Increase In Intracellular Calcium Concentration Research Articles

P2 receptor regulation of [Ca2+]i in cultured mouse mesangial cells.

Experiments were performed to establish the pharmacological profile of purinoceptors and to identify the signal transduction pathways responsible for increases in intracellular calcium concentration ([Ca(2+)](i)) for cultured mouse mesangial cells. Mouse mesangial cells were loaded with fura 2 and examined using fluorescent spectrophotometry. Basal [Ca(2+)](i) averaged 102 +/- 2 nM (n = 346). One hundred micromolar concentrations of ATP, ADP, 2',3'-(benzoyl-4-benzoyl)-ATP (BzATP), ATP-gamma-S, and UTP in normal Ca(2+) medium evoked peak increases in [Ca(2+)](i) of 866 +/- 111, 236 +/- 18, 316 +/- 26, 427 +/- 37, and 808 +/- 73 nM, respectively. UDP or 2-methylthio-ATP (2MeSATP) failed to elicit significant increases in [Ca(2+)](i), whereas identical concentrations of adenosine, AMP, and alpha,beta-methylene ATP (alpha,beta-MeATP) had no detectable effect on [Ca(2+)](i). Removal of Ca(2+) from the extracellular medium had no significant effect on the peak increase in [Ca(2+)](i) induced by ATP, ADP, BzATP, ATP-gamma-S, or UTP compared with normal Ca(2+); however, Ca(2+)-free conditions did accelerate the rate of decline in [Ca(2+)](i) in cells treated with ATP and UTP. [Ca(2+)](i) was unaffected by membrane depolarization with 143 mM KCl. Western blot analysis for P2 receptors revealed expression of P2X(2), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. No evidence of P2X(1) and P2X(3) receptor expression was detected, whereas RT-PCR analysis reveals mRNA expression for P2X(1), P2X(2), P2X(3), P2X(4), P2X(7), P2Y(2), and P2Y(4) receptors. These data indicate that receptor-specific P2 receptor activation increases [Ca(2+)](i) by stimulating calcium influx from the extracellular medium and through mobilization of Ca(2+) from intracellular stores in cultured mouse mesangial cells.

Calcium-mediated Transient Phosphorylation of Tau and Amyloid Precursor Protein Followed by Intraneuronal Amyloid-β Accumulation

Intraneuronal accumulation of hyperphosphorylated protein tau in paired helical filaments together with amyloid-beta peptide (Abeta) deposits confirm the clinical diagnosis of Alzheimer disease. A common cellular mechanism leading to the production of these potent toxins remains elusive. Here we show that, in cultured neurons, membrane depolarization induced a calcium-mediated transient phosphorylation of both microtubule-associated protein tau and amyloid precursor protein (APP), followed by a dephosphorylation of these proteins. Phosphorylation was mediated by glycogen synthase kinase 3 and cyclin-dependent kinase 5 protein kinases, while calcineurin was responsible for dephosphorylation. Following the transient phosphorylation of APP, intraneuronal Abeta accumulated and induced neurotoxicity. Phosphorylation of APP on Thr-668 was indispensable for intraneuronal accumulation of Abeta. Our data demonstrate that an increase in cytosolic calcium concentration induces modifications of neuronal metabolism of APP and tau, similar to those found in Alzheimer disease.

Resistin as an Intrahepatic Cytokine: Overexpression during Chronic Injury and Induction of Proinflammatory Actions in Hepatic Stellate Cells

Obesity and insulin resistance accelerate the progression of fibrosis during chronic liver disease. Resistin antagonizes insulin action in rodents, but its role in humans is still controversial. The aims of this study were to investigate resistin expression in human liver and to evaluate whether resistin may affect the biology of activated human hepatic stellate cells (HSCs), key modulators of hepatic fibrogenesis. Resistin gene expression was low in normal human liver but was increased in conditions of severe fibrosis. Up-regulation of resistin during chronic liver damage was confirmed by immunohistochemistry. In a group of patients with alcoholic hepatitis, resistin expression correlated with inflammation and fibrosis, suggesting a possible action on HSCs. Exposure of cultured HSCs to recombinant resistin resulted in increased expression of the proinflammatory chemokines monocyte chemoattractant protein-1 and interleukin-8, through activation of nuclear factor (NF)-kappaB. Resistin induced a rapid increase in intracellular calcium concentration, mainly through calcium release from intracellular inositol triphosphate-sensitive pools. The intracellular calcium chelator BAPTA-AM blocked resistin-induced NF-kappaB activation and monocyte chemoattractant protein-1 expression. In conclusion, this study shows a role for resistin as an intrahepatic cytokine exerting proinflammatory actions in HSCs, via a Ca2+/NF-kappaB-dependent pathway and suggests involvement of this adipokine in the pathophysiology of liver fibrosis.

A Painful Endogenous Opioid

Dynorphin A 1-17 is derived from proteolytic processing of prodynorphin and has high affinity for μ, δ, and κ opioid receptors. However, increased amounts of dynorphin A are found in models of chronic pain, and antibodies against dynorphin A alleviate, rather than potentiate, pain. Lai et al . show that a fragment of dynorphin A that has low affinity for the opioid receptors (dynorphin A 2-13 ) stimulated a transient increase in intracellular calcium concentration in cultured embryonic dorsal root ganglion (DRG) cells and in F-11 cells, a neuroblastoma-DRG hybridoma. The increase in intracellular calcium was blocked by selective inhibition of L-type or P/Q-type voltage-sensitive calcium channels. A bradykinin B2 antagonist blocked the dynorphin-induced increase in intracellular calcium, and transfection of either the bradykinin B2 or B1 receptor reconstituted dynorphin A 2-13 responsiveness in cells that had spontaneously lost the ability to respond to dynorphin A. Dynorphin A 2-13 or dynorphin A 1-17 competed for binding to B1 or B2 receptors, suggesting that dynorphin is acting as an agonist at the bradykinin receptors. In rats, intrathecal injection of dynorphin A 2-13 caused enhanced sensitivity to touch and heat, and these effects were reversed by injection of a B2 receptor antagonist. Furthermore, knockout mice lacking B2 receptors did not show hypersensitive responses in response to dynorphin A 2-13 injection. Finally, in a rat model of neuropathic pain, B2 receptor and prodynorphin transcript abundance were increased in the DRG of rats as early as day 2 after nerve injury, and treatment with a B2 antagonist after nerve injury prevented hypersensitivity responses. As discussed by Altier and Zamponi, it appears that under conditions of chronic pain, enhanced production of dynorphin in the spinal cord produces a hyperalgesia response instead of an analgesic response through the ability of dynorphin to activate spinal bradykinin receptors. J. Lai, M.-C. Luo, Q. Chen, S. Ma, L. R. Gardell, M. H. Ossipov, F. Porreca, Dynorphin A activates bradykinin receptors to maintain neuropathic pain. Nat. Neurosci. 9 , 1534-1540 (2006). [PubMed] C. Altier, G. W. Zamponi, Opioid, cheating on its receptors, exacerbates pain. Nat. Neurosci. 9 , 1465-1467 (2006). [PubMed]

Depolarization‐induced retrograde synaptic inhibition in the mouse cerebellar cortex is mediated by 2‐arachidonoylglycerol

Endocannabinoids acting on CB(1) cannabinoid receptors are involved in short- and long-term depression of synaptic transmission. The aim of the present study was to determine which endocannabinoid, anandamide or 2-arachidonoylglycerol (2-AG), is involved in depolarization-induced suppression of inhibition (DSI) in the cerebellar cortex, which is the most widely studied form of short-term depression. Depolarization of Purkinje cells in the mouse cerebellum led to an increase in intracellular calcium concentration and to suppression of the inhibitory input to these neurons (i.e. DSI occurred). Orlistat and RHC80267, two blockers of sn-1-diacylglycerol lipase, the enzyme catalysing 2-AG formation, abolished DSI by acting downstream of calcium influx. In contrast, DSI occurred also in the presence of a phospholipase C inhibitor. Intact operation of the calcium-dependent messengers calmodulin and Ca(2+)-calmodulin-dependent protein kinase II were necessary for DSI. DSI was potentiated by an inhibitor of the main 2-AG-degrading enzyme, monoacylglycerol lipase. Interference with the anandamide metabolizing enzyme, fatty acid amide hydrolase, did not modify DSI. Thus, three kinds of observations identified 2-AG as the endocannabinoid involved in DSI in the mouse cerebellum: DSI was abolished by diacylglycerol lipase inhibitors; DSI was potentiated by a monoglyceride lipase inhibitor; and DSI was not changed by an inhibitor of fatty acid amide hydrolase. Further experiments indicated that 2-AG is the endocannabinoid mediating short-term retrograde signalling also at other synapses: orlistat abolished DSI in the rat cerebellum, DSI in the mouse substantia nigra pars reticulata and depolarization-induced suppression of excitation in the mouse cerebellum.

