Carlos A. Rabito

Na+-dependent sugar transport in a cultured epithelial cell line from pig kidney.

SummaryA Na+-dependent hexose transport system with similar characteristics to that observed in the kidney is retained in a cultured epithelial cell line from pig kidney (LLC-PK1). The active transport of α methyl-d-glucoside (α MGP), a nonmetabolizable sugar, which shares the glucose-galactose transport system in kidney cells is mediated through a Na+-dependent, substrate-saturable process. The kinetic analysis of the effect of Na+ on the uptake of αMGP indicated that the Na+-sugar cotransport system is an affinity type system in which the binding of either sugar or Na+ to carrier increases the affinity for the other ligand without affecting theVmax. The sequence of selectivity for different sugars studied by the inhibition produced in the uptake of αMGP is very similar to that reported in rat kidney, rabbit kidney cortex slices, and rabbit renal brush border membrane vesicles. Phlorizin, even at very low concentration, almost completely inhibits αMGP uptake. Conversely, phloretin at the same low concentration stimulated the sugar accumulation by inhibition of efflux, probably at the level of the basolateral membrane. Sulfhydryl group inhibitors also blocked the αMGP uptake, suggesting that these groups were required for normal functioning of the sugar carrier system. This sugar transport system is an important functional marker to study the molecular events associated with the development of polarization in epithelial cells.

Distribution and characteristics of the occluding junctions in a monolayer of a cell line (MDCK) derived from canine kidney

SummaryOn solid substrates MDCK, a cell line derived from normal dog kidney, forms a confluent monolayer that is studded with “blisters”. Previous studies with this cell line suggest that these hemicysts develop as a result of active fluid accumulation between cell sheet and substratum. One factor that may determine when and how hemicysts appear only in localized sites is the interruption of occluding junctions in nonhemicyst areas. To study this possibility, we compared the permeability characteristics of the occluding junctions in hemicysts and in an uninterrupted monolayer of MDCK grown on a permeable support of collagen-coated nucleopore filter. The spontaneous electrical potential differences were small, without statistical differences between them. Relative ionic permeability coefficients were evaluated from the voltage deflections to imposed salt gradients or to a single ion substitution across both structures. The results showed that the relative permeability ratios for Na+, K+, choline+, and Cl− were the same in hemicysts and the uninterrupted monolayer. These and other results indicate that the junctional complex encircling the apical surface of a sheet of MDCK cells can provide an effective permeability barrier constituting a true occluding junction with the same properties in hemicyst and nonhemicyst areas.

Localization of the Na+-sugar cotransport system in a kidney epithelial cell line (LLC PK1).

Studies of the localization of the Na+-dependent sugar transport in monolayers of LLC PK1 cells show that the uptake of a methyl alpha-D-glucoside, a nonmetabolizable sugar which shares the glucose-galactose transport system, occurs mainly from the apical side of the monolayer. Kinetics of [3H]phlorizin binding to monolayers of LLC PK1 cells were also measured. These studies demonstrate the presence of two distinct classes of receptor sites. The class comprising high affinity binding sites had a dissociation constant (Kd) of 1.2 microM and a concentration of high affinity receptors of 0.30 mumol binding sites per g DNA. The other class involving low affinity sites had a Kd of 240 microM with the number of binding sites equal to 12 mumol/g DNA. Phlorizin binding at high affinity binding sites is a Na+-dependent process. Binding at the low affinity sites on the contrary is Na+-independent. The mode of action of Na+ on the high affinity binding sites was to increase the dissociation constant without modifying the number of binding sites. The Na+ dependence and the matching of Kd for high affinity binding sites with the Ki of phlorizin for the inhibition of methyl alpha-D-glucoside strongly suggest that the high affinity phlorizin binding site is, or is part of the methyl alpha-D-glucoside transport system. Binding studies from either side of the monolayer also show that the binding of phlorizin at the Na+ dependent high affinity binding sites occurs mainly from the apical rather than the basolateral side. The specific location of the Na+-dependent sugar transport system in the apical membrane of LLC PK1 cells is, therefore, another expression of the functional polarization of epithelial cells that is retained under tissue culture condition. In addition, since this sugar transport almost disappears after the cells are brought into suspension, it can be used as a marker to study the development of the apical membrane in this cell line.

