Received for publication May 14, 2004.
Revised July 22, 2004.
Accepted for publication July 26, 2004.

Co-Expression of Y₁, Y₂ and Y₄ Receptors in Smooth Muscle Coupled to Distinct Signaling Pathways

Abstract

Co-expression of Y₁, Y₂, and Y₄ receptors on smooth muscle cells was determined by RT-PCR and the receptors were characterized by radioligand binding, selective receptor protection, and functional analysis of signaling pathways. ¹²⁵I-PYY binding was completely inhibited by NPY and PYY, and partially inhibited by the Y₁ agonist, [Leu³¹, Pro³⁴]NPY or the Y₂ agonist, NPY_13-36. In cells where Y₁ receptors were preserved by selective receptor protection, ¹²⁵I-PYY binding was selectively inhibited by the selective Y₁ agonist or antagonist (BIBP 3226). Conversely, in cells where Y₂ receptors were preserved, ¹²⁵I-PYY binding was selectively inhibited by the Y₂ agonist or antagonist (BIIE 0246). All Y receptors activated preferentially G_i2, but only Y₂ and Y₄ receptor activated G_q. Consequently, Y₂ agonists (NPY, PYY, NPY_13-36) and the Y₄ agonist (PP) induced concentration-dependent contraction, IP₃ formation and increase in [Ca²⁺]i. Contraction induced by Y₂ and Y₄ agonists was not affected by 0 Ca₂₊, Ca₂₊ channel blockers or PTx, but was abolished by thapsigargin, U73122, or the MLCK inhibitor, ML-9. Y₂-mediated contraction was inhibited by the selective Y₂ antagonist, BIIE 0246. Insensitivity to PTx implied that the coupling to G_i did not initiate (Y₁) or contribute (Y₂ and Y₄) to contraction. All Y receptor agonists inhibited cAMP formation in a PTx-sensitive fashion. The patterns of contraction and inhibition of cAMP by various Y receptors were corroborated by selective receptor protection. The study demonstrates co-expression of Y₁, Y₂, and Y₄ receptors on smooth muscle negatively coupled to adenylyl cyclase via G_i2. Coupling of Y₂ and Y₄ receptors to G_q determines their ability to induce IP₃-dependent Ca²⁺ release and contraction.

Key words: Contraction, G proteins, Receptors, Signal transduction, cAMP, smooth muscle

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