ORIGINAL ARTICLE
Bkca opener, NS1619 pretreatment protects against shock-induced vascular hyporeactivity through PDZ-Rho GEFYRhoAYRho kinase pathway in rats Yi Hu, MD, Guangming Yang, MD, Xudong Xiao, MD, Liangming Liu, MD, PhD, and Tao Li, MD, PhD, Chongqing, China
BACKGROUND: Our previous study showed that the ischemic preconditioning and pretreatment of adenosine triphosphateYsensitive potassium channel (KATP) opener, pinacidil, may induce a good protective effect on shock-induced vascular hyporeactivity. Whether the pretreatment of opener/activator of the large-conductance calcium-activated potassium channel (Bkca), NS1619, can also induce a protective effect on vascular reactivity and play a beneficial effect on subsequent hemorrhagic shock is not clear. METHODS: With Sprague-Dawley rats subjected to hemorrhagic shock and their isolated superior mesenteric artery, the protective effect of NS1619 (0.5, 1, 2, and 4 mg/kg) pretreatment (30 minutes before hemorrhage shock) on vascular reactivity and the underlying mechanisms were observed. RESULTS: NS1619 pretreatment significantly improved the 72-hour survival of hemorrhagic shock rats, alleviated shock-induced decrease of vascular reactivity and calcium sensitivity, and increased the cardiac output and oxygen delivery. NS1619 2 mg/kg had the best effect. These protective effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity were antagonized by RhoA inhibitor, C3 transferase, and Rho kinase antagonist, Y-27632. NS1619 pretreatment up-regulated the activities of RhoA, Rho-kinase, and PDZYRho GEF (guanine nucleotide exchange factor). These effects of NS1619 pretreatment were eliminated by RhoA inhibitor, C3 transferase. CONCLUSION: Bkca opener, NS1619 pretreatment has good protective effect on vascular reactivity and calcium sensitivity, which plays a good beneficial effect on hemorrhagic shock. The mechanism may be mainly through PDZ-Rho GEFYRhoAYRho kinase pathway. Bkca channel may be a potential target for the treatment of shock-induced vascular hyporeactivity. (J Trauma Acute Care Surg. 2014;76: 394Y401. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: NS1619; vascular reactivity; hemorrhagic shock; Rho-kinase; rats.
P
revious studies, in our laboratory and those of others, have demonstrated that after severe trauma or shock, the response of peripheral blood vessels to vasoconstrictors and vasodilators is greatly reduced, which is called vascular hyporeactivity, and seriously interferes with the development, outcome, and therapy of the shock.1,2 Therefore, it is very important to study the therapy for shock-induced vascular hyporeactivity. Since the early observations found that a brief, sublethal episode of myocardial ischemia can markedly decrease the infarct size induced by sustained severe ischemia,3 a number of studies have demonstrated that ischemic preconditioning (IPC) has significant protective effects in many organs and species.4Y9 However, the application of IPC is limited in clinical settings because, actually, hemorrhage in many situations is unpredictable. Submitted: May 8, 2013, Revised: August 20, 2013, Accepted: August 22, 2013. Published online: January 6, 2014. From the State Key Laboratory of Trauma, Burns and Combined Injury (Y.H., G.Y., X.X., L.L., T.L.), Second Department of Research Institute of Surgery, and Department of Anesthesiology (Y.H.), Daping Hospital, Third Military Medical University, Chongqing, China. *L.L. and T.L. contributed equally to this work. Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jtrauma.com). Address for reprints: Tao Li, MD, PhD, and Liangming Liu, MD, PhD, Second Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Daping, Chongqing 400042, China; email: [email protected]; [email protected]. DOI: 10.1097/TA.0b013e3182aa2d98
394
Thus, pharmacologic preconditioning (PPC) represents an ideal alternative to IPC. For example, Jin et al.10 reported that adenosine pretreatment protected against myocardial ischemiareperfusion injury in children undergoing cardiopulmonary bypass (CPB) for repair of congenital heart defects. Yang et al.11 reported that sevoflurane preconditioning reduced brain infarct volumes, improved neurologic outcomes, and attenuated the neuronal cell apoptosis, which indicated that sevoflurane preconditioning could induce the protective effects against transient cerebral ischemic injuries. Our previous study showed that pretreatment of adenosine triphosphateYsensitive potassium channel (KATP) opener, pinacidil, could induce a good protective effect against shockinduced vascular hyporeactivity12 and that another major K+ channel, large-conductance calcium-activated potassium channel (Bkca) in vascular smooth muscle cell (VSMC) played an important role in the occurrence of vascular hyporeactivity following shock.13 However, whether Bkca opener, NS1619 pretreatment could induce the protection of vascular reactivity against the subsequent hemorrhagic shock and the underlying mechanisms are not known. Bkca channels are composed of tetrameric sets consisting of a pore-forming >-subunit and an auxiliary A-subunit, and they play very important roles in various physiologic processes such as neurotransmitter release, cell excitability, hormone secretion, and cardiovascular function regulation. Recent studies have shown that Bkca channel was involved in J Trauma Acute Care Surg Volume 76, Number 2
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
cardioprotection of IPC.14Y17 Xu et al.14 found Bkca channel opener, NS1619 pretreatment caused a significant reduction in infarct size in rabbit heart, and such effect was antagonized by the Bkca channel blocker, paxilline. Redel et al. reported that the activation of Bkca channels by NS1619 mimicked the cardioprotection induced by IPC.17 So we hypothesized that Bkca opener, NS1619 pretreatment could induce the protection on vascular reactivity against the subsequent hemorrhagic shock. RhoA is a molecular switch that cycles between an inactive, GDP-bound form and an active, GTP-bound form. The activity of RhoA is mainly regulated by Rho-guanine nucleotide exchange factors (Rho GEF), which promote the cycling of GDP-bound inactive RhoA toward GTP-bound active RhoA. The active, GTP-bound form triggers the activation of its effector Rho kinase, which phosphorylates the myosin phosphatase target subunit (MYPT) and thereby inhibits the activity of myosin light chain phosphatase. This inhibition results in Ca2+ sensitization of contractile proteins, which underlies the tonic component of vascular smooth muscle contraction. Previous studies in our laboratory showed that RhoAYRho kinase pathway played an important role in the regulation of vascular reactivity following hemorrhagic shock.18Y20 As mentioned earlier, we hypothesized that Bkca opener, NS1619 pretreatment could induce the protection on vascular reactivity and fight against the subsequent hemorrhagic shock and that it is possibly related to RhoAYRho kinase pathway. To confirm this hypothesis, with hemorrhagic shock rats and isolated superior mesenteric artery (SMA), the protective effects of different dosages of NS1619 on animal survival, the cardiac output (CO) and oxygen delivery (DO2) and the vascular reactivity and calcium sensitivity of SMA and its relationship to RhoA and Rho kinase, and the underlying mechanisms were observed. The main purposes were to elucidate this issue and the corresponding mechanisms and to provide more selections to prevent hemorrhage or shock-induced cardiovascular dysfunction such vascular hyporeactivity in the clinic.
Hu et al.
(MAP), exsanguinating and drug administration, two salinefilled catheters were respectively inserted into the right femoral artery and vein. To prevent the clot formation, the rats were injected with heparin (500 U/kg) through the venous catheter after catheterization. The body temperature of the rats was maintained at 37-C with a heating pad.
Experimental Protocols After the surgical procedures, different dosages of Bkca opener, NS1619 (0.5, 1, 2, and 4 mg/kg) were given intraperitoneally to the rats. Thirty minutes later after NS1619 administration, the rats were hemorrhaged by femoral artery catheter to MAP at 40 mm Hg and maintained at this MAP level for different time points (10 minutes, 0.5 hour, 1 hour, 2 hours, or 3 hours). Hemorrhagic shock rats and their isolated SMAs were used for the following experiments: (1) effects of NS1619 pretreatment on animal survival; (2) effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity; (3) effects of NS1619 pretreatment on CO, DO2 and utilization (VO2); (4) effects of RhoA and Rho kinase inhibitor or antagonist on the protective effect of NS1619 pretreatment on vascular reactivity and calcium sensitivity; (5) effect of NS1619 pretreatment on activity of Rho kinase following hemorrhagic shock and the relationship to RhoA; (6) effects of NS1619 pretreatment on activity of RhoA and Rho GEF following hemorrhagic shock in rats. The detailed protocol and methods for all variables measurement were described in supplemental materials, http://links.lww.com/TA/A347.
Statistical Analysis All data were expressed as mean (SD) of a number of experiments. The differences among experimental groups and time points were analyzed by one-way or two-way analysis of variance, followed by post hoc Tukey test. A p G 0.05 was considered significant, and p G 0.01 was considered very significant.
