inositol-1-4-5-trisphosphate and Pituitary-Neoplasms

The Ins(1,4,5)P3 regioisomers, Ins(1,4,6)P3 and Ins(1,3,6)P3, which can mimic the 1,4,5-arrangement on the inositol ring of Ins(1,4,5)P3, were examined for Ca2+ release by using four types of saponin-permeabilized cell possessing various abundances of receptor subtypes, with special reference to the relation of potency to receptor subtype. Ins(1,4,6)P3 and Ins(1,3,6)P3 were weak agonists in rat basophilic leukaemic cells (RBL cells), which possess predominantly subtype II receptors, with respective potencies of 1/200 and less than 1/500 that of Ins(1,4,5)P3 [the EC50 values were 0.2, 45 and more than 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively]. Similar rank order potencies were also evaluated for the displacement of [3H]Ins(1,4,5)P3 bound to RBL cell membranes by these regioisomers. However, they caused Ca2+ release from GH3 rat pituitary cells possessing predominantly subtype I receptors more potently; Ins(1,4,6)P3 and Ins(1,3,6)P3 evoked release at respective concentrations of only one-third and one-twentieth that of Ins(1,4,5)P3 (the EC50 values were 0.4, 1.2 and 8 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In COS-1 African green-monkey kidney cells, with the relative abundances of 37% of the subtype II and of 62% of the subtype III receptor, potencies of 1/40 and approx. 1/200 for Ins(1, 4,6)P3 and Ins(1,3,6)P3 respectively were exhibited relative to Ins(1,4,5)P3 (the EC50 values were 0.4, 15 and approx. 80 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). In HL-60 human leukaemic cells, in spite of the dominant presence of subtype I receptors (71%), similar respective potencies to those seen with COS-1 cells were exhibited (the EC50 values were 0.3, 15 and approx. 100 microM for Ins(1,4,5)P3, Ins(1,4,6)P3 and Ins(1,3,6)P3 respectively). These results indicate that these regioisomers are the first ligands that distinguish between receptor subtypes; the present observations are of significance for the future design of molecules with enhanced selectivity.

Topics: Animals; Calcium; Calcium Channels; COS Cells; HL-60 Cells; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Leukemia, Basophilic, Acute; Molecular Conformation; Pituitary Neoplasms; Rats; Receptors, Cytoplasmic and Nuclear; Stereoisomerism; Tumor Cells, Cultured

Desensitization of the cholecystokinin (CCK) octapeptide (CCK-8)-induced rise in intracellular free calcium concentration ([Ca2+]i) was characterized in GH3 cells, a pituitary tumor cell line, which are known to possess CCKB receptor subtype. The CCK-8-induced [Ca2+]i transient was reduced following the initial application of CCK-8. A similar desensitization of the CCK-8-induced response was observed following the first application of thyrotropin-releasing hormone (TRH). By contrast, the TRH-induced response was not desensitized by the preceding application of CCK-8. Desensitization of the CCK-8-induced [Ca2+]i transient was associated with diminished inositol 1,4,5-trisphosphate formation. The recovery of desensitization of the CCK-8-induced response was delayed by a phosphoserine/phosphothreonine phosphatase inhibitor, calyculin A (100 nM). The responsiveness to CCK-8 was also reduced by phorbol 12,13-dibutyrate (PDBu), and this effect of PDBu was completely abolished by preincubation with staurosporine. Staurosporine significantly attenuated the desensitization caused by preincubation with CCK-8, but this effect was too small to attribute the desensitization to the protein kinase C transduction pathway alone. It is likely that desensitization of CCK receptors involves multiple transduction pathways.

Topics: Alkaloids; Calcium; Drug Tolerance; Enzyme Activation; Inositol 1,4,5-Trisphosphate; Marine Toxins; Oxazoles; Phorbol 12,13-Dibutyrate; Phosphoric Monoester Hydrolases; Pituitary Neoplasms; Protein Kinase C; Receptors, Cholecystokinin; Sincalide; Staurosporine; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

