Ca2+ Levels (ca2+ + level)

Distribution by Scientific Domains

Kinds of Ca2+ Levels

  • intracellular ca2+ level

  • Selected Abstracts

    A Vacuolar ATPase Inhibitor, FR167356, Prevents Bone Resorption in Ovariectomized Rats With High Potency and Specificity: Potential for Clinical Application,

    Kazuaki Niikura MS
    Abstract FR167356, a novel inhibitor of vacuolar ATPase, has high potency against osteoclast V-ATPase and low potency against lysosomal V-ATPase. FR167356 is the first compound of this nature to be tested. It has the potential to be useful for clinical application. Introduction: It has been suggested that the key issue regarding the therapeutic usefulness of V-ATPase inhibitors is their selectivity. Materials and Methods: In in vitro and in vivo studies, we compared FR167356 with other vacuolar ATPase (V-ATPase) inhibitors, bafilomycin A1 and SB242784. H+ transport by various membrane vesicles was assayed by measuring uptake of acridine orange. Inhibitory activity against in vitro bone resorption was examined by measuring the Ca2+ release from cultured calvariae. In vivo, hypercalcemia was induced by retinoic acid in thyroparathyroidectomized-ovariectomized rats, and the effect on serum Ca2+ level was assessed. Ovariectomized rats were treated with FR167356 or SB242784. One week after surgery, free deoxypyridinoline levels in 24-h urine samples, which were collected from 6 h after administration of FR167356, were measured by ELISA. After 4 weeks of treatment, plasma biochemical parameters were analyzed. BMD of the distal femur metaphysis was measured with pQCT. Histomorphometric analysis of the proximal tibias was performed. Blood gases of rats treated with FR167356 were measured with a blood gas analyzer for estimating the effect of FR167356 on in vivo function of renal V-ATPase. Results: FR167356, which is distinctly different from other V-ATPase inhibitors, has a high potency against osteoclast V-ATPase and low potency against lysosomal V-ATPase. Similarly, FR167356 inhibited bone resorption in vitro when stimulated by PTH, IL-1, and IL-6. FR167356 reduced retinoic acid-induced hypercalcemia in thyroparathyroidectomized-ovariectomized rats in a dose-dependent manner. Moreover, FR167356 was shown to restore BMD of ovariectomized rats caused by the inhibition of bone resorption. Ovariectomized rats treated with FR167356 did not show adverse symptoms, whereas SB242784 caused a decrease in body weight gain and significant changes in two plasma biochemical parameters. Interestingly, FR167356 treatment did not affect blood acid-base balance; however, FR167356 inhibited renal V-ATPase with a similar potency as for osteoclast V-ATPase inhibition. Conclusion: Comparison of FR167356 with SB242784 implies that the characteristics of FR167356 may be more appropriate for clinical application as a V-ATPase inhibitor. [source]

    P2Y receptor-activating nucleotides modulate cellular reactive oxygen species production in dissociated hippocampal astrocytes and neurons in culture independent of parallel cytosolic Ca2+ rise and change in mitochondrial potential

    Stefan Kahlert
    Abstract With mixed cultures of hippocampal astrocytes and neurons, we investigated the influence of nucleotides on cytosolic Ca2+ level, generation of reactive oxygen species (ROS), and mitochondrial potential. We employed ATP and four purine/pyrimidine derivates, which are P2Y receptor subtype-preferring agonists. Stimulation with ATP, a P2Y1/2/4 receptor agonist in rat, caused a large cytosolic Ca2+ increase in astrocytes and a considerably smaller Ca2+ response in neighboring neurons. The P2Y1 receptor antagonist MRS2179 completely blocked the ATP-induced Ca2+ response in astrocytes and neurons. Application of ATP significantly reduced the mitochondrial potential in neurons, which was not inhibited by MRS2179. Interestingly, MRS2179 mediated a mitochondrial depolarization without affecting the cytosolic Ca2+ level. Stimulation with UDP, a P2Y6 receptor agonist; UTP, a P2Y2/4 receptor agonist; 2MeSATP, a P2Y1 receptor agonist; or 2MeSADP, a P2Y1/12/13 receptor agonist, evoked significant Ca2+ responses in astrocytes but small Ca2+ responses in neurons. In astrocytes, there was an inverse relationship between the amplitude of the cytosolic Ca2+ peak and the rate of ROS generation in response to nucleotide application. Activation with UDP resulted in the highest ROS generation that we detected, whereas 2MeSADP and 2MeSATP reduced the ROS generation below the basal level. 2MeSADP and UDP caused mitochondrial depolarization of comparable size. Thus, neither in astrocytes nor in neurons did the degree of mitochondrial depolarization correlate with ROS generation. Nucleotides acting via P2Y receptors can modulate ROS generation of hippocampal neurons without acutely changing the cytosolic Ca2+ level. Thus, ROS might function as a signaling molecule upon nucleotide-induced P2Y receptor activation in brain. © 2007 Wiley-Liss, Inc. [source]

    Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytes

    Stefan Kahlert
    Abstract Generation of reactive oxygen species (ROS) in brain tissue leads to neurodegeneration. The major source of ROS is the mitochondrial respiratory chain. We studied regulation of Ca2+ level, mitochondrial potential, and ROS generation in defined mixed hippocampal cell cultures exposed to glutamate (100 ,M). Recordings were made from individually identified astrocytes and neurons to compare the physiologic responses in both cell types. Neurons identified by synaptotagmin immunoreactivity were characterized functionally by the fast Ca2+ increase with K+ (50 mM) stimulation, and the astrocytes identified by glial fibrillary acidic protein (GFAP) staining had the functional characteristic of a transient Ca2+ peak in response to ATP (10 ,M) stimulation. We found that the glutamate-mediated Ca2+ response in neurons is due largely to influx of extracellular Ca2+. This is consistent with our finding that in cultured hippocampal neurons, stores depending on the activity of the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) pump had a low Ca2+ content, regardless of whether the neurons were challenged or not with K+ before applying the SERCA inhibitor cyclopiazonic acid (CPA). Astrocytes displayed a large CPA-mediated Ca2+ response, indicating a high level of Ca2+ load in the stores in astrocytes. Importantly, the rise in ROS generation due to glutamate application was cell-type specific. In neurons, glutamate induced a marked rise in generation of ROS, but not in astrocytes. In both astrocytes and neurons, the mitochondrial potential was increased in response to glutamate challenge. We conclude that in neurons, Ca2+ influx accounts for the increased ROS generation in response to glutamate. This might explain the high vulnerability of neurons to glutamate challenge compared to the vulnerability of astrocytes. The high resistance of astrocytes is accompanied by an efficient downregulation of cytosolic Ca2+, which is not found in neurons. © 2004 Wiley-Liss, Inc. [source]

    Iso-S -petasin, a hypotensive sesquiterpene from Petasites formosanus, depresses cardiac contraction and intracellular Ca2+ transients in adult rat ventricular myocytes

    Lucy B. Esberg
    ABSTRACT Petasites formosanus is an indigenous species of the medicinal plant Petasites which has been used to treat hypertension. Both S -petasin and its isoform iso-S -petasin have been shown to be the effective ingredients in P. formosanus. However, their effect on heart function has not been revealed. This study was to examine the effect of iso-S -petasin on cardiac contractile function at the myocyte level. Ventricular myocytes were isolated from adult rat hearts and were stimulated to contract at 0.5 Hz under 1.0 mm extracellular Ca2+. Contractile properties were evaluated using an lonOptix MyoCam system including peak shortening (PS), time to PS (TPS), time to 90% re-lengthening (TR90) and maximal velocity of shortening/re-lengthening (±dL/dt). Intracellular Ca2+ properties were assessed by fura-2 and presented as Ca2+ -induced Ca2+ release (CICR) and intracellular Ca2+ decay. Acute application of iso-S -petasin (10,7 to 10,4 M) elicited a concentration-dependent inhibition in PS and CICR, with maximal inhibitions of 51.0% and 31.0%, respectively. iso-S -petasin also induced a concentration-dependent inhibition of ± dL/dt without affecting TPS, TR90, baseline intracellular Ca2+ level or intracellular Ca2+ decay. Elevation of extracellular Ca2+ from 1.0 mm to 2.7 mm significantly antagonized the iso-S -petasin-induced depression in PS and CICR. These results demonstrated a direct depressant action of iso-S -petasin on ventricular contraction, which may work in concert with its antihypertensive action to reduce the cardiac load. The iso-S -petasin-induced decrease in CICR may play a role in its cardiac depressant effect. [source]

    Glutamate-induced internalization of Cav1.3 L-type Ca2+ channels protects retinal neurons against excitotoxicity

    Fengxia Mizuno
    Glutamate-induced rise in the intracellular Ca2+ level is thought to be a major cause of excitotoxic cell death, but the mechanisms that control the Ca2+ overload are poorly understood. Using immunocytochemistry, electrophysiology and Ca2+ imaging, we show that activation of ionotropic glutamate receptors induces a selective internalization of Cav1.3 L-type Ca2+ channels in salamander retinal neurons. The effect of glutamate on Cav1.3 internalization was blocked in Ca2+ -free external solution, or by strong buffering of internal Ca2+ with BAPTA. Downregulation of L-type Ca2+ channel activity in retinal ganglion cells by glutamate was suppressed by inhibitors of dynamin-dependent endocytosis. Stabilization of F-actin by jasplakinolide significantly reduced the ability of glutamate to induce internalization suggesting it is mediated by Ca2+ -dependent reorganization of actin cytoskeleton. We showed that the Cav1.3 is the primary L-type Ca2+ channel contributing to kainate-induced excitotoxic death of amacrine and ganglion cells. Block of Cav1.3 internalization by either dynamin inhibition or F-actin stabilization increased vulnerability of retinal amacrine and ganglion cells to kainate-induced excitotoxicity. Our data show for the first time that Cav1.3 L-type Ca2+ channels are subject to rapid glutamate-induced internalization, which may serve as a negative feedback mechanism protecting retinal neurons against glutamate-induced excitotoxicity. [source]

    Evolution and modulation of intracellular calcium release during long-lasting, depleting depolarization in mouse muscle

    Leandro Royer
    Intracellular calcium signals regulate multiple cellular functions. They depend on release of Ca2+ from cellular stores into the cytosol, a process that in many types of cells appears to be tightly controlled by changes in [Ca2+] within the store. In contrast with cardiac muscle, where depletion of Ca2+ in the sarcoplasmic reticulum is a crucial determinant of termination of Ca2+ release, in skeletal muscle there is no agreement regarding the sign, or even the existence of an effect of SR Ca2+ level on Ca2+ release. To address this issue we measured Ca2+ transients in mouse flexor digitorum brevis (FDB) skeletal muscle fibres under voltage clamp, using confocal microscopy and the Ca2+ monitor rhod-2. The evolution of Ca2+ release flux was quantified during long-lasting depolarizations that reduced severely the Ca2+ content of the SR. As in all previous determinations in mammals and non-mammals, release flux consisted of an early peak, relaxing to a lower level from which it continued to decay more slowly. Decay of flux in this second stage, which has been attributed largely to depletion of SR Ca2+, was studied in detail. A simple depletion mechanism without change in release permeability predicts an exponential decay with time. In contrast, flux decreased non-exponentially, to a finite, measurable level that could be maintained for the longest pulses applied (1.8 s). An algorithm on the flux record allowed us to define a quantitative index, the normalized flux rate of change (NFRC), which was shown to be proportional to the ratio of release permeability P and inversely proportional to Ca2+ buffering power B of the SR, thus quantifying the ,evacuability' or ability of the SR to empty its content. When P and B were constant, flux then decayed exponentially, and NFRC was equal to the exponential rate constant. Instead, in most cases NFRC increased during the pulse, from a minimum reached immediately after the early peak in flux, to a time between 200 and 250 ms, when the index was no longer defined. NFRC increased by 111% on average (in 27 images from 18 cells), reaching 300% in some cases. The increase may reflect an increase in P, a decrease in B, or both. On experimental and theoretical grounds, both changes are to be expected upon SR depletion. A variable evacuability helps maintain a constant Ca2+ output under conditions of diminishing store Ca2+ load. [source]

