Agonist-induced desensitization of human β3-adrenoceptors expressed in human embryonic kidney cells

β₃-Adrenoceptors are resistant to agonist-induced desensitization in some cell types but susceptible in others including transfected human embryonic kidney (HEK) cells. Therefore, we have studied cellular and molecular changes involved in agonist-induced β₃-adrenoceptor desensitization in HEK cells. Cells were treated with isoprenaline or forskolin, and following wash-out, cyclic adenosine monophosphate (cAMP) accumulation in response to freshly added agonist was quantified. Receptor and G protein expression were quantified by radioligand binding and immunoblot experiments, respectively. Treatment with isoprenaline induced a concentration- and time-dependent desensitization of cAMP accumulation in response to freshly added isoprenaline. This functional desensitization primarily consisted of reduced maximum responses with little change of agonist potency. Maximum desensitization was achieved by pre-treatment with 10 μM isoprenaline for 24 h. It was not accompanied by changes in β₃-adrenoceptor density as assessed in saturation radioligand-binding studies. The desensitization was associated with a small reduction in immunoreactivity for α-subunits for G_s and G_i1, whereas that for G_i2, G_i3, and G_q/11 was not significantly altered. In cells treated with pertussis toxin, isoprenaline-induced cAMP accumulation as well as desensitization by isoprenaline pre-treatment remained unchanged. Isoprenaline pre-treatment also reduced forskolin-induced cAMP accumulation; conversely, pre-treatment with forskolin caused a similar desensitization of isoprenaline-induced cAMP accumulation. We conclude that agonist-induced β₃-adrenoceptor desensitization in HEK cells does not involve reduced receptor numbers and small, if any, reduction of G_s expression; changes at the level of adenylyl cyclase function can fully explain this desensitization.     

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

Kinetics of desensitization and recovery from desensitization for human α4β2-nicotinic acetylcholine receptors stably expressed in SH-EP1 cells

Aim： Studies were conducted to define the kinetics of the onset of and recovery from desensitization for human α4β2- nicotinic acetylcholine receptors （nAChR） heterologously expressed in the SH-EP1 human epithelial cell line. Methods： Whole-cell patch clamp recordings were performed to evaluate α4β2-nAChR currents. Results： Application of 0.1μmol/L nicotine or 1 mmol/L acetylcholine （ACh） for I s or longer induced two phases, with time constants of -70 and -700 ms, for the onset of α4β2-nAChR desensitization. For a given duration of agonist exposure, recovery from desensitization induced by nicotine was slower than recovery from ACh-induced desensitization. Comparisons with published reports indicate that time constants for the recovery of α4β2-nAChRs from desensitization are smaller than those for the recovery of human muscle-type nAChRs from desensitization produced by the same concentrations and durations of exposure to an agonist. Moreover, the extent of human α4β2-nAChR desensitization and rate of recovery are the same, regardless of whether they are measured using whole-cell recording or based on published findings using isotopic ion flux assays; this equality demonstrates the equivalent legitimacy of these techniques in the evaluation of nAChR desensitization. Perhaps most significantly, recovery from desensitization also was best fit to a biphasic process. Regardless of whether it was fit to single or double exponentials, however, half-times for recovery from desensitization grew progressively longer with an increased duration of agonist exposure during the desensitizing pulse. Conclusion： These findings indicate the existence of α4β2-nAChRs in many distinctive states of desensitization, as well as the induction of progressively deeper states of desensitization with the increased duration of agonist exposure.     

Properties of human brain sodium channel α-subunits expressed in HEK293 cells and their modulation by carbamazepine,phenytoin and lamotrigine

Background and purpose

Voltage-activated Na⁺ channels contain one distinct α-subunit. In the brain Na_V1.1, Na_V1.2, Na_V1.3 and Na_V1.6 are the four most abundantly expressed α-subunits. The antiepileptic drugs (AEDs) carbamazepine, phenytoin and lamotrigine have voltage-gated Na⁺ channels as their primary therapeutic targets. This study provides a systematic comparison of the biophysical properties of these four α-subunits and characterizes their interaction with carbamazepine, phenytoin and lamotrigine.

Experimental approach

Na⁺ currents were recorded in voltage-clamp mode in HEK293 cells stably expressing one of the four α-subunits.

