Segmental heterogeneity of basal and aldosterone-induced electrogenic Na transport in human colon

Recent in vitro studies in human colon have demonstrated marked segmental differences in electrogenic Na transport. In the present study, the Na channel blocker amiloride was used further to characterise basal and aldosterone-induced electrogenic Na transport in isolated human distal and proximal colon. Bathed in NaCl Ringer solution, distal and proximal colon exhibited similar basal electrical properties, but the amiloride-sensitive short-circuit current (I _sc) was 200% greater in the distal than in the proximal segment. Bathed in choline-Cl Ringer solution, totalI _sc decreased by 97% in distal colon and by 88% in proximal colon, indicating that Na dependent transport process(es) account almost entirely for theI _sc in both segments. Substituting Na₂SO₄ for NaCl Ringer solution (i) increased amiloride-sensitiveI _sc by 56% (p<0.01) in distal colon but had no effect on amiloride-sensitiveI _sc in proximal colon, and (ii) decreased amiloride-insensitiveI _sc in distal and proximal colon by 52% (p<0.05) and 81% (p<0.001) respectively. After the addition of nystatin to the apical membrane, the relationship between totalI _sc and mucosal Na concentration indicated that the activity of the basolateral membrane Na pump was similar in both colonic segments. In a further series of experiments, exposure of distal colon to 1 mol/l aldosterone for 5 h increased totalI _sc by 52% (p<0.05), which reflected stimulation of its amiloride-sensitive component; in contrast, aldosterone had no effect on proximal colon. These results indicate that Na-dependent electrogenic processes (with electrogenic Na transport predominant) are present throughout human colon, but there is marked segmental variability in the Na conductive properties of the apical membrane. Apical Na entry in distal colon occurs mainly through classical amiloride and aldosterone-sensitive Na channels. In contrast, the predominant apical Na entry mechanism in proximal colon is an amiloride and aldosterone-insensitive path-way.     

Actions of the CI- channel blocker NPPB on absorptive and secretory transport processes of Na+ and CI- in rat descending colon

The effects of the Cl^- channel blocker, NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), on the transport of Na⁺ and Cl^- in the descending colon of the rat were studied in the Ussing chamber. In control tissue, NPPB administered at the mucosal side of the epithelium increased the short-circuit current (I_sc) and inhibited the unidirectional mucosa-to-serosa fluxes of Na⁺ and CI^-. In HCO^-₃-_- or Cl^--free media or in the presence of SITS (4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulphonic acid), this increase in I_sc caused by mucosal NPPB was not observed. The serosal administration of NPPB was without effect. Mhdcosal NPPB (10^--⁴ mol l^-1hd decreased the forskolin-induced increase in I_sc by only about 60%. However, the activation of the serosa-to-mucosa flux of Cl^- caused by forskolin was inhibited completely. NPPB decreased the mucosa-to-serosa fluxes of Na⁺ and Cl^- reduced additionally by forskolin. Serosal NPPB decreased I_sc and J^Na_sm, but had no effect on J^Na_sm or J^Na_sm. In HCO^-₃-free buffer the increase in I_sc induced by forskolin was inhibited completely by NPPB. The inhibition of Cl^- secretion by NPPB fits well with the capacity of the drug to block Cl^- channels. For the inhibition of neutral NaCl absorption two sites of action are discussed: an interaction with the Cl^-/HCO^-₃ exchanger or an interference with the extrusion of Cl^- through the basolateral membrane.,     

Measurement of Na K ATPase activity in segments of proximal tubules from superficial and juxtamedullary rat nephrons during antidiuresis

Summary The role of active sodium transport in the partes rectae of subcortical and juxtamedullary proximal nephrons has not yet been established. Recent knowledge of the histotopographical positions of both types of pars recta has now enabled them to be accurately distinguished and isolated from freeze-dried sections. Na K ATPase activity was measured in single dissected tubular segments by an ultramicro method. No significant difference in Na K ATPase activity in the partes rectae from subcortical and juxtamedullary nephrons could be detected.     

Active sulfate reabsorption in the proximal convolution of the rat kidney: Specificity,Na+ and HCO 3 − dependence

