Horseradish Peroxidase Accumulation Research Articles

Flow shear stress affects macromolecular accumulation through modulation of internal elastic lamina fenestrae

Background Internal elastic lamella and elastic lamellae serve as barriers for macromolecular accumulation in vascular wall. The function of internal elastic lamella in atherogenesis induced by shear stress has not been well understood yet. In the present study, internal elastic lamella remodeling and transmural accumulation of macromolecules in the arterial wall under altered shear stress were investigated. Methods The ligation of left distal carotid branches induced low shear stress in the left common carotid artery and high shear stress in the right common carotid artery of rats for 7 days. Fenestrae in internal elastic lamella were assessed by confocal microscopy. Horseradish peroxidase accumulation was determined by its transmural concentration profiles. Fibronectin expression was examined by immunohistologic staining and Western blotting. Findings Low shear stress significantly decreased the percentage of fenestrae area in internal elastic lamella from 15.35% to 8.03% in left common carotid artery, while high shear stress nearly had no influence in right common carotid artery. Comparing with less accumulation of horseradish peroxidase in right common carotid artery, horseradish peroxidase was significantly accumulated in the innermost part of the media, and fibronectin expression increased to 3.3-fold in left common carotid artery. Interpretation Our results suggested that shear stress alteration could influence the endothelial cell function as well as internal elastic lamella and elastic lamellae remodeling. Low shear stress decreased the permeability of internal elastic lamella and elastic lamellae by reducing fenestrae of them, which involved the enhanced fibronectin expression. Therefore, the increased accumulation of macromolecules in the arterial wall was observed. Our data supply new evidence on the mechanisms of atherogenesis induced by low shear stress.

Polarized membrane movements in A6 kidney cells are regulated by aldosterone and vasopressin/vasotocin.

The polarity of cell-surface membrane movements and their regulation by adrenal steroid hormones (10(-6) M aldosterone) and vasopressin or vasotocin were studied in A6 cells. This cell line is derived from the Xenopus laevis distal nephron and displays regulated Na+ reabsorption but is devoid of regulated water transport. Apical and basolateral membrane movements and their hormonal regulation were characterized by measuring the uptake of the fluid phase marker horseradish peroxidase (HRP) and the secretion of proteins on both sides of cell monolayers cultured on filters. The intracellular accumulation of HRP was visualized by electron microscopy and quantified by the measure of cell-associated peroxidase activity. The rate of intracellular HRP accumulation corresponded to 0.01 nl/minute/filter (4.7 cm2) from the apical side and was 20-32 times faster from the basolateral side. In contrast, the level of protein secretion was 3.5 times higher apically than basolaterally. Among the secreted proteins some were found to be secreted essentially apically, and others basolaterally. Vasotocin increased apical endocytosis (1.88-fold) and apical protein secretion (1.49-fold) in cells pretreated with aldosterone. Basolaterally, only the endocytosis was increased, and to a smaller extent (1.36-fold). These effects of vasotocin depended on aldosterone pretreatment and could be mimicked with forskolin and 8-bromoadenosine 3':5'-cyclic monophosphate (BrcAMP). Measurements of intracellular cAMP levels showed that there was a rankorder correlation between the induced level of intracellular cAMP and that of apical endocytosis. This study shows that vasotocin has a polarized stimulatory action on apical endocytosis and protein secretion in A6 cells, and that the mediation of this action by cAMP is aldosterone dependent.

Quantitation of fluid phase uptake in paramecium. 1. Kinetics in the cells blocked in phagocytic activity

Uptake of horseradish peroxidase (HRP) was quantified in the unicellular eukaryote Paramecium aurelia. About 80% of the total HRP accumulated within 20 min entered the cells via a fluid phase pathway as demonstrated measuring the HRP internalization in the presence of yeast mannan. The rate of HRP accumulation was concentration-dependent and was found to be linear over the range of 50–500 μg HRP per ml of the extracellular medium. During the first 10 min of exposure to HRP, the mannan-uninhibitable uptake was found to reach 1.2–1.65 ng HRP per mg protein (depending on the concentration of the marker in the medium), which corresponds to 0.68 fl per cell/min. Accumulation of HRP reached a plateau within the next 10–15 min and its intracellular uptake was 2–2.55 ng HRP per mg protein. When the phagocytic activity of the cells was blocked with 1-propranolol, the amount of cell-accumulated HRP was 1.8–2.1-fold higher than in the control and the rate of the marker uptake within the first 10 min of incubation reached 1.114 fl per cell/min.

