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Spontaneous and renal hypertensive rats: responsiveness of isolated right and left atria to noradrenaline, isoprenaline and methoxamine
Journal of the Autonomic Nervous System, 46 (1993) 1-8
1
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00
JANS 01450
Spontaneous and renal hypertensive rats: responsiveness of isolated right and left atria to noradrenaline, isoprenaline and methoxamine Marina Queiroz Amaral Turrin a, Mariano Ferrari b and Sergio De Moraes
a
Department of Pharmacology, Institute of Biomedical Sciences, University of Sdo Paulo, SFtoPaulo, Brazil, and b Institute of Clinical Medicine, University of Padua, Padua, Italy (Received 19 November 1992) (Revision received and accepted 27 April 1993)
Key words: Spontaneously hypertensive rat; Renal hypertensive rat; Isolated right and left atria; chronotropic and inotropic response; Beta- and alpha-adrenoceptor Abstract The adrenergic responsiveness of right and left atria isolated from spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR) was studied. Right atria isolated from SHR showed subsensitivity to the chronotropic effect of noradrenaline (21.3-fold at the ECs0 level, P < 0.05) and isoprenaline (12.0-fold, P < 0.05). However, atria isolated from RHR did not exhibit any significant alteration ( P > 0.05) in sensitivity to the chronotropic effect of noradrenaline or isoprenaline. Chronotropic responsiveness to theophyline was not altered in right atria isolated from SHR or RHR. avMediated inotropic responses to noradrenaline and methoxamine were blunted in left atria isolated from SHR. Left atria isolated from RHR showed supersensitivity to the inotropic effect of noradrenaline (5.4-fold at the ECs0 level, P < 0.05) and subsensitivity to the inotropic effect of methoxamine (6.0-fold, P < 0.05). It is concluded that the present results could, at least partially, explain the reduced cardiac output observed during established hypertension in SHR and the increased cardiac output observed in the initial phase of renal hypertension in RHR.
Introduction
It is generally accepted that in several forms of hypertension there is a transition from an acute hypertensive phase with an increased cardiac output to a chronic stage of established hypertension with a reduced cardiac output. In the one-clip
Correspondence to: Professor S. De Moraes, Department of Pharmacology, Institute of Biomedical Sciences, University of S~o Paulo, Cidade Universit~ria, Butantan, CEP 05508, S~o Paulo, SP, Brazil.
one-kidney model of hypertension there is a transient overshoot of angiotensin with a fast increase in peripheral resistance, cardiac output and systemic arterial blood presssure [21]. On the other hand, in established genetically-induced hypertension there is heart hypertrophy, decreased cardiac output and elevated peripheral resistance [22,34,38]. Adrenergic responsiveness plays an important role in myocardial function and its alteration might be involved in the changes of cardiac output observed during acute renal or genetically-induced hypertension. Mammalian heart tissues
contain /3- and ~-adrenoceptors that mediate chronotropic and inotropic responses [14,17,41, 42]. Supersensitivity to isoprenaline has been reported during hypertension accompained by increased cardiac output [28,35]. On the contrary, in some forms of established hypertension a decreased sensitivity to/3-adrenoceptor agonists has been found [1,19,35]. Recent studies have shown that /31-adrenoceptors were selectively reduced during cardiac hypertrophy [5,8,10,11,39,40]. However, an increase in cardiac/3-adrenoceptors number has also been reported [44]. The role of a- and /3-adrenoceptors in the control of the inotropic response during renal- or genetically-induced hypertension has not been ascertained yet. Thus, it is plausible to assume that an increased or reduced inotropic response mediated through a- and/or/3-adrenoceptors may in part be responsible for the enhanced or reduced cardiac output observed during early renal-induced or established genetically-induced hypertension. Therefore, we investigated the chronotropic and inotropic effects of some selected /3and a-adrenoceptors in right and left atria isolated from spontaneously hypertensive rats (SHR) and renal-induced hypertensive rats (RHR).
Materials and Methods
Adult male, SHR (11-13 weeks old) from the Okamoto-Aoki strain were used. Age-matched normotensive Wistar rats (NWR) were used as control. Two-month-old Wistar rats were used to obtain animals with renal-induced hypertension (one-clip-one kidney model) rats (RHR). Briefly, under ether anesthesia, the right kidney was removed after a midline ventral incision. The left renal artery was carefully exposed and dissected out. The left renal vein was untouched. A silver clip measuring 0.1 mm thick x 2 mm width x 6 mm long was used to partially fold the renal artery inducing a constriction equivalent to 0.3 mm [36]. Sham-operated rats (SOR) submitted only to right nephrectomy were used as control. The systolic arterial blood pressure (SABP) of non-anesthetised rats was measured daily using a tail-cuff method [31,46]. The mean value of a
least three consecutive readings for each animal was used. The animals were considered as hypertensive whenever their SABP was in the range of 140 to 180 mm Hg. RHR were killed between the 6th and the 12th days after surgery. SHR and RHR were killed by a sharp blow to the head and cutting of the neck vessels. Hearts were removed and right atria were set up for isometric recording of contractions in 20-ml organ baths containing Krebs-Henseleit solution of the following composition (mmol/1): NaC1, 115.0; KCI, 4.6; CAC12.2 HzO, 2.5; KHzPO4, 1.2; MgSO 4 • 7 H 20, 2.5; NaHCO 3, 25.0 and glucose, 11.0. The bathing medium was kept at 36.5°C and gassed with 95% O z / 5 % CO 2. Right atria were set up at a diastolic tension of 1 g. After an equilibration period of 1 h, with changes of the bathing medium at 15 rain intervals a stable resting rate was reached. To reduce the influence of catecholamine dissipating mechanisms, tissues were incubated with beta-estradiol (100/zM) and cocaine (1.0/zM). Full cumulative concentrationeffect curves to noradrenaline, isoprenaline or theophyline were obtained. Only one concentration-effect curve was obtained with each atrium. The agonist concentrations producing effects which were 50% of the maximum (ECs0) were calculated and presented as geometric means with 95% confidence intervals [18]. Left atrial preparations isolated from SHR and RHR were electrically-driven using platinum electrodes and a SI-10 Narco stimulator at 1 Hz, 0.63 to 1.60 ms duration and 1.2 to 1.8 V: The stimulus intensity was individually adjusted to be 20% over the limiar value, in order to reduce neurotransmitter release [3]. Isometric transducers which were connected to left atria were manipulated using microdisplacement adjusters to ensure the desired basal tension. This was defined as 95% of the optimal lenght (L 0) for maximal active tension development [25]; The preload range was between 0.6 to 0.8 g. After an equilibration period of 45 min with changes of the bathing medium at 15 min intervals, the tissues were incubated with /3-estradiol (100 ~zM), cocaine (1.0/xM) and propranolol (100 nM). Full cumulative concentration-effect curves to the inotropic effects of noradrenaline and methoxam-
3 RIGHT
ine were obtained. Only one concentration-effect curve was o b t a i n e d with each atrium. Sensitivity changes were calculated at the ECs0 level as described above and m a x i m u m contractile response was evaluated at the p e a k of the maxim u m isometric tension development. T h e m e a n wet weight of the tissues in all groups was 17.8 + 4.4 mg. F o r statistical c o m p a r i s o n we used two-way analysis of variance ( A N O V A ) . Differences between two means were tested for statistical significance using unpaired, two-tailed Student's t-test. Differences were considered as significant at P < 0.05.
