Channel Binding Sites Research Articles

On the Interaction between Amiloride and Its Putative α-Subunit Epithelial Na+ Channel Binding Site

The epithelial Na+ channel (ENaC) belongs to the structurally conserved ENaC/Degenerin superfamily. These channels are blocked by amiloride and its analogues. Several amino acid residues have been implicated in amiloride binding. Primary among these are alphaSer-583, betaGly-525, and gammaGly-542, which are present at a homologous site within the three subunits of ENaC. Mutations of the beta and gamma glycines greatly weakened amiloride block, but, surprisingly, mutation of the serine of the alpha subunit resulted in moderate (<5-fold) weakening of amiloride K(i). We investigated the role of alphaSer-583 in amiloride binding by systematically mutating alphaSer-583 and analyzing the mutant channels with two-electrode voltage clamp. We observed that most mutations had moderate effects on amiloride block, whereas those introducing rings showed dramatic effects on amiloride block. In addition, mutations introducing a beta-methyl group at this site altered the electric field of ENaC, affecting both amiloride binding and the voltage dependence of channel gating. We also found that the His mutation, in addition to greatly weakening amiloride binding, appends a voltage-sensitive gate within the pore of ENaC at low pH. Because diverse residues at alpha583, such as Asn, Gln, Ser, Gly, Thr, and Ala, have similar amiloride binding affinities, our results suggest that the wild type Ser side chain is not important for amiloride binding. However, given that some alphaSer-583 mutations affect the electrical properties of the channel whereas those introducing rings greatly weaken amiloride block, we conclude that amiloride binds at or near this site and that alphaSer-583 may have a role in ion permeation through ENaC.

Pentylenetetrazol-induced seizures affect the levels of prolyl oligopeptidase, thimet oligopeptidase and glial proteins in rat brain regions, and attenuation by MK-801 pretreatment

The regulatory mechanisms of neuropeptide-metabolizing enzymes often play a critical role in the pathogenesis of neuronal damage. A systemic administration of pentylenetetrazol (PTZ), an antagonist of GABA A receptor ion channel binding site, causes generalized epilepsy in an animal model. In the present study, we examined the involvement of prolyl oligopeptidase (POP), thimet oligopeptidase/neurolysin (EP 24.15/16) and glial proteins in PTZ-treated rat brain regions, and the suppressive effect of MK-801, a non-competitive NMDA receptor antagonist, pretreatment for their proteins. The activity of POP significantly decreased in the hippocampus at 30 min and 3 h, and in the frontal cortex at 3 h after PTZ treatment, and pretreatment with MK-801 recovered the activity in the cortex at 3 h. The activity of EP 24.15/16 significantly decreased in the hippocampus at 3 h and 1 day, and in the cortex at 3 h after the PTZ administration, whereas pretreatment with MK-801 recovered the change of the activity. The Western blot analysis of EP 24.15 showed significant decrease of the protein level in the hippocampus 3 h after the PTZ treatment, whereas pretreatment with MK-801 recovered. The expression of GFAP and CD11b immunohistochemically increased in the hippocampus of the PTZ-treated rat as compared with controls. Pretreatment with MK-801 also recovered the GFAP and CD11b expression. These data suggest that PTZ-induced seizures of the rats cause indirect activation of glutamate NMDA receptors, then decrease POP and EP 24.15/16 enzyme activities and EP 24.15 immunoreactivity in the neuronal cells of the hippocampal formation. We speculate that changes of those peptidases in the brain may be related to the levels of the neuropeptides regulating PTZ-induced seizures.

Differential Phospholipid Binding by Site 3 and Site 4 Toxins: IMPLICATIONS FOR STRUCTURAL VARIABILITY BETWEEN VOLTAGE-SENSITIVE SODIUM CHANNEL DOMAINS

