Electrically-evoked Acetylcholine Release Research Articles

Opioid pathways exert a tonic restraint in the guinea-pig isolated colon: changes after chronic sympathetic denervation.

We have studied the effects of naloxone on acetylcholine and noradrenaline release in the guinea-pig isolated distal colon, and have assessed the effect of naloxone on electrically-induced contractions of the longitudinal muscle and non-adrenergic, non-cholinergic (NANC) relaxations of the circular muscle coat. Naloxone dose-dependently increased resting and electrically-evoked acetylcholine release and electrically-evoked noradrenaline release. Naloxone was more potent in increasing resting acetylcholine release in colonic specimens obtained after chronic sympathetic denervation. Naloxone (1 microM) did not affect electrically-induced contractions of the longitudinal muscle, while it enhanced NANC relaxations of the circular muscle. The effects observed with naloxone in the present experiments suggest that opioid pathways exert a tonic restraint on neurotransmission in the guinea-pig colon. After suppression of the adrenergic inhibitory tone, the functional relevance of opioid pathways seems to be increased.

Does the release of acetylcholine in septal slices originate from intrinsic cholinergic neurons bearing p75 ntr receptors? a study using 192 IgG-saporin lesions in rats

In previous studies electrically-evoked release of acetylcholine in septal slices was demonstrated. The present experiment aimed at verifying if this release involved intrinsic neurons bearing p75 NTR receptors. Long-Evans rats sustained injections of 192 IgG-saporin into the medial septum/diagonal band of Broca (0.8 μg). Sham-operated rats served as controls. Two to 3.5 weeks later, the electrically-evoked release of acetylcholine ([ 3H]ACh) was measured in slices from the lateral septum (LS), medial septum (MS) and diagonal band of Broca (DBB). Choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity, and monoamine concentrations were measured in the septum, cortex and hippocampus. The lesion extent was also assessed by ChAT immunostaining in a separate series of rats. In the septum, the number of ChAT-positive neurons was depleted dramatically (>90% at the level of the injection site). In the hippocampus, the lesions reduced ChAT and AChE activity by 91% and 84%, respectively. In the cortex, this reduction was weaker (−55% and −47%). In the septal region, the reduction was either weak or not significant. The evoked release of acetylcholine in septal slices was not reduced, except in the slices from the LS (−64%). The effects of physostigmine and atropine confirmed the presence of autoreceptors. Our data exclude that a major part of the acetylcholine released by MS and DBB slices derived from intrinsic neurons bearing p75 NTR receptors. In the LS, part of the released acetylcholine might be from projections of such neurons located in the LS, MS and/or DBB. These data also suggest that the MS and the DBB may be the target of extrinsic cholinergic innervation that does not bear p75 NTR receptors.

Modulation of acetylcholine release from parasympathetic nerves innervating guinea-pig and human trachea by endomorphin-1 and -2

Endomorphin-1 and -2 (μ-opioid receptor agonists) produced a concentration-dependent and naloxone-sensitive inhibition of cholinergic contractile responses in guinea-pig trachea (at 10 μM, 46.1±8.0% and 33.8±8.6%, respectively). Endomorphin-1 and -2 also inhibited electrically-evoked acetylcholine release from cholinergic nerves innervating guinea-pig (at 0.1 μM, 41.8±10.9%; at 1 μM 60.1±6.3%, respectively) and human trachea (at 10 μM, 76.2±18.1%, and 77.7±14.3%, respectively). Naloxone prevented the inhibition by endomorphin-1 and -2 in both guinea-pig and human trachea, suggesting that these peptides can inhibit cholinergic, parasympathetic neurotransmission to the airways via the activation of classical opioid receptors.

Postnatal development of muscarinic autoreceptors in the rat brain: lateral and medial septal nuclei and the diagonal band of Broca

