T-channel-like pharmacological properties of high voltage-activated, nifedipine-insensitive Ca2+ currents in the rat terminal mesenteric artery

Abstract

1. Pharmacological properties of nifedipine-insensitive, high voltage-activated Ca(2+) channels in rat mesenteric terminal arteries (NICCs) were investigated and compared with those of alpha1E and alpha1G heterologously expressed in BHK and HEK293 cells respectively, using the patch clamp technique. 2. With 10 mM Ba(2+) as the charge carrier, rat NICCs (unitary conductance: 11.5 pS with 110 mM Ba(2+)) are almost identical to those previously identified in a similar region of guinea-pig, such as in current-voltage relationship, voltage dependence of activation and inactivation, and divalent cation permeability. However, these properties are considerably different when compared with alpha1E and alpha1G. 3. SNX-482(200 nM and sFTX3.3 (1 micro M), in addition to omega-conotoxin GVIA (1 micro M) and omega-agatoxin IVA (100 nM), were totally ineffective for rat NICC currents, but significantly suppressed alpha1E (by 82% at 200 nM; IC(50)=11.1 nM) and alpha1G (by 20% at 1 micro M) channel currents, respectively. A non-specific T-type Ca(2+) channel blocker nimodipine (10 micro M) differentially suppressed these three currents (by 40, 3 and 85% for rat NICC, alpha1E and alpha1G currents, respectively). 4. Mibefradil, the widely used T-type channel blocker, almost equally inhibited rat NICC and alpha1G currents in a voltage-dependent fashion with similar IC(50) values (3.5 and 0.3 micro M and 2.4 and 0.14 micro M at -100 and -60 mV, respectively). Furthermore, other organic T-type channel blockers such as phenytoin, ethosuximide, an arylpiperidine derivative SUN N5030 (IC(50)=0.32 micro M at -60 mV for alpha1G) also exhibited comparable inhibitory efficacies for NICC currents (inhibited by 22% at 100 micro M; IC(50)=27.8 mM; IC(50)=0.53 micro M, respectively). 5. These results suggest that despite distinctive biophysical properties, the rat NICCs have indistinguishable pharmacological sensitivities to many organic blockers compared with T-type Ca(2+) channels.

Figure 1

Biophysical profile of nifedipine-insensitive Ca²⁺ channel (NICC) current in rat mesenteric terminal artery. Bath; 10 mM Ba²⁺ external solution (10 μM nifedipine added). Pipette; Cs-internal solution. (A) Representative records of NICC evoked by 800 ms depolarizing step pulses from a holding potential of −80 mV at an interval of 20 s. (B) Current-voltage (I–V) relationship for rat NICC obtained from experiments as shown in A. The amplitude of NICC is normalized by the cell capacitance. (C) activation (open circles) and steady-state inactivation (evaluated by 10 s pre-conditioning pulses; filled circles) curves for rat NICC. Solid and dashed curves indicates the results of the best fits of data points by Boltzmann equation: 1/(1+exp((V_m−V_0.5)/k)), where V_m, V_0.5 and k denote the membrane potential, 50% activation or inactivation voltages, and slope factor, respectively. Activation curve is calculated by normalizing the chord conductance (the current amplitude is divided by the driving force) to its maximum. Symbols and bars indicate mean±s.e.mean from 5–8 cells. Actual traces (D) and the unitary current amplitude vs voltage relationship of single NICC activities (E) determined from four successful recordings). Pipette contained 110 mM Ba²⁺ solution supplemented with 10 μM nifedipine. Cells were bathed in 140 mM K⁺ solution (1 mM EGTA and 10 μM nifedipine added) to zero the transmembrane potential. Voltages values shown in the figure indicate the pipette potential with an inverted sign.

Figure 2

Activation and inactivation profiles of heterologously expressed α1E and α1G Ca²⁺ currents. Recording conditions are the same as in Figure 1. Actual records (A) and I–V relationships (normalized by cell capacitance) (B) of α1E (holding potential; −80 mV) and α1G (holding potential; −100 mV). (C) Activation and steady state inactivation curves evaluated as in Figure 1. Solid and dashed curves represent the best fits of data points with Boltzmann equation. Symbols and bars indicate mean±s.e.mean from 6–8 cells.

Figure 3

Channel type-specific inhibitory effects of SNX-482. Recording conditions are the same as in Figure 1 except for 400 ms voltage step pulses being used. Holding potential: −80 mV (rat NICC and α1E) and −100 mV (α1G). Depolarizing step pulses to 0 mV (rat NICC and α1E) or −20 mV (α1G) were repetitively applied every 20 s. Representative records (A) and time course (B) of the effects of SNX-482. (C) Concentration-inhibition relationship of α1E for SNX-482. Symbols and bars indicate mean±s.e.mean from eight individual experiments. Smooth solid curve is drawn according to the results of Hill analysis. (D) Comparison of the effects of 200 nM SNX-482 on rat and guinea-pig NICCs, α1E and α1G. ^**P<0.01 with ANOVA and pooled variance t-test. n=4–10.

Figure 4

Inhibitory effects of sFTX3.3 on three nifedipine-insensitive Ca²⁺ currents. Recording conditions are the same as in Figure 3. (A) Representative records. (B) Summary of inhibitory effects of 100 nM and 1 μM sFTX3.3. ^*P<0.05 with ANOVA and pooled variance t-test. n=5–6.

Figure 5

Differential inhibitory effects of nimodipine on three nifedipine-insensitive Ca²⁺ currents. Recording conditions are the same as in Figure 3. (A) Representative records. (B) Summary of inhibitory effects of 10 μM nimodipine. ^**P<0.01 with ANOVA and pooled variance t-test. n=4–7.

Figure 6

Voltage-dependent effects of mibefradil on rat NICC. Recording condition are the same as in Figure 3. Representative records (A) and the time course (B) of the effects of mibefradil on NICC evoked by depolarizing pulses to 0 mV (every 20 s) at two different holding potentials (−80 and −60 mV). (C) Concentration-inhibition curves for mibefradil at three different holding potentials. (D) Shifts of steady state inactivation curves at varying concentrations of mibefradil. The results of Hill (C) and Boltzmann (D) fitting are shown. n=5–7. In C, # and ^* indicate statistically significant differences with P values of <0.02 and <0.05, respectively, evaluated by pooled variance t-test with Bonferroni's correction.

Figure 7

Voltage-dependent effects of mibefradil on α1E and α1G. Ba²⁺ (10 mM) currents evaluated by depolarizing step pulses applied at every 20 s to 0 and −20 mV for α1E and α1G, respectively. n=5–6.

Figure 8

Effects of phenytoin and ethosuximide on rat NICC. (A) Actual records. (B) Dose-dependent effects of phenytoin. Phenytoin at concentrations higher than 100 μM was not dissolvable in the presence of 10 μM nifedipine. (C) Concentration–inhibition relationship of rat NICC for ethosuximide. Dashed curve indicates the results of Hill fitting after correcting the influence of osmolarity change on Ca²⁺ currents, which was estimated by adding varying concentrations of sucrose into the bathing solution (see inset). n=5.

Figure 9

Potent and comparable inhibition of three NI-Ca²⁺ currents by an arylpiperidine derivative SUN N5030. Recording conditions are the same as in Figure 3. (A) Actual records of rat NICC in the presence of varying concentration of SUN N5030. (B) Concentration-inhibition curves of rat NICC for SUN N5030. (C,D) Concentration-inhibition curves of α1G and α1E for SUN N5030. Sigmoid curves are drawn according to the results of Hill fitting. n= 6–13.