Using the standing droplet technique in the proximal convolution and simultaneous microperfusion of the peritubular capillaries, the decrease in luminal sulfate concentration with time and the zero net flux transtubular concentration difference of sulfate ( ) at 45 s was determined — the latter being taken as a measure of the rate of active sulfate reabsorption. Starting with 0.5 mmol/l sulfate in both perfusates the value of 0.35 mmol/l was approached exponentially with a half value time of 4.3 s. The values in the early proximal and late proximal convolution did not deviate from each other. If the Na⁺ concentration in the perfusates was reduced, the approached zero and extrapolated to a slightly negative value (c _i>c _o). When 1 mmol/l ouabain was added to the perfusates decreased by 66% (the latter experiments were performed in the golden hamster which is more sensitive to ouabain than the rat). 1 mmol/l thiosulfate diminished by 68% and 1 mmol/l molybdate by 24%. Omitting or replacing bicarbonate by HEPES or glycodiazine reduced the sulfate reabsorption significantly, while acetazolamide (0.1 mmol/l) and increasing the CO₂-pressure from 4.66 to 14.0 kPa (i.e. 5–15% CO₂) had no effect. SITS 1 mmol/l had no effect on sulfate reabsorption.The data indicate that the sulfate reabsorption is driven by a Na⁺ gradient and inhibited by thiosulfate and molybdate, i.e. molecules which have a similar tetrahedral molecule structure. The sulfate reabsorption depends in an undefined manner on the presence of bicarbonate ions.     

Renal Na+, K+-ATPase in Dahl salt-sensitive rats: K+ dependence,effect of cell environment and protein kinases

Na⁺, K⁺-ATPase in renal epithelial cells plays an important role in the regulation of Na⁺ balance, extracellular volume and blood pressure. The function of renal Na⁺, K⁺-ATPase in Dahl salt-sensitive (DS) rats, an animal model for salt-sensitive hypertension, and Dahl salt-resistant (DR) rats has been studied. In Na⁺, K⁺-ATPase partially purified from renal cortex, affinities and the Hill coefficients for Na⁺ and K⁺ activation were similar in DS and DR rats. Only one component of low ouabain affinity site was found in both strains, indicating the presence of the al isoform. Protein kinase C and cAMP-dependent protein kinase phosphorylated Na⁺, K⁺-ATPase α subunit in DS and DR rats, and the phosphorylation by protein kinase C was associated with an inhibition of enzyme activity. The kinetic parameters for K⁺ activation were also studied in a preparation of basolateral membranes and were found to be similar in DS and DR rats. In a preparation of cortical tubule cells, Na⁺, K⁺-ATPase activity was determined as ouabain-sensitive oxygen consumption (OS Qo₂). Maximal OS Qo₂, measured in Na⁺ loaded cells, was the same in DS and DR rats. The K₀₆ for K⁺ was significantly lower in DS than DR rats (0.163 ±0.042 vs. 0.447 + 0.061 mM, P < 0.05), indicating that factors regulating Na⁺, K⁺-ATPase activity in intact cells are altered in DS rats. Kinetic parameters for Na⁺ activation in cells were the same in both strains. In summary, the function of renal Na⁺, K⁺-ATPase molecule is not altered in DS rats. The intracellular systems that regulate renal Na⁺, K⁺-ATPase activity might be different in DS and DR rats.     

High salt diet down-regulates proximal tubule Na+, K+-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats: evidence of defective dopamine regulation

Nishi , A., Bertorello , A. M. & Aperia , A. 1992. High salt diet down-regulates proximal tubule Na⁺, K⁺-ATPase activity in Dahl salt-resistant but not in Dahl salt-sensitive rats; evidence of defective dopamine regulation. Acta Ptiysiol Scand 144 , 263–267. Received 26 July 1991, accepted 25 October 1991. ISSN 0001–6772. Department of Paediatrics, Karolinska Institute, Sweden We examined the regulation of Na⁺, K⁺-ATPase activity in proximal tubule segments during a high salt diet in prehypertensive Dahl salt-sensitive and salt-resistant rats. Rats were placed on normal salt or high salt diets (0.9% saline as drinking water). During the normal salt diet, Na⁺, K⁺-ATPase activity was not different between Dahl salt-sensitive and salt-resistant rats. After 2 days and 10 days on a high salt diet, Na⁺, K⁺-ATPase activity in Dahl salt-resistant rats significantly decreased when compared to Dahl salt-resistant rats on a normal salt diet (P < 0.01). The decreased Na⁺, K⁺-ATPase activity in Dahl salt-resistant rats during a high salt diet was reversed by treatment with an inhibitor of aromatic l -amino acid decarboxylase (dopamine synthesizing enzyme), benserazide. In contrast, Na⁺, K⁺-ATPase activity did not decrease during the high salt diet and benserazide had no effect on Na⁺, K⁺-ATPase activity in Dahl salt-sensitive rats. These results indicate that Dahl salt-sensitive rats do not have the capacity to down-regulate the proximal tubule Na⁺, K⁺-ATPase activity during a high salt diet. Indirect evidence suggests that the regulation of Na⁺, K⁺-ATPase activity by locally produced dopamine is absent in Dahl salt-sensitive rats.     

Tubular transport processes in proximal tubules of hypothyroid rats. Lack of relationship between thyroidal dependent rise of isotonic fluid reabsorption and Na+−K+-ATPase activity

Hypothyroid rats reconstituted with 10 g/kg b.w. per day of tri-iodothironine (T₃) for 4 days resulted in normal free T₃ and TSH levels. FT₃ levels were: 0.53±0.3 pg/ml in hypothyroid rats; 2.78±1.21 pg/ml in hormone reconstituted rats and 2.90±0.90 pg/ml in euthyroid rats. TSH levels were 3,508±513 g/ml in hypothyroid rats; 1,008±204 g/ml in reconstituted rats and 270±184 ng/ml in euthyroid rats.When hypothyroid rats were reconstituted with 50 g T₃/kg b.w. per day, TSH levels were nearly normal after 4 days (1,157±621 ng/ml). However FT₃ levels after 1–4 days were always higher than in euthyroid rats.Hypothyroid rats show a decrease in isotonic fluid reabsorption (J _v) in the proximal tubule (1.50±0.08 versus 4.96±0.23 10^–2 nl·mm^–1·s^–1 in euthyroid animals). 1 day after T₃ (10 g/kg b.w./day) injectionJ _v was increased significantly to 2.05±0.20 10^–2 nl·mm^–1·s^–1 and continued to increase during 4 days of T₃ reconstitution.When 50 g T₃/kg b.w./day was used,J _v increased to 2.75±0.07 after 1 day and to 3.10±0.42 10^–2 nl·mm^–1·s^–1 after 4 days.J _v was never reaching a value close to that of euthyroid rats because the tubular radius in hypothyroid rats (14.7±1.8 m) is less than that of euthyroid rats (19.2±0.5 m). The radius in hypothyroid rats treated with T₃ was unchanged over a 4 day course with either high or low doses of T₃.Na⁺–K⁺-ATPase activity was found to be 2.91±0.16 M P_i/h×mg protein in homogenates of kidney cortex from hypothyroid rats. Treatment of hypothyroid rats with 10 g or 50 g of T₃ resulted in an initial decrease in ATPase activity, followed by an increase to base level in hypothyroid rats with 10 g and a significantly higher level with 50 g. This decrease in ATPase activity was contrasted to the increase inJ _v.These data indicate that there is a dissociation between the effects of physiological doses of thyroid hormones on proximal tubular reabsorption and the effects of T₃ on Na⁺–K⁺-ATPase activity of kidney cortex. This leads to question the relationship between sodium transport and ATPase activity under physiological doses of thyroid hormones. An early effect of physiological doses of thyroid hormones on brush border Na⁺ permeability is suggested.     

Electron microprobe analysis of proximal tubule cellular Na,Cl and K element concentrations during acute mannitol-saline volume expansion in rats: evidence for inhibition of the Na pump

It has previously been shown that during mannitol-saline volume expansion (VE) Na transport was inhibited 50% by harvested proximal tubular fluid without a change in paracellular shunt pathway permeability to Na. To determine whether this inhibition was due to changes in cellular entry step or an effect on the pump itself, intracellular element concentrations were measured by electron microprobe X-ray ranalysis in proximal tubular cells of control (non-expanded, NE) and VE rats. Na _i , Cl _i and phosphorus _i were increased (mean±S.E.) from 19.3±0.8 to 23.4±0.6, 15.8±0.4 to 21.3±0.4 and 124.3±2.6 to 138.0±1.8 mmol · kg^–1 wet weight (P<0.001) respectively while K _i remained unchanged: 122.9±2.2 and 124.2±1.3 mmol · kg^–1 wet weight. The increases in Na _i and Cl _i were in excess of cell shrinkage produced by the hyperosmolal peritubular environment while the unchanged K _i in the face of cell shrinkage indicates and actual loss. It is concluded that mannitol-saline VE inhibits the Na pump producing a rise in Na _i and a fall in K _i .     

Regulation of Post-Tetanic Increases in the Cholinosensitivity of Neurons in the Common Snail by the Na,K Pump: The Role of Na/Ca Exchange and Calcium Mobilization

Neuroscience and Behavioral Physiology -     

The cardioplegic solution HTK: effects on membrane potential,intracellular K+ and Na+ activities in sheep cardiac Purkinje fibres

The effects of the cardioplegic solution HTK on membrane potential (E_M) and intracellular K and Na activities (a _K ⁱ , a _Na ⁱ ) were studied in sheep cardiac Purkinje fibres by means of conventional and ion-selective microelectrodes. HTK contains (mM): Na 15, K 10, Ca 0, Mg 4, histidine 180, (1) In control conditions E_M was –74.3±3.3 mV (n=25), a _K ⁱ was 116.4±4.1 mM (n=7) and a _Na ⁱ was 8.2±1.4 mM (n=15). (2) Exposure to HTK led to a depolarization to –59.7±3.6 mV (n=25) which exceeded by about 5–7 mV that induced in a Tyrode solution of 10 mM K and in a modified HTK solution supplemented by 2 mM Ca (n=6). (3) Addition of 0.5 mM barium eliminated the difference in the steady-state depolarization. (4) HTK superfusion increased a _K ⁱ to 120.1±4.4 mM (n=7) and decreased a _Na ⁱ to 3.9±0.9 mM (n=15). (5) The decrease in a _Na ⁱ was insensitive to amiloride (1 mM) and to external alkalization but was slightly increased by addition of 2 mM calcium. (6) When the calcium in Tyrode solution was lowered from 2.0 mM to 0.05 mM, a _Na ⁱ hardly decreased during subsequent exposure to unmodified HTK and it increased in the presence of 0.1 mM dihydroouabain. We propose the hypothesis (1) that the difference in membrane depolarization between HTK and a 10 mM K-Tyrode is caused by a decrease in K conductance by the HTK solution and (2) that the a _Na ⁱ decline mainly results from a coupled Ca influx via Na-Ca exchange due to a delayed washout of external calcium.This work was supported by the Deutsche Forschungsgemeinschaft, SFB 330 — Organprotektion     

Transepithelial transport of K+, NH 4 + , inorganic phosphate and water by hen (Gallus domesticus) lower intestine (colon and coprodeum) perfused luminally in vivo

The colon and coprodeum of anaesthetised laying hens was perfused luminally with solutions of varying compositions to elucidate the influence of three levels of dietary Na⁺ and of luminal fluid composition on transport of electrolytes and water. Net rates of secretion of K⁺ and absorption of NH ₄ ⁺ were increased in response to dietary Na⁺ depletion and increased luminal Na⁺ concentrations, but these fluxes were linked to Na⁺ transport and no dietary or perfusion treatment effects could be detected on them independently of effects on Na⁺ flux. Similar results were found for Na⁺-linked water absorption from isosmotic perfusates. Serosal-mucosal net osmotic flow with Na⁺-free perfusates was significantly reduced by Na⁺-depletion, but no significant dietary effect could be detected on the serosal-mucosal osmotic permeability coefficient. The reflexion coefficient was effectively unity. Mucosal-serosal inorganic phosphate flux was small. The flux was independent of Na⁺ flux and dietary Na⁺ levels, and apparently facilitated by serosalmucosal osmotic flow. The data allowed determination of the absorption/secretion in coprodeum and colon of the ions investigated, as compared to the renal excretion rate.     

The protein tyrosine kinase pathway is not involved in the regulation of K+ transport across the rat colon

The protein tyrosine kinase inhibitor, genistein, is known to activate the cystic fibrosis transmembrane regulator (CFTR) Cl^? channel and to inhibit K⁺ currents across the rat colonic epithelium. The aim of the present study is to answer the question whether these effects are involved in the regulation of transepithelial K⁺ transport. Therefore, the action of genistein on K⁺ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake and efflux of Rb⁺ in mucosa-submucosa preparations. All effects of genistein (5 × 10^?5 mol L^?1) were tested in the presence of a low concentration of forskolin (2 × 10^?7 mol L^?1), because prestimulation of the cAMP pathway has been shown to be a prerequisite for a secretory action of genistein. Forskolin caused an increase in the serosa-to-mucosa flux of Rb⁺ (J ^Rb_sm) thereby stimulating net K⁺ secretion in the proximal and distal colon. None of these effects was further enhanced after administration of genistein. Neither mucosal uptake of Rb⁺, representing mainly the activity of the H⁺-K⁺-ATPase in the distal colon, nor serosal Rb⁺ uptake, representing, e.g. the activity of the Na⁺-K⁺-2Cl^? cotransporter, were affected by genistein. Also the efflux of Rb⁺ across the apical or the basolateral membrane, an indicator for the apical and basolateral K⁺ conductance, was unchanged in the presence of genistein. These results demonstrate that the K⁺ channels inhibited by genistein are not involved in transepithelial K⁺ transport.     

Corticosteroid action on choroid plexus: Reduction in Na+—K+-ATPase activity,choline transport capacity,and rate of CSF formation

Summary Glucocorticoids have a well-known clinical effect on brain edema and intracranial hypertension, but the mechanism of action is still poorly understood. In the present report the effect of betamethasone on choroid plexus transport and CSF formation was studied. Following 5 days of daily treatment with betamethasone the CSF production rate in rabbits was reduced by 43% as measured by ventriculo-cisternal perfusion with radioactive inulin. Accordingly, the transport capacity in the choroid plexus, measured in terms of choline uptake and accumulation in vitro, and the activity of Na⁺—K⁺-ATPase decreased in both rabbit (in the lateral ventricles by 31 and 31%, respectively) and rat (by 16 and 24%, respectively). Thus, the demonstrated influence of glucocorticoids on these functions of the choroid plexus seem to be important components in their therapeutic effect on intracranial hypertension.