The ecto-nucleotidase NTPDase1 differentially regulates P2Y1 and P2Y2 receptor-dependent vasorelaxation

Abstract

Background and purpose: Extracellular nucleotides produce vasodilatation through endothelial P2 receptor activation. As these autacoids are actively metabolized by the ecto-nucleotidase nucleoside triphosphate diphosphohydrolase-1 (NTPDase1), we studied the effects of this cell surface enzyme on nucleotide-dependent vasodilatation.

Experimental approach: Vascular NTPDase expression and activity were evaluated by immunohistochemistry and histochemistry. The vascular effects of nucleotides were tested in vivo by monitoring mean arterial pressure, and in vitro comparing reactivity of aortic rings using wild-type and Entpd1(-/-) (lacking NTPDase1) mice.

Key results: The absence of NTPDase1 in Entpd1(-/-) mice led to a dramatic drop in endothelial nucleotidase activity. This deficit was associated with an exacerbated decrease in blood pressure after nucleotide injection. Following ATP injection, mean arterial pressure was decreased in Entpd1(+/+) and Entpd1(-/-) mice by 5.0 and 17%, respectively, and by 0.1 and 19% after UTP injection (10 nmole.kg(-1) both). In vitro, the concentration-response curves of relaxation to ADP and ATP were shifted to the left, revealing a facilitation of endothelial P2Y1 and P2Y2 receptor activation in Entpd1(-/-) mice. EC(50) values in Entpd1(+/+) versus Entpd1(-/-) aortic rings were 14 microM versus 0.35 microM for ADP, and 29 microM versus 1 microM for ATP. In Entpd1(-/-) aortas, P2Y1 receptors were more extensively desensitized than P2Y2 receptors. Relaxations to the non-hydrolysable analogues ADPbetaS (P2Y1) and ATPgammaS (P2Y2) were equivalent in both genotypes confirming the normal functionality of these P2Y receptors in mutant mice.

Conclusions and implications: NTPDase1 controls endothelial P2Y receptor-dependent relaxation, regulating both agonist level and P2 receptor reactivity.

Figure 1

Deficit of nucleotidase activity in Entpd1^−/− mouse vasculature in situ. (A) Aorta sections: Nucleotide hydrolysis is impaired in the wall of Entpd1^−/− vessels compared with Entpd1^+/+ aortas, which display significant ADPase and ATPase activity, as shown by the brown deposit on VSMCs and endothelial cells. The remaining ATPase activity in the adventitia is due to nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). (B) Liver sections: The deficit in nucleotidase activity in the endothelium is more obvious in the hepatic central vein. The activity in canalicules is attributable to NTPDase8 (Fausther et al., 2007). (A and B) Control immunolabelling of the endothelium (PECAM) and NTPDase1 immunolabelling was performed as described. Note that NTPDase1 is expressed on both endothelial cells and VSMCs, in agreement with the ATPase and ADPase activities in these cells. Scale bars represent 50 µm.

Figure 2

The absence of nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) reveals a potent hypotensive effect of nucleotides. (A) Time course of the vascular response on the mean arterial pressure to injections of 10 nmole·kg⁻¹ of ATP (upper panel) or UTP (lower panel) in wild-type (Entpd1^+/+) and Entpd1^−/− mice. (B) Concentration-response curves comparing the maximal decrease in mean arterial pressure (ΔMAP max) in response to i.v. injections of ATP (upper panel) or UTP (lower panel). Data are representative of the mean ± SEM of four to five experiments performed on different mice. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

The relaxing effect of nucleotides is potentiated in Entpd1^−/− aortas. The relaxing effect of nucleotides was evaluated on U46619-precontracted aortas (30 nM). ADP and ATP were used as P2Y1 and P2Y2 receptor agonists, respectively. Both ADP (A) and ATP-dependent (B) relaxations were enhanced in Entpd1^−/− aortas. Relaxations induced by the non-hydrolysable analogues ADPβS (C) or ATPγS (D) were equivalent in both genotypes.

Figure 4

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) differentially regulates the homologous desensitization of endothelial P2Y1 and P2Y2 receptors. Panel (A) represents the sequence of the protocol. Entpd1^+/+ and Entpd1^−/− aortic rings were contracted with 30 nM U46619 and challenged for a first relaxation with 10 µM of either ADPβS (white arrow) or ATPγS (black arrow). After extensive washing of the non-hydrolysable analogue, the rings were exposed respectively to ADP or ATP for 30 min (0.01 to 10 mM), washed and challenged a second time for their ability to relax in response to the same non-hydrolysable agonist (second set of arrows). Panels (B) and (C) show representative tracings of the relaxation in response to ADPβS (b) or ATPγS (C) before (left) and after (right) desensitization to 100 µM ADP (B) or ATP (C). Panels (D) and (E) summarize the effects of the various concentrations of ADP and ATP used to desensitize ADPβS and ATPγS responses, respectively. Results are expressed as percentage of the first relaxation produced before desensitization. Data are representative of the mean ± SEM of 4–10 experiments performed on different mice. *P < 0.05; **P < 0.01; ***P < 0.001.