Absence Of Extracellular Calcium Research Articles

Calcium Mobilization by Activation of M3/M5 Muscarinic Receptors in the Human Retinoblastoma

Activation of muscarinic acetylcholine receptors (mAChR) is one of the most important signal transduction pathways in the human body. In this study, we investigated the role of mAChR activation in relation to its subtypes in human retinoblastoma cell-lines (WERI-Rb-1) using Ca2+ measurement, real-time PCR, and Western Blot techniques. Acetylcholine (ACh) produced prominent [Ca2+]i transients in a repeated manner in WERI-Rb-1 cells. The maximal amplitude of the [Ca2+]i transient was almost completely suppressed by 97.3 ± 0.8% after atropine (1 µM) pretreatment. Similar suppressions were noted after pretreatments with thapsigargin (1 µM), an ER Ca2+-ATPase (SERCA) inhibitor, whereas the ACh-induced [Ca2+]i transient was not affected even in the absence of extracellular calcium. U-73122 (1 µM), a PLC inhibitor, and xestospongin C (2 µM), an IP3-receptor antagonist, elicited 11.5 ± 2.9% and 17.8 ± 1.9% suppressions, respectively. The 50% inhibitory concentration of (IC50) values for blockade of a 100 µM ACh response by pirenzepine and 4-DAMP were 315.8 and 9.1 nM, respectively. Moreover, both M3 and M5 mAChRs were prominent in quantitative real-time-PCR. Taken together, the M3/M5 subtypes appear to be the major contributor, leading to intracellular calcium mobilization from the internal store via an IP3-dependent pathway in the undifferentiated retinoblastoma cells.

Cerebellar Granule Cell Death Induced by Aluminum

Using flow cytometry of acutely isolated cerebellar granule cell neurons, we have determined the effects of Al (III) on viability, membrane potential, intracellular calcium concentration and generation of reactive oxygen species (ROS). Al (III) killed granule cells in a time- and concentration-dependent fashion when monitored by use of the DNA-binding dye, propidium iodide. The threshold concentration was about 50 micromolar, and cell death at 100 micromolar was apparent after 30 min exposure and increased over time. Cell death was accompanied by cell swelling and a decrease in membrane potential, and was not dependent on external calcium concentration. While exposure to Al (III) was accompanied by an increase in ROS and an elevation of intracellular calcium concentration, calcium chelators and ROS scavengers did not reduce cell death. The action of Al (III) was not accompanied by activation of caspase-3 or an increase in annexin-V binding, both indicators of apoptosis. In the presence of intracellular O,O'-bis(2-aminophenyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (BAPTA) and absence of extracellular calcium there was still a fluo-3 signal, which likely reflects an accumulation of intracellular Al (III). These observations suggest that the cell death is subsequent to intracellular accumulation of Al (III) and subsequent perturbation of cellular metabolism.

Augmentation of excitability in the hippocampus of juvenile rat

The short-term plasticity of synaptic transmission has usually been related to neurotransmitter release-dependent processes. In this work, we describe a calcium- and release-independent augmentation of the fiber volley (FVA) that appears during stimulation of the Wistar rat commissural/Schaffer collateral afferents at 10-Hz. Among the possible mechanisms involved in this phenomenon, an increment in sodium channel density or the facilitation of recovery from inactivation does not seem to be responsible for this effect since the depolarization rate of the somatic action potentials (APs) of CA3 pyramidal cells decreases during the 10-Hz stimulation. On the other hand, an increase in the synchronization of the APs can be observed during the very first pulses of the 10-Hz tetanus. However, the major part of the FVA occurs with any increase in synchronization of AP firing. Finally, a strong increase in the firing probability, with kinetics similar to that observed with the FVA, appears at 10-Hz stimulation when APs are induced at threshold intensities. This hyperexcitability seems to be mediated by a residual depolarization that persists for more than 100 ms after the AP. The nature of this post-spike depolarization is uncertain since it persists in the absence of extracellular calcium and was not blocked by the application of phenytoin (100 μM), and this excludes the implication of either calcium or sodium-persistent currents. Additionally, the increase of the stimulation strength did not alter this depolarization, which suggests that the presumed extracellular potassium accumulation produced after the synchronic stimulation of APs is not involved in the depolarization. Interestingly, the slow post-depolarization induced by both supra- and subthreshold pulses is well fitted by a single exponential decay with similar time constants, an indication that the tail depolarization may represent passive discharge of the membrane following an incomplete repolarization of the AP.

