characteristics of the cyclic nucleotide ... · from the adrenal medulla was twice that of the...

[CANCER RESEARCH 38, 915-920, April 1978]

Characteristics of the Cyclic Nucleotide Phosphodiesterases in aTransplantable Pheochromocytoma and Adrenal Medulla of the Rat1

Robert M. Levin and Benjamin Weiss

Department of Pharmacology, Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129

ABSTRACT

A pheochromocytoma was maintained in rats from theNew England Deaconess Hospital by giving the rats s.c.injections of isolated tumor cells. The animals were sacrificed 3 to 4 weeks after transplantation, the tumors wereexcised, and purified tumor cells were prepared. Cyclicnucleotide phosphodiesterase of the purified tumor cellswas characterized and compared with that of the adrenalmedulla.

At high concentrations of cyclic adenosine 3 :5 -mono-phosphate (cyclic AMP), the activity of phosphodiesterasefrom the adrenal medulla was twice that of the pheochromocytoma; but at low substrate concentrations, the cyclicAMP phosphodiesterase activity of the pheochromocytoma was more than 3 times that of the adrenal medulla.By contrast, the rate of hydrolysis of cyclic guanosine3 :5 -monophosphate (cyclic GMP) of the adrenal medullawas approximately 2 times that of the pheochromocytoma at all substrate concentrations studied.

The adrenal medulla and pheochromocytoma displayedbiphasic kinetics for cyclic AMP hydrolysis; the apparentMichaelis constants (Km)for the adrenal medulla (8 and130 fjLM)were significantly higher than the Km's for the

pheochromocytoma (0.9 and 18 /UM). Kinetic analysis ofphosphodiesterase activity in the subcellular componentsdemonstrated that in the pheochromocytoma all fractionscontained the high-affinity form of phosphodiesterase.However, in the adrenal medulla only the nuclear fractioncontained a high-affinity form of phosphodiesterase.

Theophylline, papaverine, cyclic GMP, trifluoperazine,and dipyridamole were relatively ineffective in inhibitingthe cyclic AMP phosphodiesterase from either the adrenalmedulla or pheochromocytoma.

Our findings that the pheochromocytoma has a greateractivity of a high-affinity cyclic AMP phosphodiesterasebut a lesser activity of cyclic GMP phosphodiesterasewhen compared with that of the normal adrenal medullasuggest that pheochromocytoma cells may have a relatively low ratio of cyclic AMP to cyclic GMP. This isconsistent with the proposition that low intracellular concentrations of cyclic AMP or a low ratio of cyclic AMP tocyclic GMP may be associated with neoplastic activity.

INTRODUCTION

Alterations in the cyclic nucleotide system have beenimplicated in the development and progression of a variety

of tumors and transformed cell lines. In general, neoplasticactivity has been associated with low intracellular concentrations of cyclic AMP2 (1,9,17, 24) and in some cases with

high intracellular concentrations of cyclic GMP (12, 27).Abnormally low concentrations of cyclic AMP may becaused either by a reduced activity of the enzyme thatcatalyzes its biosynthesis (adenylate cyclase) or by anincreased activity of the enzyme that catalyzes its hydrolysis(phosphodiesterase). There is evidence that both of thesedefects exist in certain types of cells. For example, lowactivities of adenylate cyclase have been reported in astro-cytoma cells (38), thyroid tumors (23), and hepatomas (16),and abnormally high activities of phosphodiesterase havebeen found in certain leukemic lymphocytes (13, 14).

Tumors of the adrenal medulla (pheochromocytomas)provide a particularly interesting neoplastic model for testingthe hypothesis that abnormal growth and function may beassociated with an abnormal metabolism of cyclic nucleo-tides. These tumors are both neoplastic and functional;they produce and release into the circulation large quantities of the catecholamines epinephrine and norepinephrine.Accordingly, the clinical manifestations are similar to thoseseen with high doses of catecholamines, including vascularaccidents, myocardial infarction, congestive heart failure,and malignant hypertension (15).

A rat model for pheochromocytoma has recently beendeveloped by Warren et al. (34, 35) through irradiation of astrain of rats developed at the New England DeaconessHospital. When transplanted s.c., this tumor grows rapidlyto approximately 1 cm in 30 days. The tumor-bearinganimals live between 30 and 60 days following transplantation and are characterized at the time of death by a loss ofbody weight, high blood pressure, proteinuria, and renaland cardiac lesions (34).

The treatment of choice in pheochromocytoma is surgery, but where multiple and ectopie foci exist, or in casesof malignant pheochromocytoma, surgery often providesonly temporary relief, and symptoms frequently reappear.Current pharmacological treatment also provides only atemporary reduction in the hypertensive symptoms andcannot permanently control the destructive nature of circulating catecholamines (26). The high level of catecholamines localized in the pheochromocytoma, moreover, canprotect the tumor against radiation therapy (28). Thus, thereexists a need for an effective therapeutic treatment in casesnot amenable to surgery.

Since abnormal concentrations of cyclic AMP and cyclicGMP have been associated with various forms of malignant

1 Supported by Grant CA15883 awarded by the National Cancer Institute,

Department of Health, Education and Welfare.Received April 27, 1977; accepted January 4, 1978.

2 The abbreviations used are: cyclic AMP, cyclic adenosine 3':5'-mono-phosphate; cyclic GMP, cyclic guanosine 3':5'-monophosphate; EGTA, eth-yleneglycol bis (/3-aminoethylether)-N,W'tetraacetic acid.

APRIL 1978 915

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R. M. Levin and B. Weiss

disease, one approach to the treatment of patients withpheochromocytomas may be through the alteration of thesecyclic nucleotides in the diseased tissue with specific pharmacological agents. As a first step in determining whetherthis tack may profitably be taken in reducing the growth ofpheochromocytomas, we have studied the characteristicsof the cyclic nucleotide phosphodiesterase activity of pheochromocytomas and compared those characteristics withthose of normal adrenal medulla.

MATERIALS AND METHODS

Preparation of Pheochromocytoma. The pheochromo-cytoma was maintained by injection of approximately 1 x10s tumor cells s.c. (in the intrascapular area) into 6- to 8-week-old NEDH rats. The tumor-bearing animals were sac

rificed by cervical dislocation 3 to 4 weeks after transplantation. The tumors were excised, rinsed in Hanks' balancedsalt medium, minced, and suspended in Hanks' medium

(maintained at 4Â°). The suspension was filtered twice

through glass wool to remove the debris and paniculatematter. The resulting suspension consisted of large numbers of tumor cells and erythrocytes. For purification of thetumor cells, the suspension was kept at 4Â°for 20 min,

during which time the heavier tumor cells settled to thebottom of the tube. The supernatant fluid containing theerythrocytes was discarded, and the settled tumor cellswere resuspended in Hanks' medium. The suspension was

centrifuged at 100 x g for 5 min, and the supernatant fluidwas discarded. The cells were resuspended and centrifugedas done previously. The final preparation contained approximately 95% tumor cells and 5% erythrocytes. The purifiedtumor cells were then suspended in 50 mw Tris buffer, pH7.0, containing 1 mw Mg2'; homogenized with the use of a

Polytron homogenizer; and sonically dispersed for 10 secat 200 watts with the use of a Branson cell sonifier. Eachexperiment utilized enzymes derived from a separate tumor.

Preparation of the Adrenal Medulla. The adrenals fromboth the control and tumor-bearing rats were surgicallyexised and dissected free of fat and connective tissue undera dissecting microscope as described by Guidotti and Costa(11). A slit was made in the adrenal gland, and under mildpressure applied to the side opposite the slit, the adrenalmedulla was separated from the cortex and capsule. Theadrenal medulla was dissected free of any cortex adheringto the medulla; homogenized in 50 mM Tris buffer, pH 7.0,containing 1 mM Mg2'; and sonically dispersed as de

scribed above. Each experiment utilized enzymes derivedfrom 2 adrenal medullae.

