V. G. Wilson

AGMATINE RECOGNIZES ALPHA 2-ADRENOCEPTOR BINDING SITES BUT NEITHER ACTIVATES NOR INHIBITS ALPHA 2-ADRENOCEPTORS

It has been suggested that agmatine (decarboxylated arginine) is an endogenous clonidine-displacing substance (CDS) which recognizes α2-adrenoceptor and non-adrenoceptor, imidazoline binding sites. We have examined the effect of agmatine at α2-adrenoceptor binding sites and pre- and postjunctional α2-adrenoceptors. Agmatine produced a concentration-dependent inhibition of 1 nmol/l 3H-clonidine binding to both rat (pKi–5.10+-0.05) and bovine (pKi–4.77+-0.38) cerebral cortex membranes. However, agmatine (0.1–100 μM) failed to activate pre-junctional α2-adrenoceptors regulating transmitter release in the guinea-pig isolated ileum and rat isolated vas deferens, nor did it activate post-junctional α2-adrenoceptors of the porcine isolated palmar lateral vein which mediate contraction or inhibition of forskolin-stimulated cyclic AMP formation. High concentrations of agmatine (10–30-fold the pKi at α2-adrenoceptor binding sites) failed to influence α2-adrenoceptor activation by either clonidine or UK-14304 (5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate) in any of the peripheral preparations examined. Moreover, even in a preparation where an interaction with α2-adrenoceptor binding sites on cell membranes can be demonstrated, the rat cerebral cortex, agmatine failed to inhibit forskolin-stimulated cyclic AMP in the intact tissue or affect the inhibition produced by the selective α2-adrenoceptor agonist UK-14304. Agmatine was also devoid of agonist activity in two preparations, the rat isolated thoracic aorta and the rat isolated gastric fundus, in which CDS has been reported to produce non-adrenoceptor effects. Thus, we have confirmed that agmatine recognizes α2-adrenoceptor binding sites and, therefore, is a CDS. However, since agmatine is devoid of pharmacological activity at either peripheral or central α2-adrenoceptors it can not account for earlier reports suggesting that brain-derived CDS can activate α2-adrenoceptors.

The Pseudomonas aeruginosa quorum-sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, inhibits porcine arterial smooth muscle contraction

The Pseudomonas aeruginosa quorum sensing molecule N‐(3‐oxododecanoyl)‐L‐homoserine lactone (OdDHL) has been shown to suppress cytokine production in macrophages. We have examined the effect of OdDHL and related compounds on constrictor tone of porcine blood vessels. OdDHL (1–30 μM) caused a concentration‐dependent inhibition of U46619‐induced contractions of the coronary artery through a largely endothelium‐independent mechanism, but was markedly less effective in the pulmonary artery. Quantitively similar effects to those produced by OdDHL were observed with N‐(3‐oxododecanoyl)‐L‐homocysteine thiolactone, a thiolactone derivative, while N‐3‐oxododecanamide, a lactone‐free acyl analogue, possessed 1/3rd the potency as a vasorelaxant. Neither N‐butanoyl‐L‐homoserine lactone nor L‐homoserine lactone (up to 30 μM) were active. Our findings indicate that OdDHL inhibits vasoconstrictor tone of both pulmonary and coronary blood vessels from the pig. The vasorelaxant action of OdDHL appears to be primarily determined by the N‐acyl chain length, with a minor contribution by the homoserine lactone moiety.

Molecular and pharmacological evidence for MT1 melatonin receptor subtype in the tail artery of juvenile Wistar rats

