Hideharu Hayashi

KB-R7943 Block of Ca2+ Influx Via Na+/Ca2+ Exchange Does Not Alter Twitches or Glycoside Inotropy but Prevents Ca2+ Overload in Rat Ventricular Myocytes

BACKGROUNDnThe Na(+)/Ca(2+) exchange (NCX) extrudes Ca(2+) from cardiac myocytes, but it can also mediate Ca(2+) influx, load the sarcoplasmic reticulum with Ca(2+), and trigger Ca(2+) release from the sarcoplasmic reticulum. In ischemia/reperfusion or digitalis toxicity, increased levels of intracellular [Na(+)] ([Na(+)](i)) may raise levels of intracellular [Ca(2+)] ([Ca(2+)](i)) via NCX, leading to cell injury and arrhythmia.nnnMETHODS AND RESULTSnWe used KB-R7943 (KBR) to selectively block Ca(2+) influx via NCX to study the role of NCX-mediated Ca(2+) influx in intact rat ventricular myocytes. Removing extracellular Na(+) caused [Ca(2+)](i) to rise, due to Ca(2+) influx via NCX, and this was blocked by 90% with 5 micromol/L KBR. However, KBR did not alter [Ca(2+)](i) decline due to NCX. Thus, we used 5 micromol/L KBR to selectively block Ca(2+) entry but not efflux via NCX. Under control conditions, 5 micromol/L KBR did not alter steady-state twitches, Ca(2+) transients, Ca(2+) load in the sarcoplasmic reticulum, or rest potentiation, but it did prolong the late low plateau of the rat action potential. When Na(+)/K(+) ATPase was inhibited by strophanthidin, KBR reduced diastolic [Ca(2+)](i) and abolished the spontaneous Ca(2+) oscillations, but it did not prevent inotropy.nnnCONCLUSIONSnIn rat ventricular myocytes, Ca(2+) influx via NCX is not important for normal excitation-contraction coupling. Furthermore, the inhibition of Ca(2+) efflux alone (as [Na(+)](i) rises) may be sufficient to cause glycoside inotropy. In contrast, Ca(2+) overload and spontaneous activity at high [Na(+)](i) was blocked by KBR, suggesting that net Ca(2+) influx (not merely reduced efflux) via NCX is involved in potentially arrhythmogenic Ca(2+) overload.

Sildenafil for primary and secondary pulmonary hypertension.

Sildenafil is a selective inhibitor of cyclic guanosine monophosphate‐specific phosphodiesterase type 5, an enzyme that is abundant in both lung and penile tissues. Sildenafil is widely used to dilatepenile arteries, suggesting that it may also dilate pulmonary arteries in patients with pulmonary hypertension. However, the long‐term hemodynamic effects and safety of the drug in pulmonary hypertension are not known.

Effects of a selective inhibitor of Na+/Ca2+ exchange, KB-R7943, on reoxygenation-induced injuries in guinea pig papillary muscles.

The effects of a novel agent that is reported to selectively block Ca2+ influx by Na+/Ca2+ exchange (NCX), KB-R7943, on the reoxygenation-induced arrhythmias and the recovery of developed tension after reoxygenation, were investigated in guinea pig papillary muscles. KB-R7943 dose-dependently suppressed the contracture tension during low-sodium (21.9 mM) perfusion (23+/-8% of steady-state developed tension at 10 microM vs. 56+/-11% in control; n = 6, p<0.05), but did not change action potential and contractile parameters. During the reoxygenation period after 60-min substrate-free hypoxia, KB-R7943 (10 microM) significantly decreased the incidence of arrhythmias (44 vs. 100% in control; n = 9, p <0.05) and shortened the duration of arrhythmias (16+/-11 vs. 72+/-14 s; p<0.01). KB-R7943 (10 microM) significantly enhanced the recovery of developed tension after reoxygenation (83+/-4 vs. 69+/-3% in control; p<0.05). We conclude that KB-R7943 (10 microM) selectively inhibits the reverse mode of NCX, and that it attenuates reoxygenation-induced arrhythmic activity and prevents contractile dysfunction in guinea pig papillary muscles. These results suggest that Ca2+ influx by NCX may play a key role in reoxygenation injury.

