点击显示 收起
【摘要】 Endothelin-1 (ET-1) is increased in rats on a high-salt (HS) diet and participates in salt-dependent hypertension. Afferent arterioles (AA) are important for long-term blood pressure control, and therefore we hypothesized that a HS diet would alter their responsiveness to ET-1. Sprague-Dawley rats were fed either a normal-salt (NS; 0.66% NaCl) or HS (8%) diet for 1 wk. Diameters of AA were determined in response to increasing concentrations of big ET-1, ET-1, sarafotoxin 6c (S6c), or norepinephrine (NE), using the blood-perfused juxtamedullary nephron technique. ET-1 responses were also determined during blockade of endothelin type A (ET A ) or type B (ET B ) receptors with 10 nM ABT-627 or 30 nM A-192621, respectively. Expression of ET A and ET B receptors was determined in renal microvessels. Responses of AA to big ET-1, ET-1, and S6c were significantly attenuated during a HS diet (e.g., response to 10 -10 M ET-1 in NS vs. HS rats: -52.5 ± 10.2 vs. +5.6 ± 11.3% of control diameter; P < 0.05), with no change in the responses to NE. ET B, but not ET A receptor blockade abolished the different response to ET-1 between the NS and HS groups. ET B receptor expression in renal microvessels was increased in response to HS (17.7 ± 2.4 vs. 6.6 ± 3.0% of -actin, P = 0.02), whereas ET A receptor expression was unchanged. These results suggest that the reduced vasoconstrictor response of AA to endothelin peptides during a HS diet is mediated by increased vasodilatory function of endothelial ET B receptors. By preserving renal blood flow, this may be an important mechanism to restore sodium balance during a HS diet.
【关键词】 endothelin sodium renal circulation hypertension
ENDOTHELIN -1 (ET-1), ONE OF the most potent vasoconstrictors in humans known today, is produced by the vascular endothelium and acts on two types of receptors. The endothelin type A (ET A ) receptor is located on smooth muscle cells and mediates long-lasting vasoconstriction ( 17, 37 ). Some endothelin type B (ET B ) receptors are also located on smooth muscle cells, contributing to ET-1-induced vasoconstriction ( 17, 37 ). ET B receptors are also present on endothelial cells, which limits vasoconstrictor activity by the release of nitric oxide and by clearing ET-1 from the circulation ( 10, 39 ). Since its discovery, involvement of the endothelin system has been documented in a broad spectrum of cardiovascular conditions, such as hypertension, heart failure, renal failure, and pulmonary hypertension.
More recently, there has been considerable interest in the role of endothelin in the regulation of kidney function. Interestingly, the intrarenal endothelin system is an important mediator of natriuresis, which contrasts with the overall prohypertensive effects of ET-1 in the vasculature. Inner medullary collecting duct (IMCD) cells produce high levels of ET-1 ( 24 ), which act locally on ET B receptors to inhibit tubular sodium transport and promote natriuresis ( 25, 30 ). Pharmacological blockade of ET B receptors, or disruption of the ET B receptor gene, produces a salt-sensitive phenotype ( 11, 33 ). Recent data show that tissue-specific knockout of the ET-1 gene in IMCD cells results in salt-sensitive hypertension in mice ( 1 ). Furthermore, medullary ET B receptor expression is upregulated in DOCA-salt hypertension ( 31 ), and the ET B receptor mediates an increased medullary blood flow response to big ET-1 in rats fed a high-salt (HS) diet ( 38 ). These data indicate an important role for this intrarenal signaling pathway in adjusting sodium excretion to match variations in dietary salt intake.
In contrast to the effects of ET-1 in the renal medulla, the regulation of the endothelin system in the renal vasculature by dietary sodium has not been examined. Afferent arterioles are highly specialized vessels, with profound impact on renal vascular resistance and renal blood flow. Furthermore, increases in afferent arteriolar resistance can lead to the development of arterial hypertension ( 14 ). ET-1 is a powerful vasoconstrictor of afferent arterioles ( 9, 23 ). Interestingly, it has been demonstrated that ET-1-mediated vasoconstriction of mesenteric arteries is enhanced in rats fed a HS diet ( 4 ). Because of the importance of renal vascular tone for renal function and long-term blood pressure control, we examined the effects of a HS diet on the response of afferent arterioles to endothelin peptides. In addition to these functional studies, we also determined whether a HS diet influenced the expression of ET A and ET B receptors in renal preglomerular microvessels.
METHODS
All experiments were performed with approval from the Institutional Animal Care and Use Committee at the Medical College of Georgia. Male Sprague-Dawley rats (275-300 g body wt, Charles River Laboratories, Wilmington, MA) were divided into two groups. One group received a normal-salt (NS) diet (0.66% NaCl, Harlan Laboratories, Indianapolis, IN), while the other group received a HS diet (8% NaCl). Tap water was given ad libidum. After 1 wk, videomicroscopic experiments were performed using the juxtamedullary nephron technique and preglomerular microvessels were isolated for immunoblot analyses.
