Development and application of a simple microassay for adenosine in rat plasma

Abstract

Adenosine may be a physiological modulator of vascular smooth muscle tone, sympathetic neurotransmission, renin release, and renal and cardiac function. To facilitate the elucidation of the physiological role of adenosine, a microassay for adenosine was developed that allows accurate quantitation of adenosine in 75 μl of rat plasma, thus permitting multiple determinations of plasma adenosine levels in an individual rat without inducing hemodynamic perturbations due to blood loss. The technique employs a simple and rapid extraction of plasma with a reverse-phase Sep-Pak cartridge and exploits the increased mass sensitivity of microbore high performance liquid chromatography. The assay was verified by demonstrating 1) a linear relationship between the amount of adenosine added to plasma and the amount detected by the assay, 2) a linear relationship between the rate of adenosine infusion into rats and plasma adenosine levels, and 3) the absence of measurable adenosine levels in plasma incubated with adenosine deaminase. The mean arterial plasma level of adenosine in the anesthetized rat was determined to be 119 ± 28 (SD) ng/ml (n = 10). With the use of this assay, renal venous plasma levels of adenosine were found to be elevated sixfold in two-kidney, one clip Goldblatt hypertensive rats (1 week postclipping) compared with sham-operated controls. Given the known effects of adenosine on renin release, these data support a role for endogenous adenosine as a regulator of renin release in renovascular hypertension.