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  • 1
    ISSN: 1432-2013
    Keywords: Na+-dependence ; Cl−-dependence ; Sulphate dependence ; DIDS ; Carbonic anhydrase inhibitors ; Nitrophenylglyoxal
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to measure the contraluminal bicarbonate flux in situ we applied the stopped flow capillary microperfusion technique and measured the influx of14C-bicarbonate buffer into cortical tubular cells at pH 8. It was found that the influx in percent of the starting concentration is larger at 20 mmol/l bicarbonate than at 1 mmol/l, indicating a sigmoidal type influx curve. At 20 mmol/l bicarbonate the influx was inhibited by 44%, when Na+ was replaced by choline. Replacement of gluconate by chloride or sulfate did not change H14CO 3 − influx. At this bicarbonate concentration, influx is inhibited by 10 mmol/l 4,4′-diisothiocyanato-2,2′-stilbenedisulfonate (DIDS) (22%), 5 mmol/l of the carbonic anhydrase blocker ethoxyzolamide (40%) as well as by 5 mmol/l of the arginine reagent 4-nitrophenylglyoxal (31%). At 1 mmol/l bicarbonate starting concentration, bicarbonate influx was inhibited when chloride in the perfusate was present or when sulphate was added. Replacement of sodium by choline did not change bicarbonate influx. Addition of DIDS and 8-anilino-naphthalene-1-sulfonate (5 mmol/l each) inhibited 1 mmol/l bicarbonate influx 39 and 49%, respectively. The para-aminohippurate transport blocker dipropylsulfamoyl-benzoate (probenecid), the chloride channel blocker 5-nitro-2′-(3-phenylpropylamino)-benzoate (NPPB), the SH group blocker 2-(3-hydroxymercuri-2-methoxypropyl)-carbamoyl-phenoxyacetate (mersalyl), and formate did not inhibit bicarbonate influx, at 20 and at 1 mmol/l H14CO 3 − starting concentration. The data are compatible with the assumption of 1. a contraluminal (HCO 3 − )3/Na+ cotransporter inhibitable by DIDS, carbonic anhydrase inhibitors and 4-nitrophenylglyoxal, 2. a HCO 3 − /anion exchange system, which accepts sulfate and chloride and is inhibitable by the anion exchange blockers DIDS and 8-anilino-naphthalene-1-sulfonate, and 3. a HCO 3 − influx component which could not be influenced by Na+, Cl−, nor by the inhibitors applied.
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  • 2
    ISSN: 1432-2013
    Keywords: Renal Tubule ; H+ Transport ; Sodium Dependence ; Carbonic-Anhydrase Inhibitors ; Adaptation (Acid Base Balance)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: 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.
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  • 3
    ISSN: 1432-2013
    Keywords: Renal tubule ; Phosphate transport ; pH dependence ; Micropuncture ; Microperfusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Early loops of the proximal convoluted tubule of parathyroidectomized rats (PTX-rats) were microperfused with a phosphate (4 mM) containing perfusate. With a perfusion solution of pH around 7.45 as estimated as anion deficit theP i reabsorption was two times greater than with a perfusion solution of pH around 6.85. TheP i reabsorption is reduced in PTX-rats made chronic alkalotic (PTX-cA-rats) but the same pH dependence ofP i reabsorption was found. The data indicate that the divalent phosphate is preferentially reabsorbed.
