Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.

Effects of insulin on the reversal of myocardial contractility after bupivacaine-induced cardiac asystole in vitro

Background: Insulin-glucose treatment effectively reverses severe bupivacaine (BPV)-induced myocardial depression or cardiovascular collapse in in vivo. However, the mechanisms for the recovery are poorly defined. Methods: Using the guinea pig myocardium, cumulative concentration-responses on contractile forces for insulin or insulin combined with 33 mM glucose (insulin/glucose) were measured. After achieving asystole by 500 µM BPV, different concentrations of insulin or insulin/glucose were applied to determine the recovery of stimulated contractile responses and contractions in either recirculating or non-recirculating (washout) condition. Because we did not observe any recovery from asystole with insulin treatment in the recirculating condition, further experiments were performed whether intermittent contractile responses (conduction block) could be reversed by insulin. In the washout condition, after achieving asystole, the muscles were washed with the Tyrode solution containing insulin or insulin/glucose for 60 minutes. After achieving asystole, BPV concentrations in the Tyrode solution in the presence or absence of insulin for 60 minutes were measured. Results: There were similar concentration-dependent decreases in contractility in both the insulin and insulin/glucose groups. Neither insulin nor insulin/glucose restored the stimulated contractile responses from conduction disturbance or asystole induced by BPV in the continued presence of BPV. In the BPV-washout condition, while superfusion with a control (plain Tyrode) solution for 60 minutes after achieving asystole by BPV restored contractility to approximately 60% of the baseline, time-dependent complete recovery was observed in the insulin- and insulin/glucose-treated groups. At each time period from asystole to 60 minutes, the BPV concentrations in the insulin-treated group were slightly lower than those in the control group. Conclusions: Neither insulin nor insulin/glucose treatment does not rescue Na+ channel function to reverse BPV-induced cardiac conduction block or asystole regardless of improved cardiac performance, possibly due to improved myocardial energetics in isolated in vitro animal myocardium model. These findings suggest that treatment with insulin or insulin/glucose is desirable for metabolic energy supply to the myocardium in cases of BPV-induced cardiac collapse. However, considering the importance of decreased BPV concentrations in cardiac tissues, insulin or insulin/glucose may not achieve satisfactory outcomes.

P701The role of a phosphoinositide-3 kinase inhibitor in vascular inflammation

Background/Introduction Cardiovascular inflammation is associated with endothelial damage, resulting in leukocyte trafficking and oedema formation, which results in a positive-feedback loop of inflammatory events. Purpose The phosphoinositide 3-kinase (PI3K) pathway has been shown to be upregulated in vascular disease conditions whereby its inhibitors potentially have beneficial effects. Hence this study was designed to examine the actions of PI-103, a PI3K non-selective inhibitor, on inflammatory responses. Methods Permeability assays were undertaken to quantify the effect of TNF-a in the presence and absence of PI-103 using FITC-dextran (40 kDa, 0.1 mg/ml) to measure levels of permeability in trans-wells plates using human microvascular endothelial cells (HMVEC). In vivo analysis was carried out per the UK Home Office Animals (Scientific Procedures) Act 1986. CD1 mice were anaesthetised, to test PI-103 in a zymosan-induced model of dorsal skin inflammation on neutrophil accumulation (measured by myeloperoxidase) and oedema formation (measured by Evans Blue accumulation). All data were analysed using 1-way or 2-way ANOVA with post-hoc test. Results PI-103 significantly reduced TNF-α induced microvascular endothelial cell permeability (Graph), thus impeding in vitro cytokine-induced inflammation. Whilst there was no effect on neutrophil accumulation in vivo, there was significant reduction in weight of treated areas and oedema formation, which was inhibited by PI-103 (Table 1). Effect of PI-103 in vivo Control 30 mg/kg PI-103 Tyrode solution Zymosan (μg/ml) Tyrode solution Zymosan (μg/ml) 10 30 100 10 30 100 MPO/Protein (U/mg) 0.66±0.26 1.42±0.07 1.40±0.14 1.68±0.12* 0.60±0.11 1.23±0.32 1.36±0.25 1.59±0.21* Oedema Volume (mm3) 0.00±0.00 3.26±1.76 11.70±2.21 21.50±4.67 2.20±1.53 1.05±1.05 3.27±0.74 5.52±1.58 Mice were pre-treated with PI-103 i.p. for 30 min, i.d injected with zymosan (50 μl/site) for 4 hr, followed by ex vivo MPO assay, weight and oedema volume measurements. Oedema was quantified by measuring: width (x), height (y) and depth (z) from each of the i.d. sites; volume of oedema = ((π/6)xyz). N=6 independent experiments in duplicates; *p<0.05 between Tyrode solution vs zymosan groups. Effect of PI103 in vitro on permeability Conclusions Our findings show that the PI3K non-selective inhibitor, PI-103 (as well as a PI3K α-selective inhibitor), reduces endothelial activation and inhibits inflammatory oedema formation. We conclude that there is a potential for PI3K inhibitors to act as anti-oedema agents in cardiovascular-related inflammatory conditions. Acknowledgement/Funding British Heart Foundation

