PHARMACOLOGICAL STUDIES ON THE STIMULATION OF THE PHOSPHOLIPASE A2-ACYLATION SYSTEM OF SYNAPTIC MEMBRANES OF BRAIN, BY NEUROTRANSMITTERS AND OTHER AGONISTS

Methacholine, nicotine and succinylcholine stimulated the phospholipase A2-acylation system of synaptic membranes isolated from the cerebral cortex of guinea pig. Stimulation by acetylcholine was partially blocked by atropine and by D-tuberocurarine respectively, indicating both muscarinic and nicotinic stimulation. Muscarinic stimulation by acetylcholine was greater than -isotinic stimulation, and stimulation by acetylcholine was completely blocked by a combip, or;. and n-tuberocurarine. The phospholipase A2-acylat tem was stimulated by phenylcphrine., id. Cqxoterenoi. Stimulation by noradrenaline was J-. tidlr, by phenoxybenzamine and pindalol i:spectively, indicating both 8-adrenergic and P-adrenergic ztimulation. n-Adrenergic stimulation by noradrenaline was greater than P-adrenergic-stimulation. 5 -mlation by noradrenaline was completely blocked by a combination of phenoxybenzamins and pindalol. Stimulation of both acylation and phospholipid hydrolysis, by 5-hydroxytryptamine and histamine were partially blocked by methysergide and diphenhydramine respectively. Stimulation by dopamine was blocked by halopcridol. Stimulation by y-aminobutyric acid was partially blocked by strychnine and by picrotoxin. Dichloroisoproterenol, atropine, methysergidr, diphenhydramine, strychnine, picrotoxin and eserine, at relatively high concentrations (1 mM), stimuhted the phospholipase A2-acylation system. Synergistic stimulations of both acylatior, and hydrolysis of phosphatidylcholine, were observed by adenosine combined with noradrenaline, 5-hydroxytryptamine, histamine, dopamine or yaminobutyric acid, respectively. In the presence of ATP-MgCI, synergistic stimulations of the hydrolysis of phosphatidyicholine were observed after 30 s by noradrenaline combined with 5-hydroxytryptamine, histamine, dopamine, aminobutyric acid or carbamoylcholine respectively. In the presence of GTP-MgC12 synergistic stimulations were obtained by cdrbamoylcholine combined with noradrenaline. 5-hydroxytryptamine. histamine, dopamine or y-aminobutyric acid, respectively. In the presence of ATP-MgC12 plus GTP-MgC12, stimulation by noradrenaline and one other agonist including 5-hydroxytryptamine, histamine, dopamine, y-aminobutyric acid or carbamoylcholine were close to additive.     

On the specificity of histamine H2-receptor antagonists in the rat cerebral cortex.

The effects of iontophoretically applied histamine H2-receptor antagonists and their antagonism of various amines, acetylcholine (ACh), and adenosine 5'-monophosphate (5'-AMP) were studied on spontaneously active rat cerebral cortical neurons. Metiamide selectively blocked the depressant actions of histamine. Burimamide, in amounts necessary for histamine antagonism, also antagonized the depressant effects of noradrenaline, dopamine, and 5-hydroxytryptamine. Neither antagonist affected 5'-AMP-induced depressions, but both reduced or blocked the excitatory actions of ACh. It is concluded that metiamide may be useful as a reliable antagonist of H2 receptors on cerebral cortical neurons.     

The role of calcium in monoamine-induced depression of cerebral cortical neurones

The calcium antagonists, lanthanum, verapamil and manganese, have been shown to antagonize the depressant actions of ionto-phoretically applied monoamines (noradrenaline, 5-hydroxytryptamine, dopamine and histamine), but not of γ-aminobutyric acid, on cerebral cortical neurones. Cocaine and ethanol, which also affect membrane calcium fluxes, have a similar antagonistic action. These findings suggest that calcium ions are essential for, and perhaps mediate, the characteristic depressant actions of the monoamines on cerebral cortical neurones.     

Microiontophoretic study of the effect of biogenic amines on unit activity in the rabbit visual cortex

The effect of acetylcholine, noradrenalin, and serotonin on spontaneous activity of visual cortical neurons and on their activity evoked by flashes, recorded extracellularly, was studied by microiontophoresis in unanesthetized rabbits. The ability of visual cortical neurons to respond to light does not correlate with their sensitivity to acetylcholine. This substance, which changes the spontaneous firing rate of many of the neurons tested, was less effective against their evoked activity. Noradrenalin had a powerful depressant action on both spontaneous and evoked activity of most neurons studied. Serotonin acted in different ways on the spontaneous and evoked activity of some neurons tested. It is postulated that acetylcholine mediates reticulo-cortical inputs, noradrenalin is a true inhibitory mediator in the cerebral cortex, and serotonin has a presynaptic action by preventing the liberation of natural mediators.     

Cerebral ischemia: Changes in brain choline,acetylcholine, and other monoamines as related to energy metabolism

The relationship of cerebral neurotransmitters acetylcholine (ACh), noradrenaline (NA), dopamine (DA), 5-hydroxytryptamine (5HT) to the energy state of the brain was examined in mice at various times following complete ischemia produced by decapitation, in gerbils submitted to transient global ischemia (10 min bilateral carotid artery occlusion, 5 or 30 min recirculation), and in rats 24 hr after irreversible microembolism. Ischemia caused significant reductions in brain monoamine concentrations. The alterations in NA, DA, and 5HT levels persisted during recirculation and were unrelated to energy restoration. They were accompanied by an increase in the concentrations of related metabolites, suggesting that synthesis was unable to compensate for the release of the transmitters at early post-ischemic time periods. As described for the catecholamines and 5HT, ischemia resulted in a significant decrease in ACh level, but recirculation was associated with a rapid increase in ACh concentration. Impaired synthesis and/or increased release of ACh can be responsible for the decrease in ACh concentration during ischemia. Early post-ischemic elevation of ACh may be related to the large increase in brain choline brought about by ischemia.     

Effect of Electroconvulsive Shock on the Uptake and Release of Noradrenaline and 5-Hydroxytryptamine in Rat Brain Slices

Abstract: The uptake and release of [³H]noradrenaline and [³H]-5-hydroxytryptamine (5-HT) were studied in cerebral cortex slices from rats 30 min and 24 h after a single electroconvulsive shock (ECS) and 24 h after a series of five shocks given over 10 days. Both the K _m and V _max for 5-HT uptake were lower than controls 24 h after a single ECS, whereas after 5 ECS spread over 10 days both parameters remained depressed, though only the fall in V_max was significant. Noradrenaline uptake was not altered after a single ECS, but the V_max and K _m were elevated following chronic ECS treatment. Neither ECS treatment schedule had any effect on the potassium-stimulated release of either transmitter. It is possible that the changes in monoamine uptake seen following ECS are an adaptive response to alterations in the synaptic cleft concentration of these transmitters.     

Effect of antibodies to S-100 group proteins on the electrical activity and chemical sensitivity of the cerebral cortical neurons

The influence of antibodies to proteins S-100 group on electrical activity and chemical sensitivity of rabbit cortical neurons has been investigated by means of extracellular recording and microiontophoresis. It has been found that anti-S-100 increase as a rule spontaneous discharge rate of the neurons, decrease, blockade or invert the neuronal responses to acetylcholine, noradrenaline and glutamate action. It is supposed that proteins S-100 group take part in regulation of eleitrogenic and postsynaptic (primarily glutamate- and GABA-ergic) processes of neural and glial cells in the brain.     

The oxidation of adrenaline and noradrenaline by the two forms of monoamine oxidase from human and rat brain

The selective monoamine oxidase inhibitors clorgyline and (−)-deprenyl were used to study the distribution of monoamine oxidase-A and -B (MAO-A, MAO-B) activities towards (−)-noradrenaline and (+),(−)-adrenaline in homogenates from seven different regions of human brain. The activities towards 5-hydroxytryptamine and 2-phenethylamine, which are essentially specific substrates for the A- and B-forms, respectively, under the conditions used in this work, were also determined. Noradreanline and adrenaline were substrates for both forms of the enzyme in all regions studied. The total MAO activity was found to be highest in the hypothalamus and lowest in the cerebellar cortex. Use of the selective MAO inhibitors clorgyline and (−)-deprenyl also showed adrenaline and noradrenaline to be substrates for both forms of the enzyme in rat brain. In human cerebral cortex and rat brain the two forms were found to have similar K_m-values and maximum velocities towards adrenaline. These values for the two forms were also found to be similar in human cerebral cortex when noradrenaline was used as the substrate. In contrast MAO-A showed a significantly lower K_m and a higher maximum velocity towards noradrenaline in rat brain. These results suggest that the rat may not provide a close model of the human for studies on the effects of MAO inhibitors on brain noradrenaline metabolism.     

