Elevation of intracellular calcium ions is essential for the H2O2-induced activation of SAPK/JNK but not for that of p38 and ERK in Chinese hamster V79 cells

The mitogen-activated protein kinases (MAPK), including stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), p38, and extracellular signal-related kinase (ERK), are believed to be important biomolecules in cell proliferation, survival, and apoptosis induced by extracellular stimuli. In Chinese hamster V79 cells exposed to hydrogen peroxide (H2O2), we recently demonstrated that SAPK/JNK was activated by tyrosine kinase and intracellular Ca2+ ([Ca2+]i). In this study, we report that [Ca2+]i release from intracellular stores is important in the activation of SAPK/JNK but not p38 and ERK. H2O2-induced elevation of [Ca2+]i was observed in Ca2+-free medium. Pretreatment with thapsigargin, a Ca2+-ATPase inhibition of endoplasmic reticulum (ER), did not influence H2O2-induced elevation of [Ca2+]i in the absence of external Ca2+. An intracellular Ca2+ chelator (BAPTA-AM) inhibited H2O2-induced phosphorylation of SAPK/JNK, but an extracellular Ca2+ chelator (EDTA) or a Ca2+ entry blocker (NiCl2) did not. Activation of p38 and ERK in V79 cells exposed to H2O2 was observed in the presence of these inhibitors. These results suggest that [Ca2+]i release from intracellular stores such as mitochondria or nuclei but not ER, occurred after H2O2 treatment and Ca2+-dependent tyrosine kinase-induced activation of SAPK/JNK, although [Ca2+]i was unnecessary for the H2O2-induced activation of p38 and ERK.     

Cyclosporin-A-induced effects on the free Ca2+ concentration in LLC-PK1-cells and their mechanisms

Here we have examined the effects of Cyclosporin A (CyA) on the free intracellular Ca2+ concentration ([Ca2+]i) of LLC-PK1/PKE20 cells to evaluate mechanisms of CyA nephrotoxicity using Fura-2 microspectrofluorometry or digital fluorescence video imaging. The CyA-associated changes were compared to the effects of tacrolimus (Tac), a structurally unrelated immunosuppressant with similar cellular pathways which also causes nephrotoxicity. CyA (EC50(: 1 nmol/l, n=16) and Tac (EC50: 1 nmol/l, n=5) caused a concentration-dependent increase of [Ca2+]i which was substantially attenuated by reducing the external Ca2+ concentration (10(-6) mol/l). Similarly Cyclosporin H, a non-immunosuppressive analogue of CyA, stimulated a Ca2+ influx. Nicardipine (10(-6) mol/l) reduced the CyA- and the Tac-induced Ca2+ influx to 52+/-16% (n=10) and 13+/-10% (n=13) of control respectively. Diltiazem and verapamil (10(-6) mol/l) were also effective, but flufenamate (10(-4) mol/l), Gd3+ (10(-5) mol/l) and La3+ (10(-5) mol/l) were not. In the absence of extracellular Ca2+ CyA led to a small but significant [Ca2+]i increase, indicating additional release from internal stores. Depletion of inositol-1,4,5-trisphosphate-(InsP3-) sensitive Ca2+ stores by extracellular ATP (10(4) mol/l) in low-Ca2+ solution completely suppressed the CyA-induced [Ca2+]i rise. CyA had no effect on the cellular InsP3 concentration. Furthermore, inhibition of phospholipase-Cbeta (PLCbeta) by U73122 (2x10(-5) mol/l) did not alter the CyA-stimulated [Ca2+]i rise. A direct effect of CyA on InsP3-sensitive Ca2+ stores, the InsP3 receptor, the Ca2+ content of the stores or involvement of additional stores is assumed. Incubation with CyA for 1, 12 and 24 h enhanced the rise in [Ca2+]i peak induced by ATP, arginine vasopressin (AVP) and angiotensin II. In summary, CyA stimulated a [Ca2+]i increase in LLC-PK1 cells through Ca2+ release from InsP3-sensitive stores and Ca2+ influx via a nicardipine-sensitive pathway. The CyA-mediated [Ca2+]i increase is independent of PLCbeta activity and InsP3 metabolism. CyA caused long-term enhancement of the agonist-induced rise in [Ca2+]i. The effects of CyA on Ca2+ signaling appear to be independent of its immunosuppressive action.     

