sq-23377 and Pituitary-Neoplasms

Topics: Calcium; Diazoxide; HEK293 Cells; Humans; Ionomycin; Pituitary Neoplasms; Respiration; Triazines; Verteporfin

The response of the L-type Ca2+ current (ICa,L) in pituitary GH3 cells to variations in the action potential (AP) waveform was examined using the whole-cell configuration of the patch-clamp technique. ICa,L evoked during an AP waveform exhibited an early and a late component. The early component occurred on the rising phase of the AP; the late component coincided with the falling phase. Prolonging the falling phase of the AP increased the Ca2+ charge carried by ICa,L, although the amplitude of the late ICa,L was reduced. Prolonging the peak voltage of the AP waveform, however, increased the amplitude of the late component. ICa,L inactivated during a train of AP waveforms. When Ba2+ was used as the charge carrier, current inactivation during a train of APs decreased. Likewise, ICa,L evoked by the AP templates with irregular bursting pattern was inactivated. When the repetitive firing of APs with depolarizing potentials was replayed to cells, Ca2+ entry was not only spread over the entire AP, but also occurred during the interspike voltage trajectory. After application of thyrotropin releasing hormone (TRH; 10 microM), ICa,L in response to rectangular pulses was increased and the current/voltage relation shifted slightly to more negative values. TRH (10 microM), thapsigargin (10 microM) or cyclopiazonic acid (30 microM) enhanced the late component of the AP-evoked ICa,L. TRH also attenuated the inactivation of ICa,L during a train of APs. These results indicate that in pituitary GH3 cells, the time course and kinetics of ICa,L during the AP waveforms is distinct from that evoked by rectangular voltage clamp. Changes in the shape and firing pattern of APs in GH3 cells can modulate Ca2+ influx through L-type Ca2+ channels. Ca2+ release from internal stores may affect the magnitude of AP-evoked ICa,L in these cells.

Topics: Action Potentials; Adenoma; Animals; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Dantrolene; Enzyme Inhibitors; Indoles; Ionomycin; Ionophores; Muscle Relaxants, Central; Nifedipine; Patch-Clamp Techniques; Pituitary Neoplasms; Rats; Thapsigargin; Thyrotropin; Tumor Cells, Cultured

Topics: Animals; Calcium; Ionomycin; Phosphatidylinositols; Pituitary Neoplasms; Protein Kinase C; Rats; Receptor, Serotonin, 5-HT2A; Receptors, Serotonin; Serotonin; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured

In mammosomatotropes GH3B6 cells, one of the primary responses to thyrotropin-releasing hormone (TRH) is the parallel induction of two proto-oncogenes, c-fos and jun B, which code for constituents of AP1 transcription factor. To better understand the mode of action of TRH and to look for possible functions of c-fos and jun B in these cells, we have investigated the role of different intracellular signals in the induction of each proto-oncogene on the one hand, and on prolactin (PRL) release and PRL gene expression on the other hand. Northern and dot-blot analyses revealed that the activation of protein kinase C (PKC)-, Ca(2+)- or adenylyl cyclase-dependent pathways acutely increased both c-fos and jun B transcripts. However, a gene specific responsiveness was revealed using phorbol 12-myristate 13-acetate (TPA) and several combined treatments. The simultaneous activation of PKC and Ca(2+)-dependent pathways resulted in synergistic stimulations of c-fos mRNA levels only. Consistently, ionomycin plus low doses of TPA solely reproduced the potent effect of TRH on c-fos transcripts. Data collected from TRH and TPA down-regulated cells indicated that TRH probably recruits TPA-dependent PKC isoforms for stimulating c-fos but not jun B transcripts. On the contrary, the TRH-induced stimulation of either proto-oncogene likely involves Ca(2+)-dependent mechanisms because calcium agonists and the peptide exert non-additive effects. Finally, the synergistic stimulations observed in response to TRH combined with forskolin, indicate that adenylyl cyclase-dependent mechanisms are interconnected with TRH-induced proto-oncogene expression. The overall study also reveals that among the agonists tested, the dihydropyridine Bay K 8644 and forskolin only were capable to induce a long-lasting stimulation of c-fos and jun B mRNA levels, concomitant to increased levels of PRL transcripts, as does TRH. Considering that AP1 is assumed to be involved in signal transmission from the cell surface to the nucleus, it might be thus proposed that a common stimulation of c-fos and jun B gene expression is possibly involved in the activation of the PRL gene. On the other hand, the systematic coincidence between acute PRL release and proto-oncogenes expression suggest a role for c-fos and jun B in the control of genes involved in the secretory process.

Topics: Adenylyl Cyclases; Animals; Calcium; Clone Cells; Colforsin; Cyclic AMP; Gene Expression Regulation, Neoplastic; Genes, fos; Genes, jun; Ionomycin; Neoplasm Proteins; Nuclear Proteins; Phorbol 12,13-Dibutyrate; Pituitary Gland, Anterior; Pituitary Neoplasms; Prolactin; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rats; RNA, Messenger; Signal Transduction; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

Previous observations that extracellular calcium (Ca2+) enhanced PRL mRNA levels posttranscriptionally in GH3 rat pituitary tumor cells were made using double-stranded transcription probes. The effects of Ca2+ and the Ca2+ ionophore, ionomycin, on PRL gene expression in GH3 and 235-1 cells were investigated using site- and strand-specific probes. Treatment of GH3 and 235-1 cells with 0.5 mM Ca2+ in serum-free medium specifically increased PRL mRNA levels by severalfold. In 235-1 but not GH3 cells PRL gene transcription was comparably induced by Ca2+. Use of single-stranded 5' and 3' probes revealed no antisense transcription, nor any Ca2+ effect on transcriptional elongation. Treatment with Ca2+ plus ionomycin inhibited PRL mRNA levels and gene transcription in both cell lines. Although their PRL gene transcription rates are similar, several basic differences were noted between the cell lines. The 235-1 cells exhibit a different profile of nuclear PRL pre-RNAs than GH3 cells. Also, mRNA levels for a Ca(2+)-regulated gene (GRP78) did not change in Ca(2+)-treated GH3 cells but decreased in Ca(2+)-treated 235-1 cells. Ionomycin treatment increased GRP78 mRNA levels in both cell lines. Thus, addition of extracellular Ca2+ appears to affect [Ca2+]i in 235-1 but not GH3 cells, while ionomycin affects [Ca2+]i in both cell lines. These data suggest that changing [Ca2+]i modulates PRL gene transcription. The comparative data suggest that posttranscriptional PRL regulation is Ca(2+)-regulated in GH3 cells, but is constitutive in 235-1 cells.

Topics: Animals; Base Sequence; Blotting, Northern; Calcium; DNA, Single-Stranded; Gene Expression Regulation, Neoplastic; Ionomycin; Molecular Sequence Data; Pituitary Neoplasms; Polymerase Chain Reaction; Prolactin; Rats; RNA Processing, Post-Transcriptional; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured

In GH4C1 cells, membrane depolarization induces a rapid and sustained increase in the cytosolic free calcium concentration ([Ca2+]i). In the present study we have investigated the role of [Ca2+]i in the regulation of basal intracellular pH (pHi). Depolarizing GH4C1 cells in buffer containing 0.4 mM extracellular Ca2+ decreased basal pHi from 7.02 +/- 0.04 to 6.85 +/- 0.03 (P less than 0.05). If the depolarization-induced influx of Ca2+ was inhibited by chelating extracellular Ca2+ or blocking influx through voltage-operated Ca2+ channels with nimodipine, no acidification was observed. Addition of TRH induced a rapid activation of Na+/H+ exchange in acidified cells, increasing pHi by 0.14 +/- 0.03 U. The action of TRH was blunted if extracellular Ca2+ was chelated; however, if influx of Ca2+ via voltage-operated channels was blocked by nimodipine, TRH still increased pHi. To deplete ATP, we incubated cells with 2-deoxy-D-glucose for 15-20 min and observed a decrease in basal pHi to 6.75 +/- 0.03 (P less than 0.05). No additional acidification was obtained when 2-deoxy-D-glucose-treated cells were depolarized, and no TRH-induced activation of Na+/H+ exchange was observed. Addition of ionomycin or 12-O-tetradecanoyl-phorbol-13-acetate separately to acidified cells had only modest effects on pHi; however, addition of 12-O-tetradecanoyl-phorbol-13-acetate and ionomycin together increased pHi markedly. We conclude that in GH4C1 cells, increasing [Ca2+]i reduces basal pHi through a mechanism dependent on influx of extracellular Ca2+ and independent of Na+/H+ exchange. In addition, elevation of [Ca2+]i and activation of protein kinase C act synergistically to enhance Na+/H+ exchange and increase pHi in acidified cells. Finally, normal cellular ATP is necessary for the activation of Na+/H+ exchange.

