Maria Paola Mascia

The general anesthetic propofol enhances the function of γ-aminobutyric acid-coupled chloride channel in the rat cerebral cortex

Abstract: The effect of the general anesthetic propofol on t‐[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to unwashed membrane preparations from rat cerebral cortex was studied and compared to that of other general anesthetics (pentobarbital, alphaxalone) which are known to enhance GABAergic transmission. Propofol produced a concentration‐dependent complete inhibition of [35S]TBPS binding, an effect similar to that induced by pentobarbital and alphaxalone, although these agents differ markedly in potency (alphaxalone > propofol > pentobarbital). The concomitant addition of propofol either with alphaxalone or pentobarbital produced an additive inhibition of [35S]TBPS binding, suggesting separate sites of action or different mechanisms of these drugs. Moreover, although bicuculline (0.1 μM) completely antagonized the propofol‐induced inhibition of [35S]TBPS binding, the effect of this anesthetic was not due to a direct interaction with the γ‐aminobutyric acidA (GABAA) recognition site. In fact, propofol, like alphaxalone and pentobarbital, markedly enhanced [3H]GABA binding in the rat cerebral cortex. Finally, propofol was able to enhance [3H]GABA binding in membranes previously incubated with the specific chloride channel blocker picrotoxin. Taken together these data strongly suggest that propofol, like other anesthetics and positive modulators of GABAergic transmission, might exert its pharmacological effects by enhancing the function of the GABA‐activated chloride channel.

New insight into the central benzodiazepine receptor-ligand interactions: design, synthesis, biological evaluation, and molecular modeling of 3-substituted 6-phenyl-4H-imidazo[1,5-a][1,4]benzodiazepines and related compounds.

3-Substituted 6-phenyl-4H-imidazo[1,5-a][1,4]benzodiazepines and related compounds were synthesized as central benzodiazepine receptor (CBR) ligands. Most of the compounds showed high affinity for bovine and human CBR, their K(i) values spanning from the low nanomolar to the submicromolar range. In particular, imidazoester 5f was able to promote a massive flow of (36)Cl(-) in rat cerebrocortical synaptoneurosomes overlapping its efficacy profile with that of a typical full agonist. Compound 5f was then examined in mice for its pharmacological effects where it proved to be a safe anxiolytic agent devoid of the unpleasant myorelaxant and amnesic effects of the classical 1,4-benzodiazepines. Moreover, the selectivity of some selected compounds has been assessed in recombinant α(1)β(2)γ(2)L, α(2)β(1)γ(2)L, and α(5)β(2)γ(2)L human GABA(A) receptors. Finally, some compounds were submitted to molecular docking calculations along with molecular dynamics simulations in the Cromers GABA(A) homology model.

Chronic ethanol intoxication enhances [3H]CCPA binding and does not reduce A1 adenosine receptor function in rat cerebellum

The effects of acute and chronic treatment with ethanol on the function of A1 adenosine receptor in the rat cerebellar cortex were investigated. Acute administration of ethanol (0.5-5 g/kg) had no effect on the binding of the A1-receptor agonist [3H]2-chloro-N6-cyclopentyladenosine ([3H]CCPA) or that the antagonist [3H]8-cyclopentyl-1-3-dipropylxanthine ([3H]DPCPX) in rat cerebellar cortical membranes. Rats were rendered ethanol dependent by repeated forced oral administration of ethanol (12-18 g/kg per day) for 6 days. [3H]CCPA binding was increased by 23% in cerebellar cortical membranes prepared from rats killed 3 h after ethanol withdrawal compared with saline-treated animals. The increase in [3H]CCPA binding was still apparent 12-24 h after the last ethanol administration, but was no longer detectable 3-6 days after ethanol withdrawal. In contrast, the binding of [3H]DPCPX was not modified in the cerebellar cortex of rats killed at various times after ethanol withdrawal. The acute administration of CCPA [0.25-1 mg/kg, intraperitoneally (IP)] suppressed the tremors and audiogenic seizures apparent 24 h after ethanol withdrawal. Moreover, repeated coadministration of CCPA (0.5 mg/kg, IP, four times daily) and ethanol did not prevent the generation of audiogenic seizures during withdrawal but completely prevented mortality. Finally, CCPA antagonized with similar potencies and efficacies the isoniazid-induced convulsions observed in control and ethanol-withdrawn rats. These results indicate that long-term treatment with intoxicating doses of ethanol enhances [3H]CCPA binding but does not reduce the anticonvulsant efficacy of CCPA or the function of A1 adenosine receptors.

