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Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.

Virtual screening of the commercial databases was done by using a three dimensional pharmacophore previously developed for T-type calcium channel blockers using CATALYSTtrade mark program. Biological evaluation of 25 selected virtual hits resulted in the discovery of a highly potent compound VH04 with IC(50) value of 0.10 microM, eight times as potent as the known selective T-type calcium channel blocker, mibefradil. Search for similar compounds yielded several hits with micro-molar IC(50) values and high T-type calcium channel selectivity. Based on the structure of the virtual hits, small molecule libraries with novel scaffolds were designed, synthesis and biological evaluation of which are currently in progress. This result shows a successful example of ligand based drug discovery of potent T-type calcium channel blockers.

We previously demonstrated that dextromethorphan (DM; 3-methoxy-17-methylmorphinan) analogs have neuroprotective effects, and a recent report showed that DM reduces the adverse effects of morphine and blocks alpha3beta4 nicotinic acetylcholine receptors, a major target of anti-addictive agents. Here, we investigated the effects of DM, three of its analogs (DF, 3-methyl-17-methylmorphinan; AM, 3-allyloxy-17-methoxymorphian; and CM, 3-cyclopropyl-17-methoxymorphinan) and one of its metabolites (HM; 3-methoxymorphinan), on neuronal alpha3beta4 nicotinic acetylcholine receptor channel activity expressed in Xenopus laevis oocytes, using the two-microelectrode voltage clamp technique. We found that intraoocyte injection of neuronal alpha3 and beta4 nicotinic acetylcholine receptor subunit cRNAs elicited an inward current (IACh) in the presence of acetylcholine. Co-treatment with DM, DF, AM, CM or HM inhibited IACh in a dose-dependent, voltage-independent and reversible manner. The IC50 values for DM, DF, AM, CM and HM were 19.5+/-5.2, 15.8+/-4.5, 16.3+/-1.7, 10.1+/-2.8, and 13.5+/-4.0 microM, respectively. The order of potency for the inhibition of IACh was CM>HM>DF=AM>DM in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors. The inhibitions of (IACh) by DM, DF and HM, AM and CM were non-competitive. These results indicate that AM, CM and HM could be novel non-competitive agents regulating alpha3beta4 nicotinic acetylcholine receptor channel activity.

FMLP stimulation of Xenopus oocytes expressing fMLP receptors leads to a concentration-dependent biphasic inward current. To identify the evolution of these currents we have examined the effects of blocking various cell signalling pathways. In addition we have analysed the effects of three intravenous anaesthetics on these fMLP-induced currents. Xenopus oocytes were microinjected with cRNA encoding the fMLP receptor and fMLP-stimulated (100 nM) currents measured, using two-electrode voltage-clamp (-70 mV), before and after injection of heparin (120 ng ml-1), wortmannin (1 microM), U73122 (5 microM) or buffer. Concentration-response curves were established for the action on fMLP-stimulated currents of thiopentone (5-500 microM), methohexitone (0.2-200 microM) and propofol (0.5-500 microM). Heparin significantly enhanced the fast current (p<0.05). Wortmannin had no effect on either current. U73122 inhibited only the slow current (p<0.05). All anaesthetics inhibited both currents, with the maximum inhibition for the fast/slow currents 70%/100%, 60%/60% and 100%/100% for thiopentone (IC50 147/120 microM), methohexitone (IC50 4.7/2.2 microM) and propofol (IC50 33/8 microM), respectively. We suggest (a) the slow current arises via the PLC/PKC pathway because it is reduced by the PLC inhibitor U73122, (b) the PI3K- and PLD-mediated pathways are not involved because wortmannin had no effect and (c) activation of the two conductance channels must be different because U73122 reduced the slow but not the fast current. Since both currents are decreased by all three anaesthetics, their inhibition might be mediated through an action at the agonist/receptor, although, since the slow current is consistently more sensitive than the fast, there may be additionally an action on cell signalling.

