Neurogenic mucosal bicarbonate secretion in guinea pig duodenum

Abstract

Background and purpose: To test a hypothesis that: (i) duodenal pH and osmolarity are individually controlled at constant set points by negative feedback control centred in the enteric nervous system (ENS); (ii) the purinergic P2Y(1) receptor subtype is expressed by non-cholinergic secretomotor/vasodilator neurons, which represent the final common excitatory pathway from the ENS to the bicarbonate secretory glands.

Experimental approach: Ussing chamber and pH-stat methods investigated involvement of the P2Y(1) receptor in neurogenic stimulation of mucosal bicarbonate (HCO(3)(-)) secretion in guinea pig duodenum.

Key results: ATP increased HCO(3)(-) secretion with an EC(50) of 160 nM. MRS2179, a selective P2Y(1) purinergic receptor antagonist, suppressed ATP-evoked HCO(3)(-) secretion by 47% and Cl(-) secretion by 63%. Enteric neuronal blockade by tetrodotoxin or exposure to a selective vasoactive intestinal peptide (VIP, VPAC(1)) receptor antagonist suppressed ATP-evoked HCO(3)(-) secretion by 61 and 41%, respectively, and Cl- by 97 and 70% respectively. Pretreatment with the muscarinic antagonist, scopolamine did not alter ATP-evoked HCO3(-) or Cl(-) secretion.

Conclusion and implications: Whereas acid directly stimulates the mucosa to release ATP and stimulate HCO(3)(-) secretion in a cytoprotective manner, neurogenically evoked HCO(3)(-) secretion accounts for feedback control of optimal luminal pH for digestion. ATP stimulates duodenal HCO(3)(-) secretion through an excitatory action at purinergic P2Y(1) receptors on neurons in the submucosal division of the ENS. Stimulation of the VIPergic non-cholinergic secretomotor/vasodilator neurons, which are one of three classes of secretomotor neurons, accounts for most, if not all, of the neurogenic secretory response evoked by ATP.

Figure 1

Stimulation of mucosal HCO₃⁻ and Cl⁻ (i.e. ΔIsc) secretion by ATP or ADP in guinea pig duodenum. (A) Peak HCO₃⁻ secretion evoked by ATP or ADP was concentration dependent with an EC₅₀ of 0.16 μM for ATP and an EC₅₀ of 0.33 μM for ADP. The maximal response to ATP or ADP occurred at 3 μM. (B) Stimulation of Isc as a surrogate for Cl⁻ secretion in the same preparations. Values are expressed as means ± SEM; n = 6 animals for each series of four preparations.

Figure 2

Time course for stimulation of mucosal HCO₃⁻ and Cl⁻ (i.e. ΔIsc) secretion by ATP and vehicle control in guinea pig duodenum. (A) ATP-evoked HCO₃⁻ secretion. ATP (3 μM) or vehicle control was applied to the submucosal side of the preparations at the time indicated by the arrow. HCO₃⁻ secretory responses were slow, in the range of 20–30 min for peak responses, relative to evoked changes in Isc, which reflected Cl⁻ secretion. (B) ATP evoked a rapid increase in Isc with a peak being reached within 2 min after application of ATP. Values are expressed as means ± SEM; n = 6 animals for each series of four preparations.

Figure 3

Effects of suppression of ATP hydrolysis by ARL67156, an ecto-ATPase inhibitor, on ATP-evoked mucosal HCO₃⁻ secretion and Isc, as a measure of Cl⁻ secretion. (A) Effects of 10 μM ARL67156 on HCO₃⁻ secretory responses to 0.3 or 3.0 μM ATP. ARL67156 was added to the submucosal side of the preparations 20 min before applying ATP. ARL67156 did not alter ATP-evoked secretion of HCO₃⁻. (B) Effects of 10 μM ARL67156 on Isc evoked by 0.3 or 3.0 μM ATP in the same preparations. Suppression of ATP hydrolysis by the ecto-ATPase inhibitor enhanced Isc, which reflected Cl⁻ secretory responses to ATP. Values are expressed as means ± SEM; n = 6 animals for each series of four preparations. *P < 0.05, **P < 0.01 (compared with response to ATP without pretreatment).

