Endothelial cells inhibit the vascular response to adrenergic nerve stimulation by a receptor-mediated mechanism

Rabbit central ear arteries, with and without endothelium, were perfused at a constant flow rate and the perfusion pressure was measured as an index of the vessel resistance. Transmural nerve stimulation (TNS) induced a frequency-dependent increase in perfusion pressure in all vessels that was blocked by tetrodotoxin, phentolamine, and prazosin. Removal of endothelium significantly enhanced contractions induced by TNS. The inhibitory effect of endothelium was not modified by indomethacin but was abolished by hemoglobin, indicating that endothelium-derived relaxant factor (EDRF) was the vasodilator involved. The endothelium-dependent inhibitory effect (rubbed vessel minus control vessel contractions) increased with time during the first 10-20 s after the beginning of TNS, and was frequency dependent and inhibited by low doses of phentolamine, which suggest a receptor-mediated mechanism. To analyze whether amine neurotransmitters are able to permeate the artery wall and contact the endothelial cell membrane, the passage of [3H]acetylcholine from the abluminal side to the lumen was studied in intact vessels. [3H]acetylcholine readily permeated the vessel wall, as assessed by radioautography, and appeared in the perfusion fluid at a concentration that explains the relaxation induced by perivascular acetylcholine. These data suggest that endothelial cells modulate the effect of perivascular neurotransmitters by a receptor-mediated mechanism. In the case of the sympathetic innervation, such modulation would be more relevant at low levels of transmitter release and would be minimized during intense sympathetic stimulation.     

Muscarinic acetylcholine receptors involved in the regulation of neural stem cell proliferation and differentiation in vitro

Neural stem cells (NSCs) are currently considered powerful candidates for cell therapy in neurodegenerative disorders such as Parkinson's disease. However, it is not known when and how NSCs begin to differentiate functionally. Recent reports suggest that classical neurotransmitters such as acetylcholine (Ach) are involved in the proliferation and differentiation of neural progenitor cells, suggesting that neurotransmitters play an important regulatory role in development of the central nervous system (CNS). We have shown by calcium imaging and immunochemistry that proliferation and differentiation are enhanced by M2 muscarinic Ach receptors (mAchR) expressed on the NSC surface and on their neural progeny. Moreover, atropine, an mAchR antagonist, blocks the enhancement and inhibits the subsequent differentiation of NSCs. Further understanding of this neural-nutrition role of Ach might elucidate fetal brain development, the brain's response to injury, and learning and memory.     

Role and place of cyclic nucleotides in the neuroendocrine regulation of cells and tissues

Cyclic adenosine monophosphate and cyclic guanosine monophosphate occupy one of the central places in the neuro-endocrine regulation in the highest organisms. These nucleotides mediate the effect of most hormones and neurotransmitters: peptide, polypeptide and proteinaceous, catecholamine, histamine, many prostaglandins, acetylcholine (in the case of muscarinic cholinergic receptor activation), endorphins and enkephalins. They regulate also synthesis and secretion of many hormones and neurotransmitters. Cyclic nucleotides play a key role in the functioning of organs of sense (vision, smelling and taste). Under the control of cyclic nucleotides there are main metabolic processes: glycogenolysis and lipolysis. These nucleotides have been shown to participate in the genome expression, in growth, differentiation and proliferation of cells. Accelerating or decelerating the ionic transport across the biological membranes cyclic nucleotides influence the cell bioelectric activity as well its contractility. They may also change the properties of contractile and cytoskeleton proteins, thus interfering in the processes of mobility and shape formation of cells.     

Modulating effect of angiotensin II and bradykinin on the mediator sensitivity of central neurons

Using the methods of extracellular recording of bioelectrical activity and microiontophoresis, the effect of endogenous neuropeptides (angiotensin-II and bradykinin) on the cortical neuronal sensitivity to neurotransmitters was investigated in conscious rabbits. It was found that angiotensin-II and bradykinin modified neuronal responses to neurotransmitters, increasing, decreasing and reversing their mediator sensitivity. The most prominent effect of these neuropeptides was the increase of neuronal responses to acetylcholine and noradrenaline. At the central neuronal level, endogenous neuropeptides (angiotensin-II and bradykinin) are suggested to play the role of synaptic polytransmitter neuromodulators.     

Highly sensitive muscarinic receptors in the cerebellum are coupled to prostaglandin formation

There have been relatively few studies on the effects of neurotransmitters on the synthesis of prostaglandins in the brain. We report here that acetylcholine is very effective in stimulating prostaglandin synthesis in cerebellar cortex slices incubated in vitro, i.e., 5 microM acetylcholine increased prostaglandin levels up to 3-fold over control levels. The response was saturable and dose-dependent over the range of 0.1-5 microM acetylcholine. Atropine, at a concentration of 50 nM, abolished the response. The results indicate that high-affinity muscarinic receptors in the cerebellum are coupled to prostaglandin formation. Potassium-induced depolarization or incubation with 0.1 mM histamine also significantly increased prostaglandin formation. These findings provide support for the notion that certain neurotransmitters can modulate prostaglandin levels in the mammalian brain.     

Interactive effects of serotonin and acetylcholine on neurite elongation

Serotonin (5-HT) inhibits elongation of neurites of specific identified neurons. Here we report a novel, growth-enabling action of another neurotransmitter, acetylcholine (ACh). When applied simultaneously with serotonin, ACh prevents the inhibition of Helisoma neuron B19 neurite elongation that would occur in response to application of 5-HT alone. We also report that ACh prevents the rise in growth cone Ca2+ that would occur in response to application of 5-HT alone and that ACh blocks the electrical excitatory effect of 5-HT on neuron B19. These results support the hypothesis that growth cone motility and neurite elongation can be regulated by voltage-gated Ca2+ fluxes and suggest that the dynamics of neurite morphology may be complexly regulated by an array of neurotransmitters, as is functional electrical activity.     

Classical Noncholinergic Neurotransmitters and the Vesicular Transport System for Acetylcholine

Abstract: The acetylcholine transporter exhibits such low affinity and specificity for acetylchoiine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [³H]serotonin, [³H]noradrenaline, or [³H]glutamate occurred. Serotonin, noradrenaline, and N -acetylaspartyl glutamate inhibited active transport of [³H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and γ-aminobutyric acid were shown here not to inhibit active transport of [³H]-acetylcholine. Noradrenaline was competitive with respect to [³H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [³H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.     

Adrenergic and cholinergic stimulation of arachidonate and phosphatidate metabolism in cultured astroglial cells

Primary cultures of neonatal rat brain polygonal astroglia, or cells of the C62B glioma line, were incubated with [1-¹⁴C]arachidonic acid (AA) in culture for 18 hr. In both culture systems, more than 80% of the added [1-¹⁴C]AA was taken up into cellular glycerolipids; less than 1% of the radioactivity in the cells was present in an unesterified form. When prelabeled C62B cells were stimulated with acetylcholine (ACh), there was a rapid accumulation of arachidonyl-phosphatidic acid (PA) accompanied by a liberation of [1-¹⁴C]AA. A variety of other neurotransmitters failed to activate this response in C62B glioma cells, primary astroglia generated PA and liberated [1-¹⁴C]AA in response to several neurotransmitters (i.e., ACh, norepinephrine, glutamate, and histamine) Treatment of astroglia with a combination of norephrine, ACh, and histamine resulted in a greater production of PA and free [1-¹⁴C]AA than did treatment with any one of these neurotransmitters alone. The results suggest that cultures of astroglia can respond to several different neurotransmitters with specific changes in AA and PA metabolism. Thus, a variety of neurotransmitters initiate cascades of lipid metabolism which may be of physiological significance in glial function.Special Issue dedicated to Prof. Eduardo De Robertis.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Catecholamine secretion and adenylate cyclase activation in sea urchin eggs

The role of neurotransmitters in sea-urchin eggs was investigated by studying their effect on adenylate cyclase of the egg membrane. Maximal stimulation of enzyme activity occurs in the presence of dopamine and GTP. 5-hydroxytriptamine, 5-methoxytriptamine and acetylcholine have no effect on activity, despite a decrease in intracellular cAMP level in eggs treated with 5-hydroxytriptamine antagonists as previously reported (Renaud et al., 1983). High-performance liquid chromatography (HPLC) revealed that dopamine is released from the sea-urchin egg into the external medium following fertilization.     

Interaction of dopamine and acetylcholine with an amphiphilic resorcinarene receptor in aqueous micelle system

The molecular recognition of neurotransmitters, dopamine and acetylcholine with an amphiphilic resorcinarene receptor was investigated in an aqueous sodium dodecylsulfate (SDS) micelle system by 1H NMR measurements. The interaction distances of these neurotransmitters from the hydrophilic cavity of the amphiphilic receptor were estimated based on the calculation of the ring current shift using the atomic coordinates obtained from molecular dynamics calculation.     

Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems

Brain dialysis is rapidly becoming a routine research method with a wide range of applications. Since 1982 this sampling technique is frequently used as a method to study the in vivo release of endogenous neurotransmitters such as dopamine, noradrenaline, serotonin, acetylcholine and certain amino acids. In this review most of the studies that have appeared in this field, are evaluated. Special attention was given to the question whether the neurotransmitter content in the dialysate is related to neurotransmission. Criteria such as the presence of a high tissue/dialysate concentration ratio, the sensitivity of the transmitters to membrane active compounds and the occurrence of receptor-mediated effects, are discussed. It is concluded that dopamine, noradrenaline and acetylcholine found in the dialysate are directly derived from neurotransmission, whereas the overflow of excitatory amino acid neurotransmitters is related to neurogenic as well as to metabolic events.     

Effect of indomethacin on bombesin-like immunoreactivity, somatostatin and gastrin secretion from rat stomach

In the present study the effect of indomethacin-induced prostaglandin deficiency was examined on the release of bombesin-like immunoreactivity (BLI), a putative peptidergic neurotransmitter, from the isolated perfused rat stomach. In addition, gastrin and somatostatin (SLI) secretion was determined. Pretreatment of rats with indomethacin (2 mg/kg X h) resulted in a 3-fold increase of basal BLI secretion. In response to acetylcholine (2 X 10(-6) M) BLI rose from 2,000 to 4,000 pg/min, whereas in controls BLI increased from 400 to 1,400 pg/min. While absolute values for BLI secretion were higher in indomethacin-treated stomachs the relative increase above baseline was lower (100 vs. 250%). In control rats the increase in BLI secretion in response to acetylcholine was abolished when the acidity in the gastric lumen was increased from pH 7 to pH 2. After indomethacin, however, the stimulatory effect of acetylcholine during luminal pH 7 and pH 2 was identical. The decrease of SLI by acetylcholine at luminal pH 7 was abolished in indomethacin-treated stomachs in response to 10(-6) M acetylcholine, and 2 X 10(-6) M had even a stimulatory effect on SLI secretion. Indomethacin pretreatment reduced gastrin secretion at luminal pH 7. These data demonstrate that endogenous prostaglandins exert an inhibitory tone on basal and stimulated BLI and stimulated SLI secretion in the rat stomach. It is suggested that endogenous prostaglandins also inhibit the release of a peptidergic neurotransmitter, similar to their effect on the classical neurotransmitters acetylcholine and norepinephrine.     

The comparative roles of acetylcholine and noradrenaline in regulation of spontaneous spike activity of cortical neurons

The effects of acetylcholine and noradrenaline applications on neuronal sponta-neous activity were investigated in slices of guinea-pig parietal cortex. Iontophoretic ejections of both neurotransmitters to the cortical neurons evoked the same-type slowly-developing and long-lasting increase in the rate of spike activity. The different temperature sensitivity of cholinergic and noradrenergic reactions were revealed. During the temperature shift from 32-34 degrees C to 35-36 degrees C the cholinergic effect on neuronal spike activity became extremely strong, that is why even silent at t = 32-32 degrees C neurons became to acetylcholine responsive. Temperature-dependent changes in spike reaction to acetylcholine were accompanied by stable increase in spontaneous spike activity. The noradrenergic reactions did not change with temperature in limits from 32-34 to 35-36 degrees C. In this temperature range spike reactions to glutamate, the main excitation transmitter in the cortex, remained constant. The results obtained suggest that acetylcholine is the main neurotransmitter regulating spontaneous spike activity in cortical neurons.     

Chemical signaling in plant microspore cells

Chemical signal transduction from the cell surface to organelles was studied in unicellular vegetative (Equisetum arvense) and generative (Hippeastrum hybridum pollen) microspores of plants. Neurotransmitters acetylcholine, dopamine, and serotonin, their agonists and antagonists, Na⁺, K⁺, and Ca²⁺ channel blockers, as well as forskolin and theophylline (agents increasing the intracellular level of cyclic adenosine monophosphate) were used as chemical signals. Both types of microspores exposed to neurotransmitters, their agonists, forskolin, and theophylline demonstrated growth activation, while neurotransmitter antagonists and ion channel blockers inhibited this process. No stimulating effects of neurotransmitters were observed for cells pretreated with the antagonists and ion channel blockers. Pretreatment with ion channel blockers and then by anticontractile agents (cytochalasin B or colchicine) either had no effect or increased the inhibition of microspore growth. Pathways of chemical signal transduction from the cell surface to organelles are discussed.     

Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system

Synaptic connections of neurons in the Drosophila lamina, the most peripheral synaptic region of the visual system, have been comprehensively described. Although the lamina has been used extensively as a model for the development and plasticity of synaptic connections, the neurotransmitters in these circuits are still poorly known. Thus, to unravel possible neurotransmitter circuits in the lamina of Drosophila we combined Gal4 driven green fluorescent protein in specific lamina neurons with antisera to gamma-aminobutyric acid (GABA), glutamic acid decarboxylase, a GABA(B) type of receptor, L-glutamate, a vesicular glutamate transporter (vGluT), ionotropic and metabotropic glutamate receptors, choline acetyltransferase and a vesicular acetylcholine transporter. We suggest that acetylcholine may be used as a neurotransmitter in both L4 monopolar neurons and a previously unreported type of wide-field tangential neuron (Cha-Tan). GABA is the likely transmitter of centrifugal neurons C2 and C3 and GABA(B) receptor immunoreactivity is seen on these neurons as well as the Cha-Tan neurons. Based on an rdl-Gal4 line, the ionotropic GABA(A) receptor subunit RDL may be expressed by L4 neurons and a type of tangential neuron (rdl-Tan). Strong vGluT immunoreactivity was detected in alpha-processes of amacrine neurons and possibly in the large monopolar neurons L1 and L2. These neurons also express glutamate-like immunoreactivity. However, antisera to ionotropic and metabotropic glutamate receptors did not produce distinct immunosignals in the lamina. In summary, this paper describes novel features of two distinct types of tangential neurons in the Drosophila lamina and assigns putative neurotransmitters and some receptors to a few identified neuron types.     

Synthesis, photochemistry, and biological activity of a caged photolabile acetylcholine receptor ligand

A biologically inert photolabile precursor of carbamoylcholine has been synthesized; it is photolyzed to carbamoylcholine, a well-characterized acetylcholine analogue, with a half-time of 40 microseconds at pH 7.0 and a quantum yield of 0.8. The compound, N-(alpha-carboxy-2-nitrobenzyl)carbamoylcholine, was synthesized from (2-nitrophenyl)glycine. The photolysis rates (of five compounds) and the biological activity (of two compounds) were determined, and both properties were found to depend on the nature of the substituents on the photolabile protecting group. Laser pulse photolysis at wavelengths between 308 and 355 nm was used to investigate the wavelength dependence, quantum yield, and rate of the photolysis reaction. Photolysis products were isolated by high-performance liquid chromatography and identified by chemical and spectroscopic analysis and by their ability to activate the nicotinic acetylcholine receptor. BC3H1 muscle cells containing those receptors and a cell-flow method were used in the biological assays. The approach described may be useful in the preparation and characterization of other photolabile precursors of neurotransmitters that contain amino groups. The importance of these rapidly photolyzed, inert precursors of neurotransmitters is in chemical kinetic investigations of the reactions involving diverse neuronal receptors; such studies have been hampered because the available techniques have an insufficient time resolution.     

Swim pacemaker response to bath applied neurotransmitters in the cubozoan Tripedalia cystophora

The four rhopalia of cubomedusae are integrated parts of the central nervous system carrying their many eyes and thought to be the centres of visual information processing. Rhopalial pacemakers control locomotion through a complex neural signal transmitted to the ring nerve and the signal frequency is modulated by the visual input. Since electrical synapses have never been found in the cubozoan nervous system all signals are thought to be transmitted across chemical synapses, and so far information about the neurotransmitters involved are based on immunocytochemical or behavioural data. Here we present the first direct physiological evidence for the types of neurotransmitters involved in sensory information processing in the rhopalial nervous system. FMRFamide, serotonin and dopamine are shown to have inhibitory effect on the pacemaker frequency. There are some indications that the fast acting acetylcholine and glycine have an initial effect and then rapidly desensitise. Other tested neuroactive compounds (GABA, glutamate, and taurine) could not be shown to have a significant effect.     

Corticotropin-Releasing Factor Administered Centrally, but Not Peripherally, Stimulates Hippocampal Acetylcholine Release

Abstract: In addition to corticotropin-releasing factor's well-known role in mediating hormonal and behavioral responses to stress, this peptide also reportedly affects arousal and cognition, processes that classically have been associated with forebrain cholinergic systems. Corticotropin-releasing factor stimulation of cholinergic neurons might thus provide a mechanism for this peptide's cognitive effects. To examine this possibility, the present experiments characterize the effect of corticotropin-releasing factor on cholinergic neurotransmission, using in vivo microdialysis to measure hippocampal acetylcholine release. Corticotropin-releasing factor (0.5–5.0 µg/rat intracerebroventricularly) was found to increase dialysate concentrations of acetylcholine in a dose-dependent manner in comparison with a control injection, the ovine peptide having a greater effect than the same dose of the human/rat peptide. This effect was found to be centrally mediated, independent of the peripheral effects of an exogenous corticotropin-releasing factor injection; subcutaneous injections of the peptide increased plasma concentrations of corticosterone, the adrenal hormone ultimately secreted in the rat's stress response, to the same level as did the central injections, without affecting hippocampal acetylcholine release. These results demonstrate that corticotropin-releasing factor, acting centrally, regulates hippocampal cholinergic activity, and suggest that corticotropin-releasing factor/acetylcholine interactions may underlie some of the previously identified roles of these neurotransmitters in arousal, cognition, and stress.