Isolation and characteristics of bovine pituitary secretory granules

Secretory granules containing primarily growth hormone and prolactin were isolated from bovine anterior pituitaries. Marker enzyme analysis and electron microscopy indicated that the secretory granule fraction did not contain measurable amounts of other intracellular organelles. Such isolated granules were resistant to a variety of chemical and physical challenges including variations in osmolarity, ionic strength, EGTA, sonication, boiling, etc. The only treatments that were found to routinely result in granules lysis were alkaline pH and 0.5% SDS. Nonspecific leakage of both groth hormone and prolactin was less than 9% of total hormone pool even after a 60-min incubation. The release of prolactin but not growth hormone could be increased by lowering the free calcium concentration. Conversely, 10⁻⁵ M ionophore A23187 caused a decrease in nonspecific hormone leakage. This raises the possibility that a nonexocytosis secretory pathway might be involved in pituitary hormone release. The initial secretory granule fraction was further purified using discontinuous sucrose gradient ultracentrifugation to yield a subfraction highly enriched in prolactin granules. These granules had the same stability characteristics as the original secretory granule fraction. The use of such granules should prove useful in our efforts to understand how calcium regulates cellular secretion.     

Isolated prolactin and growth hormone granules from bovine pituitary: content and stability are seasonally dependent

Secretory granules containing prolactin (PRL) and growth hormone (GH) as essentially the only proteins were isolated by centrifugation. PRL and GH varied reciprocally in the granule preparations with the seasons. During winter PRL content was lowest (20%) and GH highest (80%); during summer the converse obtained: PRL, 70% and GH,, 30%. Both hormones were in almost equal proportion during the spring. The amount of either hormone released from granules and pituitary slices was directly related to its relative content in the gland. The pattern of PRL release from secretory granules and pituitary tissue in vitro was similar to that reported for blood levels in ruminants: low during winter and high during summer. It is concluded that seasonal factors affect primarily the synthesis and/or storage of PRL and GH, and there exists a direct relationship between intracellular stores and release.     

Actions of releasing factors on isolated secretory granules mediated by calcium.

Synthetic TRH, crude hypothalamic extract and partially purified prolactin releasing factor stimulated prolactin and growth hormone release from isolated secretory granules. Somatostatin and partially purified prolactin release-inhibiting factor inhibited release of both hormones. Calcium promoted hormone release from granules; its releasing action was potentiated by TRH and ionophore A23187 but reduced by somatostatin.     

Co-localization of secretogranins/chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary

We investigated the co-localization in secretory granules of secretogranins/chromogranins, thyrotropin, and luteinizing hormone in ultra-thin frozen sections of cow anterior pituitary by double immunoelectron microscopy, using specific antibodies and protein A-gold particles of different sizes. The distribution of secretogranin II, chromogranin A, and chromogranin B (secretogranin I) was largely similar. In cells containing secretory granules of relatively small size (100-300 nm) and low electron density (identified as thyrotrophs and gonadotrophs by immunolabeling for the respective hormone) and in cells containing both small (170-250 nm) and large (300-500 nm) secretory granules of low electron density (also identified as gonadotrophs), all three secretogranins/chromogranins were detected in most if not all granules, being co-localized with the hormone. In cells containing both relatively large (400-550 nm), electron-dense granules and small, less electron-dense secretory granules (150-300 nm), identified as somatomammotrophs by double immunolabeling for growth hormone and prolactin, all three secretogranins/chromogranins were predominantly detected in the subpopulation of small, less electron-dense granules containing neither growth hormone nor prolactin. Interestingly, this granule subpopulation of somatomammotrophs was also immunoreactive for thyrotropin and luteinizing hormone. These data show that somatomammotrophs of cow anterior pituitary are highly multihormonal, in that the same cell can produce and store in secretory granules up to four different hormones and, in addition, the three secretogranins/chromogranins. Moreover, selective localization of the secretogranins/chromogranins together with thyrotropin and luteinizing hormone in a subpopulation of secretory granules of somatomammotrophs indicates the preferential co-packaging of the secretogranins/chromogranins and these hormones during secretory granule formation.     

