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Arylaminobenzoate block of the cardiac cyclic AMP-dependent chloride current
1Department of Pharmacology, University of South Carolina, School of Medicine, Columbia, South Carolina 29208, USA.
Abstract:
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel has been identified in the cardiac muscle of a number of mammalian species, including humans. The goal of this study was to begin quantifying the structural requirements necessary for arylaminobenzoate block of the CFTR channel. The cardiac cAMP-dependent Cl- current (ICl) was measured using the whole-cell arrangement of the patch-clamp technique in guinea pig ventricular myocytes during stimulation of protein kinase A with forskolin. At drug concentrations below the IC50 value for channel block, reduction of ICl by the arylaminobenzoates occurred in a strongly voltage-dependent manner with preferential inhibition of the inward currents. At higher drug concentrations, block of both the inward and outward ICl was observed. Increasing the length of the carbon chain between the benzoate and phenyl rings of the arylaminobenzoates resulted in a marked increase in drug block of the channel, with IC50 values of 47, 17, and 4 microM for 2-benzylamino-5-nitro-benzoic acid, 5-nitro-2-(2-phenylethylamino)-benzoic acid, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB), respectively. Increasing the carbon chain length further with the compound 5-nitro-2-(4-phenylbutylamino)-benzoic acid, caused no additional increase in the potency of drug block (IC50 = 4 microM). Inhibition of ICl by the arylaminobenzoates was modulated by the pH of the external solution; increasing the pH from 7.4 to 10.0 greatly weakened NPPB block, whereas decreasing the pH to 6.4 enhanced block. In addition, block of ICl was observed during intracellular dialysis of NPPB, and this action was not affected by raising the external pH.
Insights
Arylaminobenzoates block cardiac cystic fibrosis transmembrane conductance regulator (CFTR) channels. Increasing carbon chain length enhances block potency, with pH modulating inhibition effectiveness for CFTR channel research.
Area of Science:
- Cardiovascular Physiology
- Ion Channel Pharmacology
- Molecular Biology
Background:
- The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is present in mammalian cardiac muscle.
- CFTR plays a role in cardiac function, and its modulation is of therapeutic interest.
Purpose of the Study:
- To quantify the structural requirements for arylaminobenzoate compounds to block the cardiac CFTR channel.
- To investigate the voltage-dependence, pH-sensitivity, and structure-activity relationships of arylaminobenzoate CFTR channel blockers.
Main Methods:
- Whole-cell patch-clamp technique in guinea pig ventricular myocytes.
- Stimulation of protein kinase A with forskolin to activate cardiac cAMP-dependent chloride current (ICl).
- Application of various arylaminobenzoate derivatives to assess channel block.
Main Results:
- Arylaminobenzoates inhibited ICl in a voltage-dependent manner, preferentially blocking inward currents at low concentrations.
- Increasing the carbon chain length between benzoate and phenyl rings enhanced block potency, with NPPB showing an IC50 of 4 microM.
- Block was pH-sensitive, weakened at alkaline pH and enhanced at acidic pH, with intracellular application showing no pH effect.
Conclusions:
- The study elucidates the structure-activity relationship of arylaminobenzoates as CFTR channel blockers.
- Findings provide insights into the interaction of these compounds with the cardiac CFTR channel, suggesting potential for targeted drug design.