Ca2+ requirement of prostanoid but not of superoxide production by rat Kupffer cells

The release of the prostanoids prostaglandin D2 (PGD2), prostaglandin E2 (PGE2) and thromboxane induced by zymosan and phorbol ester in cultured rat Kupffer cells was found to depend on the extracellular concentration of Ca2+ to some extent. Prostanoid formation following the addition of the calcium ionophore A 23187 was totally inhibited when calcium ions were withdrawn from the medium whereas the prostanoid synthesis from added arachidonic acid was independent of Ca2+. A half-maximal rate of PGE2 release by cells treated with zymosan, phorbol ester or A23187 was obtained at 0.6-0.7 microM free extracellular Ca2+ and greater than or equal to 100 microM free Ca2+ was required to stimulate PGE2 formation maximally. The calmodulin antagonist R24571 partially inhibited the release of PGE2 elicited by zymosan and A23187 but not by phorbol ester or arachidonic acid. Verapamil and nifedipine, two calcium channel blockers, had no effect on the formation of PGE2 irrespective of the stimulus. TMB 8 [3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester] an intracellular calcium antagonist, inhibited the synthesis of PGE2 induced by zymosan and phorbol ester. The superoxide formation following the addition of zymosan and phorbol ester was not influenced by removal of calcium ions from the medium or by addition of the various calcium antagonists. The data presented here suggest that Ca2+-dependent reactions are involved in the synthesis of prostanoids induced by zymosan and phorbol ester and that both extracellular Ca2+ and mobilization of Ca2+ from intracellular stores are needed to induce maximally the production of prostanoids in cultured rat Kupffer cells.     

Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?

Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.     

Thrombin induces a calcium transient that mediates an activation of the Na+/H+ exchanger in human fibroblasts

The calcium dependence of growth factor-induced cytoplasmic alkalinization was determined in serum-deprived human fibroblasts (WS-1 cells). Intracellular pH (pHi) and intracellular calcium (Ca2+i) were measured using the fluorescent dyes 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein and fura2, respectively. Thrombin (10 nM) induced an alkalinization (0.18 +/- 0.01 pH units, n = 23) that was Na+-dependent and amiloride-sensitive, suggesting that the alkalinization was mediated by the Na+/H+ exchanger. Thrombin treatment caused a transient increase in Ca2+i (325 +/- 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Ca2+i and pHi were dependent on the concentration of thrombin. The thrombin-induced increase in Ca2+i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca2+i with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Ca2+i independent of growth factor stimulation. When Ca2+i was elevated with ionomycin, a concomitant increase in pHi was observed. The increase in pHi due to ionomycin was dependent on Na+ and sensitive to amiloride. The removal of external Ca2+i inhibited the ionomycin-induced elevation of both Ca2+i and pHi. The ionomycin-induced increases in Ca2+i and pHi were not inhibited by 8-diethylaminooctyl 3,4,5-trimethoxy-benzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Ca2+i.     

Interleukin-1 suppresses mesangial cell growth via inhibition of Ca2+ entry

We have investigated the effect of interleukin-1 (IL-1) on the cell growth and Ca2+ homeostasis of rat mesangial cells in culture. DNA synthesis measured by [3H]thymidine uptake by mesangial cells was significantly inhibited by IL-1 (10 U/ml) and the calcium channel antagonist nicardipine (5 x 10(-6) M). 45Ca2+ uptake by mesangial cells was also significantly inhibited by IL-1 and nicardipine. The above observations support the premise that IL-1 suppresses the growth of mesangial cells via inhibition of extracellular Ca2+ entry to the cytosol.     

Effects of Lead Ions on Events Associated with Exocytosis in Isolated Bovine Adrenal Medullary Cells

Lead buffers (citrate and Tiron) were used to investigate the effects of low concentrations (0.1-6 microM) of Pb2+ on stimulus-secretion coupling in isolated bovine chromaffin cells. Nicotinic agonists and high K elicit secretion by enhancing Ca2+ influx into chromaffin cells. Pb2+ inhibited the catecholamine secretion in response to 500 microM carbachol and 77 mM K+ depolarization but was without significant effect on basal secretion. Pb2+ also inhibited the influx of 45Ca occurring in response to these agents. The K0.5 values for inhibition suggest that the carbachol-evoked flux is more sensitive to Pb2+ than influx in response to a direct depolarization. When extracellular calcium was lowered in the absence of Pb2+, both secretion and 45Ca entry were reduced. The effects of Pb2+ were comparable to those of lowered Ca2+. 22Na influx through nicotinic receptor-mediated channels, measured in the presence of tetrodotoxin (2 microM) and ouabain (50 microM), was inhibited by Pb2+. The results suggest that Pb2+ inhibits exocytotic catecholamine secretion by inhibiting Ca2+ influx. The differential sensitivity to Pb2+ of K- and carbachol-evoked 45Ca flux, coupled with the 22Na measurements, indicates that Pb2+ inhibits the movement of ions through acetylcholine-induced channels as well as through voltage-sensitive calcium channels.     

Effects of Ca2+ and Mg2+ on ATP-dependent 45Ca2+ influx in A-431 human epidermoidal carcinoma cells

Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx.     

Dual pathways of calcium entry in spike and plateau phases of luteinizing hormone release from chicken pituitary cells: sequential activation of receptor-operated and voltage-sensitive calcium channels by gonadotropin-releasing hormone

It has previously been shown that, in pituitary gonadotrope cells, the initial rise in cytosolic Ca2+ induced by GnRH is due to a Ca2+ mobilization from intracellular stores. This raises the possibility that the initial transient spike phase of LH release might be fully or partially independent of extracellular Ca2+. We have therefore characterized the extracellular Ca2+ requirements, and the sensitivity to Ca2+ channel blockers, of the spike and plateau phases of secretion separately. In the absence of extracellular Ca2+ the spike and plateau phases were inhibited by 65 +/- 4% and 106 +/- 3%, respectively. Both phases exhibited a similar dependence on concentration of extracellular Ca2+. However, voltage-sensitive Ca2+ channel blockers D600 and nifedipine had a negligible effect on the spike phase, while inhibiting the plateau phase by approximately 50%. In contrast, ruthenium red, Gd3+ ions, and Co2+ ions inhibited both spike and plateau phases to a similar extent as removal of extracellular Ca2+. A fraction (35 +/- 4%) of spike phase release was resistant to removal of extracellular Ca2+. This fraction was abolished after calcium depletion of the cells by preincubation with EGTA in the presence of calcium ionophore A23187, indicating that it depends on intracellular Ca2+ stores. Neither absence of extracellular Ca2+, nor the presence of ruthenium red or Gd3+ prevented mobilization of 45Ca2+ from intracellular stores by GnRH. We conclude that mobilization of intracellular stored Ca2+ is insufficient by itself to account for full spike phase LH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

Octanol blocks fluid secretion by inhibition of capacitative calcium entry in rat mandibular salivary acinar cells

The aliphatic alcohol octanol is thought to modulate enzyme secretion from the exocrine pancreas by the inhibition of gap junction permeability. We have now investigated the effects of octanol on salivary secretion and intracellular calcium concentration ([Ca2+]i), measured in isolated perfused rat mandibular glands and in isolated mandibular acinar cells respectively. Stimulation of perfused glands with 10 microM carbachol (CCh) evoked a rapid increase in fluid secretion followed by a decrease to a sustained elevated level. Application of 1 mM octanol during CCh stimulation inhibited fluid secretion reversibly. In isolated acini, the CCh-induced [Ca2+]i increase was reversibly inhibited by the same concentration of octanol. However, octanol also inhibited the increase in [Ca2+]i in single acinar cells where gap junctions were no longer functional, indicating that octanol directly affected the intracellular Ca2+ signalling pathway. The initial increase in [Ca2+]i induced by 0.5-10 microM CCh, which is due to Ca2+ release from IP3-sensitive Ca2+ stores, was not affected by pretreatment with octanol. In contrast, CCh-, phenylephrine- or thapsigargin-induced Ca2+ entry was almost completely and reversibly inhibited by octanol. Octanol also blocked agonist-evoked Ca2+ entry in pancreatic acinar cells, and thapsigargin-evoked Ca2+ entry in fibroblasts. These data strongly suggest that octanol blocks salivary secretion from mandibular gland by the inhibition of capacitative Ca2+ entry, and raise the possibility that octanol may be a useful tool for inhibiting agonist-evoked Ca2+ entry pathways.     

