Rise In Intracellular Calcium Concentration Research Articles

Activation of skeletal muscle is controlled by a dual-filament mechano-sensing mechanism

Contraction of skeletal muscle is triggered by a transient rise in intracellular calcium concentration leading to a structural change in the actin-containing thin filaments that allows binding of myosin motors from the thick filaments. Most myosin motors are unavailable for actin binding in resting muscle because they are folded back against the thick filament backbone. Release of the folded motors is triggered by thick filament stress, implying a positive feedback loop in the thick filaments. However, it was unclear how thin and thick filament activation mechanisms are coordinated, partly because most previous studies of the thin filament regulation were conducted at low temperatures where the thick filament mechanisms are inhibited. Here, we use probes on both troponin in the thin filaments and myosin in the thick filaments to monitor the activation states of both filaments in near-physiological conditions. We characterize those activation states both in the steady state, using conventional titrations with calcium buffers, and during activation on the physiological timescale, using calcium jumps produced by photolysis of caged calcium. The results reveal three activation states of the thin filament in the intact filament lattice of a muscle cell that are analogous to those proposed previously from studies on isolated proteins. We characterize the rates of the transitions between these states in relation to thick filament mechano-sensing and show how thin- and thick-filament-based mechanisms are coupled by two positive feedback loops that switch on both filaments to achieve rapid cooperative activation of skeletal muscle.

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells. The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

Pharmacological and molecular characterization of the A-type muscarinic acetylcholine receptor from Anopheles gambiae.

Muscarinic acetylcholine receptors (mAChRs) which are G protein-coupled receptors play key roles in insect physiology. Whereas vertebrate mAChRs are important targets for pharmaceutical drugs, insect mAChRs are under-exploited by the agro-chemical industry. Moreover, insect mAChRs have been less well studied than their vertebrate counterparts. Their critical functions mean that a better knowledge of the insect mAChRs is crucial for the effort to develop a new molecular-level strategy for insect pest management. Almost all insects possess three mAChRs named A, B and C which differ according to their coupling effector systems and their pharmacological profile. The aim of this study was to characterize the A-type mAChR (mAChR-A) from Anopheles gambiae which is the major vector of malaria in order to develop new strategies in pest management. In this paper, we reported that mAChR-A is more expressed in adult mosquitoes than in larvae. Furthermore, using calcium imaging recordings, we found that the An. gambiae mAChR-A expressed in Sf9 cells is activated by specific muscarinic agonists acetylcholine, muscarine and oxotremorine M and blocked by several mAChR antagonists. Moreover, using inhibitors of phosphoinositide pathway such as Gαq/11 protein blocker, we have shown that an increased intracellular calcium concentration elicited by the acetylcholine application was mediated by PLC/IP3R pathway. As a rise in intracellular calcium concentration could lead to an increase in the insecticide target sensitivity, these results suggest that An. gambiae mAChR-A should not be only considered as a potential target for new molecules but also as a key element to optimize the efficacy of insecticide in vector control.

Elucidation of Mechanisms in Cu (II) Caused Hypercontraction of Rat Tracheal Rings.

Airway smooth muscle contraction is one of the primary factors involved in the initiation and progression of asthma which in turn is regulated by increased cytosolic Ca2+ concentration from intracellular stores and through transmembrane ion channels. Calcium-independent factors such as reactive oxygen species (ROS) generation, nitric oxide (NO) depletion and cyclooxygenase (COX) pathways also contribute to tracheal smooth muscle contraction. Studies on copper toxicity suggest significance of this essential micronutrient overdose in acute respiratory disorders, allergic asthma and ciliary motion in tracheal explants. However, the mechanism of copper caused hypercontraction upon direct exposure to tracheal smooth muscle is largely unknown. In this study we investigate the effect of copper exposure on isolated tracheal rings and relative contributions of various factors in acetylcholine-induced contractions. Results obtained suggest that rise in intracellular calcium concentration via voltage-operated Ca2+ channel (VOCC), store-operated Ca2+ channel (SOCC), stretch-activated channels (SAC) and TRP channel (transient receptor potential channel) activation is the major factor in copper-mediated hypercontraction. ROS generation or COX-dependent pathways do not appear to significantly contribute to Cu2+ caused hypercontraction.

Sortilin-derived peptides promote pancreatic beta-cell survival through CREB signaling pathway

Deterioration of insulin secretion and pancreatic beta-cell mass by inflammatory attacks is one of the main pathophysiological features of type 2 diabetes (T2D). Therefore, preserving beta-cell mass and stimulating insulin secretion only in response to glucose for avoiding the hypoglycemia risks, are the most state-of-the-art option for the treatment of T2D. In this study we tested two correlated hypothesis that 1/ the endogenous peptide released from sortilin, known as PE, that stimulates insulin secretion only in response to glucose, protects beta-cells against death induced by cytokines, and 2/ Spadin and Mini-Spadin, two synthetic peptides derived from PE, that mimic the effects of PE in insulin secretion, also provide beneficial effect on beta-cells survival. We show that PE and its derivatives by inducing a rise of intracellular calcium concentration by depolarizing the membrane protect beta-cells against death induced by Interleukin-1β. Using biochemical, confocal imaging and cell biology techniques, we reveal that the protective effects of PE and its derivatives rely on the activation of the CaM-Kinase pathway, and on the phosphorylation and activation of the transcription factor CREB. In addition, Mini-Spadin promotes beta-cell proliferation, suggesting its possible regenerative effect. This study highlights new possible roles of PE in pancreatic beta-cell survival and its derivatives as pharmacological tools against diabetes.

Apoptosis in resistance arteries induced by hydrogen peroxide: greater resilience of endothelium versus smooth muscle.

Reactive oxygen species (ROS) are implicated in cardiovascular and neurologic disorders including atherosclerosis, heart attack, stroke, and traumatic brain injury. Although oxidative stress can lead to apoptosis of vascular cells, such findings are largely based upon isolated vascular smooth muscle cells (SMCs) and endothelial cells (ECs) studied in culture. Studying intact resistance arteries, we have focused on understanding how SMCs and ECs in the blood vessel wall respond to acute oxidative stress induced by hydrogen peroxide, a ubiquitous, membrane-permeant ROS. We find that apoptosis induced by H2O2 is far greater in SMCs compared to ECs. For both cell types, apoptosis is associated with a rise in intracellular calcium concentration ([Ca2+]i) during H2O2 exposure. Consistent with their greater death, the rise in [Ca2+]i for SMCs exceeds that in ECs. Finding that disruption of the endothelium increases SMC death, we address how myoendothelial coupling and paracrine signaling attenuate apoptosis. Remarkably, conditions associated with chronic oxidative stress (advanced age, Western-style diet) protect SMCs during H2O2 exposure, as does female sex. In light of intracellular Ca2+ handling, we consider how glycolytic versus oxidative pathways for ATP production and changes in mitochondrial structure and function impact cellular resilience to H2O2-induced apoptosis. Gaining new insight into protective signaling within and between SMCs and ECs of the arterial wall can be applied to promote vascular cell survival (and recovery of blood flow) in tissues subjected to acute oxidative stress as occurs during reperfusion following myocardial infarction and thrombotic stroke.

Contribution of NAADP to Glutamate-Evoked Changes in Ca2+ Homeostasis in Mouse Hippocampal Neurons.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

Genome-wide Analyses of Chromatin State in Human Mast Cells Reveal Molecular Drivers and Mediators of Allergic and Inflammatory Diseases

Mast cells (MCs) are versatile immune cells capable of rapidly responding to a diverse range of extracellular cues. Here, we mapped the genomic and transcriptomic changes in human MCs upon diverse stimuli. Our analyses revealed broad H3K4me3 domains and enhancers associated with activation. Notably, the rise of intracellular calcium concentration upon immunoglobulin E (IgE)-mediated crosslinking of the high-affinity IgE receptor (FcεRI) resulted in genome-wide reorganization of the chromatin landscape and was associated with a specific chromatin signature, which we term Ca2+-dependent open chromatin (COC) domains. Examination of differentially expressed genes revealed potential effectors of MC function, and we provide evidence for fibrinogen-like protein 2 (FGL2) as an MC mediator with potential relevance in chronic spontaneous urticaria. Disease-associated single-nucleotide polymorphisms mapped onto cis-regulatory regions of human MCs suggest that MC function may impact a broad range of pathologies. The datasets presented here constitute a resource for the further study of MC function.

PFOA evokes extracellular Ca2+ influx and compromises progesterone-induced response in human sperm.

