Microperoxidase Research Articles

Endocytosis of CF in marginal cells of stria vascularis regulated by ROCK and MLCK signaling cascade, but not G-proteins

Objective The endocytosis of cationized feritin (CF) via a clathrin-mediated pathway is regulated by a signaling network. Marginal cells showed the active endocytosis of CF via a clathrin-mediated pathway. The internalization of receptors through this clathrin-mediated pathway is an important regulatory event in signal transduction. Numerous kinases are involved in endocytosis, and each endocytic route is subjected to high-order regulation by cellular signaling mechanisms. In this study, we investigated whether ROCK and MLCK signaling cascades and G-proteins regulate the endocytosis of CF in marginal cells of the stria vascularis.Methods CF was infused into the cochlear duct with pertussis toxin (PTX),Clostridium botulinum C3 toxin (BTX), guanosine(g-thio)-triphosphate (GTP-γS), ML-7, Y-27632. Endocytic activity was measured at 30 min after the start of infusion under an electron microscope.Results In marginal cells, CF was internalized via a clathrin-mediated pathway that depends on F-actin and microtubules (MT). Its processes were controlled by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK), but not affected by G-protein-coupled receptor (GPCR) or the RhoA signaling cascade.Conclusion Our previous study showed that the main endocytotic pathway of microperoxidase (MPO) did not depend on the Rho/ROCK molecular switch or actin/myosin motor system, but was mainly regulated by the RhoA signaling cascade. The present study results indicate that these signaling cascades regulating CF internalization completely differ from the cascades for MPO internalization.

A selective nitric oxide nanocomposite biosensor based on direct electron transfer of microperoxidase: Removal of interferences by co-immobilized enzymes

A highly selective nitric oxide (NO) biosensor was developed by immobilizing microperoxidase (MP) onto the MWCNT-poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (PTTCA) nanocomposite. Catalase (CAS) and superoxide dismutase (SOD) co-immobilized on the probe successfully protected the interferences of H 2O 2 and O 2 − during NO detection. The nanocomposite layer showed the direct electron transfer processes of the immobilized CAS, SOD, and MP simultaneously at −0.11/+0.04, −0.33/−0.27, and −0.47/−0.40 V vs. Ag/AgCl. Au nanoparticles (AuNPs) were electrodeposited on a glassy carbon surface to enhance the sensitivity of the sensor probe. The layers of CAS/SOD/MP/MWCNT-PTTCA/AuNPs were characterized using SEM, XPS, and QCM. The CAS/SOD/MP/MWCNT-PTTCA/AuNPs probe showed an excellent performance in the electrocatalytic reduction of NO which was attributed to the heme group of MP. Experimental conditions affecting the sensitivity of the biosensor were optimized in terms of pH, temperature, and applied potential. The dynamic range of NO analysis was from 1.0 to 40 μM with the detection limit of 4.3 ± 0.2 nM. The reliability of biosensor was examined for the determination of NO released from the real samples of rat liver, stomach (AGS), and intestinal (HT-29) cancer cells.

Actin filaments and microtubules regulate endocytosis in marginal cells of the stria vascularis

Conclusion. Cationized ferritin (CF) was internalized via clathrin-mediated endocytosis. This process depends on clathrin, actin filaments, and microtubules. Microperoxidase (MPO) was internalized via a clathrin- and caveolin-independent endocytic pathway, which was partially dependent on microtubules but independent of clathrin and actin filaments. Objective. We investigated the role of actin filaments and microtubules in the transport of endocytic carrier vesicles (ECVs) from the plasma membrane to the early sorting endosomes, using CF and MPO as tracers. Materials and methods. Fifty-five guinea pigs were used. The animals were divided into a CF endocytosis group and an MPO endocytosis group. These groups consisted of control, nocodazole-treated, cytochalasin (Cyt D)-treated, Cyt D + nocodazole-treated, and geldanamycin-treated subgroups. Results. For CF endocytosis, the following results were obtained. In the nocodazole experiment, in which microtubules were disrupted to form monomeric tubulin, the number of ECVs loaded with CF was greatly decreased. In the Cyt D experiment, in which the actin filaments were disrupted to form monomers, the number of ECVs labeled with CF was also greatly decreased. In the geldanamycin experiment, in which clathrin-mediated endocytosis was regulated and actin stress fibers were dissolved, the endocytosis of CF was severely inhibited. For MPO endocytosis, in the nocodazole experiment, the endocytosis of MPO was markedly suppressed.

Presence and regulation of epithelial sodium channels in the marginal cells of stria vascularis

Conclusion. This study indicates that epithelial Na+-selective channels (ENaC) recycle Na+ via clathrin-mediated endocytosis in the marginal cells of the stria vascularis and that clathrin-independent endocytosis appeared to be modulated by the amount of Na+ transported. These results suggest the presence of ENaC in the luminal membrane of marginal cells and that ENaC are an efficient pathway for the uptake of Na+ from the endolymph. Objective. The ENaC found in many transporting epithelia play a key role in the regulation of salts and water homeostasis, cellular pH, cell volume, and cell function. Both biochemical and physiological approaches have been used to identify, characterize, and quantify this important channel, but its location in the marginal cells of the stria vascularis has not been fully clarified. The aim of this study was to determine the localization and regulation of ENaC. Materials and methods. Forty healthy female guinea pigs were used: 20 for the control experiment, 10 for the amiloride experiment, and 10 for the aldosterone experiment. We perfused cationized ferritin (CF) and microperoxidase (MPO) as tracers for clathrin-mediated and clathrin-independent endocytosis, respectively, into the cochlear duct. After 30 min of endolymphatic perfusion, the tissues were fixed and CF- and MPO-loaded endosomes within the marginal cell were observed by transmission electron microscopy. The numbers of CF- and MPO-loaded endosomes were compared between the three groups. Results. In the amiloride group, the numbers of CF- and MPO-loaded endosomes decreased in comparison with the control. In the aldosterone group, the numbers of CF- and MPO-loaded endosomes decreased and increased, respectively. Recently, it has been reported that ENaC are endocytosed via clathrin-mediated endosomes and aldosterone decreases the rate of endocytosis of ENaC. In this study, the results of the aldosterone experiment were consistent with those of recent studies.

Endocytosis of microperoxidase in the marginal cells of stria vascularis

Objective Endocytosis has been thought to control entry into the cell and play a crucial role in the development, immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. We investigated the basic properties of endocytosis in the marginal cells of stria vascularis (SV) to discuss whether marginal cells have a potential to maintain the endolymph homeostasis. Methods We perfused microperoxidase (MPO), an endocytosis tracer, into the cochlear duct. After 5–60 min of endolymphatic perfusion, the tissues were fixed and the distribution of MPO within the marginal cell was observed by transmission electron microscopy. Results Endocytosis started already at 5 min after MPO perfusion. Small MPO-loaded endosomes were observed up to 30 min after MPO perfusion. The small tubulovesicular endosomes and the plasma membrane invagination were not decorated by an electron dense bristle structure. After endocytosis, MPO labeled preendosomes were quickly transported to the large vacuolar endosomes that connected with tubular endosomes. At 60 min after MPO perfusion, MPO-loaded large vesicles that have small vesicles in the lumen were observed. Conclusion The time-course of MPO-loaded endosomes was similar to that of CF-loaded endosomes in the marginal cells of SV. The strial marginal cells have vigorous endocytotic activity both in clathrin-independent and clathrin-dependent endocytosis. This high activity of endocytosis in SV seems to be needed to maintain the homeostasis of endolymph via membranous channels and/or receptors regulations.

Endocytosis in the Epithelial Cells of the Endolymphatic Sac

To investigate the properties of endocytosis of the epithelial cells in the intermediate portion of the endolymphatic sac (ES), we infused cationized ferritin (CF), microperoxidase (MPO), and horseradish peroxidase (HRP), as endocytosis tracers, into the endolymphatic space of the ES. Thirty minutes after infusion, the tissues were fixed and the distributions of HRP, MOP and CF were observed by transmission electron microscopy. The results at 30 min after tracer infusion were as follows. Concerning CF, endocytosis showed different activity depending on the area of the ES. In one area, CF was hardly observed within the epithelial cells, while in another area, it was observed within the epithelial cells. The CF-loaded vesicles were mainly coated vesicles. These results suggested that infusion of the artificial endolymphatic sac endolymph with CF may not stimulate the whole signal system of the ES. Concerning HRP, it bound to the apical membrane and was observed in the vesicles and the sorting endosomes. The diameters of these vesicles were of two types: one had a smaller diameter (less than 200 nm), while the other had a larger diameter (over 200 nm) and originated from macropinocytosis. Concerning MPO, it was much less observed in the vesicles than HRP. We discuss the different activities of endocytosis among these tracers, and suggest that a large molecular weight substance, i.e. HRP, might stimulate the fluid-phase endocytosis and/or adsorptive pinocytosis.

