Inhibition of gramicidin channel activity by local anesthetics.

Ondrias et al. ((1986) Stud. Biophys. 115, 17-22) found that dibucaine, butacaine, and tetracaine reduce the conductance of membranes containing multiple (greater than 10(6)) gramicidin channels. Similar experiments with local anesthetics (LA's) added to the bath while gently stirring showed that the inhibition developed slowly over a time course of 5-10 min. We developed a many (10-20) channel membrane technique which demonstrated that when LA's were added to the bath and the membrane was repeatedly broken and reformed, the channel occurrence frequency declined promptly. In standard single-channel membrane experiments at lower gramicidin densities, the mean single channel conductance and lifetime distributions with LA's present in the bath did not differ from the controls. The predominant channel conductance amplitude was lower by 9.1% than those of controls, but channel amplitude distributions were also modified so that the net reduction in overall population channel conductance was only about 2.0%. Channel currents showed no evidence of flicker blocks. The lifetime histograms of control and LA-exposed channel populations were both satisfactorily fit by a single-exponential function with the same mean. Thus, inhibition is due primarily to a reduction in the frequency of occurrence of conducting channels, implying a reduced concentration of active monomers in the membrane.     

Inhibition of soybean lipoxygenase-1 by n-alcohols and n-alkylthiols.

A series of n-alcohols and n-alkylthiols with carbon chains from 2 to 12 were examined for the inhibition of soybean lipoxygenase-1 (L-1). The alcohol produces a competitive inhibition, the extent of which increases with an increase in the carbon number of alkyl chain up to 8. Whereas the inhibition of the alkylthiol is noncompetitive, the extent of which is almost independent from the carbon number. From the behavior of pKi dependence on the carbon number of the alcohol, the decyl group appears to be optimum to bind to L-1. The thermodynamic analysis for the inhibition based upon van 't Hoff equation indicates positive enthalpy and entropy changes for the binding of the alcohol to the enzyme and negative enthalpy and positive to negative entropy changes for that of the alkylthiol. These observations suggest that the alcohol inhibits L-1 by binding of the hydrophobic alkyl tail to the catalytic site of the enzyme by a hydrophobic interaction. The alkylthiol inhibits by binding of the nucleophilic sulfhydryl head to a polarizable region of the enzyme and the alkyl tail to a hydrophobic region of the enzyme free from the steric hindrance as an anchor.     

Modulation of phospholipid transfer protein activity. Inhibition by local anesthetics

The transfer of phospholipid molecules between biological and synthetic membranes is facilitated by the presence of soluble catalytic proteins, such as those isolated from bovine brain which interacts with phosphatidylinositol and phosphatidylcholine and from bovine liver which is specific for phosphatidylcholine. A series of tertiary amine local anesthetics decreases the rates of protein-catalyzed phospholipid transfer. The potency of inhibition is dibucaine>tetracaine>lidocaine>procaine, an order which is compared with and identical to those for a wide variety of anesthetic-dependent membrane phenomena. Half-maximal inhibition of phosphatidylinositol transfer by dibucaine occurs at a concentration of 0.18 mM, significantly lower than the concentration of 1.9 mM required for half-maximal inhibition of phosphatidylcholine transfer activity of the brain protein. Comparable inhibition of liver protein phosphatidylcholine transfer activity is observed at 1.6 mM dibucaine. For activity measurements performed at different pH, dibucaine is more potent at the lower pH values which favor the equilibrium toward the charged molecular species. With membranes containing increasing molar proportions of phosphatidate, dibucaine is increasingly more potent. No effect of Ca²⁺ on the control transfer activity or the inhibitory action of dibucaine is noted. These results are discussed in terms of the formation of specific phosphatidylinositol or phosphatidylcholine complexes with the amphiphilic anesthetics in the membrane bilayer.     

Inhibition by pentoxifylline of procoagulant activity produced by endotoxin-active monocytes]

When appropriately stimulated, monocytes are able to initiate blood coagulation through the membrane expression of tissue factor. This procoagulant activity is thought to play a role in activating coagulation in response to inflammatory stimuli in vivo. We found that pentoxifylline, a methylxanthine derivative already reported to regulate some monocyte functions, inhibits the procoagulant activity developed by monocytes in vitro in response to endotoxin. This effect was accompanied by an early increase in intracellular levels of cyclic AMP and was mimicked by compounds that induce an increase in cyclic AMP levels. These results suggest that the suppressive effect of pentoxifylline occurs at least in part via an increase in intracellular cyclic AMP levels.     

Inhibition of mitogen-induced lymphocyte transformation by local anesthetics.

