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Urocortin Protects Against Ischemic Injury via a MAPK-dependent Pathway
BRIEF REVIEWS Urocortin Protects Against Ischemic Injury via a MAPK-dependent Pathway David S. Latchman*
Urocortin (UCN), is a peptide related to hypothalamic corticotrophin releasing hormone (CRF) and binds with high affinity to the CRF-R2 b receptor which is expressed in the heart. UCN prevents cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia as assayed by trypan blue exclusion. 3 9-OH end labeling of DNA (TUNEL), annexin-V and fluorescence activated cell sorting. The protective effect of UCN is dependent on the p42/p44 mitogen-activated protein kinase (MAPK)-pathway. UCN also reduces damage in isolated rat hearts ex vivo, subjected to regional ischemia/ reperfusion with the protective effect being observed when UCN is given either prior to ischemia or at the time of reperfusion after ischemia. Hence, UCN is a cardioprotective agent, which acts when given prior to ischemia or after ischemia at reperfusion. (Trends Cardiovasc Med 2001;11:167–169). © 2001, Elsevier Science Inc.
Urocortin (UCN) is a 40-amino-acid peptide related to the hypothalamic hormone corticotrophin releasing factor (CRF), the central mediator of the hypothalamicpituitary-adrenal axis and stress response in mammals (De Souza 1995, Plotsky 1991, Vale et al. 1981). UCN and CRF share 45% homology at the amino acid level and both are synthesized as precursors, which are subsequently processed to the mature biologically active peptides (Donaldson et al. 1996, Vaughan David S. Latchman is at the Institute of Child Health, University College London, London, United Kingdom. * Address correspondence to: David S. Latchman, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
TCM Vol. 11, No. 5, 2001
et al. 1995). Although UCN was originally identified in restricted areas of the brain, it has also been found in the placenta, lymphocytes and heart (Bamberger et al. 1998, Iino et al. 1999, Okosi et al. 1998, Petraglia et al. 1996). The CRF family of peptides binds to two types of CRF receptors, CRF-R1 and CRF-R2 (Chang et al. 1993, Kishimoto et al. 1995, Lovenberg et al. 1995). Interestingly, CRF-R2 binds UCN with a higher affinity than CRF, both in ligand binding studies (Vaughan et al. 1995) and as assayed by the effects of ligand on intracellular cAMP (Gottowik et al. 1997). In contrast, the R1 receptors show little ligand selectivity for UCN versus CRF. R2 receptors are the only type of CRF receptor found in the heart—the a form in man (Chen et al. 1993) and the b form in the rat (Stenzel et al. 1995). The CRF
family of peptides has been shown to stimulate adenylate cyclase activity in cardiac myocytes (Heldwein et al. 1996), and changes in CRF-R2 expression have been reported in the hearts of spontaneously hypertensive rats (Makino et al. 1998). The expression of CRF-R2 receptors in the heart suggests that UCN may have physiological cardiac effects. Indeed UCN, but not CRF, induced a dose-dependent increase in heart rate, cardiac output and coronary blood flow (Parkes et al. 1997). Moreover, we have previously demonstrated that the UCN mRNA and protein are expressed in the heart (Brar et al. 1999, Okosi et al. 1998). Interestingly, the expression of UCN has been shown to increase when cultured cardiac cells are exposed to stressful stimuli such as heat shock (Okosi et al. 1998) or ischemia (Brar et al. 1999). Moreover, the UCN produced is secreted into the culture medium (Brar et al. 1999). These findings raise the question as to the role that UCN may play in the response of cardiac cells to stressful stimuli. In initial experiments, we showed that conditioned medium from cardiac cells exposed to ischemia, was able to exert a protective effect against subsequent exposure to ischemia (Brar et al. 1999). This protective effect could be blocked by addition of a-helical CRF, which acts as an antagonist blocking all members of the CRF family (Brar et al. 1999). This indicated therefore that UCN released from cardiac cells in response to stress might have a protective effect. In agreement with this, UCN added to cardiac cells was able to protect them from subsequent exposure to simulated ischemia whereas CRF itself had a much less potent protective effect (Brar et al. 1999). Hence, UCN is indeed able to protect cardiac cells. Although these initial experiments were conducted by assaying total cell survival on the basis of the ability to exclude trypan blue (Brar et al. 1999), they