Measurement of blood pressure. Systolic blood pressure (SBP) was measured in conscious rats by tail-cuff plethysmography (IITC, Woodland Hills, CA) before and after 1 wk of either an NS or HS diet ( 31 ). Blood pressure was measured between 9 AM and 12 AM. SBP was averaged from three readings.
In vitro blood-perfused juxtamedullary nephron experiments. Videomicroscopic experiments were conducted in vitro using the blood-perfused juxtamedullary nephron technique, as previously described ( 21, 23 ). For each experiment, two male Sprague-Dawley rats (325-375 g at the time of the experiment), both from either the NS or HS group, were anesthetized with pentobarbital sodium (40 mg/kg ip) and prepared for videomicroscopic experiments. Perfusate blood was collected and prepared as previously described ( 21, 22 ). Briefly, blood was collected from the nephrectomized blood donor rat into a heparinized (500 U/10 ml) syringe. The plasma and erythrocyte fractions were separated and the leukocyte fraction was discarded. Plasma was filtered (0.2 µm) and combined with the erythrocytes to yield a hematocrit of 33%. The reconstituted blood was filtered through a 5-µm nylon mesh.
The right renal artery of the kidney donor was cannulated via the superior mesenteric artery and perfused with a Tyrode buffer solution containing 60 g/l bovine serum albumin (Sigma, St. Louis, MO) and a complement of L -amino acids, as described ( 21, 22 ). The rat was exsanguinated into a heparinized syringe (500 U/10 ml) via a carotid artery cannula and processed with blood collected from the blood donor rat. The perfused kidney was removed and sectioned along the longitudinal axis, leaving the intact papilla on the dorsal two-thirds portion of the kidney ( 6 ). The papilla was reflected, and the pelvic mucosa was removed to expose the renal tubules, glomeruli, and microvasculature of juxtamedullary nephrons. A portion of the renal microvasculature on the inner cortical surface was isolated by tying off branches of the renal artery.
After completion of the dissection, the Tyrode perfusate was replaced with the reconstituted blood. The blood perfusate was stirred continuously in a closed reservoir while being oxygenated with a 95% O 2 -5% CO 2 gas mixture. Perfusion pressure was monitored by a pressure cannula located within the double-barreled perfusion cannula and connected to a pressure transducer (model TRN005, Kent Scientific) linked to a chart recorder (model 201, Cole Parmer). Perfusion pressure was fixed at 100 mmHg. The inner cortical surface of the kidney was superfused with warmed (37°C) Tyrode buffer containing 10 g/l bovine serum albumin.
The perfusion chamber containing the kidney was mounted to the stage of a Nikon Eclipse E600FN microscope (Nikon, Tokyo, Japan) equipped with a Nikon water-immersion objective ( x 40). The tissue was transilluminated and viewed with a high-resolution camera (model VE1000-STI, Dage-MTI). The video image was enhanced using an image processor (MFJ-1420, MFJ Enterprises) and displayed on a video monitor while being simultaneously recorded on videotape for later analysis. Inner diameters of afferent arterioles were measured at a single site using a calibrated image-shearing monitor (model 908, Vista Electronics, Valencia, CA).
Experimental protocols. Each protocol began with a 5-min control period to ensure a stable vessel diameter and was followed by either exposure to endothelin peptides or agonists, or by exposure to NE. Due to the long washout time for endothelin peptides, only one concentration-response curve was obtained in each kidney. Afferent arteriolar responses were determined to increasing agonist concentrations ranging from 1 pM to 10 nM of ET-1, 1 pM to 100 nM of big ET-1, the immediate precursor peptide of ET-1, and from 1 pM to 10 nM of the ET B receptor-selective agonist sarafotoxin 6c (S6c) to test the function of ET B receptors only. We also tested ET B receptor function using a second approach by repeating the responses to ET-1 in the presence of the ET A receptor antagonist ABT-627 (10 nM). To test the functional response of ET A receptors only, and because there are no selective ET A receptor agonists available, ET-1 responses were also determined in the presence of the ET B receptor blocker, A-192621 (30 nM). All peptides were purchased from American Peptide (Sunnyvale, CA). Endothelin antagonists were kindly provided by Abbott Laboratories (North Chicago, IL).
Increasing concentrations of NE ranging from 10 nM to 1 µM were used to examine whether any alterations of the afferent arteriolar response during HS are specific to the endothelin system. Measurements of arteriolar diameters were made at 12-s intervals, and the sustained diameter was calculated from the average of measurements made during the final 2 min of each treatment period. Each protocol consisted of six to nine 5-min periods.
