The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-, which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca 2ϩ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC 50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4␣-phorbol 12,13-didecanoate (4␣-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4 ϩ/ϩ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4 Ϫ/Ϫ bladders. TRPV4 activation with GSK1016790A contracted TRPV4 ϩ/ϩ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4 Ϫ/Ϫ bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4␣-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4 ϩ/ϩ and TRPV4 Ϫ/Ϫ mice revealed an enhanced bladder capacity in the TRPV4 Ϫ/Ϫ mice. Infusion of GSK1016790A into the bladders of TRPV4 ϩ/ϩ mice induced bladder overactivity with no effect in TRPV4 Ϫ/Ϫ mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.Transient receptor potential (TRP) vanilloid 4 (TRPV4), a member of the TRP superfamily of cation channels, has been implicated in a number of physiological processes, including osmoregulation (Liedtke and Friedman, 2003;Mizuno et al., 2003), hearing (Tabuchi et al., 2005), thermal and mechaniThis work was supported by GlaxoSmithKline Pharmaceuticals. Article, publication date, and citation information can be found at

Efficient control of synaptic transmission requires a rapid mechanism for terminating the actions of neurotransmitters. For amino acids and monoamines, this is achieved by their uptake into the cell by specific high-affinity transporters; acetylcholine is first broken down in the extracellular space and then choline is taken up by the cell. Because ATP is hydrolysed to adenosine by membrane-bound enzymes (ectonucleotidases) that are present in most tissues, it has been assumed that these enzymes terminate the neurotransmitter actions of ATP in the brain and in the periphery. We show here, however, that stimulation of sympathetic nerves innervating the guinea-pig vas deferens releases not only neuronal ATP, but also soluble nucleotidases that break down this ATP to adenosine, indicating that inactivation of ATP is increased by nerve activity. This release of specific nucleotidases together with ATP represents a new mechanism for terminating the actions of a neurotransmitter.

1 The involvement of Rho-kinase (ROCK) in the contractile mechanisms mediating smooth muscle contraction of the rat urinary bladder was investigated using expression studies and the ROCK inhibitor Y-27632. 2 Both isoforms of ROCK (ROCK I and ROCK II) were detected in high levels in rat urinary bladder. 3 Y-27632 (10 mM) signi®cantly attenuated contractions of rat urinary bladder strips evoked by the G-protein coupled receptor agonists carbachol (58.1+10.5% at 0.3 mM) and neurokinin A (68.6+12.7% at 1 mM) without a ecting contractions to potassium chloride (10 ± 100 mM). In addition, basal tone was reduced by 47.8+2.0% by 10 mM Y-27632 in the absence of stimulation. 4 Contractions of urinary bladder strips evoked by the P2X receptor agonist a,b-methylene ATP (a,b-mATP; 10 mM) were also attenuated by Y-27632 (30.0+7.2% at 10 mM). 5 Y-27632 (10 mM) signi®cantly attenuated contractions evoked by electrical ®eld stimulation (2 ± 16 Hz). The e ect of Y-27632 on the tonic portion of the neurogenic response (4 ± 16 Hz) was not signi®cantly di erent from the e ect of atropine (1 mM) alone. 6 While the mechanism underlying the ability of Y-27632 to inhibit a,b-mATP-evoked contractions remains undetermined, the results of the present study clearly demonstrate a role for ROCK in the regulation of rat urinary bladder smooth muscle contraction and tone.

Oxytocin is known to have an antidiuretic effect, but the mechanisms underlying this effect are not completely understood. We infused oxytocin by osmotic minipump into vasopressin-deficient Brattleboro rats for five days and observed marked antidiuresis, increased urine osmolality, and increased solute-free water reabsorption. Administration of oxytocin also significantly increased the protein levels of aquaporin-2 (AQP2), phosphorylated AQP2 (p-AQP2), and AQP3 in the inner medulla and in the outer medulla plus cortex. Immunohistochemistry demonstrated increased AQP2 and p-AQP2 expression and trafficking to the apical plasma membrane of principal cells in the collecting duct, and increased AQP3 expression in the basolateral membrane. These oxytocin-induced effects were blocked by treatment with the vasopressin V2 receptor antagonist SR121463B, but not by treatment with the oxytocin receptor antagonist GW796679X. We conclude that vasopressin V2 receptors mediate the antidiuretic effects of oxytocin, including increased expression and apical trafficking of AQP2, p-AQP2, and increased AQP3 protein expression. 19: 225-232, 200819: 225-232, . doi: 10.1681 In recent years, the understanding of the molecular basis for the antidiuretic effect of vasopressin has been substantially advanced. The vasopressin V2 receptor 1 and renal aquaporin (AQP) water channels 2 have been cloned. Vasopressin has been shown to mediate both long-and short-term effects on AQP2 in the principal cells of the collecting duct. The long-term effect of vasopressin leads to increased expression of AQP2, whereas the shortterm effects involve trafficking of AQP2 to the apical membrane of the principal cells. 3 There is also evidence that vasopressin increases AQP3 protein expression on the basolateral membrane of the principal cells 4 and up-regulates the Na-K-2Cl cotransporter, 5 the initiator of the countercurrent concentrating mechanism. J Am Soc NephrolOxytocin (OT) is also known to possess antidiuretic properties. 6 In this regard, use of OT to induce labor in pregnancy has been associated with water retention and hyponatremia. 7 OT has been shown in vitro to increase osmotic water transport in microdissected renal inner medullary collecting ducts (IMCD) 8 and in vivo to cause an antidiuresis in vasopressin-deficient Brattleboro rats. 9,10 These effects were reversed by a vasopressin V2 receptor antagonist, suggesting that OT stimulation of vasopressin receptors mediates the antidiuresis. These in vitro observations were not altered by two different OT receptor antagonists. 8 The effect of OT on the long-and short-term regulation of AQP2 or the expression of AQP3 and Na-K-2Cl cotransporter has not been studied. The present study was therefore undertaken to advance the knowledge at the

