Vol. 298, Issue 2, 857-864, August 2001

Carbachol Inhibits the L-Type Ca²⁺ Current Augmented by 1,2-bis(2-Aminophenoxy)ethane-N,N,N',N'-Tetraacetic Acid in Guinea Pig Ventricular Myocytes: Calcium-Sensitivity Hypothesis for Muscarinic Inhibition

Jian-Bing Shen and Achilles J. Pappano

Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut

The L-type Ca²⁺ current [I_Ca(L)] increases with time after patch rupture in guinea pig ventricular myocytes dialyzed with pipette solutions containing 20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid ([BAPTA]_pip). I_Ca(L) progressively increases because BAPTA chelates subsarcolemmal Ca²⁺ to disinhibit cardiac adenylyl cyclase (AC) activity. We studied inhibition by carbachol (CCh) of I_Ca(L) (22-24°C). At 40 mM [BAPTA]_pip, 100 µM CCh reversibly suppressed I_Ca(L) maximally by 42%; half-maximal inhibition (20%) required 1 µM. Atropine antagonized the CCh effect on BAPTA-stimulated I_Ca(L), as did dialysis with 50 µM guanosine-5'-O-(3-thio)triphosphate. At 20, 30, and 40 mM [BAPTA]_pip, I_Ca(L) increased by 6.7 ± 1.8, 10.1 ± 1.4, and 11.3 ± 1.2 pA/pF, respectively. Inhibition by 100 µM CCh averaged 1.8 ± 0.6, 2.3 ± 0.4, and 4.1 ± 0.4 pA/pF at 20, 30, and 40 mM [BAPTA]_pip, respectively. Dialysis of the AC inhibitor 2'-dAMP (100 µM) suppressed I_Ca(L) run up in 40 mM BAPTA and its inhibition by CCh. Replacing 1.8 mM external Ca²⁺ with Ba²⁺, which lacks high-affinity regulatory sites on AC, suppressed CCh-induced inhibition. Neither I_Ca(L) run up nor its inhibition by CCh occurred when 40 mM EGTA, a slower chelator, replaced BAPTA. Our results support the AC disinhibition hypothesis for BAPTA. We propose that CCh inhibits I_Ca(L) in BAPTA by increasing either AC sensitivity to inhibition by ambient Ca²⁺ or the activity of the inhibitory guanine nucleotide binding protein.