Suppressive Effect of Carvedilol on Na+/Ca2+ Exchange Current in Isolated Guinea-Pig Cardiac Ventricular Myocytes.

BACKGROUND AND AIMS: Carvedilol ((+/-)-1-(carbazol-4-yloxy)-3-[[2-(o-methoxyphenoxy)ethyl]amino]-2-propanol), a ß-adrenoceptor-blocker, has multi-channel blocking and vasodilator properties. This agent dose-dependently improves left ventricular function and reduces mortality in patients with arrhythmia and chronic heart failure. However, the effect of carvedilol on the cardiac Na+/Ca2+ exchanger (NCX1) has not been investigated. METHODS AND RESULTS: We examined the effects of carvedilol and metoprolol, 2 ß-blockers, on Na+/Ca2+ exchange current (INCX) in guinea-pig cardiac ventricular cells and fibroblasts expressing dog cardiac NCX1. Carvedilol suppressed INCX in a concentration-dependent manner but metoprolol did not. IC50 values for the Ca2+ influx (outward) and efflux (inward) components of INCX were 69.7 and 61.5 µmol/l, respectively. Carvedilol at 100 µmol/l inhibited INCX in CCL39 cells expressing wild type NCX1 similar to mutant NCX1 without the intracellular regulatory loop. Carvedilol at 30 µmol/l abolished ouabain-induced delayed afterdepolarizations. CONCLUSION: Carvedilol inhibited cardiac NCX in a concentration-dependent manner in isolated cardiac ventricles, but metoprolol did not. We conclude that carvedilol inhibits NCX1 at supratherapeutic concentrations.

Nicorandil stimulates a Na⁺/Ca²âº exchanger by activating guanylate cyclase in guinea pig cardiac myocytes.

Nicorandil, a hybrid of an ATP-sensitive K(+) (KATP) channel opener and a nitrate generator, is used clinically for the treatment of angina pectoris. This agent has been reported to exert antiarrhythmic actions by abolishing both triggered activity and spontaneous automaticity in an in vitro study. It is well known that delayed afterdepolarizations (DADs) are caused by the Na(+)/Ca(2+) exchange current (I NCX). In this study, we investigated the effect of nicorandil on the cardiac Na(+)/Ca(2+) exchanger (NCX1). We used the whole-cell patch clamp technique and the Fura-2/AM (Ca(2+) indicator) method to investigate the effect of nicorandil on I NCX in isolated guinea pig ventricular myocytes and CCL39 fibroblast cells transfected with dog heart NCX1. Nicorandil enhanced I NCX in a concentration-dependent manner. The EC50 (half-maximum concentration for enhancement of the drug) values were 15.0 and 8.7 µM for the outward and inward components of I NCX, respectively. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), a membrane-permeable analog of guanosine 3',5'-cyclic monophosphate (cGMP), enhanced I NCX. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor (10 µM), completely abolished the nicorandil-induced I NCX increase. Nicorandil increased I NCX in CCL39 cells expressing wild-type NCX1 but did not affect mutant NCX1 without a long intracellular loop between transmembrane segments (TMSs) 5 and 6. Nicorandil at 100 µM abolished DADs induced by electrical stimulation with ouabain. Nicorandil enhanced the function of NCX1 via guanylate cyclase and thus may accelerate Ca(2+) exit via NCX1. This may partially contribute to the cardioprotection by nicorandil in addition to shortening action potential duration (APD) by activating KATP channels.

Effects of isoflavones from Sophora species on the growth and activation of a mouse macrophage-like cell line.

We investigated the effect of eleven isoflavones on the growth and activation of mouse macrophage-like Raw 264.7 cells. The study of structure-activity relationship suggests that both hydrophilic (hydroxyl) and hydrophobic (prenyl) groups within isoflavone molecules are the determinants for the induction of cytotoxic activity. When hydrophobicity was assessed by octanol-water partition coefficient (log P), the maximum cytotoxic activity was observed at a log P value above 2.5. All isoflavones did not significantly stimulate the nitric oxide (NO) production by Raw 264.7 cells, but reduced the NO production by lipopolysaccharide (LPS)-stimulated Raw 264.7 cells, at cytotoxic concentrations. Amino acid analysis in the culture medium demonstrated that isoflavones significantly inhibited the LPS-stimulated production of citrulline and asparagine. Isoflavones inhibited the LPS-stimulated NO production more efficiently than citrulline and asparagine production, possibly due to their NO scavenging activity. These data suggest that the inhibiton of LPS action by isoflavones may be coupled with their cytotoxic activity.

Stimulation of arginine consumption and asparagine production in LPS-activated macrophages.

Changes in amino acid utilization during lipopolysaccharide (LPS)-induced activation of mouse macrophage-like cells Raw264.7 were investigated. Amino acids in the medium and cell fractions were extracted by 5% trichloroacetic acid and quantitated by amino acid analyzer. Glutamine was utilized by cells at the highest rate, followed by serine and arginine, a precursor of nitric oxide (NO). When Raw264.7 cells were incubated with 10 or 100 ng/mL LPS, the consumption of arginine and the production of citrulline, nitric oxide (NO) and asparagine were significantly increased. The intracellular amino acid concentration was not significantly changed. These data suggest that arginine consumption and asparagine production might be possible markers of macrophage activation.

Effects of prenylflavanones from Sophora species on growth and activation of mouse macrophage-like cell line.

We investigated the effect of 2 flavanones and 8 chemically-defined prenylflavanones on the growth and activation of mouse macrophage-like Raw 264.7 cells. Amino acid analysis in the culture medium demonstrated the rapid consumption of serine and glutamine by Raw264.7 cells, suggesting the necessity to supplement these amino acids for the prolonged culture. Naringenin and hesperetin showed little or no cytotoxic activity. However, addition of the isoprenyl group (sophoraflavanone B, euchrestaflavanone A) or the lavandulyl and hydroxyl group (sophoraflavanone G) significantly enhanced the cytotoxic activity. The cytotoxic activity of these compounds was significantly influenced by both log P value and ionization potential. These compounds slightly, but significantly, reduced both nitric oxide (NO) and tumor necrosis factor (TNF) production by lipopolysaccharide (LPS)-stimulated Raw 264.7 cells, regardless of their cytotoxic activity. These data suggest that the macrophage inhibitory effect of prenylflavanones might not be related to their cytotoxic activity.

Effect of lignins and their precursors on nitric oxide, citrulline and asparagine production by mouse macrophage-like Raw 264.7 cells.

Lignins, tannins and flavonoids are commonly found polyphenols. Among these polyphenols, lignins, polymers of phenylpropenoids complexed with polysaccharides, were the least cytotoxic and most potently stimulated the production of nitric oxide (NO), citrulline and asparagine by mouse macrophage-like Raw 264.7 cells. The maximum production of these substances reached the level attained by lipopolysaccharide (LPS). However, epigallocatechin gallate, phenylpropenoid monomers (ferulic acid, caffeic acid) and gallic acid (component unit of tannin) were inactive. These data suggest that the macrophage-stimulation activity of polyphenols depends, at least in part, on their molecular weight or structural configuration. There was a positive relationship between the extent of asparagine production and that of NO or citrulline. Western blot analysis demonstrated that both lignins and LPS elevated the cellular level of asparagine synthetase. The present study suggests the possible link between the stimulated asparagine production and macrophage activation.