Substrate utilization in the isolated perfused cortical thick ascending limb of rabbit nephron

Isolated segments of cortical thick ascending limbs (cTAL) of rabbit kidney were perfused in vitro and the equivalent short circuit current (Isc) was measured. In a first series all substrates were removed on either side. Isc fell rapidly to 50±12% after 3 min and to 27±6% (n=5) after 10 min. This indicates that in cTAL segments Isc is strictly dependent on the presence of substrates. In series two it was tested what substrates can be utilized by the cTAL segment, and from which epithelial side [bath (b) or lumen (l)] the substrates are taken up. From the l-side only butyrate (10 mmol · l^–1) sustained the Isc at 95±2% (n=7). All other tested substrates (10 mmol · l^–1): pyruvate, acetate, -OH-butyrate,d-glucose, andl-lactate lead to a marked decline in Isc. From the b-side several substrates (5–10 mmol · l^–1) sustained the Isc:d-glucose,d-mannose, butyrate, -OH-butyrate, acetoacetate,l-lactate, acetate and pyruvate. Other compounds (1–10 mmol · l^–1): citrate, -ketoglutarate, succinate, glutamate, glutamine, propionate, caprylate and oleate did not sustain Isc. In the third series the mechanism of substrate utilization from the basolateral cell side was studied. It was shown that the Isc is a saturable function of thed-glucose,l-lactate, acetate, pyruvate or -OH-butyrate concentration with apparentK _m's between 0.05–1.0 mmol · l^–1. Several known inhibitors of sugar and of anion transport were tested at the bath side: phlorrhizin was without effect. Phloretin (500 mol · l^–1) inhibited Isc by 96%, yet its effect was not dependent on the presence of substrates on the b-side since inhibition ocurred also if the b-perfusate contained no substrate and Isc was driven by luminal butyrate. Also SITS (5 mmol · l^–1) exerted only a small inhibitory effect which was not specific since it was also observed with luminal butyrate. -Cyano-m-OH-cinnamate (10 mmol · l^–1) inhibited the Isc specifically whenl-lactate was the bath substrate. Probenecid (1 mmol · l^–1) had a similar yet less marked inhibitory effect. Thed-glucose uptake from the b-side was specifically inhibited by cytochalasin B at 5 · 10^–6 mol · l^–1. We conclude that the cTAL segment of the rabbit utilizesd-glucose and/or small anions such as pyruvate orl-lactate or acetate to energize salt reabsorption. The link between substrate availability and salt reabsorption is extremely close in this nephron segment. Substrate uptake occurs from the blood side. Sugar uptake can be inhibited by cytochalasin B andl-lactate uptake by probenecid and -cyano-m-OH-cinnamat. These data suggest that substrate uptake at the basolateral cell side occurs probably via carrier systems.Parts of this study have been presented at the 57th and 58th Tagung Deutsche Physiologische Gesellschaft, 17th Meeting Europ. Soc. Clin. Investigation, and XXIX Int. Congress Physiol. Sciences. This study was supported by Deutsche Forschungsgemeinschaft Gr 480/5-7     

Potassium activity in cells of isolated perfused cortical thick ascending limbs of rabbit kidney

The Na⁺2Cl^–K⁺ cotransporter in the apical membrane of the cortical thick ascending limb of the Henle's loop (cTAL) of rabbit nephron utilizes the electrochemical gradient for Na⁺ to transport K⁺ and Cl^– against an unfavorable electrochemical gradient from lumen to cell interior. In the present study attempts are made to measure intracellular K⁺ activity ( ) under control conditions and after inhibition of the cotransport system by furosemide (50·10^–6 mol·l^–1). 70 cTAL segments of 55 rabbits were perfused in vitro. Conventional Ling-Gerard and K⁺-selective microelectrodes were used to measure the PD across the basolateral membrane (PD_bl) as well as the PD sensed by the single barrelled K⁺-selective electrode ( ). PD_bl was –64±1 (n=65) mV and +15±1 (n=32) mV under control conditions. The positive value, significantly different from zero, indicates that is higher than predicted for passive distribution. The estimate for obtained from PD_bl and was 113±8 mmol·l^–1. Furosemide lead to the previously reported hyperpolarization of PD_bl by 17±4 (n=13) mV and to a reduction of from 15±1 to 5±1 (n=20) mV. The , obtained from this set of data, was 117±9 mmol·l^–1, and was not different from the control value. The present data indicate that is significantly above Nernst equilibrium under control conditions. The source for this above equilibrium accumulation of K⁺ stems from the carrier mediated uptake of Na⁺2Cl^– and K⁺. Consequently, the electrochemical gradient for K⁺ is rapidly reduced when the carrier is blocked by furosemide. The electrochemical gradient for K⁺, under control conditions, energizes the back leak of K⁺ from cell to lumen. This K⁺ flux is one component responsible for the lumen positive transepithelial PD.Parts of this study have been presented at the 58th Tagung Deutsche Physiologische und Deutsche Pharmakologische Gesellschaft, Mainz 1983; 67th Federation Meeting, Chicago 1983. This study was supported by Deutsche Forschungsgemeinschaft Gr. 480/5-7     

