Vesicular Transport Of Horseradish Peroxidase Research Articles

Time-related Ultrastructural Changes in an Experimental Model of Whole Brain Irradiation

TO SIMULATE THERAPEUTIC irradiation, we exposed rats to conventional fractionation (200 ± 4 cGy/d, 5 d/wk; total dose, 4000 cGy). The effects of this regimen were assessed by electron microscopic examinations of brain microvascular and parenchymal cells 15 and 90 days after irradiation. Studies of the transendothelial passage of horseradish peroxidase provided information about the functional status of the blood-brain barrier. At 15 days after irradiation, there was an increased vesicular transport of horseradish peroxidase across the intact endothelium without opening of the tight junctions, and without evidence of structural alterations of neuropil, neuronal bodies, and astrocytes. Ninety days after irradiation, well-defined ultrastructural alterations were observed, involving the microvasculature, the neuropil, the neuronal bodies, and astrocytes. The main ultrastructural feature of cortical microvessels was their collapsed aspect, associated with perivascular edema containing cell debris. Altered neurons and reactive activated astrocytes were also noticeable. These data suggest a possible association, not necessarily causal, between damage of the microvascular/glial unit of tissue injury and development of radiation-induced brain toxicity.

Time-related Ultrastructural Changes in an Experimental Model of Whole Brain Irradiation

To stimulate therapeutic irradiation, we exposed rats to conventional fractionation (200 +/- 4 cGy/d, 5 d/wk; total dose, 4000 cGy). The effects of this regimen were assessed by electron microscopic examinations of brain microvascular and parenchymal cells 15 and 90 days after irradiation. Studies of the transendothelial passage of horseradish peroxidase provided information about the functional status of the blood-brain barrier. At 15 days after irradiation, there was an increased vesicular transport of horseradish peroxidase across the intact endothelium without opening of the tight junctions, and without evidence of structural alterations of neuropil, neuronal bodies, and astrocytes. Ninety days after irradiation, well-defined ultrastructural alterations were observed, involving the microvasculature, the neuropil, the neuronal bodies, and astrocytes. The main ultrastructural feature of cortical microvessels was their collapsed aspect, associated with perivascular edema containing cell debris. Altered neurons and reactive activated astrocytes were also noticeable. These data suggest a possible association, not necessarily causal, between damage of the microvascular/glial unit of tissue injury and development of radiation-induced brain toxicity.

Brefeldin a inhibits the transcytotic vesicular transport of horseradish peroxidase in intrahepatic bile ductules isolated from rat liver

The fungal metabolite Brefeldin A (BFA) has become a valuable tool to address mechanisms of membrane transport in eukaryotic cells. The aim of the study was to investigate the action of BFA on the endocytic and transcytotic pathways in the biliary epithelium. Intrahepatic bile ductules were isolated from rat liver by collagenase digestion and mechanical separation of biliary tree from parenchymal tissue. Tissue remnants were first incubated in L-15 culture medium in absence or presence of BFA (10 or 20 mumol/L) or a BFA-inactive analog (B-36, 10 or 20 mumol/L) for 20 minutes at 37 degrees C. They were then exposed to horseradish peroxidase (HRP) (10 mg/mL) for 3 minutes at 37 degrees C and finally prepared for electron microscopy immediately (time 0) or after further 5, 10, 15, 20, 60, or 120 minutes' incubation in HRP-free medium with or without BFA. In control cells, HRP was predominantly found in regularly shaped, spherical vesicles. In the presence of BFA but not of its analog, HRP was retained in a prominent tubular juxtanuclear network. Part of this network was labeled for thiamine pyrophosphatase (TPP), a Golgi enzyme marker. A morphometric analysis of HRP-containing structures was performed to quantify the intracellular distribution of HRP. In presence of BFA, the volume density (VD = % area) of HRP-containing structures in the basolateral region was not significantly different with respect to control cells at 0 (1.08 +/- 0.11 vs. 1.32 +/- 0.11) or 5 minutes, respectively (1.33 +/- 0.19 vs. 1.40 +/- 0.13). On the contrary, VD or HRP-containing structures in the apical region at 15 minutes decreased from 1.95 +/- 0.19 in control cells to 1.12 +/- 0.20 (P < .02) in BFA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Brefeldin A inhibits the transcytotic vesicular transport of horseradish peroxidase in intrahepatic bile ductules isolated from rat liver

