Do we really know our patient population in database research? A comparison of the femoral shaft fracture patient populations in three commonly used national databases.

AIMS: While use of large national clinical databases for orthopaedic trauma research has increased dramatically, there has been little study of the differences in populations contained therein. In this study we aimed to compare populations of patients with femoral shaft fractures across three commonly used national databases, specifically with regard to age and comorbidities. PATIENTS AND METHODS: Patients were identified in the Nationwide Inpatient Sample (NIS), National Surgical Quality Improvement Program (NSQIP) and National Trauma Data Bank (NTDB). RESULTS: The distributions of age and Charleston comorbidity index (CCI) reflected a predominantly older population with more comorbidities in NSQIP (mean age 71.5; sd 15.6), mean CCI 4.9; sd 1.9) than in the NTDB (mean age 45.2; sd 21.4), mean CCI = 2.1; sd 2.0). Bimodal distributions in the NIS population showed a more mixed population (mean age 56.9; sd 24.9), mean CCI 3.2; sd 2.3). Differences in age and CCI were all statistically significant (p < 0.001). CONCLUSION: While these databases have been commonly used for orthopaedic trauma research, differences in the populations they represent are not always readily apparent. Care must be taken to understand fully these differences before performing or evaluating database research, as the outcomes they detail can only be analysed in context. TAKE HOME MESSAGE: Researchers and those evaluating research should be aware that orthopaedic trauma populations contained in commonly studied national databases may differ substantially based on sampling methods and inclusion criteria.

Recovery of Salmonella serovar Enteritidis from inoculated broiler hatching eggs using shell rinse and shell crush sampling methods.

This study compared the recovery of Salmonella from hatching eggs using 3 sampling methods (eggshell rinsing, eggshell crush following a previous rinse, and eggshell crush without previous rinse). Eggshells were drop-inoculated with approximately 10(1), 10(2), or 10(3) cfu/eggshell of Salmonella Enteritidis and allowed to dry at room temperature for 1 or 24 h. For the shell rinse groups, each inoculated egg was rinsed with buffered peptone water. These rinsed eggs were used for the shell crush with previous rinse groups, and each egg was aseptically cracked, the contents discarded, and the eggshell and membranes crushed with buffered peptone water. This same crush procedure was used for the shell crush without previous shell rinse eggs. The recovery of Salmonella 1 h after inoculation for shell rinse sampled eggs was 16% positive at 10(1), 49% at 10(2), and 93% at 10(3) cfu/eggshell challenge. For the shell crush with previous shell rinse, sampled egg recovery was 0% positive at 10(1), 3% at 10(2), and 17% at 10(3) cfu/eggshell. For the shell crush, sampled eggs had recovery of 23% positive at 10(1), 69% at 10(2), and 96% at 10(3) cfu/eggshell challenge. The recovery of Salmonella 24 h after inoculation for the shell rinse eggs was 3% positive at 10(1), 12% at 10(2), and 22% at 10(3) cfu/eggshell challenge; recovery for shell crush with previous shell rinse sampling was 2% positive at 10(1), 8% at 10(2), and 5% at 10(3) cfu/eggshell challenge; and for the shell crush sampling recovery was 2% at 10(1), 32% at 10(2), and 42% at 10(3) cfu/eggshell challenge. Eggshell crush was a more sensitive (∼10 percentage points) sampling method than eggshell rinse at both 1 and 24 h, but both methods were equally optimal when the inoculum was at 10(3) and samples were collected after 1 h. Waiting 24 h after inoculation to sample significantly lowered the recovery for both the shell rinse and shell crush sampling methods by ∼40 percentage points.

Biphenylsulfonamide endothelin receptor antagonists. 2. Discovery of 4'-oxazolyl biphenylsulfonamides as a new class of potent, highly selective ET(A) antagonists.

