External Ca Concentration Research Articles

Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells

The fractional contribution of Ca to current flow through neuronal-type nicotinic acetylcholine receptor channels was determined by quantitative fluorescence microfluorimetry using fura-2. The method, which has been applied already to several types of cells and channels is described in detail here. At -70 mV and 2 mM external Ca concentration it was found that Ca contributes 2.5% to the net current. The fractional contribution was found to be voltage dependent, increasing at negative potentials e-fold for a 110 mV potential difference. Total non-specific cation current was found to have a bell-shaped dependence on external Ca concentration peaking at 2 mM.

Factors influencing the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction

1. The effects of several conditions and agents on the twin-pulse facilitation of the release of transmitter at the mouse neuromuscular junction in low-Ca 2+ high-Mg 2+ bathing solutions were examined. 2. Twin-pulses gave two endplate potentials (epps) with first ( m1) and second ( m2) quantal contents. The ratio of m2/ m1 was taken as a measure of the degree of facilitation. 3. The mean value of this ratio was >1. Individual ratios fluctuated widely at junctions with smaller values of m1 but were focused around 1 at junctions with larger values of m1. Thus, some populations of junctions with smaller values of m1 contributed to an increment in the mean ratio. 4. The mean ratio was virtually constant irrespective of changes in the spontaneous and evoked release of transmitter at temperatures between 20 and 36°C and at external concentrations of Ca 2+ from 0.4 to 0.8 mM. 5. 4-Aminopyridine(4-AP) slightly but significantly increased this ratio with increases in m1 and m2 at temperatures of 24 and 36°C. Ouabain slightly but significantly reduced the ratio, with increases in m1 and m2. The steadiness of the ratio was maintained in the presence of caffeine, high K +, neomycin or omega-conotoxin irrespective of changes in m1 and m2, except in the case of omega-contoxin. 6. Spontaneous output at 36°C increased in the presence of 4-aminopyridine, ouabain, caffeine, high K + or neomycin. 7. These results indicate that maintenance of a stable value of the ratio of m2 to m1 is a dominant feature. The enhancement of the facilitation by 4-AP implies a contribution of another depolarization-dependent factor which may include a prolongation of the period of inflow of Ca 2+ at the terminal.

Characterization of TTX-sensitive and TTX-resistant sodium currents in small cells from adult rat dorsal root ganglia.

1. The whole-cell patch-clamp technique was used to investigate the characteristics of two types of sodium current (INa) recorded at room temperature from small diameter (13-25 microns) dorsal root ganglion (DRG) cells, isolated from adult rats and maintained overnight in culture. 2. Sodium currents were isolated pharmacologically. Internal Cs+ and external tetraethylammonium (TEA) ions were used to suppress potassium currents. A combination of internal EGTA, internal F-, a low (10 microM) concentration of external Ca2+ and a relatively high (5 mM) concentration of internal and external Mg2+ was used to block calcium channels. The remaining voltage-dependent currents reversed direction at the calculated sodium equilibrium potential. Both the reversal potential and magnitude of the currents exhibited the expected dependence on the external sodium concentration. 3. INa subtypes were characterized initially in terms of their sensitivity to tetrodotoxin (TTX). TTX-sensitive (TTXs) currents were at least 97% suppressed by 0.1 microM TTX. TTX-resistant (TTXr) INa were recorded in the presence of 0.3 microM TTX and appeared to be reduced in amplitude by less than 50% in 75 microM TTX (n = 1). 4. As in earlier studies, the peak of the current-voltage relationship, the mid-point of the normalized conductance curve and the potential (Vh) at which the steady-state inactivation parameter (h infinity) was 0.5 were found to be significantly more depolarized for the TTXr INa (by ca 10, 14 and 37 mV respectively). There was little difference in the slope at the mid-point of the normalized conductance curves (the mean slope factors were 5.1 mV for the TTXs INa and 4.9 mV for the TTXr current) but the h infinity curves for TTXr currents were significantly steeper than those for TTXs currents (mean slope factors of 3.8 and 11.5 mV respectively). Both the time to peak and the decay time constant of the peak current recorded from a holding potential of -67 mV were more than a factor of three slower for the TTXr INa than for the TTXs current. 5. However, in direct contrast to the difference in activation and decay kinetics, 'slow' TTXr INa recovered from inactivation at -67mV, or reprimed, more than a factor of ten faster than 'fast' TTXs INa. 6. The differences apparent in both the repriming kinetics of TTXs and TTXr INa at -67 mV and the kinetics of the decay phase of the peak INa are shown to be explicable largely in terms of the voltage dependence of their respective inactivation systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium mediated cytokinin action on chlorophyll synthesis in isolated embryo of Scots pine

