Evolution of the endothelin pathway drove neural crest cell diversification.

Neural crest cells (NCCs) are migratory, multipotent embryonic cells that are unique to vertebrates and form an array of clade-defining adult features. The evolution of NCCs has been linked to various genomic events, including the evolution of new gene-regulatory networks1,2, the de novo evolution of genes3 and the proliferation of paralogous genes during genome-wide duplication events4. However, conclusive functional evidence linking new and/or duplicated genes to NCC evolution is lacking. Endothelin ligands (Edns) and endothelin receptors (Ednrs) are unique to vertebrates3,5,6, and regulate multiple aspects of NCC development in jawed vertebrates7-10. Here, to test whether the evolution of Edn signalling was a driver of NCC evolution, we used CRISPR-Cas9 mutagenesis11 to disrupt edn, ednr and dlx genes in the sea lamprey, Petromyzon marinus. Lampreys are jawless fishes that last shared a common ancestor with modern jawed vertebrates around 500 million years ago12. Thus, comparisons between lampreys and gnathostomes can identify deeply conserved and evolutionarily flexible features of vertebrate development. Using the frog Xenopus laevis to expand gnathostome phylogenetic representation and facilitate side-by-side analyses, we identify ancient and lineage-specific roles for Edn signalling. These findings suggest that Edn signalling was activated in NCCs before duplication of the vertebrate genome. Then, after one or more genome-wide duplications in the vertebrate stem, paralogous Edn pathways functionally diverged, resulting in NCC subpopulations with different Edn signalling requirements. We posit that this new developmental modularity facilitated the independent evolution of NCC derivatives in stem vertebrates. Consistent with this, differences in Edn pathway targets are associated with differences in the oropharyngeal skeleton and autonomic nervous system of lampreys and modern gnathostomes. In summary, our work provides functional genetic evidence linking the origin and duplication of new vertebrate genes with the stepwise evolution of a defining vertebrate novelty.

Ubiquitin Ligase TRIM62 Regulates CARD9-Mediated Anti-fungal Immunity and Intestinal Inflammation.

CARD9 is a central component of anti-fungal innate immune signaling via C-type lectin receptors, and several immune-related disorders are associated with CARD9 alterations. Here, we used a rare CARD9 variant that confers protection against inflammatory bowel disease as an entry point to investigating CARD9 regulation. We showed that the protective variant of CARD9, which is C-terminally truncated, acted in a dominant-negative manner for CARD9-mediated cytokine production, indicating an important role for the C terminus in CARD9 signaling. We identified TRIM62 as a CARD9 binding partner and showed that TRIM62 facilitated K27-linked poly-ubiquitination of CARD9. We identified K125 as the ubiquitinated residue on CARD9 and demonstrated that this ubiquitination was essential for CARD9 activity. Furthermore, we showed that similar to Card9-deficient mice, Trim62-deficient mice had increased susceptibility to fungal infection. In this study, we utilized a rare protective allele to uncover a TRIM62-mediated mechanism for regulation of CARD9 activation.

Endothelin B receptor deficiency predisposes to pulmonary edema formation via increased lung vascular endothelial cell growth factor expression.

Endothelin (ET) may contribute to pulmonary edema formation, particularly under hypoxic conditions, and decreases in ET-B receptor expression can lead to reduced ET clearance. ET increases vascular endothelial cell growth factor (VEGF) production in vitro, and VEGF overexpression in the lung causes pulmonary edema in vivo. We hypothesized that pulmonary vascular ET-B receptor deficiency leads to increased lung ET, that excess ET increases lung VEGF levels, promoting pulmonary edema formation, and that hypoxia exaggerates these effects. We studied these hypotheses in ET-B receptor-deficient rats. In normoxia, homozygous ET-B-deficient animals had significantly more lung vascular leak than heterozygous or control animals. Hypoxia increased vascular leak regardless of genotype, and hypoxic ET-B-deficient animals leaked more than hypoxic control animals. ET-B-deficient animals had higher lung ET levels in both normoxia and hypoxia. Lung HIF-1alpha and VEGF content was greater in the ET-B-deficient animals in both normoxia and hypoxia, and both HIF-1alpha and VEGF levels were reduced by ET-A receptor antagonism. Both ET-A receptor blockade and VEGF antagonism reduced vascular leak in hypoxic ET-B-deficient animals. We conclude that ET-B receptor-deficient animals display an exaggerated lung vascular protein leak in normoxia, that hypoxia exacerbates that leak, and that this effect is in part attributable to an ET-mediated increase in lung VEGF content.

