Role of protein kinase C in the reduction of infarct size by N-methyl-1-deoxynojirimycin, an alpha-1,6-glucosidase inhibitor.

Preischaemic treatment with N-methyl-1-deoxynojirimycin (MOR-14), an alpha-1,6-glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR-14. To assess the effect of PKC inhibition on infarct size in MOR-14-treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg(-1) of MOR-14 10 min before ischaemia (17+/-2%, n=10), than in a control group (46+/-5%, n=10). This beneficial effect of MOR-14 was abolished when 5 mg kg(-1) of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR-14 injection (39+/-4%, n=10), although chelerythrine alone did not alter infarct size (43+/-4%, n=8). Further, chelerythrine had no effect on MOR-14-induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia. Immunoblot analysis of PKC in homogenates of Langendorff-perfused rabbit hearts revealed that MOR-14 significantly increased levels of PKC-epsilon in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia. Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR-14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR-14 than is inhibition of glycogenolysis.

Cooperative binding of carboxyarabinitol bisphosphate to the regulatory sites of ribulose bisphosphate carboxylase/oxygenase from spinach.

Ribulose bisphosphate carboxylase (RuBisCO) binds carboxyarabinitol bisphosphate (CABP) on its regulatory sites [Yokota, A. (1991) J. Biochem. 110, 246-252]. The characteristics of the equilibrium binding of CABP to the sites were examined by the gel-filtration method. Since RuBisCO binds CABP on the substrate sites with a dissociation constant of less than 10 pM, CABP bound exclusively to the substrate sites at less than 5 microM. Plotting the number of CABP bound to the sites other than the substrate sites against the concentration of CABP gave a typical "bumpy" curve; the binding number in the intermediate plateau at 20 to 40 microM CABP was 3.7 to 4.4 mol per mol of RuBisCO and that at the saturating concentration of CABP was 7.6 to 7.8 mol per mol of RuBisCO. The Hill plot of their relationship gave a line which bent strongly at 20 to 40 microM CABP. The best fitting of the data to the equations derived from the binding model constructed according to the reported model [Teipel, J. & Koshland, D.E., Jr. (1969) Biochemistry 8, 4656-4663] showed that the binding of CABP to the regulatory sites proceeded with positive cooperativity both before and after the plateau. The dissociation constant decreased from 31 to 14 microM by the factor of 1/1.3 in the former group and 490 to 0.7 microM by the factor of 1/8.9 in the latter with increasing binding number of CABP.

Regulation of the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase through cooperative binding of 6-phosphogluconate to its regulatory sites.

This study was attempted to elucidate the mechanism of the regulation of the turnover number on the catalytic sites by the regulatory sites of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase [Rbu(1,5)P2CO]. To this end, we analyzed the effects of the binding of 6-phosphogluconate (6-P-Gln) to the regulatory sites of the enzyme on the progress in the subsequent catalysis assayed with 0.5 mM ribulose 1,5-bisphosphate [Rbu(1,5)P2]. This concentration of Rbu(1,5)P2 hardly binds to the regulatory sites but competes with 6-P-Gln for the catalytic sites. An equilibrium binding assay showed that Rbu(1,5)P2CO bound 8 mol 6-P-Gln/mol enzyme in addition to the catalytic sites. The binding to the eight regulatory sites was positively cooperative. The biphasic reaction course, inherent in the carboxylase reaction of plant Rbu(1,5)P2CO and composed of a burst for an initial few minutes and a subsequent linear phase, became faint with increasing binding of 6-P-Gln to the sites. The change of the reaction progress from the biphasic to linear course was ascribed to the suppression of the functioning form of the enzyme from the high-activity to low-activity form and to the increased turnover number on the catalytic sites though the binding of 6-P-Gln to the regulatory sites.

Loss of antinociception induced by naloxone benzoylhydrazone in nociceptin receptor-knockout mice.

Nociceptin and nociceptin receptor, which show structural similarities to opioid peptides and opioid receptors, respectively, have been recently found to constitute a novel neuromodulatory system. In the brain, however, the physiological role of the modulation via the nociceptin receptor is still unclear. Administered nociceptin produces hyperalgesia and hypolocomotion, whereas the nociceptin receptor-knockout mice show no significant abnormalities in nociceptive thresholds and locomotion. To clarify possible involvement of the nociceptin receptor in the regulation of nociception and locomotion, we made use of the knockout mice and naloxone benzoylhydrazone (NalBzoH) identified originally as a ligand for opioid receptors. Experiments on the cultured cells transfected with the nociceptin receptor cDNA showed that NalBzoH competed with [3H]nociceptin binding and attenuated the nociceptin-induced inhibition of cAMP accumulation. Furthermore, behavioral studies demonstrated that NalBzoH completely inhibited nociceptin-induced hyperalgesia and hypolocomotion. It is therefore likely that NalBzoH can act as a potent antagonist for the nociceptin receptor in vivo. In wild-type mice, NalBzoH induced antinociception but did not affect locomotor activity. In contrast, in the knockout mice, no significant changes in nociception and locomotion were induced by NalBzoH. These results clearly suggest that the nociceptin system takes part in the physiological regulation of nociceptive thresholds but not in the basal modulation of locomotion.

N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, markedly reduced infarct size in rabbit hearts.

BACKGROUND: N-methyl-1-deoxynojirimycin (MOR-14), an alpha-glucosidase inhibitor, reduces the glycogenolytic rate by inhibiting the alpha-1,6-glucosidase of glycogen-debranching enzyme in the liver, in addition to possessing an antihyperglycemic action by blocking alpha-1,4-glucosidase in the intestine. Because the reduction of the glycogenolytic rate may be one of the mechanisms of myocardial protection in ischemic preconditioning, the compounds inhibiting myocardial alpha-1,6-glucosidase may be protective against ischemic damage. Thus, we investigated whether MOR-14 could inhibit alpha-1,6-glucosidase and reduce the infarct size in rabbit hearts without collateral circulation. METHODS AND RESULTS: MOR-14 dose-dependently decreased the alpha-1,6-glucosidase activity in rabbit heart extract. A tracer study demonstrated the myocardial uptake of a considerable amount of MOR-14 sufficient to fully inhibit alpha-1,6-glucosidase. To assess the infarct size-reducing effect of MOR-14, 54 rabbits were subjected to 30-minute coronary occlusion followed by 48-hour reperfusion. Preischemic treatment with 25, 50, and 100 mg/kg of MOR-14 dose-dependently reduced the infarct size (to 26+/-4%, 19+/-3%, and 14+/-2% of the area at risk, respectively), compared with the saline control (45+/-5%) without altering the blood pressure or heart rate. Another 40 rabbits given 100 mg of MOR-14 or saline 10 minutes before ischemia were euthanized at 10 or 30 minutes of ischemia for biochemical analysis. MOR-14 decreased the alpha-1,6-glucosidase activity to approximately 20% in vivo, reduced the glycogen breakdown, and attenuated the lactate accumulation at both 10 and 30 minutes of ischemia. CONCLUSIONS: Preischemic treatment with MOR-14 preserved glycogen, attenuated the accumulation of lactate, and reduced the myocardial infarct size by 69%. This cardioprotective effect was independent of changes of blood pressure and heart rate or regional blood flow. It may be associated with alpha-1,6-glucosidase inhibition, because MOR-14 markedly decreased the alpha-1,6-glucosidase activity in the heart.