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The effects of diabetes on β-adrenoceptor mediated responsiveness of human and rat atria
Diabetes Research and Clinical Practice 40 (1998) 113 – 122
The effects of diabetes on b-adrenoceptor mediated responsiveness of human and rat atria U8 . Deniz Dinc¸er, Arzu Onay, Nuray Arı, A. Tanju O8 zc¸elikay, V. Melih Altan * Department of Pharmacology, Faculty of Pharmacy, Uni6ersity of Ankara, Tandog˜an, 06100 Ankara, Turkey Accepted 10 March 1998
Abstract The literature on the influence of diabetes on cardiac b-adrenoceptors is still a matter of controversy. Hence, in the present study, the responsiveness of spontaneously beating right atria from streptozotocin (STZ)-diabetic rats to b-adrenoceptor agonists were compared with those from non-diabetic controls. The responsiveness of right atria from 8-week diabetic rats to the chronotropic effects of isoprenaline, noradrenaline and fenoterol was found to be unchanged. As the disease progressed, on the other hand, the diabetic atria were found to have decreased responsiveness to the chronotropic effects of noradrenaline. The pD2 value and maximum chronotropic effect of noradrenaline were decreased in 14-week diabetic right atria when compared with those from age-matched controls. A significant decrease in the maximum chronotropic response to isoprenaline with no change in pD2 value was also observed in 14-week diabetes. These results suggest that b1- but not b2-adrenoceptor mediated chronotropic responses were reduced in the right atria due to the increase in the duration of diabetes. On the other hand, the inotropic responses to b-adrenoceptor agonists were also assessed on diabetic and nondiabetic human atrial tissue. There were no significant differences in the inotropic responses to each agonists in either of the diabetic and nondiabetic human atrial tissues. The full agonist potency order was isoprenaline ] fenoterol\ noradrenaline. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetes; b-Adrenoceptors; Rat right atria; Human atrial appendage
1. Introduction * Corresponding author. Tel.: +90 312 2134478/2126805, ext. 160; fax: + 90 312 2131081; e-mail: maltan@ pharmacy.ankara.edu.tr
Chronic diabetes mellitus is known to be associated with cardiac complications in both clinical and experimental settings. Among the prominent
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defects observed in chemically induced diabetic rats are the reduced responsiveness of cardiac preparations to the inotropic and chronotropic effects of b-adrenoceptor agonists stimulation [1,2]. While the mechanisms leading to the depressed responses to b-adrenoceptor agonists of cardiac preparations from diabetic rats are not well understood, there are a number of possible explanations, one of which is the decrease in the number of cardiac b-adrenoceptors [3 – 5]. In fact, Savarase and Berkowitz [6] reported a 28% reduction in the number of b-adrenoceptors accompanied by a 24% decrease in the heart rate of STZ-diabetic rats compared to controls. b-Adrenoceptors of both b1- and b2subtypes, on the other hand, have been shown to coexist in certain cardiac tissues [7 – 10], but only b1-adrenoceptors appear to mediate the rate and tension responses in the guinea-pig [11,12], rabbit [13] and rat [14] atria. Several radioligand binding studies using human right atrium have also demonstrated a mixed b1plus b2-adrenoceptor population, although the population of each subtype varied [15,16]. In addition, it was demonstrated that in the human right atrium both b1- and b2-adrenoceptors are functionally coupled to the adenylate cyclase system [17] and both subtypes of badrenoceptors are reported to contribute to the cardiac responses of b-agonists [18]. Thus, in the human right atrium both b1- and b2adrenoceptors have been suggested to be involved in the physiological regulation of the force of contraction and/or heart rate. Although diabetes has been shown to alter the sensitivity, responsiveness and density of badrenoceptors in the heart, no data, to our knowledge, are available on the influence of diabetes on selective b2-adrenoceptor responsiveness in the heart. The aim of the present study was, therefore, to investigate the effects of diabetes on selective b1- and b2-adrenoceptor stimulation. For this purpose, an electrically driven muscle strip of the right atrial appendages from diabetic patients and spontaneously beating right atria from STZ-diabetic rats were used.
2. Materials and methods
2.1. Induction of experimental diabetes. Male Albino rats weighing 200–250 g (Ankara University Faculty of Pharmacy Animal Care Unit) were used for this study. The rats were housed two animals per cage in a room with a 12 h light:12 h dark cycle. They were given regular laboratory chow and water ad libitum throughout the experiment. Diabetes was chemically induced with streptozotocin (Sigma, St Louis, MO) 45 mg kg − 1 dissolved in citrate buffer (pH 4.5) administered as a single intravenous (i.v.) tail-vein injection under light ether anesthesia. The control rats were injected with an equivalent volume of the vehicle only. At 3 days following the STZ injection, the rats were checked for glycosuria semiquantitatively using Urine-Glucostix reagent strips (Diastix, Ames Division, Bayer Diagnostics, UK). Only rats exhibiting glycosuria were then analyzed for hyperglycaemia with Glucostix reagent strips (Glucofilm, Bayer Diagnostics, Germany) read by a Glucometer (Glucometer III, Bayer Diagnostics, France) and those with a blood glucose level ] 16.6 mM were designated diabetic. In order to investigate the time course of the development of the observed responses, diabetic rats were studied at 8 and 14 weeks after the induction of diabetes. The control rats used for comparison were age-matched to the diabetic rats. Final blood glucose levels were measured on samples taken when the rats were killed.
2.2. Rat right atrial preparations At the time of death, the hearts removed from the control and diabetic rats and immediately placed in a beaker containing ice-cold oxygenated Krebs-bicarbonate solution (pH 7.4) of the following composition (mM): NaCl, 118.4; KCl, 4.7; CaCl2, 2.5; NaHCO3, 25; MgSO4, 1.2; KH2PO4, 1.2 and glucose 11.7. After expressing blood from the heart, the ventricular tissue, fat and connective tissue were trimmed off. The atrial tissue was removed and transferred to another beaker containing cold fresh Krebs-bicarbonate solution. The right atria were then carefully dissected and
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suspended vertically in a 20-ml tissue bath of the above solution. One end of the right atria was attached to a tissue holder and the other end was mounted by silk thread to an isometric transducer (Ugo Basile, No. 7004, Varese, Italy) that was connected to a microdynamometer (Ugo Basile, Unirecord). The right atria were allowed to beat spontaneously. The tissue preparations were equilibrated at 37°C for 60 min in Krebs-bicarbonate solution aerated with a mixture of 95% O2 and 5% CO2. During this period, the Krebs-bicarbonate solution in the tissue bath was replaced every 15 min. Resting tensions of 1 g in the tissue preparations were maintained throughout the experiment.
O2 and 5% CO2 at room temperature and transported immediately to the laboratory. The preparation of the tissues was begun within 5–15 min after surgical removal. The atrial appendages were dissected without causing visible damage to the epicardium. The strips were cut to a thickness B 1 mm to facilitate diffusion of oxygen and drugs. The muscles were then prepared for tension recordings as described above and electrically stimulated via two platinum electrodes placed on the tissue at a frequency of 1.0 Hz with rectangular wave pulses of 5 ms duration at a voltage approximately 20% above the threshold using a stimulator (Grass Stimulator S44).
2.3. Human right atrial preparations
2.4. Experimental procedure
Human right atrial appendages were obtained from seven diabetic patients (five male, two female; mean age 55.8592.8 (47 – 65)) and 12 nondiabetic patients (11 male, one female; mean age 529 3.4 (36–69)) undergoing coronary artery bypass grafting. No patient suffered from acute myocardial failure; none of the patients had received catecholamines for at least three weeks before the operation. Diabetic patients had been treated with oral sulfonylureas (n=1), biguanides (n =1), insulin (n =5), calcium antagonists (n = 6), nitrovasodilators (n =7) and aspirin (n = 6). Nondiabetic patients, on the other hand, had received calcium antagonists (n =11), nitrovasodilators (n= 11), ACE inhibitors (n =1) and aspirin (n= 10). In addition, one nondiabetic patient had been treated with digitalis glycosides. Care was taken to ensure that donor patients had not received b-adrenoceptor antagonists prior to surgery. The blood glucose levels of diabetic patients had been regulated before the operation. All patients therefore underwent surgery with nearnormal blood glucose concentrations. Premedication consisted of dolantin and atropine and the operation was carried out under balanced anesthesia with fentanyl and isoflurane. In some cases N2O was added. Pancuronium was used as the muscle relaxant. Once removed, the tissue was immediately placed in a sealed vial containing Krebs-bicarbonate solution oxygenated with 95%
The tissues were allowed to equilibrate for 60 min. After ensuring steady base-line recordings, the tissues were sensitized to agonists by a brief exposure to isoprenaline (5× 10 − 6 M). The tissues were again washed several times in order to obtain steady baselines, and then a cumulative concentration –effect curve to the test agonist was performed. Agonist responses were measured as the increase in rate of the right atrium and increase in tension of the human atrial appendage. In the experiments performed on human atrial tissue, all responses were expressed as the percentage of maximum response to isoprenaline obtained in nondiabetic preparations. In all experiments, where noradrenaline was the agonist, concentration –effect curves were obtained in the presence of prazosin (5× 10 − 9 M) in order to block a-adrenoceptors. The tissues were equilibrated with prazosin for 20 min before noradrenaline was added.
2.5. Drugs The drugs used in this study were as follows: streptozotocin (Sigma), isoproteronol (Sigma), fenoterol (Sigma), noradrenaline (Sigma) and prazosin (Sigma). All the drug stock solutions were freshly prepared in distilled water and agonist serial dilutions made in 0.9% NaCl.
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Table 1 General characteristics of the rats n
Body weight (BW) (g) Blood glucose (mg dl−1)
Right atrial weight (RAW) (mg)
Ratio (RAW/BW) (×10−3)
8 Weeks Control Diabetic
8 9
307.59 5.9 173.6910.6a
105.69 0.8 403.29 5.4a
15.9 9 0.6 15.5 9 0.7
0.051 90.002 0.089 9 0.003a
14 Weeks Control Diabetic
12 12
350.0 9 8.7 201.29 9.9a
104.49 0.6 485.09 4.3a
17.4 90.9 17.0 9 0.7
0.04990.002 0.08490.002a
Values are expressed as mean 9 S.E.M. a PB0.01 significantly different from the respective controls.
2.6. Statistical analysis Statistical analysis was performed according to one-way analysis of variance (ANOVA) followed by Neuman-Keul’s test. The level of significance was at least P B0.05. Values represent the mean9 S.E.M. Agonist pD2 values (apparent agonist affinity constants; −log ED50) were calculated from each agonist concentration – effect curve by linear regression analysis of the linear portion of the curve and taken as a measure of the sensitivity of the tissues to each agonist.
3. Results
3.1. General features of diabetic and control rats The general features of control and diabetic rats are summarized in Table 1. It is evident that STZ injection into the rats resulted in a diabetic state. The diabetic rats had significantly elevated blood glucose levels compared to the age-matched control rats. The diabetic rats also exhibited a lower weight gain than the age-matched control rats, so that at the time of sacrifice, the body weights of the diabetic animals were significantly less than those of the control animals. The right atrial weights in the diabetic and corresponding control groups, on the other hand, were found to be similar. However, the right atrial weight to body weight ratio was significantly increased in the diabetic rats, indicative of relative cardiac hypertrophy in those animals.
3.2. Effects of diabetes on b-adrenoceptor agonists-induced chronotropic responses of the rat right atria The basal right atrial rate was found to be similar in 8-week diabetic rats, relative to the age-matched controls (1909 8 and 2129 12 beats/min, P= NS, respectively). 14-Week diabetes, on the other hand, caused a slight, but significant depression of the basal right atrial rate (1809 8 and 210 96 beats/min, PB0.01, respectively). Figs. 1 and 2 depict concentration–effect curves for the change in the heart rate evoked by isoprenaline, noradrenaline and fenoterol in the right atria of the diabetic rats and their age-matched controls. All b-adrenoceptor agonists produced concentration-dependent positive chronotropic effects in both the nondiabetic control and diabetic groups. The 8-week diabetic state was not observed to have any significant influence on the positive chronotropic responses to these agonists (Fig. 1). Neither the pD2 values nor the maximum rate of the right atria after these agonists were altered significantly (Table 2). The positive chronotropic response to noradrenaline in the diabetic preparations, however, was found to be significantly attenuated as the disease progressed (Fig. 2). Both the pD2 value and the maximum rate of the right atria after noradrenaline were decreased in the 14th week of the diabetes when compared with the nondiabetic control animals (Table 2).
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Fig. 1. The positive chronotropic effects of isoprenaline ("), noradrenaline ( ) and fenoterol () in the spontaneously beating right atria of control (A) and 8-week diabetic (B) rats. Values are means 9 S.E.M.
The response to isoprenaline in the rats that were diabetic for 14 weeks is shown in Fig. 2. The maximum rate of the right atria from the diabetic rats after isoprenaline was also significantly less than that observed in the nondiabetic control atria (Table 2). However, the pD2 value for isoprenaline in the diabetic right atria was not significantly different to the nondiabetic control (Table 2). On the other hand, there was no significant difference between the nondiabetic control and diabetic rats at 14 weeks to their response to fenoterol (Fig. 2). Neither the pD2 value nor the maximum chronotropic response to fenoterol was altered significantly (Table 2).
3.3. Effects of diabetes on b-adrenoceptor agonists-induced inotropic responses of human right atrial appendages The responses to the three b-adrenoceptor agonists on the human atrial tissue are shown in Fig. 3. All the agonists caused concentration-dependent positive inotropic effects in the human right atrial appendages. The right atrial appendages from the diabetic patients responded to these agonists in the same way as the controls. On the other hand, the full agonist potency order was found to be isoprenaline ]fenoterol\noradrenaline in both the control and diabetic human atrial tissues. The pD2 values are shown in Table 3.
4. Discussion The objective of this investigation was to study the effect of diabetes on selective b1- and b2adrenoceptor mediated atrial responses. Injection of STZ (45 mg kg − 1) induced a diabetic state in the rats characterized by hyperglycaemia and symptoms of uncontrolled, insulin-dependent diabetes (i.e. polyuria, polyphagia, polydipsia). All b-adrenoceptor agonists used in this study produced concentration-dependent positive chronotropic effects in the right atria from both 8- and 14-week diabetic rats and their age-matched nondiabetic controls. As is well known, the potency orders of selective full agonists are used to characterize receptors. Hence, the present results show that the sensitivity of the right atria to the effects of b1-selective agonist noradrenaline and b2-selective agonist fenoterol appears to be similar in the nondiabetic preparations since no significant changes in the pD2 values for noradrenaline and fenoterol were observed. The maximum responses were also found to be similar. However, controversy exist between the results of binding studies and those of pharmacological studies where the tissue responses have been measured. Although the coexistence of b1- and b2-adrenoceptors has been demonstrated by means of a radioligand binding study in the right atria of rats [19], only b1-adrenoceptors were suggested to mediate atrial inotropic and chronotropic re-
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Fig. 2. The positive chronotropic effects of isoprenaline ("), noradrenaline ( ) and fenoterol () in the spontaneously beating right atria of control (A) and 14-week diabetic (B) rats. Values are means 9S.E.M.
