QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online March 31, 2007
Journal of Experimental Biology 210, 1463-1471 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.001529

This Article Summary Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hille, C. Articles by Walz, B. Search for Related Content PubMed PubMed Citation Articles by Hille, C. Articles by Walz, B. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB AMMONIUM CHLORIDE DOPAMINE Social Bookmarking                         What's this?

A vacuolar-type H⁺-ATPase and a Na⁺/H⁺ exchanger contribute to intracellular pH regulation in cockroach salivary ducts

Carsten Hille^* and Bernd Walz

University of Potsdam, Institute of Biochemistry and Biology, Department of Animal Physiology, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam-Golm, Germany

Author for correspondence (e-mail: walz{at}uni-potsdam.de)

Accepted 12 February 2007

	Summary

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Cells of the dopaminergically innervated salivary ducts in the cockroach Periplaneta americana have a vacuolar-type H⁺-ATPase (V-ATPase) of unknown function in their apical membrane. We have studied whether dopamine affects intracellular pH (pH_i) in duct cells and whether and to what extent the apical V-ATPase contributes to pH_i regulation. pH_i measurements with double-barrelled pH-sensitive microelectrodes and the fluorescent dye BCECF have revealed: (1) the steady-state pH_i is 7.3±0.1; (2) dopamine induces a dose-dependent acidification up to pH 6.9±0.1 at 1 µmol l^–1 dopamine, EC₅₀ at 30 nmol l^–1 dopamine; (3) V-ATPase inhibition with concanamycin A or Na⁺-free physiological saline (PS) does not affect the steady-state pH_i; (4) concanamycin A, Na⁺ -free PS and Na⁺/H⁺ exchange inhibition with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) each reduce the rate of pH_i recovery from a dopamine-induced acidification or an acidification induced by an NH₄Cl pulse; (5) pH_i recovery after NH₄Cl-induced acidification is almost completely blocked by concanamycin A in Na⁺-free PS or by concanamycin A applied together with EIPA; (6) pH_i recovery after dopamine-induced acidification is also completely blocked by concanamycin A in Na⁺-free PS but only partially blocked by concanamycin A applied together with EIPA. We therefore conclude that the apical V-ATPase and a basolateral Na⁺/H⁺ exchange play a minor role in steady-state pH_i regulation but contribute both to H⁺ extrusion after an acute dopamine- or NH₄Cl-induced acid load.

Key words: vacuolar H⁺-ATPase, V-ATPase, NHE, BCECF, intracellular pH, dopamine, biogenic amines, insect, cockroach, Periplaneta americana, salivary glands

	Introduction

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Intracellular pH (pH_i) affects a plethora of physiological processes, such as cellular metabolism, contractility, ion channel conductivity, ion transport and cell cycle control (Madshus, 1988). Therefore, pH_i must be strictly regulated by means of several ion transport mechanisms. These mechanisms function either as acid loaders or acid extruders (Boron, 1986; Boron, 2004). One of these acid extruders is the vacuolar-type H⁺-ATPase (V-ATPase), when it is localised in the plasma membrane. V-ATPases are widely used to generate electrochemical proton gradients, i.e. they energise membranes for driving other transport processes. Plasma membrane V-ATPases have been shown to be essential in processes such as acid secretion and HCO₃^– transport in the proximal tubule and collecting duct of the kidney (Brown and Breton, 1996). They also energise fluid secretion in insect Malpighian tubules and salivary glands (Wieczorek et al., 1999; Nishi and Forgac, 2002; Zimmermann et al., 2003; Beyenbach and Wieczorek, 2006). In addition, a contribution of the V-ATPase to pH_i regulation has been described in some vertebrate epithelia (Pappas and Ransom, 1993; Stankovic et al., 1997; Granger et al., 2002; Yip et al., 2002). Although the V-ATPase has been found in a number of insect tissues (Beyenbach and Wieczorek, 2006), an involvement of the V-ATPase in pH_i regulation in insects has only been suggested for Drosophila Malpighian tubules (Bertram and Wessing, 1994). By contrast, a Na⁺/H⁺ exchange (NHE) has been proposed to play a major role in pH_i regulation in Aedes and Rhodnius Malpighian tubules (Petzel, 2000; Ianowski and O'Donnell, 2006).

The epithelial cells forming the ducts in the acinar salivary glands of the cockroach Periplaneta americana have a V-ATPase in their highly folded apical membrane (Just and Walz, 1994a) but its functional significance is unknown. The secretory acini are innervated by dopaminergic and serotonergic fibres (Baumann et al., 2002) and secrete a NaCl-rich primary saliva upon stimulation with dopamine (DA) or serotonin (Rietdorf et al., 2003). Dopaminergic fibres also innervate the ducts, and DA has been shown to cause dramatic changes in intracellular Na⁺ and K⁺ concentrations in duct cells (Lang and Walz, 2001). These findings together with those from electron probe X-ray microanalysis and capillary electrophoresis of primary and final saliva have led to the conclusion that the ducts modify primary saliva by Na⁺ reabsorption and K⁺ secretion (Gupta and Hall, 1983; Rietdorf et al., 2003). In insect epithelial tissues such as salivary glands, midgut and Malpighian tubules, an apical V-ATPase has been shown to energise apical K⁺ secretion (Maddrell and O'Donnell, 1992; Wieczorek, 1992; Zimmermann et al., 2003). Thus, the apical V-ATPase in cockroach salivary ducts might be involved in K⁺ secretion and/or in intracellular pH homeostasis.

The immediate aim of the present study has, therefore, been to study whether DA affects pH_i in duct cells and whether and to what extent the apical V-ATPase contributes to intracellular pH homeostasis. We have measured pH_i in duct cells of isolated salivary glands with double-barrelled pH-sensitive microelectrodes and microfluorometrically with the fluorescent dye BCECF and demonstrate that DA causes an intracellular acidification. We have found that V-ATPase and NHE play a minor role in steady-state pH_i regulation but contribute significantly to pH_i recovery from an acute acid load.

	Materials and methods

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
Animals and preparation
A colony of Periplaneta americana L. (Blattodea, Blattidae) was reared at 27°C under a light:dark cycle of 12 h:12 h. The animals had free access to food and water. Only male adults aged between 4 and 6 weeks were used for experiments. Salivary glands were dissected in physiological saline (PS) as described previously (Just and Walz, 1996). Small pieces of the glands consisting of one lobe with its acini and ducts were used.

