Bipotential murine hemopoietic cell line (NFS-60) that is responsive to IL-3, GM-CSF, G-CSF, and erythropoietin

NFS-60 cells were previously obtained from leukemia cells that were infected with the Cas-Br-M murine leukemia virus in vivo. We examined the proliferation and differentiation capacity of NFS-60 cells in the presence of native and recombinant (r) interleukin 3 (IL-3), recombinant granulocyte colony-stimulating factor (rG-CSF), recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and r-erythropoietin (Ep) using methylcellulose culture methods. This cell line was able to form colonies in response to each hemopoietic factor, but colony formation was rarely seen in their absence. Some populations of NFS-60 cells could differentiate into neutrophils and macrophages in the presence of IL-3 and GM-CSF. Moreover, in the presence of Ep, this cell line formed well-hemoglobinized colonies as well as nonerythroid colonies. In the presence of G-CSF, NFS-60 cells remained in the promyelocytic state. Electron microscopic studies confirmed these morphologies. A single-cell transfer experiment demonstrated that neutrophils, macrophages, and erythroblasts were derived from a single cell. It is concluded that the NFS-60 cell line is a factor-dependent, bipotential hemopoietic cell line.     

Characterization of a calmodulin-binding protein that is deficient in trifluoperazine-resistant variants of the macrophage-like cell line J774.

A calmodulin-binding protein is present in extracts of the macrophage-like mouse cell line J774 and in extracts of thioglycollate-stimulated mouse peritoneal macrophages; it is deficient in variants of J774 resistant to trifluoperazine and in resident peritoneal macrophages. The calmodulin-binding protein [CaMBP (J7)0.5] was purified from J774 and resolved from endogenous cyclic nucleotide phosphodiesterase and protein kinase activities. The protein has an apparent native Mr of 125,000-150,000 and binds calmodulin in a calcium-dependent manner with a Kd of 20 nM. It inhibits the ability of calmodulin to activate phosphodiesterase. Its sedimentation constant in glycerol gradients containing calmodulin was dependent upon the relative concentrations of calmodulin and the calmodulin-binding protein.     

Tamoxifen reduces calcium currents in a clonal pituitary cell line

The effect of the anti-estrogen Tamoxifen (Tx) on membrane electrical properties and the underlying calcium (Ca2+) conductances was examined in the clonal pituitary cell line GH3/B6 which exhibits calcium action potentials at rest. Electrophysiological recordings (109 cells) were made using either high resistance intracellular microelectrodes or the whole-cell recording (WCR) patch-clamp technique. Electrical activities of 39 spontaneously active GH3/B6 cells were recorded with sharp microelectrodes filled with 3 M KCl. The spikes were Ca2+-dependent since they were blocked by Cobalt ions (Co2+, 5 mM). When applied directly to the recorded cell, Tx (10(-7) M) inhibited action potential firing. This blockade was accompanied by a discrete hyperpolarization of the membrane potential (-2.8 +/- 2 m V) from rest (-39.5 +/- 5 m V) and a 10% increase in the input membrane resistance. The effect stopped soon after Tx removal (mean 11.4 +/- 6 sec), and Tx solvent was unable to elicit the response. Current clamp WCR with pipettes containing potassium gluconate (KGlu, 140 mM) confirmed these results (30 cells), but the addition of cell extract to KGlu was necessary to prevent rundown of the response and to obtain reproducible action potential blockade. Short (1-5 min) or long term (48 h) pretreatment with estradiol (10(-7) to 10(-5) M) did not change the response to Tx, thus indicating that its effect was not mediated through estrogen receptors. Voltage clamp WCR study of the effect of Tx (10(-7) M) using pipettes filled with cesium chloride (140 mM) showed that both fast and slow inactivating calcium conductances were inhibited (38 cells). The fast inactivating Ca2+ current was reduced by about 60-80% whereas slow inactivating Ca2+ current was completely inhibited. This action may represent one way by which the antitumoral effect of this antiestrogen is mediated.     

