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S M Parsons

Publications and source records attributed to S M Parsons.

At least 73 records · Page 4Linked to original sources
PubMed1988

Ganglioside composition of synaptic vesicles from Torpedo electric organ.

We have studied the ganglioside content and pattern of synaptic vesicles isolated from the electric organs of two species of Torpedinidae, Torpedo californica and Torpedo marmorata. The ganglioside concentrations were high relative to protein content (77 and 58 micrograms of N-acetylneuraminic acid/mg of protein, respectively), owing to the low protein-to-lipid ratio; however, they were also appreciable in relation to phospholipid (15.6 and 10.0 micrograms of N-acetylneuraminic acid/mg of phospholipid). The fact that a membrane fraction that separated from synaptic vesicles of T. californica on a controlled-pore glass-bead column and constituted the main potential source of contamination in this preparation had a lower ganglioside content and a different TLC pattern than synaptic vesicles indicated the relatively high purity of the latter. Most of the gangliosides from synaptic vesicles of both species migrated on TLC in the vicinity of standards with three or more sialic acids. Synaptosomes from T. marmorata had a higher lipid N-acetylneuraminic acid/phospholipid ratio and a different TLC pattern than synaptic vesicles. Considering these results and other data appearing recently in the literature, we suggest that reexamination of synaptic vesicles from mammalian brain for the possible presence of gangliosides is warranted.

Animals↗
PubMed1988

A further study of the neuromuscular effects of vesamicol (AH5183) and of its enantiomer specificity.

1. The effects of vesamicol (2-(4-phenylpiperidino) cyclohexanol), an inhibitor of acetylcholine storage, and its two optical isomers have been studied on neuromuscular transmission in rat and frog muscle, and on nerve conduction in frog nerve. 2. Racemic vesamicol produced a pre-block augmentation of twitch tension that also occurred in directly-stimulated muscle. This effect is thus at least partially due to an increase in muscle contractility. 3. (-)-Vesamicol was approximately 20 times more potent than (+)-vesamicol in blocking twitches elicited at 1 Hz. This degree of stereoselectivity is similar to that measured for inhibition of acetylcholine uptake by isolated synaptic vesicles. Both enantiomers were equally weak in reducing nerve action potential amplitude in frog nerve. 4. Further studies with the active isomer, (-)-vesamicol, showed that, like that produced by racemic vesamicol, the neuromuscular block was highly frequency-dependent. The block was not reversed by choline or neostigmine, but was partially reversed by 4- or 3,4-aminopyridine. 5. Preliminary electrophysiological studies showed that vesamicol reduced miniature endplate potential amplitude in rapidly-stimulated frog nerve-muscle preparations. Addition of lanthanum ions increased the frequency of miniature endplate potentials and led to the appearance of apparently normal-sized potentials amongst those of reduced amplitude. 6. The results show the close agreement between pharmacological and biochemical observations indicating the suitability of the rat diaphragm as a test model for substances of this nature. The degree of reversibility of the vesamicol-induced neuromuscular block by aminopyridines was unexpected, and it is suggested that in the presence of a drug which greatly increases release, a pool of acetylcholine is capable of being released which is not normally releasable after block of storage by vesamicol. It is also considered possible that the results from the intracellular recording studies may be explained in these terms.

Anesthetics, Local↗
PubMed1987

Quantitative autoradiography of brain binding sites for the vesicular acetylcholine transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH5183).

2-(4-Phenylpiperidino)cyclohexanol (AH5183) is a noncompetitive and potent inhibitor of high-affinity acetylcholine transport into cholinergic vesicles. It is reported here that [3H]AH5183 binds specifically and saturably to slide-mounted sections of the rat forebrain (Kd = 1.1 to 2.2 X 10(-8) M; Bmax = 286 to 399 fmol/mg of protein). The association and dissociation rate constants for [3H]AH5183 binding are 8.6 X 10(6) M-1 X min-1 and 0.18 min-1, respectively. Bound [3H]AH5183 can be displaced by nonradioactive AH5183 and by the structural analog (2 alpha,3 beta,4A beta,8A alpha)-decahydro-3-(4-phenyl-1-piperidinyl)-2- naphthalenol but not by 10 microM concentrations of the cholinergic drugs acetylcholine, choline, atropine, hexamethonium, eserine, or hemicholinium-3 or by the structurally related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, (+/-)-N-allylnormetazocine (SKF 10,047), levoxadrol, or dexoxadrol. Quantitative autoradiography reveals that [3H]AH5183 binding sites are distributed heterogenously throughout the rat forebrain and are highly localized to cholinergic nerve terminal regions. At the level of the caudate nucleus-putamen, the highest concentrations of saturable [3H]AH5183 binding (713-751 fmol/mg of protein) are found in the vertical limb of the diagonal band and the olfactory tubercle, with lesser amounts (334-516 fmol/mg of protein) in the caudate-putamen, nucleus accumbens, superficial layers of the cerebral cortex, and the primary olfactory cortex. At day 7 after transsection of the left fimbria, [3H]AH5183 binding and choline acetyltransferase activity in the left hippocampus were reduced by 33 +/- 6% and 61 +/- 7%, respectively. These findings indicate that [3H]AH5183 binds to a unique recognition site in rat brain that is topographically associated with cholinergic nerve terminals.

Acetylcholine↗
PubMed1986

Reduction of quantal size by vesamicol (AH5183), an inhibitor of vesicular acetylcholine storage.

Isolated unparalysed mouse phrenic nerve-hemidiaphragm preparations were indirectly stimulated in the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183, vesamicol). Spontaneous miniature endplate potentials were subsequently studied. They exhibited a large depression of amplitude which was more profound at higher stimulation frequencies and drug concentration. No post-junctional effects of the drug were observed. Since the drug blocks storage of acetylcholine by isolated synaptic vesicles, it is argued that the results support the theory of vesicular release of acetylcholine.

Acetylcholine↗
PubMed1986

Demonstration of a receptor in Torpedo synaptic vesicles for the acetylcholine storage blocker L-trans-2-(4-phenyl[3,4-3H]-piperidino) cyclohexanol.

Transport and storage of acetylcholine by purified Torpedo electric organ synaptic vesicles is blocked by the drug L-trans-2-(4-phenylpiperidino)cyclohexanol (AH-5183). This study sought evidence of a specific receptor for the drug. Highly tritiated L-trans-2-(4-phenyl [3,4-3H] piperidino)-cyclohexanol (L-[3H] AH5183) was synthesized. An excess of nonradioactive L-isomer competed with L-[3H]AH5183 for binding to purified Torpedo synaptic vesicles whereas nonradioactive D-isomer did so poorly. Dissociation of bound L-[3H]AH5183 was first order with a rate constant at 23 degrees C of 0.23 +/- 0.03 min-1, and association was too rapid to study. At equilibrium the amount of L-[3H]AH5183 bound at saturation varied in different vesicle preparations, but in one typical preparation specific binding of 181 +/- 15 pmol L-[3H]AH5183 per mg of synaptic vesicle protein was observed with a dissociation constant of 34 +/- 6 nM. Neither acetylcholine nor choline compete effectively with L-[3H]AH5183 for binding. The evidence suggests that about 3.7 +/- 0.3 enantioselective receptors for L-[3H]AH5183 are typically present in each cholinergic synaptic vesicle, and the L-AH5183 binding site does not recognize acetylcholine.

Acetylcholine↗
PubMed1986

Stoichiometries of acetylcholine uptake, release, and drug inhibition in Torpedo synaptic vesicles: heterogeneity in acetylcholine transport and storage.

