Cristina Porta

Identification of particular epithelial areas and cells that transport polypeptide-coated nanoparticles in the nasal respiratory mucosa of the rabbit

The active transcytosis of many different polypeptides (either presented free or adsorbed on latex nanoparticles), found in the respiratory mucosa of the upper nasal concha, has previously been shown to be proportional to the total volume of the lymphoid aggregates present in the tissue. By combining the use of fluorescent nanoparticles, flux measurements, confocal and scanning electron microscopy and conventional histology, it is shown in this paper that: (i) the areas of epithelium overlying lymphoid aggregates are the only transporting polypeptides; (ii) the respiratory epithelium in these areas consists mainly of non-ciliated microvillar cells, with numerous ciliated cells and rare mucous goblet cells at the periphery of the area only; (iii) non-ciliated microvillar cells are distinguishable in cells with well developed finger-like microvilli and cells with an irregularly pleated apical membrane, similar to that of intestinal and bronchial antigen-sampling M-cells; (iv) groups of polypeptide-coated nanospheres are found bound to this latter type of cells, demonstrating that these are the transporting cells, detected at the first stage of the transcytotic cycle.

Endocytosis inhibitors abolish the active transport of polypeptides in the mucosa of the nasal upper concha of the rabbit

An active absorption of polypeptides (elcatonin = CCT; adrenocorticotropic hormone) had been previously observed in the nasal respiratory mucosa of the rabbit. Its saturation kinetics and the parallel absence of a net transfer of other non-polypeptidic organic markers excluded the involvement of a simple pinocytosis. This absorption has been now better localized and further characterized. Unidirectional CCT fluxes (determined with radioimmunoassay) have been concomitantly monitored with transepithelial electric potential difference (Vms). Although the mucosae covering the ectoturbinal A and the lower and upper conchae displayed similar Vms, the active CCT transport was only evidenced in the upper concha. In this region cytochalasin B (which by disassembling actin microfilaments prevents the apical formation of vesicles in epithelial cells) and monensin (which prevents the split of the ligand-receptor complex in the endosomes) both eliminated the net CCT absorption, however, also permanently increasing the passive CCT junctional permeability. Aluminum fluoride (which prevents the fusion of endocytic vesicles into endosomes) and colchicine (which disrupts microtubules along which vesicles move in the cytoplasm) also permanently abolished net CCT transport, without affecting, or shortly and transiently affecting, passive permeability. On the whole these results are in favor of an active CCT transport supported by a specific vesicular transport.

Nature of the neutral Na+-Cl? coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+, Cl?/HCO 3 ? double exchange, hydrochlorothiazide-sensitive Na+-Cl? symport and Na+-K+-2Cl? cotransport are all involved

SummaryTransepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27°C, in HCO3−-free bathing media containing 10−4m acetazolamide. Whereas luminal 10−4m bumetanide or 10−4m 4-acetamido-4′-iso-thiocyanostilbene-2,2′-disulfonate (SITS) did not affect fluid absorption, 25 mm SCN− abolished it; hydrochlorothiazide (HCTZ) in the luminal medium reduced fluid absorption from 28.3±1.6 (n = 21) to 8.6±1.6 μl cm−2 hr−1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10−3m concentration; the apparent IC510 was about 2×10−4m. The residual fluid absorption, again insensitive to SITS, was completely inhibited by SCN− or bumetanide. Cl− influx at the luminal border of the epithelium, measured under the same conditions and corrected for the extracellular space and paracellular influx, proved insensitive to 10−4m bumetanide, but was slowly inhibited by 10−3m HCTZ, with maximum inhibition (about 54%) reached after a 10-min treatment; it subsequently rose again, in spite of the presence of HCTZ. However, if the epithelium, treated with HCTZ, was exposed to 10−4m bumetanide during the measuring time (45 sec), inhibition was completed and the subsequent rise of Cl− influx eliminated. Intracellular Cl− accumulation with respect to the predicted activity value at equilibrium decreased significantly upon exposure to 10−3m HCTZ, reached a minimum within 15–30 min of treatment, then rose again significantly at 60 min. Simultaneous exposure to HCTZ and bumetanide decreased the accumulation to a significantly larger extent as compared to HCTZ alone, already in 15 min, and impeded the subsequent rise. Intracellular K+ activity rose significantly within 30 min treatment with HCTZ; the increase proved bumetanide dependent.The results obtained show that Na+-Cl− symport, previously detected under control conditions, is the HCTZ-sensitive type; its inhibition elicits bumetanide-sensitive Na+-K+-2Cl− cotransport. Thus, the three forms of neutral Na+-Cl−-coupled transport so far evidenced in epithelia, Na+/H+, Cl−/HCO3−double exchange (in the presence of exogenous bicarbonate), HCTZ-sensitive Na+-Cl− symport and bumetanide-sensitive Na+-K+-2Cl− cotransport, are all present in the apical membrane of rabbit gallbladder.

