Gonadotropin-releasing hormone receptor activation of extracellular signal-regulated kinase and tyrosine kinases in transfected GH3 cells and in alphaT3-1 cells

GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.     

Treatment with drugs able to reduce iodine efflux significantly increases the intracellular retention time in thyroid cancer cells stably transfected with sodium iodide symporter complementary deoxyribonucleic acid

CONTEXT: One of the major limits of gene therapy with sodium iodide symporter (NIS), which enables cells to be subjected to radioiodine therapy, is that NIS-transfected cells rapidly release the intracellular iodine. METHODS: We transfected human anaplastic (FRO) and medullary (TT) thyroid cancer-derived cell lines that were unable to take up iodine with human NIS cDNA. The possibility of increasing the iodine retention time by treating the transfected clones with myricetin, lithium, 17-(allylamino)-17-demethoxygeldanamycin (17-AAG), and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) was explored. RESULTS: We obtained 19 FRO and 16 TT clones stably transfected with NIS. Twelve of 19 FRO and nine of 16 TT clones expressed the full-length NIS mRNA; 11 of 12 FRO and four of nine TT clones were able to take up radioiodine and correctly expressed NIS protein on the plasma membrane. Kinetic analysis of iodide uptake in the two clones (FRO-19 and TT-2) with the highest uptaking activity revealed that the plateau was reached after 30 min by FRO-19 and after 60 min by TT-2. The t(1/2) of the iodide efflux was 9 min in FRO-19 and 20 min in TT-2. The treatment of the two cell lines with four different drugs revealed that DIDS and 17-AAG, but not myricetin and lithium, significantly increased the intracellular iodide retention time in FRO-19, but not in TT-2. CONCLUSIONS: We showed that 17-AAG and DIDS prolong the retention time of (131)I in NIS-transfected thyroid tumoral cells, thus reinforcing the hope of using this approach for future clinical application, especially in patients with thyroid carcinoma who are no longer responsive to conventional therapy.     

Protein kinase Cdelta as gonadotropin-releasing hormone target isoenzyme in the alphaT3-1 gonadotrope cell line

We investigated the kinetics of gonadotropin-releasing hormone (GnRH)-induced activation of the protein kinase C (PKC) delta isoform in alphaT3-1 gonadotrope cells. Results were evaluated in subcellular fractions and whole-cell lysates using specific antibodies recognizing either non- or (trans- and auto-)phosphorylated forms of the kinase at Thr505 and Ser643 residues modulating stability and/or activation of the enzyme. Under basal conditions, and in contrast to PKC epsilon, PKC delta was mainly associated with the membrane compartment. GnRH (10(-7)M) elicited further and rapid membrane translocation and time-dependent phosphorylation at both sites of PKC delta. The neuropeptide's effects did not show a refractory period after short but successive GnRH stimulation and were abolished by the GnRH antagonist, antide. Sustained GnRH stimulation (2-6 h) provoked rapid down-regulation of PKC delta. Antide, by inhibiting the initial processes (translocation, phosphorylation), counteracted the degradation of the enzyme. Proteolytic processing of PKC delta was shown to mainly involve proteasome activity. Indeed, specific proteasome inhibitors prevented GnRH-elicited kinase depletion and induced membrane accumulation of the enzyme in a phosphorylated (Thr505, Ser643) form. Thus, GnRH may regulate time-dependent cell responses by modulating the phosphorylation/activation state of its signal transduction effector proteins, and by maintaining their appropriate expression balance via proteolytic processes involving the proteasome system.     

Molecular cloning of a complementary deoxyribonucleic acid encoding the thyrotropin-releasing hormone receptor and regulation of its messenger ribonucleic acid in rat GH cells.

