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Quantification of ANP mRNA in primary cultures of adult rat atrial myocytes by image processing: in situ
L Kordylewski1, S K Ambler, D D Doyle
1Department of Medicine, University of Chicago, Illinois, USA.
Abstract:
In primary cultures of adult rat atrial myocytes, we quantified the accumulation of atrial natriuretic peptide (ANP) mRNA in parallel with ANP secretion. ANP mRNA was quantified by image analysis of myocytes hybridized in situ with single-stranded cDNA probes generated by two successive thermal cycling procedures. In situ analysis permitted measurement of many small experimental samples in tandem while avoiding the possibility of differential extraction and processing of mRNA from sample to sample. The single-step application of 32P-labeled probes allowed processing of many parallel samples and generated intense punctate autoradiographic signals that were readily countable by image processing. Biotin-labeled probes, in conjunction with gold-labeled anti-biotin antibodies and silver intensification, gave an apparently equivalent specific signal but presented more difficulty in uniform processing of many samples and was harder to quantify by our image processing system. Measurement of ANP mRNA during atrial myocyte culture showed that ANP mRNA accumulated from undetectable levels after 1 day of culture to maximal levels by Day 8. In contrast, secretion of ANP (which is stored in atrial granules) slowly decreased, but was not abolished, during the first 5 days of culture. Subsequently, ANP secretion increased, with the increase trailing ANP mRNA accumulation by at least 24 hr.
Insights
Atrial natriuretic peptide (ANP) mRNA accumulates in rat heart cells over 8 days in culture. ANP secretion initially decreases then increases, lagging behind mRNA levels by 24 hours.
Area of Science:
- Cardiology
- Molecular Biology
- Cell Biology
Background:
- Atrial natriuretic peptide (ANP) plays a crucial role in cardiovascular homeostasis.
- Understanding the regulation of ANP synthesis and secretion is vital for cardiovascular research.
Purpose of the Study:
- To quantify atrial natriuretic peptide (ANP) mRNA accumulation in primary adult rat atrial myocytes.
- To correlate ANP mRNA levels with ANP secretion in cultured myocytes.
Main Methods:
- Primary cultures of adult rat atrial myocytes were used.
- In situ hybridization with single-stranded cDNA probes (32P-labeled) quantified ANP mRNA.
- Image analysis facilitated counting of autoradiographic signals.
- ANP secretion was measured over the culture period.
Main Results:
- ANP mRNA levels increased from undetectable to maximal by Day 8 of culture.
- ANP secretion gradually decreased during the first 5 days, then increased.
- The rise in ANP secretion lagged behind ANP mRNA accumulation by at least 24 hours.
Conclusions:
- ANP mRNA accumulation is a key regulatory step preceding changes in ANP secretion in cultured atrial myocytes.
- In situ hybridization provides a robust method for quantifying mRNA in small cell samples.
- The findings shed light on the dynamic regulation of ANP production in cardiac cells.