Proc. Nati. Acad. Sci. USAVol. 79, pp. 3656-3660, June 1982Neurobiofogy

[3H]Nitrendipine-labeled calcium channels discriminate inorganiccalcium agonists and antagonists

(nifedipine/verapamil/receptor/hypertension/angina)

ROBERT J. GOULD, KENNETH M. M. MURPHY, AND SOLOMON H. SNYDER*Departments of Neuroscience, Pharmacology and Experimental Therapeutics, Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine,72 North Wolfe Street, Baltimore, Maryland 21205Contributed by Solomon H. Snyder, March 11, 1982

ABSTRACT [3H]Nitrendipine binds with high affinity to brainmembranes with a drug specificity indicating association with sitesmediating the pharmacologic actions of dihydropyridine slow-cal-cium-channel antagonist drugs. In brain membranes, [3Hjnitren-dipine binding is absolutely dependent on the presence of calciumions. Interactions of cations with [3H]nitrendipine binding sitescorrelate with their physiologic actions at voltage-dependent cal-cium channels. Ions such as strontium and barium, which mimiccalcium physiologically, share the action of calcium in enhancing[3H]nitrendipine binding. Ions such as lanthanum and cobalt,which block the effects of calcium, can inhibit [3H]nitrendipinebinding and block the stimulating actions of calcium. The abilityto monitor the influence of ions on an agonist-antagonist contin-uum at [3H]nitrendipine binding sites provides a molecular probeto explore the regulation of cellular function by calcium and othercations.

The entry of calcium into cells via voltage-dependent channelsregulates muscle contraction and neuronal discharge (1, 2).Drugs that block these voltage-dependent calcium channelshave considerable therapeutic importance in treating angina,hypertension, and other vasoconstrictive disorders (3-5). Var-ious organic slow-calcium-channel blocking agents seem to actdifferently, with the pharmacologic actions of the dihydropyr-idine agents differing somewhat from those of drugs such asverapamil, D-600, and diltiazem (6). Recently, we (7) and others(8-10) have labeled the calcium channel binding sites for dihy-dropyridine drugs in brain and heart, using [3Hlnitrendipine.While the affinities of the dihydropyridines for these bindingsites correlate well with their pharmacologic properties, vera-pamil, D-600, and diltiazem have 'low affinities for these sites,indicating that the latter drugs act through different receptors.

Some inorganic ions, such as barium and strontium, mimicthe ability of calcium to pass through the voltage-dependentcalcium channels, while cations such as lanthanum and cobaltare calcium antagonists (11). We now report that [3H]nitrendipinebinding to 'brain membranes has an absolute- requirement forphysiological concentrations ofcalcium. Moreover, inorganic ca-tions interact with calcium in the regulation of [3H]nitrendipinebinding in an agonist-antagonist continuum.

MATERIALS AND METHODSMale Sprague-Dawley rats were killed by cervical dislocationand decapitation. Brains were removed immediately andwashed with 0.9% ice-cold NaCl, and the cerebral cortices wereseparated. Where indicated, brains were dissected into distinctanatomical regions and binding to these regions was assayed.Tissue was homogenized using a Brinkmann Polytron in 10 vol

of 50 mM Tris HCl (pH 7.7) and then centrifuged at 20,000rpm for 10 min in a Sorvall SS-34 rotor. The-pellet-was washedthree times with ice-cold 50 mM Tris-HCI (pH 7.7) and sus-pended to a final concentration of 1-4 mg (wet weight)/ml in50 mM Tris HCl. In those experiments exploring the ionic reg-ulation of [3H]nitrendipine binding, cerebral cortex or heartwas homogenized in 10 vol of 50 mM Tris HCl, pH 7.7/10 mMEDTA and immediately centrifuged at 20,000 rpm for 10 min.The pellet was suspended in 50 mM Tris-HCl, pH 7.7/10 mMEDTA and incubated on ice for 30 min. After centrifugation asbefore, the pellet was suspended in 50 mMM Tris'HCl, pH 7.7/10mM EGTA and again incubated on ice for 30 min. The pelletobtained after centrifuging was then washed three times with10 ,uM EGTA/50 mM Tris-HCl, pH 7.7, and finally resus-pended at 4 mg (wet weight)/ml in this EGTA/Tris buffer.Mixtures were incubated at 25°C as described in the figures andtables. Incubations were terminated by rapidly filtering throughGF/B glass fiber filters (Whatman). The filters were washedwith three 3-ml portions of 50 mM Tris HCl (pH 7.7), and theradioactivity retained was determined by 'liquid scintillationspectrometry using NEN-947.

[3H]Nitrendipine (85 'Ci/mmol; 1 Ci = 3.7 x 1010 becque-rels) and oxidized [3H]nitrendipine (85 Ci/mmol) were obtainedfrom New England Nuclear. Nifedipine (Pfizer), nimodipine,nisoldipine, nitrendipine (Miles), felodipine (Hassle, Molndal,Sweden), diltiazem (Tanabe, Osaka, Japan), verapamil, and D-600 (gifts ofJohn Daly) were first dissolved in absolute ethanolto 1mM and then diluted to the appropriate concentrations with50 mM Tris HCI (pH 7.7). All other chemicals were obtainedfrom standard commercial sources.

RESULTSAs previously observed (7-10), [3H]nitrendipine binding tobrain membranes is saturable. Scatchard analysis of saturationexperiments in 50 mM Tris HCI (pH 7.7) in the absence ofche-lating agents indicates a single population of binding sites hav-ing a Kd value of 137 ± 36pM and a maximal number of bindingsites (Bma) of 11.5 ± 0.6 pmoVg (wet weight). Kinetic studiesindicate a single phase for the association and dissociation of[3H]nitrendipine. The dissociation of [3H]nitrendipine frombrain membranes was examined by incubating [3H]nitrendipinewith brain membranes for 60 min and then adding 60 nM ni-fedipine and examining the residual at different time points.The-rate constant for association (k1) is 0.198 min-1 nM-1 andthe rate constant for dissociation (k-1) is 0.0462 min-'. The Kdvalue determined from the ratio k-1/k, is 230 pM, which agreeswell with results obtained by equilibrium experiments.

[3H]Nitrendipine binding increases linearly with tissue con-

Abbreviation: Bn., maximal number of -binding sites.* To whom reprint requests should be addressed.

3656

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Proc. Natl. Acad. Sci. USA 79 (1982) 3657

centration up to 8 mg (wet weight) of tissue per ml, at whichpoint it plateaus rapidly. All binding studies were carried outin the linear range. In typical experiments using 2-4 mg oftissue per ml, total binding in the presence of 0.2 nM [3H]-nitrendipine is -3,200 cpm while nonspecific binding in thepresence of 60 nM nifedipine is =600 cpm. [3H]Nitrendipinebinding is temperature dependent. With 60-min incubationperiods, specific [3H]nitrendipine binding is maximal at 250C,with only about half as much binding at0C or 370C. Subcellularfractionation experiments indicate the greatest enrichment ofspecific [3H]nitrendipine binding in crude synaptosomal frac-tions and microsomes. The subcellular distribution of [3H]-nitrendipine binding is essentially the same as that of [3H]di-hydroalprenolol binding to P-receptors assayed in these samefractions. For routine binding studies of [3H]nitrendipine, thetotal particulate fraction was used.

As observed previously, the drug specificity of the [3H]-nitrendipine binding sites indicates that they represent thesite of pharmacologic action of the dihydropyridine calciumchannel antagonists (Table 1). Of a large number of drugs ex-amined, only dihydropyridines display high potency. Felodi-pine has the greatest affinity for the binding sites with a Ki valueof -0.04 nM. Nifedipine is the least potent of the dihydropyr-idines with a Ki value of =0.38 nM, while nitrendipine, nisol-dipine, and nimodipine have intermediate potencies. Thesomewhat greater potencies for felodipine, nimodipine, andnisoldipine reported previously (7) are attributable to pipettingartifacts related to the extreme hydrophobicity of these drugs.The lower potency for dihydropyridines observed by Bellemannet al. (8) may result from an excessively high receptor concen-tration in an incubation volume of 0. 25 ml. When receptor con-centrations exceed the true Kd of the ligand, IC50 values nolonger reflect the true potency of the drug but rather the con-centration of the receptor (12). Receptor concentration in ourassays is 10 pM, substantially less than the Kd of [3H]nitren-dipine. In contrast to the high potencies of the dihydropyr-idines, verapamil, D-600, and diltiazem, which have considerablepharmacologic potencies as calcium antagonists, are weak at[3H]nitrendipine binding sites. We have failed to obtain totaldisplacement of [3H]nitrendipine by verapamil, D-600, or dil-tiazem, so that we cannot estimate molar affinities ofthese drugsfor the binding sites. For verapamil and D-600, up to 30% max-imal displacement is obtained at 100-200 nM, similar to theresults of Ehlert et al. (9).

