TOXICOLOGY AND APPLIED PHARMACOLOGY 107, 5 14-525 ( 199 I)

Differential Effect of Diltiazem and TA-3090 on Calcium Homeostasis of Neutrophils

KENNETH WONG,*‘~$ LILLIAN KWAN-YEUNG,? AND DAVID NC?

Departments of *Medicine and tPharmacolog.v, Universiry oj‘Alberta; and. the *Canadian Red Cross Society. Edmonton, .4lberta. Canada

Received April 24, 1990: accepted November 5. 1990

Differential Effect of Diltiazem and TA-3090 on Calcium Homeostasis of Neutrophils. WONG, K., KWAN-YEUNG, L., AND NG, D. (199 I) Tosicol. Appl. Pharmacol. 107, 5 14-525. The effects of diltiazem and TA-3090, an 8-chloro analog of diltiazem, on cellular responses and calcium homeostasis of human neutrophils were investigated. TA-3090, at 10 to 20 PM, enhanced lysozyme release and superoxide generation induced in neutrophils by n-formyl-methionyl-leucyl-phenyl- alanine (FMLP). Higher concentrations of TA-3090 inhibited responses at IC5Os between 70 and 85 pM. Diltiazem by comparison inhibited responses at an IC50 of about 200 pM. The two drugs had little or no effect on early signaling events; inositol I ,4,5-trisphosphate formation triggered by FMLP was not affected. Moreover, 500 pM TA-3090 or diltiazem did not significantly affect FMLP-triggered Ca2+ transients. (Cytoplasmic free Ca ‘+ levels ([Ca”]J were monitored in fura- 2-loaded neutrophils.) Diltiazem alone caused a limited influx of extracellular Ca2’ which increased basal [Ca2’li by twofold. Internal Ca2+ stores were not released. TA-3090, in contrast, induced a biphasic rise in [Ca2+li-an initial mobilization of intracellular Ca2+ stores was followed after IO- I5 min by a persistent influx of extracellular Ca2+ which increased [Ca’+], to 1.3 f 0.7 (SD) pM.

Complementary studies with semipermeabilized neutrophils showed that TA-3090 but not dil- tiazem directly released Ca’+ from intracellular stores. In TA-3090-treated cells, lactate dehydro- genase release was correlated with delayed influx of extracellular Ca 2f. The chelation of extracellular Ca2’ by EGTA prevented LDH release. Present results show that TA-3090 and diltiazem initially blocked cell signaling at steps subsequent to phospholipase C activity. With TA-3090-treated cells, elevated [Ca2+li ensuing from prolonged incubations likely activated inappropriate reactions leading to cell lysis and death. o 1991 Academic press. hc.

Human neutrophils or polymorphonuclear leukocytes are cellular mediators of the acute inflammatory reaction in viva Bacterial in- fections elicit responses from neutrophils such as chemotaxis, phagocytosis, and secretion of a gamut of autocrine factors. Phagocytosed bacteria in turn are destroyed by a combina- tion of free radicals and a host of bactericidal agents and enzymes released into phagosomes (Baggiolini and Dewald, 1985).

Rapid progress made in signal transduction mechanisms have contributed greatly to pres- ent understanding of the series of intermediary steps intervening between external signals and cellular responses of neutrophils (Nishizuka,

1986; Bet-ridge and Irvine, 1989; Snyder-man and Uhing, 1988). Stimuli such as chemotactic peptides, leukotriene B4, and complement fragments bind to specific receptors on the plasma membrane which are coupled to phos- pholipase C by guanine nucleotide binding (G) proteins (Snyder-man and Uhing, 1988). Phospholipase C in turn catalyzes the break- down of phosphatidyl4,5-bisphosphate to di- acylglycerol and inositol 1,4,%-isphosphate (Ins( 1,4,5)P3).’ The latter two species are sec-

’ Abbreviations used: [Ca*+k, cytoplasmic free calcium concentration; [Ca*+], extracellular Ca*+ concentration; 0;. superoxide anions; FMLP. n-formyl-methionyl-leucyl-

004 I-008X/9 I $3.00 Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

514

INHlLIIIlNCr crrcLI3 ur . . r . . . - y - . . . , - . - - - r , - . *n AT D*LT*AZEM AND TA-3090 515

ond messengers which activate Ca’+/phos- pholipid-dependent protein kinase C (PKC) and mobilize intracellular Ca” stores, respec- tively. Kinase C-mediated protein phosphor- ylation and Ca2+-dependent enzymatic reac- tions separately or synergistically trigger other cascades culminating in physiological re- sponses.

Several studies have reported that calcium channel blockers such as diltiazem, verapamil, and nifedipine inhibited neutrophil responses in vitro (Jouvin-Marche et al., 1983; Steiner et al., 1984; Kazanjian and Pennington, 1985; Elferink and Deierkauf, 1984; Irita et al., 1986). Generally speaking, concentrations of drugs applied in the preceding studies were greater than those required for inhibiting volt- age-operated channels or than that found in plasma. Therefore inhibition of neutrophil functions is probably not a significant effect of Ca2’ channel blockers in patients. However, these drugs may serve as pharmacological tools for elucidating signal transduction mecha- nisms in neutrophils and other cells provided they can be shown to inhibit specific site(s) rather than to effect wholesale changes in cel- lular integrity. Studies to date have not ad- dressed this question. To cast some light on this subject, we have measured and compared the effects of diltiazem and its 8-chloro con- gener, TA-3090, [(+)-(2&3x)-3-acetoxy-8- chloro-5-[(2-dimethylamino)ethyl]-2,3-dihy- dro - 2 - (4 - methoxyphenyl) - 1,5 - benzothi- azepine-4( 5H)one maleate], on physiological responses and second messenger formation in human neutrophils.

studies showed that TA-3090 suppressed Ca2’- induced contraction of isolated canine and monkey arteries at pA, values ranging from 8.34 to 7.36 (Kikkawa et al., 1988). Depending on the type of artery, diltiazem was 2 to 10 times less potent. TA-3090 bound to rat myo- cardial benzothiazepine receptors with a & of 8.8 nM while diltiazem exhibited a & of 38 nrvr (Zobrist and Mecca, 1990). TA-3090 was also more potent than diltiazem in inhibiting Ca2+ inward currents measured in smooth muscle cells of the rabbit mesenteric artery (Xiong et al., 1990). IC50 for TA-3090 was 0.03 PM, and for diltiazem, 0.3 PM.

In the present study, concentrations of dil- tiazem and TA-3090 about one or two orders of magnitude greater than those applied in the preceding experiments inhibited neutrophil responses initiated by the peptide n-formyl- methionly-leucyl-phenylalanine (FMLP). The receptor-G protein-phospholipase C linkage was not a primary target for the benzothiaze- pines since they had little or no effect on early second messenger formation induced by FMLP. Results will also show that the ben- zothiazepines at the same concentration range perturbed the Ca2+ homeostasis of neutrophils. Of interest was the finding that the mechanism and the consequence of action of TA-3090 was different from that mediated by diltiazem.

METHODS AND MATERIALS

TA-3090 is a new benzothiazepine deriva- tive with calcium channel blocking and hy- potensive properties. Basic pharmacological

phenylalanine; Ins( 1,4,5)P3, inositol 1,4,5-trisphosphate; LDH, lactate dehydrogenase: PKC, protein kinase C; EGTA, [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid; MAPTAM [bis-(2-amino-5-methyl-phenoxy)-ethane- N,N.N’,N’-tetraacetic acid tetraacetoxymethyl ester]; Me,SO, dimethyl sulfoxide: HBSS, Hanks’ balanced salt solution.

Materials. TA-3090 was supplied by Nordic Labora- tories, Inc. (Lava& PQ). The following reagents were pur- chased from Sigma Chemical Co. (St. Louis, MO): dilti- azem, N-formyl-methionyl-leucyl-phenylalanine, L-lactic acid, horse heart ferricytochrome C (type VI), cytochalasin B. Ins( I ,4,5)P3 was from Amersham Canada Ltd. (Oak- ville, Ont.). Hanks’ balanced salt solution (HBSS) was purchased from GIBCO (Grand Island, NY). When needed, Caz+-free HBSS was made from standard reagents. Fura-2/AM, the acetoxymethyl ester of fura-2, and MAP- TAM were obtained from Calbiochem (San Diego, CA). Ficoll-Paque was obtained from Pharmacia, Inc. (Dorvall, PQ). All other chemicals used were of reagent grade. FMLP was dissolved in 95% ethanol. Stock solutions of fura-2/ AM, MAPTAM, and TA-3090 were dissolved in Me$O.

516 WONG. KWAN-YEUNG. AND NG

Dilutions of these solutions were such (> 1:200) that carrier vehicle concentrations did not affect assays outlined below.

CeN isolation. Neutrophils were isolated from peripheral blood of human donors by initial separation in Ficoll- Hypaque gradients followed by lysis of contaminating erythrocytes by 0.8% NH,Cl (Wong, 1983). Washed cells were resuspended in HBSS at 1 to 2 X lo6 cells/ml. Cells examined at this stage were composed normally of >95% neutrophils.

Superoxide assay. The 0~ respiratory burst induced in neutrophils (I X lo6 cells/ml, 37°C) by FMLP was assayed by monitoring the reduction of ferricytochrome C at 550 nm as outlined previously (Wong, 1983). The rate of 0: release was calculated by dividing the linear rates of ab- sorbance change by the extinction coefficient, 21 mM-’ cm-‘. Total amount of 0; released by activated neutrophils was calculated by dividing the net absorbance change (the difference between the plateau level of Ass0 attained after 0; formation had ceased and the basal A,,,) by the ex- tinction coefficient.

