Effect of tunicamycin on IgM, IgA, and IgG secretion by mouse plasmacytoma cells

Tunicamycin, an antibiotic that prevents glycosylation of glycoproteins by blocking the formation of N-acetylglucosamine-lipid intermediates, was used to study the importance of glycosylation for the secretion of immunoglobulins by mouse plasmacytoma lines that produce immunoglobulins of different classes. Biosynthetically labeled secreted and intracellular immunoglobulins were measured by immunoprecipitation assays. Tunicamycin, at a concentration of 0.5 mug/ml produced an 81% inhibition of IgM secretion by MOPC 104E plasma cells without significantly affecting the initial rate of synthesis of intracellular IgM. No increase in the intracellular degradation of nonglycosylated IgM could be demonstrated. Tunicamycin also produced a 64% average inhibition of IgA secretion by several mouse IgA-secreting plasmacytoma lines. In contrast, despite inhibiting the incorporation of D-[14C] glucosamine into newly synthesized IgG, tunicamycin only produced a 28% average inhibition of IgG secretion, which was only slightly more than the nonspecific inhibition of secretion of the normally nonglycosylated lambda2 light chains by variant MOPC 315 plasmacytomas. These data indicate that the extent of inhibition of immunoglobulin secretion produced by tunicamycin depends on the immunoglobulin class produced by the plasma cell.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands

Summary Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/m²) was almost 3 times as high as that of ACTH. In the mature igranules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.     

Effect of tunicamycin on biosynthesis, processing and release of proopiomelanocortin-derived peptides in the intermediate lobe of the frog Rana ridibunda

The intermediate lobe of the pituitary gland synthesizes a glycoprotein, proopiomelanocortin (POMC), which is cleaved by specific proteolytic enzymes to generate several hormonal peptides. The purpose of the present study was to examine the possible role of the carbohydrate moiety in the synthesis, intracellular processing and release of POMC-derived peptides in frog (Rana ridibunda) intermediate lobe cells. In vitro incorporation of [3H]-labelled glucosamine gave rise to three major radioactive products. Trypsin digestion of each of these glycopeptides gave a single glucosamine-labelled tryptic fragment with identical chromatographic characteristics. We conclude that Rana POMC is glycosylated in only one site (its gamma-MSH region) and that intracellular processing of this prohormone gives rise to smaller glycopeptides including glycosylated gamma-MSH. Treatment with the antibiotic tunicamycin (10 micrograms/ml, 6 hr) inhibited the glycosylation of POMC but did not significantly alter the neosynthesis of the peptide moiety of the precursor. Pulse-chase experiments combined with high-performance liquid chromatography analysis of the peptides derived from POMC revealed that inhibition of glycosylation by tunicamycin had no effect on the enzymatic cleavage of the precursor nor on the release of mature peptides. Thus, it is concluded that, in the frog, glycosylation of POMC has no influence on the biosynthesis, processing and release of intermediate lobe hormones.     

Alpha-amidated peptides derived from pro-opiomelanocortin in normal human pituitary.

Normal human pituitaries were extracted in boiling water and acetic acid, and the alpha-amidated peptide products of pro-opiomelanocortin (POMC), alpha-melanocyte-stimulating hormone (alpha MSH), gamma-melanocyte-stimulating hormone (gamma 1MSH), and amidated hinge peptide (HP-N), as well as their glycine-extended precursors, were characterized by sequence-specific radioimmunoassays, gel-chromatography, h.p.l.c. and amino acid sequencing. alpha MSH and gamma 1MSH constituted 0.27-1.32% and 0.10-5.10%, respectively, of the POMC-derived products [calculated as the sum of adrenocorticotropic hormone (ACTH)-(1-39), ACTH-(1-14) and alpha MSH immunoreactivity]. alpha MSH and ACTH-(1-14) were only present in non- or mono-acetylated forms. Only large forms of gamma 1MSH and gamma 2MSH were present in partly glycosylated states. The hinge peptides were amidated to an extent two to three orders of magnitude greater than alpha MSH and gamma 1MSH. Most (99%) of the HP-N was of low molecular mass and consisted mainly of HP-N-30. The remaining part was high-molecular-mass HP-N, probably HP-N-108, although the presence of HP-N-44 could not be completely excluded. These results show that all the possible amidated POMC-related peptides are present in normal human pituitary. It also shows that cleavage in vivo at all dibasic amino acids but one, takes place at the N-terminal POMC region; the exception is at the POMC-(49-50) N-terminal of the gamma MSH sequence. The pattern of peptides produced suggests that the generation of amidated peptides is mainly regulated at the endopeptidase level.     

