Left ventricular relaxation abnormality is detectable by analysis of the relaxation time constant in patients with atrial fibrillation.

Left ventricular (LV) contractility is constantly changing during atrial fibrillation (AF), which is dependent on the force-interval relationships. However, no information has been available on LV relaxation in patients with both AF and impaired LV systolic function. LV pressure was measured with a catheter-tipped micromanometer and the time constant of isovolumic LV pressure decline (tau(bf)) was calculated with best exponential fitting from more than 10 consecutive beats. Patients with AF (5 with mitral valvular disease, 6 with idiopathic dilated cardiomyopathy, and 1 with no underlying disease) were subdivided into 2 groups: group A, with ejection fraction (EF) <0.5 (n=7); and group B, with EF > or =0.5 (n=5). Linear correlation coefficients (r) between tau and RR2, RR2/RR1, LV peak systolic pressure (peak LVP) were calculated. Although tau did not show a discrepancy between the 2 groups, tau(bf) correlated better with RR2/RR1 only in the group A patients. The relation between tau and peak LVP showed a good correlation with a steep slope (R, Deltatau/Deltapeak LVP) only in the group A patients (accentuated afterload-dependence). R was significantly different between the 2 groups. Thus, a beat-to-beat analysis of tau may be a practical and feasible way for detecting LV relaxation abnormality in patients with AF.

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.

Targeted disruption of Na+/Ca2+ exchanger gene leads to cardiomyocyte apoptosis and defects in heartbeat.

Ca(2+), which enters cardiac myocytes through voltage-dependent Ca(2+) channels during excitation, is extruded from myocytes primarily by the Na(+)/Ca(2+) exchanger (NCX1) during relaxation. The increase in intracellular Ca(2+) concentration in myocytes by digitalis treatment and after ischemia/reperfusion is also thought to result from the reverse mode of the Na(+)/Ca(2+) exchange mechanism. However, the precise roles of the NCX1 are still unclear because of the lack of its specific inhibitors. We generated Ncx1-deficient mice by gene targeting to determine the in vivo function of the exchanger. Homozygous Ncx1-deficient mice died between embryonic days 9 and 10. Their hearts did not beat, and cardiac myocytes showed apoptosis. No forward mode or reverse mode of the Na(+)/Ca(2+) exchange activity was detected in null mutant hearts. The Na(+)-dependent Ca(2+) exchange activity as well as protein content of NCX1 were decreased by approximately 50% in the heart, kidney, aorta, and smooth muscle cells of the heterozygous mice, and tension development of the aortic ring in Na(+)-free solution was markedly impaired in heterozygous mice. These findings suggest that NCX1 is required for heartbeats and survival of cardiac myocytes in embryos and plays critical roles in Na(+)-dependent Ca(2+) handling in the heart and aorta.

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.

Cellular basis for the acute inhibitory effects of IL-6 and TNF- alpha on excitation-contraction coupling.

