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.     

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Summary The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio-iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Nomarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with¹²⁵I. The basal-lateral components yielded a heterodisperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochromec reductase activities, were separated from the radio-iodine labeled components by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 × g × 1 hr after removal of the mitochondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.     

Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland

Summary A secretory granule fraction has been isolated from rat parotid by discontinuous gradient centrifugation using hyperosmotic sucrose-Ficoll solutions of low ionic strength. The secretion granule fraction comprises 25% of the total tissue -amylase activity and is judged to be of high purity, both morphologically and by its low level of contamination by enzyme activities associated with other organelles.Secretion granules were lysed by capitalizing on their lability in KCl-containing media, and the low density granule membranes were separated from residual organelle and soluble contaminants by flotation in a sucrose gradient. Residual, poorly extractable secretory contaminants of the granule membrane subfraction were selectively removed by a saponin- (10 g/ml) Na₂SO₄ (0.3m) wash, apparently with negligible disruption of granule membrane structure. Based on detailed consideration of the extent of contamination by residual mitochondria and incompletely removed secretory polypeptides, it is possible to estimate that 95% of the protein associated with the purified secretion granule membrane is bona fide granule membrane protein. Further analyses indicate that -glutamyltransferase constitutes a marker enzymatic activity shared by granule membranes and the apical domain of the plasma membrane.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretograms of radio-iodinated granule membrane polypeptides are characterized by 20–25 radioactive bands of which 5–6 are suggested to be glycoproteins by virtue of their binding of concanavalin A. The limited polypeptide composition of the secretion granule membrane (in comparison to membranes of other cellular compartments) and the high phospholipid-protein ratio (4.4 mg/mg) may reflect the functional specialization of this storage container for secretory proteins.     

ADH and phorbol ester increase immunolabeling of the toad bladder apical membrane by antibodies made to granules

Summary Polyclonal antibodies were raised to isolated toad bladder granules. On immunoblots, the anti-granule antiserum specifically stained components of isolated granules. Immunocytochemically, the anti-granule antiserum labeled the apical surface of the bladder. Immunolabeling increased at the apical surface when the bladder was exposed to antidiuretic hormone (ADH) serosally or phorbol ester (PMA) mucosally—conditions which stimulate apical granule exocytosis. The increase in granule epitopes on the apical surface was sixfold greater than the net increase in surface area.     

Ionic and permeability requirements for exocytosis in vitro in sea urchin eggs

Summary We study exocytosis in the planar isolated cortex of the egg of the sea urchinLytechinus pictus. Solutins bathing the exocytotic apparatus need not contain appreciable amounts of ions: fusion follows addition of submicromolar calcium to solutions containing only nonelectrolyte. We examine the effects of altering the granule membrane permeability to small molecules with ionophores and digitonin. Introducing holes in the secretory granule membrane to the extent of allowing free passage of small molecules does not cause seretion in vitro. We add the amphipathic compound digitonin at 12 to 15 M concentrations and demonstrate that the granule membrane can become permeable to lucifer yellow, yet that granules remain intact. Granules still undergo exocytosis after digitonin treatment at such concentrations upon subsequent addition of calcium. Higher concentrations of digitonin lead to granule content swelling and vesicle bursting. We conclude that cortical granule hydration during exocytosis is not mediated by small ionic channels.     

3H-fucose incorporation into glycoproteins of toad bladder epithelial cells.

Electron microscope autoradiography was used to detect the incorporation of 3H-fucose into glycoproteins of toad bladder epithelial cells. After short exposure to 3H-fucose, without a chase period, the Golgi regions of all four cell types were labeled. When exposure to 3H-fucose was followed by chase periods (1,3,4 and 6 hours) the apical and basal-lateral plasma membranes of granular cells were heavily labeled. Apical granules and the cytoplasm of granular cells were also labeled, suggesting that they both provide the means for glycoprotein transfer from the Golgi to the plasma membranes. The heaviest labeling in mitochondria-rich cells, after the 1- and 3-hour chase periods, was over the apical tubules, although the apical and basal-lateral plasma membranes were also heavily labeled. After 4- and 6-hour chases, the labeling of the apical tubules decreased, whereas the labeling of the plasma membranes increased, strongly suggesting that in these cells apical tubules play a major role in the transfer of glycoproteins from the Golgi to the plasma membrane. Our results demonstrate that the route of 3H-fucose incorporation into plasma membrane glycoproteins and the rate of glycoprotein synthesis and breakdown are not the same in the two major epithelial cell types in toad bladder.     

