Victor May

The role of secretory granules in peptide biosynthesis.

There are many events in the posttranslational processing of bioactive peptides that occur in secretory granules and not to any great extent in other cellular organelles and that do not appear as modifications of the structure of many conventional neurotransmitters. In addition, at least two very important steps are unique to peptide-containing granules: (1) the peptides must begin their trek to the secretory granule in the RER as a larger precursor, rather than being taken up as a finished or nearly finished product into a mature granule; (2) there is at least one crucial sorting step on the way from the RER to the secretory granule that must occur faithfully before the peptide correctly appears in the granule. As for small molecules such as the catecholamines, the posttranslational processing enzymes and any required cofactors must also be put into the granules if the final events of processing are to occur with fidelity. Many of the posttranslational processing enzymes are only beginning to be identified. It is clear from these studies on purified PAM and peptide alpha-amidation as it occurs in cells that correlating test tube studies with the functioning of secretory granules is a worthwhile, if difficult, pursuit. The unique milieu inside the granule is difficult to mimic in a test tube. Transfection of peptide-producing cells with cDNAs encoding precursors with specific alterations in processing sites offers perhaps the best way to interface the studies of secretory granules and the posttranslational processing enzymes that mediate those functions.

Transport and stability of ascorbic acid in pituitary cultures

Ascorbic acid uptake in AtT-20 tumor cells and primary cultures of rat anterior and intermediate pituitary was sodium-dependent and showed half-maximal saturation between 9 and 18 microM ascorbate. When incubated in [14C]ascorbic acid at concentrations similar to those in serum (50 microM), all of the cells concentrated ascorbate 20- to 40-fold, producing intracellular ascorbate concentrations of 1-2 mM. HPLC analyses showed that over 90% of the intracellular label comigrated with authentic ascorbic acid. Although ascorbate was rapidly oxidized in culture medium in the absence of cells, incubation of ascorbate in culture medium in the presence of cells stabilized the ascorbate substantially. Unlike systems that transport dehydroascorbic acid, the ascorbate transport systems in all three preparations were not inhibited by glucose. Thus all three systems possess similar saturable, high-affinity, sodium-dependent active transport systems for ascorbic acid.

Ability of Cofactors to Support Peptide Amidation Is Cell-Type Specific

The ability of various cofactors to substitute for ascorbate in the biosynthesis of alpha-aidated peptides from pro-ACTH/endorphin (PAE) was compared in corticotrope tumor cells (AtT-20) and in primary anterior and intermediate pituitary cultures. In all three systems, ascorbate was the most potent cofactor tested. In AtT-20 cells, dopamine, norepinephrine, epinephrine, dehydroascorbate and dihydro- and tetrahydrobiopterin supported significant alpha-amidation of joining peptide [PAE(77-94)NH2]. In contrast, amidation of joining peptide by primary corticotropes was stimulated only slightly by catecholamines and not by tetrahydrobiopterin. Neither catecholamines nor tetrahydrobiopterin stimulated peptide amidation by melanotropes. The ability of cofactors to support the synthesis of alpha-amidated peptides is cell-type specific.

Cellular Mechanisms of Peptide Processing: Focus on α-Amidation

It is now well accepted that a vast array of peptides play essential roles in intercellular communication in both the nervous and endocrine systems. Many neurons contain both a classical neurotransmitter and a peptide (Krieger, 1983; Jones & Hendry, 1986). While we know a great deal about the control mechanisms affecting neuronal synthesis, storage and secretion of classical neurotransmitters such as the catecholamines and acetylcholine, we know relatively little about the precise way in which neuronal peptides are regulated. Our lack of knowledge concerning the mechanisms by which the synthesis, storage and secretion of bioactive peptides can be manipulated stems in large part from the relative lack of knowledge about the specific enzymes involved in converting inactive peptide precursors into their final bioactive products.

