Abstract-The purpose of this study was to determine if rats lacking the ET B receptor have altered renal endothelin (ET) production and NO synthase (NOS) activity in response to high salt and if female rats are better able to control blood pressure through higher NOS activity in rats heterozygous (sl/ϩ) and homozygous (sl/sl) for ET B receptor deficiency. On normal salt (0.4% NaCl; NS), male sl/sl rats had higher systolic blood pressures compared with male sl/ϩ and female sl/ϩ and sl/sl rats. On a high salt diet (10% NaCl; HS), blood pressure in male sl/ϩ rats was significantly higher than female sl/ϩ rats. However, ET B receptor deficiency caused much larger increases in blood pressure in male and female rats. On NS, urinary ET excretion was not different between male and female of either genotype. HS significantly increased ET excretion in male and female sl/ϩ rats, but the increase was significantly less in sl/sl compared with sl/ϩ. Homogenates of inner medullary collecting duct tissue were separated into particulate and cytosolic fractions and total NOS activity measured by conversion ofFemale rats had significantly greater cytosolic NOS activity compared with male rats on NS. On HS, cytosolic NOS activity was lower in all groups compared with NS rats, whereas particulate NOS activity was significantly greater in male and female sl/ϩ rats compared with male and female sl/sl rats. These data support our hypothesis that NOS protects against rises in blood pressure in female rats and ET B receptors prevent further increases in blood pressure due to increases in renal ET production and NOS activity. , and NOS3 (endothelial NOS), have been localized to various parts of the kidney, with the greatest amount of activity found in the inner medullary collecting duct (IMCD). 1,2 The highest amount of immunoreactive ET in the body is found in the renal medulla. 3 The IMCD synthesizes 10 times more ET than any other site along the nephron, 4 and renal ET B receptors are highly concentrated in IMCD cells. 5 In the kidney, both NO and ET have been shown to inhibit tubular sodium reabsorption. 6 -8 In the thick ascending limb, there is evidence that ET B receptors serve to inhibit chloride reabsorption through an NO-dependent pathway. 9 A preliminary study has also shown that ET directly stimulates NO production in IMCD cells, possibly contributing to the paradoxical natriuretic properties of ET. 10 Additionally, rats treated chronically with an ET B receptor antagonist have elevated arterial pressures compared with control animals, and this increase in blood pressure was much greater in rats given a high salt diet compared with a low salt diet. 11 Urinary ET excretion is increased in rats given a high salt diet, suggesting that renal ET synthesis is elevated in response to a salt load. 11,12 It has been shown in both humans 13 and animals 14 that vascular NO production is greater in female than male subjects and that renal NOS3 mRNA and protein levels are higher in kidneys from female subjects than from male subjects, 1...

.) developed rescued spottinglethal rats that carry a naturally occurring deletion of the endothelin (ET) type B receptor gene resulting in a lack of functional renal ETB receptor expression. It has been shown that rats homozygous (sl/sl) for the deletion have elevated plasma ET-1 levels; thus, the purpose of this study was to determine whether this deletion would result in a downregulation of ETA receptors in renal tissue. ET-1 and ET-3 binding experiments were performed with cortex, outer medullary, and inner medullary membranes of heterozygous (sl/ϩ) and sl/sl ETB receptor-deficient rats.125 I-labeled ET-1 binding in sl/sl cortex and outer medulla was significantly lower than cortex and outer medulla from sl/ϩ rats. In contrast to sl/ϩ rats, [125 I]ET-3 binding was not detected in the cortex and outer medulla of sl/sl rats, indicating a lack of ETB receptor expression. The inner medulla of sl/ϩ rats also demonstrated an abundance of ETB receptors. Surprisingly, however, we also observed significant [ 125 I]ET-3 binding in the sl/sl inner medulla. Furthermore, ET-3 binding in the inner medulla could be blocked with an ETA receptor antagonist in sl/sl rats but not in tissue from sl/ϩ rats. These studies indicate that rats deficient in ETB receptors have decreased renal cortical and outer medullary ETA receptor number, most likely in response to elevated plasma ET-1 levels. In addition, homozygous ETB-deficient rats express a novel inner medullary ET-3 binding site.endothelin B receptor; kidney; receptor binding; spottinglethal rats IN 1988, YANAGISAWA ET AL. (33) isolated a potent vasoconstrictor produced by endothelial cells that was named endothelin (ET). Inoue and colleagues (14) determined through DNA analysis that there are three distinct genes encoding three ET isopeptides (ET-1, ET-2, and ET-3). G protein-coupled receptors were discovered and then characterized by their selective affinity to each of the ET isopeptides, location, and their biological function (18). Endothelin A (ET A ) receptors bind ET-1 with higher affinity than ET-2 or ET-3, are localized to vascular smooth muscle cells, and cause vasoconstriction. Endothelin B (ET B ) receptors are found on endothelial cells and have been shown to produce vasodilation and bind all three ET isopeptides with equal affinity. Additionally, there is evidence that ET B receptors in pulmonary endothelial cells "clear" ET-1 from the circulation (7). However, this classification of ET receptors may be oversimplified, with increasing evidence suggesting additional receptor subtypes. It is now known that there exist two pharmacologically distinct subtypes of the ET B receptor, ET B1 and ET B2 , whose biological activity is a function of their localization. ET B1 are located on the vascular endothelium and produce vasodilation upon agonist binding, whereas ET B2 are found on vascular smooth muscle cells and mediate non-ET A vasoconstriction.Within the kidney, both ET receptor subtypes have been identified in various regions differing both in number and function. ...

