Local Anesthetics Inhibit Transient Receptor Potential Vanilloid Subtype 3 Channel Function in Xenopus Oocytes.

BACKGROUND: The transient receptor potential vanilloid subtype 3 (TRPV3) channel is activated by innocuous temperature and several chemical stimuli. It is proposed to be involved in pathological pain development and is therefore considered a potential target for treating pain. Local anesthetics have been used for patients with both acute and chronic pain. Although blockage of the voltage-gated sodium channel is the primary mechanism by which local anesthetics exert their effects, they cannot be explained by this mechanism alone, especially in pathologic states such as chronic pain. Indeed, the effects of local anesthetics on multiple targets involved in the pain pathway have been reported. It has also been suggested that modulating the function of transient receptor potential (TRP) channels (eg, TRPV1 and transient receptor potential ankyrin 1 [TRPA1]) is one of the mechanisms of action of local anesthetics. However, the effects of local anesthetics on TRPV3 have not been reported. METHODS: We expressed TRPV3 in Xenopus oocytes and investigated the effects of local anesthetics on 2-aminoethoxydiphenyl borate (2APB)-induced currents using 2-electrode voltage-clamp techniques. RESULTS: Clinically used local anesthetics inhibited the 2APB-activated currents from the TRPV3 channel in a concentration-dependent manner at pharmacologically relevant concentrations with half maximal inhibitory concentration (IC50) values of 2.5 (lidocaine), 1.4 (mepivacaine), 0.28 (ropivacaine), and 0.17 (bupivacaine) mmol/L, respectively. Conversely, these local anesthetics also directly induced currents at higher concentrations, although these currents were quite small compared to the 2APB-induced currents. We found that the inhibition of TRPV3 by lidocaine is noncompetitive and independent of intracellular signaling cascades. 2APB-induced TRPV3 currents were reduced by extracellular N-(2,6-dimethylphenylcarbamoylmethyl) triethylammonium bromide (QX-314) but not by intracellular QX-314 nor benzocaine. Moreover, lidocaine showed a use-dependent block in TRPV3 inhibition. Finally, QX-314 appeared to slightly permeate the activated TRPV3 channel pore based on examination of oocytes coexpressing TRPV3 and a sodium channel. These results suggest that local anesthetics could inhibit TRPV3 channel function by extracellular interactions of their charged forms with the channel pore. CONCLUSIONS: Local anesthetics inhibited TRPV3 2APB-induced currents at pharmacologically relevant concentrations when TRPV3 was expressed in Xenopus oocytes. These effects seem to occur via an extracellular interaction between the charged form of the anesthetic with the TRPV3 channel pore. These results help to elucidate the mechanisms of action of local anesthetics.

A novel method for evaluating activity of transient receptor potential channels using a cellular dielectric spectroscopy.

Cellular dielectric spectroscopy (CDS) is a novel technology enabling pharmacological evaluation of multiple receptor types with a label-free cell-based assay. We evaluated activities of a family of ligand-gated channels, transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential ankyrin 1 (TRPA1) channels by an electrical impedance-based biosensor (CellKey™ system) using CDS. Measures of both potency (EC50) and efficacy (Emax) of these agonists with CellKey™ were almost identical to those made using the traditional Ca2+ influx assay in TRPV1- or TRPA1-expressing cells, suggesting that CellKey™ is a simpler and easier means of evaluating TRP activities.

Carvacrol inhibits the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.3, Nav1.7, and Nav1.8 expressed in Xenopus oocytes with different potencies.

