William R. Dunn

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.

The role of gap junctions in mediating endothelium‐dependent responses to bradykinin in myometrial small arteries isolated from pregnant women

Endothelium‐dependent responses were assessed in myometrial small arteries isolated from pregnant women, using pressure myography. Responses to bradykinin were unaffected by the combined presence of the nitric oxide synthase (NOS) inhibitor, N‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) and the cyclo‐oxygenase inhibitor, indomethacin (10 μM). The additional presence of clotrimazole (50 μM) attenuated, but did not abolish, vasodilator responses to bradykinin. Raising extracellular [K+] (by between 1 and 15 mM) did not evoke a vasodilator response, nor did the cannabinoids, anandamide and methanandamide. Responses to bradykinin were attenuated in the presence of the gap junction inhibitors 18‐α‐glycyrrhetinic acid (18‐α GA, 100 μM), carbenoxolone (100 μM) and palmitoleic acid (50 μM). SR141716A, the CB1 receptor antagonist attenuated responses to bradykinin, but only at high concentrations (10 μM). These results suggest that gap junctional communication is involved in the nitric oxide (NO)‐ and prostanoid‐independent vasodilator responses to bradykinin in myometrial small arteries in normal pregnancy.

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.

Pressure-Induced Myogenic Responses in Human Isolated Cerebral Resistance Arteries

BACKGROUND AND PURPOSE Pressure-induced myogenic responses have been demonstrated in cerebral resistance arteries isolated from a number of species. In the present study, we determined the response of human isolated cerebral resistance arteries to a pressure stimulus. METHODS Arteries were set up in a pressure myograph and exposed to alterations in intravascular pressure. RESULTS Human isolated cerebral resistance arteries developed spontaneous intrinsic tone in response to a pressure stimulus over the pressure range of 20 to 90 mm Hg that was not apparent in the absence of extracellular calcium. This intrinsic tone may be an inherent property of the vascular smooth muscle, since it remained after functional removal of the endothelium. CONCLUSIONS Human isolated cerebral resistance arteries spontaneously contract when exposed to raised intravascular pressure. This pressure-induced myogenic response may contribute to cerebral autoregulation of blood flow.

ATP is the predominant sympathetic neurotransmitter in rat mesenteric arteries at high pressure

Most studies of neurovascular transmission in isolated small mesenteric arteries have used either isometric recording techniques or measured vasoconstriction in vessels with no distending pressure. Here we have used pressure myography to assess the contribution of noradrenaline and ATP to sympathetic neurotransmission in rat second‐order mesenteric arteries. In arteries pressurized to 30 or 90 mmHg, activation of sympathetic axons with trains of electrical stimuli (50 pulses, 0.5–10 Hz) evoked frequency‐dependent vasoconstrictions that increased in amplitude at higher pressure. In the presence of the P2‐receptor antagonist suramin (0.1 mm), the amplitude of vasoconstrictions to trains at 2 and 10 Hz did not differ at 30 and 90 mmHg. In contrast, in the presence of the α1‐adrenoceptor antagonist prazosin (0.1 μm) vasoconstrictions at 90 mmHg were larger than those at 30 mmHg. At both pressures, the combination of prazosin and suramin virtually abolished constrictions. The purinergic component of vasoconstriction (prazosin‐resistant) was almost abolished by the L‐type Ca2+ channel antagonist nifedipine (1 μm). Increasing pressure from 30 to 90 mmHg decreased the resting membrane potential and increased the amplitude of purinergic excitatory junction potentials. These findings indicate that the contribution of ATP to neurovascular transmission increases when the pressure is raised from 30 to 90 mmHg, which is similar to the pressure second‐order mesenteric arteries experience in vivo, and that Ca2+ influx through L‐type Ca2+ channels is largely responsible for purinergic activation of the vascular smooth muscle.

Electrochemical and electrophysiological characterization of neurotransmitter release from sympathetic nerves supplying rat mesenteric arteries

Characteristic features of noradrenaline (NA) and adenosine 5′‐triphosphate (ATP) release from postganglionic sympathetic nerves in rat small mesenteric arteries in vitro have been investigated on an impulse‐by‐impulse basis. NA release was measured using continuous amperometry and ATP release was monitored by intracellular recording of excitatory junction potentials (e.j.ps). Electrical stimuli evoked transient increases in oxidation current. During trains of ten stimuli at 0.5–4 Hz there was a depression in the amplitude of oxidation currents evoked following the first stimulus in the train. The neuronal NA uptake inhibitor, desmethylimipramine (1 μM), increased the amplitude of the summed oxidation current evoked by ten stimuli at 1 Hz and slowed the decay of oxidation currents evoked by trains of ten stimuli at 1 and 10 Hz. The α2‐adrenoceptor antagonist, idazoxan (1 μM), increased the amplitudes of the oxidation currents evoked during trains of ten stimuli at 0.5–10 Hz but had no effect on the oxidation currents evoked by the first stimulus in the train. Idazoxan (1 μM) increased the amplitude of all e.j.ps evoked during trains of stimuli at 0.5 and 1 Hz. In addition, the facilitatory effect of idazoxan on e.j.ps was significantly greater than that on oxidation currents. The findings indicate that NA release from sympathetic nerves supplying small mesenteric arteries is regulated by activation of presynaptic α2‐adrenoceptors and that clearance of released NA in this tissue depends, in part, upon neuronal uptake. The different effects of idazoxan on the oxidation currents and e.j.ps may indicate that the release of NA and ATP is differentially modulated.

Remodelling and enhanced myogenic tone in cerebral resistance arteries isolated from genetically hypertensive brattleboro rats

In this study, we examined the structural and functional properties of cerebral resistance arteries isolated from normotensive (Di/N), and hypertensive (Di/H), vasopressin-deficient rats. Di/H rats had a significantly higher mean arterial blood pressure (MAP, 159 ± 3 mm Hg) than Di/N rats (125 ± 2 mm Hg). Vessels were set up in a pressure myograph, and the internal diameter and wall thickness were determined at increasing intraluminal pressures under passive (calcium-free) conditions. Arteries were then pressurized to the MAP of the animal, from which they were isolated and fixed with glutaraldehyde, embedded in araldite, sectioned and examined histologically. Under passive conditions, the middle cerebral artery (MCA) from Di/H rats had a smaller internal diameter than the MCA isolated from Di/N rats at all distending pressures. This smaller internal diameter of vessels from hypertensive rats is characteristic of eutrophic inward remodelling, whereby a similar amount of wall material is organized around a smaller lumen, without vascular growth or an alteration in artery distensibility. We have previously shown that similar structural alterations occur in mesenteric resistance arteries isolated from Di/H rats. In the presence of extracellular calcium (1.6 mmol/l), the MCA isolated from Di/H rats had significantly more intrinsic tone than the MCA isolated from Di/N rats in the pressure range of 10–110 mm Hg, although arteries from both strains had a similar myogenic index. The increased intrinsic constriction was a specific enhancement of pressure-induced tone, since responses to the thromboxane mimetic, U46619, were decreased, rather than increased, in the MCA isolated from Di/H rats. Furthermore, it is unlikely that the increased intrinsic tone in arteries isolated from Di/H rats was due to an impaired endothelial function since responses to the endothelium-dependent vasodilator, bradykinin, were enhanced in these vessels compared to arteries isolated from Di/N rats.

Purinergic transmission in blood vessels

There are nineteen different receptor proteins for adenosine, adenine and uridine nucleotides, and nucleotide sugars, belonging to three families of G protein-coupled adenosine and P2Y receptors, and ionotropic P2X receptors. The majority are functionally expressed in blood vessels, as purinergic receptors in perivascular nerves, smooth muscle and endothelial cells, and roles in regulation of vascular contractility, immune function and growth have been identified. The endogenous ligands for purine receptors, ATP, ADP, UTP, UDP and adenosine, can be released from different cell types within the vasculature, as well as from circulating blood cells, including erythrocytes and platelets. Many purine receptors can be activated by two or more of the endogenous ligands. Further complexity arises because of interconversion between ligands, notably adenosine formation from the metabolism of ATP, leading to complex integrated responses through activation of different subtypes of purine receptors. The enzymes responsible for this conversion, ectonucleotidases, are present on the surface of smooth muscle and endothelial cells, and may be coreleased with neurotransmitters from nerves. What selectivity there is for the actions of purines/pyrimidines comes from differential expression of their receptors within the vasculature. P2X1 receptors mediate the vasocontractile actions of ATP released as a neurotransmitter with noradrenaline (NA) from sympathetic perivascular nerves, and are located on the vascular smooth muscle adjacent to the nerve varicosities, the sites of neurotransmitter release. The relative contribution of ATP and NA as functional cotransmitters varies with species, type and size of blood vessel, neuronal firing pattern, the tone/pressure of the blood vessel, and in ageing and disease. ATP is also a neurotransmitter in non-adrenergic non-cholinergic perivascular nerves and mediates vasorelaxation via smooth muscle P2Y-like receptors. ATP and adenosine can act as neuromodulators, with the most robust evidence being for prejunctional inhibition of neurotransmission via A1 adenosine receptors, but also prejunctional excitation and inhibition of neurotransmission via P2X and P2Y receptors, respectively. P2Y2, P2Y4 and P2Y6 receptors expressed on the vascular smooth muscle are coupled to vasocontraction, and may have a role in pathophysiological conditions, when purines are released from damaged cells, or when there is damage to the protective barrier that is the endothelium. Adenosine is released during hypoxia to increase blood flow via vasodilator A2A and A2B receptors expressed on the endothelium and smooth muscle. ATP is released from endothelial cells during hypoxia and shear stress and can act at P2Y and P2X4 receptors expressed on the endothelium to increase local blood flow. Activation of endothelial purine receptors leads to the release of nitric oxide, hyperpolarising factors and prostacyclin, which inhibits platelet aggregation and thus ensures patent blood flow. Vascular purine receptors also regulate endothelial and smooth muscle growth, and inflammation, and thus are involved in the underlying processes of a number of cardiovascular diseases.

Hydrogen sulphide-mediated vasodilatation involves the release of neurotransmitters from sensory nerves in pressurized mesenteric small arteries isolated from rats.

Hydrogen sulphide (H2S) is a gas that has recently been shown to have biological activity. In the majority of blood vessels studied so far, H2S has been shown to cause vasorelaxation, although contractile responses have been reported. In the present study, we have made a pharmacological assessment of the effects of H2S in mesenteric small arteries isolated from rats.

No evidence for vascular remodelling during hypertension induced by chronic inhibition of nitric oxide synthase in Brattleboro rats

Objective: To examine the effects of hypertension induced by chronic inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester (l-NAME) on the structure of mesenteric resistance arteries. Design and methods: Brattleboro rats were given l-NAME (0.01-0.05 mg/ml, equivalent to 10-50 mg/kg per day) in their drinking water for 2-3 weeks. On the day of the experiment, mean arterial pressure was measured directly using an intra-arterial catheter that had been implanted 2 days previously. Isolated mesenteric resistance arteries were set up in a pressure-perfusion myograph in a calcium-free physiological solution and subsequently fixed with glutaraldehyde, embedded in Araldite, sectioned and examined histologically. Results: l-NAME administration increased mean arterial blood pressure in comparison with normotensive controls. No differences were found between the structural characteristics of mesenteric resistance arteries of first (pressurized diameter approximately 350 µm), second (approximately 270 µm) or third (approximately 200 µm) order vessels isolated from normotensive rats and rats that had been treated with l-NAME. There were no significant correlations between mean arterial blood pressure and the variables of internal diameter, media thickness and media:lumen area ratio. Conclusions: Hypertension produced by chronic inhibition of nitric oxide synthase is not associated with remodelling of mesenteric resistance arteries in Brattleboro rats.