Maximum sustainable speed, energetics and swimming kinematics of a tropical carangid fish, the green jack Caranx caballus.

Maximum sustained swimming speeds, swimming energetics and swimming kinematics were measured in the green jack Caranx caballus (Teleostei: Carangidae) using a 41 l temperature-controlled, Brett-type swimming-tunnel respirometer. In individual C. caballus [mean ±s.d. of 22·1 ± 2·2 cm fork length (L(F) ), 190 ± 61 g, n = 11] at 27·2 ± 0·7° C, mean critical speed (U(crit)) was 102·5 ± 13·7 cm s⁻¹ or 4·6 ± 0·9 L(F) s⁻¹. The maximum speed that was maintained for a 30 min period while swimming steadily using the slow, oxidative locomotor muscle (U(max,c)) was 99·4 ± 14·4 cm s⁻¹ or 4·5 ± 0·9 L(F) s⁻¹. Oxygen consumption rate (M in mg O2 min⁻¹) increased with swimming speed and with fish mass, but mass-specific M (mg O2 kg⁻¹ h⁻¹) as a function of relative speed (L(F) s⁻¹) did not vary significantly with fish size. Mean standard metabolic rate (R(S) ) was 170 ± 38 mg O2 kg⁻¹ h⁻¹, and the mean ratio of M at U(max,c) to R(S) , an estimate of factorial aerobic scope, was 3·6 ± 1·0. The optimal speed (U(opt) ), at which the gross cost of transport was a minimum of 2·14 J kg⁻¹ m⁻¹, was 3·8 L(F) s⁻¹. In a subset of the fish studied (19·7-22·7 cm L(F) , 106-164 g, n = 5), the swimming kinematic variables of tailbeat frequency, yaw and stride length all increased significantly with swimming speed but not fish size, whereas tailbeat amplitude varied significantly with speed, fish mass and L(F) . The mean propulsive wavelength was 86·7 ± 5·6 %L(F) or 73·7 ± 5·2 %L(T) . Mean ±s.d. yaw and tailbeat amplitude values, calculated from lateral displacement of each intervertebral joint during a complete tailbeat cycle in three C. caballus (19·7, 21·6 and 22·7 cm L(F) ; 23·4, 25·3 and 26·4 cm L(T) ), were 4·6 ± 0·1 and 17·1 ± 2·2 %L(T) , respectively. Overall, the sustained swimming performance, energetics, kinematics, lateral displacement and intervertebral bending angles measured in C. caballus were similar to those of other active ectothermic fishes that have been studied, and C. caballus was more similar to the chub mackerel Scomber japonicus than to the kawakawa tuna Euthynnus affinis.

Excitons in CsPbBr3 Halide Perovskite.

Excitons in Bridgman grown halide perovskite CsPbBr3 single crystals were examined using photoluminescence (PL) spectroscopy to determine the nature of the electronic states. The photoluminescence intensity was strongly temperature-dependent and depended upon the specific exciton band. At low temperatures intrinsic disorder and its related shallow below bandgap tail states determine the emission properties. Photoluminescence at low temperature revealed the presence of several strong bands at the band edge that is attributed to free or trapped/bound excitons. This PL emission results from strong electron-phonon coupling with an average phonon energy Eph of 6.5 and 27.4 meV for the emissions, comparable to that observed in other perovskites. The Huang-Rhys parameter S was calculated to be 3.81 and 1.51, indicating strong electron-phonon coupling. The interactions between electrons and phonons produce small polarons that tend to bind charge carriers and result in trapped/bound excitons. The transient photoluminescence response of each specific band was studied, and the results indicated a multiphonon recombination process. Average PL lifetimes of ∼17 ns for free excitons and ∼38 ns for trapped/bound excitons were determined. The observed edge states could be associated with native defects such as vacancies and interstitials, as well as twinning due to the cubic-to-tetragonal phase transition in CsPbBr3. Elimination of the trapping sites for binding excitons could lead to improved charge transport mobilities, carrier lifetimes, and detector properties in this system.

Magnetoamplification in a bipolar magnetic junction transistor.