Ca2+ responses in cytomegalovirus-infected fibroblasts of human origin

Cytomegalovirus (CMV) infection of fibroblasts of human origin is associated with a cascade of cellular responses (rounding, "contraction," "relaxation," and enlargement). Since in other systems these cellular responses are regulated by intracellular free Ca2+ activity ([Ca2+]i), we measured intracellular Ca2+ responses to CMV infection. At relatively high multiplicities of infection (m.o.i), an influx of Ca2+ was observed within the first hour after CMV infection (p.i.) at which time it was at its maximum rate. Both the time of occurrence and the magnitude of this Ca2+ influx were dependent on the calculated input m.o.i. In CMV-infected cells, [Ca2+]i rose gradually from 80 nM at 0 hr to 174 nM at 48 hr p.i. (about 2.7 times the [Ca2+]i found in mock-infected cells at this time). At 8 and 12 hr p.i. CMV-infected cells consistently contained a somewhat greater level of 45Ca2+ than mock-infected cells, despite the fact that there was only a small increase in [Ca2+]i between CMV and mock-infected cells in the same period. This observation suggests that there may be significant amounts of Ca2+ taken up into intracellular stores. This Ca2+ in intracellular stores may, at later times after infection, contribute to the increase in [Ca2+]i observed from 12 to 48 hr p.i. Ca2+ influx blockers, such as nifedipine and verapamil, inhibited the rise in [Ca2+]i. The increase in [Ca2+]i in response to CMV infection was shown to be dependent on the m.o.i., require infectious virus, and occur under conditions consistent with the expression of immediate-early CMV genes. The capability of inducing such Ca2+ responses was conserved among three laboratory strains of CMV. The CMV-induced Ca2+ responses may be related not only to the development of CMV cytopathology, but also to the replication of CMV, since in other studies cyclic nucleotide modulators and Ca2+ influx blockers were found to inhibit the replication of CMV.     

The cytosolic free calcium in anti-mu-stimulated human B cells is derived partly from extracellular medium and partly from intracellular stores

The inositol phospholipid metabolism and the increase in cytosolic free Ca2+ concentration ([Ca2+]i) into the cell are recognized as two important events in the anti-mu-induced B cell activation. The anti-mu stimulation caused the [3H]inositol incorporation and also a rapid increase in [Ca2+]i from 85 nM to 285 nM. This signal returned to baseline a few minutes after stimulation. By using the fluorescent indicator quin-2 we demonstrated that this [Ca2+]i uptake was derived part from extracellular medium and part from intracellular stores. Both EGTA (a calcium chelator) and TMB.8 (a drug which interferes with Ca2+ sequestration by smooth endoplasmic reticulum) partially suppressed the intracellular Ca2+ uptake and were fully inhibitory when added together. The role of Ca2+ from intracellular stores may also be evidenced in calcium-free experiments, or in permeabilized experiments using exogenous inositol 1,4,5-trisphosphate (IP3, the putative mobilizer of intracellular Ca2+). Preventing the increase in [Ca2+]i also prevents the apparition of early activation makers. These results are consistent with the hypothesis that the Ca2+ increase in B cells stimulated by anti-mu is caused by the generation of IP3 during the phosphatidyl-inositol metabolism and also by the entry of extracellular Ca2+ through the plasma membrane.     

Dissociation of unidirectional influx of external Ca2+ and release from internal stores in activated human T lymphocytes

Addition of lectin or antibody to the T cell receptor complex of human T cells results in a rapid increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This response is biphasic and results from contributions of Ca2+ from internal stores, uptake of Ca2+ across the plasma membrane and possibly a decrease in Ca2+ efflux. These responses have been linked through the activity of inositol 1,4,5-trisphosphate in releasing Ca2+ from internal stores and potentially mediating Ca2+ uptake across the plasma membrane. Following addition of phytohemagglutinin or anti-CD3 antibody to resting T cells or Jurkat cells, we have been able to dissociate the [Ca2+]i responses by loading cells with the Ca2+ chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA). In BAPTA-loaded T cells, we have shown that Ca2+ mobilized from intracellular stores following activation is effectively buffered, while stimulated Ca2+ uptake and associated changes in [Ca2+]i were relatively unaffected. In this report, we show that the sustained increase in [Ca2+]i is due to increased unidirectional influx of external Ca2+ without changes in efflux and that it is the entry of extracellular Ca2+ which is sensitive to the transmembrane potential.     

Ca(2+) sensing receptor activation by CaCl(2) increases [Ca2+]i resulting in enhanced spatial interactions with calbindin-D28k protein

The extracellular calcium ion concentration, [Ca2+]o, sensing receptor CaR is a G-protein coupled membrane receptor and it is involved in regulating cell proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a high affinity calcium-binding protein that plays important roles in modulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to [Ca2+]o and spatial interactions of CaR with calbindin-D28k in a distal tubule-like renal cell line are described. Fura-2 loaded Madin Darby bovine kidney (MDBK) cells were exposed to increasing concentrations of CaCl2 and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 caused continual increase in [Ca2+]i. The thapsigargin induced increase in [Ca2+]i observed in basal medium was eliminated by pretreatment of MDBK cells with 10 mM CaCl2 thereby suggesting the involvement of endoplasmic reticulum Ca2+ stores in increasing the [Ca2+]i. CaR was localized in the plasma membrane by using confocal microscopy. The confocal microscopy data also showed CaR and calbindin-D28k were co-localized when cells were exposed to 40 mM CaCl2. We postulate that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.     

Glutamate regulates IP3-type and CICR stores in the avian cochlear nucleus

Neurons of the avian cochlear nucleus, nucleus magnocellularis (NM), are activated by glutamate released from auditory nerve terminals. If this stimulation is removed, the intracellular calcium ion concentration ([Ca2+]i) of NM neurons rises and rapid atrophic changes ensue. We have been investigating mechanisms that regulate [Ca2+]i in these neurons based on the hypothesis that loss of Ca2+ homeostasis causes the cascade of cellular changes that results in neuronal atrophy and death. In the present study, video-enhanced fluorometry was used to monitor changes in [Ca2+]i stimulated by agents that mobilize Ca2+ from intracellular stores and to study the modulation of these responses by glutamate. Homobromoibotenic acid (HBI) was used to stimulate inositol trisphosphate (IP3)-sensitive stores, and caffeine was used to mobilize Ca2+ from Ca2+-induced Ca2+ release (CICR) stores. We provide data indicating that Ca2+ responses attributable to IP3- and CICR-sensitive stores are inhibited by glutamate, acting via a metabotropic glutamate receptor (mGluR). We also show that activation of C-kinase by a phorbol ester will reduce HBI-stimulated calcium responses. Although the protein kinase A accumulator, Sp-cAMPs, did not have an effect on HBI-induced responses. CICR-stimulated responses were not consistently attenuated by either the phorbol ester or the Sp-cAMPs. We have previously shown that glutamate attenuates voltage-dependent changes in [Ca2+]i. Coupled with the present findings, this suggests that in these neurons mGluRs serve to limit fluctuations in intracellular Ca2+ rather than increase [Ca2+]i. This system may play a role in protecting highly active neurons from calcium toxicity resulting in apoptosis.     

