Release of calcium from membranes and its relation to phagocytotic metabolic changes: A fluorescence study on leukocytes loaded with chlortetracycline

A fluorescent probe chlortetracycline was used to monitor the mobilization of intracellular divalent cations of leukocytes. When the chlortetracycline-loaded cells were stimulated with cytochalasin D or $\text{E. coli}$, a fluorescence change ascribable to the release of calcium from the intracellular hydrophobic environment was observed. The dose-response curve of the fluorescence change and that of the superoxide release of the cells were very similar. An intracellular calcium antagonist 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate inhibited both metabolic and fluorescence changes in parallel. A supposition that an intracellular mobilization of calcium ions is stimulating the metabolic change was supported.     

Spontaneous induction of superoxide release and degranulation of neutrophils in isotonic potassium medium: the role of intracellular calcium

When guinea pig peritoneal neutrophils were suspended in the isotonic medium of potassium, rubidium, and cesium ions at 37 degrees C, the cells released superoxide, while low activity was observed in the isotonic medium of sodium and lithium ions. The activity induced in the potassium medium was enhanced by potassium-ionophores, valinomycin, and gramicidin, and decreased by a potassium channel blocker, 4-aminopyridine. The superoxide-releasing activity was not affected by the presence or absence of extracellular calcium but was inhibited by an intracellular calcium antagonist-8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate(TMB-8) with the half-inhibition concentration of 50 microM. The release of granular enzymes, lysozyme and beta-glucuronidase, was also induced in the isotonic potassium medium in the absence of extracellular calcium and inhibited by TMB-8. A remarkable elevation of the intracellular free calcium concentration in neutrophils, which was monitored by quin-2 fluorescence, was found when the cells were added to the potassium medium without calcium. The elevation was inhibited by the addition of TMB-8. These observations suggest that calcium mobilization from intracellular storage sites, not an influx of calcium from the extracellular medium, causes the release of superoxide and the granular enzymes in isotonic potassium medium.     

A role for Na+/Ca2+ exchange in the generation of superoxide radicals by human neutrophils

A Na+/Ca2+ exchange mechanism has been recently described in human neutrophils that constitutes the principal pathway for Ca2+ influx into resting cells. The potential role of this system in regulating the respiratory burst in response to activation by the chemotactic tripeptide N-formyl-methionyl-leucyl-phenylalanine was explored. In the presence of 1 mM Ca2+, a variety of di- and trivalent cations suppressed the generation of O(-2) radicals in a series of decreasing efficacy: La3+ approximately Zn2+ much greater than Sr2+ approximately Cd2+ greater than Ba2+ greater than Co2+ greater than Ni2+ approximately Mg2+. This sequence is similar to their rank order of activity in inhibiting 45Ca2+ influx via Na+/Ca2+ counter-transport. Benzamil, phenamil, and 2',4'-dichlorobenzamil, analogues of amiloride which selectively block Na+/Ca2+ exchange in neutrophils, likewise suppressed the release of O(-2) with apparent Ki values of approximately 30 microM. The effect of the cations was competitive with Ca2+, while the interaction between the benzamil derivatives and Ca2+ appeared to be noncompetitive in nature. Both the divalent cations and benzamil also inhibited the rise in cytoplasmic Ca2+ as monitored by fura-2 fluorescence: these agents reduced peak cytosolic Ca2+ levels after N-formyl-methionyl-leucyl-phenylalanine stimulation to values seen in the absence of extracellular Ca2+. These results are compatible with the hypothesis that the influx of Ca2+ via Na+/Ca2+ exchange contributes to the transient elevation in intracellular free Ca2+. The polyvalent cations block the entry of critical Ca2+ ions by competing with Ca2+ for binding to the translocation site on the exchange carrier, while benzamil acts by lowering the maximal transport rate. These studies emphasize that Na+/Ca2+ exchange through its effects on cytoplasmic Ca2+ plays a major regulatory role in activation of the respiratory burst in chemotactic factor-stimulated neutrophils.     

