Koh-ichiro Kinugawa

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.

Ca2+-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.

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.

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.

Ets-1 is involved in transcriptional regulation of the chick inducible nitric oxide synthase gene in embryonic ventricular myoctyes

In order to elucidate roles of Ets family of transcription factors in transcriptional activation of inducible nitric oxide synthase (iNOS) genes, we analyzed the chick iNOS gene expression in cultured chick embryonic ventricular myocytes (CEVM). Deletional analysis and site-directed mutagenesis demonstrated that both the Ets/PEA3 site (–221 to –216 bp) and the κB site (–101 to –93 bp) of the 5′-flanking region of the chick iNOS gene were involved in the maximal activation of the lipopolysaccharide (LPS)-induced expression of the reporter (luciferase) gene, although the proximal κB site played the more essential role. Electrophoretic mobility shift assay revealed that LPS augmented the nuclear protein bindings to the Ets/PEA3 as well as κB motifs. Ets-1, one of the Ets proteins, was suggested to be bound to the Ets/PEA3 oligonucleotide. By Northern blot analysis, LPS was shown to induce iNOS mRNA in CEVM, along with a preceding increase in the levels of c-ets-1 mRNA. Ets-1 may be involved in the iNOS gene transcription in CEVM, presumably through interacting with the NF-κB.

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.