Regulation of Melanotrope ACTH/Endorphin Biosynthesis in Culturea

Intermediate pituitary (IP) melanotrope biosynthesis and secretion of pro-hCTH/ endorphin (PAEf-related peptides are primarily under neuronal control. Dopaminergic fibers inhibit melanotrope function by acting on D-2 receptors.~* Noradrenergic, peptidergic, serotonergic and GABAergic fibers have also been found within the IP lobe. It is not yet clear how melanotropes integrate the many inputs they receive to produce a response. To understand these processes, we have maintained dispersed melanotropes in a complete serum-free medium (CSFM).3*4 A comparison of melanotrope hormone production in serum-free and serum-supplemented media revealed the presence of stimulatory factors in serum-containing media. Rat IP lobes were enzymatically dispersed and cultured as previously de~cr ibed .~ Following different treatments, the cells were incubated in medium containing [3H]Pro; cell extracts and incubation media were then immunoprecipitated with affinity-purified P-endorphin antiserum for SDS-PAGE analyses. PAE mRNA levels in duplicate cultures were measured by Northern analyses using a nick-translated PAE cDN A probe. Based on measurement of cell content of immunoactive hormone after 5 days, previous studies demonstrated that maintenance of melanotropes in CSFM or in medium containing 5% rat serum (RS) produced similar result^.^ However, biosynthetic labeling studies revealed that melanotropes maintained in medium containing increasing amounts of RS exhibited higher basal secretory and biosynthetic rates than melanotropes maintained in CSFM (FIGURE 1). In CSFM, hormone biosynthesis declined 4-fold after 5 days in culture; the decline was not apparent from measurements of immunoactive hormone content because of the corresponding drop in secretory rate without serum. I f melanotropes function in a state of tonic dopaminergic inhibition, removal of the inhibitory dopaminergic inputs present in vivo would have been expected to result in increased hormone biosynthesis in culture, as is the case for lac tot rope^.^ Thus these studies suggested that melanotropes require a regulatory factor to maintain functional levels comparable to those in viva Chronic treatment of melanotropes with isoproterenol (ISO; 100 nM), a stimulatory &receptor agonist, or dBcAMP { 100 FM) increased PAE peptide biosynthesis 2to 3-fold compared to CSFM alone (FIGURE 2). The biosynthetic ability of melanotropes kept in CSFM for 5 days could be stimulated by IS0 or dBcAMP treatment. The stimulatory effects of IS0 and dBcAMP were both time and concentration dependent. While the effects of IS0 declined upon prolonged treatment, total hormone production during chronic IS0 treatment was still greater than in untreated cultures. The erects of IS0 and dBcAMP were completely blocked by the dopdmine antagonist

Transport of Ascorbic Acid into Pituitary Cultures

In 1982 Bradbury, Finnie, and Smyth described an activity in porcine pituitaries that converted the synthetic substrate D-Tyr-Val-Gly to the amidated product D-Tyr-ValNH,. Glyoxylate was detected in the reaction mixture, and mass spectra indicated that the nitrogen of the glycine residue was the source of the amide nitrogen.’ Later work showed that amidating enzyme purified from bovine pituitaries and activity from several tissues required molecular oxygen and was stimulated by copper and ascorbic acid.’” Thus, the enzyme is most likely a monooxygenase and has been called peptidylglycine a-amidating monooxygenase, or PAM. PAM activity has been localized to secretory granules in rat hypothalamus and mouse AtT-20 pituitary tumor cells?’ Primary cultures of intermediate pituitary cells lose the ability to a-amidate aMSH, but the loss can be reversed by incubating the cells in ascorbic acid:’ To continue investigating the relationship among cellular ascorbic acid concentration, amidating ability, and PAM activity, we studied ascorbic acid transport in primary cultures of rat anterior and intermediate pituitary and mouse AtT-20 tumor cells. When incubated for 7 to 9 hours in 50 pM “C-labeled ascorbic acid, all three cell preparations concentrated ascorbic acid 20to 40-fold, producing intracellular ascorbate concentrations of 1 to 2 mM, based on experimentally determined cell volumes? All three cell preparations also displayed saturable ascorbic acid uptake; half-maximal rates of ascorbic acid uptake occurred between 9 and 18 pM ascorbate. In primary cultures of intermediate pituitary, uptake of ascorbate in medium containing 5 mM sodium was less than 20% of the uptake in medium containing 156 mM sodium. When primary anterior pituitary and AtT-20 cells were incubated in medium containing 5 mM sodium, the initial rate of ascorbic acid transport was less than 10% of that measured in medium containing 156 mM sodium. In contrast to systems that transport dehydroascorbic acid, pituitary ascorbate transport was not inhibited by glucose’ (FIG. 1). HPLC analyses of extracts from cells incubated in ‘‘C-labeled ascorbic acid showed that over 90% of the intracellular label comigrated with authentic ascorbic acid.’ Although ascorbic acid was oxidized rapidly in culture media in the absence of cells, incubation of ascorbate in the presence of cells stabilized the ascorbic acid substantially.’ ‘%-labeled ascorbic acid was stable in physiological buffer containing 1 mM thiourea.’ In the presence of 1 mM thiourea, unlabeled ascorbic acid competitively inhibited the uptake of I4C-labeled ascorbic acid with a Ki that compared well with the K,,, for transport (FIG. 2). This result supports the conclusion that reduced ascorbic acid is transported into pituitary cells by a sodium-dependent active transport system.