A cation channel in the thick ascending limb of Henle's loop of the mouse kidney: inhibition by adenine nucleotides.

1. Patch-clamp single-channel current recordings were used to study the inhibition of Ca2+-activated non-selective cation channels by internal nucleotides in patches excised from basolateral membranes of the thick ascending limb of Henle's loop of the mouse kidney. 2. The application of ATP, ADP or AMP to the cytoplasmic face of excised inside-out membrane patches reduced the open-state probability of the channels (Po) in a dose-dependent way without effect upon the unitary current amplitude. Dose-response curves gave half-maximal inhibitory concentrations of 20, 21 and 2.5 microM for ATP, ADP and AMP, respectively, while the Hill coefficient was close to one in all three cases. 3. Cyclic AMP partially inhibited channel activity (Po = 35 +/- 17% of control) only at high, unphysiological concentrations (10(-3) M) while adenosine (10(-3) M) had very little effect (Po = 83 +/- 7% of control). 4. Replacement of adenine with other purines (guanine, hypoxanthine) or pyrimidine (uridine) bases very largely reduced inhibitory activity. Cyclic GMP had no effect. 5. Non-hydrolysable analogues of ATP, AMP-PNP (10(-3) M) and ATP-gamma-S (5 x 10(-4) M), were effective inhibitors of the channel (Po = 24 +/- 7 and 9 +/- 4% of control, respectively

A Ca2-activated cation-selective channel in the basolateral membrane of the cortical thick ascending limb of Henle's loop of the mouse

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Beta-adrenergic upregulation of the Na(+)-K(+)-2Cl- cotransporter in rat parotid acinar cells.

We used the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein to monitor the recovery of the intracellular pH (pHi) of rat parotid acini from an NH4(+)-induced alkaline load. This recovery was markedly inhibited by the loop diuretic bumetanide and by Cl- removal, indicating that it is largely due to NH4+ entry via the basolateral Na(+)-K(+)-2Cl- cotransporter. The rate of recovery of pHi was enhanced threefold by pretreatment (37.5 s) with isoproterenol (K1/2 = 21.5 nM) or norepinephrine (in the presence of phentolamine), and blocked by the beta 1-specific antagonist atenolol, indicating an upregulation of cotransport activity by beta 1-adrenergic stimulation. The effect of isoproterenol was prevented by protein kinase inhibitors and mimicked by cAMP analogues, and by maneuvers known to increase cytosolic cAMP levels in these cells, consistent with the involvement of protein kinase A. Physiologically, such an upregulation of the acinar Na(+)-K(+)-2Cl- cotransporter would lead to an increase in acinar chloride uptake across the basolateral membrane, and consequently, an increase in overall chloride and fluid secretion. Prevention of this upregulation by beta-blockers and possibly by other commonly used clinical agents may account for the dry mouth and dry eyes experienced by some patients taking these medications

Extracellular ATP and UTP trigger calcium entry in mouse cortical thick ascending limbs

International audienceThe effects of extracellular nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) of mouse cortical thick ascending limb (CTAL) segments were investigated using the Ca(2+)-sensitive fluorescent probe fura 2. ATP (50% effective dose, ED50, 40 microM) transiently increased [Ca2+]i, while adenosine (a P1 purinoceptor agonist), N6-cyclohexyladenosine (an A1 agonist), AMP, ADP (a P2t agonist), beta, gamma-methyleneadenosine 5'-triphosphate (a P2x agonist), or 2-methylthioadenosine 5'-triphosphate (a P2y agonist) all had little or no effect. CTAL tubules were also sensitive to UTP. The responses to 100 microM ATP and UTP were similar but not additive. Both [Ca2+]i responses were strongly inhibited by 300 microM suramin (a P2 purinoceptor antagonist). Adenosine 5'-O-(3- thiotriphosphate) and ITP were slightly less potent than ATP, while GTP and CTP had no effect. The absence of external Ca2+ or the presence of 50 microM nifedipine similarly and markedly reduced the ATP- and UTP-evoked [Ca2+]i transients. We conclude that mouse CTAL tubules possess nucleotide receptors that are equally sensitive to ATP and UTP and that transiently elevate [Ca2+]i by triggering Ca2+ entry via a nifedipine-sensitive pathway

Ion transport mechanisms in rat parotid intralobular striated ducts

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Functional evidence for a Ca2+/polyvalent cation sensor in the mouse thick ascending limb

International audienceThe effects of extracellular polyvalent cations on the cytosolic free Ca2+ concentration ([Ca2+]i) of isolated segments of the mouse nephron were investigated using fura 2 microfluorometry. Extracellular Ca2+ concentration ([Ca2+]o), gadolinium (Gd3+), and neomycin (Neo) increased the [Ca2+]i in cortical thick ascending limb (CTAL) tubules with effective doses (ED50) of approximately 3.5 mM for Ca2+, 20 microM for Gd3+, and 40 microM for Neo. This effect was reproduced by Ba2+ but not by Mg2+. High [Ca2+]o inhibited the responses to Gd3+, Neo, and Ba2+. The Gd(3+)- and Neo-evoked [Ca2+]i transients persisted in the absence of external Ca2+ and were abolished by the depletion of internal Ca2+ stores with thapsigargin (TG). The responses to rises in [Ca2+]o were similarly inhibited by TG and slightly reduced by 20 microM La3+ but not by 10 microM nifedipine. Mn2+ also mobilized a TG-sensitive internal Ca2+ store and stimulated its own entry. External Ca2+, Gd3+, and Neo induced small but significant increases in [Ca2+]i in distal convoluted tubule, cortical collecting duct, and outer medullary collecting duct segments, transiently increased [Ca2+]i in some medullary TAL (MTAL) tubules, but had no effect on descending thin limb. We conclude that a Ca(2+)-mobilizing Ca2+/polyvalent cation sensor resembling that of the parathyroid gland cells is predominantly located in the mouse CTAL but also in the MTAL and, to a lesser extent, in more distal segments

Inhibition of a small-conductance cAMP-dependent Cl- channel in the mouse thick ascending limb at low internal pH.

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Microfluorometric studies of intracellular Ca2+ and Na+ concentrations in normal human labial gland acini

International audienceThe responses of human labial salivary acini to muscarinic, adrenergic, and substance P peptidergic stimulation were studied using the fluorescent indicators fura 2 for intracellular Ca2+ concentration ([Ca2+]i) and sodium-binding benzofuran isophthalate for intracellular Na+ concentration ([Na+]i). Of the agents tested (carbachol, epinephrine, isoproterenol, and substance P) only the muscarinic agonist carbachol increased [Ca2+]i substantially above basal levels (three to fourfold; half-maximal effect approximatley 1 microM). Experiments with the Ca(2+)-adenosinetriphosphatase inhibitor thapsigargin indicated the presence of both thapsigargin-sensitive and thapsigargin-insensitive intracellular Ca2+ stores, both of which were mobilized by carbachol. [Na+]i in resting labial acini was approximately 20 mM. On stimulation with carbachol, [Na+]i rose transiently to more than three times this value and then partially recovered. This carbachol-induced rise in [Na+]i was largely blocked by bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransporter. These results are consistent with an intact muscarinic fluid secretory response in human labial acini with transepithelial Cl- secretion driven via Na(+)-K(+)-2Cl- cotransport and the secretion of fluid presumably following Cl- loss via an apical Ca(2+)-dependent anion channel, as observed in salivary acini from other species