    Depletion of membrane cholesterol eliminates the Ca2+ -activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes

    A. Shmygol
    Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl-,-cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca2+ signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19,21 rats showed that outward K+ current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol-saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K+ channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca2+ -sensitive K+ channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca2+ level ([Ca2+]i transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca2+ signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol-rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca2+ signalling and uterine contractility. [source]

    Spontaneous Ca2+ Waves in Rabbit Corpus Cavernosum: Modulation by Nitric Oxide and cGMP

    Gerard P. Sergeant PhD
    ABSTRACT Introduction., Detumescent tone and subsequent relaxation by nitric oxide (NO) are essential processes that determine the erectile state of the penis. Despite this, the mechanisms involved are incompletely understood. It is often assumed that the tone is associated with a sustained high cytosolic Ca2+ level in the corpus cavernosum smooth muscle cells, however, an alternative possibility is that oscillatory Ca2+ signals regulate tone, and erection occurs as a result of inhibition of Ca2+ oscillations by NO. Aims., The aim of this study is to determine if smooth muscle cells displayed spontaneous Ca2+ oscillations and, if so, whether these were regulated by NO. Methods., Male New Zealand white rabbits were euthanized and smooth muscle cells were isolated by enzymatic dispersal for confocal imaging of intracellular Ca2+ (using fluo-4AM) and patch clamp recording of spontaneous membrane currents. Thin tissue slices were also loaded with fluo-4AM for live imaging of Ca2+. Main Outcome Measure., Cytosolic Ca2+ was measured in isolated smooth muscle cells and tissue slices. Results., Isolated rabbit corpus cavernosum smooth muscle cells developed spontaneous Ca2+ waves that spread at a mean velocity of 65 µm/s. Dual voltage clamp/confocal recordings revealed that each of the Ca2+ waves was associated with an inward current typical of the Ca2+ -activated Cl - currents developed by these cells. The waves depended on an intact sarcoplasmic reticulum Ca2+ store, as they were blocked by cyclopiazonic acid (Calbiochem, San Diego, CA, USA) and agents that interfere with ryanodine receptors and IP3 -mediated Ca2+ release. The waves were also inhibited by an NO donor (diethylamine NO; Tocris Bioscience, Bristol, Avon, UK), 3-(5-hydroxymethyl-2-furyl)-1-benzyl indazole (YC-1) (Alexis Biochemicals, Bingham, Notts, UK), 8-bromo-cyclic guanosine mono-phosphate (Tocris), and sildenafil (Viagra, Pfizer, Sandwich, Kent, UK). Regular Ca2+ oscillations were also observed in whole tissue slices where they were clearly seen to precede contraction. This activity was also markedly inhibited by sildenafil, suggesting that it was under NO regulation. Conclusions., These results provide a new basis for understanding detumescent tone in the corpus cavernosum and its inhibition by NO. Sergeant GP, Craven M, Hollywood MA, McHale NG, and Thornbury KD. Spontaneous Ca2+ waves in rabbit corpus cavernosum: Modulation by nitric oxide and cGMP. J Sex Med **;**:**,**. [source]

    Expression and function of ryanodine receptors in rabbit penile corpus cavernosum smooth muscle cells

    ANDROLOGIA, Issue 3 2009
    H. G. Liu
    Summary This study aimed to investigate the expression and function of ryanodine receptors (RyRs) in rabbit penile corpus cavernosum smooth muscle (CCSM) cells. New Zealand white rabbit CCSM cells were cultured by primary tissue culture and identified by immunofluorescence technique. mRNA of three RyRs subunits in cultured CCSM cells was detected by reverse transcription polymerase chain reaction (RT-PCR). After excitation by noradrenaline, the concentration of Ca2+ in CCSM cells was detected by laser scanning confocal microscopy. It was found that only the RyRs1 subunit is expressed in CCSM by RT-PCR. The CCSM cells were divided into two groups: Group A, CCSM cells + 10 ,mol l,1 noradrenaline and Group B, CCSM cells + 50 ,mol l,1 procaine 10 ,mol l,1 noradrenaline. Compared with the base level and the level after noradrenaline excitation, fluorescence intensity improved 44.10 ± 6.01% in the test group A (n = 8) and 32.92 ± 4.92% (n = 8) in the procaine control group B, respectively. There were statistically significant differences between group A and group B (P < 0.01). It is concluded that RyRs1 subunit is expressed in CCSM cells and may contribute to regulating the cytoplasmic Ca2+ level. [source]

    Gating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+

    ACTA PHYSIOLOGICA, Issue 4 2006
    L. Lacinová
    Abstract Aim:, We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. Methods:, The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 ,m Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. Results:, The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ,100 mV for the Ca2+ current, but did not reach steady state even at ,150 mV for the Na+ current. Conclusions:, Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor. [source]

