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.     

Epigallocatechin-3-gallate has dual,independent effects on the cardiac sarcoplasmic reticulum/endoplasmic reticulum Ca<Superscript>2+</Superscript> ATPase

We determined the effects of epigallocatechin-3-gallate (EGCG) and epicatechin (EC), on pump turnover and Ca²⁺ transport by the cardiac form of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase (SERCA). Fluorescence spectroscopy was used to directly measure SERCA ATPase activity and to measure Ca²⁺ uptake into cardiac sarcoplasmic reticulum (SR) vesicles and microsomes derived from human embryonic kidney (HEK) cells expressing human cardiac SERCA2a. We found that EGCG reduces the maximum velocity of Ca²⁺ uptake into cardiac SR vesicles and increases the Ca²⁺-sensitivity of uptake in a concentration-dependent manner. EC is less potent than EGCG in increasing the Ca²⁺-sensitivity of uptake and does not affect maximum uptake velocity. The EGCG-dependent reduction in Ca²⁺ uptake velocity is well correlated with direct inhibition of SERCA. The effect of EGCG on the Ca²⁺-sensitivity of Ca²⁺ uptake into cardiac SR vesicles is affected by the phosphorylation status of phospholamban (PLB). When cardiac SERCA2a is expressed in HEK cells without PLB, EGCG reduces the maximum velocity of Ca²⁺ uptake but does not affect the Ca²⁺-sensitivity of uptake into microsomes derived from these cells indicating that the effect of EGCG on Ca²⁺-sensitivity requires the presence of PLB. Our results show that EGCG has dual effects on SERCA function in cardiac SR vesicles: it directly affects SERCA by reducing maximum uptake velocity; it increases the Ca²⁺-sensitivity of Ca²⁺ uptake in a manner that appears to depend on the interaction between SERCA and PLB.     

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.     

Different effects of the ionophore A-23187 and D-glucose on 45Ca2+ fluxes in isolated islets of ob/ob-mice

Fluxes of ⁴⁵Ca²⁺ were studied in β-cell rich islets of non-inbred ob/ob-mice, using LaCl₃ to wash out extracellular and superficially bound ⁴⁵Ca²⁺. The ionophore A-23187 (10,μM) increased the ⁴⁵Ca²⁺ uptake in islets both at 3 and 20 mM D-glucose, the effect being more pronounced after 10 min than after 120 min of incubation. In incubations for 120 min, 20 mM D-glucose induced a higher uptake of ⁴⁵Ca²⁺ than did A-23187. The ionophore enhanced the unidirectional efflux of ⁴⁵Ca^a+ from preloaded islets. Pretreatment of islets with 20 mM D-glucose in non-radioactive medium inhibited the subsequent D-glucose-induced ⁴⁵Ca²⁺ uptake. Similar pretreatment with A-23187 increased the subsequent ionophore-induced ⁴⁵Ca²⁺ uptake. The results suggest that A-23187 acts by catalyzing Ca²⁺ fluxes across the β-cell plasma membrane. The different effects of D-glucose and A-23187 on ⁴⁵Ca²⁺ fluxes suggest that the two agents act through different mechanisms in the β-cells.     

Modifications of Ca2+ transport induced by glutathione in sarcoplasmic reticulum membranes of frog skeletal muscle

The Ca²⁺ transport across the membrane of vesicles purified from the sarcoplasmic reticulum (SR) of frog skeletal muscle is modified by raising the concentration of the reduced form of glutathione (GSH). Passive release of Ca²⁺ is inhibited through the direct action of GSH on ryanodine receptors while active uptake is increased by a dose-dependent stimulation of Ca²⁺ pumps (Ca²⁺-ATPase). These effects are physiological since the concentrations of GSH utilised (0.01–10.0 mM) are compatible with the in vivo concentration of this antioxidant. They are independent of the external Ca²⁺ concentration and are specific for the reduced form of glutathione, since the disulphide form (GSSG) or other GSH-derivatives do not induce these effects.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Contractile activation and the effects of 2,3-butanedione monoxime (BDM) in skinned cardiac preparations from normal and dystrophic mice (129/ReJ)

