thapsigargin and Pain

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

The sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) is a critical pathway by which sensory neurons sequester cytosolic Ca(2+) and thereby maintain intracellular Ca(2+) homeostasis. We have previously demonstrated decreased intraluminal endoplasmic reticulum Ca(2+) concentration in traumatized sensory neurons. Here we examine SERCA function in dissociated sensory neurons using Fura-2 fluorometry. Blocking SERCA with thapsigargin (1 μM) increased resting [Ca(2+)](c) and prolonged recovery (τ) from transients induced by neuronal activation (elevated bath K(+)), demonstrating SERCA contributes to control of resting [Ca(2+)](c) and recovery from transient [Ca(2+)](c) elevation. To evaluate SERCA in isolation, plasma membrane Ca(2+) ATPase was blocked with pH 8.8 bath solution and mitochondrial buffering was avoided by keeping transients small (≤ 400 nM). Neurons axotomized by spinal nerve ligation (SNL) showed a slowed rate of transient recovery compared to control neurons, representing diminished SERCA function, whereas neighboring non-axotomized neurons from SNL animals were unaffected. Injury did not affect SERCA function in large neurons. Repeated depolarization prolonged transient recovery, showing that neuronal activation inhibits SERCA function. These findings suggest that injury-induced loss of SERCA function in small sensory neurons may contribute to the generation of pain following peripheral nerve injury.

Topics: Animals; Axotomy; Calcium; Calcium Signaling; Endoplasmic Reticulum; Enzyme Inhibitors; Male; Pain; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sensory Receptor Cells; Spinal Nerves; Thapsigargin

The role of intracellular calcium in acute thermal nociception was investigated in the mouse hot-plate test. Intracerebroventricular (i.c.v.) administration of TMB-8, a blocker of Ca++ release from intracellular stores, produced hypernociception. By contrast, i.c.v. pretreatment with thapsigargin, a depletor of Ca++ intracellular stores, produced an increase of the mouse pain threshold. Furthermore, non-analgesic doses of thapsigargin prevented the hypernociception produced by TMB-8. In mice undergoing treatment with heparin, an InsP3-receptor antagonist, or ryanodine, a ryanodine receptor (RyR) antagonist, a dose-dependent reduction of the pain threshold was observed. Pretreatment with D-myo inositol, compound which produces InsP3, and 4-chloro-m-cresol, a RyR agonist, induced an antinociceptive effect. The heparin hypernociception was prevented by D-myo inositol, but not by L-myo inositol, used as negative control. In the same experimental conditions, the antinociception induced by D-myo inositol was prevented by a non-hyperalgesic dose of heparin. Similarly, the reduction of pain threshold produced by ryanodine was reversed by non-analgesic doses of 4-chloro-m-cresol, whereas the antinocicpetion induced by 4-chloro-m-cresol was prevented by non-hyperalgesic doses of ryanodine. The pharmacological treatments employed did not produce any behavioral impairment of mice as revealed by the rota-rod and hole-board tests. These results indicate that a variation of intracellular calcium contents at a supraspinal level is involved in the modulation of acute thermal nociception. In particular, the stimulation of both InsP3- and Ry-receptors appears to play an important role in the induction of antinociception in mice, whereas a blockade of these receptors is involved in an hypernociceptive response to acute thermal pain.

Topics: Acute Disease; Animals; Behavior, Animal; Calcium; Calcium Channel Blockers; Calcium-Transporting ATPases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Gallic Acid; Hot Temperature; Hyperalgesia; Injections, Intraventricular; Inositol 1,4,5-Trisphosphate; Male; Mice; Pain; Pain Threshold; Postural Balance; Reaction Time; Ryanodine Receptor Calcium Release Channel; Thapsigargin

The effects produced by the intrathecal administration of dantrolene and thapsigargin, measured in several analgesic tests in the rat are described. Dantrolene decreases the release of calcium from intracellular stores and thapsigargin is able to inhibit the reticular Ca2+-ATPase, avoiding intracellular calcium storage. Dantrolene (30-300 nmol/rat) and thapsigargin (3-30 nmol/rat) reduced the nociceptive behavior (biting, scratching, licking; BSL) produced by the NK(1) receptor agonist septide (0.5 microg), without affecting the BSL induced by AMPA (2 microg) or NMDA (4 microg). Also, both drugs elicited analgesia in the tail-flick test but not in the formalin test. The antinociceptive effects induced by thapsigargin were more intense and long-lasting than those produced by dantrolene. These results seem to indicate that the intracellular modulation of calcium homeostasis could be an interesting target in order to induce spinal analgesia.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Analgesics; Animals; Behavior, Animal; Calcium; Calcium-Transporting ATPases; Dantrolene; Enzyme Inhibitors; Male; Muscle Relaxants, Central; N-Methylaspartate; Pain; Pain Measurement; Peptide Fragments; Pyrrolidonecarboxylic Acid; Rats; Rats, Wistar; Spinal Cord; Substance P; Thapsigargin

Calcium (Ca++) administered into the i.c.v. space of mice has been reported to block opioid-induced antinociception dose dependently. These studies were conducted to test the hypothesis that Ca++ i.c.v. blocks the antinociceptive effects of morphine i.c.v. as a consequence of transmembrane Ca++ influx and Ca++ release from intracellular pools. Mice were injected with voltage-sensitive Ca++ channel antagonists at a dose that did not affect morphine antinociception to determine whether this pretreatment would prevent the inhibitory effects of Ca++. Nimodipine (12 nmol i.c.v.) was ineffective in preventing the inhibitory effects of Ca++ (100 nmol i.c.v.), whereas omega-conotoxin GVIA (3.3 pmol i.c.v.) completely prevented the inhibition by Ca++ of morphine antinociception. Other experiments were conducted to determine whether blocking Ca++ release from Ca++/caffeine-sensitive microsomal pools with ryanodine would prevent the inhibitory effects of Ca++. Ryanodine (2 nmol i.c.v.) significantly attenuated the inhibition by Ca++ of morphine antinociception. Another hypothesis to be tested was that stimulation of Ca++ release from intracellular pools would, like Ca++, block morphine antinociception. Thapsigargin (0.002-30 nmol i.c.v.), which increases cytosolic Ca++ by depleting Ca++ from inositol 1,4,5-trisphosphate-sensitive microsomal pools, dose-dependently blocked the antinociceptive effects of morphine. The results of this study indicate that Ca++ blocked morphine antinociception by stimulating Ca++ influx through omega-conotoxin GVIA-sensitive channels and by stimulating Ca++ release from Ca++/caffeine-sensitive microsomal pools.

Topics: Animals; Caffeine; Calcium; Calcium Channels; Cell Compartmentation; Male; Membrane Potentials; Mice; Mice, Inbred Strains; Microsomes; Morphine; Nimodipine; omega-Conotoxin GVIA; Pain; Peptides; Ryanodine; Terpenes; Thapsigargin