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An ex vivo standardized assay to measure human platelet cGMP
- Source :
- Journal of Pharmacological and Toxicological Methods. 64:164-167
- Publication Year :
- 2011
- Publisher :
- Elsevier BV, 2011.
-
Abstract
- Introduction: Nitric oxide (NO) acts a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. The intracellular levels of cyclic guanylate monophosphate (cGMP) can be increased by NO or by inhibiting the breakdown to 5'cGMP operated by the cyclic nucleotide phosphodiesterases (PDEs). Platelets are anuclear circulating cells that are rich in both soluble guanylyl cyclase and PDEs. The purpose of this study was to standardize cGMP determination in human platelets. Methods: Fresh blood samples were obtained from a group of healthy volunteers in order to obtain washed platelets. Platelet (3 x 10(4)/mu l 5 x 10(5)/mu l) cGMP levels were measured in basal and stimulated conditions. To stimulate platelets two different NO-donors were used: sodium nitroprusside (SNP; 10, 100, 1000 mu M) or diethylamine NONOate (DEA-NONOate; 1, 10, 100 mu M). Different times of incubation were also studied (5, 15 and 30 min). As positive control has been used ODQ a well known inhibitor of guanylyl cyclase. Platelet cGMP accumulation was measured by using a standard ELISA kit using different sample dilutions. Results: The optimal stimulus was DEA-NONOate, the optimal washed platelet concentration was 5 x 10(5)/mu l, incubation time was 30 mm and dilution to be used was 1:2. Discussion: Platelets represent a valuable marker to investigate the effect of drugs interfering with the cGMP cascade. This standardized assay allows to measure ex vivo the inhibitory (PDE inhibitors) or stimulatory effect (NO donors) of drugs given in vivo to humans. (C) 2011 Elsevier Inc. All rights reserved.
- Subjects :
- Blood Platelets
Nitroprusside
GUCY1B3
medicine.medical_specialty
Time Factors
Enzyme-Linked Immunosorbent Assay
Biology
Nitric Oxide
Toxicology
In vivo
Quinoxalines
Internal medicine
medicine
Humans
Nitric Oxide Donors
Platelet
Cyclic GMP
Pharmacology
Oxadiazoles
Dose-Response Relationship, Drug
PDE5 drug design
Hydrazines
Endocrinology
Guanylate Cyclase
PDE10A
Sodium nitroprusside
Soluble guanylyl cyclase
Ex vivo
medicine.drug
Subjects
Details
- ISSN :
- 10568719
- Volume :
- 64
- Database :
- OpenAIRE
- Journal :
- Journal of Pharmacological and Toxicological Methods
- Accession number :
- edsair.doi.dedup.....989d55b935eaac76e38b928187e7d22a