Cytosolic free calcium levels increase with age in rat brain synaptosomes

Calcium homeostasis in synaptosomes is altered during ageing. The cytosolic free calcium concentration, [Ca2+]i was determined in synaptosomes and crude synaptosomal fractions from 3- and 24-month-old rats with the fluorescent indicator quin-2. The [Ca2+]i were around two times higher in 24-month-old rats than in adults, both under resting conditions and after K depolarization. This difference was still observed after incubation with an endogenous heavy metal chelator. To avoid the calcium buffering effect of quin-2, [Ca2+]i values were determined with the use of a null-point method and with fura-2. These methods confirmed the increase in [Ca2+]i with age in synaptosomes. The increase in [Ca2+]i in nerve endings may be pathologically important in brain ageing.     

Histamine H3 receptor inhibition of K+-evoked release of acetylcholine from rat cortex in vivo

Inflammation Research -     

Depolarization of the mitochondrial membrane potential increases free cytosolic calcium in synaptosomes

Intracellular calcium transients in synaptosomes, isolated from the guinea pig brain, were measured using entrapped metallochromic indicator arsenazo III. Addition of 1 microM carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP) increased rapidly the absorbance of the entrapped arsenazo III, indicating an increase in the cytosolic free calcium. The FCCP-induced increase in cytoplasmic free Ca2+ was not blocked by 200 microM verapamil, while the increment in calcium caused by 40 microM veratridine was verapamil-sensitive. The absorbance changes induced by FCCP were not significantly increased when the extracellular potassium concentration was elevated from 5.4 to 50 mM. These data indicate that in nerve endings of mammalian brain, cytoplasmic free calcium, which is essential for the release of transmitter, is increased on depolarization of major intracellular calcium buffers, mitochondria.     

Differential release of [3H]acetylcholine from the rat phrenic nerve-hemidiaphragm preparation by electrical nerve stimulation and by high potassium

Neuronal transmitter stores of the phrenic nerve were labelled under different conditions. Subsequently, transmitter release evoked by electrical nerve stimulation and by a high potassium-low sodium solution was studied. Incubation of the end-plate preparation with [³H]choline at rest led to the synthesis of [³H]acetylcholine which could not be released by electrical nerve stimulation but it was released by high potassium-low sodium solution, independent of the presence of extracellular calcium. When the end-plate preparation was labelled during stimulation at 1 Hz, prolonged periods of electrical nerve stimulation released 83% of the total releasable [³H]transmitter pool in a completely calcium-dependent manner. After exhaustion of the electrically releasable pool, high potassium-low sodium solution still caused a significant outflow. Without a preceding exhaustion of the [³H]acetylcholine pool, high potassium-low sodium solution released a similar amount in the absence of extracellular calcium or after pretreatment with the intracellular calcium chelating substance, Quin-2. When evoked transmitter release was studied at different temperatures (36, 26 and 16°C) Q 10 values of 1.6 and 1.0 were found for the release caused by electrical nerve stimulation and high potassium-low sodium solution (calcium-independent effect), respectively. After labelling during a short interval (2 min) but at a high stimulation rate (50 Hz), only 72% of the releasable [³H]transmitter could be released by electrical nerve stimulation, whereas the outflow due to the calcium-independent effect of high potassium-low sodium solution increased from 17 (labelling during stimulation at 1 Hz) to 28%.It is suggested that the calcium-independent effect of high potassium-low sodium solution reflects the release of acetylcholine from the cytoplasmic compartment, as this outflow occurred after labelling at rest and increased when cytoplasmic synthesis was enhanced by a high loading stimulation. In contrast to high potassium-low sodium solution, propagated nerve activity cannot release acetylcholine synthesized at rest (presumed to be cytoplasmic), but only [³H]acetylcholine synthesized during quantal release (presumed to be vesicular). The absolute requirement of extracellular calcium for electrically stimulated release suggests an exocytotic release mechanism. The low Q 10 value of 1.6 does not fit into the concept of a carrier- or channel-operated release mechanism. High potassium-low sodium solution triggers both calcium-dependent release (exocytosis) and calcium-independent liberation of acetylcholine, which escapes from the cytoplasmic compartment by a diffusion-like mechanism. Accordingly, it is important to consider that propagated nerve activity and high potassium-low sodium solution can cause the release of transmitter by different mechanisms from different compartments.     

