Cardiodepressant mediators are released after myocardial ischaemia: modulation by catecholamines and adenosine.

The interaction of recently characterized cardiodepressant mediators with catecholamines and adenosine after myocardial ischaemia was investigated using a model of sequential perfusion of two isolated guinea-pig hearts. Sequential perfusion was initiated after 10, 20, and 30 min (group I, II, and III) of global ischaemia in the first heart. At the onset of sequential perfusion LVdP/dtmax and min of Heart II decreased by 46 and 44% in group I, by 28 and 34% in group II, and increased by 60 and 24% in group III. Infusion of the beta1-receptor antagonist metoprolol (2.8 micromol L(-1)) into Heart II did not modulate contractile changes after 10 min of ischaemia in Heart I, prevented the attenuation of the cardiodepressant effect after 20 min of ischaemia, and completely reversed the positive inotropic effect after 30 min of ischaemia. The A1- and A2-receptor antagonists DPCPX (2 micromol L(-1)) and DMPX (20 micromol L(-1)) enhanced the positive inotropic and lusitropic effects in Heart II (LVdP/dtmax +154%, LVdP/dtmin +71%) during sequential perfusion after 30 min of ischaemia in Heart I. It is concluded that the effects of cardiodepressant mediators released after myocardial ischaemia are counteracted by a time-dependent release of catecholamines. Endogenous cardiac adenosine, in turn, attenuates the modulatory effects of catecholamines.     

Myocardial inositoltrisphosphate is depressed by dibutyryl cAMP. An experimental study in the isolated working rat heart

A possible interrelation between IP₃ and cAMP was studied in rat myocardium through circumvention of the receptor mediated stimulatory step of adenylyl cyclase by the administration of dibutyryl cAMP (db-cAMP). Changes in IP₃ and cyclic nucleotide contents were correlated to changes in contractility after 40 min of β- and α-adrenergic stimulation. Rat hearts (n= 23) were perfused with Krebs-Henseleit buffer in a modified Langendorff apparatus as a working preparation. The hearts were allocated to perfusion as control (n= 6); or with phenylephrine (10^-6 mol L^-1, n= 6); (—)-isoproterenol (10^-6 mol L^-1, n= 6); db-cAMP (2 times 10^-4 mol L^-1, n= 5). All hearts were freeze-clamped after 40 min of perfusion. Phenylephrine produced a slow increase in _max dP/dt reaching a maximal value after 10 min (P < 0.05); thereafter it decreased, reaching the control level at 30 min. Isoproterenol perfusion resulted in an early (20 s) increase in _max dP/dt (P < 0.05). Over the next 10 s _max dP/dt decreased markedly reaching an inflection point at 30 s. Thereafter only a slow increase during the rest of the perfusion was seen. Dibutyryl cAMP increased _max dP/dt slowly during the whole perfusion period reaching maximum after 40 min. Cyclic-AMP was increased by 21% after 40 min of phenylephrine perfusion while the corresponding increases by isoproterenol and db-cAMP were 131 and 105%, respectively (P < 0.05). Phenlyephrine increased IP₃ content to the same extent as isoproterenol perfusion (P < 0.05). On the other hand, a decreased IP₃ content was seen at 40 min of db-cAMP perfusion. Cyclic-AMP per se depressed basal myocardial IP₃ content in myocardial tissue. Despite normalization of contractility after 40 min of phenylephrine perfusion the IP₃ content was 27% higher than in control hearts pointing out the unclearness of the relationship between myocardial IP₃ content and the contractile system.     

Manganese and the heart: acute cardiodepression and myocardial accumulation of manganese

The aim of study was to assess acute effects of the divalent manganese ion (Mn²⁺) in an intact but isolated heart preparation. Rat hearts were perfused in the Langendorff mode at constant flow rate. Left ventricular (LV) developed pressure (LVDP), LV pressure first derivatives (LVdp/dt max and min), heart rate (HR) and aortic pressure (AoP) were recorded. Ventricular contents of high energy phosphate compounds (HEP) and Mn metal were measured at the end of experiment. Infusion of MnCl₂ for 5 min with perfusate concentrations 1–3000 μM induced an immediate depression of contractile function at and above 30 μM and negative chronotropy at and above 300 μM . These EC₅₀ values were found (μM ): LVDP 250; LVdp/dt max 160; LVdp/dp min 120; HR 1000; and increase in AoP 80. Recovery of function during a 14 min washout period was rapid and extensive, except for Mn²⁺ 3000 μM . Somewhat unexpected, Mn²⁺ 30–1000 μM raised coronary vascular resistance up to about twice the control level, whereas the vasoconstrictory response was overcome at 3000 μM . Mn²⁺ 3000 μM reduced tissue HEP. Ventricular Mn content rose stepwise for perfusate Mn²⁺ above 1 μM up to about 55 times the control level for perfusate Mn²⁺ 3000 μM . It is concluded that: acute effects of Mn²⁺ like depression of contractility and rate is rapidly reversible; and rat hearts accumulate and buffer large amounts of Mn²⁺ without affecting cardiac function or energy metabolism in the acute stage.     

