capsazepine and pyridoxal-phosphate-6-azophenyl-2--4--disulfonic-acid

Inhaled cigarette smoke (CS) triggers airway reflexes that are thought to result from the activation of lung vagal C-fiber afferents (LVCAs) via the action of reactive oxygen species in rats. We investigated the role of transient receptor potential vanilloid 1 (TRPV1) and P2X receptors in LVCA activation. Activities of LVCAs were recorded in anesthetized and artificially ventilated rats. Airway challenge of CS produced a concentration-dependent fiber stimulation. Pretreatment with dimethylthiourea [DMTU; a scavenger of hydroxyl radical (OH)], capsazepine (CPZ; a TRPV1 receptor antagonist) and iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (iso-PPADS; a P2X receptor antagonist) separately reduced the fiber responses by 64, 40 and 44%, respectively, whereas pretreatment with hexamethonium (a nicotinic acetylcholine receptor antagonist) failed to alter the response. A combination of CPZ and iso-PPADS exerted a greater inhibitory effect compared with the effect of either single pretreatment. However, a combination of DMTU, CPZ and iso-PPADS did not further reduce the fiber response compared with the combined effect of CPZ and iso-PPADS. It was concluded that both TRPV1 and P2X receptors, but not nicotinic acetylcholine receptors, participate in the stimulation of LVCAs by inhaled CS, possibly through the action of OH.

Topics: Animals; Capsaicin; Cholinergic Fibers; Humans; Hydroxyl Radical; Pyridoxal Phosphate; Rats; Reactive Oxygen Species; Receptors, Nicotinic; Receptors, Purinergic P2X; Smoke; Smoking; Thiourea; TRPV Cation Channels

The adequate stimuli and molecular receptors for muscle metaboreceptors and nociceptors are still under investigation. We used calcium imaging of cultured primary sensory dorsal root ganglion (DRG) neurons from C57Bl/6 mice to determine candidates for metabolites that could be the adequate stimuli and receptors that could detect these stimuli. Retrograde DiI labeling determined that some of these neurons innervated skeletal muscle. We found that combinations of protons, ATP, and lactate were much more effective than individually applied compounds for activating rapid calcium increases in muscle-innervating dorsal root ganglion neurons. Antagonists for P2X, ASIC, and TRPV1 receptors suggested that these three receptors act together to detect protons, ATP, and lactate when presented together in physiologically relevant concentrations. Two populations of muscle-innervating DRG neurons were found. One responded to low metabolite levels (likely nonnoxious) and used ASIC3, P2X5, and TRPV1 as molecular receptors to detect these metabolites. The other responded to high levels of metabolites (likely noxious) and used ASIC3, P2X4, and TRPV1 as their molecular receptors. We conclude that a combination of ASIC, P2X5 and/or P2X4, and TRPV1 are the molecular receptors used to detect metabolites by muscle-innervating sensory neurons. We further conclude that the adequate stimuli for muscle metaboreceptors and nociceptors are combinations of protons, ATP, and lactate.

Topics: Acid Sensing Ion Channels; Adenosine Triphosphate; Amiloride; Analysis of Variance; Animals; Calcium; Capsaicin; Cells, Cultured; Cnidarian Venoms; Dose-Response Relationship, Drug; Drug Interactions; Ganglia, Spinal; Hydrogen-Ion Concentration; Lactic Acid; Mice; Mice, Inbred C57BL; Muscle, Skeletal; Nerve Tissue Proteins; Neurons; Protons; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Receptors, Purinergic P2; Receptors, Purinergic P2X; Sodium Channels; TRPV Cation Channels

