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Raloxifene relaxes rat intrarenal arteries by inhibiting Ca²⁺ influx

¹Department of Physiology, Chinese University of Hong Kong, Hong Kong; and ²Department of Physiology and Pathophysiology, Fudan University Shanghai Medical College, Shanghai, China

Submitted 16 September 2004 ; accepted in final form 27 January 2005

	ABSTRACT

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Raloxifene may confer vascular benefits without causing estrogen-related side effects. However, its action on renal vascular circulation is unknown. This study aimed to examine the sex difference and roles of the endothelium and Ca²⁺ channels in rat renovascular relaxation to raloxifene. On isolated intralobar renal artery rings mounted in a myograph and contracted by U-46619, concentration-relaxation curves were constructed for raloxifene and contractions to CaCl₂ were studied. Changes in intracellular Ca²⁺ concentration levels ([Ca²⁺]_i) of vascular smooth muscle (VSM) were measured by fura 2 fluorescence. Raloxifene or 17-estradiol was equally effective in relaxing renal arteries from both sexes, with raloxifene being more potent than 17-estradiol. Endothelial denudation did not affect raloxifene- or 17-estradiol-induced relaxation. N^G-nitro-L-arginine methyl ester, charybdotoxin plus apamin, indomethacin, or ICI-182, 780 did not modify the effect of raloxifene. Raloxifene caused similar relaxations in rings contracted by U-46619 and high K⁺. Nifedipine attenuated the potency of raloxifene. Raloxifene reduced CaCl₂-induced contractions. K⁺ (80 mM) stimulated an increase in VSM [Ca²⁺]_i, and raloxifene attenuated this effect. Raloxifene-induced reduction of contraction and increase in VSM [Ca²⁺]_i were insensitive to ICI-182, 780. In summary, raloxifene causes relaxation in rat renal arteries; this effect is independent of a functional endothelium and is not mediated by ICI 182, 780-sensitive estrogen receptors. Raloxifene inhibited both contractions and VSM [Ca²⁺]_i in response to CaCl₂, indicating that raloxifene relaxes rat renal arteries primarily through inhibiting Ca²⁺ influx via Ca²⁺ channels. There is little sex difference in raloxifene-induced relaxation.

17-estradiol; relaxation; rat renal artery

Treatment with raloxifene, a second generation SERM in healthy postmenopausal women, enhanced flow-mediated vasodilatation (5, 21, 23, 24), increased plasma nitric oxide (NO) concentrations (21), and decreased plasma endothelin-1 levels (21). However, raloxifene therapy did not improve vascular function in postmenopausal women with coronary heart disease, whose arteries had been affected by advanced atherosclerosis (10). Raloxifene also reduced the expression of vascular cell adhesion molecule-1 in human endothelial cells (25), improved lipid profile (16, 32) and homeostatic parameters (2, 6), and lowered systemic blood pressure and arterial stiffness in postmenopausal women (7). Nongenomic signaling through estrogen receptors accounts for part of estrogen-mediated vascular actions in vitro. Raloxifene relaxed mammalian arteries (8, 28) and veins (3, 4) via both endothelium-dependent and -independent mechanisms. The former was inhibited by the classic estrogen receptor antagonist ICI-182, 780 (8), and the latter was due to direct inhibition of voltage-sensitive Ca²⁺ channels, which were insensitive to ICI-182, 780 (4, 28). Raloxifene therapy attenuated hypertension-associated endothelial dysfunction, and the underlying mechanisms may involve increased activity of endothelial nitric oxide (NO) synthase and a reduction in production of reactive oxygen species (33).

Both clinical and animal studies suggest that raloxifene and other SERMs have potentials as novel alternatives to estrogen for the treatment of menopause-related cardiovascular diseases (34). Chronic estrogen treatment augmented endothelium-dependent relaxation in perfused kidneys to a greater extent in ovariectomized female compared with male rats (17). Acute administration of estrogen lowered the elevated renovascular tone in hypertensive female rats by enhancing NO-mediated relaxation with little effect on male rats (35). These observations suggest sex-specific acute effects of estrogen on renal arteries. However, it is yet to be determined whether SERMs could exert effects similar to estrogen on renal artery tone regulation. To this end, we investigated the effects of raloxifene on the tone of isolated rat intralobar renal arteries compared with that of exogenous estrogen. Thus we specifically examined whether raloxifene-induced vascular action involved 1) the endothelium, 2) estrogen receptors, 3) inhibition of Ca²⁺ influx through Ca²⁺ channels, or 4) a sex difference.

	METHODS

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Chemicals. Acetylcholine, U-46619, N^G-nitro-L-arginine methyl ester (L-NAME), indomethacin, charybdotoxin (CTX), apamin, nifedipine, and 17-estradiol were purchased from Sigma (St. Louis, MO). ICI-182, 780 was purchased from Tocris. Raloxifene was a gift from Lilly Corporate Center (Indianapolis, IN). U-46619, raloxifene, 17-estradiol, indomethacin. and nifedipine were dissolved in DMSO and others in distilled water. Further dilution was made from a stock solution.

Blood vessel preparation. This study was approved by the Experimental Animal Ethics Committee at the Chinese University of Hong Kong. Sprague-Dawley rats of both sexes (300 g) were killed by cervical dislocation. After the abdominal cavity was opened, the kidneys were removed and placed in ice-cold Krebs solution (in mM): 119 NaCl, 4.7 KCl, 2.5 CaCl₂, 1 MgCl₂, 25 NaHCO₃, 1.2 KH₂PO₄, and 11 D-glucose. The intralobar renal arteries (mean external diameter of 366 µm) were dissected from both kidneys, and each artery was cleaned of adhering fatty tissues and cut into two ring segments, 2 mm in length. Each segment was mounted in a Multi Myograph System (Danish Myo Technology, Aarhus, Denmark), and changes in arterial tone were recorded. Briefly, two tungsten wires (each 40 µm in diameter) were inserted through the segment's lumen, and each wire was fixed to the jaws of a myograph. The organ chamber was filled with 5 ml Krebs solution and oxygenated with a 95% O₂-5% CO₂ gas mixture. Krebs solution in the chamber was maintained at 37°C using a built-in heat-exchanger device to give a pH value of between 7.3 and 7.5. Each ring was stretched initially to 2 mN, an optimal tension, and then allowed to stabilize at this baseline tone for 90 min before the start of each experiment. Each experiment was conducted using rings from different rats. In total, this study used 43 female and 42 male rats. In some arteries, the endothelium was mechanically removed by rubbing the luminal surface of the ring with small stainless steel wire. Functional removal of the endothelium was verified if the relaxant effect of acetylcholine was absent. In experiments using a high-K⁺ solution, an equimolar amount of K⁺ replaced Na⁺ to retain constant ionic strength.

Force measurement. After equilibration in Krebs solution for 30 min, each ring was first contracted by 100 nM U-46619 followed by addition of 3 µM acetylcholine to test integrity of the endothelium. The initial data showed that acetylcholine induced a maximal relaxation of 60% in concentrations between 3 and 10 µM. Only those rings that relaxed 60% in response to acetylcholine were regarded as rings with endothelia. Rings were then rinsed three times, and baseline tone was restored or readjusted.

U-46619 was used to induce steady blood vessel tone; the relaxations to cumulative concentration of raloxifene (consecutive concentration added without washout of the previous concentration) were subsequently studied. Thirty to forty minutes were required to reach a steady-state response to raloxifene. The time-matched vehicle (DMSO) control protocol was also performed. The first set of experiments determined the role of the endothelium in the effect of raloxifene. Raloxifene-induced relaxation was studied in rings with endothelia following treatment with 100 µM L-NAME (NO synthase inhibitor), CTX plus apamin [inhibitors of endothelium-derived hyperpolarizing factor (EDHF) dilation, each at 50 nM], or 3 µM indomethacin (cyclooxygenase inhibitor) to assess the involvement of endothelium-derived NO, hyperpolarizing factors, or relaxing prostanoids. The possible role of the estrogen receptor in the raloxifene-induced effect was evaluated in rings treated with 10 µM ICI-182, 780 (classic estrogen-receptor antagonist). Because L-NAME or endothelial denudation enhanced U-46619-induced contraction, the concentration of U-46619 was lowered to 50 nM to obtain an initial tone of similar amplitude to the control. For comparison, the concentration-dependent relaxant effects of 17-estradiol were examined in both female and male renal artery rings contracted by U-46619 or 80 mM K⁺.

The second series of experiments examined the effects of raloxifene in rings contracted by elevated extracellular K⁺ to assess its ability to modulate Ca²⁺ influx via voltage-sensitive Ca²⁺ channels. Each ring was contracted twice with 80 mM K⁺ at 30-min intervals. Rings were washed three times in Ca²⁺-free, 80 mM K⁺ solution containing 30 µM Na₂-EGTA, then incubated in Ca²⁺-free, 80 mM K⁺ solution (with or without raloxifene, 30-min incubation) before cumulative addition of CaCl₂. In some experiments, rings were treated with 10 µM ICI-182, 780 for 10 min before addition of raloxifene. The effect of nifedipine was tested as the control.

The last set of experiments tested the influence of nifedipine on raloxifene-induced relaxation in rings without endothelia. Addition of 10 nM nifedipine caused partial and sustained reduction (by 64.9 ± 4.8% in male rats and 71.6 ± 8.2% in female rats) of U-46619 (100 nM)-induced contractions, and raloxifene was then cumulatively applied to reduce the remaining tension in nifedipine-treated rings. Raloxifene was also examined in the control rings, but a lower concentration of U-6619 (20 nM) was used to produce vessel tone comparable to that in the presence of nifedipine (U-46619-evoked tension: 3.28 ± 1.12 mN without nifedipine and 2.38 ± 0.56 mN with nifedipine in male rats; 3.03 ± 1.42 mN without nifedipine and 2.05 ± 1.06 mN with nifedipine in female rats, n = 4, P > 0.05).

Measurement of vascular smooth muscle calcium levels. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in fura 2-loaded artery rings without endothelia using the fluorescence ratio imaging. Rings were fluorescently labeled for 1 h by incubating them with 10 µM fura 2-AM and 0.025% Pluronic F-127 in Krebs solution at room temperature. Extracellular fura 2-AM was washed off in Krebs solution. Artery rings were then perfused for 20 min with Krebs solution (37°C) at a rate of 2 ml/min to allow cleavage of intracellular fura 2-AM into active fura 2 by esterases. Because of the photosensitivity of the fura 2 molecule, precautions were taken to avoid extensive photobleaching, and the excitation light was blocked by a shutter when no fluorescence measurement was recorded.

The basic [Ca²⁺]_i imaging setup was modified from that described by Huang et al. (11). After fura 2 loading, each artery ring was cut open along its longitudinal axis and pinned onto a block of silicone elastomer (Sylgard) with the lumen side upward, which was fixed onto a base plate of the custom-made flow chamber. The base plate was then covered with a gasket and coverglass (24 x 32 mm, thickness no. 1; Menzel-Glaser, Braunschweig, Germany), and affixed by screws. There was a 1-mm gap between the vessel and coverglass to allow flow passage. This arrangement allowed free vessel movement in response to drug application. After vessel mounting, the flow chamber was placed on an inverted microscope and perfused with Krebs solution (37°C) at 2 ml/min, aided by a six-channel perfusion pump (205S; Watson-Marlow) and a custom-made basic minivalve, multichannel perfusion system.

The fura 2-loaded vessels were visualized through a Nikon CF Fluor x20 objective (numerical aperture 0.45) on an inverted Nikon Eclipse TE300 microscope. The fura 2 was excited using a collimated beam of light from a 75-W xenon arc lamp and passed through a microscope photometer D-104 (Photon Technology International) that altered wavelengths from 340 to 380 nm using an optical chopper (OC-4000, Photon Technology International). The emitted light at 510 nm was collected by a photomultiplier tube. Instrument control, data acquisition, and analysis were performed using FELIX 1.21 software (Photon Technology International). Fluorescence intensities were recorded as a function of time.

After being mounted, the arterial tissues were allowed to recover for 30 min at 37°C and then exposed for 30 min to Ca²⁺-free, 80 mM K⁺ solution containing 30 µM Na₂-EGTA. Thereafter, they were perfused with the same high-K⁺ solution supplemented with 0.1, 0.3, 1, and 3 mM CaCl₂. Tissues were then washed several times in Ca²⁺-free 80 mM K⁺ solution until baseline level was restored. Following 30 min-incubation with raloxifene (0.3–5 µM), cumulative perfusion of CaCl₂ induced a second concentration-dependent increases in [Ca²⁺]_i. The effect of 10 µM ICI-182, 780 was also tested on the raloxifene (1 µM)-induced inhibition of a rise in [Ca²⁺]_i.

Data analysis. Data are means ± SD of rings from n rats. Increases in contractile force were expressed as a percentage of the mean value of two consecutive responses to 80 mM K⁺. Cumulative concentration-response curves were analyzed by nonlinear curve fitting using GraphPad software (version 3.0). The negative logarithm of the dilator (or constrictor) concentration that caused half (pD₂ or pEC₅₀) of the maximal response (E_max) was obtained. For statistical analysis, a two-tailed Student's t-test or one-way analysis of variance followed by a Newman-Keuls test was used when more than two groups were compared. Individual concentration-response curves were also compared using a two-way analysis of variance followed by Bonferronic posttests. Statistical significance was accepted when P < 0.05.

	RESULTS

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Raloxifene-induced relaxation. Contraction with the thromboxane A₂ mimetic U-46619 or 80 mM K⁺ was insignificantly greater in male than in female arteries (4.5 ± 0.64 mN in female and 5.9 ± 0.53 mN in male, n = 8–10, for U-46619; 4.8 ± 0.73 mN in female and 6.3 ± 0.50 mN in male, n = 5–6 for high K⁺, P > 0.05).

In U-46619-contracted rings with endothelia, raloxifene induced concentration-dependent relaxation with pD₂ of 6.23 ± 0.14 (female) or 5.97 ± 0.17 (male), and the relaxation was unaltered on endothelial denudation (pD₂: 6.22 ± 0.19, P > 0.05 for female, Fig. 1A and 5.94 ± 0.13, P > 0.05 for male, Fig. 1D). The relaxant effects of raloxifene in rings from both sexes were unchanged after exposure to L-NAME, CTX plus apamin (Fig. 1, B and E, Table 1), indomethacin (Fig. 1, C and F, Table 1), or ICI-182, 780 (Fig. 1, C and F, Table 1).

View larger version (43K): [in this window] [in a new window]   Fig. 1. Raloxifene-induced relaxations in female (A–C) and male (D–F) rat renal arteries. Concentration-response curves in rings with and without endothelia (Endo; A and D), in the presence of NG-nitro-L-arginine methyl ester (L-NAME) or charybdotoxin (CTX) plus apamin (B and E), and in the presence of indomethacin or ICI-182, 780 (C and F) are shown. Values are means ± SD of 6–8 experiments.

View this table: [in this window] [in a new window]   Table 1. pD2 values for raloxifene- and 17-estradiol-induced renal artery relaxation

View larger version (27K): [in this window] [in a new window]   Fig. 2. Comparison of relaxation induced by raloxifene (A and B) and 17-estradiol (C and D) in female and male renal arteries. Relaxations in U-46619-contracted rings (A and C) and relaxations in 80 mM K+-contracted rings (B and D) are shown. Values are means ± SD of 6 experiments.

Raloxifene inhibition of CaCl₂-induced contraction. In artery rings bathed in Ca²⁺-free, 80 mM K⁺ solution, cumulative additions of CaCl₂ induced contractions (pEC₅₀: 3.21 ± 0.29 in female and 3.34 ± 0.11 in male rings, P > 0.05). Treatment with raloxifene (0.1–5 µM) diminished contractions with a progressive reduction in the maximal contraction (Fig. 3A) without affecting the contractile sensitivity to CaCl₂ in female rings without endothelia (Table 2). Similarly, the similar inhibitory effects of raloxifene were obtained in male rings (Fig. 3C, Table 2). It appears that raloxifene at concentrations higher than 1 µM was slightly more effective in suppressing CaCl₂-induced contractions in female than in male rings (Table 2). Treatment with 10 µM ICI-182, 780 did not antagonize raloxifene (1 µM)-induced inhibition of contractions to CaCl₂ in both female (Fig. 3B) and male (Fig. 3D) rings.

View larger version (30K): [in this window] [in a new window]   Fig. 3. Effects of raloxifene on CaCl2-induced contraction in male rat renal arteries in 80 mM K+ solution. Concentration-response curves in the absence and presence of different concentrations of raloxifene in rings without endothelium from female (A) and male rats (C) are shown. Lack of effect of 10 µM ICI-182, 780 (ICI) on raloxifene (1 µM)-induced inhibition of CaCl2-induced constriction in rings from female (B) and male rats (D) is also shown. Values are means ± SD of 5–6 experiments. Statistical significance was tested by 2-way ANOVA between curves. *P < 0.05 and ***P < 0.01 compared with control (without raloxifene treatment).

View larger version (15K): [in this window] [in a new window]   Fig. 4. Influence of nifedipine on raloxifene-induced renovascular relaxation. A: concentration-response curves for raloxifene in U-46619-contracted rings without endothelium in the absence and presence of 10 nM nifedipine. M, male; F, female; Nif, nifedipine. B: pD2 values for raloxifene-induced relaxation described in A. Rings were first contracted using U-46619, then partially relaxed by nifedipine, and finally exposed to raloxifene. Values are means ± SD of 5 experiments. *P < 0.05, **P < 0.01 between control and nifedipine-treated rings.

Traces in Fig. 5, A and B, show changes in VSM [Ca²⁺]_i measured as the fluorescence ratio in female renal vessels in response to CaCl₂ in 80 mM K⁺ solution before and after treatment with raloxifene (0.3 and 5 µM). The cumulative addition of CaCl₂ caused stepwise increases in [Ca²⁺]_i, and a 30-min treatment with raloxifene reduced [Ca²⁺]_i elevation (Fig. 5, A and B). Similarly, raloxifene suppressed the CaCl₂-stimulated [Ca²⁺]_i rise in male vessels without endothelia (Fig. 5, D and E). Treatment with 10 µM ICI-182, 780 did not influence the raloxifene (1 µM)-mediated inhibition of the CaCl₂-stimulated [Ca²⁺]_i rise (Fig. 5, C and F). In control experiments, nifedipine at 100 nM abolished the CaCl₂-induced rise in [Ca²⁺]_i (data not shown).

View larger version (41K): [in this window] [in a new window]   Fig. 5. Effect of raloxifene (Rf) on CaCl2-stimulated rises in intracellular Ca2+ concentration ([Ca2+]i) in arterial tissues without endothelia bathed in 80 mM K+ solution. Raloxifene (0.3 and 5 µM)-induced inhibition of [Ca2+]i increases in female (A and B) or male (D and E) renal artery tissues is shown. Lack of effect of 10 µM ICI-182, 780 on raloxifene (1 µM)-induced inhibition CaCl2-stimulated rises in [Ca2+]i in rings from female (C) and male (F) rats is also shown. Values are means ± SD of 4 experiments. Traces are average of 4 recordings from 4 animals. Statistical significance was tested by 2-way ANOVA between curves. **P < 0.01, ***P < 0.001 compared with control.

	DISCUSSION

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Raloxifene inhibited high-K⁺-induced contraction in rat renal arteries with similar effectiveness (estimated IC₅₀ of 355 nM) as observed in rat cerebral arteries (estimated IC₅₀ of 360 nM) (28). These results suggest that raloxifene exerts direct muscle relaxation, probably by functioning as a Ca²⁺ channel blocker. Indeed, raloxifene inhibited Ca²⁺ influx via Ca²⁺ channels that were also sensitive to the L-type Ca²⁺ channel blocker nifedipine, as revealed by [Ca²⁺]_i measurement in fura 2-loaded arterial rings. The potency was similar for raloxifene between relaxing CaCl₂-induced tension and inhibiting the [Ca²⁺]_i increases. Like the L-type Ca²⁺ channel blocker nifedipine, raloxifene at 5 µM nearly prevented CaCl₂-induced increases in both vessel tone and [Ca²⁺]_i. Besides, there was a slight or no difference in either the potency or sensitivity of the raloxifene-induced effect in rings contracted by U-46619 or high K⁺. Finally, partial inhibition of L-type Ca²⁺ channels by nifedipine attenuated raloxifene-induced relaxation in U-46619-contracted rings from both sexes. These data indicate that inhibition of Ca²⁺ entry via L-type Ca²⁺ channels is likely to account for raloxifene-mediated renovascular relaxation. The present study has thus provided evidence showing that raloxifene, like nifedipine, may act as an antagonist of Ca²⁺ channels in renal artery smooth muscle cells. However, it is unclear how raloxifene may directly act on Ca²⁺ channels in VSM cells if its effect is not mediated by estrogen receptors. Estrogen was demonstrated to activate Ca²⁺-activated K⁺ channels by direct interaction with the -subunit of the channel protein (29). It is yet to be elucidated whether the Ca²⁺ channel could provide such an interactive site for raloxifene. Besides, it remains to be examined whether raloxifene lowers [Ca²⁺]_i via other Ca²⁺-signaling pathways, e.g., stimulation of plasma membrane Ca²⁺-ATPase activity in VSM.

