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NO-insensitive sGCbeta1 H105F knockin mice: if NO has no place to go
© Thoonen et al; licensee BioMed Central Ltd. 2009
Published: 11 August 2009
Soluble guanylate cyclase (sGC) is a heterodimer, consisting of an alpha1- or alpha2-subunit and a beta1-subunit. Activation of sGC by NO/CO critically depends on the presence of a prosthetic ferrous heme group, linked to the axial ligand His-105 of the beta1-subunit. Removal of this heme moiety as well as its oxidation abolishes any NO-induced enzyme activation. To differentiate between sGC-dependent and sGC-independent functions of NO, and to differentiate between heme-dependent and heme-independent functions of sGC, we generated heme-deficient sGCbeta1H105F knockin (KI) mice, in which sGC retains its basal activity, but can no longer be activated by NO.
sGCbeta1H105F knockin mice were generated using a classical approach by which the sGCbeta1 allele was replaced with a mutated allele by means of homologous recombination. As such, the codon for the His-105 residue of the sGCbeta1-subunit was replaced by a codon for Phe. Initially, mice were phenotyped on a mixed background of 129S6xC57Bl/6J. Non-invasive basal systolic blood pressure (SBP) and heart rate (HR) measurements were performed in male and female wild-type (WT) and sGCbeta1H105F knockin mice with a tail-cuff pressure-recording device (Visitech BP-2000/Hatteras MC4000) and with telemetry in free living animals (DSI).
sGCbeta1H105F knockin mice showed a reduced life span, gastro-intestinal tract abnormalities and growth retardation. Basal SBP was higher in sGCbeta1H105F knockin mice than in WT mice, while HR was lower in sGCbeta1H105F knockin compared to their WT littermates. Moreover the blood pressure response to NO-donors (DETA-NO, SNP) and L-NAME was abolished, while the action of the heme-independent sGC activator Bay 58-2667 was preserved. Relaxation of precontracted aortic rings with NO-donors was completely impaired and in addition the concentration-response curve after the heme-dependent stimulator Bay 41-22172 strongly shifted to the right in KIKI aorta's vs. WT (IC50 830 nM vs 34 nM). In contrary the curve after Bay 58-2667 shifted to the left (IC50 0.26 nM vs 1.23 nM). This corroborates the hypothesis that Bay 58-2667 preferably activates the heme-free form of sGC both in vitro and in vivo.
The NO-activated state of sGC is necessary for the normal function of a number of important physiological processes in the body, such as the control of blood pressure and heart rate, normal gastro-intestinal tract function and development, and normal growth and viability.
This article is published under license to BioMed Central Ltd.