Search

The coexistence of different subtypes of voltage-dependent calcium channels (VDCC) within the same chromaffin cell (CC) and the marked interspecies variability in the proportion of VDCC subtypes that are present in the plasmalemma of the CCs raises the question on their roles in controlling different physiological functions. Particularly relevant seems to be the role of VDCCs in the regulation of the exocytotic neurotransmitter release process, and its tightly coupled membrane retrieval (endocytosis) process since both are Ca2+-dependent processes. This review is focused on the role of Ca2+ influx through L-type VDCC in the regulation of these two processes. It is currently accepted that the different VDCC subtypes (i.e., T, L, N, P/Q, R) contribute to exocytosis proportionally to their density of expression and gating properties. However, the pattern of stimulation defines a preferential role of the different subtypes of VDCC on exocytosis and endocytosis. Thus, L-type channels seem to control catecholamine release induced by prolonged stimuli while fast exocytosis in response to short square depolarizing pulses or action potentials is mediated by Ca2+ entering CCs through P/Q channels. The pattern of stimulation also influences the endocytotic process, and thus, electrophysiological data suggest the sustained Ca2+ entry through slow-inactivating L-type channels could be responsible for the activation of fast endocytosis.

Heavy metals are used in industrial applications, such as production of pesticides, batteries, alloys, and textile dyes. Excessive exposure may lead to specific disorders. The kidney is a target organ in heavy metal toxicity because of its ability to reabsorb and concentrate divalent metals. The extent of renal damage depends on the nature, the dose, and the time of exposure. In general, acute damage differs from chronic damage in its mechanism of toxicity. As a consequence, the clinical features and therapeutic approach are also different. Heavy metals in plasma exist in nondiffusible and ionized forms. The ionized form is toxic and produces acute toxicity; on the other hand the bound, inert form is conjugated with metallothionein and glutathione, which then are released into the blood by the liver and the kidney. These compounds subsequently are reabsorbed through an endocytotic process in segment S1 of the proximal tubule and can lead to chronic damage. Treatment regimens include chelation therapy, decontamination procedures, supportive care, and extracorporeal therapy. This chapter adds specific considerations for some of the most common metals.

Bioactive peptide LL-37/hCAP18, the only human member of the cathelicidin family, plays important roles in killing various pathogens, as well as in immune modulation. We demonstrate that LL-37 is internalized by human macrophages in a time-, dose-, temperature-, and peptide sequence–dependent endocytotic process. Both clathrin- and caveolae/lipid raft–mediated endocytosis pathways are involved in LL-37 internalization. We find that the P2X7 receptor (P2X7R) plays an important role in LL-37 internalization by human macrophages because significantly less internalized LL-37 was detected in macrophages pretreated with P2X7R antagonists or, more specifically, in differentiated THP-1 cells in which the P2X7R gene had been silenced. Furthermore, this P2X7R-mediated LL-37 internalization is primarily connected to the clathrin-mediated endocytosis pathway. In addition, our results demonstrate that internalized LL-37 traffics to endosomes and lysosomes and contributes to intracellular clearance of bacteria by human macrophages, coinciding with increased reactive oxygen species and lysosome formation. Finally, we show that human macrophages have the potential to import LL-37 released from activated human neutrophils. In conclusion, our study unveils a novel mechanism by which human macrophages internalize antimicrobial peptides to improve their intracellular pathogen clearance.

Under basal conditions the action potential firing rate of adrenal chromaffin cells is lower than 0.5 Hz. The maintenance of the secretory response at such frequencies requires a continuous replenishment of releasable vesicles. However, the mechanism that allows such vesicle replenishment remains unclear. Here, using membrane capacitance measurements on mouse chromaffin cells, we studied the mechanism of replenishment of a group of vesicles released by a single action potential-like stimulus (APls). The exocytosis triggered by APls (ETAP) represents a fraction (40%) of the immediately releasable pool, a group of vesicles highly coupled to voltage dependent calcium channels. ETAP was replenished with a time constant of 0.73 � 0.11 s, fast enough to maintain synchronous exocytosis at 0.2-0.5 Hz stimulation. Regarding the mechanism involved in rapid ETAP replenishment, we found that it depends on the ready releasable pool; indeed depletion of this vesicle pool significantly delays ETAP replenishment. On the other hand, ETAP replenishment also correlates with a dynamin-dependent fast endocytosis process (τ = 0.53 � 0.01 s). In this regard, disruption of dynamin function markedly inhibits the fast endocytosis and delays ETAP replenishment, but also significantly decreases the synchronous exocytosis during repetitive APls stimulation at low frequencies (0.2 and 0.5 Hz). Considering these findings, we propose a model in where both the transfer of vesicles from ready releasable pool and fast endocytosis allow rapid ETAP replenishment during low stimulation frequencies. Fil: Moya Diaz, José Abelino. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Alvarez, Yanina Daniela. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Montenegro, Mauricio Norman. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina Fil: Bayonés, Lucas. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina Fil: Belingheri, Ana Verónica. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: González-Jamett, Arlek M.. Universidad de Valparaiso; Chile Fil: Cárdenas, Ana M.. Universidad de Valparaiso; Chile Fil: Marengo, Fernando Diego. Universidad de Buenos Aires. Facultad de Ciencias Exactas. Departamento de Ecología, Genética y Evolución. Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina

Protein delivery mediated by protein transduction domains (PTD) such as the HIV-1 TAT-PTD has emerged as a promising approach for neuroprotection. The objective of this study was to generate and evaluate the neuroprotective potential of TAT fusion proteins using constructs based on Bcl-2 anti-death family proteins. A TAT-Bcl-2 construct with the loop domain deleted (TAT-Bcl-2Δloop) was tested for its ability to transduce neuronal cells and to promote survival. The potential mechanism of TAT-mediated protein internalization in neural cells was also investigated. The purified TAT-Bcl-2Δloop binds to neural cell and rat brain mitochondria, and transduces cultured neural cell lines and primary cortical neurons when used at nm concentrations. Effective internalization of TAT-Bcl-2Δloop occurs at 37°C but not at 4°C, consistent with an endocytotic process. Both cell association and internalization require interaction of TAT-Bcl-2Δloop with cell surface heparan sulfate proteoglycans. TAT-mediated protein delivery in neuronal cells occurs through a lipid raft-dependent endocytotic process, inhibited by the cholesterol-sequestering agent nystatin. Transducible loop deleted Bcl-2 increases the survival of cortical neurons following trophic factor withdrawal and also rescues neural cell lines from staurosporine-induced death. These results support the concept of using protein transduction of Bcl-2 constructs for neuroprotection.

Cortical granules are membrane-bound, electron dense cell inclusions known in oocytes of many animals, invertebrates [1-5] and vertebrates [6]. They are distributed in the peripheral ooplasm forming a monolayer beneath the plasma membrane [1,3,5]. These organelles are interpreted as Golgi complex productions [1,3,4,7,8]. In some metazoan (as crustacean), it is suggested that mitochondria take part in the origin of cortical granules [3]. Cortical granules contents differ among species (glycoproteins, mucopolysaccharide etc.) but their functions of preventing polyspermy remain similar [3,5,9,10] in the fertilization process. In planarians (Platyhelminthes, Tricladida), these inclusions are signalized for several species [4]. We present here an ultrastructural analysis of their genesis process in the marine gonochoristic triclad Sabussowia dioica where we demonstrate a novelty represented by the contribution of accessory cells in the formation of their peripheral content using an endocytotic process.

Crystal-like structures in vacuoles, precipitates in the cytoplasm and on the tonoplast membrane have been found to store remarkable amounts of Si in a number of higher plants. In most of the cases the final storage product is a SiO(2) gel. Accumulation inside the cells presumes a membrane and cytoplasm passage, driven by unknown transporters. Beside this uptake into the cytoplasm, Si-accumulating species possess a mechanism that does not involve a membrane and cytoplasm passage. Unusual small invaginations comprising the two membranes, plasmalemma and tonoplast, which enclose a small border of cytoplasm, were observed. The same cells contained vacuolar vesicles surrounded by two membranes, obviously derived from the invaginations. By energy-dispersive X-ray analysis and electron spectroscopic imaging, Si was shown in the invaginations and vacuolar vesicles. This novel endocytotic process allows the uptake of condensed, higher-molecular-weight Si compounds. In Zn hyperaccumulators, frequently SiO(2) precipitates were found in different cell compartments. Such plants showed the same invaginations and vacuolar vesicles, but Zn, colocalized with Si, was detected in these structures. Electron energy loss spectra confirmed the assumption that Zn-silicate is present in the vesicles. In the vacuoles the unstable Zn-silicate is degraded, forming SiO(2) precipitates, while the released Zn is bound to an unknown partner.

Calreticulin (CRT) is an endoplasmic reticulum chaperone protein that is involved in quality control process during protein folding and maturation. It plays an important role as a regulator of intracellular calcium homeostasis. Previously it has been shown that loss of CRT protein results in endoplasmic reticulum stress, increase in ubiquitin-proteasome activity, and resistance to apoptosis. Our preliminary studies illustrated that CRT deficient cells expresses significantly higher connexin 43 protein, however its function is significant suppressed. We showed that connexin 43 was accumulated within intracellular vesicles. Because connexin 43 function is dependent on its trafficking and localization, we hypothesized that loss of CRT function increases rate of endocytosis via caveolin dependent pathway. To test our hypothesis we measured the rate of uptake of fluorescently tagged-Wheat Germ Agglutinin, in wild type (wt) versus crt-/- mouse embryonic fibroblast cells (MEF). In crt-/- cells WGA was completely endocytosed within 30 minute incubation while in wt cells WGA was localized both on cell membrane and intracellular vesicles at 30 minute. The endocytotic process in crt -/- was significantly reduced by inhibiting proteasome activity with MG132. The caveolin-1 mRNA and protein levels were significantly increased in crt-/- cells compared to wt cells. However, there were no significant changes in clathrin protein level in wt versus crt-/- cells. Using Methyl-B-cylcodextrin to deplete cell cholesterol content, we demonstrated inhibition of endocytosis in crt-/- cells which confirms a major role for caveolin in endocytosis in these cells. Overall our data are the first to illustrate the inhibitory role of CRT in endocytotic pathways. Acknowledgment: This research was funded by Qatar National Research Fund (NPRP4-043-3-016).

Oncogene-driven adaptation of metabolism during tumorigenesis includes steps that stimulate the uptake of nutrients, especially glucose and glutamine, to sustain cell growth and proliferation. Macropinocytosis, a clathrin- and caveolin-independent endocytotic process that had previously been linked to the action of oncogenic Ras and Src, is now shown to contribute to amino acid uptake via enhanced delivery of extracellular proteins to lysosomes.

Uptake of 3,5,3'-triiodo-L-thyronine (T3) by isolated trout hepatocytes was characterized after 40 sec incubation of cells in a balanced salts medium containing [125]T3, and compared to L-thyroxine (T4) uptake (Riley and Eales, Gen. Comp. Endocrinol. 90, 31-42, 1993). T3 uptake resembled T4 uptake in several ways. There was a small (10%) diffusion component. The balance of the uptake was temperature- and energy-dependent and involved a protein carrier, but did not depend on the presence of Na+ in the medium or Na+ transport. Tyrosine and phenylalanine were ineffective competitors. Inhibition by colchicine and chloroquine indicated an endocytotic process. T3 uptake differed from T4 uptake in having a higher pH optimum (6-8) than T4 (5-6), and in having a much lower Kt (0.074 microM) than T4 (0.52 microM). T3 uptake was far more strongly inhibited than T4 uptake by TRIPROP (8% of control uptake), reverse T3 (9%), and 3,3'-diiodo-L-thyronine (9%). T4 inhibited T3 transport (Ki = 0.18 microM), but kinetic analyses indicated that the mutual inhibitions were noncompetitive, suggesting separate T3 and T4 binding sites. In conclusion, T3 uptake into isolated trout hepatocytes resembles that for T4 uptake in being an energy-dependent, carrier-mediated endocytotic process, but differs from T4 uptake in having a lower Kt, a higher pH optimum, and a greater sensitivity to inhibition by related iodothyronines. T3 and T4 uptakes may involve separate carrier systems, providing scope for individual control of T3 and T4 uptake by trout hepatocytes.

Insulin regulated aminopeptidase (IRAP) recognises "AT(4)-receptor" ligands like angiotensin IV (Ang IV) and peptidomimetics like AL-11. The metabolic stability and high affinity of [(3)H]AL-11 for catalytically active IRAP allowed its detection in Chinese hamster ovary (CHO-K1) cell membranes in the absence of chelators (Demaegdt et al., 2009). Here, we show that, contrary to [(3)H]Ang IV, [(3)H]AL-11 displays high affinity and specificity for IRAP in intact CHO-K1 cells as well. After binding to IRAP at the surface, [(3)H]AL-11 is effectively internalized by an endocytotic process. Unexpectedly, surface binding and internalization of [(3)H]AL-11 was not affected by pretreating the cells with Ang IV but declined with AL-11. In the latter case surface expression of IRAP even increased. After elimination of simpler explanations, it is proposed that metabolically stable "AT(4)-receptor" ligands undergo semi-continuous cycling between the cell surface and endosomal compartments. The in vivo efficacy of stable and unstable "AT(4)-receptor" ligands could therefore differ.

Hepatitis B virus (HBV) is a major cause of liver disease. HBV primarily infects hepatocytes by a still poorly understood mechanism. After an endocytotic process, the nucleocapsids are released into the cytoplasm and the relaxed circular rcDNA genome is transported towards the nucleus where it is converted into covalently closed circular cccDNA. Replication of the viral genome occurs via an RNA pregenome (pgRNA) that binds to HBV polymerase (P). P initiates pgRNA encapsidation and reverse transcription inside the capsid. Matured, rcDNA containing nucleocapsids can re-deliver the RC-DNA to the nucleus, or be secreted via interaction with the envelope proteins as progeny virions.

Decorin, a small interstitial dermatan sulfate proteoglycan, is turned over in cultured cells of mesenchymal origin by receptor-mediated endocytosis followed by intralysosomal degradation. Two endosomal proteins of 51 and 26 kD have been implicated in the endocytotic process because of their interaction with decorin core protein. However, heparin and protein-free dermatan sulfate were able to inhibit endocytosis of decorin in a concentration-dependent manner. After Western blotting of endosomal proteins, there was competition for binding to the 51- and 26-kD proteins between heparin and decorin. In spite of its high-affinity binding, heparin was poorly cleared from the medium of cultured cells and then catabolized in lysosomes. In contrast to decorin, binding of heparin to the 51- and 26-kD proteins was insensitive to acidic pH, thus presumably preventing its dissociation from the receptor in the endosome. Recycling of heparin to the cell surface after internalization could indeed be demonstrated.

The objective of this study was to clarify the renal uptake characteristics of oligonucleotides at a cellular level using LLC-PK1 renal epithelial cells derived from the proximal tubule. The association of [35S]-labeled 20-mer phosphodiester (PO) and phosphorothioate (PS) oligonucleotides with the monolayers of polarized LLC-PK1 cells cultured on polycarbonate filter was characterized after apical or basolateral application. The cellular association of PO and PS at both apical and basolateral membranes was time dependent and temperature dependent, and the apparent association amount of PS was larger than that of PO. The PO and PS association after apical application was saturable, with the apparent Km and Vmax values determined to be 5.4 microM and 0.14 nmol/mg protein for PO and 0.22 microM and 0.11 nmol/mg protein for PS, respectively. In contrast, almost linear kinetics were observed after basolateral application within a tested concentration range. The association was inhibited significantly by sodium azide and chloroquine, suggesting that an energy-dependent endocytotic process was involved. Internalization and subsequent transport to endosome and lysosome compartments of FITC-labeled oligonucleotides were shown by confocal laser scanning microscopy. The present study has demonstrated that both types of oligonucleotides are taken up by LLC-PK1 cells from both apical and basolateral surfaces probably via an endocytosis mechanism.

The mechanism of surfactant protein (SP)-A-mediated lipid uptake by rat type II pneumocytes was investigated. In the absence of SP-A, freshly isolated type II pneumocytes actively take up very little if any liposomes. Most of the increase with time is independent of energy or temperature but is most likely due to spontaneous exchange of labeled lipids between liposomes and cell membranes. With 5 micrograms/ml SP-A, type II cells actively take up liposomes (244 pmol dipalmitoylphosphatidylcholine.h-1.10(6) cells-1). The effect of SP-A on uptake is temperature dependent and can be abolished by ATP depletion of the cells. Coincubation with an auto-anti-idiotypic antibody against the SP-A-binding protein BP55 on the cell membrane of type II pneumocytes inhibits SP-A-mediated lipid uptake by type II cells. With increasing amounts of extracellular SP-A present, increasing amounts of liposomes are taken up and directed toward a nondegrading compartment. We suggest that SP-A-mediated surfactant lipid uptake is a receptor-mediated endocytotic process involving BP55.