Table 1. Drug specificity of [3H]nitrendipine binding to rat brainmembranes

Ki,Drug pM nH

Felodipine 38 0.90Nitrendipine 55 0.90Nisoldipine 103 0.99Nimodipine 168 1.05Nifedipine 384 0.90

IC50 values were determined by using 0.1 nM [3H]nitrendipine andsix concentrations of each inhibitor; triplicate assays were for 45 minat 24°C with 4 mg (original weight) of tissue in 4 ml of 50 mM Tris-HClbuffer (pH 7.7). Ki values were determined from the relationship Ki= IC50/(1 + L/Kd), whereL is the [3H]nitrendipine concentration andKd is the dissociation constant determined by saturation analysis inparallel experiments. Results are means of two independent deter-minations. Total and nonspecific [3H]nitrendipine binding was definedin the absence or presence of 60 nM nifedipine. Drugs that had no effecton [3H]nitrendipine binding at 1 ,tM included NAD, NADH, deaminoNAD, NADP, nicotinic acid, niacinamide, pyridine, inosine, clonidine,isoproterenol, atropine, decamethonium, morphine, theophylline, y-aminobutyric acid, cyclohexyladenosine, and phorbol dibutyrate.

Table 2. Brain region distribution of [3H]nitrendipine binding inrat and guinea pig membranes

Rat Guinea pig

Bmaxi BmaxqKd, pmol/g Kd, pmol/g

Region pM (wet weight) pM (wet weight)

Cerebral cortex 133 13 106 13Cerebellum 124 4 91 4Thalamus/hypothalamus 115 9 120 6

Striatum 129 13 126 11Olfactory bulb 100 12 112 12Hippocampus 110 15 132 14Midbrain - <1 - <1Brainstem - <1 - <1

Homogenates of brain regions pooled from two animals were assayedat 4 mg (wet weight)/ml in (total vol) 2 ml of Tris-HCl (pH 7.7). Totaland nonspecific binding, determined in the absence or presence of 60nM nifedipine, was measured at nine concentrations of [3H]nitrendipinebetween 0.005 and 2 nM. Scatchard analysis of these saturationexperiments was used to determine the Kd and Bmax values for[3H]nitrendipine in each brain region. Results are means of two in-dependent experiments.

The selectivity of the binding sites is apparent in the lack ofeffect on [3H]nitrendipine binding ofmany drugs with pyridinering systems (Table 1). In addition, oxidized [ H]nitrendipineshows no binding, further substantiating the specificity of thesesites for the dihydropyridine structure.The pattern of drug effects suggests that [3H]nitrendipine

sites cannot be equated with all calcium channels but ratherreflect some specific subpopulation. A heterogeneity ofcalciumchannels is apparent in the regional distribution of [3H]-nitrendipine binding (Table 2). In both rat and guinea pigbrain, highest binding levels occur in the cerebral cortex, stria-tum, olfactory bulb, and hippocampus, while binding in thecerebellum is only -25% as high. [3H]Nitrendipine binding inthe thalamus/hypothalamus is intermediate. Brain stem andmidbrain have detectable [3H]nitrendipine binding but levelsare inadequate for Scatchard analysis.

As previously reported, substantial binding is apparent in theheart. We have also detected displaceable binding in skeletalmuscle, as well as in several other tissues (Table 3). The potencyof nitrendipine and nifedipine in heart and skeletal muscle is

Table 3. Tissue distribution of [3H]nitrendipine binding in ratand guinea pig membranes

Binding, cpm/20 mg (wet weight)

Rat Guinea pig

Tissue Specific Nonspecific Specific Nonspecific

Heart 2,905 1,100 2,924 1,000Skeletal muscle 880 1,200 3,000 1,200Lung 183 1,600 969 1,100Kidney 319 1,500 185 1,400Ileum 14 1,400 900 400Liver 100 900 330 2,000Pancreas 164 2,000 - -

Adrenal - - 260 2,000

Homogenates of tissue pooled from two animals were assayed for[3H]nitrendipine binding at 10 mg (wet weight)/ml in (total vol) of 2ml of 50 mM Tris-HCl (pH 7.7) with 0.2 nM [3H]nitrendipine in theabsence and presence of 1 ,pM nifedipine to determine total and non-specific binding, respectively. Specific binding is defined as the dif-ference between total and nonspecific binding. Results are means ofat least two independent determinations.

