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Effects of urocortin via ion mechanisms or CRF receptors?
BBRC Biochemical and Biophysical Research Communications 336 (2005) 731–736 www.elsevier.com/locate/ybbrc
Breakthroughs and Views
Effects of urocortin via ion mechanisms or CRF receptors? Jin Tao a,b, Shengnan Li b,* a
Key Laboratory of Reproductive Medicine, Center of Human Functional Genomics, Nanjing 210029, PR China b Department of Pharmacology, Nanjing Medical University, Nanjing 210029, PR China Received 23 June 2005 Available online 26 July 2005
Abstract Urocortin (UCN), a newly isolated peptide related to hypothalamic corticotrophin releasing factor (CRF) family, had been reported to play biologically diverse roles in several systems such as cardiovascular, reproductive, appetite, stress, and inflammatory responses, etc. It was thought previously to be an endogenous agonist, producing the several actions previously attributed to CRF. But, recently, it was shown to directly reduce L-type calcium currents of acute isolated cardiac myocytes and T-type calcium currents in mouse spermatogenic cells via inhibiting calcium channel instead of binding first to its CRF-R2 receptors. UCN could also reduce the intracellular calcium in vascular smooth muscle cells via inhibiting calcium channel directly. Furthermore, UCN could increase the gene expression of ATP-sensitive potassium channels (KATP) and activate sarcolemmal ATP-sensitive potassium current during normal or hypoxia, which could be inhibited by glibenclamide, a specific KATP blocker. This review will highlight the current novel findings on the ionic mechanisms by which UCN may exert its several actions. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Urocortin; Corticotrophin releasing factor; Calcium channels; ATP-sensitive potassium channels
Urocortin (UCN) is a 40-amino-acid peptide related to the corticotrophin-releasing factor (CRF) family, which includes CRF, urotensin, and sauvagine. It was originally cloned from the rat and later in a number of mammalian species including mouse and human [1–5], and shares 45% amino acid sequence homology to CRF [6,7], which had receptors expressed in the peripheral tissues including cardiovascular system [8,9], male or female reproductive system such as placenta, ovary [10], immune system [11], and digestive system [12], etc. UCN appeared to cause a dose-dependent increase in cardiac output, coronary artery blood flow, and a decrease in blood pressure. UCN was increased in various inflammatory diseases in immune reactions, and it was found to decrease the intake of food in the digestive system [12–14]. Though the mech-
*
Corresponding author. Fax: +86 25 86863050. E-mail address: [email protected] (S. Li).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.078
anisms of UCN were not quite clear, previous reports have shown that UCN exerted these physiological properties via mitogen-activated protein kinase (MAPK)-dependent pathway [13,15,16] during ischemia/reperfusion, CRF-R2 receptors, KATP channels [1–3], respectively. Furthermore, activation of CRFR2 receptors elevated the cellular contents of cyclic AMP through the G protein–adenylate cyclase pathway [17]. UCN also stimulated the phosphorylation of cyclic AMP response element-binding protein in cells expressing CRF2 receptors, and the cyclic AMPdependent protein kinase (PKA) inhibitor blocked the formation of phosphorylated cyclic AMP response element-binding protein [18], which were all highly associated with the changes of intracellular calcium. Results in our laboratory have recently found that UCN could inhibit the calcium channel and intracellular calcium, and activate sarcolemmal I KATP in normal or hypoxia condition which could be inhibited by glibenclamide, a specific KATP blocker.
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UCN and calcium channel Previous reports (Latchman et al.) and our data have shown that UCN protected against ischemic/reperfusion injury [13,14]. Parkes et al. [19] demonstrated that UCN protected neonatal rat cardiac myocytes in vitro when administered before hypoxia or at the point of reoxygenation and protect the adult rat heart ex vivo where UCN reduced the infarct size of a perfused intact rat heart exposed to regional ischemia. These cardioprotective effects of UCN on cardiac myocytes induced by hypoxia or by ischemia/reperfusion were reported to mainly be attributed to MAPK-dependent pathway, which acted as a survival pathway in cardiac cells and other cell types [15,16,20–23]. It is well established that MAPK signaling pathway is associated with the activation of protein kinase C (PKC) in human and rabbit ventricular myocytes, leading to changes in the intracellular calcium
A
UCN 1µmol/L
Wash
[20]. Basing on these facts, it was suggested that L-type calcium channels may play some roles in UCNÕs cardioprotective effects since ischemia and hypoxia damage are highly associated with Ca2+-overload. Moreover, UCN exerted positive chronotropic and inotropic actions in the heart [19], which were thought to be associated with Ca2+, indicating a close relationship between cardioprotective effect of UCN and calcium channels. Recently, we found that UCN could inhibit the L-type calcium channels of acute isolated adult rat ventricular cells, which were not affected by astressin, the CRF receptor blocker (Fig. 1) [24]. This inhibitory effect of UCN on ICa,L could be expected to exert a cardioprotective action by reducing calcium overload via the voltage-gated calcium channels, consistent with the report that UCN protected the cardiacmyocytes against apoptosis [25], which was thought to be associated with Ca2+ overload. Inhibition of Ca2+ influx could shorten action potential duration (APD) [26], resulting in a cardioprotective action due to decrease in energy consumption via the preservation of high-energy phosphates [27,28]. In male reproductive system, we also found that UCN could reduce the T-type calcium currents in mouse spermatogenic cells directly via inhibiting the calcium channels also instead of binding CRF-R2 receptors (Fig. 2) [29]. These novel results highlight the effect of UCN on male reproductive functions such as acrosome reactions and capacUCN 1 µM
A Control 500 pA
Wash out 50 ms Control
80 70
B 50
60
40
50
% of inhibition
Inhibition % of predrug control
B
40 30 20 10
30 20 10 0 30
0 0
2
5
10
15
20
Time (min) Fig. 1. Effect of UCN on ICa,L of rat ventricular myocytes. (A) Current traces of ICa,L obtained in the absence, presence of UCN 1 lM and washout. (B) Effect of UCN 1 lM (j) and UCN 1 lM + astressin 1 lM () on ICa,L of rat ventricular myocytes (P < 0.05, n = 6) [24].
60
120
200
240
300
Time (s) Fig. 2. Effect of UCN on ICa,T of mouse spermatogenic cells. (A) Current traces of ICa,T obtained in the absence and presence of UCN 1 lM and washout. (B) Effect of UCN 1 lM (j) and UCN 1 lM + astressin 1 lM () on ICa,T of mouse spermatogenic cells (P < 0.05, n = 5) [29].
J. Tao, S. Li / Biochemical and Biophysical Research Communications 336 (2005) 731–736
itation, which were supposed to be associated with calcium channels. In vascular system, our report showed that UCN could reduce the viability of vascular smooth muscle cells via inhibiting the L-type calcium channels [30]. Pretreatment of the cells with CRF receptor blocker, astressin, did not affect the inhibitory effect of UCN on the viability of VSMC as well. Bay K 8644, the calcium channel activator [31], can promote the viability of VSMC. Pre-exposure of the cells to UCN significantly diminished the effect of Bay K 8644 (Fig. 3). Furthermore, it was found that UCN could attenuate the increase in intracellular Ca2+ fluorescence intensity induced by Bay K 8644 or KCl by using confocal laser scanning microscope (Fig. 4). These results suggest that UCN may exert the inhibitory effect via calcium chan-
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Urocortin
Control
-7 Urocortin (10 )
Bay K8644
Urocortin (10-5)
Urocortin (10-6 ) Bay K8644
A
25
140
# *
#
20
Fluoresence intensity (%)
Inhibition on the viability (% of Control)
#
15 10 5
120 100
* *
80 60
*
*
40 20
0
Astressin
UCN
UCN+Astressin
0
Control
UCN
Bay
Inhibition on the viability (% of Control)
(10 ) #
20
UCN
-6
(10 )
(10 )
-5
Fig. 4. Effects of UCN (0.1 lM) on the Ca2+ levels in VSMC in the presence of Bay K 8644 (1 lM) (#P < 0.01 vs. control; *P < 0.05 vs. Bay K 8644 0.1 lM) [30].
