The role of endocytic pathways in TGF-ß signaling.

Transforming growth factor ß (TGF-ß) superfamily consists of numerous cytokins that regulate various cellular processes. TGF-ß, the prototype of the family, signals through its cell surface serine/threonin kinase receptors and besides its role in cell differentiation, migration, adhesion etc. it is also able to induce epithelial-mesenchymal (EMT) transition via both Smad- pathway and MAPK- pathway. Among the different types of epithelial-mesenchymal transition, type II that is described to be associated with wound healing, tissue regeneration, organ fibrosis and is induced upon inflammatory stimuli. It can be triggered by secretion of growth factors such as TGF-ß, EGF. Different endocytic routes are used for the internalization of TGF-ß ligand and its receptors and these pathways can control the activity of downstream events. Internalization via clathrin-coated vesicles promotes the signaling while the caveola-mediated endocytosis plays important role in the termination of the events, although the steps of the latter event are less clear. The early endosome is considered a clue compartment in promoting the signaling. Recently published data suggest that the early endosome plays crucial role in the termination of the TGFß signaling as well. It is not only maintain a special environment for the effective signaling but can direct the internalized cargos towards degradative pathways (multivesicular bodies, lysosomes).

Caveolin-1 is transported to multi-vesicular bodies after albumin-induced endocytosis of caveolae in HepG2 cells.

Caveolae-mediated endocytosis is a highly regulated endocytic pathway that exists in parallel to other forms of clathrin-dependent and -independent endocytosis. Internalized caveolae accumulate in intermediate organelles called caveosomes. Here we addressed the further fate of internalized caveolae by inducing caveolae-mediated uptake of albumin by HepG2 cells. We followed the route of internalized caveolin-1 by immunogold labelling of ultrathin frozen sections and by Western blot analyses of purified membrane fractions. Long-term (1 and 3 hrs) albumin treatment resulted in the appearance of albumin-containing caveolae in special multi-caveolar complexes (consisting of multiple caveolae clustered together) connected to the plasma membrane and caveosome-like structures in the cytoplasm. In addition, numerous CD63 (LIMP-1) positive late endosomes/multi-vesicular bodies were found positive for caveolin-1, suggesting that upon albumin incubation, caveolin-1 is endocytosed and enters the degradative pathway. Surprisingly, the number of caveolae at the plasma membrane increased after addition of albumin. This increase was blocked by cycloheximide treatment, indicating that albumin internalization also stimulates de novo protein synthesis, which is necessary for new caveolae formation. Together, our results show that during long-term albumin uptake, caveolin-1 travels to late endosomes and is replaced by newly synthesized caveolin-1 at the plasma membrane.

Caveolae and caveolin isoforms in rat peritoneal macrophages.

Caveolea are special (highly hydrophobic) plasma membrane invaginations with a diameter of 50-100 nm. Their characteristic features are the flask- or omega-shape and the lack of basket-like coat composed of clathrin. Caveolin-an integral membrane protein-is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. In this paper we summarize the morphological and biochemical data providing strong evidence about the existence and function of caveolae in rat peritoneal macrophages. When studied electron microscopically, the surface of both resident and elicited macrophages exhibited omega- or flask-shaped plasma membrane invaginations. There was a significant difference, however, in the number of these profiles: whereas in resident cells only a small amount of them was found on the cell surface, in elicited cells they were abundantly present on the plasma membrane. Using an antibody against the VIP21/caveolin-1 isoform we showed that these plasma membrane pits were indeed caveolae. The number and the appearance of caveolae were found to be in close correlation with the functional activity of these phagocytotic cells, indicating that the formation of caveolae is a highly regulated process. Using Western blot analysis two different proteins ( approximately 29 and approximately 20 kDa)-both labelled with anti-caveolin antibodies-were identified in resident and elicited macrophages that have been isolated from rat peritoneal cavity. The approximately 20 kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29 kDa protein was labelled by both anti-VIP21/caveolin-1 and anti-caveolin-2 antibodies. The presence of the approximately 29 kDa protein was highly characteristic of resident cells, and only a small amount of approximately 20 kDa protein was detected in these cells. Elicitation has resulted in a significant increase in the amount of approximately 20 kDa protein labeled only with anit-VIP21/caveolin-1. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the cell surface of these cells. In elicited macrophages, caveolae (labelled with anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies. These data support the idea that the expression of the approximately 29 kDa (caveolin-related) protein is insufficient for caveolae formation in resident cells, it can function as a modified, macrophage-specific caveolin-2 isoform. Our results strongly suggest that caveolin-1 plays a crucial role in the formation of caveolae: it is the amount of caveolin-1 that regulates the appearance of caveolae on the plasma membrane. Studying the endocytotic processes of resident and elicited macrophages we have found that elicited macrophages bound and internalized significantly larger amounts of fluid phase marker (HRP) and immune complex (peroxidase-antiperoxidase-PAP) than resident cells. Serial section analysis, double labelled immunocytochemistry, and filipin treatment were used to demonstrate that caveolae can pinch off from the plasma membrane and can take part in endocytotic processes as alternative carriers in elicited macrophages.

