Calcium binding activity by chick intestinal brush-border membrane vesicles

Uptake of Ca²⁺ by vesicle preparation of chick intestinal brush-border membranes was rapid and extensive. With tracer quantities of Ca²⁺ uptake was complete in 10 min whereas with 2.0 mM Ca²⁺ maximum uptake by the vesicles occurred after one hour incubation. The maximum concentration of Ca²⁺ found in the vesicles was four times greater than the external Ca²⁺ concentration showing that the majority of the Ca²⁺ was membrane bound.The Ca²⁺ taken up by the vesicles was probably bound to the vesicle's interior since it was not replaced by exposure of loaded vesicles to La³⁺ (5 mM). The uptake of Ca²⁺ by the vesicles at different Ca²⁺ concentrations was analyzed and a high affinity Ca²⁺ binding site was found with an association constant for Ca²⁺ of 5×10^–5 M. More of these sites were found in the duodenum than the ileum and vitamin D increases the number of these sites.     

Release of [3H]norepinephrine from synaptic vesicles isolated from rat brain after the intracisternal administration of [3H]norepinephrine: influence of nucleotides, ions and drugs, and destabilization of transmitter storage caused by acute or chronic lithium administration

Following the intracisternal administration of [³H]norepinephrine to rats pretreated with a monoamine oxidase inhibitor, synaptic vesicles containing the radio label could be isolated from isotonically prepared microsomal fractions of rat brain. Incorporation of [³H]norepinephrine into the vesicles was reduced by pretreatment of the rats with desmethylimipramine and was also reduced if the rats had not been pretreated with a monoamine oxidase inhibitor. Incorporation of the label was totally eliminated by pretreatment with reserpine. Release in vitro of [³H]norepinephrine from the labeled vesicles was monophasic with a half-time of about 12 min at 30°C. The release was slowed by addition of adenosine 5′-triphosphate plus Mg²⁺ by a mechanism different from that of the vesicular amine uptake system; this was shown by the failure of inhibitors of adenosine triphosphate-Mg²⁺-stimulated uptake (reserpine,N-ethylmaleimide, lithium) to block the effect of adenosine triphosphate plus Mg²⁺ on release. Several other nucleotides also were able to slow the release of [³H]norepinephrine. Unlike adrenomedullary vesicles, rat brain synaptic vesicles did not show enhancement of amine release by chloride in the presence or absence of adenosine triphosphate plus Mg²⁺. The yield of labeled vesicles was substantially reduced if vesicles were prepared by hypotonic lysis of synaptosomes instead of isotonically from the microsomal fraction; the isotonic preparation appears to be superior for studies of vesicle uptake and storage properties.This preparation is readily utilizable for studies of the effects of in vivo administration of drugs thought to act on vesicular storage of catecholamines, a point illustrated by the destabilization of norepinephrine storage caused by acute or chronic lithium administration.     

Effects of Mg2+ and Ca2+ on Noradrenaline Release and Uptake in Adrenergic Nerve Granules in Different Media

Euler , U. S. V. and F. Lishajko . Effects of Mg²⁺ and Ca²⁺ on noradrenaline release and uptake in adrenergic nerve granules in different media. Acta physiol. scand. 1973. 89. 415–422. Uptake and release of noradrenaline (NA) from a suspension of storage granules from bovine splenic nerves was measured after incubation in various media and effects of added Mg²⁺ and Ca²⁺ observed. No significant differences in NA release rate were observed in different media when NA uptake was prevented by addition of potassium ferricyanide to the medium. Uptake was facilitated by phosphate ions 5–30 mM but counteracted by monovalent cations. In sucrose-phosphate media uptake of NA almost balanced spontaneous release. Mg²⁺ and Ca²⁺ had only a small action on NA release in the absence of NA uptake. In sucrose phosphate media Mg²⁺ facilitated uptake, whereas Ca²⁺ enhanced NA release. This action was associated with the formation of a calcium phosphate precipitate. No release or uptake of NA was observed during incubation at ice-water temperature, nor did Mg²⁺ or Ca²⁺ have any effect. The results tend to show that release and uptake of NA in storage granules are separate, temperature-dependent processes, influenced by mono- and divalent cations and phosphate ions.     