Metabotropic receptor-mediated Ca2+ signaling elevates mitochondrial Ca2+ and stimulates oxidative metabolism in hippocampal slice cultures

Metabotropic receptors modulate numerous cellular processes by intracellular Ca2+ signaling, but less is known about their role in regulating mitochondrial metabolic function within the CNS. In this study, we demonstrate in area CA3 of rat organotypic hippocampal slice cultures that glutamatergic, serotonergic, and muscarinic metabotropic receptor ligands, namely trans-azetidine-2,4-dicarboxylic acid, alpha-methyl-5-hydroxytryptamine, and carbachol, transiently increase mitochondrial Ca2+ concentration ([Ca2+]m) as recorded by changes in Rhod-2 fluorescence, stimulate mitochondrial oxidative metabolism as revealed by elevations in NAD(P)H fluorescence, and induce K+ outward currents as monitored by rapid increases in extracellular K+ concentration ([K+]o). Carbachol (1-1,000 microM) elevated NAD(P)H fluorescence by 相似文献   

Cyclic ADP-ribose induces a larger than normal calcium release in malignant hyperthermia-susceptible skeletal muscle fibers

Malignant hyperthermia (MH) is associated with abnormal regulation of intracellular calcium in skeletal muscle fibers. Cyclic adenosine diphosphate-ribose (cADPR) is an endogenous metabolite of beta-NAD+ that induces Ca2+ release from intracellular stores in many tissues. Microinjection of cADPR (0.5 or 1 microM) increased the intracellular resting Ca2+ concentration ([Ca2+]i) in intact swine skeletal muscle in a dose-dependent manner. However, the increase in [Ca2+]i was greater in malignant-hyperthermia-susceptible (MHS) fibers than in non-susceptible (MHN) fibers. Incubation of muscle fibers in low external Ca2+ solution or in the presence of L-type Ca2+ channel entry blockers, or intracellular microinjection of heparin or ruthenium red did not modify the effect of cADPR on [Ca2+]i. Dantrolene (50 microM), a known inhibitor of intracellular Ca2+ release, decreased resting [Ca2+]i and prevented the cADPR-induced increase in [Ca2+]i. These results provide evidence: (1) for the existence of Ca2+ release mechanisms occurring via non-ryanodine or inositol 1,4,5-trisphosphate (InsP3) receptor mechanisms; (2) that MHS skeletal muscles exhibit a higher responsiveness to cADP-ribose-induced release of Ca2+ and (3) that the ability of dantrolene to block cADP-ribose-induced release of Ca2+ could be related to its pharmacologic effect on resting [Ca2+]i.     

Acetylcholine increases intracellular Ca2+ in taste cells via activation of muscarinic receptors

Previous studies suggest that acetylcholine (ACh) is a transmitter released from taste cells as well as a transmitter in cholinergic efferent neurons innervating taste buds. However, the physiological effects on taste cells have not been established. I examined effects of ACh on taste-receptor cells by monitoring [Ca2+]i. ACh increased [Ca2+]i in both rat and mudpuppy taste cells. Atropine blocked the ACh response, but D-tubocurarine did not. U73122, a phospholipase C inhibitor, and thapsigargin, a Ca2+-ATPase inhibitor that depletes intracellular Ca2+ stores, blocked the ACh response. These results suggest that ACh binds to M1/M3/M5-like subtypes of muscarinic ACh receptors, causing an increase in inositol 1,4,5-trisphosphate and subsequent release of Ca2+ from the intracellular stores. A long incubation with ACh induced a transient response followed by a sustained phase of [Ca2+]i increase. In Ca2+-free solution, the sustained phases disappeared, suggesting that Ca2+ influx is involved in the sustained phase. Depletion of Ca2+ stores by thapsigargin alone induced Ca2+ influx. These findings suggest that Ca2+ store-operated channels may be present in taste cells and that they may participate in the sustained phase of [Ca2+]i increase. Immunocytochemical experiments indicated that the M1 subtype of muscarinic receptors is present in both rat and mudpuppy taste cells.     

Mobilization of calcium by the brief application of oxytocin and prostaglandin E2 in single cultured human myometrial cells.

