The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

THE INTERACTION OF SOLUBLE HORSERADISH PEROXIDASE WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The in vitro interaction of soluble horseradish peroxidase (HRP) with homogeneous mono layers of mouse macrophages has been studied using sensitive biochemical and cytochemical techniques. The compartmentalization of HRP in extracellular and intracellular sites has been quantitatively evaluated. A significant fraction is bound to a serum-derived layer, which coats the surface of culture vessels and may be removed by appropriate washes. Macrophages interiorize HRP as a solute in pinocytic vesicles without appreciable binding of the glycoprotein to the plasma membrane. Uptake is directly proportional to the concentration of HRP in the culture medium. 1 x 10⁶ cells ingest 0.0025% of the administered load per hr over a wide range of concentrations. Cytochemically, all demonstrable HRP is sequestered within the endocytic vesicles and secondary lysosomes of the vacuolar apparatus. After uptake, the enzymatic activity of HRP is inactivated exponentially with a half-life of 7–9 hr, until enzyme is no longer detectable. When macrophages have pinocytosed trace-labeled HRP-¹²⁵I, cell-associated isotope disappears with a t ½ of 20–30 hr and they release monoiodotyrosine-¹²⁵I into the culture medium. We were unable to obtain evidence that significant amounts of HRP (>2%) can be exocytosed after uptake, can exist intact on the cell surface, or can be digested extracellularly. It is difficult to reconcile these observations with several of the postulated mechanisms whereby macrophages are thought to play a prominent role in the induction of an immune response.     

THE INTERACTION OF PARTICULATE HORSERADISH PEROXIDASE (HRP)-ANTI HRP IMMUNE COMPLEXES WITH MOUSE PERITONEAL MACROPHAGES IN VITRO

The uptake, distribution, and fate of particulate horseradish peroxidase (HRP)-anti HRP aggregates has been studied in homogeneous monolayers of mouse macrophages in vitro. Macrophages rapidly interiorize the immune complexes after binding to the cell surface. The rate of interiorization is maximal for complexes formed in a broad zone of 4-fold antibody excess to equivalence and corresponds to a rate of 10% of the administered load/10⁶ cells per hour. This rate is 4000-fold greater than the uptake of soluble HRP. The binding and endocytosis of HRP-anti HRP by macrophages is mediated by the trypsin insensitive F_c receptor. Cytochemically, intracellular HRP is localized within membrane bound vacuoles. After uptake of HRP, the enzymatic activity is degraded exponentially with a half-life of 14–18 hr until enzyme is no longer detectable. This half-life is twice as long as that previously observed for soluble uncomplexed HRP and is related to the combination of HRP with anti-HRP rather than the absolute amounts of enzyme or antibody ingested. The half-life of HRP-¹²⁵I was 30 hr. Exocytosis of cell associated enzyme or TCA precipitable counts was not detected, nor were persistent surface complexes demonstrable. The extensive capacity of macrophages to interiorize and destroy large amounts of antigen after the formation of antibody illustrates a role of this cell in the efferent limb of the immune response.     

Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macrophages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Acid-phosphatase activity of reticular cells and macrophages in the lymph node of the rat after ingestion of mast-cell granules

Summary Acid phosphatase (ACPase) was ultracytochemically demonstrated in the lymph-node sinus reticular cells and macrophages of rats. After the uptake of horseradish peroxidase (HRP), marked ACPase activities were seen in both reticular cells and macrophages, although only sparse ACPase activity was detected in the reticular cells of the control. After the injection of HRP into the footpad, the mast cells in the regional lymph node became degranulated, and the released granules were taken up by reticular cells and macrophages. In macrophages, these taken-up mast-cell granules exhibited ACPase reaction products, whereas none of the granules taken up by reticular cells showed ACPase activity. The heparin-protamine complex was also engulfed by reticular cells and macrophages, and ACPase activity was demonstrable in the complex taken up by both types of cell. It is probable that, as is the case in macro-phages, reticular cells in the lymph-node sinuses take up and digest foreign substances through the formation of phagolysosomes, but they do not digest granules originating from the mast cells in the lymph node of the same animal.     

Reversible pinocytosis of horseradish peroxidase in lymphoid cells

A detailed study of fluid phase endocytosis of horseradish peroxidase (HRP) in rat lymph node cells (LNC) is presented in this paper. Preliminary experiments have shown that HRP was internalized by non-receptor-mediated endocytosis and interacted minimally or not at all with plasma membrane of LNC, and can then be considered as a true fluid phase marker for these cells. Kinetics of uptake of HRP was found not to be linear with incubation time at 37 degrees C and deviation from linearity can be attributed to constant exocytosis of HRP. The kinetics of exocytosis cannot be described by a single exponential process. Rather, a minimum of two exponentials is required to account for exocytosis. This suggests that at least two intracellular compartments are involved in this process. The first turns over very rapidly with a t 1/2 release of about 3 min and is saturated after 10 min of exposure with HRP. The second, which turns over very slowly, is characterized by a t 1/2 release of about 500 min and accounts for the intracellular accumulation of HRP. Similar biphasic kinetics of exocytosis were observed with unfractionated LNC, with T lymphocyte-enriched LNC and with lymphocytes purified according to their density. This suggests that most, if not all, LNC are able to release HRP and that each cell type is endowed with the two intracellular compartments. Kinetics of uptake of HRP in these two compartments indicated that they are probably filled by two endocytic pathways, at least partially independent. Taken together, these results seem to indicate that a rapid membrane recycling occurs in lymphocytes. Furthermore, the weak base ammonium chloride and the carboxylic ionophore monensin were shown in our study to inhibit fluid phase endocytosis of HRP. The inhibition was time-dependent and required a preincubation of the cells with the drugs to be observed. Our results suggest that a perturbation of the vesicular traffic or a sequestration of membranes involved in HRP uptake is induced by these drugs. Under these conditions the release of cell-associated HRP was also reduced and to the same extent as the inhibition of uptake. Distribution of HRP between the two compartments and the t 1/2 release of HRP from either compartment were not perturbed. Taken together these results seem to indicate that exocytosis is not specifically affected by these drugs. Inhibition of uptake in drug-treated cells could result from a general decrease of membrane recycling or to the formation of smaller pinocytic vesicles with a different surface to volume ratio.     

Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle

The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 degrees C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 microM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 micrograms/ml approx. 0.25 microM), is inhibited greater than or equal to 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 microM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 microM), trifluoperazine (25 microM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.     

Peroxidases enhance macrophage-mediated cytotoxicity via induction of tumor necrosis factor

Tumor necrosis factor (TNF) is a monokine which is involved in macrophage-mediated cytotoxicity (MMC). We have previously reported that peroxidases can activate thioglycollate-induced macrophages to the tumoricidal state in vitro. The present study was undertaken in an attempt to correlate peroxidase-induced MMC with production of TNF. Horseradish peroxidase (HRP) was used as the principal model for these studies. Resident and thioglycollate-induced macrophages exposed to peroxidases were examined for both MMC against 3T12 cells and production of TNF. Thioglycollate-induced macrophages exposed to HRP, bovine lactoperoxidase, or human myeloperoxidase demonstrated enhanced secretion of TNF. When exposed to HRP, both resident and thioglycollate-induced macrophages secreted significant amounts of TNF and acquired the ability to lyse 3T12 cells. However, resident macrophages were considerably less efficient in both their cytotoxic activity and TNF secretion. Macrophage-mediated cytotoxicity was eliminated by the addition of specific antisera to TNF. In addition, replacement of culture supernatants within 24 hr after exposure of the macrophages to HRP increased tumor cell killing in the absence of additional detectable TNF production, suggesting that other factors may be involved in peroxidase-induced MMC. These results indicate that TNF is intimately associated with peroxidase-induced MMC and suggest a possible role for peroxidases as immunomodulators via augmentation of macrophage capacities and functions.     

Monensin and chloroquine inhibit transfer to lysosomes of endocytosed macromolecules in cultured mouse peritoneal macrophages

The effects of the Na+/H+ ionophore monensin and the weak base chloroquine on lysosomal uptake of endocytosed macromolecules were studied in cultured mouse peritoneal macrophages using horseradish peroxidase (HRP) and ferritin as exogenous tracers. The lysosomes were first loaded with HRP using a pulse-chase protocol. The cells were then exposed to ferritin for 30 to 120 min, either in control medium or in medium containing 3 microM monensin or 50 microM chloroquine. Semiquantitative electron microscopic analyses indicated that the uptake of ferritin into HRP-labeled lysosomes was inhibited in the drug-treated cells, and that the tracer particles accumulated in endosomes. At the same time the volume density of the endosomes was increased, fourfold by monensin and threefold by chloroquine; with the latter drug there was also an increase in lysosome volume density. Further, both drugs decreased the rate of endocytosis as measured biochemically, but not in proportion to the reduction of lysosomal ferritin uptake. After withdrawal of the drugs, cell morphology returned to normal and transfer of ferritin from endosomes to HRP-labeled lysosomes was resumed. The recovery was more rapid and complete in monensin-treated than in chloroquine-treated cells. On the basis of these findings and earlier investigations demonstrating that monensin and chloroquine both raise the pH in acid cell compartments, it is suggested that the transfer of soluble and not only membrane-bound macromolecules from endosomes to lysosomes is modulated by the pH in these organelles.     

Protamine induced intracellular uptake of horseradish peroxidase and vacuolation in mouse skeletal muscle in vitro

Summary The uptake in vitro of horseradish peroxidase (HRP) in mouse skeletal muscle was examined by electron microscopy and chemical determination.In muscles exposed to an HRP solution for 60 min at +37°C, HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition HRP was found within the transverse tubules. Exposure to HRP for 30 min at +37°C followed by HRP together with a polycationic protein (protamine) for 30 min at +37°C caused an intracellular vesicular uptake of HRP. Intracellular HRP was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular HRP uptake or muscle cell vacuolation could not be detected in the absence of protamine or when the incubation temperature was + 4°C. Chemical determination of HRP uptake was in general agreement with the morphological results. The uptake of HRP in the presence of protamine was stimulated at +31°C and blocked at +4°C.The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis.     

Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications

Summary Two protein antigens, horseradish peroxidase (HRP) and ferritin, have been administered to the digestive tract of carp. Electron-microscopical observations reveal considerable absorption of both antigens in the second segment of the gut (from 70 to 95% of the total length) and also, although to a lesser extent, in the first segment (from 0 to 70% of the total length). Even when administered physiologically with food, a large amount of ferritin is absorbed by enterocytes in the second gut segment.HRP and ferritin are processed by enterocytes in different ways. HRP seems to adhere to the apical cell membrane, probably by binding to receptors, and is transported in vesicles to branched endings of lamellar infoldings of the lateral and basal cell membrane. Consequently, most of the HRP is released in the intercellular space where it contacts intra-epithelial lymphoid cells. Only small amounts of HRP become localized in secondary lysosomes of enterocytes. Ferritin does not bind to the apical cell membrane; after uptake by pinocytosis, it is present in small vesicles or vacuoles that appear to fuse with lysosome-like-bodies. In the second segment, intact ferritin ends up in the large supranuclear vacuoles (after 8 h), where it is digested slowly. Although no ferritin is found in the intercellular space, ferritin-containing macrophages are present between the epithelial cells, in the lamina propria and also to a small extent in the spleen. The transport of antigens from the intestinal lumen, through enterocytes, to intra-epithelial lymphoid cells or macrophages may have immunological implications, such as induction of a local immune response and prospectives for oral vaccination.     

Macrophages use different internalization mechanisms to clear apoptotic and necrotic cells

The present study characterized two different internalization mechanisms used by macrophages to engulf apoptotic and necrotic cells. Our in vitro phagocytosis assay used a mouse macrophage cell line, and murine L929sAhFas cells that are induced to die in a necrotic way by TNFR1 and heat shock or in an apoptotic way by Fas stimulation. Scanning electron microscopy (SEM) revealed that apoptotic bodies were taken up by macrophages with formation of tight fitting phagosomes, similar to the 'zipper'-like mechanism of phagocytosis, whereas necrotic cells were internalized by a macropinocytotic mechanism involving formation of multiple ruffles directed towards necrotic debris. Two macropinocytosis markers (Lucifer Yellow (LY) and horseradish peroxidase (HRP)) were excluded from the phagosomes containing apoptotic bodies, but they were present inside the macropinosomes containing necrotic material. Wortmannin (phosphatidylinositol 3'-kinase (PI3K) inhibitor) reduced the uptake of apoptotic cells, but the engulfment of necrotic cells remained unaffected. Our data demonstrate that apoptotic and necrotic cells are internalized differently by macrophages.     

Contribution of functional voltage-gated Na+ channel expression to cell behaviors involved in the metastatic cascade in rat prostate cancer: II. Secretory membrane activity

The secretory membrane activities of two rat prostate cancer cell lines of markedly different metastatic potential, and corresponding electrophysiological characteristics, were studied in a comparative approach. In particular, voltage-gated Na(+) channels (VGSCs) were expressed in the strongly metastatic MAT-LyLu but not in the closely related, but weakly metastatic, AT-2 cells. Uptake and release of the non-cytotoxic marker horseradish peroxidase (HRP) were used as indices of general endocytotic and exocytotic membrane activity, respectively. The amount of tracer present in a given experimental condition was quantified by light microscopic digital imaging. The uptake of HRP was an active process, abolished completely by incubating the cells at low temperature (5 degrees C) and suppressed by disrupting the cytoskeleton. Interestingly, the extent of HRP uptake into the strongly metastatic MAT-LyLu cells was almost twice that into the weakly metastatic AT-2 cells. Vesicular uptake of HRP occurred in a fast followed by a slow phase; these appeared to correspond to cytoplasmic and perinuclear pools, respectively. Importantly, the overall quantitative difference in the uptake disappeared in the presence of 1 microM tetrodotoxin which significantly reduced the uptake of HRP into the MAT-LyLu cells. There was no effect on the AT-2 cells, consistent with functional VGSC expression occurring selectively in the former. A similar effect was observed in Na(+)-free medium. The uptake was partially dependent upon extracellular Ca(2+) but was not affected by raising the extracellular K(+) concentration. We suggest that functional VGSC expression could potentiate prostate cancer cells' metastatic ability by enhancing their secretory membrane activity.     

Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.     

Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages

Lucifer Yellow CH (LY) is an excellent probe for fluid-phase pinocytosis. It accumulates within the macrophage vacuolar system, is not degraded, and is not toxic at concentrations of 6.0 mg/ml. Its uptake is inhibited at 0 degree C. Thioglycollate-elicited mouse peritoneal macrophages were found to exhibit curvilinear uptake kinetics of LY. Upon addition of LY to the medium, there was a brief period of very rapid cellular accumulation of the dye (1,400 ng of LY/mg protein per h at 1 mg/ml LY). This rate of accumulation most closely approximates the rate of fluid influx by pinocytosis. Within 60 min, the rate of LY accumulation slowed to a steady-state rate of 250 ng/mg protein per h which then continued for up to 18 h. Pulse-chase experiments revealed that the reduced rate of accumulation under steady-state conditions was due to efflux of LY. Only 20% of LY taken into the cells was retained; the remainder was released back into the medium. Efflux has two components, rapid and slow; each can be characterized kinetically as a first-order reaction. The kinetics are similar to those described by Besterman et al. (Besterman, J. M., J. A. Airhart, R. C. Woodworth, and R. B. Low, 1981, J. Cell Biol. 91:716-727) who interpret fluid-phase pinocytosis as involving at least two compartments, one small, rapidly turning over compartment and another apparently larger one which fills and empties slowly. To search for processes that control intracellular fluid traffic, we studied pinocytosis after treatment of macrophages with horseradish peroxidase (HRP) or with the tumor promoter phorbol myristate acetate (PMA). HRP, often used as a marker for fluid-phase pinocytosis, was observed to stimulate the rate of LY accumulation in macrophages. PMA caused an immediate four- to sevenfold increase in the rate of LY accumulation. Both HRP and PMA increased LY accumulation by stimulating influx and reducing the percentage of internalized fluid that is rapidly recycled. A greater proportion of endocytosed fluid passes into the slowly emptying compartment (presumed lysosomes). These experiments demonstrate that because of the considerable efflux by cells, measurement of marker accumulation inaccurately estimates the rate of fluid pinocytosis. Moreover, pinocytic flow of water and solutes through cytoplasm is subject to regulation at points beyond the formation of pinosomes.     

Vasopressin stimulates endocytosis in kidney collecting duct principal cells

In target epithelia, a vasopressin-induced water permeability increase is accompanied by the appearance of intramembranous particle (IMP) clusters, probably representing water-permeable patches, in the apical plasma membrane of responding cells. In the collecting duct principal cell, we have previously shown that these clusters are located in clathrin-coated pits. To determine whether vasopressin induces the endocytic uptake of these membrane domains in principal cells, we have examined the uptake of horseradish peroxidase (HRP) by principal cells of normal rats, vasopressin-deficient Brattleboro rats, and vasopressin-treated Brattleboro rats, following intravenous injection of HRP. By quantitative electron microscopy, principal cells of Brattleboro homozygous rats were found to take up much less HRP into cytoplasmic vesicles than normal rats, and HRP uptake was increased to normal levels in vasopressin-treated Brattleboro rats. Many invaginating coated pits at the cell surface were loaded with HRP reaction product, indicating their participation in the observed endocytosis of HRP. We conclude that vasopressin stimulates endocytosis in collecting duct principal cells. Since we have already shown that IMP clusters are found in coated pits at the cell surface, the endocytic removal of these putative water-permeable patches from the apical membrane seems to occur via a clathrin-mediated mechanism in this tissue.     

