Isolation and characterization of granules of the toad bladder

Summary The electron-dense granules that lie just below the apical plasma membrane of granular epithelial cells of toad urinary bladder contribute glycoproteins to that apical membrane. Also, exocytosis of granules (and tubules) elicited by antidiuretic hormone potentially doubles that apical surface, during the same period the transport changes characteristic of the hormonal response occur.Granules separated from other membrane systems of the cells provide the material to assess the importance of the granules as glycocalyx precursors and in hormone action. We used isosmotic media to effect preliminary separations by differential centrifugation. Then granules were isolated by centrifugation on self-forming gradients of Percoll of decreasing hypertonicity.We find qualitative and quantitative changes in protein composition and enzymic activities in the isolated fractions. The primary criterion for granule purification was electron microscopic morphology. In addition, polypeptide species found in the granule fraction are limited in number and quantity. The granules are enzymically and morphologically not lysosomal in nature. Granules may provide the glycoproteins of the apical glycocalyx but they differ from the isolated plasma membrane fraction enzymically, in protein composition and in proportion of esterified cholesterol.We conclude that the granules are not average plasma membrane precursors. Their role in the membrane properties of the toad urinary bladder may now be evaluated by characterizing permeability and other properties of the isolated organelles.     

Very low osmotic water permeability and membrane fluidity in isolated toad bladder granules

Summary Osmotic water permeability of the apical membrane of toad urinary epithelium is increased greatly by vasopressin (VP) and is associated with exocytic addition of granules and aggrephores at the apical surface. To determine the physiological role of granule exocytosis, we measured the osmotic water permeability and membrane fluidity of isolated granules, surface membranes and microsomes prepared from toad bladder in the presence and absence of VP.P _f was measured by stopped-flow light scattering and membrane fluidity was examined by diphenylhexatriene (DPH) fluorescence anisotropy. In response to a 75mm inward sucrose gradient, granule size decreased with a single exponential time constant of 2.3±0.1 sec (sem, seven preparations, 23°C), corresponding to aP _f of 5×10^–4 cm/sec; the activation energy (E _a ) forP _f was 17.6±0.8 kcal/mole. Under the same conditions, the volume of surface membrane vesicles decreased biexponentially with time constants of 0.13 and 1.9 sec; the fast component comprised 70% of the signal. Granule, surface membrane and microsome time constants were unaffected by VP. However, in surface membranes, there was a small decrease (6±2%) in the fraction of surface membranes with fast time constant. DPH anisotropies were 0.253 (granules), 0.224 (surface membrane fluidity is remarkably lower than that of surface and microsomal membranes, and (4) rapid water transport occurs in surface membrane vesicles. The unique physical properties of the granule suggests that apical exocytic addition of granule membrane may be responsible for the low water permeability of the unstimulated apical membrane.     

The participation of intracellular membranes in forming highly permeable domains in the plasma membrane of epithelial cells during the vasopressin stimulation of water transport]

Using different electron microscopic techniques, parallel studies of structural alterations in the apical membrane and specific granules of the frog urinary bladder granular cells were made. The results obtained suggest the participation of granule membranes in the formation of highly permeable domains in the apical membranes. After ADH action, the domains with high water permeability are internalized bringing cell membrane retrieval.     

Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.     

Regulation of zymogen granule exocytosis by Ca2+, cAMP, and PKC in pancreatic acinar cells

The effect of cAMP and PKC on zymogen granule exocytosis was investigated by simultaneously measuring cytosolic Ca2+ concentration ([Ca2+]c) and individual zymogen granule exocytosis in isolated mouse pancreatic acini. When acinar cells were stimulated with acetylcholine (ACh, 10 microM), exocytic events were detected through granule-attached apical membranes with [Ca2+]c rise. Application of secretin, forskolin (an adenylate cyclase activator), or PMA (a PKC activator) alone did not elicit any [Ca2+]c rise or zymogen granule exocytosis, but co-stimulation with ACh led to exocytosis in that the total number of secreted granules increased markedly without a significant difference in [Ca2+]c rises. When we evoked exocytosis by [Ca2+]c ramps, pretreatment with forskolin or PMA elicited exocytosis at lower [Ca2+]c levels. These results indicate that PKC or cAMP alone could not directly elicit zymogen granule exocytosis, but that they increase the total releasable pool by rendering zymogen granules more sensitive to Ca2+.     