sponses [10,14] in this species. The separation of Schild plots and/or difference in the pA2 values for a selective competitive antagonist, when agonists of different selectivity are used, can be taken as evidence that a certain tissue contains a heterogeneous receptor population. As Schild plots were not obtained in this study, the extent to which the b2-adrenoceptors contribute to the cardiac responses of the b-agonists in the rat can not be commented on. However, it has been demonstrated that no separation of Schild plots or no difference of pA2 values for atenolol (b1-selective antagonist) and a-methylpropranolol (b2-selective antagonist) was observed with noradrenaline and fenoterol as the agonists, indicating that a single (b1) receptor type mediates these responses [14]. Therefore, under normal physiological conditions, it seems unlikely that the stimulation of the b2adrenoceptors of the heart contributes to the regulation of the heart rate and contractility in the rat. In the presence of a pathological state, however, the role of the b2-adrenoceptor stimulation may be altered. As a matter of fact, several pathological states, including diabetes and advanced heart failure, have been shown to alter the density, sensitivity and responsiveness of the adrenoceptors in the heart [20,21]. In addition, heart disease is a major cause of morbidity and mortality in diabetic patients. In our study, 8 weeks after the injection of STZ, it was observed that chronotropic responses to b-adrenoceptor ago-
nists were unchanged in the right atria from the diabetic rats as compared with those from the control animals. However, there was a significant decrease in the chronotropic responses of the right atria from 14-week diabetic rats to noradrenaline as opposed to the controls. In addition, maximum responses to isoprenaline were lower in the diabetic rats at 14 weeks. In the 14-week diabetic rats, the decreases in maximum chronotropic responses of the right atria when stimulated by isoprenaline and noradrenaline were found to be 25 and 29%, respectively, compared to their agematched controls (Fig. 2). In contrast, responsiveness to fenoterol in the diabetic rats was still similar to the controls 14 weeks later. In the present investigation, it was also noticed that STZ-diabetes caused a depression of the basal atrial rate but a significant difference was found only in the 14-week diabetic rats compared to age-matched controls. These findings indicate that the duration of the diabetes alters the b1-adrenoceptor-mediated chronotropic responsiveness of the right atria. b2-Adrenoceptor-mediated chronotropic responsiveness, however, is not influenced by 14-week diabetes. In previous studies, it was also found that diabetes causes a decrease in the isoprenaline-induced positive chronotropic effect in the rat atria [2,22,23]. Similarly, the papillary muscles and ventricular tissues from diabetic rats show a decreased contractile response to isoprenaline [24,25]. The decrease in the
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chronotropic and inotropic effects of isoprenaline in diabetic hearts may be due to a decrease in b-adrenoceptor density [3,5,6,24]. Recently, the density of b-adrenoceptors in the ventricular muscle of diabetic rats has been shown to be decreased when expressed in terms of the number of receptors per mg of protein [26]. On the other hand, it has been demonstrated that plasma catecholamine levels are significantly increased in both experimental [27] and clinical diabetes [28]. Cardiac content of noradrenaline has also been reported to be increased in diabetic rats [27]. In addition, it was found that noradrenaline Table 2 Maximum chronotropic responses and pD2 values for b-agonists on rat right atria Maximum response
pD2 Values
7
1679 7
8.049 0.31
9
140 9 9
6.069 0.13
9
1589 15
6.59 9 0.13
8
1569 11
8.27 9 0.31
8
114 919
6.389 0.25
8
141 916
6.219 0.12
9
1769 13
8.34 9 0.53
10
138911
6.419 0.26
11
12098
6.359 0.17
8
1329 11a
7.48 9 0.39
10
979 15a
5.52 9 0.09a
n
8 Weeks Control Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective) Diabetic Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective) 14 Weeks Control Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective) Diabetic Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective)
11
123 9 13
6.279 0.20
Values are expressed as mean 9S.E.M. a PB0.05 significantly different from the respective controls.
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turnover, uptake, synthesis and release are all enhanced in diabetic cardiomyopathy [29,30]. Thus, chronically high levels of noradrenaline in diabetes may be responsible for the selective down-regulation of b1-adrenoceptors since the affinity of noradrenaline is lower for b2-adrenoceptors than for b1-adrenoceptors. This selective down-regulation of cardiac b1-adrenoceptors may therefore result in a decrease of b1-mediated cardiac stimulation. The results obtained in the present study by assessment of chronotropic responses of the right atria support this conclusion. The physiological significance of our findings that b1-adrenoceptor-mediated chronotropic responses are impaired but those mediated via b2-adrenoceptors are preserved in diabetic rats is uncertain. It is unknown at present whether b2-adrenoceptors may gain a functional role in the regulation of the heart rate of rats when b1-adrenoceptormediated responses are somewhat decreased. Because the maximum response of isoprenaline was also found to be attenuated in the diabetic atria in the present study, it seems unlikely that b2adrenoceptors play such a compensatory role in STZ-diabetes. This point, however, should be elucidated in further experiments. On the other hand, the results of this study using human atrial tissue support the findings of previous studies that b2-adrenoceptors exist in human right atrium [7,10,15,16]. Our data show that b2-selective agonist fenoterol was more potent than b1-selective agonist noradrenaline on the human right atrium. Inotropic responses to fenoterol and nonselective b-agonist isoprenaline were found to be similar. From these results b2adrenoceptors, along with b1-adrenoceptors, appear to have a functional role in mediating inotropic responses to b-adrenoceptor agonists in the human right atrial appendage. Interestingly, the diabetic atria responded to b-agonists in the same way as the controls. The right atrial appendages from the diabetic patients produced the same degree of contraction in response to isoprenaline, fenoterol and noradrenaline as did the nondiabetic control atria. Therefore, in contrast to the situation in rats, diabetes seems to have no influence on neither b1- nor b2-adrenoceptor-mediated inotropic responses in the human right
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Fig. 3. The positive inotropic effects of isoprenaline ("), noradrenaline ( ) and fenoterol () in control (A) and diabetic (B) human right atrial appendages. Responses are expressed as a percentage of the maximum response to isoprenaline in control preparations as described in the text. Values are means 9 S.E.M.
atrium. Although we have measured inotropic responses of human right atrial tissue, it would not be unreasonable to expect a decreased badrenoceptor-mediated responsiveness in diabetic patients. However, it is important to note that the patients had been treated with insulin or oral antidiabetics before surgery. Of particular importance in this regard are various studies documenting that treatment of diabetic rats with these agents normalizes cardiovascular alterations [2,31 –35]. Insulin treatment appears to be the most effective in not only preventing but also in reversing the diabetes induced-myocardial alterations. Moreover, it was also shown that the decreased number of cardiac b-adrenoceptors of diabetic rats returned to normal after the adminisTable 3 Inotropic pD2 values for b-agonists on human right atrial appendages pD2 Values Control (n =12) Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective)
7.539 0.27 5.85 9 0.21 6.89 9 0.27
Diabetic (n =7) Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective)
6.91 9 0.22 5.38 9 0.11 6.21 9 0.30
Values are expressed as mean 9 S.E.M.
tration of insulin [3]. Thus, it might be speculated that the effectiveness of cardiac b-adrenoceptormediated inotropic responses is then re-established in those patients. Alternatively, it is possible that the impaired atrial responsiveness to b-adrenoceptor agonists may be related to the duration of diabetes. Although no accurate information on the duration of the disease of the patients involved in this study could be obtained, our results demonstrating that chronotropic responses to noradrenaline and isoprenaline were decreased only in the 14-week diabetic rats but not in the 8-week diabetic animals suggest that the atrial responses worsened as the disease developed. On the other hand, insulin treatment seems to be less effective at the more chronic stages of the disease. It has been demonstrated that only a partial reversal of the cardiac function could be obtained by insulin treatment of five month diabetic rats suggesting that the cardiac changes at the chronic stages of diabetes are irreversible [36]. Hence, the possibility that the changes in badrenoceptor-mediated inotropic responses may occur in patients with long-term diabetes can not be excluded. References [1] S. Ramanadham, T.E. Tenner, Alterations in the myocardial b-adrenoceptor system of streptozotocin-diabetic rats, Eur. J. Pharmacol. 136 (1987) 377 – 389.
