Mouse Pituitary Tumor Research Articles

Relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 cell

This study tested the relationship between intact cell ATP levels and glucocorticoid receptor-binding capacity in the AtT-20 mouse pituitary tumor cell. Four treatments were used to alter intact cell ATP levels: (1) incubation with NaF, (2) incubation with 2,4-dinitrophenol, (3) alterations in culture age, and (4) incubation with dexamethasone. Each of these treatments clearly altered glucocorticoid receptor-binding capacity. Their effects on ATP levels, however, were not the same: NaF decreased ATP levels, dinitrophenol decreased ATP levels only transiently, and the last two treatments (increasing culture age and incubation with dexamethasone) produced an inverse relationship between ATP levels and receptor-binding capacity. It is concluded that, at least in the intact AtT-20 cell, receptor-binding capacity is not always directly related to ATP levels.

Peptide specificity for stimulation of corticotropin secretion: activation of overlapping pathways by the vasoactive intestinal peptide family and corticotropin-releasing factor.

The hypothalamic peptide vasoactive intestinal peptide (VIP) stimulates ACTH and endorphin secretion by the AtT20/D16 clonal strain of mouse pituitary tumor cells. The dose dependence for VIP stimulation of hormone release is biphasic, indicating that VIP is able to activate at least two classes of receptors in D16 cells (ED50 = 1.6 and 160 nM). We show that at high concentrations (ED50 greater than or equal to 150 nM), other natural peptides with primary structures homologous to that of VIP also increased ACTH secretion by D16 cells, whereas structurally unrelated peptides did not. The stimulatory actions of GH-releasing factor (GRF) and porcine heptacosapeptide with amino-terminal histidine and carboxy-terminal isoleucine amide (PHI) were mediated by high affinity VIP receptors because their effects were not additive with that of 10 nM VIP. In addition, GRF and PHI behaved as antagonists at low affinity VIP receptors; both peptides inhibited stimulation by 1 microM VIP. In contrast, glucagon and gastric inhibitory polypeptide appeared to stimulate ACTH release via low affinity VIP receptors because their effects were additive with that of 10 nM, but not 1 microM, VIP. Since all of the VIP-like peptides increased ACTH secretion only at high concentrations, they were unlikely to represent a physiological ligand for the receptor activated by high concentrations of VIP. Therefore, we determined whether cross-reactivity occurred between VIP-like peptides and corticotropin-releasing factor (CRF), a potent stimulator of ACTH secretion both in vitro and in vivo. The dose-response curve for CRF stimulation of ACTH secretion by D16 cells extended over more than a 1000-fold range of concentrations and was biphasic (ED50 = 2.6 and greater than 300 nM), indicating that CRF interacted with multiple receptor types in D16 cells. However, since the effect of 10 nM CRF was additive with that of 1 microM VIP, the CRF receptor was not the site at which high concentrations of VIP stimulated ACTH release. In contrast, the effect of 1 microM CRF was not additive with that of 1 microM VIP or other VIP-like peptides. Therefore, high concentrations of CRF and the previously recognized VIP-like peptides stimulated ACTH secretion by overlapping pathways. Comparison of the amino acid sequence of CRF with those of the VIP-like peptides showed that 18 of the 41 amino acids in CRF match a corresponding amino acid in at least 1 member of the VIP peptide family.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition of ACTH secretion in mouse pituitary tumor cells by activation of muscarinic cholinergic receptors.

Hormonally stimulated secretion of ACTH from AtT-20 mouse pituitary tumor cells is a cyclic AMP-mediated process. The presence of inhibitory cholinergic muscarinic receptors on these cells was recently reported, and in this study, the relationship between the activation of these receptors and the consequent inhibition of cyclic AMP formation and ACTH secretion was investigated. The muscarinic agent, oxotremorine, antagonized both cyclic AMP synthesis and ACTH secretion in response to corticotropin-releasing factor (CRF), vasoactive intestinal peptide, a 27-amino acid peptide with an N-terminal histidine and a C-terminal isoleucine amide, and forskolin. Other muscarinic agents, carbachol and bethanechol, had similar inhibitory effects. The cholinomimetics reduced basal (unstimulated) ACTH secretion without decreasing basal cyclic AMP levels, and also antagonized hormone release in response to cyclic AMP-independent agonists such as K+, A-23187, and phorbol ester. Scopolamine reversed the inhibitory effects of the muscarinic agents on basal and stimulated ACTH secretion and cyclic AMP formation. Increasing the extracellular calcium concentration reversed the muscarinic antagonism of basal and CRF-stimulated hormone release without affecting the cyclic AMP response. Pertussis toxin pretreatment attenuated the inhibitory effects of the muscarinic agents on forskolin-stimulated cyclic AMP synthesis and ACTH secretion as well as the inhibitory effect of carbachol on basal ACTH release. The data suggest that cyclic AMP is an essential mediator in the ACTH secretory pathway, but that an alternate cyclic AMP-independent ACTH pathway also exists in the clonal cells, and that both pathways may be modulated by a common postcholinergic receptor mechanism.

