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Journal of Physiology (2001), 537.3, pp. 735-745
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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The parietal cell in the gastric gland of the mammalian stomach secretes HCl in response to diverse molecules released via neural, endocrine and paracrine pathways. ACh released from enteric nerve terminals in the vicinity of the parietal cell binds to muscarinic M3 receptors, and increases intracellular Ca2+ in the parietal cell via stimulation of phospholipase C (Chew & Brown, 1986). Although ACh releases histamine from enterochromaffin-like (ECL) cells in the amphibian (Rangachari, 1975; Ruiz & Michelangeli, 1984; Michelangeli et al. 1987), its action in the mammalian stomach remains a matter of controversy (Prinz et al. 1993; Lindstrom et al. 1997). Gastrin binds to cholecystokinin-B receptors on ECL cells, releasing histamine via a calcium-mediated pathway (Prinz et al. 1993; Sachs et al. 1997). The histamine released from ECL cells binds to a H2 receptor on the adjacent parietal cell, leading to an increase in cAMP (Chew et al. 1980). In addition, H2 receptor activation also increases [Ca2+]i in the rabbit gastric parietal cell (Chew, 1986; Michelangeli et al. 1989). Ca2+ and cAMP activate signalling cascades that result in the stimulation of the H+-K+-ATPase by a complex mechanism that is still only vaguