NTPDase1 (CD39) controls nucleotide-dependent vasoconstriction in mouse

Abstract

Aims: Extracellular nucleotides are vasoactive molecules. The concentrations of these molecules are regulated by ectonucleotidases. In this study, we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the vasoconstrictor effect of nucleotides using Entpd1(-/-) mice.

Methods and results: Immunofluorescence, enzyme histochemistry, and HPLC analysis were used to evaluate both NTPDase expression and activity in arteries and isolated vascular smooth muscle cells (VSMCs). Vascular reactivity was evaluated in vitro and mean arterial blood pressure was recorded in anesthetized mice after nucleotide i.v. infusion. Expression of nucleotide receptors in VSMCs was determined by RT-PCR. Entpd1(-/-) mice displayed a dramatic deficit of nucleotidase activity in blood vessel wall in situ and in VSMCs in comparison to control mice. In aortic rings from Entpd1(-/-) mice, UDP and UTP induced a potent and long-lasting constriction contrasting with the weak response obtained in wild-type rings. This constriction occurred through activation of P2Y(6) receptor and was independent of other uracil nucleotide-responding receptors (P2Y(2) and P2Y(4)). UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1(-/-) mice. In addition, pressurized mesenteric arteries from Entpd1(-/-) mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. This effect was inhibited by the P2 receptor antagonist RB-2.

Conclusion: NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.

Figure 1

Deficit of nucleotidase activity in Entpd1^−/− mouse vasculature. (A) Entpd1^+/+ mouse aortic sections were analysed for expression of NTPDase1 and NTPDase2 by immunofluorescence (left panels: lum, lumen of the aorta; med, media; adv, adventitia). Purple colour on the merge panel materialize the colocalization of NTPDase1 (red) and α-actin (blue). NTPDase2 (green) is expressed by adventitial cells. (B) As shown by brown precipitates, Entpd1^+/+ mouse aortas’ sections display a broad nucleotidase activity absent from the media of Entpd1^−/− sections. (C) Similar deficit of nucleotide hydrolysis was observed in heart arteries. NTPDase2 activity is easily distinguished in the proximal adventitia of Entpd1^−/− arteries with triphosphate nucleotides as substrates (B and C).

Figure 2

NTPDase1 is the major ectonucleotidase at the surface of VSMCs. (A) Western blot analysis showing NTPDase1 expression in NTPDase1 transfected cells (COS mN1), Entpd1^+/+ VSMCs, but not in Entpd1^−/− VSMCs. The loading control with the anti-α-actin antibody revealed a band in both cultures but was absent in COS cells. (B) The nucleotide hydrolysis of Entpd1^+/+ and Entpd1^−/− VSMCs supernatants was evaluated by HPLC at the indicated time-points after addition of the indicated nucleotides (100 μmol/L). Results were normalized to spontaneous degradation of nucleotides. Data represent the mean + SEM of experiments performed in duplicates on 3–5 different cell cultures. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 3

Contractile effect of nucleotides is unmasked in Entpd1^−/− aortas. (A) While the constriction produced by depolarizing KCl (50 mmol/L) was similar in both strain of mice, (B) the vasoconstrictor effect of UDP (100 μmol/L) was greatly enhanced and more stable in Entpd1^−/− compared with wild-type aortic rings. (C–F) Nucleotides induced a sigmoid shaped dose-dependent contraction in Entpd1^−/− aortic rings (open circles) but only a weak and progressive tension in Entpd1^+/+ rings (filled circles). (G) The dose–response curve induced by U46619 was similar in both genotypes. (H ) Pharmacological inhibition of NTPDase1 with ARL 67156 potentiated the contraction induced by UDP in a dose-dependent manner in Entpd1^+/+ but not in Entpd1^−/− aortic rings. Each dose-response curve was built from the experiment performed on rings from four to seven aortas from different mice.

Figure 4

Expression of nucleotide responding receptors in aorta and VSMCs cultures. Expression of P2Y, orphan GPR17, GPR34, GPR87 as well as CysLT₁ and CysLT₂ receptors was examined in aortas and VSMCs cultures (passage 2–4) by RT–PCR. Positive controls (Ctrl) were achieved with appropriate cDNAs. Expected PCR fragment sizes are listed in Supplementary material online.

Figure 5

P2Y₆ receptor mediates uracil nucleotide-induced vasoconstriction in mouse aorta. Constriction experiments were performed in the presence of the NTPDase1 inhibitor ARL 67156 (100 μmol/L). UTP and UDP-induced an equivalent concentration-dependent contraction in P2ry2^−/− (filled triangle), P2ry4^−/− (filled square), and C57Bl/6 control (filled circle) aortic rings which was absent in P2ry6^−/− (filled diamond) rings. ATPγS-dependent contraction was partially diminished in P2ry2^−/− aortic rings. Results are expressed as the percentage of KCl (50 mmol/L)-induced contraction and represent the mean ±SEM of five independent experiments for each group of mice tested except for P2ry6^−/− that were carried out with the aortic sections from three individuals. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

In vivo effect of UDP. (A) The intravenous infusion of UDP (10 μmol/kg) induced an initial fast hypotensive response followed by a prolonged increase in blood pressure that reached a maximum between 2 and 3 min following infusion that was significantly higher in Entpd1^−/− mice. (B) The maximal increase in blood pressure was significantly higher in Entpd1^−/− for 1 and 10 μmol/kg UDP infusions. As a control, similar increase pressor effect was observed in response to angiotensin II (AgII, 100 pmol/kg). Data represent the mean ± SEM of four experiments performed on different mice. *P < 0.05, **P < 0.01.

Figure 7

Increased myogenic tone in Entpd1^−/− arteries. Third-order mesenteric arteries reactivity was evaluated with a wire myograph. (A) Uracil nucleotide-induced contractions were enhanced in Entpd1^−/− arteries. These responses were inhibited by the P2 receptor antagonist RB-2 (30 μmol/L). (B) An arteriograph was used for MT determination (see Methods). The luminal pressure was increased in 25 mmHg steps from 25 to 150 mmHg. The myogenic response (% MT) was expressed by the active (AD, determined with Ca²⁺) and passive diameters (PD, determined without Ca²⁺) such that %MT= [(PD−AD)/PD] · 100%. (C) The passive dilatation of arteries (without calcium) was equivalent in both strains of mice. Data in each panel represent the mean ± EM of measurements performed on arteries from eight different mice. Data were analysed by ANOVA followed by a Bonferroni multiple comparison post hoc test. **P < 0.01, ***P < 0.001.