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Background: The shortage of organ donors is a major challenge in lung transplantation. To expand the lung donor pool, ex-vivo lung perfusion (EVLP) has emerged as a platform for assessment and reconditioning marginal donor lungs. In this study a stable and reproducible large animal model of lipopolysaccharide (LPS) induced systemic inflammation and acute lung injury (ALI) was developed.Methods: Pigs (n=6) were anesthetized and monitored. After infusion of LPS (20 μg/kg) for 1 hour, followed by a 90-minute response period, lungs were procured and kept on ice for 2 hours, followed by 4 hours of EVLP. Pulmonary function, inflammatory biomarkers and edema formation were measured in vivo before procurement and during EVLP. Pro and anti-inflammatory cytokines were assayed in blood and in EVLP perfusate, which were collected before and every 30 minutes after LPS administration and EVLP.Results: LPS infusion resulted in significant hemodynamic instability, characterized by marked pulmonary hypertension, decreased systemic blood pressure and increased heart rate. This was associated with increased levels of TNFα, IL-10, IL-6, but no change in IL-1β. Ex vivo assessment of injured lungs showed graft dysfunction characterized by impaired gas exchange and edema formation. The inflammatory profile showed stable but elevated TNFα levels, and continuous production of interleukins during EVLP.Conclusion: We describe a reproducible large animal model of LPS-induced systemic inflammation and ALI. EVLP alone was unable to recondition severely injured lungs. These findings suggest that the EVLP platform requires adjuncts such as targeted anti-inflammatory agents to allow reconditioning of marginal donor lungs.

Ischemia-reperfusion associated with prolonged warm ischemia during donation after circulatory death (DCD) induces acute lung injury. The objective of this study was to combine ex vivo lung perfusion (EVLP) and a heat shock protein-90 inhibitor (HSP90i) to recondition DCD organs and prevent primary graft dysfunction.Pigs (55 to 65 kg) were anesthetized, ventilated, and hemodynamically monitored. Cardiac arrest was induced with potassium chloride, and animals were left nonventilated for 2 hours. Lungs were procured and perfused in an EVLP platform for 4 hours by using a cellular perfusate. In the study group, the perfusate contained HSP90i and its transport vehicle (n = 4). In the control group, the perfusate contained only the transport vehicle (n = 4). Gas exchange, airway pressures, and compliance were measured. Pulmonary edema was assessed by bronchoscopy and weight measurement. Lung biopsy samples were obtained for histologic analyses and protein expression measurements.The use of HSP90i reduced lung weight gain to 8.4 ± 3.4% vs 26.6 ± 6.2% in the control group (P.05). There was reduced edema formation. The ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen at the end of EVLP was 423 ± 65 in the study group vs 339 ± 25 mm Hg in the control group, but this difference was not statistically significant. Lactate metabolism, pulmonary vascular resistance, and pulmonary arterial pressure improved during EVLP with the use of the HSP90i.The use of HSP90i with EVLP improves the lung reconditioning process. Further research is required to confirm whether these findings translate to benefit once transplanted and observed in vivo. Successful pharmacologic inhibitors may expand the donor pool in the context of DCD donors.

Background: Primary graft dysfunction (PGD), the major complication associated with lung transplantation in the peri-operative period, is characterized by vascular and alveolar inflammation and results in hypoxemia and lung edema. Up to 30% of recipients develop the most severe form of PGD (Grade 3; PGD3), leading to 50% mortality. Animal studies suggest that neutrophils can contribute to the inflammatory process through the release of neutrophil extracellular traps(NETs). NETs are composed of DNA filaments decorated with granular proteins, which can contribute to vascular occlusion associated with PGD. Our main objective is to correlate NETosis with recipient outcomes. Methods: Clinical data and blood samples were prospectively collected from donors and recipients (n=32) pre-, intra- and post-operatively (up to 72hrs). Inflammatory markers inducing NETs’ synthesis (CRP, IL6, IL8) and markers of NETs (myeloperoxidase [MPO], MPO-DNA complexes, cell-free DNA) were quantified by ELISA. When available, histology and immunochemistry techniques were used on donor lung samples collected before transplantation to evaluate the presence of activated neutrophils and NETs. Results: In lung samples from which PGD3 developed in recipients, there was a marked increase of vascular occlusion composed of activated neutrophils and NETs before transplantation. Also, in donors and recipients pre- and intra-operatively, circulating levels of inflammatory (CRP, IL6, IL8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3. Conclusion: Elevation of these NETosis biomarkers might serve to elaborate an algorithm to better delineate the recipients at risk of developing severe PGD.

Purpose Ischemia reperfusion injury (I/R) is the major cause of primary graft dysfunction (PGD) after lung transplantation. I/R and PGD feature alveolar epithelial and endothelial damage, lung edema and inflammation. Edema resorption then depends on the restoration of alveolar epithelial integrity and the ability of alveolar cells to reabsorb Na+ (through ENaC channels) and fluid. We hypothesized that alveolar epithelial damage, and repair, are critical in PGD pathophysiology and resolution. Therefore, our aim was to identify novel biomarkers and therapeutic targets associated with I/R using cellular and animal models as well as human samples from lung transplants. Methods The impact of a protocol mimicking hypothermic ischemia and warm reperfusion was first tested on primary rat alveolar epithelial cell cultures. Then, the impact of I/R was studied in vivo using models of inflammatory stress induced by LPS infusion or after unilateral lung transplantation in pigs. Finally, lung biopsies from donor grafts were collected during lung transplantations. After defining PGD grades within the recipients, alveolar alterations were finally analyzed within donor graft tissues. Results In primary rat cell cultures, the I/R mimicking protocol impaired tight junction proteins (ZO-1), transepithelial resistance, wound repair capacity as well as expression in ENaC and CFTR channels. Treatment with a KvLQT1 K+ channel activator (R-L3) accelerated the repair rates and enhanced barrier integrity (ZO-1 staining) as well as ENaC and CFTR protein expressions. In the porcine models, an exacerbated inflammatory response was observed along with alveolar damage, lung edema and decreased ZO-1, ENaC and CFTR expressions. Our preliminary analyses also indicated that among lung transplants subsequently developing PGD, an inflammatory response and reduced ENaC, CFTR and ZO-1 expressions were already observed within biopsies from donor grafts, collected before transplantation. Conclusion Altogether our data support the hypothesis of alveolar epithelial dysfunction, after I/R injury. Based on these observations, our goal is to investigate a potential relationship between donor phenotypic factors, markers of epithelial damage/dysfunction within the grafts, inflammatory levels and the subsequent development of PGD in lung recipients.

Purpose Ischemia reperfusion (I/R) is a major cause of primary graft dysfunction (PGD) after lung transplantation. I/R and PGD feature endothelial/alveolar epithelial damage, lung edema and inflammation. Edema resorption depends on the restoration of alveolar integrity and its ability to reabsorb Na+ (ENaC) and fluid. We hypothesized that alveolar epithelial damage and repair are critical in PGD pathophysiology and resolution. Our aim is to identify novel markers and therapeutic targets associated with I/R using cellular and animal models as well as human samples from lung transplants. Methods Mimicking I/R protocol was used to evaluate alveolar epithelial barrier integrity and capacity of wound healing in primary cultures of rat's alveolar epithelial cells. These cell cultures were then treated with an activator of K+ channel (KvLQT1). In a porcine model of ischemia/ex-vivo reperfusion, an inflammatory stress was induced by i.v. infusion with LPS. Finally, samples were collected from 38 lung transplant patients. Results In primary cell cultures, we showed altered ENaC and tight junction protein (ZO-1) expression after injury. A decline in transepithelial resistance (TER) and altered alveolar wound repair rates were also observed following the mimicking I/R protocol. Treatment with K+ channel activator (R-L3) showed enhanced repair rates, barrier integrity (higher TER, ZO-1 staining) and ENaC expression. In our porcine model, lung damage and edema was observed as well as exacerbated inflammatory response and decreased ENaC expression. Preliminary data from lung transplant samples indicated an inflammatory response and decreased ENaC and ZO-1 expression in patients with PGD. Conclusion Our data support the hypothesis of alveolar epithelial dysfunction after I/R injury. We will now investigate a potential correlation between levels of inflammatory molecules and epithelial damage markers in bronchoalveolar lavages and blood samples (at different time-points) from lung transplants with various PGD scores.