Abstract. Experiments were performed to test the hypothesis that diabetes mellitus disrupts the balance between synthesis and degradation of nitric oxide (NO) in the renal cortex. Diabetes was induced by injection of streptozotocin, and sufficient insulin was provided to maintain moderate hyperglycemia for the ensuing 2 wk. Despite an 80% increase in total NO synthase activity measured by L-citrulline assay, nicotinamide adenine dinucleotide phosphate-diaphorase staining was unaltered, and no changes in NO synthase isoform protein levels or their distribution were evident in renal cortex from diabetic rats. Superoxide anion production was accelerated twofold in renal cortical slices from diabetic rats, with an associated 50% increase in superoxide dismutase activity. Western blots prepared by use of a monoclonal antinitrotyrosine antibody revealed an approximately 70-kD protein in renal cortex from sham rats, the nitrotyrosine content of which was threefold greater in cortical samples from diabetic rats. These observations indicate that the early stage of diabetes mellitus provokes accelerated renal cortical superoxide anion production in a setting of normal or increased NO production. This situation can be expected to promote peroxynitrite formation, resulting in the tyrosine nitration of a single protein of unknown identity, as well as a decline in the bioavailability of NO. These events are consistent with the postulate that oxidative stress promotes NO degradation in the renal cortex during the early stage of diabetes mellitus.

Formation of mature active neuropeptides such as substance P (SP) from their glycine extended precursors entails alpha-amidation of peptide precursors by the sequential enzymatic action of peptidylglycine alpha-monooxygenase (PAM) and peptidylamidoglycolate lyase (PGL). We reported that these two enzymes that can produce mature active neuropeptides are present in cultured bovine aortic endothelial cells (BAECs). We hypothesize that alpha-amidation of peptides occurs in endothelial cells and that these peptides are critically involved in the overall regulation of cardiovascular function. In this study, this hypothesis was tested using specific amidation inhibitors to determine their effects on the actions of SP and its glycine-extended precursor (SP-Gly). We have found that SP and SP-Gly are equipotent in stimulating nitric oxide (NO) release by BAECs. At 10(-5) M, the specific inhibitors of PAM (4-phenyl-3-butenoic acid; PBA) and PGL (5-acetamido-2,4-diketo-6-phenyl-hexanoic acid and its methyl ester) reduced NO basal release by 40, 34, and 45%, respectively. They also reduced the production of NO induced by SP-Gly by 63, 68, and 69%, respectively, but had no effect on NO production in response to either SP or acetylcholine. SP and SP-Gly also were equipotent in relaxing rat aortic segments. The vasorelaxation to SP-Gly was endothelium dependent and inhibited by the NOS antagonist L-nitroarginine methyl ester (L-NAME), but it was not affected by inhibition of prostaglandin synthesis. Inhibitors of both PAM and PGL significantly reduced the vasorelaxing actions of SP-Gly, whereas responses to SP were not affected. A cumulative infusion of PBA into the femoral artery of rabbits, at final concentrations of 2.4, 24, and 240 microM for 20 min each, increased the vascular resistance (VR), indicating the tonic production of vasodilating amidated peptide(s). This effect was maximum at 60 min after infusion (20.5 +/- 4.7 vs. 8.2 +/- 0.7 mm Hg/ml/min; p < 0.05). These results suggest that endothelial cells can produce mature SP from its SP-Gly precursor and that a product of peptide alpha-amidation tonically stimulates endothelial cell NO release to control vascular tone.

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