Electron microscopic study of the orthograde axonal transport of horseradish peroxidase in the supraoptico-neurohypophysial tract of the rat

Summary Horseradish peroxidase (HRP) has been used as a protein tracer in order to visualize the ultrastructural sites of the orthograde transport of protein macromolecules in the hypothalamo-neurohypophysial tract of the rat. After a local injection of HRP within the supraoptic nucleus, the reaction product was observed: (1) mainly in tubules of the smooth endoplasmic reticulum in the more proximal part of the axons, and (2) in granules and microvesicles of the axon terminals. Observations on thick sections clearly showed the existence of a relationship between the smooth endoplasmic reticulum containing HRP and the labeled granules or microvesicles. These data are in good agreement with previous findings showing the existence of direct continuity between tubules of the smooth endoplasmic reticulum and a fraction of the neurosecretory granules and microvesicles. This evidence further reinforces the hypothesis that the latter organelles may possibly originate locally in the axons from the tubules of the smooth endoplasmic reticulum which may therefore be proposed as a possible vehicle for a non-granular intra-axonal transport of neurosecretory material in neurosecretory neurons.     

A study of the two-way transport of horseradish peroxidase across the visceral pleura

Summary The authors report on a study of the transpleural transport of horseradish peroxidase after intrapleural and intracardiac application. Following intrapleural introduction, a retention of the marker on the apical membrane of mesothelial cells was observed, with subsequent transcellular transfer after incorporation into microvesicles. Following intracardiac injection, the marker moved out of the pulmonary capillaries across the endothelial vesicles and progressed to the pleural cavity across the intercellular spaces and mesothelial vesicles. With either route of injection, reaction product was noted in the basal lamina of the mesothelium, elastic membrane, alveolar macrophages and pneumocytes type II.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

A study of the two-way transport of horseradish peroxidase across the visceral pleura

The authors report on a study of the transpleural transport of horseradish peroxidase after intrapleural and intracardiac application. Following intrapleural introduction, a retention of the marker on the apical membrane of mesothelial cells was observed, with subsequent transcellular transfer after incorporation into microvesicles. Following intracardiac injection, the marker moved out of the pulmonary capillaries across the endothelial vesicles and progressed to the pleural cavity across the intercellular spaces and mesothelial vesicles. With either route of injection, reaction product was noted in the basal lamina of the mesothelium, elastic membrane, alveolar macrophages and pneumocytes type II.     

Block of axoplasmic transport in vitro by vinca alkaloids

The three potent antimitotic vinca alkaloids: vincristine (VCR), vinblastine (VLB), and vindesine (VDS) were compared for their effect in blocking axoplasmic transport in vitro using a desheathed preparation of the peroneal branch of cat sciatic nerve. A range of vinca alkaloid concentrations from 1–100μM was examined. The relative order of potency in blocking axoplasmic transport was VCR > VLB > VDS at a concentration of 25μM. At the higher concentrations block occurred so rapidly that a statistically significant difference between these agents could not be obtained. The relation of vinca block ot the transport mechanism is discussed.     

Histochemical studies on the uptake of horseradish peroxidase by rat kidney slices

When rat kidney slices were incubated in the presence of horseradish peroxidase, there was an energy-dependent uptake of the protein by the cells of the kidney tubules. The uptake was greatest in the proximal convoluted tubules and in the thick ascending limbs of the loops of Henle; it was abolished by cold, anoxia, 2,4-dinitrophenol, and fluoroacetate, and was more readily depressed by unfavorable metabolic conditions in the proximal convoluted tubules than in the thick ascending limbs. Protein uptake was inhibited when the kidney slices were incubated in electrolyte-free media. In sodium chloride solutions, uptake was reduced as sodium was progressively replaced by choline, and ouabain inhibited uptake in the proximal convoluted tubules, but not in the thick ascending limbs. To a limited extent, lithium could replace sodium in the incubation medium with no depression of peroxidase uptake. These results suggest that a sodium-stimulated, ouabain-sensitive ATPase may be involved in the uptake of protein by cells of the kidney tubule. The intracellular transport of peroxidase in cells of the proximal convoluted tubules was abolished by cold, anoxia, and 2,4-dinitrophenol, but it was not affected by concentrations of ouabain which inhibited the uptake of the protein.     

The uptake of horseradish peroxidase by cortical synapses in rat brain

Summary Horseradish peroxidase (HRP) was introduced directly into the cerebral cortex of adult rats, which were allowed to survive for 60 min before perfusion fixation. After the tissue had been incubated to demonstrate HRP at the LM and EM levels, blocks of cortical tissue were taken at varying distances from the injection site. These eight blocks of tissue constituted a time sequence for HRP diffusion.Qualitative examination of the presynaptic terminals showed that the most commonly encountered profiles are the plain synaptic vesicles, many of which accumulate tracer. In some terminals labelled vesicles are lined-up in tubular fashion. Other profiles commonly labelled are coated vesicles, tubular and vacuolar cisternae, and plain and coated pinocytotic vesicles.Quantitative analyses based on the number of terminals containing labelled profiles demonstrate an early rise in the rate of labelling of both plain synaptic vesicles and coated vesicles, after which synaptic vesicle labelling rises slowly towards a plateau. By contrast, there is a late parallel increase in the rate of labelling of coated vesicles and cisternae. A more detailed analysis, based on the actual numbers of labelled and total profiles within each presynaptic terminal, highlight early and late periods of rapid labelling for plain synaptic vesicles, coated vesicles and cisternae. A further aspect of HRP incorporation studied, concerns its uptake into four delineated regions of the presynaptic terminal.Our data indicate that membrane uptake into the presynaptic terminal is accomplished mainly via coated vesicles, although plain synaptic vesicles may also be involved. Coated vesicles, in turn, appear to give rise directly to plain synaptic vesicles, with some coalescing to produce vacuolar cisternae. The latter are involved in a two-way interchange of membrane with tubular cisternae, plain synaptic vesicles and coated vesicles. An additional source of plain synaptic vesicles are the tubular cisternae. Exocytosis of plain synaptic vesicles constitutes the mechanism by which transmitter is released from the presynaptic terminal.Supported by the Nuffield Foundation. We are grateful to Mr. M. Austin for help with the photography     

Protamine induced intracellular uptake of horseradish peroxidase and vacuolation in mouse skeletal muscle in vitro

Summary The uptake in vitro of horseradish peroxidase (HRP) in mouse skeletal muscle was examined by electron microscopy and chemical determination.In muscles exposed to an HRP solution for 60 min at +37°C, HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition HRP was found within the transverse tubules. Exposure to HRP for 30 min at +37°C followed by HRP together with a polycationic protein (protamine) for 30 min at +37°C caused an intracellular vesicular uptake of HRP. Intracellular HRP was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular HRP uptake or muscle cell vacuolation could not be detected in the absence of protamine or when the incubation temperature was + 4°C. Chemical determination of HRP uptake was in general agreement with the morphological results. The uptake of HRP in the presence of protamine was stimulated at +31°C and blocked at +4°C.The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis.     

Axoplasmic transport of horseradish peroxidase in single neurons of the dorsal root ganglion studied in vitro by microinjection

Summary The dependence of anterograde axoplasmic transport on cytoskeletal components was investigated using microinjection of horseradish peroxidase (HRP) into the somata of chick dorsal root ganglion cells in vitro. Microinjected HRP was transported anterogradely in the neurites and their branches; this transport was disturbed by colchicine in a drug-dependent and time-dependent manner. Cytochalasin B, a drug that depolymerizes actin, did not inhibit the transport of HRP, despite the formation of local swellings in neurites. The microinjection of polyclonal antibodies directed against tubulin and monoclonal antibodies (mAbs) against 200-kDa neurofilaments disturbed the axoplasmic transport of co-injected HRP, which then exhibited an irregular and discontinuous distribution in the axonal branches. The transport of HRP became discontinuous after the injection of anti-tubulin antibodies and led to the formation of globular deposits of HRP. Polyclonal antibodies against actin and mAbs to 160-kDa and 68-kDa neurofilaments seemed to have no effect on the axoplasmic transport of co-injected HRP. Microinjection of antibodies against tubulin induced formation of perinuclear bundles consisting of cytoskeletal components. The transport of HRP thus appears to be regulated by an intact microtubular system and cross-linker components (200-kDa neurofilaments) of the cytoskeleton. Actin and most intermediate filament proteins do not seem to play an essential role in the transport of HRP.     

A spin label study of horseradish peroxidase.

The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A.     

