BETA-ADRENOCEPTOR SUBTYPES IN YOUNG AND OLD RAT VENTRICULAR MYOCYTES - A COMBINED PATCH-CLAMP AND BINDING STUDY

1 We used electrophysiological and binding techniques to assess the pr esence of beta(1)- and beta(2)-adrenoceptors (beta(1)AR and beta(2)AR) in rat cardiac myocytes and to determine their ratio during aging. Ex periments were performed in left ventricular myocytes enzymatically di ssociated from the heart of 3-(young) or 22-month-old (old) Wistar Kyo to rats. 2 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 augme nted membrane capacitance (an index of cell size) (271.7+/-20.2 vs 164 .3+/-14.6 pF, P<0.05) compared to young rats. beta(2)AR stimulation, a chieved by superfusing myocytes with the selective beta(2)AR agonist, zinterol (10 mu M) or with (-)-isoprenaline (1 mu M) in the presence o f the selective beta(1) AR antagonist, CGP 20712A (0.1 mu M), signific antly increased L-type calcium current (I-Ca,I-L) in rat ventricular m yocytes. The percentage increase was similar in both young and old rat s, 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%, respective ly). Isoprenaline alone (beta(1)AR and beta(2)AR stimulation) caused a much smaller increase in I-Ca,I-L in old rats (58+/-12.1%) than in yo unger ones (95.3+/-8.1%) (P=0.067). 3 The number of beta AR mg(-1) pro tein, measured with saturation binding assays of the non selective bet a AR antagonist [H-3]-CGP 12177 was 1989.4+/-189.5 for 3- and of 1580. 7+/-161.5 for 22-month-old rats. Competition for [H-3]-CGP 12177 bindi ng by CGP 20712A gave biphasic curves which demonstrated two classes o f binding sites. Densities (as percentages of total beta AR density), and affinities for the two binding sites were: 80.4+/-2.2% (K-i=6.6+/- 1.3 nM) beta(1)AR and 19.6+/-2.2% (K-i=6.9+/-2.2 mu M) beta(2)AR in yo ung rats and 66.1+/-1.2% (K-i=8.3+/-1.1 nM) beta(1) AR and 33.9+/-1.2% (K-i=5.2+/-0.6 mu M) beta(1)AR in old rats. The beta(1)AR/beta(2)AR r atio was significantly (P<0.01) reduced in old rats with respect to th e younger ones. 4 By combining electrophysiological and binding measur ements, we calculated beta(1)AR and beta(2)AR densities as number of r eceptors per mu m(2) of cell surface. In old rats, beta(1)AR density w as significantly decreased compared to young rats (8.4+/-2.0 vs 15.4+/ -3.7 receptors mu m(-2), P<0.05), while beta(2)AR density remained unc hanged at both 3 and 22 months (3.8+/-0.7 and 4.2+/-1.1 receptors mu m (-2), respectively). 5 Our results demonstrate that both beta(1)AR and beta(2)AR are functionally present in rat ventricular myocytes of you ng and old rats. The decreased responsiveness to beta AR stimulation d uring aging appears to be associated with a selective reduction in the density of beta(1)AR.