Abstract We hypothesized that in female spontaneously hypertensive rats (SHR), estrogen moderates the dysfunction of arterioles by preserving nitric oxide synthesis. To this end, we conducted experiments on isolated gracilis muscle arterioles (approximately 55 μm in diameter) of 12-week-old SHR divided into four groups: females (fSHR), ovariectomized females (fSHR-OV), ovariectomized females with estrogen replacement (fSHR-OV+ES, 50 μg/kg SC 17β-estradiol benzoate every 48 hours), and males (mSHR). Arteriolar diameter in the presence of perfusion pressures of 60, 80, 100, and 120 mm Hg were obtained, and diameter changes were measured (at 80 mm Hg) in response to various concentrations of substance P (10−9 to 5×10−8 mol/L), sodium nitroprusside (10−8 to 10−6 mol/L), and A23187 (5×10−8 to 10−6 mol/L). The pressure-induced diameter of mSHR and fSHR-OV arterioles was significantly less (by approximately 10%) than that of fSHR and fSHR-OV+ES arterioles. Nω-nitro-l-arginine (10−4 mol/L), a nitric oxide synthase inhibitor, elicited a significant decrease in basal arteriolar diameter of fSHR (by approximately 19%) and fSHR-OV+ES (by approximately 17%), thereby eliminating the differences in tone among the various groups. Dilations of fSHR and fSHR-OV+ES arterioles to substance P were significantly greater (by 140% at a concentration of 5×10−8 mol/L) than those of mSHR and fSHR-OV arterioles, whereas dilations to sodium nitroprusside were not different among the groups. A23187 (a nitric oxide releaser) elicited dilations in arterioles of fSHR (5.9±1.5%, 13.0±1.8%, and 19.2±2.1%) and fSHR-OV+ES (4.3±1.0%, 10.3±2.4%, and 15.0±4.0%) but constrictions in those of mSHR (−7.5±1.6%, −25.3±39%, and −36.9±4.1%) and fSHR-OV (−2.6±1.7%, −7.4±3.3%, and −11.5±6.1%). We conclude that estrogen in fSHR is responsible for the preservation of nitric oxide synthesis in skeletal muscle arterioles, resulting in a greater modulation of pressure-induced myogenic tone than in mSHR and maintenance of nitric oxide–mediated dilations.
It has been documented that the incidence of cardiovascular diseases (hypertension, atherosclerosis) is significantly lower in premenopausal women than in age-matched men or postmenopausal women,1 2 suggesting a role for female hormones in this phenomenon. Indeed, estrogen replacement therapy in postmenopausal women has been shown to reduce markedly the risk of cardiovascular diseases,2 and essential hypertension is less frequent in premenopausal women than in men of the same age.3 A similar pattern has been found in SHR: the appearance of hypertension is delayed and less severe in female than male rats.4 The underlying mechanisms of the beneficial effect of estrogen are not fully understood. One of the hypotheses to explain these mechanisms is that estrogen affects the vascular system by promoting a lower vascular tone,5 although favorable effects of estrogen on lipid profile6 and body weight7 have also been suggested.
In support of the vascular hypothesis, in vitro studies of large vessels demonstrated a positive relationship between estrogen and basal or stimulated release of endothelium-derived NO,8 9 10 11 although there are reports contrary to this idea.12 In various tissues of guinea pigs13 and rats,14 an upregulation of NO synthase by estrogen has also been reported. In vivo studies in humans15 and animals16 17 demonstrated that estrogen can significantly increase the level of circulating nitrite and nitrate in serum, implying an enhanced release of NO into the circulation, although the source of NO has not been identified. Also, in the aorta of female normotensive rats, a greater basal and stimulated NO release has been observed than in that of the male.18 In support of these studies, it has been demonstrated that estrogen stimulates NO synthase activity in porcine uterine artery and heart and skeletal muscle,19 suggesting that this leads to reduced peripheral vascular tone.
It has been shown that endothelium-derived factors, among them NO,20 tend to counteract pressure-induced myogenic tone in skeletal muscle microvessels in normotensive21 22 but not in hypertensive23 24 rats. Recently, we also found an estrogen-dependent augmentation of NO-mediated arteriolar dilations in normotensive female compared with male rats.25 To establish the importance of estrogen-stimulated NO release in the regulation of peripheral resistance in hypertensive females, it is necessary to demonstrate whether estrogen, via NO, modulates the myogenic tone of arterioles in hypertension. In addition, a difference between NO-dependent responses of arterioles of females and males would further support a role for estrogen in the maintenance of NO synthase in vascular tissue.
