Mechanisms of chloride influx during KCl-induced swelling in the chicken retina

An increase in extracellular KCl ([KCl]_o) occurs under various pathological conditions in the retina, leading to retinal swelling and possible neuronal damage. The mechanisms of this KCl_o-induced retinal swelling were investigated in the present study, with emphasis on the Cl^– transport mechanisms. Increasing [KCl]_o (from 5 to 70 mM) led to concentration-dependent swelling in chicken retinas. The curve relating the degree of swelling to [KCl]_o was biphasic, with one component from 5 to 35 mM [KCl]_o and another from 35 to 100 mM. As Cl^– omission prevented swelling in all conditions, the effect of cotransporter or Cl^– channel blockers was examined to investigate the mechanisms of Cl^– influx. The cotransporter blockers bumetanide and DIOA reduced swelling by 68% and 76%, respectively at [KCl]_o 25 mM (K25), and by 14–17% at [KCl]_o 54 mM (K54). The Cl^– channel blockers NPPB and niflumic acid did not affect swelling at K25 but reduced it by 90–95% at K54 (NPPB IC₅₀ 60.7 µM). Furosemide showed an atypical effect, decreasing swelling by 14% at K25 and by 95% at K54 (IC₅₀ 173.9 µM). Na⁺ omission decreased swelling at K25 but not at K54. These results suggest the contribution of cotransporters to Cl^– influx at K25 and of Cl^– channels at K54. At K25, swelling was found in the ganglion cell layer and in the lower half of the inner nuclear layer. With K54, swelling occurred in all inner retinal layers. The ganglion cell layer swelling was due to both Müller cell end-foot and ganglion cell soma swelling. K54 also induced ganglion cell damage as shown by disorganized, pyknotic and refringent nuclei.     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)     

Evidence from O2 uptake measurements for Na+−K+−2 Cl− co-transport in the rabbit submandibular gland

There is evidence that the production of primary saliva by acinar cells is a consequence of Na⁺–Cl^– co-transport but more recently it has been proposed that in fact Na⁺–K⁺–2 Cl^– co-transport is responsible. The latter would be energetically more efficient and the present experiments were designed to measure the stoichiometry of acinar secretion in order to distinguish between these two mechanisms.Submandibular salivary glands from anaesthetised rabbits were isolated vascularly and oxygen consumption measured from the oxygen content of arterial inflow and venous effluent blood and the total flow through the gland. Measurements were made in the steady-state at rest and during different secretion rates induced by parasympathetic nerve stimulation. The rate of sodium transport across the acinar and ductal epithelium was determined from plasma and salivary sodium concentration and salivary flow rate.Multiple regression analysis of this data showed that 22.1 mol Na⁺ was secreted per mol O₂ consumed while 11.9 mol Na⁺ was reabsorbed per mol O₂ consumed. Since acinar secretion is energetically about twice as efficient as ductal absorption, a mechanism for Na⁺ transport other than that for tight epithelia must be involved. Na⁺K⁺–2 Cl^– co-transport is thus more likely than Na⁺–Cl^– and it is suggested that Na⁺–K⁺–2 Cl^– co-transport is the main mechanism involved in salivary acinar secretion.     

Occurrence and properties of a (Na+−K+)-activated ATPase in the mucosa of the rat intestine

Summary The existence of an ouabain-sensitive (Na⁺–K⁺)-activated ATPase system has been demonstrated in the total intestine of the rat. The (Na⁺–K⁺)-ATPase activity was about 10–15% of the total ATPase in 4 equal parts of the small intestine; in the colon about 35% of the total ATPase was (Na⁺–K⁺)-activated ATPase. The highest (Na⁺–K⁺)-ATPase activity has been observed in the first and second part of the small intestine, while in the colon the activity was 2–4 times higher than in the ileum.The (Na⁺–K⁺)-ATPase of rat colon required both Na⁺ (K _m=8.3 mmoles/l) and K⁺ (K _m=0.6 mmoles/l). Maximal activation of the (Na⁺–K⁺)-ATPase system required 2 mM Mg²⁺ at an ATP concentration of 2 mM. The pH optimum for (Na⁺–K⁺)-ATPase of rat colon was 7.5, while the Mg²⁺-activated ATPase activity had a pH optimum of 8.6. The (Na⁺–K⁺)-ATPase was inhibited by ouabain (pI ₅₀=3.6).The relation between the differences in (Na⁺–K⁺)-ATPase activity and Na⁺-absorption on different parts of the intestine is discussed.     

Ba2+ and amiloride uncover or induce a pH-sensitive and a Na+ or non-selective cation conductance in transitional cells of the inner ear

The membrane potential V _m the cytosolic pH (pH_i), the transference numbers (t) for K⁺, Cl^– and Na⁺/ non-selective cation (NSC) and the pH-sensitivity of V _m were investigated in transitional cells from the vestibular labyrinth of the gerbil. V _m, pH_i, , and the pH_i sensitivity of V _m were under control conditions were –92±1 mV (n=89 cells), pH_i 7.13±0.07 (n=11 epithelia), 0.87±0.02 (n=22), 0.02±0.01 (n=19), 0.01±0.01 (n=24) and –5 mV/pH unit (n=13 cells/n=11 epithelia), respectively. In the presence of 100 mol/l Ba²⁺ the corresponding values were: –70±1 mV (n=32), pH_i 7.16±0.08 (n=6), 0.31±0.05 (n=4), 0.06±0.01 (n=6), 0.20±0.03 (n=10) and -16 mV/pH-unit (n=15/n=6). In the presence of 500 mol/l amiloride the corresponding values were: –72±2mV (n=34), pH_i 7.00±0.07 (n=5), 0.50±0.04 (n=6), 0.04±0.01 (n=11), 0.28±0.04 (n=9) and –26 mV/pH-unit (n=20/n=5). In the presence of 20 mmol/l propionate plus amiloride the corresponding values were: –61±2 mV (n=27), pH_i 6.72±0.06 (n=5), 0.30±0.02 (n=6), 0.06±0.01 (n=5) and 0.40±0.02 (n=8), respectively. V _m was depolarized and and pH_i decreased due to (a) addition of 1 mmol/l amiloride in 150 mmol/l Na⁺ by 38±1 mV (n=8), from 0.82±0.02 to 0.17±0.02 (n=8) and by 0.13±0.01 pH unit (n=6), respectively; (b) reduction of [Na⁺] from 150 to 1.5 mmol/l by 3.3±0.5 mV (n=30), from 0.83±0.02 to 0.75±0.04 (n=9) and by 0.33±0.07 pH unit (n=4), respectively and (c) addition of 1 mmol/l amiloride in 1.5 mmol/l Na⁺ by 20±1 mV (n=11) and from 0.83±0.03 to 0.53±0.02 (n=5), respectively. These data suggest that the K⁺ conductance is directly inhibited by amiloride and Ba²⁺ and that Ba²⁺ and amiloride uncover or induce a pH-sensitive and a Na⁺/NSC conductance which may or may not be the same entity.Some of the data have been presented at various meetings and appear in abstract form in [31, 35, 37]     

The effect of secretagogues on ion conductances of in vitro perfused,isolated rabbit colonic crypts

Several secretagogues were used in this study, including those which enhance intracellular cyclic adenosine monophosphate (cAMP) production, as well as others which elevate intracellular Ca²⁺ activity and are known to increase Cl^– secretion in the intact colon and in colonic carcinoma cell lines. They were examined with respect to their effects on electrophysiological properties in isolated rabbit distal colonic crypts. Crypts were dissected manually and perfused in vitro. Transepithelial voltage (V _te), transepithelial resistance (R _te), membrane voltage across the basolateral membrane (V _bl), and fractional basolateral membrane resistance (FR _bl), were estimated. Basolateral prostaglandin E₂ (PGE₂, 0.1 mol/l), vasoactive intestinal peptide (VIP, 1 nmol/l) and adenosine (0.1 mmol/l) induced an initial depolarisation and a secondary partial repolarisation of (V _bl). In the case of adenosine, the initial depolarization of (V _bl) was by 31±2 mV (n=47).R _te fell significantly from 16.4±3.6 to 14.2±3.7 ·cm² (n= 6), andFR _blincreased significantly from 0.11±0.02 to 0.51±0.10 (n=6). In the second phase the repolarisation of (V _bl) amounted 11±2 mV (n=47) and a steadystate (V _bl) of –51±2 mV (n=47) was reached.R _te fell further and significantly to a steady-state value of 12.4±3.8 ·cm² (n=6) andFR _bl fell significantly to 0.42±0.13 (n=6). In 30% of the experiments, a transient hyperpolarisation of (V _bl) by 8±2 mV (n=14) was seen during wash out of adenosine. In the presence of adenosine, but not under control conditions, lowering of luminal Cl^– concentration from 120 to 32 mmol/l depolarised (V _bl) significantly by 8±1 mV (n=9). Basolateral ATP and ADP (0.1 mmol/l) led to a short initial depolarisation followed by a sustained and significant hyperpolarisation by 6±2 mV (n=27) and 5±4 mV (n=8), respectively. Carbachol (CCH) hyperpolarised (V _bl) in a concentration-dependent manner. At 100 mol/l (bath) the hyperpolarisation was by 14±2 mV (n=11) andFR _bl fell slightly. Neurotensin (10 nmol/l), isoproterenol (10 mol/l) and uridine 5-triphosphate (UTP, 0.1 mmol/l) had no effect. It is concluded that PGE₂, VIP and adenosine upregulate sequentially a luminal Cl^– conductance and a basolateral K⁺ conductance by increasing intracellular cAMP concentration. Ca²⁺ mobilising hormones such as ATP, ADP, and CCH increase the basolateral K⁺ conductance, while the effect on luminal Cl^– conductance appears to be very limited.     

Effect of high NaCl intake on Na+ and K+ transport in the rabbit distal convoluted tubule

Morphological studies have demonstrated that a chronic increase in distal Na⁺ delivery causes hypertrophy of the distal convoluted tubule (DCT). To examine whether high NaCl-intake also causes functional changes in the well defined DCT, we measured transmural voltage (V _T), lumen-to-bath Na⁺ flux (J _Na(LB)), and net K⁺ secretion (J _K(net)) in DCTs obtained from control rabbits and those on high NaCl-intake diets. The lumen negativeV _T was significantly greater in the high NaCl group than in the control group. The net K⁺ secretion (pmol mm^–1 min^–1) was greater in the high NaCl-intake group (54.1±13.0 vs 14.7±5.6). The K⁺ permeabïlities in both luminal and basolateral DCT membranes, as assessed by the K⁺-induced transepithelial voltage deflection inhibitable with Ba²⁺, were increased in the experimental group. The lumen-to-bath²²Na flux (pmol mm^–1 min^–1) was also greater in the experimental group (726±119 vs 396±65). TheV _T component inhibitable with amiloride was also elevated in the high NaCl-intake group. Furthermore, Na⁺–K⁺-ATPase activity of the DCT was higher in the experimental than in the control group. We conclude that high NaCl intake increases both Na⁺ reabsorption and K⁺ secretion by the DCT. This phenomenon is associated with an increased Na⁺–K⁺-ATPase activity along with increased Na⁺ and K⁺ permeabilities of the luminal membrane, and an increase in the K⁺ permeability of the basolateral membrane. Cellular mechanisms underlying these functional changes remain to be established.     

Electrophysiological study of transport systems in isolated perfused pancreatic ducts: properties of the basolateral membrane

In order to study the mechanism of pancreatic HCO ₃ ^– transport, a perfused preparation of isolated intra-and interlobular ducts (i.d. 20–40 m) of rat pancreas was developed. Responses of the epithelium to changes in the bath ionic concentration and to addition of transport inhibitors was monitored by electrophysiological techniques. In this report some properties of the basolateral membrane of pancreatic duct cells are described. The transepithelial potential difference (PD_te) in ducts bathed in HCO ₃ ^– -free and HCO ₃ ^– -containing solution was –0.8 and –2.6 mV, respectively. The equivalent short circuit current (I_sc) under similar conditions was 26 and 50 A·cm^–2. The specific transepithelial resistance (R_te) was 88 cm². In control solutions the PD across the basolateral membrane (PD_bl) was –63±1 mV (n=314). Ouabain (3 mmol/l) depolarized PD_bl by 4.8±1.1 mV (n=6) within less than 10 s. When the bath K⁺ concentration was increased from 5 to 20 mmol/l, PD_bl depolarized by 15.9±0.9 mV (n=50). The same K⁺ concentration step had no effect on PD_bl if the ducts were exposed to Ba²⁺, a K⁺ channel blocker. Application of Ba²⁺ (1 mmol/l) alone depolarized PD_bl by 26.4±1.4 mV (n=19), while another K⁺ channel blocker TEA⁺ (50 mmol/l) depolarized PD_bl only by 7.7±2.0 mV (n=9). Addition of amiloride (1 mmol/l) to the bath caused 3–4 mV depolarization of PD_bl. Furosemide (0.1 mmol/l) and SITS (0.1 mmol/l) had no effect on PD_bl. An increase in the bath HCO ₃ ^– concentration from 0 to 25 mmol/l produced fast and sustained depolarization of PD_bl by 8.5±1.0 mV (n=149). It was investigated whether the effect of HCO ₃ ^– was due to a Na⁺⁺-dependent transport mechanism on the basolateral membrane, where the ion complex transferred into the cell would be positively charged, or whether it was due to decreased K⁺ conductance caused by lowered intracellular pH. Experiments showed that the HCO ₃ ^– effect was present even when the bath Na⁺ concentration was reduced to a nominal value of 0 mmol/l. Similarly, the HCO ₃ ^– effect remained unchanged after Ba²⁺ (5 mmol/l) was added to the bath. The results indicate that on the basolateral membrane of duct cells there is a ouabain sensitive (Na⁺+K⁺)-ATPase, a Ba²⁺ sensitive K⁺ conductance and an amiloride sensitive Na⁺/H⁺ antiport. The HCO ₃ ^– effect on PD_bl is most likely due to rheogenic anion exit across the luminal membrane.     

Ca2+- and swelling-induced activation of ion conductances in bronchial epithelial cells

The present study was performed to examine Ca²⁺-dependent and cell-swelling-induced ion conductances in a polarized bronchial epithelial cell line (16HBE14o-). Whole-cell currents were measured in fast and slow whole-cell patch-clamp experiments in cells grown either on filters or on coated plastic dishes. In addition the transepithelial voltage (V _te) and resistance (R _te) were measured in confluent monolayers. Resting cells had a membrane voltage (V _m) of –36±1.1 mV (n=137) which was mainly caused by K⁺ and Cl^– conductances and to a lesser extent by a Na⁺ conductance. V _te was apical-side-negative after stimulation. Equivalent short-circuit current (I _sc = V _te/R _te) was increased by the secretagogues histamine (0.1 mmol/l), bradykinin (0.1–10 mol/l) and ATP (0.1–100 mol/l). The histamine-induced I _sc was blocked by either basolateral diphenhydramine (0.1 mmol/l, n=4) or apical cimetidine (0.1 mmol/l, n=4). In fast and slow whole-cell recordings ATP and bradykinin primarily activated a transient K⁺ conductance and hyperpolarized V _m. This effect was mimicked by the Ca²⁺ ionophore ionomycin (1 mol/l, n=11). Inhibition of the bradykinin-induced I _sc by the blocker HOE140 (1 mol/l, n=3) suggested the presence of a BK₂ receptor. The potency sequence of different nucleotide agonists on the purinergic receptor was UTP ATP > ITP > GTP CTP [,-methylene] ATP 2-methylthio-ATP = 0 and was obtained in I _sc measurements and patch-clamp recordings. This suggests the presence of a P_2u receptor. Hypotonic cell swelling activated both Cl^– and K⁺ conductances. The Cl^– conductance was only slightly inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (0.5 mmol/ l, n=3). These data indicate that 16HBE140- bronchial epithelial cells, which are known to express high levels of cystic fibrosis transmembrane conductance regulator protein, form a secretory epithelium. While hypotonic cell swelling activates both K⁺ and Cl^– channels, the Ca²⁺-induced Cl^– secretion is due mainly to activation of basolateral K⁺ channels.     

The localization of the Na+−K+-ATPase in the cells of rat kidney cortex

Summary Plasma membrane fractions of rat kidney cortex were subdivided by centrifugation on a continuous and a discontinuous sucrose gradient and by carrier free continuous electrophoresis. In the different fractions the activity of alkaline phosphatase and aminopeptidase, enzymes which are present in the brushborder membrane, as well as Mg⁺⁺-ATPase, Na⁺–K⁺-ATPase, 5-nucleotidase, acid phosphatase and glucose-6-phosphatase were determined.The distribution of alkaline phosphatase, aminopeptidase and 5-nucleotidase is identical, indicating the localization of these enzymes in the brushborder membrane. Na⁺–K⁺-ATPase does not show an identical distribution with any of the enzymes studied.Using carrier free continuous electrophoresis fractions can be obtained which are enriched in alkaline phosphatase by a factor of 15 when compared to the cortex homogenate, whereas the specific activity of Na⁺–K⁺-ATPase is reduced to one third of the starting material. On the other hand fractions can be isolated in which the specific activity of Na⁺–K⁺-ATPase is 16 times higher than in the homogenate. No enrichment of alkaline phosphatase occurs in these fractions.It is therefore concluded that the Na⁺–K⁺-ATPase is not present in the brushborder membrane nor in the lysosomes or endoplasmatic reticulum. The most probable localization of the Na⁺–K⁺-ATPase are the basal infoldings of the plasma membranes of the cells.A preliminary report has been published by Kinneet al. [28, 29].Major part of this work was done by J. E. Schmitz for his degree of M. D.     

Membrane potentials in Rana temporaria muscle fibres in strongly hypertonic solutions

Conventional microelectrode methods were used to measure variations in resting membrane potentials, E _m, of intact amphibian skeletal muscle fibres over a wide range of increased extracellular tonicities produced by inclusion of varying extracellular concentrations of sucrose. Moderate increases in extracellular tonicity to up to 2.6× normal (2.6τ) under Cl⁻ free conditions produced negative shifts in E _m that followed expectations for the K⁺ Nernst equation (E _K) applied to a perfect osmometer containing a conserved intracellular K⁺ content despite any accompanying cell volume change. In contrast, E _m remained stable in fibres studied in otherwise similar Cl⁻ containing solutions, consistent with E _m stabilization despite negative shifts in E _K through inward cation-Cl⁻ co-transport activity. Short exposures to higher tonicities (>3τ) similarly produced negative shifts in E _m in Cl⁻ free but not Cl⁻ containing solutions. However, prolonged exposures to solutions of >3τ caused gradual net positive changes in E _m in both Cl⁻ containing and Cl⁻ free solutions suggesting that these changes were independent of cation-Cl⁻ transport. Indeed, there was no evidence of cation-Cl⁻ co-transport activity in strongly hypertonic solutions despite its predicted energetic favourability, suggesting its possible regulation by E _m in muscle. Additional findings implicated a failure to maintain greatly increased transmembrane [K⁺] gradients in these E _m changes. Thus: (1) halving or doubling [K⁺]_e produced negative or positive shifts␣in E _m, respectively in isotonic or moderately hypertonic (<2.7τ), but not strongly hypertonic (>3τ) solutions; (2) subsequent restoration of isotonic extracellular conditions produced further positive changes in E _m consistent with a dilution of the depleted [K⁺]_i by fibres regaining their original resting volumes; (3) quantitative modelling similarly predicted a gradual net efflux of K⁺ as the balance between active and passive [K⁺] fluxes altered due to increased transmembrane [K⁺] gradients in hypertonic and low [K⁺]_e solutions. However, the observed positive changes in E _m in the most strongly hypertonic solutions eventually exceeded these predictions suggesting additional limitations on␣Na⁺/K⁺-ATPase activity in strongly hypertonic solutions.James A. Fraser and Kai Yuen Wong have equally contributed to this paper.     

Stimulation of colonic anion secretion by monochloramine: action sites

During inflammatory bowel disease, reactive oxygen metabolites are released by phagocytes reacting with intraluminal NH₃ to produce monochloramine (NH₂Cl). NH₂Cl is assumed to play role in the pathogenesis of inflammation-associated diarrhoea, as it is able to induce intestinal secretion. The aim of the present study was to determine the action sites of NH₂Cl in rat colonic epithelium with Ussing chamber and fura-2 experiments. In intact mucosa, NH₂Cl (5·10^–6–10^–4 mol·l^–1) evoked a concentration-dependent increase in short-circuit current (I_sc), consistent with the induction of anion secretion, as demonstrated by anion substitution and transport blocker experiments. When the apical membrane was permeabilised by the ionophore nystatin, two basolateral action sites of NH₂Cl (5·10^–5 mol·l^–1) could be identified, i.e. an increase in the K⁺ conductance and a stimulation of the Na⁺–K⁺ pump. When tissues were basolaterally depolarised by a high K⁺ concentration, the stimulation of an apical Cl^– conductance by NH₂Cl was observed. In isolated colonic crypts loaded with the Ca²⁺-sensitive fluorescent dye fura-2, NH₂Cl (5·10^–5 mol·l^–1) evoked an increase in the intracellular Ca²⁺ concentration. This increase was independent from the presence of Ca²⁺ in the extracellular medium, but was inhibited by blockade of intracellular sarcoplasmatic, endoplasmatic Ca²⁺-ATPases with cyclopiazonic acid (10^–5 mol·l^–1). The NH₂Cl-evoked Ca²⁺ release was sensitive against inhibition of ryanodine receptors with ruthenium red (5·10^–5 mol·l^–1) and against inhibition of inositol-1,4,5-trisphosphate (IP₃) receptors with 2-aminoethoxydiphenylborate (10^–4 mol·l^–1). Both blockers also inhibited the NH₂Cl-induced increase in I_sc. These results indicate that an intracellular Ca²⁺ release via ryanodine and/or IP₃ receptors is involved in oxidant stimulation of anion secretion in rat colon.     

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.     

Effect of bradykinin and histamine on the membrane voltage,ion conductances and ion channels of human glomerular epithelial cells (hGEC) in culture

The effects of bradykinin (BK) and histamine (Hist) on the membrane voltage (V _m), ion conductances and ion channels of cultured human glomerular epithelial cells (hGEC) were examined with the nystatin patch clamp technique. Cells were studied between passage 3 and 20 in a bath rinsed with Ringer-like solution at 37°C. The mean value of V _m was –41±0.5 mV (n=189). BK (10^–6 mol/l, n=29) and Hist (10^–5 mol/l, n= 55) induced a rapid transient hyperpolarization by 15±1 mV and 18±1 mV, respectively. The hyperpolarization was followed by a long lasting depolarization by 6±1 mV (BK 10^–6 mol/l) and 7±1 mV (Hist 10^–5 mol/l). The ED₅₀ was about 5×10^–8 mol/l for BK and 5×10^–7 mol/l for Hist. In the presence of both agonists, increases of outward and inward currents were observed. A change in the extracellular K⁺ concentration from 3.6 to 30 mmol/l depolarized V _m by 8±1 mV and completely inhibited the hyperpolarizing effect of both agents (n=11). Reduction of extracellular Cl^– concentration from 145 to 30 mmol/l led to a depolarization by 2 ±1 mV (n=25). In 30 mmol/l Cl^– the depolarizations induced by BK (10^–7 mol/l) and Hist (10^–6 mol/l) were augmented to 9±2 mV (n=14) and to 10±2 mV (n=11), respectively. Ba²⁺ (5 mmol/l) depolarized V _m by 19±5 mV (n=6) and completely inhibited the hyperpolarization induced by BK (10^–6 mol/l, n=3) and reduced that of Hist (10^–5 mol/l) markedly (n=3). Preincubation with the K⁺ channel blocker charybdotoxin (1–10 nmol/l) for 3 min had no significant effect on V _m, but reduced markedly the BK(10^–6 mol/l, n=11) and Hist-(10^–5 mol/l, n=6) induced hyperpolarizations. In 10 out of 31 experiments in the cell attached nystatin patch configuration big K⁺ channels with a conductance of 247±17 pS were found. The open probability of these K⁺ channels was increased 3- to 5-fold during the hyperpolarization induced by BK (10^–7 mol/l) or Hist (10^–5 mol/l, both n= 4). In excised inside/out patches this K⁺ channel had a mean conductance of 136±8.5 pS (n=10, clamp voltage 0 mV). The channel was outwardly rectifying and its open probability was increased when Ca²⁺ on the cytosolic side was greater than 0.1 mol/l. The data indicate that BK and Hist activate a and a in hGEC. The hyperpolarization is induced by the activation of a Ca²⁺-dependent maxi K⁺ channel.     

Substructure of membrane-bound Na+−K+-ATPase protein

Purified membrane-bound Na⁺–K⁺-ATPase from rat kidney outer medulla was studied by freeze-fracturing, by freeze-etching and by negative staining. Freeze-fracturing of purified Na⁺–K⁺-ATPase membranes shows intramembraneous particles with a diameter of about 100 Å. The frequency of these intramembraneous particles — as estimated from the particle densities on the two fracture faces — lies between 4700 and 5600 particles per m². Applying rotary shadowing a four partite substructure could be detected in these intramembraneous particles observed on the fracture planes. The same four partite substructure was detected in particles observed on freeze-fractured and rotary shadowed intact baso-lateral plasma membranes of the thick ascending limb of Henle's loop. Particles could be also detected on both membrane surfaces of the purified Na⁺–K⁺-ATPase. These surface particles have about the same diameter and are present at about the same frequency as those observed within the freeze-fractured membranes. Negative staining of isolated Na⁺–K⁺-ATPase membranes showed particles on both membrane surfaces with a diameter between 30 and 50 Å, at a frequency of about 19,000 per m². On aspects of membrane edges we observed structures which suggest a transmembraneous connection of the negatively stained particles on both membrane surfaces.Our results suggest that the Na⁺–K⁺-ATPase protein is composed of four units and that each unit spans the cell membrane. The native enzyme structure of the Na⁺–K⁺-ATPase protein seems to be preserved during freeze-fracturing and freeze-etching. It is proposed that the four enzyme units of the Na⁺–K⁺-ATPase complex are dissociated during the negative staining procedure.Part of this work was presented at the Frühjahrstagung of the Deutsche Physiologische Gesellschaft [6]     

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon,acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (I _sc) induced by prostaglandin E₂ (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC₅₀) at 2 mol/l from the mucosal and at 0.7 mol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 ·cm² to 136±21 ·cm² (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 mol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 mol/l 293 B had no effect on the membrane voltage across the basolateral membrane (V _bl) in non-stimulated crypt cells: –69±3 mV versus –67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba²⁺ depolarised (V _bl) significantly. However, 293 B depolarised (V _bl) significantly in the presence of 1 mol/l forskolin: –45±4mV versus –39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (V _bl) from –40±5 mV to –30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 mol/l 293 B: –75±6 mV versus –75±6 mV (n=6). Also 293 B had no effect on basal K⁺ conductance (n=4). Hence, we conclude that 293 B inhibits the K⁺ conductance induced by cAMP. This conductance is apparently relevant for Cl^– secretion and the basal K⁺ conductance is insufficient to support secretion.     

Actin-dependent activation of ion conductances in bronchial epithelial cells

Activation of Cl^– and K⁺ channels is necessary to drive ion secretion in epithelia. There is substantial evidence from previous reports that vesicular transport and exocytosis are involved in the regulation of ion channels. In the present study we examined the role of cytoskeletal elements and components of intracellular vesicle transport on ion channel activation in bronchial epithelial cells. To this end, cells were incubated with a number of different compounds which interact with either microtubules or actin microfilaments, or which interfere with vesicle transport in the Golgi apparatus. The effectiveness of these agents was verified by fluorescence staining of cellular microtubules and actin. The function was examined in ³⁶Cl^– efflux studies as well as in whole-cell (WC) patch-clamp and cell-attached studies. The cells were studied under control conditions and after exposure to (in mmol/l) ATP (0.1), forskolin (0.01), histamine (0.01) and hypotonic bath solution (HBS, NaCl 72.5). In untreated control cells, ATP primarily activated a K⁺ conductance whilst histamine and forskolin induced mainly a Cl^– conductance. HBS activated both K⁺ and Cl^– conductances. Incubation of the cells with brefeldin A (up to 100 mol/l) did not inhibit WC current activation and ³⁶Cl^– efflux. Nocodazole (up to 170 mol/l) reduced the ATP-induced WC current, and mevastatin (up to 100 mol/l) the cell-swelling-induced WC current. Neither had any effect on the WC current induced by forskolin and histamine. Also ³⁶Cl^– efflux induced by HBS, ATP, forskolin and histamine was unaltered by these compounds. Similarly, colchicine (10 mol/l) and taxol (6 mol/l) affected neither ³⁶Cl^– efflux nor WC current induced by ATP, forskolin, histamine or HBS. In contrast, depolymerisation of actin by cytochalasin D (10 mol/l) significantly attenuated ³⁶Cl^– effluxes and WC current activation by the above-mentioned agonists. Incubation with a C2 clostridial toxin (5 nmol/l) showed similar effects on WC currents. Moreover, when cytochalasin D (10 mol/l), C2 clostridial toxins (5 nmol/l), or phalloidin (10 mol/l) were added to the pipette filling solution current activation was markedly reduced. However, in excised inside-out membrane patches, cytochalasin D (10 mol/l), G-actin (10 mol/l) and phalloidin (10 mol/l) had no effect. These data suggest that actin participates in the activation of ion channels in 16HBE14o- epithelial cells and support the concept that exocytosis is a crucial step in the regulation of Cl^– and K⁺ channels in these cells.     

Membrane conductance and cell volume changes evoked by vasoactive intestinal polypeptide and carbachol in small intestinal crypts

We have used the perforated-patch whole-cell recording mode of the patch-clamp technique to monitor membrane potential and measured cell volume changes by image analysis, to determine the nature of the response to secretagogues of isolated whole guinea-pig small-intestinal crypts. Vasoactive intestinal polypeptide (VIP) produced a dose-dependent depolarisation (EC₅₀=30 nM) and an increase in membrane conductance that could be potentiated by carbachol. Similar depolarisations were observed with forskolin. The depolarisation induced by 100 nM VIP was smaller when pipette [Cl^–] was 60 mM than when it was 145 mM, suggesting an effect through Cl^– conductance activation. Carbachol alone produced a hyperpolarisation (EC₅₀=2 M). The Cl^– channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) produced a small hyperpolarization. When VIP was added in the presence of NPPB, the depolarising effect of the peptide was abolished and a hyperpolarisation was observed instead, consistent with the parallel activation of a K⁺ conductance. Both carbachol (100 M) and VIP (100 nM) induced a 25%–30% shrinkage of crypts, which was maximal 8 min after addition of the secretagogue. The induced shrinkage was sustained in the continued presence of agonist and was reversed upon washout. Shrinkage induced by the agonists was abolished by increasing extracellular K⁺ from 6 mM to 20 mM and was inhibited partially in the presence of 100 M anthracene-9-carboxylic acid in the bath. The decrease in volume induced by 100 nM VIP was totally abolished in the presence of 100 M NPPB. The results are consistent with the view that both VIP and carbachol induce secretion in small-intestinal crypts. This would lead to a net loss of K⁺ and Cl^– ions from the cytoplasm with a concomitant decrease in cell volume.     

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture

Cultured vascular smooth muscle cells from porcine aortas incubated in Na⁺-free medium rapidly release their intracellular Na⁺ contents (Na_i) (23±4% of baseline after 60 min incubation, mean ± SEM of 18 experiments). Total Na_i release was inhibited by 35–40% after addition of ouabain and by 60–70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na⁺ efflux with an IC₅₀ of about 10^–9–10^–8 M. Addition of the alpha-adrenergic agonist phenylephrine (10 M) to the cells mimicked the inhibitory action of norepinephrine on Na_i release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Na_i release. Simultaneous addition of 10 M norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Na_i decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 M) inhibited 40.0±8.1% of ouabain-sensitive Rb⁺ influx and 70.7±6.9% of bumetanide-sensitive Rb⁺ influx (mean ± SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb⁺ influx was obtained with about 5×10^–7 M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na⁺, K⁺, Cl^–]-cotransport system is able to catalyze outward Na⁺ movements (in Na⁺-free media) of a similar order of magnitude to those of the Na⁺, K⁺ pump and that alpha-adrenergic stimulation markedly inhibits Na⁺ efflux (and Rb⁺ influx) through these two transport systems.