The synthesis and structure-activity relationship (SAR) studies of a series of 4'-oxazolyl-N-(3,4-dimethyl-5-isoxazolyl)[1, 1'-biphenyl]-2-sulfonamide derivatives as endothelin-A (ET(A)) receptor antagonists are described. The data reveal a remarkable improvement in potency and metabolic stability when the 4'-position of the biphenylsulfonamide is substituted with an oxazole ring. Additional 2'-substitution of an acylaminomethyl group further increased the binding activity and provided one of the first subnanomolar ET(A)-selective antagonists in the biphenylsulfonamide series (17, ET(A) K(i) = 0.2 nM). Among the compounds described, 3 (N-(3,4-dimethyl-5-isoxazolyl)-4'-(2-oxazolyl)[1, 1'-biphenyl]-2-sulfonamide; BMS-193884) had the optimum pharmacological profile and was therefore selected as a clinical candidate for studies in congestive heart failure.

Small molecule antagonists of the bradykinin B1 receptor.

Screening Pharmacopeia's encoded combinatorial libraries has led to the identification of potent, selective, competitive antagonists at the bradykinin B1 receptor. Libraries were screened using a displacement assay of [3H]-des-Arglo-kallidin ([3H]-dAK) at IMR-90 cells expressing an endogenous human B1 receptor (Bmax = 20,000 receptors/cell, K(D) = 0.5+/-0.1 nM) or against membranes from 293E cells expressing a recombinant human B1 receptor (Bmax = 8,000 receptors/cell, K(D) = 0.5 +/- 0.3 nM). Compound PS020990, an optimized, representative member from the class of compounds, inhibits specific binding of 3H-dAK at IMR-90 cells with a KI of 6 +/- 1 nM. The compound inhibits dAK-induced phosphatidyl inositol turnover (K(Bapp) = 0.4 +/- 0.2 nM) and calcium mobilization (K(Bapp) = 17 +/- 2 nM) in IMR-90 cells. Compounds from the lead series are inactive at the B2 receptor and are > 1000-fold specific for B1 vs. a variety of other receptors, ion channels and enzymes. PS020990 and other related chemotypes therefore offer an excellent opportunity to explore further the role of B1 receptors in disease models and represent a potential therapeutic avenue.

Biphenylsulfonamide endothelin antagonists: structure-activity relationships of a series of mono- and disubstituted analogues and pharmacology of the orally active endothelin antagonist 2'-amino-N- (3,4-dimethyl-5-isoxazolyl)-4'-(2-methylpropyl)[1, 1'-biphenyl]-2-sulfonamide (BMS-187308).

Substitution at the ortho position of N-(3,4-dimethyl-5-isoxazolyl) benzenesulfonamide led to the identification of the biphenylsulfonamides as a novel series of endothelin-A (ETA) selective antagonists. Appropriate substitutions on the pendant phenyl ring led to improved binding as well as functional activity. A hydrophobic group such as isobutyl or isopropoxyl was found to be optimal at the 4'-position. Introduction of an amino group at the 2'-position also led to improved analogues. Combination of the optimal 4'-isobutyl substituent with the 2'-amino function afforded an analogue (20, BMS-187308) with improved ETA binding affinity and functional activity. Compound 20 also has good oral activity in inhibiting the pressor effect caused by an ET-1 infusion in rats. Doses of 10 and 30 micromol/kg iv 20 attenuated the pressor responses due to the administration of exogenous ET-1 to conscious monkeys, indicating that the compound inhibits the in vivo activity of endothelin-1 in nonhuman primates.

The effects of endothelin-A receptor blockade during the progression of pacing-induced congestive heart failure.

