Shaker-type Kv1 channel blockers increase the peristaltic activity of guinea-pig ileum by stimulating acetylcholine and tachykinins release by the enteric nervous system

Abstract

1 A constant intraluminal pressure system was used to evaluate the effects of Kv1 channel blockers on the peristaltic activity of guinea-pig ileum. 2 The nortriterpene correolide, a non-selective inhibitor of all Kv1 sub-types, causes progressive and sustained reduction of the pressure threshold for eliciting peristaltic contractions. 3 Margatoxin (MgTX), alpha-dendrotoxin (alpha-DTX) and dendrotoxin-K (DTX-K), highly selective peptidyl inhibitors of certain Kv1 sub-types, cause immediate reduction of the pressure threshold. This effect subsides with time, irrespective of the peptides' concentration in the bath. In preparations pretreated with saturating concentrations of MgTX, correolide further stimulates the peristaltic activity. 4 Iberiotoxin (IbTX), a selective inhibitor of the high-conductance Ca(2+)-activated K(+) (BK) channels, and charybdotoxin (ChTX), which inhibits Kv1.2 and Kv1.3 as well as BK channels, fail to stimulate the peristaltic activity. 5 Blockade of muscarinic receptors by atropine reduces, and occasionally suppresses the peristaltic activity of guinea-pig ileum. In atropine-treated preparations, correolide and MgTX retain their abilities to reduce the pressure threshold and are able to restore the peristaltic reflex in the preparations where this reflex was suppressed by atropine. 6 The stimulatory effect of correolide and MgTX in atropine-treated preparations is abolished by subsequent addition of selective antagonists of both NK1 and NK2 receptors. 7 In conclusion, blockade of Kv1, particularly Kv1.1 channels, increases the peristaltic activity of guinea-pig ileum by enhancing the release of neurotransmitters at the enteric nervous system. In contrast, stimulation of the myogenic motility by blockade of BK channels does not affect the threshold for the peristaltic reflex.

Figure 1

Effects of correolide on the peristaltic activity of a guinea-pig ileum segment. (A) Representative experiment showing the time-course of the correolide-induced reduction of the pressure threshold. (B) Concentration–response curve: Data points (means±s.e.mean, n=4 for each correolide concentration) refer to the pressure thresholds measured after 45–60 min of drug exposure.

Figure 2

Influence of perfusion flow and TTX on the effects of correolide. The peristaltic activity recorded in the presence of correolide was abolished by either stopping the intraluminal perfusion or by blocking the ENS excitability with TTX (1 μM).

Figure 3

(A) Representative experiment showing the time-course of the effects of MgTX on the peristaltic activity of guinea-pig ileum. (A) Addition of MgTX (1 nM) to the bath caused immediate reduction of the pressure threshold for eliciting a peristaltic contraction, leading to an increase in the frequency of contractions. The effect subsided partially with time in the continuous presence of the toxin, and was not restored by increasing MgTX concentration to 100 nM. (B) Effects of various peptidyl inhibitors of Kv1 and/or BK channels on the peristaltic activity. The mean pressure thresholds recorded within 5 min after administration of MgTX, α-DTX or DTX-K, or 30–45 min after the administration of IbTX or ChTX were used for quantification. The results are expressed as percent of the mean pressure threshold of the respective control, i.e. recorded during 5 min before addition of the peptide. ^*P<0.05 (paired t-test for the changes in pressure threshold elicited by each peptide).

Figure 4

The effects of MgTX and correolide in ileum segments treated with atropine (1 μM). A and B are different preparations. (A) Atropine blocked the peristaltic reflex, which was restored by addition of MgTX (10 nM). (B) Atropine increased the pressure threshold and reduced the frequency of peristaltic contractions. Addition of correolide (3 μM) reversed these effects of atropine. (C) Quantification of the effects of atropine and of MgTX or correolide in atropine-treated preparations: The experimental protocol, similar to that shown in (B), consisted of: After an initial control period (>60 min), atropine (1 μM) was applied in the bathing medium. Ten to fifteen min later, either MgTX (10 nM) or correolide (1000 nM) was added to the atropine-containing bathing medium. The columns show the mean pressure threshold (±s.e.mean) recorded during the last 10 min of the control period, after 10–15 min of exposure to atropine, and during the first 5 min of exposure to MgTX or after 45–60 min of exposure to correolide. ^*P<0.05 (paired t-test for the reduction in pressure threshold elicited by MgTX or correolide).

Figure 5

The effects of tachykinin antagonists on the peristaltic activity stimulated by correolide, on two ileum segments (A, B). At the beginning of the recordings, the preparations had been exposed to atropine (1 μM) for 15 min. Correolide (1000 nM), and subsequently GR 82334 and GR 94800 (1 μM each) were applied in the sequences indicated.