NTPDase1 Modulates Smooth Muscle Contraction in Mice Bladder by Regulating Nucleotide Receptor Activation Distinctly in Male and Female

Abstract

Nucleotides released by smooth muscle cells (SMCs) and by innervating nerve terminals activate specific P2 receptors and modulate bladder contraction. We hypothesized that cell surface enzymes regulate SMC contraction in mice bladder by controlling the concentration of nucleotides. We showed by immunohistochemistry, enzymatic histochemistry, and biochemical activities that nucleoside triphosphate diphosphohydrolase-1 (NTPDase1) and ecto-5'-nucleotidase were the major ectonucleotidases expressed by SMCs in the bladder. RT-qPCR revealed that, among the nucleotide receptors, there was higher expression of P2X1, P2Y₁, and P2Y₆ receptors. Ex vivo, nucleotides induced a more potent contraction of bladder strips isolated from NTPDase1 deficient (Entpd1^-/-) mice compared to wild type controls. The strongest responses were obtained with uridine 5'-triphosphate (UTP) and uridine 5'-diphosphate (UDP), suggesting the involvement of P2Y₆ receptors, which was confirmed with P2ry6^-/- bladder strips. Interestingly, this response was reduced in female bladders. Our results also suggest the participation of P2X1, P2Y₂ and/or P2Y₄, and P2Y₁₂ in these contractions. A reduced response to the thromboxane analogue U46619 was also observed in wild type, Entpd1^-/-, and P2ry6^-/- female bladders showing another difference due to sex. In summary, NTPDase1 modulates the activation of nucleotide receptors in mouse bladder SMCs, and contractions induced by P2Y₆ receptor activation were weaker in female bladders.

Keywords: NTPDase1; P2Y6 receptor; bladder; contraction; extracellular nucleotides; sex; smooth muscle cells.

Figure 1

Immunolocalization of ectonucleotidases in male (A) and female (B) mouse bladder. SM: smooth muscle; LP: lamina propria. EP: epithelium. bar = 60 µm. Entpd1^+/+: wild type mice. Entpd1^−/−: NTPDase1 deficient mice. 5NU: ecto-5′-nucleotidase. Experiments were done on 3 mice of each group.

Figure 1

Immunolocalization of ectonucleotidases in male (A) and female (B) mouse bladder. SM: smooth muscle; LP: lamina propria. EP: epithelium. bar = 60 µm. Entpd1^+/+: wild type mice. Entpd1^−/−: NTPDase1 deficient mice. 5NU: ecto-5′-nucleotidase. Experiments were done on 3 mice of each group.

Figure 2

Comparative analysis of ectonucleotidase activity in the bladder of Entpd1^+/+ (wild type) and Entpd1^−/− (nucleoside triphosphates diphosphohydrolase 1 (NTPDase1) deficient) female mice. SM (*): smooth muscle. bar = 60 µm. Control: no nucleotides. Adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), uridine 5′-triphosphate (UTP), uridine 5′-diphosphate (UDP), and adenosine 5′-monophosphate (AMP) were used as substrate at a concentration of 100 µM. Experiments were done on three mice of each group.

Figure 3

Hydrolysis of nucleotides in homogenates of male and female mouse bladders. Data presented are the mean ± SEM of seven experiments with homogenates from Entpd1^+/+ (wild type) and Entpd1^−/− (NTPDase1 deficient) male bladders (A) and four experiments with homogenates from male and female Entpd1^+/+ bladders (B), each in triplicate. Two symbols p < 0.01, three symbols p < 0.001. * Hydrolysis of Entpd1^−/− homogenates compared to that of wild type homogenates. n.s: not significant. ATP, ADP, UTP, UDP, and AMP were used as the substrate at a concentration of 500 µM.

Figure 4

Contractile effect of extracellular nucleotides on male and female mouse bladder strips. Data presented are mean ± SEM of 20 contraction experiments with Entpd1^+/+ (wild type) and Entpd1^−/− (NTPDase1 deficient) male bladders and 15 with Entpd1^+/+ and Entpd1^−/− female bladders. Two symbols p < 0.01, three symbols p < 0.001, and four symbols p < 0.0001. * Contraction of Entpd1^−/− mice compared to that of wild type strips and # contraction of male strips compared to female strips of the same genotype. ATP, ADP, UTP, and UDP were used at a concentration of 100 µM, 5-hydroxytryptamine (5HT) at 10 µM, and U46619 at 100 nM.

Figure 5

Gene expression profile in male and female bladder SMCs. SMCs were separated from the epithelial layer as mentioned in the “Materials and Methods” section. RNA was isolated from the SMC layer and the expression of the epithelial cell marker Krt7 and the SMC marker Myh11 (A), P2Y receptors (B), P2X receptors (C), ectonucleotidases (D), and P1 receptors (E) were quantified by RT-qPCR. Data are normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase; left panels) and ACTB (actin beta; right panels) mRNA level. Entpd1^+/+: wild type mice. Entpd1^−/−: NTPDase1 deficient mice. Data presented are the means ± SEM of three independent experiments in duplicate each with SMCs pooled from three mice.

Figure 6

Contractile effect of extracellular nucleotides on male and female strips from P2ry6^−/− mouse bladders. Data are presented as the mean ± SEM of 15 contraction experiments with strips from WT (wild type) and P2ry6^−/− (P2Y₆ receptor knock-out) male bladders and 10 with strips from WT and P2ry6^−/− female bladders. One symbol p < 0.05, 3 symbols p < 0.001. * Contraction of P2ry6^−/− bladders compared to WT; # Contraction of male strips compared to female strips of the same genotype. ATP, ADP, UTP, and UDP were used at the concentration of 100 µM, 5HT at 10 µM, and U46619 at 100 nM.

Figure 7

Effect of apyrase (Apy, 2 U/mL) (A) and the same amount of boiled apyrase (bApy) (B) on contraction induced by 100 µM ATP, ADP, UTP, or UDP on strips from male mouse bladders. Data presented are the mean ± SEM of four contraction experiments with strips from different mice. One symbol p < 0.05, two symbols p < 0.01, and three symbols p < 0.001. * Contraction in Entpd1^−/− bladders compared to contraction in WT; # Contraction in Entpd1^−/− bladders without apyrase compared to Entpd1^−/− bladders in presence of apyrase. There was no significant difference for WT bladders in presence or absence of apyrase or boiled apyrase.