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T Anagnostopoulos

Publications and source records attributed to T Anagnostopoulos.

At least 19 recordsLinked to original sources
PubMed1992

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+ (alpha Na) or Cl- (alpha Cl) 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 mumol/l, did not alter t1/2 or Vte, with regard to the paired control. Amiloride, 5 mumol/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 mumol/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.

Amiloride↗
PubMed1992

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.

Amiloride↗
PubMed1992

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.

Amiloride↗
PubMed1992

Luminal pH in the amphibian distal tubule: effects of carbonic anhydrase and carbonic anhydrase inhibitors.

To better delineate acid-base transport properties in the distal tubule (DT) of Necturus in vivo, we 1) studied the effects of peritubular (pt) isohydric increase of PCO2 and [HCO3-]pt on luminal pH (pHlu), and 2) measured the steady-state pHlu under various experimental conditions. The experiments were carried out on initial (DTi) or distal (DTd) loops of the DT in control state and then during intravenous infusion of carbonic anhydrase (CA) or CA inhibitors (CAI). In control state, isohydric increase of PCO2 and [HCO3-]pt results in transient acidification of the DTi lumen, whereas in DTd lumen the same maneuver yields sustained (plateau) acidification. Under systemic infusion of CAI, isohydric increase of PCO2 and [HCO3-]pt lowers pHlu (sustained fall of pHlu) in DTi and DTd, whereas under CA infusion both segments exhibit only transient acidification. During intravenous infusion with benzolamide DTi steady-state pHlu falls, suggesting that this maneuver inhibits a functional luminal CA, in contrast to the DTd, whose pHlu remains unaltered. Intravenous infusion of CA significantly increases steady-state DTd pHlu; by contrast, steady-state pHlu in DTi does not change. These data are consistent with the presence of functional luminal CA in the DTi, whereas the DTd segment lacks the luminal enzyme.

Acetazolamide↗
PubMed1991

Opposite modulation of ouabain cardiotoxicity by hexamethyleneamiloride and phenylephrine.

To 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 mumol/l), an inhibitor of Na/H exchange, ouabain (1 mmol/l) caused a sustained positive inotropic effect without toxicity. By contrast, phenylephrine (100 mumol/l), an alpha-adrenoceptor agonist reported to stimulate the antiporter, hastened the development of ouabain's toxicity. Neither ouabain, at a subtoxic concentration (650 mumol/l), nor phenylephrine (100 mumol/l) affected diastolic force, but in their combined presence, a substantial contracture developed and twitch contractions disappeared. Phenylephrine (30 or 100 mumol/l) or adrenaline (30 mumol/l), in the presence of a beta-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 pHi was prevented by hexamethyleneamiloride (10 mumol/l). Consistent with phenylephrine's ability to stimulate Na+ influx via the Na/H antiporter, phenylephrine (100 mumol/l) increased intracellular Na+ activity by about 3 mmol/l in ouabain (650 mumol/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 alpha-adrenoceptors may aggravate digitalis toxicity.

Amiloride↗
PubMed1991

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 greater than 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 (alpha Nai) and intracellular chloride activity (alpha Cli), were recorded during selected disturbances of peritubular fluid composition. The following results were obtained. (a) A sudden decrease of [HCO3]o leads to Vm depolarization, intracellular acidification and decrease of alpha Nai. (b) A rapid fall of [Na]o 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 alpha Cli. (d) In the functional absence of CO2/HCO3 buffer (nominally CO2-free solution plus methazolamide), the reduction of [Na]o has no effect on Vm and/or pHi. We conclude that the distal tubule basolateral cell membrane is endowed with an electrogenic chloride-independent 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.

Amiloride↗
PubMed1991

Biochemical and functional characterization of H(+)-K(+)-ATPase in distal amphibian nephron.

Because proton secretion and K+ reabsorption in the late distal tubule of amphibians are active, we evaluated whether these processes could be mediated by an H(+)-K(+)-ATPase similar to the gastric H(+)-K+ pump and to the K(+)-ATPase previously described in the terminal segments of the mammalian nephron. K(+)-stimulated ATPase activity was detected in microdissected segments of frog and Necturus nephron: its activity was high in the late distal and collecting tubules, whereas it was undetectable in the proximal convoluted tubule and early distal tubule. In frog collecting tubule, K(+)-ATPase had a high affinity for K+ (Km approximately 0.30 mM), was inhibited by vanadate, omeprazole, and the imidazopyridine Sch 28080, and was insensitive to ouabain. Furthermore, in vivo administration of Sch 28080 to anesthetized Necturus induced a significant rise of the steadystate intratubular pH in the late distal tubule, demonstrating that this drug inhibited tubular fluid acidification. It is suggested that K(+)-ATPase present in the terminal segments of amphibian nephron is similar to the gastric H(+)-K+ pump and is involved in urinary acidification.

Adenosine Triphosphatases↗
PubMed1988

Fusion of amphibian proximal convoluted cells into giant cells.

We applied the technique of renal cell fusion to the proximal convoluted tubule of Necturus kidney. Giant fused cells from this segment exhibit stable cell membrane potential, Vm, for several tens of minutes. These cells display a number of electrophysiological properties characteristic of both the apical and basolateral membranes of the proximal tubule. These include 1) significant K+ conductance, 2) no detectable Cl- conductance, 3) apical Na-glucose and Na-amino-acid electrogenic carriers, 4) basolateral ouabain-sensitive Na-K pump activity and 5) basolateral Na/HCO3 cotransport.

Animals↗
PubMed1988

Electrophysiological properties of amphibian late distal tubule in vivo.

This study was undertaken to determine the passive electrophysiological properties of the diffusive barriers of the late distal tubule (LDT) in Necturus. The transepithelial resistance (RT) determined by cable analysis was 1,130 omega.cm2, which puts the LDT in the class of "tight" epithelia. Using two different methods, we did not find significant cell-to-cell electrical coupling. The fractional apical resistance was 0.93, and it did not vary with distance from the current-injecting electrode. Relative permeabilities of K+, Na+, and Cl- during peritubular ion concentration changes were assessed by circuit analysis. The conclusions are as follows. The basolateral cell membrane is highly permeable to K+; its apparent K+ transference number is 0.78. Basolateral chloride transference was very small. Sodium removal from peritubular fluid produced depolarization, suggesting carrier-mediated electrogenic Na+ transport. The high fractional resistance of the apical cell membrane prevented assessment of apical transference numbers. However, Cl- removal from luminal fluid produced cell hyperpolarization; the underlying mechanism has not been established with certainty. The paracellular pathway does not discriminate between Na+, Cl-, and some of their substitutes; it is poorly permeable to gluconate and prefers K+ to Na+.

Algorithms↗
PubMed1988

Effects of muzolimine on the late distal tubule of necturus kidney.

The in vivo effects of the diuretic muzolimine were studied in the late distal tubule of the amphibian Necturus. Conventional and ion-selective microelectrodes were used to determine basolateral membrane potential, intracellular Cl- activity and luminal activities of Cl- and K+. Muzolimine depolarized basolateral membrane potential by about 30 mV in 1 min, in a reversible fashion. We attributed this depolarization to blockade of a K+ conductance, because the effects of muzolimine and barium on the highly K-selective basolateral membrane were not additive. In addition, muzolimine elicited a reversible increase of intracellular Cl- activity from 7.5 +/- 0.5 to 14.5 +/- 2.6 mM (concomitant to the basolateral membrane potential depolarization) and of luminal activities of Cl- from 12.4 +/- 1.5 to 22.3 +/- 2.5 mM, within approximately 1 min; both disturbances relaxed toward control values after withdrawal of the diuretic. That muzolimine increases Cl- activity in both the cell and the lumen of the late distal tubule (Cl- is accumulated in these compartments), indicates that retention of Cl- results from hindrance of the basolateral exit step rather than of apical Cl- uptake. Inasmuch as muzolimine failed to increase the luminal activity of K+, Cl- is believed to accumulate in the lumen as NaCl, not KCl.

Animals↗
PubMed1987

Effects of membrane potential changes on electrical cell-to-cell coupling in proximal tubule.

Single proximal convoluted tubules (PCT) of Necturus kidney were impaled with three microelectrodes in the sequence M1, M2, M3. M1 was used for injecting short DC current pulses, M2 for recording peritubular membrane potential, V, and M3 for injecting longer DC current steps and thereby shifting V to a new baseline potential, V'. We define the p.d. changes at M2 due to M1-induced pulses as delta V and delta V' (for baselines V and V', respectively). Our objective was to test whether delta V' was equal to delta V. The main finding is that when V depolarized by 10 to 80 mV delta V'/delta V remained close to 1.00. Care was taken to ensure that this apparent stability of the pulse ratio was not due to opposite changes of apical and basolateral membrane conductances (g(A) and g(B) respectively), to changes of the sum g(A)+g(B) compensated for by changes of the cell-to-cell junctional conductance, g(j), or to a distortion of the delta V'/delta V ratio as a function of interelectrode distance, masking voltage-dependent changes of cell membrane conductances. Hyperpolarization of V produced gradual electrical uncoupling between cells as V' became increasingly negative, by a mechanism yet to be determined.

Animals↗
PubMed1987

Cell and luminal activities of chloride, potassium, sodium and protons in the late distal tubule of Necturus kidney.

1. Double-barrelled (selective vs. conventional) microelectrodes were used to assess the steady-state activities (a) of the ions Cl-, K+, Na+ and H+ in peritubular blood capillaries (abld) and in cell (acell) and lumen (alum) of the late distal tubule (l.d.t.) of Necturus. 2. a(cell)cl, a(lum)cl and a(bld)cl were 5.5 +/- 0.3, 11.8 +/- 1.0 and 70.5 +/- 0.1 mM, respectively. They were used to compute the chemical potentials for Cl- across the three diffusive barriers of the tissue. Basolateral and apical membrane potentials were -74.3 +/- 1.1 and -60.1 +/- 2.0 mV, respectively (cell negative); the lumen was thus negative with respect to blood, by 13.6 +/- 1.5 mV. The electrochemical potential difference (e.p.d.) for Cl- of 42 mV across the apical membrane opposes Cl- absorption, implying active apical Cl- uptake, since Cl- is known to be absorbed in the l.d.t. Basolateral Cl- exit is favoured by an e.p.d. of 10 mV. 3. a(cell)K, a(lum)K and a(bld)K were 65.8 +/- 0.8, 2.5 +/- 0.1 and 2.5 +/- 0.1 mm, respectively. The electrochemical distribution of K+ indicates that K+ absorption, if present, proceeds against an adverse apical e.p.d. of 18 mV. Basolateral K+ distribution is close to its electrochemical equilibrium, suggesting high K+ permeability at this membrane. 4. a(cell)Na was 9.0 +/- 0.4 mM, a(bld)Na 71.0 +/- 0.3 mM, and a(lum)Na was approximated at about 9 mM. Diffusive Na+ entry from lumen to cell is favoured by an e.p.d. close to 65 mV. Basolateral Na+ exit must be active, since it proceeds against an e.p.d. of 130 mV. 5. Cell, luminal and blood pH were 7.14 +/- 0.03, 6.52 +/- 0.08 and 7.37 +/- 0.04, respectively. The luminal electrochemical potential of H+ is higher than that of cell (by 91 mV) and blood (by 34 mV) indicating that proton secretion into the lumen must be active. 6. The e.p.d. of each ion across the epithelium opposes, by its orientation, the established direction of net transepithelial ion transport, suggesting that the shunt pathway may serve only for back-diffusion.

Absorption↗
PubMed1986

Mechanisms of 1,25(OH)2D3-induced rapid changes of membrane potential in proximal tubule: role of Ca2+-dependent K+ channels.

