Contributions of [Ca2+]i, [Pi]i, and pHi to altered diastolic myocyte tone during partial metabolic inhibition.

Ischemia may cause increased or decreased distensibility of the left ventricle, but the cellular mechanisms involved have not been clarified. We examined the possible contributions of changes in intracellular inorganic phosphate, pH, and Ca2+ concentrations to altered diastolic function in cultured myocytes subjected to partial metabolic inhibition. Paced cultured embryonic chick and adult rabbit ventricular myocytes superfused with 20 mM 2-deoxyglucose (2DG) exhibited an increase in end-diastolic intracellular free calcium concentration ([Ca2+]i) and an upward shift in end-diastolic cell position. These results indicate that glycolytic blockade increases diastolic and systolic calcium in paced ventricular myocytes, and that this elevated diastolic calcium influences the extent of diastolic relaxation. In contrast, paced ventricular myocytes superfused with 1 mM cyanide (CN) exhibited a similar increase in end-diastolic [Ca2+]i but a decrease in end-diastolic cell position and amplitude of motion. Although changes in ATP contents were similar in both groups (2DG, -29.9%; CN, -40.1%), alterations of intracellular pH and inorganic phosphate concentrations were different. In 2DG-treated cells, pHi did not decrease significantly (7.18 +/- 0.04 to 7.12 +/- 0.11, n = 14) but in the CN group it decreased markedly within 6 min (7.18 +/- 0.04 to 6.76 +/- 0.11, n = 11, P less than 0.01). Intracellular inorganic phosphate decreased slightly in the 2DG group (-14.8%, NS) but increased in cells exposed to CN (45.7%, P less than 0.02). We conclude that while a prominent increase in diastolic [Ca2+]i occurs in rapidly paced ventricular myocytes exposed to either inhibitors of glycolysis or oxidative phosphorylation, the effects of this increase in [Ca2+]i on diastolic distensibility may be influenced by intracellular accumulation of metabolites that decrease the sensitivity of myofilament to [Ca2+]i.

Alterations in cation homeostasis in cultured chick ventricular cells during and after recovery from adenosine triphosphate depletion.

Alterations in cation homeostasis during and after recovery from myocardial ischemia may account for some of the reversible and irreversible components of myocardial cell injury. To investigate possible mechanisms involved, we exposed cultured layers of spontaneously contracting chick embryo ventricular cells to media containing 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG), and zero glucose for up to 6 h, and then allowed cultured cells to recover in serum-free culture medium for 24 h. Changes in Na, K, and Ca contents, 42K uptake and efflux, ATP content, cell water content, and lactate dehydrogenase (LDH) release were measured, and compared with changes produced by exposure to 10(-3) M ouabain and severe hypoxia. Exposure to CN and 2-DG caused marked increase in cell Na (sevenfold) and Ca (fivefold) contents, and a decrease in K content (one-fifth normal), coincident with ATP depletion to one-tenth normal levels. This produced only slight cell injury, evidenced by increased LDH release. Recovery for 24 h resulted in return to near normal values (expressed in nanomoles per milligram of protein) of Na, Ca, and ATP contents. However, there was failure of cell K content to return to normal, associated with a persistent reduced net uptake of 42K, and an increase in the rate of 42K efflux. These abnormalities in K homeostasis were associated with a decrease in cell volume and water content per milligram of protein. More marked ATP depletion (to 1/100 normal values) was produced by hypoxia plus 2-DG and zero glucose, and was associated with much more severe cell injury manifested by LDH loss. Ouabain exposure resulted in a much greater Ca gain (20-30-fold), relative to increase in Na content, than did either CN and 2-DG or hypoxia; and ouabain effects were not reversible (after a 15-fold or greater increase in Ca content was produced) and were associated with significant LDH release. We conclude that these cells are resistant to cell injury caused by moderately severe Ca overload and ATP depletion produced by exposure to CN and 2-DG. However, metabolic inhibition of ATP production produces persistent abnormalities in K homeostasis, associated with functional abnormalities.

Immediate-early gene induction and MAP kinase activation during recovery from metabolic inhibition in cultured cardiac myocytes.

