title: The relationship between density of alpha-adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels.
abstract: 1. The aim of this study was to investigate constrictor alpha-adrenoceptors in three isolated blood vessels of the pig, the thoracic aorta (TA), the splenic artery (SA) and marginal ear vein (MEV) and then compare the functional response with the densities of alpha 1- and alpha 2-adrenoceptor binding sites in these and several other porcine vascular tissues, palmar common digital artery (PCDA), palmar lateral vein (PLV) and ear artery (EA). 2. Noradrenaline (NA), phenylephrine (PE) and UK14304 (all at 0.03-10 microM) elicited concentration-dependent contractions in the TA and MEV, with a rank order of potency of UK14304 > NA > PE. UK14304 produced maximal responses which were 58% (TA) and 65% (MEV) of that of NA. In the SA, UK14304 and PE produced maximal responses which were less than 10% and 50% of the NA-induced maximal response respectively, with an order of potency of NA > PE. In the SA, NA-induced contractions were competitively antagonized by prazosin (pA2 = 8.60 +/- 0.15). Further, rauwolscine (1-10 microM) antagonized NA-induced contractions with an apparent pKB of 6.09 +/- 0.11 (n = 6), indicating an action at alpha 1-adrenoceptors. The combination of the two antagonists at concentrations selective for alpha 1- (0.1 microM) and alpha 2-adrenoceptors (1 microM) had no greater effect than either antagonist alone. This suggests that the SA expresses only post-junctional alpha 1-adrenoceptors. 3. In the TA, prazosin produced non-parallel shifts in the NA-induced CRC and this was also observed with rauwolscine, where reductions in the maximal responses were also observed. In the MEV, prazosin was largely inactive in antagonizing NA-induced contractions. In both these vessels a combination of these two antagonists had a greater effect than either alone, indicating the presence of functional alpha 1- and alpha 2-adrenoceptors. The post-junctional alpha 2-adrenoceptors in all of these vessels were resistant to prazosin, suggesting the alpha 2-adrenoceptor to be of the alpha 2A/2D subtype. The expression of functional alpha 2-adrenoceptors was MEV > TA > PLV > PCDA > SA. 4. In radioligand binding studies using TA P2 pellet membranes, [3H]-prazosin and [3H]-RX821002 ([1,4-[6,7(n)-3H] benzodioxan-2-methoxy-2-yl)-2-imidazole) labelled different high affinity sites, and in competition studies using identical membranes corynanthine displaced [3H]-prazosin with 10 fold higher affinity than rauwolscine, indicating that [3H]-prazosin was selectively binding to alpha 1-adrenoceptor sites. Further, rauwolscine displaced [3H]-RX821002 with approximately 100 fold greater affinity compared to corynanthine, which is indicative of selective alpha2-adrenoceptor binding.5. Separation of the P2 pellet into plasma membrane and mitochondrial fractions was carried out using a differential sucrose density gradient. [3H]-prazosin and [3H]-RX821002 binding sites were found in both the plasma membrane and mitochondrial fractions.6. In saturation studies all tissues produced single site saturation curves with no difference in the Kd(range 0.13-0.20nM) of the alpha1-adrenoceptor sites for [3H]-prazosin. However, there was considerable variation in Bmax of alpha 1-adrenoceptor sites; the highest density was found in the TA (397.9 =/- 52.7 fmol mg-1, n = 4), followed by the PCDA (256.7 +/- 22.7 fmol mg-1, n = 4), the PLV and SA having approximately equal density (143.6 +/- 3.9 and 159.1 +/- 7.0 fmol mg-1 respectively, n = 4 for both), followed bythe EA (91.3 +/- 10.5 fmol mg-1, n = 3) and the MEV had the lowest density (48.9 +/- 11.4 fmol mg-1,n = 3).7. In saturation studies using [3H]-RX821002, all tissues produced single site saturation curves with no differences in the Kd values (range 1.31 +/- 2.16 nM) but the highest densities were found in the TA and MEV (545.3 +/- 36.2 and 531.0 +/- 40.9 fmol mg-1 respectively), followed by the PLV (418.4 +/- 39.4 fmol mg-1), then the EA (266.3 +/- 40.0 fmol mg-1), and low densities of [3H]-RX821002 binding being found in the PCDA and SA (155.9 +/- 18.1 and 117.5 +/- 19.3 fmol mg-1 respectively).8. The pattern of binding site distribution for alpha l- and alpha 2-adrenoceptors is in reasonable agreement with functional studies carried out in these porcine vascular tissues; the TA has the highest densities of alpha 1-and alpha2-adrenoceptors; in the SA and PCDA there is a predominance (although small) of alpha l-adrenoceptor binding sites, the reverse of which is observed both in the PLV and MEV (i.e. greater density of alpha2-adrenoceptor sites). Thus, it would appear that alpha 1- and alpha2-adrenoceptor densities play a role in the expression of functional responses via these receptor subtypes; although it is interesting to note that the SA did have a small density of alpha 2-adrenoceptor binding sites, no functional response was observed after alpha2-adrenoceptor activation.
authors.affiliation: Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham.
authors.forename: I K
authors.initials: IK
authors.lastname: Wright
authors.forename: N A
authors.initials: NA
authors.lastname: Blaylock
authors.forename: D A
authors.initials: DA
authors.lastname: Kendall
authors.forename: V G
authors.initials: VG
authors.lastname: Wilson
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D001667:Binding, Competitive; D002462:Cell Membrane / Q000187:drug effects / Q000648:ultrastructure; D004146:Dioxanes / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004431:Ear, External / Q000098:blood supply; D019329:Idazoxan / Q000031:analogs & derivatives; D008297:Male; D008854:Microscopy, Electron; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism; D011224:Prazosin / Q000494:pharmacology; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects / Q000502:physiology*; D013157:Splenic Artery / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D013329:Structure-Activity Relationship; D013552:Swine; D014680:Veins / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D015016:Yohimbine / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D004146:Dioxanes; D011942:Receptors, Adrenergic, alpha; D015016:Yohimbine; C058525:2-methoxyidazoxan; D015720:5'-Nucleotidase; D011224:Prazosin; D019329:Idazoxan