AJP - Heart AJP: Gastrointestinal and Liver Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 287: H2070-H2077, 2004. First published June 24, 2004; doi:10.1152/ajpheart.00431.2004
0363-6135/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/H2070    most recent
00431.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (19)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Kukreja, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Kukreja, R. C.

Opening of Ca2+-activated K+ channels triggers early and delayed preconditioning against I/R injury independent of NOS in mice

Xiaoyin Wang, Chang Yin, Lei Xi, and Rakesh C. Kukreja

Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298-0281

Submitted 6 May 2004 ; accepted in final form 22 June 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Opening of Ca2+-activated K+ (KCa) channels has been shown to confer early cardioprotection. It is unknown whether the opening of these channels also induces delayed cardioprotection. In addition, we determined the involvement of nitric oxide synthases (NOSs), which have been implicated in cardioprotection induced by opening of mitochondrial ATP-sensitive K+ (KATP) channels. Adult male ICR mice were pretreated with the KCa-channel opener NS-1619 either 10 min or 24 h before 30 min of global ischemia and 60 min of reperfusion (I/R) in Langendorff mode. Infusion of NS-1619 (10 µM) for 10 min before I/R led to smaller infarct sizes as compared with the vehicle (DMSO)-treated group (P < 0.05). This infarct-limiting effect of NS-1619 was associated with improvement in ventricular functional recovery after I/R. The NS-1619-induced protection was abolished by coadministration with the KCa-channel blocker paxilline (1 µM). Similarly, pretreatment with NS-1619 (1 mg/kg ip) induced delayed protection 24 h later (P < 0.05). Interestingly, the NS-1619-induced late protection was not blocked by the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (15 mg/kg ip). Unlike diazoxide (the opener of mitochondrial KATP channels), NS-1619 did not increase the expression of inducible or endothelial NOS. Western blot analysis demonstrated the existence of {alpha}- and {beta}-subunits of KCa channels in mouse heart tissue. We conclude that opening of KCa channels leads to both early and delayed preconditioning effects through a mechanism that is independent of nitric oxide.

ischemia-reperfusion; nitric oxide synthase; pharmacological preconditioning; signaling; NS-1619

K+ CHANNELS ARE A UBIQUITOUS family of cell membrane proteins that play critical roles in a wide variety of physiological processes, including regulation of heart rate, muscle contraction, and neurotransmitter release, as well as cell volume regulation (43). To serve such a broad spectrum of functional roles, a diverse set of K+ channels has evolved in cells from various organs including the heart. Among them, ATP-sensitive K+ (KATP) channels have been the target of intense investigation for therapeutic applications over the past two decades (16–19, 43). As a result, numerous openers and blockers of KATP channels have been identified. It is well established that the openers of KATP channels can induce early and delayed cardioprotection against ischemia-reperfusion (I/R) injury. The blockers of KATP channels abolish the protective effects of myocardial preconditioning despite the ongoing debate on the relative importance of mitochondrial (mitoKATP) vs. sarcolemmal KATP channels and the trigger vs. end-effector roles of KATP channels in myocardial preconditioning (16, 18, 19, 27, 31, 37, 42, 49). Among the underlying mechanisms of cardioprotection with KATP channels, nitric oxide (NO), particularly that derived from inducible NO synthase (iNOS), has been identified as the key mediator of delayed cardioprotection with diazoxide, which is a putative opener of mitochondrial KATP (mitoKATP) channels (42).

Another major type of K+ channel, the so-called Ca2+-activated K+ (KCa) channel, is K+ selective with large-conductance, voltage-sensitive channels that are expressed in numerous tissue types, including the brain, heart, and smooth muscle (1, 13, 25, 29, 47). KCa channels can be activated by elevations in intracellular Ca2+ through the physiological range as well as via membrane depolarization (11, 12, 24, 30, 43). KCa channels have the biophysical characteristics of single-channel conductance of ~250 pS in symmetrical K+ (12, 30). Each KCa channel is composed of a pore-forming {alpha}-subunit and an auxiliary {beta}-subunit that has modulatory functions on the channel's opening as well as sensitivity to channel-blocking agents (12, 28, 29, 43). The cytoprotective effects of KCa channel opening were not fully recognized until very recently. This is probably due to the overwhelming interest in KATP channels regarding preconditioning and perhaps also because of the lack of highly selective openers of KCa channels before the discovery of 1,3-dihydro-1-[2-hydroxy-5-(triflu-oromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) in 1994 (32). Using this compound, Xu et al. (47) first demonstrated that opening of KCa channels leads to a significant reduction in infarct size after global I/R in isolated, perfused rabbit hearts. These authors also provided some mechanistic explanations for the NS-1619-induced cardioprotection. On the basis of the protein expression of KCa channels on mitochondrial inner membranes of guinea pig ventricular cells as well as the acceleration of mitochondrial K+ uptake (47), it was proposed that NS-1619 protects the heart against I/R injury by enhancing mitochondrial K+ uptake and in turn reducing mitochondrial Ca2+ overload in cardiomyocytes. These studies raise several important questions related to the cardioprotective effects of these channels. First, it was shown by us and others that opening of mitoKATP channels with the selective opener diazoxide induces delayed preconditioning in heart. However, it is not known whether the opening of KCa channels also induces similar, long-lasting cardioprotective effects. Furthermore, previous studies suggested that NO generation was an important component of the preconditioning effect induced by diazoxide (31). It has been proposed that diazoxide triggers signaling pathways (through activation of PKC) that lead to synthesis of iNOS. The present study was designed to target three specific aims as follows: 1) to demonstrate that opening of KCa channels with NS-1619 can induce not only early protection as found in rabbit hearts (47) but also delayed protection in mouse hearts, 2) to determine whether NS-1619-induced delayed protection is mediated by iNOS and/or endothelial NOS (eNOS), and 3) to show the existence of KCa protein isoforms in mouse cardiac tissue.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult male outbred ICR mice were purchased from Harlan (Indianapolis, IN) and used for all animal experiments, which were conducted under the guidelines for humane use and care of laboratory animals for biomedical research published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 1996). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.

