BACKGROUND
it is well established that enhanced bioavailability of endogenous nitric oxide  affords cardioprotection against ischemia and reoxygenation-induced injury  <cit> .
this important mediator is enzymatically produced by the no-synthases .
there are three nos isoforms, widely distributed through most cells and tissues, which can produce no by converting l-arginine to l-citruline in the presence of nadph, o <dig> and other co-factors  <cit> .
the endothelial nos  and neuronal nos  are constitutive and ca++-dependent whereas the expression of the inducible nos  is ca++-independent and stress-induced.
it has been documented that nnos is located in the peripheral vagal nerves, sympathetic nerves and in the autonomic control region of the central nervous system  <cit> .
in cardiomyocytes, nnos is expressed in sarcoplasmic reticulum, sarcolemma and mitochondria  <cit> .
no derived from nnos influences the beat to beat regulation of basal cardiac function, serving negative feedback control of ca++ fluxes  <cit> .
in addition, it facilitates vagal stimulation and inhibits sympathetic stimulation by exerting its effect in the central and peripheral nervous system.
no derived from enos is essential for eliciting early ischemic preconditioning   <cit> , however, there is still controversy about the role of nnos.
thus, although nnos expression is increased in the acute phase of myocardial infarction in both infarcted and non-infarcted tissues in the rat heart  <cit> , it has been reported that in the nnos knockout mice, there is no change in myocardial infarct size  <cit> .
by contrast, it appears that delayed ip  is dependent on nnos in the rabbit  <cit> , whereas the deficit of nnos reduces cerebral ischemic injury in mice  <cit> .
mitokatp channels, located in the inner mitochondrial membrane, are central for ip  <cit> .
few studies have shown a link between mitokatp and no in ip.
sasaki et al suggested that no causes the opening of mitokatp directly and indirectly  <cit>  which in turn induces blunting of the ca++ overload in mitochondria, thus contributing to the delayed ip.
it has also been reported that the opening of the mitokatp channels by diazoxide induces cardioprotection in a no-dependent manner  <cit> .
furthermore, rakhit et al have demonstrated that the exogenous no donor snap induces early ip by a cgmp-dependent mechanism and delayed ip by a cgmp-independent mechanism which is mediated by activation of pkc and mitokatp channels  <cit> .
interestingly, nakano et al suggested that snap-induced cardioprotection cannot be reproduced by endogenous no  <cit>  also implying that there are two separate pathways for no-induced cardioprotection.
our laboratory has previously demonstrated that opening of p38mapk is an obligatory step in ip that lies downstream of pkc activation  <cit> .
however, there is little information regarding the relationship of no with p38mapk in the myocardium.
thus, for example, kim et al.
showed that sodium nitroprusside activates p42/ <dig> and p38mapk in adult rat cardiomyocytes via both cgmp-dependent and cgmp-independent mechanisms  <cit> .
in another study, wang et al demonstrated that no suppressed the angiotensin-i induced activation of erk in cardiac fibroblast  <cit> .
other studies carried out in non-cardiac mitogenic cells have shown that no induced apoptosis is mediated directly by activation of mapk  <cit> .
so, the relationship of no with p38mapk remains largely undefined.
therefore, the aims of the present studies were to:  elucidate the role of nnos in ischemia/reoxygenation-induced injury and ip,  determine whether its effect is species-dependent, and  investigate the relationship of nnos with mitokatp channels and p38mapk, two key components of the intracellular transduction pathway of protection by ip.
RESULTS
myocardial viability, as assessed by the absence of necrosis and apoptosis by the end of the experimental period in aerobically incubated myocardial slices, was greater than 95% in all instances.
the role of nnos in ischemic injury
figures 1a-c show that, in the rat myocardium, the selective inhibition of nnos by trim resulted in a significant reduction in ck release and in cell necrosis and apoptosis, as compared to the mean values in the ischemia/reoxygenation alone group.
the degree of this protection was similar to administration of exogenous no but greater than the non-selective nos inhibitor l-name.
is the role of nnos species-dependent?
as seen in figures 2a-f, and in comparison with the results observed in figures 1a-c, the order of magnitude of ischemic injury for ck release and cell necrosis and apoptosis varied slightly between species, however, identical effects on nos inhibition and from the administration of exogenous no to that seen in rat myocardium  were observed for both mouse and human myocardium.
