- Email: [email protected]
Fendiline increases [Ca2+]i in Madin Darby canine kidney (MDCK) cells by releasing internal Ca2+ followed by capacitative Ca2+ entry
Life Sciences, Vol. 66, No. 1 I, pp. 1053-1062,200O Copyright 0 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0024-3205/00/S-see front matter
PIISOO24-3205(99)00670-O
FENDILINE INCREASES [Ca*+], IN MADIN DARBY CANINE KIDNEY (MDCK) CELLS BY RELEASING INTERNAL Ca”’ FOLLOWED BY CAPACITATIVE Ca” ENTRY Chung-Ren
Jan *’ , Ching-Jiunn
Tse n g ’ & Wei-Chuan
Chen2
‘Department
of Medical Education and Research, Veterans General Hospital-Kaohsiung, Kaohsiung, Taiwan 8 13 ‘Division of Urology, Ping Ttmg Christian Hospital, Ping Tung, Taiwan 900 (Received in final form October 18, 1999)
Summary The effect of fendiline, a documented inhibitor of L-type Ca2’ channels and calmodulin, on Ca2’ signaling in Madin Darby canine kidney (MDCK) cells was investigated using at 5-100 pM significantly increased [Ca”]i furaas a Ca” probe. Fendiline concentration-dependently. The [Ca”], rise consisted of an initial rise and a slow decay. External Ca2’ removal partly inhibited the Ca2’ signals induced by 25-100 pM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca” influx and internal Ca” release. In Ca2’-free medium, pretreatment with 50 pM fendiline nearly abolished the [Ca2’li rise induced by 1 PMthapsigargin, an and vice versa, pretreatment with endoplasmic reticulum Ca” pump inhibitor, thapsigargin prevented fendiline from releasing internal Ca*‘. This indicates that the internal Ca2’ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 pM, fendiline caused Mn2’ quench of fitra-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La” by 50%, implying that fendilineinduced Ca2’ influx has two components separable by La”. Consistently, 0.1 mM La” pretreatment suppressed fendiline-induced [Ca*‘], rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca” increased [Ca2’li after preincubation with 50-100 pM fendiline in Ca2’-free medium. However, 50-100 pM fendiline inhibited 1 uM thapsigargin-induced capacitative Ca2’ entry. Pretreatment with 40 pM aristolochic acid to inhibit phospholipase A2 inhibited 50 pM fendiline-induced internal Ca” release by 48%, but inhibition of phospholipase C with 2 uM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca”], in MDCK cells by releasing internal Ca*’ in a manner independent of inositol-1,4,5-trisphosphate (IP,), followed by external Ca2’ influx. Key Words:
fendiline, capacitative Ca*’ entry, MDCK cells, fura-2, Ca”
signaling
Corresponding author: C.R. Jan, Ph.D., Dept. Medical Education and Research, Veterans General Hospital-Kaohsiung, 386 Ta Chung 1st Rd., Kaohsiung, Taiwan 813. Fax: 886-7-3468056; Email: [email protected]
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Fendiline is clinically used as an anti-angina1 drug for treating coronary heart diseases (1). At the cellular level, fendiline is commonly used as an inhibitor of L-type Ca” channels and calmodulin. For example, fendiline has been shown to inhibit L channels in rat and guinea pig ventricular myocytes (2, 3), and inhibit calmodulin in several cells (1,4). Additionally, fendiline inhibits the hyperpolarization induced by bradykinin and ionomycin in canine coronary artery (5), impairs the constitutive but not the inducible nitric oxide synthase activity in the rat aorta (6), and enhances the release of endothelium-derived relaxant factor and prostacyclin from endothelial cells (7). At a single
concentration
Dictyostelium
discoideum;
of 34 pM fendiline was found to induce a [Ca”], transient in however, neither the concentration-response relationship nor the underlying mechanism of the [Ca”], rise was investigated (8). Similarly, 30-100 pM fendiline was found to increase [Ca”], in human platelets (9).
Here we have investigated the effect of fendiline on Ca*’ signaling in Madin Darby canine kidney (MDCK) cells. In this cell, we have previously shown that agonists coupled to IP, such as ATP (lo), UTP (11) and bradykinin (12) increased [Ca”], by releasing Ca2’ from the endoplasmic reticulum Ca2’ store followed by Ca” influx via a process called capacitative Ca*’ entry (I 3). Other drugs such as thapsigargin (14) and 2,5-di-tert-butylhydroquinone (15) increased [Ca”], by inhibition of the endoplasmic reticulum Ca” pump in an IP,-independent manner leading to a passive release of Ca” from the endpoplasmic reticulum followed by capacitative Ca” entry. Thus, MDCK cells provide for a good model for investigating the effect of drugs on Ca’* signaling in non-excitable cells. We report that fendiline induces a significant [Ca”], rise in fura2-loaded MDCK cells. We have established the concentration-response relationship both in the presence and absence of external Ca”, and have explored the underlying mechanism. The effect of fendiline on thapsigargin-induced capacitative Ca2’ entry has also been examined.
Materials and Methods Cell culture. MDCK cells obtained from American Type Culture Collection (CRL-6253) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 ug/ml streptomycin at 37°C in 5% CO,-containing humidified air. Ca*’ medium (pH 7.4) contained (in mM): NaCl 140; KC1 5; MgCl, 1; CaCl, 1.8; Hepes 10; glucose 5. Ca”-free medium contained no added Ca” plus 1 mM EGTA. The experimental solution contained less than 0.1% of solvent (dimethyl sulfoxide or ethanol) which did not affect [Ca”], (n=3).
Solutions.
measurements of[Ca*‘],. Trypsinized cells (lO’/ml) were allowed to recover for 1 hr in Dulbecco’s modified Eagle medium and were subsequently loaded with the ester form of fura-2, fura-2iAM (2 pM) for 30 min at 25°C. Cells were washed and resuspended in Ca” medium. Fura2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million of cells. Fluorescence was monitored with a Hitachi F-4500 spectrofluorophotometer (Japan) by continuously recording excitation signals at 340 and 380 run and the emission signal at 5 10 nm at l-s intervals. Maximum and minimum fluorescences were obtained by adding Triton X- 100 (0.1%) and EGTA (20 mM) sequentially at the end of the experiment. The fluorescence intensity of the excitation signal at 340 nm was divided by that at 380 nm and the ratio was used to calculate [Ca”], as described previously (16) assuming a K, of 155 nM. Mn” quench experiments were performed in Optical
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Ca*’ medium containing 50 pM MnCl, by continuously recording the excitation signal at 360 nm and the emission signal at 510 nm at I-s intervals and the data were presented as arbitrary fluorescence units. We have shown previously that trypsinized cells yielded qualitatively similar results as cells attached to coverslips (10, 12, 14). Chemical reagents. The reagents for cell culture were from Gibco. Fura-YAM was from Molecular Probes. U73 122 and U73343 were from RBI. All other reagents were from Sigma. Statistical analyses. All values were reported as means+SEM (n=5-6). Statistical comparisons were determined by using the Student’s paired t test, and significance was accepted when PcO.05.
