Modulation of cardiac β-adrenergic receptors by dopamine β-hydroxylase

186

Biochimica et Biophysica A cta, 1055 (1990) 186 - 188 Elsevier

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Modulation of cardiac fl-adrenergic receptors by dopamine fl-hydroxylase Pallab K. Ganguly, Shue-L. Lee and Girija Waghray Departments of Anatomy and Physiology, Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg (Canada) (Received 15 January 1990) (Revised manuscript received 16 June 1990)

Key words: fl-Adrenergic receptor; Dopamine fl-hydroxylase; (Rat)

Incubation of cardiac sarcolemma in the presence of dopamine fl-hydroxylase (DBH), a catecholamine biosynthetic enzyme, increased fl-adrenergic receptor density by 68% as measured by [3H]dihydroalprenolol (DHA) binding. The addition of DBH to plasma membranes isolated from brain, kidney, skeletal muscle, liver and intestine did not alter [3H]DHA binding. Cardiac a-receptors were unaffected under similar conditions. Since DBH is coreleased with norepinephrine, these results indicate that a functional coupling of the putative fl-adrenergic receptor with DBH may exist in cardiac muscle.

In addition to the classical physiologic responses that sympathetic stimulation is known to evoke in heart, this system also regulates and modulates the functional properties of its own receptors by releasing norepinephrine (NE) [1]. NE may increase or decrease the number of fl-adrenergic receptors, change their efficiency of coupling to biologic effectors, and even alter their structure by stimulating their covalent modification, as by phosphorylation [2-5]. Although important progress has been made in characterizing fl-adrenergic receptors on which NE acts, relatively little is known about different modulators which may be participating in the overall receptor-induced cardiac responses. Since it has now been well established that dopamine fl-hydroxylase (DBH), a catecholamine biosynthetic enzyme (EC 1.14.17.1), is secreted by the process of exocytosis from sympathetic nerve endings and coreleased with NE[6], the present report has examined the effects of DBH on fl-adrenergic receptor binding in cardiac tissue. Crude rat membranes from cardiac ventricles, skeletal muscle and intestine for identification of adrenergic receptors were prepared according to Bristow et al. [7]. Crude membranes from brain, liver and kidney were isolated by a similar procedure. Briefly, the tissues were washed, minced and then homogenized in 50 mM TrisHC1 (pH 7.4) (15 m l / g tissue) with a Polytron PT-20

Correspondence: P.K. Ganguly, Division of Cardiovascular sciences, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6.

(2 x 20 s, setting 5). The resulting homogenate was centrifuged at 40000 x g for 25 min. The resulting pellet was suspended and centrifuged again two times in the same buffer at the same speed. The final pellet was suspended in 10 mM Tris-HC1 (pH 7.4). It may be pointed out that 0.6 M KC1 was used during the isolation of crude membranes from heart, skeletal muscle and intestine to dissolve myofibrils. Purified cardiac sarcolemmal membrane was prepared according to Pitts [8]. The membrane was characterized by measuring Na+/K+-ATPase activity. To determine r-receptor binding, membranes were incubated with increasing concentrations of [aH]dihydroalprenolol (DHA) in a total volume of 0.5 ml for 30 min at 30 °C in the presence and absence of 10/~M propranolol, a-Adrenergic receptor binding was analyzed in a similar manner as above except the antagonist [aH]prazocin was used in the presence and absence of 10 /~M phentolamine hydrochloride. All incubations were terminated by rapid vacuum filtration through Whatman G F / B filters. Dopamine fl-hydroxylase was purchased from Sigma Chemical Co., St. Louis. To investigate the effects of DBH on fl-adrenergic receptors, crude membranes prepared from cardiac muscle were incubated with different concentrations of DBH (27.5, 55, 110, 220 and 440 nM) for 30 rain at 30 o C. These concentrations were selected on the basis of circulating concentrations during sympathetic nerve stimulation in vivo [9,10]. The specific binding of [3H]DHA was increased with increasing concentrations of DBH and the binding was saturated at 440 nM of

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187 TABLE I

TABLE III

Effects of dtfferent concentrations of dopamine fl-hydroxylase (DBH) on [3H]dihydroalprenolol binding (DHA) to cardiac crude membranes

Effects of dopamine fl-hydroxylase (DBH) on [3H]dihydroalprenolol binding (DHA) to purified cardiac sarcolemmal membrane

Values are means 5: S.E. of five experiments.

