INT. J. RADIAT. BIOL., 1985, VOL. 48, NO. 5, 753-760

Depression of the radioprotective effect of isoproterenol onmammalian cells in vitro after desensitization of the cAMPsystem

Y. Y. CHIRKOV, M. A. MALATSIDZE, A. R. KAZAROVand A. S. SOBOLEVt

Department of Biophysics, Biological Faculty, Moscow State University,Moscow 119899, U.S.S.R.

(Received 12 November 1984; revision received 25 April 1985;accepted I May 1985)

A short (5 min) incubation of cultured Chinese hamster fibroblasts with thespecific fl-agonist isoproterenol (I 1M) leads to an increase in the intracellularcontent of cAMP and a decrease in radiosensitivity of the cells. Prolonged (up to1 h) incubation induces a desensitization of the cAMP system to isoproterenoland causes a decrease both in the cAMP-stimulating and radioprotective effect ofisoproterenol. There were no detectable changes in the fP-adrenoreceptor numberor binding affinity of -receptors to the radiolabelled -antagonist dihy-droalprenolol in desensitized cells; cAMP-phosphodiesterase activity was alsothe same as in intact cells. It is proposed that a h incubation of the cells withisoproterenbl induces the first step of desensitization, i.e. the functional'uncoupling' of fl-receptors from adenylate cyclase. Thus, the presence of fi-receptors in cells is not enough for the realization of the radioprotective potencyof isoproterenol; an intact, non-desensitized, state of the cAMP system isobligatory.

Indexing terms: fi-adrenoreceptor, isoproterenol, desensitization, cAMP,radioprotection.

1. IntroductionStudies from this and other laboratories have indicated that the synthetic

catecholamine isoproterenol, a specific agonist of f3-adrenergic receptors, protectsmice (Langendorff and Langendorff 1972), bone marrow cells (Graevsky et al. 1981)

and cultured fibroblasts (Sobolev and Chirkov 1982) against X-irradiation. In ourprevious work (Sobolev and Chirkov 1982), we have shown that incubation of

isolated mammalian cells with isoproterenol (10-9-10- 5M) causes an increase in

intracellular cAMP content. The effects of certain concentrations of isoproterenol onthe cAMP level and radiosensitivity were found to be similar; 1 iM isoproterenolproduced the maximum effect. The fl-antagonist propranolol blocked these effects of

isoproterenol. No isoproterenol radioprotection and no influence upon cAMPsystem were noticed in experiments with transformed murine L fibroblasts, clone B-

82, deprived of f-receptors. These data showed that the radioprotective potency ofisoproterenol is realized through the fi-adrenergic mechanism. Nevertheless, therole of the cAMP system in radioprotection remained unclear. Is the elevation of

cAMP after isoproterenol treatment connected with radioprotection, or is it anindependent parallel process? In the present study we tried to answer this question.

t To whom correspondence should be addressed.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Y. Y. Chirkov et al.

The participation of cAMP in the modification of radiosensitivity by isoproter-enol can be examined in experiments with cells which possess -receptors but areincapable of responding to isoproterenol by an increase in the cAMP content. Cellsof this kind can be obtained after fl-agonist-induced desensitization of normal fl-receptor-containing cells. It is known that desensitization is a result of prolongedexposure of cells to the fl-agonist and causes a loss in the ability of the cAMP systemto respond to the fl-agonist (Su et al. 1980, Doss et al. 1981, Kassis and Fishman1982, Galant and Britt 1984).

In the present work we studied the effect of isoproterenol on the cAMP level andthe radiosensitivity of Chinese hamster fibroblasts after a short (5 min) or prolonged(up to 1 h) incubation. We determined the number of fl-receptors, their bindingaffinity to fl-antagonist dihydroalprenolol and the activity of cAMP-phosphodiesterase in desensitized and intact cells.

