Direct measurement of Na influx by23Na NMR during secretion with acetylcholine in perfused rat mandibular gland

Intracellular Na content (Na_in) in the perfused rat mandibular gland was measured by using a²³Na NMR spectroscopy at 24°C. An aqueous chemical shift reagent, dysprosium triethylenetetramine-N,N,N,N,NN-hexaacetic acid [Dy(TTHA)] was used in order to discriminate between the intracellular and the extracellular Na signal. The mandibular gland of rat was perfused arterially with a modified Krebs solution containing 10 mM Dy(TTHA). At rest, Na_in was not changed by blocking the Na⁺/K⁺ ATPase with ouabain (1 mM) and atropine (3 M), implying that, in the absence of stimulation, the spontaneous Na influx across the plasma membrane must have been negligibly small. Following onset of stimulation with acetylcholine (1 M), Na_in increased by 9.1±1.5 mmol/l intracellular fluid (mean±SEM,n=13), and remained at this level during stimulation. In the initial phase of secretion (0–5 min), about 50 mmol/min/l intracellular fluid of Na was secreted into the luminal space (estimated from the secretory rate by assuming an isotonic primary secretion) but, in spite of the higher secretion rate, Na_in increased only at an initial rate of 4.1 mmol/min/l intracellular fluid. During the steady phase of secretion (15–30 min) evoked by acetylcholine (1 M), ouabain (1 mM) caused an increment of Na_in of 44±8 mmol/l intracellular fluid (mean±SEM,n=4). From the rate of Na_in increment, the Na influx rate at the steady phase was estimated as 4.5 mmol/min/l intracellular fluid. These results suggest that the influx of Na is caused by stimulation with acetylcholine. The observed Na influx rate was about 50% of the Na secretory rate at the steady phase of secretion, estimated from the secretory rate by assuming an isotonic primary secretion. This is fully compatible with the operation of Na–K-2Cl contransport system for which one would expect a Na influx rate exactly half of the rate of Na and Cl secretion.     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

Summary The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), sprint (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p0.05). In the 51 day E group ATPase activity (0.378±0.009 mol Pi·mg^–1·min^–1) was greater than at 10 and 21 days (0.307±0.006 and 0.323±0.008 mol Pi·mg^–1·min^–1) (p0.05). No change occurred in the S group from 10 to 21 and 51 days (p0.05). Both the 21 and 51 day S (0.318±0.011 and 0.399±0.010 mol Pi·mg^–1·min^–1) and E (0.323±0.008 and 0.378±0.009 mol Pi·mg^–1·min^–1) groups had higher activity compared to the C group (0.193±0.029 and 0.172±0.031 mol Pi·mg^–1·min^–1) (p0.05). Maturation (10–51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca²⁺ binding (p0.05) while Ca²⁺ uptake ability did not change (p0.05). SR yield, Ca²⁺ binding and uptake were not altered with S training (p0.05). The E training resulted in greater Ca²⁺ uptake at 51 days compared to C and S (p0.05), with no change in Ca²⁺ binding (p0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.Supported by grants A-6449 and A-0425 from the Natural Sciences and Engineering Research Council of Canada     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Freeze-fracture study of the mechanoreceptive digital corpuscles of mice

