Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Role of neutral endopeptidase in the metabolism of endothelin.

Endothelin is a potent vasoconstrictor produced by endothelial cells. Although endothelin has been studied extensively, little is known about its metabolism in vivo. Neutral endopeptidase EC.3.4.24.11 is reported to degrade endothelin in vitro. Therefore, we studied the effect of neutral endopeptidase inhibition by SQ29,072 on plasma levels and urinary excretion of endogenous and exogenous endothelin. Injection of 30 or 60 mg/kg SQ29,072 into anesthetized rats increased the urinary excretion of endothelin nearly 14-fold. The response was maximal during the first 30 minutes of collection and lasted for 90 minutes. The larger dose of inhibitor caused a 37-43% increase (p less than or equal to 0.05) in the plasma concentration of endothelin. Only 0.20 +/- 0.04% of the total radioactivity injected as 125I-endothelin (1 microCi; 1,308 pg) into normal rats was recovered in the urine within 30 minutes. Urinary radioactivity increased to 0.54-0.63% (p less than or equal to 0.05) of the total infused in rats pretreated with SQ29,072. Chromatographic analysis of radioactivity in the urine revealed that intact endothelin accounted for only 6-9% of the total counts in control rats but 50-56% in rats pretreated with the inhibitor. We also studied the effects of another inhibitor of neutral endopeptidase, SQ28,063, on the distribution of radioactivity in the urine, kidney, and lung of rats injected with 125I-endothelin. SQ28,063 increased urinary excretion of labeled endothelin and increased total radioactivity accumulated in the lung and kidney from 157 and 105 pg to 234 and 157 pg, respectively. Intact endothelin accounted for 90% or more of the accumulated counts in both tissues. These results indicate that 1) little circulating endothelin is cleared into the urine, 2) endothelin in the urine is likely of renal origin, and 3) neutral endopeptidase EC.3.4.24.11 plays a major role in the inactivation of endothelin.

Pulmonary and renal neutral endopeptidase EC 3.4.24.11 in rats with experimental heart failure.

Congestive heart failure is characterized by avid sodium retention and a blunted renal response to exogenous and endogenous atrial natriuretic peptide. Inhibition of neutral endopeptidase EC 3.4.24.11, the main enzyme that degrades natriuretic peptides, produces a natriuretic response in different models of congestive heart failure. This raises the possibility that an increase in either the expression or activity of neutral endopeptidase is responsible for these phenomena. In the present study, we examined (1) the renal effects of SQ-28,603, a neutral endopeptidase inhibitor, in rats with moderate and severe congestive heart failure induced by an aortocaval fistula compared with sham controls, and (2) neutral endopeptidase expression and activity in the lungs and kidneys of these rats. Infusion of SQ-28,603 (40 mg/kg IV) induced a significant natriuretic response in normal rats and rats with moderate congestive heart failure. This response was blunted in rats with severe congestive heart failure. Surprisingly, renal neutral endopeptidase mRNA levels, assessed by quantitative reverse transcriptase-polymerase chain reaction; protein levels, assessed by Western blotting; and activity, assessed by gelatin gels, were comparable in all groups. Pulmonary neutral endopeptidase mRNA levels decreased by 45% in rats with severe congestive heart failure but not in rats with mild congestive heart failure. In addition, pulmonary neutral endopeptidase immunoreactivity levels and activity were significantly decreased in congestive heart failure in correlation with the severity of the disorder.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of endothelin-1 and big endothelin-1 by recombinant neutral endopeptidase EC.3.4.24.11.

