J Mol Cell Cardiol
24, 535-548
(1992)
Chronic &Blockade Transregulates Inhibitory Muscarinic M, Receptors of the Adenylyl R. Marquetant, Department of Cardiology, (Received 9 April
B. Brehm,
A, Adenosine Cyclase System
and
and R. H. Strasser
University of Heidelberg, Heidelberg, Germany
1991, accepted in revisedform 20 December 1991)
R. MARQUETANT, B. BREHM, R. H. STRASSER. Chronic &Blockade Transregulates Inhibitory A, Adenosine and Muscarinic M, Receptors of the Adenylyl Cyclase System. Journal of A4olecular and Cellular Cardiolou (1992) 24, 535-548. Chronic P-blockade has evolved to an important therapeutic strategy in medicine. Not all its therapeutic effects may be explained by its direct action on the fl-adrenergic system. We therefore investigated if chronic P-blockade in uioo or in isolated cell systems may modulate also inhibitory receptors of the adenylyl cyrlase system. Chronic treatment with metoprolol for 6 days (10 mg/day) induced an increase of P-adrenergic receptors in rat cardiac plasma membranes (53 f 8 vs 80 f 12 fmol/mg protein). Simultaneously the density of cardiac muscarinic M, receptors decreased significantly from 150 f 17 to 110 * 12 fmol/mg protein without any change of the affinity of the receptors for their agonists or antagonists. By this mechanism chronic P-blockade leads to an unexpected impairment of the muscarinic-mediated inhibition of the adenylyl cyclase. This transregulation of inhibitory receptors by chronic P-blockade was not restricted to the heart but also reduced the muscarinic receptors of rat lung (35 f 4 vs 24 f 3 fmol/mg protein). Additionally, other inhibitory receptors of the adenylyl cyclase system such as the A, adenosine receptors of rat brain were reduced by chronic P-blockade (532 i 32 vs 444 f 26 fmol/mg protein). This transregulation of A, adenosine receptors occurred only after chronic P-blockade with the active stereoisomer ( - )-metoprolol whereas the (+)-isomer was ineffective. The ability of the remaining A, adenosine receptors to form the agonist-promoted high affinity state was unaltered. Their reduction, however, was sufficient to abolish the phenylisopropyl-mediated inhibition of the adenylyl cyclase. To evaluate if this regulation of various inhibitory receptors in different organs may represent a general rellular regulation mechanism, we investigated whether this transregulation also occurred in isolated cells. Isolated smooth muscle cells derived from the vas deferens (DDT, MF-2) were cultivated in the presence of the P-blocker atenolol (10m5M) for 3 days. Chronic P-blockade in these isolated cells induced an increase of fl-adrenergic receptors and concomitantly a significant decrease of A, adenosine receptors (460 + 42 vs 368 ? 18 fmol/mg protein). The affinity of the A, adenosine receptors for their agonists and antagonists and the ability of the remaining receptors to form the agonist-promoted high affinity state remained unaltered. In contrast, the reduction of receptor density greatly impaired the adenosine-mediated inhibition of the adenylyl cyclase. These data demonstrate that chronic P-blockade leads to a functionally significant reduction of inhibitory receptors of the adenylyl cyclase system. This newly characterized transregulation of inhibitory receptors of the adenylyl cyclase system by chronic P-blockade represents a new regulation mechanism which involves several inhibitory receptor systems and occurs at the cellular level. This transregulation may explain some of the rhrrapeutic effects of the little understood chronic P-blockade. KEY WORDS: P-blockade;
Adenylyl cyclase; fl-adrenergic Heart; Smooth muscle cells;
receptors; Interaction;
Introduction P-blockade has evolved into one of the fundamental therapeutic strategies in many areas of medicine. As its main action, it is directed to one of the important signal transduction pathways, the adenylyl cyclase system. The activity of the adenylyl cyclase system
muscarinic Transregulation.
+ 14 $03.00/0
A, adenosine
receptors;
Chronic
is dually regulated by stimulation on one side and by inhibition on the other side [I]. The stimulatory pathway comprises stimulatory receptors such as P-adrenergic receptors and the stimulatory guanine nucleotide binding regulatory proteins, G,, which mediate hormonal activation to the adenylyl cyclase (2,
Part of this study was presented in abstract form in the American Please address all correspondence to: Dr. R. H. Strasser, University Cardiology. Bergheimerstr. 58, 6900 Heidelberg, Germany. 0022.2828/92/050535
receptors;
Heart Meeting of Heidelberg,
1988 and 1989. Medical Center,
0 1992 Academic
Department
Press
LImited
of
536
R. Marquetant
31. The inhibitory pathway includes inhibitory receptors such as muscarinic M2 receptors or the A, adenosine receptors which, when activated by their individual hormones, stimulate an inhibitory guanine nucleotide binding protein, Gi, to inhibit the adenylyl cyclase [I]. Moreover, the responsiveness of the adenylyl cyclase system to stimulation or inhibition respectively, is dynamically regulated. Thus, activation of individual receptors is intimately linked to their inactivation and their desensitization [4, 51. Prolonged activation of /3-adrenergic receptors leads to their desensitization [S, 7] with a decreased responsiveness upon further stimulation. Such agonist-promoted desensitization includes internalization and functional uncoupling of the receptors from the G protein. Similarly, chronic activation of inhibitory receptors, such as the muscarinic M2 or A, adenosine receptors induces a decreased responsiveness to their specific hormones [8, 91. Chronic /3blockade, on the contrary results in an increase of /3-adrenergic receptors at the cell surface, contributing to an increased sensitivity of the fl-adrenergic system to stimulation after P-blocker withdrawal [ 10, 111. To date only the isolated regulation and desensitization of individual receptor systems by their specific hormones has been well characterized. An as yet unidentified regulation of antagonistic receptor systems of the adenylyl cyclase system could further modulate the responsiveness of the adenylyl cyclase system. It is conceivable that some of the effects of chronic @-blockade might be mediated by a regulation of the inhibitory pathway of the adenylyl cyclase system. We here report that chronic P-blockade leads to a concomitant gradual and slow reversible decrease of two types of receptors which are inhibitory to the adenylyl cyclase, namely the muscarinic M, and the A, adenosine receptors. This transregulation is not restricted to the heart but was also observed in the lung and brain. Even in isoiated cells, i.e. in the absence of any reflex activities, this transregulation could be demonstrated, indicating that this newly characterized regulation process occurs at the cellular level. The reduction of inhibitory receptors has functional significance and results in the loss
et al.
of hormone-induced inhibition of the adenylyi cyclase. Thus, this transregulation of inhibitory receptors during chronic P-blockade may need to be considered for therapeutic effect with chronic P-blockade.
Materials
and
Methods
Materials [3H]DPCPX was purchased from Amersham, Braunschweig. All other radiolabelled chemicals were purchased from NEN, Dreieich. Unlabelled 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the subtype specific A, adenosine receptor antagonist [12], was from RPI, Switzerland. Guanylimidodiphosphate [Gpp(NH)p] and R-N,-phenylisopropyl adenosine (R-PIA) were purchased from Boehringer Mannheim. AF-DX 116 (11 2-(diethylamino)methyl-1-piperidinyl acethyl5, ll-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepine-6-one), the selective muscarinic M, receptor antagonist [13], was a generous gift from Thomae, Germany. Dithiothreitol (DTT) was bought from Serva, Heidelberg. Atenolol was kindly provided by ICI, Macclesfield, UK. All other chemicals were purchased from Sigma, Munich.
Animals
and cells
Male rats (body weight: 195 f 6 g) were injected daily with 10mg metoprolol in saline (0.5 ml) or 1 mg ( -)-metoprolol or 1 mg (+)-metoprolol (as indicated) for 6 days. Controls were injected with saline alone. In deep anaesthesia (Thiobarbital, 50 mg/kg, i.p.) heart, lung and brain were rapidly excised, frozen in liquid nitrogen and stored at - 80% until further use. Isolated smooth muscle cells derived from the vas deferens of hamster (DDT,, MF-2) were cultivated in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum [46].
Membrane preparation For the preparation of cardiac plasma membranes, hearts were thawed in 40 volumes of buffer A (50mM Tris-HCl, 5mM EGTA, 2 mM EGTA, pH 7.2, at 4°C) and homo-
Tramregulation
of Adenylyl
genized (Polytron, Brinkmann 10,000 U/min, 3 x 10 s). After sedimentation (35Og, 10 min, 4’C) the supernatant was filtered through two layers of cheese cloth. After sedimentation (48 000 g, 10 min, 4’C) the pelleted membranes were washed twice in 40ml buffer A, by centrifugation (48 000 g, 10 min, 4’C). The final membranes were resuspended in 50mM Tris-HCl, pH 7.6, 4’C to give a final concentration of ~0.2-0.4 mg protein/ml. In additional experiments the homogenate was defined as the supernatant of the 350 g centrifugation for 5 min at 4’C. Light vesicles were isolated by ultracentrifugation (100 000 g, 1 h, 4°C) of the first high speed supernatant. These light vesicles were resuspended in TrisHCl and stored in liquid nitrogen until further use.
The identical plasma ation was used to prepare from lung and brain.
Radioligand
membrane preparplasma membranes
binding
fl-adrenergic receptors were determined by radioligand binding using the radiolabelled /3antagonist [‘2”l]iodocyanopindolol ([‘251]CYP) [ 141. For saturation isotherms, increasing concentrations of the radioligand (15-480 PM) in 75mM Tris-HCl, 12.5mM MgCl,, pH7.5 were incubated (1 h, 3O’C) with plasma membranes (= 20-40 pg protein/tube). The incubation was stopped by rapid vacuum filtration on Whatman GF/C filters. The filters were washed (3 x 4m1, 50mM Tris-HCl, pH 7.5) and counted with a counting efficiency of 80 % . Non-specific binding was determined as the residual binding in the presence of 10e5M alprenolol and ranged between 5 and 20%. Muscarinic receptors of heart and lung were determined using the tritiated muscarinic antagonist N-methyl-[3H]scopolamine ([3H]NMS) as radioligand. In 6 to 8 point saturation curves, increasing concentrations of the radioligand [‘H]NMS (0.15-4.0 nM) were incubated (30°C, 1 h) with plasma membranes (= 30-60 pg protein/tube). The incubation was terminated by rapid vacuum filtration on Whatman GF/C filters. The filters were washed (3 x 4m1, 50mM Tris-HCl, pH 7.5, 4°C) and were counted using Aquassure (NEN) scintillation cocktail in a scintilla-
Cyclase-coupled
Receptors
537
tion counter with an efficiency of 50 % Nonspecific binding was determined in the presence of 10 - 5 M atropine and ranged between 10 and 20%. To determine the A, adenosine receptors, the A, adenosine specific radioligand [3H]-8-cyclo-pentyl-1,3-dipropylxanthine (L3H] DPCPX) was used [12]. To remove endogenous adenosine the plasma membranes were pre-incubated (15 min, 37’C) with adenosine deaminase (2.5 U/ml plasma membranes). The plasma membranes (= 20-40 kg protein/tube) were then incubated with increasing concentrations of [‘HIDPCPX (0.1-3.OnM) in 50mM Tris-HCl, pH7.5 for 1 h at 3O’C. The incubation was terminated by rapid vacuum filtration on Whatman GF/C filters. The washed filters (3 x 4m1, 50mM Tris-HCl, pH7.5, 4’C) were counted in Aquassure (NEN) scintillation cocktail with an efficiency of 50%. Non-specific binding was determined as the residual binding in the presence of lo-” M phenylisopropyladenosine (R-PIA, 10. 5 M). Non-specific binding ranged between 5 and 30%. Competition experiments were performed at a radioligand concentration of 3OOpM [‘HIDPCPX in the absence or presence of lo-“M guanvlimidodiphosphate (Gpp(NH)p) with increasing concentrations of phenylisopropyladenosine (RPIA) as indicated.