The carbohydrates of secretory granules and the glycocalyx in developing mucoid cells

Summary Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.     

Mechanism of rapid mucus secretion in goblet cells stimulated by acetylcholine

The parasympathetic control of goblet cell secretion and the membrane events accompanying accelerated mucus release were studied in large intestinal mucosal biopsies maintained in an organ culture system. The secretory response of individual goblet cells to 10(-6) M acetylcholine chloride with 3 x 10(-3) M eserine sulfate (a cholinesterase inhibitor) was assessed by light microscopy and autoradiography, by scanning and transmission electron microscopy, and by freeze-fracture. Goblet cells on the mucosal surface are unaffected by acetylcholine. In crypt goblet cells acetylcholine-eserine induces rapid fusion of apical mucous granule membranes with the luminal plasma membrane (detectable by 2 min), followed by sequential, tandem fission of the pentalaminar, fused areas of adjacent mucous granule membranes. These events first involve the most central apical mucous granules, are then propagated to include peripheral granules, and finally spread toward the most basal granules. By 60 min, most crypt cells are nearly depleted. The apical membrane, although greatly amplified by these events, remains intact, and intracellular mucous granules do not coalesce with each other. During rapid secretion membrane-limited tags of cytoplasm are observed attached to the cavitated apical cell surface. These long, thin extensions of redundant apical membrane are rapidly lost, apparently by being shed into the crypt lumen.     

A lysophospholipase specific for exocrine pancreatic cells is stored in zymogen granules and secreted into pancreatic juice

We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.     

An assessment of the role of protein kinase and zymogen granule phosphorylation during secretion by the rat exocrine pancreas.

1. The levels of protein kinase activity and zymogen granule phosphorylation were studied in the adult rat during stimulus-coupled secretion in vitro. 2. The specific activity of protein kinase associated with intact zymogen granules was 11 pmol [32P]phosphate transferred to histone per min per mg protein. Most of this activity was recovered in purified granule membranes. 2. The addition of 10(-6) M cyclic AMP to a mixture of zymogen granules and the postmicrosomal supernatant resulted in a 5-fold increase in protein kinase activity associated with zymogen granules. The adsorbed activity was eluted from granules by 0.15 M NaCl. Cyclic GMP did not promote protein kinase binding to isolated granules. 4. Incubation of tissues with carbachol (10(-5) M), pancreozymin (0.1 unit/ml), caerulein (10(-8) M) or dibutyryl cyclic AMP (2.10(-4) M) between 2.5 and 60 min did not increase the levels of protein kinase activity in isolated zymogen granules above control values. 5. Protein phosphorylation of zymogen granule membranes and granule content was not detectable in tissues incubated with carbachol, pancreozymin-C-octapeptide, or caerulein. 6. These results suggest that neither the phosphorylation of zymogen granule membrane protein nor the adsorption of protein kinase activity to zymogen granules is an obligatory step in secretion.     

76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Localization and potential role

Several experimental conditions such as antidiuretic hormone (ADH) challenge, apical treatment with phorbol myristate acetate (PMA), and mechanical stretching of the tissue are known to increase the insertion of intramembrane particle aggregates and/or granule exocytosis at the apical border of epithelial cells of amphibian urinary bladders. A constant release of 2 peptides of 76 and 14 kDa apparent molecular mass, respectively, was associated with these treatments. The localization of these 2 polypeptides was assessed by immunofluorescence and electron microscopy immunocytochemistry using fluorescent, peroxidase, and colloidal gold probes. The 76 kDa polypeptide appeared to be associated with the cell coat and with the granule content which is released at the apical cell surface. The 14 kDa peptide was also found in the cell coat, and postembedding immunocytochemistry indicates its presence in cytoplasmic subapical vesicles (aggrephores and/or granules). The migration of these 76 and 14 kDa polypeptides in SDS-polyacrylamide gel electrophoresis was modified neither by a treatment at 90 degrees C, nor by the presence or absence of calcium in the medium. Treatment with EGTA did not modify the fluorescence emission of the two peptides and, consequently, they are probably not among the major calcium binding proteins. The addition to the mucosal medium of the stretch extract or of antibodies raised against the 76 and 14 kDa peptides did not modify ADH-induced water permeability. However, a significant decrease of the hydrosmotic response to ADH occurred in subsequent stimulation-washout cycles when the anti-14 kDa peptide antiserum was applied to the mucosal bath. When the bladders were incubated with a stretch extract, we observed a slight alteration of the short-circuit current (Isc), an increase of the basal Na+ transport, and a decrease of the maximal Isc in response to ADH. The 76 kDa protein, released in the apical medium, could play a protective role in the cellular plasma membrane and could participate in the formation of the thick cell coat lining the apical membrane of the granular cells. The 14 kDa protein might be one of the proteins associated with the aggregates, but further studies will be necessary to clarify its exact role in the ADH-induced permeability modifications observed in amphibian urinary bladders.     

Actin‐dependent,Retrograde Motility of Surface‐attached Beads and Aggregating Pigment Granules in Dissociated Teleost Retinal Pigment Epithelial Cells

Teleost retinal pigment epithelial (RPE) cells contain pigment granules within apical projections which undergo actin‐dependent, bi‐directional motility. Dissociated RPE cells in culture attach to the substrate and extend apical projections in a radial array from the central cell body. Pigment granules within projections can be triggered to aggregate or disperse by the presence or absence of 1 mM cAMP. Aminated, fluorescent latex beads attached to the dorsal surface of apical projections and moved in the retrograde direction, towards the cell body. Bead rates on RPE cells with aggregating or fully aggregated pigment granules were 2.2 ± 0.5 and 2.6 ± 0.2 μm/min (mean ± SEM), respectively, similar to rates of aggregating (retrograde) pigment granule movement (2.0 ± 0.4 μm/min). Bead rates were slightly slower on cells with fully dispersed or dispersing pigment granules (1.5 ± 0.1 and 1.5 ± 0.4 μm/min). Movements of surface‐attached beads and aggregating pigment granules were closely correlated in the distal portions of apical projections, but were more independent of each other in proximal regions of the projections. The actin disrupting drug, cytochalasin D (CD), reversibly halted retrograde bead movements, suggesting that motility of surface‐attached particles is actin‐dependent. In contrast, the microtubule depolymerizing drug, nocodazole, had no effect on retrograde bead motility. The similar characteristics and actin‐dependence of retrograde bead movements and aggregating pigment granules suggest a correlation between these two processes.     

An energy-based population-balance approach to model granule growth and breakage in high-shear wet granulation processes

A mathematical model of high shear wet granulation is proposed, where granule breakage, and not growth, is the dominant process. The energy required for granule breakage is assumed to be provided by the impact of granules between themselves and the granulator parts, and the extent of granule breakage determined by the balance between the impactenergy and the work of adhesion between the agglomerating particles. A specific volume of dry powder per unit crack surface area was allowed to reattach to the surface of broken granules to account for granule growth. To verify proposed model conditions, lactose monohydrate was granulated with a relatively low amount (6%) of the binder phase, polyvinyl-pyrrolidone and water, and was added to the powder before granulation. The trend in granule size distribution during the experiment closely follwed the predicted model with an initial increase in the weight fraction of the larger granules. This increase was possibly due to extensive breakage of weaker granules and less extensive breakage, as if by attrition, of stronger granules, accompanied by the attachment of dry powder to the cracked surfaces. Eventually, larger granules experience increased impact energy and break. When excess binder is added and, higher volumes of powder reattach to the crack surface, more large granules form leading to granule overgrowth. This model highlights the importance of the probability of impact per unit time interval (ie, the rate of impact), the strength of the granules and the volume of powder that could attach to the cracked surface in high shear granulation processes where significant granule breakage is encountered. Published: August 10, 2007     

A common spectrum of polypeptides occurs in secretion granule membranes of different exocrine glands

A highly purified membrane preparation from rat parotid secretion granules has been used as a comparative probe to examine the extent of compositional overlap in granule membranes of three other exocrine secretory tissues--pancreatic, lacrimal, and submandibular--from several standpoints. First, indirect immunofluorescent studies using a polyclonal polyspecific anti-parotid granule membrane antiserum has indicated a selective staining of granule membrane profiles in all acinar cells of all tissues. Second, highly purified granule membrane subfractions have been isolated from each exocrine tissue; comparative two-dimensional (isoelectric focusing; SDS) PAGE of radioiodinated granule membranes has identified 10-15 polypeptides of identical pI and apparent molecular mass. These species are likely to be integral membrane components since they are not extracted by either saponin-sodium sulfate or sodium carbonate (pH 11.5) treatments, and they do not have counterparts in the granule content. Finally, the identity among selected parotid and pancreatic radioiodinated granule membrane polypeptides has been documented using two-dimensional peptide mapping of chymotryptic and tryptic digests. These findings clearly indicate that exocrine secretory granules, irrespective of the nature of stored secretion, comprise a type of vesicular carrier with a common (and probably refined) membrane composition. Conceivably, the polypeptides identified carry out general functions related to exocrine secretion.     

