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© 2004 European Association of Cardio-Thoracic Surgery
Long-term hypothermic lung preservation: does adenosine A1 receptor antagonism have a role in ischemic preconditioning protection?Cardiac Surgical Research, The Rayne Institute, St Thomas' Hospital, Guy's and St Thomas' NHS Trust, London SE1 7EH, UK
* Corresponding author. Tel.: +44-20-7261-0157; fax: +44-20-7928-0658 Received July 9, 2003; received in revised form November 10, 2003; accepted November 12, 2003
Ischemic preconditioning or phosphodiesterase inhibition improves lung protection during prolonged hypothermic storage. In ischemic preconditioning of cat lungs, adenosine A1 receptor antagonism was suggested as a possible mechanism. Some phosphodiesterase inhibitors (such as theophylline) are also adenosine antagonists; we showed theophylline to be particularly effective in protecting lungs. In isolated, perfused and ventilated rat lungs, we examined (1) whether synergy exists between phosphodiesterase inhibition and ischemic preconditioning and (2) whether theophylline acts both to inhibit phosphodiesterase and block adenosine receptors, by comparing its effects with enprofylline (selective phosphodiesterase inhibition) or xanthine amine congener (selective adenosine A1 receptor antagonism). In Study 1, rolipram (added to St Thomas' cardioplegia) or ischemic preconditioning before hypothermic storage (8 h) did not improve lung function during reperfusion (40 min); a combination of these treatments was also ineffective. In Study 2, lungs stored in St Thomas' cardioplegia containing enprofylline or theophylline had improved recovery of function compared to control lungs; however, xanthine amine congener was without effect. Thus, no interaction exists between phosphodiesterase inhibition and ischemic preconditioning. Adenosine A1 receptor antagonism plays no role in protecting rat lungs from the effects of prolonged hypothermic storage by either preconditioning or addition of theophylline to the storage solution.
Key Words: Rat; Lung; Preservation; Preconditioning; Adenosine receptor; Antagonists
Ischemic preconditioning occurs when short ischemic episodes protect against subsequent extended periods of ischemia [1]. Mechanisms of ischemic preconditioning have been extensively studied in the heart [2]; adenosine A1 receptor activation appears to have a major role [2] and pharmacological activation of the A1 receptor can mimic preconditioning. In the rat heart, however, this mechanism remains controversial [3]. In the lung, preconditioning protection can also be demonstrated [4,5]; however, it has been suggested that, in contrast to the heart, desensitization of adenosine A1 receptors may induce preconditioning in the cat lung [4]. Addition of phosphodiesterase (PDE) inhibitors to the hypothermic flush and storage solution of isolated lungs protects against the effects of prolonged hypothermic storage [6]; in particular, the non-selective PDE inhibitor, theophylline, is more effective than several PDE-isoenzyme selective inhibitors [6]. Interestingly, theophylline also acts as an adenosine receptor antagonist [7], so the superiority of theophylline in protecting lungs may be due to combined PDE inhibitory activity and adenosine antagonism (mimicking preconditioning [4]). To examine this, we used rat lungs undergoing long-term hypothermic storage to compare the protective effect of Rolipram, a PDE-IV selective inhibitor lacking adenosine receptor antagonist properties, to an ischemic preconditioning protocol, as well as a combination of these two treatments. Secondly, we compared the protective effects of theophylline, enprofylline (PDE inhibition selectivity [8]) and xanthine amine congener (A1 receptor antagonist selectivity [9]).
2.1. Materials Rolipram was a gift from Schering AG (Berlin, Germany). Theophylline and enprofylline were purchased from Sigma (Poole, Dorset, UK) and xanthine amine congener from ICN (Basingstoke, Hampshire, UK). Pentabarbitone was purchased from Rhone Merieux (Harlow, UK). Other chemicals were supplied by BDH Ltd (Leicestershire, UK). 2.2. Lung preparation Lungs were obtained from male Wistar rats (250–330 g). All animals received humane care in compliance with the ‘Guidance on the Operation of the Animals (Scientific Procedures) Act 1986’ published by Her Majesty's Stationary Office, London, England.The isolated, perfused lung preparation was set up as described previously [6]. Briefly, rats anesthetized by intraperitoneal injection of pentabarbitone (2 ml/kg of a 60 mg/ml solution) were tracheally intubated and ventilated (80breaths/min) with a Harvard Small Animal Ventilator. The diaphragm was removed and heparin (500IU) injected into the vena cava. The animals were then exsanguinated by withdrawal of blood from the vena cava, the thorax opened and the pulmonary artery and left atrium cannulated. The lungs were then removed and suspended in a chamber at 37 °C. Perfusion was commenced with modified bicarbonate buffer (BB, mmol/l: NaCl 118.5, KCl 3.8, KH2PO4 1.2, NaHCO3 25.0, CaCl2 2.0, MgSO4 1.2, glucose 10.0) mixed with whole rat blood (4:1 buffer/blood) to produce sanguineous BB (SBB). SBB was maintained at 37 °C in a plastic reservoir and gassed with 100% CO2 whenever pH exceeded 7.3. Perfusate, at a flow rate of 15 ml/min, was passed through a membrane de-oxygenator filled with 100% nitrogen before entering the lung; buffer leaving the lungs was returned to the reservoir and recycled. Oxygenation of the perfusate was by the isolated lungs, ventilated with room air.
The tracheal pressure (TP) applied by the ventilator was set to give a tidal volume (TV) of 2.0–2.3 ml. Positive end-expiratory pressure of 1–2cmH2O was applied. Pressure transducers were connected to the tracheal, arterial and venous cannulae. TP and TV, calculated by integration of the flow
 is the inertia and  is the differential of the flow.  and  were calculated over a 10 breath period for each timepoint examined. The difference in pressures between the pulmonary artery and venous cannulae divided by the perfusate flow rate, measured vascular resistance. The output of two flow-through oxygen electrodes (LazarLabs, CA) in the perfusion circuit before and after the lungs allowed determination of the gas-exchange. All outputs from pressure transducers, pneumotachograph and the oxygen and pH electrodes were recorded using a PowerLab 8s connected to a PowerMac (Apple Computers) computer employing the PowerLab Chart software (ADInstruments Ltd, Hastings, UK). 2.3. Experimental protocol During an initial 20 min period, lungs underwent control perfusion with SBB or the preconditioning protocol; baseline lung function parameters were measured at the end of this period. Lungs were then flushed with 30 ml of St Thomas' cardioplegia (STH) used as the storage solution (mmol/l: NaCl 110, KCl 16.0, MgCl2 16.0, CaCl2 1.2, NaHCO3 10.0, pH 7.8 at 37 °C), infused at a pressure of 30cmH2O. Flush was initially (10 ml) at 20–25 °C to reduce cold-induced vasoconstriction with the remaining 20 ml infused at 4 °C; typically, the whole flush took 150 s. Flushed lungs were stored inflated with 3 ml of room air, immersed in the storage solution and maintained at 4–6 °C throughout the storage period. After storage, lungs were re-attached to the perfusion circuit and reperfused (at 37 °C) with SBB for 40 min, using blood obtained from a second rat.2.4. Study 1 To determine the interaction between the effects of ischemic preconditioning and PDE inhibition on lung preservation, rat lungs ( per group) were randomly allocated to one of five groups: (i) control aerobic perfusion (60 min) without storage, (ii) 20 min control perfusion before flush and storage for 8 h in STH (ischemic control), (iii) two cycles of 5 min ischemia (cessation of ventilation and perfusion) and 5 min reperfusion and reventilation before flush and storage for 8 h in STH, (iv) 20 min control perfusion before flush and storage for 8 h in STH containing 30 µM Rolipram or (v) two cycles of 5 min ischemia and 5 min reperfusion before flush and storage for 8 h in STH containing 30 µM Rolipram. 2.5. Study 2 To examine the relative contribution by xanthines of adenosine antagonism or PDE inhibition to lung protection during prolonged hypothermic storage, rat lungs ( per group) were randomly allocated to one of five groups: (i) control aerobic perfusion (60 min) without storage, (ii–v) 20 min control perfusion before flush and storage for 8 h in STH alone or STH containing theophylline (3000 µM), enprofylline (3000 µM) or xanthine amine congener (10nM), respectively, followed by a further 40 min reperfusion. 2.6. Determination of lung cyclic AMP content To determine whether the xanthines were effective in maintaining tissue cAMP levels during hypothermic storage, we examined cAMP concentrations in a separate series of lungs ( per group) taken (i) immediately after 20 min perfusion with SBB, (ii)–(v) 8 h hypothermic storage in STH (control storage) or in lungs in which STH contained theophylline (3000 µM), enprofylline (3000 µM) or xanthine amine congener (10nM), respectively. Additionally, we examined the effects of preconditioning on lung cAMP levels in lungs ( per group) sampled (i) immediately after two periods of 5 min ischemia and 5 min reperfusion and (ii) after preconditioning followed by 8 h hypothermic storage in STH. Lungs were then immediately frozen in liquid nitrogen and stored at –80 °C until later assay for cAMP (as previously described [6]), using a commercially available immunoassay kit (Cayman Chemical, Ann Arbor, MI). The protein content was determined [11] and cAMP concentration expressed as pmol/mg protein. 2.7. Statistics Data are displayed as mean±standard error (SEM), with 5–6 animals/group. To compare treatment effects on lung function during reperfusion, trapezoid integration was used to calculate the area under the time-response curve (AUC) for each parameter for each animal. These values, and the actual values of the measured parameters at the end of 40 min reperfusion, were then employed for statistical comparisons between experimental groups by one-way analysis of variance (ANOVA). If this revealed significant differences, Dunnett's test was used to compare multiple values to those in control, unstored lungs. Where data deviated significantly from normality a non-parametric one-way ANOVA (Kruskal–Wallis) was applied and multiple values compared to control using Dunn's or Dunnett's test. Lung cAMP contents were analyzed using a Kruskal–Wallis ANOVA and all values compared to each other using a -test adjusted for multiple comparisons. In all tests a  value of less than 0.05 was taken as indicating significance.
3.1. Study 1 3.1.1. Lung function The values for pulmonary static compliance  transpulmonary difference in perfusate pO2 (gas exchange), airways resistance  and vascular resistance after 20 min perfusion in control (no storage) lungs are shown in Table 1A. None of the other experimental groups differed significantly from these values at this timepoint.
Storage (8 h) of lungs in STH alone resulted in a marked fall in and gas exchange and increase in and vascular resistance after 40 min of reperfusion compared to unstored lungs perfused for a total of 60 min (Fig. 1, Table 1A).
Addition of Rolipram (30 µM) to the STH solution used for storage, two periods of 5 min preconditioning ischemia and reperfusion prior to storage, and the combination of these two treatments were all without significant effect on the changes after 8 h hypothermic storage and 40 min reperfusion assessed by area under the curve analysis (Fig. 1).
3.1.2. Cyclic AMP levels
3.2. Study 2 3.2.1. Lung function Pulmonary static compliance transpulmonary difference in perfusate pO2 (gas exchange), airways resistance and vascular resistance values after 20 min perfusion in control (no storage) lungs are shown in Table 1B. Values in the other experimental groups were not significantly different at this timepoint.
Storage (8 h) of lungs in STH alone caused a marked fall in
Addition to STH of theophylline (3000 µM) or enprofylline (3000 µM) increased post-storage whereas addition of xanthine amine congener (10nM) to STH had no beneficial effect and was similar to control STH storage (Fig. 3A). Gas exchange post-storage was also improved by addition of enprofylline to the storage solution (Fig. 3B). Theophylline and enprofylline appeared to produce some improvement in post-storage; however, the variability in these values meant no differences were detected between AUCs (Fig. 3C). Vascular resistance values in lungs treated with theophylline or enprofylline were similar to that of unstored lungs whereas treatment with xanthine amine congener significantly elevated vascular resistance.
3.2.2. Cyclic AMP levels
These studies examined the linked hypotheses that protection of the lungs by theophylline involves a dual mechanism of PDE inhibition and adenosine A1 receptor antagonism, and that a potential mechanism of ischemic preconditioning in the lung is desensitization of adenosine A1 receptors [4]. Thus, from these hypotheses we predicted that (i) the protective effect of Rolipram, a selective PDE isoenzyme inhibitor, should be enhanced by combination with preconditioning, and (ii) the non-selective xanthine theophylline, should have the greatest protective effect compared to enprofylline and xanthine amine congener. In this study, neither the preconditioning protocol nor the addition of Rolipram to the flush and storage solution exerted a significant protection. Compared to previous studies [5,12], an extended storage period after the preconditioning protocol was used, whilst the dose of Rolipram was lower than the previously established optimum [6]. These changes should have been suitable to demonstrate any positive interaction between PDE inhibition and ischemic preconditioning, but we were unable to detect any interaction.
The doses of theophylline and enprofylline employed in this study (3000 µM) are considerably greater than the Preconditioning alone produced no detectable change in cAMP, nor did it prevent the fall in cAMP seen after prolonged hypothermic storage. This seems to rule out any role for changes in cAMP in the mechanism of preconditioning in the lung. However, the cAMP measurements are from whole organ samples and a change in cAMP in a single cell type critical to lung function (e.g. vascular endothelial cells) may be important. This study fails to confirm the hypothesis that protection of the lungs by theophylline is by a dual mechanism involving both PDE inhibition and adenosine A1 receptor antagonism or [4] that a potential mechanism of ischemic preconditioning in the lung was through desensitization of adenosine A1 receptors. doi:10.1016/S1569-9293(03)00274-3
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Abstract
1. Introduction
through a pneumotachograph connecting the trachea to the ventilator, were measured. Pulmonary static compliance 
and airways resistance 
and (D) vascular resistance, during 40 min reperfusion with SBB in isolated rat lungs after no storage (solid squares), 8 h storage in STH alone (solid circles), 8 h storage in STH after two periods of 5 min ischemic preconditioning (no perfusate flow or ventilation) (open triangles), 8 h storage in STH solution containing Rolipram (30 µM) (open circles), or 8 h storage in St Thomas Hospital solution containing Rolipram (30 µM) after two periods of 5 min ischemic preconditioning (open diamonds). Data are expressed as mean±SEM, 
when compared to values of non-stored tissues, by area under the curve analysis (Dunnett's test, 
or Dunn's test, gas exchange).
values for these compounds against a range of PDE isoenzyme subtypes