Oxytocin (OT) is involved in the regulation of energy metabolism and in the activation of cardioprotective mechanisms. We evaluated whether chronic treatment with OT could prevent the metabolic and cardiac abnormalities associated with diabetes and obesity using the db/db mice model. Four-week-old male db/db mice and their lean nondiabetic littermates (db/+) serving as controls were treated with OT (125 ng/kg · h) or saline vehicle for a period of 12 weeks. Compared with db/+ mice, the saline-treated db/db mice developed obesity, hyperglycemia, and hyperinsulinemia. These mice also exhibited a deficient cardiac OT/natriuretic system and developed systolic and diastolic dysfunction resulting from cardiomyocyte hypertrophy, fibrosis, and apoptosis. These abnormalities were associated with increased reactive oxygen species (ROS) production, inflammation, and suppressed 5'-adenosine monophosphate kinase signaling pathway. The db/db mice displayed reduced serum levels of adiponectin and adipsin and elevated resistin. OT treatment increased circulating OT levels, significantly reduced serum resistin, body fat accumulation (19%; P

Background Oxytocin (OT) and functional OT receptor (OTR) are expressed in the heart and are involved in blood pressure regulation and cardioprotection. Cardiac OTR signaling is associated with atrial natriuretic peptide (ANP) and nitric oxide (NO) release. During the synthesis of OT, its precursor, termed OT-Gly-Lys-Arg (OT-GKR), is accumulated in the developing rat heart. Consequently, we hypothesized that an OT-related mechanism of ANP controls cardiomyocyte (CM) hypertrophy. Methods The experiments were carried out in newborn and adult rat CM cultures. The enhanced protein synthesis and increased CM volume were mediated by a 24-h treatment with endothelin-1 or angiotensin II. Results The treatment of CM with OT or its abundant cardiac precursor, OT-GKR, revealed ANP accumulation in the cell peri-nuclear region and increased intracellular cGMP. Consequently, the CM hypertrophy was abolished by the treatment of 10nM OT or 10nM OT-GKR. The ANP receptor antagonist (anantin) and NO synthases inhibitor (l-NAME) inhibited cGMP production in CMs exposed to OT. STO-609 and compound C inhibition of anti-hypertrophic OT effects in CMs indicated the contribution of calcium–calmodulin kinase kinase and AMP-activated protein kinase pathways. Moreover, in ET-1 stimulated cells, OT treatment normalized the reduced Akt phosphorylation, prevented abundant accumulation of ANP and blocked ET-1-mediated translocation of nuclear factor of activated T-cells (NFAT) into the cell nuclei. Conclusion cGMP/protein kinase G mediates OT-induced anti-hypertrophic response with the contribution of ANP and NO. OT treatment represents a novel approach in attenuation of cardiac hypertrophy during development and cardiac pathology.

Background and purpose: Left ventricular hypertrophy (LVH) is a maladaptive process associated with increased cardiovascular risk. Regression of LVH is associated with reduced complications of hypertension. Moxonidine is an antihypertensive imidazoline compound that reduces blood pressure primarily by central inhibition of sympathetic outflow and by direct actions on the heart to release atrial natriuretic peptide, a vasodilator and an antihypertrophic cardiac hormone. This study investigated the effect of moxonidine on LVH and the mechanisms involved in this effect. Experimental approach: Spontaneously hypertensive rats were treated with several doses of moxonidine (s.c.) over 4 weeks. Blood pressure and heart rate were continuously monitored by telemetry. Body weight and water and food intake were measured weekly. Measurements also included left ventricular mass, DNA content, synthesis, fragmentation, and apoptotic/ anti-apoptotic pathway proteins. Key results: The decrease in mean arterial pressure stabilized at B � 10 mm Hg after 1 week of treatment and thereafter. Compared to vehicle-treated rats (100%), left ventricular mass was dose- and time-dependently reduced by treatment. This reduction remained significantly lower after normalizing to body weight. Moxonidine reduced left ventricular DNA content and inhibited DNA synthesis. DNA fragmentation transiently, but significantly increased at 1 week of moxonidine treatment and was paralleled by elevated active caspase-3 protein. The highest dose significantly decreased the apoptotic protein Bax and all doses stimulated anti-apoptotic Bcl-2 after 4 weeks of treatment. Conclusions and implications: These studies implicate the modulation of cardiac DNA dynamics in the control of left ventricular mass by moxonidine in a rat model of hypertension. British Journal of Pharmacology (2008) 153, 459–467; doi:10.1038/sj.bjp.0707588; published online 3 December 2007

We have previously shown that acute intravenous injections of moxonidine and clonidine increase plasma atrial natriuretic peptide (ANP), a vasodilator, diuretic and natriuretic hormone. We hypothesized that moxonidine stimulates the release of ANP, which would act on its renal receptors to cause diuresis and natriuresis, and these effects may be altered in hypertension. Moxonidine (0, 10, 50, 100 or 150 microg in 300 microl saline) and clonidine (0, 1, 5 or 10 microg in 300 microl saline) injected intravenously in conscious normally hydrated normotensive Sprague-Dawley rats (SD, approximately 200 g) and 12-14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) dose-dependently stimulated diuresis, natriuresis, kaliuresis and cGMP excretion, with these effects being more pronounced during the first hour post-injection. The actions of 5 microg clonidine and 50 microg moxonidine were inhibited by yohimbine, an alpha2-adrenoceptor antagonist, and efaroxan, an imidazoline I1-receptor antagonist. Moxonidine (100 microg) stimulated (P

Moxonidine, an imidazoline receptor agonist that acts centrally to inhibit sympathetic activity, has been shown to reduce effectively blood pressure, fasting insulin levels, and free fatty acids. In this study, we investigated the long-term effects of moxonidine treatment on cardiac natriuretic peptides (ANP and BNP) in Spontaneously Hypertensive Obese Rats (SHROBs), a rat model that resembles human Syndrome X. SHROBs expressing spontaneous hypertension, insulin resistance, and genetic obesity (weight 590 +/- 20 g, at 30 weeks) received moxonidine in chow at 4 mg/kg/day for 15 days. Moxonidine significantly reduced not only systolic blood pressure (187 +/- 6 versus 156 +/- 5 mm Hg, P < 0.05) but also plasma ANP (1595 +/- 371 versus 793 +/- 131 pg/mL, P < 0.05) and BNP (22 +/- 3 versus 14 +/- 1 pg/mL, P < 0.04), without influencing cardiac content of either peptide. Semi-quantitative PCR revealed that atrial ANPmRNA/GAPDHmRNA decreased to 39% 6 10% of pair-fed controls, P < 0.03. In left ventricles, moxonidine also decreased ANP mRNA to 69% +/- 7% and BNP mRNA to 74% +/- 6% of control, P < 0.02, but right ventricular ANP and BNP mRNA were not affected. These findings indicate that chronic inhibition of sympathetic activity with moxonidine in SHROB is associated with decreased ventricular natriuretic peptide transcription, consistent with the cardioprotective effects of moxonidine given the role of ANP and BNP as markers of cadiac disease. Moxonidine also improves the metabolic profile in these rats, thus it may be considered the drug of choice in treatment of metabolic syndrome X.

Background Acute intravenous administration of moxonidine, an imidazoline I1-receptor agonist, reduces blood pressure (BP) in normotensive and hypertensive rats, induces diuresis and natriuresis, and stimulates plasma atrial natriuretic peptide (ANP). In these studies we investigated the involvement of natriuretic peptides (ANP and brain natriuretic peptide) in the effects of chronic activation of imidazoline receptors. Methods Spontaneously hypertensive rats (SHR; 12 to 14 weeks old) received 7-day moxonidine treatment at various doses (10, 20, 60, and 120 μg/kg/h) via subcutaneously implanted osmotic minipumps. Results: Hemodynamic parameters (continuously monitored by telemetry) revealed that, compared with saline-treated rats, moxonidine dose-dependently decreased blood pressures (BPs). Maximal blood pressure lowering effect was achieved by day 4 of treatment, at which point 60 μg/kg/h reduced mean arterial pressure (MAP) by 14.5 ± 6.8 mm Hg as compared with basal levels. The decrease in MAP was influenced by a drop in both diastolic and systolic pressures. Moxonidine treatment did not alter daily urinary sodium and potassium excretions, but 120 μg/kg/h moxonidine decreased urine volume after 2 days and increased cyclic guanosine 3′5′monophosphate excretion on days 4 to 7 of treatment. Chronic moxonidine treatment dose-dependently increased plasma ANP to reach, at 120 μg/kg/h, a 40% increase (P Conclusions: ANP and BNP may be involved in the antihypertensive effect of chronic moxonidine treatment. Accordingly, natriuretic peptides may contribute to the sympatholytic and cardioprotective effects of chronic activation of imidazoline I1-receptors.

Oxytocin (OT) stimulates cardioprotection. Here we investigated heart-derived H9c2 cells in simulated ischemia–reperfusion (I–R) experiments in order to examine the mechanism of OT protection. I–R was induced in an anoxic chamber for 2 hours and followed by 2 h of reperfusion. In comparison to normoxia, I–R resulted in decrease of formazan production by H9c2 cells to 63.5 ± 1.7% (MTT assay) and in enhanced apoptosis from 1.7 ± 0.3% to 2.8 ± 0.4% (Tunel test). Using these assays it was observed that treatment with OT (1–500 nM) exerted significant protection during I–R, especially when OT was added at the time of ischemia or reperfusion. Using the CM-H2DCFDA probe we found that OT triggers a short-lived burst in reactive oxygen species (ROS) production in cells but reduces ROS production evoked by I–R. In cells treated with OT, Western-blot revealed the phosphorylation of Akt (Thr 308, p-Akt), eNOS and ERK 1/2. Microscopy showed translocation of p-Akt and eNOS into the nuclear and perinuclear area and NO production in cells treated with OT. The OT-induced protection against I–R was abrogated by an OT antagonist, the Pi3K inhibitor Wortmannin, the cGMP-dependent protein kinase (PKG) inhibitor, KT5823, as well as soluble guanylate cyclase (GC) inhibitor, ODQ, and particulate GC antagonist, A71915. In conditions of I–R, the cells with siRNA-mediated reduction in OT receptor (OTR) expression responded to OT treatment by enhanced apoptosis. In conclusion, the OTR protected H9c2 cells against I–R, especially if activated at the onset of ischemia or reperfusion. The OTR-transduced signals include pro-survival kinases, such as Akt and PKG.

Obesity and diabetes increase the risk of developing cardiovascular diseases and heart failure. These metabolic disorders are generally reflected by natriuretic peptide system deficiency. Since brain natriuretic peptide (BNP) is known to influence metabolism and cardioprotection, we investigated the effect of chronic exogenous BNP treatment on adverse myocardial consequences related to obesity and diabetes.Ten-week-old C57BL/KsJ-db/db obese diabetic mice (db/db) and their lean control littermates (db/+) were treated with BNP (0.6 μg kg(-1) h(-1)) or saline for 12 weeks (n = 10/group). Serial blood and tomography analysis were performed. Cardiac function was determined by echocardiography, and biochemical and histological heart and fat analyses were also performed.BNP treatment resulted in an average increase in plasma BNP levels of 70 pg/ml. An improvement in the metabolic profile of db/db mice was observed, including a reduction in fat content, increased insulin sensitivity, improved glucose tolerance and lower blood glucose, despite increased food intake. db/db mice receiving saline displayed both early systolic and diastolic dysfunction, whereas these functional changes were prevented by BNP treatment. The cardioprotective effects of BNP were attributed to the inhibition of cardiomyocyte apoptosis, myocardial fibrosis, cardiac hypertrophy and the AGE-receptor for AGE (RAGE) system as well as normalisation of cardiac AMP-activated protein kinase and endothelial nitric oxide synthase activities.Our results indicate that chronic BNP treatment at low dose improves the metabolic profile and prevents the development of myocardial dysfunction in db/db mice.

Principal limitation of cell therapy is cell loss after transplantation because of the interplay between ischemia, inflammation, and apoptosis. We investigated the mechanism of preconditioning of mesenchymal stem cells (MSCs) with oxytocin (OT), which has been proposed as a novel strategy for enhancing therapeutic potential of these cells in ischemic heart. In this study, we demonstrate that rat MSCs express binding sites for OT receptor and OT receptor transcript and protein as detected by RT-PCR and immunofluorescence, respectively. In response to OT (10−10 to 10−6m) treatment, MSCs respond with rapid calcium mobilization and up-regulation of the protective protein kinase B (PKB or Akt) and phospho-ERK1/2 proteins. In OT-stimulated cells, phospho-Akt accumulates intracellularly close to the mitochondrial marker cytochrome c oxidase subunit 4. Functional analyses reveal the involvement of Akt/ERK1/2 pathways in cell proliferation, migration, and protection against the cytotoxic and apoptotic effects of hypoxia and serum deprivation. In addition, OT preconditioning increases MSC glucose uptake. Genes with angiogenic, antiapoptotic, and cardiac antiremodeling properties, such as heat shock proteins (hsps) HSP27, HSP32, HSP70, vascular endothelial growth factor, thrombospondin, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, TIMP-3, and matrix metalloproteinase-2, were also up-regulated upon OT exposure. Moreover, coculture with OT-preconditioned MSC reduces apoptosis, as measured using terminal transferase dUTP nick end labeling assay in newborn rat cardiomyocytes exposed to hypoxia and reoxygenation. In conclusion, these results indicate that OT treatment evokes MSC protection through both intrinsic pathways and secretion of cytoprotective factors. Ex vivo cellular treatment with OT represents an attractive strategy aimed to maximize the biological and functional properties of effector cells.

