Isolation and culture of capillary endothelial cells from the eel, Anguilla rostrata.

There has been little development of endothelial cell cultures from vertebrates other than mammals. In this report the isolation of capillary endothelial cells from the rete mirabile of the eel, Anguilla rostrata, is described. The cells are isolated with collagenase H and dispase II. The cells are plated into fibronectin-hyaluronic acid coated flasks. The culture medium is M199 with Earle's salts supplemented with NaCl, HEPES, NaHCO(3), glutamine, pyruvate, heparin, antibiotics, endothelial cell growth supplement, and 20% serum. Cultures are incubated at 25 degrees C in humidified air. The rete mirabile contains pericytes in addition to endothelial cells. Variations in plating time, serum concentrations, and growth matrices were tried to separate the two cell types. The total number of endothelial cells and the ratio of endothelial cells to pericytes are the most important factors in obtaining pure cultures of capillary endothelial cells. Endothelial cells are isolated also from the endocardium, bulbus arteriosus, and large vessels. The initial isolates usually take 3-6 weeks to grow to confluence with subcultures taking about 2 weeks to confluence.

Pulmonary distribution of iodoantipyrine: temperature and lipid solubility effects.

In multiple indicator-dilution studies in rat and dog lungs, we have found that the distribution of iodoantipyrine (IAP) is not limited by the endothelium at a temperature > 7 degrees C but is barrier limited at the epithelium at a temperature < 15 degrees C (permeability coefficient of 6.3 x 10(-5) cm/s at 8 degrees C). IAP extraction from the vascular surface to the tissues is greater than those of antipyrine (AP) and tritiated water (THO). IAP transmittance from the alveolar surface to the vascular compartment is smaller than those of AP and THO: a lung lipid compartment, probably in the lamellar bodies of the type II cells, is more accessible to IAP than to AP or THO because IAP has a higher oil-to-water distribution coefficient. Our mathematical model takes into account these matters and also the low surface density of the type II cells: some of the IAP may bypass the lipid compartment. Lipid may affect the transit of solutes with high oil-to-water distribution coefficients in the lungs and across the alveolar-capillary barrier.

The diffusional transport of water and small solutes in isolated endothelial cells and erythrocytes.

The diffusional permeability coefficients, PD, for tritiated water (3HHO) 14C-antipyrine (AP) and 14C-iodoantipyrine (IAP) in isolated calf pulmonary artery endothelial cells and dog erythrocytes are measured with the linear diffusion technique at 11.5, 15, 20 and 37 degrees C. The PD values for both cell populations follow the sequence 3HHO > IAP > AP at each of the temperatures. PD for water is higher in the erythrocyte compared to the endothelial cells. The differences in PD for AP and IAP in the erythrocytes and endothelial cells are not dramatic and are similar to the differences seen in comparing permeation of the same solute through bilayers of different composition. A comparison of the values of PD calculated for the endothelial cells with those for isolated capillaries and the structured endothelium in whole lungs validates the use of the isolated cells as models for the endothelial cells in situ. Incubation of the endothelial cells with cis-vaccenic acid or cholesterol produces a reduction in PD for water and antipyrine. These data are analyzed in terms of Stokesian and non-Stokesian diffusion. The interpretation which best accommodates the data is that the phospholipid area of the membrane, rather than the hydrocarbon core, provides the greatest resistance to permeation for these solutes.

Endothelial and epithelial permeabilities to antipyrine in rat and dog lungs.

Temperature effects on the permeabilities of the structured endothelium and epithelium to antipyrine (AP) have been determined with the indicator dilution technique in isolated rat and dog lungs perfused between 38 and 8 degrees C. Permeability coefficients of the endothelium to AP [Pendo(AP)] from the Crone equation are smaller than values for isolated endothelial cells but close to the permeability coefficient of the interstitial epithelial plasmalemma [Pepi(AP)] obtained from physical and mathematical models. In these, tracer water is flow limited at the endothelium and the epithelium at all temperatures; AP is flow limited at the endothelium at T greater than 20 degrees C but barrier limited at the endothelium for T less than 20 degrees C and at the epithelium at all temperatures. At T less than 20 degrees C, log Pendo(AP) decreases regularly with 1/T, with a slope close to that found in cultured bovine pulmonary artery endothelial cells. At 15 degrees C, Pendo(AP) for the endothelial plasmalemma in situ is 30 X 10(-5) cm/s and is 56 X 10(-5) cm/s for the isolated cells in support of transcellular rather than paracellular passage. At T greater than 20 degrees C, log Pepi(AP) in situ decreases slightly with 1/T, with a discontinuity at T = 20 degrees C, and for T less than 20 degrees C, decreases with 1/T with a slope close to that of Pendo(AP). At 15 degrees C, Pepi(AP) is 2.8 X 10(-5) cm/s. The discontinuity may represent a change in the physical state of lipids in the interstitial plasmalemma of the epithelial cells.

