QUANTIFICATION OF ANP MESSENGER-RNA IN PRIMARY CULTURES OF ADULT-RAT ATRIAL MYOCYTES BY IMAGE-PROCESSING - IN-SITU HYBRIDIZATION TO MULTIPLE PARALLEL SAMPLES USING SINGLE-STRANDED CDNA PROBES

In primary cultures of adult rat atrial myocytes, we quantified the ac cumulation of atrial natriuretic peptide (ANP) mRNA in parallel with A NP secretion. ANP mRNA was quantified by image analysis of myocytes hy bridized in situ with single-stranded cDNA probes generated by two suc cessive thermal cycling procedures. In situ analysis permitted measure ment of many small experimental samples in tandem chile avoiding the p ossibility of differential extraction and processing of mRNA from samp le to sample. The single-step application of P-32-labeled probes allow ed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processi ng. Biotin-labeled probes, in conjunction with gold-labeled anti-bioti n antibodies and silver intensification, gave an apparently equivalent specific signal bur presented more difficulty in uniform processing o f many samples and was harder to quantify by our image processing syst em. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture t o maximal levels by Day 8. In contrast, secretion of ANP (which is sto red in atrial granules) slowly decreased, bur was not abolished, durin g the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.