LRRC52 is likely a functional component of human KSper†.

Completion of fertilization is orchestrated by various ion channels in sperm membrane. Hyperpolarization of membrane potential, an indispensable event during the capacitation process, is dominated by sperm potassium channel (KSper). In addition to sperm-specific SLO3, which forms the channel pore, the auxiliary subunit leucine-rich-repeat-containing protein 52 (LRRC52) is required to form mKSper to function under physiological conditions. However, in human sperm, although most evidence supports that hSLO3 is the pore-forming subunit, whether hLRRC52 contributes to hKSper conductance and modulates sperm function remains to be understood. Here, using an extracellular segment that is homologous between mice and humans as an antigen, we developed a polyclonal antibody designed as LID1 that specifically detected mLRRC52 and performed co-immunoprecipitation with mSLO3. Additionally, patch-clamp recordings of mouse sperm showed that, physiological activation of mKSper and sperm functions were dramatically attenuated after treatment with LID1, indicating that LID1 functionally disrupted the regulation of mLRRC52 on mKSper. Next, LID1 was used to investigate the significance of hLRRC52 for hKSper activation. As a result, hLRRC52 was expressed in human sperm and might be assembled with hSLO3. More importantly, LID1 inhibited hKSper currents and depolarized sperm membrane potential, supporting essential modulation of hLRRC52 in hKSper. Ca2+ signaling of human sperm was also compromised in the presence of LID1, which impaired sperm motility and acrosome reaction. Because LID1 specifically inhibited both mKSper and hKSper but not mCatSper or hCatSper, our results suggest that hLRRC52 functions as an important component of hKSper and regulates sperm physiological functions.

Ultrafine particulate matter pollution and dysfunction of endoplasmic reticulum Ca2+ store: A pathomechanism shared with amyotrophic lateral sclerosis motor neurons?

Increased risk of neurodegenerative diseases has been envisaged for air pollution exposure. On the other hand, environmental risk factors, including air pollution, have been suggested for Amyotrophic Lateral Sclerosis (ALS) pathomechanism. Therefore, the neurotoxicity of ultrafine particulate matter (PM0.1) (PM < 0.1 µm size) and its sub-20 nm nanoparticle fraction (NP20) has been investigated in motor neuronal-like cells and primary cortical neurons, mainly affected in ALS. The present data showed that PM0.1 and NP20 exposure induced endoplasmic reticulum (ER) stress, as occurred in cortex and spinal cord of ALS mice carrying G93A mutation in SOD1 gene. Furthermore, NSC-34 motor neuronal-like cells exposed to PM0.1 and NP20 shared the same proteomic profile on some apoptotic factors with motor neurons treated with the L-BMAA, a neurotoxin inducing Amyotrophic Lateral Sclerosis/Parkinson-Dementia Complex (ALS/PDC). Of note ER stress induced by PM0.1 and NP20 in motor neurons was associated to pathological changes in ER morphology and dramatic reduction of organellar Ca2+ level through the dysregulation of the Ca2+-pumps SERCA2 and SERCA3, the Ca2+-sensor STIM1, and the Ca2+-release channels RyR3 and IP3R3. Furthermore, the mechanism deputed to ER Ca2+ refilling (e.g. the so called store operated calcium entry-SOCE) and the relative currents ICRAC were also altered by PM0.1 and NP20 exposure. Additionally, these carbonaceous particles caused the exacerbation of L-BMAA-induced ER stress and Caspase-9 activation. In conclusion, this study shows that PM0.1 and NP20 induced the aberrant expression of ER proteins leading to dysmorphic ER, organellar Ca2+ dysfunction, ER stress and neurotoxicity, providing putative correlations with the neurodegenerative process occurring in ALS.

Changes in intracellular calcium concentration level accompany age-related inhibitions of long-term potentiation in hippocampus induced by extremely low frequency electromagnetic fields.

Extremely low frequency electromagnetic fields (ELF-EMFs) have received increasing attention and in-depth research due to their ability to affect learning and memory functions. However, its regulation and intrinsic mechanism at different ages in early developmental stages remains unclear. In this article, the regulation of 15 Hz/2 mT ELF-EMFs on long-term potentiation (LTP) persistence in the hippocampal CA1 region of Sprague-Dawley (SD) rats at early developmental stages (8-, 15-, 22-, and 29-day-old) are investigated by electrophysiological techniques. The results show that ELF-EMFs differentially inhibit LTP persistence due to age difference, and the younger the age, the more significant the inhibitory effect. Second, the inhibitory effect of ELF-EMFs on LTP persistence disappeared after the addition of 2-aminoethoxydiphenyl borate (2-APB) to block inositol-1,4,5-trisphosphate receptors (IP3 Rs) localized to intracellular calcium stores to reduce the intracellular calcium concentration ([Ca2+ ]i ), proving that the LTP persistence regulated by ELF-EMFs is associated with the IP3 Rs-mediated intracellular calcium stores. Finally, the level of [Ca2+ ]i was intervened by adjusting the extracellular calcium concentration([Ca2+ ]e ). Interestingly, the inhibitory effect of ELF-EMFs on LTP persistence in the 15-day-old group disappeared by increasing [Ca2+ ]e , whereas the inhibitory effect of ELF-EMFs on LTP persistence in the 29-day-old group appeared by decreasing [Ca2+ ]e . Our findings reveal the underlying mechanism of the effect of ELF-EMFs on synaptic plasticity in hippocampal CA1 area at early developmental stages and provide new insights into more rational application and protection of ELF-EMFs.

Ambroxol-Enhanced Frequency and Amplitude of Beating Cilia Controlled by a Voltage-Gated Ca2+ Channel, Cav1.2, via pHi Increase and [Cl-]i Decrease in the Lung Airway Epithelial Cells of Mice.

Ambroxol (ABX), a frequently prescribed secretolytic agent which enhances the ciliary beat frequency (CBF) and ciliary bend angle (CBA, an index of amplitude) by 30%, activates a voltage-dependent Ca2+ channel (CaV1.2) and a small transient Ca2+ release in the ciliated lung airway epithelial cells (c-LAECs) of mice. The activation of CaV1.2 alone enhanced the CBF and CBA by 20%, mediated by a pHi increasei and a [Cl-]i decrease in the c-LAECs. The increase in pHi, which was induced by the activation of the Na+-HCO3- cotransporter (NBC), enhanced the CBF (by 30%) and CBA (by 15-20%), and a decrease in [Cl-]i, which was induced by the Cl- release via anoctamine 1 (ANO1), enhanced the CBA (by 10-15%). While a Ca2+-free solution or nifedipine (an inhibitor of CaV1.2) inhibited 70% of the CBF and CBA enhancement using ABX, CaV1.2 enhanced most of the CBF and CBA increases using ABX. The activation of the CaV1.2 existing in the cilia stimulates the NBC to increase pHi and ANO1 to decrease the [Cl-]i in the c-LAECs. In conclusion, the pHi increase and the [Cl-]i decrease enhanced the CBF and CBA in the ABX-stimulated c-LAECs.

Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome.

In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) a few minutes later and on days 3 and 7 after scratching. An increase in [Ca2+]i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150-200 µm from the scratch border. Ca2+ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca2+]i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and ß-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca2+ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.

Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate.

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer's disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2-•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2-• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin's effects on glutamate-evoked O2-• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2-•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2-• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).

NAADP-induced intracellular calcium ion is mediated by the TPCs (two-pore channels) in hypoxia-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a form of obstructive vascular disease. Chronic hypoxic exposure leads to excessive proliferation of pulmonary arterial smooth muscle cells and pulmonary arterial endothelial cells. This condition can potentially be aggravated by [Ca2+ ] i mobilization. In the present study, hypoxia exposure of rat's model was established. Two-pore segment channels (TPCs) silencing was achieved in rats' models by injecting Lsh-TPC1 or Lsh-TPC2. The effects of TPC1/2 silencing on PAH were evaluated by H&E staining detecting pulmonary artery wall thickness and ELISA assay kit detecting NAADP concentrations in lung tissues. TPC1/2 silencing was achieved in PASMCs and PAECs, and cell proliferation was detected by MTT and BrdU incorporation assays. As the results shown, NAADP-activated [Ca2+ ]i shows to be mediated via two-pore segment channels (TPCs) in PASMCs, with TPC1 being the dominant subtype. NAADP generation and TPC1/2 mRNA and protein levels were elevated in the hypoxia-induced rat PAH model; NAADP was positively correlated with TPC1 and TPC2 expression, respectively. In vivo, Lsh-TPC1 or Lsh-TPC2 infection significantly improved the mean pulmonary artery pressure and PAH morphology. In vitro, TPC1 silencing inhibited NAADP-AM-induced PASMC proliferation and [Ca2+ ]i in PASMCs, whereas TPC2 silencing had minor effects during this process; TPC2 silencing attenuated NAADP-AM- induced [Ca2+ ]i and ECM in endothelial cells, whereas TPC1 silencing barely ensued any physiological changes. In conclusion, TPC1/2 might provide a unifying mechanism within pulmonary arterial hypertension, which can potentially be regarded as a therapeutic target.

Depressed ß-adrenergic inotropic responsiveness and intracellular calcium handling abnormalities in Duchenne Muscular Dystrophy patients' induced pluripotent stem cell-derived cardiomyocytes.

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an X-linked disease affecting male and rarely adult heterozygous females, resulting in death by the late 20s to early 30s. Previous studies reported depressed left ventricular function in DMD patients which may result from deranged intracellular Ca2+ -handling. To decipher the mechanism(s) underlying the depressed LV function, we tested the hypothesis that iPSC-CMs generated from DMD patients feature blunted positive inotropic response to ß-adrenergic stimulation. To test the hypothesis, [Ca2+ ]i transients and contractions were recorded from healthy and DMD-CMs. While in healthy CMs (HC) isoproterenol caused a prominent positive inotropic effect, DMD-CMs displayed a blunted inotropic response. Next, we tested the functionality of the sarcoplasmic reticulum (SR) by measuring caffeine-induced Ca2+ release. In contrast to HC, DMD-CMs exhibited reduced caffeine-induced Ca2+ signal amplitude and recovery time. In support of the depleted SR Ca2+ stores hypothesis, in DMD-CMs the negative inotropic effects of ryanodine and cyclopiazonic acid were smaller than in HC. RNA-seq analyses demonstrated that in DMD CMs the RNA-expression levels of specific subunits of the L-type calcium channel, the ß1-adrenergic receptor (ADRß1) and adenylate cyclase were down-regulated by 3.5-, 2.8- and 3-fold, respectively, which collectively contribute to the depressed ß-adrenergic responsiveness.

Clioquinol inhibits cell growth in a SERCA2-dependent manner.

Clioquinol has been reported to act as a potential therapy for neurodegenerative diseases and cancer. However, the underlying mechanism is unclear. We have previously reported that clioquinol induces S-phase cell cycle arrest through the elevation of calcium levels in human neurotypic SH-SY5Y cells. In this study, different types of cells were observed to detect if the effect of clioquinol on intracellular calcium levels is cell type-specific. The Cell Counting Kit-8 assay showed that clioquinol exhibited varying degrees of concentration-dependent cytotoxicity in different cell lines, and that the growth inhibition caused by it was not related to cell source or carcinogenesis. In addition, the inhibition of cell growth by clioquinol was positively associated with its effect on intracellular calcium content ([Ca2+ ]i ). Furthermore, the elevation of [Ca2+ ]i induced by clioquinol led to S-phase cell cycle arrest. Similar to our previous studies, the increase in [Ca2+ ]i was attributed to changes in the expression levels of the calcium pump SERCA2. Comparison of expression levels of SERCA2 between cell lines showed that cells with high levels of SERCA2 were more sensitive to clioquinol. In addition, analysis using UALCAN and the Human Protein Atlas also showed that the expression of SERCA2 in the corresponding human tissues was similar to that of the cells tested in this study, suggesting potential in the application of clioquinol in the future. In summary, our results expand the understanding of the molecular mechanism of clioquinol and provide an important strategy for the rational use of clioquinol.

