Biomedical subjects
Publications and source records attributed to J Bourguet.
1. ADH-induced intramembrane particle aggregates in the apical membrane of the epithelial cells are specifically related to water permeability in the epithelium. 2. Colchicine and nocadozole (both of which bind to tubulin) inhibit ADH-induced osmotic water flow in the amphibian bladder. 3. Microtubules may be involved in the translocation of the aggrephores prior to their insertion into the plasma membrane.
It has been assumed from studies in toad bladder that antidiuretic hormone (ADH)-stimulated particle delivery to the luminal membrane is mediated by particle-carrying tubular structures (aggrephores). We report studies in frog and toad urinary bladder showing that vesicles, rather than aggrephores, appear to play the major role in particle delivery in the frog and that vesicle and aggrephore delivery proceed in parallel in the toad. Our principal evidence for this view is that in the frog, transmission electron microscopy shows virtually no fused aggrephores. Supporting evidence includes the following. 1) Freeze-fracture studies show that the diameters of fusion events delivering particles can be quite small, indicating that they are formed by fused vesicles rather than fused aggrephores. 2) A significant population of small fusion events is also seen in the toad, along with larger fusion events related to both aggrephores and large vesicles. 3) Surface aggregate areas in both species are small, consistent with vesicular delivery. 4) Freeze-fracture replicas indicate delivery from shallow pits. We propose a system of transport of particles in which aggrephores act largely as intermediate storage organelles in the frog and as storage and fusion organelles in the toad.
Antimitotic drugs markedly interfere with antidiuretic response, strongly implying that cytoskeleton integrity is essential to this function. This role of the cytoskeleton in controlling the epithelial transport has been seen as a necessary step in the translocation of the water channel containing particle aggregates and in their delivery to the apical membrane. We have now reexamined the exact role of the microtubular network by appropriate time course determinations, by the use of microtubule disruptive agents that lack of the side effects of colchicine, and by trying to visualize the apparent modifications of the microtubular network that accompany water permeability alterations using immunocytochemical techniques. Our results fully confirm that after microtubular network disruption, antidiuretic hormone-induced water permeability variations undergo typical alterations consisting in both a reduction in peak net water flow and a slowing down of its onset. At the same time, the microtubular network disappears in all the epithelial cells. We also show that colchicine-induced inhibition can still be observed in the presence of a prostaglandin synthetase inhibitor and that this inhibition is most likely to occur at a post-adenosine 3',5'-cyclic monophosphate level. These data, as well as results from other series with nocodazole, indicate that the reduction of the net water flow directly results from microtubular network disruption and not from side effects of the disrupting drugs. They also show that the hydrosmotic response is only partially dependent on the microtubular network, which probably has only a guidance role in the translocation of particle aggregates and their exocytotic fusion to the apical membrane.
We took profit in this work of the discovery of a novel series of trialkylxanthines, with a biological activity greater than that of the other methylxanthines classically used as cAMP phosphodiesterase inhibitors. First, we determined their hydrosmotic activity, with the hope to find out compounds that could be used as apical agonists. Second, we employed these compounds as tools to further analyze the cellular mechanism of hydrosmotic response. Results show that propentofylline, the most potent compound in this series, exhibits a comparable activity whatever the side of administration, apical or basal. Results further show a similar mode of action in most of the studied conditions. They suggest thus that the same mechanism of action, probably an inhibition of cAMP phosphodiesterase is involved in both apical and basal stimulation. However, they reveal the existence of characteristics that are specific to the apical response. This suggest that an additional mechanism of action is possibly involved in the apically-induced hydrosmotic response.
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In the present study several aspects of the osmotic water-flow-regulation mechanism in frog urinary bladder were examined, utilizing forskolin either as a direct hydrosmotic agent or in association with vasopressin. It was found that forskolin induces a hydrosmotic effect similar to the one induced by vasopressin. This effect is rapid, reversible and dose-dependent. The half-maximally effective concentration (Ec50FSK) is 1.37 microM forskolin. No additional effect on the osmotic water flow was observed when maximal concentrations of forskolin and vasopressin were given simultaneously. Moreover, forskolin can also markedly potentiate vasopressin-induced hydrosmotic response. This potentiation was maximal with submaximal doses of vasopressin, whereas it disappeared when the hormonal concentration was increased to very high levels. Therefore, forskolin increases vasopressin potency without affecting vasopressin efficacy. The Ec50FSK for the forskolin-induced increase in vasopressin potency was 11 nmol, about two orders of magnitude lower than the Ec50FSK for the direct effect of forskolin on the osmotic water transport. On the whole, our results are compatible with a two-site model of forskolin action in frog urinary bladder: a low affinity site (Ec50FSK = 1.37 microM) that mediates the direct action of forskolin on the osmotic water flow and a high affinity site (Ec50FSK = 11 nmol), which mediates the synergic effect of forskolin with the antidiuretic hormone.
