BACKGROUND
agmatine is an endogenous polyamine that is synthesized by the decarboxylation of l-arginine by arginine decarboxylase  <cit> .
it is known to be widely but unevenly distributed in the brain and other mammalian tissues  <cit> .
agmatine has been reported to have various biological actions.
it stimulates the release of catecholamines from adrenal chromaffin cells  <cit> , insulin from pancreatic islets  <cit> , and luteinizing hormone-releasing hormone from the hypothalamus  <cit> .
also, it enhances analgesic effects of morphine  <cit> , inhibits inducible nitric oxide synthase   <cit> , and contributes to polyamine homeostasis  <cit> .
it is known that agmatine is an agonist for α2-adrenergic and imidazoline receptors  <cit> , and an antagonist for the n-methyl-d-aspartate  receptor  <cit> .
however, the precise cellular mechanisms by which agmatine acts are not yet well established.
currently, a large body of experimental evidence has demonstrated the neuroprotective effects of agmatine.
agmatine reduces infarct areas and neuronal loss in cerebral ischemic and ischemic-reperfusion injury models  <cit> .
it protects neurons from cell death after exposure to nmda and glutamate  <cit> .
it also attenuates the extent of neuronal loss following a spinal cord injury  <cit>  and shelters neurons from glucocorticoid-induced neurotoxicity  <cit>  and 1-methyl-4-phenyl- <dig> , <dig> -tetrahydropyridine-related dopaminergic toxicity  <cit> .
on the basis of these neuroprotective effects, agmatine can be presumed to have similar neuroprotective effects on retinal ganglion cells .
several molecules, including α2-adrenergic agonists  <cit> , nmda receptor antagonists  <cit>  and nos inhibitors  <cit> , have been reported to protect rgcs.
agmatine also acts as an α2-adrenergic agonist  <cit> , nmda receptor antagonist  <cit> , and suppressor of inducible nos  <cit> .
in the present investigation, we examined the protective effects of agmatine on hypoxia-induced apoptosis of rgcs by using the transformed rat rgcs   <cit> .
effects of agmatine were compared to those of brain-derived neurotrophic factor , a well-known protective neurotrophin for rgcs  <cit> .
in addition, several molecular pathways associated with these neuroprotective effects of agmatine were evaluated.
RESULTS
agmatine inhibits hypoxia-induced cell damage of rgc- <dig> cells
we first examined the effects of hypoxia on rgc- <dig> cells.
as shown in figure  <dig>  exposure to a hypoxic environment for  <dig>   <dig>  and  <dig> hours significantly increased release of lactate dehydrogenase  by  <dig> %,  <dig> %, and  <dig> %, respectively , thus demonstrating time-dependent hypoxia-induced neurotoxicity.
next, we examined the protective effects of agmatine on hypoxia-induced damage in rgc- <dig> cells.
after  <dig> and  <dig> hours of hypoxia, agmatine treatment groups did not show significant amounts of ldh release , but there were significant effects after  <dig> hours of exposure .
after  <dig> hours, the addition of  <dig> μm and  <dig> μm agmatine decreased hypoxia-induced ldh release by  <dig> % and  <dig> %, respectively .
when the protective effects of  <dig> μm agmatine were compared with those of  <dig> ng/ml bdnf, agmatine demonstrated a more powerful protective effect than that observed for bdnf .
the neuroprotective effect of agmatine against hypoxia-induced damage to rgc- <dig> cells was further studied using hoechst  <dig> and propidium iodide  double staining.
the control normoxic culture exhibited confluent hoechst-positive cells with homogeneous and compact nuclear morphology, and sparse numbers of pi-labeled cells .
exposure to hypoxia for  <dig> hours resulted in a significant loss of hoechst-positive cells and many pi-positive cells with distorted and condensed nuclei .
these changes were reduced by the addition of  <dig> μm agmatine  or  <dig> ng/ml bdnf  to the cultures, and agmatine had a greater protective effect.
agmatine protects rgc- <dig> cells from hypoxia-induced apoptosis
in order to verify whether agmatine had protective effects on hypoxia-induced apoptotic death of rgc- <dig> cells, further analyses using annexin v assay were performed.