Pure α-subunit producing tumor derived from a thyrotropic tumor: Impaired regulation of a-subunit and its mRNA by thyroid hormone

We have recently described a mouse pituitary tumor line which produces only the α-subunit of the glycoprotein hormones. This tumor line may be a useful animal model to study autonomous pituitary tumors which secrete only α-subunit. Our pure α-subunit producing tumor was derived from a thyrotropic tumor which secreted intact TSH as well as free α-subunit. Our current studies compare the regulation of α-subunit biosynthesis in a conventional thyrotropic tumor and the α-subunit producing tumor. Thyroxine or triiodothyronine administration to mice bearing the a-subunit producing tumor resulted in no change in plasma α-subunit concentration, and a 10–19% reduction in tumor α-subunit mRNA concentration that was not statistically significant. In contrast, thyroxine administration to mice bearing the thyrotropic tumor resulted in an 81% reduction in plasma α-subunit concentration, and a 75% reduction in tumor α-subunit mRNA concentration (P < 0.01). Other studies using a cDNA specific for thyrotropin-β (TSHβ) failed to detect TSHβ mRNA in the α-subunit producing tumor, while TSHβ mRNA was easily detected in the conventional thyrotropic tumor. We conclude that during the development of the a-subunit producing tumor from a thyrotropic tumor, loss of TSHβ mRNA was also associated with an impaired capacity for thyroid hormone to decrease concentrations of α-subunit mRNA.

Peptide hormone biosynthesis--recent developments.

The biosynthesis of peptide hormones is now recognized to be a highly ordered series of events. These biosynthetic events can frequently be localized to a particular subcellular organelle (Fig.1). Each organelle will be discussed in turn, to emphasize the sequential nature of the maturation process. There will be frequent references to the synthesis and processing of the corticotropin/endorphin precursor within the pituitary. This multihormone precursor is often referred to as pro-opiomelanocortin (POMC). POMC is an interesting model to study, since many post-translational modifications are manifest within its maturation products. More intriguing is the fact that processing of this multihormone precursor is tissue specific. In the pars distalis or anterior lobe of the pituitary, ACTH, β-lipotropin (β-LPH), and an N-terminal or 16 K fragment are the major biosynthetic products. In the pars intermedia or intermediate lobe processing is more complete, and α-melanotropin (α-MSH), corticotropin-like intermediate lobe peptide (CLIP), γ-LPH, various forms of acetylated endorphin, γ-MSH, and a shortened N-terminal (16 K1–49) fragment are the major products (Fig.2). More is known of this system than of many others, primarily because pituitary tissue is relatively easy to maintain in culture and lends itself well to classical pulse/chase biosynthetic experiments. Also the AtT-20 mouse pituitary tumour cell line has been an extremely useful and accurate model for anterior pituitary processing (for extensive reviews on this subject see Eipper and Mains 1980; Chrétien and Seidah 1981).KeywordsGolgi ApparatusSecretory GranulePeptide HormoneIntermediate LobeHydroxy Amino AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

The inhibitory guanine nucleotide-binding regulatory subunit of adenylate cyclase has an adenylate cyclase-independent modulatory effect on ACTH secretion from mouse pituitary tumor cells

Somatostatin inhibits agonist-stimulated cAMP synthesis and ACTH secretion from mouse pituitary tumor cells. It also decreases basal hormone release without affecting cAMP levels and inhibits ACTH secretion in response to agonists whose action is independent of prior cAMP synthesis. These inhibitory effects are attenuated by pertussis toxin, suggesting that the inhibitory guanine nucleotide-binding regulatory subunit of adenylate cyclase modulates effectors, other than adenylate cyclase, during transduction of negative hormonal signals.

