Biomedical subjects

Stanko S Stojilkovic

Publications and source records attributed to Stanko S Stojilkovic.

33 records · Page 2Linked to original sources

PubMed2004

Recovery of Ins(1,4,5)-trisphosphate-dependent calcium signaling in neonatal gonadotrophs.

Pituitary gonadotrophs express non-desensitizing gonadotropin-releasing hormone (GnRH) receptors and their activations leads to inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2+ mobilization. When added in physiological concentration range GnRH induces baseline Ca2+ oscillations, whereas in higher concentrations it induces a prolonged spike response accompanied with non-oscillatory or oscillatory plateau response. Here, we studied the recovery of calcium signaling during repetitive stimulation with short (10-30 s) GnRH pulses and variable interpulse intervals in neonatal gonadotrophs perfused with Ca2+/Na+ -containing, Ca2+ -deficient/Na+ -containing, and Ca2+ -containing/Na+ -deficient media. In Ca2+/Na+ -containing medium, baseline Ca2+ oscillations recovered without refractory period and with a time constant of approximately 20 s, whereas the recovery of spike response occurred after 25-35 s refractory period and with a time constant of approximately 30 s. During repetitive GnRH stimulation, removal of Ca2+ had only a minor effect on baseline oscillations but abolished spike response, whereas removal of Na+ slightly extended duration of baseline oscillations and considerably prolonged spike response. These results indicate that two calcium handling mechanisms are operative in gonadotrophs: redistribution of calcium within InsP3-sensitive and -insensitive pools and a sodium-dependent calcium efflux followed by calcium influx. Redistribution of Ca2+ within the cell leads to rapid recovery of InsP3-dependent pool, whereas the Na+ -dependent Ca2+ efflux pathway is activated by spike response and limits the time of exposure to elevated cytosolic Ca2+ concentrations.

Animals↗

PubMed2003

Parallelism and dissociation in the actions of an Aroclor 1260-based transformer fluid on testicular androgenesis and antioxidant enzymes.

The mechanism by which Aroclors and other polychlorinated biphenyls (PCBs) inhibit testicular androgenesis in vivo and in vitro has not been characterized. Here we studied in adult rats the effects of intratesticular (i.t.t.), intraperitoneal (i.p.) and by gavage (p.o.) administration of Pyralene, an Aroclor 1260-based transformer fluid, on testicular androgenesis and oxidative status in androgen-producing interstitial cells and liver. Pyralene markedly decreased in vitro agonist stimulated androgenesis 24 h after bilateral i.t.t.-injection (25 microg/testis), 24 h and 96 h after single i.p.-injection (10 and 50 mg/kg body weight), and 96 h after p.o.-administration (7x10 mg and 7x50 mg/kg body weight daily). Inhibited androgenesis was accompanied by changes in the activity of antioxidant enzymes (AOEs) in interstitial cells after local i.t.t.-treatment and occasionally after systemic Pyralene application. Among changes in the activity, glutathione peroxidase and catalase reflected relatively well the toxicity of Pyralene in these cells. In liver, glutathione-S-transferase (GST) and glutathione peroxidase activities were enhanced after p.o.-treatment and total glutathione (tGSH) content and lipid peroxidation (LP) were enhanced after i.p.-administration. These results indicate that Pyralene inhibits androgenesis independently of the method of its administration. The results also suggest that changes in the oxidative status in testicular milieu are not critical for Pyralene-induced inhibition of androgenesis.

Administration, Oral↗

PubMed2003

Receptor-controlled phosphorylation of alpha 1 soluble guanylyl cyclase enhances nitric oxide-dependent cyclic guanosine 5'-monophosphate production in pituitary cells.

