Short Communication: Luminal Alkalinization by the Isolated Midgut of the Tobacco Hornworm (Manduca Sexta)

Tobacco hornworms were grown from eggs or larvae purchased from Carolina Biological Supply Company (Burlington, North Carolina). The larvae were maintained at 28°C on a 16h:8h light: dark cycle and fed an artificial diet (Carolina Biological Supply Co.). Fifth-stage larvae weighing 3-5.3 g were used in all experiments.

Larvae were decapitated and a dorsal incision made to expose the midgut. The anterior, middle or posterior midgut was then cleaned of adhering tracheae and Malpighian tubules, opened, removed from the animal and mounted as a flat sheet in an Ussing type chamber. Each half of the chamber had a volume of 2.5 ml and was a modification of a design described previously (Hanrahan et al. 1984). The midgut was secured to a notched collar with cotton thread and the opening (0.196 cm²) was backed with fine nylon screen to support the tissue. The tissue was bathed bilaterally with saline which was constantly bubbled with 100 % oxygen to maintain high oxygen tension and to circulate the saline. After reaching a steady transepithelial potential difference (PD) or short-circuit current (I_sc; usually 60–90 min), the midgut was bilaterally perfused with saline at a rate of 3 ml min⁻¹ using a Gilson Minipuls peristaltic pump to ensure that any residual alkali was washed from the luminal surface. After 15 min the perfusion on the luminal side was stopped and the base secretion measured for 30 min using a pH stat technique (Radiometer PHM84 pH meter, ABU 80 autoburette and a TTT 80 titrator). Vigorous oxygenation of both sides of the epithelium was maintained throughout these procedures. Luminal alkalinization was determined by the rate of titrant (0.01 or 0.1 mol l⁻¹ HCl) added to maintain the initial pH. Since the anterior portion had the greatest rate of alkalinization (see below), transport was further explored in this section. Alkali secretion was measured for 90 min in K⁺-free saline and under short-circuit conditions. Experiments were conducted at room temperature (22–25°C).

The PD was measured with calomel electrodes connected to KC1 agar bridges which were inserted into the saline on either side of the midgut. Current was passed via silver/silver chloride electrodes connected to agar bridges. PD and I_sc, with compensation for saline resistance, were measured with a custom-made voltage clamp (Duke University Physiology Department Technical Shop) and monitored on Soltec recorders.

A saline containing haemolymph levels of ions, amino acids and carboxylic acids was used, since a saline of similar composition has been shown to maintain midgut function in vitro (Chamberlin, 1989). Control saline had the following composition (in mmol l⁻¹): Hepes, 2; MgCl₂, 5.0; CaCl₂, 1.0; KOH, 5.8; NaOH, 9.0; sodium methylsulphate, 3.0; potassium citrate, 7.7; sodium succinate, 2.8; malic acid, 5.6; glucose, 2.0; trehalose, 27.0; glutamine, 9.4; serine, 8.9; proline, 7.4; glycine, 12.8; threonine, 4.6; alanine, 3.6; polyethylene glycol (M_r 400), 140. K⁺-free saline consisted of the following (in mmol l⁻¹): Hepes, 2; MgCl₂, 5.0; CaCl₂, 1.0; NaOH, 9.0; sodium methylsulphate, 3.0; citric acid, 7.7; sodium succinate, 2.8; malic acid, 5.6; glucose, 2.0; trehalose, 27.0; glutamine, 9.4; serine, 8.9; proline, 7.4; glycine, 12.8; threonine, 4.6; alanine, 3.6; N-methyl-D-glucamine, 23.1; polyethylene glycol (M_r 400), 140. Salines were vigorously bubbled with 100% oxygen for 2 h prior to adjusting the pH to 6.7.

Table 1 shows that all three midgut sections engage in in vitro luminal alkalinization under open-circuit conditions. The rate of alkalinization is greatest in the anterior section and can be maintained at a constant rate for at least 90 min (Fig. 1). This result is consistent with the observation that the highest pH is found in the anterior and middle regions of the midgut lumen in vivo (Dow, 1984). The observation that luminal pH falls in the posterior section of the midgut in vivo may reflect a lower rate of base secretion (or H⁺ absorption), although reflux of Malpighian tubule fluid or rectal contents cannot be discounted.

The chemical nature of the secreted alkali was not investigated in this study. Secretion of ammonia is a possibility since amino acids were present in the saline and the midgut tissue can oxidize some amino acids (Chamberlin, 1987, 1989). Carbonic anhydrase is found in the epithelial cells of the midgut (Ridgway and Moffett, 1986) and therefore HCO₃⁻ may be produced and secreted. Under the conditions of the present study, exogenous CO₂ is minimal and therefore HCO₃⁻ would be synthesized from endogenously produced CO₃. The posterior midgut section consumes oxygen at a rate of 213 μmol O₂ g⁻¹ h^{− 1} (Mandel et al. 1980) and this is equivalent to a rate of 23.8μmol O₂ cm⁻² h⁻¹ using the conversion factor reported by Cioffi and Harvey (1981). With respiratory quotients ranging from 0.7 to 1, HCO₃⁻ could be produced at a rate sufficient to account for the luminal alkalinization seen in this study. The cell types responsible for this putative HCO₃⁻ transport may differ along the length of the midgut, since histochemical studies reveal that carbonic anhydrase is found predominantly in the goblet cells in the anterior and middle regions of the midgut, while this enzyme is localized in the columnar cells in the posterior region (Ridgway and Moffett, 1986).

It should be emphasized that HCO₃⁻per se may not be the species which is transported by the midgut epithelium. Secretion of OH⁻ or absorption of H⁺ could also produce luminal alkalinization. Wieczorek et al. (1989) have recently proposed that K⁺ secretion occurs via an apically located K⁺/H⁺ exchanger which is energized by the action of a H⁺ pump on the same membrane. If a Cl⁻ /OH⁻ (Cl⁻ /HCO₃⁻) exchanger were located on the apical membrane, the net result would be KOH (KHCO₃) secretion. In support of this view, Chao et al. (1989) have recently shown a Cl⁻ /HCO₃⁻ exchange mechanism in the apical membrane of midgut cells.

Luminal alkalinization is maintained (7.3±0.7 μequivcm⁻²h⁻¹, N=5) under short-circuit conditions when no transepithelial pH or electrical gradient is present, suggesting that this process is active. Under the conditions of this study, however, the passive secretion of an anion from the cell cannot be discounted (assuming an intracellular pH greater than 6.7). In contrast, if this alkalinization occurs via active transepithelial transport it may account for some of the disparity between the I_sc and ion fluxes measured under similar conditions (Chamberlin, 1990). Under open-circuit conditions, K⁺ secretion far exceeds Na⁺ absorption and Cl⁻ secretion (Chamberlin, 1990) and, under such conditions, base secretion may contribute to maintenance of electroneutrality.

Exposure to K⁺-free saline for 45 min depressed the rate of open-circuit base secretion by 66.7±1.8% (N=3). As suggested by Dow and Harvey (1988) and Dow and Peacock (1989), luminal alkalization is dependent, to a great extent, on the large K⁺-dependent transepithelial potential. The high rates of in vitro luminal alkalinization measured in this study indicate that the mechanisms of base secretion and/or acid absorption should be readily described with further experimentation.

I wish to thank J. Martin and R. B. Thomson for their critical comments. This work was supported by an Ohio University Research Challenge grant and funds from the Ohio University College of Arts and Sciences and College of Osteopathic Medicine.