Solutions and chemicals
Cockroach PS contained 160 mmol l^–1 NaCl, 10 mmol l^–1 KCl, 2 mmol l^–1 CaCl₂, 2 mmol l^–1 MgCl₂, 10 mmol l^–1 glucose and 10 mmol l^–1 Tris. The pH was adjusted to 7.4 with HCl. To induce an acute acid load in the salivary glands, 20 mmol l^–1 NH₄Cl was substituted for 20 mmol l^–1 NaCl at pH 7.4. In Na⁺-free saline, equimolar amounts of choline chloride were substituted for NaCl. BCECF/AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester], concanamycin A, DA and EIPA [5-(N-ethyl-N-isopropyl)-amiloride; all from Sigma, Deisenhofen, Germany or Invitrogen, Karlsruhe, Germany] were stored as stock solutions in small aliquots at –20°C and diluted in PS immediately before an experiment. Dimethyl sulfoxide as a solvent for BCECF/AM, concanamycin A and EIPA did not affect pH_i (N=3; data not shown). Dimethyl-trimethylsilylamine and the H⁺-ionophore I Cocktail A for pH-sensitive microelectrodes were purchased from Fluka (Buchs, Switzerland).

Microfluorometric measurements of pH_i
The pH_i in duct epithelial cells was measured microfluorometrically with the pH-sensitive fluorescent dye BCECF. Isolated gland lobes were loaded with BCECF by 10 min incubation in PS containing 0.5 µmol l^–1 BCECF/AM at room temperature. BCECF-loaded lobes were then attached to the coverslip-bottom of a custom-built recording chamber coated with the tissue adhesive Vectabond Reagent (Axxora, Grünberg, Germany) and continuously superfused with PS. The chamber was mounted on a Zeiss Axiovert 135TV inverted microscope equipped with epifluorescence optics and a Zeiss Fluar 20/0.75 objective. For fluorescence excitation, a 75 W xenon arc lamp monochromator unit (VisiChrome, Visitron, Puchheim, Germany) was connected to the microscope by a quartz fibre-optic light guide. The epifluorescence filter block in the microscope contained a 485 nm dichroic mirror and a 515–565 nm bandpass emission filter. BCECF fluorescence was excited every 10 s or 15 s for 5–40 ms at 470 nm, and for 20–160 ms at 410 nm, depending on the BCECF concentration in the cytosol. Fluorescence images were acquired and digitised with a cooled image transfer CCD camera (CoolSnap-HQ, Roper Scientific Inc., Tucson, USA) at a 12-bit resolution. Monochromator control, image acquisition and processing were carried out using MetaFluor 6.1 software (Universal Imaging Corp., Downingtown, PA, USA). pH_i was expressed as the fluorescence ratio F₄₇₀/F₄₁₀. Background fluorescence and cell autofluorescence were negligible. Because EIPA is fluorescent in UV light, BCECF fluorescence was excited at 480 nm and 450 nm (each for 5–20 ms) and the fluorescence ratio F₄₈₀/F₄₅₀ was calculated in all experiments in which EIPA was used (Lee et al., 2005). We were not able to convert the fluorescence ratio F₄₇₀/F₄₁₀ into pH_i by using the classical high-K⁺/nigericin method (Thomas et al., 1979) because the cells deteriorated rapidly in high-K⁺/nigericin solutions. Therefore, we also measured pH_i with pH-sensitive microelectrodes in order to determine resting pH_i and the magnitude of DA-induced pH changes.

pH_i measurements with pH-sensitive microelectrodes
Recordings of pH_i with pH-sensitive microelectrodes were performed as previously described (Rein et al., 2006). In brief, the active barrel of a theta-glass double-barrelled microelectrode was silanised with dimethyl-trimethylsilylamine (Munoz et al., 1983) and its tip was filled with the H⁺ sensor from behind. The sensor column was backfilled with 100 mmol l^–1 sodium citrate (pH 6.6). The reference barrel was filled with 3 mol l^–1 KCl. For electrical recordings, the two electrode barrels were connected to the inputs of a differential amplifier (V86; List Medical, Darmstadt, Germany). The potential recorded by the reference barrel was subtracted from that recorded by the active barrel. This differential signal, which indicates ion activity, and the voltage recorded from the reference barrel were monitored on a chart recorder and stored on a PC using TestPoint software (Keithley, Germering, Germany). The bath electrode was an Ag/AgCl pellet connected to the bath via a 3 mol l^–1 KCl-agar bridge. Duct epithelial cells were impaled under optical (Zeiss Stemi SV11, Jena, Germany) and electrical control. With the electrode positioned in the cytosol, the differential voltage signal is proportional to the pH_i, and the reference barrel records the basolateral membrane potential (PD_b). For calibration of the pH-sensitive microelectrodes, PS (pH 7.4), PS titrated to pH 7.9, and a Pipes-buffered PS (pH 6.9) were used. The pH-sensitive microelectrodes were calibrated immediately after a successful experiment following withdrawal of the microelectrode into the bath. The mean slope of the electrodes was 56±4 mV per pH unit (N=5).

Statistical analysis
Statistical comparisons were calculated by one-way ANOVA followed by Dunnett's tests or by Student's unpaired t-test. P<0.05 was considered significant. All analyses were performed using GraphPad Prism 4.01 (GraphPad Software, San Diego, USA). Results are given as mean ± s.e.m.

	Results

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
DA-induced pH_i changes
Microfluorometric recordings of pH_i with BCECF showed that the stimulation of isolated lobes by a brief application of 1 µmol l^–1 DA, a concentration known to stimulate fluid secretion in isolated salivary glands (Just and Walz, 1996; Rietdorf et al., 2005), caused a reversible intracellular acidification. This acidification remained stable for some minutes after DA washout, following which pH_i recovered rapidly (Fig. 1A).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Dopamine (DA)-induced changes in pHi in duct cells of isolated salivary glands as measured with BCECF (A,B) and a double-barrelled pH-sensitive microelectrode (C). (A) Treatments with 1 µmol l–1 and 100 nmol l–1 DA induce a reversible intracellular acidification, N=5. (B) Dose–response relationship of the DA-induced intracellular acidification. Experiments were performed as in A and responses were normalised to the pHi drop induced by a first 1 µmol l–1 DA control stimulus (=100%). Values are means ± s.e.m. from 4–7 experiments for each concentration. A non-linear regression according to the Hill equation was fitted through the data points. (C) Effect of DA on pHi (solid line) and basolateral membrane potential (PDb, broken line). This recording, with a double-barrelled pH-sensitive microelectrode, is representative of five independent experiments.