Degranulution assay. Neutrophils incubated at 37°C were induced to undergo degranulation by stimulation with 0.1-0.2 yM FMLP and 1 pM cytochalasin B. Lysozyme (EC 3.2.1.17) released by activated neutrophils into the extracellular medium was recovered and assayed by mea- suring the rate of lysis of Micrococcus lysodeikticus mon- itored at 450 nm as outlined previously (Wong and Chew, 1985).

Radioimmunoassay ,fbr Ins(1,4S)P3. Neutrophils preincubated IO min with 10 mM LiCl at 37°C before treatment with various agents were fixed by the addition of 5% trichloroacetic acid as outlined previously (Stmad ef al., 1986). Ether-washed extracts were processed and assayed for Ins( 1,4,5)P3 using a RIA kit purchased from Amersham Canada Ltd. This assay is based on the com- petition between unlabeled Ins( I ,4,5)P3 and a fixed quan- tity of tritium-labeled Ins( 1,4,5)P3 for binding sites on bo- vine adrenal binding protein. Maximal cross-reactivity for inositol 1,3,4,5-tetrakisphosphate and inositol 1,3,4-tris- phosphate was reported to be 6.4% and 0.22%. respectively.

Fluorometric measuremenl of [Ca”], As described re- cently in this laboratory (Parente et al., 1989) purified neutrophils (1 X 10’ cells/ml HBSS) were loaded with 1 pM fura-Z/AM (in 0.25% MezSO) for 30 min at 37°C. After washing 2X in HBSS, cells were resuspended at 1 X I O”/ml HBSS at room temperature. Aliquots ( 1 ml) were prewarmed at 37°C in disposable fluorometric cuvettes just before assays. Fura- fluorescence was continuously monitored in a Perkin-Elmer (MKF-4) fluorescence spec- trophotometer with monochromator settings of 339 nm (excitation) and 505 nm (emission). [Ca*‘]i levels were calculated using the formula

Ca*+ = KdF - F,d/(F,., - F),

where F is arbitrary fluorescent units and Kd = 224 nM is the dissociation constant for Ca’+ binding to the fura- in

an intracellular milieu (Grynkiewicz et al.. 1985). For cells suspended in buffer containing micromolar Ca*+, F,, was determined by lysing the cells with 0.1% (v/v) Triton X- 100 and F,,,,. was determined consecutively by adding to lysed cells 20 mM Tris base followed by 2-4 mM EGTA.

Measurement qf Ca’+ release in permeabilized neutro- phils. Neutrophils, 3 X IO7 cells/ml, were permeabilized by incubation with 75 pg/ml saponin at 37°C for approx- imately 30 min. The extent of penneabilization was gauged by uptake of trypan blue by cells. Permeabilized cells were washed then resuspended at 1 X 10’ cells/ml in a low Ca*+ buffer with the following constituents: NaCI, 20 mM; KCI, 100 mM; MgSO.,, 5 mM; NaHCO,, 25 mM; NaH,PO,, 0.96 mM; bovine serum albumin, 0.2%. Prior to Ca” loading, cells were incubated 5 min in 10 pM each of an- timycin A and oligomycin to inhibit Ca*+ uptake by mi- tochondria.

Ca*+ uptake into the endoplasmic reticulum of per- meabilized neutrophils was initiated by the addition of I .5 mM ATP, 10 U/ml creatine kinase, and 5 mM creatine phosphate. (The rate of Ca’+ uptake decreased with pro- longed storage of permeabilized cells at room temperature; hence permeabilized cells were used as expeditiously as possible.) The ambient Ca*’ concentration was monitored by a Ca*+-sensitive minielectrode (CAL-l and TIPCA. from World Precision Instruments, CT) connected to a pH meter and chart recorder. The electrode was calibrated using a series of Ca*+ standard solutions (CAL BUF from WPI). Ca*+ concentrations in EGTA buffers of known composition, ionic strength, and pH were calculated using the EQCAL program obtained from Biosoft (Cam- bridge, UK).

Assay ofcell viability. Cell death and plasma membrane damage were determined by the release of the cytosolic enzyme lactate dehydrogenase, LDH (EC 1.1. I .27). The presence of LDH in supernates recovered from cellular suspensions was assayed by measuring the rate of reduction at 340 nm of nicotinamide adenine dinucleotide as L-lac- tate is oxidized to L-pyruvate (Wacker ef al.. 1956).

RESULTS

Efect of TA-3090 and Diltiazem on Lysozyme and Superoxide Anion Release

Both TA-3090 and diltiazem inhibited the release of lysozyme from neutrophils incu- bated with FMLP and cytochalasin B (Fig. 1). The IC50 for TA-3090 was estimated to be 70 PM, and for diltiazem, 200 PM. Thus TA-3090 was approximately three times more potent than diltiazem in blocking this response. Nei- ther drug alone caused lysozyme release. It was

INHIBITING EFFECTS OF DILTIAZEM AND TA-3090 517

Concentration (JAM)

FIG. I. Inhibition of lysozyme release by TA-3090 and diltiazem. Neutrophils were induced to degranulate and release lysozyme by FMLP and cytochalasin B in the pres- ence of drug or Me$O vehicle (control) as described under Methods and Materials. Lysozyme activity in supernates recovered from TA-3090-treated (0) or diltiazem-treated (0) cell suspensions are expressed as percentage of control values (*SE, mean of four experiments). (*) Lysozyme released in the presence of 10 pM TA-3090 was significantly different from control levels (p < 0.05, by application of the Student paired t test).

noted that 10 PM TA-3090 significantly en- hanced lysozyme release by about 10%.