The postnatal development of the release of acetylcholine (ACh) and of presynaptic, release-inhibiting muscarinic autoreceptors was studied in the lateral septum (LS), the medial septum (MS) and the diagonal band of Broca (DB) of the rat brain. To this end, slices (350 μm thick) containing these brain regions from rats of various postnatal ages (postnatal day 3 [P3] to P16, and adult) were pre-incubated with [ 3 H ]choline and stimulated twice (S 1, S 2: 360 pulses, 3 Hz) during superfusion with physiological buffer containing hemicholinium-3 (10 μM). In addition, the activity of choline acetyltransferase (ChAT, in crude homogenates) was determined as a marker for the development of cholinergic functions. At any postnatal age, the electrically-evoked overflow of tritium from slices pre-incubated with [ 3 H ]choline was highest in the DB, followed by the MS whereas in slices containing the LS, it was only small. In all septal regions, the evoked [ 3 H ]overflow was Ca 2+-dependent and tetrodotoxin-sensitive at P3. It increased with postnatal age and reached about 60% of the corresponding adult levels at P16. Presence of the muscarinic agonist oxotremorine (1 μM) during S 2 significantly inhibited the evoked overflow of tritium beginning from P5: no significant effect was detected at P3. The ACh esterase inhibitor physostigmine (1 μM) exhibited significant inhibitory effects from P13 onwards, whereas the muscarinic antagonist atropine (1 μM) did not change the evoked ACh release. The activity of ChAT, as measured for these septal regions at various postnatal ages, correlated well with the [ 3 H ]overflow induced by electrical stimulation. In conclusion, (1) electrically-evoked release of ACh was measured for the first time in three septal subregions; (2) the postnatal development of the presynaptic cholinergic functions: ChAT activity, ACh release and muscarinic autoreceptors occurs almost synchronously in these regions of the septal complex and parallels that in the hippocampal formation; (3) as in the hippocampus, the postnatal development of autoreceptors was delayed with respect to the exocytotic release of ACh.

Effect of the cannabinoid receptor SPECT agent, AM 281, on hippocampal acetylcholine release from rat brain slices

The SPECT ligand AM 281, a less lipophilic analog of the cannabinoid receptor antagonist SR 141716A, robustly potentiated electrically-evoked release of acetylcholine from superfused hippocampal slices and prevented the inhibition of acetylcholine release by the cannabimimetic drug WIN 55212-2. These results, similar to earlier observations with SR 141716A, indicate that AM 281 is either a cannabinoid receptor antagonist or inverse agonist. Despite showing lower affinity than SR 141716A in hippocampal membrane binding experiments, AM 281 had slightly greater potency than SR 141716A in the hippocampal slice experiments, perhaps because of reduced drug absorption to slice membranes and to the apparatus.

Further evidence for the presence of cannabinoid CB1 receptors in guinea-pig small intestine.

1. CP 50,556, CP 55,940, nabilone, CP 56,667, delta 9 -tetrahydrocannabinol (THC) and cannabinol each inhibited electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation of guinea-pig small intestine in a concentration-related manner. The IC50 values of these cannabinoids, respectively 3.45, 3.46, 30.61, 162.94, 214.63, and 3913.5 nM, correlate well with previously obtained potency values for displacement of [3H]-CP 55,940 from cannabinoid binding sites. 2. Electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation were also inhibited by AM 630 (6-iodo-pravadoline) and by WIN 55,212-2 (IC50 = 1923.0 and 5.54 nM, respectively). The present finding that AM 630 is an agonist, contrasts with a previous observation that it behaves as a cannabinoid receptor antagonist in the mouse isolated vas deferens. 3. SR141716A produced dose-related parallel rightward shifts in the log concentration-response curves of CP 55,940, WIN 55,212-2, THC and AM 630 for inhibition of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation. SR141716A (1 microM) did not reverse the inhibitory effects of normorphine and clonidine on electrically-evoked contractions or potentiate the contractile response to acetylcholine. 4. Doses of naloxone and yohimbine that reversed the inhibitory effects of normorphine or clonidine on electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation did not affect the inhibitory response to WIN 55,212-2. 5. Electrically-evoked release of acetylcholine from strips of myenteric plexus-longitudinal muscle was decreased by 200 nM CP 55,940 and this inhibitory effect was almost completely reversed by 1 microM SR141716A. Acetylcholine-induced contractions were not affected by 200 nM CP 55,940. 6. These results support the hypothesis that guinea-pig small intestine contains prejunctional cannabinoid CB1 receptors through which cannabinoids act to inhibit electrically-evoked contractions by reducing release of the contractile transmitter, acetylcholine. 7. THC was found to be more susceptible to antagonism by SR141716A than CP 55,940 or AM 630, raising the possibility that guinea-pig small intestine contains more than one type of cannabinoid receptor. 8. At concentrations of 10 nM and above, SR141716A produced small but significant increases in the amplitude of electrically-evoked contractions of the myenteric plexus-longitudinal muscle preparation suggesting that this tissue may release an endogenous cannabinoid receptor agonist or that some cannabinoid receptors in this tissue are precoupled and that SR141716A can reduce the number of receptors in this state.