    Nonylphenol-induced cytosolic Ca2+ elevation and death in renal tubular cells

    Jeng-Yu Tsai
    Abstract Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca2+ levels ([Ca2+]i) and viability in Madin-Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca2+]i in a concentration-dependent manner (EC50,0.8,,M). Nonylphenol-induced Mn2+ entry demonstrated Ca2+ influx and removal of extracellular Ca2+ partly decreased the [Ca2+]i rise. The [Ca2+]i rise was inhibited by the protein kinase C activator, phorbol 13-myristate acetate (PMA) but not by L-type Ca2+ channel blockers. In Ca2+ -free medium, nonylphenol-induced [Ca2+]i rise was partly inhibited by pretreatment with 1,,M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin-induced Ca2+ release. Nonylphenol-induced Ca2+ release was unaltered by inhibition of phospholipase C. At concentrations of 5,100,,M, nonylphenol killed cells in a concentration-dependent manner. The cytotoxic effect of 100,,M nonylphenol was not affected by preventing [Ca2+]i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca2+]i increase in MDCK cells via evoking Ca2+ entry through protein kinase C-regulated Ca2+ channels, and releasing Ca2+ from endoplasmic reticulum and other stores in a phospholipase C-independent manner. Nonylphenol also killed cells in a Ca2+ -independent fashion. Drug Dev Res, 2009. © 2009 Wiley-Liss, Inc. [source]

    Role of the pro-inflammatory cytokines TNF-, and IL-1, in HIV-associated dementia

    N. A. C. H. Brabers
    Abstract Human immunodeficiency virus-1 (HIV-1)-infected and immune-activated macrophages and microglia secrete neurotoxins. Two of these neurotoxins are the pro-inflammatory cytokines tumour necrosis factor-, (TNF-,) and interleukin-1, (IL-1,), which are thought to play a major role in inducing neuronal death. Both TNF-, and IL-1, increase the permeability of the blood,brain barrier, through which subsequently HIV-infected monocytes can enter the brain. They both induce over-stimulation of the NMDA-receptor via several pathways, resulting in a lethal neuronal increase in Ca2+ levels. Additionally, TNF-, co-operates with several other proinflammatory mediators to enhance their toxic effects. Although most research has focused on the neurotoxic effects of TNF-, and IL-1, in HAD, there is also evidence that these cytokines can be neuroprotective. In this paper the effect of TNF-, and IL-1, on neuronal life and death in HAD is discussed. [source]

    NCS-1 differentially regulates growth cone and somata calcium channels in Lymnaea neurons

    Kwokyin Hui
    Abstract Local voltage-gated calcium channels, which regulate intracellular Ca2+ levels by allowing Ca2+ influx, play an important role in guiding and shaping growth cones, and in regulating the outgrowth and branching of neurites. Therefore, elucidating the mechanisms that regulate the biophysical properties of whole-cell calcium currents in the growth cones and somata of growing neurons is important to improving our understanding of neuronal development and regeneration. In this study, taking advantage of the large size of the pedal A (PeA) neurons in Lymnaea stagnalis, we compared the biophysical properties of somata and growth cone whole-cell calcium channel currents using Ba2+ and Ca2+ as current carriers. We found that somata and growth cone currents exhibit similar high-voltage activation properties. However, Ba2+ and Ca2+ currents in growth cones and somata are differentially affected by a dominant-negative peptide containing the C-terminal amino acid sequence of neuronal calcium sensor-1 (NCS-1). The peptide selectively reduces the peak and sustained components of current densities and the slope conductance in growth cones, and shifts the reversal potential of the growth cone currents to more hyperpolarized voltages. In contrast, the peptide had no significant effect on the somata calcium channels. Thus, we conclude that NCS-1 differentially modulates Ca2+ currents in the somata and growth cones of regenerating neurons, and may serve as a key regulator to facilitate the growth cone calcium channel activity. [source]

    Plasma membrane Ca2+ -ATPase in the cilia of olfactory receptor neurons: possible role in Ca2+ clearance

    Karen Castillo
    Abstract Olfactory sensory neurons respond to odorants increasing Ca2+ concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP-gated channels, activating Ca2+ -dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide-gated channels for cAMP. It has been shown that a Na+/Ca2+ exchanger (NCX) extrudes Ca2+ from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca2+ -ATPase (PMCA), and show the first evidence supporting a role in Ca2+ removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside-out cilia membrane vesicles transported Ca2+ in an ATP-dependent fashion. PMCA activity was potentiated by luminal Ca2+ (K0.5 = 670 nm) and enhanced by calmodulin (CaM; K0.5 = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca2+ transport, as expected for a CaM-modulated PMCA. The relaxation time constant (,) of the Ca2+ -dependent Cl, current (272 ± 78 ms), indicative of luminal Ca2+ decline, was increased by CE (2181 ± 437 ms), by omitting ATP (666 ± 49 ms) and by raising pH (725 ± 65 ms), suggesting a role of the pump on Ca2+ clearance. Replacement of external Na+ by Li+ had a similar effect (, = 442 ± 8 ms), confirming the NCX involvement in Ca2+ extrusion. The evidence suggests that both Ca2+ transporters contribute to re-establish resting Ca2+ levels in the cilia following olfactory responses. [source]

    Electrical and neurotransmitter activity of mature neurons derived from mouse embryonic stem cells by Sox-1 lineage selection and directed differentiation

