Membrane association of nitric oxide-sensitive guanylyl cyclase in cardiomyocytes B

Objective: Although the importance of the cyclic GMP (cGMP) signaling pathway in cardiac myocytes is well established, little is known about its regulation. Ca 2+ -dependent translocation of nitric oxide (NO) sensitive guanylyl cyclase (GC NO ) 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 GC NO bound to the cardiomyocyte membrane. Methods: Cytosolic and particulate fractions of adult rat cardiomyocytes were isolated and blotted, and their GC NO activity was assayed in parallel experiments. Results: In untreated cardiomyocytes, approximately 30% of h 1 -and a 1 -subunits of GC NO and a similar proportion of GC NO activity were found in the particulate fraction. The dependence of GC NO activity on pH, Ca 2+ , GTP and NO donor concentrations was similar in particulate and cytosolic fractions. Treatment of cardiomyocytes with the ionophore A23187 caused GC NO to translocate to the sarcolemma, increased GC NO activity in this fraction, and potentiated NO-mediated cGMP synthesis. These effects appeared to be mediated by Ca 2+ -dependent changes on the phosphorylation status of GC NO , since they were enhanced by the non-selective inhibitor staurosporine and by the selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase KN-93. The effect of drugs increasing intracellular Ca 2+ on cGMP synthesis was clearly correlated with their effects on membrane-associated GC NO activity but not with their effects on cytosol-associated GC NO . Conclusion: These results are the first evidence that 1) GC NO is associated with the cell membrane in cardiomyocytes, 2) the regulation of membrane-associated GC NO differs from that of cytosolic GC NO , and 3) membrane association may have a crucial role in determining the response of cells to NO. D 2005 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.


Introduction
Cyclic GMP (cGMP) modulates important physiological functions in the cardiovascular system as vasodilation, Ca 2+ cycling, endothelium permeability, or myocardial contrac-tility [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 (GC NO ), 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. GC NO is constituted of two subunits, a and h, and two different isoforms of the a subunit (a 1 and a 2 ) and of the h subunit (h 1 and h 2 ) have been described. The a 1 h 1 heterodimer is predominantly found in the cardiovascular system, while a 2 h 1 has been mainly found in brain.
Little is known about how GC NO 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 GC NO . The isoform a 2 h 1 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, a 1 h 1 , in the sarcolemmal region of skeletal muscle fibres [14]. This association to the particulate fraction of a 1 and h 1 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 a 1 h 1 with the membrane was unresolved. A recent study has described a protein complex formed by eNOS, Hsp90 and GC NO in endothelial cells, and that bradykinin and vascular endothelial growth factor potentiate the formation of this complex [16]. The contribution of particulate GC NO to the cell response to NO remained unknown.
In this study, we analyze how association of GC NO to membrane affects its biochemical properties in cardiomyocytes, the functional relevance of this association, and its potential regulation by Ca 2+ .

Materials and methods
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.

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].

Intracellular cGMP synthesis
After treatment, cells were stimulated for 1 minute with SNAP 100 AM (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 [ 3 H]cGMP [17].

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 Â10 À 3 , and the Ser/Thr protein phosphatase inhibitors okadaic acid 1 Â10 À 3 and cypermethrin 5 Â 10 À 4 ]. After clearing the homogenates by centrifugation at 1000 Âg for 15 min, the particulate and cytosolic fractions were obtained by centrifugation (100,000 Âg for 1 h). Membrane fractions were homogenized with buffer A plus 10% glycerol. Triton X-100 solubility of particulate GC NO 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.

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 h 1 (aminoacids 605 -619; used at 1 / 2000 dilution; Sigma) and a 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.

Modulation of the membrane association of GC NO
The effect of increased cytosolic Ca 2+ concentration on the association of GC NO to the membrane fraction was investigated by incubating cells 1 min with A23187 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 h 1 subunit and GC NO activity in cytosolic and membrane fractions.
The potential role of changes in GC NO 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, and maintained during the time of incubation with the ionophore.

Detection of GC NO phosphorylation in intact cells
Cardiomyocytes or platelets were incubated for 3 h in medium containing 0.1 mCi/ml [ 32 P]ortophosphate (Amersham), washed with Fcold_ medium, incubated for 5 min with or without 1 AM staurosporin and stimulated for 1 min with 10 AM A23187 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 Âg, and the h 1 subunit of the cytosolic and particulate fractions immunoprecipitated by incubation with Protein G-agarose beads (Amersham Biosciences) previously bound to 15 Ag of anti-h 1 antibody. Phosphorylation of the h 1 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 a 1 phosphorylation was not possible since the antibody used against this subunit did not significantly immunoprecipitate the protein.

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

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 T SEM. Nonlinear fitting was performed using SigmaPlot (SPSS Inc.).

Membrane association of GC NO in cardiomyocytes and platelets
Cytosolic and particulate fractions (100,000 Âg) of adult rat cardiomyocytes were isolated and blotted. Staining with an antibody against the h 1 subunit of the GC NO , demonstrated that 28 T 5% of h 1 (65.1 T 0.2 kDa), n = 6, was in the membrane fraction (Fig. 1A). A similar proportion of the a 1 subunit (75.7 T 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 h 1 and a 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 h 1 content. Throughout the rest of the study only the antibody against the h 1subunit was used for blot analysis.
Measurement of GC NO activity showed that 33 T 4% of the activity was associated to the particulate fraction (5.1 T 2.2 and 2.5 T 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 GC NO 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 GC NO than cardiomyocytes. Specific activity of membrane-associated GC NO , calculated by dividing GC NO activity by the densitometry of h 1 in this fraction (in arbitrary units), was similar in platelets and cardiomyocytes (Fig. 1C).
To rule-out an unspecific presence of GC NO in the membrane fraction, cardiomyocyte homogenates were extensively diluted ( medium containing 150 mM of KCl instead of sucrose, and were incubated in agitation at 4 -C for 30 min before the 100,000 Âg centrifugation. These manoeuvres had no effect on the observed membrane association of the h 1 subunit (Fig. 2C). To examine the possible presence of GC NO 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 immunos-taining for the h 1 antibody persisted in the particulate fraction (Fig. 2C).

Biochemical differences between cytosolic and particulate GC NO activity
Dependence of GC NO activity on GTP-, SNAP-, H + -and Ca 2+ -concentrations was analyzed for GC NO associated to the cytosolic and membrane fractions. Significant, although  small, differences were found for the EC 50 values for GTP (589 and 296 AM for the cytosolic and particulate activity, respectively; P < 0.05; Fig. 3A and Table 1) and for the NO donor SNAP (160 and 55 AM, 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 Ca 2+ (Fig.  4B). GC NO 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. Ca 2+ exerted a profound inhibitory effect in both cardiomyocyte fractions (no remanent GC NO activity was observed at 1 mM Ca 2+ ), that was best suited to a twocomponent model (IC 50 values calculated for the high and low affinity effects are shown in Table 1).

Effect of increasing cytosolic Ca 2+ concentration on membrane-associated GC NO
Since Ca 2+ has been proposed to be a regulating factor of the GC NO association to membrane in platelets [15], the effects of the ionophore A23187 (10 AM) and thapsigargin (0.1 AM) on this association were analyzed. Both drugs increased cytosolic Ca 2+ concentration, although according to clearly distinct patterns (Fig. 5). Addition of A23187 induced a rapid, marked and transient increase in the intracellular Ca 2+ , while thapsigargin induced a small, but sustained increase. The effects of incubating cardiomyocytes with A23187 (for 1 min) or thapsigargin (for 5 min) on NO-dependent cell synthesis of cGMP, distribution of the h 1 subunit between the particulate and the cytosolic fraction, and GC NO activity in both fractions, were analyzed. Thapsigargin did not modify cGMP synthesis induced by 0.1 mM SNAP (Fig. 6A) Fig. 6. Effect of increasing intracellular Ca 2+ concentration on NOdependent cGMP synthesis (panel A), and on h 1 immunoreactivity (panel B) and GC NO activity (panel C) measured in cytosolic and membrane fractions. After treating cardiomyocytes with no additions (Ct), thapsigargin (0.1 AM for 5 min) or A23187 (10 AM for 1 min), cGMP synthesis was measured after SNAP stimulation (panel A), or cultures were homogenized and cytosolic and membrane fractions assayed for h 1 immunoreactivity (panel B) or GC NO activity (panel C). GC NO activity was expressed as percentage of total activity in controls (cytosolic plus membrane-associated GC NO ). In panel B, a representative blot of n = 5 is shown. The amount of membrane-associated h 1 in cardiomyocytes treated with A23187 increased by 133% in this experiment (mean of 161 T 20%, respect to control, n = 5; P < 0.05). *P < 0.05 respect to control cardiomyocytes (for GC NO , respect to the control value in the same cell fraction). to membrane after homogeneization (Fig. 6B), but caused a significant decrease of GC NO activity in the cytosolic fraction (measured in the presence of Ca 2+ chelating agents; 45 T 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).
On the other hand, in cardiomyocytes treated with A23187, cGMP synthesis in response to SNAP was enhanced (152 T 17%, n = 6; P < 0.05; Fig. 6A), a slight increase in h 1 associated to the membrane was found (161 T 20%, n = 5; P < 0.05; Fig. 6B), and particulate GC NO activity was increased more than fourfold ( P < 0.05) while cytosolic GC NO activity did not increase significantly (Fig. 6C).

