Aerobic and anaerobic metabolism in smooth muscle cells of taenia coli in relation to active ion transport.

1. The O2 consumption and lactic acid production of the guinea-pig's taenia coli have been studied in relation to the active Na-K transport, in order to estimate the ratio: active Na extrusion/active K uptake/ATP hydrolysis. 2. By applying different procedures of partial metabolic ingibition, it was found that a reactivation of the active Na-K transport in K-depleted tissues could occur in an anaerobic medium, provided glucose was present and in an aerobic medium free of added metabolizable substrate. The active Na-K transport was rapidly blocked in an anaerobic-substrate free medium. 3. Readmission of K to K-depleted tissues under aerobic conditions stimulates both O2 consumption and lactic acid production. While the O2 consumption creeps up slowly and requires 50 min to reach control values, the aerobic lactic acid production increases to a maximum within 10 min and decreases again during the next 50 min to its steady-state value. 4. A reactivation of the Na-pump in K-depleted cells in a N2-glucose medium causes an immediate increase of the lactic acid production, which decreases to its control value after 60 min. The maximal increase in anaerobic lactic acid production during reactivation of the Na-K pump is a function of [K]O. The system can be cescribed with first order kinetics having a Vmax = 0-72 mumole.g-1 f. wt. min-1 and a Km = 1-1 mM. 5. By varying the glucose concentration of [K]O during reactivation of the Na-K pump, different Na-K pumping rates can be obtained. The ratios net Na extrusion/ATP or net K accumulation/ATP amount to -1-32 +/- 0-19 (36) and 1-02 +/- 0-11 (36), in the experiments with different glucose concentrations. Taking into account the interference by net passive fluxes, one can estimate a ratio:active Na transport/active K transport/ATP, of 1-7/0-8/1. This ratio is not very different from the values observed in other tissues.     

Kinetics of Thyroxine (T4) and Triiodothyronine (T3) Transport in the Isolated Rat Heart

The dynamics and kinetics of thyroid hormone transport in the isolated rat heart were examined using the modified unidirectional paired tracer dilution method. The uptake of (125)I-thyroxine ((125)I-T(4)) and (125)I-triiodothyronine ((125)I-T(3)) from the extracellular space into heart cells was measured relative to the extracellular space marker (3)H-mannitol. The thyroid hormone maximal uptake was 54.4 % for (125)I-T(4) and 52.15 % for (125)I-T(3). The thyroid hormone net uptake was 25.69 % for (125)I-T(4) and 25.49 % for (125)I-T(3). Backflux from the intracellular space was 53.17 % for (125)I-T(4) and 61.59 % for (125)I-T(3). In the presence of unlabelled thyroid hormones, (125)I-T(4) and (125)I-T(3) maximal uptakes were reduced from 10.1 to 59.74 % and from 34.6 to 65.3 %, respectively, depending on the concentration of the unlabelled hormone, suggesting a saturable mechanism of the thyroid hormone uptake by the heart cells, with K(m(T4))= 105.46 microM and the maximal rate of (125)I-thyroid hormone flux from the extracellular space to heart cells (V(max(T4))) = 177.84 nM min(-1) for (125)I-T(4) uptake, and K(m(T3)) = 80.0 microM and V(max(T3)) = 118.5 nM min(-1) for (125)I-T(3) uptake. Experimental Physiology (2001) 86.1, 13-18.     

Potassium flux in smooth muscle of frog stomach.

In frog stomach muscle fibers, normal steady-state K flux, estimated directly from 42K uptake, was 0.17 pmol/cm2 per s at 5 degrees C and 0.63 pmol/cm2 per s at 15 degrees C. Influx characteristics were studied at 5 degrees C, where backflux and diffusional delay effects are minimized. Steady-state K influx was a saturating function of external [K] over the range 0.25-11 mM [K]o; influx at normal and higher [K]o did not differ significantly. Na loading (in K-free or low K solution) strongly stimulated influx, which showed altered saturation kinetics; maximal K influx was a quasilinear function of internal [Na]. Ouabain (10(-4) M) reduced normal and stimulated K influx markedly. Ethacrynic acid (10(-3) M) caused net K loss and Na gain, but increased K influx fourfold; ouabain inhibited the stimulated influx by 50%. These results indicate that K influx depends mainly on cycling of the Na-K pump and is normally limited by Na efflux. Ethacrynic acid may stimulate another mode of pump operation, K-K exchange, and uncouple the normal operation.     

Magnesium net fluxes and distribution in rabbit myocardium in irreversible contracture

Distribution of magnesium (Mg) in heart muscle was studied by measuring fluxes of Mg and transmembrane potentials as a function of perfusate [Mg2+] after a massive increase in permeability of the sarcolemma was induced in the Langendorff prepared heart from the Nembutal-anesthetized rabbit. After onset of 0 mM [Ca2+] perfusion which produced excitation-contraction (E-C) uncoupling and mechanical arrest, action potentials recorded from subepicardial cells showed an increase in duration and decrease in amplitude, which progressed until no transmembrane potentials could be observed. Restoration of physiological salt solution perfusion after 15 min of [Ca2+]-free perfusion caused an irreversible contracture that was associated with 1) efflux of potassium (K) and myoglobin, 2) perfusate [Mg2+]-dependent flux of Mg, and 3) transmembrane potentials of 0 mV. The magnitude of net efflux of K and myoglobin during contracture was unaffected by perfusate [Mg2+]. During the first 2 min of contracture, net efflux of Mg (mumoles per gram wet muscle +/- SE) was 1.37 +/- 0.09 and 0.48 +/- 0.19 during 0 mM and 2.5 mM [Mg2+] perfusion, respectively; but a net influx of 0.56 +/- 0.23 occurred during 5 mM [Mg2+] perfusion. Total sarcoplasmic [Mg] may correspond to perfusate [Mg2+] of 3.6 mM, which was found by interpolation to prevent any net flux of Mg during contracture. 3.6 mM may, therefore, represent the upper limit of the intracellular free-ionized Mg concentration in rabbit heart.     

The secretion of alkali metal ions by the perfused cat pancreas as influenced by the composition and osmolality of the external environment and by inhibitors of metabolism and Na+, K+-ATPase activity

1. The secretion of sodium, potassium and lithium has been studied in the isolated cat pancreas, perfused with bicarbonate buffered saline solutions of varying composition and osmolality, and stimulated maximally with secretin.2. Under isosmolal conditions, when perfusate sodium chloride was replaced by sucrose, sodium secretion and potassium secretion were directly related to perfusate sodium concentration, [Na](p).3. When osmolality was varied by increasing or decreasing perfusate sodium chloride concentration, the secretion of sodium and of potassium were maximal at [Na](p) of about 120 and 80 mM respectively.4. At a given [Na](p), sodium secretion was greater under hypo-osmolal conditions than under isosmolal conditions.5. When potassium concentration was varied over the range 0-130 mM under isosmolal conditions, by adjusting perfusate NaCl concentration, the secretion of potassium and of sodium were maximal at [K](p) of about 50 and 10 mM respectively. Water flux was maximal at a [K](p) of 10-15 mM. The concentration of potassium in the secretion was almost identical with that in the perfusate over the whole concentration range.6. Replacement of perfusate sodium by lithium reduced the volume of secretion, though a small secretion was maintained even in the complete absence of sodium. The concentration of lithium in the secretion was generally slightly greater than that in the perfusate.7. Omission of potassium from the perfusate reduced secretion by about 65%. Rubidium was a complete substitute for potassium; caesium was not.8. Energy for secretion is derived largely from oxidative phosphorylation. Secretion was reduced by more than 90% under anaerobic conditions and in the presence of dinitrophenol or cyanide. Removal of glucose from the perfusate reduced secretion by more than 50% within 30 min; lactate was a complete substitute for glucose.9. Ouabain, ethacrinic acid and frusimide, known inhibitors of Na(+), K(+)-ATPase activity, all inhibited pancreatic electrolyte secretion.10. The observations are interpreted with reference to the nature of active transport processes involved in pancreatic electrolyte secretion.     

Ouabain-sensitive ion fluxes in the smooth muscle of the guinea-pig''s taenia coli.

1. Tissues with raised intracellular Na levels, produced by incubation in K-free media, were used throughout. The uptake of 42K by these Na-loaded tissues was followed for 10 min in the presence and absence of 1-37 X 10(-4) M ouabain, this being sufficient to inhibit Na pumping maximally. Subtraction of the uptake seen in the presence from that seen in the absence of ouabain gave estimates of the pumped ouabain-sensitive K uptake. 2. In Na-free (MgCl2) medium this depended on the [K]0 in a sigmoidal fashion with a half maximal [K]0 for activation of some 4mM. The maximal uptake of K was 3 m-mole/kg.min corresponding to a transmembrane flux of some 12-5 p-mole. cm-2.sec-1. 3. In the presence of Na the K activation curve became more obviously sigmoid and higher concentrations of K were needed to achieve a given active K influx. The results were well fitted by assuming that Na and K competed for two identical, non-interacting sites on the external pump face. 4. Addition of K during the efflux of 24Na into a Na-free (MgCl2) medium led to an increased rate of tracer loss. The magnitude of this increase depended on the [K] used in a hyperbolic fashion and it was abolished by addition of ouabain. The [K] causing half-maximal activation of ouabain-sensitive Na efflux was in the order of 1-2 mM. 5. When the [K] in the uptake media was 1-5 mM; Na, Li, Rb and Cs all inhibited ouabain-sensitive K uptake, the order of effectiveness being Rb greater than Cs greater than Na greater than Li. With a E1TKA10 OF 0-15 MM low concentrations of Cs and Rb were shown to stimulate K uptake. Such an effect is predicted by assuming two ion binding sites on the pump's outer face, and that the pump can translocate mixtures of K and either Rb or Cs...     

Ouabain-mediated sodium uptake and bile formation by isolated perfused rat liver.

Ouabain exhibits a dose-dependent choleretic effect in the isolated perfused rat liver. Its uptake from the perfusate into the liver is maintained against a concentration gradient and becomes clearly saturated at higher perfusate concentrations. A low extracellular sodium concentration inhibits the rate of ouabain transfer into liver cells, resulting in a marked decrease of the maximal transport rate. Dibucaine completely abolishes the uptake of the glycoside by the isolated liver. Determination of Na-22 tracer fluxes suggests that ouabain uptake is accompanied by a net flux of sodium into the cell, which seems to be due to a cotransport of sodium with ouabain rather than to the inhibition of the sinusoidal Na+ -K+ -ATPase. Sodium introduced into the cell in this way apparently is extruded into the bile canaliculi. The increase of isotonic bile flow, which is simultaneously observed, points to a dilution of the canalicular sodium gradient by water and electrolytes through an intercellular pathway. Our results present further evidence that bile secretion is controlled by transcellular sodium movements.     

Effect of pH on ionic exchange and function in rat and rabbit myocardium.

The effects of pH variation on ionic exchange and mechanical function were studied in the arterially perfused rat and rabbit septa. The pH and PCO2 of the control perfusate were 7.40 and 39 mmHg, respectively. In the rabbit septum a metabolic acidosis (pH equals 6.82, PCO2 equals 39 mmHg) caused a loss of 16% of control tension in 12 min. Na+ and K+ exchange were unaltered. A comparable respiratory acidosis (pH equals 6.81, PCO2 equals 159 mmHg) caused a 51% loss of tension in 2 min. Na+ exchange was unaltered but K+ efflux fell from 8.9 +/- 0.6 (mean +/- SE) to 4.9 +/- 0.3 mmol/kg dry wt per min (P less than 0.001, n equals 10). A net gain of K+ of 16.9 +/- 1.7 (n equals 14) mmol/kg dry wt occurred and was attributable to a delayed fall in K+ influx relative to efflux over 15 min. The net gain could not be mimicked by epinephrine administration or blocked by propranolol and was absent in the beating rat septum and the quiescent rabbit septum. These results suggest that the net uptake of K+, which appears to be dependent on a period of depolarization, and the changes of contractility are controlled by the H+ ion concentration at a cellular site whose exchange with the extracellular space is characterized by a considerable restriction of diffusion. Changes of contractility are not related to the net uptake of K+.     