Hormone-mediated Ca2+ transients in isolated renal cortical thick ascending limb cells

Peptide hormones control salt reabsorption in cortical thick ascending limb (cTAL) cells of the loop of Henle. These agonists act, in part, through alterations on intracellular Ca²⁺ ([Ca²⁺]_i). Primary cell cultures were prepared from porcine kidneys using a double antibody technique (goat antihuman Tamm-Horsfall and rabbit antigoat IgG antibodies). [Ca²⁺]_i was determined in single cells with fluorescent techniques using fura-2. Parathyroid hormone (PTH) and arginine vasopressin (AVP) transiently increased [Ca²⁺]_i in a dose-dependent manner. [Ca²⁺]_i maximally increased from 85±5 nmol/l to 608±99 nmol/l with PTH, 10^–6M, and to 766±162 nmol/l with AVP, 10^–7 M. The increment in [Ca²⁺]_i by both hormones was by intracellular Ca²⁺ release and entry through plasma membrane Ca²⁺ channels. 8-Bromoadenosine-3, 5-cyclic monophosphate (8-BrcAMP), 10^–4M, increased [Ca²⁺]_i(basal 83±3 to 427±121 nmol/l) but only from internal sources as nifedipine (10 mol), ([Ca²⁺]_i changes: 86±4 to 390±29 nmol/l) and removal of bath Ca _o ²⁺ , ([Ca²⁺]_ichanges: 84±6 to 517±142 nmol/l), were without effect on agonist-induced [Ca²⁺]_i. Thapsigargin, 1.5 mol, completely abolished the AVP- and cyclic adenosine monophosphate-(cAMP)-induced Ca²⁺ transients, and partially inhibited PTH-mediated Ca²⁺ transients by about 50%. Pretreatment with 8-BrcAMP inhibited the PTH and AVP responses likely through depletion of cAMP-sensitive Ca²⁺ stores. Activation of protein kinase C (PKC) with phorbol esters inhibited PTH and AVP responses and 8-BrcAMP-induced [Ca²⁺]_i transients. The responses partially returned following down-regulation of PKC with prolonged exposure to phorbol esters. These data suggest that PKC activation modulates agonist-induced Ca²⁺ release and entry, possibly through actions on intracellular release mechanisms. In summary, PTH and AVP stimulate Ca²⁺ signals by similar pathways involving cAMP and inositol 1,3,4-trisphosphate activity at similar sites on the endoplasmic reticulum and plasma membrane. These results suggest that peptide hormones may act through Ca²⁺ and be modulated by different pathways which may have diverse effects on cTAL function.     

Cation selectivity of the isolated perfused cortical thick ascending limb of Henle's loop of rabbit kidney

The cation permeability of the cortical thick ascending limb of Henle's loop was investigated with electrophysiological methods in the isolated perfused tubule preparation of rabbit kidney. The transepithelial specific resistance (R _T) and electrical potential difference (PD) were determined in 4 experimental groups. In group 1 (n=51) the tubules were perfused with a modified Ringer's solution on both sides of the epithelium; the PD was 7±0.4 mV lumen positive, and theR _T 34±2 ·cm². In group 2 (n=12) one of both sides of the epithelium was perfused with dilute (54 mmol/l) NaCl solutions under control conditions and in the absence of active transport. Inhibition was obtained in four different ways: low temperature (22° C), zero K⁺ solutions on both sides of the epithelium, 5·10^–5 mol/l furosemide, added to lumen perfusate, or 10^–5 mol/l ouabain added to the bathing solution. In the presence and in the absence of active transport a NaCl gradient of 154 versus 54 mmol/l induced diffusion potentials across the epithelium which were symmetrical and of nearly equal magnitude: +12, –14 and +15, –14 mV respectively. In group 3 (n=51) Na⁺ was completely replaced by choline⁺, tetraethylammonium⁺, tris-hydroxymethyl-aminomethane⁺, or Li⁺ in either bath or lumen perfusate or in both perfusates. The biionic diffusion potentials were symmetric; the replacement of Na⁺ by these cations on both sides markedly increasedR _T. Both kinds of measurements yielded a permeability sequence ofP _Na ⁺>P _Li ⁺>P _{organic cation}. In group 4 (n=17) 50 mmol/l of Na⁺ was replaced by K⁺, Li⁺, Rb⁺, or Cs⁺ on one of the sides and active transport was inhibited by furosemide or ouabain. From the membrane diffusion potentials and theR _T values in group 4 as well as in group 3 the following cation permeability sequence was calculatedP _K ⁺>P _Na ⁺>P _Rb ⁺ =P _Li ⁺>P _Cs ⁺>P _{organic cation}. It is concluded that the cortical thick ascending limb of Henle's loop has a low resistance pathway which is cation selective similar to that of leaky epithelia. Since the membrane diffusion potentials are symmetric and since they are not altered by inhibition of active transport, it is likely that this low resistance pathway is formed by a paracellular shunt.This study was supported by Deutsche Forschungsgemeinschaft. Parts of this study have been presented at the following meetings: 53rd meeting of the Deutsche Physiologische Gesellschaft, Kiel 1980; 64th Federation meetings Anaheim 1980; 28th IUPS meeting Budapest 1980.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