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim: We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes. Methods: Time‐lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. Results: At rest, when mobility was monitored for 2 min in bath with Ca²⁺, two vesicle populations were discovered: (1) non‐mobile vesicles (6.1%) with total track length (TL) < 1 μm, averaging at 0.33 ± 0.01 μm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 μm, averaging at 3.03 ± 0.01 μm (n = 20 200). ALS IgG (0.1 mg mL^?1) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca²⁺, indicating that ALS IgG vesicle mobility enhancement involves changes in Ca²⁺ homeostasis. To examine whether enhanced mobility relates to elevated Ca²⁺ activity, cells were stimulated by 1 mm ATP, a cytosolic Ca²⁺ increasing agent, in the presence (2 mm ) and in the absence of extracellular Ca²⁺. ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo. Conclusion: Amyotrophic lateral sclerosis‐IgG‐enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.     

Unidirectional Fluxes in Isolated Splenic Nerve Vesicles Measured by a Millipore Filter Technique:

Isotopically labelled noradrenaline (NA) exchange was determined in an improved preparation of NA storage vesicles isolated from bovine splenic nerve trunk. A Millipore filter technique was used which permitted analyses of unidirectional fluxes. In a Mg-ATP supplemented medium at 30 and 20° C, vesicle NA is completely exchangeable with 0.5 to 3.0 μg l-NA/ml in the medium. The kinetics are compatible with a single exponential component of exchange, and the data suggest a fully saturated Mg-ATP complexing under these conditions. Reserpine in a concentration range between 2 × 10^-8 and 2 × 10^-7 M causes more pronounced inhibition of the transfer coefficient for NA influx than for efflux. However, no net loss of vesicle NA occurs because the drug inactivates a proportional amount of the readily exchangeable NA pool. Within the concentration ranges of NA and reserpine tested, virtually complete inhibition of NA exchange can be achieved and reversal of this inhibition by elevating the NA concentration in the medium can be demonstrated. The data is consistent with the suggestion that reserpine acts via transport sites in the vesicle membrane, rather than on the intravesicular NA complex per se. This membrane stabilizing concentration range of reserpine causes no obvious deleterious effects on the vesicles at the electron microscopic level.     

Noradrenaline content correlated to matrix density in small noradrenergic vesicles from rat seminal ducts

The relation between matrix density and noradrenaline content has been investigated in a fraction of small noradrenergic storage vesicles obtained from seminal ducts of castrated rats by density gradient centrifugation. This preparation contained numerous dense-cored small vesicles with an increased diameter but with a similar appearance to those in nerve terminals in the intact vas deferens.After incubation of the vesicle fraction for 20 min at 30°C in the presence of noradrenaline, Mg²⁺ and adenosine 5'-triphosphate, about 50–60% of the vesicles with a diameter of 40–70 nm contained dense cores, in some regions of the sediment even up to 80%. This correlated well with a high level of sedimentable noradrenaline, 150% of control.Incubation of vesicles without exogenous noradrenaline in the presence of Mg²⁺ and adenosine 5'-triphosphate yielded mostly electron-lucent vesicles, and biochemically only 40% of the control level of noradrenaline was retained in the particulate fraction after centrifugation. A similar depletion of electron dense-core material was observed after incubation with tyramine, Mg²⁺ and adenosine 5'-triphosphate, where only 55% of the sedimentable noradrenaline as compared to control was retained. In this respect the small dense-cored vesicles differed markedly from the large dense-cored vesicles purified from bovine splenic nerve, where the dense cores were retained in spite of a 70–80% depletion of noradrenaline after incubation in the presence of tyramine.Incubation with 5-hydroxydopamine did not significantly increase the electron density of the small dense-cored vesicles, in contrast to the marked effect of this agent when administered in vivo. Incubation with reserpine and atractyloside did not affect either noradrenaline release or vesicle morphology significantly, whereas treatment with N-ethylmaleimide gave a 60% decrease in particulate noradrenaline content but only a moderate reduction in electron density.The results indicate that both noradrenaline and, to a lesser extent, other matrix material in the presence of Mg²⁺ and adenosine 5′-triphosphate are capable of forming electron-dense cores, and that noradrenaline is more important for dense-core formation in small noradrenergic vesicles than it is in the large vesicles.     