Characterization of a tissue kallikrein in human prolactin-secreting adenomas

Immunoreactive tissue kallikrein was co-localized with prolactin in all the eleven prolactin-secreting adenomas of the human anterior pituitary gland examined in this study. The intracellular distribution of immunoreactivity in the prolactin-secreting cells suggests that tissue kallikrein is located within the Golgi complex of these cells. Both the intracellular hormone-processing action and the kininogenase activity of tissue kallikrein may be of functional importance in human prolactinomas.     

Storage and release of acetylcholine by a clonal cell line.

The nerve cell line PC12, in its morphologically undifferentiated state, synthesizes, stores, and secretes catecholamines and acetylcholine. At least 60% of the basal level of neurotransmitter release is due to a calcium-dependent mechanism, and the rate of secretion is enhanced by increasing external potassium. A minimum of 80% of the intracellular acetylcholine and catecholamines are stored in particulate structures. The storage site for acetylcholine is dense core vesicles that can be distinguished from those containing catecholamines on the basis of vesicle density on sucrose gradients, vesicle size, and reserpine sensitivity. These results are discussed in relation to what are thought to be the early stages of synapse formation in cell culture.     

Characterization of a novel HTLV-infected cell line

A man from Chile developed an aggressive mature T cell leukemia associated with marked eosinophilia. The neoplastic lymphocytes were of T helper surface phenotype, and they expressed the p24 and p19 antigens of human T cell leukemia virus (HTLV). A cell line (ME) was established from the patient's peripheral blood cells that was initially composed of eosinophils and T and B lymphocytes. The B lymphocytes of the cell line are polyclonal and contain Epstein-Barr virus (EBV) DNA. Many of the T lymphocytes, a few of the B lymphocytes, and none of the eosinophils express HTLV p19 and p24 antigens. By 6 months of culture, the ME line no longer contained eosinophils. A variant line of ME was established; this variant (ME-2) is notable because the cells (greater than 80%) adhere tightly to the bottom of the culture flask; they do not express T lymphocyte markers, but 30% of the cells contain cytoplasmic mu heavy immunoglobulin chains. These pre-B and null lymphocytes contain p19 and p24 antigens (80% of cells), have the HTLV- I genome, and are able to transform normal T lymphocytes in vitro. We isolated a B lymphocyte clone (11A) from ME that expresses cytoplasmic immunoglobulin (70% of cells) and p19 and p24 antigens (75% of cells), contains the EBV and HTLV genomes, and can transform T lymphocytes from normal volunteers. These data show that B lymphocytes can be infected with HTLV, although no disease of HTLV-infected B lymphocytes has been reported. The T lymphocytes from normal adult peripheral blood were easily immortalized (about 70% efficiency) by cocultivation with lethally irradiated ME cells. Twenty-five of 27 of the transformant lines were composed of T lymphocytes with helper antigens, and two of the lines were of T suppressor antigen phenotype. All the cell lines that were tested constitutively produce lymphokines, including colony- stimulating factor (CSF), erythroid-potentiating activity (EPA), macrophage migration-inhibitory factory (MIF), neutrophil-inhibitory factor (NIF), and differentiation-inducing factor (DIF).     

Characterization of a new megakaryocytic cell line: the Dami cell

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryoblastic leukemia. The Dami cells grow primarily in suspension with a doubling time of 24 to 30 hours. By light and electron microscopy, the Dami cells range in size from 12 to 120 micron in diameter and have lobulated nuclei characteristic of megakaryocytes. At least 89% of the cells react with monoclonal antibodies against platelet glycoproteins (GP) Ib and IIB/IIIa, and glycophorin. The cells do not react with antibodies against lymphoid, monocyte, granulocyte, or macrophage antigens. Thirteen percent of the cells become polyploid, spontaneously achieving greater than 4N DNA ploidy levels. In response to phorbol myristate acetate (PMA), the proportion of cells with ploidy levels greater than 4N increased threefold and could be separated into discrete ploidy groups. PMA also increased the expression of GPIb, the GPIIb/GPIIIa complex,l and von Willebrand factor. Cytogenetic analysis revealed a human male hyperdiploid karyotype with a modal chromosome number of 54 to 64 and several consistent clonal chromosomal abnormalities. These included a partial deletion of chromosome 5 and a translocation involving chromosome 3. In contrast to other megakaryocytic cell lines in which only a small portion of the cells express the megakaryocytic phenotype, nearly all of the Dami cells express platelet glycoproteins. Thus, the Dami cells provide a superior model in which to study human megakaryocyte biochemistry and differentiation.     

Identification of an inhibitory factor from a Sertoli clonal cell line (TM4) that modulates adult rat Leydig cell steroidogenesis

Sertoli cell produces several biological factors that modulate Leydig cell steroidogenic function by either stimulating or inhibiting its testosterone production. We have evaluated the effect of an inhibitory factor in the spent media of a Sertoli clonal cell line (TM4) which inhibits Leydig cell steroidogenesis. The presence of such an inhibitory factor in TM4 media was bioassayed using Percoll purified Leydig cells isolated from adult rats with purity of greater than 95%. TM4 media inhibited both human chorionic gonadotropin (hCG)-stimulated testosterone and cAMP production by purified Leydig cells dose-dependently but had no apparent effect on 8-bromo-cAMP- and forskolin-stimulated testosterone production. Also it did not interfere with the binding of [¹²⁵I]hCG to Leydig cells. TM4 media inhibited cholera toxin-stimulated testosterone production as well as forskolin- and cholera toxin-stimulated cAMP production. The mechanism of action of this factor in TM4 media appears to be different from transforming growth factor (TGF-β) which inhibited both 8-bromo-cAMP- and forskolin-stimulated testosterone production and inhibited the binding of [¹²⁵I]hCG binding to Leydig cells. The inhibitory factor contained in TM4 media has been partially purified by sequential preparative anion exchange and C-18 reversed-phase high-performance liquid chromatography. In summary, the Sertoli TM4 cell line produces at least one potent inhibitory factor which decreases the responsiveness of purified Leydig cells to hCG stimulation with a dramatically different mechanism from other currently known Leydig cell inhibitory factors; this protein may serve as a valuable tool to study testicular paracrine regulation.     

Hormonal regulation of collagen synthesis in a clonal rat osteosarcoma cell line

Collagen synthesis in rat osteosarcoma cell line 17/2 (ROS 17/2) was assessed by measuring the incorporation of [3H]proline into collagenase-digestible protein and the formation of [3H]hydroxyproline. PTH and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited collagen synthesis in ROS 17/2 cells in a time- and dose-dependent manner. PTH reduced collagen synthesis after a 3-h incubation, whereas the effect of 1,25-(OH)2D3 was somewhat slower. Maximal and half-maximal inhibition of collagen synthesis occurred at approximately 1 and 0.1 nM of each hormone, respectively. At confluency, ROS 17/2 cells synthesized 96% type I and 4% type III collagen. PTH reduced the synthesis of type I, but not type III, collagen. PTH and 1,25-(OH)2D3 also reduced procollagen mRNA levels, as determined by a dot blot hybridization assay. Thus, ROS 17/2 cells are a convenient model system for studying the hormonal regulation of collagen metabolism and gene expression in a cloned cell line with the osteoblastic phenotype.     

Synthesis of opiate peptides by a clonal pituitary tumor cell line.

Clonal mouse pituitary tumor cells, AtT-20, synthesize at least four species of peptides with opiate activity. The endorphin concentration of AtT-20 cells was estimated to be 300-600 pmol/mg of protein. The two most abundant endorphins with apparent molecular weights of 1800 and 2400 were purified 300- and 24-fold, respectively; additional minor components were found with apparent molecular weights of greater than 3000 and less than 750.     

