Acetylcholine Transport: Fundamental Properties and Effects of Pharmacologic Agentsa

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Both phosphointermediate- and vacuolar-type (P- and V-type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P-type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P-type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 microM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110-kDa subunits. The V-type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 microM. The V-type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V-type ATPase revealed two Michaelis constants for MgATP of approximately 26 microM and 2.0 mM. The V-type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V-type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+,K(+)-ATPase in the synaptic vesicle preparation is demonstrated.

Section: Resultsmentioning

confidence: 99%

Characterization of the P‐Type and V‐Type ATPases of Cholinergic Synaptic Vesicles and Coupling of Nucleotide Hydrolysis to Acetylcholine Transport

Hicks

Parsons

1992

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“…The residual protein in the resulting supernatant was assayed as described in Materials andMethods. 1985, 1986;Parsons et al, 1987). It is not known whether subpopulations differing in number of copies of SV2, synaptophysin, or p65 per vesicle may also be present in isolated brain synaptic vesicles.…”

Section: Discussionmentioning

confidence: 99%

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

Floor

Feist

1989

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.

“…shown to be a potent inhibitor of vesicular ACh transport by Parson's group (Anderson et al, 1983u,b;Bahr and Parsons, 1 9 8 6~;Parsons et al, 1987), and binding studies (Bahr and Parsons, 19863) demonstrated the presence of a receptor for this drug on the cholinergic synaptic vesicles. Using another approach, Morot Abbreviations used: ACh, acetylcholine; AHS 183, 2-(4-phenylpipendino)cyclohexanoI; cetiedil, a-cyclohexyl-3-thienylacetic acid 2-(hexahydro-IH-azepin-1 -yl)ethyl ester citrate salt; NEM, N-ethylrnaleirnide; NBD-Cl, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; TCA, tnchloracetic acid.…”

Section: -(4-phenylpiperidino)cyclohexanol (Ah5 1 83) Wasmentioning

confidence: 98%

AH5183 and Cetiedil: Two Potent Inhibitors of Acetylcholine Uptake into Isolated Synaptic Vesicles from Torpedo marmorata

Diebler

Gaudry-Talarmain

1989

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)