Purinergic system in psychiatric diseases.

Psychiatric disorders are debilitating diseases, affecting >80 million people worldwide. There are no causal cures for psychiatric disorders and available therapies only treat the symptoms. The etiology of psychiatric disorders is unknown, although it has been speculated to be a combination of environmental, stress and genetic factors. One of the neurotransmitter systems implicated in the biology of psychiatric disorders is the purinergic system. In this review, we performed a comprehensive search of the literature about the role and function of the purinergic system in the development and predisposition to psychiatric disorders, with a focus on depression, schizophrenia, bipolar disorder, autism, anxiety and attention deficit/hyperactivity disorder. We also describe how therapeutics used for psychiatric disorders act on the purinergic system.

Unlocking the Potential of Purinergic Signaling in Transplantation.

Purinergic signaling has been recognized as playing an important role in inflammation, angiogenesis, malignancy, diabetes and neural transmission. Activation of signaling pathways downstream from purinergic receptors may also be implicated in transplantation and related vascular injury. Following transplantation, the proinflammatory "danger signal" adenosine triphosphate (ATP) is released from damaged cells and promotes proliferation and activation of a variety of immune cells. Targeting purinergic signaling pathways may promote immunosuppression and ameliorate inflammation. Under pathophysiological conditions, nucleotide-scavenging ectonucleotidases CD39 and CD73 hydrolyze ATP, ultimately, to the anti-inflammatory mediator adenosine. Adenosine suppresses proinflammatory cytokine production and is associated with improved graft survival and decreased severity of graft-versus-host disease. Furthermore, purinergic signaling is involved both directly and indirectly in the mechanism of action of several existing immunosuppressive drugs, such as calcineurin inhibitors and mammalian target of rapamycin inhibitors. Targeting of purinergic receptor pathways, particularly in the setting of combination therapies, could become a valuable immunosuppressive strategy in transplantation. This review focuses on the role of the purinergic signaling pathway in transplantation and immunosuppression and explores possible future applications in clinical practice.

Cytoplasmic filaments in developing and adult vertebrate smooth muscle.

An extensive study of adult and developing smooth muscle has revealed the widespread occurrence of a distinct filament with an average diameter of about 100 A (termed the 100 A filament). Unlike that of myofilaments, their appearance in longitudinal section is uniform, but in transverse section they have a round profile, occasionally exhibiting a less electron-opaque core. The 100 A filaments are almost invariably preserved under a variety of fixation procedures, whereas myofilaments, particularly the thicker filaments, are preserved inconsistently. The 100 A filaments appear to be randomly oriented throughout the cytoplasm, either singly or in small groups, although they are sometimes concentrated in the juxtanuclear region of the smooth muscle cells. The intimate association of 100 A filaments with dark bodies, in both developing and adult smooth muscle cells, may indicate that these filaments either play a role in dark body formation or, at least, constitute a part of the dark body. The 100 A filaments are conspicuous in developing smooth muscle cells and occasionally form networks or clusters; they appear to decrease in relative number as maturation proceeds, but considerable numbers are still present in adult tissue.

Freeze-fracture studies of nexuses between smooth muscle cells. Close relationship to sarcoplasmic reticulum.

The freeze-fracture appearance of the nexus was compared in the smooth muscle of guinea pig sphincter pupillac, portal vein, pulmonary artery, taenia coli, uretzr, and vas diferens, mouse vas deferens, chicken gizzard and anterior mesenteric artery, and toad stomach. Nexuses are particularly numerous in the guinea pig sphincter pupillae; they are usually oval and their average area is 0.15 mum2, although some as large as 0.6 mum2 were seen. Small aggregations of particles were observed which would not be recognizable as nexuses in thin section. What constitutes the minimum size of a nexus is discussed. It is estimated that the number of nexuses per cell in this preparation is of the order of tens rather than hundreds. All nexuses examined had 6-9-nm particles in the PF face, with corresponding 3-4-nm pits on the EF face forming a polygonal tending towards a hexagonal lattice. The nexuses are arranged in rows parallel to the main axis of the cell, usually alternating with longitudinal rows of plasmalemmal vesicles. Many nexuses in the guinea pig sphincter pupillae, chicken gizzard, and toad stomach show a close relationship with sarcoplasmic reticulum. The possibility that this may have some role in current flow across this specialized junction is discussed.

