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.     

O2 consumption,aerobic glycolysis and tissue phosphagen content during activation of the NA+/K+ pump in rat portal vein

Oxygen consumption, lactate production and tissue contents of ATP, phosphocreatine (PCr) and lactate were measured following readdition of K⁺ to K⁺-depleted rat portal veins, in order to study the energy turnover associated with Na⁺/K⁺ pumping. During incubation in K⁺-free medium at 37° C spontaneous contractions disappeared in 10–20 min. Readdition of K⁺ (5.9 mM) after 40 min K⁺-free incubation caused hyperpolarization of the cell membrane for the first 5–10 min and then gradual depolarization with return of spontaneous action potentials and contractions by 10–20 min. During the first 4–6 min after K⁺ readdition aerobic lactate production was about doubled and then gradually returned to the original level (0.17 mol/min g) at about 20 min. The increase in glycolytic rate was prevented by 1 mM ouabain. In contrast, O₂ consumption (in K⁺-free medium, 0.38 mol/min g) rose by about 10% when K⁺ was added and this increase lasted about 5 min. By 8 min after K⁺ addition the increased glycolysis and oxidative phosphorylation had accounted for each about the same amount of extra ATP generation over that extrapolated from the steady rate before K⁺ addition. The average total increase in ATP turnover in the first 8 min was 15%. During this period there was no change in the cellular content of ATP, PCr, or extractable ADP. The results indicate that Na⁺/K⁺ pumping utilizes a relatively small share of the total energy turnover in the vascular smooth muscle but is to a large extent dependent on aerobic glycolysis and therefore a major site of carbohydrate usage.     

Effects of urea on K+ fluxes and cell volume in perfused rat liver

Exposure of the perfused rat liver to a perfusate made hyperosmotic by the presence of 200 mmol l^–1glucose led, as expected, to marked, transient hepatocellular shrinkage followed by volume-regulatory net K⁺ uptake. However, even after this volume-regulatory K⁺ uptake had ceased, the liver cells are still slightly shrunken. Withdrawal of glucose from the perfusate resulted in marked transient cell swelling, net K⁺ release from the liver and restoration of cell volume. However, when the Krebs-Henseleit perfusate was made hyperosmotic by the presence of urea (20–300 mM), there was no immediate decrease in liver mass, yet a slight and persistent cell shrinkage developing 2 min after the onset of exposure to urea. Surprisingly, urea induced concentration-dependent net K⁺ efflux from the liver and removal of urea net K⁺ reuptake from the inflowing perfusate. The urea (200 mM)-induced net K⁺ release resembled that observed following a lowering of the influent [NaCl]: making the perfusate hypoosmotic (245 mosmol l^–1, by reducing influent [NaCl] by 30 mM) gave roughly the same K⁺ response as hyperosmotic exposure (505 mosmol/l) resulting from the presence of 200 mM urea. The urea-induced K⁺ efflux was not inhibited in the presence of ouabain (1 mM), or in Ca⁺⁺-free perfusion, but was modified in the presence of quinidine (1 mM) or Ba⁺⁺ (1 mM). The direction in which the liver was perfused had no effect on the urea-induced net K⁺ release. Electrophysiological studies showed that urea led to quinidine-sensitive hyperpolarization and increase in K⁺ selectivity of plasma membranes, suggesting opening of K⁺ channels in the hepatocyte plasma membrane in response to urea. The data suggest that urea, but not glucose, enters the hepatocyte as quickly as water. Furthermore, urea at high concentrations apparently leads to K⁺ efflux from the hepatocyte and cell shrinkage, possibly due to opening of K⁺ channels in the hepatocyte plasma membrane.     

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.     

Messung der K+- und Na+-Aktivität mit Mikro-Glaselektroden im Extracellulärraum des Kaninchenskeletmuskels bei Muskelarbeit

Summary Rabbits were anaesthetized with urethane and pentobarbitone sodium. The K⁺ and Na⁺ activity in the blood of the femoral vein or in the interstitial space of a skeletal muscle (vastus medialis of M. quadriceps femoris) were measured continuously by means of glass microelectrodes during stimulation of the muscles of the hindlimb. The blood flow in the femoral artery was recorded simultaneously by means of a Statham flowmeter. The K⁺ activity (at rest about 4 mmole/l) rose during the stimulation of the musculature. The course of the K⁺ increase was similar to that of the functional hyperemia. Maximum values obtained by frequent supramaximal stimulation (40 cps) were 6–6.5 mmole/l in the venous blood and 8–8.5 mmole/l in the interstitial space. The Na⁺ activity also rose from about 135 mmole/l to 136–140 mmole/l. The findings support the assumption that potassium shifts play a role in the regulation of functional hyperemia.

