Halogen substituents on the aromatic moiety of the tetracaine scaffold improve potency of cyclic nucleotide-gated channel block

A series of new tetracaine derivatives with substituents on the aromatic ring was prepared and evaluated for block of retinal rod cyclic nucleotide-gated (CNG) channels. Aromatic substitutions had little effect starting with the basic tetracaine scaffold, but electron-withdrawing substituents significantly improved the blocking potency of an octyl-tail derivative of tetracaine. In particular, halogen substitutions at either the 2- or 3-position on the ring resulted in compounds that were up to eight-fold more potent than the parent octyl-tail derivative and up to 50-fold more potent than tetracaine.     

Biphasic effect of local anesthetics on the adenosine triphosphate-dependent calcium uptake by lysed brain synaptosomes

Previous studies have shown that an adenosine triphosphate-dependent calcium uptake activity in lysed brain synaptosomes is attributable to the neuronal endoplasmic reticulum elements. The present study has examined the effects of tetracaine, lidocaine, and dibucaine on this calcium uptake process. The adenosine triphosphate-dependent uptake of 45Ca2+ was measured (in the absence and in the presence of drug) by Millipore filtration and liquid scintillation spectrometry. The local anesthetics studied exhibited a biphasic effect on 45Ca2+ uptake by lysed synaptosomes from rat brain cortex. High concentrations (5 mM tetracaine, 50 mM lidocaine, 0.6 mM dibucaine) inhibited the uptake of 45Ca2+; the order of potency for this effect was dibucaine greater than tetracaine greater than lidocaine. Lower concentrations of these local anesthetics produced either no effect on 45Ca2+ uptake (2 mM tetracaine or 30 mM lidocaine) or a stimulation of 45Ca2+ uptake (1 mM tetracaine, 10 mM lidocaine, and 0.3 mM or 0.1 mM dibucaine); the order of potency for stimulation of 45Ca2+ uptake was dibucaine greater than tetracaine greater than lidocaine.     

Inhibition of microorganisms by topical anesthetics

The effect of various topical anesthetics and their preservatives on the growth of Pseudomonas aeruginosa, Staphyloccoccus albus, and Candida albicans was investigated. The topical anesthetics were proparacaine HCl, tetracaine HCl, cocaine HCl, and benoxinate HCl. The preservatives were chlorobutanol and butyl p-hydroxybenzoate. Proparacaine inhibited C. albicans but not P. aeruginosa or S. albus. All three test organisms were inhibited to varying degrees by tetracaine, benoxinate, cocaine, chlorobutanol, and butyl p-hydroxybenzoate.     

Effects of local anesthetics on guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on beta-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2--20 mM) reduces the responsiveness of adenylate cyclase to (a) guanyl-5'-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5'-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5'-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10(-7) M. Tetracaine also competitively inhibited binding of both the antagonist [3H]dihydroalprenolol and the agonist [3H]hydroxybenzylisoproterenol to beta-adrenergic receptors. However, it was twice as potent in inhibiting [3H]hydroxybenzylisoproterenol as [3H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the beta-adrenergic receptor induced by agonists. Other local anesthetics mimicked the effects of tetracaine on adenylatecyclase and in dissociating high-affinity agonist-receptor complexes. The other of potency for both processes was dibucaine greater than tetracaine greater than bupivacaine greater than lidocaine which agrees with their relative potencies as local anesthetics. By contrast, a different order of potency was observed for competitive inhibition of [3H]dihydroalprenolol binding: dibucaine greater than tetracaine greater than greater than lidocaine greater than bupivacaine.     

