Experimental Biology and MedicineProceedings of the Society for

PSEBM

Joan Vernikos-Danellis and Clifton G. Harris IIIRat

Pituitary, Median Eminence, Heart, and Cerebral Cortex of theHydrocortisone on the Phosphodiesterase Activity of the

The Effect of in Vitro and in Vivo Caffeine, Theophylline, and

doi: 10.3181/00379727-128-331831968, 128:1016-1021.Proc Soc Exp Biol Med 

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1016 DRUGS AND PITUITARY PHOSPHODXESTERASE

that L chains are released from reduced and alkylated human IgM myeloma proteins in the absence of a dispersing agent, and that the IgM subunit might consist of 3 L chains and 2 H chains. The reasons for being able to account for only 5 combining sites on a molecule which structurally suggests 10 sites are obscure, and these findings (15, 17 ) might have some bearing on the estimation of the number of binding sites of IgM.

Summary. Purified chicken IgM anti- sheep erythrocyte antibody was mildly re- duced so that the subunits did not dissociate in propionic acid. Presumably the inter- chain disulfide bonds were intact. Compared with unreduced IgM antibody on a weight basis, these subunits had little or no hemag- glut ina ting activity . P

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6. Chan, P. C. Y. and Deutsch, H. F., J.

7. Fudenberg, H. H. and Kunkel, €3. G., J.

8. Onoue, K., Yagi, Y., Stelos, P., and Pressman,

9. Cooper, A. G., Science 157,933 (1967). 10. Burnstein, M. and Praverman, A., Compt.

Rend. 245, 2558 (1957). 11. Bencdict, A. A., in “Methods in Immunology

and Immunochemistry,” Williams, C. A. and Chase, M. W., eds., Vol. 1, p. 235. Academic Press, New York, 1967. 12. Scheidegger, J. J., Intern. Arch. Allergy 7,

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Received April 10, 1968. P.S.E.B.M., 1968, Vol. 128.

The Effect of in Vitro and in Vivo Caffeine, Theophylline, and Hydrocortisone on the Phosphodiesterase Activity of the Pituitary, Median Eminence, Heart, and Cerebral Cortex of the Rat (33183)

JOAN VERNIKOS-DANELLIS AND CLIFTON G. HARRIS, IIIl Environmental Biology Division, Ames Research Center, NASA, M o f fe t t Field, California 94035

Considerable evidence in recent years has indicated that the actions of a number of hormones, including catecholamines, vaso- pressin, and ACTH, are mediated by the cyclic nucleotide, 3’5’-adenosine monophos- phate ( 1 ) . In 1958, Sutherland and Rall ( 2 ) demonstrated that enzymatic activity in various mammalian tissues was capable of hydrolyzing cyclic 3’5’-AMP at the 3’ posi- tion. The existence of this cyclic nucleotide phosphodiesterase provided one possible

cal School, St. Louis, Missouri. 1 Present address: Washington University Medi-

means by which the steady-state level of cyclic-AMP could be regulated. Substances that inhibit this enzyme would therefore in- crease the steady-state level of cyclic-AMP and might be expected to enhance the re- sponse to those hormones whose actions are mediated by this nucleotide. Adding the methyl xanthines, caffeine, theophylline, and theobromine to various tissues in vitro inhi- bited this phosphodiesterase activity (2-5) and potentiated the cyclic-AMP mediated effects of various hormones (4,6-9). Fur- thermore, these drugs are reported to

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DRUGS AND PITUITARY PHOSPHODIESTERASE 101 7

enhance the action of catecholamines on heart function and phosphorylase activity (10,l 1), and to evoke a lipolytic response from rat adipose tissue both in vitro and in Y ~ V O (4,12).

The results of recent investigations in this laboratory have suggested a possible role for cyclic-AMP in the hypothalamic-pitui- tary response to stress (13). This interpre- tation was based on the finding that caffeine and theophylline are capable of potentiat- ing, in rats, the secretion of ACTH in re- sponse to an acute stress. I t was also noted that caffeine administration could overcome the ability of steroids to inhibit the stress- induced secretion of ACTH.

Since inhibition of the phosphodiesterase has not been demonstrated following the ad- ministration of caffeine or theophylline to animals in v im, it became necessary, in or- der to substantiate our thesis, to show that at the time the stress-induced secretion of ACTH was potentiated by the methyl xan- thines, the phosphodiesterase activity of the anterior pituitary and/or the median emi- nence was reduced. In the present study changes in the phosphodiesterase activity were measured in tissue homogenates of an- terior pituitary, medium eminence, heart, and brain cortex incubated with caffeine, theo- phylline, or hydrocortisone, or obtained from animals following their pretreatment with these drugs.

Methods. The system used for determin- ing the cyclic-3’5’-nucleotide phosphodies- terase activity was an adaptation of the two- stage assay described by Nair (14), that is, using excess 5’-nucleotidase to measure the release of inorganic phosphate. Prior to ad- dition of the substrate, 0.1 ml of tissue ho- mogenate was incubated for 15 min with the drug at 37°C. Drugs dissolved in sodium barbital buffer were added so that they were of the desired concentration in the final volume of 0.75 ml. The reaction mixture con- tained 3’5’-AMP (Sigma Chemical Co.) , 2 .O mM; MgC12, 2.0 mM; sodium barbital buffer, 4.5 mM, pH 7.8; and 0.1 ml of 2 % tissue homogenate. Controls contained tissue homogenate without the drug and boiled tissue homogenate with and without the

drug. Blanks were also run without the sub- strate. The mixture was incubated a t 37°C for 301 min. The reaction was stopped by boiling the mixture for 4 min. The mixture was then cooled, and 0.015 ml of a 5 mg/ml aqueous solution of Crotalus atrox venom (purchased from Ross Allen’s Reptile Insti- tute) was added. The mixture was incubated for another 30 min at 37OC, and the reac- tion was terminated by the addition of 0.1 ml of ice-cold 50% trichloroacetic acid (TCA) .

An adaptation of the Fiske-Subbarow method (15) was used for determining the amount of inorganic phosphate released in the incubation media. To a 0.5 ml aliquot of the TCA supernatant in Coleman cuvettes (9 X 75 mm) were added 1.4 ml of distilled water and 0.4 ml of 2.5% ammonium mo- lybdate in 5 N H2S04. The color was de- veloped by the addition of 01.2 ml of 0.25% 1 -amino-2-napthol-4-sulfonic acid. Ten min- utes later, the optical density of the result- ing solution was read at 660 mp on a ‘Cole- man colorimeter. Enzyme activity was ex- pressed as pmol of PiJmg of tissue.

The optimum pH for the phosphodies- terase assay system used in this study was obtained for each tissue homogenate by de- termining the effect of varying the pH on the rate of Pi liberation from 3’5’-cyclic AMP. Enzymatic activity was assayed as described above with a sodium barbital buffer that produced a final pH in the reac- tion mixture ranging from 7.01 to 8.9 at in- tervals of 0.4 pH unit. The cyclic phospho- diesterase showed a peak at pH 7.8 (range 7.4-8.2) with activity dropping off sharply outside this range (Fig. 1). Throughout this study, ,a pH of 7.8 was therefore used to determine phosphodies terase activity .

Female Sprague-DawIey rats, weighing 100 5 5 gm, were fed commercial rat chow and water ad libitum, and were maintained in a room at a constant temperature of 24°C. The pituitary, median eminence, brain cortex, and cardiac ventricle were re- moved and weighed immediately following decapitation. All tissues were kept on ice between excision and homogenization. A 2 % homogenate was prepared in conical ground-

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1018 DRUGS AND PITUITARY PHOSPHODIESTERASE

.01 I-

F MEDIAN 0.25 EMINENCE 0.45 CORTEX

0.30

0.25

0.20

M) 7.5 8.0 8.5 9~ PH 7.0 7.5 8.0

FIG. 1. The pH dependence of phosphodiesterase 9.0 PH 7.0 7.5 8.0 a5 91) p~

activity in various tissue homogenates.

glass homogenizers with ice-cold deionized water as diluent. Homogenates were frozen immediately and stored at -20°C until used. All homogenates were assayed within 15 min of thawing. Each homogenate pool con- sisted of tissue from at least 12 rats. Dupli- cate determinations were performed with each homogenate.

All drugs were administered subcutane- ously in a volume of 0.5 ml physiological saline (0.9%). Caffeine (2 mg/100 gm of body wt.) and theophylline (5 mg/lOO gm of body wt.) were injected 30 min before de- capitation. A suspension of hydrocortisone (free alcohol) in normal saline was injected 4 hours before the animal was sacrificed. Chronic treatment with hydrocortisone con- sisted of daily subcutaneous injections of 5 mg/100 gm of body weight for 4 days, the animals were sacrificed 24 hours following the last injection. Control groups of unin- jected animals or rats receiving normal

saline only were run with each series of experiments.

Bilateral adrenalectomy was performed under ether anesthesia. The adrenalecto- mized rats were maintained on the normal diet but with 0.9% saline in place of drink- ing water for 30 days, a t which time they were decapitated.

Resdts . Table I shows the changes in phosphodiesterase activity of homogenates from anterior pituitary, median eminence, heart, and brain cortex incubated in vitro with or without caffeine (10 mM), theo- phylline (10 mM), hydrocortisone, or dex- amethasone. The addition of caffeine and theophylline inhibited the phosphodiesterase activity in all the tissues studied. Caffeine and theophylline at 10 mM caused approx- imately 38-66% inhibition in the pituitary, median eminence, and brain cortex, while they appeared to be less effective on the heart phosphodiesterase activity. Incubation with the lower concentration of dexameth- asone reduced the phosphodiesterase ac- tivity of the anterior pituitary and brain cortex 38 and 24%, respectively, while the higher concentration had no effect on the enzyme activity in all the tissues studied. Incubation with hydrocortisone showed a 29% decrease in the enzyme activity of the pituitary while that of the median eminence and brain cortex increased by 2 2 and 18%, respectively .

Table I1 shows the effect of pretreating rats with a single subcutaneous injection of

TABLE I. Changes in Phosphodiesterase Activity Produced by Caffeine, Theophylline, Hydrocortisone, and Dexamethasone in Vitroc.

Drug Tissue phosphodiesterase activity (pmol of P, /mg of tissue/60 min -t SE) Type Conc. Anterior pituitary Median eminence Heart Brain cortex

None 0 0.113 k 0.003 (15) 0.212 k 0.005 (22) 0.024 2 0.002 (12) 0.384 2 0.009 (19)

Caffeine 10 mM 0.054 & 0.009" (4) 0.100 & 0.010' (4) 0.021 5 0.003 (4) 0.190 -c- 0.027" (4)

Theophylline 10 mM 0.039 2 0.015" (4) 0.131 & 0.005" (4) 0.015 & 0.003b (4) 0.187 0.027" (4)

Hydrocortisone 25 pg 0.080 (1) 0.165 (1) 0.025 ( I ) 0.452 (1)

Dexamethasone 50 pg 0.070 (1) 0.192 (1) 0.020 (1) 0.293 ( I )

Dexamethasone 200 pg 0.103 (1) 0.194 (1) 0.025 (1) 0.335 (1) a Differs from control value at p < 0.001.

Differs from control value at p < 0.05. Numbers in parentheses indicate the number of experiments. Homogenate for each experiment was deriyed

by pooling tissue from 10 to 12 rats. Duplicate determinations were carried out on each tissue pool.

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DRUGS AND PITUITARY PHOSPHODIESTERASE 1019

TABLE 11. Phosphodiesterase Activity in the Pituitary, Median Eminence, Heart, and Brain Cortex from Rats Pretreated with Caffeine, Theophylline, or Normal Saline.c

Phosphodiesterase activity (pmol of P,/mg of tissue/60 min -+ SE) Treatment Anterior pituitary Median eminence Heart Brain cortex

Saline 0.134 & 0.011 (7) 0.229 2 0.022 (7) 0.018 k 0.003 (7) 0.371 (2) Caffeine (2 mg/ 100 gm) 0.090 k 0.009" (8) 0.228 & 0.024 (6) 0.020 k 0.003 (6) 0.411 (3)

Theophylline (5 mg/100 gm) 0.105 k 0.007b (8) 0.284 & 0.022 (4) 0.021 k 0.003 (4) 0.493 (2) " Differs from saline control at p < 0.01.

Differs from saline control value at p < 0.05. Numbers in parentheses indicate the number of experiments. Homogenate for each experiment was derived

by pooling tissue from 10 to 12 rats. Duplicate determinations were carried out on each tissue pool.

saline, caffeine, or theophylline 30 min be- fore the phosphodiesterase activity of vari- ous tissues was determined. There was a significant decrease in the enzyme activity of the anterior pituitary only; this decrease was more pronounced with caffeine than with theophylline.

