Effects of Na-K-2Cl cotransport regulators on outflow facility in calf and human eyes in vitro.
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PURPOSE: Cultured human trabecular meshwork (TM) cells possess substantial Na-K-Cl activity, which is involved in the regulation of TM cell volume. The hypothesis in the present study was that drugs that affect the cotransporter might alter aqueous humor outflow facility (C) in the intact eye. The effects of agents and conditions known to modulate Na-K-CI cotransport activity and/or TM cell volume on C in perfused anterior segments were investigated. METHODS: Human and calf eyes were dissected and perfused, and C was determined according to standard published methods. Perfusates with modified osmolarity were used to cause alterations in TM cell volume. Cl-free perfusate and/or bumetanide (10(-5) M) was used to inhibit Na-K-Cl cotransport activity, and vasopressin (10(-7) M, 10(-8) M) was used to stimulate cotransport activity. RESULTS: In human eyes, hypo-osmotic perfusate decreased C 12%, whereas hyper-osmotic perfusate increased C 44%. These changes lasted approximately 30 minutes, after which C began to normalize. Inhibition of Na-K-Cl cotransport using Cl-free medium or bumetanide resulted in facility increases of 27% and 22%, respectively. There was an additive increase in C with bumetanide plus Cl-free media. Stimulating Na-K-Cl cotransport with 10(-8)M and 10(-7)M vasopressin resulted in 28% and 35% decreases in C, respectively. The results were similar in calf eyes: Cl-free medium or bumetanide resulted in 41% and 52% increases in C, whereas 10(-8) M and 10(-7) M vasopressin resulted in 14% and 19% decreases in C, respectively. CONCLUSIONS: Modulation of Na-K-Cl cotransport results in changes in C that may be mediated in part by cell volume changes. (+info)
Stimulation of Na+-K+-2Cl- cotransport by arsenite in ferret erythrocytes.
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1. Na+-K+-2Cl- cotransport activity was measured in ferret erythrocytes as the bumetanide-sensitive uptake of 86Rb. 2. The Na+-K+-2Cl- cotransport rate was stimulated by treating erythrocytes with sodium arsenite but not by sodium arsenate (up to 1 mM). Stimulation took an hour to develop fully. Arsenite had no effect on bumetanide-resistant 86Rb uptake. 3. In cells stored for 3 days or less, cotransport stimulation by arsenite could be described by assuming arsenite either acts at a single site (EC50, 60+/-14 microM, mean +/- S.E.M., n = 3) or that it acts at both high- (EC50, 35+/-9 microM, mean +/- S.E.M., n = 3) and low- (EC50 >2 mM) affinity sites. 4. Stimulation by 1 mM arsenite was greatest on the day of cell collection (rate about 3 times that of the control), even exceeding that produced by 20 nM calyculin A, and declined during cell storage. Addition of calyculin A to arsenite-stimulated cells resulted in further stimulation of Na+-K+-2Cl- cotransport, suggesting that arsenite and calyculin act synergistically. This was most apparent in stored cells. 5. Stimulation by 1 mM arsenite was not affected by treating cells with the mitogen-activated protein kinase inhibitors SB203580 (20 microM) and PD98059 (50 microM), but was both prevented and reversed by the kinase inhibitors staurosporine (2 microM), 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1, 50 microM) and genistein (0.3 mM), and with a combination of 10 microM A23187 and 2 mM EDTA (to reduce intracellular Mg2+ concentration). Only treatment with EDTA and A23187 prevented stimulation by the combination of 1 mM arsenite and 20 nM calyculin, whereas no treatment was able to fully reverse this stimulation once elicited. 6. Our data are consistent with arsenite stimulating (perhaps indirectly) a kinase that phosphorylates and activates the Na+-K+-2Cl- cotransporter. (+info)
Basolateral K+ channel involvement in forskolin-activated chloride secretion in human colon.
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1. In this study we investigated the role of basolateral potassium transport in maintaining cAMP-activated chloride secretion in human colonic epithelium. 2. Ion transport was quantified in isolated human colonic epithelium using the short-circuit current technique. Basolateral potassium transport was studied using nystatin permeabilization. Intracellular calcium measurements were obtained from isolated human colonic crypts using fura-2 spectrofluorescence imaging. 3. In intact isolated colonic strips, forskolin and prostaglandin E2 (PGE2) activated an inward transmembrane current (ISC) consistent with anion secretion (for forskolin DeltaISC = 63.8+/-6.2 microA cm(-2), n = 6; for PGE2 DeltaISC = 34.3+/-5.2 microA cm(-2), n = 6). This current was inhibited in chloride-free Krebs solution or by inhibiting basolateral chloride uptake with bumetanide and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid DIDS). 4. The forskolin- and PGE2-induced chloride secretion was inhibited by basolateral exposure to barium (5 mM), tetrapentylammonium (10 microM) and tetraethylammonium (10 mM). 5. The transepithelial current produced under an apical to serosal K+ gradient in nystatin-perforated colon is generated at the basolateral membrane by K+ transport. Forskolin failed to activate this current under conditions of high or low calcium and failed to increase the levels of intracellular calcium in isolated crypts 6. In conclusion, we propose that potassium recycling through basolateral K+ channels is essential for cAMP-activated chloride secretion. (+info)
beta-adrenergic agonists stimulate Na+-K+-Cl- cotransport by inducing intracellular Ca2+ liberation in crypt cells.
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Epinephrine and beta-adrenergic agonists (beta1 and beta2 for isoproterenol, beta1 for dobutamine, beta2 for salbutamol) stimulated K+ (or 86Rb) influx mediated by the Na+-K+-2Cl- cotransporter and the Na+-K+ pump in isolated colonic crypt cells. Preincubation with bumetanide abolished the epinephrine effect on the Na+-K+ pump, suggesting that the primary effect is on the cotransporter. Maximal effect was obtained with 1 microM epinephrine with an EC50 of 91.6 +/- 9.98 nM. Epinephrine-induced K+ transport was blocked by propranolol with an IC50 of 134 +/- 28.2 nM. alpha-Adrenergic drugs did not modify K+ transport mechanisms. Neither Ba2+ nor tetraethylammonium nor DIDS modified the adrenergic enhancement on the cotransporter. In addition, epinephrine did not affect K+ efflux. Dibutyryl cAMP did not alter K+ transport. Reduction of extracellular Ca2+ to 30 nM did not influence the response to epinephrine. However, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished epinephrine-induced K+ transport. Ionomycin increased Na+-K+-2Cl- cotransport activity. Moreover, epinephrine increased intracellular Ca2+ concentration in a process inhibited by propranolol. In conclusion, epinephrine stimulated the Na+-K+-2Cl- cotransporter in a process mediated by beta1- and beta2-receptors and modulated by intracellular Ca2+ liberation. (+info)
Epithelial P2X purinergic receptor channel expression and function.
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P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca(2+) influx--2 signals that stimulate secretory Cl(-) transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl(-) transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl(-) transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF. (+info)
Role of the vasopressin V(1) receptor in regulating the epithelial functions of the guinea pig distal colon.
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Vasopressin has a wide spectrum of biological action. In this study, the role of vasopressin in regulating electrolyte transport in the colon was elucidated by measuring the short-circuit current (I(sc)) as well as the Na(+), K(+), and Cl(-) flux in a chamber-mounted mucosal sheet. The cytosolic Ca(2+) concentration ([Ca(2+)](i)) was also measured in fura 2-loaded cells by fluorescence imaging. Serosal vasopressin decreased I(sc) at 10(-9) M and increased I(sc) at 10(-7)-10(-6) M. The decrease in I(sc) was accompanied by two effects: one was a decrease in the amiloride-sensitive Na(+) absorption, whereas the other was an increase in the bumetanide-sensitive K(+) secretion. The increase in I(sc) was accompanied by an increase in the Cl(-) secretion that can be inhibited by serosal bumetanide or mucosal diphenylamine-2-carboxylate. Vasopressin caused an increase in [Ca(2+)](i) in crypt cells. These responses of I(sc) and the [Ca(2+)](i) increase in crypt cells were all more potently inhibited by the vasopressin V(1) receptor antagonist than by the V(2) receptor antagonist. These results suggest that vasopressin inhibits electrogenic Na(+) absorption and stimulates electrogenic K(+) and Cl(-) secretion. In all of these responses, the V(1) receptor is involved, and the [Ca(2+)](i) increase may play an important role. (+info)
Characterization of a phosphorylation event resulting in upregulation of the salivary Na(+)-K(+)-2Cl(-) cotransporter.
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Previous studies from our laboratory have shown a close correlation between increased Na(+)-K(+)-2Cl(-) cotransporter activity and increased cotransporter phosphorylation after beta-adrenergic stimulation of rat parotid acinar cells. We demonstrate here that these effects are paralleled by an increase in the number of high-affinity binding sites for the cotransporter inhibitor bumetanide in membranes prepared from stimulated acini. We also show that the sensitivity of cotransporter fluxes to inhibition by bumetanide is the same in both resting and isoproterenol-stimulated cells, consistent with the hypothesis that beta-adrenergic stimulation and the accompanying phosphorylation result in the activation of previously quiescent transporters rather than in a change in the properties of already active proteins. In addition, we demonstrate that the increased phosphorylation on the cotransporter resulting from beta-adrenergic stimulation is localized to a 30-kDa phosphopeptide obtained by cyanogen bromide digestion. Immunoprecipitation and Western blotting experiments demonstrate that this peptide is derived from the NH(2)-terminal cytosolic tail of the cotransporter, which surprisingly does not contain the sole protein kinase A consensus site on the molecule. (+info)
Electrophysiologic characterization of an organic anion transporter cloned from winter flounder kidney (fROAT).
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The two-electrode voltage clamp technique was used to demonstrate translocation of p-aminohippurate (PAH) and related compounds such as loop diuretics in Xenopus laevis oocytes expressing the renal organic anion transporter from winter flounder kidney (fROAT). In fROAT-expressing oocytes, PAH (0.1 mM) induced a depolarization of 4.2 +/- 0.4 mV and at a holding potential of -60 mV an inward current of -22.6 +/- 3.5 nA. PAH-induced current and the current calculated from [3H]-PAH uptake were of similar magnitude. Depolarization, inward current, and current-to-uptake relation indicated exchange of the monovalent PAH with a divalent anion, possibly alpha-ketoglutarate (alpha-KG), causing net efflux of one negative charge. The kinetic analysis of PAH-induced currents revealed that translocation is dependent on membrane potential, saturable with an apparent Km of 58 +/- 8 microM, and sensitive to probenecid and furosemide. In contrast to probenecid and furosemide, the loop diuretics bumetanide, ethacrynic acid, and tienilic acid and the nephrotoxic mycotoxin ochratoxin A elicited inward currents indicating translocation through fROAT. Substrate-dependent currents provide a tool to elucidate the structure/function relationship of the renal organic anion transporter. (+info)