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Papers by Stanko Stojilkovic

Annals of the New York Academy of Sciences, 2009

G i/o protein-coupled receptors, signaling through G protein-dependent and proteinindependent pat... more G i/o protein-coupled receptors, signaling through G protein-dependent and proteinindependent pathways, have prominent effects on secretion by modulating calcium signaling and regulating the size of the releasable secretory pool, the rates of exocytosis and endocytosis, and de novo synthesis. Pituitary cells fire action potentials spontaneously, and the associated calcium influx is sufficient to maintain prolactin (PRL) release but not gonadotropin release at high and steady levels for many hours. Such secretion, termed intrinsic, spontaneous, or basal, reflects fusion of secretory vesicles triggered by the cell type-specific pattern of action potentials. In lactotrophs, activation of endothelin ET A and dopamine D 2 receptors causes inhibition of spontaneous electrical activity and basal adenylyl cyclase activity accompanied with inhibition of basal PRL release. Agonist-induced inhibition of cAMP production and firing of action potentials is abolished in cells with blocked pertussis toxin (PTX)-sensitive G i/o signaling pathway. However, agonist-induced inhibition of PRL release is only partially relieved in such treated cells, indicating that both receptors also inhibit exocytosis downstream of cAMP/calcium signaling. The PTX-insensitive step in agonist-induced inhibition of PRL release is not affected by inhibition of phosphoinositide 3-kinase and glycogen synthase kinase-3 but is partially rescued by downregulation of the G z α expression. Thus, ET A and D 2 receptors inhibit basal PRL release not only by blocking electrical activity but also by desensitizing calcium-secretion coupling.

Nature Medicine, 2003

C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylat... more C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)′ 2 -IVIG and irrelevant human monoclonal antibody. C3a-and C5a-induced thromboxane (TXB 2 ) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)′ 2 -IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)′ 2. This binding could interfere with the role of C3a and C5a in inflammation.

Journal of Neuroscience, 2013

Regulated exocytosis mediates the release of hormones and transmitters. The last step of this pro... more 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 M forskolin and 2.5-5 mM dbcAMP) these agents stimulate prolactin release, an inhibition is measured at higher concentrations (50 M forskolin and 10 -15 mM dbcAMP). By using high-resolution capacitance (C m ) measurements, we recorded discrete increases in C m , 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.

Molecular Endocrinology, 2006

Pituitary lactotrophs in vitro fire extracellular Ca 2؉dependent action potentials spontaneously ... more Pituitary lactotrophs in vitro fire extracellular Ca 2؉dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca 2؉ influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The

Role of nonselective cation channels in spontaneous and protein kinase A-stimulated calcium signaling in pituitary cells

American Journal of Physiology Endocrinology and Metabolism, May 17, 2011

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity ... more Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated Ca(2+) influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 > TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium.

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons

Biochemistry and Cell Biology, Jan 24, 2011

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult ... more Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.

Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs

Endocrinology, Apr 1, 1994

The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induc... more The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.

Spontaneous and Receptor-Controlled Soluble Guanylyl Cyclase Activity in Anterior Pituitary Cells

Molecular Endocrinology, Jul 1, 2001

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but i... more Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.

GH feedback on its own secretion at the pituitary level has been previously reported, but the mec... more GH feedback on its own secretion at the pituitary level has been previously reported, but the mechanisms involved have not been elucidated. Here we examined the autocrine/paracrine effects of GH on GH synthesis using grass carp pituitary cells as a cell model. GH receptors were identified in carp somatotrophs, and their activation by exogenous GH increased steady-state GH mRNA levels and GH production. Removal of endogenous GH by immunoneutralization using GH antiserum inhibited basal as well as stimulated GH mRNA expression induced by GHreleasing factors in fish, including GnRH, apomorphine, and pituitary adenylate cyclaseactivating polypeptide-38. Cytosolic mature GH mRNA levels were elevated by GH treatment and reduced by GH antiserum, whereas nuclear GH primary transcripts were almost undetectable after GH immunoneutralization. Inhibition of Janus kinase-2 (JAK2), phosphoinositide 3-kinase, and MAPK also abolished GH-induced steady-state GH mRNA expression. GH immunoneutralization in pituitary cells pretreated with actinomycin D induced a marked decrease in the half-life of GH mRNA, indicating that the clearance of GH transcripts could be enhanced by removing endogenous GH. These results provide evidence that GH can serve as a novel intrapituitary autocrine/paracrine factor maintaining GH gene expression in somatotrophs, and this action is mediated by JAK2/MAPK and JAK2/phosphoinositide 3-kinase cascades coupled to GH receptors.

