Mark Pullen

Upregulation of renal endothelin receptors in rats with cyclosporine A-induced nephrotoxicity

Measurement of endothelin receptors by binding assay was performed in rats treated with cyclosporine A (CYA). Cyclosporine A administration at 50 mg/kg i.p. for 4 days resulted in renal function impairment as indicated by a significant increase in serum creatinine concentration (from 0.46 +/- 0.02 to 0.61 +/- 0.03 mg/dl. P less than 0.01) and a significant decrease in 24 h creatinine clearance (from 0.65 +/- 0.04 to 0.41 +/- 0.02 ml/min per 100 g, P less than 0.01). Renal endothelin (ET) receptor density was significantly higher in CYA-treated rats (312 +/- 34 vs. 196 +/- 26 fmol/mg protein, P less than 0.01). These data support the possible involvement for endothelin in the increased renal vascular resistance associated with CYA-induced nephrotoxicity.

GW427353 (Solabegron), a Novel, Selective β3-Adrenergic Receptor Agonist, Evokes Bladder Relaxation and Increases Micturition Reflex Threshold in the Dog

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by β3-adrenergic receptors (ARs) in human bladder. Thus, the use of selective β3-AR agonists in the pharmacological treatment of overactive bladder is being explored. The present studies investigated the effects of a novel selective β3-AR agonist, (R)-3′-[[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]ethyl]amino]-[1,1′-biphenyl]-3-carboxylic acid (GW427353; solabegron) on bladder function in the dog using in vitro and in vivo techniques. GW427353 stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human β3-AR, with an EC50 value of 22 ± 6 nM and an intrinsic activity 90% of isoproterenol. At concentrations of 10,000 nM, GW427353 produced a minimal response in cells expressing either β1-ARs or β2-ARs (maximum response <10% of that to isoproterenol). In dog isolated bladder strips, GW427353 evoked relaxation that was attenuated by the nonselective β-AR antagonist bupranolol and 1-(2-ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2-propanol (SR59230A) (reported to have β3-AR antagonist activity). The relaxation was unaffected by atenolol, a selective β1-AR antagonist, or (±)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI 118551), a selective β2-AR antagonist. GW427353 increased the volume required to evoke micturition in the anesthetized dog following acetic acid-evoked bladder irritation, without affecting the ability of the bladder to void. GW427353-evoked effects on bladder parameters in vivo were inhibited by bupranolol. The present study demonstrates that selective activation of β3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.

Molecular Characterization of a Novel Human Endothelin Receptor Splice Variant

Endothelin receptors are widely distributed throughout a number of tissues. A novel ETB receptor splice variant (ETB-SVR) was identified from a human placental cDNA library. Sequence analysis indicated that the ETB-SVR is 436 amino acids long and shares 91% identity to the human ETB-R. Northern blot analysis indicated an mRNA species of 2.7 kilobases, which is expressed in the lung, placenta, kidney, and skeletal muscle. Ligand binding studies of the cloned ETB-SVR and ETB-R receptors expressed in COS cells showed that ET peptides exhibited similar potency in displacing 125I-ET-1 binding. Functional studies showed that ET-1, ET-3, and sarafotoxin 6c displayed similar potencies for inositol phosphates accumulation in ETB-R-transfected COS cells, whereas no increase in inositol phosphate accumulation was observed in ETB-SVR-transfected cells. In addition, exposure of ETB-R-transfected cells to ET-1 caused an increase in the intracellular acidification rate whereas ETB-SVR-transfected cells did not respond to ET-1. These data suggest that the ETB-SVR and ETB-R are functionally distinct and the difference in the amino acid sequences between the two receptors may determine functional coupling. Availability of cDNA clones for endothelin receptors can facilitate our understanding of the role of ET in the pathophysiology of various diseases.

Effect of nifedipine on cyclosporine A-induced nephrotoxicity, urinary endothelin excretion and renal endothelin receptor number

The aim of the present study was to determine the effect of a calcium channel blocker on renal function, urinary endothelin excretion and endothelin receptor number in rats. Administration of cyclosporine resulted in a significant impairment of renal function when measured by either [14C]inulin or 24 h creatinine clearances. This nephrotoxicity was associated with statistically significant (P less than 0.05) increases in urinary endothelin excretion and renal endothelin receptor number. Treatment with nifedipine attenuated cyclosporine A-induced renal dysfunction and reduced urinary endothelin excretion. The data provide further evidence of a role for endothelin in cyclosporine A-induced nephrotoxicity.

Transient forebrain ischemia alters acutely endothelin receptor density and immunoreactivity in gerbil brain

Pharmacologic studies suggest that endothelin (ET) plays an important role in the pathophysiology of hemorrhagic and ischemic stroke. In the gerbil, transient forebrain ischemia (10 min) resulted in profound motor deficits and a 15% reduction in ET receptor density in the hippocampus at 60 min post-reperfusion. A significant 2-fold increase in forebrain immunoreactive ET accompanied the maximum post-ischemic decrease in ET receptor density. These results suggest that the synthesis and availability of ET are increased acutely in the forebrain following transient cerebral ischemia.

Distribution of Neutral Endopeptidase Activity along the Rat and Rabbit Nephron

Neutral endopeptidase (NEP) activity was measured in various nephron segments dissected from rat and rabbit kidney. In the rat, only the proximal straight tubule and glomerulus had measurable NEP activity of 86 ± 11.3 pmol/min/mm tubule length and 5.8 ± 1.5 pmol/min/glomerulus, respectively. In the rabbit, significant activity was observed in both the proximal convoluted tubule (70.8 ± 7.2 pmol/min/mm) and proximal straight tubule (29.6 ± 2.3 pmol/min/mm) as well as in the glomerulus (12.8 ± 2.2 pmol/min/glomerulus). In the rat proximal tubule, phosphoramidon and thiorphan inhibited NEP activity, with IC50 values of 26.6 ± 6.0 and 6.9 ± 1.6 nmol/l, respectively. Incubation of rat proximal tubules with phorbol 12-myristate 13-acetate resulted in a 50% reduction in membrane-associated NEP activity. The results demonstrate that in both the rat and rabbit NEP is restricted to the glomerulus and proximal tubule. This localized distribution of NEP and its potential regulation by the protein kinase C pathway may play a key role in determining local concentrations of important regulatory peptides in the kidney.

Endothelin Receptor Subtype Distribution Predisposes Coronary Arteries to Damage

Several vasoactive drugs that lower blood pressure and increase heart rate induce regional cardiotoxicity in the dog, most frequently of right coronary arteries and right atrium. The basis for this selective damage is thought to result from local changes in vascular tone and blood flow. Administration of an endothelin receptor antagonist (ETRA, SB 209670) to dogs induced damage most frequent and severe in the right coronary artery and right atrium. Because site predisposition may correlate with distribution of vasoactive receptors, the objectives of this study were to map endothelin (ET) receptor distribution and density within regions of dog heart using both gene (mRNA) and protein expression endpoints for dog ET(A) and ET(B) receptors, and, additionally, correlate ET receptor subtype density with regional cardiac blood flow. A 10- to 15-mmHg reduction in mean arterial pressure with a concomitant increase in heart rate (10-20%), a six- and twofold increase in regional blood flow to the right and left atrium, respectively, and acute hemorrhage, medial necrosis, and inflammation were observed in the right coronary arteries and arteries of the right atrium after ETRA infusion for 5 days. Radioligand protein binding to quantify both ET receptors in normal dog heart indicated a twofold greater density of ET receptors in atrial regions versus ventricular regions. Importantly, ET receptor density in coronary arteries was markedly (about five- to sixfold) increased above that in atrial or ventricular tissues. ET receptor subtype characterization indicated ET(B) receptors were three times more prevalent in right coronary arteries compared to left coronary arteries and in situ hybridization confirmed localization of ET(B) in vascular smooth muscle. ET(A) receptor density was comparable in right and left coronary arteries. Quantitative real-time polymerase chain reaction for ET(A) and ET(B) receptor mRNA transcripts supported the site prevalence for message distribution. Consequently, the composite of protein and message expression profiles for ET(A) and ET(B) receptors indicated a disproportionate distribution of ET(B) receptors within right coronary artery of dog and this, along with functional measures of blood flow after ETRA infusion indicated a predisposition for exaggerated pharmacological responses and subsequent damage to right coronary arteries by ET and/or ETRAs.