RESULTS MATERIALS AND METHODS Animals Sprague-Dawley rats, weighing 200 g to 250 g, were used for all experiments. Animals were housed under controlled conditions (22-C, 55Y65% humidity, and 12-hour light-dark cycle) and fasted for 12 hours but allowed water ad libitum before the experiment. All experimental procedures and protocols used in this study were reviewed and approved by the Research Council and Animal Care and Use Committee of Research Institute of Surgery, Daping Hospital, the Third Military Medical University. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Instrumentation and Surgical Procedure Rats were anesthetized with an intraperitoneal injection of 30-mg/kg sodium pentobarbital and repeated intraperitoneal injections of 15 mg/kg were given as needed. The total amount of sodium pentobarbital used was not more than 50 mg/kg. For measurement of mean arterial blood pressure
Effect of NS1619 Pretreatment on Animal Survival Only 2 of 10 rats in shock control group survived over 72 hours, and their mean (SD) survival time was 37.3 (18.2) hours. NS1619 pretreatment 1 mg/kg to 4 mg/kg increased the 72-hour survival rate and prolonged the survival time. The mean (SD) survival time were 40.8 (25.8) hours, 42.5 (21.7) hours, 60 (16.3) hours, and 50.5 (23.5) hours in 0.5, 1, 2, and 4-mg/kg NS1619 pretreatment groups, respectively. NS1619 pretreatment 2 mg/kg had the best results; the 72-hour survival rate was 7 (70%) of 10 rats, and the mean (SD) survival time was 60 (16.3) hours, which were significantly higher than that in shock control group and other NS1619 pretreatment group ( p G 0.05, Fig. 1A). Based on the results of this part, 2-mg/kg NS1619 pretreatment was used in the following experiment.
Effect of NS1619 Pretreatment on Vascular Reactivity As compared with the sham-operated group, the vascular reactivity of SMA in shock group was lightly increased at
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
395
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 1. Changes of the contractile response of SMA to NE after hemorrhagic shock in rats (B) and effects of NS1619 pretreatment on them (C and D) and animal survival (A). A, n = 10; B-D, n = 8. Emax, maximal contractive tension; pD2, jlog (50% effective concentration); sham, sham operated; SHK, shock control; NS, NS1619 pretreatment; SMA, superior mesenteric artery; 0, 5, 1, 2, 4: 0.5, 1, 2, 4 mg/Kg (AYC), data represent mean (SD). *p G 0.05, **p G 0.01 as compared with sham-operated group; #p G 0.05, ##p G 0.01 as compared with shock control group; $$p G 0.01 as compared with 10 -minute shock group.
early shock (10 minutes) and then significantly decreased (Fig. 1B). The cumulative dose-response curve of SMA to norepinephrine (NE) was significantly shifted to the right (data not shown here); the Emax values of NE was significantly decreased ( p G 0.01). They were decreased by 31.1%, 52.7%, and 61.9% as compared with sham-operated group at 1 hour, 2 hours, and 3 hours after shock, respectively ( p G 0.01, Fig. 1B). A 2-mg/kg NS1619 pretreatment significantly prevented from the decrease of vascular reactivity induced by shock. The Emax values of SMA to NE were increased 1.42-fold and 1.52-fold as compared with shock control group at 2 hours and 3 hours after shock, respectively ( p G 0.05 or p G 0.01; Fig. 1C), and the pD2s were also significantly higher than those in the shock control group ( p G 0.05 or p G 0.01; Fig. 1D)
Effects of NS1619 Pretreatment on Calcium Sensitivity As the same change as vascular reactivity following hemorrhagic shock, the calcium sensitivity of SMA was decreased as shock prolonged. The cumulative dose-response curve of SMA to Ca2+ was significantly shifted to the right (data not shown here), and the Emax of SMA to Ca2+ were decreased by 24.5%, 57.3%, and 76.9% as compared with the sham-operated group at 1 hour, 2 hours, 3 hours after shock, respectively ( p G 0.05 or p G 0.01; Fig. 2A) .The calcium sensitivity of the SMA in the shock control group did not increase significantly at early shock. A 2-mg/kg NS1619 pretreatment 396
significantly prevented from the decrease of calcium sensitivity induced by shock. The Emax values of SMA to Ca2+ were increased 1.59-fold and 2.45-fold as compared with shock control group at 2 hours and 3 hours after shock, respectively. ( p G 0.01, Fig. 2B). The pD2 did not significantly change after NS1619 pretreatment as compared with the shock control group (Fig 2C).
Effects of NS1619 Pretreatment on CO, DO2, and VO2 Three hours after shock, CO was significantly decreased. It was decreased by 59.3% as compared with the shamoperated group (p G 0.01). A 2-mg/kg NS1619 pretreatment significantly increased the CO; the increase rate was 40.7% as compared with the shock control group (p G 0.05, Fig 3A). DO2 and VO2 were also significantly decreased at 3 hours after shock. The decrease rates were 68.6% and 62.7%, respectively, as compared with sham-operated group ( p G 0.01). A 2-mg/kg NS1619 pretreatment slightly increased DO2 and VO2 of hemorrhagic shock rats; the increase rates were 34.8% and 33.3% as compared with shock control group, respectively (Fig 3B and C, p 9 0.05).
Effects of Rho Kinase Antagonist Y-27632 and RhoA Inhibitor C3 Transferase on the Protective Effect of NS1619 Pretreatment in Vascular Reactivity and Calcium Sensitivity As described earlier, NS1619 pretreatment prevented the decrease of vascular reactivity and calcium sensitivity * 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 2. Changes of the contractile response of SMA to Ca2+ after hemorrhagic shock in rats (A) and effects of NS1619 pretreatment on them (B and C). Emax, maximal contractive tension; pD2, jlog (50% effective concentration); sham, sham operated; SHK, shock control; NS, NS1619 pretreatment. Data represent mean (SD), n = 8 per group.*p G 0.05, **p G 0.01 as compared with sham-operated group; ##p G 0.01 as compared with shock control group; $p G 0.05, $$p G 0.01 as compared with 10-minute shock group.
induced by shock. This protective effect of NS1619 was antagonized by Rho kinase antagonist, Y-27632, and RhoA inhibitor, C3 transferase. It appeared that the cumulative doseresponse curves of SMA to NE and Ca2+ were all significantly shifted to the right (data not shown here). The Emax values of NE and Ca2+ were significantly decreased. The decrease rates were 70.9% and 33.6% for NE and 59.5% and 41.7% for Ca2+ in NS1619 +Y-27632 or C3 transferase group as compared with NS1619 alone ( p G 0.01, Fig. 4A and B). The pD2 of NE and Ca2+ in NS1619 + Y-27632 or C3 transferase group did not significantly change as compared with NS1619 alone (data not shown here).
Effect of NS1619 Pretreatment on Activity of Rho Kinase and Its Relationship to RhoA Three hours after shock, the activity of Rho kinase (the ratio of phosphorylated MYPT1 Thr850 and MYPT1) was significantly decreased. The decrease rate was 34% as compared with the sham-operated group ( p G 0.05). NS1619 pretreatment significantly increased the activity of Rho kinase (MYPT1 phosphorylation level); the increase rate was 68% as compared with the shock control group ( p G 0.05). RhoA inhibitor, C3 transferase (5 Hg/mL) antagonized the increase effect of NS1619 pretreatment on Rho kinase activity in the SMA ( p G 0.01) (Fig. 4C and D).
Effects of NS1619 Pretreatment on the Activity of RhoA and Rho GEF Changes of the RhoA Activity Three-hour hemorrhagic shock induced a significant decrease of the activity of RhoA as compared with the shamoperated group ( p G 0.05). NS1619 pretreatment (2 mg/kg) significantly increased the activity of RhoA; the increase rate was 23% as compared with shock control group ( p G 0.01) (Fig. 5A)
Changes of the Rho GEF Activity The activities of p115-Rho GEF and LARG did not significantly change after shock. While the activity of PDZ-Rho GEF was significantly decreased. The decrease rate was 32.1% as compared with sham-operated group ( p G 0.05). NS1619 pretreatment (2 mg/kg) significantly increased PDZ-Rho GEF activity; the increase rate was 59% as compared with shock control group ( p G 0.05) (Fig. 5BYD).
DISCUSSION Previous studies have demonstrated that vascular hyporesponsiveness plays important roles in the incidence, development, and the outcome of shock and reduces the effectiveness of zvasoactive agents in the therapy of shock. Studies have
Figure 3. Effects of NS1619 pretreatment on CO, DO2, and VO2 of tissue after hemorrhagic shock in rats. Data represent mean (SD), n = 8 per group. **p G 0.01 as compared with sham-operated group; #p G 0.05 as compared with shock control group. * 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
397
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 4. Effects of Y-27632 (Rho-kinase inhibitor) and C3 transferase (RhoA-specific inhibitor) on vascular reactivity (contractile response of SMA to NE) (A) and calcium sensitivity (contractile response of SMA to calcium) (B) after NS1619 pretreatment, and effect of NS1619 pretreatment on activity of Rho kinase following hemorrhagic shock (C) and the role of C3 transferase (D). Emax, maximal contractive tension; sham, sham operated; SHK, shock control; NS, NS1619 pretreatment. A and B, n = 8 per group; C and D, n = 3 per group. Data represent mean (SD). *p G 0.05, **p G 0.01 as compared with shock control group; #p G 0.05, ##p G 0.01 as compared with NS1619 pretreatment group; $p G 0.05, as compared with sham-operated group.
