Immunopharmacology, 25 (1993) 75-82 © 1993 Elsevier Science Publishers B.V. All rights reserved 0162-3109/93/$06.00 75

IMPHAR 00615

Calcium channel blockers inhibit cellular uptake of thymidine, uridine and leucine: the incorporation of these molecules into DNA, R N A and protein in the presence of calcium channel

blockers is not a valid measure of lymphocyte activation

Matthew R. Weir a, Richard Peppler a, Diana Gomolka a and Barry S. Handwerger b'c

Divisions of a Nephrology, and b Rheumatology and Clinical Immunology, Department of Medicine, University of Maryland Hospital, Baltimore, MD, USA, and the C Medical Service, Veterans Administration Medical Center, Baltimore, MD, USA

(Received 7 March 1992; accepted 16 July 1992)

Abstract: An important role of transmembrane flux of calcium in lymphocyte activation has been previously demonstrated. Herein, we demonstrate that the calcium channel blockers verapamil and isradipine are able to inhibit in a concentration- dependent manner 3H-thymidine incorporation into DNA in phytohemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). However, verapamil and isradipine diminish PHA-stimulated thymidine incorporation into DNA to the same extent whether they are added at the beginning of culture or 4 h prior to completion of a 72-h culture. Thus, 3H-thymidine incorporation into DNA in the presence of verapamil or isradipine is not a valid measure of mitogen-induced lymphocyte proliferation. Similarly, verapamil and isradipine also inhibit PHA-stimulated incorporation of 3H-leucine into protein and 3H-uridine into RNA whether the drugs are added at the beginning of culture or 4 h prior to completion of 24-h cultures. There is no intracellular accumulation of 3H-thymidine, 3H-leucine, or 3H-uridine into 10% trichloroacetic acid-soluble molecules during inhibition with verapamil or isradipine, suggesting that these drugs impair the cellular uptake of these substances rather than directly inhibiting their incorporation into DNA, protein, or RNA, respectively. Since previous reports documenting the inhibitory effects of calcium channel blockers on lymphocyte proliferation have utilized 3H-thymidine incorporation into DNA to measure proliferation, we have re-examined the antiproliferative effects of these drugs by determining their effect on PHA-stimulated cell cycle progression, employing cytofluorometric analysis of propidium iodide-stained cells. When added at the initiation of culture, both verapamil and isradipine inhibited in a concentration-dependent manner PHA-stimulated cell cycle progression. Thus, both verapamil and isradipine are capable of inhibiting PHA-induced proliferation of human lymphocytes.

Key words: Verapamil; Isradipine; Thymidine; Proliferation

Correspondence to: M.R. Weir, Renal Division, University of Maryland Hospital, 22 S. Greene Street, Baltimore, MD 21201, USA.

Presented in part at the 9th Annual Meeting of the American Society of Transplant Physicians, May 1990, Chi- cago, IL, and the 54th Annual Meeting of the American College of Rheumatology, October, 1990, Seattle, WA, USA.

Introduction

Increased intracellular calcium has been demon- strated to be critical in lymphocyte activation (Whitney and Sutherland, 1972; Parker, 1974; Freedman et al., 1975; Diamanstein and Ulmer, 1976; Greene and Parker, 1976; Freedman, 1979; Dubois and Crupton, 1980). The relative

76

importance of intracellular shifts in calcium pools versus transmembrane flux has not been totally defined. However, the culture of lymphocytes in calcium-free medium clearly inhibits mitogen- and antigen-induced lymphocyte transformation (Whitney and Sutherland, 1972; Parker, 1974; Freedman et al., 1975; Diamanstein and Ulmer, 1976; Greene and Parker, 1976; Freedman, 1979; Dubois and Crupton, 1980), supporting a critical role of transmembrane calcium flux in lymphocyte activation.

Since calcium channel blockers inhibit calcium ion influx through passive, slow calcium channels (Antman et al., 1980) several laboratories have assessed their effect on lymphocyte activation. Previous studies have demonstrated that vera- pamil and other calcium channel blockers inhibit in vitro immunologic responses of PBMC to various mitogens and antigens (Birx et al., 1984; Grier and Mastro, 1985; McMillen et al., 1985; Wright etal., 1985; Dugas etal., 1986; Weir et al., 1987). These studies have consistently used 3H-thymidine incorporation into DNA as a mea- sure of lymphocyte activation.

In this report, we demonstrate that 3H- thymidine incorporation into DNA is not a valid assay for the measurement of the effects of calcium channel blockers on mitogen-stimulated lymphocyte proliferation, since calcium channel blockers inhibit the cellular uptake of 3H- thymidine. Similarly, 3H-leucine incorporation into protein and 3H-uridine incorporation into RNA are not valid measures of mitogen-induced protein or RNA synthesis in the presence of calcium channel blockers. However, calcium channel blockers are capable of inhibiting mitogen-stimulated lymphocyte activation as measured by analysis of cell cycle progression. Thus, calcium channel blockers do have antipro- liferative effects.

Materials and Methods

Drugs Verapamil (G.D. Searle and Co., Skokie, IL) and isradipine (Sandoz Pharmaceuticals,

E. Hanover, NJ) were diluted in RPMI 1640 to form stock solutions.

Preparation of PBMC and cell culture conditions PBMC were isolated from heparinized (preser- vative-free) venous blood of healthy adult blood donors by Ficoll-Hypaque gradient centrifuga- tion (Boyum, 1968). All cultures were performed at 37 °C in a humidified atmosphere containing 5 To CO2 - 95 ~o air. Unless otherwise stated, the cells were cultured in RPMI 1640 supplemented with 10~, heat-inactivated (56~o C for 30 rain) fetal calf serum (FCS), 2 mM glutamine, and 100 #g/ml penicillin and 100 #g/ml streptomycin (complete culture medium).

Mitogen-stimulated DNA synthesis' PBMC (1 x 105/well) were cultured in flat- bottomed 96-well microtiter plates (Costar, Cam- bridge, MA) in the presence or absence of 1.0 #g/ml PHA. The concentration of PHA was shown in preliminary experiments to be optimal. Verapamil, isradipine, or control medium was added to the cultures either at the initiation of culture (just prior to the addition of the mitogen) or 67.75 h after initiation of culture. The cultures were incubated for 72 h; during the last 4 h of culture, 0.5 #Ci of 3H-thymidine (6.7 Ci/mmol, New England Nuclear, Boston, MA) was added to each microtiter well. The cells were then harvested using a multiple channel automated cell harvester (PHD Cell Harvester, Cambridge Technology Inc., Cambridge, MA). Radioactivity incorporated into DNA was quantitated using an automated scintillation counter (Searle Mark III, Searle Laboratories, Skokie, IL).

Mitogen-stimulated protein and RNA synthesis PBMC (1 x 10S/well) were suspended in either leucine-free or uridine-free RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10~o FCS, which had been dialyzed against phos- phate-buffered saline (PBS), 2 mM glutamine, 100 U/ml penicillin, and 100 #g/ml streptomycin. Responding lymphocytes (1 x l0 s per well) were cultured in flat-bottomed 96-well microtiter

plates (Costar, Cambridge, MA) in the presence or absence of 1.0 #g/ml PHA. The concentration of stimulus was shown in preliminary experi- ments to be optimal. Verapamil, isradipine, or control medium was added to the cultures either at the initiation of culture (just prior to the addition of the mitogen) or 19.75 h after initiation of culture. The cultures were incubated for a total of 24 h; during the last 4 h of culture, 0.5 gCi of 3H-leucine (69 Ci/mmol, Amersham, Arlington Heights, IL) prepared in leucine-free media, or 0.5/~Ci of 3H-uridine (30 Ci/mmol, Amersham Arlington Heights, IL) prepared in uridine-free media was added to each microtiter well. The cells were then harvested using a multiple- channel automated cell harvester and radioactiv- ity incorporated into protein or RNA, respec- tively, was quantitated using an automated scintillation counter.

Cell viabifity PBMC (1 x 106) were cultured with 1.0 #g/ml PHA and verapamil, isradipine, or control me- dium for 24, 48, or 72 h. Cell viability was then determined by suspension of the cells in isotonic propidium iodide (Sigma, St. Louis, MO) solu- tion, with flow cytoflurometric assessment (FACS IV, Becton Dickinson, Oxnard, CA) of cells staining positively indicating cell death (Krishan, 1975).

Incorporation of 3 H-thymidine, 3 H-leucine, 3 H- uridine into trichloroacetic acid soluble and insoluble molecules PBMC (1 x 106) were cultured for 72 h in com- plete medium in 12 x 75 mm tissue culture tubes with 1.0 gg/ml PHA. Verapamil, isradipine, or control medium was added after 67.75 h of cul- ture. During the final 4 h of culture, the cells were pulsed with 0.5 gCi well of 3H-thymidine. PBMC were also cultured in either leucine-free or uridine-free culture medium for 24h with 1.0 #g/ml PHA. Verapamil, isradipine, or control medium were added after 19.75 h of culture. During the final 4 h of culture, the cells were pulsed with 0.5 gCi/well of 3H-leucine or 3H-

77

uridine, respectively, for 4 h. At the termination of culture, the cells were washed in PBS to remove any extracellular radiolabel and resus- pended in 1 cc of 10~o trichloroacetic acid (TCA). The cells were centrifuged and the super- natant and the pellet saved, prior to the pellet being resuspended in 10~o TCA and recentri- fuged. The second supernatant was combined with first and counted in a scintillation counter to assess the presence in the cells of 3H-thymidine, 3H-leucine, or 3H-uridine which had not been incorporated into macromolecules. The 10~o TCA pellet was counted to assess the incorpora- tion of the radiolabeled precursor molecules into DNA, protein, and RNA, respectively.

Mitogen-stimulated cell cycle progression PBMC were cultured for 72 h in 12 x 75 mm tissue culture tubes (Falcon, Oxnard, CA) in the presence or absence of 1.0 #g/ml PHA. Vera- pamil, isradipine, or control medium was added to the cultures just prior to the addition of PHA. After completion of incubation, the cells were washed and resuspended in a hypotonic propid- ium iodide solution (50 #g/ml in 0.1% sodium citrate). Assessment of the DNA content in the cells was carried out by flow cytofluorometry on a FACS IV (Becton-Dickinson, Oxnard, CA) to determine the percentage of cells in G1, S, and G2 + M (Krishan, 1975).

Statistical analysis All assays were performed in triplicate or qua- druplicate. Paired t-tests were used to compare the effects of specific concentrations of verapamil or isradipine with control. Statistical significance was defined as a p-value of < 0.05.

Results

In order to investigate the mechanisms underly- ing the immunosuppressive activity of verapamil and isradipine, we first confirmed previously published observations (Birx et al., 1984; Grier and Mastro, 1985; McMillen et al., 1985; Weir

78

et al., 1987) that calcium channel blockers inhibit the increase in 3H-thymidine incorporation into DNA induced by PHA stimulation of human PBMC. The subsequent experiments were de- signed to develop an understanding of the bio- logical mechanisms involved.

Inhibition of PHA-stimulated 3H-thymidine uptake into PBMC As depicted in Table I, verapamil (0.5-50 #M) and isradipine (0.5-50 #M) consistently inhib- ited in a concentration-dependent manner the increase in 3H-thymidine incorporation into DNA of PHA-stimulated PBMC. However, PHA-stimulated 3H-thymidine incorporation into DNA was inhibited to the same degree whether the drug was added at initiation of culture or just 4 h and 15 rain prior to completion of 72 h of culture, indicating that 3H-thymidine incorporation into DNA is not a valid assay for measuring the effects of these calcium channel blockers on mitogen-stimulated lymphocyte pro- liferation.

The observed inhibition of 3H-thymidine in- corporation into DNA may have resulted from either a decrease in the cellular uptake of 3H- thymidine or a block in the actual incorporation of 3H-thymidine into DNA. To address this issue, we stimulated PBMC with PHA for 72 h; verapamil and isradipine was added 4 h and 15 min prior to the termination of culture. During the last 4 h of culture we also pulsed the cells with 3H-thymidine. Radioactivity incorporated into DNA was then separated from unincorporated 3H-thymidine by precipitation of DNA with 10~o trichloroacetic acid. As shown in Table II, vera- pamil inhibited the accumulation of 3H- thymidine into the TCA-insoluble fraction with- out any concomitant increase in radiolabel in the TCA-soluble fraction. Similar results were ob- served when isradipine was added 4 h, 15 min prior to the completion of culture (data not shown). These data strongly suggest that vera- pamil and isradipine inhibit the uptake of 3H- thymidine into the cell.

T A B L E I

Inhibition of PHA-s t imula ted thymidine, leucine, and uridine incorporat ion into D N A , protein, and R N A

Verapamil (/~M) added at initiation of culture 0 0.5 5 50

Thymidine 0 a'b 7.0 -+ 3.1 15.6 _+ 3.0 c 54.8 + 4.7 ~ Leucine 0 d 0.6 _+ 0.3 5.8 _+ 3.6 32.9 _+ 3.3 c

Uridine 0 e 7.4 _+ 3.4 20.7 _+ 3.6 c 41.0 + 5.2 ~

Verapamil (pM) added 4 h, 15 m prior to harves t f

0 0.5 5 50

Thymidine 0 n 8.3 + 3.3 15.2 _+ 4.1 c 50.5 + 6.7 ~" Leu c in e 0 d 3.2-+ 1.8 3.5_+ 1.8 19.7+_6.1"

Uridine 0 ° 0.9 _+ 0.4 12.7 -+ 2.6 ~ 36.2 -+ 2.4 c

Isradipine (ktM) added at initiation o f culture 0 0.5 5 50

Thymidine 0 ~'u 11 .4+4.1 17.6 + 4.1 c

Leucine 0 d 2 .4+ 1.1 8 . 9 + 2 . 4 Uridine 0 r 8.1 _+3.0 2 1 . 4 + 2 . 2 c

Isradipine ( g M ) added 4 h 15 min harves t r 0 0.5 5

68.6_+5.1"

40.4 + 3.8 "~ 54.4 + 3.9 c

prior to

50

Thymidine 0 b 10.6 + 3.1 19.4 + 4.2 c 64.4 _+ 5.6 ~ Leucine 0 d 1.8 _+ 0.9 9.6 + 3.3 34.4 _+ 4.6 c Uridine 0 c 5.2_+2.4 21.1 +2 .4 c 39 .2+4 .8 c

a The data are expressed as the m e a n percent inhibition _+ S E M of 3H-thymidine, 3H-leucine or 3H-uridine incor-

porat ion into D N A , protein, or RNA, respectively, for three independent experiments .

b Range of baseline (no drug) cpm: 54086_+ 6677-

111740+ 10142. The inhibitory capability of drug is statistically greater

(p < 0.05) than control (no drug). d Range o f baseline (no drug) cpm: 4 0 1 4 7 + 4 2 6 4 -

163475 + 10984. Range o f baseline (no drug) cpm: 1 1 9 6 7 + 8 9 7 - 24586 _+ 2174.

f Drug was added to the cultures 15 min prior to the 3H-thymidine, 3H-leucine, 3H-uridine.

Inhibition of cellular uptake of 3H-leucine and 3 H-uridine into PHA-stimulated PBMC Subsequent studies addressed whether the verapamil- and isradipine-mediated inhibition of

TABLE II

Percent inhibition by verapamil of PHA-induced accumula- tion of thymidine, leucine, and uridine into trichloroacetic acid-soluble and trichloroacetic acid-insoluble fractions

Thymidine Verapamil (~M) TCA-soluble TCA-insoluble 0 0 a'b 0 c

O.5 4 .0±2.2 0 5 4 .5±1.5 9.7±2.2 50 2 .5±2.0 49.7±1.6

Leucine TCA-soluble TCA-insoluble

0 0 ~ 0 e

0.5 5.3±1.6 0 5 3 .7±2.4 8.0±4.2 50 3.0±2.0 20.7±3.3

Uridine TCA-soluble TCA-insoluble

0 O f 0 g 0.5 6.7±2.1 1.3± 1.2 5 17.0±3.2 13.7± 1.6 50 56.0 ± 4.0 51.0 ± 1.4

a The data represent, for three independent experiments, the mean percent inhibition ± SEM by verapamil (added 4 h, 15 min prior to culture completion) of 3H-thymidine, 3H- leucine, or 3H-uridine into 10~o TCA-soluble and 10~o TCA-insoluble fractions of PBMC at the conclusion of 72, 24 and 24 h of culture, respectively.

b Range of baseline (no drug) cpm: 210-371. Range of baseline (no drug) cpm: 22708-26421.

d Range of baseline (no drug) cpm: 133-241. e Range of baseline (no drug) cpm: 1302-3978. r Range of baseline (no drug) cpm: 2807-5994. g Range of baseline (no drug) cpm: 10494-23391.

cellular uptake of thymidine was thymidine- specific or whether a similar inhibition of uptake occurred for other molecules. To evaluate this, we examined the effects of verapamil and israd- ±pine on mitogen-stimulated incorporation of 3H- leucine into protein and 3H-uridine into RNA. As demonstrated in Table I, PHA-induced incorpo- ration of 3H-leucine into protein and 3H-uridine into RNA were inhibited in a concentration- dependent fashion by verapamil and isradipine, whether the calcium channel blockers were added at the initiation of culture, or 4 h and 15 rain prior to completion of culture.