Biosynthesis, processing and release of pro-opiomelanocortin related peptides in the intermediate lobe of the pituitary gland of the frog (Rana ridibunda)

The biosynthesis of pro-opiomelanocortin (POMC) and related peptides by the intermediate lobe of the pituitary gland was studied in the frog Rana ridibunda using the pulse-chase technique. Analysis of radioactive proteins by dodecyl sulfate polyacrylamide gel electrophoresis showed that during pulse incubations a 36,000 dalton (36K) glycosylated prohormone was synthesized. It disappeared slowly during chase incubations, giving rise to another glycosylated protein (Mr 18K), identified as the N-terminal fragment of POMC. This latter protein was secreted to the incubation medium. High performance liquid chromatography analysis of peptides synthesized during chase incubations revealed the biosynthesis of two peptides related to gamma-MSH, three peptides related to alpha-MSH, one endorphin-related and one CLIP-related peptides. These newly synthesized peptides were slowly secreted to the incubation medium. Among the alpha-MSH related peptides, only the des-N alpha-acetyl alpha-MSH form of the peptide was found to be present within the cells, in contrast to the incubation medium where the presence of des-N alpha-acetyl alpha-MSH and a modified alpha-MSH was demonstrated.     

The effect of pepstatin A, an inhibitor of the pro-opiomelanocortin (POMC)-converting enzyme, on POMC processing in mouse intermediate pituitary

In our previous studies, we have purified a unique, paired basic residue-specific, prohormone-converting enzyme from pituitary intermediate lobe secretory vesicles. This enzyme, an aspartyl protease, was shown to cleave the intermediate lobe prohormone, pro-opiomelanocortin (POMC), to adrenocorticotropin, beta-endorphin and a 16 kDa NH2-terminal glycopeptide, in vitro [(1985) J. Biol. Chem. 260, 7194-7205]. To provide some evidence that this enzyme plays a role in prohormone conversion in the intact cell, the ability of pepstatin A, an aspartyl protease inhibitor, to block POMC processing in the mouse intermediate pituitary was investigated. By the use of a radioactive pulse-chase paradigm, [3H]POMC processing was found to be inhibited by 36.4% in pepstatin A-treated intermediate lobes. This result is consistent with the inactivation of pro-opiomelanocortin-converting enzyme by pepstatin A in the intact pituitary and further supports a role of this enzyme in POMC processing in vivo.     

Effect of sodium valproate on the secretion of proopiomelanocortin derived peptides from cultured rat anterior pituitary cells

We examined effects of sodium valproate, a gamma amino butyric acid (GABA)-transaminase inhibitor, on the secretion of immunoreactive (IR)-ACTH and IR-beta-endorphin/LPH from cultured rat anterior pituitary cells to determine whether sodium valproate has a direct action on the secretion of ACTH and its related peptides from the cultured rat anterior pituitary gland. During the 3 h incubation, the basal secretion of IR-ACTH and IR-beta-endorphin/LPH decreased to 50.8% and 58.3%, respectively, of the control concentration after adding 10(-7) M sodium valproate into the incubation media and to 67.7% and 69.3%, respectively, of the control levels with 10(-8) M sodium valproate. However, sodium valproate at a concentration of 10(-6) M or 10(-9) M did not affect the basal concentration of IR-ACTH and IR-beta-endorphin/LPH. Sodium valproate at a concentration of 10(-7) M significantly attenuated the stimulated release of IR-ACTH and IR-beta-endorphin/LPH by 10(-9) or 10(-10) M of ovine corticotrophin releasing factor. These results indicate that sodium valproate could directly effect rat anterior pituitary cells to suppress both basal and stimulated release of proopiomelanocortin derived peptides and this supports the hypothesis that sodium valproate has a direct effect at the pituitary corticotroph in reducing plasma ACTH.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands. A quantitative immunocytochemical approach

Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/micron2) was almost 3 times as high as that of ACTH. In the mature granules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules' antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.     

