Description: Bladder dysfunction characterized by abnormal bladder smooth muscle (BSM) contractions is pivotal to the disease process in overactive bladder, urge incontinence, and spinal cord injury. Purinergic signaling comprises one key pathway in modulating BSM contractility, but molecular mechanisms remain unclear. Here we demonstrate, using myography, that activation of P2Y 6 by either UDP or a specific agonist (MRS 2693) induced a sustained increase in BSM tone (up to 2 mN) in a concentration-dependent manner. Notably, activation of P2Y 6 enhanced ATP-mediated BSM contractile force by up to 45%, indicating synergistic interactions between P2X and P2Y signaling. P2Y 6 -activated responses were abolished by phospholipase C (PLC) and inositol trisphosphate (IP 3 ) receptor antagonists U73122 and xestospongin C, demonstrating involvement of the PLC/IP 3 signal pathway. Mice null for Entpd1, an ectonucleotidase on BSM, demonstrated increased force generation on P2Y 6 activation (150%). Thus, in vivo perturbations to purinergic signaling resulted in altered P2Y 6 activity and bladder contractility. We conclude that UDP, acting on P2Y 6 , regulates BSM tone and in doing so selectively maximizes P2X 1 -mediated contraction forces. This novel neurotransmitter pathway may play an important role in urinary voiding disorders characterized by abnormal bladder motility.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. Extracellular UDP enhances P2X-mediated bladder smooth muscle contractility via P2Y 6 activation of the phospholipase C/inositol trisphosphate pathway.

Description: Bladder urothelium senses and communicates information about bladder fullness. However, the mechanoreceptors that respond to tissue stretch are poorly defined. Integrins are mechanotransducers in other tissues. Therefore, we eliminated β1-integrin selectively in urothelium of mice using Cre-LoxP targeted gene deletion. β1-Integrin localized to basal/intermediate urothelial cells by confocal microscopy. β1-Integrin conditional-knockout (β1-cKO) mice lacking urothelial β1-integrin exhibited down-regulation and mislocalization of α3- and α5-integrins by immunohistochemistry but, surprisingly, had normal morphology, permeability, and transepithelial resistance when compared with Cre-negative littermate controls. β1-cKO mice were incontinent, as judged by random urine leakage on filter paper (4-fold higher spotting, P 〈0.01; 2.5-fold higher urine area percentage, P 〈0.05). Urodynamic function assessed by cystometry revealed bladder overfilling with 80% longer intercontractile intervals ( P 〈0.05) and detrusor hyperactivity (3-fold more prevoid contractions, P 〈0.05), but smooth muscle contractility remained intact. ATP secretion into the lumen was elevated (49 vs. 22 nM, P 〈0.05), indicating abnormal filling-induced purinergic signaling, and short-circuit currents (measured in Ussing chambers) revealed 2-fold higher stretch-activated ion channel conductances in response to hydrostatic pressure of 1 cmH 2 O ( P 〈0.05). We conclude that loss of integrin signaling from urothelium results in incontinence and overactive bladder due to abnormal mechanotransduction; more broadly, our findings indicate that urothelium itself directly modulates voiding.—Kanasaki, K., Yu, W., von Bodungen, M., Larigakis, J. D., Kanasaki, M., Ayala de la Pena, F., Kalluri, R., Hill, W.G. Loss of β1-integrin from urothelium results in overactive bladder and incontinence in mice: a mechanosensory rather than structural phenotype.

Description: 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 ADPβS 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 μM), but is unaffected by P2Y 1 and P2Y 13 receptor antagonists. P2Y 12 R in BSM exhibits distinct pharmacological properties that are different from P2Y 12 R in platelets. After an immediate contraction, prolonged exposure to ADP causes BSM to become refractory to further ADP-mediated contraction. However, in mice lacking ectonucleotidases Entpd1 (ATP-〉ADP-〉AMP) or Nt5e (AMP-〉adenosine), or by inhibiting adenosine signaling, the refractory response was altered, resulting in repeated BSM contractions in response to repeated ADP (0.1-1 mM) stimulation. Our data indicate that P2Y 12 R undergoes slow desensitization; ADP-P2Y 12 signaling is tightly regulated by Entpd1/Nt5e activity and adenosine receptors; and ADP-adenosine signaling play an important role in modulating P2X-mediated BSM contraction. The identification of P2Y 12 R in BSM, and the current clinical availability of P2Y 12 R inhibitors, such as clopidogrel, offers potentially novel treatment strategies for bladder contractility disorders.—Yu, W., Sun, X., Robson, S. C., Hill, W. G. ADP-induced bladder contractility is mediated by P2Y 12 receptor and temporally regulated by ectonucleotidases and adenosine signaling.