Expression of P2X and P2Y receptors in the intramural parasympathetic ganglia of the cat urinary bladder

【摘要】  The distribution and function of P2X and P2Y receptor subtypes were investigated on intact or cultured intramural ganglia of the cat urinary bladder by immunocytochemistry and calcium-imaging techniques, respectively. Neurons were labeled by all seven P2X receptor subtype antibodies and antibodies for P2Y 2, P2Y 4, P2Y 6, and P2Y 12 receptor subtypes with a staining intensity of immunoreactivity in the following order: P2X 3 =P2Y 2 =P2Y 4 =P2Y 6 =P2Y 12 P2X 1 =P2X 2 =P2X 4 P2X 5 =P2X 6 =P2X 7. P2Y 1 receptor antibodies labeled glial cells, but not neurons. P2X 3 and P2Y 4 polyclonal antibodies labeled 95 and 40% of neurons, respectively. Double staining showed that 100, 48.8, and 97.4% of P2X 3 receptor-positive neurons coexpressed choline acetyl transferase (ChAT), nitric oxide synthase (NOS), and neurofilament 200 (NF200), respectively, whereas 100, 59.2, and 97.6% of P2Y 4 receptor-positive neurons coexpressed ChAT, NOS, and NF200, respectively. Application of ATP,, -methylene ATP, and uridine triphosphate elevated intracellular Ca 2+ concentration in a subpopulation of dissociated cultured cat intramural ganglia neurons, demonstrating the presence of functional P2Y 4 and P2X 3 receptors. This study indicates that P2X and P2Y receptor subtypes are expressed by cholinergic parasympathetic neurons innervating the urinary bladder. The neurons were also stained for NF200, usually regarded as a marker for large sensory neurons. These novel histochemical properties of cholinergic neurons in the cat bladder suggest that the parasympathetic pathways to the cat bladder may be modulated by complex purinergic synaptic mechanisms.

【关键词】  ATP intramural ganglia

SYMPATHETIC AS WELL AS PARASYMPATHETIC neurons and fibers are present in the pelvic ganglia or intramural ganglia of the bladder in different species ( 4, 17, 26 ). In addition to the classic transmitters norepinephrine (NE) and acetylcholine (ACh), other substances, including vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P (SP), enkephalin, somatostatin, neuropeptide Y, ATP, and nitric oxide (NO), have been identified in intramural ganglia and nerves supplying the bladder and urethra ( 17, 37, 38, 46, 47 ). These substances may act as neurotransmitters, neuromodulators, or trophic factors in the urinary bladder ( 17 ).

The role of ATP in the neural control of the bladder has attracted considerable attention because it has been identified as an excitatory cotransmitter released with ACh by parasympathetic postganglionic nerves ( 10 ) and may activate afferent nerves following release from urothelium ( 14, 22, 42 ). Previous pharmacological studies also revealed that purinergic agents have both excitatory and inhibitory effects on transmission in parasympathetic ganglia in the pelvic plexus and in the wall of the cat urinary bladder ( 3, 18, 21, 40 ). In vivo experiments revealed that low doses of ATP, ADP, AMP, and adenosine inhibited cholinergic ganglionic transmission, whereas high doses of ATP directly excited the ganglion cells. The inhibitory effects of purinergic agents were antagonized by theophylline and therefore are likely to be mediated by P1 purinergic receptors. Inhibitory effects of exogenous and endogenously released adenosine were also detected in cat bladder ganglia with intracellular recording in in vitro ganglion preparations ( 3 ). The ganglionic excitatory effects of ATP were confirmed with patch-clamp recording on cultured parasympathetic ganglion cells from neonatal rat major pelvic ganglia ( 45 ). Immunohistochemical, molecular biological, and pharmacological studies in the rat major pelvic ganglion revealed that the excitatory effects of ATP were mediated by P2X 2 receptors ( 21, 45 ). However, there are no reports of the distribution of the P2X and P2Y receptor protein in neurons in intramural ganglia of the cat bladder.

