Myocardial protection by insulin is dependent on phospatidylinositol 3-kinase but not protein kinase C or KATP channels in the isolated rabbit heart

Because tyrosine kinase blockade prevents protection by ischemic preconditioning (PC) in several species, activation of tyrosine kinase appears to be critical for cardioprotection. The tyrosine kinase's identity, however, is unknown. The present study tested whether activation of a receptor tyrosine kinase, the insulin receptor, could mimic PC and if the mechanism of protection was similar to that of PC. Isolated rabbit hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Infarct size was determined by triphenyltetrazolium staining and expressed as a percentage of the area at risk. Infarct size in control hearts was 32.6 ± 2.3 %. A 5-min infusion of insulin (5 mU/ml) followed by a 10-min washout period prior to ischemia significantly reduced infarction to 14.7 ± 2.1 % (P < 0.05). The tyrosine kinase inhibitor genistein (50 μM) given around the insulin infusion blocked protection (28.9 ± 2.8 %). However, when present during the onset of ischemia, genistein had no effect on protection triggered by insulin (14.0 ± 2.4 %; P < 0.05). Inhibition of either PKC by polymyxin B (50 μM) or K_ATP channels by 5-hydroxydecanoate (100 μM) also failed to prevent protection by insulin (17.5 ± 3.2 % and 17.6 ± 3.0 %, respectively). However, the reduction in infarct size by insulin was significantly attenuated by wortmannin (100 nM), a selective inhibitor of phosphatidylinositol 3-kinase (P13K, 28.3 ± 2.2 %). Insulin was still able to protect the heart when given only during the reperfusion period (13.2 ± 3.4 %). PC reduced infarction to 12.8 ± 2.0 % (P < 0.05). In conclusion, activation of the insulin receptor reduces infarct size in the rabbit heart even when instituted upon reperfusion. However, the mechanism of protection is quite different from that of PC and involves activation of P13K but not PKC or K_ATP channels. Received: 12 November 1998, Returned for revision: 25 November 1998, Revision received: 8 December 1998, Accepted: 10 December 1998     

Effects of chronic noradrenaline on the nitric oxide pathway in human endothelial cells

Altered endothelium-dependent vasodilation has been observed in congestive heart failure (CHF), a disease characterized by a sustained adrenergic activation. The purpose of our study was to test the hypothesis that chronically elevated catecholamines influence the nitric oxide (NO) pathway in the human endothelium. Human umbilical vein endothelial cells (HUVEC) were exposed for 7 days to a concentration of noradrenaline (NA, 1 ng/mL) similar to that found in the blood of patients with CHF. Kinetics of endothelial constitutive NO synthase (ecNOS) and inducible NO synthase (iNOS) activity, measured by [³H]L-arginine to [³H]L-citrulline conversion, and protein expression of ecNOS and iNOS, assessed by Western blot analysis, were unaffected by chronic NA treatment. Furthermore, no changes in subcellular fraction-associated ecNOS were found; this indirectly shows that chronic NA did not cause phosphorylation of the enzyme. Moreover, [³H]L-arginine transport through the plasma membrane was conserved in chronically NA-treated cells. The data demonstrate that prolonged in vitro exposure to pathologic CHF-like NA does not affect the L-arginine NO pathway in human endothelial cells. Received: 11 July 1997, Returned for revision: 13 August 1997, Revision received: 6 October 1997, Returned for 2. revision: 17 November 1997, 2. Revision received: 5 January 1998, Accepted: 26 January 1998     

Protein kinase C isoforms α, δ and θ require insulin receptor substrate-1 to inhibit the tyrosine kinase activity of the insulin receptor in human kidney embryonic cells (HEK 293 cells)

Summary Protein kinase C (PKC) isoforms are potentially important as modulators of the insulin signalling chain and could be involved in the pathogenesis of cellular insulin resistance. We have previously shown that phorbol ester stimulated PKC β1 and β2 as well as tumor necrosis factor-α (TNFα) stimulated PKC ɛ inhibit human insulin receptor (HIR) signalling. There is increasing evidence that the insulin receptor substrate-1 (IRS-1) is involved in inhibitory signals in insulin receptor function. The aim of the present study was to elucidate the role of IRS-1 in the inhibitory effects of protein kinase C on human insulin receptor function. HIR, PKC isoforms (α, β1, β2, γ, δ, ɛ, η, θ and ζ) and IRS-1 were coexpressed in human embryonic kidney (HEK) 293 cells. PKCs were activated by preincubation with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (CTPA) (10^––7 mol/l) following insulin stimulation. While PKCs α, δ and θ were not inhibitory in HEK 293 cells which were transfected only with HIR and PKC, additional transfection of IRS-1 induced a strong inhibitory effect of these PKC isoforms being maximal for PKC θ (99 ± 1.8 % inhibition of insulin stimulated receptor autophosphorylation, n = 7, p < 0.001). No effect was seen with PKC γ, ɛ, ζ and η while the earlier observed insulin receptor kinase inhibition of PKC β2 was further augmented (91 ± 13 %, n = 7, p < 0.001 instead of 45 % without IRS-1). The strong inhibitory effect of PKC θ is accompanied by a molecular weight shift of IRS-1 (183 kDa vs 180 kDa) in the sodium dodecyl sulphate polyacrylamide gel. This can be reversed by alkaline phosphatase treatment of IRS-1 suggesting that this molecular weight shift is due to an increased phosphorylation of IRS-1 on serine or threonine residues. In summary, these data show that IRS-1 is involved in the inhibitory effect of the PKC isoforms α, β2, δ and θ and it is likely that this involves serine/threonine phosphorylation of IRS-1. [Diabetologia (1998) 41: 833–838] Received: 11 February 1998 and in revised form 2 April 1998     