Because we were not able to calibrate the BCECF signals by the high K⁺/nigericin method, we also recorded steady-state pH_i and the DA-induced pH_i changes with double-barrelled pH-sensitive microelectrodes. In many of these experiments, resting pH_i appeared to be acid (pH_i<7), especially when the reference barrel of the electrode recorded poor basolateral membrane potentials (PD_b; more positive than –50 mV). This effect was more pronounced with electrodes that had a broken tip and thus, a larger tip diameter. Because we suspected that cell impalement with such poorly performing electrodes had created a H⁺ leak, we discarded all recordings associated with a PD_b more positive than –50 mV. In the remaining experiments (PD_b more negative than –50 mV), the mean steady-state pH_i was 7.3±0.1, and 1 µmol l^–1 DA induced a pH_i decrease to 6.9±0.1 (N=5; Fig. 1C). The time course of the DA-induced acidification recorded with pH-sensitive microelectrodes was almost identical to that recorded microfluorometrically with BCECF. In addition, 1 µmol l^–1 DA induced a reversible depolarisation of PD_b from –55±3 mV to –19±3 mV (N=5; Fig. 1C) as reported previously (Lang and Walz, 2001). As shown in Fig. 1C, the onset of the pH_i decrease lagged behind the onset of the depolarisation by about 20–30 s.

Effects of V-ATPase- and Na⁺-dependent acid extrusion on steady-state pH_i and pH_i recovery after DA-induced acidification
Does apical V-ATPase activity affect the DA-induced pH_i changes? In order to answer this question, we treated the salivary glands as indicated at the top of Fig. 2A. A brief application of 1 µmol l^–1 DA induced a reversible acidification and served as a control. After the pH_i had recovered to the resting level, we superfused the preparation with concanamycin A, a specific inhibitor of the V-ATPase (Dröse et al., 1993; Dröse and Altendorf, 1997). As shown in Fig. 2A, 1 µmol l^–1 concanamycin A did not affect resting pH_i or the kinetics and magnitude of the acidification induced by 1 µmol l^–1 DA. However, in the presence of concanamycin A, pH_i recovered more slowly from the DA-induced acidification. We determined the rate of the initial fast phase (1–3 min) of the pH_i recovery by a linear regression (ratio-units min^–1) and found that concanamycin A reduced the rate of pH_i recovery significantly by 33% (Fig. 2A,D; N=6, P<0.05).

View larger version (20K): [in this window] [in a new window]   Fig. 2. Recordings of the effects of 1 µmol l–1 concanamycin A (A), Na+-free (0Na+) physiological saline (PS; B) and the combined application of concanamycin A (ConA) and Na+-free PS (C) on pH changes induced by 1 µmol l–1 dopamine (DA). (D) Quantitative analysis of the rates of pHi recovery in the above experiments and in an experiment in which 1 µmol l–1 ConA and 50 µmol l–1 EIPA were applied simultaneously. Values are means ± s.e.m.; the number of experiments is given under each bar; one-way ANOVA, *P<0.05, **P<0.01.

This result suggested that the apical V-ATPase contributed to pH_i recovery but was not the sole acid extruder. Therefore, we next studied whether the duct cells had a Na⁺-dependent acid extruder by testing whether the removal of extracellular Na⁺ affected the rate of pH_i recovery. Because the DA-induced acidification required extracellular Na⁺ (C. Hille, unpublished data), bath Na⁺ was removed after DA washout (Fig. 2B). The pH_i started to recover quickly after DA washout but removal of extracellular Na⁺ during this pH_i recovery reduced its rate significantly by 70% (Fig. 2B,D; N=6, P<0.01). Re-introduction of extracellular Na⁺ restored the initial fast rate of pH_i recovery (Fig. 2B).

Thus, neither concanamycin A nor the absence of extracellular Na⁺ completely abolished the recovery from the DA-induced acidification. A prolonged bath application of 1 µmol l^–1 concanamycin A in Na⁺-free PS caused only a slight decrease in resting pH_i within 20–25 min (N=5, data not shown). However, when the ducts were stimulated with DA in the presence of 1 µmol l^–1 concanamycin A and DA was washed out with Na⁺-free PS containing concanamycin A, pH_i recovery was blocked by 95% (Fig. 2C,D; N=8, P<0.01).

In order to test, whether a NHE was responsible for the Na⁺-dependent component of pH_i recovery, we studied whether EIPA, a specific inhibitor of the NHE (Petzel, 2000; Giannakou and Dow, 2001) mimicked the above effect of Na⁺-free PS. Because EIPA is fluorescent under UV light, we recorded the fluorescence ratio F₄₈₀/F₄₅₀ to measure pH_i. Control experiments showed that the rates of pH_i recovery were the same, independently of whether pH_i was recorded using the F₄₈₀/F₄₅₀ ratio or the F₄₇₀/F₄₁₀ ratio. We found that 50 µmol l^–1 EIPA applied together with 1 µmol l^–1 concanamycin A had no effect on resting pH_i but reduced the rate of pH_i recovery from a DA-induced acidification significantly to 50% (Fig. 2D; N=5, P<0.01) of the control rate. Thus, the inhibition of the rate of pH_i recovery was significantly stronger than the inhibition caused by concanamycin A alone (Student's unpaired t-test, P<0.05), but less than in concanamycin A-containing Na⁺-free PS.

Taken together, these data indicate that the V-ATPase and an NHE play only a minor role in the regulation of steady-state pH_i and have no effect on DA-induced acidification. The V-ATPase, an EIPA-sensitive (NHE) and an unidentified EIPA-insensitive but also Na⁺-dependent acid extruder contribute to the recovery from a DA-induced acid load.

A drop in pH_i is sufficient to activate V-ATPase- and NHE-mediated acid extrusion
DA causes an increase in the intracellular Ca²⁺ concentration and possibly also in the cAMP concentration in salivary duct cells (Hille and Walz, 2006; Walz et al., 2006). Because an increase in cAMP concentration activates V-ATPase in Calliphora salivary glands (Dames et al., 2006), we have therefore asked whether a drop in pH_i is sufficient to stimulate V-ATPase-mediated outward H⁺ pumping in Periplaneta salivary duct epithelial cells. We studied this question by imposing an acid load on the cells by applying a brief (1–2 min) pulse of 20 mmol l^–1 NH₄Cl. An NH₄Cl pulse leads, in many cells, to characteristic pH_i changes (Boron and de Weer, 1976; Boron, 2004). Immediately after NH₄Cl application, pH_i increases rapidly because of an NH₃ influx and its protonation to NH₄⁺. This is typically followed by a modest drop in pH_i attributable to a slow uptake of NH₄⁺. The removal of NH₄Cl causes strong acidification because of the rapid NH₃ efflux from the cell, whereby H⁺ ions are left behind. The pH_i then recovers from this acid load to resting pH_i via the activity of H⁺ extruding and/or HCO₃^– importing transporters.

We found that, for duct epithelial cells, bath application of NH₄Cl induced only a rapid acidification without the typical initial alkalinisation (Fig. 3A,B,D). Removal of NH₄Cl boosted this acidification. These observations indicated that NH₄⁺ entered the cells faster than NH₃. A high permeability of the basolateral membrane to NH₄⁺ is not a common feature of animal plasma membranes. However, high permeabilities to NH₄⁺ have been found in mouse cerebral astrocytes (Nagaraja and Brookes, 1998), in cells from the thick ascending limb of rat kidney (Bleich et al., 1995), in Drosophila Malpighian tubules (Bertram and Wessing, 1994) and recently in blowfly salivary glands (B. Schewe and B.W., unpublished). In these cell types, NH₄Cl induces a similar rapid decrease in pH_i to that observed in our study. Although we have not studied the pathways that mediate NH₄⁺ entry, studies of other cells suggest that the Na⁺/K⁺/2Cl^– cotransporter, an NHE or K⁺ channels maybe involved (Ramirez et al., 1999; Heitzmann et al., 2000). The involvement of a Na⁺-dependent entry mechanism seems likely in our preparation, because we have observed a transient alkalinisation upon NH₄Cl application in Na⁺-free saline (see below, and Fig. 3B,C). Under these conditions, NH₃ entry must have been faster than that of NH₄⁺. Nevertheless, after NH₄Cl washout, pH_i recovered quickly, within 2–4 min (N=23; Fig. 3). The lag of 5 min before recovery from the DA-induced acidification began, in comparison with the immediate recovery from the NH₄Cl-induced acidification, probably reflects the more complex situation during DA stimulation, including changes in second messenger levels.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Recordings of the effects of (A) 1 µmol l–1 concanamycin A (ConA), (B) Na+-free physiological saline (PS), (C) 1 µmol l–1 ConA in Na+-free PS (D) and 50 µmol l–1 EIPA together with 1 µmol l–1 ConA, on pH changes induced by a brief application of 20 mmol l–1 NH4Cl to isolated salivary ducts. (E) Quantitative analysis of the rates of pHi recovery in the above experiments. Values are means ± s.e.m.; the number of experiments is given under each bar; one-way ANOVA, *P<0.05, **P<0.01.

Because pH_i recovery was not completely abolished by concanamycin A, we next investigated whether extracellular Na⁺ affected pH_i recovery. Superfusion of the preparation with Na⁺-free PS did not alter resting pH_i (Fig. 3B). However, upon bath application of NH₄Cl in Na⁺-free PS, a small transient intracellular alkalinisation could be observed before the pH_i decreased rapidly in the continuous presence of NH₄Cl (Fig. 3B,C). pH_i recovered completely in Na⁺-free PS, but at a rate of only 39% of the control rate (N=21, P<0.01; Fig. 3B,E).

Inhibition of the V-ATPase and removal of extracellular Na⁺ by bath application of 1 µmol l^–1 concanamycin A in Na⁺-free PS inhibited pH_i recovery almost completely, by 96% (N=6, P<0.01; Fig. 3C,E). The pH_i recovered, however, within 5–10 min after concanamycin A washout in Na⁺-containing PS (Fig. 3C).

Finally, we examined whether an NHE was responsible for the Na⁺-dependent component of the pH_i recovery by using EIPA as an NHE-specific inhibitor. For these experiments, we applied the acidifying NH₄Cl pulse in the presence of 1 µmol l^–1 concanamycin A to inhibit V-ATPase-mediated outward H⁺ transport. NH₄Cl washout with concanamycin A- and EIPA-containing PS (50 µmol l^–1 EIPA) resulted in a pH_i recovery at a dramatically reduced rate (N=6, P<0.01; Fig. 3D,E). Moreover, the pH_i recovered only slowly after concanamycin A and EIPA washout (Fig. 3D).

	Discussion

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 
This is the first study addressing mechanisms of intracellular pH regulation in an insect salivary gland. We are particularly interested in whether the plasma membrane V-ATPase is involved in pH_i regulation. Therefore, we have studied pH_i regulation in the dopaminergically innervated salivary ducts of P. americana, because the duct cells have a V-ATPase in their apical membrane (Just and Walz, 1994a; Walz et al., 2006) and its functional significance is unknown. Our major results are that the neurotransmitter DA causes an intracellular acidification and that the V-ATPase and an NHE contribute to H⁺ extrusion after an acute DA- or NH₄Cl-induced acid load.

Steady-state pH_i and DA-induced pH_i changes
We have recorded a mean resting PD_b of –55±3 mV and a mean resting pH_i of 7.3±0.1 with double-barrelled pH-sensitive microelectrodes. These values indicate that H⁺ is not in equilibrium across the basolateral plasma membrane. The electrochemical driving force for H⁺ can be calculated by the equation:

where DF_H is the electrochemical driving force for H⁺, [H⁺]_o and [H⁺]_i are the extracellular and intracellular H⁺ concentrations, respectively, and PD_b is the basolateral membrane potential. Positive values for DF_H favour H⁺ influx. If the above values for PD_b and for [H⁺]_i and the known [H⁺]_o (=pH 7.4) are used, we obtain a strongly inwardly directed DF_H of +49.2 mV. Thus, the cells must actively regulate their pH_i even under resting conditions.

