Oscar A. Candia

An improved assay for nanomole amounts of inorganic phosphate.

A calorimetric assay for the determination of nanomole amounts of inorganic phosphate is described. The procedure combines a very high molar extinction with color stability and insensitivity to newly released phosphate from labile organophosphates. For studies on the energetics of active ion transport in various epithelial membrane systems, we needed a dependable ultramicromethod for the determination of inorganic phosphate. The most sensitive calorimetric method, that of Itaya and Ui (1) as modified by Hess and Derr (2), has some serious drawbacks: (a) the optical density of both the blank and the samples increase with time to an objectionable level, and more importantly (b) both ATP and ADP are hydrolyzed in the presence of the reagents. Both the highly acidic conditions and the catalytic effect of molybdate contribute to the hydrolysis of labile organophosphates and result in various phosphate levels being recorded according to the length of time allowed for color development (3,4). Since our laboratory started to measure ATPase activity routinely, we settled on our slight modification of the Baginski method of Pi determination (5). This method (through the use of a citratelarsenite mixture added immediately after the molybdate reagent) is relatively sensitive, color stable, and has the advantage of being insensitive to any newly released phosphate (e.g.,

Short-circuit current related to active transport of chloride in frog cornea: effects of furosemide and ethacrynic acid.

Abstract Active transport of Cl− accounts for 90% of the short-circuit current (s.c.c.) in the isolated frog cornea. 1·10−5 M furosemide produced a 50% reversible inhibition of this s.c.c. 1·10−4 M ethacrynic acid reduced the corneal s.c.c. to 32% of the control. In the isolated frog skin epithelium furosemide had no effect on the s.c. at a concentration of 1·10−4 M and a small stimulation at a concentration of 1·10−3 M. The furosemide inhibitory effects seems to be specific for Cl−, as it also inhibits Cl− transport in the ascending limb of the loop of Henle (Burg, M.B. (1972) Proc. 5th Int. Congr. Nephrol., p. 50, Abstr.).

Lithium transport across isolated frog skin epithelium.

SummaryTransepithelial Li+ influx was studied in the isolated epithelium from abdominal skin ofRana catesbeiana. With Na+-Ringers as inside medium and Li+-Ringers as outside medium, the Li+ influx across the epithelium was 15.6 μA/cm2. This influx was considerably reduced by removal of either Na+ or K+ from the inside bath or by the addition of ouabain or amiloride. Epithelial K+ or Na+ concentration was respectively lower in epithelia bathed in K+-free Ringers or Na+-free Ringers. In conditions of negligible Na+ transport, a 20mm Li+ gradient (out→in) produced across the short-circuited epithelium a Li+ influx of 11.8 μA/cm2 and a mean short-circuit current of 10.2 μA/cm2. The same Li+ gradient in the opposite direction produced a Li+ outflux of only 1.9 μA/cm2. With equal Li+ concentration (10.3 and 20.6mm) on both sides of the epithelium, plus Na+ in the inside solution only, a stable Li+-dependent short-circuit current was observed. Net Li+ movement (out→in) was also indirectly determined in the presence of an opposing Li+ gradient. Although Li+ does not substitute for Na+ as an activator of the (Na++K+)-ATPase from frog skin epithelium, Li+ influx appears to be related to Na+−K+ pump activity. It is proposed that the permeability of the “outer barrier” to Na+ and Li+ is regulated by the electrical gradient produced by electrogenic Na+−K+ pumps located in the membrane of the deeper epithelial cells.

Effect of pH on chloride transport a across the isolated bullfrog cornea

The effect of pH on chloride fluxes and electrical parameters was studied in the isolated cornea of the frog, Rana catesbeiana. Lowering the pH from 8·6 to 7·2 produced a decrease in unidirectional and net chloride fluxes as well as in the short-circuit current and potential difference, while the electrical resistance was increased. All the effects were fully reversible when the bathing solutions were alkalized to the initial pH. It was determined that the effect was due only to the pH change on the epithelial side. Results are consistent with a pH effect on the permeability of a chloride pathway in series with the active pump.

Transport of lithium and rectification by frog skin

Abstract The isolated frog skin, bathed with Li + -Ringer (Na + -free) on the outside and Na + -Ringer on the inside, can maintain a normal potential difference (PD) and short-circuit current (s.c.c.) for more than 6. h. The s.c.c. correspondended to the Li + influx. The Na + efflux was 4% of the s.c.c. 10 −5 M ouabain depressed Li + influx and s.c.c. 10 10−5 M amiloride abolished the Li + s.c.c., while 0.1 unit/ml oxytocin stimulated it. When the inside of the skin was bathed with Li + -Ringer, PD and s.c.c. fell to zero within 2 h. The oxygen consumption of skin slices bathed in Li + -Ringer was 29% lower than controls bathed in Na + -Ringer. When the isolated frog skin is bathed in Na 2 SO 4 -Ringer it shows electrical rectification which has been correlated with the active transport of Na + . In skins transporting Li + , rectification characteristics are similar to those of skins transporting Na + . When the inner face of the skin is bathed with Li + -Ringer, rectification, PD and s.c.c. decline in a parallel fashion. It is concluded that: (1) Li + can be transported when Na + is present at the inner face. (2) Amiloride, ouabain and oxytocin affect Li + and Na + transport in a similar manner. (3) Li + transport, like Na + transport, is associated with rectification. (4) Active transport of Na + and Li + seems to depend on two different but associated proceses; one taking place at the external barrier (where rectification occurs) as shown by the effect of amiloride; and the other of an inner site related to energy requirements and affected by ouabain and Li + . (5) The cation being transported is not necessarily activating the (Na + -K + -ATPase.

Potassium flux and sodium transport in the isolated frog skin

Abstract The K + fluxes at the inside surface, and the short-circuit current (s.c.c.), were measured in a 20-cm 2 piece of the abdominal skin of the frogs, Leptodactylus ocellatus and Rana catesbeiana . The inside of the skin was perfused with Ringers solution containing 42 K + circulating in a closed system including a scintillation detector. The disaappearance of radioactive material from the Ringers solution was measured during a period greater than 3 h. A compartmental analysis was used to find out the transfer constants and unidirectional fluxes. Most of the experiments were performed under steady-state conditions. Despite the fact that the skin of L. ocellatus shows an active transport of Cl − not present in the skin of the R. catesbeiana , results in both skins were similar. The K + content of the skins was found to be in the order of 1 μequiv/cm 2 of skin surface. K + influx at the inside surface was of the order of 0.3 μequiv./cm 2 per h. The K + flux did not vary when the short-circuit condition was changed to open circuit and vice versa , or upon removal of Cl − from the Ringer. However, the Na + flux, or s.c.c. was significantly larger than the K + flux. The permeability properties for K + at the inside surface seem to be relatively independent of external electrical or ionic changes. K + flux is not directly related to the mechanism of Na + transport.

Microelectrode and short-circuiting techniques for the study of ion transport in the lens

Abstract Ion transport was studied by considering the lens as a composite asymmetrical system rather than a closed system. When the toad lens is isolated in a chamber, the anterior surface of the lens is 30 mV positive with respect to the posterior surface. Translenticular unidirectional fluxes of Na, Cl, and K were measured under short-circuit conditions. It was found that only Na is actively transported across the lens; the transport being in a posterior-anterior direction. A discrepancy between net Na flux and short-circuit-current is interpreted on the basis of Na being gained by the lens across its posterior surface and K being lost across its anterior surface. With the aid of a glass microelectrode the potential difference across the epithelial layer under the anterior capsule was determined and a larger short-circuit-current obtained. It is concluded that in the amphibian lens the movement of K appears to be secondary to electrical gradients created by an electrogenic Na pump.

Na+-K+ ATPase distribution in frog and bovine lenses

Na+-K+ ATPase activity was measured in the capsule-epithelium and decapsulated frog and bovine lens. The decapsulated lens contained approximately 20% of the whole lens activity in the frog and 30% in the bovine. These values were measured from the aqueous homogenate of the entire decapsulated lens, an approach which may have underestimated the activity by diluting the ouabain-sensitive component in the preparation. Subsequent determinations were done on separate portions of superficial (2 to 3 mm) anterior-equatorial, and posterior bovine cortex. The activities per gram of tissue were enriched with respect to the values for the entire decapsulated bovine lens. These activities were further enriched by a sucrose density gradient centrifugation. The anterior-equatorial cortical segment contained 1.6 times the activity found in the capsule-epithelium. The posterior cortex had a much smaller but statistically significant level of Na+-K+ ATPase. It is unlikely that the observed asymmetry of the anterior-equatorial segment with the posterior cortex is exclusively due to epithelial contamination for the result would require the adherence of 62% of the epithelium. Scanning electron micrographs of 6 decapsulated bovine lenses indicated an average contamination of about 9%. This asymmetry may have a physiological role in assisting the pump mechanism of the epithelium.

Microsomal (Na+ + K+)-activated ATPase from frog skin epithelium. Cation activations and some effects of inhibitors☆

A method is described for the extraction of microsomal ouabain-sensitive (a- + K+)-activated ATPase from separated frog skin epithelium. The method yields a microsomal fraction containing (Na+ K+)-stimulated activity in the range of 30- 40 nmol - mg -1 - min -1 at 26 degrees C. This portion which is also ouabain sensitive, is about half of the total activity in media containing Mg2+, Na+ and K+. These preparations also contain Mg2+-dependent or Ca2+-dependent activities which are not additive and which are not significantly affected by ouabain, Na+, K+ or Li+. The activations of the ouabain-sensitive ATPase activity by Mg2+, Na+, and K+ are similar to those described in other tissues. It is found that Li+ does not substitute for Na+ as an activator but in high concentrations does produce partial activation in the presence of Na+ with no K+. These results are pertinent to the reported observations of ouabain-sensitive Li+ flux across frog skin. It is concluded that this flux is not apparently due to a direct activating effect of Li+ on the sodium pump.

Effects of forskolin, prostaglandin F2α, and Ba2± on the short-circuit current of the isolated rabbit iris-ciliary body

To gain information on the role of cyclic AMP (cAMP), ion transport and cell membrane permeability on aqueous humor formation, agents with well-known effects on transport properties in other epithelia were tested on the isolated rabbit iris-ciliary body. Forskolin stimulated the short-circuit current (SCC) by 37.5% when added to the aqueous-side solution. Forskolin was ineffective when added to the blood-side solution or when HCO3- was absent from the bathing solutions. The effect of forskolin confirms the presence of adenylate cyclase in the ciliary epithelium and the involvement of cAMP in ion transport. In HCO3- -rich media, 5 X 10(-5) M prostaglandin F2 alpha (PGF2 alpha), produced a prompt 25% increase in the SCC when added to the aqueous-side, and a small stimulatory SCC response when added to the blood-side. No change in SCC occurred when PGF2 alpha was added to either side of a HCO3- -free bathing solution. It is implied that cAMP acts on a HCO3- dependent transport system. These results are consistent with the previously observed stimulation of the SCC by 8Br-cAMP. BaCl2, 2.5 mM, on the aqueous-side increased the SCC by 240.5%, but reduced the SCC by 26% when added to the blood-side solution. The Ba2+ effects indicate the presence of high conductance K+ channels in the basolateral membranes of both the pigmented and non-pigmented cell layers.