Paclitaxel and cyclosporine A show supra-additive antiproliferative effects on smooth muscle cells by activation of protein kinase C

We intended to establish a pharmacologic concept of synergistic antiproliferative effects on smooth muscle cells (SMC) by using paclitaxel and cyclosporine A at clinically applicable doses. Coronary SMC were incubated with paclitaxel and cyclosporine A at concentrations of 10 – 20 nmol/L and 83 – 415 nmol/L, respectively. Antiproliferative effects were assessed by cell counts, [³H]thymidine incorporation and cell cycle analysis. In addition, apoptosis was studied by cytoplasmic histone-associated DNA fragments and in vitro protein kinase C activity (PKC) was determined by immunoassay. We found paclitaxel and cyclosporine A to exert a highly supra-additive antiproliferative effect on SMC with significant reductions of cell counts (p < 0.01) and [³H]thymidine incorporation (p < 0.05). SMC were found to be arrested at the G2/M transition. This antiproliferative effect was observed in the absence of DNA fragmentation above values obtained for single compound treatment, which had virtually no impact on cell proliferation. DNA fragmentation started to increase at a drug combination comprising paclitaxel at the higher dose of 20 nmol/L. Under the treatment with both paclitaxel and cyclosporine A, PKC activity showed a 1.8-fold increase (p < 0.05) compared with untreated controls. In conclusion, PKC mediates supra-additive antiproliferative effects of paclitaxel and cyclosporine A on SMC. The data demonstrate a highly efficient pharmacologic concept for the inhibition of SMC proliferation. Further studies are needed to test this concept under in vivo conditions for the prevention of restenosis or transplant vasculopathy by systemic application of cyclosporine A – when already applied for immunosuppressive purposes – and local delivery of paclitaxel. Received: 28 August 2001, Returned for revision: 25 September 2001, Revision received: 20 November 2001, Accepted: 4 December 2001     

Regulation of the isozymes of protein kinase C in the surviving rat myocardium after myocardial infarction: distinct modulation for PKC-alpha and for PKC-delta

Objective The goal of this study was to clarify the regulation of the isozymes of protein kinase C (PKC) in the process of remodeling after myocardial infarction. Methods An in vivo model of regional myocardial infarction induced by ligation of the left anterior coronary artery in rats was used. Hemodynamic parameters and the heart and lung weights were determined 1 week and 1, 2 and 3 months after operation. In transmural biopsies from the non-ischemic left ventricular wall of the infarcted heart, PKC activity (ELISA) and the expression of its major isozymes, PKC-α, PKC-δ and PKC-ε (Westernblot analysis) were determined. Results As early as one week after myocardial infarction, heart weight and left ventricular enddiastolic pressures were significantly increased. Lung weights increased after 2 – 3 months, indicating progressive pulmonary congestion. The activity of PKC was significantly increased about 1.8-fold after 1 week, decreasing progressively in the later time course. Whereas the expression of PKC-ε did not change, PKC-α was increased after 1 month (157 %) and then returned to baseline values. In contrast, PKC-δ expression was significantly augmented after 2 and 3 months of myocardial infarction (187 %). Conclusions These data demonstrate for the first time that in the remodeling heart after myocardial infarction, a subtype-selective regulation of the PKC isozymes occurs: The upregulation of PKC-α coincides with the development of hypertrophy, whereas the extensive upregulation of PKC-δ outlasts the process of developing hypertrophy and persists in the failing heart. The trigger mechanisms for this newly characterized process remains to be elucidated. Received: 25 October 2001, Returned for revision: 3 December 2001, Revision received: 19 December 2001, Accepted: 20 December 2001     

Protection of IB-MECA against myocardial stunning in conscious rabbits is not mediated by the A1 adenosine receptor

The goal of this study was to determine whether the protective effects of the A₃AR agonist N ⁶-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (IB-MECA) against myocardial stunning are mediated by the A₁AR. Six groups of conscious rabbits underwent a sequence of six 4-minute coronary occlusion (O)/4-minute reperfusion (R) cycles for three consecutive days (days 1, 2, and 3). In vehicle-treated rabbits (group I), the recovery of systolic wall thickening (WTh) in the ischemic/reperfused region was markedly depressed on day 1, indicating the presence of severe myocardial stunning. On days 2 and 3, however, the recovery of systolic WTh was markedly accelerated, indicating the presence of late ischemic preconditioning (PC). When rabbits were pretreated with the A₁AR agonist 2-chloro-N ⁶-cyclopentyladenosine (CCPA, 100 μg/kg i.v.) or with IB-MECA (100 μg/kg i.v.) 10 min prior to the first sequence of O/R cycles on day 1 (group III and V, respectively), the recovery of systolic WTh was markedly accelerated compared to vehicle-treated animals (reflected as an ∼48 % decrease in the total deficit of systolic WTh). The magnitude of the protection afforded by adenosine receptor agonists was equivalent to that provided by late ischemic PC. Pre-treating rabbits with the A₁AR antagonist N-0861 completely blocked both the hemodynamic and the cardioprotective effects of CCPA (group IV). However, the same dose of N-0861 did not block the cardioprotective actions of IB-MECA (group VI). Importantly, N-0861 did not influence the degree of myocardial stunning in the absence of PC (group II) and it did not block the development of late ischemic PC. Taken together, these results provide conclusive evidence that the cardioprotective effects of IB-MECA are not mediated via the A₁AR, supporting the concept that activation of A₃ARs prior to an ischemic challenge provides protection against ischemia/reperfusion injury. Received: 12 February 2001, Returned for revision: 23 February 2001, Revision received: 5 March 2001, Accepted: 5 March 2001     

Protection against myocardial ischaemia/reperfusion injury by PPAR–α activation is related to production of nitric oxide and endothelin–1

Background Ligands of peroxisome proliferator–activated receptor alpha (PPAR–α) have been shown to reduce ischaemia/reperfusion injury. The mechanisms behind this effect are not well known. We hypothesized that activation of PPAR–α exerts cardioprotection via a mechanism related to nitric oxide (NO) and endothelin–1 (ET–1). Methods Five groups of anaesthetized open–chest Sprague–Dawley rats were given the PPAR–α agonist WY 14643 1 mg/kg (WY; n = 7), dimethyl sulfoxide (DMSO, vehicle for WY; n = 6), the combination of WY and the NO synthase inhibitor N–nitro–L–arginine (L–NNA, 2 mg/kg) (n = 7), L–NNA only (n = 8) or 0.9% sodium chloride (NaCl, vehicle for DMSO and L–NNA; n = 8) i.v. before a 30 min period of coronary artery occlusion followed by 2 h of reperfusion. Infarct size (IS), eNOS and iNOS protein and ET–1 mRNA expression were determined. Results There were no haemodynamic differences between the groups during the experiment. The IS was 78 ± 3% of the area at risk in the DMSO group and 77 ± 2% in the NaCl group (P = NS). WY reduced IS to 56 ± 3% (P < 0.001 vs. DMSO group). When WY was administered in combination with L–NNA the cardioprotective effect was abolished (IS 73 ± 3%, P < 0.01 vs. WY 14643). L–NNA did not affect IS per se (78 ± 2%, P = NS). The expression of eNOS but not iNOS protein in ischaemic myocardium from rats was increased in the group given WY (P < 0.05). ET–1 mRNA levels were lower in the ischaemic myocardium following WY administration. Conclusion The results suggest that the PPAR–α activation protects the rat myocardium against ischaemia/ reperfusion injury via a mechanism related to production of NO, and possibly ET–1.     

Variation in Tau, the time constant for isovolumic relaxation, along the left ventricular base-to-apex axis

Tau (τ), the time constant for isovolumic relaxation, is often used as a measure of cardiac diastolic function. However, several methods of calculating τ have been published which may produce different results and, thereby, different conclusions. The purpose of this study was to determine if the method of τ calculation effects the results when left ventricular pressure (LVP) is measured at different positions along the base-to-apex axis. In 16 dogs, we measured LVP at 6 positions along the base-to-apex axis. We calculated τ using three different methods: 1) a monoexponential model (P(_t)=[P₀–P_asym]e^At+P_asym, where t=time, P₀=LVP at t=0, P_asym is asymptotic pressure as t→∞, A is –1/τ) with a zero asymptote 2) a monoexponential model with a variable asymptote in which the monoexponential decay equation is differentiated with respect to time and substituted into the original equation so that dP/dt vs. LVP is a (–/τ), and 3) a monoexponential decay model with variable asymptote in which P_asym and A are varied until the best fit line is reached by minimizing the residual sum of squares. When τ is calculated using method 1, τ measured at the LV base is 98.01%±8.85% of the τ at the apex. If calculated using method 2, τ measured at the LV base was 75.46±39.4% of τ measured at the apex. When method 3 is used for τ calculations, base τ increases to 117.76±4.91% of the apical τ. We conclude: 1) the method used to calculate τ will effect the results and, thus, conclusions drawn from τ data. 2) When using Method 3, which appears to be the best method for τ calculation, τ increases at the LV base compared to the apex. Received: 12 November 1997, Returned for 1. revision: 1 December 1997, 1. Revision received: 5 June 1998, Returned for 2. revision: 2 July 1998, 2. Revision received: 14 August 1998, Accepted: 27 August 1998     

Blockade of A1 adenosine receptors prevents the ischaemia-induced sensitisation of adenylyl cyclase: evidence for a protein kinase C-mediated pathway

