How Areal Specification Shapes the Local and Interareal Circuits in a Macaque Model of Congenital Blindness.

There is little understanding of the structural underpinnings of the functional reorganization of the cortex in the congenitally blind human. Taking advantage of the extensive characterization of the macaque visual system, we examine in macaque the influence of congenital blindness resulting from the removal of the retina during in utero development. This effectively removes the normal influence of the thalamus on cortical development leading to an induced hybrid cortex (HC) combining features of primary visual and extrastriate cortex. Retrograde tracers injected in HC reveal a local, intrinsic connectivity characteristic of higher order areas and show that the HC receives a uniquely strong, purely feedforward projection from striate cortex but no ectopic inputs, except from subiculum, and entorhinal cortex. Statistical modeling of quantitative connectivity data shows that HC is relatively high in the cortical hierarchy and receives a reinforced input from ventral stream areas while the overall organization of the functional streams are conserved. The directed and weighted anophthalmic cortical graph from the present study can be used to construct dynamic and structural models. These findings show how the sensory periphery governs cortical phenotype and reveal the importance of developmental arealization for understanding the functional reorganization in congenital blindness.

Pentobarbital modifies the lipid raft-protein interaction: A first clue about the anesthesia mechanism on NMDA and GABAA receptors.

Recent studies have shown that anesthetic agents alter the physical properties of lipid rafts on model membranes. However, if this destabilization occurs in brain membranes, altering the lipid raft-protein interaction, remains unknown. We analyzed the effects produced by pentobarbital (PB) on brain plasma membranes and lipid rafts in vivo. We characterized for the first time the thermotropic behavior of plasma membranes, synaptosomes, and lipid rafts from rat brain. We found that the transition temperature from the ordered gel to disordered liquid phase of lipids is close to physiological temperature. We then studied the effect of PB on protein composition of lipid rafts. Our results show a reduction of the total protein associated to rafts, with a higher reduction of the NMDAR compared to the GABAA receptor. Both receptors are considered the main targets of PB. In general, our results suggest that lipid rafts could be plausible mediators in anesthetic action.

Effect of calcium hypochlorite and chloramine on blood biochemistry and sodium pentobarbital induced sleeping time in mice.

Disinfectants are chemical agents used to eradicate, deactivate or kill microorganisms. Chemical disinfectants especially chlorine compound are extensively used for water sanitization. Among these calcium hypochlorite and chloramines are commonly used now a day. Large number of chemical compounds, drugs and endogenous substances are metabolized by hepatic enzymes known as cytochrome P450 enzyme system. Many chemicals are capable of enzyme induction. Enzyme induction may change the metabolism of other drugs and endogenous substances which may alter the plasma concentration of these chemicals. To evaluate the enzyme inducing ability of calcium hypochlorite and chloramine, sleeping time induced by sodium pentobarbital was noted in mice. Normal saline was taken as negative control. Rifampicin, chloramphenicol and grapefruit juice were taken as positive control group. On completion of dosing after 4 weeks, alteration in sleep induction and recovery times was noted and compared. Histological evaluation of liver was observed. A significant decrease in sleeping time was observed in calcium hypochlorite and chloramine treated groups. Both calcium hypochlorite and chloramine caused a significant change in liver enzymes and in the values of complete blood count. In histological evaluation both caused fat deposition in the hepatocytes. It was concluded from the study that both calcium hypochlorite and chloramine were hepatic microsomal enzyme inducer.

Activation of the Rat α1ß2ε GABAA Receptor by Orthosteric and Allosteric Agonists.

