Search

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14748. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

P2X7 is an intriguing membrane receptor for the extracellular nucleotide ATP, which functions as a ligand-gated ion channel; it can activate cell membrane permeabilization and also has a wide range of downstream signaling pathways, including mediation of inflammatory responses and modulation of cell turnover. Despite recent identification of P2X7 receptor protein in the renal tract, the biological and potential pathological functions of this receptor and its signaling cascades in the kidney are not yet fully understood. P2X7 receptor protein is expressed in normal kidney development, predominantly in the condensing mesenchyme, and later in the maturing and adult derivatives of the ureteric bud. Glomerular expression of the molecule is scarce in normal kidney, but is upregulated in chronic and inflammatory conditions, suggesting a role in the inflammatory response or in repair and remodeling in these settings. P2X7 receptor expression in the adult collecting ducts of murine kidney, as well as the collecting duct cysts in autosomal recessive polycystic kidney disease, has been described and agonists of the receptor can modulate the development of renal cysts in an in vitro model of cyst formation derived from the cpk/cpk mouse. Further investigation of the function of the P2X7 receptor in normal and abnormal kidneys might lead to novel therapeutic targets in a wide range of renal diseases.

We report on the ultrastructural distribution of arginine-vasopressin (AVP) in the heart of newborn rats using pre-embedding peroxidase-antiperoxidase immunocytochemistry with a polyclonal AVP antibody for electron microscopy. Positive labelling for AVP was localized in endothelial cells of main coronary arteries and cardiac vessels of smaller diameter (microvessels). Examination of the right coronary artery showed that approximately 58% of the endothelial cells were positive for AVP. Immunoreactivity to AVP in the cytoplasm of arterial endothelium predominated in association with the membranes of granular endoplasmic reticulum and in subplasmalemmal areas. The endothelium of small vessels exhibits less endoplasmic reticulum, but still shows AVP immunoprecipitate in the cytoplasm. It is suggested that endothelial AVP may contribute to vasomotor control of the coronary circulation in the early stages of postnatal development. AVP antibody also labelled some fibroblast/fibroblast-like cells associated with the coronary arteries and microvessels; thus, these cells as well as the endothelium appear to be a source of AVP in the newborn rat heart. The functional significance of these findings is discussed.

Seven P2X receptor subunits have been cloned which form functional homo- and heterotrimers. These are cation-selective channels, equally permeable to Na(+) and K(+) and with significant Ca(2+) permeability. The three-dimensional structure of the P2X receptor is described. The channel pore is formed by the α-helical transmembrane spanning region 2 of each subunit. When ATP binds to a P2X receptor, the pore opens within milliseconds, allowing the cations to flow. P2X receptors are expressed on both central and peripheral neurons, where they are involved in neuromuscular and synaptic neurotransmission and neuromodulation. They are also expressed in most types of nonneuronal cells and mediate a wide range of actions, such as contraction of smooth muscle, secretion, and immunomodulation. Changes in the expression of P2X receptors have been characterized in many pathological conditions of the cardiovascular, gastrointestinal, respiratory, and urinogenital systems and in the brain and special senses. The therapeutic potential of P2X receptor agonists and antagonists is currently being investigated in a range of disorders, including chronic neuropathic and inflammatory pain, depression, cystic fibrosis, dry eye, irritable bowel syndrome, interstitial cystitis, dysfunctional urinary bladder, and cancer.

Adenosine 5'-triphosphate (ATP) is a cotransmitter with classical transmitters in most nerves in the peripheral and central nervous systems, although the proportions vary between tissues and species and in different developmental and pathophysiological circumstances. There was early evidence that ATP was released together with acetylcholine (ACh) from motor nerves supplying skeletal muscle, although it was considered at the time as a molecule involved in the vesicular uptake and storage of ACh. Later it was shown that in the developing neuromuscular junction, released ATP acted on P2X receptor ion channels as a genuine cotransmitter with ACh. Adenosine triphosphate was shown to be released from sympathetic nerves supplying the guinea-pig taenia coli in 1971. Soon after, the possibility was raised that ATP was coreleased with noradrenaline from sympathetic nerves to guinea-pig seminal vesicle, cat nictitating membrane and guinea-pig vas deferens. Sympathetic purinergic cotransmission has also been demonstrated in many blood vessels. Parasympathetic nerves supplying the urinary bladder use ACh and ATP as cotransmitters; ATP acts through P2X ionotropic receptors, whereas the slower component of the response is mediated by the metabotropic muscarinic receptor. Adenosine triphosphate and glutamate appear to be cotransmitters in primary afferent sensory neurons. Adenosine triphosphate, calcitonin gene-related peptide and substance P coexist in some sensory-motor nerves. A subpopulation of intramural enteric nerves provides non-adrenergic, non-cholinergic inhibitory innervation of gut smooth muscle. Three cotransmitters are involved, namely ATP, nitric oxide and vasoactive intestinal polypeptide. In recent years, studies have shown that ATP is released with ACh, noradrenaline, glutamate, gamma-aminobutyric acid, 5-hyroxytryptamine and dopamine in different subpopulations of neurons in the central nervous system.

The purinergic signalling system is one of the most ancient and arguably the most widespread intercellular signalling system in living tissues. In this review we present a detailed account of the early developments and current status of purinergic signalling. We summarize the current knowledge on purinoceptors, their distribution and role in signal transduction in various tissues in physiological and pathophysiological conditions.

