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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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sGC does not desensitize during the time course of the NO-stimulated cGMP response in human platelets. Platelets were stimulated with 300 μM GSNO at time point zero. PDE inhibitors sildenafil and EHNA (100 μM each) were administered at the time points indicated by the arrows in the inset corresponding to different states of the cGMP response (see text). After addition of PDE inhibitors, aliquots of the platelet suspension were removed into ice cold ethanol every 3 s. Extracted cGMP was then determined by RIA. For the very first data point at 0 s, basal cGMP levels were subtracted; for better comparison, the cGMP levels 3 s after addition of PDE inhibitors (i.e., 18, 33 or 63 s) were subtracted from the following cGMP levels. Thus, the y axis shows the increase in cGMP values within every 3 s. Values represent means ± SEM of hexaplicate determinations in one representative experiment out of three similar experiments.
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fig6: sGC does not desensitize during the time course of the NO-stimulated cGMP response in human platelets. Platelets were stimulated with 300 μM GSNO at time point zero. PDE inhibitors sildenafil and EHNA (100 μM each) were administered at the time points indicated by the arrows in the inset corresponding to different states of the cGMP response (see text). After addition of PDE inhibitors, aliquots of the platelet suspension were removed into ice cold ethanol every 3 s. Extracted cGMP was then determined by RIA. For the very first data point at 0 s, basal cGMP levels were subtracted; for better comparison, the cGMP levels 3 s after addition of PDE inhibitors (i.e., 18, 33 or 63 s) were subtracted from the following cGMP levels. Thus, the y axis shows the increase in cGMP values within every 3 s. Values represent means ± SEM of hexaplicate determinations in one representative experiment out of three similar experiments.

Mentions: Under substrate-depleting conditions, the actual activity of sGC cannot be determined. Therefore, we tried to bypass this problem by choosing a different experimental approach that would not generate extraordinarily elevated cGMP levels. Platelets were stimulated with GSNO and PDE inhibitors were added at different stages of the cGMP response, as indicated in the inset of Fig. 6 (simultaneous to NO, 0 s; near maximum, 15 s; within the cGMP reduction phase, 30 s; and during plateau phase, 60 s). After addition of PDE inhibitors, aliquots of the platelet suspensions were taken every 3 s (series 1: 0, 3, 6 s; series 2: 18, 21, 24 s; series 3: 33, 36, 39 s; series 4: 63, 66, 69 s after NO stimulation), and cGMP accumulation was measured. The increases in cGMP measured in the four series are shown in Fig. 6. Because of the short-term application of PDE inhibitors, depletion of substrate did not occur, and cGMP synthesis, i.e., sGC activity, was unmasked within the cGMP response. Within this time range cGMP increased linearly, and in the different series, the rates of cGMP synthesis were similar (series 1: 109 ± 23, series 2: 102 ± 7, series 3: 108 ± 6, series 4: 110 ± 20 pmol cGMP × s−1/109 platelets), indicating that the apparent NO-stimulated sGC activities were similar, regardless of the stage of the cGMP response. We conclude that sGC is not responsible for desensitizing the cGMP response.


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)

sGC does not desensitize during the time course of the NO-stimulated cGMP response in human platelets. Platelets were stimulated with 300 μM GSNO at time point zero. PDE inhibitors sildenafil and EHNA (100 μM each) were administered at the time points indicated by the arrows in the inset corresponding to different states of the cGMP response (see text). After addition of PDE inhibitors, aliquots of the platelet suspension were removed into ice cold ethanol every 3 s. Extracted cGMP was then determined by RIA. For the very first data point at 0 s, basal cGMP levels were subtracted; for better comparison, the cGMP levels 3 s after addition of PDE inhibitors (i.e., 18, 33 or 63 s) were subtracted from the following cGMP levels. Thus, the y axis shows the increase in cGMP values within every 3 s. Values represent means ± SEM of hexaplicate determinations in one representative experiment out of three similar experiments.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: sGC does not desensitize during the time course of the NO-stimulated cGMP response in human platelets. Platelets were stimulated with 300 μM GSNO at time point zero. PDE inhibitors sildenafil and EHNA (100 μM each) were administered at the time points indicated by the arrows in the inset corresponding to different states of the cGMP response (see text). After addition of PDE inhibitors, aliquots of the platelet suspension were removed into ice cold ethanol every 3 s. Extracted cGMP was then determined by RIA. For the very first data point at 0 s, basal cGMP levels were subtracted; for better comparison, the cGMP levels 3 s after addition of PDE inhibitors (i.e., 18, 33 or 63 s) were subtracted from the following cGMP levels. Thus, the y axis shows the increase in cGMP values within every 3 s. Values represent means ± SEM of hexaplicate determinations in one representative experiment out of three similar experiments.
Mentions: Under substrate-depleting conditions, the actual activity of sGC cannot be determined. Therefore, we tried to bypass this problem by choosing a different experimental approach that would not generate extraordinarily elevated cGMP levels. Platelets were stimulated with GSNO and PDE inhibitors were added at different stages of the cGMP response, as indicated in the inset of Fig. 6 (simultaneous to NO, 0 s; near maximum, 15 s; within the cGMP reduction phase, 30 s; and during plateau phase, 60 s). After addition of PDE inhibitors, aliquots of the platelet suspensions were taken every 3 s (series 1: 0, 3, 6 s; series 2: 18, 21, 24 s; series 3: 33, 36, 39 s; series 4: 63, 66, 69 s after NO stimulation), and cGMP accumulation was measured. The increases in cGMP measured in the four series are shown in Fig. 6. Because of the short-term application of PDE inhibitors, depletion of substrate did not occur, and cGMP synthesis, i.e., sGC activity, was unmasked within the cGMP response. Within this time range cGMP increased linearly, and in the different series, the rates of cGMP synthesis were similar (series 1: 109 ± 23, series 2: 102 ± 7, series 3: 108 ± 6, series 4: 110 ± 20 pmol cGMP × s−1/109 platelets), indicating that the apparent NO-stimulated sGC activities were similar, regardless of the stage of the cGMP response. We conclude that sGC is not responsible for desensitizing the cGMP response.

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