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Long-term exposure to PGE2 causes homologous desensitization of receptor-mediated activation of protein kinase A.

Malty RH, Hudmon A, Fehrenbacher JC, Vasko MR - J Neuroinflammation (2016)

Bottom Line: Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89.Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA.Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of cholera toxin, forskolin, or a stable analog of prostacyclin to activate PKA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Faculty of Science, University of Regina, Regina, SK, Canada.

ABSTRACT

Background: Acute exposure to prostaglandin E2 (PGE2) activates EP receptors in sensory neurons which triggers the cAMP-dependent protein kinase A (PKA) signaling cascade resulting in enhanced excitability of the neurons. With long-term exposure to PGE2, however, the activation of PKA does not appear to mediate persistent PGE2-induced sensitization. Consequently, we examined whether homologous desensitization of PGE2-mediated PKA activation occurs after long-term exposure of isolated sensory neurons to the eicosanoid.

Methods: Sensory neuronal cultures were harvested from the dorsal root ganglia of adult male Sprague-Dawley rats. The cultures were pretreated with vehicle or PGE2 and used to examine signaling mechanisms mediating acute versus persistent sensitization by exposure to the eicosanoid using enhanced capsaicin-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) as an endpoint. Neuronal cultures chronically exposed to vehicle or PGE2 also were used to study the ability of the eicosanoid and other agonists to activate PKA and whether long-term exposure to the prostanoid alters expression of EP receptor subtypes.

Results: Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89. After 5 days of exposure to 1 μM PGE2, administration of the eicosanoid still augments evoked release of iCGRP, but the effect is not attenuated by inhibition of PKA or by inhibition of PI3 kinases. The sensitizing actions of PGE2 after acute and long-term exposure were attenuated by EP2, EP3, and EP4 receptor antagonists, but not by an EP1 antagonist. Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA. The offset of the desensitization occurs within 24 h of removal of PGE2 from the cultures. Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of cholera toxin, forskolin, or a stable analog of prostacyclin to activate PKA.

Conclusions: Long-term exposure to PGE2 results in homologous desensitization of EP4 receptor activation of PKA, but not to neuronal sensitization suggesting that activation of PKA does not mediate PGE2-induced sensitization after chronic exposure to the eicosanoid.

No MeSH data available.


Related in: MedlinePlus

Five-day exposure of sensory neuronal cultures to PGE2 does not produce heterologous desensitization. Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity in cultures exposed to vehicle for 5 days or PGE2 (1 μM) for 5 days as indicated. a Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or CTX (1.5 μg/ml) overnight (shaded columns). b Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM forskolin for 20 min (shaded columns). c Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM cPGI2 (shaded columns) for 20 min. An asterisk indicates statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test. d The left panel represents PKA activity from cells exposed to vehicle for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated, while the right panel represents PKA activity from cells exposed to PGE2 (1 μM) for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated. After washing, the cells were re-exposed to vehicle (open columns) or 1 μM PGE2 (shaded and hatched columns) for 10 min. Asterisks indicate statistically significant differences in cells acutely exposed to vehicle versus cells exposed to PGE2. Crosses represent statistically significant differences in cells preexposed for 12 h to vehicle versus those exposed for 12 h to PGE2. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test
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Fig7: Five-day exposure of sensory neuronal cultures to PGE2 does not produce heterologous desensitization. Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity in cultures exposed to vehicle for 5 days or PGE2 (1 μM) for 5 days as indicated. a Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or CTX (1.5 μg/ml) overnight (shaded columns). b Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM forskolin for 20 min (shaded columns). c Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM cPGI2 (shaded columns) for 20 min. An asterisk indicates statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test. d The left panel represents PKA activity from cells exposed to vehicle for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated, while the right panel represents PKA activity from cells exposed to PGE2 (1 μM) for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated. After washing, the cells were re-exposed to vehicle (open columns) or 1 μM PGE2 (shaded and hatched columns) for 10 min. Asterisks indicate statistically significant differences in cells acutely exposed to vehicle versus cells exposed to PGE2. Crosses represent statistically significant differences in cells preexposed for 12 h to vehicle versus those exposed for 12 h to PGE2. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test

Mentions: Classical GPCR desensitization is mediated by receptor uncoupling from the cognate heterotrimeric G-protein and the downstream signaling pathway [13, 38]. Consequently, in sensory neurons exposed to PGE2 for 5 days, it is possible that the EP receptors are no longer coupled to the Gαs/adenylyl cyclase/PKA pathway. If receptor uncoupling mediates the loss of PGE2-induced activation of PKA, then bypassing the receptor by directly activating Gαs or adenylyl cyclases should increase PKA activity even after long-term exposure to PGE2. To test this, we examined the effects of CTX or forskolin on PKA activity after long-term exposure of sensory neuronal cultures to PGE2. When neuronal cultures treated with vehicle for 5 days were exposed to 1.5 μg/ml CTX overnight, the PKA activity increased from 0.06 ± 0.007 (vehicle) to 0.46 ± 0.01 (Fig. 7a). In a similar manner, when cultures were exposed to PGE2 for 5 days, CTX increased PKA activity from 0.05 ± 0.003 (vehicle) to 0.46 ± 0.02 (Fig. 7a). When neuronal cultures treated with vehicle for 5 days were exposed to 1 μM forskolin for 20 min, the activator of adenylyl cyclases significantly increased PKA activity from 0.06 ± 0.01 to 0.28 ± 0.04 (Fig. 7b). In cultures treated with 1 μM PGE2 for 5 days, exposure to forskolin increased PKA activity from 0.05 ± 0.01 to 0.27 ± 0.04 (Fig. 7b). Thus, the downregulation of PGE2-activated PKA appears to result from the uncoupling between PGE2 and the PKA signaling pathway at the receptor level.Fig. 7


Long-term exposure to PGE2 causes homologous desensitization of receptor-mediated activation of protein kinase A.

Malty RH, Hudmon A, Fehrenbacher JC, Vasko MR - J Neuroinflammation (2016)

Five-day exposure of sensory neuronal cultures to PGE2 does not produce heterologous desensitization. Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity in cultures exposed to vehicle for 5 days or PGE2 (1 μM) for 5 days as indicated. a Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or CTX (1.5 μg/ml) overnight (shaded columns). b Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM forskolin for 20 min (shaded columns). c Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM cPGI2 (shaded columns) for 20 min. An asterisk indicates statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test. d The left panel represents PKA activity from cells exposed to vehicle for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated, while the right panel represents PKA activity from cells exposed to PGE2 (1 μM) for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated. After washing, the cells were re-exposed to vehicle (open columns) or 1 μM PGE2 (shaded and hatched columns) for 10 min. Asterisks indicate statistically significant differences in cells acutely exposed to vehicle versus cells exposed to PGE2. Crosses represent statistically significant differences in cells preexposed for 12 h to vehicle versus those exposed for 12 h to PGE2. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test
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Related In: Results  -  Collection