The fungal metabolite Brefeldin A (BFA) has become a valuable tool to address mechanisms of membrane transport in eukaryotic cells. The aim of the study was to investigate the action of BFA on the endocytic and transcytotic pathways in the biliary epithelium. Intrahepatic bile ductules were isolated from rat liver by collagenase digestion and mechanical separation of biliary tree from parenchymal tissue. Tissue remnants were first incubated in L-15 culture medium in absence or presence of BFA (10 or 20 μmol/L) or a BFA-inactive analog (B-36, 10 or 20 μmol/L) for 20 minutes at 37°C. They were then exposed to horseradish peroxidase (HRP) (10 mg/mL) for 3 minutes at 37°C and finally prepared for electron microscopy immediately (time 0) or after further 5, 10, 15, 20, 60, or 120 minutes' incubation in HRPfree medium with or without BFA. In control cells, HRP was predominantly found in regularly shaped, spherical vesicles. In the presence of BFA but not of its analog, HRP was retained in a prominent tubular juxtanuclear network. Part of this network was labeled for thiamine pyrophosphatase (TPP), a Golgi enzyme marker. A morphometric analysis of HRP-containing structures was performed to quantify the intracellular distribution of HRP. In presence of BFA, the volume density (VD = % area) of HRP-containing structures in the basolateral region was not significantly different with respect to control cells at 0 (1.08 ± 0.11 vs. 1.32 ± 0.11) or 5 minutes, respectively (1.33 ± 0.19 vs. 1.40 ± 0.13). On the contrary, VD of HRP-containing structures in the apical region at 15 minutes decreased from 1.95 ± 0.19 in control cells to 1.12 ± 0.20 ( P < .02) in BFA-treated cells. Most striking were the changes in VD of HRP-containing elements in the intermediate (juxtanuclear) area, showing a value of 0.45 ± 0.22 in control cells against 2.59 ± 0.50 in BFA-treated cells at 15 minutes (n = 10) ( P <.01). Finally, taurocholic acid (TCA, 50 μmol/L) significantly increased the VD of HRP-labeled structures in the apical region only in the absence of BFA (2.22 ± 0.20, n = 5 vs. 1.14 ± 0.32 in BFA-treated samples at 5 minutes P < .02). These data suggest that in rat biliary epithelial cells, BFA does not interfere with fluid-phase endocytosis but inhibits the transcytotic vesicular pathway, as shown by the retention of HRP in the Golgi juxtanuclear area. These properties make BFA of interest for the study of intracellular mechanisms involved in bile ductular secretion.

Brefeldin a modifies the transcytotic vesicular transport of horseradish peroxidase (HRP) in isolated rat hepatocyte couplets and bile ductular cells A. Benedetti, L. Marucci, C. Bassotti, C. Guidarelli, G. Macarri, A. M. Jezequel, F. Orlandi. Department of Gastroenterology and Institute of Experimental Pathology, University of Ancona, School of Medicine, Ancona, Italy

Brefeldin a modifies the transcytotic vesicular transport of horseradish peroxidase (HRP) in isolated rat hepatocyte couplets and bile ductular cells A. Benedetti, L. Marucci, C. Bassotti, C. Guidarelli, G. Macarri, A. M. Jezequel, F. Orlandi. Department of Gastroenterology and Institute of Experimental Pathology, University of Ancona, School of Medicine, Ancona, Italy

Monensin action on the Golgi complex in perfused rat liver: Evidence against bile salt vesicular transport

