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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

Twenty-four hour exposure of sensory neurons in culture to 1 μM PGE2 does not alter the expression of EP receptors as measured by real-time RT-PCR. a Dorsal root ganglia were harvested and cultured for 6 days in media containing 30 ng/ml NGF. After 24-h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns), the RNA was isolated and reverse transcribed to cDNA for analysis by PCR. Each column represents the mean ± SEM of the ratio of EP receptor mRNA to GAPDH mRNA from three independent harvests of cells. b Each column represents the mean ± SEM of the % maximum immunoreactivity for EP receptors normalized to TO-PRO-3 immunoreactivity in neuronal cultures after 24 h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns) for six to eight wells of cells
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Fig3: Twenty-four hour exposure of sensory neurons in culture to 1 μM PGE2 does not alter the expression of EP receptors as measured by real-time RT-PCR. a Dorsal root ganglia were harvested and cultured for 6 days in media containing 30 ng/ml NGF. After 24-h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns), the RNA was isolated and reverse transcribed to cDNA for analysis by PCR. Each column represents the mean ± SEM of the ratio of EP receptor mRNA to GAPDH mRNA from three independent harvests of cells. b Each column represents the mean ± SEM of the % maximum immunoreactivity for EP receptors normalized to TO-PRO-3 immunoreactivity in neuronal cultures after 24 h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns) for six to eight wells of cells

Mentions: The data presented above suggest that the maintenance of PGE2 sensitization following chronic exposure to the prostanoid may be mediated by alternate EP receptors which couple to different G-proteins and activate alternate downstream signaling pathways. To examine this possibility, we measured mRNA for the EP1, EP3C, and EP4 receptors. We chose to study these receptor subtypes since our previous work suggests that EP3C and EP4 receptors contribute to acute sensitization in isolated sensory neurons [6]. Furthermore, acute sensitization by PGE2 has been proposed to be mediated through activation of EP1 receptors [36]. Six days after harvesting, sensory neuronal cultures were exposed to 1 μM PGE2 or vehicle for 24 h, and then total RNA was isolated from the treated cells and reverse transcribed to cDNA. Exposing cultures to PGE2 for 24 h did not significantly alter the amounts of mRNA for any of the EP receptors examined: EP1, EP3C, and EP4 (Fig. 3a). The levels of mRNA for the EP1 receptor normalized to mRNA for GAPDH were 0.95 ± 0.05 in control cells and 1.10 ± 0.26 after a 24-h treatment with PGE2. Levels of mRNA for EP3C and EP4 were 0.99 ± 0.14 and 1.11 ± 0.05 in control cells and 0.81 ± 0.07 and 1.03 ± 0.09, respectively, in cells treated with PGE2. Similar results were observed from neuronal cultures exposed to PGE2 for 5 days. In these cultures as in cultures treated for 24 h, long-term exposure to 1 μM PGE2 did not significantly alter mRNA to EP1, EP2, EP3, or EP4 receptors compared to cells treated with vehicle for 5 days (data not shown).Fig. 3


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)

Twenty-four hour exposure of sensory neurons in culture to 1 μM PGE2 does not alter the expression of EP receptors as measured by real-time RT-PCR. a Dorsal root ganglia were harvested and cultured for 6 days in media containing 30 ng/ml NGF. After 24-h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns), the RNA was isolated and reverse transcribed to cDNA for analysis by PCR. Each column represents the mean ± SEM of the ratio of EP receptor mRNA to GAPDH mRNA from three independent harvests of cells. b Each column represents the mean ± SEM of the % maximum immunoreactivity for EP receptors normalized to TO-PRO-3 immunoreactivity in neuronal cultures after 24 h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns) for six to eight wells of cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Twenty-four hour exposure of sensory neurons in culture to 1 μM PGE2 does not alter the expression of EP receptors as measured by real-time RT-PCR. a Dorsal root ganglia were harvested and cultured for 6 days in media containing 30 ng/ml NGF. After 24-h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns), the RNA was isolated and reverse transcribed to cDNA for analysis by PCR. Each column represents the mean ± SEM of the ratio of EP receptor mRNA to GAPDH mRNA from three independent harvests of cells. b Each column represents the mean ± SEM of the % maximum immunoreactivity for EP receptors normalized to TO-PRO-3 immunoreactivity in neuronal cultures after 24 h treatment with vehicle (lightly shaded columns) or 1 μM PGE2 (dark-shaded columns) for six to eight wells of cells
Mentions: The data presented above suggest that the maintenance of PGE2 sensitization following chronic exposure to the prostanoid may be mediated by alternate EP receptors which couple to different G-proteins and activate alternate downstream signaling pathways. To examine this possibility, we measured mRNA for the EP1, EP3C, and EP4 receptors. We chose to study these receptor subtypes since our previous work suggests that EP3C and EP4 receptors contribute to acute sensitization in isolated sensory neurons [6]. Furthermore, acute sensitization by PGE2 has been proposed to be mediated through activation of EP1 receptors [36]. Six days after harvesting, sensory neuronal cultures were exposed to 1 μM PGE2 or vehicle for 24 h, and then total RNA was isolated from the treated cells and reverse transcribed to cDNA. Exposing cultures to PGE2 for 24 h did not significantly alter the amounts of mRNA for any of the EP receptors examined: EP1, EP3C, and EP4 (Fig. 3a). The levels of mRNA for the EP1 receptor normalized to mRNA for GAPDH were 0.95 ± 0.05 in control cells and 1.10 ± 0.26 after a 24-h treatment with PGE2. Levels of mRNA for EP3C and EP4 were 0.99 ± 0.14 and 1.11 ± 0.05 in control cells and 0.81 ± 0.07 and 1.03 ± 0.09, respectively, in cells treated with PGE2. Similar results were observed from neuronal cultures exposed to PGE2 for 5 days. In these cultures as in cultures treated for 24 h, long-term exposure to 1 μM PGE2 did not significantly alter mRNA to EP1, EP2, EP3, or EP4 receptors compared to cells treated with vehicle for 5 days (data not shown).Fig. 3

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