Estrogen downregulates the number of caveolae and the level of caveolin in uterine smooth muscle.

Estrogen and progesterone, while regulating uterine functions, also regulate the number of caveolae and the level of caveolin. Large numbers of caveolae, as well as elevated expression of caveolin-1 and caveolin-2 isoforms in the myometrium of ovariectomised (OVX) rats were detected. 17beta-estradiol (E2) has a downregulating effect: the treatment of OVX rats with E2 (5 microg/animal) reduced the formation of caveolae by approx. 90%. Western blots clearly demonstrated the reduction of membrane caveolin-1 and -2 content. Progesterone treatment (2.5 mg/animal) alone did not cause any substantial change, but prevented the effect of estrogen. Control experiments showed that the quantity of Na+/K+-ATPase, a plasma membrane protein excluded from caveolae, was not downregulated by E2. The administration of the pure estrogen receptor (ERalpha) antagonist ICI 182,780 (1 mg/animal) not only compensated for the inhibitory effect of E2, but further increased the level of caveolin-1 in the myometrium of OVX rats and facilitated the formation of caveolae by approximately 70%. In contrast, the partial antagonist tamoxifen (1 mg/animal) mimicked the effect of estrogen. The amount of caveolin also changed during pregnancy. During the first half of pregnancy the expression of caveolin was suppressed, but it gradually increased until delivery. Our results indicate that the formation and number of caveolae are influenced by the physiological state of the uterus in a hormone dependent manner.

Caveolin isoforms in resident and elicited rat peritoneal macrophages.

Caveolin--an integral membrane protein--is the principal component of caveolae membranes in vivo. Multiple forms of caveolin have been identified: caveolin-1alpha, caveolin-1beta, caveolin-2 and caveolin-3. They differ in their specific properties and tissue distribution. When we studied the lysate of resident and elicited macrophages isolated from rat peritoneal cavity by Western blot analysis, we identified two different proteins (approximately 29 kDa and approximately 20 kDa) which were labelled with anti-caveolin antibodies. The approximately 20-kDa protein was labelled specifically only by anti-VIP21/caveolin-1, while the approximately 29-kDa protein was labelled by anti-VIP21/caveolin-1 and anti-caveolin-2. The presence of the approximately 29-kDa protein was characteristic of resident macrophages, and only a small amount of the approximately 20-kDa protein was detected in these cells. Elicitation resulted in a significant increase in the amount of the approximately 20-kDa protein labelled by anti-VIP21/caveolin-1 only. According to its molecular mass and antibody-specificity, this protein might be identical with the caveolin-1beta isoform. Our morphological (confocal and electron microscopical) studies have shown that in resident cells caveolin was present in the cytoplasm, in smaller vesicles and multivesicular bodies around the Golgi area. Only a very small amount of caveolae was found on the surface of these cells. In elicited macrophages, caveolae (labelled with the anti-VIP21/caveolin-1 antibody) appeared in large numbers on the cell surface, but caveolin detected by anti-caveolin-2 was also found in small vesicles and multivesicular bodies in the cytoplasm. According to these results, the absence of caveolae in resident cells can be explained by the absence of caveolin-1. The expression of the approximately 29-kDa (caveolin-related) protein in resident macrophages seems to be insufficient for caveolae formation. Elicitation significantly increased the expression of caveolin-1, and the increased amount of caveolin-1 resulted in caveolae formation on the cell surface.

