[CANCER RESEARCH 47, 3920-3928, August 1, 1987]

Sodium Dependency of Uptake of Norepinephrine and w-Iodoben/y Iguanidine intoCultured Human Pheochromocytoma Cells: Evidence for Uptake-One1

Sandford Jaques, Jr.,2 Michael C. Tobes,3and James C. Sisson

Division of Nuclear Medicine, Department of Internal Medicine, The University of Michigan Medical Center, Ann Arbor, Michigan 48109

ABSTRACT

Radioiodinated m-iodobenzylguanidine (MIBG), a scintigraphic agent

used in the detection of human pheochromocytomas, is thought to utilizethe same uptake and retention mechanism(s) as norepinephrine (NE).Using primary cultures from 16 human pheochromocytomas, we compared the uptake of MIBG to that of NE. Two different uptake systemswere identified. Both NE and MIBG were taken up by a sodium-

dependent system that was characterized by: (a) temperature dependency,(ft) high affinity, (c) low capacity, (rf) saturability, (e) ouabain sensitivity,and (/) desmethylimipramine sensitivity. However, NE and MIBG werealso taken up by a temperature-dependent, sodium-independent, apparently unsaturable system. The sodium-dependent uptake system fulfillsmany of the criteria for Uptake-one while the sodium-independent uptake

system is most likely a passive diffusion process. Competitive inhibitionstudies demonstrated that NE and MIBG share a common uptake system;a concept consistent with the linear correlation between the rate of uptakeof 1.0 Õ»MNE and that of 1.0 »«MMIBG (r = 0.942). At low concentrations, both NE and MIBG entered the tumor cells primarily by thesodium-dependent uptake system. Differential expression of the sodium-

dependent and sodium-independent uptake systems, between different

tumor cells, appears to be responsible for the variations of the kineticparameters for both NE and MIBG. These studies provide the first directcharacterization of a NE uptake mechanism in human pheochromocy-

toma cells.

INTRODUCTION

MIBG4 is a recently developed radiopharmaceutical which is

used in the detection and localization of benign and malignantpheochromocytoma (1-3). It is being evaluated as a treatmentfor malignant pheochromocytoma (4-5) and is being used,increasingly, in the treatment of neuroblastoma (6). MIBG hasalso been used to image the human heart (7) and is beingevaluated as a probe of the sympathoadrenal system (8, 9).MIBG does not appear to be metabolized within the tumortissue itself; 46- to 65-h postinjection 98% or more was recovered as the native compound (10). In vivo studies suggestedthat MIBG shares the uptake, retention, and secretion mechanisms of NE (11-13). Using bovine adrenomedullary cells as amodel system (13, 14), we recently determined that both NEand MIBG are taken up by two systems which operated independently but overlapped at high and low concentrations of theagents: (a) a sodium-dependent system which fulfilled all of thecriteria of the neuronally characterized Uptake-one system (15-22), and (¿>)a sodium-independent system which is, most likely,a simple diffusion process. Although the mechanism of uptakeinto bovine adrenomedullary cells are now well defined, we

Received 4/17/86; revised 4/22/87; accepted 5/4/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was supported by American Cancer Society Grant PDT-182, and by USPHS, NIH, National Institute of Arthritis, Metabolism andDigestive Diseases Grant AM-21477.

! To whom requests for reprints should be addressed.3 Present address: AT&T Bell Laboratories, Medical Diagnostic Systems, 480

Red Hill Rd., Middletown, NJ 07733.4 The abbreviations used are: MIBG, m-iodobenzylguanidine; NE, norepineph

rine; HEPES, A'-2-hydroxyethylpiperazine-Ar'-2-ethanesulfonic acid; H-KRG,HEPES-buffered Krebs-Ringer glucose.

know very little about the mechanisms of NE or MIBG uptakein human pheochromocytoma tissue or cells. In fact, the path-ophysiology of human pheochromocytomas has only been studied at the cellular level to a limited extent. In the past 22 years,only 25 tumors have been cultured by all other researchers (23-33). Most of these studies examined only one or two cellularcharacteristics, using cells from only one or two tumors. Recently, we described the heterogeneity of several biochemicaland morphological characteristics (cell size, catecholamine content, secretory patterns, etc.) in 18 primary cell cultures of thistumor (34).

No cell line derived from a human pheochromocytomasexists. Although NE uptake has been demonstrated in the PC 12rat pheochromocytomas cell line (35), these cells may not berepresentative of human pheochromocytoma. In fact, we foundthat there was no uptake of MIBG into the transplantabletumor (36) from which the PC 12 cell line was derived.5 In

contrast, among 400 human patients, 87% of suspected pheochromocytoma were detected with MIBG scintigraphy (37).Therefore, we chose to use primary cell cultures of humanpheochromocytoma as the most useful model to examine NEand MIBG uptake. This represents the largest existent, andmost detailed description, of a single biochemical characteristicin human pheochromocytoma cells. We report, as in bovineadrenomedullary cells, the existence of a sodium-dependentuptake system, which is probably Uptake-one, and a sodium-independent system, which is probably passive diffusion. Inaddition, these studies also demonstrate the tumor to tumorheterogeneity in the expression of the two different uptakesystems, as well as the similarity of the uptake systems to thosein "normal" bovine adrenomedullary cells.

MATERIALS AND METHODS

Chemical, Radiochemicals, and Materials. Collagenase (type I, 130to 250 units/mg; type V, 300 to 500 units/mg), DNase (200 to 300Kuntz units/mg), trypsin (9,000 to 12,000 BAEE (W-a-benzoyl-L-argi-nine ethyl ester) units/mg), /-NE, /-epinephrine, ascorbic acid, ouabain,Percoli, HEPES, and 1-0-D-arabinofuranosylcytosine were purchasedfrom Sigma Chemical Co. (St. Louis, MO). Four-well Nunc Multidishplates were obtained from Vanguard International (Neptune, NJ).Desmethylimipramine was purchased from UVS Pharmaceuticals(Tuckahoe, NY). Minimum essential medium, penicillin, and streptomycin were purchased from K. C. Biologicals (Lenexa, KS). Fetal calfserum was obtained from HyClone Laboratories (Logan, UT). Cat-A-Kits were purchased from Upjohn Diagnostic (Kalamazoo, MI). l-[ring-2,5,6-3H]NE with a specific activity of 20 to 60 Ci/mmol was obtained

from New England Nuclear (Boston, MA). Nonradiolabeled MIBGwas synthesized by the method of Wieland et al. (38). Using a radioio-dide exchange technique (39), [125I]MIBG and [I31I]MIBG were pre

pared at a specific activity of 1.5 to 4.3 Ci/mmol. The radiochemicalpurity of MIBG was greater than 98% by thin layer chromatography(11) and high pressure liquid chromatography (38) as was that of NE.5

Biocount scintillation fluid was purchased from Research ProductsInternational (Mt. Prospect, IL). All other reagents were analytic gradeor higher purity.

Cell Culture. Tumor tissue was obtained from 14 patients during

5Unpublished observations.

