Abstract We hypothesized that endothelin in addition to prostaglandin (PG)H2 may also contribute to the enhanced myogenic tone of skeletal muscle arterioles of spontaneously hypertensive (SH) rats. Changes in the diameter of isolated, cannulated arterioles (≈60 μm) from cremaster muscles of 30-week-old normotensive Wistar Kyoto (WKY) and SH rats were measured as a function of perfusion pressure (20 to 140 mm Hg). Pressure-induced constrictions were significantly enhanced between 60 to 140 mm Hg in arterioles of SH rats compared with those of WKY rats; at 80 and 140 mm Hg the normalized diameter of arterioles (expressed as a percentage of corresponding passive diameter) of SH rats was 11.0% and 15.4% less (P<.05) than that of WKY rats. After inhibition of thromboxane A2–PGH2 receptors by SQ 29,548 (10−6 mol/L), the still enhanced myogenic response of SH arterioles was eliminated by the removal of endothelium or the administration of BQ-123 (10−7 mol/L), an endothelin A (ET-A) receptor blocker, which also inhibited constrictions to exogenous ET-1 (10−11 to 5×10−10 mol/L). ET-1 elicited comparable responses in arterioles of SH and WKY rats. Thus, in SH rats the enhanced arteriolar constriction to increases in intravascular pressure seems to be due to the production of endothelium-derived constrictor factors PGH2 and endothelin.
The underlying mechanisms for maintaining an increased peripheral vascular resistance in hypertension are still not elucidated, but it is thought that the increased tone of skeletal muscle arteries and arterioles, due to the changes in their structure, contributes significantly to the elevated peripheral resistance.1 2 3
In vitro studies of arteries of various sizes demonstrated a thickening of the vascular wall in hypertension and suggested that this change is an adaptation to the elevated pressure resulting in the normalization of wall stress. It was also implied that the thickening of the smooth muscle layer3 is associated with an increased contractile capacity of the vessels. One of the primary local factors that contributes to the development of basal tone of arterioles is the myogenic mechanism4 that is especially prominent in the arterioles of skeletal muscle.5 Because this mechanism is activated by changes in transmural pressure, it was logical to assume that it is altered in the presence of an increased intravascular pressure in hypertension. Indeed, an augmented constriction of cerebral vessels,6 renal interlobular arteries,7 and arterioles of skeletal muscle8 9 and mesentery10 in response to changes in intravascular pressure has already been documented in various models of experimental hypertension. In addition, Henriksen et al found an augmented constrictor response to changes in transmural pressure in patients with essential hypertension.11
The myogenic response is intrinsic to vascular smooth muscle. Its magnitude in normotensive animals is modulated by the release of endothelium-derived dilator factors.5 In contrast, the altered function of endothelium results in an enhancement of pressure-induced arteriolar tone in hypertension. In earlier studies8 we have found that in SHR, PGH2 is one of the factors that is responsible for the enhanced myogenic tone, whereas the nature of the other endothelial factor or factors remained obscure.
Because previous studies have demonstrated an elevated level of endothelin in hypertension,12 13 and because endothelin-specific antibodies, intravenous infusion of endothelin receptor antagonists, or intravenous infusion of endothelin-converting enzyme inhibitors reduce blood pressure in genetic forms of hypertension,14 15 16 we hypothesized that endothelin is the other factor contributing to the enhancement of myogenic tone of arterioles of hypertensive rats.
Experiments were conducted on isolated arterioles (≈60 μm) of cremaster muscle of 30-week-old male normotensive WKY and SHR. The procedures and protocols were in accordance with our institutional guidelines. Systolic arterial blood pressure was measured by the tail-cuff method. Rats were anesthetized with intraperitoneal injections of sodium pentobarbital (Nembutal sodium, 50 mg/kg). The isolation procedure of cremasteric arterioles has been described previously.8 Briefly, the cremaster muscle of rats was exposed by a median incision of the scrotal sac, cleared of adhering fascia, and separated from the scrotal sac. The muscle then was cut out and placed on a Petri dish containing cold (0 to 4°C) salt solution (pH 7.4) that was composed of (in mmol/L) 145 NaCl, 5.0 KCl, 2.0 CaCl2, 1.0 MgSO4, 1.0 NaH2PO4, 5.0 dextrose, 2.0 pyruvate, 0.02 EDTA, and 3.0 3-N-morpholino-propane sulfonic acid. The muscle was pinned to the silicone bottom of the dish and allowed to equilibrate for approximately 15 minutes. After the muscle was dissected, the rats were killed by an overdose of sodium pentobarbital.
With the use of microsurgery instruments and an operating microscope (Olympus), a 1- to 2-mm length segment of a second-order arteriole branching off from the main (first-order) arteriole that supplied the muscle was isolated and then transferred to the vessel chamber. The chamber contained a pair of glass micropipettes filled with PSS (see below) at room temperature.
The inflow micropipette was connected to a silicone rubber tube linked to a pressure-servo syringe system (Living Systems Inc). The distal (outflow) pipette was equipped with a three-way stopcock. The PSS used for suffusion and perfusion of the vessels contained (in mmol/L) 118.0 NaCl, 5.0 KCl, 2.5 CaCl2, 1.0 MgSO4, 10.0 dextrose, 24.0 NaHCO3, 1.0 KH2PO4, and 0.02 EDTA; and it was equilibrated with a gas mixture of 21% O2/5% CO2, balanced with N2, at PH 7.4 (33°C). From a reservoir, the vessel chamber (15 mL) was continuously supplied with PSS at a rate of 40 mL/min.
After the vessel was mounted on the proximal pipette and secured with a suture, the PP was raised to 20 mm Hg to clear the clotted blood from the lumen. Then the other end of the vessel was mounted on the distal pipette. To flush the vessel and cannulas the system was perfused for several minutes, then the outflow cannula was closed and the PP was slowly (≈1 minute) increased to 80 mm Hg. At this time, the pressure-servo system was placed in the manual mode (ie, no automatic maintenance of PP) in order to ascertain that there were no leaks in the system. If no leaks were detected (ie, PP remained constant), the pressure-servo system was set in the automatic mode. The temperature was set to 33°C (YSI temperature controller), and the vessel was allowed to equilibrate for about 1 hour.
In all protocols, the changes in diameter of arterioles in response to increases in PP under no-flow conditions were measured with an image shearing monitor and recorded with a strip-chart recorder. Only those vessels that developed substantial spontaneous constriction to pressure (80 mm Hg) were used, because no vasoactive agent was added to the PSS. After the equilibration period PP was decreased to 20 mm Hg, and then increased, in 20 mm Hg steps, to 140 mm Hg. Each pressure step was maintained for 5 to 10 minutes to allow the vessels to reach a stable condition before the diameter of the arterioles was measured. After obtaining the pressure-diameter relationship, the pressure was lowered to 80 mm Hg, then after approximately 20 minutes, responses of arterioles to vasoactive agents were tested.
In the first protocol after control responses, the endothelium of the arterioles was removed by perfusion of the vessels with air, as previously described in detail.8 The arteriole was untied from the proximal pipette, and through the distal pipette air (≈1 mL) was injected into the lumen for 1 minute. Then the arteriole was remounted to the proximal pipette. The arteriole was then perfused with PSS for 20 minutes at a pressure of 20 mm Hg to clear the debris. The outflow stopcock was then closed and PP raised to 80 mm Hg for 30 minutes to establish a stable tone. At this pressure the efficacy of endothelial denudation was ascertained by testing the arteriolar responses to acetylcholine (5×10−8 mol/L) and sodium nitroprusside (10−7 mol/L), endothelium-dependent and -independent vasodilators, respectively, before and after the administration of the air bolus. As in previous studies,8 we found that infusion of air resulted in a loss of function of the endothelium, as indicated by the absence of dilation to acetylcholine, whereas dilation to sodium nitroprusside remained intact. After removal of the endothelium, changes in diameter in response to step increases in PP were reassessed.
