Lewis, Stephen John PHD
Principal Investigator, TITUS Group Project 3
Professor, Division of Pediatric Pulmonology, and Allergy/Immunology
Department of Pediatrics
Case Western Reserve University
I have over twenty years experience studying S-nitrosothiol pharmacology. I will assist Dr. Zaman in studying the metabolism of S-nitrosothiols in human airway pseudostratified epithelial cell culture.
A. SPECIFIC AIMS
Primary glomus cells (PGCs) within the carotid bodies (CBs) detect changes in blood pO2, pCO2 and pH, and release vesicular pools of “neurotransmitters” that activate adjacent chemoafferent nerves, which in turn, relay the information to the brain to adjust Minute Ventilation. The identities of the neurotransmitters that mediate the increases in chemoafferent activity in response to hypoxia or hypercapnia/acidosis have not been established since blockade of their target receptors does not attenuate the elicited increases in chemoafferent activity.
Our laboratory has focused on understanding the physiological roles and mechanisms of action of endogenous S-nitrosothiols (SNOs). Our published work provided evidence that (1) neurons and vascular endothelial cells contain cytoplasmic vesicles that synthesize/store SNOs, (2) these vesicles are subject to Ca2+-dependent or Ca2+-independent exocytosis, and (3) the released SNOs activate stereoselective cell-surface recognition sites (putative receptors) and by the S-nitrosylation (addition of NO+ to sulfur atoms) of functional proteins. This proposal will address the concepts that (1) PGCs contain endothelial nitric oxide synthase (eNOS)-positive cytoplasmic vesicles which synthesize and store SNOs such as S-nitroso-L-cysteine (SNO-L-CYS), S-nitroso-glutathione (GSNO), and S-nitrosocysteinylglycine, via γ-glutamyl transpeptidase (γ-GT)-mediated translation of GSNO through vesicle membranes, (2) these vesicles are subject to Ca2+-dependent exocytosis in response to stimuli that promote increases in intracellular Ca2+ such as acetylcholine (ACh) and mild hypoxia (left-hand cascade of Fig. 1) whereas more severe hypoxia, will elicit Ca2+-independent exocytosis (right-hand cascade).
Our overall objectives are to determine (1) the mechanisms by which SNOs are synthesized within vesicles of PGCs including the role of nitrite, which generates nitric oxide under hypoxia, (2) the mechanisms regulating exocytotic release of SNO-containing cytoplasmic vesicles in CBs of rats and mice, and (2) the mechanisms by which SNO-L-CYS (see Fig. 2) and related SNOs activate CB chemoafferents including, (1) S-nitrosylation of known functional proteins, (2) intracellular transport via the L-aromatic amino acid transporter (L-AT), and (3) cell-surface recognition sites linked to intracellular signal cascades. The SPECIFIC AIMS of this project are:
AIM 1: To determine (1) the localization of eNOS and fusion proteins serving vesicular exocytosis in PGCs, (2) whether SNOs exist in cytoplasmic vesicles of PGCs, and (3) sub-cellular localization of high-affinity binding sites (putative receptors) and low-affinity binding sites (putative S-nitrosylation sites) for SNO-L-CYS in the CB.
AIM 2: To characterize the importance of SNOs in the CB regulation of ventilatory control and the ventilatory responses to hypoxia/hypercapnia/acidosis, in Sprague Dawley rats and in transgenic and normal mice.
This project will provide novel insights into the role of SNOs in ventilatory control that will drive future studies directed at testing the hypothesis that free radical-induced damage to SNO function within the CB underlies the pathogenesis of hypoventilatory states associated with diseases such as diabetes, obesity and sleep apnea.