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M Tomić

Publications and source records attributed to M Tomić.

At least 19 recordsLinked to original sources
PubMed2006

Dependence of hyperpolarisation-activated cyclic nucleotide-gated channel activity on basal cyclic adenosine monophosphate production in spontaneously firing GH3 cells.

The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca(2+) signalling, and prolactin secretion in GH(3) immortalised pituitary cells. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below - 60 mV elicited a slowly activating voltage-dependent inward current (I(h)) in the majority of tested cells, with a half-maximal activation voltage of -89.9 +/- 4.2 mV and with a time constant of 1.4 +/- 0.2 s at -120 mV. The bath application of 1 mM Cs(+), a commonly used inorganic blocker of I(h), and 100 microM ZD7288, a specific organic blocker of I(h), inhibited I(h) by 90 +/- 4.1% and 84.3 +/- 1.8%, respectively. Receptor- and nonreceptor-mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8-Br-cAMP, did not affect I(h). Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of I(h) by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8-Br-cAMP, but not 8-Br-cGMP. Application of Cs(+) had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and I(h)-independent effects on spontaneous electrical activity, Ca(2+) signalling, and prolactin release. These results indicate that HCN channels in GH(3) cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials.

Adenylyl Cyclases↗
PubMed2002

Effect of Helicobacter pylori eradication on extent of duodenal gastric metaplasia and grade of gastritis.

The extent of the regression of duodenal gastric metaplasia (DGM) after the eradication of Helicobacter pylori infection is controversial. Therefore, we decided to assess the degree of DGM before, sex weeks and one year after H. pylori eradication. 105 consecutive Helicobacter pylori positive patients with endoscopically proven duodenal ulcer, with DGM and Helicobacter pylori infection were recruited for this study. The diagnosis of Helicobacter pylori infection was based on CLO-test and histology, and DGM was assessed on four bulb biopsies taken before, sex weeks and one year after Helicobacter pylori eradication. Histological assessment of Helicobacter pylori associated gastritis was performed according to the Sydney classification. Follow up study on 98 patients before, six weeks and one year after the eradication of Helicobacter pylori showed that the mean extent of DGM did not change significantly after eradication and did not differ when compared with 14 patients with persisting infection. Our results show that the inflammatory process related to Helicobacter pylori does not play the main role in the development of DGM.

Duodenal Ulcer↗
PubMed2001

Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons.

In immortalized GnRH neurons, cAMP production is elevated by increased extracellular Ca2+ and the Ca2+ channel agonist, BK-8644, and is diminished by low extracellular Ca2+ and treatment with nifedipine, consistent with the expression of adenylyl cyclase type I (AC I). Potassium-induced depolarization of GT1-7 neurons causes a dose-dependent monotonic increase in [Ca2+]i and elicits a bell-shaped cAMP response. The inhibitory phase of the cAMP response is prevented by pertussis toxin (PTX), consistent with the activation of G(i)-related proteins during depolarization. Agonist activation of the endogenous GnRH receptor in GT1-7 neurons also elicits a bell-shaped change in cAMP production. The inhibitory action of high GnRH concentrations is prevented by PTX, indicating coupling of the GnRH receptors to G(i)-related proteins. The stimulation of cAMP production by activation of endogenous LH receptors is enhanced by low (nanomolar) concentrations of GnRH but is abolished by micromolar concentrations of GnRH, again in a PTX-sensitive manner. These findings indicate that GnRH neuronal cAMP production is maintained by Ca2+ entry through voltage-sensitive calcium channels, leading to activation of Ca2+-stimulated AC I. Furthermore, the Ca2+ influx-dependent activation of AC I acts in conjunction with AC-regulatory G proteins to determine basal and agonist-stimulated levels of cAMP production.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2001

[Dermatomyositis and malignant disease--case report].

The case of patients with dermatomyositis and malignant disease has been described. The diagnosis of malignant disease was made after death of the patient, when the malignant undifferentiated cells were found in the ascites. We think that dermatomyosistis was caused by malignant disease. The similar cases of disease were described and published in the literature.

