OUP user menu

The cardiac adrenergic system in ischaemia: differential role of acidosis and energy depletion

Gregor Simonis, Rainer Marquetant, Jochen Röthele, Ruth H Strasser
DOI: http://dx.doi.org/10.1016/S0008-6363(98)00057-1 646-654 First published online: 1 June 1998

Abstract

Objective: Acute myocardial ischaemia has been shown to modulate the β-adrenergic system and to activate protein kinase C. The aim of this study was to investigate if two important components of ischaemia, i.e. energy depletion or acidosis, may contribute to these changes. Methods: Isolated rat hearts were perfused either with anoxia (in the absence of oxygen) or with cyanide in the absence of glucose as models of energy depletion with a loss of high energy phosphates. Alternatively, isolated hearts were perfused with acidic modified Krebs–Henseleit solution to induce acidosis. Results: Energy depletion induced by cyanide perfusion leads to an increase of β-adrenergic receptors (81±7 vs. 50±3 fmol/mg protein, p≤0.05) comparable to the changes observed in ischaemia, yet without any change of total adenylyl cyclase activity or protein kinase C activity. Similar, yet less pronounced changes were induced by anoxic perfusion. Acidic perfusion, in contrast, promotes a translocation of protein kinase C to the plasma membranes, suggesting its rapid activation. Additionally, an increased total forskolin-stimulated activity of adenylyl cyclase (515±16 vs. 428±17 pmol/min/mg, p≤0.05) was observed. Both were comparable to the sensitization observed in early ischaemia. In acidosis, the density of β-adrenergic receptors remained unaltered. Conclusions: These data suggest that the regulation of cardiac β-adrenergic receptors is susceptible to energy depletion, but not to acidosis, whereas the intracellular enzymes both adenylyl cyclase and protein kinase C may be regulated by intracellular acidosis. This is the first differentiation of distinct components of ischaemia modulating the β-adrenergic signal transduction pathway. Both components may be operative in concert in acute myocardial ischaemia and may contribute to the regulation of these components of signal transduction observed in acute ischaemia.

Keywords
  • Acute myocardial infarction
  • Protein kinase C
  • Heart
  • Signal transduction
  • Acidosis
  • Adenylyl cyclase
  • β-adrenergic receptors
  • Rat

Time for primary review 31 days.

1 Introduction

Acute myocardial ischaemia has been shown to lead to the sensitization of the adrenergic signal transduction system at different levels. The increased presynaptic release of endogenous catecholamines [1]meets an increased number of functionally coupled β-adrenergic receptors [2–6]. Early after the onset of ischaemia the Gs-proteins remain intact [7]and are able to transmit the signal to the adenylyl cyclase. Additionally early in acute myocardial ischaemia, the enzyme adenylyl cyclase is temporarily sensitized [3, 8], contributing to the increased intracellular cAMP levels [9–11]. In contrast, in prolonged ischaemia adenylyl cyclase activity is gradually decreased [2, 3]resulting in a decreased responsiveness of the adrenergic system in the infarcted heart. Previous studies have established, that the early sensitization at the level of adenylyl cyclase is mediated by an activation of protein kinase C in the ischaemic myocardium [12]. However, the mechanisms responsible for this activation of protein kinase C in acute myocardial ischaemia have not been identified. A potential activation of α1-adrenergic receptors by the presynaptically released norepinephrine as sole mechanism has been excluded with certainty [12].

An activation of protein kinase C may be mediated by the activation of many distinct signal transduction pathways [13]. However, it could be shown in in-vitro systems that an activation of protein kinase C may be mediated by metabolic changes such as intracellular acidosis [14].

Two important components of acute myocardial ischaemia are energy depletion with a loss of high energy phosphates [15]and the development of acidosis due to an accumulation of acidic metabolites [16, 17]. Therefore, it is conceivable that activation of protein kinase C and the changes of the adrenergic system in acute myocardial ischaemia may be mediated in part by either mechanism. To address this question, we investigated the influence of acidosis and energy depletion on the components of the cardiac adrenergic system, i.e. the density of β-adrenergic receptors, the activity of adenylyl cyclase, and the activity of protein kinase C.

2 Methods

2.1 Materials

Embedded Image and Embedded Image were purchased from New England Nuclear (Boston, MA). Embedded Image was from DuPont (Bad Homburg, FRG). Reagents for the Bradford protein assay were from Biorad (Munich, FRG). Phosphocellulose P81 was bought from Whatman (Clifton, NJ, USA). All other reagents were from Sigma (Munich, FRG). Male Wistar rats (200 g) were from Thomae (Biberach, FRG).