Calcium binding activity by chick intestinal brush-border membrane vesicles

Uptake of Ca²⁺ by vesicle preparation of chick intestinal brush-border membranes was rapid and extensive. With tracer quantities of Ca²⁺ uptake was complete in 10 min whereas with 2.0 mM Ca²⁺ maximum uptake by the vesicles occurred after one hour incubation. The maximum concentration of Ca²⁺ found in the vesicles was four times greater than the external Ca²⁺ concentration showing that the majority of the Ca²⁺ was membrane bound. The Ca²⁺ taken up by the vesicles was probably bound to the vesicle's interior since it was not replaced by exposure of loaded vesicles to La³⁺ (5 mM). The uptake of Ca²⁺ by the vesicles at different Ca²⁺ concentrations was analyzed and a high affinity Ca²⁺ binding site was found with an association constant for Ca²⁺ of 5×10^?5 M. More of these sites were found in the duodenum than the ileum and vitamin D increases the number of these sites.     

The relationship of transmitter release and storage to fine structure in a sympathetic ganglion

Summary Cat sympathetic ganglia were prepared for electron microscopy by perfusion fixation with glutaraldehyde in the presence of Mg⁺⁺. At resting boutons de passage the populations of synaptic vesicles were 6400 per bouton. The vesicle distributions displayed many of the features of spheres in close-packing. Calculations based on a vesicle close-packing hypothesis gave a figure of 8000 vesicles per bouton. In ganglia stimulated for 20 min at 20/s the vesicle populations were reduced to 25%, and to 28.5% in ganglia in which acetylcholine (ACh) synthesis was inhibited by hemicholinium (HC-3). The reduction was to 34% when stimulation was for 1 min. In ganglia stimulated for 20 min at 1/s and 4/s the vesicle populations were reduced to 44% and 46% respectively. Even following 1 min stimulation at 4/s over half the boutons showed significant loss of vesicles. ACh stores in ganglia are known not to be depleted by any of these procedures except stimulation in the presence of HC-3. The fraction of ganglionic ACh stores known to be released by stimulation for 1 min at 20/s or 4/s and by 20 min stimulation at 1/s is substantially less than the fraction of vesicles lost. The observations therefore were not readily accounted for by the vesicle theory of transmitter storage and release. They were consistent with the idea the ACh is stored in vesicles at rest, but that during maintained activity over half the bouton ACh is free in the cytoplasm. The concentration of cytoplasmic ACh was calculated to be approximately 50–150 mm 1^–1. Examination of the hypothesis that ACh may be released from this cytoplasmic pool during synaptic activation indicated an efflux of approximately 1.5–3.0 × 10^–12 M Ach cm^–2 synapsing membrane/impulse.Medical Research Associate of the Medical Research Council of Canada.     

Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum

 Ruthenium red inhibits mitochondrial Ca²⁺ uptake and is widely used as an inhibitor of ryanodine-sensitive Ca²⁺ channels that function to release Ca²⁺ from the sarcoplasmic reticulum (SR) of muscle cells. It also has effects on other Ca²⁺ channels and ion transporters. To study the effects of ruthenium red on Ca²⁺ transport into the SR of cardiac muscle cells, fluorescence measurements of Ca²⁺ uptake into cardiac SR vesicles were made. Ruthenium red significantly decreased the Ca²⁺ sensitivity of SR uptake in a dose-dependent manner at concentrations ranging from 5 μM to 20 μM. There were no significant effects of ruthenium red on the maximum velocity or the Hill coefficient of SR Ca²⁺ uptake. Received: 14 January 1998 / Received after revision: 12 March 1998 / Accepted: 16 March 1998     

α2-Aclrenoceptor agonist-mediated inhibition of [3H]noradrenaline release from rat hippocampus is reduced by 4-aminopyridine,but that caused by an adenosine analogue or co-conotoxin is not