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TABLE III

Percent inhibition of PHA-stimulated cell cycle progression of peripheral blood mononuclear cells

Calcium channel blocker (/~M)

0 0.5 5 50

Verapamil in culture for 72 h

Isradipine in culture for 72 h

Verapamil added 4 h prior to end of 72 h culture

Isradipine added 4 h prior to end of 72 h culture

0 a'b 2.5±0.5 14.7±2.1 c 55.0±4.2 c

0 1.8±1.9 11.2+1.8 c 64.6±4.8 ~

0 2.5±1.2 5.0±1.2 8.7±2.0

0 1.1±0.6 3.6±1.8 4 .2±2.0

a The data are expressed as the percent inhibition of the percentage of cells in S + G 2 + M + SEM for three indepen- dent experiments. These experiments were performed si- nmltaneously with those in Table I.

b Range of baseline (no drug) percentage of ceils entering S + G 2 + M: 38.0-42.0

° The inhibitory capability of drug is statistically greater (p < 0.05) than control (no drug).

The inhibition by verapamil and isradipine of 3H-leucine incorporation into protein and 3 H -

uridine incorporation into RNA when the drugs added at the beginning of cell culture was of lesser magnitude than the inhibition of 3 H -

thymidine incorporation into DNA. When vera- pamil was added 4 h and 15 min prior to the completion of culture, the maximal inhibition of 3H-leucine and 3H-uridine incorporation into protein though significant, was somewhat less than the maximal inhibition observed when ver- apamil and isradipine were added at the initiation of culture or when isradipine was added 4 h, 15 min prior to the completion of culture.

Verapamil and isradipine (data not shown) inhibited 3H-leucine and 3H-uridine incorpora- tion into the TCA-insoluble fraction without any concomitant increase in radiolabel in the TCA- soluble fraction added 4 h, 15 min prior to the completion culture (Table II). Thus, verapamil appears to also inhibit the cellular uptake of 3H-leucine and 3H-uridine induced by PHA.

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TABLE IV

Percent inhibition of viability of peripheral blood mononuclear cells after exposure to verapamil

Time (h PBMC exposed to PHA)

Verapamil (#M)

0 0.5 5 50

24 0 a 0 0 8.3±3.2 48 0 1.0±0.1 2.5±1.1 7.6±2.1 72 0 0.2±0.1 0 9.5+3.16

Time (h PBMC exposed to PHA)

Isradipine (/~M)

0 0.5 5 50

24 0 0 0 () 48 0 0 0 6.6 + 1.6 72 0 0.4+0.1 1.0±0.1 7.0 ±4.2 b

a The data are expressed as the mean percent inhibition _+ SEM of viability of peripheral blood mononuclear cells as determined by the exclusion of propidium iodide in three independent experiments. There were no significant changes in the cell counts in any of the cultures.

b The decrease in viability is statistically greater (p < 0.05) than control (no drug).

Since the inhibition by verapamil of 3H-leucine incorporation into protein was consistently greater when the drug was added at the initiation of culture rather than added after 19.75 h of culture, these data suggest that the inhibition of 3H-leucine incorporation when verapamil was added at t --0 was only partially due to dimin- ished cellular uptake of leucine. Similarly, israd- ipine inhibited 3H-uridine incorporation to a greater extent when added at t= 0, suggesting that there may be other mechanisms for inhibi- tion besides limitation of cellular uptake of uri- dine.

Inhibition of PHA-stimulated cell cycle progression Because 3H-thymidine incorporation into DNA is not a valid measure of lymphocyte activation when verapamil and isradipine are present in the culture, we assessed the ability of these drugs to inhibit lymphocyte proliferation by determining their effect on PHA-stimulated cell cycle progres- sion (Table III). When verapamil or isradipine was added at the initiation of culture, it exhibited a concentration-dependent ability to inhibit cell cycle progression from G~ to S + G 2 + M. In

contrast to the results reported in Table I, only minimal inhibition of cell cycle progression was noted if verapamil or isradipine was added 4 h and 15 rain prior to the completion of culture.

As shown in Table IV, the ability of verapamil and isradipine to inhibit cell cycle progression does not appear to be related to cell toxicity, as > 90~o of cells remained viable for up to 72 h in all concentrations of verapamil and isradipine tested, except for cells exposed to 50/~M vera- pamil or isradipine for 72 h, where a 19.5~o and 37.0~o decrease in cell viability was noted respec- tively.