    R. J. Lang
    Abstract Sx1TV2/16C is a mouse embryonic stem (ES) cell line in which one copy of the Sox1 gene, an early neuroectodermal marker, has been targeted with a neomycin (G418) selection cassette. A combination of directed differentiation with retinoic acid and G418 selection results in an enriched neural stem cell population that can be further differentiated into neurons. After 6,7 days post-plating (D6,7PP) most neurons readily fired tetrodotoxin (TTX)-sensitive action potentials due to the expression of TTX-sensitive Na+ and tetraethylammonium (TEA)-sensitive K+ channels. Neurons reached their maximal cell capacitance after D6,7PP; however, ion channel expression continued until at least D21PP. The percentage of cells receiving spontaneous synaptic currents (s.s.c.) increased with days in culture until 100% of cells received a synaptic input by D20PP. Spontaneous synaptic currents were reduced in amplitude and frequency by TTX, or upon exposure to a Ca2+ -free, 2.5 mm Mg2+ saline. S.s.c. of rapid decay time constants were preferentially blocked by the nonNMDA glutamatergic receptor antagonists CNQX or NBQX. Ca2+ levels within ES cell-derived neurons increased in response to glutamate receptor agonists l -glutamate, AMPA, N -methyl- d -aspartate (NMDA) and kainic acid and to acetylcholine, ATP and dopamine. ES cell-derived neurons also generated cationic and Cl, -selective currents in response to NMDA and glycine or GABA, respectively. It was concluded that ES-derived neurons fire action potentials, receive excitatory and inhibitory synaptic input and respond to various neurotransmitters in a manner akin to primary central neurons. [source]

    Calcium dynamics are altered in cortical neurons lacking the calmodulin-binding protein RC3

    Jacqueline J. W. Van Dalen
    Abstract RC3 is a neuronal calmodulin-binding protein and protein kinase C substrate that is thought to play an important regulatory role in synaptic transmission and neuronal plasticity. Two molecules known to regulate synaptic transmission and neuronal plasticity are Ca2+ and calmodulin, and proposed mechanisms of RC3 action involve both molecules. However, physiological evidence for a role of RC3 in neuronal Ca2+ dynamics is limited. In the current study we utilized cultured cortical neurons obtained from RC3 knockout (RC3,/,) and wildtype mice (RC3+/+) and fura-2-based microscopic Ca2+ imaging to investigate a role for RC3 in neuronal Ca2+ dynamics. Immunocytochemical characterization showed that the RC3,/, cultures lack RC3 immunoreactivity, whereas cultures prepared from wildtype mice showed RC3 immunoreactivity at all ages studied. RC3+/+ and RC3,/, cultures were indistinguishable with respect to neuron density, neuronal morphology, the formation of extensive neuritic networks and the presence of glial fibrillary acidic protein (GFAP)-positive astrocytes and ,-aminobutyric acid (GABA)ergic neurons. However, the absence of RC3 in the RC3,/, neurons was found to alter neuronal Ca2+ dynamics including baseline Ca2+ levels measured under normal physiological conditions or after blockade of synaptic transmission, spontaneous intracellular Ca2+ oscillations generated by network synaptic activity, and Ca2+ responses elicited by exogenous application of N-methyl- d -aspartate (NMDA) or class I metabotropic glutamate receptor agonists. Thus, significant changes in Ca2+ dynamics occur in cortical neurons when RC3 is absent and these changes do not involve changes in gross neuronal morphology or neuronal maturation. These data provide direct physiological evidence for a regulatory role of RC3 in neuronal Ca2+ dynamics. [source]

    Expression of functional NR1/NR2B-type NMDA receptors in neuronally differentiated SK-N-SH human cell line

    Marina Pizzi
    Abstract The present study demonstrates that human SK-N-SH neuroblastoma cells, differentiated by retinoic acid (RA), express functional NMDA receptors and become vulnerable to glutamate toxicity. During exposure to RA, SK-N-SH cells switched from non-neuronal to neuronal phenotype by showing antigenic changes typical of postmitotic neurons together with markers specific for cholinergic cells. Neuronally differentiated cells displayed positive immunoreactivity to the vesicular acetylcholine transporter and active acetylcholine release in response to depolarizing stimuli. The differentiation correlated with the expression of NMDA receptors. RT-PCR and immunoblotting analysis identified NMDA receptor subunits NR1 and NR2B, in RA-differentiated cultures. The NR1 protein immunolocalized to the neuronal cell population and assembled with the NR2B subunit to form functional N -methyl- d -aspartate (NMDA) receptors. Glutamate or NMDA application, concentration-dependently increased the intracellular Ca2+ levels and acetylcholine release in differentiated cultures, but not in undifferentiated SK-N-SH cells. Moreover, differentiated cultures became vulnerable to NMDA receptor-mediated excitotoxicity. The glutamate effects were enhanced by glycine application and were prevented by the NMDA receptor blocker MK 801, as well as by the NR2B selective antagonist ifenprodil. These data suggest that SK-N-SH cells differentiated by brief treatment with RA may represent an unlimited source of neuron-like cells suitable for studying molecular events associated with activation of human NR1/NR2B receptors. [source]

    Gq/11-induced intracellular calcium mobilization mediates Per2 acute induction in Rat-1 fibroblasts

    GENES TO CELLS, Issue 9 2006
    Naoyuki Takashima
    Phase resetting is one of the essential properties of circadian clocks that is required for the adjustment to a particular environment and the induction of Per1 and Per2 clock genes is believed to be a primary molecular event during this process. Although the intracellular signal transduction pathway underlying Per1 gene activation has been well characterized, the mechanisms that control Per2 up-regulation have not yet been elucidated. In our present study, we demonstrate that Gq/11 coupled receptors mediate serum-induced immediate rat Per2 (rPer2) transactivation in Rat-1 fibroblasts via intracellular Ca2+ mobilization. Stimulation of these cells with a high concentration of serum was found to rapidly increase the intracellular Ca2+ levels and strongly up-regulated rPer2 gene. rPer2 induction by serum stimulation was abrogated by intracellular Ca2+ chelation and depletion of intracellular Ca2+ store, which suggests that the calcium mobilization is necessary for the up-regulation of rPer2 gene. In addition, suppression of Gq/11 function was observed to inhibit both Ca2+ mobilization and rPer2 induction. Further, we demonstrated that endothelin-induced acute rPer2 transactivation via Gq/11-coupled endothelin receptors is also suppressed by a Gq/11 specific inhibitor. These findings together suggest that serum and endothelin utilize a common Gq/11-PLC mediated pathway for the transactivation of rPer2, which involves the mobilization of calcium from the intracellular calcium store. [source]

    Changes in the expression of plasma membrane calcium extrusion systems during the maturation of hippocampal neurons

    HIPPOCAMPUS, Issue 1 2006
    Sertac N. Kip
    Abstract Spatial and temporal control of intracellular calcium signaling is essential for neuronal development and function. The termination of local Ca2+ signaling and the maintenance of basal Ca2+ levels require specific extrusion systems in the plasma membrane. In rat hippocampal neurons (HNs) developing in vitro, transcripts for all isoforms of the plasma membrane Ca2+ pump and the Na/Ca2+ exchanger, and the major nonphotoreceptor Na+/Ca2+,K+ exchangers (NCKX) were strongly upregulated during the second week in culture. Upregulation of plasma membrane calcium ATPases (PMCAs)1, 3, and 4 mRNA coincided with a splice shift from the ubiquitous b-type to the neuron-specific a-type with altered calmodulin regulation. Expression of all PMCA isoforms increased over 5-fold during the first 2 weeks. PMCA immunoreactivity was initially concentrated in the soma and growth cones of developing HNs. As the cells matured, PMCAs concentrated in the dendritic membrane and often colocalized with actin-rich dendritic spines in mature neurons. In the developing rat hippocampal CA1 region, immunohistochemistry confirmed the upregulation of all PMCAs and showed that by the end of the second postnatal week, PMCAs1, 2, and 3 were concentrated in the neuropil, with less intense staining of cell bodies in the pyramidal layer. PMCA4 staining was restricted to a few cells showing intense labeling of the cell periphery and neurites. These results establish that all major Ca2+ extrusion systems are strongly upregulated in HNs during the first 2 weeks of postnatal development. The overall increase in Ca2+ extrusion systems is accompanied by changes in the expression and cellular localization of different isoforms of the Ca2+ pumps and exchangers. The accumulation of PMCAs in dendrites and dendritic spines coincides with the functional maturation in these neurons, suggesting the importance of the proper spatial organization of Ca2+ extrusion systems for synaptic function and development. © 2005 Wiley-Liss, Inc. [source]

    Murine TNF,ARE Crohn's disease model displays diminished expression of intestinal Ca2+ transporters

    Sylvie Huybers MSc
    Abstract Background: Patients suffering from Crohn's disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU-rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF-, levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca2+ handling problem, this study analyzed the Ca2+ homeostasis in heterozygous TNF,ARE mice (TNF,ARE/+) in comparison to wildtype littermates. Methods: Beside serum Ca2+ and vitamin D levels, the expression of Ca2+ transporters was analyzed in intestine, kidney and bone using quantitative real-time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. Results: Ca2+ transporters in duodenum (TRPV6, calbindin-D9K, PMCA1b) and kidney (TRPV5, calbindin-D28K, NCX1) showed significantly reduced mRNA expression levels in TNP,ARE/+ mice, except for renal TRPV5. In bone, only calbindin-D9K mRNA displayed a significant down-regulation. These findings were supported by declined duodenal calbindin-D9K and renal calbindin-D28K protein values. Likely, this down-regulation of Ca2+ transporters in TNP,ARE/+ mice is mediated by the 58 ± 9% reduction in serum 1,25(OH)2D3 levels. Diminished expression of Ca2+ transporters combined with unchanged serum Ca2+ levels assumes Ca2+ loss from bone to compensate for the body's overall Ca2+ shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNF,ARE/+ mice. This finding is further supported by increased total deoxypyridinoline in serum. Conclusions: Our results imply that TNF,ARE/+ mice have a disturbed Ca2+ homeostasis characterized by reduced duodenal and renal Ca2+ transporters, diminished 1,25(OH)2D3 levels, and increased bone resorption associated with profound bone abnormalities. (Inflamm Bowel Dis 2008) [source]

    Thiazide Diuretics Affect Osteocalcin Production in Human Osteoblasts at the Transcription Level Without Affecting Vitamin D3 Receptors

