ß1-Adrenergic receptors in human neuroblastoma

Brain Research, 216 (1981) 73-87 © Elsevier/North-Holland Biomedical Press

73

fll-ADRENERGIC RECEPTORS IN H U M A N N E U R O B L A S T O M A

JEFFREY A. WHITSETT*, AKIHIKO NOGUCHI, J. E. NEELY, C. L. JOHNSON and J. J. MOORE Children's Hospital Research Foundation and the Newborn Division, Department of Pediatrics, University of Cincinnati College of Medicine, 231 Bethesda Avenue, Cincinnati, Ohio 45267 (U.S.A.)

(Accepted December 4th, 1980) Key words: Neural crest tumors - - neuroblastoma - - fl-adrenergicreceptor - - (--)-[aH]dihydroal-

prenolol - - (--)-[12sI]iodohydroxybenzylpindolol- adenylate cyclase -- guanine nucleotide

SUMMARY fl-Adrenergic receptors and adenylate cyclase activity were identified and partially characterized in 10 human neuroblastoma tumors obtained from direct biopsy, from human cell lines grown in the athymic, nude mouse or from established tumor cell lines in tissue culture. Particulate membrane fractions of eight of the tumors contained fl-adrenergic receptor sites identified by the fl-adrenergie antagonists (w). [3H]dihydroalprenolol ((--)-[3H]DHA) and (--)-[t25I]iodohydroxybenzylpindolol ((--)-[125]HyP). Specific binding of high affinity sites (Bmax) varied greatly among the tumors ranging from non-detectable concentrations to approximately 130 fmol/mg of crude membrane protein. Analysis of the saturation experiments with (--)-[SH]DHA resulted in curvilinear Scatchard plots in the tumors, suggesting the presence of multiple classes of sites and there was no evidence of cooperativity in dissociation experiments with (--)-[aH]DHA. The high affinity class of sites were inhibited by catecholamines, in contrast to the ineffectiveness of catecholamines in inhibiting (--)[aH]DHA binding at the low affinity class of sites. The 1(o of the high affinity site was approximately 1-2 nM for (--)-[3H]DHA and was similar in all the tumors studied. A single class of sites was demonstrated with (--)-[125I]HYP, in several of the tumors studied, Ko approximately 120 pM, and the number of sites determined with (--)[125I]HYP was identical to the number of high affinity sites determined with (--)[aH]DHA. The properties of the receptor were characterized in several tumor lines: SK-N-MC tumor line contained both a fl-adrenergic receptor site and catecholamine * To whom reprint requests should be addressed at: Newborn Division, Department of Pediatrics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267, U.S.A.

74 sensitive adenylate cyclase activity. Agomsts competed for the fl-adrenergic sites in the order of potency (--)-Jsoproterenol 3~ (--)-norepinephrme ::-- (--)-epinephrine j . (--)-dopamine characteristic of a/51-adrenerglc receptor subtype. The affinity of the receptor for the selective agents, metroprolol (ill-selective antagomst) and zinterol (/~2selective agomst) was typical of a homogeneous fll-adrenergic site. Guanine nucleotides, guanine tnphosphate and guanyl-5'-yl-imidophosphate, decreased catecholamine affinity for the receptor site in several tumors studied and enhanced maximal catecholamine stimulated adenylate cyclase activity m the sensitive cell line. Adenylate cyclase activity in particulate fractions of most of the neuroblastoma tumors was responsive to prostaglandms (PGE1), guanine nucleotide and NaF. While most tumors contained fl-adrenerglc receptor sites only SK-N-MC was clearly catecholamine responsive as assessed by catecholamlne-sens~twe adenylate cyclase activity. Human neuroblastomas, a homogeneous tissue of neural crest origin, contained fl-adrenerglc receptor sites which varied greatly m number (Bmax)and m their ability to stimulate adenylate cyclase actwlty. Guanine nucleotides decreased agomst affimty for the receptor m the hnes tested and enhanced prostaglandin and catecholamine sensltwe actwlty in responsive tumor lines. INTRODUCTION Human neuroblastomas contain some of the synthetic and neurotransmitter properties present in more mature synaptic cells from sympathetic or parasympathetic ganglia. The presynaptic component may be represented by cells which produce a wide variety of neurotransmitters including catecholamines, acetylcholine and serotomn 1, 8,23 and might also contain auto-receptors for such hormones. Similarly a postsynaptlc component might be represented by the presence of receptors for these various neurotransmitters, and by adenylate cyclase responsive to hormonal stimulation2, 24. Neuroblastoma cell lines with muscarinic cholinergic, opiate receptors and nerve growth factor binding sitesle,25,82, as well as prostaglandin, dopamine and epinephrine sensitive adenylate cyclase zS, have been characterized - - the presence of hormonesensitwe adenylate cyclase suggesting the presence of specific receptors linked to the catalytic subunit of adenylate cyclase. While catecholamine stimulates adenylate cyclase m mouse neuroblastomas the identification of fl-adrenergic receptors, their relationship to adenylate cyclase, and the role of guanine nucleotides in mediating that relationship, have not been previously demonstrated in either mouse or human neuroblastomas. Stimulation of adenylate cyclase and inhibition of 3',5'-cyclic AMP phosphodiesterase results in the accumulation of cAMP in mouse neuroblastoma and numerous studies have indicated that cAMP may play an important role in the differentiation of this tumor resulting in increased neurite formation, tyrosine hydroxylase activity and decreased growth rate in culture24. Similarly, cAMP levels in neuroblastoma, ganglioneuroma and the sympathetic ganglion vary directly with differentiated characteristics in these tissues 7. On the basis of the increased adenylate cyclase stimulation by fl-adrenergic receptor sites and increased guanine nucleotide

75 sensitivity of receptor-cyclase interaction in experimentally differentiated mouse neuroblastoma cells, it was also suggested that the aspects of receptor-adenylate cyclase relationship may relate to the degree of differentiation of the tumorZ1,22,z4. The importance of guanine nucleotides in the activation of hormone sensitive adenylate cyclase has been extensively described in numerous systems including neuroblastoma14, 28-2s. Guanine nucleotides are thought to be an absolute requirement for hormonal activation of adenylate cyclase. Guanine nucleotide-dependent factors interact with the hormone-receptor complex, altering agonist affinity for the receptor and also interact directly with the catalytic subunit (adenylate cyclase)10,1a,14. Defects in the various components of the hormone-receptor-guanine nucleotide site-adenylate cyclase system have also been described in'hormone responsive and unresponsive 549 lymphoma clones 15. The possibility of heterogeneity in receptor number and capacity to stimulate adenylate cyclase in neuroblastoma tumor lines was therefore also investigated for catecholamines and prostaglandins. Lastly, cerebral tissues contain mixed populations of ill- and fl~-adrenergic receptors whose affinity for agonists is apparently not altered by guanine nucleotide 6. However, because of the marked heterogeneity of neuronal and non-neuronal cell types in this tissue, specific cellular location of fl-adrenergic sites and their characterlstics have not been clarified 17. fll and f12 receptors and their relative proportion in various tissues can be determined utilizing the ability of/~1- and fl2-specific adrenergic agonists and antagonists to displace radiolabeled fl-adrenerglc antagonists from these receptor sites~SA9. In the present study we have utilized classical fl-adrenergic agonists as well as the ill-specific antagonist metoprolol and the fl2-speclfiCagonist zinterol, to characterize the fl-adrenergic sites demonstrated in human neuroblastoma. METHODS AND MATERIALS [all]cAMP, [a-32p]ATP, (--)-[125I]iodohydroxybenzylpindolol ((--).[125I]HYP), 2200 Ci/mmol, and (--)-[SH]dihydroalprenolol ((--)-[aH]DHA), 56 Ci/mmol were purchased from New England Nuclear Co. Catecholamines and other reagents were obtained from Sigma Chemical Co. Guanyl-5'-yl-imidophosphate (Gpp(NH)p) was obtained from ICN, Chicago. Prostaglandins El, E2, A1, A2, F2a, D2 and Is were the kind gift of the Upjohn Co. (--)-Propranolol and (+)-propranolol were obtained from Ayerst Co. Metoprolol was the generous gift of Ciba-Geigy Corp., and zinterol from the Mead-Johnson Corp.

Tumor preparation Neuroblastomas were obtained both immediately at biopsy as well as after passage in the athymic, nude mouse (NIH Swiss background). The diagnosis of neuroblastoma was confirmed by clinical, histologic and electron microscopic characteristics of each tumor. Cell lines of human neuroblastoma SK-N-MC and SK-N-SH were obtained from Memorial Sloan-Kettering Institute for Medical Research, Cambden, and LA-N-1 and 1MR-32 were kindly provided by R. Akeson (Cincinnati, Ohio) and maintained in serial passage in vitro and nude mice. Karyotypes from these

76 hnes contain marker chromosomes and karyotypes were stable and characteristic for each line. The histology of all the tumors were characteristic ofneuroblastoma in both cell culture or nude mouse cultures. Cells for assay were grown to 75 ~ confluency in minimal essential media (Eagle) with 10 ~ fetal calf serum (Gibco), L-glutamate, and non-essential amino acids. Samples were washed extensively in an iced buffer containing 250 mM sucrose, 1 mM EGTA and 10 mM Tris.HCl (pH 7.2). Cells from 3 to 5 flasks were mechanically removed, pooled, and homogenized in the same iced buffer by three 5-sec bursts with a Tekmar Tlssuemizer (Cincinnati). The homogenate was filtered through 4 layers of gauze and centrifuged at 3000 ," g for 5 min. The pellet was dlscarded and the supernatant centrifuged for 20 min at 40,000 , g for 20 mm. The pellet was resuspended in 50 ml of the sucrose buffer and centrifuged again for 20 min at 40,000 ~ g. Th~s pellet was resuspended m the same buffer to a final membrane protein concentration of approximately 5-10 mg/ml by the method of Lowry et al. 12. Nude mouse tumors and biopsy tumors