Trinitrobenzenesulfonic acid and fluorodinitrobenzene: Probes to study local anesthetic effects in cell membranes

Summary The interaction of local anesthetics with intact erythrocytes was studied by monitoring the extent of reaction of phospholipids with trinitrobenzenesulfonic acid and fluorodinitrobenzene. Incubating erythrocytes with local anesthetics increases the amount of phosphatidylethanolamine and phosphatidylserine available for reaction with trinitrobenzenesulfonic acid and fluorodinitrobenzene. The order of potency of the local anesthetics corresponded to that reported for blocking nerve conduction: dibucaine> tetracaine>butacaine>lidocaine>procaine. Treatment of intact erythrocytes with 1mm tetracaine at 37°C allows 4–5% more of the phosphatidylethanolamine to react with trinitrobenzenesulfonic acid as compared to control cells. Treatment with tetracaine has no effect at 0°C, a temperature at which there is only limited partitioning of the anesthetic into the bilayer. Kinetic analysis of the reaction with trinitrobenzene sulfonic acid showed that the increased number of reactive phosphatidylethanolamine molecules are located mainly on the outer half of the erythrocyte membrane. Tetracaine also increases the number of phosphatidylserine and phosphatidylethanolamine molecules in the erythrocyte membrane which are available to react with the penetrating probe fluorodinitrobenzene. The reaction with PE is increased from 67 to 77% and the reaction of PS is increased from 44 to 57%. Thus tetracaine affects both halves of the lipid bilayer.     

ACCUMULATION OF ADENOSINE 3'',5''-MONOPHOSPHATE IN BRAIN SLICES: INTERACTION OF LOCAL ANAESTHETICS AND DEPOLARIZING AGENTS

Abstract— Membrane depolarizing agents such as veratridine, ouabain and high concentrations of potassium ions elicit a remarkable accumulation of cyclic AMP in brain slices incubated in vitro , and this accumulation, but not that elicited by biogenic amines, is prevented by a membrane stabilizer, cocaine. The effect of various local anaesthetics (compounds which are known to stabilize the membrane of peripheral sensory nerves) on the accumulation of cyclic AMP elicited by depolarizing agents in incubated slices of guinea pig brain has now been examined. At optimal concentrations the anaesthetics inhibited by more than 95 per cent the accumulation of cyclic AMP elicited with veratridine, ouabain, and high concentrations of potassium ions. The order of the inhibitory potency vs. veratridine was: dibucaine (ED₅₀= 9.5 ± 10⁻⁶ M) > tetracaine > cocaine (ED₅₀= 1·3 ± 10⁻⁴ M) > lidocaine > procaine (ED₅₀= 1.7 ± 10⁻³M). This order is consistent with the order of their local anaesthetic potency, but is not consonant with the order of the relative toxicity of these agents when used as spinal anaesthetics.     

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa of the adrenal cortex.

Effects of ACTH and calcium on cyclic AMP production and steroid output by the zona glomerulosa (the capsular fraction) from the rat adrenal cortex have been studied. Although high concentrations of extracellular calcium potentiated the stimulatory action of ACTH on cyclic AMP and aldosterone output, tetracaine or verapamil inhibited aldosterone output but not cyclic AMP production during ACTH-stimulation. Lanthanum reduced both aldosterone and cyclic AMP accumulation induced by ACTH. These results suggest that an extracellular calcium would be essential in stimulating the capsular steroidogenesis without involvement of the cyclic AMP system.     

Locations and dynamical perturbations for lipids of cationic forms of procaine, tetracaine, and dibucaine in small unilamellar phosphatidylcholine vesicles as studied by nuclear Overhauser effects in 1H nuclear magnetic resonance spectroscopy