Isolation of renal microvessels. Renal microvessels were isolated according to a method described previously ( 40 ). Briefly, the kidneys were perfused with a physiological salt solution containing 1% Evans blue, and the renal microvessels were separated from the rest of the cortex with the aid of sequential sieving, a digestion period, and collection under a stereomicroscope. Renal microvessels were collected, quickly frozen in liquid nitrogen, and kept frozen (-80°C) until use in Western blot analysis.
Immunoblot analysis of ET A and ET B receptor protein. Homogenates of renal microvessels were separated by electrophoresis on a 10% stacking Tris-glycine gel, and proteins were transferred electrophoretically to a nitrocellulose membrane. The primary antibodies used were rabbit anti-rat ET A receptor polyclonal antibody (1:1,000; Fitzgerald Industries International, Concord, MA) and rabbit anti-rat ET B receptor polyclonal antibody (1:200; Alomone Labs, Jerusalem, Israel). The blots were washed in phosphate-buffered saline-0.1% Tween 20 and incubated with the secondary antibody (goat anti-rabbit 1:5,000 for the ET A, 1:15,000 for the ET B receptor) conjugated to horseradish peroxidase for 1 h at room temperature and washed. Detection was accomplished by enhanced chemiluminescence Western blotting (ECL, Amersham Biosciences, Pittsburgh, PA), and blots were exposed to X-ray film (Hyperfilm-ECL, Amersham Biosciences). Band intensity was measured densitometrically, and the values were factored for -actin (Sigma).
Statistics. All data are presented as means ± SE. The effect of HS vs. NS treatment and the effect of the vasoactive agents on afferent arteriolar diameters were analyzed by two-way ANOVA. Subsequent Bonferroni analysis was performed to compare afferent arteriolar diameters between the two treatment groups at single concentrations of the various vasoactive agents. An unpaired two-tailed t -test was applied to compare the ET A and ET B receptor protein levels determined by densitometry. A value of P < 0.05 was considered statistically significant.
RESULTS
SBP was similar after 1 wk on either NS or HS diet and averaged 111 ± 5 and 110 ± 5 mmHg, respectively. In addition, baseline afferent arteriolar diameters were similar between the NS and HS groups before any of the subsequent protocols were performed ( Table 1 ).
Table 1. Baseline afferent arteriolar diameters in rats treated with NS and HS diets for 1 wk
Afferent arteriolar response to big ET-1 during NS and HS. In the NS group, big ET-1 produced vasoconstriction at a concentration of 10 -9 M and higher ( P = 0.02 for 10 -9 M compared with baseline; Fig. 1 ). At the highest concentration, 10 -7 M, a contraction of -46.3 ± 4.5% of baseline vessel diameter was achieved ( P = 0.002). In contrast, in the HS group, big ET-1 caused vasodilation from the lowest concentration of 10 -12 M ( P = 0.01 compared with baseline) to a concentration of 10 -10 M ( P = 0.03). The afferent arteriolar response reverted to vasoconstriction from 10 -9 to 10 -8 M [both not significant (NS) compared with baseline]. At the highest concentration of 10 -7 M, a contraction of -47.7 ± 12.6% of baseline diameter was achieved ( P = 0.01). The vascular responses to big ET-1 were significantly different between the NS and HS groups ( P = 0.006). Thus the HS diet resulted in a rightward shift of the concentration-response relationship.
Fig. 1. Response of afferent arterioles from rats on a normal-salt (NS; n = 4) and high-salt (HS; n = 6) diets, expressed as a percentage of control diameter, to increasing concentrations of big endothelin-1 (ET-1), the precursor peptide of ET-1. *Significant change in diameter compared with the control diameter ( P < 0.05).
Afferent arteriolar response to ET-1 during NS and HS. In vessels from rats on a NS diet, ET-1 produced afferent arteriolar vasoconstriction at concentrations of 10 -11 M and higher ( P = 0.05 for 10 -11 M compared with baseline; Fig. 2 ), resulting in a more potent effect compared with big ET-1. At the highest applied concentration of 10 -8 M, ET-1 reduced diameter by -80.5 ± 0.6% compared with baseline diameter ( P < 0.0001 compared with baseline). In the HS group, ET-1 caused vasodilation at the lower concentrations of 10 -12 to 10 -11 M (both P = 0.05). The vascular response reverted from vasodilation to vasoconstriction at the subsequent concentrations from 10 -10 to 10 -9 M (NS compared with baseline). The highest concentration of 10 -8 M reduced diameter by -68.5 ± 11.1% compared with baseline diameter ( P = 0.004). The afferent arteriolar responses to ET-1 were significantly different between the two groups, resulting in a rightward shift in the concentration-response relationship for vessels from rats treated with a HS diet ( P < 0.0001).
Fig. 2. Response of afferent arterioles from rats on NS ( n = 6) and HS ( n = 6) diets, expressed as percentage of control diameter, to increasing concentrations of ET-1. *Significant change in diameter compared with the control diameter ( P < 0.05). #Significant difference between NS and HS groups by Bonferroni post hoc analysis ( P < 0.05).
