Activity-induced internalization and rapid degradation of sodium channels in cultured fetal neurons.

A regulatory mechanism for neuronal excitability consists in controlling sodium channel density at the plasma membrane. In cultured fetal neurons, activation of sodium channels by neurotoxins, e.g., veratridine and alpha-scorpion toxin (alpha-ScTx) that enhance the channel open state probability induced a rapid down-regulation of surface channels. Evidence that the initial step of activity-induced sodium channel down-regulation is mediated by internalization was provided by using 125I-alpha-ScTx as both a channel probe and activator. After its binding to surface channels, the distribution of 125I-alpha-ScTx into five subcellular compartments was quantitatively analyzed by EM autoradiography. 125I-alpha-ScTx was found to accumulate in tubulovesicular endosomes and disappear from the cell surface in a time-dependent manner. This specific distribution was prevented by addition of tetrodotoxin (TTX), a channel blocker. By using a photoreactive derivative to covalently label sodium channels at the surface of cultured neurons, we further demonstrated that they are degraded after veratridine-induced internalization. A time-dependent decrease in the amount of labeled sodium channel alpha subunit was observed after veratridine treatment. After 120 min of incubation, half of the alpha subunits were cleaved. This degradation was prevented totally by TTX addition and was accompanied by the appearance of an increasing amount of a 90-kD major proteolytic fragment that was already detected after 45-60 min of veratridine treatment. Exposure of the photoaffinity-labeled cells to amphotericin B, a sodium ionophore, gave similar results. In this case, degradation was prevented when Na+ ions were substituted by choline ions and not blocked by TTX. After veratridine- or amphotericin B-induced internalization of sodium channels, breakdown of the labeled alpha subunit was inhibited by leupeptin, while internalization was almost unaffected. Thus, cultured fetal neurons are capable of adjusting sodium channel density by an activity-dependent endocytotic process that is triggered by Na+ influx.

Intracellular localisation of suramin, an anticancer drug, in human colon adenocarcinoma cells: a study by quantitative autoradiography.

Suramin is a polysulphonated naphthylurea currently investigated for the treatment of advanced malignancy. In the present study, we have analysed the uptake and the intracellular localisation of tritiated suramin in human colon adenocarcinoma cells (HT-29-D4), using quantitative autoradiographic techniques at the optical and electron microscopy levels. Our results show that the drug is able to enter both undifferentiated and differentiated HT-29-D4 cells. The process of suramin uptake is time-dependent, and significantly inhibited by the presence of the suramin-binding protein serum albumin in the culture medium of HT-29-D4 cells. Autoradiographic analysis revealed two distinct patterns of intracellular localisation of tritiated suramin labelling, according to the presence or absence of serum albumin. Indeed, in the absence of serum albumin, the labelling of free suramin was distributed over the nucleus, the Golgi apparatus and the mitochondria, while it was restricted to the lysosomal system when suramin was complexed with albumin. These data show that a serum factor, i.e. albumin, influences the biological activity of suramin by determining its intracellular localisation. The presence of suramin in a given compartment may account for specific effects of the drug including mitochondrial hypertrophy, altered gene expression and lysosomal perturbation.

Autoradiographic localization of tritiated suramin in polarized human colon adenocarcinoma cells.

In this report we demonstrated that [3H]suramin enters polarized human colon adenocarcinoma cells when added to the apical side of the monolayer. Using light microscopic quantitative autoradiography, we showed that suramin was accumulated in the apical cytoplasm and in the nucleus. In contrast, a weak labeling was noted in other compartments such as the basolateral cytoplasm and the intercellular space. The accumulation of suramin in the apical region of the cells is consistent with previous data showing that suramin elicited a lysosomal storage disorder in HT29-D4 cells by a mechanism of polarized endocytosis.

Co-localization of suramin and serum albumin in lysosomes of suramin-treated human colon cancer cells.

Suramin is a polysulfonated compound currently under investigation for the treatment of various types of cancer. Pharmacokinetic studies from clinical trials in humans have shown that most of the circulating drug is associated with serum albumin. The objective of the present study was to investigate the intracellular localization of suramin and serum albumin in human colon cancer cells (HT-29-D4) upon suramin treatment. For this purpose, combined gold labeling and autoradiographic methods were performed on HT-29-D4 cells grown in serum free medium containing both [3H]suramin and colloidal gold-albumin. These morphological experiments demonstrated for the first time that suramin and serum albumin were co-localized in the same cellular compartment (i.e. the lysosomal system) of the suramin-treated HT-29-D4 cells. The albumin-directed targeting of suramin in lysosomes may allow the drug to inhibit the activity of several lysosomal hydrolases, resulting in a lysosomal storage disorder.

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

Voltage-sensitive Na+ channels in the neurohypophysis of the rat as demonstrated by 125I-labelled scorpion toxin.

Fresh rat neural lobe slices were incubated in the presence of [125I] alpha-scorpion toxin (ScTX), a specific marker of Na+ channels. Quantitative electron microscope autoradiography revealed preferential, irregularly spaced labeling of the axolemma of neurosecretory axons, with a significantly higher crude specific activity than any other neuronal or non-neuronal compartment. The number of specific binding sites at the neural lobe surface was calculated to be about 23 per microns2 of axolemma.

Granulolysis in neurosecretory neurons of the rat supraoptico-posthypophyseal system.

Ultrastructural and cytochemical observations on neurosecretory neurons of the rat supraoptico-posthypophyseal systems were made under experimental conditions which resulted in striking changes in the amount of neurosecretory granules and lysosomes. Attention was focused on granulolysis. At the onset of rehydration following a 4 days water deprivation, very active autophage took place in neurosecretory axons of the neural lobe involving the marked increase in smooth endoplasmic reticulum, microvesicles and neurosecretory granules, although the latter were still very few due to previous depletion. When axonal transport was inhibited by colchicine at the onset of rehydration, granules accumulated in the perikarya while granule reloading of the neural lobe was delayed. However autophagy, although always active in axons, remained scarce in perikarya. Moreover, in the latter there was only slight evidence of crinophagy. Hypophysectomy also induced granule accumulation in the perikarya, although accompanied by little granulolysis. Images indicative of crinophagy as shown by acid phosphatase localization were few and exclusively restricted to perikarya, while autophagy occurred essentially in axons. Autophagy appeared to be the predominant process for granulolysis and might be considered here as an aspect of the general turnover of cell constituents, related to the sudden regression of hyperactivity-induced hyperthrophy, rather than as an expression of a specific regulation of an excess of secretory material.

Rickettsia conorii entry into Vero cells.

The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole. In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact. The entry occurred within 3 min after bacterium-cell contact, and R. conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol. Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed. By 12 min, 90% of bacteria were internalized and half were free in the cytosol. This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole.

Polarized distribution of gamma interferon-stimulated MHC antigens and transferrin receptors in a clonal cell line isolated from Fisher rat thyroid (FRT cells).

The characteristics of a polarized epithelial cell line and dynamics of an endogenous polarized plasma membrane constituent were studied by use of an subclone, FRT-9, from the Fisher rat thyroid cell line, FRT. Transmission electron microscopy (conventional, freeze-fracture), determination of transepithelial electrical parameters and immuno-fluorescence study, were used to establish polarity and demonstrated the basolateral distribution of transferrin receptors and the major histocompatibility complex antigens (constitutive Class I or gamma interferon-induced Class II).

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture.

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.

Expression of Ia antigens on freshly dissociated mouse thyroid follicles demonstrated by immunofluorescence.

Iak antigens were detected by indirect immunofluorescence on CBA mouse thyroid follicles. Isolated thyroid follicles, free of lymphocyte contamination, were obtained by collagenase treatment and mechanical disruption; they were then cytocentrifuged on glass slides. This material was incubated with polyclonal or monoclonal anti Iak antibodies followed by FITC-conjugated F(ab')2. Subsequent microscopic observations of these open thyroid follicles revealed that Iak antigens were spontaneously expressed. Labelling was distributed preferentially on the thyrocyte cell surface and, to a lesser extent, on intracellular organelles. Conversely, nuclei were never stained. In some cases, labelling was mottled. Ia antigens may have been previously unobserved because, beside autoimmune disorders they are present in such small amounts that detection is very difficult. The fact that Ia expression is independent of morphological signs of spontaneous autoimmune thyroiditis (particularly, lymphocyte infiltration of follicles), raises the problem of correlation between the autoimmune disorders and Ia expression.

