guanosine-diphosphate and Pain

Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the μ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-β-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.

Topics: Animals; Baclofen; Calcium Channels, L-Type; Calcium Channels, N-Type; Calcium Channels, T-Type; Dithiothreitol; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; GABA-B Receptor Agonists; gamma-Aminobutyric Acid; Ganglia, Spinal; Guanosine Diphosphate; HEK293 Cells; Humans; Nociception; Pain; Patch-Clamp Techniques; Pertussis Toxin; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Receptors, GABA-A; Receptors, GABA-B; Sensory Receptor Cells; Signal Transduction; Thionucleotides

The interstitial cells of Cajal (ICCs) are the pacemaker cells in the gastrointestinal (GI) tract. In the present study, the effects of Dangkwisoo‑san (DS) on pacemaker potentials in cultured ICCs from the small intestine of the mouse were investigated. The whole‑cell patch‑clamp configuration was used to record pacemaker potentials from cultured ICCs and the increase in intracellular Ca2+ concentration ([Ca2+i) was analyzed in cultured ICCs using fura‑2‑acetoxymethyl ester. The generation of pacemaker potentials in the ICCs was observed. DS produced pacemaker depolarizations in a concentration dependent manner in current clamp mode. The 4‑diphenylacetoxy‑N‑methyl‑piperidine methiodide muscarinic M3 receptor antagonist inhibited DS‑induced pacemaker depolarizations, whereas methoctramine, a muscarinic M2 receptor antagonist, did not. When guanosine 5'‑[β‑thio] diphosphate (GDP‑β‑S; 1 mM) was in the pipette solution, DS marginally induced pacemaker depolarizations, whereas low Na+ solution externally eliminated the generation of pacemaker potentials and inhibited the DS‑induced pacemaker depolarizations. Additionally, the nonselective cation channel blocker, flufenamic acid, inhibited the DS‑induced pacemaker depolarizations. Pretreatment with Ca2+‑free solution and thapsigargin, a Ca2+‑ATPase inhibitor in the endoplasmic reticulum, also eliminated the generation of pacemaker currents and suppressed the DS‑induced pacemaker depolarizations. In addition, [Ca2+]i analysis revealed that DS increased [Ca2+]i. These results suggested that DS modulates pacemaker potentials through muscarinic M3 receptor activation in ICCs by G protein‑dependent external and internal Ca2+ regulation and external Na+. Therefore, DS were observed to affect intestinal motility through ICCs.

Topics: Animals; Calcium-Transporting ATPases; Cells, Cultured; Diamines; Female; Gastrointestinal Motility; Guanosine Diphosphate; Interstitial Cells of Cajal; Intestine, Small; Male; Mice; Mice, Inbred BALB C; Pain; Phytotherapy; Piperidines; Plants, Medicinal; Receptor, Muscarinic M2; Receptor, Muscarinic M3; Thapsigargin; Thionucleotides

To elucidate the mechanisms of antinociception mediated by the dopaminergic descending pathway in the spinal cord, we investigated the actions of dopamine (DA) on substantia gelatinosa (SG) neurons by in vivo whole-cell patch-clamp methods. In the voltage-clamp mode (V(H)=-70mV), the application of DA induced outward currents in about 70% of SG neurons tested. DA-induced outward current was observed in the presence of either Na(+) channel blocker, tetrodotoxin (TTX) or a non-NMDA receptor antagonist, CNQX, and was inhibited by either GDP-β-S in the pipette solution or by perfusion of a non-selective K(+) channel blocker, Ba(2+). The DA-induced outward currents were mimicked by a selective D2-like receptor agonist, quinpirole and attenuated by a selective D2-like receptor antagonist, sulpiride, indicating that the DA-induced outward current is mediated by G-protein-activated K(+) channels through D2-like receptors. DA significantly suppressed the frequency and amplitude of glutamatergic spontaneous excitatory postsynaptic currents (EPSCs). DA also significantly decreased the frequency of miniature EPSCs in the presence of TTX. These results suggest that DA has both presynaptic and postsynaptic inhibitory actions on synaptic transmission in SG neurons. We showed that DA produced direct inhibitory effects in SG neurons to both noxious and innocuous stimuli to the skin. Furthermore, electrical stimulation of dopaminergic diencephalic spinal neurons (A11), which project to the spinal cord, induced outward current and suppressed the frequency and amplitude of EPSCs. We conclude that the dopaminergic descending pathway has an antinociceptive effect via D2-like receptors on SG neurons in the spinal cord.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Afferent Pathways; Animals; Barium Compounds; Chlorides; Dopamine; Dopamine Agents; Drug Interactions; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Guanosine Diphosphate; Male; Nociceptors; Pain; Patch-Clamp Techniques; Physical Stimulation; Quinpirole; Rats; Rats, Sprague-Dawley; Skin; Sodium Channel Blockers; Spinal Cord; Substantia Gelatinosa; Tetrodotoxin; Thionucleotides

