Transient Receptor Potential Channel Antagonists Research Articles

The transient receptor potential channel antagonist SKF96365 is a potent blocker of low‐voltage‐activated T‐type calcium channels

SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents. Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed. SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca(V)1, Ca(V)2 and Ca(V)3) at concentrations typically utilized to assay TRPC function (10 microM). Particularly, human Ca(V)3.1 T-type Ca channels were more potently inhibited by SKF (IC(50) approximately 560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca(V)3.1 T-type currents in a rat cerebellar PC slice preparation. SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues.

Activation by lidocaine of TRPA1 channels in the substantia gelatinosa of adult rat spinal cord

Transient receptor potential (TRP) channels, nonselective cation channels, are gated by a variety of chemical and physical stimuli. Among them, there are TRPV1 and TRPA1 channels, both of which are thought to be involved in thermosensation and nociception. TRPV1 channels are activated by noxious hot temperature, protons and capsaicin while TRPA1 channels are by noxious cold temperature, mustard oil, cinnamon oil, ginger and garlic. These TRP channels are expressed in dorsal root ganglion (DRG) neurons; their activation in the central terminals of the neurons results in an increase in the spontaneous release of L-glutamate to spinal dorsal horn, particularly substantia gelatinosa (SG; lamina II of Rexed), neurons from nerve terminals. Although local anesthetics (LAs) were recently reported to activate TRP channels which are cloned and expressed in DRG neurons, this has not been thoroughly examined yet in the CNS. We examined the effects of amine-type LAs (lidocaine, ropivacaine, prilocaine and levobupivacaine) and ester-type LAs (procaine, tetracaine and pramoxine) on glutamatergic spontaneous excitatory synaptic transmission in SG neurons of adult rat spinal cord slices by using the whole-cell patch-clamp technique. Bath-applied lidocaine dose-dependently and reversibly increased the frequency but not the amplitude of spontaneous excitatory postsynaptic current (sEPSC) recorded at a holding potential of -70 mV in SG neurons. The effect of lidocaine was unaffected by the voltage-gated Na+-channel blocker, tetrodotoxin (TTX), and the TRPV1 channel antagonist, capsazepine (Capz), but was inhibited by the non-selective TRP channel antagonist, ruthenium red (RR). In the same neuron, the TRPA1 channel agonist, allyl isothiocyanate, and lidocaine both increased sEPSC frequency. Tetracaine also increased sEPSC frequency in SG neurons in a manner insensitive to TTX and Capz while sensitive to RR, an action similar to that of lidocaine, but this action was delayed in onset by about 2.5 min compared to that of lidocaine. Like lidocaine and tetracaine, pramoxine increased sEPSC frequency; prilocaine had a tendency to do so. On the contrary, procaine, ropivacaine and levobupivacaine reduced sEPSC frequency. It is concluded that some of the amine-type and ester-type LAs activate TRPA1 channels in the SG to increase the spontaneous release of L-glutamate from nerve terminals. This action could contribute to an increase in the excitability of SG neurons.

Functional characterization of transient receptor potential channels in mouse urothelial cells

The bladder urothelium is currently believed to be a sensory structure, contributing to mechano- and chemosensation in the bladder. Transient receptor potential (TRP) cation channels act as polymodal sensors and may underlie some of the receptive properties of urothelial cells. However, the exact TRP channel expression profile of urothelial cells is unclear. In this study, we have performed a systematic analysis of the molecular and functional expression of various TRP channels in mouse urothelium. Urothelial cells from control and trpv4-/- mice were isolated, cultured (12-48 h), and used for quantitative real-time PCR, immunocytochemistry, calcium imaging, and whole cell patch-clamp experiments. At the mRNA level, TRPV4, TRPV2, and TRPM7 were the most abundantly expressed TRP genes. Immunohistochemistry showed a clear expression of TRPV4 in the plasma membrane, whereas TRPV2 was more prominent in the cytoplasm. TRPM7 was detected in the plasma membrane as well as cytoplasmic vesicles. Calcium imaging and patch-clamp experiments using TRP channel agonists and antagonists provided evidence for the functional expression of TRPV4, TRPV2, and TRPM7 but not of TRPA1, TRPV1, and TRPM8. In conclusion, we have demonstrated functional expression of TRPV4, TRPV2, and TRPM7 in mouse urothelial cells. These channels may contribute to the (mechano)sensory function of the urothelial layer and represent potential targets for the treatment of bladder dysfunction.

Transient receptor potential A1 increase glutamate release on brain stem neurons

Low temperature and noxious chemicals activate transient receptor potential (TRP) A1 channel in spinal nociceptive neurons. TRPA1 ligands are found as common ingredients of pungent spices and can trigger autonomic reflexes and alter blood pressure. Recent discovery of TRPA1 on visceral afferent pathway suggests such nonnociceptive responses may be closely related to TRPA1. To understand potential contributions of TRPA1 on autonomic response, we have tested TRPA1 agonist allyl isothiocyanate (AITC) effects on mechanically dispersed nucleus tractus solitarii neurons. AITC of 200 microM increased glutamate release in 58% of tested neurons and TRP channel antagonist ruthenium red (50 microM) blocked the AITC actions. tetrodotoxin and Cd did not eliminate AITC-evoked glutamate release increase. TRPA1 expressed in capsaicin-sensitive and insensitive neurons.

