Apical membrane endocytosis via coated pits is stimulated by removal of antidiuretic hormone from isolated,perfused rabbit cortical collecting tubule

Summary Antidiuretic hormone increases the water permeability of the cortical collecting tubule and causes the appearance of intramembrane particle aggregates in the apical plasma membrane of principal cells. Particle aggregates are located in apical membrane coated pits during stimulation of collecting ducts with ADHin situ. Removal of ADH causes a rapid decline in water permeability. We evaluated apical membrane retrieval associated with removal of ADH by studying the endocytosis of horseradish peroxidase (HRP) from an isotonic solution in the lumen. HRP uptake was quantified enzymatically and its intracellular distribution examined by electron microscopy. When tubules were perfused with HRP for 20 min in the absence of ADH, HRP uptake was 0.5±0.3 pg/min/m tubule length (n=6). The uptake of HRP in tubules exposed continuously to ADH during the 20-min HRP perfusion period was 1.3±0.8 pg/min/m (n=8). HPR uptake increased markedly to 3.2±1.1 pg/min/m (n=14), when the 20-min period of perfusion with HRP began immediately after removal of ADH from the peritubular bath. Endocytosis of HRP occurred in both principal and intercalated cells via apical membrane coated pits. We suggest that the rapid decline in cortical collecting duct water permeability which occurs following removal of ADH is mediated by retrieval of water permeable membrane via coated pits.     

The AQP2 water channel: Effect of vasopressin treatment,microtubule disruption,and distribution in neonatal rats

Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.     

Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer

Summary The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled cancanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4° C, incubated for different times in Con-A free medium at 37° C, and prepared for electron microscopy. After 1 min incubation at 37° C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37° C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.     

Evidence for the sorting of endocytic vesicle contents during the receptor-mediated transport of IgG across the newborn rat intestine

Fc receptors on the luminal membranes of intestinal epithelial cells in the neonatal rat mediate the vesicular transfer of functionally intact IgG from the intestinal lumen to the circulation. In addition, there is a low level of nonselective protein uptake, but in this case transfer does not occur. To determine whether a specialized class of endocytic vesicles could account for the selective transfer of IgG, mixtures of IgG conjugated to ferritin (IgG-Ft) and unconjugated horseradish peroxidase (HRP) were injected together into the proximal intestine of 10-d-old rats, and the cellular distribution of these two different tracers was determined by electron microscopy. Virtually all apical endocytic vesicles contained both tracers, indicating simultaneous uptake of both proteins within the same vesicle. However, only IgG-Ft bound to the apical plasma membrane, appeared within coated vesicles at the lateral cell surface, and was released from cells. HRP did not bind to the luminal membrane and was not transferred across cells but was confined to apical lysosomes as identified by acid phosphatase and aryl sulfatase activities. To test the possibility that the binding of IgG to its receptor stimulated endocytosis, HRP was used as a fluid volume tracer, and the amount of HRP taken up by cells in the presence and absence of IgG was measured morphologically and biochemically. The results demonstrate that endocytosis in these cells is constitutive and occurs at the same level in the absence of IgG. The evidence presented indicates that the principal selective mechanism for IgG transfer is the binding of IgG to its receptor during endocytosis. Continued binding to vesicle membranes appears to be required for successful transfer because unbound proteins are removed from the transport pathway before exocytosis. These results favor the proposal that IgG is transferred across cells as an IgG-receptor complex.     

Hypertonicity is involved in redirecting the aquaporin-2 water channel into the basolateral,instead of the apical,plasma membrane of renal epithelial cells