Probing the Ca2+ mobilizing properties on primary cortical neurons of a new stable cADPR mimic.

Cyclic adenosine diphosphate ribose (cADPR) is a second messenger involved in the Ca2+ homeostasis. Its chemical instability prompted researchers to tune point by point its structure, obtaining stable analogues featuring interesting biological properties. One of the most challenging derivatives is the cyclic inosine diphosphate ribose (cIDPR), in which the hypoxanthine isosterically replaces the adenine. As our research focuses on the synthesis of N1 substituted inosines, in the last few years we have produced new flexible cIDPR analogues, where the northern ribose has been replaced by alkyl chains. Interestingly, some of them mobilized Ca2+ ions in PC12 cells. To extend our SAR studies, herein we report on the synthesis of a new stable cIDPR derivative which contains the 2″S,3″R dihydroxypentyl chain instead of the northern ribose. Interestingly, the new cyclic derivative and its open precursor induced an increase in intracellular calcium concentration ([Ca2+]i) with the same efficacy of the endogenous cADPR in rat primary cortical neurons.

Physiologically detectable bisphenol A impairs human sperm functions by reducing protein-tyrosine phosphorylation.

BACKGROUND: Bisphenol A (BPA), a widely used plastic monomer and plasticizer, is detectable in blood, urine and semen of a healthy people, with concentrations ranging from 0.1 nM to 10 nM. It has been shown that in vitro exposure of BPA as low as 0.001 nM could significantly inhibited mouse sperm motility and acrosome reaction. However, it is still unclear whether BPA at those physiologically detectable concentration affects human sperm. METHODS: The effects of different concentrations of BPA (0, 10-3, 10-2, 10-1, 10, 103 nM) on sperm functions were examined, including human sperm viability, kinematic parameters, hyperactivation and capacitation. RESULTS: BPA caused a remarkable decline in human sperm viability, motility and progressive motility, hyperactivation, capacitation and progesterone-induced acrosome reaction. Mechanism studies showed that BPA could suppress the protein tyrosine phosphorylation level of human sperm, but had no effect on sperm calcium signaling. CONCLUSIONS: Physiologically detectable concentrations of BPA may impair human sperm functions via suppressing protein tyrosine phosphorylation of human sperm, implying that environmental pollution of BPA might be a factor contributing to male infertility.

The Effect of Sera from Children with Obstructive Sleep Apnea Syndrome (OSAS) on Human Cardiomyocytes Differentiated from Human Embryonic Stem Cells.

Obstructive sleep apnea syndrome (OSAS) patients suffer from cardiovascular morbidity, which is the leading cause of death in this disease. Based on our previous work with transformed cell lines and primary rat cardiomyocytes, we determined that upon incubation with sera from pediatric OSAS patients, the cell's morphology changes, NF-κB pathway is activated, and their beating rate and viability decreases. These results suggest an important link between OSAS, systemic inflammatory signals and end-organ cardiovascular diseases. In this work, we confirmed and expanded these observations on a new in vitro system of beating human cardiomyocytes (CM) differentiated from human embryonic stem cells (hES). Our results show that incubation with pediatric OSAS sera, in contrast to sera from healthy children, induces over-expression of NF-κB p50 and p65 subunits, marked reduction in CMs beating rate, contraction amplitude and a strong reduction in intracellular calcium signal. The use of human CM cells derived from embryonic stem cells has not been previously reported in OSAS research. The results further support the hypothesis that NF-κB dependent inflammatory pathways play an important role in the evolution of cardiovascular morbidity in OSAS. This study uncovers a new model to investigate molecular and functional aspects of cardiovascular pathology in OSAS.