Summary The freeze-fracture replication technique was used to study the mechanoreceptive digital corpuscles in toe pads of mice. The axon terminal plasmalemma had intramembranous particles (IMPs) at a density of 2367 ± 517 m^–2 (mean ±s.e.m.) in the P-face and 84 ± 4 m^–2 in the E-face. Particles were 10 ± 1.8 nm in diameter in the P-face and 10 ± 1.5 nm (mean ±s.d.) in the E-face. Particle-rich and particle-free areas were noted in the P-face. The lamellar cell plasmalemma had IMPs at a density of 3359 ± 224 m^–2 in the P-face and 265 ± 95 m^–2 in the E-face. Particles were 10 ± 1.4 nm in diameter in the P-face and 10 ± 1.6 nm in the E-face. Non-terminal unmyelinated fibres in the connective tissue compartment of toe pads were also examined: the P-faces of the axolemma and Schwann cell plasmalemma had IMPs at a density of 1356 ± 283 m^–2 and 1514 ± 514 m^–2, respectively, while the E-face of these membranes had only a few particles. Particles were 9 ± 1.2 nm and 10 ± 1.6 nm in diameter in the P-faces of axon and Schwann cell plasmalemmata, respectively.The results show that the IMPs in terminal axolemma and in lamellar cell plasmalemma have a much higher density than those of non-terminal axons or Schwann cells in myelinated and unmyelinated fibres. In addition, IMPs in the terminal axolemma are larger than those in non-terminal axolemma except for the nodal axolemma. It can be said that plasmalemmata of both the axon terminals and lamellar cells of digital corpuscles are specialized in terms of IMPs, suggesting that they have specific physiological properties in mechanoreceptive functions including mechano-electric transduction.     

Hydraulic permeability coefficient and sodium steady-state luminal concentration of the in vivo perfused rat distal colon

The distal colon (rat) was perfused in vivo at low rates (1–2 ml/h). Dialyzed polyethyleneglycol 4,000 (PEG) was used to vary the luminal osmotic activity (_eff). Perfusate sodium concentration of 3.7 mmol l^–1±0.9 (SD) resulted in an effluent Na⁺ of 3.3 mmol l^–1±1.4. Potassium concentrations of 15 or 25 mmol l^–1 remained unaltered. At this cationic steady-state, transmural net water flux (J _v ) was linearly correlated (r=–0.96,n=24) to the logarithm of _eff.J _v was zero at 634 mosm l^–1.L _p (l h^–1 cm^–2 mosm^–1 l) was 0.195 at _eff 85 mosm l^–1 and 0.046 at 1,050 mosm l^–1. These data characterize the distal colon as an epithelium with high capacity for salt conservation.     

Multicompartment kinetic analysis of the amiloride block of Na+ fluxes in frog skin

Previous studies have shown that the flow of Na⁺ in a multicompartment, dual Na⁺ pump frog skin model is in all hitherto tested kinetic and thermodynamic properties consistent with observations made on skin. It is shown here, that this is also true for a simulated amiloride block by which entrance of Na⁺ into a transport compartment via two parallel pathways is stopped. By the method of computer simulation, the following results were obtained.Before amiloride, steady state transmembrane influx (nEq×cm^–2×min^–1)J ⁱ=21. EffluxJ ^e=0.5 in a tight model, and 3.9 in a model which is leaky in its paracellular pathway. Total epidermal Na⁺ pool (under net flux conditions (nEq/cm²)P ⁿ=330, or 160 in a model with a stronger Na⁺ maintenance pump. The steady state time was nearly 1 h.After amiloride, rapid decrease of uptake of Na⁺ from the outside, slower release of Na⁺ to the inside under influx conditions; half time (min)t _h=2.2 ifP ⁿ=330; 0.9 ifP ⁿ=160. A new steady state was reached in 12–15 min.J ^e decreased by 45% in the tight model. In the leaky model,J ^e increased by 3%. Loss of Na⁺ from the model under net flux conditions, P ⁿ=30 nEq/cm² ifP ⁿ=330, P ⁿ=16 ifP ⁿ=160. The kinetic analysis suggests new laboratory experiments, and detailed layouts of speculative, but plausible Na⁺ current fields. These include for all compartments, values for [Na⁺]varying from 21 (P ⁿ=160) to 63 (P ⁿ=330) mM. In the Na⁺ transport compartment, [Na⁺] decreased from 5.5 to 1.4 mM after the simulated amiloride block.     

Noise analysis of cAMP-stimulated Na current in frog colon

The effects of oxytocin and cAMP on the electrogenic Na⁺-transport in the short-circuited epithelium of the frog colon (Rana esculenta, Rana temporaria) were investigated. Oxytocin (100 mU · ml^–1) elevated the shortcircuit current (I _sc) transiently by 70% whereas cAMP (1 mmol · l^–1) elicited a comparable sustained response. The mechanism of the natriferic action of cAMP was studied by analysing current fluctuations through apical Na⁺-channels induced by amiloride or CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamid). The noise data were used to calculate Na⁺-channel density (M) and single apical Na⁺-current (i _Na).i _Na-Values obtained with amiloride and CDPC were 1.0±0.1 pA (n=5) and 1.1±0.2 pA (n=6) respectively and unaffected by cAMP. On the other hand, cAMP caused a significant increase in M from 0.23±0.08 m^–2 (n=5) to 0.49±0.17 m^–2 (n=5) in the amiloride experiments. In our studies with CDPC we obtained smaller values for M in control (0.12±0.04 m^–2;n=6) as well as during cAMP treatment (0.19±0.06 m^–2;n=6). However, the cAMP-induced increase in M was also significant. We conclude that cAMP stimulates Na⁺-transport across the frog colon by activating silent apical Na⁺-channels. Thus, the mechanism of regulation of colonic Na-transport in frogs differs considerably from that in other vertebrates as mammals and birds.     

Taurocholate depolarizes rat hepatocytes in primary culture by increasing cell membrane Na+ conductance

Rat hepatocytes in primary culture were impaled with conventional microelectrodes. Addition of 5–100 mol/l taurocholate led to a slowly developing depolarization that was maximal at 50 mol/l (10.5±1.5 mV, n=15) and not reversible. The effect was Na⁺ dependent and decreased in cells preincubated with 1 mol/l taurocholate. Increasing external K⁺ tenfold depolarized the cells by 12.3±2.3 mV under control conditions and by 6.3±1.2 mV with 50 mol/l taurocholate present (n=7). Depolarization by 1 mmol/l Ba²⁺ was 7.6±0.8 mV and 6.0±0.7 mV (n=9) before and after addition of taurocholate, respectively. Cable analysis and Na⁺ substitution experiments reveal that this apparent decrease in K⁺ conductance reflects an actual increase in Na⁺ conductance: in the presence of taurocholate, specific cell membrane resistance decreased from 2.8 to 2.3 k · cm² · Na⁺ substitution by 95% depolarized cell membranes by 8.9±2.9 mV (n=9), probably due to indirect effects on K⁺ conductance via changes in cell pH. With taurocholate present, the same manoeuvre changed membrane voltages by –0.8±2.6 mV. When Na⁺ concentration was restored to 100% from solutions containing 5% Na⁺, cells hyperpolarized by 3.5±3.6 mV (n=7) under control conditions and depolarized by 4.4±2.9 mV in the presence of taurocholate, respectively. In Cl^– substitution experiments, there was no evidence for changes in Cl^– conductance by taurocholate. These results show that taurocholate-induced membrane depolarization is due to an increase in Na⁺ conductance probably via uptake of the bile acid.     

Electrical properties of amphibian urinary bladder epithelia

Summary Necturus urinary bladders were mounted in a two-compartment chamber filled with Ringer's solution and individual cells were punctured with microelectrodes from the serosal surface. By measuring the changes of transepithelial resistance and potential in response to luminal application of amiloride, and measuring the effects of amiloride on the voltage divider ratio and on the serosal cell membrane potential, two independent methods were obtained to calculate the cell membrane resistances and the resistance of the shunt path. The results from both methods agreed favourably. Th mucosal and serosal membrane resistances were found to vary respectively from 9–65 k cm² and from 1–7 k cm² under control conditions. Significant correlations were observed between the luminal membrane resistance as well as the luminal membrane zero current potential and the short circuit current, which indicate that the luminal membrane conductance consists predominantly of Na⁺-selective elements, whose density varies from bladder to bladder and determines the rate of transpithelial Na⁺ transport. Amiloride which blocks these elements increased the luminal resistance to 220 k cm². Increasing short circuit current was also correlated with increasing conductance of the serosal membrane (at fairly constant zero current potential) which points to an electrogenic mechanism of active Na⁺ transport across the serosal cell surface. The paracellular shunt path across the terminal bars plays a minor role in urinary bladder epithelium. Its resistance was estimated to be in the range of 50–125 k cm² or greater.     