Metabolite accumulation increases adenine nucleotide degradation and decreases glycogenolysis in ischaemic rat skeletal muscle

Adenine nucleotides and glycogen are degraded in skeletal muscle during no-flow ischaemia. Past investigations have ascribed these metabolic changes to the severe energetic stress which arises with the removal of exogenous substrates (principally oxygen). We tested this hypothesis by measuring the high-energy phosphagen and glycogen contents of stimulated rat hindlimb muscles (1 twitch s^?1) prior to and following 40 min of no-flow ischaemia or hypoxic perfusion without glucose (P_ao₂ = 4.6 ± 0.1 torr, plasma glucose = 0.3 ± 0.1 mmol L^?1). Both experimental protocols eliminated exogenous substrate supply; however, the maintenance of flow during hypoxic perfusion ensured the removal of metabolic by-products. A period of forty minutes of skeletal muscle ischaemia was characterized by reductions in the total adenine nucleotide pool, phosphocreatine and glycogen in the slow oxidative soleus, fast oxidative-glycolytic plantaris and the fast glycolytic white gastrocnemius. Compared to ischaemia, the total adenine nucleotide pool was higher (by 7.2–13.3 μmol g^?1 dry wt) and the glycogen content lower (by 10.0–16.6 μmol g^?1 dry wt) in skeletal muscle exposed to hypoxic perfusion without glucose. The ability of hypoxic perfusion to attenuate TAN degradation and augment glycogenolysis can be attributed to metabolic by-product removal. By limiting muscle lactate and PCO ₂ accumulation, hypoxic perfusion without glucose attenuates cellular acidification; this could in turn limit AMP deaminase activation and glycogen phosphorylase inhibition. We conclude that the ischaemia-induced alterations in adenine nucleotide and glycogen metabolism arise in response to the elimination of exogenous substrates and to the accumulation of metabolic by-products.     

Various inotropic effects of angiotensin II in post‐ischaemic rat hearts depending on ischaemic time with possible involvement of protein kinase C

Aims: The present study investigated if the inotropic effect of angiotensin II (AngII) is altered during post‐ischaemic reperfusion in hearts subjected to mild and severe ischaemia. The possible involvement of protein kinase C (PKC) in the change in the inotropic effect was also investigated. Methods: Isolated Langendorff‐perfused rat hearts were perfused under constant flow with oxygenated Krebs–Henseleit buffer and paced at 360 beats min^?1. A saline‐filled balloon catheter inserted into the left ventricle was used for measurement of contractile force. In the first series of experiments, hearts were subjected to continuous perfusion, 15‐ or 25‐min global ischaemia followed by 45‐min reperfusion. At the end of reperfusion, 0.1 μmol L^?1 AngII was infused for 5 min. In a second series of experiments, AngII was infused in hearts subjected to 25‐min ischaemia followed by 45‐min reperfusion in the absence or presence of the PKC inhibitor chelerythrine chloride (5 μmol L^?1). Results: The current study demonstrates that AngII exerts a positive inotropic effect in normoxic hearts with an increase of left ventricular developed pressure (LVDP) by 11% (P < 0.05 vs. prior to AngII infusion). In post‐ischaemic hearts subjected to 15‐min ischaemia no effect of AngII was observed. In hearts subjected to 25 min of ischaemia, however, AngII evoked a negative inotropic response with a decrease of LVDP by 18% (P < 0.05 vs. prior to AngII infusion). The negative inotropic effect of AngII was inhibited by the PKC inhibitor chelerythrine chloride. Conclusions: AngII exerts negative inotropic effect in severely injured post‐ischaemic heart, possibly through the PKC pathway.     

