Effects of pinacidil on coronary Ca(2+)-myosin phosphorylation in cold potassium cardioplegia model

The effects of the potassium (K(+)) channel opener pinacidil (Pin) on the coronary smooth muscle Ca(2+)-myosin light chain (MLC) phosphorylation pathway under hypothermic K(+) cardioplegia were determined by use of an in vitro microvessel model. Rat coronary arterioles (100-260 microm in diameter) were subjected to 60 min of simulated hypothermic (20 degrees C) K(+) cardioplegic solutions (K(+) = 25 mM). We first characterized the time course of changes in intracellular Ca(2+) concentration, MLC phosphorylation, and diameter and observed that the K(+) cardioplegia-related vasoconstriction was associated with an activation of the Ca(2+)-MLC phosphorylation pathway. Supplementation with Pin effectively suppressed the Ca(2+) accumulation and MLC phosphorylation in a dose-dependent manner and subsequently maintained a small decrease in vasomotor tone. The ATP-sensitive K(+) (K(ATP))-channel blocker glibenclamide, but not the nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine methyl ester, significantly inhibited the effect of Pin. K(+) cardioplegia augments the coronary Ca(2+)-MLC pathway and results in vasoconstriction. Pin effectively prevents the activation of this pathway and maintains adequate vasorelaxation during K(+) cardioplegia through a K(ATP)-channel mechanism not coupled with the endothelium-derived NO signaling cascade.     

Age-related differences in Na+-dependent Ca2+ accumulation in rabbit hearts exposed to hypoxia and acidification

In this study, we test the hypothesisthat in newborn hearts (as in adults) hypoxia and acidificationstimulate increased Na⁺ uptake, in part via pH-regulatoryNa⁺/H⁺ exchange. Resulting increases inintracellular Na⁺ (Na_i) alter the force drivingthe Na⁺/Ca²⁺ exchanger and lead to increasedintracellular Ca²⁺. NMR spectroscopy measuredNa_i and cytosolic Ca²⁺ concentration([Ca²⁺]_i) and pH (pH_i) inisolated, Langendorff-perfused 4- to 7-day-old rabbit hearts. AfterNa⁺/K⁺ ATPase inhibition, hypoxic hearts gainedNa⁺, whereas normoxic controls did not [19 ± 3.4 to139 ± 14.6 vs. 22 ± 1.9 to 22 ± 2.5 (SE) meq/kg drywt, respectively]. In normoxic hearts acidified using theNH₄Cl prepulse, pH_i fell rapidly and recovered,whereas Na_i rose from 31 ± 18.2 to 117.7 ± 20.5 meq/kg dry wt. Both protocols caused increases in [Ca]_i;however, [Ca]_i increased less in newborn hearts than inadults (P < 0.05). Increases in Na_i and[Ca]_i were inhibited by theNa⁺/H⁺ exchange inhibitormethylisobutylamiloride (MIA, 40 µM; P < 0.05), aswell as by increasing perfusate osmolarity (+30 mosM) immediately before and during hypoxia (P < 0.05). The data supportthe hypothesis that in newborn hearts, like adults, increases inNa_i and [Ca]_i during hypoxia and afternormoxic acidification are in large part the result of increased uptakevia Na⁺/H⁺ and Na⁺/Ca²⁺exchange, respectively. However, for similar hypoxia and acidification protocols, this increase in [Ca]_i is less in newborn thanadult hearts.

     

Effect of pH on Na induced Ca deficiency

Although it is well known that high Na concentrations induce Ca deficiency in acidic conditions, the effect of high pH on this competitive mechanism is not so well understood. The effect of Ca activity ratio (CAR) and pH on the Ca uptake of mungbeans (Vigna radiata (L.) Wilczek cv. Emerald) and Rhodes grass (Chloris gayana cv. Pioneer) in Na dominated solution cultures and in soil was investigated. Changes in pH in the alkaline range were shown not to affect the critical CAR of 0.024 (corresponding to 90% relative root length) for mungbeans grown in solution culture. Results from soil grown mungbeans confirmed those from solution culture, with a critical CAR of 0.025. A critical CAR of 0.034 was also established for soil grown Rhodes grass. The similarity of critical values established for mungbeans and Rhodes grass in solution culture and soil justifies the use of both solution culture and soil solution measurement as techniques for studying plant growth and limitations across plant species.     

Effects of cytoplasmic and luminal pH on Ca(2+) release channels from rabbit skeletal muscle

Ryanodine receptor (RyR)-Ca(2+) release channels from rabbit skeletal muscle were incorporated into lipid bilayers. The effects of cytoplasmic and luminal pH were studied separately over the pH range 5-8, using half-unit intervals. RyR activity (at constant luminal pH of 7.5) was inhibited at acidic cytoplasmic pH, with a half-inhibitory pH (pH(I)) approximately 6.5, irrespective of bilayer potential and of whether the RyRs were activated by cytoplasmic Ca(2+) (50 microM), ATP (2 or 5 mM), or both. Inhibition occurred within approximately 1 s and could be fully reversed within approximately 1 s after brief inhibition or within approximately 30-60 s after longer exposure to acidic cytosolic pH. There was no evidence of any hysteresis in the cytoplasmic pH effect. Ryanodine-modified channels were less sensitive to pH inhibition, with pH(I) at approximately 5.5, but the inhibition was similarly reversible. Steady-state open and closed dwell times of RyRs during cytoplasmic pH inhibition suggest a mechanism where the binding of one proton inhibits the channel and the binding of two to three additional protons promotes further inhibited states. RyR activity was unaffected by luminal pH in the pH range 7.5 to 6.0. At lower luminal pH (5-5.5) most RyRs were completely inhibited, and raising the pH again produced partial to full recovery in only approximately 50% of cases, with the extent of recovery not detectably different between pH 7.5 and pH 9. The results indicate that isolated skeletal muscle RyRs are not inhibited as strongly by low cytoplasmic and luminal pH, as suggested by previous single-channel studies.     

gAd-globular head domain of adiponectin increases fatty acid oxidation in newborn rabbit hearts

Adiponectin is an adipocyte-derived hormone that has a number of metabolic effects in the body, including the control of both glucose and fatty acid metabolism. The globular head domain of adiponectin, gAd, has also been shown to increase fatty acid oxidation in skeletal muscle. Within days after birth, a rapid increase in fatty acid oxidation occurs in the heart. We examined whether adiponectin or gAd plays a role in this maturation of cardiac fatty acid oxidation. Plasma adiponectin increased in newborn rabbits following birth: 1.2 +/- 0.3 microg/ml in 1-day-old, 6.8 +/- 1.8 microg/ml in 7-day-old, and 45 +/- 5 microg/ml in 6-week-old rabbits. Because plasma insulin levels decrease and remain low throughout the suckling period, and because this decrease may contribute to the maturation of fatty acid oxidation, we examined the effects of adiponectin and gAd on fatty acid oxidation in isolated perfused 1-day-old rabbit hearts in the presence or absence of 100 microunits/ml insulin. Adiponectin (10 microg/ml) did not alter fatty acid oxidation in the presence of insulin. In the absence of insulin, the addition of recombinant gAd (1.5 microg/ml) increased fatty acid oxidation compared with control (129 +/- 18 versus 66 +/- 11 nmol.g dry weight(-1).min(-1), respectively (p < 0.05). In 7-day-old hearts, where fatty acid oxidation rates were 5-fold higher than 1-day-old hearts, gAd did not alter fatty acid oxidation rates. The increase in fatty acid oxidation in 1-day-old hearts occurred independently of changes in 5'-AMP-activated protein kinase, acetyl-CoA carboxylase, or malonyl-CoA. The effect of gAd on fatty acid oxidation was reversed in the presence of 100 microunits/ml insulin. These results suggest that a decrease in plasma insulin and increase in gAd are involved in the increase of cardiac fatty acid oxidation in the immediate newborn period.     