Results
T h e r e was no differences ( P > 0.05) in resting rates of right atria isolated from N W R (277 + 6 b e a t s / m i n , n = 5), S H R (270 + 5 b e a t s / m i n , n = 5) and S O R (272 + 13 b e a t s / m i n , n = 6) groups. T h e resting rate of right atria isolated from R H R did differ f r o m the o t h e r groups (308 + 5 b e a t s / m i n , n = 6, P < 0.05). T h e m a x i m u m c h r o n o t r o p i c response of right atria isolated from S H R (noradrenaline: 64 + 3 b e a t s / m i n , n = 5 and isoprenaline: 110 + 20 b e a t s / m i n , n = 6) was red u c e d ( P < 0.05), w h e n c o m p a r e d to the maxim u m c h r o n o t r o p i c response o b t a i n e d in right atria isolated from the N W R g r o u p (noradrenaline: 175 + 5 b e a t s / m i n , n = 5 and isoprenaline: 145 + 6 b e a t s / m i n , n = 6). T h e m a x i m u m chronotropic response observed in right atria isolated from the R H R g r o u p (noradrenaline: 160 + 5 b e a t s / m i n , n = 6 and isoprenaline: 115 + 10 b e a t s / m i n , n = 6) did not differ ( P > 0.05) from that o b t a i n e d in the S O R g r o u p (noradrenaline: 135 + 4 b e a t s / m i n , n = 6 and isoprenaline: 108 + 10 b e a t s / m i n , n = 6). Fig. 1 shows that in right atria isolated from the S H R g r o u p the concentration-effect curve to the c h r o n o t r o p i c effect of noradrenaline was shifted to the right (21.3-fold at the ECs0 level, P < 0.05), w h e n c o m p a r e d to the concentration-effect curve o b t a i n e d in right atria isolated f r o m the N W R group. Fig. 2 depicted that the concentration-effect curve to the c h r o n o t r o p i c effect of isoprenaline o b t a i n e d in
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Fig. 1. Mean concentration-effect curves for the chronotropic effect of noradrenaline obtained in right atria isolated from spontaneously hypertensive rats (SHR), age-matched normotensive Wistar rats (NWR), renal hypertensive rats (RHR) and sham-operated rats (SOR). The experiments were performed in the presence of/3-estradiol (100/zM) and cocaine (1 /zM). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
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Fig. 2. Mean concentration-effect curves for the chronotropic effect of isoprenaline obtained in right atria isolated from spontaneously hypertensive rats (SHR), age-matched normotensive Wistar rats (NWR), renal hypertensive rats (RHR) and sham-operated rats (SOR). The experiments were performed in the presence of fl-estradiol (100 ~M) and cocaine (1 ~M). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
4 TABLE I
Chronotropic effect of noradrenaline and isoprenaline in right atria isolated from spontaneously hypertensive rats (SHR) and renat hypertensive rats (RHR) Group
NWR SHR SOR RHR
Noradrenaline (nM)
Isoprenaline (nM)
ECs0 (95% C.I.) a
Ratio b
ECso (95% C.I.) a
Ratio b
0.58 (0.25-1.10) 12.41 (12.10-13.0) *
21.3
0.05 (0.02-10.10) 0.60 (0.20-0.90) *
12.0
0.96 (0.67-1.36) 0.40 (0.11-1.14)
2.4
0.52 (0.26-1.15) 0.90 (0.20-1.44)
1.7
A g e - m a t c h e d normotensive Wistar rats ( N W R ) and sham-operated rats (SOR) were used as control. Shown are the results of at least five experiments a Geometric m e a n with 95% confidence Interval. b ECs0 S H R / E C 5 0 N W R or ECs0 S O R / E C 5 o R H R . * Significantly different from N W R or S O R groups ( P < 0.05).