Thus, we hypothesized that the presence of estrogen in female hypertensive rats maintains arteriolar NO synthesis, providing for a lower arteriolar pressure–induced myogenic tone and greater NO-mediated responses than in hypertensive males.
Rats were divided into four groups: (1) 12-week-old male SHR (mSHR), (2) age-matched female SHR (fSHR), (3) 12-week-old ovariectomized female SHR injected subcutaneously (0.1 mL) with 17β-estradiol benzoate (50 μg/kg, every 48 hours) for 3 weeks (fSHR-OV+ES), and (4) 12-week-old ovariectomized female SHR injected subcutaneously with vehicle for 3 weeks (fSHR-OV). Ovariectomy was performed at 9 weeks of age. Rats were anesthetized by methoxyflurane inhalation. A 1-mm incision was made on both sides of the back to expose the ovaries retroperitoneally. The ovaries were clamped and removed, the fallopian tubes were ligated, and the skin was sutured. One week after operation, rats were divided into two groups: one received estradiol replacement and the other received vehicle injection for 3 weeks.
Isolation of Gracilis Muscle Arterioles
Experiments were conducted on isolated second-order arterioles (approximately 55 μm in diameter) of rat gracilis muscle. Rats were anesthetized with intraperitoneal injections of sodium pentobarbital (50 mg/kg). The isolation procedure of arterioles has been described previously.25 Briefly, the muscle was excised and placed into a refrigerated dissecting dish containing a cold PSS (0° to 4°C) buffered with 3-(N-morpholino)propanesulfonic acid (MOPS) (pH 7.4). The solution contained (mmol/L) NaCl 145.0, KCl 5, CaCl2 2.0, MgSO4 1, NaH2PO4 1.0, dextrose 5.0, pyruvate 2.0, EDTA 0.02, and MOPS 3.0. The muscle was splayed open as a flat sheet of tissue and pinned to the bottom of the silicone-lined base of the dissecting dish. After the muscle had been dissected, blood samples were drawn from the abdominal aorta for measurement of serum estradiol concentration. The rats were euthanized by an overdose of pentobarbital sodium (150 mg/kg).
The arteriole was cleared from the adhering tissue by microscissors. The approximately 1.0-mm-long section of the arteriole was transferred to the vessel chamber containing Krebs’ bicarbonate-buffered PSS and two glass microcannulas. The proximal end of the arteriole was mounted to the inflow cannula, and the perfusion pressure was increased to 20 mm Hg with a pressure-servo syringe reservoir system (Living Systems), as described previously.25 After the arteriole was cleared of clotted blood, its distal end was mounted to the outflow cannula.
PSS, which was used to perfuse as well as suffuse the arteriole in the vessel chamber, was a Krebs’ bicarbonate buffer solution equilibrated with 21% O2/5% CO2/74% N2, pH 7.4, at 37°C. The solution contained (mmol/L) NaCl 118, KCl 5, CaCl2 2.5, MgSO4 1, KH2PO4 1, NaHCO3 24, dextrose 10, and EDTA 0.02. The perfusion system, reservoir, and vessel chamber had a total volume of 100 mL. PSS flow through the chamber was 40 mL/min. Initially, the vessel was perfused with approximately 8 μL/min at 20 mm Hg pressure for several minutes to clear it of blood. The outflow cannula was then closed and the intravascular pressure slowly increased to 80 mm Hg. The pressure-servo system was then placed in the manual mode, in which the stable pressure value indicated that the system was not leaking. Then the pressure-servo system was set in the automatic mode at 80 mm Hg, and the vessel was allowed to equilibrate for approximately 1 hour. Steady-state arteriolar diameter and peak changes in diameter were measured with an image shearing monitor (Instrumentation for Physiology & Medicine) and recorded on a chart recorder (model MC6625, Graphtec Multicorder).
In all protocols, the basal diameter of arterioles in the presence of various perfusion pressures (60, 80, 100, and 120 mm Hg) and under no-flow conditions was measured. Only those vessels that developed spontaneous tone to pressure were used, and no vasoactive agent was added to the PSS. After the equilibration period, each pressure step was maintained for 5 to 10 minutes to allow the vessels to reach a stable condition before arteriolar diameter was measured. Next, dose-dependent responses of arterioles to vasoactive agents, including substance P (10−9, 10−8, and 5×10−8 mol/L), sodium nitroprusside (10−8, 10−7, and 10−6 mol/L), the calcium ionophore A23187 (5×10−8, 5×10−7, and 10−6 mol/L), and acetylcholine (10−9, 10−8, and 5×10−8 mol/L) were obtained.
In the second protocol, after control responses were obtained, the vessel was subjected to L-NNA (10−4 mol/L), an NO synthase inhibitor, for 20 minutes. Then the basal diameter of arterioles at various perfusion pressures was reassessed. The lack of dilations to substance P (5×10−8 mol/L) and the retained responses to sodium nitroprusside (10−7 mol/L) indicated the efficacy and specificity of L-NNA.21 26
All drugs were added to the reservoir connected to the vessel chamber, and final concentrations are given. Responses to vasoactive agents were tested at a perfusion pressure of 80 mm Hg. At the conclusion of each experiment, the suffusion solution was changed to a Ca2+-free PSS that contained sodium nitroprusside (10−4 mol/L) and EGTA (1.0 mol/L). The vessel was incubated for 10 minutes, the step increases in pressure were repeated, and the passive diameter of arterioles at each pressure step was obtained.
Measurements of Serum Estradiol
After the muscle had been dissected, 5 mL blood was withdrawn from the abdominal aorta with a 10-mL syringe containing 0.1 mL heparin sodium (1000 USP U/mL). The blood sample was centrifuged immediately (3000 rpm at 4°C for 30 minutes) to obtain the plasma. The plasma was kept at −80°C for later measurement of serum estradiol concentration by a radioimmunoassay with a double-antibody estradiol kit (Diagnostic Products Corp). The samples were run in duplicate, and average numbers are reported.
All salts and chemicals were obtained from Sigma Chemical Co or Aldrich Chemical Co and were prepared on the day of the experiment. A23187 was dissolved in dimethyl sulfoxide and diluted with distilled water. 17β-Estradiol benzoate was dissolved in pure ethanol (5 mg/mL) with sesame oil as vehicle. The vehicle solutions did not affect the vascular diameters. All other drugs were dissolved in distilled water.
Data are presented as mean±SE. Changes in diameter in response to vasoactive agents or pressure were normalized to the corresponding passive diameter and expressed as percent changes. Statistical analyses were done by ANOVA (random and fixed models), followed by Tukey’s post hoc test. Student’s t tests (paired and grouped) were used for data from drug responses (within and between groups, respectively), as appropriate. A value of P<.05 was considered significant.
Body Weight, Uterus Weight, and Serum Estradiol Concentration
The Table⇓ shows the changes of body weight, uterus weight, and the ratio of uterus to body weights in fSHR, fSHR-OV, and fSHR-OV+ES. The uterus weights of fSHR-OV were significantly lower than those of fSHR and fSHR-OV+ES (P<.05). In contrast, the body weights of fSHR-OV were significantly increased compared with those of fSHR and fSHR-OV+ES (P<.05). As a result, the ratios of uterus to body weight were significantly different between fSHR-OV and fSHR or fSHR-OV+ES (P<.05). Serum 17β-estradiol concentration was significantly reduced after ovariectomy but was normal in rats with estrogen replacement (Table⇓).
Basal Arteriolar Diameter
Fig 1⇓, top, shows the arteriolar diameter of gracilis muscle arterioles from mSHR, fSHR, fSHR-OV, and fSHR-OV+ES at various perfusion pressures, expressed as a percentage of corresponding passive diameter, depicting the basal tone generated in response to pressures. Fig 1⇓ (top panel) demonstrates that the pressure-induced diameters of arterioles from mSHR and fSHR-OV are significantly smaller (ie, greater constrictions to all perfusion pressures) than those from fSHR and fSHR-OV+ES.
In the presence of L-NNA (10−4 mol/L), the basal diameter of arterioles was reduced in all groups, but more so in fSHR and fSHR-OV+ES (Fig 1⇑, bottom). Similar to L-NNA treatment, endothelium removal reduced basal diameters of vessels from all groups and eliminated the differences among them (not shown). The differences in arteriolar diameter in various rat groups during control conditions and after L-NNA administration are shown in Fig 2⇓. In fSHR and fSHR-OV+ES, the reduction of arteriolar diameter in response to L-NNA was significantly (P<.05) greater than in mSHR and fSHR-OV.
