Emiko Mori

Selective and direct inhibition of TRPC3 channels underlies biological activities of a pyrazole compound

Canonical transient receptor potential (TRPC) channels control influxes of Ca2+ and other cations that induce diverse cellular processes upon stimulation of plasma membrane receptors coupled to phospholipase C (PLC). Invention of subtype-specific inhibitors for TRPCs is crucial for distinction of respective TRPC channels that play particular physiological roles in native systems. Here, we identify a pyrazole compound (Pyr3), which selectively inhibits TRPC3 channels. Structure-function relationship studies of pyrazole compounds showed that the trichloroacrylic amide group is important for the TRPC3 selectivity of Pyr3. Electrophysiological and photoaffinity labeling experiments reveal a direct action of Pyr3 on the TRPC3 protein. In DT40 B lymphocytes, Pyr3 potently eliminated the Ca2+ influx-dependent PLC translocation to the plasma membrane and late oscillatory phase of B cell receptor-induced Ca2+ response. Moreover, Pyr3 attenuated activation of nuclear factor of activated T cells, a Ca2+-dependent transcription factor, and hypertrophic growth in rat neonatal cardiomyocytes, and in vivo pressure overload-induced cardiac hypertrophy in mice. These findings on important roles of native TRPC3 channels are strikingly consistent with previous genetic studies. Thus, the TRPC3-selective inhibitor Pyr3 is a powerful tool to study in vivo function of TRPC3, suggesting a pharmaceutical potential of Pyr3 in treatments of TRPC3-related diseases such as cardiac hypertrophy.

Transient Receptor Potential 1 Regulates Capacitative Ca2+ Entry and Ca2+ Release from Endoplasmic Reticulum in B Lymphocytes

Capacitative Ca2+ entry (CCE) activated by release/depletion of Ca2+ from internal stores represents a major Ca2+ influx mechanism in lymphocytes and other nonexcitable cells. Despite the importance of CCE in antigen-mediated lymphocyte activation, molecular components constituting this mechanism remain elusive. Here we demonstrate that genetic disruption of transient receptor potential (TRP)1 significantly attenuates both Ca2+ release-activated Ca2+ currents and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release from endoplasmic reticulum (ER) in DT40 B cells. As a consequence, B cell antigen receptor–mediated Ca2+ oscillations and NF-AT activation are reduced in TRP1-deficient cells. Thus, our results suggest that CCE channels, whose formation involves TRP1 as an important component, modulate IP3 receptor function, thereby enhancing functional coupling between the ER and plasma membrane in transduction of intracellular Ca2+ signaling in B lymphocytes.

RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca2+ channels.

The molecular organization of presynaptic active zones is important for the neurotransmitter release that is triggered by depolarization-induced Ca2+ influx. Here, we demonstrate a previously unknown interaction between two components of the presynaptic active zone, RIM1 and voltage-dependent Ca2+ channels (VDCCs), that controls neurotransmitter release in mammalian neurons. RIM1 associated with VDCC β-subunits via its C terminus to markedly suppress voltage-dependent inactivation among different neuronal VDCCs. Consistently, in pheochromocytoma neuroendocrine PC12 cells, acetylcholine release was significantly potentiated by the full-length and C-terminal RIM1 constructs, but membrane docking of vesicles was enhanced only by the full-length RIM1. The β construct beta-AID dominant negative, which disrupts the RIM1-β association, accelerated the inactivation of native VDCC currents, suppressed vesicle docking and acetylcholine release in PC12 cells, and inhibited glutamate release in cultured cerebellar neurons. Thus, RIM1 association with β in the presynaptic active zone supports release via two distinct mechanisms: sustaining Ca2+ influx through inhibition of channel inactivation, and anchoring neurotransmitter-containing vesicles in the vicinity of VDCCs.

Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells

We investigated, by using the patch clamp technique, Ca2+‐mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor‐operated Ca2+ entry channels. With nystatin‐perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 μm)‐activated TRPC6 currents (ITRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2o+) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2o+ solely inhibited TRPC7 currents (ITRPC7). Vigorous buffering of intracellular Ca2+ (Ca2i+) under conventional whole‐cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2o+, disclosing fast potentiation (EC50: ∼0.4 mm) and inhibition (IC50: ∼4 mm) of ITRPC6 and fast inhibition (IC50: ∼0.4 mm) of ITRPC7. This inhibition of ITRPC6 and ITRPC7 seems to be associated with voltage‐dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of ITRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of ITRPC6 or its acceleration by Ca2o+ probably involves phosphorylation by calmodulin (CaM)‐dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 μm), coexpression of Ca2+ ‐insesentive mutant CaM, and intracellular perfusion of the non‐hydrolysable ATP analogue AMP‐PNP and a CaMKII‐specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh‐activated TRPC7 channel activity was concentration‐dependently suppressed by nanomolar Ca2i+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of ITRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+−CaM‐dependent and ‐independent mechanisms.

Essential role of monocyte chemoattractant protein-1 in development of restenotic changes (neointimal hyperplasia and constrictive remodeling) after balloon angioplasty in hypercholesterolemic rabbits

Background—Renarrowing of dilated arterial sites (restenosis) hampers the clinical benefits of coronary angioplasty. Infiltration and activation of monocytes in the arterial wall mediated by monocyte chemoattractant protein-1 (MCP-1) might be a major cause of restenosis after angioplasty. However, there is no direct evidence to support a definite role of MCP-1 in the development of restenosis. Methods and Results—We recently devised a new strategy for anti–MCP-1 gene therapy by transfecting an N-terminal deletion mutant of the MCP-1 gene into skeletal muscles. We used this strategy to investigate the role of MCP-1 in the development of restenotic changes after balloon injury in the carotid artery in hypercholesterolemic rabbits. Intramuscular transfection of the mutant MCP-1 gene suppressed monocyte infiltration/activation in the injured arterial wall and thus attenuated the development of neointimal hyperplasia and negative remodeling. Conclusions—MCP-1–mediated monocyte infiltration is necessary in the development of restenotic changes to balloon injury in hypercholesterolemic rabbits. This strategy may be a useful and practical form of gene therapy against human restenosis.

Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCγ2 in B lymphocytes

In non‐excitable cells, receptor‐activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry‐dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5‐trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor‐activated and store‐operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cγ2 (PLCγ2)‐deficient cells. Association between receptor‐activated TRPC3 Ca2+ channels and PLCγ2, which cooperate in potentiating Ca2+ responses, was demonstrated by co‐immunoprecipitation. PLCγ2‐deficient cells displayed diminished Ca2+ entry‐induced Ca2+ responses. However, this defect was canceled by suppressing IP3‐induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCγ2 mutants demonstrated that Ca2+ entry evoked a C2 domain‐mediated PLCγ2 translocation towards the plasma membrane in a lipase‐independent manner to activate PLCγ2. Strikingly, Ca2+ entry‐activated PLCγ2 maintained Ca2+ oscillation and extracellular signal‐regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca2+ entry with PLCγ2 translocation and activation controls the amplification and co‐ordination of receptor signalling.

Molecular Characterization of Flubendiamide Sensitivity in the Lepidopterous Ryanodine Receptor Ca2+ Release Channel

Flubendiamide is a benzenedicarboxamide derivative that shows selective insecticidal activity against lepidopterous insects. The specific modulatory effects of flubendiamide on ryanodine binding in insect muscle microsomal membranes suggest that the ryanodine receptor (RyR) Ca(2+) release channel is a primary target of flubendiamide. However, the molecular mechanisms underlying the species-specific action of flubendiamide are unclear. We have cloned cDNA encoding a novel RyR from the lepidopterous silkworm RyR (sRyR) and tested the sensitivity to flubendiamide of the recombinant sRyR in HEK293 cells. Confocal localization studies and Ca(2+) imaging techniques revealed that sRyRs form Ca(2+) release channels in the endoplasmic reticulum. Importantly, flubendiamide induced release of Ca(2+) through the sRyR, but not through the rabbit RyR isoforms. Photoaffinity labeling of sRyR deletion mutants using a photoreactive derivative revealed that flubendiamide is mainly incorporated into the transmembrane domain (amino acids 4111-5084) of the sRyR. The rabbit cardiac muscle isoform RyR2 (rRyR2) and the RyR mutant carrying a replacement of the transmembrane domain (residues 4084-5084) with its counterpart sequence from rRyR2 (residues 3936-4968) were not labeled by the photoreactive compound. This replacement in the sRyR significantly impaired the responses to flubendiamide but only marginally reduced the sensitivity to caffeine, a general RyR activator. Furthermore, deletion of the N-terminal sequence (residues 183-290) abolished the responses of the sRyR to flubendiamide but not the sensitivity to caffeine. Our results suggest that the transmembrane domain plays an important role in the formation of an action site for flubendiamide, while the N-terminus is a structural requirement for flubendiamide-induced activation of the sRyR.