Afferent arteriolar response to S6c during NS and HS. In vessels from animals on a NS diet, S6c constricted afferent arterioles at concentrations of 10 -10 M and higher ( P = 0.05 for 10 -10 M compared with baseline; Fig. 3 ). At the maximum concentration of 10 -8 M, S6c reduced diameter by -76.2 ± 0.7% compared with baseline diameter ( P < 0.0001). In the vessels from animals on a HS diet, the lowest concentration of S6c, 10 -12 M, caused vasodilation; vessel diameter increased by +14.4 ± 7.1% compared with baseline ( P = 0.03). At the next higher concentration of 10 -11 M, the afferent arteriolar response reverted to vasoconstriction, as evidenced by a return of afferent arteriolar diameter to control. Subsequently, higher concentrations of S6c caused further vasoconstriction, with a maximum reduction in diameter of -62.4 ± 7.1% at a concentration of 10 -8 M ( P < 0.001). Thus S6c produced significantly less vasoconstriction in the HS vs. the NS group ( P = 0.0003).
Fig. 3. Response of afferent arterioles from rats on NS ( n = 6) and HS ( n = 6) diets, expressed as percentage of control diameter, to increasing concentrations of sarafotoxin 6c (S6c), an ET B receptor-specific agonist. *Significant change in diameter compared with the control diameter ( P < 0.05). #Significant difference between NS and HS groups by Bonferroni post hoc analysis ( P < 0.05).
Afferent arteriolar response to NE during NS and HS. In the NS group, NE caused a concentration-dependent vasoconstriction ( Fig. 4 ). The highest concentration of 10 -6 M reduced diameter by -71.8 ± 7.9% ( P < 0.001). Similarly, in vessels from animals fed a HS diet, NE produced a similar concentration-dependent reduction in diameter that was no different from responses observed in vessels from the NS group. The highest concentration of 10 -6 M reduced diameter by -73.7 ± 1.5% compared with baseline ( P < 0.001).
Fig. 4. Response of afferent arterioles from rats on NS ( n = 6) and HS ( n = 6) diets, expressed as percentage of control diameter, to increasing concentrations of NE. *Significant change in diameter compared with the control diameter ( P < 0.05).
Afferent arteriolar response to ET-1 during ET B receptor blockade. Blockade of ET B receptors had no effect on afferent arteriolar diameters in vessels from rats on a NS diet (16.7 ± 1.8 µm before vs. 16.5 ± 1.6 µm during ET B receptor blockade; NS). Similarly, ET B receptor blockade had no effect on baseline diameter in vessels from rats on a HS diet (17.5 ± 1.1 µm before vs. 17.2 ± 1.2 µm during ET B receptor blockade; NS). During ET B receptor blockade, vasoconstriction to lower concentrations of ET-1 was abolished and higher concentrations of ET-1 were needed to induce a significant vasoconstriction in vessels from rats on both NS and HS diets (both P = 0.01 for 10 -8 M compared with baseline; Fig. 5 ). There was no difference in the ET-1 response between the NS and the HS groups during ET B receptor blockade.
Fig. 5. Response of afferent arterioles from rats on NS ( n = 5) and HS ( n = 4) diets, expressed as percentage of control diameter, to increasing concentrations of ET-1 during concomitant blockade of ET B receptors with 30 nM A-192621. *Significant change in diameter compared with the control diameter ( P < 0.05).
Afferent arteriolar response to ET-1 during ET A receptor blockade. Blockade of ET A receptors had no effect on afferent arteriolar diameters in vessels from rats on a HS diet (14.5 ± 0.9 µm before vs. 14.0 ± 0.7 µm during ET A receptor blockade; NS). In the vessels from rats fed a HS diet, concentrations of ET-1 from 10 -12 to 10 -11 M led to significant vasodilation ( Fig. 6; P = 0.02 and P = 0.04, respectively, compared with baseline). At a concentration of 10 -10 M, the vascular response reverted to vasoconstriction (NS compared with baseline). The highest concentration of 10 -8 M reduced diameter by -74.2 ± 2.4% compared with baseline diameter ( P < 0.001). The initial vasodilation in vessels from rats treated with a HS diet is in contrast to what we observed previously in vessels from rats on a NS diet, where an initial vasodilatory response was not detectable ( 23 ).
Fig. 6. Response of afferent arterioles from rats on a HS ( n = 6) diet, expressed as percentage of control diameter, to increasing concentrations of ET-1 during concomitant blockade of ET A receptors with 10 nM ABT-627. *Significant change in diameter compared with the control diameter ( P < 0.05).