2.2 Perfusion of isolated rat hearts

Male Wistar rats were anaesthetized with thiopentobarbital (50 mg/kg, i.p.). After heparinization (1000 I.U. Heparin, i.v.), the hearts were rapidly removed and perfused at 37°C according to the method of Langendorff [18]at a constant flow of 4.5 ml/(min×g) wet weight, using a modified Krebs–Henseleit solution (buffer A: 125 mmol/l NaCl, 1 mmol/l MgCl2, 1.85 mmol/l CaCl2, 4 mmol/l KCl, 12 mmol/l glucose, 0.027 mmol/l Na-EDTA, 16 mmol/l NaHCO3, 0.2 mmol/l NaH2PO4). A pyruvate-free solution was chosen according to the original work of Cobbe [17]. The Krebs–Henseleit solution (buffer A) was equilibrated with 95%O2/5%CO2 resulting in a pH of 7.40. After 10 min of preperfusion for equilibration, drugs were added continuously to the perfusion media as concentrated stock solutions to give the final concentrations as indicated. Total ischaemia was induced by stop of perfusion while keeping the hearts at constant humidity and temperature (37°C) and simultaneous replacing the air of the incubation chamber with nitrogen to prevent oxygen uptake at the surface. After preperfusion acidosis was induced by perfusion with buffer B (pH, 7.0) for 3 min, followed by a perfusion for 6 min with buffer C (pH, 6.6), respectively. The pH was modified by using adjusted concentrations of NaHCO3 (3–4 mmol/l) and NaH2PO4 (0–2 mmol/l) and it was monitored continuously. For the induction of anoxia, buffer A was saturated with N2/CO2 gas at a 95%/5% ratio and 5 mmol/l sodium dithionite was added. As shown before, pO2 decreased to less than 1 mmHg using this protocol [19]. Alternatively, cyanide (final concentration: 1 mmol/l) was added to Krebs–Henseleit solution in the absence of glucose to induce energy depletion. pH of the perfusate solution was measured and monitored continuously immediately before the perfusate reaches the hearts. We did not measure pH in the effluent because of changes of the CO2/HCO3 buffer system in contact with room air. In each experiment controls and treated groups were perfused in parallel. At the end of the experiments the hearts were freeze clamped and stored at −80°C. All experimental procedures conform with the Guide for Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1985) and accord to the official approval.

2.3 Preparation of cardiac plasma membranes

Cardiac plasma membranes were prepared as described previously [3]. Briefly, hearts were homogenized three times for 10 s, 10 000 rpm, using a polytron LS10-35 (Kinematica, Luzern, Switzerland) in buffer D containing 50 mmol/l Tris-HCl, 5 mmol/l Na-EDTA, 2 mmol/l Na-EGTA (pH 7.2, 4°C). The homogenate was passed through two layers of cheese cloth and centrifuged (360×g, 10 min, 4°C). The supernatant was sedimented (45 000×g, 10 min, 4°C) and washed three times in 40 ml buffer D. The final pellet was resuspended in buffer D to give a final concentration of 2–3 mg protein/ml. Aliquots were frozen in liquid nitrogen or used directly for radioligand binding experiments. The yield of plasma membrane protein under the various conditions was comparable in all preparations.

2.4 Radioligand binding

The density of β-adrenergic receptors was determined by radioligand binding using the radiolabelled β-adrenergic receptor antagonist Embedded Image [20, 21]. The number of β-adrenergic receptors was determined in saturation experiments using increasing concentrations of Embedded Image (20–320 pmol/l) in an assay volume of 250 μl including 10–20 μg membrane protein/tube. The incubation (1 h, 30°C) was stopped by rapid vacuum filtration (Whatman GF-C) with ice-cold buffer (3×4 ml, 50 mmol/l Tris-HCl, pH 7.4).

Non-specific binding was determined as the residual binding in the presence of alprenolol (10 μmol/l). Non-specific binding accounted for up to 30% of the total binding, which is in good agreement with previously published results [2, 22].

