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.

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.

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.

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)

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.

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.

Se revisan brevemente: Los sitios de absorción del filtrado glomerular en diversos segmentos del nefrón, los sitios de acción de la hormona antidiurética (HAD), los conceptos que llevan a proponer que existen canales de agua en el túbulo renal proximal. llamados ahora Aquaporinas (AQP), algunas características biofísicas (diámetro equivalente y longitud) del filtro de selectividad de la AQP-l en el túbulo renal proximal, estructura molecular de las AQP, algunas características de las AQP- 1-5, localización de las AQP 1-4 en el nefrón y algunos ejemplos de “fisiopatología molecular”, que muestran que fallas genéticas en etapas de la acción de HAD en los túbulos colectores de los nefrones permiten describir varias formas de “diabetes insípida” renal. Hay métodos para dosar AQP-2 en orina. Esto simplificará el diagnóstico diferencial entre diversas formas de diabetes insípida. Estos hallazgos han sido posibles por los avances recientes en la biología molecular de las AQP. Sólo se citan algunas referencias críticas.