Inhibition of cardiac late sodium current (late I Na ) is a strategy to suppress arrhythmias and sodium-dependent calcium overload associated with myocardial ischemia and heart failure. Current inhibitors of late I Na are unselective and can be proarrhythmic. This study introduces GS967 (6-[4-(trifluoromethoxy), a potent and selective inhibitor of late I Na , and demonstrates its effectiveness to suppress ventricular arrhythmias. The effects of GS967 on rabbit ventricular myocyte ion channel currents and action potentials were determined. Anti-arrhythmic actions of GS967 were characterized in ex vivo and in vivo rabbit models of reduced repolarization reserve and ischemia. GS967 inhibited Anemonia sulcata toxin II (ATX-II)-induced late I Na in ventricular myocytes and isolated hearts with IC 50 values of 0.13 and 0.21 mM, respectively. Reduction of peak I Na by GS967 was minimal at a holding potential of 2120 mV but increased at 280 mV. GS967 did not prolong action potential duration or the QRS interval. GS967 prevented and reversed proarrhythmic effects (afterdepolarizations and torsades de pointes) of the late I Na enhancer ATX-II and the I Kr inhibitor E-4031 in isolated ventricular myocytes and hearts. GS967 significantly attenuated the proarrhythmic effects of methoxamine1clofilium and suppressed ischemiainduced arrhythmias. GS967 was more potent and effective to reduce late I Na and arrhythmias than either flecainide or ranolazine. Results of all studies and assays of binding and activity of GS967 at numerous receptors, transporters, and enzymes indicated that GS967 selectively inhibited late I Na . In summary, GS967 selectively suppressed late I Na and prevented and/or reduced the incidence of experimentally induced arrhythmias in rabbit myocytes and hearts.

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.

Metabolic subtlety and clinical relevance of different forms of reactive oxygen species in diabetes remain unclear. Using single knockout of Cu,Zn-superoxide dismutase (SOD1(-/-)) or Se-glutathione peroxidase-1 (GPX1(-/-)) and their double-knockout (DKO) mouse models, we determined if elevating endogenously-derived superoxide and hydroperoxide exerted distinct impacts and mechanisms on body glucose homeostasis. Whereas the three knockout groups displayed decreased plasma insulin concentrations and islet β-cells mass, only SOD1(-/-) showed decreased body weight, increased blood glucose, and blocked glucose-stimulated insulin secretion. Null of SOD1 and GPX1 elevated respective islet superoxide and hydroperoxide production, and upregulated p53 phosphorylation. Knockout of SOD1 downregulated the foxhead box A2/pancreatic and duodenal homeobox 1 pathway in a superoxide-dependent fashion at epigenetic, mRNA, and protein levels in islets, but improved insulin signaling in liver and muscle. The SOD1(-/-) mice showed more apparent pancreatitis than the GPX1(-/-) mice that were more susceptible to the cerulein-induced amylase increase. Knockout of SOD1 impaired islet function, pancreas integrity, and body glucose homeostasis more than that of GPX1. Simultaneous ablation of both enzymes did not result in additive or aggravated metabolic outcomes.

The aim of this study was to determine the role of nitric oxide (NO) in the development of salt-induced hypertension in the Brookhaven strain of Dahl rats. Six- to seven-week-old conscious salt-sensitive (S) and salt-resistant (R) rats with indwelling arterial and venous catheters received low-, normal-, and high-sodium intakes sequentially over a 16-day period, and L-arginine was infused intravenously at 2 or 4 mg.kg-1.min-1 over this time. The S rats had an impaired NO production as evidenced by a decreased urinary nitrate plus nitrite excretion. The administration of the low or high dose of L-arginine increased the whole body NO production of the S rats to that of the control R rats, and the high dose of L-arginine prevented the shift of long-term pressure-natriuresis relationship, the elevation of arterial pressure, and the increase in salt sensitivity of arterial pressure in the S rats. The sodium and water balances were not different between the age-matched R and S rats. In conclusion, a continuous infusion of L-arginine prevented both the changes in the pressure-natriuresis relationship and the development of salt-induced hypertension in Dahl S rats.

In this study aminotransferase inhibitors were used to determine the relative importance of different aminotransferases in providing nitrogen for de novo glutamate synthesis in the retina. Aminooxyacetate, which inhibits all aminotransferases, blocked de novo glutamate synthesis from H(14)CO(3)(-) by more than 60%. Inhibition of neuronal cytosolic branched chain amino acid transamination by gabapentin or branched chain amino acid transport by the L-system substrate analog, 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid, lowered total de novo synthesis of glutamate by 30%, suggesting that branched chain amino acids may account for half of the glutamate nitrogen contributed by transamination reactions. L-cycloserine, an inhibitor of alanine aminotransferase, inhibited glutamate synthesis less than 15% when added in the presence of 5 mM pyruvate but 47% in the presence of 0.2 mM pyruvate. Although high levels of pyruvate blunted the inhibitory effectiveness of L-cycloserine, the results indicate that, under physiological conditions, alanine as well as branched chain amino acids are probably the predominant sources of glutamate nitrogen in ex vivo retinas. The L-cycloserine results were also used to evaluate activity of the malate/aspartate shuttle. In this shuttle, cytosolic aspartate (synthesized in mitochondria) generates cytosolic oxaloacetate that oxidizes cytosolic NADH via malate dehydrogenase. Because L-cycloserine inhibits cytosolic but not mitochondrial aspartate aminotransferase, L-cycloserine should prevent the utilization of aspartate but not its generation, thereby increasing levels of (14)C-aspartate. Instead, L-cycloserine caused a significant decline in (14)C-aspartate. The results suggest the possibility that shuttle activity is low in retinal Müller cells. Low malate/aspartate shuttle activity may be the molecular basis for the high rate of aerobic glycolysis in retinal Müller cells.