Effects of the Actin‐Stabilizing Drug,Jasplakinolide, on Pigment Granule Motility in Isolated Retinal Pigment Epithelial (RPE) Cells of Green Sunfish,Lepomis cyanellus

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin‐disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin‐stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1–1 μM), but the drug halted movement of most pigment granules and stimulated rapid bi‐directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide‐treated cells demonstrated slow, creeping centrifugal movements and more rapid bi‐directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle‐like structures, which contained F‐actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide‐treated cells revealed a more reticulated network of actin compared to antibody‐labeled control cells. These results indicate that jasplakinolide‐induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.     

Immunocytochemical localization of ribonuclease in yolk granules of adult Rana cttesbeiana oocytes

To determine the localization of the pyrimidine-guanine sequence-specific ribonuclease in Rana catesbeiana (bullfrog) oocytes, the RNase was first isolated and used to prepare a specific rabbit antiserum. Only one protein of similar molecular size to the RNase was immunoprecipitated from ovary homogenate by the antiserum, but two bands were observed by Western blotting analysis. These two proteins were shown by further purification of antibody and Western blotting analysis to have similar antigenicity. Immunoprecipitation and Western blotting of tissue homogenates showed that the RNase was found predominantly in the ovary, but not in other tissues. The specific localization of the RNase was determined by immuno-electron microscopy of oocyte sections incubated with the specific antiserum; the yolk granules, but not other organelles, were found to contain the RNase. Most of the RNase was evenly distributed in the lateral amorphous area of the yolk granule but not in the central yolk crystal area which contains stored vitellogenin proteins. Our results indicate that the RNase is compartmentalized in the yolk granules of oocytes, which might prevent damage to cellular RNAs.     

Effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility in isolated retinal pigment epithelial (RPE) cells of green sunfish, Lepomis cyanellus

The retinal pigment epithelium (RPE) of teleosts contains pigment granules that migrate in response to changes in light condition. Dissociated, cultured RPE cells in vitro can be triggered to aggregate or disperse pigment granules by the application of cAMP or dopamine, respectively. Previous research using the actin-disrupting drug, cytochalasin D, suggested that pigment granule motility is actin dependent. To further examine the role of actin in pigment granule motility, we tested the effects of the actin-stabilizing drug, jasplakinolide, on pigment granule motility. Pigment granules in previously dispersed RPE cells remained dispersed after jasplakinolide exposure (0.1-1 microM), but the drug halted movement of most pigment granules and stimulated rapid bi-directional movements in a small subset of granules. Jasplakinolide also blocked net pigment granule aggregation and interfered with the maintenance of full aggregation. Although jasplakinolide did not block pigment granule dispersion, it did alter the motility of dispersing granules compared to control cells; rather than the normal saltatory, primarily centrifugal movements, granules of jasplakinolide-treated cells demonstrated slow, creeping centrifugal movements and more rapid bi-directional movements. Jasplakinolide also altered cell morphology; the length and thickness of apical projections increased, and enlarged, paddle-like structures, which contained F-actin appeared at the tips of projections. Actin antibody labeling of jasplakinolide-treated cells revealed a more reticulated network of actin compared to antibody-labeled control cells. These results indicate that jasplakinolide-induced disruption of the actin network compromises normal pigment granule dispersion and aggregation in isolated RPE cells, thus providing further evidence that these movements are actin dependent.     

Trans-epidermal Transport and Storage of Calcium in Holthuisana transversa (Brachyura; Sundathelphusidae) During Premoult

Holthuisana transversa reabsorbs much of its exoskeletal calcium in the last 3 days before ecdysis and stores it in circulating granules in the haemocoel and in non-circulating granules in the subepidermal connective tissue. Calcium enters the epidermal cells from the moulting fluid, probably through their apical microvilli and is either incorporated into intracellular calcium granules or exits the cell via the basolateral membranes to be used in formation of two other granule types. Intracellular granules (0.4–2 μm long) form in large masses in the apical cytoplasm of the epidermal cells. They are formed as membrane-bound vesicles by the Golgi, and calcium and organic matrix material are added from the surrounding cytoplasm. As development proceeds, lamellae appear and calcium carbonate is deposited in the matrix. Granule masses move basally and are stored in the connective tissue. Calcium is also incorporated into extracellular large granules (0.8–3.8 μm long) which are formed in narrow intercellular channels between epidermal cells. A third granule type (small granules, 0.26 μm diameter) is formed in subepidermal connective tissue cells and released into the haemolymph in very large numbers. Calcium was identified in the two larger granule types using X-ray microanalysis and significant amounts of phosphorus and potassium were also present in the large granules. A model for ion cycling between the exoskeleton and granules is presented.     

