K–STIMULATED ACETYLCHOLINE RELEASE: INHIBITION BY SEVERAL BARBITURATES AND CHLORAL HYDRATE BUT NOT BY ETHANOL, CHLORDIAZEPOXIDE OR 11-OH-Δ9-TETRAHYDROCANNABINOL

Abstract— Thiamylal, methohexital, secobarbital, amobarbital, pentobarbital, phenobarbital and barbital, and also chloral hydrate inhibited K-stimuiated acetylcholine (ACh) release from rat midbrain slices in a dose related manner. The concentrations required for 50% inhibition were related to the lipid partition coefficient. None of the barbiturates nor chloral hydrate inhibited unstimulated ACh release. Ethanol, chlordiazepoxide and 11-OH-Δ⁹-tetrahydrocannabinol were without effect on both unstimulated and stimulated ACh release. The results are discussed in terms of other effects of these drugs on the cholinergic system.     

Erythrocyte ghost--buffer partition coefficients of phenobarbital, pentobarbital, and thiopental support the pH-partition hypothesis.

The partition coefficient (lambda) between red cell ghosts and buffer has been determined for three barbiturates over a range of pH. Experimental partition coefficients were linearly proportional to the calculated degree of association of the barbiturates. Lambda was 9.5 +/- 0.52 for phenobarbital, 12.7 +/- 0.91 for pentobarbital, and 27 +/- 4.9 for thiopental in their acid forms. Lambda for all three barbiturates in their anionic forms was zero. Our data support the assumption of the pH-partition hypothesis that the dependence of lambda on pH in biological membranes behaves essentially like that in organic solvents. However, the relative magnitudes of the erythrocyte partition coefficients correlate much more closely with the physiological permeability constants than do those of organic solvents, which tend to overestimate the differences between these compounds.     

The effects of barbiturates on the metabolism of phosphatidic acid and phosphatidylinositol in rat brain synaptosomes.

Barbiturates and diphenylhydantoin inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol and phosphatidic acid, but have a relatively slight effect on the incorporation of 32P into these lipids in the absence of carbamoylcholine and no effect on 32P incorporation into phosphatidylcholine and phosphatidylethanolamine. Inhibition of the carbamoylcholine-stimulated increase was observed for pentobarbital, thiopental, phenobarbital, 5-(1,3-dimethylbutyl)-5-ethylbarbiturate, (+)- and (-)-5-ethyl-N-methyl-5-propylbarbituate and diphenylhydantoin. Similar concentrations of barbiturates and diphenylhydantoin were previously reported to inhibit the K+-stimulated Ca2+ influx, and therefore other agents that affect Ca2+ influx were tested to find whether they had any effect on 32P incorporation into these lipids. K+ (35 mM) increases 32P incorporation into phosphatidic acid, but to a smaller degree than 100 micrometer-carbamoylcholine, and its effect was inhibited by pentobarbital. Veratridine (75 micrometer) does not increase 32P incorporation into either phosphatidic acid or phosphatidylinositol, but did inhibit the carbamoylcholine-stimulated increase in 32P incorporation into phosphatidylinositol. The possible relationship between the phospholipid effect and stimulated Ca2+ influx is discussed.     

Effects of barbiturates on human platelet aggregation differ depending on their chemical structures

The effects of barbiturates on human platelet function are not fully understood. Since we have already revealed the effects and mechanisms of thiopental, thiamylal, and pentobarbital in platelets, the present study attempted to elucidate (i) the effects of other barbiturates on human platelet aggregation, (ii) the underlying mechanisms, and (iii) the structure-function relationship of barbiturates in platelets. Barbiturates, including amobarbital, butalbital, secobarbital, barbital, phenobarbital, metharbital, and primidone, were examined. Human platelet aggregation induced by adenosine diphosphate (ADP), epinephrine, and (+)-9,11-epithia-11,12-methano-thromboxane A2 (STA2), a thromboxane A2 analog, was measured using an 8-channel light-transmission aggregometer. The cytosolic free calcium concentration ([Ca2+]i) was measured by fluorometer using fura-2 loaded platelets. Inositol 1,4,5-trisphosphate (IP3) formation induced by STA2 was determined by a commercially available IP3 assay kit. Amobarbital, butalbital, and secobarbital suppressed ADP-, epinephrine- and STA2-induced platelet aggregation and the STA2-induced [Ca2+]i increase, even when Ca2+ influx was blocked by Ni2+. However, they did not affect STA2-induced IP3 formation. Barbital, phenobarbital, metharbital, and primidone (up to 1 mM) had no effect on ADP- and epinephrine-induced platelet aggregation. Thus, we conclude that amobarbital, butalbital, and secobarbital inhibit platelet aggregation by suppressing [Ca2+]i increase without affecting IP3 formation. However, these antiaggregatory effects may not have clinical importance, since the barbiturate concentrations used were higher than clinically relevant ones. The other tested barbiturates had no effects on platelet aggregation. The data indicate that the effects of barbiturates on platelet aggregation differ depending on their chemical structures.     

