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Cell Tissue Res (1999) 298:449–456 Digital Object Identifier (DOI) 10.1007/s004419900103 © Springer-Verlag 1999 REGULAR ARTICLE Mekbeb Afework · Geoffrey Burnstock Distribution of P2X receptors in the rat adrenal gland Received: 29 January 1999 / Accepted: 16 July 1999 / Published online: 19 October 1999 Abstract The distribution of each of the seven subtypes of ATP-gated P2X receptors was investigated in the adrenal gland of rat utilizing immunohistochemical techniques with specific polyclonal antibodies to unique peptide sequences of P2X1–7 receptors. A small number of chromaffin cells showed positive immunoreaction for P2X5 and P2X7, with the relative occurrence of P2X7immunoreactive chromaffin cells exceeding that of P2X5. The preganglionic nerve fibres that form terminal plexuses around some chromaffin cells showed P2X1 immunoreactivity. Intrinsic adrenal neurones were observed to be positively stained for P2X2 and P2X3 receptors. P2X2 immunoreactivity occurred in several neurones found singly or in groups in the medulla, while only a small number of neurones were immunoreactive for P2X3. Adrenal cortical cells were positively immunostained for P2X4–7. Immunoreactivity for P2X4 was confined to the cells of the zona reticularis, while P2X5–7 immunoreactivities occurred in cells of the zona fasciculata. The relative occurrence of immunoreactive cortical cells of the zona fasciculata was highest for P2X6, followed by P2X7 and then P2X5. The smooth muscle of some capsular and subcapsular blood vessels showed P2X2 immunoreactivity. The specific and widespread distribution of P2X receptor subtypes in the adrenal gland suggests a significant role for purine signalling in the physiology of the rat adrenal gland. The support of Roche Bioscience in Palo Alto, USA, is gratefully acknowledged M. Afework Department of Anatomy, Faculty of Medicine, Addis Ababa University, PO Box 9086, Addis Ababa, Ethiopia G. Burnstock (✉) Autonomic Neuroscience Institute, Royal Free and University College Medical School, Rowland Hill Street, London, NW3 2PF, UK e-mail: [email protected] Tel: +44 171 830 2948, Fax: +44 171 830 2949 Key words P2X receptor · Adrenal medulla · Adrenal cortex · Immunohistochemistry · Rat (Sprague Dawley) Introduction Several lines of evidence have shown that ATP has an extracellular role in activities such as contractile regulation of visceral muscles, neurotransmission and/or modulation, and neuroendocrine secretion (Burnstock 1972, 1997) in addition to its long-known importance as an intracellular energy source. In the adrenal gland, ATP is co-stored and co-released with catecholamines from chromaffin cells (Winkler and Westhead 1980) and is implicated in both facilitation (Chern et al. 1988; Kim and Westhead 1989) and inhibition (Chern et al. 1987) of catecholamine secretion. ATP has also been shown to be released from capsule and zona glomerulosa regions of the gland and to stimulate steroidogenesis from the adrenal zona glomerulosa (Jurányi et al. 1997) and fasciculata cells (Kawamura et al. 1991; Matsui 1991; Niitsu 1992). It is, therefore, suggested that adenine nucleotide receptors on the adrenal gland cells might play a role in regulating secretions from both medulla and cortex of the gland. P2X and P2Y receptors are the two families of adenine nucleotide receptors widely distributed among mammalian cells. P2X receptors are ligand-gated nonselective cation channels (Bean 1992) while P2Y receptors are G protein-coupled membrane proteins (Abbracchio and Burnstock 1994; Barnard et al. 1994). Seven subtypes have been cloned and characterized for the P2X family, and about nine subtypes are recognized