Objective: Acute myocardial ischaemia leads to a transient sensitisation of adenylyl cyclase which may contribute to the occurrence of malignant arrhythmias and the propagation of myocardial necrosis. It is prevented by blockade of protein kinase C (PKC) which is activated in early ischaemia as shown by its translocation from the cytosol to the plasma membranes. Translocation of PKC may also occur in ischaemic preconditioning, a process thought to be induced by activation of adenosine A₁ receptors. In this study it was investigated whether A₁ adenosine receptors may be involved in the sensitisation of adenylyl cyclase and the activation of PKC induced by ischaemia. Methods:Isolated rat hearts were perfused with the specific A₁ adenosine antagonist 8-caclopentyl-1,3-dipropylxanthine (DPCPX, 1 μM) or adenosine (1 μM) prior to ischaemia induced by stop of perfusion for 5 and 10 min. Adenylyl cyclase activity was determined in plasma membranes stimulated by forskolin or stimulated via β-receptors by isoproterenol. Total PKC activity was measured in purified plasma membranes and in the cytosolic fraction using histone III-S as a substrate. Results:Myocardial ischaemia induced a β-receptor-independent sensitisation of adenylyl cyclase (forskolin-stimulated activity 515 ± 55 vs. 384 ± 30 pmol/min/mg protein) which was completely blocked by pre-perfusion with DPCPX (385 ± 23 vs. 386 ± 24 pmol/min/mg protein). DPCPX alone did not alter the responsiveness of adenylyl cyclase to stimulation. The stimulated adenylyl cyclase activity was increased by 20% after pre-perfusion with adenosine, mimicking the ischaemia-induced sensitisation. The effect of adenosine was not augmented by additional ischaemia. PKC activity was translocated from the cytosol to the plasma membranes by acute ischaemia, indicating an activation of the enzyme. This effect was completely abolished by DPCPX. Conclusion: These data demonstrate that in the rat heart the sensitisation of adenylyl cyclase in acute myocardial ischaemia is dependent on activation of A₁ adenosine receptors. It is suggested that the sensitisation of adenylyl cyclase by adenosine or ischaemia might be mediated by an activation of PKC. Received: 23 October 1998, Returned for revision: 19 November 1998, Revision received: 8 February 1999, Accepted: 22 February 1999     

Direct biochemical evidence for eNOS stimulation by bradykinin in the human forearm vasculature

Objective: Although it has been shown recently that acetylcholine (ACh)-induced vasodilation of forearm resistance vessels is predominantly mediated by nitric oxide, direct biochemical evidence for eNOS stimulation by bradykinin (BK) in the human arterial circulation is still lacking. Therefore, the present study was designed to test the hypothesis that in the human forearm vasculature eNOS stimulation significantly contributes to BK-induced vasodilation. Methods: BK was infused in the presence and absence of the NOS inhibitor L-NMMA (8 μmol/min) into the brachial artery of 16 healthy volunteers and the effects compared to muscarinergic eNOS stimulation following acetylcholine infusion. Forearm blood flow (FBF) was measured by venous occlusion plethysmography, and plasma nitrite (NO₂ ⁻), which represents a sensitive and specific marker of regional eNOS activity, was determined in the antecubital vein and brachial artery by flow injection analysis. Nitric oxide production was calculated as product of the veno-arterial difference of NO₂ ⁻ concentration times FBF. Results: Kininergic (BK: 20, 60, 200 ng/min) as well as muscarinergic (ACh: 1, 3, 10 μg/min) stimulation resulted in a dose-dependent increase in FBF and NO₂ ⁻ in each individual. The relationship between FBF and NO production upon BK infusion was comparable to that obtained with ACh (r = 0.98; n = 64, p < 0.01). Moreover, NOS inhibition reduced both flow responses and NO production (BK: 54 and 75 %; ACh: 57 and 72 %) to a similar extent. Conclusions: These data provide direct biochemical evidence for the involvement of eNOS in bradykinin-induced vasodilation of forearm resistance vessels in humans. Received: 30 July 2002, Returned for revision: 13 August 2002, Revision received: 13 September 2002, Accepted: 24 September 2002 Correspondence to: M. Kelm, M.D.     

On the Prediction of Young’s Modulus in Calcaneal Cancellous Bone by Ultrasonic Bulk and Bar Velocity Measurements

This study evaluated two different approaches to the prediction of Young’s modulus (E) from ultrasonic velocity and density measurements in 23 cubes of cancellous bone from human calcaneae. The first approach used clinically applicable measurements of bulk velocity and bone mineral density (BMD), whilst the second involved bar velocity and apparent density, which are strictly in vitro measurements. Bulk velocities were measured with an immersion technique with 1 MHz transducers using three different transit time markers (first arrival, thresholding, zero-crossing). Bar velocities were measured in the defatted specimens using a contact technique with 37 kHz transducers in air. Volumetric BMD was derived from dual-energy X-ray absorptiometry measurements, and apparent density was measured directly. Compressive mechanical testing was used to determine E. Bulk velocity, bar velocity and E all displayed significant anisotropy, being greatest in the proximo-distal (PD) axis and least in the medio-lateral (ML) axis. Bulk velocity was dependent on the transit time marker used, with velocity differences of up to 20% observed between different markers. Bar velocities were significantly lower than bulk velocities in all directions. Both bulk and bar velocities correlated with E (r ² = 0.26–0.83, r ²= 0.36–0.81, respectively) with stronger relationships obtained when the data for the three axes were pooled. The predictive ability of bulk velocities determined using different markers was similar. In general, combining velocity and density measurements yielded improved correlations with E. Thus, strong correlations were observed between E and the product of BMD and bulk velocity² (r ²= 0.58–0.89), and the product of apparent density and bar velocity² (r ² = 0.58–0.89). These results demonstrate that clinically applicable measurements of bulk velocity and BMD are good predictors of the elastic modulus of calcaneal bone, and that bulk and bar velocity, both alone and when combined with density measurements, have a similar predictive ability for mechanical properties. Received: 21 August 1997 / Accepted: 1 June 1998     

The effects of levosimendan on the left ventricular function and protein phosphorylation in post-isschemic guinea pig hearts

The widely accepted theories for the decreased function in the stunned myocardium relate to Ca²⁺ desensitization and free radical-mediated tissue damage of the myofilaments. The aim of the present study was to examine whether the depressed contractile function and Ca²⁺ responsiveness of the stunned myocardium may be restored by a new Ca²⁺ sensitizer (levosimendan), which has been shown to improve the Ca²⁺ response of the myofilaments. The effects of levosimendan on the left ventricular function and the in vivo protein phosphorylation were examined in both the non-ischemic and the stunned myocardium. Myocardial stunning was induced in Langendorff-perfused guinea pig hearts by suspending the circulation for 8 min, followed by a 20-min reperfusion period. Perfusion of post-ischemic guinea pig hearts with levosimendan (0.03–0.48 μM, 6 min) was associated with dose- and time-dependent increases in both dP/dt_max (contractility) and dP/dt_min (speed of relaxation). When the effectiveness of levosimendan was compared in non-ischemic and post-ischemic hearts, no significant differences were noted in the relative stimulatory effects on contractility and relaxation, at any given time point (time-response curve) or concentration (dose-response curve). Perfusion of the guinea pig hearts with a high (0.3 μM) levosimendan concentration did not reveal any qualitative or quantitative difference in the phosphodiesterase inhibitory potential of the compound (elevation of tissue cyclic AMP levels and characteristics of protein phosphorylation) between the non-ischemic and the post-ischemic myocardium. However, when isoproterenol was adminstered to induce maximal in vivo phosphorylation of cardiac phosphorproteins, an attenuation of the ³²P-incorporation into troponin I was noted in the post-ischemic hearts. The decrease in isoproterenol-induced ³²P-incorporation into troponin I was associated with similar alterations in the tissue level of this protein. We conclude that the Ca²⁺ sensitizer levosimendan exerts dose- and time-dependent positive inotropic and lusitropic effects on the postischemic myocardium, lending support to the hypothesis tha Ca²⁺ desensitization of the myofibrils is involved in myocardial stunning. Received: 20 July 1998, Returned for 1. revision: 27 August 1998, 1. Revision received: 6 January 1999, Returned for 2. revision: 5 February 1999, 2. Revision received: 25 February 1999, Accepted: 3 March 1999     

Ischemic preconditioning promotes a transient,but not sustained translocation of protein kinase C and sensitization of adenylyl cyclase

Objective: Brief periods of ischemia precondition the heart and reduce the size of infarction caused by a subsequent sustained ischemia. The molecular memory of preconditioning, i.e., the molecule persistently activated by the preconditioning ischemia exhibiting protection during the infarct-inducing event, is subject to debate. Protein kinase C, adenylyl cyclase and β-adrenergic receptors are candidates for this memory, supposedly their activation persists during several cycles of ischemia and reperfusion. The goal of this study was, therefore, to determine the activation of those signaling molecules after 1 – 3 cycles of myocardial ischemia and reperfusion. Methods: Rat hearts were perfused according to the method of Langendorff with 1 – 3 cycles of ischemia (5 min) and reperfusion (10 min). In the particulate fraction of these hearts, densities of b-adrenergic receptors, activities of adenylyl cyclase, and the activities and isozyme distributions of PKC-α, PKC-β, and PKC-ɛ were determined. Results: The ischemia-induced upregulation of β-adrenergic receptors is not influenced by preconditioning. In contrast, the sensitization of adenylyl cyclase observed after 5 min of ischemia is lost after repetitive periods of ischemia and reperfusion. Translocation of protein kinase C to the particulate fraction could be shown after 1 and 2 periods of ischemia including all major cardiac isozymes, with a rapid relocation to the cytosol after every cycle of reperfusion. A third period of ischemia was unable to promote a repeated translocation of protein kinase C. Conclusion: The ischemia-induced regulation process of β-adrenergic receptors is not influenced in preconditioning. Moreover, a sustained translocation of protein kinase C and a sustained sensitization of adenylyl cyclase are obviously no prerequisite for preconditioning after various cycles of ischemia and reperfusion. Thus, those signaling molecules do not seem to be operative for the preconditioning's memory. It is suggested that the initial, synergistic burst of sensitization of the adenylyl cyclase and of protein kinase C translocation induces myocardial protection very early in ischemia and reperfusion. Received: 17 January 2002, Returned for 1. revision: 21 January 2002, 1. Revision received: 13 December 2002, Returned for 2. revision: 23 December 2002, 2. Revision received: 3 January 2003, Accepted: 8 January 2003 Parts of this study were presented at the Annual Meetings of the American Heart Association 1996 and of the American College of Cardiology 2000. The Deutsche Forschungsgemeinschaft, Bonn, supported this study with grants to R.H.S. (SFB 320) and to C.W. (W 1955/2-1). R.H.S was supported by the Hermann-Lilly-Schilling Foundation. Correspondence to: G. Simonis, M.D.     