Effect of tunicamycin on xylanase secretion in the yeast Cryptococcus albidus

The yeast Cryptococcus albidus secretes a glycosylated xylanase (48 kDa) in the culture medium in response to beta-methylxyloside as inducer. Addition of tunicamycin to the medium results in the formation of a modified xylanase (40 kDa) which is depleted in carbohydrate content and whose enzymatic activity is 2.5 times less than that of the glycosylated xylanase. The secretion of xylanase was followed under both conditions by pulse-chase experiments. The half-time of secretion of the glycosylated and nonglycosylated forms was 5 and 2 h, respectively. Cell-associated xylanase activity was not detected when the cells were treated with the antibiotic. The absence of cell wall-associated xylanase, after tunicamycin treatment, was confirmed by immunolocalization with anti-xylanase antibodies at the electron microscopic level. The results suggest that the interactions of carbohydrate moiety within the cell wall retarded the secretion of the enzyme to the medium.     

Effect of monensin on synthesis, post-translational processing, and secretion of dopamine beta-hydroxylase from PC12 pheochromocytoma cells

Monensin was used to ascertain the location in the biosynthetic pathway where the 77,000-Mr membrane-bound subunit form of dopamine beta-hydroxylase is post-translationally converted to the 73,000-Mr soluble form. Treatment with low concentrations of monensin (less than or equal to 50 nM) completely depleted the cells of the norepinephrine and dopamine, had a small effect on protein synthesis, and enhanced post-translational processing of only dopamine beta-hydroxylase which was previously synthesized and presumably packaged into neurosecretory vesicles. At these low concentrations, exit from the Golgi apparatus did not appear to be blocked since stimulated secretion of a group of high molecular weight [35S]methionine-labeled proteins was not inhibited. Treatment with higher concentrations of monensin (200 nM) prevented the secretion of the [35S] methionine-labeled proteins normally released with a secretagogue, and also prevented the secretion of [3H] mannose-labeled proteins including dopamine beta-hydroxylase. Surprisingly, a group of lower molecular weight [35S]methionine-labeled proteins was now released from monensin-treated cells. Treatment with high concentrations of monensin (greater than or equal to 200 nM) appeared to block the secretory pathway prior to the packaging step, probably in the Golgi apparatus. If the proteins were packaged prior to monensin treatment, they were released upon stimulation with secretagogues. Monensin treatment (200 nM) enabled the post-translational processing of newly synthesized dopamine beta-hydroxylase, from the 77,000-Mr to the 73,000-Mr subunit form, to go to completion. The susceptibility of this 73,000-Mr subunit form to endoglycosidase H digestion was unaltered, suggesting that dopamine beta-hydroxylase from monensin-treated cells may have the same high mannose oligosaccharide content as native dopamine beta-hydroxylase. These experiments indicate that the post-translational processing of dopamine beta-hydroxylase occurs in the Golgi apparatus and may continue in immature granules prior to their acidification.     

Post-translational processing of pro-opiomelanocortin (POMC)-derived peptides during fetal monkey pituitary development. II. beta-Lipotropin (beta-LPH)-related peptides

We have studied the post-translational processing of POMC-derived peptides during fetal monkey pituitary development using immunoassay and reverse-phase high-performance liquid chromatography (RP HPLC). Whole pituitary glands obtained from Day 50 and 55 fetal monkeys and separated lobes From Day 65 to 155 were extracted, fractionated, and analyzed for beta-melanotropin (beta-MSH), midportion beta-endorphin (beta-EP), and acetylated beta-EP immunoactivity. Separated adult pituitary lobes were analyzed for comparison. At Day 50, POMC-containing cells were located in both the anterior and intermediate pituitary lobes by immunofluorescence staining, the majority of these cells were localized in the anterior lobe. The Day 50 and 55 whole pituitaries contained predominantly beta-lipotropin (beta-LPH), gamma-lipotropin (gamma-LPH), beta-EP(1-31), and 2.2-kda beta-MSH. No acetylated products were found in Day 50 whole pituitary extracts. By Day 55, carboxy-shortened and acetylated beta-EPs were barely detectable in whole pituitary extracts. These forms were more apparent in the Day 65 separated neurointermediate lobe (NIL) extracts, and were similar to adult proportions by Day 80. The adult anterior lobe contained predominantly beta-LPH, beta-EP, and gamma-LPH. Adult NILs contained almost exclusively 2.2-kda beta-MSH, alpha-N-acetyl beta-EP(1-31) and alpha-N-acetyl beta-EP(1-27). The production of 2.2-kda beta-LPH in the monkey NIL indicates that monkey beta-LPH is different from rat beta-LPH in that it must contain the paired-basic cleavage site required for the formation of 2.2-kda beta-MSH that is known to be lacking in rat beta-LPH. Another finding was that monkey beta-EP contains a Tyr residue at position 27 as found in human beta-EP but appears to have the rat Gln substitution at position 31. The post-translational processing patterns characteristic of each lobe were well established by midterm fetal development (Day 80).     