We have demonstrated the first bipolar magnetic junction transistor using a dilute magnetic semiconductor. For an InMnAs p-n-p transistor magnetoamplification is observed at room temperature. The observed magnetoamplification is attributed to the magnetoresistance of the magnetic semiconductor InMnAs heterojunction. The magnetic field dependence of the transistor characteristics confirm that the magnetoamplification results from the junction magnetoresistance. To describe the experimentally observed transistor characteristics, we propose a modified Ebers-Moll model that includes a series magnetoresistance attributed to spin-selective conduction. The capability of magnetic field control of the amplification in an all-semiconductor transistor at room temperature potentially enables the creation of new computer logic architecture where the spin of the carriers is utilized.

Recent advances in the electrophysiological characterization of 5-HT3 receptors.

5-HT3 receptors are ligand-gated, cation-selective ion channels, mediating membrane depolarization and neuronal excitation. Established and potential therapeutic applications of selective 5-HT3 receptor antagonists, coupled with the localization of this receptor subtype within discrete areas of the CNS, have resulted in an intensification of research in this area. In this review, Jeremy Lambert and colleagues summarize recent developments in the electrophysiological characterization of 5-HT3 receptors, and comment upon the unresolved issue of 5-HT3 receptor heterogeneity.

Neurosteroids and GABAA receptor function.

In 1984, a potent and selective interaction of the steroidal anaesthetic alphaxalone with the GABAA receptor was demonstrated. Subsequent studies established that certain naturally occurring steroids were potent positive allosteric modulators of the GABAA receptor. Although peripheral endocrine glands are an important endogenous source, the brain can synthesize 'neurosteroids', and these have the potential to influence the activity of the GABAA receptor in the CNS. Systemic administration of steroids have clear behavioural effects. In this article, Jeremy Lambert and colleagues review recent advances in this field and discuss the therapeutic potential of this novel, non-genomic effect of steroids and investigate whether they may influence behaviour under physiological, or pathophysiological, conditions.

General anaesthetic action at transmitter-gated inhibitory amino acid receptors.

Research within the past decade has provided compelling evidence that anaesthetics can act directly as allosteric modulators of transmitter-gated ion channels. Recent comparative studies of the effects of general anaesthetics across a structurally homologous family of inhibitory amino acid receptors that includes mammalian GABAA, glycine and Drosophila RDL GABA receptors have provided new insights into the structural basis of anaesthetic action at transmitter-gated channels. In this article, the differential effects of general anaesthetics across inhibitory amino acid receptors and the potential relevance of such actions to general anaesthesia will be discussed.

Histochemistry defines a proteoglycan-rich layer in bovine flexor tendon subjected to bending.

Mid-substance fibrocartilage develops in bovine deep flexor tendon at the point where the tendon wraps under sesamoid bones of the foot and receives transverse compressive loading during locomotion. Fibrocartilage extends several millimeters into the tendon at this location and the proteoglycan-rich tissue stains intensely with Alcian blue. Using histochemical techniques we demonstrate the presence of aggrecan, type VI collagen, and hyaluronic acid in the extracellular matrix of this region of tendon. Biglycan staining was localized to the cells, however. Adjacent to the fibrocartilage, at the outer curvature of the tendon as it bends, the tissue resembles typical tensile tendon with dense bundles of linearly arranged collagen. Longitudinal sections revealed discrete layers of Alcian blue-stained material between the collagen bundles. We demonstrate that these layers of loose matrix also contain aggrecan, type VI collagen, and hyaluronic acid. However, the dense collagen bundles of this region are negative for these components. Transverse sections of tendon in the area adjacent to fibrocartilage show a distinct Alcian blue-stained structure surrounding vascular elements at the point where several fiber bundles come together. This is concluded to be the same structure as the Alcian blue-stained layers seen in longitudinal sections. These observations suggest that proteoglycan-rich matrices in tendon subjected to mechanical loading other than pure tension may serve multiple roles. Such matrices can not only provide compressive stiffness and separate and lubricate collagen bundles that move relative to each other, but may also protect the integrity of vasculature in tendon subjected to bending and shear.

Integration of TB-HIV services at an ANC facility in Frances Baard District, Northern Cape, South Africa.

BACKGROUND: Integrated tuberculosis-human immunodeficiency virus (TB-HIV) service delivery as part of maternal health services, including antenatal care (ANC), is widely recommended. This study assessed the implementation of collaborative TB-HIV service delivery at a hospital-based ANC service unit. METHODS: A record review of a random sample of 308 pregnant women attending the ANC service between April 2011 and February 2012 was conducted. Data were extracted from registers and patient case notes. Outcomes included the proportion of women who underwent HIV counselling and testing (HCT), CD4 count testing, antiretroviral treatment (ART), cotrimoxazole preventive treatment (CPT), TB screening and isoniazid preventive treatment (IPT). Analysis measured variations in patient characteristics associated with service delivery. RESULTS: All women underwent HCT; 80% of those who tested HIV-positive were screened for TB. Most (85.9%) of the HIV-positive women received a CD4 count. However, only 12.9% of eligible women received ART prophylaxis onsite, only 35.7% were referred for initiation of ART, only 42.3% commenced IPT and none received CPT or further investigations for TB. HIV-negative women had 2.6 higher odds (95%CI 1.3-5.3) of receiving TB screening than their HIV-positive counterparts. CONCLUSIONS: Although the identification of HIV-positive women and TB suspects was adequate, implementation of other TB-HIV collaborative activities was sub-optimal.

Contexte : Une offre de services intégrée de tuberculose et du virus de l'immunodéficience humaine (TB-VIH)­en tant qu'élément des services de santé maternelle, notamment des consultations prénatales (ANC)­est largement recommandée. Cette étude a évalué la mise en œuvre d'une offre de services intégrée TB-VIH dans une unité hospitalière de CPN.Méthodes : Les dossiers d'un échantillon aléatoire de 308 femmes enceintes qui ont fréquenté le service ANC entre avril 2011 et février 2012 ont été revus. Les données ont été extraites à partir des registres ainsi que des dossiers des patients. Les résultats attendus comprenaient la proportion de femmes bénéficiant d'un conseil et test VIH (HCT), d'un comptage des CD4, d'un traitement antirétroviral (ART), d'un traitement préventif par cotrimoxazole (CPT), d'un dépistage de TB et d'un traitement préventif par isoniazide (IPT). L'analyse a mesuré les variations des caractéristiques des patients associées à l'offre de services.Résultats : Toutes les femmes ont bénéficié du HCT et 80% de celles ayant eu un test VIH positif ont eu un dépistage de TB. La majorité (85,9%) des femmes VIH-positives ont eu un comptage des CD4. Cependant, seulement 12,9% des femmes éligibles ont reçu une prophylaxie ART sur place ; seulement 35,7% ont été référées pour une mise en route de l'ART ; seulement 42.3% ont commencé l'IPT ; et aucune n'a reçu de CPT ni d'autres investigations relatives à la TB. Les femmes VIH négatives avaient 2,6 fois (IC95% 1,3­5,3) plus de chances de bénéficier d'un dépistage de TB que leurs homologues VIH positives.Conclusions: L'identification des femmes VIH positives et de celles suspectes de TB a été satisfaisante, mais la mise en œuvre des autres activités de collaboration TB-VIH a été insuffisante.

Marco de referencia: La prestación integrada de servicios de atención de la tuberculosis (TB) y la infección por el virus de la inmunodeficiencia humana (VIH) se recomienda ampliamente como parte de los servicios que se ofrecen a las madres durante la atención prenatal (ANC). En el presente estudio se evaluó la introducción de los servicios integrados de TB y VIH en una unidad hospitalaria de ANC.Método: Se examinaron las historias clínicas de una muestra aleatoria de 308 embarazadas que acudieron al servicio de ANC entre abril del 2011 y febrero del 2012. Se extrajeron datos de los registros y las historias clínicas de las pacientes. Los criterios de evaluación fueron la proporción de mujeres en quienes se practicó la orientación y las pruebas diagnósticas del VIH (HCT), el recuento de linfocitos CD4, el tratamiento antirretrovírico (ART), el tratamiento preventivo con cotrimoxazol (CPT), la detección sistemática de la TB y el tratamiento preventivo con isoniazida (IPT). En el análisis se midieron las variaciones en las características de las pacientes asociadas con la prestación de los servicios.Resultados: Todas las mujeres recibieron HCT y en 80% que obtuvieron un resultado positivo, se practicó la detección sistemática de la TB. En la mayoría de las pacientes positivas frente al VIH se practicó el recuento de linfocitos CD4 (85,9%). Sin embargo, solo 12,9% de las mujeres aptas recibieron la profilaxis ART en el lugar de la consulta; solo 35,7% se remitieron con el fin de comenzar el ART; apenas 42,3% de las pacientes comenzaron el IPT; y ninguna recibió CPT ni tuvo investigaciones complementarias por TB. Las mujeres con resultados negativos frente al VIH exhibieron un cociente de posibilidades 2,6 veces inferior de beneficiar de la detección sistemática de la TB, en comparación con las mujeres VIH positivas.Conclusión: Se constató una detección adecuada de las mujeres positivas frente al VIH y de casos con presunción clínica de TB, pero una prestación deficiente de las demás actividades de los servicios integrados del VIH y la TB.

Familial testicular germ cell tumors (FTGCT) - overview of a multidisciplinary etiologic study.

This Review summarizes the cumulative results of the National Cancer Institute Clinical Genetics Branch Multidisciplinary Etiologic Study of Familial Testicular Germ Cell Tumors (FTGCT). Initiated 12 years ago, this protocol enrolled 724 subjects from 147 unrelated families with either ≥2 affected men (n = 90) with TGCT or a proband with bilateral TGCT and a negative family history for this cancer (n = 57). Data were collected directly from 162 subjects evaluated at the NIH Clinical Center, and 562 subjects provided information from their home communities (Field Cohort). The primary study aims included (i) ascertaining, enrolling eligible FTGCT kindred, (ii) characterizing the clinical phenotype of multiple-case families, (iii) identifying the underlying genetic mechanism for TGCT susceptibility in families, (iv) evaluating counseling, psychosocial, and behavioral issues resulting from membership in an FTGCT family, and (v) creating an annotated biospecimen repository to permit subsequent translational research studies. Noteworthy findings include (i) documenting the epidemiologic similarities between familial and sporadic TGCT, (ii) demonstrating significantly younger age-at-diagnosis for familial vs. sporadic TGCT, (iii) absence of a dysmorphic phenotype in affected family members, (iv) shifting the focus of gene discovery from a search for rare, highly penetrant susceptibility variants to the hypothesis that multiple, more common, lower penetrance genes underlie TGCT genetic risk, (v) implicating testicular microlithiasis in FTGCT risk, and (vi) observing that aberrant methylation may contribute to FTGCT risk. A clinically based, biospecimen-intensive, multidisciplinary research strategy has provided novel, valuable insights into the etiology of FTGCT, and created a research resource which will support FTGCT clinical and laboratory studies for years to come.

Modulation of human recombinant GABAA receptors by pregnanediols.

Utilising two point voltage-clamp techniques on Xenopus laevis oocytes expressing human (alpha 1 beta 1 gamma 2L) recombinant GABAA receptors, the GABA modulatory actions of six naturally occurring neurosteroids have been determined and compared with those of known positive allosteric modulators. The anaesthetic steroids 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one produced a concentration-dependent enhancement of the GABA-evoked current. The maximal enhancement of the agonist-induced response produced by these steroids was intermediate between that of pentobarbitone and diazepam, but much greater than that caused by bretazenil. For both the 5 alpha and 5 beta steroid a reduction of the 20 ketone group to form either the corresponding 20 alpha or 20 beta hydroxy steroid produced, in all cases, a reduction in potency and a decrease in the maximal effect. The relationship of steroid structure to these two parameters is considered. The influence of the alpha subtype (alpha x beta 1 gamma 2L, where x = 1, 2 or 3) for the behaviourally active 5 alpha-pregnan-3 alpha,20 alpha-diol is also determined. Although the maximal effect of the steroid is not influenced by the alpha subtype, the alpha 2-containing receptor exhibits a modest decrease (approximately 6-fold) in potency compared to alpha 1- and alpha 3-containing receptors. The results described here are discussed in relation to the distinct behavioural actions of the neurosteroids.

Pharmacological characterization of the apparent splice variants of the murine 5-HT3 R-A subunit expressed in Xenopus laevis oocytes.

The actions of 5-hydroxytryptamine3 (5-HT3) receptor agonists and antagonists have been determined on the recombinant murine 5-HT3 R-A and an apparent splice variant of this subunit, termed 5-HT3 R-AS. When expressed in Xenopus laevis oocytes, both forms of the subunit functioned as a homo-oligomeric complex and exhibited inward current responses to bath applied 5-HT. Analysis of the 5-HT concentration-response curve obtained with either homo-oligomer gave Hill coefficients greater than two, suggesting positive co-operativity within the receptor complex. The rank order of potency of a range of 5-HT3 receptor agonists [m-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT (2-Me-5-HT) > or = phenylbiguanide] was identical for both subunits. Indeed, with the exception of 2-Me-5-HT, for the agonists tested there was little difference across the subunits in either their potency, or the maximal current response that they elicited relative to 5-HT. Although 2-Me-5-HT exhibited a similar potency for both subunits, the maximal response evoked by this agonist at the 5-HT3 R-AS subunit was much reduced when compared to the 5-HT3 R-A subunit. The 5-HT-induced current mediated by either form of the subunit was inhibited by the 5-HT3 receptor selective antagonists BRL 46470, granisetron and ondansetron and the non-selective antagonists (+)-tubocurarine, metoclopramide and cocaine in a reversible and concentration-dependent manner. These antagonists did not discriminate between the subunits and their potencies were similar to those reported previously for 5-HT3 receptors native to murine neuronal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375.

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABAA receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABAA (alpha1beta2gamma2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABAA receptors. The GABA enhancing action of E-6375 on recombinant GABAA receptors was unaffected by the subtype of the alpha isoform (i.e. alphaxbeta2gamma2L; x=1-3) within the receptor, but was increased by the omission of the gamma2L subunit. Receptors incorporating beta2, or beta3, subunits were more sensitive to modulation by E-6375 than those containing the beta1 subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the beta-subunit. The various in vivo actions of general anaesthetics may be mediated by GABAA receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABAA receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.

The anaesthetic action and modulation of GABAA receptor activity by the novel water-soluble aminosteroid Org 20599.

The anaesthetic profile of a novel water-soluble aminosteroid, Org 20599 [(2 beta, 3 alpha, 5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one methanesulphonate], and the ability of the compound to allosterically regulate the activity of the GABAA receptor, have been studied in comparison to the properties of established intravenous general-anaesthetic agents. Intravenously administered Org 20599 produced a rapid onset, short duration loss of the righting reflex in mice. The anaesthetic potency of Org 20599 was comparable to that of the steroids 5 alpha-pregnan-3 alpha-ol-20-one or alphaxalone, and exceeded that of propofol, thiopentone or pentobarbitone. Org 20599 and the reference anaesthetic agents allosterically displaced the binding of [35S]-t-butylbicyclophosphorothionate (TBPS) from GABAA receptors of rat-brain membranes with the order of potency: 5 alpha-pregnan-3 alpha-ol-20-one > Org 20599 > alphaxalone > propofol > thiopentone > pentobarbitone. At human recombinant alpha 1, beta 2, gamma 2L subunit-containing GABAA receptors expressed in Xenopus laevis oocytes, the anaesthetic agents produced a concentration-dependent and reversible potentiation of the peak amplitude of GABA-evoked currents. A similar positive allosteric action of Org 20599 was observed for the GABAA receptors expressed by bovine adrenal chromaffin cells maintained in culture. The rank order of potency in the aforementioned assays was identical to that determined from the displacement of TBPS binding. At concentrations greater than those required for potentiation of GABA, the anaesthetics exhibited GABA-mimetic effects with a rank order of potency that paralleled their modulatory activity. Such direct agonism varied greatly in maximal effect between compounds. The modulatory and direct agonist actions of Org 20599 were additionally confirmed utilizing rat hippocampal neurones in culture. The results indicate Org 20599 to be a potent and short-acting intravenous anaesthetic agent in mice and suggest positive allosteric regulation of GABAA receptor function to be a plausible molecular mechanism of action for the drug.

Anesthetic activity of novel water-soluble 2 beta-morpholinyl steroids and their modulatory effects at GABAA receptors.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

Alpha-amino acid phenolic ester derivatives: novel water-soluble general anesthetic agents which allosterically modulate GABA(A) receptors.

