An experimental study of consecutive administration of ropinirole and apomorphine for emesis induction in dogs.

OBJECTIVE: To study the safety and effectiveness of consecutively administered ropinirole and apomorphine (both dopamine 2-like receptor agonists) for emesis induction in dogs. DESIGN: Prospective, crossover study design. SETTING: Institutional animal research facility. ANIMALS: Six healthy male purpose-bred Beagle dogs. INTERVENTIONS: Each dog received 4 treatments: (1) apomorphine infusion (21 µg/kg) over 30 minutes + ropinirole eye drops (3.75 mg/m2 ); (2) ropinirole infusion (108 µg/m2 ) over 30 minutes + apomorphine SC (100 µg/kg); (3) apomorphine SC (100 µg/kg) + ropinirole eye drops (7.5 mg/m2 ) after 30 minutes; and (4) ropinirole eye drops (7.5 mg/m2 ) + apomorphine SC (100 µg/kg) after 30 minutes. Infusions were administered via a catheter instrumented in the cephalic vein. Eye drops and SC injections were administered as described in the product inserts. Blood samples were taken for ropinirole and apomorphine concentration analysis before dosing and periodically following administrations. The washout period between the treatments was 5-7 days. MEASUREMENTS AND MAIN RESULTS: Number of vomits and clinical signs were recorded. Alertness and heart rate were monitored in conjunction with blood sampling. The average number of vomits varied between 4.3 and 8.8 (range 1-16) following treatments. Signs of nausea, vomiting, and lethargy were seen in all individuals without significant differences between treatments. Moderate to marked, transient increase in heart rates was detected in all treatments. Infrequent noted side effects included ocular hyperemia, blepharospasms, and muscle tremors. Prior treatment with apomorphine significantly decreased the absorption of ropinirole eye drops. CONCLUSIONS: The safety and efficacy profiles of this experimental study support that ropinirole and apomorphine could be administered consecutively in cases where the treatment using 1 substance has resulted in an incomplete evacuation of the stomach contents, and the attending veterinarian considers the use of a different agent to have benefits that outweigh the risks.

Ocular Administration of Palonosetron in the Prevention of Cisplatin-Induced Nausea and Vomiting.

Cancer treatments are frequently associated with nausea and vomiting despite greatly improved preventive medication. Administration of antinausea agents as eye drops might provide easy and rapid access to the systemic circulation for prevention of nausea and vomiting and for the treatment of breakthrough nausea, but the ocular administration route has rarely been evaluated. Palonosetron is a second-generation 5-hydroxytryptamine 3 receptor antagonist approved for prevention and treatment of chemotherapy-induced nausea and vomiting. We compared ocular administration of palonosetron to non-active vehicle eye drops and to intravenous palonosetron in the prevention of cisplatin-induced nausea and vomiting in beagle dogs. Palonosetron ocular drops at the dose of 30 µg/kg reduced cumulative nausea over time as measured with the area under the visual analog scale curve by 98% compared with the vehicle and reduced nausea-associated dog behavior by 95%. Vomiting was completely prevented with repeated palonosetron ocular dosing. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) palonosetron formulation was well tolerated locally at the palonosetron concentration of 3 mg/ml. Absorption of palonosetron from eye drops was fast. Ten minutes after ocular administration, palonosetron plasma concentrations were similar compared with intravenous administration, and remained similar for six hours. We conclude that palonosetron is rapidly absorbed into the systemic circulation from eye drops. Ocularly administered palonosetron was well tolerated in the HP-ß-CD formulation and was highly effective in the prevention of cisplatin-induced nausea and vomiting. Evaluation of the safety and efficacy of ocular administration of palonosetron is warranted in the prevention and treatment of chemotherapy-induced nausea and vomiting in clinical trials. SIGNIFICANCE STATEMENT: Palonosetron, an effective and well-tolerated antiemetic drug was rapidly absorbed into the systemic blood circulation when administered as eye drops. The achieved palonosetron blood concentrations prevented cisplatin-induced nausea and vomiting in beagle dogs. Palonosetron eye drops might provide an easy and quick method for administering palonosetron when parenteral administration is desired and intravenous administration is not feasible.

The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells.