Perfluorooctane acid (PFOA), a persistent organic pollutant, is ubiquitously present in the environment and may detrimentally affect male reproductive health. In this study, mature human sperm were invitro exposed to different concentrations of PFOA (0.25, 2.5 or 25 μg/ml) alone or in combination with progesterone (P4) to evaluate the toxicity and the potential mechanism of action. Exposure to high-dose PFOA (25 μg/ml) alone for 4 h caused a decline in capacity of human spermatozoa to penetrate synthetic mucus, with an increased production of reactive oxygen species (ROS). Furthermore, PFOA treatment (2.5 and 25 μg/ml) evoked a transient rise in intracellular calcium concentration [Ca2+]i by activating the sperm-specific CatSper channel. However, preincubation with PFOA (2.5-25 μg/ml) for 4 h significantly suppressed P4-stimulated extracellular Ca2+ influx in human spermatozoa. Moreover, PFOA pretreatment at all concentrations evaluated markedly compromised P4-induced acrosome reaction and sperm penetration into viscous medium. Taken together, these results suggest that PFOA exposure may impair human sperm function through inducing oxidative stress and disturbing P4-induced Ca2+ signaling.

MicroRNA-135a-5p reduces P2X7 -dependent rise in intracellular calcium and protects against excitotoxicity.

Excitotoxic cell death because of the massive release of glutamate and ATP contributes to the secondary extension of cellular and tissue loss following traumatic spinal cord injury (SCI). Evidence from blockage experiments suggests that over-expression and activation of purinergic receptors, especially P2X7 , produces excitotoxicity in neurodegenerative diseases and trauma of the central nervous system. We hypothesize that the down-regulation of specific miRNAs after the SCI contributes to the over-expression of P2X7 and that restorative strategies can be used to reduce the excitotoxic response. In the present study, we have employed bioinformatic analyses to identify microRNAs whose down-regulation following SCI can be responsible for P2X7 over-expression and excitotoxic activity. Additional luciferase assays validated microRNA-135a-5p (miR-135a) as a posttranscriptional modulator of P2X7 . Moreover, gene expression analysis in spinal cord samples from a rat SCI model confirmed that the decrease in miR-135a expression correlated with P2X7 over-expression after injury. Transfection of cultures of Neuro-2a neuronal cell line with a miR-135a inhibitory sequences (antagomiR-135a), simulating the reduction of miR-135a observed after SCI, resulted in the increase of P2X7 expression and the subsequent ATP-dependent rise in intracellular calcium concentration. Conversely, a restorative strategy employing miR-135a mimicked reduced P2X7 expression, attenuating the increase in intracellular calcium concentration that depends on this receptor and protecting cells from excitotoxic death. Therefore, we conclude that miR-135a is a potential therapeutic target for SCI and that restoration of its expression may reduce the deleterious effects of ATP-dependent excitotoxicity induced after a traumatic spinal cord injury.

Calcium-activated chloride channels: Potential targets for antinociceptive therapy.

The molecular identity of calcium-activated chloride channels (CaCCs) was clarified only some ten years ago when it was linked to the family of "transmembrane proteins of unknown function 16″ (TMEM16). Since then, numerous studies have been conducted both to define their role in physiology and identify their biophysical functions. For the latter, the ultrastructural description of mouse TMEM16 A was a breakthrough. CaCCs were functionally described in a number of different tissues including first-order sensory neurons. The activating rise in intracellular calcium concentration can be caused by an influx of calcium through other calcium permeable ion channels. Calcium release from intracellular stores, mediated by G-protein coupled receptors, also leads to CaCC activation. Prominent inflammatory mediators like bradykinin or serotonin stimulate CaCCs via such a mechanism. The (patho) physiological function of these ion channels renders them promising targets for antinociceptive treatment.

Understanding the pathobiology in patent ductus arteriosus in prematurity-beyond prostaglandins and oxygen.