2. Materials and methodsChinese hamster fibroblasts (B11 dii FAF-28, aneuploid clone 431) were

obtained trom the Institute of Developmental Biology, U.S.S.R. Academy ofSciences. Cells were grown in a monolayer at 37°C in medium consisting of 45 percent Eagle's medium, 45 per cent 199 medium, 10 per cent bovine serum, penicillin(100U/ml) and streptomycin (100pg/ml). Cells in log phase were detached byversene solution and suspended in Hanks' solution (106 cells/ml). The cell suspen-sion (150ml) was preincubated for 30-40min at 37°C, with stirring, and then d,l-isoproterenol (Sigma) was added to a final concentration 1 gM. After incubation withisoproterenol the suspension was divided into two aliquots, one for cAMP assay, theother diluted to 200-500 cells/ml for irradiation at 05-6-0 Gy and 2000 cells/ml fordoses up to 9'0 Gy. X-irradiation (0-5 and 10 Gy/min, 200 kV, 15 mA, filters: 05 mmCu+ l0 mm Al) was carried out under aerobic conditions in glass culture vialscontaining 1 ml of cell suspension; six vials with isoproterenol and six control vialswithout isoproterenol were irradiated at each dose level. Cell survival was defined bycounting macrocolonies according to Marcus et al. (1956). Briefly, 4ml of growthmedium containing 30 per cent bovine serum was added to each vial immediatelyafter the irradiation and 7-8 days later macrocolonies formed on the walls of the vialwere counted. Usually 120-140 macrocolonies were observed in control non-irradiated vials after plating 200 cells in suspension; isoproterenol (1 #M) had noeffect on the colony-forming capacity of control cells.

The degree of radioprotection was defined as a dose modifying factor (DMF)determined from the ratio of Do doses in the presence and absence of isoproterenol.The Do values were estimated from the regression equations of the linear part of thesurvival curves. Regression equations were computed by the method of least squaresaccording to Himmelblau (1970).

The intracellular content of cAMP was determined as described previously(Sobolev and Chirkov 1982) using a 'cAMP assay kit' (Amersham, U.K.).

For the determination of fl-receptors and cAMP-phosphodiesterase activity cellswere lysed in a 20-fold volume of 5 mM Tris-HCl (pH 8-1), containing 1 mM MgC12 ,05 mM EDTA, 2mM 2-mercaptoethanol (Serva) for 20min at 4C, and thenhomogenized in a glass-Teflon homogenizer (30 strokes of pestle). The non-lysedcells and nuclei were separated by centrifugation (800g, for 3min); the roughmembrane fraction was precipitated by re-centrifugation of lysed cells (25 000 g, for20min). The pellet was resuspended in the buffer mentioned above.

754

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Desensitization of the cAMP system and radioprotection

Equilibrium binding of 1-(propyl-4,6-[3H])-dihydroalprenolol (DHA) (Amer-sham, U.K.) (06 12 x 10- 9 M) to rough membranes from lysed cells was performedat 25°C for 30 min (Sobolev et al. 1983). Non-specific binding was estimated usingt0OM unlabelled D,L-propranolol (Sigma). The concentration curve of specificradioligand binding by membranes was linearized in Woolf co-ordinates (Sobolevet al. 1983) and the number of fl-receptors (Bmax) and the value of the dissociationconstant (Kd) were calculated.

cAMP-phosphodiesterase was assayed in the supernatant (25 000 g, for 20 min)of the lysed cells. The enzyme activity was determined as a rate of both [3 H]cAMP(Amersham) hydrolysis and [3 H]5'AMP accumulation (Sobolev et al. 1984).

The radioactivity of samples was counted as c.p.m. in a Nuclear Chicago 'Mark-2' liquid scintillation spectrometer. For counting the samples of cAMP and cAMP-phosphodiesterase assays we used a toluene-based scintillator: 4g PPO(2,5-diphenyloxazole) and 0 5 g POPOP (p-bis-[2-(5-phenyloxazolyl)]-benzene)/1 toluene. For DHA-binding assay we used a dioxane-based scintillation cocktail:5 g BPO (2-(4-biphenyl)-5-phenyloxazole), 0151 1-methylnaphthalene/0851 diox-ane. The method of internal standardization was used for quench correction.

Protein content was determined by the method of Lowry et al. (1951) with bovineserum albumin as the standard.

Statistical processing of the data was performed according to Student. All dataare presented as average values+ s.e.

