Synthesis of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2, 3-benzodiazepin-4-ones as novel and potent noncompetitive AMPA receptor antagonists.

A group of 7,8-(methylenedioxy)-1-phenyl-3,5-dihydro-4H-2, 3-benzodiazepin-4-ones was synthesized and assayed for antagonism of rat brain alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors expressed in Xenopus oocytes. The benzodiazepinones inhibited AMPA-activated membrane current responses in a manner consistent with noncompetitive, allosteric inhibition of the receptor-channel complex. The most potent compound in the series was 1-(4-aminophenyl)-7,8-(methylenedioxy)-3,5-dihydro-4H-2, 3-benzodiazepin-4-one (6), which had an IC50 of 2.7 microM. For comparison, the reference compound GYKI 52466 (2) had an IC50 of 6.9 microM. Compound 6 also had potent anticonvulsant activity in a mouse maximum electroshock-induced seizure (MES) assay: the ED50 was 2.8 mg/kg iv, whereas the ED50 for GYKI 52466 was 4.6 mg/kg iv. In contrast to a previous report, the 7,8-dimethoxy analogue of 6 was a low-potency AMPA antagonist (IC50 >100 microM) and weak anticonvulsant (ED50 >10 mg/kg iv). The benzodiazepinones described herein are potent noncompetitive AMPA receptor antagonists that could have therapeutic potential as anticonvulsants and neuroprotectants.

Compression stimuli increase the efficacy of breast pump function.

OBJECTIVE: The dynamics of milk ejection and also prolactin concentration in the blood during milk expression by means of a breast pump were studied in 82 lactating women. STUDY DESIGN: The design of the breast pump used in this study is new. It is based on the correlation with the amplitude, duration and frequency of vacuum and compression stimuli formed by the baby's mouth apparatus during the suckling process. RESULTS: It was found that during continuous co-action of the vacuum and compression stimuli at a frequency of 1 cycle per second, the rate of milk ejection from the breast changed periodically. The highest rates of milk ejection coincided with the greatest rises in intramammary pressure. When the compression stimulus was turned off, leaving only the vacuum, the period of the first rise in pressure increased 1.5-2 fold, with a 1.5-2 fold reduction in the milk ejection rate as compared with the normal function of the breast pump. The basal prolactin level on day 3 and 5 post delivery in women who used a breast pump was not significantly different from that of puerperal women who only breast-fed their babies. Prolactin concentration in the blood of puerperal women did not change within the 5-6 min of expression and stimulation with the breast pump. The prolactin level began to increase by the 10th minute, whereas by the 25th minute it exceeded the initial concentration 1.3-1.5-fold. CONCLUSION: It is suggested that by introducing a tactile component to the mechanism of the breast pump, milk secretion is enhanced, as well as stimulating the breast milk flow. This directly improves the dynamics of breast milk expression.

5-(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-diones: novel, systemically active and broad spectrum antagonists for NMDA/glycine, AMPA, and kainate receptors.

A group of 5-aza-7-substituted-1,4-dihydroquinoxaline-2,3-diones (QXs) and the corresponding 5-(N-oxyaza)-7-substituted QXs were prepared and evaluated as antagonists of ionotropic glutamate receptors. The in vitro potency of these QXs was determined by inhibition of [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) binding to N-methyl-D-aspartate (NMDA)/glycine receptors, [3H]-(S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding to AMPA receptors, and [3H]kainate ([3H]KA) binding to KA receptors in rat brain membranes. 5-(N-Oxyaza)-QXs 12a-e all have low micromolar or submicromolar potency for NMDA/glycine receptors and low micromolar potencies for AMPA and KA receptors. QXs 12a-e display 2-12-fold selectivity for NMDA/glycine receptors compared to AMPA receptors, and approximately 2-fold difference between AMPA and KA potency. In contrast to other QXs that either show high selectivity for NMDA (such as ACEA 1021) or AMPA (such as NBQX) receptors, these molecules are broad spectrum antagonists of ionotropic glutamate receptors. 7-Nitro-5-(N-oxyaza)-QX (12e) is the most potent inhibitor among 12a-e, having IC50 values of 0.69, 1.3, and 2.4 microM at NMDA, AMPA, and KA receptors, respectively. In functional assays on glutamate receptors expressed in oocytes by rat cerebral cortex poly(A+) RNA, 7-chloro-5-(N-oxyaza)-QX (12a) and 7-nitro-5-(N-oxyaza)-QX (12e) have Kb values of 0.63 and 0.31 microM for NMDA/glycine receptors, and are 6- and 4-fold selective for NMDA over AMPA receptors, respectively. 5-(N-Oxyaza)-7-substituted-QXs 12a-e all have surprisingly high in vivo potency as anticonvulsants in a mouse maximal electroshock-induced seizure (MES) model. 7-Chloro-5-(N-oxyaza)-QX (12a), 7-bromo-5-(N-oxyaza)-QX (12b), and 7-methyl-5-(N-oxyaza)-QX (12c) have ED50 values of 0.82, 0.87, and 0.97 mg/kg i.v., respectively. The high in vivo potency of QXs 12a-e is particularly surprising given their low log P values (approximately -2.7). Separate studies indicate that QXs 12a and 12e are also active in vivo as neuroprotectants and also have antinociceptive activity in animal pain models. In terms of in vivo activity, these 5-(N-oxyaza)-7-substituted-QXs are among the most potent broad spectrum ionotropic glutamate antagonists reported.

Antagonism of N-methyl-D-aspartate receptors by sigma site ligands: potency, subtype-selectivity and mechanisms of inhibition.

Recent studies propose that sigma site ligands antagonize N-methyl-D-aspartate (NMDA) receptors by either direct, or indirect mechanisms of inhibition. To investigate this question further we used electrical recordings to assay actions of seventeen structurally diverse sigma site ligands on three diheteromeric subunit combinations of cloned rat NMDA receptors expressed in Xenopus oocytes: NR1a coexpressed with either NR2A, 2B or 2C. The sigma site ligands had a wide range of potency for antagonizing NMDA receptor currents. Steady-state IC50 values ranged between approximately 0.1 to >100 microM. In all cases inhibition was non-competitive with respect to glycine and glutamate. Five structurally related sigma ligands [eliprodil, haloperidol, ifenprodil, 4-phenyl-1-(4-phenylbutyl)-piperidine and trifluperidol] were strongly selective for NR1a/2B receptors. The other drugs were weakly selective or nonselective inhibitors. There was no correlation between sigma site affinity and potency of NMDA receptor antagonism for any subunit combination. Inhibition of NR1a/2B receptors by the selective antagonists was independent of voltage whereas inhibition by the weakly selective antagonists was voltage dependent. Potency of 10 sigma ligands was cross-checked on NMDA currents in cultured rat cortical neurons. There was close correspondence between the two assay systems. Our results argue that antagonism of NMDA receptor currents by the sigma ligands tested is due to direct effects on the receptor channel complex as opposed to indirect effects mediated by sigma receptors. Inhibition occurs via sites in the NMDA receptor channel pore, or via allosteric modulatory sites associated with the NR2B subunit.

Subtype-selective antagonism of NMDA receptors by nylidrin.

The 1,4-di-substituted piperidines ifenprodil, eliprodil, CP 101,606 ((1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol ) and Ro 25-6981 ((R-(R*,S*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1- piperidinepropanol) are allosteric antagonists of NMDA receptors. Inhibition of diheteromeric NMDA receptors by this class of antagonist is characterized by pronounced selectivity for NR1/2B subunit combinations. In the current study, we assayed effects of nylidrin, a structurally-related non-piperidine, on recombinant and neuronal NMDA receptors. Nylidrin was a potent (IC50 = 0.18 microM) antagonist of NR1A/2B receptors expressed in Xenopus oocytes and was at least 150-fold weaker against NR1A/2A and NR1A/2C receptors. The blockade of NR1A/2B responses by nylidrin was not surmounted by increasing the concentrations of glutamate or glycine and was not voltage-dependent. Potency of inhibition increased approximately 3-fold upon lowering extracellular pH from 8 to 6.8. Nylidrin inhibited NMDA responses in cultured rat cortical neurons with similar potency and apparent mechanism of action as the NR1A/2B receptors. Our results suggest that nylidrin interacts with the same allosteric inhibitory site previously described for the related piperidine antagonists, and should serve as a structural lead for designing novel subtype-selective inhibitors of NMDA receptors.