GABAA receptors are a major contributor to fast inhibitory neurotransmission in the brain. The receptors are activated upon binding the transmitter GABA or allosteric agonists including a number of GABAergic anesthetics and neurosteroids. Functional receptors can be formed by various combinations of the nineteen GABAA subunits cloned to date. GABAA receptors containing the ε subunit exhibit a significant degree of constitutive activity and have been suggested to be unresponsive to allosteric agents. In this study, we have characterized the functional properties of the rat α1ß2ε GABAA receptor. We confirm that the α1ß2ε receptor exhibits a higher level of constitutive activity than typical of GABAA receptors and show that it is inefficaciously activated by the transmitter and the allosteric agonists propofol, pentobarbital, and allopregnanolone. Manipulations intended to alter ε subunit expression and receptor stoichiometry were largely without effect on receptor properties including sensitivity to GABA and allosteric agonists. Surprisingly, amino acid substitutions at the conserved 9' and 6' positions in the second transmembrane (TM2) domain in the ε subunit did not elicit the expected functional effects of increased constitutive activity and resistance to the channel blocker picrotoxin, respectively. We tested the accessibility of TM2 residues mutated to cysteine using the cysteine-modifying reagent 4-(hydroxymercuri)benzoic acid and found a unique pattern of water-accessible residues in the ε subunit.

The distribution and chemical coding of urinary bladder trigone-projecting neurons in testicular and aorticorenal ganglia in male pigs.

Combined retrograde tracing and double-labelling immunofluorescence were used to investigate the distribution and chemical coding of neurons in testicular (TG) and aorticoerenal (ARG) ganglia supplying the urinary bladder trigone (UBT) in juvenile male pigs (n=4, 12 kg. of body weight). Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of the bladder trigone under pentobarbital anesthesia. After three weeks all the pigs were deeply anesthetized and transcardially perfused with 4% buffered paraformaldehyde. TG and ARG, were collected and processed for double-labelling immunofluorescence. The expression of tyrosine hydroxylase (TH) or dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), nitric oxide synthase (NOS) and vesicular acetylcholine transporter (VAChT) were investigated. The cryostat sections were examined with a Zeiss LSM 710 confocal microscope equipped with adequate filter blocks. The TG and ARG were found to contain many FB-positive neurons projecting to the UBT (UBT-PN). The UBT-PN were distributed in both TG and ARG. The majority of them were found in the right ganglia, mostly in TG. Immunohistochemistry disclosed that the vast majority of UBT-PN were noradrenergic (TH- and/or DBH-positive). Many noradrenergic neurons contained also immunoreactivity to NPY, SOM or GAL. Most of the UBT-PN were supplied with VAChT-, or NOS- IR (immunoreactive) varicose nerve fibres. This study has revealed a relatively large population of differently coded prevertebral neurons projecting to the porcine urinary bladder. As judged from their neurochemical organization these nerve cells constitute an important element of the complex neuro-endocrine system involved in the regulation of the porcine urogenital organ function.

Babao Dan Alleviates 5-Fluorouracil-Induced Intestinal Damage via Wnt/ß-Catenin Pathway.

OBJECTIVE: To evaluate the protective function of Babao Dan (BBD) on 5-flurouracil (5-FU)-induced intestinal mucositis (IM) and uncover the underlying mechanism. METHODS: A total of 18 male mice were randomly divided into 3 groups by a random number table, including control, 5-FU and 5-FU combined BBD groups, 6 mice in each group. A single intraperitoneal injection of 5-FU (150 mg/kg) was performed in 5-FU and 5-FU combined BBD groups on day 0. Mice in 5-FU combined BBD group were gavaged with BBD (250 mg/kg) daily from day 1 to 6. Mice in the control group were gavaged with saline solution for 6 days. The body weight and diarrhea index of mice were recorded daily. On the 7th day, the blood from the heart of mice was collected to analyze the proportional changes of immunological cells, and the mice were subsequently euthanized by mild anesthesia with 2% pentobarbital sodium. Colorectal lengths and villus heights were measured. Intestinal-cellular apoptosis and proliferation were evaluated by Tunel assay and immunohistochemical staining of proliferating cell nuclear antigen, respectively. Immunohistochemistry and Western blot were performed to investigate the expressions of components in Wnt/ß-catenin pathway (Wnt3, LRP5, ß-catenin, c-Myc, LRG5 and CD44). RESULTS: BBD obviously alleviated 5-FU-induced body weight loss and diarrhea, and reversed the decrease in the number of white blood cells, including monocyte, granulocyte and lymphocyte, and platelet (P<0.01). The shortening of colon caused by 5-FU was also reversed by BBD (P<0.01). Moreover, BBD inhibited apoptosis and promoted proliferation in jejunum tissues so as to reduce the intestinal mucosal damage and improve the integrity of villus and crypts. Mechanically, the expression levels of Wnt/ß -catenin mediators such as Wnt3, LRP5, ß-catenin were upregulated by BBD, activating the transcription of c-Myc, LRG5 and CD44 (P<0.01). CONCLUSIONS: BBD attenuates the adverse effects induced by 5-FU via Wnt/ß-catenin pathway, suggesting it may act as a potential agent against chemotherapy-induced intestinal mucositis.

