Amount Of Horseradish Peroxidase Research Articles

The cellular uptake of horseradish peroxidase and its poly(lysine) conjugate by cultured fibroblasts is qualitatively similar despite a 900-fold difference in rate.

When horseradish peroxidase (HRP) is conjugated to poly(L-lysine) of molecular weight (MW) 13,000, its transport into cultured L929 fibroblasts in 1 hour at 37 degrees C is increased 918-fold. The kinetics of uptake are linear with time and concentration, reflecting a process of nonreceptor-mediated adsorptive endocytosis. Neither HRP-poly(Lys) conjugate nor free poly(Lys) of any size cause any increase in fluid phase endocytosis. All evidence indicates that the covalently bound poly(Lys) increases the binding of HRP to the cell surface and that this adequately accounts for an increased internalization occurring at a steady rate. In the absence of Ca++, both surface binding and uptake of the conjugate, but not of free HRP, are decreased. Cytochalasin B does not significantly inhibit the transport of either form of HRP. The half-lives of HRP and HRP-poly(Lys) are 7.6 and 5.6 hours, respectively, when measured over a period of 12 hours. Electron microscopic analysis of cells that have ingested comparable amounts of HRP shows that the intracellular localization of free and conjugated HRP are comparable and that both are found inside the same array of structures. Thus HRP, which binds very poorly to the cell surface and is considered a fluid-phase marker for the "contents" of pinocytotic vesicles, and HRP-poly(Lys), which binds very strongly to the cell surface and is considered a membrane marker for adsorptive endocytosis, are taken up along the same endocytotic pathway. Moreover, despite the fact that neither markers are transported by receptor-mediated endocytosis, both are seen in structures that were described as receptosomes. It appears, therefore, that the pathways utilized in receptor-mediated transport are available to all other forms of pinocytosis.

A horseradish peroxidase study of the projections from the lateral reticular nucleus to the cerebellum in the rat.

The projection from the lateral reticular nucleus (LRN) to the cerebellar cortex was studied in the rat by utilizing the retrograde transport of horseradish peroxidase (HRP). In order to study the topographic features of this projection, small amounts of HRP were injected into various sites in the cerebellar cortex. The results demonstrated that the caudal lobules of the anterior lobe vermis tend to receive afferents from the medial LRN and the rostral lobules of the vermis receive afferents from more laterally situated cells. Lobules IV and V receive inputs primarily from the magnocellular division of the LRN of both the ventromedial and dorsolateral parts of the LRN, while lobules II and III receive inputs mainly from cells which lie in the border area between the parvocellular and magnocellular division of the ventromedial part. Following injections within various areas of the posterior lobe vermis, the results indicated that lobule VIII receives the most abundant projection from the LRN and that the cells of origin are present within the parvocellular and the adjacent part of the magnocellular division throughout the rostrocaudal extent of the LRN. Following injections within lobules VI and VII, few labelled cells were found and they tended to lie within the rostral two-thirds of the magnocellular division. Little or no projection from the LRN to lobule IX was evident. The hemispheres were found to receive a modest projection from the dorsal aspect of the LRN. The projection to lobulus simplex originates mainly from the caudal two-thirds of the magnocellular division, while the projection to the ansiform and paramedian lobules originates mainly from the dorsal aspect of the rostral two-thirds of the magnocellular division. Finally, there appears to be extensive overlapping of the orgins of all three projections to the cerebellar cortex studied, and this occurs within the central area of the magnocellular division throughout the rostrocaudal extent of the LRN.

Permeability of canine submandibular glands to blood-borne horseradish peroxidase during parasympathetic secretion.

Potential permeability pathways exist between central acinar cells in submandibular glands of dogs and they are large enough to allow the passage of molecules of horseradish peroxidase (HRP) under certain conditions. During gentle submandibular secretion caused by parasympathetic nerve stimulation detectable amounts of HRP passed into the saliva as long as the concentration of HRP in the blood reaching the gland was high enough. Morphological results indicated that the passage was mainly via leaky junctions between acinar cells. It is likely that this movement was by passive permeation because it required a high concentration gradient, was slow to occur and gradually reached a peak; the actual amounts to reach the saliva under physiological conditions were small and depended on the effective concentration of HRP reaching the gland. A satisfactory reproducible secretory regime has been developed, using short periods of stimulation alternating with rest pauses. Using these conditions gradually increasing amounts of HRP reached the saliva and first samples of saliva during each stimulation period contained more HRP than subsequent samples from the same stimulation period. It is possible that contraction of myoepithelial cells, at the outset of each stimulation period, contributed to this increased movement by acting on HRP-containing fluid in spaces between themselves and the acinar cells, thereby causing an increased filtration pressure to be exerted on underlying tight junctions.

