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Calcium Entry (calcium + entry)
Selected AbstractsSmall-Molecule Inhibitors of Store-Operated Calcium EntryCHEMMEDCHEM, Issue 5 2009Zachary Abstract Molecules that inhibit store-operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the identification of selective inhibitors. Herein we document some examples of the small-molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors. Controlled variation in intracellular calcium concentration is a key component of the immune response signaling pathway in lymphocytes. Store-operated calcium entry (SOCE) in these cells provides a prolonged increase in cytoplasmic Ca2+ concentrations and ultimately leads to the production of pro-inflammatory cytokines. Molecules that inhibit SOCE could therefore be useful immunomodulating agents for the treatment of rheumatoid arthritis, psoriasis, inflammatory bowel disease, and other conditions. Although the presence of the SOCE signaling pathway in lymphocytes and other cells involved in the immune response has been known for many years, key proteins involved in SOCE were identified only recently. The identification of these proteins may further enable the identification of agents that inhibit SOCE without affecting other cellular processes. This contribution documents representative examples of the small-molecule inhibitors of SOCE that have been reported to date. Where possible, methods that were used to characterize the mechanism of action of the inhibitors are also described. [source] Further studies on the interaction of loperamide with capacitative calcium entry in Leukemic HL-60 cells,DRUG DEVELOPMENT RESEARCH, Issue 11 2006John W. Daly Abstract Loperamide at 3,10,µM has augmentative effects on calcium levels elevated by capacitative calcium entry (CCE) in leukemic HL-60 cells after release of intracellular calcium by ATP or thapsigargin (Harper et al. [1997] Proc Natl Acad Sci USA 94:14912,14917). The effect of loperamide on calcium levels was absent at a pH value of 6.8, a pH at which CCE is not active in HL-60 cells. Further investigations of HL-60 cells in recent years revealed a great reduction in the magnitude of the loperamide response. However, when preceded by a CCE blocker, namely N-methylnitrendipine (MRS 1844) or N-propargylnitrendipine (MRS 1845), loperamide caused a significant reversal of the blockade. Six structural analogs of loperamide were synthesized, but only two showed loperamide-like activity. Drug Dev. Res. 67:842,851, 2006. Published 2007 Wiley-Liss, Inc. [source] Ventricular PKC-, and humoral signaling in DOCA-Salt rats treated with labedipinedilol-ADRUG DEVELOPMENT RESEARCH, Issue 3 2003Jwu-Lai Yeh Abstract Effects of oral antihypertensive monotherapy with labedipinedilol-A, labetalol, atenolol, amlodipine, prazosin (20 mg kg,1 day,1), and short-acting nifedipine (3 mg kg,1 day,1) on DOCA-salt-induced translocation of ventricular protein kinase C-,(PKC-,), humoral signaling, and the cardiovascular system were investigated in rats for 4 weeks. The triple blocking activities of labedipinedilol-A (,/,-adrenoceptor blockade and calcium entry blockade) were compared with single blocking activities of selective drugs. Cytosolic PKC-, immunoreactivity was decreased by labedipinedilol-A, short-acting nifedipine, amlodipine, prazosin, labetalol, atenolol, and losartan. Membranous PKC-, immunoreactivity was significantly decreased by labedipinedilol-A, amlodipine, prazosin, labetalol, and atenolol. Labedipinedilol-A and prazosin more potently decreased membranous than cytosolic PKC-, expression. Labedipinedilol-A, labetalol, and atenolol effectively inhibited DOCA-salt-induced increases in angiotensin II (Ang II). All antihypertensive agents reduced endothelin-1 (ET-1) levels in urine and cardiac weight growth. Treatments with labedipinedilol-A, labetalol, atenolol, and amlodipine normalized DOCA-salt-induced ANP increases. Prazosin did not decrease ANP. Short-acting nifedipine elevated ANP. During long-term antihypertensive therapy in DOCA-salt hypertensive rats, single blockade drugs did not fully inhibit ventricular PKC-, translocation or Ang II, ET-1, and ANP humoral signaling. However, triple blockade labedipinedilol-A therapy had a wide range of ,/,-adrenergic receptor and calcium channel inhibitory activities, including diminished reflux tachycardia, inhibition of PKC-, translocation, and reduction of Ang II, ET-1, and ANP formation. Drug Dev. Res. 59:307,315, 2003. 2003 Wiley-Liss, Inc. [source] Independent signaling pathways in ATP-evoked secretion of plasminogen and cytokines from microgliaDRUG DEVELOPMENT RESEARCH, Issue 2-3 2001*Article first published online: 28 AUG 200, Kazuhide Inoue Abstract We investigated the action of ATP on the secretion of plasminogen, TNF-,, and IL-6 from microglia. ATP (10,100 ,M) stimulated the release of plasminogen from rat cultured microglia in a concentration-dependent manner with a peak response at 5,10 min after the stimulation. The release was dependent on extracellular Ca2+ and was blocked by pretreatment with oxidized ATP, a blocker of P2X7. UTP, an agonist of P2Y2, also stimulated the release of plasminogen from a subpopulation (about 20% of total cells) of cultured microglia. The release was also dependent on extracellular Ca2+, suggesting a role of stocker-operated calcium entry (SOC). ATP potently stimulated TNF-, release from 2 h after the stimulation with TNF-, mRNA expression in primary cultures of rat brain microglia. The TNF-, release was maximally elicited by 1 mM ATP and 2,- and 3,-O-(4-benzoylbenzoyl)-adenosine 5,-triphosphate (BzATP), a P2X7 selective agonist, suggesting the involvement of P2X7. This TNF-, release was correlated with a sustained Ca2+ influx. The release was inhibited by PD98059, an inhibitor of MEK1 which activates extracellular signal-regulated protein kinase (ERK), and SB203580, an inhibitor of p38 MAP kinase. However, both ERK and p38 were rapidly activated by ATP even in the absence of extracellular Ca2+. These results indicate that extracellular ATP triggers TNF-, release in rat microglia via P2X7 in a manner dependent on the sustained Ca2+ influx and via the ERK/p38 cascade independently of Ca2+ influx. ATP caused the mRNA expression and release of IL-6 in a concentration-dependent manner in MG-5. The physiological meaning of these independent release mechanisms is also discussed. Drug Dev. Res. 53:166,171, 2001. © 2001 Wiley-Liss, Inc. [source] Actions of Arachidonic Acid on Contractions and Associated Electrical Activity in Guinea-Pig Isolated Ventricular MyocytesEXPERIMENTAL PHYSIOLOGY, Issue 4 2001M. A. Mamas The actions of arachidonic acid (AA) were investigated in guinea-pig isolated ventricular myocytes. Exposure of myocytes to 10 ,M AA reduced the amplitude of contractions and calcium transients accompanying action potentials at a frequency of 1 Hz. AA (10 ,M) also reduced the amplitude of calcium currents recorded under voltage-clamp conditions. The suppression of contraction by AA was not prevented by either 10 ,M trihydroindomethicin (to inhibit cyclo-oxygenase) or 10 ,M ETYA (5,8,11,14-eicosatetraynoic acid, to inhibit AA metabolising enzymes), showing that the actions of AA appeared not to be mediated by these metabolites. The reduction of contraction by 10 ,M AA was also not prevented by the protein kinase C inhibitor, Ro31-8220 (1 ,M), showing that this pathway appeared not to be required for the observed effect. Direct effects of AA may be involved. A further action of 10 ,M AA was to suppress spontaneous electrical activity induced by either the ,-adrenergic agonist isoprenaline or the Na+ pump inhibitor, ouabain. This effect of AA on spontaneous activity might be associated with the observed reduction of calcium entry through L-type calcium channels, although additional effects of AA on calcium release from the sarcoplasmic reticulum might also be involved. [source] The Physiological Basis of Uterine Contractility: A Short ReviewEXPERIMENTAL PHYSIOLOGY, Issue 2 2001S. Wray In this review we discuss our current understanding of the cellular basis of uterine contractility, highlighting those areas requiring further study. It is clear that the basic processes of excitation-contraction coupling lie within the myometrial cell, and that these may be modified by agonists. Pacemaker acitivity, however, remains a mystery. The contribution of extracellular calcium entry to contraction is shown to be vital, whilst the role of the sarcoplasmic reticulum remains controversial. Much current experimental focus is on pathways controlling and regulating contraction, and we discuss sensitisation mechanisms and question their role in intact uterine preparations. [source] Abstract no.: 2 The influence of clozapine on tone of isolated bovine retinal arteriesFUNDAMENTAL & CLINICAL PHARMACOLOGY, Issue 3 2005Koen Boussery Aims:, It has been suggested that the atypical antipsychotic drug clozapine might be helpful in the development of new antiglaucoma agents, since it combines lowering of the intra-ocular pressure after topical instillation with vasodilation. However, the vasoactive influence of clozapine on ocular blood vessels has never been analysed. Therefore, this study aimed to evaluate whether clozapine has direct vasodilatory effects in isolated bovine retinal arteries (BRA) and to characterise pharmacologically the mechanisms involved. Methods:, Retinal arteries were isolated