Role Of Calcium Ions Research Articles

Glycosaminoglycans and Eukaryotic Cell Control

SUMMARYThe molecular properties of cell surface glycosaminoglycans are considered as well as their interactions with other ‘surface associated’ macromolecules including collagen, actomyosin and fibronectin.Experimental evidences are provided on the role of calcium ions in the modulation of cellular physiology at the cell coat level together with preliminary results suggesting the involvement of cell surface glycosaminoglycans in the maturation and in the neoplastic transformation of bone marrow leukocytes.The possible involvement of cell surface glycosaminoglycans is pellular recognition is postulated and models are presented for a direct or a protein-mediated interaction at the cell surface level.

The role of calcium ions in the synthesis and transport of noradrenaline carrier vesicles in guinea-pig sympathetic neurons in vitro.

Guinea-pig inferior mesenteric ganglia (IMG)/hypogastric nerve preparations were incubated in tissue culture media containing ethylene glycol-bis-(beta-aminoethyl ether)N,N'tetra-acetic acid (EGTA) or colchicine and examined either by fluorescence microscopy or by transmission electron microscopy. Two millimolar EGTA inhibited the accumulation of noradrenaline (NA) fluophore proximal to a crush, but did not produce an increase in the fluorescent intensity of, or the number of dense-cored vesicles (DCVs) within the neuronal perikarya. Calcium ions, but not magnesium ions, were able to block this effect of EGTA. Preparations incubated in the presence of colchicine (2.5 microgram/ml) showed a reduction in the amount of fluorescent material accumulating proximal to a crush, but an increase in both the fluorescent intensity and the number of DCVs within the neuronal perikarya. The suggestion that calcium ions are required for the synthesis of some part of the NA-containing vesicle rather than for their loading onto the axoplasmic transport mechanism is discussed.

The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN'N'-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.

Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibres.

1. Under the influence of strophantidin, Purkinje fibres exhibit transient inward current (TI) which contributes to arrhythmogenic activity. Voltage-clamp experiments were carried out to study the role of Ca ions in this phenomenon. 2. The amplitude of TI varied directly with the extracellular Ca concentration, CaO. Magnesium ions had an antagonistic effect. 3. TI was closely associated with a phasic increase in force ("aftercontraction"). Like TI, the aftercontraction was evoked by a preceding action potential or by the break of a strong depolarizing pulse. 4. TI and the aftercontraction displayed similar wave forms although peak current preceded peak force by 50--100 msec. Both transients were enhanced by increasing the strength or duration of the preceding depolarization pulse. Both events were slowed as the potential level following the pulse was displaced in the negative direction. 5. TI and the aftercontraction could be evoked in the absence of cardiotonic steroids by strongly elevating CaO. 6. Additional experiments were carried out to test the hypothesis the TI reflects an influx of Ca2+ ions. Moninhibited TI but the developed and removal of the inbibition lagged far behind the effects on the slow inward current. 7. TI could be suppressed and eventually inverted by varying the membrane potential in the positive direction. The inversion potential averaged -5mV and was not consistent with a Ca-specific pathway. The aftercontraction was more closely related to the phasic conductance change underlying the current than to thecurrent flow itself. 8. The results are consistent with the idea that an oscillatory release of Ca from an intracellular store is the primary event underlying both the aftercontraction and the conductance change which generates TI. Digitalis intoxication or very high CaO may promote such events by elevating intracellular Ca levels.

THE EFFECT OF STIMULUS INTENSITY ON α‐ADRENOCEPTOR‐MEDIATED FEED‐BACK CONTROL OF NORADRENALINE RELEASE

1 Mouse vas deferns stimulated transmurally (2.5 Hz, 2-32 V, 40-620 mA, FOR 45 s) responded with a twitch and a secondary contraction. Both responses were abolished by cinchocaine and were voltage-dependent. 2 In tissues previously incubated with (3H)-(--)-noradrenaline, stimulation also produced an increase in tritium overflow from the tissue. Phentolamine increased tritium overflow by 19% at high stimulus intensities (30 V, 600 mA) and by 130% at low stimulus intensities (11 V, 200 mA). 3 It is concluded that alpha-adrenoceptor-mediated feedback control of noradrenaline release is more marked at low stimulus intensities and that this is compatible with a role for calcium ions in this control mechanism.

