Natriuretic response to renal medullary endothelin B receptor activation is impaired in Dahl-salt sensitive rats on a high-fat diet.

Renal medullary endothelin B receptors (ET(B)) mediate sodium excretion and blood pressure (BP) control. Several animal models of hypertension have impaired renal medullary ET(B) function. We found that 4-week high-caloric diet elevated systolic BP in Dahl salt-sensitive (Dahl S) rats (126+/-2 vs. 143+/-3 mm Hg, p<0.05). We hypothesized that renal medullary ET(B) function is dysfunctional in DS rats fed a high-caloric diet. We compared the diuretic and natriuretic response to intramedullary infusion of ET(B) agonist sarafotoxin 6c (S6c) in DS rats fed either a normal or high-caloric diet for 4 weeks. Urine was collected during intramedullary infusion of saline for baseline collection followed by intramedullary infusion of either saline or S6c. We first examined the ET(B) function in DS rats fed a normal diet. S6c increased urine flow (2.7+/-0.3 microl/min during baseline vs. 5.1+/-0.6 microl/min after S6c; p<0.05; n=5) and sodium excretion (0.28+/-0.05 vs. 0.81+/-0.17 micromol/min; p<0.05), suggesting that DS rats have renal medullary ET(B) function. However, DS rats fed a high-caloric diet displayed a significant increase in urine flow (2.7+/-0.4 vs. 4.2+/-0.4 microl/min, baseline vs. S6c infusion, respectively; p<0.05, n=6), but no significant change in sodium excretion in response to S6c (0.32+/-0.06 vs. 0.45+/-0.10 micromol/min). These data demonstrate that renal medullary ET(B) function is impaired in DS rats fed a high-caloric diet, which may be contributed to the elevation of blood pressure during high-caloric feeding in this model.

ET-1 increases reactive oxygen species following hypoxia and high-salt diet in the mouse glomerulus.

AIM: This study was designed to determine whether ET-1 derived from endothelial cells contributes to oxidative stress in the glomerulus of mice subjected to a high-salt diet and/or hypoxia. METHODS: C57BL6/J control mice or vascular endothelial cell ET-1 knockout (VEET KO) mice were subjected to 3-h exposure to hypoxia (8% O2) and/or 2 weeks of high-salt diet (4% NaCl) prior to metabolic cage assessment of renal function and isolation of glomeruli for the determination of reactive oxygen species (ROS). RESULTS: In control mice, hypoxia significantly increased urinary protein excretion during the initial 24 h, but only in animals on a high-salt diet. Hypoxia increased glomerular ET-1 mRNA expression in control, but not in vascular endothelial cell ET-1 knockout (VEET KO) mice. Under normoxic conditions, mice on a high-salt diet had approx. 150% higher glomerular ET-1 mRNA expression compared with a normal-salt diet (P < 0.05). High-salt diet administration significantly increased glomerular ROS production in flox control, but not in glomeruli isolated from VEET KO mice. In C57BL6/J mice, the ETA receptor-selective antagonist, ABT-627, significantly attenuated the increase in glomerular ROS production produced by high-salt diet. In addition, chronic infusion of C57BL6/J mice with a subpressor dose of ET-1 (osmotic pumps) significantly increased the levels of glomerular ROS that were prevented by ETA antagonist treatment. CONCLUSION: These data suggest that both hypoxia and a high-salt diet increase glomerular ROS production via endothelial-derived ET-1-ETA receptor activation and provide a potential mechanism for ET-1-induced nephropathy.

ET-1 actions in the kidney: evidence for sex differences.

Hypertension and chronic kidney disease are more common in men than in premenopausal women at the same age. In animal models, females are relatively protected against genetic or pharmacological procedures that produce high blood pressure and renal injury. Overactivation or dysfunction of the endothelin (ET) system modulates the progression of hypertension or kidney diseases with the ET(A) receptor primarily mediating vasoconstriction, injury and anti-natriuresis, and ET(B) receptors having opposite effects. The purpose of this review is to examine the role of the ET system in the kidney with a focus on the inequality between the sexes associated with the susceptibility to and progression of hypertension and kidney diseases. In most animal models, males have higher renal ET-1 mRNA expression, greater ET(A) -mediated responses, including renal medullary vasoconstriction, and increased renal injury. These differences are reduced following gonadectomy suggesting a role for sex hormones, mainly testosterone. In contrast, females are relatively protected from high blood pressure and kidney damage via increased ET(B) versus ET(A) receptor function. Furthermore, ET(A) receptors may have a favourable effect on sodium excretion and reducing renal damage in females. In human studies, the genetic polymorphisms of the ET system are more associated with hypertension and renal injury in women. However, the knowledge of sex differences in the efficacy or adverse events of ET(A) antagonists in the treatment of hypertension and kidney disease is poorly described. Increased understanding how the ET system acts differently in the kidneys between sexes, especially with regard to receptor subtype function, could lead to better treatments for hypertension and renal disease. LINKED ARTICLES This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1.

Endothelin receptor A-specific stimulation of glomerular inflammation and injury in a streptozotocin-induced rat model of diabetes.

AIMS/HYPOTHESIS: Activation of endothelin receptor-A (ET(A)) increases glomerular permeability to albumin (P(alb)) and elevates pro-inflammatory markers in hyperglycaemic rats. METHODS: Male Sprague-Dawley rats were given streptozotocin (n = 32) or saline (sham; n = 32). Half of the animals in each group received the ET(A)-selective antagonist, ABT-627 (atrasentan; orally), beginning immediately after hyperglycaemia was confirmed. Glomeruli were isolated by sieving techniques and P(alb) determined from the change in glomerular volume induced by oncotic gradients of albumin. Glomerular nephrin levels were assessed by immunofluorescence, whereas urinary nephrin was measured by immunoassay. RESULTS: At 3 and 6 weeks after streptozotocin injection, proteinuria was significantly increased compared with sham controls and significantly reduced by ABT-627 treatment. P(alb) was also increased at 3 and 6 weeks post-streptozotocin. ABT-627 had no effect on P(alb) or protein excretion in sham control rats. In glomeruli isolated from hyperglycaemic rats, incubation with BQ-123, a selective ET(A) antagonist, reduced P(alb), whereas BQ-788, a selective endothelin receptor-B antagonist had no effect (n = 6 rats per group, 5-8 glomeruli per rat). Glomerular and plasma content of soluble intercellular adhesion molecule-1 and monocyte chemoattractant protein-1 were significantly increased 6 weeks after streptozotocin (ELISA). ABT-627 attenuated these increases. After 6 weeks of hyperglycaemia, glomerular nephrin content was decreased with a concurrent increase in urinary nephrin excretion. ABT-627 prevented glomerular nephrin loss in hyperglycaemic rats (n = 5-8 rats per group; eight groups). CONCLUSIONS/INTERPRETATION: These observations support the hypothesis that endothelin-1, via the ET(A) receptor, directly increases P(alb), possibly via nephrin loss, as well as early inflammation in the hyperglycaemic rat.

Contrasting effects of eplerenone and spironolactone on adrenal cell steroidogenesis.

Spironolactone and eplerenone are widely used as mineralocorticoid antagonists. Spironolactone has several nonspecific actions including inhibition of androgen receptor and steroid hormone biosynthesis. While studies have shown that eplerenone does not exhibit nonspecific actions on androgen receptor, its effects on steroid hormone production have not been reported. Herein, the effects of eplerenone (0.1-30 microM) and spironolactone (0.1-30 microM) on steroid production were examined in human adrenocortical H295R cells. Spironolactone inhibited basal production of cortisol (91%) and aldosterone (53%). Treatment of H295R cells with angiotensin II (Ang II) for 24 h increased aldosterone production by 11-fold. Spironolactone inhibited Ang II stimulation of aldosterone production by 80%. Addition of pregnenolone increased aldosterone (9-fold) and cortisol (3-fold) production. Spironolactone inhibited pregnenolone metabolism to aldosterone (67%) and cortisol (74%). The inhibitory effects of spironolactone occurred at concentrations far higher than those needed to block mineralocorticoid receptor, suggesting an action directly on the enzymes involved in steroid production. In contrast, eplerenone did not inhibit basal, Ang II, forskolin, pregnenolone-stimulated cortisol, or aldosterone production. Together, these data demonstrate that opposed to spironolactone, pharmacologic concentrations of eplerenone do not inhibit adrenal cell aldosterone or cortisol production.

