The astroglial and stem cell functions of adult rat folliculostellate cells.

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.

Ppt1-deficiency dysregulates lysosomal Ca++ homeostasis contributing to pathogenesis in a mouse model of CLN1 disease.

Inactivating mutations in the PPT1 gene encoding palmitoyl-protein thioesterase-1 (PPT1) underlie the CLN1 disease, a devastating neurodegenerative lysosomal storage disorder. The mechanism of pathogenesis underlying CLN1 disease has remained elusive. PPT1 is a lysosomal enzyme, which catalyzes the removal of palmitate from S-palmitoylated proteins (constituents of ceroid lipofuscin) facilitating their degradation and clearance by lysosomal hydrolases. Thus, it has been proposed that Ppt1-deficiency leads to lysosomal accumulation of ceroid lipofuscin leading to CLN1 disease. While S-palmitoylation is catalyzed by palmitoyl acyltransferases (called ZDHHCs), palmitoyl-protein thioesterases (PPTs) depalmitoylate these proteins. We sought to determine the mechanism by which Ppt1-deficiency may impair lysosomal degradative function leading to infantile neuronal ceroid lipofuscinosis pathogenesis. Here, we report that in Ppt1-/- mice, which mimic CLN1 disease, low level of inositol 3-phosphate receptor-1 (IP3R1) that mediates Ca++ transport from the endoplasmic reticulum to the lysosome dysregulated lysosomal Ca++ homeostasis. Intriguingly, the transcription factor nuclear factor of activated T-cells, cytoplasmic 4 (NFATC4), which regulates IP3R1-expression, required S-palmitoylation for trafficking from the cytoplasm to the nucleus. We identified two palmitoyl acyltransferases, ZDHHC4 and ZDHHC8, which catalyzed S-palmitoylation of NFATC4. Notably, in Ppt1-/- mice, reduced ZDHHC4 and ZDHHC8 levels markedly lowered S-palmitoylated NFATC4 (active) in the nucleus, which inhibited IP3R1-expression, thereby dysregulating lysosomal Ca++ homeostasis. Consequently, Ca++ -dependent lysosomal enzyme activities were markedly suppressed. Impaired lysosomal degradative function impaired autophagy, which caused lysosomal storage of undigested cargo. Importantly, IP3R1-overexpression in Ppt1-/- mouse fibroblasts ameliorated this defect. Our results reveal a previously unrecognized role of Ppt1 in regulating lysosomal Ca++ homeostasis and suggest that this defect contributes to pathogenesis of CLN1 disease.

Cell-Type-Specific Expression Pattern of Proton-Sensing Receptors and Channels in Pituitary Gland.

In mammalian cells, extracellular protons act as orthosteric and allosteric ligands for multiple receptors and channels. The aim of this study is to identify proton sensors in the rat pituitary gland. qRT-PCR analysis indicated the expression of G-protein-coupled receptor 68 gene (Gpr68) and acid-sensing ion channel (ASIC) genes Asic1, Asic2, and Asic4 in anterior pituitary cells and Asic1 and Asic2 in immortalized GH3 pituitary cells. Asic1a and Asic2b were the dominant splice isoforms. Single anterior pituitary cell RNA sequencing and immunocytochemical analysis showed that nonexcitable folliculostellate cells express GPR68 gene and protein, whereas excitable secretory cells express ASIC genes and proteins. Asic1 was detected in all secretory cell types, Asic2 in gonadotrophs, thyrotrophs, and somatotrophs, and Asic4 in lactotrophs. Extracellular acidification activated two types of currents in a concentration-dependent manner: a fast-developing, desensitizing current with an estimated EC50-value of pH 6.7 and a slow-developing, non-desensitizing current that required a higher proton concentration for activation. The desensitizing current was abolished by removal of bath sodium and application of amiloride, a blocker of ASIC channels, whereas the non-desensitizing current was amiloride insensitive and voltage dependent. Activation of both currents increased the excitability of secretory pituitary cells, consistent with their potential physiological relevance in control of voltage-gated calcium influx and calcium-dependent cellular functions.

The sex-specific patterns of changes in hypothalamic-pituitary-gonadal axis during experimental autoimmune encephalomyelitis.

Multiple sclerosis develops during reproductive years in a sex-specific manner. Various neuroendocrine changes have been described in this inflammatory, demyelinating, and debilitating disease. We here aimed to determine the extent and sex specificity of alterations in the hypothalamic-pituitary-gonadal axis in the rat model of multiple sclerosis named experimental autoimmune encephalomyelitis. During the disease course, the hypothalamic tissue showed transient upregulation of inflammatory marker genes Gfap, Cd68, Ccl2, and Il1b in both sexes, but accompanied by sex-specific downregulation of Kiss1 (in females only) and Gnrh1 (in males only) expression. In females, the expression of gonadotrope-specific genes Lhb, Cga, and Gnrhr was also inhibited, accompanied by decreased basal but not stimulated serum luteinizing hormone levels and a transient arrest of the estrous cycle. In contrast, Fshb expression and serum progesterone levels were transiently elevated, findings consistent with the maintenance of the corpora lutea, and elevated immunohistochemical labeling of ovarian StAR, a rate limiting protein in steroidogenic pathway. In males, downregulation of Gnrhr expression and basal and stimulated serum luteinizing hormone and testosterone levels were accompanied by inhibited testicular StAR protein expression. We propose that inflammation of hypothalamic tissue downregulates Kiss1 and Gnrh1 expression in females and males, respectively, leading to sex-specific changes downstream the axis.

Interactions of Pannexin1 channels with purinergic and NMDA receptor channels.

Pannexins are a three-member family of vertebrate plasma membrane spanning molecules that have homology to the invertebrate gap junction forming proteins, the innexins. However, pannexins do not form gap junctions but operate as plasma membrane channels. The best-characterized member of these proteins, Pannexin1 (Panx1) was suggested to be functionally associated with purinergic P2X and N-methyl-D-aspartate (NMDA) receptor channels. Activation of these receptor channels by their endogenous ligands leads to cross-activation of Panx1 channels. This in turn potentiates P2X and NMDA receptor channel signaling. Two potentiation concepts have been suggested: enhancement of the current responses and/or sustained receptor channel activation by ATP released through Panx1 pore and adenosine generated by ectonucleotidase-dependent dephosphorylation of ATP. Here we summarize the current knowledge and hypotheses about interactions of Panx1 channels with P2X and NMDA receptor channels. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.

The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor 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 P2X4R. 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 kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

Opposing Roles of Calcium and Intracellular ATP on Gating of the Purinergic P2X2 Receptor Channel.

P2X2 receptors (P2X2R) exhibit a slow desensitization during the initial ATP application and a progressive, calcium-dependent increase in rates of desensitization during repetitive stimulation. This pattern is observed in whole-cell recordings from cells expressing recombinant and native P2X2R. However, desensitization is not observed in perforated-patched cells and in two-electrode voltage clamped oocytes. Addition of ATP, but not ATPγS or GTP, in the pipette solution also abolishes progressive desensitization, whereas intracellular injection of apyrase facilitates receptor desensitization. Experiments with injection of alkaline phosphatase or addition of staurosporine and ATP in the intracellular solution suggest a role for a phosphorylation-dephosphorylation in receptor desensitization. Mutation of residues that are potential phosphorylation sites identified a critical role of the S363 residue in the intracellular ATP action. These findings indicate that intracellular calcium and ATP have opposing effects on P2X2R gating: calcium allosterically facilitates receptor desensitization and ATP covalently prevents the action of calcium. Single cell measurements further revealed that intracellular calcium stays elevated after washout in P2X2R-expressing cells and the blockade of mitochondrial sodium/calcium exchanger lowers calcium concentrations during washout periods to basal levels, suggesting a role of mitochondria in this process. Therefore, the metabolic state of the cell can influence P2X2R gating.

Tonotopic action potential tuning of maturing auditory neurons through endogenous ATP.

KEY POINTS: Following the genetically controlled formation of neuronal circuits, early firing activity guides the development of sensory maps in the auditory, visual and somatosensory system. However, it is not clear whether the activity of central auditory neurons is specifically regulated depending on the position within the sensory map. In the ventral cochlear nucleus, the first central station along the auditory pathway, we describe a mechanism through which paracrine ATP signalling enhances firing in a cell-specific and tonotopically-determined manner. Developmental down-regulation of P2X2/3R currents along the tonotopic axis occurs simultaneously with an increase in AMPA receptor currents, suggesting a high-to-low frequency maturation pattern. Facilitated action potential (AP) generation, measured as higher firing rate, shorter EPSP-AP delay in vivo and shorter AP latency in slice experiments, is consistent with increased synaptic efficacy caused by ATP. The long lasting change in intrinsic neuronal excitability is mediated by the heteromeric P2X2/3 receptors. ABSTRACT: Synaptic refinement and strengthening are activity-dependent processes that establish orderly arranged cochleotopic maps throughout the central auditory system. The maturation of auditory brainstem circuits is guided by action potentials (APs) arising from the inner hair cells in the developing cochlea. The AP firing of developing central auditory neurons can be modulated by paracrine ATP signalling, as shown for the cochlear nucleus bushy cells and principal neurons in the medial nucleus of the trapezoid body. However, it is not clear whether neuronal activity may be specifically regulated with respect to the nuclear tonotopic position (i.e. sound frequency selectivity). 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< 10 kHz). 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 AP generation, measured as higher firing frequency, shorter EPSP-AP delay in vivo, and shorter AP 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.

Modeling the diversity of spontaneous and agonist-induced electrical activity in anterior pituitary corticotrophs.

Pituitary corticotrophs 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 and mathematical modeling to investigate possible sources of variability in patterns of spontaneous and agonist-induced corticotroph 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.NEW & NOTEWORTHY We and others recently demonstrated that the electrical activity and calcium dynamics of corticotrophs are strikingly diverse, both spontaneously and in response to the agonists CRH and AVP. Here we demonstrate this diversity with electrophysiological measurements and use mathematical modeling to investigate its possible sources. We compare the known mechanisms of agonist-induced activity in the model, showing how the context of ionic conductances dictates the effects of agonists even when their target is fixed.

Gigantism and acromegaly due to Xq26 microduplications and GPR101 mutation.

BACKGROUND: Increased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood. METHODS: We performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly. RESULTS: We observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein-coupled receptor, was overexpressed in patients' pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone-producing cells. CONCLUSIONS: We describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.).

Role of domain calcium in purinergic P2X2 receptor channel desensitization.

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied by a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using rat, mouse, and human receptors, we show here that two processes contribute to receptor desensitization: bath calcium-independent desensitization and calcium-dependent desensitization. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of the receptor but is pronounced in cells expressing shorter versions of receptors, indicating a role of the COOH terminus in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in bath ATP and calcium concentration-dependent manners. Experiments with substitution of bath Na(+) with N-methyl-d-glucamine (NMDG(+)), a large organic cation, indicate that receptor pore dilation is a calcium-independent process in contrast to receptor desensitization. A decrease in the driving force for calcium by changing the holding potential from -60 to +120 mV further indicates that calcium influx through the channel pores at least partially accounts for receptor desensitization. Experiments with various receptor chimeras also indicate that the transmembrane and/or intracellular domains of P2X2R are required for development of calcium-dependent desensitization and that a decrease in the amplitude of current slows receptor desensitization. Simultaneous calcium and current recording shows development of calcium-dependent desensitization without an increase in global intracellular calcium concentrations. Combined with experiments with clamping intrapipette concentrations of calcium at various levels, these experiments indicate that domain calcium is sufficient to establish calcium-dependent receptor desensitization in experiments with whole-cell recordings.