Fetuin-A uptake in bovine vascular smooth muscle cells is calcium dependent and mediated by annexins

Fetuin-A is a known inhibitor of vascular calcification in vitro. In arteries with calcification, there is increased immunostaining for fetuin-A. However, vascular smooth muscle cells (VSMC) do not synthesize fetuin-A, suggesting fetuin-A may be endocytosed to exert its inhibitory effects. To examine the mechanism by which fetuin-A is taken up in bovine VSMC (BVSMC), we examined living cells by confocal microscopy and determined the uptake of Cy5-labeled fetuin-A. The results demonstrated that fetuin-A was taken up in BVSMC only in the presence of extracellular calcium, whereas phosphorus had no effect. Additional studies demonstrated the calcium-dependent uptake was specific for fetuin-A and only observed in BVSMC and osteoblasts, but not epithelial, endothelial, or adipose cells. The uptake was dose dependent, but could not be inhibited by excess unlabeled fetuin-A, suggesting a fluid phase rather than a receptor-mediated process. Fetuin-A also induced a sustained increase in intracellular calcium in BVSMC in the presence of extracellular calcium, whereas there was no increase in the absence of extracellular calcium. To further characterize the uptake, we utilized an inhibitor of annexin calcium channel activity, demonstrating inhibition of both fetuin-A uptake and intracellular calcium increase. Finally, we demonstrate that fetuin-A binds to annexin II at the cell membrane of BVSMC. In summary, our study demonstrates calcium- and annexin-dependent uptake of fetuin-A that leads to a sustained rise in intracellular calcium. This regulated uptake may be a mechanism by which fetuin-A inhibits VSMC calcification in the presence of excess calcium.

Hydrogen peroxide increases the phagocytic function of human neutrophils by calcium mobilisation

We have studied the effect of exogenous administration of hydrogen peroxide (H(2)O(2)) on phagocytic activity of human neutrophils. The treatment of cells with increasing concentrations of H(2)O(2) evoke a significant elevation of phagocytic function assayed as phagocytic index, percentage and efficiency; and was similar to that induced by the calcium mobilising agonist formyl-methionyl-leucyl-phenylalanine (fMLP). This stimulatory effect was reduced by pre-treatment of neutrophils with catalase and abolished in neutrophils loaded with the intracellular calcium quelator dimethyl BAPTA. In the absence of extracellular calcium, treatment of cells with H(2)O(2) resulted in a increase in [Ca(2+)]( i ), indicating the release of calcium from intracellular stores. H(2)O(2) abolished the typical calcium release stimulated by the physiological agonist fMLP, while depletion of agonist-sensitive calcium pools by fMLP was able to prevent H(2)O(2)-induced calcium release. We conclude that H(2)O(2) induces calcium release from agonist-sensitive stores and consequently increase the phagocytosis process.

