0 Macmillan Press Ltd,
Br. J. Pharmacol. (1992), 107, 185-188
1992
Cardiac ai-adrenoceptor densities in different mammalian species Steinfath, 2Yi-Yue Chen, 3Jan Lavick', Olaf Magnussen, Monika Nose, 'Markus Sebastian Rosswag, Wilhelm Schmitz & Hasso Scholz Abteilung Allgemeine Pharmakologie, Universitiits-Krankenhaus Eppendorf, UniversitAt Hamburg, Martinistrasse 52, D-2000 Hamburg 20, Germany 1 ax-Adrenoceptor densities were studied in cardiac membrane preparations from several mammalian species including human failing hearts under identical experiment conditions; the al-adrenoceptor antagonist, [3H]-prazosin, was used as radioligand. End-stage heart failure (NYHA IV) in human hearts was due to idiopathic dilated cardiomyopathy. 2 The ventricular a1-adrenoceptor densities were not significantly different in guinea-pig, mouse, pig, calf, and man (11 to 18 fmol mg-' protein) but about 5 to 8 fold smaller than in rat (about 90 fmol mg-' protein). Right and left ventricular receptor densities were similar in these species. 3 A sufficient amount of right and left atrial tissue was obtained from rabbit, pig, calf, and man only. The a,-adrenoceptor densities in both atria of these species were found to be at the detection limit of the method used (less than 8 fmol mg- protein). 4 The equilibrium dissociation constant (KD) was similar in all species studied ranging from 0.047 + 0.006 to 0.063 ± 0.007 nmol 1'. 5 It is concluded that differences in z1-adrenoceptor density between atria and ventricles may exist in mammalian species. The exceptionally high density of these receptors in rat ventricles seem to be a particular feature in these animals. Keywords: ax-Adrenoceptor; mammalian heart; failing human heart; end-stage idiopathic dilated cardiomyopathy; radioligand binding
Introduction The presence of al-adrenoceptors in mammalian hearts has been reported by several authors (see Wagner & Brodde, 1978; Benfey, 1980; 1990; Scholz, 1980; Endoh, 1982). It has been suggested that the a,-adrenoceptor-mediated response may become important under certain pathological conditions including ischaemia (Penny et al., 1985), abnormal thyroid function (Williams et al., 1977), dysrhythmogenesis (Sheridan et al., 1980) and hypertension (Yamada et al., 1984). ajAdrenoceptors may also be involved in other processes such as protein synthesis (Meidell et al., 1986) and cardiac hypertrophy (Simpson, 1990). Radioligand binding studies have been performed on several myocardial preparations (Williams & Lefkowitz, 1978; Rugevics & Schumann, 1987; Bevilacqua et al., 1987; Steinkraus et al., 1989; Shen et al., 1989) revealing intra- and interspecies differences in the density of cladrenoceptors which might partially be due to different experimental conditions. The present study was designed to compare the densities of cardiac m1-adrenoceptors and the equilibrium dissociation constants under identical experimental conditions in several mammalian species including the failing human heart.
Methods
Animals and patients This study was reviewed and approved by the institutional animal care and use committee. Male Wistar rats (200I
Author for correspondence. Present address: 2 Department of Pharmacology, Guangdong Medical School, Guangzhon, China. 3 Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, Louisiana, U.S.A.