It has been shown recently that polypeptide toxins that modulate the gating properties of voltage-sensitive cation channels are able to bind to phospholipid membranes, leading to the suggestion that these toxins are able to access a channel-binding site that remains membrane-restricted (Lee, S.-Y., and MacKinnon, R. (2004) Nature 430, 232-235). We therefore examined the ability of anthopleurin B (ApB), a sea anemone toxin that selectively modifies inactivation kinetics of Na(V)1.x channels, and ProTx-II, a spider toxin that modifies activation kinetics of the same channels, to bind to liposomes. Whereas ProTx-II can be quantitatively depleted from solution upon incubation with phosphatidylcholine/phosphatidylserine liposomes, ApB displays no discernible phospholipid binding activity. We therefore examined the activities of structurally unrelated site 3 and site 4 toxins derived from Leiurus and Centruroides venoms, respectively, in the same assay. Like ApB, the site 3 toxin LqqV shows no lipid binding activity, whereas the site 4 toxin Centruroides toxin II, like ProTx-II, is completely bound. We conclude that toxins that modify inactivation kinetics via binding to Na(V)1.x site 3 lack the ability to bind phospholipids, whereas site 4 toxins, which modify activation, have this activity. This inherent difference suggests that the conformation of domain II more closely resembles that of the K(V)AP channel than does the conformation of domain IV.

In vitro and in vivo characterization of a novel naphthylamide ATP-sensitive K+ channel opener, A-151892.

1. Openers of ATP-sensitive K(+) channels are of interest in several therapeutic indications including overactive bladder and other lower urinary tract disorders. This study reports on the in vitro and in vivo characterization of a structurally novel naphthylamide N-[2-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-naphthalen-1-yl]-acetamide (A-151892), as an opener of the ATP-sensitive potassium channels. 2. A-151892 was found to be a potent and efficacious potassium channel opener (KCO) as assessed by glibenclamide-sensitive whole-cell current and fluorescence-based membrane potential responses (-log EC(50)=7.63) in guinea-pig bladder smooth muscle cells. 3. Evidence for direct interaction with KCO binding sites was derived from displacement of binding of the 1,4-dihydropyridine opener [(125)I]A-312110. A-151892 displaced [(125)I]A-312110 binding to bladder membranes with a -log Ki value of 7.45, but lacked affinity against over 70 neurotransmitter receptor and ion channel binding sites. 4. In pig bladder strips, A-151892 suppressed phasic, carbachol-evoked and electrical field stimulus-evoked contractility in a glibenclamide-reversible manner with -log IC(50) values of 8.07, 7.33 and 7.02 respectively, comparable to that of the potencies of the prototypical cyanoguanidine KCO, P1075. The potencies to suppress contractions in thoracic aorta (-log IC(50)=7.81) and portal vein (-log IC(50)=7.98) were not substantially different from those observed for suppression of phasic contractility of the bladder smooth muscle. 5. In vivo, A-151892 was found to potently suppress unstable bladder contractions in obstructed models of unstable contractions in both pigs and rats with pED(35%) values of 8.05 and 7.43, respectively. 6. These results demonstrate that naphthylamide analogs exemplified by A-151892 are novel K(ATP) channel openers and may serve as chemotypes to exploit additional analogs with potential for the treatment of overactive bladder and lower urinary tract symptoms.

The low-potency, voltage-dependent HERG blocker propafenone--molecular determinants and drug trapping.

The molecular determinants of high-affinity human ether-a-go-go-related gene (HERG) potassium channel blockade by methanesulfonanilides include two aromatic residues (Phe656 and Tyr652) on the inner helices (S6) and residues on the pore helices that face into the inner cavity, but determinants for lower-affinity HERG blockers may be different. In this study, alanine-substituted HERG channel mutants of inner cavity residues were expressed in Xenopus laevis oocytes and were used to characterize the HERG channel binding site of the antiarrhythmic propafenone. Propafenone's blockade of HERG was strongly dependent on residue Phe656 but was insensitive or weakly sensitive to mutation of Tyr652, Thr623, Ser624, Val625, Gly648, or Val659 and did not require functional inactivation. Homology models of HERG based on KcsA and MthK crystal structures, representing the closed and open forms of the channel, respectively, suggest propafenone is trapped in the inner cavity and is unable to interact exclusively with Phe656 in the closed state (whereas exclusive interactions between propafenone and Phe656 are found in the open-channel model). These findings are supported by very slow recovery of wild-type HERG channels from block at -120 mV, but extremely rapid recovery of D540K channels that reopen at this potential. The experiments and modeling suggest that the open-state propafenone binding-site may be formed by the Phe656 residues alone. The binding site for propafenone (which may involve pi-stacking interactions with two or more Phe656 side-chains) is either perturbed or becomes less accessible because of closed-channel gating. This provides further evidence for the existence of gating-induced changes in the spatial location of Phe656 side chains.

