Dario Cremaschi

Transcellular ion route in rabbit gallbladder

SummaryThe intracellular potential in gallbladder epithelial cells is about −59 mV with respect to both mucosal and serosal media.It is a diffusion potential mainly due to K+; Na+ conductance seems to be very low. Entrance of Cl− into cells appears to be coupled with Na+ on a neutral carrier and exit towards blood side seems to be due to a NaCl neutral pump.

Na+ and Cl− transepithelial routes in rabbit gallbladder

SummaryThe reported experiments demonstrate that in rabbit gallbladder epithelium:a)The mucosa-cell Na+ and Cl− influxes (45 sec long) are reduced in Cl−-free and Na+-free bathing solutions respectively; transconcentration effects are observed.b)Cell Cl− labelling through the luminal membrane, with36Cl− (1 hr incubation), is nearly abolished if a Na+-free bathing solution is used; but cell Na+ labelling with22Na+ in the lumen (1 hr incubation) does not change if a Cl−-free bathing solution is employed.c)Cell Na+ and Cl− labelling through the basolateral membrane (1 hr incubation) is negligible; some Cl− labelling is obtained only when the intracellular electrical potential is artificially reduced.d)SO42− is found to abolish net water transport, to cross the epithelium, but not to enter the cell. On the basis of these data a paracellular pathway for net Cl− transport is ruled out, a Na+ and Cl− cotransport through the cell luminal barrier and a Na+ active extrusion through the basolateral membrane are suggested. No definitive conclusion about Cl− extrusion is possible.

Stimulation by HCO3- of Na+ transport in rabbit gallbladder.

SummaryBicarbonate presence in the bathing media doubles Na+ and fluid transepithelial transport and in parallel significantly increases Na+ and Cl− intracellular concentrations and contents, decreases K+ cell concentration without changing its amount, and causes a large cell swelling. Na+ and Cl− lumen-to-cell influxes are significantly enhanced, Na+ more so than Cl−. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO3−-ATPase in the brush border. The stimulation goes together with a large increase in a Na+-dependent H+ secretion into the lumen. All of these data suggests that HCO3− both activates Na+−Cl− cotransport and H+−Na+ countertransport at the luminal barrier.Thiocyanate inhibits Na+ and fluid transepithelial transport without affecting H+ secretion and HCO3−-dependent Na+ influx. It reduces Na+ and Cl− concentrations and contents, increases the same parameters for K+, causes a cell shrinking, and abolishes the lumen-to-cell Cl− influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na+-dependent and HCO3−-activated. Thus, SCN− is likely to compete for the Cl− site on the cotransport carrier and to be slowly transferred by the cotransport system itself.

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.

The nature of the neutral Na+-Cl(-)-coupled entry at the apical membrane of rabbit gallbladder epithelium: I. Na+/H+, Cl-/HCO3- double exchange and Na+-Cl- symport

SummaryCl− influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to36Cl− and3H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25mm SCN− which immediately and completely inhibits Cl− entry into the cell. Cellular influx was equal to 16.7μeq cm−2 hr−1 and decreased to 8.5μeq cm−2 hr−1 upon removal of HCO3− from the bathing media and by bubbling 100% O2 for 45 min. When HCO3− was present, cellular influx was again about halved by the action of 10−4m acetazolamide, 10−5 to 10−4m furosemide, 10−5 to 10−4m 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS), 10−3m amiloride. The effects of furosemide and SITS were tested at different concentrations of the inhibitor and with different exposure times: they were maximal at the concentrations reported above and nonadditive. In turn, the effects of amiloride and SITS were not additive. Acetazolamide reached its maximal action after an exposure of about 2 min. When exogenous HCO3− was absent, the residual cellular influx was insensitive to acetazolamide, furosemide and SITS. When exogenous HCO3− was present in the salines, Na+ removal from the mucosal side caused a slow decline of cellular Cl− influx; conversely, it immediately abolished cellular Cl− influx in the absence of HCO3−. In conclusion, about 50% of cellular influx is sensitive to HCO3−, inhibitable by SCN−, acetazolamide, furosemide, SITS and amiloride and furthermore slowly dependent on Na+. The residual cellular influx is insensitive to bicarbonate, inhibitable by SCN−, resistant to acetazolamide, furosemide, SITS and amiloride, and immediately dependent on Na+. Thus, about 50% of apical membrane NaCl influx appears to result from a Na+/H+ and Cl−/HCO3− exchange, whereas the residual influx seems to be due to Na+−Cl− contranport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counterreaction to the inhibition of the former is not clear.

