The excitation-contraction (E-C) coupling system during the development of heart can be investigated because of marked progression in electrophysiology and microfluorescence studies. During the developmental period, Ca²⁺ influx mediating the E-C coupling is mainly through the L-type Ca²⁺ channels. In the fetal period, T-type Ca²⁺ channels and the reverse mode of the Na-Ca exchange system also contribute to Ca²⁺ influx. These contributions probably reduce gradually until adulthood. The contraction of fetal cardiomyocytes has been thought to depend mainly on the Ca²⁺ influx. However, recent studies reveal that immature sarcoplasmic reticulum (SR) already works in the early fetal period. Ca²⁺ spark, a local and unitary movement of Ca²⁺, can be observed in adult cardiomyocytes by the use of a confocal microscope. On the other hand, no Ca²⁺ spark is observed in fetal cardiomyocytes. The frequency of Ca²⁺-spark evocation increases during the postnatal period. Therefore a close distance between the L-type Ca²⁺ channel and the SR Ca²⁺-release channel is essential to the establishment of the Ca²⁺ spark.

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The regulated release of ATP is a fundamental process in cell-to-cell signaling. The electrogenic translocation of ATP via an anion channel has been suggested as one possible mechanism of the release. In this review, we survey possible candidate channels for this pathway. The maxi-anion channel characterized by an exceedingly large unitary conductance has been a stray channel with regard to its function. A newly discovered property, its ATP conductivity and its activation in response to stress signals, indicates that this channel has a central role in stress-sensory transduction for cell volume regulation and tubuloglomerular feedback.

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Chronic heart failure (CHF) is associated with abnormal neurohormonal profiles and increased ventilatory response to exercise. This study determined if treatment with angiotensin II type 1 receptor antagonist, losartan, improves ventilatory efficiency and neurohormonal factors in patients with CHF. Symptom-limited cardiopulmonary exercise testing was performed after a 2-week placebo period (baseline) and after 16 weeks of treatment with losartan (40 ± 4 mg/day) in 10 patients with CHF (age 57.7 ± 3.7 years). Echocardiogram, daily physical activity (by the specific activity scale), and neurohormones were evaluated. Treatment with losartan increased left ventricular ejection fraction (baseline vs. losartan: 31 ± 3 vs. 39 ± 3%, p < 0.01) and specific activity scale score (5.3 ± 0.5 vs. 6.4 ± 0.4 METS, p < 0.05). Losartan decreased the ventilatory response to carbon dioxide production during exercise (V_E/V_CO2 slope: 34.6 ± 2.4 vs. 32.0 ± 2.2, p < 0.05). Plasma brain natriuretic peptide concentrations were decreased after therapy (301 ± 79 vs. 176 ± 53 pg/ml, p < 0.05). In summary, the results of this open-label, uncontrolled study suggest that chronic treatment with losartan may improve ventilatory efficiency and decrease plasma brain natriuretic peptide concentrations with the improvement of physical activity and left ventricular systolic function in patients with CHF.

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To understand the molecular mechanism of ischemia-induced cardiac myocyte cell death, H9c2 cells were studied by chemical hypoxia (CH), using metabolic inhibition buffer. CH suppressed the activities of caspase-3, -8, and -9. c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) were activated, whereas extracellular regulated kinase (ERK) was inactivated. Only protein kinase Cε (PKCε) among PKC isotypes was translocated to the membrane fraction implying its activation. Moreover, the administration of PKCε inhibitor suppressed the phosphorylations of JNK/p38 MAPK and reduced CH-induced cell death. An administration of JNK/p38 MAPK inhibitors also decreased CH-induced cell deaths, implying JNK/p38 MAPK's causative roles in the deaths. Collectively, this study identified a novel caspase-independent PKCε-JNK/p38 MAPK signaling module induced by CH in cardiac myocytes. Our data show that the PKCε-JNK/p38 MAPK signaling module contributes to CH-induced H9c2 cell death. This contrasts with previous notions, i.e., PKCε's protective effect against ischemic death. Thus our data suggest that PKCε can mediate alternative signals, i.e., beneficiary or deleterious signals, depending on the cell type, intensity, and/or type of injury.