Aged↗
PubMed2000

Psychotomimetics moderately affect dopamine receptor binding in the rat brain.

The hypothesis that psychotomimetics induce a rapid dopamine receptor regulation that could participate in the expression of the brain dopaminergic overactivation and in the early signs of psychotic-like behaviour, was checked by radioligand binding on rat brain cryosections. For this purpose, subchronic 7-day-d-amphetamine pretreatment was combined with acute amphetamine, phencyclidine or LSD challenge. Acute application of psychotomimetics affected only striatal and accumbens but not nigral and olfactory dopamine receptor binding after 40 min, while subchronic amphetamine expressed no effect, as revealed by two-way ANOVA. Post-hoc statistical analysis showed that only striatal and accumbens[3H]SCH 23390 binding decrease (10-12%) following phencyclidine and striatal [3H]spiperone binding increase (11%) after acute amphetamine were significant. It is assumed that such moderate dopamine receptor binding changes probably reflect the fast receptor regulation responses without important influence on a proposed drug-induced dopaminergic overactivity. The registered alterations of D1 receptor binding after phencyclidine are suggested to be capable to modify the activity of some other neural pathways in the basal ganglia and thus participate in a psychotic-like behaviour.

Amphetamine↗
PubMed2000

Characterization of calcium signaling by purinergic receptor-channels expressed in excitable cells.

ATP-gated purinergic receptors (P2XRs) are a family of cation-permeable channels that conduct Ca(2+) and facilitate voltage-sensitive Ca(2+) entry in excitable cells. To study Ca(2+) signaling by P2XRs and its dependence on voltage-sensitive Ca(2+) influx, we expressed eight cloned P2XR subtypes individually in gonadotropin-releasing hormone-secreting neurons. In all cases, ATP evoked an inward current and a rise in [Ca(2+)](i). P2XR subtypes differed in the peak amplitude of [Ca(2+)](i) response independently of the level of receptor expression, with the following order: P2X(1)R < P2X(3)R < P2X(4)R < P2X(2b)R < P2X(2a)R < P2X(7)R. During prolonged agonist stimulation, Ca(2+) signals desensitized with different rates: P2X(3)R > P2X(1)R > P2X(2b)R > P2X(4)R >> P2X(2a)R >> P2X(7)R. The pattern of [Ca(2+)](i) response for each P2XR subtype was highly comparable with that of the depolarizing current, but the activation and desensitization rates were faster for the current than for [Ca(2+)](i). The P2X(1)R, P2X(3)R, and P2X(4)R-derived [Ca(2+)](i) signals were predominantly dependent on activation of voltage-sensitive Ca(2+) influx, both voltage-sensitive and -insensitive Ca(2+) entry pathways equally contributed to [Ca(2+)](i) responses in P2X(2a)R- and P2X(2b)R-expressing cells, and P2X(7)R operated as a nonselective pore capable of conducting larger amounts of Ca(2+) independently on the status of voltage-gated Ca(2+) channels. Thus, Ca(2+) signaling by homomeric P2XRs expressed in an excitable cell is subtype-specific, which provides an effective mechanism for generating variable [Ca(2+)](i) patterns in response to a common agonist.

Animals↗
PubMed2000

Characterization of purinergic receptors and receptor-channels expressed in anterior pituitary cells.