Topics: Adenosine Triphosphate; Amiloride; Animals; Calcium; Clone Cells; Cytosol; Deoxyglucose; Drug Interactions; Energy Metabolism; Enzyme Activation; Homeostasis; Hydrogen-Ion Concentration; Hypertonic Solutions; Ionomycin; Kinetics; Pituitary Neoplasms; Potassium; Protein Kinase C; Rats; Spectrometry, Fluorescence; Tetradecanoylphorbol Acetate; Thyrotropin-Releasing Hormone

Topics: Animals; Biological Transport, Active; Calcium; Calcium Channel Blockers; Cell Line; Egtazic Acid; Hippocampus; Ionomycin; Kinetics; Phytic Acid; Pituitary Neoplasms; Potassium; Rats; Synaptosomes; Thyrotropin-Releasing Hormone

Ca2+ has been recently reported to be required for high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). In the present investigation low concentrations of the Ca2+ ionophores, A23187 and ionomycin, were found to rapidly suppress the Ca2+-dependent component of protein synthesis in GH3 cells. More ionophore was required to inhibit amino acid incorporation into protein as extracellular Ca2+ was increased. Pre-existing inhibitions of protein synthesis produced by low concentrations of ionophore at low extracellular Ca2+ concentrations were reversed by adjustment to high extracellular Ca2+. Treatment with ionophore reduced the cellular contents of polysomes and 43 S preinitiation complex to values equivalent to those found for Ca2+-depleted cells. Average ribosomal transit times were unaffected by ionophore, and treated cells retained the ability to accumulate polysomes when incubated with cycloheximide. Cell types, such as HeLa and Chinese hamster ovary, that normally display only a modest Ca2+-dependent component of protein synthesis, manifested a strong underlying Ca2+ dependence in amino acid incorporation and polysome formation following treatment with low concentrations of ionophore. Protein synthesis in GH3 or HeLa cells during recovery from heat shock and arsenite treatment was not affected by cellular Ca2+ depletion or ionophore treatment. On the basis of these results, Ca2+ ionophore is proposed to inhibit Ca2+-dependent translational initiation through facilitating the mobilization of sequestered intracellular Ca2+.

Topics: Animals; Calcimycin; Calcium; Cell Line; Cells; Ethers; Eukaryotic Cells; HeLa Cells; Humans; Ionomycin; Peptide Chain Initiation, Translational; Pituitary Gland; Pituitary Neoplasms; Polyribosomes; Protein Biosynthesis