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NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

surgery at University Hospital (Ann Arbor, MI), and single tumorswere obtained at Henry Ford Hospital (Detroit, MI) and St. JosephHospital (Ypsilanti, MI). The nine male and seven female patientsranged from 10 to 64 years of age at the time of surgery. Six of thetumors were extraadrenal and 10 were intraadrenal. Each tumor wasdiagnosed as a pheochromocytoma from multiple histological sectionsby pathologists using accepted morphological criteria.

In the operating room the intact tumor was placed in a sterile, ice-cold Ca2+,Mg2+-free Locke's solution prior to being transported to the

pathology laboratory, where the tissue was weighed and approximatelyone-fourth to one-half was retained for histological evaluation. Thebalance of the tumors were transported on ice in the Ca2+,Mg2*-freeLocke's solution to our laboratory. Transit time was usually less than

30 min from the operating room to our laboratory but was l h whenthe tumors were obtained at Henry Ford and St. Joseph Hospitals.

Tumor tissue was weighed, freed of connective tissue, includingcapsule, and minced into pieces approximately 2x2 mm. The mincedtissue was placed in a sterile beaker containing 50 to 100 ml ofCa2+,Mg2*-free Locke's solution and shaken in a Dubnoff metabolicshaker at 125 oscillations/min at 37°Cfor two 20-min periods to

remove RBC. Subsequently, the tissue was incubated with a collagenasesolution in the Ca2*,Mg2+-free Locke's solution, for four to six diges

tions of 40 to 50 min each. The collagenase solution, approximately0.05%, consisted of two parts of type V collagenase, adjusted torepresent a constant activity of 300 units/mg, and one part of type Icollagenase, adjusted to represent a constant activity of 200 units/mg.After the first digestion with collagenase, we added approximately 15%of a 0.25% trypsin solution to succeeding digestions. The digestionsolutions contained 0.05 to 0.10 mg/ml of DNase. RBC and cellulardebris were separated from the tumor cells by isopyknotic centrifugationon a preformed Percoli gradient as previously described (34). Freshlydispersed cells were plated at densities of 0.4 to 0.8 x IO6cells/well in

Nunc Multidish plates and maintained in minimal essential mediumsupplemented with 15% fetal calf serum, 100 units/ml penicillin, and100 Mg/ml streptomycin. Fibroblast growth was routinely inhibited bythe addition of 10 MM1-0-D-arabinofuranosylcytosine (40). The cellswere maintained at 37°Cin a humidified incubator (Heraeus) under a

CO2:air (5:95) atmosphere and allowed to adapt to culture for 4 daysprior to starting an experiment. After this adaptation period, themedium was changed every second or third day. Twenty-four h priorto commencing an experiment, the medium was replaced with freshmedium without l-/3-D-arabinofuranosyIcytosine.

The cells were characterized by several techniques prior to platingand/or during the course of an experimentation. Cell viability was 90%or greater by trypan blue exclusion at all times. Cell catecholaminecontent was routinely assayed by using the differential fluorometricassay technique (41) or the radioenzymatic method (42) by using theCat-A-Kit. Catecholamine content was expressed as nmol/106 cells.Periodically, the cell dispersions were evaluated by using histofluores-cence (43) to demonstrate endogenous catecholamines. All experimentsreported herein were performed 4 to 12 days postdispersion.

Incubation Conditions and Procedures. The uptake protocol has beendescribed in detail previously (13, 14). Briefly, 4 to 12 days postdisper-sion, the tissue culture plates were placed in a 37°Cwater bath or anice water bath (0.5 to 1.5'C). Fifteen min prior to commencing the

uptake study, the culture medium was removed, and the cells werepreincubated, at either temperature, with 0.5 ml H-KRG. Followingthe preincubation, the cells in each well were incubated with 0.30 mlH-KRG containing [3H]NE or [125I]MIBG or [I3'I]MIBG. At the endof the incubation period, the plates were placed on ice, the H-KRGcontaining the radiotracer was removed, and the cells were washedtwice. The radiotracer taken up by the cells was extracted with trichlo-roacetic acid and quantitated in a liquid scintillation or gamma counter,as appropriate. The 37°Cincubations were done in quadruplicate. The0°Cincubations, which reflect nonspecific binding, were done in dupli

cate. The total active uptake was determined by correcting the uptakeat 37°Cfor nonspecific uptake at 0°C.Before commencing the kinetic

studies, time course studies, using both agents at 0.5 or 1.0 MM,wereperformed in order to determine linearity of uptake and the optimaltime for performing the kinetic studies. Uptake was standardized as

pmol/106 cells/10 min, because cell numbers, as well as the incubation

time with different cell preparations, varied.Uptake Kinetics and Sodium Dependency of Uptake. Kinetic studies

were done by incubating the cells in the presence of 0.15 to 50 MMNEor MIBG to determine the Km (MM)and Vm,x(pmol/106 cells/10 min)

for each agent. The transport kinetic parameters were calculated on anApple He microcomputer by using a nonlinear regression curve-fittingpackage called ROSFIT (44). To determine the sodium dependency ofuptake, two parallel kinetic studies with a single agent, either [3H]NEor [125I]MIBG or [I3II]MIBG, were performed on the same day. Onekinetic study was performed by incubating the cells with 0.30 ml H-KRG containing the radiotracer; while in the second, complimentary,kinetic study, the cells were incubated with the same agent at identicalconcentrations in 0.30 ml zero-sodium H-KRG (13, 14). The zero-sodium H-KRG was identical to H-KRG except that the NaCl hadbeen replaced by an equimolar concentration of LiCl, i.e., 125 ITIM.Sodium-independent uptake is uptake which occurred in the zero-sodium H-KRG, and sodium-dependent uptake is uptake in the presence of NaCl less the sodium-independent uptake, i.e., uptake in thezero-sodium H-KRG. Total uptake is, therefore, the sum of the sodium-independent and sodium-dependent uptake and was measured as theuptake in H-KRG.

Ouabain and Desmethylimipramine Inhibition of Uptake. Cells wereincubated in H-KRG or zero-sodium H-KRG containing 0.5 or 20 MMconcentrations of either [3H]NE or [125I]MIBG in the presence or

absence of ouabain; the final concentration of ouabain in the incubationmedium ranged from 1.0 to 10,000 MM. Similarly, the cells wereincubated in H-KRG or zero-sodium H-KRG containing 0.5 or 20 MMconcentrations of either [3H]NE or [125I]MIBG in the presence (0.001to 1,000 MM)or absence of desmethylimipramine. The effects of des-methylimipramine, or ouabain, in the zero-sodium H-KRG directlyreflected its inhibitory capacity upon the sodium-independent uptakesystem. The inhibitory capacity of desmethylimipramine or ouabainupon the sodium-dependent uptake system was calculated by subtracting the sodium-independent uptake, at a given concentration of desmethylimipramine or ouabain, from total uptake in the presence of anidentical concentration of the inhibitor.

Competitive Inhibition of Uptake. Cells were incubated in H-KRGcontaining 0.2 to 3.5 MM[3H]NE in the presence or absence of 1.0 MMunlabeled MIBG. Cells were also incubated in H-KRG containing 0.25to 5.0 MM[I3II]MIBG in the presence or absence of 10.0 MMunlabeled

NE. These kinetic studies were analyzed by using the double reciprocalequation of Lineweaver-Burk and/or the Eadie-Hofstee equation, anda standard deviation of each parameter was calculated (45).

Data Analysis. The results of single experiments are presented unlessotherwise indicated. Each data point represents the mean of quadruplicate values ±SD. The uptake values at 1.0 MMwere determined twice,during a time course study and during subsequent kinetic studies. Thevariation in uptake velocities between these two time points (3 to 6days apart) was normally less than 10%. The inhibition experimentswith ouabain and desmethylimipramine are representative of experiments with primary cell cultures from three or more tumors. Thesignificance of differences between two points was evaluated by usingStudent's / test (46), and the significance of differences between multiple

points was evaluated by using a one-way analysis of variance in conjunction with Duncan's new multiple range test (46). Regression lines

were calculated by least squares analysis of all data points.

RESULTS

Temperature Dependency of Uptake. A typical time coursestudy is shown in Fig. 1. The uptake of 1.0 MMNE and 1.0 ^MMIBG was temperature dependent. The uptake of each agentat 37°Cwas linear to at least 20 min for all primary pheochro

mocytoma cell cultures, and was always greater than that at0°Cfor all concentrations of NE and MIBG studied. Accumulation at ()"(" was a small and constant fraction of the total

external concentration over time at all concentrations of NEand MIBG; therefore, uptake at 0°Cwas characteristic of a

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NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

160

</) 120

UJu

O 80

40

200

ISO

100

50

B. MIBG

25 50 75 50 75 100100 0 25

TIME (MIN)Fig. 1. Time and temperature dependence of uptake into a representative

primary cell culture of a human pheochromocytoma (patient 7). A, 1.0 UM NE,and »,1.0 IIM MIBG. Cells were incubated at 37'C (•)or O'C (O). Uptake was

terminated at the indicated times by removal of the incubation medium andextraction of either agent from the cells as described in "Materials and Methods."Uptake is expressed as pmol/10* cells.

-. 80

70

860

50

¿ 40

l- 30

IOCD

2

2O

10

j0 10 20 30 40 50

NE UPTAKE(PMOL/IO* CELLS/10 MIN)

Fig. 2. Relationship of the uptake velocity (pmol/1 (f cells/10 min) for 1.0 /IMNE to that of 1.0 „MMIBG as determined in primary cell cultures from 15tumors.

nonspecific process. The total uptake by the temperature-dependent process was calculated as the uptake at 37°Cless thatat 0°C.

Uptake Velocity and Cell Catecholamine Content. The totaluptake velocities (pmol/106 cells/10 min) for both NE and

MIBG at 1.0 MM was highly variable between the differentprimary cell cultures (0.2 to 46.2 for NE and 0.2 to 71.4 forMIBG). However, as shown in Fig. 2, the uptake velocity ofMIBG into the tumor cell cultures was correlated to the uptakevelocity of NE (r = 0.948) from one tumor cell preparation tothe next. The ratio of the uptake velocity of MIBG to that ofNE was 1.4:1.

Uptake of 1.0 MMMIBG into different tumor cell preparations was not correlated (r = 0.156) with cell catecholaminecontent when all cell preparations were included (Fig. 3), al

so

40

20

O 20 40 60 80 IOC

CELL CATECHOLAMINE CONTENT(NMOL/10« CELLS)

Fig. 3. Relationship of the cell catecholamine content (nmol/10* cells) at day5 in culture to the uptake velocity of (pmol/10'cells/10 min) for 1.0 »MMIBG

as determined in primary cell cultures from 14 tumors. Catecholamine content isthe sum of NE and epinephrine.

though exclusion of the two points with the highest uptake gavea better correlation (r = 0.816). Additionally, the uptake of 1.0/¿MNE did not correlate with catecholamine content either(33). The uptake velocity of 1.0 MM MIBG did not changesignificantly between days 5 to 10 of culture.5

Kinetics of Uptake. We evaluated the kinetics of both NE andMIBG uptake to determine if the total uptake for either or bothagents was a saturable process, and to determine if the kineticsfor both agents were similar among tumor cells from differentpatients. We observed four general patterns of NE and MIBGkinetics (Fig. 4). For one group, the total uptake of NE andMIBG were similar and uptake appeared to be a saturableprocess indicative of a high affinity, low capacity system (Fig.4A ). In the second group (Fig. 4B) the total uptake was morecomplex. Total uptake of MIBG took the form of a straightline superimposed upon a hyperbola and NE uptake also had aslight to moderate linear component. In the third group (Fig.4C), MIBG uptake was significantly higher than for NE, witha large linear component. NE uptake was lower than for MIBGand the linear component was barely discernible. The fourthpattern was only observed with the cells from patient 11 (Fig.4D); there was almost no uptake of NE, and the total uptakeof MIBG was significantly less at any concentration (P < 0.05)than observed in any other pheochromocytoma cell preparation.

The complex, biphasic uptake kinetics, which were mostapparent with MIBG uptake, suggested that total uptake wascomposed of a saturable and an unsaturable component. Extrapolation of the linear component to 0.0 MM,and subtractionof this component from total uptake, revealed a high affinity,saturable component. Using this technique, a linear componentof total uptake could be subtracted from most NE and MIBGtotal uptake curves (14, 47). From the derived, saturable component, the kinetic parameters of the high affinity uptakesystem were calculated by using a nonlinear curve-fitting package (44). The kinetic constants for NE and MIBG uptake inseven pheochromocytoma cell preparations are shown in Table1. The Km (MM)values for NE and MIBG ranged from 0.17 to2.80 for NE and from 0.59 to 1.65 for MIBG; the Vmax(pmol/IO6 cells/10 min) values varied from 5.2 to 79.9 for NE and

from 18.4 to 113.4 for MIBG.Sodium Dependency of Uptake. The equimolar substitution

of LiCl for NaCl in the uptake medium is a classic means ofdemonstrating the sodium dependency and sodium specificityof the Uptake-one system ( 19,22,48). Since the biphasic uptakeof NE and MIBG could have been linked to its relative sodiumdependency, we evaluated the uptake of each agent in humanpheochromocytoma cell preparations, over a wide range ofconcentrations, in H-KRG and zero-sodium H-KRG.

As shown in Fig. 5, the relative percentage of these twouptake systems depended upon both the tumor from which the

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75

60-

3 45

mog so

.5

o-

NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

1 250

5 IO 15 20

[ SUBSTRATE,/iM]

25 IO 20 30 40

[SUBSTRATE,>iM]

250

Z 2OO

3 150_ibJO

*0 IOO

50

O IO 20 30 40

[SUBSTRATElyMM]50 IO 20 30

[ SUBSTRATE,

40 50

Fig. 4. Total uptake of (3H]NE (O) and ['"IJMIBG or [I3II)MIBG (•)into primary human pheochromocytoma cell cultures, as a function of concentration. A,patient 7; B. patient 9; C, patient 10; and 11.patient II. Human pheochromocytoma cells were incubated with increasing concentrations (0.15 to SO /JM)of |'l I|Mor I' "I¡MIH(. or |'"I|MIB<; at 37*C and 0"C as described in "Materials and Methods." Total uptake was calculated as uptake at 37'C (quadruplicate values) lessuptake at 0* (duplicate values) and is expressed as pmol/106 cells/10 min.

