Limits...
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

Time course of the onset and offset of desensitization of the PGE2-induced activation of PKA in sensory neuronal cultures after chronic exposure to the eicosanoid. Each column represents the mean ± SEM of PKA activity normalized to total PKA activity for various times of exposure to 1 μM PGE2 or after removal of 1 μM PGE2. a Cultures were pretreated with vehicle (open column) or 1 μM PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure. b Cultures were pretreated with vehicle (open column) or PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure, and then, the PGE2 is removed and cells assayed at the times indicated. An asterisk indicates a statistically significant difference between PGE2-treated sensory neuronal cultures and vehicle-treated cultures using one-way ANOVA followed by Bonferroni’s post hoc test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4940832&req=5

Fig6: Time course of the onset and offset of desensitization of the PGE2-induced activation of PKA in sensory neuronal cultures after chronic exposure to the eicosanoid. Each column represents the mean ± SEM of PKA activity normalized to total PKA activity for various times of exposure to 1 μM PGE2 or after removal of 1 μM PGE2. a Cultures were pretreated with vehicle (open column) or 1 μM PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure. b Cultures were pretreated with vehicle (open column) or PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure, and then, the PGE2 is removed and cells assayed at the times indicated. An asterisk indicates a statistically significant difference between PGE2-treated sensory neuronal cultures and vehicle-treated cultures using one-way ANOVA followed by Bonferroni’s post hoc test

Mentions: The data presented above clearly show that exposing sensory neurons to PGE2 for 5 days abolishes the subsequent PGE2-induced activation of PKA. To ascertain the duration of exposure to PGE2 that is necessary to downregulate prostanoid-induced activation of PKA and to determine whether this desensitization is reversible, we examined PGE2-induced PKA activation after cultures were exposed to PGE2 for various lengths of time. To determine the time course for desensitization, sensory neuronal cultures were exposed to either vehicle for the last 5 days in culture or to 1 μM PGE2 for the last 3, 6, 12, 72 h or 5 days in culture (Fig. 6a, top panel). In all instances, PKA activity was determined after cells were maintained in culture for 12 days. Three and 6 h exposures of neuronal cultures to PGE2 resulted in a reduction in the ability of PGE2 to activate PKA by approximately 50 % (Fig. 6a). After a 12 h exposure, the PGE2-induced PKA activity is reduced by 80 %, whereas maximal inhibition is observed after 3 days of exposure (Fig. 6a). To examine whether the desensitization was reversible, sensory neurons in culture were exposed to vehicle for 36 h or to 1 μM PGE2 for 36, 33, 24, or 12 h and then to vehicle for 0, 3, 12, or 24 h, respectively (Fig. 6b, top panel), and PKA activity was measured. All cells were maintained in culture for 12 days. Exposure of sensory neurons to 1 μM PGE2 for 36 h resulted in desensitization of PGE2-induced activation of PKA (Fig. 6b), an effect we also observed after 72 h and 5 days of treatment with the eicosanoid (Fig. 6a). Three hours after the PGE2 is removed, a re-exposure to the eicosanoid did not augment PKA activity (Fig. 6b). In contrast, 12 and 24 h after removal of PGE2, PKA activation by re-exposure to the eicosanoid recovered to approximately 42 and 78 % of PGE2-activated PKA in naïve cultures (Fig. 6b). Thus, downregulation of the PGE2-induced activation of PKA is reversible and not secondary to loss of cell viability after chronic exposure to PGE2.Fig. 6


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)

Time course of the onset and offset of desensitization of the PGE2-induced activation of PKA in sensory neuronal cultures after chronic exposure to the eicosanoid. Each column represents the mean ± SEM of PKA activity normalized to total PKA activity for various times of exposure to 1 μM PGE2 or after removal of 1 μM PGE2. a Cultures were pretreated with vehicle (open column) or 1 μM PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure. b Cultures were pretreated with vehicle (open column) or PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure, and then, the PGE2 is removed and cells assayed at the times indicated. An asterisk indicates a statistically significant difference between PGE2-treated sensory neuronal cultures and vehicle-treated cultures using one-way ANOVA followed by Bonferroni’s post hoc test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4940832&req=5

Fig6: Time course of the onset and offset of desensitization of the PGE2-induced activation of PKA in sensory neuronal cultures after chronic exposure to the eicosanoid. Each column represents the mean ± SEM of PKA activity normalized to total PKA activity for various times of exposure to 1 μM PGE2 or after removal of 1 μM PGE2. a Cultures were pretreated with vehicle (open column) or 1 μM PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure. b Cultures were pretreated with vehicle (open column) or PGE2 (hatched columns) for the times indicated in the time line in the top portion of the figure, and then, the PGE2 is removed and cells assayed at the times indicated. An asterisk indicates a statistically significant difference between PGE2-treated sensory neuronal cultures and vehicle-treated cultures using one-way ANOVA followed by Bonferroni’s post hoc test
Mentions: The data presented above clearly show that exposing sensory neurons to PGE2 for 5 days abolishes the subsequent PGE2-induced activation of PKA. To ascertain the duration of exposure to PGE2 that is necessary to downregulate prostanoid-induced activation of PKA and to determine whether this desensitization is reversible, we examined PGE2-induced PKA activation after cultures were exposed to PGE2 for various lengths of time. To determine the time course for desensitization, sensory neuronal cultures were exposed to either vehicle for the last 5 days in culture or to 1 μM PGE2 for the last 3, 6, 12, 72 h or 5 days in culture (Fig. 6a, top panel). In all instances, PKA activity was determined after cells were maintained in culture for 12 days. Three and 6 h exposures of neuronal cultures to PGE2 resulted in a reduction in the ability of PGE2 to activate PKA by approximately 50 % (Fig. 6a). After a 12 h exposure, the PGE2-induced PKA activity is reduced by 80 %, whereas maximal inhibition is observed after 3 days of exposure (Fig. 6a). To examine whether the desensitization was reversible, sensory neurons in culture were exposed to vehicle for 36 h or to 1 μM PGE2 for 36, 33, 24, or 12 h and then to vehicle for 0, 3, 12, or 24 h, respectively (Fig. 6b, top panel), and PKA activity was measured. All cells were maintained in culture for 12 days. Exposure of sensory neurons to 1 μM PGE2 for 36 h resulted in desensitization of PGE2-induced activation of PKA (Fig. 6b), an effect we also observed after 72 h and 5 days of treatment with the eicosanoid (Fig. 6a). Three hours after the PGE2 is removed, a re-exposure to the eicosanoid did not augment PKA activity (Fig. 6b). In contrast, 12 and 24 h after removal of PGE2, PKA activation by re-exposure to the eicosanoid recovered to approximately 42 and 78 % of PGE2-activated PKA in naïve cultures (Fig. 6b). Thus, downregulation of the PGE2-induced activation of PKA is reversible and not secondary to loss of cell viability after chronic exposure to PGE2.Fig. 6

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