The relationship between density of α‐adrenoceptor binding sites and contractile responses in several porcine isolated blood vessels
Authors: Wright I.K. , Blaylock N.A. , Kendall D.A. , Wilson V.G. 
Published: July 2012

The aim of this study was to investigate constrictor α‐adrenoceptors in three isolated blood vessles 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 α1‐ and α2‐adrenoceptor binding sites in these and several other porcine vascular tissues, palmar common digital artery (PCDA), palmar lateral vein (PLV) and ear artery (EA).

Noradrenaline (NA), phenylephrine (PE) and UK14304 (all at 0.03–10 μm) 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 μm) antagonized NA‐induced contractions with an apparent pKB of 6.09 ±0.11 (n = 6), indicating an action at α1‐adrenoceptors. The combination of the two antagonists at concentrations selective for α1‐ (0.1 μm) and α2‐adrenoceptors (1 μm) had no greater effect than either antagonist alone. This suggests that the SA expresses only post‐junctional α1‐adrenoceptors.

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 α1‐ and α2‐adrenoceptors. The post‐junctional α2‐adrenoceptors in all of these vessels were resistant to prazosin, suggesting the α2‐adrenoceptor to be of the α2A/2D subtype. The expression of functional α2‐adrenoceptors was MEV>TA>PLV>PCDA>SA.

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 α1‐adrenoceptor sites. Further, rauwolscine displaced [3H]‐RX821002 with approximately 100 fold greater affinity compared to corynanthine, which is indicative of selective α2‐adrenoceptor binding.

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.

In saturation studies all tissues produced single site saturation curves with no difference in the Kd (range 0.13–0.20 nm) of the α1‐adrenoceptor sites for [3H]‐prazosin. However, there was considerable variation in Bmax of α1‐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 by the 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).

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).

The pattern of binding site distribution for α1‐ and α2‐adrenoceptors is in reasonable agreement with functional studies carried out in these porcine vascular tissues; the TA has the highest densities of α1‐ and α2‐adrenoceptors; in the SA and PCDA there is a predominance (although small) of α1‐adrenoceptor binding sites, the reverse of which is observed both in the PLV and MEV (i.e. greater density of α2‐adrenoceptor sites). Thus, it would appear that α1‐ and α2‐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 α2‐adrenoceptor binding sites, no functional response was observed after α2‐adrenoceptor activation.