Table I11 shows the results of an experi- ment designed to investigate whether raising the circulating level of corticosteroids by acute or chronic treatment with large doses of hydrocortisone, or lowering this level by means of long-term adrenalectomy in any way affected the phosphodiesterase activity of hypothalamic and pituitary tissue. Pre- treatment with a single dose of hydrocor- tisone 4 hours before activity was deter- mined, chronic treatment with this steroid, or adrenalectomy had little effect on the enzyme activity in either the pituitary or the median eminence.

Discussion. The distribution of the cyclic nucleotide phosphodiesterase in the order of

decreasing activity was as follows: brain cortex > median eminence > anterior pitui- tary > heart and adrenal as compared to that of 5'-nucleotidase which was: anterior pituitary > heart > adrenal > median eminence (Vernikos-Danellis and Harris, unpublished observations, 1966). The rela- tive distribution of the phosphodiesterase activity in rat tissues studied agrees, in gen- eral, with that reported by Butcher and Suth- erland ( 3 ) for the dog, and substantiates their observation that brain cortex contains the highest activity of this enzyme. Since the presence of this enzyme in the anterior pituitary gland has not previously been re- ported, it is of interest to note that not only does this gland possess such activity, but it is particularly potent in its ability to hydro- lyze cyclic-AMP as compared to heart and other endocrine organs.

The inhibition of this phosphodiesterase by the methyl xanthines in vitro, first de- scribed by Sutherland and Rall (2), has

'TABLE 111. Phosphodiesterase Activity in 30-day Adrenalectomized Rats or after Acute or Chronic Treatment with Hydrocortisone."

Phosphodiesterase activity (umol of P4/mg of tissue/60 min & SE) Treatment Anterior pituitary Median eminence Heart Brain cortex

Saline (acute) 0.134 3- 0.011 (7) 0.229 2 0.022 (7) 0.018 & 0.003 (7) 0.371 (2) Hydrocortisone (acute) 0.110 (1) 0.273 (1) 0.022 (1)

(5 mg/ 100 gin)

Saline (chronic) 0.1 10 (2) 0.211 (3) 0.022 (2) 0.352 (2) Hydrocortisone (chronic) 0.103 (3) 0.192 (2) 0.017 (1) 0.348 (2)

Normal 0.1 14 (3) 0.225 (3) 0.018 (3) 0.399 (3)

30-day Adrenalectomy (ADX) 0.112 (3) 0.231 (3) 0.023 (3) 0.425 (3)

by pooling tissue from 10 to 12 rats. Duplicate determinations were carried out on each tissue pool.

(5 mg/ 100 gm)

" Numbers in parentheses indicate the number of experiments. Homogenate for each experiment was derived

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1028 DRUGS AND PITUITARY PHOSPHODIESTERASE

been confirmed by recent studies. Butcher and Sutherland (3) found 70 and 90% inhi- bition of a purified enzyme obtained from dog heart incubated with 10 mM caffeine and theophylline, respectively. Similarly, Hynie et al. (4) reported 89% inhibition of this enzyme in rat adipose tissue with 10 mM theophylline, and Cheung ( 5 ) observed a 50% inhibition in rat brain cortex with 20 mM caffeine. Incubating homogenates with 10 mM caffeine or theophylline in this study resulted in a marked, but less profound, inhibition of enzyme activity in rat anterior pituitary ( 5 2 and 66%) median eminence (53 and 38%), and heart (13 and 38%) as com- pared to that reported by Butcher and Suth- erland (3) and Hynie et al. (4) . On the other hand, with a 10 mM concentration of these xanthines, the same degree of inhibi- tion for brain cortex was obtained as that reported by Cheung.

Pretreatment with a single dose of caffeine or theophylline was recently shown to increase the ACTH concentration of the pituitary gland 30 min after its administra- tion and to enhance the secretion of ACTH in rats after exposure to an acute stress stimulus (13). In addition, it was noted that the administration of caffeine could over- come the ability of hydrocortisone to de- press the stress-induced secretion of ACTH when such a stimulus was applied 4 hours after the injection of this steroid. This and other evidence suggested that the hypothala- mic-pituitary response to stress was possibly mediated by cyclic-AMP, and that the mechanism by which the xanthines en- hanced this response depended on their abil- ity to inhibit the cyclic nucleotide phospho- diesterase that hydrolyzes cyclic-AMP in the pituitary and/or hypothalamus.