Development of a general ligand for immunoaffinity partitioning in two phase aqueous polymer systems

The effect on the partition of erythrocytes in a two phase aqueous polymer system based on dextran T500 and polyethylene glycol (PEG) 8000 of a combination of immunoaffinity ligands, namely, rabbit immunoglobulin G (IgG) and PEG 1900-modified monoclonal IgG, was examined as a potential cell separation technique. Several hybridoma lines secreting mouse monoclonal IgG specific for the Fc receptor of rabbit IgG were raised. The monoclonal IgG was modified by cyanuric chloride attachment of PEG 1900, causing the modified antibody to partition predominantly into the PEG-rich upper phase of the systems. The PEG-modified monoclonal IgG was used as an affinity ligand in the two phase polymer system to specifically increase the partition of rabbit anti-NN glycophorin IgG. The rabbit IgG was applied together with the PEG-modified monoclonal IgG to increase the partition of human erythrocytes. The same system had no effect on the partition of rabbit erythrocytes. These experiments demonstrate that a monoclonal antibody can be modified and used as a general reagent with which to alter cell partition in two phase aqueous polymer systems in an immunologically specific manner.     

The effect of previous administration of adrenaline or isoprenaline on the activity of several hypnotics in mice]

Previous administration of adrenaline (0.5 mg/kg i.p.) and isoprenaline (10 mg/kg i.p.) enhances activity of several hypnotic drugs (pentobarbital, barbital, chloral hydrate) in mice but is without effect upon hypnotic activity of ethanol. This potentialisation is blocked by previous administration of pindolol, but not by phentolamine. Administration of SKF 525 A demonstrates that metabolism of pentobarbital is modified by this enzymatic inhibitor, which is not the case for other hypnotics.     

Cerebral Protective Effect and Radical Scavenging Action

Abstract The role of radical scavenging action in cerebral protective effect of drugs was investigated in vitro. Incubation of rat brain mitochondrial suspension with ascorbic acid and Fe²⁺ resulted in the formation of malondialdehyde and a decrease in the turbidity of the suspension, indicating that the mitochondria were peroxidatively disintegrated. Nizofenone at 10 μ m or more inhibited the peroxidative disintegration of mitochondria, and complete inhibition was observed at 100–200 μ m . The action of nizofenone was also ascertained by experiments with rat liver mitochondria. The anti-peroxidative activity of nizofenone was estimated to be approximately equivalent to that of α-tocopherol, and this property was unique. Among the cerebral protective drugs tested, thiopental was only slightly efficient, and pentobarbital, phenobarbital, and dimethyl sulfoxide had no effect. In addition, nizofenone was found to scavenge a stable free radical, diphenyl- p -picrylhydrazyl, but the barbiturates did not. These findings suggest that there is no intimate relationship between cerebral protective effect and free radical scavenging action.     

Effects of anticonvulsant drugs on calcium transport and polyphosphoinositide metabolism in rat cortical synaptosomes

1. Anticonvulsants: phenytoin, phenobarbital, carbamazepine and valium at concentrations of 10-100 microM had a significantly inhibitory effect on both K+-stimulated Ca2+-uptake and 32Pi incorporation into phospholipids of rat cortical synaptosomes. 2. Other anticonvulsant, valproic acid, at concentration upto 100 microM had no effect on these two events. 3. Our results suggest that there is a link between Ca2+-influx and polyphosphoinositide turnover in synaptosomes, and this link may relate to the inhibitory effect of these drugs on neurotransmitter release mechanisms of this preparation.     

Rat Brain Synaptosomes Prepared by Phase Partition

Synaptosomes from rat forebrain can easily be isolated by combining centrifugation with partition in an aqueous two-phase system composed of dextran T500 and polyethylene glycol 4000 in which synaptosomes have an extreme affinity for the upper phase. The fraction thus obtained has been characterized by electron microscopy and biochemical markers for synaptosomes and some other cell components. The contamination by microsomes, free mitochondria, and myelin was 4.4, 3.2, and 0.1%, respectively. The morphometric analysis of the electron micrographs shows that greater than 60% of the structures are synaptosomes. This preparation of the isolation procedure is remarkably short (less than 1 h), formance as assayed by their respiratory activities and ATP level in the absence and presence of depolarizing agents. Synaptosomes prepared by phase partition release the neurotransmitter glutamate in a Ca2(+)-dependent manner. The duration of the isolation procedure is remarkably short (less than 1 h), no ultracentrifuge is required, and the method can be applied for small- or large-scale preparations.     

Differential effect of anionic and cationic drugs on the synaptosome-associated acetylcholinesterase activity of dog brain.

The evoked effects of the negatively charged drugs phenobarbital and barbituric acid, the positively charged imipramine, perphenazine and trifluoperazine, and the neutral primidone, on the synaptosome-associated acetylcholinesterase activity were studied. A marked increase in the enzyme activity was exhibited in the presence of low concentrations (up to 3 mM) of phenobarbital, barbituric acid and primidone. Higher concentrations (up to 10 mM), however, led to a progressive inhibition of the enzyme activity. However, the activity of the enzyme was not affected by imipramine, but it was decreased by perphenazine and trifluoperazine. Arrhenius plots of acetylcholinesterase activity exhibited a break point at 23.4 degrees C for the untreated (control) synaptosomes, which was shifted to around 16 degrees C in the synaptosomes treated with the charged drugs. The allosteric inhibition by F- of acetylcholinesterase was studied in control synaptosomes and in those treated with the charged drugs. Changes in the Hill coefficients in combination with changes in Arrhenius activation energy produced by the charged drugs would be expected if it is assumed that charged drugs 'fluidize' the synaptosomal plasma membranes.     

Correlation of bacterial hyperthermic survival with anaesthetic potency

We have evaluated several local anaesthetics and hypnotics for their relative ability to influence hyperthermic cell killing. Bacterial cell survival following exposure to heat and anaesthetic was used as the assay system. The E. coli bacterium used was the unsaturated fatty acid auxotroph, K1060. It was grown at 37 degrees C in medium supplemented with oleic acid and then exposed to 47 degrees C hyperthermia in the presence of an anaesthetic. The local anaesthetics tested were procaine, lidocaine, tetracaine, and benzocaine, and the general anaesthetics were barbital and pentobarbital. The dose response for each anaesthetic was determined over a five-hour heating period. The anaesthetic concentration required during heating to halve the time for cell killing found with heat alone is 5.9 mM for procaine, 0.8 mM for lidocaine, 0.12 mM for tetracaine, 2.0 mM for benzocaine, 6.7 mM for barbital and 1.2 mM for pentobarbital. There is a direct correlation between equivalent effect doses of the local anaesthetics and published data for the relative potency of the same anaesthetics as determined by respiratory arrest in mice and by myocardial contractile force in dogs. The assay we have described would be a convenient and easy test for the interaction of these drugs with hyperthermia. The use of this interaction with hyperthermia as an adjuvant in combined radiation-hyperthermia therapy should be tested.     

Partition and counter-current distribution of membrane particles in aqueous dextran-poly(ethylene glycol) two-phase systems with special reference to synaptosomes

Aqueous two-phase systems composed of water, dextran and poly(ethylene glycol) can be used for the separation of biological particles. The adjustment of the partition of such particles between the two phases and the interface between them has been studied by using a preparation of synaptosomes (from calf brain cortex) also containing free mitochondria. The partition has been affected by variation of polymer concentrations and addition of salts, e.g. phosphates and chloride. The time for separation of the phases showed a bimodal behaviour with an initially rapid formation of bulk phases followed by a slow phase separation. The relative amount of mixed phases at the time of the transition was proportional to the amount of particles included. Counter-current distribution with moderate time for the phase separation was carried out in such way that the interface material travelled with approximately half the speed of the moving upper phase. In this way the distribution of the particles between the upper phase and the interface as well as between the interface and the lower phase could be studied in the same experiment. The heterogeneity of the synaptosome preparation was clearly demonstrated by counter-current distribution at low polymer concentrations while no separation was obtained when the system contained larger amounts of polymers. Possible reasons for this behaviour are discussed.     

Charged anaesthetics alter LM-fibroblast plasma-membrane enzymes by selective fluidization of inner or outer membrane leaflets.