Purinergic G protein-coupled receptors (P2YR) and ion-conducting receptor-channels (P2XR) are present in the pituitary. However, their identification, expression within pituitary cell subpopulations, and the ability to elevate intracellular Ca2+ concentration ([Ca2+]i) in response to ATP stimulation were incompletely characterized. Here we show that mixed populations of rat anterior pituitary cells express messenger RNA transcripts for P2Y2R, P2X2aR, P2X2bR, P2X3R, P2X4R, and P2X7R. The transcripts and functional P2Y2R were identified in lactotrophs and GH3 cells, but not in somatotrophs and gonadotrophs, and their activation by ATP led to an extracellular Ca2+-independent rise in [Ca2+]i in about 40% of cells tested. Lactotrophs and GH3 cells, but not somatotrophs, also express transcripts for P2X7R, P2X3R, and P2X4R. Functional P2X7R were identified in 74% of lactotrophs, whereas 50% of these cells expressed P2X3R and 33% expressed P2X4R. Coexpression of these receptor subtypes in single lactotrophs was frequently observed. Purified somatotrophs expressed transcripts for P2X2aR and P2X2bR, and functional receptors were identified in somatotrophs and gonadotrophs, but not in lactotrophs. Consistent with the cell-specific expression of transcripts for P2X2R and P2X3R, the expression of their functional heteromers was not evident in pituitary cells. Receptors differed in their capacities to elevate and sustain Ca2+ influx-dependent rise in [Ca2+]i during the prolonged ATP stimulation. These results indicate that the purinergic system of anterior pituitary is extremely complex and provides an effective mechanism for generating a cell- and receptor-specific Ca2+ signaling pattern in response to a common agonist.

Adenosine Triphosphate↗
PubMed2000

Local regulation of gonadotroph function by pituitary gonadotropin-releasing hormone.

Cultured rat pituitary cells and immortalized pituitary gonadotrophs (alphaT3-1 cells) express specific messenger RNA transcripts for GnRH and exhibit positive immunostaining for the GnRH peptide. Each cell type released GnRH during both static culture and perifusion, albeit in lesser amounts than cultured hypothalamic cells and GT1-7 neurons. In perifused pituitary cells, exposure to a GnRH agonist stimulated the release of GnRH as well as LH. In contrast, treatment with a GnRH receptor antagonist or with GnRH antiserum decreased basal LH release. In pituitary cell cultures, a small proportion of gonadotrophs exhibited high amplitude and low frequency baseline Ca2+ oscillations in the absence of GnRH stimulation. Such spontaneous oscillations were comparable to those induced by picomolar concentrations of GnRH and could be abolished by treatment with a GnRH antagonist. These in vitro findings indicate that locally produced GnRH causes low level activation of pituitary GnRH receptors, induces spontaneous intracellular Ca2+ oscillations, and contributes to basal LH secretion in cultured pituitary cells. In vivo, such autocrine or paracrine actions of pituitary-derived GnRH could provide a mechanism for the maintenance of optimal responsiveness of the gonadotrophs to pulses of GnRH arising in the hypothalamus. The presence and actions of GnRH in the anterior pituitary gland, the major site of expression of GnRH receptors, suggest that local regulatory effects of the neuropeptide could supplement the primary hypothalamic mechanism for the control of episodic gonadotropin secretion.

Animals↗
PubMed1999

Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs.

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca(2+)](i)) can arise from action-potential-driven Ca(2+) influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca(2+) influx to intracellular Ca(2+) release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca(2+)](i) oscillations, which rise from fluctuations in the influx of external Ca(2+) and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca(2+) stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca(2+)](i) signals. Bay K 8644, an L-type Ca(2+) channel agonist, initiated [Ca(2+)](i) signaling in quiescent cells, increased the amplitude of [Ca(2+)](i) spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca(2+) channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni(2+), a blocker of T-type Ca(2+) channels, abolished spontaneous [Ca(2+)](i) oscillations. Spiking was also abolished by the removal of extracellular Na(+) and by the addition of 10 mM Ca(2+), Mg(2+), or Sr(2+), the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca(2+) oscillator is capable of generating global Ca(2+) signals spontaneously and in response to agonist stimulation. The Ca(2+)-signaling activity of this oscillator is dependent on voltage-gated Ca(2+) influx but not on Ca(2+) release from intracellular stores.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1999

Expression of Ca(2+)-mobilizing endothelin(A) receptors and their role in the control of Ca(2+) influx and growth hormone secretion in pituitary somatotrophs.

