Ouabain-resistant mechanism of volume control and the ultrastructural organization of liver slices recovering from swellingin vitro

Summary We have studied the net extrusion of water by liver slices recovering from swelling at 1°C and have attempted to relate this to ultrastructural alterations. Special attention was paid to the ouabain-resistant extrusion of water. The restoration of many details of intracellular architecture was dissociated from the net loss of water, since an osmotic stimulus (produced by 5% inulin) caused a passive withdrawal of water with little recovery of structure. Also, a similar recovery of structure was produced during active extrusion of water in the absence and presence of ouabain, even though ouabain reduced the water extrusion by 50%. The time-course of water extrusion in the presence of ouabain was correlated with the formation of cytoplasmic vesicles. Incubation without K⁺ in the medium had similar effects to those caused by ouabain. Colchicine had little effect on water extrusion in presence or absence of ouabain except at concentrations which reduced tissue ATP levels and caused much necrosis. Cytochalasin B alone had little effect on water extrusion, but led to the accumulation of many vesicles in the cytoplasm and appeared to abolish the access of such vesicles to the canaliculi. In the presence of ouabain, cytochalasin B had a similar effect on ultrastructure, and totally prevented the ouabain-resistant water extrusion. Ni²⁺ had rather similar effects to cytochalasin B both in the presence and absence of ouabain, although to a smaller degree. The results support our previous suggestion that the ouabain-resistant water extrusion proceeds by secretion of water into cytoplasmic vesicles, followed by the exocytotic expulsion of the vesicular contents into the bile canaliculi. Microfilaments appear to play an important role in this process.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Active potassium transport coupled to active sodium transport in vesicles reconstituted from purified sodium and potassium ion-activated adenosine triphosphatase from the rectal gland of Squalus acanthias.

Vesicles containing a purified shark rectal gland (sodium + potassium)-activated adenosine triphosphatase-(NaK ATPase) were prepared by dialyzing for 2 days egg lecithin, cholate, and the NaK ATPase purified from the rectal gland of Squalus acanthias. These vesicles were capable of both Na+ and K+ transport. Studies of K+ transport were made by measuring the ATP-stimulated transport outward of 42K+ or 86Rb+. Vesicles were preloaded with isotope by equilibration at 4 degrees for 1 to 3 days. Transport of 42K+ or 86Rb+ was initiated by addition of MgATP to the vesicles. The ATP-dependent exit of either isotope was the same. Experiments are presented which show that this loss of isotope was not due to changes in ion binding but rather due to a loss in the amount of ion trapped in the vesicular volume. The transport of K+ was dependent on external Mg2+. CTP was almost as effective as ATP in stimulating K+ transport, while UTP was relatively ineffective. These effects of nucleotides parallel their effects on Na+ accumulation and their effectiveness as substrates for the enzyme. Potassium transport was inhibited by ouabain and required the presence of Na+. The following asymmetries were seen: (a) addition of external Mg2+ supported K+ transport; (b) ouabain inhibited K+ transport only if it was present inside the vesicles; (c) addition of external Na+ to the vesicles stimulated K+ transport. External Li+ was ineffective as a Na+ substitute. The specific requirement of external Na+ for K+ transport indicates that K+ exit is coupled to Na+ entry. Changes in the internal vesicular ion concentrations were studied with vesicles prepared in 20 mM NaCl and 50 mM KCl. After 1 hour of transport at 25 degrees, a typical Na+ concentration in the vesicles in the presence of ATP was 72 mM. A typical K+ concentration in the vesicles was 10 mM as measured with 42K+ or 6 mM as measured with 86Rb+. The following relationships have been calculated for Na+ transport, K+ transport and ATP hydrolysis: Na+/ATP = 1.42, K+/ATP =1.04, and Na+/K+ = 1.43. The ratio of 2.8 Na+ transported in to 2 K+ transported out is very close to the value reported for the red cell membrane. Potassium-potassium exchange similar to that observed in the red cell membrane and attributed to the Na+-K+ pump (stimulated by ATP and orthophosphate and inhibited by ouabain) was observed when vesicles were prepared in the absence of Na+. The results reported in this paper prove that the shark rectal gland NaK ATPase, which is 90 to 95% pure, is the isolated pump for the coupled transports of Na+ and K+.     

