Effects of prostaglandins on release of enzymes from lysosomes of pancreas, spleen and kidney cortex

JoAe

1nc8s V af~A&Part I, pp . 193-201, 1973

Pergamon Press

EFFECTS OF PROSTAGLANDINS ON RELEASE OF ENZYMES FROM LYSOSOMS OF PANCREAS, SPLEEN AND KIDNEY CORTEX Louis J. Ignarro, Arnold L. Oronsky and Robert J. Perper Research Department, Pharmaceuticals Division, CIBA-GEIGY'Corporation, Ardsley, New York 10502

(Received 12 October 1972; tn final form 20 December 1972) SUMMARY Prostaglandin (PG) E1, PGE 2 and PGF 2 . inhibit the release of enzymes from lysosomas of rat pancreas, spleen and kidney cortex in vitro. High concentrations of PGF I , accelerate the release of lysosomal enzymes. Theophylline, papaverine and adenosine 5'-triphosphate each potentiate the inhibition by PGE1 and PGFja , and reduce the acceleration by PGF , of lysosomal enzyme release . The dibutyryl analog of cyclic2g',5'-adenosine monophosphate inhibits, whereas cyclic 3',5'-guanosine monophosphate accelerates lysosomal enzyme release. The data suggest that inhibition of release of lysosomal enzymes by PGE1 and PGFS , might be mediated by cyclic 3',5'-adenosine monophosphate . A role for prostaglandins in the regulation of the inflammatory process has been suggested although considerable controversy exists as to whether they elicit anti-inflammatory or pro-inflammatory actions.

The recent studies of

Vane and his colleagues (1-3), on the inhibition of prostaglandin synthesis by certain anti-inflammatory drugs, suggest that Prostaglandins can mediate inflammation.

However, other studies have illustrated various anti-inflammatory

actions of prostaglandine . Prostaglandins

(PG) of the E series have been reported to be effective

in vivo , both prophylactically and therapeutically, in adjuvant polyarthritis (4,5) and in reducing edema formation after local administration of carrageenin (6) .

PGE1 inhibited in vitro the release of lyaosomal enzymes from human leuko-

cytes during phagocytosis of zymosan or immune complexes (7,8) .

PGE1 and PGE 2

were found to inhibit in vitro the allergic release of histamine from sensitised human leukocytes (9) .

All of these actions appear to be mediated by cyclic 3',

5'-adenosine monophosphate (cyclic AMP) .

Prostaglandins, via a mechanism in-

volving cyclic AMP, have been reported to modify several other inflammatory cell-mediated functions .

These include the inhibition of intracellular digestion

of phagocytosed bacteria (10), inhibition of lymphocyte-target cell cytolytic activity (11), and regulation of fibroblast proliferation (12) .

193

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Effect of Prostaglandins on Lysosomal Enzymes

Vol. 12, No. 5

Cyclic AMP and agents that elevate its levels (catecholamines, histamine, serotonin, theophylline, papaverine and adenosine 5'-triphosphate)

have been

shown to inhibit in vitro the release of enzymes from liver lysosomes (13-15) . An in vivo correlate to these findings was reported recently by Glenn (16,17) who showed that PGE 1 and PGF3Cj protect cats and dogs against rising plasma levels of a myocardial depressant factor and consequent circulatory shock, probably by inhibiting the release of lysosomel enzymes from the pancreas .

Both prostaglan-

dins were also shown to inhibit release of enzymes in vitro from liver and pancreas lysosomes (17) . In view of the reported anti-inflammatory effects of prostaglandins and their inhibitory actions on lyaosomal enzyme release in the dog and cat, we studied the effects in vitro of PGE1, PGE 2 and PGF3U on lysosomel enzyme release from pancreas, spleen and kidney cortex of the rat.

Particular attention was

focused on the apparent biphasic effect of PGFaY on lysosomel enzyme release, since high . concentrations of PGF 3 . have been reported to accelerate release of enzymes from rat liver lysosomes (18) . MATERIALS AND METHODS Rate (300 g, male Sprague Dawley, Carworth Farms) were fasted overnight (18 hr) and then decapitated and exsanguinated.

The pancreas was excised,

rinsed in cold 0.25 M sucrose-0.02 M Tris acetate, pH 7 .4 (buffer A), blotted dry, minced, weighed and homogenized (10% w/v in 15 ml of buffer A) Elvehjem-ground glass tissue grinder (600 rpm for 1 min at 0°C) .

in a Potter

The homogenate

was centrifuged at 600 g for 10 min and the supernatant was centrifuged at 1,200 g for 15 min.

