Kinetic properties and open probability of alpha7 nicotinic acetylcholine receptors.

The alpha7 nicotinic acetylcholine receptor (nAChR) has some peculiar kinetic properties. From the literature of alpha7 nAChR-mediated currents we concluded that experimentally measured kinetic properties reflected properties of the solution exchange system, rather than genuine kinetic properties of the receptors. We also concluded that all experimentally measured EC50 values for agonists must inherently be inaccurate. The aim of this study was to assess the undistorted kinetic properties of alpha7 nAChRs, and to construct an improved kinetic model, which can also serve as a basis of modeling the effect of the positive allosteric modulator PNU-120596, as it is described in the accompanying paper. Agonist-evoked currents were recorded from GH4C1 cells stably transfected with pCEP4/rat alpha7 nAChR using patch-clamp and fast solution exchange. We used two approaches to circumvent the problem of insufficient solution exchange rate: extrapolation and kinetic modeling. First, using different solution exchange rates we recorded evoked currents, and extrapolated their amplitude and kinetics to instantaneous solution exchange. Second, we constructed a kinetic model that reproduced concentration-dependence and solution exchange rate-dependence of receptors, and then we simulated receptor behavior at experimentally unattainably fast solution exchange. We also determined open probabilities during choline-evoked unmodulated and modulated currents using nonstationary fluctuation analysis. The peak open probability of 10 mM choline-evoked currents was 0.033 +/- 0.006, while in the presence of choline (10 mM) and PNU-120596 (10 muM), it was increased to 0.599 +/- 0.058. Our kinetic model could adequately reproduce low open probability, fast kinetics, fast recovery and solution exchange rate-dependent kinetics

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects

The tricyclic antidepressant desipramine inhibited the neurotoxic, kainate-induced [Ca] increases in CA1 pyramidal cells in acute hippocampal slices.

Kainate (KA), used for modelling neurodegenerative diseases, evokes excitotoxicity. However, the precise mechanism of KA-evoked [Ca2+]i increase is unexplored, especially in acute brain slice preparations. We used [Ca2+]i imaging and patch clamp electrophysiology to decipher the mechanism of KA-evoked [Ca2+]i rise and its inhibition by the tricyclic antidepressant desipramine (DMI) in CA1 pyramidal cells in rat hippocampal slices and in cultured hippocampal cells. The effect of KA was dose-dependent and relied totally on extracellular Ca2+. The lack of effect of dl-2-amino-5-phosphonopentanoic acid (AP-5) and abolishment of the response by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggested the involvement of non-N-methyl-d-aspartate receptors (non-NMDARs). The predominant role of the Ca2+-impermeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) in the initiation of the Ca2+ response was supported by the inhibitory effect of the selective AMPAR antagonist GYKI 53655 and the ineffectiveness of 1-naphthyl acetylspermine (NASPM), an inhibitor of the Ca2+-permeable AMPARs. The voltage-gated Ca2+ channels (VGCC), blocked by omega-Conotoxin MVIIC+nifedipine+NiCl2, contributed to the [Ca2+]i rise. VGCCs were also involved, similarly to AMPAR current, in the KA-evoked depolarisation. Inhibition of voltage-gated Na+ channels (VGSCs; tetrodotoxin, TTX) did not affect the depolarisation of pyramidal cells but blocked the depolarisation-evoked action potential bursts and reduced the Ca2+ response. The tricyclic antidepressant DMI inhibited the KA-evoked [Ca2+]i rise in a dose-dependent manner. It directly attenuated the AMPA-/KAR current, but its more potent inhibition on the Ca2+ response supports additional effect on VGCCs, VGSCs and Na+/Ca2+ exchangers. The multitarget action on decisive players of excitotoxicity holds out more promise in clinical therapy of neurodegenerative diseases

Protective effect of rasagiline in aminoglycoside ototoxicity.