Search for the central origin of efferent fibers in the carotid sinus nerve of the cat.

Using the horseradish peroxidase (HRP) technique, the central origin of efferent fibers in the carotid sinus nerve (CSN) was examined in the cat. To avoid false labeling due to uptake from the nerve endings in the adjacent tissues of the CSN, the critical amount of HRP was sought, which was estimated at 0.5 mg. Applying 0.5 mg of HRP to the central cut end of the CSN, many labeled cells were found in the petrosal ganglion, but no labeled cells were found in the central structure. The findings of this study indicate that the origin of the most efferent fibers in the CSN is not in the central structure, but that it may be in the extracranial part of the glossopharnygeal nerve.

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.

Ultrastructural study of a blood-muscle spindle barrier after systemic administration of horseradish peroxidase.

The permeability of the normal muscle spindle capsule to the entrance of an exogenous protein tracer was assessed by the use of horseradish peroxidase (HRP). Adult mice were injected intravenously with a solution of HRP (MW 40,000). After varying intervals, ranging from two to 240 minutes, animals were perfused aortically with fixative, and anterior tibialis muscles were removed, processed, and examined. The results were evaluated by electron microscopic cytochemical techniques, and attention was directed to the movement of tracer and its relationship to the outer and inner capsule of the muscle spindle. HRP was first demonstrated in cytoplasmic vesicles crossing the continuous capillary endothelium supplying the muscle spindle, and was then detected in the interstitial space surrounding the outer capsule. In polar regions, HRP had entered the periaxial space by two minutes, and it was seen in cytoplasmic vesicles of the attenuated outer capsule. Later, HRP abutted the sarcolemma of intrafusal fibers, and by 12.5 minutes there was evidence of tracer in T-tubules of these muscle cells. Whereas at no time was tracer observed traversing any of the numerous intracellular junctions of the capsule, it appeared that polar regions were leaky and open-ended at their distal portions. The sensory equatorial zone was considerably less permeable to the entrance of tracer. Whereas HRP was visible intially in cytoplasmic vesicles of the subjacent capillary endothelium, it was not until 12.5 minutes that racer could be demonstrated within some vesicles of equatorial outer capsule cells. At later times, a small amount of HRP was observed in the equatorial periaxial space, where it was phagocytosed and finally sequestered by cells of the inner capsule as membrane-bound lysosomal deposits. Consequently, at no time was tracer incorported into either intrafusal fibers or their sensory nerve terminals. Thus, in contract to polar regions, the muscle spindle capsule in the equatorial zone appears to be effective in preventing the indiscriminate penetration of HRP from the bloodstream. This suggests dissimilar paths of tracer movement from the microvasculature into sensory and non-sensory regions of the muscle spindle.

Lysophosphatidyl choline facilitates labeling of CNS projections with horseradish peroxidase

Previously, it has been difficult to label sensory terminations in the CNS by backfilling peripheral sensory nerves with horseradish peroxidase (HRP), apparently because the amount of HRP transported along the axons is insufficient. Labeling of central sensory projections in axolotls and frogs can be improved by agents known to disrupt membranes. Of those tested, l-α-lysophosphatidyl choline is the most effective. The addition of this detergent to the HRP solution dramatically increases both the number of axons labeled and the density of label within single axons.

Influence of the tubular flow rates on the endocytic uptake and the excretion of horseradish peroxidase by rat kidney

Summary The renal clearances of horseradish peroxidase (HRP) in diuretic and anti-diuretic rats were compared with the concentrations of HRP in the renal cortex of the same rats. The injection of mannitol, hypertonic saline, or antagonists to histamine and serotonin, together with HRP, caused diuresis. The renal clearance of HRP was 7–15% of the inulin clearance, relatively small amounts of HRP were reabsorbed into the renal cortex, and relatively large amounts were excreted in the urine. However, when HRP was injected alone causing vascular leakage and anti-diuresis, 6–10 times higher concentrations of HRP were found in the renal cortex, and only a few percent of the protein were excreted in the urine during the first 20 minutes after injection. It is suggested that the low or high endocytic uptake of HRP by the renal cortex was related to the high or low tubular flow rates during diuresis or anti-diuresis.

Cytochemistry of undamaged neurons transporting exogenous protein in vivo.

AbstractThe uptake, transport and fate of exogenous protein in undamaged cranial motor and neurosecretory neurons in mice have been investigated using enzyme cytochemical techniques for horseradish peroxidase (HRP) and lysosomal acid hydrolases. Labeling of these neuronal perikarya with HRP is accomplished by retrograde axoplasmic transport from their axon terminals following vascular injection of the protein or by direct somal‐dendritic uptake subsequent to cerebral ventriculo‐cisternal perfusion of peroxidase. Axon terminals of neurosecretory neurons innervating the posterior pituitary gland and those of cranial motoneurons can remain unexposed to HRP administered intraventricularly and, therefore, cannot directly incorporate the protein.Perikaryal organelles containing HRP reaction product include multivesicular bodies, small vacuoles, membrane‐delimited cisterns and dense bodies. The concentrations of these different types of peroxidase‐positive organelles are consistently greater in retrogradely labeled cell bodies than in perikarya which have pinocytosed the protein from the perisomal clefts, even though the amount of HRP bathing the cell bodies is much greater than the amount exposed to the axon terminals. Since the same groups of dense bodies contain lysosomal acid hydrolases as well as exogenous peroxidase, the HRP‐labeled dense bodies are indeed lysosomes. Exogenous peroxidase does not stimulate lysosome proliferation; the concentration of acid hydrolase‐positive lysosomes appears to remain the same whether or not the cells are labeled with HRP.Despite peroxidase labeling of cranial motor and neurosecretory cell bodies after intraventricular injection, the anterograde axonal transport of peroxidase in these neurons is negligible. This transport can be demonstrably increased in the neurosecretory system, but not in cranial motor nerves, in response to hyperosmolarity induced in the animals by salt loading. Under such a stimulus many axons and Herring bodies in the posterior pituitary gland contain HRP‐labeled cisterns and 1,200–2,000 Å wide dense bodies. Both types of organelles also contain acid hydrolase activity and are confluent with each other. When compared to normal controls, the concentration of acid hydrolase‐positive organelles in pituitary stalk axons and Herring bodies from osmotically stressed mice is noticeably increased. Axonal cisterns with acid hydrolase activity and those transporting peroxidase in an anterograde or a retrograde direction appear similar morphologically.Our results indicate that peroxidase pinocytosed by the neuron is eventually sequestered within perikaryal lysosomes for enzymatic degradation. Under normal conditions acid hydrolase activity and the majority of lysosomes are confined largely to dendrites and the cell body, and anterograde axonal transport of HRP is minor. In the osmotically stressed neurosecretory system, HRP appears to be carried coincidentally and anterogradely along with the movement of acid hydrolases, most likely from secondary lysosomes in the cell body. The movement of acid hydrolases down the axon is presumably increased for degradative purposes in the posterior pituitary gland. A part of the cisternal network conveying acid hydrolases and transporting peroxidase in anterograde and retrograde directions may represent a special compartment of agranular reticulum specifically involved with the lysosomal system of organelles and important in the segregation of exogenous macromolecules within the neuron for transport to or from lysosomes.

Response of peritoneal cells to horseradish peroxidase and aldehyde-fixed erythrocytes in the mouse: an electron microscope study.

In adult dd-mice, response of peritoneal free cells to either horseradish peroxidase (HRP) or glutaraldehyde-fixed erythrocytes were examined by electron microscopy. As reported previously (ABE et al., 1979), peritoneal free cells are classified into three types: type I, II and III cells. Type I cells are small lymphocytes, type II cells are medium-sized mononuclear cells, and type III cells are macrophages. The amount of HRP taken up is very large in type III cells. The cells contain numerous vacuoles filled with HRP. Type I and II cells also have vesicles containing HRP. The vesicles are fewer in type I cells than in type II. Type III cells rapidly enclose erythrocytes by thin cytoplasmic flaps and then ingest several erythrocytes. In type III cells, a labyrinth of ramifying tubes seen in the peripheral cytoplasm may serve as a reserve of the membrane system for allowing a rapid coverage and internalization of large foreign materials. A part of type II cells attach to erythrocytes and take them by a varying depth of invagination. Type I cells do not respond to erythrocytes at all. In conclusion, the three types of peritoneal cells can be differentiated by their response to foreign materials as well as by their cytologic features.

Afferent projections to the rat locus coeruleus as determined by a retrograde tracing technique.

Afferent connections to the rat locus coeruleus (LC), which contains exclusively noradrenergic neurons, have been traced using the technique of retrograde transport of horseradish peroxidase (HRP). In order to ensure accurate placement of adequate amounts of HRP in the LC, a microiontophoretic delivery technique coupled with single cell recording was employed. The use of electro physiological "landmarks" as aids in placing the injections is described. Following HRP injections into the LC, forebrain structures containing labelled neurons included the insular cortex, the central nucleus of the amygdala, the medial, lateral and magnocellular preoptic areas, the bed nucleus of the stria terminalis, and the dorsomedial, paraventricular and lateral hypothalamic areas. In the brainstem reactive neurons were observed in the central grey substance, the reticular formation, the raphe, vestibular, solitary tract and lateral reticular nuclei. In particular, the areas of catecholamine cell groups A1, A2 and A5 appeared to contain many reactive cells. Labelled neurons were also observed in the fastigial nuclei and in the marginal zones of the dorsal horns of the spinal cord. This pattern of afferent innervation supports suggestions for a role for the LC in behavioral arousal mechanisms and autonomic regulation.

Comparison of light‐microscopic methods for visualizing horseradish peroxidase granules in stained and unstained tissue

This study was directed toward a comparison of the effectiveness of three different light-microscopical methods for identifying and studying the features of cells that contain horseradish peroxidase (HRP) granules. The cells studied were located in the trigeminal ganglia and were labeled after the injection of HRP solutions into single, maxillary, canine teeth. The tissue was examined with light field, dark field, and differential interference optical systems. The results indicated that differential interference optics were especially useful in revealing the presence of intracellular HRP granules in both unstained and stained tissue, particularly when cells only contained small amounts of HRP. It was also noted that a wide variety of quantitative analyses could be performed with differential interference optics, even when using unstained tissue.

Subdivisions of the auditory cortex of the cat: the retrograde transport of horseradish peroxidase to the medial geniculate body and posterior thalamic nuclei.

AbstractEach subdivision of auditory cortex was injected with horseradish peroxidase (HRP). The amount of HRP injected was varied with the result that in some cases the diffusion of HRP was restricted while in other cases, HRP diffused over an entire cortical subdivision and encroached on adjacent subdivisions. In the case of restricted diffusion only a relatively small number of thalamic cells were “labelled”; in the case of extensive diffusion many times that number of cells were labelled. Whether relatively few or many cells, the distribution of labelled cells fell into distinctive patterns. Each pattern consisted of a focus or concentration of labelled cells in one thalamic subdivision and a sparser population of labelled cells in other thalamic subdivisions. The most striking example of a pattern is produced by injecting AI. This injection resulted in a band in the ventral division of the medial geniculate body densely populated with labelled cells; but even after the smallest injection, labelled cells were also found outside the ventral division, in the magnocellular and dorsal divisions of the medial geniculate body, and in the posterior group. (The ventral extremity of the ventral division was always free of cells and this sector is likely a separate nucleus which projects to Ep.) Other patterns are produced by injecting HRP in other cortical subdivisions: temporal injections produced a concentration in the caudal division of the medial geniculate body; insular injections resulted in a concentration in the medial sector of the posterior group; AII injections led to a concentration in the magnocellular and deep dorsal divisions of the medial geniculate body; injections in ventral Ep led to a concentration of labelled cells in the dorsal division of the medial geniculate body; finally, dorsal Ep seems to be very different from the other divisions inasmuch as injections in it resulted in labelled cells in non‐auditory thalamus–the pulvinar and lateral nuclei, as well as the auditory thalamus.These results lead to the following conclusions. The laminated and principal part of the ventral division of the medial geniculate body projects only to AI. Every other thalamic subdivision projects to two or more cortical subdivisions: the caudal division projects primarily to the temporal area with sparser projections to AII and Ep; the dorsal division projects primarily to Ep with sparser projections to AI, AII, and the temporal area; the medial sector of the posterior group projects primarily to the insular area and to the posterior ectosylvian gyrus; the magnocellular division of the medial geniculate body projects to every subdivision of auditory cortex, including AI and AII.

The projection of the locus coeruleus upon the neocortex in the macaque monkey

After injections of horseradish peroxidase into most of the functional areas of the neocortex of the cerebral hemispheres of macaque monkeys, labelled cells have been found in the locus coeruleus of the brain stem. When the injections are into the cortex of one hemisphere labelled cells are present in the locus coeruleus of both sides, but the number of cells on the contralateral side is 20–50% of those on the ipsilateral side. After injections of the same amount of horseradish peroxidase into approximately similar extents of the cortex of different functional areas in the two cerebral hemispheres the number of labelled cells is the same in the locus coeruleus of both sides. It is concluded that the locus coeruleus projects bilaterally upon most of the functional areas of the neocortex and that this projection is uniform to the different areas.

Altered renal cortical reabsorption of protein and urinary excretion of sodium in relation to vascular leakage induced by horseradish peroxidase.

The intravenous injection of horseradish peroxidase (HRP) into rats of the Sprague-Dawley strain caused vascular leakage as detectable by a 20-40% increase in the hematocrit and a 15-20% decrease in plasma protein concentration. These changes did not occur when the same amounts of HRP were injected into rats pretreated with antagonists to histamine and serotonin. After pretreatment with the antagonists, the reabsorption of HRP by the proximal tubule cells (the concentration of HRP in the total particulate fractions) showed a 77% decrease and the urinary excretion of sodium showed more than an 80% increase as compared to the values from rats treated with HRP alone. In addition, the blood clearance rate of HRP was decreased and the urinary excretion of HRP was increased after treatment with the antagonists to histamine and serotonin. Cytochemical observations of formaldehyde vapor-fixed tissue also showed the effects of vascular leakage. After the injection of HRP in physiologic or hypertonic saline, the basal infoldings of the proximal tubule cells were strongly peroxidase-positive. When the same amounts of HRP were injected after pretreatment with antagonists to histamine and serotonin, or with mannitol, the basal infoldings were not stained or were stained faintly. Rats of the Wistar/Furth strain did not show the effects of vascular leakage observed with rats of the Sprague-Dawley strains. The questions are discussed as to whether the marked depression of renal cortical HRP absorption by mannitol and hypertonic saline (J Histochem Cytochem 23:707, 1975) is related to the prevention of vascular leakage, and whether the reabsorption of both sodium and protein is increased during the leakage of serum proteins into the interstitial tissue.

A study of the dynamics of retrograde transport and accumulation of horseradish peroxidase in injured neurons

The phenomenon of retrograde intraaxonal transport of extracellular markers introduced at the level of the axon terminal has been suggested as a possible mechanism of communication between the axon terminal and the neuron cell body. We tested the hypothesis that communication after axotomy might consist of a change in the rate of uptake or of transport of material by injured neurons. Small lesions were made with a needle in one retinal quadrant of chicks and immediately afterwards horseradish peroxidase (HRP) was injected into the vitreous body of the eye. The amount of HRP accumulated by some of the neurons of the isthmo-optic nucleus (ION) which project to the damaged areas was clearly different from that of nearby cells which project to the non-damaged portions of the retina. The uninjured cells accumulated enzyme marker beginning at 3.5 h after injection. The injured neurons did not accumulate significant amounts of HRP until between 4 and 6 h after injection. Between 6.75 h and 18 h the injured cells in the ION accumulated greater amounts of HRP than cells in other regions, but by 24 h the cells of the ION in the region of injury contained distinctly less label. This pattern of enzyme accumulation was confirmed by counts of the number of HRP-positive granules within cells of chicks fixed 4, 11.75, 12.25, 27.6 and 72 h after injury. In another series of experiments, the axon terminals of the ION were first exposed to HRP, and 1 h later some of the axons were damaged with a needle. In these cases, there was no difference between the injured and control neurons in the time of first appearance of labeled cells in the ION within the first 4 h after injection of HRP. These findings suggest that injury initially results in a decrease in the uptake of the marker rather than a decrease in the rate of retrograde transport. The amount of marker found in the injured neurons later is greater than that found in the control neurons. This subsequent difference may represent an increase in the rate of uptake, transport, or both or a decrease in the rate of degradation of HRP within the cell body as a response to injury of the axon.

The permeability of muscle capillaries to horseradish peroxidase.

The main objective of this study was to determine the pathways by which horseradish peroxidase (HRP) can cross the endothelium of muscle capillaries. Specimens of mouse diaphragm were fixed for cytochemical analysis at various intervals after intervenous injection of 0.5 mg HRP, at 4 min after intervenous injection of varied amounts of HRP, and at 4 min after intervenous injections in various volumes of isotonic NaCl. Our findings indicate that endothelial junctions serve as a barrier which may allow passage of very limited amounts of HRP. They also suggest that endothelial vesicles transfer HRP from the capillary lumen to the pericapillary interstitium as well as in the reverse direction. Increasing the volume of solution injected to approximately 30% of total blood volume did not increase the amount of HRP that left the capillary lumen. Our results with HRP do not provide clearcut evidence that endothelial junctions are the site of the small pore.

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 10(6) 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-(125)I, cell-associated isotope disappears with a t (1/2) of 20-30 hr and they release monoiodotyrosine-(125)I 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.