Biomedical subjects

Stanko S Stojilkovic

Publications and source records attributed to Stanko S Stojilkovic.

At least 19 recordsLinked to original sources

PubMed2006

Pituitary cell type-specific electrical activity, calcium signaling and secretion.

All secretory anterior pituitary cells exhibit spontaneous and extracellular calcium-dependent electrical activity, but differ with respect to the patterns of firing and associated calcium signaling and hormone secretion. Thus, somatotrophs and lactotrophs fire plateau-bursting action potentials spontaneously and without coupling to calcium release from intracellular stores, which generate calcium signals of sufficient amplitude to keep steady hormone release. In these cells, both spontaneous electrical activity and basal hormone secretion can be further amplified by activation of Gq/11 and Gs-coupled receptors and inhibited by Gi/o/z-coupled receptors. In contrast, gonadotrophs fire single, high-amplitude spikes with limited ability to promote calcium influx and exocytosis, whereas activated Gq/11-coupled receptors in these cells transform single-action potential spiking into the plateau-bursting type of electrical activity and trigger periodic high-amplitude calcium signals and exocytosis of prestored secretory vesicles. Here, we review biochemical and biophysical aspects of spontaneous and receptor-controlled electrical activity, calcium signaling, and hormone secretion in pituitary cells.

Action Potentials↗

PubMed2006

Effects of testosterone on hormonal content and calcium-dependent basal secretion in female rat pituitary cells.

In vivo and in vitro effects of elevated androgens on agonist-induced gonadotropin secretion have been addressed previously. Here we investigated the effects of testosterone on hormonal content and basal (in the absence of agonists) hormone release in pituitary lactotrophs, somatotrophs and gonadotrophs from female rats. Furthermore we tested the hypothesis that testosterone action is dependent on the pattern of spontaneous and Bay K 8644 (a L-type calcium channel agonist) -induced calcium signalling. Mixed anterior pituitary cells were cultured in steroid containing or depleted media, and testosterone (1pM to 10nM) was added for 48h. Cells were studied for their spontaneous and Bay K 8644-induced calcium signalling pattern and total hormone levels (release and hormonal content). In lactotrophs, somatotrophs and gonadotrophs testosterone did not affect the pattern of spontaneous calcium signalling. Bay K 8644-induced calcium signalling and hormone release were not affected by testosterone. In both steroid-depleted and -containing medium, testosterone inhibited prolactin (PRL), luteinizing hormone (LH) and growth hormone (GH) cellular content and release in a dose-dependent manner, with IC(50)s in a sub-nanomolar concentration range. These results indicate that testosterone inhibits basal hormone release from lactotrophs, somatotrophs and gonadotrophs without affecting intracellular calcium signalling. This action of testosterone is not dependent on the presence of other steroid hormones.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed2006

Participation of the Lys313-Ile333 sequence of the purinergic P2X4 receptor in agonist binding and transduction of signals to the channel gate.

To study the roles of the Lys(313)-Ile(333) ectodomain sequence of the rat P2X(4) receptor in ATP binding and transduction of signals to the channel gate, the conserved Lys(313), Tyr(315), Gly(316), Ike(317), Arg(318), Asp(320), Val(323), Lys(329), Phe(330), and Ile(333) residues were mutated. Current recordings were done on lifted cells and ATP was applied using an ultrafast solution-switching system. The rates of wild type channel opening and closing in the presence of ATP, but not the rate of washout-induced closing, were dependent on agonist concentration. All mutants other than I317A were expressed in the plasma membrane at comparable levels. The majority of mutants showed significant changes in the peak amplitude of responses and the EC(50) values for ATP. When stimulated with the supramaximal (1.4 mm) ATP concentration, mutants also differed in the kinetics of their activation, deactivation, and/or desensitization. The results suggest a critical role of the Lys(313) residue in receptor function other than coordination of the phosphate group of ATP and possible contribution of the Tyr(315) residue to the agonist binding module. The pattern of changes of receptor function by mutation of other residues was consistent with the operation of the Gly(316)-Ile(333) sequence as a signal transduction module between the ligand binding domain and the channel gate in the second transmembrane domain.

Amino Acid Sequence↗

PubMed2006

Identification of P2X4 receptor-specific residues contributing to the ivermectin effects on channel deactivation.

Ivermectin (IVM) applied extracellularly increases the sensitivity of P2X4 receptor (P2X4R) to ATP, enhances the maximum current amplitudes, and greatly prolongs the deactivation kinetics. In this manuscript, we focused on identification of receptor-specific residues responsible for IVM effects on channel gating using the wild-type rat homomeric P2X4R, several chimeric P2X2/P2X4 receptors, and single-point P2X4R-specific mutants in the ectodomain and two transmembrane domains. Experiments with chimeric receptors revealed that the Val49-Val61 but not the Val64-Tyr315 ectodomain sequence is important for the effects of IVM on channel deactivation. Receptor-specific mutations placed in the Gly29-Val61 and Asp338-Leu358 regions showed the importance of Trp50, Val60, and Val357 residues in IVM regulation of the rate of channel deactivation, but not on the maximum current amplitude. These results suggest that the transmembrane domains and the nearby ectodomain region contribute to the effects of IVM on channel deactivation.

Adenosine Triphosphate↗

PubMed2006

ZD7288 inhibits exocytosis in an HCN-independent manner and downstream of voltage-gated calcium influx in pituitary lactotrophs.

Pituitary lactotrophs fire action potentials spontaneously and the associated voltage-gated calcium influx is sufficient to maintain high prolactin release. Here we studied the role of hyperpolarization-activated cation channels in pacemaking activity, calcium signaling, and prolactin secretion in these cells. A slowly developing and hyperpolarization-activated inward current was identified but only in a fraction of lactotrophs. The current was blocked by ZD7288, a relatively specific blocker of these channels. However, the pacemaking activity increased in ZD7288-treated cells independently of the presence of this current. This in turn facilitated voltage-gated calcium influx and transiently stimulated prolactin secretion. Sustained ZD7288 application in concentrations that are commonly used to block the hyperpolarization-activated cation channels inhibited hormone release at elevated intracellular calcium concentrations. Agonist and Bay K 8644-stimulated prolactin release was also inhibited by ZD7288, indicating that this compound attenuates the exocytotic pathway downstream of calcium influx.

Animals↗

PubMed2006

Dependence of electrical activity and calcium influx-controlled prolactin release on adenylyl cyclase signaling pathway in pituitary lactotrophs.

Pituitary lactotrophs in vitro fire extracellular Ca2+-dependent action potentials spontaneously through still unidentified pacemaking channels, and the associated voltage-gated Ca2+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 cell-permeable 8-bromo-cAMP stimulated firing of action potentials and PRL release and rescued hormone secretion in cells with inhibited ACs in an extracellular Ca2+-dependent manner, whereas 8-bromo-cGMP and 8-(4-chlorophenylthio)-2'-O-methyl-cAMP were ineffective. Protein kinase A inhibitors did not stop spontaneous and forskolin-stimulated pacemaking, VGCI, and PRL release. These results indicate that cAMP facilitates pacemaking, VGCI, and PRL release in lactotrophs predominantly in a protein kinase A- and Epac cAMP receptor-independent manner.

Adenylyl Cyclase Inhibitors↗

PubMed2006

Contribution of multidrug resistance protein MRP5 in control of cyclic guanosine 5'-monophosphate intracellular signaling in anterior pituitary cells.

The energy-dependent cyclic nucleotide cellular efflux is operative in numerous eukaryotic cells and could be mediated by multidrug resistance proteins MRP4, MRP5, and MRP8. In pituitary cells, however, the operation of export pumps and their contribution to the control of intracellular cyclic nucleotide levels were not studied previously. Here we show that cellular efflux of cyclic nucleotides was detectable in normal and immortalized GH(3) pituitary cells under resting conditions and was enlarged after concurrent stimulation of cAMP and cGMP production with GHRH, corticotropin-releasing factor, vasoactive intestinal peptide, pituitary adenylate cyclase-activating polypeptide, and forskolin. In resting and stimulated cells, the efflux pumps transported the majority of de novo-produced cGMP, limiting its intracellular accumulation in a concentration range of 1-2 microm. In contrast, only a small fraction of cAMP was released and there was a time- and concentration-dependent accumulation of this messenger in the cytosol, ranging from 1-100 microm. Stimulation and inhibition of cGMP production alone did not affect cAMP efflux, suggesting the operation of two different transport pathways in pituitary cells. The rates of cAMP and cGMP effluxes were comparable, and both pathways were blocked by probenecid and progesterone. Pituitary cells expressed mRNA transcripts for MRP4, MRP5, and MRP8, whereas GH(3) cells expressed only transcripts for MRP5. Down-regulation of MRP5 expression in GH(3) cells decreased cGMP release without affecting cAMP efflux. These results indicate that cyclic nucleotide cellular efflux plays a critical role in elimination of intracellular cGMP but not cAMP in pituitary cells and that such selectivity is achieved by expression of MRP5.

Animals↗

PubMed2006

Roles of purinergic P2X receptors as pacemaking channels and modulators of calcium-mobilizing pathway in pituitary gonadotrophs.

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.

Adenosine Triphosphate↗

PubMed2006

Hydroxyurea induces the eNOS-cGMP pathway in endothelial cells.

Hydroxyurea is a cell-cycle-specific drug that has been used to treat myeloproliferative diseases and sickle cell anemia. We have recently shown that hydroxyurea, like nitric oxide (NO)-donor compounds, increased cGMP levels in human erythroid cells. We show now that hydroxyurea increases endothelial-cell production of NO; this induction of NO in human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC) is blocked by competitive inhibitors of NO synthase (NOS), such as NG-nitro-L-arginine-methyl ester (L-NAME) and NG-nitro-L-arginine. It is dependent on cAMP-dependent protein kinase (PKA) and protein kinase B (PKB/Akt) activity. We found that hydroxyurea dose- and time-dependently induced rapid and transient phosphorylation of eNOS at Ser1177 in a PKA-dependent manner; inhibitors of PKB/Akt could partially abrogate this effect. In addition, hydroxyurea induced cAMP and cGMP levels in a dose-dependent manner, as well as levels of intracellular calcium in HUVECs. These studies established an additional mechanism by which rapid and sustained effects of hydroxyurea may affect cellular NO levels and perhaps enhance the effect of NO in myeloproliferative diseases.

Androstadienes↗

PubMed2006

Carboxyl-terminal splicing enhances physical interactions between the cytoplasmic tails of purinergic P2X receptors.

Purinergic P2X receptors are ion-conducting channels composed of three subunits, each having two transmembrane domains and intracellular amino (N) and carboxyl (C) termini. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post-transcriptional modifications on receptor architecture and function remains poorly understood. In this study, we focused on mouse pituitary P2X2 receptors. In this tissue, progressive splicing of the P2X2a C terminus generated two functional subunit variants, P2X2b and P2X2e, which exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the intersubunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C termini of P2X2a receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C termini and between C- and N termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X2b or P2X2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X2b receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in the cytoplasmic intersubunit interactions and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.

Adenosine Triphosphate↗

PubMed2006

Molecular cloning and characterization of alpha1-soluble guanylyl cyclase gene promoter in rat pituitary cells.

Soluble guanylyl cyclase is a cytosolic enzyme which catalyzes conversion of GTP to the second messenger cyclic GMP. The transcriptional regulation at the promoter levels of four soluble guanylyl cyclase subunits, termed alpha1, alpha2, beta1, and beta2, is largely unknown. In this study, we identified the transcription start site of alpha1-soluble guanylyl cyclase gene in rat pituitary cells and cloned the 3.5 kb 5'-promoter. Sequence analysis of this TATA-less promoter revealed the presence of several putative-binding sites for transcriptional factors, including CCAAT site at -41 to -32 and Sp1 site at -34 to -24. Transfection of pituitary cells with constructs of variable lengths confirmed the relevance of different promoter regions in the control of transcriptional activity. Among them, the -49 to + 156 region was critical for basal transcriptional activity. Electrophoretic mobility shift assay using nuclear proteins extracted from normal and immortalized pituitary cells indicated that the CCAAT/Sp1 site within the -49 to + 156 region was able to specifically interact with CCAAT-binding factor and Sp1. These two sites were partly overlapped and both of them conferred stimulatory effects. The in vivo recruitment of CCAAT-binding factor and Sp1 was confirmed by chromatin immunoprecipitation. These results indicate that the composite CCAAT/Sp1 cis-element contributes to the expression of alpha1-sGC subunit in resting pituitary cells.

5' Flanking Region↗

PubMed2005

Endothelin-induced, long lasting, and Ca2+ influx-independent blockade of intrinsic secretion in pituitary cells by Gz subunits.

The G protein-coupled receptors in excitable cells have prominent roles in controlling Ca2+-triggered secretion by modulating voltage-gated Ca2+ influx. In pituitary lactotrophs, spontaneous voltage-gated Ca2+ influx is sufficient to maintain prolactin release high. Here we show that endothelin in picomolar concentrations can interrupt such release for several hours downstream of spontaneous and high K+-stimulated voltage-gated Ca2+ influx. This action occurred through the Gz signaling pathway; the adenylyl cyclase-signaling cascade could mediate sustained inhibition of secretion, whereas rapid inhibition also occurred at elevated cAMP levels regardless of the status of phospholipase C, tyrosine kinases, and protein kinase C. In a nanomolar concentration range, endothelin also inhibited voltage-gated Ca2+ influx through the G i/o signaling pathway. Thus, the coupling of seven-transmembrane domain endothelin receptors to Gz proteins provided a pathway that effectively blocked hormone secretion distal to Ca2+ entry, whereas the cross-coupling to G i/o proteins reinforced such inhibition by simultaneously reducing the pacemaking activity.

Adenylyl Cyclases↗

PubMed2005

Molecular determinants of the agonist binding domain of a P2X receptor channel.

P2 purinergic receptor channel receptors (P2XRs) are a family of ligand-gated cation channels composed of two transmembrane domains, N and C termini located intracellularly, and a large extracellular loop containing the ATP binding domain. To identify regions important for binding and gating, previous experimental work was focused on mutagenesis of conserved ectodomain residues. Here, we used the known sequence and secondary structure similarities between the Lys180-Lys326 ectodomain region of P2X(4) and the class II aminoacyl-tRNA synthetases as a guide to generate a three-dimensional model of the receptor-binding site and to design mutants. The interplay between homology modeling and site-directed mutagenesis suggested that Asp280 residue of P2X(4)R coordinates ATP binding via the magnesium ion, Phe230 residue coordinates the binding of the adenine ring of ATP, and Lys190, His286, and Arg278 residues coordinate the actions of negatively charged alpha-, beta-, and gamma-phosphate groups, respectively. Until the crystal structure of the channel is solved, this model could provide a useful approach for future studies on the identification of ATP binding domain and gating of P2XRs.

Adenosine Triphosphate↗

PubMed2005

Ca2+-regulated exocytosis and SNARE function.

Secretory vesicles formed at the trans-Golgi network of neuroendocrine and endocrine cells must undergo several steps, such as translocation, docking and priming, before they are ready to fuse with the plasma membrane and deliver their cargo into the extracellular space. This process is called regulated exocytosis and is controlled by Ca(2+) (using synaptotagmin) and mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins. Recent studies from three leading laboratories reveal novel details about the mechanism by which Ca(2+) and SNAREs regulate this complex process. These findings highlight the roles of both SNAP25 (synaptosome-associated protein of 25kD), one of the SNARE proteins, and CAPS (Ca(2+)-dependent activator protein for secretion), a Ca(2+)-sensor protein, in vesicle priming, depriming and fusion.

Animals↗

PubMed2005

Radoslav K. Andjus (1926-2003): a brief summary of his life and work.

Radoslav K. Andjus was a professor of physiology and biophysics at the University of Belgrade, Serbia, from 1953 to 1992. He was an elected member of the Serbian Academy of Sciences and Arts, the International Academy of Astronautics, and the Montenegrin Academy of Sciences and Arts. He published over 190 papers in domestic and international journals and three textbooks. The main field of his research was thermophysiology. He studied hypothermia, suspended animation and resuscitation, hibernation and biological rhythms, temperature adaptation and acclimation, and cryoprotection. Professor Andjus also contributed significantly to the fields of brain metabolism, endocrinology, electroretinography, as well as biophysical modeling and theoretical biology.

Biochemistry↗

PubMed2005

Molecular dissection of purinergic P2X receptor channels.

The P2X receptors (P2XRs) are a family of ATP-gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. P2XRs are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X(1)-P2X(7). All subunits possess intracellular N- and C-termini, two transmembrane domains, and a relatively large extracellular ligand-binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of extracellular ATP leads to deactivation and resensitization of receptors. Activated P2XRs generate inward currents caused by Na(+) and Ca(2+) influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage-gated calcium entry in excitable cells. No crystal structures are available for P2XRs and these receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of P2XRs, including heteromerization, chimerization, mutagenesis, and biochemical studies.

Adenosine Triphosphate↗

PubMed2004

Identification of ectodomain regions contributing to gating, deactivation, and resensitization of purinergic P2X receptors.

The P2X receptors (P2XRs) are a family of ligand-gated channels activated by extracellular ATP through a sequence of conformational transitions between closed, open, and desensitized states. In this study, we examined the dependence of the activity of P2XRs on ectodomain structure and agonist potency. Experiments were done in human embryonic kidney 293 cells expressing rat P2X2aR, P2X2bR, and P2X3R, and chimeras having the V60-R180 or V60-F301 ectodomain sequences of P2X3R instead of the I66-H192 or I66-Y310 sequences of P2X2aR and P2X2bR. Chimeric P2X2a/V60-F301X3R and P2X2b/V60-F301X3R inherited the P2X3R ligand-selective profile, whereas the potency of agonists for P2X2a/V60-R180X3R was in between those observed at parental receptors. Furthermore, P2X2a/V60-F301X3R and P2X2a/V60-R180X3R desensitized in a P2X2aR-specific manner, and P2X2b/V60-F301X3R desensitized with rates comparable with those of P2X2bR. In striking contrast to parental receptors, the rates of decay in P2X2a/V60-F301X3R and P2X2b/V60-F301X3R currents after agonist withdrawal were 15- to 200-fold slower. For these chimeras, the decays in currents were not dependent on duration of stimuli and reflected both continuous desensitization and deactivation of receptors. Also, participation of deactivation in closure of channels inversely correlated with potency of agonists to activate receptors. The delay in deactivation was practically abolished in P2X2a/V60-R180X3R-expressing cells. However, the recovery from desensitization of P2X2a/V60-F301X3R and P2X2a/V60-R180X3R was similar and substantially delayed compared with that of parental receptors. These results indicate that both ectodomain halves participate in gating, but that the C and N halves influence the stability of open and desensitized conformation states, respectively, which in turn reflects on rates of receptor deactivation and resensitization.

Adenosine Triphosphate↗

PubMed2004

Novel aspects of growth hormone (GH) autoregulation: GH-induced GH gene expression in grass carp pituitary cells through autocrine/paracrine mechanisms.

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 GH-releasing factors in fish, including GnRH, apomorphine, and pituitary adenylate cyclase-activating 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.

Animals↗