Chlorophyll (Chl) synthesis in isolated Scots pine embryos depended on exogenous application of cytokinin (CK) and Ca2+. At a constant benzyladenine (BA) level (4.4×10-5 M) 10-4 to 10-2 M Ca2+ concentrations in mineral medium were optimum for Chl biosynthesis under both light and dark. At a zero or very low (10-6 M) concentration of external Ca2+, Chl synthesis was relatively more Ca2+-dependent in embryos cultured in darkness than in the light, which suggested that the light: (a) stimulated the transport of Ca2+ from external sources to cytosol, and/or (b) interacted with Ca2+ directly in the pathway of Chl biosynthesis. The need of external Ca2+ was evidenced in experiments with modulators of Ca2+-transport systems. The reduction of the inward current of Ca2+ from readily accessible external sites by chelating agent (ethylene glycol-bis (beta-aminoethyl ether-N,N,N'N'-tetraacetic acid, EGTA) and Ca2+-channel blockers canceled the formation of Chl. The effect of EGTA depended on the level of external Ca2+. Inhibitory action of Ca2+-channel blockers depended on their kind and concentration: at the 10-5 M concentration La3+>verapamil>nifedipine inhibited Chl formation. In the presence of Ca2+, the Ca2+-agonist A 23187 mimicked the BA effect and about 92% of Chl was synthesized as compared with the BA variant. Low concentrations of calmodulin antagonists reduced the amounts of Chl. Calmodulin was included in a second messenger system for BA action in promoting Chl biosynthesis in isolated Scots pine embryos.

Unloaded shortening after a quick release of a contracting, single fibre from crayfish slow muscle.

The time course of shortening at zero load was studied by the slack test method during tetanic contractions in isolated, single, slow muscle fibres of the crayfish. In 28 of 32 shortenings (from 14 different fibres) a biphasic shortening was seen, which consisted of an initial high-velocity phase lasting 3.3-20.8 ms and a following slow-velocity phase lasting for the entire time examined (up to 89.2 ms). Provided that the shortening occurred uniformly along the fibre length, velocity in the initial phase, V1, of the biphasic shortening was 14.4 +/- 3.4 (mean +/- SD, n = 10) microns s-1 per half sarcomere at Lo, the slack length, at 20 degrees C, while that in the second phase, V2, was 7.4 +/- 1.4 microns s-1 per half sarcomere. Lowering temperature decreased both V1 and V2 with Q10 = 1.4 for V1 and 2.0 for V2. Lowering the external Ca concentration from 15 mM, the standard, to 2 mM resulted in a tetanic tension below one-third of that at 15 mM Ca and decreased both V1 (t test; p < 0.01) and V2 (p < 0.001). Prestretching the fibre to 1.5 Lo had no significant effect on V2 (p < 0.3) but increased V1 (p < 0.001). The distance shortened during the initial high-velocity phase, LV1, was 4.0 +/- 1.8% Lo (mean +/- SD, n = 10) at 20 degrees C or about 0.14 microns per half sarcomere on average. LV1 was independent of the tetanic tension level when it was changed by lowering the external Ca concentration or temperature in the same fibre. Prestretching the fibre to 1.5 Lo, at which the sum of the active and the resting tension was lower than Po at Lo in two of three fibres, increased LV1 significantly (p < 0.001). The independency of LV1 from the tension level indicates that the initial high-velocity phase was not from shortening of some inert components in the fibre. One possibility is that the initial high-velocity phase was brought about by an acceleration of shortening by a compressive force, the origin of which has been discussed. The slow-velocity phase seemed to result from the crossbridge turnover with little exogeneous stress on myofilaments. Four different fibres exhibited an unloaded shortening with a constant velocity during the entire time examined (29.9-61.8 ms). This type of shortening had a velocity between the usual V1 and V2 values, suggesting that a compressive force accelerated the shortening during the entire time.