We were not able to construct our pH-sensitive microelectrodes as sharply as the double-barrelled K⁺- and Na⁺-sensitive microelectrodes that we used in a previous study of Periplaneta salivary duct cells (Lang and Walz, 2001). Therefore, we have recorded a PD_b that is 10 mV less than that previously obtained (–65 mV) (Lang and Walz, 2001). Assuming a leak at the site of impalement and given the above calculated inwardly directed driving force for H⁺, our pH_i is probably an underestimate but is sufficiently accurate as a quantitative supplement to our uncalibrated BCECF measurements.

The BCECF measurements have revealed that the inhibition of the apical V-ATPase with concanamycin A or the inhibition of the Na⁺-dependent acid extruder in Na⁺-free PS does not alter steady-state pH_i. If both acid extruders have a basal activity, one would have expected an acidification upon their inhibition. This result may reflect the low rate of acid loading in Periplaneta salivary ducts under resting conditions. Thus, V-ATPase and NHE seem to play a minor role in the regulation of steady-state pH_i but they are obviously important for pH_i recovery after acute intracellular acidification.

Bath application of the neurotransmitter DA causes the known depolarisation of the basolateral membrane (Lang and Walz, 2001) and, 20–30 s later, a reversible dose-dependent intracellular acidification of up to –0.4 pH units. The DA-induced acidification is half-maximal at 30 nmol l^–1 DA. This EC₅₀ value compares well with previous data. Half-maximal secretory responses in salivary glands of the cockroaches P. americana and Nauphoeta cinerea have been achieved at 110 nmol l^–1 and 88 nmol l^–1 DA, respectively (House and Smith, 1978; Just and Walz, 1996), and the DA-induced hyperpolarisation of acinar cells in the salivary glands of N. cinerea is half-maximal at 42 nmol l^–1 DA (Bowser-Riley and House, 1976).

We have not studied the H⁺ source(s) that is(are) responsible for the DA-induced acidification. However, we know that salivary duct cells contain carbonic anhydrase activity (Just and Walz, 1994b) and display dramatic changes in intracellular Na⁺, K⁺ and Ca²⁺ concentrations when they are stimulated with DA (Lang and Walz, 1999; Lang and Walz, 2001; Hille and Walz, 2006). These DA-induced changes in ion concentrations may result in increased cellular respiration, concomitant CO₂ production and a metabolic acid load. Thus, the acidification is probably a result of processes involved in saliva modification in the duct system. However, preliminary experiments indicate that increased metabolism is involved but not the only basis for the acidification. Thus, a more complex scenario in duct epithelial cells is proposed, and this will be the subject of a future study.

V-ATPase and a Na⁺-dependent transporter contribute to pH_i regulation after DA stimulation
A key result of the present work is that at least two acid extruders, viz the V-ATPase and an NHE, contribute to pH_i regulation after an acute acid load induced by a DA stimulus or an NH₄Cl pulse. The evidence for this is provided by the rate of pH_i recovery after an acid load being (1) reduced when the apical V-ATPase is specifically inhibited by bath application of the plecomacrolide antibiotic concanamycin A (Dröse et al., 1993; Dröse and Altendorf, 1997), (2) reduced in Na⁺-free PS, (3) almost completely blocked when the V-ATPase is inhibited with concanamycin A in Na⁺-free PS, and (4) partly reduced (after a DA-induced acidification) or almost completely reduced (after an NH₄Cl-induced acidification) when the V-ATPase is blocked with concanamycin A and the NHE is blocked simultaneously with the amiloride derivative EIPA (Petzel, 2000; Giannakou and Dow, 2001). Our observation that concanamycin A reduces the rate of pH_i recovery from a DA-induced acidification almost completely in Na⁺-free PS, but much less in the presence of EIPA, indicates that the duct cells must have an additional, as yet unidentified, Na⁺-dependent acid extruder that is only active after DA stimulation. Nevertheless, we cannot rule out the existence of an additional EIPA-insensitive NHE isoform activated by DA stimulation. Although NHE in Drosophila and Aedes Malpighian tubules was found to exhibit clear sensitivities to amiloride and its analogue EIPA (Petzel, 2000; Giannakou and Dow, 2001), the Aedes NHE3 was recently shown to be relatively insensitive to these drugs (Pullikuth et al., 2006).

The V-ATPase resides in the apical plasma membrane domain of the duct cells (Just and Walz, 1994a). Although we have applied the V-ATPase inhibitor concanamycin A to the bathing medium around the salivary gland preparation, concanamycin A nevertheless acts at the apical membrane. An effective inhibition of apical V-ATPase-mediated H⁺ pumping by bath application of concanamycin A has also been shown recently for blowfly salivary gland cells (Rein et al., 2006). Our finding that Na⁺-free PS mimics the pharmacological effect of EIPA indicates that the NHE is localised in the basolateral membrane of the duct cells. An exclusive basolateral localisation has also been shown for the vertebrate NHE1 isoform that is ubiquitously expressed in many epithelia (Orlowski and Grinstein, 1997; Wakabayashi et al., 1997).

The contribution of a V-ATPase to pH_i regulation has been described in several epithelia. In human eccrine sweat ducts, a V-ATPase is involved in pH_i recovery after NH₄Cl-induced acidification and probably acidifies the sweat in the duct lumen (Granger et al., 2002). An important role of V-ATPase in pH_i regulation has also been suggested for Drosophila Malpighian tubules (Bertram and Wessing, 1994), collecting ducts in rabbit kidneys (Yip et al., 2002), guinea pig inner ear (Stankovic et al., 1997) and rat hippocampal astrocytes (Pappas and Ransom, 1993).

NHE family members contribute to pH_i and cell volume regulation in many tissues, e.g. not only in vertebrate pancreatic ducts, colonic crypts and kidney collecting ducts, but also in insect Malpighian tubules (Stuenkel et al., 1988; Soleimani et al., 1994; Hasselblatt et al., 2000; Petzel, 2000).

