Inhibition of lysosomal enzyme release by catecholamines: Possible mediation by cyclic 3′, 5′-adenosine monophosphate

Life Sciences Vol . 10, Part I, pp . 1309-1315, 1971 . Printed in Great Britain

Pergamon Press

INHIBTTION OF LYS030MAL ENZi~ RELBASE BY CATECNOLAèIINE3 : POSSIBLE MEDIATION BY CYCLIC 3',5'-AD$NOSII~ MONOPHOSPHATE Louie J. Ignarro, Joeeph Slyorlca and Natalie Rrassikoff Department of Biochemistry, Geigt' Pharmaceuticals, Mvieion of CIBA-(~IGY Corporation, Ardaley, Now York 10502 (Received 26 July 1971 ; in final form 25 October 1971) Summsr~ The catacholsmines noropinephrine and epinephrine inhibit the release of enaymes from lyswomes obtained from a rat liver mitochondrial preparation. Cyclic 3',5'-adenosine monophosphate (cyclic AMP) elicits a similar action . Inhibitors of phosphodiasteraea, such as theophylline sad papaverine prevent e~yme release themselves and eahaaca the actions of added catecholeminns and cyclic AMP . Propranolol, which prevents the activation of adenyl cyclaea by catecholasd.nes, reduces the effects of the latter but not the effect of cyclic AMP. Thus, it appears that inhibition of release of lysosamal easymes by norepinaphrine and epinephrine may be mediated by cyclic AMP . Ezparimental evidence implicating catncholaminns and adrennrgic mechaaieme in the regulation or control of inflammatory reactions has accumulated during the last fear years (1-12) . Spactor and Willoughby (3) suggested that epineph rine might be a aeturally occurring anti-inflammatory hormone.

Hhalla et al .

(7) ahoaoad that epinephrine aahibits anti-inflammatory activity in carrageenin edema in non-adrenalectomisad rats and that inhilltors of monoamina osidase and catechol-0-methyl transfsrase similarly protect against inflammation .

Novever,

their studies are complicated by the fiadinge that tissue depletion of catecholsminee also inhibits carrageenin edema. Epinephrine alone or in combina-

tion with propiomasine was shorn to alleviate inflammation associated with adjuvant polyarthritis (S), to prevent ezperimentsl allergic eacephalomynlitis (9) and to inhibit circulating antibody production (10) . Ia recent studiee, Riestnrnr and Jaquee (11-12) shored that the beneficial effects of various steroidal and non-ateroidal anti-inflammatory drugs in turpentine-induced pleurisy sad traumatic pap edema ware antagonised by ß-adrenergic blocking agents such ae propraaolol.

Several recant observations support the hypothesis that cyclic 3',5'adanwiae monophosphate (cyclic AMP) mediates as a "second messenger" certain affects of catechoLaminas in leucocytes, platelets and other cells that par ticipate in the inflammatory process . Lichtanatein and Margolis (13) shored that histandna release from leucocytes in vitro could be inhibited by 1309

131 0

Release ad Lysosomal Enzymes

catecholamiaes and methylaanthiaes .

Vol. 10, No. 22

May et al . (14) and Weisematm et al . (15)

demonstrated the capacities of cyclic AMP and theophyllina to inhibit the eztrusion of lysoeamal enzymes from phagocytosiag leucocytes .

Platelet aggre-

gation, a contributing factor in inflammation, ie inhibited by cyclic AMP (16), isoprotareaol (17), prostaglandin E1

(18), and theophylliae (17) .

These

studies reveal that certain actions of catecholamines are moat likely mediated by cyclic AMP. The role of lyeoeamal enzymes is inflammation ie well established (19-21) . Considerable azperimantal evidence supports lysosame membrane stabilization as a mechanism of action of clinically effective anti-rheumatic drugs (22-26) . In wisp of the reported anti-iaflamoatory actions of catecholamines and the possible mediation of such actions by cyclic AMP, we studied the effects of catecholami.aee and cyclic AMP on lysosomes in vitro.

The data reveal the

capacities of norepiaephrine aad . apinephrine to inhibit release of enzymes from lyeosomes and suggest that such effects msy be mediated by cyclic AMP. Materials sad Methods Rat liver mitochondrial fractions rich in lysosomes were prepared as described previously (26) .

Complete details of all ezperimeatal procedures,

including measurement of lysosame membrane stabilization, assay of marker easymas sad preparation of drug solutions, have also bees described (26) . Briefly, aliquots of liver lyeoeame suspensions in sucrose-Trio acetate buffer 0 (pH 7.4) containing one or more teat campounde were incubated at 37 C for 15 min and then centrifuged (27,000 z g) .

