Desilets, Michel,Fadil, Ayman Y.2009-03-232009-03-2319941994Source: Masters Abstracts International, Volume: 33-05, page: 1450.9780315959170http://hdl.handle.net/10393/6942http://dx.doi.org/10.20381/ruor-11537In order to study $\beta$-adrenergic regulation of Na$\sp+$ transport in heart, a method was developed to directly measure intracellular free-Na$\sp+$ concentration ($\rm\lbrack Na\sp+\rbrack\sb{i}$) in ventricular myocytes freshly isolated from adult rabbit heart. The approach consisted of injecting the Na$\sp+$-sensitive fluorescent indicator SBFI by iontophoresis, and the simultaneous measurement of transmembrane ion current through the use of a single electrode voltage-clamp technique. Using this approach, steady-state $\rm\lbrack Na\sp+\rbrack\sb{i}$ was found to be 6.4 $\pm$ 1.8 mM in myocytes superfused at 37$\sp\circ$C with modified Tyrode's solution containing 2 mM Ca$\sp{2+}$. Decreasing Ca$\sp{2+}$ concentration to 0.8 mM caused an increase of $\rm\lbrack Na\sp+\rbrack\sb{i}$ to 13.0 $\pm$ 1.4 mM. The $\beta$-adrenergic agonist isoproterenol (ISO), when applied at a concentration of 0.5 $\mu$M, failed to cause any detectable change of steady-state $\rm\lbrack Na\sp+\rbrack\sb{i}$ or transmembrane current. To test whether this lack of effect also occurred at higher $\rm\lbrack Na\sp+\rbrack\sb{i}$, cells were exposed to the cation-selective ionophore gramicidin (10 nM), which induced a slow but continuous increase in $\rm\lbrack Na\sp+\rbrack\sb{i}.$ Under those conditions, ISO caused a decrease or even reversed the gramicidin-induced $\rm\lbrack Na\sp+\rbrack\sb{i}$ rise, while inducing an outward shift of the transmembrane current. These relative changes induced by ISO were not significantly affected by the removal of extracellular Ca$\sp{2+}$; $-$8.0 $\pm$ 2.5 mM/min for the ISO-induced changes in $\rm\lbrack Na\sp+\rbrack\sb{i}$ rate of rise, and 58 $\pm$ 17 pA for the transmembrane current. Under those conditions, strophanthidin (100 $\mu$M) completely inhibited the effects of ISO. Washout of intracellular Ca$\sp{2+}$ by prolonged preincubation of the myocytes in solutions containing 0 mM Ca$\sp{2+}$, 0.5 mM EGTA and 25 $\mu$M BAPTA-AM caused an important attenuation of the effects of ISO. (Abstract shortened by UMI.)187 p.Biology, Cell.Thesis