Activation of TRPV1 channels leads to stimulation of NKCC1 cotransport in the lens.

Lens ion homeostasis is crucial in maintaining water content and, in turn, refractive index and transparency of the multicellular syncytium-like structure. New information is emerging on the regulation of ion transport in the lens by mechanisms that rely on transient receptor potential vanilloid (TRPV) ion channels. We found recently that TRPV1 activation leads to Ca2+/PKC-dependent ERK1/2 signaling. Here, we show that the TRPV1 agonist capsaicin (100 nM) and hyperosmotic solution (350 vs. 300 mosM) each caused an increase of bumetanide-inhibitable Rb uptake by intact porcine lenses and Na-K-2Cl cotransporter 1 (NKCC1) phosphorylation in the lens epithelium. The TRPV1 antagonist A889425 (1 µM) abolished the increases of Rb uptake and NKCC1 phosphorylation in response to hyperosmotic solution. Exposing lenses to hyperosmotic solution in the presence of MEK/ERK inhibitor U0126 (10 µM) or the with-no-lysine kinase (WNK) inhibitor WNK463 (1 µM) also prevented NKCC1 phosphorylation and the Rb uptake responses to hyperosmotic solution. WNK463 did not prevent the increase in ERK1/2 phosphorylation that occurs in response to capsaicin or hyperosmotic solution, suggesting that ERK1/2 activation occurs before WNK activation in the sequence of signaling events. Taken together, the evidence indicates that activation of TRPV1 is a critical early step in a signaling mechanism that responds to a hyperosmotic stimulus, possibly lens shrinkage. By activating ERK1/2 and WNK, TRPV1 activation leads to NKCC1 phosphorylation and stimulation of NKCC1-mediated ion transport.

Dopamine-induced graded intracellular Ca2+ elevation via the Na+Ca2+ exchanger operating in the Ca2+-entry mode in cockroach salivary ducts.

Stimulation with the neurotransmitter dopamine causes an amplitude-modulated increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) in epithelial cells of the ducts of cockroach salivary glands. This is completely attributable to a Ca(2+) influx from the extracellular space. Additionally, dopamine induces a massive [Na(+)](i) elevation via the Na(+)K(+)2Cl(-) cotransporter (NKCC). We have reasoned that Ca(2+)-entry is mediated by the Na(+)Ca(2+) exchanger (NCE) operating in the Ca(2+)-entry mode. To test this hypothesis, [Ca(2+)](i) and [Na(+)](i) were measured by using the fluorescent dyes Fura-2, Fluo-3, and SBFI. Inhibition of Na(+)-entry from the extracellular space by removal of extracellular Na(+) or inhibition of the NKCC by 10 microM bumetanide did not influence resting [Ca(2+)](i) but completely abolished the dopamine-induced [Ca(2+)](i) elevation. Simultaneous recordings of [Ca(2+)](i) and [Na(+)](i) revealed that the dopamine-induced [Na(+)](i) elevation preceded the [Ca(2+)](i) elevation. During dopamine stimulation, the generation of an outward Na(+) concentration gradient by removal of extracellular Na(+) boosted the [Ca(2+)](i) elevation. Furthermore, prolonging the dopamine-induced [Na(+)](i) rise by blocking the Na(+)/K(+)-ATPase reduced the recovery from [Ca(2+)](i) elevation. These results indicate that dopamine induces a massive NKCC-mediated elevation in [Na(+)](i), which reverses the NCE activity into the reverse mode causing a graded [Ca(2+)](i) elevation in the duct cells.

Two distinct types of SH-groups are necessary for bumetanide and bile acid uptake into isolated rat hepatocytes.

Substances that block SH-groups were studied in respect to their effects on the uptake of the loop diuretic bumetanide and the bile acids cholate and taurocholate into isolated rat hepatocytes. SH-blockers, e.g., p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), dithiobis-nitropyridine (DTNP) and dithiobis-2-nitrobenzoic acid (DTNB) reduced bumetanide transport in a concentration-dependent manner. Inhibition of the organic mercurial PCMBS was reversed by the addition of 500 microM dithiothreitol (DTT), indicating an interaction of this substance with free SH-groups. NEM irreversibly blocked SH-groups by covalent binding and was the most effective inhibitor of bumetanide and cholate uptake. In contrast, PCMBS was the most effective inhibitor of taurocholate uptake. Photoaffinity studies with [3H]bumetanide and [3H]7,7-azotaurocholate were performed with isolated rat hepatocytes in the presence of PCMBS and DTNP. Binding of the photolabels was not reduced by SH-group blockers. Newly synthesized sulfhydryl-modifying reagents such as dithio-sulfonate-ethyl-nitrobenzoic acid (DTSNB) and dithio-octyl-nitrobenzoic acid (DTONB), are derivatives of the alkylating agent DTNB. DTSNB is regarded as a selective blocker for SH-groups in a hydrophilic environment, while DTONB is more lipophilic abd interacts with SH-groups in the transmembrane domain of transport proteins. The IC50-values of these blockers for bumetanide uptake (DTSNB 250 microM, DTONB 141 microM) and for cholate uptake (DTSNB 250 microM, DTONB 115 microM) were almost identical. These findings support the concept of a common uptake mechanism for cholate and bumetanide and indicate that two distinct moieties of SH-groups are required for the uptake of both organic anions. One of these is probably located on the outer surface and the other within the membrane of hepatocytes.

Indomethacin and the response to bumetanide.

Pretreatment of 8 normal subjects with 100 mg indomethacin decreased the response to bumetanide; cumulative 4-hr excretion of sodium due to 1.0 mg of bumetanide was reduced from 276 +/- 22.9 to 202 +/- 20.9 mEq (p less than 0.003). Effects on volume and Cl paralleled those on Na while K excretion was not affected. When response was analyzed as increment in fractional excretion over basal solute excretion determined from separate control studies, indomethacin decreased response, an effect differing from that of indomethacin on the response to furosemide. The importance and mechanism of the difference in the effect of indomethacin on bumetanide and furosemide is not known.

Sulindac inhibits bumetanide-induced sodium and water excretion.

In an open-label pilot study, we investigated the effect of sulindac on bumetanide-induced diuresis. Nine healthy volunteers were placed on diets with a standard sodium and potassium content. Each volunteer received 1 mg bumetanide orally, and blood and urine samples were collected during an 8-hour period. Urinary losses were replaced isovolumetrically with intravenous normal saline solution. Creatinine clearance and sodium and potassium excretion were compared with and without sulindac pretreatment (200 mg administered orally b.i.d. for 5 days). Sulindac pretreatment resulted in a 22% decrease in the mean cumulative sodium excretion after 3 hours (p less than 0.05) and a 21% decrease in the mean urine flow rate after 2 hours (p less than 0.05). The results suggest that sulindac has an effect on the kidney similar to that of other nonsteroidal agents.

Bumetanide-induced diuresis and natriuresis: effect of prostaglandin synthetase inhibition.

The effects on the renal actions of bumetanide of two inhibitors of prostaglandin synthetase, acetylsalicylic acid (ASA) and indomethacin, were studied in eight normal adults. Indomethacin alone resulted in a significant decrease in FENa compared to the control period. Bumetanide alone resulted in increases in urine volume, FENa, and plasma renin activity. Both ASA and indomethacin blunted the increases in urine volume and sodium excretion, although the inhibition by indomethacin was greater than that of ASA. In addition, indomethacin completely inhibited the bumetanide-induced increase in plasma renin activity. No consistent relationship between these effects and urinary prostaglandin E2 or F excretion was noted. These studies indicate that prostaglandin synthetase inhibitors may interfere with the effects of bumetanide.

Effect of probenecid on the natriuresis and renin release induced by bumetanide in man.

In a randomized crossover trial in six normal male subjects, the effect of pretreatment with probenecid on natriuresis and renin release in response to bumetanide was studied. The subjects received 120 mEq sodium and 80 mEq potassium per day. A single dose of 2 mg bumetanide was administered on the fourth morning after pretreatment with either placebo or probenecid. Creatinine and uric acid were measured in serum and urine, plasma renin activity was determined by radioimmunoassay of angiotensin I, and plasma and urine concentrations of bumetanide were measured by a highly sensitive radioimmunoassay method. Probenecid reduced both natriuresis and hyperreninemia induced by bumetanide. This effect is postulated to be due not to a direct action on sodium excretion but is probably secondary to inhibition of renal tubular secretion of bumetanide. Consequently, these findings appear to support the concept that the quantity of bumetanide delivered to the tubular lumen is an important determinant of its diuretic effect.

The anion specificity of the sodium-potassium-chloride cotransporter in rabbit kidney outer medulla: studies on medullary plasma membranes.

Plasma membrane vesicles were isolated from rabbit kidney outer medulla and employed in sodium, rubidium, and chloride flux studies. Chloride dependence and bumetanide sensitivity of (part of) the sodium and rubidium flux indicate that this plasma membrane fraction can be used to study the properties of Na-K-2Cl cotransport system present in the luminal membrane of the medullary thick ascending limb. The anion specificity of the cotransporter was investigated by determining the effect of anion replacement on sodium fluxes. When chloride was completely replaced by bromide, iodide, nitrate, or thiocyanate only bromide could effectively substitute for chloride (90% activity), whereas sodium uptake in the presence of iodide, nitrate, and thiocyanate amounted to only 25% of the sodium uptake observed in the presence of chloride. When similar replacement experiments were performed in the presence of 10 mmol/l chloride, bromide could substitute for chloride by 110%, iodide and nitrate by 60%, and thiocyanate by 70%. In the presence of 10 mmol/l bromide iodide, nitrate, and thiocyanate were similarly effective. The effect of nitrate and chloride on sodium flux was additive. Bumetanide-sensitive chloride uptake was inhibited by nitrate, the inhibition was however only partly, amounting to 60%. The results obtained are compatible with the view that the two anion binding sites of the Na-K-2Cl cotransporter can exhibit a different substrate specificity and that the transporter in addition to a 2Cl mode can also operate in a 2Br, Cl-, A- and Br-, A- mode, A- representing iodide, nitrate, or thiocyanate.