AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 292: H2634-H2642, 2007. First published February 9, 2007; doi:10.1152/ajpheart.01247.2006
0363-6135/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/H2634    most recent
01247.2006v1
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 (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jones, R. J.
Right arrow Articles by Singer, H. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jones, R. J.
Right arrow Articles by Singer, H. A.

iNOS regulation by calcium/calmodulin-dependent protein kinase II in vascular smooth muscle

Rachel J. Jones,1 David Jourd'heuil,1 John C. Salerno,2 Susan M. E. Smith,3 and Harold A. Singer1

1Center for Cardiovascular Sciences, Albany Medical Center, Albany, New York; 2Department of Biology and Physics, Kennesaw State University, Kennesaw, Georgia; and 3Department of Pathology, Emory School of Medicine, Atlanta, Georgia

Submitted 15 November 2006 ; accepted in final form 8 February 2007


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Nitric oxide synthase (NOS) expression is regulated transcriptionally in response to cytokine induction and posttranslationally by palmitoylation and trafficking into perinuclear aggresome-like structures. We investigated the effects of multifunctional calcium/calmodulin-dependent protein kinase II protein kinase (CaMKII) on inducible NOS (iNOS) trafficking in cultured rat aortic vascular smooth muscle cells (VSMCs). Immunofluorescence and confocal microscopy demonstrated colocalization of iNOS and CaMKII{delta}2 with a perinuclear distribution and concentration in aggresome-like structures identified by colocalization with {gamma}-tubulin. Furthermore, CaMKII{delta}2 coimmunoprecipitated with iNOS in a CaMKII activity-dependent manner. Addition of Ca2+-mobilizing stimuli expected to activate CaMKII; a purinergic agonist (UTP) or calcium ionophore (ionomycin) caused a general redistribution of iNOS from cytosolic to membrane and nuclear fractions. Similarly, adenoviral expression of a constitutively active CaMKII{delta}2 mutant altered iNOS localization, shifting iNOS from the cytosolic fraction. Suppression of CaMKII{delta}2 using an adenovirus expressing a short hairpin, small interfering RNA increased nuclear iNOS localization in resting cells but inhibited ionomycin-induced translocation of iNOS to the nucleus. Following addition of these chronic and acute CaMKII modulators, there were fewer aggresome-like structures containing iNOS. All of the treatments that chronically affected CaMKII activity or expression significantly inhibited iNOS-specific activity following cytokine induction. The results suggest that CaMKII{delta}2 may be an important regulator of iNOS trafficking and activity in VSMCs.

protein trafficking; inflammatory response; inducible nitric oxide synthase

THE INDUCIBLE FORM of nitric oxide (NO) synthase (iNOS) produces NO in response to vascular injury in cases where the endothelium is compromised (14, 17, 38). Macrophages and neutrophils that accumulate in the vascular wall following injury express iNOS and serve as a source both for NO released from the inflammatory cells as well as for cytokines that are capable of inducing iNOS expression in the underlying medial and neointimal vascular smooth muscle (VSM) cells (VSMCs) (10, 18, 31, 36).

Paracrine and autocrine effects of NO have important roles in vascular remodeling in response to injury (2, 8, 10, 16, 33, 38, 39). One of the most notable of these is the effect of NO on proliferation in injured vessels (33, 37, 38) and cultured cells (8, 16). The production of NO in injured vessels inhibits neointima formation, a phenomenon that is considered vasculoprotective (2, 33, 37, 39). Addition of NO to cultured VSMCs inhibits proliferation (8), and the production of iNOS in VSM has this same effect.

Recent discoveries of posttranslational modifications on iNOS raise the possibility of complex iNOS regulation separate from transcriptional regulation (13, 19, 23, 24, 28). iNOS is primarily found in the cytosolic fraction of cells, has been associated with Golgi membranes (23), and has also been identified in the nucleus of brown adipocytes under beta-adrenergic stimulation (13) and in the nuclei of rat neutrophils under quiescent conditions (28). Kolodziejska et al. (19) provided evidence for iNOS trafficking dependent on microtubules and colocalization with the microtubule organizing complex in perinuclear aggresome-like structures. Aggresomal localization of protein is usually described as a mechanism for storing excess or misfolded protein ultimately destined for degradation (7), although in the case of iNOS, evidence suggests that this compartment functions as a reservoir for latent iNOS (19). Palmitoylation of Cys3 in iNOS (28) and tyrosine phosphorylation (24) of iNOS are among the posttranslational mechanisms that affect iNOS localization. Mutations to the Cys3 palmitoylation site have been shown to cause a mislocalization of iNOS protein in C2C12 cells and obliteration of iNOS activity (28). Although it is not fully clear how iNOS trafficking is regulated, it appears that iNOS activity is dependent on proper trafficking and subcellular localization (5, 13, 19, 24, 28, 38). iNOS localization and trafficking have not been examined in VSM, where it has important physiological and pathophysiological functions.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is an ubiquitous, structurally complex multifunctional protein serine/threonine kinase. CaMKII isoform expression and function have been studied to some extent in differentiated VSM, where both {gamma}- and {delta}-isoforms have been detected (21, 25, 40) and {gamma}-isoforms linked to contractile regulation (27, 34). In undifferentiated cultured rat aortic VSMCs, the primary isoform has been shown to be the {delta}2-isoform, which has been linked to signaling pathways controlling cell migration (25, 26, 40) and proliferation (11). Limited studies have localized the CaMKII{delta}2 isoform to intracellular membranes colocalizing with endoplasmic reticulum (ER) markers in VSMCs (4) and with Golgi membranes in astrocytes (32). Furthermore, activation studies in cultured VSM indicate a close functional coupling of activated CaMKII{delta}2 with Ca2+ released from intracellular Ca2+ stores (12, 26). Ionomycin-induced activation of CaMKII in cultured VSMCs results in redistribution of both CaMKII{delta}2 and ER markers, consistent with an ER stress response. CaMKII function on intracellular membranes is not known.

