Spatiotemporal calcium signaling in a<Emphasis Type="Italic"> Drosophila melanogaster</Emphasis> cell line stably expressing a<Emphasis Type="Italic"> Drosophila</Emphasis> muscarinic acetylcholine receptor

A muscarinic acetylcholine receptor (mAChR), DM1, expressed in the nervous system of Drosophila melanogaster, has been stably expressed in a Drosophila S2 cell line (S2-DM1) and used to investigate spatiotemporal calcium changes following agonist activation. Carbamylcholine (CCh) and oxotremorine are potent agonists, whereas application of the vertebrate M1 mAChR agonist, McN-A-343, results in a weak response. Activation of S2-DM1 receptors using CCh resulted in an increase in intracellular calcium ([Ca²⁺]_i) that was biphasic. Two distinct calcium sources were found to contribute to calcium signaling: (1) internal stores that are sensitive to both thapsigargin and 2-aminoethoxydiphenyl borate and (2) capacitative calcium entry. Spatiotemporal imaging of individual S2-DM1 cells showed that the CCh-induced [Ca²⁺]_i transient resulted from a homogeneous calcium increase throughout the cell, indicative of calcium release from internal stores. In contrast, ionomycin induced the formation of a "calcium ring" at the cell periphery, consistent with external calcium influx.     

RNAi技术在转基因动物中的应用

RNAi可以作为一种有效的工具用来产生转录后沉默的效果,从而抑制特定基因的表达,已经在线虫、果蝇、小鼠、大鼠等模式生物中得到成功应用。RNAi转基因小鼠的出现,使得在哺乳动物整体水平研究靶基因的敲低成为可能。文章以RNAi转基因小鼠为代表,就转基因载体的设计策略、基因敲除与基因敲低的比较、RNAi转基因动物的优势以及目前存在的缺陷等作一总结,并展望了RNAi转基因动物对功能基因组研究的贡献以及应用前景。

     

Receptor- and calcium-dependent induced inositol 1,4,5-trisphosphate increases in PC12h cells as shown by fluorescence resonance energy transfer imaging

The production and further metabolism of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] require several calcium-dependent enzymes, but little is known about subsequent calcium-dependent changes in cellular Ins(1,4,5)P3. To study the calcium dependence of muscarinic acetylcholine receptor-induced Ins(1,4,5)P3 increases in PC12h cells, we utilized an Ins(1,4,5)P3 imaging system based on fluorescence resonance energy transfer and using green fluorescent protein variants fused with the pleckstrin homology domain of phospholipase C-delta1. The intracellular calcium concentration, monitored by calcium imaging, was adjusted by thapsigargin pretreatment or alterations in extracellular calcium concentration, enabling rapid receptor-independent changes in calcium concentration via store-operated calcium influx. We found that Ins(1,4,5)P3 production was increased by a combination of receptor- and calcium-dependent components, rather than by calcium alone. The level of Ins(1,4,5)P3 induced by the receptor was found to be half that induced by the combined receptor and calcium components. Increases in calcium levels prior to receptor activation did not affect the subsequent receptor-induced Ins(1,4,5)P3 increase, indicating that calcium does not influence Ins(1,4,5)P3 production without receptor activation. Removal of both the receptor agonists and calcium rapidly restored calcium and Ins(1,4,5)P3 levels, whereas removal of calcium alone restored calcium to its basal concentration. Similar calcium-dependent increases in Ins(1,4,5)P3 were also observed in Chinese hamster ovary cells expressing m1 muscarinic acetylcholine receptor, indicating that the observed calcium dependence is common to Ins(1,4,5)P3 production. To our knowledge, our results are the first showing receptor- and calcium-dependent components within cellular Ins(1,4,5)P3.     

Stimulus-Induced Accumulation of Inositol Tetrakis-, Pentakis-, and Hexakisphosphates in N1E-115 Neuroblastoma Cells

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.     

