Correlation of biologic data with physico-chemical properties among the vinca alkaloids and their congeners.

Displacement of [³H]vinblastine binding to tubulin by other Vinca alkaloid derivatives has been demonstrated to be a competitive process, allowing for determination of the association constant of each drug. Correlation of LD₅₀ data and anti-P-388 activity was found with log P and log K_a, according to the equations: $\text{log}\text{LD}{}_{50}\text{= 0.129 (}\text{log}\text{P)}{}^2\text{? 0.522}\text{log}\text{P ? 0.479}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 4.652}\text{log}\text{P ? 388 = 0.222 (}\text{log}\text{P)}{}^2\text{? 1.059}\text{log}\text{P ? 0.520}\text{log}\text{K}{}_{\mathrm{a}}\text{+ 5.366}$. Vincristine and desacetylvinblastine were the two most active agents in this series. That the latter drug had significant biologic activity was of considerable interest, since it is known to be a human metabolite.     

Kinetic studies on the deethylation of ethoxybenzamide: A comparative study with isolated hepatocytes and liver microsomes of rat

Kinetic parameters (K_m and V_max) of ethoxybenzamide deethylation in isolated rat hepatocytes and liver microsomes were compared. Adjustment of cofactors in microsomal deethylation, such as NADPH and Mg²⁺, to give optimum conditions, and appropriate correction of the apparent kinetic parameters for nonspecific binding and microsomal yield resulted in good agreement among the kinetic parameters of isolated hepatocytes [$\text{V}{}_{\mathrm{<mtext>max</mtext>}}\text{= 0.0863 μmole · min}{}^{\mathrm{?1}}\text{· (g liver)}{}^{\mathrm{?1}}$ and K_m = 0.459 mM] and microsomes [V_max = 0.124 μmoles · min^?1 · (gliver)^?1 and K_m = 0.378 mM].     

The effects of cadmium, manganese and aluminium on sodium-potassium-activated and magnesium-activated adenosine triphosphatase activity and choline uptake in rat brain synaptosomes

The effects of Cd²⁺, Mn²⁺ and Al³⁺ on rat brain synaptosomal sodium-potassium-activated and magnesium-activated adenosine triphosphatase (Na-K-ATPase and Mg-ATPase) activity and choline uptake were studied. All three types of metal ions inhibited Na-K-ATPase activity more markedly than Mg-ATPase activity. The rank order of inhibition of Na-K-ATPase was: $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.4 μ}\text{M}\text{) >}\text{Mn}{}_{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 955 μ}\text{m}\text{) >}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 8.3}\text{mM}$). The rank order of inhibition of Mg- was:$\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 316 μ}\text{M}\text{>}\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 5.5}\text{mM}\text{>}\text{Al}{}^{\mathrm{3+}}\text{(}\text{ic}{}_{50}\text{= 21.9}\text{mM}\text{).}\text{Al}{}^{\mathrm{3+}}$ was most potent in inhibiting synaptosomal choline uptake ($\text{ic}{}_{50}\text{= 24μ}\text{M}$ in the absence of Ca²⁺ and 123 μ.M in the presence of 1 mM Ca²⁺). $\text{Cd}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 363 μ}\text{M}\text{)}$ was a more effective inhibitor of choline uptake than $\text{Mn}{}^{\mathrm{2+}}\text{(}\text{ic}{}_{50}\text{= 1.2?1.5}\text{mM}\text{)}$ . The presence of 1 mM Ca²⁺ did not alter choline uptake, nor did it antagonize the inhibitory actions of the three metals. Our observations that Cd²⁺ and Al³⁺ inhibited synaptosomal choline uptake, but did not show parallel inhibitory effects on Na-K-ATPase activity directly contradicts the ionic gradient hypothesis. These results are also discussed in relation to the in vivo neurotoxicity of cadmium, manganese and aluminium.     

Effect of polychlorinated biphenyls on the immune responses of rhesus monkeys and mice.

