Search PubMed⌕ Search

Biomedical subjects

V May

Publications and source records attributed to V May.

At least 55 records · Page 3Linked to original sources
PubMed1989

Developmental regulation of peptidylglycine alpha-amidating monooxygenase (PAM) in rat heart atrium and ventricle. Tissue-specific changes in distribution of PAM activity, mRNA levels, and protein forms.

The high levels of peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) found in adult rat atrium led us to examine PAM expression in rat atrium and ventricle from embryonic day 14 through adulthood. Immunocytochemical studies using antisera to PAM identified cardiocytes as the major site of PAM expression in atrium and ventricle throughout development. Levels of PAM mRNA and PAM activity exhibited distinctly different developmental profiles in atrium and ventricle. Ventricular PAM mRNA and PAM activity were highest from embryonic days 14 through 18, declined at the time of birth, rose slightly during the first postnatal week, and declined toward adult levels. Atrial PAM mRNA and PAM activity were low at embryonic day 14, rose to a peak immediately before birth, declined at the time of birth, and then rose after birth. Levels of atrial PAM mRNA and PAM activity were not directly correlated at all developmental stages. Two major forms of PAM mRNA (4.2 +/- 0.1 and 3.8 +/- 0.1 kilobase(s] were identified in atrium and ventricle throughout development. The prevalence of the two forms varied with developmental stage, with atrium and ventricle containing similar forms at each stage. Western blots of atrial and ventricular membranes revealed the existence of a developmental stage-specific distribution of PAM protein among forms ranging in mass from 125 to 94 kDa. In both atrium and ventricle PAM activity was primarily soluble from embryonic days 14 through 16 and primarily particulate after birth. The role of PAM in the heart is not yet clear, but the presence of tissue-specific and developmentally regulated alterations in PAM mRNA, PAM protein, and PAM activity suggests that this peptide processing enzyme plays a key role in the heart.

Aging↗
PubMed1989

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.

Adrenocorticotropic Hormone↗
PubMed1989

Proadrenocorticotropin/endorphin production and messenger ribonucleic acid levels in primary intermediate pituitary cultures: effects of serum, isoproterenol, and dibutyryl adenosine 3',5'-monophosphate.

Long term primary rat intermediate pituitary cells cultured in complete serum-free medium (CSFM) were previously shown to exhibit a low basal secretory rate and maintain a stable cellular level of immunoactive pro-ACTH/endorphin (PAE)-related peptides. The present studies used biosynthetic labeling techniques to demonstrate that the biosynthesis of PAE-derived peptides declined as a function of the time the cultures were maintained in CSFM. Compared to hormone biosynthesis in freshly isolated cells, the ability of melanotropes maintained in CSFM to synthesize hormone declined approximately 3-fold after 5 days and 7-fold after 10 days. The endoproteolytic processing of PAE was not changed. This diminution in biosynthetic rate was not observed in serum-supplemented cultures; the rates of hormone secretion and hormone biosynthesis were both substantially higher in cultures maintained in serum-containing medium, suggesting that stimulatory factors were present in serum supplements. Chronic treatment of intermediate pituitary cultures in CSFM with 100 microM (Bu)2cAMP or 100 nM isoproterenol mimicked the effects of serum on total PAE production, hormone biosynthesis, and PAE mRNA levels. The diminished biosynthetic ability of cultures maintained in CSFM for several days could be restored by subsequent chronic (B mu)2cAMP or isoproterenol treatment. Taken together with previous information, these results suggest that melanotropes maintained in CSFM exist in a functionally basal state and that stimulatory agents, in addition to inhibitory inputs such as dopamine, exert significant regulatory control over intermediate pituitary lobe function in vivo.

Adrenocorticotropic Hormone↗
PubMed1989

Tissue specific expression of rat peptidylglycine alpha-amidating monooxygenase activity and mRNA.