Different sodium chloride cotransport systems in the apical membrane of rabbit gallbladder epithelial cells

SummaryThe kinetics of Cl− influx from the lumen to the cell and the paracellular pathway was examined in isolated rabbit gallbladder by measuring36Cl uptake (45 s) and by correcting it for the extracellular space with3H-sucrose. The paracellular fraction of the influx was studied by incubating the tissue in Na+-free saline or in solutions containing 25mm SCN−; the kinetics turned out to be hyperbolic. The cellular fraction of the influx comprised three components. The first was immediately Na+-dependent and insensitive both to exogenous and endogenous cell bicarbonate; its sigmoidal kinetics revealed the presence of a carrier with three Cl− binding sites cooperating positively with one another, with strong interaction factors. The second cellular component was immediately Na+-dependent and sensitive to endogenous cell bicarbonate; the kinetics was hyperbolic with a maximum at 20mm Cl− concentration and a substrate inhibition from 20 to 130mm; it was completely inhibited by 10−4m acetazolamide. The third cellular component was slowly Na+-dependent and slowly sensitive to exogenous bicarbonate; its kinetics was hyperbolic, without substrate inhibition in the tested Cl− concentration range. On this basis, the presence of three Na+−Cl− cotransports is suggested: i) on a single carrier without any exchange with H+ and HCO3−, ii) on a single carrier with an exchange with H+ and HCO3−, and iii) on two separate carriers in exchange with H+ and HCO3−.

Electrolyte transport across the pleura of rabbits

The amounts of Na+ and Cl- in the right pleural space of anesthetized rabbits were determined 10 and 60 min after a 2 ml hydrothorax with the following solutions: Ringer, Ringer with an inhibitor of the Na(+)-Cl- coupled transport or of the Na+/K+ pump, Ringer with gluconate instead of Cl- or with methylglucamine instead of Na+. During the 10-60 min period: (a) with Ringer Na+ and Cl- decreased (P less than 0.01) along with an iso-osmotic liquid absorption, (b) with disulfonic-stilbene (0.1 mM), amiloride (0.7 mM), acetazolamide (0.1 mM), or ouabain (0.5 mM) Na+ did not change and Cl- decreased less (P less than 0.01) than with Ringer. With gluconate-Ringer or methylglucamine-Ringer the liquid flow reversed: in the former case Cl- and, to a smaller extent, Na+ increased (P less than 0.01); in the latter only Na+ increased (P less than 0.01). These findings suggest: (1) the occurrence of a Na+/H+ and Cl-/HCO3- double exchange on the serosal side and of a Na+/K+ pump on the interstitial side of the pleural mesothelium; (2) a slow efflux from the pleural space of gluconate or methylglucamine relative to the corresponding influx of Cl- or Na+, respectively; this drags liquid into the space by osmotic gradient.

Electrical parameters in gallbladders of different species. Their contribution to the origin of the transmural potential difference.