U8 .D. Dinc¸er et al. / Diabetes Research and Clinical Practice 40 (1998) 113–122 [2] C ¸ . Karasu, Y. O8 ztu¨rk, N. Altan, N. Yildizoglu-Ari, C. Ikizler, V.M. Altan, Thyroid hormones mediated effects of insulin on alloxan diabetic rat atria, Gen. Pharmacol. 21 (1990) 735 – 740. [3] P.R. Sunderesan, V.K. Sharma, S.L. Gingold, P.S. Banerjee, Decreased beta-adrenergic receptors in rat heart in streptozotocin-induced diabetes: role of thyroid hormones, Endocrinology 114 (1984) 1358–1363. [4] J. Latifpour, J.H. Mcneill, Cardiac autonomic receptors: effect of long-term experimental diabetes, J. Pharmacol. Exp. Ther. 230 (1984) 242–249. [5] F.L. Atkins, R.T. Dowell, S. Love, b-Adrenergic receptors, adenylate cyclase activity and cardiac dysfunction in diabetic rat, J. Cardiovasc. Pharmacol. 7 (1985) 66–70. [6] J.J. Savarese, B.A. Berkowitz, b-Adrenergic receptor decrease in diabetic rat hearts, Life Sci. 25 (1979) 2075– 2078. [7] O.E. Brodde, K. Karad, H.-R. Zerkowski, N. Rohm, J.C. Reidemeister, Coexistence of b1- and b2-adrenoceptors in human heart and lung, Br. J. Pharmacol. 78 (Suppl) (1983) 72. [8] E. Carlsson, C.G. Dahlof, A. Hedberg, H. Persson, B. Tangstrand, Differentiation of cardiac chronotropic and inotropic effects of b-adrenoceptor agonists, Naunyn Schmiedebergs Arch. Pharmacol. 300 (1977) 101–105. [9] J. Wagner, B. Nick, N. Rohm, H.J. Schuman, On the coexistence of b1- and b2-adrenoceptors in various organs, Arch. Int. Pharmacodyn. 249 (1981) 26–38. [10] C. Wilson, C. Lincoln, b-Adrenoceptor subtypes in human, rat, guinea pig, and rabbit atria, J. Cardiovasc. Pharmacol. 6 (1984) 1216–1221. [11] S.R. O’Donnell, J.C. Wanstall, The importance of choice of agonist in studies designed to predict b2:b1 adrenoceptor selectivity of antagonists from pA2 values on guineapig trachea and atria, Naunyn Schmiedebergs Arch. Pharmacol. 308 (1979) 183–190. [12] K.J. Broadley, M.H. Hawthorn, Examination of cardiac b-adrenoceptor subtypes by pharmacological and radioligand binding technique, Br. J. Pharmacol. 78 (suppl) (1983) 136P. [13] B.I. Costin, S.R. O’Donnel, J.C. Wanstall, Chronotropic responses of rabbit isolated atria to b-adrenoceptor agonists are mediated by only b1-adrenoceptors, J. Pharm. Pharmacol. 35 (1983) 752–756. [14] L.J. Bryan, J.J. Cole, S.R. O’Donnell, J.C. Wanstall, A study designed to explore the hypothesis that beta-1 adrenoceptors are ‘innervated’ receptors and beta-2 adrenoceptors are ‘hormonal’ receptors, J. Pharmacol. Exp. Ther. 216 (1981) 395–400. [15] G.L. Stiles, S. Taylor, R.J. Lefkowitz, Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding, Life Sci. 33 (1983) 467 – 473. [16] P. Robberecht, M. Delhaye, G. Taton, et al., The human heart beta-adrenergic receptors. Heterogeneity of the binding sites: presence of 50% beta1- and 50% beta2adrenergic receptors, Mol. Pharmacol. 24 (1983) 169–173.
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[17] O.E. Brodde, N. O’Hara, H.R. Zerkowski, N. Rohm, Human cardiac b-adrenoceptors: both b1- and b2adrenoceptors are functionally coupled to the adenylate cyclase in right atrium, J. Cardiovasc. Pharmacol. 6 (1984) 1184 – 1191. [18] H.R. Zerkowski, K. Ikezono, N. Rohm, J.C. Reidemeister, O.E. Brodde, Human myocardial b-adrenoceptors: demonstration of both b1- and b2-adrenoceptors mediating contractile responses to b-agonists on the isolated right atrium, Naunyn Schmiedeberg’s Arch. Pharmacol. 332 (1986) 142 – 147. [19] K.P. Minneman, L.R. Hegstrand, P.B. Molinoff, Simultaneous determination of beta-1 and beta-2 adrenergic receptors in tissues containing both receptor subtypes, Mol. Pharmacol. 16 (1979) 34 – 46. [20] C.V. Jackson, G.M. McGrath, J.H. McNeill, Alterations in alpha1-adrenoceptor stimulation of isolated atria from experimental diabetic rats, Can. J. Physiol. Pharmacol. 64 (1986) 145 – 151. [21] A.J. Kaumann, Four b-adrenoceptor subtypes in the mammalian heart, Trends Pharmacol. Sci. 18 (1997) 70 – 76. [22] J.M. Foy, P.D. Lucas, Effect of experimental diabetes, food deprivation and genetic obesity on the sensitivity of pithed rats to autonomic agents, Br. J. Pharmacol. 57 (1976) 229 – 234. [23] R.K. Goyal, B. Rodrigues, J.H. McNeill, Effect of triiodothyronine on cardiac responses to adrenergic-agonists in STZ-induced diabetic rats, Gen. Pharmacol. 18 (1987) 357 – 362. [24] C.E. Heyliger, C.N. Pierce, P.K. Singal, R.E. Beamish, N.S. Dhalla, Cardiac a- and b-adrenergic receptor alterations in diabetic cardiomyopathy, Basic Res. Cardiol. 77 (1982) 610 – 618. [25] Z. Yu, J.H. McNeill, Altered inotropic responses in diabetic cardiomyopathy and hypertensive-diabetic cardiomyopathy, J. Pharmacol. Exp. Ther. 257 (1991) 64 – 71. [26] K. Kamata, T. Satoh, H. Tanaka, K. Shigenobu, Changes in electrophysiological and mechanical responses of the rat papillary muscle to a- and b- agonist in streptozotocin-induced diabetes, Can. J. Physiol. Pharmacol. 75 (1997) 781 – 788. [27] D.J. Paulson, D. Shetlar, K.E. Light, Catecholamine levels in the heart, serum and adrenals of experimental diabetic rats, Fed. Proc. 39 (1980) 637. [28] N.J. Christensen, Plasma norepinephrine and epinephrine in untreated diabetics, during fasting and after insulin administration, Diabetes 23 (1974) 1 – 8. [29] P.K. Ganguly, K.S. Dhalla, I.R. Innes, R.E. Beamish, N. Dhalla, Altered norepinephrine turnover and metabolism in diabetic cardiomyopathy, Circ. Res. 59 (1986) 684 – 693. [30] P.K. Ganguly, R.E. Beamish, K.S. Dhalla, I.R. Innes, N. Dhalla, Norepinephrine storage, distribution, and release in diabetic cardiomyopathy, Am. J. Physiol. 252 (1987) E734 – E739.
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122
[31] Y. O8 ztu¨rk, V.M. Altan, N. Yildizoglu-Ari, Effects of experimental diabetes and insulin on smooth muscle functions, Pharmacol. Rev. 48 (1996) 69–112. [32] A.R. Tahiliani, J.H. McNeill, Diabetes-induced abnormalities in the myocardium, Life Sci. 38 (1986) 959–974. [33] B. Rodrigues, J.H. McNeill, The diabetic heart: metabolic causes for the development of a cardiomyopathy, Circ. Res. 26 (1992) 913 –922. [34] A.T. O8 zc¸elikay, N. Yildizoglu-Ari, A. O8 zu¨ari, Y. O8 ztu¨rk,
.
V.M. Altan, The effect of vanadate on alloxan-diabetic rat atria, Diabetes Res. Clin. Pract. 19 (1993) 189 – 194. [35] A. O8 zu¨ari, Y. O8 ztu¨rk, N. Yildizoglu-Ari, A.T. O8 zc¸elikay, V.M. Altan, The effects glyburide and insulin on the cardiac performance in rats with noninsulin-dependent diabetes mellitus, Gen. Pharmacol. 24 (1993) 165 – 169. [36] J.H. McNeill, A.G. Tahiliani, Diabetes-induced cardiac changes, Trends Pharmacol. Sci. 11 (1986) 364 – 367.