Golli, a New Mechanism for Regulating T Cell Activation

Golli proteins, which are generated by alternative splicing from the gene that encodes myelin basic proteins (which are found only in the nervous system), are expressed not only in the nervous system but also in immune tissues. Feng et al. , who previously showed that golli negatively regulates T cell activation, uncovered a hitherto undescribed mechanism for this involving inhibition of calcium influx. When stimulated with antibody directed against CD3 (anti-CD3) or with anti-CD3 plus anti-CD28, golli-deficient T cells proliferated more than did wild-type cells; consistent with this, golli-deficient cells stimulated with anti-CD3 plus anti-CD20 produced more interleukin-2 than did wild-type cells. No differences in extracellular signal-regulated kinase (ERK) or c-Jun N-terminal kinase (JNK) activation were apparent between golli-deficient and wild-type T cells in response to anti-CD3 stimulation; however, the increase in intracellular calcium was enhanced. Calcium imaging in the presence or absence of extracellular calcium, thapsigargin, or both suggested that golli inhibited calcium influx through store-operated calcium channels. Moreover, patch-clamp analysis of golli-deficient cells revealed increased inward calcium current in response to store depletion. A portion of T cell golli protein was associated with the plasma membrane, and experiments in which cells were transfected with either wild-type golli protein or a myristoylation mutant indicated that membrane association was required for golli's inhibition of calcium influx. Thus, the authors conclude that golli acts as a negative regulator of T cell activation through a mechanism completely distinct from that of other negative regulators of T cell receptor signaling. J.-M. Feng, Y. K. Hu, L.-H. Xie, C. S. Colwell, X. M. Shao, X.-P. Sun, B. Chen, H. Tang, A. T. Campagnoni, Golli protein negatively regulates store depletion-induced calcium influx in T cells. Immunity 24 , 717-727 (2006). [PubMed]

Dopamine modulates von Willebrand factor secretion in endothelial cells via D2-D4 receptors.

von Willebrand factor (VWF) is acutely released from endothelial cells in response to numerous calcium-raising agents (e.g. thrombin, histamine) and cAMP-raising agents (e.g. epinephrine, adenosine, vasopressin). In contrast, very few inhibitors of endothelial VWF secretion have been described. The neurotransmitter dopamine is a modulator of exocytosis in several endocrine cells, and is possibly involved in the regulation of several endothelial cell functions. We therefore investigated the effect of dopamine on endothelial VWF secretion. Dopamine, D2/D3- and D4-specific agonists inhibited histamine- but not thrombin-induced VWF secretion. Expression of dopamine D2, D3 and D4 receptors was demonstrated by reverse transcription polymerase chain reaction (RT-PCR) in both human aortic (HAEC) and umbilical vein (HUVEC) endothelial cells. D2-D4 agonists did not inhibit histamine-induced rise in [Ca(2+)](i): they inhibited histamine-induced secretion even in the absence of extracellular calcium. Thus, the dopamine effects are not mediated by [Ca(2+)](i)-dependent signalling. D2/D3- and D4-specific agonists inhibited neither the rise in cAMP nor VWF secretion in response to epinephrine and adenosine, arguing against an effect on cAMP-mediated signalling. D1 and D5 receptors were not detected in HAEC or HUVEC by RT-PCR, and the D1/D5-specific agonist SKF 38 393 failed to modulate VWF secretion, arguing against a role for these receptors in endothelial exocytosis. Dopamine inhibits histamine-induced endothelial exocytosis by activating D2-D4 receptor, via a mechanism distinct from [Ca(2+)](i)-or cAMP-mediated signaling. In contrast, D1 and D5 receptors are not functionally expressed in cultured endothelial cells. Dopamine agonists may be useful as inhibitors of endothelial activation in inflammation and cardiovascular disease.

Adenosine A1 Receptors Mediate Mobilization of Calcium in Human Bronchial Smooth Muscle Cells

Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca(2+)](i)) were determined ratiometrically by imaging single cells. The nonselective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the adenosine A(1) receptor agonist, N(6)-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca(2+)](i). In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (100 nM) or N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (100 nM), selective agonists at adenosine A(2A) receptors and adenosine A(3) receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A(1) receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP(3)) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A(1) receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP(3) signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.

The cGMP/Protein Kinase G Pathway Contributes to Dihydropyridine-sensitive Calcium Response and Cytokine Production in TH2 Lymphocytes

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.