1. Inhibitors of neutral endopeptidase EC.3.4.24.11 (NEP) have been shown to attenuate the hypertensive effect of big-endothelin-1 (BET-1) in rats. To determine whether NEP converts BET-1 to endothelin-1 (ET-1), the effect of a recombinant NEP (rNEP) on BET-1 and on ET-1 was assessed in vitro. 2. Incubation of [125I]-ET-1 with 1 microgram ml-1 of rNEP resulted in degradation of the peptide within minutes. Increase in the amount of rNEP to 10 micrograms ml-1 led to total cleavage of [125I]-ET-1 within seconds. 3. Phosphoramidon (10 microM) or SQ-28,603 (100 microM) totally suppressed the degradation of [125I]-ET-1 by rNEP. 4. The degradation of [125I]-BET-1 by either 1 or 10 micrograms ml-1 of rNEP was much slower than that of [125I]-ET-1. Again, both phosphoramidon and SQ 28,603 protected the peptide from degradation. 5. Intact [125I]-ET-1 was not observed when [125I]-BET-1 was incubated with rNEP. 6. These data show that neutral endopeptidase EC.3.4.24.11 is not an endothelin converting enzyme.

Hydrolysis of iodine labelled urodilatin and ANP by recombinant neutral endopeptidase EC. 3.4.24.11.

1. Urodilatin is a 32 amino-acid peptide of similar sequence to atrial natriuretic peptide (ANP), with four additional amino-acids at the N-terminus. Although ANP and urodilatin bind to the same receptors with similar affinities, urodilatin is more active than ANP as a natriuretic agent. Previous studies, using neutral endopeptidase EC 3.4.24.11 (NEP) derived from crude membrane preparations, were inconclusive, but suggested that urodilatin was more resistant than ANP to degradation by this enzyme. In the present study, we compared the degradation rates of [125I]-urodilatin and [125I]-ANP by pure recombinant NEP (rNEP). 2. Incubation of radioactively labelled ANP with rNEP resulted in a much more rapid degradation of the peptide than that for labelled urodilatin. 3. Both phosphoramidon and SQ-28,603, potent inhibitors of NEP, completely protected both peptides from metabolism by rNEP. 4. The circular dichroism spectra of the two peptides indicate that they are very similar and exist largely in unordered or flexible conformations. 5. These results support the relative resistance of urodilatin to NEP, and indicate that urodilatin may be of use as a therapeutic agent, in conditions in which ANP is ineffective.

Glycine stimulates striatal dopamine release in conscious rats.

1. Glycine is an inhibitory neurotransmitter in the spinal cord and brainstem. The mechanism of this inhibition is via binding of glycine to specific receptors, increasing transmembrane Cl- conductance and hyperpolarizing neurones. Strychnine selectively antagonizes these effects. The role of glycinergic neurones in supraspinal regions is poorly understood. 2. Effects of glycine on release of catecholamines in the striatum were examined by microdialysis in freely-moving rats. Transcription of the genes encoding strychnine-sensitive glycine receptors was assessed in the striatum and substantia nigra, by use of reverse transcription followed by the polymerase chain reaction. 3. Glycine administered via the microdialysis probe dose-dependently increased concentrations of dopamine and its metabolites, dihydroxyphenylacetic acid and homovanillic acid, in the perfusate, indicating increased local release and metabolism of dopamine. Strychnine markedly attenuated these responses. Whereas striatal tissue did not contain mRNA for either the adult or neonatal form of strychnine-sensitive glycine receptor, nigral tissue contained a message for the adult form. 4. The results suggest that dopaminergic cells in the substantia nigra synthesize strychnine-sensitive glycine receptors and transport the receptors to terminals in the striatum. Occupation of the glycine receptors then exerts a net stimulatory effect on striatal dopamine release in vivo.

Regulation of the urinary excretion of endothelin in the rat.

