Transient Receptor Potential Canonical 3 and Nuclear Factor of Activated T Cells C3 Signaling Pathway Critically Regulates Myocardial Fibrosis.

AIMS: Cardiac fibroblasts (CFs) are emerging as major contributors to myocardial fibrosis (MF), a final common pathway of many etiologies of heart disease. Here, we studied the functional relevance of transient receptor potential canonical 3 (TRPC3) channels and nuclear factor of activated T cells c3 (NFATc3) signaling in rodent and human ventricular CFs, and whether their modulation would limit MF. RESULTS: A positive feedback loop between TRPC3 and NFATc3 drove a rat ventricular CF fibrotic phenotype. In these cells, polyphenols (extract of grape pomace polyphenol [P.E.]) decreased basal and angiotensin II-mediated Ca2+ entries through a direct modulation of TRPC3 channels and subsequently NFATc3 signaling, abrogating myofibroblast differentiation, fibrosis and inflammation, as well as an oxidative stress-associated phenotype. N(ω)-nitro-l-arginine methyl ester (l-NAME) hypertensive rats developed coronary perivascular, sub-epicardial, and interstitial fibrosis with induction of embryonic epicardial progenitor transcription factors in activated CFs. P.E. treatment reduced ventricular CF activation by modulating the TRPC3-NFATc3 pathway, and it ameliorated echocardiographic parameters, cardiac stress markers, and MF in l-NAME hypertensive rats independently of blood pressure regulation. Further, genetic deletion (TRPC3-/-) and pharmacological channel blockade with N-[4-[3,5-Bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-4-methyl-benzenesulfonamide (Pyr10) blunted ventricular CF activation and MF in l-NAME hypertensive mice. Finally, TRPC3 was present in human ventricular CFs and upregulated in MF, whereas pharmacological modulation of TRPC3-NFATc3 decreased proliferation and collagen secretion. Innovation and Conclusion: We demonstrate that TRPC3-NFATc3 signaling is modulated by P.E. and critically regulates ventricular CF phenotype and MF. These findings strongly argue for P.E., through TRPC3 targeting, as potential and interesting therapeutics for MF management.

TRPC3 channels play a critical role in the theta component of pilocarpine-induced status epilepticus in mice.

OBJECTIVE: Canonical transient receptor potential (TRPC) channels constitute a family of cation channels that exhibit a regional and cell-specific expression pattern throughout the brain. It has been reported previously that TRPC3 channels are effectors of the brain-derived neurotrophic factor (BDNF)/trkB signaling pathway. Given the long postulated role of BDNF in epileptogenesis, TRPC3 channels may be a critical component in the underlying pathophysiology of seizure and epilepsy. In this study, we investigated the precise role of TRPC3 channels in pilocarpine-induced status epilepticus (SE). METHODS: The role of TRPC3 channels was investigated using TRPC3 knockout (KO) mice and TRPC3-selective inhibitor Pyr3. Video and electroencephalography (EEG) recording of pilocarpine-induced seizures were performed. RESULTS: We found that genetic ablation of TRPC3 channels reduces behavioral manifestations of seizures and the root-mean-square (RMS) power of SE, indicating a significant contribution of TRPC3 channels to pilocarpine-induced SE. Furthermore, the reduction in SE in TRPC3KO mice is caused by a selective attenuation of pilocarpine-induced theta activity, which dominates both the preictal phase and SE phase. Pyr3 also caused a reduction in the overall RMS power of pilocarpine-induced SE and a selective reduction in the theta activity during SE. SIGNIFICANCE: Our results demonstrate that TRPC3 channels unequivocally contribute to pilocarpine-induced SE and could be a novel molecular target for new anticonvulsive drugs.

Resistance to pathologic cardiac hypertrophy and reduced expression of CaV1.2 in Trpc3-depleted mice.

Sustained elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) reprograms cardiovascular cell fate, leading to cellular hypertrophy via Ca(2+)-calmodulin/calcineurin (Cn)/NFAT activation. Accumulating evidence suggests that transient receptor potential canonical (Trpc) channels play important roles in the development of pathologic cardiac hypertrophy. Here, we demonstrated that Trpc3 mediates pathologic cardiac hypertrophy in neurohumoral elevation via direct regulation of CaV1.2 expressions. Elevated PE (phenylephrine) was maintained in mice by continuous infusion using an osmotic pump. Wild-type (WT) mice, but not Trpc3 (-/-) showed a sudden decrease in blood pressure (BP) or death following elevation of BP under conditions of elevated PE. Trpc3 (-/-) mesenteric artery showed decreased PE-stimulated vasoconstriction. Analysis of morphology, function, and pathologic marker expression revealed that PE elevation caused pathologic cardiac hypertrophy in WT mice, which was prevented by deletion of Trpc3. Interestingly, protection by Trpc3 deletion seemed to be a result of reduced cardiac CaV1.2 expressions. Basal and PE induced increased expression of protein and mRNA of CaV1.2 was decreased in Trpc3 (-/-) heart. Accordingly, altered expression of CaV1.2 was observed by knockdown or stimulation of Trpc3 in cardiomyocytes. These findings suggest that Trpc3 is a mediator of pathologic cardiac hypertrophy not only through mediating part of the Ca(2+) influx, but also through control of CaV1.2 expressions.

The role of GαO-mediated signaling in the rostral ventrolateral medulla oblongata in cardiovascular reflexes and control of cardiac ventricular excitability.

The heart is controlled by the sympathetic and parasympathetic limbs of the autonomic nervous system with inhibitory signaling mechanisms recruited in both limbs. The aim of this study was to determine the role of inhibitory heterotrimeric G proteins in the central nervous mechanisms underlying autonomic control of the heart and its potential role in arrhythmogenesis. Mice with conditional deletion of the inhibitory heterotrimeric G protein GαO in the presympathetic area of the rostral ventral lateral medulla (RVLM) were generated to determine the role of GαO-mediated signalling in autonomic control and electrophysiological properties of the heart. GαO deletion within the RVLM was not associated with changes in heart rate (HR) or the arterial blood pressure at rest (home cage, normal behavior). However, exposure to stressful conditions (novel environment, hypoxia, or hypercapnia) in these mice was associated with abnormal HR responses and an increased baroreflex gain when assessed under urethane anesthesia. This was associated with shortening of the ventricular effective refractory period. This phenotype was reversed by systemic beta-adrenoceptor blockade, suggesting that GαO depletion in the RVLM increases central sympathetic drive. The data obtained support the hypothesis that GαO-mediated signaling within the presympathetic circuits of the RVLM contributes to the autonomic control of the heart. GαO deficiency in the RVLM has a significant impact on cardiovascular responses to stress, cardiovascular reflexes and electrical properties of the heart.

The contribution of TRPC1, TRPC3, TRPC5 and TRPC6 to touch and hearing.

Transient receptor potential channels have diverse roles in mechanosensation. Evidence is accumulating that members of the canonical subfamily of TRP channels (TRPC) are involved in touch and hearing. Characteristic features of TRP channels include their high structural homology and their propensity to form heteromeric complexes which suggests potential functional redundancy. We previously showed that TRPC3 and TRPC6 double knockout animals have deficits in light touch and hearing whilst single knockouts were apparently normal. We have extended these studies to analyse deficits in global quadruple TRPC1, 3, 5 and 6 null mutant mice. We examined both touch and hearing in behavioural and electrophysiological assays, and provide evidence that the quadruple knockout mice have larger deficits than the TRPC3 TRPC6 double knockouts. Mechano-electrical transducer currents of cochlear outer hair cells were however normal. This suggests that TRPC1, TRPC3, TRPC5 and TRPC6 channels contribute to cutaneous and auditory mechanosensation in a combinatorial manner, but have no direct role in cochlear mechanotransduction.

TRPC3 channels critically regulate hippocampal excitability and contextual fear memory.

Memory formation requires de novo protein synthesis, and memory disorders may result from misregulated synthesis of critical proteins that remain largely unidentified. Plasma membrane ion channels and receptors are likely candidates given their role in regulating neuron excitability, a candidate memory mechanism. Here we conduct targeted molecular monitoring and quantitation of hippocampal plasma membrane proteins from mice with intact or impaired contextual fear memory to identify putative candidates. Here we report contextual fear memory deficits correspond to increased Trpc3 gene and protein expression, and demonstrate TRPC3 regulates hippocampal neuron excitability associated with memory function. These data provide a mechanistic explanation for enhanced contextual fear memory reported herein following knockdown of TRPC3 in hippocampus. Collectively, TRPC3 modulates memory and may be a feasible target to enhance memory and treat memory disorders.

