RESUMEN
Adenosine (Ado), a smooth muscle vasodilator and modulator of cardiac function, is taken up by many cell types via a saturable transporter, blockable by dipyridamole. To quantitate the influences of endothelial cells in governing the blood-tissue exchange of Ado and its concentration in the interstitial fluid, one must define the permeability-surface area products (PS) for Ado via passive transport through interendothelial gaps [PS(g)(Ado)] and across the endothelial cell luminal membrane (PS(ecl)) in their normal in vivo setting. With the use of the multiple-indicator dilution (MID) technique in Krebs-Ringer perfused, isolated guinea pig hearts (preserving endothelial myocyte geometry) and by separating Ado metabolites by HPLC, we found permeability-surface area products for an extracellular solute, sucrose, via passive transport through interendothelial gaps [PS(g)(Suc)] to be 1.9 +/- 0.6 ml. g(-1). min(-1) (n = 16 MID curves in 4 hearts) and took PS(g)(Ado) to be 1. 2 times PS(g)(Suc). MID curves were obtained with background nontracer Ado concentrations up to 800 micrometer, partially saturating the transporter and reducing its effective PS(ecl) for Ado. The estimated maximum value for PS(ecl) in the absence of background adenosine was 1.1 +/- 0.1 ml. g(-1). min(-1) [maximum rate of transporter conformational change to move the substrate from one side of the membrane to the other (maximal velocity; V(max)) times surface area of 125 +/- 11 nmol. g(-1). min(-1)], and the Michaelis-Menten constant (K(m)) was 114 +/- 12 microM, where +/- indicates 95% confidence limits. Physiologically, only high Ado release with hypoxia or ischemia will partially saturate the transporter.
Asunto(s)
Adenosina/metabolismo , Proteínas Portadoras/metabolismo , Vasos Coronarios/metabolismo , Endotelio Vascular/metabolismo , Animales , Espacio Extracelular/metabolismo , Glucosa/metabolismo , Cobayas , Técnicas In Vitro , Técnicas de Dilución del Indicador , Cinética , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Nucleósidos , PerfusiónRESUMEN
The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three beta-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label beta counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed.