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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: 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.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.

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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Time course of NO-induced cGMP levels and NO-induced desensitization of the cGMP response in rat aortic strips. (A) Aortic strips were stimulated with 300 μM GSNO for the times indicated. After shock freezing and homogenization of the tissue, the extracted cGMP was measured by RIA. As the amount of cGMP measured in the aortic preparations from different animals varied considerably, data are expressed as the percentage of maximal stimulation. (B) Aortic strips preincubated for 3 min with the indicated concentrations of GSNO were stimulated with 300 μM GSNO, and cGMP levels were measured after 30 s. Data represent means ± SEM of nine independent experiments performed in duplicates (A) or means ± SEM of triplicate determinations in one representative experiment out of a total of three similar experiments (B).
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fig3: Time course of NO-induced cGMP levels and NO-induced desensitization of the cGMP response in rat aortic strips. (A) Aortic strips were stimulated with 300 μM GSNO for the times indicated. After shock freezing and homogenization of the tissue, the extracted cGMP was measured by RIA. As the amount of cGMP measured in the aortic preparations from different animals varied considerably, data are expressed as the percentage of maximal stimulation. (B) Aortic strips preincubated for 3 min with the indicated concentrations of GSNO were stimulated with 300 μM GSNO, and cGMP levels were measured after 30 s. Data represent means ± SEM of nine independent experiments performed in duplicates (A) or means ± SEM of triplicate determinations in one representative experiment out of a total of three similar experiments (B).

Mentions: To see whether the observed NO-induced decrease in the cGMP response was a special feature of platelets, we chose rat aortic strips as another system; here, the cGMP system is known to play an important role in the regulation of smooth muscle tone. To prevent endogenously produced NO from affecting the sensitivity state of the cGMP system, the experiments with aortic strips were performed in the presence of the NO synthase inhibitor N-nitro-l-arginine methyl ester (200 μM). Application of GSNO (300 μM) led to a biphasic cGMP response similar to that seen in human platelets (Fig. 3 A), consisting of a fast and enormous increase in cGMP levels (150-fold; maximum after 20–30 s) followed by a rapid and pronounced decrease. Although the amount of cGMP measured in the aortic preparations from different animals varied considerably (0.5–1.5 and 100–250 pmol/mg protein under basal and stimulated conditions, respectively), the biphasic shapes of the responses were qualitatively similar.


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)

Time course of NO-induced cGMP levels and NO-induced desensitization of the cGMP response in rat aortic strips. (A) Aortic strips were stimulated with 300 μM GSNO for the times indicated. After shock freezing and homogenization of the tissue, the extracted cGMP was measured by RIA. As the amount of cGMP measured in the aortic preparations from different animals varied considerably, data are expressed as the percentage of maximal stimulation. (B) Aortic strips preincubated for 3 min with the indicated concentrations of GSNO were stimulated with 300 μM GSNO, and cGMP levels were measured after 30 s. Data represent means ± SEM of nine independent experiments performed in duplicates (A) or means ± SEM of triplicate determinations in one representative experiment out of a total of three similar experiments (B).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198829&req=5

fig3: Time course of NO-induced cGMP levels and NO-induced desensitization of the cGMP response in rat aortic strips. (A) Aortic strips were stimulated with 300 μM GSNO for the times indicated. After shock freezing and homogenization of the tissue, the extracted cGMP was measured by RIA. As the amount of cGMP measured in the aortic preparations from different animals varied considerably, data are expressed as the percentage of maximal stimulation. (B) Aortic strips preincubated for 3 min with the indicated concentrations of GSNO were stimulated with 300 μM GSNO, and cGMP levels were measured after 30 s. Data represent means ± SEM of nine independent experiments performed in duplicates (A) or means ± SEM of triplicate determinations in one representative experiment out of a total of three similar experiments (B).
Mentions: To see whether the observed NO-induced decrease in the cGMP response was a special feature of platelets, we chose rat aortic strips as another system; here, the cGMP system is known to play an important role in the regulation of smooth muscle tone. To prevent endogenously produced NO from affecting the sensitivity state of the cGMP system, the experiments with aortic strips were performed in the presence of the NO synthase inhibitor N-nitro-l-arginine methyl ester (200 μM). Application of GSNO (300 μM) led to a biphasic cGMP response similar to that seen in human platelets (Fig. 3 A), consisting of a fast and enormous increase in cGMP levels (150-fold; maximum after 20–30 s) followed by a rapid and pronounced decrease. Although the amount of cGMP measured in the aortic preparations from different animals varied considerably (0.5–1.5 and 100–250 pmol/mg protein under basal and stimulated conditions, respectively), the biphasic shapes of the responses were qualitatively similar.

Bottom Line: 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.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.

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