Loss of endothelin type B receptor function improves insulin sensitivity in rats.

High salt intake (HS) is associated with obesity and insulin resistance. ET-1, a peptide released in response to HS, inhibits the actions of insulin on cultured adipocytes through ET-1 type B (ETB) receptors; however, the in vivo implications of ETB receptor activation on lipid metabolism and insulin resistance is unknown. We hypothesized that activation of ETB receptors in response to HS intake promotes dyslipidemia and insulin resistance. In normal salt (NS) fed rats, no significant difference in body mass or epididymal fat mass was observed between control and ETB deficient rats. After 2 weeks of HS, ETB-deficient rats had significantly lower body mass and epididymal fat mass compared to controls. Nonfasting plasma glucose was not different between genotypes; however, plasma insulin concentration was significantly lower in ETB-deficient rats compared to controls, suggesting improved insulin sensitivity. In addition, ETB-deficient rats had higher circulating free fatty acids in both NS and HS groups, with no difference in plasma triglycerides between genotypes. In a separate experiment, ETB-deficient rats had significantly lower fasting blood glucose and improved glucose and insulin tolerance compared to controls. These data suggest that ET-1 promotes adipose deposition and insulin resistance via the ETB receptor.

Endothelial cells secreted endothelin-1 augments diabetic nephropathy via inducing extracellular matrix accumulation of mesangial cells in ETBR-/- mice.

Endothelin B receptor (ETBR) deficiency may contribute to the progression of diabetic nephropathy (DN) in a streptozotocin (STZ) model, but the underlying mechanism is not fully revealed. In this study, STZ-diabetic ETBR-/- mice was characterized by increased serum creatinine and urinary albumin, enhanced glomerulosclerosis, and upregulated ET-1 expression compared with STZ-diabetic WT mice. In vitro, HG conditioned media (CM) of ETBR-/- GENs promoted mesangial cell proliferation and upregulated ECM-related proteins, and ET-1 knockout in GENs or inhibition of ET-1/ETAR in mesangial cell suppressed mesangial cell proliferation and collagen IV formation. In addition, ET-1 was over-expressed in ETBR-/- GENs and was regulated by NF-kapapB pathway. ET-1/ETBR suppressed NF-kappaB to modulate ET-1 in GENs. Furthermore, ET-1/ETAR promoted RhoA/ROCK pathway in mesangial cells, and accelerated mesangial cell proliferation and ECM accumulation. Finally, in vivo experiments proved inhibition of NF-kappaB pathway ameliorated DN in ETBR-/- mice. These results suggest that in HG-exposed ETBR-/- GENs, suppression of ET-1 binding to ETBR activated NF-kappaB pathway, thus to secrete large amount of ET-1. Due to the communication between GENs and mesangial cells in diabetes, ET-1 binding to ETAR in mesangial cell promoted RhoA/ROCK pathway, thus to accelerate mesangial cell proliferation and ECM accumulation.

Diurnal pattern in skin Na+ and water content is associated with salt-sensitive hypertension in ETB receptor-deficient rats.

Impairment in the ability of the skin to properly store Na+ nonosmotically (without water) has recently been hypothesized as contributing to salt-sensitive hypertension. Our laboratory has shown that endothelial production of endothelin-1 (ET-1) is crucial to skin Na+ handling. Furthermore, it is well established that loss of endothelin type B receptor (ETB) receptor function impairs Na+ excretion by the kidney. Thus we hypothesized that rats lacking functional ETB receptors (ETB-def) will have a reduced capacity of the skin to store Na+ during chronic high-salt (HS) intake. We observed that ETB-def rats exhibited salt-sensitive hypertension with an approximate doubling in the diurnal amplitude of mean arterial pressure compared with genetic control rats on a HS diet. Two weeks of HS diet significantly increased skin Na+ content relative to water; however, there was no significant difference between control and ETB-def rats. Interestingly, HS intake led to a 19% increase in skin Na+ and 16% increase in water content (relative to dry wt.) during the active phase (zeitgeber time 16) versus inactive phase (zeitgeber time 4, P < 0.05) in ETB-def rats. There was no significant circadian variation in total skin Na+ or water content of control rats fed normal or HS. These data indicate that ETB receptors have little influence on the ability to store Na+ nonosmotically in the skin during long-term HS intake but, rather, appear to regulate diurnal rhythms in skin Na+ content and circadian blood pressure rhythms associated with a HS diet.

