Article |
Address correspondence to Doris Koesling, Abteilung für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, Universitätssr. 150, D-44780 Bochum, Germany. Tel.: 49-234-3226827. Fax: 49-234-3214521. E-mail: doris.koesling{at}ruhr-uni-bochum.de
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Abstract |
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Key Words: phosphodiesterase; cGMP; NO-sensitive guanylyl cyclase; platelets; desensitization
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Introduction |
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During the last few years, several reports indicated a possible modulatory influence of NO on the cGMP response in addition to its sGC stimulatory action. In fact, a lack of NO caused by either endothelium removal, gene disruption, or inhibition of the endothelial NO synthase has been reported to lead to increased NO sensitivities in the aortic vessels; which was paralleled by an augmentation of NO-induced cGMP levels (Moncada et al., 1991; Brandes et al., 2000). Decreased sensitivities of the NOcGMP system have been demonstrated under various conditions after treatment with NO-releasing agents (Waldman et al., 1986; Schroder et al., 1988; Ujiie et al., 1994; Filippov et al., 1997). In a recent report, Bellamy et al. (2000) showed a rapid desensitization of sGC within seconds.
It was the aim of the present study to investigate the short-term NO-dependent desensitization of the cGMP response. As the properties of the NOcGMP system in cultured cells change very rapidly during cell cultivation (Wyatt et al., 1998; unpublished data), we chose platelets and aortic smooth muscle as model systems. Here, we show that short-term preincubation of human platelets and rat aortic strips with submaximally activating S-nitrosoglutathione (GSNO) concentrations blunted the NO-induced cGMP response. In contrast to a recent report (Bellamy et al., 2000), the decrease in the cGMP response was not based on sGC desensitization, as in our experiments the enzyme remained fully activated. Our data show that within a few seconds of the NO incubation of intact platelets, an increase in PDE5 activity can be detected in the cytosolic fractions; a result that is paralleled by an increase in phosphorylation of PDE5 at Ser-92. Therefore, we postulate that activation of PDE5, not desensitization of sGC, is responsible for the NO-induced desensitization of the cGMP response.
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Results |
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Next, to investigate the possible modulation of the cGMP response by NO, we performed preincubation experiments. First, we applied submaximally effective GSNO concentrations (3, 10, and 30 µM) for 3 min (Fig. 2), a period after which the intraplatelet cGMP levels had declined to almost basal levels (Fig. 1 A). After this 3-min preincubation, platelets were stimulated with the maximally effective concentration of GSNO (300 µM; Fig. 1), and cGMP levels were determined over time. As expected, under control conditions (0 µM GSNO during preincubation), the cGMP response to 300 µM GSNO resembled that seen in Fig. 1. However, GSNO preincubation attenuated the cGMP responses in a concentration-dependent manner, when induced by maximally effective GSNO. The use of 30 µM GSNO during preincubation almost totally abrogated the NO-induced cGMP response in platelets. These data indicate a rapidly induced desensitization of the cGMP response. Furthermore, the degree of NO sensitivity of the cGMP system appears to be inversely related to the amount of NO present during preincubation of the platelets.
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Our next experiment investigated the underlying mechanism of the observed desensitization in the platelets. Intracellular cGMP levels reflect the state of activity of the cGMP-forming sGC and cGMP-degrading PDEs. Therefore, decreased synthesis or enhanced degradation should account for the desensitization of the cGMP response. To find out whether desensitization occurred on the level of sGC, NO-induced cGMP formation was determined in the presence of PDE inhibitors. As PDE5 and PDE2 are the major enzymes responsible for cGMP degradation in platelets, we chose the respective, specific inhibitors sildenafil and erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA). Under a blockade of the two PDEs, cGMP levels did not decrease after the initial rise in cGMP but rather came to an enormously elevated plateau within 60120 s (Fig. 4 A). On this plateau, cGMP levels were 10-fold higher than the maximal levels seen in the absence of PDE inhibitors (3,000 vs. 300 pmol/109 platelets; Fig. 4 A). Fig. 4 B shows NO preincubation experiments in the presence of PDE inhibitors performed similar to those in Fig. 2. GSNO preincubation (3, 10, and 30 µM for 3 min) in the presence of PDE inhibitors led to augmented cGMP levels at time point zero, since the cGMP formed during preincubation was not degraded. Although the initial rate of cGMP formation in the control sample appeared greater than the rates of the NO-preincubated samples, cGMP levels reached a similar plateau in all cases. Calculation revealed an extremely high concentration,
600 µM, of intraplatelet cGMP (see Discussion). Therefore, reduction of the substrate GTP concentration could not be ruled out.
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The fact that enhanced PDE activity was evident after breaking up the cells suggested a covalent modification as the mechanism underlying desensitization. Phosphorylation of PDE5 at Ser-92 has been shown to be associated with an increase in enzyme activity (Wyatt et al., 1998; Corbin et al., 2000). Therefore, we studied this possible phosphorylation by using a novel phospho-specific antibody against PDE5, raised against the phosphorylated Ser-92containing peptide. Fig. 8 A shows that the antibody only reacts with the phosphorylated and not with the unphosphorylated form of PDE5. Platelets were stimulated with GSNO, and at the different stages of the NO-stimulated cGMP response (Fig. 6, inset) aliquots of the platelet suspension were subjected to SDS-PAGE and blotted onto nitrocellulose (Fig. 8 B). Under nonstimulated conditions, PDE5 was not recognized. Immunoreactive signals appeared already 15 s after NO stimulation and paralleled the increased enzyme activity within a time range of 60 s (Fig. 7 A). Phosphorylation of PDE5 was detected with concentrations of GSNO as low as 3 µM (unpublished data). Thus, our data show, for the first time, the concomitant phosphorylation and activity increase of PDE5 over the course of the NO-stimulated cGMP response. In conclusion, phosphorylation of PDE5 by a yet unidentified kinase appears to be responsible for the NO-induced desensitization of the cGMP response.
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Discussion |
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The rapid desensitizing effect of NO is demonstrated by preincubating platelets or aortic strips, which reveals that the extent of the cGMP response is inversely related to the amount of NO present during the preincubation (Figs. 2 and 3 B). At high NO concentrations, the cGMP system becomes desensitized almost completely, whereas at low tissue concentrations of NO, the system retains a higher sensitivity state. Physiologically, modulation of the sensitivity of the cGMP response reflects the ability of the NOcGMP system to adapt to acute changes in NO exposition. Thus, information is not only transduced within the NOcGMP system but is also processed for adaptation to the amount of NO available.
The reduced sensitivity of the cGMP response after NO preincubation can be explained by either reduced cGMP synthesis and/or enhanced cGMP degradation. To solely monitor cGMP formation (i.e., the actual sGC activity), PDE inhibitors can be used to prevent GMP breakdown. Based mainly on this approach, i.e., measurements of cGMP accumulation in the presence of PDE inhibitors, a recent report suggested a rapid NO-induced desensitization of platelet sGC (Bellamy et al., 2000).
These results and our data (Fig. 4 A, inset) are in perfect accordance with the 10-fold elevation of NO-induced cGMP levels caused by the addition of PDE inhibitors (3,000 pmol/109 platelets equaling 1,500 pmol/mg protein, assuming a protein content of 2 mg/109 platelets; Eigenthaler et al., 1992). The plateau of the cGMP response, reached after 60 s in the presence of PDE inhibitors, indicates low cGMP-forming activity, which could be attributed to a switched-off sGC. Peculiarly, NO-preincubated platelets reached a similar plateau (Fig. 4 B) suggesting that this intracellular cGMP concentration may represent an "ultimate" level for cGMP accumulation. However, the decrease in sGC activity could also be explained by a reduction in substrate GTP. Assuming a single platelet volume of 5.2 fl (Corash et al., 1977), these peak cGMP levels correspond to an intraplatelet concentration of 600 µM (intracellular GTP levels between 400 and 800 µM have been published; Traut, 1994). Under normal conditions, sGC will cyclize GTP to cGMP, which is then hydrolyzed to GMP by PDEs. GMP will be readily phosphorylated to GDP and GTP by nucleoside mono- and diphosphate kinases, respectively. The only enzymes able to convert cGMP to GMP and hence back into the GMP/GDP/GTP metabolism are the PDEs. Production of cGMP in the presence of PDE inhibitors can be viewed as a "dead-end" for guanosine phosphates; accordingly, we see a dramatic accumulation of cGMP and a 50% reduction of intraplatelet GTP levels. Taking compartmentalization as well as protein-bound GTP into account, it is very likely that this decrease in GTP leads to a massive reduction in accessible substrate for sGC.
Realizing that substrate depletion could account for the reduction in cGMP forming activity, our next experiment was designed to avoid GTP depletion, and sGC was revealed to be fully active during the entire cGMP response. Here, sGC activities obtained at various time points during the NO-induced cGMP response were practically identical to the initial velocity measured directly after NO/sildenafil/EHNA coadministration (Fig. 6).
If sGC is not responsible, a change in PDE activity has to account for the desensitization of the cGMP response. In fact, we were able to show increased PDE activity in the cytosol of NO-incubated, intact platelets, in which sGC activity remained unaffected. Although the NO-induced increases in PDE activity were modest, they may well be sufficient to counteract cGMP synthesis. Rough calculation reveals a PDE activity in platelet cytosol of 40 pmol/s per 109 platelets at 1 µM cGMP (Fig. 7 A; 1 ml of cytosolic fraction contains 1.37 x 108 platelets). NO-stimulated cGMP formation by guanylyl cyclase ranges between 100 and 110 pmol cGMP/s per 109 platelets (Fig. 6). Thus, a 2.4-fold increase in cGMP degradation, after 60 s (Fig. 7 A), is sufficient to increase PDE activity to the level of sGC activity. The resulting net cGMP accumulation of zero explains the "plateau" phase, after 30 s, in which cGMP levels are low although guanylyl cyclase remains active (Fig. 6). In combination with the NO-induced activation, substrate-linked activation of PDE (Fig. 7 D) leads to PDE activity exceeding that of sGC, which is required for net cGMP reduction. The observed increase in PDE activity can be attributed to PDE5, as shown by sildenafil inhibition. Furthermore, the detection of enhanced PDE5 activity after disruption of cell integrity indicates a covalent modification as the mechanism underlying desensitization.
In fact, Wyatt et al. (1998) have shown that ANP-induced cGMP accumulation leads to phosphorylation of PDE5 in primary vascular smooth muscle cells concomitant with a two- to fourfold increase in the catalytic activity of the immunoprecipitated enzyme. In vitro, phosphorylation of this PDE isozyme was shown to result in a 5070% increase in catalytic activity (Corbin et al., 2000). Our experiments (Fig. 8) with a novel antibody show that phosphorylation of PDE5 occurs within the same time range as the observed increase in PDE activity (Fig. 7 A). Furthermore, phosphorylation of PDE5 was detected in a sample with a GSNO concentration as small as 3 µM. Interestingly, this was the lowest concentration that led to increased cGMP formation and desensitization of the cGMP response (Figs. 1 A and 2). In sum, our data suggest that phosphorylation of PDE5 is the underlying mechanism of the NO-induced desensitization of the cGMP response. Although it is tempting to speculate on cGKI, the kinase responsible for PDE5 phosphorylation remains to be identified. The presented data emphasize the complexity of the tightly regulated concert of cGMP synthesis and degradation in the intact cell, and underline the therapeutic importance of PDEs as pharmacological targets.
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Materials and methods |
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Phosphorylation of VASP
Platelets (5 x 108/ml) were stimulated with 300 µM GSNO; at the indicated time points, an aliquot (4.5 x 107 platelets) was removed into Laemmli buffer and heated to 95°C for 5 min. Detection of VASP phosphorylation was performed as described previously (Friebe et al., 1998).
Determination of cGMP levels in intact aortic strips
Aortas from male Wistar-Kyoto rats were cleaned of connective tissue and cut into strips of 25 mg wet weight. Before stimulation with GSNO, strips were allowed to equilibrate in the presence of 200 µM N-nitro-L-arginine methyl ester for 1 h (37°C) in Krebs-Henseleit solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, 7.5 mM glucose), pH 7.4, gassed with 95% O2 and 5% CO2. In the case of preincubation, samples were treated with the indicated GSNO concentrations for 3 min and then washed twice. Stimulation with the maximally effective GSNO concentration was performed 2 min after the second wash. To terminate the reactions, tissue was shock frozen using metal forceps precooled in liquid nitrogen. Isolation and measurement of cGMP was performed as described (Rothermund et al., 2000). Protein content was determined using the bicinchoninic acid method.
HPLC detection of GTP
After GSNO stimulation of platelets in the absence or presence of the PDE inhibitors sildenafil and EHNA (100 µM, respectively), the reactions were stopped by the addition of 0.8 M HClO4. After centrifugation (15 min at 20,000 g and 4°C), supernatants were adjusted to pH 12.0 with KOH and frozen at 80°C. After a second centrifugation step, supernatants were diluted into running buffer A (see below), and pH was adjusted to that of the running buffer. Samples were loaded onto a Mono-Q HR5/5 column (Amersham Pharmacia Biotech) and eluted with a linear gradient (buffer A: 20 mM K2HPO4, pH 8.0; buffer B: 1 mM NaCl, 20 mM K2HPO4, pH 8.0; 020% B, 240 min; flow rate 0.5 ml/min). Elution of nucleotides was monitored at 254 nm; GTP was identified by co-chromatography of 32PGTP.