The tissue specific expression of peptidylglycine alpha-amidating monooxygenase [(PAM) EC 1.14.17.3], an enzyme which catalyzes the formation of amidated bioactive peptides from their glycine-extended precursors, was examined in adult rat. Soluble and membrane-associated PAM enzymatic activities were determined, and the levels and size classes of PAM mRNA were examined by Northern blot analysis. PAM specific activity varied 1000-fold in the tissues examined, with highest levels in heart atrium, pituitary and salivary glands, and hypothalamus. The fraction of total PAM activity that was membrane associated varied from approximately 70% in heart atrium to 10% in neurointermediate pituitary lobe and thyroid gland. Levels of PAM mRNA varied over 300-fold. In the heart atrium, PAM mRNA accounts for more than 0.1% of the mRNA. For many tissues the ratio of total PAM specific activity to PAM mRNA levels was similar; however, PAM activity was higher than expected from mRNA levels in the salivary glands and lower than expected in several tissues, including heart ventricle. Three major size classes of PAM mRNA were identified among the tissues. Use of RNAse H indicated that differences in size were not due to the length of the poly(A) tail. The heart and central nervous system expressed PAM mRNA of the 4.2 kilobase (kb) and 3.8 kb size classes, while the remaining tissues expressed predominantly 3.8 kb and 3.6 kb classes; few tissues contained only one size class of PAM mRNA. The two major forms of PAM mRNA in adult heart atrium differ by the presence or absence of a 315 nucleotide segment in the protein coding region. Using a cDNA probe from within this segment, the 4.2 kb and 3.8 kb size classes of PAM mRNA in the central nervous system appeared to resemble those in the heart atrium. In the remaining tissues, a subset of PAM mRNAs in the 3.8 kb and 3.6 kb size classes hybridized with this probe, suggesting that additional forms of PAM mRNA are present.

Adrenal Glands↗
PubMed1988

Membrane-associated peptidylglycine alpha-amidating monooxygenase in the heart.

Recent investigations have shown that the heart atrium is an endocrine tissue. In the present studies, high levels of peptidylglycine alpha-amidating monooxygenase (PAM), which catalyzes the formation of bioactive alpha-amidated peptides from their glycine-extended precursors, have been found in particulate fractions from bovine and rat heart atrium; only low levels of PAM activity were present in soluble fractions. Corresponding fractions from the ventricles contained 20-fold less activity. Immunocytochemical studies demonstrated that PAM was localized primarily to atrial cardiocytes, with a distribution resembling that of atriopeptin. Following differential centrifugation of rat atrial homogenates, most of the PAM activity was associated with crude granule fractions, with lesser amounts of activity associated with crude microsomal fractions. Upon further subcellular fractionation, PAM activity in the rat atrium was found primarily with immunoactive atriopeptin in fractions enriched in secretory granules. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, antisera to purified bovine pituitary PAM identified a 113,000-dalton protein in bovine atrial microsomes and secretory granules; the protein predicted from the sequence of the cDNA encoding bovine pituitary PAM is of similar size (Eipper, B. A., Park, L. P., Dickerson, I. M., Keutmann, H. T., Thiele, E. A., Rodriguez, H., Schofield, P. R., and Mains, R. E. (1987) Mol. Endocrinol. 1, 777-790). Northern blot analysis using cDNA probes encoding bovine pituitary PAM demonstrated higher levels of PAM mRNA in heart atrium than in anterior pituitary. Rat heart contains PAM mRNA species of 3.6 and 3.8 kilobases, the smaller mRNA species corresponding in size to the PAM mRNA expressed in rat anterior pituitary.

Animals↗
PubMed1988

The role of a low pH intracellular compartment in the processing, storage, and secretion of ACTH and endorphin.