Metabotropic Ca 2+ channel-induced Ca 2+ release and ATP-dependent facilitation of arterial myocyte contraction

Voltage-gated Ca(2+) channels in arterial myocytes can mediate Ca(2+) release from the sarcoplasmic reticulum and, thus, induce contraction without the need of extracellular Ca(2+) influx. This metabotropic action of Ca(2+) channels (denoted as calcium-channel-induced calcium release or CCICR) involves activation of G proteins and the phospholipase C-inositol 1,4,5-trisphosphate pathway. Here, we show a form of vascular tone regulation by extracellular ATP that depends on the modulation of CCICR. In isolated arterial myocytes, ATP produced facilitation of Ca(2+)-channel activation and, subsequently, a strong potentiation of CCICR. The facilitation of L-type channel still occurred after full blockade of purinergic receptors and inhibition of G proteins with GDPbetaS, thus suggesting that ATP directly interacts with Ca(2+) channels. The effects of ATP appear to be highly selective, because they were not mimicked by other nucleotides (ADP or UTP) or vasoactive agents, such as norepinephrine, acetylcholine, or endothelin-1. We have also shown that CCICR can trigger arterial cerebral vasoconstriction in the absence of extracellular calcium and that this phenomenon is greatly facilitated by extracellular ATP. Although, at low concentrations, ATP does not induce arterial contraction per se, this agent markedly potentiates contractility of partially depolarized or primed arteries. Hence, the metabotropic action of L-type Ca(2+) channels could have a high impact on vascular pathophysiology, because, even in the absence of Ca(2+) channel opening, it might mediate elevations of cytosolic Ca(2+) and contraction in partially depolarized vascular smooth muscle cells exposed to small concentrations of agonists.

Modulation of ion channels by protons in retinal horizontal cells

Protons are synaptic modulators acting between photoreceptors and horizontal cells in the retina. We investigated the effects of H+ in modulating or carrying ion channel current in horizontal cells of the goldfish retina with patch-clamp. In voltage-clamp experiments, increasing the concentration of extracellular H+ (pHo = 7.8–6.0) produced reversible inhibition of inward rectifier K+ (IKir) and Ca2+ (ICa) currents and, in the absence of extracellular calcium (Ca2+o), a prominent inhibition of outward current. However, in the presence of K+ channel blockers (20 mM TEA, 10 mM 4-AP), H+ increased an inward conductance at hyperpolarized potentials (-100 mV). Characteristics of this current were similar to those of IKir, such as reversal near the K+ equilibrium potential and inhibition by 100 μM glutamate and 10 mM Cs+. Therefore, this increase in current by H+ may have been due to activation of IKir or reduced block of Kir channels by TEA and 4-AP. In the physiological range between −70 mV and −20 mV, increasing extracellular H+ also induced inward current that reversed positive to EH and that was dependent on the presence of Ca2+o. This H+-activated current may represent a population of non-selective cation channels permeable to H+. These data demonstrate multiple targets for H+-modulation of ion channels in horizontal cells and suggest potential mechanisms through which H+ could modulate synaptic transmission in the retina. Supported by NSHRF and CIHR.

Voltage Sensor for Proliferation

Hegle et al. provide evidence that a K + channel has an ion-independent signaling activity that stimulates cell proliferation. Transfection of the Drosophila ether-à-go-go (EAG) channel into mammalian cultured cells stimulated cell proliferation based on increased cell density and 5-bromo-2′-deoxyuridine (BrdU) incorporation. Stimulation of proliferation occurred in the absence of serum and in the absence of extracellular calcium. This stimulation occurred when nonconducting forms of the channel were used and was blocked by pharmacological inhibition of the p38 mitogen-activated protein kinase (MAPK) and not by inhibition of the p42 and p44 MAPKs. Cells transfected with wild-type or nonconducting EAG had increased p38 phosphorylation compared with control cells. Additional mutants of EAG that had altered voltage dependence for channel opening were used to show that the closed conformation of the channel appeared to be the conformation responsible for activation of the proliferation signal. Thus, EAG appears to have some role as a voltage-sensitive trigger for proliferation that is independent of its activity as an ion channel. A. P. Hegle, D. D. Marble, G. F. Wilson, A voltage-driven switch for ion-independent signaling by ether-à-go-go K + channels. Proc. Natl. Acad. Sci. U.S.A. 103 , 2886-2891 (2006). [Abstract] [Full Text]