Sensorineural hearing losses (SNHLs; e.g., ototoxicant- and noise-induced hearing loss or presbycusis) are among the most frequent sensory deficits, but they lack effective drug therapies. The majority of recent therapeutic approaches focused on the trials of antioxidants and reactive oxygen species (ROS) scavengers in SNHLs. The rationale for these studies was the prominent role of disturbed redox homeostasis and the consequent ROS elevation. Although the antioxidant therapies in several animal studies seemed to be promising, clinical trials have failed to fulfill expectations. We investigated the potential of rasagiline, an FDA-approved monomanine oxidase type B inhibitor (MAO-B) inhibitor type anti-parkinsonian drug, as an otoprotectant. We showed a dose-dependent alleviation of the kanamycin-induced threshold shifts measured by auditory brainstem response (ABR) in an ototoxicant aminoglycoside antibiotic-based hearing loss model in mice. This effect proved to be statistically significant at a 6-mg/kg (s.c.) dose. The most prominent effect appeared at 16kHz, which is the hearing sensitivity optimum for mice. The neuroprotective, antiapoptotic and antioxidant effects of rasagiline in animal models, all targeting a specific mechanism of aminoglycoside injury, may explain this otoprotection. The dopaminergic neurotransmission enhancer effect of rasagiline might also contribute to the protection. Dopamine (DA), released from lateral olivocochlear (LOC) fibers, was shown to exert a protective action against excitotoxicity, a pathological factor in the aminoglycoside-induced SNHL. We have shown that rasagiline enhanced the electric stimulation-evoked release of DA from an acute mouse cochlea preparation in a dose-dependent manner. Using inhibitors of voltage-gated Na+-, Ca2+ channels and DA transporters, we revealed that rasagiline potentiated the action potential-evoked release of DA by inhibiting the reuptake. The complex, multifactorial pathomechanism of SNHLs most likely requires drugs acting on multiple targets for effective therapy. Rasagiline, with its multi-target action and favorable adverse effects profile, might be a good candidate for a clinical trial testing the otoprotective indication

A nemszinaptikus nikotinikus acetilkolin és NMDA receptorok szerepe élettani körülmények között és pathológiás állapotokban = Role of nonsynaptic nicotinic acetylcholine receptors and NMDA receptors in physiological and pathophysiological conditions

A szélütés (stroke) utáni neurodegeneráció a jelenlegi morbiditási és mortalitási mutatók egyik legfontosabb tényezője. Az iszkémiás stroke kezelésében számos ígéretes gyógyszerjelölt molekula vallott kudarcot a klinikai vizsgálatokban. Ennek valószínűleg az az oka, hogy hiányosak ismereteink az iszkémiás kórképek kialakulásának mechanizmusaira vonatkozólag. A legtöbb központi idegrendszerre ható gyógyszert szinaptikusan elhelyezkedő receptorokra vagy transzporterekre fejlesztik annak érdekében, hogy igazán hatékony gyógyszereket tudjunk fejleszteni, figyelembe kell venni, hogy az extraszinaptikus receptorok és transzporterek száma jóval meghaladja a szinaptikusakét, illetve hogy nagyon sok központi idegrendszeri megbetegedés alapja a nemszinaptikus rendszer malfunkciója. Például, a szinaptikus NMDA receptorok aktivációja neuroprotektív hatást fejt ki, míg az extraszinaptikus NMDA receptor aktiváció excitotoxikus hatású. Konkrét javaslataink a gyógyszerfejlesztést illetően: Az NR2B alegységet tartalmazó NMDA receptorok szelektív gátlói (mint például a fluoxetine), és a nátriumcsatorna gátlók egyes típusai; mint neuroprotektív szerek. A nikotinikus agonisták pozitív modulátorai, amelyek a kognitív problémák kezelésében, ill. a dohányzásról való leszokás segítésében lehetnek hasznosak. | Neurodegeneration after a stroke is one of the major causes of present-day morbidity and mortality. There is a long list of neuroprotective compounds that have failed to be clinically useful in the treatment of ischaemic stroke. This is likely due, at least in part, to our inadequate knowledge regarding the core mechanisms of ischaemic diseases. Most “novel” drugs that target the CNS are designed to act on neurotransmitter receptors or transporters that are localised within synapses. To develop the most effective drugs, it is important to remember that there are extrasynaptic receptors and transporters that may outnumber those located within synapses and that, when malfunctioning, may be responsible for several symptoms of CNS disorders. For example, activation of synaptic NMDA receptors is neuroprotective, whereas stimulation of extrasynaptic NMDA receptors causes excitotoxicity. We suggest that future drug development research consider the following: Compounds that are able to selectively inhibit non-synaptic NR2B Glu receptors (such as Fluoxetine), and specific subtypes of sodium channel inhibitors as neuroprotective compounds. Positive modulators of nicotinic acetylcholine receptors. They would be potential drugs in the treatment of memory problems and in smoking cessation