In renal collecting ducts, vasopressin increases the expression of and redistributes aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical membrane, leading to urine concentration. However, basolateral membrane expression of AQP2, in addition to AQP3 and AQP4, is often detected in inner medullary principal cells in vivo. Here, potential mechanisms that regulate apical versus basolateral targeting of AQP2 were examined. The lack of AQP2-4 association into heterotetramers and the complete apical expression of AQP2 when highly expressed in Madin-Darby canine kidney cells indicated that neither heterotetramerization of AQP2 with AQP3 and/or AQP4, nor high expression levels of AQP2 explained the basolateral AQP2 localization. However, long term hypertonicity, a feature of the inner medullary interstitium, resulted in an insertion of AQP2 into the basolateral membrane of Madin-Darby canine kidney cells after acute forskolin stimulation. Similarly, a marked insertion of AQP2 into the basolateral membrane of principal cells was observed in the distal inner medulla from normal rats and Brattleboro rats after acute vasopressin treatment of tissue slices that had been chronically treated with vasopressin to increase interstitial osmolality in the medulla, but not in tissues from vasopressin-deficient Brattleboro rats. These data reveal for the first time that chronic hypertonicity can program cells in vitro and in vivo to change the insertion of a protein into the basolateral membrane instead of the apical membrane.     

Endocytic vesicles from renal papilla which retrieve the vasopressin- sensitive water channel do not contain a functional H+ ATPase

The water permeability of the kidney collecting duct epithelium is regulated by vasopressin (VP)-induced recycling of water channels between an intracellular vesicular compartment and the plasma membrane of principal cells. To test whether the water channels pass through an acidic endosomal compartment during the endocytic portion of this pathway, we measured ATP-dependent acidification of FITC-dextran-labeled endosomes in isolated microsomal fractions from different regions of Brattleboro rat kidneys. Both VP-deficient controls and rat treated with exogenous VP were examined. ATP-dependent acidification was not detectable in endosomes containing water channels from distal papilla (osmotic water permeability Pf = 0.038 +/- 0.004 cm/s). In contrast, the addition of ATP resulted in a strong acidification of renal cortical endosomes (pHmin = 5.8, initial rate = 0.18-0.25 pH U/s). Acidification of cortical endosomes was reversed with nigericin and strongly inhibited by N-ethyl-maleimide. Passive proton permeability was similar and low in both cortical and papillary endosomes from rats treated or not treated with VP. The fraction of labeled endosomes present in microsomal preparations was determined by fluorescence imaging microscopy of microsomes nonspecifically bound to poly-l-lysine-coated coverslips and was 25% in cortical preparations compared to 14% (+VP) and 9% (-VP) in papillary preparations. The fraction of cortical endosomes was enriched 1.5-fold by immunoabsorption to coverslips coated with mAbs against the bovine vacuolar proton pump. In contrast, the fraction of papillary endosomes was depleted more than twofold by immunoabsorption to identical coverslips. Finally, sections of distal papilla stained with antibodies against the lysosomal glycoprotein LGP120 showed that most of the entrapped FITC-dextran did not colocalize with this lysosomal protein. These results demonstrate that vesicles which internalize water channels in kidney collecting duct principal cells lack functional proton pumps, and do not deliver the bulk of their FITC-dextran content to lysosomes. The data suggest that the principal cell contains a specialized nonacidic apical endocytic compartment which functions primarily to recycle membrane components, including water channels, to the plasma membrane.     

The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin were localized in coated pits. The lysosomal membrane glycoprotein, lgp120 (Lewis, V., S. A. Green, M. Marsh, P. Vihko, A. Helenius, and I. Mellman, 1985, J. Cell Biol., 100: 1839-1847) was restricted to lysosomes where it co-localized with beta-glucuronidase. Endosomes, identified by preloading with HRP injected 5-15 min before rats were killed, did not contain detectable amounts of any antigen tested. The distribution of the same proteins was also determined in rats given sodium maleate, which is known to slow or reduce protein absorption by the proximal tubule and to cause vacuolation of the endocytic apparatus. After maleate treatment the distribution of microvillar and lysosomal markers was unchanged, but the coated pit markers were redistributed--gp330 was concentrated in newly formed apical vacuoles, and clathrin was diffusely distributed in the apical cytoplasm or on apical coated vesicles. These findings indicate that the membrane composition of microvilli, coated pits, endosomes, and lysosomes is distinctive in the proximal tubule cell; and that gp330, unlike other known coated pit membrane components, is not transferred to endosomes during endocytosis. After maleate treatment, the coated pits lose their clathrin coats, and the corresponding membrane is internalized.     

Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis

Cytochalasin D was found to reduce the endocytosis of ricin and the fluid phase markers [14C]sucrose and Lucifer Yellow in Vero cells without reducing the uptake of transferrin. The number of coated pits at the plasma membrane was not affected by the treatment. Cytochalasin D also reduced the endocytosis of ricin in cells where uptake of transferrin from coated pits was blocked by low cytosolic pH. Colchicine had a similar effect as cytochalasin D. Both drugs inhibited the exocytosis of ricin from the cells, and they reduced the rate by which ricin intoxicated the cells. Cytochalasin D had essentially no effect on the ability of the cells to bind transferrin, whereas colchicine reduced the binding to some extent. Epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the endocytic uptake of ricin in A431 cells both under normal culture conditions and when the coated pit/coated vesicle pathway was blocked by acidification of the cytosol. In contrast, EGF and TPA had no stimulatory effect on the uptake of transferrin at normal cytoplasmic pH, and they did not abolish the ability of low cytoplasmic pH to inhibit endocytic uptake of transferrin. The results indicate that cytochalasin D and colchicine selectively inhibit endocytic uptake from non-clathrin-coated areas of the cell membrane whereas EGF and TPA stimulate it. The data support the view that there are different endocytic mechanisms, and they indicate that at least in some cell types the non-clathrin-coated endocytosis can be modulated.     

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.     

Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.     

Fluid phase endocytosis of [125I]iodixanol in rat liver parenchymal, endothelial and Kupffer cells

Endocytosis of [125I]iodixanol was studied in vivo and in vitro in rat liver cells to determine fluid phase endocytic activity in different liver cells (hepatocytes, Kupffer cells and endothelial cells). The Kupffer cells were more active in the uptake of [l25I]iodixanol than parenchymal cells or endothelial cells. Inhibition of endocytic uptake via clathrin-coated pits (by potassium depletion and hypertonic medium) reduced uptake of [125I]iodixanol much more in Kupffer cells and endothelial cells than in hepatocytes. To gain further information about the importance of clathrin-mediated fluid phase endocytosis, the expression of proteins known to be components of the endocytic machinery was investigated. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, endothelial cells and Kupffer cells were found to express approximately fourfold more rab4, rab5 and rab7 than parenchymal cells, while clathrin was expressed at a higher level in endothelial cells than in Kupffer cells and hepatocytes. Using electron microscopy it was shown that liver endothelial cells contained approximately twice as many coated pits per membrane unit than the parenchymal and Kupffer cells, thus confirming the immunoblotting results concerning clathrin expression. Electron microscopy on isolated liver cells following fluid phase uptake of horseradish peroxidase (HRP) showed that HRP-containing organelles had a different morphology in the different cell types: In the liver endothelial cells HRP was in small, tubular endosomes, while in Kupffer cells HRP was mainly found in larger structures, reminiscent of macropinosomes. Parenchymal cells contained HRP in small vacuolar endosomes with a punctuated distribution. In conclusion, we find that the Kupffer cells and the endothelial cells have a higher pinocytic activity than the hepatocytes. The hepatocytes do, however, account for most of the total hepatic uptake. The fluid phase endocytosis in liver endothelial cells depends mainly on clathrin-mediated endocytosis, while the parenchymal cells have additional clathrin-independent mechanisms that may play an important role in the uptake of plasma membrane components. In the Kupffer cells the major uptake of fluid phase markers seems to take place via a macropinocytic mechanism.     

