PARENT SESSION
SLIDE SESSION 18: HORMONE RECEPTORS, STEROID HORMONE ACTION
Chairs: Terry Nett, Leslie Heckert, Dustin Vale-Cruz (Trainee)
Univ Ottawa-Arts Hall 257
1:30 PM-3:30 PM

EQUINE GnRH RECEPTOR KINETICS: EVIDENCE OF ACCELERATED RECEPTOR RECYCLING.

Porter, Michael¹, Allen, Matt², Nett, Terry², Clay, Colin², Sharp, Dan¹, ^{1 2}

ABSTRACT- Several studies have demonstrated slower rates of internalization for mammalian GnRH receptors (GnRH-R) compared to non-mammalian receptors. Despite high degree (>85%) of sequence homology between equine GnRH-R and other mammalian GnRH-R, in vivo data suggest insensitivity to receptor down regulation in horses. Thus the following study evaluated the rate of receptor internalization and recycling in equine pituitary gonadotropes. Internalization experiments were performed using radiolabelled D-Alanine GnRH and equine pituitary cells in primary cell culture. After 48 hours in static culture, pituitary cells were exposed to radiolabelled D-Ala GnRH and allowed to reach saturation at 4° C. Internalization of ligand-bound receptor was induced by warming cells to 37° C and terminated at set time points. Surface bound radioactivity was separated from internalized radioactivity. In Exp I, internalization was halted at 0, 20, 40, and 60 minutes where as in Exp II, internalization was halted at 0, 30, 60, 120, and 180 min. In Exp I, there was a slight decline in surface binding from time 0 to 20 minutes, which correlated with an increase in internalized receptor followed by an increase in surface binding. Steady state for surface bound ligand was reached at 60 min. and was approximately 200% greater than surface binding at time zero. The internalized radio-ligand reached steady state within 30 min. and was approximately 30% of total cell-associated ligand. Similar results were recorded in Exp II. Additionally, Scatchard plot analysis of each time point revealed an increase in receptor number and affinity over time. The endocytotic rate constant (Ke) and receptor insertion rate (Vr) were calculated for both experiments. The Ke was approximately 1000-fold slower (10-6 min-1) than published values in rats (0.012 min-1) and the Vr (467 min-1) was significantly faster than that for other GnRH-R. These data suggest that the equine pituitary gonadotropes respond to GnRH challenge by maintaining adequate concentrations of surface-bound GnRH-R.

KEY WORDS: GnRH receptor, equine, internalization, recycling