European Journal o f Pharmacology, 37 ( 1976 ) 91--95

91

© North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands

SOME A D R E N E R G I C H-BLOCKING AGENTS A F F E C T I N G LIPOLYSIS IN HUMAN ADIPOSE TISSUE IN VITRO JITKA WENKEOVA, EDUARD KUHN and MAX WENKE * Research Centre o f Metabolism and Nutrition (Head: Prof. Dr. J. Ma~ek, Dr. Sc.) o f the Institute for Clinical and Experimental Medicine (Director: Pro/'. Dr. P. M~lek, Dr.Sc.), Bud~jovick~ 800, Prague 4, and *Department o f Pharmacology, Faculty o f General Medicihe, Charles University, Albertov 4, Prague 2, Czechoslovakia

Received 6 August 1975, revised MS received 22 December 1975, accepted 22 January 1976

J. WENKEOVA, E. KUHN and M. WENKE, Some adrenergic [J-blocking agents affecting lipolysis in human adipose tissue in vitro, European J. Pharmacol. 37 (1976) 91--95. Antagonistic actions of /3-blocking drugs on isoproterenol-induced lipolysis were studied in human omental adipose tissue. Competitive interaction characterized by the following PA2 values was found: propranolol 8.7; trimepranol 8.7; practolol 7.1; H 35/25 6.0. The plot of PA2 values of these drugs for human versus rat adipose tissue is linear with slope 2.0 indicating a higher differentiation of ~-antagonist actions in human than in rat adipose tissue, Adrenergic ~-blockers Human adipose tissue

Trimepranol

Adrenergic lipolysis

Practolol

H 35/25

1. I n t r o d u c t i o n

2. Materials and m et hods

The reactivity of hum a n adipose tissue when affected by lipid mobilizing adrenomimetics differs substantially from the know n reactivity of rat adipose tissue reviewed recently (Wenkeov~[ et al., 1975a,b). Much less information is available concerning the effects of/3-blocking drugs on human adipose tissue; as yet, only the actions of propranolol have been studied more in detail qualitatively (Burns and Langley, 1969; Bray and Trygstad, 1972) and quantitatively (Wenkeov~ et al., 1970). In this paper, an a t t e m p t is made to obtain quantitative data a b o u t the effects of some other/3-antagonists on h u man adipose tissue. Of the four/3-blocking drugs used, propranolol (Levy, 1967) and trimepranol (Zakhari, 1974) blocks ~1 as well as ~= adrenergic rections (Lands et al., 1967). Plactolol acts p r e d o m i n a n t l y as a/3,-blocking drug (Vaughan Williams et al., 1973) and H 35/25 as a ~2 -antagonist (Levy, 1967).

2.1. Tissue, m e d i u m , i n c u b a t i o n , e v a l u a t i o n

Human omental adipose tissue, collected during a p p e n d e c t o m y in 7--14 years old children of both sexes, was cut into small pieces, weighed and instantly transferred for 90 min into a medium containing albumin, ascorbic acid and different concentrations and combinations of the drugs studied. The effects of these drugs on the release of free fat t y acids and in some experiments also on the release of glycerol were estimated. The technical details were described previously (Wenkeov~ et al., 1975b). In the tissue from any individual patient, the dose--response curve o f lipolytic actions of isoproterenol was estimated and the effect of some of the /~-blocking agents chosen was followed, pA2 values were calculated according to Ari~ns {1964) as the negative logarithm o f

92

J. W E N K E O V A E T AL.

the molar concentration of the antagonist shifting the dose--response curve of isoproterenol to the right by 0.3 log unit.

%FFA lo0-

ISO

--

-

--o--

!"~.-~

.... 0 .... ISO+ PC S

2. 2. Drugs and abbreviations used

t /

".'~x. ":.,. "0..

,,

/

~.~ .""

///~,

"..

liPS-..

."

50-

Isoproterenol hydrochloride (ISO; Sterling-Winthrop), propranolol hydrochloride (PP; I.C.I. ), trimepranol (TR; Spofa), practolol (PC; I.C.I.), H 35/25 (H; A.B. Hfissle). Further abbreviation: free fatty acids = FFA.