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

Sphingosine 1-phosphate protects rat liver sinusoidal endothelial cells from ethanol-induced apoptosis: Role of intracellular calcium and nitric oxide

In alcoholic liver disease, ethanol-induced damage to sinusoidal endothelial cells (SECs) appears to be important in the progression of liver damage. However, little is known about the mechanisms responsible for protection of SECs against ethanol-induced injury. To elucidate the role of sphingosine 1-phosphate (S1P), which is stored in platelets and may be released from them on their activation, we investigated the effect of S1P on rat liver SECs in primary culture. Pretreatment of cells with 1 mumol/L S1P attenuated ethanol-induced apoptosis. Electron microscopy confirmed this protective effect of S1P on damaged SECs in liver tissues after perfusion of ethanol. In the absence of ethanol, S1P increased DNA synthesis as determined via incorporation of bromodeoxyuridine. S1P also ameliorated the decreased DNA synthesis of cells induced by ethanol. Addition of S1P to cells induced an increase in intracellular calcium concentrations and NO production in cells. Western blotting revealed that S1P significantly induced the activation of endothelial NO synthase (eNOS), but not Akt, and that S1P-induced activation of eNOS was blocked by trifluoperazine, a calmodulin inhibitor. Furthermore, N(G)-nitro-L-arginine methyl ester, a NO synthase inhibitor, cancelled the effect of S1P on DNA synthesis, apoptosis, and NO production in vitro as well as the protective effect of S1P on cell damage in situ. In conclusion, the biological effect of S1P is at least partially mediated by Ca(2+)-sensitive eNOS activation and subsequent NO formation; extracellular S1P could contribute to sinusoidal protection and remodeling in alcoholic liver injury.

Isometric contraction of microvascular pericytes from mouse brain parenchyma

Pericytes were isolated and cultured from mouse cerebroparenchymal microvessels. A single pericyte clone was three-dimensionally cultured in a collagen gel by adding tensile stress, resulting in the reconstruction of narrow stringy fibers. When the contractility of these fibers was evaluated isometrically, they contracted in response to acetylcholine (ACh) 1 or noradrenaline; this was accompanied by an increase in intracellular calcium concentration ([Ca 2+] i). The fibers that were pre-contracted by ACh were completely relaxed by papaverine, which is a smooth-muscle relaxant. Moreover, the muscarinic ACh receptor-antagonist atropine depressed the [Ca 2+] i response that was induced by ACh. This study demonstrates for the first time the quantitative measurement of the contractions produced by cultured microvascular pericytes from mouse brain parenchyma.

Opposite diastereoselective activation of P2Y1 and P2Y11 nucleotide receptors by adenosine 5′‐O‐(α‐boranotriphosphate) analogues

