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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

Prostaglandin E2 and other activators of cAMP production increase PKA activity in sensory neuronal cultures. a Each point represents mean ± SEM of PKA activity normalized to total PKA after 10-min exposure to various concentrations of PGE2 from 4 to 6 independent harvests of cells maintained in the absence of added NGF. Asterisks indicate a statistically significant increase in PKA activity compared to the vehicle-treated control using one-way ANOVA followed by Bonferroni’s post hoc test. b Each column represents the mean ± SEM of PKA activity normalized to total PKA after a 10-min exposure to vehicle (V), PGE2, the EP4 receptor agonist, L902688, cPGI2, forskolin, CTX, or isoproterenol (Iso) as indicated. An asterisk indicates a statistically significant difference between PKA activation by each treatment compared to its respective vehicle control using Student’s t test
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Fig1: Prostaglandin E2 and other activators of cAMP production increase PKA activity in sensory neuronal cultures. a Each point represents mean ± SEM of PKA activity normalized to total PKA after 10-min exposure to various concentrations of PGE2 from 4 to 6 independent harvests of cells maintained in the absence of added NGF. Asterisks indicate a statistically significant increase in PKA activity compared to the vehicle-treated control using one-way ANOVA followed by Bonferroni’s post hoc test. b Each column represents the mean ± SEM of PKA activity normalized to total PKA after a 10-min exposure to vehicle (V), PGE2, the EP4 receptor agonist, L902688, cPGI2, forskolin, CTX, or isoproterenol (Iso) as indicated. An asterisk indicates a statistically significant difference between PKA activation by each treatment compared to its respective vehicle control using Student’s t test

Mentions: Previous studies have shown that exposing sensory neurons in culture to PGE2 or prostaglandin I2 (PGI2) increases the production of cAMP [6, 7]. Furthermore, inhibitors of PKA attenuate the acute sensitizing actions of PGE2 suggesting that sensitization is mediated by activation of PKA [11, 12, 26, 32]. Because cAMP has multiple downstream effectors, we measured whether exposing sensory neuronal cultures to increasing concentrations of PGE2 would directly increase PKA activity (see the “Methods” section for details). When sensory neuronal cultures were exposed to PGE2 for 10 min and PKA activity determined in cell lysates, treatment with PGE2 resulted in a concentration-dependent increase in PKA activity (Fig. 1a). The relationship between the log concentration of PGE2 and PGE2-induced PKA activity was fit to a sigmoidal curve with a correlation coefficient of 0.95 and an EC50 of 0.8 μM. The normalized PKA activity increased from 0.06 ± 0.01 for cultures treated with 0.1 μM PGE2 to 0.78 ± 0.10 for cultures exposed to 10 μM PGE2. Concentrations of 0.3, 1, 3, and 10 μM PGE2 all produced a significant increase in PKA activity compared to vehicle (Fig. 1a).Fig. 1


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)

Prostaglandin E2 and other activators of cAMP production increase PKA activity in sensory neuronal cultures. a Each point represents mean ± SEM of PKA activity normalized to total PKA after 10-min exposure to various concentrations of PGE2 from 4 to 6 independent harvests of cells maintained in the absence of added NGF. Asterisks indicate a statistically significant increase in PKA activity compared to the vehicle-treated control using one-way ANOVA followed by Bonferroni’s post hoc test. b Each column represents the mean ± SEM of PKA activity normalized to total PKA after a 10-min exposure to vehicle (V), PGE2, the EP4 receptor agonist, L902688, cPGI2, forskolin, CTX, or isoproterenol (Iso) as indicated. An asterisk indicates a statistically significant difference between PKA activation by each treatment compared to its respective vehicle control using Student’s t test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Prostaglandin E2 and other activators of cAMP production increase PKA activity in sensory neuronal cultures. a Each point represents mean ± SEM of PKA activity normalized to total PKA after 10-min exposure to various concentrations of PGE2 from 4 to 6 independent harvests of cells maintained in the absence of added NGF. Asterisks indicate a statistically significant increase in PKA activity compared to the vehicle-treated control using one-way ANOVA followed by Bonferroni’s post hoc test. b Each column represents the mean ± SEM of PKA activity normalized to total PKA after a 10-min exposure to vehicle (V), PGE2, the EP4 receptor agonist, L902688, cPGI2, forskolin, CTX, or isoproterenol (Iso) as indicated. An asterisk indicates a statistically significant difference between PKA activation by each treatment compared to its respective vehicle control using Student’s t test
Mentions: Previous studies have shown that exposing sensory neurons in culture to PGE2 or prostaglandin I2 (PGI2) increases the production of cAMP [6, 7]. Furthermore, inhibitors of PKA attenuate the acute sensitizing actions of PGE2 suggesting that sensitization is mediated by activation of PKA [11, 12, 26, 32]. Because cAMP has multiple downstream effectors, we measured whether exposing sensory neuronal cultures to increasing concentrations of PGE2 would directly increase PKA activity (see the “Methods” section for details). When sensory neuronal cultures were exposed to PGE2 for 10 min and PKA activity determined in cell lysates, treatment with PGE2 resulted in a concentration-dependent increase in PKA activity (Fig. 1a). The relationship between the log concentration of PGE2 and PGE2-induced PKA activity was fit to a sigmoidal curve with a correlation coefficient of 0.95 and an EC50 of 0.8 μM. The normalized PKA activity increased from 0.06 ± 0.01 for cultures treated with 0.1 μM PGE2 to 0.78 ± 0.10 for cultures exposed to 10 μM PGE2. Concentrations of 0.3, 1, 3, and 10 μM PGE2 all produced a significant increase in PKA activity compared to vehicle (Fig. 1a).Fig. 1

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