26] Measurements of cyclic adenosine monophosphate and cyclic guanosine monophosphate levels in polymorphonuclear leukocytes, macrophages, and lymphocytes

Abstract

This chapter describes the measurements of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in polymorphonuclear leukocytes, macrophages, and lymphocytes. It describes the technique of radioimmunoassay (RIA) that offers the most sensitive device for cyclic nucleotide measurements in these types of cells. Intracellular levels of cyclic AMP and cyclic GMP are altered in response to a variety of stimuli, including mitogens, calcium, neurohormones, prostaglandins, and oxygen radicals. Before intracellular levels of cyclic nucleotides can be determined at steady state in a cell suspension, it is essential to abruptly terminate the metabolic reactions and extract the nucleotides. This is achieved by homogenizing the cells in 8–10% trichloroacetic acid (TCA) at 0°C. Both anion- and cation-exchange resins can be used for the separation and purification of cyclic nucleotides. The samples purified with either of the chromatographic procedures are suitable for the direct quantitation of cyclic nucleotides by RIA but not for the determination of intracellular pools of radiolabeled cyclic AMP or cyclic GMP generated from radioactive adenine or guanine.