Arteriolar Responses to Vasoactive Agents
Changes in the diameter of gracilis muscle arterioles of all SHR groups in response to vasoactive agents were determined in the presence of constant intravascular pressure (80 mm Hg). Fig 3⇓ summarizes the changes in diameter in response to various doses of substance P (top) and sodium nitroprusside (bottom). Dilations of arterioles from fSHR and fSHR-OV+ES in response to substance P were significantly greater than those from mSHR and fSHR-OV. On the other hand, dilations in response to sodium nitroprusside were not significantly different in the various groups.
Fig 4⇓ shows the responses of arterioles to acetylcholine (top) and the calcium ionophore A23187 (bottom). Except at a concentration of 10−9 mol/L, arteriolar responses to acetylcholine were not different in the various groups. A23187 elicited constrictions in mSHR arterioles, as we reported previously,26 but dilations in fSHR vessels. Similarly, A23187 induced constriction of fSHR-OV arterioles and dilation of fSHR-OV+ES vessels.
The salient finding of this study is that there is a sex difference in the pressure-induced tone of skeletal muscle arterioles of SHR. This tone is significantly less in females than in males, the difference being due to the greater basal release of NO in arterioles of female SHR, which is maintained by the presence of estrogen. Our results also show that NO-mediated dilutions to agonists are greater in arterioles of female than in male SHR.
Arteriolar tone is an important determinant of peripheral resistance in both normotensive and hypertensive conditions. Previous studies demonstrated that the pressure-dependent, myogenic tone of arterioles is modulated by several factors, among them, endothelium-derived NO, signifying the importance of this substance in controlling vascular resistance.21 22 Other studies showed an enhanced myogenic arteriolar tone23 and a reduced release of endothelium-derived vasodilators in hypertension,23 24 26 which could contribute to the elevated peripheral vascular resistance.
Earlier studies of Altura27 and much circumstantial evidence from epidemiological studies1 2 3 4 5 suggest that the regulation of peripheral vascular resistance in females and males may not be the same and that this may have a role in the development of the sex differences in hypertension. The role of female hormones in blood pressure regulation has been suggested by several previous studies. In female SHR, a significant lowering of blood pressure and reversal of impaired endothelium-dependent responses have been observed during pregnancy.10 It was also found that when 17β-estradiol was administered to postmenopausal women, the circulating levels of nitrite and nitrate (the metabolites of NO) were increased.15 On the basis of these findings, one could assume that a greater release of endothelium-derived NO, caused by the chronic presence of estrogen, may be responsible for the differences in peripheral resistance and blood pressure in female versus male SHR.
In the present study, we have addressed the question of whether the pressure-induced arteriolar tone is different in male and female SHR and if so, whether this can be accounted for by a difference in NO release, possibly related to the presence of estrogen in females. We chose arterioles of rat gracilis muscle for our study because skeletal muscle is responsible for a sizable fraction of the total peripheral resistance.
To elucidate the possible role of estrogen in the observed sex differences in the development of basal tone, we ovariectomized female SHR; one group received 17β-estradiol and the other received vehicle only. The significant reduction in uterus weight and the reversal of this reduction in weight in rats receiving estrogen replacement therapy demonstrate the effectiveness of the ovariectomy. Moreover, the chronic administration of estrogen restored the plasma level of estrogen in ovariectomized female SHR (Table⇑). Ovariectomy also resulted in an increase in body weight, confirming previous findings.7
Lower Arteriolar Tone in Female SHR
At all perfusion pressures, the tone of arterioles of female SHR was significantly less (greater diameter) than that of male SHR and of female SHR whose ovaries had been removed. The finding that the arteriolar tone of ovariectomized females receiving estrogen replacement was similar to that of normal female SHR supports the conclusion that estrogen is responsible for the lower arteriolar tone in female SHR. An inhibitor of NO synthesis, L-NNA, eliminated the differences in arteriolar tone by decreasing the diameter of arterioles of the various rat groups to an identical level. Fig 2⇑ indicates that L-NNA had a significantly greater effect on arteriolar diameter of female SHR, suggesting that a greater release of NO accounts for the greater modulation of pressure-induced myogenic tone of arterioles of female than of male SHR. Our findings with ovariectomized rats and ovariectomized rats with estrogen replacement indicate that the lower arteriolar tone caused by the greater NO release corresponds with the presence of estrogen. Therefore, it seems that estrogen, by a still unknown mechanism, maintains NO synthesis in skeletal muscle arterioles of female SHR.