for the P2Y family (Abbracchio and Burnstock 1994; Burnstock and King 1996; Fredholm et al. 1997; North and Barnard 1997). Functional studies suggest the presence of both P2Y (Niitsu 1992; Currie and Fox 1996; Lim et al. 1997) and P2X receptors (Asano et al. 1995; Castro et al. 1995; Lin et al. 1995) in the adrenal gland. Moreover, the occurrence of P2X receptors in the rat adrenal gland has been 450 reported from studies recently carried out on some, but not all, of the P2X receptor subtypes using Northern blot analysis for P2X4 (Bo et al. 1995), Southern blot analysis for P2X5 (Garcia-Guzman et al. 1996) and immunohistochemistry for P2X1 and P2X2 (Vulchanova et al. 1996). In the present study, using specific polyclonal antibodies to unique peptide sequences for each of all the seven P2X receptor subtypes, we have investigated their occurrence and distribution in the adrenal gland of rat using the immunohistochemical techniques. Materials and methods Preparation of samples The study was conducted on six adult male Sprague-Dawley rats weighing 250–300 g. Principles of good laboratory animal care were followed and animal experimentation was in compliance with the specific national laws and regulations. The rats were killed by a rising concentration of carbon dioxide. The adrenal glands were removed and fixed in 4% formaldehyde containing 0.03% picric acid in 0.1 M phosphate-buffered saline (PBS), pH 7.4, for 1 h at room temperature. The adrenals were left overnight in 10% sucrose in PBS at 4°C. Frozen sections were cut at 14 µm in a cryostat (Leica CM 1800, Germany) and thaw mounted onto gelatinized slides. Preparation of antibodies The immunogens were synthetic peptides representing 15 receptor-type specific amino acids in the C-terminal part of the receptor: P2X1 , amino acid 385–399 (ATSSTLGLQENMRTS); P2X2 , amino acid 458–472 (QQDSTSTDPKGLAQL); P2X3 , amino acid 383–397 (VEKQSTDSGAYSIGH); P2X4 , amino acid 374–388 (YVEDYEQGLSGEMNQ); P2X5 , amino acid 437–451 (RENAIVNVKQSQILH); P2X6 , amino acid 357–371 (EAGFYWRTKYEEARA); P2X7 , amino acids 555–569 (TWRFVSQDMADFAIL). The peptides were covalently linked to keyhole limpet haemocyanin (KLH). Rabbits were immunized with the conjugated peptides in multiple monthly injections (performed by Research Genetics Inc., Huntsville, AL). The specificity of the P2X antibodies has been verified by immunoblotting with membrane preparations from cloned P2X1–7 receptor-expressing CHO-K1 cells or 1321N1 cells (Oglesby et al. 1999). The antibodies recognize only one protein of the expected size in the heterologous expression systems and have been shown to be receptor subtype specific. Immunoglobulin G (IgG) fractions were isolated from the preimmune and immune sera (P2X1–7 ) following the method of Harboe and Ingild (1973). The protein concentration was determined at 280 nm using an extinction factor of 1.43 for 1 mg/ml. Table 1 Summary of P2X1–7 receptor localization in the various cellular elements of the adrenal gland of the rat (n =6). The presence and absence of the labelling with the antisera are indicated by “+” and “–”, respectively. Where labelling of a cellular element by Nerve fibres in the medulla Intrinsic nerve cell bodies in the medulla Chromaffin cells Cortical cells Myocytes of capsular and subcapsular blood vessels Immunohistochemistry Immunohistochemistry for P2X receptors was carried out using the rabbit polyclonal antibodies generated as above against unique peptide sequences of each of P2X1–7 receptor subtypes and provided by Roche Bioscience, Palo Alto, CA. The sites of antibody-antigen reaction were visualized by the application of the avidin biotin technique employing a nickelintensified 3,3’-diaminobenzidine (DAB) reaction according to the protocol developed by Llewellyn-Smith et al. (1993). Endogenous peroxidase was blocked with 50% methanol and 0.4% H2 O2 for 10 min. Blocking of non-specific binding sites was achieved by incubation for 20 min with 10% normal horse serum in PBS containing 0.05% merthiolate. Sections were incubated overnight at room temperature in a humid chamber with the polyclonal antibodies against P2X1–7 receptor subtypes, at a dilution of 1 µg/ml (for P2X1–3 and P2X5–7 ) and 0.5 µg/ml (for P2X4 ) in 10% normal horse serum, found to be optimal by titration. This was followed by incubation with biotinylated donkey anti-rabbit immunoglobulin G (IgG) (Jackson Immunoresearch, PA) at a dilution of 1:500 for 1 h, and then with ExtrAvidin peroxidase conjugate (Sigma) at a dilution of 1:1500 for 1 h. After each incubation, sections were washed in PBS (3×5 min). The nickel-intensified DAB reaction was done for 5–10 min. Sections were dehydrated in graded alcohol, cleared in xylene and mounted with Eukitt (BDH). Immunoprocessed sections were studied using an Edge R 400 high-definition light microscope (Greenberg and Boyde 1997) (Edge Scientific Instrument Co., Santa Monica, CA) and photographed with Kodak TMX100 black and white film. Controls included: omission of the primary antibodies, replacement of the primary antibodies with rabbit preimmune IgG, and absorption of the primary antibodies with their respective homologous peptide antigen. Results The control experiments in which the primary antibodies were omitted or replaced by rabbit preimmune IgG or preabsorbed with the respective peptide antigen did not show any staining. In contrast, in sections which were incubated with primary antibodies, staining with specific distribution for each of the seven P2X receptor subtypes was observed, as summarized in Table 1 . Immunoreactivity for P2X1 was found in some nerve fibres located in the adrenal medulla (n =6). Some of the immunoreactive fibres were seen forming plexuses in association with regions of some chromaffin cells, while a few were seen surrounding a few blood vessels (Fig. 1). P2X2 receptor immunoreactivity occurred in myocytes of large blood vessels located in the capsule and subcapsular region of the gland (n =6; Fig. 2). Immunoreactivity for P2X2 was also observed in the intra-adrenal neurones more than one antisera occurred, the relative density of labelling in the same cellular element was rated from low (+) to high (++++). Note that comparison of the relative density is within the same cellular element, but not among the different cellular elements P2X1 P2X2 P2X3 P2X4 P2X5 P2X6 P2X7 + – – – – – ++ – – + – + – – – – – – + – – – + ++ – – – – ++++ – – – ++ +++ – 451 Fig. 1 P2X1 -immunoreactive nerve fibres (arrows ) forming plexus around chromaffin cells and blood vessels in the adrenal medulla. Scale bar 50 µm Fig. 2 P2X2 -immunoreacted adrenal gland sections showing positive immunoreactivity in the smooth muscle of capsular blood vessels (arrows ) (ca capsule, co cortex). Scale bar 50 µm Fig. 3 P2X2 -immunoreacted adrenal gland sections showing positive immunoreactivity in intrinsic adrenal neurones (arrows ) in medulla (m). Scale bar 25 µm 452 Fig. 4 P2X3-immunoreactive neurone (arrow ) in the adrenal medulla. Note the small size of this neurone as compared to those labelled with P2X2 as indicated in Fig. 3. Scale bar 25 µm Fig. 5 P2X4-immunoreactive adrenal cortical cells of zona reticularis (arrows ). Note the small size of such immunoreacted cells as compared to the large majority of the non-immunoreacted surrounding cells (r zona reticularis, m medulla). Scale bar 25 µm located in the adrenal medulla (Fig. 3). Such immunoreactive neurones