44-2C cells a neuroendocrine model to study neuropeptide, oncogene, and growth factor gene regulation.

The neuroendocrine 44-2C cells synthesize, secrete, and manifest differential regulation of calcitonin (CT), CT gene-related peptide (CGRP), neurotensin (NT), and somatostatin (SS). These cells maintain differentiated function when grown in serum-free, growth factor and hormone-deprived milieu. The cells continue to secrete CT, CGRP, NT, and SS and differentially respond to cellular secretagogues. In serum-free cultures, the cells produce biologically active acidic fibroblast growth factor (aFGF) and differentially regulate the expression of the protooncogene fos (c-fos).

The neuropeptide-synthesizing rat 44-2C cell line: regulation of peptide synthesis, secretion, 3,'5'-cyclic adenosine monophosphate efflux, and adenylate cyclase activation.

We established in culture a clonal strain (44-2C) which produces calcitonin (CT), CT gene-related peptide, neurotensin (NT), and somatostatin (SS). A compendium of experimental data detailing for this strain the differential regulation of NT, CT, and SS synthesis and secretion, adenylate cyclase activation, and cAMP efflux is presented herein. The effects of hypophysiotropic peptides, brain-gut peptides, and catecholamines are described in detail. The effects of steroid hormones, and in particular, that of the synthetic glucocorticoid, dexamethasone, are presented. The effect(s) of basic bovine fibroblast growth factor are also described. In 44-2C cells basic fibroblast growth factor selectively regulates the synthesis and secretion of CT, NT, SS, and cAMP. Moreover, basic fibroblast growth factor enhances the responsiveness of 44-2C cells to neurosecretory peptides such as rat hypothalamic GRF. We conclude that the 44-2C cells are a useful in vitro tool to study the cellular mechanism(s) controlling the differential synthesis and secretion of neuropeptides.

Fibroblast growth factor is a permissive factor mediating the cellular action of rat hypothalamic growth hormone-releasing factor.

A clonal multipeptide-secreting cell line (44-2C) was used to study the interaction of basic fibroblast growth factor (bFGF) and GRF. The experiments were carried out in cells maintained under serum-free conditions in the absence of growth factors and hormonal supplements. We have shown that in 44-2C cells, rat hypothalamic GRF (rGRF) stimulates neurotensin, calcitonin, and somatostatin secretion. bFGF is not a mitogen in the 44-2C cells, but does regulate differentiated function by a mechanism involving regulation of RNA stabilization. The results presented here show for the first time that FGF acts as a competence factor mediating the cellular action of rGRF. In FGF-pretreated cells, 4-h exposure to rGRF stimulated RNA and protein synthesis. In this cell line rGRF was biologically active at 10(-14) M, and the ED50 for rGRF-mediated RNA and protein synthesis ranged from 10(-11) to 10(-12) M. We conclude that the interaction of FGF, acting as a permissive factor, and rGRF results in a novel mechanism of action for rGRF. These findings suggest that hypophysiotropic factors affect the cellular milieu in concert with growth factors, such as bFGF.

Calcium, dexamethasone, and the antiglucocorticoid RU-486 differentially regulate neuropeptide synthesis in a rat C cell line.

The differential regulation of neurotensin (NT), calcitonin (CT), and CT gene-related peptide (CGRP) production was studied in the clonal, rat C cell-derived, 44-2C cell line. Two experimental paradigms were used: cells were incubated with maximally effective concentrations of calcium (4.0 mM); alternatively, cells were treated with the synthetic glucocorticoid, dexamethasone (DEX). The specificity of the DEX-mediated response was assessed by using the synthetic antiglucocorticoid, RU-486. Calcium was not mitogenic in 44-2C cells and did not affect cell growth. Calcium increased the secretion and cellular accumulation of NT. In contrast, calcium treatment decreased CT content and release while it diminished the levels of CT- and CGRP-specific messenger RNA (mRNA) levels. DEX (10(-8) M) inhibited cell proliferation. NT content and secretion increased after DEX treatment, and this was potentiated by the addition of calcium. DEX-treated cells showed diminished CT content and secretion. The levels of CT- and CGRP-specific mRNA were significantly reduced in DEX-treated cultures. RU-486 antagonized the action of DEX and blocked DEX-inhibited cell proliferation. Inhibition of CT secretion by DEX was blocked by RU-486; CT- and CGRP-specific mRNA levels were increased in response to treatment with equimolar or 100-fold excess concentrations of RU-486. We conclude that NT secretion as well as CT/CGRP expression and release can be differentially regulated in the 44-2C cell line.