Impairment of retrograde axonal transport in wobbler mouse motor neuron disease

The earliest horseradish peroxidase (HRP) neuronal labeling (the fastest retrograde transport) was determined by histochemical techniques at various intervals after intramuscular HRP injection in wobbler mice and normal littermates. In the clinically impaired forelimb system, the retrograde transport rate was 150-170 mm/day in wobbler mice and 170-230 mm/day in controls. However, there was no statistical difference between the two groups. The neuronal HRP accumulation at the early intervals was significantly less in wobbler mice than controls, suggesting that the amount of HRP transport was diminished in each axon. For the clinically intact hindlimb nerves, the rate was normal in wobbler mice, but the amount of neuronal HRP was significantly increased. Retrograde axonal transport appeared to be affected in a differential fashion, depending on the extent of disease.

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2–4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 × 10 −7, 1 × 10 −9, and 5 × 10 −10 M, respectively. αIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.

Fibroblasts maintain a complete endocytic pathway in the presence of lysosomotropic amines

We have investigated the effects of the lysosomotropic amines, ammonium chloride and chloroquine, on the delivery of fluid-phase pinocytic tracers to lysosomes in Chinese hamster ovary (CHO) cells. In preliminary experiments, 15 m M ammonium chloride and 0.1 m M chloroquine were found to be sufficient to give maximal protection of endocytosed material from digestion in a lysosome. In the presence of either amine at these concentrations, the generation time of CHO cells was depressed by <30% even though selective depletion of lysosomal hydrolases was observed. For cells treated with either amine for 1 or 6 days the amount of horseradish peroxidase (HRP) internalized in a 1-h pulse was ~50–70% of that of control. By cell fractionation, cells treated with amine for 2 or 6 days were found to accumulate fluorescein-dextran or HRP in lysosomes. HRP accumulation in lysosomes in amine-treated cells was also observed by electron microscopy. Little exocytosis of lysosomal HRP into the media was observed under any condition. We conclude that in long-term amine-treated CHO cells endocytic vesicle traffic is maintained.

Tracheal permeability in rats exposed to ozone. An electron microscopic and autoradiographic analysis of the transport pathway.

Exposure of rats to ozone (O3), 0.8 ppm increases the tracheal permeability to 99mTc-diethylenetriaminepentaacetate (99mTc-DTPA) about twofold but to 125I-bovine serum albumin (125I-BSA) to a lesser extent. It is generally believed that exposure to air pollutants causes perturbation of tight junctions and formation of intercellular channels for the passage of molecules from airway lumen to blood. We now report that a second mechanism, vesicular transport, is operative in the transepithelial movement of molecules, that this mechanism is speeded in tracheas of O3-exposed rats, and that there is a concurrent delay in movement of BSA from connective tissue to capillaries after O3 exposure. Horseradish peroxidase (HRP) instilled in trachea was taken up by endocytic vesicles, which could be localized in apical as well as basal regions of ciliated and nonciliated cells. A count of HRP-positive vesicles and measurement of their surface area revealed an approximate twofold increase in O3-exposed rats over that in control animals breathing clean air; this paralleled a twofold increase in transport of 99mTc-DTPA from tracheal lumen to blood. An autophagocytic process induced in tracheal epithelial cells by O3 is proposed. Despite the difference in the size of HRP and BSA, the 2 molecules migrated through common pathways and were colocalized in the luminal membranes as well as in endocytic vesicles and intercellular spaces in double labeling experiments involving simultaneous detection of HRP by cytochemistry and 125I-BSA by autoradiography. This procedure proved particularly useful in detecting a dramatic accumulation of 125I-BSA autoradiographic grains in subepithelial connective tissue and HRP accumulation in intercellular spaces and at the basal membrane-connective tissue junction in O3-exposed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Extensive intraneuronal spread of horseradish peroxidase from a focus of vasogenic edema into remote areas of central nervous system