Hyperosmotic perfusion of the beating rat heart and the role of the Na+/K+/2CI− co-transporter and the Na+/H+ exchanger

The aim of the present study was to investigate the role of the Na⁺/K⁺/2CI⁻ co-transporter and the Na⁺/H⁺ exchanger on contractile function and electrolyte regulation during hyperosmotic perfusion of the heart. Langendorff perfused rat hearts were subjected to hyperosmolal perfusion in 10-min intervals. Perfusates were made hyperosmotic by adding mannitol to the buffer (370, 450 and 600 mOsmol/kg H₂O). Cardiac contractile function was monitored with a balloon in the left ventricle (LV) coupled to a pressure transducer. Cardiac effluent was sampled repeatedly throughout and after hyperosmotic perfusion and analyzed for content of Na⁺, K⁺ and CI⁻. All three hyperosmotic perfusates initially reduced LV developed pressure (LVDP), but for 370 and 450 mOsmol/kg H₂, LVDP recovered to baseline within 4 min of perfusion. With 600 mOsmol/kg H₂, LVDP recovered slowly and was 50% below baseline after 10 min of hyperosmotic perfusion. Inhibition of the Na⁺/H⁺ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and 3-methyl-sulfonyl-4-piperidinobenzoyl-guanidine methanesulfonate (HOE 694) abolished the recovery of LVDP to the 600 mOsmol/kg H₂ perfusate, whereas inhibition of the Na⁺/K⁺/2CI⁻ co-transporter had no impact on LVDP. Potassium was taken up by the heart during hyperosmotic perfusion and this uptake was significantly reduced with inhibition of the Na⁺/H⁺ exchanger. Intracellular pH was assessed with ³¹P magnetic resonance spectroscopy and hyperosmolality induced a significant alkalosis that was dependent upon the Na⁺/H⁺ exchanger. The rat heart responds to moderate elevations in osmolality with a transient reduction in contractile function, whereas an elevation of 300 mOsmol/kg H₂ persistently reduces contractile function. The Na⁺/H⁺ exchanger, but not the Na⁺/K⁺/2CI⁻ co-transporter, is of importance in contractile recovery and electrolyte regulation during hyperosmotic perfusion in the rat heart. Received: 10 December 1998, Returned for revision: 18 January 1999, Revision received: 1 July 1999, Accepted: 19 July 1999     

Altered force-frequency relation in hypertrophic obstructive cardiomyopathy

The present study was designed to test the hypothesis that in hypertrophied myocardium of patients with hypertrophic obstructive cardiomyopathy (HOCM) a reduced contractile reserve provided by frequency dependent potentiation of force of contraction contributes to the myocardial dysfunction. Myectomy was performed in 8 HOCM patients with normal systolic left ventricular function at rest. Nonfailing myocardium from the hearts of three multiorgan donors was investigated for comparison. In thin myocardial strips we measured the inotropic effects of different stimulation frequencies (0,5–3.0Hz) at different extracellular Ca²⁺ concentrations (1.8–16.2 mmol/l). At 1.8 mmol/l extracellular Ca²⁺ concentration, increasing stimulation rates had no positive inotropic effect in HOCM myocardium, whereas in nonfailing myocardium force of contraction increased up to 3 Hz. Increasing extracellular Ca²⁺ concentrations induced a positive force-frequency relation in HOCM with a maximum at 5.4 mmol/l Ca²⁺. A further increase to 16.2 mmol/l Ca²⁺ resulted in a negative force-frequency relation in these specimens. The time to peak tension and the time to relaxation decreased at increasing stimulation frequencies at all Ca²⁺ concentrations investigated. In conclusion, in hypertrophied myocardium of HOCM patients increasing stimulation frequencies failed to have a positive inotropic effect at physiological extracellular Ca²⁺ concentrations. The induction of a positive force-frequency relation by higher Ca²⁺ concentrations suggests that an abnormal cellular Ca²⁺ handling may play an important pathophysiological role. Received: 27 December 1996, Returned for revision: 3 March 1997, Revision received: 1 September 1998, Accepted: 30 September 1998     