Silencing SERCA1b in a few fibers stimulates growth in the entire regenerating soleus muscle

The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 1 (SERCA1b) is a dominant Ca²⁺ pump in the young fibers of regenerating muscle. In vivo transfection of about 1% of the fibers with SERCA1b RNAi plasmid resulted in no apparent change in the transfected fibers, but enhanced the increase of fresh weight and fiber size in the whole regenerating rat soleus muscle, until the normal size was reached. Co-transfection of calcineurin inhibitor cain/cabin-1 with SERCA1b RNAi was sufficient to cut down the widespread growth stimulation, but the subsequent transfection of cain into the SERCA1b RNAi transfected muscle did not inhibit muscle growth. The SERCA1b RNAi preferably upregulated the expression of the NFAT reporter lacZ compared to controls when co-transfected into the fibers. Notably, perimuscular injection of interleukin-4 (IL-4) antibody but not that of an unrelevant antibody completely abolished the growth-promoting effect of SERCA1b RNAi. This indicates that silencing SERCA1b in a few fibers stimulates the calcineurin-NFAT-IL-4 pathway and fiber growth in the whole regenerating soleus. These results suggest the presence of an autocrine–paracrine coordination of growing muscle fibers, and put forward a new method to stimulate skeletal muscle regeneration.     

Positive feedback regulation of phospholipase C by vasopressin-induced calcium mobilization in WRK1 cells

In WRK1 cells vasopressin stimulates Ins(1,4,5)P3 accumulation and mobilizes intracellular calcium. These two phenomena are transient and exhibit similar time-courses. Experiments performed on intact cells or membrane preparations demonstrate that calcium may also stimulate an accumulation of inositol phosphates. This suggests a possible positive feedback regulation of the primary accumulation of Ins(1,4,5)P3 induced by vasopressin. In order to test such a possibility we studied the vasopressin-induced Ins(1,4,5)P3 accumulation, where intracellular calcium mobilization is artificially suppressed by incubating the cells with EGTA in the presence of ionomycin. Under these conditions the accumulation of Ins(1,4,5)P3 induced by 1 microM vasopressin is inhibited by around 50% when measured 5 s after stimulation. This inhibition is not due to an alteration of the VIa vasopressin receptor binding properties, a reduction of the amount of substrate available for the phospholipase C, a stimulation of the Ins(1,4,5)P3 5-phosphatase or an activation of the Ins(1,4,5,)P3 kinase. It is more likely the consequence of the suppression of calcium wave generated by Ins(1,4,5)P3 which may in its turn stimulate a phospholipase C. Different arguments favour this hypothesis: (1) calcium at an intracellular physiological concentration (0.1-1 microM) is able to stimulate a phospholipase C; (2) artificially increasing the [Ca2+]i inside the WRK1 cell induces an accumulation of Ins(1,4,5)P3; and (3) the time-course of the inhibition of Ins(1,4,5)P3 accumulation induced by an EGTA/ionomycin treatment correlates well with that of the calcium mobilization. Altogether these results suggest that Ins(1,4,5)P3 accumulation in WRK1 cells may result from two distinct mechanisms: a direct vasopressin receptor-mediated PLC activation which is independent of calcium and a calcium-mediated PLC activation related to the intracellular calcium mobilization.     

The size of inositol 1,4,5-trisphosphate-sensitive Ca2+ stores depends on inositol 1,4,5-trisphosphate concentration.

An explanation of the complex effects of hormones on intracellular Ca2+ requires that the intracellular actions of Ins(1,4,5)P3 and the relationships between intracellular Ca2+ stores are fully understood. We have examined the kinetics of 45Ca2+ efflux from pre-loaded intracellular stores after stimulation with Ins(1,4,5)P3 or the stable phosphorothioate analogue, Ins(1,4,5)P3[S]3, by simultaneous addition of one of them with glucose/hexokinase to rapidly deplete the medium of ATP. Under these conditions, a maximal concentration of either Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 evoked rapid efflux of about half of the accumulated 45Ca2+, and thereafter the efflux was the same as occurred under control conditions. Submaximal concentrations of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 caused a smaller rapid initial efflux of 45Ca2+, after which the efflux was similar whatever the concentration of Ins(1,4,5)P3 or Ins(1,4,5)P3[S]3 present. The failure of submaximal concentrations of Ins(1,4,5)P3 and Ins(1,4,5)P3[S]3 to mobilize fully the Ins(1,4,5)P3-sensitive Ca2+ stores despite prolonged incubation was not due either to inactivation of Ins(1,4,5)P3 or to desensitization of the Ins(1,4,5)P3 receptor. The results suggest that the size of the Ins(1,4,5)P3 sensitive Ca2+ stores depends upon the concentration of Ins(1,4,5)P3.     