Unique endothelin receptor binding in kidneys of ETB receptor deficient rats.

Gariepy and colleagues (Gariepy CE, Williams SC, Richardson JA, Hammer RE, and Yanagisawa M. J Clin Invest 102: 1092-1101, 1998.) developed rescued spotting-lethal 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/+) and sl/sl ETB receptor-deficient rats. 125I-labeled ET-1 binding in sl/sl cortex and outer medulla was significantly lower than cortex and outer medulla from sl/+ rats. In contrast to sl/+ rats, [125I]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/+ rats also demonstrated an abundance of ETB receptors. Surprisingly, however, we also observed significant [125I]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/+ 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.

Role of endothelin ET(B) receptors in the renal hemodynamic and excretory responses to big endothelin-1.

We determined the role of endothelin ET(B) receptor in the renal hemodynamic and excretory responses to big endothelin-1, using A-192621, a selective endothelin ET(B) receptor antagonist and the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the endothelin ET(B) receptor gene. An intravenous injection of big endothelin-1 produced a hypertensive effect, which is greater in wild-type (+/+) rats pretreated with A-192621 and in homozygous (sl/sl) rats. Big endothelin-1 markedly increased urine flow, urinary excretion of sodium and fractional excretion of sodium in wild-type rats treated with the vehicle. These excretory responses to big endothelin-1 were markedly reduced by pharmacological endothelin ET(B) receptor blockade. On the other hand, big endothelin-1 injection to the endothelin ET(B) receptor-deficient homozygous animals resulted in a small diuretic effect. When renal perfusion pressure was protected from big endothelin-1-induced hypertension by an aortic clamp, the excretory responses in vehicle-treated wild-type rats were markedly attenuated. In homozygous or A-192621-treated wild-type rats, there was a small but significant decreasing effect in urine flow. In addition, big endothelin-1 significantly elevated nitric oxide (NO) metabolite production in the kidney of wild-type rats but not in the homozygous rats. We suggest that the diuretic and natriuretic responses to big endothelin-1 consist of pressure-dependent and pressure-independent effects and that the increased NO production via the activation of endothelin ET(B) receptors in the kidney is closely related to the big endothelin-1-induced excretory responses.

Role of endothelin B receptor in the pathogenesis of ischemic acute renal failure.

This study evaluated the role of endothelin B (ET ) receptor-mediated action in the development and maintenance of ischemic acute renal failure (ARF), using the spotting-lethal ( ) rat that carries a naturally occurring deletion in the ET receptor gene. Because homozygous ( ) rats die shortly after birth due to congenital distal intestinal aganglionosis, genetic rescue of rats from the developmental defect using a dopamine-beta-hydroxylase (DbetaH)-ET transgene was performed to produce ET -deficient adult rats. Rescued homozygous (DbetaH-ET ) and wild-type (DbetaH-ET +/+) rats were subjected to ischemic ARF by clamping the renal pedicle for 45 min followed by reperfusion. At 24 h after the reperfusion, renal glomerular dysfunction and histologic damage, such as proteinaceous casts in tubuli, were markedly and observed equally in homozygous and wild-type groups, and these renal injuries gradually recovered. However, when the ischemia/reperfusion-induced renal injury was examined 7 days after the reperfusion, the recovery in homozygous ARF rats was obviously delayed compared with the wild-type animals. Two of the eight homozygous ARF rats died within 3 days after the reperfusion. Increment of renal endothelin-1 content after the ischemia/reperfusion was more marked in homozygous than in wild-type rats. Thus, ET receptor-mediated actions do not play an important role in the development of ischemic ARF but may be involved in the recovery process from ischemia/reperfusion-induced renal injury.

Abnormalities of the enteric nervous system in heterozygous endothelin B receptor deficient (spotting lethal) rats resembling intestinal neuronal dysplasia.

