Pyrimidine nucleotides potently constrict cerebral arteries by activating signal transduction pathways that influence myofilament calcium sensitivity and/or ion channels that control resting membrane potential. G-protein-coupled P2Y receptors are important to this transduction process and recent observations have implicated a prominent role for Gq, G11 and G13. To define which G-proteins control uridine triphosphate (UTP)-induced vasoconstriction, a small interference RNA (siRNA)approach was developed to induce mRNA and protein knockdown in intact cerebral arteries from humanely killed Sprague-Dawley rats. These G-proteins were cloned from rat brain cDNA (humanely killed 12- to 16-week-old Sprague Dawley rats) and siRNAs designed to specifically target Gq but not G11 or G13. Targeted siRNA (20 nM) was introduced into cerebral artery segments using reverse permeabilization and vessels were subsequently cultured in serum-free medium for up to 144 hours. During this period, arteries were removed and assayed for contractile function, mRNA (by real-time PCR) and protein (by Western blotting). A 75% reduction in Gq mRNA was observed within 72 hours post-permeabilization, a response that was sustained up to 120 hours (n=6). G11, a close family member of Gq (79% sequence identity) was unaffected (n=6). Despite reductions in Gq mRNA, protein levels measured between 96-144 hours post-permeabilization were unaffected (n=4). Consistent with the apparent lack of translational knockdown, cerebral arteries treated with Gq-targeted or scrambled siRNAs constricted similarly to UTP (0.01-100 uM) and intravascular pressure (20-100 mmHg) (n=6). Control experiments confirmed antibody specificity (anti Gq, Santa Cruz) and that Gq-targeted siRNA induced protein knockdown in cultured smooth muscle cells (n=3). Our findings demonstrate that mRNA can be successfully knocked down by siRNA in intact cerebral arteries. Selective mRNA down regulation does not, however, ensure effective protein knockdown. Ongoing experiments in intact cerebral arteries are addressing whether the absence of translational knockdown is a function of the turnover rate of the targeted protein.