INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE INTRACELLULAR MESSENGER cGMP mediates many of the most important actions of nitric oxide (NO) and atrial natriuretic peptide (ANP). NO activates cGMP synthesis by binding to a prosthetic heme group of soluble guanylyl cyclase (sGC), whereas ANP and ANP-related peptides stimulate particulate, membrane-bound guanylyl cyclases (pGCs).

cGMP has been shown to reduce vascular permeability (8, 15), cell attachment to the endothelial wall (7, 21), and myocardial reperfusion injury (2, 11). Recently, it has also been implicated in late preconditioning (14). Despite the evidence that cGMP may influence cell survival during transient myocardial ischemia, little is known about the consequences of ischemia on cGMP synthesis in myocardial cells. We recently described a marked reduction of cGMP content in rat and pig myocardium after transient sublethal ischemia (11, 25). However, other authors reported increases (3, 18) or no change (20) in cGMP content in rat hearts subjected to up to 30 min of ischemia. In cultured endothelial cells from rat hearts, simulated ischemia markedly inhibited sGC- and pGC-dependent cGMP synthesis (1). Depletion of ATP and a decrease in intracellular pH were responsible for the inhibitory effect (1). To our knowledge, the effects of ischemia on cGMP synthesis in isolated cardiomyocytes have been described in only one report (6). In this study, it was found that hypoxia potentiates NO-mediated cGMP synthesis. Neither the effects of low PO₂ on pGC-mediated cGMP synthesis nor the influence of intracellular acidosis on sGC- and pGC-dependent cGMP synthesis has been previously reported.

The aim of this study was to characterize the effects of ischemia-reperfusion on the two pathways that regulate intracellular cGMP concentration in adult rat cardiomyocytes. Changes in cGMP synthesis mediated by sGC and pGC were investigated by analyzing the response of freshly isolated adult cardiomyocytes to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) and the natriuretic factors urodilatin (Uro), ANP, and C-type natriuretic peptide (CNP). Uro is an ANP-related peptide that has been shown to reduce cardiomyocyte cell death secondary to transient ischemia in a variety of models, including the in situ pig heart (11, 25). In addition, the contributions of ATP depletion and acidosis to the inhibitory effect of ischemia on cGMP synthesis were investigated.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The care and use of animals conformed with the guidelines of the National Institutes of Health [DHHS Publication No. (NIH) 85-23, Revised 1996], and the experimental procedures were approved by the Research Commission on Ethics of Hospital Vall d'Hebron.

Cardiomyocyte isolation. Cardiomyocytes from the heart were obtained as previously described (2). Hearts from adult male Sprague-Dawley rats (300 g) were retrogradely perfused in a Langendorff system with a modified Krebs-Henseleit bicarbonate buffer (in mM: 110 NaCl, 2.6 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, and 11 glucose, pH 7.4) containing 30 µM CaCl₂ and 0.03% collagenase. After centrifugation (25 g for 3 min), cells from the pellet were subjected to a progressive normalization of Ca²⁺ levels (up to 1 mM). Rod-shaped cells were selected by gravity sedimentation in 4% albumin gradient and plated in culture dishes with medium 199-HEPES plus 4% fetal bovine serum. At the end of the procedure, culture dishes contained >85% quiescent rod-shaped cells.

Simulated ischemia and reperfusion. Cells were allocated to one of the following treatments. In normoxia, cells were incubated for 2 h in HEPES-buffered saline (in mM: 120 NaCl, 3.6 KCl, 1.2 MgSO₄, 1 CaCl₂, 20 HEPES, and 5 glucose) at pH 7.4. In simulated ischemia, cells were incubated under a 100% N₂ atmosphere in the same buffer but without glucose and at pH 6.4. The relative contributions of O₂ deprivation and acidosis to the observed effects of simulated ischemia were studied in cells exposed for 2 h to hypoxia at pH 7.4 or acidosis (pH 6.4) under normoxic conditions. The time course of recovery of cGMP synthesis after restoration of normoxia and pH 7.4 was analyzed in cultures exposed for 2 h to the different treatments. Previous studies showed that the system used to generate hypoxia reduced PO₂ in hypoxic medium to 6.5 ± 1.3 mmHg after 30 min of incubation and to 3.6 ± 1.0 mmHg after 2 h. In additional experiments, hypoxia was replaced by metabolic inhibition with 30 µM dinitrophenol (DNP) over a 40-min period.