BACKGROUND: A homozygous mutation of the endothelin B receptor (EDNRB) gene in spotting lethal (sl/sl) rats leads to Hirschsprung's disease (HSCR) with long segmented aganglionosis. However, the effects on the development of the enteric nervous system (ENS) promoted by a heterozygous mutation of the EDNRB gene are not known. The present study aimed to describe and morphometrically assess the phenotypic abnormalities of the ENS in heterozygous (+/sl) EDNRB deficient rats in comparison with homozygous (sl/sl) EDNRB deficient and wild-type (+/+) rats. METHODS: The distal small intestine, caecum, and colon were obtained from sl/sl, +/sl, and +/+ rats. To demonstrate the three dimensional organisation of the ENS, the intestinal wall was microdissected into wholemounts and incubated against the pan-neuronal marker protein gene product 9.5. Assessment of the ENS included morphometric quantification of ganglionic size and density, the number of nerve cells per ganglia, and the diameter of nerve fibre strands within both the myenteric and submucous plexus. RESULTS: Sl/sl rats were characterised by complete aganglionosis resembling the same histopathological features observed in patients with HSCR. +/sl rats revealed more subtle abnormalities of the ENS: the submucous plexus was characterised by a significantly increased ganglionic size and density, and the presence of hypertrophied nerve fibre strands. Morphometric evaluation of the myenteric plexus did not show statistically significant differences between +/sl and +/+ rats. CONCLUSIONS: In contrast with sl/sl rats, +/sl rats display non-aganglionated malformations of the ENS. Interestingly, these innervational abnormalities resemble the histopathological criteria for intestinal neuronal dysplasia (IND). Although IND has been described in several intestinal motility disorders, the concept of a clearly defined clinical-histopathological entity is still controversially discussed. The present findings support the concept of IND based on clearly defined morphological criteria suggesting a genetic link, and thus may provide a model for human IND. Furthermore, the data underline the critical role of the "gene dose" for the phenotypic effects promoted by the EDNRB/EDN3 system and confirm that the development of the ENS is not an "all or none" phenomenon.

Endothelin B receptor deficiency augments neuronal damage upon exposure to hypoxia-ischemia in vivo.

The role of functional endothelin-B (ETB)-receptors on neuronal survival upon hypoxia-ischemia (HI) has been investigated in 14-day-old ETB-receptor-deficient spotting lethal (sl/sl) and wildtype (+/+) rats. Carotid ligation followed by exposure to 8% oxygen for 2 h produced distinct cortical and hippocampal neuronal damage. Damage severity 24 h after HI was mild to intermediate in +/+ rats whereas large cortical infarcts and profound apoptosis of the hippocampus evolved in sl/sl rats. The number of apoptotic cells in the dentate 24 h after HI amounted to 30 +/- 7 cells/0.1 mm(2) in sl/sl compared to 9 +/- 3 cells/0.1 mm(2) in wildtype rats (mean +/- S.E.M., n=10-11, P=0.0093). In-vitro hypoxia (15 h) resulted in a comparable increase in cell death in primary pure neuronal hippocampal cultures from both groups (49.8 +/- 1.6% in sl/sl, 51.4 +/- 0.9% in +/+, mean +/- S.E.M., n=5, P=0.0560). To conclude, absence of functional ETB receptors is associated with an increased susceptibility to HI in-vivo, which is not intrinsic to neurons. Antagonism of ETB receptors seems not to be desirable in ischemic stroke.

Role of the astrocytic ET(B) receptor in the regulation of extracellular endothelin-1 during hypoxia.

Astrocytes are known to possess an effective endothelin (ET) eliminatory system which involves astrocytic ET(A) and ET(B) receptors and may become particularly relevant under pathophysiological conditions. The present study has therefore been designed to explore the effect of standardized hypoxia on extracellular concentrations of endothelin-1 (ET-1) and on endothelin-converting enzyme (ECE) activity in primary rat astrocytes genetically (sl/sl) or experimentally (dexamethasone) deficient in ET(B) receptors. The results revealed (1) a hypoxia-mediated decrease of extracellular ET-1 in wildtype astrocytes (+/+) that was not observed in ET(B)-deficient (sl/sl) cultures; (2) an ET receptor antagonist-induced increase in ET-1 in the media of both genotypes with further elevation upon hypoxia in +/+ cultures only; (3) augmentation of the dexamethasone-induced increase in extracellular ET-1 by hypoxia in +/+, but not in sl/sl cultures; (4) synergistic reduction of ET(B) gene transcription by hypoxia and dexamethasone; and (5) significant increases in endothelin-converting enzyme activity in the presence of hypoxia. To conclude, hypoxia stimulates astrocytic release of mature ET-1. This stimulation is (over)compensated for by increased ET-1 binding to functional ET(B) receptors. ET(B) deficiency, whether genetic or experimentally induced, impairs elimination of extracellular ET-1.

