Simultaneous Determination of Nerve-Induced Adenine Nucleotides and Nucleosides Released from Rabbit Pulmonary Artery

Abstract

Electrical field stimulation elicits the release of catecholamines, adenine nucleotides, and adenosine from the rabbit pulmonary artery in a frequency dependent manner. To enhance our ability to investigate the release of endogenous adenine nucleotides and adenosine from this and other biological preparations, a new analytical procedure has been developed. This procedure involves the use of an internal standard, 9-β-D-arabinofuranosyladenine (IS), the derivatization of ATP, ADP, AMP, adenosine (Ado), and IS with chloroacetaldehyde, the isocratic high-performance liquid chromatographic separation of these ethenopurine derivatives on an Ultron N-phenyl HPLC column, and their detection and quantitation by fluorescence spectroscopy. This procedure has enhanced sensitivity and reliability over existing procedures due to the stability of the chromatographic baseline and the use of an internal standard. When this analytical procedure was utilized to measure the adenine nucleotides and Ado that are released from the rabbit pulmonary artery in response to electrical field stimulation, it was observed that the release of endogenous ATP, ADP, AMP, and Ado exceeded that of endogenous norepinephrine. A molar ratio (6-amino purines:catecholamines) of approximately 2000:1 was obtained at a stimulation frequency of 16 Hz. This observation suggests an important extracellular role for adenine nucleotides and nucleosides in the physiology of vascular tissues.