167
have subsequently been confirmed by using assays of programmed cell death/ apoptosis (Brar et al. 2000). To do this, we utilized both annexin V surface staining, which measures the translocation of phosphatidyl serine to the outer surface of the plasma membrane that occurs early in the apoptotic process, and TUNEL labeling, which measures the DNA degradation that occurs later in the apoptotic process. In both cases, clear protective effects of UCN were observed (Brar et al. 2000). A number of studies have implicated the mitogen-activated protein kinase (MAPK) pathway as a survival pathway in both cardiac cells (Adderley and Fitzgerald 1999, Maulik et al. 1996, Sheng et al. 1997) and other cell types (Allen et al. 1999, Cano and Mahadevan 1995, Cobb and Goldsmith 1995, Downward 1998, Schaeffer and Weber 1999, Xia et al. 1995). The extracellular signalrelated kinases (EPK), belonging to one sub-family of MAP kinases, are composed of 42- and 44-kDa kinases named p42-MAPK and p44-MAPK, respectively. Phosphorylation and activation of p42MAPK and p44-MAPK is mediated by MAP kinase kinases MEK1/2. CRF has been shown to activate MAPK p42/44 in Chinese hamster ovary (CHO) cell lines transfected with the CRF-R1 and CRF-R2 receptors (Rossant et al. 1999). Moreover, the cardioprotective effect of the cytokine cardiotrophin-1 (CT-1) is mediated through gp130 receptor activation of the MEK1/2 p42/44 MAPK signaling cascade (Sheng et al. 1997). We therefore tested the effect of the MEK1/2 inhibitor PD98059 (Dudley et al. 1995) on the protective effect of UCN. The protective effect of UCN when added prior to simulated ischemia, was inhibited by PD98059 as assayed by trypan blue exclusion, annexin V staining or TUNEL labeling (Brar et al. 2000). A similar inhibition of UCN-mediated protection was also observed when we transfected cardiac cells with constructs encoding dominant negative mutants of either p42 or p44 MAPK. Hence, the p42/p44 MAPK pathway, which is activated by MEK1/2, appears to play a key role in the protective effect of UCN given prior to simulated ischemia. Evidently, however, if UCN were to have clinical potential in the treatment of myocardial infarction, it would need to have a protective effect when given at
168
reoxygenation following an ischemic episode. To assess whether UCN retains cardioprotective effects when added after a period of simulated hypoxia/ ischemia, UCN was added to the cardiac myocytes at the point of reoxygenation. Cell death was measured in the untreated cells exposed to ischemia reoxygenation by trypan blue exclusion, TUNEL and annexin V. UCN protected the cardiac cells during reoxygenation in all these assays (Brar et al. 2000). To investigate whether UCN protection at reoxygenation was mediated by the MEK-1 p42/44 MAPK signaling cascade, PD98059 was administered for 10 min prior to the addition of UCN. The cardioprotective effect of UCN, when added at reoxygenation, was inhibited by PD98059 where cell death was assessed by trypan blue, annexin V, and TUNEL (Brar et al. 2000). A similar inhibition of the protective effect of UCN at reoxygenation was also observed in cells transfected with dominant negative mutants of the p42 or p44 MAPK enzymes confirming their role in the protective effect. Having shown protective effects of UCN on simulated hypoxic/ischemic reoxygenated cardiac myocytes in vitro, we examined whether UCN had similar protective actions on the intact ischemic/ reperfused isolated heart ex vivo on a Langendorff perfusion apparatus. In these experiments UCN protected the isolated rat heart from 35 min of regional ischemia/120 min reperfusion when added 30 min prior to the stress as the percentage of infarct size to zone at risk decreased from 45.67% to 20.58%. Moreover, the addition of UCN for 30 min at reperfusion after the 35-min ischemic period reduced the percentage of infarct size to zone at risk to 32.24% (Brar et al. 2000). No significant hemodynamic changes in parameters such as heart rate, left ventricular developed pressure and coronary flow with UCN were observed compared to the control, untreated hearts. This study therefore demonstrated that UCN results in a significant reduction in infarct size when administered either 30 min prior to ischemia or from the moment of reperfusion (Brar et al. 2000). It is clear therefore that UCN can protect cardiac cells both in vitro and in the intact perfused heart from the damaging effects of ischemia/reperfusion.