Evidence of a Role for Fibroblast Transient Receptor Potential Canonical 3 Ca2+ Channel in Renal Fibrosis.

Transient receptor potential canonical (TRPC) Ca(2+)-permeant channels, especially TRPC3, are increasingly implicated in cardiorenal diseases. We studied the possible role of fibroblast TRPC3 in the development of renal fibrosis. In vitro, a macromolecular complex formed by TRPC1/TRPC3/TRPC6 existed in isolated cultured rat renal fibroblasts. However, specific blockade of TRPC3 with the pharmacologic inhibitor pyr3 was sufficient to inhibit both angiotensin II- and 1-oleoyl-2-acetyl-sn-glycerol-induced Ca(2+) entry in these cells, which was detected by fura-2 Ca(2+) imaging. TRPC3 blockade or Ca(2+) removal inhibited fibroblast proliferation and myofibroblast differentiation by suppressing the phosphorylation of extracellular signal-regulated kinase (ERK1/2). In addition, pyr3 inhibited fibrosis and inflammation-associated markers in a noncytotoxic manner. Furthermore, TRPC3 knockdown by siRNA confirmed these pharmacologic findings. In adult male Wistar rats or wild-type mice subjected to unilateral ureteral obstruction, TRPC3 expression increased in the fibroblasts of obstructed kidneys and was associated with increased Ca(2+) entry, ERK1/2 phosphorylation, and fibroblast proliferation. Both TRPC3 blockade in rats and TRPC3 knockout in mice inhibited ERK1/2 phosphorylation and fibroblast activation as well as myofibroblast differentiation and extracellular matrix remodeling in obstructed kidneys, thus ameliorating tubulointerstitial damage and renal fibrosis. In conclusion, TRPC3 channels are present in renal fibroblasts and control fibroblast proliferation, differentiation, and activation through Ca(2+)-mediated ERK signaling. TRPC3 channels might constitute important therapeutic targets for improving renal remodeling in kidney disease.

Transient receptor potential canonical type 3 channels control the vascular contractility of mouse mesenteric arteries.

Transient receptor potential canonical type 3 (TRPC3) channels are non-selective cation channels and regulate intracellular Ca2+ concentration. We examined the role of TRPC3 channels in agonist-, membrane depolarization (high K+)-, and mechanical (pressure)-induced vasoconstriction and vasorelaxation in mouse mesenteric arteries. Vasoconstriction and vasorelaxation of endothelial cells intact mesenteric arteries were measured in TRPC3 wild-type (WT) and knockout (KO) mice. Calcium concentration ([Ca2+]) was measured in isolated arteries from TRPC3 WT and KO mice as well as in the mouse endothelial cell line bEnd.3. Nitric oxide (NO) production and nitrate/nitrite concentrations were also measured in TRPC3 WT and KO mice. Phenylephrine-induced vasoconstriction was reduced in TRPC3 KO mice when compared to that of WT mice, but neither high K+- nor pressure-induced vasoconstriction was altered in TRPC3 KO mice. Acetylcholine-induced vasorelaxation was inhibited in TRPC3 KO mice and by the selective TRPC3 blocker pyrazole-3. Acetylcholine blocked the phenylephrine-induced increase in Ca2+ ratio and then relaxation in TRPC3 WT mice but had little effect on those outcomes in KO mice. Acetylcholine evoked a Ca2+ increase in endothelial cells, which was inhibited by pyrazole-3. Acetylcholine induced increased NO release in TRPC3 WT mice, but not in KO mice. Acetylcholine also increased the nitrate/nitrite concentration in TRPC3 WT mice, but not in KO mice. The present study directly demonstrated that the TRPC3 channel is involved in agonist-induced vasoconstriction and plays important role in NO-mediated vasorelaxation of intact mesenteric arteries.

Activation of endothelial transient receptor potential C3 channel is required for small conductance calcium-activated potassium channel activation and sustained endothelial hyperpolarization and vasodilation of cerebral artery.