from bovine eyes and mounted in a wire-myograph for isometric tension recording. Concentration-response curves were generated by cumulative addition of clozapine (1 nM to 10 ,M) to the organ bath. Results:, Clozapine elicited a concentration-dependent relaxation of the BRA. Removal of the endothelium of the BRA, inhibition of nitric oxide synthase with N, -nitro-L-arginine and inhibition of soluble guanylyl cyclase with ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) significantly attenuated the clozapine-response, whereas cyclo-oxygenase inhibition with indomethacin had no influence. The Ca2+ channel activator Bay k8644, the nonselective 5-hydroxytryptamine receptor antagonist methiothepin and the adenosine receptor antagonist 8-(p-sulfophenyl) theophylline also failed in affecting the clozapine-induced relaxations. Conclusion:, Clozapine clearly relaxes bovine retinal arteries in a direct way. Endothelium-derived NO is involved in this response. However, prostanoids, calcium entry blockade, 5-HT7 receptor stimulation and adenosine receptor stimulation all seem to be not involved. [source] Na,K-ATPase ,2 inhibition alters calcium responses in optic nerve astrocytesGLIA, Issue 3 2004April K. Hartford Abstract Experiments were conducted to test the effect of 1 ,M ouabain, an Na,K-ATPase inhibitor, on capacitative calcium entry (CCE) and calcium responses elicited by ATP in rat optic nerve astrocytes. In the rat, 1 ,M ouabain is sufficient to inhibit the ,2 Na,K-ATPase, but not the ,1. Immortalized astrocytes derived from Na,K-ATPase ,2 homozygous knockout (KO) mice and wild-type (WT) littermates were also used. Cytosolic calcium and sodium concentrations were measured using Fura-2 and SBFI, respectively. The magnitude of the increase in cytosolic calcium concentration during CCE was significantly greater in rat astrocytes exposed to 1 ,M ouabain. To measure calcium release from stores, cells were exposed to ATP in the absence of extracellular calcium. In astrocytes exposed to 1 ,M ouabain, a significantly greater calcium response to ATP was observed. 1 ,M ouabain was shown to inhibit ATP hydrolysis in membrane material containing Na,K-ATPase ,2 and ,1 isoforms (rat muscle) but not in membranes containing only Na,K-ATPase ,1 (rat kidney). In intact astrocytes, 1 ,M ouabain did not alter the cell-wide cytosolic sodium concentration. In mouse Na,K-ATPase ,2 KO astrocytes, the calcium increase during CCE was significantly higher than in WT cells, as was the magnitude of the calcium response to ATP. In KO astrocytes, but not WT, the cytosolic calcium increase during CCE was insensitive to 1 ,M ouabain. Taken together, the results suggest that selective inhibition of the Na,K-ATPase ,2 isoform has the potential to change calcium signaling and CCE. © 2003 Wiley-Liss, Inc. [source] Calcium influx mechanisms underlying calcium oscillations in rat hepatocytes,HEPATOLOGY, Issue 4 2008Bertina F. Jones The process of capacitative or store-operated Ca2+ entry has been extensively investigated, and recently two major molecular players in this process have been described. Stromal interacting molecule (STIM) 1 acts as a sensor for the level of Ca2+ stored in the endoplasmic reticulum, and Orai proteins constitute pore-forming subunits of the store-operated channels. Store-operated Ca2+ entry is readily demonstrated with protocols that provide extensive Ca2+ store depletion; however, the role of store-operated entry with modest and more physiological cell stimuli is less certain. Recent studies have addressed this question in cell lines; however, the role of store-operated entry during physiological activation of primary cells has not been extensively investigated, and there is little or no information on the roles of STIM and Orai proteins in primary cells. Also, the nature of the Ca2+ influx mechanism with hormone activation of hepatocytes is controversial. Hepatocytes respond to physiological levels of glycogenolytic hormones with well-characterized intracellular Ca2+ oscillations. In the current study, we have used both pharmacological tools and RNA interference (RNAi)-based techniques to investigate the role of store-operated channels in the maintenance of hormone-induced Ca2+ oscillations in rat hepatocytes. Pharmacological inhibitors of store-operated channels blocked thapsigargin-induced Ca2+ entry but only partially reduced the frequency of Ca2+ oscillations. Similarly, RNAi knockdown of STIM1 or Orai1 substantially reduced thapsigargin-induced calcium entry, and more modestly diminished the frequency of vasopressin-induced oscillations. Conclusion: Our findings establish that store-operated Ca2+ entry plays a role in the maintenance of agonist-induced oscillations