Met hamsm s o/Ageing and Development 60 ( 1991 ) 215-224 Elsewer Sclent~fic Pubhshers Ireland Ltd
215
ALTERATIONS IN f ADRENERGIC AND MUSCARINIC RECEPTORS IN AGED RAT HEART. EFFECTS OF CHRONIC ADMINISTRATION OF PROPRANOLOL AND ATROPINE
B CHEVALIER, P MANSIER, E TEIGER, F CALLENS-EL AMRANI and B SWYNGHEDAUW INSERM, U 127 H6pttal Lartbotstere 41 Bd de la Chapelle 75010 Parts (Frante) (Recetved November 20th, 1990) (Revision recewed March 26th, 1991)
SUMMARY
The car&ac responses to sympatheUc and vagal stlmulaUons are attenuated with ageing To understand these findings, the densmes of f adrenerglc (fR) and muscanmc (MR) receptors m the left ventricles have been quantltated m parallel m male Wlstar rats (4- and 24-month-old) using [125I]lodocyanoplndolol and [3H]qumuchdmyl benzllate as specific ra&ohgands The homologous regulaUon of these receptor densities was also explored after a 7-day continuous infusion of propranolol or atropine As compared to young rats, the f R and MR densmes m aged ammals were decreased (from 31 ± 2 to 23 ± 2 fmol/mg protein, P < 0 05 for fiR, from 104 ± 7 to 54 ± 3 fmol/mg protein, P < 0 001 for MR) but the &mmuUon m MR was more pronounced (-48%) than that m f R (-26%), resulting m a drop m the fiR/MR ratio Continuous infusion of propranolol or atropine up-regulated the fiR and MR denslUes (respectwely +50°,4, P < 0 01 and +33%, P < 0 05) in aged but not m young adult rats We therefore conclude 0) that the &mmutlon of the cardmc response to the sympathetic and vagal sUmulatlons during agemg may be partly explained by a decrease m the corresponding receptor density, 00 these changes are reversible and the density of these two groups of receptors can return to adult control values by chromc administration of the approprmte antagonist
Key words Rat left ventricle, Ageing, Muscarmlc receptors, f Adrenerglc receptors, Propranolol, Atropine Correspondence to Dr B Chevaher, INSERM U 127 H6pltal Lartbolslere, 41 Bd de la Chapelle, 75010 Pans, France 0047-6374/91/$03 50 Printed and Pubhshed m Ireland
© 1991 Elsevier Scientific Pubhshers Ireland Ltd
216 INTRODUCTION Ageing significantly alters cardiac performances in man even m the absence of any additional disease [1] The basic characteristics of the senescent cardlocyte are also modified, as those of the hypertrophied cardiac myocyte in response to a chronic pressure-overloading [2,3] Amongst these modifications a constant and universal finding ~s a d~mmlshed responsiveness to beta-adrenerglc modulation which is found both in senescent men and animals [4] This modification explains why during exercise the maximal heart rate declines despite an enhanced plasma catecholamlnes level m a senescent population [4] Similarly age-related diminution in chronotroplc response to atropine or vagotomy have also been reported [5,6] The possible mechanisms which may account for the drop in both sympathetic and parasympathetic responses in this condition may involve the sarcolemmal receptors. the mechanisms of transductlon, as well as the phosphorylatlon process [7--9] In this report the first level of a possible regulation has been explored and an attempt was made to address the question whether a drop in M R and OR densities could explain the diminished myocardial responsiveness to sympathetic or parasympathetic stimulation which is observed during ageing Receptor densities have been quantltated in parallel, in young adult and senescent rat left ventricles The upregulating effects of a 7-day Infusion with specific antagonist was also explored Results demonstrate a diminution In both myocardial t3-adrenerglc and muscarinlc receptor densities with ageing In the senescent left ventricle, chronic perfuslon with specific antagonists induce an up-regulation of both receptor densities, there is no longer any difference between the two groups of rat left ventricle after these treatments MATERIALS AND METHODS
Ammals Young adult male Wlstar rats ( 3 - 4 months, 310-370 g) were compared with senile ones (24--26 months, 610---740 g) Both groups were housed in plastic cages with free access to food and water and were used for experiments 1 week after their arrival
Chromc infusions of drugs The rats were anesthetized by an intraperltoneal injection of pentobarb~tal (25 mg/kg) An osmotic p u m p (model 2 M L I , Alza Corp, Palo Alto, CA) was subcutaneously placed between the second and the 13th thoracic vertebrae After a 2-cm incision was made, the p u m p was inserted and the incision was closed with sutures of 2 0-mm silk Drugs were dissolved in a solution of 0 9% N a C I - - H C I 10 m M Control animals were infused with the solvent alone Treated animals received pro-
217 pranolol at a rate of 20 mg/kg per day or atropine 100 mg/kg per day for 7 days The animals were sacrificed 16~18 h after the pump had defused the total volume and the left ventricles were homogenelzed We have verified that the pumps were empty at the end of the treatments
Cardiac membrane preparattons The left ventricle homogenates were prepared as previously described [10] Briefly, left ventricles were placed in 150 mM NaCI/5 mM T n s - - H C I (pH 7 6), at 4"C, lmmedmtely minced with scissors m 5 ml of 10 m M T n s - - H C I (pH 78) and homogemzed using Polytron (plot 2--3) After incubation for 10 mm at 4"C m the presence of 1 M KC1, the myofilaments were solubdized The homogenate was centrifuged three times at 50 000 x g for 15 m m at 4"C The final pellet was resuspended into the assay buffer (50 mM T n s - - H C i , 0 2 m M sodium metabisulfite, pH 7 8) and frozen m ahquots at - 7 0 " C The protem concentration was routinely 1 mg/ml
Radtohgand bmdlng expertments Beta adrenergtc receptor determmatton Frozen membrane suspensions (80---100 /~g) were incubated with increasing concentrations (5--150 pM) of [J25Illodocyanopmdolol ([125I]CYP) (spec act of 2000 Cl/mmol, Amersham) in a final volume of 500/~1 at room temperature during 90 m m Then 4 5 ml of assay buffer were added to reduce non-specific binding and the incubation was ended 30 mln after The filtration of each tube was performed automatically by a Skatron semiautomatic cell harvester (Skatron AS, Ller, Norway) The bound radloactwlty was determined in a Kontron GammamaUc counter (Roche B~oelectromque Kontron, Vehzy, France) w~th an efficiency of 80"/0 The non-specific binding of [125I]CYP was defined as the radioactivity bound in the presence of I/~M (-)-propranolol and usually amounted to less than 20°/,, of the total binding Mus¢arlmt receptor quantllltatlon It was performed using [~H]qulnuclldlnyl benzdate ([3HIQNB, spec act of 42--44 C1/mmol, Amersham) as a radlohgand Fractions of homogenates (80---120 ~tg) were incubated at room temperature for 150 mln m a final volume of 2 ml m presence of [3H]QNB (6--250 pM) Each incubation was terminated by automatically filtering the suspension through a GF/B glass fibre filter with a Skatron cellular collector Tntmted QNB retained on the filter was extracted for 16 h with l0 ml of scmtdlatlon fluid Radioactivity was counted m a LKB counter (75% efficiency) Specific [3H]QNB binding was experimentally determined from the difference between counts m the absence and presence of 1 /~M atropine and usually amounted to 80% of the total binding Conditions were chosen to limit total binding to less than 10% of the radioactivity m the m e d m m so that the concentration of free radlohgand did not slgmficantly change during the procedure
Calculattons and stattstteal analysts Results were normalized on the basis of the protein concentration m the
218 h o m o g e n a t e [11] a n d the r e c e p t o r n u m b e r was expressed m fmol/mg p r o t e i n Equlh b n u m b i n d i n g d a t a were a n a l y z e d using L I G A N D p r o g r a m [12] This p r o g r a m fits the b i n d i n g d a t a to the e q u a t i o n d e s c r i b i n g the law o f mass action The affinities (Kd) o f r a d l o h g a n d [I25I]CYP o r [3H]QNB, specific non-selective a n t a g o m s t s , were d e t e r m i n e d from S c a t c h a r d analyses Statistical analyses o f the effects o f a g e m g a n d t r e a t m e n t s have been p e r f o r m e d using the analysis o f variance ( p a c k a g e Systat 4 m o d u l e M G H L ) a n d the F - r a t i o d e t e r m i n a t i o n [13] RESULTS Anatomtcal data
T a b l e I shows t h a t d u r i n g senescence the left v e n t n c u l a r weight ( L V W ) p r o p o r tionally increases with the b o d y weight (BW), resulting in an u n c h a n g e d L V W / B W ratio T h e m e m b r a n e p r e p a r a h o n s f r o m b o t h e x p e r i m e n t a l g r o u p s y~eld the same a m o u n t o f p r o t e i n per g o f LV Beta adrenergw and muscarmt~ receptor densltws
Figures 1 a n d 2 represent typical S c a t c h a r d t r a n s f o r m a t i o n s o f s a t u r a t i o n exp e r i m e n t a l d a t a T a b l e II shows that the m e a n densities o f the /3-adrenergic and m u s c a n m c receptors are significantly d i m i n i s h e d by 26% a n d 48%, respecttvely, In the aged m y o c a r d m m as c o m p a r e d to that o f y o u n g a d u l t s The values (Table II) for the y o u n g e r g r o u p fit well with those p r e v i o u s l y published 30 fmol/mg protein for t h e / 3 - a d r e n o c e p t o r s as c o m p a r e d to 37 a n d 29 [14,15], 100 fmol per mg o f p r o tein for the m u s c a r m i c receptors, as c o m p a r e d to 144 a n d 130 [16,17] T h e decrease o f the m u s c a n m c r e c e p t o r density in senescent left ventricles is m o r e p r o n o u n c e d than that o f t h e / 3 - a d r e n o c e p t o r s , c o n s e q u e n t l y the muscarlnlC//3-adrenergic r e c e p t o r m e a n densities r a t i o which is a r o u n d 3 m y o u n g a d u l t rat left ventricle falls to nearly 2 m aged LV (Table II) TABLE 1 ANATOMICAL DATA HOMOGENATE PREPARATIONS Young adult (n = 16)
Aged (n = 15)
~41 ± 8 596 + 12 1 74 • 004
676 + 20*** 1055 + 23*** 1 57 4- 005
4natomttal data
Body weight (BW) m g Left ventncular weight (LVW) m mg LVW/BW m mg/g Homogenate
Protein m mgper LV Yield m mg/g fresh ussue
64 ± 4 109 + 7
Means + S E ***P < 0001 n = number of experiments
106 + 5*** 101 + 6
219
0.24
0.18
A
0.12 O
0
10
20
30
40
1251-CYP specifically bound m fmol/mg protein Fig l Effect of ageing on the/~-adrenerglc receptor dens:ty Typical Scachard representations Open symbols represented the data obtained from a young adult left ventricle and filled symbols those obtained from an aged left ventricle homogenate The spec:fic [1251]CYP bound and the K 0 values, determmed by LIGAND program, were respectively 35 fmol/mg and 22 pM m the young and 21 fmol/mg and 29 pM in the aged left ventricle
0°~.~"
0.15 r~
A
0.10
A
g~ O
0.05i o
0
. . . . . .
20
40
""-.,
60
.
80
100
3H-QNB specifically bound m fmol/mg protein F:g 2 Effect of ageing on the muscanmc receptor dens:ty Typical Scachard representat:ons Open symbols represented the data obtained from a young adult left ventricle and filled symbols those obtained from an aged left ventricle homogenate The spec:fic [3H]QNB bound and the K,j values, determined by LIGAND program, were respecUvely 82 fmol/mg and 26 pM m the young and 58 fmol/mg and 24 pM in the aged left ventricle
221) TABLE 11 B-ADRENERGIC AND MUSCARINIC RECEPTOR DENSITIES Control
A g('d
(n = 8) 31 + 2 2515 4- 268 32 4- 5
(n = 61 2t 4. 2* 286t ± 479 ~,4 + 6
Density m tmol/mg Content m fmol per LV Kd in pM
(n = 8) 104 4- 7 5252 4- 524 25 + 3
(n = 8) 54 + t*** 5349 4- 18t 27 4-
MR/BR mean denstttes ratto
t t5
2 t5
13-adrenergu receptors (BR)
Density in fmol/mg Content m fmol per LV Ka m pM Muscarmu re~eptor~ (MR)
Means 4- S E *P < 005 ***P < 0001 n = number of experiments
Effects of a chronu m[uston oj drug
The body weights o f y o u n g adult rats gain 48 4- 11 g ( P < 0 001) d u r i n g saline or 40 4- l0 g (P < 0 01) d u r i n g p r o p r a n o l o l Infusion (approx 6 g of body w t / d a y ) but the body weights of the senescent rats do not significantly change In contrast. atropine Induces a loss in body weight in both groups ( i n ) o u n g adults - 2 7 49 g , P < 0 0 5 , m aged - 7 7 + 17g, P < 0 0 0 1 ) C o n s e q u e n t l y , the LV/BW ratio i n c r e a s e s l n aged rat g r o u p from 1 50 4- 0 0 9 to 1 87 + 0 II (P < 0 0 1 ) Saline Infusions have no effect on both receptor densities (Table 111) The upregulations of ¢~-adrenerglc (+50%) a n d muscarlnlC (+33"/,,) receptors by p r o p r a n o l o l and a t r o p i n e respectively are only observed in the 24 m o n t h s old rats (Table 1II) The Kd value o f the/3-adrenerglc receptors for their specific r a d l o h g a n d ([ 125IlCYP) IS slightly increased in the y o u n g adult group, 7 days after the saline perfuslon (see Table II a n d T a b l e IllB), c o n s e q u e n t l y a significant difference between the two rat groups appears After p r o p r a n o l o l t r e a t m e n t , in the y o u n g adult group, the mean K a value is similar to that observed in c o n t r o l experiments (Table ll) b u t in the aged g r o u p a weak b u t significant Increase o f the a p p a r e n t affinity for the specific radxohgand is observed F o r the muscarsnlc receptors a n d in the y o u n g adult group, the Kd values for [3H]QNB are significantly increased either after saline or atropine perfuslon (see Table II) a n d c o n s e q u e n t l y a significant difference between the two groups of rats does also a p p e a r In the aged g r o u p of rats, the Kd values are significantly changed after the saline perfuslon (see T a b l e II) but the a t r o p i n e treatment induces a weak but significant increase o f the affinity c o n s t a n t s C o m p a r a b l e slight increases in the K d values have already been observed alter a n t a g o n i s t treatment [18--20] even, as in this paper, If the isolation procedure for m e m b r a n e s starts 16--18 h after the end of the infusion
221
TABLE 111 (A) B-ADRENERGIC A N D MUSCARINlC RECEPTOR DENSITIES AND (B) K d VALUES FOR THE [125I]CYP or [~H]QNB AFTER A 7-DAY PERFUSION WITH A SALINE SOLUTION, PROPRANOLOL OR ATROPINE
( A ) Receptor densttws (fmol/mg protems ) B-Adrenerglc receptor density Sahne Propranolol Muscanmc receptor density Sahne Atropine (B) Kd values (pM) B-Adrenerglc receptors ([1251]CYP) Sahne Propranolol Muscanmc receptors ([~HIQNB) Sahne Atropine
Young adult
Aged
29 + 2 27 ~ 3
22 ± 2÷÷ 33 + 3**
88 + 9 91 ± 5
56 ± 3÷++ 74 + 7*
61 ± 10 30 ~ 5*
38 ± 5+ 62 + 10"
47 ~ 8 44 ± 8
28 ± 3+ 40 ± 4*
Number of experiments 7 and 9 m the young adult and aged groups Means ~- S E Statlst.cal analysis of the effects of the treatments *P < 0 05. **P < 0 01 Statistical analysis of the effects of ageing +P < 0 05. ++P < 0 01, +++P < 0 001
DISCUSSION The m a m findings o f this study are the foilowmg (1) the myocardial d e n s m e s m both/3-adrenerglc a n d m u s c a n m c receptors d e n s m e s decrease d u r m g ageing, (n) this d i m i n u t i o n is reversible a n d a chronic m f u s l o n with a specific a n t a g o m s t upregulates the c o r r e s p o n d m g receptor u p to values observed m y o u n g adult
Adrenergtc receptors and sympathett~ tone durmg agemg It Is well k n o w n that the cardiovascular effects o f an adrenerglc stimulation, whether they have been o b t a i n e d by exercising or directly by lsoproterenol infusion, are a t t e n u a t e d m the senescent h u m a n a n d a m m a l [7,10] F o r example m rats the m a x i m u m rate o f force d e v e l o p m e n t after isoproterenol infusion dechnes m senescent isolated perfused m t e r v e n t n c u l a r septae [9] Such a m o d i f i c a t i o n m a y revolve one o f the several c o m p o n e n t s o f the adrenerglc cascade, i n c l u d i n g the specific sarcolemmal receptors, the t r a n s d u c t l o n mechamsms, the adenylate cyclase a n d phosphodlesterase a n d the capacity of several protems to be p h o s p h o r y l a t e d [review m 4] In this report at least the first c o m p o n e n t of this system, namely the /~R receptors are modified which by Itself can explain the physiological data b u t obviously does not rule out other modifications occurring m parallel, as m c h r o m c pressure overloading (for review see Ref 21)
222 Similar results concerning the/3 receptors have recently been reported [22] during senescence m man Other investigators found an essenually unchanged B receptors densxty but (1) they were using dlhydroalprenolol, a hgand which in our work gives an important non-specific binding, (11) thexr control adult values were obtained m 7--9-month-old rats while we used younger (2--3-month-old) controls Muscarlntc receptors and vagal a~ttwt)' m ageing Physiological and pharmacological observations mdlcated that cardmc muscarmlc responsweness decreased with age, suggesting a possible change in the concentration of cardiac muscanmc receptors The posmve chronotroplc effect of a vagotomy was nearly absent in old ammals [6] Furthermore, m humans the chronotroplc response to atropine was attenuated m old persons [5] Additional mechamsms may play a role since both the acetylchohnesterase actwlty and the threshold voltage to sUmulate the vagus nerve were dlmmashed [23] The inhibition of GTP-lsoproterenol sUmulated adenylate cyclase by carbachol (100 ~,M) was also more pronounced m the young adult (-40%) than m the aged rats (-20%) [24] The present study evidences a clear and major drop in MR density in aged left ventricles which would easaly explain the age-related dechne m chollnerglc control Such a d~mmutlon is more important than that of the /3R suggestmg a d~fferent regulation and resulting m a new unbalanced/3R/MR ratio Our result~ contradict those of Narayanan and Tucker [241 who found an unchanged left ventncular MR density m 3-, 12- and 24-month-old rats At least two differences have to be pointed out between thxs work and our finding since these authors were using a different strata of rats, the Fisher-334, and also a techmque of bmdmg with a Kd for [3H] QNB 10-fold greater than m the present report or m the majority of other pubhshed studies Effect o f a chromc mJuston with an antagom~t It has been well accepted during the last 10 years that chromc infusion with a specific antagomst up-regulates the corresponding receptor Several reports have reported an mcreased/3 receptor density after chromc treatment or xnfuslon w~th a specific antagonist [18--20] Nevertheless, it is not always the case since an acute reduction m/3-receptor number has been reported m human leucocyte both m vwo and m vitro after l-h administration of certam/3 blockers [25] Slmdar results were also reported m cultured and muscle cells with alprenolol and propranolol [261 Others found no change m/3R density m rat heart after 1 week of propranolol treatment [27,28] Very few studies have dealt with the muscanmc receptor regulation after chromc infusion of a specific antagomst [30,31] A shght mcrease m the total muscanmc receptor density has been reported m the brain after a 23-day treatment with 4 0 ~ 1 0 0 mg/kg atropine [30] It is therefore not really surprising to see no change m both 13 and muscanmc receptor denslUes after a chronic treatment w~tb the antagomst m young adult
223