We explored the stereoselective activation of the P2Y11 receptor, stably expressed and tagged with GFP, in 1321N1 cells, in comparison to its closest homologue, the P2Y1 receptor. The potency of several chiral ATP analogues was determined by measuring increases in intracellular calcium concentration ([Ca2+]i). In a series of ATP-alpha-B and ATP-alpha-S analogues, a non-bridging oxygen atom of Palpha was substituted by BH3 or sulphur, respectively, introducing a chiral center at Palpha. The pairs of diastereoisomers (A and B isomers) were each applied as purified compounds. The (B) isomers (ATP-alpha-B Sp isomers and ATP-alpha-S Rp isomers) of all derivatives tested were more potent at the P2Y11 receptor than the corresponding (A) isomers (ATP-alpha-B Rp isomers and ATP-alpha-S Sp isomers) and the parent compounds. This characteristic of the P2Y11 receptor is opposite to the behaviour of the same diastereoisomers at the P2Y1 receptor, at which the (A) isomers are more active. The distinctly opposite diastereoselective activity of ATP derivatives at the P2Y11 and the P2Y1 receptor will allow the deciphering of structural differences of the ligand recognition sites between these receptor subtypes and may aid in the development of subtype-selective agonists. Moreover, ATP-alpha-B diastereoisomers are not active at the P2Y2 receptor. Thus, they are compounds suitable for distinguishing the functional contribution of the two ATP-activated P2Y receptors, the P2Y2 and P2Y11 receptor, in physiological or pathophysiological responses of cells.

SDF-1-induced adhesion of monocytes to vascular endothelium is modulated by azelnidipine via protein kinase C inhibition

Monocyte–endothelial interaction and its modulation by chemokines play a key role in atherogenesis and inflammation. We examined the potential effects of stromal cell-derived factor (SDF-1) and azelnidipine, a novel dihydropyridine derivative, toward monocyte–endothelial interaction. Human monocytes were prepared from peripheral blood mononuclear cells obtained from healthy volunteers and pretreated with azelnidipine (1 μM) for 48 h, after which their adhesion to interleukin-1β (IL-1β)-activated human umbilical vein endothelial cells (HUVECs) was analyzed using an in vitro flow apparatus with a shear stress of 1 dyn/cm 2. In some experiments, monocytes were incubated in the presence of stromal cell-derived factor (SDF-1), a chemokine, just prior to the assay. Pre-incubation of monocytes with SDF-1 enhanced their adhesion to activated HUVECs. When monocytes were pre-incubated in the presence of azelnidipine, baseline levels as well as SDF-1-induced monocyte adhesion levels were reduced. Interestingly, the surface expressions of the adhesion molecules CD11a, CD11b, and CD36, were not changed by azelnidipine treatment. Western blotting analysis revealed that activation of protein kinase C (PKC)α was inhibited by azelnidipine treatment, while it also reduced the SDF-1-induced increase in intracellular calcium concentration ([Ca 2+] i). Further, pre-incubation of monocytes with Go6976, a potent inhibitor of PKCα, significantly reduced monocyte adhesion to HUVECs. Our results demonstrated an inhibitory action of azelnidipine toward adhesive interactions of monocytes to HUVECs, which involves inhibition of PKCα and a reduction in [Ca 2+] i. These findings imply a protective role of azelnidipine against inflammation in atherosclerosis.

Pore-Forming Pathogens Activate the Inflammasome

Pathogenic microbes introduce pores in the host plasma membrane as part of the infectious process. Gurcel et al . determined that pore-forming toxins, such as aerolysin from Aeromonas and α-toxin from Staphylococcus aureus , stimulate the SREBP (sterol response element–binding protein) transcription factors to promote membrane repair. RNA differential display analysis suggested that aerolysin increased the expression of the gene encoding SREBP-2. Indeed, exposure of cells to either aerolysin or α-toxin increased the activity of SREBP-2. In Chinese hamster ovary cells, exposure to aerolysin stimulated proteolytic processing of SREBP-1 and SREBP-2 to release these membrane-bound transcription factors from the endoplasmic reticulum and allowed translocation to the nucleus of the transcriptionally active fragment. The increase in the activity of the SREBPs was verified by an increase in the expression of target genes and an increase in total cellular cholesterol through both increased biosynthesis and uptake (the low-density lipoprotein receptor is encoded by a SREBP-responsive gene). Mutant aerolysin subunits that could not form pores were incapable of stimulating SREBP-2 activity, and SREBP-2 activation required K + efflux but did not require an increase in intracellular calcium concentration. Indeed, exposure of cells to a K + , but not a Na + , ionophore, triggered SREBP-2 activation. Decreased intracellular K + concentration is known to activate caspase 1 through activation of inflammasomes with either the IPAF or NALP3 subunits, which are intracellular pattern recognition receptors. When RNA interference was used to knock down subunits of the IPAF or NALP3 inflammasome, the activation of caspase 1 and SREBP-2 in response to aerolysin was diminished. Pharmacological inhibition or knockdown of caspase 1 also prevented SREBP-2 activation. SREBP-2 was not directly cleaved by caspase 1; instead, caspase 1 activated the known SREBP-activating pathway that involves SCAP (an escort protein) and S1P and S2P (the two enzymes responsible for SREBP proteolysis and release). Using various pharmacological and RNAi approaches, the authors showed that activation of the inflammasome caspase 1 and the SREBPs was required for cell survival after exposure to aerolysin. Saleh discusses these findings. L. Gurcel, L. Abrami, S. Girardin, J. Tschopp, F. G. van der Goot, Caspase-1 activation of lipid metabolic pathways in response to bacterial pore-forming toxins promotes cell survival. Cell 126 , 1135-1145 (2006). [Online Journal] M. Saleh, Caspase-1 builds a new barrier to infection. Cell 126 , 1028-1030 (2006). [Online Journal]