Calcium-dependent dissociation of synaptotagmin from synaptic SNARE complexes.

The formation of the synaptic core (SNARE) complex constitutes a crucial step in synaptic vesicle fusion at the nerve terminal. The interaction of synaptotagmin I with this complex potentially provides a means of conferring Ca2+-dependent regulation of exocytosis. However, the subcellular compartments in which interactions occur and their modulation by Ca2+ influx remain obscure. Sodium dodecyl sulfate (SDS)-resistant core complexes, associated with synaptotagmin I, were enriched in rat brain fractions containing plasma membranes and docked synaptic vesicles. Depolarization of synaptosomes triggered [3H]GABA release and Ca2+-dependent dissociation of synaptotagmin from the core complex. In perforated synaptosomes, synaptotagmin dissociation was induced by Ca2+ (30-300 microM) but not Sr2+ (1 mM); it apparently required intact membrane bilayers but did not result in disassembly of trimeric SNARE complexes. Synaptotagmin was not associated with unstable v-SNARE/t-SNARE complexes, present in fractions containing synaptic vesicles and cytoplasm. These complexes acquired SDS resistance when N-ethylmaleimide-sensitive fusion protein (NSF) was inhibited with N-ethylmaleimide or adenosine 5'-O-(3-thiotriphosphate), suggesting that constitutive SNARE complex disassembly occurs in undocked synaptic vesicles. Our findings are consistent with models in which the Ca2+ triggered release of synaptotagmin precedes vesicle fusion. NSF may then dissociate ternary core complexes captured by endocytosis and recycle/prime individual SNARE proteins.

Evolution of vasopressin levels in the hypothalamo-posthypophysial system of the rat during rehydration following water deprivation. Correlation with ultrastructural aspects in the posterior lobe.

Evolution of the (arginine)-vasopressin (AVP) content of the supraoptic (SON), paraventricular (PVN) and suprachiasmatic nuclei (SchN) and of the posterior lobe of the hypophysis (PLH) has been studied in rats at successive stages of rehydration after 4 days deprivation of drinking water. Particular attention has been focussed on short periods of rehydration. Evolution of the AVP content of the hypothalamo-posthypophysial system (HHS), the blood serum AVP concentration and osmolalities of serum and urine were compared. Variations of the AVP content in the different hypothalamo-hypophysial structures, are parallel. A marked depletion of AVP is observed after 2 and 4 days of dehydration. The AVP content of the PLH and of the hypothalamic nuclei shows two dramatic and short increases 15 min and 3 h after the onset of rehydration; these results are discussed in relation to the known physiological regulation mechanism of the HHS. In the PLH depleted by dehydration, reloading with neurosecretory granules (NSG) begins to be noticeable only after 24 h of rehydration, so that it does not seem to account for elevations of the AVP content occurring earlier. These could be related to a marked increase of the smooth endoplasmic reticulum (SER) network taking place in axons and nerve endings before the NSG reloading.

Cellular localization of synaptotagmin I, II, and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat.

Three isoforms of synaptotagmin, a synaptic vesicle protein involved in neurotransmitter release, have been characterized in the rat, although functional differences between these isoforms have not been reported. In situ hybridization was used to define the localization of synaptotagmin I, II, and III transcripts in the rat CNS and pituitary and adrenal glands. Each of the three synaptotagmin genes has a unique expression pattern. The synaptotagmin III gene is expressed in most neurons, but transcripts are much less abundant than the products of the synaptotagmin I and II genes. A majority of neurons in the forebrain expressed both synaptotagmin I and III mRNAs while synaptotagmin II gene expression was confined to subsets of neurons in layers IV-VI of the cerebral cortex, in the dentate granule cell region, the hilus, and the CA1-CA3 areas of the hippocampus. In the cerebellum, all three transcripts were visualized in the granule cell layer. Furthermore, synaptotagmin I probes revealed striking differences between distinct populations of neurons, as in addition to moderate labeling of granule cells, much more prominent hybridization signals were detected on scattered cell bodies likely to be Golgi interneurons. In the most caudal part of the brain, synaptotagmin II transcripts were abundant and were coexpressed with synaptotagmin III mRNAs. This pattern was found in putative motoneurons of the spinal cord, suggesting that the two isoforms might be involved in exocytosis at the neuromuscular junction. Only synaptotagmin I mRNAs were detected in the anterior and intermediate pituitary and in adrenal medullary cells. These data reveal an unexpectedly subtle segregation of the expression of synaptotagmin genes and the existence of multiple combinations of synaptotagmin isoforms which may provide diversity in the regulation of neurosecretion.

Direct effect of type 1 human immunodeficiency virus (HIV-1) on intestinal epithelial cell differentiation: relationship to HIV-1 enteropathy.

Human immunodeficiency virus (HIV)-infected patients display severe impairments of gastrointestinal functions, including diarrhea and malabsorption, even in the absence of opportunistic infections. Since HIV-1 proteins and nucleic acids have been detected in several cell types of the intestinal mucosa, it has been postulated that HIV-1 itself could alter enterocytic functions. In the present study, we analyzed the effect of HIV-1 on the differentiation process of the epithelial intestinal cell clone HT-29-D4, which mimics the maturation of enterocytes along the crypt-villus axis of the small intestine. We found that HIV-1 infection impairs cellular differentiation (i) by affecting the barrier function of the epithelium, as evidenced by a decrease in the transepithelial electrical resistance, and (ii) by inhibiting the activity of one major glucose absorption function, i.e., sodium/glucose cotransport. At the morphological level, HIV-1 infection of HT-29-D4 cells was associated with the formation of lumina, which are representative of a defect in cellular organization. These morphofunctional perturbations induced by HIV-1 could be mimicked by nocodazole, a microtubule-disrupting agent. Correspondingly, HIV-1 exposure of HT-29-D4 cells evoked a massive disruption of microtubules, as revealed by alpha-tubulin indirect immunofluorescence staining. A similar effect was observed after incubation of the cells with either recombinant gp120 or a monoclonal antibody against galactosylceramide (GalCer), the intestinal receptor for HIV-1 gp120, suggesting that the effect of HIV-1 was mediated by the binding of gp120 to GalCer. Based on these data, we propose that HIV-1 may selectively alter enterocytic functions through a direct effect on the intracellular architecture of the cells. In contrast with previous theories for HIV-1 enteropathy, our data support the concept that HIV-1 may perturb intestinal functions without necessarily infecting intestinal epithelial cells.

Developmental regulation of synaptotagmin I, II, III, and IV mRNAs in the rat CNS.

Synaptotagmin I is an abundant synaptic vesicle protein that has an essential function in mediating Ca2+-triggered neurotransmitter release. We have analyzed the distribution of four neural synaptotagmin isoforms during postnatal development of the rat CNS by in situ hybridization. Synaptotagmin I, II, III, and IV genes have distinct patterns of spatiotemporal expression except in cerebellum granule cells, where the four transcripts were detected during the formation of parallel fiber/Purkinje cell synapses. Throughout development synaptotagmin I mRNAs were widely expressed in brain, whereas synaptotagmin II transcripts were predominant in spinal cord. At all stages synaptotagmin III mRNAs were expressed uniformly in most neurons examined, although at a low level. Synaptotagmin I, II, and III gene expressions mainly increased during development and persisted in adulthood, mirroring neuronal differentiation. Conversely, synaptotagmin IV transcripts were predominant during perinatal development in a heterogeneous population of neurons and subsequently were expressed uniformly at a low level. Intense labeling was observed in the hippocampal CA3 field and in the subiculum, but not in the CA1 field, of the newborn rat. In cerebral cortex, lamina-specific labeling was detected with a high expression in cell layer V. Only a small number of Purkinje cell clusters were labeled in the flocculus and paraflocculus of the cerebellum. Heterogeneous sets of neurons expressing synaptotagmin IV gene also were observed in spinal cord. We thus speculate that synaptotagmin IV may a play a role in the development of the mammalian nervous system.