Intracellular calcium ([Ca2+]i) mobilization was studied in single cultured human myometrial cells in response to the agonists oxytocin and prostaglandin E2 (PGE2) using the fluorescent dye Fura-2. Oxytocin and PGE2 applications were associated with an increase in [Ca2+]i, although there was a marked intercell variation in the amplitude of the agonist-induced response. Removal of extracellular calcium ([Ca2+]o) reduced the oxytocin-induced rise and abolished the PGE2-induced rise in [Ca2+]i, thereby demonstrating that oxytocin but not PGE2 can mobilize intracellular stores of calcium. In nominally calcium-free medium, [Ca2+]i was not increased by PGE2 but subsequent application of oxytocin increased [Ca2+]i, thereby demonstrating that, within a single cell, calcium stores were mobilized by oxytocin and not PGE2. The intracellular calcium stores were completely depleted by a single application of oxytocin and not replenished in the absence of [Ca2+]o. Perfusion with calcium-containing medium for 100 s enabled store refilling. Cell depolarization by 140 mM-K+ caused a transient increase followed by a sustained elevation of [Ca2+]i on which were superimposed small fluctuations. Oxytocin caused an influx of calcium in cells depolarized by K+. This was more marked than that obtained with PGE2.     

Uptake of extracellular Ca2+ and not recruitment from internal stores is essential for T lymphocyte proliferation

Changes in free cytosolic calcium concentrations ([Ca2+]i) are thought to be important initiating events in the activation of T lymphocytes. Mitogen-induced increases in [Ca2+]i may result from net influx across the plasma membrane and/or release of Ca2+ from intracellular stores. In human T lymphocytes loaded with the fluorescent indicator indo-1, addition of phytohemagglutinin (PHA) or the anti-CD3 antibody UCHT-1 elicits a biphasic [Ca2+]i response. A major component of the initial transient peak was due to release from internal stores whereas the lower plateau phase was sustained by Ca2+ influx. Previous work suggested that Ca2+ influx is essential for interleukin 2 (IL 2) secretion and cell proliferation. To determine the relative effects of the initial and sustained phases of [Ca2+]i change, IL 2 secretion and cell proliferation, we introduced into the cell 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a high affinity intracellular Ca2+ chelator which neither contributes to nor interferes with the fluorescence determinations of [Ca2+]i. In cells preloaded with BAPTA, both PHA and UCHT-1 antibody failed to elicit the transient [Ca2+]i overshoot. Only the plateau phase could be observed in the presence of extracellular Ca2+. In contrast, BAPTA-loaded cells were found to be fully functional when assessed for IL 2 receptor expression, IL 2 secretion and cell proliferation. Thus, the mitogen-induced, maximal but transient increase in [Ca2+]i, contributed to mainly by release of Ca2+ from internal stores, does not appear to be essential for these T cell responses.     

Ca(2+)-independent and Ca(2+)-dependent stimulation of quantal neurosecretion in avian ciliary ganglion neurons.

1. Although it is generally agreed that Ca2+ couples depolarization to the release of neurotransmitters, hypertonic saline and ethanol (ETOH) evoke neurosecretion independent of extracellular Ca2+. One possible explanation is that these agents release Ca2+ from an intracellular store that then stimulates Ca(2+)-dependent neurosecretion. An alternative explanation is that these agents act independently of Ca2+. 2. This work extends previous observations on the action of ETOH and hypertonic solutions (HOSM) on neurons to include effects on [Ca2+]i. We have looked for Ca(2+)-independent or -dependent neurosecretion evoked by these agents in parasympathetic postganglionic neurons dissociated from chick ciliary ganglia and maintained in tissue culture. The change in concentration of free Ca2+ in the micromolar range inside neurons ([Ca2+]i) was measured with indo-1 with the use of a Meridian ACAS 470 laser scanning microspectrophotometer. 3. Elevated concentration of extracellular KCl increased [Ca2+]i and the frequency of quantal events. Also, a twofold increase in osmotic pressure (HOSM) produced a similar increase in quantal release and a significant rise in [Ca2+]i; however, the Ca2+ appeared to come from intracellular stores. 4. In contrast, ETOH stimulated quantal neurosecretion without a measurable change in [Ca2+]i. It appears the alcohol exerts its influence on some stage in the process of exocytosis that is distal to or independent of the site of Ca2+ action. 5. The effects of high [KCl]o and osmotic pressure were occlusive. This is explained in part by the observation that hypertonicity reduced Ca2+ current, but an action on Ca2+ stores is also likely.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation.

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation. The effects of combined O2/glucose deprivation were investigated on large aspiny (LA) interneurons recorded from a striatal slice preparation by means of simultaneous electrophysiological and optical recordings. LA interneurons were visually identified and impaled with sharp microelectrodes loaded with the calcium (Ca2+)-sensitive dye bis-fura-2. These cells showed the morphological, electrophysiological, and pharmacological features of large striatal cholinergic interneurons. O2/glucose deprivation induced a membrane hyperpolarization coupled to a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, this [Ca2+]i elevation was more pronounced in dendritic branches rather than in the somatic region. The O2/glucose-deprivation-induced membrane hyperpolarization reversed its polarity at the potassium (K+) equilibrium potential. Both membrane hyperpolarization and [Ca2+]i rise were unaffected by TTX or by a combination of ionotropic glutamate receptors antagonists, D-2-amino-5-phosphonovaleric acid and 6cyano-7-nitroquinoxaline-2, 3-dione. Sulfonylurea glibenclamide, a blocker of ATP-sensitive K+ channels, markedly reduced the O2/glucose-deprivation-induced membrane hyperpolarization but failed to prevent the rise in [Ca2+]i. Likewise, charybdotoxin, a large K+-channel (BK) inhibitor, abolished the membrane hyperpolarization but did not produce detectable changes of [Ca2+]i elevation. A combination of high-voltage-activated Ca2+ channel blockers significantly reduced both the membrane hyperpolarization and the rise in [Ca2+]i. In a set of experiments performed without dye in the recording electrode, either intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid or external barium abolished the membrane hyperpolarization induced by O2/glucose deprivation. The hyperpolarizing effect on membrane potential was mimicked by oxotremorine, an M2-like muscarinic receptor agonist, and by baclofen, a GABAB receptor agonist. However, this membrane hyperpolarization was not coupled to an increase but rather to a decrease of the basal [Ca2+]i. Furthermore glibenclamide did not reduce the oxotremorine- and baclofen-induced membrane hyperpolarization. In conclusion, the present results suggest that in striatal LA cells, O2/glucose deprivation activates a membrane hyperpolarization that does not involve ligand-gated K+ conductances but is sensitive to barium, glibenclamide, and charybdotoxin. The increase in [Ca2+]i is partially due to influx through voltage-gated high-voltage-activated Ca2+ channels.     

Taste receptor cell responses to the bitter stimulus denatonium involve Ca2+ influx via store-operated channels

Previous studies in rat and mouse have shown that brief exposure to the bitter stimulus denatonium induces an increase in [Ca2+]i due to Ca2+ release from intracellular Ca2+ stores, rather than Ca2+ influx. We report here that prolonged exposure to denatonium induces sustained increases in [Ca2+]i that are dependent on Ca2+ influx. Similar results were obtained from taste cells of the mudpuppy, Necturus maculosus, as well as green fluorescent protein (GFP) tagged gustducin-expressing taste cells of transgenic mice. In a subset of mudpuppy taste cells, prolonged exposure to denatonium induced oscillatory Ca2+ responses. Depletion of Ca2+ stores by thapsigargin also induced Ca2+ influx, suggesting that Ca2+ store-operated channels (SOCs) are present in both mudpuppy taste cells and gustducin-expressing taste cells of mouse. Further, treatment with thapsigargin prevented subsequent responses to denatonium, suggesting that the SOCs were the source of the Ca2+ influx. These data suggest that SOCs may contribute to bitter taste transduction and to regulation of Ca2+ homeostasis in taste cells.     