Aspects of NHE and V-ATPase activation
NHE activity is regulated by pH_i. Its activity rises with decreasing pH_i, and the existence of a H⁺ binding site for the allosteric activation of NHE activity by internal H⁺ has been postulated (Wakabayashi et al., 1997). Thus, in many NHE-expressing cell types, this transporter is not involved in the regulation of resting pH_i (Tønnessen et al., 1990; Brokl et al., 1998; Tsuchiya et al., 2001) but becomes active after an acute acid load. As shown in rat sublingual mucous acini, for example, the rate of pH_i recovery after an NH₄Cl-induced acid load increases linearly with decreasing pH_i as a result of differences in NHE activity (Zhang et al., 1992).

Little is known about the mechanisms that mediate between a pH_i decrease and the activation of the V-ATPase. In kidney proximal tubules and collecting ducts, chronic metabolic acidification induced by bath applications of NH₄Cl or CO₂ promotes the recruitment of V-ATPases from a cytoplasmic vesicular pool to the apical membrane thereby increasing the rate of H⁺ secretion (Schwartz and Al-Awqati, 1985; Chambrey et al., 1994; Sabolic et al., 1997). However, this exocytosis-based process needs several minutes to hours to become effective and the detailed molecular mechanisms are largely unknown. Carraro-Lacroix and Malnic (Carraro-Lacroix and Malnic, 2006) have recently shown that angiotensin-II-stimulated H⁺ secretion via V-ATPase in kidney proximal tubule cells occurs by a protein kinase A-independent mechanism but that protein kinase C and cytosolic Ca²⁺ play a critical role in this process. Our laboratory has recently demonstrated that, in Calliphora salivary glands, serotonin stimulates bafilomycin-sensitive V-ATPase activity, the recruitment of the V-ATPase complex V₁ to the apical membrane, the assembly of the V-ATPase V₀V₁ holoenzyme at the apical membrane and, as a result, enhanced H⁺ transport across the apical membrane into the gland lumen (Dames et al., 2006; Rein et al., 2006; Zimmermann et al., 2003). The above effects are mediated by the serotonin-induced elevation of the intracellular cAMP concentration (Dames et al., 2006). Other studies on yeast and kidney epithelial cells have shown that V-ATPase binds directly to the glycolytic enzyme aldolase and that its activity is stimulated by glucose. This suggests a coupling of V-ATPase activity to glycolysis (Kane, 1995; Lu et al., 2001; Nakamura, 2004), perhaps to adjust V-ATPase activity to the level of intracellular acidification attributable to cellular metabolism to maintain pH_i homeostasis. Clear cells of the rat epididymis express high levels of V-ATPase at their apical pole for proton secretion that, in the vas deferens, is required for sperm maturation and storage (Brown and Breton, 1996). It has also been shown that V-ATPase accumulation is regulated by a bicarbonate-activated soluble adenylyl-cyclase-dependent increase in intracellular cAMP in response to alkaline luminal pH (Pastor-Soler et al., 2003). We have found in this study that the V-ATPase in Periplaneta salivary ducts is stimulated not only by the neurotransmitter DA, but also after an NH₄Cl-induced acid load. This strongly indicates that cell types are present in which a drop in pH_i is sufficient to stimulate V-ATPase-mediated H⁺ extrusion. The simplest explanation for this observation is that the V-ATPase displays higher rates of transport just because of the enhanced availability of protons as substrate.

	List of abbreviations

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 

BCECF/AM

2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester

DA

dopamine

EIPA

5-(N-ethyl-N-isopropyl)-amiloride

NHE

Na⁺/H⁺ exchange

PD_b

basolateral membrane potential

pH_i

intracellular pH

PS

physiological saline

V-ATPase

vacuolar-type H⁺-ATPase

	Acknowledgments

 
We are grateful to Helga Liebherr for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (GRK 837 `Functional Insect Science').

	Footnotes

 
^* Present address: University of Potsdam, Institute of Chemistry, Department of Physical Chemistry, 14476 Potsdam-Golm, Germany

	References

TOP Summary Introduction Materials and methods Results Discussion List of abbreviations References

 

Baumann, O., Dames, P., Kühnel, D. and Walz, B. (2002). Distribution of serotonergic and dopaminergic fibres in the salivary gland complex of the cockroach Periplaneta americana.BMC Physiol. 2,9 .[CrossRef][Medline]

Bertram, G. and Wessing, A. (1994). Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae. J. Comp. Physiol. B 164,238 -246.[CrossRef][Medline]

Beyenbach, K. W. and Wieczorek, H. (2006). The V-type H⁺-ATPase: molecular structure and function, physiological roles and regulation. J. Exp. Biol. 209,577 -589.[Abstract/Free Full Text]

Bleich, M., Köttgen, M., Schlatter, E. and Greger, R. (1995). Effects of NH₄⁺/NH₃ on cytosolic pH and and the K⁺ channels of freshly isolated cells from the thick ascending limb of Henle's loop. Pflügers Arch. 429,345 -354.[CrossRef][Medline]

Boron, W. F. (1986). Intracellular pH regulation in epithelial cells. Annu. Rev. Physiol. 48,377 -388.[CrossRef][Medline]

Boron, W. F. (2004). Regulation of intracellular pH. Adv. Physiol. Educ. 28,160 -179.[Abstract/Free Full Text]

Boron, W. F. and de Weer, P. (1976). Intracellular pH regulation in squid giant axons caused by CO₂, NH₃, and metabolic inhibitors. J. Gen. Physiol. 67,91 -112.[Abstract/Free Full Text]

Bowser-Riley, F. and House, C. R. (1976). The actions of some putative neurotransmitters on the cockroach salivary gland. J. Exp. Biol. 64,665 -676.[Abstract/Free Full Text]

Brokl, O. H., Martinez, C. L., Shuprisha, A., Abbott, D. E. and Dantzler, D. H. (1998). Regulation of intracellular pH in proximal tubules of avian long-looped mammalian-type nephrons. Am. J. Physiol. 274,1526 -1535.

