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PubMed1995

Common cannabimimetic pharmacophoric requirements between aminoalkyl indoles and classical cannabinoids.

Aminoalkylindoles (AAIs) are structurally dissimilar from the classical cannabinoids (CCs), however, both AAIs and CCs appear to bind at the same site on the cannabinoid receptor. To obtain better insights on the structural correlation between AAIs and CCs, we have studied the conformational properties of the potent cannabimimetic AAI WIN 55212-2 and its inactive analogs using high resolution 2D NMR spectroscopy in combination with computer-assisted molecular modeling. The pharmacophoric similarities between the AAIs and the CCs were then investigated using superimposition techniques. The absolute stereochemistries of the biologically active enantiomer (-)HHC were used as superimposition points and considered as internal controls in order to test the molecular principles guiding this experiment. Our results show that the model is congruent with a superimposition in which the naphthoyl, morpholino and 3-keto groups in the AAI, respectively correspond to the side chain, cyclohexanol OH and phenolic OH of HHC. A good fit is obtained when the two biologically active antipodes are superimposed. Conversely, the fit is poor if the inactive AAI enantiomer is superimposed on the active HHC enantiomer. It can also be seen that in such an orientation a certain deviation of the C-ring from the plane of the phenol ring of the tricyclic HHC component and of the morpholinyl portion from the plane of the indole ring of WIN 55212-2 is essential for cannabimimetic activity. The inactive enantiomer WIN 55212-3 has its respective components aligned in the opposite quadrant. By comparing the stereoelectronic features of representative AAIs and CCs, we have developed a model which may help to uncover the pharmacophoric requirements of the AAIs and serve as a basis for future SAR and drug design.

Benzoxazines↗

PubMed1988

Kinetic analyses of transmitter release in neuromuscular transmission.

A majority of transmitter quanta in the nerve terminal is in a large pool of store (S), which can be utilized for release only after transformation into activated quanta (n) via two intermediate states called available quanta (A) and releasable quanta (N). Mobilization is a collective term applicable for aggregates of S----A, A----N and N----n conversions. In the present article, various electrophysiological procedures for kinetic analyses of the transmitter release in neuromuscular transmission were discussed to elucidate this not well understood process of mobilization. Especially, frequency augmentation, tetanic rundown and depression, and recovery from depression experiments were proposed to be very useful tools in identifying the drug effect on the mobilization process. Since d-tubocurarine, 2-(4-phenylpiperidino)cyclohexanol (AH5183) and Ba ions satisfied the above three criteria of drug action on mobilization, these were concluded to affect the mobilization of transmitter in neuromuscular transmission.

Animals↗

PubMed1994

Screening for agents inhibiting the mutagenicity of extracts and constituents of tobacco products.

The aim of this study was to screen for potential agents affecting the mutagenicity of tobacco products. The influence of a number of compounds which have been suggested to be antimutagenic some of which are present in tobacco products, was investigated on the mutagenicity of a cigarette smoke condensate (CSC) and, in some cases, an extract of oral Swedish moist snuff (SNUS), using a screening procedure of the Ames Salmonella/microsome assay (STY). For some of the compounds the V79/hprt mutagenicity assay with benzo[a]pyrene metabolites as mutagens was used to obtain complementary and confirmatory information on mammalian cells. The antimutagens used included two selenium compounds, sodium selenite and ebselen; the flavonoids and polyphenols, ellagic acid, (+)-catechin hydrate, scopoletin, chlorogenic acid and rutin trihydrate; the porphyrin derivatives, bovine hemin, biliverdine dihydrochloride, chlorophyllin and a plant extract containing chlorophyll; the terpenoids, beta-carotene, retinol and a mixture of the two epimers (4R) and (4S) of (1S,2E,6R,7E,11E)-cembra-2,7,11-triene-4,6-diols (CBD); and cyclohexanol and ubiquinone. Screening of antimutagenic activities using the STY involves problems with toxicity. In several cases in this study mutagenicity was decreased below the control level without signs of toxicity in the background growth of bacteria. Since the survival of mutants and slight bacteriostatic effects on the background growth cannot be determined accurately in the STY, a reduction in mutagenicity may simply be due to toxicity. Only in cases where a dose-response curve declines to a level at or above the background and then levels off, can toxicity be excluded. An antimutagenic effect determined using this test system is therefore often not sufficient for classifying a compound as antimutagenic until these findings are confirmed in other test systems and, preferably, the mechanism behind this effect is clarified. The results obtained with the selenium compounds were considered to be inconclusive since the reduction in the mutation rate declined below the background level and might only reflect the toxic effects of these compounds. For ellagic acid an almost complete inhibition of the mutagenicity of CSC and SNUS in STY was indicated. This indication of antimutagenicity was confirmed in V79 cells using two metabolites of the CSC constituent benzo[a]pyrene, i.e., trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and (+)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE). Chlorogenic acid and (+)--catechin reduced the mutagenicity of CSC and chlorogenic acid also strongly inhibited SNUS mutagenicity. Scopoletin and rutin trihydrate inhibited the mutagenicity of CSC, but showed confounding effects with SNUS.(ABSTRACT TRUNCATED AT 400 WORDS)

