Mouse Anterior Pituitary Tumor Cells Research Articles

Modulation of proopiomelanocortin gene expression by ethanol in mouse anterior pituitary corticotrope tumor cell AtT20

In humans, alcoholism is associated with a decrease in basal ACTH and cortisol levels, and blunted pituitary ACTH responses to administered corticotropin-releasing hormone (CRH) during active drinking and after long-term abstinence. Preclinical studies indicate that a persistent decrease in pituitary activation after chronic exposure to ethanol is due to a direct effect of ethanol on the corticotrope of the anterior pituitary. The present studies were undertaken to determine if ethanol has effects on proopiomelanocortin (POMC) gene transcription activity in mouse anterior pituitary corticotrope tumor cell AtT20. We measured the levels of the POMC primary nuclear RNA transcript (PT), processing intermediate, and mature mRNA in the nucleus and the levels of the POMC mRNA in the cytoplasm after treatment of AtT20 cells with 5–15mM concentrations of ethanol. After 15mM ethanol for 60 to 120min, the POMC PT levels were significantly decreased. This decreased POMC gene transcription activity was coupled with a significant reduction of the POMC cytoplasmic mRNA levels. After ethanol for 4h, however, both the decreases were no longer observed. After 8h, a decrease in the ACTH secretion in the medium was found. We further investigated if CRH or glutamate modulates the effects of ethanol on the POMC gene transcription activity. CRH at 10nM after 60min increased the POMC PT levels, and 15mM ethanol attenuated the effect of CRH on the nuclear transcription activity. Glutamate receptor proteins, including NMDA receptor subtype NR1 (but not NR2A or NR2B) and GluR2, were identified by Western immunoblot analysis in AtT20 cells, with similar sizes to those in mouse hypothalamus. The inhibitory effect of 60min ethanol at 5 to 15mM on the POMC PT levels was attenuated by 50μML-glutamate. Together, our data showed that: (1) ethanol treatment in intoxicate doses significantly inhibited POMC gene transcription activity in a dose- and time-dependent manner in AtT20 cells, and (2) the POMC gene transcription activity in response to CRH or glutamate was altered by ethanol. Our results suggest that ethanol has an inhibitory effect on the POMC gene transcription activity in the anterior pituitary corticotrope, which may contribute to the persistent decrease in pituitary activation after chronic ethanol exposure.

Effect of S(−)- and R(+)-salsolinol on the POMC gene expression and ACTH release of an anterior pituitary cell line

Tetrahydroisoquinolines (TIQs) are thought to play an important role in the process of development of alcohol dependence. Being a condensation product between the alcohol metabolite acetaldehyde and dopamine they might be involved in the balance of the opioid system as well as the reward system. Therefore, the influence of the TIQ salsolinol (SAL) on the pro-opiomelanocortin (POMC) gene expression was investigated using the AtT-20 mouse anterior pituitary tumor cell line. Our results show a significant decrease in the POMC gene expression by the S(−)-enantiomer of SAL. The basal secretion of adrenocorticotropin (ACTH) as well as the corticotropin-releasing factor (CRF)-stimulated ACTH release remained unchanged after R(+)- and S(−)-SAL treatment. Interestingly, it was clearly shown that a reduction of intracellular cAMP level occurred after the treatment of the cells with S(−)-SAL whereas R(+)-SAL did not affect the cAMP production. The obtained results suggest that S(−)-SAL is possibly involved in the establishment of the opioid deficiency in alcoholics.

Corticotropin release-inhibiting factor is preprothyrotropin-releasing hormone-(178-199).

ACTH is the major regulator of the body's adaptive response to stress and the physiological stimulus for glucocorticoid secretion. In addition to the known negative feedback regulation of ACTH by glucocorticoids, a hypothalamic corticotropin release-inhibiting factor (CRIF) that inhibits ACTH synthesis and secretion has been postulated, but not identified. We previously reported that transfection of prepro-TRH complementary DNA into the mouse anterior pituitary tumor cell line AtT-20 results in inhibition of basal and corticotropin-releasing hormone (CRH)-stimulated ACTH synthesis and secretion, suggesting that one or more of the cryptic peptides encoded within the prepro-TRH precursor has CRIF activity. To narrow the choice of peptides responsible for CRIF activity, we first deleted specific sequences within the prepro-TRH complementary DNA and transfected these constructs into AtT-20 cells. Deletion of sequences encoding amino acids 119-229 resulted in the loss of CRIF activity. Of the peptides encoded within this region, prepro-TRH-(178-199), a 22-amino acid peptide, inhibited basal and CRH-stimulated ACTH synthesis and secretion in cultured primary anterior pituitary cells. As this peptide is processed from prepro-TRH in vivo, is found in the external zone of the median eminence, and is secreted from hypothalamic slices in vitro, prepro-TRH-(178-199) fulfills the criteria for a physiological CRIF. The significance of TRH and CRIF sharing a common precursor opens new areas of research in the integrated regulation of pituitary-adrenal and pituitary-thyroid functions.

