tetrodotoxin and Acidosis

Glutamate activates peripheral group I metabotropic glutamate receptors (mGluRs) and contributes to inflammatory pain. However, it is still not clear the mechanisms are involved in group I mGluR-mediated peripheral sensitization. Herein, we report that group I mGluRs signaling sensitizes acid-sensing ion channels (ASICs) in dorsal root ganglion (DRG) neurons and contributes to acidosis-evoked pain. DHPG, a selective group I mGluR agonist, can potentiate the functional activity of ASICs, which mediated the proton-induced events. DHPG concentration-dependently increased proton-gated currents in DRG neurons. It shifted the proton concentration-response curve upwards, with a 47.3±7.0% increase of the maximal current response to proton. Group I mGluRs, especially mGluR5, mediated the potentiation of DHPG via an intracellular cascade. DHPG potentiation of proton-gated currents disappeared after inhibition of intracellular Gq/11 proteins, PLCβ, PKC or PICK1 signaling. Moreover, DHPG enhanced proton-evoked membrane excitability of rat DRG neurons and increased the amplitude of the depolarization and the number of spikes induced by acid stimuli. Finally, peripherally administration of DHPG dose-dependently exacerbated nociceptive responses to intraplantar injection of acetic acid in rats. Potentiation of ASIC activity by group I mGluR signaling in rat DRG neurons revealed a novel peripheral mechanism underlying group I mGluRs involvement in hyperalgesia.

Topics: Acetic Acid; Acid Sensing Ion Channels; Acidosis; Animals; Capsaicin; Ganglia, Spinal; Male; Methoxyhydroxyphenylglycol; Neurons; Pain; Rats, Sprague-Dawley; Receptors, Metabotropic Glutamate; Sodium Channel Blockers; Tetrodotoxin; TRPV Cation Channels

Voltage-gated Na

Topics: Acidosis; Acids; Animals; Cells, Cultured; Extracellular Space; Hydrogen-Ion Concentration; Kinetics; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats, Sprague-Dawley; Sodium Channel Blockers; Tetrodotoxin; Trigeminal Ganglion

Aspirin and its main metabolite salicylate are widely used to relieve pain, treat inflammatory diseases, and prevent ischemic stroke. Multiple pathways are responsible for the therapeutic actions exerted by these drugs. One of the pathways is targeting neuronal receptors/ion channels in the central nervous system. Correspondingly, increasing evidence has implicated acid-sensing ion channels (ASICs) in the processes of the diseases that are medicated by aspirin and salicylate. We therefore employed whole-cell patch-clamp recordings to examine the effects of salicylate as well as aspirin on ASICs in cultured cortical neurons of the rat. We recorded rapid and reversible inhibition of ASIC current by millimolar concentrations of aspirin and salicylate and found that salicylate reduced acidosis-induced membrane depolarization. These data suggest that ASICs in the cortex are molecular targets of high doses of aspirin and salicylate. In addition, the results from lactate dehydrogenase release measurement showed that high doses of aspirin and salicylate protected the cortical neuron from acidosis-induced neuronal injury. These findings may contribute to a better understanding of the therapeutic mechanisms of aspirin and salicylate actions in the brain and provide new evidence on aspirin and salicylate used as neuroprotective agents in the treatment of ischemic stroke.

Topics: Acid Sensing Ion Channels; Acidosis; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Cell Death; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Embryo, Mammalian; Nerve Tissue Proteins; Neural Inhibition; Neurons; Patch-Clamp Techniques; Propidium; Rats; Rats, Sprague-Dawley; Salicylates; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin

Acidosis accompanying cerebral ischemia activates acid-sensing ion channels (ASIC) causing increases in intracellular calcium concentration ([Ca(2+)]i) and enhanced neuronal death. Experiments were undertaken in rat cortical neurons to explore the effects of ASIC1a activation on ischemia-induced [Ca(2+)]i elevations and whole-cell currents. There was a significant contribution of ASIC1a channels to ischemia-evoked [Ca(2+)]i increases at pH 7.4, suggesting that ASIC1a channels are activated by endogenous protons during ischemia. The combination of ischemia and acidosis resulted in synergistic increases in [Ca(2+)]i and plasma membrane currents relative to acidosis or ischemia alone. ASIC1a inhibitors significantly blunted [Ca(2+)]i increases and a transient current activated by ischemia+acidosis, demonstrating that homomeric ASIC1a channels are involved. However, ASIC1a inhibitors failed to diminish a sustained current activated in response to combined ischemia and acidosis, indicating that acidosis can potentiate ischemia effects through mechanisms other than ASIC1a. The [Ca(2+)]i overload produced by acidosis and ischemia was not blocked by tetrodotoxin, 2-amino-5-phosphonopentanoic acid or nifedipine. Thus, acidosis and activation of ASIC1a channels during ischemia can promote [Ca(2+)]i overload in the absence of neurotransmission, independent of NMDA receptor or L-type voltage-gated Ca(2+) channel activation. Postsynaptic ASIC1a channels play a critical role in ischemia-induced [Ca(2+)]i dysregulation and membrane dysfunction.

