P2Y receptor-mediated Ca2+ signalling in cultured rat aortic smooth muscle cells.
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1. ATP, UTP, ADP and ADP-beta-S elicited Ca2+ -signals in cultured aortic smooth muscle cells although ADP, UDP and ADP-beta-S gave approximately 40% of the maximal response seen with ATP and UTP. Adenosine, AMP or alpha,beta-methylene-ATP had no effect. These responses were attributed to P2Y2/4 and P2Y1 receptors, which we assumed could be selectively activated by UTP and ADP-beta-S respectively. 2. The response to UTP was reduced (approximately 50%) by pertussis toxin, whilst this toxin had no effect upon the response to ADP-beta-S. This suggests P2Y2/4 receptors simultaneously couple to pertussis toxin-sensitive and -resistant G proteins whilst P2Y1 receptors couple to only the toxin-resistant proteins. 3. Repeated stimulation with UTP or ADP-beta-S caused desensitization which was potentiated by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and attenuated by staurosporine. 4. TPA completely abolished sensitivity to ADP-beta-S but the response to UTP had a TPA-resistant component. In pertussis toxin-treated cells, however, TPA could completely abolish sensitivity to UTP and so the TPA-resistant part of this response seems to be mediated by pertussis toxin-sensitive G proteins. 5. Loss of sensitivity to UTP did not occur when pertussis toxin-treated cells were repeatedly stimulated with this nucleotide, suggesting that pertussis toxin-sensitive G proteins mediate this effect. The toxin did not, however affect desensitization to ADP-beta-S. (+info)
Paired immunoglobulin-like receptor B (PIR-B) inhibits BCR-induced activation of Syk and Btk by SHP-1.
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Coligation of paired immunoglobulin-like receptor B (PIR-B) with B cell antigen receptor (BCR) blocks antigen-induced B cell activation. This inhibition is mediated in part by recruitment of SHP-1 and SHP-2 to the phosphorylated ITIMs in the cytoplasmic domain of PIR-B; however the molecular target(s) of these phosphatases remain elusive. Here we show that PIR-B ligation inhibits the BCR-induced tyrosine phosphorylation of Igalpha/Igbeta, Syk, Btk and phospholipase C (PLC)-gamma2. Overexpression of a catalytically inactive form of SHP-1 prevents the PIR-B-mediated inhibition of tyrosine phosphorylation of Syk, Btk, and PLC-gamma2. Dephosphorylation of Syk and Btk mediated by SHP-1 leads to a decrease of their kinase activity, which in turn inhibits tyrosine phosphorylation of PLC-gamma2. Furthermore, we define a requirement for Lyn in mediating tyrosine phosphorylation of PIR-B. Based on these results, we propose a model of PIR-B-mediated inhibitory signaling in which coligation of PIR-B and BCR results in phosphorylation of ITIMs by Lyn, subsequent recruitment of SHP-1, and a resulting inhibition of the BCR-induced inositol 1,4,5-trisphosphate generation by dephosphorylation of Syk and Btk. (+info)
Evidence for the involvement of a Src-related tyrosine kinase in Xenopus egg activation.
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Recently, we have purified a Src-related tyrosine kinase, named Xenopus tyrosine kinase (Xyk), from oocytes of Xenopus laevis and found that the enzyme is activated within 1 min following fertilization [Sato et al. (1996) J. Biol. Chem. 271, 13250-13257]. A concomitant translocation of a part of the activated enzyme from the membrane fraction to the cytosolic fraction was also observed. In the present study, we show that parthenogenetic egg activation by a synthetic RGDS peptide [Y. Iwao and T. Fujimura, T. (1996) Dev. Biol. 177, 558-567], an integrin-interacting peptide, but not by electrical shock or the calcium ionophore A23187 causes the kinase activation, tyrosine phosphorylation, and translocation of Xyk. A synthetic tyrosine kinase-specific inhibitor peptide was employed to analyze the importance of the Xyk activity in egg activation. We found that the peptide inhibits the kinase activity of purified Xyk at IC50 of 8 microM. Further, egg activation induced by sperm or RGDS peptide but not by A23187 was inhibited by microinjection of the peptide. In the peptide-microinjected eggs, penetration of the sperm nucleus into the egg cytoplasm and meiotic resumption in the egg were blocked. Indirect immunofluorescence study demonstrates that Xyk is exclusively localized to the cortex of Xenopus eggs, indicating that Xyk can function in close proximity to the sperm-egg or RGDS peptide-egg interaction site. Taken together, these data suggest that the tyrosine kinase Xyk plays an important role in the early events of Xenopus egg activation in a manner independent or upstream of calcium signaling. (+info)
Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.