Ion selectivity of the apical membrane Na channel in the toad urinary bladder

Summary The ion selectivity of the apical membrane Na channel in the toad urinary bladder was investigated. The electrical potential difference and resistance across the basal-lateral membrane were reduced using high concentrations of KCl in the serosal bathing medium, and gradients for various ions were imposed across the apical membrane by altering the composition of the mucosal bathing medium. Ion fluxes through the channel were measured as the transepithelial current inhibited by amiloride, a specific blocker of the channel's Na conductance. The selectivity sequence for alkali metal cations was H>Li>NaK. K, permeability was barely detectable; the selectivity for Na over K was about 1000:1. Ammonium, hydroxyl ammonium and hydrazinium ions were, like K, virtually impermeant. The results suggest that the size of the unhydrated ion is an important factor in determining permeability in this channel.     

An electron-microscope and freeze-fracture study of the egg cortex of Brachydanio rerio

Summary We have examined the cortex of the teleost (Brachydanio rerio) egg before and during exocytosis of cortical granules by scanning, transmission, and freeze-fracture electron microscopy. In the unactivated egg, the P-face of the plasma membrane exhibits a random distribution of intramembranous particles, showing a density of 959/m² and an average diameter of 8 nm. Particles over P- and E-faces of the membranes of cortical granules are substantially larger and display a significantly lower density. An anastomosing cortical endoplasmic reticulum forms close associations with both the plasma membrane of the egg and the membranes of cortical granules. Exocytosis begins with cortical granules pushing up beneath the plasma membrane to form domeshaped swellings, coupled with an apparent clearing of particles from the site of contact between the apposed membranes. A depression in the particle-free plasma membrane appears to mark sites of fusion and pore formation between cortical granules and plasma membranes. Profiles of exocytotic vesicles undergo a predictable sequence of morphological change, but maintain their identity in the egg surface during this transformation. Coated vesicles form at sites of cortical granule breakdown. Differences in particle density between cortical granules and egg plasma membranes persist during transformation of the exocytotic profiles. This suggests that constituents of the 2 membrane domains remain segregated and do not intermix rapidly, lending support to the view that the process of membrane retrieval is selective (i.e., cortical granule membrane is removed).     

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia

The present study examines the role of calcium in modulating epithelial cytomorphology by using verapamil, a calcium antagonist, and considering its effects on cytosolic granule distribution and exocytosis in toad urinary bladder. The effect of verapamil on the detection and distribution of microfilaments in toad urinary bladder using immunogold labeling techniques in toad urinary bladder epithelial cells was also examined. Verapamil, which inhibits antidiuretic hormone (ADH)-mediated water flow, increased the number, size and distribution of dense calcium-containing secretory granules in bladder epithelial cells. This calcium antagonist prevented granule exocytosis, such that, six-times the number of granules were present in verapamil-treated tissues. The normal cytomorphological changes that accompany the actions of ADH were attenuated by verapamil, including ADH-induction of microvilli. ADH increased the number of actin microfilaments as determined using protein A-gold by immunolabeling, whereas, verapamil treatment was unremarkable as compared to control. The results suggest that calcium may play a prominent role in mediating granule exocytosis and membrane fusion events that normally accompany hormone action.     

Hyperosmolality inhibits exocytosis in sea urchin eggs by formation of a granule-free zone and arrest of pore widening

Summary Hyperosmolality is known to inhibit membrane fusion during exocytosis. In this study cortical granule exocytosis in sea urchin eggs is used as a model system to determine at what step this inhibition occurs.Strongylocentrotus purpuratus eggs were incubated in hyperosmotic seawater (Na₂SO₄, sucrose or sodium HEPES used as osmoticants), the eggs activated with 20 m A23187 to trigger exocytosis, and then quick frozen or chemically fixed for electron microscopy. Thin sections and freeze-fracture replicas show that at high osmolality (2.31 osmol/kg), there is a decrease in cortical granule size, a 90% reduction in granule-plasma membrane fusion, and formation of a granulefree zone between the plasma membrane and cortical granules. This zone averages 0.64 m in thickness and prevents the majority of granules from docking at the plasma membrane. The remaining granules (10%) exhibit early stages of fusion which appear to have been stabilized; the matrix of these granules remains intact. We conclude that exocytosis is blocked by two separate mechanisms. First, the granule-free zone prevents granule-plasma membrane contact required for fusion. Second, in cases where fusion does occur, opening of the pocket and dispersal of the granule contents are slowed in hyperosmotic media.     

Purification and characterization of the apical plasma membrane of the rat pancreatic acinar cell

Summary A method is described for the rapid purification of the apical plasma membrane from the rat pancreatic acinar cell. It makes use of wheat germ agglutinin affinity chromatography to selectively bind vesicles with N-acetyl glucosamine present at their surface. Particular conditions (150 mm NaCl) had then to be used to keep membrane vesicles in the coveted orientation, i.e. as right-side-out vesicles. Due to its specific apical location in many epithelial cells, -glutamyltranspeptidase was chosen to monitor the purification procedure. The final fraction was enriched in -glutamyltranspeptidase by a factor of 75 relative to the homogenate. Na,K-ATPase, a strict basolateral membrane marker, was not detectable in the fraction. No membranes originating from other compartments, more particularly expected from zymogen granules, or from other cell types, did contaminate the preparation. As expected for an epithelial cell apical plasmalemma, lipid composition showed a very high ratio of glycolipids (37.5%). The absence of membrane-bound GP-2, and the exceptionally high specific activity of -glutamyltranspeptidase suggest that the apical membrane would not be made up by the exocytosis of secretory granule, but instead by the fusion of specialized secretory vesicles very likely originating from the constitutive secretory pathway. In conclusion, this report describes a method of obtaining a fraction highly enriched in the secretory apex of the pancreatic exocrine cell that would be directly involved in exocytosis with zymogen granules and also in local anion transport.The authors would like to thank Dr. Andrew W. Shyjan (Yale University, New Haven, CT) for his kind gift of anti-Na,K-ATPase 1 subunit, Dr. Yannick Laperche (INSERM, Hôpital Mondor, Paris) for his gift of anti--GT, and finally Mr. Gilles P. Grondin for the production of antibodies against amylase. D.L. is supported by NSERC of Canada, FCAR of Québec, and the Canadian Cystic Fibrosis Foundation.     

Modulation of the beta-adrenergic-response in cultured rat heart cells

Summary Subcellular fractionation studies in resting human neutrophils indicated a bimodal distribution for cytochrome b. A. major peak of cytochrome b co-sedimented with gelatinase under different experimental conditions. This localization was partially overlapped with specific granules (using lysozyme and lactoferrin as specific granule markers), but clearly resolved from azurophilic granules, plasma membrane, mitochondria, as well as from a novel alkaline phosphatase-rich intracellular organelle. A minor localization of cytochrome b was found in fractions enriched in both the plasma membrane marker 5-nucleotidase and alkaline phosphatase. A significant portion of ubiquinone cell content co-fractionated with the gelatinase-containing granules. After phorbol myristate acetate (PMA)-cell stimulation, cytochrome b was mobilized to fractions showing respiratory burst activity and enriched in 5-nucleotidase activity. This mobilization paralleled secretion of gelatinase and lysozyme to the extracellular medium. Furthermore, neutrophil stimulation with fluoride in the absence of cytochalasin B induced release of gelatinase and generation of superoxide anion with only minimal release of lysozyme. Preincubation of cells with the anion channel blocker 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) prevented lysozyme release, but had only a minor effect on the release of gelatinase and did not inhibit the superoxide anion generation elicited by N-formyl-methionyl-leucyl-phenylalanine or PMA. These results suggest a main location of cytochrome b in mobilizable gelatinase-containing granules, which can constitute a subpopulation of specific granules. Furthermore, these findings show that the gelatinase-containing granule is functionally involved in the respiratory burst in neutrophils and that membrane fusion between plasma membrane and the gelatinase-containing granule occurs during activation of cells.Abbreviations DIDS 4,4-diisothiocyanostilbene-2,2-disulfonic acid - FMLP N-formyl-methionyl-leucyl-phenylalanine - PMA 4-phorbol, 12-myristate, 13-acetate     

Evidence that the ability to respond to a calcium stimulus in exocytosis is determined by the secretory granule membrane: Comparison of exocytosis of injected bovine chromaffin granule membranes and endogenous cortical granules inXenopus laevis oocytes