To determine whether a low pH intracellular "sorting" step is required to route peptides into secretory granules, the effects of pH altering drugs on the biosynthesis and secretion of peptides by AtT-20 mouse corticotrope tumor cells and rat intermediate pituitary cells were examined. Doses of each drug maintaining normal protein synthesis and cell morphology, while obliterating the intracellular pH gradients detected by acridine orange fluorescence, were experimentally determined. Regions of the cell rich in secretory granules were localized by immunocytochemistry and were found to coincide with organelles with a low internal pH. Biosynthetic labeling experiments were coupled with immunoprecipitation and sodium dodecyl sulfate polyacrylamide gel analyses to examine the biosynthesis and secretion of corticotropin (ACTH(1-39], alpha-melanotropin, ACTH(18-39), beta-endorphin, gamma-melanotropin, alpha-amidated joining peptide, and the NH2-terminal region of pro-ACTH/endorphin. Chloroquine (20-40 microM) and a mixture of NH4Cl and methylamine (2-5 mM each) dissipated pH gradients but had no effect on the synthetic rate of pro-ACTH/endorphin, the extent and rate of precursor processing to smaller peptides, the rate of basal secretion of the various peptides, or the extent to which secretion of each of the peptides could be stimulated by secretagogues. Monensin (0.1-1 microM) had no discernible effect on intracellular pH gradients yet totally blocked proteolytic processing of pro-ACTH/endorphin. Thus, a monensin-blockable step occurs in peptide processing, presumably in the trans Golgi region; however, a low pH chloroquine-sensitive sorting step is not required for processing or for routing peptides to a stable storage form which can be released in response to secretagogues.

Acridine Orange↗
PubMed1988

Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity.

Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.

Animals↗
PubMed1988

Comparison of acute and chronic secretagogue regulation of proadrenocorticotropin/endorphin synthesis, secretion, and messenger ribonucleic acid production in primary cultures of rat anterior pituitary.

The acute and chronic effects of secretagogues activating cAMP-dependent pathways (CRH and cAMP) and activating cAMP-independent pathways [phenylephrine and phorbol 12-myristate 13-acetate (PMA)] on anterior pituitary function were examined in serum-free cultures. Applied acutely, PMA produced a greater stimulation of ACTH/endorphin secretion than CRH or cAMP. However, the effects of CRH and cAMP on secretion were maintained for up to 12 days, while those of PMA and phenylephrine diminished rapidly. Secretagogue effects on pro-ACTH/endorphin biosynthesis were determined by immunoprecipitation of biosynthetically labeled beta-endorphin-related peptides. Cultures exposed to CRH or cAMP and [3H]tyrosine for 12 h produced 1.7 +/- 0.2- and 1.6 +/- 0.1-fold more newly synthesized beta-endorphin-related material than control cells. Cultures exposed to phenylephrine or PMA synthesized 1.3 +/- 0.1- and 1.4 +/- 0.1-fold more peptide than control cells. Exposure of cells to CRH or cAMP for 12 days increased pro-ACTH/endorphin biosynthesis to a greater extent than the 12-h treatment (3.0 +/- 0.1- and 2.5 +/- 0.3-fold over control value, respectively). Exposure to phenylephrine or PMA for 12 days had the same effect on pro-ACTH/endorphin biosynthesis as exposure for 12 h. After acute or chronic secretagogue exposure, the cells secreted relatively more newly synthesized beta-lipotropin than beta-endorphin. Levels of pro-ACTH/endorphin mRNA in cultures treated acutely (12 h) or chronically (12 days) with CRH, cAMP, or phenylephrine changed in parallel with rates of pro-ACTH/endorphin biosynthesis. In contrast, chronic exposure to PMA stimulated biosynthesis while reducing pro-ACTH/endorphin mRNA levels. In summary, these results suggest that factors that activate cAMP-dependent pathways are more powerful stimulators of pro-ACTH/endorphin biosynthesis than factors that activate cAMP-independent pathways; the cAMP-dependent pathway may be primarily responsible for regenerating depleted hormone reserves.

Adrenocorticotropic Hormone↗
PubMed1987

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.

Animals↗
PubMed1987

Differences between in vitro and in vivo degradation of LHRH by rat brain and other organs.

