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Pharmacological evaluation of the NO/cGMP signalling system
Linköping University, Department of Medicine and Care, Pharmacology. Linköping University, Faculty of Health Sciences.
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Platelet activation and inhibition are tightly balanced processes, regulated by various endogenous molecules. In this regard, the endothelium plays a key role in mediating inhibition of platelets by releasing nitric oxide (NO) and cAMP-elevating prostaglandins.

The present study put emphasis on drugs that activate directly or modulate the NO/cGMP signalling pathway.

The specific aims were i) to compare two different NO-containing compounds; namely the S-nitrosothiol SNAP and the oxatriazole derivative GEA 3175; ii) to evaluate the mechanism of drug action of the nitrosoamine dephostatin; iii) to investigate cross-talk mechanisms between the NO/cGMP and the cAMP signalling pathways. These studies were perfom1ed using human blood platelets and iliac arteries obtained from pigs.

The present data revealed that SNAP but not GEA 3175 released detectable amounts of NO. Despite that, both compounds elevated cGMP, inhibited rises in [Ca2+]i and stimulated phosphorylation of vasodilator stimulated phosphoprotein (VASP). Moreover, the results showed that NO/cGMP-induced inhibition of Ca2+ responses, but not NO/cGMP-mediated V ASP phosphorylation, was rapidly desensitised. The results showed that an unstable NO-donor more effectively induced desensitisation.

Dephostatin, originally characterised as a protein tyrosine phosphatase (PTP) inhibitor, was found to modulate the NO/cGMP signalling in a complex way. More specifically, dephostatin is an effective NO-scavenger and surprisingly, it also serves as a source of NO and thereby induces cGMPmediated vasorelaxation. In platelets, dephostatin abolished NO/cGMP-mediated inhibition of cytosolic Ca2+, but augmented NO/cGMP-induced VASP phosphorylation.

cGMP-induced inhibition of type 3 phosphodiesterases (PDE3) enhanced adenosine-mediated inhibition of platelets. This effect was of main importance for the suppression of several platelet responses. However, inhibition of Ca2+ influx was another cGMP-specific mechanism contributing to a powerful inhibition of the platelets.

In conclusion: The present results show that release of NO from drug molecules is not a prerequisite for NO/cGMP-mediated cellular and intracellular responses. On the contrary, drug stability in terms of NO-release appears to be crucial in platelet desensitisation to NO. Therefore it is important to gain more knowledge about the NO/cGMP-signalling pathway regarding future drug design of NO-containing drugs. The findings presented in this thesis suggest that dephostatin can prove to be a very useful tool in this area of research.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press , 2005. , 82 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 919
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-31869Local ID: 17696ISBN: 91-85297-56-8 OAI: oai:DiVA.org:liu-31869DiVA: diva2:252692
Public defence
2005-11-24, Elsa Brändströmssalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-01Bibliographically approved
List of papers
1. Characterisation of GEA 3175 on human platelets: comparison with S-nitroso-N-acetyl-D,L-penicillamine
Open this publication in new window or tab >>Characterisation of GEA 3175 on human platelets: comparison with S-nitroso-N-acetyl-D,L-penicillamine
2004 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 496, no 1-3, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

By comparing the effect of two nitric oxide (NO)-containing compounds, we found that S-nitroso-N-acetyl-d,l-penicillamine (SNAP), but not GEA 3175 (1,2,3,4-Oxatriazolium,3-(3-chloro-2-metylphenyl)-5-[[(4-methylphenyl)sulfonyl]amino]-, hydroxide inner salt), released NO. Despite this, both drugs elevated cyclic guanosine 3′,5′-monophosphate (cGMP) levels in human platelets. However, SNAP was more effective after short exposure times (5 and 20 s). The compounds also inhibited thrombin-induced rises in cytosolic Ca2+. Time studies revealed that the action of SNAP rapidly declined by increasing the length of incubation (from 5 s to 30 min). This desensibilisation phenomenon mainly involved the release of Ca2+ from intracellular stores. In comparison, GEA 3175-induced inhibition of cytosolic Ca2+ signalling was much more long-lasting. The soluble guanylyl cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reversed the effect of GEA 3175 on cytosolic Ca2+. Consequently, this inhibition depends solely on the increase in cGMP. In summary, differences between GEA 3175 and SNAP were observed in NO releasing, cGMP elevating and Ca2+ suppressive properties.

Keyword
NO (Nitric Oxide), cGMP (cyclic guanosine 3′, 5′-monophosphate), Calcium, Aggregation, Platelet, SNAP (S-nitroso-N-acetyl-d, l-penicillamine), GEA 3175
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-20473 (URN)10.1016/j.ejphar.2004.06.002 (DOI)15288569 (PubMedID)
Available from: 2009-09-09 Created: 2009-09-09 Last updated: 2012-10-01Bibliographically approved
2. The nitrosoamine dephostatin interacts with nitric oxide/cGMP signalling and modulates cytosolic calcium responses in human platelets
Open this publication in new window or tab >>The nitrosoamine dephostatin interacts with nitric oxide/cGMP signalling and modulates cytosolic calcium responses in human platelets
(English)Manuscript (preprint) (Other academic)
Abstract [en]

1 The effects of the nitrosoamine dephostatin on cytosolic Ca2+ and nitric oxide (NO)/cGMP signallings in human platelets were investigated.

2 Dephostatin has been characterised as a protein tyrosine phosphatase (PTP) inhibitor, and may on that account affect platelet responses. However, western blot analysis revealed that dephostatin (1-100 µM) did not increase tyrosine-specific protein phosphorylation.

