kb-r7943 and cariporide

The cardiac Na+-Ca2+ exchanger (NCX) plays an essential role in regulating Ca2+ under physiological and pathophysiological conditions. In its forward mode of operation, which predominates under physiological conditions, it extrudes the Ca2+ that enters the cardiac myocyte on a beat-to-beat basis. During ischemia and reperfusion, increased intracellular Na+ leads to a decrease in Ca2+ efflux and enhanced Ca2+ influx via the NCX, potentially leading to Ca2+ overload, which is one of the major pathophysiological mechanisms for ischemia-reperfusion injury. Novel NCX inhibitors discovered in recent years have shown great promise in attenuating ischemia-reperfusion injury.

Topics: Aniline Compounds; Animals; Anti-Arrhythmia Agents; Cardiotonic Agents; Guanidines; Humans; Myocardial Reperfusion Injury; Phenyl Ethers; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers; Sulfones; Thiourea

Rabbit ventricular myocardium is characterized by a biphasic response to stretch with an initial, rapid increase in force followed by a delayed, slow increase in force (slow force response, SFR). The initial phase is attributed to increased myofilament Ca(2+) sensitivity, but the mechanisms of the delayed phase are only incompletely understood. We tested whether stretch-dependent stimulation of Na(+)/H(+) exchange (NHE1) and consecutive changes in pH(i) and/or [Na(+)](i) may underlie the SFR.. Isometric contractions of rabbit ventricular muscles were recorded in bicarbonate-containing Tyrode's (Tyrode) or bicarbonate-free HEPES-buffered solution (HEPES). Muscles were loaded with the Ca(2+) indicator aequorin, the pH indicator BCECF, or the Na(+) indicator SBFI and rapidly stretched from 88% (L(88)) to 98% (L(98)) of optimal length. The resulting immediate and slow increases in twitch force (1st phase and SFR) as well as changes in [Ca(2+)](i), [Na(+)](i), or pH(i) were quantified before and after inhibition of NHE1 by HOE 642 (3 microM) or reverse-mode Na(+)/Ca(2+) exchange (NCX) by KB-R 7943 (5 microM).. In both Tyrode (n=21) and HEPES (n=22), developed force increased to approximately 160% during the 1st phase followed by a further increase to approximately 205% during the SFR. The SFR was accompanied by a 21% increase of the aequorin light transient (n=4; normalized to the 1st phase) and a approximately 3 mM increase in [Na(+)](i) (n=4-7). The SFR was also associated with an increase in pH(i). However, this increase was delayed and was significant only after the SFR had reached its maximum. The delayed pH(i) increase was larger in HEPES than in Tyrode. HOE 642 and/or KB-R 7943 reduced the SFR by approximately 30-40%. In addition, HOE 642 diminished the stretch-mediated elevation of [Na(+)](i) by 72% and the delayed alkalinization.. The data are consistent with the hypothesis that SFR results from increases in [Ca(2+)](i) secondary to altered flux via NCX in part resulting from increases in [Na(+)](i) mediated by NHE1.

Topics: Animals; Bicarbonates; Calcium; Cation Transport Proteins; Guanidines; Heart Ventricles; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Proteins; Microscopy, Fluorescence; Myocardial Contraction; Myocardium; Rabbits; Sodium; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers; Stress, Mechanical; Sulfones; Thiourea

The Na(+)/Ca(2+) exchanger (NCX) is involved in myocardial ischemia-reperfusion injuries. We examined the effects of 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a potent and selective inhibitor of NCX, on myocardial ischemia-reperfusion injury models. In canine cardiac sarcolemmal vesicles and rat cardiomyocytes, SEA0400 potently inhibited the Na(+)-dependent 45Ca(2+) uptake with an IC(50) value of 90 and 92 nM, compared with 2-[2-[4-(4-nitrobenzyloxy)phenyl]isothiourea (KB-R7943, 7.0 and 9.5 microM), respectively. In rat cardiomyocytes, SEA0400 (1 and 3 microM) attenuated the Ca(2+) paradox-induced cell death. In isolated rat Langendorff hearts, SEA0400 (0.3 and 1 microM) improved the cardiac dysfunction induced by low-pressure perfusion followed by normal perfusion. In anesthetized rats, SEA0400 (0.3 and 1 mg/kg, i.v.) reduced the incidence of ventricular fibrillation and mortality induced by occlusion of the left anterior descending coronary artery followed by reperfusion. These results suggest that SEA0400 is a most potent NCX inhibitor in the heart and that it has protective effects against myocardial ischemia-reperfusion injuries.