Neutrophils become refractory to phorbol myristate acetate when treated with Ca2+-ionophore and Ca2+

Human neutrophils deprived of divalent cations by treatment with ionophore A23187 in the presence of ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) showed superoxide release when they were preincubated with calcium and then treated with the ionophore. The release was not observed when the ionophore was added first and then calcium was added more than 5 min later. The absence of the release in this case can be ascribed to a refractoriness of the cells to stimuli, because the cells did not release superoxide on stimulation with phorbol myristate acetate (PMA). The cells pretreated with either calcium or the ionophore alone did release superoxide on addition of PMA. The refractoriness of the cells to PMA depended on the concentrations of calcium and the ionophore and on the time interval between the two treatments. Calcium could be replaced with Cd2+ but not with Mg2+, Ba2+, or Sr2+. The release of granular enzymes was observed when the depleted cells were pretreated with the ionophore and then with calcium. These observations indicate that calcium has dual effects on the superoxide release of neutrophils, i.e., it stimulates the cells and makes them refractory to stimuli, depending on the time interval after the addition of the ionophore, and it also regulates the enzyme release by a different mechanism.     

Fluorescence response in chlortetracycline-loaded neutrophils measures release of Ca2+ from intracellular membrane enclosed storage sites

Chlortetracycline complexes with di- and trivalent cations resulting in an enhancement of its fluorescence emission intensity. Rabbit peritoneal neutrophils loaded with chlortetracycline gave a fluorescence response, even in the absence of extracellular Ca2+ and Mg2+, by a decrease in fluorescence intensity. The shift in the fluorescence emission maximum to lower wavelengths after the response suggested the response to be due to Ca2+ and not Mg2+ flux. The response was elicited by three mechanisms--a receptor-mediated mechanism by the chemotactic peptide, an ionophore-mediated one by lasalocid, and a detergent-mediated response by digitonin. These observations indicated that the response was due to transport of calcium across membranes in the intracellular compartments and may be physiologically significant. Whereas extracellular Ca2+ did not significantly affect the chemotactic peptide and lasalocid-mediated responses, Ca2+ inhibited the digitonin-mediated responses in a dose-dependent manner possibly due to extracellular Ca2+ flooding the cytosol through the digitonin-permeabilized plasma membrane and equilibrating the Ca2+ gradient across the intracellular membranes. The data collectively indicate that the fluorescence response is due to release of Ca2+ across intracellular membranes from a Ca2+ storage site into the cytosol.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig periotoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Fluoride-mediated activation of guinea pig neutrophils

In guinea pig peritoneal neutrophils NaF at a concentration of above 5 mM elicited a dose-dependent, delayed and sustained activation of NADPH oxidase. Unlike in human neutrophils, in guinea pig cells, this response was independent of extracellular calcium. Fura2 fluorescence measurements indicated also a fluoride-mediated moderate elevation in the level of cytosolic calcium concentration. Pretreatment of neutrophils with pertussis toxin, blocked fluoride-promoted activation of NADPH oxidase, indicating that NaF stimulation was mediated by a G protein which is a pertussis toxin substrate. NaF-elicited calcium elevation was insensitive to the toxin. Upon transfer of NaF-stimulated cells to a fluoride-free medium, superoxide release declined and calcium levels diminished. The response of the deactivated, fluoride-prestimulated guinea pig neutrophils to a secondary stimulation with phorbol myristate acetate (PMA) or fMet-Leu-Phe, was either unaffected by the previous challenge with NaF (PMA) or augmented by it (the chemotactic peptide). In parallel to the activation of NADPH oxidase, NaF also induced translocation of protein kinase C to cell membranes. This effect was also abolished by a pretreatment with pertussis toxin.     

Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.     