Adenylyl cyclase activity Adenylyl cyclase activity was determined using [a”P]ATP (=0.2pCi/tube) as substrate in a final incubation volume of lOO$ (final concentrations: 75 mM Tris-HCl, 6mM MgCl,, 1 mM DTT, 1 mM Na-EDTA, IOpM GTP, 0.1 mM ATP. 100~~ CAMP, 20mM phosphoenolpyruvat, 0.041 mg creatine kinase, z 50-100 pg plasma membranes). For stimulation, forskolin at a concentration of 2 x 10m5 M was added [15]. For inhibition of the adenylyl cyclase the muscarinic agonist oxotremorine (10 I” M- 10 ’ M) or the adenosine agonist R-PIA (lo-‘” ~-10 ‘M) was added to the incubation mixture. The incubation (10 min, 37’C) was stopped according to the method of Jakobs et al. [16]. CAMP was separated using alumina columns (0.5 x 1 cm) and counted using Cerenkov counting.
538
R. Marquetant
et al.
Protein determination
Results
Protein was measured by the method of Bradford [17j using bovine serum albumin as standard.
Effect of chronic &blocker therapy on muscarinic receptors in heart
Data analysis Saturation curves were analyzed by computer assisted techniques using non-linear, least square curve fitting techniques based on the law of mass action [ 18, 191. Statistical comparisons was performed using the Analysis of Variance and Student’s Newman Keuls test for significance.
Heart rate To determine the effect of P-blockade on heart rate, a three lead electrocardiogram (ECG) was used. The ECG was recorded before /3blockade and 1 h prior to sacrifice.
Free
[‘H]
Chronic P-blockade using metoprolol for 6 days (10 mg/day) leads to a significant increase of P-adrenergic receptors in cardiac plasma membranes (53 + 8 vs 80 f 12 fmol/mg protein) without any change in their affinity for the P-antagonist [‘251]iodocyanopindolol. As an internal control the /3-blockade-induced reduction in heart rate was documented (420 + 23 vs 340 + 35 beats/min). Concomitantly, chronic P-blockade caused a significant decrease in the density of muscarinic M2 receptors (150 f 17 vs 110 + 12 fmol/mg protein, PSO.02) without any change of the affinity of the muscarinic receptors for their antagonist [3H]N-methyl-scopolamine (K, = 338 + 48 vs 336 f 80 PM, Fig. 1). The density of muscarinic M, receptors in the intracellular pool of light vesicles was unaltered after
N-methyl-scopolamine
(PM)
FIGURE 1, Reduction of muscarinic M, receptors in the rat heart after chronic P-blocker treatment. Rats were treated with metoprolol (10 mg/day, i.p.) for 6 days. In isolated cardiac plasma membranes of the controls or treated animals the density of muscarinic receptors was determined using the radiolabelled muscarinic antagonist Nmethyl-[3H]scopolamine in saturation isotherms. Non-specific binding was determined by the residual binding in the presence of high concentrations of atropine ( 10e5M) and amounted 5-20% of total binding. Shown is the average of six sets of experiments using least square curve fitting techniques based on the mass law action (see Materials and Methods), The affinities of the receptors for their radioligand was similar in both groups (K,:338 i 48pM vs 336*80 PM).
Transregulation
of Adenylyl
chronic P-blockade treatment (data not shown). The whole homogenate, however, revealed the identical decrease of total cellular receptors which was also observed in the isolated plasma membrane preparation (data not shown). These data indicate that chronic /3blockade leads to a decrease of muscarinic M, receptors which is not due to an internalization of the receptors but to a decrease of total cellular receptors. To test the functional significance of the flblocker-mediated reduction of cardiac muscarinic M2 receptors after chronic /3blockade the inhibition of the adenylyl cyclase in response to muscarinic agonists was determined in cardiac plasma membranes before
Control
Treoted
FIGURE 2. Oxotremorine-mediated inhibition of the adenylyl cyclase activity. In cardiac plasma membranes of untreated or chronically p-blocked animals (metoprolol 10 mg/day for 6 days) maximally stimulated adenylyl cyclase activity using forskolin (2 x 10 - ‘M) and its inhibition by the muscarinic agonist oxotremorine (10 - ‘M) was investigated. Shown are the means + s E.M. of three sets of experiments with triplicate determinations at each point. Only in the membranes of untreated controls oxotremorine induced a significant (P 50.02) inhibition of adenylyl cyclase.
Cyclase-coupled
Receptors
539
and after chronic P-blockade (Fig. 2). In the controls, activation of muscarinic receptors by the muscarinic agonist oxotremorine leads to the expected dose-dependent inhibition of forskolin-stimulated adenylyl cyclase (Fig. 2). This inhibition reached a maximal extent of about 20% to 25% (Fig. 2). The subtype specific antagonist for muscarinic M, receptors, AF-DX 116 [13] was able to abolish completely the oxotremorine-mediated inhibition of cardiac adenylyl cyclase (data not shown) indicating that this inhibition is a muscarinic M2-mediated effect. After chronic treatment with the @-adrenergic antagonist metoprolol, however, the inhibition of forskolin-stimulated adenylyl cyclase activity by the muscarinic agonist oxotremorine was greatly abolished (Fig. 2). Even high concentrations of the muscarinic agonist oxotremorine only lead to a minimal inhibition of the adenylyl cyclase activity. These data indicate that chronic P-blockade induces a reduction of the inhibitory muscarinic M, receptors of’ the heart with functional significance.
Efect of chronic P-blocker therapy on muscarinic receptors in lung To address the question of whether transregulation of inhibitory receptors after @blockade may be restricted to cardiac receptors, we also determined the density of the muscarinic M2 receptors in the plasma membranes of rat lung before and after chronic treatment with metoprolol. As shown in Figure 3, the total density of muscarinic receptors in rat lung was lower than in rat heart in accordance with previously published data [20]. It could be demonstrated that these receptors are predominantly of the MT subtype (data not shown). With chronic P-blockade the number of the muscarinic M, receptors in lung decreased significantly by about 30% (35 f 4 vs 24 f 3 fmol/mg protein). The affinity of the muscarinic receptors for their antagonist N-methyl-[3H]scopolamine remained unaltered. These data demonstrate that transregulation of muscarinic receptors by chronic P-blockade is not restricted to the heart but also involves the muscarinic receptor of other organs, in this case of rat lung.
540
R. Marquetant
200
et al.
400 Free
[%I]
600
N-methyl-scopolamine
FIGURE 3. Reduction of muscarinic M, receptors chronically treated rats (compare methods and Fig. radiolabelled muscarinic antagonist N-methyl-[3H]scopolamine experiments. Non-specific binding was determined in sets of experiments with duplicate determinations at niques based on the mass action law. The affinity for
600
1000
(PM)
in lung after chronic P-blocker treatment. In the controls or 1) the density of muscarinic receptors was determined using the with increasing concentration (abscissa) in saturation the presence of atropine (10 - 5 M). Shown are the means of three each point and the best fits using least square curve fitting techthe radioligand (KD) were 194 It 25 pM (0) YS 239 f 43 pM (‘).
Effect of chronic /3-blocker therapy on A, adenosine receptors in brain To assess if such transregulation may include other inhibitory receptor systems of the adenylyl cyclase systems, we investigated even outside the cardiopulmonary system the Ai adenosine receptors derived from rat brain. The radiolabelled highly subtypeselective antagonist for Ai adenosine receptors [3H]DPCPX was used to determine the A, adenosine receptors in rat brain. [3H]DPCPX binds to the A1 adenosine receptors of plasma membranes from brain in a saturable fashion with low non-specific binding (Fig. 4). As shown in Figure 4, chronic treatment with the P-antagonist (-)-metoprolol (1 mg/d for 6 of the days) in vivo, leads to a reduction inhibitory A, adenosine receptors (532 f 32 to 444 + 26 fmol/mg protein). The affinity for the adenosine antagonist [3H]DPCPX or the adenosine agonist PIA remained unaltered
(Figs 4 and 5). Only the active stereoisomer (-)-metoprolol was able to induce this reduction of A, adenosine receptors in rat brain. Chronic treatment with the inactive isomer (+)-metoprolol failed to induce an increase of fl-adrenergic receptors in heart or brain (data not shown) or to induce the transregulation of the A, adenosine receptors in rat brain (Fig. 4). To assess the ability of the remaining A, adenosine receptors to bind their agonist with high affinity, indicating their coupling to the inhibitory G protein, Gi, agonist competition curves in the absence or in the presence of the non-hydrolysable guanine nucleotide Gpp(NH)p were determined. As shown in Figure 5, in membranes from control animals the R-PIA competition curve in the absence of the GTP-analogue is shallow and biphasic. The “high affinity state” is formed by 61 k 8% of the receptors, with a dissociation constant of KDH = 1.7 x 10ey M [Fig. 5(a)]. In
Transregulation
‘; .Q) z b 0 \E 5 E z
of Adenylyl
Cyclase-coupled
541
Receptors
-----c-P..
550-
(a)
bxxy-+
with Gpp(NH)p (Io-4h4)
Q
:~ 500-
ii 2 D y !!L
100 80 60
IO
9
8 PIA [-log
7
6
5
4
M]
FIGURE 4. Effect of chronic P-blocker treatment on the density of cerebral A, adenosine receptors. Plasma membranes were isolated from the brains of rats treated with saline (control), with the active isomer (-)-metopro101 (1 mg/day, i.p., 6 days) or with the inactive isomer ( +)-metoprolol (1 mg/day, i.p., 6 days). The density of A, adenosine receptors was determined in saturation isotherms using the radiolabelled A, adenosine selective antagonist [3H]DPCPX (see Materials and Methods). Shown are the maximal specific binding sites of four sets of experiments as determined in full saturation isotherms. The affinities for the radiolabelled antagonist (K,,) are indicated and were not significantly different. Only treatment with the active isomer ( -)-metoprolol induced a significant (P 50.02) reduction of A, adenosine receptors.