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 µM ORM-10962. Mechanistic simulations were performed by Maltsev-Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 µM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

Rapid Absorption of Naloxone from Eye Drops.

Naloxone as emergency treatment for opioid overdosing can be administered via several routes. However, the available administration methods are invasive or may be associated with incomplete or slow naloxone absorption. We evaluated pharmacokinetics and local tolerance of naloxone ocular drops in healthy beagle dogs. Naloxone administration as eye drops produced fast absorption with time to maximum plasma concentration (tmax) achieved in 14 to 28 min, high plasma exposure (Cmax 10.3 ng/mL to 12.7 ng/mL), and good bioavailability (41% to 56%). No signs of ocular irritability were observed in the scored ocular tolerability parameters, and the reactions of dogs suggesting immediate ocular discomfort after the dosing were sporadic and short lasting. Slight and transient increase in the intraocular pressure and transient decrease in the tear production were recorded. The results suggest that eye drops may provide a fast and an effective non-invasive route for naloxone administration to reverse opioid overdosing, and clinical studies in the human are warranted.

The Adenylate Cyclase Activator Forskolin Potentiates the Positive Inotropic Effect of the Phosphodiesterase Inhibitor Milrinone But Not of the Calcium Sensitizer Levosimendan nor of Its Hemodynamically Active Metabolites: An Apparent Conundrum.

ABSTRACT: OR-1855 and OR-1896 are 2 hemodynamically active metabolites of the inodilator levosimendan, with calcium sensitizing activity, but their mechanism of action is still not fully understood. It has been previously reported that the positive inotropic effect of levosimendan is not potentiated by the adenylate cyclase activator forskolin, whereas forskolin does potentiate the effects of the phosphodiesterase (PDE) inhibitor milrinone. To ascertain whether the active metabolites follow the same pattern of levosimendan, the positive inotropic effects of OR- 1855 and OR-1896 were studied in guinea-pig-isolated papillary muscle in the presence and absence of forskolin. OR-1855 and OR-1896 were also tested as inhibitors of PDE-III and PDE-IV. Our results show that 0.1 µM forskolin did not potentiate the positive inotropic effect of OR-1855 or OR-1896, as in the case of the parent compound levosimendan. As in previous studies, the positive inotropic effect of milrinone was markedly potentiated in the presence of forskolin. From these data, we propose an explanation for the divergent behavior of the calcium sensitizing drugs and PDE inhibitors.

Blockade of sodium­calcium exchanger via ORM-10962 attenuates cardiac alternans.

Repolarization alternans, a periodic oscillation of long-short action potential duration, is an important source of arrhythmogenic substrate, although the mechanisms driving it are insufficiently understood. Despite its relevance as an arrhythmia precursor, there are no successful therapies able to target it specifically. We hypothesized that blockade of the sodium­calcium exchanger (NCX) could inhibit alternans. The effects of the selective NCX blocker ORM-10962 were evaluated on action potentials measured with microelectrodes from canine papillary muscle preparations, and calcium transients measured using Fluo4-AM from isolated ventricular myocytes paced to evoke alternans. Computer simulations were used to obtain insight into the drug's mechanisms of action. ORM-10962 attenuated cardiac alternans, both in action potential duration and calcium transient amplitude. Three morphological types of alternans were observed, with differential response to ORM-10962 with regards to APD alternans attenuation. Analysis of APD restitution indicates that calcium oscillations underlie alternans formation. Furthermore, ORM-10962 did not markedly alter APD restitution, but increased post-repolarization refractoriness, which may be mediated by indirectly reduced L-type calcium current. Computer simulations reproduced alternans attenuation via ORM-10962, suggesting that it is acts by reducing sarcoplasmic reticulum release refractoriness. This results from the ORM-10962-induced sodium­calcium exchanger block accompanied by an indirect reduction in L-type calcium current. Using a computer model of a heart failure cell, we furthermore demonstrate that the anti-alternans effect holds also for this disease, in which the risk of alternans is elevated. Targeting NCX may therefore be a useful anti-arrhythmic strategy to specifically prevent calcium driven alternans.

Discovery and characterization of ORM-11372, a novel inhibitor of the sodium-calcium exchanger with positive inotropic activity.

