Papaverine - induced inhibition of phosphodiesterase activity in various mammalian tissues

Life Sciences Vol . 10, Part I, pp. 135-144, 1871 . Printed in Great Britain

Pergamon Press

PAPAVERINE -INDUCED INHIBITION OF PHOSPHODIESTERÀSE ACTIVITY IN VARIOUS MAMMALIAN TISSUES G.

PI;Sch and W.

R.

Kukovetz

Deportment of Pharmacology, University of Graz, A-8010 Graz, Austria

(Received 11 August 1970; in final form 9 December 1970) RECENT experiments revealed that the dose-dependent relaxation of Isolated circular strips of beef coronary arteries, produced by papaverine, waacleerly proceeded by a pronôunced end similarly dose-dependent Inhibitory effect on cyclic 3~,5~-rwcleotide phosphodtesterase (PDE) to this tissue which

led

us to suggest that papaverine relaxes smooth muscle by caustnganaccumulation of cyclic 3~,5~-AMP (1,2,3,4) .

Inhibition of PDE-activity by papaverine and

a variety of other smooth muscle relaxants was also found to vitro , using a purified PDE-preparation from beef heart, but was particularly pronounced In the 2000 x g supernete obtained from homogenized beef coronary arteries

Since we have found so far that papaverine was the most potent Inhibitor of PDE among numerous smooth muscle relaxants, including theophylline (1, 2, 3, 4, 5), this classical antispasmodic compound with a commonly defined ~~dtrect~~ mode of action was further studied in vitro, to determine whether similar

ef-

fects could also be obtained on the PDE-activity from guinea pig ileum which is known to be relaxed by papaverine . The experiments were extended

to

crude PDE-preparations of various other tissues of rat, including brain , epidldymal fat and 11ver In order to clarify whether the inhibitory effect of pepaverins was restricted to those organs In which It exerts its well known pharmacodynamic effects, or whether PDE-inhibition was a general property of papaverine . METHODS Preparation of PDE-extracts .

133

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Inhibition ad Phosphodiesterase

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Ileum : Five adult guinea pigs (400 - 450 g) of either sex, fed on hay and turnips, were stunned and exsanguinated . From each animal, about 4 cm of the distal ileum were removed, rinsed with cold saline, blotted on filter paper and weighed . The pieces were pooled and homogenized In a precooled mortar with washed sand and the 10-fold volume of Tris-HCI (16 x 10 2 M), containing Mg-acetate (5 x 10 3 M), pH - 7. 5. The homogenate was centrifuged at 4o C at 2000 x g for 10 min and the supernate was further diluted 20-fold

with the same medium . Aliquots of 300 ~tl, representing the supernate of 1 .5 mg tissue (wet weight) were used for the assay of PDE-activity.

Brain: Four adult female Wistar rats (300 - 350 g), fed on conventional laboratory chow and water ad tib . wero exsangulnated following light ether anesthesia . The skull was removed and the whole brain excised. Brains were pooled and processed as described above, except that before and after can-~ trtfugation the supernate was filtered through glass-wool to remove the fat. Final dilution was the same es with ileum .

Eptdidymal fat ; Two groups of 3 male fed Wistar rats each (300 - 350 g) were stunned and exsanguinated . The whole epididymal fat pads from both sides were removed, freed from connective-, vascular-, and other tissue,

diluted 5-fold with the medium described, homogenized and filtered through glass-wool. No further dilution was made because of the low PDE-activity found In preliminary experiments In this tissue. Hence, 300 ~tl-aliquots of supernate, as used for the PDE-assay represented 60 mg of epididymal fat tissue (wet weight) . Liver; Three adult fed female rats (300 - 350 g) were stunned and exsangulnated. Pieces of about 1 g of liver were excised from each animal, rinsed in saline, blotted on filter paper, weighed, pooled and processed as described for ileum, except that the final dilution was 100-fold (300 ~ I of the final supernate ~ 3 mg tissue) . Determination of PDE-activity . Some variations of the previously described method were made (4) to make it

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Inhibition of Phosphodiesterase

135

applicable for the present experiments especially with respect to the low PDEactivtty, found in adipose tissue. A detailed study of this assay will be published elsewhere (5) . The principle of the determination of PDE-activity used in the present as well as in previous studies (1, 2,