Auszugsweise vorgetragen auf der Herbsttagung der Deutschen Physiologischen Gesellschaft in Erlangen, 1970.     

HCO 3 − -dependent ACh-activated Na+ influx in sheep parotid secretory endpieces

In the present study we used the Na⁺-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na⁺ in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO ₃ ^– -free Cl^–-rich solution had a resting intracellular Na⁺ concentration ([Na⁺]_i) of 17±2 mmol/l (n=39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3×10^–7 mol/l) in a HCO ₃ ^– -free bathing solution produced no change in [Na⁺]_i or in cell volume. Changing from a Cl^–-containing HCO ₃ ^– -free bath solution to a Cl^– solution containing 25 mmol/l HCO ₃ ^– caused the endpieces to swell by 8±2 % (n=11) and the [Na⁺]_i to increase by 10±2 mmol/l (n=14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12±2 % from the volume in the HCO ₃ ^– -containing solution prior to ACh exposure, with the maximum decrease occurring after 29±7 s (n=9). This shrinkage was accompanied by a rapid and transient increase in [Na⁺]_i, the [Na⁺]_i reaching a peak at 70±5 mmol/l above the unstimulated level (n=9). Substitution of gluconate for Cl^– did not significantly alter the effects of HCO ₃ ^– on unstimulated [Na⁺]_i or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCO ₃ ^– was present. Addition of 200 mol/l dihydrogen-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (H₂-DIDS) to the gluconate/HCO ₃ ^– solution significantly reduced the peak of the ACh-induced increase in [Na⁺]_i to 34±10 mmol/l (n=4), but did not have any significant effect on the magnitude of the ACh-induced shrinkage. At 500 mol/l, H₂-DIDS abolished the ACh-induced increase in [Na⁺]_i and also significantly reduced the shrinkage due to ACh. Finally, we found that the rate of endpiece shrinkage following ACh stimulation did not depend on the presence of Cl^–.We interpret these results as indicating that sheep parotid secretory cells do not contain significant Na⁺-K⁺-2Cl^– co-transport activity and do not actively accumulate Cl^–. Rather, the mechanism of spontaneous basal secretion by these cells, in the presence of extracellular HCO ₃ ^– , is based on the accumulation of HCO ₃ ^– by the Na⁺-H⁺ exchanger. During ACh stimulation, the concentration of HCO ₃ ^– in the cytosol is also maintained by the operation of a H₂-DIDS-sensitive Na⁺-HCO ₃ ^– co-transporter. HCO ₃ ^– efflux across the apical membrane occurs via a HCO ₃ ^– conductance pathway rather than by the coupled operation of a Cl^– channel and a Cl^–-HCO ₃ ^– exchanger.     

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.     

Piretanide-dextran and piretanide-polyethylene glycol interact with high affinity with the Na+ 2 Cl− K+ cotransporter in the thick ascending limb of the loop of Henle