Uptake and intracellular sequestration of divalent cations in resting and methacholine-stimulated mouse lacrimal acinar cells. Dissociation by Sr2+ and Ba2+ of agonist-stimulated divalent cation entry from the refilling of the agonist-sensitive intracellular pool

The abilities of various divalent cations to enter the cytoplasm of mouse lacrimal acinar cells was examined under resting and agonist-stimulated conditions, by monitoring their effects on the fluorescence of cytosolic fura-2. In vitro, Ni2+, Co2+, and Mn2+ quenched the fura-2 fluorescence, whereas Sr2+, Ba2+, and La3+ produced an excitation spectrum and maximum brightness similar to Ca2+. Stimulation of mouse lacrimal acinar cells with methacholine (MeCh) caused a biphasic elevation of intracellular Ca2+ concentration [( Ca2+]i) resulting from a release of Ca2+ from intracellular pools followed by a sustained entry of extracellular Ca2+. Neither La3+ nor Ni2+ entered the cells under resting or stimulated conditions, but both blocked Ca2+ entry. Although both Co2+ and Mn2+ entered unstimulated cells, this process was not increased by MeCh. Both Sr2+ and Ba2+ were capable of supporting a sustained increase in fura-2 fluorescence in response to MeCh, indicating that these cations can enter the cells through the agonist-regulated channels. However, Sr2+, but not Ba2+, was capable of refilling the agonist-sensitive intracellular stores. These findings demonstrate dissociation of agonist-induced Ca2+ entry from intracellular Ca2+ pool refilling and thereby provide strong support for the recently modified version of the capacitative Ca2+ entry model according to which influx into the cytoplasm occurs directly across the plasma membrane and does not require a specialized cation channel directly linking the extracellular space and the intracellular Ca2+ stores.     

PGBX, a prostagandin derivative, mimics the action of the calcium ionophore A23187 on human neutrophils

The interactions have been studied of a water-soluble, polymeric derivative of prostaglandin B1, PGBX, with human polymorphonuclear leukocytes (PMN). PGBX, which is a potent ionophore of divalent cations, provoked superoxide anion (O2.-) generation and lysosomal enzyme release in cytochalasin B-treated PMN in the presence of extracellular divalent cations (Ca2+, Sr2+, Mg2+, Mn2+, Ba2+). Kinetic and dose-response studies showed that PGBX mimicked te action of ionophore A23187 in PMN. Both ionophores induced superoxide generation and release of enzymes from specific and azurophil granules (lysozyme > beta-glucuronidase) without provoking release of the cytoplasmic marker enzyme lactic dehydrogenase. In contrast, the precursor of PGBX, prostaglandin B1 (PGB1), and arachidonate did not mimic ionophore-induced stimulation of PMN. PGBX induced enzyme release both in the presence of extracellular Ca2+ and Ba2+ (both of which it translocates in model liposomes), whereas A23187 showed specificity for Ca2+ (which it translocates preferentially over Ba2+). These studies indicate that the actions of a water-soluble polymer (PGBX) derived from a naturally occurring prostaglandin (PGB1) on human neutrophils resemble those of a classical ionophore (A23187). Moreover, they provide additional evidence that increments in the intracellular levels of divalent cations may signal stimulus-secretion coupling in human neutrophils.     

Phosphorylase a activity as an indicator of neutrophil activation by chemotactic peptide