FIGURE 5. Functional coupling of A, adenosine receptors in rat brain membranes before and after chronic P-blockade. Purified rat brain membranes derived from control and treated animals (see Materials and Methods) were incubated with increasing concentrations of the adenosine agonist R-PIA at indicated concentrations (see abscissa) to compete for the binding to the A, adenosine receptor site with the radiolabelled antagonist (“300pM) in the absence or in the presence [3H]DPCPX of the stable GTP analogue Gpp(NH)p (10 -“M). The data shown are the means of three sets of experiments with duplicate determinations at each point. The lines through the data points represent the computer-derived fit utilizing non-linear least square curve fitting techniques based on the law of mass action. (a)Control: (b)Metoprolol.
the presence of the non-hydrolyzable GTP analogue Gpp(NH)p the agonist competition curve is steeper and shifted to the right. Curve analysis based on the mass law action revealed only a single side of receptors with an overall low affinity of KDL = 8.4 x 10 - 8 M for the agonist. After chronic P-blockade (lower panel, Fig. 5) the relative amount of receptors capable of forming the high affinity state (RH = 44 Z!Z3 % ) was unchanged. The affinity of KDH = 2.8 x 10eg M remained unaltered. The affinities of the agonist were also unchanged (Table 1). These data indicate that chronic P-blockade reduces the density of the
inhibitory A, adenosine receptors but it does not influence their coupling to the inhibitory G protein. To assess the functional significance of this reduction of A, adenosine receptors, the adenosine-mediated inhibition of forskolinstimulated adenylyl cyclase was determined. In the controls the forskolin-stimulated adenylyl cyclase activity was inhibited by the adenosine agonist PIA in a dose-dependent manner (Fig. 6). Maximal inhibition reached about 20 to 25% of the maximally stimulated adenylyl cyclase activity. This inhibition of adenylyl cyclase could be completely blocked by the A,
542 TABLE
1.
Influence of chronic agonist-competition adenosine receptors DDT,
MF-2
Rat
1.7 x 10-g 8.4 x 1O-8 61 +8 39* 7
KDL[MI:
RH(%): RL(%): (b)
DDT,
KDH KDL
RH(%): RL(%):
on A, and
100 --- __i ..__ i.___ j-.Trea+ed 1 1 ‘.. ---_.. :?---+T 90
After chronic P-blockade
Control
1
brain
WI:
KDH
P-blockade curves for in rat brain
cells
Control (a)
et al.
R. Marquetant
WI: PI:
MF-2
2.8 9.5 44 It 56 +
x lo-’ x 10-a 3 4
.c
cells 1.9
x 10-g
1.9
x lo-’
56 f 6 44 f 7
1.5
x 10-g
8.8 x 10-8 49 f 4 51 k7
Plasma membranes were derived from rat brain (a) or from isolated smooth muscle cells (DDT, MF-2) before (Control) or after chronic P-blocker treatment using metoprolol or atenolol, respective (see Materials and Methods). Increasing concentrations of the adenosine agonist R-PIA competed with the radiolabelled A, adenosine-specific antagonist [3H]DPCPX (-300 PM) for the binding to the A, adenosine receptor in the presence or absence of the stable GTP analogne Gpp(NH)p. The affinities and the relative amount of receptors (RH) capable of binding the agonist with high affinity (Kurt) was determined using least square curve fitting techniques based on the mass law action. The relative amount of A, adenosine receptors capable of binding the adenosine agonist PIA with high affinity (RH) was not significantly different in controls and treated animals or cells.
adenosine receptor subtype specific antagonist DPCPX (data not shown), indicating that the PIA-induced inhibition of adenylyl cyclase is an A, adenosine receptor-mediated effect. After chronic P-blocker treatment, however, the PIA-mediated inhibition of the adenylyl cyclase was greatly impaired. Maximal PIAinduced inhibition only reached a total of about 7 % . These data indicate that chronic @blockade leads to a functional transregulation of the inhibitory A, adenosine receptors with an almost complete loss of their inhibitory action. In separate experiments it could be demonstrated that the total amount of A, adenosine receptors in the whole homogenate, which includes the plasma membrane fractions and intracellular fractions, was decreased to the same extent as observed in the plasma membrane fractions (data not shown). The number of A, adenosine receptors in the so-
3 e IT
t
m---m
Control Treated
70
Old IO
9
8
7 PIA [-log
6
5
4
M]
FIGURE 6. Phenylisopropyl-mediated inhibition of adenylyl cyclase in rat brain membranes. Plasma membranes were prepared from control and P-blocker treated animals and adenylyl cyclase activity was determined as described in Materials and Methods. The data represent the mean * S.E.M. of four experiments with triplicate determinations at each point. PIA-mediated inhibition of adenylyl cyclase activity in treated rats was significantly reduced (P10.02).
called “light vesicle fraction” was unchanged (data not shown). These data demonstrate that with chronic P-blockade the total amount of cellular adenosine receptors, but not their cellular distribution, had changed.
Efect of chronic P-blocker therapy on Al adenosine receptors in isolated smooth muscle cells Since this transregulation of inhibitory receptors after chronic P-blockade could be demonstrated with two different inhibitory receptor systems in three different organs, we supposed that it may represent a general regulation mechanism of adenylyl cyclasecoupled receptors. To address the question if such general transregulation may occur at the cellular level, where complex reflex activities could be excluded with certainty, we studied the effect of chronic P-blockade on A1 adenosine receptors of isolated smooth muscle cells. The total density of A, adenosine receptors was comparable to the published observations [21, 221. Smooth muscle cells derived from the vas deferens of hamster (DDTr MF-2 cells)
Transregulation
of Adenylyl
Cyclase-coupled
r
Co
At
54:J
Receptors
(b)
1
At
FIGURE 7. Reduction of A, adenosine receptors in isolated smooth muscle cells before and after chronic P-blocker treatment. Cells were grown as described in Materials and Methods in the absence or presence of the P-blocker atenolol (10 - 5 M) for 3 days. (a) In plasma membranes of the controls or treated cells the density of P-adrenergic receptors was determined using the radiolabelled fl-adrenergic antagonist [ lz51 ] iodocyanopindolol in saturation isotherms. Nonspecific binding was determined as the residual binding in the presence of alprenolol (10m5~). Shown are the means + s E M. of SIX experiments. (b)The maximal number of A, adenosine receptors was evaluated with the A, adenosine antagonist [3H]DPCPX m six point saturation isotherms. Non-specific binding was determined in the prrsence of R-PIA (10 ~’ M). Shown are the means * S E Mof six experiments. The atenolol i:.&ced decrease In A, adenosine receptors l l (P 50.02) was significant.
kept in permanent tissue culture, were grown in the presence of atenolol(l0 - 5 M) for 3 days. As expected, the density of /3-adrenergic receptors increased significantly (95 f 9 vs 139 f 13 fmol/mg protein). Concomitantly, the inhibitory A, adenosine receptors decreased significantly by 20 to 25% (Fig. 7). The affinity of the A, adenosine receptors for their antagonists remained unaltered. The flblocker-induced changes of P-adrenergic and A, adenosine receptors were identical in cells which were grown in the absence of serum or catecholamines. The catecholamine levels were below the detection limit of the radioenzymatic assay. The ability of the remaining A, adenosine receptors to form the so-called agonist-promoted “high affinity state” of the receptors remained unchanged after chronic treatment
with metoprolol (Fig. 8). The affinity of the receptors for their agonist PIA remained unaltered [Table l(b)]. These data indicate that P-blocker treatment reduced the density of A, adenosine receptors but did not influence the ability of the residual receptors to couple IO the inhibitory Gi protein. The A, adenosinemediated inhibition was greatly abolished (45 % vs 13 % , Fig. 9). These data suggested that the reduction in receptor density was sufficient to greatly impair functionality. Moreover, these data demonstrate that functional transregulation of inhibitory receptors by chronic P-blockade occurs at the cellular level even in the absence of any reflex activity or endogenous catecholamines. This transregulation modulates total receptor number at the cellular level, but not the ability of the remaining receptors to couple to G protein.
R. Marquetant
544
et al.
40 t IO
9
8
7
PIA (-log
0 -10
-9
-8
-7
PIA (log
-6
-5
-4
MI
FIGURE 8. Functional integrity of A, adenosine receptors in isolated smooth muscle cells before and after chronic P-blockade. Purified DDT, MF-2 plasma membranes derived from control and P-blocker treated cells (see Materials and Methods) were incubated with increasing concentrations of the adenosine agonist R-PIA at indicated concentrations (see abscissa) to compete for the binding site to the A adenosine receptor with the radiolabelled antagonist [ k ]DPCPX (=3OOpM) in the absence or in the presence of the stable GTP analogue Gpp(NH)p (10-4M). The data shown are the means of three sets of experiments with duplicate determinations at each point. The lines through the data points represent the computerderived fit utilizing non-linear least square curve fitting techniques based on the law of mass action. (a)Control; (b) Treated.
Discussion The salient finding of the present study is that inactivation or blockade of a stimulatory receptor of the adenylyl cyclase system, i.e. blockade of the P-adrenergic receptor, is capable of transregulating inhibitory receptors of the adenylyl cyclase system, such as muscarinic M2 and A, adenosine receptors. This newly characterized transregulation has been shown to occur in vivo in different organs, i.e. heart, lung and brain. It could even be demonstrated that this transregulation is a new regulation mechanism which takes place
6
5
4
MI
FIGURE 9. Adenosine-mediated inhibition of adenylyl cyclase activity in DDT, MF-2 membranes. Membranes derived from control and P-blocker treated cells (atenolol, 10 5 M, 3 days) were prepared and adenylyl cyclase activity was determined as described in Materials and Methods. The data represent the means f S.E.M. of three experiments with triplicate determinations at each point. PIA-mediated inhibition of adenylyl cyclase activity in treated cells was significantly reduced after chronic P-blocker treatment (P50.01).
at the cellular level where complex reflex activities can be excluded with certainty. Furthermore, we demonstrated that this transregulation has functional significance. Thus chronic P-blockade leads to an attenuation of receptor-mediated inhibition of the stimulated adenylyl cyclase activity. Chronic P-blockade activation of muscarinic M, receptors or of A, adenosine receptors by their agonists fails to inhibit the forskolin-stimulated adenylyl cyclase. Whereas the initial studies were performed with high doses of metoprolol, we also could demonstrate that this transregulation is mediated stereospecifically by therapeutic @-blockade using different &blockers. To our knowledge this is the first description of an in vivo transregulation of two inhibitory receptor systems of the adenylyl cyclase system after chronic P-blockade and its characterization as a regulation mechanism at the cellular level. A single observation in diseased human heart suggested a decrease of muscarinic receptors after variable chronic p-
Transregulation
of Adenylyl
blockade [23]. While acute interaction of these two pathways with respect to the modulation of the acute cellular response has been well characterized [24], the chronic regulation of the density of receptors and their functional activity by the opposing receptor system was a somewhat surprising result. For enzyme regulation, however, it is well known that opposing modulators may be subjected to a co-ordinate regulation. Such transregulation may be of importance for P-blocker therapy. Thus several effects of chronic P-blocker treatment may be mediated in part by such slow transregulation of antagonistic receptor systems. In the present paper we have characterized the functional importance of this transregulation only for the inhibition of adenylyl cyclase. At present it has not been shown if muscarinic receptors other than the M, subtype may be modulated by this transregulation process. In rat heart and lung predominantly muscarinic receptors of the M, subtype are present [13, 25, 261 and mediate the carbachol and oxotremorine induced inhibition of adenylyl cyclase [27-291. These inhibitory receptors, however, may also couple to other signal transduction pathways [29]. Muscarinic M2 and A1 adenosine receptors, for example, are known to modulate several potassium channels [30, 311. This would imply that chronic P-blockade may act via this transregulation on these channels. Possibly a direct interaction of the inhibitory G protein with the ion channel may be involved in this regulation. This action could explain some of the anti-arrhythmic effects of chronic P-blockade. Other receptors which are inhibitory to the adenylyl cyclase such as a*-adrenergic receptors have been shown to activate the Na + /H + exchanger or to modulate platelet activation [32-341. Such transregulatory action of chronic P-blockade may explain some of the little understood therapeutic effects of P-blocker therapy. These include chronic antihypertensive or some of the anti-arrhythmic effects. However, in the present study the inhibitory effects on adenylyl cyclase activity of these receptor systems have been tested exclusively. On the other hand, abrupt withdrawal of chronic P-blocker treatment has been shown to lead to the so-called P-blocker withdrawal syndrome [IO, U]. The P-blocker withdrawal
Cyclase-coupled
Receptors
545
syndrome is characterized by an increased sensitivity of the adrenergic system to sympathetic stimulation. It could be speculated that the P-blocker-induced transregulation of inhibitory receptors of the adenylyl cyclase may further potentiate such sensitization of the adrenergic system. Further studies should be carried out to evaluate this possibility. The molecular mechanism of such transregulation of adenylyl cyclase-coupled receptors is not yet known. We could, however, demonstrate that transregulation is a cellular regulation mechanism which occurs both in vim, where complex reflex activity cannot be excluded, and to the same extent in isolated cells in a well defined in vitro system. Moreover, we could show that this transregulation does not involve the cellular distribution of receptors but it modulates the total density or expression of cellular receptors. These data demonstrate that internalization of receptors, which is observed in agonist-promoted desensitization processes, is not responsible for this transregulation process. Agonist-promoted desensitization, with the rapid decrease of receptors in the plasma membranes, has been characterized for both inhibitory receptors of the adenylyl cyclase system such as muscarinic M2 and A, adenosine receptors [35, 361 and stimulatory receptors such as the P-adrenergic receptors [371. Moreover, after transregulation, the remaining receptors, both muscarinic M2 and adenosine A,, are fully capable of forming the agonist-promoted high affinity state, indicating their ability to couple functionally to the G protein. Thus, uncoupling of receptors from the G protein commonly observed during early agonist-promoted desensitization of the activated receptors is not involved in the transregulation process. Functional uncoupling of receptors from one of the inhibitory G proteins has been attributed to phosphorylation of these receptors, possibly by specific kinases such as the fladrenergic receptor kinase [ 7, 38, 391. Such covalent modification of the remaining receptors seems to not be involved in the transregulation process, since functional coupling of the remaining receptors, as indicated by the formation of the agonist-promoted high affinity state of the receptors, seems to be unaltered. The reduction of receptor density was shown to be sufficient to greatly impair
546
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inhibition of adenylyl cyclase. These data suggest that transregulation of inhibitory receptors by chronic P-blockade is a process which is distinct from agonist-promoted desensitization of these receptors. 1 Also, in contrast to desensitization, which after activation of receptors occurs rapidly within minutes, the transregulation of inhibitory receptors is observed only after chronic flblockade. Several hours of P-blockade of isolated but rapidly growing cells, or even more than 1 day of in uivo administration of /3blockers, are not sufficient to promote this transregulation. This slow time course linked to the observation that the total cellular receptor density is modulated by this transregulation process suggests that processing of receptors, i.e. synthesis or degradation, may be involved in transregulation. Further studies will have to focus on this hypothesis. Previous studies have suggested that the @blocker withdrawal may only be due to a displacement of endogenous catecholamines from the @-adrenergic receptor [IO, II]. However, as shown here, both the increase of fiadrenergic receptors and the transregulation of A, adenosine receptors on isolated smooth muscle cells under chronic P-blockade occurred even in the absence of serum or catecholamines. These data indicate that the displacement of endogenous catecholamines may not be involved in this transregulation process of inhibitory receptors. They clearly demonstrate that the “empty” receptor may mediate a different signal to the cell than the occupied but blocked receptor. The mechanism responsible for this can only be speculated on. The question of whether modulation of adenylyl cyclase and the “second messenger” CAMP or coupling to the G protein is involved in this P-blocker-mediated transregulation process of inhibitory receptors, has not yet been clarified. In vivo P-blocker treatment could reduce endogenous CAMP levels. However, in isolated unstimulated smooth muscle cells, CAMP levels are extremely low, especi-
et al.
ally when grown in the absence of catecholamines, i.e. serum. It is not known if fiblocker treatment could further reduce these already low levels which range in the low detection limit of the radio-immuno assay. Therefore, it appears unlikely that CAMP levels may mediate this transregulation process. Further studies will have to focus on this question. Cross-regulation at the level of G proteins by chronic agonist treatment using P-agonists, [40] and A, adenosine agonists [U, 421 has been described recently. The intracellular signal which mediates this has been attributed to CAMP levels [40, 431. Moreover, crossregulation at the G protein level seems to involve regulation of protein synthesis. However, in these studies chronic P-blockade seems not to greatly influence inhibitory Gproteins and thus not modulate the inhibitory receptor system primarily at the G protein level. These data indicate that the regulation at the level of G proteins may be mediated by different mechanisms than the transregulation at the level of receptors. All components of the adenylyl cyclase system are cloned and sequenced. The high homology suggests that they are all individual members of a large family of similar proteins. All receptors which couple to the adenylyl cyclase are glycoproteins with seven membrane spanning regions [44]. Despite their distinct chromosomal representation [45] their similar promoter regions may be subjected to a coordinate regulation which may be the basis for this newly characterized transregulation of adenylyl cyclase coupled receptors after chronic P-blocker treatment. Acknowledgements We wish to thank U. Oehl for technical assistance and K. Schiile for help in the preparation of the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn. RHS is supported by the Hermannand Lilly-Schilling-Foundation.
References 1 GILMAN, A. G. G proteins and regulation of adenylyl cyclase. JAMA 262, 1819-1825 (1989). 2 LEFKOWITZ, R. J., CARON, M. G., STILES, G. L. Mechanism of membrane-receptor regulation. Biochemical, physiological, and clinical insights derived from the studies of the adrenergic receptors. N Engl J Med 310, 1570-1579 (1984).
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Cyclase-coupled
Receptors
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3 SI‘ILES, G. L., CARON, M. G., LEFKOWITZ, R. J. Beta-adrenergic receptors: Biochemical mechanisms [,f physiological regulation. Pharmacol Rev 64, 661-743 (1984). 4 SIRLE’I’. D. R., LEFKOWITZ, R. J. Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature 317, 124-129 (1985). 5 HARDEN, T. K. Agonist-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase Pharmacd Rev 35, 5-32 (1983). 6 STRASSER. R. H. Phosphorylation of the beta-adrenergic receptor: Mechanisms of desensitization. [n &r+n, Pho@@ation. V. K. Moudgil (ed.) pp. 199-226 Boca Raton, Florida: CRC Press (1989). 7 LEFKOWITZ, R. J., HAUSDORFF, W. P., CARON, M. G. Role of phosphorylation in desensitization of the @-adrent)ceptor. Trends Pharmacol Sci 11, 190-194 (1990). 8 KWATRA, M. M., HOSEY, M. M. Phosphorylation of the cardiac muscarinic receptor m intact chick heart and Its regulation by a muscarinic agonist. J Biol Chem 261, 12429-12432 (1986). 9 STILES. G. L. Adenosine receptors and beyond: Molecular mechanisms of physiological regulation, Chn Res 38. lo-18 (1990). IO FISHMAZ, W. H. Beta-adrenergic blocker withdrawal. Am J Cardiol 59. 26F-32F (1987). 11 PRICHARD, B. N.C., TOMLINSON, B., WALDEN, R.J., BHATTA-CHARJEF., P. The 6-adrenergic blockade xithdrawal phenomenon. J Cardiovasc Pharmacol 5, S56-S62 (1983). 12 LOHS~, M. J., KLOTZ, K-N., LINDENRORN-FOTINOS, J., REDDINGTON, M., SCHWAHE. U., OL.S~)N. R. A 8-Cyclopentyl-1.3-dipropylxanthine (DPCPX) - a selective high affinity antagonist radioligand for .4, adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 336, 204-210 (1987). 13 GI~CHETTI. A., MICHELETTI, R., MONTAGNA, E. Cardioselective profile of AF-DX116. a muscarmic M, receptor antagonist. Life Sci 38, 166331672 (1986). 14 ENCEL.. G., HOYER. D., BERTHOLD, R., WAGNER, H. [“51]Iodocyanopindolol. a new ligand for beta-adrenoceptors: Identification and quantitation of subclasses of beta-adrenocrptors in guinea pig. Naunyn-Srhmirdeberg’s Arch Pharmacol 317, 277-285 (1981). 15 SEAMOK, K.B., PADGET-. W., DALY, J.W. Forskoiin: Unique diterpene activator of adenylatt: tvrlasc in membranes and intact cells. Proc Nat1 Acad Sci USA 78, 3363-3367 (1981). 16 ,JAKOBS, K. H.. SAUR, W., SCHULTZ. G. Reduction of adenylate cyclase activity in lysates of human platelets by the cY-adrenergic component of epinephrine. J Cycl Nucl Res 2, 381-392 (1976). 17 BRADFORI). M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72, 248-254 (1976). 18 DE LEAIV, A., MUNSON, P. J., RODBARD, D. Simultaneous analysis of families of sigmoidal curves: Appltcation to bioassay and physiological dose-response curves. Am J Physiol 235. E978-El02 (1978). 19 DE LEAN, A., STADEL, J. M., LEFKOWITZ, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor, J Biol Chem 255, 7108-7117 (1980). 20 S
