V.G. Wilson

Activities of octopamine and synephrine stereoisomers on α‐adrenoceptors

1 The activities of the (‐)‐ and (+)‐forms of m‐ and p‐octopamine and m‐ and p‐synephrine on α1‐adrenoceptors from rat aorta and anococcygeus and α2‐adrenoceptors from rabbit saphenous vein were compared with those of noradrenaline (NA). 2 The rank order of potency of the (‐)‐ forms on α1‐adrenoceptors from rat aorta and α2‐adrenoceptors was NA > m‐octopamine = m‐synephrine > p‐octopamine = p‐synephrine. The two m‐compounds were 6 fold less active than NA on α1‐adrenoceptors from rat aorta and 150 fold less active on α2‐adrenoceptors. The two p‐compounds were 1,000 fold less active than NA on both α1‐adrenoceptors from rat aorta and α2‐adrenoceptors. The rank order of potency of the (‐)‐ forms on α1‐adrenoceptors from rat anococcygeus was NA = m‐synephrine > m‐octopamine > p‐octopamine = p‐synephrine. m‐Octopamine was 4 fold less active than NA and (‐)‐m‐synephrine. The two p‐compounds were 30 fold less active than NA. 3 The rank order of potency of the (+)‐ forms was NA > m‐octopamine > m‐synephrine > p‐octopamine > p‐synephrine on both α1‐ and α2‐adrenoceptors. The potency of each (+)‐ form was 1–2 orders of magnitude less than that of the (‐) counterpart, the differences being greater for the stereoisomers of synephrine than for those of octopamine on both α1‐ and α2‐adrenoceptors. 4 The yohimbine diastereoisomer antagonists, rauwolscine and corynanthine, were tested against (‐)‐NA and (‐)‐m‐octopamine‐induced contractions in both preparations. Based upon the known selectivities of these isomers for α‐adrenoceptor subtypes, it is concluded that the rat aorta contains only α1‐adrenoceptors while the rabbit saphenous vein possesses predominantly α2‐adrenoceptors. 5 Ligand binding data for the octopamine and synephrine stereoisomers at α1‐ and α2‐binding sites from rat cerebral cortex was also obtained. (‐)‐Forms were more active than (+)‐forms. The rank order of affinity of the (‐)‐forms for both α1‐ and α2‐binding sites was NA > m‐octopamine = m‐synephrine > p‐synephrine > p‐octopamine. The relative affinities of the members of the series against α1‐binding sites were very similar to their relative functional activities on rat aorta. However, the affinities of both m‐ and p‐compounds relative to that of (‐)‐NA were much greater at the α2‐binding sites than were the relative activities in rabbit saphenous vein, possibly suggesting low intrinsic efficacy. Functional antagonist responses to NA by the (‐)‐octopamine and synephrines could not, however, be demonstrated on rat aorta or rabbit saphenous vein. 6 The activities of m‐octopamine and m‐synephrine were not significantly different from each other on either α1‐adrenoceptors from rat aorta or α2‐adrenoceptors; however, m‐synephrine is more active than m‐octopamine on α1‐adrenoceptors from rat anococcygeus. Both m‐octopamine and m‐synephrine can be considered to be naturally occurring α1‐selective amines. However, if m‐ and p‐octopamine are co‐released with NA in amounts proportional to their concentration, it is concluded that their activities on α1‐ and α2‐adrenoceptors are too low to be physiologically significant.

Pharmacological analysis of postjunctional α-adrenoceptors mediating contractions to (−)-noradrenaline in the rabbit isolated lateral saphenous vein can be explained by interacting responses to simultaneous activation of α1-and α2-adrenoceptors

1 The pharmacological characteristics of the α‐adrenoceptor population in the rabbit isolated saphenous vein has been examined with (–)‐noradrenaline (NA), as principal agonist, and a number of antagonists with selectivity for either α1‐ or α2‐adrenoceptors. 2 The rank order of potency of various agonists is consistent with a population of α2‐adrenoceptors; UK‐14304 > (—)‐noradrenaline = (—)‐adrenaline > B‐HT 920 = cirazoline > phenylephrine > amidephrine, but the rank order of pA2 values for the antagonists against (—)− noradrenaline: BDF‐6143 > rauwolscine = prazosin > CH‐38083 = YM‐12617 > Wy‐26703 = phentolamine > corynanthine, is indicative of a mixed population of α1‐ and α2‐adrenoceptors or, alternatively, a new subtype with characteristics of both the α1‐ and α2‐subtypes. 3 Further evidence for two discrete populations of α‐adrenoceptors is provided by, (a) the potent but non‐competitive effect of prazosin against (—)‐noradrenaline, (b) the presence of a component of the contractions elicited by NA and phenylephrine which is resistant to the selective α2‐adrenoceptor antagonists rauwolscine and CH‐38083: these responses were inhibited by the selective α1‐adrenoceptor antagonists prazosin and YM‐12617, but not by the selective α2‐adrenoceptor antagonist BDF‐6143 and, (c) the relative potency of the yohimbine diastereoisomers rauwolscine and corynanthine against NA, phenylephrine and UK‐14304. 4 In spite of the overwhelming evidence for a population of postjunctional α2‐adrenoceptors, prazosin was similarly effective against all agonists and failed to discriminate between those with putative selectivity for α1‐ and α2‐adrenoceptors. This suggests an interaction of the effects of agonists at the two α‐adrenoceptor subtypes. 5 An attempt has been made to reconcile a number of paradoxical observations with regard to the identification of postjunctional α2‐adrenoceptors in vitro, and it is suggested that in many of the isolated blood vessels presently available for examination both subtypes reside on the same smooth muscle cell. The pharmacological consequences of multiple subtypes of receptors mediating the same response is considered.

Evidence for prazosin‐resistant, rauwolscine‐sensitive α‐adrenoceptors mediating contractions in the isolated vascular bed of the rat tail

1 The postjunctional α‐adrenoceptors mediating contractions in the isolated vascular bed of the perfused rat tail have been investigated, in the presence and absence of an increase in perfusion pressure by arginine vasopressin (AVP). 2 In the absence of AVP, bolus doses of noradrenaline (NA) and phenylephrine produced pressor responses of similar time course, while UK‐14,304 was practically inactive. Responses to noradrenaline were inhibited more by 0.05 μm prazosin than by 1 μm rauwolscine, suggesting the presence of α1‐adrenoceptors. 3 Following a sustained elevation in perfusion pressure by AVP, both UK‐14,304 and NA (the latter in the presence of 0.05 μm prazosin to inhibit α1‐adrenoceptors) elicited dose‐dependent pressor responses. The maximum response to UK‐14,304 under these conditions was approximately 30% of the maximum response to NA in the absence of prazosin and AVP. Responses to phenylephrine were not affected by the AVP‐induced increase in vascular tone. 4 In the presence of AVP, pressor responses to UK‐14,304 were resistant to 0.05 μm prazosin and susceptible to antagonism by 1 μm rauwolscine (‐log Kb 7.65 ± 0.15). Similarly, responses to NA in the presence of 0.05 μm prazosin and AVP were inhibited by 1 μm rauwolscine. This represents the first demonstration of prazosin‐resistant, rauwolscine‐sensitive α2‐adrenoceptor‐mediated responses in the vasculature of the rat tail. 5 These results suggest that in isolated vascular preparations, functional populations of postjunctional α2‐adrenoceptors may be ‘uncovered’ by the presence of AVP.

Expression of functional postjunctional α2-adrenoceptors in rabbit isolated distal saphenous artery—a permissive role for angiotensin II?

In the rabbit isolated distal saphenous artery, the population of postjunctional adrenoceptors is of the α1 variety under normal in vitro experimental conditions, based on the potency order of selective agonists and on the effects of the antagonists prazosin and rauwolscine against responses to UK‐14304. Angiotensin II (AII, 0.05 μm) however, without affecting resting baseline tension, markedly enhanced responses to UK‐14304, particularly at low concentrations. This previously unseen component of the response to UK‐14304 was resistant to prazosin (0.1 μm) but susceptible to rauwolscine (1 μm). AII would therefore appear to have a permissive role for the expression of a quiescent population of postjunctional α2‐adrenoceptors in the rabbit distal saphenous artery.

An examination of the sources of calcium for contractions mediated by postjunctional α1‐ and α2‐adrenoceptors in several blood vessels isolated from the rabbit

1 The roles of intracellular and extracellular‐derived Ca2+ in α‐adrenoceptor‐mediated contractions to noradrenaline (NA) have been investigated in several isolated blood vessels from the rabbit by examining responses in the presence of a modified Krebs‐Henseleit saline with 2.5 mm Ca2+ and a Ca2+‐buffered saline with 0.1 μm free Ca2+. 2 NA was tested in preparations of the abdominal aorta, distal saphenous artery, renal vein, lateral saphenous vein, plantaris vein and ear vein exposed to a Ca2+‐buffered saline with 0.1 μm [Ca2+]. A concentration of NA which was maximally effective in modified Krebs‐Henseleit saline, produced an initial transient contraction (ITC) followed by a relaxation towards baseline. This is evidence that α‐adrenoceptor‐mediated responses in all these blood vessels depend upon calcium from both sources. 3 The ITC was particularly pronounced in the arteries and was associated more closely with the α1‐receptor subtype. In the abdominal aorta, distal saphenous artery and renal vein the ITC can almost exclusively be attributed to an α1‐adrenoceptor (prazosin‐sensitive, rauwolscine‐resistant). In the ear vein, and to a lesser extent the plantaris vein, the ITC was mediated in part by an α2‐adrenoceptor (prazosin‐resistant, rauwolscine‐sensitive). 4 α2‐Adrenoceptors in the lateral saphenous vein largely account for the response to NA in modified Krebs‐Henseleit saline, but α1‐adrenoceptors mediate the ITC in Ca2+‐buffered saline. After selective inactivation of α1‐adrenoceptors with a combination of phenoxybenzamine and rauwolscine, responses to NA in modified Krebs‐Henseleit saline are slow in onset and there is no ITC in Ca2+‐buffered saline. 5 The possible significance of the coupling of postjunctional α2‐adrenoceptors to dual sources of Ca2+ is discussed in relation to the interaction between α‐adrenoceptor subtypes and the ease of demonstrating functional α2‐adrenoceptors in isolated blood vessels.

Effects of basal and acetylcholine‐induced release of endothelium‐derived relaxing factor on contraction to α‐adrenoceptor agonists in a rabbit artery and corresponding veins

1 The effects of an endothelium‐dependent (acetylcholine) and an endothelium‐independent (sodium nitroprusside) relaxant against noradrenaline‐induced contractions were compared in three isolated superficial blood vessels of the rabbit, the lateral saphenous vein, plantaris vein and distal saphenous artery. Both produced concentration‐related relaxations of all three vessels and were more effective against sub‐maximal than maximal contractions to noradrenaline. Transient contractions to high concentration of acetylcholine occurred only in endothelium‐intact preparations of saphenous vein and were inhibited by flurbiprofen. 2 In endothelium‐denuded preparations sodium nitroprusside was 3 times more effective than in endothelium‐intact preparations, while acetylcholine (< 3 μm) was inactive. Sensitivity was similar for each relaxant: lateral saphenous vein ≥ plantaris vein > distal saphenous artery. The similar profile of sodium nitroprusside and acetylcholine suggests that differences in susceptibility to endothelium‐derived relaxing factor (EDRF) are caused by inter‐vessel variations in the excitation‐coupling process for noradrenaline. 3 Haemoglobin inhibited acetylcholine‐induced relaxations in the endothelium‐intact preparation of the lateral saphenous vein and distal saphenous artery, which suggests a similar EDRF in each preparation and the likelihood that this is a single substance, presumably nitric oxide. 4 The influence of basal, spontaneously released EDRF on α‐adrenoceptor function was tested either by mechanical disruption of the endothelium or by adding haemoglobin to endothelium‐intact segments. Endothelial disruption slightly reduced contractions to noradrenaline (NA) in distal saphenous artery but increased response size of lateral saphenous and plantaris veins, in the latter also increasing sensitivity to NA: haemoglobin mimicked endothelial disruption. Thus, basal release of EDRF like acetylcholine and nitroprusside was more effective in the veins than in the corresponding artery. 5 In lateral saphenous vein responses to phenylephrine were enhanced by endothelial disruption, but without change in sensitivity: responses to UK‐14304, B‐HT 920 and cirazoline, which had a relatively slow speed of onset of contraction were not affected. There was no correlation between enhancement and α‐adrenoceptor sub‐type although the agonists which were enhanced all activate α1‐adrenoceptors. Competitive antagonists failed to reveal an α‐adrenoceptor subtype enhanced by endothelial disruption. However, effects of phenoxybenzamine suggest that α1‐adrenoceptors are necessary for the influence of basal EDRF.

Postjunctional α‐adrenoceptors in the rabbit isolated distal saphenous artery: indirect sensitivity to prazosin of responses to noradrenaline mediated via postjunctional α2‐adrenoceptors

1 Under normal experimental conditions, the rabbit isolated distal saphenous artery appears to contain a homogeneous population of postjunctional α1‐adrenoceptors. Prazosin competitively antagonized responses to noradrenaline (NA) with a pA2 value of 8.6, while a relatively high concentration of rauwolscine (1 μm), produced only a 2 fold rightward displacement of the NA cumulative concentration‐response curve (CCRC). 2 Despite the fact that angiotensin II (AII) was without effect on responses to NA or phenylephrine in this preparation, this peptide made responses to NA less susceptible to the antagonistic action of prazosin. This was particularly evident on the lower portion of the CCRC for NA. These results suggest that in the presence of AII, NA produces contractile responses by an action mediated through a prazosin‐resistant adrenoceptor. 3 An attempt was made to isolate a homogeneous population of postjunctional α2‐adrenoceptors by use of a receptor protection procedure involving the combination of rauwolscine and phenoxybenzamine. After the protection protocol no responses were observed to the α‐adrenoceptor agonists NA, phenylephrine or UK‐14304. In the presence of angiotensin II however, concentration‐dependent contractions were observed to each of these agonists. Under these conditions the rank order of potency, UK‐14304 > NA > phenylephrine, is consistent with that of an effect mediated through postjunctional α2‐adrenoceptors. 4 The responses to NA, after the protection protocol, in the presence of AII, were susceptible to the selective α2‐adrenoceptor antagonist, rauwolscine (1 μm), but resistant to the selective α1‐adrenoceptor antagonist prazosin (0.1 μm). Furthermore, the combination of rauwolscine (1 μm) and prazosin (0.1 μm) was no more effective in blocking responses to NA than was rauwolscine (1 μm) alone. These results are consistent with the presence of a homogeneous population of postjunctional α2‐adrenoceptors. 5 Inducing a small degree of tone with a low concentration of the selective α1‐adrenoceptor agonist, phenylephrine, markedly increased the threshold sensitivity to the selective α2‐adrenoceptor agonist UK‐14304, in a manner analogous to that seen with AII. 6 The results in the present study indicate that responses mediated via postjunctional α2‐adrenoceptors in the rabbit isolated distal saphenous artery are dependent upon a degree of vascular smooth muscle stimulation by some other receptor system. It is hypothesized that under normal experimental conditions, this function is fulfilled by stimulation of α1‐adrenoceptors, while after α1‐adrenoceptor blockade the necessary positive influence can be provided by stimulation of AII receptors. The implications for such an interaction between postjunctional α‐adrenoceptor subtypes in demonstrating prazosin‐resistant, rauwolscine‐ or yohimbine‐sensitive responses in isolated blood vessels is discussed.

An examination of the postjunctional α-adrenoceptor subtypes for (−)-noradrenaline in several isolated blood vessels from the rabbit

1 Postjunctional α‐adrenoceptors in several isolated blood vessels from the rabbit have been characterized on the basis of the relative potency of the agonists noradrenaline (NA, non‐selective), phenylephrine (α1‐selective) and UK‐14304 (α2‐selective), and the potency of antagonists rauwolscine (α2‐selective) and corynanthine (α1‐selective) against contractions elicited by NA. In addition, the potency of prazosin against NA was also assessed in the venous preparations. 2 The thoracic aorta, ear artery and left renal vein appear to possess α1‐adrenoceptors since the agonist potency order was NA > phenylephrine > UK‐14304, while corynanthine was 3–10 fold more potent than rauwolscine. 3 The ear vein appears to possess α2‐adrenoceptors. The rank order of agonist potency was UK‐14304 > NA > phenylephrine and all three agonists elicited responses of similar magnitude. Furthermore, rauwolscine was 30 fold more potent than corynanthine while prazosin failed to produce a concentration‐dependent inhibition. 4 The saphenous vein and the plantaris vein appear to possess a mixture of both subtypes since the rank order of agonist potency was UK‐14304 > NA ≫ phenylephrine, while responses elicited by UK‐14304 were smaller than those to the other agonists. However, although rauwolscine was 20 to 100 fold more potent than corynanthine in both preparations, suggestive of predominantly α2‐adrenoceptors, prazosin was either potent (saphenous vein) or relatively inactive (plantaris vein). 5 The characteristics of postjunctional α1‐ and α2‐adrenoceptors on isolated blood vessels from the rabbit are discussed in relation to the value of both the agonists, particularly NA, and the antagonists used in this study.

Evidence that the population of postjunctional-adrenoceptors mediating contraction of smooth muscle in the rabbit isolated ear vein is predominantly α2

1 Noradrenaline (NA), phenylephrine and UK‐14304 elicited concentration‐dependent contractions of the rabbit isolated ear vein of similar maximal magnitude. The rank order of potency, UK‐14304 > noradrenaline > phenylephrine, is consistent with that of an effect mediated through an α2‐subtype. 2 The potent and highly selective α1‐adrenoceptor antagonists prazosin and YM‐12617, at concentrations as high as 1 μm, produced less than a 4 fold rightward displacement of the NA concentration‐response curve. 3 The selective α2‐adrenoceptor antagonists rauwolscine, Wy‐26703 and CH‐38083 antagonized responses to noradrenaline in a competitive manner. For all three antagonists, the pA2 values were consistent with an effect at α2‐adrenoceptors. However, 0.1 μm YM‐12617 increased the potency of rauwolscine 2 fold indicating the presence of a small population of postjunctional α1‐adrenoceptors. 4 The relative antagonist potency of the yohimbine diastereoisomers rauwolscine and corynanthine against noradrenaline (rauwolscine 30 fold > corynanthine) is also consistent with an effect at α2‐adrenoceptors. 5 Contractions elicited by noradrenaline in the rabbit isolated ear vein appear to be mediated predominantly by postjunctional α2‐adrenoceptors.

Influence of angiotensin II on the α‐adrenoceptors involved in mediating the response to sympathetic nerve stimulation in the rabbit isolated distal saphenous artery

Under normal experimental conditions, sympathetic nerve‐mediated responses to electrical field stimulation in the isolated distal saphenous artery of the rabbit are sensitive to prazosin (0.1 μm) and so, by definition, are mediated by α1‐adrenoceptors. In the presence of angiotensin II (A II, 0.05 μm) however, a component of the response to nerve stimulation became resistant to prazosin. This ‘uncovered’ response was virtually abolished by the selective α2‐adrenoceptor antagonist rauwolscine (1 μm), a concentration that in the absence of A II had enhanced nerve‐mediated responses. Exposure to A II therefore, allows the clear demonstration of a role for postjunctional α2‐adrenoceptors in mediating the contractile response to sympathetic nerve stimulation in this arterial preparation.