The resultant supernatant was centrifuged at 16,500 g for 30 min

to sediment the lysosome fraction, and the sediment was resuspended gently in 2 .5 ml of 0.45 M sucrose-0.049

glycogen-0.02 M Trio acetate, pH 7 .4 (buffer B)

following 3 separate washes each with 5 ml of buffer B.

The spleen was excised,

the capsule discarded and the tissue rinsed, minced, weighed (as with pancreas) and homogenized (10% w/v in 20 ml of buffer A containing 0.01 M KC1) tissue grinder with pestle A (10 strokes manually) .

in a Dounce

The homogenate was centri-

fuged immediately after preparation as described for pancreas .

Each lysosome

sediment was resuspended in 3 ml of buffer B (containing 0 .01 M KC1) . kidneys were excised and the cortex was separated from the medulla .

The two The cortex

was minced (as with pancreas) and homogenized (10% w/v in 20 ml of buffer A) in a Potter Elvehjem-ground glass tissue grinder (600 rpm at 0°C ; operated for 30 seconds at a time, at 30 second intervals, genization) .

for a total of 180 seconds of homo-

The homogenate was centrifuged as described for pancreas . Glycogen

(and KC1 for spleen) was included in the suspending medium to prevent agglutina-

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Effect of Prostaglandins on Lysosomal Enzymes

19 5

tion of organelles .

All lysosome fraction@ contained mitochondria (cytochrome

oxidase) as well . In each experiment 0.2 ml aliquots of lysosome fractions were added to 2 ml of 0 .18 M sucrose-0.04 M Trio acetate, pH 7 .4 (buffer C) . then treated several ways .

The samples were

Some samples were centrifuged (27,000 g for 15 min)

immediately, without incubation .

These samples are termed 0 min controls and

reflect the amount of extralysosomal enzyme present in the lysosome suspension prior to incubation.

Other samples were incubated, with or without compound,

at 37 0C (while agitating at 150 cycles per min) for 30 min and then centrifuged. Incubations without compound represent 30 min controls and reflect normal enzyme release during incubation .

In order to obtain an estimate of total enzyme ac-

tivity, samples were incubated in 0.1% Triton R-100-0.04 M Trio acetate, after 10 min of vigorous agitation,

and then centrifuged.

were assayed for lysosomal marker enzymes (19) .

pH 7.4

Supernatants (1 .0 ml)

ß-glucuronidase was measured

for pancreas and kidney cortex, whereas aryl sulfatase was measured for spleen since the latter enzyme is a more accurate marker of the primary lysosome population in spleen (20) . Solutions (10-3 M) of prostaglandins in absolute ethanol were prepared and small aliquots (10 pl or less) were used in the assays . remained in solution after addition to aqueous buffer . of ethanol did not affect lysosomal enzyme release. prepared as solutions in buffer C.

Prostaglandins

The small concentration

All other compounds were

None of the compounds inhibited directly the

activity of released lysosomal enzymes . RESULTS AND DISCUSSION Table 1 illustrates the effects of PGE 1 , PGE2 and PGF2a on the release of ß-glucuronidase from pancreas lysosomes. at high concentrations .

PGE 1 and PGE 2 inhibited enzyme release

PGF2a enhanced enzyme release at high concentrations

and inhibited release at lower concentrations .

Since prostaglandins have been

reported to stimulate adenyl cyclase and thereby elevate tissue levels of cyclic AMP (10-12, 21,22) experiments were designed to elucidate whether the actions of prostaglandins on lysosomes can be attributed to cyclic AMP.

In recent studies

phosphodieaterase inhibitors (13) and adenosine 5'-triphosphate (ATP) (14), a substrate for adenyl cyclase, were each shown to enhance the inhibitory actions of catecholamines on release of enzymes from liver lysosomes,

thus suggesting

that the effect of catecholamines on lysosomes might be mediated by cyclic AMP. A similar approach was used in the present study.

Theophylline and papaverine

enhanced the action of PGE1 , but not PM=, on pancreas lysosomes (Table 1) .

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Effect of Prostaglandins on Lysosomal Enzymes

Vol. 12, No. 5

Table 1 Effect of Prostaglandins on Release of ß-Glucuronidase from Pancreas Lysosomes

% Inhibition of Release of ß-glucuronidase Prostaglandin (PG)

Molar . Concentration of Prostaglandin

PGE1 PGE 1 + Theophylline (10- SM)

10 -5

10 -6

10 -7

65

39

16

6

10-8

10 -9 0

88

61

39

21

9

PGE1 + Papaverine (10-4M)

100

74

48

30

17

PGE 1 + ATP (10-5 M)

100

76

53

31

17

PGE=

48

22

8

0

0

PGE 2 + Theophylline (10- "M)

44

25

11

0

0

51

28

7

0

0

48

21

12

0

0

(45)

(11)

42

19

8

10

31

61

37

22

8

38

70

41

20

22

68

31

18

PGE= + Papaverine (10-410 PM= + ATP (10-5M) PGF32

PGF ry + Theophylline (10-5 M) PGFsa + Papaverine (10-4M) PGF Ry + ATP (10-6 )

(15)

Data represent average values from 3 to 5 separate experiments= individual values varied by less than 15% from corresponding means. ATP signifies adenosine 5'-triphosphate . Numbers in parentheses signify % increase in enzyme release. Theophylline, papaverine or ATP alone did not inhibit enzyme release. Control values (mean f S.E .M.) for ß-glucuronidase release, expressed as extinction values (540 rim), were 0.23 t 0.02 (0 min controls), 0.88 f 0.07 (30 min controls) and 1.62 f 0.14 (total activity) . The two phosphodiesterase inhibitors reversed the lysosome labilizing action and enhanced the lysosome stabilizing action of PGFja.

ATP elicited effects that

were similar to those of theophylline and papaverine .

PGF=, with or without

added agents, consistently produced maximal inhibitory effects at 10-7 M. The prostaglandins also inhibited release of aryl sulfatase from spleen lysosomes (Table 2) .

As with pancreas lysosomes,

PGF 3a accelerated enzyme re-

lease at high concentrations and retarded release at lower concentrations .

In

addition, theophylline and ATP enhanced the effect of PGE1 but not that of PGE 2 . Theophylline and ATP also reduced the lysosome labilizing action and enhanced the stabilizing action of PGFsa" Table 3 illustrates the effects of prostaglandins on the release of ßglucuronidase from kidney cortex lysosomes.

Similar effects to those of pancreas

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Effect of Prostaglandins on Lysosomal Enzymes

197

Table 2 Effect of Prostaglandins on Release of Aryl Sulfatase from Spleen Lysosomes

Inhibition of Release of Aryl Sulfatase Prostaglandin (PG)

Molar Concentration of Prostaglandin 10-3

10 -6

10-7

PGE,

54

31

12

PGE 1 + Theophylline (10-3M)

78

50

PGE I + ATP (10-3M)

88

52

PGE _

56

PGE 2 + Theophylline (10-3 M) PGE 2 + ATP (10 -3M)

10 -8

10 -9

0

o

38

22

10

34

20

8

23

0

0

0

60

29

0

0

0

52

29

0

0

0

PGF la

(66)

(21)

38

9

0

PGF 2a + Theophylline (10-3 M)

(12)

24

52

36

16

PGF Sa f ATP (10-3 M)

(26)

14

54

32

14

Data represent average values from 3 separate experiments: individual values varied by 15% or less from corresponding means . ATP signifies adenosine 5'triphosphate . Numbers in parentheses signify ~ increase in enzyme release. Neither theophylline nor ATP alone inhibited enzyme release. Control values (mean t S.E .M.) for aryl sulfatase release expressed as extinction values (510 nm), were 0.25 t 0.01 (0 min control), 0.63 t 0.04 (30 min control) and 0.88 f 0.07 (total activity) . and spleen were obtained .

In each of the experiments with all three tissues,

theophylline, papaverine and ATP *alone, at the concentrations used, did not alter the release of lysosomal marker enzyme . Enhancement of the inhibitory actions of PGE 1 and PGF . on lysosomal enzyme release by phosphodiesterase inhibitors and ATP suggested that cyclic AMP might mediate the effect of these two prostaglandins .

In order to test this

concept, the direct effect of cyclic AMP was determined.

Dibutyryl (N 6 ,0 :'-

dibutyryl) cyclic AMP was found to inhibit release of enzymes from pancreas, spleen and kidney cortex lysosomea, whereas adenosine 5'-monophosphate was inactive (Table 4) .

The finding that high concentrations of PGF2a accelerated the

release of lysosomal enzymes is more difficult to explain in relation to the effect of dibutyryl cyclic AMP.

