Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release

Abstract

1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea.

Figure 1

Effect of exogenous vasoactive intestinal peptide (VIP) on mucus secretion in ferret trachea in vitro. (a) Concentration-response and inhibition by VIP antibody (VIP-Ab), (b) time course. Data are mean per cent change in output of macromolecules labelled in situ with ³⁵SO₄ (a marker for mucus) for 5–8 animals per group; vertical bars are one s.e.mean. ^*P<0.05, ^**P<0.01 compared with baseline control (a), or with corresponding vehicle control time point (b); #P<0.05 compared with VIP group (a).

Figure 2

Effect of endogenous vasoactive intestinal peptide (VIP) on neurogenic mucus secretion in ferret trachea in vitro. (a) Effect on cholinergic secretion. A VIP antibody (VIP-Ab, 1 : 500 dilution) was used to exclude VIP, and phentolamine, propanolol (10 μM each) and the tachykinin NK₁ receptor antagonist CP-99,994 (3 μM) were used to exclude adrenergic and tachykininergic neural influences at stimulation parameters of 2.5 or 10 Hz, 50 V, 0.5 msec for 5 min. ACh, acetylcholine (1 μM). (b) Effect on tachykininergic neural secretion. VIP antibody (1 : 500 dilution) was used to exclude VIP, and phentolamine, propanolol and atropine (10 μM each) were used to exclude adrenergic and cholinergic influences at stimulation parameters of 2.5 or 5 Hz, 50 V, 0.5 msec for 5 min. SP, substance P (1 μM). Data are mean per cent change in output of macromolecules labelled in situ with ³⁵SO₄ (a marker for mucus) for 5–7 animals per group; vertical bars are one s.e.mean. ^*P<0.05 compared with serum control group; #P<0.05, ##P<0.01 compared with sham stimulation group.

Figure 3

Effect of exogenous vasoactive intestinal peptide (VIP) on neurogenic mucus secretion in ferret trachea in vitro. (a and b) Effect on cholinergic neural secretion. Phentolamine, propanolol (10 μM each) and the tachykinin NK₁ receptor antagonist CP-99,994 (3 μM) were used to exclude adrenergic and tachykininergic influences at stimulation parameters of 2.5 (a) or 10 Hz (b), 50 V, 0.5 msec for 5 min. (c) Effect on tachykininergic neural secretion. Phentolamine, propanolol and atropine (10 μM each) were used to exclude adrenergic and cholinergic influences at stimulation parameters of 10 Hz, 50 V, 0.5 msec for 5 min. SP, substance P (1 μM). Data are mean per cent change in output of macromolecules labelled in situ with ³⁵SO₄ (a marker for mucus) for 5–7 animals per group; vertical bars are one s.e.mean. ^*P<0.05 compared with stimulation control group (a and b), or with serum control group (c).

Figure 4

Inhibition by exogenous vasoactive intestinal peptide (VIP) of cholinergic neural mucus secretion in ferret trachea in vitro, and the effect of potassium (K⁺) channel blockers. Phentolamine, propanolol (10 μM each) and the tachykinin NK₁ receptor antagonist CP-99,994 (3 μM) were used to exclude adrenergic and tachykininergic influences at stimulation parameters of 10 Hz, 50 V, 0.5 msec for 5 min. Gli: glibenclamide, ATP-sensitive K⁺ (K_ATP) channel inhibitor); apamin, small conductance calcium-activated K⁺ (SK_ca) channel blocker; IbTx: iberiotoxin, large conductance calcium-activated K⁺ (BK_ca) channel blocker. Data are mean per cent change in output of macromolecules labelled in situ with ³⁵SO₄ (a marker for mucus) for 5–6 animals per group; vertical bars are one s.e.mean. ^*P<0.05, ^**P<0.01 compared with stimulation control group; #P<0.05 compared with VIP + vehicle group.

Figure 5

Endogenous vasoactive intestinal peptide (VIP) as mediator of NANC, non-tachykininergic neural mucus secretion in ferret trachea in vitro. A VIP antibody (VIP-Ab, 1 : 500 dilution) was used to exclude VIP, and phentolamine, propanolol, atropine (10 μM) and the tachykinin NK₁ receptor antagonist CP-99,994 (3 μM) were used to exclude adrenergic, cholinergic and tachykininergic influences at stimulation parameters of 2.5 or 10 Hz, 50 V, 0.5 msec for 5 min. Data are mean per cent change in output of macromolecules labelled in situ with ³⁵SO₄ (a marker for mucus) for six animals per group; vertical bars are one s.e.mean. ^*P<0.05 compared with serum control group.

Figure 6

Effect of 10 Hz stimulation (50 V, 0.5 msec, 5 min) on acetylcholine (ACh) release by ferret tracheal strips in vitro. Tissues were pre-incubated with [³H]-choline chloride (a marker for ACh) and stimulated electrically (S). Each panel gives the rate of [³H] overflow for an individual tracheal strip. VIP, vasoactive intestinal peptide (0.1 μM); TTX, tetrodotoxin (0.1 μM); IbTx, iberiotoxin (0.1 μM).

Figure 7

Involvement of large-conductance K⁺ (BK_Ca) channels in vasoactive intestinal peptide (VIP)-inhibition of acetylcholine (ACh) release in ferret tracheal strips in vitro. IbTx, iberiotoxin (0.1 μM); BK_Ca channel blocker. Tissues were pre-incubated with [³H]-choline chloride (a marker for ACh) and stimulated electrically at 10 Hz, 50 V, 5 msec for 5 min. Data are mean per cent change in rate of [³H] overflow at a second stimulation compared with the first stimulation for 5–6 animals per group; vertical bars are one s.e.mean. ^*P<0.05 compared with Kreb's solution alone; ##P<0.01 compared with VIP+IbTx group.

Figure 8

Effect of 2.5 Hz stimulation (50 V, 0.5 msec, 5 min) on acetylcholine (ACh) release in ferret tracheal strips in vitro. Tissues were pre-incubated with [³H]-choline chloride (a marker for ACh) and stimulated electrically (S). Each panel gives the rate of [³H] overflow for an individual tracheal strip. VIP-Ab, antibody to vasocative intestinal peptide (1 : 500 dilution); TTX, tetrodotoxin (0.1 μM); α-chymotrypsin (2 u ml⁻¹).

Figure 9

Effect of endogenous vasoactive intestinal peptide (VIP) on acetylcholine (ACh) release in ferret tracheal strips in vitro. A VIP antibody (VIP-Ab, 1 : 500 dilution; a) or α-chymotrypsin (2 u ml⁻¹; b) were used to exclude VIP. Tissues were pre-incubated with [³H]-choline chloride (a marker for ACh) and stimulated electrically at 2.5 Hz, 50 V, 5 msec for 5 min. Data are mean per cent change in rate of [³H] overflow at a second stimulation compared with the first stimulation for five animals per group; vertical bars are one s.e.mean. ^*P<0.05 compared with control group.