Discussion

Several investigators have noted that calcium channel blockers exhibit antiproliferative effects on lymphocytes (Whitney and Sutherland, 1972; Parker, 1974; Freedman et al., 1975; Diaman- stein and Ulmer, 1976; Greene and Parker, 1976; Freedman, 1979; Dubois and Crupton, 1980). These prior investigative efforts have utilized 3H-thymidine incorporation into DNA as a mea-

sure of lymphocyte proliferation. Although the transmembrane flux of calcium has been shown to be critically required for lymphocyte activation (Whitney and Sutherland, 1972; Parker, 1974; Freedman et al., 1975; Diamanstein and Ulmer, 1976; Greene and Parker, 1976; Freedman, 1979; Dubois and Crupton, 1980), the mecha- nism(s) by which calcium channel blockers de- press the responsiveness of lymphocytes to mito- gens and antigens is unclear, since lymphocytes have been reported to lack voltage-gated calcium channels (Lewis and Cahalan, 1990).

Although all calcium channel blockers block calcium entry at slow voltage-sensitive channels in vascular and myocardial tissue (Antman et al., 1980), they also have numerous other effects (Endicott and Ling, 1989; Zhernig, 1990). For example, verapamil, which is a non-dihydro- pyridine calcium channel blocker, and its methoxy-derivative D600 competitively block ra- dioligand binding to el adrenergic, e2 adrenergic, and muscarinic receptors (Motulsky et al., 1983; Triggle and Swamy, 1983). Isradipine, which is a dihydropyridine calcium channel blocker, has been reported to block the transmembrane up- take of adenosine (Striessnig etal., 1985). Herein, we report that both verapamil and isra- dipine inhibit PHA-induced thymidine, leucine, and uridine incorporation into DNA, protein, and RNA respectively, whether they are added at the initiation of culture or 4 h and 15 min prior to the termination of culture. This observed inhibi- tion appears to be largely due to the inhibition of cellular uptake of these precursor molecules by verapamil and isradipine, since there was no accumulation of radiolabel in the TCA-soluble fraction. If the calcium channel blockers had inhibited the incorporation of the precursor mol- ecules into macromolecules without inhibiting their cellular uptake, unincorporated precursor molecules should have accumulated inside the cell (in the 10~o TCA-soluble fraction); this was not the case.

Because verapamil and isradipine inhibit the uptake of thymidine, leucine and uridine into the cells, the incorporation of these precursor mole-

81

cules into DNA, protein, and RNA cannot be used as a measure of mitogen- or antigen-induced lymphocyte activation. Therefore, to determine whether verapamil or isradipine actually inhibit PHA-induced lymphocyte proliferation, we eval- uated the effect of the drugs on mitogen-induced cell cycle progression as measured by cytofluoro- metric analysis of cellular DNA content. Both verapamil and isradipine exhibited a concentra- tion-dependent ability to inhibit PHA-stimulated cell cycle progression of PBMC into S + G 2 + M. Thus, both drugs truly have antiproliferative ef- fects on mitogen-stimulated human lymphocytes. The ability of verapamil and isradipine to inhibit the uptake of thymidine, leucine, and uridine into mitogen-activated lymphocytes may play an im- portant role in ability of these drugs to inhibit PHA-stimulated proliferation.

Our experimental results conflict with the re- sults of Grier and Mastro (Grier and Mastro, 1985). These investigators noted that if they separated the addition of verapamil and 3H- thymidine by 10 min, that there was no observed inhibition of radiolabel uptake. However, they performed their experiments with bovine retro- pharyngeal cells; we separated the addition of verapamil on isradipine and the various radiola- beled precursor molecules by 15 min and used human peripheral blood mononuclear cells.

The consistency of our data demonstrating inhibition of cellular uptake of all three radiola- beled precursor molecules (thymidine, leucine, and uridine) and the ability of calcium channel blockers to inhibit a broad array of afferent and efferent immunological responses (Weir et al., 1991) to a variety of mitogens and alloantigens supports our unique observations.

Acknowledgements

We wish to acknowledge the expert secretarial assistance of Teresa Chiapperelli and Donna Deluca. M.R. Weir was supported by the Na- tional Kidney Foundation of Maryland, and Designated Research Initiative Funds of the

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State of Maryland. B.S. Handwerger was sup- ported in part by the U.S. Public Health Service Grant 1 R01-AI27885 and a Merit Review grant award from the Department of Veterans Affairs.

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