There is controversy over whether nitric oxide (NO) mediates acute negative inotropic actions of cytokines including tumor necrosis factor-alpha (TNF- alpha). The reports from established laboratories have appeared inconsistent, which could be due to species differences. Thus, we tried to elucidate the mechanisms underlying negative inotropic actions of interleukin-6 (IL-6) and TNF- alpha in the same model. We studied the effects of cytokines on [Ca(2+)](i)transients (using indo-1), cell shortening (CS) (using a video motion detector) and the L-type Ca(2+)channel current (I(Ca)) (using the whole cell perforated patch clamp technique) in isolated guinea-pig ventricular myocytes. IL-6 (1000 U/ml) or TNF- alpha (500 U/ml) decreased both peak systolic [Ca(2+)](i)(IL-6: 0.43+/-0.01 to 0.40+/-0.01, n=5, P<0.05; TNF- alpha : 0.42+/-0.02 to 0.39+/-0.02, n=5, P<0.05) and the amplitude of CS (IL-6: 7.5+/-0.9 to 6.2+/-0.5 micrometers, n=5, P<0.05; TNF- alpha : 6.7+/-0.7 to 5.8+/-0.7 micrometers, n=5, P<0.05) without detectable reductions in I(Ca)(IL-6: 0.9+/-0.1 to 0.9+/-0.1 nA, n=4, N.S.; TNF- alpha : 1.1+/-0.3 to 1.1+/-0.2 nA, n=4, N.S.) within 5 min. The nitric oxide synthase (NOS) inhibitor, N(G)-monomethyl- L arginine (300 micromol/l), blocked the effects of IL-6 but not of TNF- alpha. When pretreated with 20 nmol/l isoproterenol, exposure to IL-6 decreased both I(Ca)(2.8+/-0.5 to 2. 0+/-0.3 nA) and the amplitude of CS (10.4+/-2.4 to 7.5+/-1.9 micrometer) within 5 min. TNF- alpha also clearly depressed I(Ca)(2.9+/-0.9 to 2.3+/-0.7 nA) and the amplitude of CS (7.0+/-1.4 to 5.5+/-1.3 micrometer) in beta -adrenergic stimulated cells. TNF- alpha significantly increased the content of sphingosine (product of sphingomyelin pathway) in isolated heart. The effects of low dose sphingosine (5 micromol/l) mimicked those of TNF- alpha on cardiac myocytes. IL-6 produced an acute negative inotropic effect through a NO-dependent pathway while TNF- alpha did so via a sphingomyelin-dependent pathway in isolated guinea-pig ventricular myocytes.

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.

Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes.

Previous work has demonstrated that inducible NO synthase (iNOS) can be expressed in cardiac myocytes. In this study, we investigated transcriptional regulation of the iNOS gene in these cells. Lipopolysaccharide (LPS) induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. H-89, dexamethasone, herbimycin, genistein, staurosporine, or pyrrolidine dithiocarbamate (PDTC) attenuated the iNOS induction by LPS. Forskolin, interleukin (IL)-6, tumor necrosis factor (TNF)-alpha, or interferon (IFN)-gamma enhanced the LPS-induced iNOS expression. Combined stimulation of IL-6 and TNF-alpha also induced iNOS. The 5'-upstream sequence of the rat iNOS gene contains the nuclear factor-kappa B (NF-kappa B) site, CAAT box, IFN-gamma activation site (GAS), and IFN regulatory factor (IRF) site. DNase I footprinting assay revealed that the nuclear factors binding to these elements were increased by LPS exposure. Transient transfection assay suggested that these elements were indispensable for transcriptional regulation of the iNOS induction. Electrophoretic mobility shift assay revealed that LPS or TNF-alpha increased binding activity for the NF-kappa B site. A slower-migrating complex binding to the CAAT box gave rise after exposure to LPS or forskolin. Competition assay suggested that this slower-migrating complex consisted of a heterodimer between a member of CAAT box/enhancer binding (C/EBP) protein family and cAMP responsive element binding protein (CREB). LPS or IL-6 increased binding complexes for the IRF site, which was compatible with induction of IRF-1. LPS, IL-6, or IFN-gamma induced a novel binding complex for GAS, which also existed in the 5'-flanking region of the IRF-1 gene. These data suggest that (1) iNOS induction simultaneously requires both NF-kappa B activation and IRF-1 induction, and (2) the heterodimer between C/EBP and CREB has synergistic effects on the iNOS induction via the CAAT box.

Protein kinase A and protein kinase C synergistically activate the Raf-1 kinase/mitogen-activated protein kinase cascade in neonatal rat cardiomyocytes.