Human tumor cell hnes were also grown by bilateral subcutaneous injection of minced tumors in nude mice (Harlan Inc.) to approximately 10 g per nude mouse. Animals were sacrificed by cervical dislocation, the tumors were minced and homogenized in 10 vol. of iced buffer. Crude membranes were prepared as described for the cells in culture. Fresh human tumors, obtained at biopsy, were ~mmedmtely placed in iced media, weighed and homogenized as described for tumors obtained from the nude mouse (above). Membranes were frozen in dry ice-acetone and stored at --80 °C for up to one month prior to assay. Adenylate cyclase, (--)-[3H]DHA and (--)-[125I]HYP binding were stable for several months under these conditions. Binding assays

(--)-[3H]DHA binding was determined as previously described a~,a7 in a 250/zl assay containing 100-300 #g protein, l0 mM MgCl2 and 0.2-50 nM (--)-[aH]DHA, 50 mM Tris.HCl (pH 7.4). Binding was assessed in triplicate by filtration assay after 20 min incubation at 30 °C; the assay was terminated by filtration on glass fiber filters and washed 5 times with 5 ml of the iced MgCle, Tris.HCl buffer. Non-specific binding was also assessed in triplicate in the presence of 1 /~M (-+-)-propranolol or 1 /~M (--)alprenolol which resulted in identical non-specific binding values. (--)-[1251]HYP binding was determined as described by Minneman et al. 17 in 10 mM MgC12, 50 mM Tris.HCl (pH 7.4) buffer in the presence and absence of 100/~M G T P or 100/~M Gpp (NH)p. The protein concentration ranged from 30 to 40/~g per assay and the (--)[125I]HYP varied from 25 to 300 pM for Scatchard analyses. The assay for competition experiments were performed at 50 or 100 pM (--)-[125I]HYP, near the 1(o for (--)[125I]HYP binding, at 37 °C for 30 min. The assay was terminated by rapid filtration on glass fiber filters, and washed 3 times with 4 ml of the incubation buffer at 37 °C. A longer incubation period (1 h) was required to reach equilibrium at low (--)-[125I]HYP concentrations for saturation experiments. Specific binding was proportional to protein under equilibrium conditions for both radioligands. Ko was determined from competition experiments with (_).[125I]HYp using the equation:

77 ECso KD=

[1251]HYP 1+ KD for [125I]HYP

Curvilinear Scatchard plots obtained from experiments with (--)-[SH]DHA at (--)-[aH]DHA concentrations significantly above the KD were resolved into two linear components by the method of Thakur and Rodbard as. This method uses the X and Y intercept and the slope of the Scatchard plot determined graphically at intercepts to calculate receptor binding characteristics. We assumed that the curvilinearity of the (--)-[aH]DHA Scatchards resulted from the presence of two heterogeneous, noncooperative (--)-[aH]DHA binding sites and used a Texas Instruments Model TI-59 computer and the program developed by Thakur et al. a4 to calculate the affinities (KD) and maximum binding capacity (Braax) of the two binding sites.

Adenylate cyclase Adenylate cyclase was determined at 30 °C in triplicate by a modification of the method of Salomon et al. al as previously described85,36. The assay contained 10-20/~g protein, 0.2 mM [a-3~P]ATP (2 #Ci), 4.5 mM MgCI2, 1 mM Jail]cAMP (0.01 #Ci), a creatine phosphokinase-phosphocreatine regeneration system, 0.1 ~ bovine serum albumin, 5 mM theophyUine, 12.5 mM sucrose, 0.05 mM EGTA and added hormone and guanine nucleotide. The assay mixture was preincubated for 5 min at 30 °C and the reaction initiated by the addition of labeled ATP. Basal and hormone-stimulated cAMP production was entirely linear for at least 30 min in this assay and triplicate determinations usually varied less than 5 ~o. Column recoveries of [SH]cAMP varied from 60 to 75 ~ and were not altered by theophylline. RESULTS

Identification of fl-adrenergic receptor with (--)-ESH]dihydroalprenolol (--)-[SH]Dihydroalprenol bound to particulate fractions of 8 of 10 neuroblastomas tested with the characteristics of a fl-adrenergic receptor. The characteristics of the receptor were investigated most fully in the SK-N-MC line. Specific (--)[aH]DHA binding was not altered by 10-5 M phentolamine, was stereoselective for Lstereoisomers of propranolol and isoproterenol, and was linearly related to protein. (--)-[SH]DHA binding was reversible and reached equilibrium at the lowest ligand concentration by 20 min at 30 °C and remained stable for up to 1 h thereafter. Catecholamines inhibited specific (--)-[SH]DHA binding in the order of potency (--)isoproterenol > (--)-norepinephrine = (--)-epinephrine. Specific binding increased with increasing (--)-[SH]DHA concentrations and the Scatchard analysis of (--)[SH]DHA binding at low ligand concentrations was linear in all of the tumors studied (Fig. 1). A saturable high affinity class of sites, KD = 1.8 riM, binding capacity Bmax -130 fmol/mg, was resolved from the binding curve of neuroblastoma SK-N-MC. However, at least one other class of sites is apparent from the analysis of Scatchard plots from all tumors studied if higher (--)-[SH]DHA concentrations were utilized

78 80 F I'N IX r \ 60F X I .o\O I ~ N

0 20

l 40

SK-N-MC KD=186nM °--° BMAX=130 CHRF-IOI KD=I74nM ~'"~ 8MAX=75 CHRF-96 KD=I0 B M A X = 18

I

60

\

I

80

I I% I I00 120 140

f moles [~H] DHA Bound mg1

Fig. 1. Scatchard analysis of (--)-[aH]DHA binding in tumors SK-N-MC, CHRF-101 and CHRF96 at low hgand concentrations. Total and non-specific binding were determined in triplicate at each pH]DHA concentration in crude membrane of human neuroblastomas. Specific binding was defined as total-non-specific determined in the presence of 1.0 pM (--)-propranolol The concentration of (--)-[3H]DHA was varied between 0.1 and 5 nM. (0.2-50 n M ) (Fig. 2). R e d u c t i o n o f the c o n c e n t r a t i o n o f ( - = ) - p r o p r a n o l o l to 0.1 # M o r the use o f 1 # M ( - - ) - a l p r e n o l o l to define non-specific b i n d i n g d i d n o t alter the curvilinearity o f the S c a t c h a r d plots. Because o f the curvilinear S c a t c h a r d plots for ( - - ) - [ a H ] D H A binding, suggestive o f the presence o f multiple sites or negative cooperativity, the dissociation o f b o u n d ( - - ) - p H ] D H A was studied in m o r e detail. The rate o f dissociation o f b o u n d ligand was identical in the presence a n d absence o f 10 -5 M ( - - ) - a l p r e n o l o l , suggesting t h a t c o o p e r a t i v e effects (negative) were n o t present. C a t e c h o l a m i n e s were effective inhibitors o f b i n d i n g (where present) in all o f the 70t~t\

KOI=~56nM

60 ~ ' ~

K02=84nM

BM~,X=105

I\\ 4O

2O

u

50

IO0

150

200 240 250

[3HI DIhydroolprenololbound(femtomole mg-I)

Fig. 2. Saturation experiments and Scatchard analysis of (--)-[aH]DHA binding to human neuroblastoma. Increasing concentrations of (--)-[SH]DHA (0.4-50 riM) were incubated with membrane fractions of neuroblastoma SK-N-MC(O-O), CHRF-92 (A-A), IMR-32 (O-G), SK-N-SH ( A - ~ ) and CHRF-37 ( ~ 1 ) . Tissue was obtained as listed in Table I. Total and non-specific (1 ffM (--)propranolol) binding were determined in triplicate by filtration assay as described in the methods. The curves were analyzed assuming two heterogeneous classes of sites as described by Thakur et al. a4. Two sites estimated from the computer fit are demonstrated for tumor SK-N-MC.

79

tumors studied at (--)-[aH]DHA concentrations near the KD (1-2 nM). At (--)[aH]DHA concentrations > 20 nM, where the high capacity, low affinity sites predominate, catecholamine agonists (--)-isoproterenol, (--)-epinephrine and (--)norepinephrine did not compete significantly for the low affinity (--)-[aH]DHA binding sites in either SK-N-MC and SK-N-SH, although 1 #M (-4-)-propranolol or (--)-alprenolol remained effective inhibitors, suggesting that the (--)-[aH]DHA low affinity binding does not represent specific fl-adrenergic receptor sites. This discrepancy between agonist and antagonist competition of (--)-[aH]DHA binding was not apparent with (--)-[125I]HYP. (--)-Isoproterenol (10 -5 M) and (--)-propranolol (10 -6 M) were equally effective in estimating 'non-specific' (--)-[125I]HYP binding. Two classes of binding sites were observed in all of the neuroblastomas utilizing higher (0.4-50 nM) (--)-[aH]DHA concentrations (Fig. 2). The Bmax and KD of the high affinity sites were obtained from the graphical resolution of the two sites and agreed well with those determinations from experiments performed entirely at low TABLE I

(--)-[aH]DHA binding in human neuroblastoma Binding capacity was determined from saturation experiments using crude particulate neuroblastoma membrane. The high affinity binding site was determined directly from linear Scatchard plots at low (--)-[aH]DHA concentrations or from a two site analysis of the curvilinear Scatchard plots obtained using high ligand concentrations as described in the Methods. Tumors SK-N-MC and SK-N-SH were assessed in three separate passages in the nude mouse and in cell culture without significant change.

