[Accumulation of sodium and potassium ions in oocytes of the river lamprey Lampetra fluviatilis during prespawning period].

Accumulation of Na+ and K+ ions in oocytes of the river lamprey Lampetra fluviatilis and their transport across the plasma membrane is realized by two main mechanisms--Na,K-pump and Na,K,Cl-cotransport. At the prespawning period from December to May the intracellular Na+ concentration was observed to increase from 10 to 25 mM and the K+ concentration--from 28 to 45 mM. Results obtained on isolated oocytes with aid of 204Tl radioactive label have shown that contributions of the Na,K-pump and Na,K,Cl-cotransport to potassium accumulations were close until March. In spring, the total K+ inflow almost doubled owing to activation of the Na,K-pump, whereas contribution of Na,K,Cl-cotransport did not change. It seems that an increase of the intracellular content of the main inorganic cations in oocytes resulted in parallel activation of the Na,K-pump and probably of Na/H-exchange. The biological significance of activation of these mechanisms of ion transport at the prespawning period might be due to a necessity of accumulation of Na+ and K+ ions in concentrations optimal for subsequent embryonic development.

[Transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis].

Mechanisms of transport of monovalent thallium across the membrane of oocyte of the lamprey Lampetra fluviatilis were studied by using 204Tl. Transport of Tl+ in lamprey oocytes has been shown to be realized by at least two pathways: through Na/K-pump and by the mechanism of Na,K,Cl-cotransport. In the standard Ringer solution (mM): 4 KCl, 140 NaCl, 0.5 CaCl2, 5 glucose, 10 Tris-HCl--in the presence of oubain, the coefficient of the 204Tl stationary distribution (cell/medium) was within the range of 2.3-2.5, while the time necessary to reach its 50 % value amounted to 40-5 min at 20 degrees C. In potassium-free media, transport of 204Tl via Na/K-pump was described by simple kinetis with saturation and was characterized by the value V(max) = 520 pmol/(cell x h) and K(M) = 0.3 mM. In the presence of 4 mM K+ and 0.1 mM/l Tl+, the oubain-sensitive Tl+ flow decreased to 75 pmol/(cell x h). At activation of the mechanism of Na,K,Cl-cotransport by the outer Na+ (in Na-NMDG media of different composition) the total inflow of Tl+ reached 193 +/- 20 pmol/(cell x h), while the butamenide-sensitive component--119 +/- 12 pmol/(cell x h) with K(M) for Na+ about 20 mM. In the incubation media with variable concentration of chloride ions (replacement of Cl- by NO3(-)) the total Tl+ flow reached 220 +/- 21, while via the mechanisms of Na,K,Cl-cotransport--87 +/- 8 pmol/(cell x h). Under our experimental conditions, mechanisms of active transport and Na,K,Cl-cotransport accounted for 94% of the Tl+ inflow. The potassium channels that usually are also permeable to monovalent thallium ions were not revealed.

Effects of fluoride and vanadate on K+ transport across the erythrocyte membrane of Rana temporaria.

K-Cl cotransport activity in frog erythrocytes was estimated as a Cl- -dependent component of K+ efflux from cells incubated in Cl- - or NO3- -containing medium at 20 degrees C. Decreasing the osmolality of the medium resulted in an increase in K+ efflux from the cells in a Cl- medium but not in an NO3- medium. Treatment of red cells with 5 mM NaF caused a significant decrease (approximately 50%) in K+ loss from the cells in iso- and hypotonic Cl- media but only a small decrease in K+ loss in isotonic NO3- medium. Addition of 1 mM vanadate to an isotonic Cl- medium also led to a significant reduction in K+ efflux. Similar inhibitory effects of NaF and vanadate on K+ efflux in a Cl- medium, but not in an NO3- medium were observed when the incubation temperature was decreased from 20 to 5 degrees C. Thus, under various experimental conditions, NaF and vanadate inhibited about 50% of Cl- -dependent K+ efflux from frog red cells probably due to inhibition of protein phosphatases. Cl- -dependent K+ (86Rb) influx into frog erythrocytes was nearly completely blocked (approximately 94%) by 5 mM NaF. In a NO3- medium, K+ influx was mainly mediated by the Na+,K+ pump and was unchanged in the presence of 5 mM NaF, 0.03 mM Al3+ or their combination. These data indicate that G proteins or cAMP are not involved in the regulation of Na+,K+ pump activity which is activated by catecholamines and phosphodiesterase blockers in these cells.

Kinetics of K-Cl cotransport in frog erythrocyte membrane: effect of external sodium.

In frog red blood cells, K-Cl cotransport (i.e., the difference between ouabain-resistant K fluxes in Cl and NO(3)) has been shown to mediate a large fraction of the total K(+) transport. In the present study, Cl(-)-dependent and Cl(-)-independent K(+) fluxes via frog erythrocyte membranes were investigated as a function of external and internal K(+) ([K(+)](e) and [K(+)](i)) concentration. The dependence of ouabain-resistant Cl(-)-dependent K(+) ((86)Rb) influx on [K(+)](e) over the range 0-20 mm fitted the Michaelis-Menten equation, with an apparent affinity (K(m)) of 8.2 +/- 1.3 mm and maximal velocity (V(max)) of 10.4 +/- 1.6 mmol/l cells/hr under isotonic conditions. Hypotonic stimulation of the Cl(-)-dependent K(+) influx increased both K(m) (12.8 +/- 1.7 mm, P < 0.05) and V(max) (20.2 +/- 2.9 mmol/l/hr, P < 0.001). Raising [K(+)](e) above 20 mm in isotonic media significantly reduced the Cl(-)-dependent K(+) influx due to a reciprocal decrease of the external Na(+) ([Na(+)](e)) concentration below 50 mm. Replacing [Na(+)](e) by NMDG(+) markedly decreased V(max) (3.2 +/- 0.7 mmol/l/hr, P < 0.001) and increased K(m) (15.7 +/- 2.1 mm, P < 0.03) of Cl(-)-dependent K(+) influx. Moreover, NMDG(+) Cl substitution for NaCl in isotonic and hypotonic media containing 10 mm RbCl significantly reduced both Rb(+) uptake and K(+) loss from red cells. Cell swelling did not affect the Na(+)-dependent changes in Rb(+) uptake and K(+) loss. In a nominally K(+)(Rb(+))-free medium, net K(+) loss was reduced after lowering [Na(+)](e) below 50 mm. These results indicate that over 50 mm [Na(+)](e) is required for complete activation of the K-Cl cotransporter. In nystatin-pretreated cells with various intracellular K(+), Cl(-)-dependent K(+) loss in K(+)-free media was a linear function of [K(+)](i), with a rate constant of 0.11 +/- 0.01 and 0.18 +/- 0.008 hr(-1) (P < 0.001) in isotonic and hypotonic media, respectively. Thus K-Cl cotransport in frog erythrocytes exhibits a strong asymmetry with respect to transported K(+) ions. The residual, ouabain-resistant K(+) fluxes in NO(3) were only 5-10% of the total and were well fitted to linear regressions. The rate constants for the residual influxes were not different from those for K(+) effluxes in isotonic ( approximately 0. 014 hr(-1)) and hypotonic ( approximately 0.022 hr(-1)) media, but cell swelling resulted in a significant increase in the rate constants.

Inhibition and stimulation of K+ transport across the frog erythrocyte membrane by furosemide, DIOA, DIDS and quinine.

Frog erythrocytes were incubated in iso- or hypotonic media containing 10 mmol/l Rb+ and 0.1 mmol/l ouabain and both Rb+ uptake and K+ loss were measured simultaneously. Rb+ uptake by frog red cells in iso- and hypotonic media was reduced by 30-60% in the presence of 0.01-0.1 mmol/l [(dihydroindenyl)oxy] alkanoic acid (DIOA) or 0.5-1.0 mmol/l furosemide. Furosemide inhibited K+ loss from frog erythrocytes incubated in hypotonic media but did not affect it in isotonic media. DIOA at a concentration of 0.05 mmol/l inhibited of K+ loss from frog erythrocytes in both iso- and hypotonic media. At the concentrations of 0.01 and 0.02 mmol/l DIOA significantly suppressed K+ loss in a K+-free chloride medium but not in a K+-free nitrate medium. The Cl(-)-dependent K+ loss was completely blocked at a concentration of 0.1 mmol/l DIOA and the concentration required for 50% inhibition of K-Cl cotransport was approximately 0.015 mmol/l. However, the inhibitory effect of DIOA on K-Cl cotransport was masked by an opposite stimulatory effect on K+ transport which was also observed in nitrate medium. Quinine in a concentration of 0.2-1.0 mmol/l was able to inhibit Rb+ uptake and K+ loss only in hypotonic media. In isotonic media, quinine produced a stimulation of Rb+ uptake and K+ loss. A three to five-fold activation of Rb+ uptake and K+ loss was consistently observed in frog erythrocytes treated with 0.05-0.2 mmol/l 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). In contrast, another stilbene derivative 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid (SITS) had no effect on K+ transport in the cells. Thus, of these drugs tested in the present study only DIOA at low concentrations may be considered as a selective blocker of the K-Cl cotransporter in the frog red blood cells.

Differential effects of glycolytic and oxidative metabolism blockers on the Na-K pump in erythrocytes of the frog, Rana temporaria.

This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-Cl cotransport, the frog erythrocytes were incubated in nitrate media at 20 degrees C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na(+)-K(+)-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1-2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01-0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na(+)-K(+)-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate "membrane-associated" ATP pool.

Temperature effects on ion transport across the erythrocyte membrane of the frog Rana temporaria.

Unidirectional K+ and Na+ influxes in the frog erythrocytes incubated in Cl- or NO(3)- media with 2.7 mM K+ were measured using 86Rb and 22Na as tracers. K+ influx was inhibited by 35-55% in the presence of 0.2-1.0 mM furosemide but it was unaffected by 0.1-0.2 mM bumetanide. Furosemide at a concentration of 0.5 mM had no effect on K+ loss from the frog red cells incubated in a nominally K(+)-free medium. Together with our previous studies the data support the existence of K-Cl cotransport and the absence of Na-K-2Cl cotransport in the frog erythrocyte membrane. Cell cooling from 20 to 5 degrees C caused a decrease in K+ influx and K+ efflux via the K-Cl cotransporter (3.2- and 3.7-fold, respectively) giving an apparent energy of activation (EA) of about 60 kJ/mol and Q10 value of 2.5. Only small decline (approximately 30%) in the ouabain-sensitive K+ influx was found as temperature was changed from 20 to 5-10 degrees C. Low values of Q10 (approximately 1.5) and EA (27.3 kJ/mol) were obtained for passive K+ influx in the frog erythrocytes (ouabain-insensitive in NO(3)- medium) at temperature within 5-20 degrees C. However, the temperature coefficients were greater for passive Na+ influx and passive K+ efflux (Q10 approximately 2.4-2.5 and EA approximately 56-58 kJ/mol). The temperature dependence of all ion transport components displayed discontinuities showing no changes at temperature between 5 and 10 degrees C. Thus, cooling of the frog red cells is associated with a greater decrease of Na+ influx and K+ efflux than passive and active K+ influx. These data indicate that the preservation of a relative high activity of the Na,K-pump during cell cooling and also the temperature-induced changes in the K-Cl cotransport activity and ion passive diffusion contribute to maintenance of ion concentration gradients in the frog erythrocytes at decreased temperature.

Volume regulation in red blood cells of the frog Rana temporaria after osmotic shrinkage and swelling.