The choroid plexuses secrete cerebrospinal fluid (CSF) and regulate the brain's internal environment via the blood-CSF barrier. The permeability properties of the blood-CSF interface have been studied previously in adult and immature brains, however, little is known about the development of CSF secretion and its modulation. ATP influences secretion in other epithelia via ionotropic P2X or metabotropic P2Y receptors. P2 receptors have frequently been found to be down-regulated in the postnatal period, suggesting a developmental role for purinergic and pyrimidine signalling. The present study investigated the expression of P2 receptors in lateral ventricular choroid plexus in relation to recent studies of aquaporin-1 expression and rapid expansion of the lateral ventricles in rat embryos. In the present study mRNAs for all known mammalian nucleotide receptor subtypes, except P2X(7), were identified from as early as E15. P2X(7) mRNA was detected from E18. Indications of differential expression patterns were observed for the different subtypes during development: an apparent increase in expression for P2Y(2) and P2X(7), a decline in P2X(1-2,4), no detectable difference in expression levels for P2X(6) and P2Y(12-13) and transient expression peaks for P2X(3,5) and P2Y(1,4,6,14). P2X(4,5,7) and P2Y(1,4) receptor proteins were detected immunohistochemically in the choroidal epithelium from early in development (E15 or E18). Their differing developmental profiles suggest specific roles in the development of CSF secretion that may have particular relevance for the rapid expansion of the ventricles that occurs in the embryo. P2X(5) and P2Y(6) were also detected in the developing neuropendyma from P0 and P9, respectively.

Many factors play a role in the development of atherosclerotic lesions. One of the leading risk factors for development of atherosclerosis is familial hypercholesterolemia (FH). FH is a genetic disease characterized by a deficiency, and/or mutation, of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells (EC), a high level of low density lipoprotein in the plasma, and early, spontaneous development of atherosclerosis and skin xanthoma. In this review we describe Watanabe heritable hyperlipidemic (WHHL) rabbits, which represent such an animal model for human FH. This strain of the rabbits is characterized by a genetic deficiency or mutation of functional LDL receptors and develops severe atherosclerosis, which is pathologically similar to familial homozygous hyperlipidemic patients. The most completely characterized animal model is the Watanabe rabbit, a model of homozygous and heterozygous type IIa hypercholesterolemia related to an LDL receptor deficiency. Additional manipulation such as aortic injury in this rabbit model induces the development of atherosclerotic lesions that are structurally similar to those found in humans. Thus, this model of hypercholesterolemia fulfils the above criteria set, i.e. it is able to provide new insights for a better understanding of the pathogenesis of atherosclerosis and for testing new treatment strategies.

P2X7 is an intriguing membrane receptor for the extracellular nucleotide ATP, which functions as a ligand-gated ion channel; it can activate cell membrane permeabilization and also has a wide range of downstream signaling pathways, including mediation of inflammatory responses and modulation of cell turnover. Despite recent identification of P2X7 receptor protein in the renal tract, the biological and potential pathological functions of this receptor and its signaling cascades in the kidney are not yet fully understood. P2X7 receptor protein is expressed in normal kidney development, predominantly in the condensing mesenchyme, and later in the maturing and adult derivatives of the ureteric bud. Glomerular expression of the molecule is scarce in normal kidney, but is upregulated in chronic and inflammatory conditions, suggesting a role in the inflammatory response or in repair and remodeling in these settings. P2X7 receptor expression in the adult collecting ducts of murine kidney, as well as the collecting duct cysts in autosomal recessive polycystic kidney disease, has been described and agonists of the receptor can modulate the development of renal cysts in an in vitro model of cyst formation derived from the cpk/cpk mouse. Further investigation of the function of the P2X7 receptor in normal and abnormal kidneys might lead to novel therapeutic targets in a wide range of renal diseases.

Vascular endothelial cells have been identified as a source of substance P (SP) which may act in an autocrine/paracrine fashion to bring about nitric oxide (NO)-dependent vasodilatation and mitogen-induced cell division or immunologic and inflammatory responses. Whilst SP is localised in and released from endothelial cells, an endothelial mRNA expression of SP has not previously been shown. In the present study, mRNA expression of SP in human dermal microvascular endothelial cells is demonstrated using in situ hybridisation techniques with enhancement procedures. Incubation of microvascular endothelial cells with nerve growth factor (NGF) under conditions of increased shear stress increases the mRNA expression and release of SP Endothelin (ET) release is also enhanced. These changes are pertinent to circulatory, events that may occur in Raynaud's phenomenon in systemic sclerosis.

After some early hints, cotransmission was proposed in 1976 and the 'chemical coding' gradually established for sympathetic, parasympathetic, sensory-motor, enteric and some invertebrate nerves. More recently, cotransmission has been recognised in the central nervous system. ATP appears to be a primitive signalling molecule that has been retained as a cotransmitter in every nerve type in both peripheral and central nervous systems, although the relative role of ATP varies considerably in different species and pathological conditions. In the past two years, interest has focused on the mechanisms underlying cotransmission, plasticity and differential control of cotransmitter expression.

In vitro experiments were carried out to measure the contractile responses to P(2)-receptor agonists in the greater saphenous vein isolated from patients with obliterating vascular atherosclerosis and varicose veins of the legs. In patients with varicose veins, the contractile responses of the greater saphenous vein to ATP, alpha,beta-methylene-ATP, and UTP were significantly lower than in patients with obliterating atherosclerosis, while the responses to ADP, adenosine, and 2-methylthio-ATP were similar in both groups. These data attest to the presence of P(2)-receptor-mediated contraction component in the greater saphenous vein, which are pronouncedly weakened during varicose disease.