Increased expression of Semaphorin 3A in the endothelin receptor-B null mouse model of Hirschsprung disease.

PURPOSE: Semaphorins are guidance cues for developing neurons, implicated in the determination of the migratory pathway of neural crest-derived neural precursors during enteric nervous system development. Recently, it has been reported that Semaphorin 3A (SEMA3A) expression is up-regulated in the aganglionic colon in Hirschsprung disease (HD) patients, suggesting that increased SEMA3A expression may be a risk factor for HD. Thus, the aim of our study was to determine the expression of SEMA3A using Sox10-Venus mice gut. METHODS: We harvested the gut on postnatal day 2 (P2). SOX10-Venus+/EDNRB-/- mice were compared with SOX10-Venus+/EDNRB+/+ mice as controls. QRT-PCR was performed to determine gene expression of SEMA3A (n=8). Fluorescent immunohistochemistry was performed to assess protein distribution. RESULTS: On P2, gene expression levels of SEMA3A were significantly increased in the HD group compared to controls in the proximal and distal colon (p<0.05). Laser scanning microscopy revealed SEMA3A expression was localized within the submucosa and muscle layer of the gut in both HD and controls. In HD, SEMA3A was highly expressed in the proximal and distal colon. CONCLUSIONS: In the present study, we demonstrated that SEMA3A expression is increased in the EDNRB-/- HD model on P2, suggesting that SEMA3A may interfere with ENCC migration, resulting in an absence of enteric neurons.

Functional ET(A)-ET(B) Receptor Cross-talk in Basilar Artery In Situ From ET(B) Receptor Deficient Rats.

The role of endothelin (ET)(A)-ET(B) receptor cross-talk in limiting the ET(A) receptor antagonist inhibition of ET-1 constriction is revealed by the partial or complete dependency of the ET(A) receptor antagonist inhibition on functional removal of the ET(B) receptor. Although functional removal of the ET(B) receptor is generally accomplished with ET(B) receptor antagonist, a novel approach using rats containing a naturally occurring deletion mutation in the ET(B) receptor [rescued "spotting lethal" (sl) rats; ET(B)(sl/sl)] demonstrated increased ET(A) receptor antagonist inhibition of ET-1 constriction in vena cava. We investigated whether this deletion mutation was also sufficient to remove the ET(B) receptor dependency of the ET(A) receptor antagonist inhibition of ET-1 constriction in the basilar artery. Consistent with previous reports, ET-1 plasma levels were elevated in ET(B)(sl/sl) as compared with ET(B)(+/+) rats. ET(B) receptor antagonist failed to relax the ET-1 constricted basilar artery from ET(B)(+/+) and ET(B)(sl/sl) rats. Relaxation to combined ET(A) and ET(B) receptor antagonist was greater than relaxation to ET(A) receptor antagonist in the basilar artery from ET(B)(+/+) and, unexpectedly, ET(B)(sl/sl) rats. These findings confirm the presence of ET(A)-ET(B) receptor cross-talk in the basilar artery. We speculate that mutant ET(B) receptor expression produced by alternative splicing may be sufficient to allow cross-talk.

Mucus Barriers to Microparticles and Microbes are Altered in Hirschsprung's Disease.

Mucus forms a protective hydrogel layer over the intestinal epithelium, presenting a selective and robust barrier to the uptake of particulates and microbe invasion. Disease can alter mucus production and composition, thus potentially modifying mucosal barrier properties. Hirschsprung's disease (HD) is a developmental abnormality of the nervous system often complicated by intestinal infection. An investigation of colonic mucus barrier properties in an HD animal model, endothelin receptor B mutant mice, revealed significantly reduced microsphere (passive) and microbe (active) transport rates (7-fold and 3.6-fold, respectively, in proximal colonic mucus) relative to wild-type. Transport differences were evident in both the ganglionic and aganglionic colon segments, in agreement with the risk of HD-associated enterocolitis after surgery to remove aganglionic colon segments. The development of therapies aimed at altering colonic mucus barrier properties could be explored towards preventing the onset of enterocolitis in HD.