Studies on terminal differentiation of rat renal proximal tubular cells in culture: ouabain-sensitive K and Na transport

We have studied the ontogeny of Na-K ATPase-mediated Na and K transport in rat renal proximal tubular cells using electron probe analysis. The cells were cultured from kidneys of 10-day-old, young (Y), and 40-day-old, adult (A) rats. Before an experiment cells were Na-loaded and K-depleted by incubation in K-free medium. The maximum rate of ouabain-sensitive Na and K transport was measured after reactivating the Na-K pump by transferring the cells from K-free medium to medium containing 5 mM K. In cells cultured for 2 days, ouabain-sensitive Na and K net initial transport rates were significantly higher in A than in Y cells. Between 2 and 4 days in culture there was a significant decrease in ouabain-sensitive Na and K transport rates in both Y and A cells. From 2 to 4 days of culture there was, in Y but not in A cells, a significant decrease in K/Na ratio. The decrease in K/Na ratio was due to a significant increase in Na content. After incubation in K-free medium, net intracellular solute accumulation was observed in A and Y cells cultured for 4 days but not in A and Y cells cultured for 2 days. In conclusion, maximal Na- and K-pump-mediated transport increases during terminal differentiation. This increase can be measured in cells cultured for 2 days. With longer time in culture, Na-K pump activity decreases and the difference between A and Y cells is not measurable.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of hematocrit on oxygenation of the isolated perfused rat liver

The isolated perfused rat liver is used ubiquitously for metabolic and endocrine studies of hepatic function, yet few data are available regarding the inadequacy of the oxygenation of such preparations. Moreover, the isolated rat liver is usually deprived of its arterial supply and perfused via the hepatic portal vein with low-hematocrit or cell-free solutions. To investigate the efficacy of the oxygen supply, we determined the effect of hematocrit on the relation between oxygen consumption and perfusate flow. We then attempted to define a hematocrit at which hepatic oxygenation was maximal. Livers of male rats anesthesized with pentobarbital sodium were perfused via the portal vein with fresh canine red blood cells suspended in Krebs-Ringer-bicarbonate buffer. Perfusions were carried out at various flow rates, and the relation between perfusate flow and oxygen uptake was determined. At flow rates above 100 ml X min-1 X 100 g liver-1, oxygen uptake was independent of flow but below that value was flow limited, regardless of whether the hematocrit was 10, 20, or 40%. To determine the optimal hematocrit for hepatic oxygen uptake, hepatic portal venous and hepatic venous pressures were held at 10 and 0 mmHg, respectively. The hematocrit was lowered in steps from 80 to 10%. Blood flow increased exponentially as hematocrit fell while oxygen uptake increased to a maximum at approximately 20%. It is concluded that an hematocrit of approximately 20% provides the optimal combination of blood flow and oxygen-carrying capacity while maintaining physiological perfusion pressures, e.g., 10 mmHg.     

Effects of the Potassium Channel Blocker Barium on Sodium and Potassium Transport in the Rat Loop of Henle In Vivo