Polarization of gelsolin and actin binding protein in kidney epithelial cells

Vasopressin regulates transepithelial osmotic water permeability in the kidney collecting duct and in target cells in other tissues. In the presence of hormone, water channels are inserted into an otherwise impermeable apical plasma membrane and the apical surface of these cells is dramatically remodelled. Because cytochalasin B and D greatly reduce the response of these cells to vasopressin, actin filaments are believed to participate in the events leading to an increase in transepithelial water permeability. Modulation of the actin filamentous network requires the concerted action of specific actin regulatory proteins, and in the present study we used protein A-gold immunocytochemistry to localize two important molecules, gelsolin and actin binding protein (ABP), in epithelial cells of the kidney inner medulla. Gelsolin and, to a lesser extent, ABP were concentrated in clusters in the apical cell web of principal cells of the collecting duct. Aggregates of gold particles were often associated with the cytoplasmic side of plasma membrane regions forming surface extensions or microvilli. The basolateral plasma membrane was labeled to a much lesser extent than the apical plasma membrane. In the thin limbs of Henle, ABP was localized over the apical plasma membrane in ascending limbs, but gelsolin labeling was weak in these cells. In thin descending limbs, the pattern of labeling was completely reversed, with abundant apical gelsolin labeling but only weak ABP immunolabeling. Although the significance of the distribution of actin regulatory proteins in thin limbs is unknown, the abundance and the predominantly apical polarization of both ABP and gelsolin in principal cells of the collecting duct is consistent with a role of the actin cytoskeleton in the mechanism of vasopressin actin.     

Hyperosmolarity inhibits galactosyl receptor-mediated but not fluid phase endocytosis in isolated rat hepatocytes

We have investigated the effects of hyperosmolarity induced by sucrose on the fluid phase endocytosis of the fluorescent dye lucifer yellow CH (LY) and the endocytosis of 125I-asialo-orosomucoid (ASOR) by the galactosyl receptor system in isolated rat hepatocytes. Continuous uptake of LY by cells at 37 degrees C is biphasic, occurs for 3-4 h, and then plateaus. Permeabilized cells or crude membranes do not bind LY at 4 or 37 degrees C. Intact cells also do not accumulate LY at 4 degrees C. The rate and extent of LY accumulation are concentration- and energy-dependent, and internalized LY is released from permeabilized cells. Efflux of internalized LY from washed cells is also biphasic and occurs with halftimes of approximately 38 and 82 min. LY is taken up into vesicles throughout the cytoplasm and the perinuclear region with a distribution pattern typical of the endocytic pathway. LY, therefore, behaves as a fluid phase marker in hepatocytes. LY has no effect on the uptake of 125I-ASOR at 37 degrees C. The rate of LY uptake by cells in suspension is not affected for at least 30 min by up to 0.2 M sucrose. The rate of endocytosis of 125I-ASOR, however, is progressively inhibited by increasing the osmolality of the medium with sucrose (greater than 98% with 0.2 M sucrose; Oka and Weigel (1988) J. Cell. Biochem. 36, 169-183). Hyperosmolarity completely inhibits endocytosis of 125I-ASOR by the galactosyl receptor, whereas fluid phase endocytosis of LY is unaffected. Cultured hepatocytes contained about 100 coated pits/mm of apical membrane length as assessed by transmission electron microscopy. In the presence of 0.4 M sucrose, only 17 coated pits/mm of membrane were observed, an 83% decrease. Only a few percent of the total cellular fluid phase uptake in hepatocytes is due to the coated pit endocytic pathway. We conclude that the fluid phase and receptor-mediated endocytic processes must operate via two separate pathways.     

Endocytosis in the uterine luminal and glandular epithelial cells of mice during early pregnancy