[Suramin induces lysosomal storage disorder in HT29-D4 cells during a mechanism of polarized endocytosis]. / La suramine provoque une perturbation du système lysosomal des cellules HT29-D4 au cours d'un mécanisme d'endocytose polarisée.

During the course of suramin-induced differentiation, a marked lysosomal storage disorder is observed in HT29-D4. This impairment could account for the toxic side effects of the drug during clinical trials in humans. It is shown that the perturbation is caused by a process of endocytosis of suramin-serum albumin complexes by the apical membrane of differentiated HT29-D4 cells.

Expression of the mRNA for the beta 2 subunit of the voltage-dependent sodium channel in rat CNS.

Expression of the voltage-dependent sodium channel has been analysed in adult rat central nervous system by Northern blotting and in situ hybridization. Northern blots showed that all the territories studied express beta 2 transcripts, albeit with widely varying levels (with cerebellum >> hippocampus > brain > brainstem > spinal cord). In situ hybridization confirmed that in these structures, all the neuronal cell bodies contain beta 2 mRNA; expression was particularly high in the granule cells of the cerebellum, in both pyramidal cell layer and dentate gyrus in the hippocampus, and in spinal cord motor neurons. Northern blots also showed that RNA extracted from optic nerve and cultured cortical astrocytes contained beta 2 mRNA, while it was totally absent from sciatic nerve. In situ hybridization evidenced the presence of a numerous population of beta 2-positive cells in cerebellum white matter, spinal cord white matter, and in corpus callosum, where frontal sections showed labelled cells arranged in the chain-like or row pattern typical of interfascicular oligodendrocytes. Combination of antiglial fibrillary acid protein (GFAP) immunofluorescent histochemistry with detection of beta 2 mRNA evidenced that expression of the transcripts was indeed restricted to GFAP-negative cells in white matter.

Distribution of components of the SNARE complex in relation to transmitter release sites at the frog neuromuscular junction.

At the frog neuromuscular junction, neurotransmitter release sites are regularly spaced at 1 micron intervals along the nerve terminal, directly facing postsynaptic folds which contain a high density of acetylcholine receptors. Immunostaining and laser confocal scanning microscopy were used to compare the distribution of presynaptic proteins implicated in exocytosis with that of fluorescent alpha-bungarotoxin. Syntaxin, synaptosome-associated 25 kDa protein and calcium channels were located predominantly at release sites. Synaptobrevin (vesicle-associated membrane protein) was distributed in the cytoplasm of the nerve terminal, presumably in the packets of microvesicles associated with each active zone. N-Ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment proteins (alpha beta SNAP) displayed a diffuse distribution throughout the terminal cytoplasm and also colocalized in distinct concentrated zones adjacent to the presynaptic membrane.

Redistribution of presynaptic proteins during alpha-latrotoxin-induced release of neurotransmitter and membrane retrieval at the frog neuromuscular junction.

Calcium-dependent exocytosis at the nerve terminal involves the synaptic core (SNARE) complex composed of the t-SNAREs syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), and the v-SNARE vesicle-associated membrane protein (VAMP/synaptobrevin), a stable heterotrimer which can associate with the putative calcium sensor protein, synaptotagmin. The distribution of these proteins at the frog neuromuscular junction was examined by immunofluorescent staining and confocal microscopy following exocytosis induced by alpha-latrotoxin. Experiments were performed under conditions in which synaptic vesicle recycling was either maintained in balance with exocytosis, or completely blocked, or during recovery from block of endocytosis. When endocytosis was maintained, protein distribution was essentially identical to that of unstimulated nerve terminals, in which syntaxin 1 and SNAP-25 are localized to the presynaptic active zones coincident with the postsynaptic folds that contain a high density of acetylcholine receptors (AChRs). Block of endocytosis led to complete incorporation of vesicle proteins into the plasmalemma, and t-SNARE distribution was no longer restricted to active zones. Five minutes after the onset of recovery, both synaptic vesicle proteins and t-SNARE proteins were concentrated into small spots, in a similar pattern to that obtained following endocytosis of the vital styryl dye FM1-43. These findings are consistent with a model in which following sustained exocytosis, t-SNARE trafficking involves internalization and transit via a vesicular compartment before recycling to the presynaptic plasma membrane.