Corticotropin release-inhibiting factor is preprothyrotropin-releasing hormone-(178-199)

ACTH is the major regulator of the body's adaptive response to stress and the physiological stimulus for glucocorticoid secretion. In addition to the known negative feedback regulation of ACTH by glucocorticoids, a hypothalamic corticotropin release-inhibiting factor (CRIF) that inhibits ACTH synthesis and secretion has been postulated, but not identified. We previously reported that transfection of prepro-TRH complementary DNA into the mouse anterior pituitary tumor cell line AtT-20 results in inhibition of basal and corticotropin-releasing hormone (CRH)-stimulated ACTH synthesis and secretion, suggesting that one or more of the cryptic peptides encoded within the prepro-TRH precursor has CRIF activity. To narrow the choice of peptides responsible for CRIF activity, we first deleted specific sequences within the prepro-TRH complementary DNA and transfected these constructs into AtT-20 cells. Deletion of sequences encoding amino acids 119-229 resulted in the loss of CRIF activity. Of the peptides encoded within this region, prepro-TRH-(178-199), a 22-amino acid peptide, inhibited basal and CRH-stimulated ACTH synthesis and secretion in cultured primary anterior pituitary cells. As this peptide is processed from prepro-TRH in vivo, is found in the external zone of the median eminence, and is secreted from hypothalamic slices in vitro, prepro-TRH-(178-199) fulfills the criteria for a physiological CRIF. The significance of TRH and CRIF sharing a common precursor opens new areas of research in the integrated regulation of pituitary-adrenal and pituitary-thyroid functions.

Corticotropin release inhibiting factor is encoded within prepro-TRH

Corticotropin synthesis and secretion is under negative feedback regulation by glucocorticoids. In addition a hypothalamic factor inhibiting ACTH synthesis and secretion, named corticotropin release inhibiting factor (CRIF), has been postulated but not identified. Here we report that transient transfection of a rat prepro-TRH cDNA into the mouse anterior pituitary tumor cell line AtT-20 inhibits the synthesis and secretion of both basal and CRH-stimulated ACTH. Thus, CRIF appears to be encoded by the same precursor as TRH, suggesting a coordinated regulation of pituitary-adrenal and pituitary-thyroid functions.

Corticotropin release inhibiting factor is encoded within prepro-TRH.

Corticotropin synthesis and secretion is under negative feedback regulation by glucocorticoids. In addition a hypothalamic factor inhibiting ACTH synthesis and secretion, named corticotropin release inhibiting factor (CRIF), has been postulated but not identified. Here we report that transient transfection of a rat prepro-TRH cDNA into the mouse anterior pituitary tumor cell line AtT-20 inhibits the synthesis and secretion of both basal and CRH-stimulated ACTH. Thus, CRIF appears to be encoded by the same precursor as TRH, suggesting a coordinated regulation of pituitary-adrenal and pituitary-thyroid functions.

The influence of S-(−)salsolinol treatment on the POMC gene expression in AtT20 mouse anterior pituitary tumor cells

Condensation products of endogenous amines like dopamin and acetaldehyde as the first product in the catabolic pathway of alcohol, the Tetrahydroisoquinolines (TIQ's), have become more and more interesting due to their possible role in the development of addiction. We have investigated the influence of the optical antipodes R-(+) and S-(−)Salsolinol both on the proopiomelanocortin (POMC) gene expression and on the ACTH secretion. Our results show a significant decrease of POMC gene expression caused by 1μmolar S-Salsolinol. while the ACTH release remained unchanged. The treatment with R-Salsolinol did not influence the POMC gene transcription. A combination of 1μmolar R- or S-Salsolinol and 1μmolar ascorbic acid was used to prevent the oxydation of TIQ's. In this case the POMC mRNA remained uneffected, indicating the fact, that an oxidation product of Salsolinol could be responsible for the POMC gene down regulation.

Chromogranin A does not mediate glucocorticoid inhibition of adrenocorticotropin secretion.

It has recently been proposed that chromogranin A (CgA), a 50-kilodalton acidic glycoprotein that is costored and cosecreted with hormones and neurotransmitters in a variety of tissues, mediates glucocorticoid inhibition of ACTH secretion from AtT20/D16v mouse anterior pituitary corticotroph tumor cells by an undefined autocrine mechanism. We used AtT20/D16v cells, RIAs for murine CgA and ACTH, complementary DNA probes for CgA and POMC, the precursor of ACTH, antiserum that reacts with murine CgA, highly purified bovine CgA, and synthetic rat and porcine pancreastatin, a bioactive cleavage product of CgA in some systems, to study the kinetics of the effect of glucocorticoids on CgA and ACTH synthesis and secretion and of CgA's subsequent effects on ACTH secretion. Exposure to 100 nM dexamethasone (DEX) did not alter the size of CgA or POMC messenger RNA (mRNA) transcripts but increased cell CgA mRNA content 42% by 3 h and 192% by 48 h. DEX decreased cell POMC mRNA content 22% by 6 h and 57% by 48 h. These divergent effects of DEX on steady state mRNA levels were accompanied by similar divergent effects on the production of CgA and ACTH protein. Thirty-minute exposure to 10 nM ovine CRF increased CgA and ACTH release to 300% and 360% of basal levels, respectively. One-hour DEX pretreatment inhibited CRF-stimulated CgA and ACTH release 58% and 49% at 30 min and 67% and 66% at 60 min, respectively. There was a positive correlation between CgA and ACTH release under all conditions at both times (r = 0.976 and 0.964, respectively, P < 0.001), consistent with costorage and cosecretion of the two proteins. The ratio of secreted ACTH to CgA decreased progressively with DEX treatment. Purified bovine CgA (100 nM) had little or no effect on basal or CRF-stimulated ACTH secretion from AtT20/D16v cells, 100 nM synthetic pancreastatin had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells or dispersed normal male rat anterior pituitary cells, and anti-CgA sera had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells. These results indicate: 1) that DEX stimulates CgA synthesis, whereas it inhibits POMC synthesis; 2) that CgA and ACTH are cosecreted; 3) that DEX increases CgA secretion relative to ACTH secretion, but decreases the absolute secretion of both proteins; and 4) that neither CgA nor its proteolytic product, pancreastatin, inhibits ACTH secretion. Thus, CgA does not mediate the inhibitory effect of DEX on ACTH secretion.(ABSTRACT TRUNCATED AT 400 WORDS)

Chromogranin A does not mediate glucocorticoid inhibition of adrenocorticotropin secretion

It has recently been proposed that chromogranin A (CgA), a 50-kilodalton acidic glycoprotein that is costored and cosecreted with hormones and neurotransmitters in a variety of tissues, mediates glucocorticoid inhibition of ACTH secretion from AtT20/D16v mouse anterior pituitary corticotroph tumor cells by an undefined autocrine mechanism. We used AtT20/D16v cells, RIAs for murine CgA and ACTH, complementary DNA probes for CgA and POMC, the precursor of ACTH, antiserum that reacts with murine CgA, highly purified bovine CgA, and synthetic rat and porcine pancreastatin, a bioactive cleavage product of CgA in some systems, to study the kinetics of the effect of glucocorticoids on CgA and ACTH synthesis and secretion and of CgA's subsequent effects on ACTH secretion. Exposure to 100 nM dexamethasone (DEX) did not alter the size of CgA or POMC messenger RNA (mRNA) transcripts but increased cell CgA mRNA content 42% by 3 h and 192% by 48 h. DEX decreased cell POMC mRNA content 22% by 6 h and 57% by 48 h. These divergent effects of DEX on steady state mRNA levels were accompanied by similar divergent effects on the production of CgA and ACTH protein. Thirty-minute exposure to 10 nM ovine CRF increased CgA and ACTH release to 300% and 360% of basal levels, respectively. One-hour DEX pretreatment inhibited CRF-stimulated CgA and ACTH release 58% and 49% at 30 min and 67% and 66% at 60 min, respectively. There was a positive correlation between CgA and ACTH release under all conditions at both times (r = 0.976 and 0.964, respectively, P < 0.001), consistent with costorage and cosecretion of the two proteins. The ratio of secreted ACTH to CgA decreased progressively with DEX treatment. Purified bovine CgA (100 nM) had little or no effect on basal or CRF-stimulated ACTH secretion from AtT20/D16v cells, 100 nM synthetic pancreastatin had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells or dispersed normal male rat anterior pituitary cells, and anti-CgA sera had no significant effect on basal or CRF-stimulated ACTH release from AtT20/D16v cells. These results indicate: 1) that DEX stimulates CgA synthesis, whereas it inhibits POMC synthesis; 2) that CgA and ACTH are cosecreted; 3) that DEX increases CgA secretion relative to ACTH secretion, but decreases the absolute secretion of both proteins; and 4) that neither CgA nor its proteolytic product, pancreastatin, inhibits ACTH secretion. Thus, CgA does not mediate the inhibitory effect of DEX on ACTH secretion.

Intracellular calcium and hormone secretion in clonal [formula omitted] anterior pituitary cells

Intracellular ionized Ca 2+ concentration was measured in clonal mouse anterior pituitary tumor cells with the fluorescent Ca 2+ indicator Quin-2. In control physiological solution, free cytoplasmic Ca 2+ concentration was found to be 139 ± 11 nM. Replacement of 50 mM NaCl by 50 mM KCl in the extracellular fluid caused a 29 mV depolarization and a 4.2-fold increase in the concentration of free cytoplasmic Ca 2+. Under comparable depolarizing conditions, a specific influx of 2.66 nmole of 45Ca 2+ per mg protein was detected 1 min after addition of high K +, accompanied by a marked increase in the initial rate of β-endorphin secretion. In the absence of external Ca 2+, depolarization by K + produced little or no increase in either intracellular free Ca 2+ or hormone release. Incubation of AtT-20 D16-16 cells in the secretagogue norepinephrine led to a depolarization accompanied by an increase in spontaneous action potential frequency and a marked elevation in cytosolic Ca 2+ concentration. Exposure of cells to somatostatin, an inhibitor of normone release, led to only transient decreases in burst frequency and no significant reduction in intracellular Ca 2+ levels. These results indicate that in addition to intracellular Ca 2+, other factors also control secretory activity in AtT -20 D16-16 anterlor pituitary cells.

Metallothionein-I promoter-directed expression of foreign proteins in a mouse pituitary corticotrope tumor cell line

Expressing foreign proteins in heterologous eukaryotic cells has been a powerful tool for analyzing protein structure and function. The inducible mouse metallothionein-I promoter has been particularly useful for expression studies. However, the levels of expression achieved with this promoter in heterologous eukaryotic expression systems have not equaled those observed in vivo for the metallothionein-I gene. We have constructed expression plasmids placing either the gene for chloramphenicol acetyltransferase (CAT) or the cDNA for human neuropeptide Y (NPY) under control of the mouse metallothionein-I promoter. These two expression vectors were used to transfect mouse anterior pituitary tumor cells, from which stable transformants were isolated. The resulting cell lines, Mt.NPY1a and Mt.CAT, were used to maximize functional product expression from the metallothionein-I promoter. In both cell lines, a 35-fold induction of mRNA accumulation, peptide synthesis, or CAT activity was observed.

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules.

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.

Internalization and subcellular distribution of radiolabeled somatostatin-28 in mouse anterior pituitary tumor cells.

The ACTH-secreting mouse AtT-20/D16-16 (AtT-20) tumor corticotroph possesses receptors for the tetradecapeptide somatostatin (S-14) to which the 14-amino acid N-terminal extension somatostatin-28 (S-28) also binds. When AtT-20 cells are exposed to either S-14 or S-28 for extended periods of time, a marked decrease in S-14 receptor density is observed. Since receptor down-regulation is frequently associated with internalization of ligand and/or receptor, the present study was designed to establish whether AtT-20 cells could in fact internalize S-28 and to determine the subcellular localization of internalized peptide. Cells were incubated in the presence of [Leu8, D-Trp22,125I-Tyr25] S-28 for 1, 4, and 18 h; washed with PBS; and harvested. Cell pellets were fixed, sectioned, and analyzed by light and electron microscopic autoradiography. Uptake of radiolabeled S-28 (90% of all cells) was inhibited by 80% when unlabeled S-14 was coincubated with [125I]S-28. Of the cell compartments examined, plasma membrane, secretory granules, lysosomes, Golgi apparatus, and nuclear membrane all had distinct time-dependent labeling patterns. Plasma membranes were maximally labeled 1 h after exposure to [125 I] S-28. Secretory granule and lysosomal labeling was observed within 1 h, but was maximal after 18 h of exposure. Labeling of the granule compartment preceded that of the Golgi apparatus. The nuclear compartment (membranes plus nuclei) was also labeled significantly after 18 h of incubation. However, the nuclear membrane itself was labeled after only 1 h of exposure to the ligand. The data suggest that radiolabel is transferred from the plasma membrane to the intracellular organelles as a function of exposure time. Labeling of the secretory compartment suggests that granules may bind and protect internalized peptide from lysosomal degradation. Appearance of label in the nuclear compartment suggests that S-28 (and S-14) may have effects on transcriptional activity in AtT-20 cells.

Liposome delivery of cyclic AMP-dependent protein kinase inhibitor into intact cells: specific blockade of cyclic AMP-mediated adrenocorticotropin release from mouse anterior pituitary tumor cells.

Insertion of a crude preparation of cyclic AMP (cAMP)-dependent protein kinase inhibitor (PKI) into a cloned mouse anterior pituitary cell line (AtT-20/D16-16) blocked cAMP-mediated hormone release. This was accomplished by developing a technique to incorporate PKI into multicellular cultures. The technique involved the encapsulation of the PKI into liposomes coupled to Protein A (a bacterial protein that binds to the Fc portion of antibodies). Application of such liposomes to AtT-20 cells targeted by pre-treatment with an antiserum against neural cell adhesion molecule (a cell surface glycoprotein expressed by these cells) resulted in the attachment of the liposomes onto the cell surface followed by the delivery of the liposome content into the cells. The AtT-20 cells respond to cAMP-promoting agents such as forskolin by secreting the hormone adrenocorticotropin (ACTH). Liposomes containing PKI and coupled to protein A specifically blocked cAMP-mediated ACTH release from cells treated with anti-N-CAM antibodies. In contrast, the ACTH release response to K+ or phorbol esters does not appear to involve cAMP and was not reduced by such manipulations. The specificity of PKI to block hormone release initiated by one but not by other secretagogues directly links cAMP-dependent protein kinase with the ACTH release process but suggests that there are other mechanisms also involved in stimulus-secretion coupling in corticotrophs.

Corticotropin releasing factor increases proopiomelanocortin messenger RNA in mouse anterior pituitary tumor cells.

The ability of corticotropin releasing factor (CRF) to stimulate adrenocorticotropin (ACTH) synthesis in corticotrophs was assessed by measuring total cell content of ACTH and the levels of proopiomelanocortin (POMC) mRNA in a cloned tumor cell line of the mouse anterior pituitary (AtT-20/D16-16). CRF treatment caused a time-dependent increase in POMC mRNA levels (measured using a hybridization technique) as well as elevating total ACTH content in AtT-20 cells. The increase in POMC mRNA levels preceded changes in ACTH content and slowly returned toward control levels after CRF withdrawal. The rise in POMC mRNA levels following CRF stimulation appeared to be specific since beta-actin mRNA levels were not affected by CRF treatment. Both 8-bromo-cAMP and phorbol ester increased POMC mRNA levels in AtT-20 cells, suggesting that CRF may act through different protein kinases to regulate the POMC gene. CRF appears to activate the POMC gene since treatment of the AtT-20 cells with the peptide increased the levels of an RNA species in the nuclei having the expected molecular weight of the transcript of the POMC gene. The results indicate that continued exposure of corticotrophs to CRF induces long term increases in the ACTH synthetic capacity of those cells.

Multiple mechanisms of somatostatin inhibition of adrenocorticotropin release from mouse anterior pituitary tumor cells.

The release of ACTH from a clonal cell line of the mouse anterior pituitary (AtT-20/D16-16) can be stimulated by forskolin, 8-bromo-cAMP, and K+. SRIF and its structurally related analogs are very potent inhibitors of the ACTH release response to these secretagogues. The potency of SRIF, its analogs, and somatostatin-28 to inhibit stimulated ACTH release is relatively the same for each of these three secretagogues. The mechanisms by which SRIF regulates the secretion of ACTH can be differentiated by various pharmacological manipulations. Pretreatment of AtT-20 cells with SRIF (10(-7) M) desensitizes SRIF's inhibition of forskolin but not K+ or 8-bromo-cAMP-stimulated ACTH release. Pertussis toxin pretreatment abolishes SRIF's inhibition of forskolin-stimulated ACTH release but not SRIF's inhibition of the ACTH release response to K+ or 8-bromo-cAMP. In contrast, increasing the calcium concentration in the medium reduces SRIF's inhibition of K+ but not forskolin or 8-bromo-cAMP-stimulated ACTH release. These results suggest that SRIF regulates ACTH release from AtT-20 cells through multiple mechanisms. If SRIF acts through a single receptor to produce its effects on ACTH release, then the data imply that the SRIF receptor is coupled to more than one second messenger system.

Sorting and secretion of adrenocorticotropin in a pituitary tumor cell line after perturbation of the level of a secretory granule-specific proteoglycan.

A mouse anterior pituitary tumor cell line (AtT-20) that secretes adrenocorticotropin and beta endorphin sorts the proteins it transports to the surface into two exocytotic pathways. AtT-20 cells also synthesize a secretory granule-specific sulfated molecule and secrete it on stimulation (Moore, H.-P., B. Gumbiner, and R. B. Kelly, 1983, J. Cell Biol., 97:810-817). We show here that this molecule is sensitive to proteolysis and that the residual sulfated material co-migrates with a chondroitin sulfate standard on thin-layer electrophoresis. Furthermore, this sulfated molecule is completely sensitive to chondroitinase ABC digestion. Thus the secretory granule-specific sulfated molecule is a proteoglycan with chondroitin sulfate side chains. We examined the role of proteoglycans in the sorting and secretion of adrenocorticotropin in AtT-20 cells by severely decreasing the amount of this vesicle-specific proteoglycan in two ways. First, a xyloside was used to inhibit proteoglycan biosynthesis; second, a variant of the AtT-20 cell line was isolated that synthesized little of the sulfated proteoglycan. In neither case was the sorting or secretion of adrenocorticotropin detectably altered, suggesting that the proteoglycan is not required for these processes.

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.

Characterization of a peptide alpha-amidation activity from rat anterior pituitary.

A secretory granule associated enzymatic activity from the rat anterior pituitary that can convert the synthetic peptide D-Tyr-Val-Gly into D-Tyr-Val-NH2 is described. Due to the presence of inhibitory activity in the cytosol and rough endoplasmic reticulum/Golgi apparatus fractions, the alpha-amidation activity is barely detectable in crude rat anterior pituitary homogenates. The alpha-amidation activity was primarily soluble, displayed a pH optimum of about 7.0, and showed a requirement for molecular oxygen. The activity was stimulated 7-fold by the addition of optimal concentrations of copper. Of the cofactors tested, only reduced ascorbate produced a severalfold stimulation of activity. Studies with varied D-Tyr-Val-Gly or varied monoiodo-D-Tyr-Val-Gly demonstrate Michaelis Menten kinetics with indistinguishable kinetic constants for both peptides. Upon addition of ascorbate, the apparent Km and Vmax for the synthetic substrate, as estimated from Lineweaver-Burk and Eadie-Hofstee plots, increased by 30- and 60-fold, respectively. Several alpha-melanotropin- and gamma-melanotropin-related peptides with COOH-terminal glycine residues were effective competitive inhibitors of the reaction while the corresponding alpha-amidated peptides were very poor inhibitors. The rat anterior pituitary alpha-amidation activity appears to be very similar to the alpha-amidation activity in rat intermediate and neural pituitary, and mouse anterior pituitary tumor cells.

Peptide alpha-amidation activity in mouse anterior pituitary AtT-20 cell granules: properties and secretion.

Mouse anterior pituitary corticotropic tumor cells (AtT-20/D-16v) were homogenized and subjected to subcellular fractionation. A secretory granule associated enzyme activity was detected which could convert D-Tyr-Val-Gly into D-Tyr-Val-NH2. The enzyme activity was dependent on the presence of molecular oxygen, copper ions, and reduced ascorbate. Potent endogenous inhibitors of the enzyme obscured the activity unless appropriate levels of copper ions were added. The production of radiolabeled D-Tyr-Val-NH2 from labeled D-Tyr-Val-Gly was inhibited by a wide variety of peptides possessing COOH-terminal glycine residues but not by a number of other peptides, suggesting that many peptides with COOH-terminal glycine residues can function as substrates in the alpha-amidation reaction. Kinetic studies with varied concentrations of D-Tyr-Val-Gly demonstrated Michaelis-Menten kinetics; both the Km for D-Tyr-Val-Gly and maximum velocity (Vmax) increased upon addition of ascorbate to the reaction. Under optimized assay conditions, the secretory granule pool contains enough alpha-amidation activity to alpha-amidate all the relevant peptides in granules in a small fraction of the total time required for complete biosynthetic processing. Secretion of alpha-amidation activity was stimulated along with secretion of pro-ACTH/endorphin-derived peptides upon addition of synthetic corticotropin releasing factor or 8-bromo-cAMP.