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Biomedical subjects

A Zanini

Publications and source records attributed to A Zanini.

At least 55 records · Page 3Linked to original sources
PubMed1986

Growth hormone-releasing hormone and clonidine stimulate biosynthesis of growth hormone in neonatal pituitaries.

Comparative studies were performed to verify the effect of growth hormone releasing hormone (GHRH) or clonidine (CLON), a compound thought to act via release of endogenous GHRH, in stimulating GH biosynthesis in the pituitary from neonatal and adult rats. In vitro preincubation for 1 h with GHRH (hpGRF-40, 10(-8) M) increased the incorporation of L-[3H]leucine in the electrophoretic band of GH in the pituitary from 10-day-old rats, but not in the gland from adult rats. Ex-vivo treatment with GHRH or CLON for 5 days was effective in stimulating GH biosynthesis only in the pituitary from neonatal rats. These data demonstrate that neonatal somatotropes are particularly sensitive to the GH-synthesizing activity of GHRH or a GHRH-releasing stimulus.

Age Factors↗
PubMed1986

Sulfated LH subunits and a tyrosine-sulfated secretory protein (secretogranin II) in female rat adenohypophyses: changes with age and stimulation of release by LHRH.

Sulfated proteins are present in adenohypophyseal secretory granules but their function and structure are still largely unknown. We studied these proteins in homogenates from cow and rat adenohypophyses labeled in vitro with [35S]sulfate, by one-dimensional and two-dimensional polyacrylamide gel electrophoresis followed by fluorography. We found that the heterogeneous neutral-alkaline sulfated components of approximately 22-20 kDa and approximately 20-18 kDa previously described correspond to lutropin alpha and beta subunits sulfated on carbohydrates. During development sulfated lutropin subunits were found at highest levels in anterior pituitary glands of 14-day-old female rats. Secretogranin II, an acidic tyrosine-sulfated secretory protein, whose presence in granules of gonadotrophs has been recently described, had a similar distribution during development. In the 14-day-old female rat glands luteinizing hormone-releasing hormone stimulated the in vitro release of both sulfated lutropin subunits and secretogranin II. This finding further suggests that secretogranin II might be involved in the packaging of the gonadotrophin. Immature female rat adenohypophyses provide a useful approach for studying sulfation, both on carbohydrate and on tyrosine residues, of secretory proteins.

Aging↗
PubMed1985

The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs.

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine-sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.

Animals↗
PubMed1985

In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell.

The ultrastructural localization of growth hormone and prolactin in cow anterior pituitary was studied by double immunocytochemical labeling using specific antibodies and protein A-gold particles of different sizes. The two hormones were found in specific somatotrophs and mammotrophs as well as in somatomammotropic cells which were multinucleated and predominantly arranged in clusters in the central area of the lobules. In these mixed cells the two hormones were packaged (a) in different granules of the same cell, (b) in the same granules where they were segregated in different portions of the granule content, or (c) in the same granules but evenly intermixed. The relative proportion of these three types of granules varied in somatomammotrophs of different animals. A single large Golgi complex was generally present in somatomammotrophs. Small, immature granules containing either growth hormone or prolactin or both hormones were found randomly distributed along Golgi stacks. This suggests that in these cells the two hormones are processed in the same Golgi cisternae and that mechanism(s) exist(s) to sort out the two hormones from each other.

Animals↗
PubMed1985

Secretogranins I and II: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway.

We report on the biochemical and immunological properties as well as on the cellular and subcellular distribution of two proteins, called secretogranins I and II. These proteins specifically occur in a wide variety of endocrine and neuronal cells that package and sort regulatory peptides into secretory granules. Both secretogranins take the same intracellular route as the peptides and are also sorted into secretory granules. Secretogranins I and II are biochemically and immunologically distinct proteins and differ from chromogranin A. Yet, these three proteins are similar to each other in many respects and therefore constitute one class of proteins. A remarkable feature of this protein class is a very acidic pI, brought about by a high content of acidic amino acids as well as by phosphorylation on serine and sulfation on tyrosine and O-linked carbohydrate. As a result, this class of proteins has a high net negative charge even at the acidic pH of the trans Golgi cisternae. We discuss the possibility that this property of the proteins may point to a role in the packaging of regulatory peptides into secretory granules.

Adrenal Gland Neoplasms↗
PubMed1983

Purification of a sulfated secretory protein from the adenohypophysis. Immunochemical evidence that similar macromolecules are present in other glands.