In situ hybridisation to identify P2Y(2) receptor mRNA was performed for the first time at the ultrastructural level on the thymus of adult male rats. These studies revealed transcripts for P2Y(2) receptors in cortical T cells and endothelial cells of thymic blood vessels. These transcripts are likely to be linked with the production of functional P2Y(2) receptors in these cells. In the T cells, transcripts for the P2Y(2 )receptor were localised in the cytoplasm as well as on the smooth endoplasmic reticulum and cell membrane. Dividing T cells also expressed P2Y(2 )receptor mRNA, mostly in the cytoplasm around chromosomal material. Endothelial cells displaying labels for P2Y(2 )receptor transcripts were of cortical arteries/arterioles and capillaries and of postcapillary venules in the corticomedullary junction. P2Y(2) mRNA transcripts were localised in the cytoplasm of endothelial cells, although they did not appear to be specifically associated with subcellular organelles or structures. In postcapillary venules, T cells displaying labelling for the P2Y(2) receptor were seen migrating across the P2Y(2) receptor mRNA-positive endothelium. Our findings are discussed in terms of the relationship between thymic immune cells and the endothelium. This includes the issue of immune cell trafficking into the circulation, and the ATP-related regulatory role and involvement of P2Y(2) receptors in the rat thymus.

Dorsal root ganglion (DRG) neurons respond to ATP with transient, persistent or biphasic inward currents. In contrast, the ATP responses in nodose neurons are persistent. These sustained currents are also heterogeneous, with one component being accounted for by P2X2/3 receptors, and the residual response probably mediated by P2X2 receptors, although the direct evidence for this has been lacking. In the present study, we examined the P2X receptors on DRG and nodose neurons from P2X3-deficient (P2X3-/-) mice, using whole cell voltage-clamp recording and immunohistochemistry. We found that all P2X3-/- DRG neurons lacked rapidly desensitizing response to ATP, and both DRG and nodose neurons from P2X3-null mutant mice no longer responded to alpha,beta-methylene ATP (alphabetameATP). In contrast, ATP evoked persistent inward current in 12% of DRG neurons and 84% of nodose neurons from P2X3-/- mice. This retained persistent response to ATP on nodose neurons had an EC50 for ATP of 77 microm, was antagonized by Cibacron blue and pyridoxal-5-phosphate-6-azophenyl-2',4'-disulphonic acid, potentiated by Zn2+ and acidification, but not enhanced by ivermectin or diinosine pentaphosphate. 2',3'-O-Trinitrophenyl-ATP antagonized this response with an IC50 of 8 microm. All these properties are consistent with those of recombinant P2X2 homomeric receptors. Furthermore, specific P2X2 receptor immunoreactivity detected in wild-type sensory neurons was unaltered in null mutant mice. Therefore, the alphabetameATP-insensitive persistent responses on nodose neurons are likely to be mediated by P2X2 homomers, which contribute to 60% of currents evoked by 100 microm ATP in the wild type.

Here we review the results of physiological, pharmacological, and pathophysiological studies of ATP receptors, P2 type receptors. Characteristics of two families of P2 receptors (P2X and P2Y) are presented and the possibility of using preparations affecting these receptors is discussed.

Adenosine triphosphate (ATP) has a fundamental intracellular role as the universal source of energy for all living cells. The demonstration of its release into the extracellular space and the identification and localisation of specific receptors on target cells have been essential in establishing, after considerable resistance, its extracellular physiological roles. It is now generally accepted that ATP is a genuine neurotransmitter both in the central and peripheral nervous systems. As such, there are numerous arguments which prove that the release of ATP by nerve terminals is by exocytosis. In some non-neuronal cells, however, recent evidence suggests that ATP release could also be carrier-mediated and would involve ATP-binding cassette proteins (ABC), an ubiquitous family of transport ATPases.

We have investigated the activity of naturally occurring high-performance liquid chromatography-purified diadenosine polyphosphates (Ap(n)A, n = 5-6), adenosine polyphospho guanosines (Ap(n)G, n = 5-6), and diguanosine polyphosphates (Gp(n)G, n = 5-6) under voltage-clamp conditions at recombinant rat P2X1-4 purinoceptor subtypes expressed in Xenopus laevis oocytes. At rP2X1 and rP2X3 receptors, Ap(n)As and Ap(n)Gs evoked concentration-dependent inward currents. Gp(n)Gs were not active at these receptors. At rP2X2 and rP2X4 receptors, dinucleotides did not show significant activity. For the rP2X1 receptor, Ap(n)As and Ap(n)Gs were partial agonists; for the P2X3 receptor, only Ap5G was full agonist, whereas the other tested substances were partial agonists. The rank order of potency at rP2X1 was ATPor = Ap6Aor = Ap5Aor = Ap6Gor = Ap5G, and rank order of efficacy was ATPor = Ap5Aor = Ap6AAp5GAp6G, whereas at rP2X3 the rank order of potency was ATPAp5Gor = Ap5Aor = Ap6Aor = Ap6G and the rank order of efficacy was ATP approximately Ap5Gor = Ap5A approximately Ap6Aor = Ap6G. For rP2X1 and rP2X3 it is evident that receptor agonism depended on the presence of at least one adenine moiety in the dinucleotide, while the presence of a guanine moiety had a significant impact and decreased agonist efficacy. The data suggest that naturally occurring Ap(n)As and Ap(n)Gs may play an important physiological role in different human tissues and systems by activating group I P2X receptors.

