Anthony P. D. W. Ford

Urinary bladder hyporeflexia and reduced pain-related behaviour in P2X3-deficient mice

Extracellular ATP is implicated in numerous sensory processes ranging from the response to pain to the regulation of motility in visceral organs. The ATP receptor P2X3 is selectively expressed on small diameter sensory neurons, supporting this hypothesis. Here we show that mice deficient in P2X3 lose the rapidly desensitizing ATP-induced currents in dorsal root ganglion neurons. P2X3 deficiency also causes a reduction in the sustained ATP-induced currents in nodose ganglion neurons. P2X3-null mice have reduced pain-related behaviour in response to injection of ATP and formalin. Significantly, P2X3-null mice exhibit a marked urinary bladder hyporeflexia, characterized by decreased voiding frequency and increased bladder capacity, but normal bladder pressures. Immunohistochemical studies localize P2X3 to nerve fibres innervating the urinary bladder of wild-type mice, and show that loss of P2X3 does not alter sensory neuron innervation density. Thus, P2X3 is critical for peripheral pain responses and afferent pathways controlling urinary bladder volume reflexes. Antagonists to P2X3 may therefore have therapeutic potential in the treatment of disorders of urine storage and voiding such as overactive bladder.

P2X2 knockout mice and P2X2/P2X3 double knockout mice reveal a role for the P2X2 receptor subunit in mediating multiple sensory effects of ATP

Extracellular ATP plays a role in nociceptive signalling and sensory regulation of visceral function through ionotropic receptors variably composed of P2X2 and P2X3 subunits. P2X2 and P2X3 subunits can form homomultimeric P2X2, homomultimeric P2X3, or heteromultimeric P2X2/3 receptors. However, the relative contribution of these receptor subtypes to afferent functions of ATP in vivo is poorly understood. Here we describe null mutant mice lacking the P2X2 receptor subunit (P2X2−/−) and double mutant mice lacking both P2X2 and P2X3 subunits (P2X2/P2X3Dbl−/−), and compare these with previously characterized P2X3−/− mice. In patch‐clamp studies, nodose, coeliac and superior cervical ganglia (SCG) neurones from wild‐type mice responded to ATP with sustained inward currents, while dorsal root ganglia (DRG) neurones gave predominantly transient currents. Sensory neurones from P2X2−/− mice responded to ATP with only transient inward currents, while sympathetic neurones had barely detectable responses. Neurones from P2X2/P2X3Dbl−/− mice had minimal to no response to ATP. These data indicate that P2X receptors on sensory and sympathetic ganglion neurones involve almost exclusively P2X2 and P2X3 subunits. P2X2−/− and P2X2/P2X3Dbl−/− mice had reduced pain‐related behaviours in response to intraplantar injection of formalin. Significantly, P2X3−/−, P2X2−/−, and P2X2/P2X3Dbl−/− mice had reduced urinary bladder reflexes and decreased pelvic afferent nerve activity in response to bladder distension. No deficits in a wide variety of CNS behavioural tests were observed in P2X2−/− mice. Taken together, these data extend our findings for P2X3−/− mice, and reveal an important contribution of heteromeric P2X2/3 receptors to nociceptive responses and mechanosensory transduction within the urinary bladder.

Pharmacology of P2X channels

Significant progress in understanding the pharmacological characteristics and physiological importance of homomeric and heteromeric P2X channels has been achieved in recent years. P2X channels, gated by ATP and most likely trimerically assembled from seven known P2X subunits, are present in a broad distribution of tissues and are thought to play an important role in a variety of physiological functions, including peripheral and central neuronal transmission, smooth muscle contraction, and inflammation. The known homomeric and heteromeric P2X channels can be distinguished from each other on the basis of pharmacological differences when expressed recombinantly in cell lines, but whether this pharmacological classification holds true in native cells and in vivo is less well-established. Nevertheless, several potent and selective P2X antagonists have been discovered in recent years and shown to be efficacious in various animal models including those for visceral organ function, chronic inflammatory and neuropathic pain, and inflammation. The recent advancement of drug candidates targeting P2X channels into human trials, confirms the medicinal exploitability of this novel target family and provides hope that safe and effective medicines for the treatment of disorders involving P2X channels may be identified in the near future.

