Analysis of Ejaculates from Patients with Cystic Fibrosis

Ejaculates from two men with cystic fibrosis (CF) were examined. Both had azoospermia. A considerable decrease in volume and fructose content was noted. The absolute amounts of calcium, magnesium, and zinc per ejaculate showed normal values in one of the patients but were two to three times increased in the other compared to mean values of a control group. Thus the concentrations of these cations were increased at least fivefold in both patients.

The amount of Mg²⁺- and Ca²⁺dependent ATPase was comparable to that of controls, but values were higher than in men with oligospermia. Both the divalent cations and the Mg²⁺- and Ca²⁺ -dependent ATPase curve profiles of split ejaculate fractions were atypical. Secretory granules and vesicles were plentiful in the seminal plasma of both patients while amorphous substance was practically absent.

The present findings agree with a less affected function of the prostate gland and a dysfunction of the seminal vesicles in these patients.     

Effect of cytosolic Mg2+ on mitochondrial Ca2+ signaling

Cytosolic Ca²⁺ signals are followed by mitochondrial Ca²⁺ uptake, which, in turn, modifies several biological processes. Mg²⁺ is known to inhibit Ca²⁺ uptake by isolated mitochondria, but its significance in intact cells has not been elucidated. In HEK293T cells, activation of purinergic receptors with extracellular ATP caused cytosolic Ca²⁺ signals associated with parallel changes in cytosolic [Mg²⁺]. Neither signals were affected by omitting bivalent cations from the extracellular medium. The effect of store-operated Ca²⁺ influx on cytosolic Mg²⁺ concentration ([Mg²⁺]_c) was negligible. Uncaged Ca²⁺ displaced Mg²⁺ from cytosolic binding sites, but for an equivalent Ca²⁺ signal, the change in [Mg²⁺] was significantly smaller than that measured after adding extracellular ATP. Inositol 1,4,5-trisphosphate mobilized Ca²⁺ and Mg²⁺ from internal stores in permeabilized cells. The increase of [Mg²⁺] in the range that occurred in ATP-stimulated cells inhibited mitochondrial Ca²⁺ uptake in permeabilized cells without affecting mitochondrial Ca²⁺ efflux. Therefore, the Mg²⁺ signal generated by Ca²⁺ mobilizing agonists may attenuate mitochondrial Ca²⁺ uptake.     

DIVALENT CATION MODULATION OF A-TYPE POTASSIUM CHANNELS IN ACUTELY DISSOCIATED CENTRAL NEURONS FROM WIDE-TYPE AND MUTANT DROSOPHILA

Drosophila mutants provide an ideal model to study channel-type specificity of ion channel regulation in situ. In this study, the effects of divalent cations on voltage-gated K⁺ currents were investigated in acutely dissociated central neurons of Drosophila third instar larvae using the whole-cell patch-clamp recording. Our data showed that micromolar Cd²⁺ enhanced the peak inactivating current (I_A) without affecting the delayed component (I_K). The same results were obtained in Ca²⁺-free external solution, and from slo¹ mutation, which eliminates transient Ca²⁺-activated K⁺ current. Micromolar Cd²⁺and Zn²⁺, and millimolar Ca²⁺and Mg²⁺ all shifted the steady-state inactivation curve of I_A without affecting the voltage-dependence of I_A activation, whereas millimolar Cd²⁺markedly affected both the activation and steady-state inactivation curves for I_A. Divalent cations affected I_A with different potency; the sequence was: Zn²⁺ > Cd²⁺ > Ca²⁺ > Mg²⁺. The modulation of I_A by Cd²⁺ was partially inhibited in Sh^M, a null Shaker (one of I_A-encoding genes) mutation. Taken together, the channel-type specificity, the asymmetric effects on I_A activation and inactivation kinetics, and the diverse potency of divalent cations all strongly support the idea that physiological divalent cations modulate A-type K⁺ channels through specific binding to extracellular sites of the channels.     

AlF 4 − induces Ca2+ oscillations in guinea-pig ileal smooth muscle

The effects of different compounds that inhibit the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase on the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and on the corresponding force development have been examined in smooth muscle of the longitudinal layer of the guinea-pig ileum. F^–, in the presence of Al³⁺, induced an increase of the resting force and of the amplitude of the superimposed phasic contractions. The increase of resting force was associated with an increased level of basal [Ca²⁺]_i while the phasic contractions were accompanied by concomitant oscillations in [Ca²⁺]_i. Comparable contractions could be induced by vanadate and the calmodulin antagonist calmidazolium. The oscillations of [Ca²⁺]_i and of force elicited by AlF ₄ ^– were not modified by adrenergic or cholinergic blocking agents but were inhibited by verapamil. These phasic contractions were not affected by depleting the intracellular Ca²⁺ stores with ryanodine. This finding excludes a cytosolic origin of these oscillations. However, hyperpolarization and complete depolarization of the cells inhibited the oscillations. It is concluded that AlF ₄ ^– , vanadate and calmidazolium induce cytoplasmic Ca²⁺ oscillations possibly by acting at the plasma membrane. Indeed all these substances affect by different mechanisms the isolated plasma-membrane Ca²⁺/Mg²⁺-ATPase. The generation of membrane-linked Ca²⁺ oscillations could therefore be related to an inhibition of the plasma-membrane Ca²⁺ pump resulting in an increase of [Ca²⁺]_i. This change in [Ca²⁺]_i could be responsible for the pronounced changes of the electrical and mechanical activity of this tissue.     

Ca2+ and Sr2+ activation: Comparison of cardiac and skeletal muscle contraction models

The mechanism of contraction in rabbit fast-twitch, and bovine and rabbit cardiac muscle was examined using functionally skinned fibers, ATPase activity of myofibrils, and cardiac or skeletal troponintropomyosin regulated actin heavy meromyosin. The Ca²⁺ and Sr²⁺ activation properties for the different measures of contraction were evaluated. (1) Tension in rabbit and bovine cardiac skinned fibers and rabbit cardiac myofibrillar ATPase were activated equally well by either Ca²⁺ or Sr²⁺. By contrast, rabbit adductor magnus (fast-twitch) skinned fibers required substantially higher [Sr²⁺] than [Ca²⁺] for activation, as did rabbit myofibrils from back muscle (fast-twitch). (2) Substantially more Sr²⁺ than Ca²⁺ was also required for activation of skeletal muscle actin heavy meromyosin ATPase, controlled by either the skeletal or cardiac troponin-tropomyosin complex, similar to the activation of fast-twitch muscle. (3) The absence of correlation between the divalent cation selectivity properties of actin heavy meromyosin ATPase controlled by cardiac troponin-tropomyosin and cardiac muscle tension or myofibrillar ATPase activation by Ca²⁺ and Sr²⁺ suggests that troponin, if primarily responsible for the activation of cardiac muscle, has very different in vivo and in vitro binding properties. (4) The close correlation between percentage of maximal Ca²⁺- and Sr²⁺-activated myofibrillar ATPase and tension in skinned fibers strongly justifies the use of myofibrillar ATPase, in contrast to a reconstituted troponin-tropomyosin actin heavy meromyosin ATPase system, as a biochemical measure of contraction.     

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

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

Type B monoamine oxidase and functions of Ca2+, Mg2+-dependent adenosinetriphosphatase in preparations from sarcoplasmic reticulum vesicles

Oxidative deamination of -phenylethylamine or benzylamine by type B monoamine oxidases (MAO) in preparations of sarcoplasmic reticulum vesicles from rabbit skeletal muscles is accompanied by inhibition both of active Ca²⁺ transport into the vesicles and of the activity of Ca²⁺, Mg²⁺-dependent ATPase, which is preventable by deprenil, a specific inhibitor of type B MAO. Aldehydes formed during enzymatic deamination of substrates of type B MAO may perhaps participate in the regulation of Ca²⁺, Mg²⁺-dependent ATPase, activity.Laboratory of Physicochemical Methods, Scientific-Research Institute for Biological Trials of Chemical Compounds, Ministry of the Medical Industry of the USSR. Laboratory or Biochemistry of Amines and Other Nitrogenous Compounds, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 3, pp. 283–284, March, 1977.     

Hormonal stimulation of Ca2+ and Mg2+ transport in the cortical thick ascending limb of Henle's loop of the mouse: evidence for a change in the paracellular pathway permeability

Recent studies from our laboratory have shown that in the cortical thick ascending limb of Henle's loop of the mouse (cTAL) Ca²⁺ and Mg²⁺ are reabsorbed passively, via the paracellular shunt pathway. In the present study, cellular mechanisms responsible for the hormone-stimulated Ca²⁺ and Mg²⁺ transport were investigated. Transepithelial voltages (PD_te) and transepithelial ion net fluxes (J _Na, J _Cl, J _K, J _Ca, J _Mg) were measured in isolated perfused mouse cTAL segments. Whether parathyroid hormone (PTH) is able to stimulate Ca²⁺ and Mg²⁺ reabsorption when active NaCl reabsorption, and thus PD_te, is abolished by luminal furosemide was first tested. With symmetrical lumen and bath Ringer's solutions, no Ca²⁺ and Mg²⁺ net transport was detectable, either in the absence or in the presence of PTH. In the presence of luminal furosemide and a chemically imposed lumen-to-bath directed NaCl gradient, which generates a lumen-negative PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net secretion. In the presence of luminal furosemide and a chemically imposed bath-to-lumen-directed NaCl gradient, which generates a lumen-positive PD_te, PTH slightly but significantly increased Ca²⁺ and Mg²⁺ net reabsorption. In view of the observed small effect of PTH on passive Ca²⁺ and Mg²⁺ movement, a possible interference of furosemide with the hormonal response was considered. To investigate this possibility, Ca²⁺ and Mg²⁺ transport was first stimulated with PTH in tubules under control conditions. Then active NaCl reabsorption was abolished by furosemide and the effect of PTH on J _Ca and J _Mg measured. In the absence of PD_te and under symmetrical conditions, no Ca²⁺ and Mg²⁺ transport was detectable, either in the presence or absence of PTH. In the presence of a bath-to-lumen-directed NaCl gradient, Ca²⁺ and Mg²⁺ reabsorption was significantly higher in the presence than in the absence of PTH. Finally, when active NaCl transport was not inhibited by furosemide, but reduced by a bath-to-lumen-directed NaCl gradient, PTH strongly increased J _Ca and J _Mg, whereas only a small increase in PD_te was noted. In conclusion, these data suggest that PTH exerts a dual action on Ca²⁺ and Mg²⁺ transport in the mouse cTAL by increasing the transepithelial driving force for Ca²⁺ and Mg²⁺ reabsorption through hormone-mediated PD_te alterations and by modifying the passive permeability for Ca²⁺ and Mg²⁺ of the epithelium, very probably at the level of the paracellular shunt pathway.     

Differences in magnesium and calcium effects on N-methyl-D-aspartate- and quisqualate-induced decreases in extracellular sodium concentration in rat hippocampal slices

Summary Decreases in extracellular sodium concentration ([Na⁺]o) and associated slow negative field potentials (fp's) were monitored with double barreled sodium sensitive/reference microelectrodes in area CA1 of rat hippocampal slices during iontophoretic application of the glutamate receptor agonists N-methyl-D-aspartate (NMDA) and quisqualate (quis). The effects of lowering [Ca²⁺]o on these signals were compared to those of lowering [Mg²⁺]o. Both NMDA- and quis-induced decreases in [Na⁺]o of up to 60 mM and in the fp's of up to 8 mV. Decreasing [Mg²⁺]o enhanced NMDA-induced signals, whereas quis-induced signals were unaffected. Lowering [Ca²⁺]o also enhanced NMDA signals, although somewhat less than lowering [Mg²⁺]o. This effect was still present, even when voltage dependent Na⁺ currents were blocked by 10^-7 tetrodotoxin. Interestingly, quis-induced signals could be enhanced in a low Ca²⁺ medium as well, but only when high quis concentrations were used. The results suggest that, during the sorts of large decreases of [Ca²⁺]o observed during seizure activity, activation of NMDA receptors is facilitated.Supported by a grant from the Deutsche Forschungsgemeinschaft (He 1128/2-4)     

Force-velocity relation in chemically skinned rat portal vein

Rat portal veins were chemically skinned using Triton X-100 and mounted for isometric and quick release experiments at 20°C. The skinned preparations were activated by Ca²⁺ (EGTA-buffered) in solutions containing 2 mM free-Mg²⁺ and 1 M calmodulin. Half maximal isometric force was obtained at pCa=6.2. Maximal force of the skinned preparations, at pCa=4.5, was 8.2±0.8 mN/mm² (n=6). Force-velocity relations were determined at varied Ca²⁺-concentrations. Maximal shortening velocity (V _max) was 0.10±0.01 lengths/s at pCa=4.5. At decreasing Ca²⁺-levelsV _max decreased (at pCa=6.25,V _max=0.05 l/s). At pCa =9 an increased level of free-Mg²⁺ (15mM) induces a slow and submaximal increase in tension. Force velocity relations of Mg²⁺-induced contractures were not different from those of Ca²⁺-contractures of similar magnitude (pCa=6.3). The results indicate that the degree of activation of the contractile system, as regulated by Ca²⁺ and Mg²⁺, influences the kinetic properties of the actomyosin interaction as well as the force development.     

