Journal of the autonomic nervous system最新文献

Although the emphasis in ATP research has been on postjunctional receptors, there is also evidence for presynaptic receptors regulating transmitter release in the autonomic nervous system. Recent work has attempted to identify similar mechanisms in the central nervous system. Some of the existing results can be explained by the metabolism of nucleotides to adenosine or adenosine 5′-monophosphate (AMP). However, studies of presynaptic effects using sensitive electrophysiological tests such as paired-pulse interactions indicate that nucleotides can act at presynaptic sites, but that their effects may be mediated by a release of adenosine. Results are also described which indicate that, under some conditions, nucleotides can mediate phenomena such as long-term potentiation, which probably involves a significant presynaptic element. In part these effects may involve a nucleotide-induced release of adenosine and the simultaneous activation of P1 and P2 receptors.

In this study the in vitro mouse phrenic nerve- hemidiaphragm preparation was utilized to study the release and extracellular catabolism of endogenous ATP and its action on the postsynaptic site, i.e. on the contraction force evoked by nerve stimulation. ATP, measured by the luciferin–luciferase assay, was released stimulation-dependently from the mouse hemidiaphragm in response to electrical field stimulation at 10 Hz. Blockade of the Na⁺ channel activity by tetrodotoxin inhibited the majority of the release of ATP in response to stimulation, showing that it is related to neuronal activity. The nicotinic receptor antagonists d-tubocurarine, and α-bungarotoxin and cooling the bath temperature to 7°C also reduced stimulation-induced ATP outflow, suggesting that nicotinic receptors are responsible for the part of the release of ATP that is released from postsynaptic sites in a carrier-mediated manner. Exogenous ATP (20–500 μM) added to the bath was degraded to ADP and AMP by the action of ectoATPase and ectoATPdiphosphohydrolase; the K_m and v_max values of these enzymes were 185.8 μM and 55.16 nmol/min.g respectively. However, the total amount of nucleotides ([ATP+ADP+AMP]) was increased after the addition of ATP, indicating that ATP itself promoted further adenine nucleotide release. Twitch contractions of the rat hemidiaphragm preparation evoked by low frequency electrical stimulation was blocked concentration–dependently by the non-depolarizing muscle relaxants d-tubocurarine and pancuronium. Suramin (100 μM–1 mM) reversed neuromuscular blockade by d-tubocurarine and pancuronium; i.e., it shifted their concentration–response curves to the right Taken together our data, that endogenous ATP is released by stimulation and subsequently catabolized in the hemidiaphragm preparation and that suramin inhibits ecto-ATPase activity could be interpreted as meaning that suramin prolongs the action of endogenous ATP to elicit twitch contraction, which points to a new, undefined role of ATP in neuromuscular transmission. The source of ATP is partly postsynaptic, released from the muscle in response to activation of nicotinic ACh receptors expressed on the muscle.