To investigate how cardiac myocytes recover from a brief period of ischemia, we used a metabolic inhibition (MI) model, one of the in vitro ischemic models, of chick embryo ventricular myocytes, and examined the induction of immediate-early (IE) genes mRNAs and the activity of mitogen-activated protein (MAP) kinase. We performed Northern blot analysis to study the expression of c-jun, c-fos, and c-myc mRNAs during MI using 1 mM NaCN and 20 mM 2-deoxy-d-glucose, and also during the recovery from MI of 30 min. The c-fos mRNA was induced transiently at 30 and 60 min during the recovery. The expression of c-jun mRNA was significantly augmented at 30, 60, 90, and 120 min during the recovery (3.0-, 4.7-, 2.4-, and 1.9-fold induction, respectively) and so did the expression of c-myc mRNA (1.4-, 1.7-, 1.8-, and 2.0-fold induction, respectively). In contrast, the levels of these mRNAs remained unchanged during MI. The electrophoretic mobility shift assay revealed that AP-1 DNA binding activity markedly increased at 120 min during the recovery. When the cells were pretreated with protein kinase C (PKC) inhibitors, 100 microM H-7 or 1 microM staurosporine, the induction of c-jun mRNA at 60 min during the recovery was markedly suppressed (95 or 82% reduction, respectively). The c-jun induction was partially inhibited when the cells were treated with 2 mM EGTA during MI and the recovery (42% reduction). MAP kinase activity quantified with in-gel kinase assay was unchanged during MI, but significantly increased at 5, 10, and 15 min during the recovery (3.0-, 4.1-, and 3.4-fold increase, respectively). S6 kinase activity was also augmented significantly at 15 min during the recovery. Thus, these data suggest that IE genes as well as MAP kinase may play roles in the recovery process of cardiac myocytes from MI, and that the augmentation of c-jun expression needs the activation of PKC and to some extent, [Ca2+]i.

Isoproterenol activates extracellular signal-regulated protein kinases in cardiomyocytes through calcineurin.

BACKGROUND: Extracellular signal-regulated kinases (ERKs) and calcineurin have been reported to play important roles in the development of cardiac hypertrophy. We examined here the relation between calcineurin and ERKs in cardiomyocytes. METHODS AND RESULTS: Isoproterenol activated ERKs in cultured cardiomyocytes of neonatal rats, and the activation was abolished by chelation of extracellular Ca(2+) with EGTA, blockade of L-type Ca(2+) channels with nifedipine, or depletion of intracellular Ca(2+) stores with thapsigargin. Isoproterenol-induced activation of ERKs was also significantly suppressed by calcineurin inhibitors in cultured cardiomyocytes as well as in the hearts of mice. Isoproterenol failed to activate ERKs in either the cultured cardiomyocytes or the hearts of mice that overexpress the dominant negative mutant of calcineurin. Isoproterenol elevated intracellular Ca(2+) levels at both systolic and diastolic phases and dose-dependently activated calcineurin. Inhibition of calcineurin also attenuated isoproterenol-stimulated phosphorylation of Src, Shc, and Raf-1 kinase. The immunocytochemistry revealed that calcineurin was localized in the Z band, and isoproterenol induced translocation of calcineurin and ERKs into the nucleus. CONCLUSIONS: Calcineurin, which is activated by marked elevation of intracellular Ca(2+) levels by the Ca(2+)-induced Ca(2+) release mechanism, regulates isoproterenol-induced activation of ERKs in cardiomyocytes.

Ca(2+)-growth coupling in angiotensin II-induced hypertrophy in cultured rat cardiac cells.

