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. 2004 Sep;143(1):3-7.
doi: 10.1038/sj.bjp.0705911. Epub 2004 Aug 9.

Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation

Jelena Krjukova  1 , Tomas Holmqvist, Alexander S Danis, Karl E O Akerman, Jyrki P Kukkonen
Affiliations

Phospholipase C activator m-3M3FBS affects Ca2+ homeostasis independently of phospholipase C activation

Jelena Krjukova et al. Br J Pharmacol. 2004 Sep.

Abstract

In this study, we have investigated responses to the phospholipase C (PLC) activator m-3M3FBS in SH-SY5Y human neuroblastoma cells. As measured using fura-2, m-3M3FBS caused a slowly developing - full response was obtained within 4-6 min - Ca(2+) elevation both in the presence and absence of extracellular Ca(2+), indicating Ca(2+) release from intracellular stores, putatively from endoplasmic reticulum and mitochondria. PLC activity was also measured using two methods, the classical ion-exchange separation and the more novel fluorescent real-time method. In the time frame in which m-3M3FBS caused Ca(2+) elevation (up to 7 min), no PLC activation was detected. Instead, more than 20 min were required to see any inositol phosphate generation in response to m-3M3FBS. m-3M3FBS also interfered with store-operated Ca(2+) influx and Ca(2+) extrusion. In conclusion, m-3M3FBS cannot be considered either potent or specific PLC activator.

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Figures

Figure 1
Figure 1
Ca2+ responses to muscarinic receptor stimulation (oxo-M) and m-3M3FBS. (a) Cells were stimulated with oxo-M in 1 mM extracellular Ca2+ (+Ca2+) and in nominally Ca2+-free TBM (no Ca2+ added; −Ca2+). (b) Cells were stimulated with m-3M3FBS in similar conditions. The black traces represent average responses of 30 respectively 28 cells. The grey trace displays the response of one of the individual cells in 1 mM extracellular Ca2+. The spike seen is spontaneous. (c) Cells were treated with CPA for 6 min before exposure to m-3M3FBS. CPA pretreatment was in some cases, as indicated, preceded by rotenone and oligomycin (6 min) or FCCP pretreatment (2 min). (d) Effect of m-3M3FBS on store-operated Ca2+ influx was assessed. The medium was nominally Ca2+-free TBM except for the areas marked with grey boxes, which indicate 1 mM Ca2+. In the beginning of each experiment, 1 μM thapsigargin was added, and was acting throughout the experiment. The cells in the middle trace were pretreated with 2 μM TPA for 10 min (also acting throughout the experiment). In the bottom trace, m-3M3FBS was added as indicated. The vertical calibration bars indicate change in ratio 340/380 nm (arbitrary units). The traces are averages±s.e.m. of 18–53 cells; for the sake of clarity, error bars are only shown for every third (a, b) or 10th (d) point or not at all (c). Experiments were repeated with three coverslips each.
Figure 2
Figure 2
PLC activity as measured using two different techniques. (a-d) The GFP-PH-PLCδ1 probe binds to PIP2 (membrane) and upon hydrolysis of this (i.e. generation of IP3 and diacylglycerol), the probe translocates to the cytosol. (a) Cells before stimulation. (b) Cells were stimulated with 25 μM m-3M3FBS (picture 4 min after the addition of m-3M3FBS). (c) Cells were stimulated with 10 μM oxo-M (picture 10 s after the addition of oxo-M). (d) Time curve of the fluorescence changes in an individual cell. The vertical scale indicates cytosolic fluorescence divided by the membrane fluorescence (arbitrary units), that is, IP3 production. The experiment was repeated with four coverslips. (e) Ion-exchange chromatographic separation of liberated 3H-labelled inositol phosphates. The data were normalised to the response to 10 μM oxo-M at each time point. The data are from one experiment performed in triplicate and was performed twice with similar results. The significances are as follows: ns, not significant (P>0.05); *, P<0.05; ***, P<0.001.There was no significant difference between oxo-M and m-3M3FBS+oxo-M, except for 20 min (*). (f), the effect of the PLC inhibitor U-73122. The response to m-3M3FBS and oxo-M were measured after 2 h and 20 min, respectively. For the sake of clarity, the data are normalised to the basal level (0%) and to the maximum response to either m-3M3FBS or oxo-M in the absence of U-73122 (100%). Significances as in (e).
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References

    1. BAE Y.S., LEE T.G., PARK J.C., HUR J.H., KIM Y., HEO K., KWAK J.Y., SUH P.G., RYU S.H. Identification of a compound that directly stimulates phospholipase C activity. Mol. Pharmacol. 2003;63:1043–1050. - PubMed
    1. FREDHOLM B.B., BATTIG K., HOLMEN J., NEHLIG A., ZYARTAU E.E. Actions of caffeine in the brain with special reference to factors that contribute to its widespread use. Pharmacol. Rev. 1999;51:83–133. - PubMed
    1. KUKKONEN J.P., SHARIATMADARI R., COURTNEY M.J., ÅKERMAN K.E.O. Localization of voltage-sensitive Ca2+ fluxes and neuropeptide Y immunoreactivity to varicosities in SH-SY5Y human neuroblastoma cells differentiated by treatment with the protein kinase inhibitor staurosporine. Eur. J. Neurosci. 1997;9:140–150. - PubMed
    1. LUND P.E., SHARIATMADARI R., UUSTARE A., DETHEUX M., PARMENTIER M., KUKKONEN J.P., ÅKERMAN K.E.O. The orexin OX1 receptor activates a novel Ca2+ influx pathway necessary for coupling to phospholipase C. J. Biol. Chem. 2000;275:30806–30812. - PubMed
    1. OKADA S., SHIMIZU T., YOKOTANI K. Brain phospholipase C and diacylglycerol lipase are involved in corticotropin-releasing hormone-induced sympatho-adrenomedullary outflow in rats. Eur. J. Pharmacol. 2003;475:49–54. - PubMed

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