Rat proximal tubule D-glucose transport as a function of concentration,flow, and radius

Summary From earlier microperfusion studies ofD-glucose and water reabsorption in the proximal surface nephron of the rat,D-glucose was found to be removed by a saturable carrier and by an apparent coupling with net fluid reabsorption. Equations appropriate to describe this system were developed. They incorporated carrier-mediatedD-glucose transport, net water transport, and water-coupled solute transport. Water reabsorption was assumed to be constant either per unit surface area, or per unit volume of the nephron, and the rate of carrier-transportedD-glucose was assumed constant per unit length, per unit surface area, or per unit volume of the tubule. The possibility thatD-glucose could be reabsorbed via two carrier systems was also explored analytically. It was observed from this treatment that the fraction ofD-glucose reabsorbed would change if the perfusion rate was changed. With an increase in perfusion rate, a decrease in reabsorbed fraction was seen which indicates that if net fluid reabsorption is proportional to volume, carrier-mediated sugar transport is proportional to surface area or length of the tubule. From these relationshipsJ _max, the maximal rate of carrier-transported sugar, was calculated to be 3.3×10⁻¹⁰M/cm² sec, a value comparable to that reported from other laboratories. The results of this analysis are compatible with the data obtained both by micropuncture experiments during free flow and by glucose clearance studies until theT _mG is reached. The possibility that theT _mG obtained in clearance studies is due to a decrease in the fraction of fluid reabsorbed in the proximal tubule or to a second saturable carrier is discussed. It is observed that, in either case, if load is increased by increasing the glomerular filtration rate, noT _mG would be reached, or stated another way, one would predict from the analysis thatT _mG would be proportional to glomerular filtration rate. Partially supported by NIH-AM-10779-02 and “Deutsche Forschungsgemeinschaft”. Partially presented at the XXIV International Congress of Physiological Societies, Washington, D.C., 1968. PHS post doctoral fellow 1-F2-AM-37056-01 and Dr. Henry C. and Bertha H. Buswell Fellow Dr. Henry C. and Bertha H. Buswell Fellow     

Transport of 3-O-methyl-d-glucose into mammalian pancreatic β-cells

Summary The transport and oxidation of 3-O-methyl-d-(U-¹⁴C)glucose was studied in microdissected pancreatic islets of obese-hyperglycemic mice. There was no significant production of¹⁴CO₂ during incubation for 2 h. A comparison with the uptake of sucrose and mannitol indicated that 3-O-methyl-d-glucose was uniformly distributed across the -cell plasma membrane. Externald-glucose inhibited the entry of 3-O-methyl-d-glucose and caused a significant net loss of 3-O-methyl-d-glucose from islets equilibrated with this compound. The transport of 3-O-methyl-D-glucose was also markedly reduced in the presence of phlorizin or phloretin, whereasd-mannoheptulose ord-glucosamine exerted a slight inhibition. The results support our hypothesis that the transport ofd-glucose into the pancreatic -cells is carriermediated, and indicate that 3-O-methyl-d-glucose is a non-metabolizable substrate for this carrier in the pancreatic islets. Since in contrast tod-glucose 3-O-methyl-d-glucose does not stimulte insulin release from the type of islets used, the secretagoric recognition system ford-glucose is probably not identical with the membrane transport system.     