Highly purified Torpedo electric organ synaptic vesicles form a 49 nM suspension at 1 mg protein/ml. Under active transport conditions hundreds of molecules of [3H]acetylcholine ([3H]ACh) can be accumulated per vesicle, which requires the ACh transporter to undergo multiple turnovers. The transport blocker trans-2-(4-phenylpiperidino)cyclohexanol (AH5183) has no effect on storage of endogenous ACh by vesicles. In contrast, AH5183, other blocking drugs, and nonradioactive ACh caused a rapid release of at least 30-63 molecules of newly transported [3H]ACh per vesicle. Thus AH5183 distinguishes recently transported "new" vesicular ACh from "old" endogenous ACh. l-AH5183 inhibits transport of ACh with a half-inhibitory concentration of 16 +/- 7 nM at 12 nM vesicles and 115 +/- 34 nM at 120 nM vesicles. With the assumption that AH5183 acts on a receptor in an unamplified manner about 2.7 or fewer receptors per vesicle need to be occupied to cause inhibition of ACh transport. The apparent amplification in the number of [3H]ACh molecules per vesicle that are released by AH5183 suggests that AH5183 inhibits ACh storage by an indirect mechanism that distinguishes new from old ACh.

Acetylcholine↗
PubMed1986

Acetylcholine transport and drug inhibition kinetics in Torpedo synaptic vesicles.

Steady-state initial velocity uptake of [3H]acetylcholine ([3H]ACh) by purified Torpedo electric organ synaptic vesicles was studied. Transport specific activity decreased at higher vesicle concentration. Michaelis-Menten type kinetics describe [3H]ACh active transport at constant vesicle concentration with no evidence of cooperativity or transporter heterogeneity. The ACh dissociation constant is about 0.3 mM, transport has a maximal velocity of about 1.6 nmol/min/mg protein, and both are dependent on the vesicle preparation. Nonradioactive ACh was a competitive inhibitor with respect to [3H]ACh. The potent transport inhibitor dl-trans-2-(4-phenylpiperidino)cyclohexanol (AH5183) is a non-competitive inhibitor with respect to [3H]ACh, with an inhibition constant of 41 +/- 7 nM. Inhibition by AH5183 is reversible. The results suggest that AH5183 does not bind to the ACh transporter recognition site on the outside of the vesicle membrane, and thus it might inhibit allosterically.

Acetylcholine↗
PubMed1986

Uncoupling of cholinergic synaptic vesicles by the presynaptic toxin beta-bungarotoxin.

The effect of the presynaptic neurotoxin beta-bungarotoxin (beta-BuTx) on the acetylcholine (ACh) storage system of synaptic vesicles isolated from the electric organ of Torpedo californica was studied. The toxin can totally inhibit active transport of [3H]ACh by the vesicles in a Ca2+-, time-, and concentration-dependent manner. Correlated with these effects is a 50-60% stimulation of the vesicle proton-pumping ATPase activity. The beta-BuTx-mediated transport inhibition and ATPase stimulation are antagonized by delipidated bovine serum albumin, not reversed by excess EGTA, and not mimicked by other cationic proteins or soybean or pancreatic trypsin inhibitors. The behavior is consistent with phospholipase A2 (PLA2)-dependent damage to the vesicle membrane caused by beta-BuTx, which results in uncoupling of the ATPase and ACh transporter systems. The nonneurotoxic Naja naja venom PLA2 causes similar effects, except that it is slightly more potent on a molar basis. About 100-fold more beta-BuTx is required to effect lysis of synaptic vesicles than to uncouple them. ATP is a strong inhibitor of beta-BuTx- but not of N. naja PLA2-mediated uncoupling. The observations suggest that a component of beta-BuTx toxicity in the cholinergic terminal might involve attack on synaptic vesicles or vesicle-like structures and that a nucleotide-like factor might modulate the toxicity.

Acetylcholine↗
PubMed1986

Selectivity and regulation in the phospholipase A2-mediated attack on cholinergic synaptic vesicles by beta-bungarotoxin.

The total fatty acid composition of purified Torpedo californica electric organ synaptic vesicles was determined by GLC analysis of methyl esters. Limit amounts of fatty acids released by high concentrations of either beta-bungarotoxin (beta-BuTx) or Naja naja venom phospholipase A2 (PLA2) acting in deoxycholate are reported. The time and enzyme concentration dependence for beta-BuTx- and PLA2-induced release of fatty acids from intact synaptic vesicles indicate that PLA2 is 100- to 1,000-fold more active. The Ca2+ dependence for beta-BuTx-induced release of fatty acids also was determined. ATP inhibits beta-BuTx- but not PLA2-induced release of fatty acids from vesicles in a manner that can not be ascribed only to chelation of the required Ca2+. ATP, other nucleotides, and adenosine have complex effects on beta-BuTx-induced release of fatty acids from egg yolk phosphatidylcholine dispersed in deoxycholate. The results suggest that beta-BuTx-mediated hydrolysis of the cholinergic synaptic vesicle membrane is approximately 10- to 100-fold more effective at causing uncoupling of vesicles than is PLA2 and that the enzymatic activity of beta-BuTx is subject to regulation by nucleotide-like factors.

Acetylcholine↗
PubMed1984

Ultrastructural localization of nicotinamide adenine dinucleotide phosphatase (NADPase) activity within columnar, goblet, and Paneth cells of rat small intestine.

The distribution of nicotinamide adenine dinucleotide phosphatase (NADPase) activity was examined in epithelial cells of rat small intestine. Segments of ileum were fixed with glutaraldehyde and tissue chopper sections were incubated for up to 4 hr at pH 5.0 in cytochemical media prepared with NADP as substrate. NADPase activity was found primarily within the Golgi saccules of columnar, goblet, and Paneth cells. Columnar and goblet cells showed most of the NADPase activity within the saccules which were intermediate between the cis and trans faces of the Golgi stack. Paneth cells, however, showed the heaviest staining within saccules between the intermediate and innermost saccule at the trans aspect of the Golgi stack. Both columnar cells and Paneth cells also contained spotty, and sometimes heavy, deposits of reaction product within an occasional focal area of the GERL system and within an occasional lysosome. Control experiments indicated that the Golgi-associated NADPase activity was enhanced if cells were pretreated for about 12 hr with EGTA prior to incubation. No similar enhancement was apparent if the tissues were pretreated with DMSO. Furthermore, NADPase activity within the Golgi saccules could be inhibited completely by incubating intestinal epithelial cells with NADP in the presence of sodium fluoride or L(+)-tartrate.

Animals↗
PubMed1983

Pharmacological characterization of the acetylcholine transport system in purified Torpedo electric organ synaptic vesicles.

A wide variety of pharmacologically active compounds was surveyed for effects on active transport of [3H]acetylcholine by synaptic vesicles isolated from the electric organ of Torpedo californica. In over 80 compounds tested, inhibitors of a wide range of potencies were found. The most potent inhibitor was 2-(4-phenylpiperidino)cyclohexanol (AH5183), which half-inhibited transport at 40 nM. This compound had been predicted by Marshall [Br. J. Pharmacol. 38:503-516 (1970)] to block acetylcholine storage by vesicles in vivo. The synaptic vesicle active transport system is shown to be pharmacologically distinct from other cholinergic systems. The site of action of AH5183 and other potent inhibitors is not certain, but the possibility of trivial action on the vesicle ATPase or a vesicle proton gradient was eliminated. The results constitute new evidence supporting vesicle exocytosis as the source of evoked acetylcholine release by nerve terminals. AH5183 appears to be the prototype for a new family of anticholinergics. The possibility that some drugs that exhibit secondary anticholinergic effects act in part by antagonizing acetylcholine storage is discussed.

Acetylcholine↗
PubMed1983

Inhibition of [3H]acetylcholine active transport by tetraphenylborate and other anions.