Results Fendiline increases [Ca”/i. In Ca*’ medium, fendiline at concentrations between 5- 100 PM induced a [Ca”], rise which comprised an initial rise and a gradual decay (Fig. 1A). For example, at a concentration of 100 PM, fendiline induced a peak [Caz’li of 589k13 nM (n=5) which slowly decayed in the following 300 s. At a concentration of 1 pM fendiline had little effect (not shown). At a concentration of 200 pM fendiline induced a nearly immediate rise in [Ca”], with a height of more than 1.2 PM, most likely reflecting cell membrane leakage, thus the results were not reported. The rise of the Ca*’ signal was slower in response to lower concentrations of fendiline.
C
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Fig. 1 A Concentration-dependent effects of fendiline on [Ca”], in fura-2-loaded MDCK cells. Concentration of fendiline was 100 pM in trace a, 50 PM in trace 6, 25 pM in trace c, 10 PM in trace d and 5 pM in trace e. The experiments were performed in Ca” medium. Traces are typical of 5-6 experiments. B Similar to A, except that the experiments were performed in Ca”-free medium (no added Ca*’ plus 1 mM EGTA). C Concentrationresponse plots of the fendiline response in the presence (filled circles) or absence (open circles) of external Ca*‘. Y axis: the net area under the curve (30-350 s) of the [Ca”], rise. Data are meansHEM of 5-6 experiments. *P
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b
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Fig. 2 A In Ca”-free medium, fendiline was added at 30 s followed by I pM thapsigargin
at 600 s. B Ca” influx detected by Mn*’ quench measurements. Truce a: control without addition of fendiline. Trace c: 50 uM fendiline was added at 80 s. Trace b: 0.1 mM La” was added 20 s prior to fendiline. C Dashed trace: control fendiline effect. Solid trace: 0.1 mM La3’ was added 20 s prior to fendiline. D Similar to C except that in dashed truce La” was added at the time indicated by * (80 s), and in solid trace La” was added at 320 s. The experiments in C and D were performed in Ca” medium. Traces are typical of 5-6 experiments. Internal Ca2’ release and external Ca” injlux both contribute to fendiline-induced rise in [CazcJi. Fig. 1B shows that Ca2’ removal reduced the Ca2’ signal induced by 25-100 PM fendiline by 50% both in peak value and in the area under the curve (30-350 s). The elevated phase of the [Ca”], rise induced by concentrations between lo-100 pM was abolished by Ca*’ removal. The concentration-response relationships of fendiline-induced Ca*’ signal in the presence or absence of external Ca*’ are depicted in Fig. 1C. We next examined fendiline-sensitive internal Ca*’ stores. Fig. 2A shows that in Ca2’-free medium, pretreatment with 50 uM fendiline for 600 s nearly abolished the [Ca”], rise induced by the endoplasmic reticulum Ca” pump inhibitor thapsigargin (17). Vice versa, pretreatment with thapsigargin for 370 s in Ca”-free medium prevented fendiline (50 PM) from releasing more internal Ca*’ (Fig. 3B).
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The pathway of fendiline-induced external Ca2’ influx was investigated. Mn2’ enters cells through similar pathways as Ca*‘, but quenches fura- fluorescence at all excitation wavelengths (18). Thus Ca2’ influx can be reflected by Mn” quench of fura- fluorescence. Fig. 2B shows that 50 uM fendiline induced an immediate decrease (truce c, maximum=80f6 arbitrary fluorescence units; n=6; PcO.05) in the 360 nm excitation signal compared with control (trace a). This provides direct evidence for fendiline-induced Ca2’ influx. Because we have previously shown that La” (0.1 mM) is a potent Ca2’ entry blocker in MDCK cells (10, 11, 14, 15, 19-2 l), the effect of La3’ on fendiline-induced response was explored. Fig. 2B shows that 0.1 mM La3’ added 20 s prior to fendiline inhibited fendiline-induced Mt?’ quench of fura- fluorescence by 5 1+8% (truce 6; n=6; PcO.05) in the area under the curve (80-250 s). Higher concentrations of La” induced cell aggregation and thus the results were not shown. We investigated the effect of La’* on fendiline-induced [Ca”], rise. Fig. 2C shows that pretreatment with 0.1 mM La3’ for 20 s inhibited 50 pM fendiline-induced [Ca2’li rise by retarding the rising velocity and by reducing the peak height by 38% (301+12 vs. 480+18; n=6; PcO.05). Fig. 2D depicts that addition of 0.1 mM La3’ during the rise of 50 nM fendiline-induced Ca*’ signal (at 80 s; dashed truce) immediately inhibited the response, and the area under the curve was reduced by 41+9% (80-350 s; n=6; PcO.05). However, addition of La” at 330 s had no effect (solid truce; n=6; fiO.05).