Topics: 2-Amino-5-phosphonovalerate; Acid Sensing Ion Channels; Acidosis; Animals; Brain Ischemia; Calcium; Calcium Channels, L-Type; Cerebral Cortex; Nerve Tissue Proteins; Neurons; Nifedipine; Protons; Rats; Receptors, N-Methyl-D-Aspartate; Sodium Channels; Synaptic Transmission; Tetrodotoxin

The role of extracellular acidosis in inflammatory airway diseases is not well known. One consequence of tissue acidification is the stimulation of sensory nerves via the polymodal H(+)-gated transmembrane channels ASICs and TRPV1 receptor. The present study investigated the effect of acidosis on airway basal tone and responsiveness in the guinea pig. Acidosis (pH 6.8, 10 min, 37 degrees C) significantly decreased the basal tone of tracheal rings (p<0.01 vs. paired control). Moreover, pH fall raised the maximal contraction of tracheal rings to acetylcholine (p<0.05 vs. paired control). The pH-induced relaxation of airway basal tone was inhibited by pretreatments with ASIC1a or ASIC3/ASIC2a inhibitors (0.5 mM ibuprofen, 0.1 mM gadolinium), nitric oxide synthase inhibitor (1 mM L-NAME), and guanylate cyclase inhibitor (1 microM ODQ). In contrast, the pH-induced relaxation of airway basal tone was not modified by epithelium removal or pretreatments with a TRPV1 antagonist (1 microM capsazepine), a combination of NK(1,2,3) receptor antagonists (0.1 microM each), a blocker of voltage-sensitive Na(+) channels (1 microM tetrodotoxin), a cyclooxygenase inhibitor with no activity on ASICs (1 microM indomethacin) or ASIC3 and ASIC3/ASIC2b inhibitors (10 nM diclofenac, 1 microM aspirin). Furthermore, acid-induced hyperresponsiveness to acetylcholine was inhibited by epithelium removal, capsazepine, NK(1,2,3) receptor antagonists, tetrodotoxin, amiloride, ibuprofen and diclofenac. In summary, the initial pH-induced airway relaxation seems to be independent of sensory nerves, suggesting a regulation of airway basal tone mediated by smooth muscle ASICs. Conversely, the pH-induced hyperresponsiveness involves sensory nerves-dependent ASICs and TRPV1, and an unknown epithelial component in response to acidosis.

Topics: Acid Sensing Ion Channels; Acidosis; Amiloride; Animals; Capsaicin; Enzyme Inhibitors; Guinea Pigs; Hydrogen-Ion Concentration; Male; Membrane Proteins; Muscle Contraction; Muscle Relaxation; Muscle Tonus; Nerve Tissue Proteins; Poisons; Receptors, Tachykinin; Sensory Receptor Cells; Sodium Channel Blockers; Sodium Channels; Tetrodotoxin; Trachea; TRPV Cation Channels

We investigated mechanisms of CO(2)/H(+) chemoreception in the respiratory centre of the medulla by measuring membrane potentials of pre-inspiratory neurons, which are putative respiratory rhythm generators, in the brainstem-spinal cord preparation of the neonatal rat. Neuronal response was tested by changing superfusate CO(2) concentration from 2% to 8% at constant HCO(3)(-) concentration (26 mm) or by changing pH from 7.8 to 7.2 by reducing HCO(3)(-) concentration at constant CO(2) (5%). Both respiratory and metabolic acidosis lead to depolarization of neurons with increased excitatory synaptic input and increased burst rate. Respiratory acidosis potentiated the amplitude of the neuronal drive potential. In the presence of tetrodotoxin (TTX), membrane depolarization persisted during respiratory and metabolic acidosis. However, the depolarization was smaller than that before application of TTX, which suggests that some neurons are intrinsically, and others synaptically, chemosensitive to CO(2)/H(+). Application of Ba(2+) blocked membrane depolarization by respiratory acidosis, whereas significant depolarization in response to metabolic acidosis still remained after application of Cd(2+) and Ba(2+). We concluded that the intrinsic responses to CO(2)/H(+)changes were mediated by potassium channels during respiratory acidosis, and that some other mechanisms operate during metabolic acidosis. In low-Ca(2+), high-Mg(2+) solution, an increased CO(2) concentration induced a membrane depolarization with a simultaneous increase of the burst rate. Pre-inspiratory neurons could adapt their baseline membrane potential to external CO(2)/H(+) changes by integration of these mechanisms to modulate their burst rates. Thus, pre-inspiratory neurons might play an important role in modulation of respiratory rhythm by central chemoreception in the brainstem-spinal cord preparation.