250 g), guinea-pigs (250-300 g), mice (50-60 g), rabbits (2500-3000 g), pietrain pigs (20-30 kg), and calves (about 400 kg) were killed by a blow on the head (rats, guinea-pigs, mice, rabbits) or captive-bolt pistol (pigs, calves) and bleeding from the carotid arteries. The hearts were quickly removed and dissected free of fat and large vessels. Ventricular and atrial tissues were weighed and removed into icecold incubation buffer (50mmoll- Tris-HCl, 10mmoll1' MgCl2, pH 7.5). Eight failing human hearts were obtained from patients undergoing orthotopic heart transplantation due to end-stage idiopathic dilated cardiomyopathy (NYHA class IV, 3 female, 5 male, age: 53.2 years ranging from 46 to 61 years, left ventricular ejection fraction: 16.9 ± 1.5%, cardiac index: 1.8 ± 0.21 1 min-' m2). Patients were treated with nitrates, diuretics, calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, and cardiac glycosides alone or in combination. Written informed consent was obtained from all patients before cardiac transplantation. The use of tissues obtained from human failing hearts was approved by the Ethical Committee of the University of Hamburg. After explantation the human hearts were transferred immediately to the laboratory in ice-cold aerated bathing solution (modified
Tyrode solution) containing (mmol 1-'): NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCI2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, glucose 5.0, continuously gassed with 95% 02 plus 5% CO2 at 35°C and pH 7.4. Tissue samples were frozen in liquid nitrogen and stored at - 80°C until use.
Membrane preparation and binding assay Crude membrane homogenates of each species were prepared according to the method of Baker & Potter (1980) and Baker et al. (1980) with minor modifications. Briefly, the cardiac tissues (700-800 mg) were minced and homogenized in 20
186
M. STEINFATH et al.
volumes of incubation buffer with a precooled Ultra-Turrax tissue homogenizer (Janke und Kunkel, Staufen, Germany, 3 x 10 s bursts). The suspensions were diluted with an equal volume of ice-cold 1 mol 1-' KCI solution (Baker & Potter, 1980) and left on ice for 10 min before centrifugation at 48,000 g for O min at 4TC. Pellets were resuspended and homogenized with a glass-Teflon homogenizer (Colora, Lorch, Germany) for 1 min at setting 5 and recentrifuged. This procedure was repeated twice. The final pellets were resuspended in a volume of incubation buffer that adjusted the protein content in incubation assays to 100-200 pg ml-', passed through 4 layers of gauze and kept on ice under stirring. Assays were carried out in duplicate. Aliquots of 2501il of the membrane preparations were incubated in a total volume of 1000fgl with the radioligand [3H]-prazosin (250 1Al, specific activity 76.6 Ci mmol ', New England Nuclear, Dreieich, Germany) to label myocardial a,-adrenoceptor binding sites. For saturation binding experiments, nine different concentrations of [3H]-prazosin ranging from 0.01 to 1.0 nmol 1` were used. Specific binding was calculated by subtracting nonspecific binding which was defined in the presence of 5 pmol I-I of the a-adrenoceptor antagonist phentolamine (phentolamine hydrochloride, Ciba Geigy, Basel, Switzerland) from total binding. The binding assays were incubated for 60 min at 25TC allowing complete equilibration of radioligands with the binding sites. Incubation was terminated by rapid vacuum filtration through Whatman GF/C filters (Maidstone, Great Britain) and two 5 ml washes with ice-cold incubation buffer. Filters were dried at room temperature and radioactivity was counted by liquid scintillation spectrometry. The protein content was determined by the method of Lowry et al. (1951) with bovine serum albumin (Merz und Dade, Dudingen, Switzerland) as standard. All other chemicals were of analytical or best commercial grade available. Deionized and twice distilled water was used throughout.
Statistical evaluations Values presented are means ± s.e.mean. The maximal number of binding sites (Bma) and equilibrium dissociation constant (KD) were calculated from plots according to Scatchard (1949). Statistical significance was estimated by Student's t test for unpaired observations. A P value of less than 0.05 was considered significant.
a
300 0,1
X 1500
*I 45
90
._0
E CD
0 0.
b
a)
E
._
E 'a E 0.25
0.50
0.75
[3H1-prazosin (nmol I-') Figure 1 Typical experiments showing [3H]-prazosin binding to ventricular membranes obtained from rat (a) and guinea-pig (b) showing total (0), specific (@) and non-specific (A) binding. Ordinates: [3H]-prazosin specifically bound in fmol mg'l protein. Abscissae: concentration of radioligand in nmol 1I. Inset: linear transformation of a saturation experiment according to Scatchard (1949) as bound (B) to bound per free (B/F).