The effects of diabetes on b-adrenoceptor mediated responsiveness of human and rat atria U8 . Deniz Dinc¸er, Arzu Onay, Nuray Arı, A. Tanju O8 zc¸elikay, V. Melih Altan * Department of Pharmacology, Faculty of Pharmacy, Uni6ersity of Ankara, Tandog˜an, 06100 Ankara, Turkey Accepted 10 March 1998
Abstract The literature on the influence of diabetes on cardiac b-adrenoceptors is still a matter of controversy. Hence, in the present study, the responsiveness of spontaneously beating right atria from streptozotocin (STZ)-diabetic rats to b-adrenoceptor agonists were compared with those from non-diabetic controls. The responsiveness of right atria from 8-week diabetic rats to the chronotropic effects of isoprenaline, noradrenaline and fenoterol was found to be unchanged. As the disease progressed, on the other hand, the diabetic atria were found to have decreased responsiveness to the chronotropic effects of noradrenaline. The pD2 value and maximum chronotropic effect of noradrenaline were decreased in 14-week diabetic right atria when compared with those from age-matched controls. A significant decrease in the maximum chronotropic response to isoprenaline with no change in pD2 value was also observed in 14-week diabetes. These results suggest that b1- but not b2-adrenoceptor mediated chronotropic responses were reduced in the right atria due to the increase in the duration of diabetes. On the other hand, the inotropic responses to b-adrenoceptor agonists were also assessed on diabetic and nondiabetic human atrial tissue. There were no significant differences in the inotropic responses to each agonists in either of the diabetic and nondiabetic human atrial tissues. The full agonist potency order was isoprenaline ] fenoterol\ noradrenaline. © 1998 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Diabetes; b-Adrenoceptors; Rat right atria; Human atrial appendage
1. Introduction * Corresponding author. Tel.: +90 312 2134478/2126805, ext. 160; fax: + 90 312 2131081; e-mail: maltan@ pharmacy.ankara.edu.tr
Chronic diabetes mellitus is known to be associated with cardiac complications in both clinical and experimental settings. Among the prominent
0168-8227/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved. PII S0168-8227(98)00034-5
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defects observed in chemically induced diabetic rats are the reduced responsiveness of cardiac preparations to the inotropic and chronotropic effects of b-adrenoceptor agonists stimulation [1,2]. While the mechanisms leading to the depressed responses to b-adrenoceptor agonists of cardiac preparations from diabetic rats are not well understood, there are a number of possible explanations, one of which is the decrease in the number of cardiac b-adrenoceptors [3 – 5]. In fact, Savarase and Berkowitz [6] reported a 28% reduction in the number of b-adrenoceptors accompanied by a 24% decrease in the heart rate of STZ-diabetic rats compared to controls. b-Adrenoceptors of both b1- and b2subtypes, on the other hand, have been shown to coexist in certain cardiac tissues [7 – 10], but only b1-adrenoceptors appear to mediate the rate and tension responses in the guinea-pig [11,12], rabbit [13] and rat [14] atria. Several radioligand binding studies using human right atrium have also demonstrated a mixed b1plus b2-adrenoceptor population, although the population of each subtype varied [15,16]. In addition, it was demonstrated that in the human right atrium both b1- and b2-adrenoceptors are functionally coupled to the adenylate cyclase system [17] and both subtypes of badrenoceptors are reported to contribute to the cardiac responses of b-agonists [18]. Thus, in the human right atrium both b1- and b2adrenoceptors have been suggested to be involved in the physiological regulation of the force of contraction and/or heart rate. Although diabetes has been shown to alter the sensitivity, responsiveness and density of badrenoceptors in the heart, no data, to our knowledge, are available on the influence of diabetes on selective b2-adrenoceptor responsiveness in the heart. The aim of the present study was, therefore, to investigate the effects of diabetes on selective b1- and b2-adrenoceptor stimulation. For this purpose, an electrically driven muscle strip of the right atrial appendages from diabetic patients and spontaneously beating right atria from STZ-diabetic rats were used.
2. Materials and methods
2.1. Induction of experimental diabetes. Male Albino rats weighing 200–250 g (Ankara University Faculty of Pharmacy Animal Care Unit) were used for this study. The rats were housed two animals per cage in a room with a 12 h light:12 h dark cycle. They were given regular laboratory chow and water ad libitum throughout the experiment. Diabetes was chemically induced with streptozotocin (Sigma, St Louis, MO) 45 mg kg − 1 dissolved in citrate buffer (pH 4.5) administered as a single intravenous (i.v.) tail-vein injection under light ether anesthesia. The control rats were injected with an equivalent volume of the vehicle only. At 3 days following the STZ injection, the rats were checked for glycosuria semiquantitatively using Urine-Glucostix reagent strips (Diastix, Ames Division, Bayer Diagnostics, UK). Only rats exhibiting glycosuria were then analyzed for hyperglycaemia with Glucostix reagent strips (Glucofilm, Bayer Diagnostics, Germany) read by a Glucometer (Glucometer III, Bayer Diagnostics, France) and those with a blood glucose level ] 16.6 mM were designated diabetic. In order to investigate the time course of the development of the observed responses, diabetic rats were studied at 8 and 14 weeks after the induction of diabetes. The control rats used for comparison were age-matched to the diabetic rats. Final blood glucose levels were measured on samples taken when the rats were killed.
2.2. Rat right atrial preparations At the time of death, the hearts removed from the control and diabetic rats and immediately placed in a beaker containing ice-cold oxygenated Krebs-bicarbonate solution (pH 7.4) of the following composition (mM): NaCl, 118.4; KCl, 4.7; CaCl2, 2.5; NaHCO3, 25; MgSO4, 1.2; KH2PO4, 1.2 and glucose 11.7. After expressing blood from the heart, the ventricular tissue, fat and connective tissue were trimmed off. The atrial tissue was removed and transferred to another beaker containing cold fresh Krebs-bicarbonate solution. The right atria were then carefully dissected and
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suspended vertically in a 20-ml tissue bath of the above solution. One end of the right atria was attached to a tissue holder and the other end was mounted by silk thread to an isometric transducer (Ugo Basile, No. 7004, Varese, Italy) that was connected to a microdynamometer (Ugo Basile, Unirecord). The right atria were allowed to beat spontaneously. The tissue preparations were equilibrated at 37°C for 60 min in Krebs-bicarbonate solution aerated with a mixture of 95% O2 and 5% CO2. During this period, the Krebs-bicarbonate solution in the tissue bath was replaced every 15 min. Resting tensions of 1 g in the tissue preparations were maintained throughout the experiment.
O2 and 5% CO2 at room temperature and transported immediately to the laboratory. The preparation of the tissues was begun within 5–15 min after surgical removal. The atrial appendages were dissected without causing visible damage to the epicardium. The strips were cut to a thickness B 1 mm to facilitate diffusion of oxygen and drugs. The muscles were then prepared for tension recordings as described above and electrically stimulated via two platinum electrodes placed on the tissue at a frequency of 1.0 Hz with rectangular wave pulses of 5 ms duration at a voltage approximately 20% above the threshold using a stimulator (Grass Stimulator S44).