while  <dig> hours of hypoxic exposure did not change the proportion of apoptotic cells compared with the normoxic culture, there were significant increases in apoptotic cells after  <dig> hours .
with the addition of  <dig> μm agmatine and  <dig> ng/ml bdnf, the proportion of apoptotic cells decreased .
specific caspase- <dig> activity was assessed using a caspase- <dig> assay, which could measure the cleavage of the caspase- <dig> specific substrate ac-devd-pna .
after  <dig> hours of hypoxic injury, the caspase- <dig> activity was significantly increased, and it was suppressed by  <dig> μm agmatine.
the results obtained by adding  <dig> μm agmatine were similar to those seen with  <dig> μm caspase- <dig> inhibitor z-vad-fmk.
selective suppression of jnk activation by agmatine
three mitogen-activated protein kinases , including c-jun n-terminal kinase , extracellular signal-regulated kinase,  and p <dig> kinase , were investigated using western immunoblots.
the amounts of total and phosphorylated mapks and β-actin are shown in figure  <dig>
total expression of the three mapks  and β-actin were not affected by hypoxic injury.
in addition, there were no significant changes after treatment with bdnf or agmatine.
antibodies against phospho-jnks detected two bands at  <dig> and  <dig> kda, and both bands had a similar tendency.
increases of phospho-jnks in rgc- <dig> cells became evident  <dig> hours after hypoxic injury and remained elevated for  <dig> hours .
agmatine suppressed the hypoxia-induced phosphorylation of jnks, but bdnf did not influence their phosphorylation.
antibodies against phospho-erks also detected two bands at  <dig> and  <dig> kda, and both bands were similar.
phospho-erks were not detected in the normoxic cultures.
however, they were highly expressed in rgc- <dig> cells even after  <dig> hours of hypoxia and remained elevated for  <dig> hours .
bdnf completely blocked the phosphorylation of erks for  <dig> hours, but it had no effect thereafter.
in comparison, agmatine did not significantly affect the phosphorylation of erks.
antibodies against phospho-p <dig> detected one band at  <dig> kda.
phospho-p <dig> was not detected in normoxic cultures until  <dig> hours of exposure to hypoxia, but it was evident in hypoxic cultures even after  <dig> hours and remained elevated for  <dig> hours .
bdnf only blocked the phosphorylation of p <dig> at  <dig> hours and agmatine had no effect on phospho-p <dig> levels at any time points.
thus, phospho-mapks showed different activation profiles in response to hypoxic injuries in rgc- <dig> cells; erk and p <dig> were activated relatively earlier than jnk.
bdnf inhibited the activation of erk  and p <dig> , while agmatine suppressed the activation of jnk .
suppression of nf-κb signaling by agmatine
total expression and activation of the nuclear factor-kappa b  from nuclear and cytosolic fractions were evaluated separately.
representative bands from the western immunoblots are shown in figure  <dig>  antibodies against total and phospho-nf-κb bound to their respective bands at  <dig> kda.
in nuclear fraction, total nf-κb and histone  <dig> were unaffected by hypoxic injury, and there were no changes with the addition of bdnf and agmatine.
however, phospho-nf-κb was significantly increased with  <dig> hour of hypoxia and returned to normal levels after  <dig> hours.
this increase in phospho-nf-κb was suppressed by agmatine but not by bdnf .
in comparison, in cytoplasmic fraction, there were no significant changes in levels of phospho-nf-κb and β-actin.
however, total nf-κb expression increased after  <dig> hour exposure to a hypoxic environment.
this increase was reduced by treatment with agmatine but not bdnf .
discussion
our present study demonstrates that agmatine, an endogenous polyamine with a guanidino group, prevents hypoxia-induced ldh release and apoptotic death in cultured transformed rat rgcs .
release of ldh was detected by ldh assay and the proportions of apoptotic cells were determined by annexin v and caspase- <dig> assays.
although agmatine cannot completely block cellular damage due to hypoxic injury, it has similar and even more extensive neuroprotective effects than bdnf, a well-known protective neurotrophin for rgcs  <cit> .
many molecules have been studied to rescue rgcs from glaucomatous cell death  <cit> , but there is still no drug which completely shelters rgcs from injury.
