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     

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     

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     

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.