Histamine H3 receptors regulate acetylcholine release from the guinea pig ileum myenteric plexus

The effect of selective histamine H3-receptor agonists and antagonists on the acetylcholine release from peripheral nerves was evaluated in the guinea pig longitudinal muscle-myenteric plexus preparations, preloaded with (3H)choline. In the presence of H1 and H2 blockade, histamine (10 −7–10 −4 M) and (R)-α-methylhistamine (10 −8–10 −6 M) inhibited the electrically-evoked acetylcholine release, being (R)-α-methylhistamine more active than histamine, but behaving as a partial agonist. The effect of histamine was completely reversed by selective H3-blocking drugs, thioperamide and impromidine, while only submaximal doses of (R)-α-methylhistamine were antagonized. Furthermore, thioperamide and impromidine enhanced the electrically-evoked acetylcholine release. On the contrary, the new H3-blocker, HST-7, was found substantially ineffective, both as histamine antagonist and as acetylcholine overflow enhancer. These data suggest that histamine exerts an inhibitory control on the acetylcholine release from intestinal cholinergic nerves through the activation of H3 receptors.

Dicyclomine discriminates between M1‐ and M2‐muscarinic receptors in the guinea‐pig ileum

1. The affinity of the antagonist dicyclomine for subtypes of muscarinic receptors has been assessed in the myenteric plexus-longitudinal muscle preparation of the guinea-pig. 2. Dicyclomine had a high affinity (pA2 9.13) for the neuronal M1-receptor whose activation by pilocarpine causes an increase in acetylcholine release. Dicyclomine had a low affinity for both the prejunctional M2-receptor (pA2 7.61) mediating inhibition of the electrically-evoked acetylcholine release and the postjunctional M2-receptor (pA2 7.21). 3. It is concluded that dicyclomine distinguishes between M1- and M2-muscarinic receptors in functional experiments.

Diazepam antagonizes GABA- and muscimol-induced changes of acetylcholine release in slices of guinea-pig cerebral cortex.

The acetylcholine (ACh) release was studied in superfused, electrically-stimulated slices of guinea-pig cerebral cortex. Muscimol and 4,5,6,7-tetrahydroisoxazolo (5-4-c)-pyridin-3-ol (THIP), as well as exogenous GABA, reduced the electrically-evoked ACh release and enhanced its spontaneous outflow. Picrotoxin antagonized these effects. In addition, picrotoxin and ethanolamine-O-sulphate (EOS) caused opposite changes in transmitter outflow, suggesting the existence of an exogenous GABAergic control on the cholinergic nerve endings. Neither flurazepam 6.6 X 10(-6)--3.3 X10(-5) mol/l nor diazepam 3.3 X 10(-6)--3.3 X 10(-5) mol/l by themselves affected ACh release. Diazepam prevented GABA-, muscimol- and EOS-induced changes in spontaneous and 1 Hz-evoked outflow. Ro 15-1788 3.3 X 10(*-6) mol/l abolished diazepam antagonism vs exogenous GABA. The ineffectiveness of flurazepam and diazepam on normal release (i.e. the lack of potentiation vs the endogenous GABAergic control) supports the view that "synaptic" GABA receptors acting upon the cholinergic nerve endings are not coupled with Benzodiazepine receptors. The unexpected diazepam antagonism vs exogenous GABA and GABA-like compounds can be explained with an unusual Diazepam negative cooperation with "extrasynaptic" GABA receptors, possibly present on the cholinergic terminals. Thus, the rule of benzodiazepine-GABA synergism does not seem always tenable, at least at certain pre-synaptic sites.

The effect of naloxone on opioid-induced inhibition and facilitation of acetylcholine release in brain slices.

1 The effect of morphine, methionine-enkephalin (Met-enkephalin) and D-Ala2-D-Leu5-enkephalin (DADLE) were tested on the spontaneous and electrically-evoked release of acetylcholine (ACh) from superfused slices of guinea-pig thalamus, caudate nucleus and cerebral cortex. 2 At no concentration did morphine, Met-enkephalin or DADLE modify the outflow of ACh at rest but Met-enkephalin in the presence of naloxone, reduced the resting ACh release. 3 Morphine, at a low dose (3 microM) had no effect in slices of cerebral cortex, but it enhanced the evoked release of ACh in thalamic and caudate, slices. At higher doses of morphine (10-30 microM), the ACh release evoked by electrical pulses was significantly inhibited in every area. 4 Met-enkephalin behaved like morphine in thalamic slices, whereas DADLE, a specific delta agonist, produced a slight inhibition of ACh outflow only at 10 microM. 5 Naloxone antagonized the inhibitory effect of morphine in the cerebral cortex and caudate nucleus slices. Naloxone and also spiroperidol blocked the releasing effect of morphine in caudate slices. In contrast naloxone did not affect the increase of ACh release caused by morphine and Met-enkephalin in thalamic slices. The inhibitory effect of both opioids at high doses was reversed by naloxone so that they then enhanced ACh release. 6 A two fold increase of calcium concentration in the Krebs solution prevented the inhibitory effects of morphine 10 microM. 7 It is suggested that two receptors are present in thalamic slices, one of which inhibits and the other facilitates ACh release.