Inhibitory Action of Dilazep on Histamine-Stimulated Cytosolic Ca 2+ Increase in Cultured Human Endothelial Cells Nobuyuki Okamura, Yoshiaki Shirasawa* and Youji Mitsui' Biosignal Engineering Division, Kowa Research Institute, Kowa Co., Ltd., 1-25-5 Kannondai, Tsukuba, 305, Japan 'Cell Science and Technology Division, Fermentation Research Institute, Agency of Industrial Science and Technology, Higashi, Tsukuba 305, Japan Received
October
17, 1991
Accepted
March
10, 1992
ABSTRACT Using a fluorescent Ca2+-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in cytosolic Ca21 concentration ([Ca2+]i) in cultured human endothelial cells and studied the effect of dilazep on [Ca 2+]; elevation induced by histamine. Histamine (1 uM) caused a rapid transient peak in the average [Ca 2+]; of a group of cells (approximately 102cells), followed by a decrease to a sustained elevation. Dilazep as well as diltiazem (1.0 to 100,uM) concentration-depend ently inhibited the latter sustained elevation, which was eliminated by removal of extracellular Ca2+, while the initial transient response was not changed by dilazep at concentrations up to 100 ,uM. The IC5ovalues of dilazep and diltiazem were 16 and 58 pM, respectively. The patterns of the [Ca2+]; eleva tion responses to histamine were variable among individual cells. Some single cells showed a transient peak and a sustained elevation as observed in a group of cells. Some single cells caused a repetitive spikelike elevation of [Ca2+];. Dilazep lowered the sustained elevation to the resting level and in some single cells, changed the sustained elevation to the spikelike elevation. The frequency of the spikelike [Ca2+]; elevation was also decreased by dilazep. Decrease in extracellular [Ca2+] showed the same pat tern of inhibitory actions as dilazep did. These results indicate that dilazep inhibits the extracellular Ca 2+ influx in endothelial cells. Keywords:
Endothelial
cell, Cytosolic
Ca2+, Ca2+ spiking,
Vascular endothelium plays many important roles as an active metabolic tissue to modulate the activity of adjacent smooth muscle cells and blood through the re lease of the active mediators such as von Willebrand factor, prostacyclin, tissue plasminogen activator, and endothelin. A rise in cytosolic Ca2+ concentration ([Ca2+]i) is suspected to be an important triggering event in the endothelium's responses to histamine (1, 2), thrombin (3, 4) and adenine nucleotides (5, 6), since the production and release of the endothelium derived mediators are inhibited by removal of ex tracellular Ca2+ (2, 4, 7, 8) and by an antagonist of cytosolic Ca 2+, TMB-8 (8-(N, N-diethylamino)-octyl 3,4,5-trimethoxy benzoate) (9). However, the organic Ca 2+-channel blockers are much less potent at blocking endothelial responses (7, 8, 10) than depolarization induced smooth muscle contraction.
Histamine,
Dilazep
Dilazep, a coronary and cerebral vasodilator (11, 12), not only increases peripheral blood flow but also inhib its platelet aggregation (13, 14). Although dilazep is not a typical organic Ca2+-channel blocker such as nifedi pine and diltiazem, its weak inhibitory action on Ca2+ influx has been suggested to contribute to the relaxant effect in smooth muscle (15), the negative inotropic effect in heart muscle (16) and the antiaggregatory effect in platelets (13), whereas the effects of dilazep on endothelial cells have not yet been reported. It is of worthwhile to study the effects of dilazep on cytosolic Ca 2+ mobilization which modulates the functions of endothelial cells. In the present investigation using a fluorescent Ca2+-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in [Ca2+]i in cultured human endothelial cells, both single cells and a group of cells, and studied the effect of dilazep on Ca 2+ influx in histamine-stimulated endothe lial cells. Histamine causes a typical increase in [Ca2+];
in cultured endothelial cells through the discharge of Ca2+ from the intracellular store and the influx of ex tracellular Ca 2+ (1, 2). MATERIALS AND METHODS Materials Histamine dihydrochloride, diltiazem hydrochloride, bovine serum albumin and EGTA were purchased from Sigma (St. Louis, MO). Dilazep hydrochloride was obtained from Kowa (Nagoya). Fura-2 AM and plur onic F-127 were from Molecular Probes (Junction City, OR). Fura-2 was from Dojin (Kumamoto). HEPES, trypsin and cell culture medium (MCDB151) were from Gibco (Chagrin Falls, OH). Fetal calf serum (FCS) was from Biocell (Carson, CA). Acidic fibroblast growth factor (aFGF, from bovine brain) was purchased from Toyobo (Osaka). Heparin sodium salt was from Wako (Osaka). Fibronectin (from porcine plasma) was from Itoham (Nishinomiya). All other materials were of rea gent grade. Cell culture Human vascular endothelial cell lines (HUE142-2) isolated from umbilical cord veins (17) were used at 20 30 population-doubling levels remaining before the in vitro life span of 70 population-doubling levels. Cells were cultured in MCDB 151 medium supplemented with 15% FCS, 2 ng/ml aFGF and 1 ,ug/ml heparin in flasks coated with fibronectin at 37°C in 5% CO2. Cells de tached by exposure to trypsin (0.125%) were reseeded onto the glass cover slips (Matsunami No. 1) of silicone well chambers and kept in culture for 3 4 days before use as confluent monolayers (approximately 105 cells/cm2). Cell loading with fura-2 Confluent monolayers of endothelial cells cultured in silicon well chambers were incubated for 30 min at 37°C in culture medium containing 10,uM fura-2 AM soni cated with 0.15% dimethyl sulfoxide, 0.01% pluronic F 127 and 1.5% FCS. Cell layers were washed several times with fura-2-free medium and then maintained thereafter in fresh medium for 15 min. Fluorescence measurements A silicon well chamber was placed on the thermo stated stage on an inverted microscope (Olympus, IMT 2) equipped with a X20 fluorite objective, and the cells were superfused at 1 ml/min with medium containing 145 mM NaCI, 5 mM KC1, 1 mM CaC12, 10 MM glu cose, 0.1% bovine serum albumin and 10 mM HEPES (pH 7.4) at 37°C. The solution change at the cell sur
face was approximately complete within 5 sec. The cells loaded with fura-2 were illuminated via the epifluores cence port of the microscope and a 425-nm dichroic mir ror, with alternating 340-nm and 380-nm-long pass fil ters. The mass fluorescence intensity of a group of cells (approximately 102cells) illuminated by 340 nm light (F340) and 380 nm light (F380), and the ratio of F340 to F380 (F340/F380) was continuously measured by alternating the excitation wavelength at a 3-sec interval (every 1 sec for F340, F380 and shutter). Background fluorescence (less than 10% of measured fluorescence) from the endothelial monolayers was determined before fura-2 loading and subtracted from F340 and F380 at the two wavelengths, respectively. F340/F380 was used to quantify the changes in [Ca2+]; as previously re ported (18). To determine the Ca2+ concentration, we measured F340/F380 of a series of fura-2 solutions with Ca2+ concentrations ranging from 10 nM to 1 mM for the calibration curves. The solutions were prepared essentially as described by Fabiato and Fabiato (19), by adding CaC12 to a solution of 50 mM HEPES-KOH (pH 7.14), 100 mM KCI, 1 MM MgC12, 2 mM EGTA and 1 ,uM fura-2. Although absolute values of [Ca 2+1i could be calculated based on the dissociation constant of fura-2 for Ca2+ binding (18), recent reports have suggested that the dissociation constant of fura-2 for Ca 2+ in the cytosol is different from that measured in the absence of protein (20). Thus, [Ca2+1i obtained from the calibration curves is an estimated value. In the results, we show both the measured ratio (F340/F380) and estimated [Ca2+]i, if necessary. The [Ca2+]i imaging was carried out according to the method of Tsien and Poenie (21). Cells were super fused and illuminated as described above, but the fluorescence images were collected by a silicon intensi fied target camera (Hamamatsu) and an image proces sor (PIAS LA-500) driven by a computer (NEC PC 9801 RX) via software. The excitation wavelengths were alternated at a 6-sec interval (every 2 sec for F340, F380 and shutter). The [Ca2+]i of a single cell was measured by placing a window (15 X 15 pixels) on the image of the cell by means of the software. EGTA/Ca 2+-buffer solutions containing fura-2 were used for the calibration of [Ca2+],, as described above. RESULTS Mass [Ca2+]; changes measured in a group of cells In this experiment, we monitored the mass changes of F340 and F380 in an endothelial cell monolayer. Therefore, the [Ca2+]i measured was an average value of those of many cells. Endothelial monolayer cells had a resting [Ca 211iof 0.243 ± 0.004,uM (mean ± S.E., n
= 20, ratio: 1.20 ± 0.02). Histamine (0.01 to 10 uM) elicited a concentration-dependent increase in [Ca2+], with a maximal peak response (1.31 ± 0.05 uM, ratio: 3.04 ± 0.12, n = 7) at 10 uM. We studied the effect of dilazep on the [Ca2+]i elevation induced by 1 uM hista mine which produced the submaximum responses. His tamine (1 pM), as previously reported (1, 2), caused a rapid transient peak in [Ca 2+]i of 0.92 ± 0.02 uM (ratio: 2.38 ± 0.05, n = 20), followed by decrease to a sustained elevated [Ca2+]i (approximately 0.5 u M) (Fig. 1A). This sustained elevation of [Ca2+]i was eliminated by removal of extracellular Ca2+ (Fig. 1B). Dilazep as well as diltiazem at concentrations up to 100 uM did not change the resting [Ca2+]i and the initial transient response. The peak values of transient responses in the presence of dilazep (100 uM) and diltiazem (100 uM) were 0.83 ± 0.08 u M (ratio: 2.26 ± 0.20, n = 5) and 0.85 ± 0.09 uM (ratio: 2.32 ± 0.24, n = 5), respective ly, whereas the sustained elevation of [Ca2+]i was inhib ited by dilazep and diltiazem at the concentration of 100 u M. To evaluate the inhibitory effect of dilazep on the sustained [Ca2+]i elevation, dilazep and diltiazem in concentrations from 1 to 100uM were cumulatively ap plied during the period of the sustained [Ca 2+]i eleva tion; i.e., drugs were administered 4 min after an ap plication of 1 uM histamine. Although a small spon
taneous decline of [Ca2+]i was observed in the sus tained [Ca2+]i elevation (Table 1), it was negligible as compared with the decrease in [Ca2+]i produced by dilazep and diltiazem. As shown in Fig. 2 and Table 1, dilazep as well as diltiazem concentration-dependently inhibited the sustained [Ca 2+]i elevation. The IC50 values of dilazep and diltiazem, which were corrected with the spontaneous decline of sustained [Ca2+]i eleva tion, were 16 and 58 pM, respectively. Additionally, a single application of 30 u M dilazep decreased the net increase in the ratio of the sustained elevation by 70% from 1.86 ± 0.11 to 1.46 ± 0.04 (resting: 1.29 ± 0.06, n = 5). [Ca2+]elevation measured in single cells The patterns of the [Ca 2+]i elevation responses to 1 u M histamine were variable among individual cells. Most of the single cells, 94 out of 112 investigated cells (84%), showed almost the same time-course of [Ca2+]i elevation as that observed in a group of cells, namely, a transient [Ca2+]i elevation followed by a decrease to a plateau (Fig. 3A and B). Ten cells (9%) showed only repetitive spikelike elevation of [Ca2+]i. Both the apparently fused spikes and single spikes were observed in 4 cells (4%) (Fig. 3C). Four cells (4%) failed to re spond to histamine.
Fig. 1. Histamine-induced [Ca2+]; elevation measured in a group of cells. A: A typical response in the presence of 1 mM extracellular Ca2+. The monolayer of endothelial cells was stimulated with 1 uM histamine. B: Effect of removing ex tracellular Ca2+ on histamine-stimulated sustained [Ca2+]; elevation. The monolayer of cells was perfused with medium containing either 1 mM Ca2+ or 1 mM EGTA. Each tracing shows changes in F340, F380 and the ratio (F340/F380) corre sponding to [Ca2+];measured in a group of cells. Application periods are marked by horizontal solid lines.
Table 1.
Inhibitory action of dilazep on histamine-stimulated sustained [Ca 2+]; elevation in endothelial cells
Fig. 2. Inhibitory effects of dilazep and diltiazem on the sustained [Ca 2+]i elevation induced by histamine . The monolayer of endothelial cells was perfused with medium containing dilazep (A) and diltiazem (B) at the successive concentrations of 1.0, 10 and 100,uM 4 min after an application of 1,uM histamine. Each tracing shows changes in F340, F380 and the ratio (F340/F380) corresponding to [Ca 2+1i measured in a group of cells. Application periods are marked by horizontal solid lines.
As described above, when the mass [Ca2+], changes in a group of cells was monitored, 30,uM dilazep de creased the sustained [Ca2+]i elevation to a lower level, but did not decrease it to the resting level. In contrast, measurements of [Ca 2+]i in single cells indicated that dilazep decreased the sustained [Ca2+]i elevation to the resting level and caused a spikelike [Ca2+]i elevation. An application of 30 MM dilazep decreased the sus tained elevation of [Ca2+]i to the resting level in 27 out
of 88 investigated cells (31%) (Fig. 4A) and also elicit ed the spikelike [Ca2+]i elevation in 43 cells (49%) (Fig. 4B and C). However, the sustained elevations in 6 cells (7%) were not changed by dilazep. In 8 cells (9%) which exhibited the repetitive spikelike [Ca2+]i eleva tion, dilazep decreased the frequency of spiking rather than its amplitude (Fig. 5). Similar data were obtained in a total of 48 cells using 30,uM diltiazem (data not shown).
Fig. 3. Histamine-induced [Ca2+]; elevation measured in single cells. The monolayer of endothelial cells was stimulated with 1 ,uM histamine. The changes in the ratio (F340/F380) which corre sponds to [Ca2+]i in a single cell were monitored with [Ca2+]i im aging as described in "Materials and Methods". Each tracing (A, B, C) shows typical data obtained from different single cells. Ap plication periods are marked by horizontal solid lines.
Fig. 4. Effect of dilazep on the sustained [Ca2+]; elevation in duced by histamine in single cells. The monolayer of endothelial cells was perfused with medium containing 30,uM dilazep 5 min after application of 1 uM histamine. The changes in the ratio (F340/F380) which corresponds to [Ca2+1i were monitored with the [Ca22+]; imaging method. Each tracing (A, B, C) shows typical data obtained from different single cells. Application periods are indicated by horizontal solid lines.
When the extracellular [Ca 2+] was decreased from 1 mM to 0.1 mM in the presence of 1 uM histamine, the sustained elevation of [Ca2+]i decreased to the resting level in 32 out of 52 investigated cells (62%) and changed to the spikelike elevation in 10 cells (19%). The decrease in extracellular [Ca2+] also reduced the frequency of spiking in 8 cells which showed the repeti tive spikelike elevation in the presence of histamine.
oscillation) in single endothelial cells (25, 26). Measur ing mass changes in the [Ca2+]i of a group of cells (m[Ca2+]i) is useful for evaluating the efficacy of drugs, whereas measuring changes in the [Ca2+]i of sin gle cells (s[Ca2+]i) is also important for elucidating the cellular mechanisms of the drug action. Therefore, in the present study, we measured histamine-induced [Ca2+]i changes both in single cells and a group of cells (approx. 102cells). As previously reported (1, 2), hista mine caused a rapid transient peak in m[Ca2+]i fol lowed by a decrease to a sustained elevation. Our re sults demonstrated that dilazep as well as diltiazem de creased the sustained elevation but not the transient in crease of m[Ca2+]i caused by 1,uM histamine. The sus tained elevation of m[Ca2+]i was eliminated by removal of extracellular Ca2+, indicating that the sustained elevation is produced by an extracellular Ca2+ influx, whereas the transient elevation is induced by Ca2+ release from intracellular stores (1, 2). The pres ent results suggest that dilazep as well as diltiazem in
DISCUSSION Several investigators have measured histamine induced changes in [Ca2+], in endothelial cells loaded with a Ca 2+-sensitive fluorescent dye on microcarrier beads or cover slips (2, 22-24). The mass changes in [Ca2+], measured in these cells represent average values of a group consisting of more than 103cells. The cells do not all synchronously respond to histamine since the lower concentration of histamine (0.1 3.0 ,uM) pro duces repetitive spikelike [Ca2+]i elevation (Ca2+
Fig. 5. Effect of dilazep on the spikelike [Ca2+]; elevation pro duced by histamine in single cells. The monolayer of endothelial cells was perfused with medium containing 30 ,uM dilazep 5 min after an application of 1 uM histamine. The changes in the ratio (F340/F380) which corresponds to [Ca2+1i were monitored with the [Ca2+]i imaging method. Each tracing (A, B, C) shows typical data obtained from different single cells. Application periods are indicated by horizontal solid lines.
hibits extracellular Ca2+ influx and does not affect the Ca2+ release from intracellular stores. Rotrosen and Gallin (1) have reported that 20,uM verapamil inhibits both transient and sustained increases in m[Ca2+]i pro duced by 5,uM histamine, and suggested that verapamil acts through non-Ca 2+-specific mechanisms. They treat ed a confluent monolayer of cells with EDTA and pre pared the cell suspensions for the fluorescence measure ments, whereas we measured the fluorescence of a con fluent monolayer of cells. Hamilton and Sims (2) have suggested that significant disruption of calcium homeo stasis occurs during detachment of cells from adherent cell monolayers. It might be possible that suspended cells as compared with monolayer cells are more labile and respond differently to agents. Dilazep, which is not a typical organic calcium an tagonist like diltiazem, shows only a weak inhibitory ac tion on the Ca2+ influx in smooth muscle (15). How ever, in the present study, dilazep showed approximate ly 3.5 times more potent inhibitory action on Ca2+ influx in endothelial cells than did diltiazem. From several observations that the organic Ca2+-chan
nel blockers are much less potent in blocking endothe lial responses (7, 8, 10) than in suppressing depolariza tion-induced smooth muscle contraction, and that the depolarization by extracellular high [K+] does not in crease [Ca 21]i in endothelial cells (1, 3, 27), the path ways for Ca2+ influx located on the endothelial cell membrane are distinct from the L-type voltage-depend ent Ca2+ channels. It has been also reported that Sr2+, Ba2+ and Mn2+ can substitute for Ca 2+, being able to enter the cytoplasm of endothelial cells through agonist regulated channels (22-24). Dilazep may block those divalent cation channels more effectively than diltiazem does. The patterns of s[Ca2+]i elevation responses to 1,uM histamine were variable among individual cells. Some single cells showed a transient s[Ca2+]i elevation fol lowed by a decrease to a sustained elevation as observed in a group of cells. Some cells showed a re petitive spikelike s[Ca2+]i elevation called calcium spik ing, as Jacob et al. have recently reported (25, 26). Dilazep as well as diltiazem decreased the sustained elevation of s[Ca2+]i induced by histamine to the rest ing level in 31% of the investigated cells and in 49% of the cells, they changed the sustained elevation to the repetitive spikelike elevation. The same pattern of in hibitory action was observed when the extracellular [Ca2+] was lowered from 1 mM to 0.1 mM. These re sults indicate again that dilazep and diltiazem inhibit Ca2+ influx in endothelial cells. Both drugs as well as the decrease in extracellular [Ca2+] also reduced the frequency of spiking in cells which showed the repeti tive spikelike elevation in the presence of histamine. Although calcium spiking is generated by Ca2+ release from the intracellular store, it is rather sensitive to ex tracellular Ca 2+ (25, 26). Extracellular Ca 2+ is required to refill the store. Therefore, the decreases in frequency of spiking produced by dilazep and diltiazem are due to their inhibitory actions on Ca2+ influx. From the present observations, it is obvious that the m[Ca2+]i of a group of cells represents the average of a variety of complex s[Ca2+]i events that are observed in individual cells. As mentioned above, when the m[Ca2+]i of a group of cells was monitored, 30 pM dilazep decreased the sustained m[Ca2+]i elevation to a level above the resting level. In contrast, when measur ing s[Ca2+]i in single cells, dilazep decreased the sus tained s[Ca2+]i elevation to the resting level and in some cells elicited the spikelike elevation. It is interest ing that the sustained s[Ca2+]i elevation was decreased to the resting level without staying at a level above the resting state after an application of dilazep. The [Ca 2+]i levels between the resting state and the sustained eleva tion state might be unstable. In fact, all the cells inves
tigated in the presence of dilazep showed the s[Ca2+]; of the resting state, the spikelike elevation, and the sus tained elevation which was unchanged by dilazep in a small population of cells. These results suggest that the [Ca2+]i of the sustained elevation produced by hista mine represents a stable level in the bistable model, which has been proposed for receptor-stimulated cal cium spiking (28, 29). It is also likely that the sustained [Ca2+]; elevation induced by histamine consists of fused spikes (25). So far, Ca 2+ spiking has been observed in many different kinds of eukaryotic cells (29-31) includ ing endothelial cells (25, 26, 32). The frequency of spik ing increases when the concentration of agonist is raised, whereas the amplitude and duration of spikes stay nearly the same. This invariance suggests that spikes are produced by excitation of bistable systems (28, 29), namely the sustained elevated [Ca2+]i level and the resting [Ca 2+]i level in histamine-stimulated en dothelial cells in the present study. REFERENCES
1 Rotrosen, D. and Gallin, G.: Histamine type I receptor occu pancy increasesendothelial cytosolic calcium, reduces F-actin, and promotes albumin diffusion across cultured endothelial monolayers. J. Cell Biol. 103, 2379-2387 (1986) 2 Hamilton, K.K. and Sims, P.J.: Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. J. Clin. Invest. 79, 600 608 (1987) 3 Jaffe, E.A., Grulich, J., Weksler, B.B., Hampel, G. and Watanabe, K.: Correlation between thrombin-induced pros tacyclin production and inositol trisphosphate and cytosolic free calcium levels on cultured human endothelial cells. J. Biol. Chem. 262, 8557-8565 (1987) 4 Hallam, T.J., Pearson, J.D. and Needham, L.A.: Thrombin stimulated elevation of human endothelial-cell cytoplasmic free calcium concentration causes prostacyclin production. Biochem. J. 251, 243-249 (1988) 5 Luckhoff, A. and Busse, R.: Increased free calcium in endothelial cells under stimulation with adenine nucleotides. J. Cell. Physiol. 126, 414-420 (1986) 6 Carter, T.D., Hallam, T.J., Cusack, N.J. and Pearson, J.D.: Regulation of Pty-purinoceptor-mediatedprostacyclin release from human endothelial cells by cytoplasmic calcium concen tration. Br. J. Pharmacol. 95, 1181 1190(1988) 7 Crutchley, D.J., Ryan, J.W., Ryan, U.S. and Fischer, G.H.: Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Biochim. Biophys. Acta 751, 99-107 (1983) 8 Van De Velde, V.J.S., Van Den Bossche, R.M., Bult, H. and Herman, A.G.: Modulation of prostacyclin biosynthesis by calcium entry blockers and extracellular calcium. Biochem. Pharmacol. 35, 253-256 (1986) 9 Brotherton, A.F.A. and Hoak, J.C.: Role of Ca2+ and cyclic AMP in the regulation of the production of prostacyclin by the vascular endothelium. Proc. Natl. Acad. Sci. U.S.A. 79,