Comparison of icilin- and cold-evoked responses of spinal neurones, and their modulation of mechanical activity, in a model of neuropathic pain

Cold allodynia is a poorly understood symptom of neuropathic pain. Two members of the transient receptor potential (TRP) family of proteins, TRPM8 and TRPA1, may contribute to cold somatosensation. The aim of the present study was to investigate the usefulness of icilin as a pharmacological tool to study primary afferent fibre responses to cold stimuli and to determine whether there are differences in the responses of spinal neurones to cooling of peripheral receptive fields in control versus neuropathic rats. The effects of icilin, a TRPM8 and TRPA1 agonist, on intracellular Ca2+ ([Ca2+]i) responses of small diameter adult dorsal root ganglia (DRG) neurones were determined. Icilin (10 nM–10 μM) produced a concentration-related increase in [Ca2+]i in DRG neurones, which was attenuated by the non-selective TRP channel antagonist ruthenium red (10 μM). In vivo electrophysiology in naïve, sham-operated and SNL rats demonstrated that application of ice to receptive fields evoked firing of wide dynamic range (WDR) neurones, which was significantly greater in SNL rats than naïve and sham-operated rats. Intraplantar injection of icilin did not evoke firing of WDR neurones in naïve, sham-operated or SNL rats but inhibited mechanically-evoked responses of WDR neurones in naïve and sham-operated rats, whilst facilitating mechanically-evoked responses in SNL rats. Icilin increased both innocuous (sham-operated and SNL rats) and noxious (SNL rats) receptive field sizes of WDR neurones. Our data suggests that icilin modulates the mechanosensitivity of dorsal horn neurones. The differing effects of ice and icilin on dorsal horn neurones indicate different mechanisms of action.

Conservation of Functional and Pharmacological Properties in the Distantly Related Temperature Sensors TRPV1 and TRPM8

Members of the transient receptor potential (TRP) superfamily of ion channels have now been defined as molecular transducers capable of reproducing the spectrum of temperature sensation exhibited by mammals. Because of their pivotal role in sensory transduction, many of these channels represent good targets for drug discovery. With a view to gaining further insight into the functional and pharmacological properties of these channels, we have used the whole-cell patch-clamp technique to study the human cold-sensitive menthol receptor transient receptor potential melastatin 8 (TRPM8) and compared its behavior with that of its distant relative, the heat-sensitive capsaicin-gated transient receptor potential vanilloid 1 (TRPV1). It is remarkable to find that TRPM8, in addition to its behavior as an outwardly rectifying, nonselective cation channel, shares many functional and pharmacological properties with TRPV1. TRPM8 exhibits prominent time- and voltage-dependent behavior, a property that may underlie the conserved rectification or gating mechanisms exhibited by these channels. We also show that TRPM8 is modulated by ethanol but unlike TRPV1 is insensitive to extracellular acidification. There is also significant overlap in the antagonist pharmacology of these channels with many TRPV1 antagonists such as capsazepine, N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (BCTC), (2R)-4-(3-chloro-2-pyridinyl)-2-methyl-N-[4-(trifluoromethyl)phenyl]-1-piperazinecarboxamide (CTPC), and N-(2-bromophenyl)-N'-{2-[ethyl(3-methylphenyl)amino]ethyl}-urea (SB-452533) exhibiting similar activity at TRPM8. Overall, the degree of pharmacological overlap between TRPV1 and TRPM8 has implications for the interpretation of studies conducted with these ligands to date and highlights a clear challenge for the design of selective TRP channel antagonists. Our finding that N-(3-methoxyphenyl)-4-chlorocinnamide (SB-366791), at least, represents an apparently selective antagonist for TRPV1 suggests that this goal is attainable.

Transient receptor potential and other ion channels as pharmaceutical targets in airway smooth muscle cells

Regardless of the triggering stimulus in asthma, contraction of the airway smooth muscle (ASM) is considered to be an important pathway leading to the manifestation of asthmatic symptoms. Therefore, the various ion channels that modulate ASM contraction and relaxation are particularly attractive targets for therapy. Although voltage-operated Ca2+ channels (VOCC) are the most extensively characterised Ca(2+)-permeable channels in ASM cells and are obvious pharmacological targets, blockers of VOCC have not been successful in alleviating ASM contraction in asthma. Similarly, although the Cl- and K+ channels also modulate ASM contraction and relaxation by regulating plasma membrane potential, pharmacological interventions directed against these channels have failed to abrogate ASM contraction in asthma. A large body of evidence suggests that store-operated Ca2+ channels (SOCC) and Ca(2+)-permeable second messenger-activated non-selective cation channels (NSCC) predominantly mediate ASM contraction. However, development of pharmacological interventions involving these channels has been hampered by the paucity of information regarding their molecular identity. Members of the mammalian transient receptor potential (TRP) protein family, which form voltage-independent channels with variable Ca2+ selectivity that are activated by store depletion and/or by intracellular messengers, are potential molecular candidates for SOCC and NSCC in ASM cells. While the function of TRP channels in ASM cells remains to be elucidated and there are, at present, essentially no good TRP channel antagonists, this group of proteins is a potentially valuable pharmaceutical target for the treatment of asthma.