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  • 4
    ISSN: 1432-2013
    Keywords: Renal tubule ; H+ ion secretion ; Na+ coupled transport ; Ouabain ; SITS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (α-methyl-d-glycoside), H+ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (Δc) of these substances. In the case of calcium the electrochemical potential differenceΔc +zFci Δϕ/RT) was the criterion employed. The rate of isotonic Na+-absorption (JNa) was measured with the shrinking droplet method. The effect of ouabain on the transport of these substances was tested in the golden hamster and the effect of SITS (4-acetamido-4′isothiocyanatostilbene 2,2′-disulfonic acid) was observed in rats. Ouabain (1 mM) applied peritubularly incompletely inhibited JNa (80%), but in combination with acetazolamide (0.2 mM) the inhibition was almost complete (93%). In addition, ouabain inhibited the sodium coupled (secondary active) transport processes ofl-histidine, α-methyl-d-glycoside, calcium and phosphate by more than 75%. It did not affect H+ (glycodiazine) transport and PAH transport was only slightly affected. When SITS (1 mM) was applied from both sides of the cell it inhibited H+ (glycodiazine) transport by 72% and reduced JNa by 38% when given from only the peritubular cell side. SITS (1 mM), however, had no significant affect on H+ secretion and sodium reabsorption if it was applied from only the luminal side. Furthermore it had no affect on the other transport processes tested, regardless of the cell side to which it was applied. When the HCO 3 − buffer or physically related buffers were omitted from the perfusate the absorption of Na+ was reduced by 66%, phosphate by 44%, andl-histidine by 15%. All the other transport processes tested were not significantly affected. The data are consistent with the hypothesis that the active transport processes of histidine, α-methyl-d-glycoside and phosphate, which are located in the brush border, are driven by a sodium gradient which is abolished by ouabain. This may also apply to the Na+-Ca2+ countertransport located at the contraluminal cell side. The residual Na+ transport remaining in the presence of ouabain is likely to be passively driven by the continuing H+ transport which probably is driven directly by ATP. SITS seems to inhibit the exit step of HCO 3 − from the cell and secondary to that, the luminal H+-Na+ exchange and consequently the Na+ reabsorption. In the absence of HCO 3 − buffer in the perfusates the luminal H+-Na+ exchange seems to be affected and the pattern of inhibition of the other transport processes is almost the same as with SITS. The different effects onP i reabsorption observed under these conditions might be explained by possible variations in intracellular pH.
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  • 5
    ISSN: 1432-2013
    Keywords: Pseudomonas aeruginosa toxin ; thick ascending limb of the loop of Henle ; rabbit
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract 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 (PDte) and the specific electrical transepithelial resistance (Rt) were recorded continuously. From PDte/Rt the equivalent short circuit current (Isc) was calculated. The Isc was 214±30 μA·cm−2 under control conditions, and decreased significantly to 74±34 μA·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, Rt 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.
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  • 6
    ISSN: 1432-2013
    Keywords: Lactate ; Pyruvate ; 3-hydroxybutyrate ; Acetoacetate ; Nonspecific anion channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to study the characteristic of contraluminal transport of hydrophylic small fatty acids the in situ stopped flow microperfusion technique [12] has been applied. By measuring with 4 s contact time the decrease in the contraluminal concentration of the respective radiolabelled substances the concentration dependence of the influx into the cortical cells was tested. The 4 s decrease in contraluminal concentration of chloroacetate,l-lactate,d-lactate, 3-hydroxybutyrate and acetoacetate was between 26% and 31%. For each substance the percent decrease was the same, no matter whether it was offered in a concentration of 0.1 or 10 mmol/l. Contraluminal disappearance of 0.1 mmol/ll-lactate was not influenced by 5 mmol/l H2DIDS, probenecid, phloretin, mersalyl or cyanocinnamate, but it was significantly (37%) inhibited by 5-nitro-2-(phenyl-propyl-amino) benzoate, a blocker of the nonspecific anion channel. The percent decrease in propionate uptake was somewhat larger — between 36% and 39% — but again not different at 0.01, 0.1, 1.0 and 10 mmol/l. With pyruvate the contraluminal decrease was 20% at 0.1 mmol/l and 31% at 10 mmol/l. The percent disappearance of the aromatic pyrazinoate was 38% and 34% at 0.1 and 10 mmol/l and for nicotinate 42% and 22%, respectively. The disappearance of nicotinate (0.1 mmol/l) was significantly inhibited by 10 mmol/l pyrazinoate and paraaminohippurate (PAH). The data are in agreement with the hypothesis that the hydrophilic small fatty acids traverse the contraluminal cell side by simple diffusion, possibly via the unspecific anion channel [14], pyruvate via the dicarboxylic acid pathway in a cooperative manner and pyrazinoate, as well as nicotinate, via the PAH pathway.
