Skip Navigation

Cardiovascular Research 2005 68(1):65-74; doi:10.1016/j.cardiores.2005.05.021
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Agulló, L.
Right arrow Articles by Soler-Soler, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agulló, L.
Right arrow Articles by Soler-Soler, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2005, European Society of Cardiology

Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes

Luis Agulló, David Garcia-Dorado*, Noelia Escalona, Marisol Ruiz-Meana, Maribel Mirabet, Javier Inserte and Jordi Soler-Soler

Servicio de Cardiología, Hospital Universitari Vall d'Hebron, Pg Vall d'Hebron 129, 08035 Barcelona, Spain

* Corresponding author. Tel.: +34 93 489 40 38; fax: +34 93 489 40 32. Email address: dgdorado{at}vhebron.net

Received 7 September 2004; revised 11 April 2005; accepted 18 May 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Although the importance of the cyclic GMP (cGMP) signaling pathway in cardiac myocytes is well established, little is known about its regulation. Ca2+-dependent translocation of nitric oxide (NO) sensitive guanylyl cyclase (GCNO) to the cell membrane has been recently proposed to play a role. The aim of this study was to determine the possible functional relevance of GCNO bound to the cardiomyocyte membrane.

Methods: Cytosolic and particulate fractions of adult rat cardiomyocytes were isolated and blotted, and their GCNO activity was assayed in parallel experiments.

Results: In untreated cardiomyocytes, approximately 30% of β1-and {alpha}1-subunits of GCNO and a similar proportion of GCNO activity were found in the particulate fraction. The dependence of GCNO activity on pH, Ca2+, GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187 [GenBank] caused GCNO to translocate to the sarcolemma, increased GCNO activity in this fraction, and potentiated NO-mediated cGMP synthesis. These effects appeared to be mediated by Ca2+-dependent changes on the phosphorylation status of GCNO, since they were enhanced by the non-selective inhibitor staurosporine and by the selective inhibitor of Ca2+/calmodulin-dependent protein kinase KN-93. The effect of drugs increasing intracellular Ca2+ on cGMP synthesis was clearly correlated with their effects on membrane-associated GCNO activity but not with their effects on cytosol-associated GCNO.

Conclusion: These results are the first evidence that 1) GCNO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GCNO differs from that of cytosolic GCNO, and 3) membrane association may have a crucial role in determining the response of cells to NO.

KEYWORDS Nitric oxide; Calcium (cellular); Signal transduction; Myocytes; Protein phosphorylation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Cyclic GMP (cGMP) modulates important physiological functions in the cardiovascular system as vasodilation, Ca2+ cycling, endothelium permeability, or myocardial contractility [1]. Abundant evidence indicates that cGMP can modulate cell death during ischemia-reperfusion [2–7] and cGMP has been described to mediate late preconditioning in conscious rabbits [8].

cGMP can be synthesized by two different types of guanylyl cyclases: a nitric oxide (NO)-sensitive guanylyl cyclase (GCNO), generally known as cytosolic or soluble guanylyl cyclase, and guanylyl cyclases that are integral proteins in the plasmatic membrane of the cell and can be stimulated by natriuretic peptides. GCNO is constituted of two subunits, {alpha} and β, and two different isoforms of the {alpha} subunit ({alpha}1 and {alpha}2) and of the β subunit (β1 and β2) have been described. The {alpha}1β1 heterodimer is predominantly found in the cardiovascular system, while {alpha}2β1 has been mainly found in brain.

Little is known about how GCNO is modulated in vivo. Rapid desensitisation of the signal [9] and phosphorylation by different protein kinases, as protein kinase C (PKC) [11], cyclic AMP-dependent protein kinase (PKA) [10,11] and cGMP-dependent protein kinase (PKG) [12], have been described.

Recent studies have challenged the classical concept of an exclusive cytosolic location of GCNO. The isoform {alpha}2β1 has been found tightly associated to the neuron membrane through a PSD-95 mediated interaction [13]. A histochemical study suggested the presence of the more amply distributed heterodimer, {alpha}1β1, in the sarcolemmal region of skeletal muscle fibres [14]. This association to the particulate fraction of {alpha}1 and β1 subunits has been recently demonstrated in myocardial tissue, endothelial cells and platelets [15], and in these later cells activation with ADP or collagen has been correlated with enzyme translocation to the membrane [15]. However, the mechanism of the interaction of {alpha}1β1 with the membrane was unresolved. A recent study has described a protein complex formed by eNOS, Hsp90 and GCNO in endothelial cells, and that bradykinin and vascular endothelial growth factor potentiate the formation of this complex [16]. The contribution of particulate GCNO to the cell response to NO remained unknown.

In this study, we analyze how association of GCNO to membrane affects its biochemical properties in cardiomyocytes, the functional relevance of this association, and its potential regulation by Ca2+.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
The animal protocols conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and was approved by the Research Commission on Ethics of Hospital Vall d'Hebron.

2.1 Cardiomyocyte and platelet isolation
Cardiomyocytes were obtained from adult rat hearts as previously described [17]. At the end of the procedure, culture dishes contained >85% of quiescent rod-shaped cells. Rat washed platelets were isolated from venous blood collected with sodium citrate [18].

2.2 Intracellular cGMP synthesis
After treatment, cells were stimulated for 1 minute with SNAP 100 µM (unless otherwise indicated) in the presence of 1 mM 3-isobutyl-1-methylxanthine (IBMX) as inhibitor of cGMP degradation. cGMP was quantified by radioimmunoassay using acetylated [3H]cGMP [17].

2.3 Membrane and cytosolic fractions
Following incubation with the different agents, cardiomyocytes were homogenized with a straight wall grinder (Radnoti Glass Technology) in ice-cold buffer A [in mM: Tris.HCl 50 (pH 7.4), sucrose 250, EDTA 0.1, dithiotreitol 1, plus protease inhibitors [17], the protein kinase inhibitor staurosporin 1 x 10–3, and the Ser/Thr protein phosphatase inhibitors okadaic acid 1 x 10–3 and cypermethrin 5 x 10–4]. After clearing the homogenates by centrifugation at 1000 x g for 15 min, the particulate and cytosolic fractions were obtained by centrifugation (100,000 x g for 1 h). Membrane fractions were homogenized with buffer A plus 10% glycerol. Triton X-100 solubility of particulate GCNO was assessed as described [19]. Treated platelets were centrifuged, resuspended in buffer A, frozen in liquid nitrogen, thawed slowly on ice and then homogenized with a straight wall grinder and ultracentrifuged as mentioned for cardiomyocytes.

2.4 Guanylyl cyclase activity
Guanylyl cyclase activity was determined [17] by measuring cGMP (radioimmunoassay) synthesized after incubating the particulate and cytosolic extracts with no-additions (basal) or 100 µM SNAP in assay buffer [final concentrations (in mM): Tris.HCl 50 (pH 7.4), EGTA 1, dithiotreitol 1, GTP 2.5, MgCl2 5, phosphocreatine 10, IBMX 1, plus creatine kinase (50 U/ml)] at 37 °C for 30 min. GCNO activity, calculated by substracting basal activity, was linear with respect to time in the assay period. To analyze pH dependence of GCNO activity, Tris.HCl was substituted by 30 mM PIPES (pH 6.0–6.8) or 30 mM HEPES (pH 6.8–8.0). Free Ca2+ concentrations were calculated by a modification of the RECIPC program (S. Roberston, University of Cincinnati, 1981).

2.5 Western-blotting
Proteins were separated by electrophoresis on a 10% SDS gel and transferred onto nitrocellulose membrane (Hybond-ECL, Amersham). Membranes were incubated with rabbit polyclonal antibodies to β1 (aminoacids 605–619; used at 1/2000 dilution; Sigma) and {alpha}1 (aminoacids 673–690; 1/20,000; Sigma) subunits of soluble guanylyl cyclase. A goat anti-rabbit IgG horseradish peroxidase conjugated (1/50,000; Pierce) was used as secondary antibody. Specificity of the immunostaining was assessed by displacing the corresponding bands by incubating in the presence of their respective immunization peptides (synthesized by Sigma Genosys). Quantitative chemiluminiscence detection was performed with SuperSignal West Dura Extended Substrate (Pierce) and a 16-bit cooled CCD camara system (LAS-3000, Fujifilm). Equal loading of the different samples was confirmed by Ponceau Red staining.

2.6 Modulation of the membrane association of GCNO
The effect of increased cytosolic Ca2+ concentration on the association of GCNO to the membrane fraction was investigated by incubating cells 1 min with A23187 [GenBank] or 5 min with thapsigargin. Then, cells were: a) stimulated with SNAP for determining NO-dependent cGMP synthesis, and b) homogenized for determining content of β1 subunit and GCNO activity in cytosolic and membrane fractions.

The potential role of changes in GCNO phosphorylation in this effect was investigated by analyzing its modulation by the protein kinase inhibitors staurosporin, Gö-6976, H-89, and KN-93. These drugs were added to the incubation media 4 min before stimulation with A23187 [GenBank] , and maintained during the time of incubation with the ionophore.

2.7 Detection of GCNO phosphorylation in intact cells
Cardiomyocytes or platelets were incubated for 3 h in medium containing 0.1 mCi/ml [32P]ortophosphate (Amersham), washed with ‘cold’ medium, incubated for 5 min with or without 1 µM staurosporin and stimulated for 1 min with 10 µM A23187 [GenBank] or with no drugs. At the end of the stimulation period, cells were homogenized as described, but including a phosphatase inhibitor cocktail (Sigma) in the homogeneization medium. Homogenates were fractionated by centrifugation at 100,000 x g, and the β1 subunit of the cytosolic and particulate fractions immunoprecipitated by incubation with Protein G-agarose beads (Amersham Biosciences) previously bound to 15 µg of anti-β1 antibody. Phosphorylation of the β1 subunit in the immunoprecipitates was assessed by Western-blotting and phosphor screen (Fuji Photo Film Co.) autoradiography with a red laser scanner (Typhoon 9400, Amersham Biosciences). Analysis of {alpha}1 phosphorylation was not possible since the antibody used against this subunit did not significantly immunoprecipitate the protein.

2.8 Intracellular Ca2+
Changes induced by the Ca2+ ionophore A23187 [GenBank] and by thapsigargin were monitored by ratio-fluorescence imaging in cardiomyocytes loaded with fura 2-acetoxymethyl ester (Molecular Probes) as previously described [20].

2.9 Data analysis and statistics
Differences between groups were evaluated by means of paired Student's t test when appropriated or one-way analysis of the variance. Individual comparisons between groups were performed using the Student–Newman Keuls test. Values are expressed as mean ± SEM. Nonlinear fitting was performed using SigmaPlot (SPSS Inc.).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Membrane association of GCNO in cardiomyocytes and platelets
Cytosolic and particulate fractions (100,000 x g) of adult rat cardiomyocytes were isolated and blotted. Staining with an antibody against the β1 subunit of the GCNO, demonstrated that 28 ± 5% of β1 (65.1 ± 0.2 kDa), n = 6, was in the membrane fraction (Fig. 1A). A similar proportion of the {alpha}1 subunit (75.7 ± 2.9 kDa) seemed to be associated to the particulate fraction, but the limited sensitivity of the antibody against this subunit in cardiomyocytes precluded precise analysis (Fig. 1B). Immunization peptides displaced β1 and {alpha}1 bands both in platelets and cardiomyocytes (Fig. 2A and B). However, as shown in Fig. 2, an additional band in the cytosolic fraction (of 110–115 kDa) and in the membrane fraction (of 85–90 kDa) of cardiomyocytes were also displaced. Since the identity of these other bands is unknown, they were not used for the quantification of the β1 content. Throughout the rest of the study only the antibody against the β1-subunit was used for blot analysis.


Figure 1
View larger version (73K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Presence of NO-sensitive guanylyl cyclase (GCNO) in the membrane fraction of cardiomyocytes (CM) and platelets (Plq), and estimation of its specific activity. Immunoblot analysis (panel A) of the β1-subunit in the cytosol and membrane fractions of 3 representative cardiomyocyte cultures. Panel B shows the immunoblot analysis of the {alpha}1 and β1 subunits of GCNO in cytosolic and particulate fractions obtained from 100 µg of cardiomyocytes or from 0.6–15 µg of platelets. An arrow points to the {alpha}1 band in cardiomyocytes to avoid confusion with the wider unspecific band located just above. Linearity of the densitometric measurements (panel C) was confirmed by plotting GCNO activity in each fraction versus the optical density of the β1 band for the different µg of sample ({square} cytosolic fraction, lhblk membrane fraction). Specific activity (panel C) was calculated by dividing GCNO activity by the densitometry of β1 and expressed in arbitrary units. *P<0.05 respect to the specific activity in the cytosolic fraction.

 

Figure 2
View larger version (47K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 β1 (panel A) and {alpha}1 (panel B) immunoreactivity of 65.1 kDa (for the β1 band) and 75.7 kDa (for the {alpha}1 band) were blocked by their respective immunization peptides in cytosolic and particulate fractions of platelets and cardiomyocytes. Other bands (110–115 kDa in the cytosolic fraction and 85–90 kDa in the particulate fraction) were also displaced by peptides. Membrane association of GCNO in cardiomyocytes was not altered by diluting the homogenate in a KCl containing buffer, but was completely disrupted by the presence of detergent (panel C).

 
Measurement of GCNO activity showed that 33 ± 4% of the activity was associated to the particulate fraction (5.1 ± 2.2 and 2.5 ± 1.1 pmol/mg protein x min in the cytosolic and membrane fractions, respectively, p = 6). For comparison with a cell model in which the association of GCNO to the membrane had been previously described, [15,21,22] cytosolic and membrane fractions were obtained from rat platelets (Fig. 1B). According to densitometric analysis of blots, platelets contained 100–150 times more GCNO than cardiomyocytes. Specific activity of membrane-associated GCNO, calculated by dividing GCNO activity by the densitometry of β1 in this fraction (in arbitrary units), was similar in platelets and cardiomyocytes (Fig. 1C).

To rule-out an unspecific presence of GCNO in the membrane fraction, cardiomyocyte homogenates were extensively diluted (15 times) in a homogeneization medium containing 150 mM of KCl instead of sucrose, and were incubated in agitation at 4 °C for 30 min before the 100,000 x g centrifugation. These manoeuvres had no effect on the observed membrane association of the β1 subunit (Fig. 2C). To examine the possible presence of GCNO in glycolipid-rich domains, part of the homogenates was incubated in a sucrose containing homogenization medium (standard medium) with 1% Triton X-100 (30 min at 4 °C in agitation). After this incubation, no significant immunostaining for the β1 antibody persisted in the particulate fraction (Fig. 2C).

3.2 Biochemical differences between cytosolic and particulate GCNO activity
Dependence of GCNO activity on GTP-, SNAP-, H+-and Ca2+-concentrations was analyzed for GCNO associated to the cytosolic and membrane fractions. Significant, although small, differences were found for the EC50 values for GTP (589 and 296 µM for the cytosolic and particulate activity, respectively; P<0.05; Fig. 3A and Table 1) and for the NO donor SNAP (160 and 55 µM, respectively; P<0.01; Fig. 3B and Table 1). In both cases, the concentration–response curves were biphasic, and the last value was excluded in the non-linear fitting. However, no differences were observed regarding the dependence on pH (Fig. 4A) or Ca2+ (Fig. 4B). GCNO activity was maximal at pH 7.4 for the particulate and cytosolic fractions (as previously described for the cytosolic enzyme), and the decrease in activity observed at basic or acidic pHs was identical for the two fractions. Ca2+ exerted a profound inhibitory effect in both cardiomyocyte fractions (no remanent GCNO activity was observed at 1 mM Ca2+), that was best suited to a two-component model (IC50 values calculated for the high and low affinity effects are shown in Table 1).


Figure 3
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 GCNO activity in the soluble ({circ}) and membrane (bullet) fractions of cardiomyocytes assayed at different concentrations of GTP (panel A) or SNAP (panel B). P values refer to the differences in the EC50. C, GCNO activity in the absence of GTP or SNAP for each fraction.

 

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

  		Table 1 Biochemical parameters of GCNO activity in soluble and particulate fractions

 

Figure 4
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Cytosolic ({circ}) and membrane (bullet) associated GCNO activity assayed at different pH values (panel A) or Ca2+ concentrations (panel B). C, GCNO activity when no Ca2+ was added to the assay medium for each fraction.

 
3.3 Effect of increasing cytosolic Ca2+ concentration on membrane-associated GCNO
Since Ca2+ has been proposed to be a regulating factor of the GCNO association to membrane in platelets [15], the effects of the ionophore A23187 [GenBank] (10 µM) and thapsigargin (0.1 µM) on this association were analyzed. Both drugs increased cytosolic Ca2+ concentration, although according to clearly distinct patterns (Fig. 5). Addition of A23187 [GenBank] induced a rapid, marked and transient increase in the intracellular Ca2+, while thapsigargin induced a small, but sustained increase. The effects of incubating cardiomyocytes with A23187 [GenBank] (for 1 min) or thapsigargin (for 5 min) on NO-dependent cell synthesis of cGMP, distribution of the β1 subunit between the particulate and the cytosolic fraction, and GCNO activity in both fractions, were analyzed. Thapsigargin did not modify cGMP synthesis induced by 0.1 mM SNAP (Fig. 6A) nor the proportion of β1 associated to membrane after homogeneization (Fig. 6B), but caused a significant decrease of GCNO activity in the cytosolic fraction (measured in the presence of Ca2+ chelating agents; 45 ± 11% of the activity in non-treated-cells, n = 6; P<0.05) without apparent changes in the activity associated to the particulate fraction (Fig. 6C).


Figure 5
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 5 Changes in the intracellular Ca2+ concentration after the addition of A23187 (10 µM, panel A) and thapsigargin (0.1 µM, panel B) to adult cardiomyocytes in culture. Representative recordings from three individual experiments (of a total n = 5) are shown for both drugs. Maximal fluorescence ratios reached after A23187 and thapsigargin treatments correspond to intracellular Ca2+ concentration of 0.4–2.5 µM and 200–350 nM, respectively.

 

Figure 6
View larger version (34K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 6 Effect of increasing intracellular Ca2+ concentration on NO-dependent cGMP synthesis (panel A), and on β1 immunoreactivity (panel B) and GCNO activity (panel C) measured in cytosolic and membrane fractions. After treating cardiomyocytes with no additions (Ct), thapsigargin (0.1 µM for 5 min) or A23187 (10 µM for 1 min), cGMP synthesis was measured after SNAP stimulation (panel A), or cultures were homogenized and cytosolic and membrane fractions assayed for β1 immunoreactivity (panel B) or GCNO activity (panel C). GCNO activity was expressed as percentage of total activity in controls (cytosolic plus membrane-associated GCNO). In panel B, a representative blot of n = 5 is shown. The amount of membrane-associated β1 in cardiomyocytes treated with A23187 increased by 133% in this experiment (mean of 161 ± 20%, respect to control, n = 5; P<0.05). *P<0.05 respect to control cardiomyocytes (for GCNO, respect to the control value in the same cell fraction).

 
On the other hand, in cardiomyocytes treated with A23187 [GenBank] , cGMP synthesis in response to SNAP was enhanced (152 ± 17%, n=6; P<0.05; Fig. 6A), a slight increase in β1 associated to the membrane was found (161 ± 20%, n = 5; P<0.05; Fig. 6B), and particulate GCNO activity was increased more than fourfold (P<0.05) while cytosolic GCNO activity did not increase significantly (Fig. 6C).

3.4 Role of changes in phosphorylation status as mediators of the effects of increased cytosolic Ca2+ concentration
In cells treated with A23187 [GenBank] , staurosporin at 1 µM (a concentration that inhibits Ca2+/calmodulin-dependent protein kinase or CaMK, myosin light chain kinase, PKC, PKA and PKG), potentiated the cGMP response to SNAP (165 ± 21% of the ionophore effect, n = 3; P<0.05; Fig. 7A), and induced an increase in both β1 immunostaining (138 ± 3%, n = 3; P<0.05; Fig. 7B) and GCNO activity (165 ± 21%, n = 3; P<0.05; Fig. 7C) in the particulate fraction, without significant effects on cytosolic GCNO activity. In cells not treated with A23187 [GenBank] , staurosporin did not significantly increase β1 immunostaining (results not shown) or GCNO activity in the membrane fraction (Fig. 7C). Gö-6976, an inhibitor of Ca2+-dependent PKC isozymes, at 1 µM had no significant effect on GCNO activation by A23187 [GenBank] on both cytosolic and membrane extracts (Table 2), and H-89, an inhibitor of PKA and PKG, at 10 µM inhibited the effect of A23187 [GenBank] on the cytosolic GCNO. Only KN-93, a selective CaMK inhibitor, at 30 µM had a potentiating effect on particulate GCNO activity similar to that of staurosporin (Table 2). On the other hand, the phosphatase inhibitors cypermethrin at 0.05 µM (selective inhibitor of calcineurin) or okadaic acid at 1 µM (inhibitor of PP1 and PP2A) did not block the potentiating effect of A23187 [GenBank] on membrane GCNO activity; in fact, okadaic acid enhanced it (290 ± 15% of the ionophore effect, n = 3; P<0.05).


Figure 7
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 7 Effect of staurosporin on the effect of A23187 on NO-dependent cGMP synthesis (panel A), and on β1 immunoreactivity (panel B) and GCNO activity (panel C) measured in cytosolic and membrane fractions. After treating cardiomyocytes without (Ct) or with staurosporin (Stau: 1 µM for 5 min) and then incubated in the absence (Ct) or presence of A23187 (A23: 10 µM for 1 min), cGMP synthesis was measured after SNAP stimulation (panel A), or cultures were homogenized and cytosolic and membrane fractions assayed for β1 immunoreactivity (panel B) or GCNO activity (panel C). GCNO activity was expressed as percentage of total activity in controls (cytosolic plus membrane-associated GCNO). The amount of β1 in the membrane fractions of cardiomyocytes treated with staurosporin plus A23187 (panel B) increased by 132% in this experiment respect to those cells receiving only A23187 (mean of 138 ± 3%, n = 3; P<0.05). *P<0.05 respect to control cardiomyocytes (same cell fraction, if applied). #P<0.05 respect to the effect of A23187.

 

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

  		Table 2 Effect of protein kinase inhibitors on A23187 potentiation of GCNO activity in soluble and particulate fractions

 
We were not able to detect in vivo 32P-labelling of GCNO in cardiomyocytes neither under control nor after staurosporin plus A23187 [GenBank] treatment. In platelets, incubation with the ionophore A23187 [GenBank] in the presence of staurosporin decreased phosphorylation of the β1 subunit compared to non-treated cells (to about 25% of the initial value; Fig. 8). Stimulation with A23187 [GenBank] in platelets preincubated with okadaic acid had a similar effect (Fig. 8).


Figure 8
View larger version (71K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 8 Activation with A23187 in the presence of either staurosporin or okadaic acid leads to dephosphorylation of the β1 subunit in intact platelets. Platelets were labelled with [32P]ortophosphate, stimulated with drugs, homogenized, and the β1 subunit immunoprecipitated from the cytosolic and membrane fractions obtained. 32P activity associated to the β1 subunit (panel A) or β1 immunoreactivity (panel B) of the immunoprecipitated platelet extracts are shown.

 
3.5 cGMP synthesis versus GCNO activity in cells fractions
The effects on cGMP synthesis of the different treatments assayed thorough the present manuscript were significantly correlated with their effects on membrane-associated GCNO activity, but not with the effects on GCNO activity in the cytosolic fraction (P<0.001; Fig. 9).


Figure 9
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 9 The effects of treatments on cGMP synthesis in intact cells were not significantly correlated with the effects on GCNO activity in the cytosolic fraction (panel A), but were closely correlated with the effects on GCNO in the membrane fraction (panel B). Data of cGMP synthesis shown in previous figures (Figs. 6 and 7Go, A panels) were plotted versus GCNO activity in particulate and cytosolic fractions of cardiomyocytes for each treatment (Figs. 6 and 7Go, C panels). The different measurements were expressed as percentage of their value in control cells.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This study provides the first direct evidence supporting the specific association of GCNO to the particulate fraction of cardiomyocytes. Membrane-associated GCNO activity showed similar concentration-dependence to GTP, NO donors, Ca2+ and pH than GCNO in the cytosolic location. As previously shown for the cytosolic GCNO, increasing Ca2+ concentration in the assay medium inhibited particulate GCNO. However, treating cardiomyocytes with the Ca2+ ionophore A23187 [GenBank] promoted translocation of GCNO to the membrane fraction, increased cGMP synthesis in response to stimulation of the cells with NO, and enhanced GCNO activity in this fraction when assayed in vitro. These effects were enhanced by staurosporin and the CaMK inhibitor KN-93. These results suggest that cytosolic Ca2+ concentration regulates the intracellular distribution of GCNO, and differentially regulates the activity associated to the membrane and the cytosolic fraction, probably through changes in its phosphorylation status. The observation that the effects of several treatments on NO-induced cGMP synthesis in cardiomyocytes closely correlate with their effects on GCNO activity in the particulate fraction, but not with their effects on cytosolic GCNO, suggests that membrane-associated GCNO largely determines NO-induced cGMP synthesis.

Although different data indicate that changes in GCNO activity greatly affect the function of the NO/cGMP pathway in several tissues [17,23–30], little information is available on the regulation of this enzyme. A previous study [15], demonstrated the presence of {alpha}1 and β1 subunits in the membrane fraction of rat myocardium, platelets and endothelial cells. This study described enzyme translocation to the membrane in activated platelets [15], and by means of in vitro experiments of GCNO association/dissociation to cell membranes suggested an important role of Ca2+. However, although GCNO in the particulate fraction of rat heart was responsive to NO, it was unclear whether GCNO associated to cell membrane was important in the response of the intact cell to NO.

We found that approximately 30% of the GCNO immunostaining for {alpha}1 and β1 and a similar proportion of GCNO activity were associated to the particulate fraction of cardiomyocytes. β1 association to membranes resisted extensive washing in a KCl buffer, that mimics the high intracellular potassium concentration. However, no apparent immunostaining persisted in the particulate fraction after washing in a Triton X-100 medium. This is similar to what was described by Zabel et al. [15] for the platelet particulate GCNO. Given the scarce immunostaining found in cardiomyocytes for GCNO subunits, we did not make any attempt to further analyze their subcellular location.

