The perfused gut preparation

Studies in our laboratory center on an isolated, perfused preparation of the anterior midgut developed initially by Clark and colleagues (Clark et al., 1999) and modified by Onken and colleagues (Onken et al., 2004). Briefly, for the experiments reported here, the excised gut is tied to a glass perfusion pipette at one end. The other end is left open, except that a blunt rod of appropriate dimensions is inserted partway into the lumen to support the tissue in the focal plane of a dissection microscope. This preparation offers the possibility of studying the function of a tissue in a setting in which the composition of solutions on both sides of the tissue can be controlled and in which complicating structures such as the peritrophic membrane and adjacent gut regions are absent. This preparation becomes nonfunctional within minutes after mounting, with the transepithelial electrical potential (V_te) falling to a value near zero and alkali secretion occurring at immeasurable rates. However, administration of submicromolar serotonin restores and sustains transepithelial potential values and alkali secretion for up to several hours. It is noteworthy that this is the only insect tissue shown so far to engage in extreme alkalinization in vitro.

For the experiments described herein, the hemolymph-side superfusate was Aedes saline (Clark et al., 1999) and the luminal perfusate was 100 mmol l^–1 NaCl, unless otherwise noted. In most experiments, the luminal perfusate was not buffered and contained the pH-sensitive dye m-cresol purple (0.04%). A visual indication of alkali secretion can be obtained at any point in the experiment by stopping perfusion and watching for the orange-to-purple color transition of the m-cresol purple that occurs at a pH of approximately 8.3. This method was verified with luminal pH-sensitive microelectrodes. Typically, the rate of alkali secretion is sufficiently vigorous that only a few minutes are required for this transition.

Three types of experiments using the perfused preparation are presented here: inhibitor studies, in which the inhibitor is applied in luminal or hemolymph-side perfusate and changes in the transepithelial potential and the capability to secrete alkali are measured; optical measurements of intracellular pH (pH_i) using the H⁺-sensitive dye BCECF; and microelectrode experiments in which the tissue is penetrated with intracellular glass microelectrodes for measurement of the transbasal electrical potential (V_bl).

Optical measurements of pH_i with BCECF were performed using a method modified from that of Parks and colleagues (Parks et al., 2007). In brief, the BCECF trapped in the tissue was excited at its absorption peak of 495 nm; 440 nm was used as an isobestic wavelength. Images at these wavelengths were captured digitally. The 495-to-440 nm ratios were compiled as an indication of the pH_i. At the beginning of each experiment, areas of interest were established on the CCD image of the gut. At the end of each experiment, fluorescence ratios recorded during the experiment were calibrated by superfusing the tissue successively with high-K⁺ solutions containing 5 μmol l^–1 nigericin, adjusted to cover the range of pH values 6.6–8.4.

In the microelectrode experiments, the tissue was penetrated across the hemolymph-side surface with KCl-filled microelectrodes, as in previous studies (Clark et al., 2000). From simultaneous measurements of V_bl and the transepithelial potential V_te, the transapical electrical potential (V_api) can be calculated. These measurements were combined with measurements of pH_i under similar experimental conditions to yield estimations of the electrochemical forces acting on H⁺ as it passes through the cells.

The proton electrochemical gradients

Fig. 1 shows the proton electrochemical gradients for three conditions: before serotonin stimulation, after serotonin with both luminal side and hemolymph-side buffered to pH 7.0, and after increasing the luminal pH to an in vivo-like pH of 10. The unstimulated anterior midgut tissue presented pH_i values near or slightly below neutrality (H.O., S. K. Parks, G. G. Goss and D.F.M., unpublished observations). In the unstimulated gut (Fig. 1A), with a luminal pH of 7.0, the combination of the large, inside-negative V_api (Clark et al., 2000) with the negligible transapical H⁺ activity gradient results in a substantial electrochemical gradient favoring H⁺ entry across the apical membrane. There is a similar large electrical gradient that opposes H⁺ movement from the cell to hemolymph (Clark et al., 2000).