Synergism between ouabain and monensin in neurogenic responses of guinea-pig vas deferens

Interrelations between ouabain, a Na+-K+ ATPase inhibitor, and monensin, a Na+ ionophore, on noradrenaline liberation and contractile activity were evaluated in the guinea-pig vas deferens. Monensin (1 microM) per se elicited a small contraction of the tissue. However, amplitude and time to the peak of large and sustained contractions evoked by 10 microM ouabain were potentiated and markedly shortened, respectively, by monensin. Contractions elicited by ouabain with or without monensin were prevented by 3 microM phentolamine or by pretreatment with reserpine. Contractions evoked by K+-free solution were augmented by monensin. In an HPLC study, noradrenaline outflow from the vas deferens was moderately and considerably increased by monensin (10 microM) and ouabain (100 microM), respectively. The ouabain-evoked output of noradrenaline was enhanced in the presence of monensin and the time course for maximum noradrenaline release was shortened, as was the contractile activity. This enhanced outflow after ouabain plus monensin was reserpine sensitive but not tetrodotoxin sensitive. Furthermore, this noradrenaline outflow was roughly halved in Na+-deficient medium, but was unaltered in Ca2+-free medium. These findings suggest that the synergistic effect of ouabain and monensin on noradrenaline liberation from the guinea-pig vas deferens may be due to an elevation of cytoplasmic Ca2+ concentrations, presumably resulting from a stimulation of intracellular Na+-Ca2+ exchange system, but not enhanced Ca2+ entry.     

Requirement of Cl− and Na+ for the ouabain-resistant control of cell volume in slices of rat liver

Summary The ability of liver cells to control their volume in the presence of ouabain has been studied in tissue slices that were recovering at 38°C from a period of swelling at 1°C. Morphological observations were made in conjunction with measurements of the net movements of water and ions. Extrusion of water in the presence of ouabain (2mm) was accompanied by a net loss of Na⁺ and Cl^– and by the formation of characteristic, rounded vesicles in the peri-canalicular regions of the hepatocytes; bile canaliculi were patent. When incubation was carried out in a medium in which either NO ₃ ^– or SO ₄ ^2– replaced Cl^–, ouabain-resistant water extrusion was prevented and the cytoplasmic vesicles normally found with ouabain were almost totally absent. When these slices were subsequently transferred to Cl^– medium with oubain, extrusion of intracellular water was initiated and cytoplasmic vesicles reappeared. Replacement of medium Na⁺ by Li⁺ mimicked the effects of ouabain on water and ion movements and ultrastructure. In addition, the ouabain-resistant extrusion of water and Cl^– was reduced and there was some diminution in the number of vesicles induced by ouabain. Furosemide (2mm) had little effect on water movement or ultrastructure in the absence of ouabain, but it slowed the net water loss and substantially reduced the formation of cytoplasmic vesicles in the presence of ouabain. The results show a close relationship between ouabain-resistant water extrusion and the formation of the cytoplasmic vesicles that are characteristic of treatment with ouabain. They further suggest that a cotransport of Na⁺ and Cl^– forms an important part of the mechanism underlying ouabain-resistant water extrusion and, specifically, that this cotransport may take place across the membranes of the cytoplasmic vesicles.     

Characterization and metabolism of canine proximal tubules, thick ascending limbs, and collecting ducts in suspension

Preparations of distinct nephron segments were obtained from dog kidneys by collagenase treatment. Four morphologically different tissues were isolated: glomeruli, proximal tubules, thick ascending limbs, and papillary collecting ducts. Each segment possessed a characteristic assay of membrane-bound and cytoplasmic enzymes. Specific metabolic characteristics also were found: gluconeogenesis and ammoniagenesis in proximal tubules, glycolytic aerobic metabolism in thick ascending limbs, and glycolytic anaerobic metabolism in papillary collecting ducts. The assay of Na+ -K+ ATPase, H+ -ATPase, and Ca2+ -ATPase activities in these nephron segments demonstrated a specific enrichment of Na+ -K+ ATPase in thick ascending limbs, and of H+ -ATPase in proximal tubules and papillary collecting ducts. Tubular respiration in the absence or presence of ouabain, 1,3-dicyclohexylcarbodiimide, or furosemide demonstrated that the respiration of each segment could be correlated to the activity of specific ion motive ATPases. Furthermore, a tight coupling between ion transport, ATP turnover, and substrate oxidation was demonstrated. These isolated tubular structures are thus viable and capable of transepithelial transport. Our preparation provides large amounts of defined population of tubules and are thus useful for the study of biochemical and functional heterogeneity along the nephron.     

Induction of rhythmic contraction in canine tracheal smooth muscle

Na(+)-K+ ATPase activity of the canine tracheal smooth muscle membrane is responsible for the electrogenic pumping of Na+ and K+ ions. It has been shown that this activity results in muscle relaxation. Based on the results of the current study, we suggest that prolonged electrical stimulation induces increased Na(+)-K+ ATPase activity in isolated tracheal smooth muscle. Tracheal smooth muscle pretreated with prolonged electrical stimulation developed graded mechanical activity when subsequently treated with histamine, serotonin, acetylcholine, or 80 mM K+. This increased isometric tension was interrupted by rhythmic activity, which was elicited by histamine or serotonin but not by acetylcholine or 80 mM K+ stimulation. The spontaneous phasic activity was not inhibited by atropine or propranolol but was totally inhibited by 10(-6) M ouabain. These results suggested that the relaxation phase of rhythmic contraction in response to histamine and serotonin stimulation could be the result of stimulated Na(+)-K+ ATPase activity.     

Na+ K+ ATPase activity in mast cells from the pleural and peritoneal cavities of the rat

ATPase activity in rat mast cells was studied, assuming that pleural and peritoneal mast cells are different populations. Mast cells were purified with Percoll. Enzymatic activity was found to be 36% higher in pleural cells that in peritoneal cells. Moreover, for both populations results show a Na+-K+ ATPase activity either low or slightly inhibited by ouabain.     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.     

Regulation of Na+ transport in brown adipose tissue.

In order to test the hypothesis that Na+, K+-ATPase (Na+,K+-dependent ATPase) is involved in the noradrenaline-mediated stimulation of respiration in brown adipose tissue, the effects of noradrenaline on Na+,K+-ATPase in isolated brown-fat-cell membrane vesicles, and on 22Na+ and K+ (86Rb+) fluxes across the membranes of intact isolated cells, were measured. The ouabain-sensitive fraction of the K+-dependent ATPase activity in the isolated membrane-vesicle preparation was small and was not affected by the presence of noradrenaline in the incubation media. The uptake of 86Rb+ into intact hormone-sensitive cells was inhibited by 80% by ouabain, but it was insensitive to the presence of noradrenaline. 22Na+ uptake and efflux measured in the intact cells were 8 times more rapid than the 86Rb+ fluxes and were unaffected by ouabain. This indicated the presence of a separate, more active, transport system for Na+ than the Na+,K+-ATPase. This is likely to be a Na+/Na+ exchange activity under normal aerobic conditions. However, under anaerobic conditions, or conditions simulating anaerobiosis (2 mM-NaCN), the unidirectional uptake of Na+ increased dramatically, while efflux was unaltered.     