Interactions between endothelial NOS (eNOS) or neuronal NOS (nNOS) proteins with CaMKII have been demonstrated in other cell systems. eNOS is palmitoylated, proving vital for the proper targeting of the enzyme in caveolar domains at the plasma membrane (30), and eNOS activity may be regulated by CaMKII in these domains. nNOS is also palmitoylated and interacts with scaffolding proteins such as PSD-95 necessary for nNOS trafficking to postsynaptic densities (3). nNOS interaction with PSD-95 and enzymatic activity may be regulated by CaMKII (35). To our knowledge, no one has investigated potential regulation of iNOS trafficking and/or activity by CaMKII. In the present study we reexamined the distribution of CaMKII{delta}2 in VSMCs and compared it with iNOS distribution, induced in response to cytokine stimulation. The results indicate a role for CaMKII in the regulation of iNOS trafficking and activity in VSM.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies and materials. Production and characterization of the antibody specific for CaMKII{delta}2 have been described previously (29). Monoclonal and polyclonal antibodies to iNOS and nucleolin were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-mouse and anti-rabbit secondary horseradish peroxidase-conjugated antibodies and ECL reagent were purchased from Amersham Biosciences (Little, UK). KN-93, KN-92, and ionomycin were purchased from Calbiochem (La Jolla, CA). Protein A beads were purchased from Pierce (Richmond, CA). All cell culture media and supplies were purchased from Fisher Scientific (Pittsburgh, PA). All other materials were purchased from Sigma.

Cell culture. VSMCs were enzymatically dispersed from thoracic aortas of male Sprague-Dawley rats (200–300 g) as previously described (30). Cells were cultured in DMEM/F-12 medium supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Cell confluency of between 50% and 80% was used in cell culture in passages 3–9. Before experimentation, cells were growth arrested by replacing growth media (9) with DMEM/F-12 medium supplemented with 0.4% FBS for 16 h at 37°C and 5% CO2. Use of experimental animals as a source of cells was reviewed and approved by the Albany Medical College Institutional Animal Care and Use Committee.

Induction of iNOS in VSMCs. Induction of iNOS was performed as previously described (31). Following growth arrest, fresh 0.4% FBS or 10% FBS media were added as indicated, with the following cytokine cocktail for 24 h: IL-1beta (final concentration of 20 ng/ml), IFN-{gamma} (final concentration 200 U/ml), and TNF-{alpha} (final concentration of 20 ng/ml). After this induction period, the cells were washed with 0.4% FBS growth media and used for analysis or treated with various inhibitors/activators as described in each experiment.

Cell lysates, immunoprecipitation, and immunoblotting. The CaMKII activator ionomycin was added at a concentration of 0.5 µM and incubated for 3 min. The CaMKII inhibitor KN-93 was added at a concentration of 30 µM and left on for the duration of the experiment. Cells were lysed on ice with NP-40 lysis buffer (4°C 50 mM Tris, pH 7.4, 50 mM NaF, 0.1 mM NaVO4, 0.5% NP-40, 0.1 mM phenylmethylsulfonyl fluoride, and 0.2 U/ml aprotinin) at 0.5 ml per well in a six-well plate. Immunoprecipitations consisted of a 1.5-h incubation with antibody and then a 1-h incubation with 40 µl of protein A beads.

Immunoprecipitates were resolved on a 10% SDS-PAGE gel and transferred to nitrocellulose. The membranes were blocked in 5% nonfat dry milk with Tris-buffered saline containing 0.2% Tween 20 (TBST). After the blocking was completed, the designated antibody was added to the mixture and incubated at room temperature for 1 h or 4°C overnight while being shaken at 150 rpm. The membranes were then washed three times with TBST for 10 min each and blocked again. After this, the secondary horseradish peroxidase-conjugated antibody was added and incubated for 1 h at room temperature, membrane washed three times with TBST, and developed using ECL reagent. ML-B film (Medlink Imaging; Elmsford, NY) was exposed to the membrane and developed in a SRX-101A film processor (Konica Minolta; Wayne, NJ).

Adenoviral infection and constructs. The CaMKII{delta}2 small interfering RNA (siRNA), designated here as deltakd (delta knockdown) was previously described (20). Adenovirus was added to confluent cells before serum starvation at a multiplicity of infection (MOI) of 150. This virus was incubated on the cells for 90–96 h. The adenovirus containing the constitutively active CaMKII{delta}2 mutant construct (T287D), also previously described (19), was added at an MOI of 50 and incubated for 24–48 h. The cells were then washed, serum starved overnight, and induced for iNOS expression. As a negative control, an adenovirus encoding a green fluorescent protein-specific siRNA was added to confluent cells before serum starvation at an MOI of 50. This virus was incubated for the same times as the experimental virus it was compared against and induced for iNOS expression in the same fashion.

Activity assays. Nitrite readings were taken by gas phase chemiluminescence on a nitric oxide analyzer (NOA 280; Sievers Instruments, Boulder, CO) using peak integration, and nitrite concentrations were determined using nitrite as a standard.

Immunofluorescence. Confluent VSMCs on coverslips induced to express iNOS were washed after 20–24 h, and any activators or inhibitors were added with fresh media at that time. The cells were then washed with 1x PBS and fixed with 4% paraformaldehyde for 30–40 min at room temperature, followed by three 5-min washes with 1x PBS. The cells were then permeabilized with 0.2% Triton X-100 for 5 min at room temperature and washed three times for 5 min each with 1x PBS containing 0.1% Triton X-100. Cells were then blocked with 0.5% fish gelatin for 30 min at room temperature, washed three times with 1x PBS-Triton, and then labeled with 5 µg of primary antibody for 2 h at room temperature. Cells were then washed three times with 1x PBS-Triton and labeled with secondary antibody for 2 h at room temperature. The cells were then washed three times at room temperature, and coverslips were mounted on slides, viewed, and photographed.

Cell fractionation. Cells were lysed by douncing with a pestle in a hypotonic buffer consisting of 250 mM sucrose, 10 mM MOPS, 2.5 mM EGTA, 2 mM EDTA, 0.2 mM PMSF, and 0.85 µg/ml aprotinin. The lysates were then centrifuged at 2,000 rpm for 10 min. The cell debris pellet at the bottom of the tube was discarded, and the spin was repeated. The resulting soft pellet (side of tube) was separated from the rest of the postnuclear supernatant that was carefully pipetted into 1.5-ml polycarbonate tubes. The soft pellet was washed and centrifuged twice at 10,000 rpm. At this point, the soft pellet containing nuclei was resuspended in 200 µl of the hypotonic buffer, and nuclei were lysed by pipetting with a 25-gauge needle. The resulting nuclear-enriched lysate was then centrifuged at 10,000 rpm, and the pellet was discarded. The nuclear lysate was frozen at –20°C until use. The polycarbonate tubes containing the postnuclear lysate were centrifuged at 70,000 rpm for 40 min at 4°C. The supernatant (cytosolic fraction) was removed and stored at –20°C until use, whereas the pellet was washed in 200 µl of NP-40 cell lysis buffer containing 0.1% SDS and centrifuged again at 70,000 rpm. The wash buffer was discarded, and the pellet (washed membrane fraction) was resuspended in 200 µl of hypotonic buffer (with 1 mM DTT added), homogenized through a 25-gauge needle, and frozen at –20°C until use. Fraction purity was determined by Western blotting for fraction-specific protein nucleolin (nuclear fraction), Golgi marker gm130 (membrane fraction), and beta-actin (cytosolic fraction).


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CaMKII{delta}2 and iNOS colocalize and coimmunoprecipitate in VSMCs. iNOS was induced for 24 h in growth-arrested cultured rat aortic VSMCs with a cytokine mixture containing IL-1beta (20 ng/ml), IFN-{gamma} (200 U/ml), and TNF-{alpha} (20 ng/ml). Figure 1 demonstrates the typical distribution of iNOS and CaMKII{delta}2 in VSMCs using immunofluorescence and confocal microscopy. iNOS, which was undetectable in control cells (Fig. 1C), was found to be diffusely distributed throughout the cytokine-induced cells (Fig. 1, A and B), excluding the nucleus, with a clear concentration in perinuclear regions and in structures reminiscent of Golgi-adjacent, aggresome-like structures as defined in previous studies using C2C12 (23), RT-4, human embryonic kidney 293, A-172, A-549, and RAW-264.7 cells (19). Cells stained for CaMKII{delta}2 indicated a similar distribution, although the aggresome-like distribution was not as apparent (Fig. 1, A and B). Overlays of iNOS and CaMKII{delta}2 immunofluorescence reproducibly indicated a strong degree of colocalization, particularly in the perinuclear areas.


Figure 1
View larger version (62K):
[in this window]
[in a new window]

  		Fig. 1. Inducible nitric oxide synthase (iNOS) and CaMKII{delta}2 colocalize in perinuclear regions. A: z sections of confocal images. B and C: immunofluorescence (IF) images. Vascular smooth muscle (VSM) cells (VSMCs) in A and B were induced with a cytokine cocktail for 24 h in media with no growth serum, fixed, and then stained with anti-iNOS (green) and anti-CaMKII{delta}2 (red). VSMCs in C were fixed and stained without cytokine stimulation. IF staining controls with only primary or secondary antibodies were negative (not shown).

 
Colocalization of iNOS and CaMKII{delta}2, suggested by the immunofluorescence approach, was confirmed by immunoprecipitation. CaMKII{delta}2 was immunoprecipitated from VSMCs that had been induced with cytokines, and the resolved precipitates were probed for iNOS (Fig. 2A). After induction, iNOS was coimmunoprecipitated with CaMKII{delta}2. A reverse immunoprecipitation using the iNOS antibody consistently pulled down a fraction of CaMKII{delta}2 (Fig. 2C).


Figure 2
View larger version (37K):
[in this window]
[in a new window]

  		Fig. 2. CaMKII and iNOS coimmunoprecipitate. A, top: cell lysates were immunoprecipitated with an antibody specifically recognizing CaMKII{delta}2, and the precipitates were immunoblotted with an antibody specific for iNOS (IB:iNOS). In the bottom panels, aliquots of preimmune lysates were blotted with iNOS and beta-actin antibodies to demonstrate equal levels of iNOS induction. B: immunoblots from A quantified by densitometry. Values plotted are means ± SE; n = 5 experiments; io, ionomycin. C: reverse immunoprecipitation using anti-iNOS to immunoprecipitate followed by immunoblotting with anti-CaMKII{delta}2. All samples have been stimulated with cytokines for iNOS production, except the one labeled uninduced.

 
iNOS interaction with CaMKII{delta}2 is regulated by CaMKII activity. The effect of CaMKII activity on the degree of association was tested. Acute activation of CaMKII by addition of the Ca2+ ionophore ionomycin to the cells for 3 min significantly decreased the amount of coimmunoprecipitating iNOS (Fig. 2, A and B). Pretreatment of the cells with the CaMKII inhibitor KN-93 for 30 min, but not its inactive analog KN-92, prevented the acute ionomycin-induced decrease in coimmunoprecipitating iNOS. The conditions of ionomycin treatment used here have been previously shown to activate CaMKII{delta}2 in these cells using either activity assays (1) or Western blotting with an antibody specific to the threonine 287 autophosphorylated form of the kinase (20). Similarly, the concentrations of KN-93 used in this study have been previously shown to maximally inhibit CaMKII activation in these cells (22).

