Stimulation by glucagon and PTH of Ca and Mg reabsorption in the superficial distal tubule of the rat kidney

The effects of glucagon and PTH on electrolyte reabsorption in the distal tubule were investigated in rats deprived of vasopressin, calcitonin, PTH, and glucagon. Micropunctures of distal tubule, at a late and an early site of a same nephron, have been performed in 23 rats, nine control, seven infused with glucagon (5 ng·min^–1·100 g^–1 b.w.) and seven with PTH (5 mU·min^–1·100 g^–1 b.w.). The Ca and Mg reabsorptive capacity of the distal segment was increased by glucagon and by PTH. Moreover, fractional Na and Cl reabsorption was significantly higher than in control during PTH administration. A K secretion appeared during the administration of both hormones. No phosphate net transport was observed in any group. Finally, the data presented here, together with those previously reported, indicate that the increase of Ca and Mg renal reabsorption observed with glucagon and PTH results from an effect located in both Henle's loop, where the bulk of Ca and Mg is reabsorbed, and the distal tubule.     

The effects of carbachol on water and electrolyte fluxes and transepithelial electrical potential differences of the rabbit submaxillary main duct perfusedin vitro

Summary The effect of carbachol on transepithelial potential difference and transepithelial nett electrolyte transport has been studied in the rabbit submaxillary main duct perfusedin vivo andin vitro with bicarbonate saline. The two preparations function similarly, reabsorbing Na, Cl and water and secreting K. In control ducts nett Na reabsorption was 683±55 nmol · cm^–2 · min^–1 and K secretion was 31.2±2.4 nmol · cm^–2 · min^–1. Nett water reabsorption was 970±71 nl · cm^–2 · min^–1 and the hydraulic conductivity was (14.0±1.6)×10^–6 ml · cm^–2 · s^–1 · atm^–1. The mean transepithelial potential difference was 13.1±0.8 mV (lumen negative) and, assuming no active transport of Cl, the partial conductance of the duct to Cl was (12.7±2.6)×10^–2 mho · cm^–2. Carbachol,in vivo andin vitro, caused partial depolarization of the transepithelial potential difference and reduction of nett Na and Cl reabsorption. It was without effect on duct K and HCO₃ transport.In vitro, the drug was effective in concentrations as low as 10^–7 M and perhaps lower. Atropine was able completely to block the effects of carbachol present at twice the atropine concentration. The results are consistent with the hypothesis that carbachol acts in some way to reduce the sodium conductance of the luminal face of the duct epithelial cell, this response being secondary to an undefined primary action of carbachol on the interstitial face of the cell.Preliminary accounts of this work have already been published [13, 14].     

Effect of calcium,furosemide and chlorothiazide on net volume reabsorption and basolateral membrane potential of the distal tubule

In the distal tubule of the isolated kidney of Amphiuma net volume reabsorption (split-oil droplet method) and basolateral membrane potential ( _b ) were measured. Luminal perfusion solution could be changed rapidly from 108 mmol·l^–1 NaCl plus 0.1 mmol·l^–1 calcium to solutions containing 103 or 97 mmol·l^–1 NaCl plus 3.6 or plus 7.2 mmol·l^–1 calcium. Furthermore, 10^–4 mol·l^–1 furosemide or chlorothiazide were applied luminally. (1) Addition of 7.2 mmol·l^–1 calcium hyperpolarized _b from –73.4 mV to –108.3 mV and inhibited net volume reabsorption. (2) Similarly, when furosemide was injected, _b was hyperpolarized and net volume reabsorption reduced. Application of both high calcium and furosemide further inhibited volume reabsorption. (3) The effects of chlorothiazide were similar to those of furosemide. However, when both high calcium and chlorothiazide were administered _b and volume reabsorption were almost normalized. (4) The data are consistent with the hypothesis that calcium and the diuretics interfere primarly with chloride uptake across the luminal membrane and thus reduce sodium chloride transport. When chlorothiazide in the presence of high luminal calcium almost normalized chloride transport, it is likely that its effects were by stimulating calcium transport and thus increasing intracellular calcium activity.Supported by Deutsche Forschungsgemeinschaft (Wi 328)The paper was presented, in part, at the XXVIIth Int. Congr. Physiol. Sci., Paris 1977, Proc. Vol. 13:304     

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     

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.     