Carvacrol is the predominant monoterpene in essential oils from many aromatic plants. Several animal studies showing analgesic effects of carvacrol indicate potential of carvacrol as a new medication for patients with refractory pain. Voltage-gated sodium channels (Nav) are thought to have crucial roles in the development of inflammatory and neuropathic pain, but there is limited information about whether the analgesic mechanism of carvacrol involves Nav. We used whole-cell, two-electrode, voltage-clamp techniques to examine the effects of carvacrol on sodium currents in Xenopus oocytes expressing α subunits of Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8. Carvacrol dose-dependently suppressed sodium currents at a holding potential that induced half-maximal current. The half-maximal inhibitory concentration values for Nav1.2, Nav1.3, Nav1.6, Nav1.7, and Nav1.8 were 233, 526, 215, 367, and 824 µmol/L, respectively, indicating that carvacrol had more potent inhibitory effects towards Nav1.2 and Nav1.6 than Nav1.3, Nav1.7, and Nav1.8. Gating analysis showed a depolarizing shift of the activation curve and a hyperpolarizing shift of the inactivation curve in all five α subunits following carvacrol treatment. Furthermore, carvacrol exhibits a use-dependent block for all five α Nav subunits. These findings provide a better understanding of the mechanisms associated with the analgesic effect of carvacrol.

Modulation of synaptic inputs in magnocellular neurones in a rat model of cancer cachexia.

In cancer cachexia, abnormal metabolism and neuroendocrine dysfunction cause anorexia, tissue damage and atrophy, which can in turn alter body fluid balance. Arginine vasopressin, which regulates fluid homeostasis, is secreted by magnocellular neurosecretory cells (MNCs) of the hypothalamic supraoptic nucleus. Arginine vasopressin secretion by MNCs is regulated by both excitatory and inhibitory synaptic activity, alterations in plasma osmolarity and various peptides, including angiotensin II. In the present study, we used whole-cell patch-clamp recordings of brain slices to determine whether hyperosmotic stimulation and/or angiotensin II potentiate excitatory synaptic input in a rat model of cancer cachexia, similar to their effects in normal (control) rats. Hyperosmotic (15 and 60 mmol L-1   mannitol) stimulation and angiotensin II (0.1 µmol L-1 ) increased the frequency, but not the amplitude, of miniature excitatory postsynaptic currents in normal rats; in model rats, both effects were significantly attenuated. These results suggest that cancer cachexia alters supraoptic MNC sensitivity to osmotic and angiotensin II stimulation.

Analysis of actual pressure point using the power flexible capacitive sensor during chest compression.

In chest compression for cardiopulmonary resuscitation (CPR), the lower half of the sternum is pressed according to the American Heart Association (AHA) guidelines 2010. These have been no studies which identify the exact location of the applied by individual chest compressions. We developed a rubber power-flexible capacitive sensor that could measure the actual pressure point of chest compression in real time. Here, we examined the pressure point of chest compression by ambulance crews during CPR using a mannequin. We included 179 ambulance crews. Chest compression was performed for 2 min. The pressure position was monitored, and the quality of chest compression was analyzed by using a flexible pressure sensor (Shinnosukekun™). Of the ambulance crews, 58 (32.4 %) pressed the center and 121 (67.6 %) pressed outside the proper area of chest compression. Many of them pressed outside the center; 8, 7, 41, and 90 pressed on the caudal, left, right, and cranial side, respectively. Average compression rate, average recoil, average depth, and average duty cycle were 108.6 counts per minute, 0.089, 4.5 cm, and 48.27 %, respectively. Many of the ambulance crews did not press on the sternal lower half definitely. This new device has the potential to improve the quality of CPR during training or in clinical practice.

A flexible pressure sensor could correctly measure the depth of chest compression on a mattress.

BACKGROUND: Feedback devices are used to improve the quality of chest compression (CC). However, reports have noted that accelerometers substantially overestimate depth when cardiopulmonary resuscitation (CPR) is performed on a soft surface. Here, we determined whether a flexible pressure sensor could correctly evaluate the depth CC performed on a mannequin placed on a mattress. METHODS: Chest compression was performed 100 times/min by a compression machine on the floor or a mattress, and the depth of CC was monitored using a flexible pressure sensor (Shinnosukekun) and CPRmeter(™). The depth of machine-performed CC was consistently 5cm. We compared data from the feedback sensor with the true depth of CC using dual real-time auto feedback system that incorporated an infrared camera (CPR evolution(™)). RESULTS: On the floor, the true depth of CC was 5.0±0.0cm (n=100), or identical to the depth of CC performed by the machine. The Shinnosukekun(™) measured a mean (±SD) CC depth of 5.0±0.1cm (n=100), and the CPRmeter(™) measured a depth of 5.0±0.2cm (n=100). On the mattress, the true depth of CC was 4.4±0.0cm (n=100). The Shinnosukekun(™) measured a mean CC depth of 4.4±0.0cm (n=100), and the CPRmeter(™) measured a depth of 4.7±0.1cm (n=100). The data of CPRmeter(™) were overestimated (P<.0001 between the true depth and the CPRmeter(™)-measured depth). CONCLUSION: The Shinnosukekun(™) could correctly measure the depth of CC on a mattress. According to our present results, the flexible pressure sensor could be a useful feedback system for CC performed on a soft surface.

What is the main mechanism of tramadol?

Tramadol is an analgesic that is used worldwide for pain, but its mechanisms of action have not been fully elucidated. The majority of studies to date have focused on activation of the µ-opioid receptor (µOR) and inhibition of monoamine reuptake as mechanisms of tramadol. Although it has been speculated that tramadol acts primarily through activation of the µOR, no evidence has revealed whether tramadol directly activates the µOR. During the past decade, major advances have been made in our understanding of the physiology and pharmacology of ion channels and G protein-coupled receptor (GPCR) signaling. Several studies have shown that GPCRs and ion channels are targets for tramadol. In particular, tramadol has been shown to affect GPCRs. Here, the effects of tramadol on GPCRs, monoamine transporters, and ion channels are presented with a discussion of recent research on the mechanisms of tramadol.

Tramadol and its metabolite m1 selectively suppress transient receptor potential ankyrin 1 activity, but not transient receptor potential vanilloid 1 activity.

BACKGROUND: The transient receptor potential vanilloid 1 (TRPV1) and the transient receptor potential ankyrin 1 (TRPA1), which are expressed in sensory neurons, are polymodal nonselective cation channels that sense noxious stimuli. Recent reports showed that these channels play important roles in inflammatory, neuropathic, or cancer pain, suggesting that they may serve as attractive analgesic pharmacological targets. Tramadol is an effective analgesic that is widely used in clinical practice. Reportedly, tramadol and its metabolite (M1) bind to µ-opioid receptors and/or inhibit reuptake of monoamines in the central nervous system, resulting in the activation of the descending inhibitory system. However, the fundamental mechanisms of tramadol in pain control remain unclear. TRPV1 and TRPA1 may be targets of tramadol; however, they have not been studied extensively. METHODS: We examined whether and how tramadol and M1 act on human embryonic kidney 293 (HEK293) cells expressing human TRPV1 (hTRPV1) or hTRPA1 by using a Ca imaging assay and whole-cell patch-clamp recording. RESULTS: Tramadol and M1 (0.01-10 µM) alone did not increase in intracellular Ca concentration ([Ca]i) in HEK293 cells expressing hTRPV1 or hTRPA1 compared with capsaicin (a TRPV1 agonist) or the allyl isothiocyanate (AITC, a TRPA1 agonist), respectively. Furthermore, in HEK293 cells expressing hTRPV1, pretreatment with tramadol or M1 for 5 minutes did not change the increase in [Ca]i induced by capsaicin. Conversely, pretreatment with tramadol (0.1-10 µM) and M1 (1-10 µM) significantly suppressed the AITC-induced [Ca]i increases in HEK293 cells expressing hTRPA1. In addition, the patch-clamp study showed that pretreatment with tramadol and M1 (10 µM) decreased the inward currents induced by AITC. CONCLUSIONS: These data indicate that tramadol and M1 selectively inhibit the function of hTRPA1, but not that of hTRPV1, and that hTRPA1 may play a role in the analgesic effects of these compounds.

µ-Opioid receptor activation by tramadol and O-desmethyltramadol (M1).

