Abstract
The distribution of purinergic (P2x1) receptors on smooth muscle cells in relation to autonomic nerve varicosities in the rat urinary bladder has been determined using immunofluorescence and confocal microscopy. P2x1 receptors were visualized using rabbit polyclonal antibodies against the extracellular domain of the P2x1 receptor, and varicosities were visualized using a mouse monoclonal antibody against the ubiquitous synaptic vesicle proteoglycan SV2. Two size classes of P2x1 receptor clusters were observed on the smooth muscle cells of the detrusor, namely, a large ellipse of mean long axis 1.23 ± 0.21 μm and short axis 0.92 ± 0.17 μm and a smaller spherical cluster with a mean diameter of 0.40 ± 0.04 μm. The latter occured in much greater numbers than the former in selected areas, with a density as high as 0.8 per μm2 or two orders of magnitude more than the larger-sized clusters. The large clusters are in general located beneath varicosities, with only 4.5% of P2x1 clusters not possessing an overlying varicosity. None of the small clusters was associated with varicosities. Three-dimensional reconstruction of the P2x1 and SV2 labelling at individual varicosities showed that the varicosities were immediately apposed to the P2x1 receptor clusters. On occasions, two or more small SV2-labelled varicosities about 0.7 μm in diameter each with a receptor patch were found juxtaposed to each other; these might represent the splitting up of a single large varicosity. These observations are discussed in relation to the identity of the autonomic neuromuscular junction.
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Barden, J.A., Cuthbertson, R.M., Wu, J-Z., Moseley, J.M. & Kemp, B.E. (1997) Solution structure of parathyroid hormone related protein (residues 1–34) containing an Ala substituted for an Ile in position 15 (PTHrP[Ala15]-(1-34)). Journal of Biological Chemistry 272, 29572–8.
Bennett, M.R. (1972) Autonomic Neuromuscular Transmission. Monograph of the Physiological Society. Cambridge, Cambridge University Press.
Bennett, M.R. (1996) Autonomic neuromuscular transmission at a varicosity. Progress in Neurobiology 50, 505–32.
Bennett, M.R. (1998) Transmission at sympathetic varicosities. News in the Physiological Sciences 13, 79–84.
Bennett, M.R., Farnell, L. & Gibson, W.G. (1998) On the origin of skewed distributions of spontaneous synaptic potentials in autonomic ganglia. Proceedings of the Royal Society of London B 265, 271–7.
Bennett, M.R., Farnell, L., Gibson, W.G. & Karunanithi, S. (1995) Quantal transmission at purinergic junctions: stochastic interaction between ATP and its receptors. Biophysical Journal 68, 925–35.
Bennett, M.R. & Gibson, W.G. (1995) On the contribution of quantal secretion from close-contact and loose-contact varicosities to the synaptic potentials in the vas deferens. Philosophical Transactions of the Royal Society of London B 347, 187–204.
Bennett, M.R., Robinson, J., Phipps, M.C., Karunanithi, S., Lin, Y.Q. & Cottee, L. (1996) Quantal components of spontaneous excitatory junction potentials at visualised varicosities. Journal of the Autonomic Nervous System 56, 161–74.
Berry, D., Yao, M, Barden, J.A., Balcar, V.J., Hansen, M.A., Bennett, M.R., Keogh, A. & Dos Remedios, C.G. (1998) Alterations in the expression of P2x1 receptors in failing and nondiseased human atria. Electrophoresis 19, 856–9.
Bo, X. & Burnstock, G. (1990) The effects of Bay K 8644 and nifedipine on the responses of rat urinary bladder to electrical field stimulation, β,γ-methylene ATP and acetylcholine. British Journal of Pharmacology 101, 494–498.
Bo, X. & Burnstock, G. (1992) Species differences in characteristics and distribution of [3H] α,β-methylene ATP binding sites in urinary bladder and urethra of rat, guinea-pig and rabbit. European Journal of Pharmacology 216, 59–66.
Bo, X. & Burnstock, G. (1993) Heterogeneous distributiion of [3H]α,β-methylene ATP binding sites in blood vessels. Journal of Vascular Research 30, 87–101.
Bo, X. & Burnstock, G. (1995) Characterization and autoradiographic localization of [3H] α,β-methylene adenosine 5′-triphosphate binding sites in human urinary bladder. British Journal of Urology 76, 297–302.
Boudier, J.A., Charvin, N., Boudier J.L., Fathallah, M., Tagaya, M., Takahashi, M. & Seagar, M.J. (1996) Distribution of components of the snare complex in relation to transmitter release sites at the frog neuromuscular junction. European Journal of Neuroscience 8, 545–52.