Determination of cGMP synthesis in platelets
NO-stimulated cGMP synthesis in platelets was assessed by adding the indicated PDE inhibitors (100 µM sildenafil and EHNA) either simultaneously with GSNO or 15, 30, or 60 s after addition of GSNO. Subsequently, aliquots of the platelet suspension were removed every 3 s for cGMP determination. Experiments were performed in triplicates or hexaplicates.
Measurement of PDE and sGC activities in the cytosolic fraction of platelets
Platelet suspensions were adjusted to 3 x 108 platelets/ml. Aliquots of 450 µl were equilibrated at 37°C for 10 min and stimulated with 50 µl GSNO yielding a final concentration of 300 µM. After the indicated incubation time, 500 µl of an ice cold protease inhibitor cocktail (2 µM pepstatin A, 0.4 µM benzamidine, 0.5 mM PMSF, 2 mM sodium vanadate, 1 mg/ml BSA, 4 mM DTT) was added, and the suspension was briefly sonicated (one pulse of 5 s) on ice using a Branson Sonifier B-12. After centrifugation (15 min, 4°C, 20,000 g), PDE activity in the supernatant was measured by the conversion of 32P-cGMP (synthesized from [-32P]GTP using purified sGC) to guanosine and 32P-phosphate in the presence of alkaline phosphatase at 37°C for 10 min. Reaction mixtures contained 1 µl of the supernatants, 32P-cGMP (10,00050,000 cpm), 1 µM cGMP, 12 mM MgCl2, 3 mM DTT, 0.5 mg/ml BSA, 1 U of alkaline phosphatase, and 50 mM triethanolamine/HCl, pH 7.4, in a total volume of 0.1 ml. Reactions were stopped by the addition of 900 µl ice cold charcoal suspension (20% activated charcoal in 50 mM KH2PO4, pH 2.3). After pelleting the charcoal by centrifugation, 32P-phosphate was measured in the supernatant. For the determination of sGC activity, 10 µl of the supernatant was measured in the presence of [
-32P]GTP (500,000 cpm), 300 µM GTP, 3 mM MgCl2, 3 mM DTT, 1 mM cGMP, 0.5 mg/ml BSA, 300 µM GSNO, 1 mM IBMX and a GTP-regenerating system (0.025 mg creatine kinase, 5 mM creatine phosphate), and 50 mM triethanolamine/HCl, pH 7.4, in a total volume of 0.1 ml as described previously (Friebe et al., 1996).
Generation of antiphospho-Ser92-PDE5 antibody and GST-PDE5
Antiphospho-Ser92-PDE5 antibody was raised by immunizing rabbit with keyhole limpet hemocyanin conjugate of synthetic phospho-Ser92 peptide (CTRKIS-PO3-ASEFDR). The immune serum was purified over two sequential affinity columns, CTRKISASEFDR peptide immunosorbent and the CTRKIS-PO3-ASEFDR peptide immunosorbent. Preparation of the recombinant GST-PDE5 was as described (Liu et al., 2001).
Detection of in vitrophosphorylated PDE5
200 ng of the purified nonphosphorylated and phosphorylated GSTPDE5 fusion proteins, respectively, were subjected to SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with the antiphospho-PDE5 antibody in a dilution of 1:10,000 followed by a peroxidase-coupled antirabbit antibody (Sigma-Aldrich). Detection was performed using an ECL kit (Amersham Pharmacia Biotech).
Detection of phosphorylated PDE5 in platelets
Platelet suspensions (4 x 108 platelets/ml) were stimulated with GSNO. At the indicated time points, an aliquot (3.6 x 107 platelets) was removed into Laemmli buffer and boiled for 10 min. Proteins were separated by SDS-PAGE and transferred to nitrocellulose. Membranes were incubated with phospho-PDE5specific antibody in a dilution of 1:50,000. Detection was performed as described above.
Materials
Sildenafil was a gift from Pfizer (Sandwich, UK). GSNO and monosuccinyl-tyrosyl-cGMP were obtained from Sigma-Aldrich. Anti-VASP antibody was from Alexis Biochemicals Corp. Alkaline phosphatase was purchased from Boehringer, EHNA was from Tocris Cookson. Activated charcoal was from Riedel-de Haën. [-32P]GTP (800 Ci/mmol) was purchased from DuPont, Na125I was from Amersham Pharmacia Biotech. The bicinchoninic acid protein determination kit was from Pierce Chemical Co. Chemicals used for HPLC detection of GTP were of HPLC grade.
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Footnotes |
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Acknowledgments |
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This work was supported by the Deutsche Forschungsgemeinschaft.
Submitted: 2 July 2001
Revised: 31 August 2001
Accepted: 7 September 2001
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References |
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