A characterization of ABL-101 as a potential tracer for clinical fluorine-19 MRI.

The two main challenges that prevent the translation of fluorine-19 (19 F) MRI for inflammation monitoring or cell tracking into clinical practice are (i) the relatively low signal-to-noise ratio generated by the injected perfluorocarbon (PFC), which necessitates long scan times, and (ii) the need for regulatory approval and a high biocompatibility of PFCs that are also suitable for MRI. ABL-101, an emulsion of perfluoro(t-butylcyclohexane), is a third-generation PFC that is already used in clinical trials, but has not yet been used for 19 F MRI. The objective of this study was therefore to assess the performance of ABL-101 as a 19 F MRI tracer. At magnetic field strengths of 3, 9.4 and 14.1 T, the CF3 groups of ABL-101 generated a large well-separated singlet with T2 /T1 ratios of >0.27, >0.14 and > 0.05, respectively. All relaxation times decreased with the increase in magnetic field strength. The detection limit of ABL-101 in a 0.25 mm3 voxel at 3 T, 37°C and with a 3-minute acquisition time was 7.21mM. After intravenous injection, the clearance half-lives of the ABL-101 19 F MR signal in mouse (n = 3) spleen and liver were 6.85 ± 0.45 and 3.20 ± 0.35 days, respectively. These results demonstrate that ABL-101 has 19 F MR characteristics that are similar to those of PFCs developed specifically for MRI, while it has clearance half-lives similar to PFCs that have previously been used in large doses in non-MRI clinical trials. Overall, ABL-101 is thus a very promising candidate tracer for future clinical trials that use 19 F MRI for cell tracking or the monitoring of inflammation.

Preclinical Validation of the Therapeutic Potential of Glasgow Oxygen Level Dependent (GOLD) Technology: a Theranostic for Acute Stroke.

In acute stroke patients, penumbral tissue is non-functioning but potentially salvageable within a time window of variable duration and represents target tissue for rescue. Reperfusion by thrombolysis and/or thrombectomy can rescue penumbra and improve stroke outcomes, but these treatments are currently available to a minority of patients. In addition to the utility of Glasgow Oxygen Level Dependent (GOLD) as an MRI contrast capable of detecting penumbra, its constituent perfluorocarbon (PFC) oxygen carrier, combined with normobaric hyperoxia, also represents a potential acute stroke treatment through improved oxygen delivery to penumbra. Preclinical studies were designed to test the efficacy of an intravenous oxygen carrier, the perfluorocarbon emulsion Oxycyte® (O-PFC), combined with normobaric hyperoxia (50% O2) in both in vitro (neuronal cell culture) and in vivo rat models of ischaemic stroke. Outcome was assessed through the quantification of lipid peroxidation and oxidative stress levels, mortality, infarct volume, neurological scoring and sensorimotor tests of functional outcome in two in vivo models of stroke. Additionally, we investigated evidence for any positive or negative interactions with the thrombolytic recombinant tissue plasminogen activator (rt-PA) following embolus-induced stroke in rats. Treatment with intravenous O-PFC + normobaric hyperoxia (50% O2) provided evidence of reduced infarct size and improved functional recovery. It did not exacerbate oxidative stress and showed no adverse interactions with rt-PA. The positive results and lack of adverse effects support human trials of O-PFC + 50% O2 normobaric hyperoxia as a potential therapeutic approach. Combined with the diagnostic data presented in the preceding paper, O-PFC and normobaric hyperoxia is a potential theranostic for acute ischaemic stroke.

Perfluorocarbon Enhanced Glasgow Oxygen Level Dependent (GOLD) Magnetic Resonance Metabolic Imaging Identifies the Penumbra Following Acute Ischemic Stroke.

The ability to identify metabolically active and potentially salvageable ischaemic penumbra is crucial for improving treatment decisions in acute stroke patients. Our solution involves two complementary novel MRI techniques (Glasgow Oxygen Level Dependant (GOLD) Metabolic Imaging), which when combined with a perfluorocarbon (PFC) based oxygen carrier and hyperoxia can identify penumbra due to dynamic changes related to continued metabolism within this tissue compartment. Our aims were (i) to investigate whether PFC offers similar enhancement of the second technique (Lactate Change) as previously demonstrated for the T2*OC technique (ii) to demonstrate both GOLD metabolic imaging techniques working concurrently to identify penumbra, following administration of Oxycyte® (O-PFC) with hyperoxia. Methods: An established rat stroke model was utilised. Part-1: Following either saline or PFC, magnetic resonance spectroscopy was applied to investigate the effect of hyperoxia on lactate change in presumed penumbra. Part-2; rats received O-PFC prior to T2*OC (technique 1) and MR spectroscopic imaging, which was used to identify regions of tissue lactate change (technique 2) in response to hyperoxia. In order to validate the techniques, imaging was followed by [14C]2-deoxyglucose autoradiography to correlate tissue metabolic status to areas identified as penumbra. Results: Part-1: PFC+hyperoxia resulted in an enhanced reduction of lactate in the penumbra when compared to saline+hyperoxia. Part-2: Regions of brain tissue identified as potential penumbra by both GOLD metabolic imaging techniques utilising O-PFC, demonstrated maintained glucose metabolism as compared to adjacent core tissue. Conclusion: For the first time in vivo, enhancement of both GOLD metabolic imaging techniques has been demonstrated following intravenous O-PFC+hyperoxia to identify ischaemic penumbra. We have also presented preliminary evidence of the potential therapeutic benefit offered by O-PFC. These unique theranostic applications would enable treatment based on metabolic status of the brain tissue, independent of time from stroke onset, leading to increased uptake and safer use of currently available treatment options.

The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments).

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information).

Detection of ischemic penumbra using combined perfusion and T2* oxygen challenge imaging.

BACKGROUND: Acute ischemic stroke is common and disabling, but there remains a paucity of acute treatment options and available treatment (thrombolysis) is underutilized. Advanced brain imaging, designed to identify viable hypoperfused tissue (penumbra), could target treatment to a wider population. Existing magnetic resonance imaging and computed tomography-based technologies are not widely used pending validation in ongoing clinical trials. T2* oxygen challenge magnetic resonance imaging, by providing a more direct readout of tissue viability, has the potential to identify more patients likely to benefit from thrombolysis - irrespective of time from stroke onset - and patients within and beyond the 4·5 h thrombolysis treatment window who are unlikely to benefit and are at an increased risk of hemorrhage. AIMS: This study employs serial multimodal imaging and voxel-based analysis to develop optimal data processing for T2* oxygen challenge penumbra assessment. Tissue in the ischemic hemisphere is compartmentalized into penumbra, ischemic core, or normal using T2* oxygen challenge (single threshold) or T2* oxygen challenge plus cerebral blood flow (dual threshold) data. Penumbra defined by perfusion imaging/apparent diffusion coefficient mismatch (dual threshold) is included for comparison. METHODS: Permanent middle cerebral artery occlusion was induced in male Sprague-Dawley rats (n = 6) prior to serial multimodal imaging: T2* oxygen challenge, diffusion-weighted and perfusion imaging (cerebral blood flow using arterial spin labeling). RESULTS: Across the different methods evaluated, T2* oxygen challenge combined with perfusion imaging most closely predicted 24 h infarct volume. Penumbra volume declined from one to four-hours post-stroke: mean ± SD, 77 ± 44 to 49 ± 37 mm(3) (single T2* oxygen challenge-based threshold); 55 ± 41 to 37 ± 12 mm(3) (dual T2* oxygen challenge/cerebral blood flow); 84 ± 64 to 42 ± 18 mm(3) (dual cerebral blood flow/apparent diffusion coefficient), as ischemic core grew: 155 ± 37 to 211 ± 36 mm(3) (single apparent diffusion coefficient threshold); 178 ± 56 to 205 ± 33 mm(3) (dual T2* oxygen challenge/cerebral blood flow); 139 ± 30 to 168 ± 38 mm(3) (dual cerebral blood flow/apparent diffusion coefficient). There was evidence of further lesion growth beyond four-hours (T2-defined edema-corrected infarct, 231 ± 19 mm(3) ). CONCLUSIONS: In conclusion, T2* oxygen challenge combined with perfusion imaging has advantages over alternative magnetic resonance imaging techniques for penumbra detection by providing serial assessment of available penumbra based on tissue viability.

Perfluorocarbons enhance a T2*-based MRI technique for identifying the penumbra in a rat model of acute ischemic stroke.

Accurate imaging of ischemic penumbra is crucial for improving the management of acute stroke patients. T2* magnetic resonance imaging (MRI) combined with a T2*oxygen challenge (T2*OC) is being developed to detect penumbra based on changes in blood deoxyhemoglobin. Using 100% O2, T2*OC-defined penumbra exhibits ongoing glucose metabolism and tissue recovery on reperfusion. However, potential limitations in translating this technique include a sinus artefact in human scans with delivery of 100% OC and relatively small signal changes. Here we investigate whether an oxygen-carrying perfluorocarbon (PFC) emulsion can enhance the sensitivity of the technique, enabling penumbra detection with lower levels of inspired oxygen. Stroke was induced in male Sprague-Dawley rats (n=17) with ischemic injury and perfusion deficit determined by diffusion and perfusion MRI, respectively. T2* signal change was measured in regions of interest (ROIs) located within ischemic core, T2*OC-defined penumbra and equivalent contralateral areas during 40% O2±prior PFC injection. Region of interest analyses between groups showed that PFC significantly enhanced the T2* response to 40% O2 in T2*-defined penumbra (mean increase of 10.6±2.3% compared to 5.6±1.5% with 40% O2, P<0.001). This enhancement was specific to the penumbra ROI. Perfluorocarbon emulsions therefore enhances the translational potential of the T2*OC technique for identifying penumbra in acute stroke patients.

Novel MRI detection of the ischemic penumbra: direct assessment of metabolic integrity.

We describe a novel magnetic resonance imaging technique to directly assess the metabolic integrity of penumbral tissue following stroke. For ischemically stressed tissue to be salvageable, it has to be capable of recovering aerobic metabolism (in place of anaerobic metabolism) on reperfusion. We probed ischemic brain tissue by altering the rate of oxygen delivery using a challenge of 100% oxygen ventilation. Any change from anaerobic to aerobic metabolism should alter the rate of lactate production and hence, levels of tissue lactate. Stroke was induced by permanent middle cerebral artery occlusion in rats. In Series 1 (n = 6), changes in tissue lactate during and following 100% oxygen challenge were monitored using (1)H magnetic resonance spectroscopy (MRS). Diffusion weighted imaging (DWI) and perfusion weighted imaging (PWI) were used to locate MRS voxels within the ischemic core, the homotopic contralateral striatum and within PWI/DWI mismatch (i.e. presumed penumbra). After 20 min of oxygen, lactate signal change was -16.1 ± 8.8% (mean ± SD) in PWI/DWI mismatch, +2.8 ± 5.1% in the ischemic core, and -0.6 ± 7.6% in the contralateral striatum. Return to air ventilation for 20 min resulted in a reversal, with lactate increasing by 46 ± 25.3% in the PWI/DWI mismatch, 6.6 ± 6.2% in the ischemic core, and -5 ± 11.4% in the contralateral striatum. In Series 2 (n = 6), a novel form of spectroscopic imaging was used to acquire lactate change maps to spatially identify regions of lactate change within the ischemic brain. This technique has potential clinical utility by identifying tissue that displays anaerobic metabolism capable of recovering aerobic metabolism when oxygen delivery is increased, which could provide a more precise assessment of penumbra.

Influence of 100% and 40% oxygen on penumbral blood flow, oxygen level, and T2*-weighted MRI in a rat stroke model.

Accurate imaging of the ischemic penumbra is a prerequisite for acute clinical stroke research. T(2)(*) magnetic resonance imaging (MRI) combined with an oxygen challenge (OC) is being developed to detect penumbra based on changes in blood deoxyhemoglobin. However, inducing OC with 100% O(2) induces sinus artefacts on human scans and influences cerebral blood flow (CBF), which can affect T(2)(*) signal. Therefore, we investigated replacing 100% O(2) OC with 40% O(2) OC (5 minutes 40% O(2) versus 100% O(2)) and determined the effects on blood pressure (BP), CBF, tissue pO(2), and T(2)(*) signal change in presumed penumbra in a rat stroke model. Probes implanted into penumbra and contralateral cortex simultaneously recorded pO(2) and CBF during 40% O(2) (n=6) or 100% O(2) (n=8) OC. In a separate MRI study, T(2)(*) signal change to 40% O(2) (n=6) and 100% O(2) (n=5) OC was compared. Oxygen challenge (40% and 100% O(2)) increased BP by 8.2% and 18.1%, penumbra CBF by 5% and 15%, and penumbra pO(2) levels by 80% and 144%, respectively. T(2)(*) signal significantly increased by 4.56% ± 1.61% and 8.65% ± 3.66% in penumbra compared with 2.98% ± 1.56% and 2.79% ± 0.66% in contralateral cortex and 1.09% ± 0.82% and -0.32% ± 0.67% in ischemic core, respectively. For diagnostic imaging, 40% O(2) OC could provide sufficient T(2)(*) signal change to detect penumbra with limited influence in BP and CBF.

11ß-hydroxysteroid dehydrogenase type 2 deficiency accelerates atherogenesis and causes proinflammatory changes in the endothelium in apoe-/- mice.

Mineralocorticoid receptor (MR) activation is proinflammatory and proatherogenic. Antagonism of MR improves survival in humans with congestive heart failure caused by atherosclerotic disease. In animal models, activation of MR exacerbates atherosclerosis. The enzyme 11ß-hydroxysteroid dehydrogenase type 2 (11ß-HSD2) prevents inappropriate activation of the MR by inactivating glucocorticoids in mineralocorticoid-target tissues. To determine whether glucocorticoid-mediated activation of MR increases atheromatous plaque formation, we generated Apoe(-/-)/11ß-HSD2(-/-) double-knockout (E/b2) mice. On chow diet, E/b2 mice developed atherosclerotic lesions by 3 months of age, whereas Apolipoprotein E (Apoe(-/-)) mice remained lesion free. Brachiocephalic plaques in 3-month-old E/b2 mice showed increased macrophage and lipid content and reduced collagen content compared with similar sized brachiocephalic plaques in 6-month-old Apoe(-/-) mice. Crucially, treatment of E/b2 mice with eplerenone, an MR antagonist, reduced plaque development and macrophage infiltration while increasing collagen and smooth muscle cell content without any effect on systolic blood pressure. In contrast, reduction of systolic blood pressure in E/b2 mice using the epithelial sodium channel blocker amiloride produced a less-profound atheroprotective effect. Vascular cell adhesion molecule 1 expression was increased in the endothelium of E/b2 mice compared with Apoe(-/-) mice. Similarly, aldosterone increased vascular cell adhesion molecule 1 expression in mouse aortic endothelial cells, an effect mimicked by corticosterone only in the presence of an 11ß-HSD2 inhibitor. Thus, loss of 11ß-HSD2 leads to striking atherogenesis associated with activation of MR, stimulating proinflammatory processes in the endothelium of E/b2 mice.

TNFalpha is required to confer protection in an in vivo model of classical ischaemic preconditioning.

Although Tumor Necrosis Factor alpha (TNFalpha) is used as a preconditioning mimetic in vitro, its role in ischaemic preconditioning (IPC) has not been clearly defined. Here, we propose to use an in vivo model (that takes into account the activation of leukocytes which may affect levels of TNFalpha) to demonstrate that i) TNFalpha acts as a trigger in IPC and ii) the dose-dependent nature of this cardioprotective effect of TNFalpha. Male Wistar rats were subjected to 30 min of left coronary artery occlusion (index ischaemia), followed by 24 h reperfusion. In the presence or absence of a soluble TNFalpha receptor (sTNFalpha-R), preconditioning was induced by 3 cycles of ischaemia (3 min)/reperfusion (5 min) (IPC) or various doses (0.05-4 microg/kg) of exogenous TNFalpha. Following 24 h reperfusion, infarct size (IS, expressed as % of the area at risk (AAR)) was assessed. Tissue levels of TNFalpha from the AAR, following IPC and TNFalpha stimulus were determined using Western Blot. IPC caused decrease in IS (4.5+/-1.3% vs 30.8+/-4.3% in ischaemic rats; P<0.001) and increase of TNFalpha levels following the IPC stimulus. The protective effect of IPC was abrogated in the presence of the sTNFalpha-R. In addition, exogenous TNFalpha dose-dependently reduced IS with maximal protection at a dose of 0.1 microg/kg (IS=12.6%, P<0.01 vs ischaemic). In conclusion our data provide strong evidence for a role of TNFalpha during the trigger phase of IPC. In addition, exogenous TNFalpha mimics IPC by providing a dose-dependent cardioprotective effect against ischaemia-reperfusion injury in vivo.

The in vivo effects of human urotensin II in the rabbit and rat pulmonary circulation: effects of experimental pulmonary hypertension.

In vivo haemodynamic responses to human urotensin-II were determined in two models of pulmonary hypertension: rabbits with left ventricular dysfunction following coronary artery ligation and the hypoxic rat. Effects were also examined in the presence of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Human urotensin-II increased pulmonary arterial pressure to a greater extent in ligated rabbits than their controls and L-NAME increased pulmonary pressure without significantly affecting these responses to human urotensin-II. Human urotensin-II raised right ventricular pressure slightly in control rats but not in hypoxic rats. Human urotensin-II did not constrict control rat isolated small pulmonary arteries and only induced a small constriction of these vessels in hypoxic rats. In conclusion, exogenous human urotensin-II exerts pulmonary pressor responses in vivo in rabbits and also induced small pulmonary pressor responses in control rats. Pulmonary pressor responses to urotensin-II were increased by pulmonary hypertension in rabbits but not in rats.

Pharmacological preconditioning with tumor necrosis factor-alpha activates signal transducer and activator of transcription-3 at reperfusion without involving classic prosurvival kinases (Akt and extracellular signal-regulated kinase).

BACKGROUND: We previously reported that tumor necrosis-factor-alpha (TNF-alpha) can mimic classic ischemic preconditioning (IPC) in a dose- and time-dependent manner. Because TNF-alpha activates the signal transducer and activator of transcription-3 (STAT-3), we hypothesized that TNF-alpha-induced preconditioning requires phosphorylation of STAT-3 rather than involving the classic prosurvival kinases, Akt and extracellular signal-regulated kinase (Erk) 1/2, during early reperfusion. METHODS AND RESULTS: Isolated, ischemic/reperfused rat hearts were preconditioned by either IPC or low-dose TNF-alpha (0.5 ng/mL). Western blot analysis confirmed that IPC phosphorylated Akt and Erk 1/2 after 5 minutes of reperfusion (Akt increased by 34+/-6% and Erk, by 105+/-28% versus control; P<0.01). Phosphatidylinositol 3-kinase/Akt inhibition (wortmannin) or mitogen-activated protein kinase-Erk 1/2 kinase inhibition (PD-98059) during early reperfusion abolished the infarct-sparing effect of IPC. In contrast, TNF-alpha preconditioning did not phosphorylate these kinases (Akt increased by 7+/-7% and Erk, by 17+/-14% versus control; P=NS). Neither wortmannin nor PD-98059 inhibited TNF-alpha-mediated cardioprotection. However, TNF-alpha and IPC both phosphorylated STAT-3 and the proapoptotic protein Bcl-2 antagonist of cell death (BAD) (STAT-3 increased by 58+/-17% with TNF-alpha or by 68+/-12% with IPC; BAD increased by 75+/-8% with TNF-alpha or by 205+/-20% with IPC; P<0.01 versus control), thereby activating the former and inactivating the latter. The STAT-3 inhibitor AG 490 abolished cardioprotection and BAD phosphorylation with both preconditioning stimuli. CONCLUSIONS: Activation of the classic prosurvival kinases (Akt and Erk 1/2) is not essential for TNF-alpha-induced preconditioning in the early reperfusion phase. We show the existence of an alternative protective pathway that involves STAT-3 activation specifically at reperfusion in response to both TNF-alpha and classic IPC. This novel prosurvival pathway may have potential therapeutic significance.

The role of 5-hydroxytryptamine in the control of pulmonary vascular tone in a rabbit model of pulmonary hypertension secondary to left ventricular dysfunction.

The role of 5-hydroxytryptamine (5-HT) in a rabbit model of pulmonary hypertension (PHT) secondary to left ventricular dysfunction was investigated. Following pulmonary artery catheterisation under anaesthesia, 5-HT (1-400 microg kg(-1) i.v.) was administered before and after either 5-HT(2A) receptor antagonism with ketanserin (0.5 mg kg(-1)) or infusion of the nitric oxide synthase inhibitor, l-NAME (30 micromol min(-1)). Eight week coronary artery ligated rabbits demonstrate increased mean pulmonary arterial pressure (PAP) compared to controls (17.0+/-0.5 versus 12.0+/-0.5 mmHg, P<0.001) accompanied by right ventricular hypertrophy. 5-HT alone produced a greater pulmonary pressor response in rabbits with PHT (increase of 7.5+/-1.2, n=12 c.f. 3.5+/-0.4 mmHg in shams, n=12, P<0.01). Ketanserin had no effect on basal PAP in either PHT or control rabbits but inhibited the response to 5-HT in both groups. The response to 5-HT following l-NAME was increased in both groups and was greater in rabbits with PHT (an increase of 20.1+/-2.9, n=6 c.f. 11.4+/-1.8 mmHg, n=6, P<0.05). These results suggest that the difference shown in the in vivo pulmonary response to exogenous 5-HT is mediated largely through 5-HT(2A) receptors in this model. However, activity of endogenous 5-HT at the 5-HT(2A) receptors is not responsible for maintaining the raised basal PAP through vasoconstriction in PHT rabbits once PHT has developed.

Overexpression of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension.

BACKGROUND: Increased serotonin (5-hydroxytryptamine, 5-HT) transporter activity has been observed in human familial pulmonary hypertension. METHODS AND RESULTS: We investigated pulmonary hemodynamics and the development of hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling in mice overexpressing the gene for the 5-HT transporter (5-HTT+ mice). Right ventricular pressure was elevated 3-fold in normoxic 5-HTT+ mice compared with their wild-type controls. Hypoxia-induced increases in right ventricular hypertrophy and pulmonary vascular remodeling were also potentiated in the 5-HTT+ mice. 5-HTT-like immunoreactivity, protein, and binding sites were markedly increased in the lungs from the 5-HTT+ mice. Hypoxia, however, decreased 5-HT transporter immunoreactivity, mRNA transcription, protein, and binding sites in both wild-type and 5-HTT+ mice. CONCLUSIONS: Increased 5-HT transporter expression causes elevated right ventricular pressures, and this occurs before the onset of right ventricular hypertrophy or pulmonary arterial remodeling. Hypoxia-induced remodeling is, however, increased in 5-HTT+ mice, whereas hypoxia inhibits 5-HTT expression. This provides a unique model that demonstrates differential mechanisms for familial pulmonary arterial hypertension and pulmonary arterial hypertension with hypoxemia.

Pulmonary hypertension secondary to left ventricular dysfunction: the role of nitric oxide and endothelin-1 in the control of pulmonary vascular tone.

1. Using an in vivo model of pulmonary hypertension (PHT) secondary to left ventricular dysfunction (LVD), the pulmonary arterial response to the nitric oxide synthase (NOS) blocker L-NAME (30 micromol.min(-1) i.v.) and the subsequent responses to cumulatively administered endothelin-1 (ET-1) (0.001 -- 4 nmol.kg(-1) i.v.) or big ET-1 (0.1 -- 2.0 nmol.kg(-1) i.v.) were studied. Additionally, the effect of the non-selective ET-1 receptor antagonist, SB209670, was investigated. 2. Eight weeks after coronary artery ligation or sham operation, rabbits demonstrated increased mean pulmonary arterial pressure (PAP) accompanied by right ventricular hypertrophy. 3. Blockade of NOS caused a greater increase in basal PAP (increased by 7.7 +/- 1.1 mmHg c.f. 3.8 +/- 1.0 mmHg in controls, P<0.05) and uncovered a greater pulmonary pressor response to exogenous ET-1 in rabbits with PHT (increased by 10.2 +/- 2.3 mmHg c.f. 4.9 +/- 1.0 mmHg in controls, P<0.05). 4. Big ET-1 evoked a pulmonary pressor effect, in both groups of rabbits, that was increased following blockade of NOS and was more potent in rabbits with PHT. 5. The non-selective ET-1 receptor antagonist, SB209670, reduced basal PAP (from 16.9 mmHg to 15.9 mmHg, P < 0.05) in rabbits with PHT and blocked the response to ET-1 in the presence of L-NAME. 6. In conclusion, the results demonstrate that basal NO activity masks a pulmonary pressor response to exogenously administered ET-1. An increased responsiveness to ET-1 was shown in the pulmonary arterial bed of rabbits with PHT secondary to LVD, implicating a pathophysiological role for ET-1 in this model.