H1-Receptor Antagonist Olopatadine Inhibits MUC5AC Secretion by Conjunctival Goblet Cells.

The study examined the effect of H1-receptor antagonist olopatadine on the secretory function of cultured rat conjunctival goblet cells (CGC) assessed by enzyme-linked lectin assay employing UEA-I lectin. The level of mRNA for membrane-bound protein MUC16 in histaminestimulated CGC was assayed by reverse transcription PCR in the control and after preliminary application of olopatadine. The intracellular calcium concentration [Ca2+]i was measured by the calcium colorimetric method using GENMED kits. The effects of histamine and olopatadine on p-ERK level were assessed by Western blotting. Histamine up-regulated secretion of mucin MUC5AC and expression of membrane-bound protein MUC16 in CGC. In addition, it increased both [Ca2+]i and the level of phosphorylated ERK. These effects were diminished by preliminary application of olopatadine that probably acted via the ERK signaling pathway. Thus, olopatadine reduced [Ca2+]i and down-regulated ERK phosphorylation by binding to H1-receptors, thereby inhibiting secretion of mucin from histamine-stimulated CGC.

The role of calcium-binding protein S100g (CalbindinD-9K) and annexin A10 in acute pancreatitis.

BACKGROUND: We reported that the pancreas of the interferon-regulatory factor (IRF) 2 knock-out (KO) mouse represents an early phase of acute pancreatitis, including defective regulatory exocytosis, intracellular activation of trypsin, and disturbance of autophagy. The significantly upregulated and downregulated genes in the IRF2 KO pancreas have been reported. The catalogue of gene transcripts included two types of calcium-binding proteins (S100 calcium binding protein G [S100g] and Annexin A10 [Anxa10]), which were highly upregulated in the IRF2 KO pancreas. As the intracellular calcium signal plays a pivotal role in regulatory exocytosis and its disturbance is related to pancreatitis, we then evaluated the role of S100g and Anxa10 in acute pancreatitis. METHOD: We induced cerulein-pancreatitis in wild-type mice and examined the changes in the expression of these genes by qPCR and immunohistochemistry. We constructed S100g-overexpressing or Anxa10-overexpressing AR42J cells (AR42J-S100g, AR42J-Anxa10). We examined the changes in amylase secretion, intracellular calcium ([Ca2+]i), and cell viability in these cells, when incubated with cholecystokinin (CCK). RESULTS: The expression of S100g and Anxa10 was increased in cerulean-induced pancreatitis. The acini were patchily stained for S100g and the cytosol of acini was evenly but weakly stained for Anxa10. Stimulation with 100pM CCK-8, decreased amylase secretion and inhibited the [Ca2+]i increase in AR42J-S100g cells. These effects were weak in AR42J-Anxa10 cells. Cell viability was not changed by incubation with cerulein. CONCLUSION: In cerulean pancreatitis, the expression of S100g and Anxa10 was induced in the acini. S100g may work as a Ca2+ buffer in acute pancreatitis.

Regulation of [Ca2+]i oscillations and mitochondrial activity by various calcium transporters in mouse oocytes.

