Manipulation of Intracellular Calcium in NCB-20 Cells

Journal oj”eirrochcmislr.v Raven Press, Ltd., New York 0 1992 International Society for Neurochernistry

Manipulation of Intracellular Calcium in NCB-20 Cells

Anja Garritsen and Dermot M. F. Cooper

Department of Phurmacology, University of Colorado Health Sciences Center, Denver, Colorado, U.S.A.

Abstract: A number of lines of evidence indicate that the Ca2+ and cyclic AMP signalling systems interact in NCB-20 cells. However, to date, the regulation of [Ca2+], homeostasis has not been studied in this cell line. The present study aimed to clarify our understanding of [Ca2+Ii homeostasis in these cells and to evaluate tools that manipulate [Ca”],, independently of protein kinase C effects. Bradykinin, by a B,-receptor, elevated [Ca2’Ii by a pertussis-toxin-insensitive mechanism. The BK-stimulated [Ca”], rise originated from intracellular sources, without a contribution from Ca2+ entry mechanisms. The effect of BK was precluded by pretreatment with thapsigargin and ionomycin-compounds that elevated [Ca2+], independent of phospholipase C activation. Both compounds, however, exerted effects in addi-

tion to stimulating release of Ca2+ from BK-sensitive stores; the BK-sensitive Ca2+ pool was a subset of the thapsigargin- sensitive pool; ionomycin strongly stimulates Ca2+ entry. Activation of protein kinases A and C attenuated the duration of the BK-induced rise in [Ca2+Ii, without affecting the peak [Ca2+],, suggesting interference with the BK response at a step downstream of the activation of phospholipase C . Application of these approaches should enhance the delineation of the consequences of Ca2+ mobilization on cyclic AMP accumulation. Key Words: Intracellular calcium- NCB-20 cell line-Bradykinin-Thapsigargin-Ionomy- cin. Garritsen A. and Cooper D. M. F. Manipulation of intracellular calcium in NCB-20 cells. J. Nezirochem. 59, 190- 199 ( 1992).

Interactions or “cross talk” between signal trans- duction pathways are being widely encountered, with the result that the cellular consequences of hormonal action may ramify beyond the activation of the initially targeted second messenger system. The NCB-20 cell line, a cultured cell line from mouse neuroblas- toma X 18-day Chinese hamster embryonic brain ex- plant, is becoming an instructive model in which cross talk between the cyclic AMP and Ca2+ signalling pathways can be studied. McAtee and Dawson (1989, 1990) have demonstrated that the elevation of cyclic AMP and subsequent activation of protein kinase A (PKA) reduce phospholipase C activity, inositol phosphate formation, and protein kinase C (PKC) activation by phorbol esters. On the other hand, several re- ports (Hollingsworth and Daly, 1987; Gusovsky et al., 1989; Gusovsky and Gutkind, 1990) have demonstrated that the activation of PKC in NCB-20 cells results in inhibition of cyclic AMP accumulation that is slow in onset but persistent. A more rapid, but transient, inhibition of cyclic AMP accumulation can be

evoked by bradykinin (BK) as a result of its elevation of the intracellular calcium concentration ( [Ca2+],). This inhibition appears to be due to direct inhibition of the catalytic unit of adenylate cyclase by Ca2+ (Boyajian et al., 199 1). It thus appears as though there may be a dynamic interplay between the two second messenger systems of cyclic AMP and [Ca2+], at a number of levels. To explore this interaction further, it is essential to study the physiological modulation of [Ca2+], and cyclic AMP under parallel conditions.

To date, in most studies that infer interactions between cyclic AMP and Caz+ in NCB-20 cells, Ca2+ homeostasis has not been assessed. A critical step in evaluating the modes of interaction between the Ca2+ and cyclic AMP signalling pathways in NCB-20 cells is to understand how Ca2+ homeostasis is controlled in these cells and to devise means whereby [Ca2+], can be manipulated experimentally. The mechanism of Ca2+ mobilization in NCB-20 cells by BK is consid- ered to be predominantly by inositol 1,4,5-tnsphos- phate [Ins( 1,4,5)P,]-induced release of Ca2+ from the

Received September 6, 1991; revised manuscript received No- vember 28, 199 l ; accepted December 16, 199 l .

Address correspondence and reprint requests to Dr. D. M. F. Cooper at Department of Pharmacology, University of Colorado Health Sciences Center, 4200 E 9th Avenue-Box C236, Denver, CO 80262, U.S.A.

Abbreviarions used: BK, bradykinin; BSA, bovine serum albumin; [Ca”’], , cytosolic free calcium concentration; DMEM. Dul- becco’s modified Eagle’s medium; Ins( 1,4,5)P,, inositol 1,4,5-trisphosphate; PDBu, phorbol 12,13-dibutyrate; PI, phosphoinositide; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; PTX, pertussis toxin.

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CALCIUM MOBILIZATION IN NCB-20 CELLS 191

endoplasmic reticulum (Chuang and Dillon-Carter, 1988) although other mechanisms have not been explored in any detail.

The aim of the present study was to examine the hormonal mobilization of [Ca2+], in NCB-20 cells- in terms of its kinetics and source, and the influence, if any, of either PKC or PKA thereon, and to develop means whereby [Ca2+],, which exerts a prominent in- hibitory effect on cyclic AMP accumulation (Boya- jian et al., 199 l), could be manipulated experimentally, independent of phospholipase C activation.

MATERIALS AND METHODS

Cell culture Low passage NCB-20 cells were provided by Dr. G. Daw-

son (University of Chicago, Chicago, IL, U.S.A.). Cells were grown in 75-cm2 culture flasks in Dulbecco’s modified Ea- gle’s medium (DMEM) with 5% fetal bovine serum containing 6 pg/ml of gentamicin at 37°C in an atmosphere of 10% CO, and were used 4-6 days after passage.

Intracellular calcium measurements Fiira-2 loading procedure. NCB-20 cells were detached

from culture flasks with phosphate-buffered saline (Na, HPO, 12.1 M, KH,PO, 4 mM, NaCl 130 mM, pH 7.4), containing 0.02% EDTA, and allowed to recover in DMEM, containing 0.1% bovine serum albumin (fraction V: DMEM/BSA), for 1 h at room temperature. Subse- quently, the cell suspension ( lo7 cells/ml) was mixed with 4 pMfura-2 AM and 0.02% Pluronic in DMSO (final concentration 0.5%). After incubation at room temperature for 20 min with regular swirling, the cells were centrifuged for 4 min at 1,000 rpm in a Daemon/IEC tabletop centrifuge, washed, and resuspended in DMEM/BSA. The cells were kept at room temperature until use. Immediately before as- say, an aliquot of cells (-4 X 1 O6 cells) was diluted with 3 ml of Krebs buffer (composition, in mM: NaCl 120, KCI 4.75, KHZPO, 1, NaHCO, 5, MgSO, 1.44, CaCI, 1.1, EGTA 0.1, glucose 1 1, HEPES 25, adjusted to pH 7.4 with Tris), centrifuged, resuspended in 3 ml of Krebs buffer, and transferred to a cuvette.

Fluorescence measurements. Fura-2 fluorescence was measured at 30°C (to reduce leakage of fura-2) in an H&L (Burlingame, CA, U.S.A.) series 300 spectrofluorimeter with a multiple excitation wavelength program. Excitation wavelengths alternated between 340 and 380 nm and the emitted fluorescence was collected at 500 nm. Readings were averaged and collected every 1.6 s and stored in a LOTUS 1-2-3 print file to allow later manipulation of the data.

The cell suspension was allowed to equilibrate for 1 min in a stirred, thermostatted cuvette. A base line was recorded for 1 min after which test agents were added. Additions (in general 30 p1 in an appropriate vehicle) were made with a Hamilton syringe that reached into the cuvette, permitting the continuous measurement of the fluorescence signal. Background fluorescence was not affected by this procedure.

Addition of EGTA to the cell suspension resulted in an instantaneous decrease in the ratio measured. Although the effect of EGTA could not be eliminated by several washes with buffer, addition of 1 mM MnCl, also caused an immediate decrease in the fluorescence ratio due to a drop in

fluorescence at 340 nM, without affecting the fluorescence at 380 nm. This indicated that extracellular fura-2 was re- sponsible for the effect. Approximately 6% of the total fura- 2 appeared to be present extracellularly, probably tightly bound to the cell membranes.

Calibration. At the end of experiments, the signal was calibrated as follows: in experiments in which Ca2+-containing buffer was used, 0.1% Triton X-100 was added, giving fluorescence values for the Ca2+-saturated form of fura-2 (FmaX). Subsequently, 5 mM EGTA and 30 mM Tris were added, to chelate free Ca2+ and thus give fluorescence values for the free form of fura-2 (Fmin). In Ca2+-depleted buffer (in which 1.1 mM CaCl, was replaced by 1 mM EGTA), the order was reversed i.e., after lysis of the cells, Fmin was measured, after which excess CaCI, was added to determine FmaX. In some experiments, 10 pM ionomycin was added to determine F,,, values in intact cells.

Calculation of [Ca2+li. The raw fluorescence data were corrected for autofluorescence of the cells, and, where indicated, for extracellular fura-2, and the F3,0/F380 ratio was calculated. Subsequently, ratios were converted to [Ca2+], according to the following formula:

in which R:,n and R:= are the ratios under Ca2+-free and Ca2+-saturated conditions, corrected for autofluorescence and for the difference between the viscosity of the calibration solution (which is a lysed cell suspension) and of the cytosol. The latter correction factor was determined according to the method of Poenie (1990) and from the difference between R,,, values after calibration with 10 pM ionomycin and after subsequent lysis of the cells. In both cases the average correction amounted to 0.75; this value was adopted for all experiments. The value ofJ; the ratio of the fluorescence at 380 nm of free fura-2 over that of Ca2+- bound fura-2, was determined from the calibration values, and was in general between 10 and 12. A KD value of 225 nM (Grynkiewicz et al., 1985) was used.

Calculation of the offset rate constant k The decline in [Ca”], can be described as a first-order

reaction, dCldt = k x C, in which C is the increase in [Ca2+], . Integration of this equation results in a linear relationship between [Ca2+],, t , and k In (C,) = In (Co) + k X t , in which C, is the increase in [Ca2+], at time 1, Co a theoretical starting concentration, k the offset rate constant, and t the time. In general, 930% of the total decline was used for calculation of k.

Materials The following chemicals were used in this study: DMEM

(GIBCO, Grand Island, NY, U.S.A.), fetal bovine serum (Irvine, Santa Ana, CA, U.S.A.), gentamicin (Elkins-Sinn, Cherry Hill, NJ, U.S.A.), fura-2 AM, Pluronic (Molecular Probes, Eugene, OR, U.S.A.), ionomycin (Calbiochem, San Diego, CA, U.S.A.), thapsigargin (LC Services, Woburn, MA, U.S.A.), bradykinin, forskolin, 3-isobutyl- I-methyl- xanthine, phorbol 12,13-dibutyrate (PDBu) (Sigma, St. Louis, MO, U.S.A.), pertussis toxin (PTX) (List, Campbell, CA, U.S.A. and Sigma, St. Louis, MO, U.S.A.), CGS 21680 (Dr. K. A. Jacobson, NIH, Bethesda, MD, U.S.A.), Ro 20- 1724 (Dr. K. Prasad, UCHSC, Denver, CO, U.S.A.), bradykinin, desArg9[Leu8]BK, and ~-Arg[Hyp~,Thi~.~,~-Phe’]BK (Dr. J. Stewart, UCHSC, Denver, CO, U.S.A.). All other

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192 A. GARRITSEN AND D. M. F. COOPER

chemicals were obtained from standard commercial sources and were of analytical grade.

RESULTS Calcium mobilization by bradykinin

In populations of NCB-20 cells, increasing concentrations of BK elicit progressive increases in [Ca2+], (Fig. 1). Characterization of the response to BK re- vealed that not only the amplitude of the [Ca2+Ii signal but also the kinetics of Ca2+ mobilization depend on the concentration of BK added; both the onset and offset of Ca2+ mobilization are faster at higher concentrations of BK (Fig. 2). The time between addition of the hormone and the maximal [Ca2+], was 28 2 2 s at 1 nM BK (SEM, n = 1 I), but only 8.0 * 0.2 s at 1 pM (SEM, n = 10; Fig. 2, squares). At 1 nM BK the response persisted for -90 s, compared to 60 s at 1 pM BK. The decline in [Ca"], can be described as a first-order reaction in which the change in [Ca2+], is dependent on [Ca2+], and the off-rate constant, k. At high concentrations of BK, k was significantly lower (Fig. 2, circles), indicating a faster decay of the [Ca2+], signal. This suggests that either Ca2+ reuptake into intracellular stores or Ca2+ extrusion across the plasma membrane is accelerated.

Data from a number of experiments, analogous to that presented in Fig. 1, were combined and in Fig. 3 the increase in [Ca2+Ii is plotted as a function of the

1 nM

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FIG. 1. Mobilization of [Ca2'Ii by bradykinin. The indicated concentrations of BK were added to NCB-20 cells, suspended in Ca2+-containing Krebs buffer. Fura-2 fluorescence was measured and converted to [Ca2+Ii as described in Materials and Methods. Time courses from a representative experiment are shown.

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FIG. 2. Concentrationdependent kinetics of BK-induced Ca2' mobilization. In experiments performed as described in the leg- end to Fig. 1, the time taken to reach the peak [Ca2+], (.) and the off-rate constant (O), a measure for the decline rate, are plotted as a function of the BK concentration. Data are the averages of 7-1 1 determinations from 4-8 independent experiments. Error bars represent the SEM.

BK concentration. If the peak [Ca"],, which is achieved at different times, is used, BK has a half- maximal effect at 8 nM (Fig. 3). If the area under the curve of the Ca2+ peak, which represents the balance of Ca2+ release and elimination with time, was plotted versus the BK concentration, the second dose-response curve shown is obtained, and BK exhibits a half-maximal effect at approximately 1 nM (Fig. 3).

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FIG. 3. Dose-response relationships for Ca2+ mobilization by bradykinin. The increase in [Ca2'Ii at the peak (0) or the area under the curve of the [Ca2+], peak (D) were expressed as a percentage of the maximal effect in the same experiment, averaged and plotted as a function of the BK concentration. Each data point is the average of 7-11 values out of 4-8 independent experiments. Error bars represent the SEM. 100% corresponds to an increase in [Ca2'], of 301 nM from a basal value of 92 ? 6 to a maximal value of 393 -c 31 nM (SEM, n = 11) (left ordinate) or to an area under the curve of 6,000 f 380 nM.s (right ordinate). ECS0 values, determined from Hill plots, are 8 nM (peak) and 0.9 nM (area under the curve).