300
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Fig. 3 Effects of fendiline on capacitative Ca’” entry. A Capacitative Ca** entry induced by fendiline. Truce a: 100 uM fendiline was added at 30 s followed by 3 mM CaCl, at 500 s. Truce b, similar to truce a except that 50 uM fendiline was used. Truce c (control): CaCl, was added without preincubation with fendiline. B Truce u: 1 uM thapsigargin was added at 30 s followed by CaCl, at 510 s. Truces 6, c: similar to truce a except that 50 and 100 uM fendiline were added 100 s prior to CaCl,, respectively. Traces are typical of 5-6 experiments. Fendiline induces cupucitutive Cu” entT. Because mobilization of the endoplasmic reticulum Ca” store often triggers capacitative Ca2+ entry in MDCK cells (10, 11, 12, 14, 15, 19-21) we investigated if fendiline-induced Ca*’ influx involves capacitative Ca2’ entry. Capacitative Ca2 entry was measured by addition of 3 mM Ca2’ to cells pretreated with fendiline in Ca”-free
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medium to deplete internal Ca”. Fig. 3A shows that after depletion of internal 100 and 50 pM fendiline, addition of 3 mM Ca” induced a [Ca2’], rise with 230+16 nM and 101+_9 nM, respectively (traces a, b; n=6; PcO.05). This likely due to capacitaive Ca2’ entry because addition of CaCl, to cells which with fendiline only caused a [Ca2’], rise with a peak height of 20M nM (truce
Ca” for 470 s with a net peak value of [Ca”], rise is most were not pretreated c; n=6; P
2~~~~~ thapsigargin-pretreated
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fendillne 0 0
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Fig. 4 A Solid trace: In Ca2’-free medium, 2 pM U73122 was added at 50 s followed by 10 pM
ATP at 450 s and 50 uM fendiline at 560 s, respectively. Dashed trace: control without pretreatment with U73122 and ATP. B Solid truce: in Ca*’ medium, 0.1 mM propranolol was added at 50 s (*) followed by 50 yM fendiline at 310 s. Dashed truce: 40 pM aristolochic acid was added at 50 s followed by fendiline at 500 s. The control fendiline effect is shown in A. C Cells were incubated with 1 pM thapsigargin for 40 min in Ca” medium before fluorescence measurements were started. Fendiline (50 pM) was added at 50 s. Traces are typical of 5-6 experiments. Fendiline inhibits thapsigargin-induced capacitative Ca” entry. Fendiline has been shown to inhibit L-type Ca*’ channels in cardiac cells (2, 3, 22). Because MDCK cells do not have voltagegated Ca” channels (23) we investigated whether fendiline could alter capacitative Ca2’ entry, the major Ca” influx pathway in this cell (10-12, 14, 15) induced by another agent. Fig. 3B shows that 1 pM thapsigargin induced a significant [Ca”], rise in Ca2’-free medium with a peak height ,of 260+17 nM (truce a; n=6; WO.05). After incubation with thapsigargin for 380 s addition of 3 mM CaCl, induced capacitative Ca2’ entry with a peak height of 281+19 nM (n=6; PcO.05). This thapsigargin-induced capacitative Ca2’ entry was reduced by 25f5% and 51+-5%, respectively, in the area under the curve (500-650 s) by pretreatment with 50 and 100 pM fendiline for 100 s (traces b, c; n=5; RO.05). .!I?@Y yf’irthibition of’phospholipases on fendiline-induced [Ca”], rise. We tested if inhibition of phospholipases C, A, or D could alter fendiline-induced internal Ca” release. We have previously shown that 10 pM ATP induces marked internal Ca2* release in an IP,-dependent manner (19). Fig. 4A shows that incubation with 2 pM U73122, a phospholipase C inhibitor (24) for 420 s
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prevented 10 pM ATP from releasing internal Ca” (solid trace; n=6; P
Discussion The aim of this study was to investigate the effect of fendiline on Ca*’ homeostasis in MDCK cells. We found that fendiline increases [Ca*‘], within a concentration range of 5-100 PM. It is interesting that fendiline induces considerable [Ca”], rises in MDCK cells at concentrations commonly used to inhibit L-type Ca” channels in excitable cells (2, 3, 22). Fendiline is also regarded as an inhibitor of the Ca” sensor, calmodulin (1,4). But it is not clear whether fendilineinduced [Ca2’], rise is directly coupled to its inhibition of calmodulin because we found that although other calmodulin inhibitors such as W-7, calmidazolium and clotrimazole increased [Ca”‘], in MDCK cells, trifluoperazine (5-50 uM), phenoxybenzamine (100-200 PM) and fluphenazine-N-chloroethane (2- 100 PM) did not (unpublished data). Trifluoperazine induced a rise in the mra-2 ratio signal (340/380); however, this is not a Ca2’-sensitive signal because both 340 and 380 signals were increased (not shown). Given the possibility that fendiline might directly interfere with Ca” signaling in a calmodulin-independent manner, caution must be applied in using this drug as a tool to inhibit calmodulin, especially in cases where alterations of Ca*’ signaling may have an impact on results. Our data suggest that fendiline triggers internal Ca” release and external Ca*’ influx based on two independent measurements. First, the Ca” signals induced by 25- 100 pM fendiline were reduced by 50% by Ca” removal. Note that the elevated phase of the [Ca”], rise induced by 5-50 pM fendiline was abolished by Ca” removal, suggesting that in these responses this phase was maintained by Ca” influx. However, the elevated phase of 100 uM fendiline-induced [Ca”], rise remained significant between time points of 200-350 s when the [Ca”], rise induced by lower concentrations of fendiline had recovered to baseline. This may be because that 100 uM fendiline inhibited the plasmalemmal Ca” pump leading to a slower Ca*’ efflux. We have previously reported similar phenomena in the [Ca”], rises induced by econazole and SKF96365 (20, 21). Second, Mn2’ quench experiments provide further evidence that 50 pM fendiline induced Ca*’ influx. Our findings suggest that the internal Ca*’ source for fendiline-induced [Ca”], rise is thapsigargin-sensitive endoplasmic reticulum stores because in Ca2’-free medium, pretreatment with 1 uM thapsigargin abolished 50 uM fendiline-induced [Ca2’], rise, and vice versa,
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pretreatment with fendiline prevented thapsigargin from releasing Ca*+. The question arises as to how fendiline releases Ca*’ from internal stores. We tested whether fendiline-induced internal Ca” release was mediated by elevated IP, levels. Fig. 4A shows that under the condition that IP, formation was inhibited by U73 122 (evidenced by the observation that subsequently added ATP did not increase [Ca’+],), fendiline still induced a significant [Ca2’], rise. The observation that this [Ca”], rise was smaller than control by 61% is probably because that the endoplasmic reticulum Ca’. store had been partly depleted by U73 122. Thus, it is rather unlikely that IP, formation plays a major role in mediating fendiline-induced Ca2- release. Our findings are consistent with a previous study performed in C6 glioma cells in which fendiline was found not to alter resting IP, levels (27). We found that phospholipase A, may play a role because inhibition of phospholipase A, with 40 pM aristolichic acid reduced the [Ca”]irise by 48% in peak height. Phospholipase D probably does not play a role because pretreatment with 0.1 mM propranolol to inhibit phospholipase D did not have an effect. One question is how Ca” influx occurs during fendiline stimulation. Our results suggest that fendiline induces capacitative Ca*’ entry concentration-dependently. This is consistent with the observation that fendiline induces Mn*’ quench of fura- fluorescence. This fendiline-induced Ca” influx is most likely through capacitative Ca*’ entry (i.e. fendiline does not directly open a plasmalemmal Ca*’ channel), because 50 uM fendiline failed to increase [Ca2’], after cells had been depleted with internal Ca*’ by pretreatment with 1 uM thapsigargin for 40 min in Ca2’ medium. Similarly, using the same protocol, we have previously shown that while ATP and 2,5di-tert-butylhydroquinone induced capacitative Ca*’ entry, they did not increase [Ca2’], in Ca*’ medium after cells had been depleted with internal Ca*’ with thapsigargin for 30 min, suggesting that they did not directly open a plasmalemmal Ca” channel, and the Ca*’ entry they produced required a preceding internal Ca” depletion caused by these agents (10, 1S).The Ca*’ influx induced by 50 uM fendiline appears to have two components with equal contribution separable by sensitivity to 0.1 mM La3’, because La’- suppressed fendiline-induced Mn*’ quench of furafluorescence and [Ca*+], rise. The La”-sensitive Ca*’ influx operates during the rising phase, but not the decay phase, of fendiline-induced [Ca”], rise because adding La)’ during the rising phase immediately decreased the rise while adding La ‘+ during the decay had no effect (Fig. 2D). This is consistent with our previous study that U73 I22 also induces Ca*’ influx via a La3’-sensitive and a La”-insensitive pathways (19). Interestingly, despite inducing capacitative Ca2’ entry on its own, 50-100 pM fendiline inhibits We have found thapsigargin-induced capacitative Ca*’ entry concentration-dependently. previously that econazole and SKF96365 exert similar effects on Ca2‘ signaling in MDCK cells (20, 2 1). How exactly fendiline inhibits thapsigargin-induced capacitative Ca*’ entry is unclear. One possible explanation is that fendiline depolarizes cell membrane by inhibiting K’ channels leading to a reduced driving force for Ca*‘. To test this possibility, we depolarized cells by inhibiting K- channels with tetraethylammonium (TEA; 20 mM) to see if thapsigargin-induced capacitative Ca” entry was altered. Our data suggest that TEA had no effect (not shown). One might argue that the Ca*’ influx induced by fendiline in MDCK cells is caused by an increased driving force for Ca2+ influx resulting from fendiline-induced membrane hyperpolarization by activating Ca”-dependent K’ channels and inhibiting Cl‘ channels. We examined this hypothesis by investigating the effect of valinomycin, a K’ ionophore, on [Ca2’li. Valinomycin was expected to hypcrpolarize cells by increasing K’ efflux. We found that during the 5 min of incubation with 1O-100 pM valinomycin, the resting [Ca”], did not increase (not shown). Consistently, pretreatment with 20 mM TEA and 10 pM charybdotoxin to inhibit K+ currents did not alter fendiline-induced [Ca2’li rise. Thus, it seems unlikely that fendiline-induced alterations of Ca” signaling in MDCK cells are associated with its possible effects on membrane potential.
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Collectively, we have characterized the effects of fendiline on Ca2’ signaling in MDCK cells and have investigated the underlying mechanisms. Given the complex effects of fendiline on Ca2’ signaling (i.e. inducing [Ca2’], rises per se and inhibiting thapsigargin-induced capacitative Ca2’ entry), we caution that the effect of fendiline on Ca2’ signaling should be established before using it as an inhibitor of L-type Ca” channels or calmodulin.
Acknowledgments This work was supported by grants from National Science Council (NSC89-2320-B-075B-009) Veterans General Hospital-Kaohsiung (VGHKS89-13) and VTY Joint Research Program, Tsou’s Foundation (VTY88-P3-24) to CRJ, and a grant from Ping Tut’tg Christian Hospital to WCC. We thank Chin MH for culturing cells.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
R. BAYER and R. MANNHOLD, Pharmatherapeutica 5 103- 136 (1987). H. NAWRATH, G. KLEIN, J. RUPP, J.W. WEGENER and A. SHAINBERG, J. Pharmacol. Exp. Ther. 285 546-552 (1998). 0. TRIPATHI, W. SCHREIBMAYER and H.A. TRITTHART, Br. J. Pharmacol., 108 865 869 (1993). F. OROSZ, T.Y. CHRISTOVA and J. OVADI, Mol. Pharmacol. 33 678-682 (1988). T. NAGAO, S. ILLIANO and P.M. VANHOUTTE, Br. J. Pharmacol. 107 382-386 (1992). V.B. SCHINI and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther. 261 553-559 (1992). R. BUSSE, A. LUCKHOFF, I. WINTER, A. MULSCH and U. POHL, N-S Arch. Pharmacol. 337 79-84 (1988). C. SCHLATTERER and R. SCHALOSKE, Biochem. J. 813 661-667 (1996). A. LUCKHOFF, M. BOHNERT and R. BUSSE. N-S. Arch. Pharmacol. 343 96-101 (1991). CR. JAN, C.M. HO, S.N. WU, J.K. HUANG and C.J. TSENG, Life Sci. 62 533-540 (1998). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Chin. J. Physiol. 41 59-64 (1998). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Eur. J. Pharmacol. 35 219-233 (1998). Jr. J.W. Putney and G.S.J. Bird, Cell 75 199-201 (1993). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Life Sci. 64 259-267 (1999) CR. JAN, C.M. HO, S.N. WI-J and C.J. TSENG, Eur. J. Pharmacol. 365 111-l 17 (1999). G. GRYNKIEWICZ, M. POENIE and R.Y. TSIEN, J. Biol. Chem. 260 3440-3450 (1985). 0. THASTRUP, P.T. CULLEN, B.K. DROBAK, M.R. HANLEY and A.P. DAWSON, Proc. Natl. Acad. Sci. USA 87 2466-2470 (1990). J.E. MERRITT, R. JOCOB and T.J. HALLAM, J. Biol. Chem. 264 1522-l 527 (1989). C.R. JAN, C.M. HO, S.N. WU and CJ TSENG, Life Sci. 63 895-908 (1998). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, N-S Arch. Pharmacol. 359 92-101 (1999). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Biochim Biophys Acta 1448:533-542 (1999). W. SCHREIBMAYER, 0. TRIPATHI and H.A. TRITTHART, Br. J Pharmacol. 106 15 l156 (1992). F. LANG and M. PAULMICHL M, Kidney Int. 48 1200-1205 (1995). A.K. THOMPSON, S.P. MOSTAFAPOUR, L.C. DENLINGER, J.E. BLEASDALE and S.K. FISHER, J. Biol. Chem. 266 23856-23862 (1991). M.D. ROSENTHAL, B.S. VISHWANATH and R.C. FRANSON, J. Biol. Chem. 264 1706917077 (1989).
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26. M.M. BILLAH, S. ECKEL, T.J. MULLMANN, R.W. EGAN and M.I. SIEGEL, Biochim. Biophys. Acta. 1001 l-8 (1989). 27. W.W. LIN, N-S Arch. Pharmacol. 352 679-684 (1995).