To investigate the volume regulatory ability of frog erythrocytes, we studied water and ion contents in the cells incubated in isosmotic (210 mOsm) and anisosmotic media. The cell volume decreased to 63% of the control in a hypertonic medium (+200 mM sucrose) and then gradually recovered to approximately 81% within 2 h. The regulatory volume increase (RVI) was associated with the accumulation of intracellular Na+, whereas the intracellular K+ content remained the same. Na+ (22Na) influx into the red cells increased from 1.1 +/- 0.2 in an isotonic medium to 12.4 +/- 0.4 mmol/(1 cells.h) in a hypertonic medium. The changes in both intracellular water and Na+ contents and the Na+ influx were blocked by 1 mM amiloride. Thus, it is likely that RVI observed in frog erythrocytes under hyperosmolar conditions is due to the activation of Na+/H+ exchanger. The exposure of the red cells to hypotonic media (approximately 160 mOsm) resulted in an increase of cell volume to 138% of the control value. The swollen cells gradually recovered their volume (on average by 69% in 2 h) due to losses in intracellular K+ and water. K+ (86Rb) influx in erythrocytes in the hypotonic medium (3.4 +/- 0.3 mmol/l.h) was significantly greater than that in the isotonic medium (1.4 +/- 0.03 mmol/l.h). K+ loss from frog erythrocytes incubated in a K(+)-free medium was significantly stimulated by hypotonic swelling, despite a decrease in the intracellular in K+ concentration. The regulatory volume decrease (RVD) and the changes in K+ transport across the red cell membrane in a hypotonic medium were completely abolished after C1- replacement by NO3-. The Cl(-)-dependent K+ loss in a hypotonic medium was partially (approximately 50%) inhibited by 0.05 mM DIOA ([(dihydroindenyl)oxy]alkanoic acid) and 0.5 mM quinine, but it was unaffected by 0.05 mM bumetanide. These results indicate that the swelling-activated K+, Cl- cotransport is involved in the RVD in frog erythrocytes.

Activation of the Na(+)-K+ pump in frog erythrocytes by catecholamines and phosphodiesterase blockers.

K+ and Na+ influx into frog erythrocytes incubated in standard saline was studied using 86Rb and 22Na as tracers. 10 microM isoproterenol (ISP) produced a significant increase in K+ influx for the first 15 min, which was sustained during the entire 60 min of cell incubation. Treatment of red cells with the phosphodiesterase (PDE) blockers theophylline (THEO, 1 and 5 mM) or 3-isobutyl-1-methylxanthine (IBMX, 0.5 mM) was also accompanied by an enhancement in K+ influx. A distinct additive effect on K+ influx into red cells was found when ISP and THEO or IBMX were added together. The increase in K+ transport induced by ISP plus IBMX was totally abolished by pretreatment of red cells with 0.1 mM ouabain. The ouabain-sensitive K+ influx in frog erythrocytes was elevated in the presence of ISP plus IBMX to 2.05 +/- 0.45, as compared with the control level of 0.39 +/- 0.11 mmol/L cells/hr (P < 0.001). Similar effects of ISP and IBMX on K+ influx were observed after chloride was replaced by nitrate. A dose-related increase in K+ influx into frog erythrocytes was observed at ISP concentrations of 10(-8)-10(-6) M, with a half-maximal stimulatory concentration of approximately 0.02 microM. The effects of ISP (10(-8)-10(-5) M) on K+ transport were completely abolished with 10 microM of the beta-adrenergic blocker propranolol, but alpha-adrenergic antagonists (phentolamine, prazosin, and yohimbine) did not alter the ISP-induced increase in K+ influx. The drugs tested had no effect on 22Na influx in frog red cells, but ISP produced a small decline (13%) in intracellular Na+ concentration. Thus, our study indicates that catecholamines and PDE blockers enhance K+ (86Rb) transport in frog erythrocytes mediated by Na(+)-K+ pump activity. The frog erythrocyte membrane may serve as a convenient model to investigate the hormonal modulation of the Na(+)-K+.

[Potassium ion transport in the erythrocytes of the frog Rana ridibunda]. / Transport ionov kaliia v éritrotsitakh liagushki Rana ridibunda.

The K+ transport in isolated erythrocytes from the frog Rana ridibunda has been studied using 86Rb as a tracer at the temperature of 18-20 degrees C. At physiological K+ concentration (3 mM) in C1- medium ouabain and furosemide inhibited K+ influx by approximately 42 and 47%, respectively. Furosemide had no effect on the Na+ (22Na) transport under the same conditions. The replacement of C1- by NO-3 in medium resulted in significant decrease of total K+ influx, which was not inhibited by furosemide. The ouabain- and furosemide-sensitive components of K+ influx hyperbolically increased as a function of external K+e concentration (1-90 mM) in C1- medium and calculated values of Vmax were 2.2 +/- 0.14 and 5.8 +/- 1.2 mmol/1 cells/h, respectively. K+ influx into frog erythrocytes in the presence of both ouabain and furosemide in NO-3 medium was significantly lower compared with C1- medium. C1--dependent, furosemide-insensitive component of K+ influx increased in a saturable fashion in the range of K+e concentration from 1 to 90 mM. Residual component of K+ transport in NO3 medium in the presence of the blockers was a linear function of K+e concentration and is characterized by the constant of K+ influx rate equal to 0.028 +/- 0.002 h-1. The data obtained indicate that in parallel with the Na, K-pump specific K, C1-transporter participates in K+-transport in the frog erythrocyte membrane. The latter mechanism was only partially blocked by 1 mM furosemide.