I t therefore became important to deter- mine whether the phosphodiesterase activity of these tissues was in any way altered 30 min after the administration of caffeine or theophylline to rats in vivo. The present data show that under these experimental conditions the enzyme activity is indeed de- pressed in the anterior pituitary only. I t is of interest to note that caffeine, which was found to be more effective in potentiating

the stress-induced secretion of ACTH, was also the more effective phosphodiesterase in- hibitor in vivo; whereas, theophylline was the more potent inhibitor in vitro.

The bulk of more recent evidence sug- gests that large doses of corticoids act on the hypothalamus rather than on the pituitary gland itself to depress ACTH secretion (16-20). If the antagonism of caffeine on the pituitary inhibitory properties of corti- coids was truly competitive, involving this cyclic nucleotide phosphodiesterase, then in- cubation of pituitary or hypothalamic tissue with steroids, acute or chronic treatment with hydrocortisone, or long-term adrenalec- tomy might be expected to alter the activity of this enzyme dramatically. No such change was observed in this study. The present findings support the thesis that caffeine and theophylline enhance the stress- induced secretion of ACTH by depressing pituitary phosphodiesterase activity. On the other hand, it would appear from these data that changing the levels of corticoids has no effect on the activity of this enzyme, and that, therefore, the ability of steroids to suppress hypothalamic-pi tuitary ACTH secre- tion does not involve this enzyme.

Summary. This study was undertaken to substantiate the thesis that the potentiation of the stress-induced secretion of ACTH by the methyl xanthines is due to their ability to inhibit the 3’5’-cyclic nucleotide phosphodi- esterase. The effect of various drugs on the phosphodiesterase activity of the anterior pituitary, median eminence, brain cortex, and heart of rats was determined both in vitro and in vivo. Incubation of homogenates with caffeine and t heophylline markedly reduced the phosphodiesterase activity in all tissues studied except heart, which showed a signifi- cant reduction only in the presence of theo- phylline. In contrast, pretreating the animals with a single dose of caffeine or theophylline reduced the enzyme activity of the anterior pituitary only, and caffeine was more effec- tive than theophylline. Adrenalectomy, chron- ic or acute administration of hydrocortisone, or incubation of tissue homogenates with hy- drocortisone or dexamethasone did not alter the phosphodiesterase activity of the tissues

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ENZYMES AND GLYCERIDE AFTER ALCOHOL 1021

studied. I t is concluded that the effects of caffeine and theophylline on the pituitary stress response could be attributed to their in- hibition of the phosphodiesterase activity in that tissue.

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Reoeived April 22, 1968. P.S.E.B.M., 1968, Vol. 128.

The Effect of Chronic Alcohol Administration on Enzyme Profile and Glyceride Content of Heart Muscle, Brain and Liver" (3184)

M. MARCINIAK, S. GUDBJARNASON, AND T. A. BRUCE (Introduced by R. J. Bing) Department of Medicine, Wayne State University School of Medicine and

Detroit General Hospital Research Corp., Detroit, Michigan 48207

Despite the fact that an association of alcoholism and heart disease has been known since 1873 (1), relatively few reports have appeared describing the effect of chronic alcohol administration on myocardi- al metabolism. Most of the data available on the effect of alcohol on the cardiovascular system have been derived from acute experi- ments in humans and animals ( 2 ) . Reports on the effect of chronic alcohol administra- tion on the brain have also been relatively scarce. Kinard and Hay ( 3 ) observed that chronic alcohol administration caused a dim- inution in brain cholinesterase activity. The effect of chronic alcoholism on the liver

* Work supported by U. S. Public Health Service Grant No. HE 05043, the Michigan Heart Association, and the Detroit General Hospital.

and its metabolism has, on the other hand, been studied extensively in the past (4-8).

The purpose of this study was to compare the effect of chronic alcohol administration on the glyceride content and enzyme profile of heart muscle, brain, and liver. These or- gans differ widely in alcohol-dehydrogenase content and, therefore, in their ability to oxidize ethanol.

Matmial and Methods. The study was performed on 24 mongrel dogs, divided into a control group ( N = 1 2 ) and an experimen- tal group ( N = 1 2 ) . The experimental group received 20% ethanol as the sole drinking fluid (sweetened with 1 teaspoon of Sucaryl per 2 liters of alcohol), and these animals consumed 400-600 ml of this fluid per 24 hours. The experimental and control animals

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