PIISOO24-3205(99)00670-O
FENDILINE INCREASES [Ca*+], IN MADIN DARBY CANINE KIDNEY (MDCK) CELLS BY RELEASING INTERNAL Ca”’ FOLLOWED BY CAPACITATIVE Ca” ENTRY Chung-Ren
Jan *’ , Ching-Jiunn
Tse n g ’ & Wei-Chuan
Chen2
‘Department
of Medical Education and Research, Veterans General Hospital-Kaohsiung, Kaohsiung, Taiwan 8 13 ‘Division of Urology, Ping Ttmg Christian Hospital, Ping Tung, Taiwan 900 (Received in final form October 18, 1999)
Summary The effect of fendiline, a documented inhibitor of L-type Ca2’ channels and calmodulin, on Ca2’ signaling in Madin Darby canine kidney (MDCK) cells was investigated using at 5-100 pM significantly increased [Ca”]i furaas a Ca” probe. Fendiline concentration-dependently. The [Ca”], rise consisted of an initial rise and a slow decay. External Ca2’ removal partly inhibited the Ca2’ signals induced by 25-100 pM fendiline by reducing both the initial rise and the decay phase. This suggests that fendiline triggered external Ca” influx and internal Ca” release. In Ca2’-free medium, pretreatment with 50 pM fendiline nearly abolished the [Ca2’li rise induced by 1 PMthapsigargin, an and vice versa, pretreatment with endoplasmic reticulum Ca” pump inhibitor, thapsigargin prevented fendiline from releasing internal Ca*‘. This indicates that the internal Ca2’ source for fendiline overlaps with that for thapsigargin. At a concentration of 50 pM, fendiline caused Mn2’ quench of fitra-2 fluorescence at the 360 nm excitation wavelenghth, which was inhibited by 0.1 mM La” by 50%, implying that fendilineinduced Ca2’ influx has two components separable by La”. Consistently, 0.1 mM La” pretreatment suppressed fendiline-induced [Ca*‘], rise, and adding La3+ during the rising phase immediately inhibited the signal. Addition of 3 mM Ca” increased [Ca2’li after preincubation with 50-100 pM fendiline in Ca2’-free medium. However, 50-100 pM fendiline inhibited 1 uM thapsigargin-induced capacitative Ca2’ entry. Pretreatment with 40 pM aristolochic acid to inhibit phospholipase A2 inhibited 50 pM fendiline-induced internal Ca” release by 48%, but inhibition of phospholipase C with 2 uM U73122 or inhibition of phospholipase D with 0.1 mM propranolol had no effect. Collectively, we have found that fendiline increased [Ca”], in MDCK cells by releasing internal Ca*’ in a manner independent of inositol-1,4,5-trisphosphate (IP,), followed by external Ca2’ influx. Key Words:
fendiline, capacitative Ca*’ entry, MDCK cells, fura-2, Ca”
signaling
Corresponding author: C.R. Jan, Ph.D., Dept. Medical Education and Research, Veterans General Hospital-Kaohsiung, 386 Ta Chung 1st Rd., Kaohsiung, Taiwan 813. Fax: 886-7-3468056; Email: [email protected]
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Fendiline is clinically used as an anti-angina1 drug for treating coronary heart diseases (1). At the cellular level, fendiline is commonly used as an inhibitor of L-type Ca” channels and calmodulin. For example, fendiline has been shown to inhibit L channels in rat and guinea pig ventricular myocytes (2, 3), and inhibit calmodulin in several cells (1,4). Additionally, fendiline inhibits the hyperpolarization induced by bradykinin and ionomycin in canine coronary artery (5), impairs the constitutive but not the inducible nitric oxide synthase activity in the rat aorta (6), and enhances the release of endothelium-derived relaxant factor and prostacyclin from endothelial cells (7). At a single
concentration
Dictyostelium
discoideum;
of 34 pM fendiline was found to induce a [Ca”], transient in however, neither the concentration-response relationship nor the underlying mechanism of the [Ca”], rise was investigated (8). Similarly, 30-100 pM fendiline was found to increase [Ca”], in human platelets (9).
Here we have investigated the effect of fendiline on Ca*’ signaling in Madin Darby canine kidney (MDCK) cells. In this cell, we have previously shown that agonists coupled to IP, such as ATP (lo), UTP (11) and bradykinin (12) increased [Ca”], by releasing Ca2’ from the endoplasmic reticulum Ca2’ store followed by Ca” influx via a process called capacitative Ca*’ entry (I 3). Other drugs such as thapsigargin (14) and 2,5-di-tert-butylhydroquinone (15) increased [Ca”], by inhibition of the endoplasmic reticulum Ca” pump in an IP,-independent manner leading to a passive release of Ca” from the endpoplasmic reticulum followed by capacitative Ca” entry. Thus, MDCK cells provide for a good model for investigating the effect of drugs on Ca’* signaling in non-excitable cells. We report that fendiline induces a significant [Ca”], rise in fura2-loaded MDCK cells. We have established the concentration-response relationship both in the presence and absence of external Ca”, and have explored the underlying mechanism. The effect of fendiline on thapsigargin-induced capacitative Ca2’ entry has also been examined.
Materials and Methods Cell culture. MDCK cells obtained from American Type Culture Collection (CRL-6253) were cultured in Dulbecco’s modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 ug/ml streptomycin at 37°C in 5% CO,-containing humidified air. Ca*’ medium (pH 7.4) contained (in mM): NaCl 140; KC1 5; MgCl, 1; CaCl, 1.8; Hepes 10; glucose 5. Ca”-free medium contained no added Ca” plus 1 mM EGTA. The experimental solution contained less than 0.1% of solvent (dimethyl sulfoxide or ethanol) which did not affect [Ca”], (n=3).
Solutions.
measurements of[Ca*‘],. Trypsinized cells (lO’/ml) were allowed to recover for 1 hr in Dulbecco’s modified Eagle medium and were subsequently loaded with the ester form of fura-2, fura-2iAM (2 pM) for 30 min at 25°C. Cells were washed and resuspended in Ca” medium. Fura2 fluorescence measurements were performed in a water-jacketed cuvette (25°C) with continuous stirring; the cuvette contained 1 ml of medium and 0.5 million of cells. Fluorescence was monitored with a Hitachi F-4500 spectrofluorophotometer (Japan) by continuously recording excitation signals at 340 and 380 run and the emission signal at 5 10 nm at l-s intervals. Maximum and minimum fluorescences were obtained by adding Triton X- 100 (0.1%) and EGTA (20 mM) sequentially at the end of the experiment. The fluorescence intensity of the excitation signal at 340 nm was divided by that at 380 nm and the ratio was used to calculate [Ca”], as described previously (16) assuming a K, of 155 nM. Mn” quench experiments were performed in Optical
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Ca*’ medium containing 50 pM MnCl, by continuously recording the excitation signal at 360 nm and the emission signal at 510 nm at I-s intervals and the data were presented as arbitrary fluorescence units. We have shown previously that trypsinized cells yielded qualitatively similar results as cells attached to coverslips (10, 12, 14). Chemical reagents. The reagents for cell culture were from Gibco. Fura-YAM was from Molecular Probes. U73 122 and U73343 were from RBI. All other reagents were from Sigma. Statistical analyses. All values were reported as means+SEM (n=5-6). Statistical comparisons were determined by using the Student’s paired t test, and significance was accepted when PcO.05.