Table 1 Kinetic constants for the saturable component of total NE and MIBGuptake into primary cultures of human pheochromocytoma cells

Kinetic studies were done by incubating the cells in the presence of 0.2S to SOliM of NE or MIBG to determine the K„(/IM) and V„„(pmol/106 cells/10 min)for each agent. The kinetic parameters were calculated as described in "Materialsand Methods."

NE MIBG

Patient3679101314Km1.41±0.46"1.04

±0.071.21±0.102.80

±0.260.17±0.090.71

±0.14v_76.7

±8.279.9±2.377.1±2.069.9±3.45.2±0.349.0±2.1K.0.73

±0.060.90±0.070.70±0.071.44

±0.150.59±0.131.28

±0.251.65±0.31v_„90.3

±2.6113.4±2.868.0

±2.158.9±1.418.4±0.961.5

±3.495.3±4.7

°Mean ±SD.

cell culture was obtained and the concentration of NE andMIBG in the medium. We observed two basic patterns for therelative sodium dependency of uptake of NE and MIBG. Forone group of primary cell cultures, the uptake of NE wasprimarily sodium dependent out to SO n\i (Fig. 5/4), while the

uptake of MIBG became increasingly sodium independent atconcentrations greater than 10 to 15 MM(Fig. 5B). For thesecond group of primary cell cultures, the uptake of both NEand MIBG became predominantly sodium independent at concentrations greater than 5 or 20 J/M (Fig. 5, C and D). At lowconcentrations, both agents entered primarily by the sodium-dependent uptake system. As shown in Table 2, among 11tumor cell preparations, the uptake of 1.0 /¿MNE was 91.0 to100% sodium dependent; the uptake of 1.0 #/MMIBG was 55.7to 78.1% sodium dependent among the same tumor cell preparations. The sodium-independent component, in general, increased with increasing concentrations of either NE or MIBG.

Ouabain Inhibition Studies. Ouabain, a (Na K ) YTPase inhibitor (21), should inhibit only the sodium-dependent uptakecomponent of total uptake. The effect of ouabain on the uptakeof NE and MIBG in H-KRG solution and zero-sodium H-KRGsolution is shown in Fig. 6; the primary cell cultures were frompatients 9 and 16. The sodium-dependent uptake of both NEand MIBG was inhibited in a dose-dependent manner, whilethe sodium-independent uptake of both agents was not inhibited

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NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

100 r

Fig. S. Percentage of the total uptake forthe sodium-dependent and sodium-independent uptake systems of |3H|NE and ['"IJMIBGas a function of concentration, i. patient 10,NE uptake: B. patient 10. MIBG uptake; Cpatient 14, NE uptake: and /'. patient 14,MIBG uptake. The percentage of sodium-independent uptake (O) represents uptake inzero-sodium H-KRG expressed as a percentage of the total uptake (uptake in H-KRG).The percentage of sodium-dependent uptake(•)is the total uptake (uptake in H-KRG)minus the uptake in the zero-sodium H-KRG,expressed as a percentage of the total uptake.

IO 20 30 40

[NOREPINEPHRINE,>JM]90

O IO 20 30 40 SO

[NOREPINEPHRINE./JM]

KX>r

80

¡eo

40

20

0 •

0 10 20 30 40 50[META-IODOBENZYUGUANIDINE,>JM]

100r

O IO 20 30 40 50[META- lODOBENZYLGUANIDINE.uM]

Table 2 Sodium dependency of uptake of 1.0 ¡¡MNE and MIBG into primary-cultures of human pheochromocytoma cells

Cells were incubated in the presence of 1.0 u\i NE or MIBG in H-KRG orzero-sodium H-KRG as described in "Materials and Methods."

Patient257X9101113141517V-Na°15.213.431.64.420.216.00.830.019.14.6NEV-Li*1.50.70.00.00.50.20.01.40.00.0%ofNac91.095.0100.0100.097.698.8100.095.5100.0100.0V-Na16.612.223.715.07.528.536.18.36.7MIBGV-Li11.26.77.77.02.110.716.09.22.8%ofNa59.764.675.568.278.162.355.70.070.5

"Uptake in H-KRG less uptake in zero-sodium H-KRG, expressed as pmol/10*cells/10 min.

* Uptake in zero-sodium H-KRG, expressed as pmol/10* cells/10 min.' % of V-Na = xV'Na„,. x 100.

V-Na -I-V-Li

take-one system (19-21, 49). Since the neuronal Uptake-onesystem is sodium dependent and sodium specific (15, 19-21,48), it was expected that desmethylimipramine would selectively inhibit the sodium-dependent uptake system in the cultured human pheochromocytoma cells. As shown in Fig. 7, thesodium-dependent uptake system for NE and MIBG was inhibited by concentrations of desmethylimipramine several ordersof magnitude less than required to inhibit the sodium-independent uptake system.

Competitive Inhibition of Uptake. To determine if there is ashared uptake system for MIBG and NE, we evaluated theability of each agent to inhibit the uptake of the other. Theinhibition pattern displayed with the primary tumor cells frompatient 6 (Fig. 8) is similar to that observed with the primarytumor cells from two other patients. MIBG (1.0 ¿IM)was foundto be a mixed inhibitor (VmaX;* Vmaxj,Km¡* Km¿)of [3H]NE

uptake. In contrast, 10.0 MMM was found to be a competitiveinhibitor (Vmax,= Vmaxj,Armil* Kmj of [131I]MIBG uptake.

at any concentration of ouabain. This same pattern was observed with two other primary cell cultures in which this response was evaluated. In each experiment, there appeared to bea fraction of total uptake which was sodium dependent butouabain insensitive; additionally, the sodium-dependent uptakeof MIBG was less sensitive to ouabain inhibition than that ofNE.

Desmethylimipramine Inhibition Studies. Desmethylimipramine is a selective, competitive inhibitor of the neuronal Up-

DISCUSSION

The scintigraphic agent, MIBG, which was designed as anonmetabolizable 7-emitting analogue of NE, is believed to betaken up into pheochromocytomas and other adrenergic tissueby a NE uptake pathway (11,13,14,37). Although a NE uptakepathway was postulated more than a decade ago (50), its existence in human pheochromocytoma has never been directlyinvestigated. We used primary cell cultures obtained from 16

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NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

100u

803tj

6°¡31£

40Sg¡Ü20O-.•-_-V!\\iV•«Wto

-5-{-AiT•__itl5—1

1

B

V

01234 01LOG [OUABAINjuM]

234

Fig. 6. Selective inhibition of the sodium-dependent uptake system for ('111NE and ['"IJMIBG by ouabain, a (Na*/K*)ATPase inhibition. Sodium-independent uptake (uptake in zero-sodium H-KRG, D) is expressed as a percentage ofthe total uptake (uptake in H-KRG, •).Ouabain ( 10 to 10,000 (/MI was incubatedconcurrently with either [3H1NE or |I25I]MIBG in zero-sodium H-KRG (O) or inH-KRG (•)and uptake under these conditions is expressed as a percentage ofthe total uptake..!, patient 10. Ouabain inhibited (/' < 0.05) total uptake of 20MMNE at concentrations a 10.0 MM,but had no effect (P > 0.05) on sodium-independent uptake at concentrations between I and 10,000 MM.B, patient 17.Ouabain inhibited (/' < 0.05) total uptake of 0.5 (/M MIBG at concentrationsa32 MM,but had no effect (P > 0.05) on sodium-independent uptake at concentrations between 1 and 1,000 MM.