Effects of insulin on the reversal of myocardial contractility after bupivacaine-induced cardiac asystole in vitro

Background: Insulin-glucose treatment effectively reverses severe bupivacaine (BPV)-induced myocardial depression or cardiovascular collapse in in vivo. However, the mechanisms for the recovery are poorly defined. Methods: Using the guinea pig myocardium, cumulative concentration-responses on contractile forces for insulin or insulin combined with 33 mM glucose (insulin/glucose) were measured. After achieving asystole by 500 µM BPV, different concentrations of insulin or insulin/glucose were applied to determine the recovery of stimulated contractile responses and contractions in either recirculating or non-recirculating (washout) condition. Because we did not observe any recovery from asystole with insulin treatment in the recirculating condition, further experiments were performed whether intermittent contractile responses (conduction block) could be reversed by insulin. In the washout condition, after achieving asystole, the muscles were washed with the Tyrode solution containing insulin or insulin/glucose for 60 minutes. After achieving asystole, BPV concentrations in the Tyrode solution in the presence or absence of insulin for 60 minutes were measured. Results: There were similar concentration-dependent decreases in contractility in both the insulin and insulin/glucose groups. Neither insulin nor insulin/glucose restored the stimulated contractile responses from conduction disturbance or asystole induced by BPV in the continued presence of BPV. In the BPV-washout condition, while superfusion with a control (plain Tyrode) solution for 60 minutes after achieving asystole by BPV restored contractility to approximately 60% of the baseline, time-dependent complete recovery was observed in the insulin- and insulin/glucose-treated groups. At each time period from asystole to 60 minutes, the BPV concentrations in the insulin-treated group were slightly lower than those in the control group. Conclusions: Neither insulin nor insulin/glucose treatment does not rescue Na+ channel function to reverse BPV-induced cardiac conduction block or asystole regardless of improved cardiac performance, possibly due to improved myocardial energetics in isolated in vitro animal myocardium model. These findings suggest that treatment with insulin or insulin/glucose is desirable for metabolic energy supply to the myocardium in cases of BPV-induced cardiac collapse. However, considering the importance of decreased BPV concentrations in cardiac tissues, insulin or insulin/glucose may not achieve satisfactory outcomes.

Effects of insulin on the reversal of myocardial contractility after bupivacaine-induced cardiac asystole in vitro