The effects of tunicamycin on anterior pituitary hormone producing cells in the rat

An electron microscopic study was performed to clarify the effects of tunicamycin, a glycosylation inhibitor, on rat anterior pituitary cells. Tunicamycin (10, 50, and 100 micrograms/250 g B.W.) was intraperitoneally injected into rats, which were sacrificed 24 hrs later. Protein hormone producing GH and prolactin cells, and ACTH cells which are known to have a glycosylated precursor, showed no recognizable ultrastructural changes. TSH cells and gonadotrophs, both of which secrete glycoprotein hormones consisting of alpha and beta subunits, showed remarkable dilatation of the rough endoplasmic reticulum, and decreased numbers of secretory granules. These results suggest that the role of glycosylation in TSH cells and gonadotrophs may have a different biological significance from that in ACTH cells.     

Effects of the monovalent ionophore monensin on the intracellular transport and processing of pro-opiomelanocortin in cultured intermediate lobe cells of the rat pituitary

Detailed studies on the effects of the ionophore monensin upon synthesis, maturation, and intracellular transport of pro-opiomelanocortin in cultures of rat pituitary intermediate lobe cells have been carried out. When added at concentrations larger than 5 X 10(-8) M monensin significantly inhibited protein synthesis by cultured intermediate lobe cells. Pro-opiomelanocortin synthesis was also reduced proportionally to the overall rate of protein synthesis. During pulse-chase experiments, monensin when added at a concentration of 10(-5) M at the beginning of the chase incubation completely inhibited the proteolytic processing of pro-opiomelanocortin. Using a subcellular fractionation procedure of intermediate lobe cell extracts on Percoll gradients, we were able to show that after the addition of monensin (10(-5) M), labeled pro-opiomelanocortin molecules synthesized during a 15-min pulse-incubation were recovered intact after a 2-h chase, in the fractions of the density gradient corresponding to the rough endoplasmic reticulum and Golgi elements. No maturation products or precursor molecules entered the granule fractions as observed in nontreated cells. Taken together these results strongly suggest that monensin blocks the intracellular transport of newly synthesized pro-opiomelanocortin molecules at the Golgi level and that inhibition of proteolytic processing is due to the failure of the prohormone to enter the cell compartment (probably the secretion granules) where maturation proteases are located.     

Regulation of pro-opiomelanocortin synthesis by dopamine and cAMP in the amphibian pituitary intermediate lobe

The modulation of pro-opiomelanocortin (POMC) synthesis in Xenopus laevis pituitary intermediate lobe (IL) during background adaptation and the role of dopamine and cAMP in mediating this effect were examined. Neurointermediate lobes (NILs) were pulselabeled in vitro with [3H]arginine and analyzed for POMC synthesis by acid-urea gel electrophoresis. After black background adaptation of the animal (7 days), POMC synthesis increased 5-6-fold, while after white background adaptation (7 days), POMC synthesis decreased by 76%. Dopamine (50 microM) suppressed POMC synthesis in NILs in culture. In the absence of dopamine, POMC synthesis was stimulated. Several experiments were conducted to determine the category of dopamine receptor in the X. laevis IL. A D-2 dopamine receptor agonist inhibited immunoreactive alpha-MSH release from the NIL in a D-2 antagonist-reversible manner. A D-1 receptor agonist or antagonist did not alter the release of immunoreactive alpha-MSH from the NIL. Dopamine (10 microM) inhibited forskolin-stimulated cAMP accumulation. In addition, dopamine inhibition of POMC synthesis in cultured ILs was reversed by 8-Br-cAMP. These studies suggest that white background adaptation results in stimulation of the X. laevis D-2 receptor, which reduces cAMP production and POMC synthesis. Conversely, during black background adaptation the IL D-2 receptor is not stimulated, leading to increased cAMP production and POMC synthesis.     

Expression of porcine pro-opiomelanocortin cDNA in heterologous monkey kidney cells. Biosynthesis and secretion of the prohormone without processing

Pro-opiomelanocortin is the common precursor to several pituitary hormones. These include alpha-melanotropic hormone, adrenocorticotropic hormone, beta-lipotropic hormone, and beta-endorphin. The porcine pro-opiomelanocortin cDNA was inserted downstream from the early promoter of a SV40-derived expression vector and transfected into the monkey kidney COS-1 cells. Transient expression of the pro-opiomelanocortin cDNA was observed between 48 and 70 h after transfection. Analysis of pro-opiomelanocortin-related material in COS-1 cell extracts and culture medium revealed that these cells synthesize and secrete constitutively pro-opiomelanocortin without further processing into its mature hormones. Our results suggest that COS-1 cells do not contain the necessary enzymatic machinery to process complex precursors such as pro-opiomelanocortin.     