The digestive gland of Viviparus ater was studied using histochemical and ultrastructural methods. Only one cell type was observed in the tubule epithelium of the gland. The cells are involved in an endocytotic process mediated by clathrin-coated vesicles and in the intracellular digestion of food materials (thus they can be regarded as digestive cells). The different stages of digestion and exocytotic extrusion of residual bodies into the tubule lumen were shown by electron microscopy. Very few, small mucocytes are scattered among the digestive cells. Calcium concretions, glycogen-containing cells and endocrine cells are scattered in the area of connective tissue present among the digestive tubules.

1. Serum lipoproteins play an important role in the in vivo transport of several porphyrinoid derivatives having a moderate or high degree of hydrophobicity. 2. There appears to exist a correlation between the extent of photosensitizer association with low-density lipoproteins (LDL) and the efficiency of tumour targeting by some classes of photosensitizers, such as differently sulphonated porphyrins and phthalocyanines, haematoporphyrin dialkylethers and unsubstituted phthalocyanines and naphthalocyanines. 3. In all cases, LDL-carried photosensitizers are preferentially released to malignant cells; hence, direct cell damage appears to be the major determinant of tumour damage consequent to photodynamic therapy. 4. Present evidence suggests that the LDL-associated photosensitizer is accumulated by tumour cells largely via a receptor-mediated endocytotic process. 5. Thus, the use of delivery systems for orientating a systemically injected photosensitizer towards lipoproteins has been explored; promising results have been obtained by incorporation of the dye into liposomal vesicles, oil emulsions or inclusion complexes, as well as by precomplexation of the dye with LDL. 6. Moreover, a suitable choice of the chemical constituents of the delivery system and the experimental conditions allows one to modulate the photosensitizer distribution among the different lipoproteins. 7. The occurrence of tumour-targeting strategies other than the LDL pathway is briefly discussed.

Publisher Summary This chapter describes electrical capacitance and the way it can be used to measure membrane surface area. To study exo- and endocytotic processes, it is necessary to have a parameter quantitatively related to its progress under different experimental conditions. Such information can be provided by a continuous measure of the membrane surface area as membrane is added or subtracted by the exo- or endocytotic process. Cell membrane elements such as channels, pumps, and exchangers may be directly studied through their electrophysiological effects on the membrane. Conversely, the equally important processes of exocytosis and endocytosis occur without producing clear electrical signals. The many ionic pathways of the membrane are protein molecules that reverse the membrane, and the proteins are thought to be held in a lipid matrix. The membrane can therefore be taken to be a sort of mosaic of insulating and conducting regions.

the covalent carbohydrate in the toxic lectin ricin has been modified by periodate treatment, and analysis of the products of this reaction indicates that only mannose residues are oxidized. HeLa cell toxicity, cell-surface binding, inhibition of cell-free protein synthesis, and other properties have been investigated as functions of the number of mannose residues modified. The data indicate an important role for mannose in ricin conformation and in the endocytotic process; however, mannose appears not to be essential for binding the toxin to cells.

Incubation of primary cultures of rat hepatocytes with the local anesthetics, procaine or lidocaine, had little or no effect on insulin uptake or degradation but caused an inhibition of insulin-stimulated glycogenesis. While exposure of cultures to the amines, monodansylcadaverine or CH3NH2, resulted in significant dose-dependent decreases in glycogenesis, only monodansylcadaverine (an inhibitor of receptor clustering) decreased uptake whereas CH3NH2 (a lysosomotropic agent) caused increases in both insulin uptake and degradation. When cells were treated with agents which inhibit glycolysis (NaF, 2-deoxy-D-glucose) or oxidative metabolism (2,4-dinitrophenol, carbonyl cyanide m-chlorophenyl hydrazone, NaN3, antimycin A), pronounced inhibitions of each of the bioactivities studied (syntheses of glycogen, protein, lipid) were observed, but only the glycolytic inhibitors decreased insulin uptake. These results suggest that insulin is internalized by an endocytotic process involving receptor clustering and requiring metabolic energy derived from glycolysis. The post-receptor biosynthetic processes involved in the expression of the biological activities of insulin (syntheses of glycogen, protein, lipid) require energy produced by oxidative metabolism while the degradation of insulin is carried out by nonlysosomal mechanisms which are not energy-requiring.