Effects of metabolic inhibition on conduction, Ca transients, and arrhythmia vulnerability in embryonic mouse hearts

Developing myocardium is more dependent on glycolysis than adult myocardium, yet the effects of selectively inhibiting glycolysis versus oxidative phosphorylation on embryonic heart function have not been well characterized. Accordingly, we investigated how selective metabolic inhibition affects membrane voltage and intracellular Ca (Ca(i)) transients in embryonic mouse hearts, including their susceptibility to arrhythmias. A total of 136 isolated embryonic mouse hearts were exposed to either 1) 2-deoxyglucose (2DG; 10 mM) or iodoacetate (IAA; 0.1 mM) with 10 mM pyruvate in place of glucose to selectively inhibit glycolysis or 2) the mitochondrial uncoupler protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 500 nM) with 10 mM glucose present to selectively inhibit oxidative phosphorylation. Using confocal imaging, we found that mitochondrial membrane potential monitored with tetramethylrhodamine methyl ester (200 nM) remained stable with 2DG or IAA but depolarized within 5 min after exposure to FCCP. IAA and FCCP decreased heart rate, inhibited Ca(i) transient amplitude, shortened action potential duration at 80% repolarization (APD(80)), and prolonged atrioventricular conduction time to similar extents. Although 2DG decreased heart rate and Ca(i) transient amplitude, it did not significantly affect APD(80) and AV conduction time. In addition, spontaneous arrhythmias occurred in 77 of 136 embryonic hearts (57%) after exposure to IAA (28/53) or FCCP (49/83). There were no significant differences in the types or incidence of arrhythmias induced by IAA and FCCP. These data support the idea that both glycolysis and oxidative phosphorylation play critical metabolic roles in regulating cardiac function in the embryonic mouse heart.     

Acute effects of 17beta-estradiol on myocardial pH, Na+, and Ca2+ and ischemia-reperfusion injury

Evidence suggests that 1) ischemia-reperfusion injury is due largely to cytosolic Ca²⁺ accumulation resulting from functional coupling of Na⁺/Ca²⁺ exchange (NCE) with stimulated Na⁺/H⁺ exchange (NHE1) and 2) 17-estradiol (E2) stimulates release of NO, which inhibits NHE1. Thus we tested the hypothesis that acute E2 limits myocardial Na⁺ and therefore Ca²⁺ accumulation, thereby limiting ischemia-reperfusion injury. NMR was used to measure cytosolic pH (pH_i), Na⁺ (Na), and calcium concentration ([Ca²⁺]_i) in Krebs-Henseleit (KH)-perfused hearts from ovariectomized rats (OVX). Left ventricular developed pressure (LVDP) and lactate dehydrogenase (LDH) release were also measured. Control ischemia-reperfusion was 20 min of baseline perfusion, 40 min of global ischemia, and 40 min of reperfusion. The E2 protocol was identical, except that 1 nM E2 was included in the perfusate before ischemia and during reperfusion. E2 significantly limited the changes in pH_i, Na and [Ca²⁺]_i during ischemia (P < 0.05). In control OVX vs. OVX+E2, pH_i fell from 6.93 ± 0.03 to 5.98 ± 0.04 vs. 6.96 ± 0.04 to 6.68 ± 0.07; Na rose from 25 ± 6 to 109 ± 14 meq/kg dry wt vs. 25 ± 1 to 76 ± 3; [Ca²⁺]_i changed from 365 ± 69 to 1,248 ± 180 nM vs. 293 ± 66 to 202 ± 64 nM. E2 also improved recovery of LVDP and diminished release of LDH during reperfusion. Effects of E2 were diminished by 1 µM N-nitro-L-arginine methyl ester. Thus the data are consistent with the hypothesis. However, E2 limitation of increases in [Ca²⁺]_i is greater than can be accounted for by the thermodynamic effect of reduced Na accumulation on NCE. myocardial ischemia; Na⁺/H⁺ exchange; Na⁺/Ca²⁺ exchange; nuclear magnetic resonance; ischemic biology; ion channels/membrane transport; transplantation     

Na+ effects on intracellular pH of isolated, perfused rabbit gastric mucosal surface cells.