Homogenates of brain, pituitary, liver, lung, ovary, and testes were incubated with [pyro Glu1-3,4-3H]luteinizing hormone-releasing hormone ([3H]LHRH), and the profiles of metabolites generated as a function of time were determined. After 5 min of incubation, 5 was the predominant metabolite in most homogenates. Although the profiles of metabolites varied at different time intervals, metabolites 2, 3, 4, and 5, and in some instances 7 and 9, appeared to form simultaneously and were detectable at 10 min. Neither metabolite 6 nor other larger metabolites formed initially as dominant degradation products. The findings suggest cleavage of LHRH by the simultaneous action of several endopeptidases. After a single vascular transit of [3H]LHRH, metabolites were determined in the venous blood of liver, lung, and brain of rats in vivo. There were no metabolites of [3H]LHRH in venous blood of liver and lung; however, metabolites 2-4 were present in venous blood of the brain. Incubation of rat anterior pituitary cells with [3H]LHRH yielded metabolites 1-4 but not metabolites 5 or 9 as in homogenates. Incubation of [3H]LHRH with porcine follicular granulosa cells resulted in the generation of metabolites 2-7 and 9, similar to the profile in homogenates. Thus, since homogenates contain enzymes of disrupted cells, they do not always reflect mechanisms for in vivo hydrolysis of circulating LHRH. Brain degraded 12.1% of LHRH during a single vascular transit and may account for substantial degradation of the circulating hormone.

Animals↗
PubMed1987

Alzheimer's disease: low levels of peptide alpha-amidation activity in brain and CSF.

Carboxyl terminal alpha-amidation confers biologic activity to many neuropeptides. Levels of alpha-amidating activity, peptidyl-glycine alpha-amidating monooxygenase (PAM), were reduced in the CSF of patients with dementia of the Alzheimer type (DAT) compared with healthy, age-matched controls. Repeat lumbar puncture data revealed a decline in CSF PAM activity of approximately 16% per year in DAT patients. Of the cerebral cortical regions examined, only the temporal pole showed reduced PAM activity in patients with Alzheimer's disease (AD) compared with controls. These studies may indicate selective dysfunction of neurons which normally synthesize biologically active, alpha-amidated peptides in the CNS of AD patients.

Aged↗
PubMed1986

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.

Animals↗
PubMed1986

Long term culture of primary rat pituitary adrenocorticotropin/endorphin-producing cells in serum-free medium.

Insulin, transferrin, and serum albumin were found to be essential additives for maintenance of primary rat pituitary ACTH/endorphin cells in complete serum-free defined medium (CSFM). Primary anterior pituitary cultures maintained in CSFM exhibited a 3- to 5-fold increase in cell content of ACTH/endorphin-related peptide during the first 2 weeks in culture, and this level remained stable for at least the next week. Immunocytochemical and morphometric studies indicated that the number of corticotropes increased only slightly, so that hormone content per corticotrope increased. Anterior pituitary cultures maintained in CSFM exhibited a basal secretory rate of 0.3-0.4% of cell hormone content/h throughout the 3-week period in culture, and total hormone production increased 6-fold. Primary anterior pituitary cultures maintained chronically in CSFM containing 10 nM CRF demonstrated a 15- to 20-fold increase in total ACTH/endorphin production over 3 weeks in culture. Chronic treatment with CRF brought about a sustained 6-fold increase in secretory rate (2.5% of cell hormone content/h), and corticotrope content of hormone was diminished 3-fold. Corticotropes maintained chronically in CSFM containing CRF did not increase in number and exhibited a rim of immunocytochemically identifiable hormone around the cell periphery. Anterior pituitary cultures maintained chronically in CSFM containing 100 nM dexamethasone (DEX) exhibited decreased cell hormone content and an unaltered secretory rate. In the DEX-treated cultures the number of immunocytochemically identifiable corticotropes declined, as did the staining intensity per corticotrope. Primary cultures of rat intermediate pituitary cells maintained in CSFM exhibited a 1.5- to 2-fold increase in hormone content after 1-2 weeks in culture, maintained a constant basal secretory rate of 0.4-0.5% cell content/h, and were not responsive to chronic treatment with CRF or DEX.