Water permeability of isolated endothelial cells at different temperatures.

The endothelial cells provide a potential pathway for water movement across the endothelium. The endothelial cell permeability to water can, therefore, be a factor in regulation of the rate of water movement out of the vasculature. Endothelial cells are isolated from calf pulmonary artery and cultured. The cells are removed from culture, and the diffusional water permeability is determined with the linear diffusion technique. The mean membrane permeability coefficients (PDS) determined at 20, 30, 37, and 41 degrees C are 160, 273, 304, and 387 x 10(-5) cm/s, respectively. The temperature dependence of PD is calculated with the Arrhenius equation to be 7.2 kcal/mol. If these values of PD are compared with values we have reported for the osmotic permeability coefficient (PF), the PF/PD is about one at each temperature. The values of PD in the isolated endothelial cells are compared with PD for endothelial cells estimated from whole organ studies and are similar to recently reported values.

The osmotic permeability of isolated calf pulmonary artery endothelial cells.

Osmotic permeability coefficients, PF, for water in isolated calf pulmonary artery endothelial cells determined over the temperature range 41 to 20 degrees C are 311.10(-5) cm.s-1 at 37 degrees C and 159.10(-5) cm.s-1 at 20 degrees C. The value at 37 degrees C is close to that reported earlier for the diffusional permeability coefficient, PD. The PF/PD ratio is 1.0 at 37 degrees C. The PF values are within the range of values extrapolated for filtration permeability in pulmonary endothelium. The temperature dependence expressed as the activation energy is 7.2 kcal.mol-1. The product of hydraulic conductivity, Lp (or PF) and of viscosity changes in water is not constant from 37 to 20 degrees C. These results can be interpreted to indicate a similar pathway for water whether under diffusional or osmotic gradients.

Water and nonelectrolyte permeability of isolated rat hepatocytes.

We have measured the diffusive permeability coefficients of isolated rat hepatocytes to 3H2O, [14C]urea, [14C]erythritol, [14C]mannitol, [3H]sucrose, and [3H]inulin, employing a technique previously developed for erythrocytes (Redwood et al., J. Gen. Physiol 64:706-729, 1974). Diffusion coefficients for the tracer molecules were measured in packed hepatocytes, supernatant fluid, and intracellular medium (lysed hepatocytes) and were calculated assuming one-dimensional semi-infinite diffusion through a homogeneous medium. By applying the series-parallel pathway model, the following permeability coefficients (10(-5) cm/sec) for the hepatocyte plasma membrane were obtained. 3H2O, 98.6 +/- 18.4; [14C]urea, 18.2 +/- 5.3; [14C]erythritol, 4.8 +/- 1.6; [14C]mannitol, 3.1 +/- 1.4; [3H]sucrose, 0; [3H]inulin, 0. These results indicate that isolated rat hepatocytes are highly permeable to water and polar nonelectrolytes, when compared with other transporting epithelia. This relatively high cellular permeability is consistent with a model in which nonelectrolyte permeation is via an aqueous pathway of equivalent pore diameter of 8-12 A. The finding that [14C]erythritol and [14C]mannitol cross the hepatocyte plasma membrane indicates that these molecules enter the bile canaliculus through the transcellular route. Conversely, the failure of [3H]sucrose and [3H]inulin to permeate the hepatocyte in the isolated condition supports the concept that biliary entry of these large carbohydrates, at least that fraction which cannot be accounted for by a vesicular mechanism, must occur via the transjunctional shunt pathway.

Endothelial cell permeability to water.

We have calculated diffusional permeability coefficients for tracer water and for [14C]antipyrine in endothelial cells. With these values and those from studies in whole lungs we set a range for diffusional water permeability coefficients of the intact endothelium.

Water permeability of alveolar macrophages.