Changes in the Cytoplasmic and Vacuolar pH in Intact Cells of Mung Bean Root-Tips under High-NaCl Stress at Different External Concentrations of Ca&lt;sup&gt;2+&lt;/sup&gt; Ions

Changes in the Cytoplasmic and Vacuolar pH in Intact Cells of Mung Bean Root-Tips under High-NaCl Stress at Different External Concentrations of Ca&lt;sup&gt;2+&lt;/sup&gt; Ions

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca(2+) uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 μM CaCl2 solution (pH 4.5) for 3 d. Subsequently, net Ca(2+) fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca(2+)-selective microelectrode. The kinetics of Ca(2+) uptake into cells of the root apex, for external Ca(2+) concentrations from 20 to 300 μM, were found to be quite similar for both cultivars in the absence of external Al; Ca(2+) transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 μM total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca(2+) absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca(2+) uptake over the entire Ca(2+) concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca(2+) uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 μM) caused the K m for Ca(2+) uptake to increase with each rise in Al concentration, from approx. 100 μM in the absence of Al to approx. 300 μM in the presence of 10 μM Al, while having no effect on the V max. The same Al exposures had little effect on the kinetics of Ca(2+) uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca(2+) transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca(2+)-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.

The role of the plasma-membrane Ca2+-ATPase in Ca2+ homeostasis in Sinapis alba root hairs

The regulation of cytosolic Ca(2+) has been investigated in growing root-hair cells of Sinapis alba L. with special emphasis on the role of the plasmamembrane Ca(2+)-ATPase. For this purpose, erythrosin B was used to inhibit the Ca(2+)-ATPase, and the Ca(2+) ionophore A23187 was applied to manipulate cytosolic free [Ca(2+)] which was then measured with Ca(2+)-selective microelectrodes. (i) At 0.01 μM, A23187 had no effect on the membrane potential but enhanced the Ca(2+) permeability of the plasma membrane. Higher concentrations of this ionophore strongly depolarized the cells, also in the presence of cyanide. (ii) Unexpectedly, A23187 first caused a decrease in cytosolic Ca(2+) by 0.2 to 0.3 pCa units and a cytosolic acidification by about 0.5 pH units, (iii) The depletion of cytosolic free Ca(2+) spontaneously reversed and became an increase, a process which strongly depended on the external Ca(2+) concentration, (iv) Upon removal of A23187, the cytosolic free [Ca(2+)] returned to its steady-state level, a process which was inhibited by erythrosin B. We suggest that the first reaction to the intruding Ca(2+) is an activation of Ca(2+) transporters (e.g. ATPases at the endoplasmic reticulum and the plasma membrane) which rapidly remove Ca(2+) from the cytosol. The two observations that after the addition of A23187, (i) Ca(2+) gradients as steep as-600 mV could be maintained and (ii) the cytosolic pH rapidly and immediately decreased without recovery indicate that the Ca(2+)-exporting plasma-membrane ATPase is physiologically connected to the electrochemical pH gradient, and probably works as an nH(+)/Ca(2+)-ATPase. Based on the finding that the Ca(2+)-ATPase inhibitor erythrosin B had no effect on cytosolic Ca(2+), but caused a strong Ca(2+) increase after the addion of A23187 we conclude that these cells, at least in the short term, have enough metabolic energy to balance the loss in transport activity caused by inhibition of the primary Ca(2+)-pump. We further conclude that this ATPase is a major Ca(2+) regulator in stress situations where the cytosolic Ca(2+) has been shifted from its steady-state level, as may be the case during processes of signal transduction.

Calcium‐salinity interactions affect ion transport in Chara corallina

ABSTRACTDetached internodes of Chara corallina survived in solutions containing 100 mol m−3 NaCl when the external concentration of Ca2+ was greater than 1 mol m−3. Na+ influx was roughly proportional to external Na+ up to 100 mol m−3 NaCl. Na+ influx involved two components: a Ca2+‐insensitive influx which allowed the passage of Na+ independently of external Ca2+; and a Ca2+‐inhibitable mechanism where Na+ influx was inversely proportional to external Ca2+. The Ca2+‐inhibitable Na+ influx was similar to the Ca2+‐inhibitable K+ influx. Mg2+ and Ba2+ were able to substitute for Ca2+ in partially inhibiting Na+ influx in the absence of external Ca2+. The effect of Ca2+ appears specific to Na+ and K+ influx since the effects of a Ca2+‐free solution on the influx of some other cations, anions and neutral compounds is small. It is suggested that Na+ influx via the Ca2+‐inhibitable mechanism represents Na+ leakage through K+ channels and that cell death at high salinity occurs due to a cytotoxic Na+ influx via this mechanism.