The effects of pro-opiomelanocortin peptides on cyclic AMP and tyrosinase in melanoma cells

Des-, mono-, and diacetylated melanotropin (des-, mono-, and di-Ac MSH, respectively) were compared for their dose-related effects on content of adenosine 3':5'-monophosphate (cAMP) and tyrosinase activity in the Cloudman S91 mouse melanoma tumor. Des-Ac MSH was more potent than the acetylated forms of MSH at increasing cellular levels of cAMP; mono- and di-Ac MSHs, however, were more potent than des-Ac MSH at elevating the activity of the enzyme, tyrosinase. Lysine-gamma1 MSH, a melanotropin from the amino terminus of pro-opiomelanocortin, exhibited slight stimulatory effects on tyrosinase and these actions were less than additive to those of mono-Ac MSH. Unlike their actions on amphibian skin-darkening or in mammalian behavior, neither beta-endorphin1-31 nor its derivatives, N-Ac-beta-endorphin1-27 or beta-endorphin30-31 (glycylglutamine), exhibited any influence on tyrosinase activity evoked by mono-Ac MSH in the tumor cells.     

Growth hormone-releasing peptide-2 stimulates secretion and synthesis of adrenocorticotropic hormone in mouse pituitary

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides which induce strong GH release in both animals and humans. Among them, GHRP-2 is known to stimulate GH release by acting at both hypothalamic and pituitary sites, but also induces adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRP-2 may stimulate ACTH release directly via GHRP receptor type 1a in ACTH-producing tumors. GHRP-2 increases ACTH secretion in rat in vivo, but not ACTH release from rat primary pituitary cells. In the present study, in order to elucidate the mechanism underlying ACTH secretion by GHRPs, mouse pituitary cells were stimulated by GHRP-2. GHRP receptor mRNA was expressed in the mouse pituitary, and GHRP-2 directly stimulated secretion and synthesis of ACTH in the mouse anterior pituitary cells. GHRP-2 increased intracellular cyclic AMP production. H89, a potent protein kinase A (PKA) inhibitor, and bisindolylmaleimide I, a selective protein kinase C (PKC) inhibitor, inhibited the GHRP-2-induced ACTH release, and that H89, but not bisindolylmaleimide I, inhibited the GHRP-2-induced proopiomelanocortin mRNA levels. Together, the GHRP-2-induced ACTH release was regulated via both PKA and PKC pathways in the mouse pituitary cells, while ACTH was synthesized by GHRP-2 only via the PKA pathway.     

Intracellular sites of proteolytic processing of pro-opiomelanocortin in melanotrophs and corticotrophs in rat pituitary.

A synthetic peptide (ST-1) corresponding to the cleavage site between ACTH and beta-lipotropic hormone moieties of murine pro-opiomelanocortin (POMC) was constructed and its polyclonal antibody was generated. This antiserum immunoprecipitated only POMC from extracts of AtT-20 cells. Moreover, an antiserum raised against porcine ACTH immunoprecipitated both ACTH[1-39] and POMC. When ultra-thin frozen sections of melanotrophs in rat pars intermedia were immunolabeled with anti-ST-1 followed by protein A-gold, gold particles indicating the presence of POMC were selectively found in the electron-dense secretory granules in the Golgi area. In addition, the immunolabeling was also observed in the cisternae of the Golgi apparatus and rough endoplasmic reticulum. In contrast, with a polyclonal antibody specific for alpha-melanocyte-stimulating hormone the gold particles were found exclusively in the electron-lucent secretory granules, with none seen in the electron-dense secretory granules. With anti-ACTH serum, gold particles were observed in the electron-dense and -lucent secretory granules. In corticotrophs in the pars distalis, many gold particles indicating the presence of POMC were observed in the Golgi and peripheral secretory granules, but the percentage of immunolabeling in the peripheral secretory granules varied from cell to cell. On the other hand, ACTH immunolabeling was found in almost all the secretory granules. This finding suggests that the processing of POMC in corticotrophs might occur in the relatively peripheral granules. These results suggest that the intracellular sites of POMC processing are somewhat different between melanotrophs and corticotrophs in the pituitary.