Dose-dependent effect of hydrogen peroxide on calcium mobilization in mouse pancreatic acinar cells

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.

Effects of radix salviae miltiorrhizae on endothelin-1-induced calcium changes in hepatic stellate cells

AIM: To investigate the effect of radix salviae miltiorrhizae (RSM) on the changes of [Ca]i induced by endothelin-1 (ET-1) in hepatic stellate cells (HSCs). METHODS: After preparation of RSM cream, the effects of RSM on ET-1-induced changes of HSCs [Ca]i were observed by laser scanning confocal microscopy. RESULTS: In normal buffer (including Ca, buffer A), the fluorescence intensity was enhanced accordingly with the increase of ET-1 concentration. The cumulative-response curve showed EC50 was 1.1×10 mol/L. After incubation of HSCs with ET-1 in buffer A and buffer B (absence of extracellular calcium, EGTA), the duration of calcium peak had significant difference (165.2 ± 10.1 s vs 91.0 ± 7.2 s, P 0.05). In the cells pre-incubated with RSM, KCl-induced elevation of [Ca]i was decreased, and the calcium peak value (78.0% ± 6.1% → 26.3% ± 1.2%, P < 0.01) and duration of calcium wave (70.8 ± 10.4 s → 15.9 ± 5.1 s, P < 0.01) were decreased significantly. CONCLUSION: RSM inhibits ET-1-induced depletion of intracellular calcium, which has no correlations with the influx of extracellular calcium. RSM can also inhibit KCl-induced influx of calcium, indicating its characteristic of blocking voltage-operated Ca channel.

N-(4-Trifluoromethylphenyl)amide group of the synthetic histamine receptor agonist inhibits nicotinic acetylcholine receptor-mediated catecholamine secretion

The therapeutic targeting of nicotinic receptors requires the identification of drugs that selectively activate or inhibit a limited range of nicotine acetylcholine receptors (nAChRs). In this study, we identified N-(4-trifluoromethylphenyl)amide group of the synthetic histamine receptor ligands, histamine-trifluoromethyltoluide, that act as potent inhibitors of nAChRs in bovine adrenal chromaffin cells. Catecholamine secretion induced by the nAChRs agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), was significantly inhibited by histamine-trifluoromethyltoluide. Real time carbon-fiber amperometry confirmed the ability of histamine-trifluoromethyltoluide to inhibit DMPP-induced exocytosis in single chromaffin cells. We also found that histamine-trifluoromethyltoluide inhibited DMPP-induced [Ca 2+] i and [Na +] i increases, as well as DMPP-induced inward currents in the absence of extracellular calcium. Histamine-trifluoromethyltoluide had no effect on [ 3H]nicotine binding or on calcium increases induced by high K +, bradykinin, veratridine, histamine, and benzoylbenzoyl ATP. Among the synthetic histamine receptor ligands, clobenpropit exhibited similarity. In addition, 4′-nitroacetanilide also significantly attenuated nAChR-mediated catecholamine secretion. In conclusion, the N-(4-trifluoromethylphenyl)amide group of the histamine-trifluoromethyltoluide might be the critical moiety in the inhibition of nAChR-mediated CA secretion.

