Increase In Intracellular Calcium Research Articles

Mesoporous nanosilica: A thromboprotective nanomaterial for biomedical applications

Nanosilica is widely employed in various biomedical applications because of their tailorable physiochemical properties and excellent biocompatibility. In the present study, we have evaluated interaction of nanosilica with important coagulation components, such as platelets, a highly sensitive cell found in the blood, and coagulation proteins. Mesoporous silica nanoparticles (MSNs) were prepared using sol-gel process and characterized by FESEM and TEM to find out the size and shape of the particles. Different platelet functional parameters including platelet adhesion, aggregation, activation, secretion, clot formation and clot retraction-based studies have been carried out to investigate the impact of synthesized nanosilica on the blood coagulation system. Besides, ROS generation and increase in intracellular calcium was also monitored as they play a pivotal role in regulating platelet functions. The complete detailed study revealed that MSNs neither has stimulatory action towards platelets nor do they show any effective interaction with coagulation proteins.

UVR Promotes Keratinocyte Phagocytosis and Skin Pigmentation Through TRPA1 Channels.

PurposeUltraviolet radiation (UVR) enhances skin pigmentation, which involves the production of melanin by melanocytes and subsequent transfer to keratinocytes. In the epidermis, keratinocyte phagocytosis plays a pivotal role in the process of melanosome transfer to protect DNA of epidermal cells against damage from UVR. Previous research suggested that transient receptor potential channels ankyrin 1 (TRPA1) was required for UVR-induced early melanin synthesis in melanocytes. Currently, there is no evidence that supports the detailed mechanism of TRPA1 for UVR-induced phagocytosis by keratinocytes. Here, we investigated the effect and the possible mechanisms of TRPA1 on keratinocyte phagocytosis and skin pigmentation after UVR exposure.MethodsFlow cytometry was applied to investigate the effect of TRPA1 on intracellular calcium concentration ([Ca2+]ic) and fluorescent microspheres uptake was carried out to analyze phagocytosis in HaCaT cells (human immortalized keratinocytes). Western blotting was applied to measure the protein expression of calcium/calmodulin-dependent protein kinase II (CaMKII), phosphorylated CaMKII and β-catenin after UVA/UVB exposure. Masson-Fontana staining was applied to observe the effect of XAV-939 (decreasing the expression of β-catenin) on UVB-induced skin pigmentation in guinea pigs.ResultsTRPA1 channels activated by UVR increased the [ca2+]ic and phosphorylation of CaMKII in HaCaT cells. The UVR-induced phagocytosis was regulated by TRPA1 in HaCaT cells. TRPA1 promoted the protein expression of β-catenin after UVR exposure in HaCaT cells. XAV-939, inhibiting β-catenin expression, decreased the UVB-induced skin pigmentation on in vivo guinea pig models.ConclusionTaken together, TRPA1 activated by UVR led to the increase of intracellular calcium, which promoted the phosphorylation of CaMKII, enhancing keratinocyte phagocytosis. Moreover, TRPA1 regulated the protein expression of β-catenin to exert a lightening effect on skin pigmentation. Our findings suggest that TRPA1 may be a potential therapeutic target for UVR-induced skin pigmentary diseases.

A Novel Role of the TRPM4 Ion Channel in Exocytosis.

Under physiological conditions, the widely expressed calcium-activated TRPM4 channel conducts sodium into cells. This sodium influx depolarizes the plasma membrane and reduces the driving force for calcium entry. The aberrant expression or function of TRPM4 has been reported in various diseases, including different types of cancer. TRPM4 is mainly localized in the plasma membrane, but it is also found in intracellular vesicles, which can undergo exocytosis. In this study, we show that calcium-induced exocytosis in the colorectal cancer cell line HCT116 is dependent on TRPM4. In addition, the findings from some studies of prostate cancer cell lines suggest a more general role of TRPM4 in calcium-induced exocytosis in cancer cells. Furthermore, calcium-induced exocytosis depends on TRPM4 ion conductivity. Additionally, an increase in intracellular calcium results in the delivery of TRPM4 to the plasma membrane. This process also depends on TRPM4 ion conductivity. TRPM4-dependent exocytosis and the delivery of TRPM4 to the plasma membrane are mediated by SNARE proteins. Finally, we provide evidence that calcium-induced exocytosis depends on TRPM4 ion conductivity, not within the plasma membrane, but rather in TRPM4-containing vesicles.

Olive Oil Phenols Prevent Mercury-Induced Phosphatidylserine Exposure and Morphological Changes in Human Erythrocytes Regardless of Their Different Scavenging Activity.

Phosphatidylserine (PS) translocation to the external membrane leaflet represents a key mechanism in the pathophysiology of human erythrocytes (RBC) acting as an “eat me” signal for the removal of aged/stressed cells. Loss of physiological membrane asymmetry, however, can lead to adverse effects on the cardiovascular system, activating a prothrombotic activity. The data presented indicate that structurally related olive oil phenols prevent cell alterations induced in intact human RBC exposed to HgCl2 (5–40 µM) or Ca2+ ionophore (5 µM), as measured by hallmarks including PS exposure, reactive oxygen species generation, glutathione depletion and microvesicles formation. The protective effect is observed in a concentration range of 1–30 µM, hydroxytyrosol being the most effective; its in vivo metabolite homovanillic alcohol still retains the biological activity of its dietary precursor. Significant protection is also exerted by tyrosol, in spite of its weak scavenging activity, indicating that additional mechanisms are involved in the protective effect. When RBC alterations are mediated by an increase in intracellular calcium, the protective effect is observed at higher concentrations, indicating that the selected phenols mainly act on Ca2+-independent mechanisms, identified as protection of glutathione depletion. Our findings strengthen the nutritional relevance of olive oil bioactive compounds in the claimed health-promoting effects of the Mediterranean Diet.

In Vitro Effect of Mitochondria-Targeted Triphenylphosphonium-Based Compounds (Honokiol, Lonidamine, and Atovaquone) on the Platelet Function and Cytotoxic Activity.

Introduction: Obtaining triphenylphosphonium salts derived from anticancer compounds to inhibit mitochondrial metabolism is of major interest due to their pivotal role in reactive oxygen species (ROS) production, calcium homeostasis, apoptosis, and cell proliferation. However, the use of this type of antitumor compound presents a risk of bleeding since the platelet activation is especially dependent on the mitochondrial function. In this study, we evaluated the in vitro effect of three triphenylphosphonium-based compounds, honokiol (HNK), lonidamine (LDN), and atovaquone (ATO), on the platelet function linked to the triphenylphosphonium cation by a lineal 10-carbon alkyl chain and also the decyltriphenylphosphonium salt (decylphos). Methods: Platelets obtained by phlebotomy from healthy donors were exposed in vitro to different concentrations (0.1–10 μM) of the three compounds; cellular viability, exposure of phosphatidylserine, the mitochondrial membrane potential (∆Ψm), intracellular calcium release, and intracellular ROS generation were measured. Platelet activation and aggregation were induced by agonists (adenosine diphosphate, thrombin receptor-activating peptide-6, convulxin, or phorbol-12-myristate-13-acetate) and were evaluated by flow cytometry and light transmission, respectively. Results: The three compounds showed a slight cytotoxic effect from 1 μM, and this was concomitant with a decrease in ∆Ψm and intracellular calcium increase. Only ATO produced a modest but significant increase in intra-platelet ROS. Also, the three compounds increased the exposure to phosphatidylserine in platelets expressed in platelets positive for annexin V. None of the compounds had an inhibitory effect on the aggregation or activation markers of platelets stimulated with three different agonists. Similar results were obtained with decylphos. Conclusion: Triphenylphosphonium derivatives showed slight platelet toxicity below 1 μM, probably associated with their effect on ∆Ψm and exposure to phosphatidylserine, but no significant effect on platelet activation and aggregation, making them an antitumoral alternative with a low risk of bleeding. However, future assays on animal models and human trials are required to evaluate if their effects with a low risk for hemostasis are replicated in vivo.