A sulfated secretory protein (apparent molecular weight approximately 70 000; isoelectric point approximately 4.8) recently identified as a minor component of mammotroph granules [21] has been purified, by ion-exchange chromatography on DEAE-Sephadex followed by preparative slab gel electrophoresis, from homogenates of bovine anterior pituitary glands pulse-labeled with [35S] sulfate. The homogeneity of the final product was established by one-dimensional as well as by two-dimensional gel electrophoresis followed by fluorography to reveal labeled polypeptides. Specific antibodies were generated against the purified protein. Immunodiffusion and radioimmuno-labeling of polyacrylamide gels revealed that proteins immunologically related to the adenohypophyseal sulfated component, exist in other glands, such as the neurointermediate pituitary and adrenal medulla. We envisage the possibility that the adenohypophyseal sulfated component belongs to a family of proteins which might play a widespread, though not general role in the secretory process.

Adrenal Medulla↗
PubMed1981

Characterization of adenohypophysial polypeptides by two-dimensional gel electrophoresis. I. L-[3H]leucine-labeled polypeptides.

Homogenates of cow and rat anterior pituitary slices, labeled in vitro with L-[3H]leucine, were analyzed by high-resolution two-dimensional polyacrylamide gel electrophoresis. This technique was also applied to the materials released into the chase medium from bovine anterior pituitary slices. The pattern of both total polypeptides (revealed by Coomassie-Blue staining) and L-[3H]leucine-labeled polypeptides (revealed by fluorography) was found to be more complex than previously demonstrated by different techniques. In particular, the GH band separated by one-dimensional Na-dodecylsulfate--polyacrylamide gel electrophoresis was resolved into 3--5 components; 2 of these, which were highly labeled by L-[3H]leucine, were both identified as GH by immunoprecipitation with specific anti-GH bodies. In addition, we found evidence in favor of the existence of some, previously unsuspected, 'putative' secretory proteins. In fact, besides GH and PRL, several minor components (2 with apparent Mr approximately 70 00--62 000, pI approximately 4.8; others with Mr approximately 50 000, pI between approximately 5.8 and approximately 6.8; and 1 with Mr approximately 26 000, pI approximately 5.7) were found to be synthesized at high rates and to accumulate in the medium, with different kinetics, during chase incubation.

Animals↗
PubMed1981

Characterization of adenohypophysial polypeptides by two-dimensional gel electrophoresis. II. Sulfated and glycosylated polypeptides.

Adenohypophysial sulfated and glycosylated polypeptides were studied by high-resolution two-dimensional polyacrylamide-gel electrophoresis followed by fluorography. The preparations analyzed were the following: (a) homogenates from cow and rat anterior pituitary slices labeled in vitro either with [35S]sulfate or D-[6-3H]glucosamine; (b) materials released from bovine adenohypophysis slices pulse labeled with [35S]sulfate; and (c) purified fractions of bovine prolactin granules stripped by detergent treatment of their limiting membrane. A heterogeneous family of sulfated components, almost all glycosylated, differing in their peptide moieties as well as in their isoelectric points, was revealed in the glandular tissue. The major of these components (apparent Mr approximately 70 000; pI approximately 4.8), which was also highly labeled by L-[3H]-leucine (Zanini, A., and Rosa, P. (1981) Mol. Cell. Endocrinol. 24), might be a secretory protein because it accumulates in the medium during chase incubation of bovine pituitary slices in vitro. This sulfated component, which was more concentrated in the bovine than in the rat gland, was present in purified bovine prolactin granules stripped of their limiting membrane. However, the available evidence suggests that this might not be the only subcellular location of the sulfated polypeptide in the pituitary tissue.

Animals↗
PubMed1980

Molecular organization of prolactin granules. II. Characterization of glycosaminoglycans and glycoproteins of the bovine prolactin matrix.

Prolactin (PRL) granules can be isolated from the anterior pituitary gland of adult cows in nearly 50% yield by use of a procedure previously developed for the fractionation of the rat pituitary. Treatment of the isolated bovine granules with 0.2% Lubrol PX results in the solubilization of most membranes present in the fractin but has only a limited effect on the matrices, which remain aggregated and can be recovered and purified by gradient centrifugation. These membraneless PRL granules, studied in detail by morphological and biochemical techniques, were found to contain only small amounts of contaminants (primarily growth hormone granules and small membrane fragments). SDS polyacrylamide gel electrophoresis revealed that, in comparison with other fractions isolated from the bovine pituitary, the membraneless granules have a simpler polypeptide composition including PRL (approximately 85%), growth hormone (approximately 8%), as well as approximately 13 minor bands with apparent mol wt ranging from 80,000 go 45,000. Many of these minor bands are accounted for by glycoproteins, as revealed by their binding of 125I-concanavalin A, and two of these are also stained blue by the stains-all procedure, a reaction specific for acidic glycoconjugates. Chemical analyses of the membraneless granule fractin revealed the presence of a heterogeneous mixture of complex carbohydrates. Among glycosaminoglycans, the major component is heparan sulfate, while hyaluronic acid and chondroitin sulfate ar present in smaller amounts. Moreover, some of the glycoproteins are sulfated and account for over 50% of the nondialyzable 35S radioactivity found in the fraction isolated from labeled slices. Although the concentration of glycosaminoglycans and glycoproteins is relatively low in membraneless granules, the possibility that their presence in the fraction is largely due to cross-contamination and/or artifactual adsorption could be excluded on two grounds. These are: (a) electron microscope radiautography of preparations obtained from [35S]sulfate- and D-[6-3H]glucosamine-labeled slices showed a significant labeling of PRL granules in both intact cells and membraneless granule pellets, and (b) a mixing experiment showed that membraneless granules contain very little macromolecular sulfate radiactivity adsorbed from the soluble glycoconjugates present in the pituitary homogenate.