OBJECTIVES: We sought to identify the effects of endothelin (ET) subtype-A (ET(A))) receptor blockade during the development of congestive heart failure (CHF) on left ventricle (LV) function and contractility. BACKGROUND: Congested heart failure causes increased plasma levels of ET and ET(A) receptor activation. METHODS: Yorkshire pigs were assigned to four groups: 1) CHF: 240 beats/min for 3 weeks; n=7; 2) CHF/ET(A)-High Dose: paced for 2 weeks then ET(A) receptor blockade (BMS 193884, 50 mg/kg, b.i.d.) for the last week of pacing; n=6; 3) CHF/ET(A)-Low Dose: pacing for 2 weeks then ET(A) receptor blockade (BMS 193884, 12.5 mg/kg, b.i.d.) for the last week, n=6; and 4) CONTROL: n=8. RESULTS: Left ventricle fractional shortening decreased with CHF compared with control (12+/-1 vs. 39+/-1%, p < 0.05) and increased in the CHF/ET(A) High and Low Dose groups (23+/-3 and 25+/-1%, p < 0.05). The LV peak wall stress and wall force increased approximately twofold with CHF and remained increased with ET(A) receptor blockade. With CHF, systemic vascular resistance increased by 120%, was normalized in the CHF/ET(A) High Dose group, and fell by 43% from CHF values in the Low Dose group (p < 0.05). Plasma catecholamines increased fourfold in the CHF group and were reduced by 48% in both CHF/ET(A) blockade groups. The LV myocyte velocity of shortening was reduced with CHF (32+/-3 vs. 54+/-3 microm/s, p < 0.05), was higher in the CHF/ET(A) High Dose group (39+/-1 microm/s, p < 0.05), and was similar to CHF values in the Low Dose group. CONCLUSIONS: ET(A) receptor activation may contribute to the progression of LV dysfunction with CHF.

Hypoxia stimulates human preproendothelin-1 promoter activity in transgenic mice.

Significant elevations in endothelin (ET)-1 levels accompany many diseases, but the underlying regulatory mechanisms are unclear. To investigate the in vivo regulation of human preproendothelin-1 (PPET-1), we examined the activity of the PPET-1 promoter in transgenic mice exposed to hypoxia. Mice expressing one of three PPET-1 promoter-luciferase (PPET-1/LUC) reporter transgenes (approximately 2.5 kb, 138 bp, or none of the 5'-flanking sequences of the PPET-1 gene) were generated. LUC expression was reduced in mice with a truncated 138-bp PPET-1 promoter. Exposure of mice bearing the 2.5-kb PPET-1/LUC transgene to hypoxia (10% O2 for 24 h) increased LUC expression sixfold in pulmonary tissue but only twofold in other tissues. In situ hybridization revealed the strongest transgene expression in the pulmonary vasculature and bronchiolar epithelium. These data are consistent with the hypothesis that hypoxic induction of the PPET-1 gene leads to increased pulmonary production of ET-1 in diseases associated with low O2 tension.

Inhibitors of endothelin.

With the advent of the first generation of both selective and nonselective endothelin antagonists being a relatively recent event, the manifold therapeutic potentials of these compounds are only now being explored clinically. Undoubtedly, numerous clinical utilities for these compounds will soon be realized.

Angiotensin-converting enzyme inhibition and angiotensin II subtype-1 receptor blockade during the progression of left ventricular dysfunction: differential effects on myocyte contractile processes.

Inhibition of the angiotensin-converting enzyme (ACE) in the setting of chronic left ventricular (LV) dysfunction has been demonstrated to have beneficial effects on survival and symptoms. However, whether ACE inhibition has direct effects on myocyte contractile processes and if these effects are mediated primarily through the AT1 angiotensin-II receptor subtype remains unclear. The present project examined the relationship between changes in LV and myocyte function and beta adrenergic receptor transduction in four groups of six dogs each: (1) Rapid Pace: LV failure induced by chronic rapid pacing (4 weeks; 216 +/- 2 bpm); (2) Rapid Pace/ACEI: concomitant ACE inhibition (ACEI: fosinopril 30 mg/kg b.i.d.) with chronic pacing; (3) Rapid Pace/AT1 Block: concomitant AT1 Ang-II receptor blockade [Irbesartan: SR 47436(BMS-186295) 30 mg/kg b.i.d.] with chronic pacing; and (4) CONTROL: sham controls. With Rapid Pace, the LV end-diastolic volume increased by 62% and the ejection fraction decreased by 53% from control. With Rapid Pace/ACEI, the LV end-diastolic volume was reduced by 24% and the ejection fraction increased by 26% from Rapid Pace only values. Rapid Pace/AT1 Block did not improve LV geometry or function from Rapid Pace values. Myocyte contractile function decreased by 40% with Rapid Pace and increased from this value by 32% with Rapid Pace/ACEI. Rapid Pace/AT1 Block had no effect on myocyte function when compared with Rapid Pace values. With Rapid Pace/ACEI, beta receptor density and cyclic AMP production were normalized and associated with an improvement in myocyte beta adrenergic response compared with Rapid Pace only. Although Rapid Pace/AT1 also normalized beta receptor density, cyclic AMP production was unchanged and myocyte beta adrenergic response was reduced by 15% compared with Rapid Pace only. ACE inhibition with chronic rapid pacing improved LV and myocyte geometry and function, and normalized beta receptor density and cyclic AMP production. However, AT1 Ang-II receptor blockade with chronic rapid pacing failed to provide similar protective effects on LV and myocyte geometry and function. These unique findings suggest that the effects of ACE inhibition on LV geometry and myocyte contractile processes in the setting of developing LV failure are not primarily caused by modulation of AT1 Ang-II receptor activation.