The activity of glycogen phosphorylase, an enzyme that is activated by both cAMP and calcium, was used as an indicator of the state of the cytoplasm after chemotactic stimulation of polymorphonuclear leukocytes (neutrophils). The activity of the enzyme showed a clear dependence on cytoplasmic calcium. Addition of the calcium ionophore A23187 caused a 4-5-fold increase in activity of phosphorylase a. In the absence of external Ca2+, A23187 caused only brief transient activation of phosphorylase; probably reflecting release of sequestered intracellular Ca2+. Addition of the chemotactic peptide N-formylnorleucylleucylphenylalanine (FNLLP) caused a transient 2-3-fold activation of the enzyme. The dose-dependence of activation by FNLLP showed a peak at 10(-8) M, near the Kd of the receptor for FNLLP. The phosphorylase activity peaks by 90 s and then declines, returning to basal levels by 20 min after stimulation with 10(-8) M peptide and by 60 min with 10(-7) M peptide. This finding suggests that the cells do not need to maintain elevated cytoplasmic calcium levels to exhibit stimulated locomotion. Thus, if calcium continues to modulate the motility, there either must be highly localized changes that are not detected in measures of the total cytoplasm, or the sensitivity to calcium must be variable such that basal levels are sufficient to maintain locomotion. Cells loaded with the fluorescence calcium probe quin2 (0.6 mM) in the presence or absence of external Ca2+ had elevated phosphorylase levels before addition of FNLLP. Thus, the presence of quin2 may alter the cytoplasmic Ca2+ level, and it clearly alters some aspects of the neutrophil physiology. Phosphorylase a appears to be a sensitive, nonperturbing indicator of the cytoplasmic calcium levels.     

Cyclopiazonic acid depletes intracellular Ca2+ stores and activates an influx pathway for divalent cations in HL-60 cells.

The filling state of intracellular Ca2+ stores has been proposed to regulate Ca2+ influx across the plasma membrane in a variety of tissues. To test this hypothesis, we have used three structurally unrelated inhibitors of the Ca(2+)-ATPase of intracellular Ca2+ stores and investigated their effect on Ca2+ homeostasis in HL-60 cells. Without increasing cellular inositol (1,4,5)trisphosphate levels, all three inhibitors (cyclopiazonic acid, thapsigargin, and 2,5-Di-tert-butylhydroquinone) released Ca2+ from intracellular stores, resulting in total depletion of agonist-sensitive Ca2+ stores. The Ca2+ release was relatively slow with a lag time of 5 s and a time to peak of 60 s. After a lag time of approximately 15 s, all three Ca(2+)-ATPase inhibitors activated a pathway for divalent cation influx across the plasma membrane. At a given concentration of an inhibitor, the plasma membrane permeability for divalent cations closely correlated with the extent of depletion of Ca2+ stores. The influx pathway activated by Ca(2+)-ATPase inhibitors conducted Ca2+, Mn2+, Co2+, Zn2+, and Ba2+ and was blocked, at similar concentrations, by La3+, Ni2+, Cd2+, as well as by the imidazole derivate SK&F 96365. The divalent cation influx in response to the chemotactic peptide fMLP had the same characteristics, suggesting a common pathway for Ca2+ entry. Our results support the idea that the filling state of intracellular Ca2+ stores regulates Ca2+ influx in HL-60 cells.     

Inhibition of enterocin CRL35 antibiotic activity by mono- and divalent ions

AIM: The aim of this work was to study the influence of different cations on the enterocin CRL35 activity. METHODS AND RESULTS: The antilisterial activity of enterocin CRL35 was tested by performing viability curves and measuring the dissipation of the proton motive force by fluorescent methods upon the addition of Ca2+, Mg2+, Li+, K+ and Na+ chlorides. The peptide uptake by sensitive cells was studied in the different conditions as well. The addition of calcium and magnesium chlorides (0.5-2 mmol l(-1)) induced an inhibition of the peptide activity. Potassium, sodium and lithium chlorides have an inhibitory effect as well, but at different range of concentration compared with divalent cations (50-150 mmol l(-1)). Interestingly, we found a differential protection effect among monovalent ions, KCl being almost nonprotective, meanwhile LiCl shows the stronger effect and NaCl has an intermediate effect. The ion effect depends on the pH, being more efficient in acidic media. Both mono and divalent ions inhibited the ability of the peptide to dissipate the transmembrane electric potential and pH gradient. Furthermore, the peptide uptake was also inhibited. CONCLUSIONS: The enterocin CRL35 activity is strongly dependent on the pH and the nature of the salts present in the medium. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will allow definition of the best system in which this peptide can be applied as biopreservative.     