Similar results were obtained when free- radical formation was measured. Both com- pounds inhibited superoxide (0;) formation

Concentration (pM)

by neutrophils in a dose-dependent manner (Fig. 2A). Scavenging of 0; by drugs was not a factor; control studies showed that reduction of cytochrome C by 0; generated by xan- thine-xanthine oxidase was not affected by up to 500 PM drug. As was the case in the lyso- zyme studies, TA-3090 was the more potent inhibitor; it suppressed the rate of 0; forma- tion at an IC50 of about 85 PM. Lower concentrations of TA-3090 (10 to 20 PM) sig- nificantly potentiated 0; generation by 10 to 40%.

Diltiazem inhibited rates of 0; generation at an IC50 of 200 I.LM (Fig. 2A). Like TA-3090, diltiazem potentiated neutrophil responses at lower concentrations applied in vitro. At 50 PM, diltiazem increased the control (FMLP mediated) rate by 10%. The effect was syner- gistic since 50 I.LM diltiazem alone failed to elicit any superoxide response. In the preced- ing 0: studies, similar results were obtained when the total amounts of 0; released instead of rates of release were calculated.

Further studies were carried out to deter- mine whether the effects of diltiazem and TA- 3090 were due to their blockade of Ca2’ influx. The 0; response induced by FMLP though

- ,, A* l\r B

I

0 1 10 1w Concentration (ph.!)

FIG. 2. Effect of TA-3090 and diltiazem on the rate of superoxide generation. (A) Neutrophils, suspended at 1 X lo6 cells/ml in HBSS, were stimulated with 0.2 fiM PMLP in the presence and absence of the benzothiazepines. Rates of 0; generation measured as outlined under Methods and Materials are calculated as percentage of control rates expressed by neutrophils activated with FMLP and solvent vehicle. Values plotted are the mean (&SE) of four experiments. (0) TA-3090, (0) diltiazem. Specific points indicated by (*) in (A) and (B) are significantly different from control rates of 0, generation (p < 0.05). (B) Parallel studies were carried out using cells suspended in @‘-free HBSS. Points are means (*SE) of three experiments. (0) TA-3090, (0) diltiazem.

518 WONG, KWAN-YEUNG. AND NC

maximal when triggered in the presence of millimolar extracellular Ca*+ is not dependent on the latter (Smolen et al., 1981). In agree- ment with past observations, the 0; response elicited by FMLP in neutrophils suspended in Ca2+-free medium was 50 to 60% of responses measured in millimolar Ca2+. We surmised that if the benzothiazepines were inhibiting neutrophil responses primarily by blocking Ca” entry, they would have little or no effect on 0; generation induced by FMLP in Ca*+- free medium. Results of this study (Fig. 2B) showed that diltiazem and TA-3090 main- tained their relative potencies as inhibitors. As was found with studies carried out in normal HBSS, lower concentrations of drugs en- hanced the 0; response induced by FMLP.

In the present study, further experiments focused on the inhibitory (250 PM) concen- trations of TA-3090 and diltiazem. Results are as follows.

Efect of TA-3090 on Inositol Trisphosphate Generation

Ins( 1,4,5)P3 levels in neutrophils were measured by a radioimmunoassay in order to assess the effect of the benzothiazepines on the early signal transduction steps. Results de- picted in Fig. 3 show that FMLP triggered a rapid and transient elevation of Ins( 1,4,5)P3 in agreement with previous reports (Dillon et al., 1988). TA-3090 or diltiazem alone (up to 500 PM) failed to elicit significant formation of Ins( 1,4,5)P3 in neutrophils. A concentration of TA-3090 (500 PM) which totally blocked neutrophil responses was tested for its effect on the generation of Ins( 1,4,5)P3 in FMLP- activated cells. Given the small number of ex- periments, significant differences in the level of Ins( 1,4,5)P3 in cells treated with FMLP or with the combination of FMLP and TA-3090 could not be demonstrated. It appears that diltiazem also did not greatly influence Ins( 1,4,5)P3 formation triggered by FMLP.

FIG. 3. Effect of TA-3090 and diltiazem on Ins( I ,4,5)P3 generation in neutrophils. TA-3090, diltiazem, FMLP, or combinations of agents were added to aliquots of neutro- phils (2 ml, 40 X IO6 cells/ml HBSS, 37°C); aliquots, 0.5 ml, were removed at times indicated and inactivated with TCA as outlined under Methods and Materials. Ins( I ,4,5)P3 in extracts was quantified by radioimmu- noassay. (0) resting cells. Cells incubated with 1 pM FMLP (0): 500 pM TA-3090 (A); FMLP and TA-3090, same concentrations (A); 500 NM diltiazem (m); diltiazem and FMLP (Cl). Results for diltiazem studies are the average of two experiments. All other points represent the mean (*SE) of three experiments.