Two families of purinoceptors have been identified, a P2X ionotropic ligand-gated ion channel family and a P2Y metabotropic G protein-coupled family ( 2, 34 ). Seven distinct P2X subunits (P2X 1 to P2X 7 ) have been cloned ( 13, 15 ) and shown to be expressed in primary sensory neurons ( 21, 41 ). So far, eight mammalian P2Y receptors, namely, P2Y 1, P2Y 2, P2Y 4, P2Y 6, P2Y 11, P2Y 12 ( 25, 44 ), P2Y 13 ( 16 ) and the more recent P2Y 14 receptor ( 1 ), have been cloned and shown to be activated by extracellular nucleotides.

The present study was carried out to determine the expression patterns of P2X and P2Y receptor subtypes in the intramural ganglia of cat bladder. We also examined the colocalization of P2X 3 and P2Y 4 receptors with choline acetyltransferase (ChAT), nitric oxide synthase (NOS), and neurofilament 200 (NF200) in intramural bladder neurons.

MATERIALS AND METHODS

All procedures were conducted in accordance with Institutional Animal Care and Use Committee policies at each institution [Ohio State University, University of Pittsburgh, and Home Office (UK) regulations covering regulated procedures].

Tissue preparation. Intramural ganglia were removed from the surface of the urinary bladder from deeply anesthetized ( -chloralose 60-70 mg/kg; 2% halothane) healthy aged-matched mongrel cats (of either sex; 3.5-6 kg, n = 10). Following removal of tissues, the animals were killed via overdose of anesthetic. Anesthesia was determined to be adequate for surgery by periodically testing for the absence of a withdrawal reflex to a strong pinch of a hind paw and absence of an eye blink reflex to tactile stimuli of the cornea. The ganglia was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.2) at 4°C overnight and then transferred to 20% sucrose in PBS (overnight at 4°C). Thereafter, the tissue blocks were rapidly frozen by immersion in isopentane at -70°C for 2 min. Transverse sections through the ganglia (10-µm thickness) were cut on a cryostat and thaw-mounted on poly- L -lysine-coated slides.

Antisera. The following primary antisera were used in the current studies: rabbit anti-P2Y 1, P2Y 2, P2Y 4, P2Y 6, and P2Y 12 receptors (3 µg/ml; Alomone Laboratories, Jerusalem, Israel). The immunogens used for production of polyclonal P2Y 1, P2Y 2, P2Y 4, and P2Y 12 antibody were synthetic peptides corresponding to the COOH terminal of the cloned rat P2Y 1, P2Y 2, P2Y 4, and P2Y 12 receptors, covalently linked to keyhole limpet hemocyanin. The P2Y 6 receptor antibody is produced from the COOH terminus and the P2Y 12 receptor antibody from the second intracellular loop between TM3 and TM4. The peptide sequences of the P2Y 1, P2Y 2, P2Y 4, P2Y 6, and P2Y 12 receptors are of amino acid sequence 242-258 (RALIYKDLDNSPLRRKS), 227-244 (KPAYGTTGLPRAKRKSVR), 337-350 (HEESISRWADTHQD), 311-328 (QPHDLLQKLTAKWQRQRV), and 125-142 (KTTRPFKTSNPKNLLGAK), respectively. Rabbit anti-P2X 1 -P2X 7 receptor antibodies (2.5 µg/ml) were provided by Roche (Palo Alto, CA). The other antisera used in this study as well as their respective dilutions are listed in Table 1.

Table 1. List of antisera used for immunocytochemistry

Immunocytochemistry. An indirect immunofluorescence method was used to visualize receptor expression. Briefly, the sections were washed 3 x 5 min in PBS, then preincubated with 0.2% Triton X-100 in PBS (0.1 M) for 30 min. The sections were then incubated overnight with the primary antibodies (P2X 1 -P2X 7, P2Y 1, P2Y 2, P2Y 6, and P2Y 12 ) diluted to 3 µg/ml with 10% normal horse serum (NHS) in PBS containing 0.05% merthiolate and 0.2% Triton X-100. Subsequently, the sections were incubated with Cy3-conjugated donkey anti-rabbit IgG, diluted 1:300 in 1% NHS in PBS containing 0.05% merthiolate for 1 h. All incubations were held at room temperature and separated by three 5-min washes in PBS. Slides were mounted with Citifluor (Citifluor, London, UK) and examined with fluorescence microscopy. Control experiments were performed using an excess of the appropriate homolog peptide antigen to absorb the primary antibodies and thus confirm a specific immunoreaction.