Discussion Results Typical saturation experiments of [3H]-prazosin binding to cardiac membranes are shown for the rat (Figure la) and for the guinea-pig representing the other species investigated (Figure lb), because ventricular a,-adrenoceptor densities were not significantly different (about 11 to 18 fmol mg-' protein) in guinea-pig, mouse, rabbit, pig, calf and man (failing human hearts with end-stage idiopathic dilated cardiomyopathy) but markedly smaller than in rat (about 90fmolmg-' protein, Table 1). Preparations from all the different species were saturable, revealed the characteristics of a single-site interaction as shown by linear Scatchard plots, and had similar equilibrium dissociation constants (KD Table 2) ranging from 4.7 x 10- ± 0.006 to 6.3 x 10-2 x 0.007 nmol 1-. Right and left ventricular a1-adrenoceptor densities (Table 1) as well as KD-values (Table 2) were similar in the species studied. A sufficient amount of right and left atrial tissue was obtained from the species rabbit, pig, calf, and man only. Atria derived from rat, guinea-pig, and mouse were too small for determination with the method used. Right as well as left atrial al-adrenoceptor densities in rabbit, pig, calf and man were found to be at the detection limit of less than 8 fmol mg- protein.
Many contradictory results have been published on al-adrenoceptor density in mammalian hearts with different experimental designs (Williams & Lefkowitz, 1978; Bevilacqua et al., 1987; Steinkraus et al., 1989; Shen et al., 1989). Studies comparing these receptors under identical experimental conditions in different species seem to be helpful to obtain insight into species differences. In the present study a marked difference in a,-adrenoceptor density was found between rat and other species like guinea-pig, mouse, pig, calf and man only. Similar al-adrenoceptor densities have been reported by Guicheney & Meyer (1981) in rat, Rugevics & Schumann (1987) in guinea-pig, and Kushida et al. (1988) in rabbit hearts. On the other hand, several authors found a larger number of myocardial al-adrenoceptors in rat (Han et al., 1989), guinea-pig (Karliner et al., 1979), and rabbit (Mukherjee et al., 1983). Furthermore, contradictory results have been reported for al-adrenoceptor density in the human failing hearts obtained from patients with end-stage idiopathic dilated cardiomyopathy. Kaumann (1986) found less than 10 fmol mg-' protein in ventricular preparations. Also a small number of ventricular al-adrenoceptors has been reported by Bristow et al. (1988) and Bohm et al. (1988). In contrast, Vago et al. (1989) have shown an approximately 6 fold higher density in a sarcolemma-enriched subcellular fraction. However, it seems likely that different experimental
CARDIAC al-ADRENOCEPTORS IN MAMMALIAN SPECIES
187
Table 1 Myocardial al-adrenoceptor density as shown by specifically bound (B.,,) [3H]-prazosin in right and left ventricular myocardium obtained from different mammalian species including failing human hearts (end-stage idiopathic dilated cardiomyopathy)
a,-Adrenoceptor density (fmol mg-' protein) Species Rat Guinea-pig Mouse Rabbit Pig Calf Man
Right ventricle
Left ventricle
n
9 9 8 8 7 6 8
91.5 4.8* 4.2* 16.4 2.1 2.2 15.1 ± 1.9 (+) 12.6 1.8 13.3 2.4 18.4 2.6 14.1 2.0 18.3±2.6 15.3±2.1 10.9 1.8 10.4±1.7
85.3 15.9
(+) Right and left ventricles were not distinguished because of the small size of the hearts. *P