2.5 Determination of adenylyl cyclase activity

Adenylyl cyclase activity was determined according to the method of Salomon et al. [23]. Final concentrations were 0.5 mmol/l Embedded Image (≈200 000 cpm), 75 mmol/l Tris-HCl (pH 7.5), 12.5 mmol/l MgCl2, 1 mmol/l EDTA, 100 μmol/l GTP, 1 μmol/l Dithiotreitol, 100 μmol/l cAMP, 1 mmol/l isobutylmethylxanthine, 10 units creatine kinase, 20 mmol/l phosphocreatine. For stimulation, forskolin (100 μmol/l) was used as indicated. The incubation (37°C) was started by the addition of cardiac membranes (≈60 μg protein/tube) and stopped after 10 min by the addition of 500 μl NaHCO3 (120 mmol/l) and 500 μl zinc acetate (125 mmol/l) and placing in an ice-bath. The radiolabelled cAMP was isolated according to the method of Jakobs et al. [24]using aluminium oxide columns. For quantification, Cerenkov radiation was determined (Betaszint bf 8000, Berthold, Munich, Germany) with a counting efficiency of about 50%.

2.6 Partial purification of protein kinase C

Hearts were homogenized in buffer E (20 mmol/l Tris-HCl, 250 mmol/l sucrose, 5 mmol/l EDTA, 5 mmol/l EGTA, 1 mmol/l Phenylmethyl sulfurylfluoride (PMSF), 10 mmol/l β-mercaptoethanol, 1 mmol/l benzamidine, pH 7.6) using a Brinkman polytron apparatus (3×6 s, 10 000 rpm). The homogenate was centrifuged (360×g, 10 min, 4°C). The resulting supernatant was centrifuged at 100 000×g (60 min, 4°C) in order to separate the soluble fraction from the membrane fraction. The pellet corresponding to the membrane fraction was solubilized in buffer E containing Triton X-100 at a final concentration of 0.3% by stirring on ice for 45 min at 4°C. Insoluble membrane particles were sedimented by centrifugation at 100 000×g (60 min, 4°C). Cytosolic and solubilized membrane fractions were applied to DEAE-cellulose columns (1 ml bed volume), equilibrated with buffer F (buffer E +0.1% Triton X-100). The DEAE columns were washed with 10 ml of the buffer F. Protein kinase C was eluted with 2 ml of buffer G (20 mmol/l Tris-HCl, 250 mmol/l sucrose, 400 mmol/l NaCl, 1 mmol/l PMSF, 10 mmol/l β-mercaptoethanol, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l benzamidine, 0,1% Triton X-100).

2.7 Determination of protein kinase C activity

Protein kinase C activities were determined in the cytosolic fraction and the solubilized membranes according to the method of Takai et al. [25]. The reaction mixture (50 μl) contained 20 mmol/l Tris-HCl (pH 7.6), 5 mmol/l MgCl2, 20 μmol/l [γ-32P]ATP (≈200 000 cpm) and 25 μg histone III-S. Basal activity was determined in the presence of 1 mmol/l EDTA and 1 mmol/l EGTA. Maximally stimulated protein kinase C activity was determined in the presence of 1,25 mmol/l CaCl2, 5 μg phosphatidylserine and 200 mg diacylglycerol. The reaction (5 min, 37°C) was started by the addition of 10 μg of either cytosolic or solubilized plasma membrane protein. The reaction was stopped on ice by the addition of 20 μl 25 mmol/l ATP. Aliquots of 50 μl were spotted on Whatman P81 phosphocellulose, washed (≈20 ml water/paper) and counted in a β-counter (Berthold BF 8000) using Cerenkov counts.

2.8 Determination of the adenine nucleotides

Adenine nucleotides were determined using the method of Sabina et al. [26]. The content of high energy phosphates was determined by HPLC, using different concentrations of ATP as external standards [26]. The energy charge E was calculated according to the equation of Atkinson [27]: Embedded Image

2.9 Protein determination

Protein determination was performed according to the method of Bradford [28]using bovine serum albumin as standard.

2.10 Data analysis and statistical analysis

Saturation curves were analyzed by computer assisted techniques using non-linear, least square curve fitting techniques [29, 30]. Statistical comparisons were performed using the analysis of variance and Student's Newman Keuls test for significance.

3 Results

3.1 Energy depletion

The influence of energy depletion on the components of the myocardial adrenergic system was examined in two models of isolated perfused rat heart: Anoxic perfusion and perfusion with cyanide (1 mmol/l) in the absence of glucose, both leading to a rapid decline of the intracellular high energy phosphate stores such as creatine phosphate and ATP [3, 15]. Through washout of acidic metabolites such as lactate and hydrogen ions, however, acidosis is prevented in both models. In the perfusate, the pH values were continuously monitored during the whole experiment and did not differ from baseline values.

As shown in Table 1, the energy charge in the normoxically perfused rat hearts was 0.73±0.02. After 30 min of global ischaemia, it declined to 0.21±0.09. After 30 min of cyanide perfusion, the energy charge was within the same range (0.19±0.09). The data obtained for the adenine nucleotides ATP, ADP and AMP are also given in Table 1. These data confirm that in the model used in this paper, perfusion of isolated hearts with cyanide leads to a decrease of high energy phosphates comparable to the decrease occurring in ischaemia.

View this table:
Table 1

Energy charge in rat heart

ATPADPAMPEnergy Chargep vs. control
Control (n=10)61±325±215±20.73±0.02
Ischaemia (30 min, n=8)10±722±768±120.21±0.09≤0.001
Cyanide (30 min, n=10)7±823±769±120.19±0.08≤0.001
Acidosis (3 min, n=4)56±623±121±50.67±0.050.38
Acidosis (9 min, n=4)59±423±318±30.70±0.030.64
  • Values were given as % of total adenine nucleotides detected.

The energy charges during acidic perfusion were 0.67±0.05 after 3 min of acidosis and 0.70±0.03 after 9 min of acidosis (Table 1). These data demonstrate that, in contrast, acidic perfusion as performed in this study does not lead to a significant energy depletion.

The influence of energy depletion on the density of β-adrenergic receptors is shown in Fig. 1. Cyanide perfusion for 50 min induces an increase of β-adrenergic receptors in the plasma membrane to 162% of controls (50±3 vs. 81±7 fmol/mg protein, p≤0.05). Comparable changes could also be observed after 15 min of cyanide perfusion (59±4 fmol/mg protein, p≤0.05). Similarly, energy depletion induced by anoxic perfusion increased β-adrenergic receptors to 136% of the controls (55±2 vs. 75±2 fmol/mg protein, p≤0.05). 15 min of anoxic perfusion also increased β-adrenergic receptors, but, not surprisingly, to a smaller amount than the longer time period (63±6 fmol/mg protein). The affinities of the β-adrenergic receptor for the radiolabeled antagonist Embedded Image remained unaltered: In the controls the KD was 59±2 pM, in ischaemia 62±3 pM, after 50 min of cyanide perfusion 57±5 pM and after anoxia (50 min) 60±3 pM. The yields of protein obtained in these preparations did not differ significantly. Thus, in these in-vitro-models of energy depletion, both anoxia and cyanide perfusion promote an increase in the number of β-receptors comparable to the increase observed in global ischaemia. These data suggest that energy depletion may play a pivotal role for the increase of these receptors in ischaemia.

Fig. 1

Influence of energy depletion on cardiac β-adrenergic receptors. The density of β-adrenergic receptors was determined in cardiac plasma membranes after 15 and 50 min of ischaemia, anoxia, or cyanide perfusion, using [125I]Iodocyanopindolol binding (n=6 for each condition). Anoxia and cyanide perfusion lead to a significantly increased density of receptors to the same extent as it occurs in ischaemia. Co denotes control, Is=ischaemia, Cy=cyanide, An=anoxia and * denotes p≤0.05.

To determine if anoxia and energy depletion, in addition, may induce a sensitization of adenylyl cyclase, the forskolin-stimulated adenylyl cyclase activity was determined after 15 min of anoxia or cyanide perfusion. Similar periods have been shown to induce a transient sensitization of cardiac adenylyl cyclase in globally ischaemic hearts [3]. However, neither anoxia nor cyanide perfusion promoted any change of the adenylyl cyclase activity (Fig. 2). Even prolonged anoxia or cyanide perfusion for up to 60 min had no influence on the forskolin-activated adenylyl cyclase activity (data not shown). In contrast, global ischaemia for 15 min induces a significant increase of the forskolin-stimulated adenylyl cyclase activity up to 134% of the controls, confirming in the present study the data previously shown. These results suggest that energy depletion does not greatly contribute to the changes of adenylyl cyclase activity which has been observed in ischaemic hearts.

Fig. 2

Influence of energy depletion on myocardial adenylyl cyclase activity. Adenylyl cyclase activity was determined in the particulate fraction of hearts after 15 min of ischaemia, anoxia, or cyanide perfusion (n=6 for each condition). Ischaemia leads to a sensitization of adenylyl cyclase. However, anoxia and cyanide fail to sensitize the enzyme. Data are given as % of control. The control levels did not vary significantly (see text). Co denotes control, Is=ischaemia, Cy=cyanide, An=anoxia and * denotes p≤0.05.

To test whether protein kinase C is modulated by energy depletion, protein kinase C activity was measured in the cytosolic fraction and the membrane fraction after 10 min of global ischaemia and after 5 and 10 min of cyanide perfusion (Fig. 3). Translocation of protein kinase C, as indicated by the decrease of activity in the cytosol and the concomitant increase in the membranes, occurs after 10 min of global ischaemia. In contrast, the enzyme activity or intracellular distribution is not greatly influenced by cyanide perfusion (Fig. 3). The effects of anoxia alone were not determined in those experiments. These data suggest that energy depletion per se may not contribute to the ischaemia-induced translocation and activation of protein kinase C.

Fig. 3

Cyanide perfusion does not lead to translocation of protein kinase C. Protein kinase C activity was determined in the cytosol and the plasma membranes of hearts perfused with cyanide for 5 and 10 min or in hearts subjected to ischaemia for 10 min, or in the normoxic controls (n=6 for each condition). Ischaemia leads to a decrease of cytosolic enzyme activity with a concomitant increase of the enzyme activity in the particulate, suggesting the translocation of protein kinase C. Cyanide perfusion did not translocate protein kinase C. Co denotes control, Is=ischaemia, Ac=acidosis and * denotes p≤0.05.

3.2 Acidosis

As previously established by others [31], acidic perfusion of isolated perfused rat hearts was used to simulate the ischaemia-induced acidosis in ischaemic myocardium. Similar to the reduction of pH in ischaemic myocardium [16, 17], a gradual lowering of pH in the perfusate was chosen (3 min of pH 7.0, followed by 6 min of pH 6.6). Oxygenation and glucose supplementation were continued during the whole experiment to prevent a depletion of high energy phosphates.

The effect of acidosis on β-adrenergic receptors is shown in panel A of Fig. 4. As compared to the controls (57±3 fmol/mg protein), the density of β-adrenergic receptors remains unchanged after 3 min (56±6 fmol/mg protein), 6 min (56±6 fmol/mg protein), and 9 min (55±5 fmol/mg protein) of acidic perfusion. In contrast, even a short period of ischaemia (10 min) induces a significant increase in the density of β-adrenergic receptors to 120% of the controls (67±3 fmol/mg protein, Fig. 4A), which persists and is even more pronounced upon longer periods of ischaemia (Fig. 1). The affinities of the β-adrenergic receptors for their radiolabeled antagonist Embedded Image remained unaltered under all conditions (see figure legend).

Fig. 4

Influence of acidosis on β-adrenergic receptors (panel A) and adenylyl cyclase activity (panel B). Hearts were subjected to ischaemia (10 min) or acidosis (3 min of pH 7.0 (Ac 3′), followed by 3 and 6 min of pH 6.6 (Ac 6′ and Ac 9′, n=4 for each condition). β-adrenergic receptors (Panel A) and adenylyl cyclase activity (Panel B) were determined in the particulate fraction. As shown in panel A, acidosis does not lead to an increase of the density of β-adrenergic receptors. The KD values of β-adrenergic receptors for the radiolabeled ligand Embedded Image did not differ significantly (Co: 22±6, Is 28±6, Ac 3′ 22±4, Ac 9′ 20±4 pM). In contrast, 3 min of acidosis promote a sensitization of adenylyl cyclase, which gradually declines after 6 and 9 min of acidosis (Panel B). The extent of this sensitization is comparable to the ischaemia-promoted sensitization of the enzyme. Co denotes control, Is=ischaemia, Cy=cyanide, An=anoxia and * denotes p≤0.05.

In contrast, after 3 min of acidosis the forskolin-stimulated adenylyl cyclase activity is significantly increased (control: 428±17 vs. 3 min acidosis: 515±16 pmol×mg−1 protein×min−1, Fig. 4B). This increased activity persists for 6 min of acidosis (506±26 pmol×mg−1 protein×min−1), but gradually declines after 9 min of acidosis (448±16 pmol×mg−1 protein×min−1), indicating the transient nature of this sensitization. The extent of the initial acidosis-induced sensitization of adenylyl cyclase is comparable to the transient sensitization observed during ischaemia with a maximal sensitization after 10 min of global ischaemia.