0 I

pD

II

~o

I

'

9

I

'

B

I

'

7

I

'

6

I

'

S

I

'

4

GLYCEROL

,oo,oo-

3. Results

tl@

°

3.1. Effects of practolol 50The ~, -blocking agent PC exerted a typical competitive parallel shift of the ISO dose-response curves affecting both FFA and glycerol release (see fig. 1). The pA2 values {table 1) remained consistent; neither differences in PC concentration, nor differences in the criterion estimated (FFA or glycerol release} affected the pA2 value to any extent.

3.2. Effects o f H 3 5 / 2 5 With H 35/25, which is held to possess predominantly ~2 -adrenolytic properties (Levy

"""'"'""

I

pD

tl

co

I

9

'

I

'

8

I

7

E f f e c t s o f / ] - b l o c k i n g d r u g s a n t a g o n i z i n g i s o p r o t e r e n o l lipolysis in h u m a n a d i p o s e tissue. Drug

Molar c o n c .

Criterion

n *

Practolol

1 1 1 1

10 -6 10 4 10 - s 10 -s

FFA Glycerol FFA Glycerol

7 6 7 6

H 35/25

1 × 10 -5 1 × 10 -s

FFA Glycerol

12 5

5.90 5 0 . 1 3 6.07 ± 0 . 1 8

Trimepranol

3 . 1 6 × 10 -8

FFA

6

8.71 ± 0 . 3 6

Propranolol

3 . 1 6 × 10 -8

FFA

5

8.75 ± 0.25

* n = number of patients.

I

6

'

I

5

'

I

'

4

Fig. 1. E f f e c t s o f p r a c t o l o l (PC) o n l i p o l y t i c a c t i o n s o f i s o p r o t e r e n o l (ISO) in releasing free f a t t y acids ( F F A , a b o v e ) a n d glycerol ( b e l o w ) . A b s c i s s a : logar i t h m i c scale o f m o l a r c o n c e n t r a t i o n s o f ISO ( p D = -log. c o n c . , t h u s p D ~ = c o n c . 0). O r d i n a t e : rise o f F F A a n d glycerol a b o v e t h e basal value. 100% = eff e c t o f ISO 2 x 10 -6 M; PC 6 = p r a c t o l o l 1 x 10 .6 M; PC 5 = p r a c t o l o l 1 x 10 -s M. M e a n s ± SE are given. F o r n, see table 1.

TABLE 1

x × × ×

'

PA2 ± S.E. 7.01 7.17 7.19 7.00

± 0.16 -+ 0 . 1 5 ± 0.15 ± 0.09

/]-BLOCKERS AND LIPOLYSIS IN HUMAN ADIPOSE TISSUE 7o FFA 100- --,,,0--150

PAz human AT 9

~ o ~ i

/

PP• / • TR f

/,/.,

so-

93

8

y=2x-5 /

oJ ,

II

I

pD= e0

'

I

9

8

'

I

7

'

I

'

I

6

5

'

i

'

4 7--

Z PC

FFA

lOO- ,--ib,--ISO --0o- ISO + PP Z5 ....

o ....

,so

+ TR

t~/,~, s¢ ' ~ - ~ 6

75

-

..........

I

I

!

5

6

7

pA2 rat AT . . . . . . .

.....

0 ,

pD=co

II

I

9

'

i,

I'

[

8

7

6

'

I'

I"

5

4

Fig. 2. Effects of H 3 5 / 2 5 (H 5 = H 3 5 / 2 5 1 × 10 -s M); o f p r o p r a n o l o l (P 7.5 = p r o p r a n o l o l 3.16 × 10 -8 M) a n d o f t r i m e p r a n o l ( T R 7.5 = t r i m e p r a n o l 3.16 × 10 - $ M) o n release o f f a t t y acids ( F F A ) i n d u c e d b y i s o p r o t e r e n o l (ISO). F u r t h e r d e s c r i p t i o n as in fig. 1.

and Wilkenfeld, 1969}, only weak antagonism of ISO actions was found. In accord with the presumed competitive nature of the effect, a parallel shift of the ISO curve appeared (see fig. 2); the pA2 value indicates an affinity of H 35/25 approximately 1 pA~ unit less than the affinity of PC (table 1}.