Drugs. NS-1619, which is a selective opener of KCa channels (32), and paxilline, a blocker of KCa channels (28, 36), were purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Both drugs were dissolved in DMSO (Sigma-Aldrich) for either coronary infusion or intraperitoneal administration (final DMSO concentration, <1%). N{omega}-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of all three NOS isoforms [i.e., neuronal NOS (nNOS), iNOS, and eNOS], and diazoxide, an opener of mitoKATP channels, were also purchased from Sigma-Aldrich. These chemicals were dissolved in saline before intraperitoneal injection.

Langendorff-isolated, perfused mouse heart model. The methodology of isolating the perfused mouse heart was previously described in detail (45, 46). No-flow global ischemia was attained by completely stopping the perfusate inflow. Reperfusion was achieved by reopening the buffer-perfusion line. Ventricular contractile force was continuously measured with a force-displacement transducer (model FT03; Grass) attached to the apex of the heart and was recorded and analyzed with a computerized data acquisition and analysis system (PowerLab 8SP; ADInstruments). The heart temperature was precisely maintained at 37°C throughout the experiment. Coronary flow rate was measured by timed collection of the outflow coronary perfusate.

Routes of drug administration. Intracoronary infusion or intraperitoneal injection was used as the route of drug administration depending on the experimental group (described below). Similar to the method used in our previous study (46), intracoronary infusion was accomplished through a sidearm of a three-way stopcock connected directly above the aortic cannula with a microdialysis syringe pump (model 22; Harvard). The drug infusion speed was set at 0.25 ml/min, which was ~15% of normal coronary flow rate for the mouse hearts.

Experimental groups. As illustrated in Fig. 1, mice were randomly assigned to one of 11 experimental groups (n = 6–9 mice/group). Groups 1 to 6 were used for "early protection studies," and groups 7 to 11 were designated for "delayed protection studies." Group 1 (DMSO): after 20 min of stabilization, DMSO (solvent for NS-1619 and paxilline) was infused into coronary inflow for 10 min before 30 min of ischemia and subsequent 60 min of reperfusion. Group 2 (NS): NS-1619 (10 µM) was infused for 10 min before I/R. Group 3 (NS+PX): NS-1619 and paxilline (1 µM) were coinfused for 10 min before I/R. Group 4 (PX): paxilline alone was infused for 10 min before I/R. Group 5 (DMSO-R): DMSO was infused for the first 30 min of reperfusion. Group 6 (NS-R): NS-1619 was infused for the first 30 min of reperfusion. Group 7 (DMSO-DP): mice received an intraperitoneal injection of 0.15 ml of 1% DMSO 24 h before heart isolation and I/R. Group 8 (NS-DP): mice were pretreated with NS-1619 (1 mg/kg ip) 24 h before I/R. Group 9 (NS-DP+PX): in hearts isolated from the NS-1619-pretreated mice, paxilline was infused for 10 min before I/R. Group 10 (DMSO-DP+L-NAME): DMSO was injected into the mice 24 h earlier and L-NAME (15 mg/kg ip) was administered 30 min before heart isolation and I/R. Group 11 (NS-DP+L-NAME): NS-1619 was injected 24 h earlier, and L-NAME was given 30 min before heart isolation and I/R.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1. Experimental protocols for the early and delayed protection studies (n = 6–9 mice/group). Eleven groups studied include DMSO [DMSO infusion before ischemia-reperfusion (I/R)], NS {infusion of 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)-phe-nyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS-1619) before I/R}, NS+PX (coinfusion of NS-1619 and paxilline before I/R), PX (infusion of paxilline alone before I/R), DMSO-R (DMSO infusion for first 30 min of reperfusion), NS-R (NS-1619 infusion for first 30 min of reperfusion), DMSO-DP (intraperitoneal injection of DMSO 24 h before heart isolation and I/R), NS-DP (intraperitoneal injection of NS-1619 24 h before heart isolation and I/R), NS-DP+PX (NS-1619 pretreatment 24 h before heart isolation and paxilline infusion before I/R), DMSO-DP+L-NAME [DMSO pretreatment 24 h before heart isolation and N{omega}-nitro-L-arginine methyl ester (L-NAME) 30 min before heart isolation and I/R], and NS-DP+L-NAME (NS-1619 pretreatment 24 h before heart isolation and L-NAME 30 min before heart isolation and I/R). See MATERIALS AND METHODS for additional details.

 
Measurement of myocardial infarct size. As described previously (45, 46), at the end of reperfusion, the heart was immediately removed from the perfusion apparatus, weighed, and stored overnight at –20°C. The frozen heart was cut into 6 or 7 transverse sections (~1 mm thickness) across the long axis, stained with 10% triphenyl tetrazolium chloride (TTC; Sigma-Aldrich) in phosphate buffer (pH 7.4) for 30 min at room temperature, and then fixed in 10% formaldehyde solution for at least 2 h before it was measured using a computer morphometry system (Bioquant 98). Representative pictures of the TTC-stained mouse-heart slices are shown in Fig. 2. The size of the myocardial infarction (appearing as a pale color) was quantified and calculated as percentage of risk area, which is the total area minus the cavities.



View larger version (117K):
[in this window]
[in a new window]
  		Fig. 2. Representative images of mouse heart slices stained with triphenyl tetrazolium chloride (TTC). Heart samples were selected from the following experimental groups: DMSO (A), NS (B), DMSO-DP (C), NS-DP (D), DMSO-DP+L-NAME (E), and NS-DP+L-NAME (F). See MATERIALS AND METHODS for additional details.

 
Protein extraction and Western blot analysis. As previously described (35), ventricular tissue was ground in liquid nitrogen and homogenized at 4°C in protein extraction buffer (5). The homogenate was then centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was recovered as total soluble protein. The concentration of protein from the tissue was measured using the Bradford method. Total protein (60 µg) from the heart tissue was loaded on each lane of a 10% SDS-polyacrylamide gel. Proteins were separated by electrophoresis and then transferred from the gel to a nitrocellulose membrane (Bio-Rad), which was incubated in casein-blocking buffer (Sigma-Aldrich) to block nonspecific binding sites. The membrane was subsequently incubated with either a rabbit polyclonal antibody specific to KCa-channel {alpha}-subunits (1:200 dilution; Alomone Labs; Jerusalem, Israel) or a goat polyclonal antibody specific to KCa-channel {beta}-subunits (1:100 dilution; catalog no. sc-14753; Santa Cruz). The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:250 dilution; Amersham). The membranes were developed using enhanced chemiluminescence and exposed to X-ray film. In a parallel series of experiments for determining iNOS and eNOS expression, after pretreatment with an opener of KCa or KATP channels, the cardioprotective dose of NS-1619 (1 mg/kg ip) or diazoxide (7 mg/kg ip) (42) was given to the mice in a single injection. The heart tissue samples were collected 24 h later and were homogenized at 4°C in protein extraction buffer (Sigma-Aldrich). The homogenate was then centrifuged at 10,000 g for 10 min at 4°C, and the supernatant was recovered. Protein from each sample (70 µg) was loaded on each lane of a 10% SDS-polyacrylamide gel, separated by SDS-PAGE, and transferred to a nitrocellulose membrane that was incubated in 5% nonfat dry-milk buffer to block nonspecific binding sites. The membrane was subsequently incubated with a rabbit polyclonal antibody specific to iNOS or eNOS (1:200 dilution; Santa Cruz; catalog nos. sc-650 and sc-653, respectively). The secondary antibody was a horseradish peroxidase-conjugated anti-rabbit IgG (1:250 dilution; Amersham). The membranes were developed using enhanced chemiluminescence and were exposed to X-ray film.

Statistical analysis. All data are presented as means ± SE. Statistical analysis was performed using one-way ANOVA with subsequent Student-Newman-Keuls post hoc test (for three or more groups). Unpaired t-test was used for comparison between two groups, and paired t-test was used to compare pre- and post-treatment values. A value of P < 0.05 was considered as statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Exclusions. A total of 92 hearts were subjected to the I/R protocol in the present study. Among these, 14 hearts (i.e., 15% of the 92 perfused hearts) were excluded according to the exclusion criteria described previously (45, 46), which includes exclusion of 5 hearts due to delay in cannulation time, 2 hearts due to aortic damage, and 7 hearts due to arrhythmia.

Effects of NS-1619, paxilline, and L-NAME on baseline cardiac function. As shown in Table 1, intracoronary infusion of NS-1619 or its solvent DMSO resulted in mild but consistent decreases in developed force, heart rate, and rate-force product. Such an effect on baseline cardiac function was not observed when NS-1619 was given 24 h earlier (i.e., DMSO-DP vs. NS-DP group). Paxilline had no effect on baseline functional parameters during early or delayed protection studies. Pretreatment with L-NAME resulted in profound reductions in heart rate, ventricular developed force, and rate-force product compared with other groups (Table 1).


View this table:
[in this window]
[in a new window]
  		Table 1. Preischemic baseline of cardiac function parameters after various drug pretreatments

 
Early cardioprotection by NS-1619. In these experiments, NS-1619 was infused in the absence or presence of paxilline before I/R. Infarct size was reduced in NS-1619-treated hearts (16.9 ± 1.9% in NS group; Fig. 2B) compared with the control group (i.e., 34.7 ± 2.2% in DMSO group; P < 0.05; Figs. 2A and 3). This infarct-limiting effect was associated with a significant improvement in the functional recovery at the end of reperfusion period (i.e., 70.1 ± 9.5% of preischemic baseline in NS group vs. 36.0 ± 10.5% in DMSO group; P < 0.05; Fig. 4). The NS-1619-induced protection was completely abolished by paxilline (i.e., 36.6 ± 2.5% in NS+PX group vs. 16.9 ± 1.9% in NS group; P < 0.05). Compared with DMSO-treated control mice, paxilline alone caused a larger infarct size (40.7 ± 4.2% in PX group vs. 34.7 ± 2.2% in DMSO group) and poorer functional recovery (24.6 ± 7.0% in PX group vs. 36.0 ± 10.5% in DMSO group), although these differences were not statistically significant. There was no effect of paxilline or NS-1619 on baseline coronary flow (Fig. 5). The NS-1619-treated group had a moderately increased postischemic coronary flow, although this increase was not statistically significant (P > 0.05 vs. DMSO group).



View larger version (33K):
[in this window]
[in a new window]
  		Fig. 3. Myocardial infarct sizes. Values are means ± SE (n = 6–9 mice/group). *P < 0.05 vs. all other groups in the early protection studies; {dagger}P < 0.05 vs. PX; {ddagger}P < 0.05 vs. DMSO-DP and NS-DP+PX (using ANOVA); §P < 0.05 vs. NS-DP (using unpaired t-test).