Quantitative analysis of the process and propagation of cortical granule breakdown in sea urchin eggs

Cortical granule breakdown in sea urchin eggs has been investigated with a video microscope system using Nomarski differential interference contrast optics, when induced by fertilization, microinjecting inositol 1,4,5-trisphosphate (IP3) or Ca-EGTA buffer solution into the egg, or perfusing a medium containing 1 mM Ca2+ to isolated cortices. The cortical granule increased up to 1.2 times in diameter and broke down within 40 msec. These values were almost constant among the three methods used to induce cortical granule breakdown. Upon fertilization, the cortical granule breakdown propagated over the egg surface at a speed of 3.3 microns/sec in Clypeaster japonicus eggs, which indicates that cortical granule breakdown propagated through the 3.3-microns-wide egg surface within 1 sec. In such a small area of the egg surface, however, it took much more than 1 sec for all cortical granules to break down because the maximal rate of breakdown was 7.6%/sec; that is, it took 9 sec and 18 sec for 50% and 90% respectively, of cortical granules to break down. Moreover, the rate did not simply decrease with time, and a shoulder was found during the reducing phase, which suggests that cortical granules are divided into fast and slow breakdown groups according to the responsiveness to the breakdown stimulus. The cortical granule breakdown induced by microinjecting the Ca-EGTA buffer and IP3 solutions propagated at 68 microns/sec and 35 microns/sec, respectively. The stimulus for cortical granule breakdown is discussed concerning the transient intracellular Ca2+ increase.     

The pancreatic membrane protein GP-2 localizes specifically to secretory granules and is shed into the pancreatic juice as a protein aggregate

GP-2 is the major secretory granule membrane glycoprotein of the exocrine pancreas and appears in the pancreatic juice in a modified sedimentable form. We have localized GP-2 in the rat pancreas at the electron microscopic level using affinity-purified antibodies and found it to be concentrated in the zymogen granules and in the acinar lumen. Label was also present on the apical and basolateral plasma membranes but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties reduced substantially the staining of the basolateral surface. Approximately 45% of the GP-2 in the granules was not membrane-associated but appeared instead in the granule lumen. Parallel biochemical characterization of GP-2 in isolated secretory granules demonstrated that 60% fractionated with the membranes after granule lysis while 40% remained in the content fraction. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl-phosphatidylinositol (GPI), GP-2 in the content did not enter the detergent phase upon Triton X-114 extraction; nor was it sedimentable at 200,000g, as is characteristic of the form collected in the pancreatic juice. In addition, GP-2 in the pancreatic juice was recovered in the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. These results are consistent with a life cycle for the protein that begins with synthesis of a membrane-associated precursor that can be converted by lipolytic or proteolytic cleavage to a soluble form within the zymogen granule. Further modification to a sedimentable form may then occur in the pancreatic juice.     

An ultrastructural study of the cysts in chicken ultimobranchial glands,with special reference to C-cells

Summary The ultimobranchial glands of the chicken were examined by electron microscopy and immunocytochemistry using a calcitonin antiserum. Electron microscopy confirmed the presence of C-cells, containing numerous secretory granules storing calcitonin, in the luminal lining of cyst-like structures found in these glands. These cells were furnished with prominent microvillar projections at their luminal surface, and the cytoplasm of the apical region was filled with fibril material. Furthermore, the cells contained prominent junctional complexes and desmosomes at their apico-lateral surfaces. In these C-cells, secretory granules were concentrated near the lumen and some were attached to the apical cell membrane. The luminal content of the cysts had a colloid-like and flocculent appearance, and was frequently seen attached to the cytoplasmic projections or apical cell membrane of the C-cells. Since the cysts progressively increase in volume and number with age, it is suggested that they may partly play a role in the storage of excess or unneeded hormonal products.