Showing papers on "Secretion published in 1986"

Secretion of Metalloproteinases by Stimulated Capillary Endothelial Cells

Abstract: Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.

Two roles for guanine nucleotides in the stimulus-secretion sequence of neutrophils.

Abstract: The term ’stimulus-secretion coupling‘ has, since first enunciated1, been held to involve the mobilization of cytosol Ca2+, which in turn is sufficient to trigger exocytotic secretory processes in metabolically competent cells. However, recent studies on a wide range of secretory cell types indicate that a role for Ca2+ can be obviated: examples are stimulation with phorbol ester (phorbol myristate acetate, PMA)2,3 which causes the activation of protein kinase C4 or the stimulation of platelets with collagen5. Ca2+-independent exocytosis also occurs when analogues of GTP are injected through the lumen of patch pipettes directly into the cytosol of mast cells6. The results presented here suggest that GTP analogues can activate secretory processes by actions at two distinct locations: one may be at the level of the receptor7 involving the activation of polyphos-phoinositide (PPI) phosphodiesterase8 with consequent liberation of diacylglycerol (DG)9; the other involves direct activation of the exocytotic mechanism. These conclusions are based on measurements of exocytotic secretion from permeabilized neutrophils into which we have been able to introduce, individually and in combination, Ca2+ chelators (EGTA and BAPTA), Ca2+ (buffered at micromolar concentrations with EGTA), analogues of GTP and GDP and the direct activator of protein kinase C, PMA.

Insulin-like growth factor I action on rat anterior pituitary cells: suppression of growth hormone secretion and messenger ribonucleic acid levels.

Abstract: Somatomedin preparations have previously been shown to suppress GH release. The effects of a synthetic recombinant human insulin-like growth factor analog (IGF-I; Thr 59) were, therefore, tested on long term GH secretion by male rat pituitary monolayer cell cultures grown in serum-free defined medium. IGF-I (3.25 nm) maximally suppressed basal GH secretion for up to 72 h by 66%, with an ED50 of 0.1 nm. Human pancreatic GRF-(l–44) (GHRH; 1 nm) stimulated GH secretion by 230% during 72 h. IGF-I (0.13 nm) suppressed GHRHstimulated GH secretion by 30% (P < 0.005). IGF-I (0.625 nm) completely abolished stimulation of GH by GHRH (1 nm), while higher doses of IGF-I (up to 6.5 nm) actually suppressed GH secretion even in the presence of GHRH (up to 1 nm). The depletion of intracellular GH levels in GHRH-treated cells was reversed by IGF-I (3.25 nm). PRL secretion was not altered in the same cells by IGF-I. Equivalent doses of epidermal growth factor and fibroblast growth factor did not alter basal or GHRHstimulat...

Identification of Cellular Activation Mechanisms Associated with Salivary Secretion

Abstract: In recent years, our understanding of receptor-signalling mechanisms in the salivary glands has advanced considerably. Two receptor pathways exist, one involving cAMP, which primarily regulates enzyme secretion, and another involving the hydrolysis of PIP2, which regulates Ca2+ mobilization and, subsequently, monovalent ion fluxes probably important in ion and water secretion in the intact gland. Mobilization of Ca2+ results from both the release of internal Ca2+, and from Ca2+ entry from the extracellular space. The signal for Ca2+ release appears to be (1,4,5)IP3, one of the water soluble products of PIP2 hydrolysis. The mechanism controlling Ca entry is not understood, but speculation abounds. Hydrolysis of PIP2 also produces DG, which has a messenger role in activating a specific protein kinase, the C kinase. The C kinase interacts with Ca2+ mobilization in some as yet uncharacterized way in regulating enzyme secretion.

Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide

Abstract: The presurface (preS) proteins of hepatitis B virus are structural components of the viral envelope that may play important roles in virion assembly and infectivity. They are specified by a large open reading frame that includes the coding region for the major surface (S) protein in its 3' half. Translation of the preS proteins initiates upstream from the S region, giving rise to proteins that are composed of the S domain and an additional 163 (preS1) or 55 (preS2) amino acids. Little is known about the biosynthesis and assembly of these proteins. The expression of the S and preS1 proteins was examined by transfecting cultured mammalian cells with viral DNA and injecting synthetic messenger RNA's into Xenopus oocytes. In contrast to the proteins encoded by the S region, the preS1 proteins are not detectably secreted into the culture medium. Furthermore, when the S and preS1 proteins are synthesized together, secretion of the S proteins is specifically and strongly inhibited. The results suggest a unique molecular interaction during secretion of the S and preS proteins that may be important for virus assembly.

Clathrin-coated vesicular transport of secretory proteins during the formation of ACTH-containing secretory granules in AtT20 cells.

Abstract: We have studied by electron microscopy and immunocytochemistry the formation of secretory granules containing adrenocorticotropic hormone (ACTH) in murine pituitary cells of the AtT20 line. The first compartment in which condensed secretory protein appears is a complex reticular network at the extreme trans side of the Golgi stacks beyond the TPPase-positive cisternae. Condensed secretory protein accumulates in dilated regions of this trans Golgi network. Examination of en face and serial sections revealed that "condensing vacuoles" are in fact dilations of the trans Golgi network and not detached vacuoles. Only after presumptive secretory granules have reached an advanced stage of morphological maturation do they detach from the trans Golgi network. Frequently both the dilations of the trans Golgi network containing condensing secretory protein and the detached immature granules in the peri-Golgi region have surface coats which were identified as clathrin by immunocytochemistry. Moreover both are the site of budding (or fusion) of coated vesicles, some of which contain condensed secretory protein. The mature granules below the plasma membrane do not, however, have surface coats. Immunoperoxidase labeling with an antiserum specific for ACTH and its precursor polypeptide confirmed that many of the coated vesicles associated with the trans Golgi network contain ACTH. The involvement of the trans Golgi network and coated vesicles in the formation of secretory granules is discussed.

Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung.

Abstract: The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent Multivesicular bodies and some small apical vesicles in type II cells were also labeled Secondary lysosomes of alveolar macrophages were immunoreactive Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum These observations clarify the organelles and pathways utilized in the elaboration of surfactant After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies Both lipids and proteins are present in tubular myelin Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid The role of these proteins in bronchiolar function is unknown

Some characteristics of histamine secretion from rat peritoneal mast cells stimulated with nerve growth factor.

Abstract: Nerve growth factor (NGF) isolated from mouse submandibular gland or from snake venom produced a dose-dependent release of histamine from isolated rat peritoneal mast cells. The response was almost totally dependent on the presence of extracellular calcium ions and on added phosphatidylserine or its lyso-derivative. At high concentrations, strontium ions could substitute for calcium. The process was non-cytotoxic, relatively slow, pH dependent and blocked by polyclonal antibodies to NGF. Binding of NGF to the mast cell was not dependent on added calcium. The release was unaffected by low molecular weight glucose polymers or specific quaternary ammonium salts and thus differed from that evoked by clinical dextran or polyamines. The release was not inhibited by soluble rat IgE or IgG and was unimpaired in mast cells recovered from specific pathogen free rats. As such it did not appear to be mediated through interaction with cell-fixed antibodies. The process further differed from anaphylactic histamine release in that there was no accompanying change in the intracellular level of adenosine 3',5'-cyclic monophosphate (cyclic AMP), the activated state induced by NGF was much more persistent than that evoked by antigen, and there was no cross-desensitization between the two latter stimuli. In total, these data suggest that NGF may induce secretion from rat mast cells by interaction with a specific receptor on the plasma membrane, possibly similar to that present on sensory and sympathetic neurones.

Bacterial lipopolysaccharides prime macrophages for enhanced release of arachidonic acid metabolites.

Abstract: Preincubation of resident peritoneal macrophages with 10-100 ng/ml LPS for 60 min resulted in the cells becoming primed for enhanced (three-to eightfold higher) arachidonic acid (20:4) secretion in response to a variety of triggers. The half-maximal concentration of LPS required for priming was 10 ng/ml irrespective of whether the trigger was particulate (examples: zymosan or immune complexes) or soluble (such as PMA or A23187). Similarly, the time required for half-maximal priming of macrophages was 20 min irrespective of which trigger was used. The primed state persisted for at least 30 h. LPS-priming of macrophages also affected the kinetics of 20:4 metabolite secretion. The lag phase characteristically observed when 20:4 secretion is triggered was reduced in LPS-primed cells. Furthermore, LPS-primed cells secreted 20:4 metabolites when challenged with latex beads, while unprimed cells did not. These data suggest that stimuli such as zymosan, which elicit 20:4 secretion in macrophages, promote two signals, a priming signal and a triggering signal. LPS is capable of establishing the priming signal but not the triggering signal, while latex promotes the triggering signal but is unable to prime the cells for 20:4 release. LPS did not effect the profile of 20:4 metabolites secreted in response to any of the triggers, nor did it effect the profile of products synthesized from exogenously added 20:4, suggesting that it did not regulate the 20:4 cascade at the level of either the cyclooxygenase or lipoxygenase pathways. Macrophages respond to LPS without the intervention of T lymphocytes, since the macrophages from nude mice could be primed for enhanced 20:4 secretion.

Intracellularly injected tetanus toxin inhibits exocytosis in bovine adrenal chromaffin cells

Abstract: The clostridial neurotoxins tetanus and botulinum toxin type A are known to block transmitter release from nerve terminals1–3, probably by interfering with some essential process controlling exocytosis3,4 after the entry of Ca2+ ions Although exocytosis occurs in many secretory cells, these toxins show a high specificity for neurones and the secretory response of cultured bovine adrenal medullary cells is not inhibited by exposure to medium containing tetanus or botulinum toxin type A (although it is by botulinum toxin type D)4 Here we report that when tetanus toxin and botulinum neurotoxin type A are injected intracellularly into chromaffin cells they strongly inhibit secretion, as revealed by the measurement of cell capacitance5 These results indicate that these toxins are normally ineffective in chromaffin cells because they are not bound and internalized, so do not reach their site of action Furthermore, we have localized the secretion-blocking effects of the toxin to a fragment comprising the light chain covalently linked to part of the heavy chain, suggesting that this part of the molecule contains the active site

Chloroquine and ammonium chloride prevent terminal glycosylation of immunoglobulins in plasma cells without affecting secretion.

Abstract: The generation of an acidic pH in intracellular organelles is required for several membrane and protein recycling processes. For instance, the internalization of ligands by receptor-mediated endocytosis is followed by the development of an acidic pH inside endosomes; this allows dissociation of the ligand, which is then transported to the lysosomes, from the receptor, which is recycled to the cell surface. There is evidence that part of this recycling process involves the distal region of the Golgi complex, where terminal glycosylation occurs: when the plasma membrane transferrin receptor is desialylated by neuraminidase treatment, it acquires new sialic acid molecules after endocytosis and before cell-surface re-expression. Golgi membranes have been shown to contain a proton pump and the distal Golgi cisternae appear to have an acidic content. Here, we have studied the effects of chloroquine and ammonium chloride, which raise the pH of acidic intracellular compartments, on the processing and secretion of immunoglobulins by plasma cells. Sialic acid transfer to terminal galactose residues, a reaction known to occur in the distal Golgi shortly before secretion, is completely and rapidly inhibited in the presence of these drugs, without significant modification of the secretion rate. This effect is accompanied by a dilatation of the Golgi cisternae and is not rapidly reversible.

Long-term maintenance of hepatocyte functional activity in co-culture: Requirements for sinusoidal endothelial cells and dexamethasone

Abstract: Sinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co-culture system, the effect of sinusoidal liver cells on hepatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co-cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of alpha 1-fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co-cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte-stabilizing activity was also produced by a pulmonary endothelial cell line.

Toxoplasma Modifies Macrophage Phagosomes by Secretion of a Vesicular Network Rich in Surface Proteins

Abstract: Modification of macrophage phagosomes begins shortly after formation as Toxoplasma cells secrete membranous vesicles that form a reticulate network within the vacuole. The Toxoplasma-modified compartments then resist normal endocytic processing and digestion. We have used the pronounced Ca++-dependent stability of the intraphagosomal membrane (IPM) network to purify and characterize the structural proteins of this assembly. In addition to the structural matrix, Toxoplasma secretes a discrete set of soluble proteins, including a newly described 22-kD calcium-binding protein. The IPM network adheres to intact Toxoplasma cells after host cell lysis in the presence of 1 mM Ca++; however, the network readily disperses in calcium-free buffer and was purified as vesicles that sedimented at 100,000 g. Purified IPM vesicles were specifically recognized by immune sera from mice with chronic Toxoplasma infection and consisted primarily of a 30-kD protein when analyzed by SDS PAGE. IPM network proteins share a major antigenic component located on the surface of extracellular Toxoplasma cells as shown by immunoperoxidase electron microscopy using a polyclonal antibody prepared against the IPM vesicles. Moreover, in Toxoplasma-infected macrophages, anti-IMP antibody confirmed that the extensive IPM array contains proteins also found on the Toxoplasma cell surface. Our results indicate the IMP network represents a unique structural modification of the phagosome comprised in part of Toxoplasma surface proteins.

8-Bromo-3',5'-adenosine monophosphate stimulates the endocrine activity of human cytotrophoblasts in culture.

Abstract: Cytotrophoblasts, purified from human term placentae, were cultured in the absence or presence of 8-bromo- cAMP or 8-bromo-cGMP. 8-Bromo-cAMP provoked a dose- dependent increase in the secretion of hCG and progesterone within 24 h. After 48 h, hCG secretion increased by more than 200-fold, and progesterone secretion increased nearly 5-fold. 8- Bromo-cGMP had no effect on hCG secretion. In culture in serum-supplemented medium, the mononuclear cytotrophd- blasts aggregated and fused to form syncytia. This morphological transformation was not affected by 8-bromo-cAMP. Immuno- cytochemical studies of the a- and /3-subunits of hCG in control and 8-bromo-cAMP-stimulated cultures demonstrated that the cyclic nucleotide analog promoted the synthesis of both subunits in all cellular forms, including single mononuclear cells, cell aggregates, and syncytia. In serum-free medium, the cytotroph- oblasts did not aggregate or form syncytia, yet they responded to 8-bromo-cAMP with ah increase in hCG secretion. We con- clude that the endocrine function of cytotrophoblasts can be stimulated by a cAMP-dependent mechanism which can be initiated independently of the formation of a syncytium. (

The Limitations to and Valid Use of C-Peptide as a Marker of the Secretion of Insulin

Abstract: The accuracy with which the secretion rate of insulin can be calculated from peripheral concentrations of C-peptide was investigated in conscious mongrel dogs. Biosynthetic human C-peptide and insulin were infused intraportally and their concentrations measured in the femoral artery. During steady-state infusions of C-peptide, the peripheral concentration changed in proportion to the infusion rate and the metabolic clearance rate (5.2 ± 0.3 ml/kg/min) remained constant over a wide range of plasma concentrations. Application of a two-compartment mathematical model, in which the model parameters were estimated from analysis of C-peptide decay curves after intravenous bolus injections, allowed the intraportal infusion rate of C-peptide to be derived from peripheral C-peptide concentrations, even under non-steady-state conditions. Estimates of the intraportal infusion rate based on this model were 102.4 ± 2.6% of the actual infusion rate as it was increasing and 102.3 ± 5.5% of this rate as it was falling. The peripheral C-peptide : insulin molar ratio was influenced by the rate at which equimolar intraportal infusions of C-peptide and insulin were changed. The baseline C-peptide : insulin molar ratio (4.1 ± 0.9) increased to peak values of 8.2 ± 0.6,10.3 ± 2.0, and 14.9 ±1.3 when the infusion rate was increased and then decreased rapidly. Peak values of only 5.7 ±1.2 were found if the intraportal infusion rate was changed slowly. In conclusion: (1) under steady-state conditions the secretion rate of insulin can be calculated as the product of the peripheral concentration of C-peptide and its MCR; (2) under non-steady-state conditions, however, application of more complex mathematical models, such as the two-compartment model used in the present study, allows insulin secretion rates to be accurately calculated at discrete time points; and (3) under non-steady-state conditions the C-peptide:insulin molar ratio is influenced not only by the extent of hepatic insulin extraction but by other factors, including the rate of change of insulin secretion and the clearance rate of C-peptide. Changes in this ratio should therefore not be assumed to reflect changes in hepatic insulin extraction.

Ultrastructural localization of bronchial antileukoprotease in central and peripheral human airways by a gold-labeling technique using monoclonal antibodies.

Abstract: The present report describes the ultrastructural localization of bronchial antileukoprotease (ALP) in human central and peripheral airways by using polyclonal as well as monoclonal ALP-specific antibodies in a two-step gold-labeling procedure. In the serous cells of bronchial glands, ALP could be demonstrated in secretory granules. These granules, among which 4 phenotypes could be distinguished morphologically in ultrathin sections, showed the following labeling patterns: phenotype I, which had an electron lucent, fine granular content, and phenotype II, which was homogeneously electron dense, both showed gold label over their entire area. The granules expressing zonal differences in electron density (phenotype III) showed only label in their electron-dense cores and the electron-lucent granules (phenotype IV) showed a minimal labeling. Sometimes gold particles could be observed in the rough endoplasmic reticulum and nuclear envelope, suggesting that ALP is present in these cell organelles. In the bronchiolar epithelium, ALP could be localized only in the secretory granules of Clara cells and goblet cells. These findings indicate that ALP is also synthesized in bronchioli. To our knowledge this is the first time that a well-defined protein has been described that is produced and secreted by human Clara cells.

Intracellular assembly and packaging of hepatitis B surface antigen particles occur in the endoplasmic reticulum.

Abstract: Hepatitis B surface antigen (HBsAg) particles are secreted by Chinese hamster ovary cells that are stably transfected with the S gene of hepatitis B virus. The assembly of HBsAg into cylindrical and spherical particles occurred intracellularly within the endoplasmic reticulum. HBsAg particles accumulated within large dilated areas of the endoplasmic reticulum and remained within these structures for most of the time prior to secretion from the cells. Once the particles were formed, the HBsAg polypeptides did not appear to become associated with subsequent intracellular organelle membranes or the plasma membrane. HBsAg within the dilated structures did not bind wheat germ agglutinin, indicating that its oligosaccharide chains had not yet been processed to the complex form (containing terminal sialic acid-N-acetylglucosamine residues). The oligosaccharide chains of HBsAg are processed to the complex form and can be detected on the HBsAg after secretion, but this event was not detected within cells. In addition, HBsAg was not observed on the cell surface by indirect immunofluorescence or immunoprecipitation, although immunoelectron microscopy revealed some staining at or near the cell surface. These results suggested that HBsAg was either secreted from cells without being incorporated into the plasma membrane, or that the levels of HBsAg in the plasma membrane were below the limits of detection.

Physiological mechanisms contributing to increased interleukin-1 secretion

Abstract: Interleukin-1 (IL-1) is a monocyte-derived polypeptide that mediates many host defense adaptations to environmental and infectious stresses. This investigation was intended to characterize further IL-1 activity found in human plasma following exercise (3) and to identify physiological initiators of IL-1 secretion. IL-1 activity was measured by the ability of plasma fractions to stimulate lymphocyte proliferation. This activity appeared in plasma several hours after exercise on a cycle ergometer (1 h at 60% of aerobic capacity, n = 8 subjects) and was neutralized with a specific antiserum to human IL-1. The hypothesis that IL-1 release from monocytes was initiated by phagocytosis of material from cells damaged by exercise was tested. The increase in IL-1 activity did not correlate significantly (r = 0.55) with creatine kinase activity, a marker for release of intracellular proteins into the circulation, and IL-1 secretion by monocytes was not stimulated by incubation with red blood cell lysates in vitro. Thus the stimulus for IL-1 secretion did not appear to be related to a scavenging function of monocytes. The possibility that IL-1 secretion may be mediated by stress hormones associated with exercise was examined. IL-1 secretion by monocytes was increased up to 48 +/- 18% (P less than 0.01) by addition of physiological concentrations of epinephrine in vitro. Low concentrations of hydrocortisone (1 ng/ml) also augmented IL-1 secretion by 58 +/- 20%. Higher concentrations in the physiological range had no effect, and combinations of epinephrine and hydrocortisone suppressed IL-1 secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of secretory protein translocation: ribosome-membrane interaction in endoplasmic reticulum.

Abstract: Secretory proteins are synthesized on ribosomes bound to the membrane of the endoplasmic reticulum (ER). After the selection of polysomes synthesizing secretory proteins and their direction to the membrane of the ER via signal recognition particle (SRP) and docking protein respectively, the polysomes become bound to the ER membrane via an unknown, protein-mediated mechanism. To identify proteins involved in protein translocation, beyond the (SRP-docking protein-mediated) recognition step, controlled proteolysis was used to functionally inactivate rough microsomes that had previously been depleted of docking protein. As the membranes were treated with increasing levels of protease, they lost their ability to be functionally reconstituted with the active cytoplasmic fragment of docking protein (DPf). This functional inactivation did not correlate with a loss of either signal peptidase activity, nor with the ability of the DPf to reassociate with the membrane. It did correlate, however, with a loss of the ability of the microsomes to bind ribosomes. Ribophorins are putative ribosome-binding proteins. Immunoblots developed with monoclonal antibodies against canine ribophorins I and II demonstrated that no correlation exists between the protease-induced inability to bind ribosomes and the integrity of the ribophorins. Ribophorin I was 85% resistant and ribophorin II 100% resistant to the levels of protease needed to totally eliminate ribosome binding. Moreover, no direct association was found between ribophorins and ribosomes; upon detergent solubilization at low salt concentrations, ribophorins could be sedimented in the presence or absence of ribosomes. Finally, the alkylating agent N-ethylmaleimide was shown to be capable of inhibiting translocation (beyond the SRP-docking protein-mediated recognition step), but had no affect on the ability of ribosomes to bind to ER membranes. We conclude that potentially two additional proteinaceous components, as yet unidentified, are involved in protein translocation. One is protease sensitive and possibly involved in ribosome binding, the other is N-ethylmaleimide sensitive and of unknown function.

Ca2+ and Cyclic AMP in Regulation of Intestinal Na, K, and Cl Transport

Abstract: This review is an update of the current understanding of how Ca2+ and cAMP regulate mammalian small intestinal and colonic electrolyte transport. We review the transport processes present in plasma membranes of absorptive and secretory epithelial cells and the way these processes are affected by Ca2+ and cAMP. Studies of intestinal electrolyte transport are complicated by several factors, including the presence, and often simultaneous function, of both absorptive and secretory processes, and by the presence of multiple cell types. A cultured cell line that retains the electrolyte transport properties of the intact tissue would greatly simplify investigation. However, to date neither a transporting small intestinal nor an intestinal absorptive cell line has been developed. A CI­ secreting human colonic epithelial cell line, named T-84, has been reported (1 �-17), but these studies must be interpreted with the realization that such cells do not represent the normal colon, but a cancer cell line. It is generally believed that electrolyte absorption and secretion are carried out by two separate epithelial cell types (38). The absorptive cells are thought to be present only on the villus of small intestine and on the surface of the colon, while the secretory cells are mostly present in the crypts.

The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli.

Abstract: As a first step in the detailed analysis of the mechanism of secretion of haemolysin, we sought to identify sequences or domains within haemolysin A (HlyA) that are essential for its secretion. For this purpose we examined the properties of a deletion and Tn5 insertions into the region of theHlyA gene encoding the C-terminal part of the protein, since both of these are relatively simple to generate. We showed that removal of 27 amino acids from the C-terminus of HlyA is sufficient to inhibit secretion drastically, although the residual polypeptide is still haemolytically active. Cellular fractionation studies showed that haemolytic activity does not accumulate in large amounts within the periplasmic space during normal secretion. More significantly, activity does not appear to accumulate within this compartment when the export functionshlyB andhlyD are removed. These results are consistent with a mechanism in which interaction of the C-terminus of HlyA with the secretion machinery, located in the inner membrane, is followed by direct transfer of haemolysin to the medium.

Secretion of endogenous and exogenous proteins from polarized MDCK cell monolayers.

Abstract: Confluent monolayers of MDCK (Madin-Darby canine kidney) cells provide a widely used system to study the biogenesis of epithelial cell polarity. We now report that these cells are also capable of the vectorial constitutive secretion of a major endogenous product, a glycoprotein of 81 kDa, which is released into the medium from the apical surface within 30 min of its synthesis. This release represents a bona fide exocytotic secretory process and is not the result of proteolytic cleavage of a plasma membrane-associated precursor since, in cells treated with chloroquine, a protein indistinguishable from the mature secretory product accumulated intracellularly. In contrast to the vectorial secretion of the endogenous product, a variety of exogenous exocrine and endocrine proteins synthesized in MDCK cells transfected with the corresponding genes were secreted from both the apical and basolateral surfaces. These included proteins such as rat growth hormone, chicken oviduct lysozyme, bovine gastric prochymosin, and rat salivary gland alpha 2u-globulin, which in their cells of origin are secreted via a regulated pathway, as well as the liver form of the alpha 2u-globulin and the immunoglobulin kappa chain, which are normally released constitutively. These results demonstrate the existence of secretory pathways that lead to both surfaces of MDCK cells and are accessible to the foreign secretory products. They are consistent with the operation of a sorting mechanism in which the polarized secretion of the endogenous product is effected through the recognition of signals that prevent its random distribution within the fluid phase in the cellular endomembrane system.