Key results

Na_V1.2 and Na_V1.3 subunits have a relatively slow recovery from inactivation, compared with the other subunits and Na_V1.1 subunits generate the largest window current. Lamotrigine evokes a larger maximal shift of the steady-state inactivation relationship than carbamazepine or phenytoin. Carbamazepine shows the highest binding rate to the α-subunits. Lamotrigine binding to Na_V1.1 subunits is faster than to the other α-subunits. Lamotrigine unbinding from the α-subunits is slower than that of carbamazepine and phenytoin.

Conclusions and implications

The four Na⁺ channel α-subunits show subtle differences in their biophysical properties, which, in combination with their (sub)cellular expression patterns in the brain, could contribute to differences in neuronal excitability. We also observed differences in the parameters that characterize AED binding to the Na⁺ channel subunits. Particularly, lamotrigine binding to the four α-subunits suggests a subunit-specific response. Such differences will have consequences for the clinical efficacy of AEDs. Knowledge of the biophysical and binding parameters could be employed to optimize therapeutic strategies and drug development.     

Etomidate produces similar allosteric modulation in α1β3γ and α1β3γ2L GABAA receptors

Background and Purpose

Neuronal GABA_A receptors are pentameric chloride ion channels, which include synaptic αβγ and extrasynaptic αβδ isoforms, mediating phasic and tonic inhibition respectively. Although the subunit arrangement of αβγ receptors is established as β-α-γ-β-α, that of αβδ receptors is uncertain and possibly variable. We compared receptors formed from free α1, β3 and δ or γ2L subunits and concatenated β3-α1-δ and β3-α1 subunit assemblies (placing δ in the established γ position) by investigating the effects of R-(+)-etomidate (ETO), an allosteric modulator that selectively binds to transmembrane interfacial sites between β3 and α1.

Experimental Approach

GABA-activated receptor-mediated currents in Xenopus oocytes were measured electrophysiologically, and ETO-induced allosteric shifts were quantified using an established model.

Key Results

ETO (3.2 μM) similarly enhanced maximal GABA (1 mM)-evoked currents in oocytes injected with 5 ng total mRNA and varying subunit ratios, for α1β3(1:1), α1β3δ(1:1:1) and α1β3δ(1:1:3), but this potentiation by ETO was significantly greater for β3-α1-δ/β3-α1(1:1) receptors. Reducing the amount of α1β3δ(1:1:3) mRNA mixture injected (0.5 ng) increased the modulatory effect of ETO, matching that seen with β3-α1-δ/β3-α1(1:1, 1 ng). ETO similarly reduced EC₅₀s and enhanced maxima of GABA concentration-response curves for both α1β3δ and β3-α1-δ/β3-α1 receptors. Allosteric shift parameters derived from these data depended on estimates of maximal GABA efficacy, and the calculated ranges overlap with allosteric shift values for α1β3γ2L receptors.

Conclusion and Implications

Reducing total mRNA unexpectedly increased δ subunit incorporation into receptors on oocyte plasma membranes. Our results favour homologous locations for δ and γ2L subunits in α1β3γ2/δ GABA_A receptors.     

Strong activation of ether-à-go-go-related gene 1 K+ channel isoforms by NS1643 in human embryonic kidney 293 and Chinese hamster ovary cells

Two different mechanisms leading to increased current have been described for the small-molecule human ether-à-go-go-related gene (herg) activator NS1643 [1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea]. On herg1a channels expressed in Xenopus laevis oocytes, it mainly acts via attenuation of inactivation and for rat (r) erg1b channels expressed in human embryonic kidney (HEK)-293 cells, it strongly shifts the activation curve to the left. We now investigated the NS1643 effects on erg1b channels in more detail and performed comparative experiments with rat and human erg1a in different expression systems. Significant differences were observed between expression systems, but not between the rat and human isoform. In HEK-293 or Chinese hamster ovary (CHO) cells, activation of rat erg1b channels occurred in a dose-dependent manner with a maximum current increase of 300% obtained with 10 μM NS1643. In contrast, the NS1643-induced strong leftward shift in the voltage dependence of activation further increased with higher drug concentration, needed more time to develop, and exhibited use dependence. Coexpression of KCNE1 or KCNE2 did not attenuate this NS1643 effect on erg1 channel activation and did thus not mimic the lower drug potency on this parameter observed in oocytes. NS1643 (10 μM) slowed erg1b channel deactivation and recovery from inactivation without significant changes in activation and inactivation kinetics. With the exception of accelerated activation, NS1643 affected erg1a channels similarly, but the effect was less pronounced than in erg1b or erg1a/1b channels. It is noteworthy that rerg1b and herg1a inactivation estimated from fully activated current voltage relationships were unaltered in the continued presence of 10 μM NS1643 in the mammalian expression systems, indicating qualitative differences from NS1643 effects in X. laevis oocytes.     