Yokukansan suppresses neuroinflammation in the hippocampus of mice and decreases the duration of lipopolysaccharide- and diazepam-mediated loss of righting reflex induced by pentobarbital.

Neuroinflammation is associated with the development of hypoactive delirium, which results in poor clinical outcomes. Drugs effective against hypoactive sur have not yet been established. Yokukansan has an anti-neuroinflammatory effect, making it potentially effective against hypoactive delirium. This study aimed to examine the effect of Yokukansan on the pentobarbital-induced loss of righting reflex duration extended with lipopolysaccharide (LPS)-induced neuroinflammation and diazepam-induced gamma-aminobutyric acid receptor stimulation in a mouse model. The active ingredients in Yokukansan and its anti-neuroinflammatory effect on the hippocampus were also investigated. Furthermore, we examined the in vitro anti-inflammatory effects of Yokukansan on LPS-stimulated BV2 cells, a murine microglial cell line. Findings revealed that treatment with Yokukansan significantly decreased the duration of pentobarbital-induced loss of righting reflex by attenuating the LPS-induced increase in interleukin-6 and tumor necrosis factor-alpha levels in the hippocampus. Moreover, treatment with Yokukansan significantly decreased the number of ionized calcium-binding adapter molecule-1-positive cells in the hippocampal dentate gyrus after 24 h of LPS administration. In addition, glycyrrhizic acid, an active ingredient in Yokukansan, partially decreased the duration of pentobarbital-induced loss of righting reflex. Treatment with Yokukansan also suppressed the expression of inducible nitric oxide, interleukin-6, and tumor necrosis factor mRNA in LPS-stimulated BV2 cells. Thus, these findings suggest that Yokukansan and glycyrrhizic acid may be effective therapeutic agents for treating neuroinflammation-induced hypoactive delirium.

Hepatic microsomal enzymes in man and rat: induction and inhibition by ethanol.

The feeding of ethanol increased significantly the activities of hepatic pentobarbital and benzpyrene hydroxylases in rats, and, in human volunteers, doubled pentobarbital hydroxylase activity. In vitro ethanol inhibited aniline, pentobarbital, and benzpyrene hydroxylases. These data may explain, at least in part, the increased tolerance of alcoholics to sedatives when sober, and the enhanced sensitivity to sedatives when inebriated.

Microsomal hydroxylation as measured by pentobarbital elimination in patients with idiopathic systemic lupus erythematosus.

A mechanism postulated for drug- or chemical-induced systemic lupus erythematosus (SLE) is that the chemical is covalently bound to nuclear macromolecules increasing the immunogenicity of the macromolecule. This may require metabolic activation by oxidation. There are many similarities between drug-induced and idiopathic SLE. Twelve patients with idiopathic SLE and 12 normal subjects were given 100 mg pentobarbital orally to evaluate their microsomal hydroxylating activity. Plasma pentobarbital concentration was measured by gas-liquid chromatography. Mean plasma pentobarbital half-life was 24 +/- 10 (mean +/- SD) hr in the SLE patients, which is only slightly shorter than the 26 +/- 12 hr in the control subjects. The mean apparent volume of distribution in the patients was 1.28 +/- 0.30 l/kg, which is slightly above the 1.00 +/- 0.37 l/kg in the normal subject (P less than 0.05). Mean metabolic clearance rate in the SLE patients was 0.045 +/- 0.022 l/hr/kg, which is more than the 0.028 +/- 0.008 l/hr/kg in the normal control subjects (P less than 0.02). Since the metabolic clearance rate of a drug is the proper value for evaluating metabolism rate, we conclude that patients with SLE hve an increased elimination rate for drugs or other foreign compounds that are biotransformed by microsomal oxidation and may more rapidly bioactivate chemicals to reactive compounds.

Pentobarbital elimination in patients with poor renal function.

Drug oxidation is slowed in experimental uremia in animals but has been reported to be normal or accelerated in uremic patients. Eleven normal subjects and 9 uremic patients were each given 100 mg pantobarbital orally. Several blood samples were drawn over a 36-hr period starting on the morning after the dose. Plasma pentobarbital concentration was measured by GLC. The log concentration values were graphed against time elapsed after dose for each patient, and the plasma T/2 and extrapolated value of concentration at the time of drug administration (Co) were determined. The T/2 values in normals ranged from 18 to 48 hr; mean, 26.5 +/- 9.2 (SD). In uremic patients, T/2 values are 10 to 38 hr, mean, 21.3 +/- 8.7. Four of the uremic patients had T/2 values below 18 hr (which was the lowest in the normals). The apparent volumes of distribution of pentobarbital (aVD = dose divided by Co) were 62 +/- 25 L in the normal subjects and 58 +/- 24 L in the uremic patients. The four uremic patients with the short T/2 values tended to have small apparent volumes of distribution so that the metabolic clearnce rates (aVd X 0.693 divided by half-life) were normal in 3 of them. We conclude that pentobarbital elimination is normal in renal failure. Some uremic patients have short plasma T/2 values for pentobarbital, and these more likely result from low apparent volumes of distribution with normal metabolic clearance rates than from accelerated metabolism of pentobarbital.

Pharmacokinetics of high-dose pentobarbital in severe head trauma.

High-dose pentobarbital infusion has been advocated as an effective adjunct in controlling persistent intracranial hypertension after severe head trauma in patients refractory to conventional therapy. Pentobarbital disposition was assessed in 10 adults with severe nonpenetrating head injury after an intravenous loading dose of sodium pentobarbital, 10 mg/kg, infused over 1 hour, followed by a continuous infusion at 0.5 to 3.0 mg/kg/hr provided the cerebral perfusion pressure remained greater than 50 torr. Pharmacokinetic parameters of volume of distribution at steady state (Vss), total body clearance (CL), and t1/2 for the patients with trauma were statistically compared with similar estimates reported for seven adult subjects without head injury. On discontinuation of the pentobarbital infusion, serum concentrations in the patients followed a monoexponential decline with a mean (+/- SD) t1/2 and Vss that were significantly less than values reported for the control subjects (15.6 +/- 3.9 vs. 22.3 +/- 4.0 hours and 44.0 +/- 11.7 vs. 63.4 +/- 15.2 L, respectively). However, there was no significant difference between the mean pentobarbital CL of the patients (2.0 +/- 0.7 L/hr) and the subjects (2.0 +/- 0.4 L/hr). To our knowledge this is the first report on the disposition, elimination, and intrasubject variability of high-dose pentobarbital infusion in adult patients with head trauma.

Effects of delta-9-tetrahydrocannabinol on drug distribution and metabolism. Antipyrine, pentobarbital, and ethanol.

Delta-9-tetrahydrocannabinol (THC) has been reported to inhibit drug metabolism in animals. Twenty-two hospitalized healthy volunteer subjects received THC, 60 to 180 mg/day in divided doses for 14 days. Body weight increased and plasma proteins decreased in all subjects, which is consistent with previously reported plasma volume expansion. Total bilirubin was significantly lower, while other liver function tests remained normal. A within-subject comparison of the pharmacokinetics of antipyrine, pentobarbital, or ethanol given before, during, and after THC was performed. Antipyrine plasma half-life increased during THC in 5 of 6 subjects--mean, 7.9 hr +/- 3.3 (SD) to 9.6 +/- 3.8. Pentobarbital half-life increased in 7 of 8 subjects--mean, 16.9 hr +/- 2.0 to 20.8 +/- 4.2. Blood ethanol disappearance rate decreased in 7 of 8 subjects from a mean of 0.26 mg/100 ml/min +/- 0.05 to 0.23 +/- 0.07. The effect of THC on disappearance rate of these drugs appeared to be due to a combination of: (1) increased distribution volume, due in part to expansion of extracellular fluid volume noted during THC ingestion, and (2) diminished metabolic clearance. THC also delayed absorption of pentobarbital and ethanol in several subjects. This is consistent with THC effects of slowing intestinal motility in animals. The effects of THC on absorption and drug elimination must be considered in evaluating interactions with other drugs.

The effect of age on the plasma protein binding of pentobarbitone in the mouse. A brief note.

Old mice show an increased sensitivity to parenteral pentobarbitone compared with young adult mice, despite similar plasma concentrations of the drug. One factor may be an alteration in the binding of barbiturate to the plasma protein, perhaps consequent on reduced hepatic synthesis of plasma protein with advancing age. As part of a study of the effect of age upon the pharmacological and adaptive response to barbiturates, binding of pentobarbitone to plasma proteins from mice of different ages was measured by equilibrium dialysis in the presence of a standard concentration of the drug. The total plasma protein concentration was measured and found to be the same in both age groups. The plasma protein binding of pentobarbitone was found to correlate significantly with the plasma albumin concentration, which was lower in the older mice. However, there was no significant overall difference in pentobarbitone binding between old and young mice. Pretreatment of the mice with phenobarbitone for 21 days had no effect upon the capacity for plasma protein binding of pentobarbitone in either age group. Thus, it seems unlikely that the increased sensitivity of older mice to pentobarbitone can be explained in terms of altered plasma protein binding of the drug.

Drug kinetics and alcohol ingestion.

Acute and chronic ethanol ingestion can alter both the pharmacodynamics and pharmacokinetics of other drugs. For psychotherapeutic drugs, modification of drug action by alcohol is much more important than kinetic interaction, such as ethanol induced drug metabolism. In contrast, the importance of the effects of alcohol on the kinetics of other classes of drug is incomplete. The probability and mechanism of alcohol kinetic interactions with other drugs can nevertheless be anticipated, in part, on the basis of the extent of binding of the drug to plasma proteins, the capacity of the liver for extracting the drug from blood passing through the liver and the true distribution space of the drug. Highly bound drugs with low intrinsic hepatic clearance are among the most commonly reported to have their kinetics altered by ethanol (e.g. benzodiazepines, phenytoin, tolbutamide and warfarin). Less highly bound drugs are less consistently affected (e.g. meprobamate, glutethimide, pentobarbitone and phenobarbitone). Acute administration of ethanol to laboratory animals or incubation of microsomal preparations with ethanol inhibits the mixed function oxidase activity. In the human, the elimination half-life of meprobamate, pentobarbitone and tolbutamide is increased by acute ethanol administration. Chronic administration of ethanol to rats and humans causes proliferation of the smooth endoplasmic reticulum, increase in microsomal protein content and cytochrome P450 and results in an augmentation in drug metabolising ability of the microsomes in vitro. Even though the plasma half-life of some drugs is decreased by chronic ethanol ingestion, the clinical determination of the mechanism is incomplete because few studies have measured drug metabolite levels. In addition, alcohol effects on drug distribution have not been studied very extensively. The effects of chronic alcohol ingestion on drugs with low and high hepatic extraction, high and low binding, important tissue localisation and microsomal and non-microsomal metabolism will be quite different. Systematic studies of the mechanism of alcohol kinetic interactions are needed. Such kinetic studies should be combined with pharmacodynamic measures in order to establish the clinical importance of changes in drug kinetics.

Effects of the calcium channel activator Bay K 8644 on general anaesthetic potency in mice.