3, 4, 5)

is based on the

decrease in radioactive substrate during incubation with the enzyme . This decroese can be measured since the product of the reaction, labeled 5~-AMP, in contrast to the substrate can be removed from the reaction mixture by Zn50~ Ba(OH) 2-precipitation (6) . However, in crude enzyme preparations which contain varying amounts of 5~-nucleotidase, labeled S~-AMP is further metabolized, e . g. to adenosine which is not precipitated . If the label is attached to

the adenosine moiety, this would lead to erroneously low values for PDE-activity, unless chromatographical separation of the various products is carried out. This step could be avoided by using 32P-labeled 3~,5~-AMP x, since hydrolysis of S~-AMP32 always yields 32 P~ which is also readily precipitated (5, 6). Thus, PDE-activity in crude extracts could be determined without any interference by subsequent enzymatic degradation of the product, e. g. by 5~nucleotidase . In order to estimate the possibility of using more stable labeled substrates ( TaC-, 3H-3~,5~-AMP) for the determination of PDE in crude extracts, non labeled 5~-AMP was added in high concentratlons(1 -

2

mM) which

did not yet interfere with the PDE-reaction, but to a high extent neutralized 5~-nucleotidase-activity with respect to labeled 5~-AMP. Further, the simultaneous use of 3H-, and 3ZP -labeled 3~, 5~-AMP In the same roectlon mixture In the present experiments not only allowed to measure the magnitude of the remaining error (see table 1 ), but also the influence of papaverine on PDE-, and on 5~-nucleotidase activity in parallel (see table 2 ) .

Incubation was tattled out at 37o C with 300 lil of supernate, to which 100W1 of non-labelled S~-AMP and 50 ail of distilled water (controls) or 50~It1 of papaverine-solution were added. The reaction was started by the addition 50 ~ I of the labeled substrate .

of

xA sample of 3aP-3~, 5~-AMP (spec. activity~l mCt/limole) was generously supplied by Dr. R. H. Symons, Adelaide, Austral la.

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inhibition ad Phosphodiesterase

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The final concentrations In the roaction mixturo (500 li I) wero: Tris -HCI : 9. 6 x 1Ô 2 M, pH 7. 5 Mg-acetate: 3 x 10 3 M 5'-AMP: 2 x 1 Ô3 M Pepaverine-HCI (when present): O. 53 - 10. 6 x 10 5 M The conditions of the assay (dilution of tissue supernates and time of incubation) wero chosen to obtain around 50 ~r hydrolysis at substrate concentrations of 0.5 x 10 ~ M. Experiments which will be rgported elsewhere (5) had shown, that under comparable conditions the velocity of the roaction was linear up to 50 ~ of hydrolysis of this substrate concentration. PDE-activity was calarleted from the decroase in cpm between non incubated and incubated

reaction mixturos. Inhibition by papaverine was exprossed as percent of the turnover in controls which was set at 100. Calculations wero done separately for 3H-, and 3aP~ectlvity . Al l measurements were carried out in dupl icate and the means taken for calculation. Each point used for the determination of KTvalues (table 2) and of Ki-values for papaverine (table 2 and graphs of fig. 1 and Z) Is the mean of 3 - 6 determinations . Km-values were determined graphically (8) with two different substrate concentrations (0.5 and 1 .0 x 10~ M). Papaverine-HCI was added to the supernates in concentrations of 1 - 40 Itg/ml and the Kl-values as well as the. type of PDE inhibition wero also estimated graphically (9) at the same substrate concentrations as used for the determination of Km

RESl1_TS PDE-activity In various tissues. The rolatlve amounts of PDE-activity in the supernates of the tissues investigated, calculated as nmoles of3a P-3',5'-AMP hydrolyzed by the supernate of 1 mg tissue (wet weight), or per mg protein , rospectively during 10 min of incubation at C S ] ~ O.5 x 10~ Mere shown in table 1 . The highest activity was found in brain, the lowest in adipose tis sue. It is noteworthy that PDE-activity was quite high in the supernate of guinea pig ileum and this activity most likely stems mainly from smooth muscle

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InhiMtion a~ Phosphodiesterase

137

fibers, since no PDE-activity has been found to mucosa (10) . Properatlons of guinea pig heart muscle showed an activity of 2. 7 nmoles/mIn/mg

protein

which is somewhat higher than that of proparattons of bovine coronaryarterles (2 . 1 nmoles/min/mg protein), and higher than the activity estimated in proparations of rat liver. TABLE 1 PDE-activi ty

to 2000 x g supernate of tissue homogenate tested to the pre-

sence of 2 mM 5'-AMP. [5] ~ O. 5 x 1Ô

4

M 3H/ 32P-3', 5'-AMP. Values aro

means from 3 - 5 different experiments . Incub. Organ proparatton 3', 5'-AMP hydrolyzed x w. w . / assay mg protein/nssay

rmoles / min .

