Amino Butyric Acid Type B Receptors Stimulate Neutrophil Chemotaxis during Ischemia-Reperfusion1
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免疫学杂志 2005年第11期
Abstract
Akt, or protein kinase B, has been shown to regulate a number of neutrophil functions. We sought to identify Akt binding proteins in neutrophils to provide further insights into understanding the mechanism by which Akt regulates various neutrophil functions. Proteomic and immunoprecipitation studies identified -amino butyric acid (GABA) type B receptor 2 (GABABR2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABABR2 demonstrated Akt association with the intact GABABR. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABABR2 Ab. Additionally, GABABR2 and Akt colocalization was demonstrated by confocal microscopy. A GABABR agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432 a GABABR antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABABR agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABABR2 is present in human neutrophils and that it is functionally associated with Akt. Intraventricular baclofen pretreatment in rats subjected to a stroke model showed increased migration of neutrophils to the ischemic lesion. Thus, the GABABR is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABABR potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.
Introduction
Ischemia-reperfusion injury is the underlying mechanism of common and frequently fatal illnesses such as myocardial infarction, acute renal failure, and stroke. Neutrophils play a central role in ischemia-reperfusion injury (1). Additionally, many neurological insults are accompanied by an acute inflammatory reaction due to neutrophil infiltration, the latter contributing to neuronal damage (2, 3, 4, 5, 6). Neutrophil functions such as chemotaxis, exocytosis, or superoxide burst are initiated in activated neutrophils. Activated neutrophils can contribute to tissue damage by 1) release of proteolytic enzymes, 2) release of reactive oxygen species, and 3) stimulation of proinflammatory cytokines. Therefore, identifying proteins that mediate neutrophil functional responses might allow manipulation of signal cascades and potentially modulate specific neutrophil functions (7, 8, 9, 10).
Participation of neutrophils in inflammation requires the interplay of multiple cell membrane receptors and downstream protein-protein interactions that convey information from cell surface receptors to the cell interior. One protein that is a significant regulator of neutrophil survival and function is the serine/threonine kinase Akt. Akt, also known as protein kinase B, is a cellular homologue of a viral oncogene v-Akt (11, 12, 13). Akt has been shown previously to regulate a number of neutrophil functions including, chemotaxis, respiratory burst, apoptosis, and actin polymerization (14, 15, 16, 17, 18, 19). To better understand the role of Akt in regulating various neutrophil functions, proteomic analysis was applied to Akt immunoprecipitates to identify putative Akt binding proteins. A C-terminal fragment of the -amino-butyric acid (GABA)4 type B receptor 2 (GABABR2) was identified as an Akt binding protein. The identification of a fragment of GABABR2 instead of an intact receptor could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor. However, immunoblot analysis demonstrated presence of intact GABABR2 in human neutrophils. Additionally, association and colocalization of intact GABABR2 and Akt was demonstrated by Akt and GABABR2 immunoprecipitations and confocal microscopy, respectively.
GABA is the main inhibitory neurotransmitter in the mammalian central nervous, where it exerts its effects through ionotropic (GABAA/C) receptors to produce fast synaptic inhibition and metabotropic (GABAB) receptors to produce slow, prolonged inhibitory signals. GABABR include two receptor subtypes GABABR1 and GABABR2. The GABABR is composed of dimers and ligand-induced heterodimerization of R1 and R2 subunits have been shown to be essential for GABABR trafficking and function (20, 21). Because Akt associated with the GABABR2 in human neutrophils, and GABABR agonists have been shown to stimulate directed migration in neurons, we addressed the hypothesis that the GABABR2 acts as a neutrophil chemoattractant receptor via an Akt-dependent mechanism. The present study identified for the first time the presence of functional GABABR2 in human neutrophils and its association with Akt. Additionally, baclofen, a GABABR2 agonist, stimulated Akt activation and directed neutrophil migration in a PI3K-dependent manner both in vitro and in a stroke animal model. Thus GABABR may play a significant role in the inflammatory component of ischemia-reperfusion injury.
