Sodium dependence of carnitine transport in isolated perfused adult rat hearts

In heart muscle, the intracellular carnitine concentration is approximately 40 times higher than the plasma carnitine concentration, suggesting the existence of an active transport process. At physiological serum carnitine concentrations (44 microM), 80% of total myocardial carnitine uptake occurs via a carrier-mediated transport system. The mechanism of this carrier-mediated transport was studied in isolated perfused rat hearts. Carnitine transport showed an absolute dependence on the extracellular sodium concentration. The rate of carnitine transport was linearly related to the perfusate sodium concentration at every perfusate carnitine concentration examined (15-100 microM). Total removal of extracellular sodium completely abolished the carrier-mediated transport. Decreasing the perfusate potassium concentration from a control of 5.9 to 0.6 mM stimulated transport by 35%, whereas increasing the extracellular potassium concentration from 5.9 to 25 mM reduced transport by 60%. The carrier-mediated transport was inversely proportional to the extracellular potassium concentration. Acetylcholine (10(-3) M), isoproterenol (10(-7) M), or ouabain (10(-3) did not alter the rate of carnitine transport. Addition of tetrodotoxin (10(-5) stimulated carnitine transport by about 40%, while gramicidin S (5 X 10(-6) M) decreased uptake by about 18% relative to control. The data provide evidence that carnitine transport by cardiac cells occurs by a Na+-dependent cotransport mechanism that is dependent on the Na+ electrochemical gradient.     

Mechanism of active chloride secretion by shark rectal gland: role of Na-K-ATPase in chloride transport.

The isolated rectal gland of Squalus acanthias was stimulated to secrete chloride against an electrical and a chemical gradient when perfused in vitro by theophylline and/or dibutyryl cyclic AMP. Chloride secretion was depressed by ouabain which inhibits Na-K-ATPase. Thiocyanate and furosemide also inhibited chloride secretion but ethoxzolamide, a carbonic anhydrase inhibitor, did not. Chloride transport was highly dependent on sodium concentration in the perfusate. The intracellular concentration of chloride averaged 70-80 meq/liter in intact glands, exceeding the level expected at electrochemical equilibrium and suggesting active transport of chloride into the cell. These features suggest a tentative hypothesis for chloride secretion by the rectal gland in which the uphill transport of chloride into the cytoplasm is coupled through a membrane carrier to the downhill movement of sodium along its electrochemical gradient. The latter is maintained by the Na-K-ATPase pump while chloride is extruded into the duct by electrical forces.     

Importance of bicarbonate in bile salt independent fraction of bile flow.

The bile salt independent fraction (BSIF) of canalicular bile flow from the isolated rat liver perfused with bicarbonate-free perfusate is 50% of that from the liver perfused with bicarbonate-containing perfusate. HCO3-excretion is nearly eliminated and Na+ and Cl- excretion is reduced 50%. Replacement of HCO3- into perfusate increased bile flow by 0.3 microliter/g.min without changing bile acid excretion rate. 5.5-Dimethyl-2,4-oxazolidinedione (DMO) produced a similar effect. DMO was passively distributed between bile and plasma. The data indicate that a bicarbonate transport mechanism is responsible for production of up to 50% of the BSIF. Another weak acid, N-5[5-(2-methoxyethoxy)-2-pyrimidinyl]sulfamoylbenzene (glymidine), was rapidly excreted into bile and increased bile flow by over 2.0 microliter/g.min. Glymidine is probably excreted by an independent organic anion transport mechanism, and any effect on the bicarbonate transport mechanism is obscured. Canaliculus-enriched hepatocyte membrane fractions contained no HCO3-stimulated ATPase activity. Either this enzyme is unimportant in hepatocyte bicarbonate transport or transport occurs across membranes other than the bile canalicular membrane.     

Altered hepatobiliary transport of taurocholic acid in aged rats

Hepatobiliary transport of taurocholic acid was studied in adult (3 months) and old (2 years) rats using an isolated perfused rat liver technique in order to determine the effect of age on hepatic uptake and secretion of bile acids simultaneously. The results were analyzed using a steady-state compartmental model to estimate the uptake and secretion of taurocholic acid. Hepatic secretion was decreased to a greater extent than the uptake in old rats. These changes in transport activities were associated with increases in perfusate and liver bile acid pool sizes. These results can explain the decrease in total pool size and synthesis rate of bile acids observed previously in old rats using in vivo studies. It has been suggested that the age-dependent decrease in bile acid transport capacity of the liver is secondary to the altered lipid composition of the liver plasma membranes of old rats.     

Thyroid calorigenesis in isolated, perfused rat liver: minor role of active sodium-potassium transport.

1. The effects of ouabain on hepatic oxygen uptake, cell membrane potential, and Na-K transport were examined at 37 degrees C during non-recirculating perfusion of isolated livers from fasted normal rats and rats treated with triiodothyronine (T3). The perfusate was Krebs-Ringer bicarbonate buffer containing albumin and bovine erythrocytes. 2. Treatment with T3 increased the rate of hepatic oxygen uptake by 30% (i.e. by 0-83 (micromole/min) per gram liver). 3. After shifting to perfusate containing 2-5 mM ouabain, a 4-5 mV depolarization and maximal rates of net hepatic K release and Na uptake occurred within 2 min in both thyroid states. These changes were not accompanied by any significant change in the rates of hepatic oxygen uptake. 4. T3-treatment increased the maximal, post-ouabain net flux of K by 29% (i.e. by 0-52 (muequiv/min) per gram liver). The T3-indlced increase in the net flux of Na (19%) did not achieve statistical significance. 5. In either thyroid state, the observed passive fluxes of Na and K were calculated to be balanced by active vluxes at the expense of 5-6% of the observed rate of hepatic oxygen uptake. 6. The results indicate that hyperthyroidism may enhance the rate of hepatic Na-K transport, but the energy expenditure due to this process appears to be too small to make any important contribution to thyroid calorigenesis in perfused rat liver.     

Dependence of intestinal glucose absorption on sodium, studied with a new arterial infusion technique

1. A new preparation of isolated rat jejunum plus ileum (ca. 100 cm) is described in which a saline infusate is pumped into the superior mesenteric artery, the superior mesenteric vein having been ligated.2. The arterial infusate washes out the tissue spaces: the lumen is perfused in a single pass with a segmented flow as by Fisher & Gardner (1974).3. At an arterial infusion rate of 3 ml./min, steady states are set up in the tissue fluid within 10-15 min: the compositions of the fluids bathing both sides of the mucosa can therefore be controlled.4. The rate of glucose absorption from the lumen falls only gradually when the luminal sodium is replaced by choline abruptly while the tissue fluid sodium is maintained at 144 m-equiv/l. by arterial infusion.5. The rate of glucose absorption from the lumen is unaffected by replacement of sodium in the arterial infusate by choline.6. Ouabain (10(-4) M) in an arterial infusate containing sodium 144 m-equiv/l. causes inhibition of glucose and water absorption from the lumen. There is no effect of ouabain when the arterial infusate contains sodium, 0 or 72 m-equiv/l.7. Arterial ouabain does not reverse the effects of depletion of luminal sodium. Simultaneous removal of luminal sodium and application of arterial ouabain causes faster inhibition of glucose absorption than does either treatment alone.8. Glucose absorption is more likely to depend on rate of efflux of sodium from mucosal cell to tissue fluid than on a sodium gradient at the brush border or on intracellular sodium concentration.     

Water,K+, H+, lactate and glucose fluxes during cell volume regulation in perfused rat liver