Addition of serotonin resulted in hyperpolarization of both V_bl and V_api (Clark et al., 2000); generally, the effect on V_bl is the larger of the two, so that V_te increased to lumen-negative values of up to several tens of millivolts. Serotonin also had a dramatic effect on pH_i, increasing it to a mean of 7.7 (H.O., S. K. Parks, G. G. Goss and D.F.M., unpublished observations). When the solutions on both sides of the tissue were buffered to pH 7.0, the combination of these effects increased both the gradient favoring proton entry from the lumen and that opposing proton exit across the basal membrane (Fig. 1B).

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Fig. 1.

Transepithelial profiles of the proton-motive force (in mV) calculated from the average membrane voltages and transmembrane pH gradients for (A) a control condition with mosquito saline of pH 7 on both sides of the tissue, (B) after stimulation with serotonin (pH=7 on both sides; lumen 100 mmol l^–1 NaCl) and (C) after increasing the pH of the luminal perfusate to 10. HL, hemolymph; L, lumen.

When the pH of the luminal perfusate of a serotonin-stimulated tissue was raised from 7 to 10, pH_i rose substantially to ∼8.6 (H.O., S. K. Parks, G. G. Goss and D.F.M., unpublished observations). This change dramatically increased the transbasal proton electrochemical gradient to approximately –190 mV. This value approximates the maximal pump electromotive force predicted for the V-ATPase (Moffett, 1980; Grabe et al., 2000; Luo et al., 2004). At the same time, the H⁺ electrochemical gradient across the apical membrane was essentially abolished, so that, at a luminal pH of 10, cytoplasmic H⁺ is close to electrochemical equilibrium with luminal H⁺ (Fig. 1C).

When micromolar Zn²⁺, a blocker of H⁺ channels (DeCoursey, 2003), was included in the luminal perfusate, neither the magnitude nor the rate of the change in pH_i in response to alkaline luminal perfusate was affected (H.O., S. K. Parks, G. G. Goss and D.F.M., unpublished observations). This result argues against participation of apical H⁺ channels in the mechanism of luminal alkalinization.

The transbasal processes

Transbasal processes parallel to the V-ATPase are potentially important in alkali secretion, particularly if the hemolymph is a source for bicarbonate. The presence of the V-ATPase alone cannot result in mass absorption of protons without the support of an anion transport pathway. Boudko and colleagues (Boudko et al., 2001) detected a DIDS-sensitive transbasal Cl^– efflux in a semi-intact preparation. We reported effects of hemolymph-side DIDS (0.1 mmol l^–1) as well as DPC (0.5 mmol l^–1) on V_te (Onken et al., 2004). The effects of hemolymph-side application of these two relatively nonspecific inhibitors of anion exchangers and anion channels on alkalinization has not yet been evaluated. If, in addition to Cl^– channels, there were a basal anion exchanger, it might provide HCO ^–₃ for alkali secretion. Addition of Ba²⁺ (5 mmol l^–1), an inhibitor of K⁺ channels (Nagel, 1979), to the hemolymph-side superfusate, reduces V_te by ∼26% (Onken et al., 2004). In the presence of basal K⁺ channels, the V-ATPase could serve a housekeeping role by driving the accumulation of K⁺ in the cells, thus substituting for the conventional role of the basal Na⁺/K⁺ pump, which is absent in these cells (Patrick et al., 2006). For values of V_bl of the order of those reported here, intracellular [K⁺] would be approximately 70–90 mmol l^–1, a value not unusual for insect cells.

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Fig. 2.

Model of hypothetical transport mechanisms involved in strong alkalinization based on earlier proposals (Boudko et al., 2001; Onken et al., 2004), focusing on anionic pathways in the apical membrane. CA, carbonic anhydrase.

