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Lovastatin reduces renal vascular reactivity in spontaneously hypertensive rats
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1998;11:1222–1231
Lovastatin Reduces Renal Vascular Reactivity in Spontaneously Hypertensive Rats Jian Jiang, Chen-Wen Sun, Magdalena Alonso-Galicia, and Richard J. Roman
We have reported that lovastatin attenuates the development of hypertension in spontaneously hypertensive rats (SHR). The fall in arterial pressure is associated with an elevation in renal medullary blood flow, normalization of the pressure-natriuresis relationship, and diminished hypertrophy of renal arterioles. However, the mechanism by which lovastatin alters renal vascular tone is unknown. The present study examined the effects of lovastatin on renal vascular tone and the expression of G proteins. Four-week– old SHR were chronically treated with lovastatin (20 mg/kg/day) or vehicle by gavage for 4 weeks. At the end of the study, mean arterial pressure averaged 131 6 4 (n 5 5) and 160 6 4 mm Hg (n 5 6) in lovastatin- and vehicle-treated SHR, respectively. Renal arterioles isolated from lovastatin-treated SHR were significantly less responsive to norepinephrine and vasopressin than those obtained from vehicle-treated rats (ED50: 5.0 v 1.8 3 1027 mol/L for norepinephrine, and 8.0 v
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5.2 3 10210 mol/L for vasopressin). The fall in renal vascular reactivity in lovastatin-treated SHR was associated with reduced levels of ras and rho proteins in renal arterioles, whereas the expressions of heterotrimeric G proteins (Gs, Gq, Gi) were similar in renal arterioles from vehicleand lovastatin-treated SHR. Overnight culture of renal arterioles with media containing lovastatin also diminished the expression of ras and rho proteins and the response to vasoconstrictors. These findings indicate that lovastatin diminishes the response to vasoconstrictors and the expression of small G proteins in the renal vasculature of SHR and suggest that a fall in the levels of ras and rho proteins in these vessels may contribute to the antihypertensive effects of lovastatin. Am J Hypertens 1998;11:1222–1231 © 1998 American Journal of Hypertension, Ltd. KEY WORDS:
Lovastatin, renal vascular tone, small G proteins, hypertension.
onsiderable evidence now exists that supports an association between hyperlipidemia and hypertension.1,2 The incidence of hypertension is greater in hyperlipidemic subjects and hyperlipidemia serves as an important risk factor for the development of end-organ damage
in hypertension. However, the mechanisms by which elevated levels of plasma lipids influence arterial pressure are unknown and there is very little information regarding the effects of antilipidemic therapy on blood pressure in hypertensive patients. In a recent study,3 we reported that chronic treatment of young (4-week–
Received August 28, 1997. Accepted May 6, 1998. From the Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin. This work was supported in part by grants from the National Heart Lung and Blood Institute, HL-36279 and HL-29587. Jian Jiang
was a recipient of Predoctoral Fellowship Award from the American Heart Association/Wisconsin Affiliate. Address correspondence and reprint requests to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
0895-7061/98/$19.00 PII S0895-7061(98)00143-5
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old), prehypertensive, spontaneously hypertensive rats (SHR) with the antilipidemic agent lovastatin normalized the pressure-natriuresis relation and markedly attenuated the development of hypertension. The fall in arterial pressure was associated with a marked elevation in renal medullary blood flow and diminished hypertrophy of renal arterioles. However, the mechanism by which lovastatin alters the structure and tone of renal arterioles is unknown. Lovastatin is a competitive inhibitor of the synthesis of mevalonate, the precursor for the synthesis of cholesterol and the nonsterol isoprenoids, such as the farnesyl and geranylgeranyl groups.4 Recently, it has become increasingly evident that nonsterol intermediates of mevalonate metabolism are essential for DNA synthesis and cell proliferation. For example, blockade of hepatic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity with lovastatin inhibits the S phase of DNA synthesis, leading to the arrest of cell proliferation.5– 8 The inhibitory effects of lovastatin on cell growth could be reversed by adding mevalonate, but not cholesterol, to cell culture medium. It is also apparent that the farnesyl and geranylgeranyl groups are required for the isoprenylation of a number of important signal transduction proteins. One class of these proteins includes the low-molecular– weight GTP-binding proteins ras and rho, which play an integral role in the growth signal transduction pathway in vascular smooth muscle cells.9 In addition, small G proteins participate in the regulation of vascular tone by modifying the activity of voltage-gated calcium channels10 and the calcium sensitivity of the contractile mechanism.11,12 Thus, chronic treatment of SHR with lovastatin may reduce the hypertrophy and reactivity of renal arterioles by interfering with the synthesis of mevalonate and blocking the isoprenylation of small G proteins. To test this hypothesis, young (4-week– old), prehypertensive SHR were chronically treated with lovastatin or vehicle and changes in levels of small and heterotrimeric G proteins were measured in renal arterioles using Western blot techniques. In addition, the effects of lovastatin on the vasoconstrictor response of these arterioles to vasopressin and norepinephrine were determined. MATERIALS AND METHODS General Experiments were performed on 4-weekold SHR purchased from Harlan Sprague Dawley Laboratories (Indianapolis, IN). The rats were housed in an animal care facility at the Medical College of Wisconsin that was approved by the American Association for Accreditation of Laboratory Animal Care, and had free access to food and water throughout the experiment. All protocols involving animals received prior approval from the Animal Care Committee of the Medical College of Wisconsin.
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The rats were divided into four groups. The first two groups were given lovastatin (10 mg/kg) or vehicle twice a day by gavage, whereas the other groups were treated with hydralazine (100 mg/L) or vehicle in the drinking water. Hydralazine was used as an alternative antihypertensive therapy so that we could determine whether the effects of lovastatin on renal vascular tone and on the expression of G proteins were due to a direct effect or secondary to its ability to lower blood pressure. Measurement of Arterial Pressure After 4 weeks of treatment the rats were anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (2 mg/kg) and a catheter was implanted in the femoral artery for measurement of arterial pressure. The catheter was exteriorized at the back of the neck and brought out through a stainless-steel spring and swivel device. After a 3-day recovery period, arterial pressure was recorded between 9 am and 12 pm with a pressure transducer and a computerized recording system for at least 3 h per day on 3 consecutive days while the animal was conscious in its home cage. The signals were sampled at 30 Hz, and heart rate and systolic, diastolic, and mean arterial pressures were collected at 1-min intervals and reduced to a single mean value for the recording session. After arterial pressure was measured, 1 mL of blood was collected from the femoral arterial catheter for measurements of plasma cholesterol and triglyceride concentrations. Assessment of Renal Vascular Responsiveness to Norepinephrine and Vasopressin The SHR were treated with lovastatin, hydralazine, or vehicle for 4 weeks as described above. The rats were then anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). A midline abdominal incision was made, and the left kidney was removed and placed in cold physiological salt solution (PSS), pH 7.4, which had the following composition (in mmol/ L): NaCl 119, KCl 3.3, Na2SO4 0.7, CaCl2 2.0, NaHCO3 22.8, KH2PO4 1.2, glucose 11.0, and N-2-hydroxy-ethylpiperazine-N9-2-ethanesulfonic acid 5.0. Renal interlobular arteries (, 100 mm in inner diameter) were microdissected and placed in a chamber maintained at 37°C. The vessels were bathed in PSS that was bubbled with a 93% O2-7% CO2 gas mixture. The ends of the vessel were cannulated with glass micropipettes (;50 mm, outer diameter) and tied in place with a 22-mm silk suture. Side branches were tied off. The inflow micropipette was connected to a fluid-filled reservoir containing PSS equilibrated with a 93% O2-7% CO2 gas mixture. During the experimental period, transmural pressure was controlled at 90 mm Hg by closing the outflow pipette and adjusting the pressure on the reservoir connected to the inflow pipette. Intraluminal pressure was monitored using a transducer connected
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in series with an inflow cannula. Arterial diameters were measured using a video micrometer measuring system. After a 1-h equilibration period, the viability of each vessel and the endothelium was tested by measuring the contractile response to norepinephrine (1 mmol/L) followed by the dilatory response to acetylcholine (5 mmol/L). Cumulative concentration-response curves to norepinephrine (1029 to 1025 mol/L) and vasopressin (10210 to 1028 mol/L) were then constructed. In these experiments, the drugs were added directly to the bath. In Vitro Effects of Lovastatin on Renal Vascular Tone Additional experiments were performed to determine whether the effects of lovastatin to alter renal vascular tone in SHR could be mimicked by shortterm exposure of renal arterioles to this drug in vitro. In these experiments, renal interlobular arterioles (, 100 mm in inner diameter) were microdissected from the kidneys of 4-week– old SHR and cultured overnight in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.7 g/L NaHCO3, 100 mg/mL of streptomycin, and 100 U/mL of penicillin. The vessels were divided into two groups, and lovastatin (8 mg/mL) or vehicle was added to the culture medium. Lovastatin was converted to its active dihydroxy-open acid form by hydrolysis in 0.1 mol/L NaOH at 50°C for 2 h, followed by neutralization with HCl to a pH of 7.2. After overnight exposure of the vessels to control media or media containing lovastatin, the vessels were mounted in the myograph and cumulative concentration-response curves to norepinephrine and vasopressin were constructed. To confirm that overnight organ culture of isolated renal arterioles does not alter the responsiveness of renal arterioles, we compared the responses of freshly isolated vessels from the kidneys of 4-week-old SHR to those observed in vessels cultured overnight in DMEM alone. Effects of Lovastatin on the Expression of G Proteins in the Renal Vasculature of SHR Experiments were performed to compare the expression of G proteins in preglomerular renal arterioles isolated from the kidneys of prehypertensive 4-week-old SHR and WistarKyoto (WKY) rats and from the kidneys of SHR chronically treated with lovastatin, hydralazine, or vehicle for 4 weeks. Additional experiments were also performed using renal arterioles isolated from the kidneys of 8- to 9-week– old SHR that were cultured overnight in media containing vehicle or lovastatin (8 mg/mL). In these experiments, renal arterioles were isolated using a recently described sieving procedure.13 Rats were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg) and the kidneys were flushed with 10 mL of a low-calcium Tyrode’s solution containing (in mmol/L): NaCl 145, KCl 6,
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MgCl2 1, CaCl2 0.05, HEPES 10, glucose 10, and NaHCO3 4.2. The kidneys were then flushed with 10 mL of Tyrode’s solution containing 6% albumin and 1% Evans blue dye. The kidneys were subsequently removed, hemisected, and gently pressed through a 180-mm mesh screen. The retained material was resuspended in 5 mL of Tyrode’s solution containing 1 mg/mL of each of the following: collagenase, soybean trypsin inhibitor, albumin, and dithiothreitol (DTT). The suspension was incubated for 15 min at 37°C on a shaking table and superfused with oxygen. The solution was filtered through a 75-mm screen and the retained material was resuspended in cold Tyrode’s solution. Individual Evans blue-stained arterioles were then collected using a stereomicroscope and fine forceps. The renal arterioles were frozen in liquid nitrogen and powdered using a mortar and pestle. The frozen tissue was homogenized in ice-cold 20 mmol/L Tris/ HCl, pH 8.0, containing 1 mmol/L EDTA, 1 mmol/L DTT, 0.25 mol/L sucrose, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and sonicated for 15 s. The mixture was centrifuged at 3000 g for 10 min and 9000 g for 15 min. The protein concentration of the supernatant was determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Immunoblotting Immunoblotting was performed as previously described.14 Various amounts of protein (10 – 80 mg) were loaded and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel. Nonspecific binding was blocked by placing the membrane overnight in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, 0.08% Tween-20, pH 8.0) containing 5% nonfat dry milk at 4°C. The bound antibody was detected using enhanced chemiluminescence (ECL kit, Amersham Life Science, Amersham, Buckinghamshire, UK). The blots were also probed with anti-b-actin antibody to ensure that equal amounts of protein were loaded in each lane. The rat monoclonal anti-ras p21, rabbit polyclonal anti-rhoA p21, rabbit anti-Gqa, rabbit anti-Gia, and HRP-conjugated goat anti-rabbit/rat/mouse secondary antibodies used in these experiments were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). Mouse monoclonal anti-b-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Rabbit anti-Gsa was purchased from New England Nuclear Products (NEN, Boston, MA). The primary and secondary antibodies were used at a 1:2000 dilution. Statistics Mean values 6 1 SEM are presented. The significance of the differences in mean values between and within groups was determined with an analysis of variance for repeated measures, with multiple independent factors followed by a Duncan’s multiple
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range test. A P value , .05 was considered to be significant. RESULTS Effects of Lovastatin on Arterial Pressure Chronic treatment of SHR with lovastatin or hydralazine markedly attenuated the development of hypertension. Mean arterial pressure averaged 160 6 4 mm Hg in 8- to 9-week– old SHR treated with vehicle (n 5 12), but only 131 6 4 and 135 6 6 mm Hg in SHR treated with lovastatin (n 5 5) or hydralazine (n 5 6), respectively (P , .05). Plasma cholesterol concentration was 33% lower in lovastatin-treated SHR than that seen in vehicle-treated rats (1.03 6 0.06 v 1.56 6 0.18 mmol/L, P , .05), whereas plasma triglyceride concentration was not significantly different and averaged 1.0 6 0.1 mmol/L in lovastatin-treated SHR, versus 1.1 6 0.1 mmol/L in vehicle-treated rats. Effects of Lovastatin on the Renal Vasoconstrictor Response to Norepinephrine and Vasopressin The effects of chronic treatment of SHR with lovastatin or hydralazine on the response of renal arterioles to norepinephrine (NE) are presented in Figure 1. The concentration-response curves to NE were similar in renal arterioles isolated for SHR chronically treated with hydralazine or vehicle. In contrast, renal arterioles isolated from lovastatin-treated rats were significantly less responsive to NE than those obtained from SHR treated with vehicle or hydralazine. At submaximal concentrations, renal arterioles isolated from lovastatin-treated rats exhibited significantly smaller responses to NE than the responses seen in the vessels obtained from vehicle- or hydralazine-treated SHR. The median effective dose (ED50) was fivefold higher in renal arterioles isolated from lovastatin-treated rats than those obtained from vehicle- or hydralazinetreated rats (5.0 v 1.2 3 1027 mol/L). The concentration-response relations to vasopressin are presented in Figure 2. There was no significant difference between the cumulative concentration-response curves in renal arterioles obtained from vehicle- and hydralazine-treated SHR. However, the concentration-response relation to vasopressin was significantly shifted to the right in renal arterioles obtained from SHR treated with lovastatin (ED50 5 8.0 3 10210 for lovastatin-treated SHR v 5.2 3 10210 mol/L for vehicle-treated rats). Expression of G Proteins A comparison of the expression of G proteins in renal arterioles isolated from the kidneys of 4-week-old, prehypertensive SHR and WKY rats is presented in Figure 3. The expression of the a subunits of the heterotrimeric G proteins (Gs, Gi, and Gq) and rho protein was similar in the renal arterioles isolated from these two strains. However, the level of ras protein was significantly higher in
FIGURE 1. Comparison of the concentration-response relation to norepinephrine in renal arterioles (inner diameter , 90 mm) isolated from SHR chronically treated with lovastatin, hydralazine, or vehicle. The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding value in vessels isolated from vehicletreated SHR.
renal arterioles obtained from SHR than that seen in arterioles obtained from WKY rats. The effects of chronic treatment of SHR with lovastatin on the expression of G proteins in renal arterioles are presented in Figure 4. The levels of ras and rho proteins were, respectively, 73% and 51% lower in renal arterioles isolated from lovastatin-treated SHR than in the vessels obtained from vehicle-treated SHR. In contrast, the levels of the a subunits of Gs, Gi, and Gq proteins were not significantly different in renal arterioles isolated from these two groups of rats. Hydralazine did not alter the levels of ras and rho proteins in renal arterioles (Fig. 5), even though it was equally as effective as lovastatin at lowering arterial pressure in SHR. In Vitro Effects of Lovastatin on Vascular Responsiveness and the Expression of Small G Proteins in Renal Arterioles To determine whether the effects of lovastatin to lower vascular tone in SHR were due to
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The results of these experiments (Fig. 8) indicate that overnight exposure of renal arterioles to culture media containing lovastatin reduced the expression of ras and rho proteins in renal arterioles isolated from the kidneys of SHR rats by 50% and 30%, respectively. DISCUSSION
FIGURE 2. Comparison of the concentration-response relation to vasopressin in renal arterioles (inner diameter , 90 mm) isolated from SHR chronically treated with lovastatin, hydralazine, or vehicle. The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding value in vessels isolated from vehicletreated SHR.
a direct effect or secondary to its antihypertensive action, we studied whether overnight culture of renal arterioles in media containing lovastatin would also diminish vascular responses to NE and vasopressin. The results of these experiments are summarized in Figures 6 and 7. The cumulative concentration response curves to norepinephrine and vasopressin were similar in freshly isolated renal arterioles and vessels cultured overnight in DMEM. However, renal arterioles cultured overnight in DMEM containing lovastatin were significantly less responsive to norepinephrine and vasopressin than vessels cultured in DMEM alone. The ED50 concentrations for norepinephrine and vasopressin were significantly lower in renal arterioles cultured in control media than in vessels exposed to lovastatin (1.5 v 4.5 3 1027 mol/L for norepinephrine, and 4.8 v 7.5 3 10210 mol/L for vasopressin). Additional experiments were performed to determine whether overnight exposure of renal arterioles to lovastatin alters the expression of ras and rho proteins.
Previous studies have indicated that the development of hypertension in SHR is associated with an elevation in vascular tone in preglomerular renal arterioles,15,16 a reduction in renal medullary blood flow, and a shift of the pressure natriuresis relationship toward higher pressures.17,18 More recently, chronic treatment of young SHR with lovastatin has been reported to markedly attenuate the development of hypertension.3 The fall in arterial pressure in that study was associated with an elevation in renal medullary blood flow and normalization of the pressure-natriuresis response,3 but the mechanisms involved are unknown. The present study examined whether the antihypertensive effects of lovastatin might be related to an inhibitory effect on the renal vascular response to vasoconstrictors, secondary to its action to block the isoprenylation and expression of G proteins. The results indicate that chronic treatment of SHR with lovastatin diminishes the response of renal arterioles to both norepinephrine and vasopressin. These changes in renal vascular reactivity are not simply due to the antihypertensive effects of lovastatin, as chronic treatment of SHR with another antihypertensive agent, hydralazine, had no effect on the response of renal arterioles to vasoconstrictors. Moreover, overnight exposure of renal arterioles to culture media containing lovastatin also reduced the vasoconstrictor response to norepinephrine and vasopressin. This observation supports the view that lovastatin directly alters the response of renal arterioles to vasoconstrictor agents. The mechanisms by which lovastatin diminishes renal vascular tone could potentially be related to its antilipidemic action. In the present study, plasma cholesterol concentration fell by 30% in lovastatin-treated SHR, compared with the values seen in the vehicletreated rats. However, baseline plasma cholesterol and triglyceride concentrations are relatively low in SHR compared with values reported in other strains of rats,19 and the mechanism by which small changes in plasma cholesterol concentration alter vascular tone is not intuitively obvious. Moreover, this mechanism does not explain how in vitro exposure of renal arterioles to lovastatin had an effect similar to that of the chronic treatment of SHR in inhibiting the response of renal arterioles to vasoconstrictors. Another mechanism by which lovastatin might alter vascular tone and growth is by interfering with the isoprenylation of G proteins.9 In the present study,
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FIGURE 3. (A) Western blot comparing the expression of G proteins in renal arterioles isolated from 4-week– old SHR and WKY rats. Ten micrograms of protein were loaded in each lane. Three batches of renal vessels were isolated in each group. (B) Comparison of protein levels by densitometry. Relative density is the ratio of density in a lane divided by mean density of all the bands in the same gel. * indicates a significant difference from the corresponding value in WKY rats.
chronic treatment of SHR with lovastatin significantly decreased the levels of ras and rho proteins in renal arterioles, whereas the a subunits of the major classes of heterotrimeric G proteins (Gsa, Gqa, and Gia) expressed in these arterioles were not significantly altered. The effects of lovastatin on the expression of small G proteins were not simply due to its antihypertensive actions alone, as another antihypertensive agent, hydralazine, which was equally effective at lowering blood pressure, had no effect on the expression of ras and rho proteins in renal arterioles. Similar effects on the expression of small G proteins were also seen in experiments in which renal arterioles were directly exposed to lovastatin in vitro. Together these results indicate that lovastatin has a direct effect on lowering the expression of ras and rho proteins in renal arterioles. The mechanism by which lovastatin reduces the expression of small G proteins in the renal vasculature remains to be determined; however, lovastatin may
alter the stability of normally isoprenylated small G proteins. In this regard, isoprenylation is a post-translational modification process in which an isoprenoid molecule, such as farnysyl or geranylgeranyl, is covalently attached to the carboxyl-terminus of a protein. Lovastatin inhibits the synthesis of mevalonate, which is the precursor for the synthesis of farnesyl or geranylgeranyl molecules.4 Small G proteins comprise a major class of isoprenylated proteins.9 In vascular smooth muscle (VSM) cells, two small G proteins, ras and rho p21, are expressed.9,20 Ras p21 is a key component of the growth signal transduction pathway,9,21 whereas rho p21 has been linked to the control of the contractile mechanism in VSM cells.10,20 Isoprenylation is essential for ras and rho proteins to become anchored to the plasma membrane and interact with their effector molecules.21,22 Blockade of the synthesis of the isoprenyl moieties with lovastatin has been reported to inactivate this class of signaling proteins and leads to the arrest of cell growth and prolifera-
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FIGURE 4. (A) Western blot comparing the expression of ras, rho, Gsa, Gia, and Gqa proteins in renal arterioles isolated from SHR chronically treated with lovastatin or vehicle. Ten micrograms of protein were loaded in each lane. Three batches of renal vessels were isolated from each group of rats. (B) Comparison of protein levels by densitometry. Relative density is the ratio of density in a lane divided by the mean density of all the bands in the gel. * indicates a significant difference from the corresponding values observed in vessels obtained from vehicle-treated SHR.
tion.5– 8 In vivo, lovastatin retards cell growth in a variety of situations, including tumors23 and hyperplasia24,25 of VSM cells under atherogenic conditions. Moreover, inactivation of small G proteins by lova-
FIGURE 5. Western blot comparing the expression of ras and rho proteins in renal arterioles isolated from SHR chronically treated with hydralazine or vehicle. Ten micrograms of protein were loaded in each lane. Three batches of renal vessels were isolated from each group.
statin may also reduce vascular contractility and reactivity because activation of ras has been reported to enhance Ca21 channel activity in neurons10 and reduce K1 channel activity in atrial cells.26 In VSM cells, these changes in ion channel activities lead to vasoconstriction. Ras and rho p21 have also been reported to modulate the Ca21 sensitivity of the contractile proteins in VSM cells.11,12 Therefore, the downregulation of small G protein levels and activity after lovastatin may partly explain the diminished vasoconstriction of renal preglomerular arterioles to agonists observed in the present study. Consistent with this view, simvastatin has recently been reported to reduce Ca21 influx in cultured VSM cells in response to vasopressin, an effect that can be reversed by mevalonate.27 The findings that elevations in renal vascular28 and vascular hypertrophy29 occur very early in the development of hypertension in SHR, and that these alterations often persist after normalization of arterial pres-
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FIGURE 6. Comparison of the concentration-response relation to norepinephrine in renal arterioles (inner diameter , 90 mm) freshly isolated from 4-week– old SHR versus those obtained in vessels cultured overnight in DMEM or in DMEM plus lovastatin (8 mg/mL). The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding values in renal arterioles cultured overnight with media containing vehicle.
sure by antihypertensive therapy,30,31 have suggested that signal transduction pathways regulating vascular tone and growth might play a role in the development of hypertension in SHR. A recent study has reported that the expression of a subunits of heterotrimeric G proteins (Gs, Gi, and Gq) are not significantly different in small mesenteric arteries and in the aorta of SHR and WKY rats.32 The present results are consistent with these findings and indicate that the expression of a subunits of heterotrimeric G proteins (Gs, Gi, and Gq) are not significantly different in renal arterioles obtained from the kidneys of 4-week– old SHR and WKY rats. However, the level of ras protein was markedly elevated in renal arterioles obtained from the kidneys of young SHR, compared with the levels seen in WKY rats. Because arterial pressure is barely elevated (, 10 mm Hg) at this age in SHR relative to WKY,28 the elevation in the expression of ras protein in the renal vasculature is probably not due to vascular hypertrophy induced by long-standing hypertension. The significance of the elevated expression of ras
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FIGURE 7. Comparison of the concentration-response relations to vasopressin in renal arterioles (inner diameter , 90 mm) freshly isolated from 4-week– old SHR versus those cultured overnight in DMEM, and in DMEM plus lovastatin (8 mg/mL). The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding values in renal arterioles cultured overnight with media containing vehicle.
in the renal vasculature of young SHR still remains to be determined, but given the importance of this protein in the control of vascular tone and growth, it is possible that changes in ras protein expression may contribute to the elevations in renal vascular tone and vascular hypertrophy that are seen very early in the development of hypertension in these animals. In summary, chronic treatment of SHR with lovastatin lowers arterial pressure and reduces the responsiveness of renal arterioles to vasoconstrictor agonists; this is associated with a fall in the expression of ras and rho proteins in the renal vasculature. The effects of lovastatin to diminish renal vascular tone appear to be due to a direct action, as it could be mimicked by overnight exposure of renal arterioles to culture media containing lovastatin. Overall the results of the present study indicate that lovastatin diminishes the response to vasoconstrictors and the expression of
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FIGURE 8. Western blot comparing the expression of ras and rho proteins in renal arterioles isolated from SHR that were cultured overnight in media containing vehicle or lovastatin (8 mg/mL). Five micrograms (ras) and 20 mg of protein (rho) were loaded in each lane. * indicates a significant difference from the corresponding value in renal arterioles cultured overnight with media containing vehicle.
small G proteins in the renal vasculature of SHR and suggest that a fall in the expression of ras and rho proteins in renal arterioles may contribute to the antihypertensive effects of lovastatin.
5.
Habenicht AJR, Glomset JA, Ross R: Relation of cholesterol and mevalonic acid to the cell cycle in smooth muscle and Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem 1980;225: 5134 –5140.
6.
Fairbanks KP, Witte LD, Goodman DS: Relationship between mevalonate and mitogenesis in human fibroblasts stimulated with platelet-derived growth factor. J Biol Chem 1984;259:1546 –1551.
7.
O’Donnell MP, Kasiske BL, Kim Y, et al: Lovastatin inhibits proliferation of rat mesangial cells. J Clin Invest 1993;91:83– 87.
8.
Munro E, Patel PJ, Chan P, et al: Inhibition of human vascular smooth muscle cell proliferation by lovastatin: the role of isoprenoid intermediates of cholesterol synthesis. Euro J Clin Invest 1994;24:766 –772.
9.
Hall A: The cellular functions of small GTP-binding proteins. Science 1990;249:635– 640.
10.
Collin C, Paragevnge AG, Lowry DR, Alkon DL: Early enhancement of calcium currents by H-ras oncoproteins injected into Hermissenda neurons. Science 1990; 250:1743–1745.
11.
Hirata K, Kikuchi A, Sasaki T, et al: Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 1992;267:8719 – 8722.
12.
Satoh S, Rensland H, Pfitzer G: Ras proteins increase Ca21-responsiveness of smooth muscle contraction. FEBS Lett 1993;324:211–215.
13.
Ito O, Alonso-Galicia M, Hoop KA, Roman RJ: Localization of cytochrome P-450 4A isoforms along the rat nephron. Am J Physiol 1998;274:F395–F404.
14.
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 – 685.
15.
Kost CK Jr, Herzer WA, Li P, Jackson EK: Vascular reactivity to angiotensin II is selectively enhanced in the kidneys of spontaneously hypertensive rats. J Pharmacol Exp Therap 1993;269:82– 88.
16.
Uchino K, Frohlich ED, Nishikimi T, et al: Spontaneously hypertensive rats demonstrate increased renal vascular a1-adrenergic receptor responsiveness. Am J Physiol 1991;260:R889 –R893.
17.
Roman RJ: Altered pressure-natriuresis relationship in young spontaneously hypertensive rats. Hypertension 1987;9(suppl III):III-130 –III-136.
18.
Roman RJ, Kaldunski ML: Renal cortical and papillary blood flow in spontaneously hypertensive rats. Hypertension 1988;11:657– 663.
19.
Iritani N, Fukuda E, Nara Y, Yamori Y: Lipid metabolism in spontaneously hypertensive rats (SHR). Atherosclerosis 1977;28:217–222.
20.
Kawahara Y, Kawata M, Sunako M, et al: Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product. Biochem Biophys Res Comm 1990;170:673– 683.
21.
Itoh T, Kaibuchi K, Masuda T, et al: The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase. J Biol Chem 1993; 268:3025–3028.
REFERENCES 1.
Castelli WP, Anderson K: A population at risk: prevalence of high cholesterol levels in hypertensive patients in the Framingham Study. Am J Med 1986;80(2A):23– 36.
2.
Kannel WB: Hypertension: relationship with other risk factors. Drugs 1986;109:581–585.
3.
Jiang J, Roman RJ: Lovastatin prevents the development of hypertension in spontaneously hypertensive rats. Hypertension 1997;30:968 –974.
4.
Goldstein JL, Brown MS: Regulation of the mevalonate pathway. Nature 1990;343:425– 430.
1231
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22.
lates vasopressin-stimulated Ca21 responses in rat A10 vascular smooth muscle cells. Circ Res 1994;74:173–181.
23.
Mizuno T, Kaibuchi K, Yamamoto T, et al: A stimulatory GDP/GTP exchange protein for small G p21 is active on the post-translationally processed form of c-Ki-ras p21 and rho A p21. Proc Natl Acad Sci USA 1991;88:6442– 6446.
28.
Feleszko W, Lasek W, Golab J, Jakobisiak M: Synergistic antitumor activity of tumor necrosis factor-alpha and lovastatin against MmB16 melanoma in mice. Neoplasma 1995;42:69 –74.
Imig JD, Falck JR, Gebremedhin D, et al: Elevated renal vascular tone in young spontaneously hypertensive rats. Role of cytochrome P450. Hypertension 1993;22: 357–364.
29.
Eccleston-Joyner CA, Gray SD: Arterial hypertrophy in the fetal and neonatal spontaneously hypertensive rat. Hypertension 1988;12:513–518.
24.
Soma MR, Donetti E, Parolini C, et al: HMG-CoA reductase inhibitors: in vivo effects on carotid intimal thickening in normocholesterolemic rabbits. Arterioscler Thromb 1993;13:571–578.
30.
Smeda JS, Lee RMKW, Forrest JB: Prenatal and postnatal hydralazine treatment does not prevent renal vessel wall thickening in SHR despite the absence of hypertension. Circ Res 1988;63:534 –543.
25.
Hodis HN, Mack WJ, LaBree L, et al: Reduction in carotid arterial wall thickness using lovastatin and dietary therapy. Ann Intern Med 1996;124:548 –556.
31.
26.
Yatani A, Okabe K, Polakis P, et al: Ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K1 channels. Cell 1990;61:769 –776.
Kett MM, Alcorn D, Bertram JF, Anderson WP: Enalapril does not prevent renal arterial hypertrophy in spontaneously hypertensive rats. Hypertension 1995; 25:335–342.
32.
Clark CJ, Milligan G, McLellan AR, Connell JMC: Guanine nucleotide regulatory protein levels and function in spontaneously hypertensive rat vascular smooth muscle cells. Biochem Biophys Acta 1992;1136:290 –296.
27.
Ng LL, Davies JE, Wojcikiewicz RJH: 3-Hydroxyl-3methylglutaryl coenzyme A reductase inhibitor modu-
1998;11:1222–1231
Lovastatin Reduces Renal Vascular Reactivity in Spontaneously Hypertensive Rats Jian Jiang, Chen-Wen Sun, Magdalena Alonso-Galicia, and Richard J. Roman
We have reported that lovastatin attenuates the development of hypertension in spontaneously hypertensive rats (SHR). The fall in arterial pressure is associated with an elevation in renal medullary blood flow, normalization of the pressure-natriuresis relationship, and diminished hypertrophy of renal arterioles. However, the mechanism by which lovastatin alters renal vascular tone is unknown. The present study examined the effects of lovastatin on renal vascular tone and the expression of G proteins. Four-week– old SHR were chronically treated with lovastatin (20 mg/kg/day) or vehicle by gavage for 4 weeks. At the end of the study, mean arterial pressure averaged 131 6 4 (n 5 5) and 160 6 4 mm Hg (n 5 6) in lovastatin- and vehicle-treated SHR, respectively. Renal arterioles isolated from lovastatin-treated SHR were significantly less responsive to norepinephrine and vasopressin than those obtained from vehicle-treated rats (ED50: 5.0 v 1.8 3 1027 mol/L for norepinephrine, and 8.0 v
C
5.2 3 10210 mol/L for vasopressin). The fall in renal vascular reactivity in lovastatin-treated SHR was associated with reduced levels of ras and rho proteins in renal arterioles, whereas the expressions of heterotrimeric G proteins (Gs, Gq, Gi) were similar in renal arterioles from vehicleand lovastatin-treated SHR. Overnight culture of renal arterioles with media containing lovastatin also diminished the expression of ras and rho proteins and the response to vasoconstrictors. These findings indicate that lovastatin diminishes the response to vasoconstrictors and the expression of small G proteins in the renal vasculature of SHR and suggest that a fall in the levels of ras and rho proteins in these vessels may contribute to the antihypertensive effects of lovastatin. Am J Hypertens 1998;11:1222–1231 © 1998 American Journal of Hypertension, Ltd. KEY WORDS:
Lovastatin, renal vascular tone, small G proteins, hypertension.
onsiderable evidence now exists that supports an association between hyperlipidemia and hypertension.1,2 The incidence of hypertension is greater in hyperlipidemic subjects and hyperlipidemia serves as an important risk factor for the development of end-organ damage
in hypertension. However, the mechanisms by which elevated levels of plasma lipids influence arterial pressure are unknown and there is very little information regarding the effects of antilipidemic therapy on blood pressure in hypertensive patients. In a recent study,3 we reported that chronic treatment of young (4-week–
Received August 28, 1997. Accepted May 6, 1998. From the Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin. This work was supported in part by grants from the National Heart Lung and Blood Institute, HL-36279 and HL-29587. Jian Jiang
was a recipient of Predoctoral Fellowship Award from the American Heart Association/Wisconsin Affiliate. Address correspondence and reprint requests to Richard J. Roman, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
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old), prehypertensive, spontaneously hypertensive rats (SHR) with the antilipidemic agent lovastatin normalized the pressure-natriuresis relation and markedly attenuated the development of hypertension. The fall in arterial pressure was associated with a marked elevation in renal medullary blood flow and diminished hypertrophy of renal arterioles. However, the mechanism by which lovastatin alters the structure and tone of renal arterioles is unknown. Lovastatin is a competitive inhibitor of the synthesis of mevalonate, the precursor for the synthesis of cholesterol and the nonsterol isoprenoids, such as the farnesyl and geranylgeranyl groups.4 Recently, it has become increasingly evident that nonsterol intermediates of mevalonate metabolism are essential for DNA synthesis and cell proliferation. For example, blockade of hepatic hydroxymethyl glutaryl coenzyme A (HMG-CoA) reductase activity with lovastatin inhibits the S phase of DNA synthesis, leading to the arrest of cell proliferation.5– 8 The inhibitory effects of lovastatin on cell growth could be reversed by adding mevalonate, but not cholesterol, to cell culture medium. It is also apparent that the farnesyl and geranylgeranyl groups are required for the isoprenylation of a number of important signal transduction proteins. One class of these proteins includes the low-molecular– weight GTP-binding proteins ras and rho, which play an integral role in the growth signal transduction pathway in vascular smooth muscle cells.9 In addition, small G proteins participate in the regulation of vascular tone by modifying the activity of voltage-gated calcium channels10 and the calcium sensitivity of the contractile mechanism.11,12 Thus, chronic treatment of SHR with lovastatin may reduce the hypertrophy and reactivity of renal arterioles by interfering with the synthesis of mevalonate and blocking the isoprenylation of small G proteins. To test this hypothesis, young (4-week– old), prehypertensive SHR were chronically treated with lovastatin or vehicle and changes in levels of small and heterotrimeric G proteins were measured in renal arterioles using Western blot techniques. In addition, the effects of lovastatin on the vasoconstrictor response of these arterioles to vasopressin and norepinephrine were determined. MATERIALS AND METHODS General Experiments were performed on 4-weekold SHR purchased from Harlan Sprague Dawley Laboratories (Indianapolis, IN). The rats were housed in an animal care facility at the Medical College of Wisconsin that was approved by the American Association for Accreditation of Laboratory Animal Care, and had free access to food and water throughout the experiment. All protocols involving animals received prior approval from the Animal Care Committee of the Medical College of Wisconsin.
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The rats were divided into four groups. The first two groups were given lovastatin (10 mg/kg) or vehicle twice a day by gavage, whereas the other groups were treated with hydralazine (100 mg/L) or vehicle in the drinking water. Hydralazine was used as an alternative antihypertensive therapy so that we could determine whether the effects of lovastatin on renal vascular tone and on the expression of G proteins were due to a direct effect or secondary to its ability to lower blood pressure. Measurement of Arterial Pressure After 4 weeks of treatment the rats were anesthetized with an intramuscular injection of ketamine (50 mg/kg) and xylazine (2 mg/kg) and a catheter was implanted in the femoral artery for measurement of arterial pressure. The catheter was exteriorized at the back of the neck and brought out through a stainless-steel spring and swivel device. After a 3-day recovery period, arterial pressure was recorded between 9 am and 12 pm with a pressure transducer and a computerized recording system for at least 3 h per day on 3 consecutive days while the animal was conscious in its home cage. The signals were sampled at 30 Hz, and heart rate and systolic, diastolic, and mean arterial pressures were collected at 1-min intervals and reduced to a single mean value for the recording session. After arterial pressure was measured, 1 mL of blood was collected from the femoral arterial catheter for measurements of plasma cholesterol and triglyceride concentrations. Assessment of Renal Vascular Responsiveness to Norepinephrine and Vasopressin The SHR were treated with lovastatin, hydralazine, or vehicle for 4 weeks as described above. The rats were then anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg). A midline abdominal incision was made, and the left kidney was removed and placed in cold physiological salt solution (PSS), pH 7.4, which had the following composition (in mmol/ L): NaCl 119, KCl 3.3, Na2SO4 0.7, CaCl2 2.0, NaHCO3 22.8, KH2PO4 1.2, glucose 11.0, and N-2-hydroxy-ethylpiperazine-N9-2-ethanesulfonic acid 5.0. Renal interlobular arteries (, 100 mm in inner diameter) were microdissected and placed in a chamber maintained at 37°C. The vessels were bathed in PSS that was bubbled with a 93% O2-7% CO2 gas mixture. The ends of the vessel were cannulated with glass micropipettes (;50 mm, outer diameter) and tied in place with a 22-mm silk suture. Side branches were tied off. The inflow micropipette was connected to a fluid-filled reservoir containing PSS equilibrated with a 93% O2-7% CO2 gas mixture. During the experimental period, transmural pressure was controlled at 90 mm Hg by closing the outflow pipette and adjusting the pressure on the reservoir connected to the inflow pipette. Intraluminal pressure was monitored using a transducer connected
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in series with an inflow cannula. Arterial diameters were measured using a video micrometer measuring system. After a 1-h equilibration period, the viability of each vessel and the endothelium was tested by measuring the contractile response to norepinephrine (1 mmol/L) followed by the dilatory response to acetylcholine (5 mmol/L). Cumulative concentration-response curves to norepinephrine (1029 to 1025 mol/L) and vasopressin (10210 to 1028 mol/L) were then constructed. In these experiments, the drugs were added directly to the bath. In Vitro Effects of Lovastatin on Renal Vascular Tone Additional experiments were performed to determine whether the effects of lovastatin to alter renal vascular tone in SHR could be mimicked by shortterm exposure of renal arterioles to this drug in vitro. In these experiments, renal interlobular arterioles (, 100 mm in inner diameter) were microdissected from the kidneys of 4-week– old SHR and cultured overnight in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 3.7 g/L NaHCO3, 100 mg/mL of streptomycin, and 100 U/mL of penicillin. The vessels were divided into two groups, and lovastatin (8 mg/mL) or vehicle was added to the culture medium. Lovastatin was converted to its active dihydroxy-open acid form by hydrolysis in 0.1 mol/L NaOH at 50°C for 2 h, followed by neutralization with HCl to a pH of 7.2. After overnight exposure of the vessels to control media or media containing lovastatin, the vessels were mounted in the myograph and cumulative concentration-response curves to norepinephrine and vasopressin were constructed. To confirm that overnight organ culture of isolated renal arterioles does not alter the responsiveness of renal arterioles, we compared the responses of freshly isolated vessels from the kidneys of 4-week-old SHR to those observed in vessels cultured overnight in DMEM alone. Effects of Lovastatin on the Expression of G Proteins in the Renal Vasculature of SHR Experiments were performed to compare the expression of G proteins in preglomerular renal arterioles isolated from the kidneys of prehypertensive 4-week-old SHR and WistarKyoto (WKY) rats and from the kidneys of SHR chronically treated with lovastatin, hydralazine, or vehicle for 4 weeks. Additional experiments were also performed using renal arterioles isolated from the kidneys of 8- to 9-week– old SHR that were cultured overnight in media containing vehicle or lovastatin (8 mg/mL). In these experiments, renal arterioles were isolated using a recently described sieving procedure.13 Rats were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg) and the kidneys were flushed with 10 mL of a low-calcium Tyrode’s solution containing (in mmol/L): NaCl 145, KCl 6,
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MgCl2 1, CaCl2 0.05, HEPES 10, glucose 10, and NaHCO3 4.2. The kidneys were then flushed with 10 mL of Tyrode’s solution containing 6% albumin and 1% Evans blue dye. The kidneys were subsequently removed, hemisected, and gently pressed through a 180-mm mesh screen. The retained material was resuspended in 5 mL of Tyrode’s solution containing 1 mg/mL of each of the following: collagenase, soybean trypsin inhibitor, albumin, and dithiothreitol (DTT). The suspension was incubated for 15 min at 37°C on a shaking table and superfused with oxygen. The solution was filtered through a 75-mm screen and the retained material was resuspended in cold Tyrode’s solution. Individual Evans blue-stained arterioles were then collected using a stereomicroscope and fine forceps. The renal arterioles were frozen in liquid nitrogen and powdered using a mortar and pestle. The frozen tissue was homogenized in ice-cold 20 mmol/L Tris/ HCl, pH 8.0, containing 1 mmol/L EDTA, 1 mmol/L DTT, 0.25 mol/L sucrose, and 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), and sonicated for 15 s. The mixture was centrifuged at 3000 g for 10 min and 9000 g for 15 min. The protein concentration of the supernatant was determined using the Bradford protein assay (Bio-Rad, Hercules, CA). Immunoblotting Immunoblotting was performed as previously described.14 Various amounts of protein (10 – 80 mg) were loaded and separated on a 10% sodium dodecyl sulfate-polyacrylamide gel. Nonspecific binding was blocked by placing the membrane overnight in TBS-T (10 mmol/L Tris, 150 mmol/L NaCl, 0.08% Tween-20, pH 8.0) containing 5% nonfat dry milk at 4°C. The bound antibody was detected using enhanced chemiluminescence (ECL kit, Amersham Life Science, Amersham, Buckinghamshire, UK). The blots were also probed with anti-b-actin antibody to ensure that equal amounts of protein were loaded in each lane. The rat monoclonal anti-ras p21, rabbit polyclonal anti-rhoA p21, rabbit anti-Gqa, rabbit anti-Gia, and HRP-conjugated goat anti-rabbit/rat/mouse secondary antibodies used in these experiments were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). Mouse monoclonal anti-b-actin antibody was purchased from Sigma (Sigma, St. Louis, MO). Rabbit anti-Gsa was purchased from New England Nuclear Products (NEN, Boston, MA). The primary and secondary antibodies were used at a 1:2000 dilution. Statistics Mean values 6 1 SEM are presented. The significance of the differences in mean values between and within groups was determined with an analysis of variance for repeated measures, with multiple independent factors followed by a Duncan’s multiple
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range test. A P value , .05 was considered to be significant. RESULTS Effects of Lovastatin on Arterial Pressure Chronic treatment of SHR with lovastatin or hydralazine markedly attenuated the development of hypertension. Mean arterial pressure averaged 160 6 4 mm Hg in 8- to 9-week– old SHR treated with vehicle (n 5 12), but only 131 6 4 and 135 6 6 mm Hg in SHR treated with lovastatin (n 5 5) or hydralazine (n 5 6), respectively (P , .05). Plasma cholesterol concentration was 33% lower in lovastatin-treated SHR than that seen in vehicle-treated rats (1.03 6 0.06 v 1.56 6 0.18 mmol/L, P , .05), whereas plasma triglyceride concentration was not significantly different and averaged 1.0 6 0.1 mmol/L in lovastatin-treated SHR, versus 1.1 6 0.1 mmol/L in vehicle-treated rats. Effects of Lovastatin on the Renal Vasoconstrictor Response to Norepinephrine and Vasopressin The effects of chronic treatment of SHR with lovastatin or hydralazine on the response of renal arterioles to norepinephrine (NE) are presented in Figure 1. The concentration-response curves to NE were similar in renal arterioles isolated for SHR chronically treated with hydralazine or vehicle. In contrast, renal arterioles isolated from lovastatin-treated rats were significantly less responsive to NE than those obtained from SHR treated with vehicle or hydralazine. At submaximal concentrations, renal arterioles isolated from lovastatin-treated rats exhibited significantly smaller responses to NE than the responses seen in the vessels obtained from vehicle- or hydralazine-treated SHR. The median effective dose (ED50) was fivefold higher in renal arterioles isolated from lovastatin-treated rats than those obtained from vehicle- or hydralazinetreated rats (5.0 v 1.2 3 1027 mol/L). The concentration-response relations to vasopressin are presented in Figure 2. There was no significant difference between the cumulative concentration-response curves in renal arterioles obtained from vehicle- and hydralazine-treated SHR. However, the concentration-response relation to vasopressin was significantly shifted to the right in renal arterioles obtained from SHR treated with lovastatin (ED50 5 8.0 3 10210 for lovastatin-treated SHR v 5.2 3 10210 mol/L for vehicle-treated rats). Expression of G Proteins A comparison of the expression of G proteins in renal arterioles isolated from the kidneys of 4-week-old, prehypertensive SHR and WKY rats is presented in Figure 3. The expression of the a subunits of the heterotrimeric G proteins (Gs, Gi, and Gq) and rho protein was similar in the renal arterioles isolated from these two strains. However, the level of ras protein was significantly higher in
FIGURE 1. Comparison of the concentration-response relation to norepinephrine in renal arterioles (inner diameter , 90 mm) isolated from SHR chronically treated with lovastatin, hydralazine, or vehicle. The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding value in vessels isolated from vehicletreated SHR.
renal arterioles obtained from SHR than that seen in arterioles obtained from WKY rats. The effects of chronic treatment of SHR with lovastatin on the expression of G proteins in renal arterioles are presented in Figure 4. The levels of ras and rho proteins were, respectively, 73% and 51% lower in renal arterioles isolated from lovastatin-treated SHR than in the vessels obtained from vehicle-treated SHR. In contrast, the levels of the a subunits of Gs, Gi, and Gq proteins were not significantly different in renal arterioles isolated from these two groups of rats. Hydralazine did not alter the levels of ras and rho proteins in renal arterioles (Fig. 5), even though it was equally as effective as lovastatin at lowering arterial pressure in SHR. In Vitro Effects of Lovastatin on Vascular Responsiveness and the Expression of Small G Proteins in Renal Arterioles To determine whether the effects of lovastatin to lower vascular tone in SHR were due to
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The results of these experiments (Fig. 8) indicate that overnight exposure of renal arterioles to culture media containing lovastatin reduced the expression of ras and rho proteins in renal arterioles isolated from the kidneys of SHR rats by 50% and 30%, respectively. DISCUSSION
FIGURE 2. Comparison of the concentration-response relation to vasopressin in renal arterioles (inner diameter , 90 mm) isolated from SHR chronically treated with lovastatin, hydralazine, or vehicle. The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding value in vessels isolated from vehicletreated SHR.
a direct effect or secondary to its antihypertensive action, we studied whether overnight culture of renal arterioles in media containing lovastatin would also diminish vascular responses to NE and vasopressin. The results of these experiments are summarized in Figures 6 and 7. The cumulative concentration response curves to norepinephrine and vasopressin were similar in freshly isolated renal arterioles and vessels cultured overnight in DMEM. However, renal arterioles cultured overnight in DMEM containing lovastatin were significantly less responsive to norepinephrine and vasopressin than vessels cultured in DMEM alone. The ED50 concentrations for norepinephrine and vasopressin were significantly lower in renal arterioles cultured in control media than in vessels exposed to lovastatin (1.5 v 4.5 3 1027 mol/L for norepinephrine, and 4.8 v 7.5 3 10210 mol/L for vasopressin). Additional experiments were performed to determine whether overnight exposure of renal arterioles to lovastatin alters the expression of ras and rho proteins.
Previous studies have indicated that the development of hypertension in SHR is associated with an elevation in vascular tone in preglomerular renal arterioles,15,16 a reduction in renal medullary blood flow, and a shift of the pressure natriuresis relationship toward higher pressures.17,18 More recently, chronic treatment of young SHR with lovastatin has been reported to markedly attenuate the development of hypertension.3 The fall in arterial pressure in that study was associated with an elevation in renal medullary blood flow and normalization of the pressure-natriuresis response,3 but the mechanisms involved are unknown. The present study examined whether the antihypertensive effects of lovastatin might be related to an inhibitory effect on the renal vascular response to vasoconstrictors, secondary to its action to block the isoprenylation and expression of G proteins. The results indicate that chronic treatment of SHR with lovastatin diminishes the response of renal arterioles to both norepinephrine and vasopressin. These changes in renal vascular reactivity are not simply due to the antihypertensive effects of lovastatin, as chronic treatment of SHR with another antihypertensive agent, hydralazine, had no effect on the response of renal arterioles to vasoconstrictors. Moreover, overnight exposure of renal arterioles to culture media containing lovastatin also reduced the vasoconstrictor response to norepinephrine and vasopressin. This observation supports the view that lovastatin directly alters the response of renal arterioles to vasoconstrictor agents. The mechanisms by which lovastatin diminishes renal vascular tone could potentially be related to its antilipidemic action. In the present study, plasma cholesterol concentration fell by 30% in lovastatin-treated SHR, compared with the values seen in the vehicletreated rats. However, baseline plasma cholesterol and triglyceride concentrations are relatively low in SHR compared with values reported in other strains of rats,19 and the mechanism by which small changes in plasma cholesterol concentration alter vascular tone is not intuitively obvious. Moreover, this mechanism does not explain how in vitro exposure of renal arterioles to lovastatin had an effect similar to that of the chronic treatment of SHR in inhibiting the response of renal arterioles to vasoconstrictors. Another mechanism by which lovastatin might alter vascular tone and growth is by interfering with the isoprenylation of G proteins.9 In the present study,
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FIGURE 3. (A) Western blot comparing the expression of G proteins in renal arterioles isolated from 4-week– old SHR and WKY rats. Ten micrograms of protein were loaded in each lane. Three batches of renal vessels were isolated in each group. (B) Comparison of protein levels by densitometry. Relative density is the ratio of density in a lane divided by mean density of all the bands in the same gel. * indicates a significant difference from the corresponding value in WKY rats.
chronic treatment of SHR with lovastatin significantly decreased the levels of ras and rho proteins in renal arterioles, whereas the a subunits of the major classes of heterotrimeric G proteins (Gsa, Gqa, and Gia) expressed in these arterioles were not significantly altered. The effects of lovastatin on the expression of small G proteins were not simply due to its antihypertensive actions alone, as another antihypertensive agent, hydralazine, which was equally effective at lowering blood pressure, had no effect on the expression of ras and rho proteins in renal arterioles. Similar effects on the expression of small G proteins were also seen in experiments in which renal arterioles were directly exposed to lovastatin in vitro. Together these results indicate that lovastatin has a direct effect on lowering the expression of ras and rho proteins in renal arterioles. The mechanism by which lovastatin reduces the expression of small G proteins in the renal vasculature remains to be determined; however, lovastatin may
alter the stability of normally isoprenylated small G proteins. In this regard, isoprenylation is a post-translational modification process in which an isoprenoid molecule, such as farnysyl or geranylgeranyl, is covalently attached to the carboxyl-terminus of a protein. Lovastatin inhibits the synthesis of mevalonate, which is the precursor for the synthesis of farnesyl or geranylgeranyl molecules.4 Small G proteins comprise a major class of isoprenylated proteins.9 In vascular smooth muscle (VSM) cells, two small G proteins, ras and rho p21, are expressed.9,20 Ras p21 is a key component of the growth signal transduction pathway,9,21 whereas rho p21 has been linked to the control of the contractile mechanism in VSM cells.10,20 Isoprenylation is essential for ras and rho proteins to become anchored to the plasma membrane and interact with their effector molecules.21,22 Blockade of the synthesis of the isoprenyl moieties with lovastatin has been reported to inactivate this class of signaling proteins and leads to the arrest of cell growth and prolifera-
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FIGURE 4. (A) Western blot comparing the expression of ras, rho, Gsa, Gia, and Gqa proteins in renal arterioles isolated from SHR chronically treated with lovastatin or vehicle. Ten micrograms of protein were loaded in each lane. Three batches of renal vessels were isolated from each group of rats. (B) Comparison of protein levels by densitometry. Relative density is the ratio of density in a lane divided by the mean density of all the bands in the gel. * indicates a significant difference from the corresponding values observed in vessels obtained from vehicle-treated SHR.
tion.5– 8 In vivo, lovastatin retards cell growth in a variety of situations, including tumors23 and hyperplasia24,25 of VSM cells under atherogenic conditions. Moreover, inactivation of small G proteins by lova-
FIGURE 5. Western blot comparing the expression of ras and rho proteins in renal arterioles isolated from SHR chronically treated with hydralazine or vehicle. Ten micrograms of protein were loaded in each lane. Three batches of renal vessels were isolated from each group.
statin may also reduce vascular contractility and reactivity because activation of ras has been reported to enhance Ca21 channel activity in neurons10 and reduce K1 channel activity in atrial cells.26 In VSM cells, these changes in ion channel activities lead to vasoconstriction. Ras and rho p21 have also been reported to modulate the Ca21 sensitivity of the contractile proteins in VSM cells.11,12 Therefore, the downregulation of small G protein levels and activity after lovastatin may partly explain the diminished vasoconstriction of renal preglomerular arterioles to agonists observed in the present study. Consistent with this view, simvastatin has recently been reported to reduce Ca21 influx in cultured VSM cells in response to vasopressin, an effect that can be reversed by mevalonate.27 The findings that elevations in renal vascular28 and vascular hypertrophy29 occur very early in the development of hypertension in SHR, and that these alterations often persist after normalization of arterial pres-
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FIGURE 6. Comparison of the concentration-response relation to norepinephrine in renal arterioles (inner diameter , 90 mm) freshly isolated from 4-week– old SHR versus those obtained in vessels cultured overnight in DMEM or in DMEM plus lovastatin (8 mg/mL). The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding values in renal arterioles cultured overnight with media containing vehicle.
sure by antihypertensive therapy,30,31 have suggested that signal transduction pathways regulating vascular tone and growth might play a role in the development of hypertension in SHR. A recent study has reported that the expression of a subunits of heterotrimeric G proteins (Gs, Gi, and Gq) are not significantly different in small mesenteric arteries and in the aorta of SHR and WKY rats.32 The present results are consistent with these findings and indicate that the expression of a subunits of heterotrimeric G proteins (Gs, Gi, and Gq) are not significantly different in renal arterioles obtained from the kidneys of 4-week– old SHR and WKY rats. However, the level of ras protein was markedly elevated in renal arterioles obtained from the kidneys of young SHR, compared with the levels seen in WKY rats. Because arterial pressure is barely elevated (, 10 mm Hg) at this age in SHR relative to WKY,28 the elevation in the expression of ras protein in the renal vasculature is probably not due to vascular hypertrophy induced by long-standing hypertension. The significance of the elevated expression of ras
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FIGURE 7. Comparison of the concentration-response relations to vasopressin in renal arterioles (inner diameter , 90 mm) freshly isolated from 4-week– old SHR versus those cultured overnight in DMEM, and in DMEM plus lovastatin (8 mg/mL). The 100% change in inner diameter refers to the complete closure of the vessel. * indicates a significant difference from the corresponding values in renal arterioles cultured overnight with media containing vehicle.
in the renal vasculature of young SHR still remains to be determined, but given the importance of this protein in the control of vascular tone and growth, it is possible that changes in ras protein expression may contribute to the elevations in renal vascular tone and vascular hypertrophy that are seen very early in the development of hypertension in these animals. In summary, chronic treatment of SHR with lovastatin lowers arterial pressure and reduces the responsiveness of renal arterioles to vasoconstrictor agonists; this is associated with a fall in the expression of ras and rho proteins in the renal vasculature. The effects of lovastatin to diminish renal vascular tone appear to be due to a direct action, as it could be mimicked by overnight exposure of renal arterioles to culture media containing lovastatin. Overall the results of the present study indicate that lovastatin diminishes the response to vasoconstrictors and the expression of
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FIGURE 8. Western blot comparing the expression of ras and rho proteins in renal arterioles isolated from SHR that were cultured overnight in media containing vehicle or lovastatin (8 mg/mL). Five micrograms (ras) and 20 mg of protein (rho) were loaded in each lane. * indicates a significant difference from the corresponding value in renal arterioles cultured overnight with media containing vehicle.
small G proteins in the renal vasculature of SHR and suggest that a fall in the expression of ras and rho proteins in renal arterioles may contribute to the antihypertensive effects of lovastatin.
5.
Habenicht AJR, Glomset JA, Ross R: Relation of cholesterol and mevalonic acid to the cell cycle in smooth muscle and Swiss 3T3 cells stimulated to divide by platelet-derived growth factor. J Biol Chem 1980;225: 5134 –5140.
6.
Fairbanks KP, Witte LD, Goodman DS: Relationship between mevalonate and mitogenesis in human fibroblasts stimulated with platelet-derived growth factor. J Biol Chem 1984;259:1546 –1551.
7.
O’Donnell MP, Kasiske BL, Kim Y, et al: Lovastatin inhibits proliferation of rat mesangial cells. J Clin Invest 1993;91:83– 87.
8.
Munro E, Patel PJ, Chan P, et al: Inhibition of human vascular smooth muscle cell proliferation by lovastatin: the role of isoprenoid intermediates of cholesterol synthesis. Euro J Clin Invest 1994;24:766 –772.
9.
Hall A: The cellular functions of small GTP-binding proteins. Science 1990;249:635– 640.
10.
Collin C, Paragevnge AG, Lowry DR, Alkon DL: Early enhancement of calcium currents by H-ras oncoproteins injected into Hermissenda neurons. Science 1990; 250:1743–1745.
11.
Hirata K, Kikuchi A, Sasaki T, et al: Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction. J Biol Chem 1992;267:8719 – 8722.
12.
Satoh S, Rensland H, Pfitzer G: Ras proteins increase Ca21-responsiveness of smooth muscle contraction. FEBS Lett 1993;324:211–215.
13.
Ito O, Alonso-Galicia M, Hoop KA, Roman RJ: Localization of cytochrome P-450 4A isoforms along the rat nephron. Am J Physiol 1998;274:F395–F404.
14.
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680 – 685.
15.
Kost CK Jr, Herzer WA, Li P, Jackson EK: Vascular reactivity to angiotensin II is selectively enhanced in the kidneys of spontaneously hypertensive rats. J Pharmacol Exp Therap 1993;269:82– 88.
16.
Uchino K, Frohlich ED, Nishikimi T, et al: Spontaneously hypertensive rats demonstrate increased renal vascular a1-adrenergic receptor responsiveness. Am J Physiol 1991;260:R889 –R893.
17.
Roman RJ: Altered pressure-natriuresis relationship in young spontaneously hypertensive rats. Hypertension 1987;9(suppl III):III-130 –III-136.
18.
Roman RJ, Kaldunski ML: Renal cortical and papillary blood flow in spontaneously hypertensive rats. Hypertension 1988;11:657– 663.
19.
Iritani N, Fukuda E, Nara Y, Yamori Y: Lipid metabolism in spontaneously hypertensive rats (SHR). Atherosclerosis 1977;28:217–222.
20.
Kawahara Y, Kawata M, Sunako M, et al: Identification of a major GTP-binding protein in bovine aortic smooth muscle cytosol as the rhoA gene product. Biochem Biophys Res Comm 1990;170:673– 683.
21.
Itoh T, Kaibuchi K, Masuda T, et al: The post-translational processing of ras p21 is critical for its stimulation of mitogen-activated protein kinase. J Biol Chem 1993; 268:3025–3028.
REFERENCES 1.
Castelli WP, Anderson K: A population at risk: prevalence of high cholesterol levels in hypertensive patients in the Framingham Study. Am J Med 1986;80(2A):23– 36.
2.
Kannel WB: Hypertension: relationship with other risk factors. Drugs 1986;109:581–585.
3.
Jiang J, Roman RJ: Lovastatin prevents the development of hypertension in spontaneously hypertensive rats. Hypertension 1997;30:968 –974.
4.
Goldstein JL, Brown MS: Regulation of the mevalonate pathway. Nature 1990;343:425– 430.
1231
AJH–OCTOBER 1998 –VOL. 11, NO. 10, PART 1
LOVASTATIN AND RENAL VASCULAR TONE
22.
lates vasopressin-stimulated Ca21 responses in rat A10 vascular smooth muscle cells. Circ Res 1994;74:173–181.
23.
Mizuno T, Kaibuchi K, Yamamoto T, et al: A stimulatory GDP/GTP exchange protein for small G p21 is active on the post-translationally processed form of c-Ki-ras p21 and rho A p21. Proc Natl Acad Sci USA 1991;88:6442– 6446.
28.
Feleszko W, Lasek W, Golab J, Jakobisiak M: Synergistic antitumor activity of tumor necrosis factor-alpha and lovastatin against MmB16 melanoma in mice. Neoplasma 1995;42:69 –74.
Imig JD, Falck JR, Gebremedhin D, et al: Elevated renal vascular tone in young spontaneously hypertensive rats. Role of cytochrome P450. Hypertension 1993;22: 357–364.
29.
Eccleston-Joyner CA, Gray SD: Arterial hypertrophy in the fetal and neonatal spontaneously hypertensive rat. Hypertension 1988;12:513–518.
24.
Soma MR, Donetti E, Parolini C, et al: HMG-CoA reductase inhibitors: in vivo effects on carotid intimal thickening in normocholesterolemic rabbits. Arterioscler Thromb 1993;13:571–578.
30.
Smeda JS, Lee RMKW, Forrest JB: Prenatal and postnatal hydralazine treatment does not prevent renal vessel wall thickening in SHR despite the absence of hypertension. Circ Res 1988;63:534 –543.
25.
Hodis HN, Mack WJ, LaBree L, et al: Reduction in carotid arterial wall thickness using lovastatin and dietary therapy. Ann Intern Med 1996;124:548 –556.
31.
26.
Yatani A, Okabe K, Polakis P, et al: Ras p21 and GAP inhibit coupling of muscarinic receptors to atrial K1 channels. Cell 1990;61:769 –776.
Kett MM, Alcorn D, Bertram JF, Anderson WP: Enalapril does not prevent renal arterial hypertrophy in spontaneously hypertensive rats. Hypertension 1995; 25:335–342.
32.
Clark CJ, Milligan G, McLellan AR, Connell JMC: Guanine nucleotide regulatory protein levels and function in spontaneously hypertensive rat vascular smooth muscle cells. Biochem Biophys Acta 1992;1136:290 –296.
27.
Ng LL, Davies JE, Wojcikiewicz RJH: 3-Hydroxyl-3methylglutaryl coenzyme A reductase inhibitor modu-