Using voltage-clamp procedures on Xenopus oocytes, agonist-evoked ionic currents by P2X receptors resulting from the coexpression of P2X(2) and P2X(3) subunits were compared against the agonist responses of homomeric P2X(2) and P2X(3) receptors. With the quantity of P2X(3) mRNA kept constant and quantity of P2X(2) mRNA progressively increased, expressed P2X receptors changed from a P2X(3)-like receptor to a P2X(2)-like receptor. In all cases, however, agonist-evoked responses comprised biphasic (fast and slow) currents-the former showing the properties of P2X(3) receptors and latter consistent with the presence of P2X(2) and P2X(2/3) receptors. Using desensitization procedures, the P2X(3)-like fast current was selectively removed to allow the slow current to be studied in isolation. P2X(2/3) receptors were then characterized by slowly inactivating inward currents that were reproducible within 30 s of washout and whose pharmacological profile [selective agonists, Ap(5)Aalpha,beta-methylene ATPbeta,gamma-methylene ATPUTP; antagonists, TNP-ATPsuraminor = Reactive blue-2 (RB-2)] contrasted with the profile of P2X(2) receptors (Ap(5)A, alpha,beta-methylene ATP, beta,gamma-methylene ATP, and UTP inactive; antagonists, RB-2TNP-ATPsuramin). Thus, our experiments reveal that coexpression of two P2X subunits, which of themselves can generate functional homomeric receptors, results in a complex population of heterogeneous P2X receptors-in this case P2X(2), P2X(3), and P2X(2/3) receptors. Depending on the relative levels of P2X subunit coexpression, the operational profile of the resultant P2X receptors can change from one phenotype to another. This spectrum may explain the variability of agonist responses in small sensory neurons that also express P2X(2) and P2X(3) subunits in different amounts.

ATP acts as a humoral mediator to control cell function extracellularly. The receptors that mediate the actions of ATP belong to two classes, the metabotropic P2Y receptors and the transmitter-gated, ion channel P2X receptors. This review describes the structure, distribution, function, and ligand recognition characteristics of P2X receptors, which comprise seven distinct subunits that can function as both homo- and hetero- polymers. The pharmacology of P2X receptors is complicated by marked differences between species orthologues. The current nomenclature is based largely on recombinant receptor studies and detailed knowledge of endogenous P2X receptors in native tissues is limited because of lack of good selective agonists and antagonists for each receptor type.

1. Application of ATP and alpha,beta-methylene ATP (alpha beta meATP) to voltage-clamped guinea-pig pelvic neurons produced three types of inward currents. A fast-desensitizing response was present in 5% (25/660) of neurons, 70% gave slowly-desensitizing currents, and the remainder had biphasic responses. 2. Slowly-desensitizing responses were characterized pharmacologically. The response to alpha beta meATP 100 microM was 46+/-27% (range 0--100%) of that evoked by ATP 100 microM in the same cell. Cross-desensitization indicated the presence of alpha beta meATP-sensitive and -insensitive receptors. 3. The concentration-response curve for alpha beta meATP had an EC(50) of 55 microM, and a Hill coefficient of 0.99, while at the alpha beta meATP-insensitive receptor, ATP had an EC(50) of 73 microM, with a Hill coefficient of 1.78. 4. The response to alpha beta meATP was blocked by pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS), suramin and Cibacron blue. However, the alpha beta meATP-insensitive receptor was inhibited by PPADS, but not by the other two antagonists. 5. 2'- (or 3'-) O-trinitrophenyl-ATP was 10 times more potent in inhibiting responses to alpha beta meATP than to ATP (at the alpha beta meATP-insensitive receptor). 6. Lowering extracellular pH potentiated responses to alpha beta meATP and ATP, while raising pH attenuated them. 7. Co-application of Zn(2+) (3--300 microM) inhibited the responses to alpha beta meATP and ATP, with IC(50) values of 286 and 60 microM, respectively. 8. In conclusion, unlike rat and mouse pelvic ganglion neurons, which only express P2X(2) homomers, at least three distinct P2X receptors are present in guinea-pig pelvic neurons, probably homomeric P2X(2), P2X(3) and heteromeric P2X(2/3) receptors. However, some of the novel pharmacological properties observed suggest that the guinea-pig P2X receptor subtypes may differ from their rat orthologues.

The distribution of the P2X(2) subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X(2) receptors was localised in: (i) paraventricular and supraoptic nuclei-in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis-in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X(2)-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X(2)-positive axons on P2X(2)-positive endocrine cell bodies as well as on P2X(2)-negative somata were also observed. The functional significance of these findings is discussed.

Stimulation of endothelial cells from human umbilical vein by shear stress induced release of endogenous ATP which was accompanied by an extracellular increase in the activity of enzymes degrading both ATP (ATPases) and AMP (5'-nucleotidases). The activity of soluble ATPase was progressively increased from 1.62+/-0.27 to 12.7+/-1.0 pmoles ml(-1) h(-1) after 60 min of stimulation by shear stress. The rate of [(3)H]-ATP hydrolysis in the medium was inhibited by the purinergic agents suramin, Reactive blue 2 and pyridoxalphosphate-6-azophenyl-2'4'-disulphonic acid, and remained insensitive to the classic inhibitors of ion-pumping and intracellular ATPases. Shear stress also increased the activity of 5'-nucleotidase in the medium from 2.0+/-0.5 to 27.2+/-2.8 pmoles ml(-1) h(-1). When shear stress was applied after removal of ecto-5'-nucleotidase by phosphatidylinositol-specific phospholipase C, the release of 5'-nucleotidase was drastically reduced. These results show that soluble ATPase and 5'-nucleotidase which are released during shear stress are not released from an intracellular compartment together with ATP but have an extracellular origin.

