The liver cell plasma membrane Ca2+ inflow systems exhibit a broad specificity for divalent metal ions.

1. The inflow of Mn2+ across the plasma membranes of isolated hepatocytes was monitored by measuring the quenching of the fluorescence of intracellular quin2, by atomic absorption spectroscopy and by the uptake of 54Mn2+. The inflow of other divalent metal ions was measured using quin2. 2. Under ionic conditions which resembled those present in the cytoplasmic space, Mn2+, Zn2+, Co2+, Ni2+ and Cd2+ each quenched the fluorescence of a solution of Ca2(+)-quin2. 3. The addition of Mn2+, Zn2+, Co2+, Ni2+ or Cd2+ to cells loaded with quin2 caused a time-dependent decrease in the fluorescence of intracellular quin2. Plots of the rate of decrease in fluorescence as a function of the concentration of Mn2+ reached a plateau at 100 microM-Mn2+. 4. The rate of decrease in fluorescence induced by Mn2+ was stimulated by 20% in the presence of vasopressin. The effect of vasopressin was completely inhibited by 200 microM-verapamil. Adrenaline, angiotensin II and glucagon also stimulated the rate of decrease in the fluorescence of intracellular quin2 induced by Mn2+. 5. The rate of decrease in fluorescence induced by Zn2+, Co2+, Ni2+ or Cd2+ was stimulated by between 20 and 190% in the presence of vasopressin or angiotensin II. 6. The rates of uptake of Mn2+ measured by atomic absorption spectroscopy or by using 54Mn2+ were inhibited by about 20% by 1.3 mM-Ca2+o and stimulated by 30% by vasopressin. 7. Plots of Mn2+ uptake, measured by atomic absorption spectroscopy or with 54Mn2+, as a function of the extracellular concentration of Mn2+ were biphasic over the range 0.05-1.0 mM added Mn2+ and did not reach a plateau at 1.0 mM-Mn2+. 8. It is concluded that (i) hepatocytes possess both a basal and a receptor-activated divalent cation inflow system, each of which has a broad specificity for metal ions, and (ii) the receptor-activated divalent cation inflow system is the receptor-operated Ca2+ channel.     

The measurement of Ca2+ inflow across the liver cell plasma membrane by using quin2 and studies of the roles of Na+ and extracellular Ca2+ in the mechanism of Ca2+ inflow.

1. Rates of Ca2+ inflow across the hepatocyte plasma membrane in the presence of vasopressin were estimated by using quin2. 2. Plots of the rate of Ca2+ inflow as a function of the intracellular quin2 concentration reached a plateau at about 1.7 mM intracellular quin2. Ca2+ inflow was inhibited by 60% in the presence of 400 microM-verapamil. 3. A plot of the rate of Ca2+ inflow as a function of the concentration of extracellular Ca2+ ([Ca2+]o) was biphasic. The second (slower) phase showed no sign of saturation at values of [Ca2+]o up to 5 mM. It is concluded that, in the presence of vasopressin, Ca2+ flows into the liver cell by two different processes, one of which is not readily saturated by Ca2+o. 4. The effect of the replacement of extracellular NaCl by choline or tetramethylammonium chloride on cellular Ca2+ movement was found to depend on the presence or absence of intracellular quin2. 5. In cells loaded with quin2 and incubated in the presence of choline or tetramethylammonium chloride, a small decrease in the basal intracellular free Ca2+ concentration ([Ca2+]i) was observed, and the increase in [Ca2+]i caused by the addition of vasopressin was considerably diminished when compared with cells incubated in the presence of NaCl. In cells loaded with quin2, replacement of NaCl by choline chloride caused a decrease in Ca2+ inflow in the presence of vasopressin, as measured by using quin2 or 45Ca2+ exchange, whereas no change in Ca2+ inflow was observed in the absence of vasopressin. 6. In cells not loaded with quin2, replacement of NaCl by choline chloride did not alter Ca2+ inflow either in the presence or in the absence of vasopressin. 7. It is concluded that (i) Ca2+ inflow through the basal and receptor-activated Ca2+ inflow systems does not involve the inward movement of Ca2+ in exchange for Na+ or the induction of Ca2+ inflow by intracellular Na+, and (ii) the presence of both intracellular quin2 and extracellular choline or tetramethylammonium chloride (in place of NaCl) inhibits Ca2+ inflow through the receptor-activated Ca2+ inflow system but not through the basal Ca2+ inflow system, and inhibits the release of Ca2+ from intracellular stores.     

Protamine-induced permeability changes in the neutrophil plasma membrane as the basis of activation of exocytosis.

Protamine induces a gradual change in plasma membrane permeability in rabbit neutrophils, which is evident from the increase of indol fluorescence, and the leakage of quin2 from quin2-loaded neutrophils. The influx of extracellular Ca2+ into the neutrophil provides an explanation for exocytosis which occurs in the presence of Ca2+ and protamine. The dependence of exocytosis on Ca2+ concentration follows the same pattern as is observed in neutrophils permeabilized by other means. In the absence of Ca2+, and in the presence of protamine, La3+ has an activating effect on exocytosis. At higher concentrations La3+ inhibits exocytosis that occurs in the presence of Ca2+ and protamine, as do some other metal ions. The resemblance between the membrane effects of a number of toxins, as reported in literature, and protamine-induced membrane damage suggests that they occur via the same mechanism.     

Use of manganese to discriminate between calcium influx and mobilization from internal stores in stimulated human neutrophils

Stimulation of human neutrophils with f-met-leu-phe, platelet-activating factor, or leukotriene B4 resulted in an increase in [Ca2+]i. The [Ca2+]i rise was greater in the presence than absence of external Ca2+; the component that was dependent on external Ca2+ was blocked by Ni2+, or could be reconstituted by addition of external Ca2+ following discharge of the internal Ca2+ store. These measurements of [Ca2+]i responses provide only indirect evidence for agonist-stimulated Ca2+ entry, and here we have used an alternative approach to demonstrate directly agonist-stimulated divalent cation entry. In the presence of extracellular Mn2+, f-met-leu-phe, leukotriene B4, and platelet-activating factor stimulate a quench in fluorescence of fura-2-loaded human neutrophils. This quench was due to stimulated Mn2+ influx and was blocked by Ni2+. When Mn2+ was added in the continued presence of agonist, after discharge of the internal store of Ca2+, a stimulated quench was seen; this result shows that an elevated [Ca2+]i is not needed for the stimulation of Mn2+ entry. Depolarization by high [K+] or addition of the L-type Ca2+ channel agonist, BAY-R-5417, had little or no effect on either [Ca2+]i or Mn2+ entry. These results show that agonists stimulate divalent cation entry (Ca2+ or Mn2+) by a mechanism independent of changes in [Ca2+]i and unrelated to voltage-dependent Ca2+ channels.     

Chemoattractant receptor promotion of Ca2+ influx across the plasma membrane of HL-60 cells. A role for cytosolic free calcium elevations and inositol 1,3,4,5-tetrakisphosphate production

The mechanisms by which the chemotactic peptide formyl-methyl-leucyl-phenyl-alanine stimulates Ca2+ influx across the plasma membrane were investigated in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Ca2+ influx was determined: (a) from the initial rate of Mn2+ influx, apparent from the quenching of intracellular quin2 or fura-2 fluorescence; (b) from the rate of the elevation of cytosolic free calcium, [Ca2+]i, upon readdition of Ca2+ to cells previously stimulated in the absence of extracellular Ca2+. [3H]Inositol tris-, tetrakis-, and pentakisphosphates were analyzed by a high performance liquid chromatography procedure which was optimized for the separation of inositol tetrakisphosphates, yielding three predominant isomers: inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), inositol 1,4,5,6-tetrakisphosphate, and inositol 1,3,4, 6-tetrakisphosphate. Both the kinetics and agonist dose dependence of Ca2+ influx stimulation correlated closely with the corresponding receptor-mediated variations of [Ca2+]i either in the presence or in the absence of extracellular Ca2+. Of the different inositol phosphates determined in parallel and under the same conditions, accumulation of [3H]Ins(1,3,4,5)P4 correlated best with Ca2+ influx both temporally and in its dose dependence in the presence or in the absence of extracellular Ca2+; inositol 1,3,4-trisphosphate was also correlated but to a lesser extent. Attenuations of [Ca2+]i elevations by decreasing extracellular Ca2+ or by increasing the cytosolic Ca2+ buffering capacity with quin2 led to parallel inhibition of Ca2+ influx and Ins(1,3,4,5)P4 production. In conclusion: 1) activation of Ca2+ influx by formyl-methionyl-leucyl-phenylalanine depends on the elevation of [Ca2+]i, the latter being initiated by Ca2+ mobilization from intracellular stores; 2) Ins(1,3, 4,5)P4 is a strong candidate for maintaining receptor-mediated activation of Ca2+ influx in differentiated HL-60 cells.     

Dual effects of manganese on prolactin secretion

The effect of Mn2+ (a commonly used Ca2+ antagonist) on prolactin secretion from pituitary cells was investigated. In the presence of normal extracellular Ca2+ levels (2.5mM), Mn2+ inhibited basal, TRH- and K+- stimulated prolactin secretion. The Ca2+ ionophore, A23187, partially overcame the inhibitory effect of Mn2+. However, in the presence of low extracellular Ca2+ (less than 100 microM), which decreased basal prolactin secretion and abolished any stimulatory effects of TRH or K+, a paradoxical stimulatory effect was observed with Mn2+ in the presence of A23187. In the presence of Ca2+, Mn2+ appeared to be inhibitory due to its Ca2+ antagonistic effects, but at low Ca2+ levels, intracellular stimulatory effects of Mn2+ became apparent.     

Evidence for receptor-mediated calcium entry and refilling of intracellular calcium stores in FRTL-5 rat thyroid cells.

The aim of the present study was to investigate the relationship between agonist-induced changes in intracellular free Ca2+ ([Ca2+]i) and the refilling of intracellular Ca2+ stores in Fura 2-loaded thyroid FRTL-5 cells. Stimulating the cells with ATP induced a dose-dependent increase in ([Ca2+]i). The ATP-induced increase in [Ca2+]i was dependent on both release of sequestered intracellular Ca2+ as well as influx of extracellular Ca2+. Addition of Ni2+ prior to ATP blunted the component of the ATP-induced increase in [Ca2+]i dependent on influx of Ca2+. In cells stimulated with ATP in a Ca(2+)-free buffer, readdition of Ca2+ induced a rapid increase in [Ca2+]i; this increase was inhibited by Ni2+. In addition, the ATP-induced influx of 45Ca2+ was blocked by Ni2+. Stimulating the cells with noradrenaline (NA) also induced release of sequestered Ca2+ and an influx of extracellular Ca2+. When cells were stimulated first with NA, a subsequent addition of ATP induced a blunted increase in [Ca2+]i. If the action of NA was terminated by addition of prazosin, and ATP was then added, the increase in [Ca2+]i was restored to control levels. Addition of Ni2+ prior to prazosin inhibited the restoration of the ATP response. In the presence of extracellular Mn2+, ATP stimulated quenching of Fura 2 fluorescence. The quenching was probably due to influx of Mn2+, as it was blocked by Ni2+. The results thus suggested that stimulating release of sequestered Ca2+ in FRTL-5 cells was followed by influx of extracellular Ca2+ and rapid refilling of intracellular Ca2+ stores.     

Intracellular free calcium transients induced by norepinephrine in rat aortic smooth muscle cells in primary culture

In cultured rat arterial smooth muscle cells treated with quin 2, cytosolic Ca2+ transients induced by norepinephrine were recorded microfluorometrically. In the presence or absence of extracellular Ca2+, norepinephrine induced transient and dose-dependent elevations in cytosolic Ca2+, with a similar time course, the peak levels being observed at 2 min. These transient elevations in cytosolic Ca2+ were dose-dependently inhibited by alpha-adrenergic antagonists, the order of potency being prazosin greater than phentolamine greater than yohimbine, irrespective of the presence of extracellular Ca2+. We propose that with or without extracellular Ca2+, norepinephrine activates mainly alpha-1 adrenoceptors leading to a release of Ca2+ from intracellular stores. This would explain the transient elevation in cytosolic Ca2+ in rat aortic vascular smooth muscle cells in primary culture.     