In this study reverse transcriptase‐polymerase chain reaction (RT–PCR) has been used to identify mt1 and MT2 receptor mRNA expression in the rat tail artery. The contributions of both receptors to the functional response to melatonin were examined with the putative selective MT2 receptor antagonists, 4‐phenyl‐2‐propionamidotetraline (4‐P‐PDOT) and 2‐benzyl‐N‐pentanoyltryptamine. In addition, the action of melatonin on the second messenger cyclic AMP was investigated. Using RT–PCR, mt1 receptor mRNA was detected in the tail artery from seven rats. In contrast MT2 receptor mRNA was not detected even after nested PCR. At low concentrations of the MT2 selective ligands, neither 10 nM 4‐P‐PDOT (pEC50=8.70±0.31 (control) vs 8.73±0.16, n=6) nor 60 nM 2‐benzyl‐N‐pentanoyltryptamine (pEC50=8.53±0.20 (control) vs 8.83±0.38, n=6) significantly altered the potency of melatonin in the rat tail artery. At concentrations non‐selective for mt1 and MT2 receptors, 4‐P‐PDOT (3 μM) and 2‐benzyl‐N‐pentanoyltryptamine (5 μM) caused a significant rightward displacement of the vasoconstrictor effect of melatonin. In the case of 4‐P‐PDOT, the estimated pKB (6.17±0.16, n=8) is similar to the binding affinity for mt1 receptor. Pre‐incubation with 1 μM melatonin did not affect the conversion of [3H]‐adenine to [3H]‐cyclic AMP under basal condition (0.95±0.19% conversion (control) vs 0.92±0.19%, n=4) or following exposure to 30 μM forskolin (5.20±1.30% conversion (control) vs 5.35±0.90%, n=4). Based on the above findings, we conclude that melatonin receptor on the tail artery belongs to the MT1 receptor subtype, and that this receptor is probably independent of the adenylyl cyclase pathway.

Studies on the vasoconstrictor action of melatonin and putative melatonin receptor ligands in the tail artery of juvenile Wistar rats

1 In this study we compared the vasoconstrictor activity of melatonin in rat isolated tail artery using two different recording systems, the Halpern pressure myograph and the Halpern‐Mulvany wire myograph, with the view to determining a reliable method for obtaining pharmacological data on vascular melatonin receptors. In addition, we characterized the melatonin receptor in this preparation, using analogues of melatonin, and examined the activity of various naphthalenic derivatives with biological activity in non‐vascular models of melatonin receptors. 2 Using the Halpern pressure myograph, cumulative addition of melatonin (0.1 nM to 1 μM) produced direct vasoconstriction (19.3±6.4% reduction in lumen diameter, n=5) in five of 11 pressurized segments, with pEC50 of 9.14±0.17. Similarly, non‐cumulative application of melatonin caused vasoconstriction (19.7±4.6% reduction in lumen diameter, n=7) in seven of 20 preparations examined with pEC50 of 8.74±0.26. The selective alpha2‐adrenoceptor agonist, UK‐14304 (5‐bromo‐6‐[2‐imidazolin‐2‐ylamino]‐quinoxaline bitartrate), produced vasoconstriction in all ‘melatonin‐insensitive’ preparations. 3 Melatonin (0.1 nM to 1 μM) failed to elicit isometric contractions of tail artery segments in the Halpern wire myograph, but produced concentration‐dependent potentiation of electrically‐evoked, isometric contractions (maximum effect of 150–200% enhancement) when applied either non‐cumulatively (seven of seven preparations) or cumulatively (four of seven preparations). The pEC50 value of melatonin (non‐cumulative) was 8.50±0.10 (n=7) which was not different from that obtained in the pressure myograph. All further experiments were conducted using a non‐cumulative protocol against electrically‐evoked, isometric contractions. 4 Based on the pEC50 values for the melatonin analogues examined, the pharmacological profile for the enhancement of electrically‐evoked contractions was 2‐iodomelatonin>6‐chloromelatonin(−)‐AMMTC□thinsp;5 S21634melatoninS20098>S20242S20304>6‐ hydroxymelatonin>S20932> (+) ‐AMMTC>N‐acetyl‐5‐HT. Our data suggests the vascular receptor belongs to the MEL1‐like subtype. All the indole‐based analogues of melatonin, 2‐iodomelatonin, (−)‐AMMTC, (+)‐AMMTC, S20932, 6‐chloromelatonin, 6‐hydroxymelatonin and N‐acetyl‐5‐HT, behaved as full agonists. All the naphthalenic derivatives examined, S21634, S20098, S20242 and S20304 behaved as partial agonists relative to melatonin. 5 The naphthalenic‐based antagonists, S20928 and S20929, did not modify electrically‐evoked, isometric contractions of the tail artery, but produced a parallel, rightward displacement of the melatonin concentration‐response curve. Based upon the effect of 1 μM S20928 and S20929, the estimated pKB values for these antagonists were 7.18±0.25 (n=4) and 7.17±0.25 (n=5), respectively. 6 We demonstrated that enhancement of electrically‐evoked, isometric contractions of the rat isolated tail artery (using the Halpern‐Mulvany wire myograph) is a simple and reproducible model for assessing the activity of putative agonists, partial agonists and antagonists at vascular melatonin receptors. Pharmacological characterization of the receptor suggests the presence of a MEL1‐like subtype.

Evidence for direct vasoconstrictor activity of melatonin in “pressurized” segments of isolated caudal artery from juvenile rats

SummaryResponses of isolated, 60 mmHg ‘pressurized’ segments of the distal caudal artery of adult and juvenile Wistar rats to melatonin and the selective α2-adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-ylamino]-quinoxaline bitartrate (UK-14304) were examined using the Halpern pressure myograph. Melatonin showed no direct vasoconstrictor activity in vessels from adult rats, whereas UK-14304 produced moderate vasoconstriction (pD2- 7.43+-0.09). In the presence of phenylephrine-induced tone, melatonin produced a variable but small constrictor response (< 10µm reduction in diameter) in some vessels; the response to 1 gmol/l UK-14304 was less than in the absence of tone. In vessels isolated from juvenile rats, melatonin caused concentration-dependent vasoconstriction with a maximum response about 70% of the maximum response elicited by UK-14304. Vessels from juvenile rats were more sensitive to melatonin(pD2- 9.40+-0.07) than they were to UK-14304 (pD2 -8.12+-0.14). In the presence of phenylephrine-induced tone, the vasoconstrictor responses to both melatonin and IK-14304 were markedly less; the sensitivity to melatonin was not different from that seen in the absence of tone. These findings indicate that ‘pressurized’ segments of the isolated distal caudal artery may provide a simple and convenient, functional model of melatonin receptors. The findings also appear to implicate melatonin in thermoregulatory processes in juvenile rats.

Quercetin and its major metabolites selectively modulate cyclic GMP-dependent relaxations and associated tolerance in pig isolated coronary artery

Background and purpose:  Quercetin is a major flavonoid that contributes to the reduced risk of cardiovascular disease associated with dietary ingestion of fruits and vegetables. We have pharmacologically characterized the effect of quercetin, and its sulphate and glucuronide metabolites, on vasoconstrictor and vasodilator responses in the porcine isolated coronary artery.

Comparison of the interaction of agmatine and crude methanolic extracts of bovine lung and brain with α2‐adrenoceptor binding sites

1 In the present study we have evaluated whether α2‐adrenoceptor binding sites on bovine cerebral cortex membranes labelled by [3H]‐clonidine, [3H]‐idazoxan and [3H]‐RX‐821002 can distinguish between known agonists and antagonists. This model has then been used to compare the binding profiles of the putative non‐catecholamine, clonidine‐displacing substance (CDS), agmatine and crude methanolic extracts of bovine lung and brain. 2 Saturation studies carried out in the presence and absence of noradrenaline, 10 μmol l−1, revealed that the maximum number of binding sites on bovine cerebral cortex membranes for [3H]‐idazoxan and [3H]‐RX‐821002 were approximately 60–80% greater than those for [3H]‐clonidine (62.6 fmol mg−1 protein). Rauwolscine, the selective α2‐adrenoceptor antagonist, was approximately 100 fold more potent against each of the ligands than the selective α1adrenoceptor diastereoisomer, corynanthine. Also, the pKi value for the selective α1‐adrenoceptor prazosin against each ligand was less than 6. 3 Adrenaline, UK‐14034, rauwolscine, corynanthine, RX‐811059 and prazosin produced concentration‐dependent inhibition of binding of all three 3H‐ligands. The agonists, adrenaline and UK‐14304, were approximately 5 and 10 fold less potent against [3H]‐idazoxan and [3H]‐RX‐821002, respectively, than against [3H]‐clonidine. In marked contrast, the antagonists, rauwolscine, corynanthine, RX‐811059 and prazosin exhibited a different profile, being approximately 2–3 fold more potent against sites labelled by [3H]‐RX‐821002 and [3H]‐idazoxan compared to sites labelled by [3H]‐clonidine. 4 Agmatine and histamine produced a concentration‐dependent displacement of [3H]‐clonidine, [3H]‐idazoxan and [3H]‐RX‐821002 binding to bovine cerebral cortex membranes. The pKi values for agmatine and histamine were independent of the 3H‐ligand employed, approximately 4.8 and 4.5, respectively. 5 Crude methanolic extracts of bovine brain and lung produced a concentration‐dependent inhibition of [3H]‐clonidine binding to bovine cerebral cortex membranes (>90%). Based on the volume of the extract that caused 50% inhibition of [3H]‐clonidine binding, bovine lung contains 3 fold more CDS than bovine brain. Both extracts were at least 5 fold more potent against α2‐adrenoceptor sites labelled by [3H]‐clonidine than those labelled by [3H]‐idazoxan and [3H]‐RX‐821002. 6 All three 3H‐ligands label the same population of α2‐adrenoceptor binding sites on bovine cerebral cortex membranes, but [3H]‐clonidine appears to label selectively the ‘agonist’ state of the sites: for which known agonists, adrenaline and UK‐14304, exhibit a higher affinity. Our results indicate that neither agmatine nor histamine can account for the CDS activity present in crude extracts of bovine brain and lung. Moreover, these extracts appear to possess a binding profile similar to that of adrenaline and UK‐14304, suggesting that they may possess agonist activity.

Examination of the role of inhibition of cyclic AMP in α2-adrenoceptor mediated contractions of the porcine isolated palmar lateral vein

1 We have examined the effect of elevation of cellular adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) on α1‐ and α2‐adrenoceptor‐mediated contraction of the isolated palmar lateral vein of the pig. Cellular cyclic AMP was increased by either inhibition of phosphodiesterase by rolipram, or direct activation of adenylyl cyclase by forskolin. 2 Noradrenaline (1 nm–10 μm) caused concentration‐dependent contractions of the porcine isolated palmar lateral vein (pD2 7.32 ± 0.07, n = 10). The selective α1‐adrenoceptor antagonist, prazosin (0.1 μm) and the selective α2‐adrenoceptor antagonist, rauwolscine (1 μm) caused a 10 fold rightward displacement of the concentration‐response curve and a combination of the two antagonists caused a 200 fold rightward displacement of the concentration‐response curve. The selective α2‐adrenoceptor agonist, UK‐14304, also produced concentration‐dependent contractions of the palmar lateral vein (pD2 7.70 ± 0.15, n = 5), but the maximum response was 55.5 ± 7.6% (n = 5) of that produced by noradrenaline. Prazosin (0.1 μm) failed to affect responses to UK‐14304 but rauwolscine, 1 μm, caused a 200 fold rightward displacement. The estimated pKB value for rauwolscine (8.28 ± 0.19, n = 10) is consistent with inhibition of α2‐adrenoceptors. Thus, the porcine isolated palmar lateral vein has a population of α1‐ and α2‐adrenoceptors capable of producing a contraction. 3 Rolipram, 10 μm, and forskolin, 1 μm, caused a 2–3 fold rightward displacement of the noradrenaline concentration‐response curve (CRC), but 1,9‐dideoxyforskolin, 1 μm, a forskolin analogue which does not activate adenylyl cyclase, failed to produce a significant inhibition of noradrenaline‐induced contractions. The combination of forskolin (1 μm) and rolipram (10 μm) were additive, producing a 20 fold rightward displacement of the noradrenaline CRC. 4 Responses to noradrenaline were similarly affected by a combination of rolipram (10 μm) and prazosin (0.1 μm) (isolation of α2‐adrenoceptors) and the combination of rolipram (10 μm) and rauwolscine (1 μm) (isolation of α1‐adrenoceptors), resulting in a 100 fold rightward displacement of the noradrenaline CRC. Although forskolin inhibited both α1‐ and α2‐adrenoceptor‐mediated contractions, the effects produced were not similar. In particular, noradrenaline, 0.3–3 μm, produced a significant contraction in the presence of forskolin (1 μm) and prazosin (0.1 μm) (an α2‐adrenoceptor‐mediated response) but not in the presence of forskolin (1 μm) and rauwolscine (1 μm) (an α1‐adrenoceptor‐mediated response). 5 Five minute exposure to either rolipram (10 μm) or forskolin (1 μm) elevated [3H]‐cyclic AMP of the porcine isolated palmar lateral vein by approximately 70% and 150–200%, respectively. Neither noradrenaline (1 nm − 100 μm) nor UK‐14304 (1 nm−100 μm) affected basal levels of [3H]‐cyclic AMP, but both produced a concentration‐dependent inhibition of forskolin‐stimulated [3H]‐cyclic AMP accumulation with a pKi of 7.43 ± 0.1 (n = 3) and 7.97 ± 0.18 (n = 3), respectively. The effect of noradrenaline against forskolin‐stimulated [3H]‐cyclic AMP accumulation was reversed by rauwolscine (1 μm) but not by prazosin (0.1 μm). In contrast, α2‐adrenoceptor activation did not affect rolipram‐induced elevation of [3H]‐cyclic AMP. 6 These findings indicate that α2‐adrenoceptor contractions of the porcine isolated palmar lateral vein are not produced by reduction in cellular cyclic AMP per se. It is proposed that this response involves a novel signal transduction mechanism. However, when cellular cyclic AMP has been elevated by agents that stimulate adenylyl cyclase, rather than through inhibition of phosphodiesterase, the ability of α2‐adrenoceptors to inhibit cyclic AMP formation may be of functional importance in vascular smooth muscle.

