Abstract P607: Nitric Oxide (NO) Regulates Paracellular Permselectivity in Isolated, Perfused Rat Thick Ascending Limbs through Claudin-19

About 50% of the Na reabsorbed in thick ascending limbs (TALs) traverses the paracellular pathway. The ionic selectivity of this route is regulated by claudins in the tight junctions. TALs express claudin-19 which has been reported to regulate TAL Na permeability. We showed that nitric oxide (NO) decreases Na/Cl permeability ratio (PNa/PCl) in TALs by increasing the absolute permeabilities of both ions though PCl increased more. However, whether NO affects paracellular permeability via claudin-19 is unknown. We hypothesize that NO regulates the paracellular permselectivity in TALs through this claudin. To test this we perfused TALs from Sprague Dawley rats and measured dilution potentials (a measure of permselectivity) with and without exogenously-added or endogenously-produced NO in the presence or absence of an antibody against an extracellular domain of claudin-19 or Tamm-Horsfall protein (control). Dilution potentials were generated by reducing bath NaCl from 141 to 32 mM. For the NO donor spermine NONOate (SPM): during the control period, the dilution potential was -9.3 ± 1.8 mV. After SPM (200 μM), it was -6.7 ± 1.6 mV (n = 6; p < 0.003). In the presence of the claudin-19 antibody, SPM had no significant effect on dilution potentials (claudin-19 antibody alone: -12.7 ± 2.1 mV vs claudin-19 antibody + SPM: -12.9 ± 2.4 mV; n = 6). The claudin-19 antibody alone had no effect on dilution potentials. In the presence of the Tamm-Horsfall protein, the effect of SPM was still present (Tamm-Horsfall protein antibody alone: -9.7 ± 1.0 mV; Tamm-Horsfall protein antibody + SPM: -6.3 ± 1.1 mV, p<0.006, n = 6). For experiments with endogenously-produced NO, L-arginine the substrate for NO synthase was added. During the control period, the dilution potential was -11.0 ± 1.1 mV. After L-arginine (500 μM) treatment, they were -9.0 ± 1.2 mV (n = 9; p < 0.05). In the presence of the claudin-19 antibody, L-arginine had no significant effect on dilution potentials (claudin-19 antibody alone: -10.1 ± 0.9 mV vs claudin-19 antibody + L-arginine: -10.1 ± 1.0 mV; n = 9). In the presence of the Tamm-Horsfall protein, the effect of L-arginine was still present. We conclude that the actions of NO on the paracellular permselectivity in thick ascending limbs are at least in part mediated by claudin-19.

The Volume Activated Potassium Channel KCNK5 is Up-Regulated in Activated Human T Cells, but Volume Regulation is Impaired

Background/Aims: The potential role of the two-pore domain potassium channel KCNK5 (also known as TASK-2 and K2P5.1) in activated T cell physiology has only recently been described. So far KCNK5 has been described to be up-regulated in T cells in multiple sclerosis patients and to be implicated in the volume regulatory mechanism regulatory volume decrease (RVD) in T cells. Methods: We investigated the time-dependent expression pattern of KCNK5 in CD3/CD28 activated human T cells using qPCR and Western blotting and its role in RVD using a Coulter Counter. Results: KCNK5 is highly up-regulated in CD3/CD28 activated T cells both at mRNA (after 24 h) and protein level (72 and 144 h), but despite this up-regulation the RVD response is inhibited. Furthermore, the swelling-activated Cl- permeability in activated T cells is strongly decreased, and the RVD inhibition is predominantly due to the decreased Cl- permeability. Conclusion: The up-regulated KCNK5 in activated human T cells does not play a volume regulatory role, due to decreased Cl- permeability. We speculate that the KCNK5 up-regulation might play a role in hyperpolarization of the cell membrane leading to increased Ca2+ influx and proliferation of T cells.

Effect of Aggregate Volume Fraction on the Charge Passed of Mortar and Concrete

Charge passed of mortars with different aggregate volume fractions was tested according to ASTM C1202, and the differential expression of the Ohm law was introduced to explain the relationship between charge passed data and aggregate fractions.Cl- penetration depth of the corresponding mortars and concretes was measured to illustrate the influence of the aggregate volume fraction on their chloride permeability. It was shown that the charge passed of mortar was negatively correlated with aggregate volume fractions, and high aggregate fraction bearing concrete with low charge passed did not necessarily mean low permeability. As a result, the influence of aggregate fraction on charge passed should be taken into consideration when charge passed data was employed to evaluate Cl- permeability of concrete.

Ammonium interaction with the epithelial sodium channel

The purpose of this study was to investigate the direct effect of NH3/NH[Formula: see text] on mouse epithelial Na+ channels (mENaC) expressed in Xenopusoocytes. Two-electrode voltage-clamp and ion-selective microelectrodes were used to measure the Na+ current, intracellular pH (pHi), and ion activities in oocytes expressing mENaC. In oocytes expressing mENaC, removal of external Na+reversibly hyperpolarized membrane potential by 129 ± 5.3 mV in the absence of 20 mM NH4Cl but only by 100 ± 7.8 mV in its presence. Amiloride completely inhibited the changes in membrane potential. In oocytes expressing mENaC, butyrate (20 mM) caused a decrease in pHi (0.43 ± 0.07) similar to the NH4Cl-induced pHi decrease (0.47 ± 0.12). Removal of Na+ in the presence of butyrate caused hyperpolarization that was not significantly different from that in the absence of butyrate at high pHi (in the absence of NH4Cl). Removal of external Na+ resulted in an outward current of 3.7 ± 0.8 μA (at −60 mV). The magnitude of this change in current was only 2.7 ± 0.7 μA when Na+ was removed in the presence of NH4Cl. In oocytes expressing mENaC, NH4Cl also caused a decrease in whole cell conductance at negative potential and an outward current at positive potential. In the presence of amiloride, steady-state current and the change in current caused by removal of Na+ were not different from zero. These results indicate that NH4Cl inhibits Na+ transport when mENaC is expressed in oocytes. The inhibition of voltage changes is not due to intracellular acidification caused by NH4Cl. Permeability and selectivity of ENaC to NH[Formula: see text] may play a role.

Dynamics of calcium regulation of chloride currents inXenopus oocytes

Ca-activated Cl currents are widely expressed in many cell types and play diverse and important physiological roles. The Xenopus oocyte is a good model system for studying the regulation of these currents. We previously showed that inositol 1,4,5-trisphosphate (IP3) injection into Xenopus oocytes rapidly elicits a noninactivating outward Cl current ( I Cl1-S) followed several minutes later by the development of slow inward ( I Cl2) and transient outward ( I Cl1-T) Cl currents. In this paper, we investigate whether these three currents are mediated by the same or different Cl channels. Outward Cl currents were more sensitive to Ca than inward Cl currents, as shown by injection of different amounts of Ca or by Ca influx through a heterologously expressed ligand-gated Ca channel, the ionotropic glutamate receptor iGluR3. These data could be explained by two channels with different Ca affinities or one channel with a higher Ca affinity at depolarized potentials. To distinguish between these possibilities, we determined the anion selectivity of the three currents. The anion selectivity sequences for the three currents were the same (I > Br > Cl), but I Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, after consideration of possible errors, the hypothesis that a single type of Cl channel underlies the complex waveforms of the three different macroscopic Ca-activated Cl currents in Xenopus oocytes remains a viable alternative.