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1.
Among all voltage-gated calcium channels, the T-type Ca2+ channels encoded by the Cav3.2 genes are highly expressed in the hippocampus, which is associated with contextual, temporal and spatial learning and memory. However, the specific involvement of the Cav3.2 T-type Ca2+ channel in these hippocampus-dependent types of learning and memory remains unclear. To investigate the functional role of this channel in learning and memory, we subjected Cav3.2 homozygous and heterozygous knockout mice and their wild-type littermates to hippocampus-dependent behavioral tasks, including trace fear conditioning, the Morris water-maze and passive avoidance. The Cav3.2 −/− mice performed normally in the Morris water-maze and auditory trace fear conditioning tasks but were impaired in the context-cued trace fear conditioning, step-down and step-through passive avoidance tasks. Furthermore, long-term potentiation (LTP) could be induced for 180 minutes in hippocampal slices of WTs and Cav3.2 +/− mice, whereas LTP persisted for only 120 minutes in Cav3.2 −/− mice. To determine whether the hippocampal formation is responsible for the impaired behavioral phenotypes, we next performed experiments to knock down local function of the Cav3.2 T-type Ca2+ channel in the hippocampus. Wild-type mice infused with mibefradil, a T-type channel blocker, exhibited similar behaviors as homozygous knockouts. Taken together, our results demonstrate that retrieval of context-associated memory is dependent on the Cav3.2 T-type Ca2+ channel. 相似文献
2.
Kai T. Lohbeck Ulf Riebesell Thorsten B. H. Reusch 《Proceedings. Biological sciences / The Royal Society》2014,281(1786)
Coccolithophores are unicellular marine algae that produce biogenic calcite scales and substantially contribute to marine primary production and carbon export to the deep ocean. Ongoing ocean acidification particularly impairs calcifying organisms, mostly resulting in decreased growth and calcification. Recent studies revealed that the immediate physiological response in the coccolithophore Emiliania huxleyi to ocean acidification may be partially compensated by evolutionary adaptation, yet the underlying molecular mechanisms are currently unknown. Here, we report on the expression levels of 10 candidate genes putatively relevant to pH regulation, carbon transport, calcification and photosynthesis in E. huxleyi populations short-term exposed to ocean acidification conditions after acclimation (physiological response) and after 500 generations of high CO2 adaptation (adaptive response). The physiological response revealed downregulation of candidate genes, well reflecting the concomitant decrease of growth and calcification. In the adaptive response, putative pH regulation and carbon transport genes were up-regulated, matching partial restoration of growth and calcification in high CO2-adapted populations. Adaptation to ocean acidification in E. huxleyi likely involved improved cellular pH regulation, presumably indirectly affecting calcification. Adaptive evolution may thus have the potential to partially restore cellular pH regulatory capacity and thereby mitigate adverse effects of ocean acidification. 相似文献
3.
A M Dolga N Terpolilli F Kepura I M Nijholt H-G Knaus B D'Orsi J H M Prehn U L M Eisel T Plant N Plesnila C Culmsee 《Cell death & disease》2011,2(4):e147
Exacerbated activation of glutamate receptor-coupled calcium channels and subsequent increase in intracellular calcium ([Ca2+]i) are established hallmarks of neuronal cell death in acute and chronic neurological diseases. Here we show that pathological [Ca2+]i deregulation occurring after glutamate receptor stimulation is effectively modulated by small conductance calcium-activated potassium (KCa2) channels. We found that neuronal excitotoxicity was associated with a rapid downregulation of KCa2.2 channels within 3 h after the onset of glutamate exposure. Activation of KCa2 channels preserved KCa2 expression and significantly reduced pathological increases in [Ca2+]i providing robust neuroprotection in vitro and in vivo. These data suggest a critical role for KCa2 channels in excitotoxic neuronal cell death and propose their activation as potential therapeutic strategy for the treatment of acute and chronic neurodegenerative disorders. 相似文献
4.
Background
Retinal ganglion cells expressing the photopigment melanopsin are intrinsically photosensitive (ipRGCs). These ganglion cell photoreceptors send axons to several central targets involved in a variety of functions. Within the retina ipRGCs provide excitatory drive to dopaminergic amacrine cells via glutamatergic signals and ipRGCs are coupled to wide-field GABAergic amacrine cells via gap junctions. However, the extent to which ipRGCs are coupled to other retinal neurons in the ganglion cell layer via gap junctions is unclear. Carbenoxolone, a widely employed gap junction inhibitor, greatly reduces the number of retinal neurons exhibiting non-rod, non-cone mediated light-evoked Ca2+ signals suggesting extensive intercellular coupling between ipRGCs and non-ipRGCs in the ganglion cell layer. However, carbenoxolone may directly inhibit light-evoked Ca2+ signals in ipRGCs independent of gap junction blockade.Methodology/Principal Findings
To test the possibility that carbenoxolone directly inhibits light-evoked Ca2+ responses in ipRGCs, the light-evoked rise in intracellular Ca2+ ([Ca2+]i) was examined using fura-2 imaging in isolated rat ipRGCs maintained in short-term culture in the absence and presence of carbenoxolone. Carbenoxolone at 50 and 100 µM concentrations completely abolished the light-evoked rise in [Ca2+]i in isolated ipRGCs. Recovery from carbenoxolone inhibition was variable.Conclusions/Significance
We demonstrate that the light-evoked rise in [Ca2+]i in isolated mammalian ganglion cell photoreceptors is inhibited by carbenoxolone. Since the light-evoked increase in [Ca2+]i in isolated ipRGCs is almost entirely due to Ca2+ entry via L-type voltage-gated calcium channels and carbenoxolone does not inhibit light-evoked action potential firing in ipRGCs in situ, carbenoxolone may block the light-evoked increase in [Ca2+]i in ipRGCs by blocking L-type voltage-gated Ca2+ channels. The ability of carbenoxolone to block evoked Ca2+ responses must be taken into account when interpreting the effects of this pharmacological agent on retinal or other neuronal circuits, particularly if a change in [Ca2+]i is the output being measured. 相似文献5.
Ocean acidification, one of the great global environmental issues at present, is expected to result in serious damage on marine calcareous organisms such as corals and calcifying algae, which potentially release huge amounts of CO2 from the ocean to the atmosphere. The coccolithophore, Emiliania huxleyi (Haptophyceae), which frequently produces blooms, has greatly contributed to the biological CO2 pump. This study was aimed at analyzing effects of how E. huxleyi responds to acidification. Acidification was performed by two methods, namely by just adding HCl under bubbling ordinary air at 8.2–8.4, 7.6–7.8 and 7.1–7.3 (acidification by HCl) and by bubbling with ordinary air or with increased CO2 concentration such as 406, 816 and 1,192 ppm that maintained pH of the medium at 8.0–8.3, 7.6–7.9 and 7.5–7.7 (acidification by CO2 enrichment). As a result, cell growth and cellular calcification of E. huxleyi were strongly damaged by acidification by HCl, but not by acidification by CO2 enrichment. The activities of photosystems such as F v/F m and ?PSII were not affected by any acidification conditions while photosynthetic O2 evolution was slightly stimulated. A 45Ca-radiotracer experiment revealed that Ca2+-uptake was strongly suppressed by acidification with HCl. This suppression recovered after increasing the dissolved inorganic carbon (DIC) concentration and further stimulated by an additional increase in DIC concentration. The production of storage and coccolith polysaccharides was increased by acidification by HCl and also highly stimulated by acidification with CO2 enrichment. The present study clearly showed that the coccolithophore, E. huxleyi, has an ability to respond positively to acidification with CO2 enrichment, but not just acidification. 相似文献
6.
In clinical obstetrics, magnesium sulfate (MgSO4) use is widespread, but effects on brain development are unknown. Many agents that depress neuronal excitability increase developmental neuroapoptosis. In this study, we used dissociated cultures of rodent hippocampus to examine the effects of Mg++ on excitability and survival. Mg++-induced caspase-3-associated cell loss at clinically relevant concentrations. Whole-cell patch-clamp techniques measured Mg++ effects on action potential threshold, action potential peak amplitude, spike number and changes in resting membrane potential. Mg++ depolarized action potential threshold, presumably from surface charge screening effects on voltage-gated sodium channels. Mg++ also decreased the number of action potentials in response to fixed current injection without affecting action potential peak amplitude. Surprisingly, Mg++ also depolarized neuronal resting potential in a concentration-dependent manner with a +5.2 mV shift at 10 mM. Voltage ramps suggested that Mg++ blocked a potassium conductance contributing to the resting potential. In spite of this depolarizing effect of Mg++, the net inhibitory effect of Mg++ nearly completely silenced neuronal network activity measured with multielectrode array recordings. We conclude that although Mg++ has complex effects on cellular excitability, the overall inhibitory influence of Mg++ decreases neuronal survival. Taken together with recent in vivo evidence, our results suggest that caution may be warranted in the use of Mg++ in clinical obstetrics and neonatology. 相似文献
7.
Several members of the CLC family are secondary active anion/proton exchangers, and not passive chloride channels. Among the exchangers, the endosomal ClC-5 protein that is mutated in Dent''s disease shows an extreme outward rectification that precludes a precise determination of its transport stoichiometry from measurements of the reversal potential. We developed a novel imaging method to determine the absolute proton flux in Xenopus oocytes from the extracellular proton gradient. We determined a transport stoichiometry of 2 Cl−/1 H+. Nitrate uncoupled proton transport but mutating the highly conserved serine 168 to proline, as found in the plant NO3−/H+ antiporter atClCa, led to coupled NO3−/H+ exchange. Among several amino acids tested at position 168, S168P was unique in mediating highly coupled NO3−/H+ exchange. We further found that ClC-5 is strongly stimulated by intracellular protons in an allosteric manner with an apparent pK of ∼7.2. A 2:1 stoichiometry appears to be a general property of CLC anion/proton exchangers. Serine 168 has an important function in determining anionic specificity of the exchange mechanism. 相似文献
8.
Group I metabotropic glutamate receptors (group I mGluRs; mGluR1 and mGluR5) exert diverse effects on neuronal and synaptic functions, many of which are regulated by intracellular Ca2+. In this study, we characterized the cellular mechanisms underlying Ca2+ mobilization induced by (RS)-3,5-dihydroxyphenylglycine (DHPG; a specific group I mGluR agonist) in the somata of acutely dissociated rat hippocampal neurons using microfluorometry. We found that DHPG activates mGluR5 to mobilize intracellular Ca2+ from ryanodine-sensitive stores via cyclic adenosine diphosphate ribose (cADPR), while the PLC/IP3 signaling pathway was not involved in Ca2+ mobilization. The application of glutamate, which depolarized the membrane potential by 28.5±4.9 mV (n = 4), led to transient Ca2+ mobilization by mGluR5 and Ca2+ influx through L-type Ca2+ channels. We found no evidence that mGluR5-mediated Ca2+ release and Ca2+ influx through L-type Ca2+ channels interact to generate supralinear Ca2+ transients. Our study provides novel insights into the mechanisms of intracellular Ca2+ mobilization by mGluR5 in the somata of hippocampal neurons. 相似文献
9.