1. The effects of the calcium channel activator, Bay K 8644, on the anaesthetic potencies of ethanol, argon and pentobarbitone were examined in mice. 2. Bay K 8644, at 1 mg kg-1 i.p., significantly antagonized the general anaesthetic potencies of ethanol and argon, but at 5 and 10 mg kg-1 this compound increased the general anaesthetic potency of these drugs. 3. At doses of 1, 5 and 10 mg kg-1 Bay K 8644 antagonized the anaesthetic effects of pentobarbitone. Bay K 8644, at the highest dose used, did not alter the brain concentrations of pentobarbitone or the blood concentrations of ethanol. 5. The effects of the different doses of Bay K 8644 on the actions of ethanol and of argon are compatible with the known partial agonist properties of this compound on calcium channels in vitro. 6. The actions of Bay K 8644 on the anaesthetic effects of pentobarbitone suggests that specific interactions may be involved in the anaesthetic actions of this compound.

Studies on the inhibition of clearance of organic dyes by Saramycetin.

1. Saramycetin, a polypeptide antifungal antibiotic has been found to retard the clearance of sulphobromophthalein (BSP) in man. An explanation for this observation was sought in several lower species.2. Doses of Saramycetin without effect on the other standard tests of hepatic function or on hepatic morphology profoundly altered the disposition of BSP and several other dyes in mice and dogs.3. Saramycetin strongly inhibited the hepatic enzyme which conjugates BSP to reduced glutathione, provoked a regurgitation of BSP from the liver into the bloodstream, and was anticholeretic in the dog.4. These diverse actions of Saramycetin may, in concert, explain the altered clearance of BSP. It is suggested that low doses of Saramycetin exert a pharmacological effect on certain hepatic excretory processes, whereas high doses are toxic.

A study of the factors affecting the sleeping time following intracerebroventricular administration of pentobarbitone sodium: effect of prior administration of centrally active drugs.

1 Injection of pentobarbitone sodium into a lateral cerebral ventricle of rats produced a loss of righting reflex. The duration of anaesthesia was dose-dependent.2 The optimum dose of pentobarbitone to allow study of the factors affecting the sleeping time was considered to be 650 mug injected in 25 mul water.3 In a study of the effect of age and sex on the sleeping time, the youngest rats used (88 g body weight) were found to be the most sensitive to barbiturate. Female rats were more sensitive than male animals.4 The duration of anaesthesia was not affected by induction or inhibition of hepatic drug-metabolizing enzyme activity.5 Prior administration (acute) of central nervous system depressant drugs shortened the latent period and prolonged the duration of sleep. Prior administration of stimulant drugs antagonized the effect of pentobarbitone.6 Animals withdrawn following chronic administration of a number of drugs, barbitone, barbitone/bemegride mixture, Mandrax (methaqualone: diphenhydramine; 10: 1), chlordiazepoxide, nitrazepam, chlorpromazine or ethanol, exhibited a significant tolerance to intracerebroventricularly administered pentobarbitone.7 Withdrawal of amphetamine, morphine, methyprylon or diazepam did not result in tolerance to intracerebroventricularly administered pentobarbitone.8 Chronic administration of all drugs except amphetamine and morphine induced a tolerance to intraperitoneally administered hexobarbitone (100 mg/kg).9 The usefulness of sleeping time determination following intracerebroventricular administration of pentobarbitone as an assessment of central nervous system excitability is discussed. It is concluded that this method gives a valid indication of the sensitivity of the central nervous system to barbiturate and of the level of excitability in general. The method is particularly applicable in situations where the activity of hepatic drug-metabolizing enzyme activity may be altered.

Stereoselective interaction of thiopentone enantiomers with the GABA(A) receptor.

1. As pharmacokinetic differences between the thiopentone enantiomers seem insufficient to explain the approximately 2 fold greater potency for CNS effects of (-)-S- over (+)-R-thiopentone, this study was performed to determine any enantioselectivity of thiopentone at the GABA(A) receptor, the primary receptor for barbiturate hypnotic effects. 2. Two electrode voltage clamp recording was performed on Xenopus laevis oocytes expressing human GABA(A) receptor subtype alpha1beta2gamma2 to determine relative differences in potentiation of the GABA response by rac-, (+)-R- and (-)-S-thiopentone, and rac-pentobarbitone. Changes in the cellular environment pH and in GABA concentrations were also evaluated. 3. With 3 microM GABA, the EC50 values were (-)-S-thiopentone (mean 26.0+/-s.e.mean 3.2 microM, n=9 cells) >rac-thiopentone (35.9+/-4.2 microM, n=6, P=0.1) >(+)-R-thiopentone (52.5+/-5.0 microM, n=8, P<0.02) >rac-pentobarbitone (97.0+/-11.2 microM, n=11, P<0.01). Adjustment of environment pH to 7.0 or 8.0 did not alter the EC50 values for (+)-R- or (-)-S-thiopentone. 4 Uninjected oocytes responded to >100 microM (-)-S- and R-thiopentone. This direct response was abolished by intracellular oocyte injection of 1,2-bis(2-aminophenoxy)ethane-N, N,N1,N1-tetraacetic acid (BAPTA), a Ca2+ chelating agent. With BAPTA, the EC50 values were (-)-S-thiopentone (20.6+/-3.2 microM, n=8) <(+)-R-thiopentone (36.2+/-3.2 microM, n=9, P<0.005). 5 (-)-S-thiopentone was found to be approximately 2 fold more potent than (+)-R-thiopentone in the potentiation of GABA at GABA(A) receptors expressed on Xenopus oocytes. This is consistent with the differences in potency for CNS depressant effects found in vivo.

The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study.

1. The effects of five structurally dissimilar general anaesthetics were examined in voltage-clamp recordings of agonist-evoked currents mediated by recombinant gamma-aminobutyric acid (GABA)A receptors composed of human alpha 1 beta 1 and gamma 2L subunits expressed in Xenopus laevis oocytes. A quantitative comparison of the effects of these agents was made upon recombinant glycine receptors expressed as a homo-oligomer of human alpha 1 subunits, or as a hetero-oligomer of human alpha 1 and rat beta subunits. 2. Complementary RNA-injected oocytes expressing GABAA receptors responded to bath applied GABA with an EC50 of 158 +/- 34 microM. Oocytes expressing alpha 1 and alpha 1 beta glycine receptors subsequent to cDNA injection displayed EC50 values of 76 +/- 2 microM and 66 +/- 2 microM, respectively, in response to bath applied glycine. 3. Picrotoxin antagonized responses mediated by homo-oligomeric alpha 1 glycine receptors with an IC50 of 4.2 +/- 0.8 microM. Hetero-oligomeric alpha 1 beta glycine receptors were at least 100-fold less sensitive to blockade by picrotoxin. 4. With the appropriate agonist EC10, propofol enhanced GABA and glycine-evoked currents to approximately the maximal response produced by a saturating concentration of either agonist (i.e. Imax). The calculated EC50 values were 2.3 +/- 0.2 microM, 16 +/- 3 microM and 27 +/- 2 microM, for GABAA alpha 1 beta 1 gamma 2L, glycine alpha 1 and alpha 1 beta receptors, respectively. At relatively high concentrations, propofol was observed to activate directly both GABAA and glycine receptors. 5. Pentobarbitone potentiated GABA-evoked currents to 117 +/- 8.5% of Imax with an EC50 of 65 +/- 3 microM. The barbiturate also produced a substantial enhancement of the glycine-evoked currents, Imax and EC50 values being 71 +/- 2% and 845 +/- 66 microM and 51 +/- 10% and 757 +/- 30 microM for homomeric alpha 1 and heteromeric alpha 1 beta glycine receptors respectively. At high concentrations, pentobarbitone directly activated GABAA, but not glycine, receptors. 6. The potentiation by propofol or pentobarbitone of currents mediated by alpha 1 homo-oligomeric glycine receptors was in both cases associated with a parallel sinistral shift of the glycine concentration-effect curve. The effects of binary combinations of pentobarbitone and propofol at maximally effective concentrations were mutually occlusive suggesting a common site, or mechanism, of action. 7. GABA-evoked currents were maximally potentiated by etomidate to 79 +/- 2% of Imax (EC50 of 8.1 +/- 0.9 microM). By contrast, glycine-induced currents mediated by alpha 1 and alpha 1 beta glycine receptor isoforms were enhanced only to 29 +/- 4% and 28 +/- 3% of Imax. Limited solubility precluded the calculation of EC50 values for the effect of etomidate at glycine receptors. None of the receptor isoforms examined were directly activated by etomidate. 8. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents to 69 +/- 4% of Imax, with an EC50 value of 89 +/- 6 nM. In contrast, both alpha 1 homo-oligomeric and alpha 1 beta hetero-oligomeric glycine receptors were insensitive to the action of this steroid. A direct agonist action of the steroid was discernible at GABAA, but not glycine, receptors. 9. Trichloroethanol, the active metabolite of the general anaesthetic chloral hydrate, enhanced glycine-evoked currents to 77 +/- 10% and 94 +/- 4% of Imax on alpha 1 and alpha 1 beta glycine receptors, with EC50 values of 3.5 +/- 0.1 mM and 5.9 +/- 0.3 mM respectively. On GABAA receptors, trichloroethanol had a lower maximum enhancement (52 +/- 5% of Imax), but a slightly higher potency (EC50 1.0 +/- 0.1 mM). Trichloroethanol activated neither GABAA, nor glycine, receptors. 10. The data demonstrate a variety of intravenous general anaesthetic agents, at clinically relevant concentrations, to augment preferentially GABA-evoked currents mediated by the alpha1beta1upsilon2L receptor subunit combination as compared to their effects on both alpha1 and alpha1beta glycine receptors. However, the presence on glycine receptors of lower affinity modulatory binding sites for pentobarbitone, propofol and trichloroethanol may aid in the identification of the molecular determinants of the CNS actions of these anaesthetics.

