RESUMEN
Sigma-1 receptor (S1R) is detected in different cell types and can regulate intracellular signaling pathways. S1R plays a role in the pathomechanism of diseases and the regulation of neurotransmitters. Fluvoxamine can bind to S1R and reduce the serotonin uptake of neurons and platelets. We therefore hypothesized that platelets express S1R, which can modify platelet function. The expression of the SIGMAR1 gene in rat platelets was examined with a reverse transcription polymerase chain reaction and a quantitative polymerase chain reaction. The receptor was also visualized by immunostaining and confocal laser scanning microscopy. The effect of S1R agonist PRE-084 on the eicosanoid synthesis of isolated rat platelets and ADP- and AA-induced platelet aggregation was examined. S1R was detected in rat platelets both at gene and protein levels. Pretreatment with PRE-084 of resting platelets induced elevation of eicosanoid synthesis. The rate of elevation in thromboxane B2 and prostaglandin D2 synthesis was similar, but the production of prostaglandin E2 was higher. The concentration-response curve showed a sigmoidal form. The most effective concentration of the agonist was 2 µM. PRE-084 increased the quantity of cyclooxygenase-1 as detected by ELISA. PRE-084 also elevated the ADP- and AA-induced platelet aggregation. S1R of platelets might regulate physiological or pathological functions.
Asunto(s)
Plaquetas , Agregación Plaquetaria , Adenosina Difosfato/farmacología , Animales , Plaquetas/metabolismo , Eicosanoides/metabolismo , Eicosanoides/farmacología , Humanos , Prostaglandinas/metabolismo , Prostaglandinas/farmacología , RatasRESUMEN
Hyperglycemia, hyperlipidemia, and free radicals result in platelet activation and atherogenesis. Kisspeptin (KP) is able to regulate metabolism, hemostasis, and the development of atherosclerosis. We examined whether platelet aggregation of streptozotocin-induced diabetic rats depends on the inducer type and if KP-13 and RF-9 (a kisspeptin receptor modifier) can influence platelet function. We measured the speed and the maximum of aggregation, along with the area under the curve. Serum glucose and calcium levels and urine formation of diabetic animals increased, while the body mass and platelet count decreased. Collagen was the most effective inducer of platelet aggregation. The aggregability of nondiabetic platelets was elevated in the presence of 5 × 10-8 mol/L KP-13. This effect was less expressed in diabetic animals. The effectivity of RF-9 was stronger than that of KP-13 in nondiabetic platelets, however it was ineffective in diabetic animals. RF-9 pre-treatment did not change the effects of 5 × 10-8 mol/L KP-13 in either animal group. The in vivo activation of diabetic platelets, which may be due to elevated serum calcium, induces thrombocytopenia and may lead to reduced in vitro aggregability. We could not demonstrate the antagonistic effect of RF-9 against KP-13 in isolated platelets.
Asunto(s)
Plaquetas/efectos de los fármacos , Plaquetas/fisiología , Diabetes Mellitus Experimental/fisiopatología , Kisspeptinas/farmacología , Animales , Diabetes Mellitus Experimental/metabolismo , Kisspeptinas/metabolismo , Masculino , Agregación Plaquetaria/efectos de los fármacos , Ratas , Receptores de Kisspeptina-1/metabolismo , Receptores de Neuropéptido/metabolismoRESUMEN
BACKGROUND: Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. OBJECTIVES: Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. METHODS: The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. RESULTS: One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. CONCLUSIONS: S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.