The expression and coupling of endothelin (ET) receptors were studied in rat pituitary somatotrophs. These cells exhibited periods of spontaneous action potential firing that generated high-amplitude fluctuations in cytosolic calcium concentration ([Ca(2+)](i)). The message and the specific binding sites for ET(A), but not ET(B), receptors were found in mixed pituitary cells and in highly purified somatotrophs. The activation of these receptors by ET-1 led to an increase in inositol 1,4,5-trisphosphate production and the associated rise in [Ca(2+)](i) and growth hormone (GH) secretion. The Ca(2+)-mobilizing action of ET-1 lasted for 2-3 min and was followed by an inhibition of action potential-driven Ca(2+) influx and GH secretion to below the basal levels. As in somatostatin-treated cells, the ET-1-induced inhibition of spontaneous electrical activity and Ca(2+) influx was accompanied by the inhibition of adenylyl cyclase and by the stimulation of inward rectifier potassium current. In contrast to somatostatin, ET-1 did not inhibit voltage-gated Ca(2+) channels. During prolonged agonist stimulation a gradual recovery of Ca(2+) influx and GH secretion occurred. In somatotrophs treated with pertussis toxin overnight, the ET-1-induced Ca(2+)-mobilizing phase was preserved, but it was followed immediately by facilitated Ca(2+) influx and GH secretion. Both somatostatin- and ET-1-induced inhibitions of adenylyl cyclase activity were abolished in pertussis toxin-treated cells. These results indicate that the transient cross-coupling of Ca(2+)-mobilizing ET(A) receptors to the G(i)/G(o) pathway in somatotrophs provides an effective mechanism to change the rhythm of [Ca(2+)](i) signaling and GH secretion during continuous agonist stimulation.

Adenylate Cyclase Toxin↗
PubMed1999

Expression and responsiveness of P2Y2 receptors in human endometrial cancer cell lines.

In single endometrial carcinoma HEC-1A and Ishikawa cells, ATP induced a rapid and extracellular Ca2+-independent rise in cytosolic Ca2+ concentration ([Ca2+]i) in a dose-dependent manner, with an ED50 of about 10 microM. The spike phase was followed by a sustained plateau phase that was dependent on Ca2+ influx through voltage-insensitive Ca2+ channels, whose gating was controlled by a capacitative Ca2+ entry mechanism. ADP was less potent in raising the cystolic Ca2+ concentration, and AMP and adenosine were ineffective. The order of agonist potency for this receptor was ATP = UTP > ATP-gamma-S >> ADP. Several other agonists, including beta,gamma-methylene-ATP, 2-MeS-ATP, and BzATP were ineffective. This ligand-selective profile indicates the expression of the P2Y2R subtype in endometrial cells. Accordingly, reverse transcription-PCR using P2Y2 primers amplified the expected transcript from both cell lines. The coupling of these receptors to phospholipase C was confirmed by the ability of ATP to increase inositol 1,4,5-trisphosphate and diacylglycerol productions. These receptors are also coupled to the phospholipase D-1 pathway, leading to accumulation of phosphatidic acid. Activation of P2Y2 receptors by a slowly degradable ATP analog, ATP-gamma-S, was associated with a significant suppression of cell proliferation without affecting the cellular apoptosis. These results indicate that P2Y2 receptors may participate in control of the cell cycle of endometrial carcinoma cells.

Adenosine Diphosphate↗
PubMed1998

Identification of amino acid residues contributing to desensitization of the P2X2 receptor channel.

The P2X2 receptor (P2X2R) is a member of the ATP-gated ion channels that mediate Ca2+ entry in several tissues, including the brain, adrenal medulla, and pituitary. Alternative usage of cryptic splice sites in the primary P2X2R transcript accounts for the existence of several transcript types, one of which (P2X2-2R) encodes a functional channel. P2X2-2R lacks a stretch of cytoplasmic C-terminal amino acids (Val370-Gln438) and exhibits rapid and complete desensitization, whereas P2X2R desensitizes slowly and incompletely. The role of the C terminus in P2X2R desensitization was studied by generating several channel mutants and monitoring intracellular free Ca2+ changes in transfected single GT1-7 neurons. Deletion studies indicated that the Arg371-Ile391 segment of the P2X2R is required for sustained Ca2+ influx. To identify the important residues within this segment, three contiguous amino acids were sequentially changed to alanine. Only two of these replacement mutants, at Arg371-Thr372-Pro373 and Lys374-His375-Pro376, had an enhanced rate of desensitization. Single amino acid deletions in the P2X2R C terminus and a series of insertions of wild-type sequences into the corresponding spliced site identified four residues, Pro373-Lys374-His375-Pro376, required for sustained Ca2+ influx through agonist-occupied wild-type channels. Thus, it is likely that the Pro373-Pro376 sequence of P2X2R represents a functional motif that is critical for the development of the slow desensitization profile observed in these channels. Consequently, deletion of this motif by alternative splicing provides an effective mechanism for generating a channel with controlled Ca2+ influx.

Amino Acid Sequence↗
PubMed1998

Functional role of alternative splicing in pituitary P2X2 receptor-channel activation and desensitization.

Although ATP-gated ion channel (P2XR) expression is high among anterior pituitary cells, identification of the receptor subtypes and their selective expression within subpopulations of cell types, as well as their physiological role(s), are incompletely characterized. In this study, we focused on the expression and activity of the P2X2R subtype in anterior pituitary cells. Our results indicate that the primary P2X2R gene transcript in pituitary cells undergoes extensive alternative splicing, with generation of six isoforms. Two of these isoforms encode functional channels when expressed in GT1 or HEK293 cells: the wild-type P2X2R and the spliced isoform P2X2-2R, which lacks a stretch of carboxyl-terminal amino acids (Val370-Gln438). Four other clones showed different alterations, including an interfered reading frame starting in the first transmembrane domain and a 27-amino acid deletion in the large extracellular loop. When expressed separately or in combination with wild-type channels, these clones were nonfunctional. In single cell Ca2+ current and cytosolic Ca2+ concentration ([Ca2+)i) measurements, the P2X2R and P2X2-2R had similar EC50 values for ATP and time courses for activation and recovery from desensitization but differed significantly in their desensitization rates. The spliced isoform exhibited rapid and complete desensitization, whereas the wild-type channel desensitized slowly and incompletely. The mRNAs for wild-type and spliced channels were identified in enriched somatotroph, but not gonadotroph or lactotroph fractions. Expression of a functional ATP-gated channel in somatotrophs was confirmed by the ability of ATP to increase the frequency of [Ca2+]i spikes in spontaneously active cells or initiate spiking in quiescent cells. When voltage-gated Ca2+ influx was blocked, ATP increased [Ca2+]i, with a similar profile and EC50 to those observed in GT1 cells heterologously expressing wild-type or spliced P2X2R. The ligand-selectivity profile of native channels was consistent with the presence of P2X2R in somatotrophs. Finally, the desensitization rate of P2X2R in a majority of somatotrophs was comparable to that observed in neurons coexpressing wild-type and spliced channels. These data indicate that alternative splicing of P2X2R and coexpression of P2X2R and P2X2-2R subunits provide effective mechanisms for controlled cationic influx in somatotrophs.

Adenosine Triphosphate↗
PubMed1997

Acute amphetamine and/or phencyclidine effects on the dopamine receptor specific binding in the rat brain.

Psychotic-like behaviour was induced in rats with a single i.p. injection of AMPH (20 mg/kg b.w.) and/or PCP (10 mg/kg b.w.). The D1 and D2 dopamine receptor (DAR) specific binding of [3H]SCH 23390 and [3H]spiperone, respectively, during the 120 min period upon the treatment was examined on cryosections using computerized scanning and image analysis. AMPH, alone or in combination with PCP, induced a transient decrease of the D1 receptor specific binding in the striatum (30 min; AMPH, -18%; AMPH+PCP, -31%) and nucleus accumbens (30 min; AMPH, -30%; AMPH+PCP, -40%), which was completely abolished at the 120 min point. Only AMPH persistently elevated nigral D1 receptor specific binding. PCP-induced striatal and accumbal D1 receptor down-regulation was intensive throughout the 120 min period, while in the s. nigra it was non-significant. A significant increase of the D2 receptor specific binding was observed only 30 min after the treatment in striatum (AMPH, 15%; PCP, 16%; AMPH+PCP, 13%) and n. accumbens (AMPH, 16%). These alterations of DAR specific binding may reflect a regulation of the DAR and the changes in nigrostriatal and mesolimbic DA-ergic neurotransmission during an intensive drug-induced psychotic-like behavioral expression.