Efict of TA-3090 and Diltiazem on [Ca”li of Neutrophils

In agreement with previous reports, FMLP induced a biphasic rise in [Ca2+]i (Fig. 4, traces a and d) (Andersson et al., 1986; Korchak et al.. 1988; Merritt et al., 1989). In the first min, [Ca’+]i was transiently elevated to 0.8 to 1 PM, peaking at lo-20 set; a slower rate of decline in [Ca’+]i was succeeded by a secondary rise which maintained [Ca*+]i at 0.15 to 0.3 PM for 3-4 min. The second but not the first Ca*+ transient was eliminated by the removal of ex- tracellular Ca2+ by EGTA (cf. trace a with a’, Fig. 4), thus indicating that the second plateau corresponded to an influx of extracellular Ca2+. Similar results were obtained when cells were suspended in Ca’+-free HBSS and chal- lenged with FMLP.

The initial addition of 50-500 FM TA-3090 to fura-a-loaded neutrophils produced a small, persistent elevation of [Ca”]i from about 0.1

INHIBITING EFFECTS OF DILTIAZEM AND TA-3090 519

FIG. 4. Influence of TA-3090 and dihiazem on the [Ca*+], of neutrophils cotreated with FMLP. Cells loaded with fura- as outlined under Methods and Materials were treated with TA-3090 or diltiazem, and FMLP in the order at times indicated by arrows. Each tracing is designated by a lowercase letter. The number beside each drug label indicates the final concentration in pM. In trace a’ (overlaid on trace a), EGTA (2 mM final concentration) was added to the neutrophil suspension just before FMLP. All traces shown were made using cells from a single preparation of isolated neutrophils. Results are representative of three experiments. TA, TA-3090; DIL, diltiazem.

to 0.2 j&M (this is described further below). At 500 PM TA-3090, this effect was maximal. Subsequent addition of FMLP to cells pro- duced a slightly sharper Ca2+ transient com- pared to that of the control (Fig. 4, traces b and c). The second phase of [Ca2+]i elevation followed a time course similar to that in the control trace but peak values (co.2 PM) did not exceed levels attained in FMLP-only con- trols. It appears that the combined effects of TA-3090 and FMLP were not additive with respect to the secondary plateau of [Ca2+]i.

Diltiazem, 50-500 PM, similarly raised [Ca*+]i from about 0.1 PM to <K! PM. The addition of FMLP to diltiazem-pretreated cells also resulted in more transient rises in [Ca2+]i (Fig. 4, cf. traces e and f with trace d). Like the results obtained in TA-3090 studies, the combined effects of diltiazem and FMLP were nonadditive with respect to the secondary pla- teau of heightened [Ca2+]i.

Figure 5 depicts the effect on [Ca2+]i of pro- longed incubation with TA-3090 or diltiazem

alone. TA-3090 caused a biphasic elevation of [Ca2’]i in cells (Fig. 5, trace a). As Fig. 4 already showed, 50-500 /IM TA-3090 induced a mod- est rise in [Ca2+]i. Basal [Ca2+]i of 0.09 f 0.03 /IM (SD, mean of 11 experiments) was elevated to 0.20 + 0.06 I.LM at peak values attained after 30 sec. The plateau level of [Ca2’]i maintained for about 10 min (some traces showed a slight decline) was followed by an accelerated rise in [Ca2+]i. The second plateau corresponded to [Ca’+]i of 1.33 f 0.69 PM (SD, N = 11). Re- moval of extracellular Ca2+ by EGTA elimi- nated the secondary but not the initial rise in [Ca2+]i (Fig. 5, trace b). These findings indicate that TA-3090 induced an initial release of in- ternal Ca2+ stores followed by an influx of ex- tracellular Ca2+.

In contrast, 500 NM diltiazem maximally increased [Ca’+]i in neutrophils from a basal value of 0.10 + 0.03 PM (SD, mean of five experiments) to 0.17 ? 0.04 PM in the first 2 to 3 min. The initial 70% increase in [Ca2+]i was maintained over 20 min (Fig. 5, trace c). No rise in [Ca2+li occurred when [Ca2’], was reduced to about 0.04 FM by the addition of

FIG. 5. Elevation of [Ca2+li in neutrophils subjected to prolonged treatment with TA-3090 and diltiazem. Changes in [Caz’li were followed as in Fig. 4. TA-3090 (500 FM). trace a, or diltiazem (500 yM), trace c, were added to SW pensions at arrows. In the reaction mixtures represented by traces b and d, 3 mM EGTA was added to cell suspen- sions before TA-3090 or diltiazem. Individual traces made consecutively have been overlaid and slightly displaced vertically for comparison.