The hydraulic conductivity coefficient (Lp) of alveolar macrophages, recovered by lavage from dog lungs, was determined by following volume changes induced by changes of nonpermeating solute concentrations of suspending fluid as a function of time at 20 degrees C. The volume changes were monitored as changes in absorbance of the suspended cells at 600 nm. Cell surface area was calculated from cell volume and diameter. Linear relationships between cell volume and solution osmolality changes were found over the range of 320-520 mosmol/kg; beyond these ranges the macrophages did not respond with swelling or shrinking. Lp and the filtration coefficient (Pf) were calculated from the total volume change over time. At 20 degrees C these were, respectively, 15.7 X 10(-10) cm X cmH2O-1 X s-1 and 217 X 10(-5) cm/s. Comparison of Pf and the diffusional permeability coefficient (Pd) for water of 70 X 10(-5) cm/s, yields a Pf-to-Pd ratio of 3.1. The hypothesis of water passage through aqueous membrane pores is compatible with these data. However, diffusion of water in the glycocalyx of the pericellular domain could be restricted. Pd would then be underestimated, and a falsely high ratio would be calculated. We have no evidence to support this possibility.

Altered K+ movement in liver mitochondria from alloxan diabetic rats.

Potassium movements were monitored in liver mitochondria from control and alloxan diabetic rats with a cationic electrode. There was net accumulation of K+ after Ca2+ addition to the mitochondria with the diabetic but not with the control.

Solute-excluded volumes near the Novikoff cell surface.

A differential centrifugation technique, in which all extracellular water except that intimately associated with the cell (pericellular domain) is removed, has been applied to isolated Novikoff hepatoma cells. The pericellular volumes accessible to albumin, inulin, raffinose, and sucrose were inversely related to the molecular weights of the test solutes. This phenomenon was not detectable in erythrocytes or in fat cells. Selective removal of cell surface components by enzymatic treatment produced proportional changes in the relative volumes of distribution accessible to the solutes. This discrimination in the volume accessible to each of the solutes is analogous to that obtained in gel chromatographic separation and represents, in effect, excluded volumes which are inversely related to solute size. This exclusion is associated with components of the Novikoff cell surface, including the surface coat and the microvilli that cover the Novikoff cell. These structures provide an additional level of discrimination for the Novikoff cell not seen in certain other cell types.

Effects of sulfhydryl and other reagents on the diffusional permeability of dog erythrocytes to small solutes.

The effects of p-chloromercuriphenylsulfonic acid (PCMBS), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), phloretin and thiourea on the diffusional permeability of dog erythrocytes to tritiated water and to small 14C-labeled lipophilic and hydrophilic solutes were measured at 37 degrees C by means of the linear diffusion technique. Permeability to 3HHO was significantly decreased by PCMBS but was not affected by the other reagents. The permeability to the small hydrophilic solutes acetamide and urea was decreased by phloretin and thiourea but only the permeability to acetamide was reduced to a statistically significant extent by PCMBS. The permeability to the lipophilic solutes methanol, ethanol and antipyrine was not affected by any of these agents. We interpret these results as an indication that the small lipophilic solutes probably move through lipid areas, that the small hydrophilic solutes probably move through protein associated areas in the erythrocyte membrane and that pathways for the small hydrophilic solutes are distinct from those for water. While the pathways for water may be associated with membrane protein they do not appear to be associated specifically with band 3 protein as has been suggested for human erythrocytes. Diffusional water movement through the dog erythrocyte occurs by two distinct pathways.

Membrane permeability of isolated lung cells to nonelectrolytes at different temperatures.

Membrane permeability coefficients (P0) of rabbit lung cells consisting primarily of alveolar epithelial and endothelial cells and of alveolar macrophages from dog lungs were determined for tritiated water, n-[14C]alcohols, and [14C]antipyrine over the temperature range 10 to 37 degrees C with the series-parallel pathway model. In the mixed cell preparation both the diffusional permeability to water (755 X 10(-5) cm.s-1 at 37 degrees C) and the response to temperature change (apparent activation energy, Ea, 10 kcal.mol-1) are greater than the corresponding values in the macrophages (110 X 10(-5) cm.s-1 and 4.8 kcal.mol-1, respectively). The permeability coefficients for the small alcohols (C1-C3) are similar and considerably higher than for water in both cellular preparations. The values of the permeability coefficients and the temperature dependence for antipyrine and the larger alcohols in the mixed lung cells differ from the values obtained in the macrophages. Comparison of our results with those obtained in erythrocytes and Novikoff hepatoma cells demonstrates the differences in water permeability in each cell preparation and the similarity in permeation for the more lipophilic solutes in the cell preparations. These differences may be important in the comparison of results obtained in isolated cellular systems and in intact tissues and organs.

Erythrocyte permeability to lipophilic solutes changes with temperature.