animals More Interesting is the finding that in the aged group, m which both densttIes are dtmmlshed, it is possible to up regulate the receptors by usmg an appropriate antagonist, thus reaching the values obtamed in young adults This specific up-regulation suggests that the drop m/3 and muscarimc receptor densities observed during baseline conditions in the aged heart is itself due to an homologous downregulation It has been repeatedly shown that the peak plasma catecholammes durmg exercise is age-dependent and mcreases in senescent people [4] Such an mterpretatton would m fact mean that the diminution m/3 adrenergIc and muscarlntc receptor densities cannot explam the attenuation of the cardmvascular effects of adrenergic and muscarimc stimulatmns It suggests that the explanation ts more located elsewhere either 'down the road' at the level of the transductmn mechanism, or in relation with the recently reported enhanced adenosme level [31 ] Several prewously published experiments support this hypothesis, as for example the finding that the motroptc effect of dibutyryl cAMP is diminished in old rats [9] REFERENCES 1 J L Fleg, Alterations in cardiovascular structure and function with advancing age Am J Cardtol 57 (1986) 33C ~ C 2 F C P Yln, H A Spurgeon, M L Welsfeldt and E G Lakatta, Mechanical properties of myocardram from hypertrophied rat hearts A comparison between hypertrophy induced by senescence and by aortic banding Clrc Res, 46 (1990) 292--300 3 E G Lakatta, Do hypertension and ageing slmdarly affect the myocardmm9 Circulation 75 (Suppl I) (1987) 69--77 4 C R Fllburn and E G Lakatta, Aged related alterations in the B-adrenerglc modulation of cardiac cell function In J E Johnson (ed), Aging and Cell Funttton, New York Plenum Publ Corp, 1984, pp 211--238 5 P Dauchot and J S Gravensteln, Effects of atropme on the electrocardiogram in different age groups Chn Pharmacol Ther 12(1975) 274---280 6 G J Kelhher and T S Conahan. Changes in vagal actwlty and response to muscarlmc receptor agomsts with age J Gerontol, 35 (1980) 842--894 7 N Narayanan and J A Derby, Alterations in the properties of B-adrenerglc receptors of myocardial membranes In aging impairments In agomst-receptor interactions and guanine nucleot~de regulation accompany dlmlmshed catecholamlne-responslveness of adenylate cyclase Mech Ageing Dev, 19 (1982) 127--139 8 P J Scarpace and 1 B Abrass, Beta-adrenerglc agonlst-medlated desensitization in senescent rats Mech Ageing Dev , 35 (1986) 25~-264 9 T Guarnlerl, C R FiIburn, G Zltnlk, G S Roth and E G Lakatta, Contractde and biochemical correlates of the aged heart Am J Phystol 239 (1980) H501--H508 10 B Chevaher, P Mansler, F Callens-EI Amranl and B Swynghedauw, B-Adrenerglc system is mo&fied in compensatory pressure cardiac overload in rats physiological and bmchemlcal evidence J Cardtovasc Pharmacol 4 (1989) 4 1 3 4 1 9 11 O H Lowry, N J Rosebrough, A L Farr and R J Randall, Protein measurement with the Fohn phenol reagent J Btol Chem 193 (1951) 265--275 12 P J Munson and D Rodbard. A versatile computerized approach for characterlzatmn of hgandbinding systems Anal Btochem 107 (1980) 220--239 13 J H Zar, Multlpsample hypotheses the analysis of varmnce In J H Zar (ed), Bwstattstwal Analysis, Prentice-Hall, New Jersey, 1984, pp 162--184 14 H H Ayobe and R C Tarazl, Reversal ofchanges in myocardml B receptors and motroplc respon-
224
15
16 17
18
19
20 21
22 23
24
25
26
27 28 29
30
31
slveness with regression of cardiac hypertrophy m renal hypertensive rats (RHR) Ctrt Res 54 (1984) 125--134 O A Gende. A Mattlazzl M C C M d h a n P Pedrovl C T a q m m G llambl and H E Cmgolanl. Renal hypertension impairs motroplc ~soproterenol effect without /3 receptor changes 4m J Phvstol 249 (1985) H 8 1 4 ~ H 8 1 9 J Z Fields. W R Roeske and E M o r k m Cardiac m u s c a n m c chohnerglc receptors J Btol Chem 253 (1978) 3251--3258 S P Baker. S Marchand. E O Nell. C A Nelson and P Posner Age-related changes m cardiac m u s c a n m c receptors decreased ablhty of the receptor to form a high affimtv agomst binding state J Gerontol 40 (1985) 141--146 P Gengo. A Skattebol. J F Moran. S Gallant M Hawthorn and J Tnggle Regulation by chromc drug administration of neuronal and cardmc calcmm channel, beta-adrenoreceptor and muscanm~. receptor levels Btochem Pharmatol 37 (1988)627--633 R D Aarons and P B Mohnoff. Changes m the density of beta adrenerglc receptors m rat lymphocytes, heart and lung after chromc treatment w~th propranolol J Pharmatol Exp Ther 221 (1981) 439 443 G Glaublger and R J Lefkowltz. Elevated beta-adrenerg~c receptor after chromc propranolol treatment Btochem Btophy~ Res Commun 78 ( 1 9 7 7 ) 7 2 0 ~ 7 2 5 B Chevaher and B Swynghedauw. Beta-adrenerglc receptors m hypertrophied and heart ladure In Swynghedauw. B (ed). Htpertroph, and Heart Fadure INSERM/J Llbbey Pans - - London. 1990 pp 201--216 M Bohm and E Erdmann. Influence o f age on beta adrenoreceptor down regulation m the hypertrophy and fading m y o c a r d m m Eur Heart J ( a b s t ) I1 (1990) 196 V V Frolkls V V Bezrukov. Y K Duplenko I V Shchegoleva. V G Shevtchuk and N S Verkhratsky. Acetylchohne m e t a b o h s m and chohnerg~c regulation of function m aging Gerontologta 19 (1973) 45 57 N Narayanan and M Tucker. A u t o n o m i c interactions m the ageing heart age-assocmted decrease m m u s c a n m c chohnerg~c receptor medmted inhibition o f / 3 adrenerg~c actwatlon of adenylate cyclase Mesh Agemg Dev 34 (1986) 249--259 A De Blasl. M Fratelh and O Marasco. Certain ~-blockers can decrease adrenerglc receptor number 1 Down regulation of receptor number by tertatolol and bopmdolol Clrt Res 63 (1988) 273--278 R J Huges. L C M a h a m and P A lnsel Certain /3-blockers can decrease adrenerglc receptor number II Down regulation of receptor number by alprenolol and propranolol m cultured lymp h o m a and muscle cells O r c Res 63 (1988) 279--285 S P Baker and L T Potter. Effect of propranolol on /3-adrenoceptors in rat hearts Br J Pharmaeol. 68 (1980) 8 - - 1 0 R H Kennedy and T E Donnely. Cardmc responsiveness after acute w~thdrawal of chromc propranolol treatment in rats Gen Pharmacol 13 (1982) 231 239 B C Wise. M ShojJ and J F Kuo. Decrease or mcrea~e m carding, m u s c a n m c chohnerglc receptor number m rats treated with metachohne or atropine Bto~hem Btoph~ Re~ Commun 92 (1980) 1 1 3 ~ 1 1 4 2 R Majocha and R J Baldessanm, Tolerance to an antlchohnerglc agent is paralleled by increased b m d m g to muscarlmc receptors m rat brain and increased behavioral response to a centrally actwe chohnomtmetlc Ltfe Set 35 (1984) 2247--2255 J C Dobson, R A Fentar and F D R o m a n o , Increased myocardml adenosine production and reduction of/3 adrenergJc contractde response m aged hearts Circ Re~ 66 (1990) 1381 1390
215
ALTERATIONS IN f ADRENERGIC AND MUSCARINIC RECEPTORS IN AGED RAT HEART. EFFECTS OF CHRONIC ADMINISTRATION OF PROPRANOLOL AND ATROPINE
B CHEVALIER, P MANSIER, E TEIGER, F CALLENS-EL AMRANI and B SWYNGHEDAUW INSERM, U 127 H6pttal Lartbotstere 41 Bd de la Chapelle 75010 Parts (Frante) (Recetved November 20th, 1990) (Revision recewed March 26th, 1991)
SUMMARY
The car&ac responses to sympatheUc and vagal stlmulaUons are attenuated with ageing To understand these findings, the densmes of f adrenerglc (fR) and muscanmc (MR) receptors m the left ventricles have been quantltated m parallel m male Wlstar rats (4- and 24-month-old) using [125I]lodocyanoplndolol and [3H]qumuchdmyl benzllate as specific ra&ohgands The homologous regulaUon of these receptor densities was also explored after a 7-day continuous infusion of propranolol or atropine As compared to young rats, the f R and MR densmes m aged ammals were decreased (from 31 ± 2 to 23 ± 2 fmol/mg protein, P < 0 05 for fiR, from 104 ± 7 to 54 ± 3 fmol/mg protein, P < 0 001 for MR) but the &mmuUon m MR was more pronounced (-48%) than that m f R (-26%), resulting m a drop m the fiR/MR ratio Continuous infusion of propranolol or atropine up-regulated the fiR and MR denslUes (respectwely +50°,4, P < 0 01 and +33%, P < 0 05) in aged but not m young adult rats We therefore conclude 0) that the &mmutlon of the cardmc response to the sympathetic and vagal sUmulatlons during agemg may be partly explained by a decrease m the corresponding receptor density, 00 these changes are reversible and the density of these two groups of receptors can return to adult control values by chromc administration of the approprmte antagonist
Key words Rat left ventricle, Ageing, Muscarmlc receptors, f Adrenerglc receptors, Propranolol, Atropine Correspondence to Dr B Chevaher, INSERM U 127 H6pltal Lartbolslere, 41 Bd de la Chapelle, 75010 Pans, France 0047-6374/91/$03 50 Printed and Pubhshed m Ireland
© 1991 Elsevier Scientific Pubhshers Ireland Ltd
216 INTRODUCTION Ageing significantly alters cardiac performances in man even m the absence of any additional disease [1] The basic characteristics of the senescent cardlocyte are also modified, as those of the hypertrophied cardiac myocyte in response to a chronic pressure-overloading [2,3] Amongst these modifications a constant and universal finding ~s a d~mmlshed responsiveness to beta-adrenerglc modulation which is found both in senescent men and animals [4] This modification explains why during exercise the maximal heart rate declines despite an enhanced plasma catecholamlnes level m a senescent population [4] Similarly age-related diminution in chronotroplc response to atropine or vagotomy have also been reported [5,6] The possible mechanisms which may account for the drop in both sympathetic and parasympathetic responses in this condition may involve the sarcolemmal receptors. the mechanisms of transductlon, as well as the phosphorylatlon process [7--9] In this report the first level of a possible regulation has been explored and an attempt was made to address the question whether a drop in M R and OR densities could explain the diminished myocardial responsiveness to sympathetic or parasympathetic stimulation which is observed during ageing Receptor densities have been quantltated in parallel, in young adult and senescent rat left ventricles The upregulating effects of a 7-day Infusion with specific antagonist was also explored Results demonstrate a diminution In both myocardial t3-adrenerglc and muscarinlc receptor densities with ageing In the senescent left ventricle, chronic perfuslon with specific antagonists induce an up-regulation of both receptor densities, there is no longer any difference between the two groups of rat left ventricle after these treatments MATERIALS AND METHODS
Ammals Young adult male Wlstar rats ( 3 - 4 months, 310-370 g) were compared with senile ones (24--26 months, 610---740 g) Both groups were housed in plastic cages with free access to food and water and were used for experiments 1 week after their arrival
Chromc infusions of drugs The rats were anesthetized by an intraperltoneal injection of pentobarb~tal (25 mg/kg) An osmotic p u m p (model 2 M L I , Alza Corp, Palo Alto, CA) was subcutaneously placed between the second and the 13th thoracic vertebrae After a 2-cm incision was made, the p u m p was inserted and the incision was closed with sutures of 2 0-mm silk Drugs were dissolved in a solution of 0 9% N a C I - - H C I 10 m M Control animals were infused with the solvent alone Treated animals received pro-
217 pranolol at a rate of 20 mg/kg per day or atropine 100 mg/kg per day for 7 days The animals were sacrificed 16~18 h after the pump had defused the total volume and the left ventricles were homogenelzed We have verified that the pumps were empty at the end of the treatments
Cardiac membrane preparattons The left ventricle homogenates were prepared as previously described [10] Briefly, left ventricles were placed in 150 mM NaCI/5 mM T n s - - H C I (pH 7 6), at 4"C, lmmedmtely minced with scissors m 5 ml of 10 m M T n s - - H C I (pH 78) and homogemzed using Polytron (plot 2--3) After incubation for 10 mm at 4"C m the presence of 1 M KC1, the myofilaments were solubdized The homogenate was centrifuged three times at 50 000 x g for 15 m m at 4"C The final pellet was resuspended into the assay buffer (50 mM T n s - - H C i , 0 2 m M sodium metabisulfite, pH 7 8) and frozen m ahquots at - 7 0 " C The protem concentration was routinely 1 mg/ml
Radtohgand bmdlng expertments Beta adrenergtc receptor determmatton Frozen membrane suspensions (80---100 /~g) were incubated with increasing concentrations (5--150 pM) of [J25Illodocyanopmdolol ([125I]CYP) (spec act of 2000 Cl/mmol, Amersham) in a final volume of 500/~1 at room temperature during 90 m m Then 4 5 ml of assay buffer were added to reduce non-specific binding and the incubation was ended 30 mln after The filtration of each tube was performed automatically by a Skatron semiautomatic cell harvester (Skatron AS, Ller, Norway) The bound radloactwlty was determined in a Kontron GammamaUc counter (Roche B~oelectromque Kontron, Vehzy, France) w~th an efficiency of 80"/0 The non-specific binding of [125I]CYP was defined as the radioactivity bound in the presence of I/~M (-)-propranolol and usually amounted to less than 20°/,, of the total binding Mus¢arlmt receptor quantllltatlon It was performed using [~H]qulnuclldlnyl benzdate ([3HIQNB, spec act of 42--44 C1/mmol, Amersham) as a radlohgand Fractions of homogenates (80---120 ~tg) were incubated at room temperature for 150 mln m a final volume of 2 ml m presence of [3H]QNB (6--250 pM) Each incubation was terminated by automatically filtering the suspension through a GF/B glass fibre filter with a Skatron cellular collector Tntmted QNB retained on the filter was extracted for 16 h with l0 ml of scmtdlatlon fluid Radioactivity was counted m a LKB counter (75% efficiency) Specific [3H]QNB binding was experimentally determined from the difference between counts m the absence and presence of 1 /~M atropine and usually amounted to 80% of the total binding Conditions were chosen to limit total binding to less than 10% of the radioactivity m the m e d m m so that the concentration of free radlohgand did not slgmficantly change during the procedure