Sections on which counts of P2X and P2Y receptor-positive neurons had been performed were marked and then counterstained with toluidine blue (2.5% in 0.1 M PB for 2 min followed by dehydration through increasing grades of alcohol, cleared in xylene, and cover- slipped with DPX mounting medium). This enabled the total neuron numbers to be determined by counting all neurons in the marked sections under bright-field illumination. Immunoreactive-positive and -negative neurons were counted to calculate the proportion of positive neurons.

Immunofluorescence double labeling. To demonstrate the colocalization of the P2X 3 and P2Y 4 receptor with ChAT, neuronal NOS, and a medium-molecular-weight neurofilament marker (NF200), sections were immunostained for the P2X 3 or P2Y 4 receptor, as above, then incubated with these antibodies overnight. Subsequently, the sections were incubated with FITC-conjugated donkey anti-goat IgG, or FITC-conjugated donkey anti-sheep IgG, or FITC-conjugated donkey anti-mouse IgG. All the incubations and reactions were held at room temperature and separated by 3 x 10-min washes in PBS. The sections were mounted with Citifluor and examined with fluorescence microscopy.

In colocalization studies investigating the coexpression of P2Y 4 and P2X 3 receptors, P2Y 4 receptor immunoreactivity was enhanced with tyramide amplification, which allows high sensitivity and low background specificity (Renaissance, TSA indirect, New England Nuclear). The use of TSA allows immunostaining with two rabbit antisera, as described previously ( 7, 36 ). Briefly, sections were incubated in 10% NHS in PBS for 30 min at room temperature, followed by incubation with the P2Y 4 antibody (1 µg/ml) in 10% NHS and 0.2% Triton X-100 in PBS overnight. Subsequently, the sections were incubated with biotinylated donkey anti-rabbit IgG for 1 h, with Extravidin peroxidase for 1 h, biotinylated tyramide for 8 min and then in streptavidin-FITC for 10 min. Polyclonal rabbit antibody against the P2X 3 receptor subtype was applied as a second primary antibody and detected with Cy3-conjugated donkey anti-rabbit IgG. To check for noncross reactivity, P2Y 4 receptor immunostaining using indirect TSA was performed alone on some sections, as was P2X 3 receptor indirect immunofluorescence. The localization of each marker appeared identical to the localization observed with the double staining technique, with no apparent cross-reactivity.

For immunostaining, a number of controls were performed on sections where either the primary or secondary antibody stage was omitted from the staining procedure.

Cell culture. Cat intramural bladder ganglia were visualized under a dissecting microscope, carefully removed, and transferred into ice-cold (4°C) calcium/magnesium-free HBSS (Invitrogen, Carlsbad, CA) of the following composition (in mM): 170 NaCl, 7 KCl, 1.6 Na 2 HPO 4, 6 D -glucose, and 0.01% phenol red, pH 7.3 (Invitrogen). Under a dissecting microscope, the intramural bladder ganglia were cut into smaller sections with sterile scissors and transferred into preactivated (37°C for 10 min) membrane filter-sterilized (0.2 µm 2 ) HBSS solution containing L -cysteine (2 mg/ml) and papain (14 U/ml) for 15 min at 37°C. The solution was carefully removed and replaced with membrane filter-sterilized (0.2 µm 2 ) HBSS containing dispase (8 U/ml) and Sigma blend A collagenase (1 mg/ml; Sigma, Poole, UK) and incubated at 37°C. Individual neurons were dissociated by gentle mechanical trituration with a sterile fire-polished glass pipette, over a period of between 30 and 45 min, until tissue fragments were no longer visible macroscopically.

Dissociated intramural bladder ganglia neurons were centrifuged at 200 g for 15 min, the dispase/collagenase supernatant was removed, and pellet was resuspended in neurobasal media supplemented with B27 (Invitrogen), salivary gland nerve growth factor (2.5S NGF, 10 ng/ml, Sigma) and 0.3% penicillin/streptomycin (Invitrogen) and fetal bovine serum (10%). Following another wash in growth media, the cells were plated onto sterile rat-tail collagen-coated (0.01%) 31-mm glass coverslips, at a density of 50/cm 2 on a six-well sterile culture dish and maintained at 37°C in a humidified incubator (95% O 2 -5% CO 2 ). Growth media was changed every 2 days.