3. Results and discussionWe have examined the radiosensitivity of Chinese hamster fibroblasts (figure 1).

The shoulder region extends to 15 Gy, the linear part of the survival curve beginsfrom 15-20 Gy and for the interval 20-9O0 Gy is expressed by the regressionequation:

NgNN = (0-25 + 0'03) - (0'28 + 0-02)DNo

with a regression coefficient of - 099. The calculated Do value was 154 + 011 Gy.In further experiments (time-dependence of the isoproterenol radioprotectiveeffect) cells were irradiated at doses up to 6'0 Gy in order to limit the irradiation time.

Fibroblasts were incubated with 1 M isoproterenol for 5-60 min; 5 min beforethe end of the incubation period additional isoproterenol (1 /M) was given. In orderto prevent oxidative degradation of isoproterenol during prolonged incubation withthe cells we used 0-5 mM ascorbic acid (Suet al. 1980). The presence of the ascorbicacid in the incubation medium had no influence on the cAMP level (figure 2) or theradiosensitivity of the cells (figure 3 (a), (d)).

The maximum radioprotective effect of isoproterenol (DMF = 147 + 004, dataof four independent experiments) was observed after 5 min incubation (figure 2);figure 3 (a) shows the results obtained in one of these four experiments. The slope ofthe linear part of the survival curve is observed to vary so that Do for protected cells isincreased 146-fold, whilst the extrapolation number remained just the same. Withdevelopment of desensitization, a progressive decrease in the radioprotective effectof isoproterenol was registered (figures 2,3 (b)-(d)). After 1 h incubation of the cellswith isoproterenol no radioprotection occurred (figure 3 (d)).

755

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Y. Y. Chirkov et al.

RN

Dose Gy

Figure 1. Survival of Chinese hamster fibroblasts after X-irradiation.

100

45

a:I

min

Figure 2. cAMP content and radioprotection of X-irradiated Chinese hamster fibroblasts asa function of incubation time with 1 M isoproterenol + 05 mm ascorbic acid. 0, Effectof isoproterenol on cAMP content, per cent of control. x, Radioprotective effect ofisoproterenol, DMF. 0, cAMP content during incubation with 05 mm ascorbic acid.

756

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Desensitization of the cAMP system and radioprotection

(a)

- DMF=1.46 isoproterenol

N -(nI'n~ns0021-tnln±n2).fD

control

- LgN. (0.22±0.02)-(0.30!0.03) -D \

Do=(1.47±0.14) Gy r=-0.99 '

I I I I I

(b)

- DMF=1.35 isoproterenot

19tg N (0.23±0.02)-(0.21+0.02) D

Do =(2.07 0.19)Gy r=-0.99

Do

DO=(1.54-0.16) Gy r=-0.98

I I I I I f

N

1.00

0.10

nn4

1 2 3 4 5 6Dose, Gy

(c)

- DMF=0.97 isoproterenol

" 1 N N =(0.29±0.03)-(a0.27±0.03)- D

: %4 0, Do =(1.60+0.17) Gy r= -0.97

control

t y j-I~ U - V V ~ V t - . t U

Do (1.65±0.13) Gy r= -0.98

1 2 3 4

(d)

5 6Dose, Gy

Figure 3. Survival of Chinese hamster fibroblasts X-irradiated after various periods ofincubation with I1 M isoproterenol. (a) 5 min; (b) 15min; (c) 30min; (d) 60min.0, Control. 0, Isoproterenol + 05 mM ascorbic acid. x, 0-5 mM ascorbic acid. Eachfigure represents the data of one of the four independent experiments. *P<005.

The intracellular level of cAMP reached the maximum value (185 per cent of

control) also after 5 min incubation with isoproterenol (figure 2). Longer incubation

caused a decrease in cAMP response to isoproterenol; after 1 or 3 h later no

stimulation of the cAMP system occurred even after the further addition of

isoproterenol. Thus, during the prolonged incubation of cells with isoproterenol we

NNo

1.00

0.10

DMF=1.15. DM=11 isoproterenol

I\ g N o(0.29±0.03)-(0.25±0.02)- D

- 041.73±0.14)Gy r=-0.98

control

N --=(0. 30±0.03)-(029±0.03) DN.