Calcium ions are required for the intracellular routing of insulin and its receptor.

We have studied the role of the cytosolic-free calcium concentration ([Ca2+]i) on the early and later internalization steps of insulin and its receptor. As before, we find that the rate of 125I-insulin internalization in HL60 cells remains normal when [Ca2+]i is lowered 10 times below normal resting level by the use of an intracellular Ca2+ chelator. By contrast, the subsequent intracellular steps, i.e. insulin receptor recycling and insulin degradation, are inhibited in calcium-depleted cells. Under low [Ca2+]i conditions, the association of 125I-insulin with late endosomes and lysosomes is also reduced. This suggests that calcium ions are required for fusion processes occurring at the endosomal or postendosomal stage of internalization. Thus, by regulating insulin receptor recycling and by controlling insulin degradation, Ca2+ ions play a key role in the regulation of insulin action.     

Extracellular ATP induces ion fluxes and inhibits growth of Friend erythroleukemia cells

Extracellular ATP (1 mM) inhibited the growth of Friend virus-infected murine erythroleukemia cells (MEL cells) but had no effect on dimethyl sulfoxide-induced differentiation. ATP (1 mM) also caused changes in the permeability of MEL cells to ions. There was an increased influx of 45Ca2+ from a basal level of 5 pmol/min to 18 pmol/min/10(6) cells to achieve a 2-fold increase in steady-state Ca2+ as measured at isotopic equilibration. Ca2+ influx was blocked by diisothiocyanostilbene disulfonate (DIDS), an inhibitor of anion transport. ATP also stimulated Cl- uptake, and this flux was inhibited by DIDS. The ratio of ATP stimulated Cl- to Ca2+ uptake was 1.6:1. K+ and Na+ influx were also stimulated by ATP, but phosphate uptake was inhibited; the Na+ influx dissipated the Na+ gradient and thus inhibited nutrient uptake. ATP-stimulated K+ influx was ouabain inhibitable; however, the total cellular K+ decreased due to an ATP-stimulated ouabain-resistant K+ efflux. Na+ influx and Ca2+ influx occurred by separate independent routes, since Na+ influx was not inhibited by DIDS. The effects observed were specific for ATP *K1/2 MgATP = 0.7 mM) since AMP, GTP, adenosine, and the slowly hydrolyzable ATP analogue adenyl-5'-yl imidodiphosphate were without effect. The major ionic changes in the cell were a decrease in K+ and increase in Na+; cytoplasmic pH and free Ca2+ did not change appreciably. These ATP-induced changes in ion flux are considered to be responsible for growth inhibition.     

Role of phospholipase A2 activation and calcium in CYP2E1-dependent toxicity in HepG2 cells

Previous studies suggested a role for calcium in CYP2E1-dependent toxicity. The possible role of phospholipase A2 (PLA2) activation in this toxicity was investigated. HepG2 cells that overexpress CYP2E1 (E47 cells) exposed to arachidonic acid (AA) +Fe-NTA showed higher toxicity than control HepG2 cells not expressing CYP2E1 (C34 cells). This toxicity was inhibited by the PLA2 inhibitors aristolochic acid, quinacrine, and PTK. PLA2 activity assessed by release of preloaded [3H]AA after treatment with AA+Fe was higher in the CYP2E1 expressing HepG2 cells. This [3H]AA release was inhibited by PLA2 inhibitors, alpha-tocopherol, and by depleting Ca2+ from the cells (intracellular + extracellular sources), but not by removal of extracellular calcium alone. Toxicity was preceded by an increase in intracellular calcium caused by influx from the extracellular space, and this was prevented by PLA2 inhibitors. PLA2 inhibitors also blocked mitochondrial damage in the CYP2E1-expressing HepG2 cells exposed to AA+Fe. Ca2+ depletion and removal of extracellular calcium inhibited toxicity at early time periods, although a delayed toxicity was evident at later times in Ca2+-free medium. This later toxicity was also inhibited by PLA2 inhibitors. Analogous to PLA2 activity, Ca2+ depletion but not removal of extracellular calcium alone prevented the activation of calpain activity by AA+Fe. These results suggest that release of stored calcium by AA+Fe, induced by lipid peroxidation, can initially activate calpain and PLA2 activity, that PLA2 activation is critical for a subsequent increased influx of extracellular Ca2+, and that the combination of increased PLA2 and calpain activity, increased calcium and oxidative stress cause mitochondrial damage, that ultimately produces the rapid toxicity of AA+Fe in CYP2E1-expressing HepG2 cells.     