Epidermal growth factor (EGF) stimulated the prolactin (PRL) synthesis and release from the GH4C1 cells in a dose-dependent manner. The ED50 was between 10(-11) and 10(-10) mol/l. The maximal effect was obtained at 10(-9) mol/l EGF for the release, and 10(-8) mol/l EGF for the synthesis. EGF stimulated the release of PRL from cell perfusion columns after a lag period of about 30 s. The maximal secretion of PRL occurred about 60 s after the start of stimulation. The PRL secretion declined to basal levels within 2 min. The EGF-stimulated PRL release was additive to the secretion evoked by thyrotropin-releasing hormone (TRH) and vasoactive intestinal peptide (VIP). An instantaneous increase in the intracellular concentration of free calcium, [Ca2+]i, of the GH4C1 cells was observed after the administration of EGF. EGF modified neither the basal nor the TRH-stimulated inositoltrisphosphate production in the GH4C1 cells, and EGF did not show any effect on the cyclic adenosine monophosphate production of these cells.

Topics: Animals; Calcium; Cyclic AMP; Dose-Response Relationship, Drug; Drug Synergism; Epidermal Growth Factor; Inositol 1,4,5-Trisphosphate; Pituitary Gland; Pituitary Neoplasms; Prolactin; Rats; Thyrotropin-Releasing Hormone; Time Factors; Tumor Cells, Cultured; Vasoactive Intestinal Peptide

Previous work in [3H]inositol-labelled GH3 pituitary tumor cells stimulated with thyrotropin-releasing hormone (TRH) reported the existence of at least ten distinct [3H]inositol-containing substances which were identified as different inositol mono-, bis- and tris-phosphate isomers [1]. Here a complete kinetic study of the dephosphorylation pathways of the second messenger Ins(1,4,5)P3 is reported in GH3 cell homogenates, identifying a new intermediate, Ins(4,5)P2, in the metabolism of the second messenger. in vitro results obtained with exogenous substrates are compared with in vivo results obtained measuring levels of the endogenous [3H]inositol-labelled isomers that participate in the dephosphorylation pathways of Ins(1,4,5)P3 in resting and TRH-stimulated GH3 cells. The effect of Li+ on the activity of the different phosphatases involved in these pathways is studied as well.

Topics: Animals; Cell Line; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Inositol Polyphosphate 5-Phosphatases; Kinetics; Lithium; Phosphoric Monoester Hydrolases; Phosphorylation; Pituitary Neoplasms; Rats; Thyrotropin-Releasing Hormone; Tritium; Tumor Cells, Cultured

The actions of thapsigargin (Tg), a plant sesquiterpene lactone, on Ca2+ homeostasis were investigated in digitonin-permeabilized GH4C1 rat pituitary cells. Tg (1 microM) caused a rapid and sustained increase in ambient Ca2+ concentration [( Ca2+]) and inhibited the rise in [Ca2+] induced by subsequent addition of TRH (100 nM), inositol 1,4,5-trisphosphate (IP3, 10 microM), or the nonhydrolyzable GTP analogue guanosine 5'-0-(3-thiotriphosphate) (GTP gamma S, 10 microM). However, neither IP3 nor GTP gamma S pretreatment, which themselves release sequestered Ca2+, prevented the Ca2+ accumulation induced by Tg. Pretreatment with heparin (100 micrograms/ml, 10 min), an IP3 receptor antagonist, did not affect Ca2+ accumulation induced by Tg, although it abolished the rise in [Ca2+] induced by IP3. The ability of Tg to increase [Ca2+] was dependent on added ATP. We conclude that, in GH4C1 cells, Tg acts, in part, on TRH-, IP3- and GTP gamma S-sensitive Ca2+ pools; however, Tg also acts on an ATP-dependent pool of intracellular Ca2+ which is not sensitive to TRH, IP3 or GTP gamma S, indicating a complexity of intracellular Ca2+ pools not previously appreciated in these cells.

Topics: Adenosine Triphosphate; Animals; Calcium; Calcium Channels; Cell Line; Cell Membrane Permeability; Digitonin; Guanosine 5'-O-(3-Thiotriphosphate); Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Kinetics; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Terpenes; Thapsigargin; Thyrotropin-Releasing Hormone