Inositol trisphosphate analogues induce different oscillatory patterns in Xenopus oocytes.

Agonists that utilize the calcium-mobilizing second messenger inositol(1,4,5)trisphosphate Ins(1,4,5)P3 usually generate oscillations in intracellular calcium. Such oscillations, based on the periodic release of calcium from the endoplasmic reticulum, can also be induced by injecting cells with Ins(1,4,5)P3. The mechanism responsible for oscillatory activity was studied in Xenopus oocytes by injecting them with different inositol trisphosphates. The plasma membrane of Xenopus oocytes has calcium-dependent chloride channels that open in response to calcium, leading to membrane depolarization. Oscillations in calcium were thus monitored by recording membrane potential. The naturally occurring Ins(1,4,5)P3 produced a large initial transient followed by a single transient or a burst of oscillations. By contrast, two analogues (Ins(2,4,5)P3 and Ins(1,4,5)P(S)3) produced a different oscillatory pattern made up of a short burst of sharp transients. Ins(1,3,4,5)P4 had no effect when injected by itself, and it also failed to modify the oscillatory responses to either Ins(2,4,5)P3 or Ins(1,4,5)P(S)3. Both analogues failed to induce a response when injected immediately after the initial Ins(1,4,5)P3-induced response, indicating that they act on the same intracellular pool of calcium. The existence of different oscillatory patterns suggests that there may be different mechanisms for setting up calcium oscillations. The Ins(2,4,5)P3 and Ins(1,4,5)P(S)3 analogues may initiate oscillations through a negative feedback mechanism whereby calcium inhibits its own release. The two-pool model is the most likely mechanism to describe the Ins(1,4,5)P3-induced oscillations.     

Auranofin dissociates chemotactic peptide-induced generation of inositol 1,4,5-trisphosphate from the subsequent mobilization of intracellular calcium in intact human neutrophils

Auranofin, an antiarthritic gold compound, modulates a number of chemotactic factor-induced inflammatory responses in human neutrophils. In order to unravel the mechanism involved, the present study investigated the effects of auranofin on early signal transduction events in these cells. Auranofin did not affect the chemotactic peptide (fMetLeuPhe)-induced formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), neither in the presence nor in the absence of extracellular calcium ions. In contrast, there was a progressive inhibition by auranofin on the fMet-Leu-Phe-induced mobilization of intracellular calcium. This demonstrates that auranofin can dissociate the generation of Ins(1,4,5)P3 from the subsequent release of intracellular calcium, perhaps by interfering with the intracellular binding of Ins(1,4,5)P3 to its receptor. In experiments performed in electro-permeabilized cells, however, a relatively high concentration of the drug failed to abolish the specific binding of Ins(1,4,5)P3. In addition, in the same system, auranofin also failed to abolish the Ins(1,4,5)P3-induced release of Ca2+. Consequently, auranofin-mediated dissociation of fMLP-induced Ins(1,4,5)P3 formation and intracellular calcium release can not be explained merely by an antagonistic effect of auranofin on the Ins(1,4,5)P3 receptor. Instead the interaction between auranofin and the plasma membrane seems to be an initial and important part of the mechanism by which this drug interferes with the transduction signalling system.     

Differential utilization of cyclic ADP-ribose pathway by chemokines to induce the mobilization of intracellular calcium in NK cells.