24, 535-548
(1992)
Chronic &Blockade Transregulates Inhibitory Muscarinic M, Receptors of the Adenylyl R. Marquetant, Department of Cardiology, (Received 9 April
B. Brehm,
A, Adenosine Cyclase System
and
and R. H. Strasser
University of Heidelberg, Heidelberg, Germany
1991, accepted in revisedform 20 December 1991)
R. MARQUETANT, B. BREHM, R. H. STRASSER. Chronic &Blockade Transregulates Inhibitory A, Adenosine and Muscarinic M, Receptors of the Adenylyl Cyclase System. Journal of A4olecular and Cellular Cardiolou (1992) 24, 535-548. Chronic P-blockade has evolved to an important therapeutic strategy in medicine. Not all its therapeutic effects may be explained by its direct action on the fl-adrenergic system. We therefore investigated if chronic P-blockade in uioo or in isolated cell systems may modulate also inhibitory receptors of the adenylyl cyrlase system. Chronic treatment with metoprolol for 6 days (10 mg/day) induced an increase of P-adrenergic receptors in rat cardiac plasma membranes (53 f 8 vs 80 f 12 fmol/mg protein). Simultaneously the density of cardiac muscarinic M, receptors decreased significantly from 150 f 17 to 110 * 12 fmol/mg protein without any change of the affinity of the receptors for their agonists or antagonists. By this mechanism chronic P-blockade leads to an unexpected impairment of the muscarinic-mediated inhibition of the adenylyl cyclase. This transregulation of inhibitory receptors by chronic P-blockade was not restricted to the heart but also reduced the muscarinic receptors of rat lung (35 f 4 vs 24 f 3 fmol/mg protein). Additionally, other inhibitory receptors of the adenylyl cyclase system such as the A, adenosine receptors of rat brain were reduced by chronic P-blockade (532 i 32 vs 444 f 26 fmol/mg protein). This transregulation of A, adenosine receptors occurred only after chronic P-blockade with the active stereoisomer ( - )-metoprolol whereas the (+)-isomer was ineffective. The ability of the remaining A, adenosine receptors to form the agonist-promoted high affinity state was unaltered. Their reduction, however, was sufficient to abolish the phenylisopropyl-mediated inhibition of the adenylyl cyclase. To evaluate if this regulation of various inhibitory receptors in different organs may represent a general rellular regulation mechanism, we investigated whether this transregulation also occurred in isolated cells. Isolated smooth muscle cells derived from the vas deferens (DDT, MF-2) were cultivated in the presence of the P-blocker atenolol (10m5M) for 3 days. Chronic P-blockade in these isolated cells induced an increase of fl-adrenergic receptors and concomitantly a significant decrease of A, adenosine receptors (460 + 42 vs 368 ? 18 fmol/mg protein). The affinity of the A, adenosine receptors for their agonists and antagonists and the ability of the remaining receptors to form the agonist-promoted high affinity state remained unaltered. In contrast, the reduction of receptor density greatly impaired the adenosine-mediated inhibition of the adenylyl cyclase. These data demonstrate that chronic P-blockade leads to a functionally significant reduction of inhibitory receptors of the adenylyl cyclase system. This newly characterized transregulation of inhibitory receptors of the adenylyl cyclase system by chronic P-blockade represents a new regulation mechanism which involves several inhibitory receptor systems and occurs at the cellular level. This transregulation may explain some of the rhrrapeutic effects of the little understood chronic P-blockade. KEY WORDS: P-blockade;
Adenylyl cyclase; fl-adrenergic Heart; Smooth muscle cells;
receptors; Interaction;
Introduction P-blockade has evolved into one of the fundamental therapeutic strategies in many areas of medicine. As its main action, it is directed to one of the important signal transduction pathways, the adenylyl cyclase system. The activity of the adenylyl cyclase system
muscarinic Transregulation.
+ 14 $03.00/0
A, adenosine
receptors;
Chronic
is dually regulated by stimulation on one side and by inhibition on the other side [I]. The stimulatory pathway comprises stimulatory receptors such as P-adrenergic receptors and the stimulatory guanine nucleotide binding regulatory proteins, G,, which mediate hormonal activation to the adenylyl cyclase (2,
Part of this study was presented in abstract form in the American Please address all correspondence to: Dr. R. H. Strasser, University Cardiology. Bergheimerstr. 58, 6900 Heidelberg, Germany. 0022.2828/92/050535
receptors;
Heart Meeting of Heidelberg,
1988 and 1989. Medical Center,
0 1992 Academic
Department
Press
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of
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31. The inhibitory pathway includes inhibitory receptors such as muscarinic M2 receptors or the A, adenosine receptors which, when activated by their individual hormones, stimulate an inhibitory guanine nucleotide binding protein, Gi, to inhibit the adenylyl cyclase [I]. Moreover, the responsiveness of the adenylyl cyclase system to stimulation or inhibition respectively, is dynamically regulated. Thus, activation of individual receptors is intimately linked to their inactivation and their desensitization [4, 51. Prolonged activation of /3-adrenergic receptors leads to their desensitization [S, 7] with a decreased responsiveness upon further stimulation. Such agonist-promoted desensitization includes internalization and functional uncoupling of the receptors from the G protein. Similarly, chronic activation of inhibitory receptors, such as the muscarinic M2 or A, adenosine receptors induces a decreased responsiveness to their specific hormones [8, 91. Chronic /3blockade, on the contrary results in an increase of /3-adrenergic receptors at the cell surface, contributing to an increased sensitivity of the fl-adrenergic system to stimulation after P-blocker withdrawal [ 10, 111. To date only the isolated regulation and desensitization of individual receptor systems by their specific hormones has been well characterized. An as yet unidentified regulation of antagonistic receptor systems of the adenylyl cyclase system could further modulate the responsiveness of the adenylyl cyclase system. It is conceivable that some of the effects of chronic @-blockade might be mediated by a regulation of the inhibitory pathway of the adenylyl cyclase system. We here report that chronic P-blockade leads to a concomitant gradual and slow reversible decrease of two types of receptors which are inhibitory to the adenylyl cyclase, namely the muscarinic M, and the A, adenosine receptors. This transregulation is not restricted to the heart but was also observed in the lung and brain. Even in isoiated cells, i.e. in the absence of any reflex activities, this transregulation could be demonstrated, indicating that this newly characterized regulation process occurs at the cellular level. The reduction of inhibitory receptors has functional significance and results in the loss
et al.
of hormone-induced inhibition of the adenylyi cyclase. Thus, this transregulation of inhibitory receptors during chronic P-blockade may need to be considered for therapeutic effect with chronic P-blockade.
Materials
and
Methods
Materials [3H]DPCPX was purchased from Amersham, Braunschweig. All other radiolabelled chemicals were purchased from NEN, Dreieich. Unlabelled 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), the subtype specific A, adenosine receptor antagonist [12], was from RPI, Switzerland. Guanylimidodiphosphate [Gpp(NH)p] and R-N,-phenylisopropyl adenosine (R-PIA) were purchased from Boehringer Mannheim. AF-DX 116 (11 2-(diethylamino)methyl-1-piperidinyl acethyl5, ll-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepine-6-one), the selective muscarinic M, receptor antagonist [13], was a generous gift from Thomae, Germany. Dithiothreitol (DTT) was bought from Serva, Heidelberg. Atenolol was kindly provided by ICI, Macclesfield, UK. All other chemicals were purchased from Sigma, Munich.
Animals
and cells
Male rats (body weight: 195 f 6 g) were injected daily with 10mg metoprolol in saline (0.5 ml) or 1 mg ( -)-metoprolol or 1 mg (+)-metoprolol (as indicated) for 6 days. Controls were injected with saline alone. In deep anaesthesia (Thiobarbital, 50 mg/kg, i.p.) heart, lung and brain were rapidly excised, frozen in liquid nitrogen and stored at - 80% until further use. Isolated smooth muscle cells derived from the vas deferens of hamster (DDT,, MF-2) were cultivated in Dulbecco’s Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum [46].
Membrane preparation For the preparation of cardiac plasma membranes, hearts were thawed in 40 volumes of buffer A (50mM Tris-HCl, 5mM EGTA, 2 mM EGTA, pH 7.2, at 4°C) and homo-
Tramregulation
of Adenylyl
genized (Polytron, Brinkmann 10,000 U/min, 3 x 10 s). After sedimentation (35Og, 10 min, 4’C) the supernatant was filtered through two layers of cheese cloth. After sedimentation (48 000 g, 10 min, 4’C) the pelleted membranes were washed twice in 40ml buffer A, by centrifugation (48 000 g, 10 min, 4’C). The final membranes were resuspended in 50mM Tris-HCl, pH 7.6, 4’C to give a final concentration of ~0.2-0.4 mg protein/ml. In additional experiments the homogenate was defined as the supernatant of the 350 g centrifugation for 5 min at 4’C. Light vesicles were isolated by ultracentrifugation (100 000 g, 1 h, 4°C) of the first high speed supernatant. These light vesicles were resuspended in TrisHCl and stored in liquid nitrogen until further use.
The identical plasma ation was used to prepare from lung and brain.
Radioligand
membrane preparplasma membranes
binding
fl-adrenergic receptors were determined by radioligand binding using the radiolabelled /3antagonist [‘2”l]iodocyanopindolol ([‘251]CYP) [ 141. For saturation isotherms, increasing concentrations of the radioligand (15-480 PM) in 75mM Tris-HCl, 12.5mM MgCl,, pH7.5 were incubated (1 h, 3O’C) with plasma membranes (= 20-40 pg protein/tube). The incubation was stopped by rapid vacuum filtration on Whatman GF/C filters. The filters were washed (3 x 4m1, 50mM Tris-HCl, pH 7.5) and counted with a counting efficiency of 80 % . Non-specific binding was determined as the residual binding in the presence of 10e5M alprenolol and ranged between 5 and 20%. Muscarinic receptors of heart and lung were determined using the tritiated muscarinic antagonist N-methyl-[3H]scopolamine ([3H]NMS) as radioligand. In 6 to 8 point saturation curves, increasing concentrations of the radioligand [‘H]NMS (0.15-4.0 nM) were incubated (30°C, 1 h) with plasma membranes (= 30-60 pg protein/tube). The incubation was terminated by rapid vacuum filtration on Whatman GF/C filters. The filters were washed (3 x 4m1, 50mM Tris-HCl, pH 7.5, 4°C) and were counted using Aquassure (NEN) scintillation cocktail in a scintilla-
Cyclase-coupled
Receptors
537
tion counter with an efficiency of 50 % Nonspecific binding was determined in the presence of 10 - 5 M atropine and ranged between 10 and 20%. To determine the A, adenosine receptors, the A, adenosine specific radioligand [3H]-8-cyclo-pentyl-1,3-dipropylxanthine (L3H] DPCPX) was used [12]. To remove endogenous adenosine the plasma membranes were pre-incubated (15 min, 37’C) with adenosine deaminase (2.5 U/ml plasma membranes). The plasma membranes (= 20-40 kg protein/tube) were then incubated with increasing concentrations of [‘HIDPCPX (0.1-3.OnM) in 50mM Tris-HCl, pH7.5 for 1 h at 3O’C. The incubation was terminated by rapid vacuum filtration on Whatman GF/C filters. The washed filters (3 x 4m1, 50mM Tris-HCl, pH7.5, 4’C) were counted in Aquassure (NEN) scintillation cocktail with an efficiency of 50%. Non-specific binding was determined as the residual binding in the presence of lo-” M phenylisopropyladenosine (R-PIA, 10. 5 M). Non-specific binding ranged between 5 and 30%. Competition experiments were performed at a radioligand concentration of 3OOpM [‘HIDPCPX in the absence or presence of lo-“M guanvlimidodiphosphate (Gpp(NH)p) with increasing concentrations of phenylisopropyladenosine (RPIA) as indicated.