HuR Mediates Thermogenic Metabolism in Brown Adipose Tissue Through Control of Sarco‐Endoplasmic Calcium Cycling

Obesity is classified by the World Health Organization as one of eight principle causes of preventable chronic disease and is directly associated with decreased life expectancy due to increased risk of associated comorbidities. Brown adipose tissue (BAT) is a thermogenically active, endocrine organ regulating both glucose homeostasis and heat production, and the ability to induce BAT‐mediated energy expending represents a potential therapeutic avenue to reduce obesity.We recently showed that the RNA‐binding protein HuR (Human antigen R) plays a functional role in BAT‐mediated thermogenic metabolism. RNA sequencing analysis from mice with an adipocyte‐specific deletion of HuR (Adipo‐HuR‐/‐) revealed a BAT‐specific decrease in the expression of many genes responsible for sarco‐endoplasmic reticulum (SER) calcium cycling, including SERCA1a, RyR1, RyR2, and additional RyR complex proteins such as triadin, junctin, and CASR. Canonical thermogenesis in BAT occurs through uncoupling protein 1 (UCP1). However, several UCP1‐independent energy expending cycles, including intracellular calcium transport, have also been shown to regulate thermogenesis. SER calcium cycling has recently been established as a thermogenic mechanism in beige adipose tissue, but its functional contributions to thermogenesis in BAT have not yet been fully elucidated.To determine if the HuR‐dependent changes in SER calcium cycles manifest as a functional change in SER loading/cycling in BAT, we show that decreased cytosolic intracellular calcium levels in BAT isolated from Adipo‐HuR‐/‐mice. Additionally, we show that HuR is activated acutely downstream of beta‐adrenergic stimulation in BAT, and deletion of HuR blunts adrenergic‐induced increases in intracellular calcium. Furthermore, HuR directly binds RyR2 mRNA in brown adipocytes and regulates RyR2 expression through RNA stabilization. Taken together, this growing body of work suggests an important role for HuR in mediating thermogenesis through modulation of SER calcium cycling in brown adipose tissue.

Effects of RTI‐567 on the Neuropeptide S System

Anxiety and post‐traumatic stress have increasingly become more prominent disorders and options for treatment remain limited. One of the most prescribed therapeutics are benzodiazepines. Unfortunately, these anxiolytics have negative side effects and are not as effective in the long term. The recently discovered Neuropeptide S (NPS) system may lead to an alternative, potentially improved, line of therapeutics. This system consists of a known endogenous neuropeptide, Neuropeptide S and a G‐protein‐coupled receptor termed the Neuropeptide S receptor (NPSR). Activation of this receptor leads to an increase in intracellular calcium and a mobilization of cAMP. This is associated with behavioral phenotypes such as anxiolysis, enhanced memory consolidation, and hyperlocomotion. The NPSR is highly expressed in the amygdala linking the NPS system with fear and anxiety. Previously, as part of our ongoing drug discovery program, a biased agonist (RTI‐263) was identified that preferentially enhances the calcium signaling pathway over the cAMP pathway following NPSR binding. This biased agonist also reduced hyperlocomotion or arousal observed with NPS, while maintaining the anxiolytic component. In the present study we test another compound (RTI‐567) using classic anxiolytic, locomotor, and memory testing paradigms such as the light dark box, open field, and inhibitory avoidance. Our overall goal is to identify novel synthetic agonists with reduced side effects and improved long‐term efficacy.

Traumatic-noise-induced hair cell death and hearing loss is mediated by activation of CaMKKβ.

The Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) are serine/threonine-directed protein kinases that are activated following increases in intracellular calcium, playing a critical role in neuronal signaling. Inner-ear-trauma-induced calcium overload in sensory hair cells has been well documented in the pathogenesis of traumatic noise-induced hair cell death and hearing loss, but there are no established pharmaceutical therapies available due to a lack of specific therapeutic targets. In this study, we investigated the activation of CaMKKβ in the inner ear after traumatic noise exposure and assessed the prevention of noise-induced hearing loss (NIHL) with RNA silencing. Treatment with short hairpin RNA of CaMKKβ (shCaMKKβ) via adeno-associated virus transduction significantly knocked down CaMKKβ expression in the inner ear. Knockdown of CaMKKβ significantly attenuated noise-induced hair cell loss and hearing loss (NIHL). Additionally, pretreatment with naked CaMKKβ small interfering RNA (siCaMKKβ) attenuated noise-induced losses of inner hair cell synapses and OHCs and NIHL. Furthermore, traumatic noise exposure activates CaMKKβ in OHCs as demonstrated by immunolabeling for p-CaMKI. CaMKKβ mRNA assessed by fluorescence in-situ hybridization and immunolabeling for CaMKKβ in OHCs also increased after the exposure. Finally, pretreatment with siCaMKKβ diminished noise-induced activation of AMPKα in OHCs. These findings demonstrate that traumatic-noise-induced OHC loss and hearing loss occur primarily via activation of CaMKKβ. Targeting CaMKKβ is a key strategy for prevention of noise-induced hearing loss. Furthermore, our data suggest that noise-induced activation of AMPKα in OHCs occurs via the CaMKKβ pathway.

Modulation of Metabolic Hormone Signaling via a Circadian Hormone and Biogenic Amine in Drosophila melanogaster.

In insects, adipokinetic hormone is the primary hormone responsible for the mobilization of stored energy. While a growing body of evidence has solidified the role of adipokinetic hormone (AKH) in modulating the physiological and behavioral responses to metabolic stress, little is known about the upstream endocrine circuit that directly regulates AKH release. We evaluated the AKH-producing cell (APC) transcriptome to identify potential regulatory elements controlling APC activity and found that a number of receptors showed consistent expression levels, including all known dopamine receptors and the pigment dispersing factor receptor (PDFR). We tested the consequences of targeted genetic knockdown and found that APC limited expression of RNAi elements corresponding to each dopamine receptor and caused a significant reduction in survival under starvation. In contrast, PDFR knockdown significantly extended lifespan under starvation, whereas expression of a tethered PDF in APCs resulted in significantly shorter lifespans. These manipulations caused various changes in locomotor activity under starvation. We used live-cell imaging to evaluate the acute effects of the ligands for these receptors on APC activation. Dopamine application led to a transient increase in intracellular calcium in a trehalose-dependent manner. Furthermore, coapplication of dopamine and ecdysone led to a complete loss of this response, suggesting that these two hormones act antagonistically. We also found that PDF application led to an increase in cAMP in APCs and that this response was dependent on expression of the PDFR in APCs. Together, these results suggest a complex circuit in which multiple hormones act on APCs to modulate metabolic state.

Repurposing Probenecid for the Treatment of Heart Failure (Re-Prosper-HF): a study protocol for a randomized placebo-controlled clinical trial