Locations and dynamical perturbations for lipids of local anesthetics (procaine . HCl, tetracaine . HCl, and dibucaine . HCl) in sonicated egg yolk phosphatidylcholine (PC) vesicles have been studied by 1H-1H nuclear Overhauser effect (NOE) measurements. It was found that tetracaine and dibucaine bind much strongly to the neutral lipids than does procaine and that their mobilities are lowered to such an extent that spin diffusion is transmitted (i.e., omega 2 tau c2 much greater than 1). The intermolecular NOEs between drugs and PC were more effective in the case of dibucaine than with tetracaine, indicating that dibucaine binds to the lipids more strongly than tetracaine; this order agrees well with that of anesthetic potency. However, it was only tetracaine that gave any appreciable dynamical perturbation to the PC vesicles when they were monitored by the extent of transfer of the negative NOE from alpha-methylene protons to choline methyls, olefinic methines, acyl methylenes and terminal methyl protons. This finding was interpreted as being due to the differences in the locations of these drugs in small unilamellar vesicles: (1) procaine interacts with lipids very weakly at the outer surface of the vesicles; (2) tetracaine binds to the lipids both at the outer and inner halves of the bilayer, inserting its rod-like molecule in a forest of acyl chains of PC; (3) dibucaine binds tightly to the polar head-group of PC, which resides only at the outer half of the bilayer vesicles. It was concluded that the relative order of anesthetic potency within these drugs can be correlated not with the ability to affect membrane fluidity but with the ability to bind to lipids at the polar head-group of the bilayer vesicles.     

Effect of local anesthetics on stearoyl-CoA desaturase of Tetrahymena microsomes

The effect of local anesthetics on the stearoyl-CoA desaturase activity was studied using Tetrahymena microsomal preparation. Dibucaine, tetracaine, and propranolol, a beta-blocking agent, nonspecifically inhibited the activities of NADPH-ferrihemoprotein reductase as well as of stearoyl-CoA desaturase and the terminal component, but lidocaine and procaine had no effect on these activities. The inhibitory potency was decreased in the order of dibucaine greater than propranolol greater than tetracaine much greater than lidocaine = procaine. According to the double-reciprocal plots of stearoyl-CoA desaturase, the inhibition by dibucaine appeared to be noncompetitive with respect to stearoyl-CoA as substrate. However, the activity of NADH-ferricyanide reductase was not significantly affected by concentrations of propranolol and tetracaine lower than 10mM, but by dibucaine. The terminal component, cyanide-sensitive factor, was most sensitive to local anesthetics among the microsomal electron transport components, suggesting a rate-limiting enzyme.     

Some biochemical properties of alkyl phosphotriesters of cyclic AMP.

The biochemical properties of several alkyl phosphotriesters of cyclic AMP were studied with respect to their interactions with beef heart protein kinase and cyclic nucleotide phosphodiesterase. Ethyl and propyl triesters did not enhance the phosphorylation of histone by protein kinase and methyl, ethyl, propyl and butyl triesters were poor competitors for the cyclic AMP binding site of the enzyme. However, these alkyl phosphotriesters were effective inhibitors of cyclic nucleotide phosphodiesterase with the K$\text{i}$'s arrayed in the following order: methyl > ethyl > propyl > butyl > cetyl triester. Metabolic studies with mice indicated that intraperitoneal injection of low doses of propyl triester for one week significantly increased cyclic AMP concentration.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

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.     

Inhibition of fast axonal transport in vitro by tetracaine: an increase in potency at alkaline pH, and no change in potency in calcium-depleted nerves

Some of the present in vitro experiments compare the degree of inhibition of fast axonal transport produced by tetracaine at neutral and at alkaline pH. In desheathed spinal nerves from bullfrog, 0.5 mM tetracaine reduced the quantity of [3H]leucine-labeled proteins which were transported to a ligature by 43% at pH 7.2 and by 96% at pH 8.2; separate experiments established that transport was not affected by the pH change in the absence of tetracaine. The relationship between pH and transport-blocking potency of tetracaine (pKa 8.2) is such that the local anesthetic is more potent when more uncharged form of the molecule is present; this may reflect the easier penetration across the axonal plasma membrane by the uncharged form of the tetracaine molecule. The axonal smooth endoplasmic reticulum has been attributed the function of a calcium reservoir, and it appeared possible that local anesthetics could block axonal transport by releasing calcium from this structure. However, the inhibition of transport produced by 1 mM tetracaine (pH 7.1) in sheathed nerves was approximately 80% both in nerves with a lower than normal calcium content (47% of normal) and in nerves with a normal calcium content; this result does not support the hypothesis that inhibition of axonal transport by local anesthetics is mediated by an increase in intracellular free Ca2+, but does not rule out the hypothesis either.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