Signal transduction by thyrotropin-releasing hormone (TRH) and carbamylcholine (CCH) in some cells is mediated by inositol lipid hydrolysis forming the second messengers, inositol 1,4,5-trisphosphate (I-1,4,5-P3) and 1,2-diacylglycerol, and causing elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i). In mouse thyrotropic tumor (TtT) cells, maximally effective doses of TRH caused biphasic stimulation of thyroid-stimulating hormone (TSH) secretion, whereas CCH stimulated monophasic sustained TSH secretion without a burst phase. TRH, at maximally effective doses, stimulated a rapid marked increase in I-1,4,5-P3 which was associated with a rapid elevation of [Ca2+]i to approximately 1000 nM, whereas maximally effective doses of CCH caused little increase in I-1,4,5-P3 and no burst elevation of [Ca2+]i. Both TRH and CCH caused sustained modest (to 210-280 nM) elevations of [Ca2+]i which were inhibited by voltage-sensitive channel-blocking agents and stimulated sustained hydrolysis of inositol lipids. CCH-like responses were observed when TtT cells were stimulated by low doses of TRH. In TtT cells prepared from five tumors, the ratio of the number of TRH receptors to muscarinic receptors ranged from 10 to 40:1. Lastly, CCH-like responses were observed with maximally effective doses of TRH when the TRH receptor number was down-regulated to a level similar to that of muscarinic receptors. These data suggest that the kinetic pattern of stimulated TSH secretion caused by secretagogues that use the inositol lipid signal transduction pathway is determined by the density of receptors. In particular, there appears to be a minimal number of receptor-ligand complexes which is required to generate rapidly sufficient I-1,4,5-P3 to release intracellular Ca2+ and cause a secretory burst.

Topics: Animals; Calcium; Carbachol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Mice; Pituitary Neoplasms; Receptors, Muscarinic; Second Messenger Systems; Sugar Phosphates; Thyrotropin; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured

Previous studies demonstrated a multiplicity of isomers of inositol phosphates in GH3 rat pituitary tumour cells. In order to determine their origin, we have investigated the metabolism of radiolabelled inositol phosphates (IPn) in GH3 cell homogenates by using h.p.l.c. I(1,4,5)P3 is either phosphorylated to I(1,3,4,5)P4 (in the presence of ATP) or dephosphorylated to I(1,4)P2 (in the absence of ATP). I(1,4)P2 is dephosphorylated to I(4)P (greater than 95%). I(1,3,4,5)P4 hydrolysis yields two products. By using dual-labelled [32P, 3H]I(1,3,4,5)P4 with 32P in either the 3 or the 4/5 position, we have identified the probable configuration of these isomers. The predominant (greater than 97%) IP3 formed is I(1,3,4)P3, with a minor I(1,4,5)P3 peak. Subsequent I(1,3,4)P3 hydrolysis yields two IP2 isomers [the major (approximately equal to 85%) is I(3,4)P2; the minor (approximately equal to 15%) is I(1,3)P2] and two IP isomers (the major (approximately equal to 90%) is I(3)P [L-I(1)P], the minor I(4)P). IP5 is very slowly dephosphorylated to and IP4 of undetermined isomeric configuration. We have also examined GH3 cell lipids for the presence of phosphoinositides either more highly phosphorylated than PIP2 (as potential sources of the IP4/IP5 and IP6 in these cells) or phosphorylated in positions other than 1, 4 and 5, and have been unable to find evidence of either. These data suggest two main routes of metabolism for I(1,4,5)P3 in GH3 cells: either (1) phosphorylation to I(1,3,4,5)P4, and the subsequent consecutive dephosphorylation to I(1,3,4)P3, I(3,4)P2 and finally L-I(1)P [D-I(3)P]; or (2) dephosphorylation to I(1,4)P2 and, subsequently, I(4)P.

Topics: Animals; Cell Line; Chromatography, High Pressure Liquid; Hydrolysis; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Pituitary Neoplasms; Rats; Sugar Phosphates; Tumor Cells, Cultured

TRH evoked a complex electrical membrane response in Xenopus laevis oocytes injected with either total cytosolic or poly(A)(+)-enriched RNA from GH3 pituitary cells but not in uninjected oocytes. A typical response consisted of a transient, rapid depolarizing current followed by a prolonged depolarizing current with superimposed current fluctuations. The reversal potentials of the rapid and the slow components of the response were -23.0 and -22.6 mV, respectively, and were markedly affected by CI- concentration indicating that the TRH response was mainly an increase in Cl- conductance. The response to TRH was dose dependent and was inhibited by the TRH antagonist, chlordiazepoxide. TRH caused rapid hydrolysis of labeled phosphatidylinositol 4,5-bisphosphate and a marked, prolonged increase in 45Ca2+ efflux from injected oocytes. The depolarizing response to TRH was not diminished in oocytes incubated in a Ca2(+)-free medium, but was inhibited by microinjection of EGTA. These data suggest that TRH evokes an electrophysiological response in oocytes injected with RNA from GH3 cells via activation of the same biochemical pathway that mediates its actions in GH3 cells. This pathway involves hydrolysis of phosphatidylinositol 4,5-bisphosphate, forming inositol trisphosphate that causes mobilization of cellular Ca2+. We suggest that oocytes injected with GH3 cell RNA, because of their large size and easy access to their intracellular milieu, will be a useful intact cell model in which to define the molecular details of signal transduction by TRH.