Expression of ET A and ET B receptors in renal microvessels during a NS and HS diet. To determine whether changes in receptor expression could account for the functional differences we observed, preglomerular microvessels were isolated from animals on NS and HS diets. Expression of ET A receptors in preglomerular microvessels was not different between rats treated with a HS diet or a NS diet (41.0 ± 4.8 vs. 32.4 ± 9.9% of -actin control; NS) ( Fig. 7 ). In contrast, expression of ET B receptors was increased in microvessels from rats treated with a HS diet compared with vessels from rats on a NS diet (17.7 ± 2.4 vs. 6.6 ± 3.0% of -actin control; P = 0.02).
Fig. 7. Expression of ET A and ET B receptors in preglomerular microvessels during NS ( n = 4) and HS diets ( n = 6) determined by densitometry ( top ) with respective immunoblots ( bottom ).
DISCUSSION
The main finding of the current study is that a HS diet leads to a rightward shift in the concentration-response relationship of endothelin peptides in afferent arterioles. In fact, an initial vasodilation was observed in arterioles from rats on a HS diet, but not a NS diet. These results are consistent with an enhancement of ET B receptor-mediated, endothelium-dependent vasodilation during HS. ET B receptor blockade, but not ET A receptor blockade, abrogated the vasodilatory response to ET-1 during a HS diet. In line with these functional changes, we observed an upregulation of ET B receptor protein in renal microvessels during HS. These results provide strong evidence for increased endothelial ET B receptor function in the renal microcirculation of rats on a HS diet.
Because of the importance of afferent arteriolar tone for renal function and long-term blood pressure regulation, we examined the effects of a HS diet on the response of afferent arterioles to endothelin peptides. Renal vasoconstriction is a very powerful mechanism for inducing arterial hypertension ( 14 ). Renal vascular tone, as with any other vascular bed, depends on the balance between vasoconstrictor and vasodilatory factors. Maintaining this balance in afferent arterioles, in particular, during a HS diet, might be an important mechanism guarding against the development of arterial hypertension. An enhancement of endothelial ET B receptor-mediated afferent arteriolar vasodilation may serve to restore sodium balance by increasing renal blood flow and sodium excretion during a HS diet.
We have recently documented that ET-1 is a very potent constrictor of afferent arterioles ( 23 ). In fact, the renal vasculature seems to be more sensitive to the vasoconstrictor effects of ET-1 than the systemic circulation ( 34 ). A HS diet has been shown to increase vascular expression of ET-1, although this is not a universal finding ( 20, 26, 29 ). We have previously observed that rats fed a HS diet for 2 wk had an increase in 250% ( 36 ). Because urinary endothelin derives exclusively from the kidney ( 5 ), this is consistent with increased renal production of ET-1 during a HS intake. The exact intrarenal locations where enhanced ET-1 production occurs during a HS diet remain to be clarified. However, increased local production of ET-1 in afferent arterioles would lead to vasoconstriction, if responsiveness of these vessels during a HS diet would go unchanged. In the superior mesenteric artery, constrictor responses to ET-1 are even enhanced by a high-salt intake ( 4 ), which would lead to further increases in vascular resistance in the face of enhanced ET-1 generation.
In the current study, we also investigated the effect of big ET-1 on afferent arterioles. Big ET-1 is a 38-amino acid precursor peptide, locally converted by endothelin-converting enzymes (ECE) to ET-1 (21 amino acids). Our data demonstrate that big ET-1 has less vasoconstrictor activity than equimolar concentrations of ET-1 in afferent arterioles. This may help to explain previously published in vivo data showing that a bolus injection of big ET-1 into Sprague-Dawley rats leads to a natriuretic response compared with an antinatriuretic response of a similar concentration of ET-1 ( 19 ). Local vascular ECE activity might be a rate-limiting factor for the vasoconstrictor action of big ET-1, but our study was not specifically designed to examine this in detail. The current results are also consistent with previous work suggesting that the renal circulation has relatively less ECE activity compared with the extrarenal circulation ( 32 ).
In afferent arterioles from rats on a HS diet, lower concentrations of big ET-1 led to vasodilation as opposed to vasoconstriction seen in kidneys from rats on a NS diet. With ET-1, an initial vasodilatory response was observed under HS conditions, whereas ET-1 caused strong vasoconstriction at concentrations of 10 -11 M and above in vessels from rats on a NS diet. Because vasodilation to big ET-1 and ET-1 can only be mediated by the endothelial ET B receptor, we further substantiated that observation by application of the ET B receptor-specific agonist S6c. S6c induced an initial vasodilation instead of a vasoconstrictor response in vessels from rats fed a HS diet. Blockade of ET B receptors, but not ET A receptors, abrogated the initial vasodilation to ET-1 during HS, confirming that enhanced ET B receptor function is responsible for this effect. Because only ET B receptors on endothelial cells have vasodilatory actions, these results demonstrate that endothelial ET B receptor function is enhanced during a HS diet. In addition to the initial vasodilation observed in response to big ET-1, ET-1, and S6c, vasoconstrictor responses at higher doses were also attenuated in the arterioles from rats on a HS diet. This can be due either to pronounced buffering of smooth muscle ET A /ET B receptor-mediated vasoconstriction by enhanced endothelial ET B receptor-mediated vasodilation, a reduction of smooth muscle ET A /ET B receptor-mediated vasoconstriction, or a combination of both. The fact that during ET B receptor blockade with 30 nM A-192621, which blocks both endothelial and smooth muscle ET B receptors, vasoconstriction to higher levels of endothelin peptides was similar between the NS and HS groups, indicates that ET A receptor function is probably not affected by a HS diet. In addition, ET A receptor expression was similar in vessels from rats on a NS and a HS diet.