Differential effects of ADH on sodium,chloride, potassium,calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net transports was investigated in medullary (mTAL) and cortical (cTAL) segments of the thick ascending limb (TAL) of mouse nephron, perfused in vitro. Transepithelial net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Transepithelial potential difference (PD_te) and transepithelial resistance (R_te) were measured simultaneously. cTAL segments were bathed and perfused with isoosmolal, HCO ₃ ^– containing Ringer solutions, mTAL segments were bathed and perfused with isoosmolal HCO ₃ ^– free Ringer solutions. In cTAL segments, AVP (10^–10 mol·l^–1) significantly increasedJ _Mg ²⁺ andJ _Ca ²⁺ from 0.39±0.08 to 0.58±0.10 and from 0.86±0.13 to 1.19±0.15 pmol·min^–1 mm^–1 respectively. NeitherJ _Na ⁺ norJ _Cl ^–, (J _Na ⁺: 213±30 versus 221±28 pmol·min^–1 mm^–1,J _Cl ^–: 206±30 versus 220±23 pmol·min^–1 mm^–1) nor PD_te (13.4±1.3 mV versus 14.1±1.9 mV) or R_te (24.6±6.5 cm² versus 22.6±6.4 cm²) were significantly changed by AVP. No significant effect of AVP on net K⁺ transport was observed. In mTAL segments, Mg²⁺ and Ca²⁺ net transports were close to zero and AVP (10^–10 mol·l^–1) elicited no effect. However NaCl net reabsorption was significantly stimulated by the hormone,J _Na ⁺ increased from 107±33 to 148±30 andJ _Cl ^– from 121±33 to 165±32 pmol·min^–1 mm^–1. The rise inJ _NaCl was accompanied by an increase in PD_te from 9.0±0.7 to 13.5±0.9 mV and a decrease in R_te from 14.4±2.0 to 11.2±1.7 cm². No K⁺ net transport was detected, either under control conditions or in the presence of AVP.To test for a possible effect of HCO ₃ ^– on transepithelial ion fluxes, mTAL segments were bathed and perfused with HCO ₃ ^– containing Ringer solutions. With the exception ofJ _Ca ²⁺ which was significantly different from zero (J _Ca ²⁺: 0.26±0.06 pmol·min^–1 mm^–1), net transepithelial fluxes of Na⁺, Cl^–, K⁺ and Mg²⁺ were unaffected by HCO ₃ ^– . In the presence of AVP,J _Mg ²⁺ andJ _Ca ²⁺ were unaltered whereasJ _NaCl was stimulated to the same extent as observed in the absence of HCO ₃ ^– . In conclusion our results indicate heterogeneity of response to AVP in cortical and medullary segments of the TAL segment, since AVP stimulates Ca²⁺ and Mg²⁺ reabsorption in the cortical part and Na⁺ and Cl^– reabsorption in the medullary part of this nephron segment.This study was supported by the Commission des communautés européennes, grant no. ST2J 00951 F(CD), and by Wissenschafts-ausschuß der Nato über den DAAD     

Presence of luminal K+, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney

Previous data from our laboratory have shown that active transport in the cortical thick ascending limb of Henle's loop (cTAL), as measured by the short circuit current (I_SC, A · cm^–2), requires the presence of Na⁺ and Cl^–. The data were compatible with the model of secondarily active Cl^– reabsorption involving the cotransport of Na⁺ and Cl^– across the luminal membrane. The data suggested, furthermore, that 1 Na⁺ and 2 Cl^– interact with the luminal carrier. In the present study it was tested whether this reabsorptive mechanism also requires the presence of luminal K⁺. Isolated cTAL segments (n=40) were perfused at high flow rates with a modified Ringer's solution. Removal of K⁺ from the lumen reduced I_SC significantly from 215 to 133 A·cm^–2. Addition of Ba²⁺ (10^–3 mol·l^–1) which blocks the K⁺ conductance of the luminal membrane, to the K⁺-containing lumen perfusate decreased I_SC significantly from 234 to 141 A·cm^–2. Combination of both manoeuvres: perfusion with a K⁺-free and Ba²⁺-containing solution almost abolished I_SC from a control of 237 to 56 A · cm^–2. The results are compatible with the view that in rabbit cTAL the luminal carrier interacts with all 3 ions, possibly 1 Na⁺, 2 Cl^–, and 1 K⁺. K⁺ recycles across the luminal membrane through its conductive pathway.This study was supported by Deutsche Forschungsgemeinschaft Gr. 460/5-6-2     