The relationship between lipid peroxidation, glutathione (GSH) content, and CCl₄-induced toxicity was investigated in rat hepatocytes isolated by a collagenase-perfusion technique. Two chemical initiators of lipid peroxidation, ferric ions complexed with adenosine diphosphate ($\text{ADP}\text{Fe}{}^{\mathrm{3+}}$) and diethyl maleate, were studied for comparison. CCl₄ caused a reduction of intracellular K⁺ and release of alanine aminotransferase (ALT) into the medium, but no evidence of lipid peroxidation, as measured by the absorbance of thiobarbituric acid (TBA)-reacting materials and lipid-extract diene conjugation. $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$ caused lipid peroxidation, but only a small loss of K⁺. Diethyl maleate caused a greater amount of lipid peroxidation and cell damage than did $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$. Neither response appeared to be related to the GSH content, which was reduced by diethyl maleate, but not by $\text{ADP}\text{Fe}{}^{\mathrm{3+}}$, and by CCl₄ only at the highest dose. The results suggest that lipid peroxidation is not a requisite step in CCl₄-induced toxicity in isolated hepatocytes.     

Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide: A potent and selective fluorescent inhibitor of butyrylcholinesterase

Interactions between dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (DAPA) and the cholinesterases were examined by the techniques of enzyme kinetics and fluorescence spectroscopy. When tested with partially purified enzyme preparations, DAPA was a potent inhibitor of butyrylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?7}}\text{M}\text{)}$ but not of acetylcholinesterase $\text{(}\text{IC}{}_{50}\text{= 4 × 10}{}^{\mathrm{?4}}\text{M}\text{)}$. For a detailed study of the effects of DAPA on butyrylcholinesterase (BuChE), the enzyme was purified to homogeneity from horse serum, with the aid of affinity chromatography on N-methyl acridinium. The kinetics of the inhibition of purified BuChE by DAPA were complex, having both competitive and non-competitive features, and it was not possible to estimate K_i unambiguously. Spectroscopic measurements showed that the fluorescence of the dansyl moiety was strongly affected by the binding to BuChE. With excitation at 330 nm, total fluorescence emission from bound DAPA (at 450 nm and above) was 21-fold greater than from free DAPA. In a titration experiment, this enhancement of fluorescence intensity was used to calculate that each monomer of BuChE has two apparently independent DAPA-binding sites with a K_d of 4.5 × 10^?7 M. Further measurements showed that the fluorescence emission of bound DAPA was markedly blue-shifted (to 502 nm from 570 nm in free solution) and that the fluorescence lifetime of this form was greatly prolonged (to 24 nsec from 2.7 nsec). These observations indicate that the high affinity binding sites on BuChE lock DAPA in a highly non-polar environment.     

Similar activities of nerve growth factor and its homologue proinsulin in intracellular hydrogen peroxide production and metabolism in adipocytes: Transmembrane signalling relative to insulin-mimicking cellular effects