Oocyte activation inefficiency is one of the reasons for female infertility and Ca2+ functions play a critical role in the regulation of oocyte activation. We used various inhibitors of Ca2+ channels located on the membrane, including sarcoplasmic/ endoplasmic reticulum Ca2+ATPases (SERCAs, the main Ca2+ pumps which decrease the intracellular Ca2+ level by refilling Ca2+ into the sarcoplasmic reticulum), transient receptor potential (TRP) ion channel subfamily member 7 (TRPM7, a Ca2+/Mg2+-permeable non-selective cation channel), T-type Ca2+ channels and calcium channel Orai1, to investigate their roles in [Ca2+]i oscillation patterns and mitochondrial membrane potential during oocyte activation by real-time recording. Our results showed that SERCAs, TRPM7 and T-type Ca2+ channels were important for initiation and maintenance of [Ca2+]i oscillations, which was required for mitochondrial membrane potential elevation during oocyte activation, as well as oocyte cytoskeleton stability and subsequent embryo development. Increasing the knowledge of calcium transport may provide a theoretical basis for improving oocyte activation in human assisted reproduction clinics.

Differential long-term regulation of TAS2R14 by structurally distinct agonists.

Bitter taste receptor-14 (TAS2R14) is a GPCR also expressed on human airway smooth muscle cells, which signals to intracellular [Ca2+], resulting in relaxation of the airway, and is a novel target for bronchodilators. Here, we examine long-term, agonist-promoted down-regulation of TAS2R14 expression because tachyphylaxis would be an undesirable therapeutic characteristic. Five TAS2R structurally distinct full agonists were studied to ascertain biasing away from down-regulation. Agonist exposure for 18 h caused minimal desensitization by diphenhydramine (DPD) compared with ∼50% desensitization with all other agonists. Agonists evoked ß-arrestin recruitment to TAS2R14, which was not seen with a phosphoacceptor-deficient mutant, TAS2R14-10A. All agonists except for DPD also caused subsequent TAS2R14 internalization and trafficking via early and late endosomes to down-regulation. TAS2R14-10A failed to undergo these events with any agonist. Molecular docking showed that DPD has specific interactions deep within a binding pocket that are not observed with the other agonists, which may lock the receptor in a conformation that does not internalize and therefore does not undergo down-regulation. Thus, TAS2R14 is subject to ß-arrestin-mediated internalization and subsequent down-regulation with chronic exposure to most agonists. However, by manipulating the agonist structure, biasing toward G-protein coupling but away from long-term down-regulation can be achieved.-Woo, J. A., Castaño, M., Goss, A., Kim, D., Lewandowski, E. M., Chen, Y., Liggett, S. B. Differential long-term regulation of TAS2R14 by structurally distinct agonists.

Carboxyl terminus of Hsc70-interacting protein (CHIP) promotes pulmonary artery smooth muscle cell (PASMC) proliferation via enhancement of intracellular Ca2+ concentration ([Ca2+]i).

AIMS OF THE STUDY: To investigate the effect of carboxyl terminus of Hsc70-interacting protein (CHIP) on pulmonary arterial smooth muscle cell (PASMC) proliferation and the underlying mechanism. Materials and Methods: PASMCs were harvested from distal PAs isolated from SD rat lungs and cultured. After CHIP overexpression, PASMCs were exposed to normoxia or hypoxia for 60 h. Then, PASMC proliferation, store-operated Ca2+ entry (SOCE), [Ca2+]i and the expression of TRPC1, TRPC4, and TRPC6 in PASMCs were measured. Results: CHIP overexpression promoted PASMC proliferation, SOCE, [Ca2+]i and the expression of TRPC1, TRPC4, and TRPC6. Conclusions: CHIP stimulates PASMC proliferation likely by targeting the TRPC1,4,6-SOCE-[Ca2+]i signaling pathway.

Sarco-Endoplasmic Reticulum Calcium Release Model Based on Changes in the Luminal Calcium Content.