Values are means+ S.E. of four experiments. Specific binding of [3H]DHA to the cardiac sarcolemma in the absence (control) and presence of 110 nM DBH was plotted and evaluated by LIGAND programme. The range of [3H]DHA used in the binding assay was 0.25-14.4 nM, with the non-specific binding being defined by 10 #M propranolol. Concentrations of the membrane protein was 60/~g/ml. Incubation time was 30 rain at 30 ° C. The reaction was terminated by rapid filtration technique through GF/B filter. * P < 0.05.

Concentrations of DBH (nM)

[3H]DHA binding (fmol/mg)

27.5 55 110 220 440

14 5:3 29+3 665:6 78 5:4 815:5

D B H ( T a b l e I). I n the s u b s e q u e n t e x p e r i m e n t s , 110 n M o f D B H was used in studies for r e c e p t o r activation. T h e c o n t r o l e x p e r i m e n t s with d e n a t u r e d e n z y m e p r e p a r a t i o n d i d n o t increase [ 3 H ] D H A b i n d i n g . T a b l e II shows the effects of D B H on various m e m b r a n e s i s o l a t e d f r o m heart, brain, kidney, skeletal muscle, liver a n d i n t e s t i n e a n d the values are e x p r e s s e d as p e r c e n t control. U n l i k e h e a r t m e m b r a n e , [ 3 H ] D H A b i n d i n g was u n a l t e r e d b y the a d d i t i o n of D B H in all the tissues e x a m i n e d . T h e increase in r e c e p t o r d e n s i t y with c a r d i a c m e m b r a n e s a p p e a r s to b e specific since c a r d i a c a - r e c e p t o r s were u n c h a n g e d b y the a d d i t i o n of D B H in the i n c u b a t i o n m e d i u m (control, 90 + 4; D B H - m e m b r a n e , 88 ___ 6 f m o l / m g protein). Also, a n o n s p e c i f i c protein, a l b u m i n d i d n o t increase f l - r e c e p t o r n u m b e r . A significant increase in Bm~x value of fl-adrenergic r e c e p t o r s was evident w h e n p u r i f i e d c a r d i a c s a r c o l e m m a was inc u b a t e d with D B H ; however, these results were associa t e d with a decrease in the affinity for the r e c e p t o r s ( T a b l e III). I n o r d e r to u n d e r s t a n d if the effect of D B H was m e d i a t e d b y a c o v a l e n t m o d i f i c a t i o n of the receptors, m e m b r a n e s were i n c u b a t e d with D B H , w a s h e d two times with 50 m M Tris-HC1 ( p H 7.4) a n d r e c e p t o r s assayed. D B H d i d n o t increase the b i n d i n g of [ 3 H ] D H A

TABLE II Effects of dopamine fl-hydroxylase (DBH) on [3H]dihydroalprenolol binding (DH.4) to crude membranes of various tissues

Values are means+S.E, of four experiments. Specific binding of [3H]DHA to the crude membranes was calculated in the absence (control) and presence of 110 nM DBH. The specific binding of control membranes from heart, brain, kidney, skeletal muscle, liver and intestine at 7.5 nM [3H]DHA were 68 + 5, 145 + 6, 14 + 1, 18 + 2, 89 + 10 and 4 5:0.5 fmol/mg protein, respectively. * P < 0.05. [3H]DHA binding (% of control) Heart Brain Kidney Skeletal muscle Liver Intestine

156+ 10 * 85 + 3 102+ 5 95 -t- 7 117+ 5 90+ 6

Bmax (fmol/mg of protein)

K d (nM)

