P2X receptor expression in mouse urinary bladder and the requirement of P2X₁ receptors for functional P2X receptor responses in the mouse urinary bladder smooth muscle

Abstract

1 We have used subtype selective P2X receptor antibodies to determine the expression of P2X 1 ± 7 receptor subunits in the mouse urinary bladder. In addition we have compared P2X receptor mediated responses in normal and P2X 1 receptor de®cient mice to determine the contribution of the P2X 1 receptor to the mouse bladder smooth muscle P2X receptor phenotype. 2 P2X 1 receptor immunoreactivity was restricted to smooth muscle of the bladder and arteries and was predominantly associated with the extracellular membrane. Diuse P2X 2 and P2X 4 receptor immunoreactivity not associated with the extracellular membrane was detected in the smooth muscle and epithelial layers. Immunoreactivity for the P2X 7 receptor was associated with the innermost epithelial layers and some diuse staining was seen in the smooth muscle layer. P2X 3 , P2X 5 and P2X 6 receptor immunoreactivity was not detected. 3 P2X receptor mediated inward currents and contractions were abolished in bladder smooth muscle from P2X 1 receptor de®cient mice. In normal bladder nerve stimulation evoked contractions with P2X and muscarinic acetylcholine (mACh) receptor mediated components. In bladder from the P2X 1 receptor de®cient mouse the contraction was mediated solely by mACh receptors. Contractions to carbachol were unaected in P2X 1 receptor de®cient mice demonstrating that there had been no compensatory eect on mACh receptors. 4 These results indicate that homomeric P2X 1 receptors underlie the bladder smooth muscle P2X receptor phenotype and suggest that mouse bladder from P2X 1 receptor de®cient and normal animals may be models of human bladder function in normal and diseased states.

Control of Urinary Drainage and Voiding

“…Purinergic receptors respond to nucleotides and nucleosides, like ATP, ADP, UTP, UDP, and adenosine. The primary purinergic receptor present on BSM is P2X; this has been confirmed pharmacologically by testing muscle strips for contractile responses with specific P2X 1 agonists and antagonists by immunostaining (48) and from studies of P2X 1 knockout mice (49). Recent work from our lab has confirmed the first functional evidence for a purinergic receptor Y (P2Y) in BSM-P2Y 6 (50).…”

Identification of Atropine- and P2X1Receptor Antagonist-Resistant, Neurogenic Contractions of the Urinary Bladder

“…The P2X 4 homomultimer is probably not involved, because suramin abolishes the purinergic excitatory junction potentials in the guinea pig (Hashitani and Suzuki, 1995) and genetic deletion of the P2X 1 subunit abolishes noncholinergic, neurogenic contractions and responses to exogenous ATP in the mouse (Vial and Evans, 2000) (although a crucial role of P2X 1 subunits in trafficking of P2X 4 subunits to the plasma membrane cannot be ruled out). A role for a P2X 1 ϩ 4 heteromultimer can, however, be considered.…”

“…In each case, little reversal of the inhibition was seen on washout of the antagonists. Genetic deletion of the P2X 1 receptor abolishes noncholinergic, neurogenic contractions in mouse urinary bladder (Vial and Evans, 2000), so we next investigated the effects of P2X antagonists in mouse tissue to determine whether our data are species specific. EFS (4 Hz, 0.15 ms, 20 s) elicited contractions of mouse detrusor smooth muscle of 465 Ϯ 68 mg (n ϭ 8) peak amplitude.…”

Purinergic receptors expressed in human skeletal muscle fibres

“…The following primary antibodies were used, either alone or two together (rabbit+mouse): Rabbit polyclonal anti-P2X 1 (APR-001, Alomone Labs, Israel; 1:500; previously used in the following studies [6,8,[13][14][15][16][17][18][19]); mouse polyclonal anti-P2X 4 (H00005025-B01P; Abnova, Taiwan; 1:500); rabbit polyclonal anti-P2Y 1 (APR-009, Alomone Labs; 1:500; previously used in the following studies [18,[20][21][22]); polyclonal anti-P2Y 2 (APR-010, Alomone Labs; 1:500; previously used in the following studies [6,8,18,[22][23][24]); rabbit polyclonal anti-P2Y 4 (P6497, Sigma-Aldrich, Denmark; 1:250); rabbit polyclonal anti-P2Y 11 (APR-015, Alomone Labs; 1:500; previously used in the following studies [18,[25][26][27][28][29][30]); rabbit polyclonal anti-P2Y 12 (HPA013796, Sigma-Aldrich; 1:30; previously used in the following study [31]). The P2X 1 antibody was highly specific and directed against the epitope corresponding to amino acid residues 382-399 of rat P2X 1 (human 15/18 residues identical); the P2Y 11 antibody was corresponding to amino acid residues 357-373 of human P2Y 11 ; western blots for anti-P2X 1 and anti-P2Y 11 are shown on the Alomone homepage.…”