Sodium current kinetics in freshly isolated neostriatal neurones of the adult guinea pig

Neurones of the neostriatum were freshly dispersed from the adult guinea pig brain. A fast, transient inward Na⁺ current (I _Na) was analysed using the whole-cell patch-clamp technique. Upon depolarizations, I _Na developed with a sigmoidal time course, which was described by m ³ kinetics. I _Na showed an activation threshold of about –60 mV, a peak current at –30 to –20 mV, and a reversal of polarity at +60 mV. The steady-state activation (m) curve for I _Na had a slope factor of about 9 mV with a mid-point potential of about –26 mV. The voltage dependence of the activation time constant, _m , had a bell-shaped configuration with a maximum value at –60 mV. The forward rate constant for I _Na activation ( _m ) increased as the membrane was depolarized (about 9 mV for a change in the rate constant by a factor of e) in the range between –50 mV and –20 mV. Conversely, the backward rate constant ( _m) decreased as the membrane was depolarized (about 31 mV for an e-fold change). The steady-state inactivation (h ) curve was well expressed by the Boltzmann's equation with a half-inactivation potential of –62 mV and a slope parameter of 6 mV. The time course of I _Na decay followed a second-order process, whereas the recovery from inactivation was described as a first-order process. The _h curve showed a bell-shaped configuration with a maximum value at –60 mV. The forward rate constants ( _h ) decreased as the membrane was depolarized (about 17 mV for an e-fold change) in the range between –50 mV and –20 mV. The backward rate constants ( _h) increased as the membrane was depolarized (about 10 mV for an e-fold change). There was a significant overlap between m and h curves, suggesting a steady influx of Na⁺ (window current).     

The distribution of membrane bound enzymes in the acini and ducts of the cat pancreas

Summary Enzymes activities of the Na⁺K⁺-and the HCO₃ ^–-ATPases, alkaline phosphatase, amino peptidase and 5 nucleotidase have been measured in 4 different preparations from the cat pancreas a) in the ducts including all sizes b) in ducts of three different diameters c) in that tissue, which had been dissected off from the ducts, called acini, and d) in the whole homogenate of the pancreas. The distribution of the measured enzymes shows, that the Na⁺K⁺-activity is highest in the acinar structures (mean value 0.532 M/mg Protein x h), while the ducts show nearly no Na⁺K⁺-ATPase activity. The HCO₃ ^–-ATPase, the alkaline phosphatase and the 5 nucleotidase are in the ducts between 2.4 and 3.6 times higher than in the whole organ whereas the amino peptidase does not appear to have a selective distribution. As the HCO₃ ^–-ATPase activity distribution pattern is identical with that of the secretory capacity of HCO₃ ^– as evidenced by earlier micropuncture studies, the data suggest that the HCO₃ ^–-ATPase is the main enzyme involved in the secretion of the bicarbonate buffer. Concerning the Na⁺K⁺-ATPase activity in the acinar structures we cannot contribute to its function in the enzyme secreting process.     