Calculattons and stattstteal analysts Results were normalized on the basis of the protein concentration m the
218 h o m o g e n a t e [11] a n d the r e c e p t o r n u m b e r was expressed m fmol/mg p r o t e i n Equlh b n u m b i n d i n g d a t a were a n a l y z e d using L I G A N D p r o g r a m [12] This p r o g r a m fits the b i n d i n g d a t a to the e q u a t i o n d e s c r i b i n g the law o f mass action The affinities (Kd) o f r a d l o h g a n d [I25I]CYP o r [3H]QNB, specific non-selective a n t a g o m s t s , were d e t e r m i n e d from S c a t c h a r d analyses Statistical analyses o f the effects o f a g e m g a n d t r e a t m e n t s have been p e r f o r m e d using the analysis o f variance ( p a c k a g e Systat 4 m o d u l e M G H L ) a n d the F - r a t i o d e t e r m i n a t i o n [13] RESULTS Anatomtcal data
T a b l e I shows t h a t d u r i n g senescence the left v e n t n c u l a r weight ( L V W ) p r o p o r tionally increases with the b o d y weight (BW), resulting in an u n c h a n g e d L V W / B W ratio T h e m e m b r a n e p r e p a r a h o n s f r o m b o t h e x p e r i m e n t a l g r o u p s y~eld the same a m o u n t o f p r o t e i n per g o f LV Beta adrenergw and muscarmt~ receptor densltws
Figures 1 a n d 2 represent typical S c a t c h a r d t r a n s f o r m a t i o n s o f s a t u r a t i o n exp e r i m e n t a l d a t a T a b l e II shows that the m e a n densities o f the /3-adrenergic and m u s c a n m c receptors are significantly d i m i n i s h e d by 26% a n d 48%, respecttvely, In the aged m y o c a r d m m as c o m p a r e d to that o f y o u n g a d u l t s The values (Table II) for the y o u n g e r g r o u p fit well with those p r e v i o u s l y published 30 fmol/mg protein for t h e / 3 - a d r e n o c e p t o r s as c o m p a r e d to 37 a n d 29 [14,15], 100 fmol per mg o f p r o tein for the m u s c a r m i c receptors, as c o m p a r e d to 144 a n d 130 [16,17] T h e decrease o f the m u s c a n m c r e c e p t o r density in senescent left ventricles is m o r e p r o n o u n c e d than that o f t h e / 3 - a d r e n o c e p t o r s , c o n s e q u e n t l y the muscarlnlC//3-adrenergic r e c e p t o r m e a n densities r a t i o which is a r o u n d 3 m y o u n g a d u l t rat left ventricle falls to nearly 2 m aged LV (Table II) TABLE 1 ANATOMICAL DATA HOMOGENATE PREPARATIONS Young adult (n = 16)
Aged (n = 15)
~41 ± 8 596 + 12 1 74 • 004
676 + 20*** 1055 + 23*** 1 57 4- 005
4natomttal data
Body weight (BW) m g Left ventncular weight (LVW) m mg LVW/BW m mg/g Homogenate
Protein m mgper LV Yield m mg/g fresh ussue
64 ± 4 109 + 7
Means + S E ***P < 0001 n = number of experiments
106 + 5*** 101 + 6
219
0.24
0.18
A
0.12 O
0
10
20
30
40
1251-CYP specifically bound m fmol/mg protein Fig l Effect of ageing on the/~-adrenerglc receptor dens:ty Typical Scachard representations Open symbols represented the data obtained from a young adult left ventricle and filled symbols those obtained from an aged left ventricle homogenate The spec:fic [1251]CYP bound and the K 0 values, determmed by LIGAND program, were respectively 35 fmol/mg and 22 pM m the young and 21 fmol/mg and 29 pM in the aged left ventricle
0°~.~"
0.15 r~
A
0.10
A
g~ O
0.05i o
0
. . . . . .
20
40
""-.,
60
.
80
100
3H-QNB specifically bound m fmol/mg protein F:g 2 Effect of ageing on the muscanmc receptor dens:ty Typical Scachard representat:ons Open symbols represented the data obtained from a young adult left ventricle and filled symbols those obtained from an aged left ventricle homogenate The spec:fic [3H]QNB bound and the K,j values, determined by LIGAND program, were respecUvely 82 fmol/mg and 26 pM m the young and 58 fmol/mg and 24 pM in the aged left ventricle
221) TABLE 11 B-ADRENERGIC AND MUSCARINIC RECEPTOR DENSITIES Control
A g('d
(n = 8) 31 + 2 2515 4- 268 32 4- 5
(n = 61 2t 4. 2* 286t ± 479 ~,4 + 6
Density m tmol/mg Content m fmol per LV Kd in pM
(n = 8) 104 4- 7 5252 4- 524 25 + 3
(n = 8) 54 + t*** 5349 4- 18t 27 4-
MR/BR mean denstttes ratto
t t5
2 t5
13-adrenergu receptors (BR)
Density in fmol/mg Content m fmol per LV Ka m pM Muscarmu re~eptor~ (MR)
Means 4- S E *P < 005 ***P < 0001 n = number of experiments
Effects of a chronu m[uston oj drug
The body weights o f y o u n g adult rats gain 48 4- 11 g ( P < 0 001) d u r i n g saline or 40 4- l0 g (P < 0 01) d u r i n g p r o p r a n o l o l Infusion (approx 6 g of body w t / d a y ) but the body weights of the senescent rats do not significantly change In contrast. atropine Induces a loss in body weight in both groups ( i n ) o u n g adults - 2 7 49 g , P < 0 0 5 , m aged - 7 7 + 17g, P < 0 0 0 1 ) C o n s e q u e n t l y , the LV/BW ratio i n c r e a s e s l n aged rat g r o u p from 1 50 4- 0 0 9 to 1 87 + 0 II (P < 0 0 1 ) Saline Infusions have no effect on both receptor densities (Table 111) The upregulations of ¢~-adrenerglc (+50%) a n d muscarlnlC (+33"/,,) receptors by p r o p r a n o l o l and a t r o p i n e respectively are only observed in the 24 m o n t h s old rats (Table 1II) The Kd value o f the/3-adrenerglc receptors for their specific r a d l o h g a n d ([ 125IlCYP) IS slightly increased in the y o u n g adult group, 7 days after the saline perfuslon (see Table II a n d T a b l e IllB), c o n s e q u e n t l y a significant difference between the two rat groups appears After p r o p r a n o l o l t r e a t m e n t , in the y o u n g adult group, the mean K a value is similar to that observed in c o n t r o l experiments (Table ll) b u t in the aged g r o u p a weak b u t significant Increase o f the a p p a r e n t affinity for the specific radxohgand is observed F o r the muscarsnlc receptors a n d in the y o u n g adult group, the Kd values for [3H]QNB are significantly increased either after saline or atropine perfuslon (see Table II) a n d c o n s e q u e n t l y a significant difference between the two groups of rats does also a p p e a r In the aged g r o u p of rats, the Kd values are significantly changed after the saline perfuslon (see T a b l e II) but the a t r o p i n e treatment induces a weak but significant increase o f the affinity c o n s t a n t s C o m p a r a b l e slight increases in the K d values have already been observed alter a n t a g o n i s t treatment [18--20] even, as in this paper, If the isolation procedure for m e m b r a n e s starts 16--18 h after the end of the infusion