Summary 1. To understand better the mechanisms which govern the sensitivity of secretory vesicles to a calcium stimulus, we compared the abilities of injected chromaffin granule membranes and of endogenous cortical granules to undergo exocytosis inXenopus laevis oocytes and eggs in response to cytosolic Ca²⁺. Exocytosis of chromaffin granule membranes was detected by the appearance of dopamine--hydroxylase of the chromaffin granule membrane in the oocyte or egg plasma membrane. Cortical granule exocytosis was detected by release of cortical granule lectin, a soluble constituent of cortical granules, from individual cells.2. Injected chromaffin granule membranes undergo exocytosis equally well in frog oocytes and eggs in response to a rise in cytosolic Ca²⁺ induced by incubation with ionomycin.3. Elevated Ca²⁺ triggered cortical granule exocytosis in eggs but not in oocytes.4. Injected chromaffin granule membranes do not contribute factors to the oocyte that allow calcium-dependent exocytosis of the endogenous cortical granules.5. Protein kinase C activation by phorbol esters stimulates cortical granule exocytosis in bothXenopus laevis oocytes andX. laevis eggs (Bement, W. M., and Capco, D. G.,J. Cell Biol. 108, 885–892, 1989). Activation of protein kinase C by phorbol ester also stimulated chromaffin granule membrane exocytosis in oocytes, indicating that although cortical granules and chromaffin granule membranes differ in calcium responsiveness, PKC activation is an effective secretory stimulus for both.6. These results suggest that structural or biochemical characteristics of the chromaffin granule membrane result in its ability to respond to a Ca²⁺ stimulus. In the oocytes, cortical granule components necessary for Ca²⁺-dependent exocytosis may be missing, nonfunctional, or unable to couple to the Ca²⁺ stimulus and downstream events.     

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.     

Lateral diffusion of luminal membrane components during secretion in parotid acinar cells of the rat

Summary The movements of the molecular components of the luminal plasma membrane during exocytotic secretion in parotid acinar cells were examined. For immunocytochemical study, we used an antiserum of dipeptidyl peptidase IV as a marker for the components of the luminal plasma membrane of acinar cells. In unstimulated acinar cells, dipeptidyl peptidase IV immunoreactivity is restricted to the luminal plasma membrane. However, after secretion was stimulated with a -adrenergic agonist, isoproterenol, immunostaining became detectable on the membrane of discharged granules. Freeze-fracture images showed that the density of intramembrane particles on the P-fracture leaflets of discharged granule membranes is much higher than that of undischarged granule membranes during secretion. These results suggest that in parotid acinar cells of the rat, the components of the luminal plasma membrane move laterally, during secretion, to the membranes of discharged granules.     

Subcellular fractionation of epithelial cells from toad urinary bladder. 1. Assay of marker enzymes and differential centrifugation

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5'-nucleotidase, and the transferases that donate N-acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose-6-phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate, dehydrogenase, catalase was essentially found in the high-speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl-beta-naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochrome c oxidase, acid phosphatase, acid N-acetyl-beta-glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin-resistant Mg++-ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acid N-acetyl-beta-glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugation is examined in a companion paper.     

Intramembranous response to cAMP in fetal epidermis

Summary Prekeratinized fetal epidermis may function as an osmoregulatory organ. This study shows that the structural response of fetal epidermis to cAMP (second messenger of antidiuretic hormones) is very similar to that in well characterized osmoregulatory epithelia. cAMP stimulation of unkeratinized fetal epidermis induces the appearance of aggregates of intramembranous particles. These aggregates are present in the apical plasma membrane of the periderm or outermost cell layer of the epidermis. Tubular vesicles that contain the presumptive aggregates fuse with the apical plasma membrane during cAMP stimulation. The same response to cAMP and to antidiuretic hormones has been reported in the osmoregulatory ion- and water-transporting epithelia of amphibian urinary bladder and mammalian collecting ducts. In these systems aggregates have been positively correlated with water transport. Thus, the fetal epidermis may control and regulate its water permeability.     