The relationship between free cytosolic calcium and amylase release in rat pancreatic acini

We have studied the effects of various pancreatic secretagogues on free cytosolic calcium ([Ca2+]i) and amylase release in dispersed rat pancreatic acini, to determine the role of [Ca2+]i in stimulated enzyme secretion from the exocrine pancreas. Dispersed rat pancreatic acini were loaded with the new Ca2+-sensitive fluorescent indicator, fura-2. Resting [Ca2+]i was 110 +/- 2 nM (a mean +/- S.E.). Carbachol, caerulein, bombesin, and neuromedin B and C each caused a rapid increase in [Ca2+]i; maximal increases of 100 to 400-500 nM were reached within 20s following the secretagogue addition, and this was followed by a return to a lower sustained level within 2 min. When enzyme secretion from the acini was monitored as a function of time using a perifusion system, secretagogue-induced amylase release took a biphasic pattern consisting of an initial burst phase for a several minutes and a second sustained phase during stimulation. Although sustained amylase secretion occurred at near resting [Ca2+]i, the peak [Ca2+]i correlated with the amount of stimulated amylase release as well as with the initial release, during submaximal and maximal stimulation by these agents. At supramaximal concentrations of carbachol and caerulein, amylase release, but not the increase in [Ca2+]i, was attenuated. On the other hand, in response to supramaximal concentrations of bombesin, and neuromedin B and C, both the amount of amylase released and the peak [Ca2+]i were similar to those obtained in response to maximal concentrations. From a standpoint of time course analysis of enzyme secretion, both the first burst phase and the second sustained phase were inhibited during stimulation by 10(-3) M carbachol, compared with 10(-5) M carbachol, while supramaximal stimulation by neuromedin C caused a pattern of amylase release similar to that produced by maximal stimulation. These data suggest that in pancreatic acinar cells an increase in [Ca2+]i plays an important role in stimulus-secretion coupling; however, other factors may be indispensable in regulating enzyme secretion. Furthermore, it is suggested that there is a difference in the intracellular messenger system between carbachol and caerulein, and neurotransmitters belonging to the bombesin family, especially during supramaximal stimulations.     

GABAergic mechanism in histamine H3 receptor inhibition of K+-evoked release of acetylcholine from rat cortex in vivo

Inflammation Research -     

Evaluation of autoreceptor-mediated control of [3H]acetylcholine release in rat and human neocortex

In order to assess the autoinhibitory control of endogenous acetylcholine (ACh) in rat and human neocortex, slices of these tissues were prelabelled with [³H]choline, superfused continuously and stimulated electrically using various frequencies in the presence or absence of drugs. The autoinhibitory feedback control of [³H]ACh release was operative – despite the absence of blockers of ACh esterase – at stimulation frequencies ≥3 Hz in rat and ≥6 Hz in human neocortex tissue. At these frequencies the muscarinic antagonist atropine (0.1 μM) disinhibited the release of [³H]ACh in both species. Estimation of the biophase concentration of ACh near the autoreceptor in the rat neocortex from concentration-response curves of the muscarinic agonist oxotremorine revealed that at 3 Hz about 25% of the autoreceptors were activated by endogenously released ACh. This estimation is consistent with an increase in [³H]ACh release to about 120% of control values by complete blockade of autoreceptors with atropine. The observation that in human neocortical tissue presynaptic autoinhibition of [³H]ACh release is operative at stimulation frequencies ≥6 Hz suggests that selective blockade of autoinhibition may also increase ACh release in the cortex of Alzheimer’s disease patients, without additional blockade of the enzyme acetylcholinesterase. Received: 22 September 1998 / Accepted: 27 April 1999     

Effect of K+-depolarization on the physical state of membrane lipids in rat brain synaptosomes

Laboratory of Biochemistry, Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. N. Kryzhanovskii.) Translated from Byulleten Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 1, pp. 24–27, January, 1989.     

Uptake and release of [3H]GABA by growth cones isolated from neonatal rat brain

A subcellular fraction highly enriched in neuronal growth cones was isolated from 5-day-old rat forebrain by a recently described method. The growth cone fraction was shown to have a sodium- and temperature-dependent, high-affinity (Km = 4.4 microM) uptake system for [3H]GABA. Electron microscopic autoradiography confirmed that this uptake was into growth cones since only these structures were heavily labelled with silver grains. High potassium induced the release of newly accumulated [3H]GABA from the growth cone fraction, about half of which was Ca2+-dependent. The presence of uptake and release systems for GABA in growth cones may simply reflect the development of growth cones into nerve terminals. Alternatively, these observations may indicate a role for neurotransmitter release in synaptogenesis.     

High- and low-affinity binding of [3H]acetylcholine at nicotinic cholinergic receptors in rat brain

There is both high-affinity and low-affinity nicotinic cholinergic binding of [3H]acetylcholine [( 3H]ACh) in rat brain membrane preparations. As determined by a filtration binding assay, [3H]ACh bound with Kd = 36.0 +/- 8.4 nM and Bmax = 19.4 +/- 4.5 fmol/mg protein or 3.3 +/- 0.7 fmol/mg tissue for high-affinity binding and Kd about 10(-7) to 10(-6) M and Bmax about 6-10 fmol/mg tissue or 40-60 fmol/mg protein for low-affinity binding. d-Tubocurarine (1 mM) inhibits high- as well as low-affinity binding, whereas 10 microM alpha-bungarotoxin does not compete at both binding sites. Substance P had no effect on the binding parameters of high-affinity nicotinic cholinergic binding.     

Quantitative autoradiography of nicotinic [H]acetylcholine binding sites in rat brain

Quantitative autoradiography was used to localize nicotinic [³H]acetylcholine (ACh) binding sites in rat brain. High concentrations of nicotinic [³H]ACh binding sites were observed in the anterior and medial nuclei of the thalamus, the medial habenula and the superficial layer of the superior colliculus. Moderate levels of binding sites were observed in a variety of brain regions such as the frontoparietal cortex and the hippocampus. Low levels of nicotinic ACh sites occurred throughout the hypothalamus and the primary olfactory cortex.     

Benzodiazepines decrease the release of [3H]noradrenaline and of [3H]acetylcholine in the cat superior cervical ganglion

The effects of two benzodiazepines, diazepam and clonazepam, were studied on the release of [3H]noradrenaline and of [3H]acetylcholine elicited by preganglionic stimulation of the cat isolated superior cervical ganglion and on the contractile responses evoked by either nerve stimulation or exogenous noradrenaline in the cat isolated nictitating membrane. Both 10 microM diazepam and 10 microM clonazepam reduced by approximately 50% the release of [3H]noradrenaline and of [3H]acetylcholine in the cat ganglion whereas they did not modify the contractile responses in the nictitating membrane. It is concluded that benzodiazepines are also peripheral neuroactive agents and that they exhibit a tissue-selective action within the same animal species.     

Calcium release induced by high K+ and caffeine in cultured skeletal muscle cells of embryonic chicken

To further understand the function of excitation-contraction coupling in skeletal muscle cells developing in vitro, Ca2+ transients elicited by high-K+ depolarization in the presence and absence of extracellular Ca2+ were compared with Ca2+ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K+]O with an elevation of [Ca2+]i. Although all myotubes cultured for more than 4 days exhibited Ca2+ release independent of extracellular Ca2+, only about 50% of E9I1 and E9I2 cells maintained their response to Ca(2+)-free high-[K+]O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K+]O in the absence of extracellular Ca2+. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca2+ release from caffeine-insensitive cells induced by Ca(2+)-free high-[K+]O solution could be blocked by 100-200 microM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca2+]i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.     

Inhibition of the electrically induced release of [3H]dopamine by serotonin from superfused rat striatal slices

The effect of serotonin (5-hydroxytryptamine) on the electrically induced release of [3H]dopamine from superfused slices of the rat striatum has been studied. It was observed that serotonin produced a concentration dependent decrease in the field stimulation-induced release of [3H]dopamine with the threshold concentration being 10(-6) M or lower. Methysergide, in a concentration which did not alter the evoked release, antagonized the inhibitory effect of serotonin. The present results suggest that serotonin should be added to the list of endogenous substances that can influence dopaminergic transmission in the striatum.     

Effect of hyperglycemia on the basal cytosolic free calcium level,calcium signal and tyrosine-phosphorylation in human T-cells

In this study, we investigated the effect of in vitro hyperglycemia on the function of human T-cells (Jurkat cells). Hyperglycemic conditions caused concentration-dependent elevation of basal cytosolic free calcium level and reduced calcium signal (activation capacity), either after ionomycin or monoclonal anti-CD3 antibody treatments. Similar changes were observed if cells were treated with the calcineurin inhibitor Cyclosporin-A. We found that tyrosine-phosphorylation after anti-CD3 treatment was also impaired. High glucose concentrations in the tissue culture medium are also associated with increased non-enzymatic glycation of T-cell proteins. We propose that the increased glycation of proteins involved in calcium transport and/or intracellular signal transduction in T-cells accounts for the abnormal calcium sequestration and calcium mediated signal transduction.