Endothelin(A) (ET(A)) and ET(B) receptor-mediated regulation of nitric oxide synthase 1 (NOS1) and NOS3 isoforms in the renal inner medulla.

AIM: Our laboratory and others have shown that endothelin (ET)-1 directly stimulates nitric oxide (NO) production in inner medullary collecting duct (IMCD) cells. The goal of this study was to determine which NO synthase (NOS) isoforms in IMCD are sensitive to ET-1, and the role of ET(A) and ET(B) receptor activation in vivo and in vitro. METHODS: NOS enzymatic activity and NOS isoform protein expression were examined in cultured IMCD-3 cells and isolated renal inner medulla. ET(B) receptor-deficient homozygous rats (sl/sl) have elevated levels of circulating ET-1 and lack a functional ET(B) signalling pathway in kidneys, and furthermore provides a unique model to study ET(A) receptor signalling in the renal inner medulla in vivo. RESULTS: Incubation of IMCD-3 cells with exogenous ET-1 (50 nm) resulted in ET(A)-dependent increased NOS1 protein expression in IMCD-3 cells with no effect on NOS2 or NOS3 expression. ET(B) receptor antagonism has no effect on NOS expression in IMCD-3 cells. Consistent with in vitro results, cytosolic NOS1 protein expression was significantly greater in the renal inner medulla of sl/sl rats compared with heterozygous (sl/+) controls, with no alteration in NOS3 expression. In contrast to protein expression data, NOS1- and NOS3-specific enzymatic activities decreased in the cytosolic fraction from the renal inner medulla of sl/sl compared with sl/+. CONCLUSION: These results provide evidence that both ET(A) and ET(B) receptors regulate NOS isoform activity in the renal inner medulla and specifically support the hypothesis that ET(A) receptor activation increases NOS1 expression.

Increased reactive oxygen species contributes to kidney injury in mineralocorticoid hypertensive rats.

Hypertension is associated with increased reactive oxygen species (ROS). Renal ROS production and their effects on renal function have never been investigated in mineralocorticoid hypertensive rats. In this study we hypothesized that increased ROS production in kidneys from deoxycorticosterone (DOCA)-salt rats contributes to adverse renal morphological changes and impaired renal function in DOCA-salt hypertensive rats. We also determined whether ROS-induced renal injury was dependent on blood pressure. DOCA-salt hypertensive rats exhibited a marked increase in blood pressure, renal ROS production, glomerular and tubular lesions, and microalbuminuria compared to sham rats. Treatment of DOCA-salt hypertensive rats with apocynin for 28 days resulted in attenuation of systolic blood pressure and improvement of renal morphology. Renal superoxide level in DOCA-salt rats was 215% of sham-operated rats and it was significantly decreased to 140% with apocynin treatment. Urinary protein level was decreased from 27 +/- 3 mg/day in DOCA-salt hypertensive rats to 9 +/- 2 mg/day. 28 days of Vitamin E treatment also reduced renal injury in regard to urinary protein level and renal morphology but had no effect on blood pressure in DOCA-salt rats. Increased urinary 8-isoprostane, a marker for oxidative stress, in DOCA-salt hypertensive rats (55 +/- 8 ng/day) was diminished by vitamin E treatment (24 +/- 6 ng/day). These data suggest that renal injury characteristic of mineralocorticoid hypertension is associated with oxidative stress and is partly independent of blood pressure.

