Alpha 2-adrenoceptors in the enteric nervous system: a study in alpha 2A-adrenoceptor-deficient mice

Abstract

Mammals possess three types of alpha(2)-adrenoceptor, alpha(2A), alpha(2B) and alpha(2C). Our aim was to determine the type of alpha(2)-adrenoceptor involved in the control of gastrointestinal motility. In transmitter overflow experiments, myenteric plexus longitudinal muscle (MPLM) preparations of the ileum were preincubated with [(3)H]-choline and then superfused. The alpha(2)-adrenoceptor agonist medetomidine reduced the electrically evoked overflow of tritium from preparations taken from wild type but not alpha(2A)-adrenoceptor-knockout mice. In a second series of overflow experiments, MPLM preparations were preincubated with [(3)H]-noradrenaline and then superfused. Again medetomidine reduced the electrically evoked overflow of tritium from wild type but not alpha(2A)-knockout preparations. In organ bath experiments, medetomidine reduced electrically evoked contractions of segments of the ileum from wild type but not alpha(2A)-knockout mice. In each of these three series, phentolamine antagonized the effect of medetomidine in wild-type preparations with greater potency than rauwolscine. In conscious mice, gastrointestinal transit was assessed by means of an intragastric charcoal bolus. In alpha(2A)-knockout mice, the speed of gastrointestinal transit was doubled compared to wild-type. Medetomidine, injected intraperitoneally, slowed gastrointestinal transit in wild type but not alpha(2A)-knockout mice. We conclude that the cholinergic motor neurons of the enteric nervous system of mice possess alpha(2)-heteroreceptors which mediate inhibition of acetylcholine release, of neurogenic contractions and of gastrointestinal transit. The noradrenergic axons innervating the intestine possess alpha(2)-autoreceptors. Both hetero- and autoreceptors are exclusively alpha(2A). It is the alpha(2A)-adrenoceptor which in vivo mediates the inhibition of intestinal motility by the sympathetic nervous system.

Figure 1

Tritium efflux from MPLM segments preincubated with [³H]-choline (A,B) or [³H]-noradrenaline (C,D). Segments were taken from either wild type (NMRI) or α_2A-knockout mice. After preincubation, segments were superfused and stimulated six times by either 60 pulses/1 Hz (A,B) or 30 pulses/50 Hz (C,D) (S₁ – S₆). Filled circles represent experiments without medetomidine. Empty circles represent experiments in which medetomidine was added as indicated. Each value is the mean±s.e.mean from 7 – 24 preparations.

Figure 2

Effect of medetomidine on electrically evoked tritium overflow from MPLM segments preincubated with [³H]-choline. After preincubation, segments were superfused and stimulated six times by 60 pulses/1 Hz (S₁ – S₆). Medetomidine was added at increasing concentrations before S₂ – S₆. (A) shows effect of medetomidine in segments taken from wild type or α_2A-knockout mice as indicated. (B) shows interaction of medetomidine with phentolamine and rauwolscine in preparations taken from wild type mice. Phentolamine and rauwolscine were present from the start of superfusion. Ordinates, evoked tritium overflow, calculated from S_n/S₁ ratios and expressed as a percentage of the corresponding average control ratio (no medetomidine). Each value is the mean±s.e.mean from 5 – 24 preparations. ^*Indicates significantly (P<0.05) less inhibition by medetomidine in α_2A-knockout than wild type mice. In α_2A-knockout preparations, medetomidine caused no significant change.

Figure 3

Effect of medetomidine on electrically evoked tritium overflow from MPLM segments preincubated with [³H]-noradrenaline. After preincubation, segments were superfused and stimulated six times by 30 pulses/50 Hz (S₁ – S₆). Medetomidine was added at increasing concentrations before S₂ – S₆. (A) Shows effect of medetomidine in segments taken from wild type or α_2A-knockout mice as indicated. (B) Shows interaction of medetomidine with phentolamine and rauwolscine in preparations taken from wild type mice. Phentolamine and rauwolscine were present from the start of superfusion. Ordinates, evoked tritium overflow, calculated from S_n/S₁ ratios and expressed as a percentage of the corresponding average control ratio (no medetomidine). Each value is the mean±s.e.mean from 7 – 10 preparations. ^*Indicates significantly (P<0.05) less inhibition by medetomidine in α_2A-knockout than wild type mice. In α_2A-knockout preparations, medetomidine caused no significant change.

Figure 4

Effect of medetomidine on electrically evoked contractions. Ileum segments were continuously stimulated with pairs of pulses at a frequency of 0.2 – 0.4 Hz. Medetomidine was added at increasing concentrations every 3 min. (A) shows effect of medetomidine in segments taken from wild type or α_2A-knockout mice as indicated. (B) shows interaction of medetomidine with phentolamine and rauwolscine in preparations taken from wild type mice. Phentolamine and rauwolscine were present in the medium from 30 min prior to the first dose of medetomidine. Ordinates, twitch responses, calculated from ratios of twitch amplitudes after the nth dose of medetomidine and twitch amplitudes before addition of medetomidine (t_n/t₀) and expressed as a percentage of the corresponding average control ratio (no medetomidine). Each value is the mean±s.e.mean from 9 – 17 preparations. ^*Indicates significantly (P<0.05) less inhibition by medetomidine in α_2A-knockout than wild type mice. In α_2A-knockout preparations, medetomidine caused no significant change.

Figure 5

Effect of (A) U-50488 and (B) morphine on electrically evoked contractions. Ileum segments were continuously stimulated with pairs of pulses at a frequency of 0.2 – 0.4 Hz. U-50488 or morphine was added at increasing concentrations every 3 min. Ordinates, twitch responses, calculated from ratios of twitch amplitudes after the nth dose of opioid agonist and twitch amplitudes before addition of agonist (t_n/t₀) and expressed as a percentage of the corresponding average control ratio (no agonist). Each value is the mean±s.e.mean from 4 – 10 preparations.

Figure 6

Effect of medetomidine on gastrointestinal transit in conscious wild type and α_2A-knockout mice. Mice received an intragastric bolus of charcoal suspension. Thirty minutes later, they were killed and the distance travelled by the charcoal was expressed as a percentage of total small intestine length. Medetomidine or saline was injected i.p. 20 min before the charcoal application. Each value is the mean±s.e.mean from 8 – 11 mice. ^*Indicates significant (P<0.05) inhibition by medetomidine as compared to saline.