ET B receptor expression was found to be upregulated in renal microvessels from rats on a HS diet. This suggests that increased endothelial ET B receptor protein expression may account for the enhanced functional response of endothelial ET B receptor activation in vessels from rats on a HS diet. However, our vessel preparation does not permit localization of the increased ET B receptor expression, but the functional data suggests that this occurs in the endothelium. Future studies in mice with specific knockout of endothelial ET B receptors may be able to provide a definitive answer to this question. It should be noted that in terms of regulation of ET B receptors, mice overexpressing ET-1 in the endothelium also demonstrate increased expression of ET B receptors ( 2 ). Other studies indicate, however, that the ET B receptor is downregulated when exposed to increased concentrations of ET-1 ( 3, 35 ). Therefore, the signaling pathways regulating the expression of ET B receptors under various conditions require further exploration.
To examine whether the rightward shifts in the response to endothelin peptides are an effect specific to the endothelin system, or a generalized attenuation of afferent arteriolar responsiveness to vasoconstrictors, we also examined the effect of a HS diet on the response to NE. No alteration in the vascular response to NE was detected, indicating that a HS diet selectively affects the endothelin system.
When our results are compared with the available literature, it is important to note that most studies on the effects of a HS diet on the endothelin system have been performed using the DOCA-salt model. The mineralocorticoid hypertension of the DOCA-salt model is likely to influence vascular responses to endothelin peptides differently from a HS diet alone. In addition to the hypertension, endothelial dysfunction has been documented as a hallmark feature of the DOCA-salt model ( 16 ), but it is not present in animals treated with a HS diet only ( 41 ). Furthermore, data generated in DOCA-salt rats appear to be contradictory. For example, isolated, perfused kidneys of DOCA-salt rats respond to an ET B receptor agonist with vasoconstriction and less release of nitric oxide compared with vasodilation in normotensive control rats ( 18 ), indicating reduced function of the endothelial ET B receptor. In whole-animal studies, however, we and others observed a more pronounced increase in blood pressure during ET B receptor blockade in DOCA-salt rats compared with control rats ( 15, 31 ). This would indicate enhanced endothelial ET B receptor function. Published data on the response of mesenteric arteries to ET-1 in the DOCA-salt model are also conflicting. Some authors report enhanced vasoconstrictor responses to ET-1 during DOCA-salt treatment ( 7 ), and others observe an attenuated response ( 8, 28 ). Similarly, in coronary vessels, some groups have found endothelin-induced contractions to be reduced in the DOCA-salt model ( 13 ), while others found the opposite ( 27 ). Although we lack any obvious explanation for these discrepancies, use of different rat strains may partly account for the observed differential responses.
Few studies have investigated the effect of a HS diet only, without DOCA application. Ballew et al. ( 4 ) showed an increased response to ET-1 in superior mesenteric arteries in rats treated with HS only. This is in contrast to our data, but the effects of a HS diet on vascular responses to endothelin peptides might also differ between vascular beds. Our results of enhanced ET B receptor function, however, are in accordance with data from Giardina et al. ( 12 ), who found enhanced endothelium-dependent vasodilation via ET B receptors in aortas of Sprague-Dawley rats on a HS diet.
In a previous report, we noted that ET B receptor-mediated increases in renal medullary blood flow are enhanced during a HS diet ( 38 ). Our current results indicate that vasoconstrictor responses to endothelin peptides are attenuated in afferent arterioles from rats on a HS diet and even turned into vasodilatory responses at lower peptide levels, mediated by increased endothelial ET B receptor function. Taken together, these data suggest that enhanced renal ET B receptor function may be an important antihypertensive mechanism during a HS diet, restoring sodium balance by vasodilatory effects on the afferent arteriole and by increasing medullary blood flow. In line with this notion, pharmacological blockade of ET B receptors or disruption of the ET B receptor gene has been shown to produce salt-sensitive hypertension ( 11, 33 ). Thus it should be determined in future studies whether ET B receptor function is impaired in salt-sensitive models of hypertension, such as in the DOCA model or in the Dahl salt-sensitive rat.
GRANTS
This study was supported by the Deutsche Forschungsgemeinschaft (SCHN 769/1-1 to M. P. Schneider) and by the National Institutes of Health Grants HL-74167 (E. W. Inscho and D. M. Pollock), HL-64776 (D. M. Pollock), and DK-44628 (E. W. Inscho).