The regulation of the urinary excretion of endothelin (UETV) and its clinical significance has not yet been established. The present study was designed to examine the effect of angiotensin II (A-II), arginine vasopressin (AVP), and nifedipine on UETV. Anesthetized Munich-Wistar rats were infused with low (50 ng/kg/min) and high (500 ng/kg/min) doses of A-II for 30 min. Both doses significantly increased UETV, from nondetectable (ND) levels to 155 +/- 54 (P < .03) and 450 +/- 86 fg/min (P < .001), respectively. This effect was accompanied by a significant increase in urine flow (UV), from 6 +/- 1 to 67 +/- 12 and 89 +/- 10 microL/min, and in mean arterial pressure (MAP), from 139 +/- 4 to 187 +/- 5 and 217 +/- 3 mm Hg. Infusions of A-II with its nonspecific antagonist, saralasin, resulted in a further increase in UETV to 647 +/- 126 and 782 +/- 117 fg/min (P < .002), respectively. However, infusion of A-II with its specific antagonist, losartan, completely blocked its stimulatory effect on UETV. Infusion of AVP, 10 or 100 mU/kg/h, produced increases in MAP, from 134 +/- 3 to 165 +/- 7 and 203 +/- 4 mm Hg, and in UV from 6 +/- 1 to 37 +/- 6 and 97 +/- 17 microL/min, comparable to A-II, but AVP did not have a marked effect on UETV.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary endothelin: a possible biological marker of renal damage.

Endothelin (ET) is a powerful vasoconstrictor peptide synthesized and secreted by the vascular endothelium. Significant amounts of ET are also produced by nonendothelial cells, mainly tubular-epithelial and mesangial cells. Large amounts of ET are found in the urine compared with the small amounts present in blood. Because most of the ET filtered from plasma is subject to degradation by neutral endopeptidase (EC 3.4.24.11) in the proximal tubule, urinary ET is probably of renal origin. The range of urinary ET excretion in healthy persons is 20 to 90 ng/day. The excretion of endothelin is modulated by several mechanical and chemical stimuli such as angiotensin II, phenylephrine, radiocontrast media, cyclosporine, and cis-platin. In addition, enhanced urinary ET excretion has been found in several forms of renal failure, both acute and chronic, and in diabetes mellitus. Thus, urinary ET has the potential of serving as a marker for renal disease.

Ouabain enhances the mitogenic effect of serum in vascular smooth muscle cells.

Recently ouabain has been shown to induce transcription of proto-oncogenes in different cell types. In the present study, we examined the effect of ouabain on the proliferation of cultured vascular smooth muscle cells (VSMCs). Primary aortic VSMCs of spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats, and the rat VSMC cell line A10, were used. Different concentrations of ouabain (10(-9) to 10(-5) mol/L) were added to either quiescent or proliferating cells, and the cell number, the rate of thymidine incorporation into DNA, and the transcription of c-fos and c-myc were examined. The addition of ouabain to proliferating VSMC increased the rate of thymidine incorporation into DNA in a dose-dependent manner, and induced the transcription of the proto-oncogenes within 1 h. This latter response disappeared after 24 h. The number of cells significantly increased in response to low concentrations of ouabain (10(-8) to 10(-7) mol/L), but gradually decreased in response to higher concentrations of the agent, probably due to a toxic effect. Addition of ouabain to quiescent cells, in medium without serum, did not promote cell growth by any of the parameters examined. According to a current theory, endogenous digitalis-like substances possess natriuretic and hypertensive properties, and provide the link between an excessive intake of salt and high blood pressure. The mitogenic effect of ouabain on VSMCs may be a component of this hypertensive action.

Rare renal transcription of the atrial natriuretic factor gene in rats. Demonstration by polymerase chain reaction.

