Effects of prolonged exposure to ammonia on fluid-phase,receptor-mediated,and adsorptive (non specific) endocytosis in cultured neuroblastoma cells

Summary The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.     

Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.     

Inhibition by glucocorticoids of endocytosis in a macrophage-like cell line

A macrophage-like cell line (P388D1) has been used to demonstrate that glucocorticoids inhibit the fluid-phase endocytosis of fluorescein-labeled dextran (FITC-dextran). Initial experiments demonstrated that the interaction of FITC-dextran with cells had all the features of fluid-phase uptake, ie, the amount taken up was proportional to the concentration in the medium, the uptake proceeded continuously with time and was blocked at 4 degrees C. Dexamethasone (10(-7) M) had no effect on endocytosis until 11 hours after addition of the steroid, when it inhibited the uptake of FITC-dextran by 35%. The amount of inhibition increased with longer exposure times to the hormone up to 50% after 22 hours. Although this effect on endocytosis was observed prior to any effect on growth of the cells, endocytosis as well as cell proliferation were inhibited in a dose dependent fashion. A preliminary survey of selected steroids has established that the inhibition of endocytosis was restricted to steroids of the glucocorticoid class. The key experiments were also performed using horseradish peroxidase instead of FITC-dextran with, essentially, identical results.     

Endocytosis at 0° C, 5° C,and 10° C in trophotaenial absorptive cells of goodeid embryos (Teleostei)

Summary The absorptive epithelium of the trophotaeniae of goodeid embryos is involved in the micropinocytotic uptake of protein macromolecules from the ovarian embryotrophe. Incubations of viable Xenoophorus captivus embryos in vitro with horseradish peroxidase (HRP) and/or cationized ferritin (CF) allows the tracing of the fluid-phase and receptor-mediated pathways, respectively. Effects of lowered temperature on both these endocytotic mechanisms have been investigated. At 10° C, trophotaenial absorptive cells (TACs) have a strong capacity to ingest marker proteins from double tracer media. Surface-bound ligands (CF) and solutes (HRP), taken up in primary pinocytic vesicles, are rapidly channelled to the endosomal compartment. Part of the ingested CF is segregated into dense apical tubules and small vesicles indicating that membrane recycling and transcytosis continue at 10° C. Adsorptive endocytosis of CF at 5° C proceeds at a decreased rate. After incubation periods of 30 min and 1 h, tracer molecules can be found in vesicular, tubular and vacuolar compartments of the apical endocytic zone. At 0° C, no uptake of ligand worth mentioning could be ascertained. Fluid-phase endocytosis, on the other hand, is observable at this temperature. Enzyme reaction product accumulates in flattened vacuoles rather than typical voluminous endosomes. After prolonged exposure to HRP, the epithelial junctional complex becomes leaky and the marker protein penetrates the intercellular space and the lateral lamellar membrane invaginations of TACs.Supported by the Deutsche Forschungsgemeinschaft     

The appearance of photosynthetic proteins in developing maize leaves

Soybean (Glycine max (L.) Merr.) protoplasts have been surface-labelled with cationized ferritin, and the fate of the label has been followed ultrastructurally. Endocytosis of the label occurs via the coated-membrane system. The pathway followed by the label, once it has been taken into the interior of the protoplast, appears to be similar to that found during receptor-mediated endocytosis in animal cells. Cationized ferritin is first seen in coated vesicles but rapidly appears in smooth vesicles. Labelled, partially coated vesicles are occasionally observed, indicating that the smooth vesicles may have arisen by the uncoating of coated vesicles. Structures which eventually become labelled with cationized ferritin include multivesicular bodies, dictyosomes, large smooth vesicles, and a system of partially coated reticula.Abbreviation CF cationized ferritin     

Fluid-phase endocytosis does not contribute to rapid fluid secretion in the malpighian tubules of the house cricket, Acheta domesticus.