(min . )

~ decroaae tn radioactivity

32 P

3H

15

55

S3

6. O

20

44

38

0.17

1 .2

20

40

35

0 .005

0 .1

40

49

33

per mg w. w .

per mgprot .

O. 61

9. 8

O. 37

Brain (rat) 1 . 5 mg w . w .

0.094 mg prof . Ileum (guinea pig) 1 . 5 mg w. w .

0.092 mg prof . Liver (rat)

3. O mg w. w. 0.425 mg prof. Fat (rat) 60 . O mg w. w.

2. 220 mg prat. xcalculated on the boats of 32 P-radioactivity w, w. ~ wet weight of homogen(zed tissue from which supernats/ aasaywasused . m g protein determined In duplicate by the baurot method in the 2000 x g nate that was used for the PDE assay.

super-

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The differences between the decrease in radioactivity, as determined with 32P-,

and 3H-labeled substrate to the various supernates are also shown in table 1 . They indicate the amount of labeled product that is further metabolized during incubation to the presence of 2 mM non labeled 5~-AMP .

It can be seen that these differences Increased with the time of Incubation and the concentration of the supernate, e. g. that they were inversely related with the amount of PDE-activity present to the tissue. Km-values ranged between O. 9 and 6. 7 x 10 4 M and were of the same order of magnitude when determined either with 32P-,

or with 3H-labeled substrate (table 2). TABLE 2

PDE-activity in 2000 x g supernate of tissue homogenates tested in the pre4 sence of 2 mM 5~-AMP. [5] - O. 5 or 1 . O x 1Ô M 3H/32P-3~,5~-AMP . Kmvalues were calculated from 6 - 12 determinations, and Ki -values from 26 - 44 mensurements with papaverine as inhibitor . Organ preparation Brain (rat) Ileum (guinea pig) Liver (rat) Fat (rat) x

x

Km(M)

10 1 .0 x 10

2. 2 x

5.0x10 ~ 4 1 .8x10

K1 for papaverine (M) x xx

xx

2. 5 x 10 2. 2 x 10 6.7x10 0.9x10

4 4 4

4. 2 x 10

5

5 3. 5 x 10 1 .3x10 5 5 8.7x10

4. 2 x 10

5

2. 7 x 10 5 5 1 .4x10 5 7.7x1Ô

Calculated on the basis of 32P-radioactivity

~Calculeted on the basis of 3H-radioactivity for comparison

Effect of papaverine. In the supernate of all tissues examined, papaverine exerted marked and dose-dependent Inhibitory effects on PDE-activity. The

K -values were lowest in liver and highest in adipose tissue . There was not

t much difference in the K i -values, as obtained with either 3H-, or with 32Plabeled cyclic AMP as substrate (table 2).

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InhiMtion od Phoephodiesterase

199

a

-4 .8

Ki =13 ~rM b1

-13

Ki

1 2

5

[IJ

10

>Jg/ml

Ileum (guinea-pig)

= 35 ~rM

2

5

CI1

10 Ng/ml

FIG . 1 Inhibition of PDE by papaverine [I] in the 2000 x g supernate of rat

ITver

(a), and guinea pig Ileum (b), measured at two subatrateconcentrations( 32P4 3', 5'-AMP), end determined from Dixon plots . o---o GS) ~ 0.5 x 1Ô M. r-~ CS] ~ 1 .0 x 1Ô4 M. Abszisse ( 1/v) : control rate of hydrolysis(20mtn) with the higher CS] was set ~ 1 (liver: 17.8 rmoles ; ileum: 16.5 rmoles) . Type of PDE-inhibition by papaverine. The dose-dependent inhibition of PDEactivity In the presence of 1 - 40 Itg/ml papaverine, measured at two concentrations of 32P-3', 5'-AMP is evident from the Dtxon plots, shown to flg . 1 and 2. The lowest Ki-value of 1 . 3 x 1Ô 5 M (13 IiM) was found in rat liver (fig. 1 a) where the type of Inhibition wes competitive . With guinea pig Ileum

Inhibition od Phosphadiesterase

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(fig. 1 b), a non-competitive type of inhibition with a Kt-value of 3. 5 x 10 5M (35 pM) wes obtained .

a

-15.6

2

K~ _ ~2 ~rM b1

5

10

CI7

lJg/ml

Adipose tissue (rat)

-325

10 20

Ki = 87 ~rM

CII

FIG .