Sodium channel blocking activity of AM-36 and sipatrigine (BW619C89): in vitro and in vivo evidence

Sodium channel blockers are neuroprotective against cerebral ischemia in animal models. A novel neuroprotective compound AM-36, when screened for activity at the most common receptor and ion channel binding sites, revealed activity at site 2 Na+ channels. Studies then investigated this Na+ channel blocking activity in vitro and in vivo relative to other Na+ channel blockers, including the neuroprotective agent sipatrigine (BW619C89). AM-36 inhibited batrachotoxinin (BTX)-sensitive Na+ channel binding in rat brain homogenates with an IC50 of 0.28 μM. Veratridine (100 μM)-induced neurotoxicity in murine cerebellar granule cells was completely inhibited by AM-36 (1.7 μM) compared to only partial inhibition by sipatrigine (26 μM). Veratridine-stimulated glutamate release, as measured through a microdialysis probe in the cortex of anesthetised rats, was inhibited by 90% by superfusion of AM-36 (1000 μM). In the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAo) in conscious rats, both AM-36 (6 mg/kg i.p.) and sipatrigine (10 mg/kg i.p.) 30 min post-MCAo significantly reduced cortical, but not striatal infarct volume. As the refractiveness of the striatum is likely to be dependent on the route and time of drug administration, AM-36 (1 mg/kg i.v.) was administered 3 or 5 h after MCAo and significantly reduced both cortical and striatal infarct volumes. The present studies demonstrate Na+ channel blocking activity of AM-36 both in vitro and in vivo, together with significant neuroprotection when administration is delayed up to 5 h following experimental stroke.

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.

Sodium channel blocking activity of AM-36 and sipatrigine (BW619C89): in vitro and in vivo evidence

Sodium channel blockers are neuroprotective against cerebral ischemia in animal models. A novel neuroprotective compound AM-36, when screened for activity at the most common receptor and ion channel binding sites, revealed activity at site 2 Na + channels. Studies then investigated this Na + channel blocking activity in vitro and in vivo relative to other Na + channel blockers, including the neuroprotective agent sipatrigine (BW619C89). AM-36 inhibited batrachotoxinin (BTX)-sensitive Na + channel binding in rat brain homogenates with an IC 50 of 0.28 μM. Veratridine (100 μM)-induced neurotoxicity in murine cerebellar granule cells was completely inhibited by AM-36 (1.7 μM) compared to only partial inhibition by sipatrigine (26 μM). Veratridine-stimulated glutamate release, as measured through a microdialysis probe in the cortex of anesthetised rats, was inhibited by 90% by superfusion of AM-36 (1000 μM). In the endothelin-1 (ET-1) model of middle cerebral artery occlusion (MCAo) in conscious rats, both AM-36 (6 mg/kg i.p.) and sipatrigine (10 mg/kg i.p.) 30 min post-MCAo significantly reduced cortical, but not striatal infarct volume. As the refractiveness of the striatum is likely to be dependent on the route and time of drug administration, AM-36 (1 mg/kg i.v.) was administered 3 or 5 h after MCAo and significantly reduced both cortical and striatal infarct volumes. The present studies demonstrate Na + channel blocking activity of AM-36 both in vitro and in vivo, together with significant neuroprotection when administration is delayed up to 5 h following experimental stroke.

Cavbeta-subunit displacement is a key step to induce the reluctant state of P/Q calcium channels by direct G protein regulation.

P/Q Ca(2+) channel activity is inhibited by G protein-coupled receptor activation. Channel inhibition requires a direct Gbetagamma binding onto the pore-forming subunit, Ca(v)2.1. It is characterized by biophysical changes, including current amplitude reduction, activation kinetic slowing, and an I-V curve shift, which leads to a reluctant mode. Here, we have characterized the contribution of the auxiliary beta(3)-subunit to channel regulation by G proteins. The shift in I-V to a P/Q reluctant mode is exclusively observed in the presence of beta(3). Along with the observation that Gbetagamma has no effect on the I-V curve of Ca(v)2.1 alone, we propose that the reluctant mode promoted by Gbetagamma corresponds to a state in which the beta(3)-subunit has been displaced from its channel-binding site. We validate this hypothesis with a beta(3)-I-II(2.1) loop chimera construct. Gbetagamma binding onto the I-II(2.1) loop portion of the chimera releases the beta(3)-binding domain and makes it available for binding onto the I-II loop of Ca(v)1.2, a G protein-insensitive channel. This finding is extended to the full-length Ca(v)2.1 channel by using fluorescence resonance energy transfer. Gbetagamma injection into Xenopus oocytes displaces a Cy3-labeled beta(3)-subunit from a GFP-tagged Ca(v)2.1 channel. We conclude that beta-subunit dissociation from the channel complex constitutes a key step in P/Q calcium channel regulation by G proteins that underlies the reluctant state and is an important process for modulating neurotransmission through G protein-coupled receptors.

Memantine: a therapeutic drug for Alzheimer's disease and the comparison with MK-801

Memantine is agreed officially as a therapeutic drug for moderate-to-severe Alzheimer's disease (AD) in EU and USA. Memantine is a similar uncompetitive NMDA-receptor antagonist to MK-801 and phencyclidine (PCP), and it prevents nerve cell death induced by the ischemia which induces as excessive release of glutamate. These medicines act on an ion channel binding site similar to the magnesium ion binding site. However, MK-801 and PCP cause schizophrenic symptoms, so they are not being used as a therapeutic drug for AD. Memantine does not have those toxicities and does not stimulate acetylcholine release in the cerebral cortex. Although the mechanism of the difference from memantine and MK-801 has not been made clear yet, it seems that memantine is combined and released with the ion channel depending on electric potential in the same way as the magnesium ion. Basic and clinical research will clarify the control mechanism of memantine.

Evolutionary trace analysis of scorpion toxins specific for K-channels.

Scorpion alpha-K(+) channel toxins are a large family of polypeptides with a similar structure but diverse pharmacological activities. Despite many structural and functional data available at present, little progress has been made in understanding the toxin's molecular basis responsible for the functional diversification. In this paper, we report the first complete cDNA sequences of toxins belonging to subfamily 6 and identify five new members, called alpha-KTx 6.6-6.10. By analyzing the rates of mutations that occurred in the corresponding cDNAs, we suggest that accelerated evolution in toxin-coding regions may be associated with the functional diversification of this subfamily. To pinpoint sites probably involved in the functional diversity of alpha-KTx family, we analyzed this family of sequences using the evolutionary trace method. This analysis highlighted one channel-binding surface common for all the members. This surface is composed of one conserved lysine residue at position 29 assisted by other residues at positions 10, 26, 27, 32, 34, and 36. Of them, the positions 29, 32, and 34 have been reported to be the most major determinants of channel specificity. Interestingly, another contrary surface was also observed at a higher evolutionary time cut-off value, which may be involved in the binding of ERG (ether-a-go-go-related gene) channel-specific toxins. The good match between the trace residues and the functional epitopes of the toxins suggested that the evolutionary trace results reported here can be applied to predict channel-binding sites of the toxins. Because, the side-chain variation in the trace positions is strongly linked with the functional alteration and channel-binding surface transfer of alpha-KTx family, we conclude that our findings should also be important for the rational design of new toxins targeting a given potassium channel with high selectivity.

NMDA Receptor Antagonists as Antidepressant and Antidementia Drugs: Recent Developments and Future Prospects

N-Methyl-D-aspartate (NMDA) receptor (NR) antagonists have contributed to the major advances in research of excitatory synaptic transmission, neuronal excitotoxicity, and neurodegenerative disorders. Activation of the NRs evokes a rapid Ca2+ influx into neurons, resulting in an activation of a variety of intracellular signal cascades, Ca2+ mobilization, and alterations in membrane channel activity and gene expression. The signal transduction mechanisms play a fundamental role in how the brain functions physiologically but are also at the core of neural injury and dysfunction. Evidence is emerging that abnormal NR activation underlies the development of depressive symptoms and of many forms of dementia. Correcting abnormal activity of the NRs thus represents an attractive and essential therapeutic strategy in antidepressant and antidementia medications. NR antagonism can be achieved pharmacologically through blocking the NR / complex at its various binding sites, including the NR glutamate-binding site, the ion channel binding site, the glycine co-agonist site, the polyamine binding site, and the redox modulatory site(s). Agents that have antagonistic activity at the different sites show differential structure-activity relationships, possess distinct pharmacological profiles, and therefore have different therapeutic potentials. This review summarizes current knowledge and developments concerning a variety of NR antagonists and discusses their potential as pharmacological drugs in the treatments of depression and dementia.