Potassium transport in red blood cells of frog Rana temporaria: demonstration of a K-Cl cotransport.

Pathways of K+ movement across the erythrocyte membrane of frog Rana temporaria were studied using 86Rb as a tracer. The K+ influx was significantly blocked by 0.1 mmol.l-1 ouabain (by 30%) and 1 mmol.l-1 furosemide (by 56%) in the red cells incubated in saline at physiological K+ concentration (2.7 mmol.l-1). Ouabain and furosemide had an additive effect on K+ transport in frog red cells. The ouabain-sensitive and furosemide-sensitive components of K+ influx saturated as f(K+)e with apparent Km values for external Ke+ concentration of 0.96 +/- 0.11 and 4.6 +/- 0.5 mmol.l-1 and Vmax of 0.89 +/- 0.04 and 2.8 +/- 0.4 mmol.l cells-1.h-1, respectively. The residual ouabain-furosemide-resistant component was also a saturable function of Ke+ medium concentration. Total K+ influx was significantly reduced when frog erythrocytes were incubated in NO3- medium. Furosemide did not affect K+ transport in frog red cells in NO3- media. At the same Ke+ concentration the ouabain-furosemide-insensitive K+ influx in Cl- medium was significantly greater than that in NO3- medium. We found no inhibitory effect of 1 mmol.l-1 furosemide on Na+ influx in frog red cells in Cl- medium. K+ loss from the frog erythrocytes in a K(+)-free medium was significantly reduced (mean 58%) after replacement of Cl- with NO3-. Furosemide (0.5 mmol.l-1) did not produce any significant reduction in the K+ loss in both media. The Cl(-)-dependent component of K+ loss from frog red cells was 5.7 +/- 1.2 mmol.l-1.h-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Highly selective blockade of the frog skin sodium channels by monovalent copper cations.

.5 mM Cu+ added to the mucosal side of frog skin caused rapid reversible inhibition of short-circuit current while no effect of Cu+ could be observed at the serosal side. In both cases Cu2+ was reduced to Cu+ by adding 10 mM ascorbic acid. Cu+ being similar to Na+ both in charge and crystal radius (0.096 and 0.095 nm, respectively) appears to block Na+ channels in the apical membrane. Cu2+ being of a smaller size (crystal radius 0.072 nm) was ineffective at the mucosal side causing only a rather slow irreversible inhibition of Na+ transport when added to the serosal bathing solution.

[Effect of energy metabolism on the ionic selectivity of the K+ center of the Na+/K+ pump in the skin of the frog]. / Vliianie énergeticheskogo metabolizma na ionnuiu selektivnost' K+-tsentra Na+/K+-nasosa v kozhe liagushki.

The uptake of Rb+ and Tl+ in frog skin incubated in saline containing 86Rb and 204Tl was studied. The ratio of ouabain-sensitive influxes Tl+/Rb+ = 2. Inhibition of the unidirectional transport of Na+ by rotenone or by Tl+ was not accompanied by the total decrease in the ouabain-sensitive uptake of Rb+ and Tl+. A substantial number of the Na+/K+ pumps continued to operate to maintain the intracellular ion homeostasis. The Tl+/Rb+ ratio of the cation influxes via pumps of this group was about twice as high compared to the control values. A coexistence of the two forms of Na+/K+ pumps in frog skin epithelium was postulated. They differ in both ion selectivity and the source of energy (respiration or glycolysis). The Tl+/Rb+ ratio of the ouabain-insensitive fluxes seems to be independent of the energetic metabolism.