Supernatant fractions ware assayed for

release of three soluble lysosamal marker enzymes : eulfatase and ß-glucusonidase .

acid phoephatase, aryl

These fractions, containing soluble lyeoeomal

enzymes and test campouad, pare diluted up to 20-fold prior to enzyme assay. Therefore, concentrations of test compound in the final enzyme assay solution warn lees thaw 10~M.

None of the compotmde tasted inhibited the marker

enzymes at concentrations employed is this study.

At concentrations greater

thaw 10~ M (i>i final enzyme assay), norepinephriae and epinephrine inhibited acid phosphatase only when ß-glycerophoephate was employed ae substrate. Results and Mecussion Table 1 illustrates the capacities of aorepiaaphriae and epinephrine to inhibit the release of three different marker enzymes from lysoeamee in vitro. The catacholamiane inhibited each of the marker enzymes to a slightly different eztant .

Such data may reflect the different degree to which individual enzymes

are bound to the lysosomal membrane (27) .

Although not indicated in the Table,

isoproterenol yielded similar results while phenylephrine and tyramine, phich elicit sympathomimetic effects indirectly through release of endogenous

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Release ad Lysosomal Enzymes

Vol . 10, No. 22

norepinephrine, were inactive (the racemic dl-isomers of norepinephrine and epinephrine were lees active than the 1-isomers) .

Somewhat higher concentra-

tions of catecholamines than expected were required to elicit significant in vitro effects .

Similar problems were encountered is the in vitro studies of

Lichtenstein and Margolie (13) . TABLE 1 Effect of Catecholamines on Release of Enzymes from Lysosomes

X

Inhibition of Release of Marker Enzyme Molar Concentration of Çateçholamine_

Test System

10~

10~

100

52

32

17

77

50

30

28

65

50

21

10

0

Acid Phoephatase

100

100

61

36

23

Aryl Sulfataee

100

62

45

32

2

73

57

38

20

1

10~

5 x 10-°

Acid Phosphataen

100

Aryl Sulfataee

100

ß-Glucuronidase

10-e

NOREPINEPHRINE

EPINEPHRINE

ß-Glucuronidase

Aliquots of liver lysoeome suspension is 0.18M sucrose-0 .05M Trie acetate, pH 7 :4, containing various concentra ions of norepinephrine or epinephrine were incubated at 3~C for 15 min Super and then centrifuged at 27,000 x g for 15 min at 4° C. Subnatant fractions were assayed for marker enzyme activity . strates employed for acid phosphatase, aryl sulfatase and ßglucuronidaee were, respectively, p-nitrophenyl phosphate, p-nitrocatechol sulfate and phenolphthalein glucuronide. The 1-isomers of bitartrate salts of catechoLsminee were used . Data represent average values from 3 to 6 separate eaperimeate ; values varied by lees than 10~ from corresponding means . Control values for enzyme release, expressed as eatinctiona, were : 0 .65-0 .80, acid phosphatase, 405 m4+ ; 0 .22-0.30, aryl sulfatase, 510 ni+ ; 0.50-0.62, ß-glucuronidase, 540 ~. Since adrenergic neurotransmitters are believed to stimulate adenyl cyclase via ß-receptors and to release cyclic AMP (28-30), experiments ware designed to elucidate whether the inhibition of lysosomal enzyme release by catecholamines can be attributed to cyclic AMP .

It was reasoned that inhibitare

of phoephodiesterase, an enzyme which inactivates cyclic A1~, should inhibit lysoeamal enzyme release alone and should also enhance the actions of added cyclic AMP sad catecholaminea .

Such effects would be açcounted for by an

elevation in the concentration of cyclic AMP in the teat system . papaverina

Theophylline,

(31) and zinc ion (32) are well-kaoava inhibitors of phosphodinsterase

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Release oaf Lysosomal Enzymes

Vol. 10, No. 22

TABLE 2 Effect of Cyclic AMP and Other Agents on Release of Enzymes from Lysoeomae ~ Inhibition of Release of Marker Enzyme Agent(e) Tested

Marker Enzyme

Molar Çoncentration of Test Agent 2 .5a10~

10~

5x10

10~

10~

Theophylline

BG

44

5

0

0

0

Papavnrine

BG

98

64

60

20

0

ZaCls

BG

100

100

100

52

0

cAMP

AS

100

40

13

0

0

cAMP + Theophylline (10~ M)

AS

93

58

48

38

34

oAMP

AP

100

63

35

0

0

cAMP + Papaverine (l0~ M)

AP

100

84

60

46

23

cAMP

BG

77

43

20

9

0

cAMP + ZnCls (10~ M)

BG

86

67

40

26

20

Refer to Table l'lngend for azperimantal procedures and estimations of extinction values . Abbreviations : cAMP, Cyclic 3',5'-adenosiae monophosphate ; BG, ß-glucuronidaee ; AS, aryl sulfatase ; AP, acid phosphatase . Data represent average values from 2 to 3 separate experiments ; values varied by less than 10~ from corresponding means . Two or three different marker enzymes were measured in each of these experiments and the results obtained were very similar to those reported in this Table . In the experiments with added CAMP, the inhibition of enzyme release by theophylüne, papaverine or ZnCls alone never exceeded 10~. althontgh zinc ion also inhibits adenyl cyclase.