The role of calcium ions in tetanic and post‐tetanic increase of miniature end‐plate potential frequency.

1. The role of Ca ions in transmitter release changes, during and after high frequency stimulation of the motor nerve (10--100 Hz), was examined at the frog neuromuscular junction. 2. The stimulation-induced changes in miniature end-plate potential frequency (f) resembled the changes in end-plate potential amplitude recently described by Magleby and Zengel (1975, 1976). 3. The effects of tetanic stimulation on f under inward electrochemical gradient for Ca ions were compared with those under reversed gradient and four differences were found: (a) The increase in f during the tetanus under reversed Ca gradient conditions is much smaller than with an inward Ca gradient. (b) The increase in f under reversed Ca gradient is preceded by a small decrease in f, whereas with an inward Ca gradient an immediate increase in f is observed. (c) After the termination of the tetanus with a reversed Ca gradient, there is a further increase in f, compared to a decrease with an inward Ca gradient. (d) The augmentation phase of post-tetanic potentiation was practically abolished. 4. The experimental results are explained by assuming that high frequency nerve stimulation causes an increase in transmitter release by at least two distinct processes: influx of Ca ions through the presynaptic membrane and release of Ca ions from intracellular stores. It is suggested that Na ions couple nerve activity to intracellular release of Ca.

Samarium as a tool in studies on the responsiveness of the isolated guinea pig ventral taenia coli to barium.

The responsiveness of isolated guinea pig ventral taenia coli to barium in polarized (Ringer-Tris medium) and depolarized (Ringer-KCl medium) preparations was assayed in the presence of samarium (Sm3+, 6 or 8 X 10(5) M). No difference in EC50 or maximal response was observed between polarized and depolarized preparations (p greater than 0.05). The lower Sm3+ concentration increased Ba2+ EC50 in both experimental conditions (p less than 0.05) although no alteration was observed as to the maximal response (p greater than 0.05). On the other hand, maximal tension was strongly reduced by the higher Sm3+ concentration in polarized preparations (p less than 0.01) while a potentiation of the maximum response was observed on the depolarized tissues (p less than 0.05). A possible role for calcium ions in the mediation of the effects is suggested.

Evidence that Calcium Regulates Platelet Function

It is generally believed that calcium ions play a key role in regulation of platelet function. This is based on 3 types of evidence. 1. Analogies with other cells. Calcium ions are known to trigger secretion and contraction in many cells, possibly reflecting a general role for calcium in all secretion and contraction. 2. Indirect evidence. Platelet aggregation and secretion are induced by divalent cation ionophores. The response to the ionophore A23187 is identical to that induced by other potent stimuli. 3. Direct evidence. Platelet activation can be blocked by drugs (e. g. certain local anesthetics) that block release of calcium ions from sarcoplasmic reticulum; the inhibition can be overcome by addition of extracellular calcium in the presence of a calcium ionophore. While this does not constitute definitive proof, the central role for calcium ions remains an attractive hypothesis that justifies attempts to further define calcium pools and fluxes in platelets.

The problem of sea urchin egg fertilization and its implications for biological studies.

The analysis of sea urchin egg fertilization shows that several phenomena common to other biological systems are involved: cell recognition, cell fusion, exocytosis and initiation of mitotic activity. Both the role of calcium ions in cell fusion and exocytosis and the function of the cell surface in the initiation of mitotic activity appear to have general applicability. The study of fertilization can contribute to the elucidation of these processes and, reciprocally, progress in this field can help to advance our understanding of the mechanisms of fertilization in sea urchins and other organisms.