Measurement of cGMP synthesis. After different periods of exposure to the allocated treatment, cells were stimulated for 1 min (unless otherwise indicated) with 100 µM SNAP, 1 µM Uro, 1 µM ANP, 1 µM CNP, or no drug. cGMP degradation was inhibited by addition of 1 mM 3-isobutyl-1-methylxanthine (IBMX) during the stimulation period. cGMP was quantified in cell extracts by radioimmunoassay using acetylated [³H]cGMP (2). In some experiments, a fraction of the incubation medium was collected at the end of the stimulation period to quantify the efflux of cGMP into the extracellular medium. cGMP synthesis produced by stimulation with SNAP or Uro in the different incubation conditions was expressed as a percentage of the cGMP produced after stimulation of normoxic cells (at pH 7.4) with the same drug (control plates of each batch). Before the percentage was calculated, cGMP content in nonstimulated cells (subjected to the same conditions) was subtracted from each cGMP value in the stimulated cultures. Nonstimulated cGMP was always <5% of the stimulated cGMP content.

Lactate dehydrogenase release, ATP content, and intracellular pH. Lactate dehydrogenase (LDH) activity was spectrophotometrically measured in the incubation media (2) and expressed as percentage of the total LDH content determined in the cultures after homogenization in Tris · HCl buffer. ATP content was measured in cell cultures immediately frozen in liquid nitrogen by means of the Bioluminescent Somatic Cell Assay Kit (1). Changes in intracellular pH were analyzed using a ratio-fluorescence imaging system (QuantiCell 2000, Visitech), as previously described (1). After cardiomyocytes were loaded with 1 µM 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) acetoxymethyl ester, sequential 450 nm-to-490 nm ratio images were obtained during the 120 min of exposure to the different treatments. Calibration of the BCECF ratio signal was performed in the presence of nigericin (10 µg/ml). All data on ATP content and intracellular pH were obtained in nonstimulated cells. In additional experiments, stimulation of cGMP synthesis with Uro or SNAP for 2 min did not modify ATP or pH values (data not shown).

Measurement of soluble and membrane guanylyl cyclase activities. Cell cultures were homogenized with buffer A [in mM: 50 Tris · HCl (pH 7.4), 250 sucrose, 1 EDTA, and 1 dithiothreitol] plus protease inhibitors (2), the protein kinase inhibitor staurosporine (1 × 10³ mM), and the Ser/Thr protein phosphatase inhibitors okadaic acid (1 × 10³ mM) and cypermethrin (5 × 10⁴ mM) in a Potter-Elvehjem homogenizer. After centrifugation (100,000 g for 1 h), sGC activity was determined by incubating the soluble extract with no additions (basal) or 100 µM SNAP in assay buffer [final concentrations (in mM): 50 Tris · HCl (pH 7.4), 1 EGTA, 1 dithiothreitol, 5 GTP, 4 MgCl₂, 15 phosphocreatine, and 1 3-isobutyl-1-methylxanthine] plus creatine kinase (0.2 mg/ml) at 37°C for 30 min. pGC activity was measured in the particulate fraction, rehomogenized with buffer A plus 10% glycerol, in the same assay buffer plus no additions (basal), 1 µM Uro, or 0.1% Triton X-100. Natriuretic factors have been shown to scarcely stimulate pGC after cellular homogenization (16), whereas Triton X-100 is known to elicit a marked enhancement of pGC activity (13). Reactions were terminated by addition of 1 ml of cold ethanol, and cGMP produced by enzyme activity was determined by radioimmunoassay, as described previously. In these conditions, the formation of cGMP was linear with time for 30 min. In the experiments measuring pH dependence of guanylyl cyclase activity, Tris · HCl was substituted for 30 mM PIPES (pH 6.0-6.8) or 30 mM HEPES (pH 6.8-7.4).

Data analysis and statistics. Statistical analysis was carried out with commercially available software (SPSS 8.0.0). Differences between groups were evaluated using one-way analysis of variance. Individual comparisons between groups were performed using the Student-Newman-Keuls test. A critical P value of 0.05 was used. Values are means ± SE.

Materials. Uro was kindly provided by Prof. Dr. Wolf-Georg Forssmann and Dr. Markus Meyer (Niedersächsisches Institut für Peptid-Forschung, Hannover, Germany). SNAP, ANP, CNP, DNP, IBMX, Tris, HEPES, PIPES, GTP, staurosporine, and the Bioluminescent Somatic Cell Assay Kit were obtained from Sigma, cypermethrin and okadaic acid from Calbiochem, [³H]cGMP (35 Ci/mmol) from New England Nuclear, collagenase from Serva, BCECF from Molecular Probes, plastic petri dishes from Falcon, and culture media and sera from GIBCO.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Stimulation of cGMP synthesis in normoxic cardiomyocytes. Stimulation of normoxic cardiomyocytes with 100 µM SNAP or 1 µM Uro in the presence of IBMX elicited a transient and rapid increase in the intracellular content of cGMP, with a maximum at 1 min (Fig. 1). In the extracellular medium, cGMP increased linearly with time during SNAP or Uro stimulation and reached ~30% of the intracellular concentration after 10 min. Cellular cGMP concentrations after 1 min of stimulation with SNAP or Uro were similar: 0.94 ± 0.06 and 1.12 ± 0.08 pmol/mg protein, respectively, vs. 0.04 ± 0.01 pmol/mg protein in nonstimulated cells. Responses to SNAP or Uro were not modified after 120 min of normoxic incubation: 96 ± 7 and 105 ± 15%, respectively, of preincubation values. The response to stimulation of cGMP synthesis with SNAP or Uro was significantly attenuated after a previous exposure to those agonists for 10 min (in the absence of IBMX) just before the 1 min of stimulation (49 ± 9 and 29 ± 15% of the responses to SNAP and Uro, respectively, in nonprestimulated cells, n = 3, P < 0.05).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Time and concentration dependence of cGMP stimulation by urodilatin (Uro) and S-nitroso-N-acetylpenicillamine (SNAP). A: adult rat cardiomyocytes were stimulated for 1-10 min with 1 µM Uro or 100 µM SNAP in the presence of 1 mM 3-isobutyl-1-methylxanthine (IBMX) or with IBMX alone (basal, nonstimulated). Solid line, intracellular accumulation of cGMP; dashed line, extracellular accumulation of cGMP. Extracellular cGMP in nonstimulated cells could not be detected in the period of time analyzed. B: intracellular cGMP in cell cultures stimulated for 1 min with different concentrations of Uro or SNAP in the presence of 1 mM IBMX. C, nonstimulated cells. Inset: effect of 1 min of stimulation with 0.1 and 1 µM atrial natriuretic peptide (ANP) or C-type natriuretic peptide (CNP), expressed as pmol/mg protein. Values are means ± SE of a representative experiment in A and B and 3 different experiments in inset. Mean of EC50 values calculated for Uro response from 3 different experiments was 37 ± 22 nmol/l.

Effect of simulated ischemia and reperfusion on cGMP synthesis. Simulated ischemia (hypoxia at pH 6.4) for 2 h decreases ATP content by 90% and intracellular pH to 6.42 (P < 0.05; Fig. 2) without increasing significantly LDH release with respect to normoxic incubation (14.5 ± 3.3 vs. 7.4 ± 1.2% of total LDH content, not significant). Simulated ischemia exerted a profound inhibitory effect on cGMP produced after stimulation with Uro (~50% of control after 120 min, P < 0.05; Fig. 2), ANP, or CNP (Table 1) but no significant effect on the response to SNAP (Fig. 2). Simulated reperfusion allowed complete and rapid recovery of Uro-mediated cGMP synthesis (Fig. 2).

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Effect of transient simulated ischemia (hypoxia + acidosis) on stimulated cGMP (A) and intracellular pH (pHi) and ATP levels (B) in cardiomyocytes. A: cell cultures were subjected to 30-120 min of hypoxia at pH 6.4 (or 120 min of hypoxia at pH 6.4 and 1-30 min of normoxia at pH 7.4) and stimulated for 1 min with 1 µM Uro or 100 µM SNAP in the presence of 1 mM IBMX or IBMX alone (basal). B: ATP and pHi measured in parallel cultures subjected to conditions described in A. Values are means ± SE from 4-10 different experiments. Values for stimulated cGMP were calculated by subtracting basal cGMP content from cGMP content after stimulation with SNAP or Uro in each experimental condition and are expressed as a percentage of stimulation obtained in control conditions with the same batch of cardiomyocytes. *Significant difference between groups (P < 0.05).

Effect of extracellular acidosis. Cell incubation under normoxic conditions at pH 6.4 (acidosis) reduced the intracellular pH to ~6.8 in <30 min (Fig. 3), whereas ATP content (Fig. 3) and LDH release (7.3 ± 1.5% of total cell content after 120 min) were not significantly modified. cGMP synthesis mediated by Uro decreased following a pattern very similar to that previously observed under simulated ischemia (Fig. 3). cGMP stimulation by ANP or CNP also diminished after 120 min of extracellular acidosis (Table 1). After restoration of extracellular pH, intracellular pH and the response to Uro were rapidly recovered. No changes were observed in sGC-dependent cGMP synthesis (Fig. 3).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effect of transient extracellular acidosis on stimulated cGMP (A) and intracellular pH and ATP levels (B). A: cell cultures were subjected to 30-120 min of normoxia at pH 6.4 (or 120 min of normoxia at pH 6.4 and 1-30 min of normoxia at pH 7.4) and stimulated for 1 min with 1 µM Uro or 100 µM SNAP in the presence of 1 mM IBMX or with IBMX alone (basal). B: ATP and pHi measured in parallel cultures subjected to conditions described in A. Values are means ± SE from 4-12 different experiments. *Significant difference between groups (P < 0.05).

Effect of hypoxia and metabolic inhibition at pH 7.4. Hypoxia without concomitant acidosis (pH 7.4) induced an even more profound ATP depletion than that observed during simulated ischemia (Fig. 4) without significantly altering intracellular pH (Fig. 4) or LDH release (12.2 ± 1.7%) but failed to reproduce the depressant effects of simulated ischemia on cGMP synthesis elicited by Uro (Fig. 4). Instead, a transient potentiation of cGMP synthesis in response to stimulation of pGC with Uro or sGC with SNAP was observed during the first 60 min of hypoxia. After 120 min of hypoxic incubation at pH 7.4, SNAP- and Uro-dependent cGMP synthesis returned to control levels. The response to ANP (a selective agonist of the pGC type A receptor) was also unaffected by hypoxia. On the contrary, cGMP synthesis induced by CNP (a selective agonist of the pGC type B receptor) was reduced by >50% after 120 min of hypoxia in the absence of concomitant acidosis (Table 1). Simulated reoxygenation tended to decrease transiently cGMP synthesis evoked by SNAP and had no effect on the response to Uro.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effect of transient hypoxia on stimulated cGMP (A) and pH and ATP levels (B). A: cell cultures were subjected to 30-120 min of hypoxia at pH 7.4 (or 120 min of hypoxia at pH 7.4 and 1-30 min of normoxia at pH 7.4) and stimulated for 1 min with 1 µM Uro or 100 µM SNAP in the presence of 1 mM IBMX or IBMX alone (basal). B: ATP and pHi measured in parallel cultures subjected to conditions described in A. Values are means ± SE from 4-12 different experiments. *Significant difference between groups (P < 0.05).

Guanylyl cyclase activity in soluble and particulate cell fractions. pH dependence of sGC and pGC activities was analyzed in cell fractions stimulated with 100 µM SNAP, 1 µM Uro, or 0.1% Triton X-100 at different pH values (Fig. 5A). sGC and pGC activities in cell fractions obtained from nontreated cardiomyocytes decreased when the pH of the incubation medium become acid. The decay was particularly abrupt for pGC (no activity could be detected at pH 6.4). However, regulation of sGC activity by pH seems to be complex and cannot be fully explained by the pH dependence of the enzymatic reaction. sGC activity measured at pH 6.4 was significantly higher in cells preincubated for 120 min in a normoxic medium at pH 6.4 than in cells preincubated at pH 7.4 (Fig. 5B). sGC activity was not altered by preincubation at pH 6.4 when the activity was measured at pH 7.4 (Fig. 5B). The presence of acidosis (pH 6.4) during preincubation did not influence pGC activity assayed at pH 7.4 or 6.4 (results not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Guanylyl cyclase (GC) activity in cytosolic and particulate cell fractions. A: nontreated cardiomyocytes were homogenized and centrifuged. Cell fractions were stimulated with 100 µM SNAP (soluble fraction), 1 µM Uro (particulate fraction; type A receptors), or 0.1% Triton X-100 (particulate fraction; type A and B receptors) at different pH values. B: soluble guanylyl cyclase (sGC) was isolated from cardiomyocytes previously preincubated for 120 min at pH 6.4 or 7.4 and then assayed at pH 6.4 or 7.4. Values are means ± SE of 6 experiments expressed as percentage of activity in the same conditions at pH 7.4 (22.1 ± 5.9 nmol cGMP · mg protein1 · 30 min1).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The central finding of the present study is that nonlethal simulated ischemia (hypoxia plus acidosis) substantially reduces pGC-dependent cGMP synthesis in adult rat cardiomyocytes without significantly affecting sGC-mediated cGMP synthesis. The fall of intracellular pH during simulated ischemia seemed to be mainly responsible for inhibition of the response to stimulation of pGC, although in those responses mediated by guanylyl cyclase type B receptors, energy depletion also played a significant role. A transient potentiation of the two pathways of cGMP synthesis was observed during hypoxia in the absence of extracellular acidosis.

Throughout the study, we have assumed that the intracellular accumulation of cGMP after 1 min of stimulation with exogenous SNAP or Uro in the presence of IBMX reflects sGC- and pGC-mediated cGMP synthesis, respectively. This assumption is supported by 1) the minute cGMP content in the absence of stimulation (<5% of cell content after stimulation of sGC or pGC), 2) the almost total blockade of cGMP degradation by 1 mM IBMX, and 3) the small contribution of cGMP efflux at 1 min of stimulation. This assumption is, however, invalid during more prolonged stimulation periods, because receptors rapidly desensitize and egression of cGMP increases with time. Differences in the active transport of cGMP out of the cell or in the desensitization process could explain differences in the time course of the response to stimulation of cGMP synthesis, transient in this and other studies (27) but continuous in others (12).

The effect of SNAP on cGMP synthesis in cardiomyocytes is in agreement with previous studies using NO donors (6). The results obtained with stimulation of natriuretic receptors in these cells have been less consistent. We were particularly interested in studying how simulated ischemia altered stimulation of cGMP synthesis by Uro, because this guanylyl cyclase type A receptor agonist had been found to prevent hypercontracture (11) and myocardial injury during reperfusion (25) through stimulation of cGMP synthesis. However, cardiomyocytes also express the guanylyl cyclase type B receptor (17). Although some studies (4, 17) suggested that this receptor was scarcely relevant in the regulation of cGMP synthesis in cardiomyocytes, we, as other authors (9, 24), observed synthesis of cGMP in cardiomyocytes in response to stimulation of the guanylyl cyclase type B receptor with CNP. The origin of these discrepancies is not known, but because of the apparent relevance of guanylyl cyclase type B receptors in regulation of cGMP synthesis in our cells, CNP responses were also analyzed in the different conditions tested in the present study.

Few studies have analyzed the effects of ischemia on cGMP synthesis in myocardial tissue, and the results of these studies are discrepant. It has been reported that myocardial cGMP increases after 10-25 min of ischemia in the isolated rat heart (3, 18), whereas other studies in the same model found no change (20) or a reduction (23) after 30 min of hypoxia (23) or ischemia (20). Reduced myocardial cGMP content has also been reported in the in situ rat (28) and rabbit (10) heart after 30 min of transient ischemia. We recently reported a fall in the response to Uro in isolated rat heart subjected to 40 min of transient ischemia (11) and in pig myocardium subjected to 48 min of transient coronary occlusion (25). These discrepancies could be partially explained by a heterogeneous modulation depending on the cell type. In Fig. 6, we compared the effects of ATP depletion and intracellular pH on cGMP stimulation in cardiomyocytes and microvascular coronary endothelial cells from rat heart. pGC-mediated cGMP synthesis in cardiomyocytes shows a pH dependence similar to that shown by cGMP synthesis evoked by sGC and pGC in endothelial cells, but the SNAP response in cardiomyocytes presents an unexpected resistance to intracellular acidosis. Also remarkable is the absence of effect of energy depletion in cardiomyocytes (with the exception of responses mediated by guanylyl cyclase type B receptors, as mentioned above), in contrast to the linear correlation observed in endothelial cells between ATP content and cGMP synthesis. To our knowledge, only one study has analyzed the effect of conditions simulating ischemia on the response to stimulation of cGMP synthesis in isolated cardiomyocytes (6). In agreement with our results, the authors of that study reported that a drop in the nucleotide triphosphate cell content (GTP) to <10% of the initial levels (after 60 min of hypoxia at pH 7.4) did not produce a decrease, but a substantial potentiation of cGMP synthesis, after stimulation with an NO donor.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   cGMP synthesis elicited by sGC or particulate guanylyl cyclase stimulation vs. cellular ATP content or pHi for cardiomyocytes (CM) or microvascular endothelial cells (EC) from adult rat hearts. Cells were incubated under extracellular acidosis (circles; pH dependence), 120 min of hypoxia (diamonds), or different periods of metabolic inhibition with dinitrophenol (triangles; ATP dependence) and stimulated for 1 min with 1 µM Uro (filled symbols) or 100 µM SNAP (open symbols). These results have been partially presented in Figs. 3 and 4 and in Ref. 1.

This study demonstrates for the first time the profound depressant effect of acidosis on pGC-mediated cGMP synthesis in cardiomyocytes that accounted to a large extent for the depression of cGMP synthesis observed in this synthetic pathway under simulated ischemia (hypoxia plus acidosis). There was a good correlation between the time course of inhibition of the response to Uro and the time course of intracellular pH decrease during exposure to extracellular buffer at pH 6.4. Recovery of cGMP synthesis was rapid after normalization of the extracellular pH, indicating that acidosis did not induce changes in enzyme content or irreversible alterations of protein structure. The marked dependence of pGC activity in the particulate fraction of cardiomyocytes on pH is consistent with the hypothesis that the effect of acidosis is related to the pH dependence of the enzymatic reaction, as previously shown in endothelial cells (1). The activity of sGC was also decreased at acidic pH in cytosolic fractions, although to a lesser extent. The mechanism of the resistance of the SNAP response to intracellular acidosis in intact cardiomyocytes, in contrast to the susceptibility to low pH of sGC activity measured in cell fractions, is under investigation.

The absence of a general inhibitory effect of energy depletion on cGMP synthesis in cardiomyocytes is notable. Only the responses mediated by guanylyl cyclase type B receptors were found to be sensitive to energy depletion. However, although ATP concentration determines the concentration of GTP, the substrate for sGC and pGC, a direct correlation between ATP content and cGMP synthesis cannot be presumed. ATP depletion is associated with an increase of intracellular Mg²⁺ and inorganic phosphate concentrations (22, 26). Because the substrate of sGC and pGC is Mg²⁺-GTP and phosphate stimulates pGC (22), the effect of the increase in Mg²⁺ and phosphate on cGMP synthesis tends to antagonize the effects of ATP depletion. Moreover, stimulatory and inhibitory sites for ATP have been described in pGC type A and B receptors (5, 13, 19), and ATP depletion may also affect pGC activity through modifications in its phosphorylation state (19). Further studies are needed to discern which of these mechanisms is responsible for the absence of a general effect of the fall in ATP in cardiomyocytes, or even potentiation.

In cardiomyocytes, cGMP has been found to modulate events that play an important role in the pathophysiology of injury secondary to ischemia-reperfusion. The present study demonstrates that, during ischemia, there is a reduced ability to synthesize cGMP through stimulation of pGC and that this effect is mainly mediated by the fall of intracellular pH. In these conditions, and presumably in any other situation causing an intracellular acidification, cardiomyocyte cGMP content would be strongly dependent on NO release. In addition, this study shows that the cGMP-signaling pathway can be differently modulated by ischemia in distinct myocardial cell types.