Calcium Channel Activation: a Different Type of Drug Action (Calcium Flux/Neuroblastoma/Dihydropyridines) STEPHEN B
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Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5580-5583, September 1984 Neurobiology Calcium channel activation: A different type of drug action (calcium flux/neuroblastoma/dihydropyridines) STEPHEN B. FREEDMAN* AND RICHARD J. MILLERt Department of Pharmacological and Physiological Sciences, University of Chicago, 947 East 58th Street, Chicago, IL 60637 Communicated by JosefFried, May 14, 1984 ABSTRACT Depolarization of NG108-15 (neuroblasto- plemented medium was replaced every 2 days or when re- ma-glioma) cells causes an increase in 45Ca2+ influx. This ef- quired. fect is blocked by low concentrations of dihydropyridines such 45Ca2+ Uptake Studies. Cells (passage 5-20) were subcul- as nitrendipifie and by other blockers of voltage-sensitive calci- tured onto 60-mm tissue culture plates and induced to differ- um channels such ns D-600, diltiazem, and Cd2 . Two other entiate as above. During the assay, tissue culture plates were dihydropyridines, BAY K8644 and CGP 28392, have the op- supported in an open-air waterbath at 370C. Cells were incu- posite effect. Low concentrations of these compounds enhance bated for 5 min at 37TC before the assay. Details of 45Ca2+ depolarization-induced 45Ca2+ influxes. BAY K8644 is more flux studies are given in the individual figure legends. The effective than CGP 28392. Both agents have no effect on fluxes uptake of 45Ca2+ was measured for increasing periods of measured under nondepolarizing conditions. The effects of time in Hepes (20 mM) buffered Eagle's minimal essential BAY K8644 and CGP 28392 can be inhibited by nitrendipine, medium containing 135.7 mM NaCl, 5 mM KCl, 0.44 mM D-600, diltiazem, or Cd2 . Whereas the interaction between KH2PO4, 0.34 mM Na2HPO4, 2.62 mM NaHCO3, 1.3 mM nitrendipine and BAY K8644 is shown to be competitive in CaC12, 0.81 mM MgSO4, and 5.6 mM glucose. Buffer with 50 nature, that between BAY K8644 and D-600 is shown to be mM KCl was the same except that NaCl was 90.7 mM. 45Ca2+ noncompetitive. These results indicate that dihydropyridines was added to give a final concentration of 1 /LCi/ml (1 Ci = show a variety of effects on calcium channels, ranging from 37 GBq). At the indicated times, plates were aspirated, agonistic through partially agonistic to antagonistic. More- washed immediately, and inverted to allow to dry. Cells over, the results also indicate that dihydropyridines and D-600 were solubilized with 5 ml of 0.2% sodium dodecyl sulfate exert their effects on calcium channels at different sites. and samples were removed for estimation of 45Ca uptake and protein content by fluorescence (14). The last few years have seen the development of a group of Materials. 45Ca2+ (10-40 mCi/mg of Ca2+) was obtained drugs that appear to block the flow of Ca2" through voltage- from Amersham. Nitrendipine and BAY K8644 were from sensitive calcium channels (VSCC) (1-4). For example, Miles Laboratories (New Haven, CT), D-600 from Knoll drugs such as verapamil, diltiazem, and the dihydropyridine (Ludwigshafen, F.R.G.), diltiazem from Marion Labora- nifedipine are now widely used in the treatment of angina, tories (Kansas City, MO), and CGP 28392 from'CIBA-Geigy cardiac arrhythmias, and hypertension. Elucidating the mo- (Summit, NJ). lecular basis of the action of such agents is clearly a subject of great interest at present. Calcium channel blocking drugs RESULTS AND DISCUSSION fall into a variety of chemical types (1, 4). It is quite possible We have shown that several clonal cell lines express VSCC that different drug classes block VSCC by different mecha- that are similar to those found in smooth muscle in that they nisms (5-11). Recently, a dihydropyridine (BAY K8644) has are blocked by low concentrations of dihydropyridines (13, been reported that causes smooth muscle to contract and 15). This is the case, for example, with the neuroblastoma- stimulates cardiac contractility (12). It was suggested that glioma cell line NG108-15. As can be seen in Fig. 1A, when this agent might act by opening VSCC rather than blocking these cells are subjected to depolarization by 50 mM K+ them. We now demonstrate VSCC activation by this com- they show a rapid increase in the net uptake of45Ca2+. Other pound and show that this property is shared by another dihy- studies have established that this influx is through VSCC dropyridine, CGP 28392. (13, 15). For example, it is not blocked by tetrodotoxin and is independent of the presence of external Na+. Moreover, the METHODS AND MATERIALS depolarization-induced influx is potently inhibited by dihy- Cells were grown as monolayers under 5% CO2 in Eagle's dropyridines such as nitrendipine