We used electrophysiological and binding techniques to assess the presence of β1 and β2‐adrenoceptors (β1AR and β2AR) in rat cardiac myocytes and to determine their ratio during aging. Experiments were performed in left ventricular myocytes enzymatically dissociated from the heart of 3‐(young) or 22‐month‐old (old) Wistar Kyoto rats In patch‐clamp experiments, myocytes from old rats showed a prolonged action potential duration (at −20 mV: 41.7±3.6 vs 26.2±3.1 ms; at −60 mV: 154.4 ± 17.7 vs 87.1 ±6.9 ms, P < 0.05) and an augmented membrane capacitance (an index of cell size) (271.7±20.2 vs 164.3± 14.6 pF, P < 0.05) compared to young rats. β2AR stimulation, achieved by superfusing myocytes with the selective β2AR agonist, zinterol (10 μm) or with (−)‐isoprenaline (1 μm) in the presence of the selective β1 AR antagonist, CGP 20712A (0.1 μm), significantly increased L‐type calcium current (Ica,L) in rat ventricular myocytes. The percentage increase was similar in both young and old rats, either with zinterol (26.9 ±3.6% and 24.2±2.8%, respectively) or isoprenaline plus CGP 20712A (30.4±3.7% and 22.4±4.1%, respectively). Isoprenaline alone (β1AR and β2AR stimulation) caused a much smaller increase in ICa,L in old rats (58.4±12.1%) than in younger ones (95.3±8.1%) (P=0.067) The number of βAR mg−1 protein, measured with saturation binding assays of the non selective βAR antagonist [3H]‐CGP 12177 was 1989.4± 189.5 for 3‐ and of 1580.7± 161.5 for 22‐month‐old rats. Competition for [3H]‐CGP 12177 binding by CGP 20712A gave biphasic curves which demonstrated two classes of binding sites. Densities (as percentages of total PAR density), and affinities for the two binding sites were: 80.4 ±2.2% (Ki = 6.6 ±1.3 nM) β1AR and 19.6 ±2.2% (Ki = 6.9 ±2.2 μm) β2AR in young rats and 66.1 ±1.2% (Ki = 8.3 ±1.1 nM) β1 AR and 33.9 ±1.2% (Ki = 5.2 ± 0.6 μm) β2AR in old rats. The β1AR/β2AR ratio was significantly (P<0.01) reduced in old rats with respect to the younger ones By combining electrophysiological and binding measurements, we calculated β1AR and β2AR densities as number of receptors per μm2 of cell surface. In old rats, β1AR density was significantly decreased compared to young rats (8.4±2.0 vs 15.4±3.7 receptors μm−2, P<0.05), while β2AR density remained unchanged at both 3 and 22 months (3.8 ±0.7 and 4.2 ±1.1 receptors μm−2, respectively) Our results demonstrate that both β1AR and β2AR are functionally present in rat ventricular myocytes of young and old rats. The decreased responsiveness to βAR stimulation during aging appears to be associated with a selective reduction in the density of β1AR. 1995 British Pharmacological Society

β-Adrenoceptor subtypes in young and old rat ventricular myocytes: a combined patch-clamp and binding study

CERBAI, Elisabetta
Primo
;
GUERRA, Laura
Secondo
;
VARANI, Katia;BARBIERI, Mario;BOREA, Pier Andrea
Penultimo
;
MUGELLI, Alessandro
Ultimo
1995

Abstract

We used electrophysiological and binding techniques to assess the presence of β1 and β2‐adrenoceptors (β1AR and β2AR) in rat cardiac myocytes and to determine their ratio during aging. Experiments were performed in left ventricular myocytes enzymatically dissociated from the heart of 3‐(young) or 22‐month‐old (old) Wistar Kyoto rats In patch‐clamp experiments, myocytes from old rats showed a prolonged action potential duration (at −20 mV: 41.7±3.6 vs 26.2±3.1 ms; at −60 mV: 154.4 ± 17.7 vs 87.1 ±6.9 ms, P < 0.05) and an augmented membrane capacitance (an index of cell size) (271.7±20.2 vs 164.3± 14.6 pF, P < 0.05) compared to young rats. β2AR stimulation, achieved by superfusing myocytes with the selective β2AR agonist, zinterol (10 μm) or with (−)‐isoprenaline (1 μm) in the presence of the selective β1 AR antagonist, CGP 20712A (0.1 μm), significantly increased L‐type calcium current (Ica,L) in rat ventricular myocytes. The percentage increase was similar in both young and old rats, either with zinterol (26.9 ±3.6% and 24.2±2.8%, respectively) or isoprenaline plus CGP 20712A (30.4±3.7% and 22.4±4.1%, respectively). Isoprenaline alone (β1AR and β2AR stimulation) caused a much smaller increase in ICa,L in old rats (58.4±12.1%) than in younger ones (95.3±8.1%) (P=0.067) The number of βAR mg−1 protein, measured with saturation binding assays of the non selective βAR antagonist [3H]‐CGP 12177 was 1989.4± 189.5 for 3‐ and of 1580.7± 161.5 for 22‐month‐old rats. Competition for [3H]‐CGP 12177 binding by CGP 20712A gave biphasic curves which demonstrated two classes of binding sites. Densities (as percentages of total PAR density), and affinities for the two binding sites were: 80.4 ±2.2% (Ki = 6.6 ±1.3 nM) β1AR and 19.6 ±2.2% (Ki = 6.9 ±2.2 μm) β2AR in young rats and 66.1 ±1.2% (Ki = 8.3 ±1.1 nM) β1 AR and 33.9 ±1.2% (Ki = 5.2 ± 0.6 μm) β2AR in old rats. The β1AR/β2AR ratio was significantly (P<0.01) reduced in old rats with respect to the younger ones By combining electrophysiological and binding measurements, we calculated β1AR and β2AR densities as number of receptors per μm2 of cell surface. In old rats, β1AR density was significantly decreased compared to young rats (8.4±2.0 vs 15.4±3.7 receptors μm−2, P<0.05), while β2AR density remained unchanged at both 3 and 22 months (3.8 ±0.7 and 4.2 ±1.1 receptors μm−2, respectively) Our results demonstrate that both β1AR and β2AR are functionally present in rat ventricular myocytes of young and old rats. The decreased responsiveness to βAR stimulation during aging appears to be associated with a selective reduction in the density of β1AR. 1995 British Pharmacological Society
1995
Cerbai, Elisabetta; Guerra, Laura; Varani, Katia; Barbieri, Mario; Borea, Pier Andrea; Mugelli, Alessandro
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11392/1197911
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