In vitro evidence suggests that the 'recycling' of K(+) ions through luminal K(+) channels in the thick ascending limb of the loop of Henle (TALH) is essential for the normal operation of the luminal Na(+)-K(+)-2Cl(-) co-transporter. In the present study these channels were investigated in vivo by perfusing superficial loops of Henle in anaesthetised rats with and without the K(+) channel blocker barium. Using a standard perfusate, intraluminal barium (5 mmol l(-1)) reduced sodium reabsorption (J(Na)) from 1887 +/- 50 to 1319 +/- 53 pmol min(-1) (P < 0.001). When the experiment was repeated using a low-Na(+) perfusate, designed to inhibit reabsorption in the pars recta (the initial segment of the loop of Henle), a similar reduction in J(Na) was observed (from 698 +/- 47 to 149 +/- 23 pmol min(-1), P < 0.001), strongly suggesting that the effect of barium is localised to the TALH. The magnitude of the reduction in J(Na) during blockade of K(+) channels confirms the importance of K(+) recycling in facilitating Na(+) reabsorption in the TALH in vivo. However, the reduction in J(Na) was not associated with a fall in the K(+) concentration of the fluid collected at the early distal tubule. When bumetanide, an inhibitor of the Na(+)-K(+)-2Cl(-) co-transporter, was included in the low-Na(+) perfusate, net K(+) secretion was observed. Addition of barium to this perfusate reduced, but did not abolish, the secretion, suggesting that bumetanide-induced K(+) secretion results partly from paracellular transport. Experimental Physiology (2001) 86.4, 469-474.     

Ionic transport in individual renal epithelial cells from adult and young rats

Renal epithelial cells were isolated from the outermost superficial cortex of adult and young rats. The cells, likely of proximal origin, were plated on silicon pieces, and cultured during 1-3 days. Intracellular content and concentrations of K, Na, Cl, and P, and the kinetics of change in intracellular content, after inhibition of Na-K ATPase by incubation with ouabain or in K-free medium, were measured in individual cells in small populations using electron probe analysis. In control medium, concentrations in mM were approximately: K, 130; Na, 15; Cl, 28; P, 140. After 6 h inhibition of Na-K ATPase, cells exchanged all K for Na, and the intracellular Na concentration increased to 139 mM in K-free medium. The Cl concentration increased at most to 46 mM. The sum of intracellular K + Na + Cl did not increase more than 25% after 24 h incubation in K-free medium. There were no differences in intracellular K, Na, and Cl for adult and young rat cells in similar conditions. The half-times of K efflux and Na influx after inhibition of Na-K ATPase measured in adult rat were approximately 16-20 min. In the absence of serum, in K-free medium, the half-times of K efflux and Na influx in young rat cells were approximately 30 min, significantly higher than the half-time in the presence of serum, and with ouabain, being approximately 13 min. Histograms of distributions of K and Na content showed that the cells behaved as a single functional population. Ouabain Ki was estimated to be 10(-4) M. After 24 h preincubation in K-free medium, when returned in 5 mM K-containing medium, adult rat cells recovered rapidly normal intracellular K and Na concentrations. Using this approach, expression of the kinetics ionic transport properties of renal epithelial cells during development, and the hormonal influences on terminal differentiation may be studied.     

Effect of thyroid hormone and serum on the development of Na+, K+-adenosine triphosphatase and associated ion fluxes in cultures from rat brain

The effect of culture conditions, serum supplementation or chemically defined medium and the influence of thyroid hormone were studied on the development of the Na+, K+-adenosine triphosphatase (Na+,K+-ATPase) and on the intracellular content of K+ and Na+ ions in cultures which either were greatly enriched in a neuronal cell type, the cerebellar granule cells, or contained a mixed population of cells (brain reaggregates). Foetal rat brain reaggregates displayed lower Na+,K+-ATPase activity when cultured in chemically defined medium than in the presence of serum. Supplementation of the serum-free medium with thyroid hormone resulted in a rise in the Na+,K+-ATPase activity and [3H]ouabain binding to levels similar to those found in the cultures grown in the serum-containing medium. Thyroid hormone had no significant effect on the Mg2+-ATPase activity and on the intracellular content of Na+ and K+ ions. In the granule cell-enriched cerebellar surface cultures the Na+,K+-ATPase activity was lower when the cells were grown in chemically defined medium compared with the serum-containing medium, and the intracellular Na+ to K+ ratio was higher. Thyroid hormone had no effect on the Na+,K+-ATPase activity, [3H]ouabain binding or Mg2+-ATPase activity. The hormone also failed to influence ATPase activities in cerebellar astrocytes maintained in chemically defined medium. Although thyroid hormone had no effect on the Na+,K+-ATPase activity of cultured cerebellar granule cells, treatment with the hormone resulted in a decrease in the ratio of intracellular Na+ to K+ ion content. The effect of the hormone on the Na+,K+-pump activity in live cells was therefore tested by estimating ouabain-sensitive 86Rb uptake. This was regulated as in other cell types, by the rate of Na+ entry: the Na+-ionophore monensin trebled the rate of 86Rb uptake, which was also increased (+30-100%) by 10% foetal calf serum, the maximal response being obtained by about 20 min exposure to serum. The effect was completely blocked by the Na+/H+ exchange inhibitor amiloride. The factor(s) in the serum responsible for the regulation of the Na+,K+-pump were, however, not the thyroid hormones, which failed to affect 86Rb uptake. On the basis of comparing thyroid hormone effects on the different cultures studied it was concluded that not every type of neural cell is target of the hormone action during development.     