in primary rat hepatocytes but indicate that other agonist-induced entry mechanisms must be involved to a significant extent. (HEPATOLOGY 2008.) [source] Agonist-induced calcium entry correlates with STIM1 translocationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2007Kehinde Ross The mechanisms of agonist-induced calcium entry (ACE) following depletion of intracellular calcium stores have not been fully established. We report here that calcium-independent phospholipase A (iPLA2) is required for robust Ca2+ entry in HaCaT keratinocytes following ATP or UTP stimulation. Lysophosphatidic acid (LPA), an unrelated agonist, evoked Ca2+ release without inducing robust Ca2+ entry. Both LPA and UTP induced the redistribution of STIM1 into puncta which localized to regions near or at the plasma membrane, as well as within the cytoplasm. Plasma membrane-associated STIM1 remained high for up to 10 min after UTP stimulation, whereas it had returned almost to baseline by that time point in LPA-stimulated cells. This correlated with faster reloading of the endoplasmic reticulum Ca2+ stores in LPA treated cells. Thus by differentially regulating store-refilling after agonist-mediated depletion, LPA and UTP may exert distinct effects on the duration of STIM1 localization at the plasma membrane, and thus, on the magnitude and duration of ACE. J. Cell. Physiol. 211: 569,576, 2007. © 2007 Wiley-Liss, Inc. [source] Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxinJOURNAL OF NEUROSCIENCE RESEARCH, Issue 8 2006Carmen Vale Abstract A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca2+]c), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na+. Li+ decreased the PTX-evoked rise in [Ca2+]c; replacement of Na+ by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca2+]c increase by PTX was strongly reduced after inhibition of the reverse operation of the Na+/Ca2+ exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca2+ -ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na+ -dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na+/Ca2+ exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space. © 2006 Wiley-Liss, Inc. [source] Impaired inhibitory G-protein function contributes to increased calcium currents in rats with diabetic neuropathyJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 2 2002KE Hall There is a growing body of evidence that sensory neuropathy in diabetes is associated with abnormal calcium signaling in dorsal root ganglion (DRG) neurons. Enhanced influx of calcium via multiple high-threshold calcium currents is present in sensory neurons of several models of diabetes mellitus, including the spontaneously diabetic BioBred/Worchester (BB/W) rat and the chemical streptozotocin (STZ)-induced rat. We believe that abnormal calcium signaling in diabetes has pathologic significance as elevation of calcium influx and cytosolic calcium release has been implicated in other neurodegenerative conditions characterized by neuronal dysfunction and death. Using electrophysiologic and pharmacologic techniques, the present study provides evidence that significant impairment of G-protein-coupled modulation of calcium channel function may underlie the enhanced calcium entry in diabetes. N- and P-type voltage-activated, high-threshold calcium channels in DRGs are coupled to mu opiate receptors via inhibitory G(o)-type G proteins. The responsiveness of this receptor coupled model was tested in dorsal root ganglion (DRG) neurons from spontaneously-diabetic BB/W rats, and streptozotocin-induced (STZ) diabetic rats. Intracellular dialysis with GTPgammaS decreased calcium current amplitude in diabetic BB/W DRG neurons compared with those of age-matched, nondiabetic controls, suggesting that inhibitory G-protein activity was diminished in diabetes, resulting in larger calcium currents. Facilitation of calcium current density (I(DCa)) by large-amplitude depolarizing prepulses (proposed to transiently inactivate G proteins), was significantly less effective in neurons from BB/W and STZ-induced diabetic DRGs. Facilitation was enhanced by intracellular dialysis with GTPgammaS, decreased by pertussis toxin, and abolished by GDPbetaS within 5 min. Direct measurement of GTPase activity using opiate-mediated GTPgamma[(35)S] binding, confirmed that G-protein activity was significantly diminished in STZ-induced diabetic neurons compared with age-matched nondiabetic controls. Diabetes did not alter the level of expression of mu opiate receptors and G-protein alpha subunits. These studies indicate that impaired regulation of calcium channels by G proteins is an important mechanism contributing to enhanced calcium influx in diabetes. [source] Rho A participates in the regulation