Generation of hydrogen peroxide in adipocyte plasma membrane and its intracellular metabolism and regulatory role have been shown by Mukherjee and co-workers to be a major effector system for insulin [Fedn Proc.35, 1694 (1976); Archs Biochem. Biophys.184, 69 (1977); Biochem. Pharmac.27, 2589 (1978); Fedn Proc.37, 1689 (1978); and Biochem. Pharmac.29, 1239 (1980)]. The possible involvement of this mechanism in the action of structurally similar polypeptides having some insulin-like metabolic effects was investigated. The β-subunit of nerve growth factor (2.5 S NGF, mol. wt 13,500) which has a striking structural homology with proinsulin and has been reported to exert certain insulin-like metabolic effects in its own target tissues (e.g. growing neurites and sympathetic ganglia), and the insulin-derived polypeptides, desalanine-insulin and desoctapeptide-insulin, as well as proinsulin, were examined for their effects on rat adipocytes, employing the technique of formate oxidation. Both NGF and proinsulin caused increased [¹⁴C]formate oxidation, showing similar intrinsic activities, up to a maximum of 140–160% of the basal rate; insulin increased the rate to 190–210% of the basal rate. The relative potencies of the hormones toward H₂O₂ formation and stimulation of the pentose phosphate pathway activity were: insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?11}}\text{M}\text{)}$, desalanine-insulin $\text{(}\text{EC}{}_{50}\text{: 2.5 × 10}{}^{\mathrm{?10}}\text{M}\text{)}$ , proinsulin $\text{(}\text{EC}{}_{50}\text{: 8 × 10}{}^{\mathrm{?9}}\text{M}\text{)}$, and NGF $\text{(}\text{EC}{}_{50}\text{: 10}{}^{\mathrm{?9}}\text{M}\text{)}$. The biologically inactive derivative, desoctapeptide-insulin, did not stimulate glucose oxidation, although it caused a small increase in formate oxidation, with an $\text{EC}{}_{50}\text{of}\text{5 × 10}{}^{\mathrm{?7}}\text{M}$, indicating a suboptimal level of H₂O₂ formation in the elevation of the hexose monophosphate shunt activity. 3-Amino-1,2,4,-triazole (50 mM), which irreversibly decomposes the peroxidatic compound II of the catalase: H₂O₂ complex, inhibited formate oxidation to a greater extent in the hormone-treated cells than in the control cells, whereas sodium azide, an inhibitor of the hemoprotein, catalase, completely inhibited it. The abilities of the polypeptides to stimulate H₂O₂ formation correlated with their abilities to promote lipogenesis from [U-¹⁴C]-D-glucose, as expected of insulin. The cellular GSH/GSSG ratio increased concomitantly with the stimulation of glucose oxidation via the shunt, indicating a tight coupling between these processes. The results confirm that the hydrogen peroxide production is a common basis of the metabolic actions of growth-promoting polypeptide hormones or mitogens beyond their respective receptors.     

Influence of pH on the kinetic and spectral properties of phospholipase A2 from Bitis gabonica (gaboon adder) snake venom.

The influence of pH on the kinetic parameters k_cat and $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{<mtext>b</mtext>}}$, and the spectral properties of Bitis gabonica phospholipase A₂, showed that the activity of the enzyme is controlled by groups of pK values 6·8 and 9·2. These groups were found to exhibit enthalpies of ionization (ΔH_ion) of 7·1 Kcal/mole and 6·1 Kcal/mole respectively which are characteristic of ΔH_ion of histidine and tyrosine residues. While the dissociation constant (K_ia) for reaction between enzyme and Ca²⁺ was affected by pH, the Michaelis constant (K_b) of the enzyme for the phospholipid substrate was found to be uninfluenced in the pH range tested i.e. pH 5·–9·9.0. The binding of Ca²⁺ to the enzyme is inhibited by protonation of a group ofpKca. 6·0. The ΔH_ion of — 1·6 Kcal/mole calculated for this group is in the range of the values found for carboxyl groups. The various functions of nucleophile, proton donor and Ca²⁺ binding site may be assigned to the side chains of structurally invariant residues which for the B. gabonica phospholipase A₂ are located at His-45, Tyr-25 and Asp-46 respectively. Non-competitive inhibition of the H⁺ was observed with respect to both Ca²⁺ and lecithin which probably involves an effect of the proton on free enzyme and the interconversion of the central complexes.     

Kinetic studies on the metabolism of ethylmorphine by isolated hepatocytes from adult rats

Hepatocytes of adult rats were isolated by infusion of a hyaluronidase collagenase mixture. High yields of cells excluding trypan blue were obtained. These cells, in Hank's buffer containing rat serum and 0.1% glucose, $\text{N-}\text{demethylate}\text{[}{}^3\text{H-CH}{}_3\text{-N]}$ethylmorphine. The formaldehyde initially formed is further metabolized to tritiated water. Fifteen per cent of the original metabolic activity was observed after 21 hr at 37° in 5% CO₂-air, and cumulative metabolism is linear for up to 90 min under these conditions. The K_m for the N-demethylation of [³H-CH₃-N]ethylmorphine is 50 μM, 20 per cent of the value observed for this reaction by musomal preparations. An active transport of the substrate into the cell is postulated to account for this difference.     