Eleven different secosteroids or steroids (10(-10) to 10(-8) M) were acutely and reversibly introduced in solutions delivered to the lumen of single proximal tubules of the amphibian Necturus kidney while recording basolateral cell membrane potential Vm. Seven of these molecules (1,25(OH)2D3, 25(OH)D3, 24,25(OH)2D3, 5,6-trans-25(OH)D3, 19-diol-cholesterol, estradiol and testosterone) resulted in changes of Vm (delta Vm) occurring in a few seconds, the largest delta Vm being observed with 1,25(OH)2D3, +6.5 +/- 0.75 mV (n = 19); these seven (seco)steroids, but not the four inactive sterols (vitamin D3, cholesterol, 1 alpha D3 and aldosterone) possess a hydroxyl group on at least one carbon of the C17 to C25 lateral chain of the sterol ring. The delta Vm effect was present in Na+-free or Cl-free media, but it was abolished in HCO3-free media. Depolarization of cell membrane potential by addition of glucose, 11 mM, in luminal perfusion fluid abolished the 1,25(OH)2D3-evoked delta Vm effect, suggesting dependence of the latter on the absolute value of membrane potential. Barium, a blocking agent of K+ conductances, suppressed the 1,25(OH)2D3-evoked delta Vm effect, even when the proper effects of barium of cell membrane potential were canceled by current clamp. Pretreatment with quinine, a putative blocker of Ca2+-dependent K+ channels also abolished the 1,25(OH)2D3-evoked depolarization. Such observations are consistent with the presence of Ca2+-dependent K+ channels at the apical cell membrane of the proximal tubule, these channels being inactivated by 1,25(OH)2D3 and probably by other (seco)steroids.

Animals↗
PubMed1985

Electrochemical profile of K+ and Na+ in the amphibian early distal tubule.

Double-barreled microelectrodes selective to either potassium or sodium were used to determine the transepithelial potential difference (VTE) and the intraluminal activity of potassium (alpha LuK) or sodium (alpha LuNa) in the early distal tubule (EDT) of Triturus waltlii in vivo; luminal activities were compared with the corresponding plasma ion activities, alpha PtK and alpha PtNa. The transepithelial equilibrium potentials for potassium (EK(TE] and sodium (ENa(TE] were computed from the respective transmural chemical distributions: they were used to assess the transepithelial electrochemical potential differences [(V-EK)TE and (V-ENa)TE]. By dividing the raw data into three groups of 30% total tubular length (0-30, 31-60, 61-90%), the following results were obtained. 1) VTE increases from +15 to +20 mV (lumen positive) between the first and second portion of the EDT but remains constant thereafter. 2) The alpha LuK/alpha PtK ratio decreases steadily along the EDT from 1.92 to 1.66 and then to 1.32. 3) The values of alpha LuNa/alpha PtNa in the same three subdivisions are 0.79, 0.44, and 0.45. 4) The (V-EK)TE difference is largely positive along the whole EDT: +32, +33, and +27 mV. 5) The (V-ENa)TE difference declines from +9 mV (first portion) to values statistically not different from zero in the last two thirds of the EDT.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

The effect of a disulfonic acid stilbene on proximal cell membrane potential in Necturus kidney.

The effects of 0.5 mM 4-acetamido-4'-isothiocyano-stilbene-2,2' disulfonic acid on the electrical properties of the peritubular membrane were studied in the proximal tubule of the perfused Necturus kidney. The addition of stilbene isothiocyanate disulfonic acid in peritubular perfusate resulted in an average 4.5 mV hyperpolarization with no detectable changes of peritubular membrane input conductance. The depolarization elicited by high-K media was enhanced by 18% in the presence of stilbene isothiocyanate disulfonic acid, an observation indicating that the inhibitor increased the contribution of potassium to membrane potential, presumably by decreasing anionic permeabilities. The hyperpolarizing effect of stilbene isothiocyanate disulfonic acid was abolished when peritubular bicarbonate was removed from the medium and isoosmotically replaced by chloride. These data suggest that (a) intracellular bicarbonate activity is higher than that predicted from passive distribution, (b) stilbene isothiocyanate disulfonic acid decreases P HCO3, thus hyperpolarizing the membrane, (c) chloride distribution appears to be passive when bicarbonate is removed from the peritubular perfusate. The state of Cl distribution when extracellular bicarbonate is at physiologic concentration cannot be assessed from the present data.

Animals↗
PubMed1978

[Effects of salicylate on the electrical properties of the proximal convoluted tubule of Necturus maculosus].

The effects of peritubular salicylate for chloride substitution were studied in the isolated perfused Necturus kidney. This substitution resulted in changes of cell membrane p.d., varying from tubule to tubule; the withdrawal of the test-anion invariably produced a steep and prolonged depolarization. Exposure of the tissue to salicylate brought about, in addition, electrical uncoupling of junctional membranes, which was not related to concomitant changes of membrane p.d.

Animals↗