Role of changes in phosphorylation status as mediators of the effects of increased cytosolic Ca 2+ concentration
In cells treated with A23187, staurosporin at 1 AM (a concentration that inhibits Ca 2+ /calmodulin-dependent protein kinase or CaMK, myosin light chain kinase, PKC, PKA and PKG), potentiated the cGMP response to SNAP (165 T 21% of the ionophore effect, n = 3; P < 0.05; Fig. 7A), and induced an increase in both h 1 immunostaining (138 T 3%, n = 3; P < 0.05; Fig. 7B) and GC NO activity (165 T 21%, n = 3; P < 0.05; Fig. 7C) in the particulate fraction, without significant effects on cytosolic GC NO activity. In cells not treated with A23187, staurosporin did not significantly increase h 1 immunostaining (results not shown) or GC NO activity in the membrane fraction (Fig. 7C). Gö -6976, an inhibitor of Ca 2+ -dependent PKC isozymes, at 1 AM had no significant effect on GC NO activation by A23187 on both cytosolic and membrane extracts (Table 2) After treating cardiomyocytes without (Ct) or with staurosporin (Stau: 1 AM for 5 min) and then incubated in the absence (Ct) or presence of A23187 (A23: 10 AM for 1 min), cGMP synthesis was measured after SNAP stimulation (panel A), or cultures were homogenized and cytosolic and membrane fractions assayed for h 1 immunoreactivity (panel B) or GC NO activity (panel C). GC NO activity was expressed as percentage of total activity in controls (cytosolic plus membrane-associated GC NO ). The amount of h 1 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 T 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. Table 2 Effect of protein kinase inhibitors on A23187 potentiation of GC NO   effect on particulate GC NO activity similar to that of staurosporin (Table 2). On the other hand, the phosphatase inhibitors cypermethrin at 0.05 AM (selective inhibitor of calcineurin) or okadaic acid at 1 AM (inhibitor of PP1 and PP2A) did not block the potentiating effect of A23187 on membrane GC NO activity; in fact, okadaic acid enhanced it (290 T 15% of the ionophore effect, n = 3; P < 0.05). We were not able to detect in vivo 32 P-labelling of GC NO in cardiomyocytes neither under control nor after staurosporin plus A23187 treatment. In platelets, incubation with the ionophore A23187 in the presence of staurosporin decreased phosphorylation of the h 1 subunit compared to non-treated cells (to about 25% of the initial value; Fig. 8). Stimulation with A23187 in platelets preincubated with okadaic acid had a similar effect (Fig. 8).

cGMP synthesis versus GC NO 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 GC NO activity, but not with the effects on GC NO activity in the cytosolic fraction ( P < 0.001; Fig. 9).