Statistics
Data is shown as mean ± SE. The n value shows the number of separate experiments. Values for p are calculated by the SigmaStat software using Student’s t test. A value p < 0.05 was a priori considered significant.
Results
Proteomic analysis of Akt immunoprecipitates
To understand the role of Akt in mediating a number of neutrophil functions we sought to identify Akt binding partners in human neutrophils. Neutrophil lysates were subjected to isotype control or anti-Akt immunoprecipitation. Immunoprecipitated proteins were initially resolved by high-resolution 2D-PAGE as previously described by our group (22, 23). MALDI analysis coupled with BLASTP analysis of Akt immunoprecipitates identified a C-terminal fragment of GABABR2, as an Akt binding protein. The identification of a fragment of GABABR2 instead of an intact receptor could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor. The presence of intact GABABR2 in neutrophils was documented by immunoblotting neutrophil lysates with anti-GABABR2 Ab (Sigma-Aldrich) (Fig. 1, top). These results demonstrated for the first time presence of GABABR2 in human neutrophils.
Confirmation that the GABABR2 is present in a complex with Akt in neutrophils
As the intact GABABR2 was detected in human neutrophils we next determined whether this receptor directly associated with Akt. To this end neutrophil lysates were subjected to anti-Akt-PH domain or isotype control Ab immunoprecipitations followed by immunoblotting with anti-GABABR2 Ab. Fig. 4A demonstrates an interaction between Akt and GABABR2 in anti-Akt but not isotype control precipitates. Reciprocal immunoprecipitations with anti-GABABR2 Ab confirmed association between Akt and GABABR2 (Fig. 4B). These data confirmed that Akt and GABABR were present together in a complex however, failed to demonstrate that they were functionally linked.
Discussion
It has been previously demonstrated that the brain and the immune system share common molecules and receptors (26). Additionally, psychoactive drugs, such as benzodiazepines, have been shown to modulate neutrophil functions including, intracellular calcium release, chemotaxis, and phagocytosis (27, 28). The GABAAR was recently identified as an Akt substrate in the mammalian brain, thereby linking Akt to regulation of synaptic strength (29). However, we are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that the GABABR2 is present in human neutrophils and that it is functionally associated with the signaling kinase Akt. Proteomic studies first identified a C-terminal fragment of GABABR2 as an Akt binding protein, instead of the intact receptor. This could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor, however GABABR proteolysis has not been documented. Immunoblotting studies of neutrophil lysates and confocal microscopy of anti-GABABR2-stained neutrophils documented presence of GABABR2 in neutrophils. Additionally, anti-Akt immunoprecipitates demonstrated that the intact receptor was bound to the Akt signal complex. Collectively, these results suggest that GABABR may interact with Akt via its C-terminal tail.
Akt functionally interacts with multiple signaling pathways (30, 31, 32). We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPKAPK-2, and heat shock protein 27 (30). Additionally, we demonstrated that a disruption of Akt-heat shock protein 27 interaction resulted in neutrophil apoptosis (17). To identify additional members of the Akt signal complex that regulate neutrophil functions, proteomic analyses of Akt immunoprecipitates in human neutrophils were performed. Proteomic analysis of Akt immunoprecipitates from human neutrophils identified a C-terminal fragment of GABABR2. Presence of an intact GABABR2 in human neutrophils and its association with Akt was demonstrated by immunoprecipitation studies, immunoblot analysis, and confocal microscopy. Additionally, confocal staining identified GABABR2 in the cytosol, plasma membrane, epicenter of the cell, and in the nucleus of neutrophils. Detection of GABABR2 in the nucleus was surprising; however, GABABR2 has been shown previously to interact with nuclear proteins such as transcription factors CREB2 and activating transcription factor x and factor 4 (33, 34).
Akt was shown previously to be activated in human neutrophils by fMLP, leukotriene B4, IL-8, FcR cross-linking, LPS, GM-CSF, and antineutrophil cytoplasmic Abs (18, 30, 35, 36, 37). We report for the first time, that a GABABR agonist, baclofen, stimulates Akt phosphorylation and activation in a PI3K-dependent manner, whereas CGP52432 a GABABR antagonist inhibits baclofen-stimulated Akt activation, suggesting a functional association of Akt and GABABR in human neutrophils.
Akt activation has been shown to regulate various neutrophil functions, including apoptosis, actin polymerization, chemotaxis, and superoxide burst activity (14, 15, 16, 17, 18, 19). In the current study, baclofen failed to stimulate neutrophil superoxide burst in neutrophils. Proteins that play a role in the regulation of the microtubule were identified in the GABABR-Akt signaling module and GABABR activation resulted in disassembly of the tubulin network in a PI3K-dependent manner. In neutrophils, disassembly of the microtubule network has been shown to induce development of polarity, a key feature required for chemokinesis and chemotaxis (24). Recent studies have suggested a role for the GABABR in directed migration of embryonic cortical neuron, and motility of spinal neuroblasts, thus supporting the concept that these receptors can guide directed migration or growth (25). The present study shows that GABABR selectively induces neutrophil chemotaxis via an Akt-dependent mechanism because pretreatment with LY294003 or simultaneous addition of CGP52432inhibited baclofen-stimulated neutrophil chemotaxis. Additionally, pretreatment with LY294003 inhibited baclofen-stimulated Akt phosphorylation and Akt activation. Furthermore, in vivo studies demonstrated increased migration of neutrophils into brain regions subjected to ischemic-reperfusion injury. It is possible that pharmacological inhibition of PI3K may affect multiple signaling pathways other than Akt in human neutrophils. It is also possible that other pathways such as p38 MAPK or Erk MAPK pathways may feed into the Akt pathway or may be down stream of the Akt pathway. However, those studies are beyond the scope of the current study. In human neutrophils we have previously demonstrated that fMLP-stimulated Akt activation is dependent on p38 MAPK activation (30).
These results suggest an important role for GABABR in the extent of the inflammatory response, and ultimately in the magnitude of cell loss in stroke (38, 39, 40). Increased glutamate release occurs in multiple organs subjected to ischemia (41, 42, 43) and glutamic acid decarboxylase, the critical enzyme-converting glutamate to GABA does not require oxygen, and is ubiquitously expressed in tissues such as brain, heart, kidney, and lung (44, 45). In the current study, the presence of glutamic acid decarboxylase in neutrophils was documented by Western blot analysis. Hence, we postulate that induction of GABABR-Akt mediated chemotaxis underlies a major mechanism of inflammatory regulation in tissues undergoing ischemia-reperfusion and thereby provides novel potential interventional targets for palliation of ischemic tissue injury.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants HL66358 (to J.B.K.), HL63912, P50HL60296, and HL69932 (to D.G.), and HL74296 (to E.G.). K.R.M. and J.B.K. are supported by the Department of Veterans Affairs Merit Review. This work is also supported by the American Heart Association Grant SDG 0335278N (to M.J.R.) and the Kentucky Challenge for Excellence Trust Fund.
2 Address correspondence and reprint requests to Dr. Madhavi J. Rane, University of Louisville, Baxter Research Building, 570 South Preston Street, Louisville, KY 40202. E-mail address: mrane{at}louisville.edu
3 D.G. and J.B.K. contributed equally as senior authors.
4 Abbreviations used in this paper: GABA, -amino-butyric acid; FPR, formyl peptide receptor; GABABR2, -amino-butyric acid type B receptor 2; MCAO, middle cerebral artery occlusion.
Received for publication July 28, 2004. Accepted for publication February 23, 2005.
References
Kuzuya, T., S. Hoshida, M. Nishida, Y. Kim, H. Fuji, A. Kitabatake, T. Kamada, M. Tada. 1989. Role of free radicals and neutrophils in canine myocardial reperfusion injury: myocardial salvage by a novel free radical scavenger, 2-octadecylascorbic acid. Cardiovasc. Res. 4: 323-330.
Wang, X., G. Z. Feuerstein. 2000. Role of immune and inflammatory mediators in CNS injury. Drug News Perspect. 13: 133-140.
Jean, W. C., S. R. Spellman, E. S. Nussbaum, W. C. Low. 1998. Reperfusion injury after focal cerebral ischemia: the role of inflammation and the therapeutic horizon. Neurosurgery 43: 1382-1397.
Ember, J. A., G. J. del Zoppo, E. Mori, W. S. Thomas, B. R. Copeland, T. E. Hugli. 1994. Polymorphonuclear leukocyte behavior in a nonhuman primate focal ischemia model. J. Cereb. Blood Flow Metab. 14: 1046-1054.
Kochanek, P. M., J. M. Hallenbeck. 1992. Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 23: 1367-1379.
Prestigiacomo, C. J., S. C. Kim, E. S. Connolly, Jr, H. Liao, S. F. Yan, D. J. Pinsky. 1999. CD18-mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke. Stroke 30: 1110-1117.
del Zoppo, G. J., K. J. Becker, J. M. Hallenbeck. 2001. Inflammation after stroke: is it harmful?. Arch. Neurol. 58: 669-672.(Madhavi J. Rane2,*, David)
Akt, or protein kinase B, has been shown to regulate a number of neutrophil functions. We sought to identify Akt binding proteins in neutrophils to provide further insights into understanding the mechanism by which Akt regulates various neutrophil functions. Proteomic and immunoprecipitation studies identified -amino butyric acid (GABA) type B receptor 2 (GABABR2) as an Akt binding protein in human neutrophils. Neutrophil lysates subjected to Akt immunoprecipitation followed by immunoblotting with anti-GABABR2 demonstrated Akt association with the intact GABABR. Similar results were obtained when reciprocal immunoprecipitations were performed with anti-GABABR2 Ab. Additionally, GABABR2 and Akt colocalization was demonstrated by confocal microscopy. A GABABR agonist, baclofen, activated Akt and stimulated neutrophil-directed migration in a PI3K-dependent manner, whereas CGP52432 a GABABR antagonist blocked such effects. Baclofen, stimulated neutrophil chemotaxis and tubulin reorganization in a PI3K-dependent manner. Additionally, a GABABR agonist failed to stimulate neutrophil superoxide burst. We are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that a brain-specific receptor, GABABR2 is present in human neutrophils and that it is functionally associated with Akt. Intraventricular baclofen pretreatment in rats subjected to a stroke model showed increased migration of neutrophils to the ischemic lesion. Thus, the GABABR is functionally expressed in neutrophils, and acts as a chemoattractant receptor via an Akt-dependent pathway. The GABABR potentially plays a significant role in the inflammatory response and neutrophil-dependent ischemia-reperfusion injury such as stroke.
Introduction
Ischemia-reperfusion injury is the underlying mechanism of common and frequently fatal illnesses such as myocardial infarction, acute renal failure, and stroke. Neutrophils play a central role in ischemia-reperfusion injury (1). Additionally, many neurological insults are accompanied by an acute inflammatory reaction due to neutrophil infiltration, the latter contributing to neuronal damage (2, 3, 4, 5, 6). Neutrophil functions such as chemotaxis, exocytosis, or superoxide burst are initiated in activated neutrophils. Activated neutrophils can contribute to tissue damage by 1) release of proteolytic enzymes, 2) release of reactive oxygen species, and 3) stimulation of proinflammatory cytokines. Therefore, identifying proteins that mediate neutrophil functional responses might allow manipulation of signal cascades and potentially modulate specific neutrophil functions (7, 8, 9, 10).