The sarcoplasmic/endoplasmic reticulum (SR/ER) is the main intracellular calcium (Ca2+) pool in muscle and non-muscle eukaryotic cells, respectively. The reticulum accumulates Ca2+ against its electrochemical gradient by the action of sarco/endoplasmic reticulum calcium ATPases (SERCA pumps), and the capacity of this Ca2+ store is increased by the presence of Ca2+ binding proteins in the lumen of the reticulum. A diversity of physical and chemical signals, activate the main Ca2+ release channels, i.e. ryanodine receptors (RyRs) and inositol (1, 4, 5) trisphosphate receptors (IP3Rs), to produce transient elevations of the cytoplasmic calcium concentration ([Ca2+]i) while the reticulum is being depleted of Ca2+. This picture is incomplete because it implies that the elements involved in the Ca2+ release process are acting alone and independently of each other. However, it appears that the Ca2+ released by RyRs and IP3Rs is trapped in luminal Ca2+ binding proteins (Ca2+ lattice), which are associated with these release channels, and the activation of these channels appears to facilitate that the trapped Ca2+ ions become available for release. This situation makes the initial stage of the Ca2+ release process a highly efficient one; accordingly, there is a large increase in the [Ca2+]i with minimal reductions in the bulk of the free luminal SR/ER [Ca2+] ([Ca2+]SR/ER). Additionally, it has been shown that active SERCA pumps are required for attaining this highly efficient Ca2+ release process. All these data indicate that Ca2+ release by the SR/ER is a highly regulated event and not just Ca2+ coming down its electrochemical gradient via the open release channels. One obvious advantage of this sophisticated Ca2+ release process is to avoid depletion of the ER Ca2+ store and accordingly, to prevent the activation of ER stress during each Ca2+ release event.

Activation of AMPK under Hypoxia: Many Roads Leading to Rome.

AMP-activated protein kinase (AMPK) is known as a pivotal cellular energy sensor, mediating the adaptation to low energy levels by deactivating anabolic processes and activating catabolic processes in order to restore the cellular ATP supply when the cellular AMP/ATP ratio is increased. Besides this well-known role, it has also been shown to exert protective effects under hypoxia. While an insufficient supply with oxygen might easily deplete cellular energy levels, i.e., ATP concentration, manifold other mechanisms have been suggested and are heavily disputed regarding the activation of AMPK under hypoxia independently from cellular AMP concentrations. However, an activation of AMPK preceding energy depletion could induce a timely adaptation reaction preventing more serious damage. A connection between AMPK and the master regulator of hypoxic adaptation via gene transcription, hypoxia-inducible factor (HIF), has also been taken into account, orchestrating their concerted protective action. This review will summarize the current knowledge on mechanisms of AMPK activation under hypoxia and its interrelationship with HIF.

Synergistically regulated spontaneous calcium signaling is attributed to cartilaginous extracellular matrix metabolism.

Ca2+ has been recognized as a key molecule for chondrocytes, however, the role and mechanism of spontaneous [Ca 2+ ] i signaling in cartilaginous extracellular matrix (ECM) metabolism regulation are unclear. Here we found that spontaneous Ca 2+ signal of in-situ porcine chondrocytes was [Ca 2+ ] o dependent, and mediated by [Ca 2+ ] i store release. T-type voltage-dependent calcium channel (T-VDCC) mediated [Ca 2+ ] o influx was associated with decreased cell viability and expression levels of ECM deposition genes. Further analysis revealed that chondrocytes expressed both inositol 1,4,5-trisphosphate receptor (InsP3R) and Orai isoforms. Inhibition of endoplasmic reticulum (ER) Ca 2+ release and store-operated calcium entry significantly abolished spontaneous [Ca 2+ ] i signaling of in-situ chondrocytes. Moreover, blocking ER Ca 2+ release with InsP3R inhibitors significantly upregulated ECM degradation enzymes production, and was accompanied by decreased proteoglycan and collagen type II intensity. Taken together, our data provided evidence that spontaneous [Ca 2+ ] i signaling of in-situ porcine chondrocytes was tightly regulated by [Ca 2+ ] o influx, InsP3Rs mediated [Ca 2+ ] i store release, and Orais mediated calcium release-activated calcium channels activation. Both T-VDCC mediated [Ca 2+ ] o influx and InsP3Rs mediated ER Ca 2+ release were found crucial to cartilaginous ECM metabolism through distinct regulatory mechanisms.