We show here that cyclic adenosine diphosphate-ribose (cADPR) may be a second messenger for chemokines. Extracts collected from NK cells stimulated with IL-8 for 2 min were incubated with beta-NAD for an additional 2 min (designated as IL-8 extracts). This mixture elevated the mobilization of (Ca(2+))(i) in alpha-toxin permeabilized NK cells. This activity was inhibited upon prior incubation of these cells with ruthenium red but not with heparin. Purified cADPR and not Ins 1,4,5 P(3) desensitized NK cells to the calcium mobilization effect of IL-8 extracts. Further analysis showed that ruthenium red and heparin differentially inhibit RANTES-, SDF-1alpha-, or MDC-induced calcium mobilization in IL-2-activated NK cells. Also, introduction of anti-ryanodine receptor antibody inside streptolysin O-permeabilized NK cells resulted in complete inhibition of MDC, and only partial inhibition of RANTES and SDF-1alpha-induced calcium fluxes in NK cells. Collectively, these results suggest that chemokines may utilize the cADPR/ryanodine receptor pathway as well as the Ins 1,4,5 P(3)/Ins 1,4,5 P(3) receptor signaling pathway to induce the accumulation of calcium in NK cells.     

Multiple effects of SERCA2b mutations associated with Darier's disease

Darier's disease (DD) is an autosomal dominant disorder caused by mutations in the ATP2A2 gene, encoding sarco/endoplasmic reticulum Ca2+-ATPase pump type 2b isoform (SERCA2b). Although >100 mutations in the ATP2A2 gene were identified, no apparent relation between genotype/phenotype emerged. In this work, we analyzed 12 DD-associated mutations from all of the regions of SERCA2b to study the underlying pathologic mechanism of DD and to elucidate the role of dimerization in SERCA2b activity. Most mutations markedly affected protein expression, partially because of enhanced proteasome-mediated degradation. All of the mutants showed lower activity than the wild type pump. Notably, several mutants that cause relatively severe phenotype of DD inhibited the activity of the endogenous and the co-expressed wild type SERCA2b. Importantly, these effects were not attributed to changes in passive Ca2+ leak, inositol 1,4,5-trisphosphate receptor activity, or sensitivity to inositol 1,4,5-trisphosphate. Rather, co-immunoprecipitation experiments showed that SERCA2b monomers interact to influence the activity of each other. These findings reveal multiple molecular mechanisms to account for the plethora of pathologic states observed in DD and provide the first evidence for the importance of SERCA2b dimerization in pump function in vivo.     

Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts.

The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.     

Role of PRIP-1, a novel Ins(1,4,5)P3 binding protein, in Ins(1,4,5)P3-mediated Ca2+ signaling

PRIP-1 was isolated as a novel inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] binding protein with a domain organization similar to phospholipase C-delta1 (PLC-delta1) but lacking the enzymatic activity. Further studies revealed that the pleckstrin homology (PH) domain of PRIP-1 is the region responsible for binding Ins(1,4,5)P3. In this study we aimed to clarify the role of PRIP-1 at the physiological concentration in Ins(1,4,5)P3-mediated Ca2+ signaling, as we had previously used COS-1 cells overexpressing PRIP-1 (Takeuchi et al., 2000, Biochem J 349:357-368). For this purpose we employed PRIP-1 knock out (PRIP-1-/-) mice generated previously (Kanematsu et al., 2002, EMBO J 21:1004-1011). The increase in free Ca2+ concentration in response to purinergic receptor stimulation was lower in primary cultured cortical neurons prepared from PRIP-1-/- mice than in those from wild type mice. The relative amounts of [3H]Ins(1,4,5)P3 measured in neurons labeled with [3H]inositol was also lower in cells from PRIP-1-/- mice. In contrast, PLC activities in brain cortex samples from PRIP-1-/- mice were not different from those in the wild type mice, indicating that the hydrolysis of Ins(1,4,5)P3 is enhanced in cells from PRIP-1-/- mice. In vitro analyses revealed that type1 inositol polyphosphate 5-phosphatase physically interacted with a PH domain of PRIP-1 (PRIP-1PH) and its enzyme activity was inhibited by PRIP-1PH. However, physical interaction with these two proteins did not appear to be the reason for the inhibition of enzyme activity, indicating that binding of Ins(1,4,5)P3 to the PH domain prevented its hydrolyzation. Together, these results indicate that PRIP-1 plays an important role in regulating the Ins(1,4,5)P3-mediated Ca2+ signaling by modulating type1 inositol polyphosphate 5-phosphatase activity through binding to Ins(1,4,5)P3.     

