Inhibition of synaptosomal enzymes by local anesthetics

The effects of tertiary amine local anesthetics (procaine, lidocaine, tetracaine and dibucaine) and chlorpromazine were investigated for three enzyme activities associated with rat brain synaptosomal membranes, i.e., (Na⁺ + K⁺)-ATPase (ouabain-sensitive), Mg²⁺-ATPase (ouabain-insensitive) and acetylcholinesterase. Approximately the same concentrations of each agent gave 50% inhibition of both ATPase, for example 7.9 and 10 mM tetracaine for Mg²⁺-ATPase and (Na⁺ + K⁺)-ATPase, respectively; these concentrations are 10-fold higher than required for inhibition of mitochondrial F₁-ATPase. The relative inhibitory potency of the several agents was proportional to their octanol/water partition coefficients. Acetylcholinesterase was inhibited by all agents tested, but the ester anesthetics (procaine and tetracaine) were considerably more potent than the others after correction for partition coefficient differences. For tetracaine, 0.18 mM gave 50% inhibition and showed competitive inhibition on a Lineweaver-Burk plot, but for dibucaine a mixed type of inhibition was observed, and 0.63 mM was required for 50% inhibition. Tetracaine evidently binds at the active site, and dibucaine at the peripheral or modulator site, on this enzyme.     

Vinculin phosphorylation by the src kinase: inhibition by chlorpromazine, imipramine and local anesthetics

The phosphorylation of vinculin by a highly purified $\text{src}$ kinase was stimulated by anionic phospholipids and inhibited to varying degrees, by chlorpromazine, imipramine, dibucaine and tetracaine. The drug effects are ascribed to a competitive inhibition of the activation process by their ability to interact with phospholipid.     

Inactivation and the site of labeling of F1-ATPase from Mycobacterium phlei by dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and quinacrine mustard

The F₁-ATPase from Mycobacterium phlei is inactivated by dicyclohexylcarbodiimide (DCCD), 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and quinacrine mustard. The inactivation is both time-and concentration-dependent and in the case of DCCD being more pronounced at acidic pH. The minimum inactivation half-time ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) for DCCD, NBD-Cl and quinacrine mustard was observed to be 14, 6 and 7 min, respectively. Inactivation of F₁-ATPase resulted in the incorporation of [¹⁴C]DCCD as well as [¹⁴C]NBD-Cl into α and γ subunits. The incorporation of label into α and γ subunits, utilizing [¹⁴C]NBD-Cl, was reversible by dithiothreitol. Complete inactivation, by linear extrapolation to zero activity, revealed that 4 mol [¹⁴C]DCCD and 4 mol [¹⁴C]NBD-Cl bind per mol F₁-ATPase. Kinetic and binding studies show that these probes bind to site(s) distinct from ATP-binding site in F₁-ATPase from M. phlei.     

Studies on the mechanism of action of local anesthetics on proton translocating ATPase from Mycobacterium phlei

We have measured the inhibitory potencies of local anesthetics (procaine, lidocaine, tetracaine and dibucaine) on ATP-mediated H+-translocation, Ca2+-transport and ATPase activity in membrane vesicles from Mycobacterium phlei. Procaine and lidocaine up to 1 mM concentration did not inhibit ATP-dependent H+-translocation, Ca2+-transport and ATPase activity. However, tetracaine and dibucaine at 0.2 mM concentration caused dissipation of the proton gradient, measured by the reversal of the quenching of fluorescence of quinacrine, and inhibition of active Ca2+-transport. Tetracaine (1 mM) inhibited membrane-bound ATPase activity without affecting solubilized F1-ATPase activity. Studies show that these local anesthetics do not prevent the inactivation of F0-F1 ATPase by dicyclohexylcarbodiimide (DCCD). Binding of [14C]DCCD to F0-proteolipid component remained unchanged in the presence of tetracaine indicating that DCCD and tetracaine do not share common binding sites on the F0-proteolipid sector. The inhibition of H+-translocation and membrane-bound ATPase activity by tetracaine was substantially additive in the presence of vanadate.     

Regulation of prostaglandin metabolism: activation of 15-hydroxyprostaglandin dehydrogenase by chlorpromazine and imipramine related drugs.