Growth hormone-releasing factor and fibroblast growth factor regulate somatostatin gene expression.

A multiple peptide-synthesizing clonal rat cell line was used to study the effect(s) of GRF and basic fibroblast growth factor (bFGF) on the synthesis and secretion of somatostatin (SS). The presence of SS-specific mRNA in 44-2C cells was shown morphologically by in situ hybridization. The release and cellular content of SS increased significantly after treatment with rat hypothalamic GRF (rGRF), the ED50 for rGRF stimulation of intracellular SS was 1.9 X 10(-11) M. GRF stimulated SS production in serum-supplemented and serum-free cultures. Results obtained after incubation of 44-2C cells with 125I-labeled rGRF indicated uptake and nuclear localization of rGRF by 44-2C cells. FGF stimulated the secretion and cellular content of SS. We propose that bFGF regulates the short term secretion and accumulation of SS and mediates rGRF-stimulated SS expression.

Fibroblast growth factor stabilizes ribonucleic acid and regulates differentiated functions in a multipeptide-secreting neuroendocrine cell line.

A clonal cell line (44-2C) which synthesizes and secretes somatostatin, neurotensin, calcitonin (CT), and CT gene-related peptide and transiently expresses c-fos was used to characterize the mechanism of action of basic fibroblast growth factor (bFGF). bFGF had two modes of action: 1) short term incubation of 44-2C cells with bFGF increased the cellular content of neurotensin, somatostatin, and CT; and 2) bFGF enhanced the response of the cells to rat hypothalamic GRF-mediated cAMP efflux. The long term action of bFGF was manifested by the permissive effect of the molecule. bFGF had a sustained effect on RNA synthesis, and pretreatment with bFGF for 24 h altered the time course of response of the cells to rat GRF. In this cell line the cellular action of bFGF was not mediated via protein kinase-C action. bFGF was not mitogenic in 44-2C cells. bFGF stimulated uridine incorporation without affecting thymidine incorporation. Results obtained with actinomycin-D and alpha-amanitin suggest that the above effects of bFGF can be correlated with increased RNA stability produced by bFGF.

Growth hormone (GH)-releasing factor does not regulate GH release or GH mRNA levels in GH3 cells.

Previous studies have shown that GH-releasing factor (GRF) regulates both GH production and GH mRNA levels in primary cultures of rat pituitary cells. Investigations were carried out to ascertain the ability of GRF to regulate GH production or mRNA levels in a clonal strain of rat pituitary tumor (GH3) cells. Incubation of the cells with GRF at 1-1000 nM for 4 h to 10 days did not result in a stimulation of GH or PRL production, nor did it affect the cytoplasmic levels of the corresponding mRNAs. The lack of response to GRF was not affected by dexamethasone, T3, or serum. We conclude that GH3 cells do not provide a useful model system for studies of the mechanism(s) of action of GRF on either GH release or GH gene expression.

Induction of c-fos, calcitonin gene expression, and acidic fibroblast growth factor production in a multipeptide-secreting neuroendocrine cell line.

The multipeptide-secreting 44-2C cell line maintains differentiated function when grown in a serum-free, growth factor- and hormone-deprived milieu. The cells continue to synthesize and secrete calcitonin (CT), CT gene-related peptide, neurotensin, and somatostatin and respond to cellular secretagogues such as GRF and acidic and basic fibroblast growth factor. We designed experiments to ascertain the functional role(s) of cellular factors involved in the maintenance of the differentiated state in 44-2C cells. We report here the phenotypic transformation that occurs in these cells in the course of adjustment to the serum-free state. We also show the differential increase in CT-specific mRNA, the transient induction of c-fos, and the characterization of biologically active acidic fibroblast growth factor.