Moreover, it is effective when added prior to reperfusion as well as prior to the ischemic period, indicating that it might ultimately have a clinical application. Although we have demonstrated that the protective effect of UCN requires the p42/p44 MAPK pathway, the exact mechanism of its protective effect remains unclear. We have shown that the cardioprotective effects of UCN are inhibited by blocking new protein synthesis with cycloheximide, indicating that de novo protein synthesis induced by UCN is required for its effect. It has been shown in non-cardiac cells that activation of the p42/p44 MAPK pathway is associated with enhanced synthesis of FLIP (FLICElike inhibitor protein) an inhibitor of the caspase cascade, which is necessary for apoptosis (Yeh et al. 1998). However, we have also demonstrated that the 90-kDa heat shock protein (hsp90) is induced by UCN treatment and this effect is blocked by PD98059 suggesting that the induction of cardioprotective hsps may play a role in the cardioprotective effects of UCN. Evidently, a number of agents that can protect the heart against ischemia have been described and several of these, such as CT-1, also act via the p42/p44 MAPK pathway (Sheng et al. 1997). As none of these agents have yet proved useful clinically, one may ask what is the significance of identifying yet another cardioprotective agent that acts via the p42/p44 MAPK pathway. In considering this question, it is important to realize that the utility of many protective agents is limited via the other effects which they cause such as vasodilation or hypertrophy. Indeed CT1 was originally discovered on the basis of its hypertrophic effect and was only subsequently shown to be protective [for review see Latchman (1999)]. In the case of urocortin, we have already shown that UCN does not affect the hemodynamics of the isolated heart (Brar et al. 2000) and we are currently investigating whether it is a hypertrophic agent. The identification of novel cardioprotective agents such as UCN, which act via the p42/p44 MAPK pathway, may therefore be of use if they have less undesirable side effects than other agents which protect via this pathway. Similarly, by understanding the signaling pathways activated by agents such as UCN, it may be possible to stimulate the protective pathway while blocking the pathway meTCM Vol. 11, No. 5, 2001
diating any undesirable effects. In these ways, it may be possible to take advantage of the ability of UCN to protect against ischemia/reperfusion injury when administered either prior to ischemia or at reperfusion. • Acknowledgments I thank the members of my laboratory who have worked on the UCN project for their efforts and helpful discussion, in particular, Dr. B. Brar, Dr. K. Lawrence, Dr. J. Railson, Dr. D. Schulman and Dr. A. Stephanou. I am also grateful to Dr. Dick Knight for introducing me to UCN and ongoing collaboration and to Professor Derek Yellon for ongoing collaboration in many areas including UCN. The work of my laboratory on urocortin has been funded since its inception by the British Heart Foundation and I am most grateful for their ongoing support.
References Adderley SR, Fitzgerald DJ: 1999. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem 274:5038–5046. Allen MP, Zeng C, Schneider K, et al.: 1999. Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signalling promotes gonadotropin-releasing hormone neuronal survival via extracellular signalregulated kinase (ERK) and Akt. Mol Endocrinol 13:191–201. Bamberger CM, Wald M, Bamberger AM, et al.: 1998. Human lymphocytes produce urocortin, but not corticotropin-releasing hormone. J Clin Endocrinol Metab 83:708–711. Brar BK, Jonassen AK, Stephanou AS, et al.: 2000. Urocortin protects against ischemic and reperfusion injury via a MAP-kinase dependent pathway. J Biol Chem 275:8508– 8514. Brar BK, Stephanou A, Okosi A, et al.: 1999. CRH-like peptides protect cardiac myocytes from lethal ischaemic injury. Mol Cell Endocrinol 158:55–63. Cano E, Mahadevan LC: 1995. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 20:117–122. Chang CP, Pearse RV, O’Connell S, Rosenfeld MG: 1993. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187–1195.
TCM Vol. 11, No. 5, 2001
Chen R, Lewis KA, Perrin MH, Vale WW: 1993. Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 90:8967–8971.
Parkes DG, Vaughan J, Rivier J, et al.: 1997. Cardiac inotropic actions of urocortin in conscious sheep. Am J Physiol 272:H2115– H2122.
Cobb MH, Goldsmith EJ: 1995. How MAP kinases are regulated. J Biol Chem 270:14,843–14,846.
Petraglia F, Florio P, Gallo R, et al.: 1996. Human placenta and fetal membranes express human urocortin mRNA and peptide. J Clin Endocrinol Metab 81:3807– 3810.
De Souza EB: 1995. Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology 20:789–819. Donaldson CJ, Sutton SW, Perrin MH, et al.: 1996. Cloning and characterisation of human urocortin. Endocrinology 137:2167– 2170. Downward J: 1998. Ras signalling and apoptosis. Curr Opin Gen Devel 8:49–54. Dudley DT, Pang L, Decker SJ, et al.: 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689.
Plotsky PM: 1991. Hypothalamic secretion of somatostatin and growth hormone-releasing factor into the hypophysial-portal circulation is reduced in streptozotocin diabetic male rats. Neuroendocrinology 53:433–438. Rossant CJ, Pinnock RD, Hughes J, et al.: 1999. Corticotropin-releasing factor type 1 and type 2alpha receptors regulate phosphorylation of calcium/cyclic adenosine 39,59-monophosphate response elementbinding protein and activation of p42-p44 mitogen-activated protein kinase. Endocrinology 140:1525–1536.
Gottowik J, Goetschy V, Henriot S, et al.: 1997. Labelling of CRF1 and CRF2 receptors using the novel radioligand, [3H]urocortin. Neuropharmacology 36:1439– 1446.
Schaeffer HJ, Weber MJ: 1999. Mitogen-activated protein kinases: specific messages from ubiquitous messengers. Mol Cell Biol 19:2435–2444.
Heldwein KA, Redick DL, Rittenberg MB, et al.: 1996. Corticotropin-releasing hormone receptor expression and functional coupling in neonatal cardiac myocytes and AT-1 cells. Endocrinology 137:3631–3639.
Sheng Z, Knowlton K, Chen J, et al.: 1997. Cardiotrophin 1 (CT-1) inhibition of cardiac myocyte apoptosis via a mitogen activated protein kinase dependent pathway. J Biol Chem 272:5783–5791.
Iino K, Sasano H, Oki Y, et al.: 1999. Urocortin expression in the human central nervous system. Clin Endocrinol 50:107–114.
Stenzel P, Kesterson R, Yeung W, et al.: 1995. Identification of a novel murine receptor for corticotropin-releasing hormone expressed in the heart. Mol Endocrinol 5:637–645.
Kishimoto T, Pearse RV, Lin CR, Rosenfeld MG: 1995. A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle. Proc Natl Acad Sci USA 92:1108–1112. Latchman DS: 1999. Cardiotrophin-1 (CT-1): a novel hypertrophic and cardioprotective agent. Int J Exp Pathol 80:189–196. Lovenberg TW, Liaw CW, Grigoriadis DE, et al.: 1995. Cloning and characterisation of a functionally distinct corticotropinreleasing factor receptor subtype from rat brain. Proc Natl Acad Sci USA 92:836–840. Makino S, Asaba K, Takao T, Hashimoto K: 1998. Type 2 corticotropin-releasing hormone receptor mRNA expression in the heart in hypertensive rats. Life Sci 62:515– 523. Maulik N, Watanabe M, Zu YL, et al.: 1996. Ischemic preconditioning triggers the activation of MAP kinases and MAPKAP kinase 2 in rat hearts. FEBS Lett 396:233– 237. Okosi A, Brar BK, Chan M, et al.: 1998. Expression and protective effects of urocortin in cardiac myocytes. Neuropeptides 32:167–171.