Endothelin B receptor deficiency potentiates ET-1 and hypoxic pulmonary vasoconstriction.

Endothelin (ET)-1 contributes to the regulation of pulmonary vascular tone by stimulation of the ET(A) and ET(B) receptors. Although activation of the ET(A) receptor causes vasoconstriction, stimulation of the ET(B) receptors can elicit either vasodilation or vasoconstriction. To examine the physiological role of the ET(B) receptor in the pulmonary circulation, we studied a genetic rat model of ET(B) receptor deficiency [transgenic(sl/sl)]. We hypothesized that deficiency of the ET(B) receptor would predispose the transgenic(sl/sl) rat lung circulation to enhanced pulmonary vasoconstriction. We found that the lungs of transgenic(sl/sl) rats are ET(B) deficient because they lack ET(B) mRNA in the pulmonary vasculature, have minimal ET(B) receptors as determined with an ET-1 radioligand binding assay, and lack ET-1-mediated pulmonary vasodilation. The transgenic(sl/sl) rats have higher basal pulmonary arterial pressure and vasopressor responses to brief hypoxia or ET-1 infusion. Plasma ET-1 levels are elevated and endothelial nitric oxide synthase protein content and nitric oxide production are diminished in the transgenic(sl/sl) rat lung. These findings suggest that the ET(B) receptor plays a major physiological role in modulating resting pulmonary vascular tone and reactivity to acute hypoxia. We speculate that impaired ET(B) receptor activity can contribute to the pathogenesis of pulmonary hypertension.

Exaggerated vascular and renal pathology in endothelin-B receptor-deficient rats with deoxycorticosterone acetate-salt hypertension.

BACKGROUND: Endothelin (ET)-1 plays an important role in the pathogenesis of deoxycorticosterone acetate (DOCA)-salt-induced hypertension. We evaluated the pathological role of ET(B) receptors in DOCA-salt-induced hypertension, cardiovascular hypertrophy, and renal damage by using the spotting-lethal (sl) rat, which carries a naturally occurring deletion in the ET(B) receptor gene. METHODS AND RESULTS: Homozygous (sl/sl) rats exhibit abnormal development of neural crest-derived epidermal melanocytes and the enteric nervous system, and they do not live beyond 1 month because of intestinal aganglionosis and intestinal obstruction. The dopamine ss-hydroxylase (DssH) promoter was used to direct ET(B) transgene expression in sl/sl rats to support normal enteric nervous system development. DssH-ET(B) sl/sl rats live into adulthood and are healthy, expressing ET(B) receptors in adrenal glands and other adrenergic neurons. When homozygous (sl/sl) and wild-type (+/+) rats, all of which were transgenic, were treated with DOCA-salt, homozygous rats exhibited earlier and higher increases in systolic blood pressure than did wild-type rats. Chronic treatment with ABT-627, an ET(A) receptor antagonist, completely suppressed DOCA-salt-induced hypertension in both groups. Renal dysfunction and histological damage were more severe in homozygous than in wild-type rats. Marked vascular hypertrophy was observed in homozygous rats than in wild-type rats. Renal and vascular injuries were significantly improved by ABT-627. In DOCA-salt-treated homozygous rats, there were notable increases in renal, urinary, and aortic ET-1, all of which were normalized by ABT-627. CONCLUSIONS: ET(B)-mediated actions are protective in the pathogenesis of DOCA-salt-induced hypertension. Enhanced ET-1 production and ET(A)-mediated actions are responsible for the increased susceptibility to DOCA-salt hypertension and tissue injuries in ET(B) receptor-deficient rats.

Salt-sensitive hypertension in endothelin-B receptor-deficient rats.

