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Title page for ETD etd-07232013-155120

Type of Document Dissertation
Author Jewell, Mark Langley
URN etd-07232013-155120
Title Regulation of Ca2+ channels and exocytosis by receptors for prostaglandin E2
Degree PhD
Department Pharmacology
Advisory Committee
Advisor Name Title
Dr. Rich Breyer Committee Chair
Dr. Aurelio Galli Committee Member
Dr. Dave Piston Committee Member
Dr. Heidi Hamm Committee Member
Dr. Kevin Currie Committee Member
  • amperometry
  • PGE2
  • exocytosis
  • GPCRs
  • calcium channels
Date of Defense 2013-06-27
Availability unrestricted
Adrenal chromaffin cells release catecholamines, neuropeptides and other hormones to maintain cardiovascular and metabolic homeostasis, and tune the physiological response to acute stress. As such, chromaffin cells represent a fundamental neuroendocrine component of the sympathetic nervous system. G protein coupled receptors (GPCRs) integrate a variety of autocrine / paracrine signals to precisely control catecholamine exocytosis. Systemic immune challenge or inflammatory cytokines are thought to increase local production of prostaglandin E2 in the adrenal gland where four cognate GPCRs (EP1-EP4) are expressed. Here we detail the cellular framework by which PGE2 modulates chromaffin cell function. We show mRNA for all four EP receptors (EP1- EP4) is expressed in mouse adrenal tissue. PGE2 inhibits Cav2 voltage-gated calcium channel currents (ICa) and exocytosis (changes in membrane capacitance) evoked by brief depolarizing stimuli in mouse chromaffin cells. This pathway is mediated by G protein βγ subunits (Gβγ) liberated from Gi/o-type G proteins downstream of EP3 receptor activation by PGE2. During sustained stimulation to mimic acute stress, this canonical inhibition transitions to a novel potentiation of catecholamine release. Catecholamine secretion was evoked by stimulation with 30 mM KCl and detected using carbon fiber amperometry. In chromaffin cells isolated from wild-type mice, PGE2 produced a robust potentiation of secretion that was primarily due to a significant increase in the number of vesicular fusion events (amperometric spikes). The potentiation was abolished in cells treated with pertussis toxin indicating involvement of Gi/o-type G proteins. Further, two distinct antagonists of Gβγ signaling (gallein or anti-Gβγ phosducin-like C terminus peptide) blocked the PGE2 mediated potentiation of catecholamine secretion. The potentiation was also prevented by a selective antagonist of the EP3 receptor (DG-041), and not seen in cells isolated from EP3 knockout mice. Thus, during brief stimuli, EP3 receptors suppress secretion through inhibition of ICa, but during sustained stimuli EP3 receptors potentiate evoked catecholamine secretion through a distinct pathway which also involves Gβγ subunits liberated from Gi/o-type G proteins. Taken together, our data reveal a rich, context-dependent modulation of catecholamine secretion by the inflammatory mediator PGE2, and identify a novel signaling pathway through which “inhibitory” G-proteins can potentiate neuroendocrine hormone secretion.
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