Results Fendiline increases [Ca”/i. In Ca*’ medium, fendiline at concentrations between 5- 100 PM induced a [Ca”], rise which comprised an initial rise and a gradual decay (Fig. 1A). For example, at a concentration of 100 PM, fendiline induced a peak [Caz’li of 589k13 nM (n=5) which slowly decayed in the following 300 s. At a concentration of 1 pM fendiline had little effect (not shown). At a concentration of 200 pM fendiline induced a nearly immediate rise in [Ca”], with a height of more than 1.2 PM, most likely reflecting cell membrane leakage, thus the results were not reported. The rise of the Ca*’ signal was slower in response to lower concentrations of fendiline.
C
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Fig. 1 A Concentration-dependent effects of fendiline on [Ca”], in fura-2-loaded MDCK cells. Concentration of fendiline was 100 pM in trace a, 50 PM in trace 6, 25 pM in trace c, 10 PM in trace d and 5 pM in trace e. The experiments were performed in Ca” medium. Traces are typical of 5-6 experiments. B Similar to A, except that the experiments were performed in Ca”-free medium (no added Ca*’ plus 1 mM EGTA). C Concentrationresponse plots of the fendiline response in the presence (filled circles) or absence (open circles) of external Ca*‘. Y axis: the net area under the curve (30-350 s) of the [Ca”], rise. Data are meansHEM of 5-6 experiments. *P
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b
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Fig. 2 A In Ca”-free medium, fendiline was added at 30 s followed by I pM thapsigargin
at 600 s. B Ca” influx detected by Mn*’ quench measurements. Truce a: control without addition of fendiline. Trace c: 50 uM fendiline was added at 80 s. Trace b: 0.1 mM La” was added 20 s prior to fendiline. C Dashed trace: control fendiline effect. Solid trace: 0.1 mM La3’ was added 20 s prior to fendiline. D Similar to C except that in dashed truce La” was added at the time indicated by * (80 s), and in solid trace La” was added at 320 s. The experiments in C and D were performed in Ca” medium. Traces are typical of 5-6 experiments. Internal Ca2’ release and external Ca” injlux both contribute to fendiline-induced rise in [CazcJi. Fig. 1B shows that Ca2’ removal reduced the Ca2’ signal induced by 25-100 PM fendiline by 50% both in peak value and in the area under the curve (30-350 s). The elevated phase of the [Ca”], rise induced by concentrations between lo-100 pM was abolished by Ca*’ removal. The concentration-response relationships of fendiline-induced Ca*’ signal in the presence or absence of external Ca*’ are depicted in Fig. 1C. We next examined fendiline-sensitive internal Ca*’ stores. Fig. 2A shows that in Ca2’-free medium, pretreatment with 50 uM fendiline for 600 s nearly abolished the [Ca”], rise induced by the endoplasmic reticulum Ca” pump inhibitor thapsigargin (17). Vice versa, pretreatment with thapsigargin for 370 s in Ca”-free medium prevented fendiline (50 PM) from releasing more internal Ca*’ (Fig. 3B).
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The pathway of fendiline-induced external Ca2’ influx was investigated. Mn2’ enters cells through similar pathways as Ca*‘, but quenches fura- fluorescence at all excitation wavelengths (18). Thus Ca2’ influx can be reflected by Mn” quench of fura- fluorescence. Fig. 2B shows that 50 uM fendiline induced an immediate decrease (truce c, maximum=80f6 arbitrary fluorescence units; n=6; PcO.05) in the 360 nm excitation signal compared with control (trace a). This provides direct evidence for fendiline-induced Ca2’ influx. Because we have previously shown that La” (0.1 mM) is a potent Ca2’ entry blocker in MDCK cells (10, 11, 14, 15, 19-2 l), the effect of La3’ on fendiline-induced response was explored. Fig. 2B shows that 0.1 mM La3’ added 20 s prior to fendiline inhibited fendiline-induced Mt?’ quench of fura- fluorescence by 5 1+8% (truce 6; n=6; PcO.05) in the area under the curve (80-250 s). Higher concentrations of La” induced cell aggregation and thus the results were not shown. We investigated the effect of La’* on fendiline-induced [Ca”], rise. Fig. 2C shows that pretreatment with 0.1 mM La3’ for 20 s inhibited 50 pM fendiline-induced [Ca2’li rise by retarding the rising velocity and by reducing the peak height by 38% (301+12 vs. 480+18; n=6; PcO.05). Fig. 2D depicts that addition of 0.1 mM La3’ during the rise of 50 nM fendiline-induced Ca*’ signal (at 80 s; dashed truce) immediately inhibited the response, and the area under the curve was reduced by 41+9% (80-350 s; n=6; PcO.05). However, addition of La” at 330 s had no effect (solid truce; n=6; fiO.05).