Discussion
This study provides the first direct evidence supporting the specific association of GC NO to the particulate fraction of cardiomyocytes. Membrane-associated GC NO activity showed similar concentration-dependence to GTP, NO donors, Ca 2+ and pH than GC NO in the cytosolic location. As previously shown for the cytosolic GC NO , increasing Ca 2+ concentration in the assay medium inhibited particulate GC NO . However, treating cardiomyocytes with the Ca 2+ ionophore A23187 promoted translocation of GC NO to the membrane fraction, increased cGMP synthesis in response to stimulation of the cells with NO, and enhanced GC NO 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 Ca 2+ concentration regulates the intracellular distribution of GC NO , 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 GC NO activity in the particulate fraction, but not with their effects on cytosolic GC NO , suggests that membrane-associated GC NO largely determines NO-induced cGMP synthesis.
Although different data indicate that changes in GC NO 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 a 1 and h 1 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 GC NO association/dissociation to cell membranes suggested an important role of Ca 2+ . However, although GC NO in the particulate fraction of rat heart was responsive to NO, it was unclear whether GC NO associated to cell membrane was important in the response of the intact cell to NO.
We found that approximately 30% of the GC NO immunostaining for a 1 and h 1 and a similar proportion of GC NO activity were associated to the particulate fraction of cardiomyocytes. h 1 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 GC NO . Given the scarce immunostaining found in cardiomyocytes for GC NO subunits, we did not make any attempt to further analyze their subcellular location.
Membrane-associated GC NO and cytosolic GC NO showed similar biochemical characteristics. pH-dependence of GC NO was identical for the two locations and similar to that described previously [17,31]. Membrane GC NO had a significantly lower EC 50 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 GC NO 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 Ca 2+ inhibited cytosolic GC NO . We found that membranebound GC NO was also inhibited by Ca 2+ and that in the two cell fractions GC NO showed a biphasic pattern very similar to that recently described in GC NO purified from bovine lung [34]. IC 50 values calculated for the low affinity sites for Ca 2+ were the same for cytosolic and particulate GC NO and comparable with those previously reported [34], and IC 50 values for the high affinity sites of GC NO were also similar in both cell fractions. But, besides this inhibitory effect of Ca 2+ when added to the assay medium (mediated by a direct binding of Ca 2+ to GC NO ), we observed in this study effects of increasing cytosolic Ca 2+ in intact cells that had not been previously described. These effects are persistent, and can be detected after cell homogeneization in the presence of Ca 2+ quelating agents. Importantly, the different agents used to increased cytosolic Ca 2+ concentration have different effects, suggesting distinct roles for different levels of physiological concentrations of intracellular Ca 2+ or for different subcellular location of these increases. The moderate and slow Ca 2+ increase evoked by thapsigargin did not alter GC NO activity associated to the particulate fraction, and inhibited GC NO in the cytosolic fraction, while the more marked Ca 2+ increase elicited by the Ca 2+ ionophore A23187 increased several times GC NO activity in the particulate fraction without significant effects on cytosolic GC NO . In parallel with the increase in activity in the particulate fraction, A231287 increased the amount of h 1 subunit associated to this fraction. This is similar to the translocation previously observed in activated platelets [15]. However, the change in quantity of h 1 associated to the particulate fraction was much smaller than the change observed in GC NO activity. A critical point is that for the different conditions assayed, cytosolic and membraneassociated GC NO were found to respond differentially to increased cytosolic Ca 2+ .
A potential explanation for the increase in specific activity of membrane-associated GC NO induced by A23187 is that the increase in cytosolic Ca 2+ concentration induced by the drug causes a modification in the phosphorylation status of the enzyme. Few studies have analyzed GC NO regulation by phosphorylation with conflicting results. Some studies suggested that GC NO 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 GC NO activity, while dephosphorylation of the h 1 subunit has been associated to a decrease in GC NO activity [23]. A very recent study has described, in contrast, a decrease in GC NO activity associated to an increase in GC NO phosphorylation in response to PKG activation [12]. In the present study, the protein kinase inhibitors staurosporin and KN-93 activated membrane-associated GC NO sinergically with A23187 suggesting that in cardiomyocytes intracellular Ca 2+ potentiates GC NO activity probably by promoting GC NO dephosphorylation. Although, direct evidence of GC NO dephosphorylation in response to A23187 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 suggest that regulation of GC NO 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 a 1 subunit or in some of regulatory proteins recently described: as Hsp90 [16], Hsp70 [36], or CCTD [37]) that would increase GC NO activity after phosphorylation could explain the results. To sum up, our observations suggest that the increase in cytosolic Ca 2+ induced by A23187 has two opposite effects on NO-mediated cGMP synthesis: a direct inhibitory effect and an indirect stimulatory effect mediated by h 1 dephosphorylation resulting in membrane-associated GC NO activation.
The present study provides information that strongly suggests an important functional role of GC NO localized in the membrane fraction of cardiomyocytes. Our results show that a profound inhibition of cytosolic GC NO 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 GC NO activity, but not with changes in cytosolic GC NO 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 GC NO is inhibited [16].
The association of a fraction of GC NO to cell membrane in cardiomyocytes, the important role of membrane-associated GC NO 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.