B

-2-101 23

LOG

-2-10123

Fig. 7. Inhibition of the sodium-dependent (•)and sodium-independent (O)uptake systems for |'H]NE and [I25I]MIBG by desmethylimipramine (DA//), atricyclic antidepressant and selective Uptake-one blocker. The concentration ofdesmethylimipramine ranged from 0.0001 to 1000 MM.The percentage of inhibition of the sodium-dependent and/or sodium-independent uptake is shown for:A. patient 2, 20 MM(3H]NE; and B, patient 10, 20 MM[125I]MIBG.

patients to directly study the uptake system(s) for both NE andMIBG in pheochromocytomas, making this the most extensivein vitro study of any single biochemical characteristic yet published. In this report we have shown that both NE and MIBGare transported and concentrated within these cells by twodifferent systems which were differentiated by their sodiumdependency and sodium specificity. We provide evidence forthe existence of a sodium-dependent system, which is probablyidentical with the neuronal characterized Uptake-one transportsystem (15-22), and of a sodium-independent uptake system,

ito

100

80

60

40

10

O IO 20 SO 40 90 «O TO 20 40 60 80 100 IZO

v/s v/sFig. 8. Kinetics of the inhibition of | 'I I|NK uptake by cold MIBG and the

inhibition of | " ' 11MIHi. uptake by cold NE in tumor cells of patient 6. The dataare presented by using Eadie-Hofstee plots; y represents the uptake velocity(pmol/10* cells/10 min) and S represents the substrate concentrations (MM)..1.uptake of [3H]NE in the absence (•)or presence (O) of 1.0 MMMIBG. B, uptakeof ["'IJMIBG in the absence (•)or presence (O) of 10 MMNE. The pattern of

inhibition displayed by using these cells is similar to those obtained with cellsfrom two other primary cell cultures.

which is probably passive diffusion. In addition, we observed avariable conservation and expression (heterogeneity) of thesetwo uptake systems within the population of pheochromocy-toma cells studied. Potential and observed interactions of patient medication with MIBG uptake are also considered.

The existence of two uptake systems was initially suspected,based upon kinetic studies which frequently showed a linear,unsaturated component: this was most readily observed in thekinetic studies of MIBG uptake (Fig. 4B). Two uptake systemswere separated and identified by using an equimolar substitution of LiCl for NaCl in order to demonstrate their sodiumdependency (Fig. 5). Although LiCl may have other cellulareffects (51-53), the use of LiCl to discriminate a sodium-dependent from a sodium-independent uptake system is a classic technique ( 14,15,19-21,48). The sodium-dependent uptakesystem was determined to be saturable and of high affinity andlow capacity for both NE and MIBG (Table 1). In most instances the Vmaxvalues for sodium-dependent MIBG uptakewere higher than for NE uptake, a finding consistent withobservations in bovine adrenomedullary cells (13, 14). In contrast to the sodium-dependent system, the sodium-independentuptake of NE and MIBG was linear out to at least 50 UM.Theobservation of two different kinetic patterns cannot be relatedto radiopharmaceutical impurities, since purity was greater than98% for both compounds (see "Materials and Methods"). Nei

ther would metabolism of NE or MIBG explain these observations. In patient studies, 46- to 65-h postinjection, 98 to 100%of the radioactivity recovered from tumors was native MIBG,while the remainder was free radioiodide (10). Metabolism orleakage of tracer NE is an insignificant factor in the study ofcatecholamine uptake in normal cells and tissues ( 19, 20), andthe literature indicates that the levels of catecholamine-O-methytransferase and monoamine oxidase in pheochromocytomas are the same or lower than in normal neuronal tissues(54). The association of sodium-dependent and sodium-independent uptake with the saturable and unsaturable kinetic patterns appears to represent a membrane phenomenon, since twomembrane-specific agents, ouabain and desmethyimipramine,had distinctly different effects upon the two systems.

Ouabain, a (Na+/K+)ATPase inhibitor (21 ), selectively inhibited the sodium-dependent uptake of both NE and MIBG in adose-dependent manner (Fig. 6). In contrast, even at 1.0 MM,ouabain had no effect upon the sodium-independent uptake ofeither agent. Ouabain inhibition of the sodium-dependent uptake of NE and MIBG provided evidence that the sodium-dependent catecholamine transport system is mechanistically

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NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

linked to the activity of the membrane bound (Na+/K+)ATPase

system, while the sodium-independent uptake system is not.The apparently ouabain-insensitive fraction of total uptake mayreflect decreased sensitivity of the tumor cells to this glycoside(55), but more likely reflects insufficient cellar exposure topermit the complete inhibition of the (NaYK+)ATPase, sinceouabain was coincubated with the [3H]NE and [125I]MIBG. Atime-dependent response to ouabain was observed in other cellsystems (55) and in bovine adrenomedullary cells (14).

Desmethylimipramine is a selective, competitive inhibitor ofthe membrane bound Uptake-one transport protein present inneuronal tissue (19, 20, 49) and in adrenomedullary cells (13,14). As shown in Fig. 7, desmethylimipramine inhibited sodium-dependent uptake of both NE and MIBG at concentrations several orders of magnitude less than required to inhibitthe sodium-independent uptake of these agents. Desmethylimipramine inhibited sodium-dependent uptake of both agentsat 50% inhibitory concentration (^M) values of approximately0.1 tíM,which are similar to those reported for Uptake-oneinhibition in other tissues and organs (19). Desmethylimipramine inhibition of the sodium-independent uptake was probablyrelated to nonspecific effects of this very lipophilic agent athigh concentrations (49). The selective inhibition of the sodium-dependent uptake of NE and MIBG provided further evidencethat the sodium-dependent and sodium-independent uptake areseparate and distinct systems.

These studies established that both NE and MIBG uptakeoccurred by a sodium-dependent system that was similar withrespect to saturability, kinetic parameters, and ouabain sensitivity, and was desmethylimipramine sensitive. In order todemonstrate that NE and MIBG shared the same sodium-dependent uptake transport system, we performed reciprocal,competitive inhibition studies. The competitive inhibition ofradiolabeled MIBG by "cold" NE (Fig. 8Ä)indicated a shared

uptake mechanism for MIBG and NE transport. In the reciprocal experiments, the mixed inhibition of radiolabeled NEuptake by cold MIBG (Fig. SA), indicates both a competitive(shared uptake) and a noncompetitive (indirect) effect of MIBGupon the NE uptake system. The direct, reciprocal inhibitionof one agent for the other points to a common, shared transportsystem. The noncompetitive inhibition of [3H]NE uptake by

MIBG may reflect a nonspecific interaction of the lipophilicMIBG molecule with the catecholamine transport protein (49).Further evidence of a shared uptake system is provided by theconservation of the MIBG to NE uptake ratio. Among 15primary cell cultures, in which the uptake velocities variedseveral hundredfold (Fig. 2), the correlation between uptake of1.0 UM MIBG and NE was high (r = 0.948), and the ratio ofMIBG to NE uptake (1.4:1) was similar to the 1.7:1 ratioobserved in bovine adrenomedullary cells.5

Thus, in primary cultures from 16 human pheochromocyto-mas, the sodium-dependent uptake of both NE and MIBG wasfound to fulfill the following criteria for the Uptake-one transport system: (a) temperature dependency, (b) high affinity, (c)low capacity, (d) saturability, (e) sodium dependency, (/) ouabain sensitivity, and (g) desmethylimipramine sensitivity. Energy dependency and stereospecificity of the sodium-dependentsystem are the only characteristic of the Uptake-one systemwhich, because of their scarcity, we did not investigate in theseprimary cell cultures. However, energy dependence (14) andstereospecificity5 for sodium-dependent NE and MIBG uptake

were demonstrated in bovine adrenomedullary cells. Since NEand MIBG also compete for the same transport system, theevidence strongly suggests that the sodium-dependent uptake

of both NE and MIBG in primary pheochromocytoma cellcultures is via the Uptake-one system.

NE and MIBG were also transported into human pheochromocytoma cells by a sodium-independent system which is probably passive diffusion. The characteristics of the sodium-independent uptake system, which are consistent with a diffusionmechanism (47) and/or an interaction with membrane phos-pholipids (56) are: (a) temperature dependency, (b) linear uptake as a function of increased substrate concentration (to 50UM), (c) ouabain insensitivity, and (d) decreased desmethylimipramine sensitivity. Two other characteristics of sodium-independent uptake consistent with a passive diffusion systemwere determined in bovine adrenomedullary cells; uptake wasenergy independent and linear out to 5 HIM(14). It is unlikelythat sodium-independent uptake represents membrane binding.In bovine adrenomedullary cells, acetylcholine, which stimulates exocytotic release of storage vesicles, released both agentsin similar proportion, irrespective of whether the cells wereloaded in the presence or absence of sodium.5

The NE uptake system appears to function independently ofcatecholamine synthesis. We reported a poor correlation between pHjNE uptake and cellular catecholamine levels (34).Similarly, MIBG uptake did not correlate (r = 0.156) withcellular catecholamine content at day 5 of culture (Fig. 3). Thecorrelation could be improved to 0.816 by discarding two points(patients 3 and 6), but this manipulation is difficult to justify,since cellular catecholamines were standardized to day 5 ofculture and uptake of 1.0 UMMIBG was replicable 3 to 6 daysafter the initial determination. These observations suggest thatthe uptake system(s) functions independently of catecholaminebiosynthesis and/or storage mechanisms and is consistent withthe report of a paraganglioma which concentrated MIBG invivo but did not secrete catecholamines (57).

Heterogeneous expression of basic mechanisms, such as enzyme patterns (54), different secretory patterns, and sensitivityto acetylcholine (34), have been observed in human pheochromocytoma tissue and/or cells as well as in a variety of othertumor tissues (58, 59). In these studies, heterogeneous expression of the uptake systems was apparent in the 200- to 300-fold variability in uptake velocity of 1.0 ÕÕMNE and MIBG(Fig. 2), the variability in the kinetic patterns (at least fourgeneralized kinetic patterns were observed; Fig. 4), and differentpatterns of sodium dependency (at least two patterns wereobserved; Fig. 5). The variability of these characteristics appearsto be due to variable, between tumor, conservation, and expression of both the sodium-dependent and the sodium-independentuptake systems.

Despite its variable expression, the sodium-dependent systemwas conserved, and was similar, in at least two respects, to thatreported in bovine adrenomedullary cells. First, the system wasof high affinity and saturable. The Km(UM)values, which rangedfrom 0.17 to 2.8 for NE, and from 0.59 to 1.65 for MIBG(Table 1) were similar to those determined in bovine adrenomedullary cell; i.e., 1.4 ±0.5 for NE and 1.2 ±0.1 for MIBG(14). Second, sodium dependency of 1.0 ¿IMNE and MIBGuptake (Table 2), was similar to that observed in bovine adrenomedullary cells; i.e., 91-100% compared to 90-95% for NEin bovine cells and 59.7-75.5% compared to 60-65% for MIBGin bovine cells (14).

These studies, and those in bovine adrenomedullary cells (13,14), provide insight into the mechanisms underlying scintigra-phy with MIBG. The existence of a sodium-dependent uptakesystem for NE and MIBG, which is probably Uptake-one, isdirectly documented for the first time. Since blood concentra-

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NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

lions in dogs shortly after injection (13), were no greater than0.025 MM,this suggests that the sodium-dependent system isthe major uptake route for in vivo scintigraphy. In addition, thedescription of these uptake systems provides a mechanistic basisfor understanding the known and potential interference of drugssuch as desmethylimipramine (13, 60) and cardiac glycosides.These studies with primary tumor cultures also suggest that thekinetic parameters of sodium-dependent MIBG uptake, whichvaried widely, may not define the limits of scintigraphic detection, since all of the tumors used in this study were readilyvisualized with [I3II]MIBG. Therefore, other cellular mechanisms, such as sodium-independent uptake and/or storage andretention, may be as important as the sodium-dependent uptakesystems for tumor visualization with MIBG scintigraphy.

In summary, these studies represent the largest existing invitro data base concerned with any single biochemical characteristic of primary human pheochromocytoma cell cultures.Using tumor cells from 16 patients, we have shown that NEand MIBG were transported and concentrated within theseprimary cell cultures by two different pathways which weredifferentiated by their sodium dependency and sodium specificity. The evidence supports the concept that the primary pathwayis the sodium-dependent system which is probably identicalwith the neuronally characterized Uptake-one system; the sodium-independent system appears to be passive diffusion. Consistent with observation in other tumors, but in contrast to"normal" bovine adrenomedullary cells, the relative expression

of these two uptake systems was quite variable between tumors.

ACKNOWLEDGMENTS

We thank Linder Markham for help in preparing the manuscript,James A. Baker for his excellent technical assistance, and Drs. DonaldM. Wieland, Thomas J. Mangner, and Helen Lee for synthesizing andradiolabeling MIBG. We also thank the Phoenix Memorial Laboratoryat the University of Michigan for use of their radiolabeling facilities.

REFERENCES

1. Sisson. J. C, Frager, M. S.. Valk, T. W., Gross, M. D.. Swanson, D. P.,Wieland, D. M., Tobes, M. C, Beierwaltes. W. H., and Thompson, N. W.Scintigraphic localization of pheochromocytoma. N. Engl. J. Med., 305:12-17, 1981.

2. Valk, T. W., Frager, M. E.. Gross, M. D., Sisson, J. C., Wieland, D. M.,Swanson, D. P., Mangner, T. J.. and Beierwaltes, W. H. Spectrum ofpheochromocytoma in multiple endocrine neoplasia. A scintigraphic portrayal using >3'I-metaiodobenzylguanidine. Ann. Intern. Med.. 94: 762-767,

1981.3. Shapiro. B., Copp, J. E., Sisson, J. C., Eyre, P. L., Wallis, J., and Beierwaltes,

W. H. Iodine I <1 metaiodobenzylguanidine for the locating of suspectedpheochromocytoma: experience in 400 cases. J. Nucí.Med., 26: 576-585,1985.