right atria isolated from the S H R group also presented a rightward displacement (12.0-fold, P < 0.05). However, as can be seen in Figs. 1 and 2 atria isolated from the R H R group did not exhibited any significant change, in sensitivity to the chronotropic effect of noradrenaline (2.4-fold, P > 0.05) or isoprenaline (1.7-fold, P > 0.05), when compared to atria isolated from the S O R group. These results are summarized in Table I. T h e r e was no difference in the chronotropic sensitivity to theophyline among the different experimental groups (NWR; EC50 = 0.15 m M with confidence interval of 0.06 and 0.36, n = 5; SHR; EC50 --- 0.14 m M with confidence interval of 0.07 and 0,17, n = 10; SOR; ECs0 = 0.15 m M with confidence interval of 0.05 and 0.35, n = 5; R H R ; ECs0 = 0.12 m M with confidence interval of 0.05 and 0.19, n = 5, P > 0.05). Active tension development in electricallydriven left atria was not different in any of the experimental groups (NWR: 0.31 + 0.09 g, n = 6; SHR: 0.30 + 0.08, g, n = 6; SOR: 0.39 + 0.05 g, n = 6; R H R : 0.44 ___0.05 g, n = 6, P > 0.05), Maximum responses to the inotropic effect of noradrenaline (NWR: 0.23 + 0.01 g, n = 6; SOR: 0.24 + 0.05 g, n = 6; R H R : 0.25 + 0.04 g, n = 6, P > 0.05) were- not significantly different, whilst only the maximtim response of left atria isolated from the R H R group to methoxamine was significantly different (N~v'VR: 0.26 + 0.02 g, n = 6; SOR: 0.27 _ 0.03 g, n = 6 ; R H R : 0 . 5 2 + 0.13 g, n = 6, P < 0.05). Inotropie responses of left atria isolated
from the S H R group to noradrenaline and methoxamine were undetectable (Figs. 3 a n d 4). The sensitivity of electrically, driven l e f t atria isolated from the S O R and R H R groups to the inotropic effect of noradrenaline and methoxamine was compared. Fig. 3 shows that left atria isolated from the: R H R group showed supersensi-
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Fig. 3. M e a n concentration-effect curves for the inotropic effect of noradrenaline obtained in electrical!y-driven left atria isolated from spontaneously hypertensive rats (SHR). age-matched normotensive Wistar rats (NWR), renal hypertensive rats ( R H R ) a n d sham-operated rats (SOR). The experiments were performed in the presence of cocaine (1 ~M)
and propranolol (100 nM). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
5 T A B L E I1
Inotropic effect of noradrenaline and methoxamine in the electrically-driven left atria isolated from spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR) Group
Noradrenaline (nM)
Methoxamine (nM)
ECs0 (95% C.I.) a
Ratio b
ECs0 (95% C.I.) a
Ratio b
NWR SHR
9.80 (8.01-17.90) ND c
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36.80 (9.01-65.0) ND c
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SOR RHR
3.60 (1.68-7.24) 0.66 (0.22-1.50)
5.4
1.60 (0.30-7.40) 9.60 (8.02-42.0)
6.0
Age-matched normotensive Wistar rats (WNR) and sham-operated rats (SOR) were used as control. Shown are the results of at least five experiments a Geometric mean with 95% confidence interval. b ECs0 S O R / E C s 0 R H R or ECs0 R H R / E C s o SOR. c Non-detectable. * Significantly different from SOR group ( P < 0.05).
tivity to the inotropic effect of noradrenaline (5.4-fold at the ECs0 level, P < 0.05) whereas subsensitivity to methoxamine (6.0-fold, P < 0.05) is shown in Fig. 4. These results are summarized in Table II.
Discussion The results of the present report show that the chronotropic and inotropic responsiveness to noradrenaline, isoprenaline and methoxamine are reduced or undetectable in right and left atria isolated from SHR. Furthermore, there is no change in sensitivity to the chronotropic effect of noradrenaline and isoprenaline in right atria isolated from RHR. Left atria isolated from RHR showed supersensitivity to the inotropic effect of noradrenaline and subsensitivity to methoxamine. fl-Adrenoceptors have been classified into at least three subtypes based on the differences in agonist and antagonist selectivities of the receptor-mediated responses (for a review see Ref. 47). Pharmacological studies in rat heart have shown consistently that the chronotropic effect of noradrenaline and isoprenaline is mediated preferentially by a functionally homogeneous flt-adrenoceptor population [2,16,24]. In failing human cardiac preparations a reduced sensitivity to agents that act via beta-adrenoceptors has been reported [4-8]. Consequently, it is plausible to assume that a down-regulation in the number of atrial ill-
adrenoceptors may in part be responsible for the subsensitivity to the chronotropic effect of noradrenaline and isoprenaline observed in SHR. Indeed, a selective discrete reduction of heart /3~adrenoceptors in 10-week-old SHR has been reported [27,30,45]. However, such a reduction in the number of /31-adrenoceptors would not be expected to shift to the right the concentrationeffect curves to noradrenaline and isoprenaline since, as previously reported [15], there is a large amount of spare fll-adrenoceptors in rat atrial tissue. An increase in the efficiency of the atrial catecholamine-dissipating mechanisms in SHR can also to be ruled out because the experiments were carried out in the presence of cocaine and fl-estradiol. It is well-known that the common mechanism of action of/3-adrenoceptor agonists is an adenylate cyclase-induced increase in cyclic AMP content [29,32]. Hence, a greatly attenuated fll-adrenoeeptor-induced increase in cyclic AMP content after stimulation with noradrenaline or isoprenaline could explain the subsensitivity observed in atria isolated from SHR. However, the chronotropic effect of the phosphodiesterase inhibitor theophyline was not affected in SHR or RHR, apparently excluding a reduction in the atrial accumulation of cyclic AMP as a possible mechanism to explain the observed subsensitivity to noradrenaline and isoprenaline. Recently, similar results were reported in failing human hearts [40]. Another possibility is provided by the finding that an inhibitory, sarcolemmal, GTP-binding
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Fig. 4. Mean concentration-effect curves for methoxamine obtained in electrically-driven left atria isolated from spontaneously hypertensive rats (SHR), age-matched Wistar nor-
motensive rats (WNR), renal hypertensive rats (RHR) and sham-operated rats (SOR). The experimentswere performed in the presence of cocaine (1 #M) and propranolol (100 nM). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
protein (Gi-protein) is increased in failing human myocardium [8,9,20,48]. Hence, an increase in Gi-protein might reduce /3-adrenoceptors-induced adenylate cyclase activity, thus decreasing the atrial sensitivity to the chronotropic effect of noradrenaline and isoprenaline in SHR. The a~-adrenoceptor-mediated response to noradrenaline and methoxamine is t h o t t ~ t to be caused by an activation of phospholipase C thereby inducing an enhancement in inositol-trisphosphate and diaeytglicerol [12,26,33]. In left atria isolated from SHR the inotropie response to noradrenaline and methoxamine was blunted whereas left atria isolated from RHR showed
supersensitivity to noradrenaline and subsensitivity to methoxamine. On the other hand, ~.~adrenoceptor-mediated inotropic effects have been observed in preparations isolated from diseased hearts [13,23,37]. Our results argue against the efficiency of cyclic AMP-independent agonists to maintain the heart force of contraction during the established phase of hypertension in SHR. According to Steinfath et al. [40] a subsensitivity to al-adrenoceptor stimulation might aggravate the development of heart failure, although the physiological relevance of al-adrenoceptor-mediated inotropic response is as yet unclear. Obviously, a reduced sensitivity to the chronotropic and inotropic effects of noradrenaline could, at least partially, explain the reduced cardiac output during established hypertension in SHR. Nevertheless, down-regulation of the aradrenoceptors cannot explain the diminished inotropic effect to noradrenaline and methoxamine, since recently it has been reported that an up-regulation of al-adrenoceptors occurs in failing human hearts [40]. The presence of supersensitivity to the inotropic effect of noradrenaline and subsensitivity to the inotropic effect of methoxamine in atria isolated from RHR is unexpected. It is generally accepted that noradrenaline is a non-selective postjunctional a-adrenoceptor agonist whilst methoxamine is a selective postjunctional a ladrenoceptor agonist. So far as the authors are aware, there is no evidence of the existence of postjunctional a2-adrenoceptors in rat atrial tissue. An involvement of /~-adrenoceptors in the inotropic response, to noradrenaline is ruled out by the presence of propranotol during the experiments. Probably, an impairment in one or more biochemical step(s) distally located from the a~adrenoceptor, such as the inositol trisphosphate step, could be responsible for the undetectable inotropic response observed in atria isolated from SHR. There is evidence that a]-adrenoceptor agonists also evoke their inotropic effect through an, as yet unidentified, G-protein [43]. Consequently, it is possible that like the/3-adrenoceptor system, the amount a n d / o r function of the G-proteins that transduce the a~-mediated inotropic effect
might be a l t e r e d in R H R . T h u s , the c o u p l i n g t h r o u g h G - p r o t e i n s to the effector systems might b e an i m p o r t a n t b i o c h e m i c a l defect r e s p o n s i b l e for the discrepancy in the i n o t r o p i c responses to n o r a d r e n a l i n e a n d m e t h o x a m i n e in left atria isolated from R H R . Nevertheless, the p r e s e n t report s u p p o r t s the p o i n t of view that o t h e r m e c h a nisms b e s i d e alpha- a n d b e t a - a d r e n o c e p t o r s d o w n - r e g u l a t i o n are i m p o r t a n t in the c h a n g e s of the c h r o n o t r o p i c a n d i n o t r o p i c responsiveness to a d r e n o c e p t o r agonists observed in atrial tissue isolated from S H R a n d R H R .
Acknowledgements This study was partly financially s u p p o r t e d by F i n a n c e i r a de E s t u d o s e Projetos ( F I N E P ) g r a n t 43820149/1, F u n d a ~ o de A m p a r o a Pesquisa do E s t a d o de S~o P a u l o ( F A P E S P ) g r a n t 8 6 / 2 9 8 1 - 0 ) a n d B a n c o I n t e r a m e r i c a n o de D e s e n v o l v i m e n t o ( B I D / U S P ) g r a n t 126.
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34
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sp0nse of failing heart to isoproterenol, Am. Heart J., 106 (1983) 535-540. Grollman, A.A., A survey of experimental studies in renal hypertension. In: Hypertension Mechanisms and Management. Onesti, G., Kim, K.E. and Moyer, J.H., Eds. Grune & Stratton, New York and London. pp. 584-586. Heller, L.J., Cardiac muscle mechanics from doca-and spontaneously hypertensive rats, Am. J. PhysioL., 235 (1978) H-82-H86. Jakob, H., Nawrath, H. and Rupp, J., Adrenoceptor-mediated changes of action potential and force of contraction in human isolated heart muscle, Br. J. Pharmacol., 94 (1988) 584-589. Juberg, E.N., Minneman, K.P. and Abel, P.W., ill- and /32-adrenoceptor binding and functional responses in tight and left atria of the rat heart, Naunyn-Schmiedeberg's Arch. Pharmacol., 330 (1985) 193-202. Katz, A.M., Myocardial contractility, force, velocity, length and time. In: Physiology of the Heart. Raven Press, New York (1977) p. 161. Kohl, C., Schmitz, W., Scholz, H., Toth, M., Doting, V. and Kalmar, P., Evidence for an alphal-adrenoceptormediated increase of inositol trisphosphate in the human heart, J. Cardiovasc. Pharmacol., 13 (1989) 324-327. Kuchii, M., Fukuda, K., Hano, T., Ohtani, H., Moahara, O., Nishio, I. and Masuyama, Y., Changes in cardiac /3-adrenoceptor concentrations in spontaneously hypertensive and experimental renal hypertensive rats, Jap. Circ. J., 45 (1980) 1104-1110. Kunos, G., Robertson, B., Ho Kan, W., Preiksaitis, H. and Mucci, L., Adrenergic reactivity of the myocardium in hypertension, Life Sci., 22 (1978) 847-854. Lefkowitz, R.J. and Caron, M.G., Adrenergic receptors. Models for the study of receptors and guanine nucleotide regulation proteins, J. Biol. Chem., 263 (1988) 4993-4996. Limas, C. and Limas, C.J., Reduced number of betaadrenergic receptors in the myocardium of spontaneously hypertensive rats, Biochem. Biophys. Res. Commun., 83 (1978) 710-714. Magaldi, J.B., Relatorio de estudo sobre nutrkjho realizado sobre os auspicios dos Fundos Universitarios de Pesquisa para a Defesa Nacional, Silo Paulo Medico (1944) p. 207. Navrath, H., Adrenoceptor-mediation changes of excitation and contraction in isolated heart muscle preparations, J. Cardiovasc. Pharmacol., 14 (Suppl. 3) (1989) S1-S10. Otani, H., Otani, H. and Das, D.K., al-Adrenoceptormediated phosphoinositol breakdown and inotropic responses in rat left ventricular pappilary muscle, Circ. Res., 62 (1988) 8-17. Pfeffer, M.A. and Frohlich, E.D., Hemodynamics of spontaneously hypertensive rats, Am. J. Physiol., 224 (1973) 1066-1071. Pfeffer, M.A., Pfeffer, J.M. and Frohlich, E.D., Hemodynamics of the spontaneously hypertensive rat: effect of isoproterenol, Proc. Soc. Exp. Biol. Med., 145 (1974) 1025 - 1030.
36 Schaffemburg, C.A., Device to control constrictkm of main renal artery for production of hypertension in small animals, Proc. Soc. Exp. Biol. Med., 101 (1959) 676-679. 37 Schumann, H.J., Wagner, J., Knorr, A., Reisemeister. J.C., Sadony, V. and Scramm, G., Demonstration in human atrial preparations of a~-adrenoceptors mediating positive inotropic effects. Naunyn-Schmiedeberg's Arch. Pharmacol., 302 (1978) 336-346. 38 Sen, S. and Tarazi, R.C., Cardiovascular hypertrophy in spontaneously hypertensive rats, J. Hypertension, 4 (Suppl. 3) (1986) 5123-5126. 39 Steinfath, M., Geertz, B., Schmitz, W., Scholz, H., Haverich, A., Breuil, I., Hanrath, P., Reupcke, C.. Siground, C. and Lo, H.B., Distinct down-regulation of cardiac a l- and /32-adrenoceptors in different human heart diseases, Naunyn-Schmiedeberg's Arch. Pharmacol., 343 (1991) 217-220. 40 Steinfath, M., Danielsen, W., vonder Leyen, H., Mende, U., Neumann, J., Nose, M., Reich, T., Schmitz, W., Scholz, H., Starbatty, J., Stein, B., Doting, V., Kalmar, P. and Haverich, A., Reduced a l- and fl2-adrenoceptor-mediated positive inotropic effects in human end-stage heart failure, Br. J. Pharmacol., 105 (1992) 463-469. 41 Tung, L.H., Rand, M.J., Drummer, O.H. and Louis, W.J., Positive inotropic response produced by ot-adrenoceptors in the pithed rat, J. Auton. Pharmacol., 2 (1982) 217-223. 42 Tung, L.H., Rand, M.J. and Louis, W.J., Calcium antagonists inhibit positive chronotropic responses to al-adrenoceptor mediated activation in rat isolated atria, European J. Pharmacol., 133 (1987) 177-184. 43 Vago, T., Belivacgua, M., Norbiato, G., Baldi, G., Chebat, E., Bertora, P., Baroldi, G. and Accinni. R.. Identification of al-adrenergic receptors on sarcolemma from normal subjects and patients with idiopathic dilated cardiomyopathy: characteristics and linkage to GTB-binding protein. Circ. Res., 64 (1988) 474-481. 44 Vatner, D.E., Vatner, S.F.. Fujii, A.M. and Homey, C.J.. Loss of high affinity cardiac beta-adrenergic receptors m dog with heart failure, J. Clin. Invest.. 76 (1984) 2259-2264. 45 Yamada, S., Ishima, T., Tomita, T.. Hayashi, M.. Okada. T, and Hayashi, E., Alterations in cardiac a- and 3-adrenoceptors during the development of spontaneous hypertension, J. Pharmacol. Exp. Ther.. 228 (1984) 454-460. 46 Williams, J.R., Harrison, T.R. and Grollman. A., Asimple method for determining the systolic blood pressure of the unanesthetised rat, J. Clin. Invest., 18 (1939) 373-378. 47 Zaagsma, J. and Nahorski. S.R.. Is the adipocyte betaadrenoceptor a prototype for the recently cloned atypycal /33-adrenoceptor?, Trends Pharmacol. Sci., 11 (1990)3-7. 48 Zerkowski, H.R., Ikezono. K.. Rohm, N., Reidemeister. J.C. and Brodde, O-E., Human myocardial beta-adrenoceptors: demonstration of both ill- and fl2-adrenoceptor mediating contractile responses to beta-agonists on the isolated right atrium, Naunyn-Sehmiedeberg's Arch. Pharmacol., 332 (1986) 142-147.
1
© 1993 Elsevier Science Publishers B.V. All rights reserved 0165-1838/93/$06.00
JANS 01450
Spontaneous and renal hypertensive rats: responsiveness of isolated right and left atria to noradrenaline, isoprenaline and methoxamine Marina Queiroz Amaral Turrin a, Mariano Ferrari b and Sergio De Moraes
a
Department of Pharmacology, Institute of Biomedical Sciences, University of Sdo Paulo, SFtoPaulo, Brazil, and b Institute of Clinical Medicine, University of Padua, Padua, Italy (Received 19 November 1992) (Revision received and accepted 27 April 1993)
Key words: Spontaneously hypertensive rat; Renal hypertensive rat; Isolated right and left atria; chronotropic and inotropic response; Beta- and alpha-adrenoceptor Abstract The adrenergic responsiveness of right and left atria isolated from spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR) was studied. Right atria isolated from SHR showed subsensitivity to the chronotropic effect of noradrenaline (21.3-fold at the ECs0 level, P < 0.05) and isoprenaline (12.0-fold, P < 0.05). However, atria isolated from RHR did not exhibit any significant alteration ( P > 0.05) in sensitivity to the chronotropic effect of noradrenaline or isoprenaline. Chronotropic responsiveness to theophyline was not altered in right atria isolated from SHR or RHR. avMediated inotropic responses to noradrenaline and methoxamine were blunted in left atria isolated from SHR. Left atria isolated from RHR showed supersensitivity to the inotropic effect of noradrenaline (5.4-fold at the ECs0 level, P < 0.05) and subsensitivity to the inotropic effect of methoxamine (6.0-fold, P < 0.05). It is concluded that the present results could, at least partially, explain the reduced cardiac output observed during established hypertension in SHR and the increased cardiac output observed in the initial phase of renal hypertension in RHR.
Introduction
It is generally accepted that in several forms of hypertension there is a transition from an acute hypertensive phase with an increased cardiac output to a chronic stage of established hypertension with a reduced cardiac output. In the one-clip
Correspondence to: Professor S. De Moraes, Department of Pharmacology, Institute of Biomedical Sciences, University of S~o Paulo, Cidade Universit~ria, Butantan, CEP 05508, S~o Paulo, SP, Brazil.
one-kidney model of hypertension there is a transient overshoot of angiotensin with a fast increase in peripheral resistance, cardiac output and systemic arterial blood presssure [21]. On the other hand, in established genetically-induced hypertension there is heart hypertrophy, decreased cardiac output and elevated peripheral resistance [22,34,38]. Adrenergic responsiveness plays an important role in myocardial function and its alteration might be involved in the changes of cardiac output observed during acute renal or genetically-induced hypertension. Mammalian heart tissues
contain /3- and ~-adrenoceptors that mediate chronotropic and inotropic responses [14,17,41, 42]. Supersensitivity to isoprenaline has been reported during hypertension accompained by increased cardiac output [28,35]. On the contrary, in some forms of established hypertension a decreased sensitivity to/3-adrenoceptor agonists has been found [1,19,35]. Recent studies have shown that /31-adrenoceptors were selectively reduced during cardiac hypertrophy [5,8,10,11,39,40]. However, an increase in cardiac/3-adrenoceptors number has also been reported [44]. The role of a- and /3-adrenoceptors in the control of the inotropic response during renal- or genetically-induced hypertension has not been ascertained yet. Thus, it is plausible to assume that an increased or reduced inotropic response mediated through a- and/or/3-adrenoceptors may in part be responsible for the enhanced or reduced cardiac output observed during early renal-induced or established genetically-induced hypertension. Therefore, we investigated the chronotropic and inotropic effects of some selected /3and a-adrenoceptors in right and left atria isolated from spontaneously hypertensive rats (SHR) and renal-induced hypertensive rats (RHR).
Materials and Methods
Adult male, SHR (11-13 weeks old) from the Okamoto-Aoki strain were used. Age-matched normotensive Wistar rats (NWR) were used as control. Two-month-old Wistar rats were used to obtain animals with renal-induced hypertension (one-clip-one kidney model) rats (RHR). Briefly, under ether anesthesia, the right kidney was removed after a midline ventral incision. The left renal artery was carefully exposed and dissected out. The left renal vein was untouched. A silver clip measuring 0.1 mm thick x 2 mm width x 6 mm long was used to partially fold the renal artery inducing a constriction equivalent to 0.3 mm [36]. Sham-operated rats (SOR) submitted only to right nephrectomy were used as control. The systolic arterial blood pressure (SABP) of non-anesthetised rats was measured daily using a tail-cuff method [31,46]. The mean value of a
least three consecutive readings for each animal was used. The animals were considered as hypertensive whenever their SABP was in the range of 140 to 180 mm Hg. RHR were killed between the 6th and the 12th days after surgery. SHR and RHR were killed by a sharp blow to the head and cutting of the neck vessels. Hearts were removed and right atria were set up for isometric recording of contractions in 20-ml organ baths containing Krebs-Henseleit solution of the following composition (mmol/1): NaC1, 115.0; KCI, 4.6; CAC12.2 HzO, 2.5; KHzPO4, 1.2; MgSO 4 • 7 H 20, 2.5; NaHCO 3, 25.0 and glucose, 11.0. The bathing medium was kept at 36.5°C and gassed with 95% O z / 5 % CO 2. Right atria were set up at a diastolic tension of 1 g. After an equilibration period of 1 h, with changes of the bathing medium at 15 rain intervals a stable resting rate was reached. To reduce the influence of catecholamine dissipating mechanisms, tissues were incubated with beta-estradiol (100/zM) and cocaine (1.0/zM). Full cumulative concentrationeffect curves to noradrenaline, isoprenaline or theophyline were obtained. Only one concentration-effect curve was obtained with each atrium. The agonist concentrations producing effects which were 50% of the maximum (ECs0) were calculated and presented as geometric means with 95% confidence intervals [18]. Left atrial preparations isolated from SHR and RHR were electrically-driven using platinum electrodes and a SI-10 Narco stimulator at 1 Hz, 0.63 to 1.60 ms duration and 1.2 to 1.8 V: The stimulus intensity was individually adjusted to be 20% over the limiar value, in order to reduce neurotransmitter release [3]. Isometric transducers which were connected to left atria were manipulated using microdisplacement adjusters to ensure the desired basal tension. This was defined as 95% of the optimal lenght (L 0) for maximal active tension development [25]; The preload range was between 0.6 to 0.8 g. After an equilibration period of 45 min with changes of the bathing medium at 15 min intervals, the tissues were incubated with /3-estradiol (100 ~zM), cocaine (1.0/xM) and propranolol (100 nM). Full cumulative concentration-effect curves to the inotropic effects of noradrenaline and methoxam-
3 RIGHT
ine were obtained. Only one concentration-effect curve was o b t a i n e d with each atrium. Sensitivity changes were calculated at the ECs0 level as described above and m a x i m u m contractile response was evaluated at the p e a k of the maxim u m isometric tension development. T h e m e a n wet weight of the tissues in all groups was 17.8 + 4.4 mg. F o r statistical c o m p a r i s o n we used two-way analysis of variance ( A N O V A ) . Differences between two means were tested for statistical significance using unpaired, two-tailed Student's t-test. Differences were considered as significant at P < 0.05.
Results
T h e r e was no differences ( P > 0.05) in resting rates of right atria isolated from N W R (277 + 6 b e a t s / m i n , n = 5), S H R (270 + 5 b e a t s / m i n , n = 5) and S O R (272 + 13 b e a t s / m i n , n = 6) groups. T h e resting rate of right atria isolated from R H R did differ f r o m the o t h e r groups (308 + 5 b e a t s / m i n , n = 6, P < 0.05). T h e m a x i m u m c h r o n o t r o p i c response of right atria isolated from S H R (noradrenaline: 64 + 3 b e a t s / m i n , n = 5 and isoprenaline: 110 + 20 b e a t s / m i n , n = 6) was red u c e d ( P < 0.05), w h e n c o m p a r e d to the maxim u m c h r o n o t r o p i c response o b t a i n e d in right atria isolated from the N W R g r o u p (noradrenaline: 175 + 5 b e a t s / m i n , n = 5 and isoprenaline: 145 + 6 b e a t s / m i n , n = 6). T h e m a x i m u m chronotropic response observed in right atria isolated from the R H R g r o u p (noradrenaline: 160 + 5 b e a t s / m i n , n = 6 and isoprenaline: 115 + 10 b e a t s / m i n , n = 6) did not differ ( P > 0.05) from that o b t a i n e d in the S O R g r o u p (noradrenaline: 135 + 4 b e a t s / m i n , n = 6 and isoprenaline: 108 + 10 b e a t s / m i n , n = 6). Fig. 1 shows that in right atria isolated from the S H R g r o u p the concentration-effect curve to the c h r o n o t r o p i c effect of noradrenaline was shifted to the right (21.3-fold at the ECs0 level, P < 0.05), w h e n c o m p a r e d to the concentration-effect curve o b t a i n e d in right atria isolated f r o m the N W R group. Fig. 2 depicted that the concentration-effect curve to the c h r o n o t r o p i c effect of isoprenaline o b t a i n e d in
ATRIUM
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Fig. 1. Mean concentration-effect curves for the chronotropic effect of noradrenaline obtained in right atria isolated from spontaneously hypertensive rats (SHR), age-matched normotensive Wistar rats (NWR), renal hypertensive rats (RHR) and sham-operated rats (SOR). The experiments were performed in the presence of/3-estradiol (100/zM) and cocaine (1 /zM). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
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Fig. 2. Mean concentration-effect curves for the chronotropic effect of isoprenaline obtained in right atria isolated from spontaneously hypertensive rats (SHR), age-matched normotensive Wistar rats (NWR), renal hypertensive rats (RHR) and sham-operated rats (SOR). The experiments were performed in the presence of fl-estradiol (100 ~M) and cocaine (1 ~M). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
4 TABLE I
Chronotropic effect of noradrenaline and isoprenaline in right atria isolated from spontaneously hypertensive rats (SHR) and renat hypertensive rats (RHR) Group
NWR SHR SOR RHR
Noradrenaline (nM)
Isoprenaline (nM)
ECs0 (95% C.I.) a
Ratio b
ECso (95% C.I.) a
Ratio b
0.58 (0.25-1.10) 12.41 (12.10-13.0) *
21.3
0.05 (0.02-10.10) 0.60 (0.20-0.90) *
12.0
0.96 (0.67-1.36) 0.40 (0.11-1.14)
2.4
0.52 (0.26-1.15) 0.90 (0.20-1.44)
1.7
A g e - m a t c h e d normotensive Wistar rats ( N W R ) and sham-operated rats (SOR) were used as control. Shown are the results of at least five experiments a Geometric m e a n with 95% confidence Interval. b ECs0 S H R / E C 5 0 N W R or ECs0 S O R / E C 5 o R H R . * Significantly different from N W R or S O R groups ( P < 0.05).
right atria isolated from the S H R group also presented a rightward displacement (12.0-fold, P < 0.05). However, as can be seen in Figs. 1 and 2 atria isolated from the R H R group did not exhibited any significant change, in sensitivity to the chronotropic effect of noradrenaline (2.4-fold, P > 0.05) or isoprenaline (1.7-fold, P > 0.05), when compared to atria isolated from the S O R group. These results are summarized in Table I. T h e r e was no difference in the chronotropic sensitivity to theophyline among the different experimental groups (NWR; EC50 = 0.15 m M with confidence interval of 0.06 and 0.36, n = 5; SHR; EC50 --- 0.14 m M with confidence interval of 0.07 and 0,17, n = 10; SOR; ECs0 = 0.15 m M with confidence interval of 0.05 and 0.35, n = 5; R H R ; ECs0 = 0.12 m M with confidence interval of 0.05 and 0.19, n = 5, P > 0.05). Active tension development in electricallydriven left atria was not different in any of the experimental groups (NWR: 0.31 + 0.09 g, n = 6; SHR: 0.30 + 0.08, g, n = 6; SOR: 0.39 + 0.05 g, n = 6; R H R : 0.44 ___0.05 g, n = 6, P > 0.05), Maximum responses to the inotropic effect of noradrenaline (NWR: 0.23 + 0.01 g, n = 6; SOR: 0.24 + 0.05 g, n = 6; R H R : 0.25 + 0.04 g, n = 6, P > 0.05) were- not significantly different, whilst only the maximtim response of left atria isolated from the R H R group to methoxamine was significantly different (N~v'VR: 0.26 + 0.02 g, n = 6; SOR: 0.27 _ 0.03 g, n = 6 ; R H R : 0 . 5 2 + 0.13 g, n = 6, P < 0.05). Inotropie responses of left atria isolated
from the S H R group to noradrenaline and methoxamine were undetectable (Figs. 3 a n d 4). The sensitivity of electrically, driven l e f t atria isolated from the S O R and R H R groups to the inotropic effect of noradrenaline and methoxamine was compared. Fig. 3 shows that left atria isolated from the: R H R group showed supersensi-
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Fig. 3. M e a n concentration-effect curves for the inotropic effect of noradrenaline obtained in electrical!y-driven left atria isolated from spontaneously hypertensive rats (SHR). age-matched normotensive Wistar rats (NWR), renal hypertensive rats ( R H R ) a n d sham-operated rats (SOR). The experiments were performed in the presence of cocaine (1 ~M)
and propranolol (100 nM). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
5 T A B L E I1
Inotropic effect of noradrenaline and methoxamine in the electrically-driven left atria isolated from spontaneously hypertensive rats (SHR) and renal hypertensive rats (RHR) Group
Noradrenaline (nM)
Methoxamine (nM)
ECs0 (95% C.I.) a
Ratio b
ECs0 (95% C.I.) a
Ratio b
NWR SHR
9.80 (8.01-17.90) ND c
-
36.80 (9.01-65.0) ND c
_
SOR RHR
3.60 (1.68-7.24) 0.66 (0.22-1.50)
5.4
1.60 (0.30-7.40) 9.60 (8.02-42.0)
6.0
Age-matched normotensive Wistar rats (WNR) and sham-operated rats (SOR) were used as control. Shown are the results of at least five experiments a Geometric mean with 95% confidence interval. b ECs0 S O R / E C s 0 R H R or ECs0 R H R / E C s o SOR. c Non-detectable. * Significantly different from SOR group ( P < 0.05).
tivity to the inotropic effect of noradrenaline (5.4-fold at the ECs0 level, P < 0.05) whereas subsensitivity to methoxamine (6.0-fold, P < 0.05) is shown in Fig. 4. These results are summarized in Table II.
