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Preconditioning modulates pulmonary endothelial dysfunction following ischemia-reperfusion injury in the rat lung: Role of potassium channels
Life Sciences 79 (2006) 2172 – 2178 www.elsevier.com/locate/lifescie
Preconditioning modulates pulmonary endothelial dysfunction following ischemia-reperfusion injury in the rat lung: Role of potassium channels H. Burak Kandilci a , Bülent Gümüşel a,⁎, A. Tuncay Demiryürek b , Howard Lippton c a
Department of Pharmacology, Hacettepe University, Faculty of Pharmacy, 06100, Sıhhiye, Ankara, Turkey b Department of Pharmacology, University of Gaziantep, Faculty of Medicine, 27310, Gaziantep, Turkey c Pneumosite LLC, Shreveport, Louisiana, USA Received 22 March 2006; accepted 12 July 2006
Abstract Ischemic preconditioning (IP) may protect the lung from ischemia-reperfusion (I/R) injury following cardiopulmonary by-pass and lung or heart transplantation. The present study was undertaken to investigate the role of ATP-dependent potassium channels (KATP) in IP in the isolated buffer-perfused rat lung (IBPR) under conditions of elevated pulmonary vasoconstrictor tone (PVT). Since pulmonary arterial perfusion flow and left atrial pressure were constant, changes in pulmonary arterial pressure (PAP) directly reflect changes in pulmonary vascular resistance (PVR). When compared to control value, the pulmonary vasodilator responses to histamine and acetylcholine (ACh) following 2 h of hypothermic ischemia were significantly attenuated, whereas the pulmonary vasodilator response to sodium nitroprusside (SNP) was not altered. IP in the form of two cycles of 5 min of ischemia and reperfusion applied prior to the two-hour interval of ischemia, prevented the decrease in the pulmonary vasodilator responses to histamine and ACh. Pretreatment with glybenclamide (GLB) or HMR-1098, but not 5-hydroxydecanoic acid (5-HD), prior to IP abolished the protective effect of IP. In contrast, GLB or 5-HD did not significantly alter the pulmonary vasodilator response to histamine without IP pretreatment. The present data demonstrate that IP prevents impairment of endothelium-dependent vasodilator responses in the rat pulmonary vascular bed. The present data further suggest that IP may alter the mediation of the pulmonary vasodilator response to histamine and thereby trigger a mechanism dependent on activation of sarcolemmal, and not mitochondrial, KATP channels to preserve endothelialdependent vasodilator responses and protect against I/R injury in the lung. © 2006 Elsevier Inc. All rights reserved. Keywords: Rat lung vascular bed; Ischemic preconditioning; ATP-dependent potassium channels; Sarcolemmal KATP channels; Mitochondrial KATP channels; Ischemia/reperfusion; Pulmonary vasodilation
Introduction Many studies in the heart have demonstrated that a short period of ischemia-reperfusion (I/R) protects against harmful effects of subsequent prolonged ischemia; this endogenous mechanism of protection has been termed ischemic preconditioning (IP) (Murry et al., 1986; Cleveland et al., 1996). Recent studies have reported IP increases the tolerance of lung, similar to the heart, against I/R injury (Neely and Keith, 1995; Featherstone et al., 2000; Friedrich et al., 2001; Soncul et al., 1999). I/R injury is not limited to myocytes since I/R injury ⁎ Corresponding author. Tel.: +90 312 3052081; fax: +90 312 3114777. E-mail address: [email protected] (B. Gümüşel). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.011
produces structural changes in coronary vascular endothelium resulting in endothelial damage in the myocardium (Richard et al., 1995) and induces endothelial dysfunction with increases in permeability and vascular resistance in pulmonary blood vessels (Davenpeck et al., 1993). Although the mechanism of action of IP is unclear, activation of KATP channels has been reported to contribute to the actions of IP (Mulleneim et al., 2001). Two types of KATP channels exist in the myocardium: mitochondrial and sarcolemmal KATP channels (Gross and Fryer, 1999). KATP channels are also present on both vascular smooth muscle and vascular endothelium (Standen, 2003; Chatterjee et al., 2003). The role of the mitochondrial KATP and sarcolemmal KATP channels in IP has been challenged (Gross and Fryer, 1999; Light, 1999).
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Although data have accumulated suggesting a role for mitochondrial KATP channels in cardiac IP (Garlid et al., 1997; Bernardo et al., 1999; Yellon and Downey, 2003), evidence is now available suggesting that sarcolemmal KATP channels activation may also contribute to the protective effect to IP in the heart (Toyoda et al., 2000; Light et al., 2001). The effects of IP on the pulmonary vascular bed and the possible protective role of KATP channels in I/R injury have not been investigated. The present study was undertaken to determine the contribution of subtypes of KATP channels in mediating the effects of IP on I/R-mediated injury in the IBPR. Materials and methods The experiment was performed in compliance with the “Principles of Laboratory Animal Care” formulated by the National Institutes of Health (National Institutes of Health publication no. 96 to 23, revised 1996). The experiment and animal care protocol was approved by the Ethics Committee for animal care, established in our institute (# 2001/25-4). Isolated buffer perfused rat lung (IBPR) Male Wistar rats (200–300 g) were anesthetized with thiopental (30 mg/kg, i.p.). After tracheal intubation, the chest
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was opened and heparin (200 IU) was injected into the right ventricle. The main pulmonary artery was cannulated with a stainless steel cannula via the right ventricle and the vasculature was flushed with Krebs–Henseleit solution (KHS, in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25, MgSO4 1.2 and glucose 10. The left atrium was cut and the major parts of the ventricles were removed to allow free efflux of the perfusate. The lung was inflated (4–5 ml/kg) and perfused with KHS (bubbled with 95% O2 and 5% CO2 at 37 °C) at a constant flow rate (0.03 ml/g/min) by a peristaltic pump (Gilson Model Miniplus 3, Villiers le Bel, France). Mean pulmonary arterial pressure (PAP) was measured via a pressure transducer attached to a side arm of the pulmonary arterial perfusion cannula. Changes in PAP were recorded on a computer-based data acquisition system (TDA96, Commat Ltd, Ankara, Turkey). Since pulmonary arterial perfusion flow and left atrial pressure were constant, a change in PAP reflected directly a change in pulmonary vascular resistance (PVR). Indomethacin (3 μM) was added to the perfusion solution to inhibit the cyclo-oxygenase pathway. In a control group, following a 30 min stabilization period, PAP was increased to 11.3 ± 1.1 mm Hg by an intra-arterial (i.a.) infusion of submaximal KCl (30 mM; by substituting KCl for an equal amount of NaCl in the perfusion solution) in order to create a state of elevated pulmonary vasoconstrictor tone (PVT). Intra-arterial
Fig. 1. Schematic diagram showing experimental protocol.
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bolus injections of histamine (2–200 μg/100 μl), acetylcholine (ACh) (2 μg/100 μl) and sodium nitroprusside (SNP) (5 μg/ 100 μl) were made once PVR had been elevated to a steady state. In the I/R group, after the 30 min of constant flow perfusion, the flow was stopped and the lungs were immediately immersed in cold KHS at +4 °C for 2 h (hypothermic ischemia). In the IP group, IP was performed by two successive cycles of 5 min ischemia, followed by 5 min reperfusion prior to 2 h hypothermic ischemia. After the ischemic intervals in I/R and IP groups, the lungs were reattached to the perfusion system, perfusion flow was slowly increased and the same flow rate as prior to protocols was achieved within 10 min. In IBPR perfused with KHS, basal perfusion pressure was 6.1 ± 0.3 mm Hg (n = 7). After the basal pressure was re-stabilized, PVT was actively increased by KCl (30 mM), and responses to histamine, ACh and SNP were evaluated (Fig. 1). Hypothermic ischemia did not alter KCl pressor responses (control: 11.3 ± 1.1 mm Hg; ischemia: 11.6 ± 1.3 mm Hg, p > 0.05, n = 6). IP-pretreatment did not change KCl-induced pressor responses over baseline (control: 11.3 ± 1.1 mm Hg, IP: 10.8 ± 1 mm Hg, p > 0.05, n = 6). KATP channel blocking agents and dimethysulfoxide (DMSO), vehicle for glybenclamide (GLB), did not alter the concentration of KCl necessary to produce similar increases in PAP as observed in the control experiments. The influence of blocking agents, 5-hydroxydecanoic acid (5-HD) (100 μM), GLB (3 μM), and HMR-1098 (10 μM) on the pulmonary vascular responses to IP was studied in separate groups of experiments. KATP channel blocking agents were administered to the perfusion system prior to IP protocol and given for 25 min. The concentrations of the drugs are expressed as the final concentrations in the perfusate. Responses to i.a. bolus injections of histamine, ACh and SNP were obtained after administration of the KATP channel blocking agents. The experimental protocols utilized in the present experiments are illustrated in schematic form in Fig. 1.
Results The effects of histamine, ACh and SNP on PAP were investigated in the IBPR under conditions of elevated PVT and results from these experiments are illustrated in Figs. 2 and 3. Since pulmonary arterial flow and left atrial pressure were held constant, changes in PAP directly reflect changes in PVR. When PAP was actively increased in control group, bolus injections of histamine (2–200 μg) decreased PAP in a dose-dependent manner, and single bolus injection of ACh (2 μg) also decreased PAP. The pulmonary vasodilator responses to histamine and ACh following I/R were significantly reduced when compared to control responses obtained under similar conditions (Fig. 2A and B). In a parallel group of experiments under similar conditions of elevated PVT, the group receiving IP in the form of two successive cycles of 5 min ischemia (followed by 5 min reperfusion prior to the 2 h hypothermic ischemia), bolus injections of histamine (2–200 μg) and single bolus injection of ACh (2 μg) decreased PAP. The pulmonary vasodilator responses to histamine and ACh in the control and IP groups were not significantly different, suggesting IP prevented the inhibitory effects of I/R on the pulmonary vasodilator responses to histamine and ACh (Fig. 2A and B). The pulmonary vasodilator responses to SNP in all treatment groups were not
Drugs used Histamine, ACh, SNP, diazoxide, 5-HD, GLB, indomethacin, pinacidil, DMSO were obtained from Sigma (St. Louis, MO, USA). HMR-1098 was a gift from Aventis Pharma (Germany). Commercially available heparin (Nevparin, Mustafa Nevzat, Istanbul), and thiopental sodium (Pental sodium, Ibrahim Ethem, Istanbul) were used. GLB was dissolved in DMSO solution (0.04%, v/v). Indomethacin was dissolved in equal amount of Na2CO3. The other blocking agents were dissolved in normal saline. Statistical analysis The decreases in PAP by histamine, ACh and SNP were expressed as a percentage of baseline PAP under conditions of elevated PVT. Results are expressed as mean ± S.E.M; n represents the number of animals. Data were analyzed by analysis of variance (ANOVA) followed by Student–Newman– Keuls post hoc test. A P-value of less than 0.05 was considered statistically significant.
Fig. 2. The pulmonary vasodilator responses to bolus injections of histamine (2– 200 μg) (A) and a single bolus injection of ACh (2 μg) (B) in control, IP, I/R groups of IBPR under conditions of elevated pulmonary vascular tone. Each value represents the mean ± S.E.M of five or seven experiments. ⁎Significantly different from the control group, p < 0.05.
