Biotinylated endothelin as a probe for the endothelin receptor

Biotinylated derivatives of endothelin (ET)-1 were prepared by chemical modification of ET-1 with sulfosuccinimidyl 6-(biotinamido) hexanoate. Two major biotinylated ET analogs were purified by reversed-phase high performance liquid chromatography. Edman degradation indicated that the first eluting peptide contains one biotin residue on lysine at position 9, while the second derivative contains an additional biotin residue at position 1. Competition binding studies to mouse osteoblastic cell line MC3T3-E1 using ¹²⁵I-labeled ET-1 revealed IC₅₀ values of 5, 30 and 600 nM for native ET, the mono- and the dibiotinylated ET analog, respectively. A similar order of potency was obtained when these ET derivatives were examined for stimulation of DNA synthesis in MC3T3-E1 cells. In addition, incubation of MC3T3-E1 cells with the monobiotinylated ET and subsequent addition of rhodamine-avidin resulted in an evenly distributed fluorescence over the cell surface. The fluorescence observed was completely abolished in the presence of an excess of native ET. Thus the monobiotinylated ET proves to be useful for localization of the ET receptors.     

Ligand-dependent differences in the internalization of endothelin A and endothelin B receptor heterodimers

Endothelin-1 (ET-1) is a potent vasoactive peptide that acts on endothelin A (ET(A)) and endothelin B (ET(B)) receptors. Although both receptor subtypes are co-expressed in numerous cells, little is known about their ability to form heterodimers. Here we show that both receptors were co-immunoprecipitated with an ET(B)-specific antibody using extracts from HEK293 cells stably co-expressing a fusion protein consisting of a myc-tagged ET(A) receptor and CFP (ET(A)myc.CFP) and a fusion protein consisting of an ET(B) receptor and YFP (ET(B).YFP). Co-immunoprecipitation was also observed with extracts from HEK293 cells transiently co-expressing FLAG-tagged ET(B) and myc-tagged ET(A) receptors, thereby excluding that heterodimerization is mediated by the CFP/YFP moieties. Heterodimerization was further confirmed in fluorescence resonance energy transfer (FRET) analysis of HEK293 cells transiently co-expressing ET(A)myc.CFP and ET(B).YFP receptors. FRET efficiencies were between 12 and 18% in untreated and antagonist- or ET-1-treated cells, indicating constitutive heterodimerization. Prolonged stimulation (30 min) with the ET(B) receptor-selective agonist BQ3020 decreased FRET efficiency by 50%. This decrease was not observed when internalization was inhibited by co-expression of dominant-negative K44A.dynamin I or incubation with 450 mm sucrose. Enzyme-linked immunosorbent assay and laser scanning microscopy of cell clones stably co-expressing ET(A)myc.CFP/ET(B)flag.YFP receptors revealed a slower sequestration of the ET(B)flag.YFP receptors upon stimulation with ET-1 than with BQ3020. No difference in ET-1 or BQ3020-mediated sequestration was observed with cell clones expressing ET(B)flag.YFP receptors alone. The data suggest that ET(A) and ET(B) receptors form constitutive heterodimers, which show a slower sequestration upon stimulation with ET-1 than with BQ3020. Heterodimer dissociation along the endocytic pathway only occurs upon ET(B)-selective stimulation.     

Long inverted repeats are an at-risk motif for recombination in mammalian cells

Certain DNA sequence motifs and structures can promote genomic instability. We have explored instability induced in mouse cells by long inverted repeats (LIRs). A cassette was constructed containing a herpes simplex virus thymidine kinase (tk) gene into which was inserted an LIR composed of two inverted copies of a 1.1-kb yeast URA3 gene sequence separated by a 200-bp spacer sequence. The tk gene was introduced into the genome of mouse Ltk(-) fibroblasts either by itself or in conjunction with a closely linked tk gene that was disrupted by an 8-bp XhoI linker insertion; rates of intrachromosomal homologous recombination between the markers were determined. Recombination between the two tk alleles was stimulated 5-fold by the LIR, as compared to a long direct repeat (LDR) insert, resulting in nearly 10(-5) events per cell per generation. Of the tk(+) segregants recovered from LIR-containing cell lines, 14% arose from gene conversions that eliminated the LIR, as compared to 3% of the tk(+) segregants from LDR cell lines, corresponding to a >20-fold increase in deletions at the LIR hotspot. Thus, an LIR, which is a common motif in mammalian genomes, is at risk for the stimulation of homologous recombination and possibly other genetic rearrangements.     

Prokaryotic origins for the mitochondrial alternative oxidase and plastid terminal oxidase nuclear genes

The mitochondrial alternative oxidase is a diiron carboxylate quinol oxidase (Dox) found in plants and some fungi and protists, but not animals. The plastid terminal oxidase is distantly related to alternative oxidase and is most likely also a Dox protein. Database searches revealed that the alpha-proteobacterium Novosphingobium aromaticivorans and the cyanobacteria Nostoc sp. PCC7120, Synechococcus sp. WH8102 and Prochlorococcus marinus subsp. pastoris CCMP1378 each possess a Dox homolog. Each prokaryotic protein conforms to the current structural models of the Dox active site and phylogenetic analyses suggest that the eukaryotic Dox genes arose from an ancestral prokaryotic gene.     

