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Hydrochlorothiazide enhances the apical Cl- backflux in rabbit gallbladder epithelium : Radiochemical analysis
No full textHydrochlorothiazide(HCTZ) was shown to inhibit the transepithelial NaCl transport and the apical Na+-Cl– symport and to depolarize the apical membrane potential in the rabbit gallbladder epithelium. The depolarization was likely related to the opening of a Cl– conductance. To better understand whether an apical Cl– leak is involved in the mechanism of action of HCTZ, the transapical Cl– backflux was measured radiochemically by the washout technique. The gallbladder wall, pretreated with pronase on the serosal side to homogenize the subepithelium, was loaded with 36Cl– on the luminal side; mucosal and serosal 36Cl– effluxes (J m , J s ) were then measured every 2 min. The pretreatment with pronase did not alter the membrane potentials and the selectivity of the epithelium. Under control conditions and the tissue in steady-state, J m and J s time courses were each described by two exponential decays (A,B); the rate constants, k A and k B , were 0.71 ±0.03 and 0.16±0.01 min–1, respectively, and correspondingly the half-times (t 1 2A , t 1 2B ) were 1.01±0.05 and 5.00±0.44 min (n=10); these parameters were not significantly different for J m and J s time courses. J s was always greater than J m (J s /J m =2.02±0.22 and 1.43 ±0.17 for A and B decays). Under SCN– treatment in steady-state conditions, both J m and J s time courses were described by only one exponential decay, the component B being abolished. Moreover t 1 2A was similar to that predictable for the subepithelium. It follows that it is the component B which exits the epithelial compartment. Based on the intracellular specific activity and 36Cl– J m B at 0 min time of the washout experiment, the cell-lumen Cl– backflux in steady-state was calculated to be equal to about 2 mgrmol cm–2hr–1, in agreement with the value indirectly computable by other techniques. The experimental model was well responsive to different external challenges (increases in media osmolalities; luminal treatment with nystatin). HCTZ (2.5 · 10–4 m) largely increased 36Cl– J m B . The increase was abolished by luminal treatment with 10–4 m SITS, which not only brought back the efflux time courses to the ones observed under control conditions but even increased J s /J m of the cellular component, an indication of a reduced J m B . It is concluded that HCTZ opens an apical, SITS-sensitive Cl– leak, which contributes to dissipate the intracellular Cl– accumulation and to inhibit the NaCl transepithelial transport. Moreover, the drug is likely to reduce the basal electroneutral Cl– backflux supported by Na+-Cl– cotransport, in agreement with the inhibition of the cotransport itself
Electroneutral Na+ and Cl- coupled transport across the apical membrane of epithelial cells (rabbit gallbladder)
No full textUso di microparticelle su cui sono stati adsorbiti una proteina ed un anticorpo per preparare una composizione farmaceutica somministrabile per via intranasale
No full textUso di una microparticella polimerica, su cui sono stati previamente adsorbiti una proteina ed un anticorpo, per preparare una composizione farmaceutica somministrabile per via intranasal
Use of microparticles having a protein and an antibody adsorbed thereon for preparing a pharmaceutical composition for intranasal administration
No full textUse of a microparticle having a protein and an antibody adsorbed thereon for preparing a pharmaceutical composition for intranasal administratio
Endocytosis of polypeptides in the nasal respiratory mucosa of the rabbit
No full textThe nasal respiratory mucosa of the rabbit has a leaky epithelium actively transporting polypeptides by a specific transcytosis probably involved in sampling antigens. The transfer displays saturation kinetics and is abolished by metabolic inhibitors, actin filamet and microtubule disassemblers, inhibitors of vesicle fusion, and receptor recycling;it accepts polypeptide-covered but not uncovered nanoparticles
Hydrochlorothiazide action on the apical Cl−, Ca2+ and K+ conductances in rabbit gallbladder epithelium. Presence of an apamin-sensitive, Ca2+-activated K+ conductance
No full textIn 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 degrees 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 x 10(-4) M 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 Ca(2+)-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 Vm and 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 calculate: 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
Increase in intrinsic anion conductance upon inhibition of the electroneutral Cl-/HCO3- exchanger: effect of CO2/HCO3-