Preliminary data from our laboratory indicate

that acetylcholine also accelerates release of enzymes from liver lysosomesi this is an opposite effect to that evoked by catecholamines and cyclic AMP (13) . A recent report indicated that acetylcholine, which stimulates guanyl cyclase.

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Effect of Prostaglandins on Lysosomal Enzymes

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Table 3 Effect of Prostaglandins on Release of ß-Glucuronidase from Kidney Cortex Lysosomes

Inhibition of Release of ß-Glucuronidase Prostaglandin (PG)

Molar Concentration of Prostaglandin 10 -5

10-6

PGE 1

39

21

5

0

0

PGE 1 + Theophylline (10-3M)

78

53

39

18

6

PGE =

32

18

0

0

0

PGE 2 + Theophylline (10-3M)

35

16

5

0

0

(8)

31

11

0

38

61

28

12

PGF 2a

PGF2a + Theophylline (10-3 M)

(36) 12

10 -1

10-8

10 -9

Data represent average values from 3 to 4 separate experiments; individual values varied by less than 12% from corresponding means. Numbers in parenthéses signify % increase in enzyme release . Theophylline alone did not inhibit enzyme release. Control values (mean t S.E .M.) for ß-glucuronidase release, expressed as extinction values (540 nm), were 0.096 t 0.007 (0 min control), 0.38 d:0 .02 (30 min control) and 1.22 f 0 .10 (total activity) . elevates tissue levels of cyclic 3',5'-guanosine monophosphate (cyclic GMP), and that cyclic GMP mediates certain physiologic actions of acetylcholine that are opposite to those of epinephrine (23) .

In view of the biphasic action of

PGFzx on lysosomes, cyclic GMP was tested for its capacity to enhance lysosomel enzyme release.

Cyclic GMP was found to accelerate release of enzymes from

pancreas, spleen and kidney cortex lysosomes (Table 4) .

Maximal effect was ob-

tained at 10 -5M and this action appears to be specific for the cyclic nucleotide since guanosine 5'-monophosphate (GMP) failed to enhance enzyme release.

High

concentrations of cyclic GMP inhibited enzyme release ; GNP was less active .

In

this regard, it is of interest that Beavo, Hardman and Sutherland (24) reported recently that low concentrations of cyclic GMP stimulated the rate of hydrolysis of cyclic AMP by phosphodiesterase, whereas high concentrations of cycllc .GMP inhibited cyclic AMP hydrolysis .

It is not known whether PGFXX, at high con-

centrations, can stimulate guanyl cyclase and thereby elevate tissue levels of cyclic GMP.

Therefore, the similarity in action of PGFja and cyclic GMP could

be fortuitous .

However, analogous effects of cyclic AMP and cyclic GMP on the

release of histamine from human lung tissue in vitro have been reported recently

Vol. 12, No. 5

Effect of Prostaglandins on Lysosomal Enzymes

199

Table 4 Effect of Nucleotides on Release of Enzymes from Lyaosomes of Pancreas, Spleen and Kidney Cortex

Inhibition (Increase) in Release of Lysosomal Marker Enzyme Nucleotide

Dibutyryl cyclic AMP

AMP

Cyclic GMP

(MP

Organ

Molar Concentration of Nucleotide 10 -3

10-4

10 -5

10 -6

Pancreas

70

42

24

10

Spleen

62

34

16

0

Kidney

53

31

15

0

Pancreas

0

0

0

0

Spleen

0

0

0

0

Kidney

0

0

0

0

Pancreas

36

(10)

(52)

Spleen

40

(8)

(60)

(23)

Kidney

22

(15)

(41)

(19)

Pancreas

24

8

0

0

Spleen

28

6

0

0

Kidney

15

0

0

0

(27)

Data represent average values from 2 to 4 separate experiments ; individual values varied by less than 15% from corresponding means. Abbreviations : dibutyryl cyclic AMP, N6 ,0 2 '-dibutyryl cyclic 3',5'-adenosine monophosphate ; AMP, adenosine 5'-monophosphate . cyclic GMP, cyclic 3',5'-guanosine monophosphate; GMP, guanosine 5'-monophosphate . Release of ß-glucuronidase was measured for pancreas and kidney cortex ; aryl sulfatase was measured for spleen . Refer to legends of Tables 1, 2 and 3 for actual extinction values for control and total enzyme release. (25) .

The inhibitory effect of phosphodiesterase inhibitors and ATP on PGF=-

induced enzyme release is difficult to explain unless an elevation in the level of cyclic AMP canovercome the effect of cyclic GMP.

