Yoshiaki Nonomura

Myosin II is involved in transmitter release at synapses formed between rat sympathetic neurons in culture

The presynaptic function of myosin II was studied at cholinergic synapses formed between rat superior cervical ganglion neurons in culture. Immunofluorescent staining showed that myosin II was colocalized with synaptophysin at the presynaptic nerve terminals. Antimyosin II antibody introduced into presynaptic neurons inhibited synaptic transmission. Transmission was also inhibited in a dose-dependent manner by two inhibitors of myosin light chain kinase: a peptide, SM-1, and an organic inhibitor, wortmannin. The inhibition produced by these agents was dependent on presynaptic activity. Extracellularly applied wortmannin also blocked synaptic transmission, but its effects were slower in onset. Wortmannin also decreased postsynaptic potentials and post-tetanic potentiation in intact superior cervical ganglia. These results suggest a model in which myosin light chain kinase phosphorylates myosin, and the resultant change in actin-myosin interactions is involved in neurotransmitter release.

Tetrodotoxin and Manganese Ions: Effects on Electrical Activity and Tension in Taenia Coli of Guinea Pig

Tetrodotoxin, at concentrations up to 5 x 10-6 gram per milliliter, has no effect on the spontaneous discharge in the smooth muscle of taenia coli. However, the spontaneous discharge is abolished by Mn++ at a concentration of 0.5 millimole per liter. The contraction induced by immersing the muscle in isotonic KCl solution is also suppressed in the presence of Mn++. Because Mn++ is a specific suppressor of the spike induced by Ca++ and tetrodotoxin is an inhibitor of the spike induced by Na+, we suggest that Ca++ is a charge carrier in the production of spike potential in the smooth muscle and that the entry of intervening Ca++ through the membrane acts as a trigger for the contraction of smooth muscle.

Simple and rapid purification of brevin.

Brevin or plasma gelsolin, a calcium dependent actin-binding and actin-severing protein, was purified from bovine plasma by a very rapid and simple procedure; ammonium sulfate fractionation and only one step of anion exchange column chromatography by a convenient use. It takes only 24 hrs to complete all the procedure. The purity of brevin prepared by this method was more than 95% on SDS-PAGE and total recovery was much better than previous preparation methods. This brevin preparation has about 8 isomers on 2-D PAGE and strong severing activity on F-actin under electron microscopic observation.

Potent and preferential inhibition of Ca2+/calmodulin-dependent protein kinase II by K252a and its derivative, KT5926

Effects of protein kinase inhibitors, K252a and its derivative KT5926, on Ca2+/calmodulin-dependent protein kinase II were examined. Both compounds potently inhibited Ca2+/calmodulin-dependent protein kinase II. Kinetic analyses indicated that the inhibitory effect of K252a and KT5926 was competitive with respect to ATP (Ki: 1.8 and 4.4 nM, respectively) and noncompetitive with respect to the substrates. Taken together with a previous report (Nakanishi et al. Mol. Pharmacol. 37, 482, 1990) concerning the Ki values of these compounds for ATP with various protein kinases, the results suggest that K252a and KT5926 are potent and preferential inhibitors of Ca2+/calmodulin-dependent protein kinase II.

Fine structure of the thick filament in molluscan catch muscle

Abstract 1. (1) Intact profiles of the thick filament from molluscan smooth muscle were observed by negative staining of the fresh sample, which was homogenized in a modified relaxing medium containing a high concentration of ATP. The surface of the thick filament showed clear projections of myosin heads in samples negatively stained immediately after homogenization. 2. (2) When the ATP in the homogenate was exhausted, myosin molecules bound to the surface of the thick filament were gradually removed and bound to suspended thin filaments, and the underlying periodic structure of the paramyosin core previously covered by the myosin molecules appeared. 3. (3) The profiles of the paramyosin core after complete elimination of surface myosin molecules were of two different types; the Bear-Selby net pattern type and the striated type with 14.5 run pitch. 4. (4) The thick filament was reconstructed under special conditions; myosin molecules bound to the surface of the paramyosin core or to paramyosin para-crystals appeared as projections. 5. (5) The actin-activated myosin ATPase activity, which was initiated by the addition of a mixture of ATP and actin to the myosin-paramyosin core complex, was increased slightly by increasing the ratio of paramyosin to actin plus myosin. 6. (6) The underlying striated pattern of the thick filament was revealed in sectioned material as a result of removal of myosin molecules from the surface of the core on exhaustion of ATP.