Mediatorless electrocatalytic reduction of hydrogen peroxide at graphite electrodes chemically modified with peroxidases

The electrocatalytic reduction of H 2O 2 was studied for carbonaceous electrodes modified with horse-radish peroxidase (HRP), microperoxidase (MP), and lactoperoxidase (LP). The carbonaceous electrodes were of three different graphites, carbon and glassy carbon. The peroxidase modified electrode was inserted as the working electrode in a flow through amperometric cell of the wall jet type and connected to a flow injection system. The effect of different pretreatments of the electrode surface prior to adsorption of the enzyme was investigated. Heating the electrodes in a muffle furnace at 700°C for 1.5 min was found to yield the highest currents. The electrocatalytic current for HRP-modified electrodes starts at about +600 mV vs. Ag/AgCl (pH 7.0) and reaches a maximum value at about −200 mV. For MP- and LP-modified electrodes the currents start at a lower potential (≈ 300 mV). For the best electrode material for HRP, straight calibration curves were obtained between 1 and 500 μM H 2O 2 at 0 mV. The mechanism for the electron transfer from the electrode to the adsorbed peroxidase is discussed. Deliberate modification of the electrode surface with quinoid type electroactive species was found to mediate the reaction. It is proposed that spontaneously occurring electrochemically active surface groups mediate the electron transfer to the adsorbed enzyme. However, a contribution to the observed current from a direct electron transfer cannot be ruled out.

Differences in the transport systems between cementocytes and osteocytes in rats using microperoxidase as a tracer

Microperoxidase (MP) tracer was injected intravenously into rats to investigate any differences in transport pathways of tissue fluids in the lacunae and canaliculi of cementum and bone. Light microscopically, in deep cementum lacunae, pericellular spaces contained a large amount of MP, while close to the cementum surface, the spaces contained scarcely any. In bone, MP was detected throughout all pericellular spaces. MP was detected intracellularly as granular reaction products in most cementocytes and osteocytes. Electron microscopically, MP was found in the pericellular spaces of cementum and bone lacunae, particularly on collagen fibrils and amorphous material. MP deposits were also intense along the plasma membrane of cementocytes in the deep cementum and along the innermost edge of the deep cementum matrix and bone matrix. In uptake of MP by cementocytes, although extracellular tracer was deposited extensively along the plasma membrane of the deeply positioned cementocytes, uptake by these deep cementocytes was less than that of those close to the surface. However, in bone, most osteocytes showed uniform uptake. These results suggest that the transport pathways for tissue fluids in cementum are in the pericellular spaces, but that cementum has an uneven circulation of tissue fluid. In cementum, although there seems to be a well-developed canalicular system to transport tissue fluid into the deep regions, the deep cementocytes had less endocytotic ability than those close to the surface.

Enhancement of macrophage spreading and phagocytosis by peroxidative enzymes

Macrophages stimulated by various substances exhibit altered morphology, metabolism, and enhanced phagocytosis. The present studies were done to show if peroxidative enzymes would affect macrophage spreading and phagocytosis. Resident peritoneal macrophages, collected from C57BI mice were exposed to various concentrations of peroxidases and compared with appropriate controls. Results indicated that 0.01 μM myeloperoxidase (MyPO), 0.09 μM horseradish peroxidase (HRP), O.16 μM lactoperoxidase (LPO) and 70 μM microperoxidase (MPO) significantly enhanced macrophage spreading. It was also noted that peroxidases were able to stimulate phagocytosis by increasing the number of cells with internalized zymosan at least twofold. Stimulation of these functions suggests a possible role of endogenous peroxidases as natural cell activators.

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 exhanced 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.

Tracer study on a paracellular route in experimental hydrocephalus.

Considering the possibility of a paracellular pathway for edema resolution, we studied the intracerebral movement of proteins and ionic lanthanum in rats with experimental hydrocephalus. Hydrocephalus was induced by injection of kaolin suspension into the cisterna magna. After induction of hydrocephalus, horseradish peroxidase (HRP), microperoxidase (MP), or lanthanum chloride (LaCl3) were perfused into the ventricle system. HRP and MP were localized mainly in the intercellular spaces between ependymal cells, glial cells, and in perivascular spaces and were restricted by endothelial tight junctions. Ionic lanthanum (La3+), however, penetrated these tight junctions and moved between the blood and CSF cavities by paracellular pathways. These findings indicate that in obstructive hydrocephalus, the tight junctions may constitute part of a paracellular pathway for the transendothelial movement of small solutes, although they prevent the movement of larger molecules.

Fine structure and permeability of capillaries in the stria vascularis and spiral ligament of the inner ear of the guinea pig.

The blood capillaries in the stria vascularis and the spiral ligaments of guinea pigs were studied by electron microscopy with freeze-fracture and thin section methods, including tracer experiments with horseradish peroxidase (HRP) and microperoxidase (MP). The endothelial cells of the capillaries of both tissues are connected by tight junctions, and contain about the same number of micropinocytotic vesicles. In cases of intravascular administration before fixation, both of the tracers stained the perivascular space and almost all endothelial vesicles in the stria vascularis. On the other hand, the perivascular space and many vesicles in the spinal ligament were unstained. The endothelial tight junctions in the stria vascularis prevented the penetration of HRP, but sometimes allowed the penetration of MP. Those of the spiral ligament were impermeable to both tracers. In cases of tracer administration after fixation, leakage spots of HRP from capillaries were sparsely located all over the stria vascularis. Transendothelial channels and isolated fenestrae formed by micropinocytotic vesicles were detected. It is concluded that the capillaries of the stria vascularis are similar to the muscle capillaries and to the capillaries of the elasmobranch brain, whereas those in the spiral ligament are similar to the brain capillaries of higher vertebrates.

Ultrastructural Study on the Permeability of Blood Vessels in the Inner Ear

The blood capillaries in the stria vascularis and the spiral ligament were studied by electron microscopy with freeze-fracture and thin section methods including tracer experiments of horseradish peroxidase (HRP) and microperoxidase (MP) in guinea pigs. The capillaries in both tissues, composed of non-fenestrated endothelial cells, are connected by tight junctions, and contain about the same number of micropinocytotic vesicles. After intravascular administration, both of tracers were found to have been transported to the basal lamina in the stria vascularis, but they were absent in the spiral ligament. The tight junctions between capillary endothelial cells in the stria vascularis prevented the penetration of HRP, but sometimes allowed the penertation of MP. On the other hand, that of the spiral ligament was impermeable to both tracers. Almost all micropinocytotic vesicles of the stria capillaries contained both tracers. In the spiral ligament some micropinocytotic vesicles were stained with both tracers, but others were unstined. It is concluded that the capillaries of the stria vascularis are similar to the muscle capillaries, whereas those of the spiral ligament are similar to the brain capillaries.

Modes of protein and peptide uptake in the pineal gland of the Mongolian gerbil: an ultrastructural study.

The possible existence of either a blood-brain barrier or a CSF-brain barrier was examined in the pineal gland of the Mongolian gerbil using the ultrastructural tracers, horseradish peroxidase (HRP) and microperoxidase (MP). The mechanism of protein and peptide transport within the pineal gland and its possible relationship to pineal concretions was also considered. Gerbils were injected with either MP or HRP intravenously (IV), or they received intraventricular (IVT) injections of HRP. The IV injections resulted in both MP and HRP movement through the endothelial cells of the gland by vesicular transport and by diffusion through the endothelial cells of the gland by vesicular transport and by diffusion through the endothelial intercellular junctions. Following the IVT injections, the tracer was demonstrated in the subarachnoid space as well as in the glial cells associated with the periphery of the gland. In addition, after the IVT injections, rounded enlargements of the intercellular space that resembled canaliculi were filled with reaction product. In both cases (IVT and IV), the reaction product was localized to the perivascular space, to the glial cells and pinealocytes, and to the intercellular spaces. More importantly, there was specific localization of the tracers in the vacuolated pinealocytes and in the pineal concretions. The results of this study demonstrate several significant findings: 1) neither a blood-brain barrier nor a CSF-brain barrier exists in the pineal gland of the gerbil, 2) localization of the tracers in pineal concretions indicates a relationship between these structures and protein and peptide storage within the gland, and 3) the presence of the tracers within canaliculi after the IVT injections suggests a possible mode of secretion of pineal substances into the CSF.

Vascular permeability to proteins and peptides in the mouse pineal gland.