Altered goblet cell differentiation and surface mucus properties in Hirschsprung disease.

Hirschsprung disease-associated enterocolitis (HAEC) leads to significant mortality and morbidity, but its pathogenesis remains unknown. Changes in the colonic epithelium related to goblet cells and the luminal mucus layer have been postulated to play a key role. Here we show that the colonic epithelium of both aganglionic and ganglionic segments are altered in patients and in mice with Hirschsprung disease (HSCR). Structurally, goblet cells were altered with increased goblet cell number and reduced intracellular mucins in the distal colon of biopsies from patients with HSCR. Endothelin receptor B (Ednrb) mutant mice showed increased goblet cell number and size and increased cell proliferation compared to wild-type mice in aganglionic segments, and reduced goblet cell size and number in ganglionic segments. Functionally, compared to littermates, Ednrb-/- mice showed increased transepithelial resistance, reduced stool water content and similar chloride secretion in the distal colon. Transcript levels of goblet cell differentiation factors SPDEF and Math1 were increased in the distal colon of Ednrb-/- mice. Both distal colon from Ednrb mice and biopsies from HSCR patients showed reduced Muc4 expression as compared to controls, but similar expression of Muc2. Particle tracking studies showed that mucus from Ednrb-/- mice provided a more significant barrier to diffusion of 200 nm nanoparticles as compared to wild-type mice. These results suggest that aganglionosis is associated with increased goblet cell proliferation and differentiation and subsequent altered surface mucus properties, prior to the development of inflammation in the distal colon epithelium. Restoration of normal goblet cell function and mucus layer properties in the colonic epithelium may represent a therapeutic strategy for prevention of HAEC.

Altered neuronal density and neurotransmitter expression in the ganglionated region of Ednrb null mice: implications for Hirschsprung's disease.

BACKGROUND: Hirschsprung's disease (HSCR) is a congenital condition in which enteric ganglia, formed from neural crest cells (NCC), are absent from the terminal bowel. Dysmotility and constipation are common features of HSCR that persist following surgical intervention. This persistence suggests that the portion of the colon that remains postoperatively is not able to support normal bowel function. To elucidate the defects that underlie this condition, we utilized a murine model of HSCR. METHODS: Mice with NCC-specific deletion of Ednrb were used to measure the neuronal density and neurotransmitter expression in ganglia. KEY RESULTS: At the site located proximal to the aganglionic region of P21 Ednrb null mice, the neuronal density is significantly decreased and the expression of neurotransmitters is altered compared with het animals. The ganglia in this colonic region are smaller and more isolated while the size of neuronal cell bodies is increased. The percentage of neurons expressing neuronal nNOS and VIP is significantly increased in Ednrb nulls. Conversely, the percentage of choline acetyltransferase (ChAT) expressing neurons is decreased, while Substance P is unchanged between the two genotypes. These changes are limited to the colon and are not detected in the ileum. CONCLUSIONS & INFERENCES: We demonstrate changes in neuronal density and alterations in the balance of expression of neurotransmitters in the colon proximal to the aganglionic region in Ednrb null mice. The reduced neuronal density and complementary changes in nNOS and ChAT expression may account for the dysmotility seen in HSCR.

Collecting duct-specific endothelin B receptor knockout increases ENaC activity.

Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.

Sacral neural crest-derived cells enter the aganglionic colon of Ednrb-/- mice along extrinsic nerve fibers.

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest-derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest-derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest-derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut.

Endothelin-1-induced proliferation is reduced and Ca²âº signaling is enhanced in endothelin B-deficient optic nerve head astrocytes.