BackgroundImproving contractility in heart failure with reduced ejection fraction (HFrEF) has resurfaced as a potential treatment goal. Inotropic therapy is now better understood through its underlying mechanism as opposed to the observed effect of increasing contractility. Calcitropes are a subgroup of inotropes that largely depend on the stimulation of adenylyl cyclase to transform ATP into cyclic adenosine monophosphate (cAMP). At least two clinically relevant calcitropes—istaroxime and probenecid—improve contractility through an increase in systolic intracellular calcium without activating cAMP production.Probenecid, which has been safely used clinically for decades in non-cardiac conditions, has recently been identified as an agonist of the transient receptor potential vanilloid 2 channel. Translational studies have shown that it improves calcium cycling and contractility without activating noxious pathways associated with cAMP-dependent calcitropes and can improve cardiac function in patients with HFrEF.MethodsThe Re-Prosper-HF study (Repurposing Probenecid for the Treatment of Heart Failure with Reduced Ejection Fraction) is a three-site double-blinded randomized-controlled trial that will test the hypothesis that probenecid can improve cardiac function in patients with HFrEF. Up to 120 patients will be randomized in this double-blind, placebo-controlled study that will assess whether oral probenecid administered at 1 g orally twice per day for 180 days in patients with NYHA II-III HFrEF improves systolic function (aim 1), functional status (aim 2), and self-reported health status (aim 3).DiscussionFindings from this study will provide data informing its use for improving symptomatology in patients with HFrEF as well as exploratory data for outcomes such as hospital admission rates.Trial tegistrationThe Re-Prosper HF Study (Re-Prosper HF) is registered on ClinicalTrials.gov with the identifier as NCT04551222. Registered on 9 September 2020.

Trimethylamine N-Oxide (TMAO) Impairs Purinergic Induced Intracellular Calcium Increase and Nitric Oxide Release in Endothelial Cells.

Trimethylamine N-oxide (TMAO) is a diet derived compound directly introduced through foodstuff, or endogenously synthesized from its precursors, primarily choline, L-carnitine, and ergothioneine. New evidence outlines high TMAO plasma concentrations in patients with overt cardiovascular disease, but its direct role in pathological development is still controversial. The purpose of the study was to evaluate the role of TMAO in affecting key intracellular factors involved in endothelial dysfunction development, such as reactive oxygen species, mitochondrial health, calcium balance, and nitric oxide release using bovine aortic endothelial cells (BAE-1). Cell viability and oxidative stress indicators were monitored after acute and prolonged TMAO treatment. The role of TMAO in interfering with the physiological purinergic vasodilatory mechanism after ATP stimulation was defined through measurements of the rise of intracellular calcium, nitric oxide release, and eNOS phosphorylation at Ser1179 (eNOSSer1179). TMAO was not cytotoxic for BAE-1 and it did not induce the rise of reactive oxygen species and impairment of mitochondrial membrane potential, either in the basal condition or in the presence of a stressor. In contrast, TMAO modified the purinergic response affecting intracellular ATP-induced calcium increase, nitric oxide release, and eNOSSer1179. Results obtained suggest a possible implication of TMAO in impairing the endothelial-dependent vasodilatory mechanism.

Mitochondrial calpain-5 truncates caspase-4 during endoplasmic reticulum stress

Calpains are cysteine proteases activated in response to intracellular calcium signaling. Activated calpains regulate various cellular functions by degrading substrate molecules in a site-specific manner. Although most calpains are localized in the cytosol, we previously reported that calpain-5 exists in the mitochondria. The mitochondrial calpain-5 is activated during endoplasmic reticulum (ER) stress. However, the substrate of calpain-5, as well as the physiological significance of calpain-5 activation, has not yet been elucidated. In the present study, we treated HeLa cells with A23187, tunicamycin, or hydrogen peroxide to induce intracellular calcium increase, resulting in cell death. The cells treated with A23187 or tunicamycin exhibited the activation of calpain-5 and truncation of caspase-4. The truncation of caspase-4 was inhibited by the repression of calpain-5 expression with the appropriate siRNA. Additionally, both calpain-5 and caspase-4 were observed in the mitochondria. Our study is the first to demonstrate that the activation of mitochondrial calpain-5 triggers the truncation of caspase-4, suggesting that mitochondrial calpain-5 regulates the downstream pathway of caspase-4, including cell death and the inflammatory cascade. The results of the present study provide new insights into ER-stress-related diseases such as Alzheimer's disease and cancer. These perspectives allow us to propose new therapeutic strategies such as the development of inhibitors or activators of calpain-5, which may be useful in the development of treatment for ER-stress-related diseases.