Animals↗
PubMed1980

Molecular organization of prolactin granules. III. Intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix.

The intracellular transport of sulfated glycosaminoglycans (heparan sulfate and chondroitin sulfate) and glycoproteins of the prolactin (PRL) granule matrix, as well as that of PRL, was studied using a system of double-labeled bovine anterior pituitary slices. [(35)S]sulfate was used to label sulfated macromolecules and L-[(3)H]leucine to label PRL. In membraneless granules (isolated from a PRL granule fraction after solubilization of the membrane with Lubrol PX), sulfated glycosaminoglycans and glycoproteins were considerably labeled after a 15- min pulse, while the hormone was still unlabeled. During the chase incubation, the specific radioactivity of granule PRL and the various complex carbohydrate classes first increased, reaching a peak after approximately 40 min, and then began to decline. After 4 h of chase incubation the radioactivity remaining in granule PRL and sulfated complex carbohydrates was 50-60 percent of that observed at 40 min. Thus, in pituitary mammotrophs a pool of sulfated glycoproteins and glycosaminoglycans is transported intracellularly in parallel with PRL. This finding corroborates the previous conclusion (Zanini et al., 1980 J. Cell. Biol. 86:260-272) that sulfated macromolecules are structural components of the granule matrix. The discharge of labeled PRL and complex carbohydrates from the slices to the incubation medium was also investigated. [(35)S]-glycosaminoglycans and glycoproteins were released at a rapid rate during the first 30-40 min of chase incubation, when PRL granules had not yet attained maximum specific activities. By 40 min, their release tended to level off but the radioactivity accumulating in the incubation medium was still much larger (approximately a fourfold increase) than the losses observed concomitantly in PRL granules. These discharge kinetics contrast with that of [(3)H]PRL, which was not released during the 1st h of chase incubation but then began to accumulate at a high rate in the medium, in parallel with its decrease in granules. Dopamin (5 x 10(-7) M) strongly inhibited the release of labeled PRL but had no detectable effect on the release of labeled glycosaminoglycans and glycoproteins or on the discharge of (35)S-macromolecules as revealed by SDS polyacrylamide gel electrophoresis of incubation media. Thus the releases of PRL and sulfated macromolecules have different kinetics and can be dissociated from each other. These data indicate that much of the flycosaminoglycans and glycoproteins release form pituitary slices originates from sites other than PRL granules, and that at least part of the complex carbohydrates of the PRL granule matrix might not be released with the hormone but rather remains associated with the mammotroph cells after exocytosis.

Animals↗
PubMed1980

Exercise hyperemia for the study of peripheral circulation.

Blood flow in the calf was measured during postexercise hyperemia in normal subjects and in PAD patients by means of a foot ergometer that gives direct reading of the work performed. In normals, first and peak flow increased with the rise of work load up to 100 KGM. The duration of hyperemia increased with a work load of 30 to 200 KGM. In PAD patients, first flow did not coincide with peak flow. Peak flow was lower and delayed, and the duration of hyperemia was more prolonged than in normal subjects. In patients with intermittent claudication, first flow, peak flow, and work load were higher than in patients with rest pain or impending gangrene. Exercise hyperemia appears as a useful test for screening normal limbs from those with arterial obstruction. Since in PAD patients exercise is interrupted when muscular pain appears, it is evident that the earlier the arrest of work and the appearance of pain, the greater is the involvement in the arterial tree of the leg. Therefore the exercise hyperemia test can be used also as a means of evaluating the different stages of PAD.

Adult↗
PubMed1979

Studies on rat pituitary homografts. I. In vitro biosynthesis and release of growth hormone and prolactin.