Arellano-Carbajal F Briseño-Roa L Couto A Cheung BH Labouesse M de Bono M 《PLoS genetics》2011,7(3):e1001341
Genome sequence comparisons have highlighted many novel gene families that are conserved across animal phyla but whose biological function is unknown. Here, we functionally characterize a member of one such family, the macoilins. Macoilins are characterized by several highly conserved predicted transmembrane domains towards the N-terminus and by coiled-coil regions C-terminally. They are found throughout Eumetazoa but not in other organisms. Mutants for the single Caenorhabditis elegans macoilin, maco-1, exhibit a constellation of behavioral phenotypes, including defects in aggregation, O2 responses, and swimming. MACO-1 protein is expressed broadly and specifically in the nervous system and localizes to the rough endoplasmic reticulum; it is excluded from dendrites and axons. Apart from subtle synapse defects, nervous system development appears wild-type in maco-1 mutants. However, maco-1 animals are resistant to the cholinesterase inhibitor aldicarb and sensitive to levamisole, suggesting pre-synaptic defects. Using in vivo imaging, we show that macoilin is required to evoke Ca2+ transients, at least in some neurons: in maco-1 mutants the O2-sensing neuron PQR is unable to generate a Ca2+ response to a rise in O2. By genetically disrupting neurotransmission, we show that pre-synaptic input is not necessary for PQR to respond to O2, indicating that the response is mediated by cell-intrinsic sensory transduction and amplification. Disrupting the sodium leak channels NCA-1/NCA-2, or the N-,P/Q,R-type voltage-gated Ca2+ channels, also fails to disrupt Ca2+ responses in the PQR cell body to O2 stimuli. By contrast, mutations in egl-19, which encodes the only Caenorhabditis elegans L-type voltage-gated Ca2+ channel α1 subunit, recapitulate the Ca2+ response defect we see in maco-1 mutants, although we do not see defects in localization of EGL-19. Together, our data suggest that macoilin acts in the ER to regulate assembly or traffic of ion channels or ion channel regulators. 相似文献
10.
E Ben��tez-Rangel L Garc��a M C Namorado J L Reyes A Guerrero-Hern��ndez 《Cell death & disease》2011,2(1):e113
Ion fluxes at the plasma membrane have an important role in early stages of apoptosis. Accordingly, plasma membrane depolarization and gain of Na+ and loss of K+ are initial events in apoptosis. We have studied the effect of staurosporine (STS), a well-established apoptosis inducer, on the membrane potential of HeLa cells to determine the nature of STS-activated ion conductances and their role in the activation of different caspases. We observed that STS can activate tetraethylammonium (TEA+) and 4-aminopyridine-sensitive K+ channels and flufenamic-sensitive cation channels as an early response. The combination of these ion channel inhibitors significantly reduced cytochrome c (cyt c) release and activation of caspase-9, -3 and -8. STS also induced a large reduction in the intracellular [K+] that was not blocked by the ion channel inhibitors. Our data suggest that reduction in the [K+]i is necessary but not sufficient and that ion channel inhibitors block activation of caspase-3 by two different mechanisms: the inhibitors of K+ channels by reducing cyt c release while flufenamic acid by a different, unrelated mechanism that does not involve cation channels at the plasma membrane. Our data also imply that these ion channels activated by STS are not responsible for the reduction in the [K+]i associated with apoptosis. 相似文献
11.
A Steep Dependence of Inward-Rectifying Potassium Channels on
Cytosolic Free Calcium Concentration Increase Evoked by
Hyperpolarization in Guard Cells
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Inactivation of inward-rectifying
K+ channels (IK,in) by a rise in
cytosolic free [Ca2+] ([Ca2+]i)
is a key event leading to solute loss from guard cells and stomatal
closure. However, [Ca2+]i action on
IK,in has never been quantified, nor are its
origins well understood. We used membrane voltage to manipulate
[Ca2+]i (A. Grabov and M.R. Blatt [1998]
Proc Natl Acad Sci USA 95: 4778–4783) while recording
IK,in under a voltage clamp and
[Ca2+]i by Fura-2 fluorescence
ratiophotometry. IK,in inactivation
correlated positively with [Ca2+]i and
indicated a Ki of 329 ± 31
nm with cooperative binding of four Ca2+ ions
per channel. IK,in was promoted by the
Ca2+ channel antagonists Gd3+ and calcicludine,
both of which suppressed the [Ca2+]i rise,
but the [Ca2+]i rise was unaffected by the
K+ channel blocker Cs+. We also found that
ryanodine, an antagonist of intracellular Ca2+ channels
that mediate Ca2+-induced Ca2+ release, blocked
the [Ca2+]i rise, and Mn2+
quenching of Fura-2 fluorescence showed that membrane hyperpolarization
triggered divalent release from intracellular stores. These and
additional results point to a high signal gain in
[Ca2+]i control of
IK,in and to roles for discrete
Ca2+ flux pathways in feedback control of the
K+ channels by membrane voltage.Ca2+ underlies many fundamental regulatory processes
in plants, including adaptive responses to abiotic environmental stress
(Knight et al., 1996; Russell et al., 1996; McAinsh et al., 1997) and
programmed cell death evoked by pathogen attack (Low and Merida, 1996;
Hammondkosack and Jones, 1997). Coordination of changes in
[Ca2+]i and its
integration with downstream response elements are central in coupling
stimulus input to cellular response in these processes.In stomatal guard cells, the best characterized higher-plant cell
model, major downstream targets of
[Ca2+]i and their roles
in stomatal function have been identified. Increasing
[Ca2+]i is known to
inactivate IK,in and to activate
Cl− channels, events that bias plasma membrane
transport for net efflux of osmotically active solute and a loss of
turgor, which drives stomatal closure (Blatt and Grabov, 1997).