Dissociated GABAergic retinal interneurons exhibit spontaneous increases in intracellular calcium

Early in development, before the retina is responsive to light, neurons exhibit spontaneous activity. Recently it was demonstrated that starburst amacrine cells, a unique class of neurons that secretes both GABA and acetylcholine, spontaneously depolarize. Networks comprised of spontaneously active starburst cells initiate correlated bursts of action potentials that propagate across the developing retina with a periodicity on the order minutes. To determine whether other retinal interneurons have similar "pacemaking" properties, we have utilized cultures of dissociated neurons from the rat retina. In the presence of antagonists for fast neurotransmitter receptors, distinct populations of neurons exhibited spontaneous, uncorrelated increases in intracellular calcium concentration. These increases in intracellular calcium concentration were sensitive to tetrodotoxin, indicating they are mediated by spontaneous membrane depolarizations. By combining immunofluorescence and calcium imaging, we found that 44% of spontaneously active neurons were GABAergic and included starburst amacrine cells. Whole cell voltage clamp recordings in the absence of antagonists for fast neurotransmitters revealed that after 7 days in culture, individual retinal neurons receive bursts of GABA-A receptor mediated synaptic input with a periodicity similar to that measured in spontaneously active GABAergic neurons. Low concentrations of GABA-A receptor antagonists did not alter the inter-burst interval despite significant reduction of post-synaptic current amplitude, indicating that pacemaker activity of GABAergic neurons was not influenced by network interactions. Together, these findings indicate that spiking GABAergic interneurons can function as pacemakers in the developing retina.

Changes in Purinoceptor Distribution and Intracellular Calcium Levels following Noise Exposure in the Outer Hair Cells of the Guinea Pig

Among the cells of the inner ear, the outer hair cells (OHCs) are the most important targets of noise-induced effects, being the most sensitive cell types. The aim of this study was to examine the effects of noise (50 Hz-20 kHz, 80 dB sound pressure level, 14 days) on intracellular calcium levels and on the expression pattern of purinoceptors in the membrane of the OHCs of the guinea pig and to measure the stiffness changes of the lateral membrane of these cells. In noise-exposed animals, the resting intracellular calcium concentration increased compared to nontreated animals and was slightly higher in the cells of the basal (219 +/- 29 nM: ) than in the apical (181 +/- 24 nM: ) turns of the cochlea. After application of 180 muM: adenosine triphosphate, the intracellular calcium level rose by 60 +/- 22 nM: in cells from the apical and by 44 +/- 10 nM: in cells from the basal turns, significantly less than in nontreated animals. Expression of the P(2X1), P(2X2), P(2X4), P(2X7), P(2Y1) and P(2Y4) receptor subtypes was suppressed, while expression of the P(2Y2) subtype did not decrease in either of the two preparations. In parallel with the increase in intracellular calcium concentration, the stiffness of the lateral wall of the OHCs was increased. Noise-induced changes in intracellular calcium homeostasis and subsequently in the calcium-dependent regulatory mechanisms may modify OHC lateral wall stiffness and may lead to reduction of the efficacy of the cochlear amplifier.