ET(B) receptor-deficient rats exhibit reduced contraction to ET-1 despite an increase in ET(A) receptors.

Several disease states, including hypertension, are associated with elevations in plasma endothelin-1 (ET-1) and variable changes in vascular contraction to ET-1. The spotting lethal (sl) rat carries a deletion of the endothelin-B (ET(B)) receptor gene that prevents expression of functional ET(B) receptors, resulting in elevated plasma ET-1. On a normal diet, these rats are normotensive and thus provide an opportunity to study the vascular effects of chronically elevated ET-1 in the absence of hypertension. Studies were performed in rats homozygous for the ET(B) deficiency (sl/sl; n = 8) and in transgenic rats heterozygous for the ET(B) deficiency (sl/+; n = 8). Plasma ET-1 was elevated in sl/sl rats (3.85 +/- 0.55 pg/ml) compared with sl/+ rats (0.31 +/- 0.11 pg/ml). Mean arterial blood pressure in conscious unrestrained sl/sl and sl/+ rats was 101 +/- 5 and 107 +/- 6 mmHg, respectively. Concentration-dependent contractions to ET-1 (10(-11)-10(-8) M) were reduced in mesenteric small arteries (150-250 microm) from sl/sl rats, as indicated by an approximately 10-fold increase in EC(50). A selective ET(A) antagonist, A-127722 (30 nM), abolished contraction to ET-1 in both groups, whereas a selective ET(B) antagonist had no effect. Also, ET(B) agonists (IRL-1620 and sarafatoxin 6c) produced neither contraction nor relaxation in either group, indicating that contraction to ET-1 in this vascular segment was exclusively ET(A) dependent. Despite increased plasma ET-1, protein expression of ET(A) receptors in membrane protein isolated from mesenteric small arteries was increased in sl/sl compared with sl/+ rats, as shown by Western blotting. These results indicate that, in ET(B)-deficient rats, ET(A)-induced contraction is reduced in vessels normally lacking ET(B)-mediated effects. Reduced contraction may be related to elevated plasma ET-1 and occurs in the presence of increased ET(A) receptor protein expression, suggesting an uncoupling of ET(A) receptor expression from functional activity.

Urinary excretion of vasoactive factors are correlated to sodium excretion.

BACKGROUND: The relationship between urinary vasoactive factors and sodium excretion has not been adequately addressed in humans. PROCEDURE: Excretion rates of sodium, nitrates/nitrites (NOx), cGMP, and endothelin-1 (ET-1) were measured before and after ingestion of a mixed electrolyte solution (8 oz Gatorade) while undergoing a routine cardiovascular evaluation in a sample of 51 normotensive young adults. RESULTS: Significant correlations were detected for changes in excretion between all four variables, r ranged from 0.50 to 0.86 (P < .001). Correlations were higher in African Americans than white Americans. CONCLUSIONS: The association of renal ET-1 and NO activity with sodium excretion supports the hypothesis that these factors play a role in the physiologic response to acute changes in sodium intake, particularly in African Americans.

Exercise in sickle cell anemia: effect on inflammatory and vasoactive mediators.

The aim of this study was to determine the response of inflammatory and vasoactive mediators to 3 consecutive days of exercise in African-American women with and without sickle cell anemia (SCA). Circulating inflammatory mediators [C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFalpha)] were measured before, and vasoactive mediators [endothelin-1 (ET-1), nitric oxide metabolites (NOx)] before and after each exercise bout in ten subjects with SCA and ten controls. Exercise did not affect ET-1, IL-6 or CRP concentrations (p >.05). TNFalpha was higher in SCA than controls (p < or = .0005) at all times; however, the response pattern was similar for the groups: no change from day 1 to day 2, but a decrease from day 2 to day 3 (p < or = .05). NOx increased significantly after exercise (p < or = .0001) but returned to baseline by 24 h afterward. On the 3rd day, NOx increased after exercise in SCA but not in the controls (p < or = .05). In conclusion, exercise did not cause a harmful inflammatory response in these individuals with SCA. However, NOx increased after exercise on all 3 days in SCA but appeared attenuated after 2 days in controls.

Endothelin antagonists in the treatment of renal failure.

The degree to which ET-1 participates in the pathogenesis of renal dysfunction remains poorly understood. In the kidney, ET-1 not only produces vasoconstriction, but also vasodilation in the renal medulla, stimulation of sodium reabsorption in the proximal tubule, and inhibition of reabsorption in a variety of nephron segments. It is clear that ET-1 production is increased in most forms of renal failure, although the cause of this increase is not known. Endothelin receptor antagonists, as therapeutic agents, have been investigated in a large variety of animal models, and even though there have been some promising results, there are many conflicting reports. While there is little known about the therapeutic utility of endothelin receptor antagonists in human renal failure, progress is most likely to be slow due to the complex nature of ET-1 actions and the less than promising initial studies in humans.

Endothelin-1-induced vasoconstriction is inhibited during erection in rats.

Recent evidence indicates that endothelin-1 (ET-1) might be a principal vasoconstrictor in the penis. We report that ET-1 injection into the cavernous sinuses before erection sharply reduced the magnitude of subsequent erections. Corpus cavernosum pressure-to-mean arterial pressure ratios (CCP/MAP), with maximal ganglionic stimulation, were 0.62 +/- 0.05 before ET-1 injection and 0.31 +/- 0.05 after, indicating that ET-1 acted as a vasoconstrictor. When ET-1 was injected during a maximal neurally induced erection, the ability of ET-1 to attenuate subsequent erections was diminished (CCP/MAP 0.75 +/- 0.02 before ET-1, 0.61 +/- 0.03 after). At submaximal stimulation voltages, injection of ET-1 during erection also attenuated its vasoconstrictive effect. Similarly, when ET-1 was injected during erection induced by intracavernosal injection of the nitric oxide (NO) donor NOR-1, subsequent erections were not significantly suppressed (CCP/MAP 0.53 +/- 0.04 before ET-1, 0.45 +/- 0.04 after). These findings that ET-1-induced vasoconstriction is attenuated during erection are consistent with the hypothesis that NO mediates erection both by initiating pathways that cause smooth muscle relaxation and by inhibiting the vasoconstrictive actions of ET-1.

Contrasting pharmacological ETB receptor blockade with genetic ETB deficiency in renal responses to big ET-1.

Renal clearance studies were conducted to determine the role of ET(B) receptors in the renal response to big endothelin-1 (big ET-1). Two series of experiments were conducted on Inactin-anesthetized rats to contrast acute pharmacological blockade of ET(B) receptors vs. genetic ET(B) receptor deficiency. In the first series, Sprague-Dawley rats were given either ET(B)-selective antagonist, A-192621, or vehicle (0.9% NaCl) prior to infusion of big ET-1 (10 pmol.kg(-1).min(-1)) for 60 min. A-192621 significantly increased baseline mean arterial pressure (MAP; 102 +/- 4 vs. 141 +/- 6 mmHg, P < 0.05) and urine flow rate (0.5 +/- 0.1 vs. 1.3 +/- 0.2 microl/min, P < 0.05) without any effect on glomerular filtration rate (GFR) or effective renal plasma flow (ERPF). Big ET-1 significantly increased MAP in both groups but to a higher level in rats given antagonist (120 +/- 6 vs. 169 +/- 6 mmHg, P < 0.05). Big ET-1 increased urine flow in control rats but decreased in rats given antagonist. GFR and ERPF were decreased in rats given big ET-1, an effect that was exaggerated by ET(B) blockade. Another series of experiments examined the response to big ET-1 in rats lacking functional renal ET(B) receptors, known as spotting lethal (sl) rats. Surprisingly, rats heterozygous (sl/+) for ET(B) receptor deficiency had a significantly higher baseline MAP compared with homozygous (sl/sl) rats (134 +/- 6 vs. 112 +/- 7 mmHg, P < 0.05), although other variables were similar. Big ET-1 produced no significant change in MAP in either group. Urine flow, GFR, and ERPF were significantly decreased in both groups, although these changes were much larger in sl/sl rats. These experiments indicate that the ET(B) receptor plays an important role in limiting the renal hemodynamic response to big ET-1. Furthermore, the diuretic actions of big ET-1 require a functional ET(B) receptor.