Exogenous effects and endogenous production of endothelin in cardiac myocytes: potential significance in heart failure.

Increased plasma concentrations of endothelin have been identified in patients and animals with severe congestive heart failure (CHF). However, whether and to what extent increased endothelin (ET) concentrations influence left ventricular (LV) myocyte contractility, ET-receptor subtype density, and endogenous ET production with the development of CHF remains unclear. Accordingly, myocyte contractile function, response to ET, sarcolemmal ET-receptor density, and myocyte ET production were examined in pigs following the development of pacing-induced CHF (240 beats/min, 3 wk, n = 8) and in controls (n = 8). With CHF, plasma ET increased over threefold. In the presence of ET (10-500 pM), myocyte contractility increased in a dose-dependent manner in control myocytes but decreased in CHF myocytes. For example, in the presence of 200 pM ET, velocity of shortening increased by 32.8 +/- 2.3 microns/s in controls but decreased by 8.3 +/- 2.2 microns/s with CHF. LV sarcolemmal ET-receptor density was primarily of the ETA-receptor subtype in controls (96 +/- 1.0%) and was unchanged with CHF. In quiescent myocyte preparations, control myocytes secreted ET (2.29 +/- 0.45 amol.cell-1.h-1), which was similar in CHF myocytes. These findings suggest that the production of ET may have important and potentially differential effects on contractile function with the development of CHF.

Early signal transduction by the receptor to the chemokine monocyte chemotactic protein-1 in a murine T cell hybrid.

Members of the C-C family of chemotactic cytokines promote chemotaxis and adhesion of leukocytes. In this study, we have identified a murine T cell hybrid that expresses receptors to the chemotactic cytokine monocyte chemotactic protein-1 (MCP-1). This cell line was used to examine MCP-1 receptor-mediated signal transduction events in a homologous system in the absence of interference with other receptors. Our results show that in the 3B4 M1.9 T cell hybrid, MCP-1 receptors mediate intracellular calcium mobilization and extracellular calcium import without detectable increases in total water-soluble inositol phosphates. In addition, MCP-1 regulates the tyrosine phosphorylation of specific substrates at 42 and 44 kDa and induces mobility shift of p42/44 mitogen-activated protein kinases. MCP-1-mediated calcium responses, tyrosine phosphorylation, and the electrophoretic mobility shift of p42/44 mitogen-activated protein kinases can be inhibited by pretreatment of cells with pertussis toxin, indicating a role for Gi-like G proteins in coupling the MCP-1R to signal transduction.

Mutational analysis of the endothelin type A receptor (ETA): interactions and model of selective ETA antagonist BMS-182874 with putative ETA receptor binding cavity.