Topics: Aniline Compounds; Animals; Anti-Arrhythmia Agents; Arrhythmias, Cardiac; Calcium; Cell Survival; Cells, Cultured; Dogs; Guanidines; Heart; In Vitro Techniques; Male; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Phenyl Ethers; Rats; Rats, Sprague-Dawley; Rats, Wistar; Sarcolemma; Sodium-Calcium Exchanger; Sulfones; Thiourea

Inhibition of Na(+)/H(+) (NHE) and Na(+)/Ca(2+) (NCE) exchangers prevents myocardial ischemia/reperfusion injury by preventing cardiomyocyte Ca(2+) overload. We hypothesized that it may influence ischemic/reperfused myocardium also indirectly by preventing endothelial Ca(2+) accumulation, and thereby by attenuating reperfusion-induced formation of nitric oxide (NO) and/or oxygen free radicals. Langendorff-perfused rat hearts were subjected to 30-min ischemia and 30-min reperfusion. Myocardial outflow of NO (nitrite+nitrate) and hydroxyl radical (*OH, salicylate method), and functional recoveries were followed during reperfusion. In all groups, there was a transient rise in NO and *OH outflow upon reperfusion. An inhibitor of NHE, cariporide (10 microM) [(4-Isopropyl-3-methylsulfonyl-benzoyl)-quanidine methanesulfonate], and an inhibitor of the reverse mode of NCE, KB-R7943 (5 microM) (2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate), decreased NO and *OH formation, reduced contracture, and improved the recovery of mechanical function during reperfusion, compared to the untreated hearts. The formation of NO was reduced by 40% by 100 microM N(G)-methyl-L-arginine acetate salt (L-NMMA, NO synthase inhibitor), and not affected by 50 microM L-NMMA. *OH formation, contracture, and the functional recoveries were affected neither by 50 nor by 100 microM L-NMMA. Also, the effects of cariporide and KB-R7943 were unaffected by 100 microM L-NMMA. This study shows for the first time that the inhibition of NHE and NCE attenuates post-ischemic myocardial formation of NO and *OH, suggesting that prevention of Ca(2+) overload is cardioprotective via these mechanisms. The results indicate, however, that NO synthase pathway did not interfere with the protection afforded by NHE or NCE in our model.

Topics: Animals; Coronary Circulation; Dose-Response Relationship, Drug; Enzyme Inhibitors; Free Radicals; Guanidines; Heart; Heart Rate; Hydroxyl Radical; In Vitro Techniques; Male; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocardium; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Nitrites; omega-N-Methylarginine; Perfusion; Rats; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers; Sulfones; Thiourea; Ventricular Function, Left

This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II.. Isolated cat papillary muscles were used for force, pH(i), [Na(+)](i) and [Ca(2+)](i) measurements and isolated cat myocytes for patch-clamp experiments.. In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23+/-2% (n=4, P<0.05), [Na(+)](i) by 2.2+/-0.2 mmol/l (n=4, P<0.05), and peak (but not diastolic) Ca(2+) from 0.674+/-0.11 to 0.768+/-0.13 micromol/l (n=4, P<0.05), without affecting pH(i). Force and [Na(+)](i) increase were abolished by inhibition of the Na(+)/H(+) exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na(+)](i) increase was abolished by inhibition of reverse Na(+)/Ca(2+) exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21+/-2%, n=4, P<0.05) and in [Na(+)](i) (2.4+/-0.4 mmol/l, n=4, P<0.05) that were also suppressed by TAK044 and HOE642 were induced by exogenous 5.0 nmol/l endothelin-1. KB-R7943 reverted the endothelin-1 effect on force but not on [Na(+)](i). In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (E(NCX)) to a more negative value (DeltaE(NCX): -10+/-3 and -17+/-5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642.. Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na(+)/H(+) exchanger, increases [Na(+)](i) and changes E(NCX), promoting the influx of Ca(2+) that leads to a positive inotropic effect (PIE).