Vale W, Spiess J, Rivier C, Rivier J: 1981. Characterisation of a 41-residue ovine hypothalamic peptide that stimulates secretion of corticotropin and beta-endorphin. Science 213:1394–1397. Vaughan J, Donaldson C, Bittencourt J, et al.: 1995. Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378:287– 292. Xia Z, Dickens M, Raingeaud J, et al.: 1995. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 270: 1326–1331. Yeh JH, Hsu SC, Han SH, Lai MZ: 1998. Mitogen-activated protein kinase antagonized fas-associated death domain proteinmediated apoptosis by induced FLICEinhibitory protein expression. J Exp Med 188:1795–1802.
PII S1050-1738(01)00093-7
TCM
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Urocortin (UCN), is a peptide related to hypothalamic corticotrophin releasing hormone (CRF) and binds with high affinity to the CRF-R2 b receptor which is expressed in the heart. UCN prevents cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia as assayed by trypan blue exclusion. 3 9-OH end labeling of DNA (TUNEL), annexin-V and fluorescence activated cell sorting. The protective effect of UCN is dependent on the p42/p44 mitogen-activated protein kinase (MAPK)-pathway. UCN also reduces damage in isolated rat hearts ex vivo, subjected to regional ischemia/ reperfusion with the protective effect being observed when UCN is given either prior to ischemia or at the time of reperfusion after ischemia. Hence, UCN is a cardioprotective agent, which acts when given prior to ischemia or after ischemia at reperfusion. (Trends Cardiovasc Med 2001;11:167–169). © 2001, Elsevier Science Inc.
Urocortin (UCN) is a 40-amino-acid peptide related to the hypothalamic hormone corticotrophin releasing factor (CRF), the central mediator of the hypothalamicpituitary-adrenal axis and stress response in mammals (De Souza 1995, Plotsky 1991, Vale et al. 1981). UCN and CRF share 45% homology at the amino acid level and both are synthesized as precursors, which are subsequently processed to the mature biologically active peptides (Donaldson et al. 1996, Vaughan David S. Latchman is at the Institute of Child Health, University College London, London, United Kingdom. * Address correspondence to: David S. Latchman, Institute of Child Health, University College London, 30 Guilford Street, London WC1N 1EH, UK. © 2001, Elsevier Science Inc. All rights reserved. 1050-1738/01/$-see front matter
TCM Vol. 11, No. 5, 2001
et al. 1995). Although UCN was originally identified in restricted areas of the brain, it has also been found in the placenta, lymphocytes and heart (Bamberger et al. 1998, Iino et al. 1999, Okosi et al. 1998, Petraglia et al. 1996). The CRF family of peptides binds to two types of CRF receptors, CRF-R1 and CRF-R2 (Chang et al. 1993, Kishimoto et al. 1995, Lovenberg et al. 1995). Interestingly, CRF-R2 binds UCN with a higher affinity than CRF, both in ligand binding studies (Vaughan et al. 1995) and as assayed by the effects of ligand on intracellular cAMP (Gottowik et al. 1997). In contrast, the R1 receptors show little ligand selectivity for UCN versus CRF. R2 receptors are the only type of CRF receptor found in the heart—the a form in man (Chen et al. 1993) and the b form in the rat (Stenzel et al. 1995). The CRF
family of peptides has been shown to stimulate adenylate cyclase activity in cardiac myocytes (Heldwein et al. 1996), and changes in CRF-R2 expression have been reported in the hearts of spontaneously hypertensive rats (Makino et al. 1998). The expression of CRF-R2 receptors in the heart suggests that UCN may have physiological cardiac effects. Indeed UCN, but not CRF, induced a dose-dependent increase in heart rate, cardiac output and coronary blood flow (Parkes et al. 1997). Moreover, we have previously demonstrated that the UCN mRNA and protein are expressed in the heart (Brar et al. 1999, Okosi et al. 1998). Interestingly, the expression of UCN has been shown to increase when cultured cardiac cells are exposed to stressful stimuli such as heat shock (Okosi et al. 1998) or ischemia (Brar et al. 1999). Moreover, the UCN produced is secreted into the culture medium (Brar et al. 1999). These findings raise the question as to the role that UCN may play in the response of cardiac cells to stressful