Effect of cell-substratum interaction on hemicyst formation by MDCK cells.

SummaryOn impermeable substrate MDCK cells, a cell line derived from normal dog kidney, forms a confluent monolayer that is studded with numerous hemicysts. Previous studies with this cell line suggest that these hemicysts develop as a result of active fluid accumulation between cell sheet and substratum. However, the formation of hemicysts as a multifocal phenomenon is still unexplained. The results presented here show that the hemicysts are not only expressions of active transport of solutes and water, but also of cell-substratum interaction. The increase in number and size of the hemicyst produced by dbcAMP may be explained by a decrease in the adhesive strength to substrata produced by this compound. Moreover, when the strength of the cell-substratum adhesion was increased the number of hemicysts was reduced or abolished. On the contrary, when this strength was reduced, larger hemicysts occurred, covering practically all the area available for growth. Results from cinematographic time lapse studies, showing that 90% of the area of the monolayer is able to produce hemicysts, also suggest that hemicyst formation as a multifocal phenomenon is more an expression of local variations in cell-substratum interaction than of regional changes in transepithelial active transport.

Complications of exercise and pharmacologic stress tests: differences in younger and elderly patients.

BackgroundAge characteristics of patients undergoing various types of stress tests are important because of differences in clinical background and exercise performance between the young and elderly. Adverse effects of pharmacologic agents are known to be more common in the elderly, who are less able to perform vigorous exercise stress testing. We investigated the clinical background, performance characteristics, and complication rate of various stress tests in younger (≤75 years old) and elderly (>75 years old) patient populations.MethodsA total of 3412 patients (2796 younger, 616 elderly) underwent 5 types of stress tests with (1) technetium-99m sestamibi (MIBI) single photon emission computed tomography: symptom-limited exercise (Ex, 1598 younger, 173 elderly), (2) dipyridamole infusion (0.14 mg/kg/min, 4 minutes) without exercise (D, 260 younger, 114 elderly), (3) with exercise (DEx, 339 younger, 112 elderly), (4) adenosine infusion (0.14 mg/kg/min, 5 minutes) without exercise (A, 253 younger, 101 elderly), and (5) with exercise (AEx, 346 younger, 116 elderly).ResultsSixty-seven percent of patients in the younger population were able to achieve 85% of the maximum predicted heart rate, whereas 54% of the elderly reached this level of exercise. No patient had life-threatening complications. In both the younger and elderly groups, chest discomfort, feelings of impending syncope, flushing, and fall in blood pressure occurred less frequently in DEx than D and in AEx than A. Sinus bradycardia occurred less frequently in AEx than A in the younger (1.2% vs 4.3%, P<.05) and elderly groups (0.9% vs 6.9%, P<.05). Atrioventricular block was less frequent in AEx than A in the younger group (3.2% vs 7.9%, P<.05) but not so in the elderly group (13.0% vs 17.8%, not significant). The frequency of ischemic electrocardiographic changes in DEx and AEx was very similar to that of Ex in both the younger and elderly groups, although ischemic electrocardiographic changes in D and A are known to be less frequent.ConclusionOf the elderly group who were judged to be fit to exercise to 85% of maximum predicted heart rate, nearly half failed to reach this level. In contrast, the younger patients were able to achieve this level in 67% of tests. Supplementation with modest exercise reduced most of the pharmacologically related adverse effects. The elderly group was not protected from atrioventricular block as effectively as the younger group by additional exercise in the adenosine stress test. Ischemic electrocardiographic changes in the pharmacologic stress test were as frequent as in the exercise stress test when modest supplementary exercise was added to the pharmacologic protocol. There were no deaths, myocardial infarction, or other major complications. These observations suggest that exercise and pharmacologic stress tests are safe in the elderly, including those patients more than 75 years old.

Optical, real-time monitoring of the glomerular filtration rate.

An easy and accurate assessment of the renal function is a critical requirement for detecting the initial functional decline of the kidney induced by acute or chronic renal disease. A method for measuring the glomerular filtration rate is developed with the accuracy of clearance techniques and the convenience of plasma creatinine. The renal function is measured in rats as the rate of clearance determined from time-resolved transcutaneous fluorescence measurements of a new fluorescent glomerular filtration agent. The agent has a large dose-safety coefficient and the same space distribution and clearance characteristics as iothalamate. This new approach is a convenient and accurate way to perform real-time measurements of the glomerular filtration rate to detect early kidney disease before the renal function becomes severely and irreversibly compromised.

Free radical-mediated membrane depolarization in renal and cardiac cells.

Cell membrane potential was measured with a flow cytometer by quantitating the intracellular accumulation of a fluorescent cationic carbocyanine dye. We used this system to demonstrate depolarization upon the addition of hydrogen peroxide (10-1,000 microM) and ferrous chloride (25-100 microM) to cultures of either neonatal rat myocardial or LLC-PK1 renal epithelial cells. Ferrous chloride-induced depolarization was prevented by superoxide dismutase, catalase and dimethyl sulfoxide, suggesting roles for the superoxide anion, hydrogen peroxide and the hydroxyl radical in effecting this depolarization, possibly through a Fenton-type reaction mechanism. Supplementation of either cell type with 2 microM tocopherol acid succinate during growth in tissue culture, prior to exposure to the oxidizing agent, decreased the magnitude of the depolarization in both cell types. The results are consistent with a role for tocopherols in scavenging free radical species responsible for the depolarization of the cell membrane.

Oxygen radicals and plasma membrane potential.

Plasma membrane potential is an important physiologic parameter dependent both upon cellular metabolism and upon the integrity of the cell membrane. Oxygen radicals have been shown to produce significant and early changes in the membrane potential. This data is reviewed along with the physico-chemical origins of the membrane potential and methods of its measurement.

The permeability of the membranes of experimental secondary cysts of Echinococcus granulosus to [14C]mebendazole.

Abstract The permeability of secondary E. granulosus cysts to [ 14 C]mebendazole was studied. The cysts were obtained by transplanting secondary cysts raised in mice into rats. The permeability to [ 14 C]mebendazole was established by two different experiments: uptake and washout of the drug. The cyst wall permeability to [ 14 C]mebendazole was found to be 1·33 × 10 −4 cm s −1 , which is of the same order as the diffusion permeability coefficient to water (1·88 × 10 −4 cm s −1 , Rotunno, Kammerer, Perez Esandi & Cereijido, 1974). The drug readily permeates through the cyst wall and experimental data suggest that it moves across the barrier by simple diffusion.

Morphologic and functional correlates of plasma membrane injury during oxidant exposure

Plasma membrane injury by exposure to hydrogen peroxide was examined in a renal epithelial cell line (LLC-PK1). Morphologic and functional parameters of plasma membrane integrity were studied in an attempt to eludicate the sequence of membrane alterations during the evolution of hydrogen peroxide-mediated injury. These parameters included plasma membrane potential and permeability, plasma membrane bleb formation, cellular size, and plating efficiency. Plasma membrane potential was the earliest parameter affected by hydrogen peroxide exposure. Half maximal depolarization occurred within 15-30 min of exposure to 1 mM, after 10-15 min exposure to 100 mM and after over 150 min exposure to 10 microM hydrogen peroxide. After exposure to 1 mM hydrogen peroxide, the following sequence of events was seen; increased plasma membrane blebbing (30 min), cell swelling (90-125 min) and increased plasma membrane permeability (150-240 min). After a 30 min exposure to 1 mM hydrogen peroxide, cellular plating efficiency, measured at 24 h, was reduced by 50% (P less than .001). These changes were accelerated, although their order of appearance was unchanged, at higher concentrations of hydrogen peroxide. We conclude that functional and morphologic expressions of cellular injury in this model occur in a defined sequence with plasma membrane depolarization representing the earliest marker of membrane injury during hydrogen peroxide exposure.