Evidence for endothelin involvement in the response to high salt.

Recent evidence suggests that endothelin-1 (ET-1), perhaps through the ET(B) receptor, may participate in blood pressure regulation through the control of sodium excretion. Mean arterial pressure (MAP) was continuously measured via telemetry implants in male Sprague-Dawley rats. After 1 wk of baseline measurements, rats were given either high (10%) or low (0.08%) NaCl in chow for the remainder of the experiment (n = 5 in each group). MAP was significantly increased in rats on a high-salt diet (115 +/- 2 mmHg) compared with rats on the low-salt diet (103 +/- 2 mmHg; P < 0.05). All rats were then treated with the ET(B) receptor antagonist A-192621 mixed with the food and adjusted daily to ensure a dose of 30 mg x kg(-1) x day(-1). ET(B) blockade produced an increase in MAP within a few hours of treatment and was significantly higher in rats on the high-salt diet over a 1-wk period (170 +/- 3 vs. 115 +/- 3 mmHg, P < 0.01). To determine whether the increase in MAP during A-192621 treatment was due to increased ET(A) receptor activation, all rats were then given the ET(A)-selective antagonist ABT-627 in the drinking water while a low-salt/high-salt diet and ET(B) blockade were continued. ABT-627 decreased MAP within a few hours in rats on either the high-salt (113 +/- 3 mmHg) or low-salt (101 +/- 3 mmHg) diet. These results support the hypothesis that endothelin, through the ET(B) receptor, participates in blood pressure regulation in the response to salt loading.

Enhanced endothelin-1 response and receptor expression in small mesenteric arteries of insulin-resistant rats.

Hyperinsulinemia, a primary feature of insulin resistance, is associated with increased endothelin-1 (ET-1) activity. This study determined the vascular response to ET-1 and receptor binding characteristics in small mesenteric arteries of insulin-resistant (IR) rats. Rats were randomized to control (C) (n = 32) or IR (n = 32) groups. The response to ET-1 was assessed (in vitro) in arteries with (Endo+) and without (Endo-) endothelium. In addition, arteries (Endo+) were pretreated with the ET(B) antagonist A-192621 or the ET(A) antagonist A-127722. Finally, binding characteristics of [(125)I]ET-1 were determined. Results showed that in Endo+ arteries the maximal relaxation (E(max)) to ET-1 was similar between C and IR groups; however, the concentration at 50% of maximum relaxation (EC(50)) was decreased in IR arteries. In Endo- arteries, the E(max) to ET-1 was enhanced in both groups. Pretreatment with A-192621 enhanced the E(max) and EC(50) to ET-1 in both groups. In contrast, A-127722 inhibited the ET-1 response in all arteries in a concentration-dependent manner; however, a greater ET-1 response was seen at each concentration in IR arteries. Maximal binding of [(125)I]ET-1 was increased in IR versus C arteries although the dissociation constant values were similar. In conclusion, we found the vasoconstrictor response to ET-1 is enhanced in IR arteries due to an enhanced expression of ET receptors and underlying endothelial dysfunction.

Arterial pressure response to endothelin-1 and sarafotoxin 6c in rescued endothelin-B-deficient rats.