An alpha7 nicotinic acetylcholine receptor sequence was cloned from Rhesus monkey (Macaca mulatta). This clone differs from the mature human alpha7 nicotinic acetylcholine receptor in only four amino acids, two of which are in the extracellular domain. The monkey alpha7 nicotinic receptor was characterized in regard to its functional responses to acetylcholine, choline, cytisine, and the experimental alpha7-selective agonists 4OH-GTS-21, TC-1698, and AR-R17779. For all of these agonists, the EC(50) for activation of monkey receptors was uniformly higher than for human receptors. In contrast, the potencies of mecamylamine and MLA for inhibiting monkey and human alpha7 were comparable. Acetylcholine and 4OH-GTS-21 were used to probe the significance of the single point differences in the extracellular domain. Mutants with the two different amino acids in the extracellular domain of the monkey receptor changed to the corresponding sequence of the human receptor had responses to these agonists that were not significantly different in EC(50) from wild-type human alpha7 nicotinic receptors. Monkey alpha7 nicotinic receptors have a serine at residue 171, while the human receptors have an asparagine at this site. Monkey S171N mutants were more like human alpha7 nicotinic receptors, while mutations at the other site (K186R) had relatively little effect. These experiments point toward the basic utility of the monkey receptor as a model for the human alpha7 nicotinic receptor, albeit with the caveat that these receptors will vary in their agonist concentration dependency. They also point to the potential importance of a newly identified sequence element for modeling the specific amino acids involved with receptor activation.

4-Chlorobenzo[F]isoquinoline (CBIQ) is a novel compound, here shown to activate both CFTR (cystic fibrosis transmembrane conductance regulator) Cl- ion channels and KCNN4, intermediate conductance, calcium-sensitive K+-channels, present in transporting epithelia by the use of heterologous expression systems. Earlier studies with other benzoquinolines, namely 7,8- and 5,6 benzoquinoline, showed they too could activate CFTR and KCNN4, but the evidence was only indirect. However this study also shows that CBIQ can also activate DeltaF508 CFTR, the most common mutant form of CFTR present in approximately 75% of patients with cystic fibrosis. This property is not shared with the other benzoquinolines. As activation of CFTR and KCNN4 work in unison to promote epithelial chloride secretion, CBIQ is a new chemical scaffold for developing agents that may be useful in cystic fibrosis.

The role of a voltage-dependent gate of recombinant P2X2 receptor/channel was investigated in Xenopus oocytes. When a voltage step to -110 mV was applied from a holding potential of -50 mV, a gradual increase was observed in current evoked by 30 microM ATP. Contribution of this voltage-dependent component to total ATP-evoked current was greater when the current was evoked by lower concentrations of ATP. The voltage-dependent gate closed upon depolarization, and half the gates were closed at -80 mV. On the other hand, a potential at which half the gates opened was about -30 mV or more positive, which was determined using a series of hyperpolarization steps. The results of the present study suggest that the voltage-dependent gate behavior of P2X2 receptor is not due to simple activation and deactivation of a single gate, but rather due to transition from a low to a high ATP affinity state.

The aim of this study was to determine whether the neurotrophins nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 could act as endogenous target-derived trophic factors for erection-inducing, i.e. penis-projecting major pelvic ganglion (MPG) neurons, and/or penile sensory neurons in adult rat. This was accomplished by studying the expression of NT mRNAs in the penis and their cognate receptors in the MPG and dorsal root ganglia (DRGs), and the retrograde axonal transport of radioiodinated NTs injected into the corpora cavernosa. Northern hybridization showed that NGF, BDNF, and NT-3 mRNAs are expressed in the shaft of the penis. In situ hybridization combined with usage of the retrograde tracer Fluoro-Gold showed that TrkC and p75 receptors are expressed in penis-projecting neurons of the MPG whereas the mRNAs for TrkA and TrkB receptors were undetectable. However, all the NT receptor mRNAs were expressed in penile sensory neurons of sacral level 1 (S1) DRG. (125)I-NT-3 injected into the shaft of the penis was retrogradely transported into the MPG and S1 DRG, whereas radioiodinated NGF and BDNF were transported specifically into the S1 DRG, thus confirming the existence of functional NT receptors in these penile neurons. In conclusion, these data suggest that NT-3 may act as a target-derived neurotrophic factor for both erection-inducing and penile sensory neurons, whereas NGF and BDNF may be more important for the sensory innervation of the penis.