3 Dephostatin dose-dependently (0.003-3 µM) inhibited thrombin-, thrombin-receptor activating peptide-, and ADP-stimulated rises in cytosolic free Ca2+ concentration, [Ca2+]i. in fura-2-loaded platelets. Surprisingly, higher doses of dephostatin (10-30 µM) augmented thrombin-triggered Ca2+ response. This dual action of dephostatin mainly involved modulation of Ca2+ influx.

4 The results revealed that dephostatin antagonised NO/cGMP- and prostaglandin E1/cAMP-mediated inhibition of [Ca2+]1. The action of the thrornbin-neutralising peptide hirudin was, however, unaffected.

5 Dephostatin did not affect basal or NO-mediated rises in platelet cGMP content. On the other hand, dephostatin alone directly increased the phosphorylation of ser239 on vasodilator-stimulated phosphoprotein (VASP) and markedly enhanced NO/cGMP-dependent VASP phosphorylation.

6 Cell functional studies revealed that dephostatin amplified NO-induced inhibition of platelet aggregation. Opposite to that, dephostatin diminished the inhibitory action of NO on phosphatidylserine exposure.

7 In conclusion, the results revealed that dephostatin affects a wide range of mechanisms involved in Ca2+ homeostasis in platelets. These effects are apparently not due to inhibition of PTPs. However, enhancement of VASP phosphorylation represents another important molecular mechanism of dephostatin. Considering NO signalling, the results indicate that dephostatin may be an excellent tool when elucidating the relative importance of inhibition of Ca2+ versus VASP phosphorylation.

Keyword
calcium, cGMP, dephostatin, nitric oxide, platelets, vasodilator-stimulated phosphoprotein
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-82119 (URN)
Available from: 2012-10-01 Created: 2012-10-01 Last updated: 2012-10-01Bibliographically approved
3. Dual actions of dephostatin on the nitric oxide/cGMP-signalling pathway in porcine iliac arteries
Open this publication in new window or tab >>Dual actions of dephostatin on the nitric oxide/cGMP-signalling pathway in porcine iliac arteries
2005 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 521, no 1-3, 124-132 p.Article in journal (Refereed) Published
Abstract [en]

We examined the effects of the nitrosoamine dephostatin on the nitric oxide (NO)/cyclic guanosine 3′,5′-monophosphate (cGMP)-signalling in porcine iliac arteries. Dephostatin has been characterised as a tyrosine phosphatase inhibitor, but Western blot analyses showed that dephostatin did not augment tyrosine phosphorylation of arterial proteins. However, dephostatin relaxed pre-contracted arteries, and this effect was antagonised by the soluble guanylyl cyclase inhibitor 1H[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ). Furthermore, dephostatin increased the cGMP content and the serine phosphorylation of vasodilator-stimulated phosphoprotein. Dephostatin also inhibited the relaxation induced by acetylcholine and the NO-donor S-nitroso-N-acetyl-penicillamine (SNAP). In contrast, dephostatin did not affect the NO-dependent actions of 1,2,3,4-Oxatriazolium, 3-(3-chloro-2-metylphenyl)-5-[[(4methylphenyl)sulfonyl]amino]-hydroxide inner salt (GEA 3175). Measurement of NO revealed that dephostatin accelerated the consumption of NO. In conclusion, dephostatin exerts dual effects on the NO/cGMP-signalling pathway in iliac arteries. The drug actions included scavenging of NO, but also stimulation of cGMP production. These effects were not related to inhibition of tyrosine phosphatases.

Keyword
Dephostatin, GEA 3175, Nitric oxide, Protein phosphorylation, Relaxation, SNAP
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-30909 (URN)10.1016/j.ejphar.2005.08.023 (DOI)16577 (Local ID)16577 (Archive number)16577 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2012-10-01Bibliographically approved
4. Cross-talk between adenosine and the oxatriazole derivative GEA 3175 in platelets
Open this publication in new window or tab >>Cross-talk between adenosine and the oxatriazole derivative GEA 3175 in platelets
Show others...
2005 (English)In: European Journal of Pharmacology, ISSN 0014-2999, E-ISSN 1879-0712, Vol. 517, no 3, 149-157 p.Article in journal (Refereed) Published
Abstract [en]

We examined the interplay between adenosine and the nitric oxide (NO)-containing oxatriazole derivative GEA 3175 in human platelets. The importance of cyclic guanosine 3′5′-monophosphate (cGMP)-inhibited phosphodiesterases (PDEs) was elucidated by treating the platelets with adenosine combined with either GEA 3175 or the PDE3-inhibitor milrinone. The drug combinations provoked similar cyclic adenosine 3′5′-monophosphate (cAMP) responses. On the contrary, cGMP levels were increased only in GEA 3175-treated platelets. Both drug combinations reduced P-selectin exposure, platelet adhesion and fibrinogen-binding. However, adenosine together with GEA 3175 was more effective in inhibiting platelet aggregation and ATP release. Thrombin-induced rises in cytosolic Ca2+ were suppressed by the two drug combinations. Adenosine administered with GEA 3175 was, however, more effective in reducing Ca2+ influx.

In conclusion, the interaction between adenosine and GEA 3175 involves cGMP-mediated inhibition of PDE3. The results also imply that inhibition of Ca2+ influx represent another cGMP-specific mechanism that enhances the effect of adenosine.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-30677 (URN)10.1016/j.ejphar.2005.05.019 (DOI)16282 (Local ID)16282 (Archive number)16282 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2013-09-03Bibliographically approved

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