Tramadol has been used as an analgesic for several decades. µ-Opioid receptors (µORs) are the major receptors that mediate the analgesic effects of opioids. Although µORs have been thought to be one of the sites of action of tramadol, there has been no report that directly proves whether tramadol is an agonist of µOR or not. In this study, we examined the effects of tramadol and its main active metabolite O-desmethyltramadol (M1), on the function of µORs using Xenopus oocytes expressing cloned human µORs. The effects of tramadol and M1 were evaluated using the Ca(2+)-activated Cl(-) current assay method for G(i/o)-protein-coupled receptors by using a µOR fused to G(qi5) (µOR-G(qi5)) in Xenopus oocytes. DAMGO [(D-Ala(2), N-MePhe(4), Gly(5)-ol)-enkephalin] evoked Cl(-) currents in oocytes expressing µOR-G(qi5) in a concentration-dependent manner. Tramadol and M1 also evoked Cl(-) currents in the oocytes expressing µOR-G(qi5); however, relatively higher concentrations (compared to DMAGO) were necessary to induce such currents. Tramadol and M1 had a direct effect on µORs expressed in Xenopus oocytes. Although the monoamine uptake system and several types of ligand-gated ion channels are thought to be one of the targets for tramadol, tramadol-induced antinociception may be mediated at least in part, by the direct activation of µORs.

Kisspeptin-10 potentiates miniature excitatory postsynaptic currents in the rat supraoptic nucleus.

Kisspeptin is the natural ligand of the G protein-coupled receptor -54 and plays a major role in gonadotropin-releasing hormone secretion in the hypothalamus. Kisspeptin-10 is an endogenous derivative of kisspeptin and has 10 -amino acids. Previous studies have demonstrated that central administration of kisspeptin-10 stimulates the secretion of arginine vasopressin (AVP) in male rats. We examined the effects of kisspeptin-10 on- excitatory synaptic inputs to magnocellular neurosecretory cells (MNCs) including AVP neurons in the supraoptic nucleus (SON) by obtaining in vitro whole-cell patch-clamp recordings from slice preparations of the rat brain. The application of kisspeptin-10 (100 nM-1 µM) significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The kisspeptin-10-induced potentiation of the mEPSCs was significantly attenuated by previous exposure to the kisspeptin receptor antagonist kisspeptin-234 (100 nM) and to the protein kinase C inhibitor bisindolylmaleimide I (20 nM). These results suggest that kisspeptin-10 participates in the regulation of synaptic inputs to the MNCs in the SON by interacting with the kisspeptin receptor.

The recent progress in research on effects of anesthetics and analgesics on G protein-coupled receptors.

The exact mechanisms of action behind anesthetics and analgesics are still unclear. Much attention was focused on ion channels in the central nervous system as targets for anesthetics and analgesics in the 1980s. During the 1990s, major advances were made in our understanding of the physiology and pharmacology of G protein coupled receptor (GPCR) signaling. Thus, several lines of studies have shown that G protein coupled receptors (GPCRs) are one of the targets for anesthetics and analgesics and especially, that some of them inhibit the functions of GPCRs, i.e,, muscarinic receptors and substance P receptors. However, these studies had been focused on only G(q) coupled receptors. There has been little work on G(s)- and G(i)-coupled receptors. In the last decade, a new assay system, using chimera G(i/o)-coupled receptor fused to Gq(i5), has been established and the effects of anesthetics and analgesics on the function of G(i)-coupled receptors is now more easily studied. This review highlights the recent progress of the studies regarding the effects of anesthetics and analgesics on GPCRs.

Sevoflurane inhibits the µ-opioid receptor function expressed in Xenopus oocytes.

Sevoflurane is widely used for anesthesia, and is commonly used together with opioids in clinical practice. However, the effects of sevoflurane on µ-opioid receptor (µOR) functions is still unclear. In this study, the effects of sevoflurane on µOR functions were analyzed by using Xenopus oocytes expressing a µOR fused to chimeric Gα protein G(qi5) (µOR-G(qi5)). Sevoflurane by itself did not elicit any currents in oocytes expressing µOR-G(qi5), whereas sevoflurane inhibited the [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO)-induced Cl(-) currents at clinically used concentrations. Sevoflurane did not affect the Cl(-) currents induced by AlF(4)(-), which directly led to activation of G proteins. The inhibitory effects of sevoflurane on the DAMGO-induced currents were not observed in oocytes pretreated with the protein kinase C (PKC) inhibitor GF109203X. These findings suggest that sevoflurane would inhibit µOR function. Further, the mechanism of inhibition by sevoflurane would be mediated by PKC.