The regulatory role of calcium in muscle.

The establishment of the crucial role of Ca2+ in the physiological contraction of muscle has led to the discovery of the third factor of the contractile system, i.e.. the troponin-tropomyosin system.l.2 It was once believed that the troponin-tropomyosin system would be common to all kinds of muscle. However, the discovery of a myosin-linked regulatory system in scallop striated muscle by Kendrick-Jones c’t n l . 3 has suggested some diversity in the regulatory mechanism in the animal kingdom. Indeed, the regulatory mechanism of smooth muscle appears to be entirely different from either of the above two types, although no agreement about the mechanism itself has not yet been reached. In this article we will review the regulatory mechanisms and compare them; the characteristic properties of the actomyosin system underlying such mechanisms will also be discussed.

Inhibition of Ca2+-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells

Abstract To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K + -evoked catecholamine (CA) release. The IC 50 for high K + -evoked CA release was 1μM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca 2+ -dependent exocytosis was inhibited in a dose-dependent manner (IC 50 , 1μM). These findings suggest the implication of MLCK in the Ca 2+ -triggered process in the machinery of exocytosis.

PROTEINS OF THE MYOFIBRIL

Publisher Summary This chapter focuses on the proteins of the myofibril, which is the morphological unit of the contractile mechanism of skeletal muscle. The muscle fiber consists of a number of myofibrils that are surrounded by the network of the sarcoplasmic reticulum. The myofibril is composed of those proteins that are concerned with contractile processes. This chapter describes the properties of muscle proteins from the standpoint of the contractile mechanism. The fundamental process of contraction is carried out by myosin and actin. Myosin is the major myofibrillar protein, and its interaction with actin in the presence of ATP represents the fundamental molecular basis of muscle contraction. In the sequence of chemical reactions occurring during this interaction, the breakdown of ATP, which is coupled with the conversion of chemical energy of ATP into mechanical energy, is carried out at a specified site on the myosin molecule. Actin has two important functions: (1) to activate myosin and to cooperate with it in carrying out the reaction to convert the chemical energy contained in ATP into mechanical energy and (2) to provide a place for regulatory proteins through which the physiological triggering factor, that is, calcium ion, can control the interaction of actin and myosin.

Inhibition of IgE-mediated histamine release by myosin light chain kinase inhibitors

Wortmannin, a specific inhibitor of myosin light chain kinase (MLCK) blocked IgE mediated histamine release from rat basophilic leukemia cell (RBL-2H3) and human basophils dose-dependently. Its IC50 was 20 nM for RBL-2H3 cells and 30 nM for human basophils. There was complete inhibition at the concentration of 1 microM. Wortmannin inhibited partially the A23187 induced histamine release from RBL-2H3 cells (40% inhibition at 1 microM). This inhibition was not accompanied by any significant effect on cytosolic free calcium concentration [( Ca2+]i). KT5926, another MLCK inhibitor, inhibited histamine release comparably with wortmannin and blocked to some degree the increase of [Ca2+]i in RBL-2H3 cells. Thus, the phosphorylation of myosin seems to be involved in signal transduction through Fc epsilon RI.

Direct activation by okadaic acid of the contractile elements in the smooth muscle of guinea-pig taenia coli

Summary1. Okadaic acid isolated from black sponge (Halichondria okadai), at the concentration of 10 μmol/l, caused contraction in saponin-treated skinned smooth muscle of guinea-pig taenia coli in the absence of Ca2+. In the presence of low concentration (0.3 μmol/l) of Ca2+ okadaic acid induced a greater contraction than in the absence of Ca2+ 2. Okadaic acid potentiated the contractions induced by Ca2+ and pCa2+-tension curve was shifted to the left as well as upward by 1 μmol/l okadaic acid. 3. Native actomyosin preparation (myosin B) containing calmodulinmyosin light chain kinase system and phosphatase was obtained from taenia coli. Okadaic acid (10 μmol/l) increased the actomyosin Mg2+-ATPase activity in the presence or absence of Ca2+. 4. Okadaic acid (1–100 μmol/l) had no effect on calmodulin activity as monitored by Ca2+-calmodulin activated cyclic nucleotide phosphodiesterase activity and the (Ca2+ + Mg2+)-ATPase activity of erythrocyte membranes. 5. These results suggest that okadaic acid directly activates contractile elements of smooth muscle.