The basal rate of water reabsorption and its acceleration by oxytocin, cyclic AMP (cAMP) or serosal hypertonicity in frog urinary bladders were monitored before and after exposure of the mucosal surface to sulfhydryl (SH) reactive reagents. The following observations were made: 1. N-ethylmaleimide (NEM, 10(-5)M) did not modify the basal water flux, but did potentiate the hydrosmotic response to oxytocin. At higher NEM concentrations, an increase in the basal flux was observed, while the oxytocin-induced water flux was strongly inhibited, if not, nullified. 2. Iodoacetamide (IAM, 10(-3)M) did not modify the basal water flux but did inhibit the oxytocin-, cAMP-, and serosal hypertonicity-induced increase in water permeability. Furthermore, the time course of the hydrosmotic response to oxytocin was significantly increased. 3. 5,5' dithio-bis-(2-nitrobenzoic acid) (DTNB, 10(-3)M) modified neither the basal nor the oxytocin-induced water flux when incubated at pH 8.1, but potentiated the inhibitory effect of NEM. However, at a mucosal pH of 6.5, DTNB inhibited the response to oxytocin by 30%. These results suggest that: (1) the three SH reagents affect differently the basal and the oxytocin-induced water pathways; and that (2) each of the changes in the oxytocin-induced paths occurs at a step following the hormonally-induced increase in intracellular cAMP concentration.
To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.
Antidiuretic hormone (ADH) causes the appearance of water-conducting particle aggregates in the luminal membrane of receptor cells in amphibian bladder and skin, and in the mammalian collecting duct. The aggregates originate from cytoplasmic tubules that fuse with the luminal membrane during ADH stimulation. We have studied the process of fusion and the structure of the particle aggregates by a rapid-freeze technique that renders chemical fixation and glycerol protection unnecessary. Our findings differ in some important respects from previously published work. Aggregate particles, in our study, partition equally between the external (EF) and protoplasmic (PF) membrane leaflets, rather than remaining in the protoplasmic leaflet exclusively. By including the entire population of fusion images in our survey, we have found that aggregate delivery in ADH-treated cells proceeds preferentially from small fusion images whose diameter is significantly less than the 0.12 micron characteristic of the carrier tubules themselves. We have also found that, even in unstimulated preparations, fusion images are numerous, being mostly of small diameter. ADH stimulation produces a moderate increase in the number of fusion images and a significant increase in fusion-image diameter. These findings indicate that the individual particles are mobile within the membrane, lacking interparticle linkage. In addition, contact of cytoplasmic tubules with the luminal membrane may take place even in the absence of ADH, producing small fusion images which are not associated with aggregate delivery to the luminal membrane.
Unidirectional and net water movements were determined, in frog urinary bladders, before and after glutaraldehyde fixation. Experiments were performed in three experimental conditions: in nonstimulated preparations, after the action of antidiuretic hormone (ADH) and in nonstimulated preparations to which amphotericin B was incorporated from the luminal bath. As previously observed for net water fluxes, the increase in the unidirectional water movement induced by ADH was well preserved by glutaraldehyde fixation. After correction for the effects of unstirred layers and nonosmotic pathways, the observed correlation between the ADH-induced increases in the osmotic (Pf) and diffusional (Pd) permeability coefficients was not modified by the fixative action (before glutaraldehyde: slope 11.19, r: 0.87 +/- 0.07; n = 12; after glutaraldehyde: slope 10.67, r: 0.86 +/- 0.04, n = 39). In the case of amphotericin B, delta Pf/delta Pd = 3.08 (r: 0.83 +/- 0.08), a value similar to that observed in lipid bilayers or in nonfixed toad urinary bladders. It is concluded that: The experimental approach previously employed to study water channels in artificial lipid membranes and in amphibian urinary bladders, can be applied to the glutaraldehyde-fixed frog urinary bladder. Glutaraldehyde fixation does not modify the permeability properties of the ADH-induced water channels. Any contribution of exo-endocytic processes or cell regulatory mechanisms to the observed permeability parameters can probably be excluded. Glutaraldehyde-fixed preparations are a good model to characterize these water pathways.