Adequate explanation of

these findings await analysis of the actions of prostaglandins on levels of cyclic nucleotides in lysosome fractions; such studies are now in progress . The data in this report support the earlier finding with liver tissue that high concentrations of PGFIU accelerate lysosomal enzyme release (18) . Further, our data are in agreement with earlier findings that PGE 1 and PGFja

200

Effect of Prostaglandins on Lysosomal Enzymes

Vol. 12, No. 5

inhibit lysosómal enzyme release in vitro and in vivo (16,17) .

Thus, PGF2a

appears to elicit a biphasic action on the release of lysosomal enzymes in vitro. These data support the view that prostaglandins elicit anti-inflammatory actions . Inhibition of lysosomal enzyme release, perhaps via a mechanism involving cyclic AMP, serves as a possible explanation of the anti-inflammatory actions of PGE 1 and PGF 2U . REFERENCES 1.

J.R . VANE, Nature New Biol ., 231, 232-235 (1971) .

2.

J.B . SMITH and A.L. WILLIS, Nature New Biol .,

3.

S.H . FERREIRA, S. MONCADA and J.R . VANE, Nature New Biol ., 231, 237-239

231,

235-237 (1971) .

(1971) . 4.

R.L . ASPINALL and P.S . CAMMARATA, Nature , 224,

5.

R.B . ZURIER and F. QUAGLIATA, Nature , 2 34, 304-305 (1971) .

6.

E .M. GLENN and N. ROHLOFF, Proc . Soc. Exp. Biol . Mod .,

7.

G. WEISSMANN, P. DUKOR and R.B. ZURIER, Nature New Biol ., 231, 131-135

1320-1321 (1969) . 139, 290-294 (1972) .

(1971) . 8.

G. WEISSMANN, R.B. ZURIER, P.J. SPIELER and I.M . GOLDSTEIN, J. Exp. Med., 234,

9.

149s-165s (1971) .

H.R . BOURNE, L.M. LICHTENSTEIN and K.L. MELMON, J. Immuaol., 108, 695-705 (1972) .

10 .

H.R . BOURNE, R.I . LEHRER, M.J . CLINE and K.L . MELMON, J. Clin. Invest ., 50, 920-929 (1971) .

11 .

C.S . HENNEY, R.R . BOURNE and L.M . LICHTENSTEIN, J.

Ismunol.,

108,

1526-

12 .

C.V . PEERY, G.S . JOHNSON and I. PASTAN, J. Biol . Chez+. , 246, 5785-5790

13 .

L.J . IGNARRO, J. SLYWKA and N. KRASSIKOFF, Life Sci., 10 10, Part I,

14 .

L.J . IGNARRO, N. KRASSIKOFF and J. SLYWKA, Life Sci.,

1534 (1972) . (1971) . 1315

1309-

(1971) .

(1972) .

11, Part I, 317-322

31,

15 .

L.J. IGNARRO, N. KRASSIKOFF and J. SLYWKA, Fed. Proc .,

16 .

T.M. GLENN, Fed. Proc ., 31, 545 (1972) .

17 .

T.M. GLENN, Fifth Int. Conar. Pharmscol., San Francisco, Calif., p. 83

281 (1972) .

(1972) . 18 .

R. WEINER and G. KALEY, Nature Now Biol ., 236, 46-47 (1972) .

19 .

L.J .

20 .

W.E. BOWERS and C. DE DUVE, J. Cell .

21 .

IGNARRO,

Biochem. Pharmscol., _20, 2847-2860 (1971) . Biol ., __32, 339-348 (1967) .

H R. BOURNE and K. L. DELMON, J. Pharmacol. Exp. Therap ., 178, 1-7 (1971) .

Vol . 12, No. 5

Effect of Prostaglandins on Lysosomal Enzymes

22 .

P.W. RAMWELL and J.E . SHAW, Ann. N.Y . Acad . Sci.,

23 .

W.J . GEORGE, J.B . POISON, A.G . O'TOOLE and N.D . GOLDBERG, Proc . Nat. Acad .

24.

201

180 (1971) .

Sci., 66, 398-403 (1970) .

J.A . BEAVO, J.G . HARDMAN and E.W. SUTHERLAND, J. Biol . Chem ., 246, 38413846 (1971) .

25 .

M. KALINER, R.P . ORANGE and R.F . AUSTEN, J. Ego. Med.,

136,

556-567 (1972) .