knocking out nnos
figures 3a-c demonstrate that, as opposed to wild type mice, the myocardium of nnos knockout mice could not be protected by the specific nos inhibitor trim as the mean values for ck release and cell necrosis and apoptosis were not significantly different from muscles subjected to ischemia/reoxygenation alone.
the absence of benefit when l-name was added to the myocardium of knockout nnos mice supports the thesis that although the endogenously produced no by enos may be beneficial its deficit did not cause further ischemic injury.
as seen above, again the no donor snap almost abolished the ck release and the cell death induced by ischemia and reoxygenation.
importantly, and in contrast with wild type mice, the myocardium of nnos knockout out mice could not be protected by ip since the mean values for ck release and cell necrosis and apoptosis did not differ from the values in the ischemia/reoxygenation alone group.
however, the degree of injury in the ischemia-reoxygenation alone groups was similar in both wild type and nnos knockout mice as previously reported  <cit> .
the relationship of exogenous no and nnos inhibition with mitokatp channels and p38mapk
figures 4a-c demonstrate that the reduction in ck release and in cell necrosis and apoptosis caused by the no donor snap and the inhibition of nnos with trim, was unaffected by the mitokatp channel blocker 5-hd, and that, as expected, it blocked the protection by ip.
by contrast, figures 5a-c show that the p38mapk blocker sb <dig> abolished the protective effect of exogenous no and of nnos inhibition, along with the benefit of ip.
discussion and
CONCLUSIONS
the present studies have demonstrated that nnos plays a dual role so that its presence is necessary to afford cardioprotection by ip, but at the same time its inhibition reduces myocardial ischemic/reoxygenation induced injury.
in addition, they have shown that the protection obtained by the inhibition of nnos is as potent as the exogenous administration of no and that of ip, effects that are species-independent.
importantly, the action of nnos and those of exogenous no are exerted downstream of mitokatp channels and upstream of p <dig> mapk.
the duplicity of nnos-derived effects seen in our studies can be explained, on one hand by a physiological production of no that triggers the cardioprotective intracellular mechanism of interventions such as ip, and on the other hand by a possible much greater production of no induced by ischemia/reoxygenation that is detrimental.
in this connection, other investigators have also reported in an infarct size model that nnos plays an opposite role in ischemia/reperfusion and ip  <cit>  and that the protection of delayed ip in the rabbit heart is dependent on nnos  <cit> .
since no donors such as snap also have strong cardioprotective effects, it may be speculated that the amount and cell location of no production would determine whether the final result is tissue protection or injury.
it is also interesting to note that myocardial injury  was not increased by ischemia/reoxygenation in nnos knockout mice, a finding supported by the absence of changes in infarct size in nnos knockout mice  <cit> .
by contrast, a deficit in nnos in mice reduced the degree of cerebral ischemic injury  <cit> , thus suggesting that the role of nnos in ischemia/reoxygenation could be organ-specific.
our studies locate the action of nnos downstream of the mitokatp channels since the protection induced by nnos specific inhibitor trim was blocked by 5-hd, a mitokatp channel inhibitor.
the finding that exogenous no is also acting at the same point reinforces the view that the production of endogenous no by nnos is the mechanism of protection.
indeed, other laboratories have shown that the protection induced by exogenous no is not affected by blockade of mitokatp channels with 5hd or glibenclamide  <cit>  and that the protection induced by the mitokatp channel opener diazoxide is no-dependent  <cit> , further supporting the thesis that no metabolism is a critical component of the cell survival mechanism.
the different location of the two constitutive forms of nos, nnos and enos, may also suggest that no plays a distinctive role depending on the cellular site where it is produced.
this thesis gets support from the finding that the specific inhibition of nnos was more protective than the non-specific inhibition of nos.
certainly, this is an important question that would require further investigations.
previously, our laboratory has shown that p38mapk is another critical factor of ip-induced cardioprotection located beyond the mitokatp channels  <cit> .
here we have also shown that the effect of no metabolism in the intracellular signaling pathways of cardioprotection is exerted upstream of p38mapk, as the beneficial effects of exogenous no donation and nnos inhibition were abolished by the p38mapk blocker sb <dig>  these findings are supported by the demonstration of protection of cardiac  <cit>  and non-cardiac cells  <cit> , by no through activation of p38mapk and other mapks.
an additional important finding is the identical role played by nnos in the three species studied , suggesting that the function of the enzyme has been preserved throughout evolution.
these results may have clinical implications since the findings observed from the animal studies could be extrapolated to human beings without the need for additional confirmatory investigations.