OBJECTIVES: There remain some controversies about the effect of angiotensin II on intracellular Ca2+ concentration ([Ca2+]i) in cardiac myocytes. The aim of this study was to investigate different roles of intracellular Ca2+ in the responses to angiotensin II between cardiac myocytes and nonmyocytes. METHODS: Primary cultures of neonatal rat cardiac myocytes and nonmyocytes were prepared. [Ca2+]i was measured with indo-1. Cellular growth was assayed by [3H]thymidine uptake, RNA content, [3H]phenylalanine incorporation and protein content. Induction of immediate-early gene was examined by Northern blot analysis. RESULTS: In myocytes, angiotensin II decreased [Ca2+]i transients, induced c-fos mRNA, and accelerated hypertrophy. These effects were completely suppressed by AT1 receptor blockade or protein kinase C inhibition. After chelation of extracellular Ca2+, angiotensin II caused no change in [Ca2+]i or no induction of c-fos in myocytes. Phorbol 12-myristate 13-acetate also decreased [Ca2+]i transients, caused c-fos induction, and provoked hypertrophy in myocytes. In nonmyocytes, angiotensin II increased [Ca2+]i transiently, induced c-fos mRNA and hypertrophy. These effects of angiotensin II were not fully abolished by protein kinase C inhibition. Extracellular Ca2+ chelation did not completely inhibit the effects of angiotensin II on [Ca2+]i or c-fos induction in nonmyocytes. Phorbol 12-myristate 13-acetate did not affect [Ca2+]i or cellular growth in nonmyocytes but did cause c-fos induction. CONCLUSIONS: These results suggest that angiotensin II induces cellular hypertrophy and immediate-early genes through the activation of protein kinase C in myocytes, although angiotensin II decreases [Ca2+]i transients via this signaling pathway. Induction by angiotensin II of hypertrophy and immediate-early genes in nonmyocytes may be in part mediated by a transient increase in [Ca2+]i which acts synergistically with protein kinase C activation.

Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes.

OBJECTIVES: Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. METHODS: A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique. RESULTS: PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa. CONCLUSIONS: We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.

Molecular cloning and expression of inducible nitric oxide synthase in chick embryonic ventricular myocytes.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) has been implicated to contribute to myocardial dysfunction in various settings, but considerable species differences have been noted in the levels of iNOS expression and its function in several tissues. The aim of this study was to elucidate evolutional changes in myocardial iNOS expression and function. METHODS: An iNOS cDNA clone was isolated by RT-PCR from the 10-day old cultured chick embryonic ventricular myocytes stimulated with 10 micrograms/ml of lipopolysaccharide. Expression of the iNOS mRNA was analyzed with Northern blot analysis and RNase protection assay. The iNOS activity was estimated from conversion rates of L-arginine to L-citrulline and intracellular cGMP contents were measured with radioimmunoassay. Furthermore, both [Ca2+]i (fluorescent dye indo-1) and cell contraction (video motion detector) were simultaneously recorded. RESULTS: Aside from the primer sequences, the insert (1026 bp) of the cDNA clone showed 66.4% identity at the deduced amino acid level to the human iNOS cDNAs. Northern blot analysis revealed that chicken iNOS mRNA of approximately 4.5 kb was induced by lipopolysaccharide within 6 h in the cultured myocytes. RNase protection assay also showed that lipopolysaccharide provoked 14.6 +/- 5.1-fold increases (n = 6, p < 0.05) in the iNOS mRNA signals within 6 h. The iNOS activity (+300%, P < 0.05) as well as the intracellular cGMP contents (+75%, P < 0.01) were significantly augmented in the lipopolysaccharide-stimulated cells. Both the cell contraction and [Ca2+]i were significantly reduced after the administration of a large amount (10 mM) of L-arginine in the myocytes pretreated with both lipopolysaccharide and NG-monomethyl-L-arginine (100 microM). CONCLUSION: As like as the nucleotide and amino acid sequences, the myocardial effects of the iNOS may also be evolutionary conserved.

Effects of a nonpeptide vasopressin antagonist (OPC-21268) on cytosolic Ca2+ concentration in vascular and cardiac myocytes.

A selective V1 antagonist, 1-(1-[4(3-acetylaminopropoxy)benzoyl]-4-piperidyl)-3,4-dihydro-2(1 H)- quinolinone (OPC-21268), which is nonpeptide and orally effective, has been recently synthesized. We studied the effects of vasopressin and OPC-21268 on cell contraction with a video motion detector and cytosolic Ca2+ concentration ([Ca2+]i) by using indo-1 in cultured rat vascular smooth muscle cells and cultured chick embryo ventricular myocytes. Exposure of cultured vascular smooth muscle cells to vasopressin (1-100 nM) dose-dependently produced an initial transient increase (from control level [Ca2+]i of 133.6 +/- 10.9 nM to peak [Ca2+]i of 842.7 +/- 172.8 nM at 100 nM vasopressin, p less than 0.01) and then a small sustained increase in [Ca2+]i. After pretreatment of vascular smooth muscle cells with 1 microM OPC-21268, the effects of 100 nM vasopressin on [Ca2+]i were abolished. Exposure of ventricular myocytes to 100 nM vasopressin slightly but significantly decreased peak systolic cell position (-8.7 +/- 3.7%, p less than 0.05) and also produced reductions in peak systolic [Ca2+]i (from 962.2 +/- 76.4 to 751.2 +/- 70.5 nM, p less than 0.01) within 30 seconds. Pretreatment of ventricular myocytes with OPC-21268 (1 microM) completely suppressed vasopressin-induced changes in peak systolic cell position and [Ca2+]i. These results suggest that vasopressin may increase vascular tone and may also cause a direct negative inotropic effect via V1 receptors and that this orally active V1 antagonist (OPC-21268) may have potential clinical usefulness.