Simultaneous determination of morphine and monoamine transmitters in a single mouse brain

A simple procedure for the simultaneous determination of morphine and monoamine transmitters was developed. The procedure consisted of (1) n-butanol extraction and (2) separation and quantitative determination by means of high-performance liquid chromatography combined with electrochemical detection. The maximum intracerebral concentration (210 ± 35 ng/g wet tissue) of morphine was detected 30 min after intramuscular injection (10 mg/kg), which agreed with previous research. Noradrenaline was significantly decreased by morphine injection, while dopamine and 5-hydroxytryptamine were unchanged. However, 3-methoxytyramine, a metabolite of dopamine, was increased, suggesting that the drug increased the turnover rate of dopamine. The procedure used revealed a direct correlation between pharmacokinetics (e.g., distribution of morphine) and pharmacodynamics (e.g. changes of monoamine concentrations) of the drug in vivo.     

Chemosensitivity of single smooth muscle cells to acetylcholine, noradrenaline, and histamine in vitro

Electrical responses to acetylcholine, noradrenaline, and histamine were recorded from solitary smooth muscle cells. Iontophoresis of each transmitter elicited three fast responses: a hyperpolarization, a depolarization, or a biphasic hyperpolarization-depolarization. Each transmitter activated a specific receptor since responses were specifically blocked by antagonists, two transmitters elicited different responses in solitary cells, and desensitization of response to one transmitter did not cause desensitization of responses to other transmitters. Responses were due to increased ion conductances since input resistance decreased during responses and reversal potentials were measured for depolarizing responses (-5 mV) and hyperpolarizing responses (-60 mV). Regional differences in transmitter sensitivity were mapped on solitary cells. Biphasic responses were due to simultaneous activation of receptors mediating hyperpolarizing responses and receptors mediating depolarizing responses which were segregated in the cell membrane. Noradrenaline enhanced action potential amplitude by regulation of voltage-dependent ion conductances. Finally, noradrenaline and histamine elicited periodic hyperpolarizing potentials, which may be due to increased intracellular Ca++.     

Neomycin and ruthenium red antagonism of monoaminergic depression of cerebral cortical neurones

Neomycin and ruthenium red, two agents which are known to interfere with the transport and binding of calcium, antagonize the depressant actions of noradrenaline and 5-hydroxytryptamine on cerebral cortical neurones. Ouabain and sodium azide, metabolic inhibitors, also block the action of noradrenaline. These findings support earlier suggestions that monoaminergic depression of cortical neurones involves a calcium-dependent mechanism which may be linked to a membrane ion pump.     

The nature of adrenoceptors in the guinea pig cerebral cortex: a microiontophoretic study

1. Noradrenaline, isoprenaline, and phenylephrine have been applied my microiontophoresis to neurones in the guinea pig cerebral cortex. All three compounds produced depression of neuronal firing, and all could be antagonized to some extent by phentolamine or propranolol. 2. The responses to isoprenaline were substantially reduced in size after a few applications. Noradrenaline and phenylephrine responses were partially reduced at the time of isoprenaline insensitivity, and the responses could now be blocked completely by phentolamine. 3. The results suggest that two kinds of receptors are present in the guinea pig cerebral cortex, with properties similar to alpha and beta receptors in the periphery. A single receptor with intermediate properties would not readily explain the present results. 4. The results are not consistent with the proposal that alpha receptors mediate neuronal excitation, and beta receptors inhibition in the cerebral cortex. 5. It is also suggested that the failure of some previous studies on guinea pig cortex in vitro to demonstrate the presence of beta receptors may be due to the particularly rapid desensitization of these receptors.     

Transmitter mediated regulation of energy metabolism in nervous tissue at the cellular level

The Monoamines 5-hydroxytryptamine (5-HT), noradrenaline (NA) and histamine, and the peptide Vasoactive Intestinal Polypeptide (VIP), regulate energy metabolism in nervous tissue, in addition to producing excitation and/or inhibition. These transmitters induce glycogen hydrolysis in a concentration dependent manner. The glycogen breakdown is brought about by increased cyclic AMP formation, or translocation of calcium ions to activate phosphorylase, and is partially localized in glial cells. Data from a diversity of nervous systems, including leech and snail ganglia, and rodent cortex, point towards important roles for neurons containing these transmitters in the regulation of the glycogen turnover. It is proposed that energy metabolism may be controlled within domains defined by the geometric arrangements of the neurons releasing these transmitters. The different domains may overlap temporally and spatially to coordinate energy metabolism in relation to increases in neuronal activity. The non-myelin forming glial cells, which contain glycogen whose turnover rate is altered by the transmitters, appear to be important in the local supply of energy substrate to neurons.     

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.     

Brain levels and turnover rates of presumptive neurotransmitters as influenced by administration and withdrawal of ethanol in mice

—Effects of acute or chronic administration of ethanol and its withdrawl on the steady-state levels and turnover rates of certain neurotransmitters have been investigated in mice. The influence of long-term administration of ethanol on the activities of enzymes involved in the metabolism of these transmitters has also been studied. Acute administration of ethanol or acetaldehyde or chronic administration of ethanol resulted in a decrease in the cerebral contents of acetylcholine, acetylCoA and CoA. Brain levels of 5-hydroxytryptamine, norepinephrine and choline remained unchanged after acute administration of ethanol. However, chronic administration of ethanol resulted in a decrease in the norepinephrine content without significantly affecting 5-hydroxytryptamine or choline contents. Cerebral levels of γ-aminobutyric acid increased with both acute or chronic administration of ethanol. The total incorporation of [³H]choline into acetylcholine in brain was depressed upon acute administration of ethanol. After withdrawal of ethanol for one day cerebral levels of norepinephrine returned to normal; however, γ-aminobutyric acid and acetylcholine returned to normal levels at 2 and 4 days after ethanol withdrawal, respectively. Pretreatment of mice with pyrazole, an inhibitor of alcohol dehydrogenase, prevented the ethanol-induced decrease in cerebral acetylcholine levels. The activities of cerebral choline acetyltransferase and glutamic decarboxylase were decreased after 2 weeks of chronic ethanol administration. However, the activities of acetyl cholinesterase and GABA-transaminase remained unaffected after 2 weeks of ethanol treatment     

THE EFFECT OF INJURY ON MONOAMINE CONCENTRATIONS IN THE RAT HYPOTHALAMUS

Abstract— The monoamine concentrations have been measured in four regions of the brain (hypothalamus, cortex, cerebellum and brain stem) in rats injured by either hind-limb ischaemia or scald. Both injuries produced a rapid fall in the noradrenaline concentration of the hypothalamus which recovered slowly if the injury was not fatal. This effect of injury was seen after pretreatment with a-methyl-p-tyrosine to inhibit noradrenaline synthesis, indicating an increased rate of utilization of noradrenaline after injury. These injuries did not affect the 5-hydroxytryptamine concentration in the hypothalamus, but changes were found in the concentration of this monoamine and in that of its metabolite, 5-hydroxyindole acetic acid, in the brain stem. It is concluded that these forms of injury had specific effects on the brain monoamines. The hypothalamic changes were not secondary to changes in core temperature or to hypotension or hypovolaemia and they are discussed in relation to the impairment of temperature regulation seen in the injured rat.     

Monoaminergic mechanisms in the nervous system of Lumbricus terrestris (L.)

Summary The localization and intraneuronal distribution of the monoaminergic transmitters in the nervous system of the earthworm, Lumbricus terrestris, have been investigated in detail with the aid of the histochemical fluorescence method of Falck and Hillarp.In the ventral nerve cord, many yellow fluorescent, 5-hydroxytryptamine containing neurons are found, but only few green fluorescent noradrenaline containing cell bodies, which, however, are numerous in the peripheral nervous system. There is an abundance of both fibre types in the neuropile.The 5-hydroxytryptaminergic neurons probably have a motor (possibly inhibitor) function; the adrenergic neurons in the body segments are supposed to have a receptor (exteroceptive and possibly proprioceptive) function.In the cerebral ganglion, both 5-hydroxytryptamine and noradrenaline containing neurons are found in large numbers, and there are closely packed numerous fibres of both types in the neuropile. Their function is more obscure, though an associative function can be presumed for some adrenergic neurons; smaller 5-hydroxytryptaminergic neurons might have a motor (perhaps inhibitor) function.Adrenergic sensory cells are found in the body integument, most frequently in the clitellum segments, in the prostomium, and in the roof of the buccal cavity. These cells give off varicose fibres that form a basi-epithelial network which is in communication with the green fluorescent sensory fascicles in the ventral nerve cord via the epidermal nerves, the ring nerves, and the segmental nerves. No direct adrenergic sensory-effector innervation of either circular and/or longitudinal musculature or gland cells seems to exist. No adrenergic free nerve endings in the body integument have been observed. Instead, there must be a synaptic contact with the motoneurons, either directly in the neuropile or via an interjacent neuron.No synaptic contacts have been observed in the ventral nerve cord between adrenergic or 5-hydroxytryptaminergic fibres and either the giant fibres or fluorescent or nonfluorescent perikarya.An adrenergic innervation of the pharynx musculature has been found, and sensory cells of a different type are present in and below the epithelium; here, a direct senso-motoric innervation of the pharyngeal musculature cannot be excluded. It is established that the adrenergic neurons in the stomatogastric nervous system have an exciting function on the pharynx, whereas a direct monoaminergic influence of the muscular movements of the intestine probably does not exist.Abbreviations Used A adrenaline - CA catecholamine - DA dopamine - 5-HT 5-hydroxytryptamine - MA monoamine - NA noradrenaline The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the Swedish Natural Science Research Council (99-34, 6627), and by the Swedish Medical Research Council (B67-12X-712-02A).     

Discriminatory Effects of Forskolin and EGTA on the Indirect Cyclic AMP Responses to Histamine, Noradrenaline, 5-Hydroxytryptamine, and Glutamate in Guinea-Pig Cerebral Cortical Slices

The effects of forskolin (1 microM) and EGTA (5 mM) on indirect cyclic AMP responses in slices of guinea-pig cerebral cortex were examined. Forskolin had little effect on the direct 2-chloroadenosine-stimulated cyclic AMP response. However, it completely abolished the glutamate-induced augmentation of this response. In contrast, forskolin had very little effect on the indirect cyclic AMP responses to noradrenaline, 5-hydroxytryptamine, and histamine. Conversely, rapid removal of extracellular calcium with EGTA 2 min before addition of the indirectly acting agent markedly reduced the augmentation responses produced by these latter agonists, but had little effect on the glutamate augmentation. When EGTA was added once a steady level of cyclic AMP had been achieved with the indirect agents, it was without effect on any of the responses. Thus, calcium appears to have a role in the early, but not the later, stages of the noradrenaline, 5-hydroxytryptamine, and histamine responses. A role for protein kinase C in the glutamate augmentation response was suggested, because forskolin inhibited the augmentation of the 2-chloroadenosine response produced by phorbol esters (which mimic the actions of diacylglycerol in activating protein kinase C). We conclude that there is more than one mechanism by which the augmentation of cyclic AMP responses can occur.     

The blockade of serotonin uptake into synaptosomes:relationship to an interaction with monoamine oxidase inhibitors.

To test the hypothesis that the hyperpyrexia produced by meperidine and detromethorphan in rabbits pretreated with a monoamine oxidase inhibitor is related to inhibition of neuronal uptake of serotonin (5-hydroxytryptamine (5-HT)), fluoxetine (Lilly 110140) was studied. This potent and specific 5-HT neuronal uptake blocker was administered to phenelzine-pretreated rabbits and found to produce a lethal hyperpyrexia in doses equal to or greater than 2.5 mg/kg. The order of potency in blocking 5-[14C]HT uptake into synaptosomes prepared from rabbits was: fluoxetine greater than meperidine = dextromethorphan = levorphanol greater than anileridine greater than alphaprodine greater than morphine. Since fluoxetine, meperidine, and dextromethorphan produce hyperpyrexia in phenelzine-pretreated rabbits, whereas anileridine, alphaprodine, and morphine do not, there appears to be some correlation between the hyperpyrexic response and inhibition of 5-HT uptake. The exception is levorphanol, which is not hyperpyrexic despite being equipotent with meperidine and dextromethorphan in inhibiting 5-HT uptake. The ineffectiveness of levorphanol in producing hyperpyrexia may be due to its marked depressant properties, since the addition of another depressant drug (pentobarbital) antagonized the hyperpyrexic effect of meperidine.     

Interaction between morphine and putative excitatory neurotransmitters in cortical neurons in naive and tolerant rats.

Microelectrophoretically applied morphine depressed spontaneously discharging cortical neurones of rats and blocked excitation induced by electrophoretic administrations of either acetylcholine or l-glutamate. This depressant effect and both the anti-acetylcholine and the anti-glutamate effect were naloxone antagonizable and therefore regarded as specific morphine actions. The excitatory effects of morphine were not affected by naloxone application and were classified as non-specific.In chronically morphinized rats the depressant effect of morphine on spontaneous discharge activity and also its blocking action upon acetylcholine and l-glutamate-induced excitation were almost completely abolished. The predominant response in such pre-treated animals was non-specific excitation. Acetylcholine and l-glutamate were found to be more effective in tolerant rats (supersensitivity).