Inhibition of agouti-related peptide expression by glucose in a clonal hypothalamic neuronal cell line is mediated by glycolysis, not oxidative phosphorylation

The regulation of neuroendocrine electrical activity and gene expression by glucose is mediated through several distinct metabolic pathways. Many studies have implicated AMP and ATP as key metabolites mediating neuroendocrine responses to glucose, especially through their effects on AMP-activated protein kinase (AMPK), but other studies have suggested that glycolysis, and in particular the cytoplasmic conversion of nicotinamide adenine dinucleotide (NAD+) to reduced NAD (NADH), may play a more important role than oxidative phosphorylation for some effects of glucose. To address these molecular mechanisms further, we have examined the regulation of agouti-related peptide (AgRP) in a clonal hypothalamic cell line, N-38. AgRP expression was induced monotonically as glucose concentrations decreased from 10 to 0.5 mm glucose and with increasing concentrations of glycolytic inhibitors. However, neither pyruvate nor 3-beta-hydroxybutyrate mimicked the effect of glucose to reduce AgRP mRNA, but on the contrary, produced the opposite effect of glucose and actually increased AgRP mRNA. Nevertheless, 3beta-hydroxybutyrate mimicked the effect of glucose to increase ATP and to decrease AMPK phosphorylation. Similarly, inhibition of AMPK by RNA interference increased, and activation of AMPK decreased, AgRP mRNA. Additional studies demonstrated that neither the hexosamine nor the pentose/carbohydrate response element-binding protein pathways mediate the effects of glucose on AgRP expression. These studies do not support that either ATP or AMPK mediate effects of glucose on AgRP in this hypothalamic cell line but support a role for glycolysis and, in particular, NADH. These studies support that cytoplasmic or nuclear NADH, uniquely produced by glucose metabolism, mediates effects of glucose on AgRP expression.     

Characterization of insulin-binding to the hepatoma cell line H35

We have studied the characteristics of insulin-binding to its receptor in H35 hepatoma cells. Insulin-binding was time, temperature and pH dependent. Optimum pH was 8.0-8.2. Binding at 21 degrees C reached a steady state after 90 min of incubation at a level of 30.0 +/- 2.6% per mg protein. Pre-incubation of the cells with unlabelled exogenous insulin resulted in a decrease of insulin-binding which was time and concentration dependent. Pre-incubation with 10 micrograms/ml for 24 h resulted in a decrease to 35-40% of initial binding. Scatchard plots of the binding data were curvilinear in control as well as in down regulated cells. Analysis of the Scatchard plots revealed that decrease of insulin-binding to down regulated cells was due to a decrease of insulin-binding sites, while affinity constants did not change.     

Characterization of a novel rat epididymal cell line to study epididymal function

The epididymis is an androgen-dependent organ that allows spermatozoa to become fully functional as they pass through this tissue. The specialized functions of the epididymis are mediated by interactions between epididymal epithelial cells and between epididymal cells and spermatozoa. Although the critical role of the epididymis in sperm maturation is well established, the mechanisms regulating cell-cell interactions remain poorly understood because of the lack of appropriate cell line models. We now report the characterization of a novel rat caput epididymal cell line (RCE) that was immortalized by transfecting primary cultures of rat epididymal cells with the simian virus 40 large T antigen. At the electron microscope level, the cell line was composed of epithelial principal cells with characteristics of in vivo cells; principal cells had well-developed Golgi apparatus, abundant endoplasmic reticulum cisternae, and few endosomes. RCE cells expressed the mRNAs coding for the androgen receptor, estrogen receptor alpha, and 4-ene-steroid-5-alpha-reductase types 1 and 2 as well as epididymal-specific markers Crisp-1 and epididymal retinoic acid binding protein. Epididymal retinoic acid binding protein expression was significantly induced with dihydrotestosterone, although this effect was not blocked by flutamide, suggesting that RCE cells are not androgen responsive. Neighboring cells formed tight and gap junctions characteristic of epididymal cells in vivo and expressed tight (occludin and claudin-1, -3, and -4) and gap junctional proteins (connexin-26, -30.3, -32, and -43). The RCE cell line displays many characteristics of epithelial principal cells, thus providing a model for studying epididymal cell functions.     