    D. Lajeunesse
    Abstract Besides their natriuretic and calciuretic effect, thiazide diuretics have been shown to decrease bone loss rate and improve bone mineral density. Clinical evidence suggests a specific role of thiazides on osteoblasts, because it reduces serum osteocalcin (OC), an osteoblast-specific protein, yet the mechanisms implicated are unknown. We therefore investigated the role of hydrochlorothiazide (HCTZ) on OC production by the human osteoblast-like cell line MG-63. HCTZ dose-dependently (1,100 ,M) inhibited 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]- induced OC release by these cells (maximal effect, ,40,50% and p < 0.005 by analysis of variance [ANOVA]) as measured by ELISA. This effect of HCTZ on OC release was caused by a direct effect on OC gene expression because Northern blot analysis revealed that OC messenger RNA (mRNA) levels were reduced in the presence of increasing doses of the diuretic (,47.2 ± 4.0%; p < 0.0001 by paired ANOVA with 100 ,M HCTZ). HCTZ (100 ,M) also stimulated calcium (Ca2+) uptake (8.26 ± 1.78 pmol/mg protein/15 minutes vs. 13.6 ± 0.49 pmol/mg protein/15 minutes; p < 0.05) in MG-63 cells. Reducing extracellular Ca2+ concentration with 0.5 mM EDTA or 0.5 mM ethylene glycol-bis(,-amino ethyl ether)- N,N,N',N' -tetraacetic acid (EGTA) only partly prevented the inhibitory effect of the diuretic on OC secretion (maximal effect, ,22.5 ± 6.9%), suggesting that thiazide-dependent Ca2+ influx is not sufficient to elicit the inhibition of OC secretion. Because OC production is strictly dependent on the presence of 1,25(OH)2D3 in human osteoblasts, we next evaluated the possible role of HCTZ on vitamin D3 receptors (VDR) at the mRNA and protein levels. Both Northern and Western blot analyses showed no effect of HCTZ (1,100 ,M) on VDR levels. The presence of EGTA in the culture media reduced slightly the VDR mRNA levels under basal condition but this was not modified in the presence of increasing levels of HCTZ. The OC gene promoter also is under the control of transcription factors such as Yin Yang 1 (YY1) and cFOS. Western blot analysis revealed no changes in YY1 levels in response to HCTZ either in the presence or in the absence of 0.5 mM EGTA in the culture media. In contrast, HCTZ induced a dose-dependent increase in cFOS levels (p < 0.002 by ANOVA), a situation prevented by incubation with EGTA. These studies indicate that HCTZ inhibits OC mRNA expression independently of an effect on VDR, YY1, or extracellular Ca2+ levels but involves changes in cFOS levels. As OC retards bone formation/mineralization, the inhibition of OC production by HCTZ could explain its preventive role in bone loss rate. (J Bone Miner Res 2000;15:894,901) [source]

    Influence of cardiac-specific overexpression of insulin-like growth factor 1 on lifespan and aging-associated changes in cardiac intracellular Ca2+ homeostasis, protein damage and apoptotic protein expression

    AGING CELL, Issue 6 2007
    Qun Li
    Summary A fall in circulating levels of cardiac survival factor insulin-like growth factor 1 (IGF-1) contributes to cardiac aging. To better understand the role of IGF-1 in cardiac aging, we examined the influence of cardiac IGF-1 overexpression on lifespan, cardiomyocyte intracellular Ca2+ homeostasis, protein damage, apoptosis and expression of pro- and anti-apoptotic proteins in young and old mice. Mouse survival rate was constructed by the Kaplan,Meier curve. Intracellular Ca2+ was evaluated by fura-2 fluorescence. Protein damage was determined by protein carbonyl formation. Apoptosis was assessed by caspase-8 expression, caspase-3 and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay. Pro- and anti-apoptotic proteins including Bax, p53, pp53, Bcl2, Omi/HtrA2, apoptosis repressor with caspase recruitment domain (ARC) and X-linked inhibitor of apoptosis protein (XIAP) were assessed by Western blot. Aging decreased plasma in IGF-1 levels, elevated myocyte resting intracellular Ca2+ levels, reduced electrically stimulated rise in intracellular Ca2+ and delayed intracellular Ca2+ decay associated with enhanced protein carbonyl formation, caspase-8 expression and caspase-3 activity in FVB mice, all of which with the exception of elevated resting intracellular Ca2+ were attenuated by IGF-1. Aging up-regulated expression of Bax, Bcl2 and ARC, down-regulated XIAP expression and did not affect p53, pp53 and Omi/HtrA2. The IGF-1 transgene attenuated or nullified aging-induced changes in Bax, Bcl2 and XIAP. Our data suggest a beneficial role for IGF-1 in aging-induced survival, cardiac intracellular Ca2+ homeostasis, protein damage and apoptosis possibly related to pro- and anti-apoptotic proteins. [source]

    Insensitivity to glutamate neurotoxicity mediated by NMDA receptors in association with delayed mitochondrial membrane potential disruption in cultured rat cortical neurons

    Yuki Kambe
    Abstract We have attempted to elucidate mechanisms underlying differential vulnerability to glutamate (Glu) using cultured neurons prepared from discrete structures of embryonic rat brains. Brief exposure to Glu led to a significant decrease in the mitochondrial activity in hippocampal neurons cultured for 9 or 12 days at 10 ,M to 1 mM with an apoptosis-like profile, without markedly affecting that in cortical neurons. Brief exposure to Glu also increased lactate dehydrogenase release along with a marked decrease in the number of cells immunoreactive for a neuronal marker protein in hippocampal, but not cortical, neurons. Similar insensitivity was seen to the cytotoxicity by NMDA, but not to that by tunicamycin, 2,4-dinitrophenol, hydrogen peroxide or A23187, in cortical neurons. However, NMDA was more efficient in increasing intracellular free Ca2+ levels in cortical neurons than in hippocampal neurons. Antagonists for neuroprotective metabotropic Glu receptors failed to significantly affect the insensitivity to Glu, while NMDA was more effective in disrupting mitochondrial membrane potentials in hippocampal than cortical neurons. These results suggest that cortical neurons would be insensitive to the apoptotic neurotoxicity mediated by NMDA receptors through a mechanism related to mitochondrial membrane potentials, rather than intracellular free Ca2+ levels, in the rat brain. [source]