Advanced heart failure: guideline-directed medical therapy, diuretics, inotropes, and palliative care.

Heart failure (HF) is a major cause of mortality, hospitalizations, and reduced quality of life and a major burden for the healthcare system. The number of patients that progress to an advanced stage of HF is growing. Only a limited proportion of these patients can undergo heart transplantation or mechanical circulatory support. The purpose of this review is to summarize medical management of patients with advanced HF. First, evidence‐based oral treatment must be implemented although it is often not tolerated. New therapeutic options may soon become possible for these patients. The second goal is to lessen the symptomatic burden through both decongestion and haemodynamic improvement. Some new treatments acting on cardiac function may fulfil both these needs. Inotropic agents acting through an increase in intracellular calcium have often increased risk of death. However, in the recent Global Approach to Lowering Adverse Cardiac Outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial, omecamtiv mecarbil was safe and effective in the reduction of the primary outcome of cardiovascular death or HF event compared with placebo (hazard ratio, 0.92; 95% confidence interval, 0.86–0.99; P = 0.03) and its effects were larger in those patients with more severe left ventricular dysfunction. Patients with severe HF who received omecamtiv mecarbil experienced a significant treatment benefit, whereas patients without severe HF did not (P = 0.005 for interaction). Lastly, clinicians should take care of the end of life with an appropriate multidisciplinary approach. Medical treatment of advanced HF therefore remains a major challenge and a wide open area for further research.

Sulforaphane protects myocardium from ischemia-reperfusion injury by regulating CaMKIIN2 and CaMKIIδ

Myocardial ischemia/reperfusion (I/R) injury poses a significant threat to human health. High level of reactive oxygen species (ROS) and calcium overload are the foremost causes of myocardial damage in I/R. Sulforaphane (SFN) is known for its promising antioxidant effect. Whether or not SFN has myocardial protective effect against I/R is largely unknown. This study aimed to investigate if SFN can protect myocardium from I/R injury. We found that mice or cells pre-treated with SFN showed improved cardiac functions and cell survival. SFN treatment inhibited the production of inflammatory cytokines and the increase of intracellular calcium induced by hypoxia-reperfusion (H/R), while mitochondria membrane potential was effectively maintained. Transcriptome analysis showed that CaMKIIδ expression was down-regulated by SFN treatment in I/R myocardium, while CaMKIIN2, the inhibitor of CaMKII, was upregulated. Knockdown of CaMKIIN2 not only led to increased level of total CaMKIIδ and the phosphorylated CaMKIIδ but also blocked the pro-survival effect of SFN for H/R cells. Moreover, CaMKIIN2 overexpression was sufficient to suppress CaMKIIδ activation and improve cell survival under H/R. Taken together, this study demonstrated that SFN exerts cardioprotective effect toward I/R injury through upregulating CaMKIIN2 and down-regulating CaMKIIδ.

Cyclic mechanical stretch regulates the AMPK/Egr1 pathway in tenocytes via Ca2+-mediated mechanosensing