PURPOSE: To characterize the influence of endothelin-1 (ET-1) on optic nerve head astrocyte (ONHA) proliferation and Ca²âº signaling in ONHAs lacking functional endothelin B (ETB) receptors. METHODS: ONHAs were isolated from adult wild type (WT) and transgenic spotting lethal (TSL) rats, lacking functional ETB receptors. ONHA specificity was confirmed by positive glial fibrillary acidic protein (GFAP), negative A2B5 (a marker for type II astrocytes located outside the optic nerve head) and myelin basic protein (MBP) labeling. The mitogenic effects of 10⁻7 or 10⁻9 M ET-1, or vehicle were investigated for 48 or 72 hours in WT and TSL ONHAs. Intracellular calcium levels ([Ca²âº](i)) were assessed in ONHAs loaded with fura-2 calcium indicator dye. RESULTS: ET-1-induced proliferation of TSL ONHAs was blunted at 48 hours (by 37% at 10⁻7 M and by 33% at 10⁻9 M) and 72 hours (by 117% at 10⁻7 M and by 100% at 10⁻9 M) compared with WT cells. ET-1-induced ONHA fura-2 ratio increases were significantly greater in TSL ONHAs (by 20% at 10⁻7 M and by 48% at 10⁻9 M) compared with WT ONHAs. ET-1-induced fura-2 ratio increases were blocked after pretreatment with BQ-610 (ETA antagonist) in WT and TSL ONHAs, but not by BQ-788 (ETB antagonist) in WT ONHAs. CONCLUSIONS: ET-1-induced ONHA proliferation is reduced in cells lacking functional ETB receptors, ET-1-induced [Ca²âº](i) increases are enhanced in the absence of functional ETB receptors, and ETA, but not ETB, is required for ET-1-induced [Ca²âº](i) elevation.

Endothelin as a neuroprotective factor in the olfactory epithelium.

In mammals, the olfactory sensory neurons are the only ones directly in contact with an aggressive environment. Thus, the olfactory mucosa is one of the few neuronal zones which are continuously renewed during adulthood. We have previously shown that endothelin is locally matured in the olfactory mucosa and that olfactory sensory neurons preferentially express ETB receptors, while ETA receptors are rather present in non neuronal olfactory mucosa cells. In addition to its vasoactive effect, the endothelin system is known for its pleiotropic effects including the modulation of cell population dynamics. We thus examined its potential neuroprotective effect in the olfactory mucosa using a primary culture of olfactory sensory neurons lying on non neuronal cells. While a serum deprivation led to a massive decrease of the density of olfactory sensory neurons in the primary cultures, endothelin 1 (ET-1) rescued part of the neuronal population through both ETA and ETB receptors. This effect was mainly anti-apoptotic as it reduced cleaved caspase-3 signal and nuclear condensation. Furthermore, the olfactory epithelium of ETB-deficient rats displayed increased apoptosis. These results strongly suggest that ET-1 acts as an anti-apoptotic factor on olfactory sensory neurons, directly through ETB and indirectly by limiting non neuronal cells death through ETA.

Abnormal enteric innervation identified without histopathologic staining in aganglionic colorectum from a mouse model of Hirschsprung's disease.

PURPOSE: The piebald lethal mouse with a deletion of endothelin-B receptor gene (EDNRB) is a model for Hirschsprung's disease (HD), whereas the SOX10 gene is vital for the development of intestinal neural crest-derived cells. Recently, we created a SOX10 transgenic mouse with intestinal neural crest-derived cells visible with enhanced green fluorescent protein (VENUS), that is, SOX10-VENUS(+)/EDNRB(sl/sl) to investigate intestinal innervation in HD. METHODS: SOX10-VENUS(+)/EDNRB(sl/sl) (n = 30) were compared with wild-type littermates as controls (EDNRB(s/s), n = 30). Mice were killed on days 3, 7, or 12 of age. The entire colorectum was excised, fixed with 4% paraformaldehyde, and examined using fluorescence microscopy alone without staining. RESULTS: In normoganglionic colorectum from controls, a grid network of nerve fibers/glial cells was visualized that connected smoothly with extrinsic nerve fibers running along the colorectal wall. In aganglionic colorectum from SOX10-VENUS(+)/EDNRB(sl/sl) mice, there was no grid network and more extrinsic nerve fibers than controls that invaded the colon wall becoming elongated with branching fibers. Normoganglionic colon from controls and SOX10-VENUS(+)/EDNRB(sl/sl) mice appeared the same. Innervation patterns did not change over time. CONCLUSION: This is the first time for abnormal enteric innervation in aganglionic colon in a model for HD to be visualized without staining.

Endothelial ET(B) limits vascular remodelling and development of pulmonary hypertension during hypoxia.

BACKGROUND: We hypothesised that the potential protective effects of endothelial ET(B) are important in limiting pulmonary vascular muscularisation, vasoconstriction and the development of pulmonary arterial hypertension in response to hypoxia. METHODS: EC-specific ET(B) knockout mice (EC ET(B)(-/-)) and control mice (ET(B)(f/f)) were subjected to hypobaric hypoxic (10% FiO2) or normoxic conditions for 14 days before assessment of right ventricular pressure and pulmonary vascular morphology and function. RESULTS: During normoxia, no difference in right ventricular pressure was detected between EC ET(B)(-/-) (23.7 +/- 1.7 mm Hg) and ET(B)(f/f) mice (20.2 +/- 1.5 mm Hg). Hypoxia induced an exaggerated increase in right ventricular pressure in EC ET(B)(-/-) mice (34.4 +/- 1.2 mm Hg vs. 24.6 +/- 1.4 mm Hg), accompanied by an increase in right ventricular mass. No effect was observed in ET(B)(f/f) mice. Endothelin-1 constricted pulmonary arteries from both groups, although maximum response was similar irrespective of inspired oxygen or genotype. Hypoxia increased the percentage of muscularised vessels in both groups of mice, but the percentage increase was significantly greater in EC ET(B)(-/-) mice. CONCLUSIONS: The potential protective effects of endothelial ET(B) are important in limiting pulmonary vascular muscularisation and the development of pulmonary arterial hypertension in response to hypoxia.

Inhibition of endothelin ETB receptor system aggravates neointimal hyperplasia after balloon injury of rat carotid artery.

Endothelin-1 (ET)/ET(A) receptor system has been known to play an important role in the pathogenesis of neointimal hyperplasia after endothelial injury. However, the pathological role of endothelin ET(B) receptors on neointimal hyperplasia remains to be elucidated. In the present study, we investigated the pathological role of ET(B) receptors on neointimal hyperplasia in balloon-injured rat carotid arteries by pharmacological blockade with use of 2R-(4-propoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N-(2,6-diethylphenyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (A-192621), a selective ET(B) receptor antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N,N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), a selective ET(A) receptor antagonist, and (+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic acid (J-104132), an ET(A)/ET(B) dual receptor antagonist. Moreover, the spotting-lethal rats, which carry a naturally occurring deletion in the endothelin ET(B) receptor gene, were used to examine the effects of genetic deficiency for this receptor subtype. Two weeks after balloon injury, the ratio of the neointimal to the medial area (neointima/media ratio) was determined. Treatment with A-192621 (30 mg/kg/day) for 2 weeks after injury significantly increased the neointima/media ratio in the injured artery. In contrast, ABT-627 (10 mg/kg/day) and J-104132 (10 mg/kg/day) markedly decreased the neointima/media ratio to the same extent. Furthermore, the neointima/media ratio in the injured artery of the ET(B)-deficient rat was significantly increased compared with that of the wild-type rat, and this increase was abolished by treatment with J-104132. These findings suggest that the inhibition of the ET(B) receptor system leads to an aggravation of neointimal hyperplasia after balloon injury, and the augmentation of ET(A)-mediated actions are responsible for the neointimal hyperplasia aggravated by the pharmacological blockade of ET(B) receptor or by its genetic deficiency. The antagonism of the ET(A) receptor system is essential for preventing restenosis after angioplasty.

Attenuation of experimental hepatopulmonary syndrome in endothelin B receptor-deficient rats.

Experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL) in rat is accompanied by increased lung vascular endothelial endothelin B (ETB) receptor expression and increased circulating levels of endothelin-1 (ET-1). The onset of HPS is hypothesized to be triggered by ET-1/ETB receptor activation of endothelial nitric oxide synthase (eNOS)-derived NO production in the pulmonary endothelium. However, whether functional pulmonary vascular ETB receptors are required for the development of experimental HPS is not defined. We evaluated the effects of vascular ETB receptor deficiency on the development of experimental HPS. The molecular and physiological alterations of HPS were compared in 2-wk CBDL wild-type and ETB receptor-deficient (transgenic sl/sl) rats. Relative to wild-type rats, basal hepatic and plasma ET-1 levels were elevated in sl/sl controls although, unlike wild-type animals circulating ET-1 levels, did not increase further after CBDL in sl/sl animals. In contrast to wild-type animals, ETB receptor-deficient rats did not develop increased Akt and eNOS expression and activation and did not develop gas exchange abnormalities of HPS after CBDL. There was a similar degree of pulmonary intravascular monocyte accumulation in both 2-wk CBDL sl/sl and wild-type animals. In conclusion, ETB receptor deficiency inhibits lung Akt/eNOS activation and prevents the onset of experimental HPS after CBDL. This effect is independent of inhibition of pulmonary intravascular monocyte accumulation. These results demonstrate that ET-1/ETB receptor signaling plays a key role in the initiation of experimental HPS.

Combined knockout of collecting duct endothelin A and B receptors causes hypertension and sodium retention.

The collecting duct (CD) endothelin (ET) system regulates blood pressure (BP) and Na excretion. CD-specific knockout (KO) of ET-1 causes hypertension, CD-specific KO of the ETA receptor does not alter BP, while CD-specific KO of the ETB receptor increases BP to a lesser extent than CD ET-1 KO. These findings suggest a paracrine role for CD-derived ET-1; however, they do not exclude compensation for the loss of one ET receptor by the other. To examine this, mice with CD-specific KO of both ETA and ETB receptors were generated (CD ETA/B KO). CD ETA/B KO mice excreted less urinary Na than controls during acute or chronic Na loading. Urinary aldosterone excretion and plasma renin concentration were similar during Na intake and both fell comparably during Na loading. On a normal sodium diet, CD ETA/B KO mice had increased BP, which increased further with high salt intake. The degree of BP elevation during normal Na intake was similar to CD ET-1 KO mice and higher than CD ETB KO animals. During 1 wk of Na loading, CD ETA/B KO mice had higher BPs than CD ETB KO, while BP was less than CD ET-1 KOs until the latter days of Na loading. These studies suggest that 1) CD ETA/B deficiency causes salt-sensitive hypertension, 2) CD ETA/B KO-associated Na retention is associated with failure to suppress the renin-angiotensin-aldosterone system, and 3) CD ETA and ETB receptors exerts a combined hypotensive effect that exceeds that of either receptor alone.

Deficiency in endothelin receptor B reduces proliferation of neuronal progenitors and increases apoptosis in postnatal rat cerebellum.

Endothelins regulate cellular functions in the mammalian brain through the endothelin receptors A and B (EDNRA and EDNRB). In this study, we investigated the role of EDNRB on cell proliferation in the cerebellum by using the spotting lethal (sl) rat, which carries a naturally occurring deletion in the EDNRB gene. Proliferating cells in the three genotypes, wild-type (+/+), heterozygous (+/sl) and homozygous mutant (sl/sl) rats were labelled by intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) at postnatal day 2. The density of BrdU-positive cells (per mm(2)) in the external germinal layer of sl/sl rats (Mean +/- SEM, 977 +/- 388) was significantly reduced compared to +/+ (4915 +/- 631) and +/sl (2304 +/- 557) rats. Subsequently, we examined the effects of EDNRB mutation on neural apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labelling assay. This showed that the density of apoptotic cells in the cerebella of sl/sl rats (9.3 +/- 0.5/mm(2)) was significantly more increased than +/+ rats (4 +/- 0.7). The expression of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) were measured with standard ELISA, but were unchanged in all genotypes. These results suggest that ENDRB mediates neural proliferation and have anti-apoptotic effects in the cerebellum of the postnatal rat, and that these effects are independent of changes in the expression of BDNF and GDNF. Our findings will lead to better understanding of the morphological changes in the cerebellum of Hirschsprung's disease patients with congenital EDNRB mutation.