The Interrelated Roles of Calcium Ion and Cyclic Nucleotides in Control of Secretory Processes

The Interrelated Roles of Calcium Ion and Cyclic Nucleotides in Control of Secretory Processes

The role of calcium ions in the acrosome reaction of sea urchin sperm: Regulation of exocytosis

Sperm of the sea urchins Lytechinus pictus and Strongylocentrotus purpuratus undergo the acrosome reaction when the extracellular calcium concentration (which is about 10 mM in sea water) is raised to over 50 mM. However, the concentration of extracellular calcium required to trigger the acrosome reaction can be lowered to 5–10 mM by the presence of egg jelly, ionophore A23187, or elevated extracellular pH. These acrosomal triggers appear to act by increasing the sperm's calcium permeability; this is a known property of A23187 and elevated pH (acting on other types of cells), but has not been shown before for the natural trigger, egg jelly. Even in the presence of such triggers, the acrosome reaction is blocked in a calcium-free medium or in sea water containing the following agents known to decrease calcium influx: lanthanum, magnesium, procaine, xylocaine, and lowered pH. With the possible exception of lanthanum, inhibition due to these agents can be counteracted by increasing the extracellular calcium concentration. All these data indicate that a rise in intracellular calcium by influx from the extracellular space is a necessary condition for induction of the acrosome reaction. Barium and strontium, but not magnesium, will substitute for calcium. This influx of calcium is required to initiate the acrosome reaction's first morphologically detectable step, which is membrane fusion. In a calcium-free medium the acrosomal triggers, egg jelly, A23187, and elevated pH, fail to induce fusion of the acrosomal granule membrane with the overlying plasma membrane of the sperm.

Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease.

The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease has been investigated. Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G-25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (deltaH*) and entropy (deltaS*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g-atom/mol and B. subtilis neutral protease containing 1.4 g-atoms/mol. Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions. Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 X 10(4) M-1. B. subtilis neutral protease had four independent sites. The K value for three sites was 1.1 X 10(5) M-1 and the binding constant for the other site was 1.5 X 10(3) M-1. There was little difference in total free energy change for calcium binding between these proteases. From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.

Role of calcium ion in the thermostability of alpha-amylase produced from Bacillus stearothermophilus.

In 1961, Campbell and Manning provided one clue to elucidate the molecular basis for thermostability of the enzyme from thermophile(1–3). They reported that the α-amylase of B. stearothermophilus in the native state exists in a semi-random or random coiled and well hydrated molecule with slight extent of secondary structure formed by disulfide bonds (2). This less-ordered structure was postulated as the reason for thermostability of the enzyme.

The Role of Calcium Ion in Epinephrine Activation of Lipolysis

Adipocytes were prepared by collagenase digestion of rat epididymal adipose tissue and incubated for 5, 15 or 30 minutes in Krebs-Ringer bicarbonate buffer containing albumin (40 mg/ml), glucose (1 mg/ml) and epinephrine. Calcium ion was present in some incubations at concentration of 2.5 mM and omitted from others; media with no added calcium contained 1.0 mM EGTA thereby producing a final calcium concentration of less than 10(-7) M. Glycerol release and accumulation of cyclic AMP were measured. Basal lipolysis and cell cyclic AMP levels were increased slightly but not significantly when adipocytes were incubated in calcium free media. Lipolysis could be activated with epinephrine in the absence of calcium but the sensitivity of the lipolytic response was greatly reduced; however, the maximum lipolytic response to epinephrine was not decreased in calcium free media. Similarly, incubation of adipocytes in calcium free media resulted in decreased accumulation of cyclic AMP in response to epinephrine but only when sub-maximum concentrations of the catecholamine were present. Varying the extracellular calcium concentration showed that a concentration of at least 10(-5) M was optimal for epinephrine activation of lipolysis. These observations are considered in accord with the view that activation of adenylate cyclase is facilitated by calcium ion.

The role of calcium ions in fertilization of mouse and rat eggs in vitro.