Furthermore, changes in
[Ca2+]i are associated
with ABA, CO2, and the growth hormone auxin
(Blatt and Grabov, 1997; McAinsh et al., 1997). These
[Ca2+]i signals have been
observed to oscillate (McAinsh et al., 1995; Webb et al., 1996),
characteristics that may constitute “Ca2+
signatures” to encode specific downstream responses (Berridge, 1996).
Yet, despite the evidence for
[Ca2+]i signaling in
guard cells, surprisingly little detail is known about the link between
[Ca2+]i changes and ion
channel activity at the plasma membrane or about the mechanisms
mediating such [Ca2+]i
changes. To our knowledge, in no instance have the characteristics of
ion channel regulation by Ca2+ been quantified
directly in any higher-plant cell.We recently described the coupling of membrane voltage to
[Ca2+]i, demonstrating
that hyperpolarization, whether under a voltage clamp or in the
presence of low [K+]o,
evoked [Ca2+]i increases
in guard cells, and that the voltage threshold for
[Ca2+]i rise was
profoundly altered by ABA (Grabov and Blatt, 1998). Our observations
indicated a link to Ca2+ influx across the plasma
membrane and raised questions about the efficacy of
[Ca2+]i in inactivating
IK,in and about the contributions of
intracellular Ca2+ release to the
[Ca2+]i signal. We have
used membrane voltage to experimentally manipulate
[Ca2+]i and report that
IK,in is strongly dependent on
[Ca2+]i, consistent with
a cooperative binding of four Ca2+ ions to effect
inactivation. Additional experiments indicate that voltage-evoked
[Ca2+]i increases depend
both on Ca2+ influx and on release of
Ca2+ from intracellular stores. These results
underscore the role of
[Ca2+]i as a high-gain
“switch” in the control of IK,in, and
implicate [Ca2+]i in
feedback control linking membrane voltage to the activity of the
K+ channels. 相似文献
12.
Watarai H Sekine-Kondo E Shigeura T Motomura Y Yasuda T Satoh R Yoshida H Kubo M Kawamoto H Koseki H Taniguchi M 《PLoS biology》2012,10(2):e1001255
There is heterogeneity in invariant natural killer T (iNKT) cells based on the expression of CD4 and the IL-17 receptor B (IL-17RB), a receptor for IL-25 which is a key factor in TH2 immunity. However, the development pathway and precise function of these iNKT cell subtypes remain unknown. IL-17RB+
iNKT cells are present in the thymic CD44+/− NK1.1− population and develop normally even in the absence of IL-15, which is required for maturation and homeostasis of IL-17RB−
iNKT cells producing IFN-γ. These results suggest that iNKT cells contain at least two subtypes, IL-17RB+ and IL-17RB− subsets. The IL-17RB+
iNKT subtypes can be further divided into two subtypes on the basis of CD4 expression both in the thymus and in the periphery. CD4+ IL-17RB+
iNKT cells produce TH2 (IL-13), TH9 (IL-9 and IL-10), and TH17 (IL-17A and IL-22) cytokines in response to IL-25 in an E4BP4-dependent fashion, whereas CD4− IL-17RB+
iNKT cells are a retinoic acid receptor-related orphan receptor (ROR)γt+ subset producing TH17 cytokines upon stimulation with IL-23 in an E4BP4-independent fashion. These IL-17RB+
iNKT cell subtypes are abundantly present in the lung in the steady state and mediate the pathogenesis in virus-induced airway hyperreactivity (AHR). In this study we demonstrated that the IL-17RB+
iNKT cell subsets develop distinct from classical iNKT cell developmental stages in the thymus and play important roles in the pathogenesis of airway diseases. 相似文献
13.
Ann L. Wozniak Stephen Griffin David Rowlands Mark Harris MinKyung Yi Stanley M. Lemon Steven A. Weinman 《PLoS pathogens》2010,6(9)
The hepatitis C virus (HCV) p7 protein is critical for virus production and an attractive antiviral target. p7 is an ion channel when reconstituted in artificial lipid bilayers, but channel function has not been demonstrated in vivo and it is unknown whether p7 channel activity plays a critical role in virus production. To evaluate the contribution of p7 to organelle pH regulation and virus production, we incorporated a fluorescent pH sensor within native, intracellular vesicles in the presence or absence of p7 expression. p7 increased proton (H+) conductance in vesicles and was able to rapidly equilibrate H+ gradients. This conductance was blocked by the viroporin inhibitors amantadine, rimantadine and hexamethylene amiloride. Fluorescence microscopy using pH indicators in live cells showed that both HCV infection and expression of p7 from replicon RNAs reduced the number of highly acidic (pH<5) vesicles and increased lysosomal pH from 4.5 to 6.0. These effects were not present in uninfected cells, sub-genomic replicon cells not expressing p7, or cells electroporated with viral RNA containing a channel-inactive p7 point mutation. The acidification inhibitor, bafilomycin A1, partially restored virus production to cells electroporated with viral RNA containing the channel inactive mutation, yet did not in cells containing p7-deleted RNA. Expression of influenza M2 protein also complemented the p7 mutant, confirming a requirement for H+ channel activity in virus production. Accordingly, exposure to acid pH rendered intracellular HCV particles non-infectious, whereas the infectivity of extracellular virions was acid stable and unaffected by incubation at low pH, further demonstrating a key requirement for p7-induced loss of acidification. We conclude that p7 functions as a H+ permeation pathway, acting to prevent acidification in otherwise acidic intracellular compartments. This loss of acidification is required for productive HCV infection, possibly through protecting nascent virus particles during an as yet uncharacterized maturation process. 相似文献
14.
Background
SCN5A encodes the α-subunit (Nav1.5) of the principle Na+ channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Nav1.5 channel. Pharmacological targeting of mutation-altered Na+ channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults.Methods and Results
Here, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na+ channel blockers flecainide and mexiletine. Our goal was to determine the Na+ channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na+ channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na+ channels revealed significant changes in channel biophysics, all contributing to the proband''s phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically.Significance
The results of our study provide further evidence of the grave vulnerability of newborns to Na+ channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young. 相似文献15.
16.
Background
The differential adaptations of cerebrovasculature and small mesenteric arteries could be one of critical factors in postspaceflight orthostatic intolerance, but the cellular mechanisms remain unknown. We hypothesize that there is a differential regulation of intracellular Ca2+ determined by the alterations in the functions of plasma membrane CaL channels and ryanodine-sensitive Ca2+ releases from sarcoplasmic reticulum (SR) in cerebral and small mesenteric vascular smooth muscle cells (VSMCs) of simulated microgravity rats, respectively.Methodology/Principal Findings
Sprague-Dawley rats were subjected to 28-day hindlimb unweighting to simulate microgravity. In addition, tail-suspended rats were submitted to a recovery period of 3 or 7 days after removal of suspension. The function of CaL channels was evaluated by patch clamp and Western blotting. The function of ryanodine-sensitive Ca2+ releases in response to caffeine were assessed by a laser confocal microscope. Our results indicated that simulated microgravity increased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral VSMCs, whereas, simulated microgravity decreased the functions of CaL channels and ryanodine-sensitive Ca2+ releases in small mesenteric VSMCs. In addition, 3- or 7-day recovery after removal of suspension could restore the functions of CaL channels and ryanodine-sensitive Ca2+ releases to their control levels in cerebral and small mesenteric VSMCs, respectively.Conclusions
The differential regulation of CaL channels and ryanodine-sensitive Ca2+ releases in cerebral and small mesenteric VSMCs may be responsible for the differential regulation of intracellular Ca2+, which leads to the altered autoregulation of cerebral vasculature and the inability to adequately elevate peripheral vascular resistance in postspaceflight orthostatic intolerance. 相似文献17.