Tumor SK-N-MC cell SK-N-MC mouse SK-N-SH cell SK-N-SH mouse IMR-32 mouse LA-N-I cell CHRF-37 biopsy CHRF-67 biopsy CHRF-92 biopsy CHRF-96 biopsy CHRF-101 mouse CHRF-102 biopsy ganglioneuroma

* Not detectable.

Clinical stages

(--)-[aHJDHA binding Bm~x (fmole/mgprotein)

KD (riM)

100-130

1.6--1.86

98 22

1.9 2.0

22

2.1

21

1.41

ND* IV

12

IV

ND*

II

30

1.02

IV

18

1.0

IV

75

1.7

I

49

0.9

1.1

80 hgand concentrations ( < 5.0 nM). These estimations also agreed with the number of sites determined utilizing 10-6 M (--)-lsoproterenol to define non-specific binding jn several tumors (SK-N-MC. SK-N-SH and CHRF-92). Similarly. the number of high affimty sites determined with (--)-[3H]DHA agreed well with the values obtained with (--)-[lzSI]HYP m tumors SK-N-MC and CHRF-67. Binding capacity of the h~gh affinity site varied greatly among the tumors studied (Table I) and there was no significant (--)-[3H]DHA binding to LA-N-1 cell hne or to CHRF-67 tumor. Because of the lower numbers of receptor site in some of the tumors and the curvilinear Scatchard plots obtained m all the tumors studied, it was necessary to further establish that the high affinity (--)-[3H]DHA sites in these tumors displayed the properties of a classical fl-adrenergic receptor site. (--)-[3H]DHA binding (at 2 nM) to tumors SK-N-MC, SK-N-SH, CHRF-101 and CHRF-102 was reversible, inhibition was stereoselective for (--)-~soproterenol and was also mhlbited in the order of potency (--)-lsoproterenol > (--)-epinephrine = (--)-norepmephrine.

(--J- F1251JIodohydroxybenzylpindololbinding Because of the heterogeneity of binding sites described with (--)-[3H]DHA, binding experiments were also performed with (--)-[125I]HYP. Binding characteristics were studied in detail in SK-N-MC and CHRF-92. Binding was time-dependent, reaching equilibrium within 1 h at the lowest ligand concentrations. Scatchard analyses of saturation experiments with (--)-[z25I]HYP using (--)-alprenolol or (--)isoproterenol were linear (Fig. 3, left and right), KD =- 100-120 pM for (--)-[lZSl]HYP. The number of sites defined by (--)-[125I]HYP correlated well with the high affinity class of sites determined with (--)-[3H]DHA in CHRF-92 (32 vs 30 fmol/mg) and in SK-N-MC (137 vs 130 fmol/mg). Catecholamine agomsts competed for fladrenergic receptor sites described by (--)-[I~5I]HYP in the order of potency (--)-isoproterenol > > (--)-epinephrine ---- (--)-norepinephrine > ~ (--)-dopamine, in SK120 110 100

9d E

80

~ 7o

~ ~o ~

50

g 40 o

m

/

10

f

•

9 8 7

KD = 119 pM sites = 137 r= 93

~ ~

B/F 6 ~ j - ~ 4

3o!

3

20

2 1

0

~ 100

I 20o

I 30o

[125I] HYP (pM)

I 400

0

i

I

I

20 eo 100 ~40 1so Bound fmoles/mg

I

Fig. 3. Left and right: saturation experiment and Scatchard analysis of (--)-[z2SI]HYP binding to membrane from SK-N-MC. Total and non-specific binding (1/~M (--)-propranolol) was determined in triplicate in the presence of increasing (--)-[125I]HYP concentrations after incubation at 37 °C for 1 h.

81 I00

.¢_ Q..

>-r

80

-

60

~ g

4o

0

-9

-5

-7

-3

Fig. 4. Agonist inhibition of specific (--)-[125I]HYP binding in human neuroblastoma (SK-N-MC). The agonists (--)isoproterenol (O-O), (--)norepinephrine ( ~ - ~ ) , (--)epinephrine ( ~ - ~ ) and (--)dopamine (O-G) were incubated in triplicate in increasing concentrations (10-10-10 -3 M) in assays containing neuroblastoma membrane (from nude mouse), 50 pM (--)-[laSI]HYP, 10 m M MgCIg., 50 mM Tris'HCl (pH 7.4) assessed as described in the methods.