It is generally accepted that G protein-coupled receptors stimulate soluble guanylyl cyclase (sGC)-mediated cGMP production indirectly, by increasing nitric oxide (NO) synthase activity in a calcium- and kinase-dependent manner. Here we show that normal and GH(3) immortalized pituitary cells expressed alpha(1)beta(1)-sGC heterodimer. Activation of adenylyl cyclase by GHRH, pituitary adenylate cyclase-activating polypeptide, vasoactive intestinal peptide, and forskolin increased NO and cGMP levels, and basal and stimulated cGMP production was abolished by inhibition of NO synthase activity. However, activators of adenylyl cyclase were found to enhance this NO-dependent cGMP production even when NO was held constant at basal levels. Receptor-activated cGMP production was mimicked by expression of a constitutive active protein kinase A and was accompanied with phosphorylation of native and recombinant alpha(1)-sGC subunit. Addition of a protein kinase A inhibitor, overexpression of a dominant negative mutant of regulatory protein kinase A subunit, and substitution of Ser(107)-Ser(108) N-terminal residues of alpha(1)-subunit with alanine abolished adenylyl cyclase-dependent cGMP production without affecting basal and NO donor-stimulated cGMP production. These results indicate that phosphorylation of alpha(1)-subunit by protein kinase A enlarges the NO-dependent sGC activity, most likely by stabilizing the NO/alpha(1)beta(1) complex. This is the major pathway by which adenylyl cyclase-coupled receptors stimulate cGMP production.

Alanine↗

PubMed2003

Role of nucleotide P2 receptors in calcium signaling and prolactin release in pituitary lactotrophs.

Anterior pituitary cells express nucleotide-gated G protein-coupled P2 receptors (P2YRs) and cation-conducting channels (P2XRs). However, the identification of P2 receptors subtypes and their native ligands, and the distribution and function of these receptors within the secretory and non-secretory pituitary cells has been incompletely characterized. The focus in this study was on lactotroph subpopulation of cells. ATP and ADP, but not UTP and UDP, triggered calcium signaling in a majority (85%) of lactotrophs and prolactin release in mixed pituitary cells. Consistent with the role of P2 receptors in signaling and secretion, the actions of ATP and ADP were abolished in the presence of apyrase, an ectonucleotidase. Transcripts for Gq-coupled calcium-mobilizing P2Y1R, P2Y2R, P2Y4R, and P2Y6R, as well as Gi-coupled P2Y12R, were identified in mixed anterior pituitary cells. The ligand-selectivity profile of calcium mobilization-dependent signaling and prolactin secretion and the blockade of these responses by pyridoxal 5-phosphate 6-azophenyl-2',4'-disulphonic acid indicated that P2Y1R mediates the stimulatory action of ATP and ADP. Within the channels expressed in anterior pituitary (P2X2R, P2X3R, P2X4R, and P2X7R), the P2X4R subtype provides a major pathway for calcium influx-dependent signaling and prolactin secretion. This conclusion was based on comparison of native to recombinant channels with respect to their ligand preference, sensitivity to pyridoxal 5-phosphate 6-azophenyl-2',4'-disulphonic acid, and the rates of calcium signal desensitization.

Adenosine Diphosphate↗

PubMed2003

Intracellular calcium measurements as a method in studies on activity of purinergic P2X receptor channels.

Extracellular nucleotide-activated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct small cations, including Ca2+, leading to the depolarization of cells and subsequent stimulation of voltage-gated Ca2+ influx in excitable cells. Here, we studied the spatiotemporal characteristics of intracellular Ca2+ signaling and its dependence on current signaling in excitable mouse immortalized gonadotropin-releasing hormone-secreting cells (GT1) and nonexcitable human embryonic kidney cells (HEK-293) cells expressing wild-type and chimeric P2XRs. In both cell types, P2XR generated depolarizing currents during the sustained ATP stimulation, which desensitized in order (from rapidly desensitizing to nondesensitizing): P2X3R > P2X2b + X4R > P2X2bR > P2X2a + X4R > P2X4R > P2X2aR > P2X7R. HEK-293 cells were not suitable for studies on P2XR-mediated Ca2+ influx because of the coactivation of endogenously expressed Ca2+-mobilizing purinergic P2Y receptors. However, when expressed in GT1 cells, all wild-type and chimeric P2XRs responded to agonist binding with global Ca2+ signals, which desensitized in the same order as current signals but in a significantly slower manner. The global distribution of Ca2+ signals was present independently of the rate of current desensitization. The temporal characteristics of Ca2+ signals were not affected by voltage-gated Ca2+ influx and removal of extracellular sodium. Ca2+ signals reflected well the receptor-specific EC50 values for ATP and the extracellular Zn2+ and pH sensitivities of P2XRs. These results indicate that intracellular Ca2+ measurements are useful for characterizing the pharmacological properties and messenger functions of P2XRs, as well as the kinetics of channel activity, when the host cells do not express other members of purinergic receptors.

Adenosine Triphosphate↗

PubMed2003

F(ab)'2-mediated neutralization of C3a and C5a anaphylatoxins: a novel effector function of immunoglobulins.

High-dose intravenous immunoglobulin (IVIG) prevents immune damage by scavenging complement fragments 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 (TXB2) 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.

Animals↗

PubMed2003

Dependence of purinergic P2X receptor activity on ectodomain structure.

Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X(4)R > P2X(2a)R = P2X(2b)R P2X(7)R. Chimeras having the ectodomain Ile(66)-Tyr(310) sequence of P2X(2)R and Val(61)-Phe(313) sequence of P2X(7)R in the backbone of P2X(4)R were expressed but were non-functioning channels. P2X(2a) + X(4)R and P2X(2b) + X(4)R chimeras having the Val(66)-Tyr(315) ectodomain sequence of P2X(4)R in the backbones of P2X(2a)R and P2X(2b)R were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X(2b)R and P2X(2a)R, chimeric P2X(2b) + X(4)R desensitized more rapidly than P2X(2a) + X(4)R, and the rate of desensitization of P2X(2a)+X(4)R increased by substituting its Arg(371)-Pro(376) intracellular C-terminal sequence with the Glu(376)-Gly(381) sequence of P2X(4)R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.

Adenosine Triphosphate↗

PubMed2003

Nitric oxide inhibits prolactin secretion in pituitary cells downstream of voltage-gated calcium influx.

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.

Animals↗

PubMed2002

Heteromultimerization modulates P2X receptor functions through participating extracellular and C-terminal subdomains.

P2X purinergic receptors (P2XRs) differ among themselves with respect to their ligand preferences and channel kinetics during activation, desensitization, and recovery. However, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. Here we show that homomeric receptors having the extracellular domain of the P2X(3) subunit in the P2X(2a)-based backbone (P2X(2a)/X(3)ex) mimicked two intrinsic functions of P2X(3)R, sensitivity to alphabeta-methylene ATP and ecto-ATPase-dependent recovery from endogenous desensitization; these two functions were localized to the N- and C-terminal halves of the P2X(3) extracellular loop, respectively. The chimeric P2X(2a)R/X(3)ex receptors also desensitized with accelerated rates compared with native P2X(2a)R, and the introduction of P2X(2) C-terminal splicing into the chimeric subunit (P2X(2b)/X(3)ex) further increased the rate of desensitization. Physical and functional heteromerization of native P2X(2a) and P2X(2b) subunits was also demonstrated. In heteromeric receptors, the ectodomain of P2X(3) was a structural determinant for ligand selectivity and recovery from desensitization, and the C terminus of P2X(2) was an important factor for the desensitization rate. Furthermore, [gamma-(32)P]8-azido ATP, a photoreactive agonist, was effectively cross-linked to P2X(3) subunit in homomeric receptors but not in heteromeric P2X(2) + P2X(3)Rs. These results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.

Adenosine Triphosphatases↗

PubMed2002

Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells.

It is well established that G protein-coupled receptors stimulate nitric oxide-sensitive soluble guanylyl cyclase by increasing intracellular Ca(2+) and activating Ca(2+)-dependent nitric-oxide synthases. In pituitary cells receptors that stimulated adenylyl cyclase, growth hormone-releasing hormone, corticotropin-releasing factor, and thyrotropin-releasing hormone also stimulated calcium signaling and increased cGMP levels, whereas receptors that inhibited adenylyl cyclase, endothelin-A, and dopamine-2 also inhibited spontaneous calcium transients and decreased cGMP levels. However, receptor-controlled up- and down-regulation of cyclic nucleotide accumulation was not blocked by abolition of Ca(2+) signaling, suggesting that cAMP production affects cGMP accumulation. Agonist-induced cGMP accumulation was observed in cells incubated in the presence of various phosphodiesterase and soluble guanylyl cyclase inhibitors, confirming that G(s)-coupled receptors stimulated de novo cGMP production. Furthermore, cholera toxin (an activator of G(s)), forskolin (an activator of adenylyl cyclase), and 8-Br-cAMP (a permeable cAMP analog) mimicked the stimulatory action of G(s)-coupled receptors on cGMP production. Basal, agonist-, cholera toxin-, and forskolin-stimulated cGMP production, but not cAMP production, was significantly reduced in cells treated with H89, a protein kinase A inhibitor. These results indicate that coupling seven plasma membrane-domain receptors to an adenylyl cyclase signaling pathway provides an additional calcium-independent and cAMP-dependent mechanism for modulating soluble guanylyl cyclase activity in pituitary cells.

Animals↗

PubMed2002

Ca(2+)-mobilizing endothelin-A receptors inhibit voltage-gated Ca(2+) influx through G(i/o) signaling pathway in pituitary lactotrophs.

In excitable cells, receptor-induced Ca(2+) release from intracellular stores is usually accompanied by sustained depolarization of cells and facilitated voltage-gated Ca(2+) influx (VGCI). In quiescent pituitary lactotrophs, however, endothelin-1 (ET-1) induced rapid Ca(2+) release without triggering Ca(2+) influx. Furthermore, in spontaneously firing and depolarized lactotrophs, the Ca(2+)-mobilizing action of ET-1 was followed by inhibition of spontaneous VGCI caused by prolonged cell hyperpolarization and abolition of action potential-driven Ca(2+) influx. Agonist-induced depolarization of cells and enhancement of VGCI upon Ca(2+) mobilization was established in both quiescent and firing lactotrophs treated overnight with pertussis toxin (PTX). Activation of adenylyl cyclase by forskolin and addition of cell-permeable 8-bromo-cAMP did not affect ET-1-induced sustained inhibition of VGCI, suggesting that the cAMP-protein kinase A signaling pathway does not mediate the inhibitory action of ET-1 on VGCI. Consistent with the role of PTX-sensitive K(+) channels in ET-1-induced hyperpolarization of control cells, but not PTX-treated cells, ET-1 decreased the cell input resistance and activated a 5 mM Cs(+)-sensitive K(+) current. In the presence of Cs(+), ET-1 stimulated VGCI in a manner comparable with that observed in PTX-treated cells, whereas E-4031, a specific blocker of ether-a-go-go-related gene-like K(+) channels, was ineffective. Similar effects of PTX and Cs(+) were also observed in GH(3) immortalized cells transiently expressing ET(A) receptors. These results indicate that signaling of ET(A) receptors through the G(i/o) pathway in lactotrophs and the subsequent activation of inward rectifier K(+) channels provide an effective and adenylyl cyclase-independent mechanism for a prolonged uncoupling of Ca(2+) mobilization and influx pathways.

Animals↗

PubMed2002

Purinergic P2X(2) receptor desensitization depends on coupling between ectodomain and C-terminal domain.

The wild-type P2X(2) purinergic receptor (P2X(2a)R) and its splice form lacking the intracellular Val(370)-Gln(438) C-terminal sequence (P2X(2b)R) respond to ATP stimulation with comparable EC(50) values and peak current/calcium responses but desensitize in a receptor-specific manner. P2X(2a)R desensitizes slowly and P2X(2b)R desensitizes rapidly. We studied the effects of different agonists, and of substituting the ectodomain, on the pattern of calcium signaling by P2X(2a)R and P2X(2b)R. Both receptors showed similar EC(50) values (estimated from the peak calcium response) and IC(50) values (estimated from the rate of calcium signal desensitization) for agonists, in the order 2-MeS-ATP <or= ATP <or= ATPgammaS < BzATP << alphabeta-meATP, and the IC(50) values for agonists were shifted to the right compared with their EC(50) values. Furthermore, the ATP-induced receptor-subtype specific pattern of desensitization was mimicked by high- but not by low-efficacy agonists, suggesting a ligand-specific desensitization pattern. To test this hypothesis, we generated chimeric P2X(2a)R and P2X(2b)R containing the Val(60)-Phe(301) ectodomain sequence of P2X(3)R and Val(61)-Phe(313) ectodomain sequence of P2X(7)R instead the native Ile(66)-Tyr(310) sequence. The mutated P2X(2a)+X(3)R and P2X(2b)+X(3)R exhibited comparable EC(50) values for ATP, BzATP, and alphabeta-meATP in the submicromolar concentration range and desensitized in a receptor-specific and ligand-nonspecific manner. On the other hand, the chimeric P2X(2)+X(7)R exhibited decreased sensitivity for ATP and desensitized in a receptor-nonspecific manner. These results suggest that efficacy of agonists for the ligand-binding domain of P2X(2)Rs reflects the strength of desensitization controlled by their C-terminal structures.

Adenosine Triphosphate↗

PubMed2002

Cell-type specific messenger functions of extracellular calcium in the anterior pituitary.

Calcium can serve not only as an intracellular messenger, but also as an extracellular messenger controlling the gating properties of plasma membrane channels and acting as an agonist for G protein-coupled Ca(2+)-sensing receptors. Here we studied the potential extracellular messenger functions of this ion in anterior pituitary cells. Depletion and repletion of the extracellular Ca(2+) concentration ([Ca(2+)]e) induced transient elevations in the intracellular Ca(2+) concentration ([Ca(2+)]i), and elevations in [Ca(2+)]e above physiological levels decreased [Ca(2+)]i in somatotrophs and lactotrophs, but not in gonadotrophs. The amplitudes and duration of [Ca(2+)]i responses depended on the [Ca(2+)]e and its rate of change, which resulted exclusively from modulation of spontaneous voltage-gated Ca(2+) influx. Changes in [Ca(2+)]e also affected GH and PRL secretion. The PRL secretory profiles paralleled the [Ca(2+)]i profiles in lactotrophs, whereas GH secretion was also stimulated by [Ca(2+)]e independently of the status of voltage-gated Ca(2+) influx. [Ca(2+)]e modulated GH secretion in a dose-dependent manner, with EC(50) values of 0.75 and 2.25 mM and minimum secretion at about 1.5 mM. In a parallel experiment, cAMP accumulation progressively increased with elevation of [Ca(2+)]e, whereas inositol phosphate levels were not affected. These results indicate the cell type-specific role of [Ca(2+)]e in the control of Ca(2+) signaling and secretion.

Animals↗

PubMedDate not supplied

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

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 and secretion.

Action Potentials↗

PubMedDate not supplied

Dependence of prolactin release on coupling between Ca(2+) mobilization and voltage-gated Ca(2+) influx pathways in rat lactotrophs.

Two Ca(2+)-mobilizing receptors expressed in lactotrophs, endothelin-A (ET(A)) and thyrotropin-releasing hormone (TRH), induce a rapid Ca(2+) release from intracellular stores and prolactin (PRL) secretion but differ in their actions during the sustained stimulation; TRH facilitates and ET-1 inhibits voltage-gated calcium influx (VGCI) and PRL secretion. In pertussis toxin (PTX) treated cells, ET-1-induced inhibition of VGCI was abolished and the pattern of Ca(2+) signaling was highly comparable with that observed in TRH-stimulated cells. The addition of Cs(+), a relatively specific blocker of inward rectifier K(+) channels, mimicked the effect of PTX on the pattern of ET-1-induced sustained Ca(2+) signaling, but only in about 50% of cells, and did not affect agonist-induced inhibition of PRL secretion. Extracellular Cs(+) was also ineffective in altering the TRH-induced facilitation of VGCI and PRL secretion. Furthermore, apamin and paxilline, specific blockers of Ca(2+)-activated SKand BK-type K(+) channels, respectively; E-4031, a blocker of ether a-go-go K(+) channel; and linopirdine, a blocker of M-type K(+) channel, did not affect the agonist-specific patterns of calcium signaling and PRL secretion. These results suggest that ET-1 inhibits VGCI through activation of Cs(+)-sensitive channels, presumably the Gi/o-controlled inward rectifier K(+) channels, and that this agonist also inhibits PRL release, but downstream of Ca(2+) influx. Further studies are required to identify the mechanism of sustained TRH-induced facilitation of VGCI and PRL secretion.

Animals↗