When the Malpighian tubules (Mt) of the house cricket (Acheta domesticus) are treated with dibutyryl adenosine 3', 5'-cyclic monophosphate (db-cAMP; 1 mM), which causes a doubling in secretion rate, more than 50% of the cell volume is occupied by vesicles within 420 sec of exposure. In view of the fact that the increase in vesiculation occurs concomitantly with stimulated fluid transport, we set out to determine whether the vesicles are formed as a result of fluid-phase endocytosis (pinocytosis) and subsequently used to transport fluid to the lumen as one means of increasing transport rate. We used fluorescent fluid-phase markers (Lucifer Yellow Carbohydrazide [LYCH] and Alexa 488 hydrazide) and an electron dense marker (cationized ferritin) to elucidate the degree of endocytosis that occurred with db-cAMP stimulation. We found that, although some fluid is taken into the cells of the mid-tubule via endocytosis, it does not coincide with the level of vacuolation present in stimulated tubules. The amount of LYCH transported into the primary urine by the db-cAMP-stimulated Mt decreased by 40% as compared to the unstimulated transport, and the rate of transport of LYCH was only 30% of the unstimulated tubules. In summary, our findings do not support the theory that the majority of the vesicles or vacuoles comprise intracellular, endocytotic compartments formed via a basolateral endocytotic pathway. We also found no evidence to support the functioning of vesicles or vacuoles as transcellular "shuttling" mechanisms to move fluid from the basal region to the apical membrane and into the lumen.     

Intracellular transport of transferrin- and asialoorosomucoid-colloidal gold conjugates to lysosomes after receptor-mediated endocytosis

Proteins coupled to colloidal gold particles have been widely used to visualize the uptake and intracellular transport of specific ligands by receptor-mediated endocytosis. The intracellular route of lysosome-directed ligands such as asialoglycoproteins (ASGP) are apparently unaltered by conjugation to gold, but the pathway of transferrin, a ligand that normally recycles to the cell surface, was reported to be altered by conjugation to 15-20 nm gold. In this study, we sought to determine whether a smaller transferrin-gold probe would recycle, and whether it might enter the same endosomal and lysosomal compartments as does a larger, lysosome-directed ASGP gold probe by visualizing their simultaneous uptake in human hepatoma (HepG2) cells. In the same cells, endocytosis of fluid-phase protein was followed using the soluble tracer native ferritin; lysosomal compartments were identified by acid phosphatase cytochemistry; and cell surfaces were labeled with ruthenium red or cationized ferritin. During the first 10 min of uptake at 37 degrees C, specific receptor-bound ferrotransferrin (FeTf)-8 nm gold and asialoorosomucoid (ASOR)-20 nm gold were clustered together in coated pits and entered the same coated vesicles, smooth vesicles, and tubules in the peripheral cytoplasm. At later times, however, transferrin-gold did not return to the cell surface; unlike native transferrin, this gold probe accompanied ASOR-gold into multivesicular bodies (MVB). The MVBs that contained probes were at first devoid of acid phosphatase activity, but at 30 min, enzyme activity was detected in a few MVBs. Native ferritin was present, along with gold probes, in all compartments of the endocytic pathway. We conclude that the normal intracellular pathway of transferrin is altered by its association with a colloidal gold particle.     

Effect of cholesterol and its autooxidation derivatives on endocytosis and dipeptidyl peptidases of aortic endothelial cells.

The effects of cholesterol (CHO) and cholesterol autooxidation derivatives (CAD) on the endocytosis of cationized ferritin (CF) by endothelial cells have been investigated. The effect of both substances on the activity of lysosomal enzymes dipeptidyl peptidase I (DPP I) and dipeptidyl peptidase II (DPP II) was also studied. Treatment of rats with CAD induced striking alterations in the ultrastructure of endothelial cells and makes it impossible to analyze the effect of this toxin on endocytosis processes. In contrast, CHO-treated cells displayed a good ultrastructural preservation and showed an increased ability to endocyte ferritin, as compared with controls. Both DPP I and DPP II activities increased after 3 weeks of CAD or CHO treatment. Our results indicate that although CHO damage endothelial cells, the most important effects could be attributed to CAD which usually accompanies CHO-supplemented diets.     

Endocytic response of type I alveolar epithelial cells to hypertonic stress

We present plasma membrane (PM) internalization responses of type I alveolar epithelial cells to a 50 mosmol/l increase in tonicity. Our research is motivated by interest in ATI repair, for which endocytic retrieval of PM appears to be critical. We validated pharmacological and molecular tools to dissect the endocytic machinery of these cells and used these tools to test the hypothesis that osmotic stress triggers a pathway-specific internalization of PM domains. Validation experiments confirmed the fluorescent analogs of lactosyl-ceramide, transferrin, and dextran as pathway-specific cargo of caveolar, clathrin, and fluid-phase uptake, respectively. Pulse-chase experiments indicate that hypertonic exposure causes a downregulation of clathrin and fluid-phase endocytosis while stimulating caveolar endocytosis. The tonicity-mediated increase in caveolar endocytosis was associated with the translocation of caveolin-1 from the PM and was absent in cells that had been transfected with dominant-negative dynamin constructs. In separate experiments we show that hypertonic exposure increases the probability of PM wound repair following micropuncture from 82 ± 4 to 94 ± 2% (P < 0.01) and that this effect depends on Src pathway activation-mediated caveolar endocytosis. The therapeutic and biological implications of our findings are discussed.     

Expression of chloride channel, ClC-5, and its role in receptor-mediated endocytosis of albumin in OK cells

By using Western blot and RT-PCR analyses, the expression of ClC-5, a member of the ClC family of voltage-gated chloride channels, and its mRNA was detected in OK cells. The effect of chloride channel inhibitors on receptor-mediated endocytosis of albumin was examined in OK cells and compared to that of vacuolar H(+)-ATPase inhibitors. Accumulation of fluorescein-isothiocyanate (FITC)-albumin, a receptor-mediated endocytosis marker, was inhibited by 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), a chloride channel inhibitor, in a concentration-dependent fashion. In contrast, uptake of FITC-inulin, a fluid-phase endocytosis marker, was not affected by NPPB. Other chloride channel inhibitors, 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid and diphenylamine-2-carboxylic acid, also inhibited FITC-albumin uptake. NPPB, as well as a vacuolar H(+)-ATPase inhibitor bafilomycin A(1), caused a decrease in the affinity and in the maximal velocity of FITC-albumin uptake. These results suggest that chloride channel, most likely ClC-5, plays an important role in the receptor-mediated endocytosis of albumin in OK cells.     

Potassium depletion and hypertonic medium reduce "non-coated" and clathrin-coated pit formation, as well as endocytosis through these two gates

Intracellular potassium depletion inhibits receptor-mediated endocytotic processes occurring through clathrin-coated pits. Besides the clathrin-coated pit route, flask-shaped invaginations that do not bear a typical clathrin coat have been recently implicated in receptor-mediated endocytosis of cholera toxin. These invaginations are called "non-coated" to distinguish them from the typical clathrin-coated pits. In the present study, we have investigated whether "non-coated" invaginations are sensitive, as are clathrin-coated pits, to potassium depletion and whether hypertonic medium, which inhibits receptor-mediated endocytosis, also affects "non-coated" invaginations. We found that 1) both potassium depletion and hypertonic medium reduce "non-coated" invaginations on the cell surface; 2) similar to potassium depletion, hypertonic medium markedly decreases the number of clathrin-coated pits; 3) these changes are accompanied by an inhibition of the internalization (measured morphologically) of cholera toxin-gold through "non-coated" invaginations, as well as of alpha 2-macroglobulin-gold taken up by clathrin-coated pits; and 4) in addition, both the hypertonic medium and potassium depletion inhibit the uptake of horseradish peroxidase, a marker of fluid-phase endocytosis.     

Endocytosis in guinea pig seminal vesicle epithelial cells cultivated in chemically defined medium

We have developed a chemically defined monolayer culture system for guinea pig seminal vesicle epithelial cells (SVEP). The cells appeared as a polarized monolayer with apical microvilli, tight junctions and desmosome-like junctions, and often dilated intercellular spaces. SVEP expressed epithelial-specific cytokeratins as detected immunocytochemically. Growth was obtained during the first week of culture. In this period, the cells were exposed to unconjugated horseradish peroxidase (HRP), a ricin-peroxidase conjugate (Ri-HRP), or cationized ferritin (CF). HRP was endocytosed without binding to the SVEP surface (fluid-phase endocytosis) and was found mainly in multivesicular endosomes and lysosomes. Ri-HRP and CF, however, were endocytosed following binding to the cell surface. Initially these markers were present in multivesicular endosomes, but later also in smaller tubular and vesicular endosomes, some Golgi-associated elements (but not Golgi stacks), and lysosomes. We conclude that our SVEP culture system may be useful in further studies, on e.g. hormonal regulation of endocytosis and other processes of importance for SVEP maintenance and modulation of the seminal fluid in vivo.     

Headgroup-specific exposure of phospholipids in ABCA1-expressing cells

ABCA1 has been established to be required for the efflux of cholesterol and phospholipids to apolipoproteins such as apoA-I. At present, it is unclear whether ABCA1-mediated lipid exposure is specific with regard to lipid headgroups and whether it requires calcium activation and the presence of a lipid acceptor. In the present work, we found exofacial exposure of endogenous phosphatidylserine in the absence of apoA-I to be enhanced in ABCA1-GFP expressing MDCKII and HeLa cells compared with control cells. By using C6-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) (NBD)-labeled phospholipid analogues, we observed elevated redistribution of phosphatidylserine and phosphatidylethanolamine but not of phosphatidylcholine analogues from the cytoplasmic to the exoplasmic leaflet of the plasma membrane of ABCA1-GFP expressing cells. Whereas glyburide affected neither the level of exofacial endogenous PS nor the outward movement of the amino phospholipid analogues, the latter was sensitive to intracellular Ca2+ in ABCA1-GFP expressing cells, further enhancing outward analogue redistribution with respect to control cells. Both receptor-mediated endocytosis and fluidphase endocytosis were reduced in MDCKII cells expressing ABCA1-GFP. Glyburide raised the level of receptor-mediated endocytosis in the ABCA1-GFP expressing cell to the level of control cells in the absence of glyburide. In control cells, however, fluid-phase endocytosis but not receptor-mediated endocytosis was significantly reduced upon glyburide treatment.     

Endocytosis of cationized ferritin in human resting peripheral blood by T-lymphocytes

Summary We have examined the binding and internalization of cationized ferritin in T-lymphocytes of human peripheral blood, as a model for resting cells. After 30 min of incubation only 8% of endocytotic vesicles contain cationized ferritin. T-cells internalie the equivalent of their entire surface area in approximately 54 h, a longer time than is required by non-resting cells such as PHA-stimulated human lymphocytes. These tracer experiments suggest that the endocytosis of cationized ferritin by T-lymphocytes follows a lysosome pathway similar to that described for other cell types.     

Apico-basal osmotic gradient induces transcytosis in cultured renal collecting duct epithelium

The present experiments report the existence of an apico-basal plasma membrane shuttle in cultured renal collecting duct principal cell epithelium. Apical and basal perfusion under isotonic conditions, 290 mosm phosphate-buffered saline (PBS), has no effect on the shape of the epithelium. In contrast, gradient perfusion of the epithelium with 75 mosm PBS on the apical side and 290 mosm PBS on the basal side for 10 min alters the morphology of the epithelium by causing the originally columnar epithelial cells to become lower, the intercellular spaces to dilate, and the intracellular vesicles to enlarge. Perfusion of the epithelium with isotonic PBS in the presence of electron-dense cellular markers such as gold-coupled GPCDI antibody, recognizing a glycoprotein in the plasma membrane of collecting duct cells (W.W. Minuth, G. Lauer, S. Bachman and W. Kriz, Histochemistry 80:171-182, 1984), cationized ferritin (CF), horseradish peroxidase (HRP) and native ferritin (NF) for 10 min reveals their binding at the apical plasma membrane. Little endocytosis is observable. However, after labeling the luminal side by the cellular markers and following exposure to apical hypotonicity, 75 mosm PBS for 10 min, endocytosis of all markers is enhanced to a high degree. Furthermore, the gold-coupled GPCDI antibody and cationized ferritin are transported within vesicles unidirectionally through the epithelium and are exocytosed at the basolateral aspect, indicating the retrieval and possible translocation of apical plasma membrane. In contrast, volume markers such as NF and HRP are also endocytosed under osmotic gradient exposure, but are not seen to be transcytosed. Therefore, the function of this membrane pathway seems not to be related to water reabsorption, but may be part of a cellular response as protection against the osmotic gradient.     

Multiple pathways for ligand internalization in rat hepatocytes. II: Effect of hyperosmolarity and contribution of fluid-phase endocytosis.

In a companion report (Moss and Ward: J. Cell. Physiol 149:313-318, 1991) evidence was presented for multiple pathways for insulin internalization based on differences between the internalization of insulin and that of two other ligands, asialofetuin (Afet) and epidermal growth factor (EGF), in the presence of several perturbations of endocytosis. In the present study we have explored the characteristics of three internalization pathways and the contribution of each to overall insulin uptake. Freshly isolated hepatocytes were incubated with radiolabeled ligands in the presence of hyperosmolar sucrose, treatment that is thought to inhibit the coated pit pathway of endocytosis. Insulin internalization was decreased approximately 39%, but much greater decreases were observed with Afet (86%) and EGF (62%). Competition between uptake of radiolabeled and unlabeled insulin was observed in hyperosmolar-treated cells, suggestive of endocytosis by a receptor-mediated noncoated-pit pathway. Uptake of radiolabeled insulin that persisted in the presence of hyperosmolarity and high concentrations of unlabeled insulin suggested a third uptake pathway: fluid-phase endocytosis. A rate of fluid-phase endocytosis of 7.2 microL/hr/10(6) cells was determined from the uptake of the fluid-phase marker lucifer yellow. At high insulin concentrations (greater than or equal to 250 ng/ml), fluid-phase endocytosis appears to be the predominant pathway for insulin uptake, but at lower insulin concentrations (physiological) the coated pit and noncoated pit pathways are the primary routes for insulin internalization.     

Fractionation of endocytic vesicles and glucose-transporter-containing vesicles in rat adipocytes.

We subfractionated intracellular vesicles from rat adipocytes in order to examine the subcellular distribution of endocytic vesicles or endosomes with respect to insulin-regulatable glucose-transporter (GT)-containing vesicles [James, Lederman & Pilch (1987) J. Biol. Chem. 262, 11817-11824]. Vesicles mediating fluid-phase endocytosis sedimented as a single major peak of greater density than the single distinct peak of GT-containing vesicles. This difference was also apparent during cellular insulin exposure and after insulin removal. Endocytosis of insulin and IGF (insulin-like growth factor) II was also examined. In sucrose gradients, IGF II-containing vesicles were less dense than those containing internalized insulin. Receptor-mediated endocytic vesicles were distinct from fluid-phase endocytic vesicles, but overlapped with the GT-containing vesicles. Vesicles containing internalized ligand were further fractionated by agarose-gel electrophoresis after various times of internalization. At least three different vesicle subpopulations containing the iodinated ligands were resolved after 5 min of internalization. Endocytic vesicles containing rapidly internalized insulin (1.5 min at 37 degrees C) consistently co-migrated with GT-containing vesicles. These data indicate that fluid-phase and receptor-mediated endocytosis occur via different pathways in adipocytes. Furthermore, whereas the intracellular GT-containing vesicles are distinct from fluid-phase vesicles, a rapidly labelled pool of insulin-containing vesicles consistently co-fractionated with GT-containing vesicles when separation techniques based on size, density and charge were used. This suggests that the insulin receptor may directly interact with the intracellular GT-containing vesicles after insulin-induced endocytosis.     