The present study has been performed to test for ion release from isolated perfused rat liver exposed to hypotonic perfusates. Replacement of 40 mmol/l NaCl in perfusate by 80 mmol/l raffinose leads to slight alkalinization and slight decrease of liver weight. Subsequent decrease of perfusate osmolarity by omission of raffinose results in an increase of liver weight and a parallel increase of effluent sodium, chloride and potassium activity pointing to net uptake of solute free water. While effluent chloride and sodium activities approach perfusate activities within less than 2 min, a second, 6 min lasting increase of effluent potassium activity is observed, pointing to potassium release by the liver. This transient increase of effluent potassium activity is paralleled by a decrease of liver weight. Throughout exposure to hypotonic perfusates, lactate, pyruvate and glucose release by the liver is significantly decreased and effluent pH is rendered alkaline. Readdition of 80 mmol/l raffinose leads to rapid decrease of liver weight and a parallel decrease of effluent sodium, chloride and potassium activities followed by a 10–20 min lasting decrease of effluent potassium activity, pointing to net uptake of potassium, which almost matches the net release observed before. The transient decrease of potassium activity is paralleled by an increase of liver weight, an increase of effluent glucose, lactate and pyruvate concentration and an acidification of the effluent. Similar decrease of effluent potassium activity, acidification of effluent and increase of effluent glucose, lactate and pyruvate concentration are observed, if perfusates are made hypertonic by addition of raffinose. In conclusion, both, volume regulatory decrease (VRD) and increase (VRI) can be elicited in liver and are in large part achieved by movements of potassium. Lactate and pyruvate production is decreased throughout exposure to hypotonic perfusates and enhanced following exposure to hypertonic perfusates.     

Sodium entry routes in principal and intercalated cells of the isolated perfused cortical collecting duct

Transmembrane sodium transport pathways were studied in principal and intercalated cells of the isolated perfused rabbit cortical collecting duct. Intracellular electrolyte concentrations in individual collecting duct cells were measured by electron microprobe analysis during blockage of basolateral Na-K-ATPase by ouabain and simultaneous inhibition of sodium entry across the apical and/or basolateral cell membrane. In principal cells the ouabain-induced rise in cell sodium concentration could only partially be blocked by amiloride (10^–4mol/l) in the perfusion fluid. Amiloride (10^–3mol/l) added to the bathing solution produced a further, significant reduction of sodium influx. In principal cells the ouabain-induced increase in sodium concentration was completely prevented by amiloride in the perfusion solution in combination with omission of sodium from the peritubular bathing solution. In intercalated cells ouabain caused a less pronounced increase in sodium concentration than in principal cells. Neither amiloride in the perfusate, nor amiloride in both bathing and perfusion solution, significantly reduced the ouabain-induced rise in intercalated cell sodium concentration. These results indicate that in principal cells amiloride-sensitive sodium channels constitute the predominant pathway for sodium entry across the apical cell membrane. In addition, substantial amounts of sodium enter principal cells across the basolateral cell membrane, probably via Na-H exchange. Finally, the data suggest that in intercalated cells sodium channels and the Na-H exchange are sparse or even absent.     

Uptake of ascorbic acid by freshly isolated cells and secretory granules from the intermediate lobe of ox hypophyses

Mechanically isolated cells from the intermediate lobe of ox hypophyses contained 40.6 +/- 3.7 nmol mg-1 protein (mean +/- SE, n = 5) of ascorbic acid. They accumulated radioactivity time dependently, on incubation with L-[14C]ascorbic acid in ionic medium dominated by NaCl. No definite saturation of uptake occurred when mechanically isolated cells were incubated with increasing ascorbic acid concentrations up to 0.6 mM. But if such cells were purified on a Percoll gradient, a clear saturation of uptake could be observed. Acetylsalicylic acid reduced the uptake markedly. When cells loaded with L-[14C]ascorbic acid were homogenized and placed on a Percoll gradient, the radioactivity was recovered in several subcellular fractions. Decrease of the Na+ concentration or presence of ouabain in the medium did not cause noticeable changes in uptake by non-purified cells, whereas uptake by purified cells was clearly sodium-dependent. Phloridzin inhibited uptake. Secretory granules from pars intermedia contained 40.0 +/- 3.8 nmol mg-1 protein of ascorbic acid (mean +/- SE, n = 3) and could accumulate L-[14C]ascorbic acid rapidly in a KCl-dominated medium. The uptake was not saturable with ascorbic acid concentration and was not influenced by the presence of I mM ATP + I mM Mg2+ in the medium. The concentration of copper and iron in isolated cells was comparable to that in isolated neurohypophysial nerve terminals, whereas the concentration of zinc was considerably higher in the pars intermedia cells. The concentration of Cu, Zn, Fe and Co in secretory granules from pars intermedia was higher than in secretory granules from neurohypophyses.     

Effect of graded bilirubin loads on bilirubin transport by perfused rat liver.

Features of hepatic bilirubin transport were studied with the isolated perfused rat liver. Male Wistar rats weighing 350-400 g were used as liver donors. When bilirubin was constantly infused into the perfusion medium, which contained sheep erythrocytes and 3.0 g/100 ml bovine serum albumin, the maximal excretion rate for bilirubin was 14.4 +/- 1.2 mug/min per g liver. Over a wide range of constant bilirubin infusion rates which went as high as 25.9 mug/min per g liver, there was no effect on bile flow, bile acid excretion, or the pattern of bilirubin conjugates in bile. The hepatic extraction efficiency for unconjugated bilirubin from the perfusate also remained constant averaging 26%. However, when bolus injections of bilirubin were used to produce higher levels of unconjugated bilirubin in the perfusate than could be attained during constant infusion, the disappearance rate of [14C]bilirubin from the perfusate decreased with increasing bilirubin concentrations. This was consistent with saturation of the hepatic removal of unconjugated bilirubin.     

Vitamin A1 intestinal absorption in vivo: influence of luminal factors on transport.

Intestinal absorption of [3H]retinol was studied in the unanesthetized rat. Luminal perfusate was recirculated through isolated intestinal segments with intact vascular and lymphatic circulation. Apparent saturation kinetics were found in physiological concentrations of retinol, whereas a linear relationship between the concentration and absorption rate was found at pharmacological concentrations of retinol in the perfusate. In physiological concentrations, retinol uptake in vitro by everted gut sacs was unaffected by anoxia or metabolic inhibitors and uncouplers. In vivo retinol absorption rate was decreased when sodium taurocholate concentration was raised above 5 mM, or when 2.5 mM linoleic or linolenic acids were added to the perfusate. Absorption increased markedly as the thickness of the unstirred water layer was diminished. Variations in perfusate pH from 4.5 to 8.6 did not change the retinol absorption rate. In vivo absorption of retinol in physiological concentrations is mediated by a saturable, carrier-mediated passive absorption mechanism modified by the presence of fatty acids of varying chain length.     

Water and electrolyte secretion by the perfused pancreas of the cat

1. A technique is described for perfusing the isolated cat pancreas with saline solutions.2. Single doses of secretin, although present in the perfusate for only a short time, caused a prolonged flow of pancreatic juice.3. In response to continuous secretin infusion, the preparation secreted for up to 6 hr a juice which was similar to that obtained in vivo, with the exception that the bicarbonate concentration decreased and the chloride concentration increased with time, even when the rate of secretion remained constant.4. The osmolalities of perfusate and secretion were identical over a range of 450 m-osmoles/kg, but the electrolyte concentration of the secretion was always slightly higher than that of the perfusate. Variations from perfusate isosmolality produced inverse changes in the secretion rate, over the range from 600 m-osmoles/kg, at which secretion ceased, to 150 m-osmoles/kg, at which the rate was highest. At perfusate osmolalities below 150 m-osmoles/kg secretion rapidly declined.5. Reduction in perfusate sodium chloride concentration, isosmolality being maintained with sucrose, caused a fall in secretion rate, but the sodium concentration of the juice remained constant until perfusate sodium concentration was reduced to about 70 m-equiv/l. Below this level it declined and sucrose was detected in the juice in quantities almost sufficient to account for the equiosmolality of juice and perfusate.6. Two hypotheses about the mechanism of water and electrolyte secretion by the pancreas are presented.     

Copper impairs biliary epithelial cells and induces protein oxidation and oxidative DNA damage in the isolated perfused rat liver

Copper is one of the major metals causing environmental contamination. Previous studies showed that copper induced toxic effects in isolated perfused rat liver models and these effects were associated with lipid peroxidation. Here we investigated whether effects of copper (at concentrations of 0.01, 0.03, and 0.1 mM of Cu(2+) in Krebs-Henseleit buffer perfusing the isolated rat liver for 60 min), were associated with biliary epithelial cell injury, as well as protein oxidation and oxidative DNA damage. The highest concentration of copper in perfusate (0.1 mM) did not allow complete evaluation of all parameters because it blocked portal flow within 30 min of perfusion, indicating severe microcirculatory disturbances. Further, copper decreased secretion of bile and it increased lactate dehydrogenase, aspartate transaminase, and alanine transaminase leakage into perfusate as well as liver weight in a dose-dependent manner. Biliary gamma-glutamyltransferase, an index of biliary epithelial cell integrity increased similarly at 0.01 and 0.03 mM copper concentrations in perfusate. Compared to controls, 0.01 and 0.03 mM concentrations of copper increased the amount of thiobarbituric acid reacting substances, a marker of lipid peroxidation, tissue protein carbonyl groups, an index of protein oxidation, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of oxidative DNA damage. The results suggest that toxic effects of copper in the isolated perfused rat liver may involve biliary epithelial cells and they are associated with lipid peroxidation, protein oxidation, and oxidative DNA damage.     

Canalicular bile salt-independent bile formation: concepts and clues from electrolyte transport in rat liver

Studies on canalicular electrolyte transport are reviewed with reference to the concept that hepatocellular inorganic ion secretion may provide an osmotic drive for canalicular water flow. Cellular transport of electrolytes and of some nonelectrolytes appears directly or indirectly (cotransport or potential-sensitive transport) related to the activity of Na+-K+-ATPase of the sinusoidal cell membrane, but the role of the enzyme in regulating bile flow remains undetermined. Bile secretion of the isolated rat liver continues in the absence of either Na+, K+, Cl-, or HCO-3 when these ions are replaced in the perfusion medium by other permanent ions. Transepithelial salt concentration gradients, established experimentally, cause transient changes of bile flow and dissipate very quickly. Isotopic ion equilibration between sinusoids and bile proceeds faster than between sinusoids and liver cells. Both observations indicate extensive electrolyte diffusion through a paracellular shunt pathway. This pathway appears preferentially permeable to cations, and it restricts permeation of molecules of the size of sucrose (no apparent diffusion or effects of solvent drag) or bile acids (no backleak). In promoting canalicular osmotic water flow, transepithelial concentration gradients of NaCl are less effective than those of sucrose, revealing a reflection coefficient of NaCl of 0.3. By perfusion with hypertonic medium containing sucrose, bile flow is reduced. Bile production against this opposing osmotic gradient is accomplished by an increase in biliary organic anion concentration. Inorganic ion concentrations essentially conform to a Gibbs-Donnan distribution across the canalicular epithelium, established by the presence of impermeant anions in bile. Hence, the luminal electrical potential is expected to be negative with respect to the sinusoids. It is concluded that biliary secretion of endogenous organic anions is the major osmotic driving force for canalicular bile salt-independent bile flow and that transport of inorganic ions into bile results mainly from diffusion and solvent drag.     

Effects of sodium fluxes and ouabain on brain-slice tryptophan transport

Efflux of preloaded [3H]tryptophan from rat cerebral cortex slices has been monitored into superfusion media that were altered in their sodium content. Total replacement of sodium with choline greatly increased the release of tryptophan. This release could be cancelled by re-introducing sodium into the slices. A brief exposure to ouabain, an efficient inhibitor of Na+, K+-ATPase activity, only slightly increased tryptophan efflux at the concentration of 0.1 mM, whereas at 1.0 mM it produced a similar effect as the sodium-free medium. Accordingly, when the slices were superfused in the presence of ouabain and sodium, the change of medium to sodium-free caused much greater relative enhancement of tryptophan efflux with 0.1 than 1.0 mM ouabain. Tryptophan efflux was modified by changes in sodium fluxes also in slices initially depleted of sodium ions and treated with ouabain. The results suggest that the sodium-free medium and ouabain have a similar mechanism of action in modifying the tryptophan transport, and that the cation gradients across the cell membranes are more crucial for normal amino acid transport than the functional Na+, K+-ATPase.     