Figure 4

Effects of neural blockade and pharmacological receptor antagonists on ATP-evoked mucosal HCO₃⁻ secretion and Isc, as a measure of Cl⁻ secretion. (A) Effects on ATP-evoked mucosal HCO₃⁻ secretion. ATP (3 μM) was applied to the submucosal side of the preparations in each study. Neuronal blockade with 0.5 μM TTX suppressed ATP-evoked secretion of HCO₃⁻. Blockade of purinergic P2Y₁ receptors by the selective antagonist, MRS2179 (10 μM) suppressed ATP-evoked HCO₃⁻ secretory responses. Blockade of muscarinic receptors by the selective antagonist, scopolamine (10 μM), did not alter ATP-evoked secretion of HCO₃⁻. Blockade of receptors for vasoactive intestinal peptide by the selective antagonist, VP1-AT (1.0 μM), suppressed ATP-evoked HCO₃⁻ secretory responses. (B) Effects on ATP-evoked Isc, which served as a surrogate for Cl⁻ secretion. ATP (3 μM) was applied to the submucosal side of the preparations in each case. Neuronal blockade with 0.5 μM TTX suppressed ATP-evoked increases in Isc. Blockade of purinergic P2Y₁ receptors by the selective antagonist, MRS2179 (10 μM), suppressed ATP-evoked increases in Isc. Blockade of muscarinic receptors by the selective antagonist, scopolamine (10 μM), did not alter ATP-evoked increases in Isc. Blockade of receptors for vasoactive intestinal peptide by the selective antagonist, VP1-AT (1.0 μM), suppressed ATP-evoked increases in Isc. Values are expressed as means ± SEM; n = 6 animals for each series of four preparations. ***P < 0.001 (compared with response to ATP in physiological Cl⁻).

Figure 5

Effects of Cl⁻ depletion on ATP-evoked mucosal HCO₃⁻ secretion and Isc, as a measure of Cl⁻ secretion. Gluconate was substituted for Cl⁻ in the bathing medium on both sides of the preparations. (A) ATP-evoked increases in secretion of HCO₃⁻ were suppressed by 55% in Cl⁻ depleted medium. (B) ATP-evoked increases in Isc were suppressed by 70% in Cl⁻ depleted medium. Values are expressed as means ± SEM; n = 6 animals for each series of four preparations. ***P < 0.001.

Figure 6

Effects of direct mucosal application of acid on bicarbonate secretion in guinea pig duodenum. Hydrochloric acid (1 M) in a volume of 10 μL was introduced into the 10 mL volume of the mucosal compartment of the Ussing chamber to obtain a final exposure of 10 mM HCl. The pH of the Krebs solution in the compartment was changed from pH 7.4 to pH 4.4 and the working pH of the pH-stat system was set at pH 4.4 for measurement of HCO₃⁻ secretion in response to application of HCl. (A) Exposure of the mucosa to 10 mM HCl stimulated secretion of HCO₃⁻ in a time-dependent manner. Secretory responses to 10 mM HCl were suppressed by 19.9±% when 1 μM MRS 2500, a selective P2Y₁ receptor antagonist, was present in the bathing solution. Secretory responses to 10 mM HCl were unaffected by the presence of 200 μM suramin in the bathing medium. (B) Pharmacological analysis of acid-evoked HCO₃⁻ secretion. Suppression of basal secretion (control) by 0.5 μM TTX was a reflection of blockade of spontaneous firing of secretomotor neurons. Neuronal block of intramural neurons by TTX did not alter HCO₃⁻ secretory responses to HCl. MRS 2500 (1 μM), but not 200 μM suramin, suppressed HCl-evoked secretory responses.

Figure 7

Three kinds of secretomotor neurons in the submucosal plexus of the enteric nervous system innervate the intestinal secretory glands. (A) Cholinergic secretomotor/vasodilator neurons express the immunohistochemical code for the protein, calretinin, and innervate both secretory glands and periglandular arterioles. (B) Cholinergic secretomotor/non-vasodilator neurons express the immunohistochemical code, NPY, and innervate the secretory glands, but not periglandular arterioles. (C) Non-cholinergic secretomotor/vasodilator neurons innervate both the secretory glands and periglandular arterioles. These neurons contain VIP in all species. They are the only secretomotor neurons that receive both noradrenergic and purinergic input from sympathetic postganglionic neurons. Non-cholinergic secretomotor/vasodilator neurons are responsible for stimulation of bicarbonate secretion. They express P2Y₁ purinergic receptors, are stimulated to firing threshold by ATP, and release VIP at their junctions with the secretory epithelium.