A study was made of the uptake of horseradish peroxidase (HRP) into neurons from a cryogenic cortical lesion in the mouse brain associated with vasogenic edema, following intravenous administration of the tracer. Particular emphasis was placed on the axonal spread of HRP from the primary lesion to other areas of the central nervous system. The distribution of HRP was studied by light microscopy, using highly sensitive histochemical methods, 3-144 h after the onset of the injury. Extravasated HRP was taken up into nerve cell bodies in and around the primary lesion, forming different patterns of labelling: (1) granular, (2) diffuse, and (3) a combination of granular and diffuse staining. Granularity is considered to be the result of HRP accumulation in lysosomes occurring in undamaged or slightly damaged nerve cells, whereas the diffuse, non-granular pattern presumably occurs in severely damaged neurons. Nerve cell bodies containing HRP reaction product were also found in the contralateral cortex, ipsilateral thalamus, substantia nigra, amygdala and ventral tegmental area, presumably a consequence of retrograde axonal transport of the tracer from the primary injury. HRP-containing axons were present in the corpus callosum and in the pyramidal tract of the injured hemisphere all the way down to the cervical spinal cord. Labelling of axonal terminals and preterminal axons in the ipsilateral thalamus, entopeduncular nucleus, subthalamic nucleus, substantia nigra and pons indicated anterograde transport of HRP to these regions. Thus very extensive intraneuronal spread of a macromolecular edema component takes place from a primary focal brain lesion to areas located far away from but neuroanatomically connected to this injured region. The brain thus seems to be affected by focal vasogenic edema in many more ways than are recognized at present.

Rapid effects of insulin on the cycling of the insulin receptor in a human monocyte cell line (U-937).

The insulin receptor and its regulation by insulin was studied in U-937 monocytes, a human cell line with properties similar to those of normal peripheral blood monocytes. Treatment of this cell with insulin for 8-16 h produced an overall loss in the insulin receptor, i.e., a loss of receptors from the cell surface and internal pools. In contrast, short-term insulin treatment (15-30 min) caused a reduction in cell surface receptors but an increase in the internal receptors, as judged by pronase treatment at 4 degrees C to distinguish receptor location. After the removal of insulin and pronase, the internalized receptors were rapidly reinserted back into the cell surface after warming to 37 degrees C. Further studies showed an insulin-mediated increase in fluid-phase pinocytosis as measured by horseradish peroxidase (HRP) uptake. The amount of HRP accumulation and the time course for this stimulation were similar to those for receptor internalization. These features plus other results suggest that the insulin-stimulated internalization of insulin receptors may require an acceleration in the rate of pinocytic vesicle formation.

Sources of thalamic projections in the region of vibrissal representation in the rat somatosensory cortex studied by horseradish peroxide labeling

The retrograde horseradish peroxidase (HRP) transport method was used to study the location and morphology of neuron groups in the ventrobasal complex of the thalamus projecting to the region of vibrissal representation in the somatosensory cortex in rats. Injection of HRP into a circumscribed region of the somatosensory cortex revealed the following pattern of organization of the thalamocortical relay groups of neurons. Labeled neurons were located in the ventroposterolateral nucleus of the ventrobasal complex and were associated in groups 100–120 µ in diameter. Staining of several groups, even after minimal injections of HRP, and an increase in the number of labeled cells in each group with an increase in the zone of injection of HRP in the cortex suggest the presence of both convergence and divergence of specific thalamocortical pathways. The different shapes of the relay neurons and differences in the degree of HRP accumulation by them may indicate differences in their functional role in thalamocortical integration.

The effect of nitrogen dioxide on tracheal uptake and transport of horseradish peroxidase in the guinea pig.