The inositol 1,4,5-trisphosphate receptor of cerebellum. Mn2+ permeability and regulation by cytosolic Mn2+

The inositol 1,4,5-trisphosphate receptor (InsP3R), an intracellular calcium release channel, is found in virtually all cells and is abundant in the cerebellum. We used Mn2+ as a tool to study two aspects of the cerebellar InsP3R. First, to investigate the structure of the ion pore, Mn2+ permeation through the channel was determined. We found that Mn2+ can pass through the InsP3R; the selectivity sequence for divalent cations is Ba2+ > Sr2+ > Ca2+ > Mg2+ > Mn2+. Second, to begin characterization of the cytosolic regulatory sites responsible for the Ca(2+)-dependent modulation of InsP3R function, the ability of Mn2+ to replace Ca2+ was investigated. We show that Mn2+, as Ca2+, modulates InsP3R activity with a bell-shaped dependence where the affinity of the activation site of the InsP3R is similar for both ions, but higher concentrations of Mn2+ were necessary to inhibit the channel. These results suggest that the two regulatory sites are structurally distinct. Our findings are also important for the understanding of cellular responses when Mn2+ is used to quench the intracellular fluorescence of Ca2+ indicator dyes.     

Divalent cations mimic the inhibitory effect of extracellular ionised calcium on bone resorption by isolated rat osteoclasts: further evidence for a "calcium receptor".

Osteoclast activity is thought to be regulated by calcitonin, as well as by the level of ionised calcium generated locally as a result of bone resorption. The exposure of isolated osteoclasts to elevated ambient calcium levels has been shown to lower resorptive activity and to reduce rates of enzyme release. We have attempted to determine whether these effects are mediated by a divalent cation-sensitive "calcium receptor," as has been reported for the parathyroid chief cells. Thus, we compared the effect of alkaline earth metal cations on osteoclast function using a morphometric measure of bone resorption and a spectrophotometric method for measuring the activity of the released enzyme, acid phosphatase. The exposure of resorbing osteoclasts to between 5 and 20 mM extracellular ionised calcium ([Ca2+]e) inhibited bone resorption and enzyme release to an extent similar to that seen with 0.1 to 10 microM ionomycin. The effect of combining submaximal concentrations of [Ca2+]e (15 mM) and ionomycin (0.1 microM) resulted in additivity, suggesting that the influence of [Ca2+]e on bone resorption was mediated by elevated intracellular calcium levels ([Ca2+]i). The other cations studied (Mg2+, Ba2+) were effective and elicited similar effects, although some required higher concentrations. Thus, whilst Ca2+ and Mg2+ were effective at 10 to 15 mM levels, Ba2+ was effective only at high (20 mM) concentrations. These findings are consistent with an influence of [Ca2+]e on osteoclast activity through an action on a surface membrane "calcium receptor" that can also bind other divalent cations, rather than by passive changes of [Ca2+]i with [Ca2+]e elevation.     

The effect of various stimuli and calcium antagonists on the fluorescence response of chlorotetracycline-loaded human neutrophils

Chlorotetracycline has been used in neutrophils and other cells as probe of the state of membrane-bound calcium. We report here that human neutrophils treated with chlorotetracycline response to soluble secretagogues by a prompt decrease in chlorotetracycline fluorescence. This response was observed within 2-5 s, making it one of the most immediate reactions in neutrophils to stimulation, and was obtained with three secretagogues studied: a chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, a tumor promotor (phorbol myristate acetate) and a lectin (concanavalin A). The responses of neutrophils to the three stimuli differed both quantitatively and qualitatively. The calcium EGTA, did not effect the onset of the decrease in chlorotetracycline fluorescence, suggesting that the probe was measuring changes in intracellular calcium pools. The intracellular calcium antagonists, TMb-8, W-7 and trifluoperazine, did not block, but actually augmented, the fluorescence response. All four of these calcium antagonists blocked the recovery of chlorotetracycline fluorescence which was usually observed several minutes after stimulation with N-formyl-methionyl-leucyl-phenylalanine. This suggests that recovery was dependent upon both extracellular calcium and active calmodulin. The results are consistent with the hypothesis that changes in chlorotetracycline fluorescence reflect changes in a pool of membrane-bound 'trigger calcium', the release of which is an essential first step in stimulus-response coupling in human neutrophils.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Calcium signals in neutrophils can be divided into three distinct phases

