Search Results (97)

Electrical restitution (ER) is a determinant of cardiac repolarization stability and can be measured as steady action potential (AP) duration (APD) at different pacing rates—the so-called dynamic restitution (ER_dyn) curve—or as APD changes after pre- or post-mature stimulations—the so-called standard restitution (ER_s1s2) curve. Short-term AP memory (M_s) has been described as the slope difference between the ER_dyn and ER_s1s2 curves, and represents the information stored in repolarization dynamics due to previous pacing conditions. Although previous studies have shown its dependence on ion currents and calcium cycling, a systematic picture of these features is lacking. By means of simulations with a human ventricular AP model, I show that APD restitution can be described under randomly changing pacing conditions (ER_rand) and M_s derived as the slope difference between ER_dyn and ER_rand. Thus measured, M_s values correlate with those measured using ER_s1s2. I investigate the effect on M_s of modulating the conductance of ion channels involved in AP repolarization, and of abolishing intracellular calcium transient. I show that M_s is chiefly determined by ER_dyn rather than ER_rand, and that interventions that shorten/prolong APD tend to decrease/increase M_s. Full article

Background/Objectives: Ellagic acid (EA) is a polyphenol found in several fruits and vegetables, including pomegranate, nuts and berries. It exhibits significant health benefits, mainly cardio- and vaso-protective; indeed, EA protects the myocardium against infarction and inhibits cardiac fibrosis. These beneficial effects may be, at least in part, promoted by calcium release from and uptake by the sarcoplasmic reticulum, which are crucial events for cardiac relaxation and contraction. Regardless, the exact mechanism is currently unclear. Methods: A deeper investigation of the role of EA in cardiac contractility and the underlying mechanism has been carried out by using an ex vivo model of isolated and perfused rat heart. Results and Discussion: EA perfusion (100 nM–10 µM) did not influence the coronary flow (CF), suggesting the absence of a vasoactivity, but significantly increased contractility parameters (LVDP and dP/dt). Interestingly, a more marked effect of EA on LVDP and dP/dt values was observed when it was perfused in the presence of AngII. Cyclopiazonic acid (CA) and red ruthenium (RR), specific antagonists of SERCA and RyRs, respectively, were used to explore the contribution of EA when the intracellular calcium handling was altered. In the presence of CA, EA, perfused at increasing concentrations, showed a very modest positive inotropism (significant only at 1 µM). Instead, RR, which significantly compromised all functional parameters, completely masked the effects of EA; furthermore, a marked reduction in CF and a dramatic impact on the positive inotropism occurred. Conclusions: These results demonstrate the positive inotropism of EA on isolated and perfused hearts and suggest that the RyRs may be a main target through which EA plays its effects, since inhibition with RR almost completely blocks the positive inotropism. Full article

Calcium (Ca²⁺) signaling is a fundamental regulatory mechanism controlling essential processes in the endothelium, vascular smooth muscle cells (VSMCs), and the extracellular matrix (ECM), including maintaining the endothelial barrier, modulation of vascular tone, and vascular remodeling. Cytosolic free Ca²⁺ concentration is tightly regulated by a balance between Ca²⁺ mobilization mechanisms, including Ca²⁺ release from the intracellular stores in the sarcoplasmic/endoplasmic reticulum and Ca²⁺ entry via voltage-dependent, transient-receptor potential, and store-operated Ca²⁺ channels, and Ca²⁺ elimination pathways including Ca²⁺ extrusion by the plasma membrane Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger and Ca²⁺ re-uptake by the sarco(endo)plasmic reticulum Ca²⁺-ATPase and the mitochondria. Some cell membranes/organelles are multifunctional and have both Ca²⁺ mobilization and Ca²⁺ removal pathways. Also, the individual Ca²⁺ handling pathways could be integrated to function in a regenerative, capacitative, cooperative, bidirectional, or reciprocal feed-forward or feed-back manner. Disruption of these pathways causes dysregulation of the Ca²⁺ signaling dynamics and leads to pathological cardiovascular conditions such as hypertension, coronary artery disease, atherosclerosis, and vascular calcification. In the endothelium, dysregulated Ca²⁺ signaling impairs nitric oxide production, reduces vasodilatory capacity, and increases vascular permeability. In VSMCs, Ca²⁺-dependent phosphorylation of the myosin light chain and Ca²⁺ sensitization by protein kinase-C (PKC) and Rho-kinase (ROCK) increase vascular tone and could lead to increased blood pressure and hypertension. Ca²⁺ activation of matrix metalloproteinases causes collagen/elastin imbalance and promotes vascular remodeling. Ca²⁺-dependent immune cell activation, leukocyte infiltration, and cholesterol accumulation by macrophages promote foam cell formation and atherosclerotic plaque progression. Chronic increases in VSMCs Ca²⁺ promote phenotypic switching to mesenchymal cells and osteogenic transformation and thereby accelerate vascular calcification and plaque instability. Emerging therapeutic strategies targeting these Ca²⁺-dependent mechanisms, including Ca²⁺ channel blockers and PKC and ROCK inhibitors, hold promise for restoring Ca²⁺ homeostasis and mitigating vascular disease progression. Full article