Chlorpromazine and imipramine related drugs were found to activate swine renal 15-hydroxprostaglandin dehydrogenase maximally about 2 fold. Activation by these drugs was uncompetitive with respect to both prostaglandin E₁ and NAD+. Hill plots of the rate of reaction against either substrates have a slope of approximately 1, which is not altered by the presence of the activator indicating no cooperativity between the substrate site and putative activator site. Ability of chlorpromazine and imipramine related drugs to activate this enzyme suggests that these drugs may facilitate the inactivation of prostaglandins $\text{in}\text{vivo}$.     

Influence of local and neutral anaesthetics on the polymorphic phase preferences of egg yolk phosphatidylethanolamine

(1) The polymorphic phase preferences of egg phosphatidylethanolamine have been examined in the presence of normal alcohols and alkanes of varying chain length, as well as charged amine anaesthetics. (2) It is shown that the charged anaesthetics, ethanol and butanol can stabilize a bilayer arrangement for egg phosphatidylethanolamine. In contrast, longer chain ($\text{C}\text{?6}$) normal alcohols and alkanes induce the hexagonal (H_II) phase. (3) The relative potency of local anaesthetics in vitro (chlorpromazine, dibucaine, tetracaine and procaine) is mirrored by their relative ability to stabilize bilayer structure for hydrated egg phosphatidylethanolamine. Further, the aqueous concentrations of anaesthetic required to affect phospholipid polymorphism is sensitive to the lipid composition. For example, the inclusion of 20 mol% egg phosphatidylserine in egg phosphatidylethanolamine dispersions can reduce the aqueous concentrations of dibucaine required to induce appreciable bilayer stabilization effects from 5.0 mM to 0.5 mM. (4) It is suggested that the ability of amphipatic molecules such as anaesthetics to influence phosphatidylethanolamine polymorphism arises from their molecular shape. The possibility that anaesthetic molecules may exert their effects by virtue of this shape property is raised.     

Mechanism of F1-ATPase studied by the genetic approach

E. coli F₁-ATPase has been studied mainly by the genetic approach. Mutations in either the or subunit modified the kinetics of multisite and uni-site hydrolysis of ATP. The mechanism of F₁-ATPase and the essential amino acid residues of subunits are discussed.Abbreviations used: P_i, inorganic phosphate; DCCD, dicyclohexylcarbodiimide.     

Chlorpromazine induces membrane potential depolarization and ATP loss,and inhibits microsomal ATPase in soybean (Glycine Max L.) roots

In intact soybean roots, chlorpromazine causes a depolarization of the membrane potential at low concentrations (as low as 30 μM, half-maximally at about 150 μM), and induces a marked decrease in ATP levels at higher concentrations (half-maximal at about 0.5 mM) over longer periods of time. In root microsomal suspensions, chlorpromazine inhibits an apparently specific ATPase activity component (half-maximally at about 0.3 mM). Chlorpromazine inhibits $\text{N,N′-}\text{dicyclohexylcarbodiimide}$-, diethylstilbesterol- and azide-inhibited ATPase activities. On linear sucrose gradients, chlorpromazine inhibition of ATPase activity occurs in two peaks, at 1.12 g/ml and 1.14–1.17 g/ml, which may represent a tonoplast and plasma membrane ATPase, respectively. Neither peak corresponds to the F₁ ATPase. It is unclear whether ATPase inhibition or ATP loss is the cause of the membrane potential depolarization. Clearly chlorpromazine has multiple effects which are probably unrelated to its calmodulin-inhibition activity.     

Tryptophan uptake by Mycobacterium tuberculosis H37Rv--inhibition by isoniazid.

The effect of various sub-inhibitory concentrations of isoniazid on tryptophan uptake by $\text{Mycobacterium tuberculosis}$ H₃₇Rv grown $\text{in vitro}$ and $\text{in vivo}$ was studied. Uptake, measured after 3 minutes of drug exposure was inhibited mildly by 0.1 μg/ml and 0.2 μg/ml concentration and completely by 0.3 μg/ml. However, with the minimal inhibitory concentration (MIC)⁷ of 0.5 μg/ml, not only inhibition but also a strong efflux of the preformed tryptophan pool were observed. The results are discussed in the light of the theory that isoniazid interferes with the cell wall mycolate synthesis.