The functional consequences of the differences in lipid composition and structure between the two leaflets of the plasma membrane were investigated. Fluorescence of 1,6-diphenylhexa-1,3,5-triene(DPH), quenching, and differential polarized phase fluorimetry demonstrated selective fluidization by local anaesthetics of individual leaflets in isolated LM-cell plasma membranes. As measured by decreased limiting anisotropy of DPH fluorescence, cationic (prilocaine) and anionic (phenobarbital and pentobarbital) amphipaths preferentially fluidized the cytofacial and exofacial leaflets respectively. Unlike prilocaine, procaine, also a cation, fluidized both leaflets of these membranes equally. Pentobarbital stimulated 5'-nucleotidase between 0.1 and 5 mM and inhibited at higher concentrations, whereas phenobarbital only inhibited, at higher concentrations. Cationic drugs were ineffective. Two maxima of (Na+ + K+)-ATPase activation were obtained with both anionic drugs. Only one activation maximum was obtained with both cationic drugs. The maximum in activity below 1 mM for all four drugs clustered about a single limiting anisotropy value in the cytofacial leaflet, whereas there was no correlation between activity and limiting anisotropy in the exofacial leaflets. Therefore, although phenobarbital and pentobarbital below 1 mM fluidized the exofacial leaflet more than the cytofacial leaflet, the smaller fluidization in the cytofacial leaflet was functionally significant for (Na+ + K+)-ATPase. Mg2+-ATPase was stimulated at 1 mM-phenobarbital, unaffected by pentobarbital and slightly stimulated by both cationic drugs at concentrations fluidizing both leaflets. Thus the activity of (Na+ + K+)-ATPase was highly sensitive to selective fluidization of the leaflet containing its active site, whereas the other enzymes examined were little affected by fluidization of either leaflet.     

Ethanol inhibits veratridine-stimulated sodium uptake in synaptosomes

The effects of ethanol and pentobarbital on voltage-sensitive sodium channels in whole brain (rat) synaptosomes were studied using isotopic flux measurements. Incubation of synaptosomes with ethanol or pentobarbital $\text{in}$$\text{vitro}$ inhibited veratridine-stimulated ²²Na⁺ uptake. The effect of ethanol is dose-dependent, occurs at sublethal, pharmacologically relevant concentrations and is fully reversible. These results suggest that ethanol and pentobarbital directly interfere with sodium channel function in nervous tissue. Alterations in sodium channel function may be a possible mechanism for the central nervous system (CNS) depressant action of ethanol and related compounds.     

Polyol-pathway enzymes of human brain. Partial purification and properties of aldose reductase and hexonate dehydrogenase.

Aldose reductase and hexonate dehydrogenase were isolated from human brain and partially purified. The two enzymes exhibited distinctive substrate-specificity profiles with a variety of aldoses,and aliphatic and aromatic aldehydes. Aldose reductase exhibited a high affinity for DL-glyceraldehyde (Km of 62 microM) and a low affinity (Km of 90 mM) for glucose, the physiological substrate of the polyol pathway. Hexonate dehydrogenase exhibited a relatively low affinity for D-glucuronate (Km of 4.6 mM) and a very low affinity for glucose (Km of 390 mM). Both enzymes exhibited a high specificity for NADPH, and both were inhibited competitively by NADP+. Hexonate dehydrogenase was inhibited by iodoacetate, iodoacetamide, N-ethylmaleimide and p-chloromercuribenzoate. Preincubation with 2-mercaptoethanol resulted in activation. Both enzymes were inhibited by a number of barbiturates (barbital, phenobarbital and pentobarbital) and by the central-nervous-system drugs diphenylhydantoin and ethosuccinimide. The substrate specificity and pattern of inhibition suggest that the two enzymes isolated correspond to two of four previously reported aldehyde reductases isolated from human brain.     

Inhibition by local anesthetics and barbiturates of the active transport of H+ in the synaptic vesicle membranes of the brain

The effects of local anesthetics and barbiturates on the ATP-dependent H+ transport in synaptic vesicle membranes from rat brain were studied using a fluorescent probe, acridine orange. Local anesthetics depressed the active H+ transport with the following order of potencies: tetracaine trimecaine lidocaine procaine. Respective IC50 values were 0.07, 0.28, 0.46 and 0.60 mM. The local anesthetics also disrupted the endogenous pH gradient seen in the absence of ATP. Barbiturates inhibited the active H+ transport showing IC50 values in the range of 2-5 mM except for benzobarbital and barbital characterized by IC50 values of 0.5 and 20 mM, respectively. The order of potencies was benzobarbital hexobarbital amobarbital pentobarbital phenobarbital barbital. The endogenous pH gradient was not affected by the barbiturates. The results show that local anesthetics disrupt the H+ transport by acting as permeable weak bases (uncouplers) whereas barbiturates are likely to block and anion channel which maintains electroneutrality of the H+ transport in the membrane of synaptic vesicles.     

