Membrane interactions between adjacent mucols secretion granules

In primate goblet cells, the membranes of adjacent mucous granules from contact areas which appear as extensive pentalaminar fusion sites in thin sections. In freeze-fracture replicas, the same membrane areas are smooth, except for a few 6-8-nm particles which adhere to the E face. These protein-poor membrane interaction sites are relatively long-lived, and it is proposed that further stimulus may be required to trigger membrane fission.     

Ionic currents and secretion in the exocrine glands

Exocrine glands extrude both proteins and salt. Fluid secretion is related to a modification of the membrane permeability of secreting cells. This permeability change may be measured as an increase of labelled ion fluxes or as a rise of membrane conductance. It involves Na+, K+, Cl- and Ca2+ ions. Intracellular Ca2+ acts as "second messenger" in the development of the electrical response. Recent recordings using the "patch-clamp" technique have revealed three types of ion channel activated by secretory agents. These channels are sensitive to internal Ca2+ ions. They are respectively selective to K+, Cl- and positively charged monovalent ions. Two models suggesting possible roles for these channels in the secretion process are presented. However, evaluation of such models is presently restricted by numerous uncertainties on the function of secreting cells in vivo. Information is notably lacking concerning the exact composition of the secreted fluid, and the exchanges between exocrine glands and blood circulation.     

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.     

Membrane detachment and release of the major membrane glycoprotein of secretory granules in rat pancreatic exocrine cells

The major glycoprotein of pancreatic zymogen granule membranes (GP-2) was detected in the medium of acinar cell suspensions from rat pancreas. Its release from the cells was studied in pulse-chase metabolic labeling experiments with radioactive methionine. GP-2 (apparent Mr = 80 000) was found to be processed to a form of slightly lower apparent Mr (75 000) after about 4 h chase. At about the same time this smaller form of GP-2 appeared in the medium. These results are in accordance with earlier findings in vivo. At different chase times acinar cells were extracted with Triton X-114 to separate water-soluble proteins from membrane-associated (hydrophobic) proteins. This experiment showed that GP-2 is slowly converted from a membrane-bound glycoprotein to a soluble glycoprotein after its reduction in apparent molecular mass, causing its detachment from the membrane. Further analysis indicated that the detachment process may occur at the zymogen granule membrane as well as the plasma membrane. Immunocytochemistry on ultrathin cryosections of pancreatic tissue showed that GP-2 is localized on zymogen granule membranes, plasma membranes and in the acinar lumen. Although in much smaller quantities, GP-2 is also present in the granule content. Thus, in summary, GP-2 is synthesized as a true membrane glycoprotein which is gradually processed to a soluble species and is found in the secretion.     

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.     

Ca-dependent K channels in exocrine salivary glands

In the last 15 years, remarkable progress has been realized in identifying the genes that encode the ion-transporting proteins involved in exocrine gland function, including salivary glands. Among these proteins, Ca²⁺-dependent K⁺ channels take part in key functions including membrane potential regulation, fluid movement and K⁺ secretion in exocrine glands. Two K⁺ channels have been identified in exocrine salivary glands: (1) a Ca²⁺-activated K⁺ channel of intermediate single channel conductance encoded by the KCNN4 gene, and (2) a voltage- and Ca²⁺-dependent K⁺ channel of large single channel conductance encoded by the KCNMA1 gene. This review focuses on the physiological roles of Ca²⁺-dependent K⁺ channels in exocrine salivary glands. We also discuss interesting recent findings on the regulation of Ca²⁺-dependent K⁺ channels by protein–protein interactions that may significantly impact exocrine gland physiology.     

Noc-king out exocrine and endocrine secretion

The Rab GTPase effector Noc2 was brought into the limelight by a recent publication that demonstrated its requirements at different stages of regulated exocytosis. Noc2 knockout resulted in distinct abnormalities in endocrine and exocrine cells, ranging from the accumulation of secretory granules of increased size to impairments in the regulated release of their secretory products. Explanations for these defects are beginning to emerge and they promise to reveal some of the most jealously kept secrets of regulated exocytosis.     

Recretion of enzymes and hormones by exocrine glands

The article reviews a poorly explored issue of secretive physiology-recretion from blood by glandulocites of various endocrinal glands of hydrolytic ferments and hormones that have been synthesized by digestive and endocrinal glands. The article discusses potential physiological role of the recretion function and the diagnostic significance of information obtained from analysis of recreted ferments and hormones in exosecretions.     

Changes in the size and number of secretion granules in the rat exocrine pancreas induced by feeding or stimulation in vitro

Summary The size, number and volume per cell of secretion granules in rat exocrine pancreas have been measured using stereological techniques. The changes which occur as a result of feeding starved animals (90 min) or stimulating lobular fragments in vitro with carbachol are documented. In fasted animals mean acinar cell volume was estimated as 1670 m³ and the cells contained an average of around 450 secretion granules with a corrected mean diameter of 0.70 m. They occupied around 7% of cell volume. After feeding mean cell volume was about 1300 m³ and the cells contained an average of about 190 granules per cell with a mean diameter of 0.58 m. They occupied 3% of cell volume. A shift in the size frequency distribution of granule diameters occurred as a result of feeding. In vitro experiments in which lobules were induced to secrete with carbachol (10M, 3 h, 37° C) had a similar effect. Mean cell volume was reduced from around 1760 m³ to 1360 m³, mean granule number from around 420 per cell to 180 per cell and the volume density of granules was reduced from about 8% to 3% of cell volume. There was no significant change in mean granule diameter or shift in the size-frequency distribution of granule diameters. Incubation of tissues with cycloheximide (1 mM, 3 h, 37° C) did not prevent secretion by carbachol but it prevented replacement of granules. As a consequence, depletion by carbachol was greater in the presence of cycloheximide, the granules being reduced to around 110 per cell and to only 2.5% of cell volume. We conclude that feeding causes a preferential loss of larger granules and that during secretion replacement of granules occurs. Some of these granules are smaller than those evident in the glands of starved animals.     

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.     

Neurotensin stimulates pancreatic exocrine secretion in rats

Neurotensin (NT) stimulates pancreatic exocrine secretion in dogs and humans. The purpose of this study was to examine the effect of exogenous neurotensin on pancreatic exocrine secretion in rats. Five Sprague-Dawley male rats were prepared with pancreatic, gastric and duodenal fistulas. Bile was shunted into the duodenum in order to collect pure pancreatic juice. 24 h later, neurotensin (0.05, 0.1, 0.2, 0.3, 1.0 nmol/kg) was infused intravenously in a random fashion. Pancreatic juice was collected every 10 min, and the volume was recorded and protein and bicarbonate were measured. Neurotensin stimulated, in a dose-related manner, the pancreatic secretion of water, protein and bicarbonate. Neurotensin may be involved in the physiologic control of pancreatic secretion in rats.     

Immunolocalization of potassium-chloride cotransporter polypeptides in rat exocrine glands

Potassium-chloride cotransporters (KCCs) encoded by at least four homologous genes are believed to contribute to cell volume regulation and transepithelial ion transport. We have studied KCC polypeptide expression and immunolocalization of KCCs in rat salivary glands and pancreas. Immunoblot analysis of submandibular, parotid, and pancreas plasma membrane fractions with immunospecific antibodies raised against mouse KCC1 revealed protein bands at ca 135 kDa and ca 150 kDa. Immunocytochemical analysis of fixed salivary and pancreas tissue revealed basolateral KCC1 distribution in rat parotid and pancreatic acinar cells, as well as in parotid, submandibular, and pancreatic duct cells. KCC1 or the polypeptide product(s) of one or more additional KCC genes was also expressed in the basolateral membranes of submandibular acinar cells. Both immunoblot and immunofluorescence signals were abolished in the presence of the peptide antigen. These results establish the presence in rat exocrine glands of KCC1 and likely other KCC polypeptides, and suggest a contribution of KCC polypeptides to transepithelial Cl(-) transport.     