 


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 4. Recovery of cardiac function (rate-force product) at the end of reperfusion. *P < 0.05 vs. all other groups except NS+PX (using ANOVA); n = 6–9 mice/group.

 


View larger version (38K):
[in this window]
[in a new window]
  		Fig. 5. Coronary flow rate measured before ischemia and during reperfusion. *P < 0.05 vs. preischemia; {dagger}P = 0.055 vs. preischemia (paired t-test); n = 6–9 mice/group.

 
Effects of NS-1619 when administered at reperfusion. Intracoronary infusion of NS-1619 initiated at the onset of reperfusion for 30 min marginally reduced infarct size from 34.1 ± 2.6% in the DMSO-R group to 27.5 ± 1.3% (P > 0.05; see Fig. 3). Ventricular functional recovery and coronary flow were not affected by the NS-1619 treatment compared with the DMSO-R group (see Figs. 4 and 5).

Delayed protection induced by NS-1619. Treatment with NS-1619 (1 mg/kg ip) resulted in significant cardioprotection 24 h later, i.e., infarct size was reduced from 38.8 ± 3.7% in the DMSO-DP group to 19.8 ± 2.9% in NS-DP group (P < 0.05; see Fig. 2, C and D, and Fig. 3). The delayed protective effect of NS-1619 was abolished by paxilline, which was infused for 10 min before I/R (infarct size, 34.3 ± 5.8%). The delayed infarct-limiting effect of NS-1619 was not associated with improvement in postischemic functional recovery (see Fig. 4) or coronary flow (see Fig. 5).

Effect of NOS inhibition on delayed protection. To determine the role of NO in NS-1619-induced delayed protection, the NOS inhibitor L-NAME was administered for 30 min before I/R in the NS-1619-treated mice. L-NAME further reduced infarct size from 19.8 ± 2.9% in the NS-1619-treated group to 9.4 ± 1.7% (P < 0.05; see Fig. 3). Also, L-NAME-treated control mice (DMSO-DP+L-NAME group) demonstrated significantly smaller infarct size (19.8 ± 1.1%) compared with the DMSO-DP group (38.8 ± 3.7%; P < 0.05; see Fig. 2, E and F, and Fig. 3). The infarct size was further reduced in the NS-DP+L-NAME group (9.4 ± 1.7%) compared with the DMSO-DP+L-NAME group (19.8 ± 1.1%).

Effects of NS-1619 on iNOS and eNOS expression. As shown in Fig. 6, expression of iNOS or eNOS was not enhanced in hearts 24 h after pretreatment with a cardioprotective dose of NS-1619 (1 mg/kg ip) compared with the DMSO-treated hearts. In contrast, pretreatment with the reported cardioprotective dose of diazoxide (7 mg/kg) resulted in increased iNOS and eNOS expression 24 h after the drug injection.



View larger version (42K):
[in this window]
[in a new window]
  		Fig. 6. Western blots show myocardial inducible and endothelial nitric oxide synthase (iNOS and eNOS, respectively) expression. Heart samples were collected from mice pretreated intraperitoneally with DMSO (0.15 ml), NS-1619 (1 mg/kg), or diazoxide (7 mg/kg) 24 h before heart sample collection.

 
Expression of KCa-channel isoforms in mouse cardiac tissue. To confirm the existence of KCa channels in mouse heart, we performed Western blots in ventricular tissue extracts from 10 mice using a rabbit polyclonal antibody specific to KCa-channel {alpha}-subunits. A distinct band at 55 kDa that corresponded to the predicted size for the KCa-channel {alpha}-subunit was detectable in the tissue (Fig. 7, top). Similarly, Western blots using a goat polyclonal antibody specific to KCa-channel {beta}-subunits demonstrated a single band at 31 kDa (Fig. 7, bottom).



View larger version (35K):
[in this window]
[in a new window]
  		Fig. 7. Western blots show expression of Ca2+-activated K+ (KCa) channel {alpha}- and {beta}-protein subunits in adult mouse heart tissue.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the past few years, the early (31) and delayed (40, 42) cardioprotective effects of mitoKATP-channel openers (such as diazoxide) against I/R injury have been extensively studied. However, little is known about the effects of modulating other types of K+ channels. A recent study by O'Rourke's group (47) introduced the novel concept that selective opening of the large-conductance KCa channel with NS-1619 confers a similar degree of cardioprotection as the opener of mitoKATP channels. In the present studies, we demonstrated for the first time that pretreatment with NS-1619 induces delayed cardioprotection against myocardial I/R injury in adult mice. Our results also show early cardioprotective effects of the drug as reported previously in rabbits (47). Both the early and delayed cardioprotection of NS-1619 were completely abolished by coadministration with paxilline, a selective and potent KCa-channel blocker (26, 36, 38). As shown in Fig. 3, pretreatment with paxilline alone had a tendency to exacerbate I/R injury, although the increase in infarct size was not statistically significant (i.e., 34.7 ± 2.2% in DMSO group vs. 40.7 ± 4.2% in PX group; P > 0.05). Administration of NS-1619 during reperfusion produced no significant cardioprotection (see Fig. 3), which suggests that the drug would be useful only as a "preconditioning" agent. Interestingly, the significant improvement in postischemic functional recovery was found only in conjunction with the early (and not the delayed) protection (see Fig. 4), which indicates that the components and mechanisms of protection may be different for the early phase vs. the delayed phase of protection.

Another salient finding of our study is that NS-1619-induced, delayed cardioprotection was neither blocked by the NOS inhibitor L-NAME nor associated with the increase of iNOS or eNOS enzymes (see Fig. 2, E and F, and Figs. 3 and 6). On the contrary, L-NAME pretreatment alone significantly reduced infarct size (i.e., 19.8 ± 1.1% in DMSO-DP+L-NAME group vs. 38.8 ± 3.7% in DMSO-DP group; P < 0.05). These results are in accordance with the previously reported infarct-limiting effect of L-NAME against I/R injury in rabbit heart (33) that occurred through an adenosine-dependent mechanism (44). Our results indirectly suggest the possibility of adenosine release after L-NAME treatment as indicated by decreased heart rate (a typical signature of adenosine A1-receptor activation) in the L-NAME-treated groups (see Table 1). Furthermore, despite the infarct-limiting effect of L-NAME, pretreatment with NS-1619 24 h earlier resulted in even smaller infarct size, i.e., 9.4 ± 1.7% in the NS-DP+L-NAME group vs. 19.8 ± 1.1% in the DMSO-DP+L-NAME group (P < 0.05; see Fig. 2, E and F and Fig. 3). These findings can be interpreted as 1) NS-1619 could maintain its protective effects even in the presence of an NOS inhibitor; and 2) the acute protection by L-NAME and the delayed protection by NS-1619 are additive, although the cardioprotective effects of these drugs are mediated by unique mechanisms. In addition, the Western blot results clearly showed that neither iNOS nor eNOS was induced in NS-1619-treated hearts as opposed to the diazoxide (mitoKATP-channel opener)-treated hearts, which caused robust expression of both iNOS and eNOS (see Fig. 6). Taken together, our results suggest that NS-1619 protects ischemic heart through an iNOS- and/or eNOS-independent signaling mechanism. This is indeed a surprising observation, because there is a significant body of evidence (including many studies from our own group) that demonstrates an obligatory role of iNOS and/or eNOS in mediating delayed preconditioning in heart (8–10, 20, 22, 23, 39). A large number of pharmacological agents elicit a delayed preconditioning-like effect via NOS-dependent mechanisms; these include NO donors (8, 23), endotoxin derivatives (34, 45, 46), G protein-coupled membrane receptor agonists (6, 8, 41, 48, 49), openers of mitoKATP channels (31, 42), and most recently, phosphodiesterase type-5 inhibitors (27, 35). In this respect, it appears that the KCa-channel opener NS-1619 is a unique pharmacological compound that induces delayed cardiac preconditioning without involving iNOS and eNOS. Presently we do not know about the factors that regulate the signaling mechanism of cardioprotection with the opening of KCa channels. Nevertheless, the NOS-independent feature of NS-1619-induced, late cardioprotection is interesting and requires additional investigation to delineate the signaling pathways.

In the present study, we confirmed the presence of KCa-channel protein expression in mouse cardiac tissue (see Fig. 7). Western blot analysis showed that the KCa-channel {alpha}-subunits were present in the ventricular tissue (see Fig. 7, top). These results are consistent with the protein expression of the KCa-channel {alpha}-subunits identified previously by Xu et al. (47) in samples from inner mitochondrial membranes of guinea pig ventricular myocytes. Furthermore, we confirmed the presence of {beta}-subunits of KCa channels via Western blots (Fig. 7, bottom). These results along with the blockade of cardioprotection with the selective KCa-channel blocker paxilline (see Figs. 3 and 4) suggest that protective effects of NS-1619 are most likely mediated through direct opening of KCa channels.

It was previously demonstrated that NS-1619 could open mitoKCa channels in cardiac myocytes, which in turn increased mitochondrial K+ influx, decreased excessive Ca2+ entry, and ultimately improved the efficiency of mitochondrial energy production and led to cytoprotection against I/R injury (47). Other studies also suggested that mitochondria have a high capacity for intracellular Ca2+ buffering and may regulate cytosolic Ca2+ in neurons and cardiac myocytes (4, 15), which further indicates the crucial function of mitochondria in Ca2+ homeostasis and cytoprotection. In addition to the antinecrotic protection of NS-1619 reported previously (47) and the present studies, it is logical to speculate that opening of mitoKCa channels may result in similar protection against apoptosis as was shown with diazoxide, the opener of mitoKATP channels (2). Additional studies are warranted to evaluate the potential antiapoptotic properties of NS-1619 in a suitable experimental model in which apoptosis is a prominent form of cell death.

It is notable that the KCa-channel opener NS-1619 and the channel blocker paxilline that were used in the present study may have additional actions beyond regulation of KCa channels. For instance, it was recently reported that NS-1619 could inhibit mitochondrial function in the human glioma LN229 cell line via inhibition of complex I of the mitochondrial respiratory chain (14). These studies reported an EC50 value of 3.6 ± 0.4 µM for the inhibitory effects of NS-1619 on mitochondrial membrane potential in the tumor cells. In the present studies, we used 10 µM of NS-1619, which indeed falls into this dose range. Although there is no direct evidence that NS-1619 could inhibit mitochondrial function in cardiac cells, we cannot completely rule out the possibility that the cardioprotective effect of this drug is mediated in part through inhibition of complex I of the mitochondrial respiratory chain. In fact, a recent study suggested that the cardioprotective effects of volatile anesthetics (such as halothane, isoflurane, and sevoflurane) may be mediated through inhibition of complex I of the cardiac mitochondrial respiratory chain (21). There is also evidence that paxilline inhibits sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) in vitro (7). Paxilline was shown to inhibit SERCA activity by 25 and 50% at concentrations of 5 and 50 µM, respectively, in porcine cardiac tissue. However, the contributory effect of SERCA inhibition may be negligible in the present study because we used a much lower dose of paxilline (i.e., 1 µM). In addition, we observed exacerbation of I/R injury in paxilline-treated hearts compared with the vehicle controls (see Figs. 3 and 4), which is contrary to the reported cardioprotective effects of cyclopiazonic acid, a putative SERCA inhibitor (3). These results suggest that the low-dose paxilline used in the present study predominantly blocked cardioprotection via inhibition of KCa channels rather than SERCA.