BACKGROUND: Transient receptor potential C3 (TRPC3) has been demonstrated to be involved in the regulation of vascular tone through endothelial cell (EC) hyperpolarization and endothelium-dependent hyperpolarization-mediated vasodilation. However, the mechanism by which TRPC3 regulates these processes remains unresolved. We tested the hypothesis that endothelial receptor stimulation triggers rapid TRPC3 trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for small conductance calcium-activated K(+) (SKCa) channel activation and sustained EC hyperpolarization. METHODS AND RESULTS: Pressurized artery studies were performed with isolated mouse posterior cerebral artery. Treatment with a selective TRPC3 blocker (Pyr3) produced significant attenuation of endothelium-dependent hyperpolarization-mediated vasodilation and endothelial Ca(2+) response (EC-specific Ca(2+) biosensor) to intraluminal ATP. Pyr3 treatment also resulted in a reduced ATP-stimulated global Ca(2+) and Ca(2+) influx in primary cultures of cerebral endothelial cells. Patch-clamp studies with freshly isolated cerebral ECs demonstrated 2 components of EC hyperpolarization and K(+) current activation in response to ATP. The early phase was dependent on intermediate conductance calcium-activated K(+) channel activation, whereas the later sustained phase relied on SKC a channel activation. The SKC a channel-dependent phase was completely blocked with TRPC3 channel inhibition or in ECs of TRPC3 knockout mice and correlated with increased trafficking of TRPC3 (but not SKC a channel) to the plasma membrane. CONCLUSIONS: We propose that TRPC3 dynamically regulates SKC a channel activation through receptor-dependent trafficking to the plasma membrane, where it provides the source of Ca(2+) influx for sustained SKC a channel activation, EC hyperpolarization, and endothelium-dependent hyperpolarization-mediated vasodilation.

Critical role of canonical transient receptor potential channel 7 in initiation of seizures.

Status epilepticus (SE) is a life-threatening disease that has been recognized since antiquity but still causes over 50,000 deaths annually in the United States. The prevailing view on the pathophysiology of SE is that it is sustained by a loss of normal inhibitory mechanisms of neuronal activity. However, the early process leading to the initiation of SE is not well understood. Here, we show that, as seen in electroencephalograms, SE induced by the muscarinic agonist pilocarpine in mice is preceded by a specific increase in the gamma wave, and genetic ablation of canonical transient receptor potential channel (TRPC) 7 significantly reduces this pilocarpine-induced increase of gamma wave activity, preventing the occurrence of SE. At the cellular level, TRPC7 plays a critical role in the generation of spontaneous epileptiform burst firing in cornu ammonis (CA) 3 pyramidal neurons in brain slices. At the synaptic level, TRPC7 plays a significant role in the long-term potentiation at the CA3 recurrent collateral synapses and Schaffer collateral-CA1 synapses, but not at the mossy fiber-CA3 synapses. Taken together, our data suggest that epileptiform burst firing generated in the CA3 region by activity-dependent enhancement of recurrent collateral synapses may be an early event in the initiation process of SE and that TRPC7 plays a critical role in this cellular event. Our findings reveal that TRPC7 is intimately involved in the initiation of seizures both in vitro and in vivo. To our knowledge, this contribution to initiation of seizures is the first identified functional role for the TRPC7 ion channel.

The TRPC family of TRP channels: roles inferred (mostly) from knockout mice and relationship to ORAI proteins.

Aside from entering into cells through voltage gated Ca channels and Na/Ca exchangers in those cells that express these proteins, for all cells be they excitable or non-excitable, Ca(2+) enters through channels that are activated downstream of phosphoinositide mobilization (activation of phospholipase C, PLC) and through channels that are activated secondary to depletion of internal stores. Depletion of internal stores activates plasma membrane channels known as ORAIs. Activation of PLCs activates the canonical class of transient receptor potential channels (TRPCs), and, because this activation also causes depletion of Ca(2+) stores, also ORAI based channels. Whereas the activation of ORAI is a well-accepted phenomenon, it appears that TRPC channels also participate in Ca(2+) entry triggered by store depletion with or without participation of ORAI molecules. Regardless of molecular makeup of TRPC containing channels, a plethora of studies have shown TRPCs to be important both in physiologic systems as well as in pathophysiologic phenomena. Particularly important in defining roles of TRPCs, have been studies with mice with targeted disruption of their genes, i.e., with TRPC KO mice. In this chapter we first focus on TRPCs as regulators of body functions in health and disease, and then focus on the possible make-up of the channels of which they participate. A hypothesis is set forth, whereby ORAI dimers are proposed to be regulatory subunits of tetrameric TRPC channels and serve as structural units that form ORAI channels either as dimers of dimers or trimers of dimers.