Do =(1.51+0.15 ) Gy r= -0.97

I I I I I i I

757

X

-

usu, .

r

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Y. Y. Chirkov et al.

observed a desensitization of the cAMP system to isoprotereno; after 1 h incubationcomplete desensitization had taken place (figure 2). The dynamics of both thedesensitization of the cAMP system and the reduction of isoproterenol radioprotec-tion are similar (figure 2). The scales of two y-axes are arranged so that maximumand minimum values of each curve correspond. The DMF-curve and cAMP-curverespond in an identical manner, so one curve could be drawn through both sets ofexperimental points. This indicates that the state of the cAMP system and the

realization of the radioprotective potency of isoproterenol are interdependent.It is known that catecholamine-induced desensitization of the fl-receptor-linked

adenylate cyclase, the key enzyme of the cAMP system, occurs by a two-step process(Suet al. 1980, Doss et al. 1981, Kassis and Fishman 1982). The first step results inthe 'uncoupling' of the f-receptor from adenylate cyclase due to a disturbance in theinteraction between the fl-receptor and the guanine nucleotide regulatory protein.During short-term incubation (up to 1-2 h) of cells with catecholamine this

uncoupling is expressed as a loss of responsiveness of adenylate cyclase tocatecholamine with no alteration in receptor number as assessed by a radiolabelled fl-antagonist. The second step in the process of desensitization is the reduction ofreceptor number and is observed after long-term exposure (up to 24 h). Bothphenomena cause a reduced response of the cAMP system to further stimulation by

the fl-agonist (Su et al. 1980, Doss et al. 1981, Fishman et al. 1981, Kassis and

Fishman 1982). What kind of desensitization occurs after a 1 h incubation of Chinese hamster

fibroblasts with isoproterenol? We have determined the maximum number ofspecific binding sites for [3H]dihydroalprenolol (DHA) which reflects (Su et al.1980) the number of fi-adrenergic receptors. No changes in the fl-receptor number(Bmax) were observed after 1 h incubation (figure 4). The experiments revealed no

appreciable difference in binding affinities (Kd) between membranes from controland desensitized cells (figure 4). These data indicate a first step of desensitizationafter 1 h of incubation of fibroblasts with isoproterenol.

The intracellular cAMP level is regulated by (besides adenylate cyclase) cAMP-

phosphodiesterase, the enzyme hydrolysing cAMP to 5'-AMP (Robison et al. 1971).We investigated, if a 1 h incubation of cells with isoproterenol would cause anincrease in cAMP-phosphodiesterase activity and, as a result, a decrease in the

stimulated cAMP level after catecholamine treatment. Phosphodiesterase activity inintact fibroblasts was found to be 220+0-10pmole of hydrolysed cAMP/mg ofprotein per min. Incubation of the cells with 1 M isoproterenol for 5 min or 1 h waswithout any effect on phosphodiesterase activity: 2-15 + 011 and 207 + 006 pmole ofcAMP/mg protein per min, respectively (data from four experiments).

The results obtained indicate that the loss of response of the cAMP system toisoproterenol after 1 h incubation (figure 2) is not connected with any disturbances in

the cAMP-phosphodiesterase activity or changes in the fl-receptor number.Numerous data concerning the phenomenon of catecholamine-induced desensitiz-ation of the cAMP system of various cell lines (Su et al. 1980, Doss et al. 1981,Fishman et al. 1981, Kassis and Fishman 1982, Galant and Britt 1984) suggest thatthe desensitization of Chinese hamster fibroblasts by isoproterenol (figure 2) is dueto the 'uncoupling' of desensitized ,B-receptor from adenylate cyclase. Thus, thereduction of the radioprotective effect of isoproterenol during prolonged incubationwith the cells (figures 2, 3 (a)-(d)) can be explained by the desensitization of theadenylate cyclase system because, as we have observed earlier (Sobolev and Chirkov

758

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Desensitization of the cAMP system and radioprotection

B

0 4 8 12F, nM

Figure 4. Binding of 1-[3 H]DHA to $-adrenergic receptors of cell membranes (Woolfplot). 0, Control. 0, Incubation with I M isoproterenol for 1 h. The Woolf equationis: F/B=Kd/Bmax+F/Bm..., where F is free 1-[3H]DHA; B is specifically bound I-[3 H]DHA; Bmax is the number of binding sites; and Kd the dissociation constant. Theresults of one typical experiment shown. Mean values obtained in four experiments are:for the control, Bmax= 351 + 46 fmol/mg protein; Kd = 13-4+ 22 nM; after 1 h incubationwith 1 /M isoproterenol, Bmax = 343 + 31 fmol/mg protein; Kd = 126 + 24 nm.