To define the type of BK receptor involved in Ca2+ mobilization in NCB-20 cells, a B,- and a B,-selective antagonist were evaluated for their ability to block the [Ca”], elevation elicited by BK. The effect of BK on [Ca”], was blocked by the B, antagonist D- Arg[Hyp3,Thi5>’,~-Phe7]BK (Stewart and Vavrek, 1991), with a PA, value of -8.5. The B, antagonist desArg9[Leu8]BK (Marceau and Regoli, 199 1 ) had a PA, value > 10 pM. The order of potency of these two antagonists indicates that a B, receptor mediates the effects of BK on [Ca2+Ii.

Both PTX-sensitive and -insensitive G proteins have been implicated in the effects of BK on phosphoinositide (PI) turnover (Jackson et al., 1987; Chuang and Dillon-Carter, 1988; Fasolato et al., 1988; Fu et al., 1988; McAtee et al., 1990). To determine whether a PTX-sensitive G protein mediates the BK-induced stimulation of [Ca”], , NCB-20 cells were pretreated for 18-24 h with 125 ng/ml PTX. PTX pretreatment did not significantly alter the response to BK,’ although such treatment was fully effective at blocking opiate or muscarinic receptor-mediated inhibition of cyclic AMP accumulation (not shown, see also Boyajian et al., 199 1).

The source of the Ca2+ involved in the BK-stimulated response was investigated by reducing the extracellular Ca2+ concentration to - 100 nM by adding 2.5 mM EGTA. Ca2+ mobilization induced by 1 pM BK after 15 s in low Ca2+ buffer is not significantly different from the control (Table 1): when the data are corrected for extracellular fura-2. [As described under Materials and Methods, the immediate effect of EGTA addition is to decrease the fluorescence ratio, due to the conversion of extracellular fura-2 from the Ca2+-bound into the free form]. In addition, the EC,, value of BK is unaltered in Ca2+-depleted buffer (not shown). Thus Ca” influx does not appear to play a significant role in the acute [CaZ+], mobilization induced by BK.

An additional means of determining whether Ca” influx contributes to the rise in [Ca2+], induced by BK is to assess the effect of BK on the rate of influx of Mn2+ into the cell (Wheeler et al., 1990). MnZ+, which quenches fura-2 fluorescence, enters certain cells, e.g.,

’ Cells were pretreated with PTX in six different experiments and BK was subsequently added in concentrations varying between 1 &and 1 pM, to accommodate the possibility that PTX would be effective only at submaximal or maximal concentrations of BK. As PTX-treated and control cells were separately loaded with fura-2 AM, Ca” mobilization by thapsigargin was compared in some experiments, to ensure that the overall ability of cells to release intracellular Ca” was equivalent. In control cells, 1 pM BK increased [Ca”], from 77 f 3 to 373 f 27 nM, and in PTX-treated cells from 83 * 3 to 364 34 nM. At none of the other BK concentrations was a consistent effect of PTX pretreatment observed. In addition, Ca” mobilization by thapsigargin was unaltered in PTX-treated cells.

* Prolonged exposure (for 2-3 min) to EGTA does deplete Ca2+ stores, with the result that subsequent BK responses are reduced (not shown).

TABLE 1. Effect o/clielution of’e.xtracelliilur Cu2’ (Ca2+ ex) on the fast phase of Cu2+ mobilization

by BK, thupsigurgin, and ionomycin

Basal Peak Addition CaZ+ ex [Caz+li [ ~ a ” ] , n

BK 1 pM I mM 6 0 k 2 2 5 4 f l 13 -1OOnM 71 f 2 2 8 3 f 8 10

- 1 O O n M 7 5 f 3 2 1 5 f 4 5

- 1 O O n M 8 2 f 3 4 3 0 f 2 1 5

Fura-2 fluorescence was measured as described in Materials and Methods and corrected for the presence of 6.3% extracellular fura- 2. Subsequently, [Ca2+], was calculated as described. Data are the averages of 4- 13 determinations from four independent experiments. The rapid transient elevation of [Ca”], observed after addition of 1 pM ionomycin was followed by a marked persistent elevation: [Ca”], returned to 177 f 28 nM (SEM, n = 5) .

Thapsigargn 1 pM 1 mM 64 k 4 204 f 5 4

Ionomycin 1 pM I d 6 4 f 3 4 9 4 f 9 5

platelets, by the same mechanism that facilitates Ca2+ influx (Memtt and Hallam, 1988). In such situations, in the presence of Mn2+, an increase in the rate of Ca2+ entry results in an accelerated rate of quenching of intracellular fura-2. In unstimulated NCB-20 cells, Mn2+ first causes a rapid decrease at 340 and 359 nm due to quenching of extracellular fura-2 (see Materials and Methods). Subsequently, a slower steady reduction in fluorescence at both wavelengths is observed as MnC1, enters the cell (Fig. 4A). Addition of BK induces an increase in [CaZ+], (Fig. 4A, 340 nm) but the rate of Mn2+ entry (Fig. 4A, 359 nm) is unaltered.