3. 3. Effects of propranolol and trimepranol A comparison of the blocking effects o f PP and TR in equimolar concentrations shows that these two drugs are equally effective in our experimental conditions (fig. 3, table 1). Both blocking agents are characterized by a very high pA2 value indicating an affinity approximately 50-fold that of PC.

Fig. 3. Comparison of pA2 values of ff 35/25 (H), praetolol (PC), propranolol (PP) and trimepranol (TR) in rat (abscissa) and human (ordinate) adipose tissue.

4. Discussion The pA2 value of PP 8.75, found in these experiments is consistent with that of 8.81 which was previously described for human adipose tissue (Wenkeov~ et al., 1970). The results also show that TR acts as a potent /3-adrenergic blocking agent as effectively as does PP. The pA2 value of 8.71 is comparable to the pA2 of 8.17 and 8.12 found by Zakhari (1974} for TR as an antagonist of ISO effects on cat heart rate and on cat hind limb vasodilatation respectively. At the present time, we are not able to explain the reason why PP and TR alone showed an (non significant) elevation of mean values of basal lipolysis in these experiments. No /3-mimetic c o m p o n e n t has been described as yet for the action of these two drugs (cf. e.g. Wenkeov~ et al., 1970; Zakhari, 1974). The pA2 of 7.10 found for PC shows a much closer relation to the values of 7.35, 6.14 and 6.12 described for the effect on heart rate (~1) under different conditions (Harms et al., 1974; Zakhari, 1974; Vaughan Williams et al., 1973}

94 t h a n to the pA2 of 5.20, 5.35 and 5.18 f o u n d b y t h e s a m e a u t h o r s for t h e e f f e c t o n vasodilat a t i o n or t r a c h e a l r e l a x a t i o n (/35). This s e e m s to be in general a g r e e m e n t w i t h t h e p r e s u m e d /31 - c h a r a c t e r o f adrenergic lipolysis. With H 3 5 / 2 5 , t h e s i t u a t i o n is less clear: f r o m t h e d a t a given b y L e v y and Wilkenfeld ( 1 9 6 9 ) t h e pA2 value o f less t h a n 4.90 for B1 - c h r o n o t r o p i c effects, b u t o f 6 . 2 5 - - 6 . 4 2 f o r the e f f e c t o n B2-vasodilatation can be accounted for by the method of Vaughan Williams et al. ( 1 9 7 3 ) . T h e value o f 6.00 f o u n d in the p r e s e n t w o r k s e e m s to fit m o r e with 6.2 t h a n with < 4 . 9 . T h e values of pA2 f o u n d in h u m a n a d i p o s e tissue f o r d i f f e r e n t B-blockers, differ m a r k e d l y f r o m t h e values d e s c r i b e d in rat a d i p o s e tissue. This is striking w i t h PP, in rat a d i p o s e tissue, w h e r e the pA2 values do n o t e x c e e d 7.0 (Fassina, 1 9 6 6 ; W e n k e o v ~ et al., 1970; H a r m s et al., 1974), t h u s being l o w e r t h a n in h u m a n a d i p o s e tissue b y a l m o s t t w o pA2 units. H o w e v e r , it is difficult to m a k e precise c o m p a r i s o n s as for e x a m p l e Lincov~ et al. ( 1 9 7 4 ) s h o w e d t h a t in rat a d i p o s e tissue t h e pA2 o f s o m e ~-blocking drugs change in r e l a t i o n to b o t h t h e a d r e n o m i m e t i c a n t a g o n i z e d and t h e c o n c e n t r a t i o n of b l o c k i n g a g e n t used. In view of this fact, e x p e r i m e n t s on r a t a d i p o s e tissue were t a k e n in a c c o u n t (Lincov~, u n p u b l i s h e d o b s e r v a t i o n s ) , w h e r e ISO was t h e agonist i n f l u e n c e d and w h e r e t h e ~ - a n t a g o n i s t was used in a c o n c e n t r a t i o n e v o k i n g a parallel shift similar to t h a t in comparable experiments on human adipose tissue. T h e o t h e r e x p e r i m e n t a l c o n d i t i o n s (e.g. a d d i t i o n o f ascorbic acid to t h e m e d i u m ) also allowed full c o m p a r a b i l i t y o f t h e results. D a t a o b t a i n e d in this w a y are s u m m a r i z e d in fig. 3. T h e o r d e r of p o t e n c i e s o f t h e B-blockers used r e m a i n s t h e s a m e in b o t h h u m a n and rat adip o s e tissue and t h e p l o t o f pA2 values o f t h e drugs w i t h h u m a n versus rat a d i p o s e tissue is linear. H o w e v e r t h e slope o f 2.0 s h o w s t h a t changes in pA2 values f r o m d r u g to d r u g are t w i c e as great in h u m a n as in rat a d i p o s e tissue. T h e q u e s t i o n arises w h e t h e r this finding m i g h t indicate d i f f e r e n t p r o p e r t i e s o f t h e H-receptors in h u m a n and in r a t a d i p o s e tissue. F u r t h e r