The transapical processes

Boudko–Onken hypothesis

The key features of this anion-dominated model (Fig. 2) (Boudko et al., 2001; Onken et al., 2004) are the presence of the basal V-ATPase, the absence of basal Na⁺/K⁺-ATPase ordinarily present in animal epithelia, and the presence of an apical Cl^–/HCO ^–₃ exchanger supported by cytoplasmic carbonic anhydrase (CA). A basal Cl^– channel provides an avenue for transepithelial Cl^– absorption. A putative apical anion channel could provide for transapical Cl^– recycling and/or even for HCO ^–₃ secretion. We have found that hemolymph-side Na⁺ is required for a normal V_te (in Na⁺-free solution, V_te falls from a mean of– 45 mV to <–10 mV) and for alkalinization. Amiloride applied to the hemolymph-side saline has a similar effect (Onken et al., 2004; Onken et al., 2008). The latter observations were interpreted as reflecting a transbasal Na⁺-dependent process and a transapical Na⁺-dependent component of HCO ^–₃ secretion. Note that Boudko and colleagues (Boudko et al., 2001) assume a cytoplasmic pH of 7.2, giving a transapical H⁺ gradient of 3–4 orders of magnitude. A major piece of evidence against this hypothesis was provided by our recent finding that neither methazolamide, an inhibitor of CA (Fig. 3), nor DIDS, an inhibitor of anion exchangers, nor bilateral Cl^–-free saline, affect alkalinization in the isolated and perfused anterior midgut (Onken et al., 2008).

Smith and colleagues (Smith et al., 2007) found CA in the ectoperitrophic space of the whole larval midgut of Anopheles gambiae. Although not an exclusive feature of the alkalinizing region of the midgut, the presence of CA in the midgut lumen supports the hypothesis that carbon dioxide, diffusing from the anterior midgut cells into the lumen, could be converted there to HCO₃^– and H⁺. Alkalinization could then depend solely on acid absorption (see also below). This hypothesis is indeed consistent with the findings by Boudko and colleagues (Boudko et al., 2001) and Corena and colleagues (Corena et al., 2002) that blockers of carbonic anhydrase inhibit alkalinization in semi-intact larvae or in excised midguts of Aedes aegypti. By contrast, strong alkalinization seems not to depend on ectoperitrophic carbonic anhydrase, as is demonstrated by the presence of strong alkalinization in our experiments with isolated and perfused midgut preparations where ectoperitrophic CA is removed and/or inhibited. Moreover, in our hands, CA inhibitors did not affect alkalinization in vivo. When the drugs were added together with m-cresol purple to the medium in which the larvae were maintained, all larvae still showed alkalinized anterior midguts (H.O., S. K. Parks, G. G. Goss and D.F.M., unpublished observations).

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Fig. 3.

Mean lumen negative transepithelial voltages (V_te;– s.e.m.) of six anterior midguts stimulated with serotonin (0.2 μmol l^–1) in the presence (gray bar) and absence (white bar) of luminal methazolamide (200 μmol l^–1), and photographs of a representative preparation of the anterior midgut of larval (fourth instar) Aedes aegypti at identical times after perfusion stop in the presence (right) and absence (left) of luminal methazolamide (200 μmol l^–1). [Figure reproduced from Onken and colleagues (Onken et al., 2008).]