The beginning of the last decade heralded three important and sequential developments in our understanding of cell-to-cell signalling by extracellular ATP via its cell surface receptors, the P2 purinoceptors. One major development in ATP signalling culminated in a timely review in 1991, when it was established in the clearest of terms that ATP receptors exploited discrete signal transduction pathways (Dubyak, G.R., 1991. Signal transduction by P2-purinergic receptors for extracellular ATP. Am. J. Respir. Cell. Mol. Biol. 4, 295–300; and later in Dubyak, G.R., El-Moatassim, C., 1993. Signal transduction via P2-purinergic receptors for extracellular ATP and other nucleotides. Am. J. Physiol. 265, C577–C606). Henceforth, it was universally acknowledged that some P2 purinoceptors interacted with heterotrimeric G-proteins to activate intracellular signalling cascades (metabotropic ATP receptors), whereas others contained intrinsic ion-channels (ionotropic ATP receptors). A second key development can be traced to 1992, from the discovery that ATP receptors were involved in excitatory neurotransmission in the CNS and PNS (Edwards, F.A., Gibb, A.J., Colquhoun, D., 1992. ATP receptor-mediated synaptic currents in the central nervous system. Nature 359, 144–147; Evans, R.J., Derkach, V., Surprenant, A., 1992. ATP mediates fast synaptic transmission in mammalian neurons. Nature 357, 503–505; Silinsky, E.M., Gerzanich, V., Vanner, S.M., 1992. ATP mediates excitatory synaptic transmission in mammalian neurones. Br. J. Pharmacol., 106, 762–763). Thereafter, it was accepted that ATP could play a neurotransmitter and/or modulatory role throughout the entire nervous system. The third key development stemmed from the isolation of a cDNA, from chick brain, encoding a metabotropic ATP receptor (Webb, T.E., Simon, J., Krishek, B.J., Bateson, A.N., Smart, T.G., King, B.F., Burnstock, G., Barnard, E.A., 1993. Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Lett. 324, 219–225). The cloning of a membrane protein serving as an ATP receptor ignited a widespread international interest in purinergic signalling. Investigators at University College London (UCL) — colleagues and associates of Geoffrey Burnstock — were at the forefront of this rapid phase of discovery. In this review, we highlight the UCL experience when the fields of molecular biology, physiology and cell biology converged to help advance our understanding of ATP as an extracellular signalling molecule.

Synaptosomal preparations from rat midbrain exhibit specific responses to both ATP and Ap₅A, which elicit a Ca²⁺ entrance to the presynaptic terminals. Studies of isolated single terminals showed that not all the terminals contain ionotropic receptors for nucleotides, in fact only 46% of them do. Of these, 12% responded only to the dinucleotide Ap₅A, and 20% to the mononucleotide ATP. At the presynaptic level, diinosine pentaphosphate, Ip₅I, is a good tool to specifically block dinucleotide responses, which are inhibited at low nM concentration, versus the high μM concentrations required to block ATP responses. There is evidence for a presynaptic control of mononucleotide and dinucleotide responses, mediated by metabotropic and ionotropic receptors. Stimulation of adenosine A1 receptors increases the affinity of dinucleotide receptors by five orders of magnitude, from 30 μM to 680 pM for control and in the presence of A1 agonist, respectively.

The early studies and hypotheses of Geoffrey Burnstock catalyzed intensive characterization of roles for nucleotides and P2 nucleotide receptors in neurotransmission and neuromodulation. These latter analyses have focused on the mechanisms of nucleotide release and action in the microenvironments of nerve endings and synapses. However, studies of various white blood cells, such as monocytes, neutrophils, and lymphocytes, suggest that locally released nucleotides also modulate intercellular signaling at so-called ‘immunological synapses’. This communication describes recent findings and speculations regarding nucleotide release and signaling in several key phases of the immune and inflammatory responses.

The enteric nervous system (ENS) can control gastrointestinal function independent of direct connections with the central nervous system. Enteric nerves perform this important function using multiple mechanisms of excitatory neurotransmission in enteric ganglia. Fast excitatory synaptic transmission in the autonomic nervous system (ANS) is largely mediated by acetylcholine (ACh) acting at nicotinic cholinergic receptors but in the ENS there are noncholinergic fast excitatory neurotransmitters. There are two broad types of neurons in the ENS: S neurons and AH neurons. S neurons are interneurons and motoneurons while AH neurons are sensory neurons. Three subsets of S neurons in the myenteric plexus can be distinguished on the basis of the neurotransmitters producing fast excitatory postsynaptic potentials (fEPSPs) in each subset. In one subset, fEPSPs are mediated solely by ACh acting at nicotinic cholinergic receptors. In a second subset of S neurons, ATP acting at P2X purine receptors and ACh contribute to the fEPSP while in a third subset, fEPSPs are mediated by 5-hydroxytryptamine (5-HT) acting at 5-HT₃ receptors and ACh. Some AH neurons also receive fast excitatory synaptic input. The fEPSPs recorded from AH neurons are mediated ACh and also by glutamate acting at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Multiple mechanisms of fast excitatory synaptic transmission in the ENS are likely to contribute to its capacity to regulate complex gastrointestinal functions.

Messenger RNAs and cDNAs for individual cloned P2Y₁, P2Y2 and P2Y₆ nucleotide receptors have been expressed by micro-injection into dissociated rat superior cervical sympathetic neurones and the effects of stimulting the expressed receptors on voltage-activated N-type Ca²⁺ currents and M-type K⁺ currents recorded. Both currents were reduced by stimulating all three receptors, with the following mean IC₅₀ values: P2Y₁ (agonist: ADP) – I_K(M) 6.9 nM, I_Ca 8.2 nM; P2Y₂ (agonist: UTP) – I_K(M) 1.5 μM, I_Ca 0.5 μM; P2Y₆ (agonist: UDP) – I_K(M) 30 nM, I_Ca 5.9 nM. Inhibition of I_K(M) was voltage-independent and insensitive to Pertussis toxin; inhibition of I_Ca showed both voltage-sensitive and insensitive, and Pertussis toxin-sensitive and insensitive components. It is concluded that these P2Y receptors can couple to more than one G protein and thereby modulate more than one ion channel. It is suggested that these effects on K_M and Ca_N channels may induce both postsynaptic excitory and presynaptic inhibitory responses.

Investigation of the multiple roles of extracellular nucleotides in the cochlea has developed from analysis of ATP-activated conductances in single sensory hair cells. Molecular probes such as radiolabelled ATP analogues and radiolabelled mRNA for ATP-gated ion channel subunits (P2X receptors) rapidly revealed the extensive nature of ATP signalling in this sensory organ. This has provided a foundation for physiological investigations which put extracellular nucleotides at the centre of homeostatic regulation of the driving force for sound transduction, modulation of mechanical tuning, control of cochlear blood flow and auditory neurotransmission. The purinergic signal transduction pathways associated with these processes have several novel features of significance to the broader field of purinergic neuroscience. In turn, these studies have benefited from the recent experimental advances in the field of purinergic signalling, a significant component of which is associated with the work of Professor Geoffrey Burnstock.

In addition to Professor Burnstock’s work on the short-term signaling actions of extracellular nucleotides and nucleosides, Geoff has had a long-standing interest in trophic actions of purines in development and in pathophysiological conditions which has been instrumental in encouraging my work in this area. The trophic actions of extracellular ATP, alone or in combination with polypeptide growth factors, may play an important role in brain development and may contribute to the reactive gliosis that accompanies brain injury and neurodegeneration. P2Y receptors in astrocytes are coupled to the ERK/MAPK cascade, a signal transduction mechanism crucial for cellular proliferation and differentiation. The mitogenic signaling pathway from P2Y receptors to ERK involves phospholipase D and a calcium-independent PKC isoform, PKCδ. DNA array analysis reveals a number of changes in gene expression after P2Y receptor occupancy, indicating that this methodology will be a powerful tool in understanding the mechanisms underlying the trophic actions of extracellular nucleotides and nucleosides.

αβmeATP-evoked concentration-dependent, PPADS-sensitive, desensitising, P2X receptor-mediated, constrictions of mesenteric, basilar and septal artery rings with EC₅₀ values of 1, 1 and 30 μM, respectively. In patch clamp studies on acutely dissociated artery smooth cells αβmeATP-evoked transient inward currents (τ∼100 ms) with mean current densities of ∼340, 175 and 120 pA/pF, respectively. P2X₁ receptor immunoreactivity was expressed in mesenteric and basilar arteries and this receptor subunit appears to dominate the P2X receptor phenotype in these vessels. In contrast P2X₁ receptor immunoreactivity was not detected in septal arteries and the αβmeATP sensitivity of constriction was not consistent with the involvement of P2X₁ receptors. These results suggest that not all arteries share a common P2X receptor phenotype.