The effect of the composition of the medium on the concentration of free calcium ions in the cells of the follicular wall in the common frog and in the clawed toad]

The concentration of intracellular free calcium ions in the follicle wall cells and in the follicle cells of Rana temporaria in Ringer solution is 150 +/- 10 and in the follicle wall cells of Xenopus laevis, 220 +/- 10 nM. In a chloride-free saline, its concentration in the same cells is 2.5-3 times that in Ringer solution. Voltage-dependent Ca(2+)-channel blockers diltiazem and verapamil (100 microM) reduce the level of intracellular free calcium ions in R. temporaria follicle wall cells cultivated in a chloride-free saline to 170 +/- 20 nM, which practically does not differ from the level in Ringer solution. Inhibitors (100 microM) decrease the rate of "spontaneous" maturation of R. temporaria follicle-enclosed oocytes both in chloride-free and Ringer solutions. It was concluded that an increased level of intracellular free calcium ions in the follicle cells, among other factors, may determine the stimulating effect of the medium (Ringer or chloride-free solution) on "spontaneous" maturation of follicle-enclosed amphibian oocytes. Voltage-dependent calcium channels appear to be involved in Ca2+ influx into the cells.     

Competition between Li+ and Mg2+ in neuroblastoma SH-SY5Y cells: a fluorescence and 31P NMR study.

Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.     

Calcium homeostasis during thymocyte apoptosis. I. Increase in cytosolic Ca2+ concentration at the early stage of apoptosis induced by hydrogen peroxide

The concentration of free cytosolic Ca2+ ([Ca2+]i), 45Ca2+ entry and the level of reduced glutathione (GSH) after x-irradiation in a dose of 4.5 Gy or 0.1 mM H2O2-treatment were investigated in isolated rat thymocytes during the period preceding electrophoretically detected DNA intranucleosomal fragmentation. Using fura-2 it was shown that the level of [Ca2+]i in X-irradiated thymocytes was not changed as compared with the control, while the GSH content was increased. The gradual increase in [Ca2+]i along with GSH level falling was detected in the H2O2-treated cells. 45Ca2+ entry in the cells exposed to apoptogenic stimuli was not enhanced. After addition of H2O2 to the cells previously treated with thapsigargin further [Ca2+]i increase in both normal and nominally calcium-free medium was detected. Cyclosporine A inhibited Ca2+-mobilizing effect of H2O2, but did not prevent it completely. The role of intracellular calcium depots in calcium homeostasis disturbance during oxidative stress and apoptosis is discussed.     

The strontium-induced calcium-release process and its implication in contractility of skeletal muscle of Rana ridibunda

The electrical and mechanical activities of isolated frog muscle fibres have been recorded simultaneously under conditions (chloride-free saline containing 78.5 mM strontium acetate substituting for NaCl and CaCl2) that allow the development of a tubular strontium permeability. Under voltage-clamp conditions a large part of the contraction is due to the slow inward ISr since both are inhibited by Ni (10 mM). The remaining component of contraction, which seems to be potential-dependent, is not abolished by tetracaine (40 microM) which blocks the current-dependent component. A cumulative effect of strontium, which is not observed in the presence of Ni, leads to a 60-80% reduction in contractility for an estimated [Sr]i near 3 X 10(-4) M while the ending of the contraction observed when Sr is replaced by Ba is never obtained. In contrast no cumulative effect is observed when Ca substitutes for Sr. The first evoked inward current following a caffeine contracture fails to elicit a contraction, but in Ringer 78.5 Sr, contractility is progressively restored by successive depolarizations up to an amplitude which corresponds to 25-40% of the maximum activity. In the presence of Ca instead of Sr, the restoration of contractility reaches 100%. This recovery does not occur when the inward current is blocked by Ni. After strontium loading, a calcium entry fails immediately and reversibly to induce a mechanical response while barium ions induce a progressive and irreversible block of contractility. These results suggest that the strontium entry during successive depolarizations leads to a progressive replacement of intrareticular calcium by strontium. When all the calcium ions have been substituted for by strontium ions, the contractile apparatus remains capable of being activated by intrareticular strontium.     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

Extracellular calcium and microwave enhancement of membrane conductance in snail neurons

Summary Microwave irradiation has been shown to decrease the input resistance of snail neurons. In this study, we examined the role of extracellular calcium in triggering the microwave-induced enhancement of membrane conductance. Two sets of experiments were conducted. In the first set, nerve cells were superfused using Ringer solution with added Cd²⁺ (0.9 mM) which is a known blocker of calcium channels. In the second set, cells were superfused with low Ca²⁺ (0.7 mM) Ringer solution. Microwave irradiation was conducted at 2,450 MHz for 30 min with a specific absorption rate of 13 mW/g. It was found that 7 mM to 0.7 mM lowering of Ca²⁺ in bathing solution as well as blocking of calcium channels in neuronal membrane by means of Cd²⁺ did not influence the fall in membrane resistance induced by microwave radiation. In fact, the observed changed in membrane resistance in these experiments were nearly equal to those observed for neurons superfused by normal Ringer's. Thus, these results rule out the possible contribution of external Ca²⁺ in the observed microwave effect. Experiments with high Ca²⁺ solution also support this conclusion.     

Aminophylline enhances contractility of frog skeletal muscle: an effect dependent on extracellular calcium

The effects of aminophylline (10-500 microM) on isometric twitch and tetanic forces were studied in vitro on frog semitendinosus muscle. Two hypotheses were tested: 1) that micromolar concentrations of aminophylline enhanced contractility of isolated skeletal muscle and 2) that the potentiating effect of aminophylline was dependent on the presence of extracellular calcium ions. Muscles were removed, placed in aerated Ringer solution at 20 degrees C, attached to a force transducer, and stimulated directly. Muscles in normal Ringer and aminophylline Ringer were compared throughout the frequency-force relationship from twitches to maximum tetanic force. Aminophylline increased twitch force significantly at concentrations as low as 25 microM. Over a range of stimulation frequencies, but especially at 10 and 20 Hz, aminophylline increased tetanic force. The potentiating effect of aminophylline (100 microM) was reduced or eliminated in calcium-free Ringer containing 10 mM magnesium. We conclude that aminophylline, at therapeutic concentrations, enhances muscle contractility, and the enhancement is dependent on the presence of extracellular calcium. These findings support the concept that aminophylline is effective in improving respiration in humans with airway obstruction by enhancing diaphragmatic contractility.     

Evoked transient intracellular free Ca2+ changes and secretion in isolated bovine adrenal medullary cells

When isolated bovine adrenal medullary cells are incubated with the lipid-soluble Quin 2 acetoxymethyl ester, the ester permeates the plasma membrane and enters the cytosol, where it is hydrolysed by endogenous enzymes to yield an impermeant fluorescent indicator (Quin 2) which is sensitive to Ca2+ in the 0.1 microM range. This technique permits the average intracellular free Ca2+ level ([Ca2+]i) to be determined in a suspension of adrenal medullary cells. Unstimulated cells have a [Ca2+]i of 97 +/- 4 nM (n = 69). This level seems independent of extracellular calcium in the range 0.5-2 mM. When the extracellular calcium concentration is lowered to ca. 10(-7) M, however, [Ca2+]i decreases. A transient increase in [Ca2+]i occurs when cells are challenged with either acetylcholine or a high potassium medium. The time course of the [Ca2+]i transient rises to a maximum within seconds, and decreases to basal levels over minutes. The maximum level of [Ca2+]i associated with secretion is very variable. Hexamethonium, methyoxyverapamil, and the absence of extracellular calcium block not only the secretory response but also the [Ca2+]i transient. The action of acetylcholine leading to the Ca2+]i transient is blocked when cells are suspended in a depolarizing medium. Extracellular magnesium inhibits both the [Ca2+]i transient and the secretory response evoked by acetylcholine. Secretion is, however, more sensitive to magnesium inhibition than is calcium entry. The magnitudes of the [Ca2+]i transient and the secretory response decrease as the concentration of intracellular Quin 2 increases. Measurements of the amount of indicator titrated with calcium, as a result of an acetylcholine or potassium challenge, suggest that the increase in the apparent calcium content of the cytosol might arise from two contributing sources of calcium entry.     

Calcium ions and their relation to sympathetic ganglionic transmission and to cyclic AMP levels in isolated superior cervical ganglion of the rat in vitro.

The rat superior cervical ganglion was investigated $\text{in vitro}$ by means of extracellularly recorded compound action potentials and of cyclic AMP content in relation to Ca²⁺-concentrations in the extracellular fluid. Threshold concentrations for the appearance of the potentials were lower than 1 mM. Raising the Ca²⁺-content in the Krebs solution above 40 mM led to an increase of the slow excitatory negative potential and to the appearance of the slow inhibitory positive wave, while the fast excitatory compound action potential disappeared. This effect was calcium-specific. On the other hand, cyclic AMP accumulation in the ganglion seems to be calcium-independent. While high Ca²⁺-concentrations (60 mM) in the medium led to a significant decrease of the cyclic AMP content, neurotransmitter-induced cyclic AMP accumulation occurred regardless from varying the extracellular calcium content. High external Ca²⁺ antagonized the ability of the β-adrenergic blocker propranolol in preventing isoproterenol-induced cyclic AMP accumulation to some extent. It was concluded that calcium ions are necessary for the appearance of fast and slow postganglionic compound action potentials in the ganglion. These events seemed to be independent from drug-induced changes in the ganglionic cyclic AMP content.     