Transport ofl-lysine by rat renal brush border membrane vesicles

l-³H-lysine uptake into brush border membrane vesicles was measured by a rapid filtration technique. A significant binding ofl-lysine at the vesicle interior was observed. Extrapolating initial linear uptake to zero incubation time did not indicate binding of the amino acid to the external membrane surface.Sodium stimulated thel-lysine uptake specifically. Experiments in the presence of potassium/valinomycin induced diffusion potentials, and experiments with a potential sensitive fluorescent dye documented an electrogenic uptake mechanism forl-lysine only in the presence of sodium. Sodium independent uptake proceeds via an electroneutral pathway. Transstimulation experiments show carrier mediated uptake in the presence and absence of sodium. An outwardly directed proton-gradient stimulatedl-lysine uptake in the presence and absence of sodium.Saturation ofl-lysine uptake was observed in the presence and absence of sodium. In the absence of sodium,l-lysine uptake was inhibited byl-arginine,l-cystine,l-phenylalanine andl-methionine. The sodium dependent uptake was inhibited byl-arginine andl-cysteine; small inhibition byl-phenylalanine was observed. In the presence or absence of sodium,l-lysine uptake was inhibited neither byd-lysine nor byl-glutamic acid.These results document carrier mediated transport ofl-lysine via (a) transport mechanism(s) not obligatory requiring sodium.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - Tris Tris(hydroxymethy)aminomethane - EGTA ethyleneglycol-bis-(-aminoethyl-ether)-N,N-tetraacetic acid - diamide azodicarboxylic acid[bisdimethylamide] - FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - MES 2-(N-morpholino) ethanesulfonic acid     

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na⁺-dependent system A and the Na⁺-independent system L without producing any changes in either the Na⁺-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in V _max of system A without any change in K _t. Starvation produces an increase in V _max of system L of 1.7 times without any change in K _t. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na⁺-dependent L-alanine uptake and Na⁺-independent cycloleucine uptake by small-intestinal brushborder membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.     

Hexose transport by chicken cecum during development

Hexose accumulation during development has been studied in tissue slices from chicken cecum. The age of birds ranged from 0 to 7 weeks after hatch. Ceca were divided into six portions according to their situation either proximal (PC), medial (MC) or distal (DC) to the ileocecal junction. In 0-day-old chicks all segments can accumulate 3-O-methyl-d-glucose (0.5 mmol/l) against a concentration gradient through a phloridzin-sensitive mechanism.Cumulative capacity is lower in DC than in PC and declines with development. Distal segments lose sugar transport ability 1–2 days after hatch whereas the medial region retains some concentrative ability in older birds. In 7-week chickens, PC slices have a similar cumulative ability to that of jejunum (yolk sac region). Kinetic studies showed that in PC the apparentK _m for phloridzin-sensitive transport was half that in 1-day- than in 7-week-old birds; apparentV _m increased by 50% in this time range. The ability to transport sugars by the cecum was further confirmed in isolated enterocytes from 5- to 7-week-old chickens using -methyl-d-glucoside (0.1 mmol/l) as substrate. Cell sugar concentration was greater in PC than in jejunal cells and jejunal greater than MC enterocytes. Sugar present in cells from DC was the same as in phloridzin-treated cells. It is concluded that cecal epithelium may play a significant role in the absorption of sugars during development.     

Selection of a lysine-resistant CHO-K1 mutant with reduced amino acid transport through multiple systems

High levels ofl-lysine were used to select for resistant variants of Chinese hamster ovary (CHO-K1) cells. Surviving colonies were screened for altered lysine transport and two with reduced uptake were picked. Clone CH-K^r, derived from the more severely affected colony, was analyzed in detail. In starved cells theV _max of lysine uptake in CH-K^r was half that of CHO whileK _m was unaltered. The intracellular pool of lysine, a substrate of cationic amino acid transport system y⁺, was significantly lower in CH-K^r. However, transport and pools of other amino acids, which are not substrates of y⁺, were also reduced in CH-K^r, as was the internal sodium concentration, while hexose import was increased. It appears that the mutation in CH-K^r is pleiotropic, affecting some general aspects of amino acid transport.     

Calcium binding and fluorescence measurements of dansylaziridine-labelled troponin C in reconstituted thin filaments

