Expression of alpha-subunits of G proteins in failing human heart: a reappraisal utilizing quantitative polymerase chain reaction.
A decrease in the density of the beta 1-adrenergic receptor and an increase in the functional activity of the G inhibitory protein Gi accompany human heart failure; however, the molecular and biochemical mechanisms responsible for these changes are unclear. We previously reported that the steady-state levels of the mRNAs encoding both alpha Gi-3 and alpha Gs were significantly increased in failing human heart. However, these results are not consistent with recent studies demonstrating that immunodetectable levels of alpha G proteins are not different in failing human hearts when compared with non-failing controls. In addition, analysis of the 5' flanking regions of alpha Gi and alpha Gs suggests that these two genes are unlikely to be co-regulated as their regulatory domains are quite different. Therefore, we hypothesized that the disparity between the measurements of alpha G protein gene expression and assessment of the actual levels of alpha G proteins might be due to technical limitations of the Northern blot technique utilized in previous studies for assessment of the mRNA levels; (i) cytoskeletal beta-actin mRNA was used as a standard for normalization; and (ii) only relative levels of alpha G mRNAs were measured. The recent application of the polymerase chain reaction to quantification of mRNA levels in small quantities of human heart provided the tool with which to test this hypothesis. When expressed in molecules of mRNA per microgram of total RNA, there were no differences in the levels of alpha Gi and alpha Gs mRNAs in failing human heart when compared with non-failing controls.(ABSTRACT TRUNCATED AT 250 WORDS)
Feldman
A M
AM
Department of Medicine and Physiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Ray
P E
PE
Bristow
M R
MR
D001483:Base Sequence; D004247:DNA; D019204:GTP-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism
D012333:RNA, Messenger; D004247:DNA; D019204:GTP-Binding Proteins