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Fig. 3. The pulmonary vasodilator responses to a single bolus injection of SNP (5 μg) in control, IP, I/R groups of IBPR under conditions of elevated pulmonary vascular tone. Each value represents the mean ± S.E.M of four or six experiments.
significantly different when compared to control value (Fig. 3). In order to determine the contribution of KATP channels in influencing the effects of IP on endothelium-dependent pulmonary vasodilator responses, an additional series of experiments were performed in the IBPR with IP using 5-HD, GLB and HMR-1098 under conditions of elevated PVT. The results of these studies are illustrated in Fig. 4. When compared to the pulmonary vasodilator responses to histamine (2–200 μg) in control experiments with IP, the decreases in PAP with IP in response to bolus injections of histamine in the presence of 5HD (100 μM), a mitochondrial KATP channel blocker, were not
Fig. 5. Influence of 5-HD (100 μM) (A) and GLB (3 μM) (B) on the pulmonary vascular response to i.a. administration of histamine under conditions of elevated PVT. Each value represents the mean ± S.E.M of four experiments.
Fig. 4. The effects of 5-HD (100 μM) (A), glybenclamide (GLB, 3 μM) (B), HMR-1098 (10 μM) (C) and DMSO (0.04%, v/v) (D) on pulmonary vascular responses to histamine (2–200 μg) in experiments with IP in the IBPR under conditions of elevated pulmonary vascular tone. Each value represents the mean ± S.E.M of two (for DMSO), four to six experiments. ⁎Significantly different from the IP group, p < 0.05.
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significantly altered (p > 0.05; Fig. 4A). However, following administration of GLB (3 μM), a nonselective KATP channel blocker or HMR-1098 (10 μM), a selective sarcolemmal KATP channel blocker, the pulmonary vasodilator responses to bolus injections of histamine with IP were significantly inhibited (p < 0.05, Fig. 4 B and C). DMSO (0.04%, v/v), the vehicle for GLB, did not alter pulmonary vasodilator responses (p > 0.05, Fig. 4D). In order to determine the contribution of KATP channels to the pulmonary vasodilator response to histamine under baseline conditions without IP, additional experiments were performed and results from these studies are illustrated in Fig. 5. The pulmonary vasodilator response to bolus i.a. administration of histamine remained unchanged following administration of 5-HD or GLB (p > 0.05, Fig. 5A and B, respectively). The pulmonary vasodilator response to SNP with IP was not altered by preadministration of 5-HD (100 μM), GLB (3 μM), HMR-1098 (10 μM) or DMSO (0.04%, v/v) (p > 0.05, Fig. 6). In order to confirm that the KATP channel blockers studied acted in a selective manner, additional experiments were performed using pinacidil. The pulmonary vasodilator response to a single dose of pinacidil (5 μg), an activator of sarcolemmal KATP channels, was significantly inhibited in the presence of inhibitors of sarcolemmal KATP channels, GLB (3 μM) or HMR-1098 (10 μM), but not by an inhibitor of mitochondrial KATP channels, 5-HD (100 μM) (p < 0.05, Fig. 7). Discussion The results of the present study demonstrate that I/R promotes vascular dysfunction in the rat lung by inhibiting the pulmonary vasodilator response to histamine and ACh. The pulmonary vascular response to SNP, an endothelium-independent dilator, was unaffected by I/R further suggesting that functional vascular injury from exposure to I/R may be restricted to the endothelial cell layer. IP in lungs exposed to I/R prevented this endothelial vascular impairment. Since administration of GLB or HMR-1098 abolished this protective
Fig. 6. The effects of 5-HD (100 μM), GLB (3 μM), HMR-1098 (10 μM) and DMSO (0.04%, v/v) on the pulmonary vascular response to a single bolus injection of SNP (5 μg) in experiments with IP under conditions of elevated PVT. Each value represents the mean ± S.E.M of two (for DMSO), four to six experiments.
Fig. 7. The effects of 5-HD (100 μM), GLB (3 μM) and HMR-1098 (10 μM) on the pulmonary vasodilator response to a bolus injection of pinacidil (5 μg) under conditions of elevated PVT. Each value represents the mean ± S.E.M of four or five experiments. ⁎Significantly different from the control group, p < 0.05.
effect of IP, these results suggest KATP channels contribute, in part, to the protective properties of preconditioning on endothelial vascular function. The lack of a significant inhibitory effect of 5-HD on endothelium-dependent vascular responses during IP also suggests that IP-induced endothelial protection occurs through sarcolemmal KATP rather than mitochondrial KATP channels in the pulmonary vascular bed of the rat. Data have accumulated demonstrating that pulmonary vasodilator responses to ACh and histamine, unlike those to SNP, are blocked by L-NAME, a non-selective NO synthase inhibitor (Chen and Suzuki, 1989a). Pulmonary vasodilation to histamine is predominantly mediated by endothelialderived nitric oxide (NO), however, a role for endotheliumderived hyperpolarizing factor (EDHF) in mediating vasorelaxant responses in rat pulmonary arterial conductance segments has also been established (Chen and Suzuki, 1989a,b). In order to determine the effects of I/R and IP on pulmonary vascular responses that are specifically dependent on endothelial release of NO and not EDHF, the component dependent on EDHF was reduced by increasing PVT with KCl. Based on the Nernst Equation, depolarization with KCl would be expected to reduce the capacity for vasodilation dependent on cellular potassium flux. Since histamine does not evoke full hyperpolarization in a depolarized environment (Chen and Suzuki, 1989b; Quignard et al., 1999), the contribution of EDHF to the pulmonary vasodilator response to histamine would be minimal. The increase in PVT by KCl, and therefore the level of cellular depolarization, used in present study was specifically chosen to avoid complete elimination of the pulmonary vascular response to pinacidil that depends on cellular hyperpolarization. In order to confirm the selectivity of the inhibitors of KATP channels used in the present study, and thus determine the site of action of IP in modulating endothelium-dependent vascular responses, preservation of a limited component of the pulmonary vasodilator response to pinacidil was necessary. The present data confirm the selectivity of K ATP channel agonist and antagonists used in the present study was retained despite raising PVT with KCl. As a result, the blocking agents used