Secretion and regulation of two biosynthetic enzyme activities, peptidyl-glycine alpha-amidating monooxygenase and a carboxypeptidase, by mouse pituitary corticotropic tumor cells.

Secretion of peptidyl-glycine alpha-amidating monooxygenase (PAM) activity and enkephalin convertase (a carboxypeptidase B-like enzyme) activity from AtT-20 mouse corticotrope tumor cells was compared with secretion of pro-ACTH/endorphin-derived peptides. Upon stimulation of secretion by corticotropin-releasing factor, a beta-adrenergic agonist, or barium ions, secretion of PAM activity, enkephalin convertase activity, and immunoactive hormone rose in parallel. During inhibition of stimulated secretion with glucocorticoids or cobalt ions, secretion of PAM activity, enkephalin convertase activity, and immunoactive hormone fell in parallel. No measurable secretion of cytosolic, mitochondrial, or lysosomal marker enzymes was detected, indicating that the secretion of PAM activity and enkephalin convertase activity was specific and not a result of cell damage or lysis. The kinetic characteristics, apparent mol wt, and enzymatic properties determined for the secreted enzymes were similar to those for the enzymes from secretory granules. In AtT-20 cells treated with glucocorticoids for up to 8 days, cellular levels of immunoactive ACTH/endorphin-related peptides decreased to one third of control levels. Levels of PAM activity in cell extracts declined in parallel with levels of hormone. In contrast, levels of enkephalin convertase activity, cell protein, lactate dehydrogenase, fumarase, and lysosomal carboxypeptidase did not decline in response to chronic glucocorticoid exposure.

Carbohydrate processing of thyrotropin differs from that of free alpha-subunit and total glycoproteins in microsomal subfractions of mouse pituitary tumor.

We have determined the structures of high mannose (Man) oligosaccharide units of TSH, free alpha-subunit, and non-TSH-related total glycoproteins (TP) within microsomal subfractions of mouse thyrotropic tumor. Tumor minces were incubated with D-[2-3H]Man, homogenized, and subfractionated into rough endoplasmic reticulum (RER) as well as proximal and distal smooth endoplasmic reticulum/Golgi apparatus. TSH subunits and TP were precipitated from these fractions, and high Man units released by endoglycosidase H were analyzed by paper chromatography. Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = N-acetylglucosamine) was not detected in TSH subunit precursors in any fraction, but was detected in TP. Glc1Man9GlcNAc2 accumulated in TSH with chase, but only small amounts were detected in free alpha-subunit and TP. Trimming of Man9GlcNAc2 to Man8GlcNAc2 began in RER before 1 h for all species, but the rate of Man trimming was rapid for free alpha-subunit, moderate for TSH, and slow for TP. Man8GlcNAc2 was a rate-limiting step in processing for all species in the RER. Man5GlcNAc2 was a second major rate-limiting step in processing of free alpha-subunits only and accumulated in distal smooth endoplasmic reticulum/Golgi apparatus. Other studies in progress suggest that a function of the thyrotroph is to modulate the structure of TSH oligosaccharide units during biosynthesis to achieve mature hormone with physiologically appropriate metabolic clearance and intrinsic biological activity. The present study supports this notion, since we found that the qualitative nature and kinetics of processing differ for oligosaccharide units of TSH, free alpha-subunit and TP within the microsomes.

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells.

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.

Hormonal activation of the cAMP-dependent protein kinases in AtT20 cells. Preferential activation of protein kinase I by corticotropin releasing factor, isoproterenol, and forskolin.

