FIGURE 4.

Function and molecular characteristics of uridine nucleotide receptors activated by UTP. (A,B) UTP elicits a concentration dependent Ca²⁺ response in BON. (A) Representative images of BON cells in response to 100 μM UTP perfusion. Cells responding to UTP with a Ca²⁺response are marked with an arrow; pseudocolor images based on pixel intensity (0–1500 arbitrary units). (B) Representative Ca²⁺ transients to separate applications of UTP in the concentration range of 1 nM to 5 mM. (C) Biphasic concentration-response curve to UTP for peak Ca²⁺ responses. ANOVA, p < 0.0001 (310 cells were analyzed). The apparent EC₅₀ of the first phase of the response is 11.4 μM; the EC₅₀ of the second phase is 410μM. (D) The first phase of the UTP concentration – response curve (0.1 nM–0.2 mM) is fitted with a logistic equation; symbols represent the net increase in Ca²⁺ response at each time point and the error bars are SEM (n = 15 or more cells at each concentration). (E) Representative western blots (WB) showing immunogenic bands for P2Y₄R in BON cells. (F) Representative immunogenic bands for P2Y₆R in BON, human mucosa (Muc) and isolated human EC cells from surgical specimens (hEC), and human HT-29 cells. Pre-absorption of the anti-P2Y₆R antibody with its immunogenic peptide (CP) blocked the immunogenic bands. (G) P2Y₄ is detected in BON, Muc and HT-29 cells. P2Y₂R is only detected in the human mucosa and not BON or HT-29. (H) TaqMan PCR analysis showing the relative expression of P2Y₄R and P2Y₆R normalized to OAZ as 2ˆ(–ΔCt). The C_t values are indicated inside each column, N = 6 cultures. BON cells for qPCR were tested at passage 44, and P2Y₄ expression was >P2Y₆ expression (see Results and Supplementary Figure 2).