    Serotonin and neuronal growth factors , a convergence of signaling pathways

    Daniel S. Cowen
    Abstract Monoamines, including serotonin (5-HT), have traditionally been associated with short-term signaling pathways in neurons, such as the modulation of cAMP and Ca2+ levels. In contrast, neuronal growth factors, such as neurotrophins, have been traditionally associated with signaling pathways, such as those for activation of extracellular-regulated kinase (ERK) and Akt (protein kinase B), which are known to induce long-term protective changes. It has therefore been unclear how antidepressants that increase serotonin (5-HT), induce such changes as hippocampal neuroprotection and neurogenesis. It has been hypothesized, that the actions of 5-HT may be mediated indirectly through increased synthesis of peptide growth factors. However, there is increasing evidence that some subtypes of 5-HT receptors can directly couple to activation of the ERK and Akt pathways. Such coupling suggests a more direct potential role for 5-HT in mediating the long-term actions induced by antidepressants. [source]

    The effect of short-term low-temperature treatments on gene expression in Arabidopsis correlates with changes in intracellular Ca2+ levels

    PLANT CELL & ENVIRONMENT, Issue 4 2003
    ABSTRACT The role of changes in intracellular calcium ion concentration ([Ca2+]i) in low-temperature signal transduction in plants has lately been supported by several studies. An analysis to determine whether the low-temperature-induced increase in cytosolic Ca2+ concentration ([Ca2+]cyt) could be correlated with a downstream response such as gene expression was carried out. The induction of the low-temperature-regulated gene LTI78 was used as an end point marker of the signal transduction pathway. It was found that this gene is induced by very brief low-temperature exposures and that the induction does not depend on a continuous exposure to low temperature. By altering the cooling rate, different patterns of the Ca2+ response were obtained which could be correlated with different patterns of LTI78 induction. Furthermore, reducing the Ca2+ transients by pre-treatment with the Ca2+ channel blocker La3+ also led to a reduced level of gene induction. The results show that brief exposures to low temperature results in the onset of a signalling pathway that leads to the induction of gene expression. This indicates the involvement of changes in [Ca2+]cyt in low-temperature signalling leading to LTI78 expression but the presence of multiple signalling pathways is suggested. [source]

    Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra

    Gerard P. Sergeant
    Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca2+ waves. The purpose of this study was to investigate if this activity was influenced by Ca2+ uptake into mitochondria. Spontaneous Ca2+ waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 ,m) and antimycin A (5 ,m) abolished Ca2+ waves and increased basal Ca2+ levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 ,m) and CCCP (1 ,m). Spontaneous Ca2+ waves were not inhibited by the ATP synthase inhibitor oligomycin (1 ,m), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at ,60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 ,m) also inhibited STICS. Stimulation of Ca2+ uptake into mitochondria using the plant flavonoid kaempferol (10 ,m) induced a series of propagating Ca2+ waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca2+, but was not significantly affected by the IP3 receptor blocker 2-APB (100 ,m). These data suggest that spontaneous Ca2+ waves in urethral ICC are regulated by buffering of cytoplasmic Ca2+ by mitochondria. [source]

    Improved Outcomes in Islet Isolation and Transplantation by the Use of a Novel Hemoglobin-based O2 Carrier

    J. G. Avila
    During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma-free hemoglobin-pyridoxalated (Poly SFH-P) was performed to improve O2 delivery. Rat pancreata subjected to 30-min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH-P/RPMI or RPMI (control). PO2 was increased by Poly SFH-P (381.7 ± 35.3 mmHg vs. 202.3 ± 28.2, p = 0.01) and pH maintained within physiological range (7.4,7.2 vs. 7.1,6.6, p = 0.009). Islet viability (77%± 4.6 vs. 63%± 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH-P (caspase-3: 34,714 ± 2167 vs. 45,985 ± 1382, respectively, p = 0.01). Poly SFH-P improved islet responsiveness to glucose as determined by increased intracellular Ca2+ levels and improved insulin secretion (SI 5.4 ± 0.1 vs. 3.1 ± 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH-P-treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7%± 1.8 vs. 9.8 ± 1.4, respectively, p < 0.05). O2 delivery by Poly SFH-P did not increase oxidative stress (GSH 7.1 ± 2.9 nm/mg protein for Poly SFH-P vs. 6.8 ± 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 ± 0.9 nmol/mg protein vs. 6.2 ± 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 ± 0.4 vs. 7 ± 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH-P group (AUC 8106 ± 590 vs. 10,863 ± 946, p = 0.03). Oxygenated Poly SFH-P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity. [source]

    Effects of sphingosine-1-phosphate and sphingosylphosphorylcholine on intracellular Ca2+ and cell death in prostate cancer cell lines

    A. C. M. Mulders
    Summary The sphingolipid metabolites sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) can be involved in cellular growth and apoptosis, by both receptor-dependent and -independent mechanisms. We investigated the role of S1P and SPC in intracellular Ca2+ elevation, cell proliferation and cell death in DU 145 and PC3 hormone-refractory prostate cancer cell lines. S1P and SPC increased intracellular Ca2+ levels, most likely in a receptor-independent manner. Surprisingly, both S1P and SPC did not stimulate but rather reduced cell growth through induction of apoptosis. Therefore, antagonists targeted against S1P, SPC and their receptors do not appear to be promising new approaches in the treatment of hormone-refractory prostate cancer. [source]

    Potassium channels in gastrointestinal smooth muscle

    F. Vogalis
    1 Electromechanical coupling in smooth muscle serves to coordinate the contractile activity of the syncytium. Electrical activity of smooth muscle of the gut is generated by ionic conductances that regulate and in turn are regulated by the membrane potential of smooth muscle cells. This activity determines the extent of Ca2+ entry into smooth muscle cells, and thus, the timing and intensity of contractions. 2 Potassium channels play an important role in regulating the excitability of the syncytium. The different types of K+ channel are characterized by different sensitivities to membrane potential, to intracellular Ca2+ levels and to modulation by agonists. 3 This review highlights the different types of K+ channels found in gut smooth muscle and describes their possible roles in regulating the electrical activity of the muscle. [source]

    Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis

    E. 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]