Characterization and molecular cloning of vascular neuropeptide Y receptor subtypes in pig and dog

Abstract

Cloning with subsequent in vitro and in vivo characterization of vascular neuropeptide Y (NPY) receptor subtypes in porcine and canine peripheral tissues was performed. RT-PCR with Y₁ and Y₂ receptor-specific primers, indicated expression of Y₁ receptors in both kidney and spleen of dog and pig, and expression of Y₂ receptors in pig spleen. In pig kidney, expression of Y₁ receptor mRNA was located to intrarenal arteries, as demonstrated with in situ hybridization using human probes. The cloned and sequenced canine Y₁, porcine Y₁ and Y₂ receptors revealed high homologies to previously characterized mammalian NPY receptors. Membrane and autoradiographic receptor-binding studies showed specific high-affinity binding sites for the purported Y₁-selective radioligands ¹²⁵I-[Leu³¹Pro³⁴]peptide YY (PYY) and ³H-BIBP 3226 in dog spleen, and for the putative Y₂-selective ¹²⁵I-PYY(3–36) in dog and pig spleen. In the pig in vivo, [Leu³¹Pro³⁴]PYY, administered i.v., evoked vasoconstriction in spleen and kidney, actions that were potently inhibited by the non-peptide Y₁ receptor antagonist SR 120107A. In contrast, PYY(3–36) evoked vasoconstriction only in spleen and this effect was not influenced by SR 120107A. NPY evoked renal and splenic vasoconstriction in the dog in vivo, vascular responses that were inhibited by both BIBP 3226 and SR 120107A. Furthermore, the Y₁ receptor agonist [Leu³¹Pro³⁴]NPY also caused vasoconstriction in dog kidney and spleen, whereas the putative Y₂ agonist N-acetyl[Leu²⁸Leu³¹]NPY(24–36) evoked no such vascular responses. It is concluded that the pig spleen is likely to contain Y₁ and Y₂ receptors, both involved in splenic vasoconstriction. In contrast, the Y₁ receptor seems to be the sole vascular NPY receptor subtype in pig kidney. Moreover, Y₁ receptors predominate in dog spleen and kidney. Furthermore, the cloned canine Y₁ receptor and the porcine Y₁ and Y₂ receptors show great homologies to, and possess ligand requirement profiles in accordance with, the human forms.

Introduction

The 36-amino acid peptide neuropeptide Y (NPY) was isolated from porcine brain in 1982 [1]and is one of the most abundant peptides in the central [2]and peripheral [3]nervous system. NPY is colocalized with norepinephrine in sympathetic nerve terminals [3]and is coreleased with norepinephrine, especially upon strong nerve activation. At least two NPY receptor subtypes are likely to be involved in vascular control, and they were initially pharmacologically classified on the basis of their selectivity for NPY analogues such as [Leu³¹Pro³⁴]NPY for the Y₁ receptor and C-terminal NPY fragments for the Y₂ receptor [2]. Both the Y₁ 4, 5and the Y₂ [6]receptors have now been cloned and shown to belong to the seven transmembrane G-protein-coupled family. Moreover, rat and human Y₁ (94%) and Y₂ (94%) receptors share great overall identity [7].

The Y₁ receptor was proposed to be situated mainly postjunctionally, mediating vasoconstriction, while the prejunctional Y₂ receptor inhibited norepinephrine release [2]. However, it has been suggested that Y₂ receptors also may mediate vasoconstriction, as in pig spleen [8]and, conversely, the Y₁ receptor can be prejunctional and inhibit sympathetic transmitter release, as in rabbit vas deferens [9]. The introduction of the potent non-peptide Y₁ receptor antagonists SR 120107A [10]and BIBP 3226 [11]made it possible to obtain final evidence for the involvement of endogenous NPY in sympathetic vascular control. Thus, both antagonists have been shown to potently inhibit non-adrenergic contraction evoked by high-frequency transmural sympathetic nerve stimulation in guinea pig vena cava in vitro 12, 13. By using these antagonists it was also shown that neurogenically released NPY is likely to account for a major part of the non-adrenergic sympathetic vasoconstriction evoked in various vascular beds, such as kidney, spleen and skeletal muscle, in reserpinized pigs in vivo 14, 15. The radioligand ¹²⁵I-[Leu³¹Pro³⁴]peptide YY (PYY) has been shown to possess high affinity for Y₁ receptors, and the radioligand ¹²⁵I-PYY(3–36) for Y₂ receptors in the central nervous system [16]. Furthermore, ³H-BIBP 3226 can be used as a selective non-peptide ligand for Y₁ receptors [17]. The aim of this study was to perform cloning and characterization of peripheral NPY receptors in two large experimental animals (the dog and pig), using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization to detect mRNA for Y₁ and Y₂ receptors, membrane and autoradiographic receptor binding, and parallel in vivo experiments.