Effects of antidiuretic hormone,parathyroid hormone and glucagon on transepithelial voltage and resistance of the cortical and medullary thick ascending limb of Henle's loop of the mouse nephron

The effect of antidiuretic hormone (arginine vasopressin, AVP, 10⁻¹⁰mol.l⁻¹), parathyroid hormone (PTH, 10⁻⁸ mol.l⁻⁸) and glucagon (10⁻⁸ mol.l⁻¹) on the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were tested in in vitro perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. When compared with mTAL segments (PD_te: 8.5±0.4 mV,n=16), cTAL segments displayed a high PD_te of 15.7±0.9 mV (n=11) at the beginning of perfusion experiments which reached a value of 9.4±0.6 mV (n =11) after 38±4 min perfusion. Simultaneously R_te increased significantly from 24±3 to 28±1 Ω cm² (n=11). When PTH, AVP or glucagon were added to the bath solution, PD_te increased with PTH from 10.3±0.8 to 15.2±0.8 mV (n=13), with AVP from 10.2±0.5 to 15.0±0.7 mV (n=24) and with glucagon from 11.3±1.9 to 15.3±2.1 mV (n=8). At the same time R_te decreased from 30±3 to 23±2 Ω cm², from 28±1 to 23±1 Ω cm² and from 23±2 to 18±2 Ω cm², respectively. In mTAL segments, AVP and glucagon increased PD_te from 8.4+0.5 to 13.5±0.9 mV (n=11) and from 8.8±0.6 to 12.8±0.6 mV (n=8) respectively, while R_te decreased significantly from 23±1 to 20±1 Ω cm² and from 27±3 to 21±3 Ω cm². PTH, on the other hand, had no effect on PD_te and R_te. Since the response to PTH appeared to be specific to cTAL segments, paired experiments were performed, in which AVP or glucagon were successively tested with PTH on cTAL and mTAL segments, to ascertain the specificity of the hormonal response. In cTAL segments, PTH and AVP increased the equivalent short-circuit current (I_sc=PD_te/R_te) by 82% and 86% respectively, while PTH and glucagon, in another series, increased I_sc by 95% and 81% respectively. In mTAL segments, I_sc was increased in the presence of AVP and glucagon by 88%, and 93% respectively, whereas PTH had no effect. These results indicate that Nacl reabsorption in cTAL segments is stimulated by AVP, PTH and glucagon and in mTAL segments by AVP and glucagon. The amplitude of the response to the hormones is similar in the two segments. The residual stimulation in cTAL segments, however, persists longer than in mTAL segments.     

cAMP increases the basolateral Cl−-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse

The effect of cAMP on transepithelial and transmembrane potential differences and resistances was examined in isolated in vitro perfused mouse medullary thick ascending limbs of Henle's loop (mTAL). The effects of furosemide and barium were tested. Stimulation of NaCl transport by ADH 10^–9+dbcAMP 4·10^–4+forskolin 10^–6 mol·l^–1 (paired experiments) resulted in: a) an increase in transepithelial potential difference, referenced to the grounded bath, from +6.7±0.3 mV to +12.0±0.4 mV (n=47); b) a decrease in transepithelial resistance from 25±1 cm² to 20±1 cm² (n=47); c) a depolarization of the basolateral membrane by 12 mV and of the apical membrane by 7 mV (n=36); d) a decrease in the fractional resistance of the basolateral membrane from 0.27±0.005 to 0.15±0.06 (n=12). Furosemide (10^–4 mol·l^–1) abolished the active transepithelial transport potential and hyperpolarized the basolateral membrane potential to values which were similar in both control and cAMP treated mTAL segments. Barium increased the transepithelial resistance and depolarizedPD _bl to similar values in both functional states. An increase in the fractional conductance of the basolateral membrane was also seen, if, prior to the cAMP treatment, the luminal Na⁺2Cl^–K⁺ contransport was inhibited by furosemide. Thus, we propose that stimulation of active NaCl reabsorption in the mTAL segment of the mouse by ADH, mediated via cAMP, increases primarily the basolateral chloride conductance.Supported by Deutsche Forschungsgemeinschaft Gr 480/6-2Parts of this study have been presented at the 59th Meeting of the German Physiological Society in Dortmund 1984 and at the 69th FASEB Meeting in Anaheim 1985     

Stimulation of NaCl reabsorption by antidiuretic hormone in the cortical thick ascending limb of Henle's loop of the mouse

The effect of antidiuretic hormone (ADH) on transepithelial Na⁺, Cl^–, Ca²⁺ and Mg²⁺ net fluxes (J_Na, J_Cl, J_Mg, J_Ca) was investigated in isolated perfused cortical thick ascending limb segments (cTAL) of the mouse nephron, using the microperfusion technique and the electron microprobe analysis to determine the ionic composition of the collected tubular fluid. Simultaneously, the transepithelial potential difference (PD_te) and the transepithelial resistance (R_te) were recorded. Prior to the flux measurements cTAL segments were perfused for one hour. During this equilibration period PD_te decreased significantly from +19.9±1.6 to +14.9±1.l mV and R_te increased from 30.6±3.5 cm² to 38.8±2.4 cm² (n=7), reflecting a decline in NaCl transport. After ADH was added to the bath solution at 10^–10 mol.l^–1, PD_te increased from +14.4±1.1 to +18.0±1.5 mV, accompanied by a rise in J_Na and J_Cl from 205±11 to 273±19 and from 216±12 to 283±21 pmol.min^–1.mm^–1 (n=7), respectively. J_Ca and J_Mg also increased from 0.81±0.07 to 1.50±0.12 and from 0.43±0.11 to 0.76±0.08 pmol.min^–1.mm^–1 (n=7), respectively. All these effects were fully reversible after withdrawal of the hormone. In conclusion our data indicate that ADH stimulates divalent cation transport and NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse.     

Effects of parathyroid hormone and calcitonin on Na+, Cl−, K+, Mg2+ and Ca2+ transport in cortical and medullary thick ascending limbs of mouse kidney

The effect of parathyroid hormone (PTH) on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ transport was investigated in isolated perfused cortical thick ascending limbs (cTAL) and that of human calcitonin (hCT) was tested in both cortical and medullary thick ascending limbs (mTAL) of the mouse nephron. The transepithelial ion net fluxes (J _x) were determined by electron probe analysis of the perfused and collected fluids. Simultaneously, the transepithelial voltage (PD_te) and resistance (R _te) were recorded. In cTAL segments, PTH and hCT significantly stimulated the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺. hCT generated a net K⁺ secretion towards the lumen and PTH tended to exert the same effect. Neither PD_te nor R _te were significantly altered by either PTH or hCT. However, in the post-experimental period a significant decrease in PD_te was noted. Time control experiments carried out under similar conditions revealed a significant decrease in PD_te with time, which could have masked the hormonal response. In mTAL segments, Mg²⁺ and Ca²⁺ transport was close to zero. hCT did not exert any detectable effect on either PD_te or J _Cl ^–, J _Na ⁺ J _K ⁺, J _Mg ²⁺ and J _Ca ²⁺ in these segments. In conclusion, our data demonstrate that PTH and hCT stimulate NaCl reabsorption as well as Mg²⁺ and Ca²⁺ reabsorption in the cTAL segment of the mouse. These data are in agreement with and extend data obtained in vivo in the rat.     

A cytotoxin of pseudomonas aeruginosa acts directly on the cortical thick ascending limb of henle's loop of rabbit kidney

The present study examines directly the effect of a cytotoxin of Pseudomonas aeruginosa on the in vitro perfused rabbit cortical thick ascending limb of the loop of Henle (cTAL). 25 cTAL segments were perfused at high rate. The open circuit transepithelial electrical PD (PD_te) and the specific electrical transepithelial resistance (R_t) were recorded continuously. From PD_te/R_t the equivalent short circuit current (Isc) was calculated. The Isc was 214±30 A·cm^–2 under control conditions, and decreased significantly to 74±34 A·cm^–2 60 s after the addition of toxin (2 mg·l^–1) to the lumen perfusate. Microscopic observation and photographs taken at that time clearly indicated swelling of the cTAL cells. Thereafter inhibition of active transport proceeded further, R_t fell progressively, and cells started to desquamate from the basement membrane. This effect of the toxin was dose dependent, and was half maximal at approximately 1.2 mg·l^–1. From the bath side the effect was less marked and higher doses of toxin had to be used (half maximal effect at 5 mg·l^–1). We conclude that this toxin of Pseudomonas aeruginosa exerts its toxic effect on the cTAL segment by increasing primarily the permeability of the lumen membrane.Part of this study has been presented at Spring meering Dt. Pharmakol. Ges., Mainz, 1982. This work has been supported by the Schutzkommission beim Bundesminister des Inneren, Bonn-Bad Godesberg, and by Deutsche Forschungsgemeinschaft, Gr 480/5-7     

Analogues of torasemide — structure function relationships —experiments in the thick ascending limb of the loop of Henle of rabbit nephron

The aim of the present study was to examine compounds related to torasemide with respect to their ability to block the equivalent short circuit current, corresponding to the rate of chloride reabsorption, in isolated in vitro perfused cortical thick ascending limbs of Henle of the rabbit. The torasemide molecule was modified with respect to the anionic sulfonylurea group, and the secondary amine linked to the pyridine ring. Our results indicate that only few of the tested 48 torasemide-related compounds were able to inhibit from both epithelial sides like torasemide. Only few of the tested compounds were equally effective as torasemide from the lumen side. Some analogues were acting only from the luminal side and some only from the peritubular side. The correlations between structure and potency of inhibition from the luminal side allow the following conclusions: a) The secondary amine moiety linked to the pyridine ring (toluidine in case of torasemide) can be replaced by a cycloalkylamine or, with some loss of inhibitory potency, by alkylamines. The inhibitory potency is increased with the number of C-atoms in the cycloalkylamine substituted compounds (optimum C₇ to C₈), and is also depending on the length of the alkylamines (optimum C₄). b) The secondary amine seems to be required since nitrogen cannot be replaced by –S- or –SO₂-. c) The sulfonylurea group cannot be substituted by other anionic groups such as –SO ₃ ^– or –COO^–. d) If the pyridine ring is replaced by a NO₂-substituted phenyl ring, the inhibitory potency from the luminal side is lost. However, these compounds act still (with some loss of potency) from the peritubular side. The data indicate that several of the conclusions drawn from our previous systematic surveys of chloride channel blockers and loop diuretics of the furosemide type, i.e. blockers of the Na⁺2Cl^–K⁺ carrier, hold also true for compounds related to torasemide. In addition, the pyridine ring is responsible for some specific structure activity correlations.Supported by Deutsche Forschungsgemeinschaft Gr 480/6     

Effects of glucagon on Na+, Cl−, K+, Mg2+ and Ca2+ transports in cortical and medullary thick ascending limbs of mouse kidney

The effects of glucagon on transepithelial Na⁺, Cl^–, K⁺, Ca²⁺ and Mg²⁺ net fluxes were investigated in isolated perfused cortical (cTAL) and medullary (mTAL) thick ascending limbs of Henle's loop of the mouse nephron. Transepithelial ion net fluxes (J _Na ⁺,J _Cl ^–,J _K ⁺,J _Ca ²⁺,J _Mg ²⁺) were determined by electron probe analysis of the collected tubular fluid. Simultaneously the transepithelial voltage (PD_te) and the transepithelial resistance (R _te) were recorded. In cTAL-segments (n=8), glucagon (1.2×10^–8 mol · l^–1) stimulated significantly the reabsorption of Na⁺, Cl^–, Ca²⁺ and Mg²⁺J _Na ⁺ increased from 204±20 to 228±23 pmol · min^–1 · mm^–1,J _Cl ^– from 203±18 to 234±21 pmol · min^–1 · mm^–1,J _Ca ²⁺ from 0.52±0.13 to 1.34±0.30 pmol · min^–1 · mm^–1 andJ _Mg ²⁺ from 0.51±0.08 to 0.84±0.08 pmol · min^–1 · mm^–1.J _K+ remained unchanged: 3.2±1.3 versus 4.0±1.9 pmol · min^–1 · mm^–1. Neither PD_te (16.3±1.5 versus 15.9±1.4 mV) norR _te (22.5±3.0 versus 20.3±2.6 cm²) were changed significantly by glucagon. However, in the post-experimental periods a significant decrease in PD_te and increase inR _te were noted. In mTAL-segments (n=9), Mg²⁺ and Ca²⁺ transports were close to zero and glucagon elicited no significant effect. The reabsorptions of Na⁺ and Cl^–, however, were strongly stimulated:J _Na ⁺ increased from 153±17 to 226±30 pmol · min^–1 · mm^–1 andJ _Cl ^– from 151±23 to 243±30 pmol · min^–1 · mm^–1. The rise in NaCl transport was accompanied by an increase in PD_te from 10.3±1.1 to 12.3±1.2 mV and a decrease inR _te from 19.1±2.7 to 17.8±2.0 cm². No net K⁺ movement was detectable either in the absence or in the presence of glucagon. A micropuncture study carried out in hormone-deprived rats indicated that glucagon stimulates Na⁺, Cl^–, K⁺, Mg²⁺ and Ca²⁺ reabsorptions in the loop of Henle. In conclusion our data demonstrate that glucagon stimulates NaCl reabsorption in the mTAL segment and to a lesser extent in the cTAL segment whereas it stimulates Ca²⁺ and Mg²⁺ reabsorptions only in the cortical part of the thick ascending limb of the mouse nephron. These data are in good agreement with, and extend, those obtained in vivo on the rat with the hormone-deprived model.This study was supported by the Commission des Communautés Européennes, Grant no. ST 23, 00951F (CD) and by Wissenschaftsausschuß der Nato über den DAAD     

Calcium transport across the rabbit thick ascending limb of Henle's loop perfused in vitro

Ca²⁺ transport across the cortical thick ascending limb of Henle's loop (TALH) was studied on the isolated rabbit renal tubule perfused in vitro. Both the efflux (Ke) and influx coefficient (Ki) of Ca²⁺ were determined with⁴⁵Ca at three different levels of the transtubular electrical potential difference (PDt) caused by varying the transtubular Na⁺ concentration gradient. The flux ratios,Ke/Ki, always exceeded those predicted from observed PDt by simple passive diffusion, and the net Ca²⁺ efflux was shown to occur against an electro-chemical potential. An increase in Ca²⁺ concentration in the perfusate was associated with a decrease inKe. Efflux of Ca²⁺, therefore, tended to be saturated as luminal Ca²⁺ concentration was increased.Ke (10⁻⁷ cm²/s) decreased from 4.13±0.56 to 2.02±0.30 (P<0.02) along with a decrease in PDt when 10⁻³ M NaCN was added to the bathing fluid. By contrast, 10⁻³ M iodoacetamide did not affectKe in spite of a significant decrease in PDt. Similarly, neither 10⁻⁵ M ouabain added to the bathing fluid nor 10⁻⁴ M ruthenium red, added either to the bathing fluid or the perfusate, affectedKe despite significant decreases in PDt. Addition of 10⁻⁵ M furosemide in the lumen caused a decrease inKe from 4.31±0.43 to 3.17±0.25 (P<0.01) in association with a decrease in PDt from 6.3±1.04 to 1.7±0.37 mV. These findings suggest; (a) Ca²⁺ transport across the TALH is mainly an active process requiring aerobic metabolism; (b) dissociation of Ca²⁺ and Cl⁻ transport may occur under some experimental conditions.     

Hormonal stimulation of Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse: evidence for a change in the paracellular pathway permeability

Recent studies from our laboratory have shown that in the cortical thick ascending limb of Henle's loop of the mouse (cTAL) Ca²⁺ and Mg²⁺ are reabsorbed passively, via the paracellular shunt pathway. In the present study, cellular mechanisms responsible for the hormone-stimulated Ca²⁺ and Mg²⁺ transport were investigated. Transepithelial voltages (PD_te) and transepithelial ion net fluxes (J _Na, J _Cl, J _K, J _Ca, J _Mg) were measured in isolated perfused mouse cTAL segments. Whether parathyroid hormone (PTH) is able to stimulate Ca²⁺ and Mg²⁺ reabsorption when active NaCl reabsorption, and thus PD_te, is abolished by luminal furosemide was first tested. With symmetrical lumen and bath Ringer's solutions, no Ca²⁺ and Mg²⁺ net transport was detectable, either in the absence or in the presence of PTH. In the presence of luminal furosemide and a chemically imposed lumen-to-bath directed NaCl gradient, which generates a lumen-negative PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net secretion. In the presence of luminal furosemide and a chemically imposed bath-to-lumen-directed NaCl gradient, which generates a lumen-positive PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net reabsorption. In view of the observed small effect of PTH on passive Ca²⁺ and Mg²⁺ movement, a possible interference of furosemide with the hormonal response was considered. To investigate this possibility, Ca²⁺ and Mg²⁺ transport was first stimulated with PTH in tubules under control conditions. Then active NaCl reabsorption was abolished by furosemide and the effect of PTH on J _Ca and J _Mg measured. In the absence of PD_te and under symmetrical conditions, no Ca²⁺ and Mg²⁺ transport was detectable, either in the presence or absence of PTH. In the presence of a bath-to-lumen-directed NaCl gradient, Ca²⁺ and Mg²⁺ reabsorption was significantly higher in the presence than in the absence of PTH. Finally, when active NaCl transport was not inhibited by furosemide, but reduced by a bath-to-lumen-directed NaCl gradient, PTH strongly increased J _Ca and J _Mg, whereas only a small increase in PD_te was noted. In conclusion, these data suggest that PTH exerts a dual action on Ca²⁺ and Mg²⁺ transport in the mouse cTAL by increasing the transepithelial driving force for Ca²⁺ and Mg²⁺ reabsorption through hormone-mediated PD_te alterations and by modifying the passive permeability for Ca²⁺ and Mg²⁺ of the epithelium, very probably at the level of the paracellular shunt pathway.     

[Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V₂ receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V₁ receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP ₃) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP ₃, which peaked at 200%–250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37°C, and declined to basal level after 5–10 min. Dose/response curves for InsP ₃, established at 30°C and 37° C using radioimmunoassay, showed a half-maximal stimulation of InsP ₃ production at about 1 nmol/l AVP and a maximal response at 10 nmol/l. Similar values were obtained for the response to AVP in terms of cAMP accumulation. InsP ₃ content in the presence of higher concentrations of AVP (1 mol/l) was significantly lower (P<0.001) than in the presence of 10 nmol/l AVP, giving a bell-shaped appearance to the dose/response curve at 37° C but not at 30° C. The V₂ receptor agonist, 1-deamino-[8DArg]vasopressin (dAVP) did not stimulate the formation of InsP ₃, and the V₁ receptor antagonist d(CH₂)₅[Tyr(Me)²]AVP inhibited AVP-induced InsP ₃ formation, which therefore appeared to be mediated by V₁ receptor occupancy. Under the same conditions, AVP also induced the formation of diradylglycerol via V₁ receptor activation, with an analogous dose/response curve. These results show that AVP, in addition to its well-known action through V₂ receptors, also acts on the mouse mTAL through a V₁-mediated stimulation of phospholipase C. Cyclic AMP controls this transduction pathway: dAVP (10 nmol/l), dibutyryl-cAMP (1 mmol/l and 0.1 mmol/l) and forskolin (1 mol/l) decreased the InsP ₃ formation induced by AVP. Dibutyryl-cAMP itself at 37°C also reduced the diglyceride content.     

