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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

The PGE2-induced increase of capsaicin-evoked iCGRP release from sensory neurons is attenuated by H-89 after acute exposure to the eicosanoid but not after long-term exposure. a, b Each column represents the mean ± SEM of iCGRP release as percent of total iCGRP content per well of cells maintained in the absence of added NGF. Lightly shaded columns indicate basal release whereas dark-shaded columns represent capsaicin-stimulated release of iCGRP. Cultures were treated with vehicle (a) or 1 μM PGE2 (b) for 5 days and then washed and acutely exposed to 1 μM PGE2 for 20 min in the absence or presence of 10 μM H-89, as indicated. An asterisk indicates a statistically significant difference between capsaicin-stimulated iCGRP release after exposure to vehicle versus after a 10-min exposure to PGE2 (1 μM) using one-way ANOVA followed by Bonferroni’s post hoc test. c Each column represents the mean ± SEM of cAMP content. The left panel represents cAMP content from cells exposed to vehicle for 5 days, whereas the right panel represents cAMP content from cells exposed to PGE2 (1 μM) for 5 days. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (light gray columns), or 1 μM forskolin (dark gray 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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Fig2: The PGE2-induced increase of capsaicin-evoked iCGRP release from sensory neurons is attenuated by H-89 after acute exposure to the eicosanoid but not after long-term exposure. a, b Each column represents the mean ± SEM of iCGRP release as percent of total iCGRP content per well of cells maintained in the absence of added NGF. Lightly shaded columns indicate basal release whereas dark-shaded columns represent capsaicin-stimulated release of iCGRP. Cultures were treated with vehicle (a) or 1 μM PGE2 (b) for 5 days and then washed and acutely exposed to 1 μM PGE2 for 20 min in the absence or presence of 10 μM H-89, as indicated. An asterisk indicates a statistically significant difference between capsaicin-stimulated iCGRP release after exposure to vehicle versus after a 10-min exposure to PGE2 (1 μM) using one-way ANOVA followed by Bonferroni’s post hoc test. c Each column represents the mean ± SEM of cAMP content. The left panel represents cAMP content from cells exposed to vehicle for 5 days, whereas the right panel represents cAMP content from cells exposed to PGE2 (1 μM) for 5 days. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (light gray columns), or 1 μM forskolin (dark gray columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test

Mentions: Since the acute sensitizing action of PGE2 on sensory neurons is mediated through activation of EP receptors that are part of the GPCR family [6, 34], chronic exposure to PGE2 should result in tolerance or desensitization to the sensitizing effects of this prostanoid. Previous studies, however, suggest that the ability of PGE2 to sensitize sensory neurons does not downregulate after chronic exposure to the eicosanoid [17, 24]. Consequently, we examined whether the ability of PGE2 to augment capsaicin-evoked release of iCGRP from sensory neurons downregulated after long-term exposure to the prostanoid and whether this sensitizing action was attenuated by the PKA inhibitor, H-89. When sensory neurons in culture were exposed to 30 nM capsaicin, release of iCGRP increased approximately threefold from a basal level of 3.4 ± 0.7 % of total content/10 min to 10.5 ± 1.3 % of total content/10 min (Fig. 2a). Treating cells with 1 μM PGE2 significantly augmented the capsaicin-evoked release to 15.6 ± 1.4 % of total content/10 min (Fig. 2a). Although exposure to 10 μM H-89 did not alter basal or capsaicin-stimulated release of iCGRP, it did block the ability of PGE2 to augment capsaicin-evoked release (Fig. 2a).Fig. 2


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)

The PGE2-induced increase of capsaicin-evoked iCGRP release from sensory neurons is attenuated by H-89 after acute exposure to the eicosanoid but not after long-term exposure. a, b Each column represents the mean ± SEM of iCGRP release as percent of total iCGRP content per well of cells maintained in the absence of added NGF. Lightly shaded columns indicate basal release whereas dark-shaded columns represent capsaicin-stimulated release of iCGRP. Cultures were treated with vehicle (a) or 1 μM PGE2 (b) for 5 days and then washed and acutely exposed to 1 μM PGE2 for 20 min in the absence or presence of 10 μM H-89, as indicated. An asterisk indicates a statistically significant difference between capsaicin-stimulated iCGRP release after exposure to vehicle versus after a 10-min exposure to PGE2 (1 μM) using one-way ANOVA followed by Bonferroni’s post hoc test. c Each column represents the mean ± SEM of cAMP content. The left panel represents cAMP content from cells exposed to vehicle for 5 days, whereas the right panel represents cAMP content from cells exposed to PGE2 (1 μM) for 5 days. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (light gray columns), or 1 μM forskolin (dark gray columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig2: The PGE2-induced increase of capsaicin-evoked iCGRP release from sensory neurons is attenuated by H-89 after acute exposure to the eicosanoid but not after long-term exposure. a, b Each column represents the mean ± SEM of iCGRP release as percent of total iCGRP content per well of cells maintained in the absence of added NGF. Lightly shaded columns indicate basal release whereas dark-shaded columns represent capsaicin-stimulated release of iCGRP. Cultures were treated with vehicle (a) or 1 μM PGE2 (b) for 5 days and then washed and acutely exposed to 1 μM PGE2 for 20 min in the absence or presence of 10 μM H-89, as indicated. An asterisk indicates a statistically significant difference between capsaicin-stimulated iCGRP release after exposure to vehicle versus after a 10-min exposure to PGE2 (1 μM) using one-way ANOVA followed by Bonferroni’s post hoc test. c Each column represents the mean ± SEM of cAMP content. The left panel represents cAMP content from cells exposed to vehicle for 5 days, whereas the right panel represents cAMP content from cells exposed to PGE2 (1 μM) for 5 days. Cultures were washed and then re-exposed for 10 min to vehicle (open columns), 1 μM PGE2 (light gray columns), or 1 μM forskolin (dark gray columns). An asterisk indicates a statistically significant difference from vehicle using one-way ANOVA followed by Bonferroni’s post hoc test
Mentions: Since the acute sensitizing action of PGE2 on sensory neurons is mediated through activation of EP receptors that are part of the GPCR family [6, 34], chronic exposure to PGE2 should result in tolerance or desensitization to the sensitizing effects of this prostanoid. Previous studies, however, suggest that the ability of PGE2 to sensitize sensory neurons does not downregulate after chronic exposure to the eicosanoid [17, 24]. Consequently, we examined whether the ability of PGE2 to augment capsaicin-evoked release of iCGRP from sensory neurons downregulated after long-term exposure to the prostanoid and whether this sensitizing action was attenuated by the PKA inhibitor, H-89. When sensory neurons in culture were exposed to 30 nM capsaicin, release of iCGRP increased approximately threefold from a basal level of 3.4 ± 0.7 % of total content/10 min to 10.5 ± 1.3 % of total content/10 min (Fig. 2a). Treating cells with 1 μM PGE2 significantly augmented the capsaicin-evoked release to 15.6 ± 1.4 % of total content/10 min (Fig. 2a). Although exposure to 10 μM H-89 did not alter basal or capsaicin-stimulated release of iCGRP, it did block the ability of PGE2 to augment capsaicin-evoked release (Fig. 2a).Fig. 2

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