Mechanogated (MG) ion channels play a crucial role in mechano-transduction and immune cell regulation, yet their impact on blood cancers, particularly acute lymphoblastic leukemia (ALL), remains poorly understood. This study investigates the pharmacological effects of GsMTx4, an MG channel inhibitor, in human ALL cells both in vitro and in vivo. Unexpectedly, we found that GsMTx4 remarkably increased basal calcium (Ca²⁺) levels in ALL cells through constitutive Ca²⁺ entry and enhanced store-operated Ca²⁺ influx upon thapsigargin stimulation. This increase in basal Ca²⁺ signaling promoted ALL cell viability and proliferation in vitro. Notably, chelating intracellular Ca²⁺ with BAPTA-AM reduces GsMTx4-mediated leukemia cell viability and proliferation. However, in vivo, GsMTx4 decreases cytosolic Ca²⁺ levels in Nalm-6 GFP⁺ cells isolated from mouse blood, effectively countering leukemia progression and significantly extending survival in NSG mice transplanted with leukemia cells (median survival: GsMTx4 vs. control, 37.5 days vs. 29 days, p = 0.0414). Our results highlight the different properties of GsMTx4 activity in in vitro and in vivo models. They also emphasize that Ca²⁺ signaling is a key vulnerability in leukemia, where its precise modulation dictates disease progression. Thus, targeting Ca²⁺ channels could offer a novel therapeutic strategy for leukemia by exploiting Ca²⁺ homeostasis. Full article

Ryanodine receptors (RyRs) are large intracellular Ca²⁺ release channels primarily found in muscle and nerve cells and also present at low levels in pancreatic islet endocrine cells. This review examines the role of RyRs in islet cell function, focusing on calcium signaling and hormone secretion, while addressing the ongoing debate regarding their significance due to their limited expression. We explore conflicting experimental results and their potential causes, synthesizing current knowledge on RyR isoforms in islet cells, particularly in beta and delta cells. The review discusses how RyR-mediated calcium-induced calcium release enhances, rather than drives, glucose-stimulated insulin secretion. We examine the phosphorylation-dependent regulation of beta-cell RyRs, the concept of “leaky ryanodine receptors”, and the roles of RyRs in endoplasmic reticulum stress, apoptosis, store-operated calcium entry, and beta-cell electrical activity. The relationship between RyR dysfunction and the development of impaired insulin secretion in diabetes is assessed, noting their limited role in human diabetes pathogenesis given the disease’s polygenic nature. We highlight the established role of RyR-mediated CICR in the mechanism of action of common type 2 diabetes treatments, such as glucagon-like peptide-1, which enhances insulin secretion. By integrating findings from electrophysiological, molecular, and clinical studies, this review provides a balanced perspective on RyRs in islet cell physiology and pathology, emphasizing their significance in both normal insulin secretion and current diabetes therapies. Full article