Modulation of the human ρ1 GABAA receptor by inhibitory steroids

Rationale

Modulators of the ρ1 GABA_A receptor may be useful in the treatment of visual, sleep, and cognitive disorders. Neuroactive steroids and analogues have been shown to modulate ρ1 receptor function, but the molecular mechanisms are poorly understood.

Objectives

We employed electrophysiology and voltage-clamp fluorometry to compare the actions of several neuroactive steroids and analogues on the human ρ1 GABA_A receptor.

Results

Results confirmed that P294S and T298F mutations affect modulation by steroids. The P294S mutation abolished inhibition by (3α,5β)-3-hydroxypregnan-20-one (3α5βP) while the T298F mutation eliminated inhibition by 17β-estradiol. Voltage-clamp fluorometry demonstrated that steroids differing in the presence of a charged group on C3 or nature of substituent on C17 uniquely modified fluorescence changes elicited by GABA in the extracellular domain. The I307Q mutation reversed the inhibitory effect of 3α5βP but was without effect on modulation by (3α,5β)-3-hydroxypregnan-20-one sulfate or 17β-estradiol. The effect of 3α5βP on the fluorescence change generated at Y241C was dependent on whether the steroid acted as an inhibitor or a potentiator. Further, the effect was limited to uncharged 5β-reduced steroids containing an acetyl group on C17.

Conclusions

The data demonstrate that steroids and analogues differ with respect to conformational changes elicited by these drugs as well as sensitivity to the effects of mutations. Steroids and analogues could be provisionally divided into three major groups based on their actions on the ρ1 GABA_A receptor: 5β-reduced uncharged steroids, sulfated and carboxylated steroids, and 17β-estradiol. Further division among 5β-reduced uncharged steroids was based on substituent at position C17.     

Role of GABAA/benzodiazepine receptors containing α1 and α5 subunits in the discriminative stimulus effects of triazolam in squirrel monkeys

RATIONALE: Conventional benzodiazepines (BZs), clinically used for treatment of anxiety and insomnia, bind to GABA(A) receptors containing alpha(1), alpha(2), alpha(3), or alpha(5) subunits. The role of these different GABA(A) receptor subtypes in mediating the subjective effects of BZs remains largely unknown. OBJECTIVE: The purpose of the present study was to evaluate the role of GABA(A) receptors containing the alpha(1) or alpha(5) subunits in the discriminative stimulus (DS) effects of the conventional BZ agonist triazolam. METHODS: Squirrel monkeys were trained to discriminate triazolam (0.03 mg/kg, i.v.) from vehicle under a fixed-ratio 10 schedule of food reinforcement. RESULTS: The GABA(A)/alpha(1)-preferring agonists zolpidem and zaleplon engendered responses predominantly on the triazolam lever (73-80% drug-lever responding), and the GABA(A)/alpha(1) partial agonist CL 218,872 engendered an average maximum of less than 50% triazolam-lever responding. The GABA(A)/alpha(1)-preferring antagonists beta-carboline-3-carboxylate-t-butyl ester (betaCCT) and 3-(propyloxy)-beta-carboline (3-PBC) blocked the DS effects of triazolam and zolpidem in a surmountable manner. Schild analyses for betaCCT and 3-PBC in combination with triazolam and zolpidem suggest that the interactions between these compounds were competitive in nature and mediated by a common population of receptors, presumably GABA(A)/alpha(1) receptors. In contrast, the GABA(A)/alpha(5)-preferring agonist QH-ii-66 did not engender triazolam-lever responding regardless of dose and did not alter the DS effects of triazolam when administered in combination. CONCLUSIONS: The results are consistent with GABA(A)/alpha(1) receptor involvement in mediating the DS effects of triazolam. In contrast, binding to GABA(A)/alpha(5) receptors may not play a critical role in mediating triazolam's DS effects.     