Membrane transport of vitamin B12 (cyanocobalamin; Cbl) into mammalian cells is mediated by the serum protein transcobalamin II (TCII). In mouse leukaemia L1210 cells, TCII-Cbl binds to membrane receptors in a rapid, temperature-independent step and is internalized by a slow, temperature-dependent process. To delineate the location of receptors on these cells, we have constructed a visual probe by covalently coupling purified TCII-Cbl to submicrometre latex particles (minibeads). We report here that when L1210 cells are incubated with minibeads containing TCII-Cbl at 4 degrees C and examined by scanning electron microscopy (SEM), the particles are found attached predominantly to microvilli. Incubation of the cells at 37 degrees C results in the internalization of the minibeads. As visualized by transmission electron microscopy (TEM), this endocytotic process seems to occur in clathrin-coated pits and vesicles at the cell surface.

During oocyte growth in the frog Discoglossus pictus two patterns of vitellogenesis are described. The first one consists of the transformation of multivesicular bodies into yolk platelets; the second is the result of a typical endocytotic process, as described in other species. The peculiarity in Discoglossus vitellogenesis consists of a regional difference of these features of vitellogenesis in vitellogenic oocytes: the multivesicular bodies transforming into yolk platelets are found only in the germinative area—the central portion of the animal half—whereas deep crypts with numerous endocytotic pits are found only in the vegetal half. The probable meaning of this regional difference in vitellogenic oocytes is discussed.

We have employed fluorescein and 125l-labeled elicitors of the defense response in soybeans to monitor the cellular distribution and movement of elicitors following their addition to a soybean cell suspension culture. Our results indicate that the macromolecular elicitors first bind to the cell surface and then internalize in a temperature- and energy-dependent endocytotic process. Within a few hours, virtually all of the elicitor is concentrated in the major vacuole or tonoplast of the cell. Nonspecific (control) proteins neither bound to the cell surface nor internalized in parallel assays.

The endocytotic process in cultured human RPE cells was observed after 1 min, 20 min, and 2 h incubation with cationized ferritin. Within 1 min the ferritin particles were seen to attach to the cell membrane, especially between microvilli. Uncoated and coated pits could be recognized on the cell membranes, and uncoated and coated endocytotic vesicles were found in the cytoplasm after 20 min of incubation. These vesicles were surrounded by abundant microfilaments and had no visible membranes. Loss of membrane may be an initial step in the process of developing into the irregular clumps of ferritin particles found inside the plasma membrane. With time, more irregular clumps of ferritin, smaller than the particles introduced during incubation, appeared just beneath the cell membrane. Lysosomes were adjacent to the clumps of ferritin particles associated with microtobules and finally degraded these particles. The phagolysosomes containing many particles were surrounded by many microtubules. Small ferritin particles surrounded but had not entered the rough endoplasmic reticulums, and no particles were seen either around or in the Golgi apparatus.

The acute effect of a physiological concentration (1 mU/l) of thyrotropin (TSH) on the activity of four lysosomal enzymes in the thyroid follicular lining cell has been studied by quantitative cytochemical techniques. N-acetyl-beta-glucosaminidase (NAG) activity was increased by 14% after 10 min TSH stimulation and NAG and beta-galactosidase activities were increased by 24% and 25% respectively (P less than 0.05) after 20 min stimulation and by 40% and 45% (P less than 0.05) respectively after 30 min stimulation with TSH, indicating an early processing of these carbohydrate residues in thyroglobulin. Acid phosphatase activity, an acid hydrolase unrelated to the hydrolysis of thyroglobulin, was unchanged 30 min after TSH stimulation. Leucyl-beta-naphthylaminidase (LNA) activity changed biphasically with peak activities of 7 and 25 min possibly representing an early fusion of endocytotic vesicles and lysosomes and later the release process of the thyroid hormones. The changes in LNA activity and thus membrane permeability were not reflected in the other enzyme activities studied. This may indicate that the TSH regulation of lysosomal enzyme activities could be independent to the endocytotic process, which is known to involve fusion of lysosomes and endocytotic vesicles. In conclusion we have demonstrated for the first time with physiological concentrations of TSH a specific acute regulation of some lysosomal enzyme activities which may be involved in thyroglobulin processing. Further, these effects may be independent of the changes in lysosomal membrane permeability due to formation of secondary lysosomes.