Compartmentation Of Acetylcholine Research Articles

Effects of Calyculin A and Okadaic Acid on Acetylcholine Release and Subcellular Distribution in Rat Hippocampal Formation

The mechanisms regulating the compartmentation of acetylcholine (ACh) and the relationship between transmitter release and ACh stores are not fully understood. In the present experiments, we investigated whether the inhibitors of serine/threonine phosphatases 1 and 2A, calyculin A and okadaic acid, alter subcellular distribution and the release of ACh in rat hippocampal slices. Calyculin A and okadaic acid significantly (p < 0.05) depleted the occluded ACh of the vesicular P3 fraction, but cytoplasmic ACh contained in the S3 fraction was not significantly affected. The P3 fraction is known to be heterogeneous; calyculin A and okadaic acid reduced significantly (p < 0.05) the amount of ACh recovered with a monodispersed fraction (D) of synaptic vesicles, but the other nerve terminal bound pools (E-F and G-H) were not so affected. K+-evoked ACh release decreased significantly (p < 0.01) in the presence of calyculin A and okadaic acid, suggesting that fraction D's vesicular store of ACh contributes to transmitter release. The loss of ACh from synaptic vesicle fractions prepared from tissue exposed to phosphatase inhibitors appeared not to result from a reduced ability to take up ACh. Thus, when tissue was allowed to synthesize [3H]ACh from [3H]choline, the ratio of [3H]ACh in the S3 to P3 fractions was not much changed by exposure of tissue to calyculin A or okadaic acid; furthermore, the specific activity of ACh recovered from the D fraction was not reduced disproportionately to that of cytosolic ACh. The changes are considered to reflect reduced synthesis of ACh by tissue treated with the phosphatase inhibitors, rather than an effect on vesicle uptake mechanisms. Thus, exposure of tissue to calyculin A or okadaic acid appears to produce selective depletion of tissue ACh content in a subpopulation of synaptic vesicles, suggesting that phosphatases play a role in ACh compartmentation.

Neural and non-neural acetylcholine in the rat diaphragm.

The compartmentation of acetylcholine (ACh) and of choline acetyltransferase in the rat diaphragm was analysed by measuring their contents in muscle segments containing endplates (e.p.) and endplate-free segments (non-e.p.) at different times following section of the phrenic nerve. In addition ACh release was determined before and after denervation. Freshly dissected hemidiaphragms contained about 125 pmol of ACh; more than 90% of this was localized in the e.p. portion. Between 10 and 18 h after denervation the ACh content of the e.p. portion decreased by 80% and its ACh concentration became approximately equal to that in the non-e.p. region, whose ACh content did not change. Spontaneous release of ACh was reduced by denervation and ACh release evoked by 50 mM KC1 was practically abolished. Choline acetyltransferase activity in freshly dissected preparations was about 30 nmol of ACh per gram per hour, Km 0.5 mM. About 65% of the enzyme disappeared in the first 24 h and the remaining 35% between 24 and 50 h after denervation. A different enzyme capable of ACh synthesis was found in the muscle fibres; its activity did not decrease after denervation. It is concluded that about 70% of the ACh in the diaphragm is contained in the motor nerve terminals, about 10% in the intramuscular nerve fibres and the remainder in the muscle fibres, and that about 65% of choline acetyltransferase is in the motor terminals and 35% in the nerve fibres.

Compartmentation of acetylcholine in synaptosomes

Compartmentation of acetylcholine in synaptosomes