Serotonin contracts the rat mesenteric artery by inhibiting 4-aminopyridine-sensitive Kv channels via the 5-HT2A receptor and Src tyrosine kinase

Abstract

Serotonin (5-hydroxytryptamine (5-HT)) is a neurotransmitter that regulates a variety of functions in the nervous, gastrointestinal and cardiovascular systems. Despite such importance, 5-HT signaling pathways are not entirely clear. We demonstrated previously that 4-aminopyridine (4-AP)-sensitive voltage-gated K(+) (Kv) channels determine the resting membrane potential of arterial smooth muscle cells and that the Kv channels are inhibited by 5-HT, which depolarizes the membranes. Therefore, we hypothesized that 5-HT contracts arteries by inhibiting Kv channels. Here we studied 5-HT signaling and the detailed role of Kv currents in rat mesenteric arteries using patch-clamp and isometric tension measurements. Our data showed that inhibiting 4-AP-sensitive Kv channels contracted arterial rings, whereas inhibiting Ca(2+)-activated K(+), inward rectifier K(+) and ATP-sensitive K(+) channels had little effect on arterial contraction, indicating a central role of Kv channels in the regulation of resting arterial tone. 5-HT-induced arterial contraction decreased significantly in the presence of high KCl or the voltage-gated Ca(2+) channel (VGCC) inhibitor nifedipine, indicating that membrane depolarization and the consequent activation of VGCCs mediate the 5-HT-induced vasoconstriction. The effects of 5-HT on Kv currents and arterial contraction were markedly prevented by the 5-HT2A receptor antagonists ketanserin and spiperone. Consistently, α-methyl 5-HT, a 5-HT2 receptor agonist, mimicked the 5-HT action on Kv channels. Pretreatment with a Src tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, prevented both the 5-HT-mediated vasoconstriction and Kv current inhibition. Our data suggest that 4-AP-sensitive Kv channels are the primary regulator of the resting tone in rat mesenteric arteries. 5-HT constricts the arteries by inhibiting Kv channels via the 5-HT2A receptor and Src tyrosine kinase pathway.

Figure 1

Effects of K⁺ channel inhibitors on the resting tone of mesenteric arterial rings. (a) Typical traces of mesenteric artery constriction responding to the K⁺ channel inhibitor 4-aminopyridine (4-AP, 10 mM), the Ca²⁺-activated K⁺ (BK_Ca) channel inhibitor iberiotoxin (IbTX, 120 nM), the ATP-sensitive K⁺ (K_ATP) channel inhibitor glybenclamide (Gly, 10 μM) and the inwardly rectifying K⁺ (K_ir) channel inhibitor BaCl₂ (100 μM). (b) A summary of the vasoconstrictive effects of the K⁺ channel inhibitors. TEA, tetraethylammonium. Numbers in parentheses indicate the number of arterial rings tested. *P<0.05, **P<0.01 and ***P<0.001 versus resting values.

Figure 2

The role of membrane potential (E_m) depolarization and voltage-gated Ca²⁺ channels in 5-hydroxytryptamine (5-HT)-induced mesenteric artery constriction. (a) A typical trace of mesenteric artery constriction in response to cumulative concentrations of 5-HT. (b) The effect of high KCl (70 mM) pretreatment on 5-HT-induced mesenteric artery constriction. (c) The effects of nifedipine (1 μM) on 5-HT-induced constriction. (d) The effects of the combined treatment of high KCl (70 mM) and nifedipine (1 μM) on 5-HT-induced constriction. (e) Concentration–response curves for 5-HT-induced vasoconstriction under the conditions described in (a–d); both high KCl (70 mM) and nifedipine (1 μM) pretreatment markedly suppressed 5-HT-induced mesenteric artery constriction. High KCl-induced vasoconstriction is shown (a, c) before breaks for comparison with 5-HT-induced constriction. The duration of high-KCl treatment was 10 min (note that the timescale bars are for traces after the break). **P<0.01 and ***P<0.001 versus the control. NS, not significant between all data points between the two groups.

Figure 3

The effects of 5-hydroxytryptamine (5-HT) and α-methyl 5-HT on voltage-gated K⁺ (Kv) currents. (a, e) Representative recordings of K⁺ currents in the absence and presence of 5-HT (1 μM) or α-methyl 5-HT (1 μM). The shape of the voltage pulse protocol used to elicit the Kv currents is shown as a figure inset. (b, f) Current–voltage (I–V) relationships in the absence and presence of 5-HT (1 μM) or α-methyl 5-HT (1 μM). 5-HT and α-methyl 5-HT reduced Kv currents to similar degrees. (c, d) The effect of tetraethylammonium (TEA; 1 mM) plus glybenclamide (1 μM) pretreatment on the inhibiting effect of 5-HT on the K⁺ currents. *P<0.05 versus the control. **P<0.01 versus the control.

Figure 4

The effects of ketanserin and spiperone, selective 5-hydroxytryptamine (5-HT)_2A receptor inhibitors, on the 5-HT-induced inhibition of voltage-gated K⁺ (Kv) currents and vasoconstriction. (a) Representative recordings of the Kv currents of ketanserin (100 nM)-pretreated smooth muscle cells in the absence and presence of 5-HT (1 μM). Ketanserin alone had no effect on the Kv currents (data not shown). (b) Summary of the I–V relationships of the ketanserin-pretreated cells in the absence and presence of 5-HT (1 μM). (c) Representative recordings of the Kv currents of the spiperone (10 nM)-pretreated cells in the absence and presence of 5-HT (1 μM). Spiperone alone had no effect on the Kv currents (data not shown). (d) Summary of the I–V relationships of the spiperone-pretreated cells in the absence and presence of 5-HT (1 μM). (e) Typical traces of mesenteric artery constriction in response to cumulative concentrations of 5-HT in the absence (upper panel) and presence (lower panel) of ketanserin (10 nM). (f) Concentration–response curves for 5-HT-induced vasoconstriction in the absence and presence of ketanserin (10 or 100 nM). Ketanserin blocked both 5-HT-induced Kv current inhibition and vasoconstriction. High-KCl (70 mM)-induced vasoconstrictions are shown in e before breaks for comparison with 5-HT-induced constrictions. The duration of high-KCl treatment was 10 min in all instances (note that the timescale bars are for traces after the break). *P<0.05 and ***P<0.001 versus the control. ^†P<0.05, ^††P<0.01 and ^†††P<0.001 versus the ketanserin (10 nM) group.

Figure 5

The effects of BW723C86 and anpirtoline on voltage-gated K⁺ (Kv) current and vasoconstriction. (a, c) Representative recordings of K⁺ currents in the absence and presence of BW723C86 (1 μM; a 5-HT_2B agonist) and anpirtoline (1 μM; a 5-HT_1B agonist). (b, d) I–V relationships in the absence and presence of BW723C86 (1 μM) and anpirtoline (1 μM). (e) The effects of cumulative concentrations of BW723C86 and anpirtoline. (f) The summary of e. High KCl (70 mM)-induced vasoconstrictions are shown in e before breaks for comparison with agonist-induced vasoconstrictions. The duration of high-KCl treatment is 10 min in all instances (note that the timescale bars are for traces after the break).

Figure 6

The effects of PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d]pyrimidine) on the 5-hydroxytryptamine (5-HT)-induced vasoconstriction and Kv current inhibition. (a) Concentration–response curves for 5-HT-induced vasoconstriction in the absence and presence of PP2 (5 μM) or PP3 (4-amino-7-phenylpyrazolo[3,4-d[ pyrimidine; 5 μM). (b) Representative recordings of Kv currents of the PP2 (5 μM)-pretreated smooth muscle cells with or without 5-HT (1 μM). (c) Summary of the I–V relationships of the ketanserin-pretreated cells in the absence and presence of 5-HT (1 μM). (d) Representative recordings of Kv currents of the PP3 (5 μM)-pretreated cells with or without 5-HT (1 μM). (e) Summary for the I–V relationships of the PP3-pretreated cells in the absence and presence of 5-HT (1 μM).