Studies on the synthesis of prostaglandins in the vesicular glands of essential fatty acid-deficient and hypophysectomized rats

Riochin?ica et Biophysics Acts, .(‘I Elsevier Scientific Publishing

296 (1973) 586-592 Company, Amsterdam

- Printed

in The Netherlands

BBA 56217

STUDIES

ON THE SYNTHESIS

VESICULAR

GLANDS

HYPOPHYSECTOMIZED

WEE CHONG The Normel (Received

TAN

lnstitrrte, November

OF PROSTAGLANDINS

OF ESSENTIAL

IN THE

ACID-DEFICIENT

AND

RATS

and ORVILLE University

FATTY

S. PRIVETT

ofMinnesottr,

Austin,

Minn. 55912 (U.S.A.)

I 3th, 1972)

SUMMARY

Studies are reported on the detection and itz vitro biosynthesis of prostaglandins in the vesicular glands of hypophysectomized and essential fatty acid-deficient rats. The vesicular glands of the hypophysectomized rats underwent rapid degeneration and were depleted of their stores of arachidonic acid and prostaglandins. Hypophysectomy also inhibited the in vitro conversion of arachidonic acid to prostaglandin E, with the vesicular gland of rats. The vesicular glands of essential fatty acid-deficient animals also became depleted of their stores of prostaglandins and arachidonic acid, as well as linoleic acid, and lost their activity for the h vitro conversion of exogenous arachidonic acid to prostaglandin E,.

INTRODUCTION

Although most of the biological active prostaglandins are derived from essential fatty acids’ - 5, the relationship of prostaglandin synthesis to essential fatty acid activity has not been defined3*6-9 . Recently Ziboh et aZ.9 demonstrated that the dermal symptoms of an essential fatty acid deficiency in rats can be alleviated by topical application of prostaglandins. Dermal symptoms identical to those of an essential fatty acids deficiency are produced by hypophysectomy in immature rats in spite of an abundance of essential fatty acids in the diet”,“. Hypophysectomy also impaired the it? vitro biosynthesis of prostaglandins by sheep vesicular glands”. These observations indicate a link between the biological activity of essential fatty acids and prostaglandin synthesis possibly through a function of the hypophysis. In order to obtain a further insight into the relationship of prostaglandin synthesis and the biological activity of essential fatty acids, the study reported here was undertaken to determine if there was an effect of essential fatty acid deficiency or hypophysectomy on the endogenous level and in vifro biosynthesis of prostaglandins in the vesicular glands of rats.

BIOSYNTHESIS MATERIALS

OF PROSTAGLANDINS AND

587

METHODS

Hypophysectomized and normal male rats of the Sprague-Dawley strain, 180-200 g, were obtained from the Hormone Assay Laboratory, Chicago. The animals were housed in individual cages and fed ad libitum a basic sucrose-casein diet supplemented with minerals and vitamins in the required amounts and IO %. by weight, of corn oil”. Essential fatty acid-deficient animals were obtained by feeding weanling male rats of the Sprague-Dawley strain up to 12 months a basic fat-free diet” or this diet supplemented with IO %, by wt, of hydrogenated coconut oil as the sole source of fat in the diet. Animals were killed under ether by withdrawal of blood from their aortas; the vesicular glands were excised and frozen on solid Co, when not immediately used. Endogenous prostaglandin content was determined via the ground powder technique developed especially for application to small amounts of tissue”. In this method, the glands are cut into small pieces, frozen in solid CO, and then pulverized to a fine powder in a mortar imbedded in solid CO,. These powders are used for determination of prostaglandin E content or enzymic synthesis of prostaglandins from arachidonic acid. The general procedure for determination of biosynthetic activity of the glands is as follows: The substrate, I mg of arachidonic acid, is emulsified in a 25-ml beaker with IO ml of a buffered solution consisting of o. I M NH,Cl, pH 8.5 with NH,OH, containing 20 mg of glutathione. I g of the powdered glands is added to this emulsion and the mixture is incubated at 37 “C in an atmosphere of air for I h with shaking. The reaction is stopped by lowering the pH to 3 by the addition of a solution of I M citric acid. The prostaglandins are extracted into ethyl acetate and recovered by evaporation of the solvent. The prostaglandin extract is redissolved in 5 ml of 95 “/ ethyl alcohol for analysis by ultraviolet spectrophotometry or thin-layer chromatography13.14. A control sample is prepared in the same manner except that the addition of the arachidonic acid substrate is omitted. The absorbance, at 278 nm after alkali addition, is plotted as a function of time and the absorbance at zero time is determined by extrapolation of the values. The total amount of prostaglandin E is calculated on the basis of the molar extinction coefficient (AA = 1.00 corresponds to 39.4 Llg of prostaglandin E per 3 ml of reaction mixture)13.14. Isolation and identification of the prostaglandins of the E type were carried out by thin-layer chromatography as described by Green and Samuelsson” and Ramwell and Daniels’ 6. Fatty acid composition Lipids from rat vesicular glands obtained by extraction with chloroformmethanol (2 : I, v/v) were converted to methyl esters using an HCl catalyst, as previously described17, and analyzed by gas-liquid chromatography. Gas-liquid chromatography was carried out with an apparatus equipped with a hydrogen flame detector and an 8 ft x r/8 inch column packed with IO “/, EGSS-X on Chromosorb W at 200 “C. Nitrogen was used as the carrier gas and the percent distribution of the fatty acids was obtained by proportionalities of the peak areas. Identification of the peaks was made on the basis of the retention time of known methyl esters.

$38

W. C. TAN,

0. S. PRlVETT

RESULTS

Hypophysectomy retarded growth and caused a rapid degeneration of the vesicular glands of rats (Table I). The size of the glands decreased drastically during the first 24 h after hypophysectomy and continued to decrease in weight for approximately 4 months when they appeared to level off at a weight of approximately 25 mg. The prostaglandin E content of the vesicular glands diminished simultaneously. The major decrease (85 7,:) of the prostaglandin E content of the vesicular glands came within 3 days after hypohysectomy. Thereafter, the decrease was slow and a trace of TABLE

I

EFFECT OF VESICULAR

HYPOPHYSECTOMY GLANDS OF RATS

ON THE

PROSTAGLANDIN

E CONTENT

OF THE

Zero (normal animals) I

2 3 7 14 28 I20

360

IO

‘95

~ 10*

4 3 4 6

1901 187, I83

7 x

4 5 4 5

1901 ISO! 165

3 9

/ 8 I‘), IO

I10

12 5

0.879

~ o.jo*

458

357

501

I08

0.210**

0.216 0.124 0.1 IX 0.089 0.076 0.030 0.024

426 507 760 755 333 290

53 61 67 57 IO 7

* Mean + S.D. ** Average weight of pooled glands. *** Analysis

of pooled

samples.

these compounds was still present in the vesicular glands I year after hypophysectomy. Because of the rapid degeneration of the vesicular glands, there was a period in which the concentration of prostaglandin E was greater in the glands of the hypophysectomized than in those of normal animals. Eventually the concentration, as well as the total amount of prostaglandin E, was diminished in the vesicular glands of the hypophysectomized animals as illustrated in Table I. The fatty acid composition of the lipid of the vesicular glands was also affected by hypophysectomy as illustrated in Table I I. The percentage of linoleic and arachidonic acids in the lipid increased shortly after hypophysectomy and then decreased. Little arachidonic acid was present in the glands I month after the operation, indicating impairment of the synthesis of this acid by hypophysectomy. However, the decrease in essential fatty acids was much slower than that of the prostaglandin E content. The vesicular glands of the hypophysectomized animals also lost their ability to synthesize prostaglandins as evidenced by their inability to convert arachidonic acid to prostaglandin E, as shown in Table III. These results (Table III) showed that the biosynthetic acitivity of the glands was decreased by approximately 70”; within I day and was completely absent within 7 days after hypophysectomy.

BIOSYNTHESIS TABLE

OF PROSTAGLANDINS

589

II

EFFECT OF ESSENTIAL FATTY ACID-DEFICIENCY AND HYPOPHYSECTOMY FATTY ACID COMPOSITION OF THE LIPID OF RAT VESICULAR GLANDS RATS* Fatty acid**

Hypophysectomized NOrmU

2

weeks

/ y, wt)

I month

6 months

Essential.fatty aciddeficiency I x, wt) 12

14:o

5.7

I 1.6

46.7 6.8

33.2 Il.2

1.7 18.9 14.2

4.8 20.8

3.4 21.9

3.5 20.5

4.7 56.8

3.2

24.0 _ _

14.6 _

6.9 10.9 -

_

8.8

Il.2

1.0

2.3

I.5 23.1

16: I 18:o 18:r 18:~

8.5 9.0 27.2 12.8

20:

I***

20 :

3(n-9)

_

_

20:4

2.6 I.1

* Pooled samples of 4 to 6 animals. ** Shorthand designation of fatty acids; number before colon = number of double bonds. *** Tentative identification on basis of retention time. TABLE

colon

=

chain

OF HYPOPHYSECTOMY

TO PROSTAGLANDIN

Days after hypophysectomy

No. of animals

ON THE Ez BY RAT

IN VITRO

CONVERSION

VESICULAR

number

after

Prostaglandin EZ in vitro biosynthesis

OF ARACHLDONIC

GLANDS*

(pglg tissue)

Conversion of arachidonic acid ( “/‘,yield)

4

49 i

20**

5

3

4’

15**

4

1

4

15***

3

13***

7

4 6

14 28

Normul Zero

length;

III

EFFECT ACID

months

33.6 5.6

16:0

ON THE OF ADULT

animals

Sham operated Zero

*

I.5 I.0

0

0

4

0

0

5

0

0

* I mg of arachidonic ** Mean 5 S.D. *** Analysis of pooled

acid used as the substrate

per g tissue.

samples.

The analysis of the fatty acid composition of the lipid of the vesicular glands of the animals fed the fat-free diet (Table II) showed the typical pattern of essential fatty acid deficiency generally observed in the liver. That is, depletion of linoleic and arachidonic acid with simultaneous increases in palmitoleic, oleic and 20:3 (n-9) acids. The fatty acid composition of the vesicular glands of the animals fed hydrogenated coconut oil was not determined, but the same pattern found in the fat-free group should be expected. Regardless, it was evident that the endogenous prostaglandin E content of the vesicular glands of the animals fed both diets (fat-free and hydrogenated coconut) decreased as they became older (Table IV). The vesicular glands of

W. C. TAN,

590

the animals of both groups also lost their ability to convert taglandin E, as shown in Table IV. TABLE

arachidonic

0. S. PRIVETT

acid to pros-

IV

EFFECT OF AN ESSENTIAL FATTY ACID DEFICIENCY ON VESICULAR GL.ANDS RATS. ENDOGENOUS CONTENT AND IN VITRO BIOSYNTHESIS OF PROSTAGLANDIN E FROM ARACHIDONIC ACID* ~~~~~_ __ ._ _. ~nd~~en~~lis IXet Wt of’ glmds ~rld#g~nuus Pr~st~~gl~~~d~~ CffnvefGm ictgfl i

per ~lt?ji?i~l

Pr~~st~~iundin

:g I

IT**

ijig)

/‘g/g tissue

pr

466

442

50

5

I

392

317

0

0

0.63

233

143

0

0

pr~~stagiandil~ E prr glands

nnirml

Ez**

in vitro

OF

[~S[i~ac~lid~~nir

mid to arachidonic prostagltrndin mid (pgjg tissue) E2 ( y:, j,ieldJ synthesis.frunl

Corn oil

(4 months) CoconLlt oil (3 months) Fat-free (I year)

0.95 0.8

* I mg of arachidonic acid used as substrate per g tissue. ** Average value of pooled samples of 4 to 6 animals.