Chlorpromazine (CPZ) and lidocaine were added to cultures of mouse spleen cells stimulated by concanvalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and lipopolysaccharide (LPS). Concentrations of CPZ greater than 5 x 10(-6)M and concentrations of lidocaine greater than 2 x 10(-3)M totally inhibited the mitogenic responses to all four mitogens. Minimal inhibitory concentrations of neither drug interferred with cell viability as determined by trypan blue uptake or 51Cr release. The effects were totally reversed by the removal of the drugs from the culture. Addition of the drug at intervals after mitogen exposure demonstrated that the inhibited event occurred relatively soon after exposure to the mitogen. For example, the addition of lidocaine or CPZ more than 24 hr after Con A stimulation had no effect on tritiated thymidine incorporation. Elevated concentrations of cyclic AMP, cyclic GMP (or their derivatives) or calciunown membrane active actions of these drugs and the rapid reversibility of the effect strongly support the idea that the local anesthetics act on the surface membrane of lymphocytes. Binding of radiolabeled Con A or LPS to lymphocyte membranes in the presence of lidocaine or CPZ was not inhibited. The possibility exists that CPZ and lidocaine disorganized cell membranes so as to interfere with the surface membrane elaboration or action of a second messenger, or interfere with cell-cell interactions.     

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 by nitric oxide of cytochrome P450 4A activity contributes to endotoxin-induced hypotension in rats.

Nitric oxide (NO) production during endotoxemia is associated with decreased total CYP content, CYP 1A1/2, 2B1/2, 2C6, 2C11, 3A1, and 3A2 mRNA, protein expression or activity which is prevented by NO synthase (NOS) inhibitors in rats. This study was conducted to determine if endotoxin-induced hypotension caused by NO production is mediated by inhibition of renal CYP 4A protein expression and activity. In conscious male Sprague-Dawley rats, endotoxin (10 mg/kg, ip) reduced mean arterial pressure (MAP), increased serum and renal nitrite levels, and inducible NOS (iNOS), and decreased renal CYP 4A1/A3 protein and CYP 4A activity. The selective iNOS inhibitor 1,3-PBIT (10 mg/kg, ip; 1h after endotoxin) prevented endotoxin-induced decrease in MAP, renal CYP 4A1/A3 protein level and CYP 4A activity and increase in systemic and renal nitrite production. The selective constitutive NOS (cNOS) inhibitor N(G)-nitro-L-arginine (L-NNA; 20 mg/kg, ip; 1 h after endotoxin) partially attenuated endotoxin-induced decrease in MAP. The selective CYP 4A inhibitor, aminobenzotriazole (50 mg/kg, ip; 1 h after endotoxin) diminished CYP 4A1/A3 protein level and CYP 4A activity. Aminobenzotriazole did not alter the endotoxin-induced decrease in MAP, but it reversed the effect of 1,3-PBIT in preventing endotoxin-induced fall in MAP and CYP 4A activity. These data suggest that the endotoxemia-induced increase in NO production primarily via iNOS suppresses renal CYP 4A expression and activity, and inhibition of iNOS with 1,3-PBIT restores renal CYP 4A protein and activity and MAP presumably due to increased production of arachidonic acid metabolites derived from CYP 4A.     

Inhibition of mammalian translation initiation by volatile anesthetics

Volatile anesthetics are essential for modern medical practice, but sites and mechanisms of action for any of their numerous cellular effects remain largely unknown. Previous studies with yeast showed that volatile anesthetics induce nutrient-dependent inhibition of growth through mechanisms involving inhibition of mRNA translation. Studies herein show that the volatile anesthetic halothane inhibits protein synthesis in perfused rat liver at doses ranging from 2 to 6%. A marked disaggregation of polysomes occurs, indicating that inhibition of translation initiation plays a key role. Dose- and time-dependent alterations that decrease the function of a variety of translation initiation processes are observed. At 6% halothane, a rapid and persistent increase in phosphorylation of the alpha-subunit of eukaryotic translation initiation factor (eIF)2 occurs. This is accompanied by inhibition of activity of the guanine nucleotide exchange factor eIF2B that is responsible for GDP-GTP exchange on eIF2. At lower doses, neither eIF2alpha phosphorylation nor eIF2B activity is altered. After extended exposure to 6% halothane, alterations in two separate responses regulated by the target of rapamycin pathway occur: 1) redistribution of eIF4E from its translation-stimulatory association with eIF4G to its translation-inactive complex with eIF4E-binding protein-1; and 2) decreased phosphorylation of ribosomal protein S6 (rpS6) with a corresponding decrease in active forms of a kinase that phosphorylates rpS6 (p70(S6K1)). Changes in the association of eIF4E and eIF4G are observed only after extended exposure to low anesthetic doses. Thus dose- and time-dependent alterations in multiple processes permit liver cells to adapt translation to variable degrees and duration of stress imposed by anesthetic exposure.     

Inhibition of endotoxin-induced lung inflammation by interleukin-10 gene transfer in mice

Interleukin (IL)-10 is an anti-inflammatory cytokine that has great potential for use in the treatment of inflammatory and immune illnesses. In this study, gene transfer was used to induce IL-10 transgene expression in murine lungs for treatment of endotoxin-induced lung inflammation. Gene transfer was performed with a cytomegalovirus (CMV)-IL-10 plasmid with the aid of the liposomal agents LipofectAMINE and N-[1-(2,3-dioleoyl)propyl]-N,N, N-trimethylammonium methylsulfate (DOTAP). Administration of the endotoxin caused a marked increase in lung inflammation as indicated by increased tumor necrosis factor (TNF)-alpha release and neutrophil count. Pretreatment of the mice with IL-10 plasmid with and without LipofectAMINE had no inhibitory effect on lung inflammation and IL-10 transgene expression. LipofectAMINE by itself induced lung inflammation, an effect that was not observed with DOTAP. IL-10 plasmid when codelivered with DOTAP expressed biologically active IL-10 protein and caused a reduction in endotoxin-induced inflammation. Transgene expression was observed as early as 3 h after administration, peaked at 12 h, and declined thereafter. We conclude that IL-10 gene transfer is a feasible approach for the treatment of lung inflammation.     

Inhibition of endotoxin-induced macrophage chemokine production by VIP and PACAP in vitro and in vivo

Inflammatory chemokines recruit immune cells which initiate and maintain the inflammatory response. Although such a response is necessary for the elimination of the antigen, the inflammation has to be eventually resolved. Peptides such as vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), released following antigenic stimulation, contribute to the termination of an inflammatory response primarily by inhibiting the production of proinflammatory cytokines. Here we investigated the effects of VIP and PACAP on chemokine production. We report that VIP and PACAP inhibit the expression of the macrophage-derived CXC chemokines MIP-2 and KC (IL-8), and of the CC chemokines MIP-1a, MIP-1b, MCP-1 and RANTES in vivo and in vitro. The decrease of chemokine gene expression correlates with an inhibitory effect of VIP/PACAP on NFkB binding. In an in vivo model of acute peritonitis, the inhibition of chemokine production by VIP/PACAP leads to a significant reduction in the recruitment of PMNs, macrophages and lymphocytes into the peritoneal cavity. These findings support the proposed role of VIP and PACAP as key endogenous anti-inflammatory agents, and describe a novel mechanism, i.e., the inhibition of the production of macrophage-derived chemokines.     

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.     

Inhibition of existing denitrification enzyme activity by chloramphenicol.

Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (greater than 50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log-phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.     

Structure activity relationship of lidocaine type local anesthetics.

The structure activity relationship of nine compounds were studied and compared to lidocaine. The extent of local anesthetic activity was ascertained by the tail pinch method in mice, and by the isolated frog sciatic nerve method. The effective and lethal dose in 50% of the animals was also determined. Three of the nine compounds appeared to possess significant local anesthetic activity in the $\text{in vivo}$ studies and thus were selected for further investigation $\text{in vitro}$. The $\text{in vivo}$ studies also indicated that two of the three were more toxic than lidocaine. The $\text{in vitro}$ results demonstrated that methyl substitution at positions 2,3 and 5 on the benzene ring produced a compound of slightly more anesthetic potency in an acid medium. At pH 7.8 all three compounds approached the potency of lidocaine. These data indicate that methyl substitution at other than the ortho position of the benzene ring generally results in compounds of lesser local anesthetic activity while tending to increase the toxicity.     

The mechanism of increase in the ATPase activity of sarcoplasmic reticulum vesicles treated with n-alcohols.

Treatment of sarcoplasmic reticulum vesicles with aqueous n-alcohols caused inhibition of calcium uptake and enhancement of ATPase activity. With increasing alcohol concentration, the ATPase activity reached a maximum (in the case of n-butanol, at about 350 mM) and then decreased. The effect of n-butanol was extensively studied. The purified ATPase enzyme and leaky vesicles treated with Triton X-100 or phospholipase A showed high ATPase activity in the absence of n-butanol. With increasing n-butanol concentration, their atpase activities began to decrease above about 250 mM n-butanol, without any enhancement. In the presence of ATP, the turnover rate of calcium after calcium accumulation had reached a steady level was the same as that at the initial uptake. n-Butanol did not affect these rates. Kinetic analyses of these experiments were carried out. The mechanisms of calcium transport and of increase of ATPase activity in the presence of alcohol were interpreted as follows. After calcium accumulation had reached a steady level, fast influx and efflux continued; the influx was coupled with phosphorylated enzyme (E-P) formation and most of the efflux was coupled with rephosphorylation of ATP from ADP and E-P. The observed ATPase activity is the difference between these two reactions. If alcohol molecules make the vesicles leaky, calcium ions will flow out without ATP synthesis and the apparent ATPase activity will increase. The effect of alcohols on sarcoplasmic reticulum vesicles was separated into two actions. The enhancement of ATPase activity was attributed to a leakage of calcium ions from the vesicles, while the decrease of ATPase activity at higher concentrations of alcohols was attributed to denaturation of the ATPase enzyme itself. The two effects were interpreted in terms of equilibrium binding of alcohol molecules to two different sites of the vesicles; leakage and denaturation sites. Similar analysis was carried out for various n-alcohols from methanol to n-heptanol. The apparent free energies of binding of the methylene groups of n-alcohols were evaluated to be -863 cal/mol for the leakage site, and -732 cal/mol for the denaturation site.