in this study, undifferentiated rgc- <dig> cells were used instead of differentiated rgc- <dig> cells or primary rgcs.
these immortalized cells behave differently than original rgcs, and our in vitro hypoxic model does not perfectly replicate in vivo conditions that lead to real glaucomatous injury.
however, rgc- <dig> cells, even if they are undifferentiated, have been widely used to investigate glaucomatous rgc apoptosis as a matter of convenience  <cit> .
it has been often stated that rgc- <dig> cells have similar characteristics to primary rgcs  <cit> .
the present study using rgc- <dig> cells suggests a solution to the problem, although further investigations using primary cultured rgcs or in vivo glaucoma models are needed.
various functions of agmatine have been reported  <cit> , but the precise cellular mechanisms of agmatine are not well established.
in the present study, three types of mapks and nf-κb signaling pathways were evaluated.
with hypoxic injury, phosphorylation of all three mapks and nf-κb were increased.
agmatine suppressed the hypoxia-induced activation of jnk and nf-κb, whereas bdnf inhibited the activation of erk and p <dig>  these differences might be caused by different mechanisms of action of the two molecules.
mapks are involved in highly conserved signaling pathways that regulate diverse cellular functions including cell proliferation, differentiation, migration, and apoptosis  <cit> .
they are activated through phosphorylation by distinct pathways depending on stimulus and cell type.
when activated, they can phosphorylate a wide range of substrates, including transcription factors and cytoskeletal proteins, resulting in specific cellular responses.
in the present study, agmatine regulated the activation of jnk, but not erk and p <dig>  in rgc- <dig> cells after hypoxic injury.
our results are discrepant with those of a previous report using kidney mesangial cells under high-glucose conditions, in which agmatine was involved in the erk pathway  <cit> .
however, there are no reports about agmatine's effects on mapks in the literature, and mapks have been known to work differently depending on stimuli and cell types.
furthermore, due to the implications of a report demonstrating that another antagonist of the nmda receptor mk <dig> can block the phosphorylation of mapks  <cit> , agmatine's actions as an antagonist for the nmda receptor  <cit>  suggest that it might also regulate the phosphorylation of mapks.
in the present study, we revealed that there was an activation of nf-κb in rgc- <dig> cells after hypoxic injury, and agmatine was able to suppress it.
our results are consistent with previous reports that suggest that nf-κb is activated during oxidative stress  <cit> .
however, charles et al.
<cit>  obtained a discrepant result in which the activity of nf-κb was decreased with serum-deprivation-induced apoptosis.
while oxidative stress models, including our own hypoxic model, are based on the vascular theory of glaucoma development, the serum deprivation model is based on the mechanical pressure theory  <cit> .
nf-κb signaling is presumed to have various responses according to the type of injury.
perhaps the most significant finding in this study was that both the increases in annexin v-positive cell number and caspase- <dig> activity produced by exposure of rgc- <dig> cells to hypoxia were counteracted by the addition of agmatine into the culture medium.
this suggests that agmatine may exert a neuroprotective effect by inhibiting apoptosis in the hypoxia-injured rgc- <dig> cells.
to our knowledge, this is the first report regarding the potential anti-apoptotic characteristics of agmatine in rgcs.
even though this study demonstrates that activations of jnk and nf-κb were associated with the agmatine treatment, it is still not certain whether there is a close connection between neuroprotective effects of agmatine and signaling of jnk and nf-κb.
however, it is presumed that they are related in some way.
the ability of agmatine to regulate jnk and nf-κb pathways may contribute to protecting rgcs against hypoxia-induced cell death.
further studies are needed to elucidate the precise mechanisms by which agmatine blocks apoptosis.
a deeper understanding of these mechanisms may facilitate efforts to improve the survival of rgcs from various injuries.
CONCLUSIONS
agmatine prevents hypoxia-induced ldh release and apoptotic death in transformed rgcs .
these neuroprotective effects of agmatine might be associated with the activity of jnk and nf-κb pathways.