Acquisition of Lubrol insolubility, a common step for growth hormone and prolactin in the secretory pathway of neuroendocrine cells

Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in long term cultures

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media, previously shown to affect differentially prolactin and growth hormone release. After 6 days culture, there were marked differences in the ultrastructure of both prolactin and growth hormone cells from the two groups. Morphometric data on the prolactin cells from SW-adapted eels showed a greater abundance of RER and paucity of secretory granules in cells from the low sodium medium. The size of the Golgi apparatus and the number of exocytosed secretory granules did not differ markedly between experimental groups, in contrast to previous findings on short-term cultures. Differences in the profile diameters of secretory granules are recorded between the experimental groups and the pattern differs markedly from that previously recorded for short-term cultures. The growth hormone cells from low sodium media were characterised by abundant, vesiculated RER, a prominent Golgi apparatus (in SW-adapted animals) and relatively few secretory granules. The activity of these growth hormone cells is in marked contrast to previous findings relating to short-term cultures. The shape and size of the non-granulated (stellate) cells of the RPD was again affected by the osmotic pressure of the medium.I should like to thank Mr. P.F. Hire for his photographic assistance     

Inhibitor studies with adenohypophyseal granule membrane ATPase. Evidence for a membrane environment which modulates sensitivity to inhibitors

The limiting membranes of pituitary growth hormone and prolactin secretory granules contain a Mg2+-ATPase sensitive to anions. This enzyme is in many ways similar to mitochondrial ATPase. The enzyme was potently inhibited by oligomycin (Ki 6.5 X 10(-9) M), and was much more sensitive to the inhibitor than pituitary mitochondrial ATPase (Ki 2.7 X 10(-7) M). In contrast, the enzyme activity of intact secretory granules was only sparingly inhibited by oligomycin (maximal inhibition close to 30% at 5 X 10(-4) M). However, oligomycin (5 microM) did diminish to basal levels the enhanced granule ATPase activity observed in the presence of a stimulatory anion (25 mM sodium sulfite). Other compounds known to inhibit the proton translocating mitochondrial ATPase were also tested for their ability to inhibit the secretory granule ATPase. A similar pattern of limited inhibition in granules and greater sensitivity in isolated membranes was seen with the inhibitors N,N-dicyclohexylcarbodiimide and efrapeptin. In contrast, tri-n-butyltin chloride was a potent inhibitor of the ATPase of intact granules, and the susceptibility of the enzyme to inhibition by this compound was less after isolation of membranes. These observations suggest that pituitary secretory granule membrane ATPase may have a proton pumping function similar to that of the mitochondrial enzyme. In addition, the data imply that the inhibitor binding site(s) may be masked, inaccessible, or ineffective in intact granules, but exposed (or activated) in isolated membranes. The greater sensitivity of granule ATPase to tri-n-butyltin chloride, in contrast to the greater sensitivity of membrane ATPase to the other inhibitors, indicates that the tin compound may be effective at a membrane site(s) distinct from the others, or that the mechanism of inhibition is different.     

Ultrastructural studies on prolactin and growth hormone cells in Anguilla pituitaries in vitro

Summary Eel hemi-pituitaries were cultured in vitro on high or low sodium media which are known to affect differentially prolactin and growth hormone release. Ultrastructural examination of the prolactin cells after 24 h culture showed the Golgi bodies were markedly more abundant and widely distributed in hemi-pituitaries from the low sodium medium. Secretory granule release profiles and dense bodies were also more frequent, but the percentage of the cytoplasmic volume occupied by secretory granules was lower than on the high sodium medium. RER was only slightly modified. Significant differences were noted in the shape and processes of the non-granulated (stellate) cells of the RPD, but there were only slight differences in the ultrastructure of the somatotropes.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.     

Calcium binding by subcellular fractions of bovine adrenal medulla

Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromaffin granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10^?3 M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chromaffin granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, 10^?3 M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding. Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.     

Immunocytochemical localisation of prolactin and growth hormone in the ovine pituitary

Summary Using the peroxidase-antiperoxidase immunocytochemical staining technique, prolactin and growth hormone cells have been identified and described in the ovine pituitary. The image analysing computer, Quantimet 720, was used to assess accurately the size range of the secretory granules in these cell types. The area size distributions of the prolactin and growth hormone granules are similar. An increased proportion of larger granules was observed in the prolactin cells post-partum. Serial sections stained alternately for prolactin or growth hormone confirmed that the cells contain either prolactin or growth hormone but not both.     