Radio-iodination of plasma membranes of toad bladder epithelium

Summary The present report describes high yield enzymatic radio-iodination of the apical and basal-lateral plasma membranes of toad bladder epithelium, by a procedure that does not breach the functional integrity of the epithelium, as assessed by the basal and vasopressin-sensitive short-circuit current (SCC). Restriction of the label to the membrane surface was ascertained by light and electron-microscopic autoradiographs. On the apical surface, the grains were over the glycocalyx and the plasma membrane. Analysis of the labeled glycocalyx by agarose gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), as well as enzymatic and pH-dependent hydrolysis indicated that the glycocalyx is a trichloro-acetic acid-soluble macromolecular complex of high molecular weight composed of a peptide moiety attached to large prosthetic groups (presumably carbohydrates) by O-glycosidic bonds. Analysis of the labeled apical plasma membrane components by agarose gel filtration and SDS-PAGE disclosed the presence of six major species of apparent molecular weights: 23,000, 28,000, 37,000, 44,000, 68,000, and 95,000. More than half of the membrane-associated radio-iodine was in two bands of molecular weights 37,000 and 44,000.Concentrations of vasopressin and cyclic AMP sufficient to increase the SCC significantly did not modify the extent of membrane labeling or the distribution of the label among the apical membrane components (presumably proteins) as assessed by SDS-PAGE. Iodination in the presence of amiloride inhibited incorporation but did not change the pattern of the distribution of the label among the components resolved by SDS-PAGE.Iodination of basal-lateral plasma membranes, at a yield comparable to that obtained with apical labeling, was attained after about 30 min of exposure of the intact bladder to the labeling solutions. Approximately 25% of the basal-lateral labeling was lost when the epithelial cells were harvested after collagenase treatment, implying that some iodination of the basement membrane had taken place. Less than 10% of iodination of the apical or basal-lateral surfaces was accounted for by lipid-labeling. Analysis of the labeled apical and basal-lateral species by enzymatic digestion and thin layer chromatography disclosed that virtually all the radioactivity was present as mono-iodotyrosine (MIT).     

Exocytotic events unrelated to regulation of water permeability in amphibian tight epithelia: effects of oxytocin, PMA and insulin on membrane capacitance, water and Na+ transport

We measured the effects of oxytocin on capacitance and hydroosmotic water flow in the urinary bladder of the toad Bufo marinus and the skins of Rana pipiens and Rana temporaria. Oxytocin increased capacitance in all these tissues but stimulated hydroosmotic water flow only in the urinary bladder. We also measured the effects of oxytocin and PMA on the capacitance and hydroosmotic water flow of the toad urinary bladder. Both agents produced increases in membrane capacitance that were additive, however, PMA produced a stimulation of water flow that was only a fraction of that caused by oxytocin. Comparison of the effects of PMA and insulin in toad urinary bladder showed that in contrast with PMA, insulin did not increase membrane capacitance in this tissue. Moreover, insulin stimulated Isc in the urinary bladder while PMA produced an inhibition of variable magnitude. These results suggest that: (1) oxytocin can promote the fusion with the apical membrane of cytoplasmic membranes with or without water channels; (2) oxytocin and PMA stimulate the fusion with the apical membrane of cytoplasmic membranes originating in different pools; membranes in each pool have different water permeabilities and their insertion is controlled by different signals; (3) PMA and insulin act through different mechanisms in the toad urinary bladder.     

Quantitative analyses of the constituent membranes of parotid acinar cells and of the changes evident after induced exocytosis

Summary A morphometric study has been made at the EM level of Isoproterenol (IPR) induced secretion of rabbit parotid glands in vivo. Emphasis has been placed here on the membrane content of acinar cells and the changes which occur following induced degranulation. In particular it was hoped to establish whether the preservation of zymogen granule membrane as intact electron microscopically visible subunits and the subsequent reutilisation of this membrane is a plausible hypothesis from a quantitative morphological standpoint.After two hours IPR had caused >95% depletion of granules. About 1343 m²/cell of granule limiting membrane temporarily fused with the apical plasmalemma during this time and by two hours 1158 m²/cell of this had been eliminated. Only a small increase in intracellular smooth membrane area was recorded after degranulation and we find no evidence that the zymogen granule membrane is stored indefinitely as smooth membrane fragments either in the region of the Golgi apparatus or elsewhere in the cytoplasm.IPR caused changes in RER membrane area (+37.7%, 1406 m²/cell), which is a possible, but we consider implausible relocation site of granule membrane.The possible mechanism of the removal of excess apical membrane and the ultimate fate of the zymogen granule membrane is discussed.