495 499 (1982) 10 Miller, R.C., Schoeffter, P. and Stoclet, J.C.: Insensitivity of calcium-dependent endothelial stimulation in rat isolated aorta to the calcium entry blocker, flunarizine. Br. J. Phar macol. 85, 481-487 (1985) 11 Buyniski, J.P., Losada, M., Bierwagen, M.E. and Gardier, R.W.: Cerebral and coronary vascular effects of a symmetri cal N,N'-disubstituted hexahydrodiazepine. J. Pharmacol. Exp. Ther. 181,522-528 (1972) 12 Sano, N., Katuki, S. and Kawada, M.: Enhancement of coronary vasodilator action of adenosine by 1,4-bis[3-(3,4,5 trymethoxybenzoyloxy)-propyl]-perhydro-1,4-diazepine (Dilazep I.N.N.). Arzneimittelforschung22, 1655-1658 (1972) 13 Valori, V.M., Leone, G. and Bizzi, B.: A comparative study on the effect of dilazep and dipyridamole on some platelet function. Arzneimittelforschung32, 403-405 (1982) 14 Sumiyoshi, A. and Hayashi, T.: The inhibitory effect of dilazep on in vivo accumulation of platelets onto the damaged aorta in rabbits. Thromb. Res. 29, 37-42 (1983) 15 Nakagawa, Y., Mustafa, S.J. and Gudenzi, M.: Calcium blocking activity of adenosine potentiating compounds in isolated guinea pig taenia coli and dog coronary artery. Fed. Proc. 42, 847 (1983) 16 Nakagawa, Y., Gudenzi, M. and Mustafa, S.J.: Calcium entry blocking activity of dilazep and other adenosine potenti ating compounds in guinea-pig atria. Eur. J. Pharmacol. 122, 51 58 (1986) 17 Imamura, T. and Mitsui, Y.: Heparan sulfate and heparin as a potentiator or a suppressor of growth of normal and trans formed vascular endothelial cells. Exp. Cell Res. 172, 92-100 (1987) 18 Grynkiewicz, G., Poenie, M. and Tsien, R.Y.: A new gen eration of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260, 3440-3450 (1985) 19 Fabiato, A. and Fabiato, C.: Calculator programs for com puting the composition of the solutions containing metals and ligands used experiments in skinned muscle cells. J. Physiol. (Paris) 75, 463-505 (1979) 20 Konishi, M., Olson, A., Hollingworth, S. and Baylor, S.M.: Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements. Biophys. J. 54, 1089 1104(1988) 21 Tsien, R.Y. and Poenie, M.: Fluorescence ratio imaging: a new window into intracellular ionic signaling. Trends Bio chem. Sci. 11, 450-455 (1986) 22 Schelling, W.P., Rajana, L. and Strobl-Jager, E.: Character ization of the bradykinin-stimulated influx pathway of cul tured vascular endothelial cells. J. Biol. Chem. 264, 12838 12848(1989) 23 Hallam, T.J., Jacob, R. and Merritt, J.E.: Evidence that ago nists stimulate bivalent-cation influx into human endothelial cells. Biochem. J. 255, 179-184 (1988) 24 Hallam, T.J., Jacob, R. and Merritt, J.E.: Influx of bivalent cations can be independent of receptor stimulation in human endothelial cells. Biochem. J. 259, 125-129 (1989) 25 Jacob, R., Merritt, J.E., Hallam, T.J. and Rink, T.J.: Repetitive spikes in cytoplasmiccalcium evoked by histamine in human endothelial cells. Nature 335, 40-45 (1988) 26 Jacob, R.: Calcium oscillations in endothelial cells. Cell Calcium 12, 127-134 (1991)
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October
17, 1991
Accepted
March
10, 1992
ABSTRACT Using a fluorescent Ca2+-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in cytosolic Ca21 concentration ([Ca2+]i) in cultured human endothelial cells and studied the effect of dilazep on [Ca 2+]; elevation induced by histamine. Histamine (1 uM) caused a rapid transient peak in the average [Ca 2+]; of a group of cells (approximately 102cells), followed by a decrease to a sustained elevation. Dilazep as well as diltiazem (1.0 to 100,uM) concentration-depend ently inhibited the latter sustained elevation, which was eliminated by removal of extracellular Ca2+, while the initial transient response was not changed by dilazep at concentrations up to 100 ,uM. The IC5ovalues of dilazep and diltiazem were 16 and 58 pM, respectively. The patterns of the [Ca2+]; eleva tion responses to histamine were variable among individual cells. Some single cells showed a transient peak and a sustained elevation as observed in a group of cells. Some single cells caused a repetitive spikelike elevation of [Ca2+];. Dilazep lowered the sustained elevation to the resting level and in some single cells, changed the sustained elevation to the spikelike elevation. The frequency of the spikelike [Ca2+]; elevation was also decreased by dilazep. Decrease in extracellular [Ca2+] showed the same pat tern of inhibitory actions as dilazep did. These results indicate that dilazep inhibits the extracellular Ca 2+ influx in endothelial cells. Keywords:
Endothelial
cell, Cytosolic
Ca2+, Ca2+ spiking,
Vascular endothelium plays many important roles as an active metabolic tissue to modulate the activity of adjacent smooth muscle cells and blood through the re lease of the active mediators such as von Willebrand factor, prostacyclin, tissue plasminogen activator, and endothelin. A rise in cytosolic Ca2+ concentration ([Ca2+]i) is suspected to be an important triggering event in the endothelium's responses to histamine (1, 2), thrombin (3, 4) and adenine nucleotides (5, 6), since the production and release of the endothelium derived mediators are inhibited by removal of ex tracellular Ca2+ (2, 4, 7, 8) and by an antagonist of cytosolic Ca 2+, TMB-8 (8-(N, N-diethylamino)-octyl 3,4,5-trimethoxy benzoate) (9). However, the organic Ca 2+-channel blockers are much less potent at blocking endothelial responses (7, 8, 10) than depolarization induced smooth muscle contraction.
Histamine,
Dilazep
Dilazep, a coronary and cerebral vasodilator (11, 12), not only increases peripheral blood flow but also inhib its platelet aggregation (13, 14). Although dilazep is not a typical organic Ca2+-channel blocker such as nifedi pine and diltiazem, its weak inhibitory action on Ca2+ influx has been suggested to contribute to the relaxant effect in smooth muscle (15), the negative inotropic effect in heart muscle (16) and the antiaggregatory effect in platelets (13), whereas the effects of dilazep on endothelial cells have not yet been reported. It is of worthwhile to study the effects of dilazep on cytosolic Ca 2+ mobilization which modulates the functions of endothelial cells. In the present investigation using a fluorescent Ca2+-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in [Ca2+]i in cultured human endothelial cells, both single cells and a group of cells, and studied the effect of dilazep on Ca 2+ influx in histamine-stimulated endothe lial cells. Histamine causes a typical increase in [Ca2+];
in cultured endothelial cells through the discharge of Ca2+ from the intracellular store and the influx of ex tracellular Ca 2+ (1, 2). MATERIALS AND METHODS Materials Histamine dihydrochloride, diltiazem hydrochloride, bovine serum albumin and EGTA were purchased from Sigma (St. Louis, MO). Dilazep hydrochloride was obtained from Kowa (Nagoya). Fura-2 AM and plur onic F-127 were from Molecular Probes (Junction City, OR). Fura-2 was from Dojin (Kumamoto). HEPES, trypsin and cell culture medium (MCDB151) were from Gibco (Chagrin Falls, OH). Fetal calf serum (FCS) was from Biocell (Carson, CA). Acidic fibroblast growth factor (aFGF, from bovine brain) was purchased from Toyobo (Osaka). Heparin sodium salt was from Wako (Osaka). Fibronectin (from porcine plasma) was from Itoham (Nishinomiya). All other materials were of rea gent grade. Cell culture Human vascular endothelial cell lines (HUE142-2) isolated from umbilical cord veins (17) were used at 20 30 population-doubling levels remaining before the in vitro life span of 70 population-doubling levels. Cells were cultured in MCDB 151 medium supplemented with 15% FCS, 2 ng/ml aFGF and 1 ,ug/ml heparin in flasks coated with fibronectin at 37°C in 5% CO2. Cells de tached by exposure to trypsin (0.125%) were reseeded onto the glass cover slips (Matsunami No. 1) of silicone well chambers and kept in culture for 3 4 days before use as confluent monolayers (approximately 105 cells/cm2). Cell loading with fura-2 Confluent monolayers of endothelial cells cultured in silicon well chambers were incubated for 30 min at 37°C in culture medium containing 10,uM fura-2 AM soni cated with 0.15% dimethyl sulfoxide, 0.01% pluronic F 127 and 1.5% FCS. Cell layers were washed several times with fura-2-free medium and then maintained thereafter in fresh medium for 15 min. Fluorescence measurements A silicon well chamber was placed on the thermo stated stage on an inverted microscope (Olympus, IMT 2) equipped with a X20 fluorite objective, and the cells were superfused at 1 ml/min with medium containing 145 mM NaCI, 5 mM KC1, 1 mM CaC12, 10 MM glu cose, 0.1% bovine serum albumin and 10 mM HEPES (pH 7.4) at 37°C. The solution change at the cell sur
face was approximately complete within 5 sec. The cells loaded with fura-2 were illuminated via the epifluores cence port of the microscope and a 425-nm dichroic mir ror, with alternating 340-nm and 380-nm-long pass fil ters. The mass fluorescence intensity of a group of cells (approximately 102cells) illuminated by 340 nm light (F340) and 380 nm light (F380), and the ratio of F340 to F380 (F340/F380) was continuously measured by alternating the excitation wavelength at a 3-sec interval (every 1 sec for F340, F380 and shutter). Background fluorescence (less than 10% of measured fluorescence) from the endothelial monolayers was determined before fura-2 loading and subtracted from F340 and F380 at the two wavelengths, respectively. F340/F380 was used to quantify the changes in [Ca2+]; as previously re ported (18). To determine the Ca2+ concentration, we measured F340/F380 of a series of fura-2 solutions with Ca2+ concentrations ranging from 10 nM to 1 mM for the calibration curves. The solutions were prepared essentially as described by Fabiato and Fabiato (19), by adding CaC12 to a solution of 50 mM HEPES-KOH (pH 7.14), 100 mM KCI, 1 MM MgC12, 2 mM EGTA and 1 ,uM fura-2. Although absolute values of [Ca 2+1i could be calculated based on the dissociation constant of fura-2 for Ca2+ binding (18), recent reports have suggested that the dissociation constant of fura-2 for Ca 2+ in the cytosol is different from that measured in the absence of protein (20). Thus, [Ca2+1i obtained from the calibration curves is an estimated value. In the results, we show both the measured ratio (F340/F380) and estimated [Ca2+]i, if necessary. The [Ca2+]i imaging was carried out according to the method of Tsien and Poenie (21). Cells were super fused and illuminated as described above, but the fluorescence images were collected by a silicon intensi fied target camera (Hamamatsu) and an image proces sor (PIAS LA-500) driven by a computer (NEC PC 9801 RX) via software. The excitation wavelengths were alternated at a 6-sec interval (every 2 sec for F340, F380 and shutter). The [Ca2+]i of a single cell was measured by placing a window (15 X 15 pixels) on the image of the cell by means of the software. EGTA/Ca 2+-buffer solutions containing fura-2 were used for the calibration of [Ca2+],, as described above. RESULTS Mass [Ca2+]; changes measured in a group of cells In this experiment, we monitored the mass changes of F340 and F380 in an endothelial cell monolayer. Therefore, the [Ca2+]i measured was an average value of those of many cells. Endothelial monolayer cells had a resting [Ca 211iof 0.243 ± 0.004,uM (mean ± S.E., n