The epidermal growth factor-induced calcium signal in A431 cells

Addition of epidermal growth factor (EGF) to human A431 cells causes a 2-4-fold increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured by quin-2 fluorescence. The EGF effect is rapid but transient: [Ca2+]i reaches a maximum within 30-60 s and then returns to its resting value (182 +/- 3 nM) over a 5-8-min period. The EGF-induced [Ca2+]i rise is completely dependent on extracellular Ca2+, is abolished by La3+ and Mn2+, and is not accompanied by changes in membrane potential (mean values of -64 mV). Serum also elicits a transient [Ca2+]i rise in A431 cells, but this response is not dependent on the presence of extracellular Ca2+. The tumor promoter 12-O-tetradecanoylphorbol 13-acetate completely inhibits the EGF- and serum-induced increases in [Ca2+]i without affecting basal [Ca2+]i levels. Our results, together with previous 45Ca2+ uptake data (Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20, 6280-6286), suggest that while serum factors trigger the release of Ca2+ from internal stores, EGF acts by opening a voltage-independent Ca2+ channel in the plasma membrane. The data further suggest a role for protein kinase C in attenuating the Ca2+-mobilizing mechanisms of EGF and serum.     

Platelet activating factor raises intracellular calcium ion concentration in macrophages

Peritoneal cells from thioglycollate-stimulated mice were allowed to adhere to coverglasses for 2 h to give a dense monolayer of adherent cells greater than 95% of which were macrophages. After incubation with the tetra-acetoxymethyl ester of quin2, coverglasses were rinsed with Ca2+-free saline, oriented at a 45 degree angle in square cuvettes containing a magnetically driven stir bar, and analyzed for changes in quin2 fluorescence in a spectrofluorimeter. Such fluorescence, taken as an indication of intracellular calcium ion concentration ([Ca2+]i), increased as exogenous calcium ion concentration ([Ca2+]o) was raised to 1 mM. At [Ca2+]o approximately equal to 10 microM, [Ca2+]i = 72 +/- 14 nM (n = 26); at [Ca2+]o = 1 mM, [Ca2+]i = 140-220 nM, levels not increased by N, N, N', N'-tetrakis (2-pyridylmethyl) ethylenediamine, a membrane-permeant chelator of heavy metals than can quench quin2. Addition of mouse alpha + beta fibroblast interferon, lipopolysaccharide, thrombin, collagen, vasopressin, ADP, compound 48/80, or U46619 did not change [Ca2+]i. However, addition of platelet activating factor (PAF) (2-20 ng/ml) raised [Ca2+]i by 480 nM within 1 min if [Ca2+]o = 1 mM. In the presence of 5 mM EGTA, PAF raised [Ca2+]i by 25 nM. This suggests that PAF causes influx of exogenous Ca2+, as well as releasing some Ca2+ from intracellular stores. Consistent with these results, when PAF was added to 1 mM Ca2+ in the presence of 100 microM Cd2+ or Mn2+ to block Ca2+ influx, [Ca2+]i increased by only intermediate amounts; at the times of such dampened peak response, [Ca2+]i could be raised within 1 min to normal PAF-stimulated levels by chelation of the exogenous heavy metals with diethylenetriaminepentaacetic acid. Normal PAF responses were observed in the presence of indomethacin. The lowest dose of PAF observed to raise [Ca2+]i was 0.1 ng/ml. Response of [Ca2+]i to 2-20 ng/ml PAF was transient, and second applications had no effect. The PAF response also was seen in cell suspensions. These results suggest that an increase in [Ca2+]i may be an early event in PAF activation of macrophages.     