Highly purified mitochondria from rat brain prepared by phase partition

Mitochondria and synaptosomes from adult rat forebrain can easily be separated by counter-current distribution in an aqueous two phase system composed of Dextran T500 and poly(ethylene glycol) 4000. Both particles may also be separated by a batch procedure in which the same phase system is used. Electron micrographs and enzymatic activities show a high purity of the mitochondria obtained from the dextran-rich lower phase. Electron micrographs and enzymatic activities also show that intact synaptosomes can be obtained from the poly (ethylene glycol)-rich upper phase.The mitochondria purified by this method show good ADP/O ratios, respiratory control ratios, and state 3 rates. Synaptosomes showed a state 2-state 3 transition with no recuperation to state 4.     

Barbiturates inhibit intracellular Ca2+ rise induced by thrombin in rat platelets

The effects of a number of barbiturates (anesthetic as well as anticonvulsant) on thrombin-induced calcium mobilization were tested in rat platelets using the fluorescent Ca2+ probe Fura-2. All drugs, except barbituric acid and Na-barbital, inhibited the thrombin-induced intracellular Ca2+ rise. Both the uptake of extracellular Ca2+ and the release of calcium from intracellular organelles were affected but influx was inhibited more strongly and at lower concentrations of the drugs (e.g. IC50 of thiopental was 0.83 mM for influx and 1.2 mM for intracellular release). Inhibitory potencies of the various barbiturates were markedly different. Thiopental was the most and barbital the least potent inhibitor. The order of inhibitory potency of the drugs appeared generally to follow their lipid solubility and the order of their hypnotic efficiency, with hexobarbital as the most conspicuous exception. Therefore, barbiturate treatment of cells perturbs agonist-induced calcium mobilization. This effect may be partially linked to their previously reported inhibitory action on two kinases, protein kinase C and phosphatidylinositol 4-phosphate kinase [1, 2].     

The selective effects of charged local anaesthetics on the glucagon- and fluoride-stimulated adenylate cyclase activity of rat-liver plasma membranes

The cationic local anaesthetics carbocaine and unpercaine were found to increase the fluoride-stimulated adenylate cyclase up to a maximum level; above this maximum level further increases in drug concentration inhibited the enzyme. At concentrations where this activity was stimulated, a fatty acid spin label detected an increase in bilayer fluidity, which, it is suggested, is responsible for the activation of the enzyme. A solubilized enzyme was unaffected by the drugs, a finding consistent with this proposal. These cationic drugs began to inhibit the glucagon-stimulated activity at concentrations where they activated the fluoride-stimulated activity. It is suggested that this is due to their effect on the coupling interaction between the receptor and catalytic unit. The anionic drugs, phenobarbital, pentobarbital, and salicylic acid, all inhibited the fluoride-stimulated enzyme. This may be due in part to a direct effect on the protein and in part to the interaction of the drugs with the bilayer. The drugs had small inhibitory effects on the lubrol-solubilized enzyme. The glucagon-stimulated enzyme was initially inhibited by the anionic drugs at low concentrations, then activated, and finally inhibited with increasing drug concentration. The reasons for such changes are complex, but there was no evidence from electron spin resonance studies to suggest that the elevations in activity were due to increases in bilayer fluidity.     

Neural mechanisms affecting pressor responses to angiotensins in the Pekin duck, Anas platyrhynchos

1. Administration of SQ20881 diminished pressor responses to intravenous (i.v.) injections of angiotensin I (AI), but not those to injections of angiotensin II (AII), in Pekin ducks, indicating the occurrence of a mechanism similar to the mammalian angiotensin-converting enzyme reaction. 2. Pressor responses to AII were enhanced by general anaesthesia with phenobarbital, pentobarbital, or a combination of both phenobarbital and pentobarbital. 3. Ganglionic blockade with mecamylamine enhanced the pressor responses to AII and NE, but not those to tyramine, in anaesthetized ducks. 4. It is proposed that the potentiating effects of general anaesthesia and ganglionic blockade on pressor responses were due both to a lowering of baseline blood pressure (BP) and an inhibition of the neural reflexes which normally buffer BP. Furthermore, it is suggested that the augmented responsiveness of anaesthetized, ganglion-blocked ducks shows that the pressor effect of AII is predominantly of peripheral, rather than central nervous system (CNS), origin.