Voltage -dependent suppression of calcium current by caffeine in single smooth muscle cells of the guinea-pig urinary bladder

Abstract

The suppressive action of caffeine on l,-type Ca current (Ica) in smooth muscle cells of the guinea-pig urinary bladder was investigated using the whole-cell patch clamp technique. Caffeine (5–30 mM) suppressed Ica, the effect having two phases: a rapid and transient suppression of Ica, which was followed by a sustained suppression. When intracellular Ca⁺ was strongly buffered by the Ca⁺ chelator EGTA (20 mM) or BAPTA (5 mM) in the patch pipette, the transient suppression of Ica was abolished, whereas the sustained effect remained. Similarly, inclusion of both 10 mM procaine and 1 mg/ml heparin in the patch pipette blocked the transient suppression of Ica, but did not block the sustained effect. The degree of the sustained effect of caffeine on Ica was dose-dependent with a κ_d of 20 mM. Application of the cyclic AMP analogue, 8-bromo-cyclic AMP (100 μM) or forskolin (10 μM) to the bath failed to mimick the sustained suppression of Ica, suggesting that inhibition of phosphodiesterase activity was not involved in the caffeine action. The steady-state activation curve remained unchanged by 10 mM caffeine but the steady-state inactivation curve was significantly shifted in the negative direction by 15.6 mV in 1.8 mM Ca²⁺ solution or by 10 mV in 1.8 mM Ba²⁺ solution. From these results it appears that caffeine inhibits L-type Ica via two mechanisms: (1) it releases Ca²⁺ from an internal store causing a transient Ca²⁺-mediated inactivation of the Ca channel; (2) it inhibits Ca channel via a mechanism that does not require such a Ca²⁺ release. It is possible that caffeine suppresses Ica through a preferential binding to the inactivated state of l-type Ca channel.