Konrad Löffelholz

A muscarinic inhibition of the noradrenaline release evoked by postganglionic sympathetic nerve stimulation.

Summary1.The noradrenaline output from isolated rabbit hearts perfused with Tyrode solution was estimated fluorimetrically. The postganglionic sympathetic nerves of the heart were stimulated (10 shocks/sec; 1 msec) for three 1 min periods with intervals of 10 min.2.The noradrenaline output evoked by 3 consecutive stimulation periods decreased exponentially.3.Acetylcholine (10−9–10−6 g/ml) administered continuously one min before to one min after the second stimulation caused a dose-dependent reduction of the noradrenaline output evoked by the second stimulation to as low as 19% of the normal value. Acetylcholine in the concentrations applied did not cause a noradrenaline output by itself.4.The inhibitory action of acetylcholine 10−6 g/ml was fully antagonized by atropine 10−6 g/ml, whereas hexamethonium 3×10−6 g/ml had no significant antagonistic effect.5.The noradrenaline output caused by nerve stimulation was not decreased in the presence of DMPP 10−6 g/ml. DMPP 10−5 g/ml applied 3 min before electrical nerve stimulation caused an output of noradrenaline for 2 min but did not inhibit the noradrenaline release by nerve stimulation.6.Tyramine 5×10−6 g/ml was administered to the rabbit heart for two 6 min periods at an interval of 15 min. Methacholine 7.4×10−5 g/ml or atropine 1−6 g/ml if present during the second tyramine infusion did not alter the noradrenaline output produced by tyramine.7.It is concluded that low concentrations of acetylcholine by stimulating muscarinic inhibitory receptors interfere with the noradrenaline release from the postganglionic sympathetic nerve fibres evoked by electrical nerve stimulation. The possibility of a peripheral direct interaction of the cholinergic with the adrenergic nervous system is discussed.

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 rat. The levels of free choline in blood plasma (11.4 microM), CSF (6.7 microM) and brain intracellular space (64.0 microM) were sufficiently similar to be compatible with an exchange of choline between these compartments. In contrast, the intracellular levels of glycerophosphocholine (1.15 mM) and phosphocholine (0.59 mM) in the brain were considerably higher than their CSF concentrations of 2.83 and 1.70 microM, respectively. In blood plasma, glycerophosphocholine was present in a concentration of 4.58 microM while phosphocholine levels were very low or absent (< 0.1 microM). The levels of phosphatidylcholine and lyso-phosphatidylcholine were high in blood plasma (1267 and 268 microM) but very low in cerebrospinal fluid (< 10 microM). We concluded that the transport of free choline is the only likely mechanism which contributes to the supply of choline to the brain under physiological conditions.

Role of Phospholipase D Activation in Nervous System Physiology and Pathophysiology

The signal-dependent activation of phospholipase D (PLD) has been observed in a wide variety of brain and neural-derived cells, including primary neurons and glial cells, as well as neuroblastoma, glioma, astrocytoma, and pheochromocytoma cell lines (summarized in Table 1) . Several established and putative neurotransmitters and neuromodulators and their analogues were found to activate PLD, including muscarinic cholinergic agonists, P2-purinergic agonists, histamine, endothelin, bradykinin, glutamate, insulin, and the p-opioid agonist morphiceptin . Therefore, the activation of PLD by cell surface receptor ligands is a general phenomenon . The rapid and dramatic nature of this phenomenon indicates that it may act as an intracellular signal transduction pathway . The identification of PLD activation as a novel signal transduction pathway engenders a special interest in the function of PLD in the nervous system. Brain PLD has been studied at all levels, from isolated subcellular organelles and membranes to cell cultures, brain slices, and intact brains . The present review attempts to provide an overview of these studies and to outline some possible roles of PLD and its various products in nervous system physiology and pathophysiology .

Inhibition by parasympathetic nerve stimulation of the release of the adrenergic transmitter

SummaryIsolated rabbit atria were perfused with Tyrode solution containing (+)-amphetamine. Electrical stimulation of the right postganglionic sympathetic fibres caused an output of noradrenaline which was significantly decreased by simultaneous stimulation of the vagus nerves.

Uptake and Metabolism of Choline by Rat Brain After Acute Choline Administration

Abstract: 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 μM 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 >23 μM. Net choline release and uptake were linearly dependent on the plasma choline level in the physiological range of 10‐50 μM, whereas the CSF choline level was significantly increased only at plasma choline levels of >50 μM. The bolus injection of 60 mg/kg of [3H]choline chloride caused the net uptake of > 500 μMol/g of choline by the brain as calculated from the AVD, which was reflected in a minor increase of free choline level and a long‐lasting increase of brain phosphorylcholine content, which paralleled the uptake curve. Loss of label from phosphorylcholine 30 min to 24 h after choline administration was accompanied by an increase of label in phosphatidylcholine, an indication of a delayed transfer of newly taken‐up choline into membrane choline pools. In conclusion, homeostasis of brain choline is maintained by a complex system that interrelates choline net movements into and out of the brain and choline incorporation into and release from phospholipids.

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 choline were combined. Basal hippocampal acetylcholine release in mice habituated to their environment was not affected by administration of glucose and choline. However, when hippocampal acetylcholine release was stimulated either by infusion of scopolamine (0.3microM) or by transferring the mice into a novel environment, the combination of glucose plus choline further increased acetylcholine release to a significant extent. We conclude that low doses of glucose and choline act synergistically to improve memory storage, an effect which is due to facilitation of acetylcholine release. This finding reinforces the view that central cholinergic functions are influenced under certain conditions by dietary intake of precursors.

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 containing approximately 3% of bilobalide. We conclude that ginkgo extracts can suppress hypoxia-induced membrane breakdown in the brain, and that bilobalide is the active constituent for this effect.

Autoinhibition of nicotinic release of noradrenaline from postganglionic sympathetic nerves.

Summary1.The effects of nicotine, DMPP (1,1-dimethylphenylpiperazine) and acetylcholine (plus atropine) on the isolated rabbit heart were investigated. Heart rate, amplitude of contraction, coronary flow and output of noradrenaline into the perfusate were recorded. Noradrenaline was estimated fluorimetrically.2.All nicotinic drugs evoked a dose-dependent output of noradrenaline and increased the rate and the amplitude of contraction. Increases of heart rate in response to nicotine and DMPP and increases of amplitude of contraction in response to all nicotinic drugs were clearly related to the output of noradrenaline.3.The dose-response curves of the noradrenaline output evoked by nicotine, DMPP and acetylcholine (plus atropine) were steeply rising and parallel. The concentrations of DMPP, nicotine and acetylcholine (plus atropine) needed to cause a half-maximal noradrenaline output were in the proportions 1∶ 1.1∶ 5.2 respectively.4.During infusion of nicotinic drugs the noradrenaline output rose to its maximum within 30 sec and then declined exponentially. The rate constant of the decline was not significantly different from the rate constant of the slow phase of washout of exogenously administered noradrenaline.5.Atropine (which facilitates) and hexamethonium (which blocks the noradrenaline output evoked by acetylcholine) were effective only in the first 5–10 sec of the infusion of acetylcholine.6.The threshold concentrations for nicotine or acetylcholine (plus atropine) required to cause a noradrenaline output on the one hand, and to cause autoinhibition on the other hand, were identical.7.It is concluded that nicotinic drugs cause an “explosive” release of noradrenaline from the adrenergic terminal fibres into the extraneuronal space. The rapid termination of this release is caused by sudden autoinhibition.

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 10−8−10−3m 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 formation. However, when the mice were anaesthetized with halothane (0.8 vol. %), hippocampal ACh reached significantly lower levels in AChE‐Tg mice as compared with controls. Also, the high‐affinity choline uptake (HACU) in hippocampal synaptosomes from awake hAChE‐Tg mice was accelerated but was reduced by halothane anaesthesia. Moreover, hAChE‐Tg mice displayed increased motor activity in novel but not in familiar environment and presented reduced anxiety in the elevated plus‐maze test. Systemic application of a low dose of physostigmine (100 µg/kg i.p.) normalized all of the enhanced parameters in hAChE‐Tg mice: spontaneous motor activity, hippocampal ACh efflux and hippocampal HACU, attributing these parameters to the hypocholinergic state due to excessive AChE activity. We conclude that, in hAChE‐Tg mice, hippocampal ACh release is up‐regulated in response to external stimuli thereby facilitating cholinergic neurotransmission. Such compensatory phenomena most likely play important roles in counteracting functional deficits in mammals with central cholinergic dysfunctions.

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

Abstract: 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 (<10 μM) as observed under fasting conditions, the arteriovenous difference was negative (about ‐2 μM), indicating a net release of choline from the brain of about 1.6 nmol/g/min. In rats with spontaneously high arterial blood levels (> 15 μM), the arteriovenous difference was positive, implying a marked net uptake of choline by the brain (3.1 nmol/g/min). The CSF choline concentration, which reflects changes in the extracellular choline concentration, also increased with increasing plasma levels and closely paralleled the gradually rising net uptake. Acute administration of 6, 20, or 60 mg of choline chloride/kg caused, in a dose‐dependent manner, a sharp rise of the arterial blood levels and the CSF choline, and reversed the arteriovenous difference of choline to markedly positive values. The total free choline in the brain rose only initially and to a quantitatively negligible extent. Thus, the amount of choline taken up by the brain within 30 min was stored almost completely in a metabolized form and was sufficient to sustain the release of choline from the brain as long as the plasma level remained low. We conclude that the extracellular choline concentration of the brain closely parallels fluctuations in the plasma level of choline. Moreover, the often described release of choline from the brain as reflected by the negative arteriovenous difference of brain choline is not a steady‐state phenomenon. Instead, the uptake of choline into and the release of choline from the brain seem to be in dynamic equilibrium that is closely related to the plasma choline level and, consequently, to nutritional choline uptake.