Allosteric regulation of the P2X4 receptor channel pore dilation.

Allosteric modulators of ligand-gated receptor channels induce conformational changes of the entire protein that alter potencies and efficacies for orthosteric ligands, expressed as the half maximal effective concentration (EC50) and maximum current amplitude, respectively. Here, we studied the influence of allostery on channel pore dilation, an issue not previously addressed. Experiments were done using the rat P2X4 receptor expressed in human embryonic kidney 293T cells and gated by adenosine 5'-triphosphate (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 EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) 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 P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.

Cell Type-Specific Sexual Dimorphism in Rat Pituitary Gene Expression During Maturation.

The most obvious functional differences between mammalian males and females are related to the control of reproductive physiology and include patterns of GnRH and gonadotropin release, the timing of puberty, sexual and social behavior, and the regulation of food intake and body weight. Using the rat as the best-studied mammalian model for maturation, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable Pomc profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in Gh1 and Prl profiles during embryonic to juvenile age but show the amplification of Prl expression in females and Gh1 expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized Lhb, Fshb, Cga, and Gnrhr expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental Tshb profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes.

cAMP-mediated stabilization of fusion pores in cultured rat pituitary lactotrophs.

Regulated exocytosis mediates the release of hormones and transmitters. The last step of this process is represented by the merger between the vesicle and the plasma membranes, and the formation of a fusion pore. Once formed, the initially stable and narrow fusion pore may reversibly widen (transient exocytosis) or fully open (full-fusion exocytosis). Exocytosis is typically triggered by an elevation in cytosolic calcium activity. However, other second messengers, such as cAMP, have been reported to modulate secretion. The way in which cAMP influences the transitions between different fusion pore states remains unclear. Here, hormone release studies show that prolactin release from isolated rat lactotrophs stimulated by forskolin, an activator of adenylyl cyclases, and by membrane-permeable cAMP analog (dbcAMP), exhibit a biphasic concentration dependency. Although at lower concentrations (2-10 µm forskolin and 2.5-5 mm dbcAMP) these agents stimulate prolactin release, an inhibition is measured at higher concentrations (50 µm forskolin and 10-15 mm dbcAMP). By using high-resolution capacitance (Cm) measurements, we recorded discrete increases in Cm, which represent elementary exocytic events. An elevation of cAMP leaves the frequency of full-fusion events unchanged while increasing the frequency of transient events. These exhibited a wider fusion pore as measured by increased fusion pore conductance and a prolonged fusion pore dwell time. The probability of observing rhythmic reopening of transient fusion pores was elevated by dbcAMP. In conclusion, cAMP-mediated stabilization of wide fusion pores prevents vesicles from proceeding to the full-fusion stage of exocytosis, which hinders vesicle content discharge at high cAMP concentrations.

Activation and regulation of purinergic P2X receptor channels.

Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.

Protein Tyrosine Phosphatase Receptors N and N2 Control Pituitary Melanotroph Development and POMC Expression.

The neuroendocrine marker genes Ptprn and Ptprn2 encode protein tyrosine phosphatase receptors N and N2, 2 members of protein tyrosine phosphatase receptors void of enzymatic activity, and whose function and mechanism of action have not been elucidated. To explore the role(s) of Ptprn and Ptprn2 on the hypothalamic-pituitary-adrenal axis, we used mice in which both genes were knocked out (DKO). The focus in this study was on corticotrophs and melanotrophs from the anterior and intermediate lobes of the pituitary gland, respectively. In both sexes, DKO caused an increase in the expression of the corticotroph/melanotroph genes Pomc and Tbx19 and the melanotroph-specific gene Pax7. We also found in vivo and in vitro increased synthesis and release of beta-endorphin, alpha-melanocyte-stimulating hormone, and ACTH in DKO mice, which was associated with increased serum corticosterone levels and adrenal mass. DKO also increased the expression of other melanotroph-specific genes, but not corticotroph-specific genes. The dopaminergic pathway in the hypothalamus and dopaminergic receptors in melanotrophs were not affected in DKO mice. However, hyperplasia of the intermediate lobe was observed in DKO females and males, accompanied by increased proopiomelanocortin immunoreactivity per cell. These results indicate that protein tyrosine phosphatase receptor type N contributes to hypothalamic-pituitary-adrenal function by being involved in processes governing postnatal melanotroph development and Pomc expression.