In summary, we have demonstrated that the KCa-channel opener NS-1619 induces significant early and delayed protection against myocardial infarction. A remarkable improvement in postischemic ventricular function was also associated with the early phase of protection. Coadministration with the KCa-channel blocker paxilline completely abolished both the early and the delayed protective effects of NS-1619, which indicates the selective action of this drug on KCa channels. Our results also suggest that NS-1619 induces delayed cardioprotection through a distinct signaling mechanism that is independent of iNOS and eNOS. The presence of KCa-channel protein in ventricular tissue implicates the cardioprotective effects of NS-1619 to the opening of KCa channels. Overall, our data strongly support the notion that in addition to KATP channels, KCa channels can be an important therapeutic target for pharmacological preconditioning against myocardial I/R injury and infarction.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grants HL-51045 and HL-59469 (to R. C. Kukreja). C. Yin was a postdoctoral trainee on NIH Training Grant HL-07537.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. C. Kukreja, Division of Cardiology, Box 980281, Virginia Commonwealth Univ., Richmond, VA 23298-0281 (E-mail: rakesh{at}hsc.vcu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. AdamsPR, Constanti A, Brown DA, and Clark RB. Intracellular Ca2+ activates a fast voltage-sensitive K+ current in vertebrate sympathetic neurones. Nature 296: 746–749, 1982.[CrossRef][Medline]
  2. AkaoM, Ohler A, O'Rourke B, and Marbán E. Mitochondrial ATP-sensitive potassium channels inhibit apoptosis induced by oxidative stress in cardiac cells. Circ Res 88: 1267–1275, 2001.[Abstract/Free Full Text]
  3. AvellanalM, Rodriguez P, and Barrigon S. Protective effects of cyclopiazonic acid on ischemia-reperfusion injury in rabbit hearts. J Cardiovasc Pharmacol 32: 845–851, 1998.[CrossRef][Web of Science][Medline]
  4. BabcockDF, Herrington J, Goodwin PC, Park YB, and Hille B. Mitochondrial participation in the intracellular Ca2+ network. J Cell Biol 136: 833–844, 1997.[Abstract/Free Full Text]
  5. BainesCP, Zhang J, Wang GW, Zheng YT, Xiu JX, Cardwell EM, Bolli R, and Ping P. Mitochondrial PKC{epsilon} and MAPK from signaling modules in the murine heart: enhanced mitochondrial PKC{epsilon}-MAPK interactions and differential MAPK activation in PKC{epsilon}-induced cardioprotection. Circ Res 90: 390–397, 2002.[Abstract/Free Full Text]
  6. BellRM, Smith CC, and Yellon DM. Nitric oxide as a mediator of delayed pharmacological (A1 receptor triggered) preconditioning: is eNOS masquerading as iNOS? Cardiovasc Res 53: 405–413, 2002.[Abstract/Free Full Text]
  7. BilmenJG, Wootton LL, and Michelangeli F. The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline. Arch Biochem Biophys 406: 55–64, 2002.[CrossRef][Web of Science][Medline]
  8. BolliR. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33: 1897–1918, 2001.[CrossRef][Web of Science][Medline]
  9. BolliR. The late phase of preconditioning. Circ Res 87: 972–983, 2000.[Abstract/Free Full Text]
  10. BolliR, Manchikalapudi S, Tang XL, Takano H, Qiu Y, Guo Y, Zhang Q, and Jadoon AK. The protective effect of late preconditioning against myocardial stunning in conscious rabbits is mediated by nitric oxide synthase: evidence that nitric oxide acts both as a trigger and as a mediator of the late phase of ischemic preconditioning. Circ Res 81: 1094–1107, 1997.[Abstract/Free Full Text]
  11. BraydenJE and Nelson MT. Regulation of arterial tone by activation of calcium dependent potassium channels. Science 256: 532–535, 1992.[Abstract/Free Full Text]
  12. CalderoneV. Large-conductance, Ca2+-activated K+ channels: function, pharmacology and drugs. Curr Med Chem 9: 1385–1395, 2002.[Web of Science][Medline]
  13. ChanEC and Woodman OL. Enhanced role for the opening of potassium channels in relaxant responses to acetylcholine after myocardial ischaemia and reperfusion in dog coronary arteries. Br J Pharmacol 126: 925–932, 1999.[CrossRef][Web of Science][Medline]
  14. DebskaG, Kicinska A, Dobrucki J, Dworakowska B, Nurowska E, Skalska J, Dolowy K, and Szewczyk A. Large-conductance K+ channel openers NS1619 and NS004 as inhibitors of mitochondrial function in glioma cells. Biochem Pharmacol 65: 1827–1834, 2003.[CrossRef][Web of Science][Medline]
  15. DuchenMR. Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signaling and cell death. J Physiol 516: 1–17, 1999.[Abstract/Free Full Text]
  16. GarlidKD, Paucek P, Yarov-Yarovoy V, Murray HN, Darbenzio RB, D'Alonzo AJ, Lodge NJ, Smith MA, and Grover GJ. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels: possible mechanism of cardioprotection. Circ Res 81: 1072–1082, 1997.[Abstract/Free Full Text]
  17. GhoshS, Standen NB, and Galinanes M. Evidence for mitochondrial KATP channels as effectors of human myocardial preconditioning. Cardiovasc Res 45: 934–940, 2000.[Abstract/Free Full Text]
  18. GrossGJ. The role of mitochondrial KATP channels in cardioprotection. Basic Res Cardiol 95: 280–284, 2000.[CrossRef][Web of Science][Medline]
  19. GroverGJ and Garlid KD. ATP-sensitive potassium channels: a review of their cardioprotective pharmacology. J Mol Cell Cardiol 32: 677–695, 2000.[CrossRef][Web of Science][Medline]
  20. GuoY, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, and Bolli R. The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96: 11507–11512, 1999.[Abstract/Free Full Text]
  21. HanleyPJ, Ray J, Brandt U, and Daut J. Halothane, isoflurane and sevoflurane inhibit NADH:ubiquinone oxidoreductase (complex I) of cardiac mitochondria. J Physiol 544: 687–693, 2002.[Abstract/Free Full Text]
  22. ImagawaJ, Yellon DM, and Baxter GF. Pharmacological evidence that inducible nitric oxide synthase is a mediator of delayed preconditioning. Br J Pharmacol 126: 701–708, 1999.[CrossRef][Web of Science][Medline]