Discussion The results of the present report show that the chronotropic and inotropic responsiveness to noradrenaline, isoprenaline and methoxamine are reduced or undetectable in right and left atria isolated from SHR. Furthermore, there is no change in sensitivity to the chronotropic effect of noradrenaline and isoprenaline in right atria isolated from RHR. Left atria isolated from RHR showed supersensitivity to the inotropic effect of noradrenaline and subsensitivity to methoxamine. fl-Adrenoceptors have been classified into at least three subtypes based on the differences in agonist and antagonist selectivities of the receptor-mediated responses (for a review see Ref. 47). Pharmacological studies in rat heart have shown consistently that the chronotropic effect of noradrenaline and isoprenaline is mediated preferentially by a functionally homogeneous flt-adrenoceptor population [2,16,24]. In failing human cardiac preparations a reduced sensitivity to agents that act via beta-adrenoceptors has been reported [4-8]. Consequently, it is plausible to assume that a down-regulation in the number of atrial ill-
adrenoceptors may in part be responsible for the subsensitivity to the chronotropic effect of noradrenaline and isoprenaline observed in SHR. Indeed, a selective discrete reduction of heart /3~adrenoceptors in 10-week-old SHR has been reported [27,30,45]. However, such a reduction in the number of /31-adrenoceptors would not be expected to shift to the right the concentrationeffect curves to noradrenaline and isoprenaline since, as previously reported [15], there is a large amount of spare fll-adrenoceptors in rat atrial tissue. An increase in the efficiency of the atrial catecholamine-dissipating mechanisms in SHR can also to be ruled out because the experiments were carried out in the presence of cocaine and fl-estradiol. It is well-known that the common mechanism of action of/3-adrenoceptor agonists is an adenylate cyclase-induced increase in cyclic AMP content [29,32]. Hence, a greatly attenuated fll-adrenoeeptor-induced increase in cyclic AMP content after stimulation with noradrenaline or isoprenaline could explain the subsensitivity observed in atria isolated from SHR. However, the chronotropic effect of the phosphodiesterase inhibitor theophyline was not affected in SHR or RHR, apparently excluding a reduction in the atrial accumulation of cyclic AMP as a possible mechanism to explain the observed subsensitivity to noradrenaline and isoprenaline. Recently, similar results were reported in failing human hearts [40]. Another possibility is provided by the finding that an inhibitory, sarcolemmal, GTP-binding
LEFT
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Fig. 4. Mean concentration-effect curves for methoxamine obtained in electrically-driven left atria isolated from spontaneously hypertensive rats (SHR), age-matched Wistar nor-
motensive rats (WNR), renal hypertensive rats (RHR) and sham-operated rats (SOR). The experimentswere performed in the presence of cocaine (1 #M) and propranolol (100 nM). Vertical bars represent S.E.M. and number in parentheses indicate the number of experiments.
protein (Gi-protein) is increased in failing human myocardium [8,9,20,48]. Hence, an increase in Gi-protein might reduce /3-adrenoceptors-induced adenylate cyclase activity, thus decreasing the atrial sensitivity to the chronotropic effect of noradrenaline and isoprenaline in SHR. The a~-adrenoceptor-mediated response to noradrenaline and methoxamine is t h o t t ~ t to be caused by an activation of phospholipase C thereby inducing an enhancement in inositol-trisphosphate and diaeytglicerol [12,26,33]. In left atria isolated from SHR the inotropie response to noradrenaline and methoxamine was blunted whereas left atria isolated from RHR showed
supersensitivity to noradrenaline and subsensitivity to methoxamine. On the other hand, ~.~adrenoceptor-mediated inotropic effects have been observed in preparations isolated from diseased hearts [13,23,37]. Our results argue against the efficiency of cyclic AMP-independent agonists to maintain the heart force of contraction during the established phase of hypertension in SHR. According to Steinfath et al. [40] a subsensitivity to al-adrenoceptor stimulation might aggravate the development of heart failure, although the physiological relevance of al-adrenoceptor-mediated inotropic response is as yet unclear. Obviously, a reduced sensitivity to the chronotropic and inotropic effects of noradrenaline could, at least partially, explain the reduced cardiac output during established hypertension in SHR. Nevertheless, down-regulation of the aradrenoceptors cannot explain the diminished inotropic effect to noradrenaline and methoxamine, since recently it has been reported that an up-regulation of al-adrenoceptors occurs in failing human hearts [40]. The presence of supersensitivity to the inotropic effect of noradrenaline and subsensitivity to the inotropic effect of methoxamine in atria isolated from RHR is unexpected. It is generally accepted that noradrenaline is a non-selective postjunctional a-adrenoceptor agonist whilst methoxamine is a selective postjunctional a ladrenoceptor agonist. So far as the authors are aware, there is no evidence of the existence of postjunctional a2-adrenoceptors in rat atrial tissue. An involvement of /~-adrenoceptors in the inotropic response, to noradrenaline is ruled out by the presence of propranotol during the experiments. Probably, an impairment in one or more biochemical step(s) distally located from the a~adrenoceptor, such as the inositol trisphosphate step, could be responsible for the undetectable inotropic response observed in atria isolated from SHR. There is evidence that a]-adrenoceptor agonists also evoke their inotropic effect through an, as yet unidentified, G-protein [43]. Consequently, it is possible that like the/3-adrenoceptor system, the amount a n d / o r function of the G-proteins that transduce the a~-mediated inotropic effect
might be a l t e r e d in R H R . T h u s , the c o u p l i n g t h r o u g h G - p r o t e i n s to the effector systems might b e an i m p o r t a n t b i o c h e m i c a l defect r e s p o n s i b l e for the discrepancy in the i n o t r o p i c responses to n o r a d r e n a l i n e a n d m e t h o x a m i n e in left atria isolated from R H R . Nevertheless, the p r e s e n t report s u p p o r t s the p o i n t of view that o t h e r m e c h a nisms b e s i d e alpha- a n d b e t a - a d r e n o c e p t o r s d o w n - r e g u l a t i o n are i m p o r t a n t in the c h a n g e s of the c h r o n o t r o p i c a n d i n o t r o p i c responsiveness to a d r e n o c e p t o r agonists observed in atrial tissue isolated from S H R a n d R H R .
Acknowledgements This study was partly financially s u p p o r t e d by F i n a n c e i r a de E s t u d o s e Projetos ( F I N E P ) g r a n t 43820149/1, F u n d a ~ o de A m p a r o a Pesquisa do E s t a d o de S~o P a u l o ( F A P E S P ) g r a n t 8 6 / 2 9 8 1 - 0 ) a n d B a n c o I n t e r a m e r i c a n o de D e s e n v o l v i m e n t o ( B I D / U S P ) g r a n t 126.
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