Similar to global ischaemia, 3 min of acidosis induces a significant translocation of protein kinase C activity from the cytosol to the membranes, indicating the rapid activation of the enzyme (cytosol: 89±16 vs. 73±12; membrane 117±26 vs. 159±36 pmol×mg−1 protein×min−1, p≤0.05), as shown in Fig. 5. After 6 min and 9 min of acidic perfusion, the increased protein kinase C activity in the membranes returns to control values. The extent of translocation after 3 min of acidosis is similar to the ischaemia-induced translocation. The total amount of maximally stimulated enzyme activity present in the heart does not change under these conditions (data not shown). Thus, in this model of acidosis lacking energy depletion, the translocation and activation of protein kinase C and the sensitization of adenylyl cyclase coincide like in ischaemia, whereas both are absent in energy depletion alone (Figs. 2 and 3).

Fig. 5

Acidosis leads to translocation of cardiac protein kinase C. Protein kinase C activity was determined in the cytosol and the particulate fraction of hearts subjected to ischaemia (10 min) or to acidosis (3 min of pH 7.0 (Ac 3′), followed by 3 and 6 min of pH 6.6 (Ac 6′ and Ac 9′), n=4 for each condition). Acidosis leads to an increased enzyme activity in the particulate fraction with a concomitant decrease in the cytosol, suggesting the translocation of the enzyme. This translocation after brief cardiac acidosis is comparable to the ischaemia-induced translocation of protein kinase C. During continued acidic perfusion, activity returns to control levels. Co denotes control, Is=ischaemia, Ac=acidosis and * denotes p≤0.05.

4 Discussion

Myocardial ischaemia promotes a wide array of cellular changes. Among those, energy depletion and intracellular acidosis are profound and important metabolic changes. The focus of the present study was to try to differentiate the effects of these two components of myocardial ischaemia on the adrenergic signal transduction system and on the activation of protein kinase C.

The salient findings of this study are that in myocardial energy depletion and myocardial acidosis, a differential regulation of the components of the adrenergic signal transduction system, i.e. the β-adrenergic receptors on one side and the adenylyl cyclase and protein kinase C on the other side, can be observed: The density of β-adrenergic receptors is increased during energy depletion which was induced either by anoxia and cyanide perfusion. This upregulation of β-adrenergic receptors is comparable to the increase induced by myocardial ischaemia. In contrast, neither model of energy depletion influences the effector enzymes, i.e. the activity of adenylyl cyclase or the subcellular distribution of protein kinase C. Acidic perfusion, however, leads to a sensitization of adenylyl cyclase and to a translocation, i.e. activation of protein kinase C similar to the changes observed in ischaemia. Here in contrast, the density of β-adrenergic receptors remains unchanged. These data suggest that in ischaemia, where both energy depletion and acidosis coincide and are possibly interfering, these two mechanisms may be operative synergistically: First, energy depletion may be responsible for the ischaemia-induced upregulation of β-adrenergic receptors, and second, acidosis may promote the activation of protein kinase C and the transient sensitization of adenylyl cyclase.

Energy depletion in ischaemia is caused by a dysfunction of oxidative phosphorylation and leads in consequence to a depletion of energy-rich substrates, i.e. ATP and creatine phosphate. As established by NMR spectroscopy, creatine phosphate levels decrease nearly to zero after less than 5 min of ischaemia [32]. ATP levels decrease to 70% of the initial level after 5 min of ischaemia and to 50% after 10 min, followed by a complete loss of ATP after 20 min of ischaemia [16, 31], data that could also be obtained in the present study. The energy charge of cardiac cells declines to about 0.30 after 10 min and to 0.20 after 30 min of global ischaemia, as shown by Fiolet et al. [15]and as confirmed in the present study.