Neurobiology: Gould et al..

3658 Neurobiology: Gould et al.

Table 4. Effect of cation replacement on [3Hlnitrendipinebinding

Kd, B ,Addition pM pmol/g (wet weight)

Tris-washed membranesNone 137 ± 36 11.5 ± 0.6

EGTA-washed membranesNone 176 ± 20 1.8 ± 0.30.01 mM CaCl2 190 4.80.1 mM CaC12 220 6.41mM CaCl2 188 ± 32 8.0 ± 0.71 mM SrCl2 267 9.01 mM MnCl2 219 7.91 mM CoC12 199 4.21 mM MgCl2 198 4.71 mM BaCl2 129 2.01 mM CaCl2/1 mM CoC12 174 5.01 mM CaCl2/30 uM LaCl3 180 6.01 mM CaCl2/100 uM LaCl2 196 3.0

Saturation binding experiments were carried out with Tris- andEGTA-washed. membranes in.the absence or presence of various ions.Total and nonspecific [3Hlnitrendipine binding were-determined intriplicate at nine concentrations of [3H]nitrendipine between 0.005and 2.0 nM in the absence and presence of 60 nM nifedipine. Kd andB,,,, values were determined by Scatchard analysis. Results are meansof two independent determinations or mean ± SEM of values pre-sented of three or four independent determinations.

consistent with binding to similar sites as in the brain (data notshown).

[3H]Nitrendipine binding to brain membranes is absolutelydependent on calcium (Table 4). Adding up to 10 mM calciumchloride to standard Tris HCl buffers does not alter [3H]-nitrendipine binding (data not shown). However, substantialamounts of calcium are bound to most tissue membranes. Toevaluate calcium dependence rigorously, we subjected brainmembranes to extensive washing in the presence of the che-lating agents EDTA and EGTA, which causes an 85-90% re-duction of the number of [3H]nitrendipine binding sites withno change in affinity. As little as 0.01 mM calcium increases thenumber of binding sites in the EGTA-washed membranes, andmaximal stimulation of binding occurs at 1 mM calcium withno further increase up to 10 mM calcium (Fig. 1). Interestingly,

o~' A

x 2

C..

-5 -4 -3 -2

extensive washing of heart membranes with EDTA/EGTAdoes not reduce [ H]nitrendipine binding (data not shown).

Different cations differ in their interactions with voltage-de-pendent calcium channels (11). Strontium and barium behavevery much like calcium. On the other hand, lanthanum, cobalt,copper, and manganese can antagonize the effects of calcium(13, 14).Some stimulation of [3H]nitrendipine binding is observed

with certain concentrations of all the cations examined (Fig. 1).However, the patterns vary considerably. Calcium, strontium,and manganese provide the greatest stimulation of [3H]-nitrendipine binding. However, whereas calcium and stron-tium stimulate binding at all concentrations examined, higherlevels of manganese reduce [3H]nitrendipine binding.

Like manganese, lanthanum, copper, and cobalt also stim-ulate [3H]nitrendipine binding at lower concentrations and de-press it at higher concentrations. Lanthanum is the most potentinhibitor of [3H]nitrendipine binding, followed by copper, co-balt, and then manganese. Thus, lanthanum, copper, cobalt,and manganese have both agonist and antagonist actions at[3H]nitrendipine sites, while calcium and strontium displayonly agonist activity. Magnesium and barium, like calcium andstrontium, show only stimulation of [3H]nitrendipine binding.However, maximal stimulation by magnesium and barium isonly about half as great as with calcium and strontium (Fig. 1).

The biphasic effect of ions such as lanthanum, copper, andcobalt may involve stimulation by mimicking calcium and de-pression of binding by some mechanism unrelated to calcium.In this regard, in the presence of higher concentrations of ionssuch as cobalt and lanthanum, nonspecific binding of [3H]-nitrendipine increases but there is no change in the affinity ofthe binding site for [3H]nitrendipine. Alternatively, the reduc-tion of binding may be mediated at the site where calcium nor-mally stimulates binding. To distinguish these alternatives, weexplored the interactions of various cations (Fig. 2). The stim-ulatory effects of calcium, strontium, magnesium, and bariumare additive at submaximal concentrations. The fact that no ad-ditivity occurs in the maximal stimulation by these ions indicatesthat they are acting at the same site. On the other hand, lan-thanum, copper, cobalt, and manganese can block stimulationby calcium. The specificity of this effect is apparent in that lowconcentrations of cobalt and lanthanum, which by themselvesstimulate binding, block the stimulation produced by calcium.