In the second series of experiments, after obtaining control pressure-diameter curves, the vessels were subjected to SQ 29,548 (10−6 mol/L), a TXA2-PGH2 receptor antagonist.8 16 The efficacy of this antagonist was tested by arteriolar responses to U46619 (10−8 mol/L), a TXA2-PGH2 receptor agonist, before and after the vessels were exposed to SQ 29,548. Then changes in diameter in response to step increases in PP were reassessed.
In the third group of experiments, the pressure-diameter relationship was investigated in control conditions and in the presence of BQ-123 (10−7 mol/L), a specific inhibitor of ET-A receptors.15 Then, the effect of combined administration of BQ-123 and SQ 29,548 on the pressure-diameter curve was obtained. In one group of vessels, arteriolar constrictions to ET-1 (10−11 to 5×10−10 mol/L) were assessed, while in another group of vessels from the same rat these responses were obtained in the presence of BQ-123.
All drugs were added to the reservoir connected to the vessel chamber and final concentrations are given. Responses to vasoactive agents were tested at 80 mm Hg PP. 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 mmol/L). The vessels were incubated for 10 minutes, then the step increases in pressure were repeated and the passive diameter of arterioles at each pressure step was obtained.
All salts and chemicals were obtained from Sigma Chemical Co or Aldrich Co and were prepared on the day of the experiment. Changes in diameter in response to vasoactive agents or pressure were normalized to the corresponding passive diameter and expressed as percent changes. Results are presented as mean±SEM. Only one vessel was used from each rat; n refers to the number of vessels or rats.
Statistical analyses were done by ANOVA, followed by Tukey’s post hoc test and Student’s t test (paired and grouped t tests were used for data of drug responses within and between groups, respectively) as appropriate. A value of P<.05 was considered significant.
The systolic blood pressures of WKY and SHR were 127.1±4.9 (n=7) and 212.0.±8.2 mm Hg (n=7), respectively, showing a significant increase in blood pressure in SHR compared with WKY.
In the first series of experiments, the diameter of arterioles of WKY and SHR, as a function of PP, was obtained in the presence of endothelium. In order to assess the magnitude of the pressure-induced active change in diameter (ie, active tone generated by the arteriole), after the conclusion of experiments the pressure-passive arteriolar diameter (in Ca2+-free solution, see “Methods”) relationship (Fig 1⇓, upper panel) was also obtained in each vessel. Then, the changes in diameter of active arterioles to increases in PP were normalized to the corresponding passive diameter at each pressure step. We have found that except at 20 and 40 mm Hg PPs, the passive diameters of arterioles from SHR and WKY were not significantly different from each other (Fig 1⇓, upper panel). In contrast, the pressure-diameter curve of active arterioles (Ca2+ present) of SHR started to deviate significantly (P<.05) from that of WKY at 60 mm Hg pressure and upward. That is, arterioles of SHR exhibited an enhanced constriction in response to increases in PP, compared with arterioles of WKY (Fig 1⇓, lower panel).
In order to confirm our previous findings,8 first the effect of SQ 29,548, a TXA2-PGH2 receptor blocker, on the pressure-induced behavior of arterioles was examined. SQ 29,548 did not affect pressure-diameter relationships of arterioles of WKY (Fig 2⇓, upper panel) but reduced significantly the pressure-induced enhanced arteriolar constrictions in arterioles of SHR (Fig 2⇓, lower panel). In these experiments, after removal of the endothelium, the pressure-diameter curve of WKY shifted significantly downward; that is, the constriction of the arterioles in response to pressure became significantly enhanced (Fig 2⇓, upper panel). In contrast, in SHR after removal of the endothelium, the pressure-diameter curve of arterioles shifted significantly upward, namely the constriction of vessels to increases in PP became significantly reduced (Fig 2⇓, lower panel). These findings demonstrate that in SHR arterioles PGH2 is at least in part responsible for the enhanced constriction to pressure, as suggested by previous studies,8 and that in arterioles of WKY endothelium-derived dilator factors temper myogenic constriction.