of phosphatidylserine-dependent procoagulant activity at the surface of megakaryocytic cellsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 4 2004C. Kunzelmann Summary. Once exposed at the external surface of activated platelets or apoptotic cells, phosphatidylserine, an anionic phospholipid mostly sequestered in the inner leaflet of the plasma membrane, plays essential roles in hemostasis and phagocytosis. The mechanism governing the migration of the phosphatidylserine to the exoplasmic leaflet is not yet fully understood. We have proposed that store-operated calcium entry (SOCE) constitutes a key step of this process. ERK pathway is among the elements modulating SOCE and phosphatidylserine externalization in megakaryocytic HEL cells. Here, we investigated the role of small GTPase Rho A, which may interact with the ERK pathway. Specific inhibitors of Rho A (exoenzyme C3 and toxin B) reduced both SOCE and phosphatidylserine-dependent procoagulant activity. Simultaneous inhibition of Rho A and extracellular signal-regulated kinase (ERK) pathways did not elicit further reduction with respect to each individual one. Rho A can regulate SOCE and phosphatidylserine exposure through the reorganization of actin cytoskeleton, but not through ROCK pathway. Hence, Rho A is another regulatory element for the completion of SOCE-induced phosphatidylserine transmembrane redistribution in HEL cells. [source] A new role for P2 receptors: talking with calcium-activated potassium channelsNEUROGASTROENTEROLOGY & MOTILITY, Issue 11 2007P. P. Bertrand Abstract Purinergic fast synaptic transmission may play a very subtle role in regulating the excitability of enteric circuits. That is one of the important findings in a new paper by Ren and Galligan in the current issue of this Journal. They first provide compelling evidence that P2X3 receptors (ionotropic purine receptors) are expressed by guinea-pig motor and interneurons and that these subtypes mediate the purinergic fast excitatory postsynaptic potential (EPSP). They also found that the P2X3 -mediated depolarization was often followed by a hyperpolarization. This is an intriguing finding because if the purinergic fast EPSPs are also followed by a hyperpolarization, then it could play a role in truncating bursts of synaptic potentials or in shaping periodic synaptic input. The hyperpolarization is caused by calcium entry through the P2X3 receptor which then activates a calcium-activated potassium (KCa) channel. Surprisingly, the hyperpolarization was not affected by any of the standard blockers of calcium- or voltage-activated K+ channels suggesting that a novel KCa channel is present in the enteric neurons. Such a wide-spread channel could well have an important physiological role and could be an important new drug target for regulating reflex activity in the enteric nervous system. [source] Visualization of localized store-operated calcium entry in mouse astrocytes.THE JOURNAL OF PHYSIOLOGY, Issue 3 2005Close proximity to the endoplasmic reticulum Unloading of endoplasmic reticulum (ER) Ca2+ stores activates influx of extracellular Ca2+ through ,store-operated' Ca2+ channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca2+ stores. Here, using high resolution imaging with the membrane-associated Ca2+ indicator, FFP-18, it is shown that store-operated Ca2+ entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane,ER junctions. In the absence of extracellular Ca2+, depletion of ER Ca2+ stores using cyclopiazonic acid, an ER Ca2+ -ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca2+ concentration ([Ca2+]SPM) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca2+ causes localized Ca2+ influx that first increases [Ca2+]SPM in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs, markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying ,junctional' ER. Thus, Ca2+ entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca2+ stores. [source] Phorbol esters and adenosine affect the readily releasable neurotransmitter pool by different mechanisms at amphibian motor nerve endingsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2003T. J. Searl Phorbol esters and adenosine have been proposed to interact at common sites downstream of calcium entry at amphibian motor nerve endings. We thus studied the actions and interactions of phorbol esters and adenosine using electrophysiological recording techniques in conjunction with both binomial statistical analysis and high-frequency stimulation at the amphibian neuromuscular junction. To begin this study, we confirmed previous observations that synchronous evoked acetylcholine (ACh) release (reflected as endplate potentials, EPPs) is well described by a simple binomial