Multiple Sclerosis (MS) is a chronic autoimmune disorder characterized by demyelination and neuronal damage in the central nervous system. Dysregulation of calcium homeostasis, particularly through the Store-Operated Calcium Entry-Associated Regulatory Factor (SARAF), has been implicated in MS pathogenesis. This study investigated SARAF, STIM1, and Orai1 expression patterns and their relationship to calcium homeostasis in 45 Bahraini MS patients and 45 matched healthy controls using ELISA and real-time PCR analyses. MS patients showed significantly elevated serum SARAF levels in both early (192.26 ± 47.00 pg/mL) and late MS stages (341.47 ± 96.19 pg/mL) compared to controls (129.82 ± 30.82 pg/mL; p < 0.001. SARAF expressions were markedly increased in MS patients (3.829 ± 0.04422 vs. 1 ± 0; p < 0.0001), while STIM1 (0.4324 ± 0.01471) and ORAI1 (0.2963 ± 0.02156) expressions were significantly reduced compared to the controls (p < 0.0001). Intracellular calcium levels were notably elevated in both early and late MS stages. These findings suggest that the progressive elevation of SARAF, coupled with altered STIM1 and ORAI1 expression, may serve as potential biomarkers for MS progression and represent promising therapeutic targets. Full article

Hepatocellular carcinoma (HCC), a highly aggressive liver malignancy, is often associated with disrupted calcium homeostasis. Store-operated calcium entry (SOCE), involving components such as STIM1, Orai1, and SARAF, plays a critical role in calcium signaling and cancer progression. While STIM1 and Orai1 have been extensively studied, SARAF’s role as a negative regulator of SOCE in HCC remains poorly understood. This preliminary study investigated SARAF’s effects on calcium homeostasis, proliferation, and migration in HepG2 liver cancer cells, providing initial evidence of its tumor-suppressive role. SARAF expression was modulated using siRNA knockdown and overexpression plasmids, with validation by qRT-PCR. Functional assays demonstrated that SARAF silencing increased proliferation by 50% and migration by 40% (p < 0.05), while SARAF overexpression reduced proliferation by 50% and migration by 45% (p < 0.01), highlighting its tumor-suppressive role. Intracellular calcium levels, elevated in HepG2 cells, were partially restored by SARAF overexpression, though SARAF silencing did not further disrupt calcium regulation. These findings suggest that SARAF negatively regulates proliferation and migration in HCC, potentially through its role in maintaining calcium homeostasis. SARAF represents a promising therapeutic target in HCC. Future studies should explore the downstream molecular mechanisms governing SARAF’s effects, investigate its role in other cancers, and assess its clinical potential for liver cancer therapy. Full article

Electrical stimulation of the skin has proven effective in pain management and antibacterial treatment, particularly in wound healing and counteracting the aging processes. The latter processes rely on epidermal cell migration, increased fibroblast proliferation, and upregulation of extracellular matrix protein expression. While an electrical field stimulates these processes, it is unclear how the electrical signal results in transcriptional control. Here, we postulate that the activation of voltage-gated channels, specifically voltage-gated potassium channels Kv1.3, is implicated in initiating the downstream signaling pathways that lead to increased collagen expression. We postulate that Kv1.3 and possibly calcium-activated potassium channel activity leads to the engagement of store-operated calcium channels and modulates the intracellular calcium ions distribution. In turn, changes in intracellular calcium concentration can activate calcium-generated transcriptional effectors. The Kv1.3 channel, identified via fluorescence imaging with ShK toxin (peptide), shows high-level expression in the human dermal fibroblast cell membrane. We also performed proliferation, collagen expression, and calcium imaging studies for variable electrical fields to help understand the link between the electrical stimulation, Kv1.3 channels, intracellular calcium concentration, and protein expression. Full article

