Über die Wirkung von ADP und einigen Kationen auf die Rotationsströmung in den Wurzelhaaren der Gerste (Hordeum vulgare L.)

Zusammenfassung In getrennten Versuchen wurde die Wirkung von ADP, Ca⁺⁺, Mg⁺⁺, K⁺ und Cu⁺⁺ auf die Rotationsströmung in den Wurzelhaaren der Gerste (Hordetim vulgare L.) untersucht. Das in verschiedenen Konzentrationen fortdauernd verabreichte ADP bedingte eine Stimulation der Plasmaströmung. Die Beschleunigung der Rotationsströmung war der ADP-Konzentration gegenüber umgekehrt proportional (Abb. 3).Von den untersuchten Kationen hatte nur Ca⁺⁺ (1·10^–3 Mol) eine Stimulationswirkung. Diese Stimulationswirkung wird der Aktivierung eines Enzyms bzw. eines kontraktilen Proteins mit ATPase-Eigenschaften zugeschrieben.Die Rolle von ADP und einigen Kationen bei der Stimulation der Rotation wurde dann mit Hilfe einer gemischten Behandlung untersucht. Diese bestand in der gleichzeitigen Verabreichung von ADP (1·10^–6 Mol) und CaCl₂, MgCl₂, KCl (1 · 10^–3 Mol) oder CuCl₂ (1·10^–6 Mol). Es wurde festgestellt, daß Mg⁺⁺ und Ca⁺⁺ eine antagonistische Wirkung ausüben. Ca⁺⁺ hebt die durch ADP induzierte Stimulation auf und reduziert die Rotationsgeschwindigkeit plötzlich bis auf den Kontrollwert. Die Mg⁺⁺-Wirkung bewirkt, nach einer zeitweiligen Beibehaltung der Stimulation, ebenfalls eine Abnahme der Geschwindigkeit. K⁺ hat eine ähnliche Wirkung wie Ca⁺⁺. Cu⁺⁺ beeinträchtigt die ADP-induzierte Stimulation in geringem Maße.Die gleichzeitige Einwirkung von ADP und einigen Kationen erlaubt die Aufstellung folgender Hypothese. Die Rotationsstimulation erfolgt dank dem ATP, das auf Kosten des von außen absorbierten ADP in den Mitochondrien synthetisiert wird. Die zusätzliche ATP-Synthese kann durch gleichzeitige Ca⁺⁺-Behandlung unterbunden werden. NachHanson und Mitarb, sollen Ca⁺⁺ und ADP um ein phosphoryliertes Zwischenprodukt in Kompetition treten, so daß es zu einer Ansammlung von Ca⁺⁺ und P_a in der Zelle kommt. Andererseits könnte teilweise auch die aktive, energieverbrauchende Salzabsorption die Geschwindigkeitsabnahme der Rotation bei gemischter Behandlung erklären.

---

The effect of ADP and some cations on rotational streaming in barley (Hordeum vulgare L.) root hairs

Summary The effect of ADP, Ca⁺⁺, Mg⁺⁺, K⁺, and Cu⁺⁺ upon rotational streaming within barley (Hordeum vulgäre L.) root hairs was separately studied. It was shown that various solutions of ADP may stimulate the streaming after continuous treatment. The rate increase of the rotational streaming was inverse proportional to ADP concentration (Fig. 3).From the investigated cations only Ca⁺⁺ (1·10^–3M) caused a stimulation of streaming after continuous treatment. This effect is probably due to enzymic activation of a contractile proteine which has ATPase feature.The role of ADP and of the investigated cations in the stimulation of the rotational streaming was studied by means of mixed treatment. This kind of treatment consists in a simultaneous administration of ADP (1 · 10^–6M) and CaCl₂, MgCl₂, KCl (1 · 10^–3M), or CuCl₂ (1 · 10^–6M) solutions. Ca⁺⁺ and Mg⁺⁺ showed an antagonistic action. Ca⁺⁺ brings about an immediately suppress of ADP induced stimulation. Suddenly the rate of streaming comes back to control. Mg⁺⁺ after a temporary maintaining of stimulation, also causes the lowering of the streaming. The action of K⁺ was very similar to those of Ca⁺⁺. Cu⁺⁺ changes to a little extent the stimulation caused by ADP.The simultaneous action of ADP and of the investigated cations allow us to express the following hypothesis. The stimulation of the rotational streaming after ADP treatment probably is due to ATP synthetized in mitochondria on the account of ADP. The additional synthesis of ATP can be prevented by simultaneous administration of Ca⁺⁺. According toHanson and his coworkers Ca⁺⁺ would compete with ADP for a phosphorylated intermediate product. From a such competition would result the Ca⁺⁺ and P_i accumulation. The active uptake of salts which require energy would also explain the lowering of the rotational streaming rate after the mixed treatment.