 agmatine is an endogenous polyamine that is synthesized by the decarboxylation of l-arginine by arginine decarboxylase. it is known to be widely but unevenly distributed in the brain and other mammalian tissues. agmatine has been reported to have various biological actions. it stimulates the release of catecholamines from adrenal chromaffin cells, insulin from pancreatic islets, and luteinizing hormone-releasing hormone from the hypothalamus. also, it enhances analgesic effects of morphine, inhibits inducible nitric oxide synthase, and contributes to polyamine homeostasis. it is known that agmatine is an agonist for α2-adrenergic and imidazoline receptors, and an antagonist for the n-methyl-d-aspartate receptor. however, the precise cellular mechanisms by which agmatine acts are not yet well established. currently, a large body of experimental evidence has demonstrated the neuroprotective effects of agmatine. agmatine reduces infarct areas and neuronal loss in cerebral ischemic and ischemic-reperfusion injury models. it protects neurons from cell death after exposure to nmda and glutamate. it also attenuates the extent of neuronal loss following a spinal cord injury and shelters neurons from glucocorticoid-induced neurotoxicity and 1-methyl-4-phenyl-, -tetrahydropyridine-related dopaminergic toxicity. on the basis of these neuroprotective effects, agmatine can be presumed to have similar neuroprotective effects on retinal ganglion cells. several molecules, including α2-adrenergic agonists, nmda receptor antagonists and nos inhibitors, have been reported to protect rgcs. agmatine also acts as an α2-adrenergic agonist, nmda receptor antagonist, and suppressor of inducible nos. in the present investigation, we examined the protective effects of agmatine on hypoxia-induced apoptosis of rgcs by using the transformed rat rgcs. effects of agmatine were compared to those of brain-derived neurotrophic factor, a well-known protective neurotrophin for rgcs. in addition, several molecular pathways associated with these neuroprotective effects of agmatine were evaluated.  agmatine inhibits hypoxia-induced cell damage of rgc- cells we first examined the effects of hypoxia on rgc- cells. as shown in figure exposure to a hypoxic environment for and hours significantly increased release of lactate dehydrogenase by %, %, and %, respectively, thus demonstrating time-dependent hypoxia-induced neurotoxicity. next, we examined the protective effects of agmatine on hypoxia-induced damage in rgc- cells. after and hours of hypoxia, agmatine treatment groups did not show significant amounts of ldh release, but there were significant effects after hours of exposure. after hours, the addition of μm and μm agmatine decreased hypoxia-induced ldh release by % and %, respectively. when the protective effects of μm agmatine were compared with those of ng/ml bdnf, agmatine demonstrated a more powerful protective effect than that observed for bdnf. the neuroprotective effect of agmatine against hypoxia-induced damage to rgc- cells was further studied using hoechst and propidium iodide double staining. the control normoxic culture exhibited confluent hoechst-positive cells with homogeneous and compact nuclear morphology, and sparse numbers of pi-labeled cells. exposure to hypoxia for hours resulted in a significant loss of hoechst-positive cells and many pi-positive cells with distorted and condensed nuclei. these changes were reduced by the addition of μm agmatine or ng/ml bdnf to the cultures, and agmatine had a greater protective effect. agmatine protects rgc- cells from hypoxia-induced apoptosis in order to verify whether agmatine had protective effects on hypoxia-induced apoptotic death of rgc- cells, further analyses using annexin v assay were performed. while hours of hypoxic exposure did not change the proportion of apoptotic cells compared with the normoxic culture, there were significant increases in apoptotic cells after hours. with the addition of μm agmatine and ng/ml bdnf, the proportion of apoptotic cells decreased. specific caspase- activity was assessed using a caspase- assay, which could measure the cleavage of the caspase- specific substrate ac-devd-pna. after hours of hypoxic injury, the caspase- activity was significantly increased, and it was suppressed by μm agmatine. the results obtained by adding μm agmatine were similar to those seen with μm caspase- inhibitor z-vad-fmk. selective suppression of jnk activation by agmatine three mitogen-activated protein kinases, including c-jun n-terminal kinase, extracellular signal-regulated kinase, and p kinase, were investigated using western immunoblots. the amounts of total and phosphorylated mapks and β-actin are shown in figure total expression of the three mapks and β-actin were not affected by hypoxic injury. in addition, there were no significant changes after treatment with bdnf or agmatine. antibodies against phospho-jnks detected two