4. Sisson, J. C., Shapiro, B. C., Beierwaltes, W. H., Glowniak, J. V., Nakajo,M., Mangner, T. J., Carey, J. E.. Swanson, D. P., Copp, J. E., Satterlee, W.G., and Wieland, D. M. Treatment of malignant pheochromocytomas witha new radiopharmaceutical. Trans. Assoc. Am. Physicians, 96: 209-217,1983.

5. Sisson, J. C., Shapiro, B. C.. Beierwaltes, W. H., Glowniak, J. V., Nakajo,M., Mangner, T. J., Carey, J. E., Swanson, D. P., Copp, J. E., Satterlee, W.G., and Wieland. D. M. Radiopharmaceutical treatment of malignant pheochromocytoma. J. Nucí.Med., 25: 197-206, 1984.

6. >]'I-/n«ra-Iodobenzylguanidine in therapy, diagnosis and monitoring of neuroblastoma: IV. International Workshop in Pediatrie Oncology, UniversitàCatólica, Rome, Italy, September 22-23, 1986.

7. Kline, R. C., Swanson, D. P., Wieland, D. M.. Thrall, J. H.. Gross, M. D.,Pitt, B., and Beierwaltes, W. H. Myocardial imaging in man with 1-123metaiodobenzyl-guanidine. J. Nucí.Med., 22: 129-132, 1981.

8. Sisson. J. C., Wieland, D. M., Sherman, P., Mangner, T. J., Tobes, M. C.,and Jaques, S., Jr. Metaiodobenzylguanidine as an index of the adrenergicnervous system integrity and function. J. Nucí.Med.. in press, 1987.

9. Sisson, J. C., Shapiro, B., Meyers, L., Mallet, S., Mangner, T., Wieland, D.,Glowiack, J., Sherman, P., and Beierwaltes, W. H. Metaiodobenzylguanidineto map scintigraphically the adrenergic nervous system in man. J. Nucí.Med., in press, 1987.

10. Mangner, T. J., Tobes, M. C.. Wieland, D. M., and Sisson, J. C. Metabolismof mf/a-l-131-iodobenzylguanidine in patients with metastatic pheochromocytoma: concise communication. J. Nucí.Med., 27: 37-44, 1986.

11. Wieland, D. M., Brown, L. E., Tobes, M. C., Rogers, W. L., Marsh, D. D.,Mangner, T. J., Swanson, D. P., and Beierwaltes, W. H. Imaging the primateadrenal medulla with | '•''11and ['3'I)mera-iodobenzylguanidine: concise com

munication. J. Nucí.Med., 22:358-364, 1981.12. Wieland, D. M., Brown, L. E.. Rogers, W. L., Worthington, K. C., Wu, J.,

Clinthorne, N. H., Otto, C. A., Swanson, D. P., and Beierwaltes, W. H.Myocardial imaging with a radioiodinated norepinephrine storage analog. J.Nucí.Med., 22:22-31, 1981.

13. Tobes, M. C., Jaques, S., Jr., Wieland, D. M., and Sisson, J. C. Effect ofuptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine. J. Nucí.Med., 26:897-907, 1985.

14. Jaques, S., Jr., Tobes, M. C., Sisson, J. C., Baker, J. A., and Wieland, D. M.Comparison of the sodium dependency of uptake of mffo-iodobenzylguani-dine and norepinephrine into cultured bovine adrenomedullary cells. Mol.Pharmacol., 26: 539-546, 1984.

15. Bogdanski, D. F., and Brodie. B. B. Role of sodium and potassium ions instorage of norepinephrine by sympathetic nerve endings. Life Sci., 5: 1563-1569, 1966.

16. Wakade, A. R., and Furchgott, R. F. Metabolie requirements for the uptakeand storage of norepinephrine by isolated left atrium of the guinea pig. J.Pharmacol. Exp. Ther., 163: 123-125, 1968.

17. Colburn, R. W., Goodwin, F. K., Murphy, D. L., Bunney, W. E., Jr., andDavis, J. M. Quantitative studies of norepinephrine uptake by synaptosomes.Biochem. Pharmacol., 17: 957-964, 1968.

18. Coyle, J. T., and Snyder, S. H. Catecholamine uptake by synaptosomes inhomogenates of rat brain: stereospecificity in different areas. J. Pharmacol.Exp. Ther., 170: 221-231, 1969.

19. Iverson, L. L. Uptake processes for biogenic amines. In: L. L. Iverson, S. D.Iverson, and S. H. Snyder (eds.). Handbook of Psychopharmacology, Vol. 3,pp. 381-442. New York: Plenum Press, 1975.

20. Iverson, L. The uptake of biogenic amines. In: J. H. Biel and L. G. Abood(eds.), Biogenic Amines and Physiological Membranes in Drug Therapy, pp.264-306. New York: Marcel Dekker, Inc., 1971.

21. Patón. D. M. Characteristics of uptake of noradrenaline by adrenergicneurons. In: D. M. Patón(ed.). The Mechanisms of Neuronal and Extraneu-ronal Transport of Catecholamines, pp. 49-66. New York: Raven Press,1976.

22. Graefe, K. H., and Bonish, H. The influence of the rate of perfusion on thekinetics of neuronal uptake in the rabbit isolated heart. Naunyn-Schmiede-bergs Arch. Pharmacol., 302: 275-283, 1978.

23. Cabana, B. E., Prokesch, J. C., and Christiansen, G. S. Study on thebiogenesis of catecholamines in pheochromocytoma tissue culture. Arch.Biochem. Biophys., ¡06:123-130, 1964.

24. Costero, I., Chevez, A. Z., Peralta, L., Monroy, E., and Ramon, R. Rhythmiccellular movements in tissue cultures of pheochromocytoma and adrenalmedulla. Tex. Rep. Biol. Med., 23: 213-220, 1965.

25. Misugi, K., Misugi. N., and Newton, W. J. Fine structural study of neuroblastoma, ganglioneuroblastoma and pheochromocytoma. Arch. Pathol., 86:160-169, 1968.

26. Kadin, M. E., and Bensch, K. G. Comparison of pheochromocytomas withganglion cells and neuroblasts grown in vitro: an electron microscopic andhistochemical study. Arch. Histol. Jpn. (Suppl.), 40: 61-79, 1971.

27. Tischler, A. S., Dichter, M. A., and Biales, B. Neuronal properties of culturedhuman endocrine tumor cells of proposed neural crest origin. Science (Wash.DC), / 92: 902-904, 1976.

28. Nishihira, T., Kasai, M., Hayashi, Y., Kimura, M., Matsumure, Y., Akaishi,T., Ishiguro. S., Kataoka, S.. Watanabe, H., Miura, Y.. and Haruo, S.Experimental studies on differentiation of cells originating from humanneural crest tumors in vitro and in vivo. Cell. Mol. Biol., 27: 181-196, 1980.

29. Pfragner, R.. and Walser, V. Long-term culture of human pheochromocytomas. Exp. Pathol (Jena). IS: 423-429, 1980.

30. Wilson, S. P.. Slepetis, R., Chang, K. J.. Kirshner, N., and Viveros, H.Different secretion of opioid peptides and catecholamines from cultured cellsof a human pheochromocytoma tumor. Life Sci., 29: 2257-2264, 1981.

