Gastric antisecretory role and immunohistochemical localization of cannabinoid receptors in the rat stomach

Abstract

1. The role of cannabinoid (CB) receptors in the regulation of gastric acid secretion was investigated in the rat by means of functional experiments and by immunohistochemistry. 2. In anaesthetized rats with lumen-perfused stomach, the non selective CB-receptor agonist WIN 55,212-2 (0.30 - 4.00 micromol kg(-1), i.v.) and the selective CB(1)-receptor agonist HU-210 (0.03 - 1.50 micromol kg(-1), i.v.), dose-dependently decreased the acid secretion induced by both pentagastrin (30 nmol kg(-1) h(-1)) and 2-deoxy-D-glucose (1.25 mmol kg(-1), i.v.). By contrast, neither WIN 55,212-2 (1 - 4 micromol kg(-1), i.v.) nor HU-210 (0.03 - 1.50 micromol kg(-1), i.v.) did modify histamine-induced acid secretion (20 micromol kg(-1) h(-1)). The selective CB(2)-receptor agonist JWH-015 (3 - 10 micromol kg(-1), i.v.) was ineffective. 3. The gastric antisecretory effects of WIN 55,212-2 and HU-210 on pentagastrin-induced acid secretion were prevented by the selective CB(1)-receptor antagonist SR141716A (0.65 micromol kg(-1), i.v.) and unaffected by the selective CB(2)-receptor antagonist SR144528 (0.65 - 2 micromol kg(-1), i.v.). 4. Bilateral cervical vagotomy and ganglionic blockade with hexamethonium (10 mg kg(-1), i.v., followed by continuous infusion of 10 mg kg(-1) h(-1)) significantly reduced, but not abolished, the maximal inhibitory effect of HU-210 (0.3 micromol kg(-1), i.v.) on pentagastrin-induced acid secretion; by contrast, pretreatment with atropine (1 mg kg(-1), i.v.) did not modify the antisecretory effect of HU-210. 5. Immunoreactivity to the CB(1) receptor was co-localized with that of the cholinergic marker choline acetyltransferase in neural elements innervating smooth muscle, mucosa and submucosal blood vessels of rat stomach fundus, corpus and antrum. In contrast, CB(2) receptor-like immunoreactivity was not observed. 6. These results indicate that gastric antisecretory effects of cannabinoids in the rat are mediated by suppression of vagal drive to the stomach through activation of CB(1) receptors, located on pre- and postganglionic cholinergic pathways. However, the ineffectiveness of atropine in reducing the effect of HU-210 suggests that the release of non cholinergic excitatory neurotransmitters may be regulated by CB(1) receptors.

Figure 1

Anaesthetized rat with lumen-perfused stomach. Time-course of the inhibitory effects of equiactive doses of WIN 55,212-2 (2 μmol kg⁻¹) and of HU-210 (0.3 μmol kg⁻¹) administered (↓) i.v. at the plateau of the acid secretion induced by continuous i.v. infusion of pentagastrin. In control experiments, the drug vehicle (DMSO, 0.1 ml rat⁻¹, i.v.) was administered instead of CB-receptor agonists. Values represent the mean±s.e.mean of responses in 6 – 8 animals for each experimental group. *P<0.05 and **P<0.01 vs vehicle.

Figure 2

Anaesthetized rat with lumen-perfused stomach. Effects of WIN 55,212-2 (4 μmol kg⁻¹) and HU-210 (1.5 μmol kg⁻¹) administered (↓) i.v. at the plateau of the acid secretion induced by continuous i.v. infusion of histamine. In control experiments, the drug vehicle (DMSO, 0.1 ml rat⁻¹, i.v.) was administered instead of CB-receptor agonists. Values represent the mean±s.e.mean of responses in 7 – 9 animals for each experimental group.

Figure 3

Anaesthetized rat with lumen-perfused stomach. (A) Effect of WIN 55,212-2 and HU-210 on pentagastrin-induced acid secretion (30 nmol kg⁻¹ h⁻¹). (B) Effect of SR141716A and SR144528 on the inhibitory action of either WIN 55,212-2 or HU-210 on pentagastrin-induced acid secretion (30 nmol kg⁻¹ h⁻¹). In control experiments, the drug vehicle was administered instead of CB-receptor ligands. Values represent the mean±s.e.mean of responses in 6 – 8 animals for each experimental group. n.s.=not significant.

Figure 4

Anaesthetized rat with lumen-perfused stomach. Effect of SR144528 on the inhibitory action of HU-210 on pentagastrin-induced acid secretion (30 nmol kg⁻¹ h⁻¹). In control experiments the drug vehicle was administered instead of CB-receptor ligands. Values represent the mean±s.e.mean of responses in 6 – 8 animals for each experimental group. n.s.=not significant.

Figure 5

Anaesthetized rat with lumen-perfused stomach. Effect of vagotomy (VAG), sham vagotomy (Sham), hexamethonium (HEX, 10 mg kg⁻¹ i.v. bolus, followed by 10 mg kg⁻¹ h⁻¹ continuous infusion), atropine (ATR, 1 mg kg⁻¹ i.v.) or vehicle (Ve, DMSO 0.2 ml rat⁻¹ i.v.) on the acid response to pentagastrin in the absence or in the presence of HU-210 (HU, 0.3 μmol kg⁻¹, i.v.). Values represent the mean±s.e.mean of responses in 6 – 8 animals for each experimental group. n.s.=not significant.

Figure 6

Localization of CB₁-receptor and ChAT immunoreactivity in fundus (A, A1, A2 and A3), corpus (B,C) and antrum (D,E) of rat stomach. (A) Low power micrograph of a longitudinal section through fundus. CB₁-receptor (red) and ChAT (green) immunoreactivities are colocalized (yellow) in myenteric neurons (arrow). Nonspecific labelling (*) persisted in preabsorption and omission controls. (A1) High power micrograph of the myenteric neurons seen in A (large arrow). Fibres can be observed which exhibit CB₁-receptor immunoreactivity alone (small arrow, red) or in combination with ChAT immunoreactivity (arrowhead, yellow). (A2) A single submucosal neuron exhibiting immunoreactivities to both CB₁ receptors and ChAT. Due to the diffuse nature of the CB₁-receptor immunoreactivity, a greenish hue is imparted to some of the neurons. (A3) CB₁-receptor and ChAT immunoreactivities are colocalized in nerve fibres innervating thick-walled blood vessels (short arrow and arrow head). CB₁-receptor and ChAT immunoreactivities were co-localized in fibres innervating the inner wall of the blood vessel (long arrow). (B) Myenteric neurons exhibiting CB₁-receptor immunoreactivity in the corpus (short arrow). In some neurons, CB₁-receptor immunoreactivity was colocalized with ChAT (large arrow). Most fibres terminating in the circular muscle exhibited CB₁-receptor immunoreactivity (red), but little ChAT immunoreactivity (yellow, arrowhead). (C) In the corpus, distinct CB₁-receptor-immunoreactive (short arrow) nerve fibres and fibres exhibiting both CB₁-receptor and ChAT immunoreactivity (yellow hue, long arrow) can be seen in the circular muscle layer. Some nonspecific labelling (*) is observed that persisted in omission and preabsorption controls. (D) Colocalization of CB₁-receptor and ChAT immunoreactivities in myenteric neurons (arrow) of the gastric antrum. Due to the diffuse nature of the CB₁-receptor immunoreactivity, a greenish hue is imparted to some of the neurons. CB₁-receptor-immunoreactive nerve fibres (double arrowhead) can be observed terminating in longitudinal muscle. Some nonspecific labelling (single arrow) persisted in omission and preabsorption controls. (E) In the antral submucosa, fibres immunoreactive for ChAT (green, long arrows) could be observed, but no CB₁-receptor-immunoreactive fibres were found. Some nonspecific labelling (short arrow) is observed that persisted in omission and preabsorption controls. Bar in A=50 μm; bars in A1, B – E=16 μm; bar in A2=13 μm; bar in A3=25 μm.