15 10 5
Gly
UCN
UCN+Gly
1.8 #
1.6 1.4 Absorbance (At 570 nm)
UCN
Bay K8644
# *
0
B
UCN -7
25
1.2 *
1
#
0.8
#
#
0.6 0.4 0.2 0 Control
UCN
Bay Bay+ UCN
Ver
nels directly instead of via binding first to its CRF receptors. However, it was reported that UCN stimulated Ca2+ uptake through voltage-dependant calcium channels in AtT20 cells [15]. In addition, UCN-induced increase of ANP and BNP secretion was completely abolished by a-helical CRF, a specific CRF-R2 receptor antagonist, and partially inhibited by diltiazem, another L-type calcium channel blocker in the cultured neonatal cardiomyocytes [32]. In the same paper, the authors also stated that UCN caused cAMP accumulation, which might lead to the activation of L-type calcium channels. The interpretation for the differences remains to be explored, which might be attributed to the diversity of UCNÕs action mechanisms and the different cell species/ages used for the experiments.
Ver+UCN
Fig. 3. (A) Effects of astressin (10 lM) and glibenclamide (10 lM) on VSMC viability (n = 6). #P < 0.01 vs. control, *P > 0.05 vs. UCN 0.1 lM. (B) Effects of UCN (0.1 lM), Bay K 8644 (1 lM), and Verapamil (2.5 lM) on VSMC viability (n = 6). #P < 0.01 vs. control; *P < 0.01 vs. Bay K 8644 0.1 lM [30].
UCN and KATP channel In rat isolated basilar artery, it has been reported that the UCN-induced vascular relaxation involved opening the K+ channels [5]. The role(s) of K+ channels opening
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in mediating the cardioprotective effect of UCN is not known at present. Ischemic preconditioning (IPC) is an important phenomenon in which brief periods of ischemia protect the heart against a more prolonged ischemic insult, resulting in a marked reduction in myocardial infarct size, severity of stunning, and incidence of cardiac arrhythmias. Since the discovery of ATP-sensitive potassium channels (KATP) in cardiac myocytes in 1983 [26,33], there is ample evidence indicating that KATP channels are a major contributor to cardiac protection against ischemia involving in IPC [4,8,34]. There are two populations of KATP channels in the myocardium: the mitochondrial (mito) and sarcolemmal (sarc) KATP channels. Original opinion favors a definitive role for the mitoKATP channel in IPC. Recent evidence suggests that the sarcKATP channel may in fact play a more predominant role than it was thought before. Hence, a range of sarcKATP openers have been suggested (although it has not been rigorously tested) to be important agents for cardiac ischemia. The sarcKATP have been cloned and found to be an octameric complex of four pore-forming Kir6.2 subunits and four SUR2A sulfonylurea receptors [15,19]. Heart
Vessel
Opening of sarcKATP enhances the shortening of cardiac action potential duration (APD) by accelerating phase 3 repolarization, which results in reduction in Ca2+ entry into the cell and prevention of calcium overload via L-type calcium channels [26]. The putative KATP blocker, glibenclamide, inhibits IPC protection while the opener, pinacidil, mimics IPC protection [26,34–36]. Moreover, it has been reported that glibenclamide inhibited the shortening of APD while pinacidil accelerated the shortening of APD during ischemia, resulting in a recovery of ventricular function during reperfusion [34]. Schiling et al. found that IPC resulted in an enhanced shortening of APD associated with a marked cardioprotective effect [35]. Recent molecular biology studies by transfecting KATP-deficient Cos-7 cells with KATP channel subunits suggest a possible role for sarcKATP channels in alleviating hypoxia injury and the subsequent calcium overload [15,19]. On the one hand, sarcKATP is associated with protein kinase C (PKC) since PKC activates KATP channels in cardiac and other tissues [37–39]. On the other hand, it is well established that PKC is involved in IPC with which it has been suggested to be associated with KATP channels
Urocortin (UCN)
Astressin sarcolemmal
CRF-R2
NO +
IKATP
ICa,L / L-type calcium channels (Cardiomyocytes / VSMC)
ICa,T
P42/44 MAPK
K channel
MEK 1/2
cAMP
P13K/AKT PKC
Calcium
Intracellular
overload
calcium
Mitochondrial KATP channel
VSMC viability
Coronary vasodilatation Ischemia/reperfusion cardiac damage
Vascular remodeling
Cardiovascular protection Fig. 5. Possible mechanisms of urocortinÕs effects (UCN).
Male reproduction
J. Tao, S. Li / Biochemical and Biophysical Research Communications 336 (2005) 731–736
[34,39]. Activation of stress-activated protein kinase pathways such as MAPK has been suggested to be involved in ischemic preconditioned rabbit and rat hearts. An activation of MAPK is correlated with the activation of PKC [40], implying that a connection between KATP and MAPK/PKC may exist. Latchman group have analyzed global changes in gene expression in cardiac myocytes after UCN treatment using gene chip technology [2]. They report that UCN specifically induces enhanced expression of the Kir6.1 cardiac potassium channel subunit, which showed that the cardioprotective effect of UCN both in isolated cardiac cells and in the intact heart is specifically blocked by both generalized and mitochondrial-specific KATP channel blockers, whereas the cardioprotective effect of cardiotrophin-1 is unaffected [16]. Conversely, inhibiting the Kir6.1 channel subunit greatly enhances cardiac cell death after ischemia. This is the first report of the altered expression of a KATP channel subunit induced by a cardioprotective agent and demonstrates that KATP channel opening is essential for the effect of this novel cardioprotective agent. This effect was comparable with that observed with adenosine. The cardioprotective effect of UCN was markedly attenuated by the protein kinase C inhibitor chelerythrine and by 5-hydroxydecanoate [41], an inhibitor of ATP-sensitive potassium channels, which showed the importance of KATP channel in cardiovascular protection. These results are also consistent with our recent data which indicate that urocortin can activate sarcolemmal I KATP in normal or hypoxia condition [42]. In physiological conditions, KATP are dominantly closed, but when exposed to metabolic inhibition or potassium channel openers, they can be activated contributing to shortening of action potential duration (APD) which helps to attenuate the Ca2+ overload [43–46]. Brief ischemia or hypoxia causes the opening of sarcoKATP during metabolic inhibition, attenuating the following long-term ischemia- or hypoxia-induced damage [47]. It was found that UCN protects against ischemic/reperfusion injury of cardiac myocytes via MAPK-dependent pathway [48–50]. In cultured human pregnant myometrial cells, UCN activated and phosphorylated MAPK [51,52]. However, the underlying mechanism(s) responsible for the observed cardiac effects of UCN has not been evaluated in detail. Further studies are needed to elucidate whether the cardioprotective effect of UCN involves the sarcKATP channels that are functionally associated with MAPK, PKC, and CRF receptors (Fig. 5).
Acknowledgments This work was supported by grants from Natural Science Foundation of China (No. 30371646), Natural Scientific Fund of JiangSu Provincial Education Commit-
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tee (No. 02KJB310006), and Key Subject Fund of China Ministry of Education (No. 03047).
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Breakthroughs and Views
Effects of urocortin via ion mechanisms or CRF receptors? Jin Tao a,b, Shengnan Li b,* a
Key Laboratory of Reproductive Medicine, Center of Human Functional Genomics, Nanjing 210029, PR China b Department of Pharmacology, Nanjing Medical University, Nanjing 210029, PR China Received 23 June 2005 Available online 26 July 2005
Abstract Urocortin (UCN), a newly isolated peptide related to hypothalamic corticotrophin releasing factor (CRF) family, had been reported to play biologically diverse roles in several systems such as cardiovascular, reproductive, appetite, stress, and inflammatory responses, etc. It was thought previously to be an endogenous agonist, producing the several actions previously attributed to CRF. But, recently, it was shown to directly reduce L-type calcium currents of acute isolated cardiac myocytes and T-type calcium currents in mouse spermatogenic cells via inhibiting calcium channel instead of binding first to its CRF-R2 receptors. UCN could also reduce the intracellular calcium in vascular smooth muscle cells via inhibiting calcium channel directly. Furthermore, UCN could increase the gene expression of ATP-sensitive potassium channels (KATP) and activate sarcolemmal ATP-sensitive potassium current during normal or hypoxia, which could be inhibited by glibenclamide, a specific KATP blocker. This review will highlight the current novel findings on the ionic mechanisms by which UCN may exert its several actions. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Urocortin; Corticotrophin releasing factor; Calcium channels; ATP-sensitive potassium channels
Urocortin (UCN) is a 40-amino-acid peptide related to the corticotrophin-releasing factor (CRF) family, which includes CRF, urotensin, and sauvagine. It was originally cloned from the rat and later in a number of mammalian species including mouse and human [1–5], and shares 45% amino acid sequence homology to CRF [6,7], which had receptors expressed in the peripheral tissues including cardiovascular system [8,9], male or female reproductive system such as placenta, ovary [10], immune system [11], and digestive system [12], etc. UCN appeared to cause a dose-dependent increase in cardiac output, coronary artery blood flow, and a decrease in blood pressure. UCN was increased in various inflammatory diseases in immune reactions, and it was found to decrease the intake of food in the digestive system [12–14]. Though the mech-
*
Corresponding author. Fax: +86 25 86863050. E-mail address: [email protected] (S. Li).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.07.078
anisms of UCN were not quite clear, previous reports have shown that UCN exerted these physiological properties via mitogen-activated protein kinase (MAPK)-dependent pathway [13,15,16] during ischemia/reperfusion, CRF-R2 receptors, KATP channels [1–3], respectively. Furthermore, activation of CRFR2 receptors elevated the cellular contents of cyclic AMP through the G protein–adenylate cyclase pathway [17]. UCN also stimulated the phosphorylation of cyclic AMP response element-binding protein in cells expressing CRF2 receptors, and the cyclic AMPdependent protein kinase (PKA) inhibitor blocked the formation of phosphorylated cyclic AMP response element-binding protein [18], which were all highly associated with the changes of intracellular calcium. Results in our laboratory have recently found that UCN could inhibit the calcium channel and intracellular calcium, and activate sarcolemmal I KATP in normal or hypoxia condition which could be inhibited by glibenclamide, a specific KATP blocker.
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UCN and calcium channel Previous reports (Latchman et al.) and our data have shown that UCN protected against ischemic/reperfusion injury [13,14]. Parkes et al. [19] demonstrated that UCN protected neonatal rat cardiac myocytes in vitro when administered before hypoxia or at the point of reoxygenation and protect the adult rat heart ex vivo where UCN reduced the infarct size of a perfused intact rat heart exposed to regional ischemia. These cardioprotective effects of UCN on cardiac myocytes induced by hypoxia or by ischemia/reperfusion were reported to mainly be attributed to MAPK-dependent pathway, which acted as a survival pathway in cardiac cells and other cell types [15,16,20–23]. It is well established that MAPK signaling pathway is associated with the activation of protein kinase C (PKC) in human and rabbit ventricular myocytes, leading to changes in the intracellular calcium
A
UCN 1µmol/L
Wash
[20]. Basing on these facts, it was suggested that L-type calcium channels may play some roles in UCNÕs cardioprotective effects since ischemia and hypoxia damage are highly associated with Ca2+-overload. Moreover, UCN exerted positive chronotropic and inotropic actions in the heart [19], which were thought to be associated with Ca2+, indicating a close relationship between cardioprotective effect of UCN and calcium channels. Recently, we found that UCN could inhibit the L-type calcium channels of acute isolated adult rat ventricular cells, which were not affected by astressin, the CRF receptor blocker (Fig. 1) [24]. This inhibitory effect of UCN on ICa,L could be expected to exert a cardioprotective action by reducing calcium overload via the voltage-gated calcium channels, consistent with the report that UCN protected the cardiacmyocytes against apoptosis [25], which was thought to be associated with Ca2+ overload. Inhibition of Ca2+ influx could shorten action potential duration (APD) [26], resulting in a cardioprotective action due to decrease in energy consumption via the preservation of high-energy phosphates [27,28]. In male reproductive system, we also found that UCN could reduce the T-type calcium currents in mouse spermatogenic cells directly via inhibiting the calcium channels also instead of binding CRF-R2 receptors (Fig. 2) [29]. These novel results highlight the effect of UCN on male reproductive functions such as acrosome reactions and capacUCN 1 µM
A Control 500 pA
Wash out 50 ms Control
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60
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% of inhibition
Inhibition % of predrug control
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40 30 20 10
30 20 10 0 30
0 0
2
5
10
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Time (min) Fig. 1. Effect of UCN on ICa,L of rat ventricular myocytes. (A) Current traces of ICa,L obtained in the absence, presence of UCN 1 lM and washout. (B) Effect of UCN 1 lM (j) and UCN 1 lM + astressin 1 lM () on ICa,L of rat ventricular myocytes (P < 0.05, n = 6) [24].
60
120
200
240
300
Time (s) Fig. 2. Effect of UCN on ICa,T of mouse spermatogenic cells. (A) Current traces of ICa,T obtained in the absence and presence of UCN 1 lM and washout. (B) Effect of UCN 1 lM (j) and UCN 1 lM + astressin 1 lM () on ICa,T of mouse spermatogenic cells (P < 0.05, n = 5) [29].
J. Tao, S. Li / Biochemical and Biophysical Research Communications 336 (2005) 731–736
itation, which were supposed to be associated with calcium channels. In vascular system, our report showed that UCN could reduce the viability of vascular smooth muscle cells via inhibiting the L-type calcium channels [30]. Pretreatment of the cells with CRF receptor blocker, astressin, did not affect the inhibitory effect of UCN on the viability of VSMC as well. Bay K 8644, the calcium channel activator [31], can promote the viability of VSMC. Pre-exposure of the cells to UCN significantly diminished the effect of Bay K 8644 (Fig. 3). Furthermore, it was found that UCN could attenuate the increase in intracellular Ca2+ fluorescence intensity induced by Bay K 8644 or KCl by using confocal laser scanning microscope (Fig. 4). These results suggest that UCN may exert the inhibitory effect via calcium chan-
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Urocortin
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-7 Urocortin (10 )
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Inhibition on the viability (% of Control)
(10 ) #
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(10 )
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Fig. 4. Effects of UCN (0.1 lM) on the Ca2+ levels in VSMC in the presence of Bay K 8644 (1 lM) (#P < 0.01 vs. control; *P < 0.05 vs. Bay K 8644 0.1 lM) [30].
15 10 5
Gly
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1.8 #
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nels directly instead of via binding first to its CRF receptors. However, it was reported that UCN stimulated Ca2+ uptake through voltage-dependant calcium channels in AtT20 cells [15]. In addition, UCN-induced increase of ANP and BNP secretion was completely abolished by a-helical CRF, a specific CRF-R2 receptor antagonist, and partially inhibited by diltiazem, another L-type calcium channel blocker in the cultured neonatal cardiomyocytes [32]. In the same paper, the authors also stated that UCN caused cAMP accumulation, which might lead to the activation of L-type calcium channels. The interpretation for the differences remains to be explored, which might be attributed to the diversity of UCNÕs action mechanisms and the different cell species/ages used for the experiments.
Ver+UCN
Fig. 3. (A) Effects of astressin (10 lM) and glibenclamide (10 lM) on VSMC viability (n = 6). #P < 0.01 vs. control, *P > 0.05 vs. UCN 0.1 lM. (B) Effects of UCN (0.1 lM), Bay K 8644 (1 lM), and Verapamil (2.5 lM) on VSMC viability (n = 6). #P < 0.01 vs. control; *P < 0.01 vs. Bay K 8644 0.1 lM [30].
UCN and KATP channel In rat isolated basilar artery, it has been reported that the UCN-induced vascular relaxation involved opening the K+ channels [5]. The role(s) of K+ channels opening
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in mediating the cardioprotective effect of UCN is not known at present. Ischemic preconditioning (IPC) is an important phenomenon in which brief periods of ischemia protect the heart against a more prolonged ischemic insult, resulting in a marked reduction in myocardial infarct size, severity of stunning, and incidence of cardiac arrhythmias. Since the discovery of ATP-sensitive potassium channels (KATP) in cardiac myocytes in 1983 [26,33], there is ample evidence indicating that KATP channels are a major contributor to cardiac protection against ischemia involving in IPC [4,8,34]. There are two populations of KATP channels in the myocardium: the mitochondrial (mito) and sarcolemmal (sarc) KATP channels. Original opinion favors a definitive role for the mitoKATP channel in IPC. Recent evidence suggests that the sarcKATP channel may in fact play a more predominant role than it was thought before. Hence, a range of sarcKATP openers have been suggested (although it has not been rigorously tested) to be important agents for cardiac ischemia. The sarcKATP have been cloned and found to be an octameric complex of four pore-forming Kir6.2 subunits and four SUR2A sulfonylurea receptors [15,19]. Heart
Vessel