Voluntary ethanol consumption induced by social isolation reverses the increase of α 4 /δ GABA A receptor gene expression and function in the hippocampus of C57BL/6J mice

Post-weaning social isolation (SI) is a model of prolonged mild stress characterized by behavioral and neurochemical alterations. We used SI in C57BL/6J mice to investigate the effects of ethanol (EtOH) in the free-choice drinking paradigm on gene expression and function of γ-aminobutyric acid type A receptors (GABAARs) and the role of neuroactive steroids in the actions of EtOH in the hippocampus. SI stress induced a marked reduction in hippocampal 3α-hydroxy-5α-pregnan-20-one (3α,5α-TH PROG) and was associated with molecular and functional changes of the GABAAR. The gene expression of the α4 and δ subunits was increased in the hippocampus of SI C57BL/6J mice; the expression of the γ2 subunit was decreased whereas that of the α1 did not change. Patch-clamp recordings in dentate gyrus (DG) granule cells obtained from SI C57BL/6J mice revealed a greater enhancement of tonic currents induced by α-(4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridin-3-ol (THIP) compared to that in control C57BL/6J mice. These neurochemical, molecular and functional changes observed in SI C57BL/6J mice were associated with an increased EtOH intake and EtOH preference. Nevertheless, the increase in EtOH consumption did not restore the reduction in hippocampal 3α,5α-TH PROG induced by SI. EtOH self-administration blocked the changes in gene expression of the α4 subunit but not those of the δ and γ2 subunits induced by SI. In addition, EtOH self-administration did not block the SI-induced changes in GABAAR-mediated tonic inhibition in hippocampal granule cells but increased the frequency of basal GABAergic sIPSCs in DG granule cells. We conclude that self-administration of EtOH selectively abolishes the increase of α4 subunit but not other neurochemical, molecular, and functional modifications induced by SI prolonged mild stress.

High affinity central benzodiazepine receptor ligands. Part 3: insights into the pharmacophore and pattern recognition study of intrinsic activities of pyrazolo[4,3-c]quinolin-3-ones

Novel 2-phenyl-2,5-dihydropyrazolo[4,3-c]quinolin-3-(3H)-ones (PQs) endowed with high affinity for central benzodiazepine receptor (BzR) were synthesized. In particular, 9-fluoro-2-(2-fluorophenyl)-2,5-dihydro-3H-pyrazolo[4,3-c]quinolin-3-one (2(2)) showed binding affinity in the subnanomolar concentration range and proved to be in vitro a potent antagonist. This finding allowed the nature of the hydrogen bonding receptor site H(2) to be established, as located between the N-1 nitrogen of the PQ nucleus and the ortho position of the N-2-aryl group. [35S]tert-Butylbicyclophosphorothionate ([35S]TBPS) binding assays and electrophysiological measurements of the effects on GABA-evoked Cl(-) currents at recombinant human alpha(1)beta(2)gamma(2)(L) GABA(A) receptors, expressed in Xenopus laevis oocytes, were used to assess the intrinsic activities of a large series of PQs. With the aim of extracting discriminant information and distinguishing BzR ligands with different profiles of efficacy, 51 PQ derivatives, including full and partial agonists, antagonists, and inverse agonists, were analyzed in a multidimensional chemical descriptor space, defined by the lipophilicity parameter CLOG P and 3-D molecular WHIM descriptors, by means of principal component analysis, k-nearest neighbors (k-NN) method, and linear discriminant analysis (LDA). The classification methods were applied to subsets of pairs of efficacy classes, and lipophilicity and 3-D size descriptors were detected as the discriminant variables by a stepwise linear discriminant analysis. LDA proved to be superior to k-NN, especially in classifying PQ ligands (60-84% of success in prediction ability) into categories of efficacies which were contiguous and quite overlapped in the hyperspace of variables.