40 Nglml

a

Inhibition of hDE by papaverine C 1 ] in the 2000 x g supernate of rnt brain (a), and pat epidldymal fat (b), measured as described in fig. 1 . The rate of hydrolysis at I /v ~ 1 was 23.0 rmoles with brain (J5 min), and 20 .0 rmoles with adipose tissue (40 min). 5 The K i-value found with rat brain supernate (4. 2 x 10 M or 42 ~M) was simtJar to liver and also non-competitive (fig. 2 a) . The highest Ki-value (8.7 x lÔS M or 87 ItM) , e. g. the weakest inhibitory effect of papaverine on t~DE, was obtained in the supernate of the ret epididymal fat pad. The type of tnhi-

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Inhibition of Phosphodieetergee

bition in this tissue supernate was also non-competitive , as Dixon plot (fig. 2 b).

iii Judged

from the

DISCUSSION The combination of two intermixed single labeled substrates (3H-, and 32P3~, 5~-AMP), as used in the prosent study provided a rather simple, rol labia and sensitive method to measuro PDE- and 5~-nucleotldnse-activity In crude supernates of tissues with dtfferont enzyme concentrations . From the differon ce in percent decrease to radioactivity, found between 32P and 3H after Incubation and precipitation, the amount of newly formed 5~-AMP which was further metabolized could be calculated . Thus, true PDE-activity as well as the in terference of 5~-nucleottdase and of other phosphatases could be estimated quantitatively in the same roaction mixturo.

In previous experiments it was found that the broakdown of labeled 5~-AMP , formed from labeled 3~,5~-AMP was almost completely inhibited in the prosence of 1 mM non Inhaled 5~-AMP (5) . No product Inhibition of PDE was observed in the prosence of 5~-AMP up to concentrations of 3 mM (5) . Moroover, even concentrations of 10 mM 5~-AMP did not inhibit the activity of purified PDE from brain (11). In the present experiments, 2 mM 5~-AMP were sufficient to keep the breakdown of newly formed 5~-AMP at 4 ~, in the supernate of cerobral tissue. In other supernates with lower PDE-activity which, theroforo , were incubated longer and/or less diluted, this breakdown was accordingly higher . It was found to be 16

with Ileum, 12 ~ with liver, end 33 ~, with epididymel fat supernate . It is obvious that this broakdown must be taken Into ~Ar

account tf absolute values for PDE-activity by the use of 3H- or 1 ~C-labeled substrate are to be obtained. On the other hand, this further broakdown of 3H- or 1aC-5~-AMP does not greatly interfero with the determination of K m and K I -values when 5~-nucleotidase- or phosphetase-activity is unalterod .

The Kmvalues obtained in the prosent experiments aro comparable to those estimated under differont conditions In brain and heart by various investigators (1O, 12, 13, 14).

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IahiMtion ouf Phosphodiesterase

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Confirming previous results, PDE-activity was found to be highest in brain (10, 13), and lower In adipose tissue than in liver (15) . A comparison of the amount of PDE-activity found in the present experiments in the supernates of various tissues revealed considerable differences (see table 1) showing PDEacttvtty in epididymal fat to be 1/100 of that of brain in the rat . Papaverine exerted strong inhibitory effects on PDE-activity in the supernate of all tissues tested so far. The present ftndtng that the Kt-values in one tissue were almost identical, when determined either from the changes in 3H-, or to 32P-activity, Indicates that papaverine did not Influence the activity of S~-nucleotldase or phosphatases . Papaverine in the present experiments was most potent to liver supernate where the type of PDE-Inhibition in contrast 5 to the other organ supernates was competitive (K i ~ 1 .3 x 10 M). Inhibition 5 was somewhat less pronounced in ileum end brain (Ki ~ 3. 5 - 4. 2 x 10 M) , 5 and still lower to adipose tissue supernate (Ki ' 8.7 x 10 M). The effect of papaverine on cerebral PDE was stronger than that of theophylline (Ki = 1 . 1 x lÔ4 M), but weaker than the inhibition, es reported for diethylaminoethyl1-reserpine (Ki ~ 5. 5 x 10 7 M) in this tissue (16). The present ftndtng that the apparent type of inhibition by papaverine (s different when tested on different PDE-preparations is difficult to explain. A similar observation has been made earlier (4, 5), showing that papaverine competitively inhibited

PDE in preparations from beef coronary arteries, but non-competitively in a purified myocardial enzyme preparation . It seems possible that different isoenzymes of PDE are responsible for these divergent effects of papaverine . The strong Inhibition exerted by papaverine on PDE-activity in the supernate of all tissues tested so far indicates that PDE from different sources is I tke-

wtse affected, provided that the inhibitor has free access to the enzyme .