Net ion fluxes and zero flux limiting concentrations in rat upper colon and rectum during anaesthesia-induced aldosterone liberation

Thiobutabarbital anaesthetized and abdominally operated control rats develop high endogenous plasma levels of both aldosterone and corticosterone during the course of a 12 h experiment. This effect was used as a model for examining 'acute' steroid action (i) on net ion and water fluxes and (ii) on zero flux luminal limiting concentrations in rat upper colon (proximal 50% of large intestine) and rectum (distal 40%). Experiments of both kinds consisted of 8 independent 90 min measuring periods. (i) In rectum net fluxes of Na, K, osmolytes (sum of all solutes) and water started at low levels around zero, began to rise about 2 h after plasma levels of aldosterone had increased, and reached plateau values around the 6th hour of anaesthesia. In upper colon, fluxes of Na, K, Cl, and osmolytes were high from the beginning and did not vary significantly with time. (ii) At zero flux conditions limiting concentrations of Na in the hormonally unstimulated phase of the experiment were 20 +/- 3 mM in upper colon and 22 +/- 3 mM in rectum. After maximal endogenous aldosterone liberation zero flux concentrations were 5.2 mM in upper colon and 2.2 mM in rectum, corresponding to luminal fluid to plasma ratios (LF/P) of 0.040 and 0.016, respectively. Amiloride reduced the maximal Na gradient in rectum to a LF/P of 0.3 but was not effective in upper colon and did not prevent the stimulating effect of aldosterone in this segment. Under all experimental conditions zero flow concentrations of K were higher than consistent with a solely passive distribution, indicating simultaneous passive and active secretion in both segments. In contrast to the findings of others, the luminal fluid remained isoosmolar with plasma in all zero flux experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic fluxes and permeabilities of cell membranes in rat liver

1. Intracellular ion concentrations, measured in rat liver perfused with saline solutions were, at steady state:[K](1) = 113; [Na](1) = 16.4; [Cl](1) = 25.5 m-mole l.(-1) of cells.2. Intracellular Cl concentration was measured when both [Cl](o) and membrane potential were changed. The experimental values were close to the predicted ones by the Nernst equation, indicating a passive distribution of this ion across the cell membrane.3. Fluxes were determined by means of radioactive tracers and had the following values:m(K) = 6.6; m(Na) = 12.4 and m(Cl) = 8 x 10(-12) mole cm(-2) sec(-1).4. When Na was replaced by Li in the perfusing solutions, the Na efflux was decreased by 3.3 x 10(-12) mole cm(-2) sec(-1). This was attributed to a Na-for-Na exchange (exchange-diffusion).5. A mathematical model was applied to the perfused liver. It allowed estimation of the actual fluxes across the membrane. Corrections resulting from the application of the model remain small.6. The permeability coefficients were calculated from the passive fluxes and were:P(K) = 7.6; P(Na) = 4.0; P(Cl) = 12.3 x 10(-8) cm sec(-1), corresponding to relative permeabilities of P(Na)/P(K) = 0.52 and P(Cl)/P(K) = 1.6.7. The membrane potential calculated from the Goldman equation was significantly different from the measured one. This may be accounted for by an electrogenic activity of the Na-K pump. Applying the Mullins & Noda equation, the ratio of active Na flux to active K flux becomes 3/2.     

The effect of catecholamines on Na-K transport and membrane potential in rat soleus muscle.

1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (E(M)) has been investigated in soleus muscles isolated from fed rats.2. In a substrate-free Krebs-Ringer bicarbonate buffer adrenaline (ADR) (6 x 10(-6)M) increased (22)Na efflux by 83%, (42)K influx by 34%, and E(M) by 10%. Similar effects were exerted by noradrenaline (NA), phenylephrine, salbutamol and isoprenaline. The effects of ADR on Na-K transport and E(M) were suppressed by ouabain (10(-3)M) and propranolol (10(-5)M), but not by thymoxamine (10(-5)M) or tetracaine (10(-4)M).3. Following 90 min of incubation in the presence of ADR (6 x 10(-6)M), the intracellular K/Na-ratio was increased threefold. NA produced almost the same change, and both catecholamines seem to induce a new steady-state distribution of Na and K which can be maintained for several hours in vitro.4. The effect of ADR on (22)Na efflux and E(M) could be detected at concentrations down to 6 x 10(-9) and 6 x 10(-10)M, respectively, and half-maximum increase was obtained at around 2 x 10(-8)M. NA was at least one order of magnitude less potent.5. The effect of low concentrations of ADR on (22)Na efflux was potentiated by theophylline (2 mM). When added together, dibutyryl-cyclic AMP and theophylline mimicked the action of ADR on (22)Na efflux, (42)K influx, Na/K content and E(M). Ouabain (10(-3)M) also suppressed the effect of dibutyryl-cyclic AMP and theophylline on Na-K transport.6. Following the addition of ouabain (10(-3)M), E(M) rapidly dropped from a mean of -71 to -63 mV, and then showed a slow linear fall for up to 4hr.7. The hyperpolarization induced by ADR was associated with a decrease in membrane conductance, (22)Na influx and (42)K efflux. The time course and the response to ouabain suggests that all of these effects are secondary to stimulation of the active coupled transport of Na and K.8. It is concluded that in rat soleus muscle, the active Na-K transport is electrogenic and susceptible to stimulation by catecholamines via beta-adrenoceptors. This effect is mediated by adenyl cyclase activation and may account for the increase in E(M) and the intracellular K/Na ratio.     

Effects of a medium-chain triglyceride:fish oil emulsion administered intravenously to omega3 fatty acid-depleted rats on cationic fluxes in aortic rings

The handling of 45Ca and 86Rb by aortic rings obtained from rats depleted in long-chain polyunsaturated omega3 fatty acids (second generation) was examined in vitro over 10 to 60 min incubation at either increasing concentrations of extracellular K+ (5, 3 and 60 mM) in the case of 45Ca net uptake or in the absence and presence of ouabain (50 microM) in the case of 86Rb uptake. The omega3-depleted rats were injected intravenously 120 min before sacrifice with 1.0 ml of either an omega3 fatty acid-rich medium-chain triglyceride:fish oil emulsion (MCT:FO) or a control medium-chain triglyceride:olive oil emulsion (MCT:OO). In the MCT:OO-injected rats, the rise in extracellular K+ concentration failed to stimulate 45Ca net uptake, whilst the prior injection of the MCT:FO emulsion restored the expected increase in 45Ca net uptake by aortic rings exposed to 60 mM K+. The absolute value for 86Rb net uptake after 10 or 60 min incubation and whether in the absence or presence of ouabain, which significantly decreased the uptake of 86Rb+ after 60 min incubation, only represented in the MCT:FO-injected rats 63.1+/-3.8% of the mean corresponding values found in MCT:OO-injected animals. These findings are consistent with the view that activity of cationic channels, such as the voltage-sensitive Ca2+ channel, the outflow of Ca2+ as mediated by either Na+-Ca2+ countertransport or a Ca2+-ATPase, the activity of Na+,K+-ATPase and the modality of K+ inflow by an oubain-resistant modality are all affected in aortic cells by the content of long-chain polyunsaturated omega3 fatty acids in membrane phospholipids.     