Effects of colchicine on endocytosis and cellular inactivation of horseradish peroxidase in cultured chondrocytes

Horseradish peroxidase (HRP) was used as a marker to study the effects of microtubule-disruptive drugs on uptake and cellular inactivation of exogenous material in cultures of embryonic chick chondrocytes. HRP was ingested by fluid endocytosis, and intracellular enzyme activity subsequently diminished exponentially with time. Cytochemically, reaction product for HRP was found in vesicles often located close to the dictyosomes of the Golgi complex. Colchicine and vinblastine caused disappearance of cytoplasmic microtubules and disorganization of the Golgi complex with concomitant reduction in the cellular uptake of HRP to about half of that in the controls. Lumicolchicine, on the other hand, left cell fine structure and HRP uptake unaffected. These results indicate that microtubules are of considerable importance in the process of fluid endocytosis in cultured chondrocytes although the exact mechanism remains to be elucidated. The rate of intracellular inactivation of ingested HRP was not affected by colchicine or vinblastine. Double-labeling experiments with colloidal thorium dioxide and HRP likewise indicated that fusion of endocytic vesicles and lysosomes is not dependent on intact microtubules. The total specific activities of the three lysosomal enzymes examined were weakly or not at all changed by treatment of the cultures with colchicine or vinblastine. It therefore seems unlikely that microtubular organization plays an important role in the production or degradation of lysosomal enzymes in cultured chondrocytes.     

Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.     

Peroxidase-induced enhancement of chemiluminescence by murine peritoneal macrophages

A number of substances have been shown to enhance the respiratory burst (RB) of macrophages. Many of these substances are not normally found in vivo. The present study suggests that a group of enzymes characterized as peroxidases have the ability to significantly enhance the RB and concomitant phagocytosis by murine peritoneal macrophages. Horseradish peroxidase (HRP), lactoperoxidase (LPO), and microperoxidase (MPO) can significantly augment these functions. Both resident and thioglycollate-induced macrophages exhibited enhanced chemiluminescence (CL) upon exposure to HRP, however, the effect was more pronounced with the latter. The increase in CL was correlated with an increase in production of superoxide, which was measured by reduction of cytochrome c. Horseradish peroxidase immobilized on an inert carrier, was capable of enhancing the RB suggesting that it does not have to enter the cell in order to function. Hemin, hematoheme and hematoporphyrin had little effect on macrophage stimulated CL. All of the peroxidases tested caused increased phagocytosis of opsonized zymosan. These studies indicate that peroxidases are capable of stimulating the RB, phagocytosis and possibly other macrophage functions.     

Sequential Actions of Rab5 and Rab7 Regulate Endocytosis in the Xenopus Oocyte

To explore the role of GTPases in endocytosis, we developed an assay using Xenopus oocytes injected with recombinant proteins to follow the uptake of the fluid phase marker HRP. HRP uptake was inhibited in cells injected with GTPγS or incubated with aluminum fluoride, suggesting a general role for GTPases in endocytosis. Injection of Rab5 into oocytes, as well as Rab5:Q79L, a mutant with decreased GTPase activity, increased HRP uptake. Injection of Rab5:S34N, the dominant-negative mutant, inhibited HRP uptake. Injection of N-ethylmaleimide–sensitive factor (NSF) stimulated HRP uptake, and ATPase-defective NSF mutants inhibited HRP uptake when coinjected with Rab5:Q79L, confirming a requirement for NSF in endocytosis. Surprisingly, injection of Rab7:WT stimulated both uptake and degradation/activation of HRP. The latter appears to be due to enhanced transport to a late endosomal/prelysosomal degradative compartment that is monensin sensitive. Enhancement of uptake by Rab7 appears to function via an Rab5-sensitive pathway in oocytes since the stimulatory effect of Rab7 was blocked by coinjection of Rab5:S34N. Stimulation of uptake by Rab5 was blocked by Rab5:S34N but not by Rab7:T22N. Our results suggest that Rab7, while functioning downstream of Rab5, may be rate limiting for endocytosis in oocytes.