Studies of permeability coefficients of biological barriers to members of homologous series can provide information of value in assessing barrier characteristics. To this end, we have determined the linear diffusion coefficients of tracer water (THO), [14C]antipyrine, [14C]acetamide, and n-[14C]alcohols over the range of 10-37 degrees C for dog erythrocytes (D), hemoglobin (D2), and plasma (D1). Permeability coefficients (Po) calculated with the series-parallel pathway model are higher than Po for water at 37 degrees C for all the alcohols except hexanol. Po for acetamide and antipyrine are considerably lower than Po for water at 37 degrees C. The apparent activation energies (Ea) for Po of acetamide and the C1, C2, and C3 alcohols are 10.5 kcal.mol-1, similar to the values obtained with epithelial or lipid bilayers. The Ea's for the larger alcohols are 2-4 kcal.mol-1. The Ea for Po of water is 5.3 kcal.mol-1, similar to the Ea for self-diffusion of water; Ea for antipyrine is 25 kcal.mol-1. We interpret these results to indicate heterogeneity of membrane-solute interactions or of membrane pathways in the erythrocyte for lipid-soluble molecules that is related to both lipid solubility and solute size, as we have suggested in an earlier study and confirmed experimentally in the present one.

Permeability of Novikoff hepatoma cells to water and monohydric alcohols.

The permeability coefficients of Novikoff hepatoma ascites cell membranes for tritiated water (3HHO) and for a homologous series of monohydric alcohols (methanol through hexanol) were deduced from linear diffusion coefficients by means of a series-parallel pathway model (Redwood et al. (1974) J. Gen. Physiol. 64, 706-729). Membrane permeability coefficients for 3HHO at 20, 30 and 37 degrees C were (all x 10(-5)) 97, 125, and 163 cm . s-1, respectively, and were significantly smaller than the corresponding values for the alcohols tested. In the alcohols series, ethanol had the lowest permeability coefficient 198 x 10(-5) cm . s-1 at 20 degrees C. The apparent activation energy for water permeation was 6.7 +/- 1.9 S.E. kcal . mol-1. The apparent membrane diffusion coefficients for the alcohols were a complex function of molecular properties with less diffusional membrane resistance to the alcohols in the middle of the homologous series than would have been expected on the basis of oil-water partitioning or molar volume considerations. The conventional parallel aqueous lipophilic pathway model is not consistent with the present data which can be interpreted by consideration of parallel lipophilic pathways through the Novikoff hepatoma cell membrane.

Osmotic permeability of Novikoff hepatoma cells.

The osmotic permeability coefficient (Pf) for water movement across Novikoff hepatoma cells was found to be 82 +/- 3 (S.E.) x 10(-5) cm . s-1 at 20 degrees C. The corresponding diffusional permeability coefficient for 3HHO (Pd) was 97 +/- 10 (S.E.) . 10(-5) cm . s-1, therefore the ratio Pf/Pd is close to unity. The apparent activation energy for water filtration was 10.4 +/- 0.4 (S.E.) kcal . mol-1. This value is significantly greater than the activation energy for the self diffusion of water. The product of the hydraulic permeability coefficient and the viscosity coefficient for water was temperature-dependent. However, the product of the hydraulic permeability coefficient and the viscosity coefficient for membrane lipid did not vary with temperature. These data are interpreted as evidence for water movement across a lipid membrane barrier rather than through aqueous channels.

Membrane permeability of isolated lung cells to nonelectrolytes.

Mixtures of viable endothelial and epithelial cells were separated by enzymatic digestion from rabbit lung and recovered by centrifugation. The cells were mixed with an extracellular marker and packed by centrifugation into small-diameter polyethylene tubing and pulsed with tritiated water and 14C-labeled alcohols. Calculation of diffusion coefficients for the packed cell column (D), intracellular material (D2), and extracellular fluid (D1) was based on a local steady-state one-dimensional diffusional model. Permeability coefficients were: tritiated water, 288 X 10(-5) cm s-1; methanol, 385 X 10(-5) cm s-1; ethanol, 214 X 10(-5) cm s-1; propanol, 277 X 10(-5) cm s-1; and hexanol, 1255 X 10(-5) cm s-1. The permeability coefficients of these aliphatic alcohols show a minimum at ethanol with hexanol having the highest value of all substances tested. The results support the concept of parallel aqueous and lipid pathways for small solutes in the plasma membrane. Study of the permeability properties of isolated lung cells can provide information on the cellular pathway in the transcapillary transport of water and solutes in the lung.