Homologous anterior pituitaries grafted under the kidney capsule in hypophysectomized rats were studied 30 days after transplantation. Some cells maintained the ultrastructural features peculiar to the various cell types of normotopic glands, while the others were characterized by few, small, dense granules, spherical or polymorphic, located peripherally in the cytoplasm. This picture might be due to a functional adaptation which occurs in pituitary cells still producing different hormones, once removed from central nervous system control. The major change in polypeptide hormone composition of graft homogenates relative to normotopic pituitaries is the fall in GH and PRL concentration. The in vitro incorporation of L-[3H]leucine into the two hormones and the release of radioactive GH and PRL from L-[3H]leucine-prelabeled tissue fragments are also greatly decreased. The decrease in concentration, in vitro biosynthesis, and release of GH per mg tissue protein are approximately 87, 91, and 93%, respectively. These results might be due primarily to a decrease in the number of somatotrophs and/or in their secretory activity, with relatively minor changes in GH intracellular transport and turnover. In contrast, a clear-cut fall in in vitro turnover was detected for PRL, as shown by the fact that decreases in biosynthesis and release per mg tissue protein of this hormone (approximately -95% and -99%, respectively) by far exceed the decrease in the tissue concentration (-74%). These data indicate that in in vitro secretory activity of mammotrophs is greatly reduced in the grafts with respect to the normotopic glands. Thus, the high secretory activity previously reported in hypophysectomized rats bearing pituitary grafts should be attributed to the lack of the inhibitory control of the central nervous system rather than to an increase in secretory capacity under nonrestrained conditions.

Animals↗
PubMed1979

Studies on rat pituitary homografts. II. Effects of thyrotropin-releasing hormone on in vitro biosynthesis and release of growth hormone and prolactin.

A study was made of the effect of TRH, administered in vivo by iv infusion or added in vitro to the incubation fluid, on tissue fragments prepared from rat normotopic and ectopic pituitaries. The latter were 30-day-old pituitary grafts transplanted under the kidney capsule in hypophysectomized animals. No detectable effect of TRH was found with normotopic glands. In contrast, the neurohormone produced a large increase in GH and PRL biosynthesis in the grafts, as revealed by the rise in hormone content as well as increased incorporation of L-[3H]leucine into the two hormones. These effects of TRH 1) are dose related; 2) appear after a latent period of at least 15 min; and 3) persist, although attenuated, for some time after removing the neurohormone. In vitro release of GH and PRL by tissue fragments prelabeled with L-[3H]leucine was studied by following the appearance of the radioactive hormones in the incubation fluid. Exposure to TRH produced a prompt, 2-fold or greater increase in hormone release from grafts but not from normotopic gland fragments. The possible mechanisms whereby pituitary somatotrophs and mammotrophs removed from the influence of the central nervous system increase their responsiveness to TRH stimulation are considered.

Animals↗
PubMed1976

Presence of sulfated proteoglycans in prolactin secretory granules isolated from the rat pituitary gland.

The composition of the segregated content of rat prolactin granules was investigated taking advantage of the fact that these organelles, isolated as a pure fraction, retain their structural organization after solubilization of their limiting membrane by mild detergent treatment. We found that these membraneless granules contain not only the hormone, but also a number of minor macromolecular components including sulfated glycosaminoglycans, which are labeled when pituitary slices are incubated in vitro with [35S] sulfate. In order to characterize the latter components, the isolated radioactive granules were solubilized (by treatment with either a high ionic strength solution orNaOH) and 35S-labeled acidic glycosaminoglycans precipitated by complexing with cetylpirydinium chloride. A high degree of heterogeneity was observed when the ensuing precipitates were analyzed by cellulose acetate electrophoresis: different components were found to co-migrate with authentic heparin and chondroitin sulfate A and C standards. Another component, which accounts for approx. 50% of the glycosaminoglycan-bound radioactivity, might be heparin sulfate. These acidic glycosaminoglycans are linked to peptide moieties to form proteoglycans.

Animals↗
PubMed1975

Molecular organization of rat prolactin granules. I. In vitro stability of intact and "membraneless" granules.

Studies carried out on a number of secretory cell systems suggest that the specific cytoplasmic granules in which the secretion products are stored before their release are complex organelles which can possess a distinct molecular organization. For instance, it has been reported that in some granules the segregated secretion products are organized into crystalline structures (1-3) or large intermolecular aggregates (4-8). It is likely that all phenomena of this type are favorable to the economy of the cell, in the sense that they reduce the energy required for storage of the secretion products. The prolactin (LTH) granules of the rat pituitary possess a number of morphological features which strongly suggest that the molecules(s) of their content might be arranged in a relatively stable structure. Thus, these granules are remarkably polymorphic in shape, and their membrane is usually separated from their content by a clear space. Furthermore, identifiable LTH granules devoid of their membrane are often seen in the pericapillary space, suggesting that upon discharge by exocytosis they are dissolved only slowly (9). However, no studies specifically concerned with the mechanisms of LTH storage have been reported so far. In order to obtain some information on this question, we have studied the behavior of isolated granule fractions incubated in vitro under a variety of carefully controlled experimental conditions.

Animals↗