We have localized horseradish peroxidase (HRP) in the mouse uterus after intravenous administration on days 1 and 5 of pregnancy in an effort to understand how serum proteins reach the uterine lumen. Direct movement of HRP into uterine and glandular lumina was blocked by the epithelial tight junctions on both days. In luminal and glandular epithelial cells at both times, HRP was localized in endocytic vesicles along the basolateral membranes, multivesicular bodies (mvb), elongated dense bodies below the nucleus (bdb), and many small vesicles near the apical surface of the cells. The uptake of HRP was most extensive in the luminal epithelium on day 1: the number of tracer-containing apical vesicles and bdb was largest, and there were also clusters of vesicles containing the tracer above the nucleus. Acid phosphatase was localized on day 1 in mvb and bdb in both cell types, indicating that these structures are lysosomes. It appeared that HRP followed two pathways after basolateral endocytosis by the epithelial cells: it was transported to the apical region of the cells, where it was present in small vesicles that may release their contents into the uterine or glandular lumina, or it was transported to lysosomes. To investigate whether macromolecules may be transported from the uterine lumen to the stroma, we also studied endocytosis at the apical pole of luminal epithelial cells after intraluminal injection of HRP. There was no detectable uptake of HRP from the lumen on day 1, and no tracer was detected in the intercellular spaces or basement membrane region. On day 5, a large amount of HRP was taken up from the lumen into apical endocytic vesicles, mvb, and dense bodies, but tracer was not present in the Golgi apparatus, lateral intercellular spaces, or the basement membrane region at the times studied. These observations indicate that there was no transport of luminal macromolecules to the uterine stroma on day 1, while the possibility of transport on day 5 requires further study.     

Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.     

Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells

In parotid acinar cells, horseradish peroxidase (HRP) administered via the main excretory duct is endocytosed from the apical cell surface in smooth C- or ring-shaped vesicles (Oliver, C. and A. R. Hand. 1979. J. Cell Biol. 76:207). These vesicles ultimately fuse with lysosomes adjacent to the Golgi apparatus. The present investigation extends these findings and examines the uptake and fate of intravenously injected HRP from the lateral and basal cell surfaces of resting and stimulated parotid and pancreatic acinar cells from rats and mice. Isoproterenol and pilocarpine were used to stimulate the parotid gland and the pancreas, respectively. HRP was internalized in smooth and coated vesicles primarily in areas of membrane infoldings. Both the number of coated vesicles and the amount of tracer internalized increased markedly following secretagogue administration. In both resting and stimulated cells, the HRP was rapidly sequestered in a unique system of basally located lysosomes that possess trimetaphosphatase activity, but not acid phosphatase activity. At 1-3 h after HRP administration, reaction product was also found in multivesicular bodies, vesicles, and lysosomes adjacent to the Golgi apparatus. With time, more HRP was localized in Golgi-associated lysosomes. By 6-7 h, tubules in the apical cytoplasm of stimulated cells contained HRP reaction product. When native ferritin was administered retrogradely and HRP injected intravenously, both tracers could be localized in the same lysosome after 4-5 h, indicating that material taken in from all cell surfaces mixes in Golgi-associated lysosomes. The results of this study suggest that two separate and distinct endocytic pathways exist in exocrine acinar cells: one involves membrane retrieval from the apical cell surface; and the other is a stimulation-dependent process at the lateral and basal cell surfaces.     

Morphometric analysis of coated pits and vesicles in the proximal and distal caput epididymidis

A morphometric analysis of coated and uncoated structures found in the apical portion of principal cells from both the proximal and distal caput epididymidis has been carried out. Almost all endocytic, coated vesicles are found within 1 micron of the luminal surface of principal cells and the volume fraction of these and of uncoated vesicles is much greater in the proximal caput epididymidis. A serial section analysis indicated that many coated "vesicles" are tangentially sectioned coated pits and that a complex network of interconnected vesicular and tubular structures exists in the apical cytoplasm. Efferent duct ligation has no effect on the number of size of large coated and uncoated vesicles in either the proximal or distal caput epididymidis, indicating that substances delivered to principal cells from the lumen are not required to maintain the endocytic machinery. However, this treatment does result in a considerable increase in the number of large coated vesicles associated with the basal surface of principal cells from the proximal but not the distal caput epididymidis. The volume fraction of small, presumably exocytic, coated vesicles is significantly greater in the apical cytoplasm of cells from the distal caput epididymidis in control animals. Efferent duct ligation results in a significant increase in the volume fraction of these vesicles in the proximal but not distal caput epididymidis. These results show that there are marked differences in structure among principal cells from these two regions of the epididymis and that this may reflect differences in control and function.     

Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.