Subtype-selective inhibition of N-methyl-D-aspartate receptors by haloperidol.

Previous studies indicate that haloperidol, a therapeutically useful antipsychotic drug, inhibits neuronal N-methyl-D-aspartate (NMDA) responses and has neuroprotective effects against NMDA-induced brain injury. To further characterize this inhibition, we used electrical recordings to assay the effects of haloperidol on four diheteromeric subunit combinations of cloned rat NMDA receptors expressed in Xenopus laevis oocytes: NR1A coexpressed with NR2A, NR2B, NR2C, or NR2D. Haloperidol selectively blocks NR1A/2B subunit combinations (IC50 = approximately 3 microM; maximum inhibition, approximately 85%), whereas the other subunit combinations are > or = 100-fold less sensitive (IC50 = >300 microM). Inhibition of NR1A/2B receptors is insurmountable with respect to glutamate and glycine and does not exhibit voltage dependence. The splice variant combinations NR1B/2B and NR1e/2B are also blocked by haloperidol. In oocytes from some frogs, 30-100 microM haloperidol induces potentiation of NR1A/2A receptor responses. NMDA responses in E16-17 rat cortical neurons cultured for < or = 10 days are inhibited by haloperidol at the same potency and to the extent as NR1/2B receptors (IC50 = approximately 2 microM; maximum inhibition, approximately 80%). In contrast, cells cultured for longer periods show a wide range of sensitivity. This change in pharmacology coincides with a developmental switch in subunit expression; from NR1 expressed with NR2B to NR1 coexpressed with NR2A and NR2B. Inhibition of macroscopic neuronal NMDA responses is mechanistically similar to inhibition of NR1A/2B receptors. Single-channel recordings from neurons show that antagonism is associated with a decrease in the frequency of channel openings and a shortening of mean channel open time. Collectively, our experiments indicate that haloperidol selectively inhibits NMDA receptors comprised of NR1 and NR2B subunits. Inhibition is consistent with action at a noncompetitive allosteric site that is distinct from the glutamate-, glycine-, and phencyclidine-binding sites and is probably mechanistically related to the atypical antagonist ifenprodil. Our results suggest that haloperidol can be used as a tool for investigating NMDA receptor subunit composition and can serve as a structural lead for designing novel subtype-selective NMDA receptor ligands.

Pharmacology of ACEA-1416: a potent systemically active NMDA receptor glycine site antagonist.

Excitatory amino acid receptor antagonists show potential for the treatment of ischemic stroke and head trauma. In search of novel antagonists, a series of alkyl- and alkoxyl-substituted 1, 4-dihydro-2,3-quinoxalinediones were synthesized and assayed for inhibition of glutamate receptors. We report on the pharmacological characterization of one such compound, 7-chloro-6-methyl-5-nitro-1,4-dihydro-2, 3-quinoxalinedione (ACEA-1416). Electrophysiological assays showed that ACEA-1416 is a potent antagonist of rat brain NMDA receptors expressed in Xenopus oocytes, and NMDA receptors expressed by cultured rat cortical neurons. Antagonism is via competitive inhibition at glycine co-agonist sites (Kb = 7.9 nM in oocytes, Kb = 11 nM in neurons). ACEA-1416 also antagonizes AMPA receptors, though potency is considerably lower (Kb = 3.5 microM in oocytes, Kb = 1.6 microM in neurons). Oocyte assays indicated that ACEA-1416 is weak or inactive as an antagonist at NMDA receptor glutamate binding sites (Kb > 5.9 microM) and metabotropic glutamate receptors (Kb > 57 microM). Many NMDA receptor glycine site antagonists show poor penetration of the blood-brain barrier. Systemic bioavailability of ACEA-1416 was assessed by measuring the ability of the compound to protect against electroshock-induced seizures in mice. Protective effects of ACEA-1416 had rapid onset following i.v. administration. Peak efficacy was at approximately 2 min and the biological half-time of protection was approximately 60 min. The ED50 measured at peak efficacy was approximately 1.5 mg/kg. Our results show that ACEA-1416 is a high potency systemically active NMDA receptor glycine site antagonist and a moderate potency AMPA receptor antagonist. Separate studies indicate that ACEA-1416 is efficacious as a neuroprotectant in a rat model of focal cerebral ischemia. Taken together, our results suggest that ACEA-1416 has potential for clinical development as a neuroprotectant.

