KMUP-1 inhibits L-type Ca²⁺ channels involved the protein kinase C in rat basilar artery myocytes

Abstract

This study investigated whether KMUP-1, a xanthine-based derivative, inhibits L-type Ca(2+) currents (I(Ca,L)) in rat basilar artery smooth muscle cells (RBASMCs). We used whole cell patch-clamp recording to monitor Ba(2+) currents (I(Ba)) through L-type Ca(2+) channels (LTCCs). Under voltage-clamp conditions, holding at -40 mV, KMUP-1 (1, 3, 10 μM) inhibited I(Ba) in a concentration-dependent manner and its IC(50) value was 2.27 ± 0.45 μM. A high concentration of KMUP-1 (10 μM) showed without modifying the I(Ba) current-voltage relationship. On the other hand, the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM) increase I(Ba) was inhibited by KMUP-1. Pretreatment with the PKC inhibitor chelerythrine (5 μM) intensified KMUP-1-inhibited I(Ba). However, the Rho kinase inhibitor Y-27632 (30 μM) failed to affect the I(Ba) inhibition by KMUP-1. In light of these results, we suggest that KMUP-1 inhibition of LTCCs in concentration- and voltage-dependent manners in RBASMCs may be due, at least in part, to its modulation of the PKC pathway.

Figure 1

External Ca²⁺ solution was substituted by BaCl₂ (Ba²⁺). (A) Replacement of 1.8 mM Ca²⁺ with 5 mM Ba²⁺ resulted in increases of the inward current evoked by a step from –40 to 10 mV; (B) time course of Ba²⁺ current (I_Ba) under control conditions. The peak I_Ba amplitude was plotted against time. About 12 minutes later, the current amplitude reached the steady state (n  =  6).

Figure 2

Effects of KMUP‐1 (1, 3, 10 μM) on inward I_Ba in RBASMCs. (A) Representative recordings of I_Ba evoked by a test pulse to 10 mV from a holding potential of –40 mV, before and after perfusion with KMUP‐1. (B) Bar graph showing the effects in the absence and presence of different concentrations of KMUP‐1 (n  =  6). * Denotes significant difference from control.

Figure 3

The I–V relationships of KMUP‐1 (10 μM) on inward I_Ba in RBASMCs. (A) Voltage protocol was designed to measure the I_Ba. Representative recordings of I_Ba evoked by a series of depolarizing pulse (200 ms, range from –40 to 50 mV) from a holding potential of –40 mV in the absence and presence of KMUP‐1; (B) average I–V relationship of the peak I_Ba in the absence (○) and presence (•) of KMUP‐1 (n  =  6). * Denotes significant difference from control.

Figure 4

Effects of KMUP‐1 (10 μM) on PMA (1 μM)‐induced activation of inward I_Ba in RBASMCs. (A) Representative recordings of I_Ba evoked by a test pulse to 10 mV from a holding potential of –40 mV before and after perfusion with PMA or PMA  +  KMUP‐1; (B) bar graph showing the relative I_Ba under control conditions and in the presence of PMA, KMUP‐1 or PMA  +  KMUP‐1 (n  =  6). * Denotes significant difference from control.

Figure 5

Effects of the PKC inhibitor chelerythrine (5 μM) on KMUP‐1 (10 μM)‐inhibited inward I_Ba in RBASMCs. (A) Representative recordings of I_Ba evoked by a test pulse to 10 mV from a holding potential of –40 mV before and after perfusion with chelerythrine or chelerythrine  +  KMUP‐1; (B) bar graph showing the relative I_Ba under control conditions and in the presence of chelerythrine, KMUP‐1, or chelerythrine  +  KMUP‐1 (n  =  6). * Denotes significant difference from control. ** Denotes significant difference between the two groups.

Figure 6

Effects of the Rho kinase inhibitor Y‐27632 (30 μM) on KMUP‐1 (10 μM)‐inhibited inward I_Ba in RBASMCs. (A) Representative recordings of I_Ba evoked by a test pulse to 10 mV from a holding potential of –40 mV before and after perfusion with Y‐27632 or Y‐27632  +  KMUP‐1; (B) bar graph showing the relative I_Ba under control conditions and in the presence of Y‐27632, KMUP‐1, or Y‐27632  +  KMUP‐1 (n  =  6). * Denotes significant difference from control. ** Denotes significant difference between the two groups.