My laboratory is predominantly active in two research arenas. One research area is focused on ovarian steroid regulation of intrinsic opioid analgesic pathways and sex determinants of analgesic mechanisms. This research utilizes two main models, physiological pregnancy and its hormonal simulation (hormone simulated pregnancy). Both conditions are associated with an opioid antinociception that is comprised of multiple central as well as peripheral components. Central contributors to gestational and hormonally induced antinociception include spinal m-/d-opioid receptors, a2-receptors and descending noradrenergic pathways, presumably originating in the A5-A7 cell groups of the pons. Peripheral components of gestational and ovarian steroid-induced antinociception include circulating levels of estrogen and progesterone and uterine afferent, hypogastric nerve, activity. We are presently engaged in elucidating molecular points of convergence and integration of these contributors.
My laboratory is also studying neurochemical adaptations that are causally associated with opioid tolerance formation. The guiding rubric within which this research is being conducted is that a major contributor to opioid tolerance is the emergence of new opioid receptor-coupled signaling strategies. This results from chronic morphine-induced adenylyl cyclase isoform-specific synthesis and phosphorylation. Additionally, the phosphorylation state of several other key signaling molecules is also significantly augmented following chronic morphine. These include the Gb subunit of G proteins, b-arrestin, and G protein receptor kinase2/3. As a result, there is a shift from predominantly opioid receptor Gia inhibitory to Gbg stimulatory adenylyl cyclase signaling. Notably, chronic morphine also induces the reciprocal phosphorylation and thereby regulation of two phospholipase Cb isoforms (phospholipase Cb1 and phospholipase Cb3) that has profound consequences on calcium mobilization. We are currently investigating macromolecular signaling complexes that contain opioid receptors and the influence of chronic morphine on their composition and function.
The schematic diagram shows new signaling strategies after chronic morphine treatment. Chronic morphine augments production of new adenylyl cyclase isoforms of the type II family as well as their phosphorylation. As a result, opioid signaling shifts from predominantly Gςα inhibitory to Gςα/Gβγ stimulatory.
Currently, we have broadened our investigations to include the effect of chronic morphine on other protein components of receptor G protein-coupled signaling pathways such as G protein receptor kinases and b-arrestin. Results from this line of study could provide substantial insights into how cells integrate diverse signal inputs.
The above figure illustrates autoradiographs of immunoprecipitates obtained using anti-Gβ (A) or anti GRK (B) antibodies. Lane 1, opioid naïve. Lane 2, chronic morphine-treated. Lane 3, immunoprecipitate obtained using pre-absorbed antisera. Note the increased phosphorylation of GRK (~80 kDa), β-arrestin (~45 kDa) and Gb (~3 kDa) following chronic morphine. Phosphorylation of purified Gβ (C) decreases its co-immunoprecipitation (association) with GRK (D) (see Chakrabarti et al., 2001).
Wang, L and. Gintzler, A. R. Altered m opiate receptor-G protein signal transduction following chronic morphine exposure, J. Neurochem., 68(1) 248 254, 1997.
Rivera, M. and Gintzler, A.R. Differential effect of chronic morphine on mRNA encoding adenylyl cyclase isoforms: relevance to physiological sequela of tolerance/dependence, Mol.Brain Res., 54(1): 165-169, 1998.
Chakrabarti, S., Rivera, M., Yan, S.Z., Tang, W.-J. and Gintzler, A. R., Chronic morphine augments Gbg/Gsa stimulation of adenylyl cyclase: relevance to opioid tolerance, Mol. Pharmacol., 54: 655-662, 1998a.
Chakrabarti,S., Wang, L., Tang, W.-J., and Gintzler, A.R. Chronic morphine augments adenylyl cyclase phosphorylation: relevance to altered signaling during tolerance/dependence, Mol. Pharmacol., 54: 949-953, 1998b.
Chakrabarti,S., Oppermann, M. and Gintzler, A.R. Chakrabarti,S., Oppermann, M. and Gintzler, A.R. Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling, Proc. Nat'l Acad. Sci. (USA), 98: 4209-4214, 2001.