Class
Article
College
College of Science
Department
Biology Department
Faculty Mentor
Sara M. Freeman
Presentation Type
Poster Presentation
Abstract
The neurohormone oxytocin influences many physiological pathways in animals, aiding in social behaviors, pair bonding, social recognition, and maternal behavior. A common strategy used to observe the influence of oxytocin on these behaviors is to disrupt oxytocin signaling, by blocking the oxytocin receptor (OXTR). This blockade can be achieved using antagonists which bind to the receptor and inhibit the subsequent activation of intracellular signaling pathways. We aimed to determine whether the commercially-available OXTR antagonist L-368,899 selectively binds to OXTR in coyote brains. L-368,899 selectively binds to OXTR in primates and has been used in animal behavior studies to block oxytocin-dependent behaviors. Structural homology in oxytocin and vasopressin 1a receptors (AVPR1a) results in binding promiscuity in many of the available antagonists and drugs that are used to target these receptors. Our study compares the binding selectivity of L-368,899 for OXTR over AVPR1a when used in coyote brain tissue (where these two receptors are expressed). We used 6 frozen, unfixed coyote brains (collected opportunistically) blocked coronally into 5 slabs and stored at -80°C until slicing. The blocks were then sliced at 20 micron thickness on a cryostat, mounted on microscope slides, and frozen at -80°C in sealed slide boxes packaged with desiccant packets until experimental use. These frozen and mounted coyote brain slices were subjected to competitive binding autoradiography using increasing concentrations of our antagonist L-368,899 in competition with a consistent concentration of one of two commercially-available radioligands: 1) OXTR radioligand 125I-ornithne vasotocin analog (125I-OVTA) and 2) AVPR1a radioligand 125I-linear vasopressin antagonist (125I-LVA). We quantified the binding density using a calibrated digital densitometry system and generated competition curves, which depict the selectivity of L-368,899. We found L-368,899 has a 70X greater affinity for OXTR when compared to AVPR1a. This result is also demonstrated in the Ki values that we calculated from the competition curves: 12.38 nM for OXTR and 870.7 nM for AVPR1a. While we were able to generate competition curves, unfortunately, many of our slides from the AVPR1a assay did not produce quantifiable results. In order to justify the use of this drug for in vivo studies, further experimentation is needed, although our preliminary analysis suggests L-368,899 has a high binding selectivity for the coyote OXTR.
Location
Logan, UT
Start Date
4-11-2021 12:00 AM
Included in
Pharmacological Characterization of the Oxytocin Receptor Antagonist L-368,899 for Coyote Receptors
Logan, UT
The neurohormone oxytocin influences many physiological pathways in animals, aiding in social behaviors, pair bonding, social recognition, and maternal behavior. A common strategy used to observe the influence of oxytocin on these behaviors is to disrupt oxytocin signaling, by blocking the oxytocin receptor (OXTR). This blockade can be achieved using antagonists which bind to the receptor and inhibit the subsequent activation of intracellular signaling pathways. We aimed to determine whether the commercially-available OXTR antagonist L-368,899 selectively binds to OXTR in coyote brains. L-368,899 selectively binds to OXTR in primates and has been used in animal behavior studies to block oxytocin-dependent behaviors. Structural homology in oxytocin and vasopressin 1a receptors (AVPR1a) results in binding promiscuity in many of the available antagonists and drugs that are used to target these receptors. Our study compares the binding selectivity of L-368,899 for OXTR over AVPR1a when used in coyote brain tissue (where these two receptors are expressed). We used 6 frozen, unfixed coyote brains (collected opportunistically) blocked coronally into 5 slabs and stored at -80°C until slicing. The blocks were then sliced at 20 micron thickness on a cryostat, mounted on microscope slides, and frozen at -80°C in sealed slide boxes packaged with desiccant packets until experimental use. These frozen and mounted coyote brain slices were subjected to competitive binding autoradiography using increasing concentrations of our antagonist L-368,899 in competition with a consistent concentration of one of two commercially-available radioligands: 1) OXTR radioligand 125I-ornithne vasotocin analog (125I-OVTA) and 2) AVPR1a radioligand 125I-linear vasopressin antagonist (125I-LVA). We quantified the binding density using a calibrated digital densitometry system and generated competition curves, which depict the selectivity of L-368,899. We found L-368,899 has a 70X greater affinity for OXTR when compared to AVPR1a. This result is also demonstrated in the Ki values that we calculated from the competition curves: 12.38 nM for OXTR and 870.7 nM for AVPR1a. While we were able to generate competition curves, unfortunately, many of our slides from the AVPR1a assay did not produce quantifiable results. In order to justify the use of this drug for in vivo studies, further experimentation is needed, although our preliminary analysis suggests L-368,899 has a high binding selectivity for the coyote OXTR.