Calcium transients in identified leech glial cells in situ evoked by high potassium concentrations and 5-hydroxytryptamine.

We have recorded the fluorescence of Fura-2-loaded, identified glial cells in the neuropile of the central nervous system of the leech Hirudo medicinalis using the ratio of emission at 350 nm excitation to that at 380 nm excitation as an indicator of intracellular Ca2+ concentration ([Ca2+]i). The neuropile glial cells were exposed by mechanically removing the overlying ganglionic capsule and the neuronal cell bodies and were then impaled using a microelectrode under visual control to inject Fura-2 ionophoretically. The resting Ca2+ level was measured using digitonin or Triton to permeabilize the cell membrane at different external concentrations of Ca2+; it was found to vary between 5 and 79 nmol l-1 and averaged 32 +/- 23 nmol l-1 (+/- S.D., N = 7). Raising the external K+ concentration from 4 to 20 mmol l-1 or adding 50 mumol l-1 5-hydroxytryptamine (5-HT) produced a rapid, reversible rise in [Ca2+]i. During prolonged exposure to high [K+] or 5-HT, [Ca2+]i remained high. Upon restoring normal external [K+] or removing 5-HT, [Ca2+]i returned to its initial resting value within 1-2 min. The responses of [Ca2+]i to high [K+] and 5-HT were greatly reduced in nominally Ca(2+)-free saline, suggesting that the [Ca2+]i transients required an influx of Ca2+ into the cells. In the presence of 5-HT, the rise in [Ca2+]i was accompanied by a decrease in the resistance and an increase in the responsiveness to K+ of the glial cell membrane, indicating the existence of a Ca(2+)-dependent K+ conductance elicited by 5-HT.

Triphenyltin-induced increase in the intracellular Ca2+ of dissociated mammalian CNS neuron: its independence from voltage-dependent Ca2+ channels.

To test the possibility that triphenyltin (TPT) increases the intracellular Ca2+ ([Ca2+]i) in neurons as found previously in thymocytes, the effect of TPT on [Ca2+]i was examined in rat cerebellar neurons by a flow-cytometer with fluorescent dyes. TPT at concentrations ranging from 3 x 10(-7) M to 1 x 10(-5) M dose-dependently increased the [Ca2+]i. The TPT-induced increase in [Ca2+]i was not attenuated by a Ca2+ channel blocker, suggesting that it was not dependent on voltage-dependent Ca2+ channels. As the concentration of external Ca2+ ([Ca2+]e) increased, TPT produced a more profound increase in the [Ca2+]i. However, the increase in the [Ca2+]i by TPT was observed even in nominally [Ca2+]e-free solution. These results suggest two possibilities. First, TPT may promote Ca(2+)-influx to the neuron. Secondly, TPT may affect the intracellular Ca-store sites. This study is relevant to the neurotoxicity of organotins because it has become progressively clear that sustained increases in the [Ca2+]i can activate various Ca(2+)-dependent degradative processes.

Fluoxetine-induced inhibition of synaptosomal [ 3H] 5-HT release: Possible CA 2+-channel inhibition

Fluoxetine, a selective 5-HT uptake inhibitor, inhibited 15 mM K +-induced [ 3H] 5-HT release from rat spinal cord and cortical synaptosomes at concentrations > 0.5 uM. This effect reflected a property shared by another selective 5-HT uptake inhibitor paroxetine but not by less selective uptake inhibitors such as amitriptyline, desipramine, imipramine or nortriptyline. Inhibition of release by fluoxetine was inversely related to both the concentration of K + used to depolarize the synaptosomes and the concentration of external Ca 2+. Experiments aimed at determining a mechanism of action revealed that fluoxetine did not inhibit voltage-independent release of [ 3H] 5-HT release induced by the Ca 2+-ionophore A 23187 or Ca 2+-independent release induced by fenfluramine. Moreover the 5-HT autoreceptor antagonist methiothepin did not reverse the inhibitory actions of fluoxetine on K +-induced release. Further studies examined the effects of fluoxetine on voltage-dependent Ca 2+ channels and Ca 2+ entry. Whereas fluoxetine and paroxetine inhibited binding of [ 3H] nitrendipine to the dihydropyridine-sensitive L-type Ca 2+ channel, the less selective uptake inhibitors did not alter binding. The dihydropyridine antagonist nimodipine partially blocked fluoxetine-induced inhibition of release. Moreover enhanced K +-stimulated release due to the dihydropyridine agonist Bay K 8644 was reversed by fluoxetine. Fluoxetine also inhibited the K +-induced increase in intracellular free Ca 2+ in fura-2 loaded synaptosomes. These data are consistent with the suggestion that fluoxetine inhibits K +-induced [ 3H] 5-HT release by antagonizing voltage-dependent Ca 2+ entry into nerve terminals.

Triphenyltin-Induced Increase in the Intracellular Ca2+of Dissociated Mammalian CNS Neuron: Its Independence from Voltage-Dependent Ca2+Channels

To test the possibility that triphenyltin (TPT) increases the intracellular Ca2+([Ca2+]i) in neurons as found previously in thymocytes, the effect of TPT on [Ca2+]i was examined in rat cerebellar neurons by a flow-cytometer with fluorescent dyes. TPT at concentrations ranging from 3 × 10-7M to 1 × 10-5M dose-dependently increased the [Ca2 +]i. The TPT-induccd increase in [Ca2 +]i was not attenuated by a Ca2+channel blocker, suggesting that it was not dependent on voltage-dependent Ca2+channels. As the concentration of external Ca2+([Ca2+]e) increased, TPT produced a more profound increase in the [Ca2+]i. However, the increase in the [Ca2+]i by TPT was observed even in nominally [Ca2 +]e-free solution. These results suggest two possibilities. First, TPT may promote Ca2 +-influx to the neuron. Secondly, TPT may affect the intracellular Ca-storc sites. This study is relevant to the neurotoxicity of organotins because it has become progressively clear that sustained increases in the [Ca2+]i can activate various Ca2+-dependent degradative processes.

Whole-cell K+ current across the plasma membrane of guard cells from a grass: Zea mays

Knowledge of ion fluxes in the dumbell-shaped guard cells of grass species has been limited by the difficulty of obtaining isolated epidermes or guard-cell protoplasts for use in radioactive-tracer or electrophysiological studies. We describe here a method for isolating guard-cell protoplasts from Zea mays L. Whole-cell patch clamp has been used to measure K(+)-channel current across the plasma membrane surrounding these protoplasts. Two populations of K(+)-permeable channels have been identified. Hyperpolarization of the membrane to potentials (Vm) more negative than -100 mV results in inward K(+) current through one population of channels. Inward current activation is faster than in the dicotyledon, Vicia faba L. (mean activation half-time 26 ms (Z. mays) versus 123 ms (V. faba) at Vm=-180 mV). Steady-state current density is less than in V. faba (-22 μA · cm(-2) (Z. mays) versus -40 μA · cm(-2) (V. faba) at Vm=- 180 mV in 12 mM external K(+)). Depolarization of the membrane to potentials more positive than -20 mV results in outward K(+) current through a second population of channels; these channels activate and (upon repolarization of the membrane) deactivate more slowly than in V. faba (mean activation half-time 375 ms (Z. mays) versus 187 ms (V. faba) at Vm=+ 80 mV) but result in a similar steady-state current density (23.8 μA · cm(-2) (Z. mays) versus 28.7 μA · cm(-2) (V. faba) at Vm= + 80 mV with 105 mM internal K(+)). Omission of K(+) eliminates the current. The K(+) current is sensitive to both internal and external Ca(2+) concentration: increasing internal Ca(2+) from 2 nM to 0.2 μM or increasing external Ca(2+) from 1 mM to 8.5 mM reduces the magnitude of both inward and outward current.