distribution. We then used binomial analysis to study the effects of the phorbol ester phorbol dibutyrate (PDBu, 100 nm) and adenosine (50 µm) on the binomial parameters n (the number of calcium charged ACh quanta available for release) and p (the average probability of release), where the mean level of evoked ACh release (m) =np. We found that PDBu increased m by increasing the parameter n whilst adenosine reduced m by reducing n; neither agent affected the parameter p. PDBu had no effect on either the potency or efficacy of the inhibition produced by adenosine. Subtle differences between these two agents were revealed by the patterns of EPPs evoked by high-frequency trains of stimuli. Phorbol esters increased ACh release during the early phase of stimulation but not during the subsequent plateau phase. The inhibitory effect of adenosine was maximal at the beginning of the train and was still present with reduced efficacy during the plateau phase. When taken together with previous findings, these present results suggest that phorbol esters increase the immediately available store of synaptic vesicles by increasing the number of primed vesicles whilst adenosine acts at a later stage of the secretory process to decrease the number of calcium-charged primed vesicles. [source] The developing relationship between receptor-operated and store-operated calcium channels in smooth muscleBRITISH JOURNAL OF PHARMACOLOGY, Issue 1 2002Ian McFadzean Contraction of smooth muscle is initiated, and to a lesser extent maintained, by a rise in the concentration of free calcium in the cell cytoplasm ([Ca2+]i). This activator calcium can originate from two intimately linked sources , the extracellular space and intracellular stores, most notably the sarcoplasmic reticulum. Smooth muscle contraction activated by excitatory neurotransmitters or hormones usually involves a combination of calcium release and calcium entry. The latter occurs through a variety of calcium permeable ion channels in the sarcolemma membrane. The best-characterized calcium entry pathway utilizes voltage-operated calcium channels (VOCCs). However, also present are several types of calcium-permeable channels which are non-voltage-gated, including the so-called receptor-operated calcium channels (ROCCs), activated by agonists acting on a range of G-protein-coupled receptors, and store-operated calcium channels (SOCCs), activated by depletion of the calcium stores within the sarcoplasmic reticulum. In this article we will review the electrophysiological, functional and pharmacological properties of ROCCs and SOCCs in smooth muscle and highlight emerging evidence that suggests that the two channel types may be closely related, being formed from proteins of the Transient Receptor Potential Channel (TRPC) family. British Journal of Pharmacology (2002) 135, 1,13; doi:10.1038/sj.bjp.0704468 [source] Membrane Permeabilization of a Mammalian Neuroendocrine Cell Type (PC12) by the Channel-Forming Peptides Zervamicin, Alamethicin, and GramicidinCHEMISTRY & BIODIVERSITY, Issue 6 2007Abstract Zervamicin IIB (ZER) is a 16-mer peptaibol that produces voltage-dependent conductances in artificial membranes, a property considered responsible for its antimicrobial activity to mainly Gram -positive microorganisms. In addition, ZER appears to inhibit the locomotor activity of the mouse (see elsewhere in this Issue), probably by affecting the brain. To examine whether the electrophysiological properties of the neuronal cells of the central neural system might be possibly influenced by the pore forming ZER, the present study was undertaken as a first attempt to unravel the molecular mechanism of this biological activity. To this end, membrane permeabilization of the neuron-like rat pheochromocytoma cell (PC12) by the channel-forming ZER was studied with the whole-cell patch-clamp technique, and compared with the permeabilizations of the well-known voltage-gated peptaibol alamethicin F50/5 (ALA) and the cation channel-forming peptide-antibiotic gramicidin D (GRAM). While 1,,M GRAM addition to PC12 cells kept at a membrane potential Vm=0,mV causes an undelayed gradual increase of a leak conductance with a negative reversal potential of ca. ,24,mV, ZER and ALA are ineffective at that concentration and potential. However, if ZER and ALA are added in 5,10,,M concentrations while Vm is kept at ,60,mV, they cause a sudden and strong permeabilization of the PC12 cell membrane after a delay of 1,2,min, usually leading to disintegrating morphology changes of the patched cell but not of the surrounding cells of the culture at that time scale. The zero reversal potential of the established conductance is consistent with the known aselectivity of the