Selectivity of calcium channel blockers on T- and L-type calcium currents in guinea-pig ventricular myocytes.

Both L- (CaL) and T-type (CaT) calcium channels are present in the heart (McDonald et al., 1994). They have different distribution, functional role, and electrophysiological and pharmacological properties. CaL in cardiac myocytes are blocked by classical calcium channel blockers (CCB), which are classified in different chemical classes: phenylalkylamines, dihydropyridines and benzothiazepines. T-type channels are thought to be insensitive to these drugs and to be selectively blocked by the tetralol derivative mibefradil, recently withdrawn from the market due to multiple metabolic interactions. Due to their different voltage-dependence, CaT or CaL blockade by CCB is usually studied by applying steps from more (-90 mV) or less negative (-50 mV) holding potentials (HP), respectively. Thus it is practically impossible to extrapolate from the data obtained by using voltage clamp protocols the relative selectivity of CCB under physiological conditions. Thus, we aimed to compare the CaT/CaL blocking selectivity (T/L) of several CCB by evaluating their effects on both components evoked in the same cell. A voltage-clamp protocol mimicking a normal action potential was used: HP of -90 mV, 200 ms depolarising steps to -50/+50 mV. A concentration of CCB, reported to block at least 50% of CaL evoked from a HP in the range of -50 to -30 mV, was used: 1 µM for amlodipine (AML), lacidipine (LAC) and lercanidipine (LER); 10 µM for diltiazem (DIL); 1 µM for verapamil (VER). Mibefradil (MIB, 3 µM) was used as representative CaT blocker. All drugs except MIB were dissolved in DMSO (0.1%). Methods for cell isolation and patch-clamp recordings were as described previously (Cerbai et al., 97; Koidl et al., 1997). Guinea-pig ventricular myocytes having both CaT and CaL were superfused with a Na+ and K+ free solution pre-warmed to 35°C, to abolish overlapping currents. Data are expressed as mean±s.e.m. Statistics was performed by means of ANOVA followed by the Student-Neuman-Keuls test. Probability of less than 0.05 was considered significant. Using the described voltage protocol, all CCB blocked less than 20% CaL, with the exception of LAC which reduced CaL by 61.3% of control. Surprisingly, all CCB blocked a significant amount of CaT, varying from 0.8% (DIL) to 28% (MIB). We calculated for each cell the ratio T/L between CaT and CaL blockade. As expected, MIB showed the highest T selectivity (1.3±0.1, n=5); LAC and DIL resulted to be L selective (T/L= 0.4±0.1, n=3 and 0.8±0.1, n=8, p<0.05 versus MIB). VER (0.99±0.05, n=8) and AML (0.92±0.1, n=5) were not selective, while LER showed a slight T selectivity (1.1±0.05, n=6). Thus, CCB can be differentiated also on the basis of their T/L selectivity.