Asunto(s)
Diabetes Mellitus Experimental , Animales , Ratas , Estreptozocina , Ácido Araquidónico/farmacología , Diabetes Mellitus Experimental/metabolismo , Ligandos , Células Endoteliales/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Ácidos Araquidónicos/metabolismo , Aorta/metabolismo , Eicosanoides , Ciclooxigenasa 2 , Vasodilatadores , Vasoconstrictores , Receptor Sigma-1RESUMEN
Platelets regulate cell-cell interactions and local circulation through eicosanoids from arachidonic acid. Sigma non-opioid intracellular receptor 1 (sigma-1 receptor) expressed in platelets and endothelial cells can regulate intracellular signalization. Our aim was to examine the influence of sub-chronic, in vivo-administered sigma-1 receptor ligands 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084); N-benzyl-2-[(1S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]ethan-1-amine; dihydrochloride, a new compound ((S)-L1); and N-[2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethyl]-N-propylpropan-1-amine (NE-100) on the ex vivo arachidonic acid metabolism of the platelets and aorta of male rats. The serum level of sigma-1 receptor ligands was determined by liquid chromatography-mass spectrometry. Sigma-1 receptor and cyclooxygenase gene expression in the platelets were determined by a reverse transcription-coupled quantitative polymerase chain reaction. The eicosanoid synthesis was examined using a radiolabeled arachidonic acid substrate and enzyme-linked immunosorbent assay. We confirmed the absorption of sigma-1 receptor ligands and confirmed that the ligands were not present during the ex vivo studies, so their acute effect could be excluded. We detected no changes in either sigma-1 receptor or cyclooxygenase mRNA levels in the platelets. Nevertheless, (S)-L1 and NE-100 increased the quantity of cyclooxygenases there. Both platelet and aortic eicosanoid synthesis was modified by the ligands, although in different ways. The effect of the new sigma-1 receptor ligand, (S)-L1, was similar to that of PRE-084 in most of the parameters studied but was found to be more potent. Our results suggest that sigma-1 receptor ligands may act at multiple points in arachidonic acid metabolism and play an important role in the control of the microcirculation by modulating the eicosanoid synthesis of the platelets and vessels.
Asunto(s)
Plaquetas , Receptores sigma , Animales , Aorta/metabolismo , Ácidos Araquidónicos/metabolismo , Ciclooxigenasa 2/metabolismo , Eicosanoides/metabolismo , Células Endoteliales/metabolismo , Ligandos , Masculino , Ratas , Receptores sigma/metabolismoRESUMEN
The increasing ineffectiveness of traditional antibiotics and the rise of multidrug resistant (MDR) bacteria have necessitated the revival of bacteriophage (phage) therapy. However, bacteria might also evolve resistance against phages. Phages and their bacterial hosts coexist in nature, resulting in a continuous coevolutionary competition for survival. We have isolated several clinical strains of Pseudomonas aeruginosa and phages that infect them. Among these, the PIAS (Phage Induced Antibiotic Sensitivity) phage belonging to the Myoviridae family can induce multistep genomic deletion in drug-resistant clinical strains of P. aeruginosa, producing a compromised drug efflux system in the bacterial host. We identified two types of mutant lines in the process: green mutants with SNPs (single nucleotide polymorphisms) and smaller deletions and brown mutants with large (â¼250 kbp) genomic deletion. We demonstrated that PIAS used the MexXY-OprM system to initiate the infection. P. aeruginosa clogged PIAS phage infection by either modifying or deleting these receptors. The green mutant gaining phage resistance by SNPs could be overcome by evolved PIASs (E-PIASs) with a mutation in its tail-fiber protein. Characterization of the mutant phages will provide a deeper understanding of phage-host interaction. The coevolutionary process continued with large deletions in the same regions of the bacterial genomes to block the (E-)PIAS infection. These mutants gained phage resistance via either complete loss or substantial modifications of the phage receptor, MexXY-OprM, negating its essential role in antibiotic resistance. In vitro and in vivo studies indicated that combined use of PIAS and antibiotics could effectively inhibit P. aeruginosa growth. The phage can either eradicate bacteria or induce antibiotic sensitivity in MDR-resistant clinical strains. We have explored the potential use of combination therapy as an alternative approach against MDR P. aeruginosa infection.