The effects of tetraphenylborate and other anions on the active uptake of [3H]acetylcholine by synaptic vesicles isolated from Torpedo californica electric organ were studied. Tetraphenylborate completely inhibits active uptake with a half-inhibitory concentration of 0.3 microM. Dipicrylaminate also half-inhibits at 0.3 microM, phenyldicarbaundecaborane at 14 microM, fluoride at 2 mM, thiocyanate at 3 mM, and azide at 16 mM. Tetraphenylborate had no effect on the vesicle ATPase activity or the transmembrane electric potential at low concentrations where it inhibits [3H]acetylcholine active transport. The mechanism for tetraphenylborate inhibition is uncertain, but it might be similar to that of its action as a mitochondrial uncoupler. Solubility products for the acetylcholine, choline, and potassium salts of the tetraphenylborate and dipicrylaminate anions also were measured. The inhibition results confirm the hypothesis of Marshall and Parsons [Br. J. Pharmacol. 54:333-338 (1975)] that tetraphenylborate acts on intact neuromuscular preparations to inhibit transmitter storage, and constitute new pharmacological evidence that evoked release of acetylcholine is mediated by synaptic vesicles.

Acetylcholine↗
PubMed1982

Proton gradient linkage to active uptake of [3H]acetylcholine by Torpedo electric organ synaptic vesicles.

It has been confirmed that cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit adenosine 5'-triphosphate (ATP) dependent active uptake of [3H]acetylcholine. Active uptake can be completely inhibited by low concentrations of the mitochondrial uncouplers carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, nigericin, gramicidin, valinomycin, and A 23187. Under similar conditions uncouplers stimulate the vesicle adenosinetriphosphatase (ATPase) by from 40 to 80%. ATP-supported uptake of [3H]acetylcholine increases greatly as the external pH is increased from 6.6 to 7.6 and remains approximately constant from pH 7.8 to pH 8.6. The uptake also becomes more selective for [3H]acetylcholine compared to [14C]choline as the pH is increased from 6.6 to 7.6, achieving 12-fold selectively, in a manner similar to the increase in the amount of [3H]acetylcholine taken up. Bicarbonate stimulates both the amount and selectivity of [3H]acetylcholine uptake over the lower pH range, but it has no effect over the higher pH range. Exogenous ammonium ion completely inhibits active [3H]acetylcholine uptake, with lower concentrations of ammonium ion required at higher pH values in a manner consistent with ammonia being the active species. Adenosine 5'-diphosphate and a nonhydrolyzable ATP analogue do not support active [3H]acetylcholine uptake. It is concluded that an ATPase pumps protons into the cholinergic synaptic vesicle to produce an internally acidic and positively charged proton gradient that is linked to [3H]acetylcholine uptake.

Acetylcholine↗
PubMed1982

Calmodulin stimulation of 45Ca2+ transport and protein phosphorylation in cholinergic synaptic vesicles.

Cholinergic synaptic vesicles isolated from the electric organ of Torpedo californica exhibit ATP-dependent uptake of 45Ca2+ that is stimulated by exogenous calmodulin. ATP-independent uptake also occurs, but it is only weakly stimulated by calmodulin. Saturating calmodulin decreased the Michaelis constant for ATP-dependent 45Ca2+ uptake from 52 +/- 0.4 to 12 +/- 0.2 microM and increased the maximal velocity from 3.4 +/- 0.3 to 5.2 +/- 0.5 nmol/mg of protein per min. The dose-response curve for calmodulin-dependent stimulation showed a maximal increase of 3.5-fold in the uptake rate; 0.2 microM calmodulin gave half-maximal stimulation. The activity of the vesicle-associated ATPase was unaffected. Incubation of vesicles with [gamma-32P]ATP and Ca2+ resulted in phosphorylation of four polypeptides of molecular weights about 64,000, 58,000, 54,000, and 41,000 when calmodulin was added. Vesicles that were previously phosphorylated and purified exhibited 2-fold enhanced ATP-independent uptake of 45Ca2+. Cyclic AMP could not substitute for calmodulin. The calcium transport system of the cholinergic synaptic vesicle is regulated by a calcicalmodulin-dependent protein kinase that is vesicle-associated.

Animals↗