at nanomolar concentra- minimal essential medium supplemented with 10% fetal bo- tions (nitrendipine IC50 = 6.5 nM). Other organic calcium vine serum and 2 mM glutamine. channel blockers are also effective, as are inorganic channel Cells were subcultured onto 60-mm tissue culture plates blockers such as Cd2+ and Co2+. and allowed to grow for 3 days. To induce cellular differenti- In Fig. 1A, it can be seen that addition of BAY K8644 ation (13), growth media was supplemented with 10 AuM (structure in Fig. 2) to NG108-15 cells under nondepolarizing prostaglandin E1 and 50 AM 3-isobutyl-1-methylxanthene. In conditions (5 mM K+) produces little effect on 4 Ca2+ up- parallel experiments, other workers in our laboratory have take. A small increase in uptake is observed. A quite differ- shown that this treatment will increase intracellular cyclic ent effect is seen in the presence of 50 mM K+. Fig. 1B AMP levels by 10- to 100-fold (unpublished observations). shows that, in the presence of 50 mM K+, 45Ca2+ uptake is Cells were examined visually for morphological differentia- stimulated as discussed above. The simultaneous addition of tion and were assayed for net 45Ca2+ uptake at various time BAY K8644 dramatically enhances the effect of 50 mM K+. points after treatment as indicated in the figure legends. Sup- Abbreviation: VSCC, voltage-sensitive calcium channel. *Present address: Neuroscience Research Centre, Merck Sharp & The publication costs of this article were defrayed in part by page charge Dohme, Ltd., Hertford Road, Hoddesdon, Herts EN11 9J3U, En- payment. This article must therefore be hereby marked "advertisement" gland. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 5580 Downloaded by guest on September 29, 2021 Neurobiology: Freedman and Miffer Proc. Natl. Acad Sci. USA 81 (1984) 5581 A r_ Z 0 . 15- H. Nifedipine Nitrendipine ,: 10- cu 0. + Cu tz 5- z 0 4 8 16 Time, min CGP 28 392 BAY K 8644 FIG. 2. Structures of dihydropyridines. 20. a) E 15- -6 C) ca5X0. fr_ 0 0. C) Cu u Cu Z 5- z [Agonist], M 8 12 Time, min *E35 B 30 FIG. 1. Time courses of net 45Ca2" uptake into NG108-15 cells. Cells were grown as monolayers under 10% C02 in Eagle's minimal Cu~25- essential medium supplemented with 10%o fetal bovine serum and 2 PE20 - mM glutamine. NG108-15 cells (500,000 cells per plate) were subcul- 15 tured onto 60-mm tissue culture plates and allowed to grow for 3 Cu U0 days. To induce cellular differentiation, growth medium was supple- mented with 10 ,uM prostaglandin E1 and 50 ,uM 3-isobutyl-1-meth- CE Z~ 5 ylxanthene. Supplemented medium was replaced every 2 days or when required. During the assay of 45Ca2+ uptake tissue culture plates were supported in an open air waterbath at 370C. Cells were 5 mM 50OmM 5OmM 5OmM incubated for 5 min at 370C before the assay. The uptake of 45Ca2+ K+ K+ K+ K+ was measured for increasing periods of time in Hepes (20 mM) buff- ered Eagle's minimal essential medium. Buffer with 50 mM KCI was CGP BAY prepared by adjusting Na+ reciprocally. 45Ca2+ was added at a final 28392 K8644 concentration of 1 MCi/ml. At the indicated times, plates were aspi- rated, washed immediately four times in 175 mM choline chloride/2 FIG. 3. (A) Dose-response curves for the enhancing effects of mM EGTA, and inverted to dry. Cells were solubilized with 5 ml of BAY K8644 (o) and CGP 28392 (X) upon high K+ (50 mM KCl) 0.2% sodium dodecyl sulfate and samples were removed for estima- stimulated 45Ca2` uptake into NG108-15 cells. Uptake was mea- tion of 45Ca2+ uptake and protein content by fluorescence (14). Each sured for 10 min (see Fig. 1). Results are expressed as percent maxi- point represents single experimental observations performed in du- mal response for each drug. Results are mean + SEM of triplicate plicate. Each curve is a typical experimental result that has been determinations performed on at least three separate occasions. (B) obtained on at least three separate occasions. (A) Effect of 1 AM Comparative effects of 1 AM BAY K8644 and CGP 28392 on net BAY K8644 under nondepolarizing conditions. A, 5 mM K+; o, 5 45Ca2` uptake into NG108-15 cells. Results are the mean ± SEM of mM K+ + 1 MiM BAY K8644; *, 50 mM K+. (B) Effect of 1 ILM quadruplicate determinations. Conditions are as follows, from left to BAY K8644 under depolarizing conditions. A, 5 mM K+; e, 50 mM right: 5 mM KCI; 50 mM KCI; 50 mM KCI and 1 uM CGP 28392; 50 K+; *, 50 mM K+ and 1 MM BAY K8644. mM KCI and 1 AM BAY K8644. Downloaded by guest on September 29, 2021 5582 Neurobiology: Freedman and Miller Proc. NatL Acad Sci. USA 81 (1984) In the presence of a maximally effective concentration of fect on 45Ca2" uptake into 3T3 fibroblasts under nondepolar- BAY K8644, the effect of 50 mM K+ is enhanced approxi- izing (5 mM K+) or depolarizing (50 mM K+) conditions.