Ca2+ entry via TRPC1 is essential for cellular differentiation and modulates secretion via the SNARE complex.

Properties of adipocytes, including differentiation and adipokine secretion, are crucial factors in obesity-associated metabolic syndrome. Here, we provide evidence that Ca2+ influx in primary adipocytes, especially upon Ca2+ store depletion, plays an important role in adipocyte differentiation, functionality and subsequently metabolic regulation. The endogenous Ca2+ entry channel in both subcutaneous and visceral adipocytes was found to be dependent on TRPC1-STIM1, and blocking Ca2+ entry with SKF96365 or using TRPC1-/- knockdown adipocytes inhibited adipocyte differentiation. Additionally, TRPC1-/- mice have decreased organ weight, but increased adipose deposition and reduced serum adiponectin and leptin concentrations, without affecting total adipokine expression. Mechanistically, TRPC1-mediated Ca2+ entry regulated SNARE complex formation, and agonist-mediated secretion of adipokine-loaded vesicles was inhibited in TRPC1-/- adipose. These results suggest an unequivocal role of TRPC1 in adipocyte differentiation and adiponectin secretion, and that loss of TRPC1 disturbs metabolic homeostasis.This article has an associated First Person interview with the first author of the paper.

Sigma1 Receptor Inhibits TRPC1-Mediated Ca2+ Entry That Promotes Dopaminergic Cell Death.

Regulation of Ca2+ homeostasis is essential for neuronal function and its survival. Recent data suggest that TRPC1 function as the endogenous store-mediated Ca2+ entry channel in dopaminergic cells, and loss of TRPC1 function leads to neurodegeneration; however, its regulation is not fully identified. Here we provide evidence that the sigma 1 receptor contributes to the loss of dopaminergic cells by blocking TRPC1-mediated Ca2+ entry. Importantly, downregulation of sigma 1 receptor expression significantly decreased neurotoxin-induced loss of dopaminergic cells as measured by MTT assays and caspase activity was also inhibited. Importantly, sigma 1 receptor inhibited TRPC1-mediated Ca2+ entry and silencing of sigma 1 receptor significantly restored store-dependent Ca2+ influx. Although co-immunoprecipitation failed to show an interaction between the TRPC1 and sigma 1 receptor, store depletion promoted a decrease in the sigma 1 receptor-STIM1 association. Neurotoxin-induced loss of Ca2+ entry was significantly restored in cells that had decreased sigma 1 receptor expression. Furthermore, TRPC1 or STIM1 silencing inhibited store-mediated Ca2+ entry, which was further increased upon the downregulation of the sigma 1 receptor expression. TRPC1 silencing prevented the increased neuroprotection and caspase activity observed upon the downregulation of sigma 1 receptor. Finally, sigma 1 receptor activation also significantly decreased TRPC1-mediated Ca2+ entry and lead to an increase in neurodegeneration. In contrast, addition of sigma 1 receptor antagonist prevented neurotoxin-induced neurodegeneration and facilitated TRPC1-mediated Ca2+ influx. Together these results suggest that the sigma 1 receptor is involved in the inhibition of TRPC1- mediated Ca2+ entry, which leads to the degeneration in the dopaminergic cells, and prevention of sigma 1 receptor function could protect neuronal cell death as observed in Parkinson's disease.

Using Tweetchats to Build Community Awareness and Advocacy around Alzheimer's Disease for Latinos.

