The Role Of ASICs On The Anti-nociceptive Effects Of Propofol And Chloroquine-induced Itch

Part I The role of ASICs on the anti-nociceptive effects of propofol BackgroundPropofol is widely used intravenous anesthetics for the induction and maintenance of general anesthesia. In addition to its anesthetic properties, several studies have shown that propofol has anti-nociceptive effects, including against visceral pain evoked by acetic acid, as well as the capacity to reduce inflammatory pain and depress nociceptive transmission at the spinal level. However, the molecular mechanisms responsible for these anti-nociceptive effects of propofol remain unclear.Acid-sensing ion channels (ASICs) belong to the epithelial sodium channel/degenerin (ENaC/DEG) family and are activated by extracellular protons. They are widely distributed within both the central and peripheral nervous systems. ASIC activation by protons induces sodium and/or calcium influx, giving rise to depolarization and evoking action potentials in neurons. Dorsal root ganglion (DRG) cells play an important role in the perception of nociception and highly expressed ASIC la and ASIC3. However, the regulation of propofol on ASIC is still unclear.ObjectiveThe present researched was designed to investigate the effect of propofol on the ASICs in DRG neurons and HEK293cells transfected with either ASIC1a or ASIC3.Methods1、Investigated the effect of propofol on ASICs in DRG neuron using whole-cell patch clamping.DRG neurons were isolated and placed into Petri dishes. DRG neurons in the range of15～30μm diameter were chosen to test the effect of propofol on acid-evoked currents using whole-cell patch clamping. 2、Investigated the effect of propofol on HEK193cells transfected with either ASIC la or ASICCells were transfected with pcDNA3.0constructs encoding ASIC la or ASIC3and green fluorescent protein (GFP) using LipofectamineTM2000. All recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch clamping.Result1、Effects of propofol on acid-evoked currents in rat DRG neurons An acid-evoked inward current was observed in the majority of DRG neurons. Three main current types were observed:1) a rapidly inactivating current with a fast transient phase;2) a slow-inactivating transient current followed by a small sustained component;3) a non-inactivating current with a small transient phase.Amiloride, a broad-spectrum proton-gated channel blocker, completely blocked all three types of transient currents and treatment with propofol (30μM) produced a significant reduction in the peak amplitude of acid-evoked currents (pH6.0) in DRG neurons. Under the current-clamp conditions, propofol decreased the number of action potentials induced by acid stimuli in rat DRG neurons.2、Dose-response relationship of propofol on acid-evoked currents in rat DRG neuronsThe peak amplitudes of the currents were reduced in response to to3,30, and300μM of propofol, indicating that the propofol-induced inhibition was dose-dependent.3、The pH-dependent activation of ASIC currents in response to propofol in rat DRG neuronsWe next investigated whether propofol affects the sensitivity of ASICs to H+. The result showed no apparent shift in the H+dose-response curve.4、The effects of propofol on acid-evoked currents in ASIC la and ASIC3-transfected HEK293cellsPropofol significantly inhibited both ASIC1a and ASIC3currents transfected in HEK293cells, indicating that propofol directly inhibits ASIC currents.Conclusion The present results suggested that propofol inhibits proton-gated currents in DRG neurons and this effect could partly explain the anti-nociceptive effects of propofol in primary afferent neurons.Part Ⅱ The role of ASIC3on the chloroquine-evoked itchBackgroudDRG has an important role in the perception of nociception and widely expressed ASIC1a and ASIC3. Different from ASIC la, ASIC3could be activated at slightly acidic environment and has the steady state current which produces sustained inward currents. Therefore, it has a more important role in the perception of nociception. In addition to pain, itch also is an unpleasant sensation that evokes a desire to scratch, which accompanies numerous skin and nervous system disorders. The mechanisms of itch mainly include histamine-dependent and independent pathways. The histamine-independent pathway is more important, but the mechanism is unclear. Chloroquine (CQ) has been used in animal studies and in humans as a tool to study the itch mechanisms. Recent studies have found that Mas-related G protein-coupled receptor A3(MrgprA3) and Trpvl channel involved in CQ-induced itch mechanisms. Previous studies have demonstrated that ASIC3and Trpvl are concerned with the pain caused by nociceptive mechanisms and all can be activated H+. However, whether ASIC3is also involved in CQ-evoked itch is still unclear.ObjectiveThe present researched was designed to investigate the regulation of CQ on ASIC3currents and the relationship of ASIC3channels with CQ-evoked itch.Method1、Investigated the effect of CQ on Chinese Hamster Ovary (CHO) cells transfected with ASIC3.Cells were transfected with pcDNA3.0constructs encoding ASIC3and green fluorescent protein (GFP) using LipofectamineTM2000. All recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch clamping.2、Investigated the possible binding sites of CQ on ASIC3by quick point mutation PCR technique.GLU-79or GLU-423site of ASIC3was mutated using quick point mutation PCR technique. CHO Cells were transfected with mutated ASIC3using LipofectamineTM2000. Recordings were made24to48h after transfection in GFP-positive cells using whole-cell patch3、Investigated the effect of CQ on ASIC3-like currents in DRG neuron using whole-cell patch clamping.DRG neurons were isolated and placed into Petri dishes. DRG neurons in the range of15～30μm diameter were chosen to test the effect of CQ on ASIC3-like currents in DRG neuron using whole-cell patch clamping.4, Investigated the relationship of ASIC3with CQ-evoked itch.The corresponding drugs were injected in the cheek of mice and counted the number of counted scratching with the hind limb as an indicator of itch and wiping with the forelimb as an indicator of pain.Result1、CQ selectively potentiates the sustained currents of proton gated ion channels in ASIC3-transfected CHO cells.CQ enhanced the sustained current and extended the time of desensitization of ASIC3, which was evoked in CHO cells transfected with ASIC3, however, the peak currents were not. Next, we tested the dose dependence of CQ-mediated enhancement of this sustained current. CQ selectively enhances the sustained current in a dose-dependent manner.2、CQ reset pH sensitivity of sustained ASIC3currentThe pH is dropped from7.4to6.8in steps of0.1or0.2U, with each step held for10seconds. In CHO cells transfected with ASIC3DNA, the activation curve of sustained ASIC3currents was traditional bell-shaped, which peaks at pH7.0. We tested CQ on the sustained current and found that it increases sustained ASIC3current and shifts the bell-shaped activation curve to right. In other words, CQ resets the pH sensitivity of sustained ASIC3current.3、CQ-induced potentiation of the sustained current was pH dependent and Ca2+ independent.We found that CQ dramatically enhanced the sustained component without altering the peak component. CQ-induced potentiation of the sustained current was pH dependent. We also investigated the sensitivity of CQ-induced potentiation of the sustained currents to alterations of extracellular Ca2+. The presence of either low extracellular Ca2+(0mM) or high extracellular Ca2+(3raM) has no effect on the CQ-induced potentiation of the ASIC3sustained currents, suggesting that CQ-induced enhancement of the sustained component is independent of extracellular Ca2+.4, ASIC subunit specificityTo address ASIC subunit specificity, we recorded CQ responses in CHO cells expressing different ASIC subunits. Not like the ASIC3, the homomeric channels ASIC1a was not activated by CQ.5、Mutation at Glu-79or Glu-423Prevents ASIC3Channel Activation by CQRecently, a novel nonproton ligand-sensing domain of ASIC3channels was uncovered, lined by residues around Glu-423and Glu-79of the extracellular "palm" domain of the ASIC3channel. To ascertain that CQ activates ASIC3via interactions with GMQ-sensing domain, we substituted Glu-79and Glu-423with alanine. Mutation at either Glu-79or Glu-423largerly abolished ASIC3channel activation by CQ, which support the view that CQ directly interacts with the non proton ligand.6、CQ enhances the sustain phase of acid-activated curents in DRG neuronsCQ also selectively enhances the sustained phase of acid-evoked current.7、 CQ and other compounds such as Ali, GMQ and NPFF, which are known to induce potentiation of the sustain phase of ASIC3current, induce itch in BALB/c mice.We tested BALB/c mice using the "cheek" assay, which reportedly distinguishes itch and pain responses. CQ injection produces scratching, which was largely alleviated by APETx2, a selective ASIC3channel blocker. To further confirm this result, we test the itch-associated responses of other chemicals in mice, such as Ali and GMQ, which have been reported before as the nonproton ligands to activate ASIC3. Like CQ, those compounds also evoked the scratching response. Another endogenous neuropeptide FF (NPFF), which has been proved that it enhances the sustained current of ASIC3.Unsurprisingly, it also evoked the scratching response in rat.Conclusion1、CQ selectively enhances the sustained current in a dose-dependent manner and the potentiating effect of CQ on the sustained current was pH dependent and Ca2+independent.2、Mutation at Glu-79or Glu-423prevents ASIC3channel activation by CQ.3、CQ and other compounds such as Ali, GMQ and NPFF, which are known to induce potentiating of the sustain phase of ASIC3current, induce itch in BALB/c mice and CQ-induced itch could be blocked by APET×2. These results indicated that ASIC3, a proton-gated ion channel, has the functions as an essential component of itch transduction.Part Ⅲ The effects of chloriquane on the secretion of rat ileumBackgroudApart from itch, CQ (a bitter tasting drug) is associated gastrointestinal side effects including nausea or diarrhea and the bitter taste receptors (TAS2Rs) are widely expressed in the intestinal tract. The bitter compound could evoke anion secretion in the large intestines of humans and rats. However, whether the side effect of CQ on electrolyte transport in rat ileum has the relation with TAS2Rs is still unclear.ObjectiveThe present researched was designed to investigate the effect of CQ on electrolyte transport in rat ileum and the relationship with TAS2Rs.Method1、Short-circuit current (ISC) was measured in vitro in Ussing chambers.The tissue preparations were mounted between the2halves of the Ussing chambers and investigate the effect of CQ on electrolyte transport in rat ileum.2、Investigating of CaCC-TMEM16A and bitter receptor T2R in small intestinal epithelial cells using immunohistochemistry.Results 1、CQ evoked an increase ISC in rat ileumCQ dose-dependently increased basal ISC at low concentraons (≤5×101-4M). However, it markedly decreased basal ISC at high concentrations (≥10-3M).2、The mechanisms of CQ evoked an increase in ISC in rat ileumCQ-induced increases in ISC were also abolished by CaCCs inhibitors.ConclusionChloroquine could activate TAS2Rs and induces Cl-secretion in rat ileum through CaCCs, suggesting a novel explanation for CQ-associated gastrointestinal side-effects during the treatment of malaria.