A comparison of the μ-conotoxins by [ 3H]saxitoxin binding assays in neuronal and skeletal muscle sodium channel

Sodium channels from rat brain, rat skeletal muscle, chick brain, and eel electroplax were compared by using the μ-conotoxins GIIIA, PIIIA, and StIII and [ 3H]saxitoxin. Rat skeletal muscle and eel electroplax sodium channels are equally sensitive to GIIIA, PIIIA, and StIII, displacing >90% of the [ 3H]saxitoxin binding sites in rat skeletal muscle and eel electroplax membranes and exhibiting inhibitory concentrations at half-maximal percentage specific binding (IC50) of 0.97 nM for GIIIA, 1.3 nM for PIIIA in rat skeletal muscle, and concentrations of 3.5 nM for GIIIA and 2.8 nM for PIIIA in eel electroplax. PIIIA and GIIIA at all concentrations inhibit only up to 10% of [ 3H]saxitoxin binding sites in chick brain membranes. μ-Conotoxin StIII at all concentrations inhibits only up to 10% of [ 3H]saxitoxin binding sites in rat brain membranes and displays two-site binding inhibition of the [ 3H]saxitoxin binding sites in rat skeletal muscle. PIIIA displaces >60% of the [ 3H]saxitoxin binding sites (IC50 of 44 nM) while GIIIA blocks out only 30% of the binding sites in rat brain sodium channels (IC50 of 69 nM). Thus, sodium channel subtypes can be classified into two categories: μ-conotoxin sensitive (i.e., subtypes predominantly found in rat skeletal muscle and eel electroplax) and insensitive (i.e., subtypes predominantly found in rat and chick brain).

Energetic Localization of Saxitoxin in Its Channel Binding Site

Saxitoxin (STX) selectively blocks the voltage-gated sodium channel at the outer vestibule lined by P-loops of the four domains. Neosaxitoxin has an additional -OH group at the N1 position of the 1,2,3 guanidinium (N1-OH) that interacts with domains I and IV of the Na + channel. Determination of a second toxin interaction with the channel would fix the location of STX. Gonyautoxin 2,3 and Gonyautoxin 1,4 are C-11 sulfated derivatives of saxitoxin and neosaxitoxin, respectively. We used these variants to constrain the STX docking orientation by energetically localizing the C-11 sulfate in the outer vestibule. Interactions between the C-11 sulfate and each of the four domains of the channel were determined by a systematic approach to mutant cycle analysis in which all known carboxyl groups important for site 1 toxin binding were neutralized, allowing energetic triangulation of the toxin sulfate and overcoming some limitations of mutant cycles. Toxin IC 50s were measured by two-electrode voltage clamp from Xenopus oocytes injected with the channel mRNA. Three unique types of analysis based on the coupling results localized the C-11 sulfate between domains III and IV. Combined with our previous report, the data establish the orientation of STX in the outer vestibule and confirm the clockwise arrangement of the channel domains.

L-type Ca 2+ channel modulation by dihydropyridines potentiates κ-opioid receptor agonist induced acute analgesia and inhibits development of tolerance in rats

The effect of 1,4-dihydropyridine (DHP) calcium channel blockers (CCBs), nimodipine (NIM) and lercanidipine (LDP) on the analgesic response of selective κ-opioid receptor agonists, U50,488H, PD117,302 and U69,593 was determined in male Sprague-Dawley rats using the tail-flick test. The effect of NIM on development of tolerance to U50,488H-induced analgesia and the status of brain DHP-sensitive Ca 2+ channel (L-type) binding sites in both U50,488H-naive and tolerant rats was determined using the highly selective DHP radioligand, [ 3H]PN200-110. Tolerance was induced by injecting U50,488H (25 mg/kg, i.p.) twice daily for 4 days. Intraperitoneal (i.p.) injection of κ-opioid receptor agonists produced a dose-dependent acute analgesic response. NIM (1 mg/kg; i.p.) and LDP (0.3 mg/kg; i.p.) used in the study produced no tail-flick analgesia. Administration of NIM and LDP (15 min prior) significantly potentiated the analgesia produced by three κ-opioid receptor agonists. Tolerance developed completely to the analgesic effect induced by U50,488H (25 mg/kg, i.p.) administered on the 5th day. NIM (1 mg/kg, i.p.) twice daily for 4 days not only completely inhibited the development of tolerance to analgesic response but also significantly potentiated it (supersensitivity). There was a significant up-regulation of DHP binding sites ( B max: +41%) in whole brain membranes of tolerant rats when compared to vehicle treated naive rats, implicating increased influx of Ca 2+ through L-type channels in κ-opioid tolerance. U50,488H (25 mg/kg, i.p.) and NIM (1 mg/kg, i.p.) twice daily for 4 days also resulted in an equivalent up-regulation of DHP binding sites (+36%) as that of U50,488H alone. These results strongly suggest a functional role of L-type Ca 2+ channels in the regulation of pain sensitivity, mechanism of κ-opioid analgesia and expression of tolerance.

Properties of Ionotropic Glutamate Receptors Revealed by Channel Blockade

In experiments on hippocampal and striatal neurons, it was demonstrated using specific blockers with different three-dimensional structures that the main difference between channel binding sites in the AMPA and NMDA receptors is the dissimilar location of the nucleophilic region.

Vatanidipine hydrochloride: a new long-lasting antihypertensive agent

Vatanidipine is a novel dihydropyridine (DHP)-type calcium channel blocker with slow-onset pharmacological actions, which are probably due to both its slow uptake into vascular tissues and resistance in its approach to the calcium channel binding site. Vatanidipine once incorporated into vascular tissues is not easily released, even by repeated washing, thus resulting in a long-lasting action of the agent. A slow-onset and long-lasting hypotensive action was observed in various experimental hypertensive models. Clinical trials using human subjects with essential hypertension indicated that vatanidipine exerts an antihypertensive effect with a slow onset and long duration. In spite of its potent hypotensive effect, the incidence of adverse effects by vatanidipine administration has been reported to be lower than that in cases of nitrendipine. In addition to its vasodilatory effects, vatanidipine efficiently suppressed noradrenaline release from sympathetic nerve endings, thus suggesting this agent exhibits a beneficial effect in the treatment of hypertensive patients, in which the reflex activation of peripheral sympathetic nerves is unfavourable to antihypertensive therapy. In a double-blind study, vatanidipine did not show reflex tachycardia, despite producing a potent and long-lasting hypotensive effect, in contrast to the administration of nitrendipine. In an animal study, vatanidipine exhibited a protective effect against cerebrovascular lesions, through a mechanism independent of its hypotensive effect. In addition, a renoprotective effect was also observed in experimental hypertensive models. In cholesterol-fed rabbits, vatanidipine exerted an anti-atherosclerotic action, which is probably attributable to the inhibitory action of the agent on low-density lipoprotein oxidation. In essential hypertensive patients, the plasma levels of cholesterol and triglyceride decreased after vatanidipine treatment, thus suggesting that this agent may have a therapeutic potential in preventing such vascular diseases as atherosclerosis. Taken together, vatanidipine appears to be a novel and useful antihypertensive agent, which can both prevent target-organ damage and reduce cardiovascular morbidity and mortality.

Ionotropic glutamate receptor binding and subunit mRNA expression in thalamic nuclei in schizophrenia.