520 WONG. KWAN-YEUNG. AND NG

3 mM EGTA (Fig. 5, trace d). This suggests that [Ca2+]i was elevated by the entry of ex- tracellular Ca2+. The increase in the fluores- cent signal was not due to leakage of fura- from cells because EGTA added to suspen- sions after the drug-induced Ca2+ rise had no effect. By reducing [Ca2’], from millimolar to co.04 PM, EGTA would have caused disso- ciation of any extracellular fura-2-Ca2+ com- plexes and a reduction of the signal strength. Increases in fluorescence from direct interac- tion of diltiazem or TA-3090 with fura- can also be discounted. As evidence, cell-free combination of fura- with drugs in low Ca2+ medium produced no changes in signal strength.

The time course of [Ca2+]i elevation was de- pendent on TA-3090 concentration (Fig. 6, traces d, e, and f). At 50-500 PM, TA-3090 induced small increments in [Ca2+]i in the ini- tial phase (Fig. 4). Concentration-dependent effects were more evident in the second phase. With increasing TA-3090 concentrations, the lag preceding the second rise in [Ca2+]i de- creased and the rate and extent of the rise in- creased.

The effect of Ca2+ buffering was ascertained by using the nonfluorescent, membrane per- meant, Ca2+ chelator MAPTAM (Lew et al., 1982). By loading neutrophils with increasing proportions of MAPTAM relative to fura-2, increases in [Ca2+]i as reported by fura- were effectively buffered. As results show, the pres- ence of MAPTAM dampened proportionately changes in [Ca2+]i produced by 500 ELM TA- 3090 (Fig. 6, cf. traces b and c with trace a). However, the onset of the secondary influx of extracellular Ca2+ appeared not to change.

TA-3090 Released Ca2+ from Intracellular Stores

Previous studies have shown that intracel- lular Ca2+ storage compartments remain intact in neutrophils and other tissues permeabilized with detergents (Berridge and Irvine, 1989). It

FIG. 6. Dose-dependent effect of TA-3090 on [Ca2+li and the effect of buffering on the rise in [Ca*‘]i. Studies were carried out as in Figs. 4 and 5. Traces are reproduced from the original. Trace a: neutrophils were incubated with 500 NM TA-3090; trace b: cells were preloaded for 30 min with 10 pM MAPTAM and 1 pM fura-2/AM at 37°C washed, and resuspended in fresh HBSS before treatment with 500 pM TA-3090, trace c: same as the preceding except that neutrophils were incubated for 30 min at 37°C in the presence of 100 pM MAFTAM and 1 pM fura-Z/AM, traces d, e, and f: neutrophils were treated with 500, 300, and 100 pM TA-3090, respectively; trace d: (----) is the same as trace a.

was with such systems that Ins( 1,4,5)P3 was shown to mobilize internal Ca2+ stores. Using this approach, we asked the question whether diltiazem or TA-3090 can directly release Ca2+ from such stores. Results illustrated in Fig. 7 show that TA-3090 released sequestered Ca2+ in permeabilized neutrophils in a dose-depen- dent manner. Controls showed that the Me2S0 vehicle was without effect. In contrast, similar concentrations of diltiazem (300 and 500 PM) induced little or no Ca2+ release. As a positive control Ins( 1,4,5)P3 triggered a rapid but par- tial release of sequestered Ca*+ in agreement with earlier studies (Prentki et al., 1984). As expected, the calcium ionophore, ionomycin, mediated total release of sequestered calcium.

Cytotoxicity of TA-3090 and Diltiazem

Potential cytotoxicity of benzothiazepines on neutrophils was assayed by measuring the

INHIBITING EFFECTS OF DILTIAZEM AND TA-3090 521

L_) 5 mm

FIG. 7. Effect of TA-3090 and diltiazem on Ca*’ stores in permeabilized neutrophils. Neutrophils were permeabilized by saponin and resuspended in KC1 buffer as outlined under Methods and Materials. Ambient Ca” levels were recorded over time using a Ca*+-sensitive electrode. Four separate traces are shown, each starting with the addition of 1.5 mM ATP to activate Ca*+ sequestration. Other agents were added at times shown. TA, TA-3090; DIL, diltiazem (100 or 300 pM was added); ION, ionomycin (1 PM); IP3, Ins( 1,4,5)P3 (I 0 FM). Gaps in traces occurred when the cuvette compartment was opened for the addition of reagents. For continuity, gaps have been filled in with dotted lines.

release of the cytosolic enzyme LDH into the medium (Fig. 8). The results show that over 20 min, 300 and 500 PM diltiazem had only small effects on cell viability as about 5% of total LDH was released. In contrast, similar concentrations of TA-3090 were substantially more toxic. Cell death, though, became sig- nificant only after more than 10 min of in- cubation with the drug, a point coinciding with accelerated Ca*+ influx (Fig. 5, trace a). In the inset to Fig. 8, the LDH release results for one set of cells was plotted against [Ca*+]i data ob- tained from corresponding fura- traces of the same cells. If all points are subjected to linear regression analysis, the correlation is modest (r = 0.91, this regression line is not shown in the inset). If applied only to results obtained from incubation times >lO min (when per- centage LDH release exceeded 20%), the cor- relation between LDH release and [Ca*+]i was robust (r = 0.99 as shown). A cause and effect relationship was more directly shown by the contemporary finding that LDH release was blocked in TA-3090-treated neutrophils by the addition of 3 mM EGTA. The latter effectively decreased the Ca*’ concentration in the me- dium to about 0.04 j.&M and eliminated the

chemical gradient for Ca” and thus Ca*+ in- flux.

