Recent experiments towards a model for fluid secretion in Rhodnius Upper Malpighian Tubules (UMT).

Three different methods have been used to improve a model for fluid secretion in Upper Malpighian Tubules (UMT) of the blood sucking insect Rhodnius prolixus. (I) In the first, UMT double perfusions in 5th instar Rhodnius were used to measure their fluid secretion rate. They were stimulated to secrete with 5-HT. Double perfusions allowed access separately to the basolateral and the apical cell membranes with pharmacological agents known to block different ion transport functions, namely ATPases, cotransporters and/or countertransporters and ion and water channels: ouabain, bafilomycin A1, furosemide, bumetanide, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. The basic assumption is that changes in water movement reflect changes in ion transport mechanisms. (II) Intracellular Na(+) concentrations were measured with a fluorometric method in dissected R. prolixus UMT, under several experimental conditions. (III) ATPase activities were measured in R. prolixus UMT. A tentative model for the function of the UMT cell is presented. We find that (a) at the basolateral cell membrane, fundamental is a Na(+)-K(+)-2Cl(-) cotransporter; of intermediate importance are the Na(+)-K(+)-ATPase and a ouabain-insensitive Na(+)-ATPase, ion channels and Rp-MIP water channels. (b) At the apical cell membrane, most important are a V-H(+)-ATPase; and a K(+) and/or Na(+)-H(+) exchanger.

A model for fluid secretion in Rhodnius upper Malpighian tubules (UMT).

We have measured fluid secretion rate in Rhodnius prolixus upper Malpighian tubules (UMT) stimulated to secrete with 5-OH-tryptamine. We used double perfusions in order to have access separately to the basolateral and to the apical cell membranes. Thirteen pharmacological agents were applied: ouabain, Bafilomycin A(1), furosemide, bumetanide, DIOA, Probenecid, SITS, acetazolamide, amiloride, DPC, BaCl(2), pCMBS and DTT. These agents are known to block different ion transport functions, namely ATPases, co- and/or counter-transporters and ion and water channels. The basic assumption is that water movement changes reflect changes in ion transport mechanisms, which we localize as follows: (i) At the basolateral cell membrane, fundamental are a Na(+)-K(+)-2Cl(-) cotransporter and a Cl(-)-HCO(3) (-) exchanger; of intermediate importance are the Na(+)-K(+)-ATPase, Cl(-) channels and Rp-MIP water channels; K(+) channels play a lesser role: (ii) At the apical cell membrane, most important are a K(+)-Cl(-) cotransport that is being located for the first time, a V-H(+)-ATPase; and a Na(+)-H(+) exchanger; a urate-anion exchanger and K(+) channels are less important, while Cl(-) channels are not important at all. A tentative model for the function of the UMT cell is presented.

Canales de agua (aquaporinas) y riñon / Channels water (aquaporins) and kidney

Revisamos brevemente algunos aspectos del movimiento de agua en las células renales. Primero se muestran estudios de biofísica que llevan a concluir que existen poros acuosos perforando las membranas celulares de los túbulos renales proximales; ya a poder calcular las dimensiones del filtro de selectividad de un "canal equivalente" para el paso de agua de 4.6 Å de diámetro y de una longitud equivalente de 10-20Å. Como las moléculas de agua tienen un diámetro de 3Å, sólo 4 a 6 moléculas de agua pueden acomodarse en fila india dentro del filtro de selectividad. Estudios de biología molecular han aclarado la secuencia de aminoácidos de las proteínas (de 28 kD) que forman el canal. Se denominan Aquaporinas (AQPs). Se conocen unas 10. Revisamos la estructura tridimensional de AQP-1, presente en los túbulos renales proximales. También la relación entre diabetes insípida y AQP-2, en que hay mala función de los mecanismos de concentración urinaria. Finalmente hace un árbol filogenético de las AQPs en donde se ve que hay AQPs muy antiguas en bacterias, plantas e insectos. La RP-mip una AQP de los túbulos de malpighi del Rhodnius es de las más antiguas de la escala biológica. Las AQPs de mamífero son mucho más recientes

Identification of a new water channel (Rp-MIP) in the Malpighian tubules of the insect Rhodnius prolixus.

Malpighian tubules (MT) of Rhodnius prolixus transport fluid at very high rates. To identify whether aquaporins (AQPs) are present in the MT of R. prolixus, total ribonucleic acid (RNA) was isolated from MT and used in a reverse transcription, polymerase chain reaction (RT-PCR), with two degenerate primers to highly conserved regions of the members of the AQPs family. A deoxyribonucleic acid (DNA) fragment of 370 bp was amplified; its sequence revealed a novel protein, representing a new member of the major intrinsic protein (MIP) family. The complementary DNA (cDNA) sequence of this new MIP protein was cloned by using RNA from MT and the rapid amplification of cDNA ends (RACE) technique. The cDNA had 1133 bp and the largest open reading frame coded for a protein of 286 amino acids, named R. prolixus major intrinsic protein (Rp-MIP). The hydrophobicity profile of the amino acid sequence predicts six transmembrane domains. Northern blot analysis of MT RNA showed a single transcript of about 1-1.3 kb for Rp-MIP. RT-PCR of single isolated MT and in situ hybridization analysis showed Rp-MIP transcripts in both proximal and distal segments. Expression of Rp-MIP in Xenopus laevis oocytes doubled the osmotic water permeability Pf, indicating that Rp-MIP may function as an aquaporin protein in the MT of the insect and thus may participate in urine formation in R. prolixus.

Fluid secretion in Rhodnius upper malpighian tubules (UMT): water osmotic permeabilities and morphometric studies.

We have measured the osmotic permeability of the basolateral cell membrane (Poscb) and compared it with the transepithelial permeability (Poste) to calculate the paracellular (Posp) permeability of the upper malpighian tubules (UMT) of the 5th instar of Rhodnius prolixus under several experimental conditions, namely, at rest and after stimulation to secrete with 5-HT, each under control conditions (no treatment), after treatment with pCMBS, and after addition of pCMBS and DTT. Secretion rate is negligible at rest. During stimulation mean secretion rate is 43.5 nl/cm2 sec. Secretion is severely curtailed by pCMBS and fully restored by DTT. Poscb = 9.4 (resting, control); 5.8 (control + pCMBS); 10.7 (control + pCMBS + DTT); 20.6 (stimulated, control); 14.7 (stimulated + pCMBS); 49.1 (stimulated + pCMBS + DTT) (x10?4 cm3/cm2 sec Osm). Calculated Posp are higher than the transcellular permeability, Posc, at rest and after stimulation. Electron micrograph morphometry of UMT sections show that cells significantly decrease their volume after stimulation. Lateral intercellular space (LIS) and basolateral extracellular labyrinth (BEL) are barely discernible at rest. LIS and BEL are widely dilated in stimulated UMT. Thus, ions have restricted access to the deep and narrow basolateral cell membrane indentations at rest, but they have ready access to cell membrane indentations after stimulation, because of the opening of LIS and BEL. These findings are discussed in relation to isosmotic secretion. The rate-limiting step for paracellular movement is located at the smooth septate junctions.

Length of the selectivity filter of aquaporin-1.

We have characterized the selectivity filter of the water channel aquaporin-1 (AQP1) of proximal straight tubules (PST), as an equivalent cylindrical structure with a diameter of approximately 4.5 A, where water molecules single file. We report here efforts to evaluate its length. PST were dissected from rabbit kidneys, held with pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Changes in tubule cell volume with time (dV/Adt), were monitored, on line, with an inverted microscope, a TV camera and an image processor. Osmotic permeability coefficients, Pos, and reflection coefficients (sigma s) were measured with several solutes: mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). For this purpose PST were suddenly exposed (in approximately 80 ms and for 20 s) to a hyperosmolality step (delta Cs) achieved by adding to MBS a delta Cs of 35 mOsm/kg of either R, S, M, G, A or U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the delta Cs. Pos was measured from the shrinking curves; Pos = 3000 +/- 25 microns/s with either R, S, M, G, A or U. This procedure also allowed to calculate sigma s; sigma s = 1.00 for R, S, M, G, A and U, indicating that these solutes do not penetrate the water channel. In contrast, the shrinking curves produced by a delta Cs = 35 mOsm/kg formamide (F) were 1/5th to 1/6th slower and smaller (subosmometric) than those produced by a delta Cs = 35 mOsm/kg of R, S, M, G, A or U. Furthermore, with F, cells did not remain shrunk. They recovered their original volume within 3 s. Pos (measured with F) is denoted as Pos*; Pos* = 480 +/- 30 microns/s. sigma s, formamide (denoted sigma sp) = 0.16 +/- 0.01. Use of sigma sp and Pos* values in Hill's equations for the bimodal theory of osmosis leads to n = 2-3, n being the number of water molecules single filing within the channel selectivity filter, whose length must lie within 6 to 9 A, a value lower than previous values calculated from the Pos/Pd* ratio.

Transport of water in proximal kidney tubules from whole tubules to single channels: length and section of the selectivity filter of aquaporin-1.

Proximal straight tubule (PST) were dissected from rabbit kidneys, held with crimping pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Tubule cell volume changes with time (dV/Adt) after steps in MBS osmolality (delta Cs) were monitored on line with an inverted microscope, a TV camera and an image processor. Reflection coefficients sigma and osmotic permeability coefficients, Pos, for several solutes were measured using two methods. Method 1: sigma was calculated from the delta Csiso of impermeant and permeant solutes at which (dV/Adt)t-->0 = 0 (i.e., by a null point method). It is denoted as sigma 1. sigma 1 = 1.00 for mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). With formamide (F), sigma 1, Formamide = 0.62 +/- 0.05. These findings confirm our previous value of dp = 4.5 A for the diameter of the selectivity filter of the basolateral PST cell membrane water channel AQP1. Method 2: PST were exposed for 20 s to MBS made hyperosmotic by addition of a delta Cs of 35 mOsm/kg of R, S, M, G, A and U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the osmotic challenge. Pos was measured from the shrinking curves. P(os) = 3000 +/- 25 microns/s with R, S, M, G, A and U. Method 2 also allowed to calculate sigma, denoted as sigma 2. sigma 2 = 1.00 for R, S, M, G, A and U. By contrast, the shrinking curve produced by a delta Cs of 35 mOsm/kg F was 1/5th to 1/6th slower and smaller (i.e., subosmometric) than that produced by a delta Cs of 35 mOsm/kg R, S, M, G, A and U. Furthermore, with F cells did not remain shrunk but recovered their original volume within 3 s. P(os) (measured with F) is denoted as P(os)*, P(os)* = 480 +/- 30 microns/s. sigma 2, Formamide = 0.16 +/- 0.01. Use of sigma 1, sigma 2 and P(os)* values in Hill's equations for the bimodal theory of osmosis leads to n = 2-9. Where n is the number of water molecules single filling within the channel selectivity filter, whose length must lie within 6 to 27 A, a value significantly lower than our previous value calculated from the P(os)/Pd* ratio.

Microconcreciones de calcio en túbulos de malpinghi de rhodnius: ultraestructura y morfometría / Microconcretions of calcium in malpinghian tubules of rhodnius ultrastructure and morphometry

Los Rhodnius prolixus son insectos hematófagos que no tienen mecanismos urinarios para eliminar al exterior el exceso de calcio proveniente de la dieta. Haciendo estudios morfométricos en microfotografías de túbulos de Malpighi, hemos comparado Rhodnius con ideas bajas y altas de calcio. La concentración media de Calcio medida en las miconcreciones 21 mM (Bajo Calcio) y 1.000 mM (Alto Calcio). Estos resultados indican que hay grandes diferencias en el modelo de acumulación de Calcio entre ambos grupos. La formación de miconcreciones debe ser un mecanismo para acumular Calcio sin eliminarlo

The paracellular channel for water secretion in the upper segment of the Malpighian tubule of Rhodnius prolixus.

Lumen to bath J12/C1 and bath to lumen J21/C2 fluxes per unit concentration of 19 probes with diameters (dm) ranging from 3.0-30.0 A (water, urea, erythritol, mannitol, sucrose, raffinose and 13 dextrans with dm 9.1-30.0 A) were measured during volume secretion (Jv) in the upper segment of the Malpighian Tubule of Rhodnius by perfusing lumen and bath with 14C or 3H-labeled probes. Jnet = (J12/C1-J21/C2) was studied as a function of Jv.Jv was varied by using different concentrations of 5-hydroxy tryptamine. Jnet for 3H-water was not different from Jv. We found: (i) A strong correlation between Jnet and Jv for 8 probes dm = 3.0-11.8 A (group a probes), indicating that the convective component of Jnet is more important than its diffusive component and than unstirred layers effects which are negligible. Therefore group a probes are solvent dragged as they cross the epithelium. (ii) There is no correlation between Jnet and Jv for 11 probes with dm = 11.8-30 A (group b). Therefore these probes must cross the epithelium by diffusion and not by solvent drag. (iii) In a plot of Jnet/Jv vs. dm group a probes show a steep linear relation with a slope = -0.111, while for group b probes the slope is -0.002. Thus there is a break between groups a and b in this plot. We tried to fit the data with models for restricted diffusion and convention through cylindrical or parallel slit pathways. We conclude that (i) group a probes are dragged by water through an 11.0 A-wide slit. (ii) Most of Jv must follow an extracellular noncytosolic pathway. (iii) Group b probes must diffuse through a 42 A-wide slit. (iv) A cylindrical pathway does not fit the data.