Transcriptional up-regulation of cell surface Na V 1.7 sodium channels by insulin-like growth factor-1 via inhibition of glycogen synthase kinase-3ß in adrenal chromaffin cells: enhancement of 22Na+ influx, 45Ca2+ influx and catecholamine secretion.

Insulin-like growth factor-1 (IGF-1) plays important roles in the regulation of neuronal development. The electrical activity of Na(+) channels is crucial for the regulation of synaptic formation and maintenance/repair of neuronal circuits. Here, we examined the effects of chronic IGF-1 treatment on cell surface expression and function of Na(+) channels. In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, chronic IGF-1 treatment increased cell surface [(3)H]saxitoxin binding by 31%, without altering the Kd value. In cells treated with IGF-1, veratridine-induced (22)Na(+) influx, and subsequent (45)Ca(2+) influx and catecholamine secretion were augmented by 35%, 33%, 31%, respectively. Pharmacological properties of Na(+) channels characterized by neurotoxins were similar between nontreated and IGF-1-treated cells. IGF-1-induced up-regulation of [(3)H]saxitoxin binding was prevented by phosphatydil inositol-3 kinase inhibitors (LY204002 or wortmannin), or Akt inhibitor (Akt inhibitor IV). Glycogen synthase kinase-3 (GSK-3) inhibitors (LiCl, valproic acid, SB216763 or SB415286) also increased cell surface [(3)H]saxitoxin binding by ∼ 33%, whereas simultaneous treatment of IGF-1 with GSK-3 inhibitors did not produce additive increasing effect on [(3)H]saxitoxin binding. IGF-1 (100 nM) increased Ser(437)-phosphorylated Akt and Ser(9)-phosphorylated GSK-3ß, and inhibited GSK-3ß activity. Treatment with IGF-1, LiCl or SB216763 increased protein level of Na(+) channel α-subunit; it was prevented by cycloheximide. Either treatment increased α-subunit mRNA level by ∼ 48% and accelerated α-subunit gene transcription by ∼ 30% without altering α-subunit mRNA stability. Thus, inhibition of GSK-3ß caused by IGF-1 up-regulates cell surface expression of functional Na(+) channels via acceleration of α-subunit gene transcription.

Effects of sevoflurane on voltage-gated sodium channel Na(v)1.8, Na(v)1.7, and Na(v)1.4 expressed in Xenopus oocytes.

Sevoflurane is widely used as a volatile anesthetic in clinical practice. However, its mechanism is still unclear. Recently, it has been reported that voltage-gated sodium channels have important roles in anesthetic mechanisms. Much attention has been paid to the effects of sevoflurane on voltage-dependent sodium channels. To elucidate this, we examined the effects of sevoflurane on Na(v) 1.8, Na(v) 1.4, and Na(v) 1.7 expressed in Xenopus oocytes. The effects of sevoflurane on Na(v) 1.8, Na(v) 1.4, and Na(v) 1.7 sodium channels were studied by an electrophysiology method using whole-cell, two-electrode voltage-clamp techniques in Xenopus oocytes. Sevoflurane at 1.0 mM inhibited the voltage-gated sodium channels Na(v)1.8, Na(v)1.4, and Na(v)1.7, but sevoflurane (0.5 mM) had little effect. This inhibitory effect of 1 mM sevoflurane was completely abolished by pretreatment with protein kinase C (PKC) inhibitor, bisindolylmaleimide I. Sevoflurane appears to have inhibitory effects on Na(v)1.8, Na(v)1.4, and Na(v) 1.7 by PKC pathways. However, these sodium channels might not be related to the clinical anesthetic effects of sevoflurane.