The hormonal regulation of adenylate cyclase, cAMP-dependent protein kinase activation, and adrenocorticotropic hormone (ACTH) secretion was studied in AtT20 mouse pituitary tumor cells. Corticotropin releasing factor (CRF) stimulated cAMP accumulation and ACTH release in these cells. Maximal ACTH release was seen with 30 nM CRF and was accompanied by a 2-fold rise in intracellular cAMP. When cells were incubated with both 30 nM CRF and 0.5 mM 3-methylisobutylxanthine (MIX) cAMP levels were increased 20-fold, however, ACTH release was not substantially increased beyond release seen with CRF alone. The activation profiles of cAMP-dependent protein kinases I and II were studied by measuring residual cAMP-dependent phosphotransferase activity associated with immunoprecipitated regulatory subunits of the kinases. Cells incubated with CRF in the absence of MIX showed concentration-dependent activation of protein kinase I which paralleled stimulation of ACTH release. Protein kinase II was minimally activated. When cells were exposed to CRF in the presence of 0.5 mM MIX there was still a preferential activation of protein kinase I, although 50% of the cytosolic protein kinase II was activated. Complete activation of both protein kinases I and II was seen when cells were incubated with 0.5 mM MIX and 10 microM forskolin. Under these conditions cAMP levels were elevated 80-fold. CRF, isoproterenol, and forskolin stimulated adenylate cyclase activity in isolated membranes prepared from AtT20 cells. CRF and isoproterenol stimulated cyclase activity up to 5-fold while forskolin stimulated cyclase activity up to 15-fold. Our data demonstrate that ACTH secretion from AtT20 cells is mediated by small changes in intracellular levels of cAMP and activation of only a small fraction of the total cytosolic cAMP-dependent protein kinase in these cells is required for maximal ACTH secretion.

Characterization of the AtT-20 cell's ‘repleting’ glucocorticoid receptor

Glucocorticoids deplete the AtT-20 mouse pituitary tumor cell of its glucocorticoid receptors. Once placed into steroid-free medium, however, these ‘receptor-deplete’ cells gradually regain their ability to bind glucocorticoids displaceably. The goal of this paper is to determine whether this replete binding-species is indeed a glucocorticoid receptor, and whether any precursor or modified forms are seen during repletion. Once depleted, cells take nearly 3–4 days to replete their full complement of cytosolic glucocorticoid-binding species. Scatchard analysis revealed only one binding site, of which the affinity for dexamethasone was identical to that of the native receptor. The steroid-binding specificity of the ‘replete’ binding species was exactly the same as that of the original receptor. The molecular size and surface charge density of the glucocorticoid-binding species obtained throughout the process of repletion were identical to those of the original receptor. Finally, studies in which replete cells were exposed to glucocorticoid agonists showed that the cell was able to decrease its production of ACTH, indicating that the regenerated binding species was able to function as a biologically active glucocorticoid receptor. We conclude that the replete species is a glucocorticoid receptor identical to the original, and that probably no precursor or modified forms of the repleting receptor exist.

The physico-chemical properties of the AtT-20 cell's glucocorticoid receptor during depletion

Glucocorticoid agonists decrease the number of glucocorticoid receptors in the cloned AtT-20 mouse pituitary tumor cell. To investigate whether the structure of the receptor is altered during this process, we monitored the physico-chemical properties of the nuclear and cytosolic receptors undergoing depletion. Agarose chromatography, DEAE-cellulose chromatography and sucrose gradient ultracentrifugation were employed. Cells were sampled after 2, 24, 48 and 96 h incubation with 10 nM tritiated triamcinolone acetonide. Agarose chromatography yielded, in each case, a single receptor-containing peak that had a Stokes radius of 5.8 nm. Nuclear and cytosolic glucocorticoid receptors from each preparation eluted from DEAE-cellulose as a single, symmetric peak at a KC1 concentration of 0.075 M. Sucrose gradient ultracentrifugation of all samples also yielded only a single peak. For each technique the amount of receptor recovered was inversely related to the length of intact cell incubation. Thus, depletion of the glucocorticoid receptor is not accompanied by observable changes in its size, surface charge or hydrodynamic properties. These results suggest that the first step of agonist-induced glucocorticoid receptor depletion in the AtT-20 cell involves the loss or alteration of the receptor's steroid-binding site.

Forskolin stimulates adenylate cyclase activity, cyclic AMP accumulation, and adrenocorticotropin secretion from mouse anterior pituitary tumor cells.

The effects of forskolin, an adenylate cyclase activator, were investigated on adrenocorticotropin (ACTH) secretion from AtT-20/ D16 -16 mouse pituitary tumor cells. Forskolin increased adenylate cyclase activity in these cells in the absence of added guanyl nucleotide, an effect blocked by somatostatin. Cyclic AMP synthesis and ACTH secretion increased in a concentration-dependent manner, not only in the clonal cells, but in primary cultures of rat anterior pituitary as well. Somatostatin inhibited cyclic AMP synthesis and ACTH secretion in response to forskolin. When forskolin was coapplied with corticotropin releasing factor, cyclic AMP synthesis was potentiated and ACTH secretion additive. The calcium channel blocker, nifedipine, inhibited forskolin, and 8-bromocyclic AMP stimulated ACTH secretion. These data suggest that ACTH secretion may be regulated at the molecular level by changes in cyclic AMP formation, which in turn regulate a calcium gating mechanism.