Dual gating mechanism and function of P2X7 receptor channels.

The ATP-gated P2X7 receptor channel (P2X7R) 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. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.

The opposite roles of glucocorticoid and α1-adrenergic receptors in stress triggered apoptosis of rat Leydig cells.

The stress-induced initiation of proapoptotic signaling in Leydig cells is relatively well defined, but the duration of this signaling and the mechanism(s) involved in opposing the stress responses have not been addressed. In this study, immobilization stress (IMO) was applied for 2 h daily, and animals were euthanized immediately after the first (IMO1), second (IMO2), and 10th (IMO10) sessions. In IMO1 and IMO2 rats, serum corticosterone and adrenaline were elevated, whereas serum androgens and mRNA transcription of insulin-like factor-3 in Leydig cells were inhibited. Reduced oxygen consumption and the mitochondrial membrane potential coupled with a leak of cytochrome c from mitochondria and increased caspase-9 expression, caspase-3 activity, and number of apoptotic Leydig cells was also observed. Corticosterone and adrenaline were also elevated in IMO10 rats but were accompanied with a partial recovery of androgen secretion and normalization of insulin-like factor-3 transcription coupled with increased cytochrome c expression, abolition of proapoptotic signaling, and normalization of the apoptotic events. Blockade of intratesticular glucocorticoid receptors diminished proapoptotic effects without affecting antiapoptotic effects, whereas blockade of intratesticular α(1)-adrenergic receptors diminished the antiapoptotic effects without affecting proapoptotic effects. These results confirmed a critical role of glucocorticoids in mitochondria-dependent apoptosis and showed for the first time the relevance of stress-induced upregulation of α(1)-adrenergic receptor expression in cell apoptotic resistance to repetitive IMOs. The opposite role of two hormones in control of the apoptotic rate in Leydig cells also provides a rationale for a partial recovery of androgen production in chronically stressed animals.

PI(4,5)P2-dependent and -independent roles of PI4P in the control of hormone secretion by pituitary cells.

Plasma membrane and organelle membranes are home to seven phosphoinositides, an important class of low-abundance anionic signaling lipids that contribute to cellular functions by recruiting cytoplasmic proteins or interacting with the cytoplasmic domains of membrane proteins. Here, we briefly review the functions of three phosphoinositides, PI4P, PI(4,5)P2, and PI(3,4,5)P3, in cellular signaling and exocytosis, focusing on hormone-producing pituitary cells. PI(4,5)P2, acting as a substrate for phospholipase C, plays a key role in the control of pituitary cell functions, including hormone synthesis and secretion. PI(4,5)P2 also acts as a substrate for class I PI3-kinases, leading to the generation of two intracellular messengers, PI(3,4,5)P3 and PI(3,4)P2, which act through their intracellular effectors, including Akt. PI(4,5)P2 can also influence the release of pituitary hormones acting as an intact lipid to regulate ion channel gating and concomitant calcium signaling, as well as the exocytic pathway. Recent findings also show that PI4P is not only a precursor of PI(4,5)P2, but also a key signaling molecule in many cell types, including pituitary cells, where it controls hormone secretion in a PI(4,5)P2-independent manner.

Common and female-specific roles of protein tyrosine phosphatase receptors N and N2 in mice reproduction.

Simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2, which encode the protein tyrosine phosphatase receptors N and N2, causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary-gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions.