GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.

Although µ, κ, and δ opioids activate extracellular signal-regulated kinase (ERK)/mitogen-activated protein (MAP) kinase, the mechanisms involved in their signaling pathways and the cellular responses that ensue differ. Here we focused on the mechanisms by which µ opioids rapidly (min) activate ERK and their slower (h) actions to inhibit epidermal growth factor (EGF)-induced ERK-mediated astrocyte proliferation. The µ-opioid agonists ([d-ala2, mephe4, gly-ol5] enkephalin and morphine) promoted the phosphorylation of ERK/MAP kinase within 5 min via Gi/o protein, calmodulin (CaM), and β-arrestin2-dependent signaling pathways in immortalized and primary astrocytes. This was based on the attenuation of the µ-opioid activation of ERK by pertussis toxin (PTX), the CaM antagonist, W-7, and siRNA silencing of β-arrestin2. All three pathways were shown to activate ERK via an EGF receptor transactivation-mediated mechanism. This was disclosed by abolishment of µ-opioid-induced ERK phosphorylation with the EGF receptor-specific tyrosine phosphorylation inhibitor, AG1478, and µ-opioid-induced reduction of EGF receptor tyrosine phosphorylation by PTX, and β-arrestin2 targeting siRNA in the present studies and formerly by CaM antisense. Long-term (h) treatment of primary astrocytes with [d-ala2, mephe4, gly-ol5] enkephalin or morphine, attenuated EGF-induced ERK phosphorylation and proliferation (as measured by 5′-bromo-2′-deoxy-uridine labeling). PTX and β-arrestin2 siRNA but not W-7 reversed the µ-opioid inhibition. Unexpectedly, β-arrestin-2 siRNA diminished both EGF-induced ERK activation and primary astrocyte proliferation suggesting that this adaptor protein plays a novel role in EGF signaling as well as in the opioid receptor phase of this pathway. The results lend insight into the integration of the different µ-opioid signaling pathways to ERK and their cellular responses.

In this study, vaginal mesh exposure was only associated with ΔHgb and lower BMI.

Operative and postoperative experiences were similar between groups; however, Elevate anterior/apical repairs had shorter LOS, which might reflect more aggressive discharge planning. There were no bowel or major vascular injuries, and the Prolift trocar bladder injuries did not alter the surgical procedure.