Download Distribution of P2X receptors in the rat adrenal gland

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