stimuli. In initial experiments, we showed that conditioned medium from cardiac cells exposed to ischemia, was able to exert a protective effect against subsequent exposure to ischemia (Brar et al. 1999). This protective effect could be blocked by addition of a-helical CRF, which acts as an antagonist blocking all members of the CRF family (Brar et al. 1999). This indicated therefore that UCN released from cardiac cells in response to stress might have a protective effect. In agreement with this, UCN added to cardiac cells was able to protect them from subsequent exposure to simulated ischemia whereas CRF itself had a much less potent protective effect (Brar et al. 1999). Hence, UCN is indeed able to protect cardiac cells. Although these initial experiments were conducted by assaying total cell survival on the basis of the ability to exclude trypan blue (Brar et al. 1999), they
167
have subsequently been confirmed by using assays of programmed cell death/ apoptosis (Brar et al. 2000). To do this, we utilized both annexin V surface staining, which measures the translocation of phosphatidyl serine to the outer surface of the plasma membrane that occurs early in the apoptotic process, and TUNEL labeling, which measures the DNA degradation that occurs later in the apoptotic process. In both cases, clear protective effects of UCN were observed (Brar et al. 2000). A number of studies have implicated the mitogen-activated protein kinase (MAPK) pathway as a survival pathway in both cardiac cells (Adderley and Fitzgerald 1999, Maulik et al. 1996, Sheng et al. 1997) and other cell types (Allen et al. 1999, Cano and Mahadevan 1995, Cobb and Goldsmith 1995, Downward 1998, Schaeffer and Weber 1999, Xia et al. 1995). The extracellular signalrelated kinases (EPK), belonging to one sub-family of MAP kinases, are composed of 42- and 44-kDa kinases named p42-MAPK and p44-MAPK, respectively. Phosphorylation and activation of p42MAPK and p44-MAPK is mediated by MAP kinase kinases MEK1/2. CRF has been shown to activate MAPK p42/44 in Chinese hamster ovary (CHO) cell lines transfected with the CRF-R1 and CRF-R2 receptors (Rossant et al. 1999). Moreover, the cardioprotective effect of the cytokine cardiotrophin-1 (CT-1) is mediated through gp130 receptor activation of the MEK1/2 p42/44 MAPK signaling cascade (Sheng et al. 1997). We therefore tested the effect of the MEK1/2 inhibitor PD98059 (Dudley et al. 1995) on the protective effect of UCN. The protective effect of UCN when added prior to simulated ischemia, was inhibited by PD98059 as assayed by trypan blue exclusion, annexin V staining or TUNEL labeling (Brar et al. 2000). A similar inhibition of UCN-mediated protection was also observed when we transfected cardiac cells with constructs encoding dominant negative mutants of either p42 or p44 MAPK. Hence, the p42/p44 MAPK pathway, which is activated by MEK1/2, appears to play a key role in the protective effect of UCN given prior to simulated ischemia. Evidently, however, if UCN were to have clinical potential in the treatment of myocardial infarction, it would need to have a protective effect when given at
168
reoxygenation following an ischemic episode. To assess whether UCN retains cardioprotective effects when added after a period of simulated hypoxia/ ischemia, UCN was added to the cardiac myocytes at the point of reoxygenation. Cell death was measured in the untreated cells exposed to ischemia reoxygenation by trypan blue exclusion, TUNEL and annexin V. UCN protected the cardiac cells during reoxygenation in all these assays (Brar et al. 2000). To investigate whether UCN protection at reoxygenation was mediated by the MEK-1 p42/44 MAPK signaling cascade, PD98059 was administered for 10 min prior to the addition of UCN. The cardioprotective effect of UCN, when added at reoxygenation, was inhibited by PD98059 where cell death was assessed by trypan blue, annexin V, and TUNEL (Brar et al. 2000). A similar inhibition of the protective effect of UCN at reoxygenation was also observed in cells transfected with dominant negative mutants of the p42 or p44 MAPK enzymes confirming their role in the protective effect. Having shown protective effects of UCN on simulated hypoxic/ischemic reoxygenated cardiac