Glucose-induced changes in cytoplasmic free Ca2+ concentration and the significance for the regulation of insulin release. Measurements with fura-2 in suspensions and single aggregates of mouse pancreatic beta-cells

The effects of glucose on cytoplasmic free Ca2+ concentration, [Ca2+]i, and insulin release were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Measurements of [Ca2+]i were performed in cell suspensions in a cuvette and in single cell-aggregates in a microscopic system, using fura 2 and quin 2. Insulin release was studied from indicator loaded cells in a column perifusion system. In the presence of 1.28 mM extracellular Ca2+, an increase in the glucose concentration from 0 to 20 mM had two major effects on [Ca2+]i. Initially there was a decrease, which was immediately followed by a pronounced increase. At reduced extracellular Ca2+, or when Ca2+ influx was blocked, glucose induced only a decrease in [Ca2+]i. With increasing intracellular concentrations of indicator, the effects of glucose on [Ca2+]i were markedly reduced. Changes in [Ca2+]i, similar effects being obtained in the cuvette and microfluorometric measurements, were paralleled by changes in insulin release. Insulin release from indicator loaded cells did not markedly differ from that of non-loaded controls, either with respect to rapidity or size in the response to the sugar. The addition of 20 mM glucose increased the efflux of fura 2, an effect that was not related to insulin release. Permeabilization of indicator loaded cells demonstrated a substantial amount of fura 2 bound intracellularly. Although the effects of glucose on [Ca2+]i seemed to be similar in fura 2 and quin 2 loaded cells, the demonstrated leakage and possible intracellular binding should be considered before using fura 2 for measurements in pancreatic beta-cells.     

The effect of quin2 on chemotaxis by polymorphonuclear leukocytes

Exposure of rabbit polymorphonuclear leukocytes to micromolar concentrations of quin2-AM results in high intracellular concentrations of quin2, which lead to inhibition of chemotaxis. The loading efficiency of polymorphonuclear leukocytes, being the percentage of quin2-AM which is taken up by the cells and transformed intracellularly into quin2, is very high, reaches a maximum after 30 min, is independent of the presence of extracellular Ca2+ and is fairly independent of cell concentration. As a consequence, inhibition of chemotaxis is strongly dependent on experimental conditions: with a low cell density (3 X 10(6)/ml) exposure to 20 microM quin2-AM results in complete inhibition of chemotaxis, whereas the same concentration of quin2-AM is nearly without effect when an 8-fold higher cell concentration is used. Inhibition by quin2 is dependent on extracellular Ca2+; inhibition is more pronounced in the absence of extracellular Ca2+ than in its presence. It is suggested that quin2 inhibits chemotaxis by interference with intracellular Ca2+.     

Mn2+ binding to factor VIII subunits and its effect on cofactor activity

Metal ions, such as Ca2+ and Mn2+, are necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC). Titration of EDTA-treated factor VIII with Mn2+ showed saturable binding with high affinity (K(d) = 5.7 +/- 2.1 microM) as detected using a factor Xa generation assay. No significant competition between Ca2+ and Mn2+ for factor VIII binding (K(i) = 4.6 mM) was observed as measured by equilibrium dialysis using 20 microM Ca2+ and 8 microM factor VIII in the presence of 0-1 mM Mn2+. The intersubunit affinity measured by fluorescence energy transfer of an acrylodan-labeled LC (fluorescence donor) and fluorescein-labeled HC (fluorescence acceptor) in the presence of 20 mM Mn2+ (K(d) = 53.0 +/- 17.1 nM) was not significantly different from the affinity value previously obtained in the absence of metal ion (K(d) = 53.8 +/- 14.2 nM). The sensitization of phosphorescence of Tb3+ bound to factor VIII subunits was utilized to detect Mn2+ binding to the subunits. Mn2+ inhibited the phosphorescence of Tb3+ bound to HC and LC, as well as the HC-derived A1 and A2 subunits with a relatively wide range of estimated inhibition constant values (K(i) values = 169-1147 microM), whereas Ca2+ showed no effect on Tb3+ phosphorescence. These results suggest that factor VIII cofactor activity can be generated by Mn2+ binding to site(s) on factor VIII that are different from the high-affinity Ca2+ binding site. However, like Ca2+, Mn2+ did not alter the affinity for HC and LC association. Thus, Mn2+appears to generate factor VIII cofactor activity by a similar mechanism as observed for Ca2+following its association at nonidentical sites on the protein.     