ABSTRACT Purpose Mechanical stimuli are essential for the maintenance of tendon tissue homeostasis. The study aims to elucidate the mechanobiological mechanisms underlying the maintenance of tenocyte homeostasis by cyclic mechanical stretch under high-glucose (HG) condition. Materials and methods Primary tenocytes were isolated from rat Achilles tendon and 2D-cultured under HG condition. The in vitro effects of a single bout, 2-h cyclic biaxial stretch session (1 Hz, 8%) on primary rat tenocytes were explored through Flexcell system. Cell viability, tenogenic gene expression, intracellular calcium concentration, focal adhesion kinase (FAK) expression, and signaling pathway activation were analyzed in tenocytes with or without mechanical stretch. Results Mechanical stretch increased tenocyte proliferation and upregulated early growth response protein 1 (Egr1) expression. An increase in intracellular calcium was observed after 30 min of stretching. Mechanical stretch phosphorylated FAK, calmodulin-dependent protein kinase kinase 2 (CaMKK2), and 5’ adenosine monophosphate-activated protein kinase (AMPK) in a time-dependent manner, and these effects were abrogated after blocking intracellular calcium. Inhibition of FAK, CaMKK2, and AMPK downregulated the expression of Egr1. In addition, mechanical stretch reinforced cytoskeletal organization via calcium (Ca2+)/FAK signaling. Conclusions Our study demonstrated that mechanical stretch-induced calcium influx activated CaMKK2/AMPK signaling and FAK-cytoskeleton reorganization, thereby promoting the expression of Egr1, which may help maintain tendon cell characteristics and homeostasis in the context of diabetic tendinopathy.

Lon upregulation contributes to cisplatin resistance by triggering NCLX-mediated mitochondrial Ca2+ release in cancer cells

Mitochondria are the major organelles in sensing cellular stress and inducing the response for cell survival. Mitochondrial Lon has been identified as an important stress protein involved in regulating proliferation, metastasis, and apoptosis in cancer cells. However, the mechanism of retrograde signaling by Lon on mitochondrial DNA (mtDNA) damage remains to be elucidated. Here we report the role of Lon in the response to cisplatin-induced mtDNA damage and oxidative stress, which confers cancer cells on cisplatin resistance via modulating calcium levels in mitochondria and cytosol. First, we found that cisplatin treatment on oral cancer cells caused oxidative damage of mtDNA and induced Lon expression. Lon overexpression in cancer cells decreased while Lon knockdown sensitized the cytotoxicity towards cisplatin treatment. We further identified that cisplatin-induced Lon activates the PYK2-SRC-STAT3 pathway to stimulate Bcl-2 and IL-6 expression, leading to the cytotoxicity resistance to cisplatin. Intriguingly, we found that activation of this pathway is through an increase of intracellular calcium (Ca2+) via NCLX, a mitochondrial Na+/Ca2+ exchanger. We then verified that NCLX expression is dependent on Lon levels; Lon interacts with and activates NCLX activity. NCLX inhibition increased the level of mitochondrial calcium and sensitized the cytotoxicity to cisplatin in vitro and in vivo. In summary, mitochondrial Lon-induced cisplatin resistance is mediated by calcium release into cytosol through NCLX, which activates calcium-dependent PYK2-SRC-STAT3-IL-6 pathway. Thus, our work uncovers the novel retrograde signaling by mitochondrial Lon on resistance to cisplatin-induced mtDNA stress, indicating the potential use of Lon and NCLX inhibitors for better clinical outcomes in chemoresistant cancer patients.

Acrosomal alkalinization occurs during human sperm capacitation.

Mammalian sperm capacitation is a prerequisite for successful fertilization. Capacitation involves biochemical and physiological modifications of sperm as they travel through the female reproductive tract. These modifications prepare the sperm to undergo the acrosome reaction (AR), an acrosome vesicle exocytosis that is necessary for gamete fusion. Capacitation requires an increase in both intracellular calcium ([Ca2+]i) and pH (pHi). Mouse sperm capacitation is accompanied by acrosomal alkalinization and artificial elevation of the acrosome pH (pHa) is sufficient to trigger the AR in mouse and human sperm, but it is unknown if pHa increases naturally during human sperm capacitation. We used single-cell imaging and image-based flow cytometry to evaluate pHa during capacitation and its regulation. We found that pHa progressively increases during capacitation. The V-ATPase, which immunolocalized to the acrosome and equatorial segment, is mainly responsible for the acidity of the acrosome. It is likely that the regulation of V-ATPase is at least in part responsible for the progressive increase in pHa during capacitation. Acrosome alkalinization was dependent on extracellular HCO3- and Ca2+. Inhibition of the HCO3--dependent adenylyl cyclase and protein kinase A induced significant pHa changes. Overall, alkalinization of the acrosome may be a key step in the path toward the AR.