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  • 7
    ISSN: 1432-2013
    Keywords: Cyclic GMP ; Prostaglandins ; Prostacyclins ; Thromboxane B2 ; Probenecid ; Indomethacin ; Phosphodiesterase inhibitors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent K i values) of cyclic nucleotides and prostanoids against contraluminal p-aminohippurate (PAH), dicarboxylate and sulphate transport was evaluated. Conversely the contraluminal transport rate of labelled cAMP, cGMP, prostaglandin E2, and prostaglandin D2 was measured and the inhibition by different substrates was tested. Cyclic AMP and its 8-bromo and dibutyryl analogues inhibited contraluminal PAH transport with an app. K i, PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. K i,PAH values of cGMP and its analogues are with 0.27, 0.04 and 0.05 mmol/l, considerably lower. None of the cyclic nucleotides tested interacted with contraluminal dicarboxylate, sulphate and N 1-methylnicotinamide transport. ATP, ADP, AMP, adenosine and adenine as well as GTP, GDP, GMP, guanosine and guanine did not inhibit PAH transport while most of the phosphodiesterase inhibitors tested did. Time-dependent contraluminal uptake of [3H]cAMP and [3H]cGMP was measured at different starting concentrations and showed facilitated diffusion kinetics with the following parameters for cAMP: K m=1.5 mmol/l, J max=0.34 pmol s−1 cm−1, r (extracellular/intracellular amount at steady state)=0.91; for cGMP: K m=0.29 mmol/l, J max=0.31 pmol s−1 cm−1, r=0.55. Comparison of app. K i, cGMP with app. K i, PAH of ten substrates gave a linear relation with a ratio of 1.83±0.5. All prostanoids applied inhibited the contraluminal PAH transport; the prostaglandins E1, F1α, A1, B1, E2, F2α, D2, A2 and B2 with an app. K i, PAH between 0.08 and 0.18 mmol/l. The app. K i of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1α (0.22 mmol/l) and Iloprost (0.17 mmol/l) as well as that of leukotrienes B4 (0.2 mmol/l) was in the same range, while the app. K i, PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1α (0.77 mmol/l), and 2,3-dinor-6-keto-PGF1α (0.57 mmol/l) as well as that of thromboxane Bin2 (0.36 mmol/l) was somewhat higher. None of these prostanoids inhibited contraluminal dicarboxylate transport and only PGB1, E2 and D2 inhibited contraluminal sulphate transport (app. $$K_{i,SO_4^{2--} } $$ 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed K m=0.61 mmol/l and J max of 4.26 pmol s−1 cm−1. It was inhibited by probenecid, sulphate and indomethacin. Contraluminal influx of PGD2, however, was only inhibited by probenecid. The data indicate that cyclic nucleotides as well as prostanoids are transported by the contraluminal PAH transporter. For prostaglandin E2 a significant uptake through the sulphate transporter occurs in addition. The hypothesis that prostaglandins as well as 8-bromo and dibutyryl cyclic nucleotides permeate cell membranes by simple diffusion because of their lipid solubility must be considered with reservation.
