The GTP-binding protein, Go, regulates neuronal calcium channels

In neuronal cells, opioid peptides and opiates inhibit neurotransmitter release, which is a calcium-dependent process. They also inhibit adenylyl cyclase, presumably via the membrane signal-transducing component, Gi, a guanine nucleotide-binding protein (G-protein). No causal relationship between these two events has yet been demonstrated. Besides Gi, membranes of neuronal tissues contain large amounts of Go, a G-protein with unknown function. Both G-proteins are heterotrimers consisting of alpha-, beta- and gamma-subunits; the alpha-subunits can be ADP-ribosylated by an exotoxin from Bordetella pertussis (PT), which modification inhibits receptor-mediated activation of the G-protein. It was recently shown that noradrenaline, dopamine and gamma-aminobutyric acid (GABA) inhibit the voltage-dependent calcium channels in dorsal root and sympathetic ganglia; this inhibition is mimicked by intracellular application of guanine nucleotides and blocked by PT, suggesting the involvement of a G-protein. Here we report an inhibitory effect of the opioid D-Ala2, D-Leu5-enkephalin (DADLE) on the calcium current (ICa) in neuroblastoma X glioma hybrid cells (N X G cells). Pretreatment with PT almost completely abolishes the DADLE effect. The effect is restored by intracellular application of Gi and Go. As the alpha-subunit of Go (with or without beta-gamma complex) is 10 times more potent than Gi, we propose that Go is involved in the functional coupling of opiate receptors to neuronal voltage-dependent calcium channels.     

Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission

The sympathetic nervous system regulates cardiovascular function by activating adrenergic receptors in the heart, blood vessels and kidney. Alpha2-adrenergic receptors are known to have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system; however, the individual roles of the three highly homologous alpha2-adrenergic-receptor subtypes (alpha2A, alpha2B, alpha2C) in this process are not known. We have now studied neurotransmitter release in mice in which the genes encoding the three alpha2-adrenergic-receptor subtypes were disrupted. Here we show that both the alpha2A- and alpha2C-subtypes are required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. Alpha2A-adrenergic receptors inhibit transmitter release at high stimulation frequencies, whereas the alpha2C-subtype modulates neurotransmission at lower levels of nerve activity. Both low- and high-frequency regulation seem to be physiologically important, as mice lacking both alpha2A- and alpha2C-receptor subtypes have elevated plasma noradrenaline concentrations and develop cardiac hypertrophy with decreased left ventricular contractility by four months of age.     

Interactions between enkephalin and GABA in avian retina

In addition to conventional neurotransmitters such as acetylcholine, dopamine, glycine and gamma-aminobutyric acid (GABA), a number of peptide-immunoreactive substances have recently been localized in the vertebrate retina. The functional roles of these retinal peptides and their interactions with conventional neurotransmitters are largely unknown. We have previously shown that exogenous opiates affect both the release of GABA and the firing patterns of ganglion cells in the goldfish retina, and we have now begun a systematic characterization of the opioid pathways in the chicken retina, because, among vertebrate retinas, avian retinas contain the highest concentration of enkephalins. Monoclonal antibodies specific for enkephalin have been used to demonstrate that a subpopulation of enkephalin-containing amacrine cells exists in the chicken retina. This retina also synthesizes Met-enkephalin and releases it on cell depolarization. The enkephalin-induced inhibition of GABA release in goldfish retina led us to examine whether similar interactions occur in chicken, and if so, whether enkephalins and GABA coexist in the same amacrine cells. Our results, presented here, indicate that exogenous enkephalins do indeed inhibit GABA release in the chicken retina. Surprisingly, we found that although some amacrine cells contain both enkephalin and GABA, others contain only one or the other.     

A novel analgesic dipeptide from bovine brain is a possible Met-enkephalin releaser.

It is generally accepted that morphine exerts its analgesic effect by binding to specific opiate receptors in the brain and spinal cord. Since Hughes et al. isolated and identified two endogenous pentapeptides, Met- and Leu-enkephalin, from the brain and found that they acted as agonists at opiate receptors, alpha-, beta- and gamma-endorphins, larger peptides than enkephalins and having morphine-like activity, have been identified in either the brain or pituitary of various species. Several studies have demonstrated that enkephalins possess analgesic properties and that they are distributed in the pain-mediated pathways in the central nervous system. These findings suggest that enkephalins are important neurotransmitters or neuromodulators regulating pain transmission. We now report the isolation of a novel substance which has a Met-enkephalin releasing action. Our findings suggest the possibility of a regulating mechanism for the release of endogenous opioid peptides, especially Met-enkephalin.     

GABA affects the release of gastrin and somatostatin from rat antral mucosa

gamma-Aminobutyric acid (GABA) is regarded as the major inhibitory neurotransmitter in the central nervous system of vertebrates. GABA exerts its inhibitory actions by interacting with specific receptors on pre- and postsynaptic membranes and has been shown to inhibit somatostatin release from hypothalamic neurones in vitro. Concepts of innervation of the gastrointestinal tract have been expanded by recent studies which suggest that GABAergic neurones are not confined solely to the central nervous system but may also exist in the vertebrate peripheral autonomic nervous system. Jessen and coworkers have demonstrated the presence, synthesis and uptake of GABA by the myenteric plexus of the guinea pig taenia coli, and have documented the presence of glutamic acid decarboxylase (GAD) in isolated myenteric plexus. This enzyme is responsible for the conversion of glutamic acid to GABA in GABAergic neurones. The possibility that GABA may have a role in neurotransmission or neuromodulation in the enteric nervous system of the vertebrate gut has been suggested by several investigators. Furthermore, GABA receptors have been demonstrated on elements of the enteric nervous system. The effects of GABA on gastrointestinal endocrine cell function have not been examined. We report here the effects of GABA on gastrin and somatostatin release from isolated rat antral mucosa in short-term in vitro incubations.     

An endocannabinoid mechanism for stress-induced analgesia

Acute stress suppresses pain by activating brain pathways that engage opioid or non-opioid mechanisms. Here we show that an opioid-independent form of this phenomenon, termed stress-induced analgesia, is mediated by the release of endogenous marijuana-like (cannabinoid) compounds in the brain. Blockade of cannabinoid CB(1) receptors in the periaqueductal grey matter of the midbrain prevents non-opioid stress-induced analgesia. In this region, stress elicits the rapid formation of two endogenous cannabinoids, the lipids 2-arachidonoylglycerol (2-AG) and anandamide. A newly developed inhibitor of the 2-AG-deactivating enzyme, monoacylglycerol lipase, selectively increases 2-AG concentrations and, when injected into the periaqueductal grey matter, enhances stress-induced analgesia in a CB1-dependent manner. Inhibitors of the anandamide-deactivating enzyme fatty-acid amide hydrolase, which selectively elevate anandamide concentrations, exert similar effects. Our results indicate that the coordinated release of 2-AG and anandamide in the periaqueductal grey matter might mediate opioid-independent stress-induced analgesia. These studies also identify monoacylglycerol lipase as a previously unrecognized therapeutic target.     

The glial cell glutamate uptake carrier countertransports pH-changing anions.

Uptake into glial cells helps to terminate glutamate's neurotransmitter action and to keep its extracellular concentration, [Glu]o, below neurotoxic levels. The accumulative power of the uptake carrier stems from its transport of inorganic ions such as sodium (into the cell) and potassium (out of the cell). There is controversy over whether the carrier also transports a proton (or pH-changing anion). Here we show that the carrier generates an alkalinization outside and an acidification inside glial cells, and transports anions out of the cells, suggesting that there is a carrier cycle in which two Na+ accompany each glutamate anion into the cell, while one K+ and one OH- (or HCO3-) are transported out. This stoichiometry predicts a minimum [Glu]o of 0.6 microM normally (tonically activating presynaptic autoreceptors and post-synaptic NMDA receptors), and 370 microM during brain anoxia (high enough to kill neurons). Transport of OH-/HCO3- on the uptake carrier generates significant pH changes, and may provide a mechanism for neuron-glial interaction.     

NMDA receptor agonists selectively block N-type calcium channels in hippocampal neurons.

The modulation of voltage-dependent calcium channels by various neurotransmitters has been demonstrated in many neurons. Because of the critical role of Ca2+ in transmitter release and, more generally, in transmembrane signalling, this modulation has important functional implications. Hippocampal neurons possess low-threshold (T-type) Ca2+ channels and both L- and N-type high voltage-activated Ca2+ channels. N-type Ca2+ channels are blocked selectively by omega-conotoxin and adenosine. These substances both block excitatory synaptic transmission in the hippocampus, whereas dihydropyridines, which selectively block L-type channels, are ineffective. Excitatory synaptic transmission in the hippocampus displays a number of plasticity phenomena that are initiated by Ca2+ entry through ionic channels operated by N-methyl-D-aspartate (NMDA) receptors. Here we report that NMDA receptor agonists selectively and effectively depress N-type Ca2+ channels which are involved in neurotransmitter release from presynaptic sites. The inhibitory effect is eliminated by the competitive NMDA antagonist D-2-amino-5-phosphonovalerate, does not require Ca2+ entry into the cell, and is probably receptor-mediated. This phenomenon may provide a negative feedback between the liberation of excitatory transmitter and entry of Ca2+ into the cell, and could be important in presynaptic inhibition and in the regulation of synaptic plasticity.     

Changes in hypothalamic preproenkephalin A mRNA following stress and opiate withdrawal

The median eminence of the pituitary is rich in opioid receptors, and exogenous opioids have major effects on the release of adrenocorticotropic hormone (ACTH), luteinizing hormone (LH), prolactin, growth hormone (GH) and thyrotropin. Stress results in similar changes in anterior pituitary hormone secretion. Enkephalin immunoreactivity has been reported in the medial parvocellular neurons of the hypothalamic paraventricular nucleus which project to the median eminence, the site where hypothalamic releasing factors are secreted into the portal blood and thence to the anterior pituitary gland. The endocrine response to stressful stimuli might therefore, at least in part, be mediated through the activation of hypothalamic enkephalinergic neurons. We show that two stressful stimuli, opiate withdrawal and intraperitoneal injection of hypertonic saline, both result in very rapid and marked increases in enkephalin mRNA in the parvocellular paraventricular nucleus. The activation of hypothalamic enkephalin neurons may be important in the neuroendocrine response to stress.     

Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.     

Differential regulation of PI hydrolysis and adenylyl cyclase by muscarinic receptor subtypes

Muscarinic acetylcholine receptors (mAChRs), like many other neurotransmitter and hormone receptors, transduce agonist signals by activating G proteins to regulate ion channel activity and the generation of second messengers via the phosphoinositide (PI) and adenylyl cyclase systems. Human mAChRs are a family of at least four gene products which have distinct primary structures, ligand-binding properties and patterns of tissue-specific expression. To examine the question of whether functional differences exist between multiple receptor subtypes, we have investigated the ability of each subtype to regulate PI hydrolysis and adenylyl cyclase when expressed individually in a cell lacking endogenous mAChRs. We show that the HM2 and HM3 mAChRs efficiently inhibit adenylyl cyclase activity but poorly activate PI hydrolysis. In contrast, the HM1 and HM4 mAChRs strongly activate PI hydrolysis, but do not inhibit adenylyl cyclase, and in fact can substantially elevate cAMP levels. Interestingly, the subtypes that we find to be functionally similar are also more similar in sequence. Our results indicate that the different receptor subtypes are functionally specialized.     

Selective effects of somatostatin-14, -25 and -28 on in vitro insulin and glucagon secretion

The widely occurring tetradecapeptide somatostatin (SRIF-14) has been variously implicated as a neurotransmitter, a neurohormone, a cybernin (local regulatory factor) and a hormone. In the first isolation of SRIF-14 from hypothalamic extracts and subsequent extracts of other tissues, peptides of higher molecular weight but with similar activity have been noted. Recently two such peptides have been characterized as the 28-amino acid SRIF-28 (from porcine gastro-intestinal tract and porcine and ovine hypothalamus and the 25-amino acid SRIF-25 (from ovine hypothalamus), each of which consists of an N-terminal extension of SRIF-14. We now report that SRIF-28 and SRIF-25 are more potent than SRIF-14 in the inhibition of insulin release, but that SRIF-14 preferentially inhibits glucagon release. This suggests that SRIF-28 and SRIF-25 are not mere biosynthetic precursors of SRIF-14 and that their differential release may be physiologically important.     

Enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.

Recent biochemical and immunohistochemical studies have shown that the opioid peptides, enkephalins, occur in nerve terminals and cell bodies in mammalian sympathetic ganglia1-3. Opiates and enkephalins are thought to inhibit synaptic transmission in the peripheral nervous tissues as well as in the central nervous system4-12. The mechanisms of the opiate actions, however, are not entirely clear; both pre- and postsynaptic sites of action have been proposed7-9,11,12. As acetylcholine is known to be the major neurotransmitter in the autonomic ganglia and as the mechanism of synaptic transmission is well clarified13, analysis of the peptide action could be more easily but equally usefully carried out in the peripheral synapses than in central synapses. We now report that enkephalins presynaptically inhibit cholinergic transmission in sympathetic ganglia.     

Synaptotagmin I functions as a calcium regulator of release probability

In all synapses, Ca2+ triggers neurotransmitter release to initiate signal transmission. Ca2+ presumably acts by activating synaptic Ca2+ sensors, but the nature of these sensors--which are the gatekeepers to neurotransmission--remains unclear. One of the candidate Ca2+ sensors in release is the synaptic Ca2+-binding protein synaptotagmin I. Here we have studied a point mutation in synaptotagmin I that causes a twofold decrease in overall Ca2+ affinity without inducing structural or conformational changes. When introduced by homologous recombination into the endogenous synaptotagmin I gene in mice, this point mutation decreases the Ca2+ sensitivity of neurotransmitter release twofold, but does not alter spontaneous release or the size of the readily releasable pool of neurotransmitters. Therefore, Ca2+ binding to synaptotagmin I participates in triggering neurotransmitter release at the synapse.     

Arachidonic acid induces a prolonged inhibition of glutamate uptake into glial cells

Activation of NMDA (N-methyl-D-aspartate) receptors by neurotransmitter glutamate stimulates phospholipase A2 to release arachidonic acid. This second messenger facilitates long-term potentiation of glutamatergic synapses in the hippocampus, possibly by blocking glutamate uptake. We have studied the effect of arachidonic acid on glutamate uptake into glial cells using the whole-cell patch-clamp technique to monitor the uptake electrically. Micromolar levels of arachidonic acid inhibit glutamate uptake, mainly by reducing the maximum uptake rate with only small effects on the affinity for external glutamate and sodium. On removal of arachidonic acid a rapid (5 minutes) phase of partial recovery is followed by a maintained suppression of uptake lasting at least 20 minutes. Surprisingly, the action of arachidonic acid is unaffected by cyclo-oxygenase or lipoxygenase inhibitors suggesting that it inhibits uptake directly, possibly by increasing membrane fluidity. As blockade of phospholipase A2 prevents the induction of long-term potentiation (LTP), inhibition of glutamate uptake by arachidonic acid may contribute to the increase of synaptic gain that occurs in LTP. During anoxia, release of arachidonic acid could severely compromise glutamate uptake and thus contribute to neuronal death.     

A physiological role for GABAB receptors in the central nervous system

The role of GABA in synaptic transmission in the mammalian central nervous system is more firmly established than for any other neurotransmitter. With virtually every neuron studied, the synaptic action of GABA is mediated by bicuculline-sensitive GABAA receptors which selectively increase chloride conductance. However, it has been shown that GABA has a presynaptic inhibitory action on transmitter release that is insensiive to bicuculline and is selectively mimicked by baclofen. The receptors involved in this action are referred to as GABAB receptors, to distinguish them from the classic bicuculline-sensitive GABAA receptors. In hippocampal pyramidal cells an additional postsynaptic action of GABA and baclofen has been reported that is also insensitive to GABAA antagonists, and may be mediated by GABAB receptors on the postsynaptic neuron. This action of GABA and baclofen involves an increase in potassium conductance. Synaptic activation of pathways converging on hippocampal pyramidal cells results in a slow inhibitory postsynaptic potential which involves an increase in potassium conductance, and it has been suggested that GABAB receptors might be responsible for this synaptic potential. However, to establish convincingly that GABAB receptors are physiologically important in the central nervous system, a selective GABAB antagonist is required. Here we provide this missing evidence. Using the hippocampal slice preparation, we now report that the phosphonic acid derivative of baclofen, phaclofen, is a remarkably selective antagonist of both the postsynaptic action of baclofen and the bicuculline-resistant action of GABA, and that it selectively abolishes the slow inhibitory postsynaptic potential in pyramidal cells.     

Cloning and sequence analysis of cDNA for bovine adrenal preproenkephalin

The nucleotide sequence of cloned cDNA for preproenkephalin from bovine adrenal medulla indicates that the precursor protein contains four copies of Met-enkephalin and one copy each of Leu-enkephalin, Met-enkephalin-Arg6-Phe7 and Met-enkephalin-Arg6-Gly7-Leu8, a previously undetected opioid peptide. The enkephalin and extended enkephalin sequences are each bounded by paired basic amino acid residues. Preproenkephalin may represent a multi-hormone precursor, like the corticotropin-beta-lipotropin precursor.     

Is hyperosmotic neurosecretion from motor nerve endings a calcium-dependent process?

Spontaneous liberation of neurotransmitter quanta is strongly affected by the osmotic pressure of the extracellular fluid. Elevation of the osmolarity by 20-30% increases the rate of release from motor nerve endings by more than one order of magnitude. In this respect the neuromuscular junction resembles some other secretory systems. The mechanism of this hyperosmotic neurosecretion is not yet understood; extracellular calcium ions are not directly responsible, since this effect can be produced in their absence. Recently, it has been suggested that the liberation of neurotransmitter is regulated by the intracellular concentration of free calcium ions. We have therefore examined the hypothesis that hyperosmotic neurosecretion originates from an increase in internal calcium concentration ([Ca]in). At the frog neuromuscular synapse however, it is impossible at present to estimate directly free [Ca]in; hence we used an indirect technique, which is based on two assumptions; first, the frequency of the miniature endplate potentials (m.e.p.p.s.) reflects free [Ca]in. Second, the movement of calcium ions across the presynaptic membrane is governed by the electrochemical gradient, and by the calcium conductance (g(Ca)). If hyperosmotic neurosecretion is caused by an increase in [Ca]in, then increasing g(Ca), under reversed electrochemical gradient for the calcium should cause a reduction in the effect of hyperosmotic stress on transmitter release. We report that hyperosmotic neurosecretion is dependent on [Ca]in.     

阿片肽的体内分布与功能作用浅谈

阿片肽是免疫系统中重要的调节因子,它几乎作用于所有的免疫活性细胞,对不同亚类的细胞作用不尽相同,它们的释放受应激的影响,而应激影响免疫反应,提示阿片肽可能是应激引起免疫调节的介质。