Acute effects of pentobarbital-anaesthesia on bile secretion.

Male Wistar rats were equipped with permanent catheters in the bile duct and the duodenum under ether anaesthesia, at least seven days before the experiments. By this technique, the enterohepatic circulation can be interrupted for bile collection without direct surgical intervention. 14C-Pentobarbital (26.6 mumole/100 g body wt) was injected intraperitoneally immediately before interruption of the enterohepatic circulation (NBD, Non-Bile Diverted) or after eight days of bile diversion (BD, Bile Diverted). In NBD rats, bile flow and biliary bile acid excretion were significantly reduced during the first hour after pentobarbital administration when compared to unanaesthetized controls, but markedly increased thereafter. Pentobarbital treatment slightly decreased biliary bile acid excretion in BD rats, but caused a 60% increase in bile flow. Within four hours 22.3 +/- 0.4% and 26.0 +/- 2.7% of the injected radioactivity was excreted into bile in NBD and BD rats, respectively. The calculated osmotic activity of pentobarbital and its metabolites was 47.8 +/- 5.2 microliter/mumole in NBD rats and 37.8 +/- 1.3 microliter/mumole in BD rats. Consequently, pentobarbital treatment affected the bile acid independent fraction of bile flow (BAIF). The calculated BAIF was 2.68 microliter/min/100 g body wt in unanaesthetized animals, but 4.27 microliter/min/100 g body wt in pentobarbital treated NBD rats. Corresponding values for BD rats were 1.70 and 2.38 microliter/min/100 g body wt. It is concluded that pentobarbital anaesthesia affects bile production in the rat by direct and indirect means. Firstly, pentobarbital and its metabolites are rapidly excreted into bile and exert a significant choleretic effect, thereby increasing the BAIF. Secondly, pentobarbital anaesthesia retards the exhaustion of the intestinal bile acid pool, which leads to secondary changes in the biliary excretion process.