bands at and kda, and both bands had a similar tendency. increases of phospho-jnks in rgc- cells became evident hours after hypoxic injury and remained elevated for hours. agmatine suppressed the hypoxia-induced phosphorylation of jnks, but bdnf did not influence their phosphorylation. antibodies against phospho-erks also detected two bands at and kda, and both bands were similar. phospho-erks were not detected in the normoxic cultures. however, they were highly expressed in rgc- cells even after hours of hypoxia and remained elevated for hours. bdnf completely blocked the phosphorylation of erks for hours, but it had no effect thereafter. in comparison, agmatine did not significantly affect the phosphorylation of erks. antibodies against phospho-p detected one band at kda. phospho-p was not detected in normoxic cultures until hours of exposure to hypoxia, but it was evident in hypoxic cultures even after hours and remained elevated for hours. bdnf only blocked the phosphorylation of p at hours and agmatine had no effect on phospho-p levels at any time points. thus, phospho-mapks showed different activation profiles in response to hypoxic injuries in rgc- cells; erk and p were activated relatively earlier than jnk. bdnf inhibited the activation of erk and p, while agmatine suppressed the activation of jnk. suppression of nf-κb signaling by agmatine total expression and activation of the nuclear factor-kappa b from nuclear and cytosolic fractions were evaluated separately. representative bands from the western immunoblots are shown in figure antibodies against total and phospho-nf-κb bound to their respective bands at kda. in nuclear fraction, total nf-κb and histone were unaffected by hypoxic injury, and there were no changes with the addition of bdnf and agmatine. however, phospho-nf-κb was significantly increased with hour of hypoxia and returned to normal levels after hours. this increase in phospho-nf-κb was suppressed by agmatine but not by bdnf. in comparison, in cytoplasmic fraction, there were no significant changes in levels of phospho-nf-κb and β-actin. however, total nf-κb expression increased after hour exposure to a hypoxic environment. this increase was reduced by treatment with agmatine but not bdnf. discussion our present study demonstrates that agmatine, an endogenous polyamine with a guanidino group, prevents hypoxia-induced ldh release and apoptotic death in cultured transformed rat rgcs. release of ldh was detected by ldh assay and the proportions of apoptotic cells were determined by annexin v and caspase- assays. although agmatine cannot completely block cellular damage due to hypoxic injury, it has similar and even more extensive neuroprotective effects than bdnf, a well-known protective neurotrophin for rgcs. many molecules have been studied to rescue rgcs from glaucomatous cell death, but there is still no drug which completely shelters rgcs from injury. in this study, undifferentiated rgc- cells were used instead of differentiated rgc- cells or primary rgcs. these immortalized cells behave differently than original rgcs, and our in vitro hypoxic model does not perfectly replicate in vivo conditions that lead to real glaucomatous injury. however, rgc- cells, even if they are undifferentiated, have been widely used to investigate glaucomatous rgc apoptosis as a matter of convenience. it has been often stated that rgc- cells have similar characteristics to primary rgcs. the present study using rgc- cells suggests a solution to the problem, although further investigations using primary cultured rgcs or in vivo glaucoma models are needed. various functions of agmatine have been reported, but the precise cellular mechanisms of agmatine are not well established. in the present study, three types of mapks and nf-κb signaling pathways were evaluated. with hypoxic injury, phosphorylation of all three mapks and nf-κb were increased. agmatine suppressed the hypoxia-induced activation of jnk and nf-κb, whereas bdnf inhibited the activation of erk and p these differences might be caused by different mechanisms of action of the two molecules. mapks are involved in highly conserved signaling pathways that regulate diverse cellular functions including cell proliferation, differentiation, migration, and apoptosis. they are activated through phosphorylation by distinct pathways depending on stimulus and cell type. when activated, they can phosphorylate a wide range of substrates, including transcription factors and cytoskeletal proteins, resulting in specific cellular responses. in the present study, agmatine regulated the activation of jnk, but not erk and p in rgc- cells after hypoxic injury. our results are discrepant with those of a previous report using kidney mesangial cells under high-glucose conditions, in which agmatine was involved in the erk pathway. however, there are no reports about agmatine's effects on mapks in the literature, and mapks have been known