Electron-microscopic study on the anterior pituitary gland of spontaneous dwarf rats

Summary The spontaneous dwarf rat is a novel experimental model animal on the study of pituitary dwarfism. The fine structure of the anterior pituitary cells was studied in the immature and mature dwarf rats. Pituitary glands were removed from 5-, 10-, 20-day-old immature dwarfs, adult (45 days-16 weeks) dwarfs and normal 3-month-old rats and processed for electron-microscopic observation. In the control animals, growth hormone cells were readily identified by their ultrastructural characteristics, such as the presence of numerous electron-dense secretory granules, 300–350 nm in diameter, well developed rough endoplasmic reticulum and a prominent Golgi complex. In contrast, growth hormone cells were not found in the anterior pituitary gland of the spontaneous dwarf rat at any age examined. Other pituitary cell types, i.e., luteinizing hormone/ follicle stimulating hormone, thyroid stimulating hormone, adrenocorticotropic hormone and prolactin cells, appeared similar in their fine structure to those found in the control rats. In the pituitary gland of dwarf rats, a number of polygonal cells were observed either with no or relatively few secretory granules. The rough endoplasmic reticulum was arranged in parallel cisternae and the Golgi complex was generally prominent in these cells. In addition, many were found to have abundant lysosomes. A few minute secretory granules were occasionally observed; however, the immunogold technique failed to localize growth hormone or prolactin in the granules. The nature of these cells remained obscure in this study. Since their incidence and fine structural features, other than the secretory granules, were quite similar to those of the growth hormone cells in normal rats, we postulate that these cells are dysfunctional growth hormone cells. These results suggest that the cause of the growth impairment in the spontaneous dwarf rat is due to a defect in the functional growth hormone cells in the pituitary gland, and since other pituitary cell types appeared normal, the disorder seems to be analogous to the isolated growth hormone deficiency in the human.     

Association between endocrine pancreatic secretory granules and in- vitro-assembled microtubules is dependent upon microtubule-associated proteins

By use of dark-field light microscopy, secretory granules isolated from the anglerfish endocrine pancreas were observed to attach to and release from microtubules assembled in vitro from brain homogenates. Secretory granules only bound to microtubules assembled in the presence of microtubule-associated proteins (MAPs) and not to microtubules assembled from purified tubulin. The addition of a MAP fraction to purified tubulin restored secretory granule binding. The secretory granules were released from MAP-containing microtubules by the addition of Mg-ATP but not by other nucleotides. The number of secretory granules bound to MAP-containing microtubules was increased in the presence of cyclic AMP. In addition to the associations of secretory granules with microtubules, MAP-containing microtubules also associated with each other. These laterally associated microtubules were dispersed by the addition of Mg-ATP. Electron micrographs confirmed that the associations between MAP-containing microtubules and secretory granules as well as the associations of microtubules with one another were mediated by the high molecular weight MAPs known to project from the surface of in-vitro-assembled microtubules.     

Immunocytochemical localisation of prolactin and growth hormone in the perinatal sheep pituitary

Summary The peroxidase anti-peroxidase immunocytochemical staining technique has been used to identify prolactin and growth hormone cells in pituitaries from fetal and neonatal sheep. The size of the secretory granules in these cell types has been measured using the image analysing computer Quantimet 720. The area size distributions of the fetal prolactin and growth hormone granules were compared with those in the neonate and the adult. It appears that the gestational age of the fetus may influence the size range of prolactin secretory granules.     

Release of arylsulfatase A but not B from rat mast cells by noncytolytic secretory stimuli.

The net percentage of release of arylsulfatase activity from purified rat mast cells induced by rabbit anti-rat F(ab')2 was consistently only about 1/3 that of histamine. Isoelectric focusing of the released and residual arylsulfatase activities demonstrated specific release of the A type without B and a net percentage of immunologic release of arylsulfatase A equivalent to that of histamine. When the net percentage of histamine and arylsulfatase A release were nearly maximal (88 and 76%) in response to the calcium ionophore A23187, specific release of arylsulfatase B did not occur. Thus, arylsulfatase A and not B was associated with the secretory granule released from the rat mast cell by reversed anaphylaxis or the calcium ionophore. In contrast, subcellular fractionation of water-lysed mast cells yielded arylsulfatase B with the heparin- and chymase-containing granule fraction and arylsulfatase A in the aqueous fraction comprised of cell sap and granule water eluate. It may be that arylsulfatase B resides in a minor second granule, whereas arylsulfatase A is loosely associated with the predominant secretory granule of the rat mast cell.     

