Takis Anagnostopoulos

Biionic potentials in the proximal tubule of Necturus kidney

1. Biionic potentials were studied in the proximal tubule of the doubly perfused Necturus kidney and subsequently analysed by means of an equivalent electrical circuit.

Electrophysiological study of the antiluminal membrane in the proximal tubule of Necturus: effect of inorganic anions and SCN−

1. A study has been made of the effects of anionic substitutions on the electrical potential difference (p.d.) and conductance characteristics of the antiluminal (peritubular) membrane of the proximal tubule of Necturus kidney. The tubular lumina were filled with oil in order to minimize potential and conductance contributions from luminal membrane and from paracellular shunt pathway.

Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine

SummaryTo see whether the Na/H antiporter plays a role in digitalis cardiotoxicity, we investigated the influence of modulators of Na/H exchange on the toxic effects of ouabain in isolated, paced (0.4 Hz) rat left atria. Ouabain (1 mmol/l) caused a transient positive inotropic effect followed by toxic events, including a complete loss of developed force and a gradual increase in resting force. In the presence of hexamethyleneamiloride (3 and 10 μmo1/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 μmol/ 1) an α-adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabains toxicity. Neither ouabain, at a subtoxic concentration (650 μol/l), nor phenylephrine (100 μmol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 μmol/l) or adrenaline (30 μmol/l), in the presence of a β-adrenoceptor antagonist, increased the intracellular pH by up to 0.15 pH unit, as measured using ion-selective microelectrodes in quiescent preparations. This effect on pH1 was prevented by hexamethyleneamiloride (10 μmol/l). Consistent with phenylephrines ability to stimulate Na+ influx via the Na/H antiporter, phenylephrine (100 μmol/l) increased intracellular Na+ activity by about 3 mmol/l in ouabain (650 μmol/l)-treated atria. These findings indicate that modulators of Na/H exchange affect the cardiotoxicity of digitalis glycosides and imply that the stimulation of myocardial α-adrenoceptors may aggravate digitalis toxicity.

Millimolar amiloride concentrations block K conductance in proximal tubular cells.

1 Amiloride, applied at millimolar concentrations, results in the blockade of K+ conductance in amphibian proximal convoluted cells (PCT), fused into giant cells. 2 Amiloride results directly in a blockade of K+ conductance that is not related to inhibition of the Na+‐H+ antiport, which would lower intracellular pH, adversely affecting K+ conductance. On the contrary, high amiloride concentrations promote entry of this lipophilic base in the cell, leading to higher cell pH. 3 Under voltage clamp conditions, control vs. amiloride, current‐voltage curves from PCT fused giant cells intersect at −86.2 ± 3.4 mV, a value close to the equilibrium potential for potassium. 4 Hexamethylene amiloride, 10−5 m, irreversibly depolarizes the membrane potential. 5 Barium decreased by 50% the initial slope of realkalinization, following removal of a solution containing NH4Cl, as did amiloride. In addition, these blockers reduced membrane conductance by 40%, suggesting that a fraction of the amiloride‐suppressible NH4+ efflux may be conductive. 6 Amiloride does not directly inhibit the Na+‐K+, ATPase in our preparation, contrary to the prevalent belief. 7 In vivo studies show that amiloride interferes with an apical K+ conductance but it does not alter basolateral K+ conductance.

Barium‐ or quinine‐induced depolarization activates K+, Na+ and cationic conductances in frog proximal tubular cells.

1. Frog proximal tubular cells were fused into giant cells. We measured membrane potential (Vm), its changes (delta Vm), and current‐induced voltage changes (delta psi) in single cells, during control and experimental states. Each cell served as its own control. 2. In the presence of a physiological Ringer solution, the transference number for potassium (tK) was 0.50. Barium (3 mM) reduced membrane conductance (Gm) by 50%; low‐Cl‐ solutions and low‐Na+ solutions also diminished Gm, by 52 and 30%, respectively. The association of barium and low‐NaCl solutions decreased Gm to approximately 38% of control, indicating that the impermeant substitute of a physiological ion may interact with other pathways; alternatively, blockade of steady‐state conductances may activate physiologically silent processes. 3. In an attempt to enhance the contribution of the partial K+ conductance (GK) to Gm, fused cells were exposed to low‐Cl‐ solutions, containing in addition 0.1 mM‐methazolamide, to inhibit the rheogenic Na(+)‐HCO3‐symport, and 1 microM‐amiloride, to block Na+ conductance (GNa). tK went up to 0.83. 4. The high tK preparation was challenged with barium (3 mM) or quinine (Quin, 1 mM). These blockers produced large depolarizations (approximately 60 mV), however, although Gm decreased along early‐ and mid‐depolarization, Gm plateaued and eventually it increased with larger and larger depolarization. 5. Depolarization‐associated increase in Gm reflects activation of other conductances. These are Na+, cationic, and K+ conductance(s) poorly sensitive to quinine or barium. In the presence of Ba(2+)‐ or Quin‐induced depolarization, injection of depolarizing current produces delayed increase in conductance. 6. Depolarization‐induced activation of cationic conductance (Gcat) and GNa results in enlargement of the K+ electrochemical potential difference, to about 70 mV; this difference allows recycling of K+ ions outwards, since a GK is still detected and may contribute up to 38% of the total conductance.

Basolateral electrogenic Na/HCO3 symport in the amphibian distal tubule.

This study was carried out to assess whether the amphibian distal tubule possesses a basolateral Na/ (HCO3)n>1 cotransport. The experiments were performed in the kidney of Necturus maculosus in vivo, by means of double-barreled selective microelectrodes. Basolateral membrane potential (Vm), intracellular pH (pHi), intracellular sodium activity (αNai) and intracellular chloride activity (αCli), were recorded during selected disturbances of peritubular fluid composition. The following results were obtained, (a) A sudden decrease of [HCO3]0 leads to Vm depolarization, intracellular acidification and decrease of αNai. (b) A rapid fall of [Na]0 elicits Vm depolarization and decreases pHi; these patterns are not substantially altered in the presence of millimolar amiloride concentrations or in the nominal absence of peritubular Cl. (c) An acute decrease of [Na]o does not alter αCli. (d) In the functional absence of CO2/ HCO3 buffer (nominally CO2-free solution plus methazolamide), the reduction of [Na]0 has no effect on Vm and/or pHi. We conclude that the distal tubule basolateral cell membrane is endowed with an electrogenic chlorid-eindependent Na/base carrier, mediating Na and base efflux. The blockade of this carrier by carbonic anhydrase inhibitor indicates that the cotransported base is HCO3 or a related species.

Triflocin, a novel inhibitor for the Na-HCO3 symport in the proximal tubule

1 Triflocin, applied at millimolar concentration hyperpolarizes the basolateral membrane of Necturus proximal convoluted tubular cells, in vivo. 2 Barium, 2.5 × 10−3 m, ouabain, 10−3 m, or amiloride 10−4 m, fail to prevent this hyperpolarization. 3 Triflocin has no effect on the intracellular chloride activity. 4 In physiological acid base conditions, Triflocin increases intracellular pH. 5 Upon an acute isohydric hypercapnia, Triflocin depolarizes the basolateral membrane potential. 6 It is concluded that, Triflocin inhibits the basolateral electrogenic Na‐(HCO3)n > 1 cotransport in proximal tubules.

Thiazide-sensitive Na-Cl cotransport mediates NaCl absorption in amphibian distal tubule.

To find out the mechanism(s) underlying NaCl absorption in the distal tubule of Necturus, we devised a variant of the split-drop technique. Following injection an oil column, subsequently split by a NaCl solution isotonic to plasma, a double-barrelled microelectrode (conventional/selective to Na+ or to Cl− ions) recorded Na+ (αNa) or Cl− (αCl) activity and transepithelial potential (Vte). Paired control/low-Na+ solutions yielded reabsorptive half-times (t1/2) of 0.68±0.11 min and 7.6±1.8 min respectively; corresponding Vte values were −22.2±4.0 mV and −7.6±1.9 mV. t1/2 values of control versus low-Cl− solutions were 0.77±0.32 min and 6.5±1.7 min respectively, whereas respective Vte values were not different from one another: −23.8±4.3 mV versus −18.8±5.5 mV. Nominally K+-free solutions or bumetanide, 10 μmol/l, did not alter t1/2 or Vte, with regard to the paired control. Amiloride, 5 μmol/l or 2 mmol/l, failed to decrease t1/2 or to lower Vte; apparently, the role of a Na+/H+ antiport does not contribute significantly to NaCl absorption. Furosemide, 0.1 mmol/l, reduced t1/2 by 54% with regard to the control state. Determination of t1/2 as a function of increasing hydrochlorothiazide concentrations revealed apical high- and low-affinity sites, estimated at 0.56 μmol/l and 0.115 mmol/l respectively. Taken together these observations indicate that NaCl absorption is predominantly carried out by an electroneutral Na+-Cl− cotransport.