Stimulatory and inhibitory effects of ethanol on hippocampal acetylcholine release

Using the microdialysis technique and sensitive HPLC procedures for the determination of acetylcholine (ACh) and ethanol, we investigated the release of ACh in rat hippocampus after acute ethanol administration. Systemic administration of ethanol (0.8 and 2.4 g/kg i.p.) led to peak ethanol concentrations of 21 and 42 mM in the hippocampus, respectively. The high dose caused a long-lasting inhibition of basal ACh release by up to 33%. Local infusion of scopolamine (1 microM) enhanced hippocampal ACh release up to eightfold in the presence of neostigmine (10 microM), and this stimulated release was also inhibited after systemic ethanol administration (by up to 45%). The low dose of ethanol (0…

Inhibitory and excitatory muscarinic receptors modulating the release of acetylcholine from the postganglionic parasympathetic neuron of the chicken heart.

The effects of muscarinic receptor antagonists on ACh release were studied in the absence or presence of cholinesterase (ChE) inhibition using the isolated perfused chicken heart. Presynaptic inhibitory muscarinic autoreceptor were characterized by determining the potency of various antagonists to enhance [3H]-ACh release evoked by field stimulation (3 Hz, 1 min). The order of potencies was: (±)-telenzepine > atropine > 4-DAMP > silahexocyclium > pirenzepine > hexahydro-siladifenidol > AF-DX 116. The comparison with known pA2 values for M1-, M2- and M3-receptors revealed that the presynaptic autoreceptor meets the criteria of an M1-receptor. Basal, not electrically evoked overflow of unlabe…

Ontogenetic and Pharmacological Studies on Metabotropic Glutamate Receptors Coupled to Phospholipase D Activation

The present study was aimed at characterizing the metabotropic receptor subtype which is involved in the activation of phospholipase D (PLD) by glutamate in rat hippocampal slices. We first observed that the ontogenetic profile of glutamate-induced hydrolysis of phosphoinositides and of phosphatidylcholine was strikingly similar. Both pathways were significantly activated by glutamate in tissue taken from 3-, 8- and 15-day old rats, but not in adult rats. PLD activation was strongest in slices taken from 8-day old rats. At this age, quisqualate had a higher potency for PLD activation (EC50: 0.6 microM) than 1S,3R-ACPD (EC50: 16 microM) and DHPG, a specific activator of group I mGluR, was a …

The neuronal efflux of noradrenaline: Dependency on sodium and facilitation by ouabain

Rabbit hearts were isolated after pretreatment with the MAO inhibitor pargyline and with reserpine and were perfused with 200 ng/ml noradrenaline for 1 h. During the subsequent wash-out with an amine-free solution for 2 h, the neuronal efflux of noradrenaline declined mono-exponentially with a mean halftime of 42 min. Both Na+-free solution and ouabain caused facilitation of the efflux which thereafter declined in a multi-exponential fashion. The maximum facilitation was reached after 3 min of Na+-free perfusion and 25 min after introduction of ouabain. The amount of exogenous noradrenaline accumulated in the heart was only partially released when the extracellular Na+-concentration was nor…

Cross-talk between phosphatidic acid and ceramide during ethanol-induced apoptosis in astrocytes

Background Ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid (PA) formation by phospholipase D (PLD). The present study investigates ethanol's effect on the induction of apoptosis in astrocytes and the formation of ceramide, an apoptotic signal. Evidence is presented that the formation of PA and ceramide may be reciprocally linked during ethanol exposure. Results In cultured rat cortical astrocytes, ethanol (0.3–1 %, v/v) induced nuclear fragmentation and DNA laddering indicative of apoptosis. Concomitantly, in cells prelabeled with [3H]-serine, ethanol caused a dose-dependent, biphasic increase of the [3H]-ceramide/ [3H…

Ethanol inhibits proliferation in astrocytes, an effect that was recently linked to the suppression of phosphatidic acid (PA) formation by phospholipase D (PLD). The present study investigates ethanol's effect on the induction of apoptosis in astrocytes and the formation of ceramide, an apoptotic signal. Evidence is presented that the formation of PA and ceramide may be reciprocally linked during ethanol exposure. In cultured rat cortical astrocytes, ethanol (0.3–1 %, v/v) induced nuclear fragmentation and DNA laddering indicative of apoptosis. Concomitantly, in cells prelabeled with [3H]-serine, ethanol caused a dose-dependent, biphasic increase of the [3H]-ceramide/ [3H]-sphingomyelin rat…

Role of Phospholipase D Activation in Nervous System Physiology and Pathophysiology

Enhancement of Hippocampal Acetylcholine Release by Local Ethanol Infusion

Among the multiple pathological changes in the CNS which are associated with ethanol intoxication (1), an impairment of cognitive functions is one of the most consistent findings. This impairment may be due to a damage of central cholinergic systems. Long-term administration of ethanol causes neurodegenerative changes in the cholinergic basal fore-brain neurons of rats and humans (2), and in the rat, these changes are accompanied by a reduction of the release of acetylcholine (ACh) in the hippocampal and cortical target regions of basal forebrain neurons (3). Moreover, experimental studies have documented that ethanol-induced dysfunctions of memory and learning can be ameliorated by cholino…

Effect of coronary perfusion rate on the hydrolysis of exogenous and endogenous acetylcholine in the isolated heart

1. The effect of perfusion rate on the hydrolysis of acetylcholine in isolated chicken hearts was studied by measuring both the spontaneous and the evoked output of endogenous acetylcholine into the perfusate in response to vagal stimulation and the arterio-venous difference of exogenous acetylcholine. 2. A decrease in the perfusion rate from 30 to 20 and 10 ml/min caused a graded and significant decline of both the spontaneous overflow of acetylcholine and the overflow evoked by stimulation of both vagus nerves (20 Hz, 1 ms, 40V) for 20 min. The spontaneous and evoked overflow at 30 ml/min were 2 and 3 times, respectively, the overflow at 10 ml/min. 3. Physostigmine (10−6M) raised both the…

Brain choline has a typical precursor profile.

Choline is product and precursor to both acetylcholine and membrane phospholipids, and, in the brain, is ultimately provided by the circulation. The brain is protected from excess choline and choline deprivation by a refined system of homeostatic mechanisms that maintain a level of extracellular choline that, for its role as precursor, meets saturation criteria under normal conditions. The kinetic and activity profiles of choline are typical for a biosynthetic precursor.

Muscarinic Receptor Activation Increases Efflux of Choline from Isolated Heart and Rat Cortex in Vivo. Interactions with Forskolin and IBMX

Muscarinic receptor activation modulates functions of the heart and neurotransmission in the peripheral and central nervous system. Moreover, muscarinic agonists produce changes in the metabolism of, for example, heart tissue, such as inhibition of beta-adrenoceptor-mediated cAMP accumulation, glycogenolysis and lipase activation.

Stimulatory influence of ethanol on the septohippocampal cholinergic pathway. A role for GABA receptors?

Glutamate Activates Phospholipase D in Hippocampal Slices of Newborn and Adult Rats

Phospholipase D (PLD) is activated by many neurotransmitters in a novel signal transduction pathway. In the present work, PLD activity was studied comparatively in hippocampal slices of newborn and adult rats. Basal PLD activity in adult rats was almost three times higher than in newborn rats. In newborn rats, L-glutamate and 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) time- and concentration-dependently enhanced the formation of [3H]phosphatidylpropanol ([3H]PP) and of [3H]phosphatidic acid in the presence of 2% propanol. N-Methyl-D-aspartate and kainate (both 1 mM) caused small, but significant increases (approximately 50%), whereas alpha-amino-3-hydroxy-5-methylisoxazole…

Degradation of phosphatidylethanol counteracts the apparent phospholipase D-mediated formation in heart and other organs.

Phosphatidylalcohols, such as phosphatidylethanol (PEth), are formed from phosphatidylcholine in the presence of a primary alcohol (e.g., ethanol). This 'transphosphatidylation' reaction is used as specific phospholipase D (PLD) assay. Accumulation of PEth in tissues is recognized as a reliable measure of PLD activity, as PEth is allegedly metabolically stable. The general validity of this assumption was reinvestigated in isolated rat heart, small intestine and brain slices. The half-times of 3H-PEth degradation (labelled with 3H-myristic acid and preformed by ethanol exposure for 30 min) were about 1 h in heart and small intestine, but 17 h in brain. As the formation of PEth is superimpose…

Interstitial washout and hydrolysis of acetylcholine in the perfused heart

The efflux of acetylcholine, of radioactively labelled acetylcholine and choline, into the venous effluent of the perfused chicken heart was studied to determine the kinetics of both interstitial washout and hydrolysis of acetylcholine. Stimulation of both cervical vagus nerves (e.g., for 5 s at 40 Hz) caused a release of acetylcholine, which appeared partially unhydrolyzed in the venous effluent, and reduced force of contraction and heart rate. For comparison, labelled acetylcholine or choline was infused for 5 s into the heart and again the venous efflux of either substance was determined. It was found that the kinetics of efflux of acetylcholine or choline from the interstitial space wer…

Free choline and choline metabolites in rat brain and body fluids: sensitive determination and implications for choline supply to the brain.

In the central nervous system, choline is an essential precursor of choline-containing phospholipids in neurons and glial cells and of acetylcholine in cholinergic neurons. In order to study choline transport and metabolism in the brain, we developed a comprehensive methodical procedure for the analysis of choline and its major metabolites which involves a separation step, selective hydrolysis and subsequent determination of free choline by HPLC and electrochemical detection. In the present paper, we report the levels of choline, acetylcholine, phosphocholine, glycerophosphocholine and choline-containing phospholipids in brain tissue, cerebrospinal fluid and blood plasma of the untreated ra…

Chapter 19 Muscarinic activation of phosphatidylcholine hydrolysis

Publisher Summary This chapter focuses on the muscarinic activation of phosphatidylcholine hydrolysis. The release of choline from tissues or cells is a sensitive indicator of an enhanced hydrolysis of phosphatidylcholine (PtdCho) and is easily determined by chemiluminescence. In certain cells, choline release may reflect the activity of a specific receptor-activated enzyme catalyzing PtdCho hydrolysis. A physiological role of the receptor-mediated release of choline in the brain is given by its role as biosynthetic precursor for acetylcholine (ACh) and phospholipids. When PtdCho hydrolysis is investigated to identify the phospholipase involved, the sole determination of enzymatic products …

The release of choline from phospholipids mediated by beta-adrenoceptor activation in isolated hearts.

The resting efflux of choline into the perfusate (Tyrode's solution) of isolated hearts was equal to the rate, at which choline was liberated from phospholipid degradation (Lindmar et al. 1986). Infusion of isoprenaline (2 X 10(-7) mol/l), forskolin (1-3 X 10(-6) mol/l) or 3-isobutyl-1-methylxanthine (IBMX; 3 X 10(-4) mol/l) for 40 min markedly enhanced the efflux of choline. The increase was linear during the experimental period and, in the case of isoprenaline, was blocked by 3 X 10(-7) mol/l atenolol. In the guinea-pig heart, IBMX at a threshold concentration of 10(-4) mol/l shifted the concentration-response curve for the effect of forskolin on the efflux of choline to the left by one l…

Characterization of choline efflux from the perfused heart at rest and after muscarine receptor activation.

The resting efflux of choline from perfused chicken hearts varied from 0.4 to 2.6 nmol/g min, but was constant for at least 80 min in the individual experiments. The rate of choline efflux was found to be equal to the rate of choline formation in the heart, which, from the following reasons, was essentially due to hydrolysis of choline phospholipids. Cardiac content of choline phospholipids (7,200 nmol/g) was much higher than that of acetylcholine (5.5 nmol/g). Resting release of acetylcholine was 0.016 nmol/g min and, after inhibition of cholinesterase, only about 0.1 nmol/g min. Resting efflux of choline was reduced by mepacrine, a phospholipase A2 inhibitor, by perfusion with a Ca2+-free…

The influence of long-term dietary choline deficiency on choline metabolites in the rat brain

The Homeostasis of Brain Choline

The interest in the homeostasis of brain choline is reinforced by the role of choline as immediate precursor of acetylcholine, phosphatidylcholine and other phospholipids in the brain. In order to obtain a comprehensive view of the mochanisms of homeostasis it appeared necessary to elucidate the negative arteriovenous difference of choline across the brain (net release), a phenomenon that has been known for 20 years and is present in mammals and in man. This finding prompted an intense search for a de novo synthesis of choline in the brain. We detected in anaesthetized rats a reversal of the net release into a net uptake (positive arterio-venous difference), when the plasma level of choline…

Glutamatergic activation of hippocampal phospholipase D: postnatal fading and receptor desensitization.

Abstract: Phospholipase D (PLD) activity was determined in rat hippocampal slices between postnatal days 3 and 35. After birth, basal PLD activity was low and, within 2 weeks, increased to reach a plateau that was maintained up to the adult age. Likewise the response to glutamate developed postnatally to reach a maximum at day 8, but then faded rapidly and was almost absent at day 35. Activation of PLD by 4β-phorbol 12β,13α-dibutyrate (PDB) was independent of age, whereas the effect of aluminum fluoride (AlF4−) increased to a plateau within the first week. At day 8, PLD stimulation by glutamate via metabotropic receptors involved protein kinase C activation, but was independent of Ca2+ infl…

The effect of tacrine on acetylcholine overflow in the heart

Tacrine, 10(-6) M, enhanced the acetylcholine (ACh) overflow evoked in perfused chicken hearts by field stimulation (5 Hz, 1 min) from 183 to 346 pmol g-1 min-1. Increase to the same level were observed after pretreatment with diisopropylfluorophosphate (DFP) as well as after DFP plus 10(-6) M tacrine. Tacrine, 10(-5) M, caused further enhancement with or without DFP up to 851 pmol g-1 min-1. It was concluded that 10(-6) M tacrine enhanced the ACh overflow by choline esterase inhibition, whereas 10(-5) M tacrine caused, in addition, an increase of neuronal ACh release.

Acetylcholine overflow during infusion of a high potassium-low sodium solution into the perfused chicken heart in the absence and presence of physostigmine.

1. The effect of infusion of a modified Tyrode's solution (“high K+-low Na+ solution”) into the isolated chicken heart on the content of acetylcholine in the tissue and the overflow of acetylcholine were compared to those evoked by vagal stimulation. 2. The release of acetylcholine was measured over 15-min periods of either stimulation of the vagus nerves (40 V, 1 ms) at 20 Hz or of infusion of the high K+-low Na+ solution (108 mM K+, 44 mM Na+). 3. Both stimuli caused a maximum overflow of acetylcholine in the first few minutes whether or not 10−6 M physostigmine was present. The overflow was maintained during the vagal stimulation at a constant rate of at least 35% the initial rate, where…

Effects of Norepinephrine and Cardiotrophin-1 on Phospholipase D Activity and Incorporation of Myristic Acid Into Phosphatidylcholine in Rat Heart

The present study is part of a project on phospholipase D (PLD) in cardiac hypertrophy and analyzed effects on PLD activity of two growth stimuli, norepinephrine (NE) and cardiotrophin-1 (CT-1), in incubated rat heart. Phosphatidylcholine (PC) was labeled by 3H-myristic acid. PLD produced 3H-phosphatidylethanol (3H-PEth) from 3H-PC in the presence of ethanol and maintained a basal formation of 3H-PEth. Short-term and long-term exposure to NE for 2 or 13 h, respectively, enhanced the formation of 3H-PEth, which was blocked by prazosin. Long-term pretreatment with NE or CT-1 increased the incorporation of 3H-myristic acid into PC, which was blocked by atenolol. When the 3H-PEth formation was …

Ouabain enhances release of acetylcholine in the heart evoked by unilateral vagal stimulation.

The aim of the study was to elucidate peripheral effects of ouabain on the parasympathetic innervation of the heart, effects that could contribute to the experimentally and clinically well established “vagal effect of cardiac glycosides”. The experiments were carried out with ouabain concentrations of 3×10−7 and 10−6 mol/l, which were considered “therapeutic”, as they increased force of contraction and did not elicit arrhythmias in incubated chicken atria. In atrial preparations of chickens and guinea-pigs the negative chronotropic and inotropic effects of acetylcholine (ACh) were not altered by 3×10−7 mol/l ouabain. Resting efflux of ACh from perfused chicken hearts was increased by ouabai…

Evidence for bilateral vagal innervation of postganglionic parasympathetic neurons in chicken heart

Stimulation of the cervical vagus nerves caused an output of acetylcholine (ACh) from the isolated chicken heart, which almost exclusively was released from the postganglionic neurons: (+)-tubocurarine (3 X 10(-14) M) reduced the output to 12 +/- 6% (n = 7) of the control. Stimulation of the two nerve trunks ws equally effective in releasing ACh.--Evidence that a large number of postganglionic neurons receives bilateral innervation was based on two experimental series. (1). The sum of the ACh outputs evoked by unilateral (separate) nerve stimulation of the right and the left vagus was higher than the bilaterally evoked output (100%) and increased with increasing frequencies (10, 20 and 40 H…

Choline Fluxes to and from the Rat Cerebral Cortex Studied with the “Cup Technique” in Vivo

Since MacIntosh and Oborin (11) and later Mitchell (12) introduced the “cup technique” as a mean to study acetylcholine release from the cerebral cortex in vivo, this technique has been widely used for investigating the release of various neurotransmitters in anaesthetized as well as unanaesthetized mammals (2, 13, 14). Recently we proposed the “cup technique” as a way for studying the efflux of endogenous choline (Ch) from the rat cerebral cortex (4, 5) and to estimate changes in the extracellular concentration of Ch, if we consider the fluid filling the cup as an extension of the extracellular space.

Acetylcholine overflow from isolated perfused hearts of various species in the absence of cholinesterase inhibition

1. The content of acetylcholine in the tissue and effluent of isolated hearts of various birds and mammals was determined in the absence of inhibition of cholinesterase. 2. Stimulation of both vagus nerves for 15 min at 20 Hz caused marked negative chronotropic effects in all species. Spontaneous or stimulation-induced overflow of acetylcholine into the effluents was not detected in mammals. In the avian heart, the order of spontaneous overflow was: duck = chicken > pigeon, whereas the order of evoked overflow was: chicken > pigeon > duck. The acetylcholine overflow from the cat heart was below the limit of estimation (3 pmol g−1 min−1). In the chicken heart, the evoked overflow per min (28…

Modulation of hippocampal acetylcholine release after fimbria-fornix lesions and septal transplantation in rats

Abstract Female Long–Evans rats sustained electrolytic lesions of the fimbria and the dorsal fornix causing a partial lesion of the septohippocampal pathway. Two weeks later, the rats received intra-hippocampal grafts of fetal septal cell suspensions. Nine to twelve months later, the release of acetylcholine (ACh) in the hippocampus of sham-operated, lesion-only and grafted rats was measured by microdialysis. The extent of cholinergic (re)innervation was determined by acetylcholinesterase (AChE) staining and densitometry. In both lesion-only and grafted rats, the ratio of ACh release to AChE staining intensity was increased as compared to sham-operated rats, indicating a loss of endogenous …

29 Elevated acetylcholine release in the hippocampus of transgenic mice expressing the human acetylcholinesterase

Phorbol Esters and Muscarinic Receptor Agonists Activate Phospholipase D in Heart and Brain

Phospholipase D (PLD) hydrolyzes phosphatidylcholine and thereby seems to play a key role in a novel pathway of signal transduction. PLD activity in rat hippocampal slices and atria of rat, guinea pig and chicken hearts was determined by measuring the catalytic products choline (Ch), phosphatidic acid (PA) and, in the presence of a primary alcohol, phosphatidylpropanol or phosphatidylethanol. It was found that the PLD activity was high, even under resting conditions, in both tissues, especially in the hippocampus, and that the enzyme activity could be enhanced by activation of protein kinase C and by muscarinic receptor stimulation.

Uptake and storage of choline by rat brain: influence of dietary choline supplementation.

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In th…

Regulation of Acetylcholine Synthesis and Release in the Isolated Heart

In the two decades following Loewi’s work (8,9) on the frog heart several research groups tried to use the isolated heart preparation from higher vertebrates for further investigations on synthesis, release and inactivation of acetylcholine (ACh). However the results were discouraging because the overflow of ACh during vagal stimulation was near, or below, the limit of the assay.

Chapter 17 Muscarinic receptors and cell signalling

Publisher Summary Cells have developed signal transduction mechanisms in order to communicate with the cell exterior. Acetylcholine as an external signal is recognized by nicotinic and muscarinic receptors. This chapter presents various muscarinic receptors belonging to the superfamily of G protein-coupled receptors consisting of seven transmembrane (TM) helices tightly packed in a ring-like structure and arranged in a counter-clockwise fashion. Agonist binding leads to a conformational change of the receptor, thereby activating associated G proteins. Muscarinic stimulation of G proteins leads to the activation or inhibition of ion channels, such as K + and Ca 2+ channels, the activation of…

Muscarinic mobilization of choline in rat brain in vivo as shown by the cerebral arterio-venous difference of choline.

In anesthetized rats, the choline levels of cerebrospinal fluid and plasma obtained from blood collected from peripheral vessels (carotid artery, cardiac vessels) and from the transverse sinus were determined with a radioenzymatic assay. Cortical release of choline was studied using the "cup technique." The plasma choline level of the peripheral blood (11.5 mumol/L) was lower than that of the sinus blood. The resulting cerebral arterio-venous difference of choline was negative (3.2 mumol/L) and reflected the net release of choline from the whole brain. The plasma choline levels were not different irrespective of whether the rats were anesthetized with ether, urethane, or pentobarbital. Howe…

Regulation of free choline in rat brain: dietary and pharmacological manipulations.

The present study analyses, comparatively, the kinetics of free choline in the brain of rats during dietary and pharmacological manipulations. Low-choline diet halved the choline plasma level but did not cause significant changes of CSF choline. High-choline diet, hypoxia and treatment with nicotinamide increased brain choline availability through a central site of action and increased the CSF choline concentration. CSF choline concentrations were more effectively elevated by nicotinamide treatment (20-25 microM) than by acute choline administration (13-15 microM). Increases of CSF choline, due to brain choline mobilization, were consistently associated with a net release of choline from th…

Compensatory mechanisms enhance hippocampal acetylcholine release in transgenic mice expressing human acetylcholinesterase

Central cholinergic neurotransmission was studied in learning-impaired transgenic mice expressing human acetylcholinesterase (hAChE-Tg). Total catalytic activity of AChE was approximately twofold higher in synaptosomes from hippocampus, striatum and cortex of hAChE-Tg mice as compared with controls (FVB/N mice). Extracellular acetylcholine (ACh) levels in the hippocampus, monitored by microdialysis in the absence or presence of 10(-8)-10(-3) M neostigmine in the perfusion fluid, were indistinguishable in freely moving control and hAChE-Tg mice. Muscarinic receptor functions were unchanged as indicated by similar effects of scopolamine on ACh release and of carbachol on inositol phosphate fo…

Uptake and metabolism of choline by rat brain after acute choline administration.

The present study is concerned with the uptake and metabolism of choline by the rat brain. Intraperitoneal administration of choline chloride (4-60 mg/kg) caused a dose-dependent elevation of the plasma choline concentration from 11.8 to up to 165.2 microM within 10 min and the reversal of the negative arteriovenous difference (AVD) of choline across the brain to positive values at plasma choline levels of greater than 23 microM. Net choline release and uptake were linearly dependent on the plasma choline level in the physiological range of 10-50 microM, whereas the CSF choline level was significantly increased only at plasma choline levels of greater than 50 microM. The bolus injection of …

Differential effects of hypothermia on neuronal efflux, release and uptake of noradrenaline

Isolated rabbit hearts were perfused at 34° (control), 24° or 12°C. The neuronal efflux of noradrenaline after perfusion with the amine for 1 h was depressed at 24° C (Q 10 about 5) in the presence or absence of desipramine; at 12°C the efflux was below the limit of estimation. Moderate reduction of the temperature (24° C) decreased the removal of perfused noradrenaline to about 60% of the control value and caused a 1.7-fold increase of the output of noradrenaline evoked by sympathetic nerve stimulation. It is concluded that the extremely temperature-dependent efflux of noradrenaline across the axonal membrane is not part of the release of noradrenaline evoked by nerve stimulation.

The effects of phorbol esters on choline phospholipid hydrolysis in heart and brain

The efflux of choline was determined in rat striatal slices, incubated chicken atria and perfused chicken hearts. 4 beta-Phorbol-12 beta,13 alpha-dibutyrate (PDB) and 4 beta-phorbol-12 beta-myristate, 13 alpha-acetate (PMA) were used to stimulate protein kinase C. The other phorbol esters, 4 beta-phorbol-13 alpha-acetate (PAc) and 4 alpha-phorbol-12 beta,13 alpha-didecanoate (4 alpha PDD), known to be inactive, were tested to evaluate the specificity of the responses. PDB markedly enhanced the efflux of choline in all of the three preparations. The PDB-evoked efflux of choline in incubated chicken atria was equal to the net production of choline and, therefore, was not caused by translocati…

Receptors Linked to Hydrolysis of Choline Phospholipids: the Role of Phospholipase D in a Putative Mechanism of Signal Transduction

The structure and basic functions of biomembranes are essentially determined by the lipid bilayer. In contrast, specific membrane functions, such as signal recognition and transduction and transport processes, have been preferentially attributed to proteins that are embedded in the outer or inner leaflet of this bilayer or may span the membrane up to five times or more, as in the case of receptor molecules. The segregating view of membrane protein and lipid functions may have delayed a broad research interest in the dynamic interactions between these components of the membrane. The present review is devoted to such a recently discovered interaction: the coupling of receptor activation and h…

Presence of muscarinic inhibitory and absence of nicotinic excitatory receptors at the terminal sympathetic nerves of chicken hearts.

Nicotine (2 X 10(-4) M) or acetylcholine (5.5 X 10(-4) M) in the presence of 3 X 10(-6) M atropine did not increase the rate or amplitude of contraction in isolated atria or ventricular strips of the chicken heart; both drugs also did not cause an output of noradrenaline or adrenaline and did not evoke antidromic discharges in the right sympathetic nerves of isolated perfused chicken hearts. In contrast, "high K+-solutions" evoked an output of noradrenaline and adrenaline and caused a burst of antidromic discharges. Dimethylphenylpiperazine (DMPP; 3.1 X 10(-4) M), by a tyramine-like action, elicited a small output of noradrenaline and increased rate and amplitude of contraction" but did not…

Neuronal and extraneuronal uptake and efflux of catecholamines in the isolated rabbit heart

1. Isolated rabbit hearts were perfused with (−)-noradrenaline, (−)-adrenaline and (±)-isoprenaline for various time periods (1–180 min) and then washed with an amine-free medium. The venous concentration of the amine was estimated fluorimetrically during the infusion and after its end, to study removal and efflux, respectively. 2. In untreated hearts and after pretreatment with reserpine the removal had a constant rate over 20–60 min. After pretreatment with pargyline to block monoamine oxidase (MAO), however, the removal of noradrenaline declined exponentially to zero. Inhibition of the neuronal uptake (desipramine) and chemical sympathectomy (6-hydroxydopamine) abolished the removal of n…

DMPP and the adrenergic nerve terminal: mechanisms of noradrenaline release from vesicular and extravesicular compartments.

DMPP (1,1-dimethyl-4-phenylpiperazine) in various concentrations between 1.6×10−6 M and 6.2×10−5 M was infused into isolated rabbit hearts to study the neuronal release and uptake of noradrenaline.

Phospholipase D in heart: basal activity and stimulation by phorbol esters and aluminum fluoride

Evidence for a general role of phospholipase D in signal transduction is accumulating. In the present study, the activity of the enzyme was investigated in heart tissue under basal conditions and after addition of phorbol esters or aluminum fluoride (AlF inf4 sup− ; 10 mM NaF plus 10 μM AlCl3). Atria of rats and chickens were incubated with [3H]-myristic acid in order to label preferentially phosphatidylcholine. Under basal conditions, the tissues generated choline and phosphatidic acid (PtdOH), the primary catalytic products of phospholipase D. When 0.5 or 2.0% ethanol was present, [3H]-phosphatidyl-ethanol (PETH) was rapidly formed at the expense of [3H]-PtdOH. This transphosphatidylation…

Phospholipid breakdown and choline release under hypoxic conditions: inhibition by bilobalide, a constituent of Ginkgo biloba

A marked increase of choline release from rat hippocampal slices was observed when the slices were superfused with oxygen-free buffer, indicating hypoxia-induced hydrolysis of choline-containing phospholipids. This increase of choline release was suppressed by bilobalide, an ingredient of Ginkgo biloba, but not by a mixture of ginkgolides. The EC50 value for bilobalide was 0.38 microM. In ex vivo experiments, bilobalide also inhibited hypoxia-induced choline release when given p.o. in doses of 2-20 mg/kg 1 h prior to slice preparation. The half-maximum effect was observed with 6 mg/kg bilobalide. A similar effect was noted after p.o. administration of 200 mg/kg EGb 761, a ginkgo extract con…

Effects of nicotinamide on central cholinergic transmission and on spatial learning in rats

High-dose nicotinamide (1000 mg/kg) leads to a minor increase of plasma choline but to a major increase of the choline concentrations in the intra- and extracellular spaces of the brain. In the hippocampus, the nicotinamide-induced increase in choline was associated with an increase in the release of acetylcholine under stimulated conditions. In young rats, nicotinamide in doses between 10 and 1000 mg/kg did not influence spatial learning, as tested in the Morris water maze. In old rats, low doses of nicotinamide were ineffective whereas the high dose of 1000 mg/kg even impaired spatial learning. The combined administration of choline and nicotinamide had a synergistic effect on brain choli…

Release of choline from rat brain under hypoxia: contribution from phospholipase A2 but not from phospholipase D

Moderate hypoxia induced in rats by inhalation of 10% oxygen led to an increase of the concentration of free choline in the brain and caused a large net-release of choline from the brain into the venous blood as determined by the measurement of the arterio-venous difference. In hippocampal slices from rat brain, hypoxia increased the release of choline into the superfusion medium. The activity of phospholipase D, as measured by the formation of phosphatidylpropanol in the presence of propanol, was not stimulated under these conditions. However, the mobilization of choline was completely depressed by lowering extracellular calcium and by 0.1 mM mepacrine. We conclude that hypoxia leads to a …

4-Aminopyridine antagonizes the inhibitory effect of pentobarbital on acetylcholine release in the heart

Effects of pentobarbital on acetylcholine (ACh) release, force of contraction and nervous conduction were studied in isolated heart preparations and in cervical vagus nerves, respectively. 4-Aminopyridine and tetracaine were used as pharmacological tools to eludicate the mode of action of pentobarbital. 1. 4-Aminopyridine (10−4 M) markedly increased the overflow of ACh from the isolated chicken heart evoked by electrical stimulation (1–50 Hz, 1 ms, 40 V) of the cervical vagus nerves. This effect of 4-aminopyridine was highest at low frequencies of stimulation (+ 226% at 1 Hz) and declined with increasing frequencies to reach a minimum augmentation of 22% at 30 Hz. 2. Pentobarbital and tetra…

Phospholipase D in rat myocardium: formation of lipid messengers and synergistic activation by G-protein and protein kinase C.

Activation of phospholipase D (PLD) and phosphoinositide-specific phospholipase C (PI-PLC) by fluoride, to stimulate heterotrimeric G-proteins, and by phorbol esters, to stimulate protein kinase C (PKC), was studied in rat atria. Fluoride and 4beta-phorbol-12beta,13alpha-dibutyrate (PDB), in contrast to 4beta-phorbol-13alpha-acetate (PAc), activated PLD, catalyzing the formation of [3H]-phosphatidylethanol ([3H]-PETH), [3H]-phosphatidic acid ([3H]-PA), choline and sn-1,2-diacylglycerol (DAG). Basal PLD activity was resistant to drastic changes in Ca2+ and to Ro 31-8220, a PKC inhibitor, but was decreased by genistein, an inhibitor of tyrosine kinase, and increased by vanadate, a tyrosine ph…

Upregulation of Phospholipase D Expression and Activation in Ventricular Pressure-Overload Hypertrophy

Evidence for a role of phospholipase D (PLD) in cellular proliferation and differentiation is accumulating. We studied PLD activity and expression in normal and hypertrophic rat and human hearts. In rat heart, abdominal aortic banding (constriction to 50% of original lumen) caused hypertrophy in the left ventricle (as shown by weight index and ANP expression) by about 15% after 30 days without histological evidence of fibrosis or signs of decompensation and in the right ventricle after 100 days. The hypertrophy was accompanied by small increases of basal PLD activity and strong potentiation of stimulated PLD activity caused by 4β-phorbol-12β,13α-dibutyrate (PDB) and by phenylephrine. The mR…

Chapter 23: Choline, a precursor of acetylcholine and phospholipids in the brain

Publisher Summary The plasma level of free choline is remarkably constant at about 10 pM in animals and human. Ingestion of food, especially when rich in choline or lecithin, transiently elevates the plasma choline level up to 20 pM or more. In contrast, choline-deficient diet leads to a reduction of the plasma level by about 50%. Choline is considered an essential nutrient, which is predominantly supplied as phosphatidylcholine (lecithin). For a long time, neuroscientists have been intrigued by the fact that choline is a precursor for the biosynthesis of both acetylcholine (ACh) and phospholipids. For 50 years, lecithin has been marketed in Europe as a drug that was claimed to prevent exha…

A homeostatic mechanism counteracting K+-evoked choline release in adult brain

Choline (Ch) is an essential nutrient as the biosynthetic precursor of acetylcholine (ACh) and phospholipids. Under resting conditions, the intracellular accumulation of Ch (above 10-fold), which is positively charged, is governed by the membrane potential and follows the Nernst equation. Accordingly, in synaptosomes from adult rats during depolarization, we observed a linear relationship between release of free cytoplasmic Ch and KCl concentration (2.7-120 mm). The K(+) -evoked Ch release was Ca(2+) -independent and did not originate from ACh or phospholipid hydrolysis. In superfused brain slices of adult rats, however, a K(+) -induced Ch efflux was absent. Also, under in vivo conditions, …

Release of choline in the isolated heart, an indicator of ischemic phospholipid degradation and its protection by ischemic preconditioning: No evidence for a role of phospholipase D

Abstract The release of choline as a water-soluble product of phospholipid hydrolysis was measured in the perfusate of rat hearts to monitor ischemic membrane degradation and its protection by ischemic preconditioning (IPC). Hearts were subjected to global ischemia (GI; 30 min of no-flow) followed by 60 min of reperfusion. To induce IPC, GI was preceded by four no-flow episodes of 5 min each. Deleterious consequences of GI and reperfusion, namely coronary flow reduction, incidence of arrhythmias and release of cardiac troponin T, were significantly attenuated by IPC. The release of choline increased during reperfusion in a biphasic manner: a first phase peaked immediately after GI and was f…

PRECURSOR CONTROL OF ACETYLCHOLINE SYNTHESIS AND RELEASE IN ISOLATED HEARTS ?

Adrenergic activation of phospholipase D in primary rat astrocytes.

Phospholipase D (PLD) activity was investigated in astrocytes prepared from newborn rat cerebral cortex using the transphosphatidylation assay. Basal PLD activity was measurable and was found to be enhanced by ATP, carbachol and noradrenaline. The activation by noradrenaline (EC50, 0.68 microM) was mimicked by methoxamine (EC50, 65 microM), an alpha 1-specific adrenergic agonist, and was inhibited by prazosine, an alpha 1-specific adrenergic antagonist. Clonidin, an alpha 2-adrenergic agonist, slightly lowered PLD activity whereas beta-adrenergic drugs were without effect. Experiments with mitogens indicate that PLD activation in astrocytes may be involved in the control of astrocytic cell …

Glucose plus choline improve passive avoidance behaviour and increase hippocampal acetylcholine release in mice.

The present study tests the effects of glucose and choline, the biosynthetic precursors of acetylcholine, on passive avoidance behaviour and hippocampal acetylcholine release measured by microdialysis in awake mice. Glucose (10 and 30mg/kg) or choline chloride (6-60mg/kg), given by i.p. injection immediately after training, dose-dependently enhanced retention in an inhibitory avoidance task. Combinations of low doses of glucose (10mg/kg) and choline chloride (20mg/kg) which alone were submaximally effective significantly increased retention latencies in a synergistic manner, an effect which was sensitive to atropine (0.5mg/kg). This beneficial effect vanished when higher doses of glucose or…

The role of choline in the release of acetylcholine in isolated hearts.

1. The concentrations of acetylcholine, choline and noradrenaline were estimated in the perfusate (overflow) of isolated hearts of chickens, cats, rabbits and guinea pigs. Neurotransmitter release was evoked by stimulation of both vagus nerves and by direct stimulation of the heart (field stimulation). 2. In the absence of exogenous choline and physostigmine, field stimulation at 20 Hz for 20 min caused an overflow of acetylcholine from the hearts of the 4 species investigated. During vagal stimulation, however, acetylcholine was detected only in the perfusate of the chicken heart. 3. Field stimulation for 2 min caused an overflow of 193 pmol g−1 min−1 acetylcholine and of 666 pmol g−1 min−…

Small rises in plasma choline reverse the negative arteriovenous difference of brain choline.

The concentrations of free choline in blood plasma from a peripheral artery and from the transverse sinus, in the CSF, and in total brain homogenate, have been measured in untreated rats and in rats after acute intraperitoneal administration of choline chloride. In untreated rats, the arteriovenous difference of brain choline was related to the arterial choline level. At low arterial blood levels (less than 10 microM) as observed under fasting conditions, the arteriovenous difference was negative (about -2 microM), indicating a net release of choline from the brain of about 1.6 nmol/g/min. In rats with spontaneously high arterial blood levels (greater than 15 microM), the arteriovenous diff…