Nitric oxide activates the beta 2 subunit of soluble guanylyl cyclase in the absence of a second subunit.

Previously characterized mammalian soluble guanylyl cyclases form alpha/beta heterodimers that can be activated by the gaseous messenger, nitric oxide, and the novel guanylyl cyclase modulator YC-1. Four mammalian subunits have been cloned named alpha(1), beta(1), alpha(2), and beta(2). The alpha(1)/beta(1) and alpha(2)/beta(1) heterodimeric enzyme isoforms have been rigorously characterized. The role of the beta(2) subunit has remained elusive. Here we isolate a novel variant of this subunit and show that the beta(2) subunit does not need to form heterodimers for catalytic activity because enzyme activity can be measured when it is expressed alone in Sf9 cells. In analogy to the beta(3) subunit recently isolated from the insect Manduca sexta, activity was dependent on the presence of 4 mm free Mn(2+). The EC(50) values for the NO-donor diethylamine/NO were shifted to the left by 1 order of magnitude as compared with the alpha(1)/beta(1) heterodimeric form. In the presence of the detergent Tween, NO sensitivity of beta(2) was abolished, but the enzyme could be activated by protoporphyrin IX, indicating removal of a prosthetic heme group and exchange for the heme precursor. We suggest that the beta(2) subunit is the first mammalian NO-sensitive guanylyl cyclase lacking a heterodimeric structure.

Developmental changes of nitric oxide-sensitive guanylyl cyclase expression in pulmonary arteries.

Inhaled nitric oxide (NO) is known to influence the contractile state of pulmonary arteries most likely by activation of soluble guanylyl cyclase (sGC) in smooth muscle cells. However, the cellular distribution of sGC has not been determined empirically, due to a lack of specific antibodies. Here, we describe a novel antibody directed against the beta1 subunit of sGC to study the cellular distribution of sGC in lung during development. Using the novel antibody, the enzyme was demonstrated in fetal, neonatal, and adult lungs by Western blot, showing maximum expression in neonatal lung. These data were confirmed by measurements of sGC activity. In pulmonary arteries of fetal lung sGC-beta1 immunoreactivity was present in smooth muscle cells and absent in endothelial cells. With postnatal development an increase in immunoreactivity in endothelial cells and a reciprocal decrease in smooth muscle cells was apparent. The reported changes in sGC expression likely contribute to the known age-dependent differences in response to inhaled NO.

Expression and tissue localization of soluble guanylyl cyclase in the human placenta using novel antibodies directed against the alpha(2) subunit.

The cytoplasmic or soluble forms of guanylyl cyclase (sGC) are heme-containing heterodimeric enzymes that are regulated by nitric oxide (NO) and carbon monoxide (CO). These gaseous messenger molecules are produced in the human placenta and are potential regulators of vasodilation and trophoblast invasion. The alpha(2)-subunit of sGC has only recently been shown to naturally occur in placental extracts. In the present study, two novel antibodies directed against different epitopes of the alpha(2) subunit, were generated. Western Blot analysis confirmed the presence of a 82 kDa protein, identical with alpha(2) protein overexpressed in Sf9 cells. According to RNase protection analysis the alternatively spliced alpha(2i) variant was absent from human placenta. Immunohistochemical analysis showed the presence of alpha(2) protein in syncytiotrophoblast and villous and umbilical blood vessels, which are known sites of NO production. Strong expression was observed in the extravillous (intermediate) trophoblast, where the expression of CO-generating hemeoxygenases has recently been documented. Localization of alpha(2) subunit expression suggests a role for sGC in mediating the actions of both NO and CO. The novel antibodies characterized in the present study will be powerful tools to further elucidate the role of the NO/CO/cGMP signaling pathways in pathologic states such as preeclampsia and intrauterine growth retardation.

Cloning and functional expression of the rat alpha(2) subunit of soluble guanylyl cyclase.

Nitric oxide-sensitive guanylyl cyclase is a heterodimeric enzyme consisting of one alpha and one beta subunit. Here, we clone the first alpha(2) subunit ortholog and functionally express the cDNA in Sf-9 cells. Our data indicate a high degree of conservation of the primary sequence and functional activity of the rat alpha(2) subunit.