Melatonin in elderly patients with insomnia.– A systematic review

Summary Background Melatonin is a hormone and antioxidant produced by the pineal gland of which four neurobiological roles have been claimed in the aged population: anti-ageing agent; free-radical scavenger; regulator of circadian rhythm; endogeneous sleep-inducer. The „melatonin replacement” hypothesis states that 1) the well-evidenced age-related decline contributes to insomnia and that 2) replacement with physiological doses of melatonin improves sleep. The aim of this review was to determine the evidence for the efficacy of melatonin in elderly insomniacs. Methods MEDLINE‘s database from 1990–2000 was searched with „melatonin”, „geriatrics” and „(frail)-elderly” as major subheadings. This resulted in 78 articles: only studies with empirical treatment data were reviewed (N=12). Results Six reports (abstract, research letter, retrospective case study, 3 open label studies) showed a trend towards efficacy of melatonin: sleep quality improved and in patients with Alzheimer‘s disease sundowning was reduced.?   In 6 double blind, randomised crossover trials, a total number of 95 patients (mean ages: 65–79yrs) were treated. Melatonin doses ranged from 0.5mg to 6mg; most took a single dose 30–120min before bedtime. In 3 studies a slow release form was used. Sleep quality was objectively measured by wrist actigraphy (n=4) and polysomnography (n=2), and additionally subjective sleep quality was assessed (n=2). Sleep latency decreased significantly in 4 studies. In 3 studies other measures of sleep quality (sleep efficiency, total sleep time and wake time during sleep) improved. Subjective sleep quality did not improve. No early-morning sleepiness occurred. Comparison of the studies suggests that melatonin is most effective in elderly insomniacs who chronically use benzodiazepines and/or with documented low melatonin levels during sleep. Conclusion There is sufficient evidence that low doses of melatonin improve initial sleep quality in selected elderly insomniacs. However, larger randomized controlled trials, with less strict inclusion criteria are necessary to yield evidence of effectiveness (i.e. clinical and subjective relevance) in geriatric patients who suffer from insomnia, before wide-spread use can be advocated. Received: 26 June 2001/Accepted: 10 July 2001     

Frequency dependent force generation correlates with sarcoplasmic calcium ATPase activity in human myocardium

Objective: In congestive heart failure both a decreased function of the sarcoplasmic Ca²⁺-ATPase and a negative force-frequency relationship have been shown. This study aimed to investigate a possible relationship between frequency potentiation, sarcoplasmic Ca²⁺-ATPase activity, and SERCA2 protein expression in human myocardium. Methods: Frequency potentiation was studied in electrically stimulated, isometric, left ventricular papillary muscle strip preparations (37°C, 0.5–3.0 Hz) from terminally failing (NYHA IV; n=5, dilated cardiomyopathy) and nonfailing (donor hearts, n=5) human myocardium. In the identical samples the Ca²⁺-ATPase activity (NADH coupled assay) and the protein expression of sarcoplasmic Ca²⁺-ATPase (SERCA2), phospholamban, and calsequestrin (western blot) were determined. The frequency dependent change in the force of contraction and V_max of the Ca²⁺-ATPase activity and the protein expression of SERCA2 were correlated with each other. Results: In terminally failing myocardium the force-frequency relationship was negative (2.0 Hz vs. 0.5 Hz: –0.2±0.1 ΔmN) contrasting a positive rate dependent potentiation of force in nonfailing tissue (2.0 Hz vs. 0.5 Hz: +0.8±0.2 ΔmN; p<0.01). In failing myocardium the corresponding maximal sarcoplasmic Ca²⁺-ATPase activity (V_max) was reduced significantly compared to nonfailing myocardium (174±24 vs. 296±31 nmol ATP/mg·min, p<0.01). The protein expression of SERCA2, phospholamban, and calsequestrin remained unchanged in failing myocardium. The maximal Ca²⁺-ATPase activity significantly correlated with the frequency dependent change in force of contraction (2 Hz vs. 0.5 Hz: r=0.88, p=0.001; 3 Hz vs. 0.5 Hz: r=0.84, p=0.004). No correlation between protein expression of SERCA2 and Ca²⁺-ATPase activity or change in force of contraction was observed. Conclusion: Due to a significant correlation between sarcoplasmic Ca²⁺-ATPase activity and frequency potentiation, the negative rate dependent force potentiation in human heart failure could be at least in part be attributed to decreased function of the sarcoplasmic Ca²⁺-ATPase. Received: 8 January 1998, Accepted: 2 June 1998     

Protein kinase C isoforms β 1 and β 2 inhibit the tyrosine kinase activity of the insulin receptor

Summary Downregulation of insulin receptor tyrosine kinase (IRK) activity yields to impaired insulin signalling and contributes to the pathogenesis of cellular insulin resistance. Activation of protein kinase C (PKC) by different agents is associated with an inhibition of IRK activity in various cell types. There is evidence that this effect on IRK activity might be mediated through phosphorylation of specific serine residues of the insulin receptor β -subunit. Neither the domains of the IRK where inhibiting serine phosphorylation occurs nor the PKC isoform responsible for IRK inhibition have been identified. PKC consists of a family of at least 12 isoforms. The aim of the present study was to determine which PKC isoform might be capable of IRK inhibition. The human insulin receptor and the PKC isoforms α, β 1, β 2, γ , δ , ɛ , η , θ and ζ were overexpressed in human embryo kidney fibroblasts (HEK 293 cells) in order to answer this question. PKCs were activated by preincubation with the phorbolester (TPA) (10⁻⁷ mol/l) following insulin stimulation of the cells. When the IRK was coexpressed with the PKC isoforms β 1 and β 2, a 50 ± 15.7 and 45 ± 10.1 % inhibition of tyrosine autophosphorylation of IRK was observed while coexpression with the other isoforms did not significantly modify IRK autophosphorylation. The data suggest that the PKC isoforms β 1 and β 2 might be candidates for insulin receptor inhibition. [Diabetologia (1997) 40: 863–866] Received: 3 March 1997 and in revised form: 17 April 1997     