Repolarization of striated muscle fibers by the antifatigue agent,K-Mg-aspartate

Resting membrane potentials of isolated frog sartorius muscles were measured under a variety of conditions using intracellular glass microelectrodes. Muscle cells depolarized by the addition of 5.0 or 10.0 mM KCl to the bathing Ringer solution can be repolarized some 5 to 10 mV by the substitution of an equivalent amount of K-aspartate for KCl in the presence of 2.0 mM Mg⁺⁺. The repolarization produced by this method persists when the muscle is again placed in the initial KCl solution, thus eliminating the possibility that the hyperpolarization is due to the reduction of chloride in the bathing medium. If for some reason the resting membrane potential of the muscle fibers is considerably below (less negative than) the normal level of 92 mV reported for muscles bathed in 2.5 mM Ringer solution, the substitution of 2.5 mM K-aspartate for the 2.5 mM KCl and the addition of 2.0 mM Mg-aspartate to the Ringer solution will, within 15 minutes, repolarize the fiber to the normal level. Magnesium ions alone will not produce the observed repolarization nor can it be attributed to a reduction in the activity of the potassium in the Ringer solution.     

The influence of calcium on the deformability of human granulocytes

Experiments were carried out to determine the importance of extra- and intracellular calcium for the deformability of granulocytes during filtration tests. At low calcium concentration (0.1 mM), granulocytes are more deformable than at the physiological free-calcium concentration of 1.25 mM. Increasing calcium concentrations up to 10 mM do not further impair the deformability. Parallel measurements of the intracellular calcium concentration by means of the fura fluorescence method were performed to explain this. Extracellular calcium concentrations between 1.25 mM and 10 mM had no influence on the intracellular calcium level. A lower extracellular calcium concentration (0.1 mM), however, decreased the intracellular calcium level. Therefore, the measurements of the intracellular calcium concentrations are consistent with the deformability results. Studies with the calcium entry blocker nifedipine suggested that a low intracellular calcium improves the deformability of granulocytes. It is concluded; (i) the physiological calcium concentration of 1.25 mM is stressful for isolated granulocytes, and (ii) the intracellular calcium level plays a crucial role in granulocyte deformability, i.e. the lower the intracellular calcium concentration the greater the deformability.     

Relationship between extra and intracellular sources of calcium and the contractile effect of thiopental in rat aorta

To evaluate the relationship between the vasocontractile effect of thiopental and the extra and intracellular sources of Ca2+, we analyzed both the contractile effect of the barbiturate on rat aortic rings and its ability to modify the intracellular calcium concentration in cultured rat aorta smooth muscle cells. Thiopental (10-310 microg/mL) contracted aortic rings only in the presence of extracellular Ca2+, and this effect was not blocked by verapamil or diltiazem. On the contrary, Ca2+ (0.1-3.1 mM) evoked contractions only when thiopental (100 microg/mL) was present. Although in calcium-free solution thiopental (100 microg/mL) did not contract aortic rings, it abolished the contractile effect of either phenylephrine (10(-6) M) or caffeine (10 mM). Finally, thiopental augmented the intracellular calcium concentration in cultured smooth muscle cells incubated either in the presence or absence of calcium. In conclusion, thiopental's vasocontractile effect depends on extracellular calcium influx, which is independent of L-calcium channels. The increase in intracellular Ca2+ concentration elicited by thiopental in Ca2+-free solution and its ability to block the effect of phenylephrine and caffeine suggest that this barbiturate can deplete intracellular pools of calcium. Therefore, the calcium entry pathway associated with the contractile effect of thiopental may correspond to the capacitative calcium entry model.     

