More informative abstracts revisited

Recent progress in understanding the luminal biochemistry of regulated pancreatic exocrine secretion, including acid-base interactions between acinar and duct cells and pH-dependent processes that regulate membrane trafficking (endocytosis) at the apical plasma membrane, have led to the development of in vitro models of cystic fibrosis in the rat exocrine pancreas. Based on investigations in these model systems, a unifying hypothesis is presented that proposes that pancreatic dysfunction in cystic fibrosis occurs as a result of progressive acidification of the acinar and duct lumen, which leads to secondary defects in (i) apical trafficking of zymogen granule membranes and (ii) solubilization of secretory (pro)enzymes. By directly acidifying the pH of the acinar lumen in cholescystokinin-stimulated acini, the early cytological findings observed in cystic fibrosis, including (i) massive dilatation of the acinar lumen, (ii) decreased appearance of zymogen granules, (iii) loss of the apical pole of the acinar cell, and (iv) persistent aggregation of secretory (pro)enzymes released into the luminal space, have been reproduced in primary cultures of pancreatic tissue.     

Apical endocytosis of lectins by the absorptive cells of the suckling rat jejunum in vivo

Apical endocytosis in the absorptive cells of the suckling rat jejunum was examined in vivo using the intraluminal injection of a range of different lectin-horseradish peroxidase (HRP) conjugates. Con A-HRP, PNA-HRP, LCA-HRP and RCA120-HRP bound strongly to components of the glycocalyx on the plasma membrane of the microvilli, apical coated pits and the small tubular pits at the base of the microvilli of the absorptive cells. The lectin-conjugates on the plasma membranes were endocytosed from the coated pits and small tubular pits, and then transported into coated vesicles, vesicles and small tubules. Lectin-HRP conjugates were later found within the intercellular space. In contrast, SBA-HRP and DBA-HRP bound weakly to the plasma membranes. The absorptive cells demonstrated little uptake or transepithelial transport. When WGA-HRP was injected into the intestinal lumen in vivo, the jejunum absorptive cells formed a deep and wide apical invagination at the base of the microvilli. WGA-HRP bound strongly to the components of the glycocalyx on the plasma membranes of the microvilli, the deep and wide apical invaginations, the apical coated pits and the small tubular pits. The WGA-HRP conjugate was also found in the coated vesicles, tubules, early endosomes, late endosomes, multivesicular bodies and lysosomes, and within the intercellular space. The lectin-HRP conjugate on the plasma membranes however was almost entirely transported into the early endosomes, late endosomes, multivesicular bodies and lysosomes. Therefore, lectin-HRP conjugates may bind to the glycocalyx component on the apical membrane domains, thus resulting in different membrane formations of apical endocytosis by adsorption to the apical plasma membrane specific glycoconjugates in the absorptive cells of the suckling rat jejunum. In addition, WGA induces deep and wide invaginations in the dynamic (not static) membrane domain of the apical plasma membrane in the suckling rat jejunum in vivo.     

Endocytosis in renal proximal tubules. Experimental electron microscopical studies of protein absorption and membrane traffic in isolated, in vitro perfused proximal tubules

The present study provides at least partly answers to some of the questions outlined in the introduction (see also Figs. 2 and 3): Endocytosis and intracellular transport of ferritin, HRP and insulin tracers (125I-insulin, native insulin and insulin-gold) was followed by use of EM-autoradiography, immunocytochemistry and cytochemistry. Proteins are internalized into endocytic vacuoles and transferred to the lysosomes for degradation. Tracers were not transferred to the Golgi apparatus. 125I-insulin is internalized by specific receptor mediated endocytosis from the apical plasma membrane, substantiating the hypothesis that specific endocytosis receptors are responsible for reabsorption of certain proteins. The binding sites are localized in endocytic invaginations and in the microvillus membrane. The binding sites in the invaginations are responsible for endocytosis, whereas the function of the microvilli binding sites is unclear, but they possess the ability to migrate in the plane of the microvillus membrane. Binding to specific binding sites and subsequent internalization of insulin takes place with high efficiency corresponding to more than 50% of the perfused load. Not all proteins are reabsorbed with high efficiency e.g. EGF which has similar molecular weight and pI is shown to be reabsorbed with substantially lower efficiency (about 4%). Binding and absorption efficiency of insulin may also change due to alterations in flow rate and perfused loads of protein: The load determines the magnitude of uptake and the flow rate determines the efficiency in binding and uptake. These changes are suggested to be caused by concomitant changes in the mean luminal concentration. The reabsorption process for insulin is efficient and of large capacity, and is only saturable (Michaelis-Menten kinetics) at very high concentrations of insulin. The proximal tubular internalization and degradation of 125I-insulin reach steady state rapidly. The processing can be described by a two-compartment model with t1/2 for transfer of 125I-insulin to lysosomes of 8.5 min and for lysosomal degradation of 72 min. 125I-PYY a linear peptide with similar molecular weight as EGF and insulin is not endocytosed but extracted with high efficiency (75% removed) by degradation by brush border peptidases and a substantial transtubular transport of TCA-precipitable PYY takes place by a paracellular route. A small vesicular transport of colloidal tracers was demonstrated constituting about 0.5% of the endocytosed amount. A method for covalently cross-linking insulin tracers to apical binding sites is described and evaluated. Recycling of apical binding sites was estimated to be very efficient and did not involve lysosomes or the Golgi apparatus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Influence of pH on the uptake of 5-fluorouracil into isolated tumour cells

To investigate the possible dependence of 5-fluorouracil (5FU) uptake in tumours on the intra- (pHi) and extracellular (pHe) pH, a pH gradient (deltapH) was imposed across the plasma membrane of ascites tumour cells in vitro, similar to that known to occur in some solid tumours in vivo, by incubation in media of PHe 5-8. A > or = 2:1 (intracellular/extracellular) accumulation of radiolabelled 5FU occurred after 5 min incubation of the cells with 0.5 mM 5FU at pHe of 5.0, 5.5 or 6.0. 5FU metabolism is slow under these conditions, and 5FU uptake was not affected by longer incubations up to 20 min, nor by the absence of a sodium gradient. pHi was estimated from the distribution of the weak acid, 5.5-dimethyl-2,4-oxazolidione ([14C]DMO) across the cell membrane. There was significant correlation between the intracellular/extracellular 5FU ratio and pHe (from pHe 6-8), deltapH and pHi (P < 0.02). Similar results were obtained with HT29 cells. Incubation with a drug that made plasma membranes permeable to H+ significantly decreased 5FU uptake in Lettre cells. The co-transport of 5FU may occur on a proton symport using the proton motive force of the deltapH.     

Field emission SEM, conventional TEM and HVTEM study of submandibular gland in prenatal and postnatal aging mouse

The development of acinar and ductal cells of the mouse submandibular gland was studied using field emission SEM, conventional TEM and HVTEM methods. The specimens, at 15 and 18 days of gestation and 1, 3, 7, 14, 21, 30, 90 and 180 postnatal days were fixed in 2.5% glutaraldehyde solution in 0.1 M sodium phosphate buffer (pH 7.3). At 15 and 18 days of gestation, the structure of mouse submandibular gland contains acinar and ductal cells in proliferation. The cytoplasmic organelles such as mitochondria, granular endoplasmic reticulum and Golgi apparatuses are scattered in the cytoplasm. At 18 prenatal days only several acinar cells present immature secretory granules in the apical portion. In this stage the acinar and ductal cells are enveloped by bundles of fine collagen fibrils disposed in several directions. There are also numerous capillaries located closely to the acinar cell membranes. In the aging stages of 1, 3, 7, 14, 21, and 30 postnatal days, the histo-differentiation of acinar, intercalated and ductal cell components are observed. At newborn day one the cytoplasmic organelles start to place themselves around the nucleus. Several immature secretory granules are observed at day one, however, they increase in the aging days. At postnatal day 30, the cytoplasms of acinar and ductal cells are filled with a large number of secretory granules of different sizes. The stacks of granular endoplasmic reticulum and Golgi apparatus and some vesicles and free ribosomes are noted. The intercellular membranes are attached by desmosomes and cytoplasmic interdigitations. The luminal surface shows several small projections of microvilli. An electron-dense line of basement membranes followed by fine collagen fibrils are recognized. Delicate capillaries are found in the outer surface of acinar cells. At postnatal day 90 and 180 the acinar, intercalated and striated ductal cells reveal numerous secretory granules in the apical portion. The acinar cells showed basal nuclei and the parallel arrangement of granular endoplasmic reticulum. The mitochondria are located at the base of ductal cells showing a typical pattern of cristae. In these stages the intercellular digitations of cytoplasmic protrusions and desmosomes are also noted. The cytoplasm of myoepithelial cells are seen along the cell membranes. The spongy-like structures constituting the basement membrane are followed by bundles of fine collagen fibers.     

Antagonistic effects of interferon-gamma and interleukin-4 on fibroblast cultures

The uptake of the Cd-metallothionein complex (CdMT) into LLC-PK1 cells was investigated and compared with that of CdCl2. The cells were incubated at 37 degrees C for up to 45 min with 1 microM Cd, as either CdMT or CdCl2 at pH 5.5, 6.4, or 7.4. Under all the experimental conditions described below, the accumulation of Cd from CdMT was markedly lower than that from CdCl2. Cd accumulation at pH 7.4 from CdMT increased linearly with the time of incubation, whereas Cd accumulation from CdCl2 was saturable. Metabolic inhibitors, 2,4-dinitrophenol (DNP) and carbonylcyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), and incubation at low temperature significantly decreased Cd accumulation from both Cd compounds. Coincubation with 30 microM ZnCl2, an antagonist of CdCl2 uptake, slightly decreased Cd accumulation from CdMT, but it markedly decreased that from CdCl2. Cd accumulation from CdMT at pH 5.5 was significantly higher than at pH 6.4 or 7.4, whereas Cd accumulation from CdCl2 at pH 5.5 and 6.4 was significantly lower than at pH 7.4. Although Cd accumulation from CdCl2 at pH 7.4 was significantly decreased by coincubation with 100 microM cysteine or glutathione (GSH), this decrease was not observed at pH 5.5 or 6.4. A small amount of Cd was removed, by the chelating agent EGTA, from the cell membranes after incubation with CdMT at pH 6.4 and 7.4, whereas a considerable amount of Cd was removed by EGTA after incubation with CdMT at pH 5.5 and with CdCl2 at three pHs. It appears that the CdMT complex is taken up into LLC-PK1 cells partially via an energy-dependent process, and the increase in Cd accumulation at low pH is due to the liberation of Cd. High stability and molecular size of the CdMT complex explains why it is not taken up readily into LLC-PK1 cells.     

Increased permeability of cerebral vessels to horseradish peroxidase induced by ischemia in Mongolian Gerbils

Cerebral ischemia was induced by occlusion of the left common carotic artery in adult Mongolian gerbils. The period of occlusion was 3, 6, or 18 h. Horseradish peroxidase (HRP) was intravenously injected in animals with clear neurological signs 1 h release of the clip. The HRP was allowed to circulate for 5 min. Fixation was carried out by perfusion with aldehydes. Tissue, incubated for peroxidatic activity, from the left side of the brain was treated for electron microscopy. During the postischemic period enhanced permeability was demonstrated in the brains of all animals. The amount of HRP transferred into the neuropil depended on the duration of ischemia. Thus the gerbils with 18 h occlusion showed the greatest content. The cells comprising the neuropil adjacent to vessels were studied and the degree of the pathological changes described below was increased proportionally to the time period of occlusion. The intercellular spaces, often filled with peroxidase, were expanded and the astrocytes swollen, especially the endfeet. Sometimes the astrocytes were pervious to HRP. The neurons were also swollen, but to a lesser degree than the astrocytes. No endothelial cell damage was observed. Even 18 h of occlusion did not change the plasma membranes. The intercellular spaces were free of HRP from the first luminal to the first abluminal tight junction. The cytoplasm exhibited HRP-containing vesicles of various types and shapes. Some were freely situated; others were connected to the plasma membrane and then open to the vessel lumen or to the basement membrane. Since no cell damage was demonstrated, and since no HRP was diffusely dispersed in the cytoplasm it is assumed that vesicles are responsible for the enhancement of the vesicular transport that normally occurs after intravenous injection of HRP.     

Endocytosis and exocytosis events regulate vesicle traffic in endothelial cells

We used water-soluble styryl pyridinium dyes that fluoresce at the membrane-water interface to study vesicle traffic in endothelial cells. Cultured endothelial cells derived from bovine and human pulmonary microvessels were incubated in styryl probes, washed to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that derived from plasmalemma by endocytosis were filled with the styryl dye. These vesicles were distributed throughout the cytosol as numerous particles of heterogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular albumin whereas a control protein, immunoglobulin G, had no effect. Dye uptake was abrogated by labeling at low temperatures and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyrosine kinase inhibitors (genistein and herbimycin A) prevented the albumin-induced vesicle formation. Cytochalasin B prevented vesicle redistribution indicating involvement of actin filaments in translocation of endosomes away from sites of vesicle formation. Styryl dye was lost from cells by exocytosis as evident by the disappearance of discrete fluorescent particles. N-ethylmaleimide and botulinum toxin types A and B caused cells to accumulate increased number of vesicles suggesting that exocytosis was regulated by NSF-dependent SNARE mechanism. The results suggest that phosphoinositide metabolism regulates endocytosis in endothelial cells and that extracellular albumin activates endocytosis by a mechanism involving tyrosine phosphorylation, whereas exocytosis is a distinct process regulated by the SNARE machinery. The results support the hypothesis that albumin regulates its internalization and release in vascular endothelial cells via activation of specific endocytic and exocytic pathways.     

Human cytomegalovirus glycoprotein B contains autonomous determinants for vectorial targeting to apical membranes of polarized epithelial cells

We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is vectorially transported to apical membranes of CMV-infected polarized human retinal pigment epithelial cells propagated on permeable filter supports and that virions egress predominantly from the apical membrane domain. In the present study, we investigated whether gB itself contains autonomous information for apical transport by expressing the molecule in stably transfected Madine-Darby canine kidney (MDCK) cells grown on permeable filter supports. Laser scanning confocal immunofluorescence microscopy and domain-selective biotinylation of surface membrane domains showed that CMV gB was transported to apical membranes independently of other envelope glycoproteins and that it colocalized with proteins in transport vesicles of the biosynthetic and endocytic pathways. Determinants for trafficking to apical membranes were located by evaluating the targeting of gB derivatives with deletions in the lumen, transmembrane (TM) anchor, and carboxyl terminus. Derivative gB(Delta717-747), with an internal deletion in the luminal juxtamembrane sequence that preserved the N- and O-glycosylation sites, retained vectorial transport to apical membranes. In contrast, derivatives that lacked the TM anchor and cytosolic domain (gBDelta646-906) or the TM anchor alone (gBDelta751-771) underwent considerable basolateral targeting. Likewise, derivatives lacking the entire cytosolic domain (gBDelta772-906) or the last 73 amino acids (gBDelta834-906) showed disrupted apical transport. Site-specific mutations that deleted or altered the cluster of acidic residues with a casein kinase II phosphorylation site at the extreme carboxyl terminus, which can serve as an internalization signal, caused partial missorting of gB to basolateral membranes. Our studies indicate that CMV gB contains autonomous information for apical targeting in luminal, TM anchor, and cytosolic domain sequences, forming distinct structural elements that cooperate in vectorial transport in polarized epithelial cells.     

Rapid transepithelial antigen transport in rat jejunum: impact of sensitization and the hypersensitivity reaction

BACKGROUND & AIMS: Intestine from sensitized rats develops a rapid secretory response to luminal antigen challenge that depends on activation of subepithelial mast cells. The aim of this study was to determine the timing and route of the transepithelial protein antigen transport. METHODS: Rats were sensitized to horseradish peroxidase (HRP). After 10-14 days, jejunal segments were resected, mounted in Ussing chambers, and challenged with HRP on the luminal side. RESULTS: Electron microscopy of tissue specimens fixed at 2 minutes (before mast cell activation) showed enhanced endocytic uptake of HRP in enterocytes of HRP-sensitized rats compared with ovalbumin-sensitized or saline-injected controls. At this time, HRP was distributed throughout epithelial cells and was already evident in the lamina propria. In contrast, HRP was restricted to the apical region of enterocytes in controls. At 30 minutes (after mast cell activation), in HRP-sensitized rats only, HRP was also located within tight junctions and the paracellular region between epithelial cells. Tissue conductance was increased in HRP-sensitized rats beginning 30 minutes after HRP addition and correlated with the overall flux of HRP across the tissue. CONCLUSIONS: The results show that specific sensitization enhances the initial uptake and transcytosis of antigen across intestinal epithelium. Subsequent to activation of mast cells, antigen transport is further enhanced by penetration through the paracellular pathway.     

Luminal acid increases apical cell membrane resistance in isolated Necturus antral mucosa

BACKGROUND & AIMS: The gastric mucosa must have efficient protective mechanisms to maintain physiological intracellular pH. The aim of this study was to investigate the effect of low luminal pH on apical membrane permeability. METHODS: Chambered Necturus antral mucosa was perfused with Ringer's/95% O2-5% CO2 at pH 7.25. The mucosal side was exposed to pH 4.0-2.0 with four microelectrodes placed in surface cells. Two-dimensional cable analysis was used to measure apical, basolateral, and shunt resistances. In some experiments, liquid sensor pH or Na(+)-selective microelectrodes were used. RESULTS: Luminal acidification hyperpolarized apical cell membrane potential and increased apical cell membrane resistance from 21.3 +/- 2.6 (pH 7.25) to 38.0 +/- 2.3 k omega.cm2 (pH 3.0; n = 8). The increase in apical cell membrane resistance was preceded by transient intracellular acidosis from 7.32 +/- 0.07 (pH 4.0) to 7.23 +/- 0.06 (pH 3.0; n = 6). Similar intracellular acidosis (provoked by NH4+ prepulse) failed to cause the effects observed with luminal acid. The increase in apical cell membrane resistance caused by luminal acid was eliminated when N-methyl-D-glucamine+, but not Na+, was substituted for all cations in the luminal solution. CONCLUSIONS: Luminal acidification (pH 3.0-2.0) closes apical amiloride-blockable Na+ channels. Protons are probably able to pass and even block these channels, but their effect in closing the channels does not occur intracellularly.     

Analysis of trypanosomal endocytic organelles using preparative free-flow electrophoresis

In this paper we demonstrate the power of preparative free-flow electrophoresis (FFE) for the study of endocytosis by African trypanosomes. Endocytosis of extracellular macromolecules by these parasites occurs through a specialized region of the parasite called the flagella pocket. The uptake of fluid phase markers such as horseradish peroxidase (HRP) into the various compartments of the endocytic pathway of bloodstream forms of Trypanosoma brucei brucei was manipulated by regulating the external environment (e.g., by altering the temperature of incubation). The various subcellular compartments were then separated by free-flow electrophoresis (FFE) or isopycnic density gradient centrifugation and analyzed for marker uptake. At low temperatures, HRP was found predominantly in the flagellar pocket. Increasing the temperature resulted in a time-dependent uptake of HRP into more positively charged endosomal fractions. However, little HRP activity was detected in lysosomal compartments, suggesting that either HRP had not yet entered the lysosome or was degraded immediately upon entry. Through the use of FFE we were able to identify and analyze compartments of the endosomal pathway that were not possible to identify by density gradient centrifugation alone. Although the differences in FFE separation of the endocytic compartments as seen in HRP uptake were striking, the minor changes seen within the lysosomal system were more subtle, as depicted in the protease profiles. In conclusion, we show that preparative FFE is a powerful technique for the analysis and separation of flagellar pocket-derived membranes from other endosomal and lysosomal compartments of African trypanosomes.     

Ultrastructural studies of experimental acute pancreatitis

Acute pancreatitis was studied by electron microscopy after retrograde infusion of either trypsin, and/or beta-glucuronidase into the canine pancreatic duct. Marked changes were induced by the mixture of trypsin and beta-glucuronidase. (1) The acinar cells were initially excavated from the acinar lumen and formed cystic bodies in themselves. The cystic bodies were then disrupted at their marginal membranes, and the acinar cells were filled with a large amount of fibrillar materials which originated from the contents of the cystic bodies. At this time, the luminal margin of the acinar cells completely disappeared. (2) The cellular organellas and the intracellular fibrillar materials in the acinar cells were discharged into the interstitial space through the disrupted basal lamina. Infection in the pancreatic ductal system was considered to play an important role in the pathogenesis of acute hemorrhagic pancreatitis.     

Functional characteristics and membrane localization of rat multispecific organic cation transporters, OCT1 and OCT2, mediating tubular secretion of cationic drugs

We have isolated a kidney-specific organic cation transporter, rat OCT2, which is distinct from rat OCT1 (Okuda M, Saito H, Urakami Y, Takano M and Inui K (1996) Biochem Biophys Res Commun 224:500-507). In our study, the functional characteristics and membrane localization of OCT1 and OCT2 were investigated by uptake studies using MDCK cells transfected with rat OCT1 or OCT2 cDNA (MDCK-OCT1 or MDCK-OCT2) and immunological studies. Tetraethylammonium (TEA) uptake by both MDCK-OCT1 and MDCK-OCT2 cells was markedly elevated when TEA was added to the basolateral medium, but not to the apical medium. Efflux of TEA from MDCK-OCT1 and MDCK-OCT2 cells was not changed by extracellular pH from 5.4 to 8.4, whereas TEA uptake by both transfectants was decreased by acidification of extracellular medium. Apparent Km values for TEA uptake by MDCK-OCT1 and MDCK-OCT2 cells were 38 and 45 microM, respectively. Although various hydrophilic organic cations such as 1-methyl-4-phenylpyridinium, cimetidine, quinidine, nicotine, N1-methylnicotinamide and guanidine markedly inhibited TEA uptake by both MDCK-OCT1 and MDCK-OCT2 cells, there were no significant differences in the apparent inhibition constants (Ki) against these organic cations between both transfectants. Furthermore, immunological studies using a polyclonal antibody against OCT1 revealed that OCT1 was expressed in the basolateral membranes but not in the brush-border membranes of the rat kidney. These results suggested that both OCT1 and OCT2 are basolateral-type organic cation transporters with broad substrate specificities, mediating tubular secretion of cationic drugs.     

Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2

The oncogene bcl-2 encodes a 26-kD protein localized to intracellular membranes, including the ER, mitochondria, and perinuclear membrane, but its mechanism of action is unknown. We have been investigating the hypothesis that Bcl-2 regulates the movement of calcium ions (Ca2+) through the ER membrane. Earlier findings in this laboratory indicated that Bcl-2 reduces Ca2+ efflux from the ER lumen in WEHI7.2 lymphoma cells treated with the Ca2+-ATPase inhibitor thapsigargin (TG) but does not prevent capacitative entry of extracellular calcium. In this report, we show that sustained elevation of cytosolic Ca2+ due to capacitative entry is not required for induction of apoptosis by TG, suggesting that ER calcium pool depletion may trigger apoptosis. Bcl-2 overexpression maintains Ca2+ uptake in the ER of TG-treated cells and prevents a TG-imposed delay in intralumenal processing of the endogenous glycoprotein cathepsin D. Also, Bcl-2 overexpression preserves the ER Ca2+ pool in untreated cells when extracellular Ca2+ is low. However, low extracellular Ca2+ reduces the antiapoptotic action of Bcl-2, suggesting that cytosolic Ca2+ elevation due to capacitative entry may be required for optimal ER pool filling and apoptosis inhibition by Bcl-2. In summary, the findings suggest that Bcl-2 maintains Ca2+ homeostasis within the ER, thereby inhibiting apoptosis induction by TG.     

Acetylcholine acts on M3 muscarinic receptors and induces the translocation of aquaporin5 water channel via cytosolic Ca2+ elevation in rat parotid glands

To evaluate the role of aquaporin5 (AQP5) in salivary secretion induced by cholinergic stimulation, the alteration of the distribution of AQP5 in rat parotid tissues induced by acetylcholine (ACh) was studied by immunobolt analysis. The treatment of the tissues with ACh within 1 min induced the translocation of AQP5 from intracellular membranes (ICM) to apical membranes (APM), but that for more than 5 min resulted in the converse translocation from APM to ICM. The ACh-induced increase in the amount of AQP5 in APM was inhibited by atropine, p-F-HHSiD and TMB-8, but not by methoctramine, staurosporine or H-7. The calcium ionophore A-23187 alone stimulated the translocation of AQP5 between APM and ICM. These results indicated that ACh acted on M3 muscarinic receptors and induced the translocation of AQP5 between ICM and APM, and that the cytosolic Ca2+ elevation by ACh may play a key role in this translocation in rat parotid glands.     

Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells

Sulfation is a common posttranslational modification of secretory proteins and serves as a valuable marker of constitutive and regulated secretory pathways. We investigated the cellular localization and the secretory behavior of sulfated macromolecules in the mouse pancreatic acinar cell. The major sulfated proteins of the cell were present in isolated zymogen granules, as determined by metabolic labeling with [35S]sulfate and subcellular fractionation. The sulfated proteins fell into three groups: gp300 is not secreted and is a component of the zymogen granule membrane; pancreatic lipase (56 kDa) and a 40 kDa protein are soluble and exhibit regulated secretion kinetics; and p82 is initially granule membrane associated, but is released from the cell with constitutive-like kinetics as a 75 kDa protein (p75). Secretion of p75 could be stimulated for up to 4 h after pulse labeling, presumably from immature secretory granules, but not after 6 h of chase. Treatment of cells with brefeldin A (BFA) at the start of the [35S]sulfate pulse resulted in almost total inhibition of sulfation. Addition of BFA during the chase (0-2 h) allowed normal basal and stimulated secretion of regulated secretory proteins, but reversibly inhibited the constitutive-like secretion of p75. In this case, the behavior of p75 was maintained as that of a regulated secretory protein for up to 6 h of chase. In untreated cells, immunofluorescence of p82/p75 was along the acinar lumen, and in small punctate structures in the apical cytoplasm. In BFA-treated cells, immunolabeling of p82/p75 was lost from the acinar lumen, and cytoplasmic labeling was finer and appeared to be associated with the secretory granule membranes. These data suggest a role for brefeldin A-sensitive coat formation in maturation of secretory granules after they bud from the TGN.     

Diagnosis of a case of acute chloroquine poisoning using 1H NMR spectroscopy: characterisation of drug metabolites in urine

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO2 and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO2 and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H2O2 did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H2O2, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H2O2 also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H2O2 had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H2O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used.     

Effect of E4031, a class III antiarrhythmic drug, on ischemia- and reperfusion-induced arrhythmias in isolated rat hearts

With the onset of vitellogenesis, the follicular epithelium overlying the oocyte in stick insect ovarioles becomes highly polarized and patent by formation of wide intercellular spaces. The aim of the present study was to provide experimental support to the notion that the follicular epithelium in this insect species may be involved in transcytosis. Data demonstrate that the follicular epithelium carries out sulfo-conjugation of a 85 kDa fat body derived protein by allowing it ot transit from one cell pole to another. Along the basal end, follicle cells branch into a number of cytoplasmic finger-like projections. At the opposite end facing the oocyte they taper off into lance-head shapes. Different vesicular elements are evident at both these extremities. In vivo exposure to horseradish peroxidase shows that the vesicular elements present along the apical end provide an endocytic entry. In contrast, those present along the basal end are labeled with sodium [35S]-sulfate, suggesting that they may be exocytic vesicles containing a sulfo-conjugated secretory product. In vivo exposure to sodium [35S]-sulfate caused radioactivity to appear over the Golgi apparatus and some nearby vesicles of the follicle cell cytoplasm, including the exocytic vesicles. The intracellular pathway of the follicle cells was also examined by immunogold labeling using a monoclonal antibody raised against a 85 kDa fat body derived protein. Under these conditions, gold particles were consistently detected over the Golgi apparatus and the vesicular elements lying along both poles of the follicle cell membrane. Based on this evidence, it is concluded that follicular cells in stick insect ovarioles are endowed with the ability to undergo transcytosis by providing an endocytic entry along the apical end and by releasing exocytically a sulfo-conjugated 85 kDa protein along the baso-lateral domain of the follicle cell membrane.     

Characteristics of the luminal proton pump in malpighian tubules of the ant

The active pump mechanisms involved in K+ secretion of the malpighian tubules of the ant and present in the luminal membrane were investigated on isolated, luminally perfused tubules of Formica. The specific blocker for vacuolar type ATPases, bafilomycin A1, was found to half-maximally inhibit secretion at a concentration of 10(-5) mol/l when added to the lumen. N-Ethylmaleimide reduced the calculated short circuit current (Isc) to 78 and 21% of control value when added at 5 x 10(-4) mol/l, respectively, to the lumen and the bath. Reducing luminal pH inhibited Isc with a half-maximal inhibition at a luminal pH of 4.5. Acidified omeprazole, Schering compound 28080 and vanadate (both 10(-3) and 10(-4) mol/l) inhibited Isc only partially. The present data suggest that the luminal membrane of ant malpighian tubules contains a H+ pump. This pump is only poorly bafilomycin-sensitive. Furthermore, additional active transport systems responsible for secretion may be present. Part of these results have been published as abstracts.