(1) Small cardiac myofibrillar preparations were obtained from the right ventricle of normal (129/ReJ) and dystrophic (129/ReJ dy/dy) mice and were chemically skinned in a relaxing solution by exposure to Triton X-100 (3% v/v). (2) The isometric force produced in these skinned cardiac preparations at different sarcomere lengths was measured in solutions of different [Ca²⁺] and ionic strength. The effect of the negative inotropic drug 2,3-butanedione monoxime (BDM), which is known to act at the myofibrillar level was also investigated. (3) The murine cardiac preparation from normal animals was found to develop 50% maximal force at a pCa (=–log₁₀[Ca²⁺]) of 5.59±0.08 and 5.94±0.03 (mean ±SD) under physiological (ionic equivalents concentration, I=154 mM; pH 7.10; [Mg²⁺] 1 mM) and low ionic strength (I=94 mM; pH 7.10; [Mg²⁺] 1 mM) conditions respectively. The isometric force curves were significantly shallower at low ionic strength (Hill coefficient, 1.8±0.1) than at physiological ionic strength (Hill coefficient, 2.6±0.3) and the sarcomere length effect on the force-pCa relation was markedly reduced at lower ionic strength. (4) Increasing BDM concentrations in solutions up to 100 mM reduced the maximum Ca²⁺-activated force of cardiac preparations from normal mice to less than 6% of the control values in a dose dependent fashion. BDM also rendered the cardiac preparations less sensitive to Ca²⁺ by a factor of up to 1.5 in a process which showed saturation at BDM concentrations higher than 15 mM. (5) Cardiac preparations from dystrophic animals compared with those from normal mice were significantly more sensitive to Ca²⁺ under physiological conditions, were more sensitive to the action of BDM at concentrations higher than 15 mM, changed sensitivity to Ca²⁺ less following a change in sarcomere length and in general were less affected by a decrease in ionic concentration. (6) The results indicate that dystrophy in mice affects the characteristics of both the contractile and regulatory systems of cardiac muscle and that BDM directly affects the Ca²⁺-activated contractile response possibly by binding to saturable sites on the myofilaments.     

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.     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Reversal of α1-receptor mediated relaxation in intestinal smooth muscle

The α₁-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba²⁺ concentration was increased to 10–15 mM the response to phenylephrine was a contraction, and at Ba²⁺ concentrations in between the high and low range this response was biphasic—a relaxation followed by a contractile phase. The α₂-receptor agonist clonidine did not affect the tone of the Ba²⁺ contracted preparation. When the muscle preparation was contracted by Sr²⁺ (1–20 mM) in the presence of Ca²⁺ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca²⁺, 5 mM Ba²⁺ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca²⁺ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca²⁺ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ could be reverted to the normal relaxation by the addition of 20 mM Mg²⁺. Increasing the K⁺ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba²⁺ could reverse the response of α₁ receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca²⁺.     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

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.     

Immunocytochemical evidence for in vitro release of glutamate and GABA from separate nerve terminal populations in the rat pontine nuclei

Summary A quantitative electron microscopic immunocytochemical method was used to study the synaptic handling of glutamate and GABA in slice preparations from the rat pontine nuclei. Slices were subjected to a depolarizing stimulus (55 mM K⁺, 20 min) in the presence of a physiological or low Ca²⁺concentration. Depolarization at physiological [Ca²⁺] evoked a depletion of glutamate-like immunoreactivity from nerve terminals that contain round vesicles and establish asymmetric synaptic contacts. When depolarization was induced in the presence of only 0.1 mM Ca²⁺ (10 mM Mg²⁺ added), the loss of glutamate was significantly reduced or abolished, indicative of a Ca²⁺dependent component of glutamate release. By means of a double labeling immunocytochemical method we could identify a population of nerve terminals that displayed strong GABA-like immunoreactivity, and a level of glutamate like immunoreactivity that was low but yet clearly above background level. This type of terminal contains elongated or pleomorphic vesicles and establishes symmetric synaptic contacts. In these terminals, depolarization evoked a Ca²⁺-dependent depletion of GABA like immunoreactivity, but failed to change the level of glutamate like immunoreactivity. The present study demonstrates that two different types of nerve terminal in the rat pontine nuclei contain releasable pools of glutamate and GABA, respectively, and that the GABA releasing terminals also contain a non releasable pool of glutamate. The glutamate of the latter pool could act as precursor of GABA.     

The effect of high K+ depolarization and verapamil on45Ca2+ fluxes in the canine myocardium

The effects of high K⁺ depolarization and verapamil on Ca²⁺ uptake and the total intracellular Ca²⁺ content of canine ventricular muscle strips (0.5 mm thick) were investigated. High K⁺ (96 mM) increased Ca²⁺ uptake above control and maintained this enhanced uptake throughout a 90 second measuring period. Verapamil (5×10^–6M) significantly (p<0.05) inhibited this high K⁺ stimulated uptake. However, verapamil (5×10^–6M) also had a direct effect on Ca²⁺ fluxes, causing a significant increase in both Ca²⁺ uptake (p<0.001) and total intracellular Ca²⁺ content (p<0.001) in the resting tissue. Therefore, verapamil's apparent inhibition of high K⁺ stimulated Ca²⁺ uptake may have resulted from some mechanism other than Ca²⁺ channel blockade.     

