Biomedical subjects
Publications and source records attributed to G Bottà.
The ability of cells to readjust their volume after swelling, a phenomenon known as regulatory volume decrease (RVD), is a fundamental biological achievement guaranteeing survival and function of cells under osmotic stress. This article reviews the mechanisms of RVD in mammalian cells with special emphasis on the activation of ion channels during RVD.
We investigated the transport systems that can sustain Na+ and Cl- movements across bovine gall bladder epithelium, focusing on the Na+-H+ exchanger (NHE) family and chloride conductive pathways. Experiments conducted using the fluorescent probe acridine orange (AO) with brush-border membrane vesicles (BBMV) or vesicles obtained from the total epithelium (EMV) demonstrated the presence of a Na+-H+ exchange in both preparations. The use of specific inhibitors indicated the presence of an apical NHE3 exchanger and a NHE1 isoform which should reside in the basolateral membrane. Using reverse transcriptase (RT) PCR, we identified cDNA fragments corresponding to the NHE1, NHE3, Cl--HCO3- (AE2a) transporters and to the CFTR channel. Using the patch-clamp technique, we investigated Cl- conductances on cultured epithelial cells. We found a 5 pS Cl- channel with a voltage-independent open probability, insensitive to stilbenes (SITS), Zn2+ and cAMP. The results suggest that absorption and secretion coexist in calf gall bladder epithelium. A Na+-H+-Cl--HCO3- double exchange may, at least partially, sustain the absorptive function, and a Cl- apical conductive pathway may be involved in secretion. The conductance we observed does not seem to be cAMP-regulated, unlike other mammalian gall bladders.
In guinea pig gallbladder epithelium, a secretion of fluid, secondary to an electrogenic secretion of Cl(-) and HCO(-)(3), is elicited in the presence of a high intracellular concentration of adenosine 3'-5'-cyclic monophosphate (cAMP). The aim of this study was to analyze the effects of secretagogues on the activity of anionic channels in isolated epithelial cells using the patch-clamp technique and measuring the electrical potential difference of the cellular membrane (pd(cm)). In cell-attached configuration, with the microelectrode filled with a solution of N-methylglucamine-Cl, or in inside-out configuration (symmetrical solution), it was possible to demonstrate the presence of an 18-pS Cl(-) channel with linear current/voltage (I/V) relationship and voltage independence; this channel is not activated by cAMP (cell-attached configuration). In inside-out configuration (symmetrical solution), another anionic channel with a conductance of 2.8 pS, voltage independence, and a linear I/V relationship was also identified. This channel was stimulated by cAMP (cell-attached configuration) and by PKA + ATP + cAMP (inside-out configuration). The channel was inhibited by NPPB (10(-5) M), but not by other anionic inhibitors. Measurements of the pd(cm) value suggested that in isolated cells, as in whole tissue, cAMP activates conductance for both Cl(-) and HCO(-)(3). The selectivity of the channel was gluconate < SO(2-)(4) < Cl(-) < Br(-) < I(-) < HCO(-)(3) < SCN(-) and the P(HCO(3))/P(Cl) was 2.6. Some features of the channel resemble those of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and RT-PCR performed on mRNA from isolated epithelial cells detected the presence of a CFTR homologue mRNA. The results obtained indicate that this channel is responsible for the HCO(-)(3) conductance activated by cAMP.
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 (G(Cl)), the symport inhibition might be merely due to a short circuit of the symport by G(Cl) 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 (a(i,Cl) and a(i,Na)) with selective theta microelectrodes and molecular biology methods were used. The 13Cl- 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 G(Cl) activation or abolition. After HCTZ treatment the decrease in a(i,Cl) (measured as the initial rate or in 3 min) was larger than the decrease in a(i,Na). The difference was reduced to one third in a group of epithelia in which the elicited G(Cl) was reduced to one third; moreover it was abolished in any case when G(Cl) was abolished with 10(-4) M SITS. The SITS-insensitive rate of a(i,Cl) decrease was equal to that of the a(i,Na) decrease in any case. Thus the a(i,Cl) decrease displays a component dependent on G(Cl) 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 G(Cl) 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.
Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of approximately equal to 3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.