Both thalamic and glutamatergic dysfunction have been implicated in the pathophysiology of schizophrenia. The authors examined ionotropic glutamate receptor expression in postmortem samples from patients with schizophrenia and comparison subjects, using the hypothesis that glutamate receptor expression differs in limbic nuclei of the thalamus in schizophrenia. N-Methyl-D-aspartate (NMDA), AMPA, and kainate receptor expression was determined in six thalamic nuclei from 12 subjects with DSM-III-R diagnoses of schizophrenia and eight psychiatrically normal individuals. The authors used in situ hybridization to determine NMDAR1, NMDAR2A-NMDAR2D, gluR1-gluR7, KA1, and KA2 subunit mRNA levels and receptor autoradiography to determine binding to glutamate binding sites of the three receptor subtypes and to the glycine, polyamine, and ion channel binding sites of the NMDA receptor. Glutamate receptor expression was lower at both transcriptional (NMDAR1, NMDAR2B, NMDAR2C, gluR1, gluR3, and KA2 subunit mRNAs) and posttranscriptional ([(3)H]ifenprodil and [(3)H]MDL105,519 binding to polyamine and glycine sites of the NMDA receptor) levels in the thalamus in patients with schizophrenia than in comparison subjects, but differences were most prominent in nuclei with reciprocal projections to limbic regions. Abnormalities in NMDA, AMPA, and kainate receptor expression in limbic thalamus are suggestive of the NMDA receptor hypoactivity hypothesis of schizophrenia and are consistent with diminished glutamatergic activity in the thalamus in schizophrenia. Alternatively, these results could suggest abnormal glutamatergic innervation in afferent and/or efferent regions, which are limbic structures that have been implicated in this illness. These results may provide a neurochemical anatomical substrate for antipsychotic therapies targeting ionotropic glutamate receptors.

Different arrangement of hydrophobic and nucleophilic components of channel binding sites in N-methyl- d-aspartate and AMPA receptors of rat brain is revealed by channel blockade

In order to investigate the topography of the channel binding site in (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl- d-aspartate (NMDA) types of glutamate receptors, we have compared the blocking actions of mono- and dicationic derivatives of adamantane and phenylcyclohexyl. The potencies of dicationic derivatives to block AMPA receptor channels are about 1000 times higher than those of monocationic ones, whereas NMDA receptors are equally sensitive to both mono- and dicationic derivatives. The dependence of the activity of dicationic compounds on the length of the polymethylene chain between ammonium groups has a pronounced maximum for AMPA receptor channel block. For NMDA receptor channel dicationic compounds with various internitrogen distances produce similar blocking effects. The results show that hydrophobic and nucleophilic components of the binding site are located close to each other in the NMDA receptor channel but are separated by ∼10 Å in the AMPA receptor channel.

Mechanism of vascular relaxation by thaligrisine: Functional and binding assays

In the present study we examine the mechanism by which thaligrisine, a bisbenzyltetrahydroisoquinoline alkaloid, inhibits the contractile response of vascular smooth muscle. The work includes functional studies on rat isolated aorta and tail artery precontracted with noradrenaline or KCl. In other experiments rat aorta was precontracted by caffeine in the presence or absence of extracellular Ca 2+. In order to assess whether thaligrisine interacts directly with calcium channel binding sites or with α-adrenoceptors we examined the effect of the alkaloid on [ 3H]-(+)- cis diltiazem, [ 3H]-nitrendipine and [ 3H]-prazosin binding to cerebral cortical membranes. The functional studies showed that the alkaloid inhibited in a concentration-dependent manner the contractile response induced by depolarization in rat aorta (IC 50=8.9±2.9 μM, n=5) and in tail artery (IC 50=3.04±0.3 μM, n=6) or noradrenaline induced contraction in rat aorta (IC 50=23.0±0.39 μM, n=9) and in tail artery (IC 50=3.8±0.9 μM, n=7). In rat aorta, thaligrisine concentration-dependently inhibited noradrenaline-induced contraction in Ca 2+-free solution (IC 50 = 13.3 μM, n=18). The alkaloid also relaxed the spontaneous contractile response elicited by extracellular calcium after depletion of noradrenaline-sensitive intracellular stores (IC 50 = 7.7 μM, n=4). The radioligand receptor-binding study showed that thaligrisine has higher affinity for [ 3H]-prazosin than for [ 3H]-(+)- cis-diltiazem binding sites, with Ki values of 0.048±0.007 μM and 1.5±1.1 μM respectively. [ 3H]-nitrendipine binding was not affected by thaligrisine. The present work provides evidence that thaligrisine shows higher affinity for [ 3H]-prazosin binding site than [ 3H]-(+)- cis-diltiazem binding sites, in contrast with tetrandrine and isotetrandrine that present similar affinity for both receptors. In functional studies thaligrisine, acted as an α 1-adrenoceptor antagonist and as a Ca 2+ channel blocker, relaxing noradrenaline or KCl-induced contractions in vascular smooth muscle. This compound specifically inhibits the refilling of internal Ca 2+-stores sensitive to noradrenaline, by blocking Ca 2+-entry through voltage-dependent Ca 2+-channels.