ACKNOWLEDGMENTS
We gratefully acknowledge the expert technical assistance of Anthony Cook and Shali Zhang. ABT-627 and A-192621 were kindly provided by Abbott Laboratories (North Chicago, IL).
【参考文献】
Ahn D, Ge Y, Stricklett PK, Gill P, Taylor D, Hughes AK, Yanagisawa M, Miller L, Nelson RD, Kohan DE. Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest 114: 504-511, 2004.
Amiri F, Virdis A, Neves MF, Iglarz M, Seidah NG, Touyz RM, Reudelhuber TL, Schiffrin EL. Endothelium-restricted overexpression of human endothelin-1 causes vascular remodeling and endothelial dysfunction. Circulation 110: 2233-2240, 2004.
Asada S, Kasuya Y, Sakurai T, Masaki T, Goto K. Endothelin-1-induced downregulation of ETB receptor mRNA: participation of cAMP. J Cardiovasc Pharmacol 26, Suppl 3: S272-S275, 1995.
Ballew JR, Watts SW, Fink GD. Effects of salt intake and angiotensin II on vascular reactivity to endothelin-1. J Pharmacol Exp Ther 296: 345-350, 2001.
Benigni A, Perico N, Gaspari F, Zoja C, Bellizzi L, Gabanelli M, Remuzzi G. Increased renal endothelin production in rats with reduced renal mass. Am J Physiol Renal Fluid Electrolyte Physiol 260: F331-F339, 1991.
Casellas D, Navar LG. In vitro perfusion of juxtamedullary nephrons in rats. Am J Physiol Renal Fluid Electrolyte Physiol 246: F349-F358, 1984.
De Carvalho MH, Nigro D, Scivoletto R, Barbeiro HV, de Oliveira MA, de Nucci G, Fortes ZB. Comparison of the effect of endothelin on microvessels and macrovessels in Goldblatt II and deoxycorticosterone acetate-salt hypertensive rats. Hypertension 15: I68-171, 1990.
Deng LY, Schiffrin EL. Effects of endothelin on resistance arteries of DOCA-salt hypertensive rats. Am J Physiol Heart Circ Physiol 262: H1782-H1787, 1992.
Edwards RM, Trizna W, Ohlstein EH. Renal microvascular effects of endothelin. Am J Physiol Renal Fluid Electrolyte Physiol 259: F217-F221, 1990.
Fukuroda T, Fujikawa T, Ozaki S, Ishikawa K, Yano M, Nishikibe M. Clearance of circulating endothelin-1 by ETB receptors in rats. Biochem Biophys Res Commun 199: 1461-1465, 1994.
Gariepy CE, Ohuchi T, Williams SC, Richardson JA, Yanagisawa M. Salt-sensitive hypertension in endothelin-B receptor-deficient rats. J Clin Invest 105: 925-933, 2000.
Giardina JB, Green GM, Rinewalt AN, Granger JP, Khalil RA. Role of endothelin B receptors in enhancing endothelium-dependent nitric oxide-mediated vascular relaxation during high salt diet. Hypertension 37: 516-523, 2001.
Giulumian AD, Pollock DM, Clarke N, Fuchs LC. Coronary vascular reactivity is improved by endothelin A receptor blockade in DOCA-salt hypertensive rats. Am J Physiol Regul Integr Comp Physiol 274: R1613-R1618, 1998.
Hall JE, Guyton AC, Brands MW. Pressure-volume regulation in hypertension. Kidney Int Suppl 55: S35-S41, 1996.
Hashimoto N, Kuro T, Fujita K, Azuma S, Matsumura Y. Endothelin ETB receptor-mediated action on systemic and renal hemodynamics and urine formation in deoxycorticosterone acetate-salt-induced hypertensive rats. Biol Pharm Bull 21: 800-804, 1998.
Hayakawa H, Hirata Y, Suzuki E, Kimura K, Kikuchi K, Nagano T, Hirobe M, Omata M. Long-term administration of L -arginine improves nitric oxide release from kidney in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 23: 752-756, 1994.
Haynes WG, Strachan FE, Webb DJ. Endothelin ETA and ETB receptors cause vasoconstriction of human resistance and capacitance vessels in vivo. Circulation 92: 357-363, 1995.
Hirata Y, Hayakawa H, Suzuki E, Kimura K, Kikuchi K, Nagano T, Hirobe M, Omata M. Direct measurements of endothelium-derived nitric oxide release by stimulation of endothelin receptors in rat kidney and its alteration in salt-induced hypertension. Circulation 91: 1229-1235, 1995.
Hoffman A, Grossman E, Keiser HR. Opposite effects of endothelin-1 and Big-endothelin-(1-39) on renal function in rats. Eur J Pharmacol 182: 603-606, 1990.