= 20, ratio: 1.20 ± 0.02). Histamine (0.01 to 10 uM) elicited a concentration-dependent increase in [Ca2+], with a maximal peak response (1.31 ± 0.05 uM, ratio: 3.04 ± 0.12, n = 7) at 10 uM. We studied the effect of dilazep on the [Ca2+]i elevation induced by 1 uM hista mine which produced the submaximum responses. His tamine (1 pM), as previously reported (1, 2), caused a rapid transient peak in [Ca 2+]i of 0.92 ± 0.02 uM (ratio: 2.38 ± 0.05, n = 20), followed by decrease to a sustained elevated [Ca2+]i (approximately 0.5 u M) (Fig. 1A). This sustained elevation of [Ca2+]i was eliminated by removal of extracellular Ca2+ (Fig. 1B). Dilazep as well as diltiazem at concentrations up to 100 uM did not change the resting [Ca2+]i and the initial transient response. The peak values of transient responses in the presence of dilazep (100 uM) and diltiazem (100 uM) were 0.83 ± 0.08 u M (ratio: 2.26 ± 0.20, n = 5) and 0.85 ± 0.09 uM (ratio: 2.32 ± 0.24, n = 5), respective ly, whereas the sustained elevation of [Ca2+]i was inhib ited by dilazep and diltiazem at the concentration of 100 u M. To evaluate the inhibitory effect of dilazep on the sustained [Ca2+]i elevation, dilazep and diltiazem in concentrations from 1 to 100uM were cumulatively ap plied during the period of the sustained [Ca 2+]i eleva tion; i.e., drugs were administered 4 min after an ap plication of 1 uM histamine. Although a small spon
taneous decline of [Ca2+]i was observed in the sus tained [Ca2+]i elevation (Table 1), it was negligible as compared with the decrease in [Ca2+]i produced by dilazep and diltiazem. As shown in Fig. 2 and Table 1, dilazep as well as diltiazem concentration-dependently inhibited the sustained [Ca 2+]i elevation. The IC50 values of dilazep and diltiazem, which were corrected with the spontaneous decline of sustained [Ca2+]i eleva tion, were 16 and 58 pM, respectively. Additionally, a single application of 30 u M dilazep decreased the net increase in the ratio of the sustained elevation by 70% from 1.86 ± 0.11 to 1.46 ± 0.04 (resting: 1.29 ± 0.06, n = 5). [Ca2+]elevation measured in single cells The patterns of the [Ca 2+]i elevation responses to 1 u M histamine were variable among individual cells. Most of the single cells, 94 out of 112 investigated cells (84%), showed almost the same time-course of [Ca2+]i elevation as that observed in a group of cells, namely, a transient [Ca2+]i elevation followed by a decrease to a plateau (Fig. 3A and B). Ten cells (9%) showed only repetitive spikelike elevation of [Ca2+]i. Both the apparently fused spikes and single spikes were observed in 4 cells (4%) (Fig. 3C). Four cells (4%) failed to re spond to histamine.
Fig. 1. Histamine-induced [Ca2+]; elevation measured in a group of cells. A: A typical response in the presence of 1 mM extracellular Ca2+. The monolayer of endothelial cells was stimulated with 1 uM histamine. B: Effect of removing ex tracellular Ca2+ on histamine-stimulated sustained [Ca2+]; elevation. The monolayer of cells was perfused with medium containing either 1 mM Ca2+ or 1 mM EGTA. Each tracing shows changes in F340, F380 and the ratio (F340/F380) corre sponding to [Ca2+];measured in a group of cells. Application periods are marked by horizontal solid lines.
Table 1.
Inhibitory action of dilazep on histamine-stimulated sustained [Ca 2+]; elevation in endothelial cells
Fig. 2. Inhibitory effects of dilazep and diltiazem on the sustained [Ca 2+]i elevation induced by histamine . The monolayer of endothelial cells was perfused with medium containing dilazep (A) and diltiazem (B) at the successive concentrations of 1.0, 10 and 100,uM 4 min after an application of 1,uM histamine. Each tracing shows changes in F340, F380 and the ratio (F340/F380) corresponding to [Ca 2+1i measured in a group of cells. Application periods are marked by horizontal solid lines.
As described above, when the mass [Ca2+], changes in a group of cells was monitored, 30,uM dilazep de creased the sustained [Ca2+]i elevation to a lower level, but did not decrease it to the resting level. In contrast, measurements of [Ca 2+]i in single cells indicated that dilazep decreased the sustained [Ca2+]i elevation to the resting level and caused a spikelike [Ca2+]i elevation. An application of 30 MM dilazep decreased the sus tained elevation of [Ca2+]i to the resting level in 27 out
of 88 investigated cells (31%) (Fig. 4A) and also elicit ed the spikelike [Ca2+]i elevation in 43 cells (49%) (Fig. 4B and C). However, the sustained elevations in 6 cells (7%) were not changed by dilazep. In 8 cells (9%) which exhibited the repetitive spikelike [Ca2+]i eleva tion, dilazep decreased the frequency of spiking rather than its amplitude (Fig. 5). Similar data were obtained in a total of 48 cells using 30,uM diltiazem (data not shown).
Fig. 3. Histamine-induced [Ca2+]; elevation measured in single cells. The monolayer of endothelial cells was stimulated with 1 ,uM histamine. The changes in the ratio (F340/F380) which corre sponds to [Ca2+]i in a single cell were monitored with [Ca2+]i im aging as described in "Materials and Methods". Each tracing (A, B, C) shows typical data obtained from different single cells. Ap plication periods are marked by horizontal solid lines.
Fig. 4. Effect of dilazep on the sustained [Ca2+]; elevation in duced by histamine in single cells. The monolayer of endothelial cells was perfused with medium containing 30,uM dilazep 5 min after application of 1 uM histamine. The changes in the ratio (F340/F380) which corresponds to [Ca2+1i were monitored with the [Ca22+]; imaging method. Each tracing (A, B, C) shows typical data obtained from different single cells. Application periods are indicated by horizontal solid lines.
When the extracellular [Ca 2+] was decreased from 1 mM to 0.1 mM in the presence of 1 uM histamine, the sustained elevation of [Ca2+]i decreased to the resting level in 32 out of 52 investigated cells (62%) and changed to the spikelike elevation in 10 cells (19%). The decrease in extracellular [Ca2+] also reduced the frequency of spiking in 8 cells which showed the repeti tive spikelike elevation in the presence of histamine.
