sq-23377 and sodium-binding-benzofuran-isophthalate

The relationship between cytosolic concentrations of Ca2+ (Ca2i) and Na+ (Na+i) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca(2+)-sensitive dye Fura-2 or the Na(+)-sensitive dye SBFI. Pancreatic acini showed no changes in Na+i during either transient or persistent changes in Ca2+i. Increases in Ca2+i produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na+i after a delay of 5-10 s. When Ca2+ stores were mobilized without Ca2+ influx Na+i also increased, but in acini loaded with BAPTA, a nonfluorescent Ca2+ chelator, the transient increase in Ca2+ caused by mobilization of stored Ca2+ was virtually abolished, as was the increase in Na+i. In the presence of inomycin, increases in Ca2+i were followed by increases in Na+i. Ca(2+)-dependent increases in Na+i were abolished in Na(+)-free buffer and by the presence of furosemide, a blocker of Na(+)-K(+)-2Cl- cotransport. In other studies, extracellular ATP (ATPo) produced an increase in Ca2+i and Na+i. The steady-state increase in Ca(i)2+ was reduced by increasing extracellular Na+ concentrations (Na+o in dose-dependent fashion (IC50 = 16.4 +/- 4.7 mM Na+). Likewise, increasing Na+o reduced ATPo-stimulated 45Ca2+ uptake at steady state (IC50 = 15.8 +/- 9.2 mM Na+). Changing Na+o had no effect on carbachol-stimulated increases in Ca2+i. We conclude that, in rat submandibular gland acini, ATPo promotes an increase in Ca2+i and Na+i via a common influx pathway and that, under physiologic conditions, Na+ significantly limits the ATPo-stimulated increase in Ca2+i. In the presence of carbachol, however, Na+i rises in Ca2+i-dependent fashion in submandibular gland acini via stimulation of Na(+)-K(+)-2Cl- cotransport.

Topics: Adenosine Triphosphate; Animals; Atropine; Benzofurans; Calcium; Carbachol; Chelating Agents; Cytoplasm; Egtazic Acid; Ethers, Cyclic; Fluorescent Dyes; Fura-2; Furosemide; Ionomycin; Kinetics; Male; Pancreas; Rats; Rats, Sprague-Dawley; Sodium; Submandibular Gland

The new fluorescent Na+ indicator sodium-binding benzofuran isophthalate (SBFI) was used for determination of the cytosolic free Na+ concentration, [Na+]i, in human platelets. The dye could be loaded into platelets in the form of its acetoxymethyl ester (SBFI-AM). Calibration of the fluorescence in terms of [Na+]i was done by measuring the 345/385 nm excitation ratio (emission 490 nm) at various extracellular Na+ concentrations, [Na+]o, in the presence of gramicidin D. The 345/385 intensity ratio increased almost linearly when [Na+]i was stepwise raised from 20 to 60 mM. The basal value for [Na+]i was found to be 26.0 +/- 4.5 mM (n = 15). Incubation of platelets in Na(+)-free buffer decreased [Na+]i, whereas inhibition of the (Na+ + K+)-ATPase by 0.5 mM ouabain increased [Na+]i to 56 +/- 4 mM (n = 4) within 60 min. Activation of Na+/H+ exchange by exposing platelets to propionic acid also raised [Na+]i, and a comparable effect was produced by the Na+/H+ ionophore monensin. Activation of platelets with thrombin (0.1-0.5 unit/ml) also increased the 345/385 nm intensity ratio, an effect that was not seen in Na(+)-free buffer or after raising intracellular cAMP by treatment of platelets with prostaglandin E1. On the average, [Na+]i was raised to 59.5 +/- 5.3 mM (n = 15) at 10 min after addition of thrombin without a significant decrease for further 10 min. An increase in [Na+]i was also seen when platelets were challenged with the Ca2+ ionophore ionomycin, an effect that did not occur in the absence of Na+o. Our findings confirm earlier reports which demonstrated a rise in [Na+]i in stimulated platelets and show that SBFI is a useful tool for determination of [Na+]i in resting and stimulated platelets.

Topics: Alprostadil; Benzofurans; Blood Platelets; Carrier Proteins; Cyclic AMP; Cytosol; Ethers, Cyclic; Fluorescent Dyes; Humans; Ionomycin; Monensin; Ouabain; Platelet Activation; Propionates; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Thrombin