J. WENKEOVA ET AL. e x p e r i m e n t s are n e e d e d to assess t h e influence o f d i f f e r e n t u p t a k e and d e s t r u c t i o n of the drugs. W h a t e v e r the m e c h a n i s m r e s p o n s i b l e for the d i f f e r e n c e s m a y be, it can be c o n c l u d e d t h a t , u n d e r c o n d i t i o n s described, t h e b l o c k i n g pot e n c y o f t h e B-antagonists used a p p e a r s to be for m o r e d i f f e r e n t i a t e d in h u m a n t h a n in rat a d i p o s e tissue.

Acknowledgement We are indebted to Dr. V. Holub and his team for providing us with samples of human adipose tissue, to A.B. H~issle for the specimen of H 35/25, to I.C.I. for the samples of practolol and propranolol and to Dr. V. Tr~ka for trimepranol.

References Ari~ns, E.J., 1964, Molecular Pharmacology I (Academic Press, New York--London). Bray, G.A. and O. Trygstad, 1972, Lipolysis in human adipose tissue: Comparison of human pituitary hormones with other lipolytic agents, Acta Endocrinol. 70, 1. Burns, T.W. and P.E. Langley, 1969, Differential effects of a- and ~-adrenergic blocking agents on basal and catecholamine-stimulated lipolysis in human and rat adipose tissue cells, J. Lab. Clin. Med. 74,857. Fassina, G., 1966, Effects on lipomobilisation of the ~-adrenergic blocking drugs, propranolol and INPEA, J. Pharm. Pharmacol. 18, 399. Harms, H.H., J. Zaagsma and B. Van der Wal, 1974, /3-Adrenoceptor studies. III. On the/3-adrenoceptors in rat adipose tissue, European J. Pharmacol. 25, 87. Lands, A.M., A. Arnold, J.P. McAuliff, F.P. Luduena and T.G. Brown, 1967, Differentiation of receptors activated by sympathomimetic amines, Nature 214, 597. Levy, B., 1967, A comparison of the adrenergic receptor blocking properties of 1-(4'-methylphenyl)-2-isopropylamino-propanol-HC1 and propranolol, J. Pharmacol. Exptl. Therap. 156, 452. Levy, B. and B.E. Wilkenfeld, 1969, An analysis of selective beta receptor blockade, European J. Pharmacol. 5, 227. Lincov~, D., D. Mi~ekov~, J. ~epelfk and J. Wenkeov~., 1974, Pfisobenf ~1 a t32 adrenotropnfch l~tek na lipomobilisaci, t~s. Fysiol. 23,352.

~-BLOCKERS AND LIPOLYSIS IN HUMAN ADIPOSE TISSUE Vaughan Williams, E.M., E.E. Bagwe}l and B.N. Singh, 1973, Cardiospecificity of /3-receptor blockade, Cardiovascular Res. 7,226. Wenkeovfi, J., E. Kuhn and M. Wenke, 1970, In vitro effects of isoproterenol and propranolol on human adipose tissue, Life Sci. I, 9,233. Wenkeovfi, J., E. Kuhn and M. Wenke, 1975a, Adrenergic lipolysis in human adipose tissue in

95

vitro, European J. Pharmacol. 30, 49. Wenkeovfi, J., E. Kuhn and M. Wenke, 1975b, Some adrenomimetic drugs affecting lipolysis in human adipose tissue in vitro, European J. Pharmacol. 35, 1. Zakhari, S., 1974, Structure selectivity relationships of ~-adrenoceptor antagonists in cats, European J. Pharmaco]. 29, 22.