Functional differences between somatostatin-14 (S-14) and somatostatin-28 (S-28) in atT20/D16-16 mouse pituitary tumor cells

Functional differences between somatostatin-14 (S-14) and somatostatin-28 (S-28) in atT20/D16-16 mouse pituitary tumor cells

Comparison of glucocorticoid receptor depletion and the suppression of ACTH secretion in the AtT-20 cell.

Glucocorticoids exert two effects on the cloned AtT-20 mouse pituitary tumor cell: they inhibit its production of ACTH and decrease its content of glucocorticoid receptors. In this paper the time courses of these two processes are compared to ascertain whether there is a link between them. The initial part of the time course of glucocorticoid receptor depletion was measured during an incubating of intact cells with 10 nM tritiated glucocorticoid. This showed that receptor depletion commenced at 4-6 h, but the half time of depletion was 30 h. Cell fractionation studies demonstrated that the decrease in receptor number occurred proportionately in both the nuclear and cytosolic fractions. The time course of dexamethasone suppression of ACTH secretion was measured under similar conditions. These studies showed that suppression was complete within 24 h. Comparison of these results led to the conclusion that although dexamethasone's effect on both receptor depletion and ACTH secretion is rapid, its effect on ACTH secretion is complete clearly before its effect on receptor depletion. Long term (3 week) dexamethasone incubations showed that even after dexamethasone had depleted the cell's receptor content to only a fraction of its initial value, its inhibitory effect on ACTH was undiminished. These results suggest that agonist action is not dampened with time. Taken together, these studies suggest that depletion is not a step in the mechanism of steroid-hormone action in the AtT-20 cell nor is it involved in diminishing the magnitude of agonist action. Instead, more likely, depletion is the process which removes redundant, spare glucocorticoid hormone receptor complexes.

Glucocorticoids inhibit the growth of AtT-20 mouse pituitary tumor cells

The effect of steroids on the growth of AtT-20 mouse pituitary tumor cells was investigated. It was found that under certain conditions glucocorticoids inhibit growth. Specificity studies indicated that only glucocorticoids caused this effect. Biopotency studies indicated that dexamethasone had its mid-maximal effect around 5 nM. At least part of this inhibition was caused by a decrease in the cell's ability to synthesize DNA; glucocorticoids inhibit the rate at which tritiated thymidine is incorporated into TCA-precipitable material. The magnitude of the growth-inhibiting effect depended upon the prior culture history of the cell. The effect was least on cells derived from exponentially growing cultures and most effective on cells derived from cultures that were approaching their density limit. It is concluded that glucocorticoids inhibit AtT-20 cell growth by interfering with their ability to transform from confluent cultures to ones that grow exponentially. The significance of this finding is that now two mechanisms must be considered when investigating the pathways through which glucocorticoids decrease the ACTH production of the AtT-20 cell.

Thyrotropin (TSH)-releasing hormone decreases phosphatidylinositol and increases unesterified arachidonic acid in thyrotropic cells: possible early events in stimulation of TSH secretion.

TRH stimulated the metabolism of lipids of the phosphatidylinositol (PI)-phosphatidic acid (PA) cycle and caused an increase in the level of free or unesterified arachidonic acid in mouse pituitary thyrotropic tumor (TtT) cells. In cells labeled with [32P]orthophosphate for 45 min, TRH caused a rapid specific increase in [32P]PA to 190 +/- 8% (+/- SE) of the control value at 15 sec (P less than 0.005) and in [32P]PI to 158 +/- 8% at 2 min (P less than 0.005). In cells labeled to isotopic steady state with [3H]inositol, TRH caused a decrease in [3H]PI to 92 +/- 1.8% of the control value at 1 min (P less than 0.01) and increased the level of [3H]inositolmonophosphate. In cells labeled to isotopic steady state with [14C]stearic acid, TRH caused a transient rise in [14C]diacylglycerol and a more prolonged increase in [14C]PA. In cells labeled to isotopic steady state with [3H]arachidonic acid, TRH stimulated a rise in free [3H]arachidonic acid to 210 +/- 8% of the control value at 15 sec (P less than 0.001), with a return to a level of 125 +/- 2% of the control value by 5 min. Arachidonic acid added exogenously caused efflux of 45Ca2+ from prelabeled cells and stimulated TSH secretion. Hence, in TtT cells, TRH 1) rapidly stimulated a decrease in the level of PI and increased inositolmonophosphate, diacylglycerol, and PA; and 2) caused a rapid increase in the level of free arachidonic acid. These effects may be important in stimulation of TSH secretion by TRH. Because arachidonic acid, when added exogenously, mobilized cellular Ca2+ and stimulated TSH secretion, arachidonic acid may mediate, at least in part, TRH-stimulated TSH secretion. The action of TRH on lipid metabolism in TtT cells is different from that in mammotropic pituitary cells, since TRH does not cause an increase in the level of free arachidonic acid in GH3 cells.

