CO2 chemosensing in rat oesophagus

Abstract

Background: Acid in the oesophageal lumen is often sensed as heartburn. It was hypothesised that luminal CO(2), a permeant gas, rather than H(+), permeates through the epithelium, and is converted to H(+), producing an afferent neural signal by activating chemosensors.

Methods: The rat lower oesophageal mucosa was superfused with pH 7.0 buffer, and pH 1.0 or pH 6.4 high CO(2) (P(CO2) = 260 Torr) solutions with or without the cell-permeant carbonic anhydrase (CA) inhibitor methazolamide (MTZ, 1 mM), the cell-impermeant CA inhibitor benzolamide (BNZ, 0.1 mM), the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (CPZ, 0.5 mM) or the acid-sensing ion channel (ASIC) inhibitor amiloride (0.1 mM). Interstitial pH (pH(int)) was measured with 5',6'-carboxyfluorescein (5 mg/kg intravenously) loaded into the interstitial space, and blood flow was measured with laser-Doppler.

Results: Perfusion of a high CO(2) solution induced hyperaemia without changing pH(int), mimicking the effect of pH 1.0 perfusion. Perfused MTZ, BNZ, CPZ and amiloride all inhibited CO(2)-induced hyperaemia. CA XIV was expressed in the prickle cells, with CA XII in the basal cells. TRPV1 was expressed in the stratum granulosum and in the muscularis mucosa, whereas all ASICs were expressed in the prickle cells, with ASIC3 additionally in the muscularis mucosa.

Conclusions: The response to CO(2) perfusion suggests that CO(2) diffuses through the stratum epithelium, interacting with TRPV1 and ASICs in the epithelium or in the submucosa. Inhibition of the hyperaemic response to luminal CO(2) by CA, TRPV1 and ASIC inhibitors implicates CA and these chemosensors in transduction of the luminal acid signal. Transepithelial CO(2) permeation may explain how luminal H(+) equivalents can rapidly be transduced into hyperaemia, and the sensation of heartburn.

Figure 1

Effect of luminal acid or high CO₂ challenge on interstitial pH (pH_int) and blood flow in rat oesophagus. (A) pH_int. Acid or CO₂ challenge had no effect on pH_int. (B) Blood flow. Acid or CO₂ challenge reversibly increased blood flow during the challenge period. Data are expressed as mean (SEM) (n = 6). *p < 0.05 vs pH 6.4 saline group.

Figure 2

Effect of capsazepine (CPZ) or amiloride (AML) on interstitial pH (pH_int) and blood flow during CO₂ challenge. (A) pH_int. Pretreatment with CPZ or AML followed by CO₂ challenge decreased pH_int. (B) Blood flow. CO₂ challenge-induced hyperaemia was inhibited by CPZ or AML. Data are expressed as mean (SEM) (n = 6). *p < 0.05 vs pH 6.4 saline group, †p < 0.05 vs high CO₂ group.

Figure 3

Effect of afferent denervation on interstitial pH (pH_int) and blood flow during CO₂ exposure in rat oesophagus. (A) pH_int. In capsaicin-treated rats (Cap-t), perfusion of high CO₂ saline exposure decreased pH_int. (B) Blood flow. Oesophageal CO₂ challenge-induced hyperaemia was abolished in Cap-t rats. Data are expressed as mean (SEM) (n = 6). *p < 0.05 vs pH 6.4 saline group, †p < 0.05 vs vehicle-treated (vehicle-t) + high CO₂ group.

Figure 4

Effect of carbonic anhydrase inhibitors on interstitial pH (pH_int) and blood flow during CO₂ exposure. (A) pH_int. Pretreatment with methazolamide (MTZ) followed by CO₂ challenge or luminal benzolamide (BNZ) co-perfused during CO₂ challenge decreased pH_int. (B) Blood flow. CO₂-induced hyperaemia was abolished by MTZ or BNZ. Data are expressed as mean (SEM) (n = 6). *p < 0.05 vs pH 6.4 saline group, †p < 0.05 vs high CO₂ group.

Figure 5

Expression of membrane-bound carbonic anhydrases (CAs) in rat oesophagus. No specific staining was observed for CA IV (A) and IX (B), compared with the negative control (E). CA XII was clearly localised on the plasma membrane of the basal cells (C, arrows), whereas CA XIV was observed on the membrane of the prickle cells (D). Note that oesophageal epithelium consists of stratum (St) corneum (c), St granulosum (*), St spinosum (prickle cell layer, s), St basale (basal cell layer, arrows) and lamina propria (lp) (E). Bar = 50 µm. A, B and E: conventional microscopic images; C and D: confocal microscopic images.

Figure 6

Expression of acid-sensing ion channel (ASIC) isoforms and transient receptor potential vanilloid 1 (TRPV1) in rat oesophageal mucosa. ASIC1 (A), ASIC2 (D) and ASIC3 (G) were expressed in the prickle cells. ASIC3 was also expressed in the muscularis mucosa (MM) (G). TRPV1-like immunoreactivity was observed in the granular cells (arrows) in the stratum granulosum (J). B, E, H, K: preabsorbed antibody with immunising peptide for ASIC1 (B), ASIC2 (E), ASIC3 (H) or TRPV1 (K) was reacted. C, F, I, L: positive control staining in dorsal root ganglia for ASIC1 (C), ASIC2 (F), ASIC3 (I) or TRPV1 (L) was shown. lp, lamina propria mucosa. Bar = 50 µm. A, D, G and J: confocal microscopic images, B, C, E, F, G and H: conventional microscopic images.

Figure 7

Western blot analysis for carbonic anhydrase (CA) XII, transient receptor potential vanilloid 1 (TRPV1), acid-sensing ion channel 2 (ASIC2) and ASIC3. Oesophageal mucosa (Öm) expressed CA XII (A, 55 kDa), TRPV1 (B, ~60 kDa), ASIC2 (C, 85 kDa) and ASIC3 (D, 85 kDa), whereas the oesophageal muscle layer (Ös) expressed only ASIC3. Furthermore, the mucosa (Fm) and muscle layer (Fs) of fundic stomach strongly expressed ASIC2 (C) as well as ASIC3 (D). TRPV1 was also recognised in Fs. (E) TRPV1 was recognised at ~60 kDa in Öm (open arrowhead), whereas it was ~95 kDa in dorsal root ganglia (DRGs) (filled arrowhead). Immunodetection of TRPV1 in Öm and DRGs was blocked by preabsorption with immunised peptide antigen (+P). (F) Effect of vehicle (veh) or capsaicin (Cap) treatment on the expression of TRPV1 in DRGs and Öm. The molecular mass of TRPV1 in DRGs (filled arrowhead) was reduced by Cap treatment, whereas TRPV1 in Öm (open arrowhead) had no change. One of two independent experiments is represented. β-Actin was used as an internal loading control. M, molecular marker.

Figure 8

Effect of high luminal CO₂ solution and carbonic anhydrase inhibitor on portal venous (PV) blood pH and P_CO2. PV blood was collected at t = 0, 30 and 40 min to measure PV blood pH and P_CO2. During the basal period (t = 0–30 min), PV pH and P_CO2 were stable, and luminal perfusion with a high CO₂ solution had no effect on PV pH (A) and P_CO2 (B). Pretreatment with methazolamide (MTZ, 1 mM) followed by CO₂ exposure lowered pH (A) and increased P_CO2 (B) in PV blood. Data are expressed as mean (SEM) (n = 6). *p < 0.05 vs the corresponding value at t = 0 min, †p < 0.05 vs the high CO₂ group.