Studies on the lithium transport across the red cell membrane

In studies on Li+ net-transport across the human red cell membrane following results were obtained: 1. In K+- and Na+-free choline chloride media, Li+ is transported into the erythrocytes against an electrochemical gradient. This Li+ uphill transport as well as Li+ downhill transport into the cells is inhibited by ouabain, ATP-depletion, and by external K+ and Na+. The effects of K+ and Na+ are relieved at high Li+ concentrations. 2. Ouabain-sensitive Li+ uptake, determined at 10 mM external Na+, does not obey simple Michaelis-Menten kinetics and exhibits a maximum at about pH 7. 3. Ouabain-resistant Li+ downhill transport into erythrocytes increases with rising pH. It is comprised of a saturating component and a component linearly dependent on external Li+. The linear component is partly inhibited by dipyridamole and accelerated by bicarbonate. The bicarbonate effect can be completely blocked by dipyridamole, phlorizin and phenylbutazone. 4. Li+ release is not inhibited by ouabain, ATP-depletion and external K+. It increases with external Na+ concentration, tending to saturate at 150 mM Na+. Na+-independent Li+ release is stimulated by bicarbonate. It is concluded that ouabain-sensitive Li+ uptake is mediated at the K+-site(s) of the Na+-K+ pump. Li+, K+ and Na+ appear to compete for a common site (or sites). The stimulation of Li+ transfer by bicarbonate and the inhibition by dipyridamole suggest a participation of anionic species in ouabain-resistant Li+ transfer. The Na+-dependent Li+ release and the "saturating component" of Li+ uptake are ascribed to the Na+-dependent Li+ countertransport system.     

Choline transport by lung epithelium

The uptake of [3H]choline was investigated using isolated perfused rat lungs and primary cultures of granular pneumocytes isolated by tryptic digestion of rat lungs. Metabolic products were separated from free choline by chloroform:methanol extraction and column chromatography. Tissue-associated [3H]choline increased progressively in the perfused lung, and estimated mean intracellular concentration at 2 h was 12 times the extracellular concentration (5 microM). Choline uptake was inhibited by ventilation with CO and by perfusion with the choline analog, hemicholinium-3 (HC-3). Isolated granular pneumocytes also accumulated choline against a concentration gradient by an energy-dependent process. The concentration for half-maximal uptake, after correction for the diffusion component, was estimated at 18 +/- 4 microM (mean +/- SE; n = 3), and the estimated maximal rate of uptake was 213 +/- 44 pmol/min/microliter cell water. HC-3 inhibited uptake by approximately 50% at a concentration of 10(-4) M. There was no effect on uptake when Na+ in the medium was replaced by Li+ or N-methylglucamine+. These results indicate that granular pneumocytes possess a transport system that results in accumulation of choline against a concentration gradient. The characteristics of uptake indicate that this system is similar to the low affinity choline transport system of other organs.     

The effect of cyanide on apparent potassium conductance across the peritubular cell membrane of frog proximal tubules

To test for the effect of cyanide on frog proximal renal tubules the potential difference across the peritubular cell membrane (PDpt) has been recorded continuously before and during peritubular application of 1 mmol/l cyanide using conventional microelectrodes.Before application of cyanide PDpt amounts to –61.5 ±2.2 mV in the absence of luminal substrate. Cyanide depolarizes the peritubular cell membrane by +18.8±2.3 mV/10 min in the presence and by +4.5±0.9 mV/10 min in the absence of luminal substrate. The rapid depolarization of the cell membranes to addition of glucose to luminal perfusate is not significantly influenced by exposure to cyanide, whereas the influence of altered peritubular potassium concentration (from 3 to 9 mmol/l) is significantly reduced from +15.2±1.7 mV to +8.7±1.8 mV. Following exposure to cyanide the lumped resistance of the luminal and peritubular cell membranes increases significantly by 36±7%/6 min, and the cellular core resistance significantly by 14±6%/6 min. As a result, cyanide markedly decreases the peritubular potassium conductance, depolarizes the cell membranes and reduces the driving force for sodium coupled transport processes. Thus cyanide fully mimicks the effects of ouabain, although cyanide in contrast to ouabain is expected to deplete the cells from ATP. In conclusion ATP/ADP is not likely to play a major role in the regulation of sodium coupled transport processes and peritubular potassium conductance in amphibian proximal tubules.