were found singly as well as in groups and belong to both small and large types of neurones described in the gland (Unsicker et al. 1978; Afework and Burnstock 1994). Intrinsic neurones in the medulla also showed positive immunoreactivity for P2X3 antibody (n =6). However, unlike the P2X2 immunoreactivity described above, the P2X3 -immunoreactive neurones were encountered only occasionally and only as small single cells located in the medulla (Fig. 4). Adrenal cortical cells gave positive immunoreaction for P2X4 in some small-sized dispersed cells of the zona reticularis (Fig. 5; n =6). In contrast, P2X5 immunoreactivity was found in cortical cells of zona fasciculata (Fig. 6; n =6), and in chromaffin cells (Fig. 7). P2X6 immunoreaction was also found in cortical cells of the zona fasciculata (Fig. 8; n =6), whereas P2X7 immunoreaction was seen in both chromaffin cells (Fig. 9 ) and cortical cells of the zona fasciculata (Fig. 10; n =6). The relative number and staining intensity of immunoreactivities for P2X5–7 in the zona fasciculata was highest for P2X6 followed by P2X7 and then P2X5. P2X5- and P2X7 -immunoreactive chromaffin cells were seen occasionally dispersed in the medulla. The relative occurrence of P2X7immunoreactive chromaffin cells was higher than that of P2X5-positive chromaffin cells (Table 1). Discussion The failure of immunoreactive staining in the adrenal gland sections in which the primary antibodies were omitted, replaced by rabbit preimmune IgG or preabsorbed with an excess of the respective homologous peptide antigens confirms that the positive immunoreactivities we observed were due to the presence of the P2X receptors towards which the primary antibodies were di- 453 Fig. 6 P2X5-immunoreactive adrenal cortical cells of zona fasciculata (arrows ) (f zona fasciculata, g zona glomerulosa). Scale bar 50 µm Fig. 7 P2X5-immunoreactivity in chromaffin cells of medulla (arrows ). Scale bar 25 µm Fig. 8 P2X6-immunoreactive adrenal cortical cells of zona fasciculata (arrows) (g zona glomerulosa, f zona fasciculata). Scale bar 50 µm 454 Fig. 9 Adrenal gland section showing P2X7 immunoreactivity in chromaffin cells (arrows) in medulla. Scale bar 25 µm Fig. 10 P2X7 immunoreactivity in adrenal cortical cells of zona fasciculata (arrows ) (g zona glomerulosa, f zona fasciculata). Note that the relative number and intensity of immunoreactive adrenal cortical cells of the zona fasciculata is highest for P2X6 (Fig. 8) followed by P2X7 (Fig. 10) and then P2X5 (Fig. 6). Scale bar 50 µm rected. In the present study, the immunolocalization of P2X receptors was not confined to only the cell membranes, where functional receptors would be expected to be located, but was also found in the cytoplasm of the cell bodies and nerve fibres. Such a pattern of P2X receptor localization has also previously been observed in other studies (Lê et al. 1998; Llewellyn-Smith and Burnstock 1998). The precise sites of localization and significance of the intracytoplasmic receptors, however, await future investigation. In the present study with antibodies to the seven P2X receptor subtypes, a small number of dispersed chromaffin cells were found to be immunoreactive to P2X5 and P2X7 , although the presence of immunoreactivities for P2X1 and P2X2 in the adrenal chromaffin cells of rat has previously been reported (Vulchanova et al. 1996). Our finding that only a small number of chromaffin cells pos- sess P2X receptors is consistent with reports from functional studies characterizing the receptors in the rat chromaffin cells as P2Y (Lim et al. 1997), and with little response to ATP in rat, as distinct from guinea-pig, adrenal chromaffin cells (Liu et al. 1999). However, the occurrence