Enhancement of Excitatory Synaptic Transmission in Spiny Neurons After Transient Forebrain Ischemia

Spiny neurons in the neostriatum are highly vulnerable to ischemia. Enhancement of excitatory synaptic transmissions has been implicated in ischemia-induced excitotoxic neuronal death. Here we report that evoked excitatory postsynaptic currents in spiny neurons were potentiated after transient forebrain ischemia. The ischemia-induced potentiation in synaptic efficacy was associated with an enhancement of presynaptic release as demonstrated by an increase in the frequency of miniature excitatory postsynaptic currents (mEPSCs) and a decrease in the paired-pulse ratio. The amplitude of inward currents evoked by exogenous application of glutamate did not show significant changes after ischemia, suggesting that postsynaptic mechanism is not involved. The ischemia-induced increase in mEPSCs frequency was not affected by blockade of voltage-gated calcium channels, but it was eliminated in the absence of extracellular calcium. Bath application of ATP P2X receptor antagonist pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) significantly reduced mEPSC frequency in ischemic neurons but had no effects on the control ones. Furthermore, the inhibitory effect of PPADS on ischemic neurons was abolished in Ca2+-free external solution. These results indicate that excitatory synaptic transmissions in spiny neurons are potentiated after ischemia via presynaptic mechanisms. Activation of P2X receptors and the consequent Ca2+ influx might contribute to the ischemia-induced facilitation of glutamate release.

Protein kinase activity is associated with CD63 in melanoma cells

BackgroundThe tetraspan protein CD63, originally described as a stage-specific melanoma antigen but also present in a number of normal cells, regulates melanoma cell growth in nude mice, motility in serum containing media, and adhesion to several extracellular matrix proteins. CD63 has been reported to associate with β1 and β2 integrins, but the mechanism of signal transduction by CD63 is not clear. This study examined whether CD63 is associated with protein kinase and can transmit signals in melanoma cells.MethodsImmunoprecipitation and radiolabeling were used to test for association of protein kinase activity with CD63. Adhesion of cells to monoclonal antibodies immobilized to microtiter plates was used to examine the ability of CD63 to transmit signals.ResultsCD63 was capable of transmitting a signal in melanoma cells that required extracellular calcium. In the absence of extracellular calcium at the time of binding to the CD63 mAb, the cell was no longer responsive to stimulation by CD63. Immunoprecipitation studies demonstrated protein kinase activity associated with CD63, and phosphoamino acid analysis revealed that most of this protein kinase activity was due to serine kinase activity.ConclusionThe current study suggests that serine protein kinase activity associated with CD63 may play a role in signaling by CD63 in melanoma cells.

IL-4 inhibits calcium transients in bovine trachealis cells by a ryanodine receptor-dependent mechanism.

IL-4 and IL-13 have important roles in the pathogenesis of asthma. A novel finding was that brief exposure of airway smooth muscle cells to IL-4 inhibited carbachol-stimulated calcium transients. We hypothesized that IL-4 inhibits transients by decreasing calcium store content and tested this by measuring the effects of IL-4 on transients induced by a nonspecific ionophore. Bovine trachealis cells were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca2+]i) were measured in single cells by digital microscopy. Stimulation (S1) with carbachol (10 microM) caused rapid, transient increases in [Ca2+]i to 1299 +/- 355 nM (n=5). After recovery of calcium stores, stimulation (S2) of the same cells with ionomycin (10 microM), in the absence of extracellular calcium, also increased [Ca2+]i to give S2/S1 ratio of 1.03 +/- 0.29. However, after 20 min of IL-4 (50 ng/ml), but not IL-13, ionomycin transients were decreased to 0.50 +/- 0.16 (S2/S1, P=0.02, n=6). IL-4 did not inhibit transients with ryanodine receptor calcium release channels (RyR) blocked by ryanodine (200 microM) (S2/S1=1.01+/-0.11) but still did in the presence of 8-bromo cyclic ADP-ribose, an antagonist of cyclic ADP-ribose (cADPR) signaling at RyR (S2/S1=0.48+/-0.13). Together, findings suggest that IL-4 decreases intracellular calcium stores by mechanisms dependent on RyR, but not on cADPR signaling.