Membrane-associated GCNO and cytosolic GCNO showed similar biochemical characteristics. pH-dependence of GCNO was identical for the two locations and similar to that described previously [17,31]. Membrane GCNO had a significantly lower EC50 value for GTP than the cytosolic, but the difference was small and its physiological relevance is doubtful. The difference in sensitivity to the NO donor SNAP was more clear and similar to that described previously in heart extracts [15]. However, a very recent study [21] has suggested that contaminating myoglobin in cytosolic extracts from heart tissue neutralizes a significant part of the NO released by NO donors. According to this, our results may underestimate GCNO activity in the cytosol. Specific activity in cytosol could be thus higher than in the particulate fraction, as observed in platelets.

As previously described [32,33], in the present study Ca2+ inhibited cytosolic GCNO. We found that membrane-bound GCNO was also inhibited by Ca2+ and that in the two cell fractions GCNO showed a biphasic pattern very similar to that recently described in GCNO purified from bovine lung [34]. IC50 values calculated for the low affinity sites for Ca2+ were the same for cytosolic and particulate GCNO and comparable with those previously reported [34], and IC50 values for the high affinity sites of GCNO were also similar in both cell fractions. But, besides this inhibitory effect of Ca2+ when added to the assay medium (mediated by a direct binding of Ca2+ to GCNO), we observed in this study effects of increasing cytosolic Ca2+ in intact cells that had not been previously described. These effects are persistent, and can be detected after cell homogeneization in the presence of Ca2+ quelating agents. Importantly, the different agents used to increased cytosolic Ca2+ concentration have different effects, suggesting distinct roles for different levels of physiological concentrations of intracellular Ca2+ or for different subcellular location of these increases. The moderate and slow Ca2+ increase evoked by thapsigargin did not alter GCNO activity associated to the particulate fraction, and inhibited GCNO in the cytosolic fraction, while the more marked Ca2+ increase elicited by the Ca2+ ionophore A23187 [GenBank] increased several times GCNO activity in the particulate fraction without significant effects on cytosolic GCNO. In parallel with the increase in activity in the particulate fraction, A231287 increased the amount of β1 subunit associated to this fraction. This is similar to the translocation previously observed in activated platelets [15]. However, the change in quantity of β1 associated to the particulate fraction was much smaller than the change observed in GCNO activity. A critical point is that for the different conditions assayed, cytosolic and membrane-associated GCNO were found to respond differentially to increased cytosolic Ca2+.

A potential explanation for the increase in specific activity of membrane-associated GCNO induced by A23187 [GenBank] is that the increase in cytosolic Ca2+ concentration induced by the drug causes a modification in the phosphorylation status of the enzyme. Few studies have analyzed GCNO regulation by phosphorylation with conflicting results. Some studies suggested that GCNO phosphorylation increases its activity. Both in vitro phosphorylation by PKC and PKA [11] and in vivo phosphorylation by PKA [35] have been described to increase GCNO activity, while dephosphorylation of the β1 subunit has been associated to a decrease in GCNO activity [23]. A very recent study has described, in contrast, a decrease in GCNO activity associated to an increase in GCNO phosphorylation in response to PKG activation [12]. In the present study, the protein kinase inhibitors staurosporin and KN-93 activated membrane-associated GCNO sinergically with A23187 [GenBank] suggesting that in cardiomyocytes intracellular Ca2+ potentiates GCNO activity probably by promoting GCNO dephosphorylation. Although, direct evidence of GCNO dephosphorylation in response to A23187 [GenBank] could not be obtained, the evidence obtained in platelets supports this hypothesis. However, the fact that the protein phosphatase inhibitor okadaic acid also enhanced the response to A23187 [GenBank] suggest that regulation of GCNO activity by phosphorylation may be complex. A protein phosphatase activated by phosphorylation, as found in chromaffin cells [23], or an additional regulatory site (in the {alpha}1 subunit or in some of regulatory proteins recently described: as Hsp90 [16], Hsp70 [36], or CCT{eta} [37]) that would increase GCNO activity after phosphorylation could explain the results. To sum up, our observations suggest that the increase in cytosolic Ca2+ induced by A23187 [GenBank] has two opposite effects on NO-mediated cGMP synthesis: a direct inhibitory effect and an indirect stimulatory effect mediated by β1 dephosphorylation resulting in membrane-associated GCNO activation.

The present study provides information that strongly suggests an important functional role of GCNO localized in the membrane fraction of cardiomyocytes. Our results show that a profound inhibition of cytosolic GCNO do not significantly affect the cell response to SNAP, while activation of the particulate fraction markedly increases it. As shown in Fig. 9, cGMP synthesis in the entire cell correlates well with changes in membrane GCNO activity, but not with changes in cytosolic GCNO activity. This is in agreement with recent results in bovine aortic endothelial cells, indicating a decrease cell-response to NO stimulation when the formation of a membrane-associated protein complex between eNOS, HSP90 and GCNO is inhibited [16].

The association of a fraction of GCNO to cell membrane in cardiomyocytes, the important role of membrane-associated GCNO on the cell response to NO, and the fact that the regulation of its activity differs from that of the cytosolic enzyme, may be of great relevance for the better understanding of pathophysiological conditions in which the NO/cGMP-pathway is compromised, and in the design of new therapies for these conditions.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
Supported by Fondo Investigaciones Sanitarias (Grant 01/3008 and RECAVA), and Comisión Interministerial de Ciencia y Tecnología (Grant SAF2002/0759). We thank Angeles Rojas for her excellent technical work.