Rat pituitary GH cells have been used extensively to study the biochemical actions of TRH on lactotropic cells. To investigate the structure and regulation of the rat TRH receptor (rTRHR), we have cloned its cDNA from GH4C1 cells. Using the polymerase chain reaction with degenerate primers and pools of cloned cDNAs from a GH4C1 cDNA library, a fragment sharing high similarity to the mouse thyrotrope TRHR (mTRHR) was identified. Conventional library screening with this fragment was used to isolate a single cDNA. mRNA synthesized in vitro from this cDNA was injected into Xenopus oocytes, and a characteristic conductance response to TRH was detected by voltage clamp recording. DNA sequence analysis revealed a molecule of 412 amino acid residues, with 96% similarity to the mTRHR. However, in contrast to the mTRHR, the rTRHR had an additional 19 amino acid residues at its carboxy-terminus. A mRNA of about 4 kilobases was identified in GH3 cells. Regulation of the rTRHR mRNA concentration was studied in GH3 cells. Steady state rTRHR mRNA levels were decreased to 30% of the control level by incubation with TRH for 48 h and increased 4-fold by incubation with dexamethasone for 12 h. Southern blot analysis of genomic DNA from GH3 cells gave a simple banding pattern consistent with a single copy gene. We conclude that the rTRHR shares high primary sequence similarity to the mTRHR, but the rTRHR has an extension of 19 amino acids at its carboxy-terminus, which is lacking in the mTRHR.     

The lack of gonadotrophin-releasing hormone (GnRH) receptor desensitisation in alphaT3-1 cells is not due to GnRH receptor reserve or phosphatidylinositol 4,5-bis-phosphate pool size

The phospholipase C (PLC)-activating gonadotrophin-releasing hormone (GnRH) receptor is thought not to rapidly desensitise in alphaT3-1 cells. This extremely unusual characteristic raises the concern that it might be a feature of the cell type, rather than the receptor per se. Here we have used video imaging to establish whether the effects of endogenous PLC-activating G-protein coupled receptors (GPCRs) on Ca2+ ion concentration [Ca2+]i desensitise in these cells. Oxytocin, endothelin-1, methacholine, and UTP all caused [Ca2+]i increases which underwent rapid homologous desensitisation in that they were transient and responses to repeat stimuli were attenuated whereas subsequent responses to GnRH were not. To test whether receptor reserve obscures functional desensitisation of GnRH receptors, a photoaffinity antagonist (Pant-1), was used to effect a partial and irreversible receptor blockade. UV crosslinking in medium with 1000 nM Pant-1 reduced GnRH receptor number to 20 +/- 5% and reduced maximal buserelin-stimulated [3H]IP(X) accumulation to 57 +/- 5%, demonstrating removal of receptor reserve. In control alphaT3-1 cells the initial rate of GnRH-stimulated [3H]IP(X) accumulation was maintained for at least 5 min and GnRH caused a sustained increase in Ins(1,4,5)P3 mass (confirming the resistance of GnRH receptors to desensitisation) and Pant-1 pre-treatment reduced the magnitude of these responses without altering their temporal profiles. In alphaT3-1 cells stably transfected with recombinant human muscarinic receptors (alphaT3-1/M3), responses to methacholine were characteristic of desensitising GPCRs (transient Ins(1,4,5)P3 and curvilinear [3H]IP(X) responses) and were unaltered by Pant-1. To test the relevance of phospholipid pool size, alphaT3-1/M3 cells were pre-treated with GnRH or methacholine in medium with LiCl (to deplete PtdIns(4,5)P2 pools). These pre-treatments reduced subsequent responses to methacholine and GnRH comparably, indicating access to a shared PtdIns(4,5)P2 pool. Partial depletion of this pool (GnRH pre-treatment in medium with LiCl) reduced the magnitude of the [3H]IP(X) and Ins(1,4,5)P3 responses to methacholine and GnRH, without altering their temporal profiles. Thus the GnRH receptor does not undergo rapid homologous desensitisation in alphaT3-1 cells in spite of the fact that they can desensitise other endogenous (and recombinant) PLC-activating GPCRs, and the lack of desensitisation cannot be attributed to the existence of GnRH receptor reserve or access to an atypically large or rapidly re-cycled PtdIns(4,5)P2 pool. This unique functional characteristic (mammalian GnRH receptors are the only PLC-activating GPCRs known not to rapidly desensitise) almost certainly therefore reflects the atypical structure of these receptors (mammalian GnRH receptors are the only PLC-activating GPCRs known to lack C-terminal tails).     