Phosphodiesterase Activity. Cyclic AMP phosphodiesterase activity was measured by the luciferin-luciferasemethod as previously described (37). Each reaction vesselcontained 50 mM glycylglycine buffer (pH 8.0), 25 mwammonium acetate, 3 mw MgCI2, 2 Â¿igmyokinase, 1 ^9pyruvate kinase, 400 /Â¿Mcyclic AMP, 100 Â¿Â¿MCaCL, and thephosphodiesterase preparation in a total volume of 160 n\.5'-AMP standards were incubated under the same condi

tions as the phosphodiesterase so that corrections couldbe made for any influence the compounds under studymight have on the assay system.

Cyclic GMP phosphodiesterase was measured by themethod of Fertel and Weiss (7). In this assay, the 5'-GMP

formed from the hydrolysis of cyclic GMP is reacted withATP in the presence of guanylate kinase. The decline ofATP is quantitated by the luciferin-luciferase reaction and isproportional to the 5'-GMP formed.

Preparation of Activator. Activator was purified frombovine brain by the method described by Teo ef al. (29).Briefly, fresh bovine brains were obtained from a localslaughterhouse and homogenized in 0.1 M Tris-HCI buffercontaining 1 mM Mg24, pH 7.5. The homogenate was

centrifuged at 3000 x g for 20 min, and the activator in thesupernatant fluid was purified by ammonium sulfate precipitation, DEAE-cellulose anion-exchange chromatography,

and column chromatography with the use of Sephadex G100. Homogeneity of the activator preparation was confirmed by use of analytical gel electrophoresis; the molecular weight was estimated to be approximately 18,000.

Activator Activity. Activator activity was assessed by itsability to increase the activity of an activator-deficient phos

phodiesterase prepared from bovine brain according to themethod of Teo ef a/. (29). One unit of activator is defined asthe amount of activator necessary to produce 50% of themaximum activation of this activator-deficient phosphodi

esterase attainable under standard experimental conditionsdescribed above.

Assay of Catecholamines. Sections of pheochromocy-

toma (approximately 100 mg of tissue) or the entire adrenalmedulla from control rats were homogenized in 4 ml of cold0.4 N HCIO;, and centrifuged, and the supernatant fluid wasfrozen until the assay was performed. Urine specimens (24-

hr) were collected from individual rats in bottles containing1 ml of 1.2 N HCIO;,. The samples were centrifuged, and thesupernatant fluid was frozen until assayed. At the time theanimal was sacrificed, blood was collected and centrifuged.Two ml of plasma were added to 1 ml 0.8 N perchloric acid.The samples were centrifuged, and the supernatant fluidwas stored until assayed.

Immediately prior to the assay of the catecholamines, 1ml of 0.5 M Tris, pH 9.0, was added to each sample, and thepH was adjusted to 8.3. The catecholamines were adsorbedonto alumina at pH 8.2 to 8.4 and eluted with 2 ml of 0.1 Nacetic acid. Approximately 60% of the catecholamines wererecovered by this procedure. The concentration of thepurified catecholamines was determined by the trihydrox-

yindole method as described by Chang (3).Protein Concentration. Protein concentrations were

measured by the method of Lowry ef a/. (22).Materials. The breeder rats and samples of the tumor

were most generously supplied by Dr. Shields Warren andRosanna Chute of the New England Deaconess Hospital.Trifluoperazine was generously supplied by Smith Kline andFrench Laboratories, Philadelphia, Pa.; theophylline, cyclicGMP, cyclic AMP, and papaverine were purchased fromSigma Chemical Co., St. Louis, Mo.; dipyridamole was fromBoehringer Ingelheim, Ltd. Ridgefield, Conn, pyruvatekinase, myokinase, and phosphoenolpyruvate were fromBoehringer Mannheim, Mannheim, Germany; firefly luciferinand luciferase were from E. l. du Pont de Nemours andCo., New Brunswick, N. J.; Hanks' balanced salt medium

was from Microbiological Associates, Bethesda, Md.; and

916 CANCER RESEARCH VOL. 38



Phosphodiesterases in Pheochromocytoma

EGTA from Eastman Organic Chemicals, Rochester, N. Y.Other reagents were obtained from general commercialsources.

