Naunyn-Schmiedeberg's

Archivesof Pharmacology

Naunyn-Schmiedeberg's Arch. Pharmacol. 300, 11 - 17 (1977)

9 by Springer-Verlag 1977

Effect of Catechol-O-Methyl-Transferase (COMT) Inhibition on the Vascular and Metabolic Responses to Noradrenaline, Isoprenaline and Sympathetic Nerve Stimulation in Canine Subcutaneous Adipose Tissue ERIK BELFRAGE, BERTIL B. FREDHOLM, and SUNE ROSELL Department of Pharmacology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden

Summary. The effect of COMT-inhibitors (U-0521, H 22/54) on lipolysis and on vascular responses induced by noradrenaline, isoprenaline and nerve stimulation in canine adipose tissue in situ has been studied. C O M T inhibition, before or after/~-adrenoceptor blockade, did not influence the a-adrenoceptor mediated vasoconstriction due to nerve stimulation ( 2 5 Hz). Vasoconstriction due to close i.a. noradrenaline was unaffected at a dose of 2 x 10 - ~o moles but at a dose of 10 .9 moles the peak vasoconstriction was enhanced but the duration of the response was not changed. Prior fl-adrenoceptor blockade did not alter the responses to U-0521. After ~-adrenoceptor blockade with Hydergine ( 1 2 0 - 3 0 0 gg i.a.) a vasodilatation was induced by noradrenaline 2 x 10-to mole. This response and that produced by isoprenaline (2 x 10 -12 or 10 -l~ moles) were enhanced after U-0521-treatment, but the vasodilating response to nerve stimulation (2 Hz) after ~-adrenoceptor blockade was unaffected. Lipolysis induced by nerve stimulation or noradrenaline in vivo was increased 50 ~ and that induced by isoprenaline was increased 1 0 0 ~ by C O M T inhibition. U-0521 did not alter basal lipolysis in vitro but enhanced lipolysis induced by 0.1 I-tM noradrenaline significantly, both in the presence and in the absence of theophylline. The present results shows that C O M T influences the effective concentration of isoprenaline more than that of noradrenaline. In the vasculature the effects of exogenous noradrenaline is influenced to a larger degree than those induced by nerve stimulation. However, C O M T blockade increased lipolysis induced by equieffective doses of exogenous noradrenaline and nerve stimulation to an equal extent. These results are in agreement with the idea that C O M T Send offprint requests to E. Belfrage at the above address

is of physiological importance mainly at receptor sites that are not in close contact with sympathetic nerve endings and supports the idea that vascular ~-adrenoceptors are innervated receptors. Vascular fl-adrenoceptors on the other hand seem to be humoral receptors unrelated to sympathetic nerve endings and preferentially stimulated by circulating noradrenaline. The fl-adrenoceptors on the fat cells may be of both types, i.e. innervated and humoral.

Key words."Adipose tissue - Adrenoceptors - Blood flow - C O M T - Lipolysis - Nerve stimulation Noradrenaline.

INTRODUCTION The role of catechol-O-methyl-transferase (COMT) as a determinant of the catecholamine concentration in the biophase appears to vary between tissues, with the type of catecholamine and with the mode of administration (see Review by Guldberg and Marsden, 1975). In densely innervated tissue such as the cat nictitating membrane (Trendelenburg et al., 1971) and cat papillary muscle (Kaumann, 1970) inhibition of C O M T results in a potentiation of the effects of noradrenaline only if the neuronal uptake mechanism is inhibited. This suggests that neuronal uptake is the dominating inactivating mechanism in such tissues where the neuroeffector gap is narrow. On the other hand, in the rabbit aorta which has a wide neuroeffector gap, COMT-inhibition causes a small potentiation of the NA-induced vasoconstriction even when the neuronal uptake mechanism is left intact (Levin and Furchgott, 1970). It is possible that C O M T may be of importance as a determinant of the NA concentration in those tissues where the distance between nerve terminals and the adrenoceptors is large.

