Pieter H. Van der Graaf

Analysis of α1L‐adrenoceptor pharmacology in rat small mesenteric artery

To illuminate the controversy on α1A‐ or α1L‐adrenoceptor involvement in noradrenaline‐mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype‐selective α1‐adrenoceptor agonists and antagonists under different experimental conditions. The agonist potency order in rat SMA was: A61603 >> SKF89748‐A > cirazoline > noradrenaline > ST‐587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29–8.80) indicating the involvement of α1L‐ rather than α1A‐adrenoceptors. The putative α1L‐adrenoceptor antagonist JTH‐601, but not the α1B‐adrenoceptor antagonist chloroethylclonidine (10 μM) antagonized noradrenaline‐induced contractions of SMA. The potency of the selective α1D‐adrenoceptor antagonist BMY 7378 against noradrenaline (pA2=6.16±0.13) and of the selective α1A‐adrenoceptor antagonist RS‐17053 against noradrenaline (pKB=8.35±0.10) and against the selective α1A‐adrenoceptor agonist A‐61603 (pKB=8.40±0.09) were too low to account for α1D‐ and α1A‐adrenoceptor involvement. The potency of RS‐17053 (pKB/pA2s=7.72–8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619. Selective protection of a putative α1A‐adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS‐17053 (pA2=8.25±0.06 against A61603). Combined concentration‐ratio analysis demonstrated that tamsulosin, which does not discriminate between α1A‐ and α1L‐adrenoceptors, and RS‐17053 competed for binding at the same site in the SMA. In summary, data obtained in our experiments in rat SMA indicate that the α1‐adrenoceptor mediating noradrenaline‐induced contraction displays a distinct α1L‐adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that α1L‐adrenoceptors represent an affinity state of the α1A‐adrenoceptor in functional assays. Furthermore, there is no co‐existing α1A‐adrenoceptor in the SMA.

Analysis of α1-adrenoceptors in rabbit lower urinary tract and mesenteric artery

In this study, we have investigated the effects of a series of alpha1-adrenoceptor antagonists on the phenylephrine-mediated contractions of rabbit isolated prostate, urethra, trigone and mesenteric artery. With the exception of RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dim ethyl-1 H-indole-3-ethanamine hydrochloride), the antagonists displayed the lowest potency in the urethra. Catecholamine uptake1 and uptake2 appeared not to be the cause for the low pK(B)/pA2 values obtained in the urethra because cocaine and corticosterone had no effect on the potency of phenylephrine in this tissue. The low potencies displayed by prazosin. RS-17053 and HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)amino )propyl)benzene-acetonitrile fumarate) suggest that the functional receptors in all four tissues belong to the alpha(1L)-adrenoceptor class. Whether or not the significant between-tissue differences in antagonist potencies are due to heterogeneity of this receptor class remains to be elucidated.

[Arg8]Vasopressin-induced responses of the human isolated coronary artery: effects of non-peptide receptor antagonists

Contractions induced by [Arg8]vasopressin (vasopressin) and the effect of nonpeptide vasopressin receptor antagonists were studied in the human isolated coronary artery. Vasopressin induced contraction of coronary artery segments with a high pD2 (9.25) but a low Emax (11.8% of the response to 100 mM K+). This response was not affected by removal of the endothelium. Contraction was antagonized by the vasopressin V1 receptor antagonist SR 49059 ((2S) 1-[(2R 3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene- sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2- carboxamide) (pA2: 9.76). OPC-31260 ([5-dimethylamino-1-(4-(2-methylbenzoylamino)benzoyl)-2,3,4,5-tetr ahydro-1H- benzazepine]: vasopressin V2 receptor antagonist) and OPC-21268 (1-(1-[4-(3-acetylaminopropoxy) benzoyl]-4-piperidyl)-3,4- dihydro-2(1H)-quinolinone: reported vasopressin V1 receptor antagonist) were less potent antagonists of vasopressin-induced contractions (pA2: 7.31 and 5.6, respectively). The antagonist potency order (SR 49059 > OPC-31260 > OPC-21268) corresponds to the reported affinity order for the human cloned vasopressin V1 receptor. Therefore, the vasopressin V1 receptor antagonist SR 49059, but not OPC-21268, appears to be an appropriate tool to investigate further the role of vasopressin in pathological processes involving coronary vasoconstriction in humans.

Characterization of receptors mediating contraction of the rat isolated small mesenteric artery and aorta to arginine vasopressin and oxytocin

1 The exact nature of the receptor subtype(s) involved in the action of arg‐vasopressin (AVP) on the rat aorta and small mesenteric artery (SMA) is controversial. Therefore, we have studied the effects of the selective V1A receptor antagonists, OPC 21268 and SR 49059, and the oxytocin (OT) receptor antagonist, atosiban, on the AVP‐ and OT‐induced contractions of the two vessels. 2 AVP and OT displayed similar intrinsic activities in the rat aorta and SMA, but AVP was ∼130 fold and ∼500 fold more potent than OT, respectively. In the rat aorta, Hill slopes (nH) were similar for OT and AVP. However, in rat SMA, the OT concentration‐effect (E/[A]) curve was significantly steeper than the AVP E/[A] curve (nH,=3.3±0.20, 2.3±0.15; P<0.001). 3 In the aorta OPC 21268, SR 49059 and atosiban competitively antagonized the AVP and OT E/[A] curves. Except for atosiban and SR 49059 against AVP, competitive antagonism was also observed in the SMA. Atosiban caused concentration‐dependent steepening of the AVP E/[A] curve, whereas SR 49059 decreased the upper asymptote. 4 Schild analysis yielded affinities indicative of V1A receptor involvement in both vessels: pKB/pA2=9.20–9.48, 7.56–7.71 and 6.19–6.48 for SR 49059, OPC 21268 and atosiban, respectively. 5 Neither AVP nor OT relaxed U46619 pre‐contracted aorta or SMA in the presence of SR 49059, suggesting no interference of a vasodilatory component. 6 Despite predominant involvement of V1A receptors in both vessels, the different Hill slopes of AVP and OT E/[A] curves as well as the steepening of the AVP E/[A] curves by atosiban are indicative of receptor heterogeneity in the rat SMA.

Characterisation of the 5-HT receptor potentiating neurotransmission in rabbit bladder

We have investigated the effects of 5-hydroxytryptamine (5-HT) on electrically induced contractions of rabbit isolated bladder. Electrical field stimulation evoked twitch contractions which were potentiated by 5-HT (0.3-10 microM). The potentiating effect of 5-HT was inhibited by ondansetron (pA2 9.2) and granisetron (pA2 9.1) but not by methysergide or SB 204070 ((1-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxan-5-carboxylate hydrochloride). This suggests that the potentiating effect of micromolar concentrations of 5-HT on neuromuscular transmission in rabbit isolated bladder is mediated by 5-HT3 receptors. The receptors involved in the response to lower concentrations of 5-HT, observed in some tissues, remain to be characterised.

Functional characterisation of the pharmacological profile of the putative α1B-adrenoceptor antagonist, (+)-cyclazosin

We studied the functional pharmacological profile of (+)-cyclazosin, which has been characterised as a selective, high-affinity (pKi = 9.68) alpha1B-adrenoceptor ligand in binding experiments with rat liver membranes. The pKa/pA2 values for antagonism of contractions mediated via alpha1A/L-adrenoceptors of rat small mesenteric artery, alpha1B-adrenoceptors of rat aorta and alpha1B-adrenoceptors of rat spleen were 7.78 +/- 0.04, 6.86 +/- 0.07 and 7.96 +/- 0.08, respectively. Furthermore, in mouse spleen, which is also regarded as an alpha1B-adrenoceptor preparation, (+)-cyclazosin displayed low potency and did not act as a competitive antagonist. Thus, in contrast with results obtained in radioligand binding experiments, (+)-cyclazosin does not behave as a selective alpha1B-adrenoceptor antagonist in functional tissues. Whether this discrepancy has consequences for the classification of alpha1-adrenoceptors requires further investigation.

Exposure and characterization of the action of noradrenaline at dopamine receptors mediating endothelium‐independent relaxation of rat isolated small mesenteric arteries

1 Previously, we reported that noradrenaline (NA), in addition to its α1‐adrenoceptor‐mediated contractile effect, may relax the rat small mesenteric artery (SMA) in order to account for steep Schild plots obtained with compounds classified as α1‐adrenoceptor antagonists. In this study, a relaxant action of NA has been exposed in the rat isolated, endothelium‐denuded SMA precontracted by the thromboxane A2‐mimetic, U46619. 2 NA, but not the selective α2‐adrenoceptor agonist, UK14304, produced concentration‐dependent contraction of the SMA (pEC50 = 5.7 ± 0.1). After precontraction with 0.1 μm U46619, 10 nM – 30 μm NA produced a further contraction (pEC50 = 6.1 ± 0.2), while higher concentrations of NA produced small, but significant, relaxant responses. 3 In the presence of 1 μm prazosin, 0.1–30 μm NA produced concentration‐dependent relaxation (pIC50 = 5.9 ± 0.1) after precontraction with 0.1 μm U46619. The NA relaxation concentration‐effect curve was completely inhibited by 1 μm of the β/β‐adrenoceptor antagonist, timolol. However, when the concentration of prazosin was increased by 10 fold (10 μm), NA once again produced concentration‐ dependent relaxation (pIC50 = 4.5 ± 0.2). This relaxation concentration‐effect curve was not blocked by a 10 fold higher concentration of timolol (10 μm), nor by the presence of idazoxan (10 μm), cyanopindolol (10 μm), NG‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μm), indomethacin (10 μm) or sulpiride (1 μm). However, haloperidol (10 μm) and ±‐SCH‐23390 (10 nM) produced significant inhibition of the relaxation, suggesting the involvement of dopamine D1 receptors. 4 Dopamine also produced concentration‐dependent relaxation following U46619 precontraction (pIC50 = 5.4 ± 0.1) which was significantly inhibited by haloperidol and (±)‐SCH‐23390. Pretreatment with 10 μm phenoxybenzamine for 60 min produced a significant inhibition of the dopamine and NA relaxation curves and application of the operational model of agonism yielded estimates of the affinity (pKA = 5.3 ± 0.2 and 4.4 ± 0.2) and efficacy (log τ = 0.06 ± 0.11 and 0.01 ± 0.10) for dopamine and NA, respectively, at D1receptors. 5 HV723 (0.1 and 1 μm), a ligand that yielded a Schild plot slope parameter of unity as an antagonist of NA in the contractile assay, produced concentration‐dependent inhibition of the NA‐mediated relaxation (pA2∼8). 6 The results of this study indicate that NA can activate D1receptors mediating relaxation in the rat SMA at concentrations which were encountered in our previous receptor classification experiments using competitive α1‐adrenoceptors; antagonists.

Analysis of the action of idazoxan calls into question the reliability of the rat isolated small mesenteric artery assay as a predictor for α1-adrenoceptor-mediated pressor activity

We have studied the effects of idazoxan in rat aorta and small mesenteric artery. In the aorta, idazoxan behaved as a partial agonist (pKA=6.30). Prazosin produced rightward shift (pA2=9.88) and steepening of the idazoxan curve. In contrast, idazoxan had no effect of basal tension in the mesenteric artery, but shifted the noradrenaline curve to the right in a parallel manner (pA2=6.12). The selective al-adrenoceptor agonist, indanidine, also behaved as a partial agonist in the aorta and produced no significant contractions of the small mesenteric artery. Since idazoxan and indanidine have been reported to raise blood pressure in the pithed rat via an action at vascular α1-adrenoceptors, these results call into question the reliability of the small mesenteric artery assay as a predictor for α1-adrenoceptor-mediated pressor activity in vivo.

The α1-adrenoceptor agonist, SDZ NVI-085, behaves as a potent, competitive antagonist of 5-hydroxytryptamine-induced contraction of rat aorta

In this study we investigated the actions of SDZ NVI-085 ((-)-(4aR,10aR)-3,4,4a,5,10,10a-hexahydro-6-methoxy-4-methyl -9-(methylthio)-2H- naphth[2,3-b)-1,4-oxazine hydrogen malonate), previously classified and employed as a selective alpha 1-adrenoceptor agonist, in the rat isolated aorta assay. It was shown that, in addition to its alpha 1-adrenoceptor agonistic action, SDZ NVI-085 behaves as a competitive antagonist of 5-hydroxytryptamine (5-HT)-induced contraction of rat aorta (pKB = 8.13 +/- 0.08). The structurally related alpha 1-adrenoceptor agonist, SK & F 89748-A (l-1,2,3,4-tetrahydro-8-methoxy-5-(methylthio)-2-naphthalenamine hydrochloride), produced inhibition of the 5-HT response only at the highest concentration tested (1 microM) with an associated pA2 value of 6.0 +/- 0.1. These findings suggest that the affinity of SDZ NVI-085 for 5-HT2 receptors is considerably higher than for alpha 1-adrenoceptors, which may have implications for its use as a pharmacological tool.

Benextramine acts as an irreversible noncompetitive antagonist of U46619-mediated contraction of the rat small mesenteric artery

We have studied the effects of benextramine on the U46619 (11 alpha,9 alpha-epoxymethano-15S-hydroxy-prosta-5Z,13E-dienoic acid)-mediated contraction of the rat isolated small mesenteric artery. U46619 (10 nM-10 microM) produced a concentration-dependent contraction of the small mesenteric artery. The selective prostanoid TP receptor antagonist, SQ 30,741 ([1S-[1 alpha,2 alpha(5Z),3 alpha,4 alpha]]-7- [[[[[(oxaheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hep t-2-yl]-5- heptenoic acid; 1 microM), produced a parallel, rightward shift of the U46619 curve with an associated pA2 value of 7.43 +/- 0.09. Treatment of tissues with 100 microM benextramine depressed the maximum response to U46619 in a time-dependent manner. However, neither SQ 30,741 (10 microM) nor U46619 (10 microM) incubation significantly protected against this effect. Thus, benextramine acts as an irreversible noncompetitive antagonist of U46619. The mechanism of this action is not yet clear.