We continue investigations on receptors and channels expressed in the anterior pituitary gland and their roles in signaling, gene transcription, and hormone secretion. Our collaborative work with Dr. Arthur Sherman was focused on modeling the diversity of spontaneous and agonist-induced electrical activity in cultured corticotrophs. These cells fire action potentials spontaneously and in response to stimulation with corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and such electrical activity is critical for calcium signaling and calcium-dependent adrenocorticotropic hormone secretion. These cells typically fire tall, sharp action potentials when spontaneously active, but a variety of other spontaneous patterns have also been reported, including various modes of bursting. There is variability in reports of the fraction of corticotrophs that are electrically active, as well as their patterns of activity, and the sources of this variation are not well understood. The ionic mechanisms responsible for CRH- and AVP-triggered electrical activity in corticotrophs are also poorly characterized. We use electrophysiological measurements in single cultured corticotrophs and mathematical modeling to investigate possible sources of variability in patterns of spontaneous and agonist-induced electrical activity. In the model, variation in as few as two parameters can give rise to many of the types of patterns observed in electrophysiological recordings of corticotrophs. We compare the known mechanisms for CRH, AVP, and glucocorticoid actions and find that different ionic mechanisms can contribute in different but complementary ways to generate the complex time courses of CRH and AVP responses. In summary, our modeling suggests that corticotrophs have several mechanisms at their disposal to achieve their primary function of pacemaking depolarization and increased electrical activity in response to CRH and AVP. This finding is consistent with critical roles of electrical activity in function of these cells. In collaboration with the same group, we also examined common and diverse elements of ion channels and receptors underling electrical activity in secretory pituitary cells: corticotrophs, melanotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. All these cell types are electrically excitable, and voltage-gated calcium influx is the major trigger for their hormone secretion. Along with hormone intracellular content, G-protein-coupled receptor and ion channel expression can also be considered as defining cell type identity. While many aspects of the developmental and activity dependent regulation of hormone and G-protein-coupled receptor expression have been elucidated, much less is known about the regulation of the ion channels needed for excitation-secretion coupling in these cells. We compare the spontaneous and receptor-controlled patterns of electrical signaling among endocrine pituitary cell types, including insights gained from mathematical modeling. We argue that a common set of ionic currents unites these cells, while differential expression of another subset of ionic currents could underlie cell type-specific patterns. We demonstrate these ideas using a generic mathematical model, showing that it reproduces many observed features of pituitary electrical signaling. Mapping these observations to the developmental lineage suggests possible modes of regulation that may give rise to mature pituitary cell types. In collaboration with Dr. Zvi Naor, we also studied bleb formation in native pituitary gonadotrophs and gonadotroph derived LbetaT2 cells. The blebs appear within twominutes at a turnover rate of 2-3 blebs/min and last for at least 90min. Four experiments revealed that formation of the blebs requires active ERK1/2 and RhoA-ROCK but not active c-Src. Although the following ligands stimulate ERK1/2 in LbetaT2 cells: EGF>GnRH>PMA>cyclic adenosine monophosphate (cAMP), they produced little or no effect on bleb formation as compared to the robust effect of gonadotropin-releasing hormone (GnRH), the native agonist of these cells (GnRH>PMA>cAMP>EGF). These findings indicating that ERK1/2 is required but not sufficient for bleb formation possibly due to compartmentalization. Members of the above-mentioned signalosome are recruited to the blebs, some during bleb formation (GnRHR, c-Src, ERK1/2, focal adhesion kinase, paxillin, and tubulin), and some during bleb retraction (vinculin), while F-actin decorates the blebs during retraction. Fluorescence intensity measurements for these proteins across the cells showed higher intensity in the blebs vs. intracellular area. Moreover, GnRH induces blebs in primary cultures of rat pituitary cells and isolated mouse gonadotrophs in an ERK1/2-dependent manner. The novel signalosome-bleb pathway suggests that as with the signalosome, the blebs are apparently involved in cell migration. Hence, we have extended the potential candidates which are involved in the blebs life cycle in general and for the GnRH receptor in particular. We also continued our investigations on purinergic P2X receptor channel focusing on their functions. In collaboration with Dr. Ivan Milenkovic, we studied the role of these channels in electrical activity of maturing auditory neurons. Using slice recordings before hearing onset and in vivo recordings with iontophoretic drug applications after hearing onset, we show that cell-specific purinergic modulation follows a precise tonotopic pattern in the ventral cochlear nucleus of developing gerbils. In high-frequency regions, ATP responsiveness diminished before hearing onset. In low-to-mid frequency regions, ATP modulation persisted after hearing onset in a subset of low-frequency bushy cells (characteristic frequency<10kHz). Down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, thus suggesting a high-to-low frequency maturation pattern. Facilitated action potential generation, measured as higher firing frequency, shorter EPSP-AP delay in vivo, and shorter spike latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. Finally, by combining recordings and pharmacology in vivo, in slices, and in human embryonic kidney 293 cells, it was shown that the long-lasting change in intrinsic neuronal excitability is mediated by the P2X2/3R. We also studied allosteric regulation of P2X4 channels by ivermectin (IVM) in collaboration with Dr. Anmar Khadra. Treatment with IVM increases the efficacy of ATP to activate P2X4 channel, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the channel pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4 channels. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R channel kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.
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