ATP and its metabolite adenosine act synergistically to mobilize intracellular calcium via the formation of inositol 1,4,5-trisphosphate in a smooth muscle cell line.

Interactions between ATP and adenosine on the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and mobilization of intracellular calcium were investigated in the smooth muscle cell line DDT1 MF-2. Activation of adenosine A1 receptors with adenosine or cyclopentyladenosine (CPA) or of nucleotide receptors with ATP increased both Ins(1,4,5)P3 formation and intracellular calcium concentrations. The A1 receptor-induced Ins(1,4,5)P3 formation (EC50 10 nM) was antagonized by the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and by pretreatment of the cells with pertussis toxin (PTX). ATP-stimulated Ins(1,4,5)P3 formation (EC50 21 microM) was attenuated, but still present, after PTX treatment. ATP and CPA had supraadditive effects on Ins(1,4,5)P3 accumulation and CPA increased ATP-induced Ins(1,4,5)P3 accumulation in a concentration-dependent manner with an EC50 of 3 nM, a concentration which per se had little or no effect on Ins(1,4,5)P3 accumulation. ATP (EC50 4 microM) and CPA (EC50 4 nM) both increased intracellular calcium levels. The effect of ATP was partially sensitive to PTX treatment, whereas the effect of CPA was blocked both by PTX and by DPCPX. Concentrations of ATP and CPA that by themselves were insufficient to raise intracellular calcium were able to do so when combined. The synergy between ATP and CPA on the mobilization of intracellular calcium was abolished after treatment of cells with PTX or when DPCPX was included in the experiment. Since ATP was metabolized by ecto-enzymes to ADP, AMP, and adenosine, we also examined whether adenosine formed from ATP could enhance the ATP effects on Ins(1,4,5)P3 accumulation. Indeed, the addition of the A1 receptor antagonist DPCPX or removal of endogenous adenosine by inclusion of adenosine deaminase in the experimental medium significantly attenuated the ATP response, and the two treatments did not have additive effects. The present study thus demonstrates that in a clonal cell line two types of receptors increase phospholipase C activity, but via different pathways; nucleotide receptors appeared to act via partially PTX-insensitive, and A1 receptors via PTX-sensitive G-proteins. ATP and CPA are not only able per se to induce formation of Ins(1,4,5)P3 and mobilize intracellular calcium, but they also act synergistically. Finally, it is demonstrated that endogenous adenosine, possibly formed from the rapid breakdown of ATP, can significantly enhance some ATP effects.     

Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in alpha T3-1 gonadotropes

Despite no evidence for desensitization of phospholipase C-coupled gonadotropin-releasing hormone (GnRH) receptors, we previously reported marked suppression of GnRH-mediated Ca(2+) responses in alphaT3-1 cells by pre-exposure to GnRH. This suppression could not be accounted for solely by reduced inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) responses, thereby implicating uncoupling of Ins(1,4,5)P(3) production and Ca(2+) mobilization (McArdle, C. A., Willars, G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a homologous and heterologous desensitization of Ca(2+) signaling in alphaT3-1 cells that is coincident with a rapid (t((12)) < 20 min), marked, and functionally relevant loss of type I Ins(1,4,5)P(3) receptor immunoreactivity and binding. Furthermore, using an alphaT3-1 cell line expressing recombinant muscarinic M(3) receptors we show that the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of Ins(1,4,5)P(3) receptor immunoreactivity. These data highlight a potential role for rapid Ins(1,4,5)P(3) receptor down-regulation in homologous and heterologous desensitization and in particular suggest that this mechanism may contribute to the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues.     