The inhibitory effect of an adenosine analogue, R-PIA, and an α₂-adrenoceptor agonist, UK 14,304, on [³H]NA efflux from field-stimulated rat hippocampal slices was examined. The effect of 0.1 μm UK 14,304 was mimicked by 30 nM w-conotoxin and by 10 μM cadmium chloride, inhibitors of N- and L-type Ca²⁺ channels. R-PIA (1 μM) had no effect per se, but caused a clear-cut inhibition after blockade of the pre-synaptic α₂-receptor by yohimbine. 4-Aminopyridine (4-AP) caused a dose-dependent increase in evoked transmitter release. At 30 μM 4-AP did not affect the actions of ω-conotoxin or cadmium chloride. The pre-synaptic effect of R-PIA was similarly unaffected by 30 μM 4-AP. The presynaptic effect of UK 14,304 was virtually abolished by 4-AP (30 μM). The effect of UK 14,304 (0.1 μM) could be partly restored by reducing the Ca²⁺ concentration during treatment with 4-AP (22% inhibition compared to 42% with normal Ca²⁺). The magnitude of increase in evoked [³H]NA efflux by yohimbine (1 μM) was decreased by 4-AP in a concentration-dependent manner from 142% increase in controls to 21 % at 100 μM 4-AP. The present results indicate that NA release is reduced by somewhat different mechanisms by pre-synaptic α₂- and adenosine Aj-receptors. Furthermore, the results indicate that pre-synaptic A_rreceptors on hippocampal NA neurons do not primarily regulate 4-AP-dependent potassium channels, but they might act directly on a Ca²⁺ conductance.     

Choline uptake, acetylcholine synthesis and release by Limulus abdominal ganglia

Isolated ganglia from the ventral nerve cord of the horseshoe crab, Limulus polyphemus, were incubated in [³H]choline and subsequently analyzed for choline uptake, conversion of choline to acetylcholine and the release of the newly synthesized acetylcholine. The ganglia readily accumulated radioactivity when incubated in Chao's solution containing 2 μM [³H]choline. The rate of uptake was 0.08 pmols/min/mg tissue and 26% of the [³H]choline taken up during a 1 h period was converted to [³H]acetylcholine. A 15 min exposure of ganglia to 90 mM K⁺ prior to incubation in [³H]choline caused an 89% increase in choline uptake and a 150% increase in its conversion to [³H]acetylcholine. The presence of unlabeled acetylcholine in the uptake medium inhibited both uptake and conversion of [³H]choline.There was a 5-fold increase in the efflux of radioactivity when ganglia incubated in 2 μM [³H]choline were superperfused with 90 mM K⁺. The increased efflux of radioactivity was Ca²⁺-dependent and was inhibited by Mg²⁺ (44%) and by Co²⁺ (72%). Similarly, addition of veratridine caused a Ca²⁺ and Na⁺-dependent release of radioactivity from prelabeled ganglia. Analysis of the superperfusate revealed that virtually all of the released radioactivity was [³H]acetylcholine.The abdominal ganglia of Limulus take up choline at micromolar concentrations, convert substantial amounts of it to acetylcholine and possess a depolarization-triggered, Ca²⁺-requiring mechanism for the specific release of acetylcholine. These results give further support to the view that the abdominal ganglia of Limulus contain a population of cholinergic terminals.     

Tetrodotoxin-resistant release of3H-noradrenaline from the mouse vas deferens by high intensity electrical stimulation

Vasa deferentia of mice were preincubated with ³H-noradrenaline and then superfused with a medium containing cocaine 10 μM and phentolamine 30 μM. The tetrodotoxin-resistant outflow of tritium evoked by high intensity electrical field stimulation (0.5 Hz, 200 mA current strength, 2 ms pulse width) was studied and, in some experiments, compared with the tetrodotoxin-sensitive outflow evoked by low intensity electrical field stimulation (0.5 Hz, 50 mA, 1 ms). In contrast to the outflow evoked by low intensity stimulation, the outflow evoked by high intensity stimulation was increased in Na⁺-free medium, and was only partly dependent on the external Ca²⁺ concentration. The Ca²⁺-dependent fraction consisted mainly of ³H-noradrenaline. Again, in contrast to the outflow caused by stimulation at low intensity, that caused by stimulation at high intensity was not reduced by Mg²⁺ 20 mM, Co²⁺ 5 mM or normorphine 40 or 100 μM, and was not enhanced by tetraethylammonium 5 mM or 4-aminopyridine 1 mM. It is concluded that high intensity electrical stimulation elicits a tetrodotoxin-resistant, calciumdependent release of noradrenaline which differs in mechanism from the release elicited by action potentials.     