Inhalation of H2O2 is known to evoke bradypnea followed by tachypnea, which are reflexes resulting from stimulation by reactive oxygen species of vagal lung capsaicin-sensitive and myelinated afferents, respectively. This study investigated the pharmacological receptors and chemical mediators involved in triggering these responses. The ventilatory responses to 0.2% aerosolized H2O2 were studied before and after various pharmacological pretreatments in anesthetized rats. The initial bradypneic response was reduced by a transient receptor potential vanilloid 1 (TRPV1) receptor antagonist [capsazepine; change (Delta) = -53%] or a P2X purinoceptor antagonist [iso-pyridoxalphosphate-6-azophenyl-2',5'-disulphonate (PPADS); Delta = -47%] and was further reduced by capsazepine and iso-PPADS in combination (Delta = -78%). The initial bradypneic response was reduced by a cyclooxygenase inhibitor (indomethacin; Delta = -48%), ATP scavengers (apyrase and adenosine deaminase in combination; Delta = -50%), or capsazepine and indomethacin in combination (Delta = -47%), was further reduced by iso-PPADS and indomethacin in combination (Delta = -75%) or capsazepine and ATP scavengers in combination (Delta = -83%), but was not affected by a lipoxygenase inhibitor (nordihydroguaiaretic acid) or by any of the various vehicles. No pretreatment influenced delayed tachypnea. We concluded that 1) the initial bradypneic response to H2O2 results from activation of both TRPV1 and P2X receptors, possibly located at terminals of vagal lung capsaicin-sensitive afferent fibers; 2) the functioning of the TRPV1 and P2X receptors in triggering the initial bradypnea is, in part, mediated through the actions of cyclooxygenase metabolites and ATP, respectively; and 3) these mechanisms do not contribute to the H2O2-evoked delayed tachypnea.

Topics: Adenosine Deaminase; Adenosine Triphosphate; Animals; Apyrase; Arachidonic Acid; Capsaicin; Cyclooxygenase Inhibitors; Hydrogen Peroxide; Hyperventilation; Hypoventilation; Indomethacin; Lipoxygenase Inhibitors; Male; Oxidants; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Receptors, Purinergic P2; Receptors, Purinergic P2X; Reflex; Respiratory Mechanics; TRPV Cation Channels

Vanilloid (VR1) and purinergic (P2X) receptors are found in cranial afferent neurons in nodose ganglia and their central terminations within the solitary tract nucleus (NTS), but little is known about their function. We mechanically dissociated dorsomedial NTS neurons to preserve attached native synapses and tested for VR1 and P2X function primarily in spindle-shaped neurons resembling intact second-order neurons. All neurons (n = 95) exhibited spontaneous glutamate (EPSCs) and GABA (IPSCs)-mediated synaptic currents. VR1 agonist capsaicin (CAP; 100 nm) reversibly increased EPSC frequency, effects blocked by capsazepine. ATP (100 microm) increased EPSC frequency, actions blocked by P2X antagonist pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid (PPADS; 20 microm). In all CAP-resistant neurons, P2X agonist alphabeta-methylene-ATP (alphabeta-m-ATP) increased EPSC frequency. Neither CAP nor alphabeta-m-ATP altered EPSC amplitudes, kinetics, or holding currents. Thus, activation of VR1 and P2X receptors selectively facilitated presynaptic glutamate release on different NTS neurons. PPADS and 2',3'-O-(2,4,6-trinitrophenyl)-ATP blocked alphabeta-m-ATP responses, but P2X1-selective antagonist NF023 (8,8'-[carbonylbis (imino-3,1-phenylene carbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid) did not. The pharmacological profile and transient kinetics of ATP responses are consistent with P2X3 homomeric receptors. TTX and Cd(2+) did not eliminate agonist-evoked EPSC frequency increases, suggesting that voltage-gated sodium and calcium channels are not required. In nodose ganglia, CAP but not alphabeta-m-ATP evoked inward currents in slow conducting neurons and the converse pattern in myelinated, rapidly conducting neurons (n = 14). Together, results are consistent with segregation of glutamatergic terminals into either P2X sensitive or VR1 sensitive that correspondingly identify myelinated and unmyelinated afferent pathways at the NTS.

Topics: Adenosine Triphosphate; Afferent Pathways; Animals; Capsaicin; Cell Separation; Excitatory Postsynaptic Potentials; Glutamic Acid; In Vitro Techniques; Male; Neural Inhibition; Neurons; Nodose Ganglion; Patch-Clamp Techniques; Presynaptic Terminals; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Receptors, Drug; Receptors, Purinergic P2; Solitary Nucleus