Renal synthesis of a peptide homologous to atrial natriuretic factor (ANF) has been demonstrated. The aim of the present study was to determine if transcription of the ANF gene occurs in the kidney. Rat renal RNA was extracted from whole kidneys, and, separately, from the cortex and outer and inner medulla of rat kidneys. Probing with rat ANF-cDNA did not reveal a detectable message in Northern blot analysis, even when large quantities of RNA were used at low stringency hybridization conditions. Therefore, reverse transcription (RT) followed by 35 cycles of polymerase chain reaction (PCR) was used to search for the renal message for ANF. Two 21-mer primers encompassing the 450 base pairs (bp) of the coding region of the gene were used. Each cycle consisted of annealing at 56 degrees C, extension at 72 degrees C, and denaturation at 94 degrees C. The PCR product was proven to be identical to the ANF gene by high stringency hybridization, which revealed the expected 450-bp hybrid band. Furthermore, the sequence of this product was identical to that of the coding region of the ANF gene. We used an RNA-specific PCR to obtain this band as a single reaction product. We conclude that the transcript of the ANF gene exists in the kidney, at extremely low levels. The low abundance of the RNA message raises major concerns about its physiologic relevance. Direct evidence for the translation of this transcript, and its quantification and localization, is still required to determine its significance.

Neutral endopeptidase inhibition increases the urinary excretion and plasma levels of endothelin.

The pathway for removal of the circulating potent vasoconstrictor peptide, endothelin (ET), is unclear. To determine the contribution of neutral endopeptidase (NEP) to ET metabolism, urinary excretion (UETV) and plasma levels of ET (P(ET)) were measured after infusion of the NEP inhibitor (NEP-I), SQ 29,072 (30 mg/kg [n = 10] and 60 mg/kg [n = 6]), in anesthetized Munich Wistar rats. Both doses significantly increased UETV at 30, 60, and 90 minutes; the maximal effect was obtained 30 minutes after infusion, and the response was longer in rats pretreated with the higher dose of the inhibitor. P(ET) increased 36% and 55% (P less than or equal to .05) at 30 and 120 minutes after injection of the larger dose of SQ 29,072. We also studied the effect of NEP-I on the excretion of exogenous ET after the infusion of 125I-ET (1 microCi) in rats pretreated with either NEP-I or vehicle. In rats treated with the lower dose of the inhibitor, urinary radioactivity increased 2.1- and 1.5-fold (P less than or equal to .05 v control) after 30 and 60 minutes, respectively. After the higher dose of NEP-I, urinary radioactivity increased 2.7- and 1.7-fold (P less than or equal to .05). The distribution of the urinary radioactivity as defined by high-performance liquid chromatography (HPLC) showed that intact labeled ET accounts for only 6% to 9% of the total counts recovered in urine from control rats, while the remainder was either free iodine or other products of hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Histologic atrial myolysis is associated with atrial fibrillation after cardiac operation.

BACKGROUND: Postoperative atrial fibrillation after cardiac operation is common. Despite the identification of risk factors associated with postoperative atrial fibrillation, the pathophysiologic mechanisms remain unclear. Myolysis has been recently described to be associated with maintenance of atrial fibrillation in experimental animals. In this study, we attempted to identify histopathologic changes in atria that might predict the development of postoperative atrial fibrillation, and specifically address its association with myolysis. METHODS: Right appendicular atrial tissue was sampled before and after cardiopulmonary bypass from 60 patients in sinus rhythm who underwent elective coronary artery bypass grafting. RESULTS: Fifteen patients (25%) developed postoperative atrial fibrillation. Histopathologic abnormalities were found in most patients (52 of 60). However, only myolysis and lipofuscin levels were found to be an independent histologic finding associated with the development of postoperative atrial fibrillation. Electron microscopy showed that myolytic vacuoles were not membrane bound, and were associated with lipofuscin deposits. Neither mitochondrial pathology nor apoptosis was detected in the atria before or after operation. CONCLUSIONS: Abnormalities in biopsies before cardiopulmonary bypass can indicate the susceptibility to develop postoperative atrial fibrillation. This implies that the status of the atrium before cardiopulmonary bypass is a major determinant in the development of this common complication.

Short- and long-term swimming exercise training increases myocardial insulin-like growth factor-I gene expression.

UNLABELLED: OBJECTIVES. We investigated the effect of short- and long-term swimming exercise, with or without insulin-like growth factor (IGF)-I administration, on the expression of myocardial IGFs and contractile proteins. METHODS: Sprague-Dawley male rats (n=36) were subjected to swimming exercise for 2 or 6 weeks. IGF-I (0.5mg/rat) was administered continuously for 1 week, using alzet osmotic pumps. Control groups remained sedentary. IGF-I, IGF-I receptor (IGF-IR), IGF-II, skeletal alpha-actin (sk-actin), and beta myosin heavy chain (beta MHC) mRNAs were measured using Northern blot analysis and RT-PCR. RESULTS: A significant 2-fold increase in myocardial IGF-I mRNA was found after 2 and 6 weeks of swimming in both IGF-I treated and untreated rats (p<0.001). IGF-IR mRNA was significantly (p<0.05) increased after 6 weeks of training only in the IGF-I treated animals. IGF-II mRNA remained unchanged at all time points. While beta MHC mRNA was significantly decreased (p=0.003) at 2 and 6 weeks, sk-actin mRNA remained unchanged. CONCLUSIONS: Short- and long-term swimming exercise training increase myocardial expression of IGF-I mRNA. Exogenous administration of IGF-I, during the first week of the exercise session, did not produce any effect on myocardial IGF-I but was associated with increased IGF-IR signal after the long-term exercise training. These data suggest a relationship between IGF-I expression and cardiac adaptation to exercise training.

Functional alpha 3-glycine receptors in rat adrenal.

The adrenal medulla contains high-affinity strychnine binding sites, presumed to be receptors for glycine. In this study, glycine injection (400 pmol) via a cannula attached to a microdialysis probe increased in vivo concentrations of catecholamines in the adrenal microdialysate in anesthetized rats. Strychnine perfusion (20 pmol/20 min) blocked these responses. To identify receptors potentially mediating this effect, we tested for RNA transcripts of the three known alpha subunits of strychnine binding site, using the reverse transcription-polymerase chain reaction. Only mRNA encoding the alpha 3 isoform was found in the rat adrenal. The findings suggest that in the rat adrenal, glycine stimulates catecholamine release by binding to strychnine binding sites and that those sites probably contain the alpha 3 isoform.

Spontaneous hyperplasia of the ventral lobe of the prostate in aging genetically hypertensive rats.

Recent studies have shown that the prostatic autonomic innervation takes part in its homeostasis and growth. Other works showed that spontaneously hypertensive rats (SHR) show excessive sympathetic activity, accompanied by lower urinary tract symptoms, increased growth capacity of prostatic stromal cells, and increased levels of androgens and their receptors. Furthermore, young SHR were reported to present incipient stages of benign prostatic hyperplasia (BPH). The aim of the present study was to examine whether this strain indeed develops spontaneous BPH with age, and can thus serve as a genuine natural model for this disorder. For this purpose, ventral lobes of prostates of one-year-old, male SHR and their normotensive counterparts, Wistar Kyoto (WKY) rats, were examined histopathologically, and the degree of hyperplasia was evaluated according to a score-chart protocol (histoscore). SHR exhibited severe adenomatous spontaneous BPH, characterized by piling-up of epithelial cells, with papillary formations, accompanied by a mild increase in the amount of fibrocytes and smooth muscle cells in the stroma. This was reflected by histoscore values of 38 +/-2. Thickening of prostatic arterioles also was noted, as well as mild chronic inflammatory exudate. WKY rats did not show any of these features of BPH despite their age (histoscore 17 +/- 3, significantly different from that of SHR). We conclude that SHR can serve as a rodent model for the spontaneous development of BPH with age, most probably due to the excessive neuroendocrine activity characteristic of this rat strain.

Effects of senescence and citral on neuronal vacuolar degeneration in rat pelvic ganglia.

