ADP‐induced bladder contractility is mediated by P2Y ₁₂ receptor and temporally regulated by ectonucleotidases and adenosine signaling

Abstract

Purinergic signaling comprises one key pathway in modulating bladder smooth muscle (BSM) contractility, disorders of which become highly prevalent with aging. ADP was first observed to modulate BSM contractility >40 yr ago, yet the underlying molecular mechanism still remains unclear. Here, we demonstrate, using myography, that ADP and ADP␤S dose-dependently induce mouse BSM contraction, and ADP-induced BSM contraction is blocked by a selective P2Y 12 receptor (P2Y 12 R) antagonist, PSB 0739 (25 M), but is unaffected by P2Y 1 and P2Y 13 receptor antagonists. Lower urinary tract symptoms (LUTS), including bladder overactivity, urinary urgency and frequency, urinary incontinence, and bladder pain, afflict millions of people and severely compromise quality of life and independence. A recent study reported that LUTS were highly prevalent among men and women aged Ͼ40 yr, with Ͼ70% experiencing some symptoms (1). The etiology of LUTS is extremely poorly understood, and treatment options are few and of limited efficacy.Bladder smooth muscle (BSM) plays an essential role during urinary filling and voiding, with highly coordinated phases of relaxation and contraction. Consequently, myogenic disorders are frequently involved in LUTS, as may be seen with the development of significant noncholinergic and non-P2X 1 receptor-mediated contraction in diabetic bladder dysfunction (2), yet knowledge of the underlying mechanisms remains poorly understood (3).BSM contractility is, in part, regulated by purinergic signaling. Extracellular purines like ATP and UTP can be released either as neurotransmitters at neuromuscular junctions or from somatic cells in response to environmental stress, and they bind to P2X (1-7) and P2Y (1,2,4,6,(11)(12)(13)(14) receptors to elicit downstream signaling. ATP/UTP can be further sequentially converted into ADP/UDP and ultimately to adenosine by ectonucleotidases to activate P2Y receptors and adenosine receptors (P1) correspondingly. Thus, ectonucleotidases temporally and spatially regulate the availability of purines to activate P1 and P2 receptors (4).The functional significance of purinergic signaling in regulating bladder and other organs had been shown Ͼ40 yr ago, in experiments with neuronal release or exogenously applied purines causing smooth muscle

An ex vivo bladder model with detrusor smooth muscle removed to analyse biologically active mediators released from the suburothelium

“…Thus, if enzymes that degrade nucleotides and nucleosides are available on either side of the urothelium, ATP will be degraded to ADP, which in turn will be degraded to AMP and then to adenosine. Previous studies have documented that ATP and ADP cause DSM contractions via stimulation of P 2X1 and P 2Y12 receptors on smooth muscle cells, respectively (Burnstock, 2014;Yu et al 2014), whereas NAD (Breen et al 2006) and ADO (Burnstock, 2014) cause DSM relaxation. Therefore, having the ability to measure simultaneously excitatory and inhibitory mediators during different stages of bladder filling will provide new insights into how the urothelium contributes to regulation of storage of urine during filling and initiation of voiding.…”

“…Previous studies have documented that ATP and ADP cause DSM contractions via stimulation of P 2X1 and P 2Y12 receptors on smooth muscle cells, respectively (Burnstock, ; Yu et al . ), whereas NAD (Breen et al . ) and ADO (Burnstock, ) cause DSM relaxation.…”

Targetable purinergic receptors P2Y12 and A2b antagonistically regulate bladder function

“…However, both P2Y 12 and A2B mRNA are well expressed in human bladder according to NCBI data. Interestingly, ADPβS has long been known to induce significant human BSM contraction, suggesting an important role of the P2Y 12 receptor in regulating human bladder activity (12,(22)(23)(24). Likewise, Northern blot and quantitative PCR analyses suggest high expression of A2b receptors in the human bladder wall (51,52).…”

“…ADP was reported to be a potent agonist for inducing BSM contraction, and ADPβS (an analog of ADP) was found to be as potent as P2X 1 receptor agonist α,β-meATP in inducing human BSM contraction (12,22,23). Using pharmacological tools, we recently demonstrated that ADP-induced bladder contractility is mediated by P2Y 12 receptor and that contraction was temporally regulated by ectonucleotidases and adenosine signaling (24). Adenosine, which originated from the conversion of ADP by Nt5E, was reported to relax BSM by activating adenosine A2 receptors.…”

Reduced Expression of P2Y2 Receptor and Acetylcholinesterase at Neuromuscular Junction of P2Y1 Receptor Knock-out Mice

“…ATP is co-stored with other neurotransmitters, such as acetylcholine (ACh), in a ratio of about 1:5, in which ATP is found to stabilize ACh and co-released from the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Upon pre-synaptic neural stimulation, ATP is released and triggers a series of signal transduction, which cross-talks with other co-released neurotransmitter-stimulated signaling pathways, and results in regulating post-synaptic gene expressions (Noguchi et al 2013;Yuan et al 2013;Jacob et al 2014;Yu et al 2014).…”