DISCUSSION

Present results have shown that TA-3090 and diltiazem produced multiple effects on neutrophil responses and physiology. Dilti- azem and especially TA-3090 exerted a bi- phasic effect on 0; generation and lysozyme release. Lower concentrations of drugs poten- tiated responses induced by FMLP while higher concentrations inhibited these re- sponses (Figs, 1 and 2). The potentiation of responses occurring at lo-20 PM TA-3090 or 50 PM diltiazem was not characterized further in this study.

At concentrations > 50 I.LM, both TA-3090 and diltiazem inhibited neutrophil responses with TA-3090 being the more potent inhibitor. The IC50 of 200 I.LM observed for diltiazem inhibition of 0; generation was lower than the IC50 of 450 j.&M reported by Irita et al. (1986). The difference may reside in Irita et al.‘s use of phorbol my&ate acetate rather than FMLP to stimulate neutrophils. In con-

522 WONG, KWAN-YEUNG, AND NC

I . _1 0 5 10 15 20

Mln incubation

FIG. 8. LDH release in TA-3090/diltiazem-treated neu- trophils. Neutrophils (2 ml, at 2 X IO6 cells/ml HBSS, 37°C) were treated with reagents indicated: OS-ml aliquots were removed at times shown and immediately centrifuged for i min in an Eppendorf microfuge. Collected supemates were assayed for LDH activity as described under Methods and Materials. Results were calculated as percentage of total activity released from resting cells sonicated with three IO-set bursts at 60 W (Brausonic 15 IO sonicator). Cells were incubated with 300 pM TA-3090 (0). 500 pM TA- 3090 (O), 2 mM EGTA and 500 PM TA-3090 (0) 300 pM diltiazem (A), and 500 pM diltiazem (A). Results are the average of two separate experiments except for 500 yM TA-3090-treated cells where points represent the means (*SE) of three experiments. In one of the latter experi- ments. fura-2-loaded neutrophils were treated with TA- 3090: the LDH activity of one aliquot was measured and the changes in fluorescence of another aliquot were fol- lowed for 20 min. The percentage of LDH released and [Ca2+li values corresponding to the same incubation time were plotted as shown in the inset. Linear regression anal- ysis ofthe right-most four points yielded a correlation coef- ficient of 0.99.

firmation of previous reports, the inhibitory concentrations of these agents were much greater than those needed to block voltage- sensitive Ca2+ channels (Irita et al.. 1986; Schwartz et al., 1988; Xiong et al., 1990) or to antagonize Ca*+-dependent muscle con- traction (Kikkawa et al., 1988). The finding that neutrophil responses induced in the ab- sence of extracellular Ca*+ were inhibited by the two benzothiazepines argues against Ca*’ channel blockade as the mechanism of action. Moreover, the possible involvement of voltage sensitive Ca*+ channels in the action of the benzothiazepine is rendered moot by studies showing the absence of such channels in neu-

trophils (Andersson et al.. 1986; Von Tschar- ner et al., 1986; Berger and Birx, 1988).

At 50 to 500 PM, TA-3090 and diltiazem, in the short term (incubation periods < 10 min), compromised selective systems in neu- trophils. Both agents had little or no effect on the early signal transduction steps involving the binding of FMLP and the activation of phospholipase C. As dose-response results (Figs. 1 and 2) show, 500 FM of either drug totally inhibited 0~ production and enzyme release but modulated the FMLP-induced Ca*+ transients subtly. It appears that in the presence of TA-3090 or diltiazem, the decline of [Ca*‘]i after peaking was more rapid, re- sulting in sharper transients. The finding that the benzothiazepines did not significantly in- hibit Ins( 1,4,5)P3 formation in FMLP-stim- ulated cells corroborated the Ca2+ studies. Drug inhibition of the G-protein coupling re- ceptors to phospholipase C can be discounted as noticeable suppression of Ca2+ transients and Ins( 1,4,5)P3 formation would have been seen. The evidence suggests that the initial site(s) of inhibition for both TA-3090 and dil- tiazem involved signaling steps which follow phospholipase C activity. For example, Ca*+- dependent protein kinase C or calmodulin- dependent reactions might be blocked by the drugs.