Participation of neutrophils in inflammation requires the interplay of multiple cell membrane receptors and downstream protein-protein interactions that convey information from cell surface receptors to the cell interior. One protein that is a significant regulator of neutrophil survival and function is the serine/threonine kinase Akt. Akt, also known as protein kinase B, is a cellular homologue of a viral oncogene v-Akt (11, 12, 13). Akt has been shown previously to regulate a number of neutrophil functions including, chemotaxis, respiratory burst, apoptosis, and actin polymerization (14, 15, 16, 17, 18, 19). To better understand the role of Akt in regulating various neutrophil functions, proteomic analysis was applied to Akt immunoprecipitates to identify putative Akt binding proteins. A C-terminal fragment of the -amino-butyric acid (GABA)4 type B receptor 2 (GABABR2) was identified as an Akt binding protein. The identification of a fragment of GABABR2 instead of an intact receptor could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor. However, immunoblot analysis demonstrated presence of intact GABABR2 in human neutrophils. Additionally, association and colocalization of intact GABABR2 and Akt was demonstrated by Akt and GABABR2 immunoprecipitations and confocal microscopy, respectively.
GABA is the main inhibitory neurotransmitter in the mammalian central nervous, where it exerts its effects through ionotropic (GABAA/C) receptors to produce fast synaptic inhibition and metabotropic (GABAB) receptors to produce slow, prolonged inhibitory signals. GABABR include two receptor subtypes GABABR1 and GABABR2. The GABABR is composed of dimers and ligand-induced heterodimerization of R1 and R2 subunits have been shown to be essential for GABABR trafficking and function (20, 21). Because Akt associated with the GABABR2 in human neutrophils, and GABABR agonists have been shown to stimulate directed migration in neurons, we addressed the hypothesis that the GABABR2 acts as a neutrophil chemoattractant receptor via an Akt-dependent mechanism. The present study identified for the first time the presence of functional GABABR2 in human neutrophils and its association with Akt. Additionally, baclofen, a GABABR2 agonist, stimulated Akt activation and directed neutrophil migration in a PI3K-dependent manner both in vitro and in a stroke animal model. Thus GABABR may play a significant role in the inflammatory component of ischemia-reperfusion injury.
Statistics
Data is shown as mean ± SE. The n value shows the number of separate experiments. Values for p are calculated by the SigmaStat software using Student’s t test. A value p < 0.05 was a priori considered significant.
Results
Proteomic analysis of Akt immunoprecipitates
To understand the role of Akt in mediating a number of neutrophil functions we sought to identify Akt binding partners in human neutrophils. Neutrophil lysates were subjected to isotype control or anti-Akt immunoprecipitation. Immunoprecipitated proteins were initially resolved by high-resolution 2D-PAGE as previously described by our group (22, 23). MALDI analysis coupled with BLASTP analysis of Akt immunoprecipitates identified a C-terminal fragment of GABABR2, as an Akt binding protein. The identification of a fragment of GABABR2 instead of an intact receptor could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor. The presence of intact GABABR2 in neutrophils was documented by immunoblotting neutrophil lysates with anti-GABABR2 Ab (Sigma-Aldrich) (Fig. 1, top). These results demonstrated for the first time presence of GABABR2 in human neutrophils.
Confirmation that the GABABR2 is present in a complex with Akt in neutrophils
As the intact GABABR2 was detected in human neutrophils we next determined whether this receptor directly associated with Akt. To this end neutrophil lysates were subjected to anti-Akt-PH domain or isotype control Ab immunoprecipitations followed by immunoblotting with anti-GABABR2 Ab. Fig. 4A demonstrates an interaction between Akt and GABABR2 in anti-Akt but not isotype control precipitates. Reciprocal immunoprecipitations with anti-GABABR2 Ab confirmed association between Akt and GABABR2 (Fig. 4B). These data confirmed that Akt and GABABR were present together in a complex however, failed to demonstrate that they were functionally linked.