nonetheless, it is worth emphasizing that the present studies were performed in an in vitro model and therefore care must be taken when extrapolating the present results to clinical conditions.
however, despite the differences between atrial and ventricular samples, the myocardium from the mouse and rat ventricles and from the human right atrial appendage, responded in a similar manner and hence the different source of myocardium should not represent a problem for the interpretation of the results.
during cardiac surgery, myocardial ischemia is induced in a controlled manner and therefore it would be possible to enhance cardioprotection by manipulation of the no metabolism.
however, in the clinical setup, the heart may be subjected to stresses that can result in the expression of the inducible nos  and it would be important to elucidate the role of the constitutive nos isoforms, nnos and enos, in the pathophysiology of ischemia/reperfusion-induced injury under such conditions.
it is worth emphasizing that, although the activity of nnos has been shown to be increased in the late phase of delayed ip <cit> , to the best of our knowledge, the activity of the enzyme has not been determined in early ip.
indeed, it would be important to determine the activity of all nos isoforms in future studies.
in conclusion, here we have shown that nnos is a critical factor playing a dual role by participating in myocardial ischemia/reoxygenation injury, and at the same time, by promoting myocardial protection.
these results are of clinical relevance for the design of therapeutic interventions aimed at reducing the myocardial damage caused by ischemia/reoxygenation.

 it is well established that enhanced bioavailability of endogenous nitric oxide affords cardioprotection against ischemia and reoxygenation-induced injury. this important mediator is enzymatically produced by the no-synthases. there are three nos isoforms, widely distributed through most cells and tissues, which can produce no by converting l-arginine to l-citruline in the presence of nadph, o and other co-factors. the endothelial nos and neuronal nos are constitutive and ca++-dependent whereas the expression of the inducible nos is ca++-independent and stress-induced. it has been documented that nnos is located in the peripheral vagal nerves, sympathetic nerves and in the autonomic control region of the central nervous system. in cardiomyocytes, nnos is expressed in sarcoplasmic reticulum, sarcolemma and mitochondria. no derived from nnos influences the beat to beat regulation of basal cardiac function, serving negative feedback control of ca++ fluxes. in addition, it facilitates vagal stimulation and inhibits sympathetic stimulation by exerting its effect in the central and peripheral nervous system. no derived from enos is essential for eliciting early ischemic preconditioning, however, there is still controversy about the role of nnos. thus, although nnos expression is increased in the acute phase of myocardial infarction in both infarcted and non-infarcted tissues in the rat heart, it has been reported that in the nnos knockout mice, there is no change in myocardial infarct size. by contrast, it appears that delayed ip is dependent on nnos in the rabbit, whereas the deficit of nnos reduces cerebral ischemic injury in mice. mitokatp channels, located in the inner mitochondrial membrane, are central for ip. few studies have shown a link between mitokatp and no in ip. sasaki et al suggested that no causes the opening of mitokatp directly and indirectly which in turn induces blunting of the ca++ overload in mitochondria, thus contributing to the delayed ip. it has also been reported that the opening of the mitokatp channels by diazoxide induces cardioprotection in a no-dependent manner. furthermore, rakhit et al have demonstrated that the exogenous no donor snap induces early ip by a cgmp-dependent mechanism and delayed ip by a cgmp-independent mechanism which is mediated by activation of pkc and mitokatp channels. interestingly, nakano et al suggested that snap-induced cardioprotection cannot be reproduced by endogenous no also implying that there are two separate pathways for no-induced cardioprotection. our laboratory has previously demonstrated that opening of p38mapk is an obligatory step in ip that lies downstream of pkc activation. however, there is little information regarding the relationship of no with p38mapk in the myocardium. thus, for example, kim et al. showed that sodium nitroprusside activates p42/ and p38mapk in adult rat cardiomyocytes via both cgmp-dependent and cgmp-independent mechanisms. in another study, wang et al demonstrated that no suppressed the angiotensin-i induced activation of erk in cardiac fibroblast. other studies carried out in non-cardiac mitogenic cells have shown that no induced apoptosis is mediated directly by activation of mapk. so, the relationship of no with p38mapk remains largely undefined. therefore, the aims of the present studies were to: elucidate the role of nnos in ischemia/reoxygenation-induced injury and ip, determine whether its effect is species-dependent, and investigate the relationship of nnos with mitokatp channels and p38mapk, two key components of the intracellular transduction pathway of protection by ip.  myocardial viability, as assessed by the absence of necrosis and apoptosis by the end of the experimental period in aerobically