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Transient receptor potential ankyrin 1 (TRPA1) is known as one of the nociceptors expressed in sensory neurons. It also plays a role in non-neural cells in inflammatory sites. However, the regulatory mechanisms for the reactivity of TRPA1 in these cells under inflammatory conditions are not clear. To clarify these mechanisms, we examined the effects of inflammatory cytokines (interleukin [IL]-1α, IL-1β and tumor necrosis factor α [TNFα]) on TRPA1 reactivity and expression in the endogenously TRPA1-expressing lung tumor cell line A549. Treatment with IL-1α, but not IL-1β or TNFα, increased the number of cells responding to allyl isothiocyanate, a TRPA1 agonist, in a dose- and time-dependent manner. The IL-1α-induced increase of TRPA1 responsiveness was inhibited by an extracellular-regulated kinase (Erk) inhibitor (PD98059) but not by inhibitors of c-Jun kinase, p38 mitogen-activated protein kinase or phosphatidylinositol-3 kinase. Phosphorylation of Erk gradually increased at 24 h after its transient induction in cells treated with IL-1α. IL-1α increased the TRPA1 levels on biotinylated cell surface proteins. These results suggest that IL-1α enhances the translocation of TRPA1 to the plasma membrane via the activation of Erk in A549. TRPA1 may have a pathophysiological role in non-neural lung cells under inflammatory conditions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable, nonselective cation channel mainly expressed in a subset of nociceptive neurons. TRPA1 functions as a cellular sensor detecting mechanical, chemical, and thermal stimuli. Because TRPA1 is considered to be a key player in nociception and inflammatory pain, TRPA1 antagonists have been developed as analgesic agents. Recently, by utilizing species differences, we identified the molecular basis of the antagonistic action of A967079, one of the most potent mammalian TRPA1 antagonists. Here, we show a unique effect of A967079 on TRPA1 from diverse vertebrate species, i.e. it acts as an agonist but not as an antagonist for chicken and frog TRPA1s. By characterizing chimeric channels of human and chicken TRPA1s, as well as point mutants, we found that a single specific amino acid residue located within the putative fifth transmembrane domain was involved in not only the stimulatory but also the inhibitory actions of A967079. AP18, structurally related to A967079, exerted similar pharmacological properties to A967079. Our findings and previous reports on species differences in the sensitivity to TRPA1 antagonists supply useful information in the search for novel analgesic medicines targeting TRPA1., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. The beta3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in beta3-null mice to determine the role of the beta3 subunit in catecholamine release from the adrenal chromaffin system. Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The beta3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in beta3-null mice, while patch-clamp recordings showed a significant reduction in Ca(2+)-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production. In conclusion, our results indicate the importance of the beta3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells., (Copyright 2010. Published by Elsevier Inc.)

We investigated the endogenous control through vesicular contents of voltage-dependent Ca2+ channels (VDCCs) in cultured porcine adrenal chromaffin cells. To examine paracrine regulation of VDCCs, catecholamine release was monitored amperometrically together with patch-clamp recording under culture conditions at different cell densities. A depolarizing pulse evoked Ca(2+)- (ICa) and Ba(2+)-currents (IBa) in Ca(2+)- and Ba(2+)-containing solutions, respectively. In cells cultured at high density, stop-flow of the external solution decreased the I(Ba) concomitant with a sustained increase of amperometric current (Iamp), but not in cells at low density, suggesting the endogenous modulation of VDCCs in a paracine fashion. The degree of the prepulse facilitation was similar regardless of the flow condition. Application of noradrenaline (NA), ATP, methionine-enkephalin (ENK) or protons decreased IBa. The extent of the prepulse facilitation of the endogenous VDCC inhibition was similar to those induced by NA and ATP. GDPbetaS, pertussis toxin (PTX), blockers for alpha-adrenoceptors and P2-purinoceptors significantly reduced the endogenous VDCC inhibition. These results suggest that VDCCs are regulated by vesicular substances in a paracrine fashion, at least by noradrenaline and ATP, through activation of alpha-adrenoceptors and P2-purinoceptors, respectively, in porcine adrenal chromaffin cells.