Several studies suggest that bile salts are transported from the basolateral to the canalicular membrane of hepatocytes by a vesicular pathway, possibly in part via the Golgi complex. To test this hypothesis, the present study examined, in the perfused rat liver, the influence of the Na+ ionophore monensin on the biliary secretion of taurocholate and biliary lipids. The effects of the drug have been checked by the study of the ultrastructural modifications of the Golgi complex, secretion of horseradish peroxidase, and bile salt uptake. An infusion of monensin (1, 3, or 5 μmol/L) into the liver induced considerable swelling of the Golgi complex within 5 minutes. After a bolus injection of horseradish peroxidase during monensin infusion, the biliary secretion of the protein was delayed (1 μmol/L monensin) and markedly reduced (5 μmol/L monensin). Bile salt uptake was virtually unchanged except with 5 μmol/L monensin. This suggests that monensin has the same effects on the subcellular traffic in the perfused liver as in cultured cells. After a bolus injection of taurocholate (0.25, 5.0, or 8.5 μmol/100 g body wt) during monensin infusion, the pattern of biliary secretion of the bile salt was identical to that of controls. During continuous infusion of taurocholate, a 10-minute monensin infusion (1 or 3 μmol/L) had no effect on the biliary secretion of taurocholate and on the secretion of lecithin and cholesterol induced by taurocholate. High concentrations (5 μmol/L) or prolonged infusions (20 minutes) of monensin decreased the biliary secretion of bile salts but corresponded to a marked decrease of taurocholate uptake. In summary, the Na+ ionophore monensin altered the Golgi complex and the vesicular transport of horseradish peroxidase, whereas taurocholate biliary secretion was not influenced unless taurocholate uptake by the liver was markedly decreased. It may be concluded that taurocholate and biliary lipid secretion, under these conditions, does not depend essentially on pathways involving acidic transporting vesicles and particularly the trans-Golgi complex.

Evidence for vesicular transport of horseradish peroxidase across endoneurial vessels of the sciatic nerve in normal mice.

The permeability of the endoneurial blood vessels of the sciatic nerve to horseradish peroxidase (HRP) was investigated in mice. The animals were killed 2,5, or 30 min after an i.v. injection of the protein, and the distribution of HRP in the sciatic nerve was studied by light and electron microscopy. HRP was frequently found in endothelial vesicles that were located near both the luminal and the abluminal side of the endothelium. The intercellular junctions appeared to be closed to HRP. The perivascular and interstitial macrophages in the endoneurium accumulated HRP by endocytosis. The results are interpreted as evidence for vesicular transport of HRP across endoneurial vessels to interstitial and perivascular macrophages.

Vesicular Transport of Horseradish Peroxidase during Chronic Bile Duct Obstruction in the Rat†

The vesicular transport system for biliary secretion of plasma-derived proteins was investigated in rats with chronic bile duct obstruction. Horseradish peroxidase, previously demonstrated to be a suitable tracer for vesicular transport, was employed in these studies. Both the time course of horseradish peroxidase secretion into bile and the morphological events in its uptake, transport, and biliary secretion were found to proceed in a manner essentially identical to that of sham-operated control animals. In addition, fragmentation of hepatocytes leading to sloughing into bile of large pieces of cytoplasm bearing horseradish peroxidase-containing endocytic transport vesicles frequently was observed in the cholestatic animals. These data suggest that the vesicular transport system for the secretion into bile of plasma-derived proteins remains intact and functional during chronic bile duct obstruction and that another mechanism, possibly fragmentation and solubilization of hepatocyte membranes followed by regurgitation of proteins released from endocytic vesicles, may be responsible for the elevation of biliary proteins within plasma seen during cholestasis.

Effects of cytochalasin D and colchicine on the uptake, translocation, and biliary secretion of horseradish peroxidase and [14C]sodium taurocholate in the rat

The roles of microfilaments and microtubules in the hepatocellular uptake, translocation, and biliary excretion of horseradish peroxidase and [14C]sodium taurocholate were investigated using the microfilament inhibitor cytochalasin D and the microtubule inhibitor colchicine. In separate studies, horseradish peroxidase and [14C]taurocholate were injected separately as a bolus into rat portal veins after treatment with cytochalasin D or colchicine, and bile was collected and analyzed for the presence of horseradish peroxidase and [14C]taurocholate. Cytochalasin D treatment depressed bile flow by ~50% and decreased the biliary secretion of [14C]taurocholate in direct proportion to bile flow. Horseradish peroxidase secretion into bile was unaffected, and total biliary protein secretion was decreased only slightly. Because of the depression of bile secretion, concentrations in bile of horseradish peroxidase and total biliary protein increased significantly. Consistent with reported observations of cytochalasin D, decreases in microfilaments and dilated bile canaliculi were observed by electron microscopy; however, the vesicular transport of horseradish peroxidase as observed using electron microscopy cytochemistry appeared to be normal and unaffected by cytochalasin D treatment. Colchicine, in contrast, had minimal effect on bile flow and did not diminish the biliary secretion of [14C]taurocholate. Colchicine inhibited both the total amount of horseradish peroxidase secreted into bile as well as the rate of its secretion in comparison with control and cytochalasin D-treated animals. Cellular morphology was consistent with published observations for colchicine, which included a marked decrease in microtubules. In addition, after electron microscopy cytochemistry there was a paucity of horseradish peroxidase-containing vesicles within the hepatocytes, suggesting that colchicine interfered with the vesicular transport of horseradish peroxidase. Collectively, the data suggest that (a) the mechanism used by hepatocytes for the secretion of bile acids is independent of the vesicular transport of biliary proteins and is dependent upon intact microfilaments and (b) such vesicular transport of protein into bile requires an intact and functioning microtubular network.