The permeability of fenestrated capillaries in the mouse pineal gland to proteins and peptides was demonstrated by means of ultrastructural tracers. Horseradish peroxidase (HRP) and microperoxidase (MP) were injected intravenously and allowed to circulate for approximately 30 s, 1 min, 5 min, 1 or 2 h. The tissue was then fixed by vascular perfusion or by immersion with aldehydes. In all experiments a pronounced extravasation of HRP and MP occurred. Transendothelial vesicular transport seemed to have occurred across the fenestrated capillaries. The most pronounced tracer labeling of vesicles was found after 1 min of MP- or HRP-circulation. The vesicles were uncoated and more than 70% of the HRP- and MP-containing vesicles exhibited diameters between 50 and 110 nm. Furthermore, three other transcapillary pathways taken by the tracers are suggested: 1) via intercellular junctions, 2) through fenestrae and 3) via channels formed by fusion of vesicles with the luminal and abluminal cell membranes. Based on these results, it is assumed that the capillaries in the mouse pineal gland also permeable to peptides synthesized and secreted by the pineal gland.

Permeability of muscle capillaries to microperoxidase.

In this study we attempted to identify a morphologic counterpart of the small pore of muscle capillaries. The existence of such a pore has been postulated by physiologists to explain the permeability of muscle capillaries to small macromolecules. We injected mice intravenously with microperoxidase (MP) and fixed specimens of diaphragm at intervals of 0-250 s after the injection to localize the tracer by electron microscopy. The small size of MP (1,900 mol wt and 20 A molecular diameter [MD]) ensures its ready passage through the small pore since the latter is thought to be either a cylindrical channel 90 A in diameter or a slit 55 A wide. MP appears in the pericapillary interstitium within 30 s of initiation of its intravenous injection. The patterns of localization of MP observed within clefts between adjacent capillary endothelial cells indicate that some endothelial junctions are permeable to this tracer. Although small vesicles transfer MP across the endothelium, we do not believe that the vesicles transfer substantial amounts of MP into the pericapillary interstitium. We did not obtain evidence that MP crosses the endothelium of capillaries through channels formed either by a single vesicle or by a series of linked vesicles opening simultaneously at both surfaces of the endothelial cell. From our observations we conclude that some endothelial junctions of capillaries are permeable to MP, and that these permeable junctions are a plausible morphologic counterpart of the small pore.

Dissociation of CO from carboxyhemoglobin.

The reaction between carboxyhemoglobin and reduced microperoxidase (MP): Hb4(CO)4 + 4MP=Hb4 + 4MPCO, recently reported by us, has been further studied. By generating species Hb4(CO), Hb4(CO)2, and Hb(CO)3 in the stopped flow cuvette by the reaction of dithionite with the species of the general formula Hb4(O2)x(CO)y(x + y=4) in the presence of microperoxidase it has been possible to determine the stepwise CO dissociation rate constants l4, l3, l2, and l1. The overall CO dissociation rate constant l, which is the same in this system as l4, is not affected by 2,3-diphosphoglyceric acid. The activation energy of the reaction is 21,400 cal in 15-25 degrees range. The ratio deltal/deltapH is approximately 3 in 6.5 to 7.5 pH range. The kinetic data indicate that, compared to HbO2, the contribution to the cooperativity of the dissociation rate constants of carboxyhemoglobin is greatly reduced. The ligand-dependent differences in the reactions of Hb with CO, O2, and NO suggest that in the combination reactions the ligand plays an active role in the rate-limiting step.

A comparative study of four cytochemical detection methods of concanavalin A binding sites on the cell membrane

Four different electron cytochemical methods to detect concanavalin A (ConA) binding sites on the plasma membrane of mouse fibroblasts were compared in this study. The ConA binding sites were made visible either by adding ConA, followed by horseradish peroxidase (HRP) or hemocyanin (HC), or by marking the sites with complexes of ConA with ferritin (Fer) or with micro-peroxidase (MP). HC and Fer are directly visible in the electron microscope; HRP and MP are detected by their electron-dense reaction product with diaminobenzidin and H 2O 2. Differences in sensitivity of the ConA binding sites for the different markers were found and resulted in a tentative interpretation of the labelling reactions. All experiments suggested that normal and transformed murine fibroblasts both have plasma membranes in which the binding sites can move equally well and can be induced to form clusters. These results are discussed in relation with the hypothesis that differences in clustering of ConA sites between normal and transformed cells are responsible for differences in the agglutinability by ConA of these cells.