300
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Fig. 3 Effects of fendiline on capacitative Ca’” entry. A Capacitative Ca** entry induced by fendiline. Truce a: 100 uM fendiline was added at 30 s followed by 3 mM CaCl, at 500 s. Truce b, similar to truce a except that 50 uM fendiline was used. Truce c (control): CaCl, was added without preincubation with fendiline. B Truce u: 1 uM thapsigargin was added at 30 s followed by CaCl, at 510 s. Truces 6, c: similar to truce a except that 50 and 100 uM fendiline were added 100 s prior to CaCl,, respectively. Traces are typical of 5-6 experiments. Fendiline induces cupucitutive Cu” entT. Because mobilization of the endoplasmic reticulum Ca” store often triggers capacitative Ca2+ entry in MDCK cells (10, 11, 12, 14, 15, 19-21) we investigated if fendiline-induced Ca*’ influx involves capacitative Ca2’ entry. Capacitative Ca2 entry was measured by addition of 3 mM Ca2’ to cells pretreated with fendiline in Ca”-free
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medium to deplete internal Ca”. Fig. 3A shows that after depletion of internal 100 and 50 pM fendiline, addition of 3 mM Ca” induced a [Ca2’], rise with 230+16 nM and 101+_9 nM, respectively (traces a, b; n=6; PcO.05). This likely due to capacitaive Ca2’ entry because addition of CaCl, to cells which with fendiline only caused a [Ca2’], rise with a peak height of 20M nM (truce
Ca” for 470 s with a net peak value of [Ca”], rise is most were not pretreated c; n=6; P
2~~~~~ thapsigargin-pretreated
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fendillne 0 0
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Fig. 4 A Solid trace: In Ca2’-free medium, 2 pM U73122 was added at 50 s followed by 10 pM
ATP at 450 s and 50 uM fendiline at 560 s, respectively. Dashed trace: control without pretreatment with U73122 and ATP. B Solid truce: in Ca*’ medium, 0.1 mM propranolol was added at 50 s (*) followed by 50 yM fendiline at 310 s. Dashed truce: 40 pM aristolochic acid was added at 50 s followed by fendiline at 500 s. The control fendiline effect is shown in A. C Cells were incubated with 1 pM thapsigargin for 40 min in Ca” medium before fluorescence measurements were started. Fendiline (50 pM) was added at 50 s. Traces are typical of 5-6 experiments. Fendiline inhibits thapsigargin-induced capacitative Ca” entry. Fendiline has been shown to inhibit L-type Ca*’ channels in cardiac cells (2, 3, 22). Because MDCK cells do not have voltagegated Ca” channels (23) we investigated whether fendiline could alter capacitative Ca2’ entry, the major Ca” influx pathway in this cell (10-12, 14, 15) induced by another agent. Fig. 3B shows that 1 pM thapsigargin induced a significant [Ca”], rise in Ca2’-free medium with a peak height ,of 260+17 nM (truce a; n=6; WO.05). After incubation with thapsigargin for 380 s addition of 3 mM CaCl, induced capacitative Ca2’ entry with a peak height of 281+19 nM (n=6; PcO.05). This thapsigargin-induced capacitative Ca2’ entry was reduced by 25f5% and 51+-5%, respectively, in the area under the curve (500-650 s) by pretreatment with 50 and 100 pM fendiline for 100 s (traces b, c; n=5; RO.05). .!I?@Y yf’irthibition of’phospholipases on fendiline-induced [Ca”], rise. We tested if inhibition of phospholipases C, A, or D could alter fendiline-induced internal Ca” release. We have previously shown that 10 pM ATP induces marked internal Ca2* release in an IP,-dependent manner (19). Fig. 4A shows that incubation with 2 pM U73122, a phospholipase C inhibitor (24) for 420 s
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prevented 10 pM ATP from releasing internal Ca” (solid trace; n=6; P
Discussion The aim of this study was to investigate the effect of fendiline on Ca*’ homeostasis in MDCK cells. We found that fendiline increases [Ca*‘], within a concentration range of 5-100 PM. It is interesting that fendiline induces considerable [Ca”], rises in MDCK cells at concentrations commonly used to inhibit L-type Ca” channels in excitable cells (2, 3, 22). Fendiline is also regarded as an inhibitor of the Ca” sensor, calmodulin (1,4). But it is not clear whether fendilineinduced [Ca2’], rise is directly coupled to its inhibition of calmodulin because we found that although other calmodulin inhibitors such as W-7, calmidazolium and clotrimazole increased [Ca”‘], in MDCK cells, trifluoperazine (5-50 uM), phenoxybenzamine (100-200 PM) and fluphenazine-N-chloroethane (2- 100 PM) did not (unpublished data). Trifluoperazine induced a rise in the mra-2 ratio signal (340/380); however, this is not a Ca2’-sensitive signal because both 340 and 380 signals were increased (not shown). Given the possibility that fendiline might directly interfere with Ca” signaling in a calmodulin-independent manner, caution must be applied in using this drug as a tool to inhibit calmodulin, especially in cases where alterations of Ca*’ signaling may have an impact on results. Our data suggest that fendiline triggers internal Ca” release and external Ca*’ influx based on two independent measurements. First, the Ca” signals induced by 25- 100 pM fendiline were reduced by 50% by Ca” removal. Note that the elevated phase of the [Ca”], rise induced by 5-50 pM fendiline was abolished by Ca” removal, suggesting that in these responses this phase was maintained by Ca” influx. However, the elevated phase of 100 uM fendiline-induced [Ca”], rise remained significant between time points of 200-350 s when the [Ca”], rise induced by lower concentrations of fendiline had recovered to baseline. This may be because that 100 uM fendiline inhibited the plasmalemmal Ca” pump leading to a slower Ca*’ efflux. We have previously reported similar phenomena in the [Ca”], rises induced by econazole and SKF96365 (20, 21). Second, Mn2’ quench experiments provide further evidence that 50 pM fendiline induced Ca*’ influx. Our findings suggest that the internal Ca*’ source for fendiline-induced [Ca”], rise is thapsigargin-sensitive endoplasmic reticulum stores because in Ca2’-free medium, pretreatment with 1 uM thapsigargin abolished 50 uM fendiline-induced [Ca2’], rise, and vice versa,