Localization of a Mg2+-activated ATPase in the plasma membrane of Trypanosoma cruzi

The Wachstein and Meisel incubation medium was used to detect ATPase activity in epimastigote, spheromastigote (amastigote), and bloodstream trypomastigote forms of Trypanosoma cruzi. Reaction product, indicative of enzyme activity, was associated with the plasma membrane covering the cell body and the flagellum of the parasite. No reaction product was found in the portion of the plasma membrane lining the flagellar pocket. The plasma membrane-associated ATPase activity was not inhibited by ouabain or oligomycin, was detected in incubation medium without K+, was inhibited by prolonged glutaraldehyde fixation, and its activity was diminished when Mg2+ was omitted from the incubation medium. The Ernst medium was used to detect Na+-K+-ATPase activity in T. cruzi. No reaction product indicative of the presence of this enzyme was detected. Reaction product indicative of 5'-nucleotidase was not detected in T. cruzi. Acid phosphatase activity was detected in lysosomes. Those results indicate that a Mg2+-activated ATPase is present in the plasma membrane of T. cruzi and that it can be used as an enzyme marker, provided that the mitochondrial and flagellar ATPases are inhibited, to assess the purity of plasma membrane fractions isolated from this parasite.     

Sodium-stimulated ATPase in Streptococcus faecalis.

We measured Na+-stimulated ATPase activity in a mutant of Streptococcus faecalis defective in the generation of proton motive force. The activity in membrane vesicles was 62.1 +/- 5.9 nmol of phosphate produced per min per mg of protein when cells were grown on medium containing 0.12 M Na+. Activity decreased as the concentration of Na+ in the growth medium decreased. The decrease in enzyme activity corresponded to the decrease in transport activity for Na+ in both whole cells and membrane vesicles. The effects of pH on both activities were identical. Thus, it is suggested that Na+ movement is mediated by this enzyme. Sodium extrusion and ATPase activity in the wild-type strain were markedly lower than those observed in the mutant strain. Elevated activities of both Na+ extrusion and Na+-stimulated ATPase could be detected in the wild-type strain when cells were grown in the absence of proton motive force. Thus, we propose that the level of ATPase is increased by dissipation of the proton motive force.     

Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H(+)-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na(+)-K(+)-ATPase (Mayahara et al. 1980) and gastric H(+)-K(+)-ATPase (Fujimoto et al. 1986). K(+)-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na(+)-K(+)-ATPase and Ca2(+)-ATPase. The K(+)-independent NPPase activity was diminished by the inhibitors of H(+)-ATPase such as N-ethylmaleimide (NEM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H(+)-ATPase which plays a role in acidification.     

Calcium transport and calcium-ATPase activity in human lymphocyte plasma membrane vesicles

We have studied Ca transport and the Ca-activated Mg-ATPase in plasma membrane vesicles prepared from normal human lymphocytes. Membrane vesicles that were exposed to oxalate as a Ca-trapping agent accumulated Ca in the presence of Mg2+ and ATP. ADP, AMP, GTP, UTP, ITP, TTP, or CTP did not substitute for ATP in energizing uptake. The Vmax for Ca uptake was 2.4 pmol of Ca/micrograms of protein/min, and the Km values for Ca and ATP were 1.0 and 80 microM, respectively. One microM A23187, added initially, completely inhibited net Ca uptake and, if added later, caused the release of Ca accumulated previously. Cyanide, oligomycin, ouabain, or varying Na+ or K+ concentrations had no effect on Ca uptake. A Ca-activated ATPase was present in the same membrane vesicles, which had a Vmax of 25 pmol of Pi/micrograms of protein/min at a free Ca concentration of 4-5 microM. This Ca-ATPase had Km values for Ca and ATP of 0.6 and 90 microM, respectively. These kinetic parameters were similar to those observed for uptake of Ca by the vesicles. The Ca-ATPase activity was insensitive to azide, oligomycin, ouabain, or varying Na+ or K+ concentrations. No Ca-activated hydrolysis of GTP or UTP was observed. Both Ca transport and the Ca-ATPase activity of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-treated lymphocyte plasma membranes were stimulated 2-fold by a cytoplasmic component (calmodulin) that was purified 500-fold from lymphocyte cytoplasm. Thus, human lymphocyte plasma membranes have both a Ca transport activity and a Ca-stimulated ATPase activity with similar substrate affinities and specificities and similar sensitivities to calmodulin.     