Ca2+与NO在过氧化氢所致正常成年大鼠肠系膜微血管通透性增高中的作用

 目的 观察内皮细胞Ca2+浓度及NO生成在过氧化氢所致正常成年大鼠肠系膜微血管通透性增高中的作用。方法 通过测定在体大鼠肠系膜微血管静水传导性观察微血管通透性变化。采用钙荧光指示剂(Fura 2-AM)、NO荧光指示剂(DAF-2 DA)标记在体微血管内皮细胞,并应用荧光显微镜检测细胞内钙或NO的荧光信号,观察H2O2作用下内皮细胞内钙离子浓度([Ca2+]i)、NO的变化。结果 H2O2可增加正常成年大鼠微血管通透性（正常对照的6.13±0.87倍,P<0.01）,同时增加微血管内皮细胞[Ca2+]i（714.58±144.70 nmol/L,P<0.01）,并促进内皮细胞NO的生成（正常对照荧光强度的1034.3%±44.3%,P<0.01）。Ca2+通道阻滞剂氯化镧可抑制H2O2所引起的微血管通透性增加（P<0.01）及内皮细胞[Ca2+]i升高（P<0.01）。NOS抑制剂AP-Cav-1可抑制H2O2所引起的微血管通透性增加（P<0.01）,但对H2O2的Ca2+增加作用无影响。结论 H2O2所致的通透性增加与细胞内Ca2+增加、NO的产生增多有关。     

Synergistic effects of ATP on oxytocin-induced intracellular Ca2+ response in mouse mammary myoepithelial cells

An increase in intracellular Ca2+ ([Ca2+]i) is essential for mammary myoepithelial cells to contract, leading to milk ejection during lactation. In this study, the intracellular signaling leading to the increase in [Ca2+]i in cultured myoepithelial cells from mouse lactating mammary glands was investigated using fura-2 fluorescence ratiometry. [Ca2+]i increased in cultured myoepithelial cells in response to either oxytocin (1 nM) or ATP (10 microM), and the cells then contracted. These [Ca2+]i responses were diminished by treatment with an inhibitor of phospholipase C (> or = 1 microM U73122). Intracellular application of inositol 1,4,5-trisphosphate (IP3: 10 or 100 microM) increased [Ca2+]i. Pretreatment with pertussis toxin (PTX: 0.1 or 1 microgram/ml) inhibited the [Ca2+]i response to ATP, but had less of an effect on the response to oxytocin. These results indicate that oxytocin and purinergic receptors are coupled to PTX-insensitive and PTX-sensitive G proteins, respectively, and that their activation leads to the increase in [Ca2+]i through the release of Ca2+ from IP3-sensitive intracellular stores via the inositol-phospholipid signaling pathway. Furthermore, we found that the [Ca2+]i responses to oxytocin at physiological doses (0.01-0.1 nM) were augmented in the presence of a sub-responsive dose of ATP (1 microM). The activation of purinergic receptors may facilitate myoepithelial cell contraction in milk-ejection responses.     

Evidence for P2Y-type ATP receptors on the serosal membrane of frog skin epithelium

The present study presents the first evidence for P2Y-type adenosine 5'-triphosphate (ATP) receptors on the basolateral membranes of frog skin epithelial cells. Cytosolic calcium ([Ca2+]i) was measured with fura-2 and Calcium-Green-1 using epifluorescence microscopy and confocal laser scanning microscopy respectively. In the presence of Ca2+ in the solutions ATP increased [Ca2+]i. The increase in [Ca2+]i was due to the agonist activity of ATP and not to the activity of the potential products of ATP metabolism, i.e. adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP) or adenosine, as shown by a comparison of the magnitude of the increases in [Ca2+]i caused by the various compounds. The rise in [Ca2+]i was predominantly monophasic at low ATP concentrations (below 100 microM). At higher concentrations the initial spike was followed by a plateau phase. In the absence of Ca2+ in the extracellular solution ATP caused Ca2+ release from intracellular stores. This could be inhibited by pre-treatment of the tissue with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum calcium ATPase. The nucleotide uridine 5'-triphosphate (UTP) had similar effects on [Ca2+]i although the plateau level of the [Ca2+]i response was higher with this P2Y agonist. Confocal laser scanning microscopy showed that all cell layers of the epithelium responded to ATP. Our data indicates that serosal ATP acts on serosal P2Y-type receptors in frog skin epithelium. This is the first evidence of a phospholipase C-coupled receptor in this tissue.     