Chloride and sodium transport across bovine tracheal epithelium: Effects of secretagogues and indomethacin

The regulation of ion transport in bovine tracheal epithelium was studied in vitro. In the absence of exogenous midifiers of ion transport, average values for transepithelial electrical potential difference (_t), short-circuit-current (I _sc) and tissue resistance (R _t) were 35.4 mV (lumen negative), 5.4 Eq·h^–1·cm^–2 and 187 ·cm² respectively; net Cl secretion (3.2 Eq·h^–1·cm^–2) and net Na absorption (1.3 Eq·h^–1·cm²) accounted for 82% of theI _sc. Amiloride reduced (₁) andI _sc, and increasedR _t. The values of (_t),R _t andI _sc obtained following addition of theophylline, epinephrine or prostaglandin E₁ (PGE₁) were not different from control values. Theophylline aldo did not alter Na and Cl fluxes but it increased tissue cAMP content 3-fold. Indomethacin did not affect (_t) but it increasedR _t and net Na absorption, and decreasedI _sc and net Cl secretion; it did not significantly reduce tissue cAMP. When added to indomethacin-treated tissues, epinephrine restoredI _sc,R _t and Na and Cl fluxes to control levels and increased tissue cAMP 3-fold. Similary, when PGE₁ was added to indomethacin-treated tissues,I _sc andR _t were restored to control levels.We conclude that: (1) bovine tracheal epithelium, like its canine counterpart, absorbs Na and secretes Cl; the two tissues differ, however, in two ways: the spontaneous rate of Na absorption is higher in bovine trachea and the spontaneous rate of Cl secretion cannot be further increased in bovine trachea by secretagogues; (2) Cl secretion and Na absorption in bovine trachea are normally regulated by endogenous prostaglandins; (3) although cAMP may mediate changes in ion transport, a strict correlation between tissue cAMP content and Na and Cl transport rates is not evident; and (4) Na absorptive and Cl secretory rates are reciprocally related suggesting that both processes are present in the same cells.     

Water handling in Caco-2 cells: effects of acidification of the medium

Caco-2 cells were cultured on permeable supports. At confluence the minute-by-minute net water movement (J _w) was automatically recorded. Simultaneously, unidirectional [¹⁴C]mannitol, ²²Na⁺, and/or ³⁶Cl^– fluxes and transepithelial resistances were measured. The water and mannitol permeabilities went progressively down between 9 and 16 days after seeding and then stabilized. In this last condition the hydrostatic permeability coefficient (P _hydr) was 2.67±0.31 cm s^–1 while the osmotic permeability coefficient (P _osm) was 0.0017±0.0004 cm s^–1. P _hydr but not P _osm was dependent on the temperature and on the presence of Na⁺ in the medium. A net secretory J _w was observed 16 days after seeding, in the absence of any osmotic, hydrostatic or chemical gradient. This secretory J _w was associated with net Cl^– (1.43±0.43 equiv h^–1 cm^–2) and Na⁺ (1.05±0.35 equiv h^–1 cm^–2) secretions. Amiloride reduced, in open-circuit conditions, both Na⁺ and Cl^– apical to basal fluxes, thus enhancing the net Na⁺ and Cl^– exit. Acidification of the medium (pH 6.2) reversibly increased water and mannitol permeabilities in 10-day-old cultures. In 16-day-old cultures the same shift in medium pH did not change mannitol permeability, while stimulating water secretion. These results, obtained in the absence of supracellular structures (villae, crypts) and subepithelial components (muscular, vascular and conjunctive tissues) indicate that paracellular and transport-associated water pathways are sensitive to changes in the pH of the medium in Caco-2 cell layers.     