The glucocorticoid-induced receptor (GIR) is an orphan G-protein-coupled receptor awaiting pharmacological characterization. GIR was originally identified in murine thymoma cells, and shows a widespread, yet not completely complementary distribution in mouse and human brain. Expression of the mouse GIR gene is modulated by dexamethasone in the brain and periphery, suggesting that GIR function is directly responsive to glucocorticoid signals. The rat GIR was cloned from rat prefrontal cortex by our group and was shown to be up-regulated following chronic amphetamine. The physiological role of GIR in the rat is not known at present. In order to gain a clearer understanding of the potential functions of GIR in the rat, we performed a detailed mapping of GIR mRNA expression in the rat brain. GIR mRNA showed widespread distribution in forebrain limbic and thalamic structures, and a more restricted distribution in hindbrain areas such as the spinal trigeminal nucleus and the median raphe nucleus. Areas with moderate to high levels of GIR include olfactory regions such as the nucleus of olfactory tract, hippocampus, various thalamic nuclei, cortical layers, and some hypothalamic nuclei. In comparison with previous studies, significant regional differences exist in GIR distribution in mouse and rat brain, particularly in the thalamus, striatum and in hippocampus at a cellular level. Overall, the expression of GIR in rat brain more closely approaches that seen previously in human than mouse, suggesting that rat models may be more informative for understanding the role of GIR in glucocorticoid physiology and glucocorticoid-related disease states. GIR mRNA distribution in the rat indicates a potential role of this receptor in the control of feeding and ingestive behavior, regulation of stress and emotional behavior, learning and memory, and, drug reinforcement and reward.

Corticotropin-releasing-factor (CRF) containing systems and monoaminergic afferents of the central amygdaloid nucleus (Ce) are crucial players in central nervous stress responses. For functional analyses of specific roles of these systems, numerous mouse models have been generated which lack or overexpress individual signal transduction components. Since data concerning system morphologies in murine brain are rarely available, mouse studies are usually designed and interpreted based on previous findings in rats, although interspecies differences are frequent. In the present study, in situ hybridization for CRF mRNA and correlative immunocytochemistry for CRF and monoaminergic afferents revealed numerous CRF mRNA-reactive neurons in the lateral Ce subnucleus (CeL) codistributed with dense dopaminergic fiber plexus in mice as has been demonstrated in rats. However, while in rats the lateral capsular Ce (CeLc) displays only scarce CRF immunoreactive (CRF-ir) innervation, particularly dense CRF-ir fiber plexus were observed in the CeLc in mice, with differences in labeling densities between different strains. CRF-ir terminal fibers overlap with the moderate serotonergic innervation of this subnucleus in mice. Additionally, CRF mRNA-reactive neurons were found immediately dorsal to the amygdala in the region of the interstitial nucleus of the posterior limb of the anterior commissure/amygdalostriatal transition area in both species. In mice, this region displayed dense CRF-ir fiber plexus, with variations between the strains. The results indicate that in mice and rats dopaminergic afferents represent the primary monoaminergic input to the CRF neurons in the CeL. In mice only, CRF-ir afferents provide dense innervation of CeLc neurons. Since the CeLc lacks dopaminergic input in both species but possesses moderate serotonergic afferents, CRF/serotonin interactions may occur selectively in mouse CeLc. The observed interspecies and interstrain differences in CRF input and CRF/monoaminergic interactions may influence the interpretation of findings concerning Ce functions in stress and fear in mouse models.

Available IBDV vaccines based on classical IBDV strains do not induce full protection in the presence of maternally derived antibodies (MDA) against variant strains. The aim of this study was the generation of an IBDV chimera between both classical and variant strains. Characterization of the generated chimeric IBDV (D78-Chim) was performed in both cell culture and susceptible chickens using the classical vaccine strain D78 for comparison. Growth kinetics on cell culture showed that the D78-Chim replicates comparable to the live vaccine strain D78. Administration of the D78-Chim to SPF chickens resulted in equally high virus neutralizing antibodies titres against both, classical and variant IBDV strains. In vivo experiments in commercial chickens revealed that D78-Chim and D78 induced comparable bursal damage in presence of maternally derived antibodies. Both D78-Chim and D78 viruses were able to breakthrough a similar level of MDA.

The modulation of ionotropic gamma-aminobutyric acid (GABA) receptors (GABA-gated Cl(-) channels) by a group of natural and synthetic flavonoids was studied in electrophysiological experiments. Quercetin, apigenin, morine, chrysin and flavone inhibited ionic currents mediated by alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors expressed in Xenopus laevis oocytes in the micromolar range. alpha(1)beta(1)gamma(2s) GABA(A) and rho(1) GABA(C) receptors differ largely in their sensitivity to benzodiazepines, but they were similarly modulated by different flavonoids. Quercetin produced comparable actions on currents mediated by alpha(4)beta(2) neuronal nicotinic acetylcholine, serotonin 5-HT(3A) and glutamate AMPA/kainate receptors. Sedative and anxiolytic flavonoids, like chrysin or apigenin, failed to potentiate but antagonized alpha(1)beta(1)gamma(2s) GABA(A) receptors. Effects of apigenin and quercetin on alpha(1)beta(1)gamma(2s) GABA(A) receptors were insensitive to the benzodiazepine antagonist flumazenil. Results indicate that mechanism/s underlying the modulation of ionotropic GABA receptors by some flavonoids differs from that described for classic benzodiazepine modulation., (Copyright 2003 Elsevier Science B.V.)