Caveolae can be alternative endocytotic structures in elicited macrophages.

The aim of the present study was to identify omega-shaped plasma membrane invaginations, abundantly present in the plasma membrane of elicited macrophages, as caveolae. We have used an antibody against the major component of the caveolar coat (VIP21/caveolin), and the omega-shaped vesicles were found to be labeled with anti-VIP21 providing evidence that these structures were really caveolae. Filipin that had been shown to affect caveolae was used to investigate the possible endocytotic role of these structures in elicited macrophages. When caveolae were selectively inhibited by filipin, the rate of both fluid-phase and receptor-mediated endocytosis has been decreased. These data together with our results obtained from serial sectioning support that in elicited macrophages caveolae can pinch off from the plasma membrane and can function as alternative carriers in the endocytotic processes of these cells.

Early endocytotic steps in elicited macrophages: omega-shaped plasma membrane vesicles at their cell surface.

Fluid-phase and receptor-mediated endocytosis were studied in Freund's adjuvant elicited macrophages. These cells were found to bind and internalize significantly larger amounts of peroxidase-antiperoxidase (PAP) immune complex than resident macrophages. Similarly the rate of the fluid-phase uptake was higher in elicited cells. When studying the early steps of endocytotic processes, omega-shaped plasma membrane pits (d approximately 90 nm) were found at the macrophage cell surface. Although occurring occasionally in resident cells, their number was highly increased after elicitation in 30% of the macrophage cell population. The different morphology of these cells coincided with a lower endocytotic activity and a very strong ecto Ca(2+)-ATPase reaction. The present findings indicate that the elicited macrophage population is heterogenous and consists of different subclasses.

[Behavior of adamantinoma of the long bones based on long-term follow up studies]. / Csöves csontok adamantinomáinak viselkedése hosszútávú utánkövetés alapján.

Authors report on the long time (in average 12 years, range: 5-36 years) follow-up results of 5 cases of adamantinoma, localized on the tibia. In one case recurrence was found very late, 20 and 36 years after the primary wide resection, and resection was repeatedly performed. Because of problems of differential diagnosis in one case the tumor was excised intralesionally (curettage + plasty with cancellous bone). 7 years later the persistence of the process was found only. 1 patient died in consequence of pulmonary metastasis 9 years after the primary operation. Wide resection is suggested both for the removal of the primary tumor and the recurrences, appearing very late. For the reconstruction of the bone autologous fibula is proposed. Adamantinoma is thought to be a low malignity tumor, the outcome of which cannot be predicted from the clinical and histological findings. Considering the late recurrences and metastasis a long range, minimally 10 years, following of the patient is thought to be necessary.

Follow-up evaluation of anomalies of the vesicoureteric junction.

Anomalies of the vesicoureteric junction are important, particularly obstruction and reflux, as they may predispose to urinary tract infection. Over a 5-year period, 52 babies were referred with dilatation of the urinary tract detected antenatally or/and postnatally by ultrasound. Sixteen had an anomaly of the vesicoureteric junction: 9 had vesicoureteric reflux, 3 had ureteroceles, 1 had urethral stenosis with secondary reflux and 3 had stenosis of the vesicoureteric junction. Ten patients underwent 14 surgical procedures. The mean time to reconstructive surgery was 9.3 months. Ultrasonography showed regression of the dilatation in all patients who underwent surgery. Seven patients with minor dilatation are still under observation. In only 1 case was there loss of renal parenchyma. With conservative medical treatment the patients are 1 year old before reconstructive surgery is undertaken; with reflux, however, progression may indicate earlier surgery.

The human neutrophil FcRIII (CD16) on the plasma membrane can be replenished from an intracellular source.

The internalization of peroxidase-antiperoxidase (PAP) immune complexes by human neutrophil granulocytes was studied at an ultrastructural level. PAP initially bound to the plasma membrane at 4 degrees C accumulated in endosomes within 5 min of internalization. By that time, all of the ligand bound to the plasma membrane had been removed from the cell surface and the cells were able to bind newly added PAP. Pre-embedding labelling of FcRIII on these cells showed that this receptor was present on the cell surface, indicating involvement of FcRIII in the rebinding of PAP in neutrophils. The origin of FcRIII present on the plasma membrane after Fc receptor-mediated internalization of PAP was investigated in another series of experiments. Incubation of the cells with pronase eliminated the epitope on the Fc receptor recognized by anti-FcRIII. After the pronase treatment hardly any Fc receptors were detected on the plasma membrane. However, incubation of the cells for only 5 min in a protease-free medium after the pronase treatment led to an abundance of FcRIII on the plasma membrane of the neutrophils. These findings support the hypothesis that FcRIII on the plasma membrane of human neutrophil granulocytes is replenished from an internal source of free Fc receptors and suggest that at least some of the receptors present on the cell surface after the binding and internalization of PAP originate from this source in the cytoplasm.

[Pancreatic pseudocysts in children]. / Pancreas pseudocysták gyermekkorban.

Pancreas pseudocysts in childhood. On the basis of experience obtained with 9 patients treated over 31 years the authors publish the first comprehensive study in Hungary on the pancreas pseudocyst in childhood. Having reviewed their series and the medical literature main features and the possible prevention and management of this disease is outlined. Review of these patients with ultrasonography and chemistry tests is advised in order to follow-up the recovery and to screen the error in the glucose metabolism.

Effect of acidic extracellular pH on the receptor-mediated endocytosis of peroxidase-antiperoxidase (PAP) immune complex in rat peritoneal macrophages.

The effect of acidic extracellular medium on the uptake of peroxidase-antiperoxidase (PAP) immune complex in rat peritoneal macrophages was studied with the electron microscope. The acidic extracellular medium resulted in acidification of the cytosol in a relatively short period of time as was shown with 5(6) carboxyfluorescein as indicator. At pH 5.5 PAP was internalized. Some of the ligand was found in endosomes; however, a considerable quantity was still present at the cell surface after 5 min internalization. By 30 min the surface was cleared of PAP and the ligand was detectable almost exclusively in endosomes. PAP was never found in lysosomes which were identified by previous loading with cationized ferritin. We conclude that a pH shift of the cytoplasm results in inhibition of endosome-lysosome fusion.

Slow regeneration of immune complex binding sites on the macrophage surface after previous digestion with pronase.

Peroxidase-antiperoxidase (PAP) binding sites on the rat peritoneal macrophage surface were damaged by pronase digestion and the reappearance of functionally intact receptors was investigated morphologically and spectrophotometrically. Pronase digestion decreased the PAP binding ability of macrophages to about 40% of the original value. Removing the pronase the regeneration of ligand binding was very protracted with only about 60% intact receptors even after 30 min. From these findings we conclude that the recycling of internalized Fc receptors greatly contribute to the replenishment of receptors on the cell surface.

Reappearance of immune complex binding sites on macrophages after internalization and its inhibition by monensin.

Receptor-mediated endocytosis of IgG and immune complexes in macrophages is terminated with digestion of the ligand in lysosomes. However, there are controversial data on whether Fc receptors are degraded together with the ligand or recycled to the cell surface. In the present study, rat peritoneal macrophages were incubated at 4 degrees C with rat peroxidase-antiperoxidase (PAP) complex for 1 h, washed and warmed up to 37 degrees C for different time periods and reincubated with new PAP at 4 degrees C. In another series of experiments, the cells were preincubated with 50 nM monensin, then cooled to 4 degrees C and reincubated with PAP in the presence of monensin. The cells were fixed and processed for electron microscopy at different stages of the experiments. Quantitative data were obtained by measuring PAP-binding membrane lengths on electron micrographs (morphometry) and by determining surface-bound PAP with spectrophotometry. In macrophages which had bound PAP at 4 degrees C and were warmed up for 5 min, the PAP was cleared from the cell surface and was found in endosome-like structures. When reincubated with PAP at 4 degrees C, such cells again bound the ligand on the cell surface, mainly in labyrinthic invaginations of the plasma membrane (synonyms: lacunae, caveolar indentations). Macrophages which had been warmed up for longer periods (30 and 60 min) showed the bound ligand all along the plasma membrane. Treatment of cells with monensin did not affect internalization of PAP, however, it decreased the ligand binding ability of macrophages considerably. These findings led us to assume an Fc receptor replenishment from a cytoplasmic pool.

