RESUMEN
We reported previously that the rat submandibular gland is able to release nanovesicles capable to hydrolyse millimolar concentrations of ATP, ADP and AMP in vitro. Here, we show that rat saliva also contains nanovesicles with the ability to hydrolyse ATP. Our aim was to identify and characterize vesicular nucleotidases by using kinetic, immunological and in silico approaches. Nucleotidase activity in the absence or presence of specific inhibitors allowed us to assume the participation of NTPDase1, -2 and -3, together with ecto-5'-nucleotidase, confirmed using specific antibodies. At neutral pH, initial ATPase activity would be mostly due to NTPDase2, which was thereafter inactivated, leaving NTPDase1 and NTPDase3 to hydrolyse ATP and ADP with an efficacy ATPase/ADPase around 2. Ecto-5'nucleotidase would be mainly responsible for AMP hydrolysis and adenosine accumulation. We proposed a kinetic model for NTPDase2 as a tool to isolate and analyse the turnover of this enzyme in the presence of different ATP concentrations, including those expected in extracellular media. Our study characterizes the ectonucleotidases carried by extracellular vesicles which contribute to modulate ATP and adenosine concentrations in the oral cavity, essential players in purinergic signalling.
Asunto(s)
5'-Nucleotidasa/metabolismo , Vesículas Extracelulares/metabolismo , Boca/metabolismo , Pirofosfatasas/metabolismo , Saliva/enzimología , Proteínas y Péptidos Salivales/metabolismo , Transducción de Señal , Adenosina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Ratas , Ratas WistarRESUMEN
BACKGROUND: Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. OBJECTIVE: To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. METHODS: Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgADP or MgAMP. RESULTS: Histamine increased the release of vesicles with nucleotidase activity in a concentration dependent manner. JNJ7777120 significantly reduced this effect. Vesicular nucleotidases obtained in the absence or presence of histamine promoted Pi production from ATP, ADP and AMP. CONCLUSION: The results show a relationship between histamine and the regulation of purinergic signaling, which could be important in the modulation of inflammatory processes.