Contraluminal transport of organic cations in the proximal tubule of the rat kidney. I. Kinetics of N1-methylnicotinamide and tetraethylammonium, influence of K+, HCO3-, pH; inhibition by aliphatic primary, secondary and tertiary amines and mono- and bisquaternary compounds.

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 N1-[3H]methylnicotinamide (NMeN+) and [3H]tetraethylammonium (TEA+) at different concentrations and contact times was measured and the following parameters evaluated: Km,NMeN = 0.54 mmol/l, Jmax,NMeN = 0.4 pmol s-1 cm-1; Km,TEA = 0.16 mmol/l, Jmax,TEA = 0.8 pmol s-1 cm-1. TEA+ inhibited NMeN+ transport and NMeN+ the uptake of TEA+. Thereby, the Ki values for inhibition correspond closely to the Km 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 HCO3-, 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. Ki values) of homologous series of primary, secondary, tertiary and hydroxy amines as well as of mono- and bisquaternary ammonium compounds against NMeN+ transport was tested. The inhibitory potency increased in the sequence methyl less than ethyl less than propyl less than butyl and primary less than secondary less than tertiary amines less than quaternary ammonium compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney. VIII. Transport of corticosteroids.

Using the stop-flow peritubular capillary microperfusion method contraluminal transport of corticosteroids was investigated (a) by determining the inhibitory potency (apparent Ki 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 17 alpha- and 6 beta-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. Ki, PAH of 0.07 mmol/l). Acetylation of the OH-group in position 21 of 11-deoxycorticosterone, introduction of an additional hydroxy group in position 17 alpha to yield 11-deoxycortisol or in position 11 to yield corticosterone brings the app. Ki, 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 6 beta position reduces or abolishes it. Cortisol and its derivatives prednisolone, dexamethasone and cortisone exert similar inhibitory potencies (app. Ki, PAH 0.12-0.27 mmol/l). But again, omission of the 21-OH group in cortisone or addition of a 6 beta-OH group reduces or even abolishes the inhibitory potency against PAH transport. The interaction of corticosterone was not changed when 11 beta, 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. Ki, PAH 0.02 mmol/l). When the A ring of corticosterone is saturated and reduced to 3 alpha, 11 beta-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. Ki, PAH very much. Glucuronidation, however, reduces it (app. Ki, PAH approximately 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Contraluminal p-aminohippurate transport in the proximal tubule of the rat kidney. VII. Specificity: cyclic nucleotides, eicosanoids.

Using the stop-flow peritubular capillary microperfusion method the inhibitory potency (apparent Ki 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. Ki,PAH of 3.4, 0.63 and 0.52 mmol/l. The respective app. Ki,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 N1-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: Km = 1.5 mmol/l, Jmax = 0.34 pmol S-1 cm-1, r (extracellular/intracellular amount at steady state) = 0.91; for cGMP: Km = 0.29 mmol/l, Jmax = 0.31 pmol S-1 cm-1, r = 0.55. Comparison of app. Ki,cGMP with app. Ki,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, F1 alpha, A1, B1, E2, F2 alpha, D2, A2 and B2 with an app. Ki,PAH between 0.08 and 0.18 mmol/l. The app. Ki of the prostacyclins 6,15-diketo-13,14-dihydroxy-F1 alpha (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. Ki,PAH of the prostacyclins PGI2 (0.55 mmol/l), 6-keto-PGF1 alpha (0.77 mmol/l) and 2,3-dinor-6-keto-PGF1 alpha (0.57 mmol/l) as well as that of thromboxane B2 (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. Ki,SO4(2-) 5.4, 11.0, 17.9 mmol/l respectively). Contraluminal influx of labelled PGE2 showed complex transport kinetics with a mixed Km = 0.61 mmol/l and Jmax 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Contraluminal bicarbonate transport in the proximal tubule of the rat kidney.

In order to measure the contraluminal bicarbonate flux in situ we applied the stopped flow capillary microperfusion technique and measured the influx of 14C-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 H14CO3- 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-amino-hippurate 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 H14CO3- starting concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraluminal transport of small aliphatic carboxylates in the proximal tubule of the rat kidney in situ.

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/l L-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.

Contraluminal transport of hexoses in the proximal convolution of the rat kidney in situ.

In order to study contraluminal hexose transport, concentration and time-dependent influx of 3H-2-deoxy-D-glucose from the interstitium into cortical tubular cells has been measured. The influx curves fit to a two parameter kinetics (Km 1.3 +/- 0.2 mmol/l, Jmax 0.67 +/- 0.16 pmol/s X cm) plus an additional diffusion term (with P = 6 X 10(-8) cm2/s) and a distribution ratio extracellular to intracellular amount of 2-deoxy-D-glucose of 1:0.6. Since the extracellular to intracellular free water space as estimated from morphological data was 1:2, one must conclude that glucose has only free access to 1/3 of the cell water. The intracellularly accessible space was augmented when the tubules were preperfused for 10 s with hypotonic saline. Thereby an increase of the compartment into which diffusion occurs was revealed and a final rupture of this intracellular compartment at 1/4 isotonic solutions was observed. Total replacement of ions in the peritubular perfusate by mannitol did not change 2-deoxy-D-glucose influx, indicating that it is Na+-independent. By adding isotonic concentrations of the respective sugars to the capillary perfusate, three degrees of inhibition of 2-deoxy-D-glucose influx could be revealed: strong inhibition by D-glucose, methyl-beta-D-glucoside, D-mannose, 3-O-methyl-D-glucose, 2-deoxy-D-galactose, methyl-beta-D-galactoside and 6-deoxy-D-glucose, moderate inhibition by D-galactose, L-glucose, L-mannose and D-fructose, no or borderline inhibition by methyl alpha-D-glucoside, 2-deoxy-methyl-alpha-D-galactoside, 1-thio-beta-D-glucose, 1-thio-beta-D-galactose, 5-thio-alpha-D-glucose, myo-inositol and mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraluminal phosphate transport in the proximal tubule of the rat kidney.

In order to study the characteristics of contraluminal phosphate transport the stopped flow microperfusion technique [13] has been applied. By measuring the time-dependent decrease of interstitial 33Pi concentration at different starting concentrations a simple diffusion kinetics with a permeability coefficient of 7.5 +/- 1.0 X 10(-8) cm2 s-1 was found. Such a kinetic was so far only observed with 2-deoxy-D-glucose. This substance, however, is transported in addition by facilitated diffusion as was seen by paraaminohippurate, methylsuccinate and sulfate. The contraluminal transport of phosphate was inhibited by H2-DIDS (5 mmol/l). It was, however, not influenced by omission of Na+ from the perfusates, by addition of sulfate (150 mmol/l), methylsuccinate (50 mmol/l), arsenate (50 mmol/l), the Hg-compound mersalyl (5 mmol/l), high and low phosphate diet and pH changes between 6.0 and 8.0. The data indicate that phosphate, which is reabsorbed from the lumen by a Na+-dependent transport system, leaves the cell by a rather unspecific contraluminal diffusion pathway.

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 (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 mu A.cm-2 under control conditions, and decreased significantly to 74 +/- 34 mu A.cm-2 60 s after the addition of toxin (2 mg.1(-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.1(-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.1(-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.

Bicarbonate reabsorption in the papillary collecting duct of rats.

Using the technique of capillary perfusion and simultaneous luminal stop flow microperfusion the reabsorption of bicarbonate and glycodiazine from the papillary collecting duct was evaluated. Starting with equal H14CO3- and 3H-glycodiazine concentrations in the luminal and peritubular perfusates, the decrease in the luminal concentration at 10 and 45 s contact time was measured. In control rats with 25 mmol/l HCO3- in the perfusates the rate of HCO3- reabsorption calculated from the 10 s values was 0.34 nmol cm-2 s-1. In acute metabolic acidosis, the rate of bicarbonate reabsorption was 2,3 times higher. In metabolic alkalosis, the rate of bicarbonate absorption dropped to 13% of the control values. Also the 45 s values of acidotic and alkalotic animals differed significantly from each other. With 25 mmol/l glycodiazine in both perfusates the rate of buffer reabsorption as calculated from the 10 s values was 0.76 nmol cm-2 s-1 in control rats and did not deviate significantly from this value in acidotic and alkalotic animals. In control rats the bicarbonate reabsorption in % was the same, no matter whether both luminal and capillary perfusate contained 25 mmol/l bicarbonate or 10 mmol/l. In acidotic rats the rate of HCO3- reabsorption did not change significantly if all Na+ in the perfusates was replaced by choline (0.88 versus 0.79 nmol cm-2 s-1 at 25 mmol/l HCO3-). When in acidotic rats. 0.1 mmol/l acetazolamide or 1 mmol/l SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) was added to both perfusates the rate of HCO3- reabsorption dropped by 75 and 58%, respectively. A potassium deficient diet for one week and DOCa administration had no influence on the bicarbonate reabsorption of rats which were on standard diet. The data indicate that (1) the buffer reabsorption from the papillary collecting duct is rather due to H+ ion secretion than to buffer anion reabsorption. (2) The adaptation to metabolic acidosis and alkalosis is specific for bicarbonate and not seen with glycodiazine. (3) Within the concentration range tested the HCO3- reabsorption rises linearly with the HCO3- concentration. (4) The HCO3- reabsorption in the papillary collecting duct is Na+-independent, it can be inhibited by acetazolamide and SITS, but is not influenced by K+-deficient diet plus DOCA.

Sodium reabsorption in the papillary collecting duct of rats. Effect of adrenalectomy, low Na+ diet, acetazolamide, HCO-3-free solutions and of amiloride.