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Cats; Electrophysiology; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Glycopeptides; Guanidines; In Vitro Techniques; Losartan; Muscle Contraction; Papillary Muscles; Patch-Clamp Techniques; Peptides, Cyclic; Sodium-Calcium Exchanger; Sulfones; Thiourea

Since the Na(+)-H(+) exchanger (NHE) is not the only pathway of Na(+) influx into cardiomyocytes during ischemia/reperfusion, we hypothesized that blockade of Na(+)-Ca(2+) exchanger (NCX) may be a more efficient strategy than is NHE inhibition for protecting the myocardium from infarction. To test this hypothesis, we compared KB-R7943 (KBR), a novel selective NCX blocker, with cariporide, a selective NHE blocker, with regard to their protective effects against infarction. In isolated rabbit hearts, infarction was induced by 30-min global ischemia/2-h reperfusion, and infarct size was determined by tetrazolium staining and expressed as a percentage of area at risk (%IS/AR). Hearts received no drugs, or were infused with cariporide (1 microM) for 10 min or KBR (0.3 or 10 microM) for 5 min before ischemia or after the onset of reperfusion. Protein level of NCX was assessed by Western blotting. Cariporide infusion before ischemia significantly reduced %IS/AR from 63.9 +/- 2.9% to 20.2 +/- 3.0%, but its infusion upon reperfusion failed to achieve a significant protection (%IS/AR = 53.8 +/- 4.6%). In contrast, KBR infusion similarly reduced infarct size both when infused before ischemia (%IS/AR = 33.3 +/- 6.3% and 21.9 +/- 4.7% by 0.3 and 10 microM KBR, respectively) and when infused for only 5 min after reperfusion (%IS/AR = 35.3 +/- 7.1% and 31.5 +/- 2.1% by 0.3 and 10 microM KBR, respectively). Protein levels of NCX after 30-min ischemia and 30-min ischemia/30-min reperfusion were similar to baseline values in both untreated controls and hearts treated with 0.3 microM KBR upon reperfusion. These results suggest that lethal reperfusion injury is more efficiently suppressed by blockade of the NCX than by blockade of the NHE.

Topics: Animals; Guanidines; Hemodynamics; In Vitro Techniques; Male; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Myocytes, Cardiac; Rabbits; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers; Sulfones; Thiourea

Our objective was to assess the participation of Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX) on systolic and diastolic alterations of myocardial stunning. Isolated perfused rat hearts were submitted to 20 min of global ischemia (Is) followed by 30 min of reperfusion (R). This protocol was repeated after treatment before ischemia and/or early in R. with HOE 642 1 microM, a specific blocker of NHE-1 and KB-R7943 1 microM the novel inhibitor of the reverse mode of NCX. In control ischemic hearts the contractility assessed through +dP/dtmax recovered approximately 60%. When the NHE blockade was performed before is or early in R the postischemic recovery reached 100%. The blockade of the reverse mode of NCX only improved significantly the recovery when administered before is and early in R (95 +/- 7%). The ischemic contracture decreased when the treatment with both blockers was performed before Is. During R the increase of end diastolic pressure (EDP) observed in control ischemic hearts (at 30 min of R, EDP value was 44 +/- 4 mmHg) diminished significantly by NHE (24 +/- 6 and 12 +/- 2 mmHg when the blocker was administered before or after Is) and NCX blockade performed before and after is (12 +/- 6 mmHg). These results indicate that the activation of the reverse mode of NCX secondary to the NHE activation during ischemia and reperfusion is the mechanism responsible for the Ca2+ overload involved in the diminution of contractility that characterizes myocardial stunning.

Topics: Animals; Anti-Arrhythmia Agents; Guanidines; Myocardial Contraction; Myocardial Ischemia; Myocardial Stunning; Rats; Sodium-Calcium Exchanger; Sodium-Hydrogen Exchangers; Sulfones; Thiourea; Time Factors