α-Latrotoxin (αLTX) causes exhaustive release of neurotransmitters from nerve terminals in the absence of extracellular Ca²⁺ (Ca²⁺_e). To investigate the mechanisms underlying this effect, we loaded mouse neuromuscular junctions with BAPTA-AM. This membrane-permeable Ca²⁺-chelator demonstrates that Ca²⁺_e-independent effects of αLTX require an increase in cytosolic Ca²⁺ (Ca²⁺_cyt). We also show that thapsigargin, which depletes Ca²⁺ stores, induces neurotransmitter release, but inhibits the effect of αLTX. We then studied αLTX’s effects on Ca²⁺_cyt using neuroblastoma cells expressing signaling-capable or signaling-incapable variants of latrophilin-1, a G protein-coupled receptor of αLTX. Our results demonstrate that αLTX acts as a cation ionophore and a latrophilin agonist. In model cells at 0 Ca²⁺_e, αLTX forms membrane pores and allows the influx of Na⁺; this reverses the Na⁺-Ca²⁺ exchanger, leading to the release of stored Ca²⁺ and inhibition of its extrusion. Concurrently, αLTX stimulates latrophilin signaling, which depletes a Ca²⁺ store and induces transient opening of Ca²⁺ channels in the plasmalemma that are sensitive to inhibitors of store-operated Ca²⁺ entry. These results indicate that Ca²⁺ release from intracellular stores and that Ca²⁺ influx through latrophilin-activated store-operated Ca²⁺ channels contributes to αLTX actions and may be involved in physiological control of neurotransmitter release at nerve terminals. Full article

Mucociliary clearance, the ability of the respiratory tract to protect the integrity of the airways through the mechanical removal of potentially harmful substances, is of enormous importance during intensive care treatment. The present study aimed to evaluate the influence of clinically relevant inotropic agents on mucociliary clearance. The particle transport velocity (PTV) of isolated murine tracheae was measured as a surrogate for mucociliary clearance in the presence of dobutamine, epinephrine, and milrinone. Inhibitory substances were applied to elucidate the signal transduction cascades and the value and origin of calcium ions which provoke alterations in mucociliary clearance function. Dobutamine, epinephrine, and milrinone increased the PTV in a dose-dependent manner with half maximal effective concentrations of 75.7 nM, 87.0 nM, and 13.7 µM, respectively. After the depletion of intracellular calcium stores, no increase in PTV was observed after administering any of the three inotropic agents. While dobutamine and epinephrine activated β-adrenergic receptors, epinephrine used both the phospholipase C (PLC) and protein kinase A (PKA) pathway to promote the release of intracellular Ca²⁺. However, dobutamine primarily acted on the PKA pathway, having only a minor influence on the PLC pathway. The induced changes in PTV following milrinone administration required both the PKA and PLC pathway, although the PKA pathway was responsible for most of the induced changes. In conclusion, the common inotropic agents dobutamine, epinephrine, and milrinone increase murine PTV in a concentration-dependent manner and ultimately release Ca²⁺ from intracellular calcium stores, suggesting the function of changes in mucociliary clearance in the respiratory tract. Full article

Lectins are proteins that specifically recognize carbohydrates on cell membranes, triggering several cellular events such as apoptosis of cancer-transformed cells; however, the mechanisms of action remain incompletely understood. Our research group has reported that a concentrated fraction of Tepary bean lectins (Phaseolus acutifolius; TBLF) exhibits the concentration-dependent induction of apoptosis in colon cancer cells by caspase activation. It is well established that an increase in cytoplasmic calcium ([Ca²⁺]_i) initiates intracellular signals involved in processes such as exocytosis, gene transcription, apoptosis, cell cycle regulation, and muscle contraction, among others. Furthermore, dysregulated calcium signaling has been implicated in various diseases, including certain neurological disorders and cancer. In this study, we aim to demonstrate the effects of native TBLF lectins and a recombinant lectin (rTBL-1) on calcium mobility in breast cancer cells (MCF-7) and non-cancerous cells (MCF-12F). Both TBLF and rTBL-1 increased intracellular calcium concentrations and mobilized calcium from intracellular stores in a concentration-dependent manner; however, the two cell lines exhibited differential responses. While MCF-12F cells restored cytoplasmic calcium concentration, MCF-7 cells maintained a high intracellular calcium concentration. This strongly suggests that lectins can elicit differential cellular responses in cancer and non-cancer cells due to variations in their intrinsic mechanisms of calcium homeostasis. Finally, we demonstrated that TBLF and rTBL-1 can differentially alter Metabolic Cellular Activity (MCA) as a direct measure of cell viability (CVi) in both cell lines. These findings strengthen the evidence of the therapeutic potential of Tepary bean lectins. Undoubtedly, further studies will be necessary to elucidate the mechanisms underlying their applications. Full article