Summary The direct binding of Ca²⁺ to reconstituted thin filaments containing troponin C and the 5-dimethylaminonaphthalene-1-sulphonylaziridine (DANZ) fluorescent analogue of troponin C (TnC _DANZ ) was measured (25° C) at three Mg²⁺ concentrations. Biphasic Scatchard plots were found for all binding curves reflecting the binding of Ca²⁺ to high- and low-affinity sites of troponin. The binding of Ca²⁺ to the high-affinity sites had a greater sensitivity to Mg²⁺ (K _Mg=1×10⁴ m ^–1) than the low-affinity sites (K _Mg=1.2×10³ m ^–1). The fluorescence change of thin filaments reconstituted with TnC _DANZ was titrated with Ca²⁺ in the same solutions used for binding assays. The Ca²⁺-dependent fluorescence change had nearly the same sensitivity to Mg²⁺ (K _Mg=9.4×10² m ^–1) as did Ca²⁺ binding to the low-affinity sites. The Ca²⁺ concentration at the midpoint of the fluorescence change was about 0.3 log units less than at the midpoint for Ca²⁺ binding to the low-affinity sites. A similar relationship between the fluorescence change and Ca²⁺ binding to the low-affinity sites of isolated TnC _DANZ was measured (4° C). From these results the binding of Ca²⁺ to either low-affinity site is concluded to produce the fluorescence change. In comparison with the low-affinity sites of isolated troponin and troponin-tropomyosin complex, the low-affinity sites of reconstituted thin filaments were consistently lower in Ca²⁺ affinity.     

Sugar transport in isolated rat kidney papillary collecting duct cells

d-Glucose is an important substrate of energy metabolism and osmolyte synthesis in the renal papillary collecting duct. In order to characterize the cellular entry ofd-glucose in this tubular segment, collecting duct cells were isolated from rat kidney papilla and the rate ofd-glucose uptake was measured indirectly by monitoring thed-glucose-dependent O₂ uptake in the presence of the uncoupler CCCP.d-Glucose uptake was found to be sodium-independent and not sensitive to phlorizin even at a concentration of 10^–3 M. Uptake was, however, completely inhibited by 10^–5 M cytochalasin B and 10^–4 M phloretin. The apparentK _i for cytochalasin B was 1.5×10^–6 M and for phloretin 2.0×10^–5 M. Studies on the substrate specificity revealed that at 1 mMd-mannose is taken up and metabolized to the same extent asd-glucose. A 50-fold higher concentration of 2-deoxy-d-glucose and 2-amino-2-deoxy-d-glucose inhibitedd-glucose uptake completely whereas -methyl-d-glucoside,d-allose, andd-galactose were without effect. Under conditions whered-glucose utilization was maximally stimulated an apparentK _m of 1.2 mM and aV _max of 1 mmold-glucose/g protein hour was found ford-glucose uptake.These results indicate that thed-glucose uptake into papillary collecting duct cells is probably mediated by a transport system similar to the one found in basal-lateral membranes of pelarized renal, intestinal, and liver cells as well as in nonpolarized fat cells and erythrocytes.Supported by grant DFG Gr 916/1-1     

Micropuncture and microperfusion study ofl-glucose secretion in rat kidney

Summary Studies with the free flow micropuncture technique have shown that the ratio of TF/P_l-glucose to TF/P_Inulin in proximal tubular fluid, in distal tubular fluid, and in more than half of the final urine samples measured was greater than one, which suggests thatl-glucose was actively secreted. Studies with the microperfusion technique confirmed this finding and showed thatl-glucose was secreted by the proximal tubules. A maximum rate of secretion was reached at a plasma concentration of 4 mM. The tubular secretion ofl-glucose was augmented by the presence of 16.6 mMd-glucose in tubular lumen and inhibited by 10^–4 M phlorizin. Kinetic analysis showed that theV _max values forl-glucose secretion in the absence and in the presence ofd-glucose are 5.0×10^–10 and 6.3×10^–10 mol×cm^–2×sec^–1 respectively which were very close to the value reported for theV _max ford-glucose reabsorption. However, theK _m forl-glucose secretion was 3.1 mM and was reduced to 1.6 mM whend-glucose was present in the perfusion fluid. TheK _m ford-glucose reabsorption has been reported to be 0.6 mM (8). The results of this investigation were interpreted as being consistant with the hypothesis thatl-glucose secretion andd-glucose reabsorption share the same carrier system.On sabbatical leave from Univ. Louisville, School of Medicine and supported by a NIAMD Special Fellowship (1-F3-AM-32,720-01) and a research grant of USPHS (AMO2217-10).     