Characterization of a new monoclonal anti-glypican-3 antibody specific to the hepatocellular carcinoma cell line,HepG2

AIM To characterize the antigen on HepG2 cell that is specifically recognized by a new monoclonal antibody raised against human liver heparan sulfate proteoglycan(HSPG), clone 1E4-1D9.METHODS The antigen recognized by m Ab 1E4-1D9 was immunoprecipitated and its amino acid sequence was analyzed LC/MS. The transmembrane domain, number of cysteine residues, and glycosylation sites were predicted from these entire sequences. Data from amino acid analysis was aligned with glypican-3(https://www.ebi.ac.uk/Tools/msa/clustalo/). The competitive reaction of mA b 1E4-1D9 and anti-glypican-3 on HepG2 cells was demonstrated by indirect immunofluorescence and analyzed by flow cytometry. Moreover, co-immunoprecipitation of mA b 1E4-1D9 and anti-glypican-3 was performed in HepG2 cells by Western immunoblotting. The recognition by mA b 1E4-1D9 of a specific epitope on solid tumor and hematopoietic cell lines was studied using indirect immunofluorescence and analyzed by flow cytometry.RESULTS Monoclonal antibody 1E4-1D9 reacted with an HSPG isolated from human liver and a band of 67 kD was detected under both reducing and non-reducing conditions. The specific antigen pulled down by m Ab 1E4-1D9, having a MW of 135 kD, was analyzed. The results showed two sequences of interest, gi30722350(1478 amino acid) and gi60219551(1378 amino acid). In both sequences no transmembrane regions were observed. Sequence number gi30722350 was 99.7% showed a match to FYCO1, a molecule involved in induction of autophagy. Sequence number gi60219551 contained 15 cysteines and 11 putative glycosylation sites with 6 predicted N-glycosylation sites. It was also matched with all PDZ domain proteins. Moreover, it showed an 85.7% match to glypican-3. Glypican-3 on HepG2 cells competitively reacted with both phycoerythrin-conjugated anti-glypican-3 and mA b 1E4-1C2 and resulted in an increase of double-stained cell population when higher concentration of m Ab 1E4-1D9 was used. Moreover, antigens precipitated from HepG2 cell by anti-glypican-3 could be detected by mA b 1E4-1D9 and vice versa. The recognition of antigens, on other solid tumor cell lines, by m Ab 1E4-1D9 was studied. The results demonstrated that m Ab 1E4-1D9 reacted with Huh7, HepG2, HT29, MCF7, SW620, Caco2, B16F1, U937, K562 and Molt4 cells. It was also found to be weakly positive to SW1353 and HL60 and negative to H460 and Hela cell lines. CONCLUSION All findings show that mA b 1E4-1D9 specifically recognizes glypican-3. Moreover, a new partner molecule of glypican-3, FYCO1 is proposed based on the results from co-precipitation studies.     

Characterization of a temperature-sensitive beta-endorphin-secreting transformed endometrial cell line

An endometrial cell line (HRE-H9) was established and characterized to study the mechanism by which gene expression of POMC-derived peptides is controlled in uterine tissues. The HRE-H9 cell line was isolated by transforming primary rabbit endometrial cell cultures, derived from hCG-treated pseudopregnant rabbits, with a temperature-sensitive A209 mutant (tsA209) simian virus 40 at a permissive temperature (33 C). The resulting cells exhibited temperature sensitivity in growth and synthesis of immunoreactive beta-endorphin (ir-beta END). The ir-beta-END present in the cell extracts and culture media was assayed by a specific beta END RIA. Sephadex G-50 gel filtration chromatography of the transformed cell extracts showed three peaks of beta END immunoreactivity. The first peak eluted at the void volume, the second peak coeluted with the beta-lipotropin standard, and the third peak coincided with the porcine beta END standard, ir-beta-END was also detectable in endometrial culture media, suggesting that the transformed endometrial cells secreted POMC-derived peptides. Our data indicate that the tsA209 mutant virus-transformed endometrial cell line provides a suitable model for study of the synthesis and regulation of POMC-derived peptides in extrapituitary tissues.     