Ikeda T, Ohta H, Okada M, Kawai N, Nakao R, Siegl PK, Kobayashi T, Maeda S, Miyauchi T, Nishikibe M. Pathophysiological roles of endothelin-1 in Dahl salt-sensitive hypertension. Hypertension 34: 514-519, 1999.
Inscho EW, Carmines PK, Navar LG. Juxtamedullary afferent arteriolar responses to P1 and P2 purinergic stimulation. Hypertension 17: 1033-1037, 1991.
Inscho EW, Carmines PK, Navar LG. Prostaglandin influences on afferent arteriolar responses to vasoconstrictor agonists. Am J Physiol Renal Fluid Electrolyte Physiol 259: F157-F163, 1990.
Inscho EW, Imig JD, Cook AK, Pollock DM. ETA and ETB receptors differentially modulate afferent and efferent arteriolar responses to endothelin. Br J Pharmacol 146: 1019-1026, 2005.
Kohan DE. Endothelin synthesis by rabbit renal tubule cells. Am J Physiol Renal Fluid Electrolyte Physiol 261: F221-F226, 1991.
Kohan DE, Padilla E, Hughes AK. Endothelin B receptor mediates ET-1 effects on cAMP and PGE 2 accumulation in rat IMCD. Am J Physiol Renal Fluid Electrolyte Physiol 265: F670-F676, 1993.
Lariviere R, Thibault G, Schiffrin EL. Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats. Hypertension 21: 294-300, 1993.
Millette E, de Champlain J, Lamontagne D. Contribution of endogenous endothelin in the enhanced coronary constriction in DOCA-salt hypertensive rats. J Hypertens 21: 115-123, 2003.
Nguyen PV, Parent A, Deng LY, Fluckiger JP, Thibault G, Schiffrin EL. Endothelin vascular receptors and responses in deoxycorticosterone acetate-salt hypertensive rats. Hypertension 19: II98-II104, 1992.
Ohkita M, Wang Y, Nguyen ND, Tsai YH, Williams SC, Wiseman RC, Killen PD, Li S, Yanagisawa M, Gariepy CE. Extrarenal ET B plays a significant role in controlling cardiovascular responses to high dietary sodium in rats. Hypertension 45: 940-946, 2005.
Plato CF, Pollock DM, Garvin JL. Endothelin inhibits thick ascending limb chloride flux via ET B receptor-mediated NO release. Am J Physiol Renal Physiol 279: F326-F333, 2000.
Pollock DM, Allcock GH, Krishnan A, Dayton BD, Pollock JS. Upregulation of endothelin B receptors in kidneys of DOCA-salt hypertensive rats. Am J Physiol Renal Physiol 278: F279-F286, 2000.
Pollock DM, Opgenorth TJ. ET A receptor-mediated responses to endothelin-1 and big endothelin-1 in the rat kidney. Br J Pharmacol 111: 729-732, 1994.
Pollock DM, Pollock JS. Evidence for endothelin involvement in the response to high salt. Am J Physiol Renal Physiol 281: F144-F150, 2001.
Rabelink TJ, Kaasjager KA, Boer P, Stroes EG, Braam B, Koomans HA. Effects of endothelin-1 on renal function in humans: implications for physiology and pathophysiology. Kidney Int 46: 376-381, 1994.
Sakurai T, Morimoto H, Kasuya Y, Takuwa Y, Nakauchi H, Masaki T, Goto K. Level of ETB receptor mRNA is down-regulated by endothelins through decreasing the intracellular stability of mRNA molecules. Biochem Biophys Res Commun 186: 342-347, 1992.
Sasser JM, Pollock JS, Pollock DM. Renal endothelin in chronic angiotensin II hypertension. Am J Physiol Regul Integr Comp Physiol 283: R243-R248, 2002.
Seo B, Oemar BS, Siebenmann R, von Segesser L, Luscher TF. Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels. Circulation 89: 1203-1208, 1994.
Vassileva I, Mountain C, Pollock DM. Functional role of ET B receptors in the renal medulla. Hypertension 41: 1359-1363, 2003.
Verhaar MC, Strachan FE, Newby DE, Cruden NL, Koomans HA, Rabelink TJ, Webb DJ. Endothelin-A receptor antagonist-mediated vasodilatation is attenuated by inhibition of nitric oxide synthesis and by endothelin-B receptor blockade. Circulation 97: 752-756, 1998.
White SM, Imig JD, Kim TT, Hauschild BC, Inscho EW. Calcium signaling pathways utilized by P2X receptors in freshly isolated preglomerular MVSMC. Am J Physiol Renal Physiol 280: F1054-F1061, 2001.
Zhao X, Pollock DM, Zeldin DC, Imig JD. Salt-sensitive hypertension after exposure to angiotensin is associated with inability to upregulate renal epoxygenases. Hypertension 42: 775-780, 2003.
作者单位：1 Vascular Biology Center and Departments of 2 Physiology and 3 Surgery, Medical College of Georgia, Augusta, Georgia