Diabetes mellitus is a metabolic syndrome that has grown globally to become a significant public health challenge. Hypothesizing that the plasma membrane protein, transient receptor potential ankyrin-1, is a pivotal target in insulin resistance, we investigated the mechanism of action of cinnamaldehyde (CIN), an electrophilic TRPA1 agonist, in skeletal muscle, a primary insulin target. Specifically, we evaluated the effect of CIN on insulin resistance, hepatic glycogen accumulation and muscle and adipose tissue glucose uptake. Furthermore, the in vitro role of CIN in glucose uptake and intracellular signaling was determined in insulin-resistant rats whose calcium influx was analyzed. Moreover, the serum lipid profile was assessed following short-term CIN treatment in rats, and lipid tolerance was analyzed. The effects of CIN on insulin resistance were mediated by TRPA1, with downstream signaling involving the activation of PI3-K, MAPK, PKC, as well as extracellular calcium and calcium release from intracellular stores. Additionally, cytoskeleton integrity was required for the complete action of CIN on glucose uptake in muscle. CIN also ameliorated the serum lipid profile and improved triglyceride tolerance following acute vivo exposure. Full article

Intracellular organelles are common to eukaryotic cells and provide physical support for the assembly of specialized compartments. In skeletal muscle fibers, the largest intracellular organelle is the sarcoplasmic reticulum, a specialized form of the endoplasmic reticulum primarily devoted to Ca²⁺ storage and release for muscle contraction. Occupying about 10% of the total cell volume, the sarcoplasmic reticulum forms multiple membrane contact sites, some of which are unique to skeletal muscle. These contact sites primarily involve the plasma membrane; among these, specialized membrane contact sites between the transverse tubules and the terminal cisternae of the sarcoplasmic reticulum form triads. Triads are skeletal muscle-specific contact sites where Ca²⁺ channels and regulatory proteins assemble to form the so-called calcium release complex. Additionally, the sarcoplasmic reticulum contacts mitochondria to enable a more precise regulation of Ca²⁺ homeostasis and energy metabolism. The sarcoplasmic reticulum and the plasma membrane also undergo dynamic remodeling to allow Ca²⁺ entry from the extracellular space and replenish the stores. This process involves the formation of dynamic membrane contact sites called Ca²⁺ Entry Units. This review explores the key processes in biogenesis and assembly of intracellular membrane contact sites as well as the membrane remodeling that occurs in response to muscle fatigue. Full article

Calcium is an important second messenger that is involved in almost all cellular processes. Disruptions in the regulation of intracellular Ca²⁺ levels ([Ca²⁺]_i) adversely impact normal physiological function and can contribute to various diseased conditions. STIM and Orai proteins play important roles in maintaining [Ca²⁺]_i through store-operated Ca²⁺ entry (SOCE), with STIM being the primary regulatory protein that governs the function of Orai channels. STIM1 and STIM2 are single-pass ER-transmembrane proteins with their N- and C-termini located in the ER lumen and cytoplasm, respectively. The N-terminal EF-SAM domain of STIMs senses [Ca²⁺]_ER changes, while the C-terminus mediates clustering in ER-PM junctions and gating of Orai1. ER-Ca²⁺ store depletion triggers activation of the STIM proteins, which involves their multimerization and clustering in ER-PM junctions, where they recruit and activate Orai1 channels. In this review, we will discuss the structure, organization, and function of EF-hand motifs and the SAM domain of STIM proteins in relation to those of other eukaryotic proteins. Full article

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca²⁺ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6–8 days in vitro, DIV) and long-term (20–22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca²⁺ concentration in hippocampal neurons. Ca²⁺ responses to the E protein are due to Ca²⁺ release from intracellular stores at the ER. Moreover, E protein-induced Ca²⁺ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca²⁺ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca²⁺, probably acting like a viroporin, thus producing Ca²⁺ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients. Full article