Endocrinology, Apr 1, 2000

Cultured rat pituitary cells and immortalized pituitary gonadotrophs (␣T3-1 cells) express specif... more Cultured rat pituitary cells and immortalized pituitary gonadotrophs (␣T3-1 cells) express specific messenger RNA transcripts for GnRH and exhibit positive immunostaining for the GnRH peptide. Each cell type released GnRH during both static culture and perifusion, albeit in lesser amounts than cultured hypothalamic cells and GT1-7 neurons. In perifused pituitary cells, exposure to a GnRH agonist stimulated the release of GnRH as well as LH. In contrast, treatment with a GnRH receptor antagonist or with GnRH antiserum decreased basal LH release. In pituitary cell cultures, a small proportion of gonadotrophs exhibited high amplitude and low frequency baseline Ca 2ϩ oscillations in the absence of GnRH stimulation. Such spontaneous oscillations were comparable to those induced by pico-molar concentrations of GnRH and could be abolished by treatment with a GnRH antagonist. These in vitro findings indicate that locally produced GnRH causes low level activation of pituitary GnRH receptors, induces spontaneous intracellular Ca 2ϩ oscillations, and contributes to basal LH secretion in cultured pituitary cells. In vivo, such autocrine or paracrine actions of pituitary-derived GnRH could provide a mechanism for the maintenance of optimal responsiveness of the gonadotrophs to pulses of GnRH arising in the hypothalamus. The presence and actions of GnRH in the anterior pituitary gland, the major site of expression of GnRH receptors, suggest that local regulatory effects of the neuropeptide could supplement the primary hypothalamic mechanism for the control of episodic gonadotropin se-

Differential Expression of Ionic Channels in Rat Anterior Pituitary Cells

Molecular Endocrinology, Aug 1, 2001

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific pattern... more Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.

Mol Endocrinol, 2005

Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but... more Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but their biophysical properties and roles in spontaneous and receptor-controlled electrical activity have not been characterized. Here we focused on extracellular ATP actions in gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the Ca2+-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, nondesensitizing, and slow deactivating currents. In contrast, ATP induced nonoscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not Ca2+-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations and LH release in an extracellular Ca2+-dependent manner. These inositol 1,4,5-triphosphate-dependent oscillations were facilitated, slowed, or stopped, depending of ATP concentration, the time of its application, and the level of Ca2+ content in intracellular stores. These results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion.

Nitric Oxide Inhibits Prolactin Secretion in Pituitary Cells Downstream of Voltage-Gated Calcium Influx

Endocrinology, Jul 1, 2013

The coupling between nitric oxide (NO)-cGMP signaling pathway and prolactin (PRL) release in pitu... more The coupling between nitric oxide (NO)-cGMP signaling pathway and prolactin (PRL) release in pituitary lactotrophs has been established previously. However, the messenger that mediates the action of this signaling pathway on hormone secretion and the secretory mechanism affected, calcium dependent or independent, have not been identified. In cultured pituitary cells, basal PRL release was controlled by spontaneous voltage-gated calcium influx and was further enhanced by depolarization of cells and stimulation with TRH. Inhibition of constitutively expressed neuronal NO synthase decreased NO and cGMP levels and increased basal PRL release. The addition of a slowly releasable NO donor increased cGMP levels and inhibited basal PRL release in a time-dependent manner. Expression of inducible NO synthase also increased NO and cGMP levels and inhibited basal, depolarization-induced, and TRH-induced PRL release, whereas inhibition of this enzyme decreased NO and cGMP production and recovered PRL release. None of these treatments affected spontaneous and stimulated voltage-gated calcium influx. At basal NO levels, the addition of permeable cGMP analogs did not inhibit PRL secretion. At elevated NO levels, inhibition of cGMP production and facilitation of its degradation did not reverse inhibited PRL secretion. These experiments indicate that NO inhibits calcium-dependent PRL secretion in a cGMP-independent manner and downstream of voltage-gated calcium influx.

Gonadotropin-releasing hormone neurons Intrinsic pulsatility and receptor-mediated regulation

Trends in Endocrinology and Metabolism, Jul 31, 1994

The pulsatile pattern of gonadotropin-releasing hormone (GnRH) release from the hypothalamus is d... more The pulsatile pattern of gonadotropin-releasing hormone (GnRH) release from the hypothalamus is driven by a functionally interconnected and synchronized network of GnRH neurons termed the GnRH pulse generator. Several recent observations have revealed that immortalized GnRH neurons can generate an episodic pattern of GnRH release when cultured in the absence of other cell types. The in vitro operation of the pulse generator depends on the development of synaptic contacts among GnRH neurons, the electrical properties of individual GnRH neurons, and the GnRH-induced modulation of its secretory mechanism. The expression o f several other receptors by GnRH neurons provides the means for integrated regulation of pulse generator activity from without the network by agonists including glutamate, GABA, endothelin, and catecholamines.

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature... more Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature of this event is poorly understood. We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perifused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently-labelled atrial natriuretic peptide cargo from ~2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of ~10% of vesicles/cell, with different unloading/loading time-course of the two fluorescent probes. In cell-attached studies, discrete changes in membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that in hypotonicity-stimulated lactotrophs transient vesicle fusion mediates hormone release.

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

Biology of Reproduction, 2015

The most obvious functional differences between mammalian males and females are related to the co... more 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.

Participation of voltage-sensitive calcium channels in pituitary hormone release

The Journal of biological chemistry, Jan 15, 1988

The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of ra... more The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of rat anterior pituitary cells. The basal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH), and adrenocorticotropin (ACTH) secretion were independent of extracellular Ca2+ concentration ([Ca2+]e). In contrast, the basal levels of growth hormone (GH) and prolactin (PRL) release showed dose-dependent increases with elevation of [Ca2+]e, and were abolished by Ca2+-channel antagonists. Under Ca2+-deficient conditions, BaCl2 mimicked the effects of calcium on PRL and GH release but with a marked increase in potency, and also increased basal LH and FSH release in a dose-dependent manner. In the presence of normal [Ca2+]e, depolarization with K+ maximally increased cytosolic [Ca2+] ([Ca2+]i) from 100 to 185 nM and elevated LH, FSH, TSH, ACTH, PRL, and GH release by 7-, 5-, 4-, 3-, 2-, and 1.5-fold, respectively. These effects of KCl were abolished in Ca2+-de...

Role of Domain Calcium in Purinergic P2X2 Receptor Channel Desensitization

American journal of physiology. Cell physiology, Jan 11, 2015

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompan... more Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied with a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using, rat, mouse and human receptors, here we show that two processes contribute to receptor desensitization: bath calcium-independent and -dependent. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of receptor, but is pronounced in cells expressing shorter versions of receptors, indicating a role of C terminal in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in ATP- and bath calcium-concentration dependent manner. Experiments with substitution of bath Na(+) with NMDG(+), a large organic cation, indicate that receptor pore dilation is a calcium-independent p...

P2X receptor channels in endocrine glands

Wiley interdisciplinary reviews. Membrane transport and signaling, 2013

The endocrine system is the system of ductless glands and single cells that synthetize hormones a... more The endocrine system is the system of ductless glands and single cells that synthetize hormones and release them directly into the bloodstream. Regulation of endocrine system is very complex and ATP and its degradable products ADP and adenosine contribute to its regulation acting as extracellular messengers for purinergic receptors. These include P2X receptors, a family of ligand-gated ion channels which expression and roles in endocrine tissues are reviewed here. There are seven mammalian purinergic receptor subunits, denoted P2X1 through P2X7, and the majority of these subunits are also expressed in secretory and non-secretory cells of endocrine system. Functional channels have been identified in the neuroendocrine hypothalamus, the posterior and anterior pituitary, the thyroid gland, the adrenals, the endocrine pancreas, the gonads and the placenta. Native channels are capable of promoting calcium influx through its pore in both excitable and non-excitable cells, as well as of in...

Biophysical basis of pituitary cell type-specific Ca2+ signaling-secretion coupling

Trends in endocrinology and metabolism: TEM

All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical act... more All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical activity. Somatotrophs and lactotrophs fire plateau-bursting action potentials, which generate Ca2+ signals of sufficient amplitude to trigger hormone release. Gonadotrophs also fire action potentials spontaneously, but as single, high-amplitude spikes with limited ability to promote Ca2+ influx and secretion. However, Ca2+ mobilization in gonadotrophs transforms single spiking into plateau-bursting-type electrical activity and triggers secretion. Patch clamp analysis revealed that somatotrophs and lactotrophs, but not gonadotrophs, express BK (big)-type Ca2+-controlled K+ channels, activation of which is closely associated with voltage-gated Ca2+ influx. Conversely, pituitary gonadotrophs express SK (small)-type Ca2+-activated K+ channels that are colocalized with intracellular Ca2+ release sites. Activation of both channels is crucial for plateau-bursting-type rhythmic electrical activity...

Annals of the New York Academy of Sciences, 2009

G i/o protein-coupled receptors, signaling through G protein-dependent and proteinindependent pat... more G i/o protein-coupled receptors, signaling through G protein-dependent and proteinindependent pathways, have prominent effects on secretion by modulating calcium signaling and regulating the size of the releasable secretory pool, the rates of exocytosis and endocytosis, and de novo synthesis. Pituitary cells fire action potentials spontaneously, and the associated calcium influx is sufficient to maintain prolactin (PRL) release but not gonadotropin release at high and steady levels for many hours. Such secretion, termed intrinsic, spontaneous, or basal, reflects fusion of secretory vesicles triggered by the cell type-specific pattern of action potentials. In lactotrophs, activation of endothelin ET A and dopamine D 2 receptors causes inhibition of spontaneous electrical activity and basal adenylyl cyclase activity accompanied with inhibition of basal PRL release. Agonist-induced inhibition of cAMP production and firing of action potentials is abolished in cells with blocked pertussis toxin (PTX)-sensitive G i/o signaling pathway. However, agonist-induced inhibition of PRL release is only partially relieved in such treated cells, indicating that both receptors also inhibit exocytosis downstream of cAMP/calcium signaling. The PTX-insensitive step in agonist-induced inhibition of PRL release is not affected by inhibition of phosphoinositide 3-kinase and glycogen synthase kinase-3 but is partially rescued by downregulation of the G z α expression. Thus, ET A and D 2 receptors inhibit basal PRL release not only by blocking electrical activity but also by desensitizing calcium-secretion coupling.

Nature Medicine, 2003

C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylat... more C3b and C4b. We tested the hypothesis that exogenous immunoglobulin molecules also bind anaphylatoxins C3a and C5a, thereby neutralizing their pro-inflammatory effects. Single-cell calcium measurements in HMC-1 human mast cells showed that a rise in intracellular calcium caused by C3a and C5a was inhibited in a concentration-dependent manner by IVIG, F(ab)′ 2 -IVIG and irrelevant human monoclonal antibody. C3a-and C5a-induced thromboxane (TXB 2 ) generation and histamine release from HMC-1 cells and whole-blood basophils were also suppressed by exogenous immunoglobulins. In a mouse model of asthma, immunoglobulin treatment reduced cellular migration to the lung. Lethal C5a-mediated circulatory collapse in pigs was prevented by pretreatment with F(ab)′ 2 -IVIG. Molecular modeling, surface plasmon resonance (SPR) and western blot analyses suggested a physical association between anaphylatoxins and the constant region of F(ab)′ 2. This binding could interfere with the role of C3a and C5a in inflammation.

Journal of Neuroscience, 2013

Regulated exocytosis mediates the release of hormones and transmitters. The last step of this pro... more 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 M forskolin and 2.5-5 mM dbcAMP) these agents stimulate prolactin release, an inhibition is measured at higher concentrations (50 M forskolin and 10 -15 mM dbcAMP). By using high-resolution capacitance (C m ) measurements, we recorded discrete increases in C m , 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.

Molecular Endocrinology, 2006

Pituitary lactotrophs in vitro fire extracellular Ca 2؉dependent action potentials spontaneously ... more Pituitary lactotrophs in vitro fire extracellular Ca 2؉dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca 2؉ influx (VGCI) is sufficient to maintain basal prolactin (PRL) secretion high and steady. Numerous plasma membrane channels have been characterized in these cells, but the mechanism underlying their pacemaking activity is still not known. Here we studied the relevance of cyclic nucleotide signaling pathways in control of pacemaking, VGCI, and PRL release. In mixed anterior pituitary cells, both VGCI-inhibitable and -insensitive adenylyl cyclase (AC) subtypes contributed to the basal cAMP production, and soluble guanylyl cyclase was exclusively responsible for basal cGMP production. Inhibition of basal AC activity, but not soluble guanylyl cyclase activity, reduced PRL release. In contrast, forskolin stimulated cAMP and cGMP production as well as pacemaking, VGCI, and PRL secretion. Elevation in cAMP and cGMP levels by inhibition of phosphodiesterase activity was also accompanied with increased PRL release. The AC inhibitors attenuated forskolin-stimulated cyclic nucleotide production, VGCI, and PRL release. The

Role of nonselective cation channels in spontaneous and protein kinase A-stimulated calcium signaling in pituitary cells

American Journal of Physiology Endocrinology and Metabolism, May 17, 2011

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity ... more Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated Ca(2+) influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 > TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium.

Autocrine regulation of calcium influx and gonadotropin-releasing hormone secretion in hypothalamic neurons

Biochemistry and Cell Biology, Jan 24, 2011

Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult ... more Gonadotropin-releasing hormone (GnRH) receptors are expressed in hypothalamic tissues from adult rats, cultured fetal hypothalamic cells, and immortalized GnRH-secreting neurons (GT1 cells). Their activation by GnRH agonists leads to an overall increase in the extracellular Ca2+-dependent pulsatile release of GnRH. Electrophysiological studies showed that GT1 cells exhibit spontaneous, extracellular Ca2+-dependent action potentials, and that their inward currents include Na+, T-type and L-type Ca2+ components. Several types of potassium channels, including apamin-sensitive Ca2+-controlled potassium (SK) channels, are also expressed in GT1 cells. Activation of GnRH receptors leads to biphasic changes in intracellular Ca2+ concentration ([Ca2+]i), with an early and extracellular Ca2+-independent peak and a sustained and extracellular Ca2+-dependent plateau phase. During the peak [Ca2+]i response, electrical activity is abolished due to transient hyperpolarization that is mediated by SK channels. This is followed by sustained depolarization and resumption of firing with increased spike frequency and duration. The agonist-induced depolarization and increased firing are independent of [Ca2+]i and are not mediated by inhibition of K+ currents, but by facilitation of a voltage-insensitive and store depletion-activated Ca2+-conducting inward current. The dual control of pacemaker activity by SK and store depletion-activated Ca2+ channels facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process accounts for the autoregulatory action of GnRH on its release from hypothalamic neurons.

Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs

Endocrinology, Apr 1, 1994

The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induc... more The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.

Spontaneous and Receptor-Controlled Soluble Guanylyl Cyclase Activity in Anterior Pituitary Cells

Molecular Endocrinology, Jul 1, 2001

Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but i... more Nitric oxide (NO)-dependent soluble guanylyl cyclase (sGC) is operative in mammalian cells, but its presence and the role in cGMP production in pituitary cells have been incompletely characterized. Here we show that sGC is expressed in pituitary tissue and dispersed cells, enriched lactotrophs and somatotrophs, and GH(3) immortalized cells, and that this enzyme is exclusively responsible for cGMP production in unstimulated cells. Basal sGC activity was partially dependent on voltage-gated calcium influx, and both calcium-sensitive NO synthases (NOS), neuronal and endothelial, were expressed in pituitary tissue and mixed cells, enriched lactotrophs and somatotrophs, and GH(3) cells. Calcium-independent inducible NOS was transiently expressed in cultured lactotrophs and somatotrophs after the dispersion of cells, but not in GH(3) cells and pituitary tissue. This enzyme participated in the control of basal sGC activity in cultured pituitary cells. The overexpression of inducible NOS by lipopolysaccharide + interferon-gamma further increased NO and cGMP levels, and the majority of de novo produced cGMP was rapidly released. Addition of an NO donor to perifused pituitary cells also led to a rapid cGMP release. Calcium-mobilizing agonists TRH and GnRH slightly increased basal cGMP production, but only when added in high concentrations. In contrast, adenylyl cyclase agonists GHRH and CRF induced a robust increase in cGMP production, with EC(50)s in the physiological concentration range. As in cells overexpressing inducible NOS, the stimulatory action of GHRH and CRF was preserved in cells bathed in calcium-deficient medium, but was not associated with a measurable increase in NO production. These results indicate that sGC is present in secretory anterior pituitary cells and is regulated in an NO-dependent manner through constitutively expressed neuronal and endothelial NOS and transiently expressed inducible NOS, as well as independently of NO by adenylyl cyclase coupled-receptors.

GH feedback on its own secretion at the pituitary level has been previously reported, but the mec... more GH feedback on its own secretion at the pituitary level has been previously reported, but the mechanisms involved have not been elucidated. Here we examined the autocrine/paracrine effects of GH on GH synthesis using grass carp pituitary cells as a cell model. GH receptors were identified in carp somatotrophs, and their activation by exogenous GH increased steady-state GH mRNA levels and GH production. Removal of endogenous GH by immunoneutralization using GH antiserum inhibited basal as well as stimulated GH mRNA expression induced by GHreleasing factors in fish, including GnRH, apomorphine, and pituitary adenylate cyclaseactivating polypeptide-38. Cytosolic mature GH mRNA levels were elevated by GH treatment and reduced by GH antiserum, whereas nuclear GH primary transcripts were almost undetectable after GH immunoneutralization. Inhibition of Janus kinase-2 (JAK2), phosphoinositide 3-kinase, and MAPK also abolished GH-induced steady-state GH mRNA expression. GH immunoneutralization in pituitary cells pretreated with actinomycin D induced a marked decrease in the half-life of GH mRNA, indicating that the clearance of GH transcripts could be enhanced by removing endogenous GH. These results provide evidence that GH can serve as a novel intrapituitary autocrine/paracrine factor maintaining GH gene expression in somatotrophs, and this action is mediated by JAK2/MAPK and JAK2/phosphoinositide 3-kinase cascades coupled to GH receptors.

Endocrinology, Apr 1, 2000

Cultured rat pituitary cells and immortalized pituitary gonadotrophs (␣T3-1 cells) express specif... more Cultured rat pituitary cells and immortalized pituitary gonadotrophs (␣T3-1 cells) express specific messenger RNA transcripts for GnRH and exhibit positive immunostaining for the GnRH peptide. Each cell type released GnRH during both static culture and perifusion, albeit in lesser amounts than cultured hypothalamic cells and GT1-7 neurons. In perifused pituitary cells, exposure to a GnRH agonist stimulated the release of GnRH as well as LH. In contrast, treatment with a GnRH receptor antagonist or with GnRH antiserum decreased basal LH release. In pituitary cell cultures, a small proportion of gonadotrophs exhibited high amplitude and low frequency baseline Ca 2ϩ oscillations in the absence of GnRH stimulation. Such spontaneous oscillations were comparable to those induced by pico-molar concentrations of GnRH and could be abolished by treatment with a GnRH antagonist. These in vitro findings indicate that locally produced GnRH causes low level activation of pituitary GnRH receptors, induces spontaneous intracellular Ca 2ϩ oscillations, and contributes to basal LH secretion in cultured pituitary cells. In vivo, such autocrine or paracrine actions of pituitary-derived GnRH could provide a mechanism for the maintenance of optimal responsiveness of the gonadotrophs to pulses of GnRH arising in the hypothalamus. The presence and actions of GnRH in the anterior pituitary gland, the major site of expression of GnRH receptors, suggest that local regulatory effects of the neuropeptide could supplement the primary hypothalamic mechanism for the control of episodic gonadotropin se-

Differential Expression of Ionic Channels in Rat Anterior Pituitary Cells

Molecular Endocrinology, Aug 1, 2001

Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific pattern... more Secretory anterior pituitary cells are of the same origin, but exhibit cell type-specific patterns of spontaneous intracellular Ca2+ signaling and basal hormone secretion. To understand the underlying ionic mechanisms mediating these differences, we compared the ionic channels expressed in somatotrophs, lactotrophs, and gonadotrophs from randomly cycling female rats under identical cell culture and recording conditions. Our results indicate that a similar group of ionic channels are expressed in each cell type, including transient and sustained voltage-gated Ca2+ channels, tetrodotoxin-sensitive Na+ channels, transient and delayed rectifying K+ channels, and multiple Ca2+ -sensitive K+ channel subtypes. However, there were marked differences in the expression levels of some of the ionic channels. Specifically, lactotrophs and somatotrophs exhibited low expression levels of tetrodotoxin-sensitive Na+ channels and high expression levels of the large-conductance, Ca2+ -activated K+ channel compared with those observed in gonadotrophs. In addition, functional expression of the transient K+ channel was much higher in lactotrophs and gonadotrophs than in somatotrophs. Finally, the expression of the transient voltage-gated Ca2+ channels was higher in somatotrophs than in lactotrophs and gonadotrophs. These results indicate that there are cell type-specific patterns of ionic channel expression, which may be of physiological significance for the control of Ca2+ homeostasis and secretion in unstimulated and receptor-stimulated anterior pituitary cells.

Mol Endocrinol, 2005

Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but... more Anterior pituitary cells release ATP and express several subtypes of purinergic P2 receptors, but their biophysical properties and roles in spontaneous and receptor-controlled electrical activity have not been characterized. Here we focused on extracellular ATP actions in gonadotrophs from embryonic, neonatal, and adult rats. In cells from all three age groups, the Ca2+-mobilizing agonist GnRH induced oscillatory, hyperpolarizing, nondesensitizing, and slow deactivating currents. In contrast, ATP induced nonoscillatory, depolarizing, slowly desensitizing, and rapidly deactivating current, indicating that these cells express cation-conducting P2X channels but not Ca2+-mobilizing P2Y receptors. The amplitudes of P2X current response and the rates of receptor desensitization were dependent on ATP concentration. The biophysical and pharmacological properties of P2X currents were consistent with the expression of P2X2 subtype of channels in these cells. ATP-induced rapid depolarization of gonadotrophs lead to initiation of firing in quiescent cells, an increase in the frequency of action potentials in spontaneously active cells, and a transient stimulation of LH release. ATP also influenced GnRH-induced current and membrane potential oscillations and LH release in an extracellular Ca2+-dependent manner. These inositol 1,4,5-triphosphate-dependent oscillations were facilitated, slowed, or stopped, depending of ATP concentration, the time of its application, and the level of Ca2+ content in intracellular stores. These results indicate that, in gonadotrophs, P2X receptors could operate as pacemaking channels and modulators of GnRH-controlled electrical activity and secretion.

Nitric Oxide Inhibits Prolactin Secretion in Pituitary Cells Downstream of Voltage-Gated Calcium Influx

Endocrinology, Jul 1, 2013

The coupling between nitric oxide (NO)-cGMP signaling pathway and prolactin (PRL) release in pitu... more The coupling between nitric oxide (NO)-cGMP signaling pathway and prolactin (PRL) release in pituitary lactotrophs has been established previously. However, the messenger that mediates the action of this signaling pathway on hormone secretion and the secretory mechanism affected, calcium dependent or independent, have not been identified. In cultured pituitary cells, basal PRL release was controlled by spontaneous voltage-gated calcium influx and was further enhanced by depolarization of cells and stimulation with TRH. Inhibition of constitutively expressed neuronal NO synthase decreased NO and cGMP levels and increased basal PRL release. The addition of a slowly releasable NO donor increased cGMP levels and inhibited basal PRL release in a time-dependent manner. Expression of inducible NO synthase also increased NO and cGMP levels and inhibited basal, depolarization-induced, and TRH-induced PRL release, whereas inhibition of this enzyme decreased NO and cGMP production and recovered PRL release. None of these treatments affected spontaneous and stimulated voltage-gated calcium influx. At basal NO levels, the addition of permeable cGMP analogs did not inhibit PRL secretion. At elevated NO levels, inhibition of cGMP production and facilitation of its degradation did not reverse inhibited PRL secretion. These experiments indicate that NO inhibits calcium-dependent PRL secretion in a cGMP-independent manner and downstream of voltage-gated calcium influx.

Gonadotropin-releasing hormone neurons Intrinsic pulsatility and receptor-mediated regulation

Trends in Endocrinology and Metabolism, Jul 31, 1994

The pulsatile pattern of gonadotropin-releasing hormone (GnRH) release from the hypothalamus is d... more The pulsatile pattern of gonadotropin-releasing hormone (GnRH) release from the hypothalamus is driven by a functionally interconnected and synchronized network of GnRH neurons termed the GnRH pulse generator. Several recent observations have revealed that immortalized GnRH neurons can generate an episodic pattern of GnRH release when cultured in the absence of other cell types. The in vitro operation of the pulse generator depends on the development of synaptic contacts among GnRH neurons, the electrical properties of individual GnRH neurons, and the GnRH-induced modulation of its secretory mechanism. The expression o f several other receptors by GnRH neurons provides the means for integrated regulation of pulse generator activity from without the network by agonists including glutamate, GABA, endothelin, and catecholamines.

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature... more Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature of this event is poorly understood. We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perifused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently-labelled atrial natriuretic peptide cargo from ~2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of ~10% of vesicles/cell, with different unloading/loading time-course of the two fluorescent probes. In cell-attached studies, discrete changes in membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that in hypotonicity-stimulated lactotrophs transient vesicle fusion mediates hormone release.

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

Biology of Reproduction, 2015

The most obvious functional differences between mammalian males and females are related to the co... more 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.

Participation of voltage-sensitive calcium channels in pituitary hormone release

The Journal of biological chemistry, Jan 15, 1988

The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of ra... more The role of extracellular Ca2+ in pituitary hormone release was studied in primary cultures of rat anterior pituitary cells. The basal levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyrotropin (TSH), and adrenocorticotropin (ACTH) secretion were independent of extracellular Ca2+ concentration ([Ca2+]e). In contrast, the basal levels of growth hormone (GH) and prolactin (PRL) release showed dose-dependent increases with elevation of [Ca2+]e, and were abolished by Ca2+-channel antagonists. Under Ca2+-deficient conditions, BaCl2 mimicked the effects of calcium on PRL and GH release but with a marked increase in potency, and also increased basal LH and FSH release in a dose-dependent manner. In the presence of normal [Ca2+]e, depolarization with K+ maximally increased cytosolic [Ca2+] ([Ca2+]i) from 100 to 185 nM and elevated LH, FSH, TSH, ACTH, PRL, and GH release by 7-, 5-, 4-, 3-, 2-, and 1.5-fold, respectively. These effects of KCl were abolished in Ca2+-de...

Role of Domain Calcium in Purinergic P2X2 Receptor Channel Desensitization

American journal of physiology. Cell physiology, Jan 11, 2015

Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompan... more Activation of P2X2 receptor channels (P2X2Rs) is characterized by a rapid current growth accompanied with a decay of current during sustained ATP application, a phenomenon known as receptor desensitization. Using, rat, mouse and human receptors, here we show that two processes contribute to receptor desensitization: bath calcium-independent and -dependent. Calcium-independent desensitization is minor and comparable during repetitive agonist application in cells expressing the full size of receptor, but is pronounced in cells expressing shorter versions of receptors, indicating a role of C terminal in control of receptor desensitization. Calcium-dependent desensitization is substantial during initial agonist application and progressively increases during repetitive agonist application in ATP- and bath calcium-concentration dependent manner. Experiments with substitution of bath Na(+) with NMDG(+), a large organic cation, indicate that receptor pore dilation is a calcium-independent p...

P2X receptor channels in endocrine glands

Wiley interdisciplinary reviews. Membrane transport and signaling, 2013

The endocrine system is the system of ductless glands and single cells that synthetize hormones a... more The endocrine system is the system of ductless glands and single cells that synthetize hormones and release them directly into the bloodstream. Regulation of endocrine system is very complex and ATP and its degradable products ADP and adenosine contribute to its regulation acting as extracellular messengers for purinergic receptors. These include P2X receptors, a family of ligand-gated ion channels which expression and roles in endocrine tissues are reviewed here. There are seven mammalian purinergic receptor subunits, denoted P2X1 through P2X7, and the majority of these subunits are also expressed in secretory and non-secretory cells of endocrine system. Functional channels have been identified in the neuroendocrine hypothalamus, the posterior and anterior pituitary, the thyroid gland, the adrenals, the endocrine pancreas, the gonads and the placenta. Native channels are capable of promoting calcium influx through its pore in both excitable and non-excitable cells, as well as of in...

Biophysical basis of pituitary cell type-specific Ca2+ signaling-secretion coupling

Trends in endocrinology and metabolism: TEM

All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical act... more All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical activity. Somatotrophs and lactotrophs fire plateau-bursting action potentials, which generate Ca2+ signals of sufficient amplitude to trigger hormone release. Gonadotrophs also fire action potentials spontaneously, but as single, high-amplitude spikes with limited ability to promote Ca2+ influx and secretion. However, Ca2+ mobilization in gonadotrophs transforms single spiking into plateau-bursting-type electrical activity and triggers secretion. Patch clamp analysis revealed that somatotrophs and lactotrophs, but not gonadotrophs, express BK (big)-type Ca2+-controlled K+ channels, activation of which is closely associated with voltage-gated Ca2+ influx. Conversely, pituitary gonadotrophs express SK (small)-type Ca2+-activated K+ channels that are colocalized with intracellular Ca2+ release sites. Activation of both channels is crucial for plateau-bursting-type rhythmic electrical activity...