   Notes
 
* Ajay Shah (King's College, London) served as Guest Editor for this manuscript

Time for primary review 19 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

  1. Casadei B., Sears C.E. Nitric-oxide-mediated regulation of cardiac contractility and stretch responses. Prog Biophys Mol Biol (2003) 82:67–80.[CrossRef][ISI][Medline]
  2. Padilla F., Garcia-Dorado D., Agulló L., Barrabés J.A., Inserte J., Escalona N., et al. Intravenous administration of the natriuretic peptide urodilatin at low doses during coronary reperfusion limits infarct size in anesthetized pigs. Cardiovasc Res (2001) 51:592–600.[Abstract/Free Full Text]
  3. D'Souza S.P., Yellon D.M., Martin C., Schulz R., Heusch G., Onody A., et al. B-type natriuretic peptide limits infarct size in rat isolated hearts via KATP channel opening. Am J Physiol Heart Circ Physiol (2003) 284:1592–1600.
  4. Ockaili R., Salloum F., Hawkins J., Kukreja R.C. Sildenafil (Viagra) induces powerful cardioprotective effect via opening of mitochondrial KATP channels in rabbits. Am J Physiol Heart Circ Physiol (2002) 283:1263–1269.
  5. Ahluwalia A., Foster P., Scotland R.S., McLean P.G., Mathur A., Perretti M., et al. Antiinflammatory activity of soluble guanylate cyclase: cGMP-dependent down-regulation of P-selectin expression and leukocyte recruitment. Proc Natl Acad Sci U S A (2004) 101:1386–1391.[Abstract/Free Full Text]
  6. Izumi T., Saito Y., Kishimoto I., Harada M., Kuwahara K., Hamanaka I., et al. Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-{kappa}B activation and alleviates myocardial ischemia/reperfusion injury. J Clin Invest (2001) 108:203–213.[CrossRef][ISI][Medline]
  7. Hempel A.M., Friedrich M., Schlüter K.D., Forssmann W.G., Kuhn M., Piper H.M. ANP protects against reoxygenation-induced hypercontracture in adult cardiomyocytes. Am J Physiol (1997) 273:244–249.
  8. Kodani E., Xuan Y.T., Takano H., Shinmura K., Tang X.L., Bolli R. Role of cyclic guanosine monophosphate in late preconditioning in conscious rabbits. Circulation (2002) 105:3046–3052.[Abstract/Free Full Text]
  9. Bellamy T.C., Garthwaite J. Sub-second kinetics of the nitric oxide receptor, soluble guanylyl cyclase, in intact cerebellar cells. J Biol Chem (2001) 276:4287–4292.[Abstract/Free Full Text]
  10. Kostic T.S., Tomic M., Andric S.A., Stojilkovic S.S. Calcium-independent and cAMP-dependent modulation of soluble guanylyl cyclase activity by G protein-coupled receptors in pituitary cells. J Biol Chem (2002) 277:16412–16418.[Abstract/Free Full Text]
  11. Zwiller J., Revel M.O., Malviya A.N. Protein kinase C catalyzes phosphorylation of guanylate cyclase in vitro. J Biol Chem (1985) 260:1350–1353.[Abstract/Free Full Text]
  12. Murthy K.S. Modulation of soluble guanylate cyclase activity by phosphorylation. Neurochem Int (2004) 45:845–851.[CrossRef][ISI][Medline]
  13. Russwurm M., Wittau N., Koesling D. Guanylyl cyclase/PSD-95 interaction. Targeting of the nitric oxide-sensitive {alpha}2β1 guanylyl cyclase to synaptic membranes. J Biol Chem (2001) 276:44647–44652.[Abstract/Free Full Text]
  14. Feussner M., Richter H., Baum O., Gossrau R. Association of soluble guanylate cyclase with the sarcolemma of mammalian skeletal muscle fibers. Acta Histochem (2001) 103:265–277.[CrossRef][ISI][Medline]
  15. Zabel U., Kleinschnitz C., Oh P., Nedvetsky P., Smolenski A., Müller H., et al. Calcium-dependent membrane association sensitizes soluble guanylyl cyclase to nitric oxide. Nat Cell Biol (2002) 4:307–311.[CrossRef][ISI][Medline]
  16. Venema R.C., Venema V.J., Ju H., Harris M.B., Snead C., Jilling T., et al. Novel complexes of guanylate cyclase with heat shock protein 90 and nitric oxide synthase. Am J Physiol Heart Circ Physiol (2003) 285:669–678.
  17. Agulló L., Garcia-Dorado D., Escalona N., Ruiz-Meana M., Inserte J., Soler-Soler J. Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes. Am J Physiol Heart Circ Physiol (2003) 284:2170–2176.
  18. Massaguer A., Engel P., Perez-del-Pulgar S., Bosch J., Pizcueta P. Production and characterization of monoclonal antibodies against conserved epitopes of P-selectin. Tissue Antigens (2000) 56:117–128.[CrossRef][ISI][Medline]
  19. García-Cardeña G., Oh P., Liu J., Schnitzer J.E., Sessa W.C. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci U S A (1996) 93:6448–6453.[Abstract/Free Full Text]
  20. Ruiz-Meana M., Garcia-Dorado D., Lane S., Pina P., Inserte J., Mirabet M., et al. Persistence of gap junction communication during myocardial ischemia. Am J Physiol Heart Circ Physiol (2001) 280:2563–2571.