All these results underline the importance of the continuous release of NO in the regulation of vascular resistance in both normotensive and hypertensive conditions. The modulatory effect of NO on arteriolar myogenic tone in female rats could be even more pronounced in vivo, since blood flow exerts a shear force on the endothelium, stimulating NO synthesis.28 Also, agonist- and shear stress–induced release of NO was shown to be impaired in arterioles of male SHR.26 28 All these findings support the hypothesis that the diminished role of NO in male SHR contributes to the elevated peripheral resistance. By the same token, the presence of NO may temper the increase in peripheral resistance and blood pressure in female SHR.
Our findings correspond to those of previous studies showing that estrogen enhances endothelium-dependent dilator responses in vessels of various species, including canine coronary arteries,29 rat aorta,9 and rabbit femoral artery.30 In contrast, in rabbit aorta, an enhanced constriction to arachidonic acid in the presence of 17β-estradiol was observed.31 Our previous study suggests that unlike in conduit vessels, the dilator response to acetylcholine in arterioles of male SHR is mediated primarily by endothelium-derived hyperpolarizing factor (EDHF) rather than NO.26 Also, we found no difference in dilation to acetylcholine of arterioles of male Wistar-Kyoto rats and SHR26 and in the present study between male and female SHR (Fig 4⇑, top). If indeed, in arterioles of male SHR, a greater proportion of the response to acetylcholine is mediated by EDHF than by NO, then our findings suggest that EDHF is not affected by hypertension. Rather, it may in fact compensate for the reduction in the role of NO in the response to acetylcholine. However, dilation to substance P, a response mediated exclusively by NO, was found to be greatly reduced in male SHR.26 In arterioles of female SHR and estradiol-treated ovariectomized SHR, dilations to all concentrations of substance P were significantly greater than in arterioles of male SHR and ovariectomized female SHR. The similar responses of arterioles of all SHR groups to sodium nitroprusside, an agent that affects smooth muscle directly, rules out the possibility of an altered reactivity of arteriolar smooth muscle to NO.
To further characterize the difference in arteriolar endothelium of female and male SHR, we used the calcium ionophore A23187, which is known to induce dilation by stimulation of NO synthesis via a non–receptor-mediated mechanism. A23187 induced dilation in arterioles of female SHR and estradiol-treated ovariectomized female SHR, whereas it elicited constrictions in those of male SHR and ovariectomized female SHR, indicating that the release of NO in response to A23187 also depends on the presence of estrogen. Together, these findings support the hypothesis that there is a greater NO release from the endothelium of skeletal muscle arterioles of female SHR.
Previously, as in the present study, we found that hypertension not only impairs NO synthesis in arterioles of male SHR but also diverts prostaglandin synthesis to the increased production of prostaglandin H2 and that A23187 evokes an endothelium-dependent arteriolar constriction mediated by prostaglandin H2.23 26 In the present study, in female SHR, ovariectomy converted the dilation to A23187 to constriction, and estradiol replacement restored the dilator response to this agent. One of the explanations for the observed difference can be that estrogen maintains NO synthesis in female SHR, which prevents the production or overcomes the effect of prostaglandin H2.
The exact mechanism of how estrogen stimulates NO release is not yet understood. Estrogen may activate muscarinic receptors,29 31 or after interacting with its endothelial cell receptors,32 it may stimulate NO synthase to produce more NO, as shown previously in rat aorta.18 Also, estrogen was shown to attenuate the contraction of rabbit coronary arteries to endothelin-1 via an endothelium-independent mechanism, possibly by affecting calcium influx.33 Hence, the mechanism of action of estrogen in modulating endothelium-dependent responses could be quite different in different vascular beds. When one compares the results of various studies, however, it seems important to make a distinction between the acute and chronic effects of estrogen, which can be quite disparate.
In conclusion, we found that the regulation of arteriolar tone by the pressure-induced myogenic mechanism and endothelium-derived NO is clearly different in skeletal muscle arterioles of female and male SHR. The greater role of NO in the regulation of vascular tone in females may have important consequences for our understanding of the control of resistance and blood pressure by local mechanisms in normotensive and hypertensive conditions in both females and males.
Selected Abbreviations and Acronyms
|PSS||=||physiological salt solution|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported by grants from the National Institutes of Health (HL-46813, P01-HL-43023) and the American Heart Association, New York State Affiliate (950117). We gratefully acknowledge Dr Carl I. Thompson for his expert technical advice and help with the radioimmunoassay for serum 17β-estradiol.
- Received August 19, 1996.
- Revision received October 3, 1996.
- Accepted December 9, 1996.
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