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.
The L-type Ca2+ current [I(Ca(L))] increases with time after patch rupture in guinea pig ventricular myocytes dialyzed with pipette solutions containing > or =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 degrees C). At 40 mM [BAPTA]pip, 100 microM CCh reversibly suppressed I(Ca(L)) maximally by 42%; half-maximal inhibition (20%) required 1 microM. Atropine antagonized the CCh effect on BAPTA-stimulated I(Ca(L),) as did dialysis with 50 microM 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 microM 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 microM) 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.
Shen
J B
JB
Department of Pharmacology, University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
Pappano
A J
AJ
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020746:Calcium Channels, L-Type / Q000187:drug effects*; D020013:Calcium Signaling / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology*; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology*; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000494:pharmacology*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques
D000067956:Adenylyl Cyclase Inhibitors; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D018118:Chloride Channels; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D004533:Egtazic Acid; D002217:Carbachol; D017868:Cyclic AMP-Dependent Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid