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The cellular concentrations of Na, K, and C1 have been measured in kidney slices of the amphibian, Necturus maculosus. Permeability coefficients have been determined for Na, K, C1, Rb, Cs, and choline, from studies both of the uptake of radioactive isotopes and the rate of cell swelling in anisotonic solutions. The results of both methods were found to agree well. Measurements were also made of electrical potential differences across the peritubular face of the kidney cells using bathing solutions in which the electrolyte composition and concentrations could be varied. The data obtained are consistent with a model cell in which the potential difference arises as a result of differences in Na permeability relative to K on the two faces of the cell. The intracellular Na concentration is considered to be regulated by a Na-K coupled pump located at the peritubular face of the cell.Electrical potential gradients have been demonstrated between the interstitial fluid and the lumen of the proximal tubule of the kidney of the amphibian, Necturus rnaculosus, by Giebisch (1, 2), Whittembury (3), and Whittembury and Windhager (4). These authors have also shown that the electrical potential difference across the peritubular face of the cell is larger than that across the luminal face. The relationship between electrical potential differences and ionic permeabilities has been studied in nerve by Hodgkin and Katz (5). The present studies are concerned with similar relationships in Necturus proximal tubule cells.For this purpose we have used kidney slices. The permeability of kidney slices to a variety of small ions and molecules has been studied using a combination of radioactive techniques and studies of cell swelling. It is possible to make chemical analyses of the slices before and after exposure to permeant molecules and to measure potential differences in the slices under the same conditions. The combination of the present results with those obtained in situ has enabled us to put forward a hypothetical model kidney cell which 689

Stopped flow microperfusion technique ( Am. J. Physiol. 195: 563, 1958) was used to study water movement across the proximal tubular wall of Necturus kidney. In 23 experiments, net water movement was measured from perfusion solutions containing 50, 62.5, 75 and 100 mEq. NaCl/1. which were made isosmotic with Necturus plasma by addition of mannitol. Water movement was shown to depend upon luminal NaCl concentration. Studies of the relationship between net solute flux and water flux demonstrated a linear relationship: net water flux (mµl/sec.) = 9.4 x net solute flux + 0.003. Net water flux is statistically zero when net solute flux is zero. Under these experimental conditions no force is important for water movement other than that arising from solute movement. It is concluded that net movement of Na has taken place up an electrochemical potential gradient, indicating active transport of this ion. Furthermore, movement of water from the tubule is considered to be passive since net water flux may be accounted for quantitatively in terms of osmotically induced forces arising from net solute movement.

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.

The electrical potential profile of the isolated toad skin was recorded, in vitro, by impalement with micropipette-electrodes, when both sides of the skin were bathed with sulfate-Ringer. The outer side of the skin was some 110 mv negative with respect to the inner side. Upon impalement from the outer side, two main positive steps of 40 to 70 my each were found to form the skin potential. The site of measurement of each potential difference was permanently marked in the tissue during recording, by deposition of carmine from the micropipette tip using iontophoresis. Serial histological sections of the skin were prepared and search was then made of the carmine deposits 2 to 6/~ in size, under phase contrast microscopy. By this method the main steps were located at the outer and the inner sides of the stratum germinativum cells. The 9c resistances between the micropipette tip and the bathing solutions were measured during the recording of each potential difference. The resistance at the outer side of the stratum germinativum cells, of 1.09 kilohm, cm ~, was larger than that at their inner side, of 0.30 kilohm, cm e. The stratum germinativum cells maintained a potential difference of --34 mv during short-circuiting of the skin.A m p h i b i a n skins are convenient for the study of several processes involved in ion transport. For better interpretation of these processes further knowledge of the differences in electrical potential, one of the forces driving ions across the skin structures, is necessary. It is well known that the outer side of the skin is negative with respect to the inner side (24). Recent recordings with micropipettes indicate that the skin electrical potential profile is formed by steps (19,5,20), but disagree in their location a n d number. Thus Ottoson et al. (19) recorded one electrical potential step and interpreted it as localized at the basement m e m b r a n e ; Engbaek a n d Hoshiko (5, 8) found two steps a n d suggested that one was located at the outer a n d one at the inner face of the stratum g e r m i n a t i v u m cells; a n d Scheer a n d M u m b a c h (20) described one step at the epithelium a n d one at the tela subcutanea. Frazier (6) working with the toad bladder, a preparation that behaves in m a n y ways as the skin, 795

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Slices from the cortex corticis of the guinea pig kidney were immersed in a chilled solution without K and then reimmersed in warmer solutions. The Na and K concentrations and the membrane potential V,,, were then studied as a function of the Na and K concentrations of the reimmersion fluid. It was found that Na is extruded from the cells against a large electrochemical potential gradient. Q0 for net Na outflux was -2.5. At bath K concentrations larger than 8 mM the behavior of K was largely passive. At the outset of reimmersion (Vm > Ej) K influx seemed secondary to Na extrusion. Na extrusion would promote K entrance, being limited and requiring the presence of K in the bathing fluid. At bath K concentrations below 8 m, K influx was up an electrochemical potential gradient. Thus a parallel active K uptake is apparent. Qo0 for net K influx was -2.0. Dinitrophenol inhibited net Na outflux and net K influx, Q o became <1.1 for both fluxes. The ratio between these fluxes varied. Thus at the outset of reimmersion the net Na outflux to net K influx ratio was > 1. After 8 minutes it was < 1.