log cation concentration

FIG. 1. Ionic regulation of [3H]nitrendipine binding to rat cortical membranes. Total and nonspecific [3H]nitrendipine binding to EGTA-washedmembranes were determined intriplicate in the absence and presence of 60 nM nifedipine by using 0.2 nM [3Hlnitrendipine, 8 mg (original weight)of membranes in 2 ml (final vol) of 50 mM Tris HCl (pH 7.7), and various concentrations of various cations added as chlorides. (A) Cations wereCa+2 (o), Sr+2 (o), Mg+2 (A),.and Ba+2 (o). (B) Cations were Ca+2 (o), Mn+2 (A), Co+2 (o), Cu+2 (o), and La`3 (O). Specific binding is defined asthe difference between total and nonspecific binding and was determined at each ionic concentration. Results are mean ± SEM of three indepen-dent experiments.

Proc. Natl. Acad. Sci. USA 79 (1982)

Proc. Natl. Acad. Sci. USA 79 (1982) 3659

¢ -. , , .-eU

-5 -4 -3 -2log CaCl2 concentration

-5 -4 -3 -2

FIG. 2. Ionic interactions with [3H]nitrendipine binding. Calciumstimulation curves were constructed by determining total and non-specific binding in the absence and presence of 60 nM nifedipine byusing 0.2 nM [3H]nitrendipine and various calcium concentrations.The effects of Sr+2 (A), Ba"2 (B), Mn"2 (C), C0+2 (D), Cu+2 (E), and La"3(F) on the stimulation curves were examined by adding these ions aschloride salts to (final concentration) 0 mM (e), 0.1 mM (0), 1 mM(n), or 10 mM (A). Results are means of triplicate determinations froma representative experiment, which was replicated at least twice.

Manganese (10 mM) blocks calcium stimulation of binding, in-dicating that the modest reduction of [3H]nitrendipine pro-duced by manganese involves blockage of calcium-sensitivesites.

Potencies of stimulation and depression of [3H]nitrendipinevary among the ions. Calcium and manganese are the most po-

tent stimulators. While strontium shares with calcium the great-est maximal stimulation of [3H]nitrendipine binding, it isslightly less potent than calcium in eliciting this stimulation.

Scatchard analysis of ionic effects on [3H]nitrendipine bind-ing indicates that all observed stimulation or inhibition of[3H]nitrendipine binding alters the number of binding siteswith little change in affinity (Table 4). Moreover, the blockadeby lanthanum and cobalt of calcium stimulation of [3H]-nitrendipine binding involves numbers of sites rather than af-finity (Fig. 3 and Table 4).

DISCUSSIONThe properties of [3H]nitrendipine binding sites described hereand elsewhere (7-10) indicate that these are the sites at whichdihydropyridine calcium antagonists exert their pharmacologicactions. The relative potencies of various dihydropyridines incompeting for the binding sites parallel their pharmacologicpotencies, whereas numerous other drugs are very weak or in-active (Table 1). The pharmacologic evidence that dihydropyr-idines have a different site of action from drugs such as vera-

pamil, D-600, and diltiazem (6) is supported by the weak activityof these latter drugs at [3H]nitrendipine sites (7-10). It is wellestablished that the pharmacologic influences of dihydropyri-dines on muscle contraction involve blockage ofvoltage-depen-dent slow calcium channels. The fact that the molar affinitiesof the dihydropyridines for [3H]nitrendipine binding sites areessentially the same as potencies in influencing calcium chan-

0.20

0)0)

-.Cla

000.10 - \\_

0 2 4 6 8

[3H]Nitrendipine bound, pmol/g (wet weight)

FIG. 3. Scatchard analysis of ionic interactions. Saturation bind-ing experiments were carried out on EGTA-washed membranes in theabsence of ions (i) or in the presence of 1 mM CaCl2/0.1 mM LaCl3(A), 1 mM CaCl2/0.03 mM LaCl3 (A), 1 mM CaCl2/1 mM CoC12 (0), or1 mM CaCl2 alone (A). Total and nonspecific [3H]nitrendipine bindingwere determined in triplicate at nine concentrations of [3H]nitrendipinebetween 0.005 and 2.0 nM in the absence and presence of 60 nM ni-fedipine, respectively. Data presented are from a representative ex-periment that was replicated two or three times with similar results.

nels indicates that sites labeled by [3H]nitrendipine are asso-ciated with the voltage-dependent slow calcium channels.