Department of Animal Science, College of Agriculture, Kyoto University, Kyoto, Japan (Received 14th April 1975) In attempts to define suitable conditions for the in-vitro fertilization of mammalian eggs, it has been shown that the osmolality of the medium affects the fertilization rate of mouse and hamster eggs (Miyamoto & Chang, 1973b), and the pH values of the medium affect the fertilization rate of hamster eggs (Bavister, 1969; Miyamoto et al., 1974), mouse eggs (Iwamatsu & Chang, 1971; Miyamoto et al., 1974) and rat eggs (Miyamoto et al., 1974). Iwamatsu & Chang (1971) showed that calcium ions affect the capacitation of spermatozoa and fertilization of mouse eggs in vitro in the presence of bovine follicular fluid. It has recently been shown that guinea-pig spermatozoa fail to fertilize eggs in calcium-free media (Yanagimachi & Usui, 1974). The achievement of fertilization in vitro by epididymal spermatozoa of mice (Toyoda et al., 1971) and

The interference of some cytostatics on morphine-induced analgesia.

Abstract The influence of some cytostatic agents (the antibiotic daunomycin, the polypeptide peptikemio and the nitrosourea derivative BCNU) on the antinociceptive effect of morphine is here investigated. It is demonstrated that daunomycin and peptikemio significantly enhance morphine-induced analgesia, while on the other hand, BCNU is ineffective. Daunomycin neither prevents morphine binding to blood proteins nor facilitates its access to the brain. A role of calcium ions in daunomycin potentiating effect is suggested.

201) - Action of cardiac steroids on the intestinal smooth muscle in relation to changes in electrical properties of membrane and the role of calcium ion

201) - Action of cardiac steroids on the intestinal smooth muscle in relation to changes in electrical properties of membrane and the role of calcium ion

Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid.

1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.

The role of calcium ions in initiating transformation of lymphocytes.

TRANSFORMATION of lymphocytes by antigens and by nonspecific mitogens (lectins and anti-immunoglobulin sera) is a consequence of the binding of the mitogen to specific receptors on the cell surface1–3. The receptors rapidly form clusters (‘patches’) which may aggregate into polar ‘caps’4 at higher mitogen concentration. These events initiate a series of biochemical changes—enhanced uptake of ions and metabolites1, increased cyclic GMP5, and increased metabolism of phosphatidyl inositol and other phospholipids (ref. 6 and V. C. M., M. J. Hayman and M. J. C., unpublished)—which culminate in DNA synthesis and mitosis 48–72 h later. Although the sequence, relative importance and control of these events is not yet defined, there is evidence for a primary role for Ca2+. Phaseolus vulgaris phytohaemagglutinin (PHA) initiates a slow accumulation of Ca2+ by lymphocytes7,8 and its stimulation of DNA synthesis is inhibited by citrate9, EDTA (ref. 9) or EGTA (ref. 10). The inhibition is reversed by Ca2+. It has also been shown that inhibition by the low concentrations (1.4 mM) of EGTA used did not prevent binding of PHA (ref. 11). Although these results showed Ca2+ to be an important factor in initiation of transformation it was not clear whether influx of Ca2+ was the only or even the main consequence of the binding of PHA.

Vasopressin release from the isolated neurohypophysis induced by a calcium ionophore, X-537A.

TEN years ago observations on isolated neurohypophyses prompted the suggestion that action potentials in the hypothalamo-neurohypophysial tract release neurohypophysial hormones by promoting calcium influx and that the appearance of free calcium ions somewhere in the neurosecretory terminals provides the immediate stimulus for hormone extrusion1–3. Some time later it was argued that the role of calcium ions in stimulus-secretion coupling in this system and in many other secretory cells was to set in motion a common secretory mechanism, exocytosis4. Very recently the evidence supporting this view has been strengthened by light microscopic observations showing extrusion of secretory granules from mast cells on intracellular injection of calcium ions5. Meanwhile, electron microscopic6–11 and chemical12–16 evidence has borne out the conjecture that neurohypophysial hormones are secreted by exocytosis, and the case for calcium-influx as the initiating event has been strengthened by a number of observations set out in recent reviews15,16. These reviews make the further point that the neurohypophysis may be a useful model system for studying stimulus-secretion coupling in nerve cells generally: the involvement of calcium in release of neural transmitters is well-known and may have a similar explanation17,18.