While this paper was in preparation, Trtner et el . (20) have reported that papaverine inhibited PDE-activity in homogenates of uterine-, vascular-, and striated muscle tissue in vitro, and O~Dea et al . (21) have described similar effects on PDE-activity of rat cerebral tissue .

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Inhibition a~f Phosphodiestelrase

1~l3

The pharmacodynamic actions of papaverine aro by far moro I tmited than those of the much weaker PDE-inhibitor theophylline . Whereas theophylline mimics most of the effects of adronerglc beta receptor stimulation (17), or, more generally, the effects of increased formation of cyclic AMP to various tissues, the actions of papaverine aro mainly conüned to smooth muscle . To our know-

ledge there is no evidence that papaverine markedly stimulates cardiac contraction and rate, glycogenolysis in liver or Itpolysis in adipose tissue to an extent that would be compatible to its inhibitory effect on PDE in the supernates of these tissues. The most likely explanation at prosent would be that theophylline is able to penetrate the cellular membranes of many tissues moro easily than papaverine, and that the latter in vtvo has no access to this enzyme . In view of the ample evidence which favours the concept that cyclic 3~,5~-AMP not only mediates a number of well known metabolic effects (17), but also mechanical stimulation of the heart (18), and rolaxation of smooth muscle (1, 3, 4, 19), produced by certain drugs and hormones, one would expect that tissues which do not respond to papaverine would also fail to show increases to the concentration of cyclic 3~,5~-AMP. REFERENCES 1 . W. R. KUKOVETZ, H. JUAN and G. PASCH, Naunyn- Schmiedebergs Arch. Pharmak. 264 , 262 (1969) . 2. G. PÜCH, H. JUAN and W. R. KUKOVETZ, Naunyn-Schmiedebergs Arch. Pharmak. 264 , 293 (1969) . W. 3. R. KUKOVETZ, G. PÖCH, and H. JUAN, Abstracts of the Fourth International Congross of Pharmacology p. 270. Schwabe fs Co. , Basel (1969) . 4. W. R. KUKOVETZ and G. P1SCH, Naunyn-Schmiedebergs Arch. Pharmak. 189 (1970) . 5. G. PÖCH, Naunyn-Schmiedebergs Arch. Pharmak. in press (1970) . 8. G. KRISHNA,, B. WEISS and B. B. BRODIE, J. Pharmacol. exp. Ther. 1~ , 379 (1968) . 7. R. H. SYMONS, Blochern. Biophys. Res. Commun. 38, 80? (1970) .

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8 . H. LINEWEAVER and D. BURK, J. Amer. Chem . Soc. 56, 658 (1934) . 9. M. DIXON, Biochem.J. 55, 170 (1953) . 10 .G . I . DRl.W1M01~ and S. PERROT-YEE, J. Biol . Chem . 236, i 1 126 (1961) . 11 . W. Y. CHEUNG, Btochim. Biophys. Acta ~ , 303 (1969) . 12 . W. Y. CHEUNG, Biochemistry 6 , 1079 (1967) .

13 . R. W. BUTCHER and E. W . SUTHERLAND, J. Btol . Chem . 237 , 1244 (1962) . 14 . K. G. NAIR, Biochemistry 5, 150 (1966) . 15 . B. MILLER-OERLINGHAUSEN, U. SCHWABE, A. HASSELBLATT, and F. H. SCHMIDT, Life Scl. 7 II

, 593 (1968) .

16 . F. HONDA and H. IMAMURA, Biochim. Btophys. Acta 1~, 267 (1968) . 17 . G. A. ROBISON, R. W. BUTCHER and E. W. SUTHERLAND, Ann. N. Y. Acad . Sci. ~:1+, 703 (1967) . 18 . W. R. KUKOVETZ and G. PÖCH, Naunyn-Schmiedebergs Arch . Pharmak. 2~ , 236 (1970) . 19 . P. F. MOORE, L. C. IORIO, J. M. McMANUS, J. Pharm. Pharmacol . 368 (1968) . 20.L . TRINER, Y. VLLLIEMOZ, I . SCHWARTZ, and G. G. NAHAS, Biochem . Biophys. Res. Commun . 4~0 , 64 (1970) . 21 . R. F. O~DEA, M. K. HADDOX, N. D. GOLDBERG, Fed. Proc . 29 , 473 (1970) .