Intracellular calcium mobilization of the aganglionic intestine in the endothelin B receptor gene-deficient rat.

BACKGROUND AND AIM: Up to now, numerous reports have analyzed the pathogenesis of Hirschsprung's disease (HD) by means of physiologic, pathologic, or molecular biologic methods. However, very little is still known about the smooth muscle cell itself. The endothelin B receptor gene-deficient (EDNRB(-/-)) rat, which is suitable for research of HD, has an aganglionic segment of the total colon. Our purpose is to investigate the myogenic mechanisms using simultaneous measurements of the intracellular Ca2+ concentration ([Ca2+]i) and tension and reverse transcriptase polymerase chain reaction for L-type Ca2+ channel (L-VOC) expression. METHODS: The muscle strips of the rat distal colon were loaded with a Ca2+ indicator dye, fura-PE3/AM, for 3 to 4 hours. The changes in the fluorescence intensity of Ca2+-fura-PE3 complex of the strips were monitored with a front surface fluorometer (CAM-230). The fluorescence intensities at 340- and 380-nm excitation and their ratio (F340/F380) were recorded as the level of [Ca2+]i. The comparison of L-VOC alpha1c subunit messenger RNA (mRNA) expression in both wild and homozygous rat was performed by reverse transcriptase polymerase chain reaction. RESULTS: The peak levels of force development induced by carbachol were 139.1% +/- 5.0% in EDNRB(-/-) rat, whereas the peak levels were 242.1% +/- 27.7% in EDNRB(+/+) rat. The changes in the [Ca2+]i elevation induced by carbachol were 101.7% +/- 12.2% in the homozygous rat, whereas these were 143.8% +/- 8.9% in the wild-type rat. Both results in the homozygous rat significantly decreased in comparison with those of the wild rat (P < .05). The expression of the L-VOC channel mRNA also decreased in the homozygous rat. CONCLUSIONS: This is the first report to show the [Ca2+]i mobilization in the smooth muscles of the rat model of HD. The decrease in both [Ca2+]i and force development was thus considered to be due to the decrease in the Ca2+ channel expression.

Involvement of the endothelin ET(B) receptor in gender differences in deoxycorticosterone acetate-salt-induced hypertension.

1. In the present study, we investigated the role of endothelin ET(B) receptors in gender differences in the development of deoxycorticosterone acetate (DOCA)-salt-induced hypertension by using the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the ET(B) receptor gene. 2. In wild-type rats, the elevation of systolic blood pressure (SBP) by DOCA-salt treatment for 4 weeks was extremely lower in females than in males, but this gender difference was partially attenuated in ovariectomized (OVX) animals. These alterations of SBP corresponded with vascular superoxide () production. 3. In homozygous (sl/sl) group, the SBP of male, intact female and OVX rats was markedly elevated by DOCA-salt treatment to the same extent, indicating that the gender difference in DOCA-salt-induced hypertension was abolished by the genetic ET(B) receptor deficiency. There were similar increases in the vascular endothelin (ET)-1 content in the three DOCA-salt-treated animal groups, but vascular production in male and OVX rats was much higher than that in intact females. 4. Daily oral administration of ABT-627, an ET(A) receptor antagonist, to sl/sl rats for 2 weeks suppressed the DOCA-salt-induced hypertension more efficiently in intact female rats than in male animals. 5. Thus, vascular oxidative stress is related, at least in part, to differences in the development of DOCA-salt-induced hypertension between male and female rats, but this gender difference is abolished by the genetic ET(B) receptor deficiency, suggesting that ET(B) receptor-mediated vasoprotective actions contribute to the gender differences seen. In addition, in both sexes, vascular ET-1 overproduction and the ET(A) receptor-mediated action seem to be responsible for the enhanced susceptibility to DOCA-salt hypertension in genetic ET(B) receptor deficiency.