Okech–Patrick hypothesis

The key features of this cation-dominated model (Fig. 4) are the presence of apical Na⁺/2H⁺ exchanger, Na⁺-coupled amino acid transporter and apical Na⁺/K⁺-ATPase (Okech et al., 2008; Patrick et al., 2006). The postulated existence of an apical Na⁺/2H⁺ exchanger is logical as such an exchanger could exploit the large transapical proton motive force (Fig. 1B). If the luminal pH is 7, a transapical H⁺ gradient favorable for H⁺ absorption exists under the conditions of our experiments (see above); this could drive Na⁺/2H⁺ exchange, as long as the cytoplasmic [Na⁺] is low. If the gut becomes alkaline, this gradient ultimately disappears as the luminal pH approaches 10 (Fig. 1C). However, luminal amiloride (200 μmol l^–1), a general inhibitor of such exchangers, did not affect alkalinization (Fig. 5) (Onken et al., 2008). A K⁺/2H⁺ exchanger is postulated in the lepidopteran midgut, another insect tissue that also develops strong alkalinization (Azuma et al., 1995). If such an exchanger were present in the mosquito, the gradient would be even more favorable. However, increased luminal K⁺ did not affect luminal alkalinization in the perfused preparations (Onken et al., 2008).

The location of the Na⁺/K⁺-ATPase on the apical membrane is an unusual expression pattern for this pump, which is generally expected to have a basal location. As freshwater mosquito larvae such as Aedes and Anopheles consume a low-Na⁺ diet, it might serve in vivo to deliver Na⁺ to the gut lumen at the proximal end of the gut to support the Na⁺-dependent nutrient-absorption processes. Even more striking is that the Na⁺/K⁺-ATPase is located in the apical membrane in exactly the region of high luminal alkalinity and the hypothesis arises that it could serve as an ATP-driven Na⁺/H⁺ exchanger. It has been known that the ATPase can accept H⁺ instead of Na⁺ and/or K⁺ (Polvani and Blostein, 1988). However, when we tested this hypothesis by addition of ouabain (5 mmol l^–1), a specific inhibitor of the Na⁺/K⁺ ATPase, to the luminal perfusate, there was no significant effect on V_te, and alkalinization was not impaired (Onken et al., 2009).

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Fig. 4.

Model of hypothetical transport mechanisms involved in strong alkalinization and amino acid absorption based on earlier proposals (Okech et al., 2008; Patrick et al., 2006), focusing on cationic pathways in the apical membrane.

Okech and colleagues (Okech et al., 2008) found amino acid transporters in the anterior midgut by using immunohistochemistry. The presence of such transporters is indeed confirmed by electrophysiological measurements in which a significant increase in V_te is observed when individual amino acids are added to the luminal perfusate (S. Izeirovski, S. B. Moffett, D.F.M. and H.O., personal communication). The effect of glutamine, which gives the largest response of the amino acids present in the Aedes saline, is shown in Fig. 6. Interestingly, amino acid transport, as indicated by changes of V_te, is stimulated by serotonin. In our recent experiments, the luminal perfusate did not include amino acids, but alkalinization was nevertheless observed. Therefore, the presence of luminal nutrients might facilitate alkali secretion but is not a requirement for it. By contrast, if amino acids are eliminated from the hemolymph-side superfusate, the V_te drops significantly and alkali secretion is retarded (S. Izeirovski, S. B. Moffett, D.F.M. and H.O., personal communication).

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Fig. 5.

Mean lumen negative transepithelial voltages (V_te;– s.e.m.) of five anterior midguts stimulated with serotonin (0.2 μmol l^–1) in the presence (gray bar) and absence (white bar) of luminal amiloride (200 μmol l^–1), and photographs of a representative preparation of the anterior midgut of larval (fourth instar) Aedes aegypti at identical times after perfusion stop in the presence (right) and absence (left) of luminal amiloride (200 μmol l^–1). [Figure reproduced from Onken and colleagues (Onken et al., 2008).]

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Fig. 6.

Representative time-course of the lumen negative transepithelial voltage (V_te) of the anterior midgut of larval (fourth instar) Aedes aegypti in the presence of hemolymph-side mosquito saline and luminal 100 mmol l^–1 NaCl. After mounting of the tissue, V_te declines but successively recovers after addition of serotonin (0.2 μmol l^–1) to the hemolymph-side bath and glutamine (10 mmol l^–1) to the luminal perfusate. Washout of serotonin in the presence of luminal glutamine indicates that the effect of luminal glutamine is stimulated by serotonin.