Previous studies have demonstrated that the high basal level of transcription of the rat PRL gene in pituitary tumor GH3 cells is dependent on [CA2+]e. In the present study, we have extended these findings by examining the effects of the Ca2+ ionophores, A23187 and ionomycin, on [Ca2+]i, and on PRL mRNA levels and glucose-regulated protein (GRP) mRNA levels in GH3 cells cultured in a low Ca2+, serum-free medium (SFM). Using digital imaging microscopy of individual Fura 2-loaded GH3 cells in SFM plus 0.4 mM CaCl2, extranuclear and nuclear [Ca2+] were both about 70 nM. Addition of 600 nM ionomycin increased these levels by 10-fold within minutes, and by about 45-fold after 120 min. As previously published, addition of 0.4 mM CaCl2 to GH3 cells cultured in SFM significantly increased PRL mRNA, and had little or no effect on GRP78 and GRP94 mRNA after 16 h. Addition of 0.4 mM CaCl2 plus 100 nM A23187 significantly increased GRP78 and GRP94 mRNA. Surprisingly, the Ca2+ ionophore significantly inhibited PRL gene expression below that obtained in 0.4 mM CaCl2 without A23187. This same pattern of stimulation of GRP78 gene expression, but inhibition of PRL gene expression, was observed with 125 and 600 nM ionomycin. Both Ca2+ ionophores had no effect on histone 3 mRNA, and A23187 depressed PRL gene expression at a concentration (50 nM) that did not affect protein synthesis. Although A23187 reproducibly lowered PRL mRNA levels, it slightly inhibited its degradation in cells in which RNA synthesis was blocked by actinomycin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Calcimycin; Calcium; Calcium Chloride; Carrier Proteins; Endoplasmic Reticulum Chaperone BiP; Extracellular Space; Gene Expression Regulation; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Ionomycin; Membrane Proteins; Molecular Chaperones; Pituitary Neoplasms; Prolactin; Rats; RNA, Messenger; Tumor Cells, Cultured

Receptor-mediated regulation of prolactin synthesis by 1,25-dihydroxycholecalciferol (1,25(OH)2D3) in the pituitary cell strain GH4C1 is dependent on the concentration of extracellular calcium. We have now investigated the actions of 1,25(OH)2D3 on cytosolic free calcium concentrations [( Ca2+]i) in these cells using the fluorescent indicator quin2. Basal resting [Ca2+]i was unchanged in cells treated with 1 nM 1,25(OH)2D3 either acutely (from 0 to 15 min) or for periods of up to 48 h. However, the initial peak of the biphasic change in [Ca2+]i induced by thyrotropin-releasing hormone (TRH) was enhanced more than twofold in cells pretreated for 24 or 48 h with 1,25(OH)2D3. This 1,25(OH)2D3-enhanced calcium response was restricted to the initial phase of TRH action; the secondary plateau phase was unaffected. Neither the affinity nor number of TRH receptors nor the early time course of [3H]MeTRH binding to GH4C1 cells were affected by pretreatment with 1,25(OH)2D3. Because TRH binding was not altered, four sites along the intracellular signal transduction pathway of TRH action were examined. Neither protein kinase C activation nor inositol polyphosphate accumulation were enhanced in response to TRH, in 1,25(OH)2D3 pretreated cells, indicating that phosphatidylinositol hydrolysis was unchanged by pretreatment. A low concentration of ionomycin was used to probe the size of the nonmitochondrial intracellular calcium pool that is sensitive to TRH. Ionomycin was not able to mobilize more calcium from 1,25(OH)2D3 pretreated cells, indicating that TRH-responsive intracellular calcium stores were probably not enhanced by pretreatment. Chelation of extracellular calcium, however, did eliminate enhancement of the TRH response in 1,25(OH)2D3-pretreated cells. We conclude that 1,25(OH)2D3 modulates acute dynamic changes in [Ca2+]i induced by TRH without affecting basal [Ca2+]i. The mechanism of the enhanced response of 1,25(OH)2D3-pretreated cells to TRH appears to depend upon a postreceptor event independent of phosphatidylinositol hydrolysis that involves increased calcium conductance at the level of the plasma membrane. A less likely explanation involves enhancement of intracellular calcium stores in an ionomycin-resistant, EGTA-sensitive, TRH-mobilizable reservoir.