The effects of extracellular Na+ on intracellular pH were studied by perfusing BCECF loaded gastric mucosal surface cells adherent to glass coverslips held in a spectrophotofluorometer. Removal of Na+ from a NaCl Ringer perfusate (pH 7.4) resulted in progressive intracellular acidification, which was partially blocked by amiloride. An H+ conductance did not appear to be present. Acidification induced either by Na+ removal or by a NH4 prepulse was reversed by extracellular Na+, but this effect was not completely prevented by amiloride. Amiloride significantly, but not completely, inhibited Na22 uptake by gastric mucosal surface cells. The data suggest that extracellular Na+ maintains intracellular pH of gastric mucosal surface cells through amiloride-sensitive and -insensitive pathways. In the absence of extracellular Na+, cellular acidification seemed to be partially due to Na+/H+ exchange.     

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes

Much less is known about the contributions of the Na⁺/Ca²⁺ exchanger (NCX) and sarcoplasmic reticulum (SR) Ca²⁺ pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca²⁺ decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I_NCX) and the Ca²⁺ transient. We found that the integral of I_NCX, an indicator of SR Ca²⁺ content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca²⁺ extrusion by NCX (V_NCX) was linear with [Ca²⁺] and did not indicate saturation kinetics until [Ca²⁺] reached 1–3 µM for each age group. There was a significantly greater time delay between the peaks of I_NCX and the Ca²⁺ transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content     

Effects of pH, Ca2+, temperature, and protease pretreatment on interkingdom fusion.

The incubation of carrot protoplasts and cultured Xenopus cells in a protease solution has been shown to enhance their subsequent interkingdom fusion by a high pH/high Ca2+ method. The effects of Ca2+ concentration, pH, and temperature on the frequency of heterokaryon formation have also been studied. Potentially viable heterokaryons have been repeatedly produced at high frequencies (consistently greater than 10%), far exceeding those so far achieved in PEG-mediated fusion. Cell aggregates are readily dispersed after this method of fusion, permitting the accurate estimation of fusion frequencies.     

温度和胞内钠、ATP及酸碱度对心室肌细胞钠钙交换电流的调节

心肌缺血损伤过程中,胞内Na^＋、ATP及pH都出现明显变化。钠／钙交换对心肌细胞的钙平衡起重要的调节作用。本实验采用膜片钳全细胞记录豚鼠心室肌细胞钠／钙交换电流,研究温度和胞内Na^＋、ATP及pH对钠／钙交换双向电流的影响。结果表明,温度从22℃升至34℃,钠／钙交换电流增大约4倍,而pH值的改变对钠／钙交换双向电流没有明显的影响。在22～24℃时,同时耗竭胞内ATP和胞内酸化对钠／钙交换双向转运功能影响程度小;而在34—37℃时,同时耗竭胞内ATP和胞内酸化能抑制钠／钙交换双向电流的外向和内向成分,且内向成分抑制程度高于外向成分抑制程度。表明同时耗竭胞内ATP和胞内酸化对钠／钙交换的作用具有温度依赖性。胞内Na^＋超载能使钠／钙交换电流的外向成分增加,但不增加或减少内向电流（即正向转运）成分。因此,胞内酸化及耗竭胞内ATP损伤细胞排钙机制和胞内钠超载通过钠／钙反向交换引起钙内流是引起心肌细胞钙超载的两个独立的重要因素。