Somatocrinin stimulates adenylate cyclase-Ns regulatory subunit in a GH3 cell-line: comparison with VIP.

We used a GH3 cell-line to compare the effects of rat GRF (rGRF) and VIP on the adenylate cyclase activity and to determine on what subunit the site of action of these two peptides is. In the GH3 cell-line, VIP was more potent than rGRF to stimulate adenylate cyclase activity. The stimulatory effects of rGRF and forskolin were additive. Cholera toxin decreased the apparent potency of these peptides and pertussis toxin reversed the inhibition by somatostatin of their adenylate cyclase stimulation. We conclude that rGRF acts on the regulatory subunit Ns, different from the regulatory subunit Ni on which somatostatin is suggested to be acting and that, in the GH3 cells, rGRF stimulates adenylate cyclase through VIP-preferring sites.

GRF (somatocrinin) stimulates release of neurotensin, calcitonin and cAMP by a rat C cell line.

We reported previously that GRF stimulates release of neurotensin (NT) by a clonal line of rat C cells (44-2 C). We report here that GRF stimulates calcitonin (CT) and cAMP release in these cells. For release experiments, replicate cultures are incubated for 5-180 min in serum-free defined medium. CT, NT and cAMP are measured by RIA. At a maximally effective concentration of GRF (.1-1.0 nM), there is a 2-3 fold stimulation of CT release at 60-90 min with peak release at 180 min. In contrast, GRF causes a rapid 4-6 fold increase of NT release within 5-15 min. In 44-2 C cells there is a 4-40 fold stimulation of cAMP release by GRF. We conclude that in 44-2 C cells GRF stimulates release of NT and cAMP and show for the first time the effect of this peptide on release of CT.

Somatostatin inhibits growth hormone-releasing factor-stimulated adenylate cyclase activity in GH, cells.

GH3 cells were used to study the effect of rat growth hormone-releasing factor on adenylate cyclase activity and its interaction with somatostatin. Rat GRF stimulates adenylate cyclase activity (ED5 0 = 6 X 10(-8) M) and somatostatin-14 inhibits this GRF-stimulated activity in a non-competitive manner without affecting the basal enzyme levels. Inhibition by somatostatin-14 is observed at concentrations as low as 10(-11) M and the half-maximal effect is obtained with 10(-8) M. Somatostatin-28 is more potent than SS-14 and has an ED5 0 of 3 X 10(-11) M. VIP is more active than rat GRF in stimulating adenylate cyclase activation. We conclude that in GH3 cells rat GRF behaves as a partial VIP agonist by interacting with VIP-preferring receptors and its effects are inhibited by somatostatin.

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s).

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.

Effects of forskolin and cholera toxin on cyclic AMP release in a neurotensin-secreting rat C-cell line.

The effects of forskolin and cholera toxin on the regulation of cAMP release were studied in a neurotensin-secreting rat C-cell line. The interaction of these agents with norepinephrine, a potent neurotensin secretagogue, was also investigated. Forskolin stimulated cAMP release 10(2)-10(3) fold while it increased neurotensin release 2-3 fold. Cholera toxin caused a 10(2)-10(3) fold increase in cAMP release and had no effect on neurotensin release. We conclude that the 44-2 C-cells provide a new model for studying the regulation of the concomitant (via forskolin) or independent (via cholera toxin) secretion of cyclic AMP and/or neurotensin.

GRF is a highly potent activator of adenylate cyclase in the normal human, bovine and rat pituitary: interaction with somatostatin.

The effect of GRF adenylate cyclase activation was studied in normal human, bovine and rat pituitary tissues. Human GRF (hGRF) activates adenylate cyclase in normal human pituitary membrane preparations in a concentration dependent manner (ED5 0 = 10(-11) M). In bovine pituitary cells hGRF stimulates GH secretion into the medium (ED5 0 = 7 X 10(-12) M) and activates adenylate cyclase (ED5 0 = 10(-11) M). In normal rat pituitary cells in monolayer culture, rat GRF (rGRF) stimulates adenylate cyclase (ED5 0 = 3 X 10(-11) M). In normal human pituitary membrane preparations and in normal rat pituitary cells in culture, somatostatin inhibits GRF-stimulated adenylate cyclase in a non-competitive manner, while it does not affect basal (i.e. non-stimulated) adenylate cyclase levels. VIP, a peptide which is structurally homologous to hGRF and rGRF is a weak GRF-agonist and activates adenylate cyclase in human and rat pituitary preparations at concentrations greater than 10 nM.

The first Flaviviridae symposium.

The first annual Flaviviridae Symposium, sponsored by ICN Pharmaceuticals Inc., was held in Lyon, France, on 9 October, 1997, to communicate current understanding on the Flaviviridae. This multidisciplinary symposium attracted over 300 international delegates and presentations covered virology, viral pathogenesis, potential therapies and strategies for vaccine development. The symposium reviewed the research area that may lead to the discovery and design of human and veterinary medicines against members of this virus family.

Myelin basic protein present in the acid extracts of rat hypothalami releases insulin and glucagon from isolated rat pancreatic islets.

The insulinotropic and glucagon-releasing activity of acid extracts of rat hypothalami were tested in two bioassay systems using short-time incubation of isolated rat pancreatic islets and a rat insulinoma (RIN) cell line. Release of insulin and glucagon into the incubation medium was measured by radioimmunoassay. The major insulin-releasing and glucagon-releasing activity eluted in a broad zone in Sephacryl S-200 gel filtration in 30% acetic acid corresponding to the molecular weights between approximately 10 and 40 kD. The highest activity was eluted in a zone corresponding to 14 kD and was purified to homogeneity by means of two steps of reversed-phase HPLC. Amino acid analysis and SDS polyacrylamide gel electrophoresis indicated that the biologically active material was the rat small (myelin) basic protein characterized previously by Dunkley & Carnegie (1974). The purified rat hypothalamic material showed insulinotropic and glucagon-releasing activity indistinguishable from that of purified porcine myelin basic protein, and lost its insulinotropic activity when incubated with anti-myelin basic protein immunosorbent. We conclude that the major insulin-releasing and glucagon-releasing activity in acid extracts of the high-molecular-weight fractions of rat hypothalami is myelin basic protein.

Growth hormone-releasing factor regulates growth hormone mRNA in primary cultures of rat pituitary cells.

A peptide with high intrinsic activity for specifically stimulating the secretion of immunoreactive growth hormone (GH; somatotropin) has been characterized and reproduced by total synthesis. This peptide, human pancreatic growth hormone-releasing factor, 44-amino-acid form (hpGRF1-44-NH2), was isolated from a tumor localized in the pancreas of a patient with acromegaly. We report here the effect of this growth hormone-releasing factor (GRF) on GH release and the GH mRNA levels in monolayer cultures of rat pituitary. The cytoplasmic dot hybridization technique was used to examine the effect of GRF on GH mRNA levels. Incubation of rat pituitary cultures with GRF for 72 hr resulted in a 2- to 2.5-fold increase in GH mRNA levels, and the maximal levels of stimulation were achieved at GRF concentrations as low as 1 fM. GRF did not stimulate prolactin release, nor did it affect specific prolactin mRNA levels in the pituitary cultures. We conclude that GRF is a potent and specific GH secretagogue that also affects specifically GH mRNA levels in normal pituitary cells.

Cloning and sequence analysis of cDNA for the precursor of human growth hormone-releasing factor, somatocrinin.

Molecular cloning has established the primary structures of two precursors of the human pancreas growth hormone-releasing factor (hpGRF-44), somatocrinin. Both polypeptides contain the sequence of hpGRF-44 flanked by basic processing sites. Furthermore, the precursors include a putative signal sequence and a carboxyl-terminal amidation signal for hpGRF-44. The two forms of mRNA code for pre-pro-GRF-107 and pre-pro-GRF-108. Pre-pro-GRF-108 differs from pre-pro-GRF-107 by the insertion of a serine in the carboxyl-terminal portion of the precursor. In vitro translation of tumor poly(A)+ RNA followed by immunoprecipitation with GRF-specific antiserum and gel electrophoresis showed the molecular weight of preprosomatocrinin to be approximately 13,000, which is in good agreement with the molecular weight deduced from the sequences of the cDNA clones.