The clinical importance of the endothelial NO pathway is well accepted. NO protects against the development of atherosclerosis (30), and selective endothelial dysfunction is believed to be an early and pathogenic event in atherosclerosis (18). Treatment with raloxifene augmented flow-mediated vasodilatation (5, 21, 23, 24) in menopausal women probably through increasing NO production (21). Raloxifene acutely relaxed rabbit coronary arteries (8) and improved coronary perfusion in the ischemic rat heart (15) by involving NO, and it also triggered nontranscriptional signaling pathways, leading to stimulation of endothelial NO synthase in human endothelial cells (26). However, our study did not show involvement of the endothelium because the relaxant effects of raloxifene or 17-estradiol were identical in rings with and without endothelia. Inhibition of NO synthase by L-NAME did not influence relaxation. Furthermore, relaxation responses to raloxifene remained unchanged after treatment with CTX plus apamin or with indomethacin. Similarly, the endothelium did not contribute to estrogen- or raloxifene-mediated cerebrovascular relaxation (22, 28). Estrogen failed to affect NO release and NO synthase activity in nonpregnant ewe renal arteries (31). Last, acute treatment with raloxifene did not modify endothelial NO-dependent renal artery relaxation responses to acetylcholine (data not shown). Although chronic raloxifene treatment upregulated the NO function (21, 33), the present results suggest that endothelium-derived relaxing factors do not contribute to the acute relaxing responses to raloxifene, at least in rat renal arteries.

Sex differences in blood vessel tone described in humans or experimental animals are likely caused by direct vascular effects of sex hormones. The present results show that there was a slight sex difference in the relaxing effects of raloxifene in rings contracted by a receptor-dependent constrictor (U-46619) but not by a receptor-independent constrictor (high K⁺). This small discrepancy may be caused by a slight difference in initial tension induced by U-46619 between male (5.9 ± 0.53 mN) and female (4.5 ± 0.64 mN) rings. In the same preparations, 17-estradiol induced similar relaxation in the rings from both sexes. Similarly, in rabbit coronary arteries, the relaxation response to estrogen (13) or raloxifene (9) showed no sex difference.

Nongenomic effects of raloxifene may be mediated through activation of estrogen receptors. The selective estrogen-receptor antagonist ICI-182, 780 did not antagonize raloxifene-induced relaxation or raloxifene-induced inhibition of contraction and the VSM [Ca²⁺]_i increase in rings from either sex exposed to CaCl₂. In contrast, this blocker attenuated endothelium-dependent coronary artery relaxation to raloxifene (8) without affecting the endothelium-independent effect (8, 22, 28). ICI-182, 780 also inhibited raloxifene-stimulated NO formation in human endothelial cells (26). Similarly, ICI-182, 780 antagonized only the endothelium-dependent relaxation to tamoxifen, another SERM (9). Taken together, it appears that the raloxifene-induced endothelium-mediated effect involves ICI-182, 780-sensitive estrogen receptors, whereas the direct muscle-relaxing action is probably unrelated to classic estrogen receptor stimulation.

In conclusion, we have provided experimental evidence to show a principal mechanism by which raloxifene relaxes the rat intralobar renal arteries. Both raloxifene and 17-estradiol relax renal arteries through inhibiting Ca²⁺ entry mechanisms, probably via L-type Ca²⁺ channels. This effect is independent of a functional endothelium or ICI-182, 780-sensitive estrogen receptors, and it is equivalent in both sexes. Raloxifene induced significantly greater renovascular relaxation than 17-estradiol at submaximal concentrations. Similar observations were also made in rabbit coronary arteries (8). However, chronic and in vivo genomic effects of raloxifene may differ. Raloxifene is clinically used to treat menopausal women, but the present data show that raloxifene is equally effective in causing renovascular relaxation in both sexes. However, chronic and genomic action of raloxifene on renovasculature may differ. Long-term oral intake of 60 mg/day of raloxifene in women is expected to result in a mean maximal plasma concentration of 1.36 µg/l raloxifene (2.7 nM) (14). In the present study, the minimal concentration of raloxifene needed to cause vasorelaxation is >10 nM. The efficacy of raloxifene-induced action would be reduced in in vitro studies that exclude the involvement of circulating hormones and dilator factors. Therefore, renal effects of raloxifene deserve further study in vivo where interactions with other circulating factors or its genomic effects may enhance its vascular action.

	GRANTS

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This work is supported by grants from the Research Grants Council of Hong Kong (CUHK 4366/03M and 4362/04M). F. P. Leung is a postdoctoral fellow supported by these grants.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y. Huang, Dept. of Physiology, Faculty of Medicine, Chinese Univ. of Hong Kong, Hong Kong, China (e-mail: yu-huang{at}cuhk.edu.hk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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