Dependence of smooth muscle tone upon pulsatility in the iliac artery of the anaesthetised pig.

The aim of the study was to examine features of the myogenic response of a conduit artery to the presence and absence of pulsatile pressure. The iliac arteries of 16 anaesthetised pigs (10 in control conditions, 6 under sympathetic blockade) were instrumented with flowmeter, sonomicrometry crystals for diameter measurement, a micro-tip manometer for pressure measurement and snares placed proximally and distally to the crystals to isolate a test segment from the remainder of the arterial system. When the snares were tightened to occlude the test segment, systemic arterial pressure remained constant. There was a large shift in the pressure-diameter relationship, in that there was a rapid decline in test segment pressure for the same diameter. This indicated arterial wall smooth muscle relaxation in response to removal of pulsatility of arterial pressure. The difference in mean pressure between pulsatility present and absent was significant (p < 0.0001, paired t test, n = 10). Before proximal and distal occlusion, test segment pressure was (mean ± SD) 92.26 ± 12.39 mmHg, whereas after distal and proximal occlusion at the same diameter, it was 42.34 ± 10.87 mmHg. We conclude that in the presence of pulsatile pressure, there is a large proportion of arterial wall smooth muscle tone related to stretch of the arterial wall during the cardiac cycle, indicating that, under normal pulsatile pressure conditions, much of the normal tone can be attributed to the pulsatile component of the arterial myogenic response.

Guidelines for reporting experiments involving animals: the ARRIVE guidelines.

British Journal of Pharmacology (BJP) is pleased to publish a new set of guidelines for reporting research involving animals, simultaneously with several other journals; the 'ARRIVE' guidelines (Animals in Research: Reporting In Vivo Experiments). This editorial summarizes the background to the guidelines, gives our view of their significance, considers aspects of specific relevance to pharmacology, re-states BJP's guidelines for authors on animal experiments and indicates our commitment to carrying on discussion of this important topic. We also invite feedback via the British Pharmacological Society website.

Themed section: Advances in nutritional pharmacology. Editorial.

A themed section in this issue of Br J Pharmacol, on 'Advances in Nutritional Pharmacology', provides a valuable and timely update on progress in this area. The value of dietary components to improvement in health and, particularly, to prevention of cardiovascular disease and cancer, is frequently reported in the media and therefore often captures the attention of the wider public. Understanding the pharmacological mechanisms by which nutritional elements confer their health benefits enables us to keep the public informed, but also aids in the identification of new targets for drug development. In recent years there has been significant progress in this field. Four rapidly developing areas are reviewed. Vosper (2009) covers the identification of a receptor for niacin and the subsequent development of selective agonists as lipid lowering agents. Wu-Wong (2009) describes the development of new Vitamin D analogues for the treatment of cardiovascular disease. de Roos et al. (2009) provide detailed insight into how omega-3 fatty acids, also known as longchain n-3 polyunsaturated fatty acids (PUFAs) protect against cardiovascular disease. Zhou et al. (2009) cover the mechanisms underlying the beneficial effects of resveretrol in protection against cancer. These reviews are complimented by three key original articles focusing on endogenous mechanisms of weight control involving endocannabinoids (Izzo et al., 2009), a circulating protein, the soluble leptin receptor (Zhang & Scarpace, 2009) and a treatment, zinc plus cyclo-(His-Pro) (CHP), known to increase insulin metabolism (Song et al., 2009).

Mast cells, peptides and cardioprotection - an unlikely marriage?

1 Mast cells have classically been regarded as the 'bad guys' in the setting of acute myocardial ischaemia, where their released contents are believed to contribute both to tissue injury and electrical disturbances resulting from ischaemia. Recent evidence suggests, however, that if mast cell degranulation occurs in advance of ischaemia onset, this may be cardioprotective by virtue of the depletion of mast cell contents that can no longer act as instruments of injury when the tissue becomes ischaemic. 2 Many peptides, such as ET-1, adrenomedullin, relaxin and atrial natriuretic peptide, have been demonstrated to be cardioprotective when given prior to the onset of myocardial ischaemia, although their physiological functions are varied and the mechanisms of their cardioprotective actions appear to be diverse and often ill defined. However, one common denominator that is emerging is the ability of these peptides to modulate mast cell degranulation, raising the possibility that peptide-induced mast cell degranulation or stabilization may hold the key to a common mechanism of their cardioprotection. 3 The aim of this review was to consolidate the evidence implying that mast cell degranulation could play both a detrimental and protective role in myocardial ischaemia, depending upon when it occurs, and that this may underlie the cardioprotective effects of a range of diverse peptides that exerts physiological effects within the cardiovascular system.

Mast cell degranulation--a mechanism for the anti-arrhythmic effect of endothelin-1?

BACKGROUND AND PURPOSE: The aim of this study was to investigate whether the previously reported anti-arrhythmic effect of endothelin-1 (ET-1) is mediated by degranulation of cardiac mast cells prior to myocardial ischaemia. EXPERIMENTAL APPROACH: Male Sprague-Dawley rats received either ET-1 (1.6 nmolxkg(-1)) in the presence or absence of disodium cromoglycate (DSCG; 20 mgxkg(-1)xh(-1)) prior to coronary artery occlusion (CAO). In separate experiments rats were given compound 48/80 (50 microgxkg(-1)) to compare the effects of ET-1 with those of a known mast cell degranulator. Ischaemia-induced ventricular arrhythmias were detected through continuous monitoring of a lead I electrocardiogram. After 30 min of CAO, the hearts were removed and mast cell degranulation determined by histological analysis. A parallel series of sham groups were performed to determine the direct effects of ET-1 and compound 48/80 on mast cell degranulation in the absence of ischaemia. KEY RESULTS: ET-1 and compound 48/80 both exerted profound anti-arrhythmic effects, significantly reducing the total number of ventricular ectopic beats (P < 0.001) and the incidence of ventricular fibrillation (P < 0.05). These anti-arrhythmic effects were abolished by concomitant DSCG infusion prior to CAO. In sham animals ET-1 and compound 48/80 both induced mast cell degranulation (P < 0.001), an effect which was abolished by DSCG, confirming their ability to induce degranulation of mast cells. CONCLUSIONS AND IMPLICATIONS: These results demonstrate for the first time that when given prior to ischaemia ET-1 mediates its anti-arrhythmic effects, at least in part, via cardiac mast cell degranulation.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1alpha(betaS*, deltaS*), 2alpha, 6alpha, 8beta-(R*), 8a alpha]]-1,2,6,7,8,8a-Hexahydro-beta, delta, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice.

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1alpha(betaS*,deltaS*),2alpha,6alpha,8beta-(R*),8a alpha]]-1,2,6,7,8,8a-hexahydro-beta,delta,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 +/- 1.9% versus 16.6 +/- 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 +/- 2.9% versus 23.4 +/- 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC(50), 6.37 +/- 0.37) compared with both vehicle-treated (-logEC50, 5.81 +/- 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 +/- 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 +/- 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

Activation of mouse protease-activated receptor-2 induces lymphocyte adhesion and generation of reactive oxygen species.

BACKGROUND AND PURPOSE: Protease-activated receptor-2 (PAR-2) is expressed on lymphocytes and endothelial cells, and plays a significant role in inflammatory reactions. Since leukocyte-endothelial cell interaction and reactive oxygen species (ROS) generation are hallmarks of the development of inflammation, the effects of PAR-2 activation by trypsin on lymphocyte adhesion and ROS generation was examined utilising PAR-2 wild type and knockout (PAR-2-/-) mice. EXPERIMENTAL APPROACH: Lymphocyte adhesion to the luminal surface of mouse isolated aortae was measured using 51Cr-labelled leukocytes and ROS generation from isolated lymphocytes was quantified using chemiluminescence. KEY RESULTS: Trypsin induced adhesion of lymphocytes when added exogenously to the endothelial surface of the aorta for 30 min. Similarly, increased lymphocyte adhesion was also observed when mice were injected with trypsin intravenously 24 h prior to the adhesion assay, an effect which was partly ICAM-1 mediated. Trypsin also increased ROS generation from isolated mouse lymphocytes in a dose-dependent manner. The increase in lymphocyte adhesion and ROS production in response to trypsin were abolished in PAR-2-/- mice indicating a PAR-2 dependent mechanism. Superoxide dismutase had a greater inhibitory effect in PAR-2-/- mice compared to wild type mice when lymphocytes were stimulated with PMA but not trypsin. CONCLUSIONS AND IMPLICATIONS: The present study indicates that activation of PAR-2 may be an important factor in modulating lymphocyte adhesion and ROS generation. The results have implications for developing anti-inflammatory strategies.

The effect of NCX4016 [2-acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester] on the consequences of ischemia and reperfusion in the streptozotocin diabetic rat.

The aim of this study was to assess the effect of chronic administration of NCX4016 [2 acetoxy-benzoate 2-(2-nitroxymethyl)-phenyl ester], a nitric oxide-releasing aspirin derivative on the consequences of coronary artery occlusion in streptozotocin-diabetic rats. Rats were made diabetic by injection of streptozotocin (60 mg kg(-1)) and received insulin (2.5 U kg(-1) s.c.) daily for 4 weeks. Animals received vehicle (1 ml kg(-1) polyethylene glycol), aspirin (65.2 mg kg(-1)), NCX4016 (60 mg kg(-1)), or (iv) NCX4016 (120 mg kg(-1)) orally, once daily for the last 5 days before coronary artery occlusion (CAO). One hour after the last dose, pentobarbital-anesthetized rats were subjected to CAO for 30 min followed by 120-min reperfusion. Neither drug significantly modified initial hemodynamics or plasma glucose levels compared with vehicle treatment in either nondiabetic or diabetic rats. Neither drug modified the total ventricular premature beat (VPB) count in normal animals, although NCX4016, but not aspirin, reduced the total VPB count and the incidence of ventricular tachycardia in diabetic rats. In nondiabetic animals, both aspirin and NCX4016 reduced infarct size. However, in diabetic rats, infarct size was reduced only by the larger dose of NCX4016 (120 mg kg(-1)) but not by aspirin or the lower dose of NCX4016. These results demonstrate that the cardioprotective effects of NCX4016 are reduced in the presence of diabetes compared with the effects seen in nondiabetic animals. In summary, the present study confirms the protective effect of NCX4016 against ischemia-reperfusion injury in the normal rat heart and demonstrates for the first time its protective effect in the heart of streptozotocin-diabetic rats.

Anti-arrhythmic and electrophysiological effects of the endothelin receptor antagonists, BQ-123 and PD161721.

The effects of the endothelin ET(A), (BQ-123) and endothelin ET(A/B) (PD161721) receptor antagonists were investigated on ischaemia-induced arrhythmias and on the maximum following frequency. The study was carried out in Langendorff perfused rat hearts subjected to coronary artery occlusion in which the severity of arrhythmias, coronary perfusion pressure and heart rate were measured. The % incidence of ischaemia-induced irreversible ventricular fibrillation (ventricular fibrillation) was reduced significantly from 58%, in control rat hearts, to 0% (at 10(-7) and 10(-6) M PD161721 and 10(-6) M BQ-123 P<0.05). Maximum following frequency was measured in guinea-pig isolated atria. In the presence of normal extracellular [K(+)], BQ-123 and PD161721, at 10(-6) M, significantly decreased the maximum following frequency from 9.0+/-0.7 to 7.2+/-0.4 and from 8.3+/-0.4 to 6.7+/-0.3 Hz, respectively (P<0.05). These effects were not potentiated by raising the extracellular [K(+)] with the exception of 10(-9) M PD161721. In contrast, lignocaine's ability to reduce the maximum following frequency was greater in elevated (e.g. at 1.7x10(-4) M from 8.4+/-0.3 to 2.5+/-0.6 Hz) than in normal [K(+)] (from 9.0+/-0.3 to 4.9+/-0.5 Hz). In conclusion, both BQ-123 and PD161721 had an anti-fibrillatory effect in isolated rat hearts that may be due, at least in part, to an ability to reduce the maximum following frequency. This latter effect is unlikely to be due to Na(+) channel blockade since it was not markedly potentiated by elevation of extracellular [K(+)].

Inflammation as a key event in the development of neointima following vascular balloon injury.

1. The present review discusses the current evidence to implicate leucocytes as key players in the development of neointima in arteries that have been subjected to balloon angioplasty injury. 2. There is substantial clinical evidence that leucocytes are activated after angioplasty, as determined by increased plasma levels of both leucocyte granulation products and soluble leucocyte and endothelial cell adhesion molecules. 3. Experimental evidence to implicate leucocytes in neointimal formation comes from studies that demonstrate leucocyte accumulation occurs within the vascular wall soon after injury and that induction of leukopenia prevents neointimal formation. 4. The evidence implicating specific adhesion molecules and cytokines in the key events leading to neointimal formation is discussed.