incubated myocardial slices, was greater than 95% in all instances. the role of nnos in ischemic injury figures 1a-c show that, in the rat myocardium, the selective inhibition of nnos by trim resulted in a significant reduction in ck release and in cell necrosis and apoptosis, as compared to the mean values in the ischemia/reoxygenation alone group. the degree of this protection was similar to administration of exogenous no but greater than the non-selective nos inhibitor l-name. is the role of nnos species-dependent? as seen in figures 2a-f, and in comparison with the results observed in figures 1a-c, the order of magnitude of ischemic injury for ck release and cell necrosis and apoptosis varied slightly between species, however, identical effects on nos inhibition and from the administration of exogenous no to that seen in rat myocardium were observed for both mouse and human myocardium. knocking out nnos figures 3a-c demonstrate that, as opposed to wild type mice, the myocardium of nnos knockout mice could not be protected by the specific nos inhibitor trim as the mean values for ck release and cell necrosis and apoptosis were not significantly different from muscles subjected to ischemia/reoxygenation alone. the absence of benefit when l-name was added to the myocardium of knockout nnos mice supports the thesis that although the endogenously produced no by enos may be beneficial its deficit did not cause further ischemic injury. as seen above, again the no donor snap almost abolished the ck release and the cell death induced by ischemia and reoxygenation. importantly, and in contrast with wild type mice, the myocardium of nnos knockout out mice could not be protected by ip since the mean values for ck release and cell necrosis and apoptosis did not differ from the values in the ischemia/reoxygenation alone group. however, the degree of injury in the ischemia-reoxygenation alone groups was similar in both wild type and nnos knockout mice as previously reported. the relationship of exogenous no and nnos inhibition with mitokatp channels and p38mapk figures 4a-c demonstrate that the reduction in ck release and in cell necrosis and apoptosis caused by the no donor snap and the inhibition of nnos with trim, was unaffected by the mitokatp channel blocker 5-hd, and that, as expected, it blocked the protection by ip. by contrast, figures 5a-c show that the p38mapk blocker sb abolished the protective effect of exogenous no and of nnos inhibition, along with the benefit of ip. discussion and  the present studies have demonstrated that nnos plays a dual role so that its presence is necessary to afford cardioprotection by ip, but at the same time its inhibition reduces myocardial ischemic/reoxygenation induced injury. in addition, they have shown that the protection obtained by the inhibition of nnos is as potent as the exogenous administration of no and that of ip, effects that are species-independent. importantly, the action of nnos and those of exogenous no are exerted downstream of mitokatp channels and upstream of p mapk. the duplicity of nnos-derived effects seen in our studies can be explained, on one hand by a physiological production of no that triggers the cardioprotective intracellular mechanism of interventions such as ip, and on the other hand by a possible much greater production of no induced by ischemia/reoxygenation that is detrimental. in this connection, other investigators have also reported in an infarct size model that nnos plays an opposite role in ischemia/reperfusion and ip and that the protection of delayed ip in the rabbit heart is dependent on nnos. since no donors such as snap also have strong cardioprotective effects, it may be speculated that the amount and cell location of no production would determine whether the final result is tissue protection or injury. it is also interesting to note that myocardial injury was not increased by ischemia/reoxygenation in nnos knockout mice, a finding supported by the absence of changes in infarct size in nnos knockout mice. by contrast, a deficit in nnos in mice reduced the degree of cerebral ischemic injury, thus suggesting that the role of nnos in ischemia/reoxygenation could be organ-specific. our studies locate the action of nnos downstream of the mitokatp channels since the protection induced by nnos specific inhibitor trim was blocked by 5-hd, a mitokatp channel inhibitor. the finding that exogenous no is also acting at the same point reinforces the view that the production of endogenous no by nnos is the mechanism of protection. indeed, other laboratories have shown that the protection induced by exogenous no is not affected by blockade of mitokatp channels with 5hd or glibenclamide and that the protection induced by the mitokatp channel opener diazoxide is no-dependent, further supporting the thesis that no metabolism is a critical component of the cell survival mechanism. the different location of the two constitutive forms of nos, nnos and enos, may also suggest that no plays a distinctive role depending on the cellular site where it is produced. this thesis gets support from the finding that the specific inhibition of nnos was more protective than the non-specific inhibition of nos. certainly, this is an important question that would require further investigations. previously, our laboratory has shown that p38mapk is another critical factor of ip-induced cardioprotection