Schistosoma mansoni: preparation, characterization of (Na+ + K+)ATPase from tegument and carcass

The preparation and some biochemical properties of a (Na+ + K+)ATPase from male adult Schistosoma mansoni are described. After incubation in a membrane disruption medium, the tegument and carcass of the worms were separated and treated to obtain fractions enriched in (Na+ + K+)ATPase. The activity of the tegumental ouabain sensitive (Na+ + K+)ATPase at 37 C was 20.3 mumole Pi X mg-1 protein X hr-1 and represented 32% of the total ATPase activity. The (Na+ + K+)ATPase prepared from the carcass had a lower specific activity (3.7 mumole Pi X mg-1 protein X hr-1) but a higher relative activity (55%). Similar concentrations of Na+ and K+ activated the enzymes from both sources, and both enzymes were inhibited by similar concentrations of calcium. However, the enzyme from carcass was ten times more sensitive to ouabain than the enzyme from tegument. Comparison with results obtained on the (Na+ + K+)ATPase of human heart showed that the enzymes from the worms were more resistant to ouabain. The half maximal inhibitory concentration of dihydroouabain compared to that of ouabain was also different in the enzymes from human and worm. We conclude that (1) there exists at least one structural difference between the (Na+ + K+)ATPase of S. mansoni and that of the human host, and (2) it is useful to separately study the enzymes from tegument and carcass because they differ in sensitivity to cardiac glycosides.     

K+ transport in resting rat hind-limb skeletal muscle in response to paraxanthine, a caffeine metabolite

This study tested the hypothesis that paraxanthine, a caffeine metabolite, stimulates skeletal muscle potassium (K+) transport by an increase in Na+ -K+ ATPase activity. The unidirectional transport of K+ into muscle (J(in)K) was studied using a perfused rat hind limb technique. Using 12 hind limbs, we examined the response to 20 min of paraxanthine perfusion (0.1 mM), followed by 20 min perfusion with 0.1 mM paraxanthine and 5 mM ouabain (n = 5) to irreversibly inhibit Na+ -K+ ATPase activity. Paraxanthine stimulated J(in)K by 23+/-5% within 20 min. Ouabain abolished the paraxanthine-induced stimulation of J(in)K, suggesting the increase in K+ uptake was due to activation of the Na+ -K+ ATPase. To confirm the role of the Na+ -K+ ATPase, 14 hind limbs were perfused for 20 min with 5 mM ouabain prior to 20 min perfusion with 0.1 mM paraxanthine and 5 mM ouabain (n = 6). Ouabain alone resulted in a 41+/-7% decrease in J(in)K within 15 min. Inhibition of ouabain-sensitive J(in)K prevented the paraxanthine-induced increase in J(in)K. Hind limbs (n = 3) were also perfused with 0.1 mM paraxanthine for 60 min to examine the response to longer duration paraxanthine perfusion. The paraxanthine-induced increase in J(in)K continued for the entire 60 min. In another series, hind limbs were perfused with 0.01 (n = 9), 0.1 (n = 9), or 0.5 (n = 6) mM paraxanthine for 15 min. There was no concentration-dependent relationship between J(in)K and paraxanthine concentration, and 0.01, 0.1, and 0.5 mM paraxanthine increased J(in)K similarly (25+/-5, 22+/-4, and 27+/-6%, respectively). The effect of paraxanthine on J(in)K could not be reversed by subsequent perfusion with paraxanthine-free perfusate. Caffeine (0.05-1.0 mM) had no effect on K+ transport. It is concluded that paraxanthine increases J(in)K in resting skeletal muscle by stimulating ouabain-sensitive Na+ -K+ ATPase activity.     