The proximal straight tubule (PST) basolateral cell membrane water channel: selectivity characteristics.

Proximal straight tubules (PST) were dissected from rabbit kidneys, held by crimping pipettes in a chamber and bathed in a buffered isosmotic (295 mOsm/kg) solution containing 200 mM mannitol (MBS). Changes in tubule diameter were monitored on line with an inverted microscope, TV camera and image processor. The PST were then challenged for 20 sec with MBS made 35 mOsm/kg hyperosmotic by addition of either NaCl, KCl, mannitol (M), glycerol (G), ethylene glycol (E), glycine (g), urea (U), acetamide (A) or formamide (F). With NaCl, KCl, M, G, E, g, U, and A, tubules shrunk osmometrically within 0.5 sec and remained shrunk for as long as 20 sec without recovering their original volume (sometimes A showed some recovery). PST barely shrunk with F and quickly recovered their original volume. The permeability coefficients were 0 microns/sec (NaCl, M, g, E and U), 1 micron/sec (A), 84 microns/sec (F) and 0.02 micron/sec (G). The reflection coefficients sigma = 1.0 (NaCl, KCl, M, G, E, g and U), 0.95 (A) and 0.62 (F). Similar sigma values were obtained by substituting 200 mOsm/kg M in MBS by either NaCl, KCl, G, E, g, U, a or F. The olive oil/water partition coefficients are 5 (M), 15 (U), 85 (A) and 75 (F) (all x 10(-5)). Thus, part of F permeates the cell membrane through the lipid bilayer. The probing molecules van der Waals diameters are 7.4 x 8.2 x 12.0 (M), 3.6 x 5.2 x 5.4 (U), 3.8 x 5.2 x 5.4 (A) and (3.4 x 4.5 x 5.4 (F) A.(ABSTRACT TRUNCATED AT 250 WORDS)

Water and urea diffusive permeabilities in isolated proximal tubule cells.

We measured the water (3H2O) and urea ([14C]urea) diffusive permeabilities (Pd) in intact proximal tubule cells (PTC). Isolated rabbit PTC were packed in polyethylene tubes to measure the diffusion coefficients for 3H2O and [14C]urea at 14 degrees C. Pd values for water and urea were estimated from the diffusion coefficients in intact cells, in extracellular and in intracellular media using a model of parallel and series diffusion in the packed PTC. Pd was 65 +/- 8 and 18 +/- 3 microns/s for water and urea, respectively. We examined the effect of different pharmacological agents on the Pd values for water and for urea. p-Chloromercuribenzenesulfonate at 2 mM markedly inhibited the Pd of water and urea. Phloretin (1 mM) inhibited the Pd for urea, whereas it increased Pd for water. KMnO4 (30 microM) markedly inhibited urea Pd but did not alter water Pd. The values for energy of activation of Pd for water and for urea were 2.9 +/- 2 and 7.3 +/- 4 kcal/mol, respectively. These results show that water and urea do not share the same pathway across the PTC membrane.

The fate of calcium in the diet of Rhodnius prolixus: storage in concretion bodies in the Malpighian tubules.

We have investigated the fate of the large amounts of calcium ingested by Rhodnius prolixus in its meals of blood. 45Ca2+ injected into the haemolymph or fed to fifth-stage Rhodnius reared on rabbits is accumulated at high concentrations in the cells of the upper Malpighian tubules; very little is excreted from the body This 45Ca2+ accumulation goes on continuously for at least 12 days and the rate of uptake is increased several-fold within 3-4 days of a meal. The extent of calcium accumulation in tubule cells is correlated with the presence of intracellular membrane-bound concretion bodies, which are therefore likely sites of calcium deposition. X-ray diffraction showed that the calcium deposits are non-crystalline. Tubules from rabbit-fed fifth-stage Rhodnius contain 410 mmol l-1 calcium; in those from chicken-fed insects the calcium concentration is over 1 mol l-1; and in those fed in vitro on heparinised low-K+ sheep blood the calcium concentration is only 21 mmol l-1. The concentration of calcium in the haemolymph in all these insects was 8 mmol l-1 and its activity determined by an ion-selective electrode was 2.5 mmol l-1. 45Ca2+ deposited in the tubules is readily exchangeable, but the efflux preferentially passes to the haemolymph side of the tubule epithelium. The ability to sequester calcium in the Malpighian tubules may prevent calcium from interfering with reabsorptive processes in the rectum.

