METHODOLOGICAL VARIABLES DURING ANALYSIS OF IN-VIVO CEREBELLAR CYCLIC-GMP, AN INDIRECT MARKER OF NITRIC-OXIDE RELEASE

In light of the recent recognition of the physiological significance o f nitric oxide, there is considerable interest in the methodological v ariables that can confound the results of the cerebellar cGMP analysis from in vivo experiments. In this study, using male Swiss Webster mic e, the effect of such methodological variables as 1) weight of the ani mals; 2) tissue extraction procedures used in radioimmunoassay for cGM P; and 3) the commercial source of the assay kit on, harmaline-, penty lenetetrazole- or SNAP-induced increase in cerebellar cGMP in vivo wer e evaluated. Results indicate that mice in the 15- to 19-g weight rang e are most sensitive and best suited for in vivo drug effects on cereb ellar cGMP. Furthermore, for the extraction of cerebellar cGMP, use of ice-cold 0.5N hydrochloric acid and subsequent dilution of the sample in assay buffer is the simplest and fastest method. Present data also indicate that the source of the radioimmunoassay kit has a significan t effect on the cerebellar cGMP results. Based on the present results, the protocol developed and the guidelines drawn are timely and of hig h practical significance for research in the area of pharmacology of n itric oxide.