U.S. Latinos are 1.5 times more likely to develop Alzheimer's disease (AD) than non-Latino Whites. To raise awareness of and action around this rising public health issue, Salud America!, a national Latino health advocacy network, organized three #SaludTues tweetchats on Twitter between 2018 and 2020. For the three Alzheimer's tweetchats ─Aug. 14, 2018, June 6, 2019, and Oct. 6, 2020─Salud America! partnered with global groups that advocate for AD solutions in Latino and other communities. We analyzed the three tweetchats' #Saludtues hashtag usage, participant demographics, and other metrics using Symplur analytics software. For the first tweetchat in 2018, there were 579 tweets with a total of 3.89 million impressions; the second tweetchat in 2019 had a bigger impact with 704 tweets with 5.72 million impressions; the third tweetchat had the biggest impact with 932 tweets and 6.62 million impressions. Most tweetchat participants were from states with large Latino populations, and most tweets indicated positive sentiment related to increasing awareness of solutions to AD issues among Latinos. The three Alzheimer's-focused #SaludTues tweetchats particularly served as unique testing grounds for the fast dissemination and increasingly exposed many people to the issue of AD and the need to advocate for the Latino community.

Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Alterations in Ca2+ homeostasis affect neuronal survival. However, the identity of Ca2+ channels and the mechanisms underlying neurotoxin-induced neuronal degeneration are not well understood. In this study, the dopaminergic neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridium ions (MPP+)/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which mimic Parkinson's disease (PD), induced neuronal degeneration by decreasing store-mediated Ca2+ entry. The function of the transient receptor potential canonical (TRPC)-1 channel was decreased upon exposure to the neurotoxins, followed by a decrease in TRPC1 expression. Similar to neurotoxins, samples from patients with PD exhibited attenuated TRPC1 expression, which was accompanied by a decrease in autophagic markers and a subsequent increase in apoptosis markers. Furthermore, exposure to neurotoxins attenuated PKC phosphorylation, decreased expression of autophagic markers, and increased apoptosis in SHSY-5Y neuroblastoma cells, which was again dependent on TRPC1. Prolonged neurotoxin treatment attenuated the binding of NF-κB to the TRPC1 promoter, which resulted in a decrease in TRPC1 expression, thereby attenuating autophagy and activating cell death. Restoration of TRPC1 expression rescued the effects of the dopaminergic neurotoxins in neuroblastoma cells by increasing Ca2+ entry, restoring NF-κB activity, and promoting autophagy. Overall, these results suggest that dopaminergic neurotoxins initially decreased Ca2+ entry, which inhibited the binding of NF-κB to the TRPC1 promoter, thereby inhibiting TRPC1 expression and resulting in cell death by preventing autophagy.-Sukumaran, P., Sun, Y., Antonson, N., Singh, B. B. Dopaminergic neurotoxins induce cell death by attenuating NF-κB-mediated regulation of TRPC1 expression and autophagy.

Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Neutrophil extracellular trap (NET) formation constitutes an important extracellular antimicrobial function of neutrophils that plays a protective role in bacterial pneumonia. Formation of reactive oxygen species (ROS) such as highly diffusible hydrogen peroxide (H2O2) is a hallmark of oxidative stress during inflammatory lung conditions including pneumonia. However, the impact of exogenous ROS on NET formation and the signaling pathway involved in the process is not completely understood. Here we demonstrate that the ROS-sensing, nonselective, calcium-permeable channel transient receptor potential melastatin 2 (TRPM2) is required for NET formation in response to exogenous H2O2. This TRPM2-dependent H2O2-mediated NET formation involved components of autophagy and activation of AMPK and p38 MAPK, but not PI3K and AKT. Primary neutrophils from Trpm2-/- mice fail to activate this pathway with a block in NET release and a concomitant decrease in their antimicrobial capacity. Consequently, Trpm2-/- mice were highly susceptible to pneumonic infection with Klebsiella pneumoniae owing to an impaired NET formation and high bacterial burden despite increased neutrophil infiltration in their lungs. These results identify a key role of TRPM2 in regulating NET formation by exogenous ROS via AMPK/p38 activation and autophagy machinery, as well as a protective antimicrobial role of TRPM2 in pneumonic bacterial infection.-Tripathi, J. K., Sharma, A., Sukumaran, P., Sun, Y., Mishra, B. B., Singh, B. B., Sharma, J. Oxidant sensor cation channel TRPM2 regulates neutrophil extracellular trap formation and protects against pneumoseptic bacterial infection.

Inhibition of L-Type Ca2+ Channels by TRPC1-STIM1 Complex Is Essential for the Protection of Dopaminergic Neurons.

Loss of dopaminergic (DA) neurons leads to Parkinson's disease; however, the mechanism(s) for the vulnerability of DA neurons is(are) not fully understood. We demonstrate that TRPC1 regulates the L-type Ca2+ channel that contributes to the rhythmic activity of adult DA neurons in the substantia nigra region. Store depletion that activates TRPC1, via STIM1, inhibits the frequency and amplitude of the rhythmic activity in DA neurons of wild-type, but not in TRPC1-/-, mice. Similarly, TRPC1-/- substantia nigra neurons showed increased L-type Ca2+ currents, decreased stimulation-dependent STIM1-Cav1.3 interaction, and decreased DA neurons. L-type Ca2+ currents and the open channel probability of Cav1.3 channels were also reduced upon TRPC1 activation, whereas increased Cav1.3 currents were observed upon STIM1 or TRPC1 silencing. Increased interaction between Cav1.3-TRPC1-STIM1 was observed upon store depletion and the loss of either TRPC1 or STIM1 led to DA cell death, which was prevented by inhibiting L-type Ca2+ channels. Neurotoxins that mimic Parkinson's disease increased Cav1.3 function, decreased TRPC1 expression, inhibited Tg-mediated STIM1-Cav1.3 interaction, and induced caspase activation. Importantly, restoration of TRPC1 expression not only inhibited Cav1.3 function but increased cell survival. Together, we provide evidence that TRPC1 suppresses Cav1.3 activity by providing an STIM1-based scaffold, which is essential for DA neuron survival.SIGNIFICANCE STATEMENT Ca2+ entry serves critical cellular functions in virtually every cell type, and appropriate regulation of Ca2+ in neurons is essential for proper function. In Parkinson's disease, DA neurons are specifically degenerated, but the mechanism is not known. Unlike other neurons, DA neurons depend on Cav1.3 channels for their rhythmic activity. Our studies show that, in normal conditions, the pacemaking activity in DA neurons is inhibited by the TRPC1-STIM1 complex. Neurotoxins that mimic Parkinson's disease target TRPC1 expression, which leads to an abnormal increase in Cav1.3 activity, thereby causing degeneration of DA neurons. These findings link TRPC1 to Cav1.3 regulation and provide important indications about how disrupting Ca2+ balance could have a direct implication in the treatment of Parkinson's patients.

The TRPC1 Ca2+-permeable channel inhibits exercise-induced protection against high-fat diet-induced obesity and type II diabetes.

The transient receptor potential canonical channel-1 (TRPC1) is a Ca2+-permeable channel found in key metabolic organs and tissues, including the hypothalamus, adipose tissue, and skeletal muscle. Loss of TRPC1 may alter the regulation of cellular energy metabolism resulting in insulin resistance thereby leading to diabetes. Exercise reduces insulin resistance, but it is not known whether TRPC1 is involved in exercise-induced insulin sensitivity. The role of TRPC1 in adiposity and obesity-associated metabolic diseases has not yet been determined. Our results show that TRPC1 functions as a major Ca2+ entry channel in adipocytes. We have also shown that fat mass and fasting glucose concentrations were lower in TRPC1 KO mice that were fed a high-fat (HF) (45% fat) diet and exercised as compared with WT mice fed a HF diet and exercised. Adipocyte numbers were decreased in both subcutaneous and visceral adipose tissue of TRPC1 KO mice fed a HF diet and exercised. Finally, autophagy markers were decreased and apoptosis markers increased in TRPC1 KO mice fed a HF diet and exercised. Overall, these findings suggest that TRPC1 plays an important role in the regulation of adiposity via autophagy and apoptosis and that TRPC1 inhibits the positive effect of exercise on type II diabetes risk under a HF diet-induced obesity environment.