Classical transient receptor potential 1 and 6 contribute to hypoxic pulmonary hypertension through differential regulation of pulmonary vascular functions.

Hypoxic pulmonary hypertension is characterized by increased vascular tone, altered vasoreactivity, and vascular remodeling, which are associated with alterations in Ca(2+) homeostasis in pulmonary arterial smooth muscle cells. We have previously shown that classical transient receptor potential 1 and 6 (TRPC1 and TRPC6) are upregulated in pulmonary arteries (PAs) of chronic hypoxic rats, but it is unclear whether these channels are essential for the development of pulmonary hypertension. Here we found that pulmonary hypertension was suppressed in TRPC1 and TRPC6 knockout (Trpc1(-/-) and Trpc6(-/-)) mice compared with wild-type after exposure to 10% O(2) for 1 and 3 weeks. Muscularization of pulmonary microvessels was inhibited, but rarefaction was unaltered in hypoxic Trpc1(-/-) and Trpc6(-/-) mice. Small PAs of normoxic wild-type mice exhibited vasomotor tone, which was significantly enhanced by chronic hypoxia. Similar vasomotor tone was found in normoxic Trpc1(-/-) PAs, but the hypoxia-induced enhancement was blunted. In contrast, there was minimal vascular tone in normoxic Trpc6(-/-) PAs, but the hypoxia-enhanced tone was preserved. Chronic hypoxia caused significant increase in serotonin-induced vasoconstriction; the augmented vasoreactivity was attenuated in Trpc1(-/-) and eliminated in Trpc6(-/-) PAs. Moreover, the effects of 3-week hypoxia on pulmonary arterial pressure, right ventricular hypertrophy, and muscularization of microvessels were further suppressed in TRPC1-TRPC6 double-knockout mice. Our results, therefore, provide clear evidence that TRPC1 and TRPC6 participate differentially in various pathophysiological processes, and that the presence of TRPC1 and TRPC6 is essential for the full development of hypoxic pulmonary hypertension in the mouse model.

Canonical transient receptor potential 3 channels regulate mitochondrial calcium uptake.

Mitochondrial Ca(2+) homeostasis is fundamental to regulation of mitochondrial membrane potential, ATP production, and cellular Ca(2+) homeostasis. It has been known for decades that isolated mitochondria can take up Ca(2+) from the extramitochondrial solution, but the molecular identity of the Ca(2+) channels involved in this action is largely unknown. Here, we show that a fraction of canonical transient receptor potential 3 (TRPC3) channels is localized to mitochondria, a significant fraction of mitochondrial Ca(2+) uptake that relies on extramitochondrial Ca(2+) concentration is TRPC3-dependent, and the up- and down-regulation of TRPC3 expression in the cell influences the mitochondrial membrane potential. Our findings suggest that TRPC3 channels contribute to mitochondrial Ca(2+) uptake. We anticipate our observations may provide insights into the mechanisms of mitochondrial Ca(2+) uptake and advance understanding of the physiological role of TRPC3.

A TRPC1 protein-dependent pathway regulates osteoclast formation and function.

Ca(2+) signaling is essential for bone homeostasis and skeletal development. Here, we show that the transient receptor potential canonical 1 (TRPC1) channel and the inhibitor of MyoD family, I-mfa, function antagonistically in the regulation of osteoclastogenesis. I-mfa null mice have an osteopenic phenotype characterized by increased osteoclast numbers and surface, which are normalized in mice lacking both Trpc1 and I-mfa. In vitro differentiation of pre-osteoclasts derived from I-mfa-deficient mice leads to an increased number of mature osteoclasts and higher bone resorption per osteoclast. These parameters return to normal levels in osteoclasts derived from double mutant mice. Consistently, whole cell currents activated in response to the depletion of intracellular Ca(2+) stores are larger in pre-osteoclasts derived from I-mfa knock-out mice compared with currents in wild type mice and normalized in cells derived from double mutant mice, suggesting a cell-autonomous effect of I-mfa on TRPC1 in these cells. A new splice variant of TRPC1 (TRPC1ε) was identified in early pre-osteoclasts. Heterologous expression of TRPC1ε in HEK293 cells revealed that it is unique among all known TRPC1 isoforms in its ability to amplify the activity of the Ca(2+) release-activated Ca(2+) (CRAC) channel, mediating store-operated currents. TRPC1ε physically interacts with Orai1, the pore-forming subunit of the CRAC channel, and I-mfa is recruited to the TRPC1ε-Orai1 complex through TRPC1ε suppressing CRAC channel activity. We propose that the positive and negative modulation of the CRAC channel by TRPC1ε and I-mfa, respectively, fine-tunes the dynamic range of the CRAC channel regulating osteoclastogenesis.

Do canonical transient receptor potential channels mediate cholinergic excitation of cortical pyramidal neurons?

Activation of M1-type muscarinic acetylcholine receptors excites neocortical pyramidal neurons, in part by gating a nonselective cation conductance that produces calcium-dependent 'afterdepolarizing potentials' (ADPs) following short trains of action potentials. Although the identity of the cation conductance mediating the ADP is not known, previous work has implicated canonical transient receptor potential (TRPC) channels, specifically the TRPC5 and TRPC6 subtypes. Using pharmacological and genetic approaches, we tested the role of TRPC channels in generating cholinergic ADPs in layer 5 pyramidal neurons in the mouse medial prefrontal cortex (mPFC). A variety of compounds that block TRPC channels, including 2-aminoethoxydiphenyl borate, flufenamic acid, lanthanum, SKF-96365, and Pyr-3, had little, if any, impact on cholinergic ADPs. Similarly, genetic deletion of several TRPC subunits, including TPRC1, TRPC5, and TRPC6 (single knockouts), or both TRPC5 and TRPC6 together (double knockout), failed to reduce the amplitude of cholinergic ADPs. These data suggest that TRPC5 and TRPC6 subunits are not required for cholinergic excitation of layer 5 pyramidal neurons in the mouse mPFC and that the focus of future work should be expanded to test the involvement of other potential ionic effectors.

The in vivo regulation of heart rate in the murine sinoatrial node by stimulatory and inhibitory heterotrimeric G proteins.

Reciprocal physiological modulation of heart rate is controlled by the sympathetic and parasympathetic systems acting on the sinoatrial (SA) node. However, there is little direct in vivo work examining the role of stimulatory and inhibitory G protein signaling in the SA node. Thus, we designed a study to examine the role of the stimulatory (Gαs) and inhibitory G protein (Gαi2) in in vivo heart rate regulation in the SA node in the mouse. We studied mice with conditional deletion of Gαs and Gαi2 in the conduction system using cre-loxP technology. We crossed mice in which cre recombinase expression was driven by a tamoxifen-inducible conduction system-specific construct with "Gαs floxed" and "Gαi2 floxed" mice. We studied the heart rate responses of adult mice compared with littermate controls by using radiotelemetry before and after administration of tamoxifen. The mice with conditional deletion of Gαs and Gαi2 had a loss of diurnal variation and were bradycardic or tachycardic, respectively, in the daytime. In mice with conditional deletion of Gαs, there was a selective loss of low-frequency power, while with deletion of Gαi2, there was a loss of high-frequency power in power spectral analysis of heart rate variability. There was no evidence of pathological arrhythmia. Pharmacological modulation of heart rate by isoprenaline was impaired in the Gαs mice, but a muscarinic agonist was still able to slow the heart rate in Gαi2 mice. We conclude that Gαs- and Gαi2-mediated signaling in the sinoatrial node is important in the reciprocal regulation of heart rate through the autonomic nervous system.

Knockout of the Trpc1 gene reveals that TRPC1 can promote recovery from anaphylaxis by negatively regulating mast cell TNF-α production.