of P2X receptors has been suggested in the chromaffin cells from both bovine (Castro et al. 1995; Lin et al. 1995) and guinea-pig adrenal gland (Asano et al. 1995). The precise functional roles of the P2X5 and P2X7 receptors in the adrenal chromaffin cells await further investigation. However, previous studies have implicated P2X7 receptors in the induction of apoptosis (Di Virgilio et al. 1989; Surprenant et al. 1996; Collo et al. 1997; Rassendren et al. 1997), so it is possible that this receptor may also be involved in similar apoptotic events in the adrenal chromaffin cells. P2X5 receptors appear to be associated with proliferation and/or differentiation in 455 stratified epithelium (Gröschel-Stewart et al. 1999), perhaps indicating a similar role in the adrenal gland. As the P2X1 -immunoreactive nerve fibres observed in the adrenal medulla formed terminal plexuses around the chromaffin cells, it appears that these fibres belong to the cholinergic preganglionic nerve fibres that control the secretion from the chromaffin cells (Parker et al. 1993). The occurrence of P2X receptors on preganglionic nerve fibres suggests that purines may act as prejunctional modulators for the release of transmitters in the adrenal gland, in addition to their possible action as cotransmitters and/or modulators of adrenal medullary cells. Similarly, the presence of P2X2 and P2X3 immunoreactivity in the intrinsic neurones of the gland may imply a functional role for purines on the activities of these neurones which are known to contain several neurotransmitters, and which innervate local blood vessels and the adrenal cortex (Dagerlind et al. 1990; Afework and Burnstock 1994; Oomori et al. 1994). The present study has revealed a substantial number of cortical cells of adrenal gland of the rat that possess P2X4–7 receptor subtypes. As mentioned above, the implication of P2X7 receptors in apoptosis suggests that some of the P2X7 -labelled cortical cells may be in the process of apoptosis. In addition, the localization of this receptor along with the P2X4–6 in a large number of cortical cells suggests that P2X receptors may be involved in steroidogenesis, in addition to the P2Y receptors that have been already implicated from functional studies (Niitsu 1992). It is also possible that purines are involved in other cellular activities in addition to steroidogenesis and secretion from the cortical cells. P2X2 receptors were localized in the smooth muscle of blood vessels in the capsular region. It is known that blood flow in the adrenal gland has a direct relationship with the secretory activities of both the adrenal cortex (Vinson and Hinson 1992) and medulla (Sparrow and Coupland 1987; Faraci et al. 1989), and is actively regulated. One of the regulatory mechanisms of blood flow in the gland could be through ATP. In the control of blood vessel tone, ATP released as co-transmitter from perivascular sympathetic nerves acts on P2X receptors on the smooth muscle of blood vessel wall to produce vasoconstriction (Burnstock 1997). In addition, ATP released from endothelial cells by sheer stress and hypoxia is believed to act, via P2Y receptors, on endothelial cells to release nitric oxide with subsequent vasodilatation (Burnstock 1997). The finding of P2X2 immunoreactivity in the myocytes of blood vessels located in the capsular and subcapsular region of the gland is, therefore, in agreement with the role of P2X receptors on blood vessel wall as described above and indicates the involvement of ATP in the control of blood flow in the adrenal gland. Acknowledgements We are grateful to Prof. U. Gröschel-Stewart for her helpful discussion while conducting the study. The editorial assistance of Mr. R. Jordan is gratefully acknowledged. References Abbracchio MP, Burnstock G (1994) Purinoceptors: are there families of P2x and P2y purinoceptors? Pharmacol Ther 64:445– 475 Afework M, Burnstock G (1994) Colocalization of neuropeptides and NADPH-diaphorase in the intra-adrenal neuronal cell bodies and fibres of the rat. Cell Tissue Res 280:291–295 Asano T, Otsuguro K-I, Ohta T, Sugawara T, Ito S, Nakazato Y (1995) Characteristics of ATP-induced catecholamine secretion from adrenal chromaffin cells of the guinea-pig. Comp Biochem Physiol 112C:101–108 Barnard EA, Burnstock G, Webb TE (1994) G protein-coupled receptors for ATP and other nucleotides: a new receptor family. Trends Pharmacol Sci 15:67–70 Bean BP (1992) Pharmacology and electrophysiology of ATP-activated ion channels. Trends Pharmacol Sci 13:87–90 Bo X, Zhang Y, Nassar M, Burnstock G, Schoepfer R (1995) A P2X purinoceptor cDNA conferring a novel pharmacological profile. FEBS Lett 375:129–133 Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509– 581 Burnstock G (1997) The past, present and future of purine nucleotides as signalling molecules. Neuropharmacology 36:1127– 1139 Burnstock G, King B (1996) Numbering of cloned P2 purinoceptors. Drug Dev Res 38:67–71 Castro E, Mateo J, Tome AR, Barbosa RM, Miras-Portugal MT, Rosario LM (1995) Cell-specific purinergic receptors coupled to Ca2+ entry and Ca2+ release from internal stores in adrenal chromaffin cells. J Biol Chem 270:5098–5106 Chern YJ, Herrera M, Kao LS, Westhead E (1987) Inhibition of catecholamine secretion from bovine chromaffin cells by adenine nucleotides and adenine. J Neurochem 48:1573–1576 Chern YJ, Kim KT, Slaky LL, Westhead E (1988) Adenosine receptors activate adenylate cyclase and enhance secretion from bovine adrenal chromaffin cells in the presence of forskolin. J Neurochem 50:1484–1493 Collo G, Neidhart S, Kawashima E, Kosco-Vilbois M, North RA, Buell G (1997) Tissue distribution of the P2X7 receptor. Neuropharmacology 36:1277–1283 Currie KPM, Fox AP (1996) ATP serves as a negative feedback inhibitor of voltage-gated Ca2+ channel currents in cultured bovine adrenal chromaffin cells. Neuron 16:1027–1036 Dagerlind A, Goldstein M, Hökfelt T (1990) Most ganglion cells in the rat adrenal medulla are noradrenergic. Neuroreport 1:137–140 Di Virgilio F, Bronte V, Collavo D, Zanovello P (1989) Responses of mouse lymphocytes to extracellular ATP. Lymphocytes with cytotoxic activity are resistant to permeabilizing effects of ATP. J Immunol 143:1955 Faraci FM, Chilian WM, Williams JK, Heistad DD (1989) Effects of reflex stimuli on blood flow to the adrenal medulla. Am J Physiol 257:H590–H596 Fredholm BB, Abbracchio MP, Burnstock G, Dubyak GR, Harden TK, Jakobson KA, Schwabe U, Spedding M, Williams M (1997) Towards a revised nomenclature for P1 and P2 receptors. Trends Pharmacol Sci 18:79–82 Garcia-Guzman M, Soto F, Laube B, Stühmer W (1996) Molecular cloning and functional expression of a novel rat heart P2X purinoceptor. FEBS Lett 388:123–127 Greenberg GL, Boyde A (1997) Convenient and controllable direct-view 3D imaging in conventional light microscopes: approaches via illumination and inspection. Proc R Microsc Soc 32:87–101 Gröschel-Stewart U, Bardini M, Robson T, Burnstock G (1999) Localisation of P2X5 and P2X7 receptors by immunohistochemistry in rat stratified squamous epithelia. Cell Tissue Res 296:599–605 Harboe N, Ingild A (1973) Immunization, isolation of immunoglobulins, estimation of antibody titre. Scand J Immunol Suppl 1:161–164 456 Jurányi Z, Orsó E, Jánssy A, Szaly KS, Sperlágh B, Windisch K, Vinson GP, Vizi ES (1997) ATP and [3 H]noradrenaline release and the presence of ecto-Ca2+ -ATPases in the capsuleglomerulosa