Chloride transport in human placental microvillus membrane vesicles

³⁶Cl uptake by microvillus brush border membrane vesicles prepared from normal term human placenta has been studied using an ion exchange column assay and a novel method to correct for vesicle recovery. Uptake of this anion, which was largely into an osmotically active space, was time dependent with a half-time of approximately 4 min at 4°C. DIDS at a maximal inhibitory concentration inhibited³⁶Cl uptake by approximately 40%; furosemide (10^–4 M) showed a similar degree of inhibition and the effects of the two drugs appeared not to be additive. Anions including Cl, Br, I, NO₃, salicylate and SCN reduced³⁶Cl uptake, the latter two being the most potent; gluconate was without effect. Inhibition by maximal inhibitory concentrations of DIDS and the anions SCN and salicylate was not additive. Neither the DIDS-sensitive nor the SCN-sensitive component of³⁶Cl uptake was influenced by membrane potential; in contrast the inhibitor-insensitive component was increased by an inside positive membrane potential. These findings suggest that under the conditions employed an electroneutral anion exchange system is responsible for approximately half of the chloride flux across this plasma membrane.     

Adenosine Triphosphate Dependent Calcium Uptake by Subcellular Fractions from Bovine Neurohypophyses

Bovine neurohypophyses were fractionated by differential and density gradient ultracentrifugation and the Ca²⁺ uptake and ATPase activities in the microsomal, mitochondrial and secretory granule fractions were studied. The microsomal and mitochondrial fractions accumulated Ca²⁺ in the presence of ATP. The accumulation by the latter per mg protein was at least twice as large as by the former. This Ca²⁺ accumulation was accompanied by liberation of inorganic phosphate (P_i). In the presence of sodium azide (2 mM) Ca²⁺ uptake and P_i liberation were inhibited in the mitochondrial, but not in the microsomal fraction. Further studies of the microsomal fraction revealed that the ATP-dependent Ca²⁺ uptake and P_i liberation activities were temperature and pH-dependent and required Mg²⁺. Both activities were stimulated by very low concentrations of Ca²⁺ (1–10 μM) and were inhibited by EGTA (2 mM). N-ethyl-maleimide (2 mM) inhibited both the Ca²⁺ uptake and ATPase activities of the microsomal fraction. These results suggest the presence of a membrane ATPase that is stimulated by both Ca²⁺ and Mg²⁺. It is suggested that the observed Ca²⁺ uptake activities are involved in maintaining a low axoplasmic free Ca²⁺ concentration, thus playing an important role in the release mechanism of vasopressin by the neuro-secretory terminals.     

Release of 3H-noradrenaline from the rat vas deferens under various in vitro conditions

The release of ³H-(-)-noradrenaline (NA) from rat vas deferens in vitro was examined under various experimental conditions. It was found that in normal and reserpinized vas deferens the release of NA evoked by (+)-amphetamine (5 times 10^?6 M) or low external Na⁺ (26 mM) was antagonized by imipramine methiodide and desipramine, inhibitors of the NA uptake, but was not dependent on the presence of Ca²⁺ in the medium and was not antagonized by the potent local anaesthetic agent bethoxycaine. The release evoked by veratridine in reserpinized tissue was antagonized by the uptake inhibitors but was in normal tissue only partially inhibited in presence of Ca²⁺ but almost completely in absence of Ca²⁺. The release by high K⁺ (117 mM) + low Na⁺ (26 mM) in normal tissue was dependent on the presence of Ca²⁺ and was antagonized by the muscarinic agonists carbacholine and metacholine and by high concentrations of desipramine. In the reserpinized vasa the corresponding release was not dependent on Ca²⁺ and was not antagonized by the muscarinic agents but was inhibited by high concentrations of desipramine.     