Intracellular ATP modulates desensitization of acetylcholine receptors controlling chloride current in Lymnaea neurons.

Chloride current activated by nicotinic acetylcholine receptors (AChR) was examined in dialysed voltage-clamp neurons of Lymnaea stagnalis. Fast superfusion of acetylcholine (ACh) evoked an inward current rapidly rising to a peak followed by a decline due to desensitization. When adenosine triphosphate with Mg2+ (MgATP, 2-10 mM) was added intracellularly the peak of the ACh-induced current was increased and its decay was slowed down. ATP without Mg2+ did not affect desensitization. Mg2+ alone accelerated desensitization. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, increased the desensitization rate and decreased the peak current. MgATP after arsenate wash-out restored the initial characteristics of the response; a mixture of glycolytic substrates had a similar effect. A non-hydrolysable analogue of ATP, adenosine [gamma-thio]triphosphate mimicked ATP action after arsenate removal but was weaker; another non-hydrolysable analogue, adenylyl imidodiphosphate, did not affect desensitization at all. Intracellular treatment of the neurons with alkaline phosphatase accelerated current decay. The data suggest that a change in intracellular ATP concentration modulates AChR desensitization via an enzymatic process that might be phosphorylation of AChR or some associated protein(s). Involvement of Ca2+ homeostasis cannot be excluded. The results are compared with the data obtained on vertebrate tissues under conditions promoting phosphorylation.

Intracellular ATP modifies the voltage dependence of the fast transient outward K+ current in Lymnaea stagnalis neurones.

1. The action of intracellular ATP on the fast transient outward K+ current (A-current) was studied in dialysed voltage-clamped Lymnaea stagnalis neurones. 2. When introduced intracellularly in millimolar concentrations ATP caused a shift of the steady-state inactivation curve along the voltage axis in the direction of positive potentials and decreased A-current at all test voltages. 3. Intracellular treatment with an inhibitor of ATP synthesis, sodium arsenate, led to the opposite changes. The action of arsenate was not reversed upon its removal. After wash-out of arsenate ATP restored the initial voltage dependence. 4. Addition of Mg2+ to the solution weakened the action of ATP in proportion to the Mg2+: ATP concentration ratio. On the other hand, in neurones pretreated with arsenate, Mg2+ did not affect the ATP action. 5. When a mixture of glycolytic substrates was applied after arsenate wash-out the activation and inactivation curves shifted towards positive voltages. A substrate of oxidative phosphorylation was ineffective in the same conditions. 6. Non-hydrolysable analogues of ATP, adenosine-5'-O-gamma-thiotriphosphate and adenylyl imidodiphosphate, did not mimic the ATP action. This means that the ATP effect is mediated by some enzymatic process(es). 7. Elevation of total cytosolic Ca2+ concentration as well as intracellular application of agents increasing intracellular free Ca2+ reduced A-current amplitude but failed to alter its voltage dependence. Therefore, ATP action cannot be related to activation of Ca2+ transport. 8. Treatment of the neurones with alkaline phosphatase evoked a shift of the inactivation voltage dependence towards hyperpolarizing potentials and increased the A-current amplitudes at all test voltages. 9. The data indicate that a change in intracellular ATP concentration modulates the A-current voltage dependence. The effect of ATP is probably the result of phosphorylation of a channel protein or some associated proteins, but lowering of free Mg2+ concentration cannot be excluded. The possible physiological significance of the phenomenon is discussed.