The ductus arteriosus (DA) is probably the most intriguing vessel in postnatal hemodynamic transition. DA patency in utero is an active state, in which prostaglandin E2 (PGE2) and nitric monoxide (NO), play an important role. Since the DA gets programmed for postnatal closure as gestation advances, in preterm infants the DA frequently remains patent (PDA). PGE2 exposure programs functional postnatal closure by inducing gene expression of ion channels and phosphodiesterases and anatomical closure by inducing intimal thickening. Postnatally, oxygen inhibits potassium and activates calcium channels, which ultimately leads to a rise in intracellular calcium concentration consequently inducing phosphorylation of the myosin light chain and thereby vasoconstriction of the DA. Since ion channel expression is lower in preterm infants, oxygen induced functional vasoconstriction is attenuated in comparison with full term newborns. Furthermore, the preterm DA is more sensitive to both PGE2 and NO compared to the term DA pushing the balance toward less constriction. In this review we explain the physiology of DA patency in utero and subsequent postnatal functional closure. We will focus on the pathobiology of PDA in preterm infants and the (un)intended effect of antenatal exposure to medication on both fetal and neonatal DA vascular tone.

Histamine causes influx via T-type voltage-gated calcium channels in an enterochromaffin tumor cell line: potential therapeutic target in adverse food reactions.

The P-STS human ileal neuroendocrine tumor cells, as a model for gut enterochromaffin cells, are strongly and synergistically activated by histamine plus acetylcholine (ACh), presumably via histamine 4 receptors, and weakly activated by histamine alone. Sensing these signals, enterochromaffin cells could participate in intestinal intolerance or allergic reactions to food constituents associated with elevated histamine levels. In this study we aimed to analyze the underlying molecular mechanisms. Inhibition by mepyramine and mibefradil indicated that histamine alone caused a rise in intracellular calcium concentration ([Ca2+]i) via histamine 1 receptors involving T-type voltage-gated calcium channels (VGCCs). Sensitivity to histamine was enhanced by pretreatment with the inflammatory cytokine tumor necrosis factor-α (TNF-α). In accordance with the relief it offers some inflammatory bowel disease patients, otilonium bromide, a gut-impermeable inhibitor of T-type (and L-type) VGCCs and muscarinic ACh receptors, efficiently inhibited the [Ca2+]i responses induced by histamine plus ACh or by histamine alone in P-STS cells. It will take clinical studies to show whether otilonium bromide has promise for the treatment of adverse food reactions. The cells did not react to the nutrient constituents glutamate, capsaicin, cinnamaldehyde, or amylase-trypsin inhibitors and the transient receptor potential channel vanilloid 4 agonist GSK-1016790A. The bacterial product butyrate evoked a rise in [Ca2+]i only when added together with ACh. Lipopolysaccharide had no effect on [Ca2+]i despite the presence of Toll-like receptor 4 protein. Our results indicate that inflammatory conditions with elevated levels of TNF-α might enhance histamine-induced serotonin release from intestinal neuroendocrine cells. NEW & NOTEWORTHY We show that histamine synergistically enhances the intracellular calcium response to the physiological agonist acetylcholine in human ileal enterochromaffin tumor cells. This synergistic activation and cell activation by histamine alone largely depend on T-type voltage-gated calcium channels and are inhibited by the antispasmodic otilonium bromide. The cells showed no response to wheat amylase-trypsin inhibitors, suggesting that enterochromaffin cells are not directly involved in nongluten wheat sensitivity.

Potent PDZ-Domain PICK1 Inhibitors that Modulate Amyloid Beta-Mediated Synaptic Dysfunction

Protein interacting with C kinase (PICK1) is a scaffolding protein that is present in dendritic spines and interacts with a wide array of proteins through its PDZ domain. The best understood function of PICK1 is regulation of trafficking of AMPA receptors at neuronal synapses via its specific interaction with the AMPA GluA2 subunit. Disrupting the PICK1-GluA2 interaction has been shown to alter synaptic plasticity, a molecular mechanism of learning and memory. Lack of potent, selective inhibitors of the PICK1 PDZ domain has hindered efforts at exploring the PICK1-GluA2 interaction as a therapeutic target for neurological diseases. Here, we report the discovery of PICK1 small molecule inhibitors using a structure-based drug design strategy. The inhibitors stabilized surface GluA2, reduced Aβ-induced rise in intracellular calcium concentrations in cultured neurons, and blocked long term depression in brain slices. These findings demonstrate that it is possible to identify potent, selective PICK1-GluA2 inhibitors which may prove useful for treatment of neurodegenerative disorders.

TrpC5 regulates differentiation through the Ca2+/Wnt5a signalling pathway in colorectal cancer.

Transient receptor potential channel 5 (TrpC5) is a member of the TrpC subgroup, and it forms a receptor-activated, non-selective Ca2+ channel. The architecture of the TrpC5 channel is poorly understood. In the present study, we report that TrpC5 is a key factor in regulating differentiation in colorectal cancer (CRC). Through a study of specimens from a large cohort of patients with CRC, we found that TrpC5 was highly expressed and its cellular level correlated with tumour grade. We showed further that up-regulated TrpC5 caused a robust rise in intracellular calcium concentration [Ca2+]i, increased Wnt5a expression and the nuclear translocation of β-catenin, leading to a reduction in cancer differentiation and an increase in cancer cell stemness. Notably, patients with tumours that expressed high levels of TrpC5 showed significantly poorer disease-free and overall survival. Therefore, our findings suggest that TrpC5 is an independent adverse prognostic factor for death in CRC, reducing differentiation through the Ca2+/Wnt5a signalling pathway.

In silico approach to ascertain the calcium dependent role of Plasmodium falciparum SERA5

The P. falciparum serine repeat antigen (PfSERA5) is the most abundantly expressed protein in the parasitophorous vacuole during the asexual blood stage and serves as both drug and vaccine target. The processed central fragment (56 KDa) of PfSERA5 is implicated to play an important role in parasite exit (egress) during schizont rupture from erythrocytes. Structural characterization of its enzymatic domain supports protease-like function for this central domain. The understanding of exact functional role of PfSERA5 in parasite egress remains unconfirmed as recent studies also indicate an indispensable non-catalytic role for PfSERA5 putative enzyme domain in the blood stage. No structural insight into PfSERA5 prodomain is available. Structure prediction of PfSERA5 prodomain using in silico approach in our study, showed it to have structural similarity with calcium-binding proteins. An earlier observation of steep rise in intracellular calcium concentration as an important factor in egress makes the prodomain calcium-binding role significant. The implication of calcium on structure and activity of PfSERA5 putative enzyme domain is also unknown, and such information would aid to substantiating any calcium-dependent effects on PfSERA5. To understand this, we performed molecular dynamic (MD) simulation both in the presence and absence of calcium. MD results show secondary structure conformational differences in local regions of protein structure. Our results support calcium to be an important parameter for stability and function of PfSERA5. This computational assessment suggest a need to design future experiments like calcium-dependent inhibition studies to reveal exact functional role of PfSERA5 in parasite egress.

Virus and calcium: an unexpected tandem to optimize insecticide efficacy.

The effective control of insect pests is based on the rational use of the most efficient and safe insecticide treatments. To increase the effects of classical insecticides and to avoid the ability of certain pest insects to develop resistance, it is essential to propose novel strategies. Previous studies have shown that calcium-dependent phosphorylation/dephosphorylation is now considered as a new cellular mechanism for increasing the target sensitivity to insecticides. Because it is known that virus entry is correlated with intracellular calcium concentration rise, this report attempts to present the most important data relevant to the feasibility of combining an insect virus such as baculovirus or densovirus with an insecticide. In this case, the insect virus is not used as a bioinsecticide but acts as a synergistic agent able to trigger calcium rise and to activate calcium-dependent intracellular signalling pathways involved in the increase of the membrane receptors and/or ion channels sensitivity to insecticides. This virus-insecticide mixture represents a promising alternative to optimize the efficacy of insecticides against insect pests while reducing the doses.

Activity-dependent regulation of calcium and ribosomes in the chick cochlear nucleus

Cochlea removal results in the death of 20–30% of neurons in the chick cochlear nucleus, nucleus magnocellularis (NM). Two potentially cytotoxic events, a dramatic rise in intracellular calcium concentration ([Ca2+]i) and a decline in the integrity of ribosomes are observed within 1h of deafferentation. Glutamatergic input from the auditory nerve has been shown to preserve NM neuron health by activating metabotropic glutamate receptors (mGluRs), maintaining both normal [Ca2+]i and ribosomal integrity. One interpretation of these results is that a common mGluR-activated signaling cascade is required for the maintenance of both [Ca2+]i and ribosomal integrity. This could happen if both responses are influenced directly by a common messenger, or if the loss of mGluR activation causes changes in one component that secondarily causes changes in the other. The present studies tested this common-mediator hypothesis in slice preparations by examining activity-dependent regulation of [Ca2+]i and ribosomes in the same tissue after selectively blocking group I mGluRs (1-Aminoindan-1,5-dicarboxylic acid (AIDA)) or group II mGluRs (LY 341495) during unilateral auditory nerve stimulation. Changes in [Ca2+]i of NM neurons were measured using fura-2 ratiometric calcium imaging and the tissue was subsequently processed for Y10B immunoreactivity (Y10B-ir), an antibody that recognizes a ribosomal epitope. The group I mGluR antagonist blocked the activity-dependent regulation of both [Ca2+]i and Y10B-ir, but the group II antagonist blocked only the activity-dependent regulation of Y10B-ir. That is, even when group II receptors were blocked, stimulation continued to maintain low [Ca2+]i, but it did not maintain Y10B-ir. These results suggest a dissociation in how calcium and ribosomes are regulated in NM neurons and that ribosomes can be regulated through a mechanism that is independent of calcium regulation.

