Involvement of nitric oxide in the reflex relaxation of the stomach to accommodate food or fluid.

The fundus of the guinea-pig stomach actively dilates in response to low increases in intragastric pressure. This physiological response, now called adaptive relaxation, accommodates the intake of liquid or food. It is independent of external innervation, resistant to ganglion blockade, but reflex in origin. The nerves involved are neither adrenergic nor cholinergic in nature. Non-adrenergic, non-cholinergic (NANC) nerves have now been recognized in many parts of the gastrointestinal tract and have recently been linked with release of nitric oxide (NO) on electrical stimulation. Here we show that adaptive relaxation in isolated stomach of the guinea pig is mediated by a NANC neurotransmitter substance indistinguishable from NO derived from L-arginine. This is substantiated by inhibition of adaptive relaxation by NG-monomethyl-L-arginine or N omega-nitro-L-arginine methyl ester, both inhibitors of NO synthesis, and by methylene blue, an inhibitor of soluble guanylate cyclase. There are two distinct neuronal pathways signalling NO-dependent adaptive relaxation, as evidenced by tetrodotoxin sensitivity. The first is a local reflex arc, the afferent fibres of which sense changes in intragastric pressure. The second is stimulated by an agonist for ganglionic nicotinic receptors. Thus, the functional significance of NO release from NANC nerves in the stomach is to bring about adaptive relaxation through a reflex response to increases in intragastric pressure.     

Vascular endothelial cells synthesize nitric oxide from L-arginine

Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue and for the inhibition of platelet aggregation and platelet adhesion attributed to endothelium-derived relaxing factor. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay, chemiluminescence or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.     

Calcitonin gene-related peptide acts as a novel vasodilator neurotransmitter in mesenteric resistance vessels of the rat

Systemic blood pressure is controlled by changes in the resistance of the peripheral vascular bed for example in the mesenteric blood vessels. The tone of peripheral blood vessels is primarily maintained by sympathetic vasoconstrictor nerves. Although vasodilator innervation has been identified in certain isolated elastic arteries, it is not known whether vasodilator nerves contribute to the regulation of the peripheral resistance vessels. We present pharmacological evidence for the existence of nonadrenergic, noncholinergic (NANC) vasodilator nerves in the mesenteric resistance vessel of the rat and that the resistance is controlled by not only sympathetic vasoconstrictor nerves but also NANC vasodilator nerves. We also show that the neurogenic vasodilation was selectively abolished by depleting endogenous calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide, from perivascular nerves. This indicates that CGRP is a novel vasodilator neurotransmitter and may play a role in control of the total peripheral resistance of systemic circulation through a local reflex mechanism.     

Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor

Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.     

Hyperpolarization and relaxation of arterial smooth muscle caused by nitric oxide derived from the endothelium

Stimulation of the endothelial lining of arteries with acetylcholine results in the release of a diffusible substance that relaxes and hyperpolarizes the underlying smooth muscle. Nitric oxide (NO) has been a candidate for this substance, termed endothelium-derived relaxing factor. But there are several observations that argue against the involvement of NO in acetylcholine-induced hyperpolarization. First, exogenous NO has no effect on the membrane potential of canine mesenteric arteries. Second, although haemoglobin (believed to bind and inactivate NO (refs 11-15)) and methylene blue (which prevents the stimulation of guanylate cyclase) inhibit relaxation, neither has an effect on hyperpolarization. Finally, nitroprusside, thought to generate NO in vascular smooth muscle, relaxes rat aorta without increasing rubidium efflux. Nevertheless, nitrovasodilators, nitroprusside and nitroglycerin cause hyperpolarization in some arteries. NO might therefore be responsible for at least part of the hyperpolarization induced by acetylcholine. We now report that hyperpolarization and relaxation evoked by acetylcholine are reduced by NG-monomethyl-L-arginine, an inhibitor of NO biosynthesis from L-arginine. Thus NO derived from the endothelium can cause hyperpolarization of vascular smooth muscle, which might also contribute to relaxation by closing voltage-dependent calcium channels. Our findings raise the possibility that hyperpolarization might be a component of NO signal transduction in neurons or inflammatory cells.     

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.     

Endogenous nitric oxide release required for long-term synaptic depression in the cerebellum.

Conjunctive stimulation of climbing and parallel fibres in the cerebellum evokes a long-term depression of parallel-fibre Purkinje-cell transmission, a phenomenon implicated as the cellular mechanism for cerebellar motor learning. It is suspected that the increase in cyclic GMP concentration that occurs after activation of climbing fibres is required to evoke long-term depression. Excitatory amino acids are known to cause the release of nitric oxide (NO), resulting in elevation of the cGMP level in the cerebellum. Here we report that endogenous NO is released after stimulation of climbing fibres, that long-term depression evoked by conjunctive stimulation of parallel and climbing fibres is blocked by haemoglobin (which strongly binds NO) or L-NG-monomethyl-arginine (an inhibitor of NO synthase), and that exogenous NO or cGMP can substitute for the stimulation of climbing fibres to cause long-term depression in rat cerebellar slices. These results demonstrate that the release of endogenous NO is essential for the induction of synaptic plasticity in the cerebellum.     

Axonal transport of vasoactive intestinal peptide in sciatic nerve.

Dense plexuses of neurones containing immunoreactive vasoactive intestinal peptide (VIP) have been found in discrete areas of the central nervous system and in peripheral organs, including the gastrointestinal tract, pancreas and urogenital system. In many of these locations VIP is concentrated in nerve endings, where it can be released by high K+ concentrations in a Ca2+-dependent manner. VIP release may also be provoked by electrical stimulation of nerves, for example the vagus. VIP thus shows some of the features of neurotransmitter or neuromodulator substances. The presence of immunoreactive VIP in the fine terminal varicosities as well as in the cell bodies of neurones suggests that it might be transported from the perikaryon, where it is presumably formed, to the nerve endings, through the axonal transport system. Such transport would be in keeping with a role for the peptide as a neurohumor or neurohormone. We report here that VIP accumulates in constricted rat sciatic nerves in a manner suggesting fast, anterograde axonal flow.     

Regulation of endothelium-derived nitric oxide production by the protein kinase Akt.

Endothelial nitric oxide synthase (eNOS) is the nitric oxide synthase isoform responsible for maintaining systemic blood pressure, vascular remodelling and angiogenesis. eNOS is phosphorylated in response to various forms of cellular stimulation, but the role of phosphorylation in the regulation of nitric oxide (NO) production and the kinase(s) responsible are not known. Here we show that the serine/threonine protein kinase Akt (protein kinase B) can directly phosphorylate eNOS on serine 1179 and activate the enzyme, leading to NO production, whereas mutant eNOS (S1179A) is resistant to phosphorylation and activation by Akt. Moreover, using adenovirus-mediated gene transfer, activated Akt increases basal NO release from endothelial cells, and activation-deficient Akt attenuates NO production stimulated by vascular endothelial growth factor. Thus, eNOS is a newly described Akt substrate linking signal transduction by Akt to the release of the gaseous second messenger NO.     

Localization of nitric oxide synthase indicating a neural role for nitric oxide.

Nitric oxide (NO), apparently identical to endothelium-derived relaxing factor in blood vessels, is also formed by cytotoxic macrophages, in adrenal gland and in brain tissue, where it mediates the stimulation by glutamate of cyclic GMP formation in the cerebellum. Stimulation of intestinal or anococcygeal nerves liberates NO, and the resultant muscle relaxation is blocked by arginine derivatives that inhibit NO synthesis. It is, however, unclear whether in brain or intestine, NO released following nerve stimulation is formed in neurons, glia, fibroblasts, muscle or blood cells, all of which occur in proximity to neurons and so could account for effects of nerve stimulation on cGMP and muscle tone. We have now localized NO synthase protein immunohistochemically in the rat using antisera to the purified enzyme. We demonstrate NO synthase in the brain to be exclusively associated with discrete neuronal populations. NO synthase is also concentrated in the neural innervation of the posterior pituitary, in autonomic nerve fibres in the retina, in cell bodies and nerve fibres in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells. These prominent neural localizations provide the first conclusive evidence for a strong association of NO with neurons.     

Ultrastructural localization of choline acetyltransferase in vascular endothelial cells in rat brain

Furchgott and Zawadski have shown that acetylcholine (ACh) does not act directly on the smooth muscle of blood vessel walls, but rather via receptors on the endothelial cells lining the lumen, to release an endothelium-derived relaxing factor (EDRF). As it is very unlikely that neurotransmitter released from the periarterial nerves, which are confined to the adventitial-medial border, diffuses all the way through the medial muscle coat before acting on endothelial cells to release EDRF to produce vasodilatation, this discovery has been regarded as an indication of a pathophysiological mechanism, rather than a physiological one (see refs 2, 3). ACh is rapidly degraded in the blood by acetylcholinesterase, so that ACh must be released locally to be effective on endothelial cells. Here we demonstrate the immunocytochemical localization of choline acetyltransferase in endothelial cells of small brain vessels, which is consistent with the view that the ACh originates from endothelial cells that can synthesize and store it. We suggest that release of ACh following damage to endothelial cells during ischaemia contributes to a pathophysiological mechanism of vasodilation which protects that segment of vessel from further damage as well as brain cells from hypoxia.     

太湖湖水及沉积物磷释放对藻类生长潜力研究

在太湖湖水中添加不同的氮、磷营养物进行藻类生长潜力测试，试验结果表明：单独添加ＨＰＯ２－４和同时添加ＨＰＯ２－４、ＮＯ－３的所有浓度组均能显著促进斜生栅列藻（Ｓｃｅｎｄｅｓｍｕｓｏｂｌｉｑüｕｓ）和羊角月芽藻（Ｓｅｌｅｎａｓｔｒｕｍｃａｐｒｉｃｏｒｎｕｔｕｍ）的增长，而单独添加ＮＯ－３和ＮＨ＋４对二种藻类不但均无促进、并有一定抑制作用，表明了磷是太湖藻类生长的限制因子。在所试验条件下，厌氧状态（ＤＯ为０～２．５ｍｇ／Ｌ）下沉积物释放后滤液能促进斜生栅列藻增长，但对羊角月芽藻无促进效应；好氧状态下（ＤＯ为７．５～８．０ｍｇ／Ｌ）沉积物释放后滤液均不能促进二种藻类增长。     

Control of enzyme secretion by non-cholinergic, non-adrenergic nerves in guinea pig pancreas

Depolarization of pancreatic cells by exposure to high potassium solutions is associated with release of amylase. In the guinea pig, but not the mouse or cat, this Ca-dependent amylase secretion is resistant to atropine blockade, thus Scheele and Haymovits concluded that the enzyme secretion evoked by K depolarization does not involve release of transmitter from intrapancreatic nerves but is a consequence of Ca uptake into acinar cells mediated by the membrane depolarization. This hypothesis is inconsistent with current concepts of stimulus--secretion coupling in electrically non-excitable cells. The observation of Scheele and Haymovits could, however, also be explained by the release of a non-cholinergic, secretomotor transmitter as a consequence of the depolarization of intrapancreatic nerves. By adapting the technique of electrical field stimulation of isolated pancreatic segments to our studies of amylase secretion, we have now been able to demonstrate both cholinergic and non-cholinergic, non-adrenergic secretomotor nerves in the guinea pig pancreas. Excitation of the non-cholinergic nerves stimulates amylase secretion by a different intracellular coupling mechanism from that activated by cholinergic nerves or by peptides belonging to the cholecystokinin, gastrin or bombesin families.     

Octopamine.

Octopamine is highly concentrated in neurones of several invertebrate species. Unlike in mammals, octopaminergic neurones in invertebrates are spatially separated from catecholaminergic neurons. In identified nerve cells of Aplysia, however, this amine coexists with other putative neurotransmitters. Octopamine is synthesized in nerves from tyrosine and tyramine and metabolised mainly by monoamine oxidase. When lobster nerves are depolarized, octopamine is liberated by a Ca2+-dependent process. A specific adenylate cyclase is stimulated by octopamine in several invertebrates to activate phosphorylase in the cockroach, induce a light-flash in firefly lattern or inhibit rhythm contractions in locust muscle. All of these observations provide compelling evidence that octopamine is a neurotransmitter in invertebrates. In mammals octopamine is localised in nerves in peripheral tissues and brain where it seems to coexist with noradrenaline, the catecholamine being present in much higher concentrations. Octopamine is released from nerves together with noradrenaline and it may under certain conditions modify the actions of the adrenergic neurotransmitter. Octopamine is present in unusually high concentrations in certain neurological and hepatic diseases and may have a pathophysiological role.     

Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. Synaptic vesicles are loaded with neurotransmitter by means of specific vesicular transporters. Here we show that expression of BNPI, a vesicle-bound transporter associated with sodium-dependent phosphate transport, results in glutamate uptake by intracellular vesicles. Substrate specificity and energy dependence are very similar to glutamate uptake by synaptic vesicles. Stimulation of exocytosis--fusion of the vesicles with the cell membrane and release of their contents--resulted in quantal release of glutamate from BNPI-expressing cells. Furthermore, we expressed BNPI in neurons containing GABA (gamma-aminobutyric acid) and maintained them as cultures of single, isolated neurons that form synapses to themselves. After stimulation of these neurons, a component of the postsynaptic current is mediated by glutamate as it is blocked by a combination of the glutamate receptor antagonists, but is insensitive to a GABA(A) receptor antagonist. We conclude that BNPI functions as vesicular glutamate transporter and that expression of BNPI suffices to define a glutamatergic phenotype in neurons.     

Vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin

Vertebrates achieve internal homeostasis during infection or injury by balancing the activities of proinflammatory and anti-inflammatory pathways. Endotoxin (lipopolysaccharide), produced by all gram-negative bacteria, activates macrophages to release cytokines that are potentially lethal. The central nervous system regulates systemic inflammatory responses to endotoxin through humoral mechanisms. Activation of afferent vagus nerve fibres by endotoxin or cytokines stimulates hypothalamic-pituitary-adrenal anti-inflammatory responses. However, comparatively little is known about the role of efferent vagus nerve signalling in modulating inflammation. Here, we describe a previously unrecognized, parasympathetic anti-inflammatory pathway by which the brain modulates systemic inflammatory responses to endotoxin. Acetylcholine, the principle vagal neurotransmitter, significantly attenuated the release of cytokines (tumour necrosis factor (TNF), interleukin (IL)-1beta, IL-6 and IL-18), but not the anti-inflammatory cytokine IL-10, in lipopolysaccharide-stimulated human macrophage cultures. Direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxaemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF amounts, and prevented the development of shock.     

Reaction mechanism determines NMDA receptor response to repetitive stimulation

At central excitatory synapses, N-methyl-D-aspartate (NMDA) receptors, which have a high affinity for glutamate, produce a slowly rising synaptic current in response to a single transmitter pulse and an additional current after a second, closely timed stimulus. Here we show, by examining the kinetics of transmitter binding and channel gating in single-channel currents from recombinant NR1/NR2A receptors, that the synaptic response to trains of impulses is determined by the molecular reaction mechanism of the receptor. The rate constants estimated for the activation reaction predict that, after binding neurotransmitter, receptors hesitate for approximately 4 ms in a closed high-affinity conformation before they either proceed towards opening or release neurotransmitter, with about equal probabilities. Because only about half of the initially fully occupied receptors become active, repetitive stimulation elicits currents with distinct waveforms depending on pulse frequency. This high-affinity/low-efficiency activation mechanism might serve as a link between stimulation frequency and the directionality of the ensuing synaptic plasticity.     

Release of endogenous excitatory amino acids from turtle photoreceptors

Responses to light are transmitted from photoreceptors to second-order retinal neurons by chemical synapses that may use an excitatory amino acid (EAA) as the neurotransmitter. This hypothesis is based primarily on the pharmacological actions of EAA agonists and antagonists on the membrane potentials and light responses of second-order neurons. But the release of endogenous EAAs, which is a critical criterion for the identification of EAAs as transmitters, has not been demonstrated. Here we report the use of outside-out membrane patches excised from rat hippocampal neurons to detect the release of EAAs from synaptic terminals of isolated turtle photoreceptors. Electrical stimulation of or application of lanthanum chloride to photoreceptors induced an increase in the frequency of opening of 50-pS channels in the patches. These channels were identified as the class of glutamate-activated channels that are also gated by aspartate and NMDA (N-methyl-D-aspartate). In several photoreceptor-patch pairs, spontaneous channel activity was observed near the synaptic terminals. These results provide strong evidence to support the hypothesis that both rods and cones of the turtle use an EAA as their neurotransmitter.     

Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms

Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.     

一氧化氮参与附睾上皮细胞感染后的宿主防御

 以金黄色葡萄球菌Staphylococcus aureus感染附睾上皮细胞的体外感染模型,分别采用RT-PCR和western blot等 方法研究一氧化氮（NO）在附睾上皮细胞宿主防御中的作用。结果表明附睾上皮感染后上调iNOS RNA和蛋白水平的表达,并产生 大量的NO,采用iNOS的抑制剂L NMMA和AGE预处理后,NO水平显著降低,与空白处理组相比,抑制剂处理组的S aureus数量显著增加,这表明NO参与了附睾上皮细胞的宿主防御过程,在附睾上皮细胞的宿主防御中具有重要的作用。