GRK2 and arrestin2 regulate UTP-mediated arterial contraction ANOVA as indicated, with appropriate post hoc testing (GraphPad Prism, San Diego, CA, USA). 3. Results 3.1 Characterization of UTP-mediated contractions in mesenteric arteries Concentration-dependent contractions of third-order mesenteric arteries (Figure 1A and B) were induced by UTP addition, with maximal contraction seen at UTP concentrations ≥50 mM (EC50 195 28 mM). Repeated addition of maximal UTP (100 mM, 5 min) concen- trations, interspersed by 5 min wash periods produced robust arterial contractions with little evidence of tachyphylaxis (data not shown). These ﬁndings are consistent with those previously obtained for H1 histamine receptor-mediated Ca2+ responses,15 where desensitiza- tion is masked by the presence of signiﬁcant cell/tissue receptor reserve with respect to the observed response. To uncover any potential loss of UTP-induced contractile responsiveness, an amended protocol was applied where arteries were challenged with an approximate EC50 concentration of UTP (30 mM) for 5 min before (R1) and after (R2) the addition of a maximal UTP Figure 1 Characterization of UTP-stimulated contractions of third-order mesenteric arteries. (A) Representative trace showing K+-induced con- traction followed by washout and concentration-dependent UTP (0 – 100 mM)-stimulated contractions (B) Cumulative concentration-dependent UTP- stimulated arterial contraction data shown as means + SEM for arterial preparations from at least four animals. (C – E) Desensitization protocol: arteries were exposed to 30 mM UTP (R1, for 1 min) challenge, followed by 5 min washout, maximal UTP (Rmax ¼ 100 mM, 1 min) challenge, followed by a variable period of 2 – 15 min washout before a further 30 mM UTP (R2, 1 min) exposure. Representative traces from single arteries with either a 5 min (C ) or 15 min (D) Rmax-to-R2 washout period are shown. (E) Time-course of resensitization of the contractile response to UTP expressed as a percent change in R2 relative to R1 (means + SEM for arterial preparations from at least four animals).