Brown, D. and Breton, S. (1996). Mitochondria-rich, proton-secreting epithelial cells J. Exp. Biol. 199,2345 -2358.[Abstract]

Carraro-Lacroix, L. R. and Malnic, G. (2006). Signaling pathways involved with the stimulatory effect of angiotensin II on vacuolar H⁺-ATPase in proximal tubule cells. Pflügers Arch. Eur. J. Physiol. 452,728 -736.[CrossRef][Medline]

Chambrey, R., Paillard, M. and Podevin, R.-A. (1994). Enzymatic and functional evidence for adaptation of the vacuolar H⁺-ATPase in proximal tubule apical membranes from rats with chronic metabolic acidosis. J. Biol. Chem. 269,3243 -3250.[Abstract/Free Full Text]

Dames, P., Zimmermann, B., Schmidt, R., Rein, J., Voß, M., Schewe, B., Walz, B. and Baumann, O. (2006). cAMP regulates plasma membrane vacuolar-type H⁺-ATPase assembly and activity in blowfly salivary glands. Proc. Natl. Acad. Sci. USA 103,3926 -3931.[Abstract/Free Full Text]

Dröse, S. and Altendorf, K. (1997). Bafilomycins and concanamycins as inhibitors of V-ATPases and P-ATPases. J. Exp. Biol. 200,1 -8.[Abstract]

Dröse, S., Bindseil, K. U., Bowman, E. J., Siebers, A., Zeeck, A. and Altendorf, K. (1993). Inhibitory effect of modified bafilomycins and concanamycins on P- and V-type adenosine triphosphatases. Biochemistry 32,3902 -3906.[CrossRef][Medline]

Giannakou, M. E. and Dow, G. A. T. (2001). Characterization of Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family. J. Exp. Biol. 204,3703 -3716.[Abstract/Free Full Text]

Granger, D., Marsolais, M., Burry, J. and Laprade, R. (2002). V-type H⁺-ATPase in the human eccrine sweat duct: immunolocalization and functional demonstration. Am. J. Physiol. 282,1454 -1460.

Gupta, B. J. and Hall, T. A. (1983). Ionic distribution in dopamine-stimulated NaCl fluid-secreting cockroach salivary glands. Am. J. Physiol. 244,176 -186.

Hasselblatt, P., Warth, R., Schulz-Baldes, A., Greger, R. and Bleich, M. (2000). pH regulation in isolated in vitro perfused rat colonic crypts. Pflügers Arch. 441,118 -124.[CrossRef][Medline]

Heitzmann, D., Warth, R., Bleich, M., Henger, A., Nitschke, R. and Greger, R. (2000). Regulation of the Na⁺2Cl^–K⁺ cotransporter in isolated rat colon crypts. Pflügers Arch. 439,378 -384.[CrossRef][Medline]

Hille, C. and Walz, B. (2006). Dopamine-induced graded intracellular Ca²⁺ elevation via the Na⁺-Ca²⁺ exchanger operating in the Ca²⁺-entry mode in cockroach salivary ducts. Cell Calcium 39,305 -311.[CrossRef][Medline]

House, C. R. and Smith, R. K. (1978). On the receptors involved in the nervous control of salivary secretion by Nauphoeta cinerea Oliver. J. Physiol. Lond. 279,457 -471.[Abstract/Free Full Text]

Ianowski, J. P. and O'Donnell, M. J. (2006). Electrochemical gradients for Na⁺, K⁺, Cl^– and H⁺ across the apical membrane in Malpighian (renal) tubule cells of Rhodnius prolixus. J. Exp. Biol. 209,1964 -1975.[Abstract/Free Full Text]

Just, F. and Walz, B. (1994a). Immunocytochemical localization of Na⁺/K⁺-ATPase and V-H⁺-ATPase in the salivary gland of the cockroach, Periplaneta americana. Cell Tissue Res. 278,161 -170.[Medline]

Just, F. and Walz, B. (1994b). Localization of carbonic anhydrase in the salivary glands of the cockroach, Periplaneta americana. Histochemistry 102,217 -277.

Just, F. and Walz, B. (1996). The effects of serotonin and dopamine on salivary secretion by isolated cockroach salivary glands. J. Exp. Biol. 199,407 -413.[Abstract]

Kane, P. M. (1995). Disassembly and reassembly of the yeast vacuolar H⁺-ATPase in vivo. J. Biol. Chem. 270,17025 -17032.[Abstract/Free Full Text]

Lang, I. and Walz, B. (1999). Dopamine stimulates salivary duct cells in the cockroach Periplaneta americana.J. Exp. Biol. 202,729 -738.[Abstract]

Lang, I. and Walz, B. (2001). Dopamine-induced epithelial K⁺ and Na⁺ movements in the salivary ducts of Periplaneta americana. J. Insect Physiol. 47,465 -474.[CrossRef][Medline]

Lee, J. E., Nam, J. H. and Kim, S. J. (2005). Muscarinic activation of Na⁺-dependent ion transporters and modulation by bicarbonate in rat submandibular gland acini. Am. J. Physiol. 288,822 -831.

Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A. and Gluck, S. L. (2001). Interaction between aldolase and vacuolar H⁺-ATPase. J. Biol. Chem. 276,30407 -30413.[Abstract/Free Full Text]

Maddrell, S. H. P. and O'Donnell, M. J. (1992). Insect Malpighian tubules: V-ATPase action in ion and fluid transport. J. Exp. Biol. 172,417 -429.[Abstract/Free Full Text]

Madshus, I. H. (1988). Regulation of intracellular pH in eukaryotic cells. Biochem. J. 250, 1-8.[Medline]

Munoz, J. L., Deyhimi, F. and Coles, J. A. (1983). Silanization of glass in the making of ion-sensitive microelectrodes. J. Neurosci. Methods 8, 231-247.[CrossRef][Medline]

Nagaraja, T. N. and Brookes, N. (1998). Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes. Am. J. Physiol. 274,883 -891.

Nakamura, S. (2004). Glucose activates H⁺-ATPase in kidney epithelial cells. Am. J. Physiol. 287,97 -105.

Nishi, T. and Forgac, M. (2002). The vacuolar H⁺-ATPases – nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 3, 94-103.[CrossRef][Medline]

Orlowski, J. and Grinstein, S. (1997). Na⁺/H⁺ exchangers of mammalian cells. J. Biol. Chem. 272,22373 -22376.[Free Full Text]

Pappas, C. A. and Ransom, B. R. (1993). A depolarization-stimulated, bafilomycin-inhibitable H⁺ pump in hippocampal astrocytes. Glia 9, 280-291.[CrossRef][Medline]

Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R. and Breton, S. (2003). Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling. J. Biol. Chem. 278,49523 -49529.[Abstract/Free Full Text]

Petzel, D. H. (2000). Na⁺/H⁺ exchange in mosquito Malpighian tubules. Am. J. Physiol. 279,1996 -2003.