Receptor-mediated endocytosis of phosphodiester oligonucleotides in the HepG2 cell line: evidence for non-conventional intracellular trafficking

Having identified an oligonucleotide (ON) receptor in the HepG2 cell line, we have re-examined here the kinetics of ON uptake, subcellular distribution and intracellular localisation in these cells, at concentrations relevant for the study of a receptor-dependent process. Kinetic parameters of ON endocytosis were comparable with those of the receptor-mediated endocytosis tracer, transferrin (uptake equilibrium, saturation with concentration, specific competition and rapid efflux) and were clearly distinct from those of fluid-phase endocytosis. By analytical subcellular fractionation, particulate ON showed a bimodal distribution after 2 h of uptake, with a low-density peak superimposed on the distribution of endosomes, and a high-density peak overlapping lysosomes. After an overnight chase, only the high-density peak remained, but it could be dissociated from lysosomes, based on its refractoriness to displacement upon chloroquine-induced swelling. After 2 h of uptake at 300 nM ON-Alexa, a punctate pattern was resolved, by confocal microscopy, from those of transferrin, of a fluid-phase tracer, and of vital staining of lysosomes by LysoTracker. At 3 µM ON-Alexa, its pattern largely overlapped with the fluid-phase tracer and LysoTracker. Taken together, these data suggest that ON may be internalised at low concentrations by receptor-mediated endocytosis into unique endosomes, then to dense structures that are distinct from lysosomes. The nature of these two compartments and their significance for ON effect deserve further investigation.     

Membrane retrieval in epithelial cells of isolated thyroid follicles.

Follicles from rat and pig thyroid glands were isolated by digestion with collagenase. The epithelial cells of isolated follicles maintain their structural and functional polarity as shown by incorporation of 3H-leucine and autoradiography. To trace the fate of surface membrane, isolated follicles were opened, stimulated with thyrotropin and incubated for various time intervals with cationized ferritin (CF), uncharged dextran, native ferritin (NF), and latex spheres (0.5 mum in diameter) which were either pre-coated with CF or added together with CF. Uncharged dextran and native ferritin did not bind to the luminal cell membrane, were taken up in small amounts and accumulated in lysosomes; anionic NF was not found in Golgi cisternae in contrast to uncharged dextran which occassionally reached a few Golgi stacks. CF bound rapidly and in clusters to the luminal plasmalemma, preferentially to coated pits, was taken up by endocytosis, accumulated in lysosomes after 5 min and reached the Golgi cisternae after 30 min. Latex spheres were taken up by engulfment through fusion of microvilli and reached the lysosomes. CF particles coating the latex spheres may detach at this station and reach the Golgi cisternae. The findings show that the route of small tracers depends on the charge of the tracer, in agreement with results obtained by Farquhar [8]. Vesicles carrying NF can be traced to lysosomes only, whereas vesicles containing uncharged dextran or - more conspicuously -CF also fuse with Golgi membranes. Large tracers (latex beads) reach only the lysosomes, but CF taken up with them may move to Golgi cisternae.     

Evidence for a decreased membrane recycling in the cells of renal proximal tubules exposed to high concentrations of ferritin

Summary The present study was performed to investigate whether membrane recycling via the dense apical tubules in cells of renal proximal tubules could be modified after exposure to large amounts of cationized ferritin. Proximal tubules in the rat kidney were microinfused in vivo with cationized ferritin for 10 or 30 min and then fixed with glutaraldehyde by microinfusion, or proximal tubules were microinfused with ferritin for 30 min and then fixed 2 h thereafter. The tubules were processed for electron microscopy, and the surface density and the volume density of the different cell organelles involved in endocytosis were determined by morphometry. The morphometric analyses showed that after loading of the endocytic vesicles with ferritin the surface density of dense apical tubules decreased to about 50% of the original value. However, 2 h later when ferritin had accumulated in the lysosomes the surface density of dense apical tubules had returned to control values. Furthermore, cationized ferritin was virtually absent from the Golgi region, indicating that the Golgi apparatus in these cells does not participate in membrane recycling. In conclusion, the present study shows that membrane recycling in renal proximal tubule cells can in part be inhibited by loading the endocytic vacuoles with ferritin.