channels formed. This sudden permeabilization does not occur within 10,20,min at Vm=0,mV, in accordance with the known voltage dependency of ZER and ALA channel formation in artificial lipid membranes. The permeabilizing action of these peptaibols on the culture as a whole is further supported by K+ -release measurements from a PC12 suspension with a K+ -selective electrode. Further analysis suggested that the permeabilizing action is associated with extra- or intracellular calcium effects, because barium inhibited the permeabilizing effects of ZER and ALA. We conclude, for the membrane of the mammalian neuron-like PC12 cell, that the permeabilizing effects of the peptides ZER and ALA are different from those of GRAM, consistent with earlier studies of these peptides in other (artificial) membrane systems. They are increased by cis -positive membrane potentials in the physiological range and may include calcium entry into the PC12 cell. [source] Small-Molecule Inhibitors of Store-Operated Calcium EntryCHEMMEDCHEM, Issue 5 2009Zachary Abstract Molecules that inhibit store-operated calcium entry (SOCE) are potentially useful immunomodulating agents. The identification of proteins involved in this pathway may further enable the identification of selective inhibitors. Herein we document some examples of the small-molecule inhibitors of SOCE that have been reported to date. We also describe methods that were used to characterize the mechanism of action of these inhibitors. Controlled variation in intracellular calcium concentration is a key component of the immune response signaling pathway in lymphocytes. Store-operated calcium entry (SOCE) in these cells provides a prolonged increase in cytoplasmic Ca2+ concentrations and ultimately leads to the production of pro-inflammatory cytokines. Molecules that inhibit SOCE could therefore be useful immunomodulating agents for the treatment of rheumatoid arthritis, psoriasis, inflammatory bowel disease, and other conditions. Although the presence of the SOCE signaling pathway in lymphocytes and other cells involved in the immune response has been known for many years, key proteins involved in SOCE were identified only recently. The identification of these proteins may further enable the identification of agents that inhibit SOCE without affecting other cellular processes. This contribution documents representative examples of the small-molecule inhibitors of SOCE that have been reported to date. Where possible, methods that were used to characterize the mechanism of action of the inhibitors are also described. [source] Itraconazole-mediated inhibition of calcium entry into platelet-activating factor-stimulated human neutrophils is due to interference with production of leukotriene B4CLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2007H. C. Steel Summary The primary objective of this study was to probe the involvement of leukotriene B4 (LTB4) in itraconazole (0·1,5 µM)-mediated inhibition of Ca2+ uptake by chemoattractant-activated human neutrophils. Following exposure of the cells to platelet-activating factor (PAF, 200 nM), LTB4 was measured by immunoassay, while neutrophil cytosolic Ca2+ concentrations were determined by a fura-2/AM-based spectrofluorimetric procedure. Activation of neutrophils was accompanied by an abrupt and sustained (for about 1 min) elevation in cytosolic Ca2+ which was associated with increased generation of LTB4, both of which were attenuated significantly by itraconazole at 0·5 µM and higher. The inhibitory effect of the anti-mycotic on Ca2+ uptake by PAF-activated cells was mimicked by an LTB4 antibody, as well as by LY255283 (1 µM) and MK886 (0·5 µM), an antagonist of LTB4 receptors and an inhibitor of 5,-lipoxygenase-activating protein, respectively, while addition of itraconazole to purified 5,-lipoxygenase resulted in inhibition of enzyme activity. A mechanistic relationship between itraconazole-mediated inhibition of LTB4 production and Ca2+ influx was also supported by the observation that pulsed addition of purified LTB4 to PAF-activated neutrophils caused substantial restoration of Ca2+ uptake by cells treated with the anti-mycotic. Taken together, these observations suggest that the potentially beneficial anti-inflammatory interactions of itraconazole with activated neutrophils result from interference with production of LTB4, with consequent attenuation of a secondary LTB4 -mediated wave of Ca2+ uptake by the cells. [source] Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosisCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 3 2005E. Janas Summary Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca2+ levels and downstream apoptotic signalling. [source] | ||||||||||||||||