Renal tubular biochemistry during acute and chronic metabolic alkalosis in the dog.

Acute metabolic alkalosis was induced in dogs by the infusion of sodium bicarbonate, 0.3 M. Chronic alkalosis was induced by chloride restriction and the administration of sodium bicarbonate and furosemide. In a third group of dogs, potassium was added to the regimen to prevent frank potassium depletion. Plasma bicarbonate ranged from 29.0 to 32.9 mM. In all three dog groups, renal ammoniagenesis fell by over 30%, which was consistent with a decrease in the renal uptake of glutamine. Glutamate was released in the renal vein and alanine production was decreased. Total production of ammonia was lowest in the animals given a potassium supplement where muscle potassium decreased much less than in the other chronic animals. Urinary ammonia excretion was very low in all three animal groups; this was related to an alkaline urine. However, this relationship was not entirely consistent and the low excretion of ammonia could also be related to decreased ammonia production by the renal tubular cell. In the renal cortical tissue (freeze-clamped), the concentration of glutamate did not change and that of alpha-ketoglutarate rose only in the animals supplemented with potassium. Malate rose in all groups. In all animals, renal tissue concentration of lactate and citrate rose while citrate excretion increased. We feel that glycolysis could play an important role in renal metabolism during acute and chronic metabolic alkalosis. We have proposed a unified theory to explain the metabolic changes that occur in lactate and citrate metabolism during metabolic alkalosis with a depressing effect on ammoniagenesis. Although citrate could be generated in the mitochondria from pyruvate, its oxidation is probably inhibited with exit and accumulation in the cytosol.

pH-dependent binding of peroxidase-antiperoxidase /PAP/ immune complex to Fc receptors of macrophages.

Rat peritoneal macrophages were incubated at 4 degrees C in media of varying pH-s containing rat peroxidase-antiperoxidase /PAP/ immune complex. Two binding maxima were found at pH 5.5 and 7.0, resp. There was a sharp decrease in binding between 5.5 and 5.0. The peak at pH 7.0 was found to be trypsin-sensitive.

Receptor-mediated pinocytosis of IgG and immune complex in rat peritoneal macrophages: an electron microscopic study.

The uptake mechanism of homologous IgG and immune complex, and the participation of coated vesicles in this process were studied in rat peritoneal macrophages. Peroxidase-antiperoxidase (PAP) immune complex produced in rat, and purified rat IgG adsorbed to gold particles (IgG-Au) were used as ligands. Freshly collected peritoneal macrophages were preincubated with the ligands at 4 degrees C, washed, warmed up to 37 degrees C, maintained in a serum-free culture medium for 5 sec to 30 min and subsequently fixed for electron microscopy. In the IgG-Au experiments, acid phosphatase reaction was also applied to identify lysosomes, and ruthenium red to trace membranes exposed to the extracellular space. At the end of the preincubation period PAP and IgG were found randomly distributed on the external surface of the plasma membrane. After warming up the cells to 37 degrees C, the ligands bound to the plasma membrane showed a tendency to move towards deep labyrinthic invaginations of the cell surface from where they were internalized via coated pits and coated vesicles. In the initial period, these structures seemed to be the primary carriers of the ligands. In the period between 5 and 10 min, ligands were concentrated in vacuoles (endosomes) located in the deeper cytoplasm, while after 30 min, they were present in large lysosome-like or multivesicular bodies, which were found to be acid phosphatase positive.

Influence of protein synthesis inhibitors on neutral red-induced autophagocytosis in chicken pancreatic acinar cells.

Administration of neutral red to chicks resulted in an appearance of autophagic vacuoles in the pancreatic exocrine cells, containing mainly fragments of the endoplasmic reticulum, as well as Golgi elements, mitochondria and zymogen granules. The time course of the morphological changes was followed. The translational inhibitors cycloheximide and emetine did not induce injury, even they were equally found to exert a dose dependent, temporary, preventive effect against autophagocytosis induced by neutral red. This protection might be related to the capability of the drugs for stabilizing the polyribosomes and thereby preserving the structural integrity of the granular endoplasmic reticulum membrane.