Topics: Animals; Calcium; Hydrolysis; Inositol 1,4,5-Trisphosphate; Lipids; Membrane Potentials; Oocytes; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Pituitary Neoplasms; RNA, Messenger; RNA, Neoplasm; Thyrotropin-Releasing Hormone; Tumor Cells, Cultured; Xenopus laevis

Thyrotropin-releasing hormone (TRH) increases rapidly two potential intracellular signals, inositol trisphosphate (IP3) and free cytosolic calcium ([Ca2+]i), for stimulated prolactin release and synthesis in GH4C1 rat pituitary cells. We have examined the temporal relationships between TRH-enhanced formation of inositol phosphates and TRH-elevated [Ca2+]i. TRH-enhanced IP3 content was closely paralleled by the initial phase of TRH-elevated [Ca2+]i. To investigate receptor-effector coupling for these rapid actions of TRH, we examined their dependence on receptor number in five GH4C1 variant strains containing 0-2.6 X 10(5) receptor sites/cell. We found that receptor number (up to 1.7 X 10(5)/cell) was limiting for TRH-enhanced IP3 formation as well as for both the initial burst and plateau phases of TRH-elevated [Ca2+]i. The ED50 for rapid (5 s) TRH-stimulated IP3 formation was higher than for other sustained TRH actions in these cells, and we postulated that the initial TRH receptor interactions occur with rapid dissociation kinetics. To test this hypothesis, we performed rapid dilution experiments following a 1-s stimulation and found that TRH-stimulated IP3 formation decreased within 4 s of dilution and disappeared within 60 s at which time fresh TRH could restimulate IP3 formation. We conclude that receptor occupancy is the limiting step for TRH-stimulated IP3 formation and elevated [Ca2+]i and that maximal TRH action requires multiple rapid interactions between TRH and its receptor.

Topics: Animals; Calcium; Cell Line; Dose-Response Relationship, Drug; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Sugar Phosphates; Thyrotropin-Releasing Hormone; Time Factors

Thyrotropin-releasing hormone (TRH) stimulated a rapid rise in inositol trisphosphate (IP3) formation and prolactin release from 7315c tumor cells. The potencies (half-maximal) of TRH in stimulating IP3 formation and prolactin release were 100 +/- 30 and 140 +/- 30 mM, respectively. Pretreatment of the cells with pertussis toxin (for up to 24 h) had no effect on either process. Pretreatment of the cells with cholera toxin (30 nM for 24 h) also failed to affect basal or TRH-stimulated IP3 formation. TRH was also able to stimulate IP3 formation with a half-maximal potency of 118 +/- 10 nM in a lysed cell preparation of 7315c cells; the TRH-stimulated formation of IP3 was enhanced by GTP. 5'-Guanosine gamma-thiotriphosphate (GTP gamma S) and 5'-guanylyl imidodiphosphate (Gpp(NH)p), nonhydrolyzable analogs of GTP, stimulated IP3 formation in the absence of TRH with half-maximal potencies of 162 +/- 50 and 7500 +/- 4300 nM, respectively. In contrast to the lack of effect of pertussis toxin on the TRH receptor system, treatment of 7315c cells with pertussis toxin for 3 h or longer completely abolished the ability of morphine, an opiate agonist, to inhibit either adenylate cyclase activity or prolactin release. During this 3-h treatment, pertussis toxin was estimated to induce the endogenous ADP ribosylation of more than 70% of Ni, the inhibitory GTP-binding protein. GTP gamma S and Gpp(NH)p inhibited cholera toxin-stimulated adenylate cyclase activity (presumably by acting at Ni) with half-maximal potencies of 25 +/- 9 and 240 +/- 87 nM, respectively. Finally, Gpp(NH)p was also able to inhibit the [32P]ADP ribosylation of Ni with a half-maximal potency of 300 nM. These results suggest that a novel GTP-binding protein, distinct from Ni, couples the TRH receptor to the formation of IP3.