N-MC (Fig. 4). This pattern of potency was confirmed by the dose-response of adenylate cyclase to catecholamines in the SK-N-MC line (Fig. 5), and thus indicated the predominance of a fll-adrenergic receptor subtype in this tumor. The ill-selective antagonist metoprolol, and fl2-selective agonist zinterol, were also used to clarify the possible presence of a mixed/31 and r2 population of adrenergic sites in SK-N-MC (Fig. 6). Metoprolol (KD = 0.54 #M) was approximately 20 times more effective than zinterol (KD = 11.0 /~M) in inhibiting specific (--)-[125I]HYP binding, and the steepness of the inhibition curves (Hill coefficient) was nearly 1.0 in these experiments, suggesting the presence of a homogeneous receptor subtype population. The affinities of these agents were typical of a fll-adrenergic receptor subtype previously reported by this laboratoryaa. g

loo

o

80

isoproterenol • epinephrine ! noreplnephrme

dopamme

o

E

~

6O

20

go

-9

-8

-7

-6

-5

-4

log [agomst] (M)

Fig. 5. Catecholamine-sensitive adenylate cyclase in neuroblastoma SK-N-MC. Adenylate cyclase activity was assessed in triplicate in human neuroblastoma obtained from the nude mouse. The data is representative of three separate dose response experiments for each agonist. Adenylate cyclase activity was assessed in the presence of 10-4 M GTP.

82

J00 .¢ Q_

>- 80

-it H

-~

60

~.

4o

metoprolol~

g ~

o

-8

-7

-6

-5

-4

log concentration (M]

Fig. 6. Inhibition of (--)-[t25I]-HYP binding by E/i-specificmetoprolol, and /~-specifie zinterol. Metoprolol and zinterol (10-°-10-4 M) were incubated with neurobtastoma membrane (SK-N-MC from nude mouse), 100 pM (--)-[125I]-HYP, 10-5 M GTP, and buffer as described in the methods. Inhibition of specific(--)-[I~t]HYP binding (10 pM (--)isoproterenol) is plotted vs the concentration of the competing hgands. Non-specific binding (maximal competition with (--)-[I~bI]HYP) was identical among (--)-isoproterenol, zinteroi or metoprolol. The figure represents the mean of three separate experiments with each ligand, at each point. Hill plots of this data were linear, and the steepness coefficientswere approximately 1.0 in the presence of GTP.

Guanosine triphosphate Guanosine triphosphate (GTP) or guanyl-5'-yl-imidophosphate (Gpp(NH)p) decreased agonist potency for the inhibition of specific (--)-[12Sl]HYP binding approximately 12-fold in cell line MC, significantly increasing the Ko for (--)-isoproterenol from 0.030 :E 0.009 # M (n : 3) in the absence of G T P to 0.36 ~ 0.08 p M (m 4- S.D., n : 5, P < 0.01) in its presence, and also increased the Hill coefficient for this interaction from 0.6 to 0.90. G T P (10-5 M) did not significantly alter agonist potency or increase the Hill coefficient from approximately 0.6--0.7 in tumor CHRF-92. However, in the presence of the non-hydrolyzable analog, G p p ( N H ) p (100 #M), the Hill coefficient was increased to 0.9 and agonist potency for (--)-isoproterenol was also significantly reduced in CHRF-92, (KD : 0.18 :l: 0.04, n = 3) like that observed in SK-N-MC line.

Adenylate cyclase Adenylate cyclase activity was determined in crude membranes from 12 of the human neurobtastomas (Table II). While basal activity, fluoride and hormone sensitivity varied greatly among the tumors, the number of fl-adrenergic receptor sites and hormone sensitivity were maintained in the SK-N-SH and SK-N-MC lines whether grown in tissue culture or in the nude mouse. These characteristics were stable to at least 3 passages in culture and the nude mouse. Catecholamine-stimulated adenylate cyclase was observed in SK-N-MC hne and maximal activation required the presence of guanine nucleotides. G T P and Gpp(NH)p were both effective in enhancing hormone sensitive activity in the responsive tumor lines (SK-N-MC). (--)-Propranolol (10 -5 M) had no effect on basal adenylate cyctase in any cell line. Adenylate cyclase was stimulated by GTP, G p p ( N H ) p and fluoride in

83 all t u m o r s studied (Table II), a n d p r o s t a g l a n d i n E1 (PGE1)-sensitive adenylate cyclase was present in all t u m o r s except the S K - N - M C cell line. I n general, g u a n i n e nucleotides increased the activity o f p r o s t a g l a n d i n sensitive adenylate cyclase. However, PGEl-sensitive adenylate cyclase was n o t present in the S K - N - M C cell line in cell cultures in the presence a n d absence of g u a n i n e nucleotide. I n contrast, PGE1 stimulation was clearly seen in samples o f S K - N - M C cell line g r o w n in the n u d e mouse. I n the S K - N - S H cell line p r o s t a g l a n d i n sensitivity was d e m o n s t r a t e d in the order o f potency PGE1 :

12 :> E~ > A1, As, F~a a n d D2.

TABLE II

Prostaglandin- and catecholamine-sensitive adenylate cyclase activity in human neuroblastoma Adenylatecyclase activity (pmol/mg/min) was determined in triplicate in crude, washed neuroblastoma membranes during 15 min incubations at 30 °C. Agents were added to give final concentrations of 10-5 M GTP, 5 x 10-5 M (--)-epinephrine, 2.5 × 10-~ M PGE1, and 10 mM NaF. Gpp(NH)p (10-5 M) also markedly stimulated activity in all tumors and an apparent increase in PGE1, and catecholamine activation was also observed in sensitive tumors with this analog. Basal determinations were performed in sextuplicate.

