Coexpression of Y1, Y2, and Y4 receptors in smooth muscle coupled to distinct signaling pathways.

Coexpression of Y1, Y2, and Y4 receptors on smooth muscle cells was determined by reverse transcription-polymerase chain reaction, and the receptors were characterized by radioligand binding, selective receptor protection, and functional analysis of signaling pathways. 125I-peptide YY (PYY) binding was completely inhibited by neuropeptide Y (NPY) and PYY, and partially inhibited by the Y1 agonist [Leu31, Pro34]NPY or the Y2 agonist NPY13-36. In cells where Y1 receptors were preserved by selective receptor protection, 125I-PYY binding was selectively inhibited by the Y1 agonist or antagonist BIBP 3226 [(R)-N2-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]-D-arginine-amide]. Conversely, in cells where Y2 receptors were preserved, 125I-PYY binding was selectively inhibited by the Y2 agonist or antagonist BIIE 0246 [(S)N2-[1-[2-[4-[(R,S)-5,11-dihydro-6(66H)-oxodibenz[b,e]azepin-11-y]-1piperazinyl]-2-oxoethyl]cyclopentyl]acetyl]-N-[2-[1,2-dihydro-35(4H)-dioxo-1,2-diphenyl-3H-1,2,4-triazol-4-yl]ethyl]-argininamide]. All Y receptors activated preferentially Gi2, but only Y2 and Y4 receptors activated Gq. Consequently, Y2 agonists (NPY, PYY, and NPY13-36) and the Y4 agonist (pancreatic polypeptide) induced concentration-dependent contraction, inositol 1,4,5-trisphosphate (IP3) formation, and increase in cytosolic free Ca2+. Contraction induced by Y2 and Y4 agonists was not affected by 0 Ca2+, Ca2+ channel blockers, or pertussis toxin (PTx), but it was abolished by thapsigargin, U73122 [1-(6-(17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-25-dione], or the myosin light chain kinase inhibitor ML-9 [1-(5-chloronaphthalene-1-sulfonyl)homopiperazine, HCl]. Y2-mediated contraction was inhibited by the selective Y2 antagonist BIIE 0246. Insensitivity to PTx implied that the coupling to Gi did not initiate (Y1) or contribute (Y2 and Y4) to contraction. All Y receptor agonists inhibited cAMP formation in a PTx-sensitive manner. The patterns of contraction and inhibition of cAMP by various Y receptors were corroborated by selective receptor protection. The study demonstrates coexpression of Y1, Y2, and Y4 receptors on smooth muscle negatively coupled to adenylyl cyclase via Gi2. Coupling of Y2 and Y4 receptors to Gq determines their ability to induce IP3-dependent Ca2+ release and initiate contraction.