Studies on the mechanism of dog heart (Na+ + K+)-ATPase inhibition by D,L-aspartic acid and its K+ and Mg2+ salts.

d,l-aspartic acid as well as its K⁺ and Mg²⁺ salts have been found to be inhibitory to Na⁺ + K⁺)-ATPase activity of the dog heart microsomal fraction enriched with sarcolemma. According to intensity of the inhibitory effect (noncompetitive) they form the following sequel: mono-K⁺-aspartate $\text{?}\text{d,l}\text{-aspartic acid}\text{<}\text{mono-Mg}{}^{\mathrm{2+}}\text{-}\text{aspartate}\text{<}\text{mixture}$ of K⁺ and Mg²⁺-aspartate in a molar ratio of 1:1 (K⁺, Mg²⁺-ASP). The latter mixture is widely used as an agent in cardiac failure. For (Na⁺ + K⁺)-ATPase the salient effects of d,l-aspartic acid and/or its K⁺ and Mg²⁺ salts were: (i) decrease in V for ATP as substrate with unchanged K_m; (ii) for Na⁺ as an allosteric modifier of (Na⁺ + K⁺)-ATPase activity a decrease in V without any alteration in n as a measure of cooperativity between activating sites; (iii) for K⁺ a decrease in V and n as well as an increase in K_0.5. In the presence of Na⁺ and ATP the high affinity of the enzyme for K⁺ became reduced by d,l-aspartic acid, lowering at the same time the K_0.5 value.Effects like these have also been described for ouabain. The present data show that K⁺ and Mg²⁺ salts of d,l-aspartic acid act at a similar locus as does ouabain.     

Mechanisms by which quipazine,a putative serotonin receptor agonist,alters brain 5-hydroxyindole metabolism

Quipazine (2-[1-piperazinyl] quinoline maleate) was shown to increase serotonin and decrease 5-hydroxyindoleacetic acid concentrations in whole brain, several brain regions, and the spinal cord of rats 1 hr after its administration (10 mg/kg, i.p.). In animals with transected spinal cords, quipazine induced stronger activation of extensor reflexes than 5-hydroxytryptophan, chlorimipramine, or Lilly 110140. This response could be blocked by methiothepin. In slices of rat cerebral cortex, quipazine inhibited the uptakes of [³H]-serotonin (EC₅₀ = 10^?6 M) and [³H]-norepinephrine ($\text{EC}{}_{50}\text{= 2 × 10}{}^{\mathrm{?6}}\text{m}$); it was equipotent with Lilly 110140 in inhibiting serotonin uptake, but less potent than chlorimipramine ($\text{EC}{}_{50}\text{= 10}{}^{\mathrm{?7}}\text{m}$). Quipazine administration to rats did not inhibit monoamine oxidase activity, and actually elevated brain tryptophan levels. These observations suggest that the effects of quipazine on brain serotonin and 5-hydroxyindoleacetic acid concentrations could have been caused by direct activation of central serotonin receptors (which would secondarily decrease impulse flow along serotonergic neurones), or by the inhibition of serotonin reuptake, or by both mechanisms.     

Secretagogue-induced transport of H+ and K+ by in vitro amphibian gastric mucosa

The effects of histamine, pentagastrin (PG), urecholinc (UC) amd dibutyryl cyclic AMP (cAMP) on the chambered fundic mucosa from Rana catesbeiana were studied to determine the H⁺ and K⁺ transport characteristics of the in vitro preparation. The three secretagogues (histamine, PG and UC) elicited very similar responses—a sustained steady-state secretion of H⁺ within 15—30 min and a transitory efflux of K⁺ that returned to control level within 60 min. Inhibition of H⁺ transport of the secretagogue-stimulated mucosa by 2.5 × 10^?4 Mp-chloromercuribenzene sulfonic acid (pCMBS) caused an increased efflux of K⁺. Replacement of pCMBS by β-mercaptoethanol (2 × 10^?3 M) rapidly restored the H⁺ transport, with a concomitant decrease of K⁺ efflux, to the pre-stimulated control level. The effects of cAMP were qualitatively similar to those of other secretagogues except that there was a delayed peak response for both H⁺ and K⁺. Our data are compatible with a K⁺ conductive pathway and a membrane-recycyling mechanism for K⁺in exchange for H⁺ at the secretory membrane of the gastric cells. Thiocyanate inhibited H⁺ transport, and the effect appeared to be due to inhibition of the transport and coupling of K⁺ to the $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange mechanism.     

Adenosine receptor interactions and anxiolytics

$\text{[}{}^3\text{H}\text{]-N}{}^6\text{-}\text{cyclohexyladenosine}$ and [³H]-1,3-diethyl-8-phenylxanthine label the A₁ subtype of adenosine receptor in brain membranes. The affinities of methylxanthines in competing for A₁ adenosine receptors parallel their potencies as locomotor stimulants. The adenosine agonist N⁶-(-phenylisopropyl) adenosine is a potent locomotor depressant. Both diazepam and $\text{N}{}^6\text{-(}\text{l}\text{-}\text{phenylisopropyl}\text{)}\text{adenosine}$ cause locomotor stimulation in a narrow range of subdepressant doses. Combined stimulant doses of the two agents depress motor activity, as do larger doses of either one, given separately.Evidence supporting and against the hypothesis that some of the actions of benzodiazepines are mediated via the adenosine system is reviewed. A number of compounds interact with both systems, probably because of physico-chemical similarities between adenosine and diazepam. It is concluded that of the four classic actions of benzodiazepines, the sedative and muscle relaxant (but not anxiolytic or anticonvulsant) actions could possibly be mediated by adenosine.     

The role of lipid,free radical initiator,and oxygen on the kinetics of lipid peroxidation

The relationship between the concentration of unsaturated lipid, free radical initiator, and oxygen concentration on the kinetics of lipid peroxidation was determined. The rate of lipid peroxidation was studied with the thiobarbituric acid (TBA), diene conjugation (DC), and ferrithiocyanate (Fe-SCN) methods. The rate of peroxidation was half-order with respect to unsaturated lipid, initiator, and oxygen. The half-order relationship could be expressed as: $\text{rate}\text{= (}\text{fk}{}_1\text{k}{}_2\text{k}{}_3\text{k}{}_6{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{azobisisobutyronitrile}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$$\text{(}\text{RH}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{O}{}_2\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. The half-order relationship was found with linoleic (18:2), linolenic (18:3), and arachidonic (20:4) acids. A linear relationship existed between the logarithm of unsaturation and the rate of peroxidation. No peroxidation of linolenic acid was indicated when the DC method was employed, but was when the TBA and Fe-SCN methods were used.     

Hepatobiliary dysfunction and inhibition of adenosine triphosphatase activity in bile canaliculi-enriched fractions following in vivo mirex, photomirex, and chlordecone exposures

Hepatobiliary dysfunction was induced by dietary exposure of male Spraque-Dawley rats to mirex (M), photomirex (PM), or chlordecone (CD). The possible relationship to ATPase activities of a bile canaliculi-enriched fraction (BCEF) also was examined employing a dosing regimen which either did (100 ppm for 15 days) or did not impair (10 ppm for 15 days) hepatobiliary function. The 1-hr cumulative biliary excretion of phenolphthalein glucuronide was significantly inhibited by all of the organochlorine analogs at 100 ppm (38, 28, and 36%, by M, PM, and CD, respectively) while bile flow was significantly increased (1.98-, 1.86-, and 1.96-fold, respectively) in bile duct-cannulated, intact animal preparations. Animals treated at levels of 10 ppm were without similar effect. Oligomycin-insensitive Mg²⁺-ATPase (OI MATPase) represented the major ATPase activity of BCEF followed by oligomycin-sensitive MATPase and $\text{Na}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}\text{-ATPase}$. A positive correlation (r = 0.91) was found between OI MATPase activity and biliary excretory function in control, M, and PM but not in CD-treated rats. This apparent disparity was due to “washing-out” of CD during membrane isolation because of its relatively high water solubility as suggested by the ratios of octanol:water partition coefficients of 20:1:0.6 for CD:M:PM. Liver:bile ratios observed for the organochlorines which approximate their octanol:water partition coefficients discount the possibility that competitive phenomena underlie hepatobiliary dysfunction produced by the chlorocarbon analogs studied. Instead, the hepatobiliary dysfunction may be more closely associated with the inhibition of BCEF ATPase activities.     

Effect of diphenylhydantoin on the uptake and catabolism of l-[3H]norepinephrine in vitro in rat cerebral cortex tissue

The effect of diphenylhydantoin on the accumulation of [³H]norepinephrine in vitro was examined in brain slices prepared from rat cerebral cortex. High concentrations of diphenylhydantoin (10^?3 M) caused a significant reduction in the 5-min accumulation of [³H]norepinephrine. On the other hand, 10^?5–10^?4 M diphenylhydantoin facilitated the 20-min accumulation of [³H]norepinephrine. This facilitative action of diphenylhydantoin was (1) associated with a reduction in oxidative catabolism of [³H]norepinephrine and (2) abolished by the 2-hr pretreatment of rats with 100 mg/kg of nialamide (i.p.). The inhibitory action of diphenylhydantoin on the oxidative catabolism of [³H]norepinephrine was observed in both whole and lyzed crude synaptosomal preparations. When diphenylhydantoin and pargyline were compared, it was found that pargyline $\text{(}\text{id}{}_{50}\text{= 1.5 × 10}{}^{\mathrm{?6}}\text{M}\text{)}$ was 37 times more effective than diphenylhydantoin $\text{(}\text{id}{}_{50}\text{= 5.5 × 10}{}^{\mathrm{?5}}\text{M}\text{)}$ in inhibiting the oxidative deamination of [³H]norepinephrine. These results suggest that diphenylhydantoin alters norepinephrine metabolism in cerebral cortex slices by an inhibitory action on (1) monoamine oxidase activity and (2) the neuronal uptake system.     

Interaction of [3H]flunitrazepam with the benzodiazepine receptor: Evidence for a ligand-induced conformation change

The interaction of [³H]flunitrazepam with benzodiazepine receptors in rat brain homogenates was studied in the presence of 2 μM endogenous GABA at 0° at pH 7.2. Equilibrium binding experiments showed a dominant component of high affinity with an equilibrium dissociation constant K = 0.86 ± 0.07 nM which accounted for 75% of total binding and another component of lower affinity (K ? 30 nM). The dissociation kinetics of the [³H]flunitrazepam complex at the high affinity site were strictly monophasic with a rate constant k_off = (7.7 ± 0.3) × 10^?4/sec. The association kinetics with the high affinity sites were studied with ligand concentrations [L]₀ in large excess over binding sites. The kinetics were in accordance with a single exponential with a reaction rate τ^?1. In the higher concentration range [L]₀ ? 10 nM, τ^?1 as a function of [L]₀ deviated from linearity and started to level off. The data are compatible with a two-step mechanism where R and L rapidly combine to form a pre-complex RL which then slowly isomerizes to the final complex C:

where $\text{K}{}_1\text{= (}\text{[}\text{R}\text{][}\text{L}\text{]}\text{([}\text{RL}\text{]}\text{)}$ and $\text{[}\text{RL}\text{]}\text{[}\text{C}\text{]}\text{=}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{= k}{}_2$. Nonlinear parameter estimation yielded K₁24.2 ± 7.1 nM, k₂ = (2.8 ± 0.5) × 10^?2/sec and k_?2 = (9 ± 2) × 10^?4/sec. The isomerization step might reflect a ligand-induced conformation change of the high affinity site which is involved in the potentiation of GABA-ergic transmission produced by the benzodiazepines.