Active NaCl transport in the cortical thick ascending limb of Henle's loop of the mouse does not require the presence of bicarbonate

The aim of the present study was to investigate whether bicarbonate buffer (CO₂ + HCO ₃ ^– ) is required to sustain maximal NaCl transport in the cortical thick ascending limb of Henle's loop (cTAL) of the mouse. Transepithelial Na⁺ and Cl^– net fluxes (J _Na, J _Cl, pmol min^–1 mm^–1), measured by electron microprobe analysis, were similar irrespective of the presence or absence of CO₂ + HCO ₃ ^– in luminal and bathing solutions J _NaCl with CO₂ + HCO ₃ ^– =203±25 pmol min^–1 mm^–1; J NaCl without CO₂ + HCO ₃ ^– =213±13 pmol min^–1 mm^–1, n=14). Furthermore the transepithelial potential difference, V _te, the transepithelial resistance, R _te, and the basolateral membrane potential, V _bl, were unaffected by CO₂ + HCO ₃ ^– . In the absence of CO₂ + HCO ₃ ^– , V _te was +17.0±1.7 mV(n=9) (lumen positive), R _te was 28±2 cm² (n=9) and V _bl was –76±4 mV (n=6). In the presence of CO₂ + HCO ₃ ^– , V _te, R _te and V _bl were +15.9±1.5 mV, 29±1 cm² and –73±5 mV, respectively. 4-Acetamido-4-isothiocyanatostilbene-2,2-disulphonic acid (SITS; 0.1 mmol l^–1) and amiloride (1 mmol l^–1) added to the (CO₂ + HCO ₃ ^– )-containing lumen perfusate were without effect on V _te and R _te. Finally, the effect of furosemide (0.1 mmol l^–1) on V _te and V _bl in the presence of CO₂ + HCO ₃ ^– was investigated. Furosemide reversibly decreased V _te from +13.7±1.1 mV to +1.7±0.7 mV (n=6) and hyperpolarized V_bl from –70±1 to –89±3 mV (n=5), suggesting passive distribution of Cl^– across the basolateral membrane. In conclusion, these data suggest that active NaCl transport in the cTAL of the mouse does not require the presence of CO₂ + HCO ₃ ^– .     

Measurements of macula densa cell volume changes in isolated and perfused rabbit cortical thick ascending limb. II. Apical and basolateral cell osmotic water permeabilities

Cortical thick ascending limbs containing macula densa plaques were dissected and perfused in vitro. Macula densa cell osmotic water permeability of the apical and basolateral membranes were measured by setting up osmotic steps across them in less than 0.1 s and following the ensuing time-dependent cell volume changes. The results of this study are in accordance with the view that the macula densa cells have a relatively low permeability to water. Apical and basolateral osmotic water permeabilities are 2.4 and 30.4 times 10^-4 cm³ s^-1 osMolar^-1 cm^-2 basement membrane area, respectively. No infoldings were taken into consideration. These water permeabilities were not affected by maximal and supramaximal doses of vasopressin. This paper provides new insight into the physiological behaviour of this small, and almost inaccessible, sensing epithelial disc of cells which improves the understanding of its participation in the juxtaglomerular feedback response.     

The time course of intracellular calcium movements in single human umbilical vein smooth muscle cells

Intracellular Ca²⁺ ([Ca²⁺]_i) was measured in single isolated human umbilical vein smooth muscle cells. Stimulation with histamine, in the absence of external Ca²⁺, mobilised Ca²⁺ from intracellular stores. When repeated brief applications of agonist were used, the time to onset, amplitude and rate of rise of the Ca²⁺ transients were found to change. Two components could often be discerned in the rising phase of the transients, an initial slow pacemaker and a second faster and larger component. Following the first histamine-activated transient the basal level of [Ca²⁺]_i was invariably lower than that prior to stimulation. This lower value was maintained whilst the cell remained in Ca²⁺-free solution, but could be returned to a higher level if the cell was exposed to external Ca²⁺. When the mobilisation of the intracellular store was reduced to undetectable levels, re-exposure to Ca²⁺-containing medium reactivated responses. In the absence of external Ca²⁺, continuous application of histamine activated a series of transient increases in intracellular Ca²⁺, which decreased progressively in amplitude and rate of rise. The interval between transients also increased. These findings are discussed in terms of the activation of inositol trisphosphate-sensitive intracellular Ca²⁺ stores and their sensitivity to cytoplasmic Ca²⁺ and intrasarcoplasmic reticulum Ca²⁺.