Serum IL-6, TNFα, p55 srTNFα, p75 srTNFα, srIL-2α Levels and Disease Acitivity in Systemic Lupus Erythematosus

The aim of this study was to determine whether the levels of serum cytokines IL-6 and TNFα and of the soluble receptors p55 srTNFα, p75 srTNFα and srIL-2α are valuable markers of disease activity in patients with systemic lupus erythematosus (SLE) compared with the established parameters of anti-dsDNA, C3, C4 and CH₅₀. Forty patients with SLE, 19 ambulatory and 21 hospitalised, were included in this study. On the day of blood sampling a clinical examination was performed and SLEDAI and ECLAM disease activity scores were used to assess disease activity. Nineteen patients had inactive disease and 21 patients had active disease. Thirteen patients from the second group developed nephritis. In these patients the blood sampling and disease activity assessment were performed twice (at presentation and 6 months after treatment). Serum levels of cytokines and soluble receptors were measured by ELISA. Serum levels of cytokines and soluble receptors of patients with active disease were significantly higher than in patients with inactive disease (IL-6 p = 0.0004, TNFαp = 0.0015, srIL-2αp<0.0001, p55 srTNFαp<0.0001, p75 srTNFαp<0.0001). Serum soluble receptor levels of patients with inactive disease were higher than those of healthy controls (p55 srTNFαp<0.0001, p75 srTNFαp = 0.0002, srIL-2αp = 0.0012). No significant difference was found for TNFα and IL-6 (TNFαp = 0.015, IL-6 p = 0.019). Serum TNFα levels and especially srIL-2α, p55 srTNFα and p75 srTNFα levels correlated strongly with SLEDAI and ECLAM disease activity scores, anti-dsDNA, C3, C4 and CH₅₀ (p<0.0001). Serum soluble receptor (srIL-2α, p55 srTNFα, p75 srTNFα) levels were higher in patients with nephritis before treatment and decreased significantly 6 months after treatment (p = 0.005). The same trend was noticed with SLEDAI and ECLAM disease activity scores (p = 0.005) and anti-dsDNA (p = 0.008). In contrast, no significant differences were observed for C3 and C4 levels before and after treatment, which suggests that soluble receptors of cytokines are more sensitive markers of disease activity than C3 or C4 in predicting improvement. Serum levels of srIL-2α, p55 srTNFα and p75 srTNFα could provide useful information about disease activity in SLE patients, especially in cases where the other markers do not. Received: 3 March 1998 / Accepted: 13 July 1998     

Contribution of I<Subscript>K,ADO</Subscript> to the negative dromotropic effect of adenosine

Objective Despite the pathophysiological and therapeutic significance of the negative dromotropic effect of adenosine, its underlying ionic mechanism, and specifically the role of the adenosine-activated K⁺ current (I_K,ADO) is not experimentally defined. Therefore, we studied the contribution of I_K,ADO to the negative dromotropic effect of adenosine. Methods Effects of adenosine on single atrioventricular nodal and left atrial myocytes from rabbits were studied using the whole cell configuration of the patch clamp technique. Complementary experiments were done in rabbit and guinea pig isolated hearts instrumented to measure the atrium-to-His bundle interval. Results In contrast to its effect in atrial myocytes, Ba²⁺ selectively and completely blocked I_K,ADO at membrane potentials from −70 to 0 mV in atrioventricular nodal myocytes and abolished the adenosine-induced leftward shift of the reversal membrane potential. Ba²⁺ alone did not significantly prolong the A–H interval, but markedly attenuated the A–H interval prolongation caused by adenosine. In guinea pig heart, EC₅₀ values (pD₂± SEM) for adenosine-induced atrium-to-His bundle interval prolongation were 3.3 μmol/L (5.48 ± 0.04) and 13.2 μmol/L (4.88 ± 0.05, P < 0.001) in the absence and presence of Ba²⁺, respectively. Despite species-dependent differences in sensitivities to adenosine (guinea pig > rabbit), the relative contribution of adenosine-activated K⁺ current to the atrium-to-His bundle interval prolongation was nearly identical. In guinea pig hearts it ranged from 37.8 % (P = 0.013) to 72.5 % (P < 0.001) at 2 to 6 μmol/L adenosine, respectively. Conclusion I_K,ADO contributes significantly to the negative dromotropic effect of adenosine, but predominantly at relatively high concentrations of the nucleoside. Received: 16 November 2001/Returned for 1. revision: 12 December 2001/1. Revision received: 20 December 2001/Returned for 2. revision: 2 January 2002/2. Revision received: 14 January 2002/Accepted: 21 January 2002