Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

Intrinsic nitric oxide and superoxide production regulates descending vasa recta contraction

Descending vasa recta (DVR) are 12- to 15-μm microvessels that supply the renal medulla with blood flow. We examined the ability of intrinsic nitric oxide (NO) and reactive oxygen species (ROS) generation to regulate their vasoactivity. Nitric oxide synthase (NOS) inhibition with Nω-nitro-l-arginine methyl ester (l-NAME; 100 μmol/l), or asymmetric NG, NG-dimethyl-l-arginine (ADMA; 100 μmol/l), constricted isolated microperfused DVR by 48.82 ± 4.34 and 27.91 ± 2.91%, respectively. Restoring NO with sodium nitroprusside (SNP; 1 mmol/l) or application of 8-Br-cGMP (100 μmol/l) reversed DVR vasoconstriction by l-NAME. The superoxide dismutase mimetic Tempol (1 mmol/l) and the NAD(P)H inhibitor apocynin (100, 1,000 μmol/l) also blunted ADMA- or l-NAME-induced vasoconstriction, implicating a role for concomitant generation of ROS. A role for ROS generation was also supported by an l-NAME-associated rise in oxidation of dihydroethidium that was prevented by Tempol or apocynin. To test whether H2O2 might play a role, we examined its direct effects. From 1 to 100 μmol/l, H2O2 contracted DVR whereas at 1 mmol/l it was vasodilatory. The H2O2 scavenger polyethylene glycol-catalase reversed H2O2 (10 μmol/l)-induced vasoconstriction; however, it did not affect l-NAME-induced contraction. Finally, the previously known rise in DVR permeability to 22Na and [3H]raffinose that occurs with luminal perfusion was not prevented by NOS blockade. We conclude that intrinsic production of NO and ROS can modulate DVR vasoactivity and that l-NAME-induced vasoconstriction occurs, in part, by modulating superoxide concentration and not through H2O2 generation. Intrinsic NO production does not affect DVR permeability to hydrophilic solutes.