The effect of 5 and 15 ppm of nitrogen dioxide (NO2) on airway transepithelial permeability to horseradish peroxidase (HRP) (molecular weight, approximately 40,000 daltons) was studied in the guinea pig. Age- and sex-matched guinea pigs (n = 34) were exposed to NO2 for 2 and for 14 days. In the control guinea pigs, 0.05% of the intratracheally instilled dosage HRP was present in the plasma within 10 min, as detected by radioimmunoassay, and increased thereafter at amean rate of 0.0016%/min. After exposure to NO2, there was a significant increase in the rate of plasma HRP accumulation (p < 0.05) with the maximal increase detected in the group exposed to 15 ppm of NO2 for 2 days. The morphologic correlates of increased permeability to HRP appear to be (a) an increase in pinocytotic activity within the hyperplastic globlet cells, and (b) transjunctional transport by leaky tight junctions that was noted only in animals exposed to 15 ppm for 14 days (p < 0.01). Our data suggests that pinocytotic vesicular transport of proteins in the secretory cells of the airway may be an important mechanism for the movement of exogenous nonlipid soluble macromolecules across the epithelial barrier.

Effects of insulin-induced hypoglycemia on cerebrovascular permeability to horseradish peroxidase.

The effects of hypoglycemia on cerebrovascular permeability to a protein, horseradish peroxidase (HRP), were studied in mice given 3 or 8 units of crytalline zinc insulin intraperitoneally. HRP (10 mg in 0.1 ml saline) was injected intravenously 15 to 20 minutes prior to sacrifice. Both mildly and severely hypoglycemic groups of mice showed a drastic reduction in the normal transit of HRP across cerebral arterioles. The number of normally-stained vessel segments and of HRP-filled endothelial vesicles decreased in insulin-treated mice. In the brains of severely hypoglycemic mice, however, increased parenchymal HRP accumulation occurred. A ruptured blood vessel was found in the center of one-fourth of the focal exudates examined. Electron microscopic examination revealed thrombin, sometimes extending through the vessel wall, and hemorrhage, yet inter-endothelial tight junctions remained intact. Seizures were associated with severe hypoglycemia in 6 out of 10 mice with serum glucose levels below 40 mg/100 ml following 8 units of insulin, but the number of focal exudates per brain was similar in all 10 mice. We conclude that insulin-induced hypoglycemia is associated with decreased HRP transit across cerebral arterioles, and that severe insulin shock is also accompanied by actual rupture of vessel walls and extravasation of blood and HRP into the parenchyma of the brain.

Uptake and retrograde axonal transport of horseradish peroxidase in regenerating facial motor neurons of the mouse.

Uptake and retrograde axonal transport of intravenously injected horseradish peroxidase (HRP) was studied during regeneration after a crush injury of the facial nerve of the mouse. The circulation time of HRP was 12 to 24 h. HRP injected immediately after the crush diffused into injured axons in the crushed region and accumulated subsequently in perikarya of facial neurons in the brain stem. After a time interval of 1 h or 5 days between the crush and the injection only a faint HRP accumulation occurred in a few facial neurons. After an interval of 7 days a moderate number of neurons had incorporated the tracer, while after more than 9 days the HRP activity in the regenerating neurons was more pronounced than in the contralateral neurons. Ultrastructurally, muscles of the vibrissae showed denervated subneural apparatuses 6 days after the crush. 8 days after the crush regenerating axon terminals containing small clusters of synaptic vesicles, dense cored vesicles and some HRP-labelled vesicles, were found over some gutters and after 10 to 13 days all examined gutters contained axon terminals with large numbers of synaptic vesicles and some HRP-containing vesicles. More than one axon terminal profile was seen in the same synaptic gutter. 32 and 64 days after the crush the neuromuscular junctions had regained a more mature appearance. The calibre spectra of the crushed facial nerves still showed a shift towards smaller diameters 134 days after the crush, at a time when a slight increase in HRP activity in the facial neurons persisted.