Fine structure of smooth muscle cells grown in tissue culture.

The fine structure of smooth muscle cells of the embryo chicken gizzard cultured in monolayer was studied by phase-contrast optics and electron microscopy. The smooth muscle cells were irregular in shape, but tended to be elongate. The nucleus usually contained prominent nucleoli and was large in relation to the cell body. When fixed with glutaraldehyde, three different types of filaments were noted in the cytoplasm: thick (150-250 A in diameter) and thin (30-80 A in diameter) myofilaments, many of which were arranged in small bundles throughout the cytoplasm and which were usually associated with dark bodies; and filaments with a diameter of 80-110 A which were randomly orientated and are not regarded as myofilaments. Some of the aggregated ribosomes were helically arranged. Mitochondria, Golgi apparatus, and dilated rough endoplasmic reticulum were prominent. In contrast to in vivo muscle cells, micropinocytotic vesicles along the cell membrane were rare and dense areas were usually confined to cell membrane infoldings. These cells are compared to in vivo embryonic smooth muscle and adult muscle after treatment with estrogen. Monolayers of cultured smooth muscle will be of particular value in relating ultrastructural features to functional observations on the same cells.

[3H]adenosine triphosphate: release during stimulation of enteric nerves.

The isolated taenia coli of the guinea pig takes up tritiated adenosine, adenosine monophosphate, adenosine diphosphate, and adenosine triphosphate, in preference to tritiated inosine and adenine. After uptake, [(3)H]adenosine is converted and retained primarily as [(3)H]adenosine triphosphate. Tritium is released from taenia coli treated with [(3)H]adenosine upon activation of the nonadrenergic inhibitory nerves. These results are consistent with the previous evidence that adenosine triphosphate may be the transmitter from the nerves.

Expression of P2X5 receptors in the mouse CNS.

P2X receptors are ATP-gated cationic channels composed of seven known subunits (P2X1-7) which are involved in different functions in neural tissue. The present study investigates the P2X5 receptor expression pattern in the mouse CNS using immunohistochemistry and in situ hybridization histochemistry. The specificity of the immunostaining has been verified by pre-absorption, Western blot and in situ hybridization methods. Heavy P2X5 receptor immunostaining was observed in the mitral cells of the olfactory bulb; cerebral cortex; globus pallidum, anterior cortical amygdaloid nucleus, amygdalohippocampal area of subcortical telencephalon; anterior nuclei, anteroventral nucleus, ventrolateral nucleus of thalamus; supraoptic nucleus, ventromedial nucleus, arcuate nucleus of hypothalamus; substantia nigra of midbrain; pontine nuclei, mesencephalic trigeminal nucleus, motor trigeminal nucleus, ambiguous nucleus, inferior olive, hypoglossal nucleus, dorsal motor vagus nucleus, area postrema of hindbrain; Purkinje cells of cerebellum; and spinal cord. The identification of extensive P2X5 receptor immunoreactivity and mRNA distribution within the CNS of the mouse demonstrated here is consistent with a role for extracellular ATP acting as a fast neurotransmitter.

Abnormalities in neuromuscular junction structure and skeletal muscle function in mice lacking the P2X2 nucleotide receptor.

ATP is co-released in significant quantities with acetylcholine from motor neurons at skeletal neuromuscular junctions (NMJ). However, the role of this neurotransmitter in muscle function remains unclear. The P2X2 ion channel receptor subunit is expressed during development of the skeletal NMJ, but not in adult muscle fibers, although it is re-expressed during muscle fiber regeneration. Using mice deficient for the P2X2 receptor subunit for ATP (P2X2(-/-)), we demonstrate a role for purinergic signaling in NMJ development. Whereas control NMJs were characterized by precise apposition of pre-synaptic motor nerve terminals and post-synaptic junctional folds rich in acetylcholine receptors (AChRs), NMJs in P2X2(-/-) mice were disorganized: misapposition of nerve terminals and post-synaptic AChR expression localization was common; the density of post-synaptic junctional folds was reduced; and there was increased end-plate fragmentation. These changes in NMJ structure were associated with muscle fiber atrophy. In addition there was an increase in the proportion of fast type muscle fibers. These findings demonstrate a role for P2X2 receptor-mediated signaling in NMJ formation and suggest that purinergic signaling may play an as yet largely unrecognized part in synapse formation.

Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells.

In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.

Purinergic receptors: their role in nociception and primary afferent neurotransmission.

The recent discovery of a P2X purinoceptor (a ligand-gated ion channel triggered by ATP) that is selectively expressed by small-diameter sensory neurons has led to the exploration of the sources of ATP involved in the initiation of different types of nociception and pain, including sympathetic nerves, endothelial cells and tumour cells. In addition, the anti-nociceptive actions of adenosine via prejunctional P1(A1) purinoceptors in the spinal cord and the pain-enhancing actions of adenosine via P1(A2) purinoceptors in the periphery have generated great interest in the development of P1 agonists and antagonists, as well as P2X antagonists as potential analgesic drugs.

P2X receptors in peripheral neurons.

P2X receptors are a family of ligand-gated ion channels, activated by extracellular ATP. The seven subunits cloned (P2X1-7) can assemble to form homomeric and heteromeric receptors. Peripheral neurons of neural crest origin (e.g. those in dorsal root, trigeminal, sympathetic and enteric ganglia) and placodal origin (e.g. those in nodose and petrosal ganglia) express mRNAs for multiple P2X subunits. In this review, we summarize the molecular biological, electrophysiological and immunohistochemical evidence for P2X receptor subunits in sensory, sympathetic, parasympathetic, pelvic and myenteric neurons and adrenomedullary chromaffin cells. We consider the pharmacological properties of these native P2X receptors and their physiological roles. The responses of peripheral neurons to ATP show considerable heterogeneity between cells in the same ganglia, between ganglia and between species. Nevertheless, these responses can all be accounted for by the presence of P2X2 and P2X3 subunits, giving rise to varying proportions of homomeric and heteromeric receptors. While dorsal root ganglion neurons express predominantly P2X3 and rat sympathetic neurons express mainly P2X2 receptors, nodose and guinea-pig sympathetic neurons express mixed populations of P2X2 and heteromeric P2X2/3 receptors. P2X receptors are important for synaptic transmission in enteric ganglia, although their roles in sympathetic and parasympathetic ganglia are less clear. Their presence on sensory neurons is essential for some processes including detection of filling of the urinary bladder. The regulation of P2X receptor expression in development and in pathological conditions, along with the interactions between purinergic and other signalling systems, may reveal further physiological roles for P2X receptors in autonomic and sensory ganglia.

Coexpression of rat P2X2 and P2X6 subunits in Xenopus oocytes.

Transcripts for P2X(2) and P2X(6) subunits are present in rat CNS and frequently colocalize in the same brainstem nuclei. When rat P2X(2) (rP2X(2)) and rat P2X(6) (rP2X(6)) receptors were expressed individually in Xenopus oocytes and studied under voltage-clamp conditions, only homomeric rP2X(2) receptors were fully functional and gave rise to large inward currents (2-3 microA) to extracellular ATP. Coexpression of rP2X(2) and rP2X(6) subunits in Xenopus oocytes resulted in a heteromeric rP2X(2/6) receptor, which showed a significantly different phenotype from the wild-type rP2X(2) receptor. Differences included reduction in agonist potencies and, in some cases (e.g., Ap(4)A), significant loss of agonist activity. ATP-evoked inward currents were biphasic at the heteromeric rP2X(2/6) receptor, particularly when Zn(2+) ions were present or extracellular pH was lowered. The pH range was narrower for H(+) enhancement of ATP responses at the heteromeric rP2X(2/6) receptor. Also, H(+) ions inhibited ATP responses at low pH levels (

P2X3 knock-out mice reveal a major sensory role for urothelially released ATP.

The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X(3) receptor knock-out (P2X(3)-/-) and wild-type control (P2X(3)+/+) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X(3). P2X(3)-positive staining was completely absent in the subepithelial plexus of the P2X(3)-/- mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder-pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X(3)+/+ and P2X(3)-/- mice, although P2X(3)-/- bladder had an increased capacity compared with that of the P2X(3)+/+ bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X(3)-/- mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X(3)+/+, but not P2X(3)-/-, were rapidly activated by intravesical injections of P2X agonists (ATP or alpha,beta-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2',3'-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2',4'-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X(3) receptors on a subpopulation of pelvic afferent fibers.

Inhibitory effects of some purinergic agents on ecto-ATPase activity and pattern of stepwise ATP hydrolysis in rat liver plasma membranes.

Inhibitory effects of various purinergic compounds on the Mg(2+)-dependent enzymatic hydrolysis of [(3)H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis-Menten kinetics with K(m) for ATP of 368+/-56 microM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap(4)A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC(50), 4.570)>Reactive blue 2 (4.297)&z.Gt;Ap(4)A (3. 268)>pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (2. 930). Slowly hydrolyzable P2 agonists ATPgammaS, ADPbetaS, alpha, beta-methylene ATP and beta,gamma-methylene ATP as well as the diadenosine polyphosphates Ap(3)A and Ap(5)A did not exert any inhibitory effects on the enzyme activity at concentration ranges of 10(-4)-10(-3) M. Thin-layer chromatography analysis of the formation of [(3)H]ATP metabolites indicated the presence of other enzyme activities on liver surface (ecto-ADPase and 5'-nucleotidase), participating in concert with ecto-ATPase in the nucleotide hydrolysis through the stepwise reactions ATP-->ADP-->AMP-->adenosine. A similar pattern of sequential [(3)H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap(4)A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [(3)H]ATP in the presence of Reactive blue 2 only followed the pattern ATP-->ADP, with formation of the subsequent metabolites AMP and adenosine being virtually eliminated. These data suggest that although nucleotide-binding sites of ecto-ATPase are distinct from those of P2 receptors, some purinergic agonists and antagonists can potentiate cellular responses to extracellular ATP through non-specific inhibition of the ensuing pathways of purine catabolism.

Steady-state binding of [3H]ATP to rat liver plasma membranes and competition by various purinergic agonists and antagonists.

Steady-state analysis of nucleotide-binding sites on rat liver plasma membranes was carried out using 3H-labelled ATP as radioligand under complete inhibition of ecto-ATPase activity by excess EDTA. Binding of [3H]ATP to the membranes is saturable, reversible and apparently involves one population of specific binding sites with Kd of about 90 nM and binding capacity (Bmax) of 15 pmol/mg protein. A broad spectrum of purinergic agonists and antagonists was examined as potential inhibitors of the measured binding. The displacement studies showed the following rank order of inhibitory potency for [3H]ATP-binding sites (pIC50 values in parentheses): ATPgammaS (7.49)>2-MeSATP (7.18)>ATP (6.91)>ADPbetaS (6.64)>/=ADP (6.56)>>RB2 (6.14)>>suramin (5.40)>>Ap4A (4. 57)>alpha,beta-MeATP (4.19)>/=beta,gamma-MeATP (3.97). AMP, adenosine, Ap5A, PPADS, beta-glycerophosphate as well as non-adenine nucleoside triphosphates GTP, UTP and CTP did not exert any effect on the measured binding at concentration ranges of 10-6-10-4 M. In order to ascertain whether ATP and its analogues are capable of interacting with the same binding domain, 2-MeSATP and ADP were treated as alternative ligands that could compete with unlabelled ATP for its binding sites. A 2-fold increase of Kd value for ATP-receptor interaction was observed in the presence of 2-MeSATP (60 nM) or ADP (250 nM) without any modulation of Bmax value, confirming that inhibitory effects of these compounds are competitive in nature. These studies demonstrate that ATP and its analogues are able to interact with a single binding domain on liver plasma membranes, which may be identified as ligand-binding component of P2 purinoceptors of the P2Y1 subtype.

Purine-mediated signalling in pain and visceral perception.

Receptor subtypes for purines have been identified in a variety of tissues, increasing interest in the roles of purine-mediated signalling in pathophysiological processes. Growing evidence supports the involvement of one of the purinoceptor subtypes, P2X3, in nociception. In this article, recent studies of purine-mediated nociception and visceral pain will be discussed. Furthermore, a novel hypothesis is proposed for purine-mediated mechanosensory transduction where ATP released during distension from epithelial cells lining tubes (such as ureter and gut) and sacs (such as the bladder) acts on P2X3 receptors on a subepithelial nerve plexus to initiate impulses that are relayed via the spinal cord to pain centres in the brain.

Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors.

In the past five years, an extended series (P2Y1-n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y1-n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.

