Hypoxic chemotransmission in the rat carotid body (CB) is mediated in part by ATP acting on P2X urinoceptors. Here we use RT-PCR, cloning, and sequencing techniques to show P2X2 and P2X3 receptor expression in petrosal neurons (PN). Confocal immunofluorescence indicated co-localization of P2X2 and P2X3 protein in many petrosal neurons and CB afferent terminals in situ. Petrosal and nodose neurons share a similar developmental origin (i.e. the ectodermal placodes) and GDNF is a required survival factor for both the neuronal groups. However, in the present study the profiles of P2X receptor subunit expression in the petrosal and nodose neurons differed from each other. The PNs showed a slight increase in the expression of P2X2 and P2X3 mRNA by P-21, whereas in nodose neurons the corresponding expression levels peaked by P-9, but fell significantly by P-21. However, in both cases the expression levels for the P2X2 mRNA were higher than those of P2X3 mRNA and the expression profile for both the P2X subunits developed along a parallel time course. Previous developmental studies have indicated that expression levels of various autoreceptors on type I cells (e.g. DA D2-receptors, A2a and A2b adenosine receptors) change during the first week of postnatal life, as the animal breathes normoxic air. In the present study, the possibility that oxygen tension might regulate P2X receptor expression on type I cells was examined. RT-PCR was employed to compare expression levels of the P2X subunits in isolated type I cells grown under different 0₂ tensions. Compared to nonnoxic (20% 02) cell clusters grown under chronic hypoxia ( 6% O₂). Immunofluorescence studies cells grown under CHox. Thus, ATP and P2X2/P2X3 purinoceptors play an important role under normal conditions and following exposure to chronic hypoxia. / Thesis / Master of Science (MSc)

Adenosine triphosphate (ATP) is well recognized for its role as the primary cellular energy currency. However, studies dating back to 1929 have reshaped our understanding of ATP as not only an energy source, but also as a signaling agent. Among the most important of these discoveries are animal purinergic receptors (P2X and P2Y receptors) that perceive extracellular ATP (eATP), primarily in the nervous system. Though eATP is an established receptor agonist in animals and applied poorly hydrolyzable ATP analogs have numerous effects on growth in plants, eATP is not widely accepted as a signal in plants where no purinoceptor has been identified. Here, enriched outside-out plasma membrane vesicles were isolated and proteins labeled with a radioactive ATP analog (8N₃ATP[α²³P]) to identify a putative purinoceptor-like receptor. We used etiolated seedlings to capture proteins from plant tissue that was actively growing and used sodium carbonate washes to separate peripheral and integral membrane proteins. With this method, we have generated lists of plasma membrane ATP binding proteins, and therefore possible eATP receptors. Ectoapyrases are phosphohydrolases thought to regulate eATP in both animals and plants. Here, we also investigated the expression and role of the candidate ectoapyrases AtAPY1 and AtAPY2 in guard cells and stomatal responses. AtAPY1 and AtAPY2 transcript and protein expression was confirmed in guard cells. Early genetic studies using an apy2 knock out with induced RNAi-silencing of APY1 suggest a role for these apyrases in stomatal regulation. In response to treatment with five hours light, the apyrase-suppressed line features wider stomatal aperture when compared to WS wild-type. / text