The role of α1 and α5 subunit-containing GABAA receptors in motor impairment induced by benzodiazepines in rats

Benzodiazepines negatively affect motor coordination and balance and produce myorelaxation. The aim of the present study was to examine the extent to which populations of γ-aminobutyric acid A (GABAA) receptors containing α1 and α5 subunits contribute to these motor-impairing effects in rats. We used the nonselective agonist diazepam and the α1-selective agonist zolpidem, as well as nonselective, α1-subunit and α5-subunit-selective antagonists flumazenil, βCCt, and XLi093, respectively. Ataxia and muscle relaxation were assessed by rotarod and grip strength tests performed 20 min after intraperitoneal treatment. Diazepam (2 mg/kg) induced significant ataxia and muscle relaxation, which were completely prevented by pretreatment with flumazenil (10 mg/kg) and βCCt (20 mg/kg). XLi093 antagonized the myorelaxant, but not the ataxic actions of diazepam. All three doses of zolpidem (1, 2, and 5 mg/kg) produced ataxia, but only the highest dose (5 mg/kg) significantly decreased the grip strength. These effects of zolpidem were reversed by βCCt at doses of 5 and 10 mg/kg, respectively. The present study demonstrates that α1 GABAA receptors mediate ataxia and indirectly contribute to myorelaxation in rats, whereas α5 GABAA receptors contribute significantly, although not dominantly, to muscle relaxation but not ataxia.     

No functional β_3-adrenergic receptors expressed in rat skeletal muscle cells

目的：研究原代培养的大鼠骨骼肌细胞中是否存在功能性β３肾上腺素受体（β３ＡＲ）．方法：利用柱层析方法测定异丙肾上腺素（Ｉｓｏ），β３ＡＲ激动剂ＣＧＰ１２１７７Ａ和β３ＡＲ拮抗剂ＳＲ５９２３０Ａ对培养骨骼肌细胞环磷腺苷（ｃＡＭＰ）生成作用．结果：Ｉｓｏ剂量依赖性刺激骨骼肌细胞ｃＡＭＰ的生成，ＥＣ５０为１５１ｎｍｏｌ·Ｌ－１．普萘洛尔０．１μｍｏｌ·Ｌ－１抑制Ｉｓｏ刺激的ｃＡＭＰ的生成，ＫＢ值为３４７ｎｍｏｌ·Ｌ－１．ＣＧＰ１２１７７Ａ无刺激ｃＡＭＰ生成作用，但可抑制Ｉｓｏ的作用．ＳＲ５９２３０Ａ１０ｎｍｏｌ·Ｌ－１不能抑制Ｉｓｏ刺激ｃＡＭＰ的产生．结论：大鼠骨骼肌细胞中不存在功能性β３ＡＲ或至少不与腺苷酸环化酶耦联     

Functional effects of polyamines via activation of human β1- and β2-adrenoceptors stably expressed in CHO cells

Polyamines mediate acute metabolic effects and cardiac hypertrophy associated with β-adrenoceptor stimulation. They may also modulate β-adrenoceptors, causing functional responses in rat atria and tracheal smooth muscle. The aim of this study was to determine whether polyamines interact with human β(1)- and β(2)-adrenoceptors and the functional consequences of such an interaction. Chinese hamster ovary (CHO) cells stably transfected with human β(1)- and β(2)-adrenoceptors were used to evaluate the effect of polyamines binding to β-adrenoceptors, cAMP production and morphological changes, which were pharmacologically validated by investigating the effects of the β-adrenoceptor agonists, isoproterenol and salbutamol. Polyamines interacted with human β(1)- and β(2)-adrenoceptors, as shown by the displacement of [(125)I]iodocyanopindolol in the binding assay. Putrescine showed higher affinity to β(1)- than β(2)-adrenoceptors. Spermidine and spermine produced partial displacement (approximately 50%) and, at the highest concentration, the effect was reversed. Putrescine and spermine acutely increased cAMP and, in a serum-free medium, induced a stellate-like form in cells, which was inhibited by propranolol, a β-blocker. A 10 to 15 h incubation with putrescine produced a spindle-like form and spatial organization via β-adrenoceptor activation, evidenced by the antagonizing effect by propranolol and lack of effect in wild-type CHO cells. Additionally, it decreased cell proliferation independently of β-adrenoceptor activation. Spermine caused cell death via fetal bovine serum-dependent and -independent mechanisms. The results suggest that putrescine may act as a non-selective and low affinity agonist of human β(1)- and β(2)-adrenoceptors, eliciting morphological changes. These findings may be of importance in physiology and in diseases involving β-adrenoceptor functionality.     

Modulation of motor activity by α1 and α2 stimulation in mice

Summary The influence of two -adrenoceptor agonists, clonidine and B-HT 920, on motor activity was tested in mice. Both, clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[4,5-d]-azepine) in the dose range 30–300 g/kg s.c. equieffectively inhibited exploratory activity. On the other hand only clonidine, which stimulates ₂- and ₂-adrenoceptors increased locomotor activity in mice treated with reserpine (5 mg/kg) and apomorphine (3 mg/kg) in the doses of 0.3 and 1 mg/kg i.p. The highly selective ₂-agonist B-HT 920 was ineffective under these conditions up to 30 mg/kg i.p. It is concluded, that in mice sedative -adrenoceptors are of the ₂- and excitatory of the ₁-type.     

Stable overexpression of human β2-adrenergic receptors in mammalian cells

Summary Human ₂-adrenergic receptors were overexpressed in chinese hamster ovary (CHO) and human epitheloid carcinoma (HeLa) cells. Stable expression in these cells was achieved by transfection of a vector containing the cDNAs for the human ₂-adrenergic receptor as well as for dihydrofolate reductase. By stepwise increases of the concentration of methotrexate — an inhibitor of dihydrofolate reductase — the expression in CHO cells could be increased to levels of almost 200 pmol/mg membrane protein, which is more than 1% of the total membrane protein. In contrast, overexpression of the receptors in HeLa cells by the same technique led to cell death.The receptors produced in overexpressing CHO cells were correctly processed and were fully functional with respect to their ligand binding and signalling properties. The adenylyl cyclase activity of membranes from these cells responded with extremely high sensitivity to the -adrenergic receptor agonist isoproterenol. The receptors could be purified from these membranes to apparent homogeneity by solubilization and chromatography on a single affinity column. Thus, the expression system described here allows the preparation of human ₂-adrenergic receptors in quantities sufficient for pharmacological and biochemical investigations.     

β-Adrenoceptor subtypes in sections of rat and guinea-pig kidney

Summary The present autoradiographical study examines the distribution of the two -adrenoceptor subtypes in sections of rat and guinea-pig kidney. The radioligand [¹²⁵Iodo]-(-)-cyanopindolol was used for the labelling of -adrenoceptors and the selective -adrenoceptor blocking agents ICI 89-406 (₁-antagonist) and ICI 118-551 (₂-antagonist) were utilized to differentiate both subclasses unequivocally. -Adrenoceptors in rat kidney were found to be almost exclusively ₁. They were located mainly on glomeruli and to a lesser extent on the straight part of the distal tubules and on the cortical portion of the collecting ducts. Some ₂-adrenoceptors were localized around the corticomedullary junction. Grain localization in the autoradiograms was absent in the inner medulla and papilla. Glomeruli and distal tubules of the guinea-pig kidney also possess only ₁-adrenoceptors, but, in contrast to the rat, extremely high concentrations of ₂-adrenoceptors were associated with the straight part of the proximal tubules in the cortex and possibly with the cortical portion of the collecting duct. Labelling was not detected on the proximal convoluted tubule in either species.     

Comparison of α1A- and α1B-adrenoceptor coupling to inositol phosphate formation in rat kidney

We have compared the coupling mechanisms of rat renal _1A- and _1B-like adrenoceptors to inositol phosphate formation. The experiments were performed in parallel in native renal tissue preparations and in those where _1B-adrenoceptors had been inactivated by treatment with 10 mol/l chloroethylclonidine for 30 min at 37°C; renal slices were used in most experiments but isolated renal cells were also used in some cases. The Ca²⁺ chelating agent, EGTA (5 mmol/l), reduced noradrenaline-stimulated inositol phosphate formation in native but enhanced it in chloroethylclonidine-treated renal slices. The inhibitory effect of EGTA was not mimicked by 100 nmol/l nifedipine. Inactivation of 87% of cellular G_i by 16–20 h treatment with 500 ng/ml pertussis toxin did not significantly affect noradrenaline-stimulated inositol phosphate formation in isolated renal cells but abolished the inhibitory effect of chloroethylclonidine. The adenylate cyclase activator, forskolin (20 mol/l), inhibited noradrenaline-stimulated inositol phosphate formation in native and chloroethylclonidine-treated slices, and the inhibitory effects of chloroethylclonidine treatment and forskolin were additive. We conclude that in rat kidney inositol phosphate formation via _1B-like adrenoceptors may involve the influx of extracellular Ca²⁺ and a pertussis toxin-sensitive G-protein but is insensitive to inhibition by forskolin. In contrast _1A-like adrenoceptor-mediated inositol phosphate formation does not require the presence of extracellular Ca²⁺ or of G_i and is sensitive to inhibition by forskolin. In comparison to published data from other model systems we further conclude that the signaling mechanisms of ₁-adrenoceptor subtypes may depend on their cellular environment.     

Disrupting androgen production of Leydig cells by resveratrol via direct inhibition of human and rat 3β-hydroxysteroid dehydrogenase

Resveratrol is a polyphenol produced by several plants. It has been demonstrated that it has anti-inflammatory, antitumor, and anti-diabetic effects in animal models. However, its side effects are generally unclear. In the present study, we reported that resveratrol inhibited luteinizing hormone-stimulated androgen production in rat immature Leydig cells. Further analysis demonstrated that it was a competitive inhibitor of rat and human 3β-hydroxysteroid dehydrogenase with IC₅₀ values of 3.87 ± 0.06 and 8.48 ± 0.04 μM, respectively. The inhibition on 3β-hydroxysteroid dehydrogenase was specific since it did not inhibit another hydroxysteroid dehydrogenase 17β-hydroxysteroid dehydrogenase 3 at the highest concentration (100 μM) tested. In conclusion, resveratrol potentially interferes with androgen biosynthesis of rat Leydig cells.     

The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide directly blocks human ether ��-go-go-related gene potassium channels stably expressed in human embryonic kidney 293 cells

Background and purpose:

Results from several studies point to voltage-gated Na⁺ channels as potential mediators of the immobility produced by inhaled anaesthetics. We hypothesized that the intrathecal administration of tetrodotoxin, a drug that blocks Na⁺ channels, should enhance anaesthetic potency, and that concurrent administration of veratridine, a drug that augments Na⁺ channel opening, should reverse the increase in potency.

Experimental approach:

We measured the change in isoflurane potency for reducing movement in response to a painful stimulus as defined by MAC (minimum alveolar concentration of anaesthetic required to abolish movement in 50% of subjects) caused by intrathecal infusion of various concentrations of tetrodotoxin into the lumbothoracic subarachnoid space of rats, and the change in MAC caused by the administration of a fixed dose of tetrodotoxin plus various doses of intrathecal veratridine.

Key results:

Intrathecal infusion of tetrodotoxin (0.078–0.63 µM) produced a reversible dose-related decrease in MAC, of more than 50% at the highest concentration. Intrathecal co-administration of veratridine (1.6–6.4 µM) reversed this decrease in a dose-related manner, with nearly complete reversal at the highest veratridine dose tested.

Conclusions and implications:

Intrathecal administration of tetrodotoxin increases isoflurane potency (decreases isoflurane MAC), and intrathecal administration of veratridine counteracts this effect in vivo. These findings are consistent with a role for voltage-gated Na⁺ channel blockade in the immobility produced by inhaled anaesthetics.     

Benzodiazepine-induced spatial learning deficits in rats are regulated by the degree of modulation of α1 GABAA receptors

Despite significant advances in understanding the role of benzodiazepine (BZ)-sensitive populations of GABA_A receptors, containing the α₁, α₂, α₃ or α₅ subunit, factual substrates of BZ-induced learning and memory deficits are not yet fully elucidated. It was shown that α₁-subunit affinity-selective antagonist β-CCt almost completely abolished spatial learning deficits induced by diazepam (DZP) in the Morris water maze. We examined a novel, highly (105 fold) α₁-subunit selective ligand—WYS8 (0.2, 1 and 10 mg/kg), on its own and in combination with the non-selective agonist DZP (2 mg/kg) or β-CCt (5 mg/kg) in the water maze in rats. The in vitro efficacy study revealed that WYS8 acts as α₁-subtype selective weak partial positive modulator (40% potentiation at 100 nM). Measurement of concentrations of WYS8 and DZP in rat serum and brain tissues suggested that they did not substantially cross-influence the respective disposition. In the water maze, DZP impaired spatial learning (acquisition trials) and memory (probe trial). WYS8 caused no effect per se, did not affect the overall influence of DZP on the water-maze performance and was devoid of any activity in this task when combined with β-CCt. Nonetheless, an additional analysis of the latency to reach the platform and the total distance swam suggested that WYS8 addition attenuated the run-down of the spatial impairment induced by DZP at the end of acquisition trials. These results demonstrate a clear difference in the influence of an α₁ subtype-selective antagonist and a partial agonist on the effects of DZP on the water-maze acquisition.