Stanko Stojilkovic - Academia.edu (original) (raw)

Papers by Stanko Stojilkovic

Research paper thumbnail of Multiple Roles of Gi/o Protein-coupled Receptors in Control of Action Potential Secretion Coupling in Pituitary Lactotrophs

Annals of the New York Academy of Sciences, 2009

Research paper thumbnail of F(ab)′2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins

Research paper thumbnail of cAMP-Mediated Stabilization of Fusion Pores in Cultured Rat Pituitary Lactotrophs

Journal of Neuroscience, 2013

Research paper thumbnail of Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs

Molecular Endocrinology, 2006

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of Novel Aspects of Growth Hormone (GH) Autoregulation

Research paper thumbnail of Local Regulation of Gonadotroph Function by Pituitary Gonadotropin-Releasing Hormone

Endocrinology, Apr 1, 2000

Research paper thumbnail of 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.

Research paper thumbnail of Roles of Purinergic P2X Receptors as Pacemaking Channels and Modulators of Calcium-Mobilizing Pathway in Pituitary Gonadotrophs

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.

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of Hypotonicity and Peptide Discharge from a Single Vesicle Abbreviated title: Hypotonicity and peptide discharge

Research paper thumbnail of 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.

Research paper thumbnail of 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...

Research paper thumbnail of 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...

Research paper thumbnail of 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...

Research paper thumbnail of 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...

Research paper thumbnail of Multiple Roles of Gi/o Protein-coupled Receptors in Control of Action Potential Secretion Coupling in Pituitary Lactotrophs

Annals of the New York Academy of Sciences, 2009

Research paper thumbnail of F(ab)′2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins

Research paper thumbnail of cAMP-Mediated Stabilization of Fusion Pores in Cultured Rat Pituitary Lactotrophs

Journal of Neuroscience, 2013

Research paper thumbnail of Dependence of Electrical Activity and Calcium Influx-Controlled Prolactin Release on Adenylyl Cyclase Signaling Pathway in Pituitary Lactotrophs

Molecular Endocrinology, 2006

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of Novel Aspects of Growth Hormone (GH) Autoregulation

Research paper thumbnail of Local Regulation of Gonadotroph Function by Pituitary Gonadotropin-Releasing Hormone

Endocrinology, Apr 1, 2000

Research paper thumbnail of 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.

Research paper thumbnail of Roles of Purinergic P2X Receptors as Pacemaking Channels and Modulators of Calcium-Mobilizing Pathway in Pituitary Gonadotrophs

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.

Research paper thumbnail of 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.

Research paper thumbnail of 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.

Research paper thumbnail of Hypotonicity and Peptide Discharge from a Single Vesicle Abbreviated title: Hypotonicity and peptide discharge

Research paper thumbnail of 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.

Research paper thumbnail of 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...

Research paper thumbnail of 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...

Research paper thumbnail of 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...

Research paper thumbnail of 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...