Mechanisms of adrenomedullin-induced vasodilation in the rat kidney.

To explore the mechanisms of adrenomedullin-induced vasorelaxation, we tested the effects of adrenomedullin on renal function in rats in vivo and measured the release of endothelium-derived nitric oxide from isolated perfused rat kidney (using a chemiluminescence assay) and the diameters of the glomerular arterioles in the hydronephrotic kidney. Adrenomedullin decreased blood pressure in a dose-dependent manner (3 nmol/kg: -29 +/- 2% [SEM]; P < .01) and slightly increased the glomerular filtration rate and urinary sodium excretion (+108%; P < .05). These changes were associated with significant increases in urinary excretion of cyclic AMP (+54%; P < .05). Adrenomedullin decreased renal vascular resistance (10(-7) mol/L adrenomedullin: -41 +/- 2%; P < .001) and increased release of nitric oxide (+5.1 +/- 0.7 fmol/min per gram kidney weight; P < .001) in the isolated kidney. This increase in nitric oxide release was abolished by the inhibitor NG-monomethyl-L-arginine, and it also reversed the decrease in renal vascular resistance seen with adrenomedullin. Renal responses of deoxycorticosterone acetate-salt hypertensive rats to adrenomedullin were significantly smaller than those of control rats for both release of nitric oxide (10(-7) mol/L adrenomedullin: +0.8 +/- 0.2 fmol/min per gram kidney weight; P < .01 versus control) and renal vasodilation (-28 +/- 6%; P < .05). Videomicroscopic analysis revealed that adrenomedullin increased the diameters of both afferent and efferent arterioles (3 nmol/kg: +11%; P < .05). Thus, adrenomedullin-induced renal vasodilation is partially endothelium dependent and is attenuated in deoxycorticosterone acetate-salt hypertension, probably due to endothelial damage.

Estimation of the secretion rate of atrial natriuretic peptide from the coronary sinus in coronary artery disease.

Although atrial natriuretic peptide (ANP) is known to be secreted through the coronary sinus into the systemic circulation, its actual secretion rate has not been thoroughly investigated. The immunoreactive ANP concentrations in plasma samples from the ascending aorta and coronary sinus in 11 patients with the coronary artery disease were measured and the coronary sinus flow rate using the continuous thermodilution method was simultaneously determined at the time of sampling. These variables were also determined during the intravenous infusion of synthetic alpha-human ANP at 0.025 microgram/kg.min in 7 of the 11 patients. In the basal state, the plasma concentration of ANP was 61 +/- 6 (standard error) pg/ml in the aorta and 541 +/- 40 pg/ml in the coronary sinus, and the coronary sinus flow index was 57.3 +/- 12.3 ml/min.m2. Thus, the secretion rate of ANP was determined to be 14.4 +/- 2.8 ng/min.m2. The secretion rate of ANP correlated significantly with the plasma concentration of ANP in the aorta (r = 0.65, p less than 0.05). The ANP infusion, which decreased pulmonary artery wedge pressure from 8.0 +/- 0.6 to 6.3 +/- 0.4 mm Hg (p less than 0.01), elevated the plasma concentrations of ANP in the aorta and coronary sinus by 701% (p less than 0.001) and 33% (p less than 0.05), respectively, and decreased the secretion rate of ANP by 40% (p less than 0.05). These results suggest that the circulating plasma concentration of ANP may reflect the secretion rate of ANP and that an increase in circulating ANP directly or indirectly reduces ANP secretion.

Doppler echocardiographic-determined changes in left ventricular diastolic filling flow velocity during the lower body positive and negative pressure method.

Changes in parameters of left ventricular (LV) diastolic filling flow obtained with Doppler echocardiography during the lower body positive and negative pressure method were analyzed in 15 patients (12 with coronary artery disease and 3 with dilated cardiomyopathy). Lower body pressure was altered at 5 steps (+20, +10, 0, -20 and -40 mm Hg vs atmospheric pressure). Pulmonary capillary wedge pressure measured with a balloon-tipped catheter was changed proportionally with lower body pressure during the procedures (p less than 0.01). Mean systemic arterial pressure was changed slightly during lower body positive pressure and negative pressure of -40 mm Hg. Heart rate was almost unchanged except at lower body pressure of -40 mm Hg. The peak velocity of LV early diastolic filling flow was changed with pulmonary capillary wedge pressure in an almost parallel fashion during the procedures (p less than 0.01). The peak velocity of LV late diastolic filling flow showed smaller changes than that of early diastolic filling flow. Changes in pulmonary capillary wedge pressure correlated positively with changes in the peak velocity of LV early diastolic filling flow (r = 0.759, p less than 0.01), but not with changes in the peak velocity of LV late diastolic filling flow (r = 0.039, not significant) during lower body negative pressure of -20 mm Hg. These data suggest that left atrial pressure is one of the important determinants of LV early diastolic filling flow in this acute clinical setting and that LV late diastolic filling flow is less sensitive to changes in left atrial pressure than LV early diastolic filling flow.

Effects of contrast media on calcium transients and motion in cultured ventricular cells.

To investigate the mechanisms of the negative inotropic effects of contrast media, we superfused spontaneously contracting cultured chick embryo ventricular cells with Renografin-76 and iohexol (12% solutions), and hypertonic sucrose during simultaneous measurement of [Ca2+]i transients (indo-1) and motion (video-motion detector system). Exposure to contrast agents caused a significant reduction of contractility, with Renografin-76 having a much greater effect on amplitude of motion than iohexol. Renografin-76 significantly depressed [Ca2+]i transient amplitude, whereas iohexol had no effect. Addition of Ca2+ to correct for calcium binding by Renografin-76 completely reversed its depression of [Ca2+]i transients but only partially reversed the negative inotropic effects. Hypertonic sucrose caused a significant decrease in contraction amplitude, with no significant effects on [Ca2+]i transient amplitude. We conclude that the marked negative inotropic effect of Renografin-76 is caused by both calcium binding and hypertonicity. The less marked depression of contractility produced by iohexol likely is a result of hypertonicity and not caused by alteration of [Ca2+]i.

Evidence that reverse Na-Ca exchange can trigger SR calcium release.

Several results suggest that the Na-Ca exchange can function as a trigger promoting SR Ca release and ensuing contractions. First, if the Ca current was the sole trigger for contraction we would expect the relationship between triggered contractions and voltage to be similar to the relationship between Ca current and contraction. When Na is present in the pipette this is not observed. Between -40 and +10 mV the relationships between contractions and voltage and current and voltage are similar. At potentials positive to 10 mV the Ca current declines as expected but contractions either decline much more slowly or continue to increase depending upon the concentration of intracellular Na. In addition, we have observed that contractions can be activated when Ca current is largely or completely blocked. Since these contractions are sensitive to the presence of ryanodine and thapsigargin they appear to be triggered by Na-Ca exchange. Also, contractions that are activated in the presence of nifedipine are sensitive to the Na-Ca exchange inhibitor XIP. Finally, rapid removal of extracellular Na apparently stimulates enough reverse exchange triggering of SR Ca release without affecting the SR content. It is clear that the shape of the shortening voltage relationship depends upon the concentration of dialyzing Na. This is likely to occur for two reasons. Either the shape of the shortening voltage relationship depends upon the extent to which Na-Ca exchange contributes a trigger for SR Ca release or alternatively the shape of the shortening voltage relationship depends upon SR Ca content. The latter is known to depend upon the Na concentration. In addition it is now established that the gain of SR Ca release is influenced by SR content. However, we studied triggered contractions in the absence of a Na gradient when the only available trigger is the Ca current. We measured triggered contractions over a range of voltages between -30 and +60 mV. Between each measurement we reestablished the Na gradient and activated a series of conditioning pulses to standardize the SR Ca content. Just before a test pulse we removed extracellular Na and activated either 3 or 6 pulses to produce two different SR Ca loads (in the absence of a Na gradient entering Ca cannot be extruded and therefore changes the SR Ca content). Regardless of the number of prepulses in the absence of a Na gradient the shortening voltage relationship was similar and bell shaped. From this we conclude that the shape of the relationship between shortening and voltage does not depend upon SR Ca content. Therefore, we conclude that the asymmetry in the shortening voltage relationship that depends upon intracellular Na is due to a contribution of reverse Na-Ca exchange. It is too early to say what the physiological significance (if any) of triggering by reverse exchange actually is. However, it does seem likely that it might provide a powerful inotropic mechanism. For example intracellular Na might be expected to change with heart rate and to be elevated at higher heart rates. Presumably this increased intracellular Na would tend to favor triggering by reverse exchange and would therefore enhance contractility at a time when it would be most required.

Effects of a nonpeptide angiotensin II receptor antagonist (CV-11974) on [Ca2+]i and cell motion in cultured ventricular myocytes.

We studied the effects of angiotensin II and CV-11974 (a newly synthesized angiotensin II receptor antagonist) on cell contraction and [Ca2+]i in cultured neonatal rat ventricular myocytes. Exposure of these cells to 10 nM angiotensin II significantly decreased peak systolic cell position (60.1 +/- 3.3% of the control, P < 0.01) and peak systolic [Ca2+]i (from 1111 +/- 250 to 572 +/- 143 nM, P < 0.05) within 60 s. Pretreatment of ventricular myocytes with CV-11974 (10-100 nM) completely suppressed the angiotensin II-induced changes in peak systolic cell position and [Ca2+]i. These results suggest that CV-11974 inhibits cardiac angiotensin II receptors.

Selectivity of felodipine for depolarized ventricular myocytes: a study at the single-cell level.

We studied the effects of felodipine (a second-generation dihydropyridine Ca2+ channel blocker) on excitation-contraction coupling (E-C coupling) in single isolated guinea-pig ventricular myocytes, using the whole-cell perforated patch-clamp technique or the Ca indicator, indo-1. Felodipine inhibited both L-type Ca2+ channel currents (ICa) and cell contractions in a concentration-dependent manner (10 pM to 100 nM) when we used a holding potential of -80 mV or -40 mV. The potency of felodipine was sharply dependent on a holding potential. Namely, use of a more depolarized holding potential markedly increased the potency of felodipine for inhibition of ICa and cell contraction. Next we current-clamped cells and obtained the resting membrane potential of -82 +/- 8 mV. When cells were current-injected at 0.1 Hz, exposure to 10 nM felodipine slightly but significantly diminished the amplitude of cell contractions (7.2 +/- 1.6 to 6.7 +/- 1.7 microns, P < 0.05) within 10 min. When cells were field stimulated, exposure of cells to 10 nM felodipine also slightly diminished the amplitude of cell shortening (5.1 +/- 2.0 to 4.6 +/- 1.9 microns, P < 0.05) and [Ca2+]i transients. We observed clear voltage-dependent blockade of E-C coupling by felodipine in ventricular myocytes. Thus, therapeutic concentrations (1-10 nM) of felodipine could inhibit E-C coupling in depolarized ventricular myocytes, which might simulate an ischemic or failing heart.

Chronic total occlusion of the left main coronary artery.

We report 3 patients with chronic total occlusion of the left main coronary artery, which is considered to be very rare. In all three cases, coronary arteriograms showed a total occlusion of the left main coronary artery with good collaterals from the intact right coronary arteries. All of the patients underwent successful coronary artery bypass surgery; two of the cases were followed up for more than 10 years after the surgery. The Japanese literature is reviewed, and a comparison of foreign and Japanese cases is discussed.

Mechanism of protective effects of Ca++ channel blockers on energy deprivation contracture in cultured ventricular myocytes.

To examine mechanisms of the protective effects of Ca++ channel blockers on energy deprivation contracture, we measured cystolic calcium ion concentration ([Ca++]i) (Indo-1 fluorescence), development of contracture (video motion detector) and ATP contents during exposure of cultured chick embryo ventricular cells to 1 mM cyanide (CN) and 20 mM 2-deoxyglucose (2-DG). The time periods required for [Ca++]i to reach 50% of [Ca++]i transient ([Ca++]i-50) and contracture were determined after exposure to 1) CN + 2-DG alone, 2) CN + 2-DG simultaneous with 1 microM verapamil (V-sim) and 3) verapamil followed by CN + 2-DG (V-pre). Time periods required to reach [Ca++]i-50 under these conditions were 4.2 +/- 0.4 min (CN + 2-DG alone), 3.8 +/- 0.4 min (NS vs. CN + 2-DG alone) (V-sim) and 6.4 +/- 1.1 min (P less than .05 vs. CN + 2-DG alone) (V-pre), respectively. Time periods required for contracture development were 4.4 +/- 0.3 min (CN + 2-DG alone), 4.4 +/- 0.6 min (NS vs. CN + 2-DG alone) (V-sim) and 9.3 +/- 1.2 min (P less than .05 vs. CN + 2-DG alone) (V-pre). Three minutes after metabolic inhibition, ATP contents declined from 32.3 +/- 0.7 nmol/mg of protein to 4.2 +/- 1.0 in CN + 2-DG alone, to 4.5 +/- 0.9 (NS vs. CN + 2-DG alone) with V-sim and to 8.3 +/- 2.2 (P less than .05 vs. CN + 2-DG alone) with V-pre.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanisms of reoxygenation injury in cultured ventricular myocytes.

To investigate factors contributing to reperfusion and reoxygenation myocardial injury, we exposed layers of cultured chick ventricular myocytes to severe hypoxia for up to 3 hours in the presence of 20 mM 2-deoxyglucose, zero glucose, and 5 mM pyruvate, and then exposed the myocytes to reoxygenation. Lactate dehydrogenase (LDH) release was moderately increased during 3 hours of hypoxia but was increased markedly during reoxygenation. Coincident changes in intracellular calcium concentration ([Ca2+]i) and cell motion were also measured during hypoxia and reoxygenation. During hypoxia, [Ca2+]i increased to more than 1 microM, and with reoxygenation, [Ca2+]i abruptly decreased slightly but remained elevated more than 1 microM. Cells developed a stable rigor after 30 minutes of hypoxia. Reoxygenation caused a marked hypercontracture within 5 minutes. Pretreatment of myocytes with either 2,3-butanedione monoxime, which inhibits Ca2(+)-dependent force development, or cyanide inhibited reoxygenation hypercontracture. LDH release after reoxygenation was also significantly reduced in the presence of 2,3-butanedione monoxime. Treatment of myocytes with superoxide dismutase and catalase during hypoxia also resulted in a decrease in LDH release during reoxygenation. We conclude that an abrupt increase in [Ca2+]i during reoxygenation does not account for reoxygenation injury. However, in the presence of elevated [Ca2+]i, reoxygenation and the resulting probable resynthesis of ATP causes [Ca2+]i-dependent myofilament crossbridge cycling, and the resulting hypercontracture contributes to myocyte damage. The generation of oxygen free radicals after reoxygenation also appears to contribute to cell injury in this system.

Detection of La3+ influx in ventricular cells by indo-1 fluorescence.

We exposed indo-1-loaded cultured embryonic chick ventricular cells to 0.03-1.0 mM extracellular lanthanum concentration ([La3+]o) and simultaneously measured cell contractile motion and the 410/480 nm fluorescence intensity ratio. After exposure to La3+, ventricular cells stopped contracting and relaxed within seconds, and the 410/480 fluorescence ratio increased. The increase in the 410/480 signal was related to [La3+]o but was not affected by short exposures to zero extracellular calcium concentration ([Ca2+]o) or caffeine, suggesting that the fluorescence was not caused by a La3+-induced increase in intracellular calcium concentration ([Ca2+]i) but rather to increased intracellular lanthanum concentration ([La3+]i). In vitro studies confirmed that indo-1 fluorescence was sensitive to La3+. The increase in [La3+]i in 0.1 mM [La3+]o was directly related to intracellular sodium concentration ([Na+]i), suggesting that La3+ entered cells via Na+-La3+ exchange. In contrast to ventricular cells, which have a functionally distinct Na+-Ca2+ exchange system, exposure of indo-1-loaded cultured bovine endothelial cells to La3+ failed to produce an increase in [La3+]i. These results indicate that exposure of ventricular cells to 0.1-1.0 mM [La3+]o results in a [La3+]i greater than 250 nM within 1 min. Therefore, changes in myocardial 45Ca2+ fluxes and contents induced by La3+ cannot be ascribed solely to extracellular La3+ effects.