Topics: Acidosis; Animals; Animals, Newborn; Barium; Calcium; Carbon Dioxide; Chemoreceptor Cells; Hydrogen; Hydrogen-Ion Concentration; Magnesium; Medulla Oblongata; Membrane Potentials; Neurons; Potassium Channels; Rats; Rats, Wistar; Respiratory Center; Synapses; Tetrodotoxin

The chemosensitive response of locus coeruleus (LC) neurones to changes in intracellular pH (pH(i)), extracellular pH (pH(o)) and molecular CO(2) were investigated using neonatal rat brainstem slices. A new technique was developed that involves the use of perforated patch recordings in combination with fluorescence imaging microscopy to simultaneously measure pH(i) and membrane potential (V(m)). Hypercapnic acidosis (15 % CO(2), pH(o) 6.8) resulted in a maintained fall in pH(i) of 0.31 pH units and a 93 % increase in the firing rate of LC neurones. On the other hand, isohydric hypercapnia (15 % CO(2), 77 mM HCO(3)(-), pH(o) 7.45) resulted in a smaller and transient fall in pH(i) of about 0.17 pH units and an increase in firing rate of 76 %. Acidified Hepes (N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid)-buffered medium (pH(o) 6.8) resulted in a progressive fall in pH(i) of over 0.43 pH units and an increase in firing rate of 126 %. Isosmotic addition of 50 mM propionate to the standard HCO(3)(-)-buffered medium (5 % CO(2), 26 mM HCO(3)(-), pH(o) 7.45) resulted in a transient fall in pH(i) of 0.18 pH units but little increase in firing rate. Isocapnic acidosis (5 % CO(2), 7 mM HCO(3)(-), pH(o) 6.8) resulted in a slow intracellular acidification to a maximum fall of about 0.26 pH units and a 72 % increase in firing rate. For all treatments, the changes in pH(i) preceded or occurred simultaneously with the changes in firing rate and were considerably slower than the changes in pH(o). In conclusion, an increased firing rate of LC neurones in response to acid challenges was best correlated with the magnitude and the rate of fall in pH(i), indicating that a decrease in pH(i) is a major part of the intracellular signalling pathway that transduces an acid challenge into an increased firing rate in LC neurones.

Topics: Acidosis; Animals; Animals, Newborn; Carbon Dioxide; Electrophysiology; Extracellular Space; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; Hypercapnia; Kinetics; Locus Coeruleus; Membrane Potentials; Microscopy, Fluorescence; Neurons; Patch-Clamp Techniques; Pons; Propionates; Rats; Rats, Sprague-Dawley; Tetrodotoxin

Activity-related shifts in intracellular pH (pHi) can exert potent neuromodulatory actions. Different states of neuronal activity of thalamocortical neurons were found to differentially modulate pHi. Tonic activity evoked by injection of depolarizing current led to a reversible rise in [H+]i which was nearly abolished in the presence of TTX. Block of voltage-gated calcium channels with I mM Ni2+ reduced the [H+]i transients related to tonic activity. Rhythmic activation of burst discharges caused changes of [H+]i which were decreased by TTX, whereas I mM Ni2+ almost abolished the [H+]i transients. The present results show that different forms of neuronal activity can lead to intracellular acidification caused by different mechanisms, i.e. Na+ and Ca2+ influx through sodium and Ca2+ channels, respectively, and the subsequent activation of a Ca2+/H+ pump. The resulting acidosis is suggested to reduce further Ca2+ influx and prevent excessive neuronal excitation.

Topics: Acidosis; Animals; Calcium Channels; Electric Stimulation; Electrophysiology; Fluoresceins; Fluorescent Dyes; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Neurons; Patch-Clamp Techniques; Rats; Rats, Long-Evans; Sodium Channels; Tetrodotoxin; Thalamus

The response to hypercapnic acidosis (2-8% CO2, bath pH 7.8-7.2) was examined in whole cell recordings from neonatal (P1 to P5) rat Locus coeruleus (LC) neurones in the in vitro brainstem-spinal cord preparation exposed to low Ca2+ (0.2 mM)-high Mg2+ (5 mM). This medium suppressed chemical synaptic transmission and resulted in a pattern of subthreshold oscillations of membrane potential and rhythmic burst discharge which was synchronized throughout the network. The oscillation was suppressed, and the discharge of individual neurones desynchronized, by the gap junction uncoupler, carbenoxolone, indicating that in low Ca2+-high Mg2+ LC neurones form an electrically coupled network. Switching from 2 to 8% CO2 decreased the oscillation amplitude and increased its frequency. The oscillation was suppressed by external Cd2+ and by TTX. but persisted during injection into the cell soma of QX-314. We conclude that in LC neurones acidosis increases the frequency of a Ca2+- and Na+-dependent dendritic oscillator which is synchronized by gap junction coupling throughout the network. This coupling is retained during acidosis.

Topics: Acidosis; Animals; Cadmium; Calcium; Cells, Cultured; Culture Media; Hydrogen-Ion Concentration; Hypercapnia; Lidocaine; Locus Coeruleus; Magnesium; Membrane Potentials; Neurons; Rats; Rats, Sprague-Dawley; Tetrodotoxin

Topics: Acidosis; Action Potentials; Animals; Dogs; Electrophysiology; Endocardium; Hyperkalemia; Hypoxia; Membrane Potentials; Papillary Muscles; Purkinje Fibers; Solutions; Tetrodotoxin; Verapamil