Elevation of cytosolic [Ca2+] due to intracellular Ca2+ release retards carbachol stimulation of divalent cation entry in rat parotid gland acinar cells

This study examines the activation of divalent cation entry into rat parotid gland acinar cells by using Mn2+ as a Ca2+ surrogate cation. Following muscarinic-cholinergic stimulation of dispersed parotid acini with carbachol (10 microM), the onset of internal Ca2+ release (cytosolic [Ca2+], [Ca2+]i, increase) and the stimulation of Mn2+ entry (increase in fura2 quenching) are not simultaneously detected. [Ca2+]i elevation, due to intracellular release, is detected almost immediately following carbachol addition and peak [Ca2+]i increase occurs at 6.0 +/- 0.8 sec. However, there is an interval (apparent lag) between carbachol addition and the detection of stimulated Mn2+ entry. This apparent lag is decreased from 26 +/- 3.1 sec to 9.2 +/- 1.5 sec when external Mn2+ ([Mn2+]0) is increased from 12.5 to 500 microM. It is not decreased further with increase in [Mn2+]0 from 500 microM to 1 mM (9.8 +/- 2.1 sec), although both intracellular free Mn2+ and [Mn2+-fura2]/[fura2] increase. Thus, at [Mn2+]0 < 500 microM, the observed lag time is partially due to a limitation in the magnitude of Mn2+ entry. Furthermore, neither peak [Ca2+]i nor the time required to reach peak [Ca2+]i is significantly altered by [Mn2+]0 (12.5 microM to 1 mM). At every [Mn2+]0 tested (i.e., 12.5 microM-1 mM), the apparent lag is significantly greater than the time required to reach peak [Ca2+]i. However, when carbachol stimulation of the [Ca2+]i increase is attenuated by loading the acini with the Ca2+ chelator, 2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA), there is no detectable lag in carbachol stimulation of Mn2+ entry (with 1 mM [Mn2+]0). Importantly, in BAPTA-loaded acini, carbachol stimulates Mn2+ entry via depletion of the internal Ca2+ pool and not via direct activation of other divalent cation entry mechanisms. Based on these results, we suggest that the apparent lag in the detection of carbachol stimulation of Mn2+ entry into parotid acinar cells is due to a retardation of Mn2+ entry by the initial increase in [Ca2+]i, due to internal release, which most likely occurs proximate to the site of divalent cation entry.     

Calcium influx factor (CIF) as a diffusible messenger for the activation of capacitative calcium entry in Xenopus oocytes.

Acid extracts of thapsigargin-treated Xenopus oocytes revealed Ca2(+)-dependent Cl- currents by microinjection into Xenopus oocytes. These currents were detected in highly purified fractions by carrying out a sequence of purification steps including gel filtration chromatography and high performance thin layer chromatography. The nature of the membrane currents evoked by the highly purified fractions were carried by chloride ions as blockade by the selective chloride channel blocker 1 mM niflumic acid. Injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) eradicated the current activities, indicating that the current responses are completely Ca2(+)-dependent. Moreover, the currents were sensitive to the removal of extracellular calcium, indicating the dependence on calcium entry through plasma membrane calcium entry channels. These results elucidate that the highly purified fractions aquired by thapsigargin-stimulated oocytes is an authentic calcium influx factor (CIF). Thus, the detection of increased CIF production from thapsigargin treatment in Xenopus oocytes would give strong support for the existence of CIF as a diffusible messenger for the activation of capacitative calcium entry pathways in Xenopus oocytes.     

Change in the activity of calcium ions during illumination of a suspension of visual cell outer segment fragments]

Changes in the activity of calcium ions in the medium containing outer fragments suspension of bovine eye retina rods have been studied by the method of calcium-selective electrodes. Illumination of the suspension increases calcium ion activity in the incubation medium. Photoinduced yield of calcium ions depends on Ca+2 concentration: it equals 0.11+/-0.015 M Ca2+/1m rodopsin in the medium containing 0.1 mM CaCl2 and 0.046+/-0.002Ca2+/1M rodopsin in the medium containing 0.05 mM CaCl2. In the medium containing more than 10(-4) M CaCl2 both an increase and a decrease of Ca2+ ions have been observed.     

Involvement of cellular calcium incorporated from the medium in production of inositol trisphosphate in A-431 cells

The property of intensive 45Ca2+ uptake by A-431 human epidermoidal carcinoma cells was indicated to be an influx, not binding to the cell surface, since the two apparent dissociation constants (Kd) between 45Ca2+ and cells were almost the same when measured in either the presence or absence of 1 mM [ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA); these constants were approximately 5-10 x 10(-6) and 1 x 10(-4) M, respectively, which are much higher than the chelating constant of EGTA for Ca2+ (approximately 10(-11) M). Furthermore, addition of A23187, a calcium ionophore, rapidly released the 45Ca2+ incorporated into cells at both 37 degrees C and 0 degrees C. The 45Ca2+ associated with the cells was slowly released or exchanged when cells were incubated in medium depleted of Ca2+, or in that containing 1 mM non-radioactive Ca2+. The ability of A-431 cells to respond to extracellular ATP by elevating their level of intracellular calcium ions, as well as by producing inositol trisphosphate (InsP3), was suppressed in cells depleted of cellular calcium. These data suggest that calcium ions are extensively incorporated or exchanged with those outside the cells, maintained as stored calcium, and involved in production of InsP3, when A-431 cells are stimulated by ATP to trigger the signal transduction system.     

