The role of ATP signalling in response to mechanical stimulation studied in T24 cells using new microphysiological tools.

The capacity to store urine and initiate voiding is a valued characteristic of the human urinary bladder. To maintain this feature, it is necessary that the bladder can sense when it is full and when it is time to void. The bladder has a specialized epithelium called urothelium that is believed to be important for its sensory function. It has been suggested that autocrine ATP signalling contributes to this sensory function of the urothelium. There is well-established evidence that ATP is released via vesicular exocytosis as well as by pannexin hemichannels upon mechanical stimulation. However, there are still many details that need elucidation and therefore there is a need for the development of new tools to further explore this fascinating field. In this work, we use new microphysiological systems to study mechanostimulation at a cellular level: a mechanostimulation microchip and a silicone-based cell stretcher. Using these tools, we show that ATP is released upon cell stretching and that extracellular ATP contributes to a major part of Ca2+ signalling induced by stretching in T24 cells. These results contribute to the increasing body of evidence for ATP signalling as an important component for the sensory function of urothelial cells. This encourages the development of drugs targeting P2 receptors to relieve suffering from overactive bladder disorder and incontinence.

Inhibitory Effects of Urothelium-related Factors.

The urothelium of the bladder has long been recognized as a protective barrier between detrusor and urine. In recent years, it has become more evident that the urothelium plays a role as an active source of mediators. The urothelium can release neurotransmitters and modulators such as acetylcholine, ATP, nitric oxide, prostaglandins and neuropeptides. They exert both excitatory and inhibitory effects in modulating urinary tract motility. In addition, several studies have reported the existence of an urothelium-derived unknown inhibitory factor in the urinary bladder. By the use of a new serial cascade superfusion bioassay on guinea pig ureter, recent studies confirm that the guinea pig bladder urothelium releases a substance with inhibitory bioactivity, which was resistant to treatment with nitric oxide synthase inhibitor and cyclooxygenase inhibitor and to adenosine A1/A2 receptor blockade. Lately, a marked and quickly inactivated novel release of PGD2 from the bladder urothelium was discovered, together with localization of prostaglandin D synthase therein. PGD2 was found to have an inhibitory influence on nerve-induced contractions in guinea pig urinary bladder and on spontaneous contractions in the out-flow region. An altered release of excitatory and inhibitory factors is likely to play an important part in bladder motility disturbances, of which the prostanoids are a notable group. Due to the fact that the bladder is relaxed 99% of the time, not only excitatory mechanisms in the bladder are necessary to study, but also inhibitory mechanisms need considerable attention, which will contribute to the discovery of new targets to treat bladder motility disorders.

Cascade bioassay evidence for the existence of urothelium-derived inhibitory factor in Guinea pig urinary bladder.

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1-5 µM in the presence of scopolamine 5-30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.