Endothelin (ET) receptor antagonism is a potential therapeutic intervention in the treatment of vascular diseases. To elucidate the mechanism of antagonist-ET receptor complex formation, the interactions of four chemically distinct antagonists were investigated using a combination of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane domain was critical for high-affinity, subtype-selective binding to the A subtype of ET (ETA) receptors [Krystek et al. (1994) J. Biol. Chem. 269, 12383-12386]. Affinities of the constrained cyclic pentapeptide BQ-123, the pyrimidinylbenzenesulfonamide bosentan, the indancarboxlic acid SB 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SB 209670. BMS-182874 binding affinity was decreased 10-fold in Tyr129Phe and Tyr129trp ET receptors. These data indicate a role of aromatic interactions in the binding of these antagonists to ETA receptors an, in the case of BMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported by structure-activity data with analogs of BMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of BMS-182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ETA-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of BMS-182874 in the ETA receptor are proposed as a starting point for further delineation of interactions that underlie antagonist-ETA receptor complex formation.

A role for endothelin ETA receptors in regulation of renal function in spontaneously hypertensive rats.

While there is evidence to suggest that endothelin-1 is involved in regulation of kidney function and blood pressure, the importance of endothelin ETA receptors in this area has not been clearly defined. The novel, non-peptide endothelin ETA receptor antagonist, BMS-182874, (5-(dimethylamino)-N-(3,4- dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide) was used to examine effects of endothelin ETA receptor blockade on renal function in spontaneously hypertensive rats. Preliminary studies were conducted to determine an effective dose of BMS-182874. Infusion of BMS-182874 (10 mumol/kg/min, i.v.) inhibited effects of exogenous endothelin-1 on glomerular filtration rate, renal blood flow, and mean arterial pressure in Sprague-Dawley rats. Administration of BMS-182874 (10 mumol/kg/min, i.v.) to anesthetized, male, spontaneously hypertensive rats decreased renal blood flow by approximately 50% (1.2 +/- 0.11 ml/min/100 g body weight) compared to vehicle (2.7 +/- 0.23). There was no effect of BMS-182874 on glomerular filtration rate (0.5 +/- 0.05 ml/min/100 g body weight; vehicle: 0.7 +/- 0.06). Mean arterial pressure decreased significantly after BMS-182874 (123 +/- 3.8 mm Hg; vehicle: 162 +/- 4.8). Urine flow and renal vascular resistance were unchanged by BMS-182874. Endothelin ETA receptor density was increased approximately 50% in spontaneously hypertensive rat kidneys compared to normotensive kidneys, with no change in equilibrium dissociation constant. Endothelin ETB receptor density and equilibrium dissociation constant were similar in the two rat strains. Plasma immunoreactive endothelin was higher in hypertensive (5.9 +/- 0.31 fmol/ml) than normotensive rats (2.8 +/- 0.15). The results suggest endothelin ETA receptors may play a role in the regulation of renal function in this model of hypertension.

Cloning and expression of an endothelin receptor subtype B from human prostate that mediates contraction.

Recent evidence suggests a role for endothelin (ET) in contraction of human prostate [J. Urol. 149:495-499 (1993)]. Although both ETA and ETB receptors have been shown to mediate contraction of smooth muscle, the molecular identity of the contractile ETB receptor is controversial. The aim of this study was to examine the receptor subtype that mediates ET-induced contraction in prostate from patients with benign prostatic hyperplasia. Saturation binding with 125I-ET-1 and 125I-ET-3 in prostate stromal cells (PSC) indicated the presence of receptors with subnanomolar affinity for these radioligands, with equivalent receptor densities. Inhibition of specific 125I-ET-1 or 125I-ET-3 binding in PSC revealed a rank order of potency of ET-1 - ET-3 = sarafotoxin S6c >> BQ-123. These data are consistent with a predominance of ETB receptors in PSC. The functional effects of ET stimulation of PSC were examined in a collagen gel contraction assay. ET-1 and ET-3 caused contraction of underlying collagen gel matrices with EC50 values of 0.4 +/- 0.04 and 0.7 +/- 0.2 nM, respectively. To determine the molecular nature of the contractile ETB receptor in PSC, reverse transcription-polymerase chain reactions were conducted with oligonucleotide primers to the 5' and 3' ends of the coding sequence of the full length human ETB receptor. DNA sequence analysis of the 1.3-kilobase DNA product showed 99% homology to other human ETB receptor cDNAs. The encoded protein has a deduced amino acid sequence identical to that of other human ETB receptors, with the exception of two conservative substitutions. Expression of the PSC ETB cDNA in COS-7 cells resulted in a binding profile similar to that observed in parent cells. Polymerase chain reaction analysis revealed the presence of prepro-ET-1 mRNA in PSC. Collectively, these data indicate that PSC from patients with benign prostatic hyperplasia express ETB receptors that mediate ET-induced contraction.

Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors.

The endothelin receptors, ETA and ETB, are G protein-coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype-specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383-12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB correspond to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET-1, a 160-fold increase in ET-3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS-182874. ET-1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET-3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS-182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype-selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126 Ala ETA variant, ET-1 and ET-3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype-selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

BMS-182874 is a selective, nonpeptide endothelin ETA receptor antagonist.

BMS-182874 [5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide] is a recently discovered, low molecular weight, nonpeptide endothelin (ET) receptor antagonist. BMS-182874 competitively inhibited the binding of [125I]ET-1 to ETA receptors in rat vascular smooth muscle A10 (VSM-A10) cell membranes (Ki = 61 nM) and in CHO cells stably expressing the human ETA receptor (Ki = 48 nM), but was a weak inhibitor at ETB receptors (Ki > 50 microM) and non-ET receptors. BMS-182874 inhibited ET-1-stimulated inositol phosphate accumulation (KB = 75 nM) and calcium mobilization (KB = 140 nM) without suppressing the maximal responses in VSM-A10 cells. BMS-182874 was a competitive antagonist of force development elicited by stimulation of ETA, but not other, receptors in isolated blood vessels such as the rabbit carotid artery (KB = 520 nM). The apparent discrepancy between efficacy in cell and tissue models was likely related to the high degree of protein binding exhibited by BMS-182874. When administered either orally (ED50 = 30 mumol/kg) or intravenously (ED50 = 24 mumol/kg) to conscious, normotensive rats, BMS-182874 blunted the pressor response to exogenous ET-1. These data demonstrate that BMS-182874 is a competitive, selective and orally active ETA receptor antagonist that will be useful in understanding the role of ET in normal and disease states.

Comparison of a novel ETA receptor antagonist and phosphoramidon in renal ischemia.

Infusion (0.46 mumol/kg/min) of the endothelin (ET)-converting-enzyme inhibitor, phosphoramidon (P), protected function and structure after 30 min renal ischemia in rats more than treatment (5 mumol/kg/min) with the ETA receptor antagonist, BMS-182874 (B). The glomerular filtration rate (GFR; 0.7 +/- 0.12 ml/min) and renal plasma flow (RPF) decreased approximately 40% at 2 h reflow versus controls (C: 1.2 +/- 0.12). B weakly protected the GFR (0.8 +/- 0.07 ml/min); P restored it (1.1 +/- 0.05). Both compounds reduced tubular injury at 2 h reflow; P ameliorated glomerular changes. At 24 h the GFR (0.6 +/- 0.06 ml/min) and RPF decreased 67% versus C (1.8 +/- 0.08). B did not protect the GFR and RPF. P partially protected the GFR (0.9 +/- 0.07 ml/min) but not RPF, and reduced tubular injury. The results suggest that both ETA and non-ETA receptors mediate ET-induced changes in ischemic renal failure.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB.

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

Urinary incontinence in younger women.

Urinary incontinence (UI) in younger adult (30 to 59 years) women is an underdiagnosed and undertreated problem, with social and economic ramifications. This article highlights the risk factors for UI brought about by life choices, challenges, and changes: onset of sexual activity, career choices, travel, pregnancy, childbirth, and menopause. Suggestions are made concerning possible preventative strategies and identifying the need for research. Included are hard-to-assemble normal data and experienced-based strategies used in the evaluation process, which are useful to the generalist NP and the continence specialist.

Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.

The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.