American Society of Anesthesiologists

Fig. 5.

$Cicaprost induces a protein kinase A–dependent GluR1 channel phosphorylation. (A and B) Rat embryonic spinal cord cultures were treated with 1 µm cicaprost (n = 5), 20 µm 8-bromo-cyclic adenosine monophosphate (n = 3; A), or 0.1 µm EP2/EP4 agonists (Ono-AE-259-01 and Ono-AE-329; n = 3; B) for 20 min. Western blot analysis showing GluR1 protein level after stimulation in membrane fractions. Densitometry of the GluR1 signals corrected for heat-shock protein (HSP) 90 loading and normed to the control is shown in the lower panel. Data are shown as mean ± SEM (n = 3–5). One-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, ***P < 0.001. (C) Intrathecal (IT) injection of cicaprost (10 µm) in rats resulted in translocation of GluR1 but not GluR2 subunits to the membrane shown by Western blot analysis of membrane fractions. Densitometry of the GluR1 and GluR2 signals corrected for pan Cadherin loading and normed to the control is shown in the lower panel. Data shown as mean ± SEM (n = 3). Two-way ANOVA with Bonferoni post hoc test, *** P < 0.001. (D) Rats were injected with 100 µl zymosan (12.5 mg/ml) in the hind paw (intraperitoneal) and the L4-L5 spinal dorsal horn was extracted after 6 h. Western blot analysis shows GluR1 phosphorylation at Serine 845 (pSer845) and a translocation of GluR1 to the membrane. The densitometric analysis is shown in the panel below. Data are shown as mean ± SEM (n = 4). Two-tailed Student t test: *P < 0.05. (E) Spinal cords of adult mice were dissected, sliced to 500 µm at a vibratome, and stimulated to measure the released glutamate of the cells in the supernatant. Cicaprost 1 µm (n = 58 slices) and 50 mm KCl (n = 13) increased glutamate release. This effect was blocked by the selective PKA inhibitor H89 (10 µm, n = 18). Data shown as mean ± SEM. One-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, **P < 0.01, *** P < 0.001. (F) Same as C except that spinal cord slices from IP knockout were used (n = 10). Data are shown as mean ± SEM. One-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, **P < 0.01.$

Cicaprost induces a protein kinase A–dependent GluR1 channel phosphorylation. (A and B) Rat embryonic spinal cord cultures were treated with 1 µm cicaprost (n = 5), 20 µm 8-bromo-cyclic adenosine monophosphate (n = 3; A), or 0.1 µm EP2/EP4 agonists (Ono-AE-259-01 and Ono-AE-329; n = 3; B) for 20 min. Western blot analysis showing GluR1 protein level after stimulation in membrane fractions. Densitometry of the GluR1 signals corrected for heat-shock protein (HSP) 90 loading and normed to the control is shown in the lower panel. Data are shown as mean ± SEM (n = 3–5). One-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, ***P < 0.001. (C) Intrathecal (IT) injection of cicaprost (10 µm) in rats resulted in translocation of GluR1 but not GluR2 subunits to the membrane shown by Western blot analysis of membrane fractions. Densitometry of the GluR1 and GluR2 signals corrected for pan Cadherin loading and normed to the control is shown in the lower panel. Data shown as mean ± SEM (n = 3). Two-way ANOVA with Bonferoni post hoc test, *** P < 0.001. (D) Rats were injected with 100 µl zymosan (12.5 mg/ml) in the hind paw (intraperitoneal) and the L4-L5 spinal dorsal horn was extracted after 6 h. Western blot analysis shows GluR1 phosphorylation at Serine 845 (pSer845) and a translocation of GluR1 to the membrane. The densitometric analysis is shown in the panel below. Data are shown as mean ± SEM (n = 4). Two-tailed Student t test: *P < 0.05. (E) Spinal cords of adult mice were dissected, sliced to 500 µm at a vibratome, and stimulated to measure the released glutamate of the cells in the supernatant. Cicaprost 1 µm (n = 58 slices) and 50 mm KCl (n = 13) increased glutamate release. This effect was blocked by the selective PKA inhibitor H89 (10 µm, n = 18). Data shown as mean ± SEM. One-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, **P < 0.01, *** P < 0.001. (F) Same as C except that spinal cord slices from IP knockout were used (n = 10). Data are shown as mean ± SEM. One-way ANOVA with Newman–Keuls multiple comparison test, *P < 0.05, **P < 0.01.

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