Calcium mobilization by thapsigargin As a prelude to evaluating the nature and fraction

of the intracellular Caz+ stores accessed by BK, the effects of the microsomal Ca2+-ATPase inhibitor thapsigargin on [Caz+], were explored. Thapsigargh is frequently used to deplete nonmitochondrial Ca2+ se- questering stores, as it specifically blocks Ca2+ uptake into the endoplasmic reticulum (Thastrup, 1990). The net effect of the compound is a release of intracellular Ca” without a concomitant rise in inositol phosphates (Jackson et al., 1988; Thastrup, 1990).

Thapsigargin evokes a relatively slow rise in [Caz+Ii, as compared to BK, which returns after 2-3 min to a new steady-state level, which is slightly above starting [Ca2+Ii (Fig. 5 , solid line). The initial Ca2+ release is independent of extracellular Ca2+ (Table I ) , as expected from the proposed mechanism of action of the compound (Thastrup, 1990). The sustained elevation is presumably due to the influx of Ca2+, as it can be eliminated by the chelation of extracellular Ca” (Fig. 5 , dashed line). Interestingly, thapsigargin does not alter the rate of Mn2+ influx into NCB-20 cells in an experiment similar to that described above for BK (see Fig. 4A and B).

Thapsigargh is potent, showing a half-maximal effect at 8 nM, when the increase in [Ca2+], at the peak

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FIG. 4. Effects of bradykinin, thapsigargin, and ionomycin on Mn" entry. MnCI, (1 mM) was added at the first arrow; 1 pM BK (A), 1 pM thapsigargin (B), and 1 pM ionomycin (C) were added after 1 min. Solid lines represent fluorescence at 359 nm. dotted lines the fluorescence at 340 nm. The former wavelength was used because it is the wavelength at which the fluorescence was independent of the calcium concentration. The latter wavelength was used to verify that the additions elevated [Ca"], .

was measured (Fig. 6). Although the peak [Ca"], levels, reached after addition of a maximal concentration of thapsigargin, were lower than those elicited by a maximal concentration of BK, total [Ca2+], increased by thapsigargin, when expressed as the area under the curve of the [Ca2*], peak, was 184 k 17% (SEM, n = 6 ) of that increased by BK. This could be due either to a reduction in the reuptake and/or extrusion of excess [Ca2+], or to the release of Ca2+ from additional, BK-insensitive pools or combinations thereof.'

An experimentally valuable consequence of the action of thapsigargin is that because it inhibits reuptake of Ca2+ into recruitable stores, pretreatment of cells with this compound is anticipated to perturb subse-

It should be noted that, given the fast decline in [Ca2'], after stimulation with high concentrations of BK, the possibility exists that the time resolution of our measurements is inadequate to esti- mate the maximal [Ca"], elicited by BK.

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FIG. 5. Effect of thapsigargin on [Ca2+li. Thapsigargin (1 pM) was added to NCB-20 cells suspended in 1 mM Cazf-containing Krebs buffer. At the indicated time (first arrow) 2.5 mM EGTA (dashed line) or Krebs buffer (solid line) was added to chelate extracellular Ca2+. Thapsigargin (1 pM) was added 15 s later (second arrow). Time courses are from a representative experiment. [Ca2'Ii values were corrected for the presence of 6.3% extracellular fura-2.

quent hormonal effects (Law et al., 1990). Indeed, stimulation of NCB-20 cells with increasing concentrations of thapsigargin progressively precludes subsequent [Ca2+Ii mobilization by BK, after pretreatment with 3 nM thapsigargin -50% of the original BK response is seen (see Fig. 7A and B). Increasing the thapsigargin concentration to 10 nM (Fig. 7C) or 30 nM (Fig. 7D) results in a decrease in the BK response to - 12 and 4%, respectively, of the control. This indicates that all BK-sensitive intracellular Ca2+ stores are depleted by thapsigargin. However, after a maximal concentration of BK ( 1 pM), thapsigargin still elevates [Ca2+Ii (see Fig. 7E and F). Thus, thapsigargin releases [Ca"], from BK-sensitive and -insensitive stores.

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FIG. 6. Dose-response relationship for Ca2' mobilization by thapsigargin. The increase in [Ca"], at the indicated thapsigargin concentrations was expressed as a percentage of the maximum and the data from two experiments were averaged. 100°/~ corresponds to an average increase in [Ca"], from a basal of 100 to 285 nM.

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FIG. 7. Relationship between thapsigargin- and BK-sensitive Ca2' stores. NCB-20 cells were suspended in Ca2'-containing Krebs buffer and thapsigargin was added (first arrow) in the following concentrations: (A) dimethyl sulfoxide alone, (8) 3 nM, (C) 10 nM, (D) 30 nM. In each case, 1 pM BK was added after 3 min (second arrow). In a separate experiment (E and F) the relationship between BK-sensitive and thapsigargin-sensitive Ca2+ pools was further explored. E Addition of 10 nM thapsigargin was followed by addition of 1 pM BK. F 1 pM BK was added first, followed by 10 nM thapsigargin. The results are from representative experiments that were repeated with virtually identical results.

Calcium elevation induced by ionomycin The Ca2+ ionophore ionomycin was used in an-

other approach to modulate [Ca''], without activation of phospholipase C (Albert and Tashjian, 1986). Ionomycin has a dual effect. First, ionomycin releases Ca2+ from intracellular stores, resulting in a rapid transient elevation of [Ca"], (Fig. 8). As shown in Table 1, and as suggested by Fig. 8, this initial effect is

largely independent of extracellular Ca2+, as the ionomycin-induced increase in [Ca2+], in Ca2+-depleted buffer is 8 1% of the value measured in 1 mM Ca2+- containing buffer (see Fig. 8A and B). Following the rapid elevation of [Ca2+],, a marked sustained elevation is observed (Fig. 8A). The magnitude of the sustained elevation is dependent on the ionomycin concentration (Fig. 9) and on the extracellular Ca" concentration (see Fig. 8A-C). If ionomycin is added after MnCl,, there is an instantaneous quenching of intracellular fura-2 (Fig. 4C), demonstrating that ionomycin enhances the entry of Mn2+ (and thus Ca2+) as expected from a divalent cation ionophore.

To probe the relationship between ionomycin- and BK-induced Ca2+ mobilization, cells were pretreated with increasing concentrations of ionomycin and subsequently stimulated with 1 pMBK (Fig. 9). The ability of BK to mobilize [Ca2+], was compromised by treatment with ionomycin in concentrations as low as 10 nM (Fig. 9C) and abolished by treatment with 2 100 nM ionomycin (Fig. 9D and E). However, if 1 pM BK was added to the cells first, followed by ionomycin, only the rapid transient phase of the ionomycin response was attenuated, whereas the second sustained phase was unaffected (Fig. 9F). Effect of PKA and PKC activation on the BK response

BK elevates [Ca2+], via the stimulation of phospholipase C-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate. One of the products of this reaction, diacylglycerol, activates PKC. The possible role of PKC activation in Ca2+ mobilization in NCB-20 cells was evaluated. Desensitization of PKC by over- night treatment of cells with 1 pM PDBu did not alter the response to 1 pLM BK. Treatment for 2 or 10 min with 1 pM PDBu did not change either resting [Ca''], levels or the peak [Ca2+], elicited by 1 pM BK. How- ever, this acute treatment attenuated the response to BK the rate constant for the decrease in [Ca2+Ii, k, increased from 0.062 rt_ 0.006 to 0.090 k 0.01 1 6' (SEM, n = 4, p < 0.05 in a paired t test) (Fig. 10A). This effect was not observed in PKC-desensitized cells (not shown).

A similar phenomenon was observed after activation of PKA. McAtee and Dawson (1990) reported

FIG. 8. Effect of ionomycin on [ca2+1,; role of extracellular Ca2+. NCB-20 cells were resuspended in 1 mM Ca2' containing Krebs buffer. The following additions were made (first arrow): (A) none, (6) 3 mM EGTA (C) 4 mM CaCI, . After 15 s, 1 pM ionomycin was added (second arrow). [Ca2+], values were corrected for the presence of 6.3% extracellular fura-2. The results are from a representative experiment that was repeated twice with similar results.

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FIG. 9. Relationship between Ca2+ mobilization by bradykinin and ionomycin. NCB-20 cells were suspended in Ca'+-containing Krebs buffer. Increasing amounts of ionomycin were added (first arrow): (A) dimethyl sulfoxide alone, (8) 1 nM, (C) 10 nM, (D) 100 nM, (E) 300 nM. After 3 min, 1 pM BK was added (second arrow). In F the sequence was reversed; first 1 pM BK was added, and after 3 min 300 nM ionomycin was added. Data are from two representative experiments (A-D and E and F were done in parallel) that were repeated twice with similar results.

that activation of cyclic AMP-dependent protein kinase reduces BK-induced inositol phosphate production. To evaluate the consequences of such an interaction for the effect of BK on [Ca2+Ii, cyclic AMP levels were elevated by pretreatment of the cells for 10 min with the phosphodiesterase inhibitor Ro 20- 1724 (400 pM), forskolin (10 p M ) , and CGS 2 1680 (1 pM), a selective adenosine A, receptor agonist. Although this treatment did not affect basal [Ca2+], (Fig. IOB), Ca2+ mobilization stimulated by BK was modified. Although the amplitude of the [Ca2+Ii signal was similar to the control, the duration of the Ca2+ peak was significantly shortened (Fig. 10B). In the control situa- tion [Ca2+Ii had returned to 10% above resting levels after 52 f 1 s (SEM, n = 5). After addition of forskolin, Ro 20-1724, and CGS 21680 in parallel experiments, the response lasted for only 42 k 1 s (SEM, n = 5). As after PKC activation, the rate constant for the decrease in [Ca2+Ii was 0.084 f 0.003 s-' in cells with high cyclic AMP levels, compared to 0.063 f 0.002 s-' (SEM, n = 5 ) in control cells ( p < 0.01, t test). This suggests that either the intracellular reuptake and/or the extrusion of Ca2+ across the plasma

membrane, following the stimulus, is accelerated by protein kinase activation or that protein kinase activation blocked additional Ca2' release.