The role of the endothelin-B receptor (ET(B)) in vascular homeostasis is controversial because the receptor has both pressor and depressor effects in vivo. Spotting lethal (sl) rats carry a naturally occurring deletion in the ET(B) gene that completely abrogates functional receptor expression. Rats homozygous for this mutation die shortly after birth due to congenital distal intestinal aganglionosis. Genetic rescue of ET(B)(sl/sl) rats from this developmental defect using a dopamine--hydroxylase (DBH)-ET(B) transgene results in ET(B)-deficient adult rats. On a sodium-deficient diet, DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats both exhibit a normal arterial blood pressure, but on a high-sodium diet, the former are severely hypertensive. We find no difference in plasma renin activity or plasma aldosterone concentration between salt-fed wild-type, DBH-ET(B);ET(B)(+/+) or DBH-ET(B);ET(B)(sl/sl) rats, and acute responses to intravenous L-NAME and indomethacin are similar between DBH-ET(B);ET(B)(sl/sl) and DBH-ET(B);ET(B)(+/+) rats. Irrespective of diet, DBH-ET(B);ET(B)(sl/sl) rats exhibit increased circulating ET-1, and, on a high-sodium diet, they show increased but incomplete hypotensive responses to acute treatment an ET(A)-antagonist. Normal pressure is restored in salt-fed DBH-ET(B);ET(B)(sl/sl) rats when the epithelial sodium channel is blocked with amiloride. We conclude that DBH-ET(B);ET(B)(sl/sl) rats are a novel single-locus genetic model of severe salt-sensitive hypertension. Our results suggest that DBH-ET(B);ET(B)(sl/sl) rats are hypertensive because they lack the normal tonic inhibition of the renal epithelial sodium channel.

Enhanced blood pressure sensitivity to DOCA-salt treatment in endothelin ET(B) receptor-deficient rats.

The role of endothelin ET(B) receptor-mediated action in the development and maintenance of deoxycorticosterone acetate (DOCA)-salt-induced hypertension was evaluated using the spotting-lethal (sl) rat which carries a naturally occurring deletion in the ET(B) receptor gene. Homozygous (sl/sl) rats treated with DOCA-salt for 1 week exhibited an earlier onset of hypertension than heterozygous (sl/+) and wild-type (+/+) rats (systolic blood pressure, SBP; 156.7+/-3.4 versus 128.8+/-5.3 and 132.9+/-3.7 mmHg, respectively). Four weeks after the start of DOCA-salt treatment, homozygous rats developed marked hypertension, with a SBP of 206. 0+/-4.5 mmHg, compared with 184.8+/-10.7 mmHg in heterozygous and 164.3+/-4.8 mmHg in wild-type rats. Cardiovascular hypertrophy and renal dysfunction observed after 4-weeks treatment with DOCA-salt were more severe in homozygous rats, compared to wild-type and heterozygous animals. These evidences support strongly the view that ET(B) receptor-mediated actions are a protective factor in the pathogenesis of DOCA-salt-induced hypertension.

Phenotype, intestinal morphology, and survival of homozygous and heterozygous endothelin B receptor--deficient (spotting lethal) rats.

BACKGROUND/PURPOSE: Spotting lethal (sl) rats, a model for Hirschsprung's disease, recently have been found to carry a deletion in the endothelin B (ET(B)) gene, causing functional lack of ET(B) receptors. The ET(B) receptor mediates, together with and in counterbalance to the ET(A) receptor, endothelin actions on vessels, cell proliferation, and migration. The authors investigated the effect of homozygosity (sI/sI) or heterozygosity (+/sl) on phenotype, intestinal morphology, and survival. METHODS: Weight, circumference, and serum albumin were measured. Histological tests of major organs and immunoperoxidase reaction for Peripherin, glial fibrillary acid protein (GFAP), and S-100 in small and large intestine were performed. Peripherin-immunostained sections of colon and jejunum were analyzed morphometrically. Screening for sepsis included search for enterocolitis, bacterial infection, endotoxin, and iNOS mRNA. RESULTS: Sl/sl rats died within 4 weeks of life, showing an early and a later death group. Serum albumin levels were decreased in sl/sl rats, whereas signs of sepsis were rare. Immunostaining uncovered alterations in nerve and glial cells in the whole gut of sl/sl rats, and to a subtle degree also in +/sl rats, which appear clinically normal. Morphometric quantification yielded statistically significant alterations in sl/sl rats only. No obvious abnormalities were found in other organs. CONCLUSIONS: Sl/sl rats die from malnutrition rather than sepsis, too early for ischemic complications to occur. Rats of the later death group are a suitable model for studying the ET8 receptor in vivo. Subtle abnormalities in the enteric nervous system of heterozygous rats underline the critical role of the "gene dose" for functional compensation.