Ca2+ influx and protein scaffolding via TRPC3 sustain PKCβ and ERK activation in B cells

Ca2+ signaling mediated by phospholipase C that produces inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DAG) controls lymphocyte activation. In contrast to store-operated Ca2+ entry activated by Ins(1,4,5)P3-induced Ca2+ release from endoplasmic reticulum, the importance of DAG-activated Ca2+ entry remains elusive. Here, we describe the physiological role of DAG-activated Ca2+ entry channels in B-cell receptor (BCR) signaling. In avian DT40 B cells, deficiency of transient receptor potential TRPC3 at the plasma membrane (PM) impaired DAG-activated cation currents and, upon BCR stimulation, the sustained translocation to the PM of protein kinase Cβ (PKCβ) that activated extracellular signal-regulated kinase (ERK). Notably, TRPC3 showed direct association with PKCβ that maintained localization of PKCβ at the PM. Thus, TRPC3 functions as both a Ca2+-permeable channel and a protein scaffold at the PM for downstream PKCβ activation in B cells.

Successful and optimized in vivo gene transfer to rabbit carotid artery mediated by electronic pulse

Several gene transfer methods, including viral or nonviral vehicles have been developed, however, efficacy, safety or handling continue to present problems. We developed a nonviral and plasmid-based method for arterial gene transfer by in vivo electronic pulse, using a newly designed T-shaped electrode. Using rabbit carotid arteries, we first optimized gene transfer efficiency, and firefly luciferase gene transfer via electronic pulse under 20 voltage (the pulse length: Pontime 20 ms, the pulse interval: Poff time 80 ms, number of pulse: 10 times) showed the highest gene expression. Exogenous gene expression was detectable for at least up to 14 days. Electroporation-mediated gene transfer of E. coli lacZ with nuclear localizing signal revealed successful gene transfer to luminal endothelial cells and to medial cells. Histological damage was recognized as the voltage was increased but neointima formation 4 weeks after gene transfer was not induced. In vivo electroporation-mediated arterial gene transfer is readily facilitated, is safe and may prove to be an alternative form of gene transfer to the vasculature.

Preoperative Elevation of Serum C-Reactive Protein as an Independent Prognostic Indicator of Colorectal Carcinoma

PurposeThe preoperative elevation of serum C-reactive protein (CRP) is thought to be a prognosticator of carcinomas of the digestive tract. We conducted this study to investigate the clinical importance of the preoperative elevation of serum CRP in patients with colorectal carcinoma (CRC).MethodsWe investigated the correlation between an elevated preoperative serum CRP level and the clinicopathologic factors, including prognosis, of 116 patients who underwent resection of CRC.ResultsForty-seven (40.5%) patients had an elevated serum CRP value preoperatively (group H) and 69 (59.5%) did not (group L). There were significant differences in the tumor size, proportion of poorly differentiated tumors, depth of invasion, lymph node metastasis, lymphatic invasion, and tumor stage between the two groups. Survival was significantly lower in group H than in group L (P < 0.0001). Multivariate analysis showed that the preoperative elevation of serum CRP (P = 0.0007), as well as poor differentiation (P = 0.027) and advanced tumor stage (P = 0.007) were independent prognostic factors in patients with CRC.ConclusionWe found the preoperative elevation of serum CRP to be an independent prognostic indicator of CRC.