RESULTS

Functional Characteristics of Pheochromocytoma. Thefunctional nature of the pheochromocytoma was determined by analyzing the total catecholamine content of thetumor, adrenal medulla, urine, and plasma. The concentration of catecholamines in the normal adrenal medulla was95 nmol total catecholamine per mg protein, which is about4 times greater than that of the pheochromocytoma (29nmol/mg protein). This is not surprising when one considers the large amount of connective tissue in the wholetumor. The tumor was functional, however, since it releasedlarge quantities of catecholamines into the circulation asevidenced by the high concentrations of free catecholamines in the plasma (1.2 Â±0.2 nmol/ml) and urine (15 Â±6nmol/24 hr) of tumor-bearing animals. By contrast, the

concentration of catecholamines in plasma of control ratswas less than 0.2 nmol/ml, and the quantity excreted inurine was 1.6 Â±0.4 nmol/24 hr. These results are consistentwith the biochemical characterization of the tumor reportedby Warren and Chute (34) and Chalfie and Perlman (2).

Kinetic Properties of Phosphodiesterase Isolated fromthe Adrenal Medulla and Pheochromocytoma. Chart 1compares the activities of cyclic AMP phosphodiesterase inadrenal medulla and pheochromocytoma. At high concentrations of cyclic AMP, the phosphodiesterase activity ofthe adrenal medulla was twice that of the pheochromocytoma. However, as the concentration of cyclic AMP decreased, the ratio of phosphodiesterase activity of adrenalmedulla to that of the pheochromocytoma also decreaseduntil, at concentrations of cyclic AMP less than 50 Â¿Â¿M,thephosphodiesterase activity of the adrenal medulla fell belowthat of the pheochromocytoma. At substrate concentrationsof 1 H.M, the pheochromocytoma had about 3 times greaterphosphodiesterase activity than did the adrenal medulla.

By contrast, the rate of hydrolysis of cyclic GMP wasapproximately 2 times higher in the ac'renal medulla than

in the pheochromocytoma at all concentrations of cyclicGMP studied (Chart 2).

The phosphodiesterase activities of adrenal medullaefrom control and tumor-bearing rats were similar.

Kinetic analyses of the hydrolysis of cyclic AMP andcyclic GMP in homogenates of adrenal medulla and pheochromocytoma are shown in Charts 3 and 4. The adrenalmedulla and pheochromocytoma both displayed biphasickinetic properties for cyclic AMP hydrolysis, resulting in 2apparent Michaelis constants (Km's), each having a differentmaximum velocity (Vmax). However, the Km's for the phos

phodiesterase of the pheochromocytoma were substantiallylower than those for the adrenal medulla. This wouldaccount for the relatively high activity of the enzyme isolated from the pheochromocytoma when low concentrations of cyclic AMP were used. The hydrolysis of cyclicGMP by both the adrenal medulla and pheochromocytomadisplayed linear kinetics and had approximately the sameaffinity constants.

Effect of Phosphodiesterase Activator and EGTA on

so-

4.5->-5 i 4.0-

3.5-

50 100 150 200 250 300 350 400 450 500

CYCLIC AMP (pMI

Chart 1 Cyclic AMP phosphodiesterase activity of adrenal medulla andpheochromocytoma. Cyclic AMP phosphodiesterase activity of homogenatesof adrenal medulla and purified pheochromocytoma cells was determined asdescribed in "Materials and Methods." The substrate concentration ranged

from 0.5 to 500 Â¡J.M.Each point is the mean of 4 separate experiments.Bars, S.E.

HÃŒÂ« E

t o.K> 5I"a Ã•a Ã¯ PHEOCHROMOCrTOMA

0 50 KXJ ISO 20O 250 300 35O 400 450 SCOCYCLIC GMP ()<M)

Chart 2. Cyclic GMP phosphodiesterase activity of adrenal medulla andpheochromocytoma. Cyclic GMP phosphodiesterase activity of homogenates of adrenal medulla and purified pheochromocytoma cells was determined as described in "Materials and Methods." The substrate concentration

ranged from 0.5 to 500 UM. Each point is the mean of 4 separate experiments.Bars, S.E.