Ca2+- and voltage-dependent inactivation of the expressed L-type Ca(v)1.2 calcium channel

Ca2+-dependent regulation of the ion current through the alpha1Cbeta2aalpha2delta-1 (L-type) calcium channel transiently expressed in HEK 293 cells was investigated using whole cell patch clamp method. Ca2+ or Na+ ions were used as a charge carrier. Intracellular Ca2+ was either buffered by 10 mM EGTA or 200 microM Ca2+ was added into non-buffered intracellular solution. Free intracellular Ca2+ inactivated permanently about 80% of the L-type calcium current. The L-type calcium channel inactivated during a depolarizing pulse with two time constants, tau(fast) and tau(slow). Free intracellular calcium accelerated both time constants. Effect on the tau(slow) was more pronounced. About 80% of the channel inactivation during brief depolarizing pulse could be attributed to a Ca2+-dependent mechanism and 20% to a voltage-dependent mechanism. When Na+ ions were used as a charge carrier, the L-type current still inactivated with two time constants that were 10 times slower and were virtually voltage-independent. Ca2+ ions stabilized the inactivated state of the channel in a concentration-dependent manner.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

Osmotic strength differentiates between two types of calcium transport pathways regulating catecholamine secretion from cultured bovine chromaffin cells

Calcium transport and catecholamine secretion was measured in cultured bovine chromaffin cells. Calcium ions which entered the cells following stimulation with either nicotine or 50 mM KCl (high potassium) triggered catecholamine release, but then inactivated the secretory process. The nicotine and the high potassium-induced calcium transport mechanisms were mechanistically distinct, but functionally dependent on each other. The specific evidence is that whereas the high potassium-induced Ca2+ influx was found to be inhibited by hyperosmotic medium, the nicotine-stimulated calcium influx was unaffected under these conditions. High potassium and nicotine-stimulated catecholamine release were also differently affected by hyperosmotic medium. While potassium-stimulated catecholamine release was profoundly inhibited by hyperosmolarity, nicotine-stimulated release was only moderately inhibited. Sequential treatments of cells with nicotine and high potassium, under isotonic physiological conditions, indicate that there is a functional, biochemical communication between the otherwise mechanistically distinct calcium channels. Calcium ions which were found to inactivate these channels may be the basis for such communication.     

Calcium permeability changes and neurotransmitter release in cultured rat brain neurons. I. Effects of stimulation on calcium fluxes