Evidence for TRH-induced influx of extracellular Ca2+ in pituitary GH4C1 cells

The aim of the study was to investigate the relationship between thyrotropin-releasing hormone (TRH)-induced changes in intracellular free Ca2+ ([Ca2+]i), and influx of extracellular Ca2+ in Fura 2 loaded pituitary GH4C1 cells. Stimulating the cells with TRH in a Ca(2+)-containing buffer induced a biphasic change in [Ca2+]i. First, a transient increase in [Ca2+]i, followed by a sustained phase. In cells stimulated with TRH in a Ca(2+)-free buffer, the transient increase in [Ca(2+)]i was decreased (p less than 0.05), and the sustained phase was totally abolished. Addition of Ni2+ prior to TRH blunted the component of the TRH-induced transient increase in [Ca2+]i dependent on influx of Ca2+. In the presence of extracellular Mn2+, TRH stimulated quenching of Fura 2 fluorescence. This quenching was blocked by Ni2+. The results indicate that both the TRH-induced transient increase in [Ca2+]i as well as the sustained phase in [Ca2+]i in GH4C1 cells is dependent on influx of extracellular Ca2+.     

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.     

Characterization of the bradykinin-stimulated calcium influx pathway of cultured vascular endothelial cells. Saturability, selectivity, and kinetics

Measurement of fura-2 fluorescence and 45Ca2+ uptake was used to evaluate Ca2+ influx in cultured bovine aortic endothelial cells (BAECs) stimulated by bradykinin (BK). The BK-stimulated influx pathway was characterized with respect to its 1) sensitivity to extracellular Ca2+, 2) inhibition by membrane depolarization, and 3) permeability to Ba2+ and Sr2+. The results indicate that the activity of the influx pathway is a saturable function of extracellular Ca2+ and that membrane depolarization inhibits Ca2+ influx by changing the apparent affinity and maximal capacity of the pathway for Ca2+. Fura-2 fluorescence was used to compare the profiles for BK-stimulated changes in cytosolic Ca2+, Sr2+, and Ba2+ (Ca2+i, Ba2+i, and Sr2+i). Addition of Ca2+ and Sr2+ to Ca2+-depleted cells in the presence of BK produced a transient increase in Ca2+i and Sr2+i. Following the peak of the response, Ca2+i and Sr2+i declined within 2 min to a steady elevated level. Blockade of influx by the addition of La3+ at the peak of the response to Ca2+ and Sr2+ immediately reduced Ca2+i and Sr2+i to basal levels. Addition of Ba2+ to Ca2+-depleted cells in the presence of BK produced an increase in Ba2+i which continued to rise with time to a steady level. Addition of La3+ after Ba2+, however, did not reduce Ba2+i. These results suggest that 1) Ca2+ and Sr2+ (but not Ba2+) are sequestered by intracellular mechanisms and that the declining phase of the Ca2+ and Sr2+ response reflects a time and divalent cation-dependent inactivation of the influx pathway. The inactivation of the influx pathway was further demonstrated by measuring the kinetics of BK-stimulated 45Ca2+ uptake into BAECs. The results of these experiments demonstrate that BK stimulates a 100- to 150-fold increase in Ca2+ permeability of the BAEC but that the influx pathway turns off or inactivates within 2 min. The magnitude of the flux, the voltage sensitivity, and the ability to conduct Ca2+, Sr2+, and Ba2+ are suggestive of a channel mechanism.     

Stimulation of non-selective cation channels providing Ca2+ influx into platelets by platelet-activating factor and other aggregation inducers.

