Expression, regulation and functional aspects of the NPY Y1 receptors in rat

Abstract

The distribution of the NPY receptor subtypes, mainly of Y1 and Y2 receptors, has been studied extensively by others employing pharmacological ligand binding techniques. The major aim of this thesis was to study in detail the distribution of Y1-receptors (Y1Rs) in rat and mouse with antisera against the C-terminus (CT) of the rat Y1R using an immunohistochemical technique with enhanced sensitivity (tyramide signal amplification = TSA). The specificity of the CT antisera was supported by several control experiments. The regulation of NPY receptors was studied in the hippocampus in an animal model with kindling induced seizures and in the testis following hypophysectomy and testosterone substitution.

In the central nervous system (CNS) of the rat and wild type mouse (WT, a wide -distribution of Y1R-like immunoreactivity (Y1R-LI) was observed in almost an brain regions, mostly localized in neuronal processes and cell bodies. In addition, dot-like staining, most likely representing axons or axon terminals, was seen in several tracts and nuclei. Y1R-LI was also observed in trigeminal fibers innervating the mystacial vibrissae between embryonic day (E) 16.5 and E 18, but not at older ages, where Y1R-LI was confined to cell bodies of the trigeminal ganglion. This Y1R-LI was specific in the sense that it was absent in adjacent sections following preadsorption of the antibody with 10-5M of the antigenic peptide, and that it could not be observed in Y1R knock-out (KO) mice. The importance of multiple specificity controls was demonstrated with two other antisera against the N-terminus (NT) of the rat Y1R In the CNS, these NT antisera exhibited a labeling pattern different from the CT antiserum. The staining appeared to represent reaction with another unknown protein rather than genuine Y1Rs since the antisera labeled sections of Y1R KO mice in a similar way as of WT mice and rats although they did not exhibit staining following preadsorption with their antigenic peptide. Following lesion of the sciatic nerve, the spinal cord and the brain also the CT antiserum appeared to label another epitope other than genuine Y1Rs in degenerating axons. This was indicated by the fact that it stained degenerating axons in sciatic nerves also of Y1R KO mice.

Region and time-wise differential changes were observed in the levels of Y1, Y2 (Y2R) and Y5 (Y5R) receptor mRNA in hippocampus and some associated regions following rapid kindling. The levels of NPYand Y5R mRNAs were generally increased, of Y1R mRNA decreased and of Y2R mRNA decreased in some and increased in some other regions.

Y1R-LI was observed in vascular smooth muscle cells of mostly superficial cerebral blood vessels. NPY immunoreactive JR) fibers were found abutting on large Y1R positive arteries, but not on small Y1R-IR artetioles. On the other hand, many central NPY-IR interneurons were observed near small Y1R positive arterioles.

Y1R-LI and mRNA were observed in vascular smooth muscle cells in the female and male urogenital tracts. In testis, expression of Y1R-LI and mRNA in vascular smooth muscle cells was shown to be dependent on presence of testosterone since Y1R-LI and mRNA signals only were observed when hypophysectomized rats received testosterone substitution. Similarly, the Y1R mediated vasoconstriction, decreasing testicular blood flow (TBF) by -40% upon intratesticular injection of the Y1R agonist, [Leu31' pro34]NPY or NPY, was only observed in hypophysectomized rats that had received testosterone.