The experiments on the Ca*+ homeostasis of neutrophils revealed more pharmacological differences between TA-3090 and diltiazem. As results show (Figs. 4-6) diltiazem per se produced a limited elevation of [Ca*+]i by me- diating entry of extracellular Ca*+. The per- sistence of the new steady-state level of [Ca*+]i suggests that diltiazem had altered the con- ditions of equilibrium. Enhanced rates of dif- fusion of extracellular Ca*+ into cells and/or depressed rates of Ca*+ extrusion might be consequences of drug action. However, the basis for diltiazem action on [Ca’+]i awaits further studies.

In contrast, TA-3090 induced a biphasic re- sponse-mobilization of internal Ca2+ was followed by a sustained influx of extracellular

INHIBITING EFFECTS OF DILTIAZEM AND TA-3090 523

Ca*+. Chemically TA-3090 is more lipophilic than diltiazem. This property may allow TA- 3090 to cross the plasma membrane to interact with intracellular target sites insensitive to dil- tiazem. In line with this theory was the finding that TA-3090 but not diltiazem directly re- leased Ca*+ sequestered in microsomal frac- tions in semipermeabilized neutrophils (Fig. 7).

Differences in drug-mediated perturbation of [Ca*‘]i were manifested in cytotoxicity as- says. As shown, the time course of LDH release in TA-3090-treated cells was correlated with the secondary influx of extracellular Ca*’ and sustained elevation of [Ca*+]i (Fig. 8). The suppression of LDH release by EGTA indi- cates that cell killing was dependent on mas- sive entry of extracellular Ca*+. Nonspecific permeabilization of the plasma membrane by TA-3090 was not likely since this conjecture predicts that LDH release should be insensitive to extracellular Ca*’ levels. According to Boobis et al. ( 1989) and Orrenius et al. (1989), sustained elevation of [Ca*+]i catalyzes inap- propriate activation of proteases, phospholi- pases, endonucleases, and breakdown of the cytoskeleton. The culmination of their effects is the breakdown of cellular structure and cell death. The extent of the rise in [Ca*+]i appears to be a critical parameter since diltiazem, which induced a modest but sustained increase in [Ca*+]i, was substantially less toxic.

The effect of TA-3090 on [Ca*+]i raised in- teresting questions concerning the regulation of this cation. Two conjectures can be ad- vanced for the biphasic nature of increases in [Ca2+]i: (a) the initial release of internal Ca*+ directly or indirectly regulated the entry of ex- tracellular Ca*+, or (b) TA-3090 acted directly on the plasma membrane to trigger a delayed Ca*+ influx.

The first model implies the existence of sec- ond messenger-activated Ca*+ channels in the plasma membrane of neutrophils. Previously Von Tscharner and co-workers (1986) dem- onstrated the presence of Ca*+-activated cation channels from patch clamping studies of neu-

trophil membranes. However, studies with in- tact neutrophils showed a lack of correlation between [Ca*‘]i and the opening of membrane cation channels (Andersson et al., 1986; Kor- chak et al., 1988; Nasmith and Grinstein, 1987). Present results also did not support a Ca*+-activated mechanism. As evidence, both TA-3090 and diltiazem initially increased [Ca*+]i to comparable levels, but a secondary influx of Ca*+ occurred only in TA-3090- treated cells (Fig. 5). In a different approach, dampening of the initial rise in [Ca*+]i by MAPTAM was found not to deter or delay the onset of the secondary influx of Ca*+ (Fig. 6).

Other possible second messengers are Ins( 1,4,5)P3 and its metabolites. Recently Ir- vine and Moor (1987) proposed that Ins( 1,4,5)P3 and inositol 1,3,4,5-tetrakisk- phosphate may catalyze Ca*+ influx in nonex- citable cells. In this study, it is unlikely that TA-3090 acted via inositol phosphates since this reagent did not induce the formation of Ins( 1,4,5)P3, the precursor of inositol 1,3,4,5- tetrakiskphosphate (Fig. 3).

A cause and effect relationship may exist between the two phases of [Ca2’]i elevation. Putney (1986) has postulated that the emp- tying of intracellular Ca2’ stores might furnish a signal for channel opening and the refilling of stores with extracellular Ca*+. He also pro- posed that extracellular Ca*+ enters into in- ternal pools via a pathway which bypasses the cytoplasm. The latter idea is not supported by recent evidence (Taylor, 1990). However, the concept that mobilization of intracellular Ca*’ regulates entry of extracellular Ca*+ still per- tains. On the basis of this hypothesis, TA- 3090-mediated, persistent release of Ca*+ from internal stores resulted in persistent influx of extracellular Ca*+. Unregulated influx of Ca*+ did not occur in diltiazem-treated cells since internal Ca*+ was not released. The preceding models notwithstanding, a simpler possibility that TA-3090 may directly open membrane- bound cation channels exists. The biochemical

524 WONG, KWAN-YEUNG. AND NG

rationale for TA-3090 effects awaits further clarification and study.

ACKNOWLEDGMENTS

This work was supported by research grants from the Medical Research Council of Canada and Nordic Labo- ratories. The authors thank Dr. Denis Garceau of Nordic Laboratories for his consultation.

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