Intracellular traffic of the ecto-nucleotide pyrophosphatase/phosphodiesterase NPP3 to the apical plasma membrane of MDCK and Caco-2 cells: apical targeting occurs in the absence of N-glycosylation.

Glycosylation was considered the major signal candidate for apical targeting of transmembrane proteins in polarized epithelial cells. However, direct demonstration of the role of glycosylation has proved difficult because non-glycosylated apical transmembrane proteins usually do not reach the cell surface. Here we were able to follow the targeting of the apical transmembrane glycoprotein NPP3 both when glycosylated and non-glycosylated. Transfected in polarized MDCK and Caco-2 cells, NPP3 was exclusively expressed at the apical membrane. The transport kinetics of the protein to the cell surface were studied after metabolic (35)S-labeling and surface immunoprecipitation. The newly synthesized protein was mainly targeted directly to the apical surface in MDCK cells, whereas 50% transited through the basolateral surface in Caco-2 cells. In both cell types, the basolaterally targeted pool was effectively transcytosed to the apical surface. In the presence of tunicamycin, NPP3 was not N-glycosylated. The non-glycosylated protein was partially retained intracellularly but the fraction that reached the cell surface was nevertheless predominantly targeted apically. However, transcytosis of the non-glycosylated protein was partially impaired in MDCK cells. These results provide direct evidence that glycosylation cannot be considered an apical targeting signal for NPP3, although glycosylation is necessary for correct trafficking of the protein to the cell surface.

Stimulation of liver RNA and protein breakdown in endotoxemic rats: role of glucocorticoids.

In systemic or localized acute inflammation, liver ribosomal RNA (rRNA) and protein contents increase. We first determined whether changes in RNA, more specifically rRNA, and protein breakdown rates were involved in the accumulation of both types of macromolecules 24 h after induction of endotoxemia. Liver RNA and protein contents were enhanced by 35 and 19%, respectively, in the endotoxemic rats. RNA and protein degradation rates measured during in situ cyclic perfusions of the livers were significantly higher in the endotoxemic rats than in the controls (42 and 46%, respectively). In order to check that the stimulation of RNA and protein degradation corresponded to an activation of the hepatocyte autophagic pathway, the fractional cytoplasmic volume (FCV) of autophagosomes, digestive autophagic vacuoles and dense bodies was measured by morphometry in electron microscopy. The FCV of the sum of these lysosomal structures was significantly increased in the endotoxemic rats. We next tried to identify the factor(s) responsible for the high breakdown rates. The increase in macromolecular degradation did not result from reduced portal amino acid supply. The effects of dexamethasone, interleukin-6, interleukin-1beta, and tumor necrosis factor alpha on RNA degradation were then investigated in primary cultures of hepatocytes isolated from control rats. Only dexamethasone stimulated RNA breakdown. Finally, pretreatment of endotoxemic rats with RU 38486, a glucocorticoid receptor antagonist, completely abolished the stimulation of RNA degradation observed in the sham-gavaged LPS-treated rats. Our data suggest an important role of glucocorticoids in the high levels of RNA and protein breakdown in endotoxemic rats.

Down-regulation of liver RNA breakdown by turpentine administration in the starved rat: autophagy and relevant factors.

OBJECTIVE AND DESIGN: To determine whether the inhibition of RNA breakdown observed in ad libitum fed rats 24 h after turpentine administration still occurs in inflamed rats fasted for 24 h and to examine the mechanism and factors involved. METHODS: RNA breakdown was measured during cyclic in situ perfusion of livers by the accumulation of [14C] cytidine after in vivo RNA labelling. Autophagic activity was determined by the morphometric analysis of lysosomal structures. RESULTS: The decrease in RNA breakdown (53%) observed in the inflamed rats was accompanied by a 38% drop in the fractional cytoplasmic volume of initial and digestive autophagic vacuoles. Among amino acids, only the portal levels of glutamate were significantly enhanced by 83%. In vivo suppression of glucocorticoid activity using RU 38486 in inflamed rats did not affect the inhibition of RNA breakdown. CONCLUSIONS: The results show that turpentine-induced inflammation in fasted rats inhibits RNA degradation as well as autophagy and that glucocorticoids do not seem to be involved.

Identification of B10, an alkaline phosphodiesterase of the apical plasma membrane of hepatocytes and biliary cells, in rat serum: increased levels following bile duct ligation and during the development of cholangiocarcinoma.

Alkaline phosphodiesterase (APDE) is associated with the cellular plasma membrane of many organs. Several isoforms are also detected in normal human serum and their respective amounts vary in liver diseases but their significance is unknown. The aims of this study were: 1) to identify a serum form of B10, an APDE exclusively localized at the apical pole of the plasma membrane of rat hepatocytes and biliary cells; 2) to gain insight into its origin; and 3) to investigate its behavior, in two liver diseases in which an abnormal membrane expression of B10 has been reported, namely cholestasis and cholangiocarcinoma. A soluble form of B10 was immunoprecipitated from normal rat serum, which amounted to 13% of total serum APDE activity. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the size of the serum enzyme was 125 kd, which is slightly lower than that found in the plasma membrane (130 kd). In bile, a 120-kd and a 130-kd form was found. A sixfold and fivefold increase of B10 APDE activity was observed in the serum of bile duct-ligated rats and in the Long-Evans Cinnamon (LEC) rats which spontaneously develop cholangiocarcinoma. The molecular size of the form present in serum was unchanged. A threefold increase was also observed in LEC rats which had not yet developed a cholangiocarcinoma. In conclusion, we identified a soluble form of B10 in normal rat serum. The increase in serum B10 in the experimental and pathological conditions investigated does not seem to result from passage of the biliary form to the serum but seems to be caused by increased cleavage of the membrane form. Its rise early during the onset of cholangiocarcinoma suggests that B10 in the serum might be a marker of carcinogenesis and/or be involved in the development of cholangiocarcinoma.

Rotavirus is released from the apical surface of cultured human intestinal cells through nonconventional vesicular transport that bypasses the Golgi apparatus.

Rotaviruses are nonenveloped viruses that infect enterocytes of the small intestine and cause severe infantile gastroenteritis. It was previously thought that rotavirus exits cells by lysis, but this behavior does not match the local pathogenesis of the virus. In this study, we have investigated the release of the simian rotavirus strain (RRV) from the polarized intestinal Caco-2 cells. We found that RRV is released almost exclusively from the apical pole of Caco-2 cells before any cells lyse. Using confocal laser scanning microscopy and drugs that inhibit vesicular transport, we studied the RRV transport route from the endoplasmic reticulum (ER) to the apical side of intestinal cells. We demonstrated that RRV exits from the ER through a carbonyl cyanide m-chlorophenylhydrazone-sensitive vesicular transport. RRV staining was never found within the Golgi apparatus or lysosomes, suggesting that the RRV intracellular pathway does not involve these organelles. This finding was confirmed by treatment with monensin or NH4Cl, which do not affect release of RRV. Electron microscopic analysis revealed RRV containing small smooth vesicles in the apical area and free virions outside the cell in the brush border, consistent with a vesicular vectorial transport of virus. These results may provide, for the first time, a cellular explanation of the pathogenesis of rotavirus.

Biochemical and molecular identification of distinct forms of alkaline phosphodiesterase I expressed on the apical and basolateral plasma membrane surfaces of rat hepatocytes.

We have identified B10, a plasma membrane protein previously defined by a monoclonal antibody, as an alkaline phosphodiesterase I (APDE) expressed in the plasma membrane of rat hepatocytes and enterocytes, with a restricted apical distribution. B10 complementary DNA (cDNA) was cloned from a rat intestinal library screened with a polyclonal antibody directed to the hepatic protein. Two distinct B10 clones with an open reading frame of 2,625 bp were obtained that differed only by 12 bases in the coding region. One B10 clone had a single base difference with gp130RB13-6 cDNA, which was recently cloned in rat fetal brain. B10/gp130RB13-6 had 50% identity at the amino acid level with the plasma cell antigen PC-1, an APDE cloned in the mouse and in human. Anti-B10 antibodies immunoprecipitated 34% of the APDE activity in liver plasma membranes and over 95% of the APDE activity in intestinal cells. Most of the remaining activity in hepatocytes (44%) could be immunoprecipitated by antibodies directed to PC-1. APDE activity immunoprecipitated with anti-B10 antibodies was found in the apical rat liver plasma membrane fractions on a sucrose gradient whereas most of the remaining APDE activity was associated with the basolateral fractions, which contained PC-1. By immunofluorescence, B10 was localized to the apical surfaces of hepatocytes and enterocytes whereas PC-1 was present on the basolateral surfaces of hepatocytes. B10/gp130RB13-6 and rat PC-1 are a unique example of distinct molecules having similar enzymatic activity but different apical/basolateral location, and possibly different functions.

Expression of cytokine-dependent adhesion molecules in postreperfusion biopsy specimens of liver allografts.

BACKGROUND/AIMS: Allogeneic recognition of donor cells by host T lymphocytes requires the expression of cytokine-dependent molecules, such as class II major histocompatibility antigens, intracellular adhesion molecule 1 (ICAM-1), and lymphocyte function-associated antigen 3 (LFA-3). In the liver, activation of Kupffer cells after ischemic injury during the transplantation procedure may result in an early induction of cytokine-dependent molecules. METHODS: The pattern of induction of ICAM-1, HLA-DR, HLA-DQ, and LFA-3 was investigated in 30 postreperfusion surgical biopsy specimens of liver allografts by an immunohistochemical technique. RESULTS: Two patterns of induction were observed: focal or diffuse. On hepatocytes, ICAM-1 was induced in 22 cases (11 focal, 11 diffuse), HLA-DR in 18 cases (13 focal, 5 diffuse), HLA-DQ in 13 cases (3 focal, 10 diffuse), and LFA-3 in 1 case (focal). On bile duct cells, HLA-DR was expressed in 19 cases, associated with HLA-DQ in 7 cases. No induction of ICAM-1 and LFA-3 was detected. Compared with the other patients, the group of patients with diffuse postoperative hepatocellular induction of ICAM-1 was characterized by higher preharvesting serum transaminase levels in the donor (P < or = 0.001), suggestive of preoperative ischemic injury, and increased incidence of acute graft rejection (P = 0.04). CONCLUSIONS: Preoperative warm ischemia may modify the immunogenicity of liver allografts.

Expression of complement-regulatory proteins in normal and UW-preserved human liver.

BACKGROUND/AIMS: Somatic cells are protected against complement-mediated injury by specialized membrane proteins, known as complement-regulatory proteins (CRP). The knowledge of the pattern of CRP expression in the liver is important to evaluate the role of complement-mediated injury in graft rejection. METHODS: We determined the distribution of four main CRP: membrane cofactor protein (MCP), decay accelerating factor (DAF), protectin, and complement receptor 1 (CR1) in 30 histologically normal livers, 13 samples from University of Wisconsin cold-storage solution (UW)-preserved tissue and 17 postoperative biopsies of UW-preserved allografts. RESULTS: In normal liver, hepatocytes expressed only MCP. Bile duct cells were reactive for MCP and protectin. Sinusoidal endothelial cells expressed MCP and protectin but displayed no or faint expression of DAF. Endothelial cells of portal vessels and centrilobular veins expressed high levels of DAF, MCP, and protectin. No expression of CR1 was observed. No change in CRP expression was usually detected after UW preservation, except for protectin, induced on hepatocytes in 9 samples of UW-preserved liver tissue and in 9 allografts. CONCLUSIONS: Hepatocytes and sinusoidal endothelial cells, which have a defective expression of CRP, might be at risk for complement-mediated injury. However, this risk is not aggravated after UW preservation.

Cooperation between platelets and neutrophils for paf-acether (platelet-activating factor) formation.

Association of platelets and neutrophils is frequently observed within thrombi or inflammatory sites. Interactions between these two cell populations have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether, which is synthesized by activated platelets and neutrophils. Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelet and neutrophil cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 x 10(6)/ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 +/- 2.5 ng/ml paf-acether. Human washed platelets (3 x 10(8)/ml) stimulated with thrombin (1 IU/ml) formed 0.60 +/- 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as did platelets and neutrophils separately (10.90 +/- 4.25 ng/ml, n = 6, P less than .001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled (10.24 +/- 3.81 ng/ml paf-acether vs. 5.30 +/- 2.23, P less than .05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to an increase of biosynthesis of paf-acether by neutrophils (13.86 +/- 2.26 ng/ml paf-acether vs. 5.76 +/- 0.38, P less than .05, n = 3). These results indicate that a cooperation between platelets and neutrophils exists for paf-acether formation. The phenomenon depends on a platelet-derived soluble factor, possibly lyso paf-acether. This cell-to-cell interaction is of interest since paf-acether is formed by and acting on platelets and neutrophils and represents a molecular basis for potent amplification of inflammatory reactions.

Quantitation of paf-acether by release of endogenous platelet serotonin assessed by liquid chromatography with electrochemical detection.

A new method to quantitate paf-acether (paf) was developed. It is based on the measurement of serotonin released from washed rabbit platelets challenged with paf. Platelets (1 X 10(8)/ml) were exposed with or without stirring to various concentrations of paf (26-130 pM) at 37 degrees C or at room temperature. Supernatants were submitted to a 4-min liquid chromatography run and serotonin was measured by electrochemical detection. We quantitated paf from three different biological sources, human neutrophils, mouse peritoneal macrophages, and cultured mast cells, comparing a classical method, i.e., platelet aggregation with the electrochemical detection of endogenous serotonin. We found similar results since, when compared with the aggregation method, the results differed by 12 to 47%. The sensitivity of both methods was 26 pM. The between-day variation coefficient was 23 and 14% (n = 12) for the aggregation method and the serotonin release, respectively, whereas the within-day variation coefficient for serotonin quantitation was less than 5% (n = 12). The superiority of the new method lies in its simplicity, the economy of platelets, and its possibility of automation. It can be applied to any agonist or any mechanism capable of releasing serotonin from platelets and more generally when a simple and fast method for measuring serotonin is desirable.

Interaction of the Paf antagonist WEB 2086 and its hetrazepine analogues with human platelets and endothelial cells.

1. Intact platelets and confluent human umbilical vein endothelial cells bound [3H]-Paf-acether (platelet activating factor, [3H]-Paf) at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA). 2. [3H]-Paf binding to platelets was inhibited in a concentration-dependent manner by WEB 2086. An excess of WEB 2086 indicated the presence of specific, saturable Paf binding which reached a maximum of 28.3 +/- 3.7 fmol [3H]-Paf per 5 x 10(7) platelets. In platelets, different hetrazepines (WEB 2098, 2105, but not 2118) also inhibited [3H]-Paf binding in a concentration-dependent manner. 3. WEB 2086 partially displaced platelet-bound [3H]-Paf in a concentration-dependent manner reaching a plateau at 400 nM WEB 2086. No further displacement was observed when WEB 2086 and an excess of unlabelled Paf were added together. 4. The hetrazepines inhibited platelet aggregation. Platelet aggregation IC50 values correlated well with the IC50 values of the hetrazepines against [3H]-Paf binding (r2 = 0.99). WEB 2086 shifted the Paf dose-response curve rightwards in a parallel manner. Tested against platelet aggregation the pA2 obtained for WEB 2086 was 7.9. 5. WEB 2086 inhibited [3H]-Paf binding to endothelial cells in a concentration-dependent manner. WEB 2086 also inhibited the Paf-mediated cytosolic calcium increase in endothelial cells with an IC50 value of 23.1 +/- 10.4 nM as compared with an IC50 of 21.6 +/- 10.4 nM WEB 2086 for platelet aggregation. 6. These results demonstrate an inhibition of [3H]-Paf binding to platelets and endothelial cells by different hetrazepines, most probably at the Paf receptor level.

The role of extracellular Ca2+ and Na+ in paf-acether-induced human platelet activation.

The dependence of paf-acether (paf)-induced human platelet activation on extracellular Ca2+ and Na+ was examined by quantitating aggregation, secretion and thromboxane (Tx) formation in the presence of physiological and/or low concentrations of Ca2+ and/or Na+. In the presence of 2 mM Ca2+ and 140 mM Na+, paf induced a dose-dependent reversible aggregation and less than 25% of [14C]serotonin release. These responses were insensitive to aspirin or Tx antagonist SQ 29548 treatment and negligible amounts of Tx were formed. In low Ca2+ buffer, paf induced irreversible aggregation and the 14C-serotonin release could exceed 60%. These increases in platelet response were associated with the formation of Tx and were suppressed by aspirin and SQ 29548 treatments, or by substituting NaCl with N-methylglucamine hydrochloride. Thus, in low Ca2+ medium, Tx synthesis is favored during platelet activation and is dependent on Na+ concentrations. A decrease in extracellular Na+ inhibited the paf-acether-induced Tx synthesis observed in low Ca2+ medium but not that induced by the Tx direct precursor, arachidonic acid (AA). Therefore, the increase observed in low Ca2+ medium, no longer seen when the Na+ level is decreased is not related to an impairment of the cyclooxygenase activity but rather implicates an effect on the activity of phospholipase A2. A decrease in extracellular Na+ (2 mM Ca2+ present), inhibited [14C]serotonin release induced by paf from platelets which had, or had not been, treated with aspirin. In this medium, the AA-induced release reaction was also affected whereas Tx formation was not altered, thus suggesting that other mechanisms involved in platelet response apart from Tx synthesis are dependent on extracellular Na+.

Reduced sensitivity of human platelets to PAF-acether following ticlopidine intake.

We investigated in 9 patients the effect of a 7-day treatment by Ticlopidine (250 mg b.i.d.) on washed platelets activation by PAF-acether in comparison with adenosine 5'-diphosphate (ADP) and arachidonic acid (AA). Aggregations induced by ADP were totally suppressed upon drug administration. AA-induced aggregations were partly but significantly inhibited (p less than 0.05). Responses of platelets to PAF-acether before treatment differed from patient to patient. A paired Student's test and a two-way analysis of variance showed a significant inhibitory effect of Ticlopidine treatment on PAF-acether-induced aggregation. The inhibitory effect of Ticlopidine or its metabolite(s) was evidenced after platelet washing procedure, suggesting a persistent effect of this drug on platelet after administration of the drug has been stopped.

Hispidulin, a natural flavone, inhibits human platelet aggregation by increasing cAMP levels.

Hispidulin, a natural flavone, and theophylline inhibited platelet aggregation triggered by adenosine-5'-monophosphate, arachidonic acid, paf-acether and collagen. Hispidulin was 100-fold more potent than theophylline. A threshold concentration of PGE1 did not modify the anti-aggregatory effect of hispidulin but potentiated the effect of theophylline. A threshold concentration of hispidulin had no effect on the inhibitory action of theophylline. Hispidulin (100 microM) and theophylline (10 mM) increased the control cAMP level in platelets 4-fold. A threshold concentration of PGE1 had a small effect on hispidulin-induced cAMP levels but increased the theophylline-induced cAMP levels 3-fold. Theophylline (10 mM)-induced cAMP levels were not modified by hispidulin. We demonstrate a correlation between the inhibition of platelet aggregation and the increase in cAMP levels induced by hispidulin. These data suggest that hispidulin could inhibit platelet aggregation by elevating cAMP levels by a mechanism different from that of theophylline or PGE1.

Exercise- and allergen-induced asthma do not change the production of Paf-acether by neutrophils and platelets.

Paf-acether, whose role has been suggested in asthma, is a mediator released by stimulated neutrophils, platelets and other cells. Neutrophils and platelets are activated in vivo during exercise or allergen-induced asthma. Upon in vitro stimulation, macrophages from mice treated with an inflammatory stimulus, such as thioglycoccollate, release less paf-acether than macrophages from non-treated mice. We hypothesized that upon in vitro activation platelets and neutrophils should produce less paf-acether after exercise- or allergen-induced asthma. To test this hypothesis, we measured the production of paf-acether by neutrophils and platelets obtained before, 15 and 75 min after exercise in seven normal subjects and five asthmatic subjects with exercise-induced asthma, and in five other asthmatic subjects after specific challenge with Dermatophagoides Pteronyssinus. Purified neutrophils and washed platelets were incubated independently for 10 min at 37 degrees C with no specific activator, with a platelet activator (thrombin, 1 IU.ml-1), a neutrophil activator (opsonized zymosan, 1 mg.ml-1), and both together. We found no significant difference between asthmatic and normal subjects in the amount of paf-acether synthesized by platelets or neutrophils and no fall in the production of paf-acether after exercise- or allergen-induced asthma. However, our method may lack sensitivity in detecting partial activation of these cells and is based on the assumption that changes in peripheral blood cells are representative of changes of these cells in lungs.

Inhibition by ticlopidine of Paf-acether-induced in vitro aggregation of rabbit and human platelets.

Ticlopidine was incubated in vitro with rabbit or human washed platelets and aggregations were triggered by submaximal concentrations of adenosine-5'-diphosphate (ADP), arachidonic acid (AA) and Paf-acether (platelet-activating factor), the mediators of the three known pathways of platelet activation. Inhibition of Paf-acether-induced rabbit platelet aggregation was proportional to the concentrations of Ticlopidine used. The same range of inhibition by Ticlopidine was observed when aggregations were triggered by the two other agonists. Human platelet aggregation induced by Paf-acether was also inhibited by Ticlopidine. Inhibition was increased when platelets were rendered insensitive to ADP and AA. Our results show that Ticlopidine inhibits human and rabbit platelet aggregation triggered by Paf-acether through a mechanism not related to the inhibition of the ADP and prostaglandin pathways.