Using the shrinking droplet method and simultaneous perfusion of the peritubular capillaries the isotonic reabsorption of Ringer's solution from the papillary collecting ducts was measured. Under control conditions the volume reabsorption from the papillary collecting ducts was Jv +/- SE = 2.6 +/- 0.1 . 10(-5) cm3 . cm-2 . s-1. In rats which were on low Na+ diet, Jv increased to 127%, and in adrenalectomized animals it decreased to 34% of the control value. Three hours after a;ocatopm pf a;dpsterpme om tje adrenalectomized animals Jv was partially restored to 63% of control rats. Amiloride 10(-4) M, added to the luminal perfusate, produced a strong inhibition of Jv (to 32% of control). Acetazolamide, 10(-4) M, added to both perfusates, reduced Jv very strongly (to 40% of control), while omission of bicarbonate reduced it only to 77% of control. Acetazolamide, added to bicarbonate-free perfusates, did not result in a significant further reduction of Jv. The data indicate that the Na+ reabsorption from the papillary collecting duct is controlled by mineralocorticoids. Furthermore, they suggest the existence of two transport mechanisms in the luminal cell membrane: 1. An amiloride-sensitive entry step and 2. an entry step via a Na+-H+-countertransport mechanism, the latter being less important.

Coupling between proximal tubular transport processes. Studies with ouabain, SITS and HCO3-free solutions.

The rate of active transport by the proximal renal tubule of amino acid (L-histidine), sugar (alpha-methyl-D-glycoside), H+ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (deltac) of these substances. In the case of calcium the electrochemical potential difference (delta + zF-CIdeltaphi/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 of L-histidine, alpha-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%, and L-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, alpha-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 on Pi reabsorption observed under these conditions might be explained by possible variations in intracellular pH.

Effect of parathyroid hormone and cyclic adenosine 3',5'-monophosphate on isotonic fluid reabsorption: polarity of proximal tubular cells.

Isotomic fluid reabsorption (JV) of rat renal proximal tubules was examined by the shrinking droplet method in combination with simulatneous perfusion of blood capillaries. Sensitivity of JV measurement was improved by using each punctured tubule for control measurements: 1) Parathyroid hormoen (PTH) on the contraluminal cell side reduced JV in a dose-response behavior. The maximal inhibition was achieved at a PTH concentration of 10(-5) M, the half maximal inhibition at a concentration of 3 X 10(-9) M. PTH on the luminal cell side had a small inhibitory effect. 2) Cyclic AMP inhibited JV preferentially when applied to the luminal cell side. On the luminal cell side, both cyclic AMP and dibutyryl cyclic AMP inhibited JV in a similar dose-dependent behavior. Concentrations of both nucleotides as low as 10(-10) M had a definite inhibitory effect. Tested at a high concentration, N6-butyryl cyclic AMP was almost as effective as cyclic AMP. Deoxy cyclic AMP, 5' AMP, cyclic guanosine monophosphate (cyclic GMP), dibutyryl cyclic GMP had no effect. ATP inhibited JV to a very small extent. 3) The reduction of JV after administration of PTH and dibutyryl cyclic AMP was not additive. The similar inhibitory effect of PTH at the contraluminal cell face and of cyclic AMP at the luminal cell face suggests the following sequence of events in the mediation of the action of PTH: 1) activation of adenylate cyclase by PTH in the contraluminal cell membrane, and 2) action of the generated cyclic AMP on the luminal cell membrane. The interaction of cyclic AMP and the luminal cell membrane is initiated at the luminal cell surface.

Effect of cyclic nucleotides on the isotonic fluid reabsorption in the proximal convoluted tubule of rat kidney.

In order to reveal the sidedness of the cyclic AMP action on renal tubules the effect of cyclic nucleotides on isotonic fluid reabsorption was examined in proximal convoluted rat kidney tubules by using advanced micropuncture techniques. Cyclic AMP inhibited isotonic fluid reabsorption preferentially when applied on the luminal cell side. This finding coincides with a predominant distribution of a membrane bound cyclic AMP dependent protein kinase in a luminal cell membrane fraction. On the luminal cell side cyclic AMP and dibutyryl cyclic AMP inhibited isotonic fluid reabsorption in a similar dose dependent behaviour. Concentrations of both nucleotides as low as 10-10M had a definite inhibitory effect. Tested at a high concentration N6-butyryl cyclic AMP was almost as effective as cyclic AMP. Deoxy cyclic AMP, 5' AMP, cyclic GMP and dibutyryl cyclic GMP had no effect. ATP inhibited isotonic fluid reabsorption to a very small extent. The data indicate that the observed inhibition of isotonic fluid reabsorption is specific for cyclic AMP and that the interaction of cyclic AMP and the luminal cell membrane is initiated at the luminal cell surface.