DISCUSSION

In populations of NCB-20 cells, BK transiently elevates [Ca2+], in a dose-dependent manner with an ECSo value in the low nanomolar range. Not only the magnitude of the [Ca2+], rise but also the kinetics of the response depend on the BK concentration; both the time-to-peak and the duration of the response decrease with increasing BK concentrations. A similar change in the kinetics of the BK response has been observed in populations of PC12 cells (Fasolato et al., 1988). The faster decay of the signal may be the result of the activation of Ca2+ extrusion or reuptake at

I 1 t 01 I I

400

300

200

100

0

0 60 120 180 240 300

I ' 1 u-

60 660 720 TIME (S)

FIG. 10. Effect of cyclic AMP elevation and PKC activation on the BK-induced [Ca"], peak. NCB-20 cells were suspended in 1 mM Ca2+-containing Krebs buffer. A dimethyl sulfoxide (solid line) or 1 f l PDBu (dotted line) was added for 2 min prior to addition of 1 pM BK. [Ca"], and off-rate constants were calculated as described in Materials and Methods. Off-rate constants were 0.056 s-' and 0.087 s-' for control and PDBu-treated, respectively. 8: 400 pM Ro 20-1724, 10 f l forskolin, and 1 pM CGS 21680 (dotted line) or vehicle (solid line) were added. After 10 min, 1 pM BK was added. [Ca2'], and off-rate constants were calculated as described in Materials and Methods. Off-rate constants were 0.067 5-l and 0.092 s-' for basal and stimulated cyclic AMP conditions, respectively. The results are from a representative experiment that was repeated twice with similar results.

J. Neiirocliern.. Vol. 59. No. 1. 1992


higher [Ca2+],, e.g., via Ca2+-calmodulin dependent mechanisms (Thastrup, 1990). Such possibilities could be addressed in single cell studies.

In a number of tissues and cell lines (Farmer and Burch, 199 l), including NCB-20 cells (Chuang and Dillon-Carter, 1988), the bradykinin-B, receptor has been implicated in Ca2+ mobilization. Our results, showing that a B, antagonist, but not a B, antagonist, blocks the BK-induced increase in [Ca2+],, confirm that the BK receptor coupled to Ca2+ mobilization in NCB-20 cells is a B, receptor.

The presumed G protein that couples the BK receptor to CaZ+ mobilization in NCB-20 cells is not PTX sensitive, as has been observed in other neuronal cell lines (Jackson et al., 1987; Fasolato et al., 1988). This finding contrasts with the partial inhibition by PTX treatment of PI turnover in NCB-20 cells, reported by Chuang and Dillon-Carter (1988) and McAtee and Dawson ( 1990). The former group measured inositol monophosphate accumulation over 45 min in the presence of 60 mM LiC1-conditions that are not readily comparable with [Ca2+], measurements. How- ever, in the latter studies, PI turnover was measured under conditions close to those used in our experiments. This apparent discrepancy might suggest that [Ca2+], can be fully stimulated by submaximal levels of inositol phosphates.

NCB-20 cells are of neuronal origin and earlier studies had shown that 50 M K C l could stimulate 45Ca2+ uptake, via dihydropyridine-sensitive Ca2+ channels (Freedman et al., 1984). Such 45Ca2+ uptake, however, does not necessarily imply changes in [Ca2+],. Indeed, with our methods, we were unable to find evidence for any contribution to [Ca2+], by voltage- gated ion channels."

In many excitable (Schlegel et al., 1987; Fasolato et al., 1988) and nonexcitable cells (Jackson et al., 1988; Putney, 1990; Wheeler et al., 1990), hormones induce a rapid transient increase in [Ca2+], followed by a sustained elevation. The persistent effect requires extracellular Ca2+, and is presumed to be due to the modulation of ion channel activity and PKC activation in excitable cells (Dufy et al., 1987) and the result ofa yet to be defined mechanism that may involve inositol polyphosphates in nonexcitable cells (Putney, 1990). Extracellular Caz+ does not appear to play a role in the acute BK-induced rise in [Ca2+], in NCB-20 cells, as following chelation of extracellular Ca2+ with EGTA, neither the peak height, kinetics, nor dose-response relationship were altered.

Alternative means to manipulate resting and BK-

~ ~~

A number of tactics were attempted to detect cryptic contributions of voltage-gated ion channels to [Ca2'],, including the addition of 30 M K C I and agents affecting the activity of voltage-gated Ca2+ channels, such as BAY K 8644 ( 1 p M ) and nifedipine ( 1 p M ) . None of these treatments altered resting or BK-stimulated [Ca"], .

stimulated [Ca2+], , independent of the activation of phospholipase C, were explored using thapsigargin and ionomycin. Thapsigargin specifically blocks microsomal Ca2+-ATPases, thus preventing the uptake of Ca2+ by Ins( 1,4,5)P,-sensitive stores (Thastrup, 1990). Initially, thapsigargin increases [Ca"], , presumably as a result of a passive leak of Ca2+ from the endoplasmic reticulum. The kinetics of the thapsigargin-evoked [Ca2+], rise differ from the BK response; the onset and offset rate are slow compared to BK and the signal did not decay faster at higher thapsigargin concentrations. Furthermore, as in other cell types (Thastrup, 1990), thapsigargin appears to have a small effect on Ca2+ influx. A small persistent elevation of [Ca2+], is noticeable, which can be blocked by EGTA. The fact that neither thapsigargin nor BK ac- celerates the rate of MnZ+ entry suggests that there is no direct relationship between the intracellular release of Ca2+ and Mn2+ influx.