Pharmacological pleiotropism of the human recombinant α1A-adrenoceptor : implications for α1-adrenoceptor classification

Three fully‐defined α1‐adrenoceptors (α1A, α1B and α1D) have been established in pharmacological and molecular studies. A fourth α1‐adrenoceptor, the putative α1L‐adrenoceptor, has been defined in functional but not molecular studies, and has been proposed to mediate contraction of human lower urinary tract tissues; its relationship to the three fully characterized α1‐adrenoceptors is not known. In the present study, binding affinities were estimated by displacement of [3H]‐prazosin in membrane homogenates of Chinese hamster ovary (CHO‐K1) cells stably expressing the human α1A‐, α1B‐ and α1D‐adrenoceptors and were compared with affinity estimates obtained functionally in identical cells by measuring inhibition of noradrenaline (NA)‐stimulated accumulation of [3H]‐inositol phosphates. For the α1A‐adrenoceptor, binding studies revealed a pharmacological profile typical for the classically defined α1A‐adrenoceptor, such that prazosin, RS‐17053, WB4101, 5‐methylurapidil, Rec15/2739 and S‐niguldipine all displayed subnanomolar affinity. A different profile of affinity estimates was obtained in inositol phosphates accumulation studies: prazosin, WB4101, 5‐methylurapidil, RS‐17053 and S‐niguldipine showed 10 to 40 fold lower affinity than in membrane binding. However, affinity estimates were not ‘frameshifted’, as tamsulosin, indoramin and Rec15/2739 yielded similar, high affinity estimates in binding and functional assays. In contrast, results from human α1B‐ and α1D‐adrenoceptors expressed in CHO‐K1 cells gave antagonist affinity profiles in binding and functional assays that were essentially identical. A concordance of affinity estimates from the functional (inositol phosphates accumulation) studies of the α1A‐adrenoceptor in CHO‐K1 cells was found with estimates published recently from contractile studies in human lower urinary tract tissues (putative α1L‐adrenoceptor). These data show that upon functional pharmacological analysis, the cloned α1A‐adrenoceptor displays pharmacological recognition properties consistent with those of the putative α1L‐adrenoceptor. Why this profile differs from that obtained in membrane binding, and whether it explains the α1L‐adrenoceptor pharmacology observed in many native tissues, requires further investigation.

Immunocytochemical localization of P2X3 purinoceptors in sensory neurons in naive rats and following neuropathic injury

P2X3 purinoceptor cellular distribution was studied in rat sensory neurons in naive animals and following peripheral nerve injury using immunohistochemical methods. Specific antiserum was raised in rabbits and characterized by Western blot, absorption assays and labeling of recombinant receptors. In naive animals, P2X3 immunoreactivity was present predominantly in a subpopulation of small-diameter sensory neurons in dorsal root ganglia. In the spinal cord, immunoreactivity was observed in the superficial laminae of the dorsal horn. Following a chronic constriction injury to the sciatic nerve, the number of P2X3 positive small and medium diameter neurons increased in dorsal root ganglia when compared with sham-operated animals. In addition, the spinal cord immunoreactivity increased in magnitude on the side ipsilateral to the ligated nerve, consistent with up-regulation of receptors in presynaptic terminals of the primary sensory neurons.

P2X RECEPTORS AND THEIR ROLE IN FEMALE IDIOPATHIC DETRUSOR INSTABILITY

PURPOSE It is clear from previous studies that adenosine triphosphate is released as a contractile co-transmitter with acetylcholine from parasympathetic nerves supplying the mammalian bladder but the physiological significance of ligand gated purinergic P2X receptors in human bladder innervation has not been adequately investigated. We examined the role of these receptors in female patients with idiopathic detrusor instability. MATERIALS AND METHODS Female patients with idiopathic detrusor instability were recruited for cystoscopy and bladder biopsy with ethics approval. Control tissue was obtained from age and sex matched patients with a urodynamically proved stable bladder. We obtained 4 bladder biopsies per patient from the posterior wall. Samples were analyzed in an organ bath for functional studies of the detrusor muscle to assess the purinergic contribution to its contraction. In addition, we performed quantitative analysis using reverse transcriptase-polymerase chain reaction and immunohistochemical localization of P2X receptors. RESULTS In patients with idiopathic detrusor instability detrusor P2X2 receptors were significantly elevated, while other P2X receptor subtypes were significantly decreased. A purinergic component of nerve mediated contractions was not detected in control female bladder biopsy specimens but there was a significant component in unstable bladder specimens. It was particularly prominent at stimulation frequencies of 2 to 16 Hz. which are likely to be most relevant physiologically. Approximately 50% of nerve mediated contractions were purinergic in idiopathic detrusor instability cases. CONCLUSIONS In patients with idiopathic detrusor instability there is abnormal purinergic transmission in the bladder, which may explain symptoms. This pathway may be a novel target for the pharmacological treatment of overactive bladder.

Capsaicin receptor VR1 and ATP-gated ion channel P2X3 in human urinary bladder.

Objectives To determine the presence, distribution and molecular forms of the vanilloid receptor VR1, and confirm the presence and distribution of the ATP‐gated ion channel P2X3 in the human urinary bladder.

Localization of P2X1 purinoceptors by autoradiography and immunohistochemistry in rat kidneys.

P2 receptors have been identified in rat kidney by autoradiography, using the radioligand [3H]α,β-methylene ATP, and by immunohistochemistry, using a polyclonal antibody to the P2X1 purinoceptor. They have been localized to the vascular smooth muscle of intrarenal arteries, including arcuate and interlobular arteries, and afferent arterioles, but not glomeruli, postglomerular efferent arterioles, or renal tubules. We conclude that at least some of the P2 receptors present on vascular smooth muscle are of the P2X1 subtype. The functional significance of these findings in the vascular control of the kidney is discussed.

Localization of ATP-gated P2X2 and P2X3 receptor immunoreactive nerves in rat taste buds.

P2X receptors have been suggested to play a role in the transduction of sensory signals such as pain and sound. In the present study, polyclonal antibodies against P2X1 to P2X6 receptors were used to localize P2X receptors in circumvallate and fungiform papillae of rats. Nerve fibres innervating the taste buds stained intensely with P2X3 receptor antibodies. P2X3 receptor-positive nerves were observed in the intra- and subgemmal regions. The nerve fibres were also stained with P2X2 receptor antibodies, but the intensity was much lower. The distribution of P2X2 receptor immunoreactivity overlaps with that of P2X3. These results suggest that ATP might be a neurotransmitter in taste reception cells in the taste buds, where it transducts the taste signals to the afferent taste nerves by activating P2X receptors at the synapses. This is the first experiment indicating such a role for ATP, although supplementary functional studies are required.

Overexpression of NGF in mouse urothelium leads to neuronal hyperinnervation, pelvic sensitivity and changes in urinary bladder function

NGF has been suggested to play a role in urinary bladder dysfunction by mediating inflammation, as well as morphological and functional changes, in sensory and sympathetic neurons innervating the urinary bladder. To further explore the role of NGF in bladder sensory function, we generated a transgenic mouse model of chronic NGF overexpression in the bladder using the urothelium-specific uroplakin II (UPII) promoter. NGF mRNA and protein were expressed at higher levels in the bladders of NGF-overexpressing (NGF-OE) transgenic mice compared with wild-type littermate controls from postnatal day 7 through 12-16 wk of age. Overexpression of NGF led to urinary bladder enlargement characterized by marked nerve fiber hyperplasia in the submucosa and detrusor smooth muscle and elevated numbers of tissue mast cells. There was a marked increase in the density of CGRP- and substance P-positive C-fiber sensory afferents, neurofilament 200-positive myelinated sensory afferents, and tyrosine hydroxylase-positive sympathetic nerve fibers in the suburothelial nerve plexus. CGRP-positive ganglia were also present in the urinary bladders of transgenic mice. Transgenic mice had reduced urinary bladder capacity and an increase in the number and amplitude of nonvoiding bladder contractions under baseline conditions in conscious open-voiding cystometry. These changes in urinary bladder function were further associated with an increased referred somatic pelvic hypersensitivity. Thus, chronic urothelial NGF overexpression in transgenic mice leads to neuronal proliferation, focal increases in urinary bladder mast cells, increased urinary bladder reflex activity, and pelvic hypersensitivity. NGF-overexpressing mice may, therefore, provide a useful transgenic model for exploring the role of NGF in urinary bladder dysfunction.