Membrane potential governs calcium influx into microvascular endothelium: integral role for muscarinic receptor activation.

In resistance arteries, coupling a rise of intracellular calcium concentration ([Ca(2+)]i) to endothelial cell hyperpolarization underlies smooth muscle cell relaxation and vasodilatation, thereby increasing tissue blood flow and oxygen delivery. A controversy persists as to whether changes in membrane potential (V(m)) alter endothelial cell [Ca(2+)]i. We tested the hypothesis that V(m) governs [Ca(2+)]i in endothelium of resistance arteries by performing Fura-2 photometry while recording and controlling V(m) of intact endothelial tubes freshly isolated from superior epigastric arteries of C57BL/6 mice. Under resting conditions, [Ca(2+)]i did not change when V(m) shifted from baseline (∼-40 mV) via exposure to 10 μM NS309 (hyperpolarization to ∼-80 mV), via equilibration with 145 mm [K(+)]o (depolarization to ∼-5 mV), or during intracellular current injection (±0.5 to 5 nA, 20 s pulses) while V(m) changed linearly between ∼-80 mV and +10 mV. In contrast, during the plateau (i.e. Ca(2+) influx) phase of the [Ca(2+)]i response to approximately half-maximal stimulation with 100 nm ACh (∼EC50), [Ca(2+)]i increased as V(m) hyperpolarized below -40 mV and decreased as V(m) depolarized above -40 mV. The magnitude of [Ca(2+)]i reduction during depolarizing current injections correlated with the amplitude of the plateau [Ca(2+)]i response to ACh. The effect of hyperpolarization on [Ca(2+)]i was abolished following removal of extracellular Ca(2+), was enhanced subtly by raising extracellular [Ca(2+)] from 2 mm to 10 mm and was reduced by half in endothelium of TRPV4(-/-) mice. Thus, during submaximal activation of muscarinic receptors, V(m) can modulate Ca(2+) entry through the plasma membrane in accord with the electrochemical driving force.

Amlodipine protects rat ventricular cardiomyoblast H9c2 From hypoxia-induced apoptosis and restores oxidative balance by Akt-1-dependent manner.

Hypoxia-induced rise in intracellular calcium concentration is a causative agent of apoptosis and oxidative damage in cardiomyocytes. We examined the efficacy of calcium channel blocker amlodipine in preventing hypoxia-induced apoptosis in H9c2 cells and restoring oxidative balance. H9c2 cells were exposed to hypoxia (0.5% oxygen) to evaluate the efficacy of amlodipine in restoring cellular calcium levels. Cellular markers of apoptosis (Bax/Bcl2 and caspase-3, -7, and -9) and pro-survival markers (Akt/p-Akt levels) were evaluated under hypoxia. Redox damage was evaluated by assessing markers of oxidative damage, that is, glutathione reduced, glutathione oxidized, lipid peroxidation, reactive oxygen species, and manganese superoxide dismutase activity. Cellular adenosine triphosphate (ATP) pool and AMPKα levels were measured to evaluate regulation of metabolism under hypoxia. Amlodipine treatment at 25 nM prevented apoptosis and restored cellular calcium levels and oxidative damage in cardiomyocytes. Stabilization of caspase-3, -7, and -9 along with restoration of Akt/p-Akt levels depicted pro-survival efficacy of amlodipine. Also, restoration of cellular ATP and AMPKα levels indicates that amlodipine prevents cardiomyocytes from hypoxia-induced metabolic stress. Amlodipine thus protects H9c2 cells from hypoxia-induced apoptosis by regulating Akt/p-Akt-mediated caspase-3, -7, and -9 activation and restoring cellular ATP and redox status.