12

In canine subcutaneous adipose tissue catecholamines induce several different effects. In the vascular smooth muscle, e-adrenoceptors mediate vasoconstriction and /?-adrenoceptors vasodilatation (Ngai et al., 1966). On the fat cells/Ladrenoceptor stimulation causes increased lipolysis (see Fredholm, 1970). Based on studies on the effects of chronic denervation or cocaine it has been proposed that the vascular ~-adrenoceptors are innervated receptors in close contact with nerve terminals, while the vascular /3-adrenoceptors are humoral receptors unrelated to the nerve terminals (Rosell and Belfrage, 1975; Belfrage and Rosell, 1976). The same authors also suggested that the fat cell /Ladrenoceptors may be innervated. However, other findings have been taken as evidence that fat cells are not directly innervated (Fredholm, 1970). In order to further clarify the innervation of the various adrenoceptors in adipose tissue the present study has examined the effect of COMT-inhibition on vascular and lipolytic responses caused by sympathetic nerve stimulation and by intra arterial noradrenaline and isoprenaline.

Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)

Injection of Vasoactive Substances. In 10 dogs close intra-arterial injections of noradrenaline [( )arterenol HC1, Sigma], isoprenaline [()Isoprenaline bitartrate)] or terbutaiine (Terbutaline sulfate, Draco) were given. In these experiments the adipose tissue on both sides were used, one serving as control and the other being treated with the COMT inhibitor U-0521 (1.5-2.5 nag i.a.). There was no difference between the sides in resting resistance or in basal glycerol outflow (Figs. 2 and 3). Mean tissue weights were 37 g (range 24-54) on the control and 35 g (23-57) on the U-0521 treated side. A mixture of dihydroergotalkaloids (dihydroergocornin, dihydroergocryptin, dihydroergocristin: Hydergine, Sandoz) 120300 gg i.a. was given as an s-receptor blocking agent in some of the experiments. A dose of 5 0 - 100 pg was given every hour to assure a complete c~-adrenoceptor blockade. Hydergine had no effect on basal [ipolysis and did not influence the vasodilator or the lipolytic effects of isoprenaline. All drugs were administered via a side arm of the arterial drop counter. The injection volume was 0.2 ml. Ascorbic acid, 20 pg/ml, was added to the solutions of noradrenaline and isoprenaline. Ascorbic acid solution (0.2 ml) had no vascular effects. Sympathetic Nerve Stimulation. In 9 dogs with a mean adipose tissue weight of 45 g (23-83) the distal end of the sectioned nerve was placed on a bipolar silver electrode and stimulated for 4 min at 2 - 5 Hz with impulses of supramaximal intensity (12V) and duration (2 ms), delivered by a Grass model $4 stimulator. Vascular and lipolytic responses were obtained before and 10-20 min after administration of U-0521 (1.5-2.5 mg i.a.) or H 22/54 (5 mg i.a.). Lipolysis in vitro. Subcutaneous adipose tissue was removed from

METHODS The experiments were conducted on 19 female mongrel dogs weighing 11 - 23 kg, anesthetized with sodium pentobarbital (30 mg/ kg with supplement as necessary). Tracheotomy was performed and most of the dogs were mechanically ventilated with a Braun Melsungen model 74052 respirator. To prevent coagulation heparin (2500 IU/kg) was administered i.v. Subcutaneous adipose tissue in the left and the right inguinal region was isolated from skin and other surrounding tissues according to the method described by Rosell (1966). This provides two almost identical adipose tissue preparations, each supplied by one artery, one vein and a nerve containing sympathetic fibres. The nerve to the adipose tissue was sectioned on both sides in all experiments. Arterial blood from a femoral artery was diverted to the adipose tissue. A drop recorder was included in the arterial loop. The venous outflow was directed to a femoral vein. Systemic blood pressure was measured in a carotid artery with a Statham P23AC transducer and recorded together with blood flow on a Grass model 7B polygraph. The resting blood flows was in the normal range reported earlier for this tissue (Ngai et al., 1966). Vasodilatation was calculated from the per cent decrease in resistance (ram Hg x 100 g x min • ml- 1) and vasoconstriction from the per cent decrease in conductance (ml x min- 1 x 100 g- 1 x mm Hg 1). Thus, both vasoconstrictions and vasodilatations are expressed in percentages between 0 and 100. Venous blood samples from the adipose tissue vein was collected into ice-cooled plastic tubes. After centrifugation plasma was taken for determination of glycerol (Laurell and Tibbling, 1966). Arterial samples were taken from the carotid artery and the glycerol release rate was calculated by multiplying the A-V difference by plasma flow in m l x m i n -1 • -1. Net release of glycerol (gmol x 100 g-~) was obtained by subtracting the basal glycerol release from the total amount released during and following an injection of drug or a nerve stimulation.