Discussion
It has been previously demonstrated that the brain and the immune system share common molecules and receptors (26). Additionally, psychoactive drugs, such as benzodiazepines, have been shown to modulate neutrophil functions including, intracellular calcium release, chemotaxis, and phagocytosis (27, 28). The GABAAR was recently identified as an Akt substrate in the mammalian brain, thereby linking Akt to regulation of synaptic strength (29). However, we are unaware of the association of GABABR with Akt in any cell type. The present study shows for the first time that the GABABR2 is present in human neutrophils and that it is functionally associated with the signaling kinase Akt. Proteomic studies first identified a C-terminal fragment of GABABR2 as an Akt binding protein, instead of the intact receptor. This could have resulted from degradation of the intact receptor during sample preparation or due to physiological proteolytic cleavage of the intact receptor, however GABABR proteolysis has not been documented. Immunoblotting studies of neutrophil lysates and confocal microscopy of anti-GABABR2-stained neutrophils documented presence of GABABR2 in neutrophils. Additionally, anti-Akt immunoprecipitates demonstrated that the intact receptor was bound to the Akt signal complex. Collectively, these results suggest that GABABR may interact with Akt via its C-terminal tail.
Akt functionally interacts with multiple signaling pathways (30, 31, 32). We have shown previously that Akt exists in a signal complex with p38 MAPK, MAPKAPK-2, and heat shock protein 27 (30). Additionally, we demonstrated that a disruption of Akt-heat shock protein 27 interaction resulted in neutrophil apoptosis (17). To identify additional members of the Akt signal complex that regulate neutrophil functions, proteomic analyses of Akt immunoprecipitates in human neutrophils were performed. Proteomic analysis of Akt immunoprecipitates from human neutrophils identified a C-terminal fragment of GABABR2. Presence of an intact GABABR2 in human neutrophils and its association with Akt was demonstrated by immunoprecipitation studies, immunoblot analysis, and confocal microscopy. Additionally, confocal staining identified GABABR2 in the cytosol, plasma membrane, epicenter of the cell, and in the nucleus of neutrophils. Detection of GABABR2 in the nucleus was surprising; however, GABABR2 has been shown previously to interact with nuclear proteins such as transcription factors CREB2 and activating transcription factor x and factor 4 (33, 34).
Akt was shown previously to be activated in human neutrophils by fMLP, leukotriene B4, IL-8, FcR cross-linking, LPS, GM-CSF, and antineutrophil cytoplasmic Abs (18, 30, 35, 36, 37). We report for the first time, that a GABABR agonist, baclofen, stimulates Akt phosphorylation and activation in a PI3K-dependent manner, whereas CGP52432 a GABABR antagonist inhibits baclofen-stimulated Akt activation, suggesting a functional association of Akt and GABABR in human neutrophils.
Akt activation has been shown to regulate various neutrophil functions, including apoptosis, actin polymerization, chemotaxis, and superoxide burst activity (14, 15, 16, 17, 18, 19). In the current study, baclofen failed to stimulate neutrophil superoxide burst in neutrophils. Proteins that play a role in the regulation of the microtubule were identified in the GABABR-Akt signaling module and GABABR activation resulted in disassembly of the tubulin network in a PI3K-dependent manner. In neutrophils, disassembly of the microtubule network has been shown to induce development of polarity, a key feature required for chemokinesis and chemotaxis (24). Recent studies have suggested a role for the GABABR in directed migration of embryonic cortical neuron, and motility of spinal neuroblasts, thus supporting the concept that these receptors can guide directed migration or growth (25). The present study shows that GABABR selectively induces neutrophil chemotaxis via an Akt-dependent mechanism because pretreatment with LY294003 or simultaneous addition of CGP52432inhibited baclofen-stimulated neutrophil chemotaxis. Additionally, pretreatment with LY294003 inhibited baclofen-stimulated Akt phosphorylation and Akt activation. Furthermore, in vivo studies demonstrated increased migration of neutrophils into brain regions subjected to ischemic-reperfusion injury. It is possible that pharmacological inhibition of PI3K may affect multiple signaling pathways other than Akt in human neutrophils. It is also possible that other pathways such as p38 MAPK or Erk MAPK pathways may feed into the Akt pathway or may be down stream of the Akt pathway. However, those studies are beyond the scope of the current study. In human neutrophils we have previously demonstrated that fMLP-stimulated Akt activation is dependent on p38 MAPK activation (30).