Dopaminergic innervation of the spinal cord is largely derived from the brain. To understand the cellular mechanisms of antinociception mediated by descending dopaminergic pathways, we examined the actions of dopamine (DA) on nociceptive transmission by using behavioural studies and whole-cell patch-clamp recordings from substantia gelatinosa (SG) neurones in the spinal cord. Intrathecal administration of DA increased the mechanical nociceptive threshold and this effect was mimicked by a D2-like receptor agonist, quinpirole, but not by a D1-like receptor agonist, SKF 38393. In current-clamp mode of patch-clamp recordings, bath application of DA hyperpolarized the membrane potential of SG neurones and suppressed action potentials evoked by electrical stimulation of a dorsal root. In voltage-clamp mode, DA induced an outward current that was resistant to TTX, was blocked by the addition of Cs+ or GDP-beta-S in the pipette solution, and was inhibited in the presence of Ba+. The DA-induced current reversed its polarity at a potential close to the equilibrium potential of the K+ channel calculated from the Nernst equation. The DA-induced outward current was mimicked by quinpirole, but not by SKF 38393. The DA-induced outward current was suppressed by a D2-like receptor antagonist, sulpiride, but not by a D1-like receptor antagonist, SCH 23390. In contrast, DA did not cause any significant change in amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs). These results indicate that DA mainly acts on postsynaptic SG neurones to induce an outward current via G-protein-mediated activation of K+ channels through D2-like receptors. This may be a possible mechanism for antinociception by the descending dopaminergic pathway.

Topics: Action Potentials; Analgesics; Animals; Barium; Cesium; Dopamine; Dopamine Agonists; Dopamine Antagonists; Dose-Response Relationship, Drug; Guanosine Diphosphate; Male; Motor Activity; Neural Inhibition; Neurons; Nociceptors; Pain; Potassium Channels; Quinpirole; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Signal Transduction; Spinal Cord; Stress, Mechanical; Substantia Gelatinosa; Sulpiride; Thionucleotides; Time Factors

Patch-clamp recordings from small-diameter rat dorsal root ganglion (DRG) neurons maintained in culture demonstrated preferential inhibition by ATP of high-voltage-activated, but not low-voltage-activated, Ca2+ currents (I(Ca)). The rank order of agonist potency was UTP > ADP > ATP. ATP depressed the omega-conotoxin GVIA-sensitive N-type current only. Pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and 2'-deoxy-N6-methyladenosine 3',5'-bisphosphate tetraammonium, two P2Y1 receptor antagonists, almost abolished the ATP-induced inhibition. Both patch-clamp recordings and immunocytochemistry coupled with confocal laser microscopy indicated a colocalization of functional P2X3 and P2Y1 receptors on the same DRG neurons. Because the effect of ATP was inhibited by intracellular guanosine 5'-O-(2-thiodiphosphate) or by applying a strongly depolarizing prepulse, P2Y1 receptors appear to block I(Ca) by a pathway involving the betagamma subunit of a G(q/11) protein. Less efficient buffering of the intracellular Ca2+ concentration ([Ca2+]i) by reducing the intrapipette EGTA failed to interfere with the ATP effect. Fura-2 microfluorimetry suggested that ATP raised [Ca2+]i by a Galpha-mediated release from intracellular pools and simultaneously depressed the high external potassium concentration-induced increase of [Ca2+]i by inhibiting I(Ca) via Gbetagamma. Adenosine 5'-O-(2-thiodiphosphate) inhibited dorsal root-evoked polysynaptic population EPSPs in the hemisected rat spinal cord and prolonged the nociceptive threshold on intrathecal application in the tail-flick assay. These effects were not antagonized by PPADS. Hence, P2Y receptor activation by ADP, which is generated by enzymatic degradation of ATP, may decrease the release of glutamate from DRG terminals in the spinal cord and thereby partly counterbalance the algogenic effect of ATP.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Analgesia; Analgesics; Animals; Calcium; Calcium Channels, N-Type; Cells, Cultured; Excitatory Postsynaptic Potentials; Ganglia, Spinal; GTP-Binding Protein alpha Subunits, Gq-G11; Guanosine Diphosphate; Injections, Spinal; Neurons; Pain; Patch-Clamp Techniques; Potassium; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Purinergic P2; Receptors, Purinergic P2X3; Receptors, Purinergic P2Y1; Thionucleotides