BACKGROUND AND PURPOSE: The lack of selective sodium-calcium exchanger (NCX) inhibitors has hampered the exploration of physiological and pathophysiological roles of cardiac NCX 1.1. We aimed to discover more potent and selective drug like NCX 1.1 inhibitor. EXPERIMENTAL APPROACH: A flavan series-based pharmacophore model was constructed. Virtual screening helped us identify a novel scaffold for NCX inhibition. A distinctively different NCX 1.1 inhibitor, ORM-11372, was discovered after lead optimization. Its potency against human and rat NCX 1.1 and selectivity against other ion channels was assessed. The cardiovascular effects of ORM-11372 were studied in normal and infarcted rats and rabbits. Human cardiac safety was studied ex vivo using human ventricular trabeculae. KEY RESULTS: ORM-11372 inhibited human NCX 1.1 reverse and forward currents; IC50 values were 5 and 6 nM respectively. ORM-11372 inhibited human cardiac sodium 1.5 (INa ) and hERG KV 11.1 currents (IhERG ) in a concentration-dependent manner; IC50 values were 23.2 and 10.0 µM. ORM-11372 caused no changes in action potential duration; short-term variability and triangulation were observed for concentrations of up to 10 µM. ORM-11372 induced positive inotropic effects of 18 ± 6% and 35 ± 8% in anaesthetized rats with myocardial infarctions and in healthy rabbits respectively; no other haemodynamic effects were observed, except improved relaxation at the lowest dose. CONCLUSION AND IMPLICATIONS: ORM-11372, a unique, novel, and potent inhibitor of human and rat NCX 1.1, is a positive inotropic compound. NCX inhibition can induce clinically relevant improvements in left ventricular contractions without affecting relaxation, heart rate, or BP, without pro-arrhythmic risk.

Long-term effects of Na+ /Ca2+ exchanger inhibition with ORM-11035 improves cardiac function and remodelling without lowering blood pressure in a model of heart failure with preserved ejection fraction.

AIMS: Heart failure with preserved ejection fraction (HFpEF) is increasingly common but there is currently no established pharmacological therapy. We hypothesized that ORM-11035, a novel specific Na+ /Ca2+ exchanger (NCX) inhibitor, improves cardiac function and remodelling independent of effects on arterial blood pressure in a model of cardiorenal HFpEF. METHODS AND RESULTS: Rats were subjected to subtotal nephrectomy (NXT) or sham operation. Eight weeks after intervention, treatment for 16 weeks with ORM-11035 (1 mg/kg body weight) or vehicle was initiated. At 24 weeks, blood pressure measurements, echocardiography and pressure-volume loops were performed. Contractile function, Ca2+ transients and NCX-mediated Ca2+ extrusion were measured in isolated ventricular cardiomyocytes. NXT rats (untreated) showed a HFpEF phenotype with left ventricular (LV) hypertrophy, LV end-diastolic pressure (LVEDP) elevation, increased brain natriuretic peptide (BNP) levels, preserved ejection fraction and pulmonary congestion. In cardiomyocytes from untreated NXT rats, early relaxation was prolonged and NCX-mediated Ca2+ extrusion was decreased. Chronic treatment with ORM-11035 significantly reduced LV hypertrophy and cardiac remodelling without lowering systolic blood pressure. LVEDP [14 ± 3 vs. 9 ± 2 mmHg; NXT (n = 12) vs. NXT + ORM (n = 12); P = 0.0002] and BNP levels [71 ± 12 vs. 49 ± 11 pg/mL; NXT (n = 12) vs. NXT + ORM (n = 12); P < 0.0001] were reduced after ORM treatment. LV cardiomyocytes from ORM-treated rats showed improved active relaxation and diastolic cytosolic Ca2+ decay as well as restored NCX-mediated Ca2+ removal, indicating NCX modulation with ORM-11035 as a promising target in the treatment of HFpEF. CONCLUSION: Chronic inhibition of NCX with ORM-11035 significantly attenuated cardiac remodelling and diastolic dysfunction without lowering systemic blood pressure in this model of HFpEF. Therefore, long-term treatment with selective NCX inhibitors such as ORM-11035 should be evaluated further in the treatment of heart failure.

Novel Na+/Ca2+ Exchanger Inhibitor ORM-10962 Supports Coupled Function of Funny-Current and Na+/Ca2+ Exchanger in Pacemaking of Rabbit Sinus Node Tissue.

BACKGROUND AND PURPOSE: The exact mechanism of spontaneous pacemaking is not fully understood. Recent results suggest tight cooperation between intracellular Ca2+ handling and sarcolemmal ion channels. An important player of this crosstalk is the Na+/Ca2+ exchanger (NCX), however, direct pharmacological evidence was unavailable so far because of the lack of a selective inhibitor. We investigated the role of the NCX current in pacemaking and analyzed the functional consequences of the If-NCX coupling by applying the novel selective NCX inhibitor ORM-10962 on the sinus node (SAN). EXPERIMENTAL APPROACH: Currents were measured by patch-clamp, Ca2+-transients were monitored by fluorescent optical method in rabbit SAN cells. Action potentials (AP) were recorded from rabbit SAN tissue preparations. Mechanistic computational data were obtained using the Yaniv et al. SAN model. KEY RESULTS: ORM-10962 (ORM) marginally reduced the SAN pacemaking cycle length with a marked increase in the diastolic Ca2+ level as well as the transient amplitude. The bradycardic effect of NCX inhibition was augmented when the funny-current (If) was previously inhibited and vice versa, the effect of If was augmented when the Ca2+ handling was suppressed. CONCLUSION AND IMPLICATIONS: We confirmed the contribution of the NCX current to cardiac pacemaking using a novel NCX inhibitor. Our experimental and modeling data support a close cooperation between If and NCX providing an important functional consequence: these currents together establish a strong depolarization capacity providing important safety factor for stable pacemaking. Thus, after individual inhibition of If or NCX, excessive bradycardia or instability cannot be expected because each of these currents may compensate for the reduction of the other providing safe and rhythmic SAN pacemaking.

Complex electrophysiological remodeling in postinfarction ischemic heart failure.

Heart failure (HF) following myocardial infarction (MI) is associated with high incidence of cardiac arrhythmias. Development of therapeutic strategy requires detailed understanding of electrophysiological remodeling. However, changes of ionic currents in ischemic HF remain incompletely understood, especially in translational large-animal models. Here, we systematically measure the major ionic currents in ventricular myocytes from the infarct border and remote zones in a porcine model of post-MI HF. We recorded eight ionic currents during the cell's action potential (AP) under physiologically relevant conditions using selfAP-clamp sequential dissection. Compared with healthy controls, HF-remote zone myocytes exhibited increased late Na+ current, Ca2+-activated K+ current, Ca2+-activated Cl- current, decreased rapid delayed rectifier K+ current, and altered Na+/Ca2+ exchange current profile. In HF-border zone myocytes, the above changes also occurred but with additional decrease of L-type Ca2+ current, decrease of inward rectifier K+ current, and Ca2+ release-dependent delayed after-depolarizations. Our data reveal that the changes in any individual current are relatively small, but the integrated impacts shift the balance between the inward and outward currents to shorten AP in the border zone but prolong AP in the remote zone. This differential remodeling in post-MI HF increases the inhomogeneity of AP repolarization, which may enhance the arrhythmogenic substrate. Our comprehensive findings provide a mechanistic framework for understanding why single-channel blockers may fail to suppress arrhythmias, and highlight the need to consider the rich tableau and integration of many ionic currents in designing therapeutic strategies for treating arrhythmias in HF.

Inotropic effect of NCX inhibition depends on the relative activity of the reverse NCX assessed by a novel inhibitor ORM-10962 on canine ventricular myocytes.

