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  • br Material and methods br


    Material and methods
    Results and discussion
    Conclusions Prostanoid-E receptor selective antagonists that inhibit EP2 or EP4 receptor activities may be used as a pharmacological strategy to limit cyst formation and ADPKD progression. Our study follows on from our previous observations of the regulation of cAMP mediated cystogenesis in human ADPKD cells. Here we indicate major differences in EP2 and EP4 receptor expression and regulation in IMCD-3 cells. Our study highlights that the mechanism of PGE2-mediated cAMP formation and cystogenesis in IMCD-3 Biotin-HPDP is EP4 dependent and contrasts that observed in human ADPKD cells. As a consequence of this, as well as the observed differences in cell distribution of EP2 and EP4 receptors in these different systems, these data suggest that careful selection of a suitable model system in order to test blockade of prostanoid E receptors on cyst formation in vivo will be necessary.
    Acknowledgments We gratefully acknowledge the funding of the Children's Hospital Foundation. We thank Lijuan Chen for her technical assistance. We thank Dr. Leonidas Tsiokas for his comments and making his laboratory available for some of the experiments described in this study. We also thank the cDNA Ressource Center (University of Missouri-Rolla, for providing the EP receptor expression vectors
    Introduction Prostaglandins are neuroactive substances involved in the regulation of gonadotrophin-releasing hormone (GnRH) secretion in the hypothalamus (Ojeda et al., 1989). An in vivo study using ovarian steroid-primed ovariectomized (OVX) rats showed that prostaglandin E2 (PGE2) injection into either into the preoptic area (POA) or medio-basal hypothalamus induced the secretion of luteinizing hormone (Ojeda et al., 1977). In vitro studies using fragments of the medio-basal hypothalamus showed that PGE2 stimulates GnRH secretion (Bigdeli and Snyder, 1978, Ojeda et al., 1979), whereas inhibition of PGE2 synthesis suppressed catecholamine-induced GnRH secretion (Ojeda et al., 1979, Ojeda et al., 1982, Heaulme and Dray, 1984). Collectively, these reports indicate that PGE2 stimulates GnRH secretion by activating GnRH neurons. PGE2 can activate four subtypes of G protein-coupled receptor (prostaglandin E receptor [EP]1-4) linked to distinct intracellular signaling pathways (Coleman et al., 1994, Sugimoto and Narumiya, 2007). PGE2-mediated activation of EP1 drives an increase in intracellular free Ca2+ (Katoh et al., 1995, Sugimoto and Narumiya, 2007), whereas EP2 and EP4 combine with stimulatory G proteins to increase intracellular cAMP levels in response to PGE2 (Sugimoto and Narumiya, 2007, Clasadonte et al., 2011). In contrast, EP3 decreases intracellular cAMP levels via inhibitory G proteins (Sugimoto and Narumiya, 2007, Clasadonte et al., 2011). The differential responses of several types of neurons to PGE2 (Sekiyama et al., 1995, Jennings and Mawe, 1998, Ibrahim et al., 1999, Baba et al., 2001, Ferri et al., 2005) have been attributed to their expression of G protein-coupled receptors. In GnRH neurons, PGE2 acts directly, as these neurons express PGE2 receptor mRNA and protein (Rage et al., 1997, Clasadonte et al., 2011). An electrophysiological study revealed that PGE2 and an EP2 agonist induce membrane depolarization of GnRH neurons regardless of the stage of estrous cycle (Clasadonte et al., 2011). Thus, the direct excitatory action of PGE2 on GnRH neurons is responsible, at least in part, for the stimulatory effect of PGE2 on GnRH release, irrespective of estrogen concentration. In contrast to the direct effect of PGE2 on the excitability of GnRH neurons, its influence on synaptic transmission remains unclear. As reported for other neurons, PGE2 may modulate synaptic transmission to GnRH neurons (Sekiyama et al., 1995, Sang et al., 2005, Lu et al., 2007). Although GnRH neurons have been reported to have relatively few synapses (Witkin et al., 1995), synaptic regulation appears to be involved in the modulation of GnRH neuron function. GnRH neurons have glutamatergic synaptic architectures (Witkin et al., 1995, Kiss et al., 2003), express glutamate receptors (Eyigor and Jennes, 1996), and undergo ion-channel responses to glutamate, AMPA, kainate, or NMDA (Spergel et al., 1999). Importantly, the somatic spine density in GnRH neurons is estrogen responsive (Chan et al., 2011), suggesting synaptic involvement in GnRH neurons, possibly as part of an estrogen-sensitive positive feedback mechanism. Moreover, the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) are altered during the estrous cycle and dependent on the estrogen environment (Tada et al., 2013).