Rabbit neutrophils were loaded with the fluorescence probe indo-1 and cytosolic free calcium levels were monitored during chemotactic peptide (fMet-Leu-Phe) stimulation. The fMet-Leu-Phe-induced calcium signal consisted of three consecutive phases: (1) an initial peak that was independent of extracellular calcium, (2) a secondary shoulder that required extracellular calcium but was totally blocked by hyperosmolality and (3) a final plateau of elevated calcium that was dependent on extracellular calcium but insensitive to hyperosmolality.     

Regulation of 22Na+ transport by calcium in an established kidney epithelial cell line.

The role of calcium in regulating the Na+ channel in an established kidney epithelial cell line has been examined. Extracellular calcium was inhibitory to Na+ uptake, and a Dixon plot of the initial Na+ uptake rate in the presence of Ca2+ was nonlinear, suggesting a mixed pattern of inhibition. Similar patterns of inhibition were also observed for other divalent cations, including Ba2+, Mg2+, and Mn2+. In contrast elevated concentrations of intracellular calcium resulted in a stimulation of Na+ entry. This intracellular effect was specific to calcium, with Mg2+ and Mn2+ appearing much less effective. Lineweaver-Burk plots of Na+ influx in calcium-loaded and unloaded cells were linear, suggesting that under both conditions a single system transported Na+. Although Na+ entry was stimulated by intracellular Ca2+, the cells did not exhibit other counter transport phenomena reported with cell types in which a Na+/Ca2+ exchange system is operative. Thus, the results indicate that calcium acts as an allosteric regulator of Na+ transport by the Na+ channel.     

Effect of divalent cations of the amphetamine-induced stimulation of [3H]catechol synthesis in the striatum.

Abstract— The effects of divalent cations on the stimulation of [³H]catechol formation in striatal slices induced by d-amphetamine was studied in order to determine the role of calcium in this action of amphetamine. In the absence of any divalent cations in the medium, amphetamine did not significantly stimulate [³H]catechol synthesis in striatal slices, but it produced a marked stimulation of synthesis when calcium (1.25 mm ) was added to the medium. In the presence of calcium (1.25 mm ), high concentrations of magnesium (15mm ), other divalent cations (2.5 mm ) such as barium, strontium, manganese and cobalt, as well as verapamil, inhibited the amphetamine-induced stimulation. When the slices were incubated in medium containing no divalent cations, the addition to the medium of either strontium, cobalt, zinc, or magnesium (2.5 mm ) could not support the amphetamine-induced stimulation of [³H]catechol synthesis, while the addition of barium resulted in a significant stimulation of synthesis. In contrast, the stimulation produced by amphetamine in the presence of manganese was comparable to that observed when calcium had been added to the medium. Since amphetamine did not alter the specific activity of [³H]tyrosine in the tissue in the presence of any of the divalent cations tested, the amphetamine-induced stimulation of [³H]catechol synthesis was probably due to an increase in tyrosine hydroxylase activity. Calcium and manganese were also able to support the stimulation of [³H]catechol synthesis in striatal slices induced by high potassium concentration. However, compared to the effects with amphetamine, manganese was much less effective than calcium in supporting the stimulation induced by high potassium concentration. These results show that specific divalent cations can support the stimulation of catechol synthesis induced by amphetamine in striatal slices, and suggest that the entry of these specific ions into cells, presumably dopamine neurons, is involved in this action.