Water and total CO2 reabsorption along the rat proximal convoluted tubule

Micropuncture experiments were performed on rat kidney to evaluate the profile of water and total CO₂ reabsorption along the proximal tubule. Three to eight samples were collected along the same nephron and the puncture-to-glomerulus distances were measured for each site. In Munich rats with accessible glomeruli, the water reabsorption rate was found to be constant all along the first five millimeters of proximal tubule. In Sprague Dawley rats with no accessible glomerulus, the same observation was made for these five millimeters, and the water reabsorption rate per mm along this segment was found to be a function of the glomerular filtration rate. For the two last millimeters of tubule accessible at the kidney surface, the water reabsorption rate was found to decrease in 5 out of the 21 tubules studied and ranged from 0.15–3.5 nl·min^–1·mm^–1.In Sprague Dawley rats the fall in the luminal total CO₂ concentration (CO₂)_t along the tubular length was nearly constant (21 mmole·l^–1) between Bowman's capsule and the end proximal tubule, irrespective of the plasma (CO₂)_t value. The distance needed to reach half-maximum total CO₂ reabsorption varied from 1.1–1.9 mm from one tubule to another, as a function of the total CO₂ filtered load. These data suggest that the tubular length involved in avid bicarbonate reabsorption increases as a function of the filtered load and that in the first millimeters of tubule, bicarbonate reabsorption depends on a rapidly saturable mechanism. However, no close relationship was found between total CO₂ movement or the calculated transepithelial chloride gradient on the one hand and water reabsorption along the convoluted proximal tubule on the other.     

Renal function and concentrating ability in a desert rodent: The gundi(Ctenodactylus vali)

Clearance and cortical micropuncture experiments were carried out on non diuretic gundis. In this species, the kidney has a long and well developed papilla but, unlike other desert rodents, the vascular organization of the outer medulla is very simple. After withdrawal of water supply for either 24 h or 3 days before the experiments, the urine osmolality was only 1,361±57,n=9, before and 1,136±89 mosmol ·kg^–1 during anesthesia. The GFR per 100 g B. W. (0.450 ml ·min^–1) is lower than in the rat studied under similar conditions. With regard to electrolytes the tubular handling of Na, Ca, K and Mg is similar to that observed for another desert rodent, psammomys obesus. For P, massive reabsorption (more than 30% of the filtered load) takes place along the distal convoluted tubule. The relatively poor concentrating ability of the gundi's kidney is not due to a lack of medullary recycling of urea since a net addition of urea to short loops of Henle is observed in this species. Physiological and morphological observations concerning the gundi and other desert rodent species suggest that the vascular bundle development in the outer medulla might affect the renal response to water deprivation.     

The role of extracellular sodium on heart muscle energetics

A study has been made of changing external sodium concentration [Na]_e, over the range 75 to 200 mmol · l^–1, on contractile parameters and heat production in isolated, arterially perfused, interventricular rabbit septa.-The observed changes in maximum rate of contraction with [Na]_e, either in the presence of a constant external Ca concentration [Ca]_e or in the presence of a constant [Na] _e ² /[Ca]_e ratio, paralleled those observed for tension development (T). On the other hand the maximal rate of relaxation and the ratio increased. While the ratio between active heat production and developed tension remained unaltered (0.111±0.003 mJ · mN^–1 · g^–1 dry weight), resting heat production increased with [Na] _e ² with a slope of 95±18 mW · g^–1 · mol^–2 · l². Under resting conditions, a decrease in [Na]_e of 50 mmol · l^–1 induced a fall in⁴²K uptake of about 16 nmol · s^–1 · g^–1 without changes in⁴²K efflux, suggesting that such an intervention depresses K influx. If the depressed K influx, induced by a decrease in [Na]_e of 50 mmol · l^–1 is associated with a decrease in Na–K pump activity, a fall in resting heat production of about 0.64 mW · g^–1 would be expected. This represent 56% of the calculated change in the resting heat production, 1.14±0.22 mW · g^–1 (mean ± one confidence interval), suggesting that some process in addition to a depressed Na-K pump activity may be altered by changes in [Na]_e.Supported by the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Argentina     