Na+/Ca2+ exchanger (NCX) is the main Ca2+ transporter in cardiac myocytes. Its inhibition could be expected to exert positive inotropic action by accumulation of cytosolic Ca2+ ([Ca2+]i). However, we have observed only a marginal positive inotropic effect upon selective inhibition of NCX, which was enhanced when forward activity was facilitated. Here we attempted to clarify the underlying mechanism of the limited inotropic action of selective NCX inhibition by a novel inhibitor ORM-10962 on canine ventricular myocytes. 1µM ORM-10962 reduced the Ca2+ content of sarcoplasmic reticulum (SR) when the reverse NCX was favoured, while SR Ca2+ content was increased by ORM-10962 under conditions favouring the forward activity, like elevation of [Ca2+]i. L-type Ca2+ current (ICa) was not affected by 1µM ORM-10962 in the absence of SR Ca2+ release, while ICa was suppressed by ORM-10962 during normal Ca2+ cycling. The apparent degree of forward NCX inhibition was dependent on the elevation of [Ca2+]i, suggesting that an increased driving force of forward NCX can also limit the accumulation of [Ca2+i]. We concluded that in healthy myocardium the possible positive inotropic potential of NCX inhibition is considerably weaker than it was expected earlier by theoretical assumptions. The underlying mechanism may involve the autoregulation of Ca2+ handling and/or the preserved inducibility of forward NCX by high [Ca2+]i. This limitation of selective NCX inhibition seen in undiseased myocardium requires further studies in failing heart, which may allow correct evaluation of the potential therapeutic value of selective NCX inhibitors in the treatment of heart failure.

The Effect of a Novel Highly Selective Inhibitor of the Sodium/Calcium Exchanger (NCX) on Cardiac Arrhythmias in In Vitro and In Vivo Experiments.

BACKGROUND: In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients, cell shortening and in experimental arrhythmias. The level of selectivity of the novel inhibitor on several major transmembrane ion currents (L-type Ca2+ current, major repolarizing K+ currents, late Na+ current, Na+/K+ pump current) was also determined. METHODS: Ion currents in single dog ventricular cells (cardiac myocytes; CM), and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 µg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts. RESULTS: ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 µM, did not modify significantly the magnitude of ICaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 µM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts. CONCLUSIONS: The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis, in vivo, however, its effect on I/R induced arrhythmias is still uncertain.

ORM-3819 promotes cardiac contractility through Ca(2+) sensitization in combination with selective PDE III inhibition, a novel approach to inotropy.

This study is the first pharmacological characterization of the novel chemical entity, ORM-3819 (L-6-{4-[N'-(4-Hydroxi-3-methoxy-2-nitro-benzylidene)-hydrazino]-phenyl}-5-methyl-4,5-dihydro-2H-pyridazin-3-one), focusing primarily on its cardiotonic effects. ORM-3819 binding to cardiac troponin C (cTnC) was confirmed by nuclear magnetic resonance spectroscopy, and a selective inhibition of the phosphodiesterase III (PDE III) isozyme (IC50=3.88±0.3 nM) was revealed during in vitro enzyme assays. The Ca(2+)-sensitizing effect of ORM-3819 was demonstrated in vitro in permeabilized myocyte-sized preparations from left ventricles (LV) of guinea pig hearts (ΔpCa50=0.12±0.01; EC50=2.88±0.14 µM). ORM-3819 increased the maximal rate of LV pressure development (+dP/dtmax) (EC50=8.9±1.7 nM) and LV systolic pressure (EC50=7.63±1.74 nM) in Langendorff-perfused guinea pig hearts. Intravenous administration of ORM-3819 increased LV+dP/dtmax (EC50=0.13±0.05 µM/kg) and improved the rate of LV pressure decrease (-dP/dtmax); (EC50=0.03±0.02 µM/kg) in healthy guinea pigs. In an in vivo dog model of myocardial stunning, ORM-3819 restored the depressed LV+dP/dtmax and improved % segmental shortening (%SS) in the ischemic area (to 18.8±3), which was reduced after the ischaemia-reperfusion insult (from 24.1±2.1 to 11.0±2.4). Our data demonstrate ORM-3819 as a potent positive inotropic agent exerting its cardiotonic effect by a cTnC-dependent Ca(2+)-sensitizing mechanism in combination with the selective inhibition of the PDE III isozyme. This dual mechanism of action results in the concentration-dependent augmentation of the contractile performance under control conditions and in the postischemic failing myocardium.

Effect of levosimendan therapy on myocardial infarct size and left ventricular function after acute coronary occlusion.