Quercetin and its principal metabolites, but not myricetin, oppose lipopolysaccharide-induced hyporesponsiveness of the porcine isolated coronary artery

BACKGROUND AND PURPOSE Quercetin is anti‐inflammatory in macrophages by inhibiting lipopolysaccharide (LPS)‐mediated increases in cytokine and nitric oxide production but there is little information regarding the corresponding effect on the vasculature. We have examined the effect of quercetin, and its principal human metabolites, on inflammatory changes in the porcine isolated coronary artery.

α2‐Adrenoceptor‐mediated contractions of the porcine isolated ear artery: evidence for a cyclic AMP‐dependent and a cyclic AMP‐independent mechanism

1 The aim of this study was to determine the conditions under which the α2‐adrenoceptor agonist UK14304 produces vasoconstriction in the porcine isolated ear artery. 2 UK14304 (0.3 μm) produced a small contraction of porcine isolated ear arteries which was 7.8±3.3% of the response to 60 mm KC1. Similar sized contractions were obtained after precontraction with either 30 nm angiotensin II, or 0.1 μm U46619 (8.2±1.8% and 10.2±2.6% of 60 mm KC1 response, respectively). However, an enhanced α2‐adrenoceptor response was uncovered if the tissue was precontracted with U46619, and relaxed back to baseline with 1–2 μm forskolin before the addition of UK14304 (46.9±9.6% of 60 mm KC1 response). 3 The enhanced responses to UK14304 in the presence of U46619 and forskolin were not inhibited by the α1‐adrenoceptor antagonist prazosin (0.1 μm), but were inhibited by the α2‐adrenoceptor antagonist rauwolscine (1 μm), indicating that the enhanced responses were mediated via postjunctional α2‐adrenoceptors. 4 In the presence of 0.1 μm U46619 and 1 mm isobutylmethylxanthine (IBMX), 1 μm forskolin produced an increase in [3H]‐cyclic AMP levels in porcine isolated ear arteries. Addition of 0.3 μm UK14304 prevented this increase. 5 The enhanced UK14304 response was dependent upon the agent used to relax the tissue. After relaxation of ear arteries precontracted with 10 nm U46619 and relaxed with forskolin the UK14304 response was 46.9±9.6% of the 60 mm KC1 response, and after relaxation with sodium nitroprusside (SNP) the response was 24.8±3.3%. However, after relaxation of the tissue with levcromakalim the UK14304 response was only 8.2±1.7%, which was not different from the control response in the same tissues (12.2±5.6%). An enhanced contraction was also obtained after relaxation of the tissue with the cyclic AMP analogue dibutyryl cyclic AMP (23.2±1.3%) indicating that at least part of the enhanced response to UK14304 is independent of the ability of the agonist to inhibit cyclic AMP production. 6 Relaxation of U46619 contracted ear arteries with SNP could be inhibited by the NO‐sensitive guanylyl‐cyclase inhibitor 1H‐[1,2,4] oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) indicating that production of cyclic GMP is necessary for the relaxant effect of SNP. However, ODQ had no effect on the relaxation of tissue by forskolin, suggesting that this compound does not act via production of cyclic GMP. Biochemical studies showed that while forskolin increases the levels of cyclic AMP in the tissues, SNP had no effect on the levels of this cyclic nucleotide. 7 In conclusion, enhanced contractions to the α2‐adrenoceptor agonist UK14304 can be uncovered in porcine isolated ear arteries by precontracting the tissue with U46619, followed by relaxation back to baseline with forskolin, SNP or dibutyryl cyclic AMP before addition of UK14304. There was a greater contractile response to UK14304 after relaxation with forskolin than with SNP or dibutyryl cyclic AMP, suggesting that cyclic AMP‐dependent and‐ independent mechanisms are involved in the enhancement of the UK14304 response.