the inguinal region in two dogs and immediately put into KrebsRinger phosphate buffer pH 7.4 containing 3 ~ bovine serum albumine (Fraction V, Sigma). Pieces of about 300 mg were removed from the tissue and preincubated in the buffer for 30 min at 37~C. The pieces were then removed from the solution, weighed and put into 3 ml of incubation buffer containing the test substances. Incubations were carried out for 60 min at 37~ in a water bath shaking at 160 cycles per minute. Lipolysis was quantitated by assay of glycerol release (Laurell and Tibbling, 1966).

COMT-Inhibitors. H 22/54 (c~-propyl-3-4, dihydroxy phenylacetamide, Hgssle) has been shown to have COMT-inhibitory properties (Carlsson et al., 1963). We found that H 22/54 (5 mg close i.a.) almost completely inhibited the 3H-normetanephrine overflow after sympathetic nerve stimulation in subcutaneous adipose tissue prelabelled with 200 gCi 3H noradrenaline (New England nuclear). However, the total 3H-overflow during and after sympathetic nerve stimulation was not altered in 5 dogs indicating that H 22/54 does not alter transmitter release in adipose tissue although the compound has been shown to inhibit noradrenaline synthesis (Carlsson et al., 1963). U-0521 (3',4',-dihydroxy-2-methylpropiophenone, Upjohn) has been shown to be a competitive inhibitor of COMT (Giles and Miller, 1967). We tested the drug using the COMT-dependent noradrenaline-dopamine assay in vitro described by Coyle and Henry (1973). 1 pg/ml completely inhibited the o-methylation of dopamine. In dog adipose tissue in situ the fraction of 3H noradrenaline and deaminated catechols in the venous blood, sampled 2 20 min after an intraarterial injection of 50 gCi 3H noradrenaline, increased by about 7 0 ~ after treatment with 2 mg U-0521 dose i.a. Thus, the doses of H 22/54 and U-0521 used in the in situ experiments as well as the concentration of U-0521 used in vitro appears to be high enough to ensure a high degree of COMTinhibition. Statistics. Results were evaluated by Student's t-test for paired observations.

E, Belfrage et ai. : Role of COMT in Adipose Tissue

13

Table I. Peak vasoconstrictor effects of noradrenaline (NA) and sympathetic nerve stimulation (NS) during control conditions and after inhibition of COMT. Results are expressed as mean • S.E.M. Dose

N

~ Decrease in conductance control

COMT-inhibition

Significance

COMTinhibitor

NA NA NA (fi-biockade)

2 x 10 - m moles ]0 -9 moles ]0 -9 moles

7 8 4

26 4- 5 44 _+ 7 46 _+ 12

30 • 4 50 • 7 52 _+ 8

N.S. P < 0.05 P < 0.05

U-0521 U-0521 U-0521

NS NS (fi-blockade) NS

2 Hz 2 Hz 5 Hz

6 4 4

57 • 7 54 + 8 85 • 4

60 • 8 57 + 6 82 • 3

N.S. N.S. N.S.

U-0521 U-0521 H 22/54

RESULTS Vasoconstriction COMT-inhibition (1.5-2.5 mg U-0521 or 5 mg H 22/ 54 i.a.) did not influence the peak vasoconstrictor response to sympathetic nerve stimulation (Table 1). After nerve stimulation vascular resistance fell below control values indicating a reactive hyperaemia, which appeared at the same time before and after U-0521treatment (Fig. 1). Thus, COMT-inhibition did not prolong the vasoconstrictor effects of nerve stimulation. When 2 x 10 -l~ moles of noradrenaline was given by close intraarterial injection there was a small increase in resistance. Treatment with U-0521 did not significantly alter the vasoconstrictor effect of noradrenaline (Fig. 2). However, when a 5 times higher dose ofnoradrenaline was injected, U-0521 significantly potentiated the increase in resistance (Fig. 2, Table 1). The duration of the vasoconstriction was not changed. The lack of effect of COMT-inhibition on the vasoconstriction induced by nerve stimulation and by the low dose of noradrenaline could be due to a potentiation of/~-adrenoceptor mediated vasodilatation large enough to counteract the ~-adrenoceptor mediated vasoconstriction. However, pretreatment with the fl-adrenoceptor blocking agent propranolol (500 gg i.s.) did not alter the effects of U-0521 on vasoconstrictor responses (Table 1), i.e. a small but significant increase in the vasoconstriction induced by 109 moles of noradrenaline but no effect on that induced by nerve stimulation at 2 Hz. Vasodilatation

After c~-adrenoceptor blockade by Hydergine both noradrenaline injection and nerve stimulation induced vasodilatation rather than vasoconstriction. The vasodilatation induced by 2• 10 -1~ moles noradrenaline was significantly potentiated by the U-0521 treatment (Fig. 3, Table 2). However, U-0521 did not significantly alter the vasodilatation induced by

GLYCEROL 1 IJmoles,mi6,1OlJ~[ 40J, 9 i~:i:i~:i:::i::::i:i '~ :::::::::::::::::::::::::::::: i \ 9

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RESISTANCE per cent 300. :iI J

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Fig. i. Time course of vasoconstriction and glycerol release during and following sympathetic nerve stimulation (NS) (for 4 rain) at 2 Hz. N = 6. The resting resistances in PRUloo (peripheral resistance units in m m H g x ]00 g • -~) were 13 • 2 (control) and 20 _+ 4 (U-0521), respectively, and differed significantly from each other (P < 0.05). Black dot (O) indicate a significant (P < 0.05) difference between U-0521 and control. The figures are given as mean • S.E.M.

sympathetic nerve stimulation at 2 Hz (Table 2). Close intraarterial injection of isoprenaline resulted in vasodilatation and this was significantly potentiated by the U-0521 treatment both at 2• - n and at 10 -1~ moles of isoprenaline (Fig. 3, Table 2). Both the peak and duration of the vasodilator response were increased at both dose levels (Fig. 3). Lipolysis in vivo and in vitro

The peak rate of glycerol release after both sympathetic nerve stimulation at 2 Hz and injection of 10-9moles of noradrenaline was increased after U-0521-treatment (Figs. I and 2). The peak lipolytic

Naunyn-Schmiedeberg's Arch. Pharmacol. 300 (1977)

14 ~Tcm

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Fig. 2 Time course of vasoconstriction and glycerol retease following close-arterial injection of noradrenaline (NA). Resting resistance in PRUa0o was 22 • 8 (control) and 22 _+ 8 (U-0521) at 2 • 10-1~ moles (N = 7) and 21 _4- 4 (control) and 21 _+ 4 (U-0521) at 10 -9 moles (N = 8), respectively. For further details see legend to Figure 1

0

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min

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Fig. 3 Time course of vasodilatation and glycerol release following close-arterialinjection of noradrenaline (NA) after c~-adrenoceptor blockade and following isoprenaline (ISO). Resting resistance in PRU100 was 12 i 2 (control) and 15 + 4 (U-0521) at NA 2x 10 -1~ moles (N = 6), 21 _+ 5 (control) and 21 i 4 (U-0521) at ISO 2x 10 -11 moles (N = 10) and 21 _+ 3 (control) and 21 • 4 (U-0521) at tSO 10 -t~ moles (N = 10), respectively. For further details see legend to Figure 1

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Table 2. Peak vasodilatation induced by isoprenaline (ISO), noradrenaline (NA) and sympathetic nerve stimulation (NS) during control conditions and after treatment with U-0521. Results are expressed as mean _+ S.E.M. Dose

ISO ISO NA (c~-blockade) NS (c~-blockade)

2x10 - n moles 10 -1~ moles 2x 10 -1~ moles 2 Hz

N

10 10 6 4

r e s p o n s e to i n j e c t i o n o f 1 0 - 1 ~ isoprenaline w a s a l s o p o t e n t i a t e d (Fig. 3). T h e n e t g l y c e r o l r e l e a s e i n d u c e d by n e r v e s t i m u l a t i o n a n d n o r a d r e n a l i n e inj e c t i o n w e r e e a c h i n c r e a s e d 50 ~ b y t r e a t m e n t w i t h e i t h e r U - 0 5 2 1 o r H 22/54 a n d U - 0 5 2 1 i n c r e a s e d t h e l i p o l y t i c effect o f i s o p r e n a l i n e m o r e t h a n 1 0 0 ~ ( T a b l e 3). U - 0 5 2 1 d i d n o t i n f l u e n c e t h e l i p o l y s i s ind u c e d b y i n j e c t i o n o f t e r b u t a l i n e (2 exp.).