DISCUSSION

The present study shows that hypophysectomy has a drastic effect on the vesicular glands of rats. The glands lose their ability to synthesize prostaglandins of the E-type, and the endogenous stores of these compounds are gradually depleted. The presence of traces of prostaglandins approximately I year after hypophysectomy in the absence of synthesis indicates that the turnover of these compounds is slow in the vesicular glands of hypophysectomized animals. Hypophysectomy also inhibited the interconversion of linoleic acid in the vesicular glands which became depleted of their stores of arachidonic acid. The initial increase in the percentage of linoleic and arachidonic acid may be explained, in part, on the effect of hypopllysectomy on the relative rates of catabolism of the individual fatty acids ” . However the decrease in endogenous prostaglandins in the vesicular glands of hypophysectbmized rats does not appear to be due to a lack of precursor acids because their loss is much faster than the decrease in arachidonic acid. Therefore, it appears that the decrease in endogenous prostaglandins in the hypophysectomized animals is due to the absence of the synthesis of these compounds as evidenced by the inability of the glands of these animals to convert arachidonic acid to prostaglandin E,. The turnover of prostaglandins was also slow in the vesicular glands of the essential fatty acid-deficient animals inasmuch as they contained an appreciable amount of these compounds long after depletion of essential fatty acids from the tissues. Moreover, the glands of these animals lost their ability to synthesize prostaglandin E, from arachidonic acid long before there was a large decrease in the endogenous prostaglandins. It may seem paradoxical that the biosynthesis of prostaglandins in the vesicular glands is impaired by an essential fatty acid deficiency. However, there are a number of

BIOSYNTHESIS

OF PROSTAGLANDINS

59’

similarities between the effects of hypophysectomy and an essential fatty acid deficiency. Ahluwalia et a1.r9 have shown that an essential fatty acid deficiency causes atrophy of the pituitary gland of rabbits. Hence, if the hypophysis is involved in the effects produced on prostaglandin synthesis by hypophysectomy, it also may be similarly involved in an essential fatty acid deficiency. It is pertinent in this connection that an essential fatty acid deficiency also causes atrophy of the testes2’. Another similarity between the effect of hypophysectomy and an essential fatty acid deficiency is in the development of dermal lesions on the tail and feet”” ‘. One of the most active sites of prostaglandin synthesis is in the skin3321. In another study soon to be reported (Tan, W. C., and Privett, 0. S., manuscript in preparation), we have demonstrated that the synthesis of prostaglandins is impaired in tail skin of both essential fatty acid deficient and hypophysectomized rats. Hence, because of the parallel of the effects of hypophysectomy and an essential fatty acid deficiency on prostaglandin synthesis, it appears that the biological activity of essential fatty acids may, in part, be related to the synthesis of prostaglandins through a function of the hypophysis. The mechanism of the reaction is not known. There is obviously an abundance of essential fatty acids in the vesicular glands of hypophysectomized animal at the time there is an impairment of its biosynthetic activity to convert arachidonic acid to prostaglandin E,. Although the effect could be due to the general atrophy of the glands, this possibility seems unlikely inasmuch as the glands lost most of their biosynthetic activity for the conversion of arachidonic acid to prostaglandin E, within 24 h. Thus, loss of biosynthetic activity might well be associated with the dissipation of the gonadotrophins. In the case of the essential fatty acid-deficient animal, the absence of biosynthetic activity is due to either the absence or dormancy of the synthetase system. Dormancy could be caused by impairment of the function of the pituitary gland based on the observation of the effect of an essential fatty acid deficiency on this gland in the rabbit thereby giving a condition resembling hypophysectomy. However, atrophy of the vesicular glands in the essential fatty acid-deficient animals was not nearly so great as in the hypophysectomized animals. Another possibility is that the synthetase system is absent (due to an effect of essential fatty acid deficiency on protein synanimal inasthesis22’23). A similar condition could exist in the hypophysectomized much as enzyme synthesis could be inhibited by an effect on RNA production as suggested by Goswami et al. 24 in the testes. Accordingly, all of the elements are present suggesting that prostaglandin synthesis is likely to be controlled by a feedback mechanism involving the hypophysis. ACKNOWLEDGMENTS

Supported in part by P.H.S. Research Grant No. AM-04942 from the National Institutes of Health, P.H.S. Research Grant No. HL-08214 from the Program Projects Branch Extramural Programs, National Heart and Lung Institute, and The Hormel Foundation. REFERENCES I Bergstrom, 4006-4008

S., Danielsson,

H.,

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D. and

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W. C. TAN, 0. S. PRIVETT

592

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(Marinetti,