shown that IPC and PPC have good protective effects on shock- or ischemia-induced organ damage including the heart, kidney, liver, stomach, intestine, brain, and so on.4Y9,12,21 Recent
studies have shown that Bkca channel was involved in the cardioprotective effect of IPC.14Y17 Our recent study showed that IPC and pinacidil pretreatment significantly restored the
Figure 5. Effects of NS1619 pretreatment on activity of RhoA, p115-Rho GEF, PDZ-Rho GEF, and LARG following hemorrhagic shock (AYD). Sham, sham operated; SHK, shock control; NS, NS1619 pretreatment; RLU, relative luminescence units. mant-GTP, 2¶-(or-3¶)-O-(N-Methylanthraniloyl)Yguanosine- 5¶-triphosphate, trisodium salt. Data represent mean (SD), n =4 per group. *p G 0.05, as compared with sham-operated group; #p G 0.05, as compared with shock control group. 398
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
decreased vascular reactivity following hemorrhagic shock.12,21 However, whether the pretreatment of Bkca channel opener, NS1619 improves shock-induced vascular hyporeactivity and by which play antishock effect are not known. The present study showed that the pretreatment of Bkca opener NS1619 significantly improved shock-induced decrease of vascular reactivity and calcium sensitivity as well as increased the CO, DO2, and animal survival. The protective effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity were antagonized by RhoA and Rho kinase antagonist/inhibitor. Meanwhile, NS1619 pretreatment up-regulated the activities of RhoA, Rho kinase, and PDZ-Rho GEF. It was suggested that Bkca opener, NS1619 pretreatment has good protective effect on vascular reactivity and calcium sensitivity and by which plays a good beneficial effect on hemorrhagic shock. PDZ-Rho GEFYRhoAYRho kinase pathway may play an important role in this process. Bkca channels are distributed in many types of tissue cells and play very important roles in various physiologic processes such as neurotransmitter release, cell excitability, hormone secretion, and cardiovascular function regulation. NS1619 [1,3Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)2H-benzimidazol -2-one] is one of Bkca channel openers and belongs to synthetic benzimidazolone derivative.22 As the opener of Bkca channels, NS1619 have potential therapeutic implications in many diseases such as hypertension, coronary artery spasm, urinary incontinence, and several neurologic disorders, and so on. Recent studies showed that NS1619 pretreatment could induce the cardioprotection and neuroprotection in rats, mice, and canines.14Y17,23 In the present study, the results showed that NS1619 pretreatment not only improved the vascular reactivity but also increased the CO. The results suggested that NS1619 pretreatment plays beneficial effects on hemorrhagic shock not only coming from the improvement of vascular reactivity but also coming from the cardioprotection. The mechanisms of the protection afforded by NS1619 pretreatment have not been well understood. Most researchers thought that NS1619 pretreatmentYinduced protection is caused by the opening of Bkca channel. The opening of Bkca channel causes membrane hyperpolarization, which reduces the voltage-dependent Ca2+ influx by promotion K+ efflux and thus prevents Ca2+ overload; in contrast, the opening of mitochondrial Bkca channel improves mitochondrial adenosine triphosphate production and decreases the production of reactive oxygen species and Ca2+ overload in the mitochondria, thus playing beneficial effect. Recently, Gaspar et al.23 reported that NS1619 pretreatment induced the neuroprotection by activating the PI3KYAktYGsk3Asignaling axis and inhibiting the activation of caspases 3 and 7. In our present study, we found that the protective effect of NS1619 pretreatment on vascular reactivity after hemorrhagic shock was related to PDZ-Rho GEFYRhoAYRho kinase pathway. However, how NS1619 pretreatment activates PDZ-Rho GEF is unclear. NS1619 pretreatment could induce vasodilatation and mimics IPC.14,23 Studies showed that protein kinase C (PKC) and protein tyrosine kinase (PTK) are the important signal molecules involved in the mechanism of IPC.24,25 Meanwhile, a numerous studies reported that PKC and PTK could regulate the activity of Rho GEF.26,27 Thus, it is speculated that
Hu et al.
NS1619 pretreatment activation of PDZ-Rho GEF is possibly related to PKC and/or PTK. Of course, this speculation needs further confirmation. As the main regulator of RhoA activity, Rho GEFs include three isoformers as follows p115-Rho GEF, PDZRho GEF, and leukemia-associated Rho-specific GEF or LARG.28,29 Because of the multiplicity of Rho GEFs, it has been suggested that Rho GEFs involved in the activation of RhoA are probably different under different pathologic conditions. Christophe et al.30 showed that control of RhoA signaling through p115-Rho GEF is central to the development of angiotensin IIYdependent hypertension. Wirth et al.31 demonstrated that G>12-G>13/LARG signaling pathway played important roles in salt-induced hypertension. Our present study indicated that the activity of PDZ-Rho GEF was significantly decreased following shock, while the activities of p115-Rho GEF and LARG did not significantly change. NS1619 pretreatment significantly increased the activity PDZ-Rho GEF, but not p115-Rho GEF and LARG. The increase of the activity of PDZ-Rho GEF was positively correlated with the increase of Rho kinase and RhoA activities induced by NS1619 pretreatment. These results suggested that PDZ-Rho GEF might be the main isoformer that participates in the activation of RhoAYRho kinase pathway induced by NS1619 pretreatment (Fig. 6). To observe the roles of Rho kinase and RhoA in the NS1619-induced protective effect, we selected Y-27632 and C3 transferase as an inhibitor of Rho kinase and RhoA, respectively. Y-27632 inhibits Rho kinase by competing with adenosine triphosphate for binding to the catalytic site of Rho kinase.32 Our previous studies showed that incubation of SMA rings with 1 10j5 mol/L of Y-27632 for 10 minutes could significantly antagonize the effects of Rho kinase. Therefore, in the present study, 1 10j5 mol/L of Y-27632 was selected. C3 transferase inhibits RhoA proteins by adenosine diphosphate ribosylation on asparagine 41 in the effector-binding domain of the GTPase and blocks its translocation to the membrane.33 The major limitation of using C3 transferase is that it is not cell permeable. Therefore, we used cell-permeable C3 transferase in this study, which comprises a proprietary cell-penetrating moiety. This moiety allows transport through the plasma membrane rapidly and efficiently and is released in the cytosol; thereby, C3 transferase can freely diffuse within the cell and inactivate RhoA. Our previous study and present study all demonstrated that incubation of SMA rings with 5-mg/mL cellpermeable C3 transferase for 6 hours could significantly antagonize the effects of RhoA. Although PPC was suggested to be a safe method of eliciting protection against ischemia-reperfusion injury, their clinical application has been restricted in many situations, in which the hemorrhage is unpredictable. However, some scheduled surgical operations with possible hemorrhage are suitable for the pretreatment intervention. Thus, NS1619 pretreatment could be used in the preoperative preparation to offer protection against the surgical operationYinduced severe hemorrhage. In addition, NS1619 pretreatment has been demonstrated to induce cardioprotection and neuroprotection.14,15,17,23 Therefore, NS1619 pretreatment may have great potentials to be used in organ transplantation, cardiopulmonary bypass surgery and cerebral surgery, and so on.
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
399
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 6. A schematic signal transduction of RhoAYRho kinase pathway in the regulation of vascular reactivity after NS1619 pretreatment. NS1619 pretreatment could induce a light vasodilation and mimics the IPC, which evokes the activation of endogenous protection mechanisms. The endogenous protective substances such as adenosine activate Rho GEF via G protein coupled receptor and then activate RhoA. RhoA activates Rho kinase and then increases the phosphorylation of MLC20 and increase the calcium sensitivity and vascular reactivity via inhibition of myosin light chain phosphatase. GPCR, G protein coupled receptor; RhoGDI, guanine nucleotide dissociation inhibitor of RhoA; MLCP, myosin light chain phosphatase; MLC20, 20-kD myosin light chain; C3, C3 transferase, the inhibitor of RhoA, which can inhibit RhoA activity and block RhoA translocation to the membrane;Y-27632, a selective antagonist of Rho kinase.
Although this study showed that NS1619 pretreatment could improve vascular reactivity and calcium sensitivity after hemorrhagic shock and that the PDZ-Rho GEFYRhoAYRho kinase pathway played an important role in this process, there are still some issues that need to be answered in the future. First, this study was limited to the small animals, and whether the protective effect of NS1619 pretreatment would be duplicated in large animals, even in human beings, needs further investigation. Second, the preliminary mechanism of the protective effect of NS1619 pretreatment on shock-induced vascular hyporeactivity was investigated in this study, but the detailed mechanism of how NS1619 pretreatment activates PDZ-Rho GEF also needs further exploration. Third, NS1619 was shown to inhibit L-type calcium channels and voltagedependent channels.34,35 Moreover, NS1619 can also induce Ca2+ release from intracellular pools.36 Therefore, further study is needed to confirm whether the protective effect of NS1619 pretreatment on vascular reactivity is related to calcium channel and calcium release.
play antishock effect. PDZ-Rho GEFYRhoAYRho kinase pathway plays important role in the protective effect of NS1619 pretreatment on vascular reactivity. Among three Rho GEFs, PDZ-Rho GEF is the only one involved in the protective effect of NS1619 pretreatment. Bkca may be a potential target to treat shock-induced vascular hyporeactivity. AUTHORSHIP Y.H. performed the experiment, the data collection, and manuscript preparation. G.Y. and X.X. participated in the animal study and data analysis. T.L. and L.L. conceived of the study, participated in the design and coordination, and edited the manuscript. All authors read and approved the final manuscript.