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We have used digital fluorescence imaging techniques to explore the interplay between mitochondrial Ca2+ uptake and physiological Ca2+ signaling in rat cortical astrocytes. A rise in cytosolic Ca2+ ([Ca2+]cyt), resulting from mobilization of ER Ca2+ stores was followed by a rise in mitochondrial Ca2+ ([Ca2+]m, monitored using rhod-2). Whereas [Ca2+]cyt recovered within approximately 1 min, the time to recovery for [Ca2+]m was approximately 30 min. Dissipating the mitochondrial membrane potential (Deltapsim, using the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazone [FCCP] with oligomycin) prevented mitochondrial Ca2+ uptake and slowed the rate of decay of [Ca2+]cyt transients, suggesting that mitochondrial Ca2+ uptake plays a significant role in the clearance of physiological [Ca2+]cyt loads in astrocytes. Ca2+ signals in these cells initiated either by receptor-mediated ER Ca2+ release or mechanical stimulation often consisted of propagating waves (measured using fluo-3). In response to either stimulus, the wave traveled at a mean speed of 22.9 +/- 11.2 micrometer/s (n = 262). This was followed by a wave of mitochondrial depolarization (measured using tetramethylrhodamine ethyl ester [TMRE]), consistent with Ca2+ uptake into mitochondria as the Ca2+ wave traveled across the cell. Collapse of Deltapsim to prevent mitochondrial Ca2+ uptake significantly increased the rate of propagation of the Ca2+ waves by 50%. Taken together, these data suggest that cytosolic Ca2+ buffering by mitochondria provides a potent mechanism to regulate the localized spread of astrocytic Ca2+ signals. (+info)
The neoglycoprotein mannose-bovine serum albumin, but not progesterone, activates T-type calcium channels in human spermatozoa.
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The neoglycoproteins alpha-D-mannose-bovine serum albumin (mannose-BSA) and N-acetyl-alpha-D-glucosamine-BSA (glucNAc-BSA) were shown to rapidly increase intracellular free calcium ([Ca2+]i) in human spermatozoa. The increase in [Ca2+]i induced by these neoglycoproteins accounts for the known ability of these compounds to induce the acrosome reaction in human spermatozoa. Our data support the hypothesis that mannose-BSA, but not progesterone, activates T-type Ca2+ channels in human spermatozoa for the following reasons: (i) the capacity of mannose-BSA to increase [Ca2+]i was inhibited by the specific T-type Ca2+ channel blocker mibefradil (IC50 = 10(-6) mol/l) while progesterone was not inhibited by 10(-5) M mibefradil; (ii) the effect of mannose-BSA to elevate [Ca2+]i was inhibited more potently by Ni2+ (IC50 = 0.1 mmol/l) than Cd2+ (IC50 = 0.5 mmol/l), whereas the effect of progesterone to elevate [Ca2+]i was inhibited equally by Ni2+ and Cd2+ (IC50 = 0.25 mmol/l); (iii) the effects of mannose-BSA and progesterone to increase [Ca2+]i were greater than additive. These data support the idea that mannose-BSA and progesterone were activating distinct Ca2+ channels, one of which was a T-type Ca2+ channel activated by mannose-BSA whereas the Ca2+ channel that was activated by progesterone has yet to be defined at the molecular level. (+info)
Male accessory sex gland secretions affect oocyte Ca2+ oscillations during in-vitro fertilization in golden hamsters.
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To evaluate the effect of male accessory sex gland secretions on Ca2+ oscillations of oocytes, epididymal or ejaculated spermatozoa recovered from uteri were used to inseminate oocytes. Ca2+ oscillations were measured by Fura 2 fluorescence imaging (F340/F380). We showed that although Ca2+ oscillations induced by ejaculated spermatozoa had a pattern similar to those induced by epididymal spermatozoa, the amplitude of the first Ca2+ transient in the former group was significantly higher (P < 0.05) and the duration was significantly longer (P < 0.01). Oocytes inseminated with ejaculated spermatozoa recovered from uteri from males had ampullary glands or ventral prostates removed showed significantly lower Ca2+ oscillations compared to the controls (P < 0.05, P < 0.01 respectively). Moreover, the relative area of the first Ca2+ transient in treatment groups was significantly smaller than the control. In addition, a significantly higher percentage of oocytes (52%) inseminated by spermatozoa from males with all accessory sex glands removed showed non-oscillatory Ca2+ transients, compared to the controls (5%, P < 0.05). These results indicate that accessory sex gland secretions can affect Ca2+ oscillations. The differences between Ca2+ oscillations induced by epididymal and uterine spermatozoa from males with all accessory sex glands removed suggest that uterine factors may also influence this process. (+info)
Spatiotemporal dynamics of inositol 1,4,5-trisphosphate that underlies complex Ca2+ mobilization patterns.
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Inositol 1,4,5-trisphosphate (IP3) is a second messenger that elicits complex spatiotemporal patterns of calcium ion (Ca2+) mobilization and has essential roles in the regulation of many cellular functions. In Madin-Darby canine kidney epithelial cells, green fluorescent protein-tagged pleckstrin homology domain translocated from the plasma membrane to the cytoplasm in response to increased concentration of IP3. The detection of translocation enabled monitoring of IP3 concentration changes within single cells and revealed spatiotemporal dynamics in the concentration of IP3 synchronous with Ca2+ oscillations and intracellular and intercellular IP3 waves that accompanied Ca2+ waves. Such changes in IP3 concentration may be fundamental to Ca2+ signaling. (+info)
The insulin receptor tyrosine kinase domain in a chimaeric epidermal growth factor-insulin receptor generates Ca2+ signals through the PLC-gamma1 pathway.
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The receptors for insulin (IR) and epidermal growth factor (EGFR) are members of the tyrosine kinase receptor (TKR) family. Despite homology of their cytosolic TK domains, both receptors induce different cellular responses. Tyrosine phosphorylation of insulin receptor substrate (IRS) molecules is a specific IR post-receptor response. The EGFR specifically activates phospholipase C-gamma1 (PLC-gamma1). Recruitment of substrate molecules with Src homology 2 (SH2) domains or phosphotyrosine binding (PTB) domains to phosphotyrosines in the receptor is one of the factors creating substrate specificity. In addition, it has been shown that the TK domains of the IR and EGFR show preferences to phosphorylate distinct peptides in vitro, suggesting additional mechanisms of substrate recognition. We have examined to what extent the substrate preference of the TK domain contributes to the specificity of the receptor in vivo. For this purpose we determined whether the IR TK domain, in situ, is able to tyrosine-phosphorylate substrates normally used by the EGFR. A chimaeric receptor, consisting of an EGFR in which the juxtamembrane and tyrosine kinase domains were exchanged by their IR counterparts, was expressed in CHO-09 cells lacking endogenous EGFR. This receptor was found to activate PLC-gamma1, indicating that the IR TK domain, in situ, is able to tyrosine phosphorylate substrates normally used by the EGFR. These findings suggest that the IR TK domain, in situ, has a low specificity for selection and phosphorylation of non-cognate substrates. (+info)