Glutathione deficiency intensifies ischaemia‐reperfusion induced cardiac dysfunction and oxidative stress

The efficacy of glutathione (GSH) in protecting ischaemia‐reperfusion (I‐R) induced cardiac dysfunction and myocardial oxidative stress was studied in open‐chest, stunned rat heart model. Female Sprague–Dawley rats were randomly divided into three experimental groups: (1) GSH‐depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg^–1, i.p.) 24 h prior to I‐R, (2) BSO injection (4 mmol kg^–1, i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg^–1, i.v.) infusion 1 h prior to I‐R, and (3) control (C), receiving saline treatment. Each group was further divided into I‐R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by ?50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia‐reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and –dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I‐R, whereas ±dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I‐R. For BSO + AT125, ±dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I‐R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I‐R. Rate‐pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I‐R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I‐R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole‐body GSH homeostasis.     

In vivo heat shock preconditioning mitigates calcium overload during ischaemia/reperfusion in the isolated, perfused rat heart

Heat shock (HS) pretreatment of the heart is effective in mitigating the deleterious effects of ischaemia/reperfusion. The main objective of this study was to determine whether the beneficial effect of HS is associated with the preservation of intracellular Ca²⁺ handling in the ischaemic/reperfused, isolated rat heart. Twenty-four hours after raising body core temperature to 42 °C for 15 min, rat hearts were perfused according to Langendorff and subjected to 30 min ischaemia followed by 20 min reperfusion. Cyclic changes of cytoplasmic calcium ion [Ca²⁺_i] levels were measured by surface fluorometry using Indo-1 AM. Reperfused HS hearts showed improved recovery of contractile function compared with control hearts: end-diastolic pressure: 45±11 vs. 64±22 mm Hg; developed pressure: 72±12 vs. 41±20 mm Hg; maximum rate of pressure increase (+dP/dt_max): 1,513±305 vs. 938±500 mm Hg/s; maximum rate of pressure decrease (–dP/dt_max): –1,354±304 vs. –806±403 mm Hg/s. HS hearts displayed a significantly lower end-diastolic cytosolic [Ca²⁺] ([Ca²⁺]_i) after reinstallation of flow. The dynamic parameters of the Ca²⁺_i transients, i.e. the maximum rate of increase/decrease (±dCa²⁺_i/dt_max) and amplitude, did not differ between reperfused control and HS hearts. The novel finding of this study is that improved performance of the HS-preconditioned heart after an ischaemic insult is associated with a reduced end-diastolic Ca²⁺_i load, and most likely, preserved Ca²⁺ sensitivity of the myocardial contractile machinery.     

Carbachol-induced increase in inositol trisphosphate (IP3) content is attenuated by adrenergic stimulation in the isolated working rat heart

The interrelated responses of concomitant adrenergic and muscarinic receptor stimulation on second messengers and mechanical activity in the isolated perfused working rat heart were studied. The hearts were perfused with Krebs-Henseleit buffer in a modified Langendorff apparatus. The hearts were perfused with noradrenaline (10^-6 mol L^-1, n= 20), with carbachol (3 times 10^-7 mol L^-1n= 11) or with noradrenaline plus carbachol (n= 20) in the above-mentioned concentrations. The hearts were frozen at 20 s, 30 s and 40 min after addition of noradrenaline and noradrenaline plus carbachol and at 20 s and 40 min after addition of carbachol. Five hearts were freeze-clamped directly after preperfusion and another five hearts after 40 min of perfusion and used as controls. Myocardial cAMP increased at 20 s and 40 min after noradrenaline perfusion. In contrast to this cAMP was unchanged at 20 s and decreased at 40 min after perfusion with noradrenaline plus carbachol. IP₃ content increased after 20 s of carbachol- and after 40 min of noradrenaline perfusion (P < 0.05). However, noradrenaline plus carbachol did not induced any significant increase in IP₃ content after 20 s and 30 s, but after 40 min a decrease below basal level was found (P < 0.05). Noradrenaline stimulation attenuated muscarinic agonist induced IP₃ formation. A reciprocity existed in that noradrenaline induced IP₃ formation was attenuated by carbachol. No direct relationship was observed between the IP₃ response and contractility, also valid for cAMP. Hence, a significant increase in contractility was found in spite of a blunted cAMP response probably not accounted for by an an α₁-adrenergic effect alone.     