Topics: Adenosine Diphosphate Ribose; Adenylate Cyclase Toxin; Adenylyl Cyclases; Animals; Cell Line; Female; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Guanylyl Imidodiphosphate; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Pertussis Toxin; Pituitary Neoplasms; Prolactin; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Thionucleotides; Thyrotropin-Releasing Hormone; Type C Phospholipases; Virulence Factors, Bordetella

Thyrotropin-releasing hormone (TRH), like numerous other Ca2+-mobilizing agonists, has been found to stimulate polyphosphoinositide hydrolysis in responsive cells. The present studies further clarify the mechanism of action of this peptide hormone by demonstrating direct in vitro effects of TRH on polyphosphoinositide hydrolysis in GH3 pituitary cell membranes. Membranes from [3H]myoinositol-labeled cells were found to generate inositol bis- and tris- but not monophosphate upon incubation. Inositol polyphosphate generation was stimulated 2-3-fold by nanomolar concentrations of TRH in a reaction which was potentiated by micromolar concentrations of GTP; hormone-stimulated hydrolysis observed in the absence of GTP was fully antagonized by guanosine 5'-O-(2-thiodiphosphate). Guanosine 5'-O-(3-thiotriphosphate), Ca2+, and sodium fluoride also activated phosphoinositide hydrolysis in vitro. Stimulated inositol polyphosphate generation was accompanied by stimulated 1,2-diacylglycerol formation. Evidence that both phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 4-phosphate served as substrates for the activated phosphoinositide phosphodiesterase is presented. Pretreatment of GH3 cells with cholera or pertussis toxin did not influence stimulated hydrolysis in membranes. It is concluded that the TRH receptor directly regulates polyphosphoinositide hydrolysis in GH3 cell plasma membranes by a GTP-dependent process. The GTP dependence does not appear to be mediated through a cholera or pertussis toxin substrate and may involve a novel GTP-binding protein (NP).

Topics: Animals; Cell Line; Cell Membrane; Cholera Toxin; Diglycerides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hydrolysis; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pituitary Gland; Pituitary Neoplasms; Rats; Thionucleotides; Thyrotropin-Releasing Hormone

A number of clonal cell lines derived from a rat pituitary tumour, collectively termed GH cells, have retained a range of differentiated cell functions, including their ability to secrete the hormones prolactin and growth hormone in response to stimuli such as thyrotropin-releasing hormone (TRH). The mechanisms underlying this release process involve, at least in part, an increase in cytosolic free calcium levels, and the cells have proved useful as a model system in studies of receptor-controlled calcium mobilization. The initial response of the cells to the addition of TRH now appears to be the interaction of the occupied TRH receptor with a GTP-binding protein. A sophisticated signalling system is then activated which initially involves the phosphodiesteratic hydrolysis of phosphatidylinositol 4,5-bisphosphate to 1,2-diacylglycerol and inositol 1,4,5-trisphosphate. Both of these products are important intracellular messengers, and their formation leads to a plethora of biochemical and electrical changes which culminate in the biphasic release of hormone from the cell. The changes in cytosolic free calcium that occur following TRH addition follow a complex temporal pattern. Within 1 s, the concentration starts to increase from a resting level, in the range 100-150 nmol l-1, to a peak value of around 1 mumol l-1 which is attained within 6-8 s. This 'spike' of calcium is almost exclusively derived from intracellular stores, probably the endoplasmic reticulum, in response to the formation of inositol 1,4,5-trisphosphate. With high concentrations of the peptide, the cytosolic free calcium concentration declines promptly, due to the activation of a protein kinase C-mediated extrusion and/or sequestration process. This inhibitory phase is less marked at low agonist concentrations but, in all cases, is superseded by a second increase in free calcium, which is due to the stimulated influx of the cation through dihydropyridine-sensitive calcium channels. These biphasic changes in calcium, in concert with the activation of protein kinase C, appear sufficient to regulate prolactin secretion.