31. Pfragner, R., Sadjak, A., and Walser, V. The effect of nerve growth factor(NGF) on the catecholamine contents of two human pheochromocytomas intissue culture. Exp. Pathol. (Jena), 26: 21-31, 1984.

32. Tischler, A. S., Lee, H. C., Perlman. R. L., Costopoulos, D., Slayton, V. W.,and Bloom. S. R. Production of "ectopie" vasoactive intestinal peptide-likeand neurotensin-like immunoreactivity in human pheochromocytoma cellcultures. J. Neurosa., 4: 1389-1404. 1984.

33. Tischler, A. S., Allen, J. M., Costopoulos, D., and Bloom. S. R. Increasedcontent of neuropeptide Y in human pheochromocytoma cell cultures. J.Clin. Endocrinol. Metab., 61: 303-305, 1985.

34. Jaques. S., Jr.. and Tobes, M. C. Morphologic and biochemical variability oftissue and cultured cells from human pheochromocytoma. J. Cell Physiol.,128: 261-270. 1986.

35. Greene, L. A., and Rein, G. Release, storage and uptake of catecholaminesby a clonal cell line of nerve growth factor (NGF) responsive pheochromocytoma cells. Brain Res., 129: 247-263, 1977.

36. Warren, S.. Grozdev, L., Gates, O., and Chute, R. N. Radiation-inducedadrenal medullary tumors in the rat. Arch. Pathol., 82: 115-118. 1966.

37. Shapiro, B., Copp, J. E., Sisson. J. C., Eyre, P. L.. Wallis, J., and Beierwaltes.W. H. Iodine-131 metaiodobenzylguanidine for the locating of suspected

3927

Research. on September 5, 2020. © 1987 American Association for Cancercancerres.aacrjournals.org Downloaded from

NE AND MIBG UPTAKE IN HUMAN PHEOCHROMOCYTOMA CELLS

pheochromocytoma: experience in 400 cases. J. Nucí.Med.. 26: 576-585,1985.

38. Wieland, D. M., Wu, J., Brown, L. E., Mangner. T. J., Swanson, D. P., andBeierwaltes, W. H. Radiolabeled adrenergic neuron-blocking agents: adrenomedullary imaging with [I3ll]iodobenzylguanidine. J. Nucí.Med.. 21: 349-

353, 1980.39. Mangner, T. J., Wu, J. L.. and Wieland, D. M. Solid-phase exchange

radioiodination of aryliodides. Facilitation by ammonium sulfate. J. Org.Chem., 47: 1484-1488, 1982.

40. Kilpatrick, D. L., Ledbetter, F. H., Carson, K. A., Kirshner, A. G.. Slepetis,K . and Kirshner, N. Stability of bovine adrenal medulla cells in culture. J.Neurochem., 35:679-692, 1980.

41. Anton, A. H.. and Sayre, D. F. A study of the factors affecting the aluminumoxide trihydroxy indole procedure for the analysis of catecholamines. J.Pharmacol. Exp. Ther., 138: 360-375, 1962.

42. Peuler, J. D., and Johnson, G. A. Simultaneous single isotope radioenzymaticassay of plasma norepinephrine, epinephrine and dopamine. Life Sci., 21:625-636, 1977.

43. De La Torre, J. C., and Surgeon. J. W. A methodological approach to rapidand sensitive monoamine histofluorescence using a modified glyoxylic acidtechnique: the SPG method. Histochemistry. 48: 81-93, 1976.

44. Greco, W. R., Priore, R. L., Sharma, M., and Korytnyk, W. ROSFIT: anenzyme kinetics nonlinear regression curve fitting package for a microcomputer. Comput. Biomed. Res., IS: 39-45, 1982.

45. Zivin. J. A., and Wand. D. R. Minireview. How to analyze binding, enzymeand uptake data: the simplest case, a single phase. Life Sci., 3: 1407-1422,1982.

46. Steel, R. D. G., and Torrie, J. H. Principles and Procedures for Statistics.New York: McGraw-Hill Book Co., 1960.

47. Stahl, S. M., and Meltzer, H. Y. A kinetic and pharmacologie analysis of 5-hydroxytryptamine transport by human platelets and platelet storage granules: comparison with central serotonergic neurons. J. Pharmacol. Exp.Ther., 205: 118-132, 1978.

48. Iverson, L. L.. and Kravitz. E. A. Sodium dependence of transmitter uptakeof adrenergic nerve terminals. Mol. Pharmacol.. 2: 360-362. 1966.

49. Maxwell, R. M., Ferris, R. M., and Burcsu, J. E. Structural requirements forinhibitions of noradrenaline uptake by phenethylamine derivatives, desipra-mine, cocaine and other compounds. In: D. M. Paton (ed.). The Mechanismsof Neuronal and Extraneuronal Transport of Catecholamines, pp. 95-153.New York: Raven Press, 1976.

50. Anderson, B. G., Beierwaltes, W. H., Harrison, T. S., Ansari, A. N., Buswink,A. A., and Ice, R. D. Labeled dopamine concentration in pheochromocyto-mas. J. Nucí.Med., 14: 781-809, 1973.

51. Forn, J. Lithium and cyclic AMP. In: F. N. Johnson (ed.), Lithium Researchand Therapy, pp. 485-497. New York: Academic Press, Inc., 1975.

52. Hallcher, L. M., and Sherman, W. R. The effects of lithium ion and otheragents on the activity of myo-inositol-1-phosphatase from bovine brain. J.Biol. Chem., 255: 10896-10901, 1980.

53. Nordenberg, J. M., Kaplansky, M., Beery, E., Klein, S., and Beitner, R. Theeffects of lithium on the activities of phosphofructokinase and phosphoglu-comutase and on glucose-1,6-diphosphate levels in rat muscles, brain andliver. Biochem. Pharmacol., 31: 1025-1031, 1982.

54. Feldman, J. M., Blalock, J. A., Zern, R. T., and Wells, S. A. The relationshipbetween enzyme activity and the catecholamine content and secretion ofpheochromocytomas. J. Clin. Endocrino!. Metab., 49:451-455, 1979.

55. Schwartz, A., Lindenmayer, G. E., and Allen, J. C. The sodium-potassiumadenosine triphosphatase: pharmacological, physiological and biochemicalaspects. Pharmacol. Rev., 27: 3-134, 1975.

56. Shaefer, G. Guanidines and biguanidines. Pharmacol. Ther., 8: 275-295,1979.

57. Smit, A. J., VanEssen, L. H., Hollema. H., Muskiet, A. A. J., and Piers, D.A. mffa-["3']lodobenzylguanidine uptake in a nonsecreting paraganglioma.J. Nucí.Med., 25: 984-986, 1984.

58. Weber, G. Key enzymes and tumor cell heterogeneity. Antibiot. Chemother.,28: 53-61, 1980.

59. Weinhouse, S. New dimensions in the biology of cancer. Cancer (I'hila.), 45:2975-2980, 1980.

60. Nakajo, M., Shapiro. B., Sisson, J. C., Swanson, D. P., and Beierwaltes, W.H. Salivary gland accumulation of m?to-['3ll]iodobenzylguanidine. J. Nucí.Med., 25: 2-6, 1984.

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mSodium Dependency of Uptake of Norepinephrine and

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