RESUMEN
AIMS: Caffeine is a methylxanthine with multiple actions in vascular smooth muscle cells (VSMCs), including the increase in the intracellular Ca2+ (iCa2+) concentration by the activation of ryanodine receptors (RyRs). The present study aimed at investigating the participation of Ca2+-influx through different Ca2+-channels on the transient contraction (TC) induced by caffeine in mice mesenteric arteries. MAIN METHODS: Second-order of mesenteric arteries was isolated from male Swiss mice. Vessels without functional endothelium were stimulated with caffeine (10â¯mM). The caffeine-induced TC was evaluated after the incubation of artery rings for 30â¯min with the following drugs: nifedipine (10⯵M), a Cav1.2 blocker; 2-aminoethoxydiphenyl borate (2-APB; 10⯵M) and ruthenium red (RuR; 10⯵M), transient receptor potential (TRPs) channels blockers; capsazepine (10⯵M) and HC067047 (10⯵M), TRPV1 and TRPV4 antagonists, respectively; paxilline (1⯵M), a selective BKCa blocker; and SKF-96365 (30⯵M), an Orai blocker. Ca2+-fluorescence measurements were also performed on the investigated arteries. KEY FINDINGS: The TC induced by caffeine was partially dependent on Ca2+-influx. However, the blockage of Cav1.2 increased the TC while reduced the iCa2+ signal. Similar results were observed after the blockage of TRPs or BKCa. Therefore, caffeine promoted Ca2+-influx via TRPs and Cav1.2, and hyperpolarization through the activation of BKCa, inducing negative feedback of TC. SIGNIFICANCE: Our results indicate an alternative mechanism for the control of VSMCs contraction in resistance arteries. The evidence of the negative feedback of contraction via TRP-Cav1.2-BKCa provides a new perspective for understanding the mechanism involved in the vascular responses triggered by caffeine.
Asunto(s)
Cafeína/farmacología , Canales de Calcio Tipo L/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Arterias Mesentéricas/efectos de los fármacos , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Canales Catiónicos TRPV/metabolismo , Vasoconstricción/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
The inhibitory effect of the flavonoid dioclein was assessed on purified vascular cyclic nucleotide phosphodiesterase isoforms (EC 3.1.4.17, PDE1-5) in comparison with 8-methoxymethyl-isobutylmethylxanthine (8-MM-IBMX) and vinpocetine which are currently used as PDE1 inhibitors. The mechanism underlying the vasorelaxant effect of dioclein was investigated in human saphenous vein. Dioclein inhibited PDE1 more selectively than vinpocetine and 8-MM-IBMX, with IC(50) values of 2.47+/-0.26 and 1.44+/-0.35 microM, respectively in basal- and calmodulin-activated states. Dioclein behaved as a competitive inhibitor for cGMP hydrolysis by PDE1 in basal- and calmodulin-activated states (K(i)=0.62+/-0.14 and 0.55+/-0.07 microM, respectively), indicating this inhibitory effect to be independent of calmodulin interactions. In addition, dioclein induced a concentration-dependent relaxation of human saphenous vein which was independent on the presence of functional endothelium (EC(50) values of 7.3+/-3.1 and 11+/-2.7 microM, respectively with and without endothelium). 8-MM-IBMX relaxed human saphenous vein with an EC(50)=31+/-16 microM, whereas vinpocetine did not cause any vasorelaxation at concentrations up to 100 microM. Rp-8-pCPT-cGMPS, which inhibits cGMP-dependent protein kinase (PKG), blocked the vasodilator effect of dioclein, whereas H-89, which is a cAMP-dependent protein kinase (PKA) inhibitor, had a minor inhibitory effect. Our data show that dioclein is a potent calmodulin-independent selective inhibitor of PDE1 and that inhibition of PDE1 is involved in the PKG-mediated vasorelaxant effect of dioclein in human saphenous vein. Furthermore, dioclein may represent a new archetype to develop more specific PDE1 inhibitors.