Coupling of metabolic CO2 production to ion transport in isolated rat thick ascending limbs and collecting tubules

Metabolic CO₂ production from appropriate [U-¹⁴C]-labelled substrates (eitherl-lactate ord-glucose) was measured in single pieces of tubule as previously described (Le Bouffant et al. 1984). Changing the incubate osmotic pressure by mannitol addition resulted in an increase in oxidative metabolism which was more marked in outermedullary segments (MAL and MCT) than in cortical segments (CAL and CCT). Availability of metabolic substrate was not rate limiting under these conditions because FCCP addition (1 mol·l^–1) produced a marked rise in CO₂ production in these structures.Ouabain (1 mmol·l^–1) decreased by more than 50% the CO₂ production by CAL, MAL, CCT and MCT samples, indicating that the larger part of oxidative metabolism was coupled to active Na transport. Furosemide addition (10^–5 mol·l^–1) to CAL and MAL samples, or amiloride addition (10^–4 mol·l^–1) to CCT and MCT samples reduced the rate of CO₂ production to an extent almost similar to that obtained with ouabain, an observation suggesting that apical entry of Na⁺ was present in these non-perfused tubules.Finally, the effects of changing the concentration of either K⁺ or Cl^– was tested in CAL samples. K⁺ suppression greatly depressed the rate of CO₂ production. Replacement of chloride with sulfate also decreased this rate to an extent similar to that observed with furosemide. The CO₂ production increased in a sigmoid way (apparentK _a=41 mmol·l^–1, Hill coefficient=2.12) as a function of [Cl^–] in the incubate, suggesting that oxidative metabolism was coupled to bath chloride via the Cl^–-requiring Na entry along the 1 Na⁺–1K⁺–2Cl^– luminal contrasport system.     

Effect of acetylcholine on time-independent currents in sheep cardiac Purkinje fibers

Voltage-clamp experiments were carried out in sheep Purkinje fibers in order to find an explanation for the prolongation of the action potential, the positive shift of the plateau, the hyperpolarization of the maximum diastolic potential and the increase in rate of diastolic depolarization, occurring in the presence of acetylcholine (Ach).In the presence of Ach the instantaneous current-voltage relation is shifted in the inward direction for potentials positive to –75 mV, while the opposite shift is obtained for more negative potentials; the results suggest a decrease in background conductance.The contribution of K, Cl, Na and Ca to the Ach sensitive current was studied by varying K₀ concentration or adding 20 mmol·l^–1 Cs, by omitting Cl or Na, and by changing the Ca concentration.In 20 mmol·l^–1 Cs the apparent reversal potential of the Ach sensitive current is –50 mV, as compared to –75 mV in normal Tyrode. The component of the Ach sensitive current, which is suppressed by Cs, shows inward going rectification. In different K₀ concentrations the reversal potential of the Ach sensitive current is changed; the shift obeys the theoretical change in equilibrium potential of K. The results are consistent with a decrease in K background current by Ach (inward and outward rectifier).In Cl free media the Ach sensitive current is not decreased excluding a major contribution of Cl ions. The Ach effect also persists in Na free media; the reversal potential of the Ach sensitive current is slightly shifted in the hyperpolarizing direction. These results indicate that active electrogenic pumps (Na or Na–Ca) do not play an important role; they are in accord with a reduction in inward Na background current by Ach. The shift of the current-voltage relation by Ach was greater the lower the Ca_o concentration; the mechanism is not clear.The inward shift of the current at –40 mV was dependent on the Ach concentration. Half-maximum effect was obtained at 3·10^–7 mol·l^–1 Ach; the Hill coefficient was 1.12.It is concluded that Ach interacts in a one to one reaction with a muscarinic receptor and reduces the background current mainly carried by K (inward and outward rectifier), and less by Na (and probably Ca).Supported by F.G.W.O. Belgium 3.0087.74     

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.     