Chronic treatment of the VSMCs with KN-93 alone after the iNOS induction phase also significantly enhanced the CaMKII{delta}2-iNOS interaction (Fig. 3), suggesting that the interaction was also sensitive to basal activity of the kinase (i.e., unstimulated by acute addition of ionomycin). As expected, suppression of CaMKII{delta}2 expression before iNOS induction using a siRNA specific for the {delta}-isoform reduced both the content of CaMKII{delta}2 in the cells and proportionately decreased coimmunoprecipitating iNOS (Fig. 3). The results of the immunoprecipitation experiments confirm the immunofluorescence colocalization result and suggest that the association of iNOS and CaMKII{delta}2 may be regulated by the state of CaMKII activation.


Figure 3
View larger version (40K):
[in this window]
[in a new window]

  		Fig. 3. iNOS interaction with CaMKII{delta}2 is enhanced following treatment with the CaMKII inhibitor KN-93. VSMCs were infected with an adenovirus encoding small interfering RNA (siRNA) targeting CaMKII{delta}2 or a control siRNA for green fluorescent protein (SIGFP). After 96 h, the cells were serum starved overnight and then induced for iNOS expression for 24 h. After this, KN-93 was added for 16 h to the appropriate well with fresh media. A: iNOS from a CaMKII{delta}2 immunoprecipitation. The membrane was then stripped and reprobed for CaMKII{delta}2. The bottom panel shows preimmune lysates. B: densitometric analysis of the blots in A; inclusive of at least three experiments. The overall level of cellular iNOS remained constant in these experiments; the average suppression of CaMKII was 69% (as quantified by densitometry; not shown).

 
iNOS and CaMKII localization are affected by CaMKII activity. The distribution of iNOS in the VSMCs shown in Fig. 1 includes concentration in a perinuclear area consistent with previously reported aggresome-like structures (19). Since aggresomes have been previously shown to colocalize with {gamma}-tubulin in microtubule organizing centers, we compared iNOS and {gamma}-tubulin distribution in the VSMCs (Fig. 4A). A strong degree of overlap in staining is consistent with the interpretation of iNOS concentration in an aggresomal-like compartment. Inhibition of CaMKII activity with KN-93 after the iNOS induction phase enhanced localization of iNOS in the aggresome-like structures (Fig. 4C). CaMKII was also confirmed to colocalize in this compartment, and levels of the protein also increased with lowered kinase activity. iNOS localization was quantified in Fig. 5, with 57% of control cytokine-stimulated cells demonstrating concentrated iNOS immunoreactivity in aggresome-like structures, whereas 75% of the cells pretreated with KN-93 demonstrated this pattern.


Figure 4
View larger version (64K):
[in this window]
[in a new window]

  		Fig. 4. Changes in iNOS localization dependent on CaMKII{delta}2. A: iNOS (green) and {gamma}-tubulin (red) colocalization by immunofluorescence; {gamma}-tubulin is known to colocalize to aggresomes, adjacent to the Golgi. B–D: iNOS (green) and CaMKII{delta}2 (red) immunofluorescence. Images from B and C were from the experiment demonstrating iNOS localization in VSMCs (B) or cells to which KN-93 was added for 4 h (C). VSMCs in D were infected with an adenovirus encoding a siRNA for CaMKII{delta}2 for 96 h. CaMKII{delta}2 knockdown visualized cell to cell was variable, as indicated in the two adjacent cells. Interestingly, cells with CaMKII{delta}2 knockdown had a different pattern of iNOS localization (diffuse/nuclear) when compared with the cells expressing higher amounts of CaMKII{delta}2 (aggresome-like). All cells shown in A–D have been stimulated with cytokines for iNOS production.

 

Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 5. CaMKII affects aggresome-like localization of iNOS. VSMCs were either infected with an adenovirus encoding either siRNA for CaMKII{delta}2 (deltakd), constitutively active CaMKII (CAdelta), or a control siRNA for GFP (control). After 96 h, the cells were serum starved overnight and then induced for iNOS expression. Alternatively, cells were induced for iNOS for 24 h and then exposed acutely to activators of CaMKII:UTP or ionomycin for 3 min or CaMKII inhibitor KN-93 for 4 h. Cells expressing iNOS and demonstrating a noticeable aggresome-like accumulation of iNOS were compared against the total number of iNOS-expressing cells. The results are a compilation of at least 4 separate experiments showing means ± SE. All samples have been stimulated with cytokines for iNOS production.

 
As an alternative to pharmacological inhibition of CaMKII activity, we evaluated the consequences of siRNA suppression of CaMKII on iNOS localization. VSMCs were infected with an adenovirus encoding a siRNA for CaMKII{delta}2 (deltakd) for 96 h, and the cells were then induced for iNOS, fixed, and stained. On a cell-to-cell basis, CaMKII{delta}2 knockdown using this protocol was variable between cells, as shown between the two cells in Fig. 4D. Interestingly, those cells in which CaMKII{delta}2 expression and staining were most suppressed demonstrated diffuse iNOS localization (upper cell), whereas cells with more CaMKII{delta}2 demonstrated aggresomal-like iNOS localization. This result was also quantified in Fig. 5. Following CaMKII{delta}2 knockdown, significantly fewer cells demonstrated iNOS localization in aggresomes when compared with control cells (57% vs. 31%, respectively). On the basis of the opposing effects of CaMKII activity inhibition compared with lack of expression, we propose that inactive CaMKII{delta}2 may facilitate recruitment to or stabilization of iNOS to aggresome-like structures, perhaps through formation of a protein complex.

Because inhibition of CaMKII activity enhanced aggresomal-like localization of iNOS, we tested the effect of CaMKII activation on this localization. In cells stimulated with either a pharmacological (ionomycin) or physiological (UTP) Ca2+-mobilizing stimulus, significantly fewer cells demonstrated this pattern of localization (Fig. 5). Furthermore, cells infected with an adenovirus encoding a constitutively active form of CaMKII (T287D) exhibited a similar decrease in aggresome-like localization of iNOS. These results coupled with the results from Fig. 2 suggest that the complex between iNOS and CaMKII dissociates when CaMKII is activated.

iNOS content in VSMC fractions. Cellular fractionations were performed to determine iNOS content in the nuclear, membrane, and cytosolic fractions of VSMCs. iNOS protein distribution was quantified by immunoblotting aliquots from the fractions, and relative content was calculated after adjustment for fraction volume (Table 1). In control cells, iNOS is concentrated in cytosolic and membrane fractions, as expected from the immunofluorescence localization approach. Chronic treatment with the CaMKII inhibitor KN-93 after iNOS induction decreased nuclear and membrane fraction iNOS in favor of a cytosolic distribution, supporting the aggresomal data shown in Fig. 5. Conversely, acute activation of CaMKII with ionomycin or UTP and overexpression of constitutively active CaMKII{delta}2 (CAdelta) increased nuclear and membrane localization of iNOS while decreasing cytosolic content. Thus the changes in nuclear- and membrane-associated iNOS following perturbations in CaMKII activity mirror (reciprocally) changes in cytosolic/aggresomal iNOS localization.


View this table:
[in this window]
[in a new window]

  		Table 1. iNOS distribution in VSM cell fractions

 
Suppression of CaMKII{delta}2 expression with siRNA (deltakd) also resulted in a marked redistribution of iNOS to the nuclear and membrane fractions. Addition of ionomycin to these CaMKII-deficient cells had no further effects on iNOS localization (Table 1). This result also mirrors the aggresomal data in shown in Fig. 5, and supports the idea that CaMKII protein and activity regulates iNOS trafficking in VSMCs.

CaMKII affects iNOS activity in VSM. To determine whether CaMKII could regulate iNOS activity, nitrite accumulation was measured in the cultures following iNOS induction as a function of manipulations (KN-93, deltakd, and constitutively active CaMKII{delta}2), resulting in altered CaMKII expression or activity (Fig. 6A). Nitrite accumulation in noninduced cells was low, and the induced nitrite accumulation was blocked by treatment with aminoguanidine (data not shown), suggesting that the measured products were primarily derived from iNOS-dependent production of NO. Total iNOS protein content was not significantly affected by the experimental treatments (Fig. 6B). Manipulation of CaMKII activity by treatment with KN-93 after the iNOS induction phase or by expressing a constitutively active CaMKII{delta}2 (CAdelta) significantly inhibited iNOS activity. Suppression of CaMKII{delta}2 by treatment with siRNA also inhibited iNOS activity. Because the assay measures long-term accumulation of nitrite, it is not sensitive to acute activation of CaMKII, and short-term addition of ionomycin had no effect nitrite accumulation.


Figure 6
View larger version (42K):
[in this window]
[in a new window]

  		Fig. 6. CaMKII-dependent modulation of iNOS activity. VSMCs were infected with an adenovirus encoding either siRNA for CaMKII{delta}2 (deltakd), constitutively active CaMKII (CAdelta), or a control siRNA for GFP. After 96 h, the cells were serum starved overnight and then induced for iNOS expression. Fresh medium was added at 24 h after incubation with KN-93, ionomycin, or a vehicle (DMSO); 16 h after that, the media were analyzed for nitrite accumulation. After nitrite analysis, the cells were lysed and run on an SDS-PAGE gel, and the iNOS amount was quantified for overall activity analysis using a standard curve. All samples have been stimulated with cytokines for iNOS production.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Posttranslational modifications that control function and localization have been found on each of the NOS enzymes (23, 24, 13, 19, 28). In particular, lipid modifications and trafficking of both eNOS and nNOS to specific subcellular microdomains have been found to be important components of activity and function. CaMKII has been reported to regulate both eNOS and nNOS activity (6, 32). In the case of nNOS, CaMKII may affect its trafficking to postsynaptic densities by regulating nNOS interaction with scaffolding proteins such as PSD-95 (32). When compared with eNOS and nNOS, iNOS trafficking is less completely characterized, and little is known about stimuli and posttranslational events that influence its localization and activity. Several recent studies have indicated the importance of palmitoylation in iNOS processing and activation (23). We show here that CaMKII{delta}2 influences iNOS localization and activity in cultured VSMCs.

iNOS and CaMKII{delta}2 were found to colocalize (Fig. 1) and coimmunoprecipitate (Fig. 2) in primary VSMCs. Immunofluorescence and confocal microscopy indicated the presence of iNOS concentrated in a perinuclear distribution and colocalizing with {gamma}-tubulin, consistent with previous reports describing iNOS localization in aggresome-like structures (19). The distribution is also consistent with previous reports localizing iNOS to Golgi membranes, where it is palmitoylated, a modification required for activity (23). CaMKII{delta}2 subcellular localization has not been studied carefully, although previous reports from this lab using VSMC (4) and studies in astrocytes (32) suggest a concentration of the kinase in perinuclear areas, associated with ER and Golgi markers. Coimmunoprecipitation of iNOS and CaMKII{delta}2 suggest their localization in a complex, although at this time it is not known whether or not this is a direct interaction or an interaction involving other unidentified proteins. On the basis of studies of nNOS regulation, it is tempting to speculate that a protein scaffold analogous to PSD-95 may be involved in formation of a multiprotein complex including iNOS and CaMKII.

