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CRF2 receptor expression in the rat cardiovascular system Evidence is emerging that the function of CRF and the related peptide, urocortin-1, is to mediate an adaptive or 'stress coping' cardiovascular response, especially vasodilatation. Recently, urocortin-2 (Ucn2) has been identified that displays about 1000 fold selectivity for the CRF-2 receptor over the CRF-1 sub-type. We have previously shown Ucn2 is a potent vasodilator of human isolated vessels ( Wiley & Davenport, 2004 ) and in conscious rats in vivo, Ucn2 caused dose dependent tachycardia and hypotension with mesenteric and hindquarters vasodilatation (Gardiner et al., 2005). CRF-1 mRNA has been detected mainly in brain, whereas CRF-2 mRNA is more abundant in heart but the precise identity of cells expressing these receptors is not yet known. Our aim was to characterised the CRF2 receptors in rat heart and brain by radioligand binding using [125I]-antisauvagine 30, a radioligand selective for CRF 2 receptors artificially expressed in cell lines (Higelin et al., 2001) and immunocytochemistry using receptor specific selective antisera. Male Sprague-Dawley rats (350-450g) were killed by exposure to increasing concentration of CO2. Saturation, kinetic and competition binding assays were carried out on sections (30 m m) of rat brain and heart (Davenport & Kuc, 2005) that were pre-incubated for 15min in 50mM Tris buffer (100mM NaCl, 10mM MgCl and 0.3% BSA, pH 7.4). For saturation analysis sections were incubated with increasing concentrations of [125I]-antisauvagine 30 (2pmol l-1-2nmol l-1) for 30min. Sections were incubated with 0.2nM [125I]-antisauvagine 30 in incubation buffer for increasing time periods (0-60min) in association assays and for 30min in dissociation and competition assays. For all assays sections were washed in Tris-HCl buffer at 4 ° C for 10min (for dissociation assays wash times were 0-120min) and counted. Competition assays were carried out as above in the presence of increasing concentrations (2pM-10μM) of Ucn2. In all experiments non-specific binding was determined using 1 μM Ucn1. Data were analysed using the KELL suite of programs (Biosoft, Cambridge, UK. Data for each experiment are mean ± s.e.mean, n=3-6 animals). Sections (30μm) of rat tissues were also subjected to immunocytochemical analysis using CRF2 receptor antibody (SC-1826, Santa Cruz, scbt.com) as previously described (Kuc, 2002). High affinity, saturable and specific [125I]-antisauvagine 30 binding was detected with a single site preferred to a two site model in the both rat heart (K D 0.1 ± 0.01nM; B max 0.9 ± 0.08 fmol mg -1 protein; K obs 0.14 ± 0.017 min -1,t 1/2 5.08 ± 0.64 min; K -1 0.01 ± 0.002 min -1,t 1/2 73.8 ± 14.2 min) and brain (KD 0.2 ± 0.03nM; Bmax 5.5 ± 0.62 fmol mg-1 protein; Kobs 0.04 ± 0.007 min-1,t1/2 19 ± 2.5 min; K-1 0.003 ± 0.0002 min -1,t1/2 230 ± 22 min). Human Ucn2 competed for [125I]-antisauvagine 30 binding monophasically with a one site fit preferred over a two site model in both rat heart (KD 12.6 ± 4.2nM; Bmax 0.90 ± 0.17 fmol mg-1 protein) and brain (KD 25.0 ± 3.8nM; Bmax 1.75 ± 0.30 fmol mg-1 protein). In peripheral tissues, CRF2 immunoreactivity localised principally to the smooth muscle of intramyocardial, mesenteric and skeletal (hindquarters) vessels as well as the myocardium of the heart and to intracerebral vessels in the brain. The results from the competition assays show in native rat tissues, [125I]-antisauvagine 30 bound with a single affinity in agreement with the radioligand being selective for CRF 2. In agreement with our previous functional studies, CRF2 receptors localised to smooth muscle of the vasculature consistent with Ucn2 mediating vasodilatation via a direct action on this sub-type.
Davenport A.P. & Kuc R.E. (2005). Methods Mol Biol. 306, 93-120. |