Abstract
The L-type Ca2+ current [ICa(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). ICa(L)progressively increases because BAPTA chelates subsarcolemmal Ca2+ to disinhibit cardiac adenylyl cyclase (AC) activity. We studied inhibition by carbachol (CCh) of ICa(L)(22–24°C). At 40 mM [BAPTA]pip, 100 μM CCh reversibly suppressed ICa(L) maximally by 42%; half-maximal inhibition (20%) required 1 μM. Atropine antagonized the CCh effect on BAPTA-stimulated ICa(L), as did dialysis with 50 μM guanosine-5′-O-(3-thio)triphosphate. At 20, 30, and 40 mM [BAPTA]pip, ICa(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 ICa(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 ICa(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 ICa(L) in BAPTA by increasing either AC sensitivity to inhibition by ambient Ca2+ or the activity of the inhibitory guanine nucleotide binding protein.
Footnotes
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This work was supported by U.S. Public Health Service Grant HL-13339.
- Abbreviations:
- mAChR
- muscarinic acetylcholine receptor
- ICa(L)
- L-type calcium current
- IBa(L)
- L-type barium current
- IK(Ach)
- inwardly rectifying K+current
- AC
- adenylyl cyclase
- P-site
- purine site
- BAPTA
- 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- PKA
- protein kinase A
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- Giα
- inhibitory guanine nucleotide binding protein
- [Ca]sm
- subsarcolemmal calcium
- τ1
- time constant of fast inactivating component
- τ2
- time constant of slow inactivating component
- CCh
- carbachol
- ACh
- acetylcholine
- ISO
- isoproterenol
- I-V
- current-voltage
- PDE
- phosphodiesterase
- CaM
- Ca/calmodulin
- ICl(cAMP)
- cAMP-induced Ca− current.
- Received December 18, 2000.
- Accepted May 9, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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