To elucidate the mechanism of the receptor-stimulated Ca2+ entry into human platelets, the influence of Ca(2+)-mobilizing agonists on plasma membrane potential (Em) has been studied. Em changes were registered using potentiometric probe 3,3'-dipropyl-2,2'-thiadicarbocyanine iodide. The agonist effect on Em varied from hyperpolarization to slight and slow rise. On the contrary, after loading of platelets with intracellular Ca2+ indicator quin2, platelet-activating factor (PAF), thrombin, vasopressin, ADP and thromboxane-A2-mimetic U46619 cause substantial transient membrane depolarization. Similar effects were observed after platelet loading with other Ca2+ chelators fura-2 and indo-1. Agonist-induced depolarization considerably reduced if quin2-loaded platelets were suspended in isoosmotic choline-containing medium. Using Ba2+ as a substitute of Ca2+, we have demonstrated that in choline-containing medium PAF-induced Ba2+ entry into platelets results in membrane depolarization. Dependence on Ba2+ concentration and depolarization kinetics correlates with the dose dependence and kinetics of Ba2+ entry detected by quin2 fluorescence. The agonists also stimulate considerable Na+, Li+ and Cs+ inward currents into platelets. Na(+)-dependent depolarization is 2-5-fold suppressed by extracellular Ca2+ [median inhibitory concentration (IC50) approximately 0.3 mM]. Ni2+ and Cd2+ at similar concentrations block Ca2+ entry and agonist-induced Na2+ current (IC50 for both cations approximately 50 microM). Agonist-induced depolarization is blocked by the adenylate cyclase stimulator prostaglandin E1 and the protein kinase C stimulator phorbol ester. It is concluded that agonists stimulate Ca2+ entry into human platelets via receptor-operated channels which are not strictly selective toward divalent cations and are permeable to Na+, Li+ and Cs+.     

Extracellular ATP elevates intracellular free calcium in rat parotid acinar cells

The effect of extracellular ATP on intracellular free Ca2+ was characterized in quin2-loaded parotid acinar cells. ATP specifically increased the intracellular Ca2+ concentration six-fold above a basal level of 180 nM. Of other purine nucleotides tested, only adenylylthiodiphosphate (ATP gamma S) had significant activity. ATP and the muscarinic agonist carbachol increased intracellular Ca2+ even in the absence of extracellular Ca2+. Both agonists stimulated K+ release, which was followed by reuptake of K+, even in the continued presence of agonist. In the absence of Mg2+, ATP was much more potent but no more efficacious in elevating intracellular Ca2+, suggesting that ATP4- is the active species. The effect of ATP was reversed by removal with hexokinase, arguing against a role for an active contaminant of ATP and against a non-specific permeabilizing effect of extracellular ATP. Lactate dehydrogenase release was unaffected by a maximally effective concentration of ATP. These observations are consistent with a possible neurotransmitter role for ATP in the rat parotid gland.     

Interaction of monoclonal antiparathyroid antibody with Ca2+ agonistic actions of Mn2+ in normal human parathyroid cells

Effects of the monoclonal antiparathyroid antibodies G11 and E11 on Mn2+ interaction with individual normal human parathyroid cells were studied. At 0.5mM Ca2+, 3mM Mn2+ induced a rapid transient increase in cytoplasmic Ca2+ [Ca2+i] followed by quenching of the fluorescence from the Ca2+ indicator fura-2 as Mn2+ entered into the cells. Whereas the antibody E11 had no effects, treatment with G11 abolished the Ca2+i transient and considerably delayed the entry of Mn2+. The results support the presence of a cation-sensitive receptor mechanism on parathyroid cells and indicate that the antibody G11 not only blocks the interaction between Ca2+ and this receptor mechanism but also that of Mn2+.     

Is intracellular Ca2+ the trigger for oxygen radical production by polymorphonuclear leucocytes?

The aim of this paper is critically to evaluate the existing evidence for the role of intracellular Ca2+ in polymorphonuclear leucocyte (PMN) activation and in particular in oxygen radical production. Indirect experiments are based on the manipulation of extracellular Ca2+, measurement of 45Ca fluxes, employing pharmacological agents such as Ca2+-ionophores and intracellular Ca2+ antagonists and monitoring chlortetracycline fluorescence. Experiments of this type do not provide the necessary definitive evidence that an increase in intracellular Ca2+ is the trigger for PMN activation. Recent direct measurements of intracellular free Ca2+ using the Ca2+-activated photoprotein, obelin, and the Ca2+-sensitive fluorescent indicator, quin 2, have provided evidence for the existence of two distinct mechanisms of activation, one triggered by a rise in intracellular Ca2+ and the other independent of a rise in intracellular Ca2+. The source of the Ca2+ for the former mechanism is mainly extracellular but can also come from an intracellular Ca2+ store.