Anoxia and cyanide perfusion promote an energy depletion similar to ischaemia, as shown in Table 1, data which are in good agreement with those of Fiolet et al. [15]. It has been established previously that in both models, only a mild intracellular acidosis develops [15]. Since acidic metabolites are continuously removed during perfusion, the extent of acidosis in anoxia or after cyanide perfusion is much smaller than in ischaemia: 5 min of anoxia or cyanide perfusion reduce the tissue pH by only 0.05, 20 min only by 0.18 and 0.23, respectively [33]. Furthermore, the intracellular pH changes only marginally in anoxia: After 30 min, it falls only by 0.04 [34]. In contrast, ischaemia for 20 min produces a quite pronounced acidosis with a decrease of the myocardial pH by 1.3 [16]. Thus, as established previously, anoxic and cyanide perfusion are a valuable model to imitate predominantly the energy depletion in ischaemia without any significant decrease of intracellular pH.

Acidosis in the ischaemic myocardium occurs rapidly. Starting from a baseline intracellular pH of about 7.2, 3 min of ischaemia lead to a decrease in pH to 6.7, further falling to pH 6.5 after 5 min and to pH 6.25 after 10 min of ischaemia [16]. Therefore, stepwise reduction of pH during perfusion was chosen to mimic the continuous decrease of pH in ischaemia [16]without loss of high energy phosphates. In metabolic acidosis (extracellular pH, 6.7), Jelicks et al. [31]have shown that intracellular ATP levels and oxydative phosphorylation are not impaired in glucose-perfused hearts. Williamson et al. [35]demonstrated that a prolonged, more severe acidosis (15 min, pH 6,6), leads only to minor changes of the energy charge. It could be confirmed in the present study that short periods of acidosis do not lead to significant changes of the energy charge or of the concentration of high-energy phosphates (Table 1). Therefore we propose that in the model of acidosis used in this study, energy depletion is of minor importance.

A limitation of the present study is that the intracellular pH was not determined. However, as shown by Jelicks et al, the intracellular pH quickly follows the extracellular pH, i.e. the pH of the perfusate solution, with little delay [31]. Since a more abrupt decrease of extracellular pH led to an immediate cardiac arrest in pilot experiments (data not shown), a stepwise acidification was used for the present study. Furthermore, comparison with recent data shows that activation of protein kinase C and adenylyl cyclase occur in the same time course as in ischaemia [36]. These data suggest that the models used in this study are comparable to those individual elements of acute myocardial ischaemia. It is emphasized, however, that in myocardial ischaemia in vivo, both components are also interdependent variables, caused by the lack of perfusion, and cannot be fully separated.

A further limitation of the present study is that all measurements were performed in plasma membranes derived from intact myocardial tissue. Thus, the presumption is that cardiomyocytes are primarily responsible for the changes observed here. However, it has not been determined at the cellular level to which extent other cells such as fibroblasts may be involved.

In previous studies, we and others have demonstrated the sensitization of adenylyl cyclase [3, 5, 38], the translocation of protein kinase C [12, 39]and increased densities of β-adrenergic receptors [3, 38]to occur in early myocardial ischaemia.

The time frames for the increase of β-adrenergic receptors in ischaemic hearts and for the sensitization of the adenylyl cyclase or the translocation of protein kinase C are different. The increase of receptors occurs somewhat slower and persists even after 50–60 min of ischaemia [3, 5]. In contrast, the sensitization of adenylyl cyclase is observed early after few minutes following the onset of ischaemia, but is only transient [3]. Also translocation and activation of protein kinase C has been shown to occur after 5–10 min of ischaemia and to decline later in ischaemia [37]. Therefore, in the present study these parameters were determined at time points when their maximal changes were expected.

The mechanisms responsible for the increase in β-adrenergic receptors during myocardial ischaemia or energy depletion have not yet been clarified at the molecular level. Although local catecholamine levels are elevated in myocardial ischaemia [40, 41], β-adrenergic receptor internalization, which is an energy dependent process, does not occur under these pathophysiological conditions. In contrast, an increased density of β-adrenergic receptors in the membrane is observed. As we could show previously, energy depletion by cyanide perfusion greatly impairs the agonist-promoted receptor internalization [21]. Similar data were obtained in isolated cells [42]. These findings suggest that energy depletion may be the leading mechanism for the sensitization of β-adrenergic receptors during acute myocardial ischaemia. Additionally, ischaemia leads to a rapid subtype-specific increase of mRNA for β1-adrenergic receptors [43]. These data suggest, that in addition to impaired receptor internalization, transcription of mRNA for the β-adrenergic receptors may be enhanced, both contributing to the net increase of β-adrenergic receptors in ischaemia and in energy-depletion. Chronic hypoxia has been shown also in isolated neonatal rat cardiac myocytes and in other organs to promote an increased density of β-adrenergic receptors with an increase both at the protein and at the mRNA level [44, 45]. In these models it has not been determined, which component of hypoxia may be responsible and whether energy depletion alone may suffice to induce an increase of β-adrenergic receptors.

Previously, we could demonstrate that the sensitization of the adenylyl cyclase in early myocardial ischaemia may be a consequence of the activation of protein kinase C [12]. In ischaemia and, as shown in the present paper, in acidosis the sensitization of the adenylyl cyclase was associated with the early activation of protein kinase C. Whether acidosis might be able to induce sensitization of adenylyl cyclase in the absence of protein kinase C activation could not been evaluated since it coincided. However, it has been shown previously that blockade of the activation of protein kinase C in ischaemia completely prevents sensitization of adenylyl cyclase [12], suggesting the pivotal role of protein kinase C in this sensitization process. Similarly, in adipocytes and in isolated neonatal rat cardiomyocytes, the link between activation of protein kinase C and the sensitization of adenylyl cyclase has been established [46, 47]. Moreover, in isolated systems it has been shown that phosphorylation of adenylyl cyclase by protein kinase C directly induces a sensitization of adenylyl cyclase [48].

The molecular mechanisms involved in the activation of cardiac protein kinase C during myocardial acidosis have not been determined so far. However, McFadden et al. have demonstrated in vitro that lowering of pH leads to autophosphorylation of protein kinase C [14]. This process occurs independent of the classic protein kinase C activators, calcium and phospholipids and appears to indicate an activation of protein kinase C [49]. It cannot be determined, if in ischaemia acidosis may be the only responsible mechanism leading to the activation of protein kinase C, since in the ischaemic heart the occurrence of intracellular acidosis and the influx of calcium cannot be separated. The patterns of sensitization of adenylyl cyclase and of activation of protein kinase C are quite comparable in ischaemia and in acidosis. Therefore, it is conceivable that acidosis as an intimate component of ischaemia plays a central role in modulating those enzyme activities in the ischaemic heart.

It has to be mentioned that the protein kinase C activities were obtained using Histon-IIIs as a substrate. So, preferentially the calcium-dependent isoforms of protein kinase C were assayed. However, preliminary data show that acute myocardial ischaemia leads to a translocation of all major cardiac isoforms [37]. Therefore, it seems reliable to use this ‘classic’ assay protocol [25].

At this point, it might be worth noting that the stoichiometry of translocation may not reflect the extent of activation of the enzyme. In this study, as in the literature [50–53], activation of protein kinase C is documented by its translocation from the cytosol to the plasma membranes. However, it has been shown that very different physiological and pharmacological stimuli of protein kinase C mediate quite a similar extent of translocation to the plasma membrane [50, 54–56]. These data suggest that translocation is a qualitative, but not quantitative parameter of protein kinase C activation. Thus, although the extent of translocation is comparable in ischaemia and in early acidosis, it cannot be decided with certainty whether acidosis and ischaemia lead to different extents of protein kinase C activation.

The present study characterizes that the two most evident metabolic components of ischaemia, i.e. energy depletion and intracellular acidosis differentially regulate β-adrenergic receptors, adenylyl cyclase, and protein kinase C. Energy depletion induces an increase of β-adrenergic receptors, yet fails to activate adenylyl cyclase and protein kinase C. Acidosis on the other side leads to the activation of protein kinase C and subsequent sensitization of cardiac adenylyl cyclase activity without affecting the density of β-adrenergic receptors. In global ischaemia, both acidosis and energy depletion coincide leading to both the increased density of β-adrenergic receptors and the activation of protein kinase C and adenylyl cyclase. To our knowledge, this is the first characterization of different components of ischaemia, proposing their distinct contribution to the activation of the adrenergic system in acute myocardial ischaemia.

Acknowledgements

Parts of this study were presented at the Annual Meetings of the American Heart Association 1994 and 1996.

This study was supported by the Deutsche Forschungsgemeinschaft, Bonn, (SFB 320). R.H.S is supported by the Hermann-and-Lilly-Schilling Foundation.

We are indebted to Ms. Ritva Pennanen for excellent secretarial help in preparing the manuscript, to Ms. Ulrike Oehl for helpful technical assistance, and to Dr. Renate Ihl-Vahl, Dr. Mathias M. Borst and Dr. Martin Braun for helpful discussion.

References

View Abstract