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Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme.

Mullershausen F, Russwurm M, Thompson WJ, Liu L, Koesling D, Friebe A - J. Cell Biol. (2001)

Bottom Line: Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases.We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase.Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

View Article: PubMed Central - PubMed

Affiliation: Abteilung für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, D-44780 Bochum, Germany.

ABSTRACT
Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

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Related in: MedlinePlus

HPLC analysis of intraplatelet GTP levels under NO-stimulated conditions in the absence and presence of PDE inhibitors. After preincubation for 10 min with DMSO (control) or the combination of sildenafil and EHNA (100 μM each), platelets were stimulated with 300 μM GSNO for 60 s. Subsequently, reactions were stopped by the addition of HClO4 (final concentration 0.7 M). After precipitation of proteins and adjustment of the pH, nucleotides were separated on a Mono-Q column as described under Materials and methods. (A) Original trace data shows elution of GTP at 13.3% buffer B (see Materials and methods). (B) Bars show the quantitative analysis as means ± SEM from four independent experiments.
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fig5: HPLC analysis of intraplatelet GTP levels under NO-stimulated conditions in the absence and presence of PDE inhibitors. After preincubation for 10 min with DMSO (control) or the combination of sildenafil and EHNA (100 μM each), platelets were stimulated with 300 μM GSNO for 60 s. Subsequently, reactions were stopped by the addition of HClO4 (final concentration 0.7 M). After precipitation of proteins and adjustment of the pH, nucleotides were separated on a Mono-Q column as described under Materials and methods. (A) Original trace data shows elution of GTP at 13.3% buffer B (see Materials and methods). (B) Bars show the quantitative analysis as means ± SEM from four independent experiments.

Mentions: To find out whether NO-induced cGMP formation in the presence of PDE inhibitors leads to a significant reduction of the substrate GTP, we measured intraplatelet GTP concentrations using HPLC. Original traces of the GTP peaks of NO-stimulated platelets in the absence (control; dotted line) and presence of both PDE inhibitors (100 μM each; solid line) are shown in Fig. 5 A. Clearly, the inhibition of PDEs in NO-stimulated platelets led to a 50% reduction of the intracellular GTP concentration, as shown in the statistical analysis in Fig. 5 B. Because of this massive loss of available GTP in the stimulated platelets in the presence of PDE inhibitors, kinetic analysis of cGMP synthesis is not appropriate.


Rapid nitric oxide-induced desensitization of the cGMP response is caused by increased activity of phosphodiesterase type 5 paralleled by phosphorylation of the enzyme.

Mullershausen F, Russwurm M, Thompson WJ, Liu L, Koesling D, Friebe A - J. Cell Biol. (2001)

HPLC analysis of intraplatelet GTP levels under NO-stimulated conditions in the absence and presence of PDE inhibitors. After preincubation for 10 min with DMSO (control) or the combination of sildenafil and EHNA (100 μM each), platelets were stimulated with 300 μM GSNO for 60 s. Subsequently, reactions were stopped by the addition of HClO4 (final concentration 0.7 M). After precipitation of proteins and adjustment of the pH, nucleotides were separated on a Mono-Q column as described under Materials and methods. (A) Original trace data shows elution of GTP at 13.3% buffer B (see Materials and methods). (B) Bars show the quantitative analysis as means ± SEM from four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198829&req=5

fig5: HPLC analysis of intraplatelet GTP levels under NO-stimulated conditions in the absence and presence of PDE inhibitors. After preincubation for 10 min with DMSO (control) or the combination of sildenafil and EHNA (100 μM each), platelets were stimulated with 300 μM GSNO for 60 s. Subsequently, reactions were stopped by the addition of HClO4 (final concentration 0.7 M). After precipitation of proteins and adjustment of the pH, nucleotides were separated on a Mono-Q column as described under Materials and methods. (A) Original trace data shows elution of GTP at 13.3% buffer B (see Materials and methods). (B) Bars show the quantitative analysis as means ± SEM from four independent experiments.
Mentions: To find out whether NO-induced cGMP formation in the presence of PDE inhibitors leads to a significant reduction of the substrate GTP, we measured intraplatelet GTP concentrations using HPLC. Original traces of the GTP peaks of NO-stimulated platelets in the absence (control; dotted line) and presence of both PDE inhibitors (100 μM each; solid line) are shown in Fig. 5 A. Clearly, the inhibition of PDEs in NO-stimulated platelets led to a 50% reduction of the intracellular GTP concentration, as shown in the statistical analysis in Fig. 5 B. Because of this massive loss of available GTP in the stimulated platelets in the presence of PDE inhibitors, kinetic analysis of cGMP synthesis is not appropriate.

Bottom Line: Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases.We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase.Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

View Article: PubMed Central - PubMed

Affiliation: Abteilung für Pharmakologie und Toxikologie, Medizinische Fakultät, Ruhr-Universität Bochum, D-44780 Bochum, Germany.

ABSTRACT
Most of the effects of the signaling molecule nitric oxide (NO) are mediated by cGMP, which is synthesized by soluble guanylyl cyclase and degraded by phosphodiesterases. Here we show that in platelets and aortic tissue, NO led to a biphasic response characterized by a tremendous increase in cGMP (up to 100-fold) in less than 30 s and a rapid decline, reflecting the tightly controlled balance of guanylyl cyclase and phosphodiesterase activities. Inverse to the reported increase in sensitivity caused by NO shortage, concentrating NO attenuated the cGMP response in a concentration-dependent manner. We found that guanylyl cyclase remained fully activated during the entire course of the cGMP response; thus, desensitization was not due to a switched off guanylyl cyclase. However, when intact platelets were incubated with NO and then lysed, enhanced activity of phosphodiesterase type 5 was detected in the cytosol. Furthermore, this increase in cGMP degradation is paralleled by the phosphorylation of phosphodiesterase type 5 at Ser-92. Thus, our data suggest that NO-induced desensitization of the cGMP response is caused by the phosphorylation and subsequent activity increase of phosphodiesterase type 5.

Show MeSH
Related in: MedlinePlus