Antigen-mediated mast cell (MC) degranulation is the critical early event in the induction of allergic reactions. Transient receptor potential channels (TRPC), particularly TRPC1, are thought to contribute to such MC activation. To explore the contribution of TRPC1 in MC-driven allergic reactions, we examined antigen-mediated anaphylaxis in Trpc1⁻/⁻ and WT mice, and TRPC1 involvement in the activation of MCs derived from the bone marrow (BMMCs) of these mice. In vivo, we observed a similar induction of passive systemic anaphylaxis in the Trpc1⁻/⁻ mice compared to WT controls. Nevertheless, there was delayed recovery from this response in Trpc1⁻/⁻ mice. Furthermore, contrary to expectations, Trpc1⁻/⁻ BMMCs responded to antigen with enhanced calcium signaling but with little defect in degranulation or associated signaling. In contrast, antigen-mediated production of TNF-α, and other cytokines, was enhanced in the Trpc1⁻/⁻ BMMCs, as were calcium-dependent events required for these responses. Additionally, circulating levels of TNF-α in response to antigen were preferentially elevated in the Trpc1⁻/⁻ mice, and administration of an anti-TNF-α antibody blocked the delay in recovery from anaphylaxis in these mice. These data thus provide evidence that, in this model, TRPC1 promotes recovery from the anaphylactic response by repressing antigen-mediated TNF-α release from MCs.

Bisphenol A delays the perinatal chloride shift in cortical neurons by epigenetic effects on the Kcc2 promoter.

Bisphenol A (BPA) is a ubiquitous compound that is emerging as a possible toxicant during embryonic development. BPA has been shown to epigenetically affect the developing nervous system, but the molecular mechanisms are not clear. Here we demonstrate that BPA exposure in culture led to delay in the perinatal chloride shift caused by significant decrease in potassium chloride cotransporter 2 (Kcc2) mRNA expression in developing rat, mouse, and human cortical neurons. Neuronal chloride increased correspondingly. Treatment with epigenetic compounds decitabine and trichostatin A rescued the BPA effects as did knockdown of histone deacetylase 1 and combined knockdown histone deacetylase 1 and 2. Furthermore, BPA evoked increase in tangential interneuron migration and increased chloride in migrating neurons. Interestingly, BPA exerted its effect in a sexually dimorphic manner, with a more accentuated effect in females than males. By chromatin immunoprecipitation, we found a significant increase in binding of methyl-CpG binding protein 2 to the "cytosine-phosphate-guanine shores" of the Kcc2 promoter, and decrease in binding of acetylated histone H3K9 surrounding the transcriptional start site. Methyl-CpG binding protein 2-expressing neurons were more abundant resulting from BPA exposure. The sexually dimorphic effect of BPA on Kcc2 expression was also demonstrated in cortical neurons cultured from the offspring of BPA-fed mouse dams. In these neurons and in cortical slices, decitabine was found to rescue the effect of BPA on Kcc2 expression. Overall, our results indicate that BPA can disrupt Kcc2 gene expression through epigenetic mechanisms. Beyond increase in basic understanding, our findings have relevance for identifying unique neurodevelopmental toxicity mechanisms of BPA, which could possibly play a role in pathogenesis of human neurodevelopmental disorders.

Canonical transient receptor channel 5 (TRPC5) and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms.

Seizures are the manifestation of highly synchronized burst firing of a large population of cortical neurons. Epileptiform bursts with an underlying plateau potential in neurons are a cellular correlate of seizures. Emerging evidence suggests that the plateau potential is mediated by neuronal canonical transient receptor potential (TRPC) channels composed of members of the TRPC1/4/5 subgroup. We previously showed that TRPC1/4 double-knockout (DKO) mice lack epileptiform bursting in lateral septal neurons and exhibit reduced seizure-induced neuronal cell death, but surprisingly have unaltered pilocarpine-induced seizures. Here, we report that TRPC5 knockout (KO) mice exhibit both significantly reduced seizures and minimal seizure-induced neuronal cell death in the hippocampus. Interestingly, epileptiform bursting induced by agonists for metabotropic glutamate receptors in the hippocampal CA1 area is unaltered in TRPC5 KO mice, but is abolished in TRPC1 KO and TRPC1/4 DKO mice. In contrast, long-term potentiation is greatly reduced in TRPC5 KO mice, but is normal in TRPC1 KO and TRPC1/4 DKO mice. The distinct changes from these knockouts suggest that TRPC5 and TRPC1/4 contribute to seizure and excitotoxicity by distinct cellular mechanisms. Furthermore, the reduced seizure and excitotoxicity and normal spatial learning exhibited in TRPC5 KO mice suggest that TRPC5 is a promising novel molecular target for new therapy.