% Decrease in resistance

Significance

control

U-0521

53 69 44 48

62 72 56 53

! 4 ___ 2 +_ 4 • 11

_+ 3 • 2 i 4 • 11

P < 0.01 P < 0.05 P < 0.05 N.S.

To determine whether the increased lipolytic r e s p o n s e in v i v o w a s c a u s e d by i n h i b i t i o n o f C O M T in t h e a d i p o s e tissue o r by i n c r e a s e d access o f a g o n i s t s d u e to C O M T i n h i b i t i o n in t h e b l o o d vessel wall, t h e effects o f U - 0 5 2 1 o n n o r a d r e n a l i n e i n d u c e d l i p o l y s i s in v i t r o w a s s t u d i e d . A s s h o w n in T a b l e 4, U - 0 5 2 1 (1 g g / m l ) i n c r e a s e d t h e lipolysis i n d u c e d b y 10 - 7 M n o r a d r e n a l i n e in v i t r o b o t h in the a b s e n c e a n d in t h e

E. BeIfrage et al. : Role of COMT in Adipose Tissue

15

Table 3. Net glycerol release in vivo following isoprenaline (ISO), noradrenaline (NA) and sympathetic nerve stimulation (NS) during control conditions and after inhibition of COMT. Results are expressed as mean _+ S.E.M.

ISO NA NS NS

Dose

N

Glycerol control

gmoles x 100 g-1 COMT-inhibition

Significance

COMT-inhibitor

10 -~~ moles 10 -9 moles 2 Hz 5 Hz

8 8 6 4

16.7 10.4 10.0 7.2

35.1 17.0 15.4 9.1

P P P P

U-0521 U-0521 U-0521 H 22/54

+ 5.0 _+ 3.5 • 1.6 • 1.7

Table 4. Glycerol release in vitro induced by noradrenaline, with and without addition of theophylline, during control conditions and during COMT-inhibition with U-0521 (1 F.g/mi). Results are expressed as mean of 5 - 10 observations _-+ S.E.M. Noradrenaline Glycerol release (gMoles x g-1 • h-1) concentration no Theophylline + Theophylline 1 m M control 0

U-0521

U-0521

0.27__+0.02 0.27__+0.05 1.25__0.21

~, /

N.S. /0 7 M

control

0.26+0.05 -\

0.57+0.1I /-

P < 0.05

\

1.36_+0.15

/

N.S. 1.33+0.12 -\

1.99+0.26 /-

P < 0.05

presence of 1 mM theophylline indicating that tissue COMT may be of importance. U-0521 had no lipolytic effect and did not alter the lipotysis induced by theophylline in the concentration used. DISCUSSION In the present study, it was found that COMTinhibition increased the vascular effects of intraarterial noradrenaline mediated by/Ladrenoceptors to a larger extent than those mediated by c~-adrenoceptors but did not alter the vascular effects of sympathetic nerve stimulation, mediated by either type of adrenoceptor. On the other hand, there was an equal potentiation by COMT-inhibition of the lipolysis induced by arterial noradrenaline and by nerve stimulation. Thus, the importance of COMT in affecting the noradrenaline concentrations in adipose tissue seems to vary between the different adrenoceptors and depends on the mode of access to the adrenoceptors. Two main factors may govern the importance of COMT. First the sensitivity of the tissue for noradrenaline, i.e. the concentration needed for an effect, is of great importance (Trendelenburg et al., 1971). Thus, the relative importance of COMT may be smaller at high concentrations of noradrenaline since in the intact tissue COMT has been shown to be saturable with high

_+ 5.9 _+ 5.3 • 1.6 _+ 1.8

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