These results suggest an important role for GABABR in the extent of the inflammatory response, and ultimately in the magnitude of cell loss in stroke (38, 39, 40). Increased glutamate release occurs in multiple organs subjected to ischemia (41, 42, 43) and glutamic acid decarboxylase, the critical enzyme-converting glutamate to GABA does not require oxygen, and is ubiquitously expressed in tissues such as brain, heart, kidney, and lung (44, 45). In the current study, the presence of glutamic acid decarboxylase in neutrophils was documented by Western blot analysis. Hence, we postulate that induction of GABABR-Akt mediated chemotaxis underlies a major mechanism of inflammatory regulation in tissues undergoing ischemia-reperfusion and thereby provides novel potential interventional targets for palliation of ischemic tissue injury.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Institutes of Health Grants HL66358 (to J.B.K.), HL63912, P50HL60296, and HL69932 (to D.G.), and HL74296 (to E.G.). K.R.M. and J.B.K. are supported by the Department of Veterans Affairs Merit Review. This work is also supported by the American Heart Association Grant SDG 0335278N (to M.J.R.) and the Kentucky Challenge for Excellence Trust Fund.
2 Address correspondence and reprint requests to Dr. Madhavi J. Rane, University of Louisville, Baxter Research Building, 570 South Preston Street, Louisville, KY 40202. E-mail address: mrane{at}louisville.edu
3 D.G. and J.B.K. contributed equally as senior authors.
4 Abbreviations used in this paper: GABA, -amino-butyric acid; FPR, formyl peptide receptor; GABABR2, -amino-butyric acid type B receptor 2; MCAO, middle cerebral artery occlusion.
Received for publication July 28, 2004. Accepted for publication February 23, 2005.
References
Kuzuya, T., S. Hoshida, M. Nishida, Y. Kim, H. Fuji, A. Kitabatake, T. Kamada, M. Tada. 1989. Role of free radicals and neutrophils in canine myocardial reperfusion injury: myocardial salvage by a novel free radical scavenger, 2-octadecylascorbic acid. Cardiovasc. Res. 4: 323-330.
Wang, X., G. Z. Feuerstein. 2000. Role of immune and inflammatory mediators in CNS injury. Drug News Perspect. 13: 133-140.
Jean, W. C., S. R. Spellman, E. S. Nussbaum, W. C. Low. 1998. Reperfusion injury after focal cerebral ischemia: the role of inflammation and the therapeutic horizon. Neurosurgery 43: 1382-1397.
Ember, J. A., G. J. del Zoppo, E. Mori, W. S. Thomas, B. R. Copeland, T. E. Hugli. 1994. Polymorphonuclear leukocyte behavior in a nonhuman primate focal ischemia model. J. Cereb. Blood Flow Metab. 14: 1046-1054.
Kochanek, P. M., J. M. Hallenbeck. 1992. Polymorphonuclear leukocytes and monocytes/macrophages in the pathogenesis of cerebral ischemia and stroke. Stroke 23: 1367-1379.
Prestigiacomo, C. J., S. C. Kim, E. S. Connolly, Jr, H. Liao, S. F. Yan, D. J. Pinsky. 1999. CD18-mediated neutrophil recruitment contributes to the pathogenesis of reperfused but not nonreperfused stroke. Stroke 30: 1110-1117.
del Zoppo, G. J., K. J. Becker, J. M. Hallenbeck. 2001. Inflammation after stroke: is it harmful?. Arch. Neurol. 58: 669-672.(Madhavi J. Rane2,*, David)