1982), radioprotection of cells by isoproterenol is realized through the fl-adrenergic

receptors linked to adenylate cyclase.The major conclusion from this work is that the availability of /-receptors in cells

is not enough for the realization of the radioprotective potency of isoproterenol. Anintact, non-desensitized cAMP system is obligatory.

ReferencesDoss, R. C., PERKINS, J. P., and HARDEN, T. K., 1981, Recovery of beta-adrenergic receptors

following long term exposure of astrocytoma-cells to catecholamine. Journal ofbiological Chemistry, 256, 12281-12286.

FISHMAN, P. H., MALLORGA, P., and TALLMAN, J. F., 1981, Catecholamine-induced

desensitization of adenylate cyclase in rat glioma C6 cells. Evidence for a specificuncoupling of beta-adrenergic receptors from a functional regulatory component ofadenylate cyclase. Molecular Pharmacology, 20, 310-318.

GALANT, S. P., and BRITT, S., 1984, Uncoupling of the beta-adrenergic receptor as amechanism of in vitro neutrophil desensitization. Journal of Laboratory and clinicalMedicine, 103, 322 332.

759

P

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.

Desensitization of the cAMP system and radioprotection

GRAEVSKY, E. Y., SOBOLEV, A. S., SMIRNOVA, I. B., CHIRKOV, Y. Y., DONTSOVA, G. V., andGRAEVSKAYA, E. E., 1981, Study of the role of beta-adrenoreceptors in the radioprotec-tive effect of isoproterenol. Radiobiologiya, 21, 688 693 (in Russian).

HIMMELBLAU, D. M., 1970, Process Analysis by Statistical Methods (New York, London,Sidney, Toronto: Wiley).

KASSIS, S., and FISHMAN, P. H., 1982, Different mechanisms of desensitization of adenylatecyclase by isoproterenol and prostaglandin E1 in human fibroblasts. Role of regulatorycomponents in desensitization. Journal of biological Chemistry, 257, 5312-5318.

LANGENDORFF, H., and LANGENDORFF, M., 1972, Experimentelle Untersuchungen zum

Wirkungsmechanismus strahlenresistenzerhohender Substanzen. Strahlentherapie,143, 432-443.

LOWRY, . H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J., 1951, Proteinmeasurement with the Folin phenol reagent. Journal of biological Chemistry, 193,265 275.

MARCUS, P. I., CIECIURA, S. J., and PucK, T. T., 1956, Clonal growth in vitro of epithelial cellsfrom normal human tissues. Journal of experimental Medicine, 104, 615 621.

ROBISON, G. A., BUTCHER, N. W., and SUTHERLAND, E. W., 1971, Cyclic AMP (New York:Academic Press).

SOBOLEV, A. S., and CHIRKOV, Y. Y., 1982, Beta-adrenergic mechanism of mammalian cellsradioprotection by isoproterenol. Strahlentherapie, 158, 747-751.

SOBOLEV, A. S., ROSENKRANZ, A. A., and KAZAROV, A. R., 1983, A study of mechanisms ofpost-irradiation changes in adenylate cyclase activity. International Journal of RadiationBiology, 44, 31-39.

SOBOLEV, A. S., CHIRKOV, Y. Y., TERTOV, V. V., and KAZAROV, A. R., 1984, Effects of

radioprotectors on the cAMP and cGMP systems. Radiation and environmentalBiophysics, 23, 79-93.

SU, Y.-F., HARDEN, T. K., and PERKINS, J. P., 1980, Catecholamine-specific desensitization ofadenylate cyclase. Evidence for a multistep process. Journal of biological Chemistry, 255,7410-7419.

760

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y H

ochs

chul

bibl

ioth

ek D

arm

stad

t on

11/2

5/14

For

pers

onal

use

onl

y.