Cation exchange and glycoside binding in cultured rat heart cells.

The Na/K-exchange characteristics, ouabain-binding kinetics, and Na pump turnover rates of synchronously contracting monolayers of neonatal rat myocardial cells were studied. The cells exchange Na rapidly (T1/2 = 35 s) with a mean Na flux of approximately 25 (pmol/cm2)/s. The half time (T1/2) of K exchange is much longer (12 min); the mean K flux is 13 (pmol/cm2)/s. Active Na/K transport, as measured by K influx, is relatively ouabain sensitive, and 10(-6) M ouabain produces half-maximal inhibition. Ouabain (10(-2)M) inhibits 60% of the Na efflux and 75% of the K influx. The cells bind [3H]ouabain rapidly (T1/2 = 8 min), but release it very slowly (T1/2 = 11 h), and both the amount bound and the rate of binding were inversely proportional to extracellular K. Specific [3H]ouabain binding demonstrates saturation reaching a maximum of 1.6 x 10(6) molecules per cell at 2 x 10(-7) M [3H]ouabain. From cell surface area and ouabain-sensitive flux measurements, the Na pump density was calculated at 720/micrometer2 with an individual pump turnover rate of 50/s. Thus the studies indicate that despite their neonatal origin, the behavior of the Na pump in these cells is very similar to that in other mammalian tissues.     

The effect of ouabain on ion transport across isolated sheep rumen epithelium.

1. The net flux of Na from the lumen to the blood side of isolated sheep rumen epithelium was reduced to zero in the third 30 min period after treatment with ouabain. 2. The net flux of K from blood to lumen side of the epithelium was reduced from 0-34 to 0-13 mumole/cm2. hr in the third 30 min period after ouabain treatment. 3. The net flux of Cl from lumen to blood side of the epithelium was reduced from 1-4 to 0-72 mumole/cm2. hr in the third 30 min period after ouabain treatment. 4. The results support the existence of a Cl pump independent of Na transport and also a K pump in sheep rumen epithelium.     

Some properties of a preparation of rat colon perfused in vitro through the vascular bed

1. A technique is described for the perfusion of the isolated colon of the rat, involving the infusion of an appropriate fluid through both the inferior and the superior mesenteric arteries. During neither the preparation nor the subsequent perfusion is the colon without an adequate supply of oxygen. The preparation remains histologically intact and metabolically viable and is capable of actively transporting ions for up to 5 hr.2. The addition of at least 3 g albumin/100 ml. perfusate is necessary to prevent the formation of large quantities of serosal exudate. With erythrocytes added to the vascular perfusate the preparation appears to be adequately oxygenated as judged by measurements of the rate of glycolysis. The mean rate of oxygen utilization over 4 hr is 9.2 +/- 0.3 (4) mumole. hr(-1).g(-1) fat free dry weight.3. Ion transport rates approaching those found in vivo are found only after the administration of an antihistamine substance to the colon donor rat before operation. In the absence of an antihistamine substance there appears to be an ultrafiltration of the plasma fluid into the lumen.4. Vasodilatory substances accumulate in the recycled perfusate. In a ;single pass' perfusion, the transport capacity of the preparation decreases at high perfusion pressures. It is suggested that this is due to some form of autoregulation whereby perfusate is shunted away from the epithelium into deeper layers as the pressure is increased.5. With CO(2) absent from the vascular infusate there is an increase in the net lumen to blood flux of total CO(2). This increased flux is accompanied by an equivalent amount of cation, comprising Na(+) and K(+) in the ratio of 12:1.6. The presence of ammonium in the lumen, a physiological constituent of the contents of rat distal colon in vivo, has a marked inhibitory effect upon the secretion of CO(2) into the contents of the lumen of the colon.