ATP-dependent potassium channels and mitochondrial permeability transition pores play roles in the cardioprotection of theaflavin in young rat

Previous studies have confirmed that tea polyphenols possess a broad spectrum of biological functions such as anti-oxidative, anti-bacterial, anti-tumor, anti-inflammatory, anti-viral and cardiovascular protection activities, as well as anti-cerebral ischemia-reperfusion injury properties. But the effect of tea polyphenols on ischemia/reperfusion heart has not been well elucidated. The aim of this study was to investigate the protective effect of theaflavin (TF1) and its underlying mechanism. Young male Sprague-Dawley (SD) rats were randomly divided into five groups: (1) the control group; (2) TF1 group; (3) glibenclamide + TF1 group; (4) 5-hydroxydecanoate (5-HD) + TF1 group; and (5) atractyloside + TF1 group. The Langendorff technique was used to record cardiac function in isolated rat heart before and after 30 min of global ischemia followed by 60 min of reperfusion. The parameters of cardiac function, including left ventricular developing pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), maximal differentials of LVDP (±LVdP/dt _max) and coronary flow (CF), were measured. The results showed: (1) compared with the control group, TF1 (10, 20, 40 μmol/l) displayed a better recovery of cardiac function after ischemia/reperfusion in a concentration-dependent manner. At 60 min of reperfusion, LVDP, ±LVdP/dt _max and CF in the TF1 group were much higher than those in the control group, whereas left ventricular end-diastolic pressure (LVEDP) in the TF1 group was lower than that in the control group (P < 0.01). (2) Pretreatment with glibenclamide (10 μmol/l), a K_ATP antagonist, completely abolished the cardioprotective effects of TF1 (20 μmol/l). Also, most of the effects of TF1 (20 μmol/l) on cardiac function after 60 min of reperfusion were reversed by 5-HD (100 μmol/l), a selective mitochondria K_ATP antagonist. (3) Atractyloside (20 μmol/l), a mitochondrial permeability transition pore (mPTP) opener, administered at the beginning of 15 min of reperfusion completely abolished the cardioprotection of TF1 (20 μmol/l). The results indicate that TF1 protects the rat heart against ischemia/reperfusion injury through the opening of K_ATP channels, particularly on the mitochondrial membrane, and inhibits mPTP opening.     

β3‐Adrenoceptors modulate left ventricular relaxation in the rat heart via the NO‐cGMP‐PKG pathway

Aims: Using a model of isolated and Langendorff‐perfused rat heart we analysed whether activation of β₃‐adrenergic receptors (β₃‐ARs) influences ventricular lusitropic performance. We also focused on the NOS/NO/cGMP/PKG cascade as the signal transduction mechanism. Methods: Hearts were treated with increasing concentrations (from 10^?12 to 10^?6 m ) of BRL₃₇₃₄₄, a selective β₃‐AR agonist, and cardiac performance was evaluated by analysing both lusitropic parameters and coronary motility. Cardiac preparations were also perfused with BRL₃₇₃₄₄ in the presence of either isoproterenol (ISO) or nadolol, or pertussis toxin (PTx), or selective inhibitors of the NOS/NO/cGMP/PKG pathway. Results: BRL₃₇₃₄₄ caused a significant concentration‐dependent reduction in (LVdP/dt)_min, a decrease in half time relaxation significant starting from 10^?12 m , and an increase in (LVdP/dt)_max/(LVdP/dt)_min ratio (T/?t). BRL₃₇₃₄₄ abolished the ISO‐mediated positive lusitropism. β₃‐AR‐dependent effects on relaxation were insensitive to β₁/β₂‐AR inhibition by nadolol (100 nm ), and were abolished by G_i/o protein inhibition by PTx (0.01 nm ). NO scavenging by haemoglobin (10 μm ), and nitric oxide synthase (NOS) inhibition by NG‐monomethyl‐l ‐arginine (10 μm ) revealed the involvement of NO signalling in BRL₃₇₃₄₄ response. Pre‐treatment with inhibitors of either soluble guanylate cyclase (ODQ; 10 μm ) or PKG (KT₅₈₂₃; 100 nm ) abolished β₃‐AR‐dependent negative lusitropism. In contrast, anantin (10 nm ), an inhibitor of particulate guanylate cyclase, did not modify the effect of BRL₃₇₃₄₄ on relaxation. Conclusion: Taken together, our findings provide functional evidence for β₃‐AR modulation of ventricular relaxation in the rat heart which involves PTx‐sensitive inhibitory Gi protein and occurs via an NO‐cGMP‐PKG cascade. Whether the effects of β₃‐AR stimulation on lusitropism are beneficial or detrimental remains to be established.     

