Life Sciences Vol. 10, Part I, pp. 1255-1280, 1971 .

Pergamon PressPrinted in Great Brítaín

ADENYLATE CYCLASE AND PHOSPHODIESTERASE ACTIVITY OF NORMAL AND SV40 VIRUS-

TRANSFORMED HAMSTER ASTROCYTES IN CEDI CULTURE

Benjamin Weiss, Harvey M. Shein* and Roger Snyder

(Received 10 June 1971; in final form li October 1971)

Laboratory of Preclinical Pharmacology, National Institute of Mental Health,

Saint Elizabeths Hospital, Washington, D .C . 20032, McLean Hospital Research

Laboratory, Belmont, Massachusetts, and Department of Psychiatry, Harvard

Medical School, Boston, Massachusetts .

Sumna ry

Adenylate cyclase and phosphodlesterase activity were determinedin pure . cultures of astrocytes derived from newborn hamster brains .The activities of both enzymes were of the same order of magnitudeas that found in whole cerebrum of newborn hamsters . Phosphodies-terase activity of SV40 virus-transformed newborn hamster astrocyteswas similar to that of normal newborn hamster astrocytes . Adenylatecyclase activity of the virally transformed cells was less thanone-half that of the normal astroeytes . The results show that nor-mal and neoplastic astrocytes contain the enzymatic maehlneryrequired to synthesize and hydrolyze adenosine 3',5'-monophosphate(cyclic 3',5'-AMP) . The findings suggest a role for cyclic 3',5'-AMP In astrocyte function and indicate a possible mechanism forcartmunicatlon of neurons with astrocytes.

Cyclic 3',5'-adenosine monophosphate (cyclic 3',5'-AMP) madlates the

effects of many bloehemically active molecules in both animal and bacterial

systems (1, 2, 3) . Adenylate cyclase, the enzyme that catalyzes the conversion

of adenosine triphosphate to cyclic 3',5'-AMP (4) Is activated by various

hormones and neurotransmitters and Is one of the principal factors governing

the intracellular concentration of the cyclic nucleotide (1-6) . Cyclic nucleo-

tide phosphodlesterase, which hydrolyzes cyclic 3',5'-AMP to 5'-AMP (7) probably

also plays a role in regulating the intracellular concentration of cyclic 3',5'-

AMP . Thus, the relative activities of these two enzymes are a critical deter-

minant of cellular function generally and probably also of the function of brain

* Supported In part by U .S .P.H.S. Research Grant NB 06610

1259

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Cyclic AMP and Aetrocyte Fonction

Vol. 10, No. 21

cells .

It has been reported that the concentration of cyclic 3',5'-AMP in brain

1256

Cyclic AMP and Astrocyte Function

Vol. 10, No. 21

AMP, I .e ., phosphodlesterase activity, was also proportional to the time of

Incubation and to the concentration of tissue .

0

o_ 8a

I

I

I

I

50 100 150 200f~.GM PROTEIN

0W

MU_

UrU

0 I

I

I

I

10 20 30 40INCUBATION TIME lminl

FIG . 1

FIG . 2

Figure 1

Fonnatlon of cyclic 3',5'-AMP from ATP to normal newborn hamster

astrocytes as a function of tissue concentration . The tissue samples

were prepared and assayed for adenylate cyclase activity as described

In Methods . Tissues were Incubated for 10 minutes In the presence of

10 mM NaF . Each point represents the mean value of 3 experiments .

Figure 2 Formation of cyclic 3',5'-AMP from ATP in normal newborn hamster

astrocytes as a function of Incubation time . Ttssue samples were

prepared and assayed for adenylate cyclase activity in the presence

of 10 mM NaF as described in Methods . Each sample contained 100 ~gm

protein . Each point represents the mean value of 3 experiments .

~~ 20 2wm ó

â0

E~ 16E 1 .6óEc

W 12 Iz

Vol . 10, No. 21

Cyclic AMP and Astrocyte Function

1255

A portion of the cell suspension was analyzed in a hemocytometer,to determine

call number and to compare rates of cell growth .

The ce hs were then washed

twice with Trls-HCI buffer (0 .05 M) pH 7 .4 containing sufficient NaCI to make

the solution Isotonic and were collected by centrifuging at 200 x g 'for 5 min .