A new double-barrelled,ionophore-based microelectrode for chloride ions

A new Cl^– selective microelectrode based on the ionophore 5,10,15,20-tetraphenyl-21H,23H-porphin manganese(III) chloride is presented which discriminates better against HCO ₃ ^– and several organic anions than electrodes containing the Corning 477913 ion-eschanger. Using a redesigned construction procedure, fine-tip double-barrelled microelectrodes were produced which had slopes of –52.4±0.6 mV (SE,n=24), resistances of about 7·10¹¹ and a selectivity coefficient logK _{Cl, HCO3} ^pot of –1.40±0.03. Some electrodes showed a small unexplained sensitivity to pH>7.6. When used to puncture cells of isolated S3 segments of rabbit renal proximal tubule during perfusion with HCO ₃ ^– Ringer solution, the electrodes gave a membrane potential of –69.8±1.5 mV and an intracellular Cl^– activity, [Cl^–]_i, of 35.3±2.6 mmol/l. Upon switching bath and lumen perfusions to Cl^–-free solutions the residual [Cl^–]_i dropped to 1.20±0.03 mmol/l, while in similar measurements with ion-exchanger electrodes the residual [Cl^–]_i dropped only to 10.9±0.5 mmol/l. These observations demonstrate the superiority of the new electrode and prove that previously determined high [Cl^–]_i values in Cl^–-free ambient solutions reflect interference problems rather than non-exchangeable intracellular chloride.     

Einfluß von Aldosteron auf den Natriumtransport in den Sammelrohren der Säugetierniere

Zusammenfassung Durch Mikropunktion und -perfusion der Vasa recta an der freigelegten Nierenpapille von Wistarratten wurde es möglich, die schwankenden Harnstoff- und Natriumkonzentrationen im Interstitium des Nierenmarks zu beseitigen und definierte Versuchsbedingungen für die Sammelrohre zu schaffen.An diesen funktionell isolierten Sammelrohrabschnitten wurdenin situ sowohl die Gleichgewichtskonzentrationsdifferenz bei fehlendem Nettosubstanz-und -volumenfluß (C _Na) als auch der Nettonatriumtransport ( _Na) bei gleicher Natriumkonzentration auf beiden Seiten der Sammelrohrwand gemessen. Es konnte gezeigt werden, daß unter diesen Versuchsbedingungen, bei denen die Tiere in Antidiurese sind, die Natriumrückresorption aus den Sammelrohren isoton abläuft.Die Versuche wurden an vier Tiergruppen durchgeführt: an adrenalektomierten Tieren, an normal ernährten Tieren, an salzarm ernährten Tieren und an normal ernährten Tieren, die zusätzlich Aldosteron bekamen. _{Na iso} war bei adrenalektomierten Tieren 1,2·10^–5 bei normal ernährten Tieren 3,1·10^–5, bei salzarm ernährten Tieren 4,1·10^–5 und bei normal ernährten Tieren unter Aldosteronsubstitution 4,2·10^–5 Äq·mm^–2·sec^–1. Die entsprechenden C _Na werte waren 4, 31, 98, 93 mÄq/l.Unter der Annahme, daß _{Na iso} die Transportkapazität des Systems angibt und daß C _Na bei gegebenem _{Na iso} umgekehrt proportional der Leckpermeabilität für Natriumionen ist, kann man aus den vorliegenden Daten schließen, daß Aldosteron am Sammelrohr nicht nur die innere Transportkapazität für Na erhöht, sondern auch die Leckpermeabilität für Na herabsetzt.     

Electrical properties and electrolyte transport in bovine tracheal epithelium: Effects of ion substitutions,transport inhibitors and histamine

The active transport of Na⁺ and Cl^– across the tracheal epithelium of the cow was investigated in vitro, using the short-circuit technique, by means of ion substitutions, transport inhibitors and by measuring²²Na and³⁶Cl fluxes. Under short-circuit conditions, short-circuit current (i _o) was 168±5A cm^–2 (mean±SEM,n=30), i.e. 6.2±0.2 Eq h^–1cm^–2 and resistance (R) was 248±10cm². Net Na⁺ flux toward the submucosa (J _{Na net} ^L-S ) and net Cl^– flux toward the lumen (J _{Cl net} ^S-L ) were of the same magnitude, i.e. 2.7±0.2 and 2.9±0.2 Eq h^–1 cm^–2, respectively. The permeability coefficients were 3.6·10^–6 forP _Na and 7.8·10^–6 cm s^–1 forP _Cl. Under open-circuit conditions, the transepithelial electrical potential difference () was 43±2 mV (lumen negative,n=20).J _{Na net} ^L-S andJ _{Cl net} ^S-L were close to zero.Bilateral substitution of Cl^– with SO ₄ ^2– or isethionate, or administration of furosemide 5·10^–3M or bumetanide 10^–4M in the submucosal bathing medium produced a 40 to 50% decrease ini ₀; furosemide abolishedJ _{Cl net} ^S-L . Bilateral substitution of Na⁺ with choline or Mg²⁺, or addition of ouabain 10^–4M to the submucosal bath abolishedi ₀;J _{Na net} ^L-S andJ _{Cl net} ^S-L were suppressed by ouabain. Amiloride 10^–4M in the luminal bath reducedi ₀ by 23% and diminishedJ _{Na net} ^L-S by 80%. Histamine 10^–4M, added to the submucosal bathing medium, reducedJ _{Na net} ^L-S and increasedJ _{Cl net} ^S-L , under short-circuit conditions. In open-circuit conditions, histamine had little effect on ion fluxes. This substance had no effect on the electrical properties, as shown previously.These results are consistent with the model proposed by Silva et al. [20] for a Cl^–-secreting, Na⁺-reabsorbing epithelium.Supported by the Swiss National Foundation (SNF), grant no. 3.588-0.79     

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.     

Abnormal erythrocyte sodium leak in a subset of essential hypertensive patients

Summary We measured the ouabain- and bumetanide-resistant Na⁺ efflux in Mg²⁺-sucrose medium (passive Na⁺ leak) in erythrocytes from 30 normotensive controls and 72 essential hypertensive patients. The mean values (±SEM) of the rate constant of Na⁺ leak (k_pNa) were not significantly different between normotensives and hypertensives. Nevertheless, using the 95% confidence limits of the k_pNa (in 10^–3.h^–1) in the normotensive group as a cut-off point, 7 (9.7%) essential hypertensives exhibited increased values (58.96±10.12) when compared with the other 65 patients (23.86±0.74). revealing increased passive Na⁺ permeability in the former (leak + hypertensives). Na⁺ fluxes depending on the Na⁺-K⁺ pump, outward Na⁺-K⁺ cotransport, and Na⁺-Li⁺ countertransport were also measured in fresh erythrocytes from the same 72 patients. Three of them (4.2%) exhibited decreased values of ouabain-sensitive Na⁺ efflux and 6 (8.3%) of bumetanide-sensitive Na⁺ efflux, while 8 patients (11.1%) showed increased values of Li⁺-stimulated Na⁺ efflux and, finally, 48 patients (59.7%) did not present any evident abnormality in these Na⁺ transport systems. No differences were observed between leak + hypertensives and the remaining 65 patients when both basal erythrocyte Na⁺ content and clinical parameters of hypertension were compared. However, Na⁺ efflux depending on the outward Na⁺-K⁺ cotransport was significantly higher in the leak + hypertensive subset (299.43±43.18 vs 181.52±10.76 µmol.(l cells.h)^–1;P=0.0078), suggesting a compensatory phenomenon. Enhancement of Na⁺ permeability detected in 3% to 16% of essential hypertensives may be implicated in the pathogenesis of primary hypertension.Abbreviations ATPase adenosine triphosphatase - Dcat difference between the external Na⁺ concentration after incubation at 37° C and at zero time - k_pNa rate constant of passive Na⁺ leak - Leak + hypertensive essential hypertensive patient with abnormally high erythrocyte Na⁺ leak - MOPS 4-morpholinopropanesulfonic acid - OBR ouabain- and bumetanide-resistant - PRA plasma renin activity - sPRA plasma renin activity stimulated after furosemide infusion - SEM standard error of the mean Supported in part by Grant 87/1078 of the Fondo de Investigaciones Sanitarias de la Seguridad Social and Grant PA85/0168 of the Comisión Asesora de Investigación Científica y Técnica     

Differential expression of β1, β3 and β4 integrins in sarcomas of the small,round, blue cell category

Integrins are a large and complex family of membrane spanning heterodimeric cell surface glycoproteins mediating cell/cell and cell/matrix interactions. Small, round, blue cell sarcomas (SRBCS) are a group of poorly differentiated tumours of various and in part uncertain histogenesis displaying similar cytomorphology. Among them are rhabdomyosarcomas (RMS), ganglioneuroblastomas [(G)NB], primitive peripheral neuroectodermal tumours (pPNET) and Ewing's sarcomas (ES). Thirty-two SRBCS were studied immunohistochemically for the distribution of 1, 3 and 4 integrins in situ. We found complex and to some extent differential patterns of 1, 3 and 4 integrin subunit expression in different types of SRBCS: all of the sarcomas studied were consistently 1⁺, 4^–, 2^–. Four of nine RMS were completely negative for all other integrin subunits studied while one RMS was 5⁺ throughout and three RMS were focally 5⁺. Three RMS expressed the 6 and v chains. In contrast to RMS, pPNET and ES, all of which were 1^–, 3^–, (G)NB were 3⁺ and frequently co-expressed 1. The eight pPNET and seven ES studied showed a similarily restricted integrin profile that was limited to the expression of 1 and 5 in nearly all cases. In summary, RMS were 1⁺, 1^–, 3^– and heterogeneously expressed 5 and 6. (G)NB were generally 1⁺, 1⁺, 3⁺, 5^–, 6^–. pPNET and ES were 1⁺, 1^–, 3^–, 5⁺, 6^–. The data illustrate a complex expression pattern of various integrins in SRBCS, a differential expression pattern of some of the integrin subunits among different types of SRBCS and almost identical integrin profiles in pPNET and ES.This paper is dedicated to Prof. Dr. Dres. h.c. Wilhelm Doerr on the occasion of his 80^th birthday     

Influence of glucose absorption on ion activities in cells and submucosal space in goldfish intestine

Mucosal glucose addition evokes in goldfish intestinal epithelium a fast depolarization of the mucosal membrane potential (_mc = 12 mV) followed by a slower repolarization (_mc = –7 mV). The intracellular sodium activity, a_iNa⁺, rises from 13.2±2.4 meq/l by 6.7±0.5 meq/l within 5 min, a_iCl^– rises about 3 meq/l above the control value of 37.7±2.2 meq/l, while a_iK is constant (97.7 ±7.4 meq/l). The potassium activity measured in the submucosal interstitium near the basal side of the cells (a_iK⁺) is 5.2±0.2 meq/l in non-absorbing tissue compared to 4.2 meq/l in the bathing solution and shows a transient increase due to glucose absorption (1.1±0.1 meq/l).In chloride-free media a_sK⁺=4.2±0.1 meq/l and _mc hyperpolarizes by –13 mV. The depolarization due to glucose absorption increases (_mc = 14.1 ± 1.4 mV) and the repolarization ( _mc ^repol ) disappears. In addition, a_iNa⁺ rises from 16.3±2.4 meq/l by 9.9±1.5 meq/l within 5 min, a_iK⁺ remains constant and equal to the value in chloride containing solutions (88.5±2.8 meq/l); a_sK⁺ increases transiently (1.1±0.1 meq/l).Serosal Ba²⁺ (5 mM) depolarizes _mc (+14.2±1.0 mV) and abolishes the repolarization. Increased serosal or mucosal potassium activity depolarizes _mc and abolishes the repolarization.These effects are discussed in terms of changes of ion activities, the basolateral potassium conductance, the influence of intracellular Ca²⁺, the functional state of the Na/K-pump, and modulation of membrane permeabilities by extracellular potassium.