Piretanide blocks the Na⁺ 2Cl^– K⁺ cotransporter protein in the thick ascending limb (TAL) of the loop of Henle reversibly. When tested from the luminal side in isolated perfused cTAL segments it leads to a half maximal inhibition (IC₅₀) of the equivalent short circuit current (I_sc) at a concentration of 10^–6 mol/l. From the basolateral side it has no effect on I_sc up to 10^–4 mol/l. The present study was designed to search for high affinity blockers of the Na⁺ 2Cl^– K⁺ cotransporter with large molecular weight in an attempt to use these macromolecules for antibody-labelling or affinity separation of this transport-protein. Amino-ethyl-dextran or amino-ethyl-polyethylene glycol (M.W. 5kd) were coupled to isothiocyanato-piretanide (ISO-PIR) at room temperature in DMSO. The resulting compounds dextran-sulfonylurea-piretanide (PIR-DEX) and polyethylene glycol-sulfonylurea-piretanide (PIR-PEG) (M.W. 5.38kd) were purified and tested in isolated perfused cTAL segments. IC₅₀ values for ISO-PIR, PIR-DEX and PIR-PEG were estimated from dose response curves after their addition to the lumen or bath perfusate, respectively. ISO-PIR, PIR-DEX and PIR-PEG acted from the lumen side at 3·10^–6, 6·10^–6 and 2·10^–6 mol/l. The inhibitory effect was easily reversible. From the basolateral side no effect for any compound was seen at up to 10^–4 mol/l. In clearance experiments PIR-DEX was given to female Wistar rats as an i.v. bolus (25 mol/kg) and the diuretic urine was collected. After dialysis (exclusion limit 2.5kd) the dialysed urine and the dialysate were tested in isolated perfused cTAL segments. The dialysates had no effect on I_sc, but the dialysed urine inhibited I_sc by 35% from the luminal side. The present data show: High molecular derivatives of piretanide with dextran or polyethylene glycol moieties block the Na⁺ 2Cl^– K⁺ cotransporter in cTAL segments at roughly the same low concentration as piretanide itself. Our data exclude a metabolism of these piretanide compounds in the kidney. Since these macromolecular probes can probably not enter the cell their inhibitory effect indicates that the binding site for piretanide diuretics on the Na⁺ 2Cl^– K⁺ cotransporter is exposed on the surface of the luminal cell membrane.This study was supported by Deutsche Forschungsgemeinschaft Gr 480/9     

Potassium and sodium shifts during in vitro isometric muscle contraction,and the time course of the ion-gradient recovery

Intracellular potassium ([K⁺]_i), interstitial potassium ([K⁺]_inter), intracellular sodium ([Na⁺]_i), and resting membrane potential (RMP) were measured before and after repetitive stimulation of mouse soleus and EDL (extensor digitorum longus) muscles. At rest, RMP was –69.8 mV for soleus and –74.9 mV for EDL (37°C). [K⁺]_i was 168 mM and 182 mM, respectively. In soleus, free [Na⁺]_i was 12.7 mM. After repetitive stimulation (960 stimuli) RMP had decreased by 11.9 mV for soleus and by 18.2 mV for EDL. [K⁺]_i was reduced by 32 mM and 48 mM, respectively, whereas [K⁺]_inter was doubled. In soleus [Na⁺]_i had increased by 10.6 mM, demonstrating that the [K⁺]_i-decrease is three times higher than the [Na⁺]_i-increase. It is concluded that this difference reflects different activity induced movements of Na and K, and that the difference is not due to the Na/K pumping ratio. The possible involvement of the potassium loss in muscle fatigue is discussed. After stimulation RMP recovered with a time constant of 0.9 min for soleus and 1.5 min for EDL. Within the first minutes after stimulation the intracellular potassium concentration increased by 20.4 mM/min for soleus and 21.7 mM/min for EDL. Free [Na⁺]_i decreased with less than 10 mM/min. The mechanisms underlying the different rate of changes are discussed.Parts of this work have been published in preliminary form (Juel and Sjøgaard 1984)     

Membrane potential measurements of transitional cells from the crista ampullaris of the Gerbil

Transitional cells of the crista ampullaris were impaled with microelectrodes in order to record the membrane potential (PD) and to investigate membrane properties. In control solution the PD was –87±1 mV (n=103). This value is not significantly different from –83±2 mV (n=24) measured in Cl^– free solution. [Cl^–] steps from 150 to 15 mmol/l (n=24) depolarized the membrane by about 2 mV, indicating a minor Cl^– conductance. The transference number for K⁺ was 0.75±0.01 (n=79) obtained from the PD responses to K⁺ steps from 3.6 to 25 mmol/l. The cell membrane depolarized and the amplitude of PD responses to [K⁺] steps was reduced by Ba²⁺ (2·10^–6 to 10^–3 mol/l), quinidine (10^–3 mol/l), quinine (10^–3 mol/l), Rb⁺ (20 mmol/l), Cs⁺ (20 mmol/l), NH₄ ⁺ (20 mmol/l) and Tl⁺ (0.5 mmol/l), whereas tetraethylammonium (TEA, 20 mmol/l) had no effect. The dose-response curve for Ba²⁺ in the presence of 3.6 mmol/l K⁺ was shifted to the right by approximately three decades in the presence of 25 mmol/l K⁺ and by a factor of about 4 in the presence of 135 mmol/l gluconate as a substitute for Cl^–. Transitional cells were depolarized by ouabain, suggesting the presence of (Na⁺+K⁺-ATPase.This work was supported by grants from the Deafness Research Foundation to PhW and the National Institute of Health (NS 19490) to DCM     

Torasemide inhibits NaCl reabsorption in the thick ascending limb of the loop of Henle

Torasemide (1-isopropyl-(4-(3-methylphenylamino)pyrid-3-yl)urea) is a new diuretic. The present study examines the effects of this substance in the isolated perfused thick ascending limb (TAL) of mouse and rabbit kidney. In cortical TAL segments of the rabbit, torasemide added to the lumen perfusate led to a fall in equivalent short circuit current (= transepithelial voltage divided by transepithelial resistance, which corresponds to the rate of chloride reabsorption) with a half maximal inhibition concentration of 3 · 10^–7 mol/l. This effect was accompanied by a hyperpolarization of the luminal and basolateral membrane from –78 to –81 mV and from –72 to –81 mV, respectively. A similar hyperpolarization of both membrane voltages was also observed in medullary TAL segments of the mouse. Torasemide, added to the basolateral perfusate of cortical TAL segments of the rabbit, also inhibited the equivalent short circuit current. However, 3 · 10^–5 mol/l were necessary for a half maximal inhibition. The fall in the equivalent short circuit current was accompanied by a significant increase in transepithelial resistance from 34 to 38 cm², by an increase in the fractional resistance of the basolateral membrane, and by a hyperpolarization mainly of the basolateral membrane. Again, similar results were obtained in the medullary TAL segment of the mouse.The strong inhibitory effect of torasemide from the lumen side can be explained by an interference with the Na⁺ 2Cl^–K⁺ carrier in the luminal membrane. In fact, torasemide apparently is structurally related to furosemide. The weaker effect of torasemide from the peritubular side can, at least in part, be explained as an interference with chloride channels present in the basolateral membrane. Torasemide is also structurally related to chloride channel blockers such as diphenylamine-2-carboxylate.Supported by Deutsche Forschungsgemeinschaft DFG Gr 480/6-4 and 6-5     

Effect of extracellular volume expansion on renal cortical and medullary Na+−K+-ATPase

Summary The role of renal Na⁺–K⁺-ATPase in the acute changes in sodium reabsorption caused by isotonic volume expansion was evaluatedin vivo andin vitro in the rat and the dog. Duringin vivo volume expansion with isotonic saline in the rat, renal medullary Na⁺–K⁺-ATPase specific activity increased, while the simultaneously determined cortical Na⁺–K⁺-ATPase specific activity and kinetics remained unchanged. Furthermore, experimentsin vitro failed to demonstrate a circulating inhibitor of renal Na⁺–K⁺-ATPase both in plasma dialysates from volume-expanded rats and in plasma dialysates concentrated 20-fold by ultrafiltration from volume-expanded dogs. These results suggest that the decreased proximal tubular reabsorption of sodium during volume expansion is not mediated by inhibition of renal cortical Na⁺–K⁺-ATPase. The acute increment in medullary Na⁺–K⁺-ATPase observed could represent an adaptive response to increased sodium reabsorption by the loops of Henle, and raises the possibility that this enzyme may participate in relatively rapid adjustments in the transport of sodium by the renal tubule.     

Relation between extracellular [K+], membrane potential and contraction in rat soleus muscle: modulation by the Na+-K+ pump

An increased extracellular K⁺ concentration ([K⁺]₀) is thought to cause muscle fatigue. We studied the effects of increasing [K⁺]₀ from 4 mM to 8–14 mM on tetanic contractions in isolated bundles of fibres and whole soleus muscles from the rat. Whereas there was little depression of force at a [K⁺]₀ of 8–9 mM, a further small increase in [K⁺]₀ to 11–14 mM resulted in a large reduction of force. Tetanus depression at 11 mM [K⁺]_o was increased when using weaker stimulation pulses and decreased with stronger pulses. Whereas the tetanic force/resting membrane potential (E _M) relation showed only moderate force depression with depolarization from –74 to –62 mV, a large reduction of force occurred whenE _M fell to –53 mV. The implications of these relations to fatigue are discussed. Partial inhibition of the Na⁺-K⁺ pump with ouabain (10^–6 M) caused additional force loss at 11 mM [K⁺]₀. Salbutamol, insulin, or calcitonin gene-related peptide all stimulated the Na⁺-K⁺ pump in muscles exposed to 11 mM [K⁺ ₀] and induced an average 26–33% recovery of tetanic force. When using stimulation pulses of 0.1 ms, instead of the standard 1.0-ms pulses, force recovery with these agents was 41–44% which was significantly greater (P < 0.025). Only salbutamol caused any recovery ofE _M (1.3 mV). The observations suggest that the increased Na⁺ concentration difference across the sarcolemma, following Na⁺-K⁺ pump stimulation, has an important role in restoring excitability and force.     

Short-term and long-term stimulation of Na+−H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney

The effect of unilateral nephrectomy on Na⁺–H⁺ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70–75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted toV _max, whereas theK _m for Na⁺ was similar in control and compensatory growing kidney. The increase of Na⁺–H⁺ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na⁺–H⁺ exchange. The late, but not the initial stimulations of Na⁺–H⁺ exchange were associated with elevated activities of cortical (Na⁺+K⁺)-ATPase indicating that changes in antiporter activity precede those in the (Na⁺+K⁺)-pump. The early stimulation of Na⁺–H⁺ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na⁺–H⁺ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na⁺–H⁺ exchanger may be regulated by phosphorylation-dephosphorylation events. In support, the concentration of a well known protein kinase C activator, 1,2-diacylglycerol, in the cortical tissue of the remnant kidney increased up to 100% within 5 min following unilateral nephrectomy and preceded the increase in Na⁺–H⁺ exchange. The early stimulation of Na⁺–H⁺ exchange may be a trigger in initiating the kidney growth.     

Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

Über das Membranpotential des Meerschweinchenvorhofes nach Hypothermie

Zusammenfassung An isolierten Vorhöfen des Meerschweinchenherzens wurden der Extracellulärraum (ER), die intracellulären Na⁺-und K⁺-Konzentrationen ([Na_i], [K]_i) und das membranpotential (MP) vor, während und nach Unterkühlung der Präparate gemessen. Das Wiedererwärmen der Vorhöfe erfolgte bei 35° C in Tyrode-Lösungen mit einem K⁺-Gehalt ([K]_e) von 0–21,6 mM/l [K]_e.Der Extracellulärraum (ER) wurde als Inulinraum gemessen. Bei 35° C betrug er etwa 350 ml/kg, bei 4–6° C etwa 300 ml/kg.Der intracelluläre K⁺-Gehalt der Vorhöfe sank während der Unterkühlung ab und nahm bei Wiedererwärmen der Präparate zu. Die intracelluläre Na⁺-Konzentration stieg während der Hypothermie an und fiel während des Wiedererwärmens ab.Das Membranpotential nahm bei Unterkühlung der Vorhöfe ab. Wiedererwärmen verursachte einen raschen Anstieg des Membranpotentiales. In Tyrodelösung mit 21,6 bzw. 10,8 mM/l [K]_e war das Membranpotential innerhalb der ersten 10 min des Wiedererwärmens signifikant mehr negativ als das entsprechende K⁺-Gleichgewichtspotential (E_K). Bei Weidererwärmen der Vorhöfe in einer Tyrodelösung mit 5,4 mM/l [K]_e waren Membranpotential und K⁺-Gleichgewichtspotential in den ersten 10 min gleich hoch. In K⁺-freier Tyrodelösung war das Ansteigen, des Membranpotentiales bei Wiedererwärmen nicht von einer gleichzeitigen Veränderung von [Na]_i begleitet. Unter diesen Bedingungen erreichte das Membranpotential wahrscheinlich nicht die Höhe des K⁺-Gleichgewichtspotentiales.Aus den Versuchsergebnissen wird geschlossen, daß das Membranpotential des Meerschweinchenvorhofes nach Hypothermie von einer elektrogenen Na⁺-Pumpe mit bestimmt werden kann. Die Leistung des elektrogenen Na⁺-Transportes ist von [K]_e und wahrscheinlich von [Na]_i abhängig.     

(Na+K+)-activated ATPase in human cornea

Summary Distribution and principal characteristics of (Na⁺K⁺)-activated ATPase in human cornea were investigated.(Na⁺K⁺)-ATPase was present in both epithelium and endothelium, whereas the corneal stroma did not exhibit significant enzyme activity.In homogenates specific activity of the (Na⁺K⁺)-ATPase was 2.3-fold higher in endothelium than in epithelium. Calculation of total enzyme activity revealed a 6.1-fold higher content of (Na⁺K⁺)-ATPase in the epithelium.In the epithelium a 7-fold enrichment of (Na⁺K⁺)-ATPase compared to the homogenate was obtained in the 150–1500×g _av fraction. Maximum enrichment in the endothelium was 3.5-fold and was achieved in the 1500–2500×g _av fraction. Both fractions showed, however, the same specific activity.The pH-optimum of (Na⁺K⁺)-ATPase in the 150–1500×g _av fraction ranged from 8.0–8.2 in both epithelium and endothelium.In the epithelial 150–1500×g _av fraction the apparentK _m-values were 4.0 mM for Na⁺, 2.8 mM for K⁺ and 0.12 mM for Mg²⁺ · ATP in equimolar concentrations.The inhibition constant of epithelial (Na⁺K⁺)-ATPase for ouabain was determined asK _i=3.3×10^–7 M.The present data support the view that control of corneal hydration in man is a function of both endothelium and epithelium.     

Solubilisation and reconstitution of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel into liposomes suitable for patch clamp studies

Sarcoplasmic reticulum (SR) membrane vesicles have been prepared from rabbit skeletal muscle and solubilised using K⁺ cholate. Solubilised membrane proteins were reconstituted into small asolectin liposomes by dialysis against cholate-free solution. Large liposomes were produced by freezing and thawing at –80°C and room temperature, respectively. The liposomes were assayed for the SR K⁺ channel using the patch clamp technique. Channel density was modulated by varying protein: lipid ratios during reconstitution. Channels inserted into the membrane with a preferred orientation. The solubilised and reconstituted channel behaves ohmically over the holding potential range ±70 mV and has a conductance of 178.4±4.4 pS (mean ± SE,n=37) in 200 mM KCl. The channel has a selectivity sequence of K⁺>NH ₄ ⁺ >Rb⁺>Na⁺ and K⁺ conductance is blocked by hexamethonium and decamethonium. The opening probability of the reconstituted channel is voltage dependent. The conductance and gating characteristics displayed by the solubilised and reconstituted channel correlate well with those previously observed following the fusion of native SR membrane vesicles with planar phospholipid bilayers.     

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.     

Renal proximal tubular buffer-(glycodiazine) transport

Summary Using the stop flow microperfusion technique with simultaneous capillary perfusion the secretory rate of H⁺ ions in the proximal tubule was evaluated by measuring the level flow reabsorption as well as the static head concentration difference of³H labelled glycodiazine. At ambient glycodiazine concentration of 21 mmol/l the level flow reabsorption is in the same range as that of bicarbonate. In the early proximal loops the reabsorption is 20% greater than in the late proximal loops. The carbonic anhydrase inhibitors acetazolamide and 3,4-methylenedioxyphenyl-sulfonamide (both 10^–4 M) as well as furosemide (10^–3 M) inhibit the glycodiazine reabsorption 43%, 27% and 22% respectively. Thiocyanate (2 · 10^–2 M), however, exerted only an insignificant inhibition (12%). When Na⁺ in the ambient perfusion solutions was replaced by Li⁺ or choline⁺ the glycodiazine transport was strongly reduced. Ouabain (5 · 10^–2 M) inhibited too, but amiloride (10^–3 M) had no effect on glycodiazine transport.The glycodiazine transport was 28% reduced in metabolic alkalosis and to a smaller although significant extent (17%) in metabolic acidosis; it was unchanged in chronic hypercapnia. In chronic K⁺ depletion the glycodiazine reabsorption was accelerated by 12% only in the early proximal loops. Chronic parathyroidectomy as well as acute substitution with parathyroid hormone had no effect on the glycodiazine absorption. The main conclusions are: Proximal H⁺ transport proceeds with suitable buffers. Although independent of HCO₃ ^– and carbonic anhydrase, it could be partially inhibited by CA inhibitors. H⁺ transport is supposed to proceed as countertransport with Na⁺ ions. In chronic alkalosis the H⁺ transport is reduced.