A major unanswered question in pulmonary physiology is: how do oxygen sensitive cells detect a reduction in oxygen tension and transduce this signal into altered cell function and ultimately into altered function of the organism? In mammal, the carotid body mediates the respiratory response (hyperventilation) to reduced O2 tension (hypoxia). It is now generally accepted that the type I (glomus) cells are the O2-sensitive cells in the carotid body. The type I cells communicate information concerning the O2 status of arterial blood to closely apposed sensory fibers by release of neurotransmitter. Although a number of neurotransmitters/peptides have been identified in the type I cells, there is growing evidence the catecholamine neurotransmitter dopamine mediates information transfer between type I cells and primary sensory afferents during hypoxia. A critical aspect of O2 chemosensitivity is the precise coupling of neurotransmitter synthesis with the prevailing level of hypoxia. Findings from biochemical studies revealed that the activity (Vmax) of tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of dopamine, is enhanced by hypoxia. Our laboratory demonstrated recently that this was due to an increase in TH gene expression which involved an increase in the rate of TH gene transcription. This latter study was performed in pheochromocytoma (PC 12) cells which we have established as an appropriate model system to study the molecular and cellular mechanisms involved in O2 chemosensitivity. In the proposed studies we shall attempt to identify the signal transduction and genomic mechanisms that mediate increased transcription of the TH gene during hypoxia. We hypothesize that specific interactions between elements on the TH gene and hypoxia activated protein factors are responsible for enhanced transcription of the TH gene during hypoxia in O2 sensitive cells. We also hypothesize that the signal transduction system that mediates this response involves cyclic nucleotide (cAMP and cGMP) and proto-oncogene (immediate early gene; fos/jun families) signal transduction systems. The proposed experiments are performed in the PC 12 model system, intact carotid body, cultured carotid body type I cells and transgenic mice.
The specific aims are: 1) Identify the cis-elements on the TH gene that regulate transcription during hypoxia, 2) Identify interactions among cis-acting elements on the TH gene and trans-acting regulatory factors (proteins) during hypoxia, 3) Determine if cyclic nucleotide second messenger systems are involved in regulation of TH gene transcription during hypoxia. Determine if the immediate early gene products (Jun and Fos) are involved in regulating transcription of the TH gene during hypoxia, and 4) Determine in transgenic mice if hypoxia stimulates transcription of the TH gene in the intact carotid body by cis-trans interactions identified in the PC12 model cell line system. This final specific aim will allow us to verify our findings on gene regulatory elements from the PC12 clonal cell line system in the carotid body of intact mice.
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