Antimutagenic Agents↗

PubMed1990

[3H]vesamicol binding in human brain cholinergic deficiency disorders.

We measured the binding of the vesicular acetylcholine transport blocker [3H]vesamicol (2-[4-phenylpiperidino] cyclohexanol; AH-5183) to autopsied frontal cortex and amygdala of patients from 4 disorders having a marked brain cholinergic reduction, namely Alzheimer's disease, Parkinson's disease with dementia, dominantly inherited olivopontocerebellar atrophy and Down's syndrome. Although mean activity of the specific cholinergic marker enzyme choline acetyltransferase (ChAT) was markedly reduced by about 60% in frontal cortex in the 4 patient groups and by 80% or greater in amygdala of the Alzheimer's and Down's syndrome patients, [3H]vesamicol binding density was, on average, either normal or only slightly reduced as compared with the controls. This discrepancy suggests that in human brain [3H]vesamicol binding is either not preferentially localized to cholinergic nerve endings or, in these cholinergic deficiency syndromes, a substantial proportion of the vesamicol binding sites persist on cholinergic nerve terminals despite loss of ChAT activity.

Acetylcholine↗

PubMed1992

Routes of acetylcholine leakage from cytosolic and vesicular compartments of rat motor nerve terminals.

Acetylcholine efflux at the rat neuromuscular junction was assayed following blockage of ACh transport into synaptic vesicles by 2-(4-phenylpiperidino) cyclohexanol (AH5183). [2H4]Choline was used as a labeled precursor. AH5183 completely blocked ACh efflux from the cytosolic compartment but had comparatively less effect on release from the unlabeled vesicular pool. Tissue [2H4]ACh levels increased after AH5183 addition due to cytosolic ACh retention. Thus, ACh in the non-vesicular pool (calculated to be 34% of the total ACh) may efflux solely via the AH5183-sensitive ACh transporter inserted into the terminal membrane. ACh released from the vesicular fraction was about 100-fold more than could be accounted for by miniature end-plate potentials; possible causes of this overestimate are discussed.

Acetylcholine↗

PubMed1992

Acetylcholine synthesis is modulated by acetylcholine content of cytosolic fraction but not by that of releasable fraction.

Synthesis and release of acetylcholine (ACh) in the rat hippocampal slices were examined to clarify the mechanism of modulation of ACh synthesis. Treatment with 2-(4-phenylpiperidino)cyclohexanol (AH5183, 50 microM), an inhibitor of ACh transport from cytosol to synaptic vesicles, inhibited the increase in ACh content of the membrane-bound fraction which is readily releasable, but did not affect the cytosolic ACh content. Under these conditions, the total ACh content reached a plateau value. These results indicate that ACh synthesis is modulated by cytosolic ACh content but not by the vesicular fraction.

Acetylcholine↗

PubMed1976

Ligand-induced structural changes in amylose partially complexed with iodine.

The influence of complexing agents such as methanol, ethanol, 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, cyclohexanol and 2-octanol on the formation of a blue coloured amylose - iodine complex (pH 4.8), under suboptimum concentrations of iodine and in the absence of potassium iodide, is studied by recording the absorbance at 640 nm. A drop in absorbance at 640 nm accompanied by a blue shift in the spectrum (640-580 nm) was observed at higher concentrations of the complexing agents. This behaviour of amylose partially complexed with iodine appears to be due to ligand-induced structural changes in the amylose chain. The fall in absorbance at 640 nm observed when the temperature of amylose - iodine complex in the presence of complexing agents is raised, and the subsequent regeneration of the absorbance on cooling, indicates the possible helix to random coil transition of the amylose chain in an aqueous system.

