Endogenous nucleotides have widespread actions in the cardiovascular system, but it is only recently that the P2X and P2Y receptor subtypes, at which they act, have been identified and subtype-selective agonists and antagonists developed. These advances have greatly increased our understanding of the physiological and pathophysiological functions of P2X and P2Y receptors, but investigation of the clinical usefulness of selective ligands is at an early stage. Nonetheless, the evidence considered in this review demonstrates clearly that various cardiovascular disorders, including vasospasm, hypertension, congestive heart failure and cardiac damage during ischemic episodes, may be viable targets. With further development of novel, selective agonists and antagonists, our understanding will continue to improve and further therapeutic applications are likely to be discovered.
ATP and UDP constrict rat intrapulmonary arteries, but which receptors mediate these actions is unclear. Here, we used selective agonists and antagonists, along with measurements of P2Y receptor expression, to characterize the receptor subtypes involved. Isometric tension was recorded from endotheliumdenuded rat intrapulmonary artery rings (i.d. 200 -500 m) mounted on a wire myograph. Expression of P2Y receptor subtype expression was determined by using reverse transcriptionpolymerase chain reaction with receptor-specific oligonucleotide primers. The selective P2Y 1 agonist (N)-methanocarba-2-methylthioadenosine-5Ј-O-diphosphate (MRS2365) induced small, concentration-dependent contractions that were inhibited by the P2Y 1 antagonist N 6 -methyl-2Ј-deoxyadenosine-3Ј,5Ј-bisphosphate (MRS2179). Contractions evoked by ATP were unaffected by MRS2179, but inhibited by approximately one-third by the P2Y 12 antagonist N 6 -(2-methylthiomethyl)-2-(3,3,3-trifluoropropylthio)dichloro-methylene ATP (AR-C69931MX). Combined blockade of P2X1 and P2Y 12 receptors virtually abolished the response to ATP. ADP also evoked contractions that were abolished by AR-C69931MX. The selective P2Y 6 receptor agonist 3-(2-oxo-2-phenylethyl)-UDP (PSB 0474) evoked concentrationdependent contractions and was approximately three times more potent than UDP, but the P2Y 14 agonist UDP-glucose had no effect. Contractions evoked by UDP were inhibited by the P2Y 6 receptor antagonist N,NЈ-1,4-butanediylbis-NЈ-(3-isothiocyanatophenyl)thiourea (MRS2578), but not the cysteinyl leukotriene 1 (CysL 1 ) antagonist 3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid (MK571). Higher concentrations of MRS2578 inhibited contractions to KCl, so they were not studied further. mRNA for P2Y 1 , P2Y 6 , and P2Y 12 receptors was identified. Our working model is that P2Y 12 and P2X1 receptors are present in rat intrapulmonary arteries and together mediate ATP-induced vasoconstriction. Contractile P2Y 6 , but not P2Y 14 or CysLT 1 , receptors are also present and are a major site through which UDP evokes constriction.
Background: Phosphodiesterase-5 inhibitors (PDE5i) are the first line of drugs used for the treatment of erectile dysfunction. PDE5i inhibits the activities of phosphodiesterase-5 (PDE5) found in corpus cavernosum reducing production of guanosine monophosphate (GMP) that results in vasodilation of blood vessels thereby prolonging penile erection. Various counterfeit drugs adulterated with unknown levels of PDE5i pose a danger with reported undesirable adverse effects on patients. Methods: We developed an assay to detect counterfeit drugs containing PDE5i using spectrophotometry based on a colour change of malachite green from blue to green when exposed to inorganic phosphate (Pi) measured at 630 nm. Initially, a standard graph of a known Pi concentration was established ranging from 0 to 20 µM followed by a dual biochemical reaction system of converting GMP from cGMP by PDE5, generating Pi from the first reaction’s product by calf intestinal alkaline phosphatase (CIAP), and then measuring Pi using an equation derived from the generated standard curve. Dilutions of CX1A, a counterfeit drug sample adulterated with the PDE5i sildenafil, were prepared via ultrasonication and filtration for assay testing and evaluation. Results: Sil was detected from the two separate samples comprised of low and high dilutions of CX1A quantified as 0.673% and 0.407%, respectively, based on a standard curve of pure sildenafil established from 0.1 nM to 300 µM. Conclusions: This PDE5i assay that we developed has the potential to become an alternative analytical method for detecting PDE5i showing comparable results when evaluated using HPLC.
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