Intracellular Mg2+ modulates the A-current and its blockage by catechol in isolated Lymnaea neurons.

The effects of intracellular Mg2+ (2-8 mM) upon the transient outward current (the A-current) under normal conditions and under catechol-induced blockage were studied in molluscan neurons by using the voltage-clamp and intracellular dialysis techniques. Identified giant Lymnaea stagnalis L. neurons were investigated at room temperature (20-22 degrees C). When applied intracellularly, Mg2+ caused both time- and dose-dependent shifts of the voltage dependence of the steady-state activation and inactivation of the A-current to more negative membrane potentials. Upon external application, catechol suppressed (5-6 mM) or eliminated (9-10 mM) the A-currents, slowed down the current decay and shifted the activation and inactivation curves to more positive membrane voltages. Intracellular Mg2+ decreased the blocking ability of extracellularly applied catechol, whereas catechol antagonized the Mg2(+)-induced negative shift of the steady-state activation and inactivation curves of the A-currents.

Generation of twitch tension in frog atrial fibers by Na/Ca exchange.

Electrical and mechanical responses of frog atrial trabeculae were studied simultaneously using the double-sucrose gap method. Action potentials and twitch tension could be successively generated in fibers in which the slow inward calcium channel current was not observed. As a rule, this could be obtained in the course of a long experiment (3 to 4 hours). Peak tension was shown to increase monotonically with membrane potential in these preparations. In preparations with the slow inward current the total peak tension could be separated into two components. The first component (tonic) monotonically increased with the membrane potential and was probably related to Na/Ca exchange (Horackova 1984). The potential dependency of the second (phasic) component correlated with that of the slow inward calcium current. Only the tonic but not the phasic component could be observed in preparations without the presence of the slow inward calcium current. The tonic component prevailed when both the slow inward current and phasic tension were greatly reduced by nifedipine. Long experiments, long depolarizing clamp pulses, a metabolic inhibitor 2,4-dinitrophenol, inhibitors of Na/K pump ouabain and AR-L57, toxins promoting intracellular sodium accumulation (aconitine, scorpion toxin) were all shown to increase the tonic tension, but not the slow inward current; they induced a transition from biphasic tension-voltage curve into a monotonically increasing one. We concluded that these procedures and agents greatly stimulate Ca influx via Na/Ca exchange. These results show that Na/Ca exchange can function as a reserve system of Ca2+ used for contraction, thus supporting the heart function, especially under unfavourable metabolic conditions.

The Cole-Moore effect in nodal membrane of the frog Rana ridibunda: evidence for fast and slow potassium channels.

The K conductance (gK) kinetics were studied in voltage-clamped frog nodes (Rana ridibunda) in double-pulse experiments. The Cole-Moore translation for gK--t curves associated with different initial potentials (E) was only observed with a small percentage of fibers. The absence of the translation was found to be caused by the involvement of an additional, slow, gK component. This component cannot be attributed to a multiple-state performance of the k channel. It can only be accounted for by a separate, slow K channel, the fast channel being the same as the n4 K channel in R. pipiens. The slow K channel is characterized by weaker sensitivity to TEA, smaller density, weaker potential (E) dependence, and somewhat more negative E range of activation than the fast K channel. According to characteristics of the slow K system, three types of fibers were found. In Type I fibers (most numerous) the slow K channel behaves as and n4 HH channel. In Type II fibers (the second largest group found) the slow K channel obeys the HH kinetics within a certain E range only; beyond this range the exponential decline of the slow gK component is preceded by an E-dependent delay, its kinetics after the delay being the same as those in Type I fibers. In Type III fibers (rare) the slow K channel is lacking, and it is only in these fibers that the Cole-Moore translation of the measured gK--t curves can be observed directly. The physiological role of the fast and slow K channel in amphibian nerves is briefly discussed.