The renin-angiotensin and sympathetic nervous systems play critical interlinked roles in the development of left ventricular hypertrophy, fibrosis, and dysfunction. These studies investigated the hemodynamic and cardiac effects of monoblockade and coblockade of renin-angiotensin and sympathetic nervous systems. Stroke-prone spontaneously hypertensive rats (16 weeks old; male; n=12 per group) received the sympatholytic imidazoline compound, moxonidine (2.4 mg/kg per day); the angiotensin-receptor blocker eprosartan (30 mg/kg per day), separately or in combination; or saline vehicle for 8 weeks, SC, via osmotic minipumps. Blood pressure and heart rate were continuously measured by radiotelemetry. After 8 weeks, in vivo cardiac function and structure were measured by transthoracic echocardiography and a Millar conductance catheter, and the rats were then euthanized and blood and heart ventricles collected for various determinations. Compared with vehicle, the subhypotensive dose of moxonidine resulted in lower ( P P P

Objective:Obesity and diabetes enhance the risk of developing cardiovascular diseases and heart failure. The heart/cardiac oxytocin (OT) system was discovered by our group and shown to regulate cardiovascular cell survival pathways and cardioprotection. OT is also involved in the regulation of cardi

Imidazoline I1-receptors are present in the heart and may be involved in atrial natriuretic peptide (ANP) release. The following studies investigated whether moxonidine (an antihypertensive imidazoline I1-receptor and alpha2-adrenoceptor agonist) acts directly on the heart to stimulate ANP release, and to characterize the receptor type involved in this action. Perfusion of rat (200-225 g) isolated hearts with moxonidine (10(-6) and 10(-5) M), for 30 min, resulted in ANP release (83+/-29 and 277+/-70 ng/30 min, above basal, respectively), significantly (P0.01) different from perfusion with buffer (-6+/-31 ng/30 min). ANP release stimulated by moxonidine (10(-6) M) was inhibited by co-perfusion with the antagonists, AGN192403 (imidazoline I1-receptor), phenoxybenzamine (alpha2alpha1-adrenoceptors), and prazosin (alpha1alpha2-adrenoceptors), but increased by rauwolscine (alpha2-adrenoceptors). Perfusion with 10(-5) M brimonidine (full alpha2-adrenoceptor agonist) inhibited moxonidine-stimulated ANP release. Similarly, moxonidine (10(-6) M) tended to reduce coronary flow, but significantly increased coronary flow in the presence of brimonidine, which was vasoconstrictive when perfused alone. Coronary flow was reduced by 10(-5) M each, brimonidineclonidinemoxonidine; while similar bradycardia was observed with clonidine and moxonidine, but not with brimonidine. In conclusion, these results argue in favor of moxonidine acting primarily on imidazoline I1-receptors to release ANP, with both alpha2-adrenoceptor and imidazoline I1-receptors exerting inhibitory inter-relation. In contrast, the coronary vasodilatory effect of moxonidine requires full activation of alpha2-adrenoceptor. The sympatholytic and ANP-releasing effects of moxonidine appear to be mediated by cardiac imidazoline receptors that may be differentially localized. Most importantly, moxonidine can stimulate ANP release from the heart without contribution of the central nervous system.

We have previously shown that acute intravenous injections of moxonidine and clonidine increase plasma atrial natriuretic peptide (ANP), a vasodilator, diuretic and natriuretic hormone. We hypothesized that moxonidine stimulates the release of ANP, which would act on its renal receptors to cause diuresis and natriuresis, and these effects may be altered in hypertension. Moxonidine (0, 10, 50, 100 or 150 microg in 300 microl saline) and clonidine (0, 1, 5 or 10 microg in 300 microl saline) injected intravenously in conscious normally hydrated normotensive Sprague-Dawley rats (SD, approximately 200 g) and 12-14-week-old Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) dose-dependently stimulated diuresis, natriuresis, kaliuresis and cGMP excretion, with these effects being more pronounced during the first hour post-injection. The actions of 5 microg clonidine and 50 microg moxonidine were inhibited by yohimbine, an alpha2-adrenoceptor antagonist, and efaroxan, an imidazoline I1-receptor antagonist. Moxonidine (100 microg) stimulated (P0.01) diuresis in SHR (0.21+/-0.04 vs 1.16+/-0.06 ml h(-1) 100 g(-1)), SD (0.42+/-0.06 vs 1.56+/-0.19 ml h(-1) 100 g(-1)) and WKY (0.12+/-0.04 vs 1.44+/-0.21 ml h(-1) 100 g(-1)). Moxonidine-stimulated urine output was lower in SHR than in SD and WKY. Moxonidine-stimulated sodium and potassium excretions were lower in SHR than in SD, but not WKY, demonstrating an influence of strain but not of pressure. Pretreatment with the natriuretic peptide antagonist anantin (5 or 10 microg) resulted in dose-dependent inhibition of moxonidine-stimulated urinary actions. Anantin (10 microg) inhibited (P0.01) urine output to 0.38+/-0.06, 0.12+/-0.01, and 0.16+/-0.04 ml h(-1) 100 g(-1) in SD, WKY, and SHR, respectively. Moxonidine increased (P0.01) plasma ANP in SD (417+/-58 vs 1021+/-112 pg ml(-1)) and WKY (309+/-59 vs 1433+/-187 pg ml(-1)), and in SHR (853+/-96 vs 1879+/-229 pg ml(-1)). These results demonstrate that natriuretic peptides mediate the urinary actions of moxonidine through natriuretic peptide receptors.