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  • 8
    ISSN: 1432-2013
    Keywords: Transport kinetics ; Distribution ratio ; Driving forces ; Hydrophobicity plot ; Choline ; Acetylcholine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to study the characteristics of contraluminal organic cation transport from the blood site into proximal tubular cells the stopped-flow capillary perfusion method was applied. The disappearance of N 1-[3H]methylnicotinamide (NMeN+) and [3H]tetraethylammonium (TEA+) at different concentrations and contact times was measured and the following parameters evaluated: K m,NMeN = 0.54 mmol/l, J max,NMeN = 0.4 pmol s−1 cm−1; K m,TEA = 0.16 mmol/l, J max,TEA = 0.8 pmol s−1 cm−1. TEA+ inhibited NMeN+ transport and NMeN+ the uptake of TEA+. Thereby, the K i values for inhibition correspond closely to the K m values for uptake. Similar inhibitory potencies of ten organic cation against TEA+ and NMeN+ transport provide further evidence for a common transport system. Omission of HCO 3 − , or Na+ and addition of K+ (with or without Ba2+) reduce NMeN+ transport, while omission of K+ (with or without valinomycin) or addition of thiocyanate has no effect. Since the manoeuvres that depolarize contraluminal electrical potential difference reduce NMeN+ transport, cell-negative electrical potential difference is suggested as a driving force for contraluminal organic cation transport from the interstitium into the cell. Furthermore, the inhibitory potency (app. K i values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquarternary ammonium compounds against NMeN+ transport was tested. The inhibitory potency increased in the sequence methyl 〈 ethyl 〈 propyl 〈 butyl and primary 〈 secondary 〈 tertiary amines 〈 quarternary ammonium compounds. With the amines a reversed correlation between K i,NMeN and the octanol/water partition coefficient (log octanol) is seen. With quarternary ammonium compounds the inhibitory potency decreases with increasing molecular size: tetrabutyl- 〉 tetrapentyl- 〉 tetrahexyl- 〉 tetraheptyl 〉 tetraoctylammonium. Introducing two OH groups into triethylamine reduces the inhibitory potency while introduction of two OH groups into diethylamine or three OH groups into triethylamine abolishes the inhibitory potency as a result of reduced hydrophobicity. With choline (trimethylethanolamine) and its analogues the reversed correlation between K i,NMeN and log octanol was also seen. Molecules with a similar hydrophobic moiety to those of the monoammonium compounds, but with two ammonium groups, showed only a small or no inhibitory potency against NMeN+ transport. The data indicate that (a) hydrophobic moieties are important for the interaction with the contraluminal organic cation transporter, and (b) the size of the molecule can be a limiting factor. The reduced or missing interaction of the bisquarternary compound might be caused either by the second charge and/or reduced hydrophobicity and/or too large size of a molecule.
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  • 9
    ISSN: 1432-2013
    Keywords: d-Glucose ; Microperfusion ; Proximal Tubule ; Active Reabsorption ; Kinetic Study ; d-Glucose ; Mikroperfusion ; proximaler Tubulus ; aktive Reabsorption ; kinetische Studien
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Zusammenfassung Proximale Konvolute von Rattennieren wurden bei fehlendem Nettofluß von Natriumionen und Wasser kontinuierlich mit Lösungen perfundiert, die eined-Glucosekonzentration zwischen 0,5 und 2,0 mmol/l enthielten. Der Abfall der intraluminalend-Glucosekonzentration entlang eines Konvolutes wurde durch Absaugen der perfundierten Lösung in abnehmender Entfernung von der Perfusionsstelle verfolgt. Die pro innere Tubulusoberfläche und Zeit transportierted-Glucosemenge wird mit Abnahme der intraluminalen Glucosekonzentration kleiner. Dieses Verhalten läßt sich durch eine 2-parametrige Gleichung analog der Michealis-Menten-Kinetik beschreiben. Es errechnet sich eine maximale Transportrate,V max, von 6 · 10−10 mol · cm−2 · sec−1 und eine Halbsättigungskonzentration,K m, von 0,6 mmol/l. Die so beschriebene aktive Resorption und die von uns gefundene passive Permeabilität des proximalen Konvolutes fürd-Glucose reichen, nach angestellten Computerberechnungen zu schließen, allein nicht aus, um den Nettoglucosetransport der Gesamtniere unter Freiflußbedingungen quantitativ zu beschreiben.
    Notes: Summary The proximal convoluted tubule of rat kidney was continuously perfused with a steady state solution containing 0.5 to 2.0 mM ofd-glucose. The gradual decrease of intraluminald-glucose concentration was investigated with repeated collections of perfusate from the same tubule whereby the sequence of punctures proceeded towards the site of perfusion. The rate ofd-glucose transport per unit area decreased with decreasing intraluminald-glucose concentration. This relationship could be expressed by a two parameter system corresponding to the Michaelis-Menten equation. It was found that the local maximal transport rateV max equals 6×10−10 mol×cm−2×sec−1 andK m equals 0.6 mM. Our data on active resorption and passive permeability ofd-glucose in the proximal convolution have been subjected to computer analysis. The sum of both components ofd-glucose transport alone as measured under the condition of zero netflux of sodium chloride and water did not match the amount of net glucose transport found for the whole kidney under free-flow-conditions.
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  • 10
    ISSN: 1432-2013
    Keywords: Corticosteroids ; Membrane transport ; Diffusion of corticosteroids ; Renal transport of p-aminohippurate and corticosteroids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Using the stop-flow peritubular capillary microperfusion method contraluminal transport of corticosteroids was investigated (a) by determining the inhibitory potency (apparent K i values) of these compounds against p-aminohippurate (PAH), dicarboxylate (succinate) and sulphate transport and (b) by measuring the transport rate of radiolabelled corticosteroids and its inhibition by probenecid. Progesterone did not inhibit contraluminal PAH influx but its 17α- and 6β-hydroxy derivatives inhibited with an app. Ki of 0.36 mmol/l. Introduction of an OH group in position 21 of progesterone, to yield 11-deoxycorticosterone, augments the inhibitory potency considerably (app. K i, PAH of 0.07 mmol/l). Acetylation of the OH-group in position 21 of 11deoxycorticosterone, introduction of an additional hydroxy group in position 17 α to yield 11-deoxycortisol or in position 11 to yield corticosterone brings the app. K i, PAH back again into the range of 0.2–0.4 mmol/l. Acetylation of corticosterone or introduction of a third OH group to yield cortisol does not change the inhibitory potency, but, omission of the 21-OH group or addition of an OH group in the 6β position reduces or abolishes it. Cortisol and its derivatives prednisolone, dexamethasone and cortisone exert similar inhibitory potencies (app. K i, PAH 0.12–0.27 mmol/l). But again, omission of the 21-OH group in cortisone or addition of a 6β-OH group reduces or even abolishes the inhibitory potency against PAH transport. The interaction of corticosterone was not changed when 11β, 18-epoxy ring (aldosterone) was formed. On the other hand, the interaction was considerably augmented if the 11-hydroxy group was changed to an oxo group in 11-dehydrocorticosterone (app. K i, PAH 0.02 mmol/l). When the A ring of corticosterone is saturated and reduced to 3α, 11β-tetrahydrocorticosterone the inhibitory potency is not changed very much. But if more than four OH or oxo groups are on the pregnane skeleton or if the OH in position 21 is missing, the inhibitory potency decreases drastically (app. Ki, PAH 0.7–1.7 mmol/l). Introduction of a 21-ester sulphate into corticosterone, cortisol and cortisone does not change app. K i, PAH very much. Glucuronidation, however, reduces it (app. Ki, PAH ≈ 1.2 mmol/l). None of the tested corticosteroids interacts, in concentrations applicable, with dicarboxylate transport and only the sulphate esters interact with sulphate transport. Radiolabelled cortisol, d-aldosterone, 11-dehydrocorticosterone, and corticosterone are rapidly transported into proximal tubular cells. With the latter three compounds no sign of saturation and no transport inhibition with probenecid could be seen. Only with cortisol was a shift toward saturation observed. In addition, cortisol transport could be inhibited by probenecid. The data indicate that corticosteroids interact with the contraluminal renal PAH transporter, whereby hydroxylation in position 21 augments, and hydroxylation in the 6β or 3α, 17β position reduces interaction. However, as tested so far, simple diffusion seems to prevail when corticosteroids cross the cell membrane. Sulphation makes corticosteroids also a substrate for the sulphate transporter.
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