As illustrated in Table 2

these agents inhibited the release of enzymes from lysosomee .

At concentrations

greater than 2 .5 x 10~ M, ZnCls did not stabilize but rather labilized lyeoeomes (uapubliahed observations) .

Cyclic AMP exhibited a similar effect and this was

enhanced by the concomitant addition of phoaphodiesterase inhibitors .

Pre-

liminary data indicate that ATP also stabilizes lysosomes, possibly by com~ersion to cyclic AMP since phoaphodiesterase inhibitors potentiate the actions of ATP (AMP and ADP were much less active) .

The synergistic effects of combinations of

epinephrine or norepinephrine with phoaphodiesterase inhibitors are presented in Table 3 .

Theophylline, papaverine and ZnCls , at concentrations which elicit

very small or negligible effects by themselves, poteatiated the actions of the catecholamiaes . Propraaolol, a drug which antagonizes ß-adrenergic receptors (33) and inhibits the stimulation of adenyl cyclase by catecholaminea

(34-35), reduced

the action of norepinephrine but not that of cyclic AMP (Table 3) .

Practolol,

a sew antagonist of ß-receptors (36), reduced the effect of epinephrine but not that of cyclic AMP.

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Release ad Lysosomal Enzymes

1313

It is important to recognise that, u in previous studies, a crude liver lysosama fraction, contaminated With mitochondria, Was employed is the present experiments .

Measurements of ATP, adeayl cyclise and phosphodiasterase in

lysosamal membraane have not bean reported ; such studies are is progress in our laboratory .

It ie possible, and perhaps likely, that the biochemical machinery

for generation of cyclic AMP resides in extra-lysosamal fractions sad that the cyclic AMP generated then exerts its effects on the lysoscmal membrane . TABLE 3 Effects of Various Agents on Catecholamine- and Cyclic AMP-Induced Inhibition of Lysosomal Enzyme Release 76 Inhibition of Release of Marker Enzyme Agents) Tested

~ r~

Molar Concentration of E, KE or cAMP 10~

5x101

a

10

10~

10~

E

AS

100

75

43

33

0

E + Theophyllina (10~ M)

AS

100

%

67

60

40

E + Papaverine (10~ M)

AS

100

90

75

68

51

E

BG

66

47

34

27

0

E + Practolol (5a10-° M)

BG

61

6

0

0

0

NE

BG

66

44

18

6

0

BG

82

66

48

40

27

NE + Theophylline 2~ + Papaverine

(10~ M)

BG

81

72

48

40

15

NE + Propraaglol (5a10i M)

BG

34

13

0

0

0

cAMP

AP

100

75

17

0

0

oAMP + Propranolol (5xl0 M)

AP

100

79

13

0

0

cAMP + Practolol (5xl0 M)

AP

83

72

16

0

0

(10~ M)

Refer to Table 1 legend for experimental procedures and estimations of eatinction .valuas . The substrate used for AP Was ß-glycerophosphate . Abbreviations : E, 1-epinephrine bitartrate ; NE, 1-norepiaephrine bitartrata ; cAl~, cyclic 3',5'-adenosine monophosphate ; A3, aryl sulfatase ; BG, ß-glucuronidaee ; AP, acid phoephatue . Data represent average values from 2 to 5 separate experiments ; values varied by no more than lOx from corresponding means . TWO or three different markers Ware measured in each experiment and the results were very simiPropranolol or practolol alone did not inhibit lar to those presented here . the release of marker enzymes . Ia summary,

the data presented in this report reveal that the catechol-

amines norepinephrine and epinephrine inhibit the release of enzymes from lysosomas in

tro and that such actions may be mediated by endogenous cyclic AMP.

Thus, the anti-inflammatory actions reported for catncholaminee might be due partially to their capacity to inhibit release of lysosamal enzymes .

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Release

oaf

Lysosomal Enzymes

Vol, 10, No. 22 .

Ackaowledgments The authors soould lilts to thaak Dr . William D . Cash and Dr . Barbara Petrack for their valuable advice and criticism throughout these studies . References 1.

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Release ad Lysosomal Enzymes

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