Interaction between 16α,17α-cyclopropane progesterone and progesterone receptors in rat uterus

Binding of³H-16α, 17α-cyclopropanoprogesterone (CPG) and³H-progesterone (PG) to progesterone receptors in soluble fraction of rat uterus is studied. It is shown that CPG and PG specifically bind to the protein with similar affinity and binding capacity. Unlabeled PG competitively inhibits the binding of CPG, and unlabeled CPG competitively inhibits the binding of PF with the same efficiency. Dissociation of CPG- and PG-receptor complexes is characterized by the same dissociation constant. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 5, pp. 532–534, May, 1998     

The effects of exogenous amino acids on the relaxant responses of pig urethral smooth muscle evoked by stimulation of the inhibitory nitrergic nerves

Inhibitory innervation of urethral smooth muscle is mediated partly through release of NO. We investigated the mechanisms involved in the supply of the substrate l-arginine to NO synthase by examining the relaxant response of the muscle to electrical field stimulation (EFS) and the effects of addition of amino acids to the bathing medium. Relaxant responses persisted during hours of repetitive stimulation but were enhanced rapidly by addition of l-arginine (the arginine paradox). Addition of l-lysine (competes with l-arginine for transport on the y⁺ carrier) and l-glutamine (competing on the y⁺L carrier) attenuated the enhancement. Enhancement persisted after washing but was reversed by application of l-lysine, suggesting that exogenous l-arginine fills an intracellular pool and that l-lysine can trans-stimulate its efflux from the pool. After prolonged depolarization in high-K⁺, Na⁺-free solution the relaxant response became purely nitrergic. Addition of l-arginine during the exposure continued to enhance the subsequent responses but l-glutamine added with l-arginine, could no longer reduce this enhancement. The results show the arginine paradox in inhibitory nerves and suggest the involvement of y⁺ and y⁺L carriers in the transport of l-arginine.     

Effect of rabbit duodenal mRNA on phosphate transport in Xenopus laevis oocytes: dependence on 1,25-dihydroxy-vitamin-D3

Expression of Na⁺-dependent transport of phosphate (P_i) was analysed in Xenopus laevis oocytes after injection of poly(A)-rich RNA isolated from the duodenal mucosa of rabbits with increased levels of 1,25-(OH)₂-VitD₃ (injection of vitamin D₃ or low-P_i diet) or from control animals. In parallel, the effect of elevated levels of vitamin D₃ was studied in isolated duodenal brush-border membrane vesicles. In brush-border membrane vesicles, the rate of Na⁺-P_i cotransport was found to be doubled after 1,25-(OH)₂-VitD₃ injections while Na⁺ — d-glucose cotransport (measured as a control) was not altered. In X. laevis oocytes, Na⁺-dependent P_i uptake was increased after injection of poly(A)-rich RNA isolated from duodenal mucosa of animals with increased levels of 1,25-(OH)₂-VitD₃ but not after injection of poly(A)-rich RNA isolated from control animals; between the two groups of mRNA no difference in the expression of the Na⁺ — d-glucose transport system was observed. Sucrose density gradient fractionation suggests that mRNA species related to the increased Na⁺-dependent P_i uptake are of average chain lengths between 2×10³ and 3×10³ bases (2–3 kb). It is concluded that in duodenal enterocytes 1,25-(OH)₂-VitD₃ increases the content of mRNA species of 2–3 kb that might be involved either directly in Na-P_i cotransport or at least in controlling its activity.     