located beyond the mitokatp channels. here we have also shown that the effect of no metabolism in the intracellular signaling pathways of cardioprotection is exerted upstream of p38mapk, as the beneficial effects of exogenous no donation and nnos inhibition were abolished by the p38mapk blocker sb these findings are supported by the demonstration of protection of cardiac and non-cardiac cells, by no through activation of p38mapk and other mapks. an additional important finding is the identical role played by nnos in the three species studied, suggesting that the function of the enzyme has been preserved throughout evolution. these results may have clinical implications since the findings observed from the animal studies could be extrapolated to human beings without the need for additional confirmatory investigations. nonetheless, it is worth emphasizing that the present studies were performed in an in vitro model and therefore care must be taken when extrapolating the present results to clinical conditions. however, despite the differences between atrial and ventricular samples, the myocardium from the mouse and rat ventricles and from the human right atrial appendage, responded in a similar manner and hence the different source of myocardium should not represent a problem for the interpretation of the results. during cardiac surgery, myocardial ischemia is induced in a controlled manner and therefore it would be possible to enhance cardioprotection by manipulation of the no metabolism. however, in the clinical setup, the heart may be subjected to stresses that can result in the expression of the inducible nos and it would be important to elucidate the role of the constitutive nos isoforms, nnos and enos, in the pathophysiology of ischemia/reperfusion-induced injury under such conditions. it is worth emphasizing that, although the activity of nnos has been shown to be increased in the late phase of delayed ip, to the best of our knowledge, the activity of the enzyme has not been determined in early ip. indeed, it would be important to determine the activity of all nos isoforms in future studies. in conclusion, here we have shown that nnos is a critical factor playing a dual role by participating in myocardial ischemia/reoxygenation injury, and at the same time, by promoting myocardial protection. these results are of clinical relevance for the design of therapeutic interventions aimed at reducing the myocardial damage caused by ischemia/reoxygenation. 
the cardioprotection seen with nnos inhibition was as potent as that of ip, however, in nnos knockout mice the cardioprotective effect of non-selective nos and selective nnos inhibition and also that of ip was blocked while the benefit of exogenous no remained intact. ventricular myocardial slices from rats and wild and nnos knockout mice, and right atrial myocardial slices from human were subjected to min ischemia and min reoxygenation. additional studies revealed that the cardioprotection afforded by exogenous no and by inhibition of nnos were unaffected by the mitokatp channel blocker 5-hd, although it was abrogated by p38mapk blocker sb therefore, the aims of these studies were to: investigate the role of nnos in ischemia/reoxygenation-induced injury and ip, determine whether its effect is species-dependent, and elucidate the relationship of nnos with mitokatp channels and p38mapk, two key components of ip transduction pathway. the role of nnos is species-independent and exerted downstream of the mitokatp channels and upstream of p38mapk. nnos plays a dual role in ischemia/reoxygenation in that its presence is necessary to afford cardioprotection by ip and its inhibition reduces myocardial ischemic injury.
 nitric oxide is cardioprotective and a mediator of ischemic preconditioning. endothelial nitric oxide synthase is protective against myocardial ischemic injury and a component of ip but the role and location of neuronal nitric oxide synthase remains unclear. therefore, the aims of these studies were to: investigate the role of nnos in ischemia/reoxygenation-induced injury and ip, determine whether its effect is species-dependent, and elucidate the relationship of nnos with mitokatp channels and p38mapk, two key components of ip transduction pathway.  ventricular myocardial slices from rats and wild and nnos knockout mice, and right atrial myocardial slices from human were subjected to min ischemia and min reoxygenation. specimens were randomized to receive various treatments. both the provision of exogenous no and the inhibition of endogenous no production significantly reduced tissue injury, an effect that was species-independent. the cardioprotection seen with nnos inhibition was as potent as that of ip, however, in nnos knockout mice the cardioprotective effect of non-selective nos and selective nnos inhibition and also that of ip was blocked while the benefit of exogenous no remained intact. additional studies revealed that the cardioprotection afforded by exogenous no and by inhibition of nnos were unaffected by the mitokatp channel blocker 5-hd, although it was abrogated by p38mapk blocker sb  nnos plays a dual role in ischemia/reoxygenation in that its presence is necessary to afford cardioprotection by ip and its inhibition reduces myocardial ischemic injury. the role of nnos is species-independent and exerted downstream of the mitokatp channels and upstream of p38mapk. 