Tumor SK-N-MC cell SK-N-MC mouse SK-N-SH cell SK-N-SH mouse IMR-32 mouse LA-N-1 cell CHRF-37 mouse CHRF-67 biopsy CHRF-79 biopsy CHRF-91 biopsy CHRF-92 biopsy CHRF-96 biopsy CHRF-101 mouse CHRF-101 biopsy CHRF-102 biopsy

Basal

GTP

PGE1

PGE1 (GTP)

Epi

Epi( GTP) F

8.3

9.0

9.6

8.0

14.8"

20.0*

43.6

10.0

10.8

17.3

34.3

24.8*

30.9*

78.8

6.6

7.8

11.4

36.8

6.8

9.4

82.0

5.6

6.3

19.0

39.0

7.2

8.9

34.4

2.4

3.1

6.7

13.6

4.1

5.1

42.4

2.8

8.9

1.0

1.0

NT

NT

37.7

2.4

2.8

6.4

10.9

4.0

2.5

20.4

29.2

55 2

43.8

81.6

30.8

54.2

97.2

6.3

16.6

8.3

26.9

6.0

19.8

55.0

13.9

18.3

21.0

31.8

15.0

19.3

54.8

36.0

61.0

67.0

104.0

37.4

65.0

103.0

8.2

12.2

10.3

19.2

9.4

11.5

34.6

6.6

39.2

18.8

65.4

7.2

41.9

59.1

20.0

26.0

36.9

47.9

18.7

31.8

60.7

9.9

22.0

16.7

52.0

10.3

28.5

72.6

* (--)-Epinephrinesensitive, P < 0.001 by paired t-test, n = 3 separate experiments; NT = not tested. Stimulation by PGE1 was significant in all tumors studied except SK-N-MC cell lines, P < 0.01.

84 DISCUSSION The present study. (1) identifies and partially characterizes fll-adrenerglc receptor s~tes, catecholamine and prostaglandin-sensltive adenylate cyclase in human neuroblastomas; (2) demonstrates guanine nucleotlde interactions with fl-adrenergic receptor, decreasing agomst affimty and enhancing catecholamine sensitive adenylate cyclase activity in a sensitive line tumor; (3) demonstrates the interaction of guanine nucleotides with prostaglandin sensitive adenylate cyclase; (4) demonstrates that a relatively homogeneous tissue of neural crest origin contains fll-adrenergic receptor sites, and lastly (5) demonstrates that the properties of fl-receptor were maintained in two cell lines during cell culture and passage in the nude mouse. The binding of (--)-[lzSI]HYP and (--)-[3H]DHA (high affinity site) to particulate fractions of human neuroblastoma fulfills the criteria of a fl-adrenergic receptor site: the high affimty binding site was saturable, reversible, stereoselectwe, and shows an order of affinity for agonists (--)-isoproterenol > (--)-epmeprhme = (--)-norepinephrine indicative of the predominance of a {St subtype by Lands' criteria 9. The affinities of zinterol and metoprolol for (--)-[125I]HYP binding m SKN-MC were characteristic of binding to a fll-adrenergic site 1s,19, and were similar to that reported for fll-adrenerglc receptor sites in rabbit lung recently reported from this laboratory 36. The complex Scatchard analyses of (--)-[3H]DHA binding in all the tumors studied suggest the presence of multiple binding sites. Negative cooperativlty of (--)[3H]DHA binding to the frog erythrocyte 11 has been previously suggested, but there was no evidence of such interaction in the present study. Complex binding of (--)[aH]DHA has been reported previously and it has also been recently suggested that the lower affinity site may represent non-receptor binding 5,38. I11support of this is the present finding that catecholamine agonists were potent competitors of(--)-a[H]DHA binding at low (--)-[aH]DHA concentrations (near the KD 1-2 nM), but were not effective at the high (--)-[3H]DHA concentrations (10-50 nM) where a greater proportion of binding is to the low affinity sites. These binding sites were effectwely demonstrated m the presence of (--)-propranolol or (--)-alprenolol, but not in the presence of catecholamines. It is therefore apparent that 'specific' binding is overestimated m this system when 1/~M or even 0. l/~M (±)-propranolol or (--)-alprenolol is used to define non-specific binding, and we suggest caution in using high (--)-[3H]DHA concentrations and this conventional definition of non-specific binding in this tissue. The use of low (--)-[3H]DHA concentrations to define fl-adrenergic receptor site was useful in the estimation of the high affinity site with the ligand. A slmdar use of low ligand concentrations to minimize complex (--)-[3H]DHA binding m leukocytes had also been recently reported 5. Guanosine triphosphate (GTP) and its non-hydrolyzable analog Gpp(NH)p, markedly decreased agonist potency for fl-adrenergic sites in several human neuroblastomas tested, confirming the effects of guanine nucleotldes on fl-adrenergic receptors previously described in the $49 lymphoma cell and its clones 1~,15. The effect of guanine nucleotides on agonlst affimty and adenylate cyclase has been reported in