Next, we investigated the possible contribution of ET to the pressure-induced responses of arterioles by using BQ-123, a known ET-A receptor blocker.15 18 In normotensive rats BQ-123 did not affect the pressure-induced arteriolar tone (Fig 3⇓, upper panel). In contrast, in arterioles of SHR, BQ-123 significantly reduced the arteriolar constrictions in response to step increases in PP (Fig 3⇓, lower panel). The administration of additional SQ 29,548 did not elicit significant changes in the pressure-induced tone in arterioles from WKY, whereas it further reduced significantly the myogenic tone of vessels from SHR (Fig 3⇓, upper and lower panels, respectively).
To further assess the effect of ET on basal diameter of arterioles, as a function of pressure, we expressed the changes in diameter of arterioles isolated from SHR as percent of control diameter in the presence of BQ-123 (Fig 4⇓).
In arterioles of both strains of rats SQ 29,548 eliminated the constrictions to U46619, known to be a specific agonist of PGH2-TXA2 receptors (Fig 5⇓, upper panel). Administration of ET-1 elicited dose-dependent constrictions of arterioles that were not significantly different in the two strains of rats. The efficacy of BQ-123 to block ET receptors is indicated by the finding that even responses to the highest concentration of ET-1 were completely eliminated by BQ-123 (Fig 5⇓, lower panel).
The salient findings of this study are that isolated skeletal muscle arterioles of SHR exhibit a significantly greater constriction to increases in intraluminal pressure than those of normotensive rats and that this enhanced myogenic response is due to the presence of endothelium-derived PGH2 and ET.
Previous studies demonstrated changes in the structure of the vascular wall of large vessels in hypertension.1 2 3 The observed thickening of the vascular wall was suggested to be responsible for the reduced diameter and enhanced contractility of smooth muscle.3 Because the large conduit vessels are responsible for only a fraction of the total peripheral resistance, in order to explain the increase in peripheral resistance in hypertension similar changes in the structure and/or function of arterioles had to be surmised.
It was logical to investigate first the myogenic mechanism of arterioles in hypertension, because it is specifically sensitive to changes in intraluminal pressure that elicit changes in the diameter of arterioles. As in the present study, many previous studies have been in cremasteric arterioles, because micropressure measurements in in vivo preparations showed an elevated intravascular pressure in these vessels in hypertensive rats compared with normotensive rats.19 20 Also, in these arterioles, both in vivo9 and in vitro,8 9 an enhanced constriction of arterioles in response to increases in intravascular pressure was found. Similar findings were reported in cerebral,6 renal,7 and mesenteric10 arteries from SHR, as well.
In cremasteric arterioles Falcone et al9 found that the augmented pressure-induced response was neither structural in origin nor mediated by α-receptors. We have also observed in the current and previous8 studies that the passive diameter of WKY and SHR arterioles (in Ca2+-free solution) was not significantly different in the in vivo pressure range, suggesting that structural differences in the arteriolar wall are not sufficient to explain the differences in their contractile activity. Thus, we hypothesized that alterations in the function of arteriolar endothelium are responsible for the observed changes. We found, indeed, that after removal of the endothelium, arterioles of hypertensive rats no longer exhibited enhanced constriction to pressure. These findings suggested that the contractile activity of vascular smooth muscle arterioles of hypertensive rats is greater than that of normotensive rats only because of the presence of endothelium-derived constrictor factors.
One of these factors that we could identify previously is PGH2. This finding was confirmed by the present study, which showed that the PGH2 receptor blocker SQ 29,654 significantly reduced (but did not eliminate) the enhanced response of hypertensive arterioles to pressure (Fig 2⇑, lower panel). These and previous findings21 22 together suggest that hypertension elicits an alteration in arachidonic acid metabolism, resulting in an enhanced synthesis of PGH2 in arteriolar endothelium.
