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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

Exposing sensory neuronal cultures to PGE2 or to the EP4 receptor agonist L902688 for 5 days desensitizes the agonist-induced increase in PKA activity. a Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures grown in the absence of added NGF and treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (closed columns) or 10 μM PGE2 (hatched column). b Each column represents mean ± SEM of total specific activity of PKA after exposure to 10 μM cAMP from cultures treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed then re-exposed for 10 min to vehicle (open columns) or 1 μM PGE2 (closed columns). c Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures treated with vehicle, 300 nM L902688, or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 300 nM L902688 (closed columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test
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Fig5: Exposing sensory neuronal cultures to PGE2 or to the EP4 receptor agonist L902688 for 5 days desensitizes the agonist-induced increase in PKA activity. a Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures grown in the absence of added NGF and treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (closed columns) or 10 μM PGE2 (hatched column). b Each column represents mean ± SEM of total specific activity of PKA after exposure to 10 μM cAMP from cultures treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed then re-exposed for 10 min to vehicle (open columns) or 1 μM PGE2 (closed columns). c Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures treated with vehicle, 300 nM L902688, or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 300 nM L902688 (closed columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test

Mentions: Although PGE2-induced sensitization of sensory neurons after long-term exposure to the eicosanoid does not appear to be PKA dependent, the question remains whether the ability of PGE2 to increase PKA activity downregulates with chronic exposure to the prostanoid. To examine this directly, we determined whether 1 μM PGE2 could increase PKA activity in neuronal cultures treated with the eicosanoid for 5 days. As observed in previous experiments, when sensory neuronal cultures were exposed to vehicle for 5 days and then challenged with 1 μM PGE2 for 10 min, the eicosanoid caused a significant increase in PKA activity from 0.06 ± 0.003 to 0.52 ± 0.1 (Fig. 5a). In contrast, when cultures are exposed to 1 μM PGE2 for 5 days and then re-exposed to the eicosanoid, there was no significant increase in PKA activity (PKA activity was 0.07 ± 0.0003, Fig. 5a). Increasing the concentration of PGE2 10-fold caused a small, but not significant, increase in PKA activity (0.14 ± 0.01) in cultures exposed to PGE2 for 5 days (Fig. 5a). The total specific PKA activity after exposure to 10 μM cAMP was not affected by the long-term exposure to PGE2 suggesting that the downregulation of PGE2-activated PKA was not caused by any decrease in the overall kinase activity (Fig. 5b).Fig. 5


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)

Exposing sensory neuronal cultures to PGE2 or to the EP4 receptor agonist L902688 for 5 days desensitizes the agonist-induced increase in PKA activity. a Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures grown in the absence of added NGF and treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (closed columns) or 10 μM PGE2 (hatched column). b Each column represents mean ± SEM of total specific activity of PKA after exposure to 10 μM cAMP from cultures treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed then re-exposed for 10 min to vehicle (open columns) or 1 μM PGE2 (closed columns). c Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures treated with vehicle, 300 nM L902688, or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 300 nM L902688 (closed columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test
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Related In: Results  -  Collection

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Fig5: Exposing sensory neuronal cultures to PGE2 or to the EP4 receptor agonist L902688 for 5 days desensitizes the agonist-induced increase in PKA activity. a Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures grown in the absence of added NGF and treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (closed columns) or 10 μM PGE2 (hatched column). b Each column represents mean ± SEM of total specific activity of PKA after exposure to 10 μM cAMP from cultures treated with vehicle or PGE2 (1 μM) for 5 days as indicated. Cultures were washed then re-exposed for 10 min to vehicle (open columns) or 1 μM PGE2 (closed columns). c Each column represents the mean ± SEM of the treatment-stimulated PKA activity normalized to total PKA activity from cultures treated with vehicle, 300 nM L902688, or PGE2 (1 μM) for 5 days as indicated. Cultures were washed and then re-exposed for 10 min to vehicle (open columns) or 300 nM L902688 (closed columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test
Mentions: Although PGE2-induced sensitization of sensory neurons after long-term exposure to the eicosanoid does not appear to be PKA dependent, the question remains whether the ability of PGE2 to increase PKA activity downregulates with chronic exposure to the prostanoid. To examine this directly, we determined whether 1 μM PGE2 could increase PKA activity in neuronal cultures treated with the eicosanoid for 5 days. As observed in previous experiments, when sensory neuronal cultures were exposed to vehicle for 5 days and then challenged with 1 μM PGE2 for 10 min, the eicosanoid caused a significant increase in PKA activity from 0.06 ± 0.003 to 0.52 ± 0.1 (Fig. 5a). In contrast, when cultures are exposed to 1 μM PGE2 for 5 days and then re-exposed to the eicosanoid, there was no significant increase in PKA activity (PKA activity was 0.07 ± 0.0003, Fig. 5a). Increasing the concentration of PGE2 10-fold caused a small, but not significant, increase in PKA activity (0.14 ± 0.01) in cultures exposed to PGE2 for 5 days (Fig. 5a). The total specific PKA activity after exposure to 10 μM cAMP was not affected by the long-term exposure to PGE2 suggesting that the downregulation of PGE2-activated PKA was not caused by any decrease in the overall kinase activity (Fig. 5b).Fig. 5

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