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pretreatment with fendiline prevented thapsigargin from releasing Ca*+. The question arises as to how fendiline releases Ca*’ from internal stores. We tested whether fendiline-induced internal Ca” release was mediated by elevated IP, levels. Fig. 4A shows that under the condition that IP, formation was inhibited by U73 122 (evidenced by the observation that subsequently added ATP did not increase [Ca’+],), fendiline still induced a significant [Ca2’], rise. The observation that this [Ca”], rise was smaller than control by 61% is probably because that the endoplasmic reticulum Ca’. store had been partly depleted by U73 122. Thus, it is rather unlikely that IP, formation plays a major role in mediating fendiline-induced Ca2- release. Our findings are consistent with a previous study performed in C6 glioma cells in which fendiline was found not to alter resting IP, levels (27). We found that phospholipase A, may play a role because inhibition of phospholipase A, with 40 pM aristolichic acid reduced the [Ca”]irise by 48% in peak height. Phospholipase D probably does not play a role because pretreatment with 0.1 mM propranolol to inhibit phospholipase D did not have an effect. One question is how Ca” influx occurs during fendiline stimulation. Our results suggest that fendiline induces capacitative Ca*’ entry concentration-dependently. This is consistent with the observation that fendiline induces Mn*’ quench of fura- fluorescence. This fendiline-induced Ca” influx is most likely through capacitative Ca*’ entry (i.e. fendiline does not directly open a plasmalemmal Ca*’ channel), because 50 uM fendiline failed to increase [Ca2’], after cells had been depleted with internal Ca*’ by pretreatment with 1 uM thapsigargin for 40 min in Ca2’ medium. Similarly, using the same protocol, we have previously shown that while ATP and 2,5di-tert-butylhydroquinone induced capacitative Ca*’ entry, they did not increase [Ca2’], in Ca*’ medium after cells had been depleted with internal Ca*’ with thapsigargin for 30 min, suggesting that they did not directly open a plasmalemmal Ca” channel, and the Ca*’ entry they produced required a preceding internal Ca” depletion caused by these agents (10, 1S).The Ca*’ influx induced by 50 uM fendiline appears to have two components with equal contribution separable by sensitivity to 0.1 mM La3’, because La’- suppressed fendiline-induced Mn*’ quench of furafluorescence and [Ca*+], rise. The La”-sensitive Ca*’ influx operates during the rising phase, but not the decay phase, of fendiline-induced [Ca”], rise because adding La)’ during the rising phase immediately decreased the rise while adding La ‘+ during the decay had no effect (Fig. 2D). This is consistent with our previous study that U73 I22 also induces Ca*’ influx via a La3’-sensitive and a La”-insensitive pathways (19). Interestingly, despite inducing capacitative Ca2’ entry on its own, 50-100 pM fendiline inhibits We have found thapsigargin-induced capacitative Ca*’ entry concentration-dependently. previously that econazole and SKF96365 exert similar effects on Ca2‘ signaling in MDCK cells (20, 2 1). How exactly fendiline inhibits thapsigargin-induced capacitative Ca*’ entry is unclear. One possible explanation is that fendiline depolarizes cell membrane by inhibiting K’ channels leading to a reduced driving force for Ca*‘. To test this possibility, we depolarized cells by inhibiting K- channels with tetraethylammonium (TEA; 20 mM) to see if thapsigargin-induced capacitative Ca” entry was altered. Our data suggest that TEA had no effect (not shown). One might argue that the Ca*’ influx induced by fendiline in MDCK cells is caused by an increased driving force for Ca2+ influx resulting from fendiline-induced membrane hyperpolarization by activating Ca”-dependent K’ channels and inhibiting Cl‘ channels. We examined this hypothesis by investigating the effect of valinomycin, a K’ ionophore, on [Ca2’li. Valinomycin was expected to hypcrpolarize cells by increasing K’ efflux. We found that during the 5 min of incubation with 1O-100 pM valinomycin, the resting [Ca”], did not increase (not shown). Consistently, pretreatment with 20 mM TEA and 10 pM charybdotoxin to inhibit K+ currents did not alter fendiline-induced [Ca2’li rise. Thus, it seems unlikely that fendiline-induced alterations of Ca” signaling in MDCK cells are associated with its possible effects on membrane potential.
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Collectively, we have characterized the effects of fendiline on Ca2’ signaling in MDCK cells and have investigated the underlying mechanisms. Given the complex effects of fendiline on Ca2’ signaling (i.e. inducing [Ca2’], rises per se and inhibiting thapsigargin-induced capacitative Ca2’ entry), we caution that the effect of fendiline on Ca2’ signaling should be established before using it as an inhibitor of L-type Ca” channels or calmodulin.
Acknowledgments This work was supported by grants from National Science Council (NSC89-2320-B-075B-009) Veterans General Hospital-Kaohsiung (VGHKS89-13) and VTY Joint Research Program, Tsou’s Foundation (VTY88-P3-24) to CRJ, and a grant from Ping Tut’tg Christian Hospital to WCC. We thank Chin MH for culturing cells.
References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
R. BAYER and R. MANNHOLD, Pharmatherapeutica 5 103- 136 (1987). H. NAWRATH, G. KLEIN, J. RUPP, J.W. WEGENER and A. SHAINBERG, J. Pharmacol. Exp. Ther. 285 546-552 (1998). 0. TRIPATHI, W. SCHREIBMAYER and H.A. TRITTHART, Br. J. Pharmacol., 108 865 869 (1993). F. OROSZ, T.Y. CHRISTOVA and J. OVADI, Mol. Pharmacol. 33 678-682 (1988). T. NAGAO, S. ILLIANO and P.M. VANHOUTTE, Br. J. Pharmacol. 107 382-386 (1992). V.B. SCHINI and P.M. VANHOUTTE, J. Pharmacol. Exp. Ther. 261 553-559 (1992). R. BUSSE, A. LUCKHOFF, I. WINTER, A. MULSCH and U. POHL, N-S Arch. Pharmacol. 337 79-84 (1988). C. SCHLATTERER and R. SCHALOSKE, Biochem. J. 813 661-667 (1996). A. LUCKHOFF, M. BOHNERT and R. BUSSE. N-S. Arch. Pharmacol. 343 96-101 (1991). CR. JAN, C.M. HO, S.N. WU, J.K. HUANG and C.J. TSENG, Life Sci. 62 533-540 (1998). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Chin. J. Physiol. 41 59-64 (1998). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Eur. J. Pharmacol. 35 219-233 (1998). Jr. J.W. Putney and G.S.J. Bird, Cell 75 199-201 (1993). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Life Sci. 64 259-267 (1999) CR. JAN, C.M. HO, S.N. WI-J and C.J. TSENG, Eur. J. Pharmacol. 365 111-l 17 (1999). G. GRYNKIEWICZ, M. POENIE and R.Y. TSIEN, J. Biol. Chem. 260 3440-3450 (1985). 0. THASTRUP, P.T. CULLEN, B.K. DROBAK, M.R. HANLEY and A.P. DAWSON, Proc. Natl. Acad. Sci. USA 87 2466-2470 (1990). J.E. MERRITT, R. JOCOB and T.J. HALLAM, J. Biol. Chem. 264 1522-l 527 (1989). C.R. JAN, C.M. HO, S.N. WU and CJ TSENG, Life Sci. 63 895-908 (1998). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, N-S Arch. Pharmacol. 359 92-101 (1999). C.R. JAN, C.M. HO, S.N. WU and C.J. TSENG, Biochim Biophys Acta 1448:533-542 (1999). W. SCHREIBMAYER, 0. TRIPATHI and H.A. TRITTHART, Br. J Pharmacol. 106 15 l156 (1992). F. LANG and M. PAULMICHL M, Kidney Int. 48 1200-1205 (1995). A.K. THOMPSON, S.P. MOSTAFAPOUR, L.C. DENLINGER, J.E. BLEASDALE and S.K. FISHER, J. Biol. Chem. 266 23856-23862 (1991). M.D. ROSENTHAL, B.S. VISHWANATH and R.C. FRANSON, J. Biol. Chem. 264 1706917077 (1989).
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26. M.M. BILLAH, S. ECKEL, T.J. MULLMANN, R.W. EGAN and M.I. SIEGEL, Biochim. Biophys. Acta. 1001 l-8 (1989). 27. W.W. LIN, N-S Arch. Pharmacol. 352 679-684 (1995).