Although the maximal peak [Ca2+], elicited by BK was higher than that elicited by thapsigargin, the [Ca"], rise integrated over time induced by thapsigargin was almost twofold larger than that induced by BK. This does not necessarily mean that thapsigargin releases more Ca2+-it could also be due to altered rates of reuptake. However, whereas stimulation of NCB-20 cells with thapsigargin in submaximal concentrations precludes a subsequent response to BK, additional Ca2+ can be released by thapsigargin after stimulation with a maximal concentration of BK. This indicates that, in agreement with previous studies (Jackson et al., 1988; Verma et al., 1990), BK-sensitive Ca2+ stores are a subset of thapsigargin-sensitive stores in NCB-20 cells.

Ionomycin elevates [Ca"], in yet another manner. The predominant effect of this ionophore in low concentrations is to release Ca2+ rapidly from intracellular pools, which is superficially more similar to the effect of BK than is the action of thapsigargin. In higher concentrations, ionomycin causes substantial Ca2+ influx, which is obvious in the presence of MnCl,. Stimulation with ionomycin prevents a further response to BK, suggesting that all BK-sensitive stores are depleted by the ionophore. After maximally effective concentrations of BK, ionomycin still elevates [Ca2+],, although the response is largely modified; the rapid rise, associated with intracellular Ca2+ release, is abolished and a slow component, associated with the ionophoretic activity of ionomycin, at the plasma membrane, is predominantly observed. Thus, like thapsigargin, ionomycin affects BK-sensitive Ca2+ stores, but also other means of ICa2+], elevation.

The manipulations described above can be used to investigate cross talk mechanisms between cyclic AMP and Ca2+ signalling pathways. Changes in [Ca"], can be predicted, whereas cyclic AMP accumulation and PI turnover are measured under highly

J. Nerrrochon.. Ibl. SY, No. I , l Y Y 2


comparable conditions; in addition, the effect on [Ca2+], of manipulations that affect PI turnover or cyclic AMP accumulation can be established. For exarn- ple, as mentioned earlier, elevation of cyclic AMP reduces the formation of inositol phosphates (McAtee and Dawson, 1990); whether [Ca2+], was affected was not clear in that study. The present data indicate that elevation of cyclic AMP modifies the response to BK. However, because the peak [Ca2+Ii in PKA-activated samples were virtually identical to the control, the initial Ins( 1 ,4,5)P3-induced release appears not to be the target of cyclic AMP action. Elevation of cyclic AMP mainly expedites the decay of the [Ca2+Ii signal and the BK response was shorter lived. A similar shorten- ing of the response, without an effect on the peak [Ca2+],, is observed after activation of PKC with PDBu.

Several explanations may exist for the observation that activation of protein kinases reduces the duration of the BK response. Activation of PKA can stimulate Ca2+-ATPases in the plasma membrane and in the endoplasmic reticulum, thus promoting the decline in [Ca2+Ii (Carafoli, 199 1; Thastrup, 1990). Moreover, the Ins( 1,4,5)P3 receptor can be phosphorylated, resulting in reduced Ca2+ release (Supattapone et al., 1988) or PKA activation may block Ca2+-induced Ca2+ release (Finch et al., 1991). Finally, Ca2+-induced phospholipase C activation may be inhibited, which would result in reduced PI turnover as demonstrated by McAtee and Dawson (1990) and affect the maintenance of the [Ca2+], signal rather than the initial release.

In conclusion, NCB-20 cells appear to provide a powerful model system for the study of cross talk between the cyclic AMP and Ca2+ signalling systems; the effects of hormones on Ca" release are not com- plicated by additional contributions of ion channel activity, although both PKA and PKC can modify the kinetics of the [Ca2+], rise. The studies described dem- onstrate that agents such as thapsigargin and ionomycin can be used to mimic actions of hormones at ele- vating [Ca2+], without activating phospholipase C; on the other hand, pretreatment with these agents precludes hormonal elevation of [CaZ+], while allowing any other actions mediated by hormones to occur. Application of these strategies in this cell line should accelerate the detailed delineation of the consequences of Ca" mobilization on cyclic AMP accumulation, which had previously been proposed to be a negative effect, by virtue of a Ca'+-inhibitable adenylylcyclase expressed in these cells (Boyajian et al., 1991).

Acknowledgmenk We thank Glenda Tate for cell culture work, Dr. R. A. Harris (UCHSC) for the use of the H&L spectrofluorimeter, Dr. J. Stewart (UCHSC) for BK and its antagonists, and Dr. P. Mollard for critical comments on the manuscript. The work was supported by NIH grant NS 28389.

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Manipulation of Intracellular Calcium in NCB-20 Cells