Changes in total and quantal release of acetylcholine in the mouse diaphragm during activation and inhibition of membrane ATPase.

Okazaki

Japanese Journal of Pharmacology

1993

ABSTRACT-Acetylcholine (ACh) release from the motor nerve terminal in the streptozocin-induced diabetic state was studied in mouse phrenic nerve-diaphragm muscle preparations. Electrically evoked re lease of 3H-ACh from the preparation preloaded with 3H-choline was measured during two consecutive periods of stimulation (S1 and S2). In diabetic mice, the amount of 3H-ACh release during S2 was decreased, and the evoked ACh release declined more steeply with successive stimulation periods than in normal ddY mice. The decrease in release was restored when the presynaptic autoreceptors were stimulated by ac cumulating ACh under the irreversible inhibition of junctional cholinesterase by methanesulfonyl fluoride. This effect was abolished by the administration of (+)-tubocurarine (5 tiM). In diabetic mice, the biphasic (acceleration and suppression) effect by succinylcholine on evoked ACh release was caused at 3 to 10-fold lower concentrations than in normal mice. The degree of enhancement of resting 3H-overflow by suc cinylcholine (10 and 30 ttM) was greater in the diabetic state. These results indicated that in the diabetic state, the decrease in evoked ACh release interferes with its presynaptic action on inducing further release (positive feedback modulation) via the presynaptic nicotinic ACh receptor (n-AChR). The presynaptic hypersensitivity to succinylcholine may be due to the augmentation of presynaptic n-AChR sensitivity caused by the reduction of evoked ACh release in the diabetic state.

Section: Discussionmentioning

confidence: 99%

Ca2+-Induced Increase in Oxidative Metabolism of Dissociated Mammalian Brain Neurons: Effect of Extract of Ginkgo Biloba Leaves

Okazaki

Japanese Journal of Pharmacology

1993

The Journal of Cell Biology

“…Neurotransmitters are released from nerve terminals in two ways: by the continuous leakage of individual molecules across the nerve terminal membrane (23,33,41,50) and by synchronous secretion of few thousands molecules in packages, or quanta (14,15,20). Quantal secretion causes the changes in membrane potential that excite the postsynaptic cell, and transmission at a synapse is critically dependent upon this mechanism of release.…”

mentioning

confidence: 99%

Temporal coincidence between synaptic vesicle fusion and quantal secretion of acetylcholine.

Torri-Tarelli¹,

Grohovaz²,

Fesce³

et al. 1985

150

We applied the quick-freezing technique to investigate the precise temporal coincidence between the onset of quantal secretion and the appearance of fusions of synaptic vesicles with the prejunctional membrane. Frog cutaneous pectoris nerve-muscle preparations were soaked in modified Ringer's solution with 1 mM 4-aminopyridine, 10 mM Ca 2+, and 10 -4 M d-Tubocurarine and quick-frozen 1-10 ms after a single supramaximal shock. The frozen muscles were then either freeze-fractured or cryosubstituted in acetone with 13% OsO4 and processed for thin section electron microscopy. Temporal resolution of <1 ms can be achieved using a quick-freeze device that increases the rate of freezing of the muscle after it strikes the chilled copper block (15°K) and that minimizes the precooling of the muscle during its descent toward the block. We minimized variations in transmission time by examining thin sections taken only from the medial edge of the muscle, which was at a fixed distance from the point of stimulation of the nerve. The ultrastructure of the cryosubstituted preparations was well preserved to a depth of 5-10/~m, and within this narrow band vesicles were found fused with the axolemma after a minimum delay of 2.5 ms after stimulation of the nerve. Since the total transmission time to this edge of the muscle was ~3 ms, these results indicate that the vesicles fuse with the axolemma precisely at the same time the quanta are released.Freeze-fracture does not seem to be an adequate experimental technique for this work because in the well-preserved band of the muscle the fracture plane crosses, but does not cleave, the inner hydrophobic domain of the plasmalemma. Fracture faces may form in deeper regions of the muscle where tissue preservation is unsatisfactory and freezing is delayed.Neurotransmitters are released from nerve terminals in two ways: by the continuous leakage of individual molecules across the nerve terminal membrane (23,33,41,50) and by synchronous secretion of few thousands molecules in packages, or quanta (14,15,20). Quantal secretion causes the changes in membrane potential that excite the postsynaptic cell, and transmission at a synapse is critically dependent upon this mechanism of release. The vesicle hypothesis holds that each quantum is confined within one of the synaptic vesicles that populate the nerve terminal and is released by exocytosis when the membrane of that vesicle fuses with the axolemma at one of the many specialized regions called "active zones" (8,13,51).A number of workers have shown that at frog neuromuscular junctions vesicles fuse with the axolemma of stimulated 1386 terminals and some of the remaining vesicles become labeled with extracellular tracers (9)(10)(11)25). Although these observations indicate that vesicles fuse with and are recovered from the axolemma when quanta are actively secreted, the slowness of chemical fixation precludes demonstrating that vesicle fusion and transmitter release are coincident. The images of fusion seen after chemical fixation represent...

“…The spontaneous release of ACh includes that released quantally which causes m.e.p.ps and that released non-quantally to cause a small depolarization of the endplate region equivalent to that produced by about 10-8M ACh in the frog (Katz & Miledi, 1977), 10-7M in mice (Vyskocil et al, 1983) and 6 x 10-9M in rats (Smith, 1984). In unstimulated tissues, the amount released non-quantally from the nerve is about ten times greater than that released quantally (Miledi et al, 1983 & Vyskocil, 1979;Dolezal & Tucek, 1983;Stanley & Drachman, 1983) and is from the cytoplasm of the nerve (Molenaar et al, 1987 (Adams et al, 1986). In this regard, Jenkinson & Nicholls (1961) and Takeuchi (1963) reported abolition by Mg2+ of the local contracture normally found after ACh.…”

Section: Methodsmentioning

confidence: 99%

Accumulation of extracellular calcium at the endplate of mouse diaphragm after ecothiopate in vitro

Burd

Ferry

Smith

1989

British J Pharmacology

Experiments were carried out to investigate the accumulation from the extracellular medium of 45Ca2+ by the endplate region of skeletal muscle. Mouse diaphragm muscle was incubated in physiological saline labelled with 45Ca at 37°C for periods of up to 1.5 h. The muscle was divided into junctional and non‐junctional portions and the Ca from the extracellular fluid accumulated at the endplate determined from the 45Ca content of the portions. The accumulation of extracellular Ca at the endplate region of muscles incubated in physiological saline alone was nil, but there was accumulation in the presence of the anticholinesterase ecothiopate iodide 0.5 × 10−6 m (ECO). Stimulation of the phrenic nerve at 0.02 Hz caused no further increase in accumulation, but reduced the amount of spontaneous fasciculation. In tetrodotoxin (TTX) 10−6 m, the accumulation was halved, and in 3.5 mM Mg2+ the accumulation was nil. Carbachol 10−4 m resulted in an accumulation of Ca similar to that in ECO. It is concluded that there was an accumulation of extracellular Ca following excitation of the nerve by stimulation at a low frequency and during the spontaneous fasciculations, and about half of the accumulation of extracellular Ca after ECO in the experiments was due to the postsynaptic action of ACh released non‐quantally from the nerve terminals.