Our observations on the ionic regulation of [3H]nitrendipinebinding further support the notion that [3H]nitrendipine bindsto sites linked to calcium channels (Table 4 and Fig. 1). Inter-estingly, we did not detect calcium dependence of [3H]ni-trendipine binding in heart membranes, which is surprisingbecause most properties of the [3H]nitrendipine binding sitesare essentially identical in heart and brain (7-9). Since calciumbinds firmly to various intracellular components, it is possiblethat in the heart even treatment with chelating agents has failedto remove all calcium. This may also explain the failure of ex-tensive treatment with chelating agents to reduce [3H]ni-trendipine binding in brain to <10% of original levels.The most striking finding ofthe present study is that the abil-

ity of various cations to stimulate or inhibit [3H]nitrendipinebinding or to block the stimulatory actions ofcalcium correlateswith their known agonist or antagonist profiles at calcium chan-nels (13, 14). For instance, strontium and barium are knowncalcium agonists and mimic the effects ofcalcium in stimulating[3H]nitrendipine binding (Fig. 1). Lanthanum and cobalt, clas-sical calcium antagonists, reduce [3H]nitrendipine binding andblock the stimulatory actions ofcalcium (Figs. 1 and 2). The factthat inorganic calcium antagonists, including manganese, co-balt, copper, and lanthanum, also can stimulate [3H]nitrendipinebinding at low concentrations suggests that these ions possessagonist as well as antagonist properties at calcium channels.

The physiological relevance of the ion effects we have ob-served is apparent from correlations with electrophysiologicdata (13). Thus, in blocking calcium currents, lanthanum is-=10 times more potent than cobalt, which is in turn :3 timesmore potent than manganese (13). This is similar to the relative

Neurobiology: Gould et al.

3660 Neurobiology: Gould et al.

potencies of these ions in decreasing [3H]nitrendipine binding(Fig. 1). Also, maximal stimulation of [3H]nitrendipine bindingby cations is related to the ionic crystal radius of the ions. Max-imal stimulation occurs at a diameter corresponding to that ofcalcium with substantially lesser effects for ions with smaller orlarger diameters (Fig. 4), which fits nicely with observations(15) that ionic crystal radii are related to actions at calciumchannels.

Ionic currents through the calcium channel involve bindingto and mobility through the channel (2, 15). Potent inorganiccalcium-channel blocking agents, such as lanthanum, show highbinding but very low mobility. Ions such as manganese, withboth high binding affinity and some mobility, not only blockcalcium currents but can also carry current themselves, reflect-ing partial agonist activity. Thus, blockade of current throughthe calcium channel corresponds well with inhibition of[3H]nitrendipine binding by the inorganic calcium antagonists.

2

x

._

U

ca04~0ar8.a4

1.51-

1

0.51-

0.5 1.0Crystal radius, A

FIG. 4. Maximal stimulation of [3H]nitrendipine binding vs. ioniccrystal radii of divalent cations.

Conceivably, the stimulation-inhibition duality ofion effects on[3H]nitrendipine binding relates to differential binding andmobility through the calcium channel.

Calcium is a virtually universal regulator ofmembrane prop-erties and metabolism. Analyzing in detail the regulation ofcal-cium channels in neurophysiologic studies is hampered by tech-nical difficulties and the use of intact organs, precludingrigorous manipulation of the ionic environment. [3H]Nitrendi-pine binding may thus provide a molecular probe to character-ize how calcium regulates cellular function.We thank Drs. D. J. Triggle and Stephen Hurt of New England

Nuclear for helpful discussion and synthesis of [3H]nitrendipine andoxidized [3H]nitrendipine, Gregory Mack for valuable technical assis-tance, and Dawn C. Hanks for expert manuscript preparation. Thiswork was supported by U.S. Public Health Service Grants DA-00266,MH-18501, and NS-16375; a grant of the McKnight Foundation; andU.S. Public Health Service Research Scientist Award DA-00074 toS. H. S., Training Grant GM-073090 to K. M. M. M., and Training GrantMH-15330 to R.J.G.

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Mn2+

Mg2+ *Ba2+

Proc. Natt Acad. Sci. USA 79 (1982)