Changes in GABAA Receptor Gene Expression Induced by Withdrawal of, but Not by Long-Term Exposure to, Ganaxolone in Cultured Rat Cerebellar Granule Cells

The effects of ganaxolone, a synthetic analog of the endogenous neuroactive steroid allopregnanolone, on the function and expression of GABAA receptors were determined. Electrophysiological recordings demonstrated that ganaxolone potentiated with a potency and efficacy similar to those of allopregnanolone the Cl−currents evoked by GABA at recombinant human GABAAreceptors (comprising α1β2γ2L or α2β2γ2L subunit assemblies) expressed in Xenopus oocytes. Exposure of cultured rat cerebellar granule cells to 1 μM ganaxolone for 5 days had no effect on the abundance of mRNAs encoding the α1, α2, α3, α4, α5, γ2L, or γ2S subunits of the GABAA receptor. Withdrawal of ganaxolone after such long-term treatment, however, induced an increase in the abundance of α2, α4, and α5 subunit mRNAs and a decrease in the amounts of α1, γ2L, and γ2S subunit mRNAs. These changes were maximal 3 to 6 h after drug withdrawal and were reversible, being no longer apparent after 24 h. These results suggest that long-term exposure of cerebellar granule cells to ganaxolone does not affect the sensitivity of the GABAAreceptor to several positive modulators. Nevertheless, the reduction in the amounts of the α1 and γ2 subunit mRNAs together with the increase in the abundance of the α4 subunit mRNA induced by abrupt discontinuation of long-term treatment with ganaxolone suggest that withdrawal of this drug might result in a reduced response to classic benzodiazepines.

The anesthetic potencies of alkanethiols for rats : Relevance to theories of narcosis

Meyer and Overton suggested that anesthetic potency correlates inversely with lipophilicity. Thus, MAC times the olive oil/gas partition coefficient equals an approximately constant value of 1.82 ± 0.56 atm (mean ± sd). MAC is the minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects. Although MAC times the olive oil/gas partition coefficient also equals an approximately constant value for normal alkanols from methanol through octanol, the value (0.156 ± 0.072 atm) is 1/10th that found for conventional anesthetics. We hypothesized that substitution of sulfur for the oxygen in n-alkanols would decrease their saline/gas partition coefficients (i.e., decrease polarity) while sustaining lipid/gas partition coefficients. Further, we hypothesized that these changes would produce products of MAC times olive oil partition coefficients that approximate those of conventional anesthetics. To test these predictions, we measured MAC in rats, and saline and olive oil solubilities for the series H(CH2)nSH, comparing the results with the series H(CH2)nOH for compounds having three to six carbon atoms. As hypothesized, the alkanethiols had similar oil/gas partition coefficients, 1000-fold smaller saline gas partition coefficients, and MAC values 30 times greater than for comparable alkanols. Such findings are consistent with the notion that the greater potency of many alkanols (greater than would be predicted from conventional inhaled anesthetics and the Meyer-Overton hypothesis) results from their greater polarity. Implications The in vivo anesthetic potency of alkanols and alkanethiols correlates with their lipophilicity and hydrophilicity.

Effects of propofol, pentobarbital and alphaxalone on t-[35S]butylbicyclophosphorothionate binding in rat cerebral cortex.

The effects of propofol, pentobarbital, alphaxalone, etomidate and diazepam on t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding to membrane preparations from rat cerebral cortex were studied in the absence of gamma-aminobutyric acid (GABA). Addition of low concentrations (3-10 microM) of propofol to washed membrane preparations (devoid of GABA) markedly enhanced [35S]TBPS binding (maximal enhancement, 85%), whereas higher concentrations (50-100 microM) inhibited this parameter. Diazepam also enhanced [35S]TBPS binding in this preparation (maximal enhancement, 38%). In contrast, pentobarbital, alphaxalone and etomidate decreased [35S]TBPS binding in a concentration-dependent manner. The propofol-induced increase in [35S]TBPS binding in washed membranes was completely reversed by the addition of GABA at a concentration (0.3 microM) that alone did not modify [35S]TBPS binding (78% increase with 10 microM propofol alone, 33% decrease in the additional presence of GABA). The ability of GABA to reverse the effect of propofol on [35S]TBPS binding in washed membranes was shared by pentobarbital (200 microM) and alphaxalone (3 microM); etomidate (20 microM) only partially antagonized the effect of propofol. Diazepam at a concentration (30 microM) that alone had no effect on [35S]TBPS binding failed to modify the propofol-induced increase in [35S]TBPS binding, whereas at a concentration (3 microM) that alone increased [35S]TBPS binding the effect of diazepam was additive with that of propofol. The addition of bicuculline to washed membranes failed to abolish the increase in [35S]TBPS binding induced by propofol or diazepam, but completely antagonized the effects of pentobarbital, alphaxalone and etomidate.(ABSTRACT TRUNCATED AT 250 WORDS)

The Anesthetic Potency of Propanol and Butanol Versus Propanethiol and Butanethiol in α1 Wild Type and α1(s267q) Glycine Receptors

Although similar in shape and size, and although differing only by substitution of a sulfur atom for an oxygen atom, propanethiol and butanethiol differ markedly from propanol and butanol in their in vivo potency and physical properties. Recent theories of narcosis suggest that anesthetics may act by enhancing the effect of inhibitory agonists, such as glycine, on their receptors. We tested whether propanol, butanol, propanethiol, and butanethiol enhance the effect of glycine on &agr;1 glycine receptors expressed in Xenopus laevis oocytes in a manner that reflects the in vivo differences found for potencies. As anticipated, we found an immediate parallel between in vivo (rat minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects) and in vitro (recombinant receptor) effects. All four compounds enhanced the effect of glycine on wild type receptors, and the extent of enhancement for a given minimum alveolar concentration-multiple was approximately the same for all compounds. We also found that propanethiol, butanethiol, propanol, and butanol did not affect, or minimally affected, the action of glycine in anesthetic resistant mutants in which the amino acid serine at position 267 was replaced by glutamine [&agr;1(S267Q)]. Implications The in vivo potencies of propanethiol, butanethiol, propanol, and butanol correlate with their capacities to enhance the effect of glycine on &agr;1 glycine receptors expressed in Xenopus laevis oocytes. These results support the notion that a protein mediates anesthetic action.

Design, Synthesis, and Biological Evaluation of Imidazo[1,5-a]quinoline as Highly Potent Ligands of Central Benzodiazepine Receptors

A series of imidazo[1,5-a]quinoline derivatives was designed and synthesized as central benzodiazepine receptor (CBR) ligands. Most of the compounds showed high CBR affinity with Ki values within the submicromolar and subnanomolar ranges with interesting modulations in their structure-affinity relationships. In particular, fluoroderivative 7w (Ki = 0.44 nM) resulted in the most potent ligand among the imidazo[1,5-a]quinoline derivatives described so far. Overall, these observations confirmed the assumption concerning the presence of a large though apparently saturable lipophilic pocket in the CBR binding site region interacting with positions 4 and 5 of the imidazo[1,5-a]quinoline nucleus. The in vivo biological characterization revealed that compounds 7a,c,d,l,m,q,r,w show anxiolytic and antiamnestic activities without the unpleasant myorelaxant side effects of the classical 1,4-BDZ. Furthermore, the effect of 7l,q,r, and 8i in lowering lactate dehydrogenase (LDH) release induced by ischemia-like conditions in rat brain slices suggested neuroprotective properties for these imidazo[1,5-a]quinoline derivatives.