DISCLOSURE This work was supported by the National Natural Science Foundation of China (81071540), Major State Basic Research Program of China (2012CB518101), and Project of State Key Laboratory of Trauma, Burns and Combined Injury (SKLZZ200901, SKLZZ200912).
CONCLUSION
REFERENCES
The pretreatment of Bkca opener, NS1619 could improve the vascular reactivity and cardiac function and by which
1. Liu LM, Ward JA, Dubick MA. Hemorrhage-induced vascular hyporeactivity to norepinephrine in select vasculatures of rats and the roles of nitric oxide and endothelin. Shock. 2003;19:208Y214.
400
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
2. Liu LM, Dubick MA. Hemorrhagic shock-induced vascular hyporeactivity in the rat: relationship to gene expression of nitric oxide synthase, endothelin-1, and select cytokines in corresponding organs. J Surg Res. 2005;125:128Y136. 3. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemia myocardium. Circulation. 1986; 74:1124Y1136. 4. Rodrigo GC, Samani NJ. Ischemic preconditioning of the whole heart confers protection on subsequently isolated ventricular myocytes. Am J Physiol Heart Circ Physiol. 2008;294:524Y531. 5. Jia RP, Zhu JG, Wu JP, Xie JJ, Xu LW. Experimental study on early protective effect of ischemic preconditioning on rat kidney graft. Transplant Proc. 2009;41:69Y72. 6. Suzuki S, Inaba K, Konno H. Ischemic preconditioning in hepatic ischemia and reperfusion. Curr Opin Organ Transplant. 2008;13:142Y147. 7. Bobryshev P, Bagaeva T, Filaretova L. Ischemic preconditioning attenuates gastric ischemia-reperfusion injury through involvement of glucocorticoids. J Physiol Pharmacol. 2009;60:155Y160. 8. Pang CY, Neligan P, Zhong A, Xu H, Forrest CR. Effector mechanism of adenosine in acute ischemic preconditioning of skeletal muscle against infarction. Am J Physiol. 1997;273:887Y895. 9. Gu¨lu¨zar Y, Abdullah TD, Bu¨lent GHoward L. Ischemic preconditioning modulates ischemia-reperfusion injury in the rat lung: role of adenosine receptors. Eur J Pharmacol. 2007;556:144Y150. 10. Jin Z, Duan W, Chen M, Yu S, Zhang H, Feng G, Xiong L, Yi D. The myocardial protective effects of adenosine pretreatment in children undergoing cardiac surgery: a randomized controlled clinical trial. Eur J Cardiothorac Surg. 2011;39:90Y96. 11. Yang QZ, Yan WJ, Li X, Hou LH, Dong H, Wang Q, Dong HL, Wang SQ, Zhang X, Xiong LZ. Activation of canonical Notch signaling pathway is involved in the ischemic tolerance induced by sevoflurane preconditioning in mice. Anesthesiology. 2012;117:996Y1015. 12. Xu J, Li T, Yang G, Liu LM. Pinacidil pretreatment improves vascular reactivity after shock through PKC> and PKCD in rats. J Cardiovasc Pharmacol. 2012;59:514Y522. 13. Zhou R, Liu LM, Hu DY. Involvement of Bkca > subunit tyrosine phosphorylation in vascular hyporesponsiveness of superior mesenteric artery following hemorrhagic shock in rats. Cardio Res. 2005;68:327Y335. 14. Xu W, Liu Y, Wang S, McDonald T, Van EJ, Sidor A, O’Rourke B. Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science. 2002;298:1029Y1033. 15. Shintani Y, Node K, Asanuma H, Sanada S, Takashima S, Asano Y, Liao Y, Fujita M, Hirata A, Shinozaki Y, et al. Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts. J Mol Cell Cardiol. 2004;37:1213Y1218. 16. Malinska D, Mirandola SR, Kunz WS. Mitochondrial potassium channels and reactive oxygen species. FEBS Lett. 2010;584:2043Y2048. 17. Redel A, Lange M, Jazbutyte V, Lotz C, Smul TM, Roewer N, Kehl F. Activation of mitochondrial large-conductance calcium-activated K+ channels via protein kinase A mediates desflurane-induced preconditioning. Anesth Analg. 2008;106:384Y391. 18. Xu J, Liu LM. The role of calcium desensitization in vascular hyporeactivity and its regulation following hemorrhagic shock in the rat. Shock. 2005; 23:576Y581. 19. Li T, Liu L, Xu J, Yang GM, Ming J. Changes of Rho-kinase activity after hemorrhagic shock and its role in shock-induced biphasic response of vascular reactivity and calcium sensitivity. Shock. 2006;26:504Y509. 20. Li T, Fang YQ, Yang GM, Xu J, Zhu Y, Liu LM. Effects of the balance in activity of RhoA and Rac1 on the shock-induced biphasic change of vascular reactivity in rats. Ann Surg. 2011;253:185Y193.
Hu et al.
21. Hu Y, Li T, Tang XF, Chen K, Liu LM. Effects of ischemic preconditioning on vascular reactivity and calcium sensitivity after hemorrhagic shock and their relationship to the RhoAYRhokinase pathway in rats. J Cardiovasc Pharmacol. 2011;57:231Y239. 22. Srinivas G, Deepthi N, Xiaoping X. Large-conductance, calcium-activated potassium channels: structural and functional implications. Pharmacol Ther. 2006;110:103Y116. 23. Gaspar T, Katakam P, Snipes JA, Kis B, Domoki F, Bari F, Busija DW. Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels. J Neurochem. 2008;105:1115Y1128. 24. Yun N, Kim SH, Lee SM. Differential consequences of protein kinase C activation during early and late hepatic ischemic preconditioning. J Physiol Sci. 2012;62(3):199Y209. 25. Tang XL, Kodani E, Takano H, Hil M, Shinmura K, Vondriska TM, Ping P, Bolli R. Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning. Am J Physiol Heart Circ Physiol. 2003;284(4):H1441YH1448. 26. Chikumi H, Fukuhara S, Gutkind JS. Regulation of G proteinYlinked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase. J Biol Chem. 2002;277(14):12463Y12473. 27. Peng J, He F, Zhang C, Deng X, Yin F. Protein kinase C-> signals P115RhoGEF phosphorylation and RhoA activation in TNF->Yinduced mouse brain microvascular endothelial cell barrier dysfunction. J Neuroinflammation. 2011;8:28. 28. Fukuhara S, Chikumi H, Gutkind JS. RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene. 2001;20:1661Y1668. 29. Jaiswal M, Gremer L, Dvorsky R, Haeusler LC, Cirstea IC, Uhlenbrock K, Ahmadian MR. Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G-protein signaling (RGS)Ycontaining Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZRhoGEF (PRG), and leukemia-associated RhoGEF (LARG). J Biol Chem. 2011;286:18202Y18212. 30. Guilluy C, Bre´geon J, Toumaniantz G, Rolli-Dekinderen M, Retaillean K, Lourfrani L, Henrion D, Scalbert E, Bril A, Torres RM, et al. The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nat Med. 2010;16(2):183Y190. 31. Wirth A, Benyo´ Z, Lukasova M, Leutgeb B, Wettschureck N, Gorbey S, Orsy P, Horva´th B, Marser-Glath C, Greiner E, et al. G12-G13LARGYmediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat Med. 2008;14: 64Y68. 32. Somlyo AP, Somlyo AV. Signal transduction by G protein, Rho kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol. 2000;522:177Y185. 33. Aktories K, Wilde C, Vogelsges ang M. Rho-modifying C3-like ADPribosyltransferases. Rev Physiol Biochem Pharmacol. 2004;152:1Y22. 34. Edwards G, Niederste-Hollenberg A, Schneider J, Noack T, Weston AH. Ion channel modulation by NS 1619, the putative Bkca channel opener, in vascular smooth muscle. Br J Pharmacol. 1994;113:1538Y1547. 35. Park WS, Kang SH, Son YK, Kim N, Ko JH, Kim HK, Ko EA, Kim CD, Han J. The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes. Biochem Biophys Res Commun. 2007;362:31Y36. 36. Yamamura H, Ohi Y, Muraki K, Watanabe M, Imaizumi Y. BK channel activation by NS-1619 is partially mediated by intra-cellular Ca2+ release in smooth muscle cells of porcine coronary artery. Br J Pharmacol. 2001;132:828Y834.
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
401
Bkca opener, NS1619 pretreatment protects against shock-induced vascular hyporeactivity through PDZ-Rho GEFYRhoAYRho kinase pathway in rats Yi Hu, MD, Guangming Yang, MD, Xudong Xiao, MD, Liangming Liu, MD, PhD, and Tao Li, MD, PhD, Chongqing, China
BACKGROUND: Our previous study showed that the ischemic preconditioning and pretreatment of adenosine triphosphateYsensitive potassium channel (KATP) opener, pinacidil, may induce a good protective effect on shock-induced vascular hyporeactivity. Whether the pretreatment of opener/activator of the large-conductance calcium-activated potassium channel (Bkca), NS1619, can also induce a protective effect on vascular reactivity and play a beneficial effect on subsequent hemorrhagic shock is not clear. METHODS: With Sprague-Dawley rats subjected to hemorrhagic shock and their isolated superior mesenteric artery, the protective effect of NS1619 (0.5, 1, 2, and 4 mg/kg) pretreatment (30 minutes before hemorrhage shock) on vascular reactivity and the underlying mechanisms were observed. RESULTS: NS1619 pretreatment significantly improved the 72-hour survival of hemorrhagic shock rats, alleviated shock-induced decrease of vascular reactivity and calcium sensitivity, and increased the cardiac output and oxygen delivery. NS1619 2 mg/kg had the best effect. These protective effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity were antagonized by RhoA inhibitor, C3 transferase, and Rho kinase antagonist, Y-27632. NS1619 pretreatment up-regulated the activities of RhoA, Rho-kinase, and PDZYRho GEF (guanine nucleotide exchange factor). These effects of NS1619 pretreatment were eliminated by RhoA inhibitor, C3 transferase. CONCLUSION: Bkca opener, NS1619 pretreatment has good protective effect on vascular reactivity and calcium sensitivity, which plays a good beneficial effect on hemorrhagic shock. The mechanism may be mainly through PDZ-Rho GEFYRhoAYRho kinase pathway. Bkca channel may be a potential target for the treatment of shock-induced vascular hyporeactivity. (J Trauma Acute Care Surg. 2014;76: 394Y401. Copyright * 2014 by Lippincott Williams & Wilkins) KEY WORDS: NS1619; vascular reactivity; hemorrhagic shock; Rho-kinase; rats.
P
revious studies, in our laboratory and those of others, have demonstrated that after severe trauma or shock, the response of peripheral blood vessels to vasoconstrictors and vasodilators is greatly reduced, which is called vascular hyporeactivity, and seriously interferes with the development, outcome, and therapy of the shock.1,2 Therefore, it is very important to study the therapy for shock-induced vascular hyporeactivity. Since the early observations found that a brief, sublethal episode of myocardial ischemia can markedly decrease the infarct size induced by sustained severe ischemia,3 a number of studies have demonstrated that ischemic preconditioning (IPC) has significant protective effects in many organs and species.4Y9 However, the application of IPC is limited in clinical settings because, actually, hemorrhage in many situations is unpredictable. Submitted: May 8, 2013, Revised: August 20, 2013, Accepted: August 22, 2013. Published online: January 6, 2014. From the State Key Laboratory of Trauma, Burns and Combined Injury (Y.H., G.Y., X.X., L.L., T.L.), Second Department of Research Institute of Surgery, and Department of Anesthesiology (Y.H.), Daping Hospital, Third Military Medical University, Chongqing, China. *L.L. and T.L. contributed equally to this work. Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jtrauma.com). Address for reprints: Tao Li, MD, PhD, and Liangming Liu, MD, PhD, Second Department of Research Institute of Surgery, Daping Hospital, Third Military Medical University, Daping, Chongqing 400042, China; email: [email protected]; [email protected]. DOI: 10.1097/TA.0b013e3182aa2d98
394
Thus, pharmacologic preconditioning (PPC) represents an ideal alternative to IPC. For example, Jin et al.10 reported that adenosine pretreatment protected against myocardial ischemiareperfusion injury in children undergoing cardiopulmonary bypass (CPB) for repair of congenital heart defects. Yang et al.11 reported that sevoflurane preconditioning reduced brain infarct volumes, improved neurologic outcomes, and attenuated the neuronal cell apoptosis, which indicated that sevoflurane preconditioning could induce the protective effects against transient cerebral ischemic injuries. Our previous study showed that pretreatment of adenosine triphosphateYsensitive potassium channel (KATP) opener, pinacidil, could induce a good protective effect against shockinduced vascular hyporeactivity12 and that another major K+ channel, large-conductance calcium-activated potassium channel (Bkca) in vascular smooth muscle cell (VSMC) played an important role in the occurrence of vascular hyporeactivity following shock.13 However, whether Bkca opener, NS1619 pretreatment could induce the protection of vascular reactivity against the subsequent hemorrhagic shock and the underlying mechanisms are not known. Bkca channels are composed of tetrameric sets consisting of a pore-forming >-subunit and an auxiliary A-subunit, and they play very important roles in various physiologic processes such as neurotransmitter release, cell excitability, hormone secretion, and cardiovascular function regulation. Recent studies have shown that Bkca channel was involved in J Trauma Acute Care Surg Volume 76, Number 2
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
cardioprotection of IPC.14Y17 Xu et al.14 found Bkca channel opener, NS1619 pretreatment caused a significant reduction in infarct size in rabbit heart, and such effect was antagonized by the Bkca channel blocker, paxilline. Redel et al. reported that the activation of Bkca channels by NS1619 mimicked the cardioprotection induced by IPC.17 So we hypothesized that Bkca opener, NS1619 pretreatment could induce the protection on vascular reactivity against the subsequent hemorrhagic shock. RhoA is a molecular switch that cycles between an inactive, GDP-bound form and an active, GTP-bound form. The activity of RhoA is mainly regulated by Rho-guanine nucleotide exchange factors (Rho GEF), which promote the cycling of GDP-bound inactive RhoA toward GTP-bound active RhoA. The active, GTP-bound form triggers the activation of its effector Rho kinase, which phosphorylates the myosin phosphatase target subunit (MYPT) and thereby inhibits the activity of myosin light chain phosphatase. This inhibition results in Ca2+ sensitization of contractile proteins, which underlies the tonic component of vascular smooth muscle contraction. Previous studies in our laboratory showed that RhoAYRho kinase pathway played an important role in the regulation of vascular reactivity following hemorrhagic shock.18Y20 As mentioned earlier, we hypothesized that Bkca opener, NS1619 pretreatment could induce the protection on vascular reactivity and fight against the subsequent hemorrhagic shock and that it is possibly related to RhoAYRho kinase pathway. To confirm this hypothesis, with hemorrhagic shock rats and isolated superior mesenteric artery (SMA), the protective effects of different dosages of NS1619 on animal survival, the cardiac output (CO) and oxygen delivery (DO2) and the vascular reactivity and calcium sensitivity of SMA and its relationship to RhoA and Rho kinase, and the underlying mechanisms were observed. The main purposes were to elucidate this issue and the corresponding mechanisms and to provide more selections to prevent hemorrhage or shock-induced cardiovascular dysfunction such vascular hyporeactivity in the clinic.
Hu et al.
(MAP), exsanguinating and drug administration, two salinefilled catheters were respectively inserted into the right femoral artery and vein. To prevent the clot formation, the rats were injected with heparin (500 U/kg) through the venous catheter after catheterization. The body temperature of the rats was maintained at 37-C with a heating pad.
Experimental Protocols After the surgical procedures, different dosages of Bkca opener, NS1619 (0.5, 1, 2, and 4 mg/kg) were given intraperitoneally to the rats. Thirty minutes later after NS1619 administration, the rats were hemorrhaged by femoral artery catheter to MAP at 40 mm Hg and maintained at this MAP level for different time points (10 minutes, 0.5 hour, 1 hour, 2 hours, or 3 hours). Hemorrhagic shock rats and their isolated SMAs were used for the following experiments: (1) effects of NS1619 pretreatment on animal survival; (2) effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity; (3) effects of NS1619 pretreatment on CO, DO2 and utilization (VO2); (4) effects of RhoA and Rho kinase inhibitor or antagonist on the protective effect of NS1619 pretreatment on vascular reactivity and calcium sensitivity; (5) effect of NS1619 pretreatment on activity of Rho kinase following hemorrhagic shock and the relationship to RhoA; (6) effects of NS1619 pretreatment on activity of RhoA and Rho GEF following hemorrhagic shock in rats. The detailed protocol and methods for all variables measurement were described in supplemental materials, http://links.lww.com/TA/A347.
Statistical Analysis All data were expressed as mean (SD) of a number of experiments. The differences among experimental groups and time points were analyzed by one-way or two-way analysis of variance, followed by post hoc Tukey test. A p G 0.05 was considered significant, and p G 0.01 was considered very significant.
RESULTS MATERIALS AND METHODS Animals Sprague-Dawley rats, weighing 200 g to 250 g, were used for all experiments. Animals were housed under controlled conditions (22-C, 55Y65% humidity, and 12-hour light-dark cycle) and fasted for 12 hours but allowed water ad libitum before the experiment. All experimental procedures and protocols used in this study were reviewed and approved by the Research Council and Animal Care and Use Committee of Research Institute of Surgery, Daping Hospital, the Third Military Medical University. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health.