BACKGROUND: Levosimendan is an inotropic agent with cardioprotective and vasodilating properties used for the management of acutely decompensated heart failure. We studied the effects of levosimendan treatment on the size of myocardial infarction (MI) and left ventricular (LV) function in experimental pig model of post MI heart failure. METHODS: After occlusion of the left anterior descending (LAD) coronary artery, animals received levosimendan 5 mg/kg/day orally for 8 weeks (n=7) or no treatment (n=18). One week after stopping treatment, transthoracic echocardiography, CT scan and positron emission tomography were performed to evaluate myocardial function, perfusion and oxidative metabolism. Histology was used to confirm the size of MI and features of LV remodelling. RESULTS: The size of MI was significantly smaller in the levosimendan group than in the controls (12±13% vs 27±15% of the LV, p=0.03). End-diastolic volume (EDV) and end-systolic volume (ESV) were smaller in the levosimendan than in the control group (EDV 161±29 mL vs 245±84 mL, p=0.06; ESV 81±18 mL vs 149±67 mL, p=0.03), whereas ejection fraction tended to be higher in the levosimendan group (50±6% vs 41±8%, p=0.06). CONCLUSIONS: Eight weeks of levosimendan therapy after recent LAD occlusion decreases the size of MI and leads to better preservation of LV function as well as reduced LV remodelling.

Levosimendan alone and in combination with valsartan prevents stroke in Dahl salt-sensitive rats.

The effects of levosimendan on cerebrovascular lesions and mortality were investigated in models of primary and secondary stroke. We aimed to determine whether the effects of levosimendan are comparable to and/or cumulative with those of valsartan, and to investigate whether levosimendan-induced vasodilation has a role in its effects on stroke. In a primary stroke Dahl/Rapp rat model, mortality rates were 70% and 5% for vehicle and levosimendan, respectively. Both stroke incidence (85% vs. 10%, P<0.001) and stroke-associated behavioral deficits (7-point neuroscore: 4.59 vs. 5.96, P<0.001) were worse for vehicle compared to levosimendan. In a secondary stroke model in which levosimendan treatment was started after cerebrovascular incidences were already detected, mean survival times were 15 days with vehicle, 20 days with levosimendan (P=0.025, vs. vehicle), 22 days with valsartan (P=0.001, vs. vehicle), and 31 days with levosimendan plus valsartan (P<0.001, vs. vehicle). The respective survivals were 0%, 16%, 20% and 59%, and the respective incidences of severe lesions were 50%, 67%, 50% and 11%. In this rat model, levosimendan increased blood volume of the cerebral vessels, with significant effects in the microvessels of the cortex (∆R=3.5±0.15 vs. 2.7±0.17ml for vehicle; P=0.001) and hemisphere (∆R=3.2±0.23 vs. 2.6±0.14ml for vehicle; P=0.018). Overall, levosimendan significantly reduced stroke-induced mortality and morbidity, both alone and with valsartan, with apparent cumulative effects, an activity in which the vasodilatory effects of levosimendan have a role.

Exacerbation of acute kidney injury by bone marrow stromal cells from rats with persistent renin-angiotensin system activation.

Hypertension and persistent activation of the renin-angiotensin system (RAS) are predisposing factors for the development of acute kidney injury (AKI). Although bone-marrow-derived stromal cells (BMSCs) have shown therapeutic promise in treatment of AKI, the impact of pathological RAS on BMSC functionality has remained unresolved. RAS and its local components in the bone marrow are involved in several key steps of cell maturation processes. This may also render the BMSC population vulnerable to alterations even in the early phases of RAS pathology. We isolated transgenic BMSCs (TG-BMSCs) from young end-organ-disease-free rats with increased RAS activation [human angiotensinogen/renin double transgenic rats (dTGRs)] that eventually develop hypertension and die of end-organ damage and kidney failure at 8 weeks of age. Control cells (SD-BMSCs) were isolated from wild-type Sprague-Dawley rats. Cell phenotype, mitochondrial reactive oxygen species (ROS) production and respiration were assessed, and gene expression profiling was carried out using microarrays. Cells' therapeutic efficacy was evaluated in a rat model of acute ischaemia/reperfusion-induced AKI. Serum urea and creatinine were measured at 24 h and 48 h. Acute tubular damage was scored and immunohistochemistry was used for evaluation for markers of inflammation [monocyte chemoattractant protein (MCP-1), ED-1], and kidney injury [kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL)]. TG-BMSCs showed distinct mitochondrial morphology, decreased cell respiration and increased production of ROS. Gene expression profiling revealed a pronounced pro-inflammatory phenotype. In contrast with the therapeutic effect of SD-BMSCs, administration of TG-BMSCs in the AKI model resulted in exacerbation of kidney injury and high mortality. Our results demonstrate that early persistent RAS activation can dramatically compromise therapeutic potential of BMSCs by causing a shift into a pro-inflammatory phenotype with mitochondrial dysfunction.