In the search for a novel water-soluble general anesthetic agent the activity of an alpha-amino acid phenolic ester lead, identified from patent literature, was markedly improved. In addition to improving in vivo activity in mice, good in vitro activity at GABA(A) receptors was also conferred. Within the series of compounds good enantioselectivity for both in vitro and in vivo activity was found, supporting a protein-mediated mechanism of action for anesthesia involving allosteric modulation of GABA(A) receptors. alpha-Amino acid phenolic ester 19, as the hydrobromide salt Org 25435, was selected for clinical evaluation since it retained the best overall anesthetic profile coupled with improved stability and water solubility. In the clinic it proved to be an effective intravenous anesthetic in man with rapid onset of and recovery from anesthesia at doses of 3 and 4 mg/kg.

Ketamine potentiates 5-HT3 receptor-mediated currents in rabbit nodose ganglion neurones.

The interaction of ketamine with the 5-HT3 receptor of rabbit nodose ganglion neurones is described. Ketamine (3-30 microM) enhanced 5-HT3 receptor-mediated currents recorded under voltage-clamp conditions. This action did not appear to be related to the known effect of ketamine of inhibiting 5-HT uptake.

An electrophysiological investigation of the properties of a murine recombinant 5-HT3 receptor stably expressed in HEK 293 cells.

1. The pharmacological and biophysical properties of a recombinant 5-HT3 receptor have been studied by use of patch-clamp techniques applied to HEK 293 cells stably transfected with the murine 5-HT3 R-A cDNA. 2. At a holding potential of -60 mV, 77% of cells investigated responded to ionophoretically applied 5-HT with an inward current. Such currents were unaffected by methysergide (1 microM), or ketanserin (1 microM), but were antagonized in a concentration-dependent and reversible manner by the selective 5-HT3 receptor antagonist, ondansetron (IC50 = 440 pM) and the non-selective antagonists (+)-tubocurarine (IC50 = 1.8 nM) and metoclopramide (IC50 50 nM). 3. The 5-HT-induced current reversed in sign (E5-HT) at approximately -2mV and exhibited inward rectification. The influence of extra- and intracellular ion substitutions upon E5-HT indicates the 5-HT-evoked current to be mainly mediated by a mixed monovalent cation conductance. 4. Calcium and magnesium (0.1-10 nM) produced a concentration-dependent, voltage-independent, inhibition of the 5-HT-induced response. Zinc (0.3-300 microM) exerted a biphasic effect with low concentrations enhancing, and high concentrations depressing, the 5-HT-evoked current. 5. Fluctuation analysis of inward currents evoked by a low (1 microM) concentration of 5-HT suggests the current to be mediated by the opening of channels with a conductance of 420 fS. 6. The pharmacological and biophysical properties of the 5-HT3 R-A are similar to those previously described for 5-HT3 receptors native to murine neuroblastoma cell lines, with the exception that the function of the recombinant receptor was enhanced by low concentrations of zinc. This observation suggests that the properties of the native receptor are not completely represented by the 5-HT3 R-A subunit alone.

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.

Subunit-dependent interaction of the general anaesthetic etomidate with the gamma-aminobutyric acid type A receptor.

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant gamma-aminobutyric acidA (GABAA) receptor subunits. 2. Currents mediated by recombinant receptors with the ternary subunit composition alpha x beta y gamma 2L (where x = 1,2,3 or 6 and y = 1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the alpha and beta subunit isoforms. 3. For the beta 2-subunit containing receptors tested, the EC50 for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 microM) was little affected by the nature of the alpha subunit present within the hetero-oligomeric complex. However, replacement of the beta 2 by the beta 1 subunit produced a 9-12 fold increase in the etomidate EC50 (6 to 11 microM) for all alpha-isoforms tested. 4. For alpha 1, alpha 2 and alpha 6, but not alpha 3-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for beta 2- than for beta 1-subunit containing receptors. This was most clearly exemplified by receptors composed of alpha 6 beta 1 gamma 2L compared to alpha 6 beta 2 gamma 2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28 +/- 2% and 169 +/- 4% of the maximal GABA response, respectively. 5. For alpha 1 subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the beta subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one was marginally higher for beta 1 rather than the beta 2 subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms. 6. The GABA-mimetic action of etomidate was supported by beta 2- but not beta 1-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either beta isoform. For beta 2-subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the alpha isoform. 7. It is concluded that the subtype of beta-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the beta isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the alpha-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.