Instrumentation and Surgical Procedure Rats were anesthetized with an intraperitoneal injection of 30-mg/kg sodium pentobarbital and repeated intraperitoneal injections of 15 mg/kg were given as needed. The total amount of sodium pentobarbital used was not more than 50 mg/kg. For measurement of mean arterial blood pressure
Effect of NS1619 Pretreatment on Animal Survival Only 2 of 10 rats in shock control group survived over 72 hours, and their mean (SD) survival time was 37.3 (18.2) hours. NS1619 pretreatment 1 mg/kg to 4 mg/kg increased the 72-hour survival rate and prolonged the survival time. The mean (SD) survival time were 40.8 (25.8) hours, 42.5 (21.7) hours, 60 (16.3) hours, and 50.5 (23.5) hours in 0.5, 1, 2, and 4-mg/kg NS1619 pretreatment groups, respectively. NS1619 pretreatment 2 mg/kg had the best results; the 72-hour survival rate was 7 (70%) of 10 rats, and the mean (SD) survival time was 60 (16.3) hours, which were significantly higher than that in shock control group and other NS1619 pretreatment group ( p G 0.05, Fig. 1A). Based on the results of this part, 2-mg/kg NS1619 pretreatment was used in the following experiment.
Effect of NS1619 Pretreatment on Vascular Reactivity As compared with the sham-operated group, the vascular reactivity of SMA in shock group was lightly increased at
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
395
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 1. Changes of the contractile response of SMA to NE after hemorrhagic shock in rats (B) and effects of NS1619 pretreatment on them (C and D) and animal survival (A). A, n = 10; B-D, n = 8. Emax, maximal contractive tension; pD2, jlog (50% effective concentration); sham, sham operated; SHK, shock control; NS, NS1619 pretreatment; SMA, superior mesenteric artery; 0, 5, 1, 2, 4: 0.5, 1, 2, 4 mg/Kg (AYC), data represent mean (SD). *p G 0.05, **p G 0.01 as compared with sham-operated group; #p G 0.05, ##p G 0.01 as compared with shock control group; $$p G 0.01 as compared with 10 -minute shock group.
early shock (10 minutes) and then significantly decreased (Fig. 1B). The cumulative dose-response curve of SMA to norepinephrine (NE) was significantly shifted to the right (data not shown here); the Emax values of NE was significantly decreased ( p G 0.01). They were decreased by 31.1%, 52.7%, and 61.9% as compared with sham-operated group at 1 hour, 2 hours, and 3 hours after shock, respectively ( p G 0.01, Fig. 1B). A 2-mg/kg NS1619 pretreatment significantly prevented from the decrease of vascular reactivity induced by shock. The Emax values of SMA to NE were increased 1.42-fold and 1.52-fold as compared with shock control group at 2 hours and 3 hours after shock, respectively ( p G 0.05 or p G 0.01; Fig. 1C), and the pD2s were also significantly higher than those in the shock control group ( p G 0.05 or p G 0.01; Fig. 1D)
Effects of NS1619 Pretreatment on Calcium Sensitivity As the same change as vascular reactivity following hemorrhagic shock, the calcium sensitivity of SMA was decreased as shock prolonged. The cumulative dose-response curve of SMA to Ca2+ was significantly shifted to the right (data not shown here), and the Emax of SMA to Ca2+ were decreased by 24.5%, 57.3%, and 76.9% as compared with the sham-operated group at 1 hour, 2 hours, 3 hours after shock, respectively ( p G 0.05 or p G 0.01; Fig. 2A) .The calcium sensitivity of the SMA in the shock control group did not increase significantly at early shock. A 2-mg/kg NS1619 pretreatment 396
significantly prevented from the decrease of calcium sensitivity induced by shock. The Emax values of SMA to Ca2+ were increased 1.59-fold and 2.45-fold as compared with shock control group at 2 hours and 3 hours after shock, respectively. ( p G 0.01, Fig. 2B). The pD2 did not significantly change after NS1619 pretreatment as compared with the shock control group (Fig 2C).
Effects of NS1619 Pretreatment on CO, DO2, and VO2 Three hours after shock, CO was significantly decreased. It was decreased by 59.3% as compared with the shamoperated group (p G 0.01). A 2-mg/kg NS1619 pretreatment significantly increased the CO; the increase rate was 40.7% as compared with the shock control group (p G 0.05, Fig 3A). DO2 and VO2 were also significantly decreased at 3 hours after shock. The decrease rates were 68.6% and 62.7%, respectively, as compared with sham-operated group ( p G 0.01). A 2-mg/kg NS1619 pretreatment slightly increased DO2 and VO2 of hemorrhagic shock rats; the increase rates were 34.8% and 33.3% as compared with shock control group, respectively (Fig 3B and C, p 9 0.05).
Effects of Rho Kinase Antagonist Y-27632 and RhoA Inhibitor C3 Transferase on the Protective Effect of NS1619 Pretreatment in Vascular Reactivity and Calcium Sensitivity As described earlier, NS1619 pretreatment prevented the decrease of vascular reactivity and calcium sensitivity * 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 2. Changes of the contractile response of SMA to Ca2+ after hemorrhagic shock in rats (A) and effects of NS1619 pretreatment on them (B and C). Emax, maximal contractive tension; pD2, jlog (50% effective concentration); sham, sham operated; SHK, shock control; NS, NS1619 pretreatment. Data represent mean (SD), n = 8 per group.*p G 0.05, **p G 0.01 as compared with sham-operated group; ##p G 0.01 as compared with shock control group; $p G 0.05, $$p G 0.01 as compared with 10-minute shock group.
induced by shock. This protective effect of NS1619 was antagonized by Rho kinase antagonist, Y-27632, and RhoA inhibitor, C3 transferase. It appeared that the cumulative doseresponse curves of SMA to NE and Ca2+ were all significantly shifted to the right (data not shown here). The Emax values of NE and Ca2+ were significantly decreased. The decrease rates were 70.9% and 33.6% for NE and 59.5% and 41.7% for Ca2+ in NS1619 +Y-27632 or C3 transferase group as compared with NS1619 alone ( p G 0.01, Fig. 4A and B). The pD2 of NE and Ca2+ in NS1619 + Y-27632 or C3 transferase group did not significantly change as compared with NS1619 alone (data not shown here).
Effect of NS1619 Pretreatment on Activity of Rho Kinase and Its Relationship to RhoA Three hours after shock, the activity of Rho kinase (the ratio of phosphorylated MYPT1 Thr850 and MYPT1) was significantly decreased. The decrease rate was 34% as compared with the sham-operated group ( p G 0.05). NS1619 pretreatment significantly increased the activity of Rho kinase (MYPT1 phosphorylation level); the increase rate was 68% as compared with the shock control group ( p G 0.05). RhoA inhibitor, C3 transferase (5 Hg/mL) antagonized the increase effect of NS1619 pretreatment on Rho kinase activity in the SMA ( p G 0.01) (Fig. 4C and D).
Effects of NS1619 Pretreatment on the Activity of RhoA and Rho GEF Changes of the RhoA Activity Three-hour hemorrhagic shock induced a significant decrease of the activity of RhoA as compared with the shamoperated group ( p G 0.05). NS1619 pretreatment (2 mg/kg) significantly increased the activity of RhoA; the increase rate was 23% as compared with shock control group ( p G 0.01) (Fig. 5A)
Changes of the Rho GEF Activity The activities of p115-Rho GEF and LARG did not significantly change after shock. While the activity of PDZ-Rho GEF was significantly decreased. The decrease rate was 32.1% as compared with sham-operated group ( p G 0.05). NS1619 pretreatment (2 mg/kg) significantly increased PDZ-Rho GEF activity; the increase rate was 59% as compared with shock control group ( p G 0.05) (Fig. 5BYD).
DISCUSSION Previous studies have demonstrated that vascular hyporesponsiveness plays important roles in the incidence, development, and the outcome of shock and reduces the effectiveness of zvasoactive agents in the therapy of shock. Studies have
Figure 3. Effects of NS1619 pretreatment on CO, DO2, and VO2 of tissue after hemorrhagic shock in rats. Data represent mean (SD), n = 8 per group. **p G 0.01 as compared with sham-operated group; #p G 0.05 as compared with shock control group. * 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
397
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 4. Effects of Y-27632 (Rho-kinase inhibitor) and C3 transferase (RhoA-specific inhibitor) on vascular reactivity (contractile response of SMA to NE) (A) and calcium sensitivity (contractile response of SMA to calcium) (B) after NS1619 pretreatment, and effect of NS1619 pretreatment on activity of Rho kinase following hemorrhagic shock (C) and the role of C3 transferase (D). Emax, maximal contractive tension; sham, sham operated; SHK, shock control; NS, NS1619 pretreatment. A and B, n = 8 per group; C and D, n = 3 per group. Data represent mean (SD). *p G 0.05, **p G 0.01 as compared with shock control group; #p G 0.05, ##p G 0.01 as compared with NS1619 pretreatment group; $p G 0.05, as compared with sham-operated group.
