Regulation of vascular soluble guanylate cyclase

Release of nitric oxide (NO) from vascular endothelial cells causes relaxation of arterial blood vessels, resulting in a decrease of blood pressure. The effect of NO is mediated by activation of soluble guanylate cyclase (sGC), an enzyme that converts GTP to the messenger molecule cyclic GMP (cGMP). Activation of downstream signaling pathways by cGMP causes decreased Ca2+ sensitivity and thereby relaxation of smooth muscle cells. Recently, we discovered an as yet unidentified cytosolic protein in porcine coronary arteries, which significantly increases the activity of maximally NO-stimulated sGC. First attempts to enrich this sGC activating factor (sGC-AF) yielded a protein mixture of actin, gelsolin, and annexin A6. These Proteins are part of the contractile apparatus or are closely related to it. Interestingly, further purification led to loss of sGC-AF activity. Thus, it is assumed that an essential component was removed. In this research project we aim to identify the sGC activating factor (sGC-AF) and all essential components and to study the tissue distribution and biological role of this protein. In order to obtain peptide sequences that will allow identification of the sGC-AF we will establish a method to isolate the complete sGC-AF from porcine coronary artery cytosols. The constituents of sGC-AF will then be analyzed by mass spectrometry. A bioassay with NO- stimulated purified sGC established in our laboratory will be used for detection of activity. Subsequently sGC-AF will be cloned, expressed in a suitable system, and purified to homogeneity. The mode of action of sGC-AF will be studied in enzyme assays with purified sGC. To clarify the physiological role of sGC-AF in the vasculature, we will change the expression of the protein in cultured cells and mouse aortas. In these experiments the biological activity of sGC-AF will be assessed by determination of tissue cGMP levels and NO- induced relaxation of blood vessels. If time permits, we will study genetic mouse models with conditional smooth muscle-specific overexpression and knockout of sGC-AF to clarify the contribution of this protein to blood pressure regulation in vivo. The proposed work is expected to elucidate a novel pathway of regulating NO/cGMP signaling in blood vessels and perhaps other tissues that express sGC. Besides contributing to our knowledge about smooth muscle physiology, the results may pave the way for the development of a new class of drugs that enhance vascular relaxation in coronary artery disease and other cardiovascular disorders associated with endothelial dysfunction and impaired NO signaling.