It has been shown that testicular germ cell development is critically dependent upon somatic cell activity but, conversely, the extent to which germ cells normally regulate somatic cell function is less clear. This study was designed, therefore, to examine the effect of germ cell depletion on Sertoli cell and Leydig cell transcript levels. Mice were treated with busulphan to deplete the germ cell population and levels of mRNA transcripts encoding 26 Sertoli cell-specific proteins and 6 Leydig cell proteins were measured by real-time PCR up to 50 days after treatment. Spermatogonia were lost from the testis between 5 and 10 days after treatment, while spermatocytes were depleted after 10 days and spermatids after 20 days. By 30 days after treatment, most tubules were devoid of germ cells. Circulating FSH and intratesticular testosterone were not significantly affected by treatment. Of the 26 Sertoli cell markers tested, 13 showed no change in transcript levels after busulphan treatment, 2 showed decreased levels, 9 showed increased levels and 2 showed a biphasic response. In 60% of cases, changes in transcript levels occurred after the loss of the spermatids. Levels of mRNA transcripts encoding Leydig cell-specific products related to steroidogenesis were unaffected by treatment. Results indicate (1) that germ cells play a major and widespread role in the regulation of Sertoli cell activity, (2) most changes in transcript levels are associated with the loss of spermatids and (3) Leydig cell steroidogenesis is largely unaffected by germ cell ablation.

Renin can be detected in cardiovascular and other tissues but it disappears after bilateral nephrectomy indicating that tissues can take up or bind renal renin from the circulation. If renin uptake is the result of specific binding, plasma prorenin may be a natural antagonist of tissue directed renin-angiotensin systems. To investigate if specific prorenin/renin uptake occurs in rat tissues, binding studies were performed, with rat microsomal membrane preparations using recombinant rat prorenin metabolically labeled with 35S-methionine as a probe. A high affinity binding site for both renin and prorenin was identified. Affinities for prorenin and renin were approximately 200 and 900 pmol/L, respectively. Binding was reversible, saturable, and pH and temperature dependent. The relative binding capacities of membranes from various rat tissues were as follows (fmol/mg): renal cortex (55), liver (54), testis (63), lung (31), brain (18), renal medulla (15), adrenal (17), aorta (7), heart (4), and skeletal muscle (1). Bound prorenin was displaced by rat and human renin or prorenin but not by the prosequence of rat prorenin, angiotensin I or II, rat or human angiotensinogen, the renin inhibitor SQ30697, atrial natriuretic factor, amylase, insulin, bovine serum albumin, hemoglobin, heparin, lysozyme, ovalbumin, cytochrome C, pepsin, pepsinogen, ribonuclease A, mannose-6-phosphate, alpha-methyl mannoside, gonadotropin releasing hormone, or an antibody to hog renin binding protein. these results demonstrate specific binding of prorenin to a site in rat tissues, herein named ProBP, that also binds renin. It is possible that differences in prorenin/renin binding capacity determine the activity of tissue-directed renin-angiotensin systems and that prorenin is a natural antagonist. Alternatively, a prorenin/renin receptor may have been identified that may function by transducing an intracellular signal.

Conventional indicator dilution techniques for measuring body fluid volume are laborious, expensive, and highly invasive. Bioimpedance spectroscopy (BIS) may be a useful alternative due to being rapid, minimally invasive, and allowing repeated measurements. BIS has not been reported in mice; hence we examined how well BIS estimates body fluid volume in mice. Using C57/Bl6 mice, the BIS system demonstrated <5% intermouse variation in total body water (TBW) and extracellular (ECFV) and intracellular fluid volume (ICFV) between animals of similar body weight. TBW, ECFV, and ICFV differed between heavier male and lighter female mice; however, the ratio of TBW, ECFV, and ICFV to body weight did not differ between mice and corresponded closely to values in the literature. Furthermore, repeat measurements over 1 wk demonstrated <5% intramouse variation. Default resistance coefficients used by the BIS system, defined for rats, produced body composition values for TBW that exceeded body weight in mice. Therefore, body composition was measured in mice using a range of resistance coefficients. Resistance values at 10% of those defined for rats provided TBW, ECFV, and ICFV ratios to body weight that were similar to those obtained by conventional isotope dilution. Further evaluation of the sensitivity of the BIS system was determined by its ability to detect volume changes after saline infusion; saline provided the predicted changes in compartmental fluid volumes. In summary, BIS is a noninvasive and accurate method for the estimation of body composition in mice. The ability to perform serial measurements will be a useful tool for future studies.