Hepatic handling of bile salts and protein in the rat during intrahepatic cholestasis

17 α-Ethynyl estradiol-induced cholestasis was used to study the relationship of protein to bile salt transport in liver. The biliary secretion of horseradish peroxidase was unaltered in treated animals despite a 56% reduction in bile flow. Cytochemistry confirmed that estradiol caused no alteration in the handling of tracer. In a second study, the peak biliary secretion of [14C]taurocholate was reduced by ~46% in treated animals. The kinetics of 125I-cholylglycylhistamine, a bile salt derivative, were identical to those of taurocholate in control and cholestatic animals. Taurocholate and cholylglycylhistamine secretion were markedly reduced in control animals during competition with unlabeled taurocholate. Quantitative electron microscopic autoradiography with 125I-cholylglycyihistamine revealed a high concentration of grains over the endoplasmic reticulum and the Golgi complex including associated lysosomes and vesicles. These data demonstrate that estradiol markedly inhibits bile salt transport, but not vesicular transport of horseradish peroxidase. Furthermore, estradiol may alter the movement of bile salts through these organelles.

Blood-spinal cord barrier response to transection

The intergrity of the blood-spinal cord barrier (BSB) distal to a surgical transection was evaluated at the light and electron microscopic levels, using the tracers Evans blue albumin (EBA) and horseradish peroxidase (HRP). At the light microscopic level, the orange fluorescence of the EBA was detected as far as 0.45 mm distal to the transection. The tracer was not only diffusely distributed in the neuropil but also was apparently taken up by ventral horn neurons and glial cells. A feathery halo of EBA extending beyond their walls indicated that there had occurred some leakage of EBA from intrinsic vessels into the surrounding cord tissue. The microvasculature was evaluated at the ultrastructural level for evidence of changes in permeability to HRP. Pinocytosis and vesicular transport of the protein were apparent in some endothelial cells. In addition, there was limited evidence for leakage across tight junctions. After 1 day the barrier selectivity appeared to be restored and vesicular transport of HRP was not found. Endothelial pinocytosis, however, appeared enhanced for as long as 7 days following injury.

Effect of colchicine and S, S, S-tributyl phosphorotrithioate (DEF) on the biliary excretion of sucrose, mannitol and horseradish peroxidase in the rat

The purpose of this study was to demonstrate the effect of colchicine and S, S, S-tributyl phosphorotrithioate (DEF) on vesicular transport of horseradish peroxidase (HRP) into bile at the same time that permeability changes occur in the canalicular membrane. Anesthetized Sprague-Dawley rats were surgically prepared with a bile duct cannulae. Four hours after colchicine pretreatment (10 mg/kg, i.p.), biliary excretion of intravenously injected sucrose and mannitol was increased, while that of HRP was decreased. Administration of these marker compounds from the bile side by segmented retrograde intrabiliary injection (SRII) (40 μl washed in with 110 μl saline) resulted in decreased biliary excretion of all three markers in colchicine-pretreated rats. Twenty minutes after DEF pretreatment by intrabiliary administration (40 μl of DEF washed in with 31 μl of saline and held in place for 0.5 min), the biliary excretion of intravenously injected sucrose and mannitol was decreased but that of HRP was increased; when administered by SRII, the biliary excretion of all three marker compounds was increased. These results demonstrate that colchicine, in addition to having blocked vesicular transport (HRP), also increased canalicular permeability to sucrose and mannitol. DEF, on the other hand, decreased canalicular permeability to sucrose and mannitol and enhanced vesicular transport of HRP.

Morphologic effect of dimethyl sulfoxide on the blood-brain barrier.