Br. J. Pharmacol. (1992), 107, 185-188
1992
Cardiac ai-adrenoceptor densities in different mammalian species Steinfath, 2Yi-Yue Chen, 3Jan Lavick', Olaf Magnussen, Monika Nose, 'Markus Sebastian Rosswag, Wilhelm Schmitz & Hasso Scholz Abteilung Allgemeine Pharmakologie, Universitiits-Krankenhaus Eppendorf, UniversitAt Hamburg, Martinistrasse 52, D-2000 Hamburg 20, Germany 1 ax-Adrenoceptor densities were studied in cardiac membrane preparations from several mammalian species including human failing hearts under identical experiment conditions; the al-adrenoceptor antagonist, [3H]-prazosin, was used as radioligand. End-stage heart failure (NYHA IV) in human hearts was due to idiopathic dilated cardiomyopathy. 2 The ventricular a1-adrenoceptor densities were not significantly different in guinea-pig, mouse, pig, calf, and man (11 to 18 fmol mg-' protein) but about 5 to 8 fold smaller than in rat (about 90 fmol mg-' protein). Right and left ventricular receptor densities were similar in these species. 3 A sufficient amount of right and left atrial tissue was obtained from rabbit, pig, calf, and man only. The a,-adrenoceptor densities in both atria of these species were found to be at the detection limit of the method used (less than 8 fmol mg- protein). 4 The equilibrium dissociation constant (KD) was similar in all species studied ranging from 0.047 + 0.006 to 0.063 ± 0.007 nmol 1'. 5 It is concluded that differences in z1-adrenoceptor density between atria and ventricles may exist in mammalian species. The exceptionally high density of these receptors in rat ventricles seem to be a particular feature in these animals. Keywords: ax-Adrenoceptor; mammalian heart; failing human heart; end-stage idiopathic dilated cardiomyopathy; radioligand binding
Introduction The presence of al-adrenoceptors in mammalian hearts has been reported by several authors (see Wagner & Brodde, 1978; Benfey, 1980; 1990; Scholz, 1980; Endoh, 1982). It has been suggested that the a,-adrenoceptor-mediated response may become important under certain pathological conditions including ischaemia (Penny et al., 1985), abnormal thyroid function (Williams et al., 1977), dysrhythmogenesis (Sheridan et al., 1980) and hypertension (Yamada et al., 1984). ajAdrenoceptors may also be involved in other processes such as protein synthesis (Meidell et al., 1986) and cardiac hypertrophy (Simpson, 1990). Radioligand binding studies have been performed on several myocardial preparations (Williams & Lefkowitz, 1978; Rugevics & Schumann, 1987; Bevilacqua et al., 1987; Steinkraus et al., 1989; Shen et al., 1989) revealing intra- and interspecies differences in the density of cladrenoceptors which might partially be due to different experimental conditions. The present study was designed to compare the densities of cardiac m1-adrenoceptors and the equilibrium dissociation constants under identical experimental conditions in several mammalian species including the failing human heart.
Methods
Animals and patients This study was reviewed and approved by the institutional animal care and use committee. Male Wistar rats (200I
Author for correspondence. Present address: 2 Department of Pharmacology, Guangdong Medical School, Guangzhon, China. 3 Department of Pharmacology and Therapeutics, Louisiana State University Medical Center, Shreveport, Louisiana, U.S.A.
250 g), guinea-pigs (250-300 g), mice (50-60 g), rabbits (2500-3000 g), pietrain pigs (20-30 kg), and calves (about 400 kg) were killed by a blow on the head (rats, guinea-pigs, mice, rabbits) or captive-bolt pistol (pigs, calves) and bleeding from the carotid arteries. The hearts were quickly removed and dissected free of fat and large vessels. Ventricular and atrial tissues were weighed and removed into icecold incubation buffer (50mmoll- Tris-HCl, 10mmoll1' MgCl2, pH 7.5). Eight failing human hearts were obtained from patients undergoing orthotopic heart transplantation due to end-stage idiopathic dilated cardiomyopathy (NYHA class IV, 3 female, 5 male, age: 53.2 years ranging from 46 to 61 years, left ventricular ejection fraction: 16.9 ± 1.5%, cardiac index: 1.8 ± 0.21 1 min-' m2). Patients were treated with nitrates, diuretics, calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, and cardiac glycosides alone or in combination. Written informed consent was obtained from all patients before cardiac transplantation. The use of tissues obtained from human failing hearts was approved by the Ethical Committee of the University of Hamburg. After explantation the human hearts were transferred immediately to the laboratory in ice-cold aerated bathing solution (modified
Tyrode solution) containing (mmol 1-'): NaCl 119.8, KCl 5.4, CaCl2 1.8, MgCI2 1.05, NaH2PO4 0.42, NaHCO3 22.6, Na2EDTA 0.05, ascorbic acid 0.28, glucose 5.0, continuously gassed with 95% 02 plus 5% CO2 at 35°C and pH 7.4. Tissue samples were frozen in liquid nitrogen and stored at - 80°C until use.
Membrane preparation and binding assay Crude membrane homogenates of each species were prepared according to the method of Baker & Potter (1980) and Baker et al. (1980) with minor modifications. Briefly, the cardiac tissues (700-800 mg) were minced and homogenized in 20
186
M. STEINFATH et al.
volumes of incubation buffer with a precooled Ultra-Turrax tissue homogenizer (Janke und Kunkel, Staufen, Germany, 3 x 10 s bursts). The suspensions were diluted with an equal volume of ice-cold 1 mol 1-' KCI solution (Baker & Potter, 1980) and left on ice for 10 min before centrifugation at 48,000 g for O min at 4TC. Pellets were resuspended and homogenized with a glass-Teflon homogenizer (Colora, Lorch, Germany) for 1 min at setting 5 and recentrifuged. This procedure was repeated twice. The final pellets were resuspended in a volume of incubation buffer that adjusted the protein content in incubation assays to 100-200 pg ml-', passed through 4 layers of gauze and kept on ice under stirring. Assays were carried out in duplicate. Aliquots of 2501il of the membrane preparations were incubated in a total volume of 1000fgl with the radioligand [3H]-prazosin (250 1Al, specific activity 76.6 Ci mmol ', New England Nuclear, Dreieich, Germany) to label myocardial a,-adrenoceptor binding sites. For saturation binding experiments, nine different concentrations of [3H]-prazosin ranging from 0.01 to 1.0 nmol 1` were used. Specific binding was calculated by subtracting nonspecific binding which was defined in the presence of 5 pmol I-I of the a-adrenoceptor antagonist phentolamine (phentolamine hydrochloride, Ciba Geigy, Basel, Switzerland) from total binding. The binding assays were incubated for 60 min at 25TC allowing complete equilibration of radioligands with the binding sites. Incubation was terminated by rapid vacuum filtration through Whatman GF/C filters (Maidstone, Great Britain) and two 5 ml washes with ice-cold incubation buffer. Filters were dried at room temperature and radioactivity was counted by liquid scintillation spectrometry. The protein content was determined by the method of Lowry et al. (1951) with bovine serum albumin (Merz und Dade, Dudingen, Switzerland) as standard. All other chemicals were of analytical or best commercial grade available. Deionized and twice distilled water was used throughout.
Statistical evaluations Values presented are means ± s.e.mean. The maximal number of binding sites (Bma) and equilibrium dissociation constant (KD) were calculated from plots according to Scatchard (1949). Statistical significance was estimated by Student's t test for unpaired observations. A P value of less than 0.05 was considered significant.
a
300 0,1
X 1500
*I 45
90
._0
E CD
0 0.
b
a)
E
._
E 'a E 0.25
0.50
0.75
[3H1-prazosin (nmol I-') Figure 1 Typical experiments showing [3H]-prazosin binding to ventricular membranes obtained from rat (a) and guinea-pig (b) showing total (0), specific (@) and non-specific (A) binding. Ordinates: [3H]-prazosin specifically bound in fmol mg'l protein. Abscissae: concentration of radioligand in nmol 1I. Inset: linear transformation of a saturation experiment according to Scatchard (1949) as bound (B) to bound per free (B/F).
Discussion Results Typical saturation experiments of [3H]-prazosin binding to cardiac membranes are shown for the rat (Figure la) and for the guinea-pig representing the other species investigated (Figure lb), because ventricular a,-adrenoceptor densities were not significantly different (about 11 to 18 fmol mg-' protein) in guinea-pig, mouse, rabbit, pig, calf and man (failing human hearts with end-stage idiopathic dilated cardiomyopathy) but markedly smaller than in rat (about 90fmolmg-' protein, Table 1). Preparations from all the different species were saturable, revealed the characteristics of a single-site interaction as shown by linear Scatchard plots, and had similar equilibrium dissociation constants (KD Table 2) ranging from 4.7 x 10- ± 0.006 to 6.3 x 10-2 x 0.007 nmol 1-. Right and left ventricular a1-adrenoceptor densities (Table 1) as well as KD-values (Table 2) were similar in the species studied. A sufficient amount of right and left atrial tissue was obtained from the species rabbit, pig, calf, and man only. Atria derived from rat, guinea-pig, and mouse were too small for determination with the method used. Right as well as left atrial al-adrenoceptor densities in rabbit, pig, calf and man were found to be at the detection limit of less than 8 fmol mg- protein.
Many contradictory results have been published on al-adrenoceptor density in mammalian hearts with different experimental designs (Williams & Lefkowitz, 1978; Bevilacqua et al., 1987; Steinkraus et al., 1989; Shen et al., 1989). Studies comparing these receptors under identical experimental conditions in different species seem to be helpful to obtain insight into species differences. In the present study a marked difference in a,-adrenoceptor density was found between rat and other species like guinea-pig, mouse, pig, calf and man only. Similar al-adrenoceptor densities have been reported by Guicheney & Meyer (1981) in rat, Rugevics & Schumann (1987) in guinea-pig, and Kushida et al. (1988) in rabbit hearts. On the other hand, several authors found a larger number of myocardial al-adrenoceptors in rat (Han et al., 1989), guinea-pig (Karliner et al., 1979), and rabbit (Mukherjee et al., 1983). Furthermore, contradictory results have been reported for al-adrenoceptor density in the human failing hearts obtained from patients with end-stage idiopathic dilated cardiomyopathy. Kaumann (1986) found less than 10 fmol mg-' protein in ventricular preparations. Also a small number of ventricular al-adrenoceptors has been reported by Bristow et al. (1988) and Bohm et al. (1988). In contrast, Vago et al. (1989) have shown an approximately 6 fold higher density in a sarcolemma-enriched subcellular fraction. However, it seems likely that different experimental
CARDIAC al-ADRENOCEPTORS IN MAMMALIAN SPECIES
187
Table 1 Myocardial al-adrenoceptor density as shown by specifically bound (B.,,) [3H]-prazosin in right and left ventricular myocardium obtained from different mammalian species including failing human hearts (end-stage idiopathic dilated cardiomyopathy)
a,-Adrenoceptor density (fmol mg-' protein) Species Rat Guinea-pig Mouse Rabbit Pig Calf Man
Right ventricle
Left ventricle
n
9 9 8 8 7 6 8
91.5 4.8* 4.2* 16.4 2.1 2.2 15.1 ± 1.9 (+) 12.6 1.8 13.3 2.4 18.4 2.6 14.1 2.0 18.3±2.6 15.3±2.1 10.9 1.8 10.4±1.7
85.3 15.9
(+) Right and left ventricles were not distinguished because of the small size of the hearts. *P
