We continue investigations on receptors and channels expressed in anterior pituitary cells and their roles in signaling, gene expression and hormone secretion. Hypothalamic GnRH is the primary regulator of reproduction in vertebrates, acting via the G protein-coupled GnRH receptor (GnRHR) in pituitary gonadotrophs to control synthesis and release of gonadotropins. To identify elements of the GnRHR-coupled gene network, GnRH was applied in a pulsatile manner for 6 hours to a mixed population of perifused pituitary cells from cycling females, mRNA was extracted, and RNA sequencing analysis was performed. This revealed 83 candidate-regulated genes, including a large number coding for secreted proteins. Most notably, GnRH induces a >600-fold increase in expression of dentin matrix protein-1 (Dmp1), one of five members of the small integrin-binding ligand N-linked glycoprotein gene family. The Dmp1 response is mediated by the GnRHR, not elicited by other hypothalamic releasing factors, and is about 20-fold smaller in adult male pituitary cells. The sex-dependent Dmp1 response is established during peripubertal period and independent of the developmental pattern of Gnrhr expression. In vitro, GnRH-induced expression of this gene is coupled with release of DMP1 in extracellular medium through the regulated secretory pathway. In vivo, pituitary Dmp1 expression in identified gonadotrophs is elevated postovulatory. Cell signaling studies revealed that GnRH-induction of Dmp1 is mediated by the protein kinase C signaling pathway and reflects opposing roles of ERK1/2 and p38 MAPK;in addition, the response is facilitated by progesterone. These results establish that DMP1 is a novel secretory protein of female rat gonadotrophs, the synthesis and release of which are controlled by the hypothalamus through the GnRHR signaling pathway. This advance raises intriguing questions about the intrapituitary and downstream effects of this new player in GnRH signaling. The other main focus in our investigations is on structural and functional characterization of two ATP-gated P2X receptor subtypes, P2X7 and P2X4, expressed in pituitary lactotrophs. The P2X7 receptor operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. In previous work, we showed that the same receptor is capable of exhibiting sensitization and pore dilation (leading to the formation of biphasic currents and cell death) and/or desensitization (leading to a decline in current amplitude and cell life signaling) during sustained application of orthosteric agonists, depending on their concentrations. This was done in part by developing a 12-state Markov model consisting of nave, desensitized, and sensitized/dilated states that reproduced whole-cell current recordings generated experimentally. In this model, we assumed that the occupancy of one or two ATP-binding sites of nave receptors favored transition from open to desensitized states, whereas the occupancy of the third binding site favored receptor sensitization. More recently, we extend this modeling study by examining how this model cell behaves under various ionic conditions in the medium, including those that contain the large organic cation N-methyl-D-glucamine or divalent cations such as calcium. We illustrate how the two main gating patterns behave under these ionic conditions and determine why the shift in reversal potential and the dilation of the channels are accompanied paradoxically by a decrease in the total conductance during voltage ramp protocols. The model adds more evidence to our previous hypothesis, suggesting that dilation is masking desensitization. Our results also indicate that the allosteric sites through which divalent cations inhibit P2X7R should be extracellularly located. We also studied the influence of allostery on channel pore dilation using the rat P2X4 receptor expressed in HEK-293T cells and gated by ATP in the presence and absence of ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the agonist potency and kinetics of receptor deactivation. The corresponding kinetic model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically-induced sensitization of P2X4 receptor thus provides sustained signaling during prolonged and repetitive ATP stimulation. P2X receptors consist of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. We used cysteine and alanine scanning mutagenesis of the rat P2X4 receptor V47-V61 and K326-N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closed-open-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, I332, I333, T335, I337, and N338 in channels undergoing a transition from an open-to-desensitized state;residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating is stimulatory in all reactive V47-V61 mutants and inhibitory in the majority of reactive K326-N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition are located within the lumen of the extracellular vestibule and toward the central vestibule;however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region.
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