Validation of a technique to measure leukocyte adhesion to arterial segments: effects of drug treatments.

Adhesion and transmigration of leukocytes into arterial walls occurs after vascular injury and may play a role in the development of atherosclerosis and restenosis. This protocol presents a simple, rapid method for quantifying leukocyte adhesion to artery segments ex vivo. The procedure involves isolating leukocytes from rabbit whole blood and labelling with the gamma-emitting isotope 51Cr. Labelled leukocytes are added to open rings of subclavian artery taken from the same rabbit. After gamma counting, percentage leukocyte adhesion can be calculated with reference to a sample containing a quantity of labelled leukocytes equivalent to that which was added to the artery. Leukocyte adhesion was increased by L-NAME, thrombin and increasing incubation time and decreased by low temperatures. In addition, leukocyte adhesion was found to be increased following a vascular stretch injury performed in vitro. This protocol offers a number of advantages: the rapidity of the leukocyte isolation and labelling; the small quantity of leukocytes required; the ability to use autologous leukocytes; the applicability to whole arteries and arteries injured in vitro or in vivo, allowing the effects of vascular injury on leukocyte adhesion to be studied.

Short-term local delivery of an inhibitor of Ras farnesyltransferase prevents neointima formation in vivo after porcine coronary balloon angioplasty.

BACKGROUND: Mitogenic stimuli present at the site of coronary arterial balloon injury contribute to the progression and development of a restenotic lesion, many signaling through a common pathway involving the small G protein p21(ras). Our aim was to demonstrate in biochemical studies that farnesyl protein transferase inhibitor III (FPTIII) is an inhibitor of p21(ras) processing and that when it is given locally in vivo at the site of coronary balloon injury in a porcine model, it can inhibit neointima formation. METHODS AND RESULTS: FPTIII (1 to 25 micromol/L) concentration-dependently reduced p21(ras) levels in porcine coronary artery smooth muscle cell membranes. FPTIII also prevented p42/p44 MAPK activation and DNA synthesis in response to platelet-derived growth factor in these cells at a concentration of 25 micromol/L. Application of 25 micromol/L FPTIII locally for 15 minutes to balloon-injured porcine coronary arteries in vivo prevented neointima formation assessed at 4 weeks, reduced proteoglycan deposition, and inhibited adventitial hypertrophy. Coronary arteries from FPTIII-treated pigs had no deterioration in contraction or in endothelium-dependent relaxation. CONCLUSIONS: The study demonstrates in the pig that short-term local delivery of inhibitors of p21(ras)-dependent mitogenic signal transduction prevents restenosis after balloon angioplasty.

The effects of endothelin-1 on ischaemia-induced ventricular arrhythmias in rat isolated hearts.

We have shown previously that a small bolus dose of endothelin-1, given intravenously before coronary occlusion, exerts a marked antiarrhythmic effect in anaesthetised rats. The aim of the current study was to determine whether or not this is due to a direct effect of endothelin-1 on the heart by assessing the antiarrhythmic effect of endothelin-1 against occlusion-induced arrhythmias in rat isolated hearts. Rat isolated hearts were perfused in Langendorff mode (constant flow) and subjected to coronary artery occlusion for 30 min. Coronary perfusion pressure and a surface electrocardiogram (ECG) were monitored throughout the experiment. In the first series of studies, the effects of three 5-min infusions of endothelin-1 (0.1-10 nM), given prior to coronary occlusion, were assessed. A second series of hearts was given a single bolus dose of endothelin-1 (10 pmol) 5 min prior to ischaemia. A third series of experiments was performed using a modified (low K+) Krebs Henseleit solution to increase the incidence of ischaemia-induced ventricular fibrillation (VF). In these hearts, endothelin-1 (0.1 or 2 pmol) was administered as a bolus injection 5 min before ischaemia. Infusion of endothelin-1 prior to ischaemia did not modify the incidence or severity of arrhythmias at any of the concentrations used. Bolus administration of endothelin-1 (10 pmol) in hearts perfused with Kreb's Henseleit solution containing normal K+ (4.4 mM) was found to cause a small rise in coronary perfusion pressure, with no preceding depressor response. Under these conditions, endothelin-1 exerted only a very moderate reduction in arrhythmias, by reducing the arrhythmia count in the 21-30-min post-occlusion period. Furthermore, in hearts perfused with low K+ solution, bolus injection of endothelin-1, in a dose that either had no effect on coronary perfusion pressure (0.1 pmol) or produced a significant vasodilator effect with no significant pressor effect (2 pmol), had no effect on ventricular fibrillation. Thus, in concentrations that are sufficient to exert effects on the coronary vasculature, endothelin-1 fails to modify arrhythmias in an isolated heart preparation. These results suggest that the antiarrhythmic effects of endothelin-1 previously observed in vivo are not due to a direct effect on either the myocardium or the coronary blood vessels.

Inhibition by leukocyte depletion of neointima formation after balloon angioplasty in a rabbit model of restenosis.

OBJECTIVE: The aim of the current study was to examine neointima formation in balloon injured left subclavian artery of rabbits subjected to two different methods of leukocyte depletion at the time of injury. METHODS: Angioplasty of the left subclavian artery was performed in leukopenic male New Zealand White rabbits. Depletion of circulating leukocytes was induced by either mustine hydrochloride or an antibody against leukocyte common antigen (anti-LCA) before angioplasty. Left and right subclavian arteries were removed 28 days after injury for morphological analysis and measurement of neointimal size. At the same time, leukocytes were isolated from autologous rabbit blood for 51Cr-labelling for assessment of leukocyte adhesion to injured and non-injured artery segments. RESULTS: Leukopenia decreased neointima formation in injured arteries (neointimal area was 0.09+/-0.03 mm(2) in mustine-treated arteries, n=8, vs. 0.56+/-0.07 mm(2) in control arteries, n=7; P<0.001 and 0.07+/-0.01 mm(2) in anti-LCA treated arteries, n=9, vs. 0.22+/-0.04 mm(2) in non immune serum-treated arteries, n=9; P<0.001). Adventitial fibrosis was also significantly (P<0.05) decreased by both leukopenic interventions. Neither medial nor adventitial area was modified in any of the groups. No differences in leukocyte adhesion were observed between injured and non-injured arteries in any of the experimental groups at the 28 day time point. CONCLUSION: These results suggest that leukocytes play a major role in the development of two of the major characteristics of the response to balloon injury, namely formation of neointima and adventitial fibrosis, that currently limit the success of clinical angioplasty. Elucidation of the fine mechanisms involved in leukocyte-mediated injury may lead to the discovery of novel therapeutic targets for the prevention of restenosis.

Sarafotoxin 6c protects against ischaemia-induced cardiac arrhythmias in vivo and in vitro in the rat.

The aim of this study was to investigate whether the endothelin-B- (ETB) receptor agonist sarafotoxin 6c (S6c) can protect against ischaemia-induced cardiac arrhythmias. Arrhythmias were induced by a 30 min period of coronary artery occlusion in pentobarbitone-anaesthetized male rats, or in Langendorff-perfused rat hearts. Rats or rat hearts were administered a bolus dose of vehicle or S6c (0.8 nmol/kg i.v. or 10(-8) M into the coronary circulation, respectively) 5 min before the onset of ischaemia. In vivo administration of S6c significantly reduced the incidence of ventricular fibrillation (VF) from 59% to 13% and the number of premature ventricular beats. This effect was associated with a transient fall in mean arterial blood pressure. In isolated hearts, S6c reduced significantly both the incidences of ventricular tachycardia (VT) and VF while having no statistically significant effect on coronary perfusion pressure. This is the first report to show that stimulation of ETB-receptors, with a bolus dose of S6c, has an antiarrhythmic effect on rat hearts both in vivo and in vitro, suggestive of a direct effect on the myocardium.

A rapid, quantitative method for measuring leukocyte adhesion to normal and balloon-injured arteries in vitro.

Many of the currently available techniques for quantifying leukocyte adhesion require monolayers of cells and are therefore unsuitable for use in ex vivo arterial tissue. Here we describe a rapid method to measure adhesion of leukocytes to intact artery strips and to determine the effect of artery injury on adhesiveness of leukocytes with and without activation. Leukocytes were isolated from rabbit blood, labelled with 51Cr, and added to the luminal face of the left and right subclavian arteries derived from the same animal. In some experiments the endothelium was removed before addition of leukocytes and in another series of experiments the artery was injured by inflating a balloon catheter within the lumen in vitro before leukocyte addition. After washing, the adhesion of labelled leukocytes was quantified by gamma counting. To determine localization of the leukocytes, some arteries were fixed in situ and examined microscopically, with confirmation of leukocyte identification by enzyme cytochemistry. The adhesion of leukocytes increased progressively during 60 min and was inhibited by reducing the temperature to 4 degrees C. Adhesion was increased by the nitric oxide synthase inhibitor L-NAME. Stretching the artery wall in vitro using a balloon catheter increased leukocyte adhesion within 1 h after injury. In contrast, this did not occur following simple arterial denudation. Histological examination of stained en face preparations and transverse sections of the subclavian arteries revealed loosely adherent granulocytic leukocytes on the endothelial surface. This technique is straightforward and allows accurate and rapid measurement of autologous leukocyte adhesion to normal and pathologically altered arteries ex vivo.

Correlation of leukocyte adhesiveness, adhesion molecule expression and leukocyte-induced contraction following balloon angioplasty.

1. The aim of this study was to examine the changes in leukocyte adhesion and leukocyte-induced contraction in balloon-injured rabbit subclavian artery and to correlate these changes with vessel morphology and expression of adhesion molecules on the injured arteries. 2. Rabbits were anaesthetized and their left subclavian arteries were injured by balloon inflation and withdrawal followed by sacrifice at 2, 24, 48 h or 8 days after injury. The left and right subclavian arteries were removed and leukocytes were isolated from autologous rabbit blood. Leukocyte-induced contraction was measured in 5-HT precontracted artery rings and leukocyte adhesion was measured using (51)Cr-labelled leukocytes. Immunocytochemistry using paraffin-embedded tissue was employed to detect changes in the expression of adhesion molecules on injured arteries. 3. Autologous leukocytes caused a contraction of rabbit subclavian artery rings, which was prevented by L-NAME (10(-3) M). Balloon-induced injury abolished the contractile response to leukocytes, which correlated with loss of carbachol-induced relaxation 4. Balloon injury markedly enhanced the adhesiveness of the subclavian artery for leukocytes, most notably at 24 and 48 h after injury (1.7 and 1.8 fold respectively). Increased leukocyte adhesion at these two time points correlated with an upregulation of E-selectin, P-selectin and VCAM-1 expression on the remaining endothelium of the injured artery. 5. Vessel morphology revealed that balloon inflation had induced an infiltration of inflammatory cells into the vessel wall, the greatest increase being seen at 24 h after injury. 6. It is concluded that an increase in the expression of E-selectin, P-selectin and VCAM-1 following balloon-induced injury leads to enhanced leukocyte adhesion and migration into the injured vessel.

Endothelin and ischaemic arrhythmias-antiarrhythmic or arrhythmogenic?

OBJECTIVE: The aim of this study was to investigate the influence of endogenously released and exogenously applied endothelin-1 (ET-1) on ischaemia-induced arrhythmias. METHODS: Ischaemia was induced in pentobarbitone-anaesthetised rats by ligation of a coronary artery for 30 min. To determine the role of endogenous ET-1 in ischaemic arrhythmias, either the ETA receptor antagonist BQ123 (50 micrograms/kg/min, i.v.; n = 10) or the ETB receptor antagonist PD161721 (0.1 or 1 mg/kg i.v.; n = 10 per group) was administered before the onset of ischaemia. To assess the influence of exogenous ET-1 on arrhythmias, ET-1 (1.6 nmol/kg i.v.) was administered 5 min before ischaemia in the absence (n = 12) or presence of BQ123 (n = 10) or PD161721 (n = 10). The total number of ventricular ectopic beats (VEB's) were counted and expressed as median (Q1-Q3) and the incidence of ventricular fibrillation (VF) and ventricular tachycardia (VT) in each group was determined. Mean arterial blood pressure (MABP) and heart rate (HR) were measured. RESULTS: In control animals (n = 20), the incidence of VF was 65% and the total VEB count was 2775 (1870-4041). Both BQ123 and the higher dose of PD161721 reduced the VEB count to 654 (348-1489; P < 0.05) and 782 (432-1153; P < 0.05) respectively. Only PD161721 reduced the incidence of VF (to 10%; P < 0.05). Administration of ET-1 reduced VEB's to 1530 (1204-2017); P < 0.05) and the incidence of VF to 17% (P < 0.05). Neither PD161721 nor BQ123 modified this antiarrhythmic effect of ET-1, but rather enhanced the reduction in arrhythmias. Before occlusion, ET-1 caused a transient fall in MABP (from 107 +/- 3 to 63 +/- 3 mmHg; P < 0.05). PD161721, but not BQ123, partially blocked this effect. Upon occlusion, MABP fell in control animals (from 106 +/- 4 to 67 +/- 4 mmHg at 1 min post-occlusion; P < 0.05). This was significantly attenuated by ET-1, although neither of the antagonists were able to block this effect of ET-1. CONCLUSIONS: ET-1 released endogenously during ischaemia is arrhythmogenic whereas exogenous application of ET-1 may, under certain conditions, be antiarrhythmic.

Role of nitric oxide and free radicals in the contractile response to non-preactivated leukocytes.

Previous studies from our laboratory have shown that nitric oxide (NO) can reduce the release of free radicals from activated leukocytes. The aim of this study was to assess the role of endothelium-derived nitric oxide and leukocyte-derived free radicals in the contractile response to non-preactivated leukocytes. Vessel tension studies were performed in rabbit endothelium-intact aortic vessel rings precontracted with 5-hydroxytryptamine (1 microM). Addition of leukocytes isolated from rabbit blood were added to the rings in increasing concentrations (10(3)-10(6) cell ml(-1)) under control conditions and in the presence of L-nitroarginine methyl ester (L-NAME 1 mM), D-NAME (1 mM), or superoxide dismutase (100 U ml(-1)). The responses to superoxide radical (generated by xanthine plus xanthine oxidase, X/XO), hydrogen peroxide, hypochlorite and peroxynitrite were also assessed. The nature of the free radicals released from non-activated isolated leukocytes, zymosan-stimulated leukocytes (in whole blood) and isolated vessel rings was assessed using luminol-enhanced chemiluminescence. Cumulative addition of leukocyte suspensions to aortic rings caused a concentration-dependent contractile response which was abolished by preincubation of the vessel ring with L-NAME. D-NAME and superoxide dismutase were without effect. All the free radicals tested produced a relaxation of the precontracted aortic ring. The response to X/XO was not affected by superoxide dismutase, but abolished by catalase. The responses to hydrogen peroxide and hypochlorite were both found to be dependent upon the presence of endothelium and NO. The response to peroxynitrite was endothelium-independent and was blocked by methylene blue. While the main free radical released from unstimulated leukocytes and vessel rings was superoxide, the main radical released from activated leukocytes was found to be hypochlorite. These results suggest that the vascular contraction seen in response to non-preactivated leukocytes is due to inhibition, by NO, of the release of free radicals from the leukocytes when activated by contact with the vascular endothelium, thus allowing co-released vasoconstrictor substances to exert their effect.

Modulation of endothelin-1 release by a transmissible factor from ischemic myocardium.

The aim of this study was to detect the possible release from the ischemic rabbit myocardium of a factor capable of modulating the release of endothelin-1 (ET-1) from the peripheral vasculature. Isolated rabbit hearts were perfused on a Langendorff apparatus so that part of the coronary effluent was pumped directly into the arterial supply of isolated ears. Mean ET-1 outflow from ears perfused with fresh Krebs was 4.33 +/- 0.72 pg/min; from ears perfused with coronary effluent it was 84.3% less. This still represented net ET-1 production in the ear (venous/arterial ET-1 concentration ratio (V/A) = 3.9 +/- 1.2). Myocardial ischemia (30 min) caused a large reduction in ET-1 outflow from the ears and changed net production to net loss (V/A = 0.32 +/- 0.17). Within 2 min of reperfusion the V/A had risen to 6.3 +/- 2.2 and the ET-1 outflow from the ears rose 16-fold. Neither 30-min coronary occlusion nor 2-min reperfusion altered ET-1 concentrations in the coronary effluents. After 60-min reperfusion, ET-1 concentration in coronary effluent rose by 230%. During myocardial ischemia and reperfusion there was no change in pO2, pCO2 or pH in the perfusate supplying the ears. Therefore the changes in ET-1 handling by the ear vasculature must have been induced by a transmissible factor released from the heart.