Alcohols↗

PubMed1982

Purification and characterization of a nicotinamide adenine dinucleotide-dependent secondary alcohol dehydrogenase from Candida boidinii.

From the yeast Candida boidinii grown on glucose a new secondary alcohol dehydrogenase was purified 426-fold by heat treatment, column chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose Cl-6b, and gel filtration on Sephacryl S-300. The purified enzyme was homogeneous as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was found to be 150000 by sedimentation equilibrium as well as by gel filtration. The enzyme appears to be composed of four identical subunits (Mr=38000) as determined by SDS-gel electrophoresis. The enzyme catalyzes the oxidation of isopropanol to acetone in the presence of NAD+ as an electron acceptor. The Km values were found to be 0.099 mM for isopropanol and 0.14 mM for NAD+. Besides isopropanol also other secondary alcohols like butan-2-ol, pentan-2-ol, pentan-3-ol, hexan-2-ol, cyclobutanol, cyclopentanol, and cyclohexanol served as a substrate and were oxidized to the corresponding ketones. Isopropanol seems to be the best substrate for this enzyme which we therefore call isopropanol dehydrogenase. Primary alcohols are not oxidized by the enzyme. The optimum pH for enzymatic activity in the oxidation reaction was found to be 9.0, the optimal temperature is 45 degrees C. The isoelectric point of the isopropanol dehydrogenase was found to be pH 4.9. The enzyme is inactivated by mercaptide-forming reagents and chelating agents, 2-mercaptoethanol is an inhibitor. Zinc ions appear necessary for enzyme production.

Alcohol Oxidoreductases↗

PubMed1992

The pharmacology of vesamicol: an inhibitor of the vesicular acetylcholine transporter.

1. Vesamicol (2-[4-phenylpiperidino] cyclohexanol) inhibits the transport of acetylcholine into synaptic vesicles in cholinergic nerve terminals. 2. Recent pharmacological studies of the effects of vesamicol on skeletal neuromuscular transmission have revealed a pattern of activity for the compound consistent with the neurochemical observation of the mechanism of action of the compound. 3. Pharmacological manipulation of vesicular acetylcholine transport has been used to investigate the recycling and mobilization of synaptic vesicles within cholinergic nerve terminals. 4. In addition to its effects on vesicular acetylcholine transport, vesamicol also possesses some sodium channel and alpha-adrenoceptor blocking activity. 5. Vesamicol clearly represents a unique tool for investigating presynaptic mechanisms in cholinergic nerve terminals.

Acetylcholine↗

PubMed1994

Effects of vesicular acetylcholine uptake blockers on frequency augmentation-potentiation in frog neuromuscular transmission.

Vesamicol inhibits the vesicular loading of acetylcholine molecules. The effects of vesamicol and similarly acting compounds on neuromuscular transmission in frogs were investigated to determine whether these inhibitors-inhibit the frequency augmentation-potentiation of transmitter release. Various vesicular acetylcholine transport blockers suppressed the stimulation frequency-related release parameter, k, in a dose-dependent manner. Artane, cetiedil, chloroquine, ethodin, quinacrine, vesamicol and its benzyl-analogue, 2-(4-benzylpiperidino)cyclohexanol, had strong effects, while those of aminacrine, chlorpromazine, fluphenazine, imipramine, pyrilamine and thioridazine were weak. A significant correlation was observed between the biochemically reported values of IC50 and the electrophysiological inhibitory potencies on k at 20 microM. Contrary to expectations from the biochemical data, however, vesamicol and its benzyl-analogue showed equipotent inhibitory actions on the electrophysiological frequency augmentation-potentiation relation. Low sensitivity and low selectivity of the frequency augmentation-potentiation for vesamicol and its benzyl-analogue lead us to conclude that the vesicular acetylcholine transporter is not the site of the electrophysiological action of vesamicol and similarly acting chemicals.

Acetylcholine↗

PubMed1993

Stereoselective determination of the active metabolites of a new anti-inflammatory agent (CS-670) in human and rat plasma using antibody-mediated extraction and high-performance liquid chromatography.