Adenylyl cyclase activity Adenylyl cyclase activity was determined using [a”P]ATP (=0.2pCi/tube) as substrate in a final incubation volume of lOO$ (final concentrations: 75 mM Tris-HCl, 6mM MgCl,, 1 mM DTT, 1 mM Na-EDTA, IOpM GTP, 0.1 mM ATP. 100~~ CAMP, 20mM phosphoenolpyruvat, 0.041 mg creatine kinase, z 50-100 pg plasma membranes). For stimulation, forskolin at a concentration of 2 x 10m5 M was added [15]. For inhibition of the adenylyl cyclase the muscarinic agonist oxotremorine (10 I” M- 10 ’ M) or the adenosine agonist R-PIA (lo-‘” ~-10 ‘M) was added to the incubation mixture. The incubation (10 min, 37’C) was stopped according to the method of Jakobs et al. [16]. CAMP was separated using alumina columns (0.5 x 1 cm) and counted using Cerenkov counting.
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et al.
Protein determination
Results
Protein was measured by the method of Bradford [17j using bovine serum albumin as standard.
Effect of chronic &blocker therapy on muscarinic receptors in heart
Data analysis Saturation curves were analyzed by computer assisted techniques using non-linear, least square curve fitting techniques based on the law of mass action [ 18, 191. Statistical comparisons was performed using the Analysis of Variance and Student’s Newman Keuls test for significance.
Heart rate To determine the effect of P-blockade on heart rate, a three lead electrocardiogram (ECG) was used. The ECG was recorded before /3blockade and 1 h prior to sacrifice.
Free
[‘H]
Chronic P-blockade using metoprolol for 6 days (10 mg/day) leads to a significant increase of P-adrenergic receptors in cardiac plasma membranes (53 + 8 vs 80 f 12 fmol/mg protein) without any change in their affinity for the P-antagonist [‘251]iodocyanopindolol. As an internal control the /3-blockade-induced reduction in heart rate was documented (420 + 23 vs 340 + 35 beats/min). Concomitantly, chronic P-blockade caused a significant decrease in the density of muscarinic M2 receptors (150 f 17 vs 110 + 12 fmol/mg protein, PSO.02) without any change of the affinity of the muscarinic receptors for their antagonist [3H]N-methyl-scopolamine (K, = 338 + 48 vs 336 f 80 PM, Fig. 1). The density of muscarinic M, receptors in the intracellular pool of light vesicles was unaltered after
N-methyl-scopolamine
(PM)
FIGURE 1, Reduction of muscarinic M, receptors in the rat heart after chronic P-blocker treatment. Rats were treated with metoprolol (10 mg/day, i.p.) for 6 days. In isolated cardiac plasma membranes of the controls or treated animals the density of muscarinic receptors was determined using the radiolabelled muscarinic antagonist Nmethyl-[3H]scopolamine in saturation isotherms. Non-specific binding was determined by the residual binding in the presence of high concentrations of atropine ( 10e5M) and amounted 5-20% of total binding. Shown is the average of six sets of experiments using least square curve fitting techniques based on the mass law action (see Materials and Methods), The affinities of the receptors for their radioligand was similar in both groups (K,:338 i 48pM vs 336*80 PM).
Transregulation
of Adenylyl
chronic P-blockade treatment (data not shown). The whole homogenate, however, revealed the identical decrease of total cellular receptors which was also observed in the isolated plasma membrane preparation (data not shown). These data indicate that chronic /3blockade leads to a decrease of muscarinic M, receptors which is not due to an internalization of the receptors but to a decrease of total cellular receptors. To test the functional significance of the flblocker-mediated reduction of cardiac muscarinic M2 receptors after chronic /3blockade the inhibition of the adenylyl cyclase in response to muscarinic agonists was determined in cardiac plasma membranes before
Control
Treoted
FIGURE 2. Oxotremorine-mediated inhibition of the adenylyl cyclase activity. In cardiac plasma membranes of untreated or chronically p-blocked animals (metoprolol 10 mg/day for 6 days) maximally stimulated adenylyl cyclase activity using forskolin (2 x 10 - ‘M) and its inhibition by the muscarinic agonist oxotremorine (10 - ‘M) was investigated. Shown are the means + s E.M. of three sets of experiments with triplicate determinations at each point. Only in the membranes of untreated controls oxotremorine induced a significant (P 50.02) inhibition of adenylyl cyclase.
Cyclase-coupled
Receptors
539
and after chronic P-blockade (Fig. 2). In the controls, activation of muscarinic receptors by the muscarinic agonist oxotremorine leads to the expected dose-dependent inhibition of forskolin-stimulated adenylyl cyclase (Fig. 2). This inhibition reached a maximal extent of about 20% to 25% (Fig. 2). The subtype specific antagonist for muscarinic M, receptors, AF-DX 116 [13] was able to abolish completely the oxotremorine-mediated inhibition of cardiac adenylyl cyclase (data not shown) indicating that this inhibition is a muscarinic M2-mediated effect. After chronic treatment with the @-adrenergic antagonist metoprolol, however, the inhibition of forskolin-stimulated adenylyl cyclase activity by the muscarinic agonist oxotremorine was greatly abolished (Fig. 2). Even high concentrations of the muscarinic agonist oxotremorine only lead to a minimal inhibition of the adenylyl cyclase activity. These data indicate that chronic P-blockade induces a reduction of the inhibitory muscarinic M, receptors of’ the heart with functional significance.
Efect of chronic P-blocker therapy on muscarinic receptors in lung To address the question of whether transregulation of inhibitory receptors after @blockade may be restricted to cardiac receptors, we also determined the density of the muscarinic M2 receptors in the plasma membranes of rat lung before and after chronic treatment with metoprolol. As shown in Figure 3, the total density of muscarinic receptors in rat lung was lower than in rat heart in accordance with previously published data [20]. It could be demonstrated that these receptors are predominantly of the MT subtype (data not shown). With chronic P-blockade the number of the muscarinic M, receptors in lung decreased significantly by about 30% (35 f 4 vs 24 f 3 fmol/mg protein). The affinity of the muscarinic receptors for their antagonist N-methyl-[3H]scopolamine remained unaltered. These data demonstrate that transregulation of muscarinic receptors by chronic P-blockade is not restricted to the heart but also involves the muscarinic receptor of other organs, in this case of rat lung.
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R. Marquetant
200
et al.
400 Free
[%I]
600
N-methyl-scopolamine
FIGURE 3. Reduction of muscarinic M, receptors chronically treated rats (compare methods and Fig. radiolabelled muscarinic antagonist N-methyl-[3H]scopolamine experiments. Non-specific binding was determined in sets of experiments with duplicate determinations at niques based on the mass action law. The affinity for
600
1000
(PM)
in lung after chronic P-blocker treatment. In the controls or 1) the density of muscarinic receptors was determined using the with increasing concentration (abscissa) in saturation the presence of atropine (10 - 5 M). Shown are the means of three each point and the best fits using least square curve fitting techthe radioligand (KD) were 194 It 25 pM (0) YS 239 f 43 pM (‘).
Effect of chronic /3-blocker therapy on A, adenosine receptors in brain To assess if such transregulation may include other inhibitory receptor systems of the adenylyl cyclase systems, we investigated even outside the cardiopulmonary system the Ai adenosine receptors derived from rat brain. The radiolabelled highly subtypeselective antagonist for Ai adenosine receptors [3H]DPCPX was used to determine the A, adenosine receptors in rat brain. [3H]DPCPX binds to the A1 adenosine receptors of plasma membranes from brain in a saturable fashion with low non-specific binding (Fig. 4). As shown in Figure 4, chronic treatment with the P-antagonist (-)-metoprolol (1 mg/d for 6 of the days) in vivo, leads to a reduction inhibitory A, adenosine receptors (532 f 32 to 444 + 26 fmol/mg protein). The affinity for the adenosine antagonist [3H]DPCPX or the adenosine agonist PIA remained unaltered
(Figs 4 and 5). Only the active stereoisomer (-)-metoprolol was able to induce this reduction of A, adenosine receptors in rat brain. Chronic treatment with the inactive isomer (+)-metoprolol failed to induce an increase of fl-adrenergic receptors in heart or brain (data not shown) or to induce the transregulation of the A, adenosine receptors in rat brain (Fig. 4). To assess the ability of the remaining A, adenosine receptors to bind their agonist with high affinity, indicating their coupling to the inhibitory G protein, Gi, agonist competition curves in the absence or in the presence of the non-hydrolysable guanine nucleotide Gpp(NH)p were determined. As shown in Figure 5, in membranes from control animals the R-PIA competition curve in the absence of the GTP-analogue is shallow and biphasic. The “high affinity state” is formed by 61 k 8% of the receptors, with a dissociation constant of KDH = 1.7 x 10ey M [Fig. 5(a)]. In
Transregulation
‘; .Q) z b 0 \E 5 E z
of Adenylyl
Cyclase-coupled
541
Receptors
-----c-P..
550-
(a)
bxxy-+
with Gpp(NH)p (Io-4h4)
Q
:~ 500-
ii 2 D y !!L
100 80 60
IO
9
8 PIA [-log
7
6
5
4
M]
FIGURE 4. Effect of chronic P-blocker treatment on the density of cerebral A, adenosine receptors. Plasma membranes were isolated from the brains of rats treated with saline (control), with the active isomer (-)-metopro101 (1 mg/day, i.p., 6 days) or with the inactive isomer ( +)-metoprolol (1 mg/day, i.p., 6 days). The density of A, adenosine receptors was determined in saturation isotherms using the radiolabelled A, adenosine selective antagonist [3H]DPCPX (see Materials and Methods). Shown are the maximal specific binding sites of four sets of experiments as determined in full saturation isotherms. The affinities for the radiolabelled antagonist (K,,) are indicated and were not significantly different. Only treatment with the active isomer ( -)-metoprolol induced a significant (P 50.02) reduction of A, adenosine receptors.