The tramadol metabolite O-desmethyl tramadol inhibits substance P-receptor functions expressed in Xenopus oocytes.

Tramadol has been widely used as analgesic. O-Desmethyl tramadol (ODT) is one of the main metabolites of tramadol, having much greater analgesic potency than tramadol itself. Substance P receptors (SPR) are well known to modulate nociceptive transmission within the spinal cord. In this study, we investigated the effects of ODT on SPR expressed in Xenopus oocytes by examining SP-induced Ca(2+)-activated Cl(-) currents. ODT inhibited the SPR-induced Cl(-) currents at pharmacologically relevant concentrations. The protein kinase C (PKC) inhibitor bisindolylmaleimide I did not abolish the inhibitory effects of ODT on SP-induced Ca(2+)-activated Cl(-) currents. The results suggest that the tramadol metabolite ODT inhibits the SPR functions, which may be independent of activation of PKC-mediated pathways.

Allyl isothiocyanates and cinnamaldehyde potentiate miniature excitatory postsynaptic inputs in the supraoptic nucleus in rats.

Allyl isothiocyanates (AITC) and cinnamaldehyde are pungent compounds present in mustard oil and cinnamon oil, respectively. These compounds are well known as transient receptor potential ankyrin 1 (TRPA1) agonists. TRPA1 is activated by low temperature stimuli, mechanosensation and pungent irritants such as AITC and cinnamaldehyde. TRPA1 is often co-expressed in TRPV1. Recent study showed that hypertonic solution activated TRPA1 as well as TRPV1. TRPV1 is involved in excitatory synaptic inputs to the magnocellular neurosecretory cells (MNCs) that produce vasopressin in the supraoptic nucleus (SON). However, it remains unclear whether TRPA1 may be involved in this activation. In the present study, we examined the role of TRPA1 on the synaptic inputs to the MNCs in in vitro rat brain slice preparations, using whole-cell patch-clamp recordings. In the presence of tetrodotoxin, AITC (50µM) and cinnamaldehyde (30µM) increased the frequency of miniature excitatory postsynaptic currents without affecting the amplitude. This effect was significantly attenuated by previous exposure to ruthenium red (10µM), non-specific TRP channels blocker, high concentration of menthol (300µM) and HC-030031 (10µM), which are known to antagonize the effects of TRPA1 agonists. These results suggest that TRPA1 may exist at presynaptic terminals to the MNCs and enhance glutamate release in the SON.

Analysis of the effects of anesthetics and ethanol on mu-opioid receptor.

G protein-coupled receptors, in particular, Ca(2+)-mobilizing G(q)-coupled receptors have been reported to be targets for anesthetics. Opioids are commonly used analgesics in clinical practice, but the effects of anesthetics on the opioid mu-receptors (muOR) have not been systematically examined. We report here an electrophysiological assay to analyze the effects of anesthetics and ethanol on the functions of muOR in Xenopus oocytes expressing a muOR fused to chimeric Galpha protein G(qi5) (muOR-G(qi5)). Using this system, the effects of halothane, ketamine, propofol, and ethanol on the muOR functions were analyzed. In oocytes expressing muOR-G(qi5), the( )muOR agonist DAMGO ([D-Ala(2),N-MePhe(4),Gly-ol]-enkephalin) elicited Ca(2+)-activated Cl(-) currents in a concentration-dependent manner (EC(50) = 0.24 microM). Ketamine, propofol, halothane, and ethanol themselves did not elicit any currents in oocytes expressing muOR-G(qi5), whereas ketamine and ethanol inhibited the DAMGO-induced Cl(-) currents at clinically equivalent concentrations. Propofol and halothane inhibited the DAMGO-induced currents only at higher concentrations. These findings suggest that ketamine and ethanol may inhibit muOR functions in clinical practice. We propose that the electrophysiological assay in Xenopus oocytes expressing muOR-G(qi5) would be useful for analyzing the effects of anesthetics and analgesics on opioid receptor function.