2.3. Human right atrial preparations
2.4. Experimental procedure
Human right atrial appendages were obtained from seven diabetic patients (five male, two female; mean age 55.8592.8 (47 – 65)) and 12 nondiabetic patients (11 male, one female; mean age 529 3.4 (36–69)) undergoing coronary artery bypass grafting. No patient suffered from acute myocardial failure; none of the patients had received catecholamines for at least three weeks before the operation. Diabetic patients had been treated with oral sulfonylureas (n=1), biguanides (n =1), insulin (n =5), calcium antagonists (n = 6), nitrovasodilators (n =7) and aspirin (n = 6). Nondiabetic patients, on the other hand, had received calcium antagonists (n =11), nitrovasodilators (n= 11), ACE inhibitors (n =1) and aspirin (n= 10). In addition, one nondiabetic patient had been treated with digitalis glycosides. Care was taken to ensure that donor patients had not received b-adrenoceptor antagonists prior to surgery. The blood glucose levels of diabetic patients had been regulated before the operation. All patients therefore underwent surgery with nearnormal blood glucose concentrations. Premedication consisted of dolantin and atropine and the operation was carried out under balanced anesthesia with fentanyl and isoflurane. In some cases N2O was added. Pancuronium was used as the muscle relaxant. Once removed, the tissue was immediately placed in a sealed vial containing Krebs-bicarbonate solution oxygenated with 95%
The tissues were allowed to equilibrate for 60 min. After ensuring steady base-line recordings, the tissues were sensitized to agonists by a brief exposure to isoprenaline (5× 10 − 6 M). The tissues were again washed several times in order to obtain steady baselines, and then a cumulative concentration –effect curve to the test agonist was performed. Agonist responses were measured as the increase in rate of the right atrium and increase in tension of the human atrial appendage. In the experiments performed on human atrial tissue, all responses were expressed as the percentage of maximum response to isoprenaline obtained in nondiabetic preparations. In all experiments, where noradrenaline was the agonist, concentration –effect curves were obtained in the presence of prazosin (5× 10 − 9 M) in order to block a-adrenoceptors. The tissues were equilibrated with prazosin for 20 min before noradrenaline was added.
2.5. Drugs The drugs used in this study were as follows: streptozotocin (Sigma), isoproteronol (Sigma), fenoterol (Sigma), noradrenaline (Sigma) and prazosin (Sigma). All the drug stock solutions were freshly prepared in distilled water and agonist serial dilutions made in 0.9% NaCl.
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Table 1 General characteristics of the rats n
Body weight (BW) (g) Blood glucose (mg dl−1)
Right atrial weight (RAW) (mg)
Ratio (RAW/BW) (×10−3)
8 Weeks Control Diabetic
8 9
307.59 5.9 173.6910.6a
105.69 0.8 403.29 5.4a
15.9 9 0.6 15.5 9 0.7
0.051 90.002 0.089 9 0.003a
14 Weeks Control Diabetic
12 12
350.0 9 8.7 201.29 9.9a
104.49 0.6 485.09 4.3a
17.4 90.9 17.0 9 0.7
0.04990.002 0.08490.002a
Values are expressed as mean 9 S.E.M. a PB0.01 significantly different from the respective controls.
2.6. Statistical analysis Statistical analysis was performed according to one-way analysis of variance (ANOVA) followed by Neuman-Keul’s test. The level of significance was at least P B0.05. Values represent the mean9 S.E.M. Agonist pD2 values (apparent agonist affinity constants; −log ED50) were calculated from each agonist concentration – effect curve by linear regression analysis of the linear portion of the curve and taken as a measure of the sensitivity of the tissues to each agonist.
3. Results
3.1. General features of diabetic and control rats The general features of control and diabetic rats are summarized in Table 1. It is evident that STZ injection into the rats resulted in a diabetic state. The diabetic rats had significantly elevated blood glucose levels compared to the age-matched control rats. The diabetic rats also exhibited a lower weight gain than the age-matched control rats, so that at the time of sacrifice, the body weights of the diabetic animals were significantly less than those of the control animals. The right atrial weights in the diabetic and corresponding control groups, on the other hand, were found to be similar. However, the right atrial weight to body weight ratio was significantly increased in the diabetic rats, indicative of relative cardiac hypertrophy in those animals.
3.2. Effects of diabetes on b-adrenoceptor agonists-induced chronotropic responses of the rat right atria The basal right atrial rate was found to be similar in 8-week diabetic rats, relative to the age-matched controls (1909 8 and 2129 12 beats/min, P= NS, respectively). 14-Week diabetes, on the other hand, caused a slight, but significant depression of the basal right atrial rate (1809 8 and 210 96 beats/min, PB0.01, respectively). Figs. 1 and 2 depict concentration–effect curves for the change in the heart rate evoked by isoprenaline, noradrenaline and fenoterol in the right atria of the diabetic rats and their age-matched controls. All b-adrenoceptor agonists produced concentration-dependent positive chronotropic effects in both the nondiabetic control and diabetic groups. The 8-week diabetic state was not observed to have any significant influence on the positive chronotropic responses to these agonists (Fig. 1). Neither the pD2 values nor the maximum rate of the right atria after these agonists were altered significantly (Table 2). The positive chronotropic response to noradrenaline in the diabetic preparations, however, was found to be significantly attenuated as the disease progressed (Fig. 2). Both the pD2 value and the maximum rate of the right atria after noradrenaline were decreased in the 14th week of the diabetes when compared with the nondiabetic control animals (Table 2).
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Fig. 1. The positive chronotropic effects of isoprenaline ("), noradrenaline ( ) and fenoterol () in the spontaneously beating right atria of control (A) and 8-week diabetic (B) rats. Values are means 9 S.E.M.
The response to isoprenaline in the rats that were diabetic for 14 weeks is shown in Fig. 2. The maximum rate of the right atria from the diabetic rats after isoprenaline was also significantly less than that observed in the nondiabetic control atria (Table 2). However, the pD2 value for isoprenaline in the diabetic right atria was not significantly different to the nondiabetic control (Table 2). On the other hand, there was no significant difference between the nondiabetic control and diabetic rats at 14 weeks to their response to fenoterol (Fig. 2). Neither the pD2 value nor the maximum chronotropic response to fenoterol was altered significantly (Table 2).
3.3. Effects of diabetes on b-adrenoceptor agonists-induced inotropic responses of human right atrial appendages The responses to the three b-adrenoceptor agonists on the human atrial tissue are shown in Fig. 3. All the agonists caused concentration-dependent positive inotropic effects in the human right atrial appendages. The right atrial appendages from the diabetic patients responded to these agonists in the same way as the controls. On the other hand, the full agonist potency order was found to be isoprenaline ]fenoterol\noradrenaline in both the control and diabetic human atrial tissues. The pD2 values are shown in Table 3.
4. Discussion The objective of this investigation was to study the effect of diabetes on selective b1- and b2adrenoceptor mediated atrial responses. Injection of STZ (45 mg kg − 1) induced a diabetic state in the rats characterized by hyperglycaemia and symptoms of uncontrolled, insulin-dependent diabetes (i.e. polyuria, polyphagia, polydipsia). All b-adrenoceptor agonists used in this study produced concentration-dependent positive chronotropic effects in the right atria from both 8- and 14-week diabetic rats and their age-matched nondiabetic controls. As is well known, the potency orders of selective full agonists are used to characterize receptors. Hence, the present results show that the sensitivity of the right atria to the effects of b1-selective agonist noradrenaline and b2-selective agonist fenoterol appears to be similar in the nondiabetic preparations since no significant changes in the pD2 values for noradrenaline and fenoterol were observed. The maximum responses were also found to be similar. However, controversy exist between the results of binding studies and those of pharmacological studies where the tissue responses have been measured. Although the coexistence of b1- and b2-adrenoceptors has been demonstrated by means of a radioligand binding study in the right atria of rats [19], only b1-adrenoceptors were suggested to mediate atrial inotropic and chronotropic re-
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Fig. 2. The positive chronotropic effects of isoprenaline ("), noradrenaline ( ) and fenoterol () in the spontaneously beating right atria of control (A) and 14-week diabetic (B) rats. Values are means 9S.E.M.
sponses [10,14] in this species. The separation of Schild plots and/or difference in the pA2 values for a selective competitive antagonist, when agonists of different selectivity are used, can be taken as evidence that a certain tissue contains a heterogeneous receptor population. As Schild plots were not obtained in this study, the extent to which the b2-adrenoceptors contribute to the cardiac responses of the b-agonists in the rat can not be commented on. However, it has been demonstrated that no separation of Schild plots or no difference of pA2 values for atenolol (b1-selective antagonist) and a-methylpropranolol (b2-selective antagonist) was observed with noradrenaline and fenoterol as the agonists, indicating that a single (b1) receptor type mediates these responses [14]. Therefore, under normal physiological conditions, it seems unlikely that the stimulation of the b2adrenoceptors of the heart contributes to the regulation of the heart rate and contractility in the rat. In the presence of a pathological state, however, the role of the b2-adrenoceptor stimulation may be altered. As a matter of fact, several pathological states, including diabetes and advanced heart failure, have been shown to alter the density, sensitivity and responsiveness of the adrenoceptors in the heart [20,21]. In addition, heart disease is a major cause of morbidity and mortality in diabetic patients. In our study, 8 weeks after the injection of STZ, it was observed that chronotropic responses to b-adrenoceptor ago-
nists were unchanged in the right atria from the diabetic rats as compared with those from the control animals. However, there was a significant decrease in the chronotropic responses of the right atria from 14-week diabetic rats to noradrenaline as opposed to the controls. In addition, maximum responses to isoprenaline were lower in the diabetic rats at 14 weeks. In the 14-week diabetic rats, the decreases in maximum chronotropic responses of the right atria when stimulated by isoprenaline and noradrenaline were found to be 25 and 29%, respectively, compared to their agematched controls (Fig. 2). In contrast, responsiveness to fenoterol in the diabetic rats was still similar to the controls 14 weeks later. In the present investigation, it was also noticed that STZ-diabetes caused a depression of the basal atrial rate but a significant difference was found only in the 14-week diabetic rats compared to age-matched controls. These findings indicate that the duration of the diabetes alters the b1-adrenoceptor-mediated chronotropic responsiveness of the right atria. b2-Adrenoceptor-mediated chronotropic responsiveness, however, is not influenced by 14-week diabetes. In previous studies, it was also found that diabetes causes a decrease in the isoprenaline-induced positive chronotropic effect in the rat atria [2,22,23]. Similarly, the papillary muscles and ventricular tissues from diabetic rats show a decreased contractile response to isoprenaline [24,25]. The decrease in the
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chronotropic and inotropic effects of isoprenaline in diabetic hearts may be due to a decrease in b-adrenoceptor density [3,5,6,24]. Recently, the density of b-adrenoceptors in the ventricular muscle of diabetic rats has been shown to be decreased when expressed in terms of the number of receptors per mg of protein [26]. On the other hand, it has been demonstrated that plasma catecholamine levels are significantly increased in both experimental [27] and clinical diabetes [28]. Cardiac content of noradrenaline has also been reported to be increased in diabetic rats [27]. In addition, it was found that noradrenaline Table 2 Maximum chronotropic responses and pD2 values for b-agonists on rat right atria Maximum response
pD2 Values
7
1679 7
8.049 0.31
9
140 9 9
6.069 0.13
9
1589 15
6.59 9 0.13
8
1569 11
8.27 9 0.31
8
114 919
6.389 0.25
8
141 916
6.219 0.12
9
1769 13
8.34 9 0.53
10
138911
6.419 0.26
11
12098
6.359 0.17
8
1329 11a
7.48 9 0.39
10
979 15a
5.52 9 0.09a
n
8 Weeks Control Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective) Diabetic Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective) 14 Weeks Control Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective) Diabetic Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective)
11
123 9 13
6.279 0.20
Values are expressed as mean 9S.E.M. a PB0.05 significantly different from the respective controls.
119
turnover, uptake, synthesis and release are all enhanced in diabetic cardiomyopathy [29,30]. Thus, chronically high levels of noradrenaline in diabetes may be responsible for the selective down-regulation of b1-adrenoceptors since the affinity of noradrenaline is lower for b2-adrenoceptors than for b1-adrenoceptors. This selective down-regulation of cardiac b1-adrenoceptors may therefore result in a decrease of b1-mediated cardiac stimulation. The results obtained in the present study by assessment of chronotropic responses of the right atria support this conclusion. The physiological significance of our findings that b1-adrenoceptor-mediated chronotropic responses are impaired but those mediated via b2-adrenoceptors are preserved in diabetic rats is uncertain. It is unknown at present whether b2-adrenoceptors may gain a functional role in the regulation of the heart rate of rats when b1-adrenoceptormediated responses are somewhat decreased. Because the maximum response of isoprenaline was also found to be attenuated in the diabetic atria in the present study, it seems unlikely that b2adrenoceptors play such a compensatory role in STZ-diabetes. This point, however, should be elucidated in further experiments. On the other hand, the results of this study using human atrial tissue support the findings of previous studies that b2-adrenoceptors exist in human right atrium [7,10,15,16]. Our data show that b2-selective agonist fenoterol was more potent than b1-selective agonist noradrenaline on the human right atrium. Inotropic responses to fenoterol and nonselective b-agonist isoprenaline were found to be similar. From these results b2adrenoceptors, along with b1-adrenoceptors, appear to have a functional role in mediating inotropic responses to b-adrenoceptor agonists in the human right atrial appendage. Interestingly, the diabetic atria responded to b-agonists in the same way as the controls. The right atrial appendages from the diabetic patients produced the same degree of contraction in response to isoprenaline, fenoterol and noradrenaline as did the nondiabetic control atria. Therefore, in contrast to the situation in rats, diabetes seems to have no influence on neither b1- nor b2-adrenoceptor-mediated inotropic responses in the human right
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Fig. 3. The positive inotropic effects of isoprenaline ("), noradrenaline ( ) and fenoterol () in control (A) and diabetic (B) human right atrial appendages. Responses are expressed as a percentage of the maximum response to isoprenaline in control preparations as described in the text. Values are means 9 S.E.M.
atrium. Although we have measured inotropic responses of human right atrial tissue, it would not be unreasonable to expect a decreased badrenoceptor-mediated responsiveness in diabetic patients. However, it is important to note that the patients had been treated with insulin or oral antidiabetics before surgery. Of particular importance in this regard are various studies documenting that treatment of diabetic rats with these agents normalizes cardiovascular alterations [2,31 –35]. Insulin treatment appears to be the most effective in not only preventing but also in reversing the diabetes induced-myocardial alterations. Moreover, it was also shown that the decreased number of cardiac b-adrenoceptors of diabetic rats returned to normal after the adminisTable 3 Inotropic pD2 values for b-agonists on human right atrial appendages pD2 Values Control (n =12) Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective)
7.539 0.27 5.85 9 0.21 6.89 9 0.27
Diabetic (n =7) Isoprenaline (nonselective) Noradrenaline (b1-selective) Fenoterol (b2-selective)
6.91 9 0.22 5.38 9 0.11 6.21 9 0.30
Values are expressed as mean 9 S.E.M.
tration of insulin [3]. Thus, it might be speculated that the effectiveness of cardiac b-adrenoceptormediated inotropic responses is then re-established in those patients. Alternatively, it is possible that the impaired atrial responsiveness to b-adrenoceptor agonists may be related to the duration of diabetes. Although no accurate information on the duration of the disease of the patients involved in this study could be obtained, our results demonstrating that chronotropic responses to noradrenaline and isoprenaline were decreased only in the 14-week diabetic rats but not in the 8-week diabetic animals suggest that the atrial responses worsened as the disease developed. On the other hand, insulin treatment seems to be less effective at the more chronic stages of the disease. It has been demonstrated that only a partial reversal of the cardiac function could be obtained by insulin treatment of five month diabetic rats suggesting that the cardiac changes at the chronic stages of diabetes are irreversible [36]. Hence, the possibility that the changes in badrenoceptor-mediated inotropic responses may occur in patients with long-term diabetes can not be excluded. References [1] S. Ramanadham, T.E. Tenner, Alterations in the myocardial b-adrenoceptor system of streptozotocin-diabetic rats, Eur. J. Pharmacol. 136 (1987) 377 – 389.