Habitual dietary calcium intake and body weight in 7–10 year old children

PurposeSeveral recent observational studies detected inverse associations between dietary calcium intake and body weight. It was demonstrated that low calcium diets lead to an increase in intracellular calcium concentrations, which in turn act to promote body fat deposition, reduce lipolysis and reduce thermogenesis. Most of the studies have been conducted on adults, however, it was recently demonstrated that longitudinal calcium intake is negatively associated with children's body fat levels. The purpose of the current study is to investigate possible associations between habitual calcium intake and body weight in a group of 7–10 years old children.Design/methodology/approachEighty‐five children, 21 boys and 64 girls (mean age: 9.2±0.9) were recruited from 12 primary schools in the London area. Dietary intake was measured using the 7‐day weighed inventory method. Body weight and height measurements were also recorded.FindingsData suggested that girls have significantly lower intakes of calcium than boys and that 48 per cent of boys and 38 per cent of girls were overweight (above the 91st centile). However, there were no significant correlations between body weight or body mass index (BMI) and habitual intake of dietary calcium in this age group, which is in contrast with the results of similar studies conducted in adults.Originality/valueOne explanation could be that the possible effect of calcium on adiposity and body weight is more pronounced in adulthood than in childhood. It is important for future studies to measure levels of body fat in children together with body weight in conjunction with calcium intake in order to elucidate the original hypothesis.

Pucker Up: Sour Taste Receptors Revealed

Although the receptors for most taste modalities have been identified, the mechanisms by which sour and salty tastes are detected have remained elusive. Ishimaru et al. show that two members of the transient receptor potential (TRP) family, PKD1L3 and PKD2L1, are expressed in particular regions of taste tissue (in situ mRNA hybridization and reverse transcription polymerase chain reaction analysis) and that their expression does not overlap with that of TRPM5, a downstream effector in some forms of taste perception. Antibodies raised against PKD2L1 showed that the protein was present at the taste pore, the predicted location of a taste receptor. Coexpression of tagged versions of the two proteins in human embryonic kidney (HEK) 293 cells showed that both proteins had to be coexpressed for abundant surface expression, and coimmunoprecipitation experiments indicated that the proteins interacted. Electrophysiological analysis and calcium imaging showed that HEK293 cells expressing the two proteins responded to acids, such as citric acid, HCl, and malic acid, with a rapidly inactivating current and with an increase in intracellular calcium concentration. The response did not appear to be mediated solely by a change in pH, because citric acid was more potent at activating the channel than HCl at the same pH. Y. Ishimaru, H. Inada, M. Kubota, H. Zhuang, M. Tominaga, H. Matsunami, Transient receptor potential family members PKD1L3 and PKD2L1 form a candidate sour taste receptor. Proc. Natl. Acad. Sci. U.S.A. 103 , 12569-12574 (2006). [Abstract] [Full Text]

Aryl Hydrocarbon Receptor- and Calcium-dependent Induction of the Chemokine CCL1 by the Environmental Contaminant Benzo[a]pyrene

Polycyclic aromatic hydrocarbons (PAHs) are widely distributed immunotoxic environmental contaminants well known to regulate expression of pro-inflammatory cytokines such as interleukine-1beta and tumor necrosis factor-alpha. In the present study, we demonstrated that the chemokine CCL1, notably involved in cardiovascular diseases and inflammatory or allergic processes, constitutes a new molecular target for PAHs. Indeed, exposure to PAHs such as benzo[a]pyrene (BP) markedly increased mRNA expression and secretion of CCL1 in primary human macrophage cultures. Moreover, intranasal administration of BP to mice enhanced mRNA levels of TCA3, the mouse orthologue of CCL1, in lung. CCL1 induction in cultured human macrophages was fully prevented by targeting the aryl hydrocarbon receptor (AhR) through chemical inhibition or small interfering RNA-mediated down-modulation of its expression. In addition, BP and the potent AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin were found to enhance activity of a CCL1 promoter sequence containing a consensus xenobiotic-responsive element known to specifically interact with AhR. Moreover, 2,3,7,8-tetrachlorodibenzo-p-dioxin triggered AhR binding to this CCL1 promoter element as revealed by chromatin immunoprecipitation experiments and electrophoretic mobility shift assays. In an attempt to further characterize the mechanism of CCL1 induction, we demonstrated that BP was able to induce an early and transient increase of intracellular calcium concentration in human macrophages. Inhibition of this calcium increase, using the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester or the calcium store-operated channel inhibitor 2-aminoethoxydiphenyl borate, fully blocked CCL1 up-regulation. Taken together, these results bring the first demonstration that PAHs induce expression of the chemokine CCL1 in an AhR- and calcium-dependent manner.