Opening of sarcKATP enhances the shortening of cardiac action potential duration (APD) by accelerating phase 3 repolarization, which results in reduction in Ca2+ entry into the cell and prevention of calcium overload via L-type calcium channels [26]. The putative KATP blocker, glibenclamide, inhibits IPC protection while the opener, pinacidil, mimics IPC protection [26,34–36]. Moreover, it has been reported that glibenclamide inhibited the shortening of APD while pinacidil accelerated the shortening of APD during ischemia, resulting in a recovery of ventricular function during reperfusion [34]. Schiling et al. found that IPC resulted in an enhanced shortening of APD associated with a marked cardioprotective effect [35]. Recent molecular biology studies by transfecting KATP-deficient Cos-7 cells with KATP channel subunits suggest a possible role for sarcKATP channels in alleviating hypoxia injury and the subsequent calcium overload [15,19]. On the one hand, sarcKATP is associated with protein kinase C (PKC) since PKC activates KATP channels in cardiac and other tissues [37–39]. On the other hand, it is well established that PKC is involved in IPC with which it has been suggested to be associated with KATP channels
Urocortin (UCN)
Astressin sarcolemmal
CRF-R2
NO +
IKATP
ICa,L / L-type calcium channels (Cardiomyocytes / VSMC)
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P42/44 MAPK
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MEK 1/2
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P13K/AKT PKC
Calcium
Intracellular
overload
calcium
Mitochondrial KATP channel
VSMC viability
Coronary vasodilatation Ischemia/reperfusion cardiac damage
Vascular remodeling
Cardiovascular protection Fig. 5. Possible mechanisms of urocortinÕs effects (UCN).
Male reproduction
J. Tao, S. Li / Biochemical and Biophysical Research Communications 336 (2005) 731–736
[34,39]. Activation of stress-activated protein kinase pathways such as MAPK has been suggested to be involved in ischemic preconditioned rabbit and rat hearts. An activation of MAPK is correlated with the activation of PKC [40], implying that a connection between KATP and MAPK/PKC may exist. Latchman group have analyzed global changes in gene expression in cardiac myocytes after UCN treatment using gene chip technology [2]. They report that UCN specifically induces enhanced expression of the Kir6.1 cardiac potassium channel subunit, which showed that the cardioprotective effect of UCN both in isolated cardiac cells and in the intact heart is specifically blocked by both generalized and mitochondrial-specific KATP channel blockers, whereas the cardioprotective effect of cardiotrophin-1 is unaffected [16]. Conversely, inhibiting the Kir6.1 channel subunit greatly enhances cardiac cell death after ischemia. This is the first report of the altered expression of a KATP channel subunit induced by a cardioprotective agent and demonstrates that KATP channel opening is essential for the effect of this novel cardioprotective agent. This effect was comparable with that observed with adenosine. The cardioprotective effect of UCN was markedly attenuated by the protein kinase C inhibitor chelerythrine and by 5-hydroxydecanoate [41], an inhibitor of ATP-sensitive potassium channels, which showed the importance of KATP channel in cardiovascular protection. These results are also consistent with our recent data which indicate that urocortin can activate sarcolemmal I KATP in normal or hypoxia condition [42]. In physiological conditions, KATP are dominantly closed, but when exposed to metabolic inhibition or potassium channel openers, they can be activated contributing to shortening of action potential duration (APD) which helps to attenuate the Ca2+ overload [43–46]. Brief ischemia or hypoxia causes the opening of sarcoKATP during metabolic inhibition, attenuating the following long-term ischemia- or hypoxia-induced damage [47]. It was found that UCN protects against ischemic/reperfusion injury of cardiac myocytes via MAPK-dependent pathway [48–50]. In cultured human pregnant myometrial cells, UCN activated and phosphorylated MAPK [51,52]. However, the underlying mechanism(s) responsible for the observed cardiac effects of UCN has not been evaluated in detail. Further studies are needed to elucidate whether the cardioprotective effect of UCN involves the sarcKATP channels that are functionally associated with MAPK, PKC, and CRF receptors (Fig. 5).
Acknowledgments This work was supported by grants from Natural Science Foundation of China (No. 30371646), Natural Scientific Fund of JiangSu Provincial Education Commit-
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tee (No. 02KJB310006), and Key Subject Fund of China Ministry of Education (No. 03047).
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