fraction of the rat adrenal gland. J Endocrinol 153:105–114 Kawamura M, Matsui T, Niitsu A, Kondo T, Ohno Y, Nakamichi N (1991) Extracellular ATP stimulates steroidogenesis in bovine adrenocortical fasciculata cells via P2 purinoceptors. Jpn J Pharmacol 56:543–545 Kim KT, Westhead E (1989) Cellular responses to Ca2+ from extracellular and intracellular sources are different as shown by simultaneous measurements of cytosolic Ca2+ and secretion from bovine chromaffin cells. Proc Natl Acad Sci U S A 86:9881–9885 Lê K-T, Villeneuve P, Ramjaun AR, McPherson PS, Beaudet A, Séguéla P (1998) Sensory presynaptic and widespread somatodendritic immunolocalization of central ionotropic P2X receptors. Neuroscience 83:177–190 Lim W, Kim SJ, Yan HD, Kim J (1997) Ca2+ -channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells. Pflugers Arch Eur J Physiol 435:34–42 Lin LF, Bott MC, Kao L-S, Westhead EW (1995) ATP stimulated catecholamine secretion: response in perfused adrenal glands and a subpopulation of cultured chromaffin cells. Neurosci Lett 183:147–150 Llewellyn-Smith IJ, Burnstock G (1998) Ultrastructural localization of P2X3 receptors in rat sensory neurones. Neuroreport 9:2545–2550 Llewellyn-Smith IJ, Pilowsky P, Minson JB (1993) The tungstatestabilized tetramethylbenzidine reaction for light and electron microscopic immunochemistry and for revealing biocytinfilled neurons. J Neurosci Methods 46:27–40 Liu M, Dunn PM, King BF, Burnstock G (1999) Rat chromaffin cells lack P2X receptors while those of the guinea-pig express a P2X receptor with novel pharmacology. Br J Pharmacol 128:61–68 Matsui T (1991) Biphasic rise caused by extracellular ATP in intracellular calcium concentration in bovine adrenocortical fasciculata cells. Biochem Biophys Res Commun 178:1266–1272 Niitsu A (1992) Calcium is essential for ATP-induced steroidogenesis in bovine adrenocortical fasciculata cells. Jpn J Pharmacol 60:269–274 North RA, Barnard EA (1997) Nucleotide receptors. Curr Opin Neurobiol 7:346–357 Oglesby IB, Lachnit WG, Burnstock G, Ford APDW (1999) Subunit specificity of polyclonal antisera to the carboxy terminal regions of P2X receptors, P2X1 through P2X7 . Drug Dev Res 47:189–195 Oomori Y, Okuno S, Fujisawa H, Iuchi H, Ishikawa K, Satoh Y, Ono K (1994) Ganglion cells immunoreactive for catecholamine-synthesising enzymes, neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland. Cell Tissue Res 275:201–213 Parker TL, Kesse WK, Mohamed AA, Afework M (1993) The innervation of the mammalian adrenal gland. J Anat 183: 265–276 Rassendren F, Buell GN, Virginio C, Collo G, North RA, Surprenant A (1997) The permeabilizing ATP receptor, P2X7 . Cloning and expression of a human cDNA. J Biol Chem 272: 5482–5486 Sparrow RA, Coupland RE (1987) Blood flow to the adrenal gland of the rat: its distribution between the cortex and the medulla before and after haemorrhage. J Anat 155:51–61 Surprenant A, Rassendren F, Kawashima E, North RA, Buell G (1996) The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7 ). Science 272:735–738 Unsicker K, Kabura-Flüh O, Zwarg U (1978) Different types of small granule-containing cells and neurones in the guinea-pig adrenal medulla. Cell Tissue Res 189:109–130 Vinson GP, Hinson JP (1992) Blood flow and hormone secretion in the adrenal gland. In: James VHT (ed) The adrenal gland, 2nd edn. Raven Press, New York, pp 71–86 Vulchanova L, Arvidsson U, Riedl M, Wang J, Buell G, Surprenant A, North RA, Elde R (1996) Differential distribution of two ATP-gated ion channels (P2x receptors) determined by immunocytochemistry. Proc Natl Acad Sci USA 93:8063– 8067 Winkler H, Westhead E (1980) The molecular organization of adrenal chromaffin granules. Neuroscience 5:1803–1823