Fumiaki Hata

Effect of ascorbic acid on release of acetylcholine from synaptic vesicles prepared from different species of animals and release of noradrenaline from synaptic vesicles of rat brain

Abstract Low concentrations of L-ascorbic acid caused release of acetylcholine from isolated synaptic vesicles (rat, guinea-pig and rabbit) in the presence of 2mM ATP, 2 mM MgCl2 and 10−5 M CaCl. The half maximum effect was obtained with about 2 to 2.5 ωM L-ascorbic acid, and the effect was inhibited by addition of 1mM EGTA. The release of noradrenaline from rat synaptic vesicles was also enhanced by L-ascorbic acid, but the concentration for half maximal stimulation was about 20 ωM, indicating that noradrenaline release was less sensitive to L-ascorbic acid than acetylcholine release. The physiological function of L-ascorbic acid in the brain is discussed in relation to release of transmitters.

Changes in brain muscarinic acetylcholine receptors and behavioral responses to atropine and apomorphine in chronic atropine-treated rats

Abstract Chronic blockade of cholinergic transmission with atropine resulted in a decrease in atropine-induced activity in the rats, whereas apomorphine - induced locomotion was enhanced. Maximal binding of 3 H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, to homogenate of cerebral cortex, striatum and hippocampus was significantly higher in chronic atropine-treated rats than in control animals. No difference was observed in K D value of the specific 3 H-QNB binding or in ID 50 value of oxotremorine in inhibiting 3 H-QNB binding. No change in the specific binding of 3 H-spiroperidol, a dopaminergic antagonist, was observed in those three regions of brains of chronic atropine-treated rats when it was compared with that of control animals. The role of brain muscarinic acetylcholine receptors in behavioral responses is discussed relating an effect of dopaminergic neurons on cholinergic activities.

Experimental evidence and dynamic aspects of spare receptor

Abstract Data obtained in radioligand - binding studies on guinea pig and mouse ileum were compared with those on the contractile responses to the muscarinic agonists methacholine (MCh), oxotremorine, bethanechol (BCh) and pilocarpine. Guinea pig ileum has a large amount of binding sites for 3 H-quinuclidinyl benzilate (QNB), a muscarinic antagonist, (102 fmoles/mg tissue), whereas mouse ileum has a small amount of these binding sites (19 fmoles/mg tissue). MCh, oxotremorine and BCh were full agonists in the contractile responses ofboth ilea, but pilocarpine was full agonist only in guinea pig ileum. After treatment with dibenamine (0.15 mM) (for 15 min at 30°C) guinea pig ileum exhibited maximal responses to MCh, oxotremorine and BCh, but not to pilocarpine, although the ED 50 values for the agonists in the muscle were increased by this treatment. The amount of binding sites of 3 H-QNB in dibenamine-treated guinea pig ileum was 55% of that in untreated ileum. When the amount of 3 H-QNB-binding sites in mouse ileum was decreased to 54% by dibenamine-treatment for 15 min, the maximal contractions induced by MCh, oxotremorine and BCh were also decreased to some degree. In low Ca 2+ medium, pilocarpine became a partial agonist in the contractile responses of guinea pig ileum. The following conclusions were drawn from these findings: 1. 1. The spare receptors proposed by Stephenson actually exist, and change in the amount of spare receptors, which can be brought about by dibenamine-treatment, or in low Ca 2+ medium, can change the ED 50 value of agonists. 2. 2. In a single tissue, the amount of spare receptors for different agonists differ, probably depending on the efficacy of the agonists. 3. 3. For a single agonist, the amount of spare receptors in a tissue differs from species to species, presumably depending on the total amounts of receptors.

Increase in response and number of α-adrenoceptors of rat vas deferens on brief pretreatment with an α-agonist

Abstract The contractile response of rat vas deferens to epinephrine (E) was enhanced by preincubation with E. After pretreatment with E, there was no significant change in the ED50 of E but there was a significant increase in the maximum response (39%). There was no change in the cholinergic response after the pretreatment. α-Adrenoceptors in control and E-treated tissues were assessed by measuring [3H]WB-4101 binding. Treatment with E resulted in a 27% increase in the number of α-adrenoceptors, but no change in muscarinic acetylcholine receptors measured with [3H] quinuclidinyl benzilate. These results are discussed in relation to the response of α-adrenoceptors to the agonist.

FACTORS REQUIRED FOR Ca-SENSITIVE ACETYLCHOLINE RELEASE FROM CRUDE SYNAPTIC VESICLES

Abstract— Studies were made on intracellular factors required for the release of ACh from crude synaptic vesicles prepared from rat brain. A factor stimulating ACh release in the presence of calcium ions was found in the cell sap.

STUDIES ON SOLUBLE PROTEINS RELEASED FROM THE SYNAPTIC VESICLES OF RAT BRAIN CORTEX

Abstract— The soluble proteins released from the synaptic vesicles of rat cerebral cortex were studied. One fraction (D4) of these proteins was released in parallel with release of acetylcholine when synaptic vesicles were incubated at 37°C for 10 min in isotonic medium. Another fraction (Dj) was liberated from synaptic vesicles when their membranes were ruptured by mild treatment under hyposmotic conditions and freeze‐thawing after release of D1 fraction. Fractions D1 and D2 contained 12 and 9 per cent, respectively, of the total protein in the synaptic vesicles. Some properties of these fractions were investigated by zone electrophoresis and ultracentrifugation, and by measuring their binding capacities for [14C]acetylcholine and various enzyme activities related to acetylcholine metabolism.

Action of colchicine on cholinergic and adrenergic mechanisms in guinea pig vas deferens.

Abstract Guinea pig vas deferens responds to externally applied acetylcholine (ACh) or noradrenaline (NA) by a small rapid contraction (phasi phase) and then a large contraction (tonic phase). The phasic phase was not affected by removal of external Ca2+, but tonic phase depended on external Ca2+. At lower temperatures the two components became larger and detectable separately. The tonic phase induced by ACh at low temperature (at 20°C) was greatly depressed by brief treatment with colchicine (0.5 μM – 5 μM), although the tonic phase at high temperature (at 37°C) was not affected. Na-induced contraction (phasic or tonic phase) was not changed by the colchicine-treatment. High K+ (40 mM)-contracture, which in many cases consisted of a single phase and depended on external Ca2+, was also not affected by brief treatment with colchicine. Culture of vas deferens for 3 days in the presence of colchicine, increased the phasic phase of ACh- and NA-induced contractions significantly, but reduced the tonic phase of contractions induced by ACh and NA. Colchicine also reduced high K+-contracture, the decrease depending on the period of culture with colchicine. Organ culture with colchicine did not affect the amounts of m-ACh and α-Ad receptors or the IC50 value of ACh and NA on 3H-ligand binding. These results suggest that colchicine specifically interacts with some steps in m-ACh and α-Ad receptor-responsor (e.g. ionophore) coupling without affecting the receptor number or affinity of the receptors for agonists. The mechanisms of action of colchicine are discussed in relation to m-ACh and α-Ad receptor functions.