The main metabolites of (+-)-2-[4-(2-oxocyclohexylidenemethyl)phenyl]propionic acid (CS-670), a new pro-drug anti-inflammatory agent of the 2-arylpropionic acid type, have one or two chiral centres arising from reduction of the oxocyclohexylidene moiety in addition to an original chiral centre in the propionic acid moiety. To determine these metabolites stereoselectively, antibody-mediated extraction was investigated as a stereoselective clean-up method prior to chiral HPLC. Immunoglobulin G, which recognizes each stereoisomeric cyclohexanol moiety, was coupled to cyanogen bromide-activated Sepharose 4B to prepare re-usable immobilized antibody, and its specificity was improved by examination of a washing process after charging of samples. Plasma extracted with the immobilized antibody column was derivatized with a chiral reagent to separate the enantiomers of the propionic acid moiety by HPLC. This newly developed analytical method clarified the stereoselective biotransformation of the pro-drug to pharmacologically active forms in humans and rats related to reduction of the oxocyclohexylidene moiety and chiral inversion in the propionic acid moiety.

Animals↗

PubMedDate not supplied

Role of alcohol P-450-oxygenase (APO) in microsomal ethanol oxidation.

The form of liver microsomal cytochrome P-450 induced by chronic administration of ethanol to rabbits, designated as P-450ALC or P-450 isozyme 3a, has been purified to homogeneity as judged by several criteria, including NH2- and COOH-terminal amino acid sequence determination. The reconstituted alcohol-P-450 oxygenase (APO) system containing P-450ALC and NADPH-cytochrome P-450 reductase catalyzes the oxidation of a variety of primary and secondary alcohols to aldehydes and ketones, including methanol, ethanol, n-propanol, n-butanol, 2-butanol, n-pentanol, and cyclohexanol. Other purified P-450 cytochromes, including isozymes 2, 3b, 3c, 4, and 6, are much less active than P-450ALC in the oxidation of alcohols. That P-450ALC functions in ethanol oxidation in liver microsomal membranes as well as in the reconstituted system was shown by immunochemical experiments involving inhibition by sheep anti-P-450ALC antibodies. We conclude that P-450ALC is the predominant ethanol-oxidizing cytochrome present after induction by chronic alcohol administration and that the other P-450 cytochromes have low but significant activity in both control and ethanol-induced animals.

Alcohols↗

PubMed1991

Functionalization of hydroxy compounds with nitrilotriacetic acid for technetium-99m chelation: excretory properties of the radiolabelled chelates.

Substituted monoanilides of nitrilotriacetic acid (NTA) have gained much popularity in recent years as an important class of ligands for technetium-99m (99mTc) radiopharmaceutical preparations used in liver imaging and function studies. We were interested in investigating the properties of the corresponding ester analogues of this important class of ligands and for this study cyclohexanol was selected as a hydroxy component, which on condensation with nitrilotriacetic acid in the presence of acetic anhydride, furnished the monoester, N-cyclohexyloxycarbonylmethyl iminodiacetic acid 4 and the corresponding diester 5. Phenol on similar condensation produced mainly the diester, N,N-di(phenyloxycarbonylmethyl) aminoacetic acid 2, with traces of the corresponding monoester 7. A reinvestigation of the well known condensation reaction of aniline with nitrilotriacetic acid revealed that in addition to the reported monoanilide, N-phenylcarbamoylmethyl imino diacetic acid 3, the corresponding dianilide 6 was also produced in appreciable amount. The ester ligands 2, 4, 5 after 99mTc chelation exhibited good in vitro and in vivo stabilities. The biodistribution characteristics of these radiolabelled esters and amides were very similar showing thereby that esterification with NTA could be an effective method for converting alcohols to 99mTc-radiopharmaceuticals without generating any unusual properties because of the ester linkage. Residual radiopharmaceutical concentration after i.v. administration of these amide and ester 99mTc chelates at 30 min in blood, urine, liver, kidney and intestine were correlated with their lipophilicities and during this correlation it was observed that in addition to lipophilicity the anionic strength of these chelates is also an important determinant in governing their biodistribution. The ester ligand 4 after 99mTc chelation showed ultrafast hepatobiliary kinetics and was therefore compared in a rabbit model with a standard hepatobiliary radiopharmaceutical 99mTc-N-(p-butylphenylcarbamoyl methyl) iminodiacetic acid (99mTc-BIDA) by gamma-camera scintigraphy to investigate the potential of the former for clinical studies.

Animals↗

PubMed1994

Capillary electrophoresis with chiral selectors: optimization of separation and determination of thermodynamic parameters for binding of tioconazole enantiomers to cyclodextrins.

A systematic approach is outlined for optimization of enantiomeric separations in free solution capillary electrophoresis using chiral mobile-phase additives. Maximum electrophoretic mobility difference between the enantiomers occurs when the concentration of free selector is equal to the reciprocal of the average binding constant. General equations and data analysis methods are presented to relate mobilities to equilibrium constants in simple and competitive binding equilibria and used to determine thermodynamic parameters for host-guest complexation of tioconazole enantiomers with a range of cyclodextrin selectors. Selectivities are found to be in the reverse order of binding constants in the series dimethyl-beta-cyclodextrin (K1 = 6.9 x 10(3) M-1, alpha = 1.10) to hydroxypropyl-beta-cyclodextrin (K1 = 0.72 x 10(3) M-1, alpha = 1.29). For beta-cyclodextrin (K1 = 1.32 x 10(3)M-1, alpha = 1.20), delta H zero provides the dominant contribution to binding but delta delta H zero and T delta delta S zero terms give comparable contributions to the selectivity. Addition of alcohol does not affect the selectivity, but allows displacement of the optimum separation conditions to higher cyclodextrin concentration through either competitive binding (with cyclohexanol) or preferential solvation of reactants (with methanol).

Antifungal Agents↗

PubMed1991

Characterization of the R-state insulin hexamer and its derivatives. The hexamer is stabilized by heterotropic ligand binding interactions.

1H NMR and UV-visible electronic absorption studies have been performed to investigate the effects of anions and cyclic organic molecules on the interconversion of the T- and R-conformational states (Kaarsholm et al., 1989) of hexameric M (II)-substituted insulin in solution (M = Zn or Co.). Two ligand binding processes that stabilize the R-state conformation of the M(II)-substituted insulin hexamer [M(II)-R6] have been distinguished: (i) The binding of neutral organic molecules to the six, crystallographically identified, protein pockets in the Zn(II)-R6 insulin hexamer (Derewenda et al. 1989) generate homotropic site-site interactions that stabilize the R-state. Cyclohexanol, phenol, 4-nitrophenol, and 4-hydroxymethylbenzoate are shown to bind at these sites. (ii) The coordination of singly charged anions that are able to gain access to the two HisB10 coordinated metal ions of the M(II)-R6 hexamer stabilizes the R-state. Adducts of the M(II)-R6 hexamer are formed, thereby, in which the solvent-accessible fourth coordination position of the M(II) ion is replaced by a competing anion. Binding to these two classes of sites introduces strong heterotropic interactions that stabilize the R-state. UV-visible spectral data and apparent affinity constants for the adducts formed by the Co(II)-R6 hexamer with a wide range of anionic ligands are presented. The Co(II)-R6 adducts have a strong preference for the formation of pseudotetrahedral Co(II) centers. The HCO3- and pyridine-2-thiolate ions form Co(II)-R6 adducts that are proposed to possess pentacoordinate Co(II) geometries. The relevance of the Co(II)-R6 complexes to carbonic anhydrase catalysis and zinc enzyme model systems is discussed.

Biopolymers↗

PubMed1983

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes.

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.

Alcohol Dehydrogenase↗

PubMed1988

Sidedness and chemical and kinetic properties of the vesamicol receptor of cholinergic synaptic vesicles.

Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which when occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of [3H]vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N'-dicyclo-hexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles is resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. [3H]Vesamicol binding depends on deprotonation of a group of pKa1 = 6.26 +/- 0.03 and protonation of a group of pKa2 = 10.60 +/- 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analogue dl-trans-4-oxo-4-[5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl )-1- naphthalenyl]amino]butanoic acid (TPNB) is an effective inhibitor of AcCh active transport with an IC50 value of (51 +/- 8) x 10(-9) M. At 23 degrees C, [3H]vesamicol bound to the receptor at a rate of (1.74 +/- 0.06) x 10(5) M-1 s-1, and excess unlabeled vesamicol displaced a low concentration of bound [3H]vesamicol at 0.29 +/- 0.01 min-1. At 0 degrees C, 10 microM unlabeled vesamicol displaced 36 +/- 2% of a low concentration of bound [3H]vesamicol at 0.16 +/- 0.02 min-1 and 64 +/- 2% at 0.013 +/- 0.001 min-1. One micromolar unlabeled vesamicol behaved similarly.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗

PubMed1988

Cholinergic synaptic vesicle heterogeneity: evidence for regulation of acetylcholine transport.

Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 +/- 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 +/- 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