Amphetamine↗
PubMed1997

Wortmannin-sensitive and -insensitive steps in calcium-controlled exocytosis in pituitary gonadotrophs: evidence that myosin light chain kinase mediates calcium-dependent and wortmannin-sensitive gonadotropin secretion.

In cultured rat pituitary cells, increases in the cytosolic calcium concentration ([Ca2+]i) and LH release are induced by activation of GnRH receptors as well as by nonreceptor-mediated stimuli. Treatment of pituitary cells with the myosin light chain kinase (MLCK) inhibitor, wortmannin, attenuated GnRH-induced LH release. Wortmannin also reduced the LH responses to nonreceptor-mediated elevation of [Ca2+]i by ionomycin and activation of voltage-sensitive Ca2+ channels by Bay K 8644 or high K+, as well as Ca2+-induced LH release in permeabilized pituitary cells. The [Ca2+]i responses to these stimuli were unaltered in wortmannin-treated pituitary cells, indicating that this compound inhibits a Ca2+-dependent step in exocytosis without affecting Ca2+ signaling. In perifused pituitary cells, the GnRH-induced early spike phase of LH release was not affected by wortmannin, whereas the subsequent plateau phase was almost completely inhibited. No significant changes in GnRH-induced phospholipase D activity and diacylglycerol production were observed in wortmannin-treated pituitary cells during the sustained phase of agonist stimulation. Wortmannin also had no effect on LH responses to the protein kinase C activator, phorbol 12-myristate 13-acetate, further indicating that the attenuation of agonist-induced LH release is not related to inhibition of the diacylglycerol/protein kinase C pathway. In addition, agonist-induced LH release was attenuated by two other MLCK inhibitors, MS-347a and KT5926. These data suggest that MLCK mediates the downstream effects of Ca2+ on exocytosis, an action supported by the finding of wortmannin-sensitive phosphorylation of a 20-kDa protein in pituitary cells and alphaT3-1 gonadotrophs treated with GnRH, K+, and Bay K 8644. This protein was coprecipitated from pituitary extracts with a specific antibody to nonmuscle myosin IIB and comigrated with 20-kDa smooth muscle myosin light chain on SDS-PAGE. These results demonstrate that Ca2+ controls exocytosis through an initial wortmannin-insensitive step and a sustained wortmannin-sensitive step and suggest that the latter event in the cascade of cellular responses is dependent on phosphorylation of nonmuscle myosin IIB light chain by MLCK.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Expression of purinergic receptor channels and their role in calcium signaling and hormone release in pituitary gonadotrophs. Integration of P2 channels in plasma membrane- and endoplasmic reticulum-derived calcium oscillations.

The role of ATP as a positive feedback element in Ca2+ signaling and secretion was examined in female rat pituitary gonadotrophs. ATP and ADP, but not AMP or adenosine, induced a dose- and extracellular Ca2+-dependent rise in [Ca2+]i in identified gonadotrophs in a Mg2+- and suramin-sensitive manner. ATP, adenosine-5'-O-(3-thiotriphosphate), adenosine-5'-O-(1-thiotriphosphate), 2-methylthio-ATP, and 3'-O-(4-benzoyl)benzoyl-ATP were roughly equipotent in rising [Ca2+]i in gonadotrophs, while ADP was effective only at submillimolar concentration range, and none of these compounds permeabilized the cells. On the other hand, alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, and UTP were unable to induce any rise in [Ca2+]i. This pharmacological profile is consistent with expression of P2X2 and/or P2X5 purinergic receptor channels. Patch-clamp experiments showed that ATP induced an inward depolarizing current in gonadotrophs clamped at -90 mV, associated with an increase in [Ca2+]i. The ATP-induced [Ca2+]i response was partially inhibited by nifedipine, a blocker of voltage-sensitive Ca2+ channels (VSCC), but was not affected by tetrodotoxin, a blocker of voltage-sensitive Na+ channels. Thus, the P2-depolarizing current itself drives Ca2+ into the cell, but also activates Ca2+ entry through VSCC. In accord with this, low [ATP] induced plasma membrane-dependent [Ca2+]i oscillations in quiescent cells, and increased the frequency of spiking in spontaneously active cells. ATP-induced Ca2+ influx also affected agonist-induced and InsP3-dependent [Ca2+]i oscillations by increasing the frequency, base line, and duration of Ca2+ spiking. In addition, ATP stimulated gonadotropin secretion and enhanced agonist-induced gonadotropin release. ATP was found to be secreted by pituitary cells during agonist stimulation and was promptly degraded by ectonucleotidase to adenosine. These observations indicate that ATP represents a paracrine/autocrine factor in the regulation of Ca2+ signaling and secretion in gonadotrophs, and that these actions are mediated by P2 receptor channels.

Adenosine Triphosphate↗
PubMed1995

Effects of endothelin-1 on Ca2+ signaling and secretion in parathyroid cells.

It has been previously reported that parathyroid cells express endothelin (ET) receptors and secrete ET-1 in an extracellular Ca2+ concentration ([Ca2+]e)-dependent manner. Here, we examined the effects of ET-1 on intracellular signaling and parathyroid hormone (PTH) secretion in dispersed bovine parathyroid (bPT) cells, which comprise several cell types including epithelial and endothelial cells, in two cell lines, the rat parathyroid epithelial (PT-r) and the bovine parathyroid endothelial (BPE-1) cells. An RNA-polymerase chain reaction analysis revealed that both ETA and ETB receptors are expressed in bovine parathyroid tissue and BPE-1 cells, and only the ETA receptor is expressed in PT-r cells. PT-r cells also expressed an inositol 1,4,5-trisphosphate (Ins[1,4,5]P3) receptor, and ionomycin induced an increase in the intracellular Ca2+ concentrations ([Ca2+]i) in a Ca(2+)-deficient medium, indicating the presence of an operative intracellular Ca2+ pool in these cells. In cells bathed in 1 mM [Ca2+]e, ET-1 induced a rapid and transient increase in the Ins(1,4,5)P3 production, which was associated with a similar profile of increase in [Ca2+]i and with a peak response of about 800 nM. No changes in the profile of [Ca2+]i responses were observed in ET-1-stimulated cells in the presence of Ca2+ channel blockers, or in Ca(2+)-deficient medium, indicating that Ca2+ mobilization was not associated with Ca2+ entry. Furthermore, a sustained stimulation with ET-1 induced a decrease in [Ca2+]i below the prestimulatory level in a large population of cells, and the percentage of the cell population that shows the sustained decrease of [Ca2+]i increased in higher ET-1 concentrations. [Ca2+]i in PT-r cells was also controlled by a [Ca2+]e-dependent mechanism that changed [Ca2+]i from 28 to 506 nM in a 0.1-3 mM concentration range with an EC50 of 1.2 mM, which is comparable to that reported for bPT cells. In the same range of [Ca2+]e, PTH secretion from bPT cells was inhibited with an IC50 of 1 mM, and ET-1 increased PTH release in a dose-dependent manner but without affecting the IC50 for the [Ca2+]e-dependent inhibition. Thus, the parathyroid epithelial cells appear to respond to ET-1 in a unique way, and the ET autocrine system can be regarded as a possible mechanism to modulate the sensitivity of [Ca2+]e-dependent PTH release.

Animals↗