An improved isolated, left ventricular ejecting, murine heart model

 An improved, isolated, left ventricular-ejecting, murine heart model is described and evaluated. Special attention was paid to the design and impedance characteristics of the artificial aortic outflow tract and perfusate composition, which contained glucose (10 mM plus insulin) and pyruvate (1.5 mM) as substrates. Temperature of the isolated perfused hearts was maintained at 38.5 °C. During antegrade perfusion (preload 10 mm Hg, afterload 50 mm Hg, 2.5 mM Ca²⁺) proper design of the aortic outflow tract provided baseline values for cardiac output (CO), left ventricular developed pressure (LVDP) and the maximum first derivative of left ventricular pressure (LV dP/dt _max) of 11.1±1.7 ml min^–1, 83±5 mm Hg and 6283±552 mm Hg s^–1, respectively, resembling findings in the intact mouse. During 100 min normoxic antegrade perfusion CO declined non-significantly by less than 10%. Varying pre- and afterloads resulted in typical Frank-Starling relationships with maximal CO values of 18.6±1.8 ml min^–1 at pre- and afterload pressures of 25 and 50 mm Hg, respectively. Left ventricular function curves were constructed at free [Ca²⁺] of 1.5 and 2.5 mM in the perfusion medium. Significantly higher values for CO, LVDP and LV dP/dt _max and LV dP/dt _min were obtained at 2.5 mM Ca²⁺ at all loading conditions investigated. Phosphocreatine and creatine levels remained stable throughout the perfusion period. Despite a small but significant decline in tissue ATP content, the sum of adenine nucleotides did not change during the normoxic perfusion period. The tissue content of glycogen increased significantly. Received: 28 April 1998 / Received after revision and accepted: 10 September 1998     

Relationship between ischaemic time and ischaemia/reperfusion injury in isolated Langendorff‐perfused mouse hearts

Myocardial functional recovery and creatine kinase (CK) release following various periods of ischaemia were investigated in isolated mouse hearts. The hearts were perfused in the Langendorff mode with pyruvate‐containing Krebs–Hensleit (KH) buffer under a constant perfusion pressure of 80 mmHg, and were subjected to either continuous perfusion or to 5, 15, 20, 25, 30, 45 or 60 min of global ischaemia followed by 45 min of reperfusion. In hearts subjected to ischaemic periods of 5, 15 or 20 min, there was a transient reduction in the left ventricular (LV) dP/dt max during the early phase of reperfusion, while the recovery at the end of reperfusion reached a level similar to that in hearts subjected to continuous perfusion. In hearts subjected to longer ischaemic periods, i.e. 25, 30, 45 or 60 min, the decrease in the cardiac performance was more pronounced and persistent, with significantly lower recovery in LV dP/dt max and higher LV end diastolic pressure (LVEDP) at the end of reperfusion than in the non‐ischaemic hearts. There were no significant differences in the recoveries in coronary flow or in heart rate (HR) between groups. Similarly to the functional recovery, the release of CK showed a clear ischaemic length‐related increase. In conclusion, the Langendorff‐perfused isolated mouse heart could be a valuable model for studies of myocardial ischaemia/reperfusion injury. Future studies using gene‐targeted mice would add valuable knowledge to the understanding of myocardial ischaemia/reperfusion injury.     

Interaction between 3α-hydroxy-5α-pregnan-20-one and carbachol in the control of neuronal excitability in hippocampal slices of female rats in defined phases of the oestrus

The effects of 3α-hydroxy-5α-pregnan-20-one (allopregnanolone) and carbachol on CA1 and dentate gyrus action potentials were studied in hippocampus slices in premature, follicular and luteal phase rats. A 0.5 nL droplet of allopregnanolone (12.5 μmol L^?1), carbachol (5 μmol L^?1) or a mixed solution of 12.5 μmol L^?1 allopregnanolone and 5 μmol L^?1 carbachol was applied locally onto the stratum oriens-pyramidale or granular layer. The amplitude of CA1 population spike (POPSP) was reduced by allopregnanolone (?38 ± 3%) and carbachol (?21 ± 4%) in the luteal phase slices. The mixture of allopregnanolone and carbachol doubled this inhibition (?77 ± 6%). The inhibition caused by allopregnanolone and the mixture of allopregnanolone and carbachol in CA1 was significantly larger in the luteal phase than in the follicular phase (P = 0.02 and 0.0002). In the granular layer of the dentate gyrus, these inhibitions showed no significant difference between the phases. Neither in CA1 nor in the dentate gyrus did the carbachol inhibition differ between the phases. Perfusion with 5–10 μmol L^?1 carbachol caused an increasing inhibition of the POPSP during the first few minutes. Thereafter the inhibition gradually diminished and was replaced by a facilitation. The local allopregnanolone inhibition was enhanced by simultaneous carbachol perfusion. Picrotoxin (100 μmol L^?1) substantially reduced the allopregnanolone but not the carbachol inhibition. Atropine (10 μmol L^?1) blocked the carbachol response, but not the allopregnanolone inhibition. Perfusion with a mixed solution of picrotoxin and atropine reduced, but did not block, the inhibition caused by local application of allopregnanolone or by the mixture of allopregnanolone and carbachol. Our data suggest that neuroprogestine modulators of the GABA_A-receptor-mediated inhibition may play a significant role in the control of the cholinergic excitation in the hippocampus.     

Induction and functional characterization of β 2-microglobulin (β2-μ)-free HLA class I heavy chains expressed by β2-μ-deficient human FO-1 melanoma cells

The frequent loss of β2-microglobulin (β₂-μ) in malignant cells has stimulated interest in the functional characteristics of β₂-μ-free HLA class I heavy chains, since this information contributes to assess the impact of β₂-μ abnormalities on the interaction of malignant cells with immune cells. Therefore, the present study has investigated the ability of β₂-μ-free HLA class I heavy chains to modulate NK cell-mediated lysis of melanoma cells and to present melanoma-associated antigen (MAA)-derived peptides to HLA class I-restricted, MAA-specific cytotoxic T lymphocytes (CTL). β₂-μ-free HLA class I heavy chains were induced on B₂m null FO-1 cells by sequential incubation with IFN-α for 48 h at 37 °C and for 24 h at 26 °C. Transfection of cells with a wild-type H-2L^d gene (FO-1L^d) enhanced the induction of β₂-μ-free HLA class I heavy chains under such experimental conditions. β₂-μ-free HLA class I heavy chains expressed on the cell membrane did not protect the B₂m null FO-1 cells from NK cell-mediated lysis. Furthermore, FO-1 cells which express β₂-μ-free HLA-A2 heavy chains following transfection with a wild-type HLA-A2 gene were not lysed by HLA-A2-restricted, MAA-specific CTL lines and clones. These results indicate that association with β₂-μ is required for interaction of HLA class I molecules with NK inhibitory receptors and for peptide presentation to CTL.     

Engineering the binding properties of the T cell receptor:peptide:MHC ternary complex that governs T cell activity

The potency of a T cell is determined in large part by two interactions, binding of a cognate peptide to the MHC, and binding of the T cell receptor (TCR) to this pepMHC. Various studies have attempted to assess the relative importance of these interactions, and to correlate the corresponding binding parameters with the level of T cell activity mediated by the peptide. To further examine the properties that govern optimal T cell activity, here we engineered both the peptide:MHC interaction and the TCR:pepMHC interaction to generate improved T cell activity. Using a system involving the 2C TCR and its allogeneic pepMHC ligand, QL9–L^d, we show that a peptide substitution of QL9 (F5R), increased the affinity and stability of the pep–L^d complex (e.g. cell surface t_1/2-values of 13 min for QL9–L^d versus 87 min for F5R–L^d). However, activity of peptide F5R for 2C T cells was not enhanced because the 2C TCR bound with very low affinity to F5R–L^d compared to QL9–L^d (K_D = 300 μM and K_D = 1.6 μM, respectively). To improve the affinity, yeast display of the 2C TCR was used to engineer two mutant TCRs that exhibited higher affinity for F5R–L^d (K_D = 1.2 and 6.3 μM). T cells that expressed these higher affinity TCRs were stimulated by F5R–L^d in the absence of CD8, and the highest affinity TCR exhibited enhanced activity for F5R compared to QL9. The results provide a guide to designing the explicit binding parameters that govern optimal T cell activities.     