A study was made of the effects of La(3+) on neuronal alpha 2 beta 4 nicotinic acetylcholine receptors expressed in Xenopus oocytes. La(3+) by itself (up to 10 microM) did not elicit significant membrane currents. However, La(3+) reversibly inhibited the ionic currents induced by acetylcholine (IC(50)=13.5+/-4.3 microM). When La(3+) and acetylcholine were simultaneously applied onto an oocyte, the level of inhibition of the acetylcholine response was the same as when the oocyte was first preincubated with La(3+) and then exposed to acetylcholine plus La(3+). In the presence of La(3+), the EC(50) decreased from 43.8+/-6.4 to 26.5+/-5.1 microM, suggesting a small increase in the affinity of acetylcholine for the receptors through a noncompetitive mechanism. The inhibition of acetylcholine response was independent of the membrane potential. From these results we conclude that La(3+) regulates nicotinic receptors, reversibly and noncompetitively, presumably by inhibiting allosterically the receptor through interactions at an external domain of the receptor complex.

Although convulsions due to local anesthetic systemic toxicity are thought to be due to inhibition of GABA(A) receptor-linked currents in the central nervous system, the mechanism of action remains unclear. We therefore examined the effects of local anesthetics on gamma-aminobutyric acid (GABA)-induced currents using recombinant GABA(A) receptors with specific combinations of subunits. Murine GABA(A) receptors were expressed by injection of cRNAs encoding each subunit into Xenopus oocytes. The effects of local anesthetics (lidocaine, bupivacaine, procaine and tetracaine) on GABA-induced currents of receptors expressing different subunit combinations (alpha1beta2, alpha1beta2gamma2s, alpha4beta2gamma2s and beta2) were examined via the two electrode voltage clamp method. At alpha1beta2, alpha1beta2gamma2s and alpha4beta2gamma2s GABA(A) receptors, all local anesthetics inhibited GABA-induced currents in a dose-dependent manner. The presence of the gamma2s subunit resulted in a greater inhibition by all local anesthetics, but the presence of the alpha4 subunit resulted in less inhibition. At beta2 homomeric receptors, local anesthetics directly induced an outward current similar to that of picrotoxin. These data indicated that (1) the alpha and gamma subunits of GABA(A) receptors modulated the inhibitory effects of local anesthetics on GABA(A) function, and (2) local anesthetics can activate the beta2 subunit and may block the GABA(A) receptor channel pore.

The interactions of sulfonylureas and a novel anti-diabetic drug, nateglinide, with rat renal organic anion transporter (rOAT1) expressed in Xenopus laevis oocytes were studied. Uptake of p-aminohippurate via rOAT1 was markedly inhibited by glibenclamide and nateglinide, and moderately by chlorpropamide and tolbutamide. The inhibition constant values (K(i)) for chlorpropamide, glibenclamide, tolbutamide and nateglinide were 39.5, 1.6, 55.5 and 9.2 microM, respectively. Kinetic analysis showed that the inhibition of p-aminohippurate uptake by glibenclamide was competitive. Sulfonylureas examined and nateglinide did not show a trans-stimulation effect on [14C]p-aminohippurate efflux from rOAT1-expressing oocytes. There was no stimulation of [3H]glibenclamide uptake via rOAT1. These findings suggested that sulfonylureas and nateglinide interact with rOAT1, but these drugs are not translocated via the transporter.

The use of aminoglycoside antibiotics is limited by side effects, the most critical of which are vestibular and cochlear toxicity. Recent evidence indicates that these effects result from an excitotoxic process mediated, at least in part, through a polyamine-like activation of NMDA receptors. This study investigated whether these positive modulatory effects of aminoglycosides at NMDA receptors are dissociable from their antibacterial properties. A group of structurally related apramycin derivatives was evaluated for the ability to enhance [3H]dizocilpine binding to rat brain membranes, and for the ability to augment agonist responses on recombinant (NR1A/2B) NMDA receptors expressed in Xenopus oocytes. Based on the antibacterial potencies of these derivatives against Staphylococcus aureus and Escherichia coli, it is concluded that there is no correlation between the ability of an aminoglycoside to produce a positive modulation of NMDA receptors and minimum inhibitory antibacterial concentrations. These findings indicate that it may be possible to develop an aminoglycoside antibiotic with reduced potential for ototoxicity.