We have recently identified imidazoline I(1)-receptors in the heart. In the present study, we tested regulation of cardiac I(1)-receptors versus alpha(2) -adrenoceptors in response to hypertension and to chronic exposure to agonist. Spontaneously hypertensive rats (SHR, 12-14 weeks old) received moxonidine (10, 60, and 120 microg/kg/h s.c.) for 1 and 4 weeks. Autoradiographic binding of (125)I-paraiodoclonidine (0.5 nM, 1 h, 22 degrees C) and inhibition of binding with epinephrine (10(-10)-10(-5) M) demonstrated the presence of alpha(2)-adrenoceptors in heart atria and ventricles. Immunoblotting and reverse transcription-polymerase chain reaction identified alpha(2A)-alpha(2B)-, and alpha(2C), and -adrenoceptor proteins and mRNA, respectively. However, compared with normotensive controls, cardiac alpha(2) -adrenoceptor kinetic parameters, receptor proteins, and mRNAs were not altered in SHR with or without moxonidine treatment. In contrast, autoradiography showed that up-regulated atrial I(1)-receptors in SHR are dose-dependently normalized by 1 week, with no additional effect after 4 weeks of treatment. Moxonidine (120 microg/kg/h) decreased B(max) in right (40.0 +/- 2.9-7.0 +/- 0.6 fmol/unit area; p0.01) and left (27.7 +/- 2.8-7.1 +/- 0.4 fmol/unit area; p0.01) atria, and decreased the 85- and 29-kDa imidazoline receptor protein bands, in right atria, to 51.8 +/- 3.0% (p0.01) and 82.7 +/- 5.2% (p0.03) of vehicle-treated SHR, respectively. Moxonidine-associated percentage of decrease in B(max) only correlated with the 85-kDa protein (R(2) = 0.57; p0.006), suggesting that this protein may represent I(2)-receptors. The weak but significant correlation between the two imidazoline receptor proteins (R(2) = 0.28; p0.03) implies that they arise from the same gene. In conclusion, the heart possesses I(1)-receptors and alpha(2)-adrenoceptors, but only I(1)-receptors are responsive to hypertension and to chronic in vivo treatment with a selective I(1)-receptor agonist.

The effect of treatment with moxonidine (120 mg/kg/h sc, 4 weeks) on cardiac I(1)-receptors and natriuretic peptide synthesis was evaluated in spontaneously hypertensive rats (SHR). I(1)-receptor protein (85 kD) was up-regulated in SHR atria, and normalized in right and left atria by moxonidine. Similarly, moxonidine normalized atrial and ventricular atrial natriuretic peptide messenger RNA (mRNA) and brain natriuretic peptide mRNA. This study shows that cardiac I(1)-receptors are functional, being regulated by hypertension and by chronic exposure to agonist, and that cardiac natriuretic peptides may be regulated by I(1)-receptor-mediated mechanisms.

We have recently uncovered the presence of an oxytocin system in the heart and found that oxytocin is a physiological regulator of atrial natriuretic peptide (ANP), a diuretic, natriuretic and vasodilator cardiac hormone. However, dynamic changes in these systems during gestation, when mechanisms of volume and pressure homeostasis are altered, are not clear. Accordingly, ANP, oxytocin and oxytocin receptors were evaluated in rat hearts and plasma at three stages of gestation (7, 14 and 21 days) and at 2 and 5 days postpartum. Compared with non-pregnant controls, plasma ANP was elevated in mid-gestation, but significantly decreased at term (21 days), to increase again postpartum. Right and left atrial ANP mRNA levels were not altered throughout gestation but increased by 1.5- to 2-fold postpartum (P

