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Figure 1 Inhibition of m2 mAChR-mediated [Ca2+]i increase by sphingosine kinase inhibitors. Carbachol (100  M)-induced [Ca2+]i increases were determined in m2 mAChR-expressing HEK-293 cells pretreated for 10 min, without (A) (Control) and with (B) 30 M DHS, (C) 15 M DMS or (D) 30 M N-acetylsphingosine (N-Ac-SP). The addition of carbachol is indicated by arrows.
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 | Figure 2 Concentration-dependent inhibition of m2 and m3 mAChR-induced [Ca2+]i increases by DHS and DMS. [Ca2+]i increases induced by 100 and 0.3 M carbachol were determined in m2 (upper panel) and m3 (lower panel) mAChR-expressing HEK-293 cells, pretreated for 10 min with the indicated concentrations of DMS ( ) or DHS ( ). Mean  SEM of three independent experiments, each carried out at least in duplicate.
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Figure 3 Influence of DHS and DMS on m2 and m3 mAChR-mediated PLC stimulation. Following 10 min treatment without (Control) and with 30 M DHS or 15 M DMS, formation of [3H]IP3 (upper panels) and total [3H]inositol phosphates ([3H]IPs, lower panels) was determined in m2 and m3 mAChR-expressing HEK-293 cells in the absence (open bars) and presence (solid bars) of 100 M (m2 mAChR) or 0.3 M carbachol (m3 mAChR). Mean SEM (n=4) from one experiment, representative of at least two similar experiments.
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 | Figure 4 Influence of receptor stimulation on DHS-induced inhibition of m2 and m3 mAChR-mediated [Ca2+]i increases. Increase in [Ca2+]i by the indicated concentrations of carbachol was determined in m2 (upper panel) and m3 (lower panel) mAChR-expressing HEK-293 cells pretreated for 10 min with ( ) and without ( ) 30 M DHS. MeanSEM of three (m2 mAChR) or eight (m3 mAChR) independent experiments, each carried out at least in duplicate. *Significantly different from cells not treated with DHS (p <0.05, two-tailed paired t-test).
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Figure 5 Stimulation of [3H]SPP formation by m2 and m3 mAChRs. Formation of [3H]SPP from [3H]sphingosine was determined in m2 and m3 mAChR-expressing HEK-293 cells for the indicated periods in the absence and presence of 100 M (m2 mAChR) or 0.3 M (m3 mAChR) carbachol. Values are expressed as percentage increase relative to the radioactivity in unstimulated cells and are meanSEM of three or more independent experiments, each carried out at least in triplicate. Incubation with carbachol did not alter the uptake of [3H]sphingosine. *Significantly different from unstimulated cells (p <0.05, two-tailed paired t-test).
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 | Figure 6 Fura-2 imaging of single HEK-293 cells injected with SPP and IP3. [Ca2+]i was measured in adherent m2 mAChR-expressing HEK-293 cells pretreated with PTX (75 ng/ml, 16 h). In the panels, several individual fluorescence traces representative of between 8 and 40 recordings are shown. Arrows indicate time points of injection. Injection buffer solution contained (A) vehicle (control), (B) 1 mM SPP, (C) 100 M SPP, (D) 10 M SPP, (E) 1 mM SPP after pretreatment for 30–120 s with 5 mM EGTA, or (F) 1 mM IP3.
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