shown that IPC and PPC have good protective effects on shock- or ischemia-induced organ damage including the heart, kidney, liver, stomach, intestine, brain, and so on.4Y9,12,21 Recent
studies have shown that Bkca channel was involved in the cardioprotective effect of IPC.14Y17 Our recent study showed that IPC and pinacidil pretreatment significantly restored the
Figure 5. Effects of NS1619 pretreatment on activity of RhoA, p115-Rho GEF, PDZ-Rho GEF, and LARG following hemorrhagic shock (AYD). Sham, sham operated; SHK, shock control; NS, NS1619 pretreatment; RLU, relative luminescence units. mant-GTP, 2¶-(or-3¶)-O-(N-Methylanthraniloyl)Yguanosine- 5¶-triphosphate, trisodium salt. Data represent mean (SD), n =4 per group. *p G 0.05, as compared with sham-operated group; #p G 0.05, as compared with shock control group. 398
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
decreased vascular reactivity following hemorrhagic shock.12,21 However, whether the pretreatment of Bkca channel opener, NS1619 improves shock-induced vascular hyporeactivity and by which play antishock effect are not known. The present study showed that the pretreatment of Bkca opener NS1619 significantly improved shock-induced decrease of vascular reactivity and calcium sensitivity as well as increased the CO, DO2, and animal survival. The protective effects of NS1619 pretreatment on vascular reactivity and calcium sensitivity were antagonized by RhoA and Rho kinase antagonist/inhibitor. Meanwhile, NS1619 pretreatment up-regulated the activities of RhoA, Rho kinase, and PDZ-Rho GEF. It was suggested that Bkca opener, NS1619 pretreatment has good protective effect on vascular reactivity and calcium sensitivity and by which plays a good beneficial effect on hemorrhagic shock. PDZ-Rho GEFYRhoAYRho kinase pathway may play an important role in this process. Bkca channels are distributed in many types of tissue cells and play very important roles in various physiologic processes such as neurotransmitter release, cell excitability, hormone secretion, and cardiovascular function regulation. NS1619 [1,3Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)2H-benzimidazol -2-one] is one of Bkca channel openers and belongs to synthetic benzimidazolone derivative.22 As the opener of Bkca channels, NS1619 have potential therapeutic implications in many diseases such as hypertension, coronary artery spasm, urinary incontinence, and several neurologic disorders, and so on. Recent studies showed that NS1619 pretreatment could induce the cardioprotection and neuroprotection in rats, mice, and canines.14Y17,23 In the present study, the results showed that NS1619 pretreatment not only improved the vascular reactivity but also increased the CO. The results suggested that NS1619 pretreatment plays beneficial effects on hemorrhagic shock not only coming from the improvement of vascular reactivity but also coming from the cardioprotection. The mechanisms of the protection afforded by NS1619 pretreatment have not been well understood. Most researchers thought that NS1619 pretreatmentYinduced protection is caused by the opening of Bkca channel. The opening of Bkca channel causes membrane hyperpolarization, which reduces the voltage-dependent Ca2+ influx by promotion K+ efflux and thus prevents Ca2+ overload; in contrast, the opening of mitochondrial Bkca channel improves mitochondrial adenosine triphosphate production and decreases the production of reactive oxygen species and Ca2+ overload in the mitochondria, thus playing beneficial effect. Recently, Gaspar et al.23 reported that NS1619 pretreatment induced the neuroprotection by activating the PI3KYAktYGsk3Asignaling axis and inhibiting the activation of caspases 3 and 7. In our present study, we found that the protective effect of NS1619 pretreatment on vascular reactivity after hemorrhagic shock was related to PDZ-Rho GEFYRhoAYRho kinase pathway. However, how NS1619 pretreatment activates PDZ-Rho GEF is unclear. NS1619 pretreatment could induce vasodilatation and mimics IPC.14,23 Studies showed that protein kinase C (PKC) and protein tyrosine kinase (PTK) are the important signal molecules involved in the mechanism of IPC.24,25 Meanwhile, a numerous studies reported that PKC and PTK could regulate the activity of Rho GEF.26,27 Thus, it is speculated that
Hu et al.
NS1619 pretreatment activation of PDZ-Rho GEF is possibly related to PKC and/or PTK. Of course, this speculation needs further confirmation. As the main regulator of RhoA activity, Rho GEFs include three isoformers as follows p115-Rho GEF, PDZRho GEF, and leukemia-associated Rho-specific GEF or LARG.28,29 Because of the multiplicity of Rho GEFs, it has been suggested that Rho GEFs involved in the activation of RhoA are probably different under different pathologic conditions. Christophe et al.30 showed that control of RhoA signaling through p115-Rho GEF is central to the development of angiotensin IIYdependent hypertension. Wirth et al.31 demonstrated that G>12-G>13/LARG signaling pathway played important roles in salt-induced hypertension. Our present study indicated that the activity of PDZ-Rho GEF was significantly decreased following shock, while the activities of p115-Rho GEF and LARG did not significantly change. NS1619 pretreatment significantly increased the activity PDZ-Rho GEF, but not p115-Rho GEF and LARG. The increase of the activity of PDZ-Rho GEF was positively correlated with the increase of Rho kinase and RhoA activities induced by NS1619 pretreatment. These results suggested that PDZ-Rho GEF might be the main isoformer that participates in the activation of RhoAYRho kinase pathway induced by NS1619 pretreatment (Fig. 6). To observe the roles of Rho kinase and RhoA in the NS1619-induced protective effect, we selected Y-27632 and C3 transferase as an inhibitor of Rho kinase and RhoA, respectively. Y-27632 inhibits Rho kinase by competing with adenosine triphosphate for binding to the catalytic site of Rho kinase.32 Our previous studies showed that incubation of SMA rings with 1 10j5 mol/L of Y-27632 for 10 minutes could significantly antagonize the effects of Rho kinase. Therefore, in the present study, 1 10j5 mol/L of Y-27632 was selected. C3 transferase inhibits RhoA proteins by adenosine diphosphate ribosylation on asparagine 41 in the effector-binding domain of the GTPase and blocks its translocation to the membrane.33 The major limitation of using C3 transferase is that it is not cell permeable. Therefore, we used cell-permeable C3 transferase in this study, which comprises a proprietary cell-penetrating moiety. This moiety allows transport through the plasma membrane rapidly and efficiently and is released in the cytosol; thereby, C3 transferase can freely diffuse within the cell and inactivate RhoA. Our previous study and present study all demonstrated that incubation of SMA rings with 5-mg/mL cellpermeable C3 transferase for 6 hours could significantly antagonize the effects of RhoA. Although PPC was suggested to be a safe method of eliciting protection against ischemia-reperfusion injury, their clinical application has been restricted in many situations, in which the hemorrhage is unpredictable. However, some scheduled surgical operations with possible hemorrhage are suitable for the pretreatment intervention. Thus, NS1619 pretreatment could be used in the preoperative preparation to offer protection against the surgical operationYinduced severe hemorrhage. In addition, NS1619 pretreatment has been demonstrated to induce cardioprotection and neuroprotection.14,15,17,23 Therefore, NS1619 pretreatment may have great potentials to be used in organ transplantation, cardiopulmonary bypass surgery and cerebral surgery, and so on.
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
399
J Trauma Acute Care Surg Volume 76, Number 2
Hu et al.
Figure 6. A schematic signal transduction of RhoAYRho kinase pathway in the regulation of vascular reactivity after NS1619 pretreatment. NS1619 pretreatment could induce a light vasodilation and mimics the IPC, which evokes the activation of endogenous protection mechanisms. The endogenous protective substances such as adenosine activate Rho GEF via G protein coupled receptor and then activate RhoA. RhoA activates Rho kinase and then increases the phosphorylation of MLC20 and increase the calcium sensitivity and vascular reactivity via inhibition of myosin light chain phosphatase. GPCR, G protein coupled receptor; RhoGDI, guanine nucleotide dissociation inhibitor of RhoA; MLCP, myosin light chain phosphatase; MLC20, 20-kD myosin light chain; C3, C3 transferase, the inhibitor of RhoA, which can inhibit RhoA activity and block RhoA translocation to the membrane;Y-27632, a selective antagonist of Rho kinase.
Although this study showed that NS1619 pretreatment could improve vascular reactivity and calcium sensitivity after hemorrhagic shock and that the PDZ-Rho GEFYRhoAYRho kinase pathway played an important role in this process, there are still some issues that need to be answered in the future. First, this study was limited to the small animals, and whether the protective effect of NS1619 pretreatment would be duplicated in large animals, even in human beings, needs further investigation. Second, the preliminary mechanism of the protective effect of NS1619 pretreatment on shock-induced vascular hyporeactivity was investigated in this study, but the detailed mechanism of how NS1619 pretreatment activates PDZ-Rho GEF also needs further exploration. Third, NS1619 was shown to inhibit L-type calcium channels and voltagedependent channels.34,35 Moreover, NS1619 can also induce Ca2+ release from intracellular pools.36 Therefore, further study is needed to confirm whether the protective effect of NS1619 pretreatment on vascular reactivity is related to calcium channel and calcium release.
play antishock effect. PDZ-Rho GEFYRhoAYRho kinase pathway plays important role in the protective effect of NS1619 pretreatment on vascular reactivity. Among three Rho GEFs, PDZ-Rho GEF is the only one involved in the protective effect of NS1619 pretreatment. Bkca may be a potential target to treat shock-induced vascular hyporeactivity. AUTHORSHIP Y.H. performed the experiment, the data collection, and manuscript preparation. G.Y. and X.X. participated in the animal study and data analysis. T.L. and L.L. conceived of the study, participated in the design and coordination, and edited the manuscript. All authors read and approved the final manuscript.
DISCLOSURE This work was supported by the National Natural Science Foundation of China (81071540), Major State Basic Research Program of China (2012CB518101), and Project of State Key Laboratory of Trauma, Burns and Combined Injury (SKLZZ200901, SKLZZ200912).
