Motoaki Bessho

Differential extractability of creatine phosphate and ATP from cardiac muscle with ethanol and perchloric acid solution

To compare the extractability of creatine phosphate with that of ATP by alcohol extraction, both compounds were extracted from normal perfused rat heart tissues by using various stepwise concentrations of ethanol and 0.4 M HClO4. Powdered samples (6-15 mg wet wt) from the freeze-clamped tissues were homogenized in 2 ml of the ethanol solutions. After centrifugation, the supernatant was removed; each centrifuged sediment was rehomogenized with 2 ml of 0.4 M HClO4 and centrifuged. The supernatant was neutralized with 0.4 m KHCO3. The same powdered samples were directly homogenized with 2 ml of 0.4 M HClO4 and treated in the same manner. Only a small amount of ATP in the tissues was extracted by an 85% or higher concentration of ethanol. Further, about 13% of the tissue ATP was not extractable by the subsequent perchloric acid extraction. In contrast to ATP, creatine phosphate in the tissues was partially extracted by 95% ethanol and nearly all of the tissue creatine phosphate was extracted by 70% ethanol. The total creatine phosphate obtained by 70% ethanol and by subsequent perchloric acid extraction was significantly higher than that obtained by direct perchloric acid extraction. From these results, it was concluded that the extractability of creatine phosphate in the tissue by alcohol extraction is clearly different from that of ATP. Additionally, the stepwise extraction is recommended as a useful method for the extraction of energy metabolites in perfused rat heart tissue.

Effects of geranygeranylacetone on gastrointestinal secretion in rats

The effects of geranylgeranylacetone (GGA), a new acyclic polyisoprenoid with a novel antiulcer action on gastrointestinal secretion were studied in rats. Intraduodenal administration of GGA (1-30 mg/kg) dose-relatedly reduced the gastric acid secretion caused by pentagastrin, histamine or insulin. On the other hand, GGA (3-30 mg/kg i.d.) dose-relatedly increased pancreatic secretion but did not affect biliary secretion. The above-mentioned findings seem consistent with the further findings that GGA depressed a plasma gastrin level enhanced by insulin while in increased the basal level of plasma secretin. These pharmacological features found in the present studies may partially, at least, account for the mechanism of GGA antiulcer action.

NAD and NADH values in rapidly sampled dog heart tissues by two different extraction methods

To clarify the most quantitative extraction method for the determination of NAD and NADH in dog heart tissues, both pyridine dinucleotides were extracted from normal and ischemic heart tissues by the Klingenberg method and the Karp method and determined by bacterial luciferase. Tissues from normal beating hearts were sampled by a specially developed freeze-clamping device in 120 ms to minimize ischemic NADH production during sampling. Samples were obtained from both the subendocardium and the subepicardium of the frozen heart tissues. In the Klingenberg method, NAD and NADH were separately extracted with 0.6 M HClO4 and 0.5 M KOH in 50% ethanol, respectively. Both pyridine dinucleotides were simultaneously extracted with 70% ethanol in 0.01 M phosphate buffer in the Karp method. The mean values of NAD and NADH in the normal tissues were 5.08 +/- 0.84 and 0.18 +/- 0.10 nmol/mg protein, respectively, with a NAD/NADH ratio of 25-30 by the Klingenberg method. While the values by the Karp method were 4.37 +/- 0.68 and 0.09 +/- 0.04 nmol/mg protein, with a NAD/NADH ratio of 55-65. The efficiency of extraction of both pyridine dinucleotides by the Karp method was lower than that by the Klingenberg method in all tested samples and states of the tissues. These results suggest that the Klingenberg method is preferable for the extraction of both pyridine dinucleotides from dog heart tissues and that the mean NAD/NADH ratio in normal dog heart tissues is 25-30.

Rabeprazole treatment attenuated Helicobacter pylori-associated gastric mucosal lesion formation in Mongolian gerbils

Background and Aim: Although rabeprazole (RPZ), a proton pump inhibitor, has been reported to have a bactericidal effect on Helicobacter pylori (H. pylori), no studies have been conducted regarding the effect of RPZ on gastric mucosal lesion formation caused by this bacterium. In the present study, we investigated the effect of RPZ on H. pylori‐associated gastric mucosal lesion formation.