[Ultrastructural aspects of lanthanum penetration in guinea gallbladder in function of the absorption flow of water]. / Aspectos ultraestructurales de la penetración de lantano en vesícula biliar de cobayo en función del flujo de absorción de agua.

It is controversial whether water crosses the paracellular pathway in leaky epithelia (i.e. gall bladder). Therefore, this paper explores whether variations in the absorptive volume flux (Jv) are correlated with different degrees of penetration of the extracellular marker La3+ at two osmolalities namely 300 and 100 mOsm/kg, which allow to vary Jv. Guinea pig gall bladders were perfused in vitro with oxygenated fluids containing 1 and/or 10 mM La3+, at physiological pH and temperature. Tissue samples were then prepared for transmission electron microscopy. In the micrographs, penetration of La3+ beginning at the apical end of the space was measured along the intercellular space toward its basal end. Two parameters were measured, namely degree of penetration (in microns) and number of intercellular spaces penetrated by La3+. Jv is twice as large in hyposmotic condition than in the isosmotic preparations. Different degrees of inter(para)cellular La3+ penetration were observed which keep a direct relation with Jv. La3+ was not detected in the cytoplasm which showed good preservation, also at 100 mOsm/kg, this indicate adequate cell membrane integrity. These results show that La3+ penetration varies with Jv, indicating a paracellular water flow.

Solvent drag of sucrose during absorption indicates paracellular water flow in the rat kidney proximal tubule.

Single convoluted proximal tubules of the rat kidney were lumen perfused in situ with isosmotic solutions containing C14-sucrose and H3-inulin as tracers, to evaluate whether the extracellular marker sucrose is entrained by water during proximal tubular reabsorption. Inulin was used as volume marker. The absorptive rate was varied by using as luminal perfusion fluids either a solution made up of (in mmole/l) 120 NaCl, 5 glucose, 25 NaHCO3 and altering the perfusion rate, or a solution containing 110 NaCl and 70 raffinose. Js, the net sucrose efflux is found to be a function of the net volume flow, Jv, such that at Jv = 0, Js is very small and at high rates of Jv, Js is over 60-fold the value observed at low Jv values. In addition, the transported to luminal sucrose concentrations decreased with Jv in a hyperbolic manner. Unstirred layers affect the diffusive component of Js, but only to a small extent. Therefore, the large remaining dependency of Js with Jv must be due to drag of sucrose by water, within the paracellular pathway. This leads to the conclusion that water flows through the paracellular pathway during absorption in the rat proximal tubule, in addition to transcellular water flow. Using equations for molecular sieving and the measured value of sigma s for sucrose of 0.76-0.91, it is calculated that the pathway where entrainment of solute by water occurs must be 1.0-1.1 nm wide. This calculation is only tentative since sigma s depends on the as yet unknown relative contribution of transcellular and paracellular pathways to transepithelial water osmotic permeability.

Diffusive water permeability in isolated kidney proximal tubular cells: nature of the cellular water pathways.

The diffusive water permeability (Pd) of the plasma membrane of proximal kidney tubule cells was measured using a 1H-NMR technique. The values obtained for the exchange time (Tex) across the membrane were independent of the cytocrit and of the Mn2+ concentration (in the range 2.5 to 5 mM). At 25 degrees C the calculated Pd value was (per cm2 of outer surface area without taking into account membrane invaginations) 197 +/- 17 microns/sec. This value equals 22.3 +/- 1.9 microns/sec when the invaginations are taken into account. Cell exposure to 2.5 mM parachloromercuribenzenesulfonic acid, pCMBS, (for 20 to 35 min) reduced Pd to 45% of its control value. Five mM dithiothreitol, DTT, reverted this effect. The activation energy for the diffusive water flux was 5.2 +/- 1.0 kcal/mol under control conditions. It increased to 9.1 +/- 2.2 kcal/mol in the presence of 2.5 mM pCMBS. Using our previous values for the osmotic water permeability (Pos) in proximal straight tubular cells the Pos/Pd ratio equals 18 +/- 1, under control conditions, and 3.2 +/- 0.3 in the presence of pCMBS. These experimental results indicate the presence of pathways for water, formed by proteins, crossing these membranes, which are closed by pCMBS. Assuming laminar flow (within the pore), from Pos/Pd of 13 to 18 an unreasonably large pore radius of 12 to 15 A is calculated which would not hinder cell entry of known extracellular markers. Alternatively, for a single-file pore, 11 to 20 would be the number of water molecules which would be in tandem inside the pore.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of transcellular and transepithelial water osmotic permeabilities (Pos) in the isolated proximal straight tubule (PST) of the rabbit kidney.