To probe further whether the interaction between CaMKII and iNOS is functional, we evaluated the effects of manipulating CaMKII activity or content on iNOS localization and activity. Acute activation of the kinase with a Ca2+ ionophore, ionomycin, decreased interaction between CaMKII{delta}2 and iNOS, an effect blocked by the CaMKII inhibitor KN-93, but not its inactive analog. The latter results strongly suggest that the ionomycin effects on localization were, in fact, attributable to CaMKII activity and not, for example, a nonspecific stress response that might disrupt ER or Golgi structure. The fact that prolonged incubation with KN-93 alone after the iNOS induction phase enhanced iNOS/CaMKII{delta}2 interaction suggests that basal CaMKII activity in growing cells plays a function in iNOS targeting.

Interestingly, CaMKII activation with ionomycin or expression of a constitutively active mutant of CaMKII{delta}2 decreased the number of cells exhibiting a distinct aggresome-like structure localized with iNOS and CaMKII. Conversely, addition of an inhibitor of CaMKII activity appears to increase iNOS localization in such structures (Figs. 4 and 5). A similar phenomenon of reduced aggresome-like localization of iNOS was observed in cells lacking CaMKII{delta}2 following suppression with siRNA. Thus both CaMKII activity and content may affect trafficking of iNOS into the aggresome-like structure. Although aggresomes are usually thought of as sites of misfolded protein accumulation and degradation, it has been proposed that, in the case of iNOS, the structure may serve as a reservoir for functional iNOS (19).

Cell fractionation was used as an alternative approach to assess CaMKII-dependent changes in intracellular iNOS distribution. Acute activation of CaMKII with ionomycin or a G protein receptor-coupled agonist, UTP, increased the relative content of iNOS in the membrane and nuclear fractions (Table 1). A similar effect was observed following overexpression of constitutively active CaMKII{delta}2. Conversely, exposure to KN-93 caused an increase in cytosolic iNOS, with reductions in the nuclear and membrane fractions. Results using this approach paralleled those from the immunofluorescence approach, suggesting iNOS localized in aggresome-like structures might be recovered in the cytosolic fraction. Together, these approaches indicate that the activity of CaMKII modulates iNOS localization.

There are several reports suggesting nuclear NOS localization in other cell systems (13, 28), similar to what we have observed here in primary VSMCs following expression of constitutively active CaMKII of activation of the endogenous kinase with Ca2+-mobilizing stimuli. Although the functional significance of nuclear localized NOS is not clear, it is possible that locally generated NO could differentially affect the redox environment or posttranslational modification of proteins through nitrosylation in specific subcellular domains and thereby affect specific cellular functions. High concentrations of nuclear NO in VSMC could affect gene transcription or cell proliferation, resulting in VSMC cytostasis or cytotoxicity.

Conventional thought dictates that iNOS activity is largely unregulated following induction and similar or the same regardless of the cellular compartment (15). If this were the case, then CaMKII-dependent changes in iNOS localization would not modulate iNOS activity. However, iNOS activity measured by accumulation of nitrate in the cell culture medium was decreased under conditions of CaMKII activation or depletion shown to affect iNOS localization. At the present time, we cannot propose a specific molecular model linking CaMKII activity to regulation of iNOS trafficking and activity. Our general interpretation of the data is that regulated CaMKII activity is important for iNOS trafficking and consequently activity: inhibiting CaMKII activity changes the pattern of trafficking, including accumulation of a CaMKII/iNOS complex in aggresome-like structures with consequent decreases in iNOS activity; depleting CaMKII disrupts normal iNOS trafficking and activation, perhaps interfering with posttranslational modification and activation by palmitoylation; and overexpressing constitutively active CaMKII disrupts normal trafficking through dysregulated CaMKII activity. A common mechanistic link, consistent with proposed mechanisms of nNOS regulation, may be through CaMKII activity-regulated interactions of iNOS with protein complexes involved in vesicular transport. If aggresome-like structures contain a pool of activatable iNOS, as proposed (19), CaMKII activation could provide a mechanism for mobilizing this pool.

However, certain factors could complicate this interpretation. One includes the possibility that the overexpression of a constitutively active multifunctional kinase or knockdown of CaMKII by siRNA could have "off target" effects. In addition, the possibility of acute modulation of iNOS activity by CaMKII was not addressed in these studies. Immediate changes in iNOS activity are difficult to assess with the assay used, and, therefore, any changes in activity due to acute and transient activation of CaMKII would not be readily detected. Both eNOS and nNOS have been suggested to be CaMKII substrates (30, 35), but this has not yet been evaluated, or at least reported, in the case of iNOS. Although our results suggest that CaMKII-dependent regulation of iNOS trafficking may affect the active pool of iNOS, we cannot yet rule out direct effects of CaMKII on iNOS activity independent of localization per se.

iNOS can be upregulated in VSM in response to acute injury (strokes, stent placement, balloon catheter, and vascular transplants), infection and sepsis, and chronic maladies such as hypertension. VSM-localized iNOS is generally considered vasculoprotective due to inhibitory effects of NO on VSMC migration and proliferation. The results of these studies suggest for the first time that, like eNOS and nNOS, efficient NO production by iNOS is coordinated, in part, by CaMKII activity-dependent regulation of iNOS trafficking, affecting enzymatic activity in a biologically important cell type. We speculate that involvement of CaMKII in iNOS trafficking shown here may be analogous to the relatively well-defined function of the kinase in regulating nNOS localization and activity in postsynaptic densities. Additional studies are warranted to better understand the molecular mechanisms underlying the interaction physical and functional interactions between CaMKII and iNOS and the biological significance of this with respect to VSM cell responses to injury.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a fellowship from National Institutes of Health (T32-HL-07194) for R. J. Jones and in part by National Heart, Lung, and Blood Institute Grant R01-HL-49426 to H. A. Singer.