Receptor activity modifying proteins interaction with human and porcine calcitonin receptor-like receptor (CRLR) in HEK-293 cells

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM), two closely related peptides, initiate their biological responses through their interaction with calcitonin receptor-like receptor (CRLR). The CRLR receptor phenotype can be determined by coexpression of CRLR with one of the three-receptor activity modifying proteins (RAMPs). In this report, we characterized the pharmacological properties of the human or porcine CRLR with individual RAMPs transiently expressed in human embroynic kidney cell line (HEK-293). Characterization of RAMP1/human or porcine CRLR combination by radioligand binding ([125I] hαCGRP) and functional assay (activation of adenylyl cyclase) revealed the properties of CGRP receptor. Similarly characterization of RAMP2/human or porcine CRLR and RAMP3/human or porcine CRLR combination by radioligand binding ([125I]rADM) and functional assay (activation of adenylyl cyclase) revealed the properties of ADM (22–52) sensitive-ADM receptor. In addition, porcine CRLR/RAMP2 or 3 combination displayed specific high affinity [125I] hαCGRP binding also. Also, co-transfection of porcine CRLR with RAMPs provided higher expression level of the receptor than the human counterpart. Thus the present study along with earlier studies strongly support the role of RAMPs in the functional expression of specific CRLRs.

SB 234551, a novel endothelin-A receptor antagonist, unmasks endothelin-induced renal vasodilatation in the dog

Infusion of endothelin-1 (ET-1) into conscious, chronically instrumented dogs (10 ng/kg.min i.v.) resulted in a significant increase in mean arterial pressure and significant reductions in renal plasma flow, glomerular filtration rate, and sodium excretion. Intravenous infusion of SB 209670 (30 micrograms/kg.min, i.v.) abolished these responses, whereas infusion of SB 234551 (30 micrograms/kg.min, i.v.) resulted in significant increases in renal plasma flow and urinary sodium excretion. These data indicate that SB 234551 can unmask ETB receptor-induced renal vasodilatation and inhibition of sodium reabsorption.

Identification and function of putative ETB receptor subtypes in the dog kidney.

Summary: Binding studies performed in dog kidney, lung, and spleen using [125I]endothelin (ET) 3 and a series of ETB-selective ligands indicated the presence of two subtypes of ETB receptors, an IRL-1620-sensitive (putative ETB1) and an IRL-1620-insensitive (putative ETB2) receptor. The IRL-1620-sensitive (but not IRL-1620-insensitive) ETB receptors displayed similar pharmacology to the cloned human ETB receptor and a high affinity for the ETB receptor antagonist RES-701. ETB1 receptors predominated in the dog kidney and lung and ETB2 receptors predominated in the dog spleen. Stimulation of ETB receptors in dogs in vivo inhibited sodium reabsorp-tion, as ET-1 infusion in the presence of the ETA antagonist BQ123, but not the mixed ETA/ETB receptor antagonist, (±)SB 209670, resulted in increased sodium excretion. Furthermore, infusion of the ETB-selective agonist Sarafotoxin S6c (S6c) resulted in an increase in sodium excretion, a response that was attenuated by infusion of RES-701. These data indicate that the putative ETB1 receptor may mediate ET-induced inhibition of tubule sodium reabsorption in the dog. In addition, we observed no putative ETB2 receptor-mediated renal vasoconstriction, consistent with the apparent low abundance of this subtype in the dog kidney.