Background: Insulin-glucose treatment effectively reverses severe bupivacaine (BPV)-induced myocardial depression or cardiovascular collapse in in vivo. However, the mechanisms for the recovery are poorly defined. Methods: Using the guinea pig myocardium, cumulative concentration-responses on contractile forces for insulin or insulin combined with 33 mM glucose (insulin/glucose) were measured. After achieving asystole by 500 µM BPV, different concentrations of insulin or insulin/glucose were applied to determine the recovery of stimulated contractile responses and contractions in either recirculating or non-recirculating (washout) condition. Because we did not observe any recovery from asystole with insulin treatment in the recirculating condition, further experiments were performed whether intermittent contractile responses (conduction block) could be reversed by insulin. In the washout condition, after achieving asystole, the muscles were washed with the Tyrode solution containing insulin or insulin/glucose for 60 minutes. After achieving asystole, BPV concentrations in the Tyrode solution in the presence or absence of insulin for 60 minutes were measured. Results: There were similar concentration-dependent decreases in contractility in both the insulin and insulin/glucose groups. Neither insulin nor insulin/glucose restored the stimulated contractile responses from conduction disturbance or asystole induced by BPV in the continued presence of BPV. In the BPV-washout condition, while superfusion with a control (plain Tyrode) solution for 60 minutes after achieving asystole by BPV restored contractility to approximately 60% of the baseline, time-dependent complete recovery was observed in the insulin- and insulin/glucose-treated groups. At each time period from asystole to 60 minutes, the BPV concentrations in the insulin-treated group were slightly lower than those in the control group. Conclusions: Neither insulin nor insulin/glucose treatment does not rescue Na+ channel function to reverse BPV-induced cardiac conduction block or asystole regardless of improved cardiac performance, possibly due to improved myocardial energetics in isolated in vitro animal myocardium model. These findings suggest that treatment with insulin or insulin/glucose is desirable for metabolic energy supply to the myocardium in cases of BPV-induced cardiac collapse. However, considering the importance of decreased BPV concentrations in cardiac tissues, insulin or insulin/glucose may not achieve satisfactory outcomes.

Intermittent Hypoxia Inhibits Na+-H+ Exchange-Mediated Acid Extrusion Via Intracellular Na+ Accumulation in Cardiomyocytes

Background/Aims: Intermittent hypoxia (IH) has been shown to exert preconditioning-like cardioprotective effects. It also has been reported that IH preserves intracellular pH (pHi) during ischemia and protects cardiomyocytes against ischemic reperfusion injury. However, the exact mechanism is still unclear. Methods: In this study, we used proton indicator BCECF-AM to analyze the rate of pHi recovery from acidosis in the IH model of rat neonatal cardiomyocytes. Neonatal cardiomyocytes were first treated with repetitive hypoxia-normoxia cycles for 1-4 days. Cells were then acid loaded with NH4Cl, and the rate of pHi recovery from acidosis was measured. Results: We found that the pHi recovery rate from acidosis was much slower in the IH group than in the room air (RA) group. When we treated cardiomyocytes with Na+-H+ exchange (NHE) inhibitors (Amiloride and HOE642) or Na+-free Tyrode solution during the recovery, there was no difference between RA and IH groups. We also found intracellular Na+ concentration ([Na+]i) significantly increased after IH exposure for 4 days. However, the phenomenon could be abolished by pretreatment with ROS inhibitors (SOD and phenanathroline), intracellular calcium chelator or Na+-Ca2+ exchange (NCX) inhibitor. Furthermore, the pHi recovery rate from acidosis became faster in the IH group than in the RA group when inhibition of NCX activity. Conclusions: These results suggest that IH would induce the elevation of ROS production. ROS then activates Ca2+-efflux mode of NCX and results in intracellular Na+ accumulation. The rise of [Na+]i further inhibits the activity of NHE-mediated acid extrusion and retards the rate of pHi recovery from acidosis during IH.

Relaxant effects of aqueous cold extracts of Carum copticum on smooth muscle

Background: The objective was to study the nature of relaxant effect of cold aqueous extracts of Carum copticum on rat alimentary tract and uterus.Methods: The aqueous cold extracts of Carum copticum (omum) whole seeds (OE1) or ground seeds (OE2) was prepared by leaving them (10g to 100mL) in distilled water for 4 hrs. Rat isolated tissues like stomach, duodenum, jejunum or uterus 4 cm pieces were mounted in a 20mL bath containing Tyrode solution or De Jalons at 37°C with continuous aeration. The effect of Carum copticum extracts and thymol on spontaneous activity of intestine and on acetylcholine (ACh), Histamine (H), Barium chloride (BaCl2) induced contractions or Adrenaline produced (Adr) relaxations were observed. Thymol content of omum was estimated by HPLC.Results: Omum extracts decreased or abolished spontaneous contractions of intestine but did not antagonize the contractions induced by ACh, H or relaxation induced by adrenaline. Thymol did not show any such effect. The stimulant action of BaCl2 was antagonized by aqueous cold extracts of omum similar to Papaverine.Conclusions: The relaxant effect of Carum copticum on rat intestine appears to be a direct effect on smooth muscle like papaverine.