Local L-type Calcium Channel Signaling in Anterior Pituitary Gonadotropes

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 DESCRIPTION (provided by applicant): Mammalian reproduction requires intercommunication within the hypothalamic-pituitary-gonadal (HPG) axis. Defects within the HPG axis lead to abnormal reproductive cycles and infertility. For ovulation to occur, gonadotropin-releasing hormone (GnRH) from the hypothalamus stimulates gonadotrope cells in the anterior pituitary gland to acutely release luteinizing hormone (LH), known as the "LH surge". This LH secretion regulated by GnRH is mandatory for reproduction and involves specific interactions and signaling events. Therefore, understanding the molecular mechanisms underlying GnRH-induced LH secretion by gonadotropes has important implications for clinical manipulation of female reproduction. Calcium (Ca2+) is one of most versatile and universal signaling molecules that controls cellular processes, and activates important factors for fertility including key signal transduction molecules, mitogen-activated protein kinases (MAPK), that ultimately leads to LH transcription in gonadotrope cells. Previous research has provided indirect evidence that specific MAPK pathways, ERK and JNK, are activated by either a localized Ca2+ release through L-type Ca2+ channels, or by global Ca2+ signals released from intracellular stores in the endoplasmic reticulum, respectively. Importantly, the phosphorylation (i.e., activation) of ERK is the key signal required for enhanced LH synthesis and the LH surge. My goal is to investigate novel mechanistic molecular components and structures necessary for transducing GnRH receptor activation to these localized Ca2+ microdomains and ultimately activation of ERK. Specifically, I hypothesize that GnRH leads to ERK activation via highly localized Ca2+ microdomains produced by L-type Ca2+ channels, which are regulated by reactive oxygen species (ROS) and actin dynamics. Using a unique Ca2+ imaging technique combining electrophysiology and total internal reflection fluorescence (TIRF) microscopy, I will record Ca2+ influx through L-type Ca2+ channels with high spatiotemporal resolution. I will also use protein immunoblotting to measure ERK activation as phosphorylated ERK (pERK). In Specific Aim 1, I will test the hypothesis that reactive oxygen species (ROS) modulate local L-type Ca2+ channel function and ERK activity. In Specific Aim 2, I will test the hypothesis that that reactive oxygen species (ROS) modulate local L-type Ca2+ channel function and ERK activity. In each Specific Aim, I include preliminary data as well as proposed experimental plans for each mechanism that influences localized Ca2+ activity. This proposal attempts to better understand specific molecular mechanisms critical for reproduction, and therefore directly supports the NICHD mission statement "to ensure that every person is born healthy and wanted, [and] that women suffer no harmful effects from reproductive processes."