Water was added to the washed pellet and the suspension was gently hamogentzed

with a Teflon pestle. The disrupted cells were then centrifuged at 30,000 x g

for 30 minutes . The supernatant fluid was saved for assay of phosphodlesterase

activity . The pellet was resuspended in 0 .05 M Trls HC1 buffer, pH 7 .4 and

assayed for adenylate cyclase activity .

Adenylate cyclase activity was determined by the method of Krishna, Weiss,

and Brodle (17) using unlabeled cyclic 3',5'-AMP to prevent the hydrolysis of

the newly-formed radioactive cyclic nucleotide (18) . Each sample contained the

tissue sample (about 400 pg protein), 1 mM ~8-14C~ ATP (0 .5 ~Ci), 10 mM NaF,

3 mM "carrier" cyclic 3',5'-AMP, and 3 mM Mgt all in 100 W1 of 50 mM Trls HCI

buffer, pH 7.4 . The samples were incubated at 30°C in a Dubnoff Metabolic

Shaker.

Phosphodiesterase activity was measured according to the procedure described

by Butcher and Sutherland (7) with slight mod ificattons (19) .

Each sample con-

twined the tissue preparation (about 300 pg protein), 1 mM cyclic 3',5'-AMP,

100 pg alkaline phosphatase from Eschericha toll, and 3 mM Mgt in 1 ml of 50

mM Tris HC1 buffer, pH 8.0. Blanks contained everything except the substrate

(cyclic 3',5'-AMP) . The samples were incubated for up to 30 minutes at 37C .Inorganic phosphate released from the hydrolysis of . 5'-AMP was determined by the

procedure described by Basil et al . (20) .

Results and Discussion

Figure 1 shows the formation of cyclic 3',5'-AMP from ATP with Incroasing

amounts of homogenate derived from culturod normal hamster astrocytes, and

Figuro 2 shows the formation of cyclic 3',5'-AMP as a function of the time of

incubation. As may be seen, the reactions aro linear with respect to time of

Incubation and concentration of tissue protein . The hydrolysis of cyclic 3',5'-

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G~clic AMP and Astrocyte Ftinction

Vol . 10, No. 21

AMP, i .e ., phosphodlesterase activity, was also proportional to the time of

incubation and to the concentration of tissue .

I

I

1

I

10 20 30 40INCUBATION TIME (min)

FIG . 1

FIG . 2

Figure 1

Formation of cyclic 3',5'-AMP from ATP in normal newborn hamster

astrocytes as a function of tissue concentration . The tissue samples

were prepared and assayed for adenylate cyclase activity as described

in Methods . Tissues ware incubated for 10 minutes In the presence of

10 mM NaF . Each point represents the mean value of 3 experiments .

Figure 2 Formation of cyclic 3',5'-AMP from ATP in normal newborn hamster

astrocytes as a function of Incubation time . Tissue samples were

prepared and assayed for adenylate cyclase activity in the presence

of 10 mM NaF as described in Methods . Each sample contained 100 pgm

protein . Each point represents the mean value of 3 experiments .

.É 20.dç 2óâPEE~ 16 1.6

óEc

w 12 120

0 w

o_ 8 ó 0.8wa o.i[ï a;,~ 4 in'- 0.4U_ M

UI I I I

U_

U O50 100 150 200 U

0r

~e.GM PROTEIN U

Vol. 10, No . 21

Cyclic AMP and Astrocyte Funcrion

1257

calculated òn a protein basis, the astrocytes, which were derived from the

activity of whole cerebrum of neonatal hamsters, but about twice the activity

of neonatal hamster ftbroblasts ;~:,.. .;Fhosphodiesterase activity of astròcytas,

ftbroblasts, and cerebrum was similar .

For cooperative purposes wa have Included

data on adenylate cyclase and phosphodtesterasé activity of adult hamster

cerebrum . The adult cerebrum exhibited more than twice the adenylate cyclase

activity and more than 3 times the phosphodlesterase activity of newborn hamster

cerebrum . This postnatal increase in adenylate cyclase and phosphodiesterase

activity of hamster cerebrum is similar to that noted previously for the rat

cerebrum (21) .

TABLE 1

COMPARISON OF ADENYLATE CYCLASE AND PHOSPHODIESTERASE ACTIVITY OF WHOLE CEREBRUM

ASTROCYTES, AND FIBROBLASTS OF THE HAMSTER

Cell cultures

determineda

b

c

Table 1 comparas the activities of adenylate cyclase and phosphodiesterase

in pure cultures of normal neonatal hamster astrocytes and ftbroblasts with

those of the whole neonatal hamster cerebrum . When the enzyme activities were

neonatal hamster cerebrum, had less than one-half of the adenylate cyclase

were prepared as previously described . Enzyme

as described In the text .

pmoles cyclic 3',5'-AMP formed/mg protein/min t S .E .

nmoles cyclic 3',5'-AMP hydrolyzed/mg protein/min t S .E .

P < 0 .01 compared with astrocytes .

activities were

TISSUE ADENYLATE CYCLASE a PHOSPHODIESTERASEb

Newborn cerebrum 225 t 23 11 t 1

Adult cerebrum 583 ± 50 37 t 4

Newborn astrocytes 91 t 9 11 t 1

Newborn ftbroblasts 49 t 4c 11 t 2

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Ggclic AMP and Astrocyte Ftinction

Vol . 10, No. 21

It should be emphasized that the phosphodiesterase activity measured with

high substrate concentrations (I :e ., 1 mM) may reflect the sum of the actlvitles

of different forms of phosphodtesterese . For it has been reported that several

tissues, Including brain, possess multiple forms of phosphodiesterase activity ;

at least one with a relatively high Michaelas constant (Km), about 10~M, and

one with a relatively low Km, between 10-5 and 10~M (22-24) .

It Is likely

that the low Km enzyme Is more important physiologically since the concentration

of cyclic 3',5'-AMP in astrocytes is very low, in the order of 2 Gunoles of cyclic

3',5'-AMP per Kg of tissue (Weiss, Strada, and Klein, unpublished data) . Using

a newly-developed mtcroassay (25), we are currently studying whether pure

cultures of astrocytes also possess more than one form of phosphodiesterase

activity .

In Table II we compare the actlvitles of adenylate cyclase and phospho-

dlesterase of normal newborn hamster astrocytes with that of SV40 virus-trans-

formed newborn hamster astrocytoma cells. No significant differences in the

activity of phosphodtesterase were found between the groups of cells . However,

the SV40 transformed hamster astrocytes had only about one-third the adenylate

cyclase activity of the normal hamster astrocytes . The reduced activity in the

SV40-virus-transformed astrocytes as compared to the normal hamster astrocytes

is similar to the previously reported observation by Burk (26) of reduced

adenylate cyclase activity In a poiyoma virus-transformed cell line.

TABLE 2

ADENYLATE~CYCLASE AND PHOSPHODIESTERASE ACTIVITY OF ASTROCYTES AND VIRALLY-

TRANSFORMED ASTROCYTOMA CELLS OF THE HAMSTER

Cultures of normal astrocytes and SV40 transformed astrocytoma cells were preparedas previously described in the text.a

pmoles cyclic 3',5'-AMP formed/mg protein/min t S .E .

T SSUE ADENYLATE CYCLASEa PHOSPHODIESTERASE

Normal Newborn Astrocytes

Newborn Astrocytama Cells

75 ± 9

29 t lc

7 .0 ± 1 .3

10 .8 t 1 .2

Vol . 10, No. 21

Ggclic AMP and Astrocyte Fwncüon

1259

bnmoles cyclic 3',5'-AMP hydrolyzed/mg protein/min ± S .E .

cP < 0.001 compared with normal astrocytes .

The present results demonstrate that pure cultures of normal or neoplasttc

astrocytes derived from neonatal hamster contain the enzymatic machinery

required to synthesize and degrade cyclic 3',5',-AMP . Moreover, the data tndicatP

that the activities of adenylate cyclase and phosphodlesterase of neonatal

hamster astrocytes are of the same order of magnitude as that of whole neonatal

hamster cerebrum and thus suggest a functional role for cyclic 3',5'-AMP in

normal astrocytes . Since neurotransmitters present in brain neurons can increase

the concentration of cyclic 3',5'-AMP in brain tissue, probably by stimulating

adenylate cyclase, the present finding of adenylate cyclase activity in normal

astrocytes supports the concept that neurons in brain may communicate with

astrocytes by a neurotransmitter-stimulated activation of an adenylate cyclase

system present in astrocytomas .

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Cyclic AMP and Astrocyte Function

Vol . 10, No . 21

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