FIGURE 5. Functional coupling of A, adenosine receptors in rat brain membranes before and after chronic P-blockade. Purified rat brain membranes derived from control and treated animals (see Materials and Methods) were incubated with increasing concentrations of the adenosine agonist R-PIA at indicated concentrations (see abscissa) to compete for the binding to the A, adenosine receptor site with the radiolabelled antagonist (“300pM) in the absence or in the presence [3H]DPCPX of the stable GTP analogue Gpp(NH)p (10 -“M). The data shown are the means of three sets of experiments with duplicate determinations at each point. The lines through the data points represent the computer-derived fit utilizing non-linear least square curve fitting techniques based on the law of mass action. (a)Control: (b)Metoprolol.
the presence of the non-hydrolyzable GTP analogue Gpp(NH)p the agonist competition curve is steeper and shifted to the right. Curve analysis based on the mass law action revealed only a single side of receptors with an overall low affinity of KDL = 8.4 x 10 - 8 M for the agonist. After chronic P-blockade (lower panel, Fig. 5) the relative amount of receptors capable of forming the high affinity state (RH = 44 Z!Z3 % ) was unchanged. The affinity of KDH = 2.8 x 10eg M remained unaltered. The affinities of the agonist were also unchanged (Table 1). These data indicate that chronic P-blockade reduces the density of the
inhibitory A, adenosine receptors but it does not influence their coupling to the inhibitory G protein. To assess the functional significance of this reduction of A, adenosine receptors, the adenosine-mediated inhibition of forskolinstimulated adenylyl cyclase was determined. In the controls the forskolin-stimulated adenylyl cyclase activity was inhibited by the adenosine agonist PIA in a dose-dependent manner (Fig. 6). Maximal inhibition reached about 20 to 25% of the maximally stimulated adenylyl cyclase activity. This inhibition of adenylyl cyclase could be completely blocked by the A,
542 TABLE
1.
Influence of chronic agonist-competition adenosine receptors DDT,
MF-2
Rat
1.7 x 10-g 8.4 x 1O-8 61 +8 39* 7
KDL[MI:
RH(%): RL(%): (b)
DDT,
KDH KDL
RH(%): RL(%):
on A, and
100 --- __i ..__ i.___ j-.Trea+ed 1 1 ‘.. ---_.. :?---+T 90
After chronic P-blockade
Control
1
brain
WI:
KDH
P-blockade curves for in rat brain
cells
Control (a)
et al.
R. Marquetant
WI: PI:
MF-2
2.8 9.5 44 It 56 +
x lo-’ x 10-a 3 4
.c
cells 1.9
x 10-g
1.9
x lo-’
56 f 6 44 f 7
1.5
x 10-g
8.8 x 10-8 49 f 4 51 k7
Plasma membranes were derived from rat brain (a) or from isolated smooth muscle cells (DDT, MF-2) before (Control) or after chronic P-blocker treatment using metoprolol or atenolol, respective (see Materials and Methods). Increasing concentrations of the adenosine agonist R-PIA competed with the radiolabelled A, adenosine-specific antagonist [3H]DPCPX (-300 PM) for the binding to the A, adenosine receptor in the presence or absence of the stable GTP analogne Gpp(NH)p. The affinities and the relative amount of receptors (RH) capable of binding the agonist with high affinity (Kurt) was determined using least square curve fitting techniques based on the mass law action. The relative amount of A, adenosine receptors capable of binding the adenosine agonist PIA with high affinity (RH) was not significantly different in controls and treated animals or cells.
adenosine receptor subtype specific antagonist DPCPX (data not shown), indicating that the PIA-induced inhibition of adenylyl cyclase is an A, adenosine receptor-mediated effect. After chronic P-blocker treatment, however, the PIA-mediated inhibition of the adenylyl cyclase was greatly impaired. Maximal PIAinduced inhibition only reached a total of about 7 % . These data indicate that chronic @blockade leads to a functional transregulation of the inhibitory A, adenosine receptors with an almost complete loss of their inhibitory action. In separate experiments it could be demonstrated that the total amount of A, adenosine receptors in the whole homogenate, which includes the plasma membrane fractions and intracellular fractions, was decreased to the same extent as observed in the plasma membrane fractions (data not shown). The number of A, adenosine receptors in the so-
3 e IT
t
m---m
Control Treated
70
Old IO
9
8
7 PIA [-log
6
5
4
M]
FIGURE 6. Phenylisopropyl-mediated inhibition of adenylyl cyclase in rat brain membranes. Plasma membranes were prepared from control and P-blocker treated animals and adenylyl cyclase activity was determined as described in Materials and Methods. The data represent the mean * S.E.M. of four experiments with triplicate determinations at each point. PIA-mediated inhibition of adenylyl cyclase activity in treated rats was significantly reduced (P10.02).
called “light vesicle fraction” was unchanged (data not shown). These data demonstrate that with chronic P-blockade the total amount of cellular adenosine receptors, but not their cellular distribution, had changed.
Efect of chronic P-blocker therapy on Al adenosine receptors in isolated smooth muscle cells Since this transregulation of inhibitory receptors after chronic P-blockade could be demonstrated with two different inhibitory receptor systems in three different organs, we supposed that it may represent a general regulation mechanism of adenylyl cyclasecoupled receptors. To address the question if such general transregulation may occur at the cellular level, where complex reflex activities could be excluded with certainty, we studied the effect of chronic P-blockade on A1 adenosine receptors of isolated smooth muscle cells. The total density of A, adenosine receptors was comparable to the published observations [21, 221. Smooth muscle cells derived from the vas deferens of hamster (DDTr MF-2 cells)
Transregulation
of Adenylyl
Cyclase-coupled
r
Co
At
54:J
Receptors
(b)
1
At
FIGURE 7. Reduction of A, adenosine receptors in isolated smooth muscle cells before and after chronic P-blocker treatment. Cells were grown as described in Materials and Methods in the absence or presence of the P-blocker atenolol (10 - 5 M) for 3 days. (a) In plasma membranes of the controls or treated cells the density of P-adrenergic receptors was determined using the radiolabelled fl-adrenergic antagonist [ lz51 ] iodocyanopindolol in saturation isotherms. Nonspecific binding was determined as the residual binding in the presence of alprenolol (10m5~). Shown are the means + s E M. of SIX experiments. (b)The maximal number of A, adenosine receptors was evaluated with the A, adenosine antagonist [3H]DPCPX m six point saturation isotherms. Non-specific binding was determined in the prrsence of R-PIA (10 ~’ M). Shown are the means * S E Mof six experiments. The atenolol i:.&ced decrease In A, adenosine receptors l l (P 50.02) was significant.
kept in permanent tissue culture, were grown in the presence of atenolol(l0 - 5 M) for 3 days. As expected, the density of /3-adrenergic receptors increased significantly (95 f 9 vs 139 f 13 fmol/mg protein). Concomitantly, the inhibitory A, adenosine receptors decreased significantly by 20 to 25% (Fig. 7). The affinity of the A, adenosine receptors for their antagonists remained unaltered. The flblocker-induced changes of P-adrenergic and A, adenosine receptors were identical in cells which were grown in the absence of serum or catecholamines. The catecholamine levels were below the detection limit of the radioenzymatic assay. The ability of the remaining A, adenosine receptors to form the so-called agonist-promoted “high affinity state” of the receptors remained unchanged after chronic treatment
with metoprolol (Fig. 8). The affinity of the receptors for their agonist PIA remained unaltered [Table l(b)]. These data indicate that P-blocker treatment reduced the density of A, adenosine receptors but did not influence the ability of the residual receptors to couple IO the inhibitory Gi protein. The A, adenosinemediated inhibition was greatly abolished (45 % vs 13 % , Fig. 9). These data suggested that the reduction in receptor density was sufficient to greatly impair functionality. Moreover, these data demonstrate that functional transregulation of inhibitory receptors by chronic P-blockade occurs at the cellular level even in the absence of any reflex activity or endogenous catecholamines. This transregulation modulates total receptor number at the cellular level, but not the ability of the remaining receptors to couple to G protein.
R. Marquetant
544
et al.
40 t IO
9
8
7
PIA (-log
0 -10
-9
-8
-7
PIA (log
-6
-5
-4
MI
FIGURE 8. Functional integrity of A, adenosine receptors in isolated smooth muscle cells before and after chronic P-blockade. Purified DDT, MF-2 plasma membranes derived from control and P-blocker treated cells (see Materials and Methods) were incubated with increasing concentrations of the adenosine agonist R-PIA at indicated concentrations (see abscissa) to compete for the binding site to the A adenosine receptor with the radiolabelled antagonist [ k ]DPCPX (=3OOpM) in the absence or in the presence of the stable GTP analogue Gpp(NH)p (10-4M). The data shown are the means of three sets of experiments with duplicate determinations at each point. The lines through the data points represent the computerderived fit utilizing non-linear least square curve fitting techniques based on the law of mass action. (a)Control; (b) Treated.
Discussion The salient finding of the present study is that inactivation or blockade of a stimulatory receptor of the adenylyl cyclase system, i.e. blockade of the P-adrenergic receptor, is capable of transregulating inhibitory receptors of the adenylyl cyclase system, such as muscarinic M2 and A, adenosine receptors. This newly characterized transregulation has been shown to occur in vivo in different organs, i.e. heart, lung and brain. It could even be demonstrated that this transregulation is a new regulation mechanism which takes place
6
5
4
MI
FIGURE 9. Adenosine-mediated inhibition of adenylyl cyclase activity in DDT, MF-2 membranes. Membranes derived from control and P-blocker treated cells (atenolol, 10 5 M, 3 days) were prepared and adenylyl cyclase activity was determined as described in Materials and Methods. The data represent the means f S.E.M. of three experiments with triplicate determinations at each point. PIA-mediated inhibition of adenylyl cyclase activity in treated cells was significantly reduced after chronic P-blocker treatment (P50.01).
at the cellular level where complex reflex activities can be excluded with certainty. Furthermore, we demonstrated that this transregulation has functional significance. Thus chronic P-blockade leads to an attenuation of receptor-mediated inhibition of the stimulated adenylyl cyclase activity. Chronic P-blockade activation of muscarinic M, receptors or of A, adenosine receptors by their agonists fails to inhibit the forskolin-stimulated adenylyl cyclase. Whereas the initial studies were performed with high doses of metoprolol, we also could demonstrate that this transregulation is mediated stereospecifically by therapeutic @-blockade using different &blockers. To our knowledge this is the first description of an in vivo transregulation of two inhibitory receptor systems of the adenylyl cyclase system after chronic P-blockade and its characterization as a regulation mechanism at the cellular level. A single observation in diseased human heart suggested a decrease of muscarinic receptors after variable chronic p-
Transregulation
of Adenylyl
blockade [23]. While acute interaction of these two pathways with respect to the modulation of the acute cellular response has been well characterized [24], the chronic regulation of the density of receptors and their functional activity by the opposing receptor system was a somewhat surprising result. For enzyme regulation, however, it is well known that opposing modulators may be subjected to a co-ordinate regulation. Such transregulation may be of importance for P-blocker therapy. Thus several effects of chronic P-blocker treatment may be mediated in part by such slow transregulation of antagonistic receptor systems. In the present paper we have characterized the functional importance of this transregulation only for the inhibition of adenylyl cyclase. At present it has not been shown if muscarinic receptors other than the M, subtype may be modulated by this transregulation process. In rat heart and lung predominantly muscarinic receptors of the M, subtype are present [13, 25, 261 and mediate the carbachol and oxotremorine induced inhibition of adenylyl cyclase [27-291. These inhibitory receptors, however, may also couple to other signal transduction pathways [29]. Muscarinic M2 and A1 adenosine receptors, for example, are known to modulate several potassium channels [30, 311. This would imply that chronic P-blockade may act via this transregulation on these channels. Possibly a direct interaction of the inhibitory G protein with the ion channel may be involved in this regulation. This action could explain some of the anti-arrhythmic effects of chronic P-blockade. Other receptors which are inhibitory to the adenylyl cyclase such as a*-adrenergic receptors have been shown to activate the Na + /H + exchanger or to modulate platelet activation [32-341. Such transregulatory action of chronic P-blockade may explain some of the little understood therapeutic effects of P-blocker therapy. These include chronic antihypertensive or some of the anti-arrhythmic effects. However, in the present study the inhibitory effects on adenylyl cyclase activity of these receptor systems have been tested exclusively. On the other hand, abrupt withdrawal of chronic P-blocker treatment has been shown to lead to the so-called P-blocker withdrawal syndrome [IO, U]. The P-blocker withdrawal
Cyclase-coupled
Receptors
545
syndrome is characterized by an increased sensitivity of the adrenergic system to sympathetic stimulation. It could be speculated that the P-blocker-induced transregulation of inhibitory receptors of the adenylyl cyclase may further potentiate such sensitization of the adrenergic system. Further studies should be carried out to evaluate this possibility. The molecular mechanism of such transregulation of adenylyl cyclase-coupled receptors is not yet known. We could, however, demonstrate that transregulation is a cellular regulation mechanism which occurs both in vim, where complex reflex activity cannot be excluded, and to the same extent in isolated cells in a well defined in vitro system. Moreover, we could show that this transregulation does not involve the cellular distribution of receptors but it modulates the total density or expression of cellular receptors. These data demonstrate that internalization of receptors, which is observed in agonist-promoted desensitization processes, is not responsible for this transregulation process. Agonist-promoted desensitization, with the rapid decrease of receptors in the plasma membranes, has been characterized for both inhibitory receptors of the adenylyl cyclase system such as muscarinic M2 and A, adenosine receptors [35, 361 and stimulatory receptors such as the P-adrenergic receptors [371. Moreover, after transregulation, the remaining receptors, both muscarinic M2 and adenosine A,, are fully capable of forming the agonist-promoted high affinity state, indicating their ability to couple functionally to the G protein. Thus, uncoupling of receptors from the G protein commonly observed during early agonist-promoted desensitization of the activated receptors is not involved in the transregulation process. Functional uncoupling of receptors from one of the inhibitory G proteins has been attributed to phosphorylation of these receptors, possibly by specific kinases such as the fladrenergic receptor kinase [ 7, 38, 391. Such covalent modification of the remaining receptors seems to not be involved in the transregulation process, since functional coupling of the remaining receptors, as indicated by the formation of the agonist-promoted high affinity state of the receptors, seems to be unaltered. The reduction of receptor density was shown to be sufficient to greatly impair
546
R. Marquetant
inhibition of adenylyl cyclase. These data suggest that transregulation of inhibitory receptors by chronic P-blockade is a process which is distinct from agonist-promoted desensitization of these receptors. 1 Also, in contrast to desensitization, which after activation of receptors occurs rapidly within minutes, the transregulation of inhibitory receptors is observed only after chronic flblockade. Several hours of P-blockade of isolated but rapidly growing cells, or even more than 1 day of in uivo administration of /3blockers, are not sufficient to promote this transregulation. This slow time course linked to the observation that the total cellular receptor density is modulated by this transregulation process suggests that processing of receptors, i.e. synthesis or degradation, may be involved in transregulation. Further studies will have to focus on this hypothesis. Previous studies have suggested that the @blocker withdrawal may only be due to a displacement of endogenous catecholamines from the @-adrenergic receptor [IO, II]. However, as shown here, both the increase of fiadrenergic receptors and the transregulation of A, adenosine receptors on isolated smooth muscle cells under chronic P-blockade occurred even in the absence of serum or catecholamines. These data indicate that the displacement of endogenous catecholamines may not be involved in this transregulation process of inhibitory receptors. They clearly demonstrate that the “empty” receptor may mediate a different signal to the cell than the occupied but blocked receptor. The mechanism responsible for this can only be speculated on. The question of whether modulation of adenylyl cyclase and the “second messenger” CAMP or coupling to the G protein is involved in this P-blocker-mediated transregulation process of inhibitory receptors, has not yet been clarified. In vivo P-blocker treatment could reduce endogenous CAMP levels. However, in isolated unstimulated smooth muscle cells, CAMP levels are extremely low, especi-
et al.
ally when grown in the absence of catecholamines, i.e. serum. It is not known if fiblocker treatment could further reduce these already low levels which range in the low detection limit of the radio-immuno assay. Therefore, it appears unlikely that CAMP levels may mediate this transregulation process. Further studies will have to focus on this question. Cross-regulation at the level of G proteins by chronic agonist treatment using P-agonists, [40] and A, adenosine agonists [U, 421 has been described recently. The intracellular signal which mediates this has been attributed to CAMP levels [40, 431. Moreover, crossregulation at the G protein level seems to involve regulation of protein synthesis. However, in these studies chronic P-blockade seems not to greatly influence inhibitory Gproteins and thus not modulate the inhibitory receptor system primarily at the G protein level. These data indicate that the regulation at the level of G proteins may be mediated by different mechanisms than the transregulation at the level of receptors. All components of the adenylyl cyclase system are cloned and sequenced. The high homology suggests that they are all individual members of a large family of similar proteins. All receptors which couple to the adenylyl cyclase are glycoproteins with seven membrane spanning regions [44]. Despite their distinct chromosomal representation [45] their similar promoter regions may be subjected to a coordinate regulation which may be the basis for this newly characterized transregulation of adenylyl cyclase coupled receptors after chronic P-blocker treatment. Acknowledgements We wish to thank U. Oehl for technical assistance and K. Schiile for help in the preparation of the manuscript. This study was supported by the Deutsche Forschungsgemeinschaft, Bonn. RHS is supported by the Hermannand Lilly-Schilling-Foundation.
References 1 GILMAN, A. G. G proteins and regulation of adenylyl cyclase. JAMA 262, 1819-1825 (1989). 2 LEFKOWITZ, R. J., CARON, M. G., STILES, G. L. Mechanism of membrane-receptor regulation. Biochemical, physiological, and clinical insights derived from the studies of the adrenergic receptors. N Engl J Med 310, 1570-1579 (1984).
Tramregulation
of Adenylyl
Cyclase-coupled
Receptors
547
3 SI‘ILES, G. L., CARON, M. G., LEFKOWITZ, R. J. Beta-adrenergic receptors: Biochemical mechanisms [,f physiological regulation. Pharmacol Rev 64, 661-743 (1984). 4 SIRLE’I’. D. R., LEFKOWITZ, R. J. Molecular mechanisms of receptor desensitization using the beta-adrenergic receptor-coupled adenylate cyclase system as a model. Nature 317, 124-129 (1985). 5 HARDEN, T. K. Agonist-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase Pharmacd Rev 35, 5-32 (1983). 6 STRASSER. R. H. Phosphorylation of the beta-adrenergic receptor: Mechanisms of desensitization. [n &r+n, Pho@@ation. V. K. Moudgil (ed.) pp. 199-226 Boca Raton, Florida: CRC Press (1989). 7 LEFKOWITZ, R. J., HAUSDORFF, W. P., CARON, M. G. Role of phosphorylation in desensitization of the @-adrent)ceptor. Trends Pharmacol Sci 11, 190-194 (1990). 8 KWATRA, M. M., HOSEY, M. M. Phosphorylation of the cardiac muscarinic receptor m intact chick heart and Its regulation by a muscarinic agonist. J Biol Chem 261, 12429-12432 (1986). 9 STILES. G. L. Adenosine receptors and beyond: Molecular mechanisms of physiological regulation, Chn Res 38. lo-18 (1990). IO FISHMAZ, W. H. Beta-adrenergic blocker withdrawal. Am J Cardiol 59. 26F-32F (1987). 11 PRICHARD, B. N.C., TOMLINSON, B., WALDEN, R.J., BHATTA-CHARJEF., P. The 6-adrenergic blockade xithdrawal phenomenon. J Cardiovasc Pharmacol 5, S56-S62 (1983). 12 LOHS~, M. J., KLOTZ, K-N., LINDENRORN-FOTINOS, J., REDDINGTON, M., SCHWAHE. U., OL.S~)N. R. A 8-Cyclopentyl-1.3-dipropylxanthine (DPCPX) - a selective high affinity antagonist radioligand for .4, adenosine receptors. Naunyn-Schmiedeberg’s Arch Pharmacol 336, 204-210 (1987). 13 GI~CHETTI. A., MICHELETTI, R., MONTAGNA, E. Cardioselective profile of AF-DX116. a muscarmic M, receptor antagonist. Life Sci 38, 166331672 (1986). 14 ENCEL.. G., HOYER. D., BERTHOLD, R., WAGNER, H. [“51]Iodocyanopindolol. a new ligand for beta-adrenoceptors: Identification and quantitation of subclasses of beta-adrenocrptors in guinea pig. Naunyn-Srhmirdeberg’s Arch Pharmacol 317, 277-285 (1981). 15 SEAMOK, K.B., PADGET-. W., DALY, J.W. Forskoiin: Unique diterpene activator of adenylatt: tvrlasc in membranes and intact cells. Proc Nat1 Acad Sci USA 78, 3363-3367 (1981). 16 ,JAKOBS, K. H.. SAUR, W., SCHULTZ. G. Reduction of adenylate cyclase activity in lysates of human platelets by the cY-adrenergic component of epinephrine. J Cycl Nucl Res 2, 381-392 (1976). 17 BRADFORI). M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Anal Biochem 72, 248-254 (1976). 18 DE LEAIV, A., MUNSON, P. J., RODBARD, D. Simultaneous analysis of families of sigmoidal curves: Appltcation to bioassay and physiological dose-response curves. Am J Physiol 235. E978-El02 (1978). 19 DE LEAN, A., STADEL, J. M., LEFKOWITZ, R. J. A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled beta-adrenergic receptor, J Biol Chem 255, 7108-7117 (1980). 20 S