Amino terminal domains of the NMDA receptor are organized as local heterodimers

The N-methyl-D-aspartate (NMDA) receptor, an obligate heterotetrameric assembly organized as a dimer of dimers, is typically composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Despite the crucial role that the NMDA receptor plays in the nervous system, the specific arrangement of subunits within the dimer-of-dimer assemblage is not conclusively known. Here we studied the organization of the amino terminal domain (ATD) of the rat GluN1/GluN2A and GluN1/GluN2B NMDA receptors by cysteine-directed, disulfide bond-mediated cross-linking. We found that GluN1 ATDs and GluN2 ATDs spontaneously formed disulfide bond-mediated dimers after introducing cysteines into the L1 interface of GluN2A or GluN2B ATD. The formation of dimer could be prevented by knocking out endogenous cysteines located near the L1 interface of GluN1. These results indicate that GluN1 and GluN2 ATDs form local heterodimers through the interactions in the L1-L1 interface and further demonstrate a dimer-of-heterodimer arrangement in GluN1/GluN2A and GluN1/GluN2B NMDA receptors.     

Therapeutic potential of endothelin receptor antagonists for chronic proteinuric renal disease in humans

Diabetes and arterial hypertension continue to be the main causes of chronic renal failure in 2010, with a rising prevalence in part due to the worldwide obesity epidemic. Proteinuria is a main feature of chronic renal disease and mediated by defects in the glomerular filtration barrier and is as a good predictor of cardiovascular events. Indeed, chronic renal disease due to glomerulosclerosis is one of the important risk factors for the development of coronary artery disease and stroke. Glomerulosclerosis develops in response to inflammatory activation and increased growth factor production. Preclinical and first preliminary clinical studies provide strong evidence that endogenous endothelin-1 (ET-1), a 21-amino-acid peptide with strong growth-promoting and vasoconstricting properties, plays a central role in the pathogenesis of proteinuria and glomerulosclerosis via activation of its ET_A subtype receptor involving podocyte injury. These studies have not only shown that endothelin participates in the disease processes of hypertension and glomerulosclerosis but also that features of chronic renal disease such as proteinuria and glomerulosclerosis are reversible processes. Remarkably, the protective effects of endothelin receptors antagonists (ERAs) are present even on top of concomitant treatments with inhibitors of the renin–angiotensin system. This review discusses current evidence for a role of endothelin for proteinuric renal disease and podocyte injury in diabetes and arterial hypertension and reviews the current status of endothelin receptor antagonists as a potential new treatment option in renal medicine.     

Correlation between apolipoprotein B and endothelin-1-induced vasoconstriction in humans

Low-density lipoprotein (LDL) concentrations have been linked to altered responses to endogenous vasodilators and vasoconstrictors. We evaluated retrospectively the relationship between LDL and vasoconstrictor (endothelin-1, phenylephrine) responsiveness of the forearm vasculature in 15 elderly healthy volunteers with apolipoprotein B and LDL levels in the normal range. In contrast to phenylephrine, changes in forearm vascular resistance induced by endothelin-1 were correlated with apolipoprotein B, LDL, and total cholesterol concentrations in women but not in men. These findings might suggest that lipids may increase vascular tone through both impaired endothelial vasodilation and increased vasoconstriction to endothelin-1 at least in women.     

Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment

Background

Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment.

Methods

Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β₁-adrenergic receptor blocker atenolol for twelve weeks.

Results

The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele.

Conclusions

Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.     

Phenotypic variation in a family with mutations in two Hirschsprung-related genes (RET and endothelin receptor B)

Hirschsprung disease is a congenital malformation affecting 1 in 5000 live births. The absence of parasympathetic neuronal ganglia (Meissner, Auerbach) in the hindgut results in poor coordination of peristaltic movement, and a varying degree of constipation. Four different genes have been implicated in the pathogenesis of Hirschsprung disease: the RET tyrosine kinase receptor gene; one of its ligands, the glial cell line-derived neurotrophic factor (GDNF) gene; the endothelin receptor B (EDNRB) gene; and its ligand, endothelin-3 (EDN3). Recently, combinations of mutations in two of these genes (RET and GDNF) have been reported in Hirschsprung patients. We report a family with missense mutations in both the RET gene (R982C) and the EDNRB gene (G57S). In this family, three out of five members have the two mutations, but only one, a boy, has the Hirschsprung disease phenotype. This illustrates the complexity of the molecular background of Hirschsprung disease. Received: 23 January 1998 / Accepted: 24 March 1998     

Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans

1. Download : Download high-res image (238KB)
2. Download : Download full-size image