221
TABLE 111 (A) B-ADRENERGIC A N D MUSCARINlC RECEPTOR DENSITIES AND (B) K d VALUES FOR THE [125I]CYP or [~H]QNB AFTER A 7-DAY PERFUSION WITH A SALINE SOLUTION, PROPRANOLOL OR ATROPINE
( A ) Receptor densttws (fmol/mg protems ) B-Adrenerglc receptor density Sahne Propranolol Muscanmc receptor density Sahne Atropine (B) Kd values (pM) B-Adrenerglc receptors ([1251]CYP) Sahne Propranolol Muscanmc receptors ([~HIQNB) Sahne Atropine
Young adult
Aged
29 + 2 27 ~ 3
22 ± 2÷÷ 33 + 3**
88 + 9 91 ± 5
56 ± 3÷++ 74 + 7*
61 ± 10 30 ~ 5*
38 ± 5+ 62 + 10"
47 ~ 8 44 ± 8
28 ± 3+ 40 ± 4*
Number of experiments 7 and 9 m the young adult and aged groups Means ~- S E Statlst.cal analysis of the effects of the treatments *P < 0 05. **P < 0 01 Statistical analysis of the effects of ageing +P < 0 05. ++P < 0 01, +++P < 0 001
DISCUSSION The m a m findings o f this study are the foilowmg (1) the myocardial d e n s m e s m both/3-adrenerglc a n d m u s c a n m c receptors d e n s m e s decrease d u r m g ageing, (n) this d i m i n u t i o n is reversible a n d a chronic m f u s l o n with a specific a n t a g o m s t upregulates the c o r r e s p o n d m g receptor u p to values observed m y o u n g adult
Adrenergtc receptors and sympathett~ tone durmg agemg It Is well k n o w n that the cardiovascular effects o f an adrenerglc stimulation, whether they have been o b t a i n e d by exercising or directly by lsoproterenol infusion, are a t t e n u a t e d m the senescent h u m a n a n d a m m a l [7,10] F o r example m rats the m a x i m u m rate o f force d e v e l o p m e n t after isoproterenol infusion dechnes m senescent isolated perfused m t e r v e n t n c u l a r septae [9] Such a m o d i f i c a t i o n m a y revolve one o f the several c o m p o n e n t s o f the adrenerglc cascade, i n c l u d i n g the specific sarcolemmal receptors, the t r a n s d u c t l o n mechamsms, the adenylate cyclase a n d phosphodlesterase a n d the capacity of several protems to be p h o s p h o r y l a t e d [review m 4] In this report at least the first c o m p o n e n t of this system, namely the /~R receptors are modified which by Itself can explain the physiological data b u t obviously does not rule out other modifications occurring m parallel, as m c h r o m c pressure overloading (for review see Ref 21)
222 Similar results concerning the/3 receptors have recently been reported [22] during senescence m man Other investigators found an essenually unchanged B receptors densxty but (1) they were using dlhydroalprenolol, a hgand which in our work gives an important non-specific binding, (11) thexr control adult values were obtained m 7--9-month-old rats while we used younger (2--3-month-old) controls Muscarlntc receptors and vagal a~ttwt)' m ageing Physiological and pharmacological observations mdlcated that cardmc muscarmlc responsweness decreased with age, suggesting a possible change in the concentration of cardiac muscanmc receptors The posmve chronotroplc effect of a vagotomy was nearly absent in old ammals [6] Furthermore, m humans the chronotroplc response to atropine was attenuated m old persons [5] Additional mechamsms may play a role since both the acetylchohnesterase actwlty and the threshold voltage to sUmulate the vagus nerve were dlmmashed [23] The inhibition of GTP-lsoproterenol sUmulated adenylate cyclase by carbachol (100 ~,M) was also more pronounced m the young adult (-40%) than m the aged rats (-20%) [24] The present study evidences a clear and major drop in MR density in aged left ventricles which would easaly explain the age-related dechne m chollnerglc control Such a d~mmutlon is more important than that of the /3R suggestmg a d~fferent regulation and resulting m a new unbalanced/3R/MR ratio Our result~ contradict those of Narayanan and Tucker [241 who found an unchanged left ventncular MR density m 3-, 12- and 24-month-old rats At least two differences have to be pointed out between thxs work and our finding since these authors were using a different strata of rats, the Fisher-334, and also a techmque of bmdmg with a Kd for [3H] QNB 10-fold greater than m the present report or m the majority of other pubhshed studies Effect o f a chromc mJuston with an antagom~t It has been well accepted during the last 10 years that chromc infusion with a specific antagomst up-regulates the corresponding receptor Several reports have reported an mcreased/3 receptor density after chromc treatment or xnfuslon w~th a specific antagonist [18--20] Nevertheless, it is not always the case since an acute reduction m/3-receptor number has been reported m human leucocyte both m vwo and m vitro after l-h administration of certam/3 blockers [25] Slmdar results were also reported m cultured and muscle cells with alprenolol and propranolol [261 Others found no change m/3R density m rat heart after 1 week of propranolol treatment [27,28] Very few studies have dealt with the muscanmc receptor regulation after chromc infusion of a specific antagomst [30,31] A shght mcrease m the total muscanmc receptor density has been reported m the brain after a 23-day treatment with 4 0 ~ 1 0 0 mg/kg atropine [30] It is therefore not really surprising to see no change m both 13 and muscanmc receptor denslUes after a chronic treatment w~tb the antagomst m young adult
223