Characterization of a transformed ovine lymphatic endothelial cell line

OBJECTIVE: To develop a non-tumor-derived stable lymphatic endothelial cell line that exhibits rapid growth rate without serum and exogenous growth factors, while still maintaining key features characteristics of the non-transformed lymphatic endothelium. METHODS: Lymphatic endothelial cells were isolated from ovine mesenteric lymphatic vessels, grown to confluence and transfected with SV40 DNA using the calcium phosphate method. The resulting cell line was characterized using morphological, immunocytochemical, flow cytometric analysis, and immunoprecipatitation and Western blotting methods. RESULTS: The resulting cell line (sheep lymphatic endothelial transformed cell line, SLET-1) underwent rapid proliferation in the absence of growth factors and reduced concentrations of serum. In addition, key morphological and functional properties of the non-transformed lymphatic endothelium were retained. These include the ability to form confluent monolayer cultures, the expression of the lymphatic endothelial-specific VEGFR-3, FLT-4) tyrosine kinase receptor, the biosynthesis and secretion of von Willebrand factor and plasminogen activators. In addition, SLET-1 cells express cell surface antigens found on LEC that may act as antibody targets in various immune reactions. Monolayer cultures of the SLET-1 cells incubated with endothelial cell-growth factor formed tubular structures, indicating the retention of the capacity to differentiate. CONCLUSION: The SLET-1 cell line retained key morphological and functional properties characteristic of the non-transformed lymphatic endothelium. The ability to form capillary-like tubular structures provides an important cell line for defining the role of specific proteins that are involved in the lymphagiogenic (formation of new lymphatic vessels) process. Thus, this transformed lymphatic endothelial cell line provides an in citro model that may have widespread utility in studying regulatory mechanisms of lymphatic endothelial cell function and differentiation.     

Substance P enhances cholinergic receptor desensitization in a clonal nerve cell line.

Substance P inhibits carbamylcholine-induced 22Na+ uptake in the clonal cell line PC12. This inhibition is noncompetitive with agonist but competitive with Na+. Octahydrohistrionicotoxin (H8-HTX) also exhibits this same pattern of inhibition. Moreover, both substance P and H8-HTX are very effective in enhancing agonist-induced receptor desensitization. Local anesthetics, such as QX222, also cause inhibition that is competitive with Na+, but they have only marginal effects on desensitization. Because substance P and H8-HTX cannot by themselves cause desensitization, their action is dependent on and synergistic with the action of agonist. Furthermore, substance P and H8-HTX do not appear to compete for the same site as QX222, which is thought to bind to the ion channel. Finally, substance P can stabilize the desensitized state of the receptor even when added subsequent to the actual desensitization and removal of agonist. Thus, substance P does not require open ion channels for binding and may modulate the activity of the receptor-ionophore complex by binding to a distinct regulatory site.     

Characterization, growth, and differentiation of a human myeloid leukemia cell line, TI-1 cell.