Selective damage to sensorimotor perivascular nerves in the mesenteric vessels of diabetic rats.

The perivascular innervation of the superior mesenteric artery and vein was examined using immunohistochemical and immunoassay techniques in rats 8 weeks after induction of diabetes with streptozotocin (STZ). Increased density of innervation and fluorescence intensity was noted for substance P- and calcitonin gene-related peptide-immunoreactive nerves in the diabetic vessels. A slight increase in the density of vasoactive intestinal polypeptide-immunoreactive nerve fibers innervating the mesenteric artery was also noted. However, there was no change in the density of neuropeptide Y- and dopamine beta-hydroxylase-immunoreactive nerve fibers, although the fluorescence intensity of neuropeptide Y-immunoreactive nerve fibers was reduced in diabetic rat vessels. Immunoassays showed that the levels of substance P- and calcitonin gene-related peptide were increased > 10-fold in the diabetic mesenteric vein, while levels of neuropeptide Y and vasoactive intestinal polypeptide were unchanged. In summary, there is a marked increase in nerve fibers containing sensory neuropeptides in mesenteric vessels of STZ-induced diabetic rats, which, in view of the reported impaired sensorimotor function in these vessels, is likely to reflect a neuropathic change.

Vasoactive intestinal polypeptide-like immunoreactive nerves in diabetic penis. A comparison between streptozotocin-treated rats and man.

Vasoactive intestinal polypeptide (VIP) has been demonstrated by immunofluorescence histochemistry in nerves in human and rat penile tissue. A reduction in VIP-like immunoreactivity in nerves was revealed in tissue from streptozotocin-diabetic rats and a human diabetic with impotence. These results suggest that an impairment in the VIP-ergic innervation in penile tissue may be an important factor in the development of impotence in diabetes. They also support the view that the streptozotocin-treated rat is a useful experimental model for diabetic autonomic neuropathy.

Purinoceptors: are there families of P2X and P2Y purinoceptors?

There has been an exponential growth in interest in purinoceptors since the potent effects of purines were first reported in 1929 and purinoceptors defined in 1978. A distinction between P1 (adenosine) and P2 (ATP/ADP) purinoceptors was recognized at that time and later, A1 and A2, as well as P2x and P2y subclasses of P1 and P2 purinoceptors were also defined. However, in recent years, many new subclasses have been claimed, particularly for the receptors to nucleotides, including P2t, P2z, P2u(n) and P2D, and there is some confusion now about how to incorporate additional discoveries concerning the responses of different tissues to purines. The studies beginning to appear defining the molecular structure of P2-purinoceptor subtypes are clearly going to be important in resolving this problem, as well as the introduction of new compounds that can discriminate pharmacologically between subtypes. Thus, in this review, on the basis of this new data and after a detailed analysis of the literature, we propose that: (1) P2X(ligand-gated) and P2Y(G-protein-coupled) purinoceptor families are established; (2) four subclasses of P2X-purinoceptor can be identified (P2X1-P2X4) to date; (3) the variously named P2-purinoceptors that are G-protein-coupled should be incorporated into numbered subclasses of the P2Y family. Thus: P2Y1 represents the recently cloned P2Y receptor (clone 803) from chick brain; P2Y2 represents the recently cloned P2u (or P2n) receptor from neuroblastoma, human epithelial and rat heart cells; P2Y3 represents the recently cloned P2Y receptor (clone 103) from chick brain that resembles the former P2t receptor; P2Y4-P2Y6 represent subclasses based on agonist potencies of newly synthesised analogues; P2Y7 represents the former P2D receptor for dinucleotides. This new framework for P2 purinoceptors would be fully consistent with what is emerging for the receptors to other major transmitters, such as acetylcholine, gamma-aminobutyric acid, glutamate and serotonin, where two main receptor families have been recognised, one mediating fast receptor responses directly linked to an ion channel, the other mediating slower responses through G-proteins. We fully expect discussion on the numbering of the different receptor subtypes within the P2X and P2Y families, but believe that this new way of defining receptors for nucleotides, based on agonist potency order, transduction mechanisms and molecular structure, will give a more ordered and logical approach to accommodating new findings. Moreover, based on the extensive literature analysis that led to this proposal, we suggest that the development of selective antagonists for the different P2-purinoceptor subtypes is now highly desirable, particularly for therapeutic purposes.