Calreticulin expression in spinal motoneurons of the rat

We have examined the expression of calreticulin in rat spinal motoneurons in order to reveal the occurrence and distribution of Ca²⁺-storage organelles in these neurons. Calreticulin, the non-muscle equivalent of calsequestrin, is the low-affinity, high-capacity calcium-binding protein responsible for intracompartmental Ca²⁺-storage in a number of different cell types. The results of the present immunohistochemical study show that all spinal motoneurons express calreticulin at approximately the same level; no significant differences in cytoplasmic immunostaining intensity were observed between different motoneuron pools or between small and large spinal motoneurons. Immunoelectron microscopy revealed that the intracellular localization of calreticulin within spinal motoneurons was confined to the endoplasmic reticulum and to spherical or pleiomorphic, frequently ‘coated’ vesicles with a diameter ranging between 120 and 150 nm. Some of these vesicles may represent the so-called calciosomes, the intracellular Ca²⁺-storage vesicles described in liver cells and in cerebellar Purkinje cells. The molecular components responsible for the uptake and release of Ca²⁺ from the Ca²⁺-storage organelles in spinal motoneurons still remain to be identified.     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

The effect of Ca2+ on the metarhodopsin I–II transition

Summary The effect of Ca²⁺ on kinetics and equilibrium of the Meta I–II transition was studied in rhodopsin-digitonin-solutions using flash-photometry. With increasing Ca²⁺-concentration the Meta I–II-equilibrium is shifted to Meta I. The pH-dependence of the Meta I–II equilibrium is suppressed by Ca²⁺. To obtain the same effect as with bivalent cations about the 10-fold concentration of univalent ions is required. Ca²⁺-ions have also an effect on the rate of equilibrating Meta I–II: with increasing Ca²⁺-concentration the rate-constants of the rapid and slow component decrease and become equal to the value at pH 8. This observation can be described as an inhibition of the catalytic effect of protons by Ca²⁺. Similar results are obtained with Mg²⁺, whereas K⁺ and Na⁺ are practically ineffective. In the presence of the Ca²⁺-blocking agents verapamil (Isoptin^®) and D-600 the rate of equilibrating Meta I–II is reduced. These and several former observations can be explained by a model in which the Meta I–II transition is coupled with the separation of negative fixed changes, which can be clamped by Ca²⁺.This work was supported by the Deutsche Forschungsgemeinschaft project No. Em 18/1-4     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes

Our objectives were to investigate regulation of intracellular ionised Mg²⁺ concentration ([fMg²⁺]_i) in cardiac muscle and cardiac Na⁺/Mg²⁺ antiport stoichiometry. [fMg²⁺]_i was measured at 37°C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na⁺ and Ca²⁺ free solutions containing 30 mM Mg²⁺ for 15 min more than doubled [fMg²⁺]_i from its basal level (0.75 mM). Re-addition of Na⁺ caused [fMg²⁺]_i to fall exponentially with time to basal level, the rate increasing linearly with [Na⁺]. Log(recovery rate) increased linearly with log([Na⁺]), the slope of 1.06 (95% confidence limits, 0.94–1.17) suggesting one Na⁺ ion is exchanged for each Mg²⁺. [fMg²⁺]_i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg²⁺] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min^–1) with a Q₁₀ of 2.2. It was completely inhibited by 200 M imipramine but was unaffected by 20 M KB-R7943 or 1 M SEA0400, suggesting the Na⁺ /Ca²⁺ antiporter is not involved. Membrane depolarisation by increasing superfusate [K⁺] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na⁺ containing solutions more than threefold. We conclude [fMg²⁺]_i recovery is by Mg²⁺ efflux on a 1 Na⁺:1 Mg²⁺ antiport.     