Calcium imaging of cultured cat bladder ganglion neurons. Cultured cat intramural ganglia neurons (2-5 days following plating) were incubated with the fluorescent Ca 2+ indicator, fura 2-acetoxymethyl (AM) (5 µM, Invitrogen) in HBSS containing bovine serum albumin (5 mg/ml) for 30 min at 37°C in an atmosphere of 5% CO 2. Cells were washed in HBSS (containing in mM; 138 NaCl, 5 KCl, 0.3 KH 2 PO 4, 4 NaHCO 3, 2 CaCl 2, 1 MgCl 2, 10 HEPES, 5.6 glucose, pH 7.35, 310 mosmol/kgH 2 O), transferred to a perfusion chamber and mounted onto an epifluorescence microscope (Olympus, IX70). Measurement of intracellular Ca 2+ concentration ([Ca 2+ ] i ) was performed by ratiometric imaging of fura-2-AM at 340 and 380 nm (100 Hz) and the emitted light monitored at 510 nm. The fluorescence ratio F340/F380 was calculated and acquired by C-Imaging systems (Compix), and background fluorescence was subtracted. All test agents were bath applied (flow rate = 1.5 ml/min). Data were obtained from a minimum of five independent cultures, unless stated otherwise.

Photomicroscopy. Images of immunofluorescence labeling were taken with the Leica DC 200 digital camera (Leica, Heerbrugg, Switzerland) attached to a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany). Images were imported into a graphics package (Adobe Photoshop 5.0). The two-channel readings for green and red fluorescence were merged by using Adobe-Photoshop 5.0.

Analysis. All analyses were performed blind using x 20 objective magnification. P2X and P2Y receptor expression in ganglia was determined by counting all P2X and P2Y receptor-positive neurons in every sixth section throughout the ganglia from each animal. Scores for P2X and P2Y immunostaining were made by using a personal, subjective, graded scale varying from -, undetectable staining; +, weak staining but distinguishable from background, or scattered cells with moderate intensity staining; ++, moderate intensity staining in over 30% of cells; +++, very intense immunoreactivity in over 30% of cells. This analysis is a purely personal evaluation based on extensive experience and not meant as a quantitative analysis.

To calculate percentages of P2X 3 and P2Y 4 receptor colocalization with cytochemical markers, four randomly selected ganglia sections were chosen for each pair of markers for each animal. For each section, counts were made of the number of positive neurons for P2X 3 or P2Y 4 receptor, the number of positive neurons for the other marker, and the number of neurons expressing both antigens and percentages were calculated.

RESULTS

P2X and P2Y receptor staining in the intramural ganglia of the cat urinary bladder. The polyclonal antibodies for the seven P2X and four P2Y receptor subtypes labeled neurons in the intramural ganglia of the cat urinary bladder with differing intensity ( Table 2; Figs. 1 and 2 ). In control experiments, no signal was observed when the preimmune sera were used. Antibodies for all P2X receptor subtypes produced neuronal staining. P2X 3 receptor staining was the most intense and was detected in 95% of the neurons ( Fig. 1 C ). The staining was evenly distributed throughout the cytoplasm of these cells. The expression of P2X 1, P2X 2, and P2X 4 receptors ( Fig. 1, A, B, D ) in the intramural ganglia was lower than that of P2X 3 receptors, but higher than that of P2X 5, P2X 6, or P2X 7 receptors ( Fig. 1, E, F, G ). The intensity of staining of the seven P2X receptors in the intramural ganglia was in the order of P2X 3 P2X 1 =P2X 2 =P2X 4 P2X 5 =P2X 6 =P2X 7.

Table 2. Comparison of intensity of immunoreactivities for P2X 1-7 and P2Y 1,2,4,6,12 receptors on neurons in intramural ganglia of the cat urinary bladder

Fig. 1. Localization of P2X receptor subtype immunoreactivity in the intramural ganglia of the cat urinary bladder. A : P2X 1 receptor. B : P2X 2 receptor. C : P2X 3 receptor. D : P2X 4 receptor. E : P2X 5 receptor. F : P2X 6 receptor. G : P2X 7 receptor. Scale bar = 50 µm.

Fig. 2. Localization of P2Y receptor subtype immunoreactivity in the intramural ganglia of the cat urinary bladder. A : control. B : P2Y 1 receptor. C : P2Y 2 receptor. D : P2Y 4 receptor. E : P2Y 6 receptor. F : P2Y 12 receptor. Scale bar = 50 µm.

Neurons in the intramural ganglia of the cat urinary bladder were shown to express P2Y 2, P2Y 4, P2Y 6, and P2Y 12 receptor subtypes ( Table 2; Fig. 2 ), while P2Y 1 receptor antibodies labeled glial cells, but not neurons. P2Y 2, P2Y 4, P2Y 6, and P2Y 12 receptor polyclonal antibodies labeled over 50% of the neurons with high intensity ( Fig. 2, C, D, E, F ). The staining was evenly distributed throughout the cytoplasm of these cells and positively labeled cells were randomly distributed throughout the ganglia. Figure 2 A shows a control with no significant background staining after displacement with the relevant P2Y receptor peptide.

Coexpression of P2X 3 receptors with ChAT, NOS, and NF200. Double-labeling or colocalization studies showed that all (100%) of the P2X 3 receptor neurons in the intramural ganglia of the cat urinary bladder expressed ChAT immunoreactivity ( Table 3; Fig. 3, A - C ). Only about 48.8% of the P2X 3 receptor neurons expressed NOS immunoreactivity. However, 98.1% of NOS-immunoreactive neurons were also immunoreactive for P2X 3 receptor ( Table 3; Fig. 3 D ). A majority (97.4%) of the P2X 3 receptor positive neurons exhibited NF200 immunoreactivity ( Table 3; Fig. 3 E ).

Table 3. Colocalization of P2X 3, or P2Y 4 receptor immunoreactivity with ChAT, or NOS, or NF200 in intramural ganglia of the cat urinary bladder

Fig. 3. Double staining to show colocalization (yellow/orange) of P2X 3 or P2Y 4 receptor immunoreactivity (red) with ChAT, or NOS, or NF200 (green) in intramural ganglia of the cat urinary bladder. A - C : double staining for P2X 3 -IR and ChAT-IR. D : double staining for P2X 3 -IR and NOS-IR. E : double staining for P2X 3 -IR and NF200-IR. F - H : double staining for P2Y 4 -IR and ChAT-IR. I : double staining for P2Y 4 -IR and NOS-IR. J : double staining for P2Y 4 -IR and NF200-IR. K : double staining for P2X 3 -IR (red) and P2Y 4 -IR (green). Scale bar = 50 µm.

Coexpression of P2Y 4 receptors with ChAT, NOS, and NF200. A double immunofluorescence method revealed all (100%) of neurons positive for P2Y 4 receptor colocalized with ChAT. On the other hand, only 37.5% of ChAT- immunoreactive neurons exhibited P2Y 4 receptor immunoreactivity ( Table 3; Fig. 3, F - H ); 59.2% of P2Y 4 receptor neurons expressed NOS immunoreactivity, whereas 45.7% of NOS-immunoreactive neurons were also immunoreactive for P2Y 4 receptor ( Table 3; Fig. 3 I ). Double immunofluorescence showed that 97.6% of P2Y 4 receptor neurons were also NF200 immunoreactive. Conversely, 32.3% of NF200-immunoreactive neurons coexpressed P2Y 4 receptors ( Table 3; Fig. 3 J ).

Colocalization of P2X 3 with P2Y 4 receptors. In the intramural ganglia of the cat urinary bladder, P2X 3 receptor immunoreactivity was very often coexpressed with P2Y 4 receptor immunoreactivity. About 36.8% of P2X 3 receptor neurons were also found to be P2Y 4 receptor immunoreactive. Conversely, 100% of P2Y 4 -immunoreactive neurons coexpressed P2X 3 receptor ( Fig. 3 K ). In general, a greater number of neurons appeared to display P2X 3 rather than P2Y 4 receptors ( Figs. 1 C and 2 D ).

Calcium imaging of cultured cat bladder intramural ganglion neurons. Calcium-imaging techniques were used to determine whether purinergic agonists evoked functional responses in cultured cat intramural ganglia neurons. In this series of experiments, bath-applied ATP (10 µM) and uridine 5'-triphosphate (UTP; 10 µM) produced a rapid increase in [Ca 2+ ] i in cultured intramural neurons ( Fig. 4, A and C ). The response typically reached a peak 1 min post-ATP/UTP application and fully recovered to baseline within 2-3 min post-agonist application. Approximately 58 and 62% tested responded with an elevation in [Ca 2+ ] i following application of UTP (10 µM, 163/108 neurons; n = 5 independent cultures) and ATP (10 µM, 13/21 neurons; n = 2 independent cultures), respectively. Bath application of, -methylene ATP (, -meATP; Fig. 4, B and D; 10 µM), a potent activator of P2X 1 and P2X 3 receptors, also caused a rapid increase in a subpopulation of intramural neurons (12.6%, 11/87 neurons; n = 5 independent cultures). It is possible that the low percentage of neurons responding to, -meATP may have been due to rapid desensitization of the P2X 1 and P2X 3 receptors ( 27 ) or because the culture medium contained phenol red, which is known to antagonize P2X 1 and P2X 3 receptors ( 28 ). All the neurons that were responsive to, -meATP (10 µM) were also responsive to UTP. ATP S ( Fig. 4 E; 10 µM), a particularly active agonist at P2X 5 receptors, evoked an increase in [Ca 2+ ] i in a subpopulation of intramural neurons (50%; 10/20 neurons; n = 3 independent cultures). Figure 4 F shows a representative cultured cat intramural neuron loaded with the calcium indicator fura 2-AM.

Fig. 4. Cultured cat intramural ganglia neurons express functional P2X and P2Y responses. A : representative changes in [Ca 2+ ] i in cultured intramural cat ganglia neurons following bath-application of ATP (10 µM) and UTP (10 µM). B :, -meATP (10 µM) an activator of the ligand-gated ion channel, P2X 3, evoked an increase in [Ca 2+ ] i in a subpopulation of cat intramural neurons. C : UTP (10 µM) an activator of the metabotropic purinergic receptors, P2Y, also evoked an increase in [Ca 2+ ] i in a subpopulation of cultured intramural neurons. D : all cultured cat intramural ganglia neurons that responded to, -meATP (10 µM) also responded to UTP (10 µM). E : ATP S (10 µM) also evoked elevation of [Ca 2+ ] i demonstrating the presence of P2Y 2/4 receptors within intramural ganglia neurons. F : representative cultured cat intramural neuron loaded with the calcium indicator fura 2-AM.

DISCUSSION

In the present experiments, immunohistochemical method techniques revealed that neurons in the intact intramural parasympathetic ganglia of the cat urinary bladder express a variety of P2X and P2Y purinergic receptor subtypes. Purinergic agonists (ATP, UTP, and, -meATP) activated receptors in cultured dissociated ganglion cells, eliciting an increase in [Ca 2+ ] i. A full receptor characterization of the cultured ganglia was not carried out so that while it is assumed that receptor expression between the native and cultured neurons are the same, the presence of some differences cannot be ruled out. However, as the data obtained from this study are consistent with the results of earlier experiments ( 3, 18, 40, 45 ), the probability of native and cultured neurons having comparable receptor expression seems high. Our results indicate that purinergic agents can modulate synaptic transmission and alter the excitability of neurons in bladder and pelvic parasympathetic ganglia. The presence of multiple subtypes of purinergic receptors in bladder ganglia raises the possibility that purines may have complex synaptic modulatory functions in these ganglia.

We found that 95 and 40% of neurons in the intramural ganglia of the cat urinary bladder were P2X 3 and P2Y 4 receptor immunoreactive, respectively, and intensely stained. Approximately 37% of P2X 3 receptor-positive neurons coexpressed P2Y 4 receptors. Conversely, 100% of P2Y 4 receptor-immunoreactive neurons coexpressed the P2X 3 receptors. This means that most P2X 3 and P2Y 4 receptors are located in the same neurons. Apart from P2X 3 and P2Y 4 receptors, immunoreactivity for P2X 1,2,4-7 and P2Y 2, P2Y 6, and P2Y 12 receptor subtypes was shown to be present on neurons in the intramural ganglia, whereas P2Y 1 receptors were localized to glial cells in the ganglia.

In our previous study of cat dorsal root ganglia, it was noted that P2Y 1 receptor expression was very low on neurons but there was significant labeling of glial cells ( 35 ). Similarly, P2Y 1 receptor expression and function have been reported on glial cells in a study of mouse superior cervical ganglia ( 12 ). There is widespread expression of P2Y receptors on glial cells, and it has been suggested that they might have important roles in neuron-glial interactions ( 11 ).

The staining for P2X 3 and P2Y 4 receptors, the two of the most heavily expressed P2 receptor subtypes, was also linked with the expression of other cytochemical markers: ChAT, NOS, and NF200. ChAT is the enzyme responsible for the synthesis of ACh and anti-ChAT has been the specific antiserum of choice for the localization of ACh ( 23, 43 ). The present results show that all (100%) of the neurons exhibiting P2X 3 and P2Y 4 receptor immunoreactivity in the intramural ganglia of the cat urinary bladder expressed ChAT immunoreactivity. This indicates that P2X 3 and P2Y 4 receptors are localized on parasympathetic cholinergic postganglionic neurons in the bladder ganglia of the cat.

P2X 3 receptors, previously thought to be selectively expressed in sensory neurons, have been shown recently to be localized also on neurons on some parasympathetic ganglia, notably rat otic, sphenopalatine, and submandibular ganglia ( 31 ). The present study showing expression of P2X 3 receptors on parasympathetic neurons in cat intramural ganglia is consistent with these findings. In sensory neurons ATP elicits a depolarization by eliciting fast- and slow-inactivating inward currents. The fast-inactivating ATP currents are mediated by homomeric P2X 3 receptors, whereas the slow-desensitising currents are mediated by heteromeric P2X 2/3 receptors ( 7, 9 ). ATP also evokes responses in nociceptive sensory nerves through metabotropic P2Y receptors ( 24, 36, 39 ). The P2Y receptors couple through G proteins to various second messenger pathways, mediating slower metabotropic responses. The increases in [Ca 2+ ] i in bladder ganglion cells elicited by, -meATP are likely mediated by P2X receptors, whereas the responses to UTP are attributable to activation of P2Y receptors. The response to ATP could be mediated by activation of both types of receptors. In contrast to the prominent expression of P2X 3 receptors in cat bladder ganglia, P2X 2 receptors are highly expressed in the rat major pelvic ganglion and mediate the rapidly activating and slowly inactivating ATP-induced inward currents ( 21, 45 ).

The small percentage of neurons that responsed to, -meATP might reflect that the receptor was desensitized, and P2X 3 receptors are known to desensitize rapidly ( 27 ); however, the possibility that the cultured neurons expressed a different complement of P2X receptor subtypes should also be considered, which might lead to variations in the sensitivity to, -meATP.

NO has been identified as a neuronal messenger in the lower urinary tract ( 5 ). The enzyme responsible for the synthesis of NO from L -arginine, NOS, has been detected by using an antibody directed against NOS ( 8 ). NOS immunoreactivity has been detected in 45% of the intramural neurons of the guinea pig urinary bladder ( 37, 38, 46, 47 ). It has been suggested that NO may be involved in the relaxation activity in the bladder base during micturition ( 37, 38, 46, 47 ). Our study has demonstrated the 48 and 59% of neurons expressing P2X 3 and P2Y 4 receptor immunoreactivity, respectively, also exhibited NOS immunoreactivity. This subpopulation of parasympathetic neurons may have special functions in the bladder mediated by the corelease of ACh and NO.

NF200 is a marker of both A -fiber sensory neurons, which play an important role in nociception, and for the large-diameter neurons known to have myelinated axons and to be predominantly responsive to mechanical stimuli ( 29, 33 ). However, we found that most of the neurons in the intramural ganglia of the cat urinary bladder staining for P2X 3 receptors and ChAT were NF200 positive (see Table 3 ). Double immunofluorescence showed that about 97% of P2X 3 and P2Y 4 receptor-immunoreactive neurons were NF200 immunoreactive. This is an unexpected finding because bladder parasympathetic postganglionic neurons are thought to have unmyelinated axons and therefore should not express a marker for neurons with myelinated axons. This antibody has been successfully used in a previous study ( 35 ), although the percentages of P2X and P2Y receptor subtypes that were NF200 immunoreactive were significantly less. The possibility that the observed high percentages of P2X and P2Y receptors that colocalized with NF200 were due to nonspecific staining cannot be ignored.

The presence of purinergic receptors at sites of cholinergic transmission in bladder parasympathetic ganglia raises the question of the physiological significance of these receptors. The urinary bladder has two functions: to store and periodically release urine. These functions are regulated by complex nervous control originating in the central nervous system and passing through ganglionic synapses in the pelvic plexus and in the bladder wall ( 20, 30 ). The peripheral ganglia have integrative as well as relay functions and are involved in coordinating sympathetic and parasympathetic inputs to the bladder ( 19 ). In addition to classic transmitters NE and ACh, other putative neurotransmitters or neuromodulators, including VIP, SP, CGRP, enkephalin, somatostatin, neuropeptide Y, ATP, and NO, have been identified in intramural ganglia and in nerves supplying the bladder and urethra of various species ( 17, 32, 37, 38, 46, 47 ) indicating that transmission in the efferent pathways to the bladder is complex.

Previous pharmacological studies revealed that ATP administered by intra-arterial injection to cat parasympathetic bladder ganglion cells in vivo can excite or inhibit synaptic transmission depending on the dose ( 18, 40 ), whereas adenosine, AMP, and ADP have an inhibitory effect. The ganglionic-inhibitory effects of ATP and adenosine are blocked by theophylline and therefore must be mediated by P1 receptors. Stimulation of preganglionic nerves in an in vitro cat bladder ganglion preparation produced a hyperpolarization of the ganglion cells that was elicited by an adenosine-like substance acting on P1 purinergic receptors ( 3 ). It is not known whether adenosine is released directly by preganglionic nerves or produced by the metabolism of ATP released by nerve stimulation.

ATP might arise from several sources in the ganglia. It is known that axons of the parasympathetic ganglion cells release ATP as well as ACh at terminals in the bladder smooth muscle ( 10 ). Thus ATP might also be released from the soma and dendrites of parasympathetic ganglion cells and act in an auto-feedback manner on to the purinergic receptors on the same cells. In addition, because parasympathetic ganglia in the cat bladder receive inputs from various types of axons including 1 ) parasympathetic preganglionic axons in the pelvic nerve arising in the sacral spinal cord, 2 ) lumbar sympathetic preganglionic and postganglionic axons in the hypogastric nerve, 3 ) sympathetic postganglionic axons from the caudal sympathetic chain ganglia travelling in the pelvic nerve, and 4 ) afferent axons originating in the lumbosacral dorsal root ganglia and passing through the pelvic and hypogastric nerves to the bladder ganglia ( 20, 30 ), the purinergic receptors in the bladder ganglionic cells might be activated by transmitters released from multiple neural pathways.

Purinergic mechanisms in cat bladder ganglia could have a significant impact on ganglionic function because synaptic transmission in these ganglia occurs with a low safety factor and is very sensitive to homosynaptic and heterosynaptic modulatory mechanisms that inhibit or facilitate transmission ( 19 ). Other ganglia such as the rat major pelvic ganglion where cholinergic synaptic transmission occurs with a high safety factor ( 19 ) might be less susceptible to purinergic modulation.

In conclusion, the presence of various subtypes of purinergic receptors in the cat bladder ganglia as well as at other sites in the bladder including the smooth muscle ( 10 ), afferent nerves, and urothelial cells lining the bladder lumen ( 6 ) indicates that purinergic mechanisms have the potential for exerting a broad influence on the neural regulation of micturition in the cat.

GRANTS

This work was supported by National Institutes of Health Grant DKR-0157284 and a grant from Roche (to L. A. Birder).

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作者单位：1 Autonomic Neuroscience Centre, Royal Free and University College Medical School, London, United Kingdom; 2 Department of Neurobiology, Third Military Medical University, Chongqing; Departments of 3 Medicine and 4 Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;