Measurements of the water osmotic permeabilities of apical and basolateral membranes of PST cells and of the transepithelial permeability have been carried out using a very fast method with high temporal and spatial resolution. At 25 degrees C the values obtained are: 80.8 +/- 11.9 x 10(-4) cm3/s osmol cm2 of apical (luminal) surface area and 90.1 +/- 13.0 x 10(-4) cm3/s osmol cm2 of basement membrane area (no membrane invaginations taken in account). These values are higher than previously published values due to the use of a faster and more accurate volume measuring and recording system. The transepithelial water osmotic permeability at 25 degrees C is 77 +/- 11 in units of 10(-4) cm3/s osmol cm2 basement membrane area. The transcellular water osmotic permeability is 32 +/- 7 (same units), leaving a paracellular contribution of 45 +/- 10 (same units). In the presence of 2.5 mM parachloromercuribenzenesulfonate (pCMBS) the apical permeability is reduced with an incubation of 10-15 min to 23% of its control value and the basolateral permeability to 8% of its control value (after 25 min) but the transepithelial permeability is only reduced to about 1/2 of the control value. This leaves a transcellular permeability of 6 x 10(-4) cm3/s osmol cm2 of basement membrane area and a paracellular contribution of 33 +/- 6 (same units). These results indicate a significant contribution of the paracellular pathway to the transepithelial water osmotic permeabilities in PST.

Pathways for water absorption and physiological role of the lateral interspaces in the kidney tubule.

Possible routes for water and salt flow and the most likely theories that describe coupling between water and salt flow across leaky epithelia are presented. The osmotic theories seem the most likely ones. However, several of the theories have weaknesses that render them unsatisfactory, in particular because of the possibility of paracellular water flow in these epithelia. Puzzling are the findings that measurements of the cellular water osmotic permeability give figures that are too low for some of the exclusively transcellular theories to work. If these observations hold in the future, it may be shown that part of the water moves through paracellular pathways in these leaky epithelia. This view is supported by the observation that large extracellular markers are dragged by volume flow. Finally, experimental evidence is reviewed indicating that changes in the luminal area concentration may modulate the functional state of the nephron junctional complexes.

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump.

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.

Channels for water flow in epithelia: characteristics and regulation.

Pos and Pd, osmotic and diffusive water permeability coefficients of isolated rabbit proximal tubule (PT) cells (expressed per cm2 of real cell membrane area, in microns/sec) are: Pos 396; Pd, 22; Pos/Pd, 18 (control), and after exposure to parachloromercuribenzenesulfonate (pCMBS): Pos, 32; Pd, 10; Pos/Pd, 3. The sulfhydryl reagent dithiothreitol (DTT) reverts pCMBS action. The activation energies (kcal/mol) are Pos, 3.2 (control); 9.2 (pCMBS); Pd, 5.2 (control) and 9.1 (pCMBS). Thus water channels pierce the control plasma membrane and are reversibly closed by pCMBS. High control PT permeabilities are comparable with those of amphibian urinary bladder and collecting tubules (CT) stimulated with antidiuretic hormone (ADH), and low PT (pCMBS) values with those of CT in the resting state, respectively. Transcellular permeability is regulated in PT by the state of sulfhydryl groups and in CT by ADH induced insertion (or, no ADH, suppression) of water channels. In PT (a) large extracellular markers are dragged by water flow indicating extracellular solute-water interaction, (b) transepithelial Pos is much higher than transcellular Pos. Therefore water also flows paracellularly, in addition to the transcellular flow. In PT paracellular permeability is increased if urea in lumen is higher than in blood. It is reduced in the reverse situation. In CT paracellular permeability is virtually zero (resting condition). It may be increased by high lumen urea. Thus, paracellular permeability (which is significant in control PT and zero in CT) can be regulated by changes in the transepithelial urea concentrations. Transcellular permeability depends on the number of channels/cm2 epithelium, their probability of being opened and their individual permeability.(ABSTRACT TRUNCATED AT 250 WORDS)