A single cell model of myocardial reperfusion injury: changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes.

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 μM carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 ± 2.0 to 14.0 ± 3.4 mM (means ± S.E., p < 0.01) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 ± 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 ± 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 ± 0.04 to 6.31 ± 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 μM) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca+]i declined. In hypercontracted cells, [Na+]i increased as much as in ‘Ca2+ wave’ cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 μM), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.

The importance of glycolytically-derived ATP for the Na+/H+ exchange activity in guinea pig ventricular myocytes.

The cardiac subtype of Na+/H+ exchanger (NHE-1) plays an important role in the regulation of intracellular pH (pHi) and also can be a major route for Na+ influx. Although intracellular ATP is required for the optimal function of NHE-1, the regulation of the exchanger by ATP is less well characterized. This study was designed to investigate which intracellular ATP generated by oxidative phosphorylation or by glycolysis is dominant for the activation of NHE-1 in intact cardiac myocytes. Isolated guinea pig ventricular myocytes were loaded with the pHi-sensitive fluorescent indicator, 2′-7′-bis(carboxyl)-5′,6′-carboxy fluorescein (BCECF), and exposed to 20 mM 2-deoxyglucose (2-DG) or 2 mM sodium cyanide (CN) to inhibit glycolysis or oxidative phosphorylation, respectively. The activity of NHE-1 was estimated with pHi recovery following transient application of 15 mM NH4Cl (NH4Cl prepulse). After the NH4Cl prepulse, pHi decreased from 7.00 ± 0.03 (mean ± S.E.) to 6.60 ± 0.06 and recovered to 6.94 ± 0.13 at 10 min (n = 7). The pHi recovery was suppressed in the presence of 2-DG (6.67 ± 0.05, p < 0.01, n = 7), but was not changed in the presence of CN (6.88 ± 0.18, n = 6). Since there was no difference in the intrinsic H+ buffering power, the estimation of the net acid efflux demonstrated that the activity of NHE-1 was significantly depressed in 2-DG. The inhibitory effect of 2-DG was not due to more extensive depletion of global intracellular ATP or secondary to the change in either intracellular Na+ or Ca2+ concentration. We concluded that ATP generated by glycolysis rather than by oxidative phosphorylation is essential to activate NHE-1 in ventricular myocytes.

Effects of an endothelin receptor antagonist TAK-044 on myocardial energy metabolism in ischemia/reperfused rat hearts

The purpose of this study was to investigate the effects of an endothelin-receptor antagonist TAK-044 on functional defects and metabolic derangement in myocardial ischemia/reperfusion injury. We sequentially measured high-energy phosphate metabolites and intracellular pH by phosphorus magnetic resonance spectroscopy during 35-min global ischemia followed by 60-min reperfusion in Langendorff-perfused rat hearts. TAK-044 (initial loading by 3 mg/kg followed by perfusion with 100 nM solution) was administered in two different ways: before ischemia or immediately after reperfusion. In addition, we investigated the effects of TAK-044 on functional defects and metabolic alterations induced by hydrogen peroxide (200 microM, 30 min). The recoveries of left ventricular developed pressure after reperfusion in TAK-044 groups (51 +/-12% in TAK-I, 61 +/- 12% in TAK-R) were better than in control (10 +/- 5% in control; p < 0.01). Increases in left ventricular end-diastolic pressure (LVEDP) in TAK-044 groups (22 +/- 5 mm Hg in TAK-I, 24 +/- 5 mm Hg in TAK-R) were less than in control (38 +/- 3 mm Hg; p < 0.01). Adenosine triphosphate (ATP) (33 +/- 5% in TAK-I, 28 +/- 4% in TAK-R) in TAK-044 groups were higher than in control (13 +/- 3%; p < 0.01). The creatine phosphokinase (CPK) release during reperfusion in TAK-044 groups (3.3 +/- 1.5 IU/g wet wt/60 min in TAK-I, 3.5 +/- 2.5 IU/g wet wt/60 min in TAK-R) were lower than in control (13.8 +/- 3.9 IU/g wet wt/60 min; p < 0.05). In contrast, TAK-044 did not attenuate the myocardial injury induced by hydrogen peroxide. TAK-044, even if administered simultaneous with coronary reperfusion, attenuated myocardial ischemia/ reperfusion injury. The energy-preservative effect of TAK-044 could be associated with the good functional recovery in ischemia/reperfused rat hearts.

Involvement of CCAAT/enhancer-binding protein in regulation of the rat serine:pyruvate/alanine:glyoxylate aminotransferase gene expression

In the rat liver, transcription of the serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT) gene occurs from two sites, +1 and +66, in exon 1, resulting in the formation of two mRNAs, one for a precursor of mitochondrial SPT/AGT and the other for peroxisomal SPT/AGT, respectively. In this study, we attempted to characterize the downstream promoter responsible for generation of peroxisomal SPT/AGT. The minimal downstream promoter was confined to the +21–+90 region. We demonstrated that C/EBPα and C/EBPβ bound around the downstream start site (+66) contribute to the promoter activity. The downstream promoter activity is also regulated positively by a short inverted repeat, located 20–30 bp upstream of the downstream start site, through a protein factor(s) bound to this region. On the other hand, the sequence just downstream of the start site may negatively regulate the promoter activity.

Role of high-energy phosphate metabolism in hydrogen peroxide-induced cardiac dysfunction

This study was undertaken to clarify the role of high-energy phosphate metabolism in hydrogen peroxide-induced cardiac dysfunction using phosphorus and fluorine nuclear magnetic resonance spectroscopy. The exposure of a Langendorff-perfused heart to hydrogen peroxide (200-400 μmol/L, 8 min) provoked biphasic contractile dysfunction characterized by a transient depression of left ventricular developed pressure during the administration of hydrogen peroxide and a delayed elevation of left ventricular end-diastolic pressure after the washout of hydrogen peroxide. The initial phase of cardiac dysfunction correlated well with the accumulation of sugar phosphates (r = 0.89, p < 0.01). Furthermore, we demonstrated that glibenclamide, a potent inhibitor of the ATP-sensitive K+ channel, attenuated the initial depression of developed pressure. On the other hand, the delayed elevation of end-diastolic pressure correlated well with the total ATP depletion (r = 0.96, p < 0.01). However, ATP loss was supposed to be a mere result from the increased ATP consumption corresponding to a rise in intracellular free Ca2+ (from the control value of 315 ± 23 nmol/L to 708 ± 104 after the administration of hydrogen peroxide, p < 0.01), which also paralleled the elevation of end-diastolic pressure. Thus glycolytic inhibition and intracellular Ca2+ overload are independently responsible for the biphasic contractile dysfunction induced by hydrogen peroxide.

Insulin inhibits coronary endothelial cell calcium entry and coronary artery relaxation.

Hyperinsulinemia is closely related to coronary artery disease. Endothelial cells are important for the control of vascular tone, and dysfunction of endothelial cells has been implicated in coronary artery disease. The direct effects of insulin on coronary endothelial cells are nonetheless unknown. In this study, the acute effects of high-dose insulin were investigated on agonist-induced intracellular Ca 2+ concentration ([Ca 2+ ] i ) in porcine coronary endothelial cells and coronary relaxation. Bradykinin (10 n M) and cyclopiazonic acid (100 &mgr;M), an inhibitor of the endoplasmic reticulum Ca 2+ -ATPase, provoked large increases in [Ca 2+ ] i in coronary endothelial cells. This increase was dose-dependently inhibited by a 10-min preincubation with high doses of insulin (10, 30, 100 mU/ml). Under Ca 2+ -free conditions, bradykinin and cyclopiazonic acid provoked transient, small increases in [Ca 2+ ] i . These increases were not affected by pretreatment with insulin (100 mU/ml). Bradykinin (1, 10, 100, 1,000 n M) and cyclopiazonic acid (10 &mgr;M) significantly relaxed porcine coronary artery rings precontracted with histamine (1 &mgr;M). The vasodilator effects of bradykinin and cyclopiazonic acid were dose-dependently inhibited by insulin. These acute effects were not observed at physiologic concentrations. Our data indicate that high-dose insulin inhibits agonist-induced Ca 2+ response in coronary endothelial cells and attenuates agonist-induced coronary vasodilatation. The study suggests that hyperinsulinemia might be associated with coronary artery disease via derangement of endothelial Ca 2+ -dependent functions.