Effects of intermittent lower limb ischaemia on coronary blood flow and coronary resistance in pigs

Aim: Intermittent limb ischaemia prior to cardiac ischaemia is a cardioprotective stimulus. This study was to investigate whether this peripheral stimulus had any effects on basal coronary blood flow and resistance, and to explore its potential mechanisms by studying the effect of femoral nerve transection and Katp blockade by glibenclamide. Methods: Remote ischaemic preconditioning (rIPC) was induced by four 5‐min cycles of lower limb ischaemia. Coronary resistance was measured using standard formulae and coronary blood flow in the left anterior descending artery (LAD) by a flow probe. In experiment 1, coronary ischaemia was induced by inflation of a cuff placed around the mid‐LAD, and inflated until cessation of flow. Left ventricular (LV) function was assessed using dp/dt and Tau at 1 and 30 min of ischaemia. Experiment 1: 20 pigs were randomized to control (n = 6), rIPC (n = 7) or femoral nerve transection + rIPC (n = 7) groups. The femoral nerve was transected before the rIPC protocol. All data were collected at fixed heart rates of 120 bpm. Coronary resistance was decreased and flow was increased significantly by the rIPC stimulus (P = 0.003, P = 0.016, paired t‐test), and these changes were preserved after femoral nerve transection. Experiment 2: 19 pigs were randomized to control (n = 5), rIPC (n = 8) or glibenclamide‐treated rIPC (n = 6) groups. Data were collected at baseline, and during incremental pacing between 120 and 180 bpm. Results: Experiment 1: Coronary resistance was decreased and flow was increased significantly by rIPC stimulus (P = 0.003, P = 0.016, paired t‐test), and these changes were preserved after femoral nerve transaction. rIPC was associated with superior LV function (dp/dt_max) at 30 min, compared with controls and the rIPC + femoral nerve transaction group. Experiment 2: Coronary resistance was significantly lower, and LAD flow was significantly higher in rIPC group (P < 0.0001, P = 0.0008, two‐way anova ). These effects were reversed in the glibenclamide group. Conclusion: The rIPC stimulus leads to reduced coronary resistance and increased flow. This effect, while modified by glibenclamide appears to be a generic effect of remote ischaemia rather than a direct preconditioning effect.     

Endothelin‐1‐induced proliferation of human endothelial cells depends on activation of K+ channels and Ca2+ influx

Aims: Endothelin‐1 (ET‐1) promotes endothelial cell growth. Endothelial cell proliferation involves the activation of Ca²⁺‐activated K⁺ channels. In this study, we investigated whether Ca²⁺‐activated K⁺ channels with big conductance (BK_Ca) contribute to endothelial cell proliferation induced by ET‐1. Methods: The patch‐clamp technique was used to analyse BK_Ca activity in endothelial cells derived from human umbilical cord veins (HUVEC). Endothelial proliferation was examined using cell counts and measuring [³H]‐thymidine incorporation. Changes of intracellular Ca²⁺ levels were examined using fura‐2 fluorescence imaging. Results: Characteristic BK_Ca were identified in cultured HUVEC. Continuous perfusion of HUVEC with 10 nmol L^?1 ET‐1 caused a significant increase of BK_Ca open‐state probability (n = 14; P < 0.05; cell‐attached patches). The ET_B‐receptor antagonist (BQ‐788, 1 μmol L^?1) blocked this effect. Stimulation with Et‐1 (10 nmol L^?1) significantly increased cell growth by 69% (n = 12; P < 0.05). In contrast, the combination of ET‐1 (10 nmol L^?1) and the highly specific BK_Ca blocker iberiotoxin (IBX; 100 nmol L^?1) did not cause a significant increase in endothelial cell growth. Ca²⁺ dependency of ET‐1‐induced proliferation was tested using the intracellular Ca²⁺‐chelator BAPTA (10 μmol L^?1). BAPTA abolished ET‐1 induced proliferation (n = 12; P < 0.01). In addition, ET‐1‐induced HUVEC growth was significantly reduced, if cells were kept in a Ca²⁺‐reduced solution (0.3 mmol L^?1), or by the application of 2 aminoethoxdiphenyl borate (100 μmol L^?1) which blocks hyperpolarization‐induced Ca²⁺ entry (n = 12; P < 0.05). Conclusion: Activation of BK_Ca by ET‐1 requires ET_B‐receptor activation and induces a capacitative Ca²⁺ influx which plays an important role in ET‐1‐mediated endothelial cell proliferation.     