Cytoplasmic calcium oscillations: a two pool model

Cytosolic calcium oscillations induced by a wide range of agonists, particularly those which stimulate phosphoinositide metabolism, are the result of a periodic release of stored calcium. The formation of inositol 1,4,5 trisphosphate (Ins(1,4,5)P3) seems to play an important role because it can initiate this periodic behaviour when injected or perfused into a variety of cells. A two pool model has been developed to explain how Ins(1,4, 5)P3 sets up these calcium oscillations. It is proposed that Ins(1,4,5)P3 acts through its specific receptor to create a constant influx of primer calcium (Ca2+p) made up of calcium released from the Ins(1,4,5)P3-sensitive pool (ISCS) together with an influx of external calcium. This Ca2+p fails to significantly elevate cytosolic calcium because it is rapidly sequestered by the Ins(1,4,5)P3-insensitive (IICS) stores of calcium distributed throughout the cytosol. Once the latter have filled, they are triggered to release their stored calcium through a process of calcium-induced calcium release to give a typical calcium spike (Ca2+s). In many cells, each Ca2+s begins at a discrete initiation site from which it then spreads through the cell as a wave. The two pool model can account for such waves if it is assumed that calcium released from one IICS diffused across to excite its neighbours thereby setting up a self-propagating wave based on calcium-induced calcium release.     

Genetic mouse models for behavioral analysis through transgenic RNAi technology

Pharmacological inhibitors and knockout mice have developed into routine tools to analyze the role of specific genes in behavior. Both strategies have limitations like the availability of inhibitors for only a subset of proteins and the large efforts required to construct specific mouse mutants. The recent emergence of RNA interference (RNAi)-mediated gene silencing provides a fast alternative that can be applied to any coding gene. We established an approach for the efficient generation of transgenic knockdown mice by targeted insertion of short hairpin (sh) RNA vectors into a defined genomic locus and studied the efficiency of gene silencing in the adult brain and the utility of such mice for behavioral analysis. We generated shRNA knockdown mice for the corticotropin-releasing hormone receptor type 1 (Crhr1), the leucine-rich repeat kinase 2 (Lrkk2) and the purinergic receptor P2X ligand-gated ion channel 7 (P2rx7) genes and show the ubiquitous expression of shRNA and efficient suppression of the target mRNA and protein in the brain of young and 11-month-old knockdown mice. Knockdown mice for the Crhr1 gene exhibited decreased anxiety-related behavior, an impaired stress response, and thereby recapitulate the phenotype of CRHR1 knockout mice. Our results show the feasibility of gene silencing in the adult brain and validate knockdown mice as new genetic models suitable for behavioral analysis.     

Characterization of inositol 1,4,5-trisphosphate-stimulated calcium release from rat cerebellar microsomal fractions. Comparison with [3H]inositol 1,4,5-trisphosphate binding.

The abilities of D-myo-inositol phosphates (InsPs) to promote Ca2+ release and to compete for D-myo-[3H]-inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) binding were examined with microsomal preparations from rat cerebellum. Of the seven InsPs examined, only Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 stimulated the release of Ca2+. Ca2+ release was maximal in 4-6 s and was followed by a rapid re-accumulation of Ca2+ into the Ins(1,4,5)P3-sensitive compartment after Ins(1,4,5)P3, but not after Ins(2,4,5)P3 or Ins(4,5)P2. Ca2+ re-accumulation after Ins(1,4,5)P3 was also faster than after pulse additions of Ca2+, and coincided with the metabolism of [3H]Ins(1,4,5)P3. These data suggest that Ins(1,4,5)P3-induced Ca2+ release and the accompanying decrease in intraluminal Ca2+ stimulate the Ca2+ pump associated with the Ins(1,4,5)P3-sensitive compartment. That this effect was observed only after Ins(1,4,5)P3 may reflect differences in either the metabolic rates of the various InsPs or an effect of the Ins(1,4,5)P3 metabolite Ins(1,3,4,5)P4 to stimulate refilling of the Ins(1,4,5)P3-sensitive store. InsP-induced Ca2+ release was concentration-dependent, with EC50 values (concn. giving half-maximal release) of 60, 800 and 6500 nM for Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 respectively. Ins(1,4,5)P3, Ins(2,4,5)P3 and Ins(4,5)P2 also competed for [3H]Ins(1,4,5)P3 binding, with respective IC50 values (concn. giving 50% inhibition) of 100, 850 and 13,000 nM. Comparison of the EC50 and IC50 values yielded a significant correlation (r = 0.991). These data provide evidence of an association between the [3H]Ins(1,4,5)P3-binding site and the receptor mediating Ins(1,4,5)P3-induced Ca2+ release.     