Phosphodiesterases S-sulfhydration contributes to human skeletal muscle function.

The increase in intracellular calcium is influenced by cyclic nucleotides (cAMP and cGMP) content, which rating is governed by phosphodiesterases (PDEs) activity.Despite it has been demonstrated a beneficial effect of PDEs inhibitors in different pathological conditions involving SKM, not much is known on the role exerted by cAMP-cGMP/PDEs axis in human SKM contractility. Here, we show that Ssulfhydration of PDEs modulates human SKM contractility in physiological and pathological conditions. Having previously demonstrated that, in the rare human syndrome Malignant Hyperthermia (MH), there is an overproduction of hydrogen sulfide (H2S) within SKM contributing to hyper-contractility, here we have used MH negative diagnosed biopsies (MHN) as healthy SKM, and MH susceptible diagnosed biopsies (MHS) as a pathological model of SKM hypercontractility. The study has been performed on MHS and MHN human biopsies after diagnosis has been made and on primary SKM cells derived from both MHN and MHS biopsies. Our data demonstrate that in normal conditions PDEs are S-sulfhydrated in both quadriceps’ biopsies and primary SKM cells. This post translational modification (PTM) negatively regulates PDEs activity with consequent increase of both cAMP and cGMP levels. In hypercontractile biopsies, due to an excessive H2S content, there is an enhanced Ssulfhydration of PDEs that further increases cyclic nucleotides levels contributing to SKM hyper-contractility. Thus, the identification of a new endogenous PTM modulating PDEs activity represents an advancement in SKM physiopathology understanding.

Beyond the plasma membrane—how G proteins help direct protein translation

Activation of G protein coupled receptors (GPCRs) lead to a series of events that can result in changes in the cell such as growth, migration, and release of exocytotic vesicles. These events are mediated by the second messengers (i.e. cAMP, Ca2+, diacyglycerol) that are generated by G protein activity. For example, activation of Gαq by acetylcholine, dopamine or other neurotransmitters causes an increase in intracellular calcium that changes the properties of specific transcription factors to impact cell properties.

Low Intensity Electromagnetic Fields Act via Voltage-Gated Calcium Channel (VGCC) Activation to Cause Very Early Onset Alzheimer's Disease: 18 Distinct Types of Evidence.

Electronically generated electromagnetic fields (EMFs), including those used in wireless communication such as cell phones, Wi-Fi and smart meters, are coherent, producing very high electric and magnetic forces, which act on the voltage sensor of voltage-gated calcium channels to produce increases in intracellular calcium [Ca2+]i. The calcium hypothesis of Alzheimer’s disease (AD) has shown that each of the important AD-specific and nonspecific causal elements is produced by excessive [Ca2+]i. [Ca2+]i acts in AD via excessive calcium signaling and the peroxynitrite/oxidative stress/inflammation pathway, which are each elevated by EMFs.An apparent vicious cycle in AD involves amyloid-beta protein (Aβ) and [Ca2+]i. Three types of epidemiology suggest EMF causation of AD, including early onset AD. Extensive animal model studies show that low intensity EMFs cause neurodegeneration, including AD, with AD animals having elevated levels of Aβ, amyloid precursor protein and BACE1. Rats exposed to pulsed EMFs every day are reported to develop universal or near universal very early onset neurodegeneration, including AD; these findings are superficially similar to humans with digital dementia. EMFs producing modest increases in [Ca2+]i can also produce protective, therapeutic effects. The therapeutic pathway and peroxynitrite pathway inhibit each other. A summary of 18 different findings is provided, which collectively provide powerful evidence for EMF causation of AD. The author is concerned that smarter, more highly pulsed “smart” wireless communication may cause widespread very, very early onset AD in human populations.