PARENT SESSION

(15) GnRH ENGAGEMENT OF THE ACTIN CYTOSKELETON UNDERLIES ACTIVATION OF MAP KINASE AND LEADS TO DYNAMIC CELLULAR MOVEMENTS IN THE ANTERIOR PITUITARY GLAND.

Navratil, Amy¹, Knoll, J. Gabriel¹, Tobet, Stuart¹, Clay, Colin¹, ¹ Colorado State University, Ft. Collins, CO

ABSTRACT- Central to reproductive function is the binding of GnRH to specific receptors located on gonadotrope cells of the anterior pituitary gland. Recently, we have found that GnRH binding leads to rapid and transient cytoskeletal rearrangements in the gonadotrope derived T3-1 cell line. Consistent with engagement of the actin cytoskeleton, GnRH induced changes in cytoarchitecture were blocked by 1 M jasplakinolide and latrunculin B and adenoviral mediated overexpression of dominant-negative RhoGTPase. These treatments also attenuated GnRH induced activation of MAP kinase. Thus, a functional link exists between GnRH signaling to the actin cytoskeleton and MAP kinase. Based on both sucrose density gradient separation and co-localization of GnRH receptor with the lipid raft associated GM1 ganglioside, we suggest that this functional link occurs in the context of discrete, cholesterol enriched membrane microdomains termed lipid rafts. To address the degree of conservation of these events in bona fide pituitary cells, ovine pituitaries were dissociated, treated with 100 nM GnRH and examined by confocal microscopy. Consistent with the T3-1 model, GnRH treatment led to transient formation of distinct cellular processes by selected ovine pituitary cells. To determine if a similar phenomenon was evident in cells in intact tissue, murine pituitaries were harvested, embedded in 8% low-melt agarose and then sliced (200 M) using a vibrating microtome. Glass shards coated with a carbocyanine dye (DiI) were placed into pituitary slices. Quantitative measurements of pituitary cell movement were obtained by live video microscopy. Data was acquired 30 min prior to and after administration of 100 nM GnRH. After GnRH, selected DiI labeled cells displayed a 96% increase in net distance moved and 38% increase in rate of movement. These data were independently confirmed using adenoviral delivery of GFP to visualize cells. These data are the first to directly demonstrate hormone-induced movement of endocrine cells in the anterior pituitary gland. We hypothesize that GnRH responsive cells move to maximize apposition to vascular endothelium thus gaining more immediate access to the bloodstream for hormone release.

KEY WORDS: GnRH Receptor, Cytoskeleton, Pituitary, Signaling