Dexmedetomidine preconditioning ameliorates kidney ischemia-reperfusion injury.

Kidney ischemia-reperfusion (I/R) injury is a common cause of acute kidney injury. We tested whether dexmedetomidine (Dex), an alpha2 adrenoceptor (α2-AR) agonist, protects against kidney I/R injury. Sprague-Dawley rats were divided into four groups: (1) Sham-operated group; (2) I/R group (40 min ischemia followed by 24 h reperfusion); (3) I/R group + Dex (1 µg/kg i.v. 60 min before the surgery), (4) I/R group + Dex (10 µg/kg). The effects of Dex postconditiong (Dex 1 or 10 µg/kg i.v. after reperfusion) as well as the effects of peripheral α2-AR agonism with fadolmidine were also examined. Hemodynamic effects were monitored, renal function measured, and acute tubular damage along with monocyte/macrophage infiltration scored. Kidney protein kinase B, toll like receptor 4, light chain 3B, p38 mitogen-activated protein kinase (p38 MAPK), sirtuin 1, adenosine monophosphate kinase (AMPK), and endothelial nitric oxide synthase (eNOS) expressions were measured, and kidney transciptome profiles analyzed. Dex preconditioning, but not postconditioning, attenuated I/R injury-induced renal dysfunction, acute tubular necrosis and inflammatory response. Neither pre- nor postconditioning with fadolmidine protected kidneys. Dex decreased blood pressure more than fadolmidine, ameliorated I/R-induced impairment of autophagy and increased renal p38 and eNOS expressions. Dex downregulated 245 and upregulated 61 genes representing 17 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, in particular, integrin pathway and CD44. Ingenuity analysis revealed inhibition of Rac and nuclear factor (erythroid-derived 2)-like 2 pathways, whereas aryl hydrocarbon receptor (AHR) pathway was activated. Dex preconditioning ameliorates kidney I/R injury and inflammatory response, at least in part, through p38-CD44-pathway and possibly also through ischemic preconditioning.

Selective Na(+) /Ca(2+) exchanger inhibition prevents Ca(2+) overload-induced triggered arrhythmias.

BACKGROUND AND PURPOSE: Augmented Na(+) /Ca(2+) exchanger (NCX) activity may play a crucial role in cardiac arrhythmogenesis; however, data regarding the anti-arrhythmic efficacy of NCX inhibition are debatable. Feasible explanations could be the unsatisfactory selectivity of NCX inhibitors and/or the dependence of the experimental model on the degree of Ca(2+) i overload. Hence, we used NCX inhibitors SEA0400 and the more selective ORM10103 to evaluate the efficacy of NCX inhibition against arrhythmogenic Ca(2+) i rise in conditions when [Ca(2+) ]i was augmented via activation of the late sodium current (INaL ) or inhibition of the Na(+) /K(+) pump. EXPERIMENTAL APPROACH: Action potentials (APs) were recorded from canine papillary muscles and Purkinje fibres by microelectrodes. NCX current (INCX ) was determined in ventricular cardiomyocytes utilizing the whole-cell patch clamp technique. Ca(2+) i transients (CaTs) were monitored with a Ca(2+) -sensitive fluorescent dye, Fluo-4. KEY RESULTS: Enhanced INaL increased the Ca(2+) load and AP duration (APD). SEA0400 and ORM10103 suppressed INCX and prevented/reversed the anemone toxin II (ATX-II)-induced [Ca(2+) ]i rise without influencing APD, CaT or cell shortening, or affecting the ATX-II-induced increased APD. ORM10103 significantly decreased the number of strophanthidin-induced spontaneous diastolic Ca(2+) release events; however, SEA0400 failed to restrict the veratridine-induced augmentation in Purkinje-ventricle APD dispersion. CONCLUSIONS AND IMPLICATIONS: Selective NCX inhibition - presumably by blocking rev INCX (reverse mode NCX current) - is effective against arrhythmogenesis caused by [Na(+) ]i -induced [Ca(2+) ]i elevation, without influencing the AP waveform. Therefore, selective INCX inhibition, by significantly reducing the arrhythmogenic trigger activity caused by the perturbed Ca(2+) i handling, should be considered as a promising anti-arrhythmic therapeutic strategy.

Efficacy of selective NCX inhibition by ORM-10103 during simulated ischemia/reperfusion.

In this study we evaluated the effects of selective Na+/Ca2+ exchanger (NCX) inhibition by ORM-10103 on the [Ca2+]i transient (CaT), action potential (AP), and cell viability in isolated canine ventricular cardiomyocytes exposed to a simulated ischemia/reperfusion protocol performed either alone (modeling moderate low-flow ischemia) or with simultaneous strophantidine challenge (modeling more severe low-flow ischemia). CaTs were monitored using a Ca2+-sensitive fluorescent dye, APs were recorded by intracellular microelectrodes, and anaerobic shifts in cellular metabolism were verified via monitoring native NADH fluorescence. Simulated ischemia increased the NADH fluorescence, reduced the amplitudes of the AP and CaT and induced membrane depolarization. APs moderately shortened, CaTs prolonged. Diastolic [Ca2+]i ([Ca2+]iD) level increased significantly during ischemia and further elevated following strophantidine application. Reperfusion normalized the NADH level, the amplitude of the AP and duration of the [Ca2+]i transient, but only partially restored action potential triangulation and the amplitude of the CaT. [Ca2+]iD decreased in untreated, but further increased in strophantidine-treated cells. 10 µM ORM-10103 significantly reduced the ischemic [Ca2+]i raise in both untreated and strophantidine-treated cells. During reperfusion ORM-10103 decreased [Ca2+]i and eliminated its diastolic elevation in untreated and strophantidine-treated cardiomyocytes. Following the application of ORM-10103 the detrimental effect of ischemia/reperfusion on cell viability and the reperfusion-induced increase in AP and CaT variabilities were substantially reduced, but ischemia-induced shifts in AP morphology were barely influenced. In conclusion, selective NCX inhibition by ORM-10103 is highly effective against ischemia/reperfusion induced pathologic alterations in [Ca2+]i homeostasis, however, it fails to normalize untoward arrhythmogenic changes in AP morphology.

Extracellular membrane vesicles from umbilical cord blood-derived MSC protect against ischemic acute kidney injury, a feature that is lost after inflammatory conditioning.

BACKGROUND: Mesenchymal stromal cells (MSC) are shown to have a great therapeutic potential in many immunological disorders. Currently the therapeutic effect of MSCs is considered to be mediated via paracrine interactions with immune cells. Umbilical cord blood is an attractive but still less studied source of MSCs. We investigated the production of extracellular membrane vesicles (MVs) from human umbilical cord blood derived MSCs (hUCBMSC) in the presence (MVstim) or absence (MVctrl) of inflammatory stimulus. METHODS: hUCBMSCs were cultured in serum free media with or without IFN-γ and MVs were collected from conditioned media by ultracentrifugation. The protein content of MVs were analyzed by mass spectrometry. Hypoxia induced acute kidney injury rat model was used to analyze the in vivo therapeutic potential of MVs and T-cell proliferation and induction of regulatory T cells were analyzed by co-culture assays. RESULTS: Both MVstim and MVctrl showed similar T-cell modulation activity in vitro, but only MVctrls were able to protect rat kidneys from reperfusion injury in vivo. To clarify this difference in functionality we made a comparative mass spectrometric analysis of the MV protein contents. The IFN-γ stimulation induced dramatic changes in the protein content of the MVs. Complement factors (C3, C4A, C5) and lipid binding proteins (i.e apolipoproteins) were only found in the MVctrls, whereas the MVstim contained tetraspanins (CD9, CD63, CD81) and more complete proteasome complex accompanied with MHCI. We further discovered that differently produced MV pools contained specific Rab proteins suggesting that same cells, depending on external signals, produce vesicles originating from different intracellular locations. CONCLUSIONS: We demonstrate by both in vitro and in vivo models accompanied with a detailed analysis of molecular characteristics that inflammatory conditioning of MSCs influence on the protein content and functional properties of MVs revealing the complexity of the MSC paracrine regulation.