonstrated in intact small mesenteric arteries from 20- to 24-week-old SHR constricted with high K⁺.¹⁷ Our data suggest that marked (>50%) changes in media:lumen ratio are required to observe significant increases in active tension in response to a direct activator of contraction, because mesenteric vessels from 5-week and femoral vessels from 5- and 20-week animals exhibited 35% to 40% increases in media:lumen ratio without enhancement of the tension developed to maximal Ca²⁺ (see Fig 1). Or it may be that a combination of remodeling and growth, seen only in mesenteric arteries from 20-week-old SHR, which may result in a more efficient arrangement of muscle cells around the lumen, is required for this response. When dose-response curves were constructed, the sensitivity to Ca²⁺ was not altered in permeabilized mesenteric arteries from SHR, indicating that this was not the explanation for the increased maximal tension in response to Ca²⁺ in these arteries. In fact, only femoral arteries from 20-week-old hypertensive rats exhibited an increase in Ca²⁺ sensitivity compared with normotensive animals. No previous studies have investigated Ca²⁺ sensitivity in permeabilized femoral arteries in hypertension. However, in larger intact femoral arteries from the SHRSP, no difference in myofilament Ca²⁺ sensitivity was observed when membrane depolarization was achieved with K⁺.²⁴ These differences may reflect the size of femoral artery used or the different protocols. Certainly, in permeabilized preparations, no difference in Ca²⁺ sensitivity of the contractile apparatus has been found in coronary⁹ or tail¹² arteries of the SHRSP or in intact small arteries from the mesenteric^{20 25 26} bed of SHR. In contrast, Soloviev and Bershtein¹⁰ demonstrated increased Ca²⁺ sensitivity in permeabilized aorta and portal vein smooth muscle from SHR compared with WKY, suggesting differences between large conduit and small resistance arteries.

Hypertension did not affect the Ca²⁺ sensitization induced by GTP, ET-1, or NE in -toxin–permeabilized mesenteric arteries. This is an important observation, as increased sensitivity to NE²⁰ and decreased vascular smooth muscle sensitivity to ET-1²⁷ have been reported in intact mesenteric small arteries from SHR compared with WKY controls. This observation suggests that the differences in reactivity observed in intact mesenteric arteries to these agonists are not caused by an abnormality of the processes regulating myofilament contractility but may be due to altered Ca²⁺ handling. Indeed, there is evidence that smooth muscle Ca²⁺ homeostasis is compromised in hypertensive animal models.²⁸

Our results in permeabilized mesenteric arteries are in contrast to the reported enhanced GTPS- and 5-hydroxytryptamine–induced Ca²⁺ sensitization in permeabilized coronary arteries from SHRSP.⁹ An explanation for this difference is not immediately apparent, although it most probably reflects differences in the mechanisms coupling agonists to contraction in smooth muscle and/or differences in the vascular beds studied. The coronary arteries used by Satoh et al⁹ were smaller (approximately 140 µm) and exhibited no morphological differences compared with WKY control vessels, although the blood pressure of the animals was similar to our study. However, in agreement with our results, there was no difference in the sensitivity to Ca²⁺ in these coronary arteries, suggesting that the abnormality lay upstream of the contractile proteins and their immediate regulatory proteins, myosin light chain kinase and phosphatase. Also, these authors reported a decrease in desensitization of contraction in response to Ca²⁺ in coronary arteries from SHRSP compared with WKY, suggesting that the balance between these two mechanisms may be disturbed in these vessels in hypertension,⁹ which could result in an apparent increase in agonist-induced Ca²⁺ sensitization.

Endothelin-1– and NE-induced Ca²⁺ sensitization was poor in -toxin–permeabilized renal and femoral arteries compared with mesenteric arteries. This finding was surprising, as intact renal and femoral arteries constrict to NE and ET-1 in vitro.^{23 29} It is unlikely that this effect was due to differences in efficiency of -toxin permeabilization, as the tension developed to maximal Ca²⁺ showed a positive relationship with the thickness of the arterial wall (see Fig 1), such that tension developed by the thicker-walled femoral arteries was greater than that of the thinner-walled mesenteric arteries. Any inefficiency of permeabilization would be expected to occur in the thicker-walled vessels, due to restricted diffusion of -toxin, and this occurrence is not supported by our data. Another explanation is that ET-1– and NE-induced contraction in femoral and renal vessels is predominantly dependent on Ca²⁺, whereas in mesenteric arteries, modulation of the Ca²⁺ sensitivity of the myofilaments by these agonists may play a more substantial role. Therefore, in addition to heterogeneity in the contribution of pharmacomechanical and electromechanical coupling to the response to agonists, dependent on the size of vessel studied,^{30 31} there may be heterogeneity between arteries from different vascular beds. These differences between arteries may underlie the unequal contribution of organ resistance to the elevated total peripheral resistance reported in renovascular and genetic hypertension.³² However, further studies are required to test this possibility.

In conclusion, we have shown that the sensitivity of the myofilaments to Ca²⁺ and agonist-induced Ca²⁺ sensitization are not different in small arteries from peripheral vascular beds in SHR compared with WKY, despite increased blood pressure and marked structural modifications. These results are in contrast to the enhanced 5-hydroxytryptamine–induced Ca²⁺ sensitivity in coronary arteries from SHRSP⁹ and do not support a role for increased vascular smooth muscle Ca²⁺ sensitivity or agonist-induced Ca²⁺ sensitization in the maintenance of elevated peripheral resistance in genetic hypertension.

	Selected Abbreviations and Acronyms

 

CSA = cross-sectional area ET-1 = endothelin-1 NE = norepinephrine SHR = spontaneously hypertensive rat(s) SHRSP = spontaneously hypertensive stroke-prone rats WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
This study was funded by the British Heart Foundation. We are grateful to Stuart J. Bund for performing the blood pressure measurements and to Ashley S. Izzard for discussion of the manuscript.

Received August 23, 1996; first decision September 18, 1996; accepted January 21, 1997.

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