No full textThe electroneutral Cl(-)/HCO(3)(-) exchange, present at the apical membrane of rabbit gallbladder epithelium, apparently is converted into a stilbene- and dipyridamole-sensitive, nonrectifying, approximately 5-pS anion channel after the exchange is directly inhibited (inhibitors tested: hydrochlorothiazide (HCTZ), phlorizin, phenylglyoxal and diphenylamine-2-carboxylic acid (DPC)). In intact tissue, in the absence of CO(2)/HCO(3)(-) in the media, the opening of these channels causes an approximately 7-mV depolarization of the apical membrane. This has been shown to be a constant index of the total Cl(-) conductance (G(Cl)) activated. The effect of exogenous and endogenous CO(2)/HCO(3)(-) on the depolarization has now been investigated in the intact tissue by conventional microelectrodes. The anion exchange has been measured radiochemically. The presence of exogenous or endogenous CO(2)/HCO(3)(-) reduces the depolarization induced by HCTZ, phlorizin and DPC from approximately 7 to 3 mV, but 10(-4) mol/l acetazolamide restores the full depolarization. Response time, S(0.5) and Hill number are unchanged in each case. The way of bicarbonate replacement is irrelevant. The depolarization generated by phenylglyoxal, which covalently binds to the transport site of the exchanger and prevents HCO(3)(-) binding, is unaffected by CO(2)/HCO(3)(-) presence. HCO(3)(-) binding to the transport site is suggested to partially hinder the conversion of the exchanger into the channel
Inhibitors of the Cl-/HCO3- exchanger activate an anion channel with similar features in the epitelial cells of rabbit gallbladder : patch-clamp analysis
No full textThe stilbene- and dipyridamole-sensitive Cl-conductance (GCl), non-additively activated by some inhibitors of the Cl-/HCO3- exchanger (hydrochlorothiazide, phlorizin, phenylglyoxal) after the exchanger inhibition in the apical plasma membrane of rabbit gallbladder epithelium, has been investigated by patch-clamp technique with cell-attached and inside-out configurations. No Cl- channels were observed under basal conditions or after treatment with 2.5 x 10(-4) mol/l 8-Br-cAMP or hydrochlorothiazide (HCTZ) on the cytosolic side. Conversely, with 2.5 x 10(-4) mol/l HCTZ or 2 mmol/l phlorizin in the pipette, a non-rectifying Cl- channel with about 5 pS conductance and 0.3-0.4 voltage-independent open probability was observed; it was inhibited by 10(-4) - 5 x 10(-4) mol/l SITS, 10(-4) mol/l furosemide or 0.6 x 10(-4) mol/l dipyridamole; the effects of HCTZ and phlorizin were not additive. Open probability increased from 0 (after seal formation) to a maximum of 0.3-0.4 reached in 7-9 min. Similar results were obtained with both configurations. With the cell-attached configuration, HCTZ added to the bath did not activate Cl- channels in the patch. The channel was shown to exclude cations, to be selective for Cl-, but also conductive for gluconate (PGluc/PCl = 0.18). On this basis, it is concluded that: (1) GCl, activated either by HCTZ or phlorizin, has the same underlying anion channels, (2) the channels can be activated only on the external side of the membrane, also in the absence of cytoplasm, without cellular mediations or effects at a distance along the membrane, (3) the channels are inhibited by the same drugs which inhibit the very small intrinsic anion conductance of the exchanger, (4) they are either related to a slow conversion of inhibited exchangers into channels or, less probably, they are parallel to the exchanger and slowly activated by intra-membrane (or membrane-bound) mediators in their turn activated by near, inhibited exchangers
Nature of the neutral Na+-Cl- coupled entry at the apical membrane of rabbit gallbladder epithelium: IV. Na+/H+,Cl-/HCO3- double exchange, hydrochlorothiazide-sensitive Na+-Cl- symport and Na+-K+-2Cl- cotransport are all involved
No full textTransepithelial fluid transport was measured gravimetrically in rabbit gallbladder (and net Na+ transport was calculated from it), at 27 degrees C, in HCO(3-)-free bathing media containing 10(-4) M acetazolamide. Whereas luminal 10(-4) M bumetanide or 10(-4) M 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 microliters cm-2 hr-1 (n = 10), i.e., to about 30%. This maximum effect was already obtained at 10(-3) M concentration; the apparent IC50 was about 2 x 10(-4) M. 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(-4) M bumetanide, but was slowly inhibited by 10(-3) M 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(-4) M 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(-3) M 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
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