Dimethyl sulfoxide (DMSO) opens the blood-brain barrier of mice to the enzymatic tracer horseradish peroxidase. A single injection of horseradish peroxidase in 10 to 15 percent DMSO into the tail vein along with 10 to 15 percent DMSO delivered intraperitoneally allowed horseradish peroxidase to fill the extracellular clefts throughout the brain within 2 hours. In the absence of DMSO, peroxidase failed to enter brain parenchyma except through the circumventricular organs. Opening of the blood-brain barrier by DMSO is reversible. Dimethyl sulfoxide stimulated the pinocytosis of horseradish peroxidase by the cerebral endothelium; the peroxidase was then directed to lysosomal dense bodies for degradation. Vesicular transport of horseradish peroxidase from the luminal to the abluminal wall of the endothelial cell was not observed. Dimethyl sulfoxide did not alter the morphology of endothelial cells or brain parenchyma.

Vesicular transport of horseradish peroxidase by ependymal cells of the medulla oblongata

Vesicular transport of horseradish peroxidase by ependymal cells of the medulla oblongata

Vascular permeability alterations to horseradish peroxidase in experimental brain injury

Protein uptake and transport within the brain stem vasculature of mechanically brain injured cats was studied by means of both light and electron microscopy utilizing intravenously injected horseradish peroxidase as the protein tracer. In animals sustaining low grade head injuries not of sufficient intensity to elicit either microscopic, intraparenchymal hemorrhages or subtle, neuropathological responses, peroxidase extravasation was noted both in the vascular walls and in the surrounding parenchyma of the ventromedial aspect of the brain stem. At the ultrastructural level as early as 3 min after brain injury, occasional arterioles, venules and capillaries displayed peroxidase leakage. In serial sections large endothelial segments of these vessels revealed the peroxidase reaction product within numerous vesicles which often shared continuity with tubular and vacuolar profiles. Such vesicular activity apparently moved the peroxidase from the luminal surface to extrude it into the basal lamina. From the perivascular basal lamina, the reaction product flooded the interstices of the surrounding brain stem parenchyma where occasional neural, glial and pericytic elements incorporated the peroxidase within coated invaginations, vesicles, tubules and vacuoles. In that protein leakage was consistently observed despite the apparent integrity of both the endothelial tight junctions and their cell membranes, it is concluded that the vesicular transport of horseradish peroxidase across the endothelia of the brain stem vasculature represents a possible mechanism of blood-brain barrier dysfunction in mechanical brain injury.

Vesicular transport of horseradish peroxidase from brain to blood in segments of the cerebral microvasculature in adult mice

The transfer of protein from the cerebral ventricles to the parenchymal blood-stream in mice was studied by electron microflapy. After perfusion with the protein tracer horseradish peroxidase (HRP; M.W. approx. 40,000) through the cerebral ventricles, the tracer penetrated the ependymal lining of the ventricles and was found in the extracellular space of the neuropil close to the ependyma. HRP was also seen in the vascular basement membrane and in endothelial vesicles opening at the abluminal endothelial surface, or situated within the endothelial cells in segments of the microvasculature (mostly small arterioles). In some of these segments HRP was also seen on the luminal surface of the endothelia and in surface-connected vesicles. The junctions connecting adjacent endothelial cellls were never penetrated by HRP. It is concluded that vesicular transport of HRP across the endothelium of the cerebral microvasculature represents a possible mechanism for protein removal from brain extracellular space.

Vasogenic Edema in the Injured Spinal Cord: Possible Role of Microtubules in Endothelial Vesicular Transport of HRP

The genesis of edema in the central nervous system (CNS) parallels changes in the blood-brain barrier (BBB). The anatomical seat of the BBB is located in the endothelium of the CNS. The presence of interendothelial tight junctions and the low rate of pinocytotic (vesicular) activity constitute the main features of the BBB. Based on the fate of intravascular horseradish peroxidase (HRP), mechanisms of edema genesis have been elucidated. A vesicular mechanism appears to play a role in the formation of edema in the injured (compression) spinal cord.Following spinal cord compression injury (200 gm/cm2-5 min), circulating HRP traversed the endothelium and entered extracellular spaces of the cord parenchyma (Fig. 1). Most microvessels in the injured and adjacent areas exhibited an intensified endothelial vesicular uptake of HRP (Figs. 1, 3). In areas of intense endothelial vesicular activity, the frequency of microtubules (MT) appeared to be increased (Figs. 2, 3).