   FOOTNOTES
 

Address for reprint requests and other correspondence: H. A. Singer, Center for Cardiovascular Sciences, Albany Medical College, 43 New Scotland Ave., Albany, NY 12208 (e-mail: singerh{at}mail.amc.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
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Abraham ST, Benscoter H, Schworer CM, Singer HA. In situ Ca2+ dependence for activation of Ca2+/calmodulin-dependent protein kinase II in vascular smooth muscle cells. J Biol Chem 271: 2506–2513, 1996.[Abstract/Free Full Text]
  2. Banning AP, Groves PH, Buttery LDK, Wharton J, Rutherford RAD, Black P, Winkler F, Polak JM, Lewis MJ, Drexler H. Reciprocal changes in endothelial and inducible nitric oxide synthase expression following carotid angioplasty in the pig. Atherosclerosis 145: 17–32, 1999.[CrossRef][Web of Science][Medline]
  3. Brenman JE, Chao DS, Gee SH, McGee AW, Craven SE, Santillano DR, Wu Z, Huang F, Xia H, Peters MF, Froehner SC, Bredt DS. Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and alpha1-syntrophin mediated by PDZ domains. Cell 84: 757–767, 1996.[CrossRef][Web of Science][Medline]
  4. Caran N, Johnson LD, Jenkins KJ, Tombes RM. Cytosolic targeting domains of gamma and delta calmodulin-dependent protein kinase II. J Biol Chem 276: 42514–42519, 2001.[Abstract/Free Full Text]
  5. Daniliuc S, Bitterman H, Rahat MA, Kinarty A, Rosenzweig D, Lahat N. Hypoxia inactivates inducible nitric oxide synthase in mouse macrophages by disrupting its interaction with alpha-actinin 4. J Immunol 171: 3225–3232, 2003.[Abstract/Free Full Text]
  6. Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R. Phosphorylation of Thr495 regulates Ca2+/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88: E68–E75, 2001.[CrossRef][Web of Science][Medline]
  7. Garcia-Mata R, Gao Y, Sztul E. Hassles with taking out the garbage: aggravating aggresomes. Traffic 3: 388–96, 2002.[CrossRef][Web of Science][Medline]
  8. Garg UC, Hassid A. Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83: 1774–1777, 1989.[Web of Science][Medline]
  9. Geisterfer AAT, Peach MJ, Owens GK. Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells. Circ Res 62: 749–756, 1988.[Abstract/Free Full Text]
  10. Geng YJ, Almqvist M, Hansson GK. cDNA cloning and expression of inducible nitric oxide synthase from rat vascular smooth muscle cells. Biochim Biophys Acta 1218: 421–424, 1994.[Medline]
  11. Ginnan R, Pfleiderer PJ, Pumiglia K, Singer HA. PKC-{delta} and CaMKII-{delta}2 mediate ATP-dependent activation of ERK1/2 in vascular smooth muscle. Am J Physiol Cell Physiol 286: C1281–C1289, 2004.[Abstract/Free Full Text]
  12. Ginnan R, Singer HA. CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle. Am J Physiol Cell Physiol 282: C754–C761, 2002.[Abstract/Free Full Text]
  13. Giordano A, Tonello C, Bulbarelli A, Cozzi V, Cinti S, Carruba MO, Nisoli E. Evidence for a functional nitric oxide synthase system in brown adipocyte nucleus. FEBS Lett 514: 135–140, 2002.[CrossRef][Web of Science][Medline]
  14. Hansson GK, Geng YJ, Holm J, Hardhammar P, Wennmalm A, Jennische E. Arterial smooth muscle cells express nitric oxide synthase in response to endothelial injury. J Exp Med 180: 733–738, 1994.[Abstract/Free Full Text]
  15. Jagnandan D, Sessa WC, Fulton D. Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS. Am J Physiol Cell Physiol 289: C1024–C1033, 2005.[Abstract/Free Full Text]
  16. Johansson R, Persson K. Phenotypic modulation of cultured bladder smooth muscle cells and the expression of inducible nitric oxide synthase. Am J Physiol Regul Integr Comp Physiol 286: R642–R648, 2004.[Abstract/Free Full Text]
  17. Joly GA, Schini VB, Vanhoutte PM. Balloon injury induces nitric oxide synthase activity in rat carotid arteries. J Cardiovasc Pharmacol 20, Suppl 12: S151–S154, 1992.[Web of Science][Medline]
  18. Koide M, Kawahara Y, Tsuda T, Yokoyama M. Cytokine-induced expression of an inducible type of nitric oxide synthase gene in cultured vascular smooth muscle cells. FEBS Lett 318: 213–217, 1993.[CrossRef][Web of Science][Medline]
  19. Kolodziejska KE, Burns AR, Moore RH, Stenoien DL, Eissa NT. Regulation of inducible nitric oxide synthase by aggresome formation. Proc Natl Acad Sci USA 102: 4854–4859, 2005.[Abstract/Free Full Text]
  20. Lu KK, Armstrong SE, Ginnan R, Singer HA. Adhesion-dependent activation of CaMKII and regulation of ERK activation in vascular smooth muscle. Am J Physiol Cell Physiol 289: C1343–C1350, 2005.[Abstract/Free Full Text]
  21. Marganski WA, Gangopadhyay SS, Je HD, Gallant C, Morgan KG. Targeting of a novel Ca2+/calmodulin-dependent protein kinase II is essential for extracellular signal-regulated kinase-mediated signaling in differentiated smooth muscle cells. Circ Res 97: 541–549, 2005.[Abstract/Free Full Text]
  22. Muthalif MM, Benter IF, Uddin MR, Malik KU. Calcium/calmodulin-dependent protein kinase IIalpha mediates activation of mitogen-activated protein kinase and cytosolic phospholipase A2 in norepinephrine-induced arachidonic acid release in rabbit aortic smooth muscle cells. J Biol Chem 271: 30149–30157, 1996.[Abstract/Free Full Text]