Inositol trisphosphate, calcium and muscle contraction

The identity of organelles storing intracellular calcium and the role of Ins(1,4,5)P3 in muscle have been explored with, respectively, electron probe X-ray microanalysis (EPMA) and laser photolysis of 'caged' compounds. The participation of G-protein(s) in the release of intracellular Ca2+ was determined in saponin-permeabilized smooth muscle. The sarcoplasmic reticulum (SR) is identified as the major source of activator Ca2+ in both smooth and striated muscle; similar (EPMA) studies suggest that the endoplasmic reticulum is the major Ca2+ storage site in non-muscle cells. In none of the cell types did mitochondria play a significant, physiological role in the regulation of cytoplasmic Ca2+. The latency of guinea pig portal vein smooth muscle contraction following photolytic release of phenylephrine, an alpha 1-agonist, is 1.5 +/- 0.26 s at 20 degrees C and 0.6 +/- 0.18 s at 30 degrees C; the latency of contraction after photolytic release of Ins(1,4,5)P3 from caged Ins(1,4,5)P3 is 0.5 +/- 0.12 s at 20 degrees C. The long latency of alpha 1-adrenergic Ca2+ release and its temperature dependence are consistent with a process mediated by G-protein-coupled activation of phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P2) hydrolysis. GTP gamma S, a non-hydrolysable analogue of GTP, causes Ca2+ release and contraction in permeabilized smooth muscle. Ins(1,4,5)P3 has an additive effect during the late, but not the early, phase of GTP gamma S action, and GTP gamma S can cause Ca2+ release and contraction of permeabilized smooth muscles refractory to Ins(1,4,5)P3. These results suggest that activation of G protein(s) can release Ca2+ by, at least, two G-protein-regulated mechanisms: one mediated by Ins(1,4,5)P3 and the other Ins(1,4,5)P3-independent. The low Ins(1,4,5)P3 5-phosphatase activity and the slow time-course (seconds) of the contractile response to Ins(1,4,5)P3 released with laser flash photolysis from caged Ins(1,4,5)P3 in frog skeletal muscle suggest that Ins(1,4,5)P3 is unlikely to be the physiological messenger of excitation-contraction coupling of striated muscle. In contrast, in smooth muscle the high Ins(1,4,5)P3-5-phosphatase activity and the rate of force development after photolytic release of Ins(1,4,5)P3 are compatible with a physiological role of Ins(1,4,5)P3 as a messenger of pharmacomechanical coupling.     

Tyrosine protein kinase activity of the EGF receptor is required to induce activation of receptor-operated calcium channels

We have investigated the effects of epidermal growth factor (EGF) on calcium ion channels in A431 epidermoid carcinoma cells. We have found that: -1- EGF stimulates Ca2+ channels. -2- EGF stimulated Ca2+ channels are voltage independent, exhibit a low conductance (8 pS) and a bursting multichannels activity (BMC). -3- Activation of the tyrosine-kinase function of the EGF receptor is required to generate Ca2+ current. -4- Inositol (1,4,5) triphosphate (Ins (1,4,5) P3) and EGF have similar effect on the channel activation. These results suggest that: stimulation of tyrosine-kinase activity of the EGF receptor, production of Ins (1,4,5)P3 and calcium entry via voltage independent channels are important connected steps in mediating the mitogenic effect of this growth factor.