Ultrastructural localisation of type I (neuronal) and type III (endothelial) isoforms of nitric oxide synthase in perivascular nerve fibres (axons) and endothelial cells was studied in the Wistar rat cerebral basilar artery, using monoclonal antibodies either to type I or type III nitric oxide synthase and post-embedding colloidal-gold immunocytochemistry. Labelling signal (gold particles) for type I and type III nitric oxide synthase was localised both in axons and endothelial cells. In the axon profiles, labelling for either type I or type III nitric oxide synthase was localised in the axoplasm and the lumen and/or membrane of small agranular synaptic vesicles. In the endothelial cells, labelling for either type-I or type-III nitric oxide synthase was predominantly in the cytoplasm. The present qualitative data extends our previous study of cerebrovascular nerve fibres and endothelial cells employing monoclonal antibodies; the localisation of nitric oxide synthase in a subpopulation of synaptic vesicles in nitric oxide synthase-positive cerebrovascular nerves suggests that vesicular mechanisms may be involved in the production/release of nitric oxide.

Physiological and pharmacological studies have shown that ATP has potent effects on developing chick skeletal muscle. These effects have previously been shown to be developmentally regulated, and the responses were characteristic of activation of the P2X ligand-gated ion-channel family of ATP receptors. Here, using immunohistochemistry, we describe the expression patterns of two members of the P2X receptor family, P2X5 and P2X6, during development of skeletal muscle in the chick embryo. These receptors were first expressed at early stages of skeletal muscle development, and expression disappeared immediately before the stage at which fusion of myoblasts to form myotubes occurs. P2X5 was also demonstrated in nerves supplying developing skeletal muscle, in some dorsal root ganglion cells, and in dorsal and ventral spinal cord. No expression of the other five members of the P2X family were demonstrated in developing skeletal muscle.

1. The antagonist activity of a series of diinosine polyphosphates (IpnI, where n=3, 4, 5) was assessed against ATP-activated inward currents at rat P2X(1-4) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2. Diinosine polyphosphates were prepared by the enzymatic degradation of their corresponding diadenosine polyphosphates (e.g., Ap5A into Ip5I) using 5'-adenylic deaminase, and purified using reverse-phase chromatography. 3. Against ATP-responses at rP2X1 receptors, the potency order for antagonism was (pIC50): Ip5I (8.5)Ip4I (6.3)Ip3I (4.5). Ip5I (10-100 nM) caused a concentration-dependent rightwards displacement of the ATP concentration-response curve without reducing the maximum ATP effect. However, the Schild plot was non-linear which indicated Ip5I is not a competitive antagonist. Blockade by micromolar concentrations of Ip5I was not surmountable. Ip4I also behaved as a non-surmountable antagonist. 4. Against ATP-responses at rP2X3 receptors, the potency order for antagonism was (pIC50): Ip4I (6. 0)Ip5I (5.6)Ip3I (4.5). Blockade by Ip4I (pA2, 6.75) and Ip5I (pA2, 6.27) was surmountable at micromolar concentrations. 5. Diinosine polyphosphates failed to inhibit ATP-responses at rP2X2 receptors, whereas agonist responses at rP2X4 were reversibly potentiated by Ip4I and Ip5I. None of the parent diadenosine polyphosphates behave as antagonists at rP2X1 - 4 receptors. 6. Thus, Ip5I acted as a potent and relatively-selective antagonist at the rP2X1 receptor. This dinucleotide pentaphosphate represents a high-affinity antagonist for the P2X1 receptor, at which it acts in a competitive manner at low (/=100 nM) concentrations but has more complex actions at higher (100 nM) concentrations.

1 The modulatory activity of extracellular H+ and Zn2+ was examined on ATP-responses at rat P2X1 (rP2X1) and rat P2X3 (rP2X3) receptors expressed in Xenopus oocytes and studied under voltage-clamp conditions. 2 Superfused ATP (0.03-30 microM, at pH 7.5) evoked inward currents at rP2X1 receptors (EC50 value, 300+/-7 nM). ATP potency was reduced 2 fold at pH 6.5, and 6 fold at pH 5.5, without altering the maximum ATP effect. Alkaline conditions (pH 8.0) did not alter ATP activity. 3 Superfused ATP (0.01 - 300 microM, at pH 7. 5) evoked inward currents at rP2X3 receptors (EC50 value, 1.8+/-0.3 microM). ATP activity was affected only at pH 5.5, reducing agonist potency 15 fold without altering the maximum ATP effect. 4 Extracellular Zn2+ inhibited ATP-responses at rP2X1 receptors in a time-dependent manner, a 20 min pre-incubation being optimal (IC50 value, 1.0+/-0.2 microM). However, the Zn2+ effect was pH-independent, suggesting Zn2+- and H+-inhibition of ATP-responses occur through independent processes. 5 Extracellular Zn2+ weakly potentiated ATP-responses at rP2X3 receptors (EC50 value, 11+/-1 microM). The Zn2+ effect was dependent on pre-incubation time and, with 20 min pre-incubation periods, Zn2+ potentiated then inhibited ATP-responses in a concentration-dependent, but pH-independent, manner. 6 In summary, ATP activity at rP2X1 receptors was decreased by both extracellular H+ and Zn2+ and their effects were additive. ATP activity at rP2X3 receptors was less sensitive to H+-inhibition and, in contrast, was potentiated by Zn2+ in a pH-independent manner. These differential effects may help distinguish P2X1 and P2X3 receptors in whole tissues.

The pattern of distribution and colocalization of nitric oxide synthase and the calcium-binding protein calretinin in myenteric neurons and nerve fibers were examined in the human small intestine from preterm fetuses (14-17 weeks of gestation), normal adults (mean age 50 years old), old age (mean age 80 years old), and Crohn's disease patients (mean age 30 years old) using NADPH-diaphorase histochemistry and immunohistochemical techniques. In all age groups investigated, NADPH-diaphorase-reactive and calretinin-immunoreactive neurons and nerve fibers were seen throughout the myenteric plexus. The highest proportion of NADPH-diaphorase-reactive neurons was found in the myenteric ganglia of old age intestines (56% of protein gene product-immunoreactive neurons) followed by fetal intestines (41%) and Crohn's intestine (30%) compared with intestines of control adults (20%). A similar trend was observed for calretinin-immunoreactive neurons where the highest proportion of immunoreactive neurons was found in the myenteric ganglia of old age intestines (28% of protein gene product-immunoreactive neurons), followed by fetal intestines (22%), and Crohn's intestines (18%) compared with intestines of control adults (9%). A colocalization of NADPH-diaphorase activity and calretinin immunoreactivity was only seen in the myenteric neurons of fetal intestines (2% of NADPH-diaphorase-reactive neurons were also calretinin-immunoreactive). The pattern of distribution of NADPH-reactive and calretinin-immunoreactive neurons in the myenteric ganglia of fetal intestine differs from that of the other age groups. In the fetal intestine, the myenteric neurons containing either calretinin or NADPH-diaphorase are distributed through out the myenteric ganglia with no specific orientation to one another. In the intestines of control adult, Crohn's, and old age patients, single large calretinin-immunoreactive neurons are surrounded by a number of small NADPH-diaphorase-positive neurons, with this feature being more prominent in intestines of old-age and Crohn's disease patients. In summary, a high number of both NADPH-diaphorase-reactive and calretinin-immunoreactive neurons were seen in the myenteric ganglia of fetal, old age, and Crohn's intestines; we discuss that there may be a role for nitric oxide and calretinin in the process of development, aging, and pathological changes in the human intestine associated with alteration in the calcium homeostasis in the myenteric neurons.

In order to investigate whether purinoceptors are involved in the physiological renewal and regeneration of epithelia, we used immunohistochemical techniques on fresh frozen sections of various stratified squamous epithelial tissues (cornea, tongue, soft palate, oesophagus, vagina and footpad) of the rat and specific polyclonal antibodies to unique peptide sequences of P2X1-7 receptors. Only two of the antibodies, anti-P2X5 and anti-P2X7, reacted with epithelial structures. P2X5 immunoreactivity was mainly associated with the membranes of the proliferating and differentiating cell layers (spinous and granular layer) in both keratinised and non-keratinised epithelia and growing hair follicles. In contrast, P2X7 immunoreactivity was clearly associated with the keratinisation process, the staining being most intense in the upper keratinised and the exfoliated layers. These findings suggest, for the first time, that P2X5 and P2X7 receptors play an important role in the physiological turnover of continuously regenerating cells, and further, raise the possibility that they represent novel targets for the development of pharmacological tools of potential benefit for diseases of epithelial dysfunction.

Since the discovery of the vasorelaxant properties of nitric oxide and the vasoconstrictor effect of endothelin-1, there have been many studies of the distribution and functional significance of these agents in various vascular beds. In the coronary and pulmonary circulation nitric oxide and endothelin-1 actions have been largely investigated in terms of an imbalance between the opposing effects of these vasoactive agents leading to pathophysiological conditions. This article review functional and immunocytochemical studies with emphasis on the ultrastructural localization of nitric oxide synthase and endothelin-1 in the coronary and pulmonary vascular beds. Localization of nitric oxide synthase (type III or I or II) has been shown in endothelial cells, smooth muscle, and perivascular nerves of the coronary and pulmonary vascular beds and in the neurons, nerve fibers, and the small granule-containing cells within cardiac ganglia. Endothelin-1 was mainly localized in subpopulations of coronary and pulmonary endothelial cells. These immunocytochemical studies provide information about the sources of nitric oxide and endothelin-1 that contribute to the vasomotor control of cardiac and pulmonary circulation under normal and pathophysiological conditions.

The aim of our study was to investigate the changes that occur in functional responses of the golden hamster mesenteric arterial bed after: 1) 8 wk of hibernation and 2) 2 hr after arousal from hibernation. Age-matched and cold-exposed hamsters were used as controls. At 8 wk after hibernation there was an increase in sensitivity of vasoconstrictor responses to sympathetic nerve stimulation but no significant difference in constrictor responses to norepinephrine, alpha,beta-methylene ATP, uridine 5'-triphosphate or KCl (studied in unconstricted preparations), or in endothelium-dependent vasodilatation to acetylcholine and uridine 5'-triphosphate (in methoxamine-preconstricted preparations) compared with the control groups. In contrast, in the arousal from hibernation group, sympathetic vasoconstriction was similar to that in the control groups, and the maximal response to exogenous norepinephrine, and responses to alpha,beta-methylene ATP were augmented. These results suggest that there is an augmentation of sympathetic neurotransmission of golden hamster mesenteric arteries at 8 wk after hibernation, which appears to be due to pre- rather than postjunctional changes. This is reversed with arousal from hibernation, when the sensitivity of sympathetic contractile responses is not different from that of the controls. However, an increase in maximal constrictor responses to norepinephrine suggests that postjunctional changes may occur in sympathetic neurotransmission during arousal.