It is now generally accepted that ADH-induced increase in water permeability in responsive epithelia is associated with the insertion of specific structures in the apical membrane of epithelial cells. Up to now, these structures have only been recognized in freeze-fractured preparations and their chemical nature is still unknown. In this study, we used the label-fracture method (Pinto da Silva and Kan, J. Cell Biol., 99, 1156-1161, 1984) to investigate the distribution of wheat germ agglutinin (WGA) on the luminal plasma membrane of freeze-fractured frog urinary bladder epithelial cells. With label-fracture, the cytochemical markers are seen superimposed with the conventional high resolution image of the E face. Label-fracture of tissue treated for 15 min with WGA and subsequently labeled with colloidal gold coated with ovomucoid showed uniform distribution of gold particles along the exoplasmic fracture face. Stereomicrographs show that the gold label is under the fracture face as it is attached to the outer surface of the membrane. Preincubation of the bladder with WGA for 3 hr induced a segregation of the intramembranous particles of the apical plasma membrane. In this condition, we observed a co-distribution of WGA-gold complexes with the segregated particles on the E face. This indicates that WGA-binding sites are located on glycoproteins which probably comprise the large intramembranous particles dispersed on the exoplasmic faces of freeze-fractured luminal membranes. In contrast, the numerous small intramembrane particles observed on P faces remained evenly distributed even after exposure to WGA and are, therefore, unrelated to WGA receptor sites. After WGA treatment, ADH still induced the formation of aggregates inside the smooth domains. A few WGA-binding sites appeared to be associated to these aggregates.
The apical plasma membrane of epithelial cells of frog and toad urinary bladder is subject to large modifications during the induction of water permeability by the antidiuretic hormone. A better characterization of the apical membrane is necessary for a clear understanding of the mechanisms of hormone action. Towards this end, apical material was extracted by enzymatic treatment and by incubation with detergent. Proteolytic enzyme alone had little effect under our conditions. A pretreatment with several glycosidases (alpha-mannosidase or endo-beta-N-acetylglucosaminidase H) increased the hydrolytic action of papain, elastase, proteinase K or Staphylococcus aureus V8 protease and allowed the detection of a major 76 kD in SDS gel electrophoresis. The n-octyl-beta-D-glucopyranoside (0.2%) led to the extraction after 150 mn of 1 to 5 micrograms proteins per cm2 of amphibian urinary bladder apical surface. The extracted proteins migrated as several bands on SDS gels. One of them probably corresponds to the 76 kD fragment obtained after proteolysis. The absence of alteration of the water permeability after extraction and the good preservation of the ultrastructure are evidence for the localisation of the 76 kD at the apical membrane surface. This protein may be the best candidate as antigen to raise antibodies against the apical surface of amphibian urinary bladder epithelial cells.
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8-Bromoadenosine 3',5'-cyclic monophosphate [an analogue of adenosine 3',5'-cyclic monophosphate (cAMP), the intracellular mediator for antidiuretic hormone (ADH) action] induces, in frog urinary bladder, an increase in water permeability that is rapidly and reversibly inhibited by cellular acidification. The effect of CO2 bubbling on the simultaneously observed intramembranous particle aggregates, which probably represent water channels, depended on the time that elapsed after changing medium pH: 3 min of CO2 bubbling depressed the water flux by 70%, whereas the membrane surface occupied by the aggregates remained unchanged. On the contrary, after 9-15 min of CO2 bubbling, both the water flux and the surface area occupied by the aggregates were strongly reduced. These results can be interpreted by accepting two post-cAMP levels of action for cellular acidification: 1) the channels themselves that, as previously suggested by ADH experiments at low temperature, would shift their structure from an "open" to a "closed" state, and 2) the mechanism that controlled the aggregates' plug in and removal.
The onset and offset of the hydrosmotic responses elicited by different agonists in frog urinary bladder, frog skin and isolated frog skin epithelium were studied and compared. The removal of the frog skin supporting layers accelerated the onset of the reactions to oxytocin and 8-bromo-cyclic AMP (8-Br-cAMP), which, however, remained slower than the onset in the urinary bladder. The offset of these actions was not affected by corion removal and was also slower than in the bladder. A marked asymmetry between onset and offset of the hydrosmotic response was observed when the isolated skin epithelium was stimulated with 8-Br-cAMP. The results show that the increases in water permeability elicited by oxytocin and cyclic-AMP derivatives in frog skin and frog urinary bladder, although attributable to the same general mechanisms, present different specific characteristics.
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The ADH-induced water fluxes and the associated appearance of intramembranous particle aggregates in the luminal membrane of frog urinary bladders have been correlated in a time course study. Plots of the onset and reversal of the oxytocin-induced hydrosmotic response were sigmoidal in shape, symmetrical and slowed by low temperature to the same degree. Parallel freeze-fracture studies showed that the mean size distribution of the aggregates was constant at different temperatures and at different times during hormonal stimulation and washout. No qualitatively different picture of aggregate formation was detected at low temperature: this suggests that the insertion and removal of individual aggregates into or from the apical plasma membrane is a rather rapid process, both at 20 and at 6.5 degrees C. As in the case of water permeability, both aggregate appearance and disappearance were similarly slowed by lowering the temperature. A similar time-course study of the inhibition of the hydrosmotic response by acidification of the medium was also made. In this case, lowering the incubation temperature induced a clear dissociation between net water flow and the surface area occupied by the aggregates. For the first time, a low water permeability was found associated with a high aggregate surface area in the apical membrane, indicating that cellular acidification induces an impairment of aggregate function rather than a reduction of surface area.