SummaryAmphotericin B enhances Na+ conductance of the mucosal membrane of gallbladder epithelial cells and in such a way it modifies the brush border electromotive force. On this basis a method to measure cell and shunt resistances by comparing changes of the mucosal membrane potential (Vm) and of the transmural p.d. (Vms) is developed. This method is applied in gallbladders of different vertebrate species (i.e. rabbit, guinea pig, goose, tortoise, toad, trout). The two tested mammals, rabbit and guinea pig, exhibited a lower shunting percentage (89–93%) than the nonmammals (96–97%), but this fact did not bring about a homogeneous positiveVms. This means that shunting percent contributes, but it is not the only source of differences inVms, in accordance with that reported by Gelarden and Rose (J. Membrane Biol.19:37, 1974). Moreover, mammals exhibited a lower luminal resistance and a lower ratio between luminal and basolateral resistance than nonmammals. Possible causes of these differences are discussed.

Active transport of polypeptides in rabbit nasal mucosa: Possible role in the sampling of potential antigens

Transepithelial pathways of macromolecule transport have been studied in vitro in rabbit nasal respiratory mucosa, maintained at 27° C. Transepithelial electrical potential difference, short-circuit current and resistance were 3.4±0.5mV (submucosa positive), 65.0±6.7 μA cm−2 and 52.1±5.6 Ω cm−2 respectively (n=15). These electrical characteristics are those of a leaky epithelium allowing macromolecules to permeate paracellularly. A detailed permeation study of a polypeptide (elcatonin, Mw=3362) was also undertaken. Elcatonin mucosa-submucosa (Jms) and submucosa-mucosa (Jsm) fluxes were measured by radioimmunoassay. With 10 μg/ml elcatonin, Jms was significantly larger than Jsm for the whole 120-min period of observation; net flux showed a maximum in the first 30 min (Jms=13.6±1.0 ng cm−2 h−1, Jsm=1.4±0.1 ng cm−2 h−1, n=10). Jms fell towards the value of Jsm if the temperature was reduced to 4°C or if the mucosa was simultaneously treated with 0.1 mM dinitrophenol and 3 mM monoiodoacetate. Jms and Jnet followed saturation kinetics with increasing elcatonin concentrations. Adrenocorticotropic hormone (Mr=4500) produced a similar pattern to elcatonin. However, Jms and Jsm were not significantly different from each other at any time either for [3H]sucrose (Mw=342) or for [14C]polyethyleneglycol-4000 (Mw=4000) when present in the bathing medium at 500 μM concentration. The results show active transport of polypeptides in parallel with passive permeation (possibly through leaky intercellular junctions). Active transport does not appear to be related to nonspecific pinocytosis but to receptor-mediated endocytosis. The latter may be important for the sampling of potential antigens from the nasal lumen.

Post‐natal development of amiloride sensitive sodium transport in pig distal colon.

1. Both electrophysiological properties and unidirectional Na and Cl fluxes have been determined across distal colons taken from pigs during early post‐natal development. 2. The transmural potential difference (Vms) was 5 mV in the new‐born and 10 mV in the 4 day old colon. The short circuit current (Scc) showed a three to sixfold increase during the first 10 days of post‐natal life. The microvillar membrane potential (Vm) fell from about ‐45 mV in the new‐born to ‐40 mV in the 4 day old colon. 3. Amiloride had no effect on Vms, Scc or Vm, measured in the new‐born animal. It reduced Vms and Scc, caused a hyperpolarization of Vm and increased the microvillar membrane/basolateral membrane resistance ratio (Rm/Rs) in colons taken from older animals. 4. The Scc of distal colons taken from new‐born and 1 day old pigs was only half that predicted from unidirectional measurements of Na flux. This discrepancy, which could not be completely accounted for by net CL absorption, disappeared in the older animals. 5. Net transport of Na doubled during the first 24 h of post‐natal life. Part of this transport took place through an amiloride sensitive, non‐electrogenic, pathway. 6. It is suggested that Na uses mainly a non‐electrogenic pathway to cross the mucosa of the new‐born pig. This pathway is replaced by an electrogenic amiloride sensitive mechanism in older animals. Aldosterone is thought to initiate these changes in Na tranport.