In vivo, in gracilis muscle of normotensive cats, Ekelund at al18 found no role for ET in the basal tone or the mediation of the myogenic response of arterioles. The current study confirms their findings. In a genetic model of hypertension,14 however, ET-specific antibodies or intravenous infusion of ET-A and ET-B receptor antagonists reduced blood pressure, suggesting a role for ET in the maintenance of high blood pressure. In addition, in borderline hypertension in humans an increased basal concentration of plasma ET was reported.12 13 On the basis of these studies and because in SHR removal of the endothelium resulted in a greater attenuation of the myogenic tone than the administration of SQ 29,548 alone (Fig 2⇑, lower panel), we surmised that ET may also contribute to the enhanced pressure-induced constriction of arterioles.
The ET-A receptor antagonist BQ-123 significantly reduced pressure-induced constrictions of arterioles of hypertensive rats, ie, it elicited dilations of 5% to 30%, which seemed to be in parallel with increases in pressure (Fig 4⇑), whereas it did not significantly affect diameters of vessels of normotensive rats. Also, dose-dependent constrictor responses to ET-1 were similar (and completely blocked by BQ-123) in arterioles of both groups of rats (Fig 5⇑). Thus, we concluded that ET, produced in the endothelium, augments the myogenic mechanism of arterioles in hypertension, whereas synthesis of this constrictor factor is negligible in normotensive rats. These findings correspond to the recent demonstration of expression of ET-1 in small arteries from humans with essential hypertension23 and an elevated level of immunoreactive ET-1 in the plasma of hypertensive humans. Interestingly, ET-1 was also significantly higher in hypertensive black females and males, which have been shown to have a greater prevalence for hypertension than white persons.24
Combined application of ET-A and PGH2 receptor antagonists eliminated completely the enhanced pressure-induced constrictions in hypertensive rats (Fig 3⇑), suggesting that PGH2 and ET together are responsible for the enhancement of the myogenic response.
The reasons for alterations in the function of endothelium in hypertension that lead to an enhanced release of PGH2 and ET are at present uncertain. A role for oxygen-derived free radicals that could be produced in hypertension has been suggested as the possible cause of the enhanced production of PGH2,25 26 27 which may also be linked to the diminished synthesis of endothelium-derived nitric oxide, also observed in arterioles of spontaneously hypertensive rats.22 All these alterations favor vasoconstriction, which could lead to chronic increases in wall shear stress.28 Furthermore, increases in shear stress may stimulate ET release, as suggested by previous studies,29 30 which then could further enhance the myogenic mechanism. Thus, a vicious cycle can develop, leading to further increases in peripheral resistance and systemic blood pressure.
It is of interest also that in the absence of endothelium the myogenic tone of WKY arterioles became significantly enhanced, suggesting an important role for endothelium-derived vasodilator factors in reducing the pressure-induced tone of these arterioles. One of these factors is likely to be nitric oxide, since previous studies showed that inhibition of nitric oxide synthesis elicits significant increases in basal arteriolar tone but only in arterioles of normotensive rats.28 Thus, the balance of endothelium-derived dilator and constrictor factors seems to be an important determinant of arteriolar tone.
In conclusion, we found an endothelium-dependent augmentation of the pressure-induced constriction of arterioles of genetically hypertensive rats. This enhanced myogenic response of hypertensive arterioles is due to the enhanced synthesis of PGH2 and ET, which seems to be negligible in normotensive arterioles. It seems, therefore, that an altered endothelial mechanotransduction in skeletal muscle arterioles could be an important factor in the development of increased peripheral resistance in hypertension.
Selected Abbreviations and Acronyms
|PSS||=||physiological salt solution|
|SHR||=||spontaneously hypertensive rats|
This work was supported by grants from the National Institutes of Health HL-46813, PO1 HL 43023, and OTKA T-023863. We wish to thank Miriam Nunez for her excellent secretarial assistance.
Reprint requests to Akos Koller, MD, PhD, Department of Physiology, New York Medical College, Valhalla, NY 10595.
- Received January 17, 1997.
- Revision received February 13, 1997.
- Accepted May 20, 1997.
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