A significant part of the morbidity in elderly men involves pelvic organs and their autonomic neural regulation. Environmental stimuli also impair the structure and function of pelvic organs. One of these factors is citral, a widely-used cosmetic fragrance constituent, which causes severe prostatic hyperplasia in rats. In this study, we assessed the effect of topical administration of citral (30 days) on the morphology of pelvic ganglia (PG) in young adult and old Wistar rats. Neuronal vacuolar degeneration with preserved nuclei of PG neurons was observed in untreated senescent, but not young rats. Citral significantly increased the rate of vacuolated neurons in old rats (from 3 to 14%), but only slightly in young ones (from 0 to 0.5-0.3%). Similar lesions were not found in inferior cervical or celiac ganglia, in either group. This shows that environmental stimuli enhance age-related processes of vacuolar neuronal degeneration in PG, and may contribute to the dysfunction of pelvic organs in the elderly.

Potential preoperative markers for the risk of developing atrial fibrillation after cardiac surgery.

Postoperative atrial arrhythmias after cardiac surgical procedures are common, with a reported overall incidence of approximately 50%. The pathophysiological mechanisms responsible for atrial fibrillation after a cardiac procedure remain unclear, although several clinical studies published during the past decade have identified certain preoperative risk factors associated with postoperative atrial fibrillation. In this study, we attempted to identify the histopathological changes in atrial cardiomyocytes that might predict the development of atrial fibrillation during the postoperative period. Atrial tissue from 60 patients was sampled before and after a cardiopulmonary bypass. Fifteen patients (25%) developed postoperative atrial fibrillation. The only clinical independent risk factor for the development of postoperative atrial fibrillation was chronic obstructive pulmonary disease (COPD) (P = .037). Histologically, there were 3 findings in the atrial myocardium that were more common in patients who developed postoperative atrial fibrillation: (1) vacuolation size (P = .017), (2) vacuolation frequency (P = .0136), and (3) lipofuscin content (P = .013). The identification of these histological markers for the development of postoperative atrial fibrillation may contribute not only to our understanding of the underlying pathophysiology that leads to postoperative atrial fibrillation but also to a method of preventing this troublesome complication of cardiac surgery.

Placental shelf - a common, typically transient and benign finding on early second-trimester sonography.

OBJECTIVE: Placental shelves are believed to represent circumvallate placentae. It is thought that circumvallate placenta may be associated with adverse perinatal outcome when present at delivery. The objective of this study was to determine the prevalence, persistence and significance of placental shelves detected in the early second trimester. METHODS: In 152 consecutive anomaly scans performed between 13 and 16 weeks of gestation, special attention was directed to placental structure and the presence of a placental shelf. When present, a mid-gestation scan was performed to verify if the finding persisted. If so, a third-trimester scan was performed. Delivery charts were reviewed for all cases initially diagnosed with a placental shelf, recording any placenta-related complications. RESULTS: In 17 of 152 (11.2%) early second-trimester scans a placental shelf was detected. In three of these 17 cases the shelf persisted to the 20-22-week scan. In the two cases that presented for the third-trimester scan the shelf was no longer present. In all 17 cases the perinatal outcome was good. CONCLUSIONS: In our study group early second-trimester placental shelves rarely persisted to mid-gestation and never to the third trimester. There were no placenta-related perinatal problems. Early second-trimester placental shelf appears to be a common, benign and transient sonographic finding.

Discrepancy between the development of tolerance to bupivacaine in extradural and spinal anaesthesia in rabbits.

We gave equal groups of rabbits seven extradural (500 micrograms kg-1) or intrathecal (250 micrograms kg-1) injections of bupivacaine, at 24-h intervals. A decrease in the duration of motor block was observed after the extradural injections. The intrathecal injections exerted a reproducible effect. An additional regimen was tested in which five doses of bupivacaine 125 micrograms kg-1 were administered intrathecally after a loading dose of 250 micrograms kg-1, when the animals showed partial recovery from the previous dose; there was no decrease in the effect. The absence of tolerance to intrathecal bupivacaine implies that tachyphylaxis to extradural local anaesthetics results from a decrease in availability of the drug to the neural target, rather than a diminution in effect at the site of action.