  23. Navarro-Lerida I, Corvi MM, Barrientos AA, Gavilanes F, Berthiaume LG, Rodriguez-Crespo I. Palmitoylation of inducible NOS at Cys3 is required for proper intracellular traffic and nitric oxide synthesis. J Biol Chem 279: 55682–55689, 2004.[Abstract/Free Full Text]
  24. Pan J, Burgher L, Szczepanik AM, Ringheim GE. Tyrosine phosphorylation of inducible nitric oxide synthase: implications for potential post-translational regulation. Biochem J 314: 889–894, 1996.[Web of Science][Medline]
  25. Pauly RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, Crow MT. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation 91: 1107–1115, 1995.[Abstract/Free Full Text]
  26. Pfleiderer PJ, Lu KK, Crow MT, Keller RS, Singer HA. Modulation of vascular smooth muscle cell migration by calcium/calmodulin-dependent protein kinase II-{delta}2. Am J Physiol Cell Physiol 286: C1238–C1245, 2004.[Abstract/Free Full Text]
  27. Rokolya A, Singer HA. Inhibition of CaM kinase II activation and force maintenance by KN-93 in arterial smooth muscle. Am J Physiol Cell Physiol 278: C537–C545, 2000.[Abstract/Free Full Text]
  28. Saini R, Patel S, Saluja R, Sahasrabuddhe AA, Singh MP, Habib S, Bajpai VK, Dikshit M. Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies. J Leukoc Biol 79: 519–528, 2006.[Abstract/Free Full Text]
  29. Schworer CM, Rothblum LI, Thekkumkara TJ, Singer HA. Identification of novel isoforms of the delta subunit of Ca2+/calmodulin-dependent protein kinase II. Differential expression in rat brain and aorta. J Biol Chem 268: 14443–14449, 1993.[Abstract/Free Full Text]
  30. Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T. Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem 271: 6518–6522, 1996.[Abstract/Free Full Text]
  31. Spink J, Cohen J, Evans TJ. The cytokine responsive vascular smooth muscle enhancer of inducible nitric oxide synthase. J Biol Chem 270: 29541–29547, 1995.[Abstract/Free Full Text]
  32. Takeuchi Y, Yamamoto H, Fukunaga K, Miyakawa T, Miyamoto E. Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization. J Neurochem 74: 2557–2567, 2000.[CrossRef][Web of Science][Medline]
  33. Taquchi J, Abe J, Ozakaki H, Takuwa Y, Kurokawa K. L-Arginine inhibits neointimal formation following balloon injury. Life Sci 53: PL387–PL392, 1993.[CrossRef][Medline]
  34. Wang Q, Patton WF, Chiang ET, Hechtman HB, Shepro D. Filamin translocation is an early endothelial cell inflammatory response to bradykinin: regulation by calcium, protein kinases, and protein phosphatases. J Cell Biochem 62: 383–396, 1996.[CrossRef][Web of Science][Medline]
  35. Watanabe Y, Song T, Sugimoto K, Horii M, Araki N, Tokumitsu H, Tezuka T, Yamamoto T, Tokuda M. Post-synaptic density-95 promotes calcium/calmodulin-dependent protein kinase II-mediated Ser847 phosphorylation of neuronal nitric oxide synthase. Biochem J 372: 465–471, 2003.[CrossRef][Web of Science][Medline]
  36. Xie QW, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-gamma and bacterial lipopolysaccharide. Trans Assoc Am Physicians 106: 1–12, 1993.[Medline]
  37. Yan Z, Hansson GK. Overexpression of inducible nitric oxide synthase by neointimal smooth muscle cells. Circ Res 82: 21–29, 1998.[Abstract/Free Full Text]
  38. Yan Z, Yokota T, Zhang W, Hansson GK. Expression of inducible nitric oxide synthase inhibits platelet adhesion and restores blood flow in the injured artery. Circ Res 79: 38–44, 1996.[Abstract/Free Full Text]
  39. Yogo K, Shimokawa H, Funakoshi H, Kandabashi T, Miyata K, Okamoto S, Egashira K, Huang P, Akaike T, Takeshita A. Different vasculoprotective roles of NO synthase isoforms in vascular lesion formation in mice. Arterioscler Thromb Vasc Biol 20: 396–100, 2000.
  40. Zhang S, Yang Y, Kone BC, Allen JC, Kahn AM. Insulin-stimulated cyclic guanosine monophosphate inhibits vascular smooth muscle cell migration by inhibiting Ca/calmodulin-dependent protein kinase II. Circulation 107: 1539–1544, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
P. Wang, G.-H. Liu, K. Wu, J. Qu, B. Huang, X. Zhang, X. Zhou, L. Gerace, and C. Chen
Repression of classical nuclear export by S-nitrosylation of CRM1
J. Cell Sci., October 15, 2009; 122(20): 3772 - 3779.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
P. Ponnuswamy, E. Ostermeier, A. Schrottle, J. Chen, P. L. Huang, G. Ertl, B. Nieswandt, and P. J. Kuhlencordt
Oxidative Stress and Compartment of Gene Expression Determine Proatherosclerotic Effects of Inducible Nitric Oxide Synthase
Am. J. Pathol., June 1, 2009; 174(6): 2400 - 2410.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
M. Z. Mercure, R. Ginnan, and H. A. Singer
CaM kinase II{delta}2-dependent regulation of vascular smooth muscle cell polarization and migration
Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1465 - C1475.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/6/H2634    most recent
01247.2006v1
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 (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jones, R. J.
Right arrow Articles by Singer, H. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jones, R. J.
Right arrow Articles by Singer, H. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.