Acetylcholine and ATP are excitatory cotransmitters in parasympathetic nerves. We used P2X 1 receptor antagonists to further characterize the purinergic component of neurotransmission in isolated detrusor muscle of guinea pig urinary bladder. In the presence of atropine (1 M) and prazosin (100 nM), pyridoxalphosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (PPADS) (0.1-100 M) and suramin (1-300 M) inhibited contractions evoked by 4 Hz nerve stimulation in a concentration-dependent manner (IC 50 of 6.9 and 13.4 M, respectively). Maximum inhibition was 50 -60%, which was unaffected by coadministration of the ectonucleotidase inhibitor ARL67156

1 Field stimulation of the sympathetic nerves of the guinea-pig isolated vas deferens with trains of pulses for 20 s at 1-8 Hz produced characteristic biphasic contractions. The effect of the novel ectoATPase inhibitor, 6-N,N-diethyl-D-13,y-dibromomethyleneATP (ARL 67156, formerly known as FPL 67156), on the magnitude of the initial, predominantly purinergic peak of this response was studied in order to determine the influence of enzymatic degradation of adenosine 5'-triphosphate (ATP) on its action as a neurotransmitter. 2 The peak magnitude of the response to nerve stimulation was significantly increased in a concentration-dependent manner by ARL 67156 (5-100 rM) and the size of the neurogenic response at 4 Hz was approximately doubled in the presence of ARL 67156 (100 gM). 3 ARL 67156 (100 gM) has a rapid onset of action. The enhancing effect on neurogenic contractions was maximal after 10 min, was well maintained for at least 30 min and was rapidly reversed, with responses returning to control levels 10 min after washout.4 The neurogenic contraction in the presence of prazosin (0.1 gM) was purely purinergic, as it was abolished by the P2-purinoceptor antagonist, PPADS (100 /M). ARL 67156 (100 /M) produced a similar degree of enhancement of neurogenic responses in the absence and presence of prazosin, supporting the view that the enhancing effects of ARL 67156 on neurogenic contractions result from potentiation of the action of ATP.5 Exogenous ATP and a,,B-methyleneATP produced rapid transient contractions. Responses to ATP were increased in magnitude and duration in the presence of ARL 67156 (100 gM), whereas those to the stable analogue, cx,fl-methyleneATP were not significantly affected.6 Contractions to exogenous noradrenaline (10 gM) and KCl (40 mM) were significantly enhanced by ARL 67156 (100 gM), but this potentiation was abolished by PPADS (100 pM). Therefore, this effect of the ecto-ATPase inhibitor may be due to a build up of endogenous ATP, increasing the sensitivity of the smooth muscle to other agonists. 7 It is concluded that ARL 67156 potentiates the action of ATP, and that when ATP acts as a neurotransmitter its postjunctional actions are greatly attenuated by enzymatic degradation.

Two endogenous receptors for the potent smooth musclestimulating peptide neuromedin U (NmU) have recently been identified and cloned. Pharmacological, binding, and expression studies were conducted in an attempt to determine the receptor(s) involved in the smooth muscle-stimulating effects of NmU. The NmU peptides caused a concentration-dependent contraction of canine isolated urinary bladder. NmU did not have this same effect in the urinary bladder from rat, guinea pig, rabbit, mouse, or ferret. Although NmU had no effect on canine uterus it did cause contraction of canine stomach, ileum, and colon. As well as causing contraction of canine bladder in vitro, NmU administered systemically resulted in a significant increase in urinary bladder pressure in vivo. High-affinity binding sites for NmU were identified in canine bladder. The four NmU peptides porcine NmU-8, rat NmU-23, human NmU-25, and porcine NmU-25 displaced 125 I-NmU-25 binding with similar K i values (0.08 -0.24 nM). A different binding profile was revealed in human embryonic kidney-293 cells transiently expressed with the canine NmU-2 receptor where porcine NmU-8 (K i ϭ 147.06 nM) was much less potent than the other NmU peptides. Using TaqMan, expression of NmU-1 was detected in human urinary bladder, small intestine, colon, and uterus. Expression of NmU-2 was much lower or absent in these human tissues and undetectable in canine bladder and stomach. The results of this study reveal significant species differences in the activity of NmU. The contractile activity in human and canine smooth muscle seems to be mediated by the recently cloned NmU-1 receptor.

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