Abstract-NO plays a role in the regulation of blood pressure through its effects on renal, cardiovascular, and central nervous system function. It is generally thought to freely diffuse through cell membranes without need for a specific transporter. The water channel aquaporin-1 transports low molecular weight gases in addition to water and is expressed in cells that produce or are the targets of NO. Consequently, we tested the hypothesis that aquaporin-1 transports NO.In cells expressing aquaporin-1, NO permeability correlated with water permeability. NO transport was reduced by 71% by HgCl 2 , an inhibitor of aquaporin-1. Transport of NO by aquaporin-1 saturated at 3 mol/L NO and displayed a K 1/2 (the concentration of NO that produces half of the maximum transport rate) of 0.54 mol/L. Reconstitution of purified aquaporin-1 into lipid vesicles increased NO influx by 316%. In endothelial cells, lowering aquaporin-1 expression with a small interfering RNA (siRNA) blunted aquaporin-1 expression by 54% and NO release by 44%. We conclude that NO transport by aquaporin-1 may allow cells to control intracellular NO levels and effects. NO transport by aquaporin-1 may play a role in central nervous system, vascular and renal function, and consequently blood pressure. Disruption of NO transport by aquaporin-1 offers an alternate cause for diseases currently explained by inadequate NO bioavailability.

We have reported that nitric oxide (NO) inhibits thick ascending limb (THAL) chloride absorption (J(Cl(-))). NaCl transport in the THAL depends on apical Na(+)-K(+)-2Cl(-) cotransporters, apical K(+) channels, and basolateral Na(+)-K(+)-ATPase. However, the transporter inhibited by NO is unknown. We hypothesized that NO decreases THAL J(Cl(-)) by inhibiting the Na(+)-K(+)-2Cl(-) cotransporter. THALs from Sprague-Dawley rats were isolated and perfused. Intracellular sodium ([Na(+)](i)) and chloride concentrations ([Cl(-)](i)) were measured with sodium green and SPQ, respectively. The NO donor spermine NONOate (SPM) decreased [Na(+)](i) from 13.5 +/- 1.2 to 9.6 +/- 1.6 mM (P < 0.05) and also decreased [Cl(-)](i) (P < 0.01). We next tested whether NO decreases Na(+)-K(+)-2Cl(-) cotransporter activity by measuring the initial rate of Na(+) transport. In the presence of SPM in the bath, initial rates of Na(+) entry were 49.6 +/- 6.0% slower compared with control rates (P < 0.05). To determine whether NO inhibits apical K(+) channel activity, we measured the change in membrane potential caused by an increase in luminal K(+) from 1 to 25 mM using a potential-sensitive fluorescent dye. In the presence of SPM, increasing luminal K(+) concentration depolarized THALs to the same extent as it did in control tubules. We then tested whether a change in apical K(+) permeability could affect NO-induced inhibition of THAL J(Cl(-)). In the presence of luminal valinomycin, which increases K(+) permeability, addition of SPM decreased THAL J(Cl(-)) by 41.2 +/- 10.4%, not significantly different from the inhibition observed in control tubules. We finally tested whether NO alters the affinity or maximal rate of Na(+)-K(+)-ATPase by measuring oxygen consumption rate (QO(2)) in THAL suspensions in the presence of nystatin in varying concentrations of Na(+). In the presence of 10.5 mM Na(+), nystatin increased QO(2) to 119.1 +/- 19.2 and 125.6 +/- 23.4 nmol O(2). mg protein(-1). min(-1) in SPM- and furosemide-treated tubules, respectively. In the presence of 145 mM extracellular Na(+), nystatin increased QO(2) by 104 +/- 7 and 94 +/- 20% in NO- and furosemide-treated tubules, respectively. We concluded that NO decreases THAL J(Cl(-)) by inhibiting Na(+)-K(+)-2Cl(-) cotransport rather than inhibiting apical K(+) channels or the sodium pump.

Abstract-The proximal nephron reabsorbs 60% to 70% of the fluid and sodium and most of the filtered bicarbonate via Na/H exchanger 3. Enhanced proximal nephron transport is implicated in hypertension. Our findings show that a fructose-enriched diet causes salt sensitivity. We hypothesized that fructose stimulates luminal Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. Na/H exchange was measured in rat proximal tubules as the rate of intracellular pH (pH i ) recovery in fluorescent units/s. Replacing 5 mmol/L glucose with 5 mmol/L fructose increased the rate of pH i recovery (1.8±0.6 fluorescent units/s; P<0.02; n=8). Staurosporine, a protein kinase C inhibitor, blocked this effect. We studied whether this effect was because of the addition of fructose or removal of glucose. The basal rate of pH i recovery was first tested in the presence of a 0.6-mmol/L glucose and 1, 3, or 5 mmol/L fructose added in a second period. The rate of pH i recovery did not change with 1 mmol/L but it increased with 3 and 5 mmol/L of fructose. Adding 5 mmol/L glucose caused no change. Removal of luminal sodium blocked pH i recovery. With 5.5 mmol/L glucose, angiotensin II (1 pmol/L) did not affect the rate of pH i recovery (change, -1.1±0.5 fluorescent units/s; n=9) but it increased the rate of pH i recovery with 0.6 mmol/L glucose/5 mmol/L fructose (change, 4.0±2.2 fluorescent units/s; P<0.02; n=6). We conclude that fructose stimulates Na/H exchange activity and sensitizes the proximal tubule to angiotensin II. This mechanism is likely dependent on protein kinase C. These results may partially explain the mechanism by which a fructose diet induces