Ca2+ channels in the apical membrane of the toad urinary bladder

Previously (Van Driessche et al. 1987) we showed that small inward (mucosa towards serosa) oriented shortcircuit currents (I _sc) were recorded through the toad urinary bladder when the mucosal side was exposed to Ca²⁺ free solutions containing K⁺, Na⁺ (+amiloride), Cs⁺ or Rb⁺ as main cation. This current component is inhibitable by micromolar concentrations of mucosal La³⁺ and divalent cations (Ca²⁺, Cd²⁺) and is considerably elevated by oxytocin (0.1 U/ml). The present study demonstrates that the addition of 50 nmol/l Ag⁺ to the mucosal medium during oxytocin treatment caused an additional large increase of the La³⁺-sensitiveI _sc component. The power density spectrum of the fluctuation in current contained a Lorentzian component which was enhanced by oxytocin treatment. The Lorentzian component disappeared as a consequence of the administration of mucosal Ag⁺. In experiments with Ca²⁺, Ba²⁺ or Mg²⁺ as principal mucosal cation, the La³⁺-sensitiveI _sc component was negligible under control conditions and during oxytocin treatment. Mucosal Ag⁺ (40 nmol/l) elicited a large inward oriented current which was blockable by the calcium channel blockers, La³⁺ and Cd²⁺. Also the organic calcium entry blockers, nicardipine and verapamil (10 mol/l) depressed the inward current considerably. Noise analysis of the currents carried by divalent cations showed a La³⁺-sensitive noise component. Oxytocin-Ag⁺ activated currents could not be recorded in the absence of the divalent cations or small inorganic cations, e.g. with solutions which contained N-methyld-glucamine (NMDG) as main mucosal cation.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

The intracellular Ca2+ concentration optimal for T cell activation is quite different after ionomycin or CD3 stimulation

The relationship between the initial increase of intracellular Ca²⁺ concentration ([Ca²⁺]_i) (measured at the single-cell level with an imaging system) and the ensuing proliferation was examined in a human T cell clone stimulated by a phorbol ester in combination with ionomycin, thapsigargin or an anti-CD3 mAb (monoclonal antibody against the CD3 molecule, UCHT1). From the responses to various ionomycin concentrations, one can define a range of [Ca²⁺]_i values (400–900 nM) which appears optimal for T cell proliferation; lower [Ca²⁺]_i values are suboptimal, higher values are cytotoxic. It was then examined if the [Ca²⁺]_i requirements were similar following anti-CD3 stimulation. [Ca²⁺]_i oscillations elicited by a concentration of UCHT1 (1/1,000) optimal for mitogenicity fall precisely within the 400–900 nM range. However, very low concentrations of UCHT1 (1/100,000) which evoke barely detectable [Ca²⁺]_i responses still cause the cells to proliferate. The possibility that the lower [Ca²⁺]_i requirements observed following anti-CD3 stimulation was due to [Ca²⁺]_i oscillations was tested under conditions which prevented the appearance of these oscillations. It turns out that an oscillatory Ca²⁺signal is not more mitogenic than a sustained augmentation of [Ca²⁺]_i. Finally, it was examined if overstimulation via CD3 could have toxic consequences similar to those elicited after ionomycin overstimulation. Large transient [Ca²⁺]_i responses can be observed following anti-CD3 stimulation in appropriate conditions, and namely in T cells pretreated with interleukin-2. These [Ca²⁺]_i augmentations are not cytotoxic. A role for the plasmalemmal Ca²⁺ pump in the prevention of cytotoxicity can be demonstrated. In conclusion, the correspondence between the [Ca²⁺]_i response and cell proliferation is entirely different following stimulation by ionomycin and by anti-CD3. In addition, cell proliferation evoked by very low UCHT1 concentration might reveal the existence of a yet unidentified activation pathway.     

Divalent cations selectively alter the voltage dependence of inactivation of A-currents in chick autonomic neurons

A-currents were studied in acutely dissociated chick autonomic neurons. Switching from salines containing 4 mM Mg²⁺ to salines containing 4 mM Ca²⁺ caused a large positive shift in the voltage dependence of steady-state inactivation, but had no effect on the voltage dependence or kinetics of activation, deactivation, the rate at which channels became inactivated, or the rate at which channels recovered from inactivation. This effect saturated with increasing concentrations of Ca²⁺. Other group IIA divalent cations were less effective than Ca²⁺, in the order Ca²⁺ >Ba²⁺ >Mg²⁺=Sr²⁺. Application of 4 mM Mg²⁺ partially antagonized the effects of 4 mM Ca²⁺. The results suggest that divalent cations selectively alter the voltage dependence of steady-state inactivation by acting at sites accessible from the outside of the cell.