  23. JonesWK, Flaherty MP, Tang XL, Takano H, Qiu Y, Banerjee S, Smith T, and Bolli R. Ischemic preconditioning increases iNOS transcript levels in conscious rabbits via a nitric oxide-dependent mechanism. J Mol Cell Cardiol 31: 1469–1481, 1999.[CrossRef][Web of Science][Medline]
  24. KitazonoT, Faraci FM, Taguchi H, and Heistad DD. Role of potassium channels in cerebral blood vessels. Stroke 26: 1713–1723, 1995.[Abstract/Free Full Text]
  25. KnausHG, Schwarzer C, Koch RO, Eberhart A, Kaczorowski GJ, Glossmann H, Wunder F, Pongs O, Garcia ML, and Sperk G. Distribution of high conductance Ca2+-activated K+ channels in rat brain: targeting to axons and nerve terminals. J Neurosci 16: 955–963, 1996.[Abstract/Free Full Text]
  26. KnausHG, McManus OB, Lee SH, Schmalhofer WA, Garcia-Calvo M, Helms LM, Sanchez M, Giangiacomo K, Reuben JP, and Smith AB. Tremorgenic indole alkaloids potently inhibit smooth-muscle high conductance calcium activated potassium channels. Biochemistry 33: 5819–5828, 1994.[CrossRef][Medline]
  27. KukrejaRC, Ockaili R, Salloum F, Yin C, Hawkins J, Das A, and Xi L. Cardioprotection with phosphodiesterase-5 inhibition: a novel preconditioning strategy. J Mol Cell Cardiol 36: 165–173, 2004.[CrossRef][Web of Science][Medline]
  28. LonglandCL, Dyer JL, and Michelangeli F. The mycotoxin paxilline inhibits the cerebellar inositol 1,4,5-trisphosphate receptor. Eur J Pharmacol 408: 219–225, 2000.[CrossRef][Web of Science][Medline]
  29. LuT, Katakam PV, VanRollins M, Weintraub NL, Spector AA, and Lee HC. Dihydroxyeicosatrienoic acids are potent activators of Ca2+-activated K+ channels in isolated rat coronary arterial myocytes. J Physiol 534: 651–667, 2001.[Abstract/Free Full Text]
  30. NelsonMT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799–C822, 1995.[Abstract/Free Full Text]
  31. OckailiR, Emani VR, Okubo S, Brown M, Krottapalli K, and Kukreja RC. Opening of mitochondrial KATP channel induces early and delayed cardioprotective effect: role of nitric oxide. Am J Physiol Heart Circ Physiol 277: H2425–H2434, 1999.[Abstract/Free Full Text]
  32. OlesenSP, Munch E, Moldt P, and Drejer J. Selective activation of Ca2+-dependent K+ channels by novel benzimidazolone. Eur J Pharmacol 251: 53–59, 1994.[CrossRef][Web of Science][Medline]
  33. PatelVC, Yellon DM, Singh KJ, Neild GH, and Woolfson RG. Inhibition of nitric oxide limits infarct size in the in situ rabbit heart. Biochem Biophys Res Commun 194: 234–238, 1993.[CrossRef][Web of Science][Medline]
  34. PengJ, Lu R, Deng HW, and Li YJ. Involvement of alpha-calcitonin gene-related peptide in monophosphory lipid A-induced delayed preconditioning in rat hearts. Eur J Pharmacol 436: 89–96, 2002.[CrossRef][Web of Science][Medline]
  35. SalloumF, Yin C, Xi L, and Kukreja RC. Sildenafil induces delayed preconditioning through inducible nitric oxide synthase-dependent pathway in mouse heart. Circ Res 92: 595–597, 2003.[Abstract/Free Full Text]
  36. SanchezM and McManus OB. Paxilline inhibition of the alpha-subunit of the high-conductance calcium-activated potassium channel. Neuropharmacology 35: 963–968, 1996.[CrossRef][Web of Science][Medline]
  37. SatoT, Sasaki N, Seharaseyon J, O'Rourke B, and Marbán E. Selective pharmacological agents implicate mitochondrial but not sarcolemmal KATP channels in ischemic cardioprotection. Circulation 101: 2418–2423, 2000.[Abstract/Free Full Text]
  38. StrobaekD, Christophersen P, Holm NR, Moldt P, Ahring PK, Johansen TE, and Olesen SP. Modulation of the Ca2+-dependent channel, hslo, by the substituted diphnylurea NS 1608, paxilline and internal Ca2+. Neuropharmacology 35: 903–914, 1996.[CrossRef][Web of Science][Medline]
  39. TakanoH, Manchikalapudi S, Tang XL, Qiu Y, Rizvi A, Jadoon A, Zhang Q, and Bolli R. Nitric oxide synthase is the mediator of late preconditioning against myocardial infarction in conscious rabbits. Circulation 98: 441–449, 1998.[Abstract/Free Full Text]
  40. TakashiE, Wang Y, and Ashraf M. Activation of mitochondrial KATP channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res 85: 1146–1153, 1999.[Abstract/Free Full Text]
  41. Tejero-TaldoMI, Gursoy E, Zhao TC, and Kukreja RC. {alpha}-Adrenergic receptor stimulation produces late preconditioning through inducible nitric oxide synthase in mouse heart. J Mol Cell Cardiol 34: 185–195, 2002.[CrossRef][Web of Science][Medline]
  42. WangY, Kudo M, Xu M, Ayub A, and Ashraf M. Mitochondrial KATP channel as an end effector of cardioprotection during late preconditioning: triggering role of nitric oxide. J Mol Cell Cardiol 33: 2037–2046, 2001.[CrossRef][Web of Science][Medline]
  43. WickendenAD. K+ channels as therapeutic drug targets. Pharmacol Ther 94: 157–182, 2002.[CrossRef][Web of Science][Medline]
  44. WoolfsonRG, Patel VC, Neild GH, and Yellon DM. Inhibition of nitric oxide synthesis reduces infarct size by an adenosine-dependent mechanism. Circulation 91: 1545–1551, 1995.[Abstract/Free Full Text]
  45. XiL, Jarrett NC, Hess ML, and Kukreja RC. Essential role of inducible nitric oxide synthase in monophosphoryl lipid A-induced late cardioprotection: evidence from pharmacological inhibition and gene knockout mice. Circulation 99: 2157–2163, 1999.[Abstract/Free Full Text]
  46. XiL, Salloum F, Tekin D, Jarrett NC, and Kukreja RC. Glycolipid RC-552 induces delayed preconditioning-like effect via iNOS-dependent pathway in mice. Am J Physiol Heart Circ Physiol 277: H2418–H2424, 1999.[Abstract/Free Full Text]
  47. XuW, Liu Y, Wang S, McDonald T, Van Eyk JE, Sidor A, and O'Rourke B. Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science 298: 1029–1033, 2002.[Abstract/Free Full Text]
  48. ZhaoT, Xi L, Chelliah J, Levasseur JE, and Kukreja RC. Inducible nitric oxide synthase mediates delayed myocardial protection induced by activation of adenosine A1 receptors: evidence from gene-knockout mice. Circulation 102: 902–907, 2000.[Abstract/Free Full Text]
  49. ZhaoTC and Kukreja RC. Late preconditioning elicited by activation of adenosine A3 receptor in heart: role of NF{kappa}B, iNOS and mitochondrial KATP channel. J Mol Cell Cardiol 34: 263–277, 2002.[CrossRef][Web of Science][Medline]



