Inhibitory mechanism of xestospongin-C on contraction and ion channels in the intestinal smooth muscle

Abstract

1. Xestospongin-C isolated from a marine sponge, Xestospongia sp., has recently been shown to be a membrane-permeable IP(3) receptor inhibitor. In this study we examined the effects of this compound on smooth muscle from guinea-pig ileum. 2. In guinea-pig ileum permeabilized with alpha-toxin, xestospongin-C (3 microM) inhibited contractions induced by Ca(2+) mobilized from sarcoplasmic reticulum (SR) with IP(3) or carbachol with GTP, but not with caffeine. 3. In intact smooth muscle tissue, xestospongin-C (3-10 microM) inhibited carbachol- and high-K+-induced increases in [Ca(2+)](i) and contractions at sustained phase. 4. It also inhibited voltage-dependent inward Ba(2+) currents in a concentration-dependent manner with an IC(50) of 0.63 microM. Xestospongin-C (3-10 microM) had no effect on carbachol-induced inward Ca(2+) currents via non-selective cation channels; but it did reduce voltage-dependent K+ currents in a concentration-dependent manner with an IC(50) of 0.13 microM. 5. These results suggest that xestospongin-C inhibits the IP(3) receptor but not the ryanodine receptor in smooth muscle SR membrane. In intact smooth muscle cells, however, xestospongin-C appears to inhibit voltage-dependent Ca(2+) and K+ currents at a concentration range similar to that at which it inhibits the IP(3) receptor. Xestospongin-C is a selective blocker of the IP(3) receptor in permeabilised cells but not in cells with intact plasma membrane.

Figure 1

Effects of xestospongin-C (Xest-C, 3 and 10 μM) on transient contractions due to Ca²⁺ release from the sarcoplasmic reticulum in α-toxin-skinned muscle fibres. Prior to the experiment the muscle had been conditioned by adding 20 mM caffeine. We then induced contractions with IP₃ (30 μM), GTP (100 μM)+carbachol (10 μM) and caffeine (3 mM) twice to demonstrate the reproducibility of these responses. (A) Typical tracing of the effects of 3 μM xestospongin-C on transient contraction induced by 30 μM IP₃ in Ca²⁺-free solution. (B) Typical tracing of the effects of 3 μM xestospongin-C on a transient contraction induced by 10 μM carbachol in the presence of 100 μM GTP. (C) Typical tracing of the effects of 3 μM xestospongin-C on a transient contraction induced by 3 mM caffeine. (D) The area of force oscillations during a 15 min period was used for quantitative assessment of the effects of caffeine, carbachol and IP₃, and the summarized data of (A), (B) and (C) is shown (n=6 each). **P < 0.01 vs control. 100% represents the first control response before the addition of xestospongin-C.

Figure 2

Effects of xestospongin-C on carbachol-stimulated [Ca²⁺]_i (upper tracing) and muscle tension (lower tracing) in ileal smooth muscle. When [Ca²⁺]_i and muscle tension induced with carbachol reached a steady state level, the vehicle (ethanol 0.2%) (A), 3 (B) or 10 (C) μM xestospongin-C and 10 μM verapamil were added sequentially. (D) Summarized data on (B) and (C) (n=5 and 6, respectively). In (D), 100% represents carbachol-induced increases in [Ca²⁺]_i and force at steady state before the addition of xestospongin-C. Values were obtained 25 min after the addition of xestospongin-C. **P < 0.01 vs control.

Figure 3

Effects of xestospongin-C on high-K⁺-stimulated [Ca²⁺]_i (upper tracing) and muscle tension (lower tracing) in ileal smooth muscle. When [Ca²⁺]_i and muscle tension induced with high K⁺ reached a steady state level, 3 (A) or 10 (B) μM xestospongin-C and 10 μM verapamil were added sequentially. (C) Summarized data on (A) and (B) (n=6). In (C), 100% represents high K⁺-induced increases in [Ca²⁺]_i and force at steady state before the addition of xestospongin-C. Values were obtained 20–30 min after the addition of 3 μM xestospongin-C and 15 min after the addition of 10 μM xestospongin-C. **P < 0.01 vs control.

Figure 4

Effects of xestospongin-C on I_Ba through Ca²⁺ channels in single guinea-pig ileal smooth muscle cells. I_Ba was elicited by 80 ms depolarisation from −60 to 0 mV at 0.1 Hz. (A) Typical tracings of I_Ba in the presence or absence of 3 μM xestospongin-C. (B) Changes in peak amplitude of I_Ba with time. Xestospongin-C (3 μM) was applied during the period indicated by a horizontal bar. Time 0 min indicates the start of I_Ba recording under conditions in which the K⁺ current was blocked by internal diffusion of Cs from the recording pipette for about 3 min after the patch membrane was ruptured. (C) Effects of various concentrations of xestospongin-C on I_Ba (n=4–6).

Figure 5

Typical tracing of the effects of xestospongin-C on carbachol-induced inward currents in ileal smooth muscle cell. Once a whole cell had been prepared, the current was clamped at −40 mV. When the carbachol (50 μM)-induced inward current reached steady state, xestospongin-C (3 μM) was added to the bathing medium (A). Summarized data on the effects of xestospongin-C at various concentrations are presented in (B) (n=4–6).

Figure 6

Effects of xestospongin-C on outward K⁺ currents recorded from ileal smooth muscle cells. The cells were held at −60 mV, and test depolarisation with a duration of 900 ms was applied from −40 to +50 mV in increments of 10 mV. A pipette containing 0.05 mM EGTA was used to record currents. (A) Effect of 1 μM xestospongin-C on whole-cell currents. Summarized data on the effects of xestospongin-C (0.03–3 μM) at various concentrations on whole-cell currents (n=6) are presented in (B), in which cells had been pretreated with xestospongin-C for 10 min.