Mitochondrial Modulation of Ca2+-Induced Ca2+-Release in Rat Sensory Neurons

Ca2+ -induced Ca2+ -release (CICR) from ryanodine-sensitive Ca2+ stores provides a mechanism to amplify and propagate a transient increase in intracellular calcium concentration ([Ca2+]i). A subset of rat dorsal root ganglion neurons in culture exhibited regenerative CICR when sensitized by caffeine. [Ca2+]i oscillated in the maintained presence of 5 mM caffeine and 25 mM K+. Here, CICR oscillations were used to study the complex interplay between Ca2+ regulatory mechanisms at the cellular level. Oscillations depended on Ca2+ uptake and release from the endoplasmic reticulum (ER) and Ca2+ influx across the plasma membrane because cyclopiazonic acid, ryanodine, and removal of extracellular Ca2+ terminated oscillations. Increasing caffeine concentration decreased the threshold for action potential-evoked CICR and increased oscillation frequency. Mitochondria regulated CICR by providing ATP and buffering [Ca2+]i. Treatment with the ATP synthase inhibitor, oligomycin B, decreased oscillation frequency. When ATP concentration was held constant by recording in the whole cell patch-clamp configuration, oligomycin no longer affected oscillation frequency. Aerobically derived ATP modulated CICR by regulating the rate of Ca2+ sequestration by the ER Ca2+ pump. Neither CICR threshold nor Ca2+ clearance by the plasma membrane Ca2+ pump were affected by inhibition of aerobic metabolism. Uncoupling electron transport with carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone or inhibiting mitochondrial Na+/Ca2+ exchange with CGP37157 revealed that mitochondrial buffering of [Ca2+]i slowed oscillation frequency, decreased spike amplitude, and increased spike width. These findings illustrate the interdependence of energy metabolism and Ca2+ signaling that results from the complex interaction between the mitochondrion and the ER in sensory neurons.

Heterogeneous CPA sensitivity of spontaneous excitation in smooth muscle of the rabbit urethra

1. To investigate the role of intracellular Ca stores in generating spontaneous excitation of the urethra, the effects of cyclopiazonic acid (CPA) on spontaneous contractions, transient increases in intracellular calcium concentration ([Ca2+]i; Ca transients) and depolarizations were examined in smooth muscles of the rabbit urethra. 2. In about 90% of circular smooth muscle (CSM) preparations, CPA (10 microM) increased the amplitude of spontaneous contractions by about 180% and reduced their frequency to some 25% of control values (CPA-resistant), while it readily abolished the contractions in the remaining preparations. 3. In about 70% of CSM preparations, CPA prevented the generation of spontaneous depolarizations termed slow waves, but increased their amplitude and duration in the remainder. CPA also prevented the generation of spontaneous Ca transients in about 40% of CSM preparations, while increasing their amplitude and duration in the remaining preparations. In CPA-resistant preparations that had been exposed to nicardipine (1 microM), subsequent CPA invariably abolished residual spontaneous depolarizations or Ca transients. CPA abolished caffeine-induced Ca transients in Ca-free solutions, suggesting that it effectively depleted intracellular Ca stores. 4. Longitudinal smooth muscles generated spontaneous action potentials, which had a shape distinct from that of slow waves in CSM. Spontaneous action potentials were abolished by nicardipine but not CPA. 5. Transmural nerve stimulation increased the frequency of Ca transients to give a sustained rise in [Ca2+]i, but inhibited their generation after blocking alpha-adrenoceptors with phentolamine (1 microM). These nerve-evoked responses were preserved in preparations that had been exposed to CPA. Similarly, both in control and CPA-treated CSM preparations, spontaneous Ca transients were accelerated by noradrenaline (NAd, 1 microM) and were suppressed by 3-morpholino-sydnonimine (SIN-1, 10 microM), a nitric oxide (NO) donor. 6. In conclusion, CSM of the urethra generates spontaneous activity, which depends on Ca release from intracellular Ca stores. However, after blocking this primary pacemaking mechanism, L-type Ca channel-dependent action potentials may drive CSM. Irrespective of the origin of pacemaking, neurally-released NAd and NO are capable of modulating spontaneous excitation.