The inhibitory effects of opioids on voltage-dependent calcium channels (VDCCs) were investigated in cultured porcine adrenal chromaffin cells using whole-cell patch clamp technique. The effects of the opioid on [Ca(2+)](i) increase and catecholamine secretion induced by high K(+) were also examined in single cells by fura-2 microfluorimetry and amperometry. A depolarizing pulse to 0 mV (test pulse) from a holding potential of -80 mV evoked an inward barium current (I(Ba)), which was reversibly inhibited by methionine-enkephalin. This inhibitory effect of methionine-enkephalin was abolished by naloxone. Selective agonists of opioid receptor subtypes (DAMGO: mu, DPDPE: delta, U50488: kappa) dose-dependently inhibited I(Ba). In inhibitory potency, the order was DAMGO>U50488>DPDPE. These agonists applied sequentially produced a reversible I(Ba) inhibition in the same cells. The inhibitory effect of DAMGO on I(Ba) almost disappeared in the presence of omega-conotoxin GVIA but not omega-agatoxin IVA plus nifedipine. Application of a conditioning prepulse to +100 mV prior to the test pulse partly retrieved the I(Ba) inhibition by DAMGO, suggesting the involvement of voltage-sensitive components in opioid-induced VDCC inhibition. Intracellular application of GDPbetaS or GTPgammaS as well as pretreatment with pertussis toxin significantly reduced the extent of I(Ba) inhibition induced by DAMGO. DAMGO reversibly inhibited the [Ca(2+)](i) increase and catecholamine release induced by high K(+). RT-PCR revealed the expression of mu-, delta- and kappa-opioid receptor mRNAs in cultured adrenal chromaffin cells. These results suggest that porcine adrenal chromaffin cells possess mu-, delta- and kappa-opioid receptors and activation of opioid receptors mainly inhibits N-type VDCCs via pertussis toxin-sensitive G-proteins.

Abstract: The β subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming α1 subunits. The β3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in β3-null mice to determine the role of the β3 subunit in catecholamine release from the adrenal chromaffin system. Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The β3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in β3-null mice, while patch–clamp recordings showed a significant reduction in Ca2+-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production. In conclusion, our results indicate the importance of the β3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells. [Copyright &y& Elsevier]

The inhibitory effects of opioids on voltage-dependent calcium channels (VDCCs) were investigated in cultured porcine adrenal chromaffin cells using whole-cell patch clamp technique. The effects of the opioid on [Ca2+]i increase and catecholamine secretion induced by high K+ were also examined in single cells by fura-2 microfluorimetry and amperometry. A depolarizing pulse to 0 mV (test pulse) from a holding potential of −80 mV evoked an inward barium current (IBa), which was reversibly inhibited by methionine-enkephalin. This inhibitory effect of methionine-enkephalin was abolished by naloxone. Selective agonists of opioid receptor subtypes (DAMGO: μ, DPDPE: δ, U50488: κ) dose-dependently inhibited IBa. In inhibitory potency, the order was DAMGO>U50488>DPDPE. These agonists applied sequentially produced a reversible IBa inhibition in the same cells. The inhibitory effect of DAMGO on IBa almost disappeared in the presence of ω-conotoxin GVIA but not ω-agatoxin IVA plus nifedipine. Application of a conditioning prepulse to +100 mV prior to the test pulse partly retrieved the IBa inhibition by DAMGO, suggesting the involvement of voltage-sensitive components in opioid-induced VDCC inhibition. Intracellular application of GDPβS or GTPγS as well as pretreatment with pertussis toxin significantly reduced the extent of IBa inhibition induced by DAMGO. DAMGO reversibly inhibited the [Ca2+]i increase and catecholamine release induced by high K+. RT-PCR revealed the expression of μ-, δ- and κ-opioid receptor mRNAs in cultured adrenal chromaffin cells. These results suggest that porcine adrenal chromaffin cells possess μ-, δ- and κ-opioid receptors and activation of opioid receptors mainly inhibits N-type VDCCs via pertussis toxin-sensitive G-proteins. [Copyright &y& Elsevier]