Topics: Animals; Calcitriol; Calcium; Cell Line; Cytosol; Ethers; Inositol Phosphates; Ionomycin; Pituitary Neoplasms; Protein Kinase C; Rats; Receptors, Neurotransmitter; Receptors, Thyrotropin-Releasing Hormone; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

Thyrotropin-releasing hormone (TRH) induces rapid and transient conversion of protein kinase C (Ca2+/phospholipid-dependent enzyme) from a soluble to a particulate-bound form in GH4C1 rat pituitary cells. Ionomycin (200 nM), a calcium ionophore, had no effect by itself on the subcellular distribution of protein kinase C. However, pretreatment of the cells with 200 nM ionomycin inhibited by greater than 50% the ability of TRH to cause translocation of protein kinase C from the cytosol to the particulate cell fraction. Inhibition by ionomycin required that the cells be incubated with the ionophore for at least 10 s before TRH addition. Ionomycin pretreatment did not alter the kinetics of TRH-induced protein kinase C redistribution. Incubation of the cells with 43 mM potassium prior to TRH addition almost completely reversed the inhibition induced by ionomycin. We propose that the mechanism by which ionomycin attenuates TRH action on protein kinase C may involve the capacity of the ionophore to empty the intracellular calcium reservoir which normally releases calcium into the cytosol in response to TRH. Our result provides evidence that the rise in intracellular calcium, which accompanies diacylglycerol formation following TRH action on polyphosphatidylinositide hydrolysis, may be required to achieve maximal conversion of protein kinase C to its presumed active, membrane-bound form in these cells.

Topics: Animals; Biological Transport; Cell Line; Ethers; Ionomycin; Kinetics; Pituitary Neoplasms; Potassium; Protein Kinase C; Thyrotropin-Releasing Hormone

The 7315c prolactin-secreting tumor cell was used as a model of a normal pituitary cell in order to study the enhancement by adenosine 3',5'-cyclic monophosphate (cAMP) of calcium-evoked hormone release. Forskolin and, by implication, cAMP had little effect on basal hormone release during a 10-min incubation period. Ionomycin and a high potassium concentration, treatments which enhanced the cytosolic calcium concentration, increased hormone release. When cells were exposed to forskolin prior to and during a challenge with either ionomycin or high potassium, a synergistic effect on prolactin release was observed. 8-Bromoadenosine 3',5'-cyclic monophosphate mimicked forskolin in enhancing ionomycin-evoked prolactin release while having little effect of its own on hormone release. Forskolin did not alter the increase in cytosolic calcium concentration elicited by either ionomycin or high potassium, nor did it increase the potency of ionomycin in enhancing prolactin release. The calcium channel antagonist, D-600, did not alter ionomycin-induced release or its enhancement by forskolin; D-600 blocked potassium-induced prolactin release. Ionomycin had no effect on basal cAMP synthesis by tumor cells and inhibited slightly the forskolin-induced increase in nucleotide synthesis. The results suggest that cAMP acts, at a site distal to the entry of calcium into the cytosol, to enhance the amount of prolactin released in response to an increase in the cytosolic calcium concentration.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Aminoquinolines; Animals; Calcium; Cell Line; Colforsin; Cyclic AMP; Cytosol; Ethers; Ionomycin; Pituitary Neoplasms; Potassium; Prolactin; Rats

In the GH4C1 strain of rat pituitary cells, ionomycin, a divalent cation ionophore, induces a rapid and transient spike in cytosolic free Ca2+ concentrations [( Ca2+]i) similar to that induced by the Ca2+-mobilizing hormone thyrotropin-releasing hormone (TRH). To test directly the hypothesis that ionomycin causes the spike in [Ca2+]i by altering cellular Ca2+ stores, we have measured ionomycin-induced changes in 45Ca2+ fluxes and have compared these to previously characterized changes induced by TRH. Ionomycin (half-maximal concentration = 30 nM) rapidly (within 1 min) induced a release into the medium of 50-60% of cell-associated 45Ca2+, paralleling the spike in [Ca2+]i. The ionomycin-induced 45Ca2+ efflux was greater than with TRH, and TRH did not induce further 45Ca2+ efflux in the presence of ionomycin. Ionomycin pretreatment blocked induction of the spike in [Ca2+]i elicited by TRH but did not alter basal or TRH-induced enhancement of inositol phosphate levels. These results provide evidence that the spike in [Ca2+]i induced by ionomycin or TRH is produced largely by release of Ca2+ into the cytosol from the same intracellular pool, followed by rapid extrusion of the released Ca2+ into the extracellular space. However, unlike TRH, ionomycin appears to release cellular Ca2+ directly, acting as an ionophore, without the generation of known second messengers.

