Shuji Hasegawa

Polymerization of the adenosine 5'-diphosphate-ribose moiety of nicotinamide-adenine dinucleotide by nuclear enzyme. I. Enzymatic reactions.

Abstract [8- 14 C]ATP was incorporated by a rat-liver nuclear preparation into acid-insoluble material in the presence of NMN. Experiments using variously labeled ATP and NMN or NAD revealed that the ADP-ribose moiety of NAD was incorporated into the acid-insoluble reaction product. NAD was formed from ATP in the presence of NMN and was an immediate precursor of the acid-insoluble reaction product. The reaction involving the incorporation of NAD had an optimum pH of 8 and required neither a divalent cation nor a sulfhydryl compound. The reaction was effectively inhibited by nicotinamide but not by isonicotinic acid hydrazide. The contribution of NADase in the nuclei towards this enzymatic activity was suggested.

Post-natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture.

1. The whole‐cell voltage clamp technique was used to record Ba2+ currents in voltage‐sensitive Ca2+ channels in mouse flexor digitorum brevis muscles developing in situ from day 1 to 30 after birth. Effects of denervation and tissue culture on the Ca2+ channel currents were also studied. 2. The muscle fibres in newborn mice showed two distinct types of Ca2+ channel currents, a low‐threshold transient current and a high‐threshold sustained current. 3. The specific amplitude of the transient current was 2.7 +/‐ 1.7 (S.D.) A/F in response to ‐30 mV test pulses in medium containing 30 mM‐Ba2+ on day 1 after birth. The transient current decreased progressively in the post‐natal days and became undetectable by day 17. In contrast, the specific amplitude of the sustained current in response to +20 mV test pulses increased 4‐fold from 6.9 A/F on day 1 to 27.7 A/F on day 30. 4. The disappearance of the transient current could not be accounted for by either shifts in voltage dependence of activation and inactivation or changes in activation and inactivation times of the two types of current during development. 5. Denervating muscle fibres on day 8 after birth did not prevent the disappearance of the transient current. Denervating them on day 17 did not allow reappearance of the transient current. However, the increase of the sustained current was suppressed by the denervation either on day 8 or day 17. 6. In muscle fibres isolated on day 8 after birth and cultured thereafter, the transient current did not disappear until day 19 in culture (27 days after birth), while the sustained current was maintained at the level on day 8. 7. In muscle fibres isolated on day 17, when the transient current had become undetectable, and cultured thereafter, the transient current did not reappear until day 15 in culture (32 days after birth), while the sustained current was maintained at a level similar to that on day 17. 8. We conclude that innervation has little influence on the developmental disappearance of the transient Ca2+ channel current in mouse muscle fibres, and suggest that some influencing factors from surroundings other than the nerve may be required for the disappearance of the functional transient channels.

The polymerization of adenosine 5'-diphosphate-ribose moiety of NAD by nuclear enzyme. II. Properties of the reaction product.

Abstract The radioactive acid-insoluble material, Product I, which was formed by rat-liver nuclear preparation from labeled NAD or labeled ATP with nicotinamide mononucleotide, has been partially purified. It has a peak at 3 S with sucrose density-gradient centrifugation, and a density of around 1.57 with Cs 2 SO 4 equilibrium density-gradient centrifugation. Product I was converted into the acid-soluble form by snake-venom phosphodiesterase. Product I was not digested by micrococcal nuclease, spleen phosphodiesterase, potato phosphodiesterase, potato nucleotide pyrophosphatase, spleen NAD nucleosidase, or trypsin. The radioactive acid-soluble substance, Product II, which was released by venom-phosphodiesterase digestion, was isolated from a Dowex 1 formate column. Product II showed absorption maxima at 261, 259, and 258 mμ under alkaline neutral and acidic conditions, respectively. The molar ratio of adenine (as adenosine), ribose, phosphorus and periodate consumption was 1:2:2:1. All phosphates were converted to inorganic phosphate by acid-phosphatase digestion. Structures of Products I and II were proposed.

Postnatal induction and neural regulation of inward rectifiers in mouse skeletal muscle.

The whole-cell voltage-clamp technique was used to examine developmental changes of inward rectifier currents in fibres of the flexor digitorum brevis muscle acutely isolated from mice on postnatal day 0 (PO) to P36. Neither a steady-state component (Is−s) nor a slowly activated component (Irise) of inward rectifier currents were observed in fibres of P0 and P4 mice. Both Is−s and Irise became apparent between days P8 and PI6. The specific amplitudes of Is−s and Irise measured at a test-pulse potential of −100 mV at 20 mM extracellular K+ ([K+]0) increased to their respective plateau values of −68 ±10 and −15 ±7 μA/cm2 at P20. In fibres denervated on day P4 the developmental increase of Is−s was suppressed, its specific amplitude at P20 being one-tenth of that in the corresponding normal fibres. Irise did not appear in P4-denervated fibres throughout the development. In muscle fibres denervated at P16 or P20, the specific amplitudes of Is−s and Irise decreased, reaching the levels of P4-denervated fibres in 2–4 days after denervation. We conclude that Is−s and Irise develop within 3 weeks after birth, and suggest that innervation plays a key role in their induction.