Hydrogen sulphide regulates calcium homeostasis in microglial cells

Hydrogen sulphide (H2S), which is produced endogenously from L-cysteine in mammalian tissues, has been suggested to function as a neuromodulator in the brain. However, the role of H2S in microglial cells is unclear. In this study, the effect of exogenous and endogenous H2S on intracellular calcium homeostasis was investigated in primary cultured microglial cells. Sodium hydrosulphide (NaHS), a H2S donor, caused a concentration-dependent (0.1-0.5 mM) increase in intracellular calcium concentration ([Ca2+]i). This effect was significantly attenuated in the presence of a calcium-free extracellular solution, Gd3+ (100 microM), a nonselective Ca2+ channel blocker, or thapsigargin (2 microM), an inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase. These observations suggest that the increase in [Ca2+]i in response to H2S involves both calcium influx across the plasma membrane and calcium release from intracellular stores. The H2S-induced calcium elevation is partly attenuated by H-89, a selective cAMP-dependent protein kinase (PKA) inhibitor, but not by U73122, a phospholipase C (PLC) inhibitor, and chelerythrine, a selective protein kinase C (PKC) inhibitor, suggesting the involvement of cAMP/PKA, but not PLC/PKC/phosphoinositol-3,4,5-inositol (IP3) pathway. Using RT-PCR, only cystathionine gamma-lyase (CSE), a H2S producing enzyme, was detected in primary cultures of microglia. Lowering endogenous H2S level with, D,L-propargylglycine and beta-cyano-L-alanine, two CSE inhibitors, significantly decreased [Ca2+]i, suggesting that endogenous H2S may have a positive tonic influence on [Ca2+]i homeostasis. These findings support the possibility that H2S may serve as a neuromodulator to facilitate signaling between neurons and microglial cells.

When a sperm meets an egg: Block to polyspermy

Embryonic development is initiated after the fertilizing spermatozoon enters the egg and triggers a series of events known as egg activation. Activation results in an increase in intracellular calcium concentration, cortical granule exocytosis (CGE), cell cycle resumption and recruitment of maternal mRNA. CGE is an evolutionary developed mechanism that causes modification of the zona pellucida to prevent penetration of additional spermatozoa, ensuring successful egg activation and embryo development. The egg CGE is a unique and convenient mammalian model for studying the different proteins participating at the membrane fusion cascade, which, unlike other secretory cells, occurs only once in the egg's lifespan. This article highlights a number of proteins, ascribed to participate in CGE and thus the block to polyspermy. CGE can be triggered either by a calcium dependent pathway, or via protein kinase C (PKC) activation that requires a very low calcium concentration. In a recent study, we suggested that the filamentous actin (F-actin) at the egg's cortex is a dynamic network. It can be maneuvered towards allowing CGE by activated actin associated proteins and/or by activated PKC and its down stream proteins, such as myristoylated alanine-rich C kinase substrate (MARCKS). MARCKS, a protein known to cross-link F-actin in other cell types, was found to be expressed and colocalized with actin in non-activated MII eggs. We further demonstrated MARCKS dissociation from actin after activation by ionomycin, a process that can lead to the breakdown of the actin network, thus allowing CGE. The more we know of the intricate process of CGE and of the proteins participating in it, the more the assisted reproductive procedures might benefit from that knowledge.