Spinal nociceptive transmission is mediated by glutamate and neuropeptides such as substance P (SP) and neurokinin A (NKA). The neuropeptide-mediated excitatory postsynaptic potentials (EPSPs) had a slow onset and long duration. Here, we demonstrate SP- and NKA-mediated excitatory postsynaptic currents (EPSCs) in dorsal horn neurons of young rats using whole-cell patch-clamp recording techniques. After complete blockade of glutamate receptor-mediated currents, we observed a small residual EPSC. The residual EPSCs exhibited temporal summation in response to a train of stimulation (six pulses delivered at 10-50 Hz). High intensity stimulation (the same or greater than the stimulation threshold for nociceptive fibers in vivo) was required for evoking these summated EPSCs. Summated EPSCs were attenuated or abolished by capsaicin pretreatment, which depletes SP and NKA from presynaptic terminals; SP and NKA pretreatment; NK(1) or NK(2) receptor antagonists; and inhibition of postsynaptic G proteins. EPSCs were neither blocked by a metabotropic glutamate receptor antagonist nor a gamma-aminobutyric acid(B) receptor antagonist. The summated EPSCs were also sensitive to voltage-gated calcium channel antagonists or mu-opioid receptor activation by DAMGO. The present study provides electrophysiological evidence that suggests the possible contribution of SP and NKA to sensory synaptic transmission between primary afferent fibers and dorsal horn neurons.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Analgesics, Opioid; Animals; Calcium Channel Blockers; Calcium Channels, P-Type; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; GABA Antagonists; Guanosine Diphosphate; In Vitro Techniques; Neurokinin A; omega-Conotoxin GVIA; Pain; Patch-Clamp Techniques; Phosphinic Acids; Posterior Horn Cells; Propanolamines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P; Synaptic Transmission; Thionucleotides

Recent evidence strongly suggests that the hyperalgesia induced by agents acting directly on the primary afferent is mediated by stimulatory G-proteins and the cAMP second messenger system. In this study, we used the Randall-Selitto paw-pressure device to study hyperalgesia that develops in the streptozotocin-diabetic rat. Subcutaneous injection of streptozotocin in male Sprague-Dawley rats induced hyperglycemia and glucosuria detectable within 24 h of injection. A decrease in mechanical nociceptive threshold in the hindpaw was detected after one week. Intradermal injection of indomethacin, a cyclooxygenase inhibitor, had no significant effect on nociceptive threshold; and prostaglandin E2, which produces hyperalgesia by a direct action on the primary afferent, decreased nociceptive threshold similarly in streptozotocin-diabetic and control rats. Guanosine 5'-O-(2-thiodiphosphate), which blocks stimulatory G-proteins, attenuated the prostaglandin E2-hyperalgesia in both streptozotocin-diabetic and control rats, but had no effect on baseline nociceptive threshold in either group. Intradermal injection of either 2',5'-dideoxyadenosine, an inhibitor of adenylate cyclase, or phosphodiesterase, which degrades cAMP, increased mechanical nociceptive threshold in streptozotocin-diabetic rats whilst not affecting mechanical nociceptive threshold in the control rats. Intradermal injection of 8-bromo cAMP, a membrane-permeable analog of cAMP, produced hyperalgesia of significantly greater magnitude in the streptozotocin-diabetic rats than the control rats. Intradermal injection of N6-cyclopentyl adenosine, an A1-type adenosine agonist, which can activate an inhibitory G-protein and decrease cAMP production, also increased nociceptive thresholds in streptozotocin-diabetic rats. This effect was blocked by pertussis toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenosine; Adenylate Cyclase Toxin; Animals; Diabetes Mellitus, Experimental; Dideoxyadenosine; Dinoprostone; GTP-Binding Proteins; Guanosine Diphosphate; Hyperalgesia; Indomethacin; Male; Mechanoreceptors; Pain; Pertussis Toxin; Rats; Rats, Sprague-Dawley; Sensory Thresholds; Thionucleotides; Virulence Factors, Bordetella