Kazuro Sugishita

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.

Thoracic aortic calcification measured by X-ray computed tomography vs age and risk factors of arteriosclerosis

The purpose of this study was to examine possible correlations among age, arteriosclerotic risk factors, and specific sites of calcification in the thoracic aorta as detected by X-ray computed tomography (CT). A total of 80 patients (mean age 59±9 years, 50 M/30 Fe) included 34 patients with ischemic heart disease, 32 with chest pain syndrome, 5 with valvular heart disease, and 9 with other diseases. The thoracic aortic calcification score, based on X-ray CT images, is the sum of the length (cm) of calcification detected in 1-cm-interval horizontal cross-sections. Differences in calcification were compared for patients with and without hypertension, diabetes, and hyperlipemia. Calcification occurred more often in the external left arch wall (52 cases), followed by the lower arch wall (50 cases). Calcification in the ascending aorta was detected in only 18 cases. Aortic calcification score ranged from 0 to 103.3 points with a mean of 8.8±14.9 points, showing a significant correlation (r=0.48,p<0.01) with patient age. However, there was no significant difference in ascending and descending aorta between sites of cross-sections. Calcification score was higher in patients with hypertension or diabetes. This difference, though significant, was very small. Moreover, patient age did have some correlation with calcification score, but the presence of ischemic heart disease and gender had no effect.

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.