In intact tissue, DAGO ([D-Ala2, MePhe4, Gly-ol5]enkephalin; 10(-5) M; mu-ligand; addition on the serosal side) stimulated D-glucose absorption and D-glucose-dependent variations in short-circuit current (delta Isc,glu); naloxone (10(-6) M) antagonized these effects. DADLE ([D-Ala2, D-Leu5]enkephalin, mainly a delta-ligand; 10(-5) M) and (pCl-Phe4)-DPDPE ([D-pen2, p-chloro-Phe4, D-Pen5]enkephalin, a more selective delta-ligand; 10(-5) M) did not significantly stimulate delta Isc,glu (addition on the serosal side). In the absence of the muscularis and myenteric plexus or using intact tissue treated with tetrodotoxin (TTX; 3 x 10(-7) M), DAGO was unable to increase delta Isc,glu. Addition of DAGO to the mucosal side did not induce any variations in delta Isc,glu. In conclusion, DAGO is able to increase D-glucose absorption by interacting with mu-receptors located in the myenteric plexus.
In intact tissue, [D-Ala2,MePhe4,Gly-ol5]enkephalin (10(-5) M; mu-ligand), diminished short-circuit current (Isc) and increased water, Na+ and Cl- net fluxes in vitro under open circuit conditions; it also inhibited L-valine absorption and L-valine-dependent variations of short-circuit current (delta Isc,val). Naloxone (10(-6) M) antagonized these effects. In the absence of the muscularis and myenteric plexus this enkephalin or morphine (mu-ligand) reduced Isc and delta Isc,val. These enkephalin effects occurred at different times. Different concentrations of enkephalin were tested for their effects on delta Isc,val. [D-Ala2,D-Leu5]enkephalin (mainly a delta-ligand) significantly decreased Isc but not delta Isc,val. The reduction of L-valine absorption does not depend on the effects on basal ion transport. Interaction of opioids with mu-receptors located in the submucosal plexus and/or in the epithelial cell accounts for this reduction. This enkephalin effect seems to be at least partially under the control of the myenteric plexus.
Transepithelial 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.
In rabbit gallbladder epithelium, a Na+/H+, Cl-/HCO3- double exchange and a Na(+)-Cl- symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na+, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na+ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (delta pH) was imposed [( H+]in greater than [H+]out, PD = 0), the addition of Na-gluconate to the external solution caused delta pH dissipation at a rate that followed saturation kinetics. Amiloride (10(-4) M) reduced the delta pH dissipation rate. Taken together, these data indicate the presence of Na+ and H+ conductances in addition to an amiloride-sensitive, electroneutral Na+/H+ exchange. An inwardly directed [Cl-] gradient (PD = 0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl-/OH- exchange. When both [Na+] and [Cl-] gradients (outwardly directed, PD = 0) were present, fluorescence quenching reached a maximum 20-30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na+] gradient alone or the same [Na+] gradient with Cl- at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na+ and Cl- concentration gradients and hydrochlorothiazide (5 x 10(-4) M). The decrease was not influenced by an inhibitor of Cl-/OH- exchange (10(-4) M furosemide) or of Na(+)-K(+)-2Cl- symport (10(-5) M bumetanide). We conclude that a Na+/H+ exchange and a Na(+)-Cl- symport are present and act simultaneously. This suggests that in intact tissue the Na(+)-Cl- symport is also likely to work in parallel with the Na+/H+ exchange and does not represent an induced homeostatic reaction of the epithelium when Na+/H+ exchange is inhibited.
We studied the influence on ionic basal transport (Na+ and Cl-) and L-valine transport of two enkephalins which are not metabolized and act in delta and mu receptors respectively. Transports have been indirectly determined measuring the transepithelial electric potential and the short circuit current. DADLE does not significantly influence ion and amino-acid transport, while DAGO alters both of them in the presence of the myenteric plexus (muscle layers present) or inhibits only L-valine transport in the absence of the plexus (muscle layers removed).