Spatial organization and dynamic properties of neurotransmitter release sites in the enteric nervous system

Synaptic communication requires an efficient coupling of vesicle fusion to release neurotransmitter and vesicle retrieval to repopulate the synapse. In synapses of the CNS many proteins involved in exocytosis, endocytosis and refilling of vesicles have been identified. However, little is known about the organization and functioning of synaptic contacts in the enteric nervous system (ENS). We used fluorescent antibodies against presynaptic proteins (synaptobrevin, synaptophysin, synaptotagmin and bassoon) to identify synaptic contacts not only in guinea-pig enteric ganglia but also in the interconnecting fiber strands. Staining patterns were not altered by colchicine (100 μM), ruling out a contribution of protein transport at the time of fixation. Active release sites at fiber intersections and around neuronal cell bodies were labeled with FM1-43 (10 μM) by high K⁺ or electric field stimulation (EFS). During a second round of EFS, vesicles were reused, as reflected by dye loss. Destaining rates increased with stimulus frequency (2–30 Hz), reaching a maximum at about 15 Hz, likely caused by synaptic depression at higher frequencies. Tetrodotoxin (TTX, 1 μM) as well as nominally zero external Ca²⁺ (2 mM EGTA) prevented all destaining. The readily releasable pool (RRP, a subset of vesicles docked at the membrane and ready to fuse upon [Ca²⁺]_i increase) can be specifically released by a hypertonic challenge (500 mM sucrose). We measured this pool to be ∼27% of the total recycling pool, remarkably similar to synapses in the CNS. In whole-mount preparations, FM1-43 also reliably labeled active release sites in ganglia, fiber strands and in muscle bundles. The staining pattern indicated that the presynaptic antibodies mainly labeled active sites. The presence of numerous release sites suggests information processing capability within interconnecting fibers. With FM imaging, enteric synaptic function can be monitored independent of any postsynaptic modulation. Although electron microscopy data suggest that ENS synapses may not be as specialized as hippocampal synapses, remarkably similar release properties were measured.     

Block of synaptic vesicle exocytosis without block of ca2+-influx. An ultrastructural analysis of the paralysing action of habrobracon venom on locust motor nerve terminals

The venom of the wasp Habrobracon hebetor presynaptically blocks excitatory but not inhibitory neuromuscular transmission at locust skeletal muscle. Its mode of action on excitatory motor nerve terminals has been studied at the retractor unguis muscle of Schistocerca by means of ultrastructural stereology. Paralysed and unparalysed preparations, either resting or stimulated for 7 min at 20 Hz, were compared. Paralysis does not cause structural damage to the nerve terminals but prevents the depletion of vesicles occurring upon nerve stimulation in the controls. Prolonged paralysis leads to an increase in the number and the size of vesicles resulting in an increase of total membrane per terminal cross-section by about 33% after 2 days. Stimulation causes swelling of mitochondria both in controls and in paralysed preparations, resulting from a rise of intraterminal [Ca²⁺] as is indicated by the absence of the swelling if extracellular Ca²⁺ is replaced by Mg²⁺. In addition, stimulation leads to a reduction of vesicle size, an increase in the area of axolemma and in the number of cisternae and of profiles of the smooth endoplasmic reticulum in controls but not in paralysed preparations. However, neither in controls nor in paralysed preparations is the total amount of membrane per teminal cross-section affected by stimulation. Under paralysis, vesicles tend to stick to the presynaptic membrane.It is concluded that Habrobracon venom does not block the depolarization-dependent Ca²⁺-influx into the nerve terminal and that it is unlikely to interfere with some transmitter-related process. Rather, the venom seems to block vesicle exocytosis itself. The results lend further support to the view that in insect neuromuscular synapses exocytosis is the mechanism whereby transmitter quanta are released.     

Calcium-antagonists and islet function

Summary The modality of Ba²⁺-induced insulin release was investigated in the isolated perfused rat pancreas. The insulinotropic action of Ba²⁺ was antagonized by Ca²⁺, Mg²⁺ and verapamil, and enhanced by EGTA, theophylline, glucose and cytochalasin B. Likewise the net uptake of¹³³Ba²⁺ by isolated islets was inhibited by Ca²⁺, Mg²⁺ and verapamil. Glucose increased¹³³Ba²⁺ net uptake, but only when sufficient Ba²⁺ had accumulated in the islets. Theophylline failed to affect¹³³Ba²⁺ net uptake. These data suggest that (i) Ba²⁺-induced insulin release is dependent on the accumulation of this cation in the B-cell; (ii) Ba²⁺ inward transport in the B-cell occurs through a verapamil-sensitive channel characterized by competition between Ba²⁺, Ca²⁺ and Mg²⁺; and (iii) the enhancing effect of theophylline upon insulin release could be due to an intracellular translocation of alkaline-earth cations rather than to an increase in their net uptake. The present findings also support the idea that insulin release can be triggered by the accumulation of suitable divalent cations in a critical site of the B-cell, leading to the activation of a cytochalasin B-responsive effector system.     