Effect of alpha1-adrenergic stimulation of Cl- secretion and signal transduction in exocrine glands (Rana esculenta)

In the present work, the effect of stimulation of alpha-adrenergic receptors on Cl- secretion via exocrine frog skin glands was investigated. The alpha-adrenergic stimulation was performed by addition of the adrenergic agonist noradrenaline in the presence of the beta-adrenergic antagonist propranolol. In the presence of propranolol, noradrenaline had no effect on the cellular cAMP content. The Cl- secretion was measured as the amiloride-insensitive short circuit current (ISC). Addition of noradrenaline induced a biphasic increase in the ISC. The increase in ISC coincided with an increase in the net 36Cl- secretion. The noradrenaline-induced increase in ISC was dose-dependent with an EC50 of 13 +/- 0.3 microM. Epifluorescence microscopic measurements of isolated, fura-2-loaded frog skin gland acini were used to characterize the intracellular calcium ([Ca2+]i) response. Application of noradrenaline induced a biphasic [Ca2+]i response, which was dose-dependent with an EC50 of 11 +/- 6 microM. The Ca2+ plateau unlike the peak-response was sensitive to removal of Ca2+ from the extracellular medium. The noradrenaline-induced increase in the Cl- secretion as well as in [Ca2+]i was sensitive to the alpha1-adrenergic antagonist prazosine. Ryanodine and caffeine had no effect on [Ca2+]i indicating that the release was independent of ryanodine-sensitive Ca2+ stores. Noradrenaline mediated a significant increase in the cellular inositol 1,4,5-trisphosphate (IP3) content suggesting that the signal transduction pathway leading to the noradrenaline-induced increase in Ca2+ involved IP3 and a release of Ca2+ from IP3-sensitive stores.     

Sphingosine-1-phosphate-induced intracellular Ca2+ mobilization in human endothelial cells.

The authors have studied the effect of sphingosine-1-phosphate (S1P) on Ca2+ release from intracellular stores in cultured human umbilical vein endothelial cells (HUVECs). In the presence of extracellular Ca2+, S1P increased intracellular Ca2+ concentration ([Ca2+]i) and this increase was partially inhibited by La3+ (1 microM), indicating that S1P induces Ca2+ influx from extracellular pool and Ca2+ release from intracellular stores. S1P increased [Ca2+]i concentration dependently in Ca2+-free extracellular solution. The Hill coefficient (1.7) and EC50 (420 nM) was obtained from the concentration-response relationship. When caffeine depleted Ca2+ store in the presence of ryanodine, S1P did not induce intracellular Ca2+ release. Furthermore, the Ca2+-induced Ca2+ release inhibitors ruthenium red or dantrolene completely inhibited S1P-induced intracellular Ca2+ release. S1P-induced intracellular Ca2+ release was inhibited by the phospholipase C (PLC) inhibitors neomycin and U73312, or the inositol 1,4,5-triphosphate (IP3)-gated Ca2+ channel blocker aminoethoxybiphenyl borane (2-APB). In contrast, S1P-induced intracellular Ca2+ release was not inhibited by the mitochondrial Ca2+ uptake inhibitor CCCP or the mitochondrial Ca2+ release inhibitor cyclosporin A. These results show that S1P mobilizes Ca2+ from intracellular stores primarily via Ca2+-induced and IP3-induced Ca2+ release and this Ca2+ mobilization is independent of mitochondrial Ca2+ stores.