Triadin (Trisk 95) Overexpression Blocks Excitation-Contraction Coupling in Rat Skeletal Myotubes

To identify the function of triadin in skeletal muscle, adenovirus-mediated overexpression of Trisk 95 or Trisk 51, the two major skeletal muscle isoforms, was induced in rat skeletal muscle primary cultures, and the physiological behavior of the modified cells was analyzed. Overexpression did not modify the expression level of their protein partners ryanodine receptor, dihydropyridine receptor, and the other triadin. Caffeine-induced calcium release was also unaffected by triadin overexpression. Nevertheless, in the absence of extracellular calcium, depolarization-induced calcium release was almost abolished in Trisk 95 overexpressing myotubes (T95 myotubes), and not modified in Trisk 51 overexpressing myotubes (T51 myotubes). This was not because of a modification of dihydropyridine receptors, as depolarization in presence of external calcium still induced a calcium release, and the activation curve of dihydropyridine receptor was unchanged, in both T95 and T51 myotubes. The calcium release complex was also maintained in T95 myotubes as Trisk 95, ryanodine receptor, dihydropyridine receptor, and Trisk 51 were still co-localized. The effect of Trisk 95 overexpression on depolarization-induced calcium release was reversed by a simultaneous infection with an antisense Trisk 95 adenovirus, indicating the specificity of this effect. Thus, the level of Trisk 95 and not Trisk 51 is important on regulating the calcium release complex, and an excess of this protein can lead to an inhibition of the physiological function of the complex.

Clathrin‐independent internalization of the human histamine H1‐receptor in CHO‐K1 cells

The aim of the present study was to investigate the cellular pathway involved in histamine-stimulated internalization of the human H1-receptor in CHO-K1 cells expressing N-terminal myc-tagged H1-receptor (Myc-H1) or N-terminal myc-C-terminal green fluorescent protein (Myc-GFP H1) versions of the receptor. Studies of 3H-mepyramine binding and histamine-stimulated 3H-inositol phosphate accumulation in these cells showed that the Myc-H1 and Myc-GFP H1-receptors had identical pharmacology to the wild-type H1-receptor. The Myc-H1-receptor was rapidly internalized in CHO-K1 cells following stimulation with histamine (0.1 mM). This response occurred within 15 min, and could be prevented by the quaternary H1-receptor antagonist alpha-pirdonium. Similar data were obtained with the Myc-GFP H1-receptors. Internalization of the Myc-GFP H1-receptor was maintained in the absence of extracellular calcium and was not inhibited by the CAM kinase II inhibitor KN-62 (10 microM). Phorbol dibutyrate, an activator of protein kinase C, was also able to stimulate internalization of the H1-receptor. However, inhibition or downregulation of protein kinase C (which significantly modified histamine-stimulated inositol phosphate responses) was without effect on the internalization of the H1-receptor stimulated by histamine. Hypertonic sucrose did not prevent histamine-induced internalization of the Myc-GFP H1-receptor, but was able to attenuate internalization of transferrin via clathrin-mediated endocytosis in the same cells. In contrast, preincubation of cells with filipin or nystatin, which disrupts caveolae and lipid rafts, completely inhibited the histamine-induced internalization of the Myc-GFP H1-receptor, but was without effect on the sequestration of transferrin. The H1-receptor and cholera toxin subunit B were colocalized under resting conditions at the cell surface. Immunohistochemical studies with an antibody to caveolin-1 confirmed that this protein was also localized predominantly to the plasma membrane. However, following stimulation of CHO-Myc-GFP H1 cells with histamine, there was no evidence for internalization of caveolin-1 in parallel with the H1-receptor. These data provide strong evidence that the H1-receptor is internalized via a clathrin-independent mechanism and most likely involves lipid rafts.