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in the present experiments were efficacious in teasing out the differential contribution of subtypes of KATP channels to I/R injury and IP. Histamine dilated the pulmonary vascular bed in all groups studied; however, pulmonary vasodilator responses to histamine were only reduced following I/R. By using different endothelium-dependent agonists to evoke pulmonary vasodilation in the present study, the present authors speculate that I/R impairs endothelial vascular function indiscriminately and thus promotes deleterious effects on pulmonary vascular endothelial cells in a non-specific manner. Since the inhibitory effect of I/R on pulmonary vasodilator responses to both ACh and histamine acting on muscarinic and histaminergic receptors, respectively, was prevented by IP, the present data suggest the ability of IP to protect endothelial vascular function occurs independent of receptor population and may occur at a site similar to I/Rmediated endothelial dysfunction. Therefore, the present results using histamine and ACh was taken as evidence that IP attenuates endothelial dysfunction in the pulmonary vascular bed of the rat. In the present study the pulmonary vasodilator response to SNP was not altered by any treatments, demonstrating that endothelium-dependent, but not endothelium-independent, injury occurred in response to I/R. Results of the present study are consistent with previous work demonstrating that selective endothelial dysfunction occurs in the presence of endothelial cell injury from I/R (DeFily and Chilian, 1993; Richard et al., 1994; Kaeffer et al., 1996). The protective actions of IP-pretreatment on endothelial vascular function were prevented by GLB as well as HMR1098, in the pulmonary vascular bed. In contrast, the protective actions of IP-pretreatment on endothelial vascular function was not altered by 5-HD. The present data suggest that the activation of sarcolemmal KATP channels, but not mitochondrial KATP channels, mediates the endothelial cytoprotective properties of IP. Biochemical pathways involved in anti-ischemic actions mediated by activation of sarcolemmal KATP channels have been proposed. KATP channel activation produces endothelial hyperpolarization and through a change in membrane potential, induces endothelial Ca2+ entry (Adams et al., 1989; Janigro et al., 1993) which will augment NO production (Luckhoff and Busse, 1990). The contribution of NO in IP has been well documented (Parratt and Vegh, 1994; Kuo and Chancellor, 1995; Nandagopal et al., 2001). An alternative interpretation includes mitochondrial potassium influx and cellular potassium efflux (opening of KATP channels) that triggers the generation of reactive oxygen species (ROS) during IP (Krenz et al., 2002). ROS are postulated to be downstream mediators of protein kinase C activation in promoting endothelial protection (Laude et al., 2002). A predominant role for mitochondrial KATP in IP during I/R injury has been also well documented (Broadhead et al., 2004; Pain et al., 2000; Garlid et al., 1997). Taken together, work by others suggests several mechanisms for IPinduced protection (Krenz et al., 2002; Holmuhamedov et al., 1999). Although pulmonary vascular responses to histamine tended to be decreased by 5-HD but not to a significant level, the present data may provide limited evidence to suggest that
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selective inhibition of mitochondrial KATP channels during IP may also alter endothelium-dependent vasodilator responses in the pulmonary vascular bed of the rat. The pulmonary vasodilator response to histamine under baseline conditions was not altered by 5-HD and GLB whereas following IP, the same degree of pulmonary vasodilation in response to histamine was inhibited by GLB and HMR-1098 but not by 5-HD. It is possible that IP alters the mediation of the pulmonary vasodilator response to histamine by becoming dependent on activation of sarcolemmal KATP channels and thereby offers protection against I/R and the development of endothelial vascular dysfunction. The present data suggest that IP triggers rather than preserves vasodilation mediated by activation of sarcolemmal KATP channels and represents a novel regulatory mechanism of endothelial cytoprotection during I/R in the pulmonary vascular bed. In summary, the present data demonstrate that I/R selectively impair endothelium-dependent pulmonary vasodilator responses. The non-selective impairment of endotheliumdependent vasodilator responses in the rat lung is prevented by IP. The present data further suggest that activation of sarcolemmal KATP channels mediates the protective actions of IP on endothelial-dependent vasodilation in the rat lung during I/R injury. It is possible that I/R and IP may act on similar KATP channels; however, further studies are needed to clarify these opposing mechanisms on endothelial function. Acknowledgements This work was supported by a grant from Hacettepe University (# 00.01.201.002). This paper was presented in part at FASEB Experimental Biology, 2004, Washington D.C., USA. The authors acknowledge assistance by the Ark-LA-Tex Research Group. References Adams, D.J., Barakeh, J., Laskey, R., Van Breemen, C., 1989. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB Journal 3 (12), 2389–2400. Bernardo, N.L., D'Angelo, M., Okubo, S., Joy, A., Kukreja, R.C., 1999. Delayed ischemic preconditioning is mediated by opening of ATP-sensitive potassium channels in the rabbit heart. American Journal of Physiology 276, H1323–H1330. Broadhead, M.W., Kharbanda, R.K., Peters, M.J., MacAllister, R.J., 2004. KATP channel activation induces ischemic preconditioning of the endothelium in humans in vivo. Circulation 110 (15), 2077–2082. Chatterjee, S., Al-Mehdi, A.B., Levitan, I., Stevens, T., Fisher, A.B., 2003. Shear stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells. American Journal of Physiology. Cell Physiology 285 (4), C959–C967. Chen, G., Suzuki, H., 1989a. Direct and indirect actions of acetylcholine and histamine on intrapulmonary artery and vein muscles of the rat. Japanese Journal of Physiology 39, 51–65. Chen, G., Suzuki, H., 1989b. Some electrical properties of the endotheliumdependent hyperpolarization recorded from rat arterial smooth muscle cells. Journal of Physiology (London) 410, 91–106. Cleveland Jr., J.C., Wollmering, M.M., Meldrum, D.R., Rowland, R.T., Rehring, T.F., Sheridan, B.C., Harken, A.H., Banerjee, A., 1996. Ischemic preconditioning in human and rat ventricle. American Journal of Physiology 271, H1786–H1794.