A rapid-cross-sectioning and freeze-clamping device for the beating canine heart

A new sampling method of cross-sectioning the canine heart in situ was developed. A mechanical device, driven by spring power, enabled cross-sectioning of a short-axis plane of the beating canine heart (4 mm thick) with high speed rotating blades, at a pre-determined phase of the cardiac cycle, and instantaneous freeze-clamping (2.4 mm thick) with pre-cooled aluminum blocks, all within 120 ms. By this method, the anatomical structures of the sample were well preserved. Transmural metabolism and flow distribution were instantaneously fixed and high resolution of the two-dimensional redox state was obtained by application of NADH fluorescence photography. Micro-samplings from the desired portion of the cross-sectional slice were possible at -190 degrees C. NADH fluorescence of the samples did not increase from the surface to 1.2 mm in depth, confirming that there was no ischemic artifact. With the present technique, a heart sample in which transmural metabolism, and the redox state, are fixed and visualized is attainable, thus providing a new tool for the study of myocardial ischemia.

Continuous inhibition of poly(ADP-ribose) polymerase does not reduce reperfusion injury in isolated rat heart.

Abstract: Poly(ADP-ribose) polymerase (PARP), an enzyme that is important to the regulation of nuclear function, is activated by DNA strand breakage. In massive DNA damage, PARP is overactivated, exhausting nicotinamide adenine dinucleotide and leading to cell death. Recent studies have succeeded in reducing cellular damage in ischemia/reperfusion by inhibiting PARP. However, PARP plays an important part in the DNA repair system, and its inhibition may be hazardous in certain situations. We compared the short-time inhibition of PARP against continuous inhibition during ischemia/reperfusion using isolated rat hearts. The hearts were reperfused after 21 minutes of ischemia with a bolus injection of 3-aminobenzamide (3-AB) (10 mg/kg) followed by continuous 3-AB infusion (50 &mgr;M) for the whole reperfusion period or for the first 6 minutes or without 3-AB. At the end of reperfusion, contractile function, high-energy phosphate content, nicotinamide adenine dinucleotide content, and infarcted area were significantly preserved in the 3-AB 6-minute group. In the 3-AB continuous group, these advantages were not apparent. At the end of reperfusion, PARP cleavage had significantly proceeded in the 3-AB continuous group, indicating initiation of the apoptotic cascade. Thus, continuous PARP inhibition by 3-AB does not reduce reperfusion injury in the isolated rat heart, which may be because of acceleration of apoptosis.

Hemoglobin vesicle improves recovery of cardiac function after ischemia-reperfusion by attenuating oxidative stress in isolated rat hearts.

Abstract Hemoglobin vesicle (HbV) could be a useful blood substitute in emergency medicine. The aim of this study was to clarify the effects of HbV on cardiac function after ischemia–reperfusion (I/R) ex vivo. Isolated rat hearts were perfused according to the Langendorff method. An ischemia–reperfusion group (n = 6) was subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. HbV (hemoglobin, 0.33 g/dL) was perfused before ischemia–reperfusion for 10 minutes (HbV group, n = 6). Hemodynamics were monitored, and tissue glutathione contents were measured. The redox state of reactive thiols in cardiac tissues was assessed by the biotinylated iodoacetamide labeling method. Left ventricular developed pressure was significantly recovered in the HbV group after 30 minutes of reperfusion (56.3 ± 2.8 mm Hg vs. ischemia–reperfusion group 27.0 ± 8.0 mm Hg, P < 0.05). Hemodynamic changes induced by HbV were similar to those observed when NG-nitro-L-arginine methyl ester was perfused for 10 minutes before ischemia–reperfusion (L-NAME group). The oxidized glutathione contents of cardiac tissues significantly decreased, and biotinylated iodoacetamide labeling of thiols was maintained in both the HbV and the L-NAME groups. HbV improved the recovery of cardiac function after ischemia–reperfusion in isolated rat hearts. This mechanism is dependent on functional protection against thiol oxidation.