Adrenocorticotropic Hormone↗
PubMed1985

Regulation of peptide amidation in cultured pituitary cells.

The intermediate lobe of the pituitary contains the alpha-amidated peptide alpha-melanotropin and high levels of a copper and ascorbate-dependent peptidylglycine alpha-amidating monooxygenase (PAM) capable of converting peptides terminating in -X-Gly into amidated products (-X-NH2). As reported previously, the ability of cultured intermediate pituitary cells to produce alpha-amidated alpha-melanotropin declined rapidly. A decline in PAM activity assayed in vitro under optimized conditions failed to account quantitatively for the lack of production of alpha-amidated product, while a 100-fold decline in cellular levels of ascorbate could account for the lack of production of alpha-amidated product. Incubation of intermediate pituitary cultures with ascorbate partially restored the ability of the cells to produce alpha-amidated product without significantly increasing the level of PAM activity. In intermediate pituitary cultures made competent to produce alpha-melanotropin by addition of ascorbate, the actual extent of amidation occurring was modulated by the presence of specific secretagogues (bromocriptine or corticotropin-releasing factor). Cultured anterior pituitary cells showed a similar rapid 3-fold decline in PAM activity assayed in vitro under optimized conditions. Cellular levels of ascorbate also declined rapidly to levels 100-fold below those in the intact anterior pituitary. The addition of ascorbate to the anterior pituitary cultures rapidly restored the enzyme activity assayed in vitro to the levels in the initial cell suspension. Thus, production of amidated product peptide may be regulated by cellular levels of ascorbate, by cellular levels of PAM activity, and by the concentration of specific secretagogues to which the cells are exposed.

Animals↗
PubMed1983

[Bone and phosphoro-calcium metabolism in reflex sympathetic dystrophy].

A combined study organised by the French Society of Rheumatology was devoted to the investigation of bone and phosphoro-calcium metabolism in cases of reflex sympathetic dystrophy. The following observations were made: the usual phosphoro-calcium parameters are not altered, apart from a slight elevation of the urinary calcium in multifocal forms of the disease, during the 3rd and 4th months; the level of PTH, studied in 11 patients, was normal in each case; the examination of 8 bone biopsies, one performed in the 7th week and six others performed during the 3rd and 4th months of the disease, showed, initially, invasion of the spongy tissue by oedema, signs of marrow stress and bone stress, with a reduction in the number of osteoblasts, without any marked alteration of bone remodelling. At a later stage, the biopsy shows intense bone remodelling with hyperosteoclastosis and hyperosteoblastosis and the formation of irregular bone tissue which later becomes lamellar. Electron microscopic study of two biopsies revealed signs of acellular demineralisation with normal appearance of the osteoblasts and osteoclasts.

Bone and Bones↗
PubMed1983

[The joint in algodystrophy. Joint fluid, synovium, cartilage].

The joint fluid in cases of algodystrophy was studied in 17 samples. It is poor in cells (275/mm3 with a mean of 15% polymorphs). The protein level was studied 4 times and was equal to or greater than 40 g/l, i.e. double the normal value. A detailed histological examination of 33 biopsies of synovial tissue reveals a typical appearance of the synovium in algodystrophy, defined by 6 features, 2 negative and 4 positive: absence of hyperplasia of the synovial lining, absence of any real inflammatory cell infiltrate, presence of a marked hypervascularity, an oedema of the chorion, especially marked in the first weeks of the disease, hyperplasia of the walls of some arterioles, and fibrosis of the chorion which becomes more marked as the disease progresses and which is predominantly in the deeper layers. The authors recall that articular cartilage can show superficial alterations of structure with areas of fibrosis.

Biopsy↗