Adrenoceptor agonists play an important role in cardiac hypertrophy. In cardiomyocytes, activation of alpha- and beta-adrenoceptors induces a variety of hypertrophic responses via activation of protein kinase C (PKC) and protein kinase A (PKA), respectively. Although PKC evokes activation of the Raf-1 kinase (Raf-1)/mitogen-activated protein (MAP) kinase cascade, PKA has been shown to inhibit the activation of Raf-1 and MAP kinases induced by growth factors in various cell types. The present study was performed to elucidate the role of PKA and PKC in cardiomyocyte hypertrophy. PKA activators such as forskolin (FSK), isobutylmethylxanthine, dibutyryl cAMP and isoproterenol, significantly activated Raf-1 and MAP kinases with a peak at 2 and 8 min, respectively, followed by an increase in protein synthesis in cardiac myocytes. Similar responses were observed when cardiomyocytes were stimulated with PKC activators such as 12-O-tetradecanoylphorbol-13-acetate (TPA), angiotensin II, phenylephrine and mechanical stretch. After depleting extracellular Ca2+ with EGTA, FSK did not activate MAP kinases, while down-regulation of PKC by long exposure with TPA did not influence FSK-induced MAP kinase activation. Furthermore, FSK and TPA synergistically activated Raf-1. Similar synergistic activation of MAP kinases was observed when other PKC activators were added to cardiac myocytes with FSK at the same time. In conclusion, unlike other cell types, PKA activates Raf-1 and MAP kinases followed by an increase in protein synthesis in cardiac myocytes.

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.

Cardiac inducible nitric oxide synthase negatively modulates myocardial function in cultured rat myocytes.

Recent work has demonstrated that endotoxin or cytokines induce nitric oxide synthase in heart or cardiac myocytes. We investigated the functional significance of inducible nitric oxide synthase (iNOS) in indo 1-loaded beating myocytes with regard to intracellular Ca2+ concentration ([Ca2+]i) and cell contraction. Lipopolysaccharide (LPS; 10 micrograms/ml) time dependently induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. Nitrite concentration in the medium and intracellular guanosine 3',5'-cyclic monophosphate (cGMP) contents after 24-h exposure to LPS increased in proportion to the levels of iNOS induction in these cells. Myocytes treated with both NG-monomethyl-L-arginine and LPS for 24 h expressed iNOS protein, but nitrite production was significantly inhibited. Subsequent perfusion with 100-fold molar excess L-arginine of these myocytes elicited decreases in peak systolic [Ca2+]i (790 +/- 42 to 551 +/- 27 nM, P < 0.05), relative amplitude of cell contraction (100 to 72.4 +/- 5.5%, P < 0.05), and spontaneous beating rate (146 +/- 13 to 85 +/- 22 beats/min, P < 0.05). Pretreatment with methylene blue or KT-5823 inhibited these negative myocardial effects. These results suggest that LPS induces iNOS in cardiac myocytes and that the increased nitric oxide produced by iNOS has cardiac depressant effects through the activation of cGMP-dependent protein kinase.

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.

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.

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.

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.

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.

Nitric oxide-mediated effects of interleukin-6 on [Ca2+]i and cell contraction in cultured chick ventricular myocytes.

Cytokines have significant roles in some cardiovascular disorders, but direct myocardial effects of cytokines remain to be elucidated. In the present study, we examined both the early and delayed effects of interleukin-6 (IL-6) on cultured chick embryo ventricular myocytes. Exposure of these cells to human recombinant IL-6 significantly decreased peak systolic [Ca2+]i (71.0 +/- 0.6% of the control value) and the amplitude of cell contraction (66.0 +/- 7.4% of the control value) within a few minutes. Pretreatment with NG-monomethyl-L-arginine (L-NMMA) or methylene blue completely inhibited the IL-6-induced early changes. Subsequent addition of L-arginine reversed the effects of L-NMMA. The levels of cGMP were significantly increased after 30 minutes of exposure to IL-6 (134.4 +/- 9.1% of the control value). Pretreatment with L-NMMA or EGTA significantly inhibited the IL-6-induced early elevation of cGMP. These results suggest that IL-6 acutely decreases intracellular Ca2+ transients and depresses cell contraction by nitric oxide (NO)-cGMP-mediated pathway. Therefore, IL-6 may enhance the Ca(2+)-dependent constitutive NO synthase activity in cardiac myocytes. On the other hand, 24-hour exposure to IL-6 also increased the levels of cGMP (159.0 +/- 22.8% of the control value) regardless of pretreatment with EGTA. These delayed increases in cGMP were also shown to be coupled with decreases in intracellular Ca2+ transients and the amplitude of cell contraction. Thus, IL-6 may induce Ca(2+)-independent NO synthase in cardiac myocytes. Together with the previous reports that have suggested the possible roles of IL-6 in myocardial stunning or endotoxic shock, this negative inotropic effect of IL-6 may contribute to these clinical settings.