     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Nucleus Basal Body Connector of Dunaliella: Threshold Concentration of Calcium Necessary for in vitro Contraction*

Detergent-isolated flagellar apparatuses of the flagellate green alga Dunaliella bioculata retain remnants of the nucleus (the karyoskeleton) which are linked to the basal bodies by the centrin-containing nucleus basal body connectors (NBBC). Such complexes were subjected to different calcium concentrations (1 × 10^?9 M ? 5 × 10^?4 M Ca²⁺) and the distance between the basal bodies and the karyoskeleton was measured by light microscopy. The threshold concentration of Ca²⁺ for NBBC contraction was determined to be around 5 × 10^?8 M Ca²⁺. At > 10^?7 M Ca²⁺ NBBC were maximally contracted and the distance between the basal bodies and the karyoskeleton was only about 50% of the initial distance. Using a polyclonal antibody generated against centrin (Salisbury et al., 1984), the NBBC were visualized by indirect immunofluorescence in both the extended and contracted state. Our results demonstrate that in vitro contraction of centrin-containing filaments in green algae is initiated at about the same free Ca²⁺ concentration in three different centrin-containing basal apparatus components (i.e. the distal connecting fibre, the flagellar transitional region and the NBBC).     

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles

Summary The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca⁺⁺ accumulation in SR vesicles at detergent concentrations below 10^–3 M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca⁺⁺ accumulation in the concentration range between 10^–4 M and 2.5 × 10^–2 M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca⁺⁺-ATPase activity up to a 10^–3 M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.Abbreviations SR sarcoplasmic reticulum - SDS sodium dodecylsulphate - DTT dithiothreitol - EGTA ethyleneglycoltetraacetate - PEP phosphoenolpyruvate     

Effects of Cd++ on short-circuit current across epithelial membranes

Summary Cadmium ion (Cd⁺⁺) significantly increased potential difference (PD) and short-circuit current (SCC) across isolated frog skin when added to the outside Ringer's solution at 10^–4, 10^–3 and 5×10^–3 m concentration. Resistance was reduced by 10^–4 m Cd⁺⁺ but not significantly changed by the higher concentrations. When SCC was first stimulated by vasopressin, 10^–4 and 10^–3 m Cd⁺⁺ produced additive stimulation which was reversible by washing with Cd⁺⁺-free Ringer's. If SCC was first stimulated by Cd⁺⁺, further stimulation by vasopressin was additive with 10^–4 m Cd⁺⁺ but completely inhibited by 10^–3 m Cd⁺⁺. Elevating the calcium ion (Ca⁺⁺) concentration of the outer Ringer's from 10^–3 m to 5×10^–3 m or 10^–2 m prior to Cd⁺⁺ treatment did not reduce the magnitude of SCC stimulation by Cd⁺⁺. Removal of Ca⁺⁺ from the outside Ringer's with 2×10^–3 m EDTA increased SCC as predicted. Subsequent addition of 5×10^–3 m Cd⁺⁺ drastically reduced SCC below control levels while equimolar concentrations of Cd⁺⁺ and EDTA reduced SCC only to control levels. These results suggest that Cd⁺⁺ interacts with the components of the apical plasma membranes of epithelial cells which are associated with the stimulation of SCC by vasopressin and Ca⁺⁺ removal and may be a useful probe for elucidating these components.     

The thermodynamic efficiency of the Ca2+-Mg2+-ATPase is one hundred percent

The thermodynamic efficiency of the Ca²⁺-Mg²⁺-ATPase of skeletal sarcoplasmic reticulum has been evaluated by comparing the Ca²⁺ gradient established with the ATP/(ADP*P_i) ratio. The evaluation was made at an external Ca²⁺ level (4.7 × 10^–8 M) which is below theK _m value of 7 × 10^–8 M. The Mg-ATP and phosphate concentrations were held constant (0.1 mM) and the ADP concentration was varied. Maximal uptake to an internal free Ca²⁺ concentration of 17 mM was observed at infinite ATP/(ADP*P_i) ratio (absence of ADP). This corresponds to a [Ca²⁺]_i/[Ca²⁺]₀ gradient of 3.6 × 10⁵. A Ca²⁺ gradient one-half as large was observed at an ATP/(ADP*P_i) ratio of 3.5 × 10³ M^–1. The square of the Ca²⁺ gradient is shown to be proportional to the ATP/(ADP*P_i) ratio, for finite values of the latter. The proportionality constant is identical to the equilibrium constant for hydrolysis of ATP (9.02 × 10⁶ M) under these conditions (0.1 mM Mg²⁺, 30°C). The intrinsic thermodynamic efficiency of the pump is shown to be 100%, with a maximal uncertainty of 3%. The efficiency is lower under less optimal conditions, when the pump is inhibited and passive leak processes compete.Dedicated to Prof. Philip George, University of Pennsylvania, whose instruction, research, and example made this contribution possible.     

Calcium and ATP regulation of the oscillatory torsional movement in a Triton model of Physarum plasmodial strands

A segment of the plasmodial strand of Physarum polycephalum, when hung vertically, shows oscillatory torsional movements that accompany shortening and elongation, i.e., contraction and relaxation in the vertical direction. Reactivation of such movements was achieved in a fresh, demembranated model system through the use of Triton X-100 and chemically defined physiological solutions. Oscillatory rotations were reactivated in solutions containing 0.5 mM Mg-ATP and 10⁻⁶–10⁻⁴ of free Ca²⁺. Lowering the Ca²⁺ concentration to 10⁻⁸ M brought about unidirectional rotations with concomitant vertical elongation of the strands, i.e., relaxation. Such unidirectional rotations were also observed either when Mg-ATP concentrations was raised to 5 mM, or when Mg-ATP was replaced with Mg-pyrophosphate. Higher concentrations of free Ca²⁺ than 10⁻⁴ M similarly caused unidirectional rotations, but there was no concomitant vertical elongation. These data mean that the oscillatory contraction and relaxation require a factor which is activated at Ca²⁺ concentration above 10⁻⁶ M and inactivated at 10⁻⁸ M. The findings that Mg-pyrophosphate or high concentrations of Mg-ATP-induced relaxation suggest that the actomyosin is dissociated in the relaxation phase. In the light of the present study, it is not likely that the periodic movement in the plasmodia is caused by any oscillatory activities in either the ATP-producing system or the plasma membrane.     

Two calcium currents in the somatic membrane of mollusc neurons

Two new types of calcium channels were discovered during research in ionic currents in the somatic membrane ofHelix pomatia neurons, using an intracellular perfusion technique. Apart from the principal calcium current described in the literature with a holding potential of about –110 mV, an additional calcium current was observed activated at depolarizations of –40 to –80 mV and was not reduced when the cell was perfused with solutions containing fluoride anions. The kinetics of this current were well described in the context of the Hodgkin and Huxley model with a time constant of activation of 6–8 msec and of inactivation of 300–600 msec. It increased in amplitude as the Ca⁺⁺ rose in the cellular environment but was reduced by extracellular addition of the Ca⁺⁺ antagonists Co⁺⁺, Ni⁺⁺, and Cd⁺⁺, and the organic blockers nifedipine and verapamil. The association constants of these substances with corresponding channels determined from the maximum of the current-voltage relationship were 2 (Ca⁺⁺), 3 (Co⁺⁺), 0.06 (nifedipine), and 0.2 mM (verapamil). The properties detected in this component of calcium conductance are compared with those of calcium channels in other excitatory formations and its possible functional role is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 5, pp. 627–633, September–October, 1985.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Decondensation of sperm nuclei of australian marsupials: Effects of air drying and of calcium and magnesium

Spermatozoa of six species of Australian marsupials have been studied. The nucleus is highly unstable when compared with those of eutherian mammals. When thin films of spermatozoa in buffered saline are air-dried on glass slides, the nucleus disintegrates and flattens, leaving the acrosome, midpiece, and tail intact. This spreading of the nucleus can be inhibited by seminal plasma proteins and by bovine serum albumin, but is potentiated by detergents. The nucleus also decondenses spontaneously in the presence of high concentrations (>0.25M) of calcium and magnesium salts, leaving the head membranes, acrosome, midpiece, and tail intact. This is inhibited by EDTA. In some species, certain areas of the nucleus appear more resistant t o Ca⁺⁺/Mg⁺⁺ treatment, and the initial stages of decondensation are uneven. Ultrastructurally the Ca⁺⁺/Mg⁺⁺ dispersed chromatin shows a moderately fine, branching, fibrillar structure, interspersed with dense granules. Treatment with disulphide bond cleaving agents together with detergents results in rapid and complete dispersal of the chromatin and acrosome, and slow digestion of midpiece and tail structures. Treatment with HCl, NaCl, KCl, EDTA, detergents, and sucrose has no effect on nuclear integrity, but treatment with NaOH (0.9–1.0M) results in complete digestion of the whole sperm. These findings are discussed in the light of evolutionary differences between marsupial and eutherian mammals in terms of sperm structure and composition.     

Ca2+ ion reversibly inhibits the cytoplasmic streaming ofNitella

Summary When Ca²⁺, K⁺ or Cl^– was injected iontophoretically into the cytoplasm of intactNitella cell, only Ca²⁺ reversibly inhibited the cytoplasmic streaming. However, when an extremely large current was used, the cytoplasmic streaming was reversibly inhibited irrespective of the ion species. This inhibition may be due to a transient increase of free Ca²⁺.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Stimulation of lymphocyte receptor capping by the ionophore monensin

Summary The carboxylic ionophore monensin has a biphasic effect on antibody-induced Thy-1 cap formation. At higher concentrations, 5×10^–6–5×10^–5 m monensin causes a significant inhibition of receptor capping similar to that previously found with the Ca²⁺ selective ionophore A23187. At lower concentrations, 5×10^–8–5×10^–7 m capping is stimulated. It is concluded that capping at lower ionophore concentrations is a secific response to the ability of monensin to induce a rise in intracellular Na⁺, which indirectly elevates intracellular Ca²⁺ activity. This in turn activates the contractile machinery required for the aggregation of surface receptors into capped structures. At higher concentrations monensin acts as a nonspecific detergent, which causes detrimental structural alterations in some of the membrane components involved in the capping process.     

Carbamylcholine-induced morphological changes and spatial dynamics of [Ca2+]c in Harderian glands of guinea pigs: calcium-dependent lipid secretion and contraction of myoepithelial cells

To determine whether lipid-secreting cells have cytosolic Ca²⁺ concentration ([Ca²⁺]_c)-related secretory mechanisms, morphological changes and intracellular calcium dynamics of Harderian glands of guinea pigs stimulated by secretagogs were studied by electron microspy and Fura-2/AM digital image analysis. Control glandular cells contained large lipid vacuoles that were bordered by multi-layered membranes. Rough-surfaced endoplasmic reticulum, mitochondria, and smooth-surfaced endoplasmic reticulum may be involved in lipid vacuole formation. Myoepithelial cells surrounded alveoli. After carbamylcholine (CCh, 10^–6, 10^–5, and 10^–3 M) stimulation, lipid materials within the membranous structures were frequently discharged by an exocytotic mechanism. Conspicuous deformation of glandular cells caused by vigorous contraction of myoepithelial cells was observed in isolated alveoli after 10^–6M CCh stimulation, whereas the deformaties of glandular tissues perfused via vessels were small even after 10^–3M CCh stimulation. Connective tissue between glandular alveoli inhibited unbridled myoepithelial-cell contraction. Fura-2/AM digital imaging analysis revealed that CCh stimulation caused an increase in [Ca²⁺]_c in isolated alveoli. The morphological reactions and changes in [Ca²⁺]_c were prevented by atropine. When extracellular calcium ions were absent, enhanced extrusion of lipid vacuoles, myoepithelial-cell contraction, and a rise in [Ca²⁺]_c after CCh stimulation were not observed. Nicotine and catecholamines had no effect on the secretion or on the dynamics of [Ca²⁺]_c. It can be concluded that acetylcholine elicits exocytosis in glandular cells and contraction of the myoepithelial cells of Harderian glands, accompanied by an increase in [Ca²⁺]_c. The dynamics of [Ca²⁺]_c of the gland alveoli are mostly dependent on extracellular Ca²⁺.     

2,6-Diisopropylphenol,a general anesthetic,inhibits glutamate action on rat synaptosomes

2,6-diisopropylphenol (propofol), a general intravenous anesthetic, inhibits the glutamate-dependent Ca²⁺ entry in rat synaptosomes with an approximate IC₅₀ of 3.0×10^–5 M.Propofol, at concentrations above 10^–6M, also inhibits the ATP-dependent uptake of glutamate in the presence of Ca²⁺, with an approximate IC₅₀ of 3.5×10^–5M, while it only has a slight inhibitory effect on the release of glutamate. The ouabain-insensitive synaptosomal ATPase is strongly inhibited by propofol, with an IC₅₀ of about 2.5×10^–6M, at concentrations which do not affect the luciferase system.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Participation of Ca2+ in cessation of cytoplasmic streaming induced by membrane excitation inCharaceae internodal cells

Summary The mechanism of the cessation of cytoplasmic streaming upon membrane excitation inCharaceae internodal cells was investigated.Cell fragments containing only cytoplasm were prepared by collecting the endoplasm at one cell end by centrifugation. In such cell fragments lacking the tonoplast, an action potential induced streaming cessation, indicating that an action potential at the plasmalemma alone is enough to stop the streaming.The active rotation of chloroplasts passively flowing together with the endoplasm also stopped simultaneously with the streaming cessation upon excitation. The time lag or interval between the rotation cessation and the electrical stimulation for inducing the action potential increased with the distance of the chloroplasts from the cortex. The time lag was about 1 second/15 m, suggesting that an agent causing the rotation cessation is diffused throughout the endoplasm.Using internodes whose tonoplast was removed by replacing the cell sap with EGTA-containing solution (tonoplast-free cells,Tazawa et al. 1976), we investigated the streaming rate with respect to the internal Ca²⁺ concentration. The rate was roughly identical to that of normal cells at a Ca²⁺ concentration of less than 10^–7 M. It decreased with an increase in the internal Ca²⁺ concentration and was zero at 1 mM Ca²⁺.The above results, together with the two facts that Ca²⁺ reversibly inhibits chloroplast rotation (Hayama andTazawa, unpublished) and the streaming in tonoplast-free cells does not stop upon excitation (Tazawa et al. 1976), lead us to conclude that a transient increase in the Ca²⁺ concentration in the cytoplasm directly stops the cytoplasmic streaming. Both Ca influxes across the resting and active membranes were roughly proportional to the external Ca²⁺ concentration, which did not affect the rate of streaming recovery. Based on these results, several possibilities for the increase in Ca²⁺ concentration in the cytoplasm causing streaming cessation were discussed.     

Ca2+-activated Cl− channel in plasmalemma ofNitellopsis obtusa

Summary The mechanisms of Cl^–-channel activation in the plasmalemma ofNitellopsis obtusa was studied by measuring both the transient inward current under voltage clamp and Cl^– efflux during the action potential. 9-anthracenecarboxylic acid (A-9-C) at 1.0mm inhibited both the transient inward current and the Cl^– efflux, but did not uncouple the sudden cessation of the cytoplasmic streaming. Since this excitation-cessation coupling is caused by a transient increase in the cytoplasmic Ca²⁺ concentration, these results suggest that A-9-C inhibited not the Ca²⁺ channel but specifically the Cl^– channel. The following results were found between the Ca²⁺-channel activation and the Cl^–-channel activation: (1) The Ca²⁺-channel blocker La³⁺ uncoupled the excitation-cessation coupling and inhibited both the transient inward current and the Cl^– efflux, although the Cl^–-channel blocker A-9-C did not affect the excitation-cessation coupling. (2) The Cl^– efflux was greatly reduced by depletion of Ca²⁺ from the external solution and restored by an increase in the external Ca²⁺ concentration. (3) An increase in the external ionic, strength which increases Ca²⁺ entry (T. Shiina & M. Tazawa,J. Membrane Biol. 96:263–276, 1987) enhanced the Cl^– efflux. (4) Mg²⁺, which cannot pass through the Ca²⁺ channel, reduced both the transient inward current and the Cl^– efflux. (5) Although Sr²⁺ can pass through the plasmalemma Ca²⁺ channel, Cl^–-channel activation by Sr²⁺ was only partial. These findings support the hypothesis that voltage-dependent Ca²⁺-channel activation, which increases the free Ca²⁺ concentration in the cytoplasm, is necessary for the subsequent Cl^–-channel activation.