Reabsorption of monocarboxylic acids in the proximal tubule of the rat kidney

The transport ofd-lactate across the epithelium of the late proximal convolution was investigated by two methods: 1. by measuring the zero net flux transtubular concentration difference (c _tt,45s) and the permeability (P) ofd-lactate and calculating from both the transtubular active transport rate (J _lac ^act ). 2. By measuring the 3.5 s efflux ofd-lactate from the tubular lumen, while blood was flowing through the capillaries. The 3.5 s efflux comprises two components, one going through the brush border (J _lac ^bb ) and one going the paracellular pathway (J _lac ^paracell =P _lac·c _{lac lumen}). Both,J _lac ^act andJ _lac ^bb ofd-lactate gave the sameK _m 1.9 and 1.7 mmol/l and the same maximal transport rate 3.2 and 2.9 pmol cm^–1 s^–1. TheK _i ofl-lactate tested againstJ _lac ^act andJ _lac ^bb ofd-lactate was also the same: 1.1 and 1.0 mmol/l. These data indicate that under our experimental conditions only the flux through the brush border seems to be rate limiting and thatd-lactate uses the same transport system asl-lactate.When Na⁺ was omitted from the perfusatesJ _lac ^act disappeared completely, whileJ _lac ^bb was reduced by 64%. These data reflect the Na⁺ dependence of thed-lactate transport through the brush border. Variation of intra-and extracellular pH by raisingpCO₂, omitting HCO ₃ ^– from the perfusates or adding acetazolamide had no effect on the transport ofd-lactate when -ketoglutarate was used as fuel. However, when acetate was used as fuel, intracellular acidosis brought the reducedJ _lac ^act back to the values obtained with -ketoglutarate as fuel. It is suggested that this is an effect on a contraluminal transport step.Probenecid (5 mmol/l) and phloretin (0.25 mmol/l) inhibitedJ _lac ^act significantly.J _lac ^bb , however, was only inhibited by probenecid when acetate was used as fuel. These data indicate that both compounds act on thed-lactate exit at the contraluminal cell side, but that probenecid acts in addition at the luminal cell side. SITS (1 mmol/l) augmentedJ _lac ^bb when acetate was used as fuel and is similar to the effect of lowering intracellular pH as described above. The SH reagents mersalyl (1.0 mmol/l) and maleolylglycine (1 mmol/l) did not influenceJ _lac ^bb .     

Oral administration ofl-arginine, a nitric oxide precursor, decreases nociceptive sensitivity in rats

Tail-flick test was used to evaluate the effect of orally administeredl-arginine on nociceptive sensitivity of albino rats, which produced a) analgesia at 30 min postadministration lasting about 1.5 h (100 mg/kg); b) short-term analgesia (50 mg/kg); and c) no analgesic effect (250 mg/kg).d-Arginine (100 mg/kg) did not affect the nociceptive sensitivity. A significant NO increase took place in cerebral cortex at 30 min postadministration ofl-arginine in the given doses. At 1 h postadministration ofl-arginine in doses of 50 and 250 mg/kg, cortical NO content was lower than that in control animals. Analgesic effect ofl-arginine is presumably related to additional synthesis of NO. This effect seems to be not directly produced by NO, but is realized via other transmitter systems. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 11, pp. 498–501, November, 1997     

Sodium dependence of the amino acid transport in the proximal convolution of the rat kidney

Summary With the technique of stop flow microperfusion with simultaneous capillary microperfusion the zero net flux transtubular concentration differences (c) of labelled amino acids which are equivalent to their active transport rates were measured. Alll-amino acids tested (phenylalanine, histidine, aminobicycloheptane-carboxylic acid, aminoisobutyric acid; lysine, ornithine, arginine; aspartic acid; proline and glycine) showed a considerable c, i.e. active transport rate. When, however, the ambient sodium was replaced by choline the c values dropped to zero. An analysis of the Na⁺ dependence of the ornithine transport revealed that the sodium-dependence is of the mixed type, i.e. thatK _m decreased andV _max increased with increasing Na⁺ concentration to the same extent.In contrast to other biological systems no mutual interaction between the Na⁺-dependentd-glucose andl-histidine transport could be observed.Incidental to these studies it was observed that the active transport rate ofd-histidine was in the range of 40% of that of thel-isomer while ford-phenylalanine it was only in the range of 10% of the active transport of thel-isomer. Furthermore it was found that thel-aspartic acid transport was already saturated at a luminall-aspartic acid concentration of 0.05 mmol/l while that ofl-phenylalanine was not saturated even at a luminal concentration of 9 mmol/l.     

Effects of chronic morphine intake on binding of NAD and GABA-benzodiazepine agonists to synaptic membranes

Nine weeks of compulsory morphine drinking decreased the specific binding of³H-muscimol to GABA receptors and¹⁴C-NAD to rat brain synaptic membranes and increased the synaptosomal uptake of¹⁴C-GABA. These effects of morphine on the GABA-benzodiazepine receptor complex were reversed by excessive doses of vitamin B₃. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 3, pp. 295–301, March, 1999     

The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells

The effect of insulin-like growth factor-I on amino acid transport was studied by measuring the uptake of tritiated L-proline in the cultured human osteoblast-like SaOS-2 cells. The uptake of L-proline was supported by both transport system A, ASC and Gly and by Na⁺-dependent amino acid transport system A, and by Na⁺-independent system L. The initial rate of total L-proline uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (K _m) and maximum velocity (V _max) values of 1.87 mM and 8.89 nmol⋅(mg protein)⁻¹⋅(3 min)⁻¹, respectively. Na⁺-dependent L-proline uptake was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Kinetic analysis showed that insulin-like growth factor-I enhanced transport activity by increasing the V _max of transport without significant changes in the affinity (K _m) of the carrier for the substrate. The increase in transport activity was significantly reduced by cycloheximide. The stimulated increment above basal L-proline uptake was completely inhibited by α-(methylamino) isobutyric acid, suggesting that only system A was affected by insulin-like growth factor-I. Na⁺-dependent L-proline uptake was also stimulated by insulin-like growth factor-II and insulin-like growth factor-I analogues. The insulin-like growth factor-I-stimulated L-proline uptake was inhibited by one of its binding protein, insulin-like growth factor binding protein-4, in a concentration-dependent manner. Received: 15 January 1996/Accepted 21 February 1996     

Molecular specificity of tubular reabsorption ofl-proline

In microperfusion experiments the reabsorption of³H and¹⁴C labelledl-proline by two recently defined transport systems (one with high capacity and low affinity, the other one having the opposite characteristics) was measured in vivo et situ on addition of several amino acids and some N-methylated derivatives.The high capacity system is apparently an unspecific system for neutral amino acids. The methylation of the amino group does not change the affinity to the system. The affinity decreases in the order phenylalanine >glutamine>alanine>proline, hydroxyproline >glycine.The low capacity system seems to be a specific reabsorption mechanism for imino acids like proline, hydroxyproline, sarcosine and N-methylalanine. Common neutral amino acids are not accepted.The different characteristics of both transport systems are also demonstrated by the finding that the affinity of phenylalanine for the high capacity system is about 5 times higher but its affinity for the low capacity system is about 50 times lower than the affinity for proline.Parts of this work were presented at the 50th Meeting of the German Physiological Society at Göttingen, FRG, October 1978 [26]     

Coupling between proximal tubular transport processes

Summary The rate of active transport by the proximal renal tubule of amino acid (l-histidine), sugar (-methyl-d-glycoside), H⁺ ions (glycodiazine), phosphate and para-aminohippurate was evaluated by measuring the zero net flux concentration difference (c) of these substances. In the case of calcium the electrochemical potential differencec +zFc_i /RT) was the criterion employed. The rate of isotonic Na⁺-absorption (J_Na) 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-4isothiocyanatostilbene 2,2-disulfonic acid) was observed in rats.Ouabain (1 mM) applied peritubularly incompletely inhibited J_Na (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, -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 J_Na 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, -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.     

Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

A6 epithelia, a cell line originating from the distal tubular part of the kidney ofXenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (T _c), short-circuit current (I _sc) and transepithelial conductance (G _t) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na⁺ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease ofT _c described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na⁺ uptake by replacing apical Na⁺ byN-methyl-d-glucamine (NMDG⁺) did not affect cell volume under isotonic conditions. An increase ofT _c by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na⁺ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na⁺ uptake or Na⁺ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K⁺ channels by 5 mM Ba²⁺ elicited a significant increase inT _c of 16.3% above control. Quinine, a potent blocker of volume-activated K⁺ channels, did not changeT _c significantly. Basolateral hypotonicity elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na⁺ uptake as well as after stimulating apical Na⁺ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K⁺ efflux at the basolateral side with Ba²⁺ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K⁺, whereas the effects of Ba²⁺ and quinine are most likely due to the blockage of the basolateral K⁺ pathway.