E. Frömter

Rheogenic sodium-bicarbonate cotransport in the peritubular cell membrane of rat renal proximal tubule

The mechanism of bicarbonate transport across the peritubular cell membrane was investigated in rat kidney proximal tubules in situ by measuring cell pH and cell Na+ activity in response to sudden reduction of peritubular Na+ and/or HCO3−. The following observations were made: 1. sudden peritubular reduction of either ion concentration produced the same transient depolarizing potential response; 2. bicarbonate efflux in response to peritubular reduction of bicarbonate was accompanied by sodium efflux; 3. sodium efflux in response to peritubular sodium removal was accompanied by cell acidification indicating bicarbonate efflux; 4. all aforementioned phenomena were inhibited by SITS (10−3 mol/l) except for a small SITS-independent sodium efflux and depolarization which occurred in response to peritubular sodium removal and was not accompanied by cell pH changes; 5. bicarbonate efflux and accompanying potential changes in response to reduction of peritubular bicarbonate virtually vanished in sodium-free solutions. From these observations we conclude that bicarbonate efflux proceeds as rheogenic sodium-bicarbonate cotransport with a stoichiometry of bicarbonate to sodium greater than 1. The question which of the charged species of the bicarbonate buffer system moves cannot yet be decided. Attempts to determine the stoichiometry from the SITS-inhibitable initial cell depolarization and from the SITS-inhibitable initial fluxes suggest a stoichiometry of 3 HCO3−: 1 Na+. In addition to sodium-dependent bicarbonate flux, evidence was obtained for a sodium-independent transport system of acids or bases which is able to regulate cell pH even in sodium-free solutions.

Cl− channel inhibition by glibenclamide is not specific for the CFTR-type Cl− channel

As long as the question of which channels are responsible for cAMP-mediated epithelial Cl− secretion remains unsolved, it is still important to search for specific inhibitors that might help to relate macroscopic to microscopic events. Following the report by Sheppard and Welsh (J Gen Physiol 100: 573, 1992) that glibenclamide inhibits whole-cell Cl− currents in genetically manipulated fibroblasts expressing the cystic fibrosis transmembrane conductance regulator (CFTR), we have studied the effect of glibenclamide on different types of Cl− channels of HT29 and T84 cells at the single-channel level. Our results confirm that micromolar concentrations of glibenclamide inhibit the linear, low-conductance Cl-channel, which appears to represent CFTR and show that the inhibition results from a typical flicker block. However, the same concentrations of glibenclamide inhibit also the outwardly rectifying intermediate conductance Cl− channel which, potentially, may contribute to transepithelial Cl− secretion.

Pathways of NH3/NH4+ permeation across Xenopus laevis oocyte cell membrane

While acid loading with extracellular NH4Cl solutions usually first alkalinizes the cells through NH3 influx, and acidifies only when NH4Cl is removed, Xenopus oocytes became immediately acidic upon NH4Cl addition and the cells did not acidify further upon its removal. Since NH4Cl solutions also collapsed the membrane potential (Vm) and resistance (Rm), we conclude that primarily NH4+entered the cells where it liberated H+, with NH3 being trapped in intracellular lipid stores. To identify the NH4+permeation pathway we have used K+ channel blockers (Ba2+, Cs+, tetraethylammonium, quinidine), various cation transport inhibitors (ouabain, bumetanide, amiloride) and other inhibitors, some of which block non-selective cation channels (La3+, diphenylamine-2-carboxylate, and p-chloromercuribenzoate). However, only the latter substances partially prevented the collapse of Vm and Rm. This suggests, that NH4+passes through non-selective cation channels. In accordance with the voltage dependence and/or stretch activation of such channels NH4+fluxes appeared to be asymmetric. NH4+influx, which depolarized and swelled the cells, was large and acidified rapidly, while the efflux, which repolarized and shrank the cells, was slow and alkalinized only slowly.

Basolateral proteinase‐activated receptor (PAR‐2) induces chloride secretion in M‐1 mouse renal cortical collecting duct cells

1 Using RT‐PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase‐activated receptor (PAR‐2) and demonstrated its presence in native renal epithelial and in cultured M‐1 mouse cortical collecting duct (CCD) cells. 2 We investigated the effects of a PAR‐2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M‐1 cells using patch‐clamp, intracellular calcium (fura‐2) and transepithelial short‐circuit current (ISC) measurements. 3 In single M‐1 cells, addition of AP elicited a concentration‐dependent transient increase in the whole‐cell conductance. Removal of extracellular Na+ had no effect while removal of Cl− prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+‐free pipette solution completely abolished the electrical response to AP. 4 In confluent monolayers of M‐1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5 The ISC response to AP was reduced in the presence of the Cl− channel blocker diphenylamine‐2‐carboxylic acid on the apical side and abolished in the absence of extracellular Cl−. 6 Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole‐cell currents or ISC. 7 In conclusion, our data suggest that AP or trypsin stimulates Cl− secretion by Ca2+‐activated Cl− channels in M‐1 CCD cells by activating basolateral PAR‐2.

Cell pH of rat renal proximal tubule in vivo and the conductive nature of peritubular HCO 3 ? (OH?) exit

Intracellular pH (pHc) was measured on surface loops of rat kidney proximal tubules under free-flow conditions in vivo using fine tip double-barrelled pH microelectrodes based on a neutral H+ ligand. The microelectrodes had Nernstian slopes and a resistance of the order of 1012 Ω. By using a driven shield feed back circuit the response time to pH jumps was lowered to around 1 s. At a peritubular pH of 7.42 and a luminal pH of 6.68 ± 0.13 (n=27), pHc was 7.17 ± 0.08 (n=19). Perfusing the peritubular capillaries suddenly with bicarbonate Ringer solutions of plasma-like composition which were equilibrated with high or low CO2 pressures, acidified or respectively alkalinized the cells rapidly as expected from the high CO2 permeability of the cell membranes. Such data allowed us to calculate the cytoplasmic buffering power of the tubular cells. Sudden peritubular perfusion with Ringer solution containing only 3 mmol/l of HCO3− at constant physiological CO2 pressure led to a similar fast cell acidification which indicated that the peritubular cell membrane is also highly permeable for bicarbonate or OH− (H+). The latter response was completely blocked by the stilbene derivative SITS at the concentration of 10−3 mol/l. The observations indicate first that pHc of rat proximal tubule is more acidic than was previously thought on the basis of distribution studies of weak acids, second that intracellular bicarbonate concentration is around 13 mmol/l and third that bicarbonate exit across the peritubular cell membrane is a passive rheogenic process via a conductive pathway which can be inhibited by SITS. The latter point confirms the conclusion which we had derived previously from membrane potential measurements in response to changing peritubular bicarbonate concentrations.

Stoichiometry of the rat kidney Na+-HCO3- cotransporter expressed in Xenopus laevis oocytes.

Abstract The rat kidney Na+-HCO3– cotransporter (rkNBC) was expressed in Xenopus laevis oocytes and transport via rkNBC was studied with the patch-clamp technique in giant inside/out (i/o) or outside/out (o/o) membrane patches. The current/voltage (I/V) relation(s) of individual patches was(were) determined in solutions containing only Na+ and HCO3– as permeable ions. The current carried by rkNBC (INBC) was identified by its response to changing bath Na+ concentration(s) and quantified as the current blocked by 4,4’-diisothiocyanatostilbene disulfonate (DIDS). The stoichiometric ratio (q) of HCO3– to Na+ transport was determined from zero-current (reversal) potentials. The results and conclusions are as follows. First, DIDS (250 µmol/l) blocks INBC irreversibly from both the extracellular and the intracellular surface. Second, in the presence of Na+ and HCO3– concentration gradients similar to those which rkNBC usually encounters in tubular cells, q was close to 2. The same value was also observed when the HCO3– concentration was 25 mmol/l throughout, but the Na+ concentration was either high (100 mmol/l) or low (10 mmol/l) on the extracellular or intracellular surface of the patch. These data demonstrate that in the oocyte cell membrane rkNBC works with q=2 as previously observed in a study of isolated microperfused tubules (Seki et al., Pflügers Arch 425:409, 1993), however, they do not exclude the possibility that in a different membrane and cytoplasmic environment rkNBC may operate with a different stoichiometry. Third, in most experiments bath application of up to 2 mmol/l ATP increased the DIDS-inhibitable conductance of i/o patches by up to twofold with a half saturation constant near 0.5 mmol/l. This increase was not associated with a change in q, nor with a shift in the I/V relationship which would suggest induction of active transport (pump current). Since the effect persisted after ATP removal and was not observed with the non-hydrolysable ATP analogue AMP-PNP, it is possible that rkNBC is activated by phosphorylation via protein kinases that might adhere to the cytoplasmic surface of the membrane patch.

Evidence for rheogenic sodium bicarbonate cotransport in the basolateral membrane of oxyntic cells of frog gastric fundus

AbstractIonic conductance properties of the basolateral cell membrane of oxyntic cells were studied in frog gastric fundus in vitro. After mounting the fundus in a modified Ussing chamber the serosal connective tissue was dissected off and individual oxyntic cells were punctured from the serosal surface with microelectrodes. Under resting conditions the membrane potential averaged −56.9, SD±9.5 mV (n=63), cytoplasm negative. Lowering or raising serosal HCO3− concentration from 17.8 to 6 or 36 mmol/l respectively at constant

Axial heterogeneity of sodium-bicarbonate cotransport in proximal straight tubule of rabbit kidney.

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