Acute intravenous administration of moxonidine, an imidazoline I1-receptor agonist, reduces blood pressure (BP) in normotensive and hypertensive rats, induces diuresis and natriuresis, and stimulates plasma atrial natriuretic peptide (ANP). In these studies we investigated the involvement of natriuretic peptides (ANP and brain natriuretic peptide) in the effects of chronic activation of imidazoline receptors.Spontaneously hypertensive rats (SHR; 12 to 14 weeks old) received 7-day moxonidine treatment at various doses (10, 20, 60, and 120 microg/kg/h) via subcutaneously implanted osmotic minipumps.Hemodynamic parameters (continuously monitored by telemetry) revealed that, compared with saline-treated rats, moxonidine dose-dependently decreased blood pressures (BPs). Maximal blood pressure lowering effect was achieved by day 4 of treatment, at which point 60 microg/kg/h reduced mean arterial pressure (MAP) by 14.5 +/- 6.8 mm Hg as compared with basal levels. The decrease in MAP was influenced by a drop in both diastolic and systolic pressures. Moxonidine treatment did not alter daily urinary sodium and potassium excretions, but 120 microg/kg/h moxonidine decreased urine volume after 2 days and increased cyclic guanosine 3'5'monophosphate excretion on days 4 to 7 of treatment. Chronic moxonidine treatment dose-dependently increased plasma ANP to reach, at 120 microg/kg/h, a 40% increase (P.01) above that of corresponding saline-treated SHR, with a concomitant increase in left and right atrial ANP mRNA (more than twofold). Plasma BNP increased by 120 microg/kg/h moxonidine (11.0 +/- 1.1 v 16.5 +/- 1.9 pg/mL, P.002) without significant increases in atrial and ventricular BNP mRNA.ANP and BNP may be involved in the antihypertensive effect of chronic moxonidine treatment. Accordingly, natriuretic peptides may contribute to the sympatholytic and cardioprotective effects of chronic activation of imidazoline I1-receptors.

International audience; OBJECTIVE: Measurement of capillary-alveolar permeability to fluorescein isothiocyanate-dextran (FITC-D) (molecular mass, 71,300 daltons) by a sequential bronchoalveolar lavage (BAL) technique. DESIGN: Animal research. SETTING: The Department of Physiology at a scientific and medical university. SUBJECTS: Nine anesthetized and mechanically ventilated dogs. INTERVENTIONS: Two separate experiments were performed in each subject-an initial control experiment followed by an oleic acid-induced lung injury. The indicator was administered at constant blood concentration before serial BAL including eight fluid instillation-recovery cycles. MEASUREMENTS: Plasma to BAL solute clearance at saturation (capillary-alveolar clearance at saturation, mL/min) was calculated and normalized to lavage fluid volume (measured by 1251 serum albumin dilution) to obtain a transport rate (TR) constant. MAIN RESULTS: TR for FITC-D70 was 4.0+/-0.8 and 46.1+/-18.1 x 10(-5) x min(-1) in control and injured lung, respectively (p < .02). Capillary-alveolar clearance of FITC-D70 was not affected by the lavage procedure itself. TR reflected essentially epithelial permeability in normal lung and combined epithelial and endothelial permeability in injured lung. A significant correlation was found between cardiac output and TR in injured lung. CONCLUSIONS: Saturation BAL allowed us to estimate capillary-alveolar macromolecule permeability in vivo in dogs. Further study may allow bedside evaluation of lung injury by BAL in patients.

Background Distortion of the pressure wave by a liquid-filled catheter-transducer system leads most often to an overestimation in systolic arterial blood pressure in pulmonary and systemic circulations. The pressure distortion depends on the catheter-transducer frequency response. Many monitoring systems use either mechanical or electronic filters to reduce this distortion. Such filters assume, however, that the catheter-transducer frequency response does not change over time. The current study aimed to study the changes with time of the catheter-transducer frequency response and design a flush procedure to reverse these changes back to baseline. Methods An in vitro setup was devised to assess the catheter-transducer frequency response in conditions approximating some of those met in a clinical environment (slow flushing, 37 degrees C, 48-h test). Several flush protocols were assessed. Results Within 48 h, catheter-transducer natural frequency decreased from 17.89 +/- 0.36 (mean +/- SD) to 7.35 +/- 0.25 Hz, and the catheter-transducer damping coefficient increased from 0.234 +/- 0.004 to 0.356 +/- 0.010. Slow and rapid flushing by the flush device built into the pressure transducer did not correct these changes, which were reversed only by manual fast flush of the transducer and of the catheter. These changes and parallel changes in catheter-transducer compliance may be explained by bubbles inside the catheter-transducer. Conclusions Catheter-transducer-induced blood pressure distortion changes with time. This change may be reversed by a manual fast flush or "rocket flush" procedure, allowing a con. stant correction by a filter.