The permeability of neuronal membranes to Ca2+ is of great importance for neurotransmitter release. The temporal characteristics of Ca2+ fluxes in intact brain neurons have not been completely defined. In the present study 45Ca2+ was used to examine the kinetics of Ca2+ influx and efflux from unstimulated and depolarized rat brain neurons in culture. Under steady-state conditions three cellular exchangeable Ca2+ pools were identified in unstimulated cells: 1) a rapidly exchanging pool (t1/2 = 7 s) which represented about 10% of the total cellular Ca2+ and was unaffected by the presence of Co2+, verapamil, or tetrodotoxin; 2) a slowly exchanging pool (t1/2 = 360 s) which represented 42% of the total cellular Ca2+ and was inhibited by Co2+, but not by verapamil or tetrodotoxin; 3) a very slowly exchanging pool (t1/2 = 96 min) which represented 48% of the total cell Ca2+ was observed only in the prolonged efflux experiments. The rate of exchange of 45Ca2+ in the unstimulated cells was dependent on the extracellular Ca2+ concentration (half-saturation at 70 microM). Depolarization of the neurons with elevated K+ causes a rapid and sustained 45Ca2+ uptake. The cellular Ca2+ content increased from 56 nmol/mg protein in unstimulated cells to 81 nmol/mg protein during 5 min of depolarization. The kinetics of the net 45Ca2+ uptake by the stimulated neurons was consistent with movement of the ion with a first order rate constant of 0.0096 s-1 (t1/2 = 72 s) into a single additional compartment. The other cellular Ca2+ pools were apparently unaffected by stimulation. The stimulated 45Ca2+ uptake was inhibited by Co2+ and by the Ca2+ channel blocker verapamil but not by the Na+ channel blocker tetrodotoxin. Ca2+ uptake into this compartment was dependent on the extracellular Ca2+ concentration (half-saturation at 0.80 mM Ca2+). Predepolarization of the cells with high K+ for 10-60 s prior to the addition of the radioactive calcium did not alter the rate of 45Ca2+ incorporation into the stimulated cells. It is concluded that the rapidly exchanging, the slowly exchanging, and the depolarization-induced Ca2+ pools observed in intact brain neurons are physically as well as kinetically distinct from each other. In addition, the depolarization-induced component observed in stimulated cells represents movement of the Ca2+ ions through a single class of voltage-sensitive Ca2+ channels. These Ca2+ channels are inhibited by Co2+ ions and by verapamil and are not inactivated during depolarization of the brain neurons.     

Na+-Ca2+ exchange activity in rat skeletal myotubes: effect of lithium ions

The whole-cell patch-clamp technique coupled with intracellular [Ca2+] measurements was used to investigate the sodium-calcium exchange mechanism in rat skeletal muscle cells in primary culture. Replacing external Na+ ions with Li+ or N-methyl-D-glucamine (NMDG+) ions generated outward currents which were correlated with significant increases of free cytosolic-calcium concentration. These results strongly argue for a functional Na+-Ca2+ exchange mechanism working in its reverse mode. Moreover, the outward currents were sensitive to the new compound KB-R7943 (10 microM), which has been shown to be a potent inhibitor of the sodium-calcium exchanger. Outward Na+-Ca2+ exchange current densities were reduced in the presence of external Li+ as compared to those measured in the presence of NMDG+. After replacing internal sodium by lithium ions, rapid changes of external lithium concentrations generated sarcolemmal currents which were accompanied by subsequent variations of intracellular calcium activity. The currents were dependent on extracellular Li+ with a half-maximal activation at 67 mM and a Hill coefficient of 2.9. This work shows that the Na+-Ca2+ exchanger is able to significantly influence the myoplasmic calcium concentration of cultured rat myotubes. On the other hand, our results suggest that Li+ ions may substitute Na+ ions to catalyse an electrogenic Li+/Ca2+ counter transport.     

Energy and calcium ion dependence of proteolysis during sporulation of Bacillus subtilis cells.

Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.     

Detection and localization of a Ca2+-ATPase activity in Toxoplasma gondii

Toxoplasma gondii, the agent causing toxoplasmosis, is an obligate intracellular protozoan parasite. A calcium signal appears to be essential for intracellular transduction during the active process of host cell invasion. We have looked for a Ca2+-transport ATPase in tachyzoites and found Ca2+-ATPase activity (11-22 nmol Pi liberated/mg protein/min) in the tachyzoite membrane fraction. This ATP-dependent activity was stimulated by Ca2+ and Mg2+ ions and by calmodulin, and was inhibited by pump inhibitors (sodium orthovanadate or thapsigargin). We used cytochemistry and X-ray microanalysis of cerium phosphate precipitates and immunolabelling to find the Ca2+, Mg2+-ATPase. It was located mainly in the membrane complex, the conoid, nucleus, secretory organelles (rhoptries, dense granules) and in vesicles with a high calcium concentration. Thus, Toxoplasma gondii possesses Ca2+-pump ATPase (Ca2+, Mg2+-ATPase) as do eukaryotic cells.