Whether SSTR subtype regulates DARPP-32 phosphorylation is not known was restricted to cell membrane

Figure 9 schematically illustrates our findings with each enzyme specifically expressed by particular cell types. NTPD1 is the major ectonucleotidase responsible for degrading ATP within the vasculature and our data clearly show that it is prominently expressed in endothelial cells within bladder. It has been shown to play a key role in hemostasis and thrombosis with complex effects on platelet aggregation. It is likely therefore that its presence in vascular elements within the bladder is not specific to this tissue. The presence of NTPD1 and NT5E in the cell membranes of smooth muscle cells suggests important functional roles related to muscle contraction and relaxation during the voiding cycle. Indeed, concerted actions are probable given what is known of urinary bladder smooth muscle physiology. To initiate voiding, parasympathetic nerves release ATP to stimulate bladder smooth muscle contraction through P2X1 receptors. NTPD1, also present on these membranes, has approximately equal affinities for ATP and ADP and is therefore able to rapidly catalyze the production of AMP. Following the contractile phase of voiding, NTPD1 and NT5E acting coordinately could rapidly convert ATP to adenosine in order to not only effect cessation of P2X1- mediated muscle contraction, but to facilitate muscle relaxation through A2b receptors. Relaxation is clearly a prerequisite for accommodating the next filling cycle. Support for this hypothesis comes from studies showing that adenosine receptor, A2b is abundantly expressed in detrusor, and further, that adenosine inhibits detrusor contraction elicited through carbachol, electrical field stimulation, acetylcholine and potassium. This model suggests that ATP is responsible not only for the contractile phase but also via NTPD1/NT5E activity, the relaxant phase of the micturition cycle and could therefore be considered a ‘dual effector’. NTPD2 shows an interesting partial distribution in lamina propria, being present in the deeper layer adjacent to the detrusor. Within detrusor, NTPD2-positive cells circumferentially surround and are in close proximity to smooth muscle bundles. Our data show clearly these cells are unlikely to be typical fibroblasts or nerve fibers. In fact based on their branched morphology, network patterning, location around muscle bundles and their intimate association with neurons, we considered the possibility that they might be interstitial cells of Cajal. Despite multiple attempts with different antibodies we were unable to immunostain for the ICC marker, c-kit. c-kit positive ICC, or pacemaker cells, have been convincingly demonstrated in bladders from a number of species including human, dog, pig, and guinea pig, however studies on bladders of mice are mixed. Lagou et al. demonstrated ICC in mouse bladder by morphology and by pharmacological stimulation, however ICC were c-kit negative. Likewise, Pezzone et al. also failed to detect c-kit in mouse bladder despite finding c-kit positive cells in the ureter. In contrast to this McCloskey et al. were able to show c-kit positive cells in mouse bladder. Despite our inability to colocalize c-kit with NTPD2, there remains a strong circumstantial case for considering that these cells may be ICC. There are other possibilities also. They may represent a subgroup of myofibroblasts which do not express aSMA. Cells matching this description were identified as myofibroblasts by Liu et al. Dranoff et al. noted that NTPD2 was present in a novel compartment of fibroblasts in liver, namely portal fibroblasts which surround intrahepatic bile ducts. In another study, myofibroblasts were found to exhibit close contacts with nerve varicosities in electron micrographs and the authors speculated that myofibroblasts and their attached axonal varicosities could collectively function as bladder stretch receptors. Our data show similar intimate contacts between NTPD2-positive cells and neurons. Since this ectoenzyme is highly specific for ATP over ADP it’s primary function is likely to be in attenuating ATP signaling and generating ADP/ UDP ligands for stimulating P2Y receptors in a cellular compartment that lies close to and surrounds the detrusor. It is possible given not only its proximity to smooth muscle but also to nerve fibers that it may play an important role in degrading ATP released as a neurotransmitter from efferent neurons. Fig. 9 shows the cross-boundary distribution of NTPD2-positive cells in both lamina propria and between muscle bundles in the detrusor. NTPD3 is uniquely localized to the urothelium and interestingly does not appear to present in umbrella cells but occurs in cell membranes of the intermediate and basal cell layers. This explains the observed difference in the kinetics of ATP hydrolysis on the luminal and serosal surfaces. Urothelial stretch-induced ATP secretion lumenally, is likely to have autocrine signaling effects on the apical membranes of umbrella cells while basal release suggests paracrine effects serosally. A primary signaling target for the urothelium is afferent neurons which are closely apposed to and penetrate between basal cells of the lamina propria and even urothelium. The signaling pathways activated by these interactions may influence such diverse phenomena as intracellular Ca2+ signaling action potentials and exocytosis of other potent mediators like nitric oxide, acetylcholine and prostaglandins. NTPD3 likely modulates the strength or duration of this stimulus on the cells in which it is expressed i.e. urothelium. Indeed if the primary target for basally secreted ATP is cells in the stroma or detrusor, its role might be to limit the potential for self-stimulation.