Brain, K.L. & Bennett, M.R. (1997) Calcium in sympathetic varicosities of mouse vas deferens during facilitation, augmentation and autoinhibition. Journal of Physiology 502, 521–36.
Brain, K.L., Cottee, L.J. & Bennett, M.R. (1997) Varicosities of single sympathetic nerve terminals possess syntaxin zones and different synaptotagmin N-terminus labelling following stimulation. Journal of Neurocytology 26, 491–500.
Bramich, N.J. & Brading, A.F. (1996) Electrical properties of smooth muscle in the guinea-pig urinary bladder. Journal of Physiology 492, 185–98.
Brown, C., Burnstock, G. & Cocks, T. (1979) Effects of adenosine 5′-triphosphate (ATP) and β,γ-methylene ATP on the rat urinary bladder. British Journal of Pharmacology 65, 97–102.
Buckley, K. & Kelly, R.G. (1985) Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. Journal of Cell Biology 100, 1284–94.
Burnstock, G. (1986) Autonomic neuromuscular junctions: current developments and future directions. Journal of Anatomy 146, 1–30.
Burnstock, G., Cocks, T., Kasakov, L. & Wong, H.K. (1978) Direct evidence for ATP release from non-adrenergic, non-cholinergic (“purinergic”) nerves in the guinea-pig taenia coli and bladder. European Journal of Pharmacology 49, 145–9.
Collo, G., North, R.A., Kawashima, E., Merlo-Pich, E., Neidhart, S., Surprenant, A. & Buell, G. (1996) Cloning of P2x5 and P2x6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. Journal of Neuroscience 16, 2495–507.
Ferrero, J.D., Cocks, T. & Burnstock, G. (1980) A comparison between ATP and bradykinin as possible mediators of the responses of the smooth muscle to nonadrenergic non-cholinergic nerves. European Journal of Pharmacology 63, 295–302.
Fuji, K. (1988) Evidence for adenosine triphosphate as an excitatory transmitter in guinea-pig, rabbit and pig urinary bladder. Journal of Physiology 404, 39–52.
Gabella, G. (1995) The structural relations between nerve fibres and muscle cells in the urinary bladder of the rat. Journal of Neurocytology 24, 159–87.
Gabella, G. & Uvelius, B. (1990) Urinary bladder of rat: fine structure of normal and hypertrophic musculature. Cell and Tissue Research 262, 67–79.
Gosling, J.A. & Dixon, J.S. (1975) The structure and innervation of smooth muscle in the wall of the bladder neck and proximal urethra. British Journal of Urology 47, 549–58.
Hansen, M.A., Barden, J.A., Balcar, V.J., Keay, K.A. & Bennett, M.R. (1997) Structural motif and characteristics of the extracellular domain of P2X receptors. Biochemical and Biophysical Research Communications 236, 670–5.
Hirst, G.D., Choate, J.K., Cousins, H.M., Edwards, F.R. & Klemm, M.F. (1996) Transmission by postganglionic axons of the autonomic nervous system: the importance of the specialized neuroeffector junction. Neuroscience 73, 7–23.
Horch, W.H.L. & Sargent, P.B. (1995) Perisynaptic surface distribution of multiple classes of nicotinic acetylcholine receptors on neurons in the chicken ciliary ganglion. Journal of Neuroscience 15, 7778–95.
Horch, W.H.L. & Sargent, P.B. (1996) Synaptic and extrasynaptic distribution of two distinct populations of nicotinic acetylcholine receptor clusters in the frog cardiac ganglion. Journal of Neurocytology 25, 67–76.
Hoyes, A.D., Barber, P. & Martin, B.G. (1975) Comparative ultrastructure of the nerves innervating the muscle of the body of the bladder. Cell and Tissue Research 164, 133–44.
Hoyes, A.D., Bourne, R. & Martin, G.H. (1976) Innervation of the muscle of the bladder in the rat. British Journal of Urology 48, 43–53.
Hoyle, C.H. & Burnstock, G. (1985) Atropine-resistant excitatory junction potentials in rabbit bladder are blocked by alpha, beta-methylene ATP. European Journal of Pharmacology 114 , 239–4 0.
Husted, S., SjØgren, C. & Andersson, K.E. (1980) Mechanisms of the responses to non-cholinergic, non-adrenergic nerve stimulation and to ATP in isolated rabbit urinary bladder: evidence for ADP evoked prostaglin release. Acta Pharmacology and Toxicology 47, 84–92.
Inoue, R. & Brading, A.F. (1990) The properties of the ATP-induced depolarization and current in single cells isolated from the guinea-pig urinary bladder. British Journal of Pharmacology 100, 619–25.
Isenberg, G., Ganitkevich, V.Y.A. & Schneider, P. (1992) Ca2+ influx through voltage-and purinoceptor-operated channels estimated from [Ca2+]c signals (myocytes from guinea-pig urinary bladder). Advances in Experimental Medicine and Biology 311, 369–71.
Kasakov, L. & Burnstock, G. (1983) The use of slowly degradable analog, alpha, beta-methylene ATP, to produce desensitisation of the P2-purinoceptor: effect on non-adrenergic, non-cholinergic responses of the guinea-pig urinary bladder. European Journal of Pharmacology 86, 291–4.
Kura, H., Obara, K. & Yabu, H. (1992) Contractile responses to electrical field stimulation and ATP in guinea-pig urinary bladder. Comparative Biochemistry and Physiology C: Comparative Biochemistry and Toxicology 102, 193–7.
Levin, R.M., Shofer, F.S. & Wein, A.J. (1980) Cholinergic, adrenergic and purinergic response of sequential strips of rabbit urinary bladder. Journal of Pharmacology and Experimental Therapeutics 212, 536–40.
Levin, R.M., Zderic, S.A., Ewalt, D.H., Duckett, J.W. & Wein, A.J. (1991) Effects of pregnancy on muscarinic receptor density and function in the rabbit urinary bladder. Pharmacology 43, 69–77.
Luff, S.E. (1996) Ultrastructure of sympathetic axons and their structural relationship with vascular smooth muscle. Anatomy and Embryology 193, 515–31.
Macleod, G.T., Lavidis, N.A. & Bennett, M.R. (1994) Calcium dependence of quantal secretion from visualized sympathetic nerve varicosities on the mouse vas deferens. Journal of Physiology 480, 61–70.
Maggi, C.A. (1991) Omega conotoxin and prejunctional modulation of the biphasic response of the rat isolated urinary bladder to single pulse electrical field stimulation. Journal of Autonomic Pharmacology 11, 295–304.
Matthews-Bellinger, J. & Salpeter, M.M. (1978) Distribution of acetylcholine receptors at frog neuromuscular junctions with a discussion of some physiological implications. Journal of Physiology 279, 197–213.
Nakayama, S. (1993) Effects of excitatory neurotransmitters on Ca2+ channel current in smooth muscle cells isolated from guinea-pig urinary bladder. British Journal of Pharmacology 110, 317–25.
North, R.A. (1996) P2x receptors: a third major class of ligand-gated ion channels. CIBA Foundation Symposium 198, 91–105.
Parija, S.C., Raviprakash, V. & Mishra, S.K. (1991) Adenosine-and α,β-methylene ATP-induced differential inhibition of cholinergic and non-cholinergic neurogenic responses in rat urinary bladder. British Journal of Pharmacology 102, 396–400.
Schneider, P., Hopp, H.H. & Isenberg, G. (1991) Ca2+ influx through ATP-gated channels increments ([Ca2+]i) and inactivates ICa in myocytes from guinea-pig urinary bladder. Journal of Physiology 440, 479–96.
Surprenant, A., Rassendren, F., Kawashima, E., North, R.A. & Buell, G. (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2x receptor (P2x7). Science 272, 735–8.
Theobald, R. J. Jr. (1995) Purinergic and cholinergic components of bladder contractility and flow. Life Sciences 56, 445–54.
Turner, W.H. & Brading, A.F. (1997) Smooth muscle of the bladder in the normal and the diseased state: pathophysiology, diagnosis and treatment. Pharmacology and Therapeutics 75, 77–110.
Usune, S., Katsuragi, T. & Furukawa, T. (1996) Effects of PPADS and suramin on contractions and cytoplasmic Ca2+ changes evoked by AP4A, ATP and α,β-methylene ATP in guinea-pig urinary bladder. British Journal of Pharmacology 117, 698–702.
Valera, S., Hussy, N., Evans, R.J., Adami, N., North, R.A., Surprenant, A. & Buell, G. (1994) A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP. Nature 371, 516–9.
Vulchanova, L., Arvidsson, V., Riedl, M., Wang, J., Buell, G., Surprenant, A., North, R.A. & Elde, R. (1996). Differential distribution of two ATP-gated channels (P2x receptors) determined by immunocytochemistry. Proceedings of the National Academy of Sciences of the USA 93, 8063–7.
Zhao, M., Bo, X., Neely, C.F. & Burnstock, G. (1996). Characterization and autoradiographic localization of [3H] α,β-methylene ATP binding sites in cat urinary bladder. General Pharmacology 27, 509–12.
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Hansen, M.A., Balcar, V.J., Barden, J.A. et al. The distribution of single P2x1-receptor clusters on smooth muscle cells in relation to nerve varicosities in the rat urinary bladder. J Neurocytol 27, 529–539 (1998). https://doi.org/10.1023/A:1006908010642
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DOI: https://doi.org/10.1023/A:1006908010642