TGFß-induced epithelial-to-mesenchymal transition in prostate cancer cells is mediated via TRPM7 expression.

Growth factors, such as the transforming growth factor beta (TGFß), play an important role in promoting metastasis of prostate cancer, thus understanding how TGFß could induce prostate cancer cell migration may enable us to develop targeted strategies for treatment of advanced metastatic prostate cancer. To more clearly define the mechanism(s) involved in prostate cancer cell migration, we undertook a series of studies utilizing non-malignant prostate epithelial cells RWPE1 and prostate cancer DU145 and PC3 cells. Our studies show that increased cell migration was observed in prostate cancer cells, which was mediated through epithelial-to-mesenchymal transition (EMT). Importantly, addition of Mg2+ , but not Ca2+ , increased cell migration. Furthermore, TRPM7 expression, which functions as an Mg2+ influx channel, was also increased in prostate cancer cells. Inhibition of TRPM7 currents by 2-APB, significantly blocked cell migration in both DU145 and PC3 cells. Addition of growth factor TGFß showed a further increase in cell migration, which was again blocked by the addition of 2-APB. Importantly, TGFß addition also significantly increased TRPM7 expression and function, and silencing of TRPM7 negated TGFß-induced cell migration along with a decrease in EMT markers showing loss of cell adhesion. Furthermore, resveratrol, which decreases prostate cancer cell migration, inhibited TRPM7 expression and function including TGFß-induced cell migration and activation of TRPM7 function. Together, these results suggest that Mg2+ influx via TRPM7 promotes cell migration by inducing EMT in prostate cancer cells and resveratrol negatively modulates TRPM7 function thereby inhibiting prostate cancer metastasis.

TRPC Channels and Parkinson's Disease.

Parkinson's disease (PD) is a common neurodegenerative disorder, which involves degeneration of dopaminergic neurons that are present in the substantia nigra pars compacta (SNpc) region. Many factors have been identified that could lead to Parkinson's disease; however, almost all of them are directly or indirectly dependent on Ca2+ signaling. Importantly, though disturbances in Ca2+ homeostasis have been implicated in Parkinson's disease and other neuronal diseases, the identity of the calcium channel remains elusive. Members of the transient receptor potential canonical (TRPC) channel family have been identified as a new class of Ca2+ channels, and it could be anticipated that these channels could play important roles in neurodegenerative diseases, especially in PD. Thus, in this chapter we have entirely focused on TRPC channels and elucidated its role in PD.

Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression: IMPLICATIONS FOR THYROID CANCER CELL MIGRATION AND PROLIFERATION.

The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells.

Resveratrol activates autophagic cell death in prostate cancer cells via downregulation of STIM1 and the mTOR pathway.

Resveratrol (RSV), a natural polyphenol, has been suggested to induce cell cycle arrest and activate apoptosis-mediated cell death in several cancer cells, including prostate cancer. However, several molecular mechanisms have been proposed on its chemopreventive action, the precise mechanisms by which RSV exerts its anti-proliferative effect in androgen-independent prostate cancer cells remain questionable. In the present study, we show that RSV activates autophagic cell death in PC3 and DU145 cells, which was dependent on stromal interaction molecule 1 (STIM1) expression. RSV treatment decreases STIM1 expression in a time-dependent manner and attenuates STIM1 association with TRPC1 and Orai1. Furthermore, RSV treatment also decreases ER calcium storage and store operated calcium entry (SOCE), which induces endoplasmic reticulum (ER) stress, thereby, activating AMPK and inhibiting the AKT/mTOR pathway. Similarly, inhibition of SOCE by SKF-96365 decreases the survival and proliferation of PC3 and DU145 cells and inhibits AKT/mTOR pathway and induces autophagic cell death. Importantly, SOCE inhibition and subsequent autophagic cell death caused by RSV was reversed by STIM1 overexpression. STIM1 overexpression restored SOCE, prevents the loss of mTOR phosphorylation and decreased the expression of CHOP and LC3A in PC3 cells. Taken together, for the first time, our results revealed that RSV induces autophagy-mediated cell death in PC3 and DU145 cells through regulation of SOCE mechanisms, including downregulating STIM1 expression and trigger ER stress by depleting ER calcium pool.

Cholesterol-induced activation of TRPM7 regulates cell proliferation, migration, and viability of human prostate cells.

Cholesterol has been shown to promote cell proliferation/migration in many cells; however the mechanism(s) have not yet been fully identified. Here we demonstrate that cholesterol increases Ca(2+) entry via the TRPM7 channel, which promoted proliferation of prostate cells by inducing the activation of the AKT and/or the ERK pathway. Additionally, cholesterol mediated Ca(2+) entry induced calpain activity that showed a decrease in E-cadherin expression, which together could lead to migration of prostate cancer cells. An overexpression of TRPM7 significantly facilitated cholesterol dependent Ca(2+) entry, cell proliferation and tumor growth. Whereas, TRPM7 silencing or inhibition of cholesterol synthesis by statin showed a significant decrease in cholesterol-mediated activation of TRPM7, cell proliferation, and migration of prostate cancer cells. Consistent with these results, statin intake was inversely correlated with prostate cancer patients and increase in TRPM7 expression was observed in samples obtained from prostate cancer patients. Altogether, we provide evidence that cholesterol-mediated activation of TRPM7 is important for prostate cancer and have identified that TRPM7 could be essential for initiation and/or progression of prostate cancer.

Canonical transient receptor potential channel 2 (TRPC2): old name-new games. Importance in regulating of rat thyroid cell physiology.

In addition to the TSH-cyclic AMP signalling pathway, calcium signalling is of crucial importance in thyroid cells. Although the importance of calcium signalling has been thoroughly investigated for several decades, the nature of the calcium channels involved in signalling is unknown. In a recent series of investigations using the well-studied rat thyroid FRTL-5 cell line, we showed that these cells exclusively express the transient receptor potential canonical 2 (TRPC2) channel. Our results suggested that the TRPC2 channel is of significant importance in regulating thyroid cell function. These investigations were the first to show that thyroid cells express a member of the TRPC family of ion channels. In this review, we will describe the importance of the TRPC2 channel in regulating TSH receptor expression, thyroglobulin maturation, intracellular calcium and iodide homeostasis and that the channel also regulates thyroid cell proliferation.

Inhibition of store-operated calcium entry in microglia by helminth factors: implications for immune suppression in neurocysticercosis.

BACKGROUND: Neurocysticercosis (NCC) is a disease of the central nervous system (CNS) caused by the cestode Taenia solium. The infection exhibits a long asymptomatic phase, typically lasting 3 to 5 years, before the onset of the symptomatic phase. The severity of the symptoms is thought to be associated with the intensity of the inflammatory response elicited by the degenerating parasite. In contrast, the asymptomatic phase shows an absence of brain inflammation, which is presumably due to immunosuppressive effects of the live parasites. However, the host factors and/or pathways involved in inhibiting inflammation remain largely unknown. Recently, using an animal model of NCC in which mice were intracranially inoculated with a related helminth parasite, Mesocestoides corti, we reported that Toll-like receptor (TLR)-associated signaling contributes to the development of the inflammatory response. As microglia shape the initial innate immune response in the CNS, we hypothesized that the negative regulation of a TLR-induced inflammatory pathway in microglia may be a novel helminth-associated immunosuppressive mechanism in NCC. METHODS AND RESULTS: Here we report that helminth soluble factors (HSFs) from Mesocestoides corti inhibited TLR ligation-induced production of inflammatory cytokines in primary microglia. This was correlated with an inhibition of TLR-initiated upregulation of both phosphorylation and acetylation of the nuclear factor κB (NF-κB) p65 subunit, as well as phosphorylation of JNK and ERK1/2. As Ca2+ influx due to store-operated Ca2+ entry (SOCE) has been implicated in induction of downstream signaling, we tested the inhibitory effect of HSFs on agonist-induced Ca2+ influx and specific Ca2+ channel activation. We discovered that HSFs abolished the lipopolysaccharide (LPS)- or thapsigargin (Tg)-induced increase in intracellular Ca2+ accumulation by blocking the ER store release and SOCE. Moreover, electrophysiological recordings demonstrated HSF-mediated inhibition of LPS- or Tg-induced SOCE currents through both TRPC1 and ORAI1 Ca2+ channels on plasma membrane. This was correlated with a decrease in the TRPC1-STIM1 and ORAI1-STIM1 clustering at the plasma membrane that is essential for sustained Ca2+ entry through these channels. CONCLUSION: Inhibition of TRPC1 and ORAI1 Ca2+ channel-mediated activation of NF-κB and MAPK pathways in microglia is likely a novel helminth-induced immunosuppressive mechanism that controls initiation of inflammatory response in the CNS.

The Relationship Between Pre-Pandemic Comorbidities With SARS-CoV-2 Infections and Hospitalizations in Black Versus White Military Service Members: A Retrospective Study.

INTRODUCTION: Comorbidities such as hypertension, diabetes mellitus, asthma, and cardiovascular conditions have been reported to worsen the clinical progression of coronavirus disease 2019 (COVID-19) and related hospitalizations. Furthermore, the COVID-19 pandemic has disproportionately affected the historically marginalized groups, i.e., Black, Hispanic, and Asian individuals have substantially higher rates of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection, COVID-19 hospitalization, and death compared to White individuals. Despite these findings in civilian populations, the impact of comorbidities and race in SARS-CoV-2 infection and COVID-19 hospitalizations in military populations is unknown. We evaluated the relationship of pre-selected pre-pandemic comorbidities and race with SARS-CoV-2 infections and COVID-19 hospitalizations in U.S. military service members (SMs). MATERIALS AND METHODS: We conducted a systematic review of Military Health System beneficiaries' records by accessing the Defense Medical Epidemiological Database. Our inclusion criteria were being an active duty SM and having at least one pre-COVID-19 pandemic comorbidity. Retired as well as uninfected healthy active duty SMs and beneficiaries were excluded from the study. A total population of 1.334 million active duty SM records was drawn from Defense Medical Epidemiological Database. The data were stratified, by race (primary outcome), as well as gender, age, and military service branches (secondary outcomes). RESULTS: We found higher trends in SARS-CoV-2 infection and COVID-19 hospitalization rates in Black compared to White SMs. This seamless inequality was also seen in other viral infections affecting SMs including human immunodeficiency virus and viral hepatitis. We hypothesized this disparity to some extent be associated with the presence of pre-pandemic comorbidities that is affecting this military subpopulation. Supporting our hypothesis, we found trends toward the higher pre-pandemic prevalence of diabetes mellitus, asthma, hypertension, and ischemic heart disease, in Black compared to White military SMs, especially in Black older male adults. CONCLUSION: Our results highlight the role of pre-pandemic comorbidities and race likely enhancing the frequency of SARS-CoV-2 infections and COVID-19 hospitalizations in military SMs. These preliminary findings underscore the need for future retrospective studies using additional Military Health System data bases reporting data on this military subpopulation, especially in the setting of future pathogens outbreaks or pandemics affecting military populations.

Canonical transient receptor potential channel 2 (TRPC2) as a major regulator of calcium homeostasis in rat thyroid FRTL-5 cells: importance of protein kinase C δ (PKCδ) and stromal interaction molecule 2 (STIM2).

Mammalian non-selective transient receptor potential cation channels (TRPCs) are important in the regulation of cellular calcium homeostasis. In thyroid cells, including rat thyroid FRTL-5 cells, calcium regulates a multitude of processes. RT-PCR screening of FRTL-5 cells revealed the presence of TRPC2 channels only. Knockdown of TRPC2 using shRNA (shTRPC2) resulted in decreased ATP-evoked calcium peak amplitude and inward current. In calcium-free buffer, there was no difference in the ATP-evoked calcium peak amplitude between control cells and shTRPC2 cells. Store-operated calcium entry was indistinguishable between the two cell lines. Basal calcium entry was enhanced in shTRPC2 cells, whereas the level of PKCß1 and PKCδ, the activity of sarco/endoplasmic reticulum Ca(2+)-ATPase, and the calcium content in the endoplasmic reticulum were decreased. Stromal interaction molecule (STIM) 2, but not STIM1, was arranged in puncta in resting shTRPC2 cells but not in control cells. Phosphorylation site Orai1 S27A/S30A mutant and non-functional Orai1 R91W attenuated basal calcium entry in shTRPC2 cells. Knockdown of PKCδ with siRNA increased STIM2 punctum formation and enhanced basal calcium entry but decreased sarco/endoplasmic reticulum Ca(2+)-ATPase activity in wild-type cells. Transfection of a truncated, non-conducting mutant of TRPC2 evoked similar results. Thus, TRPC2 functions as a major regulator of calcium homeostasis in rat thyroid cells.

Functional coupling of TRPC2 cation channels and the calcium-activated anion channels in rat thyroid cells: implications for iodide homeostasis.

The initial step in a synthesis of thyroid hormones is the uptake of iodide from the circulation. Iodide (I(-)) is transported into thyroid cells via a Na(+)/I(-) symporter (NIS), which is electrogenic and thus sensitive to alterations in membrane potential (V(m)). I(-) is then released to the lumen of thyroid follicles where the hormones are synthesised and stored. The mechanisms of I(-) release to follicle lumen are poorly characterised. Our whole-cell voltage clamp recordings revealed the presence of a Ca(2+) activated Cl(-) current (CaCC) in Fisher rat thyroid cell line 5 (FRTL-5). Transcripts of anoctamin 1 (ANO1) and anoctamin 10 (ANO10), putative molecular constituents of CaCC, were detected. The anion channels underlying CaCC are highly permeable to I(-). Both niflumic acid (NFA) and 2-aminoethyl diphenylborinate (2-APB), antagonists of CaCC and transient receptor potential channels, respectively, inhibited CaCC. Canonical transient receptor potential channel 2 (TRPC2) is the only TRPC member present in FRTL-5 cells. The activation rate of CaCC was markedly slower in shTRPC2 knock-down cells, indicating that Ca(2+) entry via TRPC2 contributes to CaCC activation. The uptake of iodide was enhanced and the resting V(m) was more depolarised in TRPC2 knock-down cells. We suggest that the interplay between TRPC2 and ANO1 may have dual effects on iodide transport, modulating I(-) release via ANO channels and I(-) uptake via the V(m) sensitive NIS.

Targeting alarmin release reverses Sjogren's syndrome phenotype by revitalizing Ca2+ signalling.