Stimulation of prostaglandin E2 release from cultured rabbit gastric cells by sodium deoxycholate.

Although bile salts are irritants in the gastric mucosa, their effects on prostaglandin (PG) release have not been well studied. We investigated the effects of bile salts on PGE2 release and the possible mechanisms involved. Cultured rabbit gastric mucous epithelial cells were studied. PGE2 was measured by radioimmunoassay. Intracellular free Ca2+ concentration was measured with Ca2+ fluorescent dye indo-1 AM. Dihydroxy bile salts, such as chenodeoxycholate and deoxycholate (DC), dose-dependently increased PGE2 release, while non-dihydroxy bile salts did not. Since agents involved in the cellular signal transduction system have been reported to play important roles in PG release, the possible involvement of Ca2+, calmodulin, and protein kinase C (PKC) in DC-induced PGE2 release was studied. Deprivation of Ca2+ from the medium blocked DC-induced PGE2 release. Lanthanum (La3+), which displaced surface-bound Ca2+, suppressed DC-induced PGE2. However, BAPTA (a chelator of intracellular Ca2+) did not decrease it. Neither calmodulin inhibitors nor PKC inhibitors altered DC-induced PGE2 release. DC increased intracellular free Ca2+ concentrations. This effect was blocked by deprivation of Ca2+ from the medium. Quinacrine (a phospholipase A2 inhibitor) blocked DC-induced PGE2 release. These results suggest that in cultured rabbit gastric cells, deoxycholate stimulates PGE2 release mainly through the influx of extracellular Ca2+.

P2 purinergic receptor regulation of mucus glycoprotein secretion by rabbit gastric mucous cells in a primary culture.

BACKGROUND/AIMS: Physiological regulation of gastric mucus secretion has not been well studied. The present study investigated the effects of adenosine 5'-triphosphate (ATP), a P2 purinergic receptor agonist, and its analogues on gastric mucus secretion using gastric mucous cells in a primary culture. METHODS: A monolayer culture of gastric mucous cells from adult rabbits were prepared after enzyme digestion. Mucus secretion was estimated from the release of [3H]glucosamine from prelabeled cells. Intracellular calcium concentration ([Ca2+]i) was monitored by a Ca(2+)-sensitive probe, indo-1. Prostaglandin E2 (PGE2) in the media was measured by an enzyme-linked immunoassay. RESULTS: ATP significantly stimulated mucus secretion by these cells at nontoxic doses in a dose-dependent fashion. The order of potency of ATP analogues stimulating mucus secretion was alpha beta-methylene ATP > ATP > 2-methylthio ATP, whereas adenosine, a P1 purinergic receptor agonist, had no effect. ATP also induced an elevation of [Ca2+]i in a dose-dependent fashion. The efficacy of ATP analogues to increase [Ca2+]i showed a similar potency to their actions on mucus secretion. ATP increased PGE2 at relatively higher concentrations, whereas indomethacin did not block ATP-induced increase of mucus secretion. CONCLUSIONS: These results suggest that ATP stimulates mucus secretion by gastric mucous cells through P2 purinergic receptors; this appears to be mediated by intracellular calcium not by endogenous PGE2.