This article has been cited by other articles:


Home page
 Anesth. Analg.Home page
A. Redel, M. Lange, V. Jazbutyte, C. Lotz, T. M. Smul, N. Roewer, and F. Kehl
Activation of Mitochondrial Large-Conductance Calcium-Activated K+ Channels via Protein Kinase A Mediates Desflurane-Induced Preconditioning
Anesth. Analg., February 1, 2008; 106(2): 384 - 391.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
A. Heinen, A. K. S. Camara, M. Aldakkak, S. S. Rhodes, M. L. Riess, and D. F. Stowe
Mitochondrial Ca2+-induced K+ influx increases respiration and enhances ROS production while maintaining membrane potential
Am J Physiol Cell Physiol, January 1, 2007; 292(1): C148 - C156.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
I. Bak, I. Lekli, B. Juhasz, N. Nagy, E. Varga, J. Varadi, R. Gesztelyi, G. Szabo, L. Szendrei, I. Bacskay, et al.
Cardioprotective mechanisms of Prunus cerasus (sour cherry) seed extract against ischemia-reperfusion-induced damage in isolated rat hearts
Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1329 - H1336.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
N. Ahmad, Y. Wang, K. H. Haider, B. Wang, Z. Pasha, O. Uzun, and M. Ashraf
Cardiac protection by mitoKATP channels is dependent on Akt translocation from cytosol to mitochondria during late preconditioning
Am J Physiol Heart Circ Physiol, June 1, 2006; 290(6): H2402 - H2408.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. F. Stowe, M. Aldakkak, A. K. S. Camara, M. L. Riess, A. Heinen, S. G. Varadarajan, and M.-T. Jiang
Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation
Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H434 - H440.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
B. O'Rourke, S. Cortassa, and M. A. Aon
Mitochondrial Ion Channels: Gatekeepers of Life and Death
Physiology, October 1, 2005; 20(5): 303 - 315.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
D. D. Gutterman
Mitochondria and Reactive Oxygen Species: An Evolution in Function
Circ. Res., August 19, 2005; 97(4): 302 - 304.
[Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
C.-M. Cao, Q. Xia, Q. Gao, M. Chen, and T.-M. Wong
Calcium-Activated Potassium Channel Triggers Cardioprotection of Ischemic Preconditioning
J. Pharmacol. Exp. Ther., February 1, 2005; 312(2): 644 - 650.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
287/5/H2070    most recent
00431.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (19)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, X.
Right arrow Articles by Kukreja, R. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, X.
Right arrow Articles by Kukreja, R. C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.