Hypophysiotropic neurons in the goldfish hypothalamus demonstrated by retrograde transport of horseradish peroxidase

Summary The horseradish-peroxidase (HRP) technique was used to visualize the cell bodies of axons projecting to the goldfish pituitary. Following intravenous injections of HRP, HRP reaction products were observed in axons of the rostral pars distalis, proximal pars distalis, neurointermediate lobe, pituitary stalk and in axons coursing from the pituitary into the hypothalamus. HRP-labelled cells in the brain were localized in two regions only — the nucleus preopticus (NPO) pars magnocellularis and pars parvocellularis, and the nucleus lateralis tuberis (NLT) of the hypothalamus. These observations suggest that the NPO and NLT are the source of the neurosecretory innervation of the goldfish pituitary.     

Titration study of guaiacol oxidation by horseradish peroxidase.

Titration of guaiacol by hydrogen peroxide in the presence of a catalytic amount of horseradish peroxidase shows that the reduction of hydrogen peroxide proceeds by the abstraction of two electrons from a guaiacol molecule. In the same way, it can be demonstrated that 0.5 mol of guaiacol can reduce, at low temperature, 1 mol of peroxidase compound I to compound II. Moreover, the reaction between equal amounts of compound I and guaiacol at low temperature produces the native enzyme. A reaction scheme is proposed which postulates that two electrons are transferred from guaiacol to compound I giving ferriperoxidase and oxidized guaiacol with the intermediary formation of compound II. The direct two-electron transfer from guaiacol to compound I without a dismutation of product free radicals must be considered as an exception to the general mechanism involving a single-electron transfer.     

Receptor-mediated uptake of horseradish peroxidase in innervated and denervated skeletal muscle

The in vitro uptake of [3H]inulin and horseradish peroxidase (HRP) has been studied in innervated and 6 days denervated extensor digitorum longus muscle of the mouse. Both markers were taken up at a higher rate in denervated muscle. The increase in uptake after denervation was, however, larger for HRP than for [3H]inulin. After 2 h incubation at 37 degrees C, pH 7.3, in the presence of equimolar concentrations of HRP and [3H]inulin (approx. 2.1 microM), the uptake of HRP was approx. 8 times as great as the uptake of [3H]inulin in the same innervated muscles. In denervated muscle the HRP uptake was approx. 19 times as great as the [3H]inulin uptake in the same muscles. Various possible explanations of these differences in uptake have been considered and tested experimentally. [3H]Inulin uptake in skeletal muscle has previously been shown to obey bulk kinetics. The present investigation shows the HRP uptake to obey saturation kinetics. The HRP uptake shows dependency on divalent cations and is reduced if incubation is carried out at pH 6.4. The uptake of HRP, when used at a low, non-saturating concentration (10 micrograms/ml approx. 0.25 microM), is inhibited greater than or equal to 60% by yeast mannan (0.1 mg/ml), ribonuclease B (0.1 mg/ml, approx. 7.4 microM), mannose (30 mM), monodansylcadaverine (1 mM), chloroquine (100 microM), trifluoperazine (25 microM) or maleic acid (2 mM). It is concluded that HRP is taken up in innervated and denervated skeletal muscle by a process of receptor-mediated endocytosis and that this uptake is under neurotrophic control.     

Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.     

Ultrastructural organization of the geniculo-cortical neurons demonstrated by retrograde axonal transport of horseradish peroxidase

The ultrastructural organization of geniculo-cortical relay neurons projecting to the primary auditory cortex (field 22) was studied in the cat by the method of retrograde axonal transport of horseradish peroxidase. The labeled neurons appear to be a medium-sized cells containing a large amount of profiles of vacuolar systems as well as organelles involved in catabolic processes. These cells differ from small unmarked neurons in having a narrow band scanty cytoplasm. Only a few axo-somatic synapses are found on the labeled neurons, the majority of afferent impulsation is perceived by their dendrites. The function of relay neurons is discussed.     

A horseradish peroxidase study on the mammillothalamic tract in the rat

The mammillary projections to the anterior thalamic nuclei were investigated in the rat, using the horseradish peroxidase (HRP) method. Pars centralis of the medial mammillary nucleus projects to the medial portion of the ateromedial nucleus (AM). Pars medialis (Mm) of the medial mammillary nucleus sends fibers to the ipsilateral AM and sparsely to the medial portion of the contralateral side. The ventral and dorsal portions of Mm project to the anterior and posterior portions of AM, respectively. The pars latralis (Ml) and pars posterior (Mp) of the medial mammillary nucleus send fibers predominantly to the ipsilateral anteroventral nucleus and sparsely to the contralateral side. A slight difference between Ml and Mp projections was observed. The lateral mammillary nucleus projects bilaterally to the anterodorsal nucleus.