Depolarization-induced Ca entry via Na-Ca exchange triggers SR release in guinea pig cardiac myocytes.

In mammalian heart muscle, Ca entry through L-type Ca channels is thought to be the primary trigger for the sarcoplasmic reticulum (SR) Ca release, which initiates contraction. The results of this study show that, in guinea pig myocytes with a normal internal Na (10 mM Na in pipette), another trigger mechanisms for SR release and contraction exists. A crucial feature of these experiments was the ability to change rapidly the extracellular environment of a single myocyte so that alterations of intracellular Ca and SR Ca load were minimized for each solution change. We found the following results. 1) A switch to Na-free solution 50 ms before depolarization led to an increase of phasic contraction without increasing L-type Ca current (Ica) or Ca loading of the SR. 2) Although rapid application of 20 microM nifedipine 3 s before a + 10-mV pulse blocked ICa completely, 43 +/- 11 (SE) % of the phasic contraction remained. Similar results were obtained by rapid switching to 150 microM Cd to block ICa. 3) Phasic contraction and ICa had different voltage dependence. With steps to positive potentials there was little ICa but still a substantial phasic contraction. 4) Under action potential conditions, 64.6 +/- 7.9% of the control phasic contraction remained after switching to 20 microM nifedipine to block ICa. 5) The contraction remaining with nifedipine was unaffected by adding 100 microM Ni. Because 100 microM Ni blocks T-type Ca channels, this shows that Ca entry via T-type Ca channels is not involved in triggering SR release. 6) The phasic contraction remaining after a rapid switch to nifedipine was blocked completely by adding 5 mM Ni. Because this concentration of Ni is known to block the Na-Ca exchange, this result suggests that the exchange plays a role in triggering SR release. Taken together, the present results indicate that depolarization-induced Ca entry on the Na-Ca exchange is able to trigger SR release and phasic contraction. This explanation can account for increased phasic contraction after a rapid switch to Na-free solution, persistence of a phasic contraction in the complete absence of ICa, substantial phasic contraction at positive test potentials where there is no ICa, and abolition of nifedipine-resistant contraction by 5 mM Ni.

Variable effects of endothelin-1 on [Ca2+]i transients, pHi, and contraction in ventricular myocytes.

We examined the effects of endothelin-1 (ET-1) on intracellular free calcium concentration ([Ca2+]i) transients, intracellular pH (pHi), and cell contraction in both embryonic and neonatal as well as in adult ventricular myocytes. Exposure of chick ventricular myocytes to ET-1 (10 nM) significantly decreased both peak systolic and end-diastolic [Ca2+]i (from 949 +/- 43 to 628 +/- 59 nM and from 230 +/- 13 to 162 +/- 8 nM, respectively; P < 0.05, n = 12). The amplitude of cell contraction was also decreased during exposure to 10 nM ET-1 (81.7 +/- 1.2% of control, P < 0.01, n = 12). Exposure to 10 nM ET-1 slightly decreased pHi (-0.055 +/- 0.020 U; P < 0.05). Exposure of cultured neonatal rat ventricular myocytes to ET-1 (10 nM) produced similar effects. Responses of adult rabbit ventricular myocytes to ET-1 were dramatically different from those of embryonic or neonatal ventricular myocytes. Exposure to 10 nM ET-1 increased the amplitude of cell contraction to 159 +/- 32% of control (P < 0.01) without an increase in [Ca2+]i transients. ET-1 also increased pHi (+0.081 +/- 0.047 U; P < 0.01). These results indicate that ET-1 produces a negative inotropic effect by decreasing [Ca2+]i transients and induces a slight intracellular acidosis in immature ventricular myocytes. However, ET-1 causes a positive inotropic effect in adult ventricular myocytes via an intracellular alkalinization, rather than by an increase in the [Ca2+]i transient. Thus the response of myocytes to vasoactive peptides may vary with development and/or species.