BACKGROUND: Primary Sjogren's syndrome (pSS) is a systemic autoimmune disease that is embodied by the loss of salivary gland function and immune cell infiltration, but the mechanism(s) are still unknown. The aim of this study was to understand the mechanisms and identify key factors that leads to the development and progression of pSS. METHODS: Immunohistochemistry staining, FACS analysis and cytokine levels were used to detect immune cells infiltration and activation in salivary glands. RNA sequencing was performed to identify the molecular mechanisms involved in the development of pSS. The function assays include in vivo saliva collection along with calcium imaging and electrophysiology on isolated salivary gland cells in mice models of pSS. Western blotting, real-time PCR, alarmin release, and immunohistochemistry was performed to identify the channels involved in salivary function in pSS. RESULTS: We provide evidence that loss of Ca2+ signaling precedes a decrease in saliva secretion and/or immune cell infiltration in IL14α, a mouse model for pSS. We also showed that Ca2+ homeostasis was mediated by transient receptor potential canonical-1 (TRPC1) channels and inhibition of TRPC1, resulting in the loss of salivary acinar cells, which promoted alarmin release essential for immune cell infiltration/release of pro-inflammatory cytokines. In addition, both IL14α and samples from human pSS patients showed a decrease in TRPC1 expression and increased acinar cell death. Finally, paquinimod treatment in IL14α restored Ca2+ homeostasis that inhibited alarmin release thereby reverting the pSS phenotype. CONCLUSIONS: These results indicate that loss of Ca2+ signaling is one of the initial factors, which induces loss of salivary gland function along with immune infiltration that exaggerates pSS. Importantly, restoration of Ca2+ signaling upon paquinimod treatment reversed the pSS phenotype thereby inhibiting the progressive development of pSS.

Direct Outreach in Bars and Clubs to Enroll Cigarette Smokers in Mobile Cessation Services: Exploratory Study.

BACKGROUND: Cigarette smoking and alcohol use are well known to be concomitant behaviors, but there is a lack of studies related to recruitment of smokers for mobile cessation services at places where alcohol is consumed, such as bars and clubs. Adapting recruitment strategies to expand the reach of cessation programs to where tobacco users are located may help decrease the health-equity gap in tobacco control by improving reach and enrollment of underserved smokers residing in low-income and rural areas who are not reached by traditional cessation services. OBJECTIVE: The purpose of this exploratory study was to assess the feasibility of direct outreach in bars, clubs, and restaurants to recruit smokers to Quitxt, our mobile smoking cessation service. Quitxt is delivered through SMS text messaging or Facebook Messenger. METHODS: We collaborated with an advertising agency to conduct in-person recruitment of young adult smokers aged 18-29 years, focusing on urban and rural Spanish-speaking Latino participants, as well as English-speaking rural White and African American participants. Street team members were recruited and trained in a 4-hour session, including a brief introduction to the public health impacts of cigarette smoking and the aims of the project. The street teams made direct, face-to-face contact with smokers in and near smoking areas at 25 bars, clubs, and other venues frequented by young smokers in urban San Antonio and nearby rural areas. RESULTS: The 3923 interactions by the street teams produced 335 (8.5%) program enrollments. Most participants were English speakers with a mean age of 29.2 (SD 10.6) years and smoked a mean of 8.5 (SD 6.2) cigarettes per day. Among users who responded to questions on gender and ethnicity, 66% (70/106) were women and 56% (60/107) were Hispanic/Latino. Among users ready to make a quit attempt, 22% (17/77) reported 1 tobacco-free day and 16% (10/62) reported maintaining cessation to achieve 1 week without smoking. The response rate to later follow-up questions was low. CONCLUSIONS: Direct outreach in bars and clubs is a useful method for connecting young adult cigarette smokers with mobile cessation services. However, further research is needed to learn more about how mobile services can influence long-term smoking cessation among those recruited through direct outreach, as well as to test the use of incentives in obtaining more useful response rates.

TRPC2: of mice but not men.

Relatively little is known in regard to the physiological significance of TRPC2 and its regulation or interaction with other calcium regulating signalling molecules. In rodents, however, the importance of TRPC2 is indisputable. In mice, transcripts for TRPC2 have been found in testis, sperm, in neurons in the vomeronasal organ, and both in the dorsal root ganglion and in the brain. In rats, TRPC2 is thought to be expressed exclusively in the vomeronasal organ. In mice, TRPC2 is of importance in regulating both sexual and social behaviour. In sperm, TRPC2 is of importance in the acrosome reaction. This review will summarize the known physiological effects of TRPC2 channels, and the regulation of the function of the channel. In addition, some new preliminary data on the role of TRPC2 in rat thyroid cells will be presented.