It is well known that activation of the cloned kappa-opioid receptor by nanomolar concentrations of U50488H (trans-(+/-)-3, 4-dichloro-N-methyl-N-(2-[1-pyrrolidinyl]cyclohexyl-benzeneacetamide) , a selective kappa-opioid receptor agonist, leads to the opening of GIRK1 channels. In this study, we demonstrate that the cloned kappa-opioid receptor functionally couples to GIRK1/GIRK2 channels (G-protein-coupled inwardly rectifying K(+) channels), mimicking the probable heteromultimeric state of neuronal GIRK channels. We also show that micromolar concentrations of U50488H reduce GIRK1/GIRK2 current through direct GIRK1/GIRK2 channel block in a voltage-independent manner (IC(50)=70.28+/-3.68 microM). Similarly, it was found that propoxyphene, methadone, and naloxone also can block GIRK1/GIRK2 current. In contrast, elevated concentrations of morphine (up to 1 mM) did not cause channel block. The related inwardly rectifying K(+) channel, IRK1, was not affected by elevated concentrations of these drugs. We conclude that nanomolar concentrations of opioid receptor ligands activate GIRK1/GIRK2 channels through a receptor-mediated pathway, while micromolar concentrations of some opioid receptor ligands inhibit GIRK1/GIRK2 channels by direct channel block.

The effects of chronic administration of testosterone on alpha 2-adrenoceptor expression in male hamsters were investigated in order to explore the selectivity of testosterone regulation towards the alpha 2-adrenoceptor subtypes. A homogeneous population of alpha 2-adrenoceptors was identified with [3H]RX821002 binding in adipocytes, colocytes and liver, whereas the alpha 2-adrenoceptor sites identified in kidney and brain were heterogeneous. Competition studies with alpha 2-adrenoceptor ligands characterized the presence of the alpha 2A-adrenoceptor subtype in adipocytes, colocytes, kidney and brain homogenates and of the alpha 2B-adrenoceptor subtype in kidney and liver. RNase protection assay with a selective hamster alpha 2A-adrenoceptor cRNA probe confirmed the expression of alpha 2A-adrenoceptor mRNA in adipocytes, colocytes, kidney and brain. Testosterone treatment did not modify the alpha 2-adrenoceptor densities whatever the subtype, except for the adipocyte alpha 2A-adrenoceptor, which was significantly increased. These results demonstrate that testosterone only up-regulates the adipocyte alpha 2A-adrenoceptor.

Pharmacological modulation of human sodium current was examined in Xenopus oocytes expressing human heart Na+ channels. Na+ currents activated near -50 mV with maximum current amplitudes observed at -20 mV. Steady-state inactivation was characterized by a V1/2 value of -57 +/- 0.5 mV and a slope factor (k) of 7.3 +/- 0.3 mV. Sodium currents were blocked by tetrodotoxin with an IC50 value of 1.8 microM. These properties are consistent with those of Na+ channels expressed in mammalian myocardial cells. We have investigated the effects of several pharmacological agents which, with the exception of lidocaine, have not been characterized against cRNA-derived Na+ channels expressed in Xenopus oocytes. Lidocaine, quinidine and flecainide blocked resting Na+ channels with IC50 values of 521 microM, 198 microM, and 41 microM, respectively. Use-dependent block was also observed for all three agents, but concentrations necessary to induce block were higher than expected for quinidine and flecainide. This may reflect differences arising due to expression in the Xenopus oocyte system or could be a true difference in the interaction between human cardiac Na+ channels and these drugs compared to other mammalian Na+ channels. Importantly, however, this result would not have been predicted based upon previous studies of mammalian cardiac Na+ channels. The effects of DPI 201-106, RWJ 24517, and BDF 9148 were also tested and all three agents slowed and/or removed Na+ current inactivation, reduced peak current amplitudes, and induced use-dependent block. These data suggest that the alpha-subunit is the site of interaction between cardiac Na+ channels and Class I antiarrhythmic drugs as well as inactivation modifiers such as DPI 201-106.