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Preconditioning modulates pulmonary endothelial dysfunction following ischemia-reperfusion injury in the rat lung: Role of potassium channels H. Burak Kandilci a , Bülent Gümüşel a,⁎, A. Tuncay Demiryürek b , Howard Lippton c a
Department of Pharmacology, Hacettepe University, Faculty of Pharmacy, 06100, Sıhhiye, Ankara, Turkey b Department of Pharmacology, University of Gaziantep, Faculty of Medicine, 27310, Gaziantep, Turkey c Pneumosite LLC, Shreveport, Louisiana, USA Received 22 March 2006; accepted 12 July 2006
Abstract Ischemic preconditioning (IP) may protect the lung from ischemia-reperfusion (I/R) injury following cardiopulmonary by-pass and lung or heart transplantation. The present study was undertaken to investigate the role of ATP-dependent potassium channels (KATP) in IP in the isolated buffer-perfused rat lung (IBPR) under conditions of elevated pulmonary vasoconstrictor tone (PVT). Since pulmonary arterial perfusion flow and left atrial pressure were constant, changes in pulmonary arterial pressure (PAP) directly reflect changes in pulmonary vascular resistance (PVR). When compared to control value, the pulmonary vasodilator responses to histamine and acetylcholine (ACh) following 2 h of hypothermic ischemia were significantly attenuated, whereas the pulmonary vasodilator response to sodium nitroprusside (SNP) was not altered. IP in the form of two cycles of 5 min of ischemia and reperfusion applied prior to the two-hour interval of ischemia, prevented the decrease in the pulmonary vasodilator responses to histamine and ACh. Pretreatment with glybenclamide (GLB) or HMR-1098, but not 5-hydroxydecanoic acid (5-HD), prior to IP abolished the protective effect of IP. In contrast, GLB or 5-HD did not significantly alter the pulmonary vasodilator response to histamine without IP pretreatment. The present data demonstrate that IP prevents impairment of endothelium-dependent vasodilator responses in the rat pulmonary vascular bed. The present data further suggest that IP may alter the mediation of the pulmonary vasodilator response to histamine and thereby trigger a mechanism dependent on activation of sarcolemmal, and not mitochondrial, KATP channels to preserve endothelialdependent vasodilator responses and protect against I/R injury in the lung. © 2006 Elsevier Inc. All rights reserved. Keywords: Rat lung vascular bed; Ischemic preconditioning; ATP-dependent potassium channels; Sarcolemmal KATP channels; Mitochondrial KATP channels; Ischemia/reperfusion; Pulmonary vasodilation
Introduction Many studies in the heart have demonstrated that a short period of ischemia-reperfusion (I/R) protects against harmful effects of subsequent prolonged ischemia; this endogenous mechanism of protection has been termed ischemic preconditioning (IP) (Murry et al., 1986; Cleveland et al., 1996). Recent studies have reported IP increases the tolerance of lung, similar to the heart, against I/R injury (Neely and Keith, 1995; Featherstone et al., 2000; Friedrich et al., 2001; Soncul et al., 1999). I/R injury is not limited to myocytes since I/R injury ⁎ Corresponding author. Tel.: +90 312 3052081; fax: +90 312 3114777. E-mail address: [email protected] (B. Gümüşel). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.07.011
produces structural changes in coronary vascular endothelium resulting in endothelial damage in the myocardium (Richard et al., 1995) and induces endothelial dysfunction with increases in permeability and vascular resistance in pulmonary blood vessels (Davenpeck et al., 1993). Although the mechanism of action of IP is unclear, activation of KATP channels has been reported to contribute to the actions of IP (Mulleneim et al., 2001). Two types of KATP channels exist in the myocardium: mitochondrial and sarcolemmal KATP channels (Gross and Fryer, 1999). KATP channels are also present on both vascular smooth muscle and vascular endothelium (Standen, 2003; Chatterjee et al., 2003). The role of the mitochondrial KATP and sarcolemmal KATP channels in IP has been challenged (Gross and Fryer, 1999; Light, 1999).
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Although data have accumulated suggesting a role for mitochondrial KATP channels in cardiac IP (Garlid et al., 1997; Bernardo et al., 1999; Yellon and Downey, 2003), evidence is now available suggesting that sarcolemmal KATP channels activation may also contribute to the protective effect to IP in the heart (Toyoda et al., 2000; Light et al., 2001). The effects of IP on the pulmonary vascular bed and the possible protective role of KATP channels in I/R injury have not been investigated. The present study was undertaken to determine the contribution of subtypes of KATP channels in mediating the effects of IP on I/R-mediated injury in the IBPR. Materials and methods The experiment was performed in compliance with the “Principles of Laboratory Animal Care” formulated by the National Institutes of Health (National Institutes of Health publication no. 96 to 23, revised 1996). The experiment and animal care protocol was approved by the Ethics Committee for animal care, established in our institute (# 2001/25-4). Isolated buffer perfused rat lung (IBPR) Male Wistar rats (200–300 g) were anesthetized with thiopental (30 mg/kg, i.p.). After tracheal intubation, the chest
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was opened and heparin (200 IU) was injected into the right ventricle. The main pulmonary artery was cannulated with a stainless steel cannula via the right ventricle and the vasculature was flushed with Krebs–Henseleit solution (KHS, in mM): NaCl 118, KCl 4.7, CaCl2 2.5, KH2PO4 1.2, NaHCO3 25, MgSO4 1.2 and glucose 10. The left atrium was cut and the major parts of the ventricles were removed to allow free efflux of the perfusate. The lung was inflated (4–5 ml/kg) and perfused with KHS (bubbled with 95% O2 and 5% CO2 at 37 °C) at a constant flow rate (0.03 ml/g/min) by a peristaltic pump (Gilson Model Miniplus 3, Villiers le Bel, France). Mean pulmonary arterial pressure (PAP) was measured via a pressure transducer attached to a side arm of the pulmonary arterial perfusion cannula. Changes in PAP were recorded on a computer-based data acquisition system (TDA96, Commat Ltd, Ankara, Turkey). Since pulmonary arterial perfusion flow and left atrial pressure were constant, a change in PAP reflected directly a change in pulmonary vascular resistance (PVR). Indomethacin (3 μM) was added to the perfusion solution to inhibit the cyclo-oxygenase pathway. In a control group, following a 30 min stabilization period, PAP was increased to 11.3 ± 1.1 mm Hg by an intra-arterial (i.a.) infusion of submaximal KCl (30 mM; by substituting KCl for an equal amount of NaCl in the perfusion solution) in order to create a state of elevated pulmonary vasoconstrictor tone (PVT). Intra-arterial
Fig. 1. Schematic diagram showing experimental protocol.
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bolus injections of histamine (2–200 μg/100 μl), acetylcholine (ACh) (2 μg/100 μl) and sodium nitroprusside (SNP) (5 μg/ 100 μl) were made once PVR had been elevated to a steady state. In the I/R group, after the 30 min of constant flow perfusion, the flow was stopped and the lungs were immediately immersed in cold KHS at +4 °C for 2 h (hypothermic ischemia). In the IP group, IP was performed by two successive cycles of 5 min ischemia, followed by 5 min reperfusion prior to 2 h hypothermic ischemia. After the ischemic intervals in I/R and IP groups, the lungs were reattached to the perfusion system, perfusion flow was slowly increased and the same flow rate as prior to protocols was achieved within 10 min. In IBPR perfused with KHS, basal perfusion pressure was 6.1 ± 0.3 mm Hg (n = 7). After the basal pressure was re-stabilized, PVT was actively increased by KCl (30 mM), and responses to histamine, ACh and SNP were evaluated (Fig. 1). Hypothermic ischemia did not alter KCl pressor responses (control: 11.3 ± 1.1 mm Hg; ischemia: 11.6 ± 1.3 mm Hg, p > 0.05, n = 6). IP-pretreatment did not change KCl-induced pressor responses over baseline (control: 11.3 ± 1.1 mm Hg, IP: 10.8 ± 1 mm Hg, p > 0.05, n = 6). KATP channel blocking agents and dimethysulfoxide (DMSO), vehicle for glybenclamide (GLB), did not alter the concentration of KCl necessary to produce similar increases in PAP as observed in the control experiments. The influence of blocking agents, 5-hydroxydecanoic acid (5-HD) (100 μM), GLB (3 μM), and HMR-1098 (10 μM) on the pulmonary vascular responses to IP was studied in separate groups of experiments. KATP channel blocking agents were administered to the perfusion system prior to IP protocol and given for 25 min. The concentrations of the drugs are expressed as the final concentrations in the perfusate. Responses to i.a. bolus injections of histamine, ACh and SNP were obtained after administration of the KATP channel blocking agents. The experimental protocols utilized in the present experiments are illustrated in schematic form in Fig. 1.
Results The effects of histamine, ACh and SNP on PAP were investigated in the IBPR under conditions of elevated PVT and results from these experiments are illustrated in Figs. 2 and 3. Since pulmonary arterial flow and left atrial pressure were held constant, changes in PAP directly reflect changes in PVR. When PAP was actively increased in control group, bolus injections of histamine (2–200 μg) decreased PAP in a dose-dependent manner, and single bolus injection of ACh (2 μg) also decreased PAP. The pulmonary vasodilator responses to histamine and ACh following I/R were significantly reduced when compared to control responses obtained under similar conditions (Fig. 2A and B). In a parallel group of experiments under similar conditions of elevated PVT, the group receiving IP in the form of two successive cycles of 5 min ischemia (followed by 5 min reperfusion prior to the 2 h hypothermic ischemia), bolus injections of histamine (2–200 μg) and single bolus injection of ACh (2 μg) decreased PAP. The pulmonary vasodilator responses to histamine and ACh in the control and IP groups were not significantly different, suggesting IP prevented the inhibitory effects of I/R on the pulmonary vasodilator responses to histamine and ACh (Fig. 2A and B). The pulmonary vasodilator responses to SNP in all treatment groups were not
Drugs used Histamine, ACh, SNP, diazoxide, 5-HD, GLB, indomethacin, pinacidil, DMSO were obtained from Sigma (St. Louis, MO, USA). HMR-1098 was a gift from Aventis Pharma (Germany). Commercially available heparin (Nevparin, Mustafa Nevzat, Istanbul), and thiopental sodium (Pental sodium, Ibrahim Ethem, Istanbul) were used. GLB was dissolved in DMSO solution (0.04%, v/v). Indomethacin was dissolved in equal amount of Na2CO3. The other blocking agents were dissolved in normal saline. Statistical analysis The decreases in PAP by histamine, ACh and SNP were expressed as a percentage of baseline PAP under conditions of elevated PVT. Results are expressed as mean ± S.E.M; n represents the number of animals. Data were analyzed by analysis of variance (ANOVA) followed by Student–Newman– Keuls post hoc test. A P-value of less than 0.05 was considered statistically significant.
Fig. 2. The pulmonary vasodilator responses to bolus injections of histamine (2– 200 μg) (A) and a single bolus injection of ACh (2 μg) (B) in control, IP, I/R groups of IBPR under conditions of elevated pulmonary vascular tone. Each value represents the mean ± S.E.M of five or seven experiments. ⁎Significantly different from the control group, p < 0.05.
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Fig. 3. The pulmonary vasodilator responses to a single bolus injection of SNP (5 μg) in control, IP, I/R groups of IBPR under conditions of elevated pulmonary vascular tone. Each value represents the mean ± S.E.M of four or six experiments.
significantly different when compared to control value (Fig. 3). In order to determine the contribution of KATP channels in influencing the effects of IP on endothelium-dependent pulmonary vasodilator responses, an additional series of experiments were performed in the IBPR with IP using 5-HD, GLB and HMR-1098 under conditions of elevated PVT. The results of these studies are illustrated in Fig. 4. When compared to the pulmonary vasodilator responses to histamine (2–200 μg) in control experiments with IP, the decreases in PAP with IP in response to bolus injections of histamine in the presence of 5HD (100 μM), a mitochondrial KATP channel blocker, were not
Fig. 5. Influence of 5-HD (100 μM) (A) and GLB (3 μM) (B) on the pulmonary vascular response to i.a. administration of histamine under conditions of elevated PVT. Each value represents the mean ± S.E.M of four experiments.
Fig. 4. The effects of 5-HD (100 μM) (A), glybenclamide (GLB, 3 μM) (B), HMR-1098 (10 μM) (C) and DMSO (0.04%, v/v) (D) on pulmonary vascular responses to histamine (2–200 μg) in experiments with IP in the IBPR under conditions of elevated pulmonary vascular tone. Each value represents the mean ± S.E.M of two (for DMSO), four to six experiments. ⁎Significantly different from the IP group, p < 0.05.
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significantly altered (p > 0.05; Fig. 4A). However, following administration of GLB (3 μM), a nonselective KATP channel blocker or HMR-1098 (10 μM), a selective sarcolemmal KATP channel blocker, the pulmonary vasodilator responses to bolus injections of histamine with IP were significantly inhibited (p < 0.05, Fig. 4 B and C). DMSO (0.04%, v/v), the vehicle for GLB, did not alter pulmonary vasodilator responses (p > 0.05, Fig. 4D). In order to determine the contribution of KATP channels to the pulmonary vasodilator response to histamine under baseline conditions without IP, additional experiments were performed and results from these studies are illustrated in Fig. 5. The pulmonary vasodilator response to bolus i.a. administration of histamine remained unchanged following administration of 5-HD or GLB (p > 0.05, Fig. 5A and B, respectively). The pulmonary vasodilator response to SNP with IP was not altered by preadministration of 5-HD (100 μM), GLB (3 μM), HMR-1098 (10 μM) or DMSO (0.04%, v/v) (p > 0.05, Fig. 6). In order to confirm that the KATP channel blockers studied acted in a selective manner, additional experiments were performed using pinacidil. The pulmonary vasodilator response to a single dose of pinacidil (5 μg), an activator of sarcolemmal KATP channels, was significantly inhibited in the presence of inhibitors of sarcolemmal KATP channels, GLB (3 μM) or HMR-1098 (10 μM), but not by an inhibitor of mitochondrial KATP channels, 5-HD (100 μM) (p < 0.05, Fig. 7). Discussion The results of the present study demonstrate that I/R promotes vascular dysfunction in the rat lung by inhibiting the pulmonary vasodilator response to histamine and ACh. The pulmonary vascular response to SNP, an endothelium-independent dilator, was unaffected by I/R further suggesting that functional vascular injury from exposure to I/R may be restricted to the endothelial cell layer. IP in lungs exposed to I/R prevented this endothelial vascular impairment. Since administration of GLB or HMR-1098 abolished this protective
Fig. 6. The effects of 5-HD (100 μM), GLB (3 μM), HMR-1098 (10 μM) and DMSO (0.04%, v/v) on the pulmonary vascular response to a single bolus injection of SNP (5 μg) in experiments with IP under conditions of elevated PVT. Each value represents the mean ± S.E.M of two (for DMSO), four to six experiments.
Fig. 7. The effects of 5-HD (100 μM), GLB (3 μM) and HMR-1098 (10 μM) on the pulmonary vasodilator response to a bolus injection of pinacidil (5 μg) under conditions of elevated PVT. Each value represents the mean ± S.E.M of four or five experiments. ⁎Significantly different from the control group, p < 0.05.
effect of IP, these results suggest KATP channels contribute, in part, to the protective properties of preconditioning on endothelial vascular function. The lack of a significant inhibitory effect of 5-HD on endothelium-dependent vascular responses during IP also suggests that IP-induced endothelial protection occurs through sarcolemmal KATP rather than mitochondrial KATP channels in the pulmonary vascular bed of the rat. Data have accumulated demonstrating that pulmonary vasodilator responses to ACh and histamine, unlike those to SNP, are blocked by L-NAME, a non-selective NO synthase inhibitor (Chen and Suzuki, 1989a). Pulmonary vasodilation to histamine is predominantly mediated by endothelialderived nitric oxide (NO), however, a role for endotheliumderived hyperpolarizing factor (EDHF) in mediating vasorelaxant responses in rat pulmonary arterial conductance segments has also been established (Chen and Suzuki, 1989a,b). In order to determine the effects of I/R and IP on pulmonary vascular responses that are specifically dependent on endothelial release of NO and not EDHF, the component dependent on EDHF was reduced by increasing PVT with KCl. Based on the Nernst Equation, depolarization with KCl would be expected to reduce the capacity for vasodilation dependent on cellular potassium flux. Since histamine does not evoke full hyperpolarization in a depolarized environment (Chen and Suzuki, 1989b; Quignard et al., 1999), the contribution of EDHF to the pulmonary vasodilator response to histamine would be minimal. The increase in PVT by KCl, and therefore the level of cellular depolarization, used in present study was specifically chosen to avoid complete elimination of the pulmonary vascular response to pinacidil that depends on cellular hyperpolarization. In order to confirm the selectivity of the inhibitors of KATP channels used in the present study, and thus determine the site of action of IP in modulating endothelium-dependent vascular responses, preservation of a limited component of the pulmonary vasodilator response to pinacidil was necessary. The present data confirm the selectivity of K ATP channel agonist and antagonists used in the present study was retained despite raising PVT with KCl. As a result, the blocking agents used
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in the present experiments were efficacious in teasing out the differential contribution of subtypes of KATP channels to I/R injury and IP. Histamine dilated the pulmonary vascular bed in all groups studied; however, pulmonary vasodilator responses to histamine were only reduced following I/R. By using different endothelium-dependent agonists to evoke pulmonary vasodilation in the present study, the present authors speculate that I/R impairs endothelial vascular function indiscriminately and thus promotes deleterious effects on pulmonary vascular endothelial cells in a non-specific manner. Since the inhibitory effect of I/R on pulmonary vasodilator responses to both ACh and histamine acting on muscarinic and histaminergic receptors, respectively, was prevented by IP, the present data suggest the ability of IP to protect endothelial vascular function occurs independent of receptor population and may occur at a site similar to I/Rmediated endothelial dysfunction. Therefore, the present results using histamine and ACh was taken as evidence that IP attenuates endothelial dysfunction in the pulmonary vascular bed of the rat. In the present study the pulmonary vasodilator response to SNP was not altered by any treatments, demonstrating that endothelium-dependent, but not endothelium-independent, injury occurred in response to I/R. Results of the present study are consistent with previous work demonstrating that selective endothelial dysfunction occurs in the presence of endothelial cell injury from I/R (DeFily and Chilian, 1993; Richard et al., 1994; Kaeffer et al., 1996). The protective actions of IP-pretreatment on endothelial vascular function were prevented by GLB as well as HMR1098, in the pulmonary vascular bed. In contrast, the protective actions of IP-pretreatment on endothelial vascular function was not altered by 5-HD. The present data suggest that the activation of sarcolemmal KATP channels, but not mitochondrial KATP channels, mediates the endothelial cytoprotective properties of IP. Biochemical pathways involved in anti-ischemic actions mediated by activation of sarcolemmal KATP channels have been proposed. KATP channel activation produces endothelial hyperpolarization and through a change in membrane potential, induces endothelial Ca2+ entry (Adams et al., 1989; Janigro et al., 1993) which will augment NO production (Luckhoff and Busse, 1990). The contribution of NO in IP has been well documented (Parratt and Vegh, 1994; Kuo and Chancellor, 1995; Nandagopal et al., 2001). An alternative interpretation includes mitochondrial potassium influx and cellular potassium efflux (opening of KATP channels) that triggers the generation of reactive oxygen species (ROS) during IP (Krenz et al., 2002). ROS are postulated to be downstream mediators of protein kinase C activation in promoting endothelial protection (Laude et al., 2002). A predominant role for mitochondrial KATP in IP during I/R injury has been also well documented (Broadhead et al., 2004; Pain et al., 2000; Garlid et al., 1997). Taken together, work by others suggests several mechanisms for IPinduced protection (Krenz et al., 2002; Holmuhamedov et al., 1999). Although pulmonary vascular responses to histamine tended to be decreased by 5-HD but not to a significant level, the present data may provide limited evidence to suggest that
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selective inhibition of mitochondrial KATP channels during IP may also alter endothelium-dependent vasodilator responses in the pulmonary vascular bed of the rat. The pulmonary vasodilator response to histamine under baseline conditions was not altered by 5-HD and GLB whereas following IP, the same degree of pulmonary vasodilation in response to histamine was inhibited by GLB and HMR-1098 but not by 5-HD. It is possible that IP alters the mediation of the pulmonary vasodilator response to histamine by becoming dependent on activation of sarcolemmal KATP channels and thereby offers protection against I/R and the development of endothelial vascular dysfunction. The present data suggest that IP triggers rather than preserves vasodilation mediated by activation of sarcolemmal KATP channels and represents a novel regulatory mechanism of endothelial cytoprotection during I/R in the pulmonary vascular bed. In summary, the present data demonstrate that I/R selectively impair endothelium-dependent pulmonary vasodilator responses. The non-selective impairment of endotheliumdependent vasodilator responses in the rat lung is prevented by IP. The present data further suggest that activation of sarcolemmal KATP channels mediates the protective actions of IP on endothelial-dependent vasodilation in the rat lung during I/R injury. It is possible that I/R and IP may act on similar KATP channels; however, further studies are needed to clarify these opposing mechanisms on endothelial function. Acknowledgements This work was supported by a grant from Hacettepe University (# 00.01.201.002). This paper was presented in part at FASEB Experimental Biology, 2004, Washington D.C., USA. The authors acknowledge assistance by the Ark-LA-Tex Research Group. References Adams, D.J., Barakeh, J., Laskey, R., Van Breemen, C., 1989. Ion channels and regulation of intracellular calcium in vascular endothelial cells. FASEB Journal 3 (12), 2389–2400. Bernardo, N.L., D'Angelo, M., Okubo, S., Joy, A., Kukreja, R.C., 1999. Delayed ischemic preconditioning is mediated by opening of ATP-sensitive potassium channels in the rabbit heart. American Journal of Physiology 276, H1323–H1330. Broadhead, M.W., Kharbanda, R.K., Peters, M.J., MacAllister, R.J., 2004. KATP channel activation induces ischemic preconditioning of the endothelium in humans in vivo. Circulation 110 (15), 2077–2082. Chatterjee, S., Al-Mehdi, A.B., Levitan, I., Stevens, T., Fisher, A.B., 2003. Shear stress increases expression of a KATP channel in rat and bovine pulmonary vascular endothelial cells. American Journal of Physiology. Cell Physiology 285 (4), C959–C967. Chen, G., Suzuki, H., 1989a. Direct and indirect actions of acetylcholine and histamine on intrapulmonary artery and vein muscles of the rat. Japanese Journal of Physiology 39, 51–65. Chen, G., Suzuki, H., 1989b. Some electrical properties of the endotheliumdependent hyperpolarization recorded from rat arterial smooth muscle cells. Journal of Physiology (London) 410, 91–106. Cleveland Jr., J.C., Wollmering, M.M., Meldrum, D.R., Rowland, R.T., Rehring, T.F., Sheridan, B.C., Harken, A.H., Banerjee, A., 1996. Ischemic preconditioning in human and rat ventricle. American Journal of Physiology 271, H1786–H1794.
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