The spotting lethal rat carries a naturally occurring deletion of the endothelin-B- (ET(B)) receptor gene that prevents expression of functional ET(B)-receptors. Gariepy and colleagues used tissue-specific ET(B) transgene expression to support normal enteric nervous system development. To determine functional consequences of ET(B)-receptor deficiency, studies were conducted to characterize the pressor response to endothelin-1 (ET-1) and the ET(B) agonist, sarafotoxin 6c (S6c) in transgenic rats homozygous for the ET(B)-deficiency (sl/sl). Similar transgenic rats heterozygous for the ET(B) deficiency were used as controls (sl/+). All rats were anesthetized with Inactin (100 mg/kg, i.p.) and a tracheostomy performed. The right carotid artery and right jugular veins were catheterized for measuring mean arterial pressure (MAP) and infusion of peptides, respectively. Following baseline measurement of MAP, hexamethonium was infused (10 mg/kg) to block sympathetic reflex responses. After a 10-15 min stabilization period, ET-1 or S6c was infused at 0, 1, 0.3 and 1.0 nmol/kg at 10 min intervals. MAP in the two groups of anesthetized rats was similar during the baseline period. The sl/+ rats showed a classic dose-dependent pressor response to ET-1; a transient vasodilation followed by prolonged vasoconstriction. In contrast, the vasodilation was absent in sl/sl rats. Furthermore, ET-1 was more potent in sl/sl compared to the sl/+ rats. The response to S6c was qualitatively similar to ET-1 in the sl/+ rats. However, the sl/sl rats also had a significant pressor response to the ET(B) agonist, S6c. These studies provide in vivo evidence that the rescued ET(B)-deficient rat lacks functional vasodilatory ET(B) responses in response to ET-1 but retains the vasoconstrictor response to ET(B)-receptor agonists.

Shear stress-mediated NO production in inner medullary collecting duct cells.

Recent evidence suggests that nitric oxide (NO) within the inner medullary collecting duct (IMCD) functions to regulate sodium and water reabsorption. Because fluid shear stress has been shown to increase NO production in endothelial and vascular smooth muscle cells, experiments were designed to determine whether a similar mechanism exists in IMCD cells. Cultured IMCD-3 cells derived from murine IMCD were subjected to 60 min of pulsatile shear stress. Nitrite production (2,3-diaminonaphthalene fluorometric assay) increased 12-, 16-, and 23-fold at 3.3, 10, and 30 dyn/cm(2), respectively, compared with static control cultures. Preincubation with the non-isoform-specific NO synthase inhibitor nitro-L-arginine methyl ester reduced nitrite production by 83% in response to 30 dyn/cm(2). Western blotting and immunofluorescence analysis of static IMCD-3 cell cultures revealed the expression of all three NO synthase isoforms (NOS-1 or neuronal NOS, NOS-2 or inducible NOS, and NOS-3 or endothelial NOS) in IMCD-3 cultures. These results indicate that NO production is modulated by shear stress in IMCD-3 cells and that fluid shear stress within the renal tubular system may play a role in the regulation of sodium and water excretion by control of NO production in the IMCD.

Endothelin inhibits thick ascending limb chloride flux via ET(B) receptor-mediated NO release.

Endothelin-1 (ET-1) inhibits transport in various nephron segments, and the thick ascending limb of the loop of Henle (TALH) expresses ET-1 receptors. In many tissues, activation of ET(B) receptors stimulates release of NO, and we recently reported that endogenous NO inhibits TALH chloride flux (J(Cl)). However, the relationship between ET-1 and NO in the control of nephron transport has not been extensively studied. We hypothesized that ET-1 decreases NaCl transport by cortical TALHs through activation of ET(B) receptors and release of NO. Exogenous ET-1 (1 nM) decreased J(Cl) from 118.3 +/- 15.0 to 62.7 +/- 13.6 pmol. mm(-1). min(-1) (48.3 +/- 8.2% reduction), whereas removal of ET-1 increased J(Cl) in a separate group of tubules from 87.6 +/- 10.7 to 115.2 +/- 10.3 pmol. mm(-1). min(-1) (34.5 +/- 6.2% increase). To determine whether NO mediates the inhibitory effects of ET-1 on J(Cl), we examined the effect of inhibiting of NO synthase (NOS) with N(G)-nitro-L-arginine methyl ester (L-NAME) on ET-1-induced changes in J(Cl). L-NAME (5 mM) completely prevented the ET-1-induced reduction in J(Cl), whereas D-NAME did not. L-NAME alone had no effect on J(Cl). These data suggest that the effects of ET-1 are mediated by NO. Blockade of ET(B) receptors with BQ-788 prevented the inhibitory effects of 1 nM ET-1. Activation of ET(B) receptors with sarafotoxin S6c mimicked the inhibitory effect of ET-1 on J(Cl) (from 120.7 +/- 12.6 to 75.4 +/- 13.3 pmol. mm(-1). min(-1)). In contrast, ET(A) receptor antagonism with BQ-610 did not prevent ET-1-mediated inhibition of TALH J(Cl) (from 96.5 +/- 10.4 to 69.5 +/- 8.6 pmol. mm(-1). min(-1)). Endothelin increased intracellular calcium from 96.9 +/- 14.0 to 191.4 +/- 11.9 nM, an increase of 110.8 +/- 26.1%. We conclude that exogenous endothelin indirectly decreases TALH J(Cl) by activating ET(B) receptors, increasing intracellular calcium concentration, and stimulating NO release. These data suggest that endothelin acts as a physiological regulator of TALH NO synthesis, thus inhibiting chloride transport and contributing to the natriuretic effects of ET-1 observed in vivo.

Receptor-specific influence of endothelin-1 in the erectile response of the rat.

Specific receptor antagonists were used to examine the role of endothelin-1 (ET-1) in the erectile response of the rat. In these studies, intact rats were cannulated to permit the continuous recording of mean arterial pressure (MAP) and intracavernosal pressure (CCP). Erection was induced by electrical stimulation of the autonomic ganglion, which regulates blood flow to the penis. The animals were subjected to intracavernosal injection with vehicle only (Cont) or with an antagonist to the endothelin-A receptor (ET(A)) or to the endothelin-B receptor (ET(B)). Blockade of the ET(A) or the ET(B) had no effect on the erectile response (CCP/MAP) during maximal ganglionic stimulation. When ET-1 was injected into Cont rats, there was a marked vasoconstriction with a sharp rise in MAP and a decline in CCP as the cavernosal arterioles constricted and limited inflow. The injection of the ET(A) antagonist prevented the vasoconstriction after ET-1 injection into Cont rats, whereas blockade of the ET(B) had no effect on the vasoconstrictive effect to ET-1. Similar results were obtained during submaximal ganglionic stimulation. With minimal levels of ganglionic stimulation, ET-1 injection led to a moderated degree of vasodilation in the presence of the ET(A) antagonist. The ET(B) antagonist failed to alter the CCP response during minimal stimulation, but it did have a marked effect on the MAP response to ET-1 injection. The results of these studies confirm that cavernosal tissue of the rat penis is highly responsive to ET-1. However, the failure of the ET-1 antagonists to affect penile erection in response to ganglionic stimulation reflects a minimal role of ET-1 in the erectile response in the rat.

Renal endothelin in hypertension.

Due to the potent vasoconstrictor action of endothelin-1 and its synthesis throughout the vasculature and other tissues, most investigators believe that it is an active participant in the pathogenesis of hypertension. However, the autocrine and paracrine nature of the endothelin system has made its role difficult to define. In recent years, it has become apparent that endothelin-1 contributes to the regulation of renal salt and water excretion and that it is a major contributor to the hypertension associated with salt-dependency. Evidence suggests that endothelin-1 within the renal medulla is activated in conditions of salt loading and inhibits reabsorption of sodium in a nitric oxide-dependent manner. Blockade of endothelin A receptors lowers arterial pressure in animal models of salt-dependent hypertension. Furthermore, circulating levels of endothelin-1 are generally higher in African-Americans compared to white Americans as is the prevalence of salt-dependent hypertension. Therefore, it would appear that use of endothelin A-selective receptor antagonists should be targeted to those individuals at risk for salt-dependent hypertension. Blockade of endothelin B receptors would not be desirable because of their important role in eliminating a salt load.