Local anesthetics-induced inhibition of chloroplast electron transport

The effects of local anesthetics on photosynthetic activity of pea chloroplasts were investigated in order to elucidate the role of Ca2+ in photosynthetic electron transport. Dibucaine, benzocaine and tetracaine were found to inhibit the O2-evolving activity. The inhibitory effect decreases in the order dibucaine greater than benzocaine greater than tetracaine greater than trimecaine similarly as does the potency to inhibit propagation of excitation in nerve fibre. As demonstrated in experiments with artificial donors and acceptors, the site of inhibition is the water-splitting site of PSII. The inhibitory power of the anesthetics grows with increasing ionic strength of the incubating mixture (by adding NaCl or MgCl2) and with pH; this is explained by occurrence of the neutral form of amine. At low concentrations the charged anesthetic acts as a protonofore; however, the inactivation of water splitting is not due to the protonophoric effect. The incubation is followed by the disappearance of ESR signal IIs. The role of Ca2+ and Ca2+-binding protein in PSII electron transport and its localization are discussed.     

A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity.

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.     

Pituitary thyrotropin-releasing hormone receptors: local anesthetic effects on binding and responses

Pharmacological agents are widely used to probe the mechanism of action of TRH. A number of these drugs behave as local anesthetics at high concentrations. The effect of local anesthetics on the binding of [3H]Me-TRH to specific receptors was studied using the GH4C1 line of rat pituitary tumor cells. [3H]Me-TRH binding was inhibited by classical local anesthetics with the order of potency (IC50 values): dibucaine (0.37 mM) greater than tetracaine (1.2 mM) greater than lidocaine (3.3 mM) greater than procaine and benzocaine (greater than 10 mM). IC50 values for other drugs with local anesthetic properties that inhibited [3H]Me-TRH were: 100 microM trifluoperazine, 100 microM imipramine, 170 microM chlorpromazine, 300 microM verapamil, and 700 microM propranolol. Inhibition by tetracaine and verapamil increased as the pH was raised from 6 to 8.5, indicating that the free base form of the amine drugs was the inhibitory species, and the local anesthetic effect was greater at 37 C than at 24 C or 0 C. [3H]Me-TRH binding to receptors in isolated membranes was inhibited to the same extent as binding to receptors on intact cells. Local anesthetics were 3- to 20-fold less potent at inhibiting [3H]Me-TRH to digitonin-solubilized receptors than binding to intact cells. In contrast, the potency of chlordiazepoxide, a putative TRH antagonist, to inhibit [3H]Me-TRH binding was equal using cells and solubilized receptors (IC50 = 10 microM). Local anesthetics inhibited TRH-stimulated PRL release and also inhibited basal PRL secretion and secretion stimulated by two nonhormonal secretagogues, (Bu)2cAMP and a phorbol ester.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of side chain modification on the activity of propyl cyclohexaneacetate as an attractant to the German cockroach

Propyl cyclohexaneacetate, a synthetic attractant to the German cockroach, Blattella germanica, appears to consist of a head (cyclohexane ring) and a tail (ester linkage). The tail was modified as regards a number of structural parameters, and the change in activity was interpreted in terms of the corresponding receptor site.Irrespective of the position and direction of the ester linkage, six atoms were optimum for the side chain. The activity increased when the terminal methyl group was replaced with chlorine, and decreased when changed into methylene. The methyl branch in the alcohol unit depressed the activity. The order of attraction among the esters with six atom side chain was as follows: propyl cyclohexaneacetate > cyclohexylmethyl butanoate ≧ ethyl 3-cyclohexylpropanoate ≧ cyclohexyl pentanoate > butyl cyclohexanecarboxylate = 2-cyclohexylethyl propanoate.Ethers, ketones and hydrocarbons which were derived from the esters with six atom side chains by replacing either or both of the carbonyl groups and ether oxygen with methylene(s) were inferior to the parent esters. Their relative activities were in the following order: pentyl cyclohexyl ether > propyl 2-cyclohexylethyl ether > butyl cyclohexyl-methyl ether = pentyl cyclohexyl ketone = butyl cyclohexylmethyl ketone = 6-cyclohexylhexane. The SAR in respect of the ester group resembled that in the muscarinic activity of acetylcholine.     

