Enhanced Inhibition of L-type Ca 2+ Current by  3 -Adrenergic Stimulation in Failing Rat Heart Zhu-Shan Zhang, Heng-Jie Cheng, Katsuya Onishi, 1 Nobuyuki Ohte, 2 Thomas Wannenburg, and Che-Ping Cheng Wake Forest University School of Medicine, Winston-Salem, North Carolina Received May 16, 2005; accepted August 30, 2005 ABSTRACT  3 -adrenergic receptors (AR) have recently been identified in mammalian hearts and shown to be up-regulated in heart fail- ure (HF).  3 -AR stimulation reduces inotropic response asso- ciated with an inhibition of L-type Ca 2+ channels in normal hearts; however, the effects of  3 -AR activation on Ca 2+ chan- nel in HF remain unknown. We compared the effects of  3 -AR activation on L-type Ca 2+ current (I Ca,L ) in isolated left ventric- ular myocytes obtained from normal and age-matched rats with isoproterenol (ISO)-induced HF (4 months after 340 mg/kg s.c. for 2 days). I Ca,L was measured using whole-cell voltage clamp and perforated-patch recording techniques. In normal myo- cytes, superfusion of 4-[-[2-hydroxy-(3-chlorophenyl)ethyl- amino]propyl]phenoxyacetate (BRL-37,344; BRL), a  3 -AR ag- onist, caused a dose-dependent decrease in I Ca,L with maximal inhibition (21%, 1.1  0.2 versus 1.4  0.1 nA) (p  0.01) at 10 -7 M. In HF myocytes, the same concentration of BRL pro- duced a proportionately greater inhibition (31%) in I Ca,L (1.1  0.2 versus 1.6  0.2 nA) (p  0.05). A similar inhibition of I Ca,L was also observed with ISO (10 -7 M) in the presence of a  1 - and  2 -AR antagonist, nadolol (10 -5 M). Inhibition was abolished by the  3 -AR antagonist (S)-N-[4-[2-[[3-[3-(acetami- domethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]ben- zenesulfonamide (L-748,337; 10 -6 M), but not by nadolol. The inhibitory effect of BRL was attenuated by a nitric-oxide syn- thase (NOS) inhibitor, N G -nitro-L-arginine methyl ester (10 -4 M), and was prevented by the incubation of myocytes with pertus- sis toxin (PTX; 2 g/ml, 36°C, 6 h). In conclusion,  3 -AR acti- vation inhibits L-type Ca 2+ channel in both normal and HF myocytes. In HF,  3 -AR stimulation-induced inhibition of Ca 2+ channel is enhanced. These effects are likely coupled with PTX-sensitive G-protein and partially mediated through a NOS- dependent pathway.  1 -,  2 -, and  3 -adrenergic receptors have been found to be present in mammalian hearts and shown to modulate cardiac contractility by a variety of mechanisms.  1 - and  2 -AR stim- ulation of L-type Ca 2+ channel are mediated by a cAMP/ protein kinase A-signaling mechanism and coupled with G s proteins (Skeberdis et al., 1997; Xiao et al., 1999; Zhang et al., 2001).  2 -AR also couples with G i protein (Xiao et al., 1999). Recently,  3 -AR was identified in mammalian hearts, including human, dog, rat, and guinea pig (Gauthier et al., 1996; Kitamura et al., 2000; Cheng et al., 2001; Dincer et al., 2001).  3 -AR stimulation inhibits cardiac contractility via a G i protein pathway and by a mechanism coupled with the nitric-oxide synthase (NOS) system (Gauthier et al., 1996, 1998; Seppet, 2003). The negative inotropic effect of  3 -AR stimulation is associated with alterations of action potentials (Gauthier et al., 1996) and decreased Ca 2+ transient (Kita- mura et al., 2000). BRL-37,344, a selective  3 agonist, inhib- its L-type Ca 2+ channels and attenuates intracellular Ca 2+ transients in canine ventricular myocytes with an associated dose-dependent decrease in contractility (Cheng et al., 2001). A similar effect on basal I Ca,L is partly abolished by N G -nitro- L-arginine methyl ester (L-NAME) (Au and Kwan, 2002). Recently, several studies have reported that  3 -ARs are up-regulated in the failing human heart (Moniotte et al., 2001), in the canine models of HF (Cheng et al., 2001), as well as in diabetic rat hearts (Dincer et al., 2001). HF is associated with selective down-regulation of  1 -AR and a marked in- This study was supported in part by grants from the National Institutes of Health (HL45258 and HL53541) and the American Heart Association (9640189N). 1 Current affiliation: First Department of Internal Medicine, Mie Univer- sity School of Medicine, Tsu City, Japan. 2 Current affiliation: Department of Internal Medicine and Pathophysiol- ogy, Nagoya City University Graduate School of Medical Sciences, Mizuho-cho, Mizuho-ku, Japan. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.089672. ABBREVIATIONS: AR, adrenergic receptor; NOS, nitric-oxide synthase; BRL, 4-[-[2-hydroxy-(3-chlorophenyl)ethyl-amino]propyl]phenoxyacetate (BRL-37,344); I Ca,L , L-type Ca 2+ current; L-NAME, N G -nitro-L-arginine methyl ester; HF, heart failure; LV, left ventricular; ISO, isoproterenol; PTX, pertussis toxin; L-748,337, (S)-N-[4-[2-[[3-[3-(acetamidomethyl)phenoxy]-2-hydroxypropyl]amino]ethyl]phenyl]benzenesulfonamide; ICI, ()-1- [2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol (ICI-118,551); Nad, nadolol; NO, nitric oxide. 0022-3565/05/3153-1203–1211$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 315, No. 3 Copyright © 2005 by The American Society for Pharmacology and Experimental Therapeutics 89672/3062995 JPET 315:1203–1211, 2005 Printed in U.S.A. 1203 at ASPET Journals on July 20, 2018 jpet.aspetjournals.org Downloaded from