oscillation) in single endothelial cells (25, 26). Measur ing mass changes in the [Ca2+]i of a group of cells (m[Ca2+]i) is useful for evaluating the efficacy of drugs, whereas measuring changes in the [Ca2+]i of sin gle cells (s[Ca2+]i) is also important for elucidating the cellular mechanisms of the drug action. Therefore, in the present study, we measured histamine-induced [Ca2+]i changes both in single cells and a group of cells (approx. 102cells). As previously reported (1, 2), hista mine caused a rapid transient peak in m[Ca2+]i fol lowed by a decrease to a sustained elevation. Our re sults demonstrated that dilazep as well as diltiazem de creased the sustained elevation but not the transient in crease of m[Ca2+]i caused by 1,uM histamine. The sus tained elevation of m[Ca2+]i was eliminated by removal of extracellular Ca2+, indicating that the sustained elevation is produced by an extracellular Ca2+ influx, whereas the transient elevation is induced by Ca2+ release from intracellular stores (1, 2). The pres ent results suggest that dilazep as well as diltiazem in
DISCUSSION Several investigators have measured histamine induced changes in [Ca2+], in endothelial cells loaded with a Ca 2+-sensitive fluorescent dye on microcarrier beads or cover slips (2, 22-24). The mass changes in [Ca2+], measured in these cells represent average values of a group consisting of more than 103cells. The cells do not all synchronously respond to histamine since the lower concentration of histamine (0.1 3.0 ,uM) pro duces repetitive spikelike [Ca2+]i elevation (Ca2+
Fig. 5. Effect of dilazep on the spikelike [Ca2+]; elevation pro duced by histamine in single cells. The monolayer of endothelial cells was perfused with medium containing 30 ,uM dilazep 5 min after an application of 1 uM histamine. The changes in the ratio (F340/F380) which corresponds to [Ca2+1i were monitored with the [Ca2+]i imaging method. Each tracing (A, B, C) shows typical data obtained from different single cells. Application periods are indicated by horizontal solid lines.
hibits extracellular Ca2+ influx and does not affect the Ca2+ release from intracellular stores. Rotrosen and Gallin (1) have reported that 20,uM verapamil inhibits both transient and sustained increases in m[Ca2+]i pro duced by 5,uM histamine, and suggested that verapamil acts through non-Ca 2+-specific mechanisms. They treat ed a confluent monolayer of cells with EDTA and pre pared the cell suspensions for the fluorescence measure ments, whereas we measured the fluorescence of a con fluent monolayer of cells. Hamilton and Sims (2) have suggested that significant disruption of calcium homeo stasis occurs during detachment of cells from adherent cell monolayers. It might be possible that suspended cells as compared with monolayer cells are more labile and respond differently to agents. Dilazep, which is not a typical organic calcium an tagonist like diltiazem, shows only a weak inhibitory ac tion on the Ca2+ influx in smooth muscle (15). How ever, in the present study, dilazep showed approximate ly 3.5 times more potent inhibitory action on Ca2+ influx in endothelial cells than did diltiazem. From several observations that the organic Ca2+-chan
nel blockers are much less potent in blocking endothe lial responses (7, 8, 10) than in suppressing depolariza tion-induced smooth muscle contraction, and that the depolarization by extracellular high [K+] does not in crease [Ca 21]i in endothelial cells (1, 3, 27), the path ways for Ca2+ influx located on the endothelial cell membrane are distinct from the L-type voltage-depend ent Ca2+ channels. It has been also reported that Sr2+, Ba2+ and Mn2+ can substitute for Ca 2+, being able to enter the cytoplasm of endothelial cells through agonist regulated channels (22-24). Dilazep may block those divalent cation channels more effectively than diltiazem does. The patterns of s[Ca2+]i elevation responses to 1,uM histamine were variable among individual cells. Some single cells showed a transient s[Ca2+]i elevation fol lowed by a decrease to a sustained elevation as observed in a group of cells. Some cells showed a re petitive spikelike s[Ca2+]i elevation called calcium spik ing, as Jacob et al. have recently reported (25, 26). Dilazep as well as diltiazem decreased the sustained elevation of s[Ca2+]i induced by histamine to the rest ing level in 31% of the investigated cells and in 49% of the cells, they changed the sustained elevation to the repetitive spikelike elevation. The same pattern of in hibitory action was observed when the extracellular [Ca2+] was lowered from 1 mM to 0.1 mM. These re sults indicate again that dilazep and diltiazem inhibit Ca2+ influx in endothelial cells. Both drugs as well as the decrease in extracellular [Ca2+] also reduced the frequency of spiking in cells which showed the repeti tive spikelike elevation in the presence of histamine. Although calcium spiking is generated by Ca2+ release from the intracellular store, it is rather sensitive to ex tracellular Ca 2+ (25, 26). Extracellular Ca 2+ is required to refill the store. Therefore, the decreases in frequency of spiking produced by dilazep and diltiazem are due to their inhibitory actions on Ca2+ influx. From the present observations, it is obvious that the m[Ca2+]i of a group of cells represents the average of a variety of complex s[Ca2+]i events that are observed in individual cells. As mentioned above, when the m[Ca2+]i of a group of cells was monitored, 30 pM dilazep decreased the sustained m[Ca2+]i elevation to a level above the resting level. In contrast, when measur ing s[Ca2+]i in single cells, dilazep decreased the sus tained s[Ca2+]i elevation to the resting level and in some cells elicited the spikelike elevation. It is interest ing that the sustained s[Ca2+]i elevation was decreased to the resting level without staying at a level above the resting state after an application of dilazep. The [Ca 2+]i levels between the resting state and the sustained eleva tion state might be unstable. In fact, all the cells inves
tigated in the presence of dilazep showed the s[Ca2+]; of the resting state, the spikelike elevation, and the sus tained elevation which was unchanged by dilazep in a small population of cells. These results suggest that the [Ca2+]i of the sustained elevation produced by hista mine represents a stable level in the bistable model, which has been proposed for receptor-stimulated cal cium spiking (28, 29). It is also likely that the sustained [Ca2+]; elevation induced by histamine consists of fused spikes (25). So far, Ca 2+ spiking has been observed in many different kinds of eukaryotic cells (29-31) includ ing endothelial cells (25, 26, 32). The frequency of spik ing increases when the concentration of agonist is raised, whereas the amplitude and duration of spikes stay nearly the same. This invariance suggests that spikes are produced by excitation of bistable systems (28, 29), namely the sustained elevated [Ca2+]i level and the resting [Ca 2+]i level in histamine-stimulated en dothelial cells in the present study. REFERENCES
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