Phosphodiesterase Activity of Adrenal Medulla and Pheochromocytoma. Table 1 shows that neither the purifiedphosphodiesterase activator nor EGTA had any influenceon cyclic AMP phosphodiesterase activity of adrenal medulla or pheochromocytoma over a wide range of substrateconcentrations. These treatments also failed to influencethe hydrolysis of cyclic GMP in adrenal medulla and pheochromocytoma (data not shown).

Subcellular Distribution of Cyclic AMP Phosphodiesterase. The subcellular distribution of cyclic AMP phosphodiesterase is shown in Table 2. There were no markeddifferences between the adrenal medulla and pheochromocytoma in the distribution of phosphodiesterase activity inany of the subcellular fractions. At 400 /XM substrate concentration, the adrenal medulla had about twice the specificactivity of the pheochromocytoma. In the adrenal medulla,the nuclear fraction displayed biphasic kinetics similar tothat of the crude homogenate. All other fractions of theadrenal medulla displayed linear kinetics with Km's ranging

from 50 to 240 Â¿Â¿M.In the pheochromocytoma, all subcellu-

APRIL 1978 917




lar fractions displayed biphasic kinetics similar to that seenin the crude homogenates.

Effect of Inhibitors on Phosphodiesterase Activity. Several pharmacological agents were examined for their abilityto inhibit cyclic AMP phosphodiesterase prepared fromadrenal medulla and pheochromocytoma (Table 3). Theo-phylline, papaverine, and cyclic GMP were competitiveinhibitors and showed no pronounced differences in their

ADRENAL MEDULLA

Vmax' = 0.8 Â±0.1

Km = 8.0 Â±0.8 ,. M

! Vmax' = 6.2 Â±1.0

Km = 130 Â±23 pM

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

"CYCLIC AMP |,,Ml

PHEOCHROMOCYTOMA

-0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 08 0.9 1.0

"CYCLIC AMP ((4M)

Chart 3. Kinetic analysis of cyclic AMP phosphodiesterase activity of theadrenal medulla and pheochromocytoma. The cyclic AMP phosphodiesterase activity of homogenates of adrenal medulla (A] and purified pheochromocytoma (B) cells was plotted according to the method of Lineweaver andBurk (21). The lines of best fit were drawn by linear regression analysis.Substrate concentrations ranged from 0.5 to 500 MM Each point is the meanof 4 separate experiments. *, nmol cyclic AMP hydrolyzed per mg protein

per min.

ability to inhibit either phosphodiesterase preparation. Tri-fluoperazine, a potent inhibitor of phosphodiesterase frombrain (20, 32, 36), and dipyridamole, an agent effectiveagainst phosphodiesterase of platelets (25) and lymphocytes (W. N. Hait and B. Weiss, unpublished observations),showed a mixed type of inhibition of the phosphodiesteraseof both adrenal medulla and pheochromocytoma.

ADRENAL MEDULLA

Â±> '

Vmax' = 1.7 Â±0.4

Km - 27 Â±4 /iM

B

0.025 0 0.05 0.1 0.15 0.2 0.25

"CYCLIC GMP (,,M|

Chart 4. Kinetic analysis of cyclic GMP phosphodiesterase activity of theadrenal medulla and pheochromocytoma. The cyclic GMP phosphodiesterase activity of homogenates of adrenal medulla (Ai and purified pheochromocytoma (B) cells was plotted according to the method of Lineweaver andBurk (21). The lines of best fit were drawn by linear regression analysis.Substrate concentrations ranged from 0.5 to 500 AIM.Each point is the meanof 4 separate experiments. *, nmol cyclic AMP hydrolyzed per mg protein

per min.

Table 1Effect of phosphodiesterase activator and EGTA on cyclic AMP phosphodiesterase activity of rat adrenal medulla and

pheochromocytomaThe effect of the calcium-dependent phosphodiesterase activator (10 units/sample) and EGTA (0.3 mw) on cyclic

AMP phosphodiesterase activity of homogenates of the adrenal medulla and pheochromocytoma was determined asdescribed in the text.

Cyclic AMP phosphodiesterase activity(nmol cyclic AMP hydrolyzed/mg protein/min)

Cyclic AMP(MM)1

10100

400Adrenal

medullaControl0.16

Â±0.02"

0.43 Â±0.021.7 Â±0.14.4 Â±0.8Activator0.14

Â±0.010.39 Â±0.011.6 Â±0.14.4 Â±0.8EGTA0.16

Â±0.010.37 Â±0.021.5 Â±0.14.5 Â±0.6PheochromocytomaControl0.44

Â±0.010.71 Â±0.021.5 Â±0.11.8 Â±0.4Activator0.43

Â±0.020.69 Â±0.031.6 Â±0.11.8 Â±0.4EGTA0.42

Â±0.020.69 Â±0.021.5 Â±0.11.8 Â±0.2

" Mean Â±S.E. of 4 determinations.




Phosphodiesterases in Pheochromocytoma

Table 2Subcellular distribution of cyclic AMP phosphodiesterase in adrenal medulla and pheochromocytoma

Each homogenate was centrifugea successively at 900 x g for 10 min, 10,500 x g for 20 min, and 100,000 x g for 60 min. Eachparticulate fraction was washed by resuspending in Tris-HCI buffer and recentrifuging. The resulting washed particulate fractions wereresuspended in Tris-HCI buffer and assayed for protein content and cyclic AMP phosphodiesterase activity with the use of 400 MMcyclicAMP as substrate for determination of the total phosphodiesterase activity and the specific activity. Each value represents the mean of 3separate experiments.

% of total recoveredphosphodiesterase

activity

Specific activity (nmol cyclicAMP hydrolyzed/mg pro

tein/mm)

Michaelis constants"

FractionHomogenate900

x g(nuclear)10,500xg(mitochondrial)100,000

xg(microsomal)Supernatant

fluidAdrenal

medulla100356356Pheochromocytoma1004112542Adrenal

medulla3.9

Â±0.2*2.8Â±0.21.9Â±0.11.0

Â±0.13.9

Â±0.3Pheochro

mocytoma1.7

Â±0.10.8Â±0.11.7Â±0.21.0

Â±0.12.0

Â±0.1AdrenalKm,

(MM)89medullaKm,(MM)10012024011050PheochromocytomaKm,(MM)0.92.91.31.10.6Km,(MM)1650231418

" The Km'swere determined by kinetic analysis with the use of substrate concentrations ranging from 0.5 to 500 MM.b Mean Â±S.E.

Table 3Effect of several phosphodiesterase inhibitors on

phosphodiesterase activity of adrenal medulla andpheochromocytoma

Cyclic AMP phosphodiesterase activity of homogenates of adrenal medulla and purified pheochromocytoma was measured in thepresence of various phosphodiesterase inhibitors. Enzyme activitywas determined in triplicate at 7 different inhibitor concentrationsfor each of 4 substrate concentrations. The data were plottedaccording to the method of Dixon (5), and the K,'s were determined

from the median point at which the lines intersected.

InhibitorTrifluoperazine

DipyridamoleCyclic GMPPapaverineTheophyllineAdrenal

medulla(K,)60

100150260650Pheochromocy

toma(Ki)175

400270270850

DISCUSSIONThe major points made in this study are: (a) normal

adrenal medulla had a higher maximum rate of hydrolysis(Vmax)Â°fcyclic AMP phosphodiesterase than the pheochromocytoma cells; and (b) the pheochromocytoma cells canhydrolyze low (and perhaps physiological) concentrationsof cyclic AMP at a far greater rate than can the normaladrenal medulla; and (c) this apparently is due to thepresence in the pheochromocytoma cells of a Iow-Km(high-affinity) form of cyclic AMP phosphodiesterase not found inthe normal adrenal medulla. Thus, although both the pheochromocytoma and adrenal medulla displayed biphasickinetics for cyclic AMP phosphodiesterase activity, theapparent Km's for cyclic AMP hydrolysis in the pheochromocytoma were significantly lower (between 5- and 10-fold)than those of the normal adrenal medulla.

Kinetic analysis of the cyclic AMP phosphodiesteraseactivity of the subcellular fractions demonstrated a difference in the distribution of the high- and low-affinity formsof phosphodiesterase. In the adrenal medulla, the high-affinity form of phosphodiesterase was localized in thenuclear fraction, whereas all fractions of the pheochromocytoma contained the Iow-Km form of phosphodiesterase.