Calcium permeability of neuronal nicotinic acetylcholine receptor channels in PC12 cells

The conductance properties of neuronal nicotinic acetylcholine receptors (neuronal nAChR channels) in PC12 cells were studied using single channel and whole cell gigaohm seal voltage clamp techniques. Acetylcholine receptor agonists were applied using external pipettes. Neuronal nAChR channels were selective for monovalent cations, and were impermeable to anions. Based on measurements of shifts in reversal potential with changes in external Na and K ion concentrations and evaluation of the GHK constant field relation, P K/P Na was 1.45 (ignoring Ca permeability). P Ca/P K was 1.75 based on the shift in reversal potential seen when raising the external Ca concentration from 2 to 50 mM (holding the external Na ion concentration constant), and determining the best fit to the extended GHK constant field relation using ionic activities. This value is larger than that determined for the muscle-type nAChR channel. Evaluation of the extended GHK current equations using these permeability coefficients indicated that at subthreshold voltages approximately 5% of total current through neuronal nAChR channels will be carried by Ca ions. Thus, these channels may be important mediators of activity-dependent Ca influx in the nervous system.

The effects of aluminum and calcium on the growth and nutrition of selected ectomycorrhizal fungi of jack pine

The influence of Al and Ca on the growth, in axenic culture, of three ectomycorrhizal fungi associated with jack pine (Pinus banksiana Lamb.) was examined. Isolates of Hebeloma crustuliniforme (St. Amans) Quél., Rhizopogon rubescens (Tul.) Tulasne, and Suillus tomentosus (Kauff.) Singer, Snell &amp; Dick were obtained from basidiocarps collected in jack pine stands of different ages. Basidiocarps and the soil surrounding them were analyzed for Al and Ca, as well as other elements. Each fungal species was grown for 4 weeks in nutrient solutions containing 37, 185, 370, or 740 μM Al combined in a factorial design with 25, 125, 250, or 500 μM Ca and maintained at pH 3.8. Growth of all three fungal species was reduced at 370 μM Al. Significant interaction was found between fungal species and Al treatment for all six elements measured in mycelial tissue. Stepwise increments in external Al concentration resulted in reduced mycelial concentrations of Ca, Mg, and K, and increased mycelial concentrations of Al, P, and Fe in H. crustuliniforme. High external Al levels resulted in reduced mycelial concentrations of all elements measured except Al and P in R. rubescens. In contrast, for S. tomentosus the same external Al levels increased the mycelial concentrations of all elements except Ca. The response of the three ectomycorrhizal fungi to Ca also differed. Growth of H. crustuliniforme was stimulated by stepwise increments in external Ca concentrations from 25 to 500 μM. Increments in calcium had no effect on the growth of R. rubescens. High external levels of Ca acted synergistically with high external Al concentrations to reduce growth by S. tomentosus. Unlike the other two species, the response of S. tomentosus to Al and Ca could not have been predicted from the soil and basidiocarp analyses. Alterations in Ca to Al ratios of soils may influence the succession of ectomycorrhizal fungi on conifer root systems. Key words: ectomycorrhizae, jack pine, aluminum, calcium, basidiocarps.

A transient outward current dependent on external calcium in rat cerebellar granule cells

The outward potassium current of rat cerebellar granule cells in culture was studied with the whole-cell patch-clamp method. Two voltage-dependent components were identified: a slow current, resembling the classical delayed rectifier current, and a fast component, similar to an IA-type current. The slow current was insensitive to 4-aminopyridine and independent of external Ca2+, but significantly inhibited by 3 mM tetraethylammonium. The fast current was depressed by external 4-aminopyridine, with an ED50 = 0.7 mM, and it was abolished by removal of divalent cations from the external medium. The sensitivity of the transient outward current to different divalent cations was investigated by equimolar substitution of Ca2+, Mn2+ and Mg2+. In 2.8 mM Mn2+, the transient potassium conductance was comparable to that in 2.8 mM Ca2+, while in 2.8 mM Mg2+ the transient component was drastically reduced, as in the absence of any divalent cations. However, when Ca2+ was present, Mg2+ up to 5 mM had no effect. The transient current increased with increasing concentrations of external Ca2+, [Ca2+]o, and the maximum conductance vs. [Ca2+]o curve could be approximated by a one-site model. In addition, the current recorded with 5.5 mM BAPTA in the intracellular solution was not different from that recorded in the absence of any Ca2+ buffer. These results suggest that divalent cations modulate the potassium channel interacting with a site on the external side of the cell membrane.