Changes in acetylcholine and noradrenaline sensitivity of chick smooth muscle wholly innervated by sympathetic nerve during development

Developmental changes in sensitivity of the isolated expansor sedumdariorum muscle of posthatching chicks to noradrenaline (NA), Acetylcholine (ACh) and some other drugs were investigated. This muscle responded to both NA and ACh in early life. The sensitivity to ACh decreased progressively with increasing age and disappeared on day 40 after hatching, however, a corresponding elevation of cholinesterase activity was not observed. The sensitivity to NA remained at the same level during the period of 2-60 days after hatching. The Contractile action of ACh on this muscle was not affected by d-tubocurarine, hexamethonium or phentolamine, but was completely abolished by atropine. These results suggest that there are at least two kinds of responsive sites on the expansor secumdariorum muscle in the new-born chick and that the sites sensitive to ACh degenerate progressively during the developmental processes. The cholinergic sensitive sites of this muscle in the new-born chick may be muscarinic.

Multiple effects of interferon on myogenesis in chicken myoblast cultures

Effects of chicken interferon on the differentiation of chicken skeletal muscle in vitro were examined. Continuous treatment of chicken myoblast culture with 200 IU/ml of interferon (10 IU/mg protein) resulted in significant inhibition of cell fusion and subsequent myotube formation. However, treatment of myoblast culture with 2 to 200 IU/ml of interferon increased activities of creatine kinase and myokinase in 4- or 6-day cultured muscle cells in a dose-dependent fashion. The effect of interferon on myokinase was less than on creatine kinase. Three-fold increase in creatine kinase activity induced by interferon was not accompanied by the accelerated transition of creatine kinase isozyme from BB- to MM-type. On the other hand, accumulation of acetylcholinesterase in interferon-treated cells at day 6 was suppressed to nearly half the level of control cells. Rates of actin and myosin synthesis in 4-day cultures estimated by pulse-labelling with [35S]methionine were also suppressed to 85% of control cultures. However, a proportion of 35S-labelled actin and myosin in labelled proteins associated with glycerinated cells was not changed by interferon treatment. These results indicate that partially purified interferon has multiple effects on the process of the myogenic differentiation of chicken myoblast in vitro.

Partial purification and characterization of neurotrophic substance affecting tetrodotoxin sensitivity of organ-cultured mouse muscle

From mouse spinal cord homogenate, we isolated a trophic substance which reverses the post-denervation decrease in tetrodotoxin sensitivity of action potential in organ-cultured extensor digitorum longus muscle of mouse and characterized its physicochemical properties. The trophic substance was separated from macromolecules in homogenate by gel filtration on Biogel P2 column. The partially purified trophic substance was heat-stable, acid-stable and alkaline-labile. The trophic activity was destroyed by lyophilization at neutral pH but not at acidic pH. The trophic activity was abolished by incubation with pronase or leucine aminopeptidase, but not by trypsin, chymotrypsin, thermolysin or carboxypeptidase A. The trophic substance passed through an ultrafiltration membrane UM10 freely. A small part of the trophic activity passed through a UM2 or UM05, and the rest was retained on the membranes. The trophic substance adsorbed on CM-Sephadex at pH 7.2 but passed through DEAE-Sephadex at pH 8.4. These results suggest that the trophic substance regulating tetrodotoxin sensitivity of action potential in mouse skeletal muscle is a peptide with a rather low molecular weight of less than 10,000 and that while the N-terminus of the peptide is free, the C-terminus is probably blocked. This peptide differs from other trophic substances reported previously by other investigators.

Effects of spinal cord and other tissue extracts on resting and action potentials of organ-cultured mouse skeletal muscle.

Summary The trophic effects of spinal cord extract on resting potential, action potential and sensitivity of action potential to tetrodotoxin (TTX) in organ-cultured mouse skeletal muscle were examined. Spinal cord extract blocked decrease in the maximal rates of rise and fall of the action potential caused by cultivation and also partially prevented decrease in its TTX-sensitivity and in the resting potential. In another experiment, spinal cord extract was added to organ cultures of skeletal muscle that had been previously denervated for 3 days in vivo. Spinal cord extract partially reversed post-denervation fall in the maximal rate of rise of action potential and its TTX-sensitivity. Extracts of non-nervous tissues (kidney, spleen, pancreas and adrenal gland) were ineffective. These results indicate that mouse spinal cord appears to possess neurotrophic substance(s) regulating membrane properties of mouse skeletal muscle.

Lesion of the nucleus basalis magnocellularis does not affect cerebral cortical blood flow in rats

The effects of a unilateral ibotenic acid lesion of the nucleus basalis magnocellularis (NBM) on blood flow of the cerebral cortex and striatum were studied at 2, 4, 8 and 16 weeks after the lesion in conscious rats. In the cerebral cortex, no side-to-side difference in blood flow was observed, though cholinergic enzyme activity was markedly reduced on the side of the lesion. The results suggest that NBM lesion produces disturbance of cholinergic neurons in the cerebral cortex without significant alteration of blood flow.

Up-regulation of type II adenylyl cyclase mRNA in kindling model of epilepsy in rats.

The expression level of type II adenylyl cyclase mRNA (ACII) was analyzed by northern blotting in amygdaloid kindled rats. Remarkable increases in ACII mRNA were observed in the bilateral cerebral cortex and hippocampus at 24 h after the last generalized seizure. The elevated expression level in the hippocampus persisted for 4 weeks on the stimulated side. There were no changes in expression level in single-stimulated and partially-kindled states. These results suggest that the involvement of ACII might have an effect on the mechanisms of seizure generalization and the maintenance of persistent epileptogenesis rather than on the acquisition process.