myocytes in vitro, we examined whether UCN had similar protective actions on the intact ischemic/ reperfused isolated heart ex vivo on a Langendorff perfusion apparatus. In these experiments UCN protected the isolated rat heart from 35 min of regional ischemia/120 min reperfusion when added 30 min prior to the stress as the percentage of infarct size to zone at risk decreased from 45.67% to 20.58%. Moreover, the addition of UCN for 30 min at reperfusion after the 35-min ischemic period reduced the percentage of infarct size to zone at risk to 32.24% (Brar et al. 2000). No significant hemodynamic changes in parameters such as heart rate, left ventricular developed pressure and coronary flow with UCN were observed compared to the control, untreated hearts. This study therefore demonstrated that UCN results in a significant reduction in infarct size when administered either 30 min prior to ischemia or from the moment of reperfusion (Brar et al. 2000). It is clear therefore that UCN can protect cardiac cells both in vitro and in the intact perfused heart from the damaging effects of ischemia/reperfusion.
Moreover, it is effective when added prior to reperfusion as well as prior to the ischemic period, indicating that it might ultimately have a clinical application. Although we have demonstrated that the protective effect of UCN requires the p42/p44 MAPK pathway, the exact mechanism of its protective effect remains unclear. We have shown that the cardioprotective effects of UCN are inhibited by blocking new protein synthesis with cycloheximide, indicating that de novo protein synthesis induced by UCN is required for its effect. It has been shown in non-cardiac cells that activation of the p42/p44 MAPK pathway is associated with enhanced synthesis of FLIP (FLICElike inhibitor protein) an inhibitor of the caspase cascade, which is necessary for apoptosis (Yeh et al. 1998). However, we have also demonstrated that the 90-kDa heat shock protein (hsp90) is induced by UCN treatment and this effect is blocked by PD98059 suggesting that the induction of cardioprotective hsps may play a role in the cardioprotective effects of UCN. Evidently, a number of agents that can protect the heart against ischemia have been described and several of these, such as CT-1, also act via the p42/p44 MAPK pathway (Sheng et al. 1997). As none of these agents have yet proved useful clinically, one may ask what is the significance of identifying yet another cardioprotective agent that acts via the p42/p44 MAPK pathway. In considering this question, it is important to realize that the utility of many protective agents is limited via the other effects which they cause such as vasodilation or hypertrophy. Indeed CT1 was originally discovered on the basis of its hypertrophic effect and was only subsequently shown to be protective [for review see Latchman (1999)]. In the case of urocortin, we have already shown that UCN does not affect the hemodynamics of the isolated heart (Brar et al. 2000) and we are currently investigating whether it is a hypertrophic agent. The identification of novel cardioprotective agents such as UCN, which act via the p42/p44 MAPK pathway, may therefore be of use if they have less undesirable side effects than other agents which protect via this pathway. Similarly, by understanding the signaling pathways activated by agents such as UCN, it may be possible to stimulate the protective pathway while blocking the pathway meTCM Vol. 11, No. 5, 2001
diating any undesirable effects. In these ways, it may be possible to take advantage of the ability of UCN to protect against ischemia/reperfusion injury when administered either prior to ischemia or at reperfusion. • Acknowledgments I thank the members of my laboratory who have worked on the UCN project for their efforts and helpful discussion, in particular, Dr. B. Brar, Dr. K. Lawrence, Dr. J. Railson, Dr. D. Schulman and Dr. A. Stephanou. I am also grateful to Dr. Dick Knight for introducing me to UCN and ongoing collaboration and to Professor Derek Yellon for ongoing collaboration in many areas including UCN. The work of my laboratory on urocortin has been funded since its inception by the British Heart Foundation and I am most grateful for their ongoing support.