12-O-tetradecanoyl-phorbol-13-acetate-induced acth secretion in pituitary tumor cells

The ability of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to stimulate secretion of immunoreactive ACTH from a clonal strain of mouse pituitary tumor cells (AtT-20/D16-16), was investigated. The secretory response to TPA was concentration-and time-dependent. TPAs action on AtT-20 cells was cyclic AMP independent. TPA, even at maximally effective concentrations, had an additive effect on ACTH secretion when co-applied with other agonists such as corticotropin releasing factor, vasoactive intestinal peptide, or (-)-isoproterenol. Secretion of ACTH in response to TPA was reduced by lowering extracellular calcium concentration or in the presence of the calcium channel blocker, nifedipine. Indomethacin and dexamethasone, both blocked TPA stimulated secretion; arachidonic acid, in high concentrations, had a small stimulatory effect on ACTH release. The data suggest that TPA induced secretion in pituitary tumour cells may result from the previously reported ability of the phorbol ester to stimulate the activity of phospholipase A 2, and to the subsequent biosynthesis of prostaglandins.

ACTH secretion in mouse pituitary tumor cells in culture: Inhibition of CRF-stimulated hormone release by somatostatin

Synthetic corticotropin-releasing factor (CRF) stimulates ACTH secretion in the clonal mouse pituitary cell strain AtT20/D16v (D16) in a dose-dependent manner with a half-maximal effect at 2×10 −9M. A single dose of 5×10 −9M CRF maximally stimulates the rate of ACTH secretion during the initial two hrs of treatment. During the period of maximal CRF stimulation intracellular hormone concentration declines progressively to a nadir at 4 hrs. During the ensuing 24 hrs of incubation intracellular hormone levels in CRF-stimulated cells increase gradually toward control values. Somatostatin (SRIF) inhibits the secretory response to CRF. This action of SRIF is dose-dependent with a half-maximal effect at 1×10 −9M and results in decreased maximal ACTH secretion with little effect on the ED 50 for CRF.

A subclass of proteins and sulfated macromolecules secreted by AtT-20 (mouse pituitary tumor) cells is sorted with adrenocorticotropin into dense secretory granules.

The AtT-20 cell, a mouse pituitary tumor line that secretes adrenocorticotropin and beta-endorphin, sorts the proteins it externalizes into two exocytotic pathways. Cells that are labeled with [35S]methionine or [35S]sulfate can be shown to transport three acidic polypeptides (65,000, 60,000, and 37,000 mol wt) and at least two sulfated macromolecules into storage secretory granules. When the cells are stimulated by the secretagogue 8-bromo-cAMP, these polypeptides are coordinately secreted with mature adrenocorticotropin into the culture medium. In contrast, a completely different set of secreted polypeptides and sulfated macromolecules does not enter a storage form and is transported to the cell surface more rapidly. Their secretion from the cells is constitutive and does not require the presence of secretagogues. These molecules, like a viral membrane glycoprotein described previously (Gumbiner, B., and R. B. Kelly, 1982, Cell, 28:51-59) are not found in isolated secretory granules and therefore must reach the cell surface in a different exocytotic vesicle. The segregation of a subclass of secretory macromolecules into the secretory granules, despite the existence of another potential secretory pathway, suggests that these molecules have specific functions related to regulated hormone secretion or storage. Presumably all of the proteins secreted by the regulated secretory granule pathway share some common property that targets them to the secretory granule.