animals More Interesting is the finding that in the aged group, m which both densttIes are dtmmlshed, it is possible to up regulate the receptors by usmg an appropriate antagonist, thus reaching the values obtamed in young adults This specific up-regulation suggests that the drop m/3 and muscarimc receptor densities observed during baseline conditions in the aged heart is itself due to an homologous downregulation It has been repeatedly shown that the peak plasma catecholammes durmg exercise is age-dependent and mcreases in senescent people [4] Such an mterpretatton would m fact mean that the diminution m/3 adrenergIc and muscarlntc receptor densities cannot explam the attenuation of the cardmvascular effects of adrenergic and muscarimc stimulatmns It suggests that the explanation ts more located elsewhere either 'down the road' at the level of the transductmn mechanism, or in relation with the recently reported enhanced adenosme level [31 ] Several prewously published experiments support this hypothesis, as for example the finding that the motroptc effect of dibutyryl cAMP is diminished in old rats [9] REFERENCES 1 J L Fleg, Alterations in cardiovascular structure and function with advancing age Am J Cardtol 57 (1986) 33C ~ C 2 F C P Yln, H A Spurgeon, M L Welsfeldt and E G Lakatta, Mechanical properties of myocardram from hypertrophied rat hearts A comparison between hypertrophy induced by senescence and by aortic banding Clrc Res, 46 (1990) 292--300 3 E G Lakatta, Do hypertension and ageing slmdarly affect the myocardmm9 Circulation 75 (Suppl I) (1987) 69--77 4 C R Fllburn and E G Lakatta, Aged related alterations in the B-adrenerglc modulation of cardiac cell function In J E Johnson (ed), Aging and Cell Funttton, New York Plenum Publ Corp, 1984, pp 211--238 5 P Dauchot and J S Gravensteln, Effects of atropme on the electrocardiogram in different age groups Chn Pharmacol Ther 12(1975) 274---280 6 G J Kelhher and T S Conahan. Changes in vagal actwlty and response to muscarlmc receptor agomsts with age J Gerontol, 35 (1980) 842--894 7 N Narayanan and J A Derby, Alterations in the properties of B-adrenerglc receptors of myocardial membranes In aging impairments In agomst-receptor interactions and guanine nucleot~de regulation accompany dlmlmshed catecholamlne-responslveness of adenylate cyclase Mech Ageing Dev, 19 (1982) 127--139 8 P J Scarpace and 1 B Abrass, Beta-adrenerglc agonlst-medlated desensitization in senescent rats Mech Ageing Dev , 35 (1986) 25~-264 9 T Guarnlerl, C R FiIburn, G Zltnlk, G S Roth and E G Lakatta, Contractde and biochemical correlates of the aged heart Am J Phystol 239 (1980) H501--H508 10 B Chevaher, P Mansler, F Callens-EI Amranl and B Swynghedauw, B-Adrenerglc system is mo&fied in compensatory pressure cardiac overload in rats physiological and bmchemlcal evidence J Cardtovasc Pharmacol 4 (1989) 4 1 3 4 1 9 11 O H Lowry, N J Rosebrough, A L Farr and R J Randall, Protein measurement with the Fohn phenol reagent J Btol Chem 193 (1951) 265--275 12 P J Munson and D Rodbard. A versatile computerized approach for characterlzatmn of hgandbinding systems Anal Btochem 107 (1980) 220--239 13 J H Zar, Multlpsample hypotheses the analysis of varmnce In J H Zar (ed), Bwstattstwal Analysis, Prentice-Hall, New Jersey, 1984, pp 162--184 14 H H Ayobe and R C Tarazl, Reversal ofchanges in myocardml B receptors and motroplc respon-
224
15
16 17
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24
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26
27 28 29
30
31
slveness with regression of cardiac hypertrophy m renal hypertensive rats (RHR) Ctrt Res 54 (1984) 125--134 O A Gende. A Mattlazzl M C C M d h a n P Pedrovl C T a q m m G llambl and H E Cmgolanl. Renal hypertension impairs motroplc ~soproterenol effect without /3 receptor changes 4m J Phvstol 249 (1985) H 8 1 4 ~ H 8 1 9 J Z Fields. W R Roeske and E M o r k m Cardiac m u s c a n m c chohnerglc receptors J Btol Chem 253 (1978) 3251--3258 S P Baker. S Marchand. E O Nell. C A Nelson and P Posner Age-related changes m cardiac m u s c a n m c receptors decreased ablhty of the receptor to form a high affimtv agomst binding state J Gerontol 40 (1985) 141--146 P Gengo. A Skattebol. J F Moran. S Gallant M Hawthorn and J Tnggle Regulation by chromc drug administration of neuronal and cardmc calcmm channel, beta-adrenoreceptor and muscanm~. receptor levels Btochem Pharmatol 37 (1988)627--633 R D Aarons and P B Mohnoff. Changes m the density of beta adrenerglc receptors m rat lymphocytes, heart and lung after chromc treatment w~th propranolol J Pharmatol Exp Ther 221 (1981) 439 443 G Glaublger and R J Lefkowltz. Elevated beta-adrenerg~c receptor after chromc propranolol treatment Btochem Btophy~ Res Commun 78 ( 1 9 7 7 ) 7 2 0 ~ 7 2 5 B Chevaher and B Swynghedauw. Beta-adrenerglc receptors m hypertrophied and heart ladure In Swynghedauw. B (ed). Htpertroph, and Heart Fadure INSERM/J Llbbey Pans - - London. 1990 pp 201--216 M Bohm and E Erdmann. Influence o f age on beta adrenoreceptor down regulation m the hypertrophy and fading m y o c a r d m m Eur Heart J ( a b s t ) I1 (1990) 196 V V Frolkls V V Bezrukov. Y K Duplenko I V Shchegoleva. V G Shevtchuk and N S Verkhratsky. Acetylchohne m e t a b o h s m and chohnerg~c regulation of function m aging Gerontologta 19 (1973) 45 57 N Narayanan and M Tucker. A u t o n o m i c interactions m the ageing heart age-assocmted decrease m m u s c a n m c chohnerg~c receptor medmted inhibition o f / 3 adrenerg~c actwatlon of adenylate cyclase Mesh Agemg Dev 34 (1986) 249--259 A De Blasl. M Fratelh and O Marasco. Certain ~-blockers can decrease adrenerglc receptor number 1 Down regulation of receptor number by tertatolol and bopmdolol Clrt Res 63 (1988) 273--278 R J Huges. L C M a h a m and P A lnsel Certain /3-blockers can decrease adrenerglc receptor number II Down regulation of receptor number by alprenolol and propranolol m cultured lymp h o m a and muscle cells O r c Res 63 (1988) 279--285 S P Baker and L T Potter. Effect of propranolol on /3-adrenoceptors in rat hearts Br J Pharmaeol. 68 (1980) 8 - - 1 0 R H Kennedy and T E Donnely. Cardmc responsiveness after acute w~thdrawal of chromc propranolol treatment in rats Gen Pharmacol 13 (1982) 231 239 B C Wise. M ShojJ and J F Kuo. Decrease or mcrea~e m carding, m u s c a n m c chohnerglc receptor number m rats treated with metachohne or atropine Bto~hem Btoph~ Re~ Commun 92 (1980) 1 1 3 ~ 1 1 4 2 R Majocha and R J Baldessanm, Tolerance to an antlchohnerglc agent is paralleled by increased b m d m g to muscarlmc receptors m rat brain and increased behavioral response to a centrally actwe chohnomtmetlc Ltfe Set 35 (1984) 2247--2255 J C Dobson, R A Fentar and F D R o m a n o , Increased myocardml adenosine production and reduction of/3 adrenergJc contractde response m aged hearts Circ Re~ 66 (1990) 1381 1390