105 + 10 176 + 13 *

0.87 + 0.07 3.45 + 0.15 *

Control With DBH

i n d i c a t i n g that a close i n t e r a c t i o n b e t w e e n D B H a n d m e m b r a n e r e c e p t o r s is p e r h a p s necessary for D B H - i n d u c e d r e c e p t o r a c t i v a t i o n a n d it is u n l i k e l y that a covalent m o d i f i c a t i o n of the r e c e p t o r s is r e s p o n s i b l e for such efects. T o p r o v i d e a n insight into the f u n c t i o n a l implic a t i o n s for these b i o c h e m i c a l results, isolated rat hearts were p e r f u s e d a c c o r d i n g to the p r o c e d u r e of L a n g e n d o r f f [11] with D B H in the presence a n d a b s e n c e of i s o p r o t e r e n o l (0.01/~M). D B H p o t e n t i a t e d the effect of i s o p r o t e r e n o l a n d b o t h c a r d i a c c o n t a c t i l e force as well as r a t e of force d e v e l o p m e n t ( d F / d t ) were significantly i n c r e a s e d as c o m p a r e d to c o n t r o l hearts p e r f u s e d o n l y with 0.01 /LM i s o p r o t e r e n o l ( T a b l e IV). Since D B H is a large p r o t e i n , it is p o s s i b l e that a c o m p o n e n t m a y leave the c o r o n a r y c i r c u l a t i o n a n d b e c o m e s a s s o c i a t e d with the s a r c o l e m m a of the v e n t r i c u l a r myocytes. T h e p r e s e n t findings i n d i c a t e that in a d d i t i o n to acting as a s y n t h e t i c e n z y m e for N E [12], D B H m a y serve as a m o d u l a t o r of fl-adrenergic receptors in the c a r d i a c tissue. T h e p o s s i b l e f u n c t i o n a l role of D B H u n d e r p h y s i o l o g i c a l c o n d i t i o n s with i n t a c t n o r a d r e n e r g i c m e c h a n i s m s in the heart, however, r e m a i n s to b e established. N o n e t h e l e s s , the i n t e r a c t i o n s b e t w e e n fl-receptors a n d D B H m a y p r o v i d e i n f o r m a t i o n c o n c e r n i n g the

TABLE IV Cardiac contractile force and rate of force deoelopment (dF/dt) after perfusion with 54 nM dopamine fl- hydroxylase alone and in the presence of 10 nM isoproterenol

Values are means+ S.E. of five experiments. Hearts were initially equilibrated for 15 min in the absence of DBH to obtain basal values. DBH and isoproterenol were added later in the perfusion medium. * P < 0.05.

DBH Isoproterenol DBH + isoproterenol

Contractile force (% increase)

d F/d t (% increase)

50+6 59 + 2 * 68 + 4 *

47+5 65 + 7 * 83 + 5 *

188 possibility of involvement of ' s p a r e receptors' in cardiac tissue. At any rate, these results are unlikely to be due to an artifact associated with the experimental protocol employed in this study because (1) the effect of D B H was specific in cardiac muscle and was absent in other tissues such as brain, kidney, liver, skeletal muscle and intestine, (2) cardiac a-adrenergic receptors were unaffected by D B H and finally, (3) purified sarcolemmal membranes exhibited similar response. Since washing abolishes the effect of D B H , it is apparent that a close interaction between D B H and m e m b r a n e receptors is necessary for the observed increase in Brnax value. It m a y be pointed out that a considerable b o d y of evidence now suggests that circulating concentrations of D B H m a y serve as an index of the activity of the sympathetic nerves [11,12]. E n h a n c e d sympathetic activation which is often associated with heart failure m a y lead to down-regulation of/3-adrenergic receptors [7]. It is possible, therefore, that D B H u n d e r such circumstances m a y act as an i m p o r t a n t m o d u l a t o r potentiating the effect of N E on cardiac muscle. In fact, our preliminary experiments using L a n g e n d o r f f ' s preparation support this view. Structural h o m o l o g y between D B H and /~-adrenergic receptors [13], further indicates that D B H m a y act as an endogenous positive inotropic agent. This study was supported b y a grant from the M a n i t o b a Heart and Stroke Foundation. P.K. G a n g u l y

is a Scholar of the Heart Canada.

and Stroke Foundation,

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