Electrical correlates of secretion in endocrine and exocrine cells

Many types of secretory cells including neurons and cells of endocrine and exocrine glands show changes in electrical potential and resistance when secretion is stimulated. These electrical correlates result from the movement of ions across the cell membrane through specific ion-selective channels. In neurons and certain endocrine cells (such as pancreatic beta cells and certain cells of the anterior pituitary), these channels are voltage dependent and open transiently upon depolarization leading to action potentials. Thus some endocrine cells are electrically excitable, a property previously held to occur only in nerve and muscle. In other nonexcitable endocrine and exocrine cells (such as the pancreas and parotid), ion channels are responsive to either occupancy of specific membrane receptors or changes in intracellular metabolites and second messengers. Ion fluxes through these latter channels also lead to changes in the electrical potential and resistance, but these changes are generally more sustained and action potentials are not seen. The entry of Ca2+ through both voltage-dependent and voltage-independent ion channels plays a major role in the activation of secretion via exocytosis.     

Post-translational amino acid racemization in the frog skin peptide deltorphin I in the secretion granules of cutaneous serous glands

The dermal glands of the South American hylid frog Phyllomedusa bicolor synthesize and expel huge amounts of cationic, alpha-helical, 24- to 33-residue antimicrobial peptides, the dermaseptins B. These glands also produce a wide array of peptides that are similar to mammalian hormones and neuropeptides, including a heptapeptide opioid containing a D-amino acid, deltorphin I (Tyr-DAla-Phe-Asp-Val-Val-Gly NH2). Its biological activity is due to the racemization of L-Ala2 to D-Ala. The dermaseptins B and deltorphins are all derived from a single family of precursor polypeptides that have an N-terminal preprosequence that is remarkably well conserved, although the progenitor sequences giving rise to mature opioid or antimicrobial peptides are markedly different. Monoclonal and polyclonal antibodies were used to examine the cellular and ultrastructural distributions of deltorphin I and dermaseptin B in the serous glands by immunofluoresence confocal microscopy and immunogold-electron microscopy. Preprodeltorphin I and preprodermaseptins B are sorted into the regulated pathway of secretion, where they are processed to give the mature products. Deltorphin I, [l-Ala2]-deltorphin I and dermaseptin B are all stored together in secretion granules which accumulate in the cytoplasm of all serous glands. We conclude that the L- to D-amino acid isomerization of the deltorphin I occurs in the secretory granules as a post-translational event. Thus the specificity of isomerization depends on the presence of structural and/or conformational determinants in the peptide N-terminus surrounding the isomerization site.     

Calcium release and internal calcium regulation in acinar cells of exocrine glands

Conclusion Recent results call for a reinterpretation of the mechanisms underlying the recruitment of intracellular Ca²⁺ in exocrine glands. One new hypothesis suggested by these developments is that InsP₃-sensitive channels liberate Ca²⁺ ions from secretory vesicles, as illustrated in Fig. 5.     

Distribution of S-100 protein and its subunits in bovine exocrine glands

 The distribution of S-100 protein and its α- and β-subunits in bovine exocrine glands was studied by indirect immunohistochemistry. The entire spectrum of salivary glands, glands of the respiratory tract, intestinal glands, male and female genital glands, and skin glands was examined. S-100 and its β-subunit were identified in most serous secretory cells of mixed salivary glands, although secretory acini in some serous glands remained unreactive for these antigens. Mucous cells were constantly negative; mucoid cells were positive in the lacrimal and Harderian gland. The α-subunit of S-100 protein was identified in serous cells but the staining reaction was faint. Subunits of S-100 showed a characteristic distribution along the excretory duct systems of compound glands: S-100 and the β-subunit were present in intercalated duct epithelium, while striated duct epithelium stained for S100-α. Therefore, it is suggested that S100-α is related to resorption and secretion in striated ducts, while S100-β may govern acinar exocytosis and probably regulates proliferation and differentiation of glandular cells. Differing staining intensities for S-100 and its subunits in secretory cells of exocrine glands most probably indicate functional differences with regard to secretory activity and the cell cycle. Accepted: 11 February 1997     

Postnatal development of biliary and pancreatic exocrine secretion in piglets

1. The secretory responses of bile and exocrine pancreas were studied in various aged piglets. 2. At 3 days old the bile and exocrine pancreas could be reacted by various stimulations. The response by secretin was the same as that in the 28 day old. 3. Protein concentration in pancreatic juice by CCK-8 increased steeply after 6 days old, but the ratio of amylase to protein rose abruptly at 28 days old. 4. These findings indicate that (1) the secretory capacity of bile and pancreatic juice developed predominantly at an early period of postnatal life; (2) the formation of bile acids and pancreatic digestive enzymes developed gradually during the suckling period.