NO upregulation of a cyclic nucleotide-gated channel contributes to calcium elevation in endothelial cells

We investigated whether nitric oxide (NO) upregulates a cyclic nucleotide-gated (CNG) channel and whether this contributes to sustained elevation of intracellular calcium levels ([Ca(2+)](i)) in porcine pulmonary artery endothelial cells (PAEC). Exposure of PAEC to an NO donor, NOC-18 (1 mM), for 18 h increased the protein and mRNA levels of CNGA2 40 and 50%, respectively (P < 0.05). [Ca(2+)](i) in NO-treated cells was increased 50%, and this increase was maintained for up to 12 h after removal of NOC-18 from medium. Extracellular calcium is required for the increase in [Ca(2+)](i) in NO-treated cells. Thapsigargin induced a rapid cytosolic calcium rise, whereas both a CNG and a nonselective cation channel blocker caused a faster decline in [Ca(2+)](i), suggesting that capacitive calcium entry contributes to the elevated calcium levels. Antisense inhibition of CNGA2 expression attenuated the NO-induced increases in CNGA2 expression and [Ca(2+)](i) and in capacitive calcium entry. Our results demonstrate that exogenous NO upregulates CNGA2 expression and that this is associated with elevated [Ca(2+)](i) and capacitive calcium entry in porcine PAEC.     

An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding

The extreme thermal stabilization achieved by the introduction of a disulfide bond (G8C/N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease from Bacillus stearothermophilus[Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997) J Biol Chem272, 11152-11156] was attributed to the fixation of the loop region 56-69. In this study, the role of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfolding and autoproteolysis kinetics of pWT and G8C/N60C was analyzed by fluorescence spectroscopy, far-UV CD spectroscopy and SDS/PAGE. First-order rate constants (kobs) were evaluated by chevron plots (ln kobs vs. GdnHCl concentration). The kobs of unfolding showed a difference of nearly six orders of magnitude (DeltaDeltaG# = 33.5 kJ.mol(-1) at 25 degrees C) between calcium saturation (at 100 mM CaCl2) and complete removal of calcium ions (in the presence of 100 mM EDTA). Analysis of the protease variant W55F indicated that calcium binding-site III, situated in the critical region 56-69, determines the stability at calcium ion concentrations between 5 and 50 mM. In the chevron plots the disulfide bridge in G8C/N60C shows a similar effect compared with pWT as the addition of calcium ions, suggesting that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autoproteolysis. Owing to the presence of the disulfide bridge, the DeltaDeltaG# is 13.2 kJ.mol(-1) at 25 degrees C and 5 mM CaCl2. Non-linear chevron plots reveal an intermediate in unfolding probably caused by local unfolding of the loop 56-69. The occurrence of this intermediate is prevented by calcium concentrations of > 5 mM, or the introduction of the disulfide bridge G8C/N60C.     

Conformational changes in human prothrombin as detected by antibody populations

The amino-terminal peptides of human prothrombin corresponding to residues 1-51 and 52-156 have been isolated from a thrombin digest of prothrombin fragment 1. The products of digestion were purified by means of barium citrate and ammonium sulfate precipitations, followed by gel filtration and hydroxyapatite chromatographies. They were identified by their molecular sizes as well as their amino acid compositions. Peptides 1-51 (F1A) and 52-156 (F1B) were used as affinity ligands for the isolation of antibody populations from antisera that were elicited against human prothrombin or prothrombin fragment 1. These antibody populations displayed restricted specificity for the respective ligands as shown by competitive radioimmunoassays. They were used to study the conformational changes in prothrombin and fragment 1. The F1A-specific antibody populations detected a conformational change which is stabilized by calcium ions and which has a transition midpoint at approximately 0.2 mM calcium ion concentration. The F1B-specific antibody populations identified a different conformational change which is destabilized by calcium ions and which has a transition midpoint at approximately 0.5 mM calcium.     

Calcium Transport by Primary Cultured Neuronal and Glial Cells from Chick Embryo Brain

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca²⁺ and 5.4 mM-K⁺. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 MM. The effect of high external potassium was to increase the V_max value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl₂, but 0.5 μM-tet-rodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.