The inhibitory effects of oxytocin on distal colonic contractile activity in rabbits are enhanced by ovarian steroids

Aims: To study the effects of oxytocin on isolated rabbit distal colon and the regulation of ovarian steroids by its action. Methods: Muscle strips parallel to either the circular or the longitudinal fibres were excised and suspended in tissue chambers containing 5 mL Krebs solution (37 °C) and bubbled continuously with 95% O₂ and 5% CO₂. The effects of oxytocin on isometric spontaneous contractile responses were recorded. The effects of atosiban, tetrodotoxin, Mg²⁺, progesterone and oestradiol on the oxytocin‐induced response were also examined. Results: Oxytocin (1, 10 and 100 nmol L^?1) dose dependently decreased the area under the contraction curve of distal colonic smooth muscle strips. The oxytocin receptor antagonist atosiban blocked the oxytocin (10 nmol L^?1)‐caused responses in a dose‐dependent manner. Tetrodotoxin (10 μmol L^?1) had no effect on the oxytocin‐induced response. Mg²⁺‐free Krebs solution attenuated the oxytocin‐induced response, but oestradiol (0.1 μmol L^?1) or progesterone (0.1 μmol L^?1) increased the oxytocin‐induced response. Conclusion: These results suggest that oxytocin inhibits the contractile motility of the distal colon, which is regulated by Mg²⁺ and ovarian steroids.     

Role of aspirin in modulating myocardial ischemic reperfusion injury

The role of low-dose aspirin (3 mg/kg, i.v.) in attenuating ischemic reperfusion injury was studied in a canine model. Regional ischemia for 40 min was produced by temporary occlusion of the left anterior descending coronary artery and thereafter reperfusion instituted for 3 h. Mean arterial pressure (MAP), heart rate (HR), left ventricular end diastolic pressure (LVEDP), positive (+) LV dP/dt _max and negative (–) LV dP/dt _max were monitored alongwith myocardial adenosine triphosphate (ATP), creatine phosphate (CP), glycogen and lactate. Following reperfusion, there was a significant fall in (i) MAP, (ii) (+) LV dP/dt _max and (iii) (–) LV dP/dt _max. LVEDP was corrected after about 2h of reperfusion. Replenishment of only myocardial CP occurred, without any change in ATP and glycogen, although lactate accumulation was corrected.Aspirin administered 15 min before reperfusion (posttreatment) caused normalisation of LVEDP within 15 min and prevented any deterioration in (–) LV dP/dt _max, although it had no effect on MAP and (+) LV dP/dt _max. After 3h of reperfusion (post-treatment), myocardial ATP, CP, glycogen and lactate contents became normal. The number of premature ventricular complexes was significantly reduced after aspirin treatment. The present study indicates that low-dose aspirin post-treatment can ameliorate at least some of the deleterious consequences of reperfusion injury of the myocardium.     

Evaluation of habitual physical activity from a week's heart rate monitoring in French school children

Habitual physical activity (HPA) was studied in 30 boys and 34 girls aged 6–11 years. All the children performed a shuttle run test (SRT) to assess maximal heart frequency (f _cmaxSRT) and to evaluate maximal oxygen uptake (VO_2maxSRT). Heart rate (f _c) was measured continuously from Monday to Sunday, using a heart rate counter. The time spent at f _c greater than 140 beats · min^–1 (t _fc>140) and at f _c greater than 160 beats · min^–1 (t _fc>160) permitted HPA to be evaluated. The daily heart rate (f _cd) and the percentage of heart rate reserve (%f _crd) were calculated to evaluate the metabolic activity. In the boys and girls, f _cd and %f _crd varied little with age. The metabolic activity varied in a rhythmical way during the week and was higher during school days than during free days (P < 0.001). The children were more active during school days (d _s) than during the free days (d _f). This observation was particularly marked in the boys having t _fc>140 being twice as high during d _s compared to d _f [t _fc>140, d _s 85 (SD 25), d _f 40 (SD 26) min; t _fc>160, d _s 36 (SD 19), d _f 16 (SD 13) min]. During d _s t _fc>160was greater in the boys than in the girls (P < 0.01) . The same held for t _fc>140and % f _crd from the age of 9 years (P < 0.001) . It was during the recreation periods that the differences between the boys and the girls were observed (P < 0.01). There was no significant difference between the boys and the girls during lessons, in the evening and during d _f (% f _crd 26–28%, t _fc>14035–45 min, t _fc>16010–18 min). In contrast, the children who were physically active in a sports club, had less spontaneous physical activity and %f _crd, t _fc>140, t _fc>160and VO_2maxSRT were identical to those of the other children.