CONCLUSION
REFERENCES
The pretreatment of Bkca opener, NS1619 could improve the vascular reactivity and cardiac function and by which
1. Liu LM, Ward JA, Dubick MA. Hemorrhage-induced vascular hyporeactivity to norepinephrine in select vasculatures of rats and the roles of nitric oxide and endothelin. Shock. 2003;19:208Y214.
400
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Trauma Acute Care Surg Volume 76, Number 2
2. Liu LM, Dubick MA. Hemorrhagic shock-induced vascular hyporeactivity in the rat: relationship to gene expression of nitric oxide synthase, endothelin-1, and select cytokines in corresponding organs. J Surg Res. 2005;125:128Y136. 3. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemia myocardium. Circulation. 1986; 74:1124Y1136. 4. Rodrigo GC, Samani NJ. Ischemic preconditioning of the whole heart confers protection on subsequently isolated ventricular myocytes. Am J Physiol Heart Circ Physiol. 2008;294:524Y531. 5. Jia RP, Zhu JG, Wu JP, Xie JJ, Xu LW. Experimental study on early protective effect of ischemic preconditioning on rat kidney graft. Transplant Proc. 2009;41:69Y72. 6. Suzuki S, Inaba K, Konno H. Ischemic preconditioning in hepatic ischemia and reperfusion. Curr Opin Organ Transplant. 2008;13:142Y147. 7. Bobryshev P, Bagaeva T, Filaretova L. Ischemic preconditioning attenuates gastric ischemia-reperfusion injury through involvement of glucocorticoids. J Physiol Pharmacol. 2009;60:155Y160. 8. Pang CY, Neligan P, Zhong A, Xu H, Forrest CR. Effector mechanism of adenosine in acute ischemic preconditioning of skeletal muscle against infarction. Am J Physiol. 1997;273:887Y895. 9. Gu¨lu¨zar Y, Abdullah TD, Bu¨lent GHoward L. Ischemic preconditioning modulates ischemia-reperfusion injury in the rat lung: role of adenosine receptors. Eur J Pharmacol. 2007;556:144Y150. 10. Jin Z, Duan W, Chen M, Yu S, Zhang H, Feng G, Xiong L, Yi D. The myocardial protective effects of adenosine pretreatment in children undergoing cardiac surgery: a randomized controlled clinical trial. Eur J Cardiothorac Surg. 2011;39:90Y96. 11. Yang QZ, Yan WJ, Li X, Hou LH, Dong H, Wang Q, Dong HL, Wang SQ, Zhang X, Xiong LZ. Activation of canonical Notch signaling pathway is involved in the ischemic tolerance induced by sevoflurane preconditioning in mice. Anesthesiology. 2012;117:996Y1015. 12. Xu J, Li T, Yang G, Liu LM. Pinacidil pretreatment improves vascular reactivity after shock through PKC> and PKCD in rats. J Cardiovasc Pharmacol. 2012;59:514Y522. 13. Zhou R, Liu LM, Hu DY. Involvement of Bkca > subunit tyrosine phosphorylation in vascular hyporesponsiveness of superior mesenteric artery following hemorrhagic shock in rats. Cardio Res. 2005;68:327Y335. 14. Xu W, Liu Y, Wang S, McDonald T, Van EJ, Sidor A, O’Rourke B. Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science. 2002;298:1029Y1033. 15. Shintani Y, Node K, Asanuma H, Sanada S, Takashima S, Asano Y, Liao Y, Fujita M, Hirata A, Shinozaki Y, et al. Opening of Ca2+-activated K+ channels is involved in ischemic preconditioning in canine hearts. J Mol Cell Cardiol. 2004;37:1213Y1218. 16. Malinska D, Mirandola SR, Kunz WS. Mitochondrial potassium channels and reactive oxygen species. FEBS Lett. 2010;584:2043Y2048. 17. Redel A, Lange M, Jazbutyte V, Lotz C, Smul TM, Roewer N, Kehl F. Activation of mitochondrial large-conductance calcium-activated K+ channels via protein kinase A mediates desflurane-induced preconditioning. Anesth Analg. 2008;106:384Y391. 18. Xu J, Liu LM. The role of calcium desensitization in vascular hyporeactivity and its regulation following hemorrhagic shock in the rat. Shock. 2005; 23:576Y581. 19. Li T, Liu L, Xu J, Yang GM, Ming J. Changes of Rho-kinase activity after hemorrhagic shock and its role in shock-induced biphasic response of vascular reactivity and calcium sensitivity. Shock. 2006;26:504Y509. 20. Li T, Fang YQ, Yang GM, Xu J, Zhu Y, Liu LM. Effects of the balance in activity of RhoA and Rac1 on the shock-induced biphasic change of vascular reactivity in rats. Ann Surg. 2011;253:185Y193.
Hu et al.
21. Hu Y, Li T, Tang XF, Chen K, Liu LM. Effects of ischemic preconditioning on vascular reactivity and calcium sensitivity after hemorrhagic shock and their relationship to the RhoAYRhokinase pathway in rats. J Cardiovasc Pharmacol. 2011;57:231Y239. 22. Srinivas G, Deepthi N, Xiaoping X. Large-conductance, calcium-activated potassium channels: structural and functional implications. Pharmacol Ther. 2006;110:103Y116. 23. Gaspar T, Katakam P, Snipes JA, Kis B, Domoki F, Bari F, Busija DW. Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels. J Neurochem. 2008;105:1115Y1128. 24. Yun N, Kim SH, Lee SM. Differential consequences of protein kinase C activation during early and late hepatic ischemic preconditioning. J Physiol Sci. 2012;62(3):199Y209. 25. Tang XL, Kodani E, Takano H, Hil M, Shinmura K, Vondriska TM, Ping P, Bolli R. Protein tyrosine kinase signaling is necessary for NO donor-induced late preconditioning against myocardial stunning. Am J Physiol Heart Circ Physiol. 2003;284(4):H1441YH1448. 26. Chikumi H, Fukuhara S, Gutkind JS. Regulation of G proteinYlinked guanine nucleotide exchange factors for Rho, PDZ-RhoGEF, and LARG by tyrosine phosphorylation: evidence of a role for focal adhesion kinase. J Biol Chem. 2002;277(14):12463Y12473. 27. Peng J, He F, Zhang C, Deng X, Yin F. Protein kinase C-> signals P115RhoGEF phosphorylation and RhoA activation in TNF->Yinduced mouse brain microvascular endothelial cell barrier dysfunction. J Neuroinflammation. 2011;8:28. 28. Fukuhara S, Chikumi H, Gutkind JS. RGS-containing RhoGEFs: the missing link between transforming G proteins and Rho? Oncogene. 2001;20:1661Y1668. 29. Jaiswal M, Gremer L, Dvorsky R, Haeusler LC, Cirstea IC, Uhlenbrock K, Ahmadian MR. Mechanistic insights into specificity, activity, and regulatory elements of the regulator of G-protein signaling (RGS)Ycontaining Rho-specific guanine nucleotide exchange factors (GEFs) p115, PDZRhoGEF (PRG), and leukemia-associated RhoGEF (LARG). J Biol Chem. 2011;286:18202Y18212. 30. Guilluy C, Bre´geon J, Toumaniantz G, Rolli-Dekinderen M, Retaillean K, Lourfrani L, Henrion D, Scalbert E, Bril A, Torres RM, et al. The Rho exchange factor Arhgef1 mediates the effects of angiotensin II on vascular tone and blood pressure. Nat Med. 2010;16(2):183Y190. 31. Wirth A, Benyo´ Z, Lukasova M, Leutgeb B, Wettschureck N, Gorbey S, Orsy P, Horva´th B, Marser-Glath C, Greiner E, et al. G12-G13LARGYmediated signaling in vascular smooth muscle is required for salt-induced hypertension. Nat Med. 2008;14: 64Y68. 32. Somlyo AP, Somlyo AV. Signal transduction by G protein, Rho kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol. 2000;522:177Y185. 33. Aktories K, Wilde C, Vogelsges ang M. Rho-modifying C3-like ADPribosyltransferases. Rev Physiol Biochem Pharmacol. 2004;152:1Y22. 34. Edwards G, Niederste-Hollenberg A, Schneider J, Noack T, Weston AH. Ion channel modulation by NS 1619, the putative Bkca channel opener, in vascular smooth muscle. Br J Pharmacol. 1994;113:1538Y1547. 35. Park WS, Kang SH, Son YK, Kim N, Ko JH, Kim HK, Ko EA, Kim CD, Han J. The mitochondrial Ca2+-activated K+ channel activator, NS 1619 inhibits L-type Ca2+ channels in rat ventricular myocytes. Biochem Biophys Res Commun. 2007;362:31Y36. 36. Yamamura H, Ohi Y, Muraki K, Watanabe M, Imaizumi Y. BK channel activation by NS-1619 is partially mediated by intra-cellular Ca2+ release in smooth muscle cells of porcine coronary artery. Br J Pharmacol. 2001;132:828Y834.
* 2014 Lippincott Williams & Wilkins
Copyright © 2014 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
401