5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid and its derivatives inhibit ionotropic gamma-aminobutyric acid receptors by binding to the 4'-ethynyl-4-n-propylbicycloorthobenzoate site

Acyclic noncompetitive antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors, bearing an ester or ether linkage, were designed, synthesized, and assayed for their inhibition of the specific binding of [3H]4'-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a radiolabeled noncompetitive antagonist, to rat brain and housefly head membranes. 5-[4-(3,3-Dimethylbutoxycarbonyl)phenyl]-4-pentynoic acid (DBCPP), a butyl benzoate analogue, was found to competitively inhibit the binding of [3H]EBOB in rat brain membranes, with an IC50 of 88 nM. The potency conferred by the p-substituent decreased in the order C(triple bond)C(CH2)2COOH > C(triple bond)C(CH2)2COOCH3 > C(triple bond) CH > Br. Pentyl phenyl ethers were equally potent compared with butyl benzoates, while phenyl pentanoates and benzyl butyl ethers were less pont. These compounds were generally less active in housefly head membranes than in rat brain membranes. The introduction of an isopropyl group into the 1-position of the 3,3-dimethylbutyl group of a butyl benzoate and two benzyl butyl ethers caused an increase in potency in housefly GABA receptors, whereas this modification at the corresponding position of other compounds led to an unchanged or decreased potency. In the case of rat receptors, this modification resulted in a decrease in potency except for a phenyl pentanoate. To confirm that DBCPP interferes with GABA receptor function, we performed whole-cell patch clamp experiments with rat dorsal root ganglion neurons in the primary culture. Repeated co-applications of GABA and DBCPP suppressed GABA-induced whole-cell currents with an IC50 of 0.54 microM and a Hill coefficient of 0.7. These findings indicate that DBCPP and its derivatives inhibit ionotropic GABA receptors by binding to the EBOB site and that there might be structural difference in the noncompetitive antagonist-binding site between rat and housefly GABA receptors.     

Use of polymyxin B, levallorphan, and tetracaine to isolate novel envelope mutants of Escherichia coli.

Mutants of Escherichia coli were isolated by their resistance to the bacteriocidal effects of the membrane-active drugs polymyxin B, levallorphan, and tetracaine. The mutants were examined for additional changes in cellular physiology evoked by the lesions; many polymyxin-resistant strains had a concomitant increased sensitivity to anionic detergents, and several strains of each type had concomitant alterations in generation time and morphology. Mutants of each class (polymyxin resistant, tetracaine resistant, and levallorphan resistant) were transduced into recipient strains. The levallorphan resistance site (lev) was located at approximately 9 min on the E. coli chromosome. Polymyxin (pmx) and tetracaine (tec) resistance loci were also transduced. The lev and tec strains had a slight prolongation of generation time, in contrast with their isogenic wild-type strains. The tec transductant produced long filaments in the absence of tetracaine and had an altered colonial morphology, it reverted at high frequency, with the morphological abnormalities reverting along with the tetracaine resistance. The pmx transductant had an increased sensitivity to levallorphan and to anionic detergents. In contrast, both lev and tec mutants were more resistant to acriflavine than was the wild type or the pmx transductant. The pmx, lev, and tec loci differed in sensitivity to mitomycin C; the lev strain was more resistant, the tec strain was more sensitive, and the pmx strain was much more sensitive than the wild type. There was no difference in sensitivity to several other dyes and detergents, colicins, or T bacteriophage between the transductant and isogenic wild-type strains. Thus, lev, tec, and pmx loci confer more subtle alterations in the permeability barrier than do lipopolysaccharide-deficient mutants previously studied.