Endothelin b receptor deficiency is associated with an increased rate of neuronal apoptosis in the dentate gyrus.

The dentate gyrus retains neuronal proliferative potential throughout life. Using immature endothelin B receptor-deficient (sl/sl) rats, a rabbit model of pneumococcal meningitis and autopsy brains from humans who died from pneumococcal meningitis, we explored the role of endothelin B receptors in physiological and pathological neuronal apoptosis in the dentate gyrus. At postnatal days 3-4, the rate of apoptosis in the dentate gyrus was high in all rats, declining to low levels in wild-type rats (+/+) on days 14 and 22, but remaining high in both homozygous (sl/sl) and heterozygous (sl/+) endothelin B receptor-deficient rats. Increased apoptosis was not significantly compensated for by neuronal proliferation. Hippocampal neuronal cultures also exhibited genotype-dependent apoptosis with the highest rate in neurons from homozygous endothelin B receptor-deficient (sl/sl) rats. In rabbit and human pneumococcal meningitis, increased apoptosis in the dentate gyrus was associated with loss of neuronal endothelin B receptor immunoreactivity. In conclusion, endothelin B receptors appear to act as neuronal survival factors in the dentate gyrus in rodents and man, both during postnatal development and under pathological conditions.

Heterozygous knock-Out of ET(B) receptors induces BQ-123-sensitive hypertension in the mouse.

Homozygous knock-out of ET(A) or ET(B) receptor genes results in lethal developmental phenotypes in the mouse. Such deleterious phenotypes do not occur in heterozygous littermates. However, it remains to be determined whether mice partially defective in ET(A) or ET(B) receptors display significant alterations in their responses to exogenous or endogenous endothelin-1 (ET-1). Furthermore, the anesthetized ET(B) (+/-) knock-out mice showed a significantly higher mean arterial blood pressure than the ET(A) (+/-) knock-out or their wild-type littermates. The pressor response to ET-1 but not to a selective ET(B) agonist, IRL-1620, was significantly reduced in the ET(A) (+/-) knock-out mice. In ET(B) (+/-) knock-out mice, the pressor effect of IRL-1620 was more markedly altered than those induced by ET-1. In wild-type mice, both ET(A) and ET(B) receptors were found to be involved in the pressor effect of ET-1, as confirmed by the significant and specific antagonism induced by either BQ-123 (ET(A) antagonist) or BQ-788 (ET(B) antagonist). Also, ET(A)-selective or mixed ET(A)/ET(B)- but not ET(B)-selective antagonists reversed the hypertensive state of the ET(B) (+/-) knock-out mice to the level of wild-type littermates. Finally, radiolabeled ET-1 plasmatic clearance was altered in ET(B) (+/-) but not ET(A) (+/-) knock-out mice when compared with wild-type animals. Thus, heterozygous knock-out of ET(B) receptors results in a hypertensive state, suggesting an important physiological role for that particular receptorial entity in opposing the endogenous ET-1-dependent pressor effects in the mouse.

Spatially restricted hypopigmentation associated with an Ednrbs-modifying locus on mouse chromosome 10.