85 most tissues with a number of hormone agonists; however, the observed decrease in agonist potency in neural crest tissue presently reported contrasts with the lack of effect of GTP on fl-adrenergic receptor sites in cerebral cortex4,~. GTP (100 #M) was not effective in decreasing agonist potency in CHRF-92; however, agonist potency was reduced to that observed in SK-N-MC when assessed in the presence of 100 #M Gpp(NH)p, suggesting that the lack of GTP effect was related to rapid GTP hydrolysis during the incubation rather than to changes in the properties of the fl-adrenergic receptor or guanine nucleotide-dependent sites. The prostaglandin-sensitive adenylate cyclase presently described in human neuroblastoma was enhanced by guanine nucleotide and was also similar to that described in previous studies in mouse and human neuroblastomaZ,2~, e2. In the present study guanine nucleotides enhanced prostaglandin-sensitive adenylate cyclase in all tumors, but did not enhance catecholamine-sensitive adenylate cyclase in tumors other than SK-N-MC, suggesting that prostaglandin and catecholamine receptors are independently linked to adenylate cyclase by guanine nucleotide-dependent factors in neuroblastoma. It is also possible that the low fl-adrenergic binding capacity in some of the tumors was not sufficient for activation of adenylate cyclase, or, lastly, that the fl-adrenergic receptor system in neuroblastoma is less stable to membrane preparation than is the prostaglandin receptor system. Tumor SK-N-MC was the only tumor with catecholamine-sensitive adenylate cyclase activity and it is interesting that this cell line is entirely cholinergic and lacking the enzymes required to synthesize catecholamines2. The lack of inhibition of adenylate cyclase activity by (--)-propranolol in all tumors supports the conclusion that enzyme activity was not already activated by endogenous catecholamine and further extensive wash procedures also failed to alter the pattern of activation in the tumors studied. In general, the activities of hormone-sensitive adenylate cyclase and fl-receptors were similar in SK-N-SH and SK-N-MC cell lines whether in the nude mouse or the cell culture. However, lack of PGE1 sensitivity (with and without GTP and Gpp(NH)p) in the SK-N-MC line in cell cultures was observed repeatedly, suggesting that prostaglandin receptors are absent or uncoupled to the catalytic unit in this cell line. PGE1 stimulated the adenylate cyclase in the SK-N-MC line grown in the nude mouse; activation was enhanced by GTP and Gpp(NH)p. This contrasted with the presence of a prostaglandin response in SK-N-SH cell line maintained both in nude mouse and cell culture and in all other tumors studied, fl-Adrenergic-sensitive adenylate cyclase was present in SK-N-MC whether maintained in the nude mouse or in cell culture. It is not clear whether the nude mouse vascular tissue contributes to the prostaglandin responsivity or whether the PGE1 sensitivity of the tumor is altered when grown in the nude mouse as compared to cell culture. No significant flz-adrenergic component was detected in studies of the tumors grown in the nude mouse as might be expected if mouse vascular tissues were a major component of the fl-adrenergic receptors in the crude membrane. Prasad et al. have demonstrated previously that when mouse neuroblastoma NBP2 is differentiated by cAMP exposure in vitro that the adenylate cyclase system has enhanced sensitivity to PGE1 in the presence of GTP, and suggested a relationship

86 between differentmtlon a n d guanine n u c l e o t l d e - d e p e n d e n t interactions between the r e c e p t o r a n d adenylate cyclase 22. However, m the present study there ~s no evidence t h a t the G T P r e q m r e m e n t s for m a x i m a l a c t w a t i o n by PGE1 o r c a t e c h o l a m i n e is a differentiated feature o f the various n e u r o b l a s t o m a lines. In fact, G T P e n h a n c e d P G E I sensitwe a d e n y l a t e cyclase activity m all t u m o r s studied, as well as e n h a n c i n g c a t e c h o l a m i n e sensitwity m S K - N - M C cell line (a r a p i d l y g r o w i n g a n d m o r p h o l o g i cally undifferentiated cell line). Similarly, the G T P effects on prostaglandln-sensltlve adenylate cyclase in C H R F - 1 0 2 , a highly dxfferentmted g a n g h o n e u r o m a , were identical to those in less differentiated n e u r o b l a s t o m a s , a n d a relationship between cell differentiation a n d g u a n i n e n u c l e o t l d e - d e p e n d e n t adenytate cyclase activation is n o t apparent. ACKNOWLEDGEMENT S u p p o r t e d by C h i l d r e n ' s H o s p i t a l R e s e a r c h F o u n d a t i o n .

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