  21. Wykes V., Garthwaite J. Membrane-association and the sensitivity of guanylyl cyclase-coupled receptors to nitric oxide. Br J Pharmacol (2004) 141:1087–1090.[CrossRef][ISI][Medline]
  22. Kempfert J., Behrends. Analysis of nitric oxide-sensitive guanylyl cyclase in human platelets before and after aggregation. Platelets (2003) 14:429–435.[CrossRef][ISI][Medline]
  23. Ferrero R., Rodríguez-Pascual F., Miras-Portugal M.T., Torres M. Nitric oxide-sensitive guanylyl cyclase activity inhibition through cyclic GMP-dependent dephosphorylation. J Neurochem (2000) 75:2029–2039.[CrossRef][ISI][Medline]
  24. Baltrons M.A., Saadoun S., Agulló L., García A. Regulation by calcium of the nitric oxide/cyclic GMP system in cerebellar granule cells and astroglia in culture. J Neurosci Res (1997) 49:333–341.[CrossRef][ISI][Medline]
  25. Agulló L., Garcia-Dorado D., Escalona N., Inserte J., Ruiz-Meana M., Barrabés J.A., et al. Hypoxia and acidosis impair cGMP synthesis in microvascular coronary endothelial cells. Am J Physiol Heart Circ Physiol (2002) 283:917–925.
  26. Papapetropoulos A., Abou-Mohamed G., Marczin N., Murad F., Caldwell R.W., Catravas J.D. Downregulation of nitrovasodilator-induced cyclic GMP accumulation in cells exposed to endotoxin or interleukin-1 beta. Br J Pharmacol (1996) 118:1359–1366.[ISI][Medline]
  27. Klöss S., Bouloumié A., Mülsch A. Aging and chronic hypertension decrease expression of rat aortic soluble guanylyl cyclase. Hypertension (2000) 35:43–47.[Abstract/Free Full Text]
  28. Laber U., Kober T., Schmitz V., Schrammel A., Meyer W., Mayer B., et al. Effect of hypercholesterolemia on expression and function of vascular soluble guanylyl cyclase. Circulation (2002) 105:855–860.[Abstract/Free Full Text]
  29. Mülsch A., Oelze M., Klöss S., Mollnau H., Töpfer A., Smolenski A., et al. Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta. Circulation (2001) 103:2188–2194.[Abstract/Free Full Text]
  30. Sinnaeve P., Chiche J.D., Nong Z., Varenne O., Van Pelt N., Gillijns H., et al. Soluble guanylate cyclase {alpha}1 and β1 gene transfer increases NO responsiveness and reduces neointima formation after balloon injury in rats via antiproliferative and antimigratory effects. Circ Res (2001) 88:103–109.[Abstract/Free Full Text]
  31. Grabow M., Chakraborty G., Ledeen R.W. Characterization of guanylyl cyclase in purified myelin. Neurochem Res (1996) 21:457–462.[ISI][Medline]
  32. Parkinson S.J., Jovanovic A., Jovanovic S., Wagner F., Terzic A., Waldman S.A. Regulation of nitric oxide-responsive recombinant soluble guanylyl cyclase by calcium. Biochemistry (1999) 38:6441–6448.[CrossRef][ISI][Medline]
  33. Serfass L., Carr H.S., Aschenbrenner L.M., Burstyn J.N. Calcium ion downregulates soluble guanylyl cyclase activity: evidence for a two-metal ion catalytic mechanism. Arch Biochem Biophys (2001) 387:47–56.[CrossRef][ISI][Medline]
  34. Kazerounian S., Pitari G.M., Ruiz-Stewart I., Schulz S., Waldman S.A. Nitric oxide activation of soluble guanylyl cyclase reveals high and low affinity sites that mediate allosteric inhibition by calcium. Biochemistry (2002) 41:3396–3404.[CrossRef][ISI][Medline]
  35. Kostic T.S., Andric S.A., Stojilkovic S.S. Receptor-controlled phosphorylation of {alpha}1 soluble guanylyl cyclase enhances nitric oxide-dependent cGMP production in pituitary cells. Mol Endocrinol (2004) 18:458–470.[Abstract/Free Full Text]
  36. Balashova N., Chang F.J., Lamothe M., Sun Q., Beuve A. Characterization of a novel type of endogenous activator of soluble guanylyl cyclase. J Biol Chem (2005) 280:2186–2196.[Abstract/Free Full Text]
  37. Hanafy K.A., Martin E., Murad F. CCTeta, a novel soluble guanylyl cyclase-interacting protein. J Biol Chem (2004) 279:46946–46953.[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
K. S. Murthy
Inhibitory Phosphorylation of Soluble Guanylyl Cyclase by Muscarinic m2 Receptors via G{beta}{gamma}-Dependent Activation of c-Src Kinase
J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 183 - 189.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
D. Garcia-Dorado, A. Rodriguez-Sinovas, M. Ruiz-Meana, J. Inserte, L. Agullo, and A. Cabestrero
The end-effectors of preconditioning protection against myocardial cell death secondary to ischemia-reperfusion
Cardiovasc Res, May 1, 2006; 70(2): 274 - 285.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Agulló, L.
Right arrow Articles by Soler-Soler, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Agulló, L.
Right arrow Articles by Soler-Soler, J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?