Topics: Animals; Calcium; Cell Line; Diglycerides; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Phosphatidylinositol Phosphates; Phosphatidylinositols; Pituitary Gland; Pituitary Neoplasms; Prolactin; Protein Kinase C; Rats; Receptors, Cell Surface; Thyrotropin-Releasing Hormone

The effect of decreasing the concentration of receptors for thyrotropin-releasing hormone (TRH) on the surface of cloned rat pituitary (GH3) cells on TRH-stimulated inositol trisphosphate (Ins-P3) formation was investigated. Incubation of cells with dibutyryl cAMP (Bt2cAMP) for 16 h caused a decrease in [3H] TRH binding to intact cells to a minimum level 37 +/- 9.1% of control. Scatchard analysis of the concentration dependency of [3H]TRH binding showed that the effect of Bt2cAMP was to lower the receptor concentration without affecting its affinity for TRH. Similar decreases in [3H]TRH binding were found in cells incubated with 8-bromo-cAMP, cholera toxin, and sodium butyrate and, as shown previously, with TRH. In cells incubated with 1 mM Bt2cAMP for 16 h, but not for 1 h, the maximum TRH-induced increase in Ins-P3 was inhibited to 25 +/- 3.2% of that in control cells. Inhibition of TRH-induced Ins-P3 formation was also observed in cells treated with 8-bromo-cAMP, cholera toxin, and sodium butyrate for 16 h, and with TRH for 48 h. Inhibition of TRH-induced Ins-P3 formation and lowering of TRH receptor concentration caused by Bt2cAMP occurred in parallel with increasing doses of Bt2cAMP; at 16 h of exposure, half-maximal effects occurred with 0.3 mM Bt2cAMP. The concentration dependency of TRH-induced Ins-P3 formation was the same in control and Bt2cAMP-treated cells; half-maximal effects occurred with 10 nM TRH. These data demonstrate that decreases in TRH receptor concentration caused by several agents that act via different mechanisms are associated with reduced stimulation of Ins-P3 formation and suggest that the TRH receptor is tightly coupled to stimulation of hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Bucladesine; Butyrates; Butyric Acid; Cell Line; Cholera Toxin; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Pituitary Neoplasms; Rats; Receptors, Cell Surface; Receptors, Thyrotropin-Releasing Hormone; Sugar Phosphates; Thyrotropin-Releasing Hormone

Thyrotropin-releasing hormone (TRH) stimulation of prolactin secretion from GH3 cells, cloned rat pituitary tumor cells, is associated with 1) hydrolysis of phosphatidylinositol 4,5-bisphosphate to yield inositol trisphosphate (InsP3) and 2) elevation of cytoplasmic free Ca2+ concentration [( Ca2+]i), caused in part by mobilization of cellular calcium. We demonstrate, in intact cells, that TRH mobilizes calcium and, in permeabilized cells, that InsP3 releases calcium from a nonmitochondrial pool(s). In intact cells, TRH caused a loss of 16 +/- 2.7% of cell-associated 45Ca which was not inhibited by depleting the mitochondrial calcium pool with uncoupling agents. Similarly, TRH caused an elevation of [Ca2+]i from 127 +/- 6.3 nM to 375 +/- 54 nM, as monitored with Quin 2, which was not inhibited by depleting mitochondrial calcium. Saponin-permeabilized cells accumulated Ca2+ in an ATP-dependent manner into a nonmitochondrial pool, which exhibited a high affinity for Ca2+ and a small capacity, and into a mitochondrial pool which had a lower affinity for Ca2+ but was not saturated under the conditions tested. Permeabilized cells buffered free Ca2+ to 129 +/- 9.2 nM when incubated in a cytosol-like solution initially containing 200 to 1000 nM free Ca2+. InsP3, but not other inositol sugars, released calcium from the nonmitochondrial pool(s); half-maximal effect occurred at approximately 1 microM InsP3. Ca2+ release was followed by reuptake into a nonmitochondrial pool(s). These data suggest that InsP3 serves as an intracellular mediator (or second messenger) of TRH action to mobilize calcium from a nonmitochondrial pool(s) leading to an elevation of [Ca2+]i and then to prolactin secretion.

Topics: Animals; Calcimycin; Calcium; Calcium-Transporting ATPases; Cell Line; Clone Cells; Cytoplasm; Drug Interactions; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mitochondria; Pituitary Neoplasms; Prolactin; Rats; Sugar Phosphates; Thyrotropin-Releasing Hormone; Uncoupling Agents