Alternative Splicing of cGMP-Dependent Protein Kinase I and Nitrate Tolerance
To the Editor:
A major point of the recent study by Gerzanich et al1 is that angiotensin II–induced “alternative splicing of cGKI [cGMP-dependent protein kinase I] represents a novel mechanism for reducing sensitivity to NO/cGMP” (page 805).
This conclusion was based on the authors’ finding of “a large increase in cGKIβ mRNA and a decrease in cGKIα mRNA” (page 805) in basilar arteries of angiotensin II–hypertensive versus control rats (their Figure 5). The cGKIα and cGKIβ proteins differ only in their amino-terminal regions (approximately the first 100 amino acids), which are encoded by alternatively spliced exons of the cGKI gene.2 Alternative splicing resulting in upregulation of cGKIβ, which is less sensitive to cGMP activation than cGKIα, might well be a mechanism contributing to nitrate tolerance of vascular smooth muscle. The authors claim to measure relative mRNA levels for cGKIα and cGKIβ by reverse transcription (RT)-PCR and Northern blot analysis using probes unique for each isoform mRNA. However, our inspection of the PCR primers used in this study and of the rat genomic cGKI locus revealed that the amplicons detected in the PCR experiments do not correspond to the known mRNA/cDNA sequence of the cGKIα or cGKIβ isoform but to intronic DNA sequences of the rat cGKI locus. Similarly, the probe used for Northern blot detection of the cGKIα mRNA corresponds to intronic DNA, and only part of the cGKIβ probe is complementary to the cGKIβ mRNA, the rest corresponding to intronic sequences. It is not clear why the authors could detect “mRNA” with probes that are located in introns. It cannot be excluded that the RNA samples used for RT-PCR analysis were contaminated with genomic DNA (a control for DNA contamination was not shown), or that the signals were derived from aberrant splicing of the cGKI primary transcript. Anyway, we want to point out that the “mRNA” signals detected by RT-PCR (Figure 5) have nothing to do with the known mRNA/cDNA sequences of the cGKIα and cGKIβ isoforms. Therefore, an important experiment of the study has to be reinterpreted. In summary, we feel that the study of Gerzanich et al1 does not demonstrate angiotensin II–induced “alternative splicing of cGKI α and β isoforms” (page 810) as a potential mechanism for nitrate tolerance.
Gerzanich V, Ivanov A, Ivanova S, Yang JB, Zhou H, Dong Y, Simard JM. Alternative splicing of cGMP-dependent protein kinase I in angiotensin-hypertension: novel mechanism for nitrate tolerance in vascular smooth muscle. Circ Res. 2003; 93: 805–812; published online before print September 25, 2003, 10.1161/01.RES.0000097872.69043.A0.