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Fig7: Five-day exposure of sensory neuronal cultures to PGE2 does not produce heterologous desensitization. Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity in cultures exposed to vehicle for 5 days or PGE2 (1 μM) for 5 days as indicated. a Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or CTX (1.5 μg/ml) overnight (shaded columns). b Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM forskolin for 20 min (shaded columns). c Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 1 μM cPGI2 (shaded columns) for 20 min. An asterisk indicates statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test. d The left panel represents PKA activity from cells exposed to vehicle for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated, while the right panel represents PKA activity from cells exposed to PGE2 (1 μM) for 12 h in the absence or presence of 10 μM H-89 or 1 μM BIM-I as indicated. After washing, the cells were re-exposed to vehicle (open columns) or 1 μM PGE2 (shaded and hatched columns) for 10 min. Asterisks indicate statistically significant differences in cells acutely exposed to vehicle versus cells exposed to PGE2. Crosses represent statistically significant differences in cells preexposed for 12 h to vehicle versus those exposed for 12 h to PGE2. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test
Mentions: Classical GPCR desensitization is mediated by receptor uncoupling from the cognate heterotrimeric G-protein and the downstream signaling pathway [13, 38]. Consequently, in sensory neurons exposed to PGE2 for 5 days, it is possible that the EP receptors are no longer coupled to the Gαs/adenylyl cyclase/PKA pathway. If receptor uncoupling mediates the loss of PGE2-induced activation of PKA, then bypassing the receptor by directly activating Gαs or adenylyl cyclases should increase PKA activity even after long-term exposure to PGE2. To test this, we examined the effects of CTX or forskolin on PKA activity after long-term exposure of sensory neuronal cultures to PGE2. When neuronal cultures treated with vehicle for 5 days were exposed to 1.5 μg/ml CTX overnight, the PKA activity increased from 0.06 ± 0.007 (vehicle) to 0.46 ± 0.01 (Fig. 7a). In a similar manner, when cultures were exposed to PGE2 for 5 days, CTX increased PKA activity from 0.05 ± 0.003 (vehicle) to 0.46 ± 0.02 (Fig. 7a). When neuronal cultures treated with vehicle for 5 days were exposed to 1 μM forskolin for 20 min, the activator of adenylyl cyclases significantly increased PKA activity from 0.06 ± 0.01 to 0.28 ± 0.04 (Fig. 7b). In cultures treated with 1 μM PGE2 for 5 days, exposure to forskolin increased PKA activity from 0.05 ± 0.01 to 0.27 ± 0.04 (Fig. 7b). Thus, the downregulation of PGE2-activated PKA appears to result from the uncoupling between PGE2 and the PKA signaling pathway at the receptor level.Fig. 7

Bottom Line: Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89.Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA.Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of cholera toxin, forskolin, or a stable analog of prostacyclin to activate PKA.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, Faculty of Science, University of Regina, Regina, SK, Canada.

ABSTRACT

Background: Acute exposure to prostaglandin E2 (PGE2) activates EP receptors in sensory neurons which triggers the cAMP-dependent protein kinase A (PKA) signaling cascade resulting in enhanced excitability of the neurons. With long-term exposure to PGE2, however, the activation of PKA does not appear to mediate persistent PGE2-induced sensitization. Consequently, we examined whether homologous desensitization of PGE2-mediated PKA activation occurs after long-term exposure of isolated sensory neurons to the eicosanoid.

Methods: Sensory neuronal cultures were harvested from the dorsal root ganglia of adult male Sprague-Dawley rats. The cultures were pretreated with vehicle or PGE2 and used to examine signaling mechanisms mediating acute versus persistent sensitization by exposure to the eicosanoid using enhanced capsaicin-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) as an endpoint. Neuronal cultures chronically exposed to vehicle or PGE2 also were used to study the ability of the eicosanoid and other agonists to activate PKA and whether long-term exposure to the prostanoid alters expression of EP receptor subtypes.

Results: Acute exposure to 1 μM PGE2 augments the capsaicin-evoked release of iCGRP, and this effect is blocked by the PKA inhibitor H-89. After 5 days of exposure to 1 μM PGE2, administration of the eicosanoid still augments evoked release of iCGRP, but the effect is not attenuated by inhibition of PKA or by inhibition of PI3 kinases. The sensitizing actions of PGE2 after acute and long-term exposure were attenuated by EP2, EP3, and EP4 receptor antagonists, but not by an EP1 antagonist. Exposing neuronal cultures to 1 μM PGE2 for 12 h to 5 days blocks the ability of PGE2 to activate PKA. The offset of the desensitization occurs within 24 h of removal of PGE2 from the cultures. Long-term exposure to PGE2 also results in desensitization of the ability of a selective EP4 receptor agonist, L902688 to activate PKA, but does not alter the ability of cholera toxin, forskolin, or a stable analog of prostacyclin to activate PKA.

Conclusions: Long-term exposure to PGE2 results in homologous desensitization of EP4 receptor activation of PKA, but not to neuronal sensitization suggesting that activation of PKA does not mediate PGE2-induced sensitization after chronic exposure to the eicosanoid.

No MeSH data available.


Related in: MedlinePlus