to work differently depending on stimuli and cell types. furthermore, due to the implications of a report demonstrating that another antagonist of the nmda receptor mk can block the phosphorylation of mapks, agmatine's actions as an antagonist for the nmda receptor suggest that it might also regulate the phosphorylation of mapks. in the present study, we revealed that there was an activation of nf-κb in rgc- cells after hypoxic injury, and agmatine was able to suppress it. our results are consistent with previous reports that suggest that nf-κb is activated during oxidative stress. however, charles et al. obtained a discrepant result in which the activity of nf-κb was decreased with serum-deprivation-induced apoptosis. while oxidative stress models, including our own hypoxic model, are based on the vascular theory of glaucoma development, the serum deprivation model is based on the mechanical pressure theory. nf-κb signaling is presumed to have various responses according to the type of injury. perhaps the most significant finding in this study was that both the increases in annexin v-positive cell number and caspase- activity produced by exposure of rgc- cells to hypoxia were counteracted by the addition of agmatine into the culture medium. this suggests that agmatine may exert a neuroprotective effect by inhibiting apoptosis in the hypoxia-injured rgc- cells. to our knowledge, this is the first report regarding the potential anti-apoptotic characteristics of agmatine in rgcs. even though this study demonstrates that activations of jnk and nf-κb were associated with the agmatine treatment, it is still not certain whether there is a close connection between neuroprotective effects of agmatine and signaling of jnk and nf-κb. however, it is presumed that they are related in some way. the ability of agmatine to regulate jnk and nf-κb pathways may contribute to protecting rgcs against hypoxia-induced cell death. further studies are needed to elucidate the precise mechanisms by which agmatine blocks apoptosis. a deeper understanding of these mechanisms may facilitate efforts to improve the survival of rgcs from various injuries.  agmatine prevents hypoxia-induced ldh release and apoptotic death in transformed rgcs. these neuroprotective effects of agmatine might be associated with the activity of jnk and nf-κb pathways. 
agmatine is an endogenous polyamine formed by the decarboxylation of l-arginine. although total expression of mapks and nf-κb was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. expression and phosphorylation of mitogen-activated protein kinases and nuclear factor-kappa b were investigated by western immunoblot analysis. cell viability was determined by lactate dehydrogenase assay and apoptosis was examined by annexin v and caspase- assays. this observed cell loss was due to apoptotic cell death, as established by annexin v and caspase- assays. our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in rgc- cells and that its effects may act through the jnk and nf-κb signaling pathways. after hours of hypoxic culture, the ldh assay showed % cell loss, which was reduced to % and % when agmatine and bdnf were administered, respectively. we investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells. the effects of agmatine were compared to those of brain-derived neurotrophic factor, a well-known protective neurotrophin for retinal ganglion cells.
 agmatine is an endogenous polyamine formed by the decarboxylation of l-arginine. we investigated the protective effects of agmatine against hypoxia-induced apoptosis of immortalized rat retinal ganglion cells. rgc- cells were cultured in a closed hypoxic chamber with or without agmatine. cell viability was determined by lactate dehydrogenase assay and apoptosis was examined by annexin v and caspase- assays. expression and phosphorylation of mitogen-activated protein kinases and nuclear factor-kappa b were investigated by western immunoblot analysis. the effects of agmatine were compared to those of brain-derived neurotrophic factor, a well-known protective neurotrophin for retinal ganglion cells.  after hours of hypoxic culture, the ldh assay showed % cell loss, which was reduced to % and % when agmatine and bdnf were administered, respectively. this observed cell loss was due to apoptotic cell death, as established by annexin v and caspase- assays. although total expression of mapks and nf-κb was not influenced by hypoxic injury, phosphorylation of these two proteins was increased. agmatine reduced phosphorylation of jnk and nf-κb, while bdnf suppressed phosphorylation of erk and p  our results show that agmatine has neuroprotective effects against hypoxia-induced retinal ganglion cell damage in rgc- cells and that its effects may act through the jnk and nf-κb signaling pathways. our data suggest that agmatine may lead to a novel therapeutic strategy to reduce retinal ganglion cell injury related to hypoxia. 