Spectrofluorimetric and image recordings of spontaneous and lectin-induced cytosolic calcium oscillations in Jurkat T cells

Intracellular variations in Ca 2+ concentrations have been measured in single Jurkat T lymphocyte variants (77 6.8 and E6.1) using Fura-2 as a probe. Under basal conditions, the cytosolic Ca 2+ level is stable but some cells show spontaneous Ca 2+ oscillations (frequency, 0.30 ± 0.06 Hz). These oscillations are sensitive to the external concentration of Ca 2+ since they can no longer be observed when the bathing solution is replaced (superfusion) with a Ca 2+-free medium or when a Ca 2+ chelator (EGTA) is added. various changes in the cytosolic concentration of Ca 2+ ([Ca 2+] i) can be observed when the cells are exposed to the mitogenic lectin phytohemagglutinin (PHA, 80 nM). For instance, in the case of non-oscillating cells, the lectin induces either a rapid increase in [Ca 2+] i that is followed by a sustained response (plateau) or it triggers Ca 2+ spikes. In the case of experiments done in Ca 2+-free medium, only the initial spike was observed. In the case of spontaneously oscillating cells, PHA induces a rapid increase in [Ca 2+] i that is followed by a piateau where oscillations are absent. In every case, the PHA-dependent Ca 2+ response is abrogated in a Ca 2+-free medium. Computer simulations based on the model of Goldbeter et al. [27] show that the various Ca 2+ responses of Jurkat cells are related to the cytosolic level of free Ca 2+. Video imaging analyses show that the cellular Ca 2+ responses are not homogeneous whether the observations are made in spontaneously oscillating Jurkat cells or when they are exposed to PHA.

Salinity-calcium interactions on growth and ionic concentration of Citrus plants

Two-year-old Navel orange scions (Citrus sinensis (L.) Osbeck) budded to either Cleopatra mandarin (C. reticulata) and Troyer citrange (C. sinensis × P. trifoliata) rootstocks were used in this experiment. Cleopatra manda in rootstock was considered more tolerant to salinity than Troyer citrange, and this property was attributed to a greater capacity to exclude chloride ions. Plants were grown under glasshouse conditions and supplied with nutrient solution containing either no or 45 mM NaCl. Calcium concentration was increased from 3 to 30 mM. Sodium, potassium, calcium and chloride concentrations in plant organs were analyzed after 90 days of treatment. Supplemental Ca was found to mitigate the adverse effects of salinity on plant growth, defoliation or leaf injury. Chemical analysis indicated that in plants grafted on Troyer citrange Ca restricted uptake and subsequent translocation of Na to the leaves and increased K concentration in both roots and leaves. However, in Cleopatra mandarin-grafted plants increasing Ca levels seemed to reduce transport of Na from roots to leaves, and Na accumulation in roots was associated with reduced concentration of K in this rootstock. Organ chloride analysis showed that Cl accumulation in leaves of plants grafted on both rootstocks was reduced when external Ca concentration increased, whereas Cl concentration in roots remained constant or increased. The data of distribution of Cl in plants showed that a high external Ca level increased Cl accumulation in the basal stem and roots, and reduced the transport of Cl from roots to leaves.

Voltage dependence of desensitization to carbamylcholine in frog muscle fibers reverses in low-calcium solutions

1. The effect of reduction in external Ca concentration on the voltage dependence of desensitization to carbamylcholine was studied in frog muscle fibers to determine whether local increase in cytosolic free Ca2+ from inward movement through agonist-activated receptor channels is a factor producing increased desensitization rate, as has been proposed. A critical feature is whether or not the relation of desensitization rate with voltage decreases in a regular manner as potential is increased to levels above ECa. 2. A conventional two-electrode voltage clamp procedure was used for voltage control of the postjunctional membrane of K-depolarized muscle fibers, and a standard 1-mM solution of carbamylcholine was applied to voltage-clamped endplates by local superperfusion from a micropipette. Measurements of average sarcomere length were also made in separate experiments to detect persistent raised levels of internal Ca2+ in the high-K test media. 3. In high-K test solutions containing 1.8 mM Ca, desensitization rate decreased in a regular monotonic relation with increase in potential through the range from -40 to +30 mV, giving an exponential voltage sensitivity factor of 0.016 mV-1. 4. By contrast, desensitization onset rate in high-K test media containing Ca ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (CaEGTA) buffer (calculated free Ca2+ = 61 nM) showed a nonmonotonic, reversing relation with potential characterized by a decrease in rate between -40 and -20 mV and an increase of rate with increase in potential from -20 to +30 mV.(ABSTRACT TRUNCATED AT 250 WORDS)