Atrial natriuretic peptide has no direct effect on proximal tubule sodium and water reabsorption

Infusion of ANP has been shown to increase the urinary excretion of sodium and water. However it is still controversial in which tubular segment sodium reabsorption is inhibited. To clarify this problem we have performed in vivo and in vitro studies to examine the direct effect of ANP on rat proximal tubules. The in vivo effect of ANP has been tested by using the micropuncture technique and in particular the shrinking droplet method that allows each investigated tubule to serve as its own control. Addition of either low (10^–9 M) or high (2×10^–6 M) concentrations of ANP to the luminal perfusate resulted in no significant change in isotonic fluid reabsorption (J _v). The same holds when the proximal tubules were perfused on both the tubular and peritubular side, with modified Ringer solution containing 10^–9 M ANP. To examine possible in vitro effects of ANP we prepared highly purified proximal tubule suspension derived from rat renal cortex and monitored oxygen consumption (QO₂) that is tightly coupled to sodium transport in this segment. Synthetic ANP, either at low (10^–9 M) or at high (10^–6 M) concentrations, did not affect basal rate of tubular respiration. Moreover the peptide hormone (10^–9 M) did not inhibit nystatin stimulated and ouabain sensitive QO₂. These results indicate that the enhancement of renal sodium excretion induced by ANP is not related to a direct inhibition of sodium transport in the proximal tubule.     

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.     

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

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PD_te), transepithelial resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of the lumen membrane (FR₁), and the cellular activities for Cl^–, Na⁺, and K⁺ (a _x ^cell ) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10^–4, adenosine 10^–4, and forskolin 10^–6 mol · l^–1) on these parameters were recorded and compared to nonstimulated state (NS). PD_te increased from –1.9±0.2 mV to –11.0±0.9 mV (n=51). PD_bI depolarized from –86±1 to –74±1.4 mV (n=52). R_te fell from 29±2.8 to 21±2 cm² (n=23), and FR₁ fell from 0.96±0.005 to 0.79±0.04 (n=9).a _K+ ^cell was constant (123±13 versus 128±17 mmol · 1^–1) (n=6), buta _Cl– ^cell -fell significantly from 48±4 to 41±3 mmol · l^–1 (n=7).a _Na+ ^cell increased from 11±2.1 to 29.5±6.6 mmol · l^–1 (n=4). In series 2 the conductivity properties were examined by rapid K⁺, and Cl^– concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K⁺ led to a hyperpolarization of PD_bI with a mean slope of 28±1.3 mV/decade (n=9) (as compared to 19 mV/decade for S-state). Reduction of lumen Cl^– led to very little depolarization of the lumen membrane PD in NS-state: 6.5±2.3 mV/decade (n=4) (as compared to 13 mV/decade for S-state). In series 3 the effects of furosemide (7 · 10^–5 mol l^–1, bath) were examined in NS and S tubules. In NS RGT segments furosemide had no effect on PD_bI or PD_te;a _Cl– ^cell fell slowly after furosemide with an initial rate of 0.33 mmol · l^–1 s^–1, as compared to 1.5 mmol · l^–1 · s^–1 for S-state. The increase ina _Cl– ^cell after removal of furosemide from NS to S-states was examined in the presence of furosemide. Despite the presence of furosemide stimulation was accompanied by a fall in R_te, FR₁, anda _Cl– ^cell . From these data we conclude that (a) stimulation by cyclic AMP increases the Cl^–-conductance of the apical cell membrane at least by a factor of 10, that (b) in the NS-state the Na⁺2Cl^–K⁺ carrier can be triggered to work at rates similar to the S state by loweringa _Cl– ^cell , and that (c) the increase in apical Cl^–-conductance is the primary event in cyclic AMP mediated stimulation of NaCl secretion.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-1, and by NIH Grant AM 34208     

Chloride reabsorption in the rabbit cortical thick ascending limb of the loop of Henle

The question was investigated whether the chloride reabsorption in the cortical thick ascending limb (cTAL) of rabbit kidney is primary or secondary active, i.e. whether it depends on sodium. Isolated cTAL segments were perfused in vitro at high flow rates (10–20 nl·min^–1) with identical modified Ringer's solutions on both sides of the epithelium. The modified Ringer's solution contained sodium and/or organic cations (tris-hydroxymethyl-aminomethane, tetraethylammonium, choline) and 150 mmol·l^–1 chloride. Transepithelial electrical potential difference (PD) and transepithelial specific resistance (R _T) were directly measured, and used to calculate the short circuit current (I_sc) under three types of experimental conditions. In group 1 (n=8), the tubules were first perfused with solutions containing 150 mmol·l^–1 sodium. The PD was +9.6±0.4 mV (relative to the tubule lumen). Then sodium was replaced by an organic cation whereby the PD fell slightly to +8.2±1.6 mV. This PD disappeared when furosemide was added to the lumen perfusate, ouabain added to the bath, or temperature decreased to 295°K (22° C). To test whether the removal of sodium was successfull in this series, the sequence of perfusions was reversed in the second group (n=31). First the perfusion system was thoroughly rinsed with a sodium-free solution. Then a tubule was mounted and perfused under seemingly sodium-free conditions. The PD was only + 2.6 ±0.3 mV, corresponding to an I_sc of 29±3 A·cm^–2. When sodium was gradually added, PD and I_sc increased steeply with a half maximal response at 3.4 mmol·l^–1. The maximal I_sc was 258 A·cm^–2. In the last series (n=13), the efforts to make the system sodium-free were even more rigorous. The perfusion system in this series was made from sodium-free glass and the bath exchange rate was increased. The PD in the absence of sodium was +0.6±0.2 mV. These data indicate that the apparent sodium-independence in series 1 as well as in previous reports on this issue is artefactual and is caused by a small amount of sodium which is left in the system after the replacement by sodium-free solutions. A kinetic analysis of series 2 and 3 revealed that all active chloride reabsorption by the cortical thick ascending limb of Henle's loop is sodium-dependent suggesting that sodium and chloride are cotransported in the luminal membrane of this nephron segment.This study was supported by Deutsche Forschungsgemeinschaft. Parts of this study have been presented at the 28th IUPS meeting Budapest 1980     

Renal handling ofl-histidine studied by continuous microperfusion and free flow micropuncture in the rat

Renal tubular reabsorption ofl-histidine (His) was measured in vivo et situ by continuous microperfusion and free flow micropuncture of single proximal convoluted tubules of the rat kidney. The reabsorption is shown to be saturable. A permeability coefficient (P) of <29 m² · s^–1, a maximum reabsorption rate (J _max) of 2.75±1.05>J _max>1.97±0.86 (SEM) nmol · m^–1 · s^–1 and an affinity constant (K _m) of 13.8±4.2>K _m>10.9±4.0 (SEM) mol · l^–1 (lower values forP=29 m² · s^–1, higher values forP=0) were calculated from the microperfusion data. Using these constants and taking backflux of His and water reabsorption into account a good fit with the concentration profile of His along the proximal tubule — measured by free flow micropuncture — was obtained.Varying the buffered pH-values of the perfusion fluids (5.0 or 7.4) influenced neither the active reabsorption nor passive permeability of His. This indicates that the charge of the imidazol group of His does not play a significant role in His reabsorption. Further experiments showed that the addition of 20 mmol · l^–1 l-arginine — a strong inhibitor of the reabsorption system for dibasic ammino acids — did not have a significant effect on the reabsorption ofl-histidine. It is concluded, therefore, that His is reabsorbed by a system for neutral amino acids. Non ionic diffusion does not play an important role for His reabsorption.Part of this work was presented at the 51st meeting of the German Physiological Society in Kiel, 1979 [15]     

Proximal tubular transport and urinary excretion of sodium after renal denervation in sodium depleted rats

The effect of unilateral renal denervation on renal handling of water, sodium and potassium was studied with clearance and micropuncture techniques in sodium depleted anaesthetized rats in the nondiuretic state. In clearance experiments renal denervation resulted in a +140 and +320% increase in urine flow and potassium excretion, but sodium excretion of innervated (I) and denervated (D) kidneys was similar (I: 12.0±2.0, D: 14.0±3.6 nM·min^–1·g^–1; NS). However, upon the loop diuretic furosemide (1 mg·kg^–1), a marked denervation natriuresis was observed (I: 2.8±0.9, D: 5.9±1.0 M·min^–1;P<0.05) and denervation diuresis and kaliuresis persisted, too (+95 and +60%, respectively). Micropuncture results revealed that fractional reabsorption of filtrate to late proximal puncture site was depressed by renal denervation from 62 to 49% while no change in time control rats was seen (64±2 vs. 64±1%; NS). In micropuncture experiments besides augmented urine flow (+82%) from D kidneys also a small denervation natriuresis was present (I: 21.6±6.4, D: 29.2±7.0 nM·min^–1;P<0.05). It is concluded that the lack or marked attenuation of denervation natriuresis in sodium depleted rats were the result of an almost complete compensatory distal reabsorption of the excess sodium (but not of water and potassium) leaving the proximal tubule after denervation. The distal adaptive response can be overcome by furosemide.     

Mechanism of hydrogen ion transport in the diluting segment of frog kidney

Transepithelial H⁺ transport was studied in diluting segments of the isolated-perfused kidney ofrana esculenta. The experiments were performed in controls as well as in K⁺-adapted and Na⁺-adapted animals (exposed to 50 mmol/l KCl or NaCl, resp. for at least 3 days). Conventional and single-barreled, liquid ion-exchanger H⁺-sensitive microelectrodes were applied in the tubule lumen to evaluate transepithelial H⁺ net flux (J _te ^H ) as well as limiting transepithelial electrical and H⁺ electrochemical potential differences (PD _te ,E _te ^H ) and luminal pH at zero net flux conditions. The measurements were made in absence (control) and presence of furosemide (5·10^–5 mol/l) or amiloride (10^–3 mol/l). E _te ^H (lumen positive vs ground) was 19±3 mV in controls, 43±3 mV in K⁺ adapted but about zero in Na⁺ adapted animals. Using the correspondingPD _te -values, steady state luminal pH of 7.63±0.05, 7.13±0.05 and 8.02±0.02 was calculated for the respective groups of animals (peritubular pH 7.80). In parallel, significant secretoryJ _te ^H (from blood to lumen) was found in controls (14±2 pmol·cm^–2·S^–1) which was stimulated by K⁺ adaptation (61±8 pmol·cm^–2·s^–1) but reversed in direction by Na⁺-adaptation (–8±1 pmol·cm^–2·s^–1). Amiloride inhibited secretoryJ _te ^H . Elimination of the lumen positivePD _te by furosemide did not affect significantlyE _te ^H andJ _te ^H in control and K⁺ adapted animals but abolished reabsorptiveJ _te ^H in Na⁺ adapted animals.We conclude that in frog diluting segment H⁺ secretion is an active, amiloride-sensitive, furosemide-insensitive transport process. The data are consistent with luminal Na⁺/H⁺ exchange. The activity of this system depends critically on the metabolic state of the animal.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)This work was supported by österr. Forschungsrat, Proj. No.: 4366 and by Dr. Legerlotz Stiftung     

“Run-down” of the Ca current during long whole-cell recordings in guinea pig heart cells: role of phosphorylation and intracellular calcium

We examined by a statistical approach the decrease of the Ca current (run-down) during long-lasting recordings with the whole-cell patch-clamp technique in guinea pig ventricular myocytes. The results are as follows. (1) Run-down of the Ca current (I _Ca) occurs in three phases (T1–T3). T1 (38±19 min,n=135) and T3 (35±17 min,n=23) are characterized by a slow rate of decay ofI _Ca [90±20 and 60±20 nA·cm^–2·min^–1, respectively]. T1 and T3 are separated by T2 (6±4 min,n=135) during which the current decays quickly [1200±230 nA·cm^–2·min^–1]. Between the onsets of T1 and T3,I _Ca decreases from 11±3 to 3.5±1 A/cm². (2) Normalized current-voltage relationship, reversal potential and voltage-dependencies of steady-state activation and inactivation ofI _Ca are globally shifted toward more negative potentials during the run-down process by 10–15 mV. (3)I _Ca3 measured during T3 retains the pharmacological properties (blockade by D600, NiCl₂ and CoCl₃, increase by isoprenaline and insensitivity to tetrodotoxin) of the originalI _Ca. (4) Intracellular perfusion of the nonhydrolysable ATP analogue AMP-PNP does not prevent the occurrence of T2, suggesting that a phosphorylation-dephosphorylation process is not involved in the fast run-down ofI _Ca. (5) With 0.1 mM EGTA in the pipette, addition of 3 mM ATP significantly prolongsI _Ca survival. No improvements are obtained by increasing the ATP concentration to 10 mM or replacing ATP with creatine phosphate. With 3 mM ATP present, increasing the EGTA concentration to 10–20 mM doublesI _Ca survival time. EGTA alone (10 mM) is less effective than the mixture 3 mM ATP-0.1 mM·EGTA. Intracellular perfusion with a cytoplasmic extract considerably prolongs T2 and the overallI _Ca survival. (6) The results are consistent with the hypothesis that run-down ofI _Ca can partially be explained by a rise in intracellular Ca concentration and a loss of high energy compounds. Beneficial effect of ATP might include an increased capability of the cells to either extrude or sequester intracellular Ca, and a protection against enzymatic proteolysis.Recipient of successive fellowships from the Simone et Cino del Duca and Alexander von Humboldt FoundationsThis work was supported by the Deutsche Forschungsgemeinschaft, SFB 246, Project A1     

Mechanisms of oxalate absorption and secretion across the rabbit distal colon

To further evaluate the mechanisms of oxalate (Ox^2–) transport in the intestine the following studies were performed using isolated, short-circuited segments of the rabbit distal colon (DC). In control buffer, the DC absorbed Ox^2– (net Ox^2– flux, J _Net ^Ox =5.4±0.7 pmol · cm^–2 · h^–1). Replacement of Na⁺ with N-methyl-d-glucamine (NMDG⁺) abolished Ox^2– absorption by decreasing mucosal to serosal Ox^2– flux (J _ms ^Ox ), without affecting Cl^– transport, while gluconate substitution for Cl^– did not affect J _Net ^Ox or net Na⁺ flux (J _Net ^Na ). Addition of Na⁺ to the serosal side of tissues bathed by NMDG⁺ buffer increased J _ms ^Ox 40% without altering mucosal to serosal Cl^– flux (J _ms ^Cl ). Serosal amiloride or dimethyl amiloride (10^–3 M) abolished J _Net ^Ox by decreasing J _ms ^Ox , it increased serosal to muscosal Cl^– flux (J _sm ^Cl ) and it gradually inhibited short-circuit current (I _sc). Mucosal amiloride (10^–4 M) abolished I _sc but had no effect on Ox^2– or Cl^– fluxes. Serosal 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 10^–6 M) reduced J _ms ^Ox by 20% and J _Net ^Ox by 43% without affecting J _ms ^Cl or J _Net ^Cl . Dibutyryl cyclic adenosine monophosphate (dB-cAMP, 5×10^–4 M, both sides) stimulated Ox^2– secretion (J _Net ^Ox = –12.6±3.3 pmol · cm^–2 · h^–1). The dB-cAMP-induced secretion of Ox^2– and Cl^– were fully abolished by serosal furosemide (10^–4 M) and partially inhibited (35%) by 5×10^–4 M mucosal NPPB [5-nitro-2-(3-phenylpropylamino)-benzoic acid], a putative Cl^– channel blocker. It is proposed that: (1) basal absorption of Ox^2–, but not Cl^–, is dependent upon a previously undescribed basolateral Na⁺-H⁺ exchanger that may be coupled to a DIDS-sensitive, basolateral anion exchange system that mediates Ox^2– flux; (2) the DC secretes Ox^2– in response to dB-cAMP by a mechanism that is indistinguishable from the pathway for Cl^– secretion.