References Adderley SR, Fitzgerald DJ: 1999. Oxidative damage of cardiomyocytes is limited by extracellular regulated kinases 1/2-mediated induction of cyclooxygenase-2. J Biol Chem 274:5038–5046. Allen MP, Zeng C, Schneider K, et al.: 1999. Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signalling promotes gonadotropin-releasing hormone neuronal survival via extracellular signalregulated kinase (ERK) and Akt. Mol Endocrinol 13:191–201. Bamberger CM, Wald M, Bamberger AM, et al.: 1998. Human lymphocytes produce urocortin, but not corticotropin-releasing hormone. J Clin Endocrinol Metab 83:708–711. Brar BK, Jonassen AK, Stephanou AS, et al.: 2000. Urocortin protects against ischemic and reperfusion injury via a MAP-kinase dependent pathway. J Biol Chem 275:8508– 8514. Brar BK, Stephanou A, Okosi A, et al.: 1999. CRH-like peptides protect cardiac myocytes from lethal ischaemic injury. Mol Cell Endocrinol 158:55–63. Cano E, Mahadevan LC: 1995. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 20:117–122. Chang CP, Pearse RV, O’Connell S, Rosenfeld MG: 1993. Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain. Neuron 11:1187–1195.
TCM Vol. 11, No. 5, 2001
Chen R, Lewis KA, Perrin MH, Vale WW: 1993. Expression cloning of a human corticotropin-releasing-factor receptor. Proc Natl Acad Sci USA 90:8967–8971.
Parkes DG, Vaughan J, Rivier J, et al.: 1997. Cardiac inotropic actions of urocortin in conscious sheep. Am J Physiol 272:H2115– H2122.
Cobb MH, Goldsmith EJ: 1995. How MAP kinases are regulated. J Biol Chem 270:14,843–14,846.
Petraglia F, Florio P, Gallo R, et al.: 1996. Human placenta and fetal membranes express human urocortin mRNA and peptide. J Clin Endocrinol Metab 81:3807– 3810.
De Souza EB: 1995. Corticotropin-releasing factor receptors: physiology, pharmacology, biochemistry and role in central nervous system and immune disorders. Psychoneuroendocrinology 20:789–819. Donaldson CJ, Sutton SW, Perrin MH, et al.: 1996. Cloning and characterisation of human urocortin. Endocrinology 137:2167– 2170. Downward J: 1998. Ras signalling and apoptosis. Curr Opin Gen Devel 8:49–54. Dudley DT, Pang L, Decker SJ, et al.: 1995. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 92:7686–7689.
Plotsky PM: 1991. Hypothalamic secretion of somatostatin and growth hormone-releasing factor into the hypophysial-portal circulation is reduced in streptozotocin diabetic male rats. Neuroendocrinology 53:433–438. Rossant CJ, Pinnock RD, Hughes J, et al.: 1999. Corticotropin-releasing factor type 1 and type 2alpha receptors regulate phosphorylation of calcium/cyclic adenosine 39,59-monophosphate response elementbinding protein and activation of p42-p44 mitogen-activated protein kinase. Endocrinology 140:1525–1536.
Gottowik J, Goetschy V, Henriot S, et al.: 1997. Labelling of CRF1 and CRF2 receptors using the novel radioligand, [3H]urocortin. Neuropharmacology 36:1439– 1446.
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