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The cell-volume regulation by swelling-activated Cl⁻ current (I_Cl,swell) was studied in guinea pig ventricular myocytes, using a microscopic video-image analysis. We have previously shown that in ventricular cells depolarized in high-K⁺ ([K⁺]_o >45 mM) solution, an activation of the cyclic AMP-dependent Cl⁻ current (I_Cl,cAMP) leads to cell swelling. We first investigated the mechanism underlying the I_Cl,cAMP-independent recovery (shrinkage) of the swollen cells. They shrank when the membrane potential (V_m) was made negative to the equilibrium potential of Cl⁻ (E_Cl) by lowering [K⁺]_o or [Cl⁻]_o in the high-K⁺ solution. This shrinkage was attenuated by the inhibitors (DIDS, glibenclamide, furosemide) of swelling-activated Cl⁻ current (I_Cl,swell). These findings suggested an involvement of I_Cl,swell in the observed isosmotic cell shrinkage. On the other hand, an application of hyposmotic (70% of control) solution to the cells at normal [K⁺]_o (E_Cl>V_m) induced a cell swelling, and the swollen cells underwent a slight but definite spontaneous cell shrinkage during hyposmotic challenge, indicating the operation of the mechanism of regulatory volume decrease (RVD). This RVD was pronounced at low [Cl⁻]_o, at which E_Cl was much more positive than V_m. On the contrary, when the hyposmotic solution was applied to the cells at high [K⁺]_o, at which E_Cl was negative to V_m, the cells swelled vigorously and monotonically without showing RVD, the swelling being much greater than that seen at normal [K⁺]_o. Both the RVD at normal [K⁺]_o and the extra cell swelling at high [K⁺]_o were suppressed by DIDS. These results suggest that I_Cl,swell activated by cell swelling can shrink or inflate the cardiac cells under hyposmotic as well as isosmotic conditions, depending on V_m and E_Cl.

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Effects of oxidative stress on intact human erythrocytes were investigated using tert-butyl hydroperoxide (tBHP). Exposure of erythrocytes to tBHP caused a marked decrease in filterability in a time-dependent manner. Erythrocytes exposed to tBHP also show an increase in mean corpuscular volume and a remarkable formation of methemoglobin (met-Hb) without any appearance of hemichromes that form Heinz bodies. High performance liquid chromatography demonstrated that the tBHP-treated erythrocytes exhibited an apparent decrease in the membrane phospholipid, phosphatidylethanolamine (PE). The decrease in PE was inhibited by pretreatment with ascorbate, but not with verapamil. SDS-polyacrylamide gel electrophoresis of the tBHP-treated erythrocyte membrane showed a degradation of spectrin, band 3, band 4.2, and band 4.5, accompanied by the appearance of low–molecular-weight products. The degradation of the membrane proteins was not prevented by pretreatment with verapamil or ascorbate. However, the pretreatment with verapamil but not with ascorbate revealed significant inhibition of the tBHP-induced impairment in filterability in the presence of extracellular Ca²⁺. Thus, the present study shows that verapamil, a potent drug in reperfusion therapy, plays an important role in protection against oxidative injury, based on a close linkage among decreased filterability, met-Hb formation, and impaired membrane integrity.

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We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019–2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14–16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-β hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.

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Hyperthermia and hypoxia influence ventilation and metabolism; however, their synergistic effects remain unanswered. We hypothesized that an enhancement of ventilation induced by hyperthermia is competitive with hypoxic hypometabolism. We then examined the relationship of body temperature, hypoxia, and respiration in conscious mice, measuring minute ventilation (V_E), aerobic metabolism, and arterial blood gases. All parameters were measured at two different body temperatures (BTs), approximately 37°C (normothermia) and 39°C (hyperthermia), under both normoxia (room air inhalation) and hypoxia (7% O₂ inhalation). Under normoxia, V_E and O₂ consumption (V_O2) were lower at hyperthermia than at normothermia, and the V_E–V_O2 ratio remained constant. Pa_CO2 values were normal at both BTs under normoxia. Hypoxic gas inhalation increased V_E, which reached a peak in 2 min, then decreased at both BTs. V_E remained at a higher level during hyperthermia than during normothermia throughout the 10 min experiment. V_O2 decreased during hypoxia at both BTs. Hypoxia increased the V_E–V_O2 ratio because of relatively high V_E with respect to the decreased V_O2, which means hyperventilation. At hypoxia under hyperthermia, serious hyperventilation occurred with a further increase in V_E. The augmented ventilation may be due to the thermal stimulus and a lowered thermoregulatory set point for hypoxia. Thus hyperthermia reduces ventilation and metabolism to maintain normocapnia; as a result, thermogenesis is reduced under normoxia. Hyperthermia augments hyperventilation induced by hypoxia, leading to severe hypoxic hypocapnia. Thermal stimuli may impair the adjustment of ventilation and metabolism when O₂ is limited.

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Pontamine sky blue (PSB) is used in brain studies to mark the position of microelectrode and micropipette tips. However, few studies have been made on the effects of PSB on neurons; therefore we examined these effects. When puffed on isolated sensory ganglion cells of rats, PSB increased membrane conductance, depolarized membrane potential, and reduced the amplitude of action potentials. When dripped on frog sympathetic ganglion, much like hexamethonium, PSB decreased the amplitude of compound action potentials of the postganglionic strand. A bath application of PSB to sartorius muscle fibers that had been treated with tetrodotoxin depolarized the membrane potential and increased the frequency and amplitude of miniature end-plate potentials. All these effects were reversible. When injected into the rat's pontine part corresponding to the location of the canine pontine defecation reflex center, PSB produced repetitive colorectal contractions and irreversibly abolished them in response to anal-canal stimulation. The excitatory and blocking effects of PSB and its staining ability make it a useful tool for examining the functions of focal brain parts.

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Sargassum horneri is an edible marine brown alga distributed along the seacoast of Japan. Here we examined effects on the water-soluble (ethanol-insoluble) extracts (EIS) from Sargassum horneri on ion transports across the isolated rat colonic mucosa set in Ussing chambers. The nonpolysaccharide fraction of EIS (EIS-2) significantly decreased short-circuit current (I_sc) across the mucosa, and increased the tissue conductance (G_t). The half-maximal effect of EIS-2 was obtained at 20 μg/ml. In contrast, the polysaccharide fraction of EIS (EIS-1; 100 μg/ml) had little effect on I_sc and G_t. The effect of EIS-2 depended on the presence of Cl⁻ and HCO₃⁻ but not K⁺ in the bathing solution. These results suggest that EIS-2 stimulates Cl⁻ absorption in the colonic mucosa. The EIS-2-induced changes in I_sc and G_t were inhibited by 3-(1-[p-chlorobenzyl]-5-[isopropyl]-3-t-butylthioindol-2-yl)-2,2-dimethyl-propanoic acid sodium (MK-886; 10 μM), a 5-lipoxygenase-activating protein inhibitor, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; 100 μM), a Cl⁻ channel blocker. EIS-2 attenuated the prostaglandin E₂ (0.5 μM)-increased I_sc, and the half-maximal effect of EIS-2 was obtained at 50 μg/ml. The present study suggests that the EIS-2 stimulates Cl⁻ absorption mediated by basolateral leukotriene-sensitive Cl⁻ channels and apical Cl⁻/HCO₃⁻ exchanger in the rat colonic mucosa.

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The spatial resolution of near infrared spectroscopic imaging in brain function mapping studies needs to be improved. Most near infrared spectroscopic imaging systems use optical fibers that are arranged like a lattice. The light source and the detector have one-on-one correspondence at intervals of about 3 cm. In this study, we apply several detectors against one source to improve spatial resolution. We assume that a local absorber within a strong scattering medium is a model for local cerebral activation. When we calculate absorbance, which is used to spectroscopically calculate hemoglobin concentration, the peak position shifts away from above the position where an absorber is located. As far as absorbance is calculated, we cannot obtain spatial information about the absorber even if many detectors are used against one source. When we calculate the difference between detected light intensities, however, we demonstrate that an absorber projected onto the measuring surface has an influence directly above it. We predict this property from a light diffusion equation and also prove it experimentally through measurements of a uniform resinous phantom with an absorber. This is one of the basic principles supporting the achievement of higher spatial resolutions with near infrared spectroscopic imaging.

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An organ-level assessment of the total Ca²⁺ handled in the excitation-contraction coupling in a beating heart has been accomplished in canine left ventricles (LVs). This approach combines the intramyocardial Ca²⁺ recirculation fraction (RF) with the cardiac O₂ consumption for the excitation-contraction coupling. The RF has conventionally been obtained from the exponential decay of the postextrasystolic (PES) potentiation of myocardial contractility. However, in canine LVs, the PES contractility in terms of E_max (end-systolic pressure–volume ratio) has been shown to decay generally in alternans under both physiological and pathological conditions. Nevertheless, the RF can be obtained from the exponential decay component in the PES E_max alternans decay. We expected that the same Ca²⁺ assessment could be applied to the human heart. As the first step, we investigated whether the PES E_max would decay in alternans or exponentially in patient LVs. We retrospectively analyzed 13 patient cases that had stable regular beats unexpectedly interrupted by a spontaneous extrasystole followed by a PES compensatory pause during their diagnostic examination. These patients had either mitral regurgitation, old myocardial infarction, or dilated cardiomyopathy. Their LV E_max decayed consistently in alternans within the first several PES beats. These E_max alternans decays resemble those reported in canine LVs. This finding suggests for the first time the applicability of the same organ-level RF assessment method developed for canine hearts to human hearts.

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