The transapical Cl- influx and transepithelial Na+ transport were measured in rabbit gallbladder. Only 11.7% of the transported Na+ was found to be accompanied by HCO3-. 10(-4) M SITS eliminated the HCO3- dependent fraction of Cl- influx (50%) but did not significantly alter intracellular Na+ activity and Na+ transport. Exposure to HCO3-free salines or to 10(-4) M acetazolamide about halved Cl- influx and Na+ transport. 25 mM SCN- reduced Cl- influx to zero, decreased intracellular Na+ activity, but only halved Na+ transport which under these conditions was abolished only in the absence of HCO3-. Exposure to a Cl- -free saline produced effects similar to those caused by SCN-. These results suggest that when Cl-/HCO3- exchange is inhibited at the apical membrane, Na+/H+ exchange and transepithelial Na+ transfer are unmodified if HCO3- is available for transport. The permanent uncoupling of the exchangers and the elevated transepithelial transport of Na+ are not due to an increased activity of the parallel Na+-Cl- cotransport but to a redirection of HCO3- flux toward the basolateral side.
The apical membranes of rabbit gallbladder epithelial cells were isolated by treating the homogenate with Ca2+ or Mg2+ and centrifuging the suspension in Percoll gradient. In this way brush-border membranes were obtained with enrichment factors ranging between 10 and 20 and yields of 15-30%. A second method is described with which membranes were isolated, without any preliminary treatment, first by differential centrifugation, then with Percoll gradient; the final membrane enrichment was over 15, however the yield was very low (3%). Many possible enzymatic markers of the apical plasma membrane were investigated: L-gamma-glutamyltransferase, alkaline phosphatase, leucine aminopeptidase, sucrase. The first appears to be that of choice. Apical membrane fraction could be also evidenced by autofluorescence or by labeling with Lotus tetragonolobus lectin. Preliminary experiments showed that apical plasma membranes isolated in this way form vesicles.
Cl- influx at the luminal border of the epithelium of rabbit gallbladder was measured by 45-sec exposures to 36Cl- and 3H-sucrose (as extracellular marker). Its paracellular component was evaluated by the use of 25 mM SCN- which immediately and completely inhibits Cl- entry into the cell. Cellular influx was equal to 16.7 mu eq cm-2 hr-1 and decreased to 8.5 mu eq 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(-4) M acetazolamide, 10(-5) to 10(-4) M furosemide, 10(-5) to 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS), 10(-3) M 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- cotransport on a single carrier. Whether both components are simultaneously present or the latter represents a cellular homeostatic counter-reaction to the inhibition of the former is not clear.
In the epithelium of rabbit gallbladder, in the nominal absence of bicarbonate, intracellular Cl- activity is about 25 mM, about 4 times higher than intracellular Cl- activity at the electrochemical equilibrium. It is essentially not affected by 10(-4) M acetazolamide and 10(-4) M 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonate (SITS) even during prolonged exposures: it falls to the equilibrium value by removal of Na+ from the lumen without significant changes of the apical membrane potential difference. Both intracellular Cl- and Na+ activities are decreased by luminal treatment with 25 mM SCN-; the initial rates of change are not significantly different. In addition, the initial rates of change of intracellular Cl- activity are not significantly different upon Na+ or Cl- entry block by the appropriate reduction of the concentration of either ion in the luminal solution. Luminal K+ removal or 10(-5) M bumetanide do not affect intracellular Cl- and Na+ activities or Cl- influx through the apical membrane. It is concluded that in the absence of bicarbonate NaCl entry is entirely due to a Na+-Cl- symport on a single carrier which, at least under the conditions tested, does not cotransport K+.
A new method is described to prepare theta-microelectrodes with tips up to 0.15 micron diameter controlled under scanning electron microscope. K+- and Na+-sensitive resins were tested. Method features are the following: i) hard drying of the glass, ii) rehydration of one channel and weak wetting of the other with a three-methylchlorosilane solution before pulling, iii) simultaneous presence of water and silane in the two channels during pulling, iv) gradual silanization from the tip to the shank. Selective and conventional channels did not affect each other and no displacements of resins were observed. The change of potential difference of the selective channel was more than -50 mV/decade. Apical membrane potentials and cell Na+ and K+ activities of the epithelial cells of rabbit gall-bladder (cell diameter: 5-10 micron) were measured with these theta-microelectrodes and with single-barrel microelectrodes of similar tip size: results obtained were not significantly different.