Many factors play important roles in the development of atherosclerotic lesions. The leading risk factor for atherosclerosis is familial hypercholesterolaemia (FH). FH is a genetic disease characterized by a deficiency of receptors for low density lipoprotein (LDL) on the plasmalemma of endothelial cells, a high level of serum LDL, and early development of atherosclerosis and skin xanthoma. Watanabe and colleagues have developed a line of rabbits with unprovoked hypercholesterolaemia, increased blood level of LDL, pronounced atherosclerosis and skin xanthoma. These Watanabe Heritable Hyperlipidaemic (WHHL) rabbits possess an inheritable mutation of one gene, similar to that in human FH. The morphogenesis of atherosclerosis in patients with FH is characterized by multifocal deposit of lipids in the stromal cells of thymus, spleen, skin, interstitial and parenchymatous cells of kidneys and the presence of some single foam cells in aorta. The manifestation of atherosclerotic lesions in WHHL rabbits increases progressively with age but the presence of atherosclerotic lesions in newborn WHHL rabbits suggest that the process may commence in utero. Moreover, the main mass of plasma cholesterol in WHHL rabbits is first found in LDL and to a lesser degree in lipoproteins of intermediate density. This is contrary to diet-induced atherosclerosis in rabbits where the main mass of serum cholesterol is found in very low density beta-lipoproteins. Thus the distribution of cholesterol among lipoprotein fractions differs from that in WHHL rabbits. Atherosclerotic damage of arteries in WHHL rabbits goes through several stages. During the progression of intimal damage, lipid and foam cell deposits are found in the internal surface together with developing plaques and increased content of lipids in the tunica media. Calcification often follows this process. The main factors initiating atherosclerosis in WHHL rabbits are adhesion of leukocytes and platelets to endothelial cells and the accumulation of lipids in the aortic wall. The deposits of lipids in macrophages and intimal smooth muscle cells in WHHL rabbits occurs mostly at the expense of cytoplasmic neutral lipid particles with some accumulation in lysosomes. Hypertension as a risk factor increases the area of atherosclerotic damage in all arterial vessels in WHHL rabbits, particularly in the thoracic and abdominal aorta. Morphogenesis of the development of atherosclerosis in WHHL and diet-induced atherosclerosis in rabbits was similar, but differs from rats with heritable hypercholesterolaemia. Damage or loss of endothelial cells can predispose the atherosclerotic vessels to vasospasm and can leave vessels unprotected against vasoconstrictor stimuli. The development of the WHHL model has not only given insight into the mechanisms of development of familial hypercholesterolaemia but has also provided a model for assessing various therapeutic approaches for the prevention and treatment of atherosclerosis.

An intronless open reading frame encoding a protein (361aa in length) was isolated from a rat genomic library probed with a DNA fragment from rat heart. This protein showed 83% sequence identity with the human P2Y4 (hP2Y4) receptor and represents a homologue of the human pyrimidinoceptor. However, the rP2Y4 receptor is not selective for uridine nucleotides and, instead, shows an agonist potency order of ITP = ATP = ADP(pure) = UTP = ATPgammaS = 2-MeSATP = Ap4AUDP(pure). ADP, ATPgammaS, 2-MeSATP and UDP are partial agonists. Thus, in terms of agonist profile, rP2Y4 is more like the P2U receptor subtype. The rP2Y4 receptor was reversibly antagonized by Reactive blue 2 but not by suramin which, otherwise, inhibits the hP2Y2 receptor (a known P2U receptor). Thus, rP2Y4 and the P2Y2 subtype appear to be structurally distinct forms of the P2U receptor (where ATP and UTP are equi-active) but can be distinguished as suramin-insensitive and suramin-sensitive P2U receptors, respectively.

1. Purinergic and cholinergic components of parasympathetic neurotransmission and contractile responses to exogenous alpha,beta-methylene ATP, acetylcholine, substance K, substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and capsaicin have been investigated in the urinary bladder of hibernating hamsters (4 weeks), cold exposed (4 weeks) and age-matched controls. 2. Electrical field stimulation (EFS) evoked increased frequency-dependent contractions in the detrusor strips from hibernating hamsters compared with those obtained from cold-exposed and age-matched animals. Tetrodotoxin (10(-6) M) completely blocked the frequency-dependent contractions in all groups. 3. The purinergic component of the parasympathetic neurotransmission was not affected in hibernating and cold-exposed animals while the cholinergic component was increased with respect to age-matched animals. The neurogenic response to EFS, still present after incubation with atropine (10(-6) M) and suramin (10(-4) M), was attenuated by indomethacin (10(-6) M) and blocked by tetrodotoxin (10(-6) M). 4. Exogenous administration of alpha,beta-methylene ATP elicited a significantly reduced contraction in strips from hibernating and cold-exposed hamsters relative to age-matched animals. The contractile response to exogenous acetylcholine was greater in the detrusors from hibernating hamsters than in cold-exposed and age-matched animals. Substance K elicited reduced contractions in preparations from hibernating animals compared with cold-exposed and control animals. Calcitonin gene-related peptide, vasoactive intestinal polypeptide, substance P and capsaicin did not elicit any relaxant or contractile response either at resting tone or in carbachol (5 x 10(-7) M)-precontracted tissues. 5. In summary, our findings indicate that 4 weeks of hibernation can significantly increase neurogenic responses in the hamster urinary bladder. This appears to be due to an increase in postjunctional responses to acetylcholine. In contrast, there was a decrease of the postjunctional responses to the parasympathetic cotransmitter ATP and also to the sensory-motor neurotransmitter substance K.

1. Sympathetic neurotransmission and noradrenaline content of the tail artery of Donryu rats fed for 2 months with a cholesterol-supplemented diet enriched with 4% cholesterol, 1% cholic acid, 0.5% thiouracil (CCT), were examined. 2. Total serum cholesterol level of CCT fed rats (7.05 +/- 1.77 mg ml(-1), n = 8) was significantly greater than lab-chow fed controls (2.58 +/- 0.32 mg ml(-1), n = 8). Low density lipoprotein level was also significantly increased in CCT-fed (1.79 +/- 0.26 mg ml(-1), n = 8) compared with control fed rats (1.35 +/- 0.25 mg ml(-1), n = 8) but plasma levels of triglyceride and high density lipoproteins did not differ significantly between the two groups. 3. Contractile responses of the arterial rings to transmural nerve stimulation (65 V, 0.1 ms, 4-64 Hz, 1 s), were markedly attenuated in the CCT fed animals compared with the controls. This reduction involved the noradrenergic rather than purinergic component of sympathetic transmission. 4. Vasoconstrictor responses to exogenous noradrenaline (0.01-300 microM) and adenosine 5'-triphosphate (0.3-1000 microM) were unaffected by CCT diet, indicating prejunctional alteration of sympathetic neurotransmission during CCT-induced hyperlipidaemia. 5. The noradrenaline content of the tail arteries of CCT fed animals (2.64 +/- 0.36 ng mg(-1), n = 6) was significantly lower than that of controls (3.82 +/- 0.32 ng mg(-1), n = 6). 6. These findings show that chronic treatment of Donryu rats with a cholesterol-supplemented diet led to altered levels of circulating lipid fractions accompanied by attenuated sympathetic noradrenergic neurotransmission and reduced noradrenaline content of the rat tail artery.

Hepatic arterial infusion (HAI) chemotherapy as treatment for human colorectal liver metastases is promising, but not entirely satisfactory. Improved drug delivery during HAI may be achieved by manipulating the different control mechanisms of normal versus tumour blood vessels. The peptidergic/aminergic innervation of vessels in normal liver and in two animal models of liver metastasis (Lister Hooded rat with syngeneic MC28 sarcoma; athymic (nude) rat with human HT29 carcinoma) was investigated to assess the suitability of these models for future pharmacological studies. Normal liver and metastases were studied immunohistochemically for the presence of protein gene product 9.5 (PGP), neuropeptide Y (NPY), tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and substance P (SP). Perivascular innervation was also examined by transmission electron microscopy. In Lister rat normal livers, perivascular immunoreactive nerve fibres containing PGP, NPY, TH, CGRP and SP were observed around the interlobular blood vessels near the hilum and in the portal tracts. The highest density was seen for PGP, followed in decreasing order, by NPY, TH, CGRP and SP. VIP-immunoreactive nerves were absent. No immunoreactive nerves were observed in the hepatic lobule. In athymic rat livers, the pattern of innervation was similar, except that SP immunoreactivity was more sparse. No perivascular immunoreactive nerves were observed in either MC28 or HT29 tumours. Electron microscopy confirmed the absence of perivascular nerves. Smooth muscle cells were not observed in tumour blood vessel walls. These results are comparable with previous observations on human liver metastases and suggest that the animal models may be suitable for pharmacological studies on vascular manipulation of HAI chemotherapy.

Long-term interactions between sympathetic and sensory-motor nerves have been shown in several tissues. Previous investigations in this laboratory have demonstrated an increase in cardiac sensory-motor innervation after neonatal sympathectomy by guanethidine and an increase of perivascular sympathetic neurotransmission after neonatal treatment by capsaicin. The present study evaluated the effects of sensory-motor denervation on sympathetic neurotransmission in the heart. Newborn rats were injected with capsaicin or its vehicle (Tween 80). Sympathetic neurotransmission was studied in isolated atria driven at a constant rate (4 Hz) by measuring cardiac responses to electrical field stimulation, in the presence of atropine 1 microM. Inotropism of tyramine, norepinephrine and calcitonin gene-related peptide was also tested. Neonatal capsaicin treatment did not affect cardiac responses to trains of an increasing number (2-32) of field pulses. Moreover, inotropic responses to tyramine did not differ between control, capsaicin- and Tween 80-treated preparations. Neither maximal effect nor pD2 values were significantly different between the groups. Similarly, the inotropism of calcitonin gene-related peptide was comparable in all groups of atrial preparations. In marked contrast to earlier papers on blood vessels, this study shows a lack of effect of sensory-motor denervation by neonatal capsaicin treatment on cardiac sympathetic neurotransmission. The different neuronal plasticity of vascular and cardiac sensory innervation will be discussed. The present results also indicate that capsaicin-induced sensory-motor denervation is not associated with changes in cardiac responsiveness to calcitonin gene-related peptide.