Pullikuth, A. K., Aimanova, K., Kang'ethe, W., Sanders, H. R. and Gill, S. S. (2006). Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti. J. Exp. Biol. 209,3529 -3544.[Abstract/Free Full Text]

Ramirez, M., Fernandez, R. and Malnic, G. (1999). Permeation of NH₃/NH₄⁺ and cell pH in colonic crypts of the rat. Pflügers Arch. 438,508 -515.[CrossRef][Medline]

Rein, J., Zimmermann, B., Hille, C., Lang, I., Walz, B. and Baumann, O. (2006). Fluorescence measurements of serotonin-induced V-ATPase-dependent pH changes at the luminal surface in salivary glands of the blowfly Calliphora vicina. J. Exp. Biol. 209,1716 -1724.[Abstract/Free Full Text]

Rietdorf, K., Lang, I. and Walz, B. (2003). Saliva secretion and ionic composition of saliva in the cockroach Periplaneta americana after serotonin and dopamine stimulation, and effects of ouabain and bumetamide. J. Insect Physiol. 49,205 -215.[CrossRef][Medline]

Rietdorf, K., Blenau, W. and Walz, B. (2005). Protein secretion in cockroach salivary glands requires an increase in intracellular cAMP and Ca²⁺ concentrations. J. Insect Physiol. 51,1083 -1091.[CrossRef][Medline]

Sabolic, I., Brown, D., Gluck, S. L. and Alper, S. L. (1997). Regulation of AE1 anion exchanger and H⁺-ATPase in rat cortex by acute metabolic acidosis and alkalosis. Kidney Int. 51,125 -137.[Medline]

Schwartz, G. J. and Al-Awqati, Q. (1985). Carbon dioxide causes exocytosis of vesicles containing H⁺ pumps in isolated perfused proximal and collecting tubules. J. Clin. Invest. 75,1638 -1644.[Medline]

Soleimani, M., Singh, G., Bizal, G. L., Gullans, S. R. and McAteer, J. A. (1994). Na⁺/H⁺ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. J. Biol. Chem. 269,27973 -27978.[Abstract/Free Full Text]

Stankovic, K. M., Brown, D., Alper, S. L. and Adams, J. C. (1997). Localization of pH regulating proteins H⁺ ATPase and Cl^–/HCO₃^– exchanger in the guinea pig inner ear. Hear. Res. 114, 21-34.[CrossRef][Medline]

Stuenkel, E. L., Machen, T. E. and Williams, J. A. (1988). pH regulatory mechanisms in rat pancreatic ductal cells. Am. J. Physiol. 254,925 -930.

Thomas, J., Buchsbaum, R., Zimniak, A. and Racker, A. (1979). Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18,2210 -2218.[CrossRef][Medline]

Tønnessen, T. I., Sandvig, K. and Olsnes, A. S. (1990). Role of Na⁺-H⁺ and Cl^–-HCO₃^– antiports in the regulation of cytosolic pH near neutrality. Am. J. Physiol. 258,1117 -1126.

Tsuchiya, Y., Hayashi, H. and Suzuki, Y. (2001). Na⁺-dependent recovery of intracellular pH from acid loading in mouse colonic crypt cells. Tohoku J. Exp. Med. 193,1 -11.[CrossRef][Medline]

Wakabayashi, S., Shigekawa, M. and Pouyssegur, J. (1997). Molecular physiology of vertebrate Na⁺/H⁺ exchangers. Physiol. Rev. 77, 51-74.[Abstract/Free Full Text]

Walz, B., Baumann, O., Krach, C., Baumann, A. and Blenau, W. (2006). The aminergic control of cockroach salivary glands. Arch. Insect Biochem. Physiol. 62,141 -152.[CrossRef][Medline]

Wieczorek, H. (1992). The insect V-ATPase, a plasma membrane proton pump energizing secondary active transport: Molecular analysis of electrogenic potassium transport in the tobacco hornworm midgut. J. Exp. Biol. 172,335 -343.[Abstract/Free Full Text]

Wieczorek, H., Brown, D., Grinstein, S., Ehrenfeld, J. and Harvey, W. R. (1999). Animal plasma membrane energization by proton-motive V-ATPases. BioEssays 21,637 -648.[CrossRef][Medline]

Yip, K.-P., Tsuruoka, S., Schwartz, G. J. and Kurtz, I. (2002). Apical H⁺/base transporters mediating bicarbonate absorption and pH_i regulation in the OMCD. Am. J. Physiol. 283,1098 -1104.

Zhang, G. H., Cragoe, E. J. and Melvin, J. E. (1992). Regulation of cytosolic pH in rat sublingual mucous acini at rest and during muscarinic stimulation. J. Membr. Biol. 129,311 -321.[Medline]

Zimmermann, B., Dames, P., Walz, B. and Baumann, O. (2003). Distribution and serotonin-induced activation of vacuolar-type H⁺-ATPase in the salivary glands of the blowfly Calliphora vicina. J. Exp. Biol. 206,1867 -1876.[Abstract/Free Full Text]

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    Twitter    What's this?

This article has been cited by other articles:

				C. Rotte, J. Witte, W. Blenau, O. Baumann, and B. Walz Source, topography and excitatory effects of GABAergic innervation in cockroach salivary glands J. Exp. Biol., January 1, 2009; 212(1): 126 - 136. [Abstract] [Full Text] [PDF]

				C. Hille and B. Walz Characterisation of neurotransmitter-induced electrolyte transport in cockroach salivary glands by intracellular Ca2+, Na+ and pH measurements in duct cells J. Exp. Biol., February 15, 2008; 211(4): 568 - 576. [Abstract] [Full Text] [PDF]

This Article Summary Figures Only Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hille, C. Articles by Walz, B. Search for Related Content PubMed PubMed Citation Articles by Hille, C. Articles by Walz, B. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB AMMONIUM CHLORIDE DOPAMINE Social Bookmarking                         What's this?