Boris Musset Melania Capasso Vladimir V. Cherny Deri Morgan Mandeep Bhamrah Martin J. S. Dyer Thomas E. DeCoursey 《The Journal of biological chemistry》2010,285(8):5117-5121
Voltage-gated proton channels and NADPH oxidase function cooperatively in phagocytes during the respiratory burst, when reactive oxygen species are produced to kill microbial invaders. Agents that activate NADPH oxidase also enhance proton channel gating profoundly, facilitating its roles in charge compensation and pHi regulation. The “enhanced gating mode” appears to reflect protein kinase C (PKC) phosphorylation. Here we examine two candidates for PKC-δ phosphorylation sites in the human voltage-gated proton channel, HV1 (Hvcn1), Thr29 and Ser97, both in the intracellular N terminus. Channel phosphorylation was reduced in single mutants S97A or T29A, and further in the double mutant T29A/S97A, by an in vitro kinase assay with PKC-δ. Enhanced gating was evaluated by expressing wild-type (WT) or mutant HV1 channels in LK35.2 cells, a B cell hybridoma. Stimulation by phorbol myristate acetate enhanced WT channel gating, and this effect was reversed by treatment with the PKC inhibitor GF109203X. The single mutant T29A or double mutant T29A/S97A failed to respond to phorbol myristate acetate or GF109203X. In contrast, the S97A mutant responded like cells transfected with WT HV1. We conclude that under these conditions, direct phosphorylation of the proton channel molecule at Thr29 is primarily responsible for the enhancement of proton channel gating. This phosphorylation is crucial to activation of the proton conductance during the respiratory burst in phagocytes. 相似文献
18.
Objectives
Anthrax infection is associated with devastating cardiovascular sequelae, suggesting unfavorable cardiovascular effects of toxins originated from Bacillus anthracis namely lethal and edema toxins. This study was designed to examine the direct effect of lethal toxins on cardiomyocyte contractile and intracellular Ca2+ properties.Methods
Murine cardiomyocyte contractile function and intracellular Ca2+ handling were evaluated including peak shortening (PS), maximal velocity of shortening/ relengthening (± dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise measured as fura-2 fluorescent intensity (ΔFFI), and intracellular Ca2+ decay rate. Stress signaling and Ca2+ regulatory proteins were assessed using Western blot analysis.Results
In vitro exposure to a lethal toxin (0.05 – 50 nM) elicited a concentration-dependent depression on cardiomyocyte contractile and intracellular Ca2+ properties (PS, ± dL/dt, ΔFFI), along with prolonged duration of contraction and intracellular Ca2+ decay, the effects of which were nullified by the NADPH oxidase inhibitor apocynin. The lethal toxin significantly enhanced superoxide production and cell death, which were reversed by apocynin. In vivo lethal toxin exposure exerted similar time-dependent cardiomyocyte mechanical and intracellular Ca2+ responses. Stress signaling cascades including MEK1/2, p38, ERK and JNK were unaffected by in vitro lethal toxins whereas they were significantly altered by in vivo lethal toxins. Ca2+ regulatory proteins SERCA2a and phospholamban were also differentially regulated by in vitro and in vivo lethal toxins. Autophagy was drastically triggered although ER stress was minimally affected following lethal toxin exposure.Conclusions
Our findings indicate that lethal toxins directly compromised murine cardiomyocyte contractile function and intracellular Ca2+ through a NADPH oxidase-dependent mechanism. 相似文献19.
The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K
I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. 相似文献
20.
Varga Z Juhász T Matta C Fodor J Katona É Bartok A Oláh T Sebe A Csernoch L Panyi G Zákány R 《PloS one》2011,6(11):e27957