Stretch and anesthetic dependency of atrial natriuretic peptide release demonstrated by an ultrastructural assay

Using an ultrastructural assay developed to quantify the secretion of atrial natriuretic peptide-containing granules, release of the hormone, in response to different degrees of atrial distension, is directly demonstrated at the cellular level. The ultrastructural assay developed uses an in situ tannic acid perfusion technique to arrest the exocytosis of atrial granules in the anesthetized rat. Secretory granules, which retain the capacity to undergo exocytosis throughout a 30-minute tannic acid perfusion, accumulate at the cell surface in a state of fusion with the plasma membrane, with the core contents retained. Quantification of arrested granules thus provides a measure of the rate of granule release and allows the responses to different stimuli to be assessed. By altering the height of the perfusate, perfusion pressure and hence the degree of distension of the right atrium can be increased, and this causes a proportional rise in the release of secretory granules from individual myocytes. An anesthetic regime incorporating fentanyl citrate was found to increase significantly the rate of granule release, and this was further augmented by atrial distension. Quantification of the numbers of cytoplasmic granules under the same conditions did not reveal a reduction in granules. This is thought to be because only a small pool of granules is recruited for exocytosis, and granule production may continue during the perfusion period. Our assay of atrial secretory granule release allows the effect of a variety of stimulatory and inhibitory agents to be assessed directly at the cellular level and provides an independent comparison with previous biochemical data from whole animal and isolated organ studies. © 1993 Wiley-Liss, Inc.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.     

Chemiosmotic lysis and insulin secretion: studies of isolated granules, intact and permeabilised rat islets of Langerhans

The possible involvement of chemiosmotic lysis of secretory granules in the exocytosis of insulin from pancreatic beta cells was investigated by comparing insulin release from isolated secretory granules, from intact islets of Langerhans, and from electrically permeabilised islets. Lysis of isolated granules was stimulated by ATP in the presence of Mg2+. ATP-induced granule lysis was pH and temperature dependent and was inhibited by collapsing the pH gradient across the granule membrane by removal of permeant anions, or by increasing the extragranular osmolarity. However, insulin secretion from intact islets in response to glucose, a phosphodiesterase inhibitor or a Ca2+ ionophore was only partially inhibited by anion replacement, while Ca2+ -induced insulin release from electrically permeabilised islets was not affected by altering the extragranular or intragranular pH. These results suggest that studies of the stability of isolated granules in vitro do not necessarily relate to insulin release from whole cells, and do not support a major role for chemiosmotic lysis of secretory granules in the exocytotic release of insulin.     

HORMONE STORAGE GRANULES IN THE BEEF ANTERIOR PITUITARY : I. Isolation, Ultrastructure, and Some Biochemical Properties

A method is described for isolation of relatively large quantities of large and small hormone storage granules from the beef adenohypophysis. The hormone storage granules are highly purified, as indicated by ultrastructural and biochemical criteria. The average size of large granules is 400 mµ and of small granules is 220 mµ. The large granules contain growth hormone and prolactin; the small granules contain high concentrations of follicle-stimulating, luteinizing, and thyroid-stimulating hormones. An alkaline protease with a pH optimum of 8.3 is associated with the small granule fraction.     

Divalent cation inhibition of hormone release from isolated adenohypophysial secretory granules

Divalent cations inhibited in vitro release of growth hormone (GH) and prolactin (PRL) from bovine adenohypophysial secretory granules. Zinc, nickel, and cadmium were most potent, exerting 50% inhibition of protein release near 0.1 mM; relative potency was Ni2+ greater than or equal to Zn2+ greater than Cd2+ much greater than Mn2+ greater than Co2+ greater than Cu2+ much greater than Mg2+ greater than Ca2+. The pH optimum for inhibition, 8.0, was lower than that for stimulation of release by thiols. EDTA augmented release and reversed metal inhibition. Both immunoassay and polyacrylamide gel electrophoresis results indicated that metals inhibited both PRL and GH release in a dose-related fashion, and that PRL was more sensitive to all cations tested. With zinc present, known stimulators of release (reduced glutathione, ATP, and bicarbonate) restored GH release, but only ATP restored PRL release. Bicarbonate potently stimulated GH release, but only affected PRL when Mg2+ and ATP were present. We suggest that divalent cations influence GH and PRL release in a reversible fashion and at multiple sites. Some loci may be common to both lactotrope and somatotrope granules; however, the different sensitivities to metals and differential reversal by stimulators of release indicate that metal-protein interactions may also be specific for either granule, or for the hormones themselves.