Changes in Ca2+ ion activity within unrestrained rat's hippocampus perfused with alcohol or acetaldehyde

In the freely moving rat, the kinetics of Ca²⁺ ion activity were determined at circumscribed sites in the hippocampus, which was perfused with ethanol, tertiary-butyl alcohol or acetaldehyde. Initially, a region in CA1 or other cell field of the dorsal hippocampus was prelabelled by microinjection of⁴⁵Ca²⁺ through a permanently implanted guide tube. Then the tip of a concentric push-pull cannula assembly was lowered through the guide tube to the labelled site, and an isotonic artificial cerebrospinal fluid was repeatedly perfused at a rate of 25 μ1/min. Each perfusion was timed for 5.0min with a 5.0 min interval between each. Once the washout curve of⁴⁵Ca²⁺ activity had begun to approach its asymptote, ordinarily in the midpoint of a series of perfusions, an isotonic solution of ethanol (188–942 mM), tertiary-butyl alcohol (12–580 mM) or acetaldehyde (10–98 mM) was added to the fourth perfusate. Thereafter, the hippocampal site was again perfused with the normal cerebrospinal fluid for the remainder of the experiment. Although the lowest concentration of ethanol exerted no effect on⁴⁵Ca²⁺ ion activity, an intermediate concentration caused mixed effects in either enhancing or suppressing the efflux into the perfusate of this cation. The highest concentration of ethanol produced in most experiments an initial suppression in Ca²⁺ ion efflux which was followed frequently by an elevation in the release of⁴⁵Ca²⁺. Similar changes in Ca²⁺ ion activity were produced by tertiary-butyl alcohol, but the magnitude of its effect was generally less than that of ethanol, suggesting that its effect on brain tissue differs from that of ethanol. Acetaldehyde evoked an intense and concentration-dependent enhancement of Ca²⁺ ion efflux from the perfused tissue at all of the sites in the hippocampus examined.These results suggest that in the unrestrained rat ethanol could unbind Ca²⁺ ions from hippocampal membranes or retard their uptake into cells of the hippocampus. The dual excitatory and inhibitory effect of ethanol on Ca²⁺ ion activity corresponds to the electrophysiological effects of this alcohol and could alter neurotransmitter release from neurons in this subcortical structure. The mechanism of action of acetaldehyde is envisaged to be due to its affinity to membrane sulfhydryl groups which alters protein conformation and thus interferes with both Ca²⁺channels and Ca²⁺ binding properties.     

Some aspects of calcium uptake by human myometrial mitochondria and microsomes relevant to relaxation

Calcium uptake by mitochondria and microsomes isolated from the human myometrum was studied at physiological Ca⁺⁺ concentrations. The initial rates as well as the maximum velocity of Ca uptake by mitochondria were 10–20 times higher than those by microsomes. The Ca⁺⁺ concentration for half-maximal transport in the mitochondria and microsomes was about 1 μM and 0.5 μM, respectively. The Ca uptake capacity of mitochondria measured after 20 min of uptake (1 μM Ca⁺⁺ in the medium) was 10–30 times higher than that of microsomes. The capacity but not the initial rates of Ca uptake by microsomes was increased in the presence of 5 mM oxalate. There was only minor differences in the Ca uptake kinetics of subcellular fractions isolated from the pregnant and non-pregnant myometria. The results of this study reinforce the argument for a domineering role of mitochondria in the relaxation of the human myometrium.     

A comparative study of the calcium accumulation by mitochondria and microsomes isolated from the smooth muscle of the guinea-pig taenia coli

Summary The calcium uptake by mitochondria and microsomes isolated from the guinea-pig taenia coli was studied at physiological Ca²⁺ concentrations, buffered by Ca-EGTA mixtures. The Ca accumulation by the mitochondria was measured from the difference between the amount of Ca taken up in the presence and in the absence of a specific mitochondrial inhibitor. The Ca uptake by the microsomes was determined in a solution containing oxalate and a mitochondrial inhibitor. It was calculated from the difference in Ca uptake measured with and without ATP. By using this procedure, the necessity of extensive purification of the isolated fractions was avoided.The (Ca²⁺) for half-maximal transport in the mitochondria is 7×10^–6 M. At (Ca²⁺) lower than 2×10^–7 M, Ca is taken up in an energy-dependent way.In the microsomes the apparentK _m for Ca is 7×10^–7 M. Accumulation is still stimulated by ATP at a (Ca²⁺) as low as 4×10^–8 M.The results show that the rate of Ca uptake by the cell organelles corresponding to the microsomal vesicles is sufficiently fast to explain the speed of relaxation of the taenia coli. The results also suggest that these cell organelles are more important than the mitochondria in regulating the cytoplasmic Ca concentration.     

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     

An analysis of the mechanisms underlying the non-quantal release of acetylcholine at the mouse neuromuscular junction

The hyperpolarization produced by the application of 10^?5 M d-tubocurarine to the end-plate of mouse diaphragm-phrenic nerve preparation pretreated with an irreversible anticholinesterase was studied. This hyperpolarization has been attributed to a non-quantal release of acetylcholine by the nerve terminals. The usual hyperpolarization effect of about 10 mV was mimicked by adding 10^?7 M acetylcholine to the bath which contained 15mM Ca²⁺ to block the non-quantal release. The hyperpolarization effect was maximal (9.2 ± 0.8mV) in saline solution with the normal concentrations of Ca²⁺ (2mM) and K⁺ (5mM). Reducing [Ca²⁺] in the bath decreased the hyperpolarization effect. In Ca-free solution with 4.10^?4 M ethleneglycoltetra-acetate the hyperpolarization effect was3.8 ± 0.6mV. An increase of [Ca²⁺] also reduced the hyperpolarization effect and it was absent in 15mM of Ca²⁺. When the external concentration of Ca²⁺ was kept constant (2mM), both decreasing and increasing K⁺ concentration from 5 mM diminished the hyperpolarization effect. However, the decrease at elevated K⁺ concentrations was due to a reduction of membrane resistance and when the hyperpolarization effect was corrected for the change in size of the miniature endplate potential, it was unchanged in solutions with increased [K⁺] up to 13 mM. No traces of non-quantal release were observed during repetitive stimulation of the phrenic nerve in a medium containing 0.3 mM Ca²⁺. The hyperpolarization effect was not found in denervated muscles that lacked signs of neuromuscular transmission. The hyperpolarization effect was blocked by botulinum toxin while some evoked release of ACh was present. The time required for the block by 1 μg/ml toxin was shorter than for 0.25 μg/ml. The application of α-bungarotoxin produced a hyperpolarization of the membrane in the junctional region. The addition of d-tubocurarine failed to produce the hyperpolarization effect. The hyperpolarization effect was also absent when muscles were incubated with the desensitization potentiating drug, SKF-525A. The hyperpolarization effect was unchanged by replacement of Na⁺ with guanidine and was absent following the replacement of Na⁺ with arginine.The data suggest the channel responsible for the hyperpolarization effect is the same as that giving the endplate potential. The results are in agreement with the hypothesis that the hyperpolarization effect reflects a sustained non-quantal release of acetylcholine from the nerve terminal into the synaptic cleft.     

The influence of calcium on morphology and histamine content of isolated intact rat mast cell granules

Histamine is released by “sequential exocytosis” in mast cells. The exocytosis involves fusion of the plasma membrane with the perigranular membrane and further fusions of adjacent perigranular membranes. To study a possible direct effect of Ca²⁺ on granule membrane fusions, mast cell granule suspensions were prepared from sonicated rat mast cells. With the sonication method used, more than 60% of the granules obtained were found to be homogeneous, electron dense and surrounded by a perigranular membrane, when observed in the electron microscope. These granules correspond to normal, histamine-containing granules found in untreated mast cells and are therefore named “intact” granules. The other granules were swollen, less electron dense and without a perigranular membrane. These “changed” granules are formed during the histamine release process. Aliquots of the granule suspension were incubated in 0.34 M sucrose buffered with 10 mM HEPES, pH 7.0, containing different concentrations of CaCl₂, MgCl₂ (10 mM, 1 mM, 100 μM, 10 μM) or NaCl (10 mM). Only with the highest concentration (10 mM) of Ca²⁺ or Mg²⁺ was it possible to visualize an apposition of the perigranular membranes of “intact”, normal granules. No elimination of the individual membrane structures could be observed at the place of membrane contact. Thus, we found no signs of membrane fusions. The histamine content was lower in the suspensions incubated with lower concentrations of these ions or with 10 mM NaCl. Ca²⁺ and Mg²⁺ in high concentrations seemed to stabilize the perigranular membranes instead of initiating histamine release. Therefore, changes in the Ca²⁺-ion concentration per se do not explain the membrane fusions seen in mast cells during “sequential exocytosis”.