Effects of magnesium on45Ca uptake and release at different sites in rabbit aortic smooth muscle

Effects of 1.5 mM Mg²⁺ on muscle tension and on⁴⁵Ca uptake and release at different sites in the rabbit aortic media-intimal layer were investigated. The sustained contraction induced by either 10^?6 M norepinephrine (NE) or 60 mM K⁺ was not affected by 1.5 mM Mg²⁺ in the presence of 1.5 mM Ca²⁺. However, the contractions elicited with NE or K⁺ in 0.03 mM Ca²⁺-containing solution were inhibited by 1.5 mM Mg²⁺ by 67% and 27%, respectively. Total⁴⁵Ca uptake measured in the presence of either 1.5 mM or 0.03 mM Ca²⁺ was not affected by 1.5 mM Mg²⁺. The rate of residual⁴⁵Ca uptake (⁴⁵Ca uptake followed by a wash in La³⁺-containing solution at low temperature) measured in the presence of 1.5 mM Ca²⁺ was slightly lower in the presence of 1.5 mM Mg²⁺. However, the increase in rate of residual⁴⁵Ca uptake induced by NE or the net increase in the residual⁴⁵Ca uptake induced by K⁺ was not decreased by 1.5 mM Mg²⁺. The residual⁴⁵Ca uptake measured in the presence of 0.03 mM Ca²⁺ was reduced to 64% and 24% of controls by addition of 1.5 mM Mg²⁺ or Sr²⁺, respectively. A part of the residual⁴⁵Ca was released by NE. Uptake of⁴⁵Ca at this NE-affected Ca²⁺ site did not take place in the presence of 1.5 mM Mg²⁺ when the Ca²⁺ concentration of the medium was 0.03 mM. However, this⁴⁵Ca uptake component was only partially inhibited when the Ca²⁺ concentration of the medium was 1.5 mM. The NE-induced increase in⁴⁵Ca efflux was not inhibited by 1.5 mM Mg²⁺. From these results, Mg²⁺ appears to be a weak antagonist for both Ca²⁺ entry into the vascular smooth muscle cell and Ca²⁺ binding at a high affinity intracellular site.     

Ethanol inhibits NMDA-induced toxicity and trophism in cultured cerebellar granule cells

In cerebellar granuleCell cultures, glutamate and N-methyl-D-aspartate (NMDA) caused either neurotoxic or trophic effects, depending on the developmental stage of the neurones. Ethanol (100 mM) partly inhibited delayed neurotoxicity induced by the excitatory amino acids (25μM glutamate for 15 min or 100/tM NMDA for 30 min) assessed 24 h after the incubations in mature cultures in the absence of Mg^2t. Glycine (5 μM) potentiated the toxicity of glutamate and the ethanol inhibition, and was routinely added in these experiments. The viability of neurones in the presence of 25 mM K⁺ and 0.8 mM Mg^2t was not impaired when maintained in 40–50 mM ethanol for the whole culture period of 7 days. However, ethanol almost completely inhibited the trophic effects of NMDA on developing cultures in 12.5 mM Kμ0.8 mM Mg²⁺ medium. Glutamate (25 μM) and NMDA (100μM) potently induced ⁴⁵Ca²⁺ uptake by granule cells from day 2 in vitro onward. Sixty-five per cent of the 15-min ⁴⁵Ca^2f influx induced by glutamate and 80% of that induced by NMDA were inhibited by ethanol (100 mM). MK-801 (a non-competitive antagonist of NMDA receptors; 100 nM) completely inhibited the toxic and trophic actions of glutamate and NMDA, as well as the ⁴⁵Ca²⁺ influx induced by NMDA, but only 80% of the ⁴⁵Ca^2f influx induced by glutamate. These results show that the toxic and trophic actions of glutamate are mediated mainly by Ca²⁺ influx through NMDA receptors. Both of these actions and the underlying Ca²⁺ influx are significantly inhibited by ethanol at pharmacological concentrations (< 100 mM), although the mechanisms of inhibition still need further study.