U8 .D. Dinc¸er et al. / Diabetes Research and Clinical Practice 40 (1998) 113–122 [2] C ¸ . Karasu, Y. O8 ztu¨rk, N. Altan, N. Yildizoglu-Ari, C. Ikizler, V.M. Altan, Thyroid hormones mediated effects of insulin on alloxan diabetic rat atria, Gen. Pharmacol. 21 (1990) 735 – 740. [3] P.R. Sunderesan, V.K. Sharma, S.L. Gingold, P.S. Banerjee, Decreased beta-adrenergic receptors in rat heart in streptozotocin-induced diabetes: role of thyroid hormones, Endocrinology 114 (1984) 1358–1363. [4] J. Latifpour, J.H. Mcneill, Cardiac autonomic receptors: effect of long-term experimental diabetes, J. Pharmacol. Exp. Ther. 230 (1984) 242–249. [5] F.L. Atkins, R.T. Dowell, S. Love, b-Adrenergic receptors, adenylate cyclase activity and cardiac dysfunction in diabetic rat, J. Cardiovasc. Pharmacol. 7 (1985) 66–70. [6] J.J. Savarese, B.A. Berkowitz, b-Adrenergic receptor decrease in diabetic rat hearts, Life Sci. 25 (1979) 2075– 2078. [7] O.E. Brodde, K. Karad, H.-R. Zerkowski, N. Rohm, J.C. Reidemeister, Coexistence of b1- and b2-adrenoceptors in human heart and lung, Br. J. Pharmacol. 78 (Suppl) (1983) 72. [8] E. Carlsson, C.G. Dahlof, A. Hedberg, H. Persson, B. Tangstrand, Differentiation of cardiac chronotropic and inotropic effects of b-adrenoceptor agonists, Naunyn Schmiedebergs Arch. Pharmacol. 300 (1977) 101–105. [9] J. Wagner, B. Nick, N. Rohm, H.J. Schuman, On the coexistence of b1- and b2-adrenoceptors in various organs, Arch. Int. Pharmacodyn. 249 (1981) 26–38. [10] C. Wilson, C. Lincoln, b-Adrenoceptor subtypes in human, rat, guinea pig, and rabbit atria, J. Cardiovasc. Pharmacol. 6 (1984) 1216–1221. [11] S.R. O’Donnell, J.C. Wanstall, The importance of choice of agonist in studies designed to predict b2:b1 adrenoceptor selectivity of antagonists from pA2 values on guineapig trachea and atria, Naunyn Schmiedebergs Arch. Pharmacol. 308 (1979) 183–190. [12] K.J. Broadley, M.H. Hawthorn, Examination of cardiac b-adrenoceptor subtypes by pharmacological and radioligand binding technique, Br. J. Pharmacol. 78 (suppl) (1983) 136P. [13] B.I. Costin, S.R. O’Donnel, J.C. Wanstall, Chronotropic responses of rabbit isolated atria to b-adrenoceptor agonists are mediated by only b1-adrenoceptors, J. Pharm. Pharmacol. 35 (1983) 752–756. [14] L.J. Bryan, J.J. Cole, S.R. O’Donnell, J.C. Wanstall, A study designed to explore the hypothesis that beta-1 adrenoceptors are ‘innervated’ receptors and beta-2 adrenoceptors are ‘hormonal’ receptors, J. Pharmacol. Exp. Ther. 216 (1981) 395–400. [15] G.L. Stiles, S. Taylor, R.J. Lefkowitz, Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding, Life Sci. 33 (1983) 467 – 473. [16] P. Robberecht, M. Delhaye, G. Taton, et al., The human heart beta-adrenergic receptors. Heterogeneity of the binding sites: presence of 50% beta1- and 50% beta2adrenergic receptors, Mol. Pharmacol. 24 (1983) 169–173.
121
[17] O.E. Brodde, N. O’Hara, H.R. Zerkowski, N. Rohm, Human cardiac b-adrenoceptors: both b1- and b2adrenoceptors are functionally coupled to the adenylate cyclase in right atrium, J. Cardiovasc. Pharmacol. 6 (1984) 1184 – 1191. [18] H.R. Zerkowski, K. Ikezono, N. Rohm, J.C. Reidemeister, O.E. Brodde, Human myocardial b-adrenoceptors: demonstration of both b1- and b2-adrenoceptors mediating contractile responses to b-agonists on the isolated right atrium, Naunyn Schmiedeberg’s Arch. Pharmacol. 332 (1986) 142 – 147. [19] K.P. Minneman, L.R. Hegstrand, P.B. Molinoff, Simultaneous determination of beta-1 and beta-2 adrenergic receptors in tissues containing both receptor subtypes, Mol. Pharmacol. 16 (1979) 34 – 46. [20] C.V. Jackson, G.M. McGrath, J.H. McNeill, Alterations in alpha1-adrenoceptor stimulation of isolated atria from experimental diabetic rats, Can. J. Physiol. Pharmacol. 64 (1986) 145 – 151. [21] A.J. Kaumann, Four b-adrenoceptor subtypes in the mammalian heart, Trends Pharmacol. Sci. 18 (1997) 70 – 76. [22] J.M. Foy, P.D. Lucas, Effect of experimental diabetes, food deprivation and genetic obesity on the sensitivity of pithed rats to autonomic agents, Br. J. Pharmacol. 57 (1976) 229 – 234. [23] R.K. Goyal, B. Rodrigues, J.H. McNeill, Effect of triiodothyronine on cardiac responses to adrenergic-agonists in STZ-induced diabetic rats, Gen. Pharmacol. 18 (1987) 357 – 362. [24] C.E. Heyliger, C.N. Pierce, P.K. Singal, R.E. Beamish, N.S. Dhalla, Cardiac a- and b-adrenergic receptor alterations in diabetic cardiomyopathy, Basic Res. Cardiol. 77 (1982) 610 – 618. [25] Z. Yu, J.H. McNeill, Altered inotropic responses in diabetic cardiomyopathy and hypertensive-diabetic cardiomyopathy, J. Pharmacol. Exp. Ther. 257 (1991) 64 – 71. [26] K. Kamata, T. Satoh, H. Tanaka, K. Shigenobu, Changes in electrophysiological and mechanical responses of the rat papillary muscle to a- and b- agonist in streptozotocin-induced diabetes, Can. J. Physiol. Pharmacol. 75 (1997) 781 – 788. [27] D.J. Paulson, D. Shetlar, K.E. Light, Catecholamine levels in the heart, serum and adrenals of experimental diabetic rats, Fed. Proc. 39 (1980) 637. [28] N.J. Christensen, Plasma norepinephrine and epinephrine in untreated diabetics, during fasting and after insulin administration, Diabetes 23 (1974) 1 – 8. [29] P.K. Ganguly, K.S. Dhalla, I.R. Innes, R.E. Beamish, N. Dhalla, Altered norepinephrine turnover and metabolism in diabetic cardiomyopathy, Circ. Res. 59 (1986) 684 – 693. [30] P.K. Ganguly, R.E. Beamish, K.S. Dhalla, I.R. Innes, N. Dhalla, Norepinephrine storage, distribution, and release in diabetic cardiomyopathy, Am. J. Physiol. 252 (1987) E734 – E739.
U8 .D. Dinc¸er et al. / Diabetes Research and Clinical Practice 40 (1998) 113–122
122
[31] Y. O8 ztu¨rk, V.M. Altan, N. Yildizoglu-Ari, Effects of experimental diabetes and insulin on smooth muscle functions, Pharmacol. Rev. 48 (1996) 69–112. [32] A.R. Tahiliani, J.H. McNeill, Diabetes-induced abnormalities in the myocardium, Life Sci. 38 (1986) 959–974. [33] B. Rodrigues, J.H. McNeill, The diabetic heart: metabolic causes for the development of a cardiomyopathy, Circ. Res. 26 (1992) 913 –922. [34] A.T. O8 zc¸elikay, N. Yildizoglu-Ari, A. O8 zu¨ari, Y. O8 ztu¨rk,
.
V.M. Altan, The effect of vanadate on alloxan-diabetic rat atria, Diabetes Res. Clin. Pract. 19 (1993) 189 – 194. [35] A. O8 zu¨ari, Y. O8 ztu¨rk, N. Yildizoglu-Ari, A.T. O8 zc¸elikay, V.M. Altan, The effects glyburide and insulin on the cardiac performance in rats with noninsulin-dependent diabetes mellitus, Gen. Pharmacol. 24 (1993) 165 – 169. [36] J.H. McNeill, A.G. Tahiliani, Diabetes-induced cardiac changes, Trends Pharmacol. Sci. 11 (1986) 364 – 367.