We have used the varied expressivity of white spotting (hypopigmentation) observed in intrasubspecific crosses of Ednrb(s) mice (Mayer Ednrb(s)/Ednrb(s) and C3HeB/FeJ Ednrb(s)/Ednrb(s)) to analyze the effects of modifier loci on the patterning of hypopigmentation. We have confirmed that an Ednrb(s) modifier locus is present on mouse Chromosome 10. This locus is now termed k10, using the nomenclature established by Dunn in 1920. The k10(Mayer) allele is a recessive modifier that accounts for almost all of the genetic variance of dorsal hypopigmentation. Using intercross analyses we identified a second allele of this locus or a closely linked gene termed k10(C3H). The k10(C3H) allele is semidominant and is associated with the penetrance and expressivity of a white forelock phenotype similar to that seen in Waardenburg syndrome. Molecular linkage analysis was used to determine that the k10 critical interval was flanked by D10Mit10 and D10Mit162/D10Mit122 and cosegregates with mast cell growth factor (Mgf). Complementation crosses with a Mgf(Sl) allele (a 3-5-cM deletion) confirm the semidominant white forelock feature of the k10(C3H) allele and the dorsal spotting feature of K10(Mayer) allele. MgF was assessed as a candidate gene for k10(Mayer) and k10(C3H) by sequence and genomic analyses. No molecular differences were observed between the Mayer and C57BL/6J alleles of MgF; however, extensive genomic differences were observed between the C3HeB/FeJ and C57BL/6J alleles. This suggests that alteration of MgF expression in C3H mice may account for the k10(C3H) action on white forelock hypopigmentation. Crosses of Ednrb(s) with Kit(WJ-2) (the receptor for MGF)-deficient mice confirmed the hypothesis that synergistic interaction between the Endothelin and MGF signaling pathways regulates proper neural crest-derived melanocyte development in vivo.

Endothelin B receptor-deficient rats as a subtraction model to study the cerebral endothelin system.

Endothelins, due to their potent vasoactivity and mitogenicity, appear to play an important role in the brain, where all components of the endothelin system, peptides, receptors and converting enzyme, are expressed. To further elucidate the role of the cerebral endothelin system, astrocytes and cerebral vessels from sl/sl rats, devoid of functional endothelin B receptors, have been employed. Astrocytes from sl/sl rats display the following abnormalities as compared to wild-type (+/+) cells: (i) elevated basal extracellular endothelin-1 levels; (ii) exclusive presence of functional endothelin A receptors; (iii) increased extracellular endothelin-1 levels upon endothelin A receptor blockade; (iv) augmented basal endothelin-converting enzyme activity; (v) altered calcium response to endothelin-1. The basilar artery of sl/sl rats shows an enhanced constricting response to endothelin-1 and fails to dilate in response to endothelin-3, shifting the endothelin vasomotor balance to constriction. In conclusion, endothelin B receptors may be essential for restricting extracellular endothelin-1 levels in the brain, as well as for a balanced cerebral vasomotor action of endothelins.

Both ET(A) and ET(B) receptors are involved in mitogen-activated protein kinase activation and DNA synthesis of astrocytes: study using ET(B) receptor-deficient rats (aganglionosis rats).

Endothelin (ET) is known to be a potent mitogen in astrocytes. However, the contribution and signalling pathway of ET(A) and/or ET(B) receptor to the proliferation of astrocytes remain unclear. We investigated ET-induced DNA synthesis in astrocytes using ET(B) receptor-deficient mutant rats (aganglionosis rats: sl/sl). Western blotting with anti-ET receptor subtype-specific antibodies and Scatchard analysis of binding revealed that ET(B) receptor expression in astrocytes depended on gene dosage (+/+: sl/+: sl/sl=2: 1:0), whereas ET(A) receptor expression was unchanged among the three genotypes. ET-1 (10 nM) stimulated [3H]thymidine incorporation and mitogen-activated protein kinase (MAP kinase) activity not only in +/+ via both ET(A) and ET(B) receptors, but also in sl/sl astrocytes via ET(A) receptor with about half the extent of those observed in +/+ astrocytes. Treatment with pertussis toxin (PTX) suppressed the ET-1-induced increases in the incorporation and MAP kinase activity in +/+, but not sl/sl astrocytes, indicating that the ET(B) receptor-, but not the ET(A) receptor-, mediated pathway to DNA synthesis involves PTX-sensitive G proteins, e.g. Gi and/or Go (Gi/o). In +/+ astrocytes, ET-1 (1 nM) stimulated cAMP accumulation, and the ET(B) receptor-selective agonist IRL 1620 (1 nM) suppressed 10 microM forskolin-induced cAMP accumulation, suggesting Gs coupling to the ET(A) receptor and Gi/o coupling to the ET(B) receptor. On the other hand, ET-1 did not increase cAMP accumulation in sl/sl astrocytes, although ET-1 (1 nM) suppressed the forskolin-induced response, suggesting Gi/o coupling to the ET(A) receptor. Our results suggest the possibility that the selectivity of G protein for ET(A) receptor is changed from Gs to Gi/o in ET(B) receptor-deficient astrocytes.