Aniseed oil increases glucose absorption and reduces urine output in the rat

Anise (Pimpinella anisum) has been used as a traditional aromatic herb in many drinks and baked foods because of the presence of volatile oils in its fruits commonly known as seeds. Hot water extracts of the seeds have been used also in folk medicine for their diuretic and laxative effect, expectorant and anti-spasmodic action, and their ability to ease intestinal colic and flatulence. The aim of this work was to study the effect of aniseed oil on transport processes through intestinal and renal epithelia and determine its mechanism of action. The essential oils were extracted from the seeds by hydrodistillation and analyzed by gas chromatography. Aniseed oil enhanced significantly glucose absorption from the rat jejunum and increased the Na+-K+ ATPase activity in a jejunal homogenate in a dose dependent manner. The oil, however, exerted no effect on water absorption from the colon and did not alter the activity of the colonic Na+-K+ ATPase. When added to drinking water, it reduced the volume of urine produced in the rat and increased the activity of the renal Na+-K+ ATPase even at extremely low concentrations. It was concluded that aniseed oil increases glucose absorption by increasing the activity of the Na+-K+ ATPase and consequently the sodium gradient needed for the sugar transport. Its anti-diuretic effect is also mediated through a similar mechanism in the kidney whereby a stimulation of the Na+-K+ pump increases tubular sodium reabsorption and osmotic water movement. The colonic Na+-K+ ATPase was however, resistant to the oil.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

TNF-alpha down-regulates the Na+-K+ ATPase and the Na+-K+-2Cl-cotransporter in the rat colon via PGE2

TNF-alpha is believed to play a pivotal role in the pathogenesis of inflammatory bowel diseases which have diarrhea as one of their symptoms. This work studies the effect of the cytokine on electrolyte and water movements in the rat distal colon using an intestinal perfusion technique and attempts to determine its underlying mechanism of action. TNF-alpha inhibited net water and chloride absorption, down-regulated in both surface and crypt colonocytes the Na+-K+-2Cl- cotransporter, and reduced the protein expression and activity of the Na+-K+ ATPase. Indomethacin up-regulated the pump and the cotransporter in surface cells but not in crypt cells, and in its presence, TNF-alpha could not exert its effect, suggesting an involvement of PGE2 in the cytokine action. The effect of TNF-alpha on the pump and symporter was studied also in cultured Caco-2 cells in isolation of the effect of other cells and tissues, to test whether the cytokine acts directly on intestinal cells. In these cells, TNF-alpha and PGE2 had a similar effect on the pump expression and activity as that observed in crypt cells but were without any effect on the Na+-K+-2Cl- cotransporter. It was concluded that the effect of the cytokine on colonocytes is mediated via PGE2. By inhibiting the Na+-K+ ATPase, it reduces the Na+ gradient needed for NaCl absorption, and by down-regulating the expression of the Na+-K+-2Cl- symporter, it reduces basolateral Cl- entry and luminal Cl- secretion. The inhibitory effect on absorption is more significant than the inhibitory effect on secretion resulting in a decrease in net electrolyte uptake and consequently in more water retention in the lumen.     

Effect of inhibition of Na+-K+ ATPase on the prostacyclin generation of cultured human vascular endothelial cells

Prostacyclin (PGI2) generation of cultured human vascular endothelial cells (VEC) was observed coincidentally with the increase of 45Ca net influx. Ca ionophore A23187 enhanced not only PGI2 generation and 45Ca net influx but also 45Ca efflux. PGI2 generation was completely abolished by the pretreatment with Ca++ immobilizer, TMB-8. A Na+-K+ ATPase inhibitor, ouabain increased 45Ca net influx, but decreased 45Ca efflux, and enhanced PGI2 generation. These observation indicate that PGI2 generation of VEC may be regulated by not only Ca++ but also Na+, and it was suggested that enhanced PGI2 generation by ouabain might be derived from the increased cytosolic Ca++concentration by the decreased Ca++ efflux, and it was considered to be originated from the suppression of Na+-Ca++ exchange systems by the increased intracellular Na+ concentration via inhibition of Na+-K+ ATPase activity by ouabain. Enhancement of PGI2 generation of VEC by the increased ouabain like substances (OLS) in hypertension is suspected to be beneficial on the maintenance of vascular homeostasis.