This difference in the high- and Iow-Km phosphodiester-ases between adrenal medulla and pheochromocytomacells may explain our observation that the rate of hydrolysisof cyclic AMP in the tumor is greater than that of theadrenal medulla when measured at low substrate concentrations but lower at high substrate concentrations. Similarfindings have been reported by Clark ef al. (4) and Hickie eral. (18) for rat hepatomas. Both groups report an increasedactivity of a Iow-Km form of cyclic AMP phosphodiesterasein the neoplasm compared to that of normal liver. Thesestudies show again the complexity of the cyclic nucleotidephosphodiesterase system and emphasize the need forstudying phosphodiesterase activity over a wide range ofsubstrate concentrations when different tissues are beingcompared.

Phosphodiesterase exists in multiple forms with varyingphysiological and pharmacological characteristics, such assubstrate specificity (19, 30), stability (32, 33), and responseto activator and inhibitors (8, 20, 32, 33, 36). One form ofphosphodiesterase is particularly sensitive to an endogenous calcium-dependent activator of phosphodiesterase (33,36). Uzunov ef al. (31) demonstrated the presence of thisactivator-sensitive phosphodiesterase in adrenal medullaafter separating the phosphodiesterase isozymes by poly-acrylamide gel electrophoresis. In our experiments, theaddition of activator or EGTA (which would inhibit theactivity of the calcium-dependent activable phosphodiesterase) failed to affect the phosphodiesterase activity of homogenates of adrenal medulla or pheochromocytoma atany concentration of cyclic AMP. This suggests that themajor form of phosphodiesterase present in the adrenalmedulla or pheochromocytoma is not activated by thiscalcium-dependent activator nor is the enzyme alreadyactivated. Our results, which are consistent with the observations of Egrie and Siegel (6), who also failed to demonstrate an activable form of phosphodiesterase in adrenalmedulla, suggest that the activator-sensitive form of phosphodiesterase does not contribute significantly to the totalphosphodiesterase activity observed at any concentrationof substrate.

Since the multiple forms of phosphodiesterase can be

APRIL 1978 919




differentially inhibited by drugs (8, 36), it may be possible tofind a pharmacological agent specific for the high-affinityform of phosphodiesterase which is present in the pheo-chromocytoma but not in the adrenal medulla. In a preliminary attempt to find such a drug, we chose compoundsknown to be effective inhibitors of phosphodiesterase inother tissues and which may have different mechanisms ofaction: papaverine and theophylline, which are competitiveinhibitors of phosphodiesterase (36); trifluoperazine, whichis a potent inhibitor of phosphodiesterase of brain (36) andwhich acts by preventing the activation of phosphodiesterase (20); and dipyridamole, which has been shown to be aparticularly effective inhibitor of phosphodiesterase of lymphocytes (W. N. Hait and B. Weiss, unpublished observation). Unfortunately, none of these pharmacological agentsexamined so far were particularly effective inhibitors of thephosphodiesterase isolated from either the adrenal medullaor pheochromocytoma, although the enzyme from the adrenal medulla was somewhat more sensitive to inhibition bytrifluoperazine and dipyridamole.

In summary, we have confirmed the growth pattern andfunctional activity of a transplantable rat pheochromocytoma and characterized the activity of cyclic nucleotidephosphodiesterase isolated from purified pheochromocytoma cells and compared it with the phosphodiesteraseactivity of the adrenal medulla. Our demonstrations thatpheochromocytoma cells have a lower activity of cyclicGMP phosphodiesterase and a higher activity of the high-affinity cyclic AMP phosphodiesterase when compared withthat of the adrenal medulla are consistent with the proposition that tumor growth may be associated with either a reduced intracellular concentration of cyclic AMP or a decreased ratio of cyclic AMP to cyclic GMP.

ACKNOWLEDGMENTS

We thank Thomas Wallace for his excellent technical assistance.

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1978;38:915-920. Cancer Res Robert M. Levin and Benjamin Weiss the Rata Transplantable Pheochromocytoma and Adrenal Medulla of Characteristics of the Cyclic Nucleotide Phosphodiesterases in

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