The active transport of polypeptides in the rabbit nasal mucosa is supported by a specific vesicular transport inhibited by cytochalasin D

We have previously demonstrated that polypeptides (elcatonin and ACTH) can be actively absorbed across the rabbit nasal mucosa. In this paper we show that elcatonin is also transported when it is adsorbed onto microspheres (diameter: 0.5 micron). whereas the elcatonin-uncovered microspheres do not display any net transport. Cytochalasin D (0.1 microgram/ml) abolishes the net absorption of elcatonin presented either alone or adsorbed. At the same concentration the inhibitor does not affect cellular active ion transports (and hence metabolism); although it increases intercellular ion and elcatonin permeability, it does not affect intercellular and paracellular permeability of the elcatonin-covered microspheres. Altogether, these results show that polypeptide transport is supported by a specific vesicular transfer inhibited by cytochalasin D by disassembly of the actin cytoskeleton, probably at the apical border of the cell.

Relationship between polypeptide transcytosis and lymphoid tissue in the rabbit nasal mucosa

It has been suggested that the specific transcytosis of polypeptides, demonstrated in rabbit nasal mucosa (upper concha), is involved in antigen sampling at the airway entry. To test this hypothesis, unidirectional transepithelial fluxes of carbocalcitonin (CCT Mw = 3362) from the mucosal to the submucosal side, and vice versa, were measured by radioimmunoassay every 30 min for 120 min and, from the difference, net absorption was determined in the upper concha and septum mucosae. The exposed mucosae were examined by quantitative histology; isolated scattered lymphoid cells/mm2 and volumes of lymphoid infiltrates and aggregates were quantified. CCT absorption was observed in the mucosae of the upper concha and septum provided that aggregates were present, being proportional to aggregate volume. No relationship was noted with isolated scattered lymphoid cells and infiltrates. Passive permeability was unaffected by lymphoid tissue. On this basis, the antigen sampling hypothesis seems to be at least partially substantiated.

DIFFERENT KINDS OF POLYPEPTIDES AND POLYPEPTIDE-COATED NANOPARTICLES ARE ACCEPTED BY THE SELECTIVE TRANSCYTOSIS SHOWN IN THE RABBIT NASAL MUCOSA

The specific transcytosis of polypeptides, demonstrated in the nasal respiratory mucosa of the rabbit, seems to be involved in antigen sampling at the airway entry, since absorption has been shown only to occur if lymphoid aggregates are present beneath the epithelium and to be proportional to aggregate volume. Nanoparticles and many polypeptides besides the two previously tested (i.e. carbocalcitonin (CCT) and adrenocorticotropic hormone) should be transportable, in agreement with the vesicular transcytosis and antigen sampling hypothesis. Thus unidirectional mucosa-submucosa and opposite fluxes (Jms, Jsm) and the corresponding net fluxes (Jnet) of uncoated or polypeptide-coated polystyrene nanospheres (diameter: about 0.5 micrometer) have been measured with the aid of spectrophotometry and quantitative dark-field microscopy. No net transport has been observed for uncoated beads, whereas it has always been shown for polypeptide-coated beads, although to different extents. The selectivity sequence for the polypeptides tested is as follows: BSA congruent with enkephalin << anti-BSA IgG congruent with IgA congruent with CCT congruent with insulin </=anti-insulin IgG. With the exception of BSA and enkephalin-coated beads, whose Jnet is very small, in all the other cases the apparent affinities for receptors seem to be equal or similar; just over 6% polypeptide coating on the nanosphere is sufficient to elicit maximal transport; finally, transport seems to require many cooperating binding sites between the single nanosphere and receptors or one or many non-cooperating binding sites, but with a threshold number of polypeptide molecules adsorbed on the nanosphere to reach a minimal binding probability.

Apical Na+-Cl- symport in rabbit gallbladder epithelium: a thiazide-sensitive cotransporter (TSC).

Abstract. Cl− apically enters the epithelium of rabbit gallbladder by a Na+-Cl− symport, sensitive to hydrochlorothiazide (HCTZ). Since HCTZ also activates an apical SITS-sensitive Cl− conductance (GCl), the symport inhibition might be merely due to a short circuit of the symport by GCl rather than to a direct action of HCTZ on the symporter. To examine whether the symport is directly inhibited by HCTZ and whether the symporter belongs to the family of thiazide-sensitive cotransporters (TSC), radiochemical measurements of the apical Cl− uptake, electrophysiological determinations of intracellular Cl− and Na+ activities (ai,Cl and ai,Na) with selective theta microelectrodes and molecular biology methods were used. The 36Cl− uptake proved to be a measurement of the apical unidirectional Cl− influx (Jmc) and of the symport only (without backflux components), with measuring times of 45 sec under all experiment conditions; its inhibition by HCTZ was unaffected by GCl activation or abolition. After HCTZ treatment the decrease in ai,Cl (measured as the initial rate or in 3 min) was larger than the decrease in ai,Na. The difference was reduced to one third in a group of epithelia in which the elicited GCl was reduced to one third; moreover it was abolished in any case when GCl was abolished with 10−4m SITS. The SITS-insensitive rate of ai,Cl decrease was equal to that of the ai,Na decrease in any case. Thus the ai,Cl decrease displays a component dependent on GCl activation and a second component dependent on symport inhibition. Using the RT-PCR technique a cDNA fragment was obtained that was 99% identical to the corresponding region of the rabbit renal TSC isoform. The results indicate that in rabbit gallbladder epithelium HCTZ displays a dual action, namely GCl activation and Na+-Cl− symport inhibition. This Na+-Cl− symporter is the first TSC found to be functionally expressed in a nonrenal or nonrenal-like epithelium.

Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive, Ca2+-activated K+ conductance

In the rabbit gallbladder epithelium, hydrochlorothiazide (HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na+-Cl− symport, to depolarize the apical membrane potential and to enhance the cell-to-lumen Cl− backflux (radiochemically measured), this increase being SITS-sensitive. To better investigate the causes of the depolarization and the Cl− backflux increase, cells were punctured with conventional microelectrodes on the luminal side (incubation in bicarbonate-free saline at 27°C) and the apical membrane potential (Vm) was studied either with prolonged single impalements or with a set of short multiple impalements. The maximal depolarization was of 3–4 mV and was reached with 2.5 × 10−4m HCTZ. It was significantly enhanced by reducing luminal Cl− concentration to 30 mm; it was abolished by SCN−, furosemide, SITS; it was insensitive to DPC. SITS converted the depolarization into a hyperpolarization of about 4 mV; this latter was apamin, nifedipine and verapamil sensitive. It was concluded that HCTZ concomitantly opens apical Cl− and (probably) Ca2+ conductances and, indirectly, a Ca2+-sensitive, apamin inhibitable K+ conductance: since the intracellular Cl− activity is maintained above the value predicted at the electrochemical equilibrium, the opening of the apical Cl− conductance depolarizes Vmand enhances Cl− backflux. In the presence of apamin or verapamil, to avoid the hyperpolarizing effects due to HCTZ, the depolarization elicited by this drug was fully developed (7–10 mV) and proved to be Ca2+ insensitive. On this basis and measuring the transepithelial resistance and the apical/basolateral resistance ratio, the Cl− conductance opened by HCTZ has been estimated and the Cl− backflux increase calculated: it proved to be in the order of that observed radiochemically. The importance of this Cl− leak to the lumen in the overall inhibition of the transepithelial NaCl transport by HCTZ has been evaluated.

Lubricating effect of sialomucin and hyaluronan on pleural mesothelium

Coefficient of kinetic friction (μ) between rabbit visceral and parietal pleura, sliding in vitro at physiological velocities and load, increases markedly after blotting mesothelial surface with filter paper; this increase is only partially reduced by wetting blotted mesothelium with Ringer solution. Given that mesothelial surface is covered by a thick coat with sialomucin and hyaluronan, we tested whether addition of sialomucin or hyaluronan solution after blotting lowers μ more than Ringer alone. Actually, these macromolecules lowered μ more than Ringer, so that μ was no longer significantly higher than its preblotting value. Moreover, Ringer addition, after washout of macromolecule solution, increased μ, in line with their dilution. These findings indicate that mesothelial blotting removes part of these molecules from the coat covering mesothelial surface, and their relevance for pleural lubrication. Transmission electron micrographs of pleural specimens after mesothelial blotting showed that microvilli were partially or largely removed from mesothelium, consistent with a substantial loss of macromolecules normally entrapped among them.

Evidence for Na+–glucose cotransporter in type I alveolar epithelium

Functional evidence of Na+–glucose cotransport in rat lung has been provided by Basset et al. (J. Physiol. 384:325–345, 1987). By autoradiography [3H]phloridzin binding has been found confined to alveolar epithelial type II cells in mouse and rabbit lungs (Boyd, J. Physiol. 422: 44P, 1990). In this research we checked by immunofluorescence whether Na+–glucose cotransporter (SGLT1) is also expressed in alveolar type I cells. Lungs of anesthetized rats and lambs were fixed by paraformaldehyde, perfused in pulmonary artery, or instilled into a bronchus, respectively. Tissue blocks embedded in paraffin or frozen were sectioned. Two specific anti-SGLT1 antibodies for rat recognizing aminoacid sequence 402–420, and 546–596 were used in both species. Bound primary antibody was detected by secondary antibody conjugated to fluorescein isothiocianate or Texas red, respectively. In some sections cellular nuclei were also stained. In rats alveolar type I cells were identified by fluorescent Erythrina cristagalli lectin. Sections were examined by confocal laser-scanning microscope. Both in rats and lambs alveolar epithelium was stained by either antibody; no labeling occurred in negative controls. Hence, SGLT1 appears to be also expressed in alveolar type I cells. This is functionally relevant because type I cells provide 95–97% of alveolar surface, and SGLT1, besides contributing to removal of lung liquid under some circumstances, keeps low glucose concentration in lining liquid, which is useful to prevent lung infection.