A new capillary tube system for measuring the uptake and release of materials from cultured cells

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: Separation Of 3h Choline and 3h-achmentioning

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

“…3H -choline and 3H-ACh in each perfusate were separat ed into individual fractions by a modified choline kinase method (9,11). A 500-pl aliquot of perfusate was treated for 15 min with 20 it of choline kinase (10 mU/ml) in the presence of 125 pl of 200 mM sodium phosphate buffer (pH 8.0), 50 p1 of 10 mM ATP and 10 MM MgCl2.…”

Section: Separation Of 3h-choline and 3h--a Chmentioning

confidence: 99%

Aconitine-Induced Increase and Decrease of Acetylcholine Release in the Mouse Phrenic Nerve-Hemidiaphragm Muscle Preparation

Okazaki

Japanese Journal of Pharmacology

1994

ABSTRACT-The effect of aconitine on acetylcholine (ACh) release from motor nerve terminals in the mouse phrenic nerve-diaphragm muscle preparation was studied by a radioisotope method. Both electrical stimulation-evoked release and spontaneous release of 3H-ACh from the preparation preloaded with 3H choline were measured. The change in the muscle tension was simultaneously recorded in the same prepara tion. Aconitine (0.1 ttM) increased electrically evoked 3H-ACh release, while at higher concentrations (0.3 3 pM) it decreased the evoked release and muscle tension. High concentrations of aconitine (3 30 pM) caused a concentration-dependent increase in spontaneous 3H-ACh release. All these effects were sup pressed by tetrodotoxin. The aconitine-induced spontaneous release consisted of two different components: a Ca"-dependent phasic release that was inactivated within a few minutes and a Ca" -independent, long lasting release at a low level. The depression of the Ca" -dependent quantal release seems attributable to the decline of Ca" influx into the nerve rather than inactivation of sodium channels. We conclude that aconitine increases and then decreases electrical stimulation-evoked ACh release from the motor nerve through prolonged activation of sodium channels. Further activation of the channels enhances spontaneous release and the subsequent complete inactivation of the quantal release may be due to block of Ca" influx.

Regulation of presynaptic cellular function

McGee

1980

Mol Cell Biochem

Experiments from several different laboratories are reviewed in which clonal neuronal cell lines are being used to study neuronal cellular functions. Primary emphasis is placed on two cell lines, the neuroblastoma X glioma hybrid clone NG108-15 and the pheochromocytoma clone PC12. These particular cell lines are useful because they display many of the properties normally associated with differentiated neurons. The properties which have been studied include: the regulation of adenylate cyclase and the receptors which activate or inhibit its activity, regulation of the cholinergic properties of NG 108-15 and both adrenergic and cholinergic properties of PC12, the response of PC12 to nerve growth factor, and the regulation of synaptogenesis between NG 108-15 cells and cultured muscle. The goal of the review is to not only summarize the information obtained with these two cell lines but also to emphasize the types of research in which clonal cell lines may be most useful in the future.