Intracellular calcium ions facilitate dengue virus entry into endothelial cells and compromise endothelial barrier integrity

Objective: To investigate the involvement of Ca2+ in dengue virus (DENV)-infected human umbilical vein endothelial cells (HUVECs) and the disruption of endothelial integrity. Methods: HUVECs were infected with DENV-2 in the presence of intracellular Ca2+ or endoplasmic reticulum Ca2+ chelators. Virus infectivity was measured by focus-forming assay and quantitative RT-PCR. Intracellular Ca2+ was measured using Fluo-4-AM dye. VE-cadherin and focal adhesion kinase (FAK) expressions were investigated by immunofluorescence and immunoblotting assays, respectively. Results: DENV infection increased intracellular cytosolic Ca2+ levels and caused disassembly of the adherens junction protein, VEcadherin as evidenced by decreased VE-cadherin expression at the periphery of DENV-2 infected HUVECs. Depletion of intracellular Ca2+ stores, particularly those of the endoplasmic reticulum Ca2+, significantly decreased DENV yield in HUVECs. Decreased virus yield following the depletion of intracellular Ca2+ was caused by the inhibition of viral entry into HUVECs and not the inhibition of viral binding or attachment. DENV-2 infection also resulted in Ca2+- dependent activation of FAK. Conclusions: Intracellular Ca2+ is required for the early phases of DENV infection in endothelial cells. Increased cytosolic Ca2+ levels in endothelial cells during DENV infection activated FAK, disrupted adherens junctions and compromised barrier integrity. Thus, Ca2+ plays an important role in DENV infection in endothelial cells.

S100 proteins: An emerging cynosure in pregnancy & adverse reproductive outcome

S100 proteins are calcium (Ca2+)-binding proteins and these have an important function in progression, manifestation and therapeutic aspects of various inflammatory, metabolic and neurodegenerative disorders. Based on their involvement in intracellular or extracellular regulatory effects, S100 proteins are classified into three subgroups: one subgroup is specialized in exerting only intracellular effects, other performs both intracellular and extracellular functions and the third subgroup members only display extracellular regulatory effects. S100 proteins are expressed particularly in vertebrates and have cell-specific expression. Functionally, S100 proteins act through their surface receptors and regulate cell functions in autocrine or paracrine mode. Receptor for advanced glycation end products (RAGEs) and toll-like receptor 4 are the main surface receptors. S100 proteins participate in the regulation of cellular differentiation, proliferation, apoptosis and inflammation along with Ca2+ homeostasis, energy metabolism and cellular migration, and perform the respective functions through their interaction with transcription factors, nucleic acids, enzymes, receptors, cytoskeleton system, etc. Currently, their role in adverse pregnancy outcomes and compromised reproductive health is being explored. These proteins are present in amniotic fluid, endometrium tissue and foetal brain; therefore, it is quite likely that alterations in the expression levels of S100 family members will be affecting the particular function they are involved in and ultimately affecting the pregnancy in adverse manner. The current review discusses about an association of S100 proteins in pregnancy disorders such as endometriosis, intrauterine growth retardation and miscarriage.

Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients

BACKGROUND: Transient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19+ B cells, CD56bnght and CD56dim cell populations from CFS/ME patients. RESULTS: TRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56bright TRPM3 35.72 % ± 7.37; CD56dim 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19+ B cells (1.56 ± 0.191) and CD56bright NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19+ B lymphocytes. CD56bright NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients. CONCLUSIONS: The results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.

The effect of carbamylcholine on the depolarization-induced change of intracellular calcium in the outer hair cells of guinea pig cochlea / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Auditory signal can be amplified by the organ of Corti and be detected in the auditory cortex. Outer hair cells (OHCs) play a pivotal role in this amplification process. The signal transduction mechanism of OHC relys on the precise control of intracellular calcium ([Ca2+]i). Membrane depolarization, induced by electrical stimulation or extracellular high concentration of KCl solution, increases [Ca2+]i through the opening of voltage-gated Ca2+ channel or the release from intracellular Ca2+ store which is stimulated by second messenger system. Efferent stimulation seems to inhibit the electromotility of OHC and modify the amplification process. Acetylcholine is the most promising neurotransmitter released from efferent synapse. In this study, we aimed to observe the effect of carbamylcholine (a non-hydrolyzable, non-selective cholinergic agonist) on the change of [Ca2+]i, and the modification of depolarization-induced [Ca2+]i increase in isolated OHCs of the guinea pig cochlea using the fluorescent indicator fluo-3. MATERIALS AND METHOD: Outer hair cells were isolated from the guinea pig cochlea and incubated in HBSS containing 5 M fluo-3 for 30 min. The [Ca2+]i was measured under inverted microscope equipped with epifluorescence system. The images were analysed and [Ca2+]i was calculated using Fmax, Fmin and Kd obtained from in vivo and in vitro calibration. RESULTS: [Ca2+]i increased by extracellular carbamylcholine application (1mM) in OHCs. KCl solution could induce [Ca2+]i increase when used more than 25mM and the responses were concentration-dependent. Preincubation of carbamylcholine, however, did not modify the depolarization-induced [Ca2+]i increase. CONCLUSION: Through these results, we speculate that the acetylcholine, released from the efferent synapse, can induce local increase of [Ca2+]i, but acetylcholine can not regulate the depolarization-induced [Ca2+]i in isolated OHCs of the guinea pig cochlea.