Characterization of the gonadotrophin-releasing hormone calcium response in single alpha T3-1 pituitary gonadotroph cells.

Intracellular calcium ([Ca2+]i) was measured in single immortalized gonadotroph alpha T3-1 cells using dual wavelength fluorescence microscopy combined with dynamic video imaging. Gonadotrophin-releasing hormone (GnRH, 10(-8) M) produced a biphasic rise in [Ca2+]i which could be abolished by a GnRH antagonist. The initial calcium transient was complete within seconds while the smaller secondary plateau phase lasted several minutes. The calcium spike was reduced by nifedipine (10(-6) M), a calcium channel blocker, and thapsigargin (10(-6) M) which inhibits inositol 1,4,5-trisphosphate (IP3) mediated release of [Ca2+]i but abolished by the intracellular calcium antagonist TMB-8 (10(-6) M). The secondary phase was reduced following pretreatment with either nifedipine or the protein kinase C (PKC) antagonist, H-7 (10(-6) M). The PKC agonist PMA (phorbol 12-myristate 13-acetate, 10(-6) M) produced a small rise in basal [Ca2+]i and abolished the GnRH calcium response. The initial calcium response to GnRH therefore involves both an IP3-mediated rise in cytosolic calcium due to the release from intracellular stores and an influx of extracellular calcium through second messenger-operated calcium channels. In contrast the secondary calcium response mainly involves the influx of extracellular calcium through PKC-activated calcium channels.     

Constitutive and regulated membrane expression of aquaporin 1 and aquaporin 2 water channels in stably transfected LLC-PK1 epithelial cells.

The aquaporins (AQPs) are a family of homologous water-channel proteins that can be inserted into epithelial cell plasma membranes either constitutively (AQP1) or by regulated exocytosis following vasopressin stimulation (AQP2). LLC-PK1 porcine renal epithelial cells were stably transfected with cDNA encoding AQP2 (tagged with a C-terminal c-Myc epitope) or rat kidney AQP1 cDNA in an expression vector containing a cytomegalovirus promoter. Immunofluorescence staining revealed that AQP1 was mainly localized to the plasma membrane, whereas AQP2 was predominantly located on intracellular vesicles. After treatment with vasopressin or forskolin for 10 min, AQP2 was relocated to the plasma membrane, indicating that this relocation was induced by cAMP. The location of AQP1 did not change. The basal water permeability of AQP1-transfected cells was 2-fold greater than that of nontransfected cells, whereas the permeability of AQP2-transfected cells increased significantly only after vasopressin treatment. Endocytotic uptake of fluorescein isothiocyanate-coupled dextran was stimulated 6-fold by vasopressin in AQP2-transfected cells but was only slightly increased in wild-type or AQP1-transfected cells. This vasopressin-induced endocytosis was inhibited in low-K+ medium, which selectively affects clathrin-mediated endocytosis. These water channel-transfected cells represent an in vitro system that will allow the detailed dissection of mechanisms involved in the processing, targeting, and trafficking of proteins via constitutive versus regulated intracellular transport pathways.     

Inhibition of cAMP accumulation by intracellular calcium mobilization in C6-2B cells stably transfected with substance K receptor cDNA.

C6-2B rat glioma cells were stably transfected with substance K receptor cDNA and used to study interactions between cAMP and Ca2+ signaling pathways. Activation of the newly expressed receptors by substance K increased the intracellular free Ca2+ concentration, as monitored by single-cell fura-2 imaging, and markedly inhibited agonist-stimulated cAMP accumulation. Blockade of intracellular Ca2+ mobilization abolished the substance K receptor-mediated inhibition of isoproterenol-induced cAMP production. Phosphodiesterase inhibitors, down-regulation or inhibition of protein kinase C, and pertussis toxin failed to prevent substance K-induced inhibition of agonist-stimulated cAMP accumulation. An increased intracellular Ca2+ concentration caused by either calcium ionophores or activation of endogenous bradykinin receptors was found to markedly reduce cAMP production in wild-type cells. These results demonstrate that elevated intracellular Ca2+ concentration can negatively modulate agonist-stimulated adenylate cyclase activity in C6-2B glioma cells.