A cell line (TI-1) has been established from the peripheral blood of a patient with acute myeloid leukemia (M2). A typical TI-1 cell displayed many abnormalities of its chromosomes, but not the Philadelphia (Ph1) chromosome. Light and electron microscopic examination and histochemical analysis indicated that the TI-1 cells were undifferentiated blast cells, but immunologic marker studies suggested that these cells had myeloid characteristics. The proliferation of TI-1 cells was dependent on the concentration of fetal bovine serum (FBS). Their doubling time was 13.8 hours when they were cultured in a medium containing 10% FBS. Phorbol-12 myristate 13-acetate (PMA) induced the TI-1 cells to differentiate into monocyte-like cells, as judged by their morphologic similarity to monocytes, their adhesion to the culture dish, and their increase of both nitroblue tetrazolium (NBT)-reducing ability and nonspecific esterase (NSE)-activity. PMA significantly inhibited the proliferation and DNA synthesis of TI-1 cells in a dose-dependent manner. The PMA-induced differentiation was significantly inhibited by the protein kinase C inhibitors (H-7, staurosporine). Hemin induced the TI-1 cells to differentiate into erythroid cells. The number of hemoglobin-producing cells and hemoglobin production was increased by hemin treatment. Hemin also inhibited the proliferation of the TI-1 cells. Thus, the TI-1 cell represents a bipotent, granulo-monocytoid, and erythroid cell line. The TI-1 cell line will be a useful model for monocytoid and erythroid differentiation.     

Adenosine inhibits prolactin and growth hormone secretion in a clonal pituitary cell line

Although purine nucleosides have been shown to regulate the secretion of several peptide and steroid hormones, effects on pituitary hormone release have not been reported. We show here that in the clonal GH4C1 pituitary cell line maximal concentrations of adenosine (greater than or equal to 50 microM) inhibited PRL and GH secretion by 40%. Adenosine deaminase abolished the inhibitory effect of adenosine but not that of SRIF or (-)N6(R-2-phenylisopropyl)adenosine (PIA), a nonhydrolyzable adenosine analog. Furthermore, this enzyme increased basal secretion by 50%, and analysis of the incubation medium by HPLC demonstrated that the cells secreted biologically effective concentrations of adenosine. These results indicate that adenosine produced in culture tonically inhibits hormone release. In other target cells, adenosine inhibition is mediated by two types of binding sites: an extracellular Ri-site requiring an intact ribose moiety or an intracellular P-site requiring an intact purine ring. Four lines of evidence indicate that in GH4C1 cells, adenosine acts at an Ri-site. PIA, an Ri-site-specific agonist, was a potent inhibitor of hormone release (ED50 = 30 nM). Theophylline, an Ri-site antagonist, competitively inhibited the action of PIA (Ki = 2.4 microM). 3) 2'5'-Dideoxyadenosine, a P-site-specific agonist, did not inhibit PRL release even at a concentration of 1 mM. 4) Dipyridamole, an adenosine uptake inhibitor, did not reduce adenosine inhibition. In addition to its effect on basal secretion, PIA inhibited stimulation of hormone release by vasoactive intestinal peptide and TRH. PIA also reduced vasoactive intestinal peptide-stimulated cAMP accumulation by 75%, consistent with its action to inhibit adenylate cyclase via Ri receptors in other targets. Since PIA inhibition of PRL release and cAMP accumulation was not additive with the effects of SRIF and carbamyl choline, these inhibitors may act via a common rate-limiting step. Our results demonstrate that adenosine activates an Ri-type of adenosine receptor in GH4C1 cells and that the production of adenosine under normal culture conditions causes autocrine inhibition of secretion.     

Nerve growth factor-mediated induction of tyrosine hydroxylase in a clonal pheochromocytoma cell line.

We have established a clonal cell line, PC-G2, from an experimentally induced rat pheochromocytoma. Administration of nerve growth factor to PC-G2 causes a 4- to 8-fold induction in the specific activity of tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine,tetrahydropteridine:oxygen oxidoreductase(3-hydroxylating); EC 1.14.16.2]. The response is elicited in a dose-dependent fashion, at concentrations above 0.1 microgram/ml. Antiserum to nerve growth factor inhibited the induction of tyrosine hydroxylase. Dexamethasone enhances the nerve growth factor-mediated elevation of tyrosine hydroxylase. After 3--4 days of exposure to nerve growth factor the maximal induction of tyrosine hydroxylase is seen, although a significant increase can be observed after 24 hr. In contrast to the PC-12 cell line (derived from the same tumor), in which neurite outgrowth occurs in response to nerve growth factor, there is no morphological change or alteration in growth rate of PC-G2 cells after exposure to nerve growth factor.