Topics: Animals; Calcium; Cell Line; Ethers; Ionomycin; Kinetics; Pituitary Neoplasms; Rats; Thyrotropin-Releasing Hormone

In clonal rat pituitary cells (GH cells), thyrotropin-releasing hormone (TRH) induced a pattern of changes in cytosolic free calcium concentrations [( Ca2+]i) composed of two phases: an acute spike phase to micromolar levels which decayed (t1/2 = 8 s) to a near-basal concentration and then rose to a prolonged plateau phase of elevated [Ca2+]i (as measured using Quin 2). Closely following these changes in [Ca2+]i, TRH stimulated a rapid "spike phase" of pronounced, but brief, enhancement of the rate of prolactin and growth-hormone secretion and then a "plateau phase" of prolonged enhancement. These two phases were dissociated using two classes of pharmacologic agents: the ionophore ionomycin, and a calcium channel antagonist nifedipine. Ionomycin (100 nM) specifically blocked (less than 90%) the spike phase of TRH action by rapidly emptying the TRH-regulated reservoir of cellular Ca2+ to generate a TRH-like spike in [Ca2+]i; nifedipine inhibited (less than 50%) the plateau phase of TRH-induced changes in [Ca2+]i and hormone secretion by preventing Ca2+ influx through voltage-dependent Ca2+ channels. These agents demonstrated that the TRH-induced spike in [Ca2+]i in GH cells is caused by release of an ionomycin-sensitive pool of cellular Ca2+ with a small component (10%) due to influx of extracellular Ca2+. The TRH-induced plateau in [Ca2+]i is due to influx of extracellular Ca2+, about half of which enters through voltage-dependent calcium channels and half of which enters via nifedipine/verapamil-insensitive influx. The TRH-induced spike in [Ca2+]i led to a burst in hormone secretion, and the plateau in [Ca2+]i produced a prolonged enhancement of secretion; the spike and plateau phases were generated independently by TRH. A spike in [Ca2+]i is necessary, but not sufficient, to induce burst release of hormone, while the prolonged rate of hormone secretion is intimately related to the steady-state [Ca2+]i.

Topics: Animals; Calcium; Cell Line; Cytosol; Ethers; Growth Hormone; Ionomycin; Kinetics; Nifedipine; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Verapamil

SRIF inhibits ACTH secretion by AtT20/D16v (D16) mouse pituitary cells stimulated by high (50 mM) extracellular concentrations of K+ or by divalent cation ionophores. Although stimulation of ACTH secretion by K+ requires extracellular Ca+2, the response is invariant over medium Ca+2 concentrations of 0.003-1 mM; with Ca+2 concentrations from 1-5 mM there is a dramatic amplification of the secretory response. SRIF at concentrations of 10(-8) M completely inhibits the secretory response to K+ at Ca+2 concentrations between 0.2 and 1 mM; with increasing medium Ca+2 above 1 mM there is a progressive attenuation of SRIF-inhibition. At concentrations of 5 mM, Ca+2 alone can serve as an ACTH secretagogue. The ionophore ionomycin stimulates ACTH secretion in a Ca+2-dependent manner with a half-maximal effect at 5 X 10(-6) M ionomycin. The secretory response to ionomycin and to X537A is inhibited by at least 50% by SRIF. The secretory response to K+ is accompanied by a rapid and sustained increase in 45Ca+2 uptake, whereas the ionophores ionomycin, X537A, and A23187 increase Ca+2 efflux. SRIF does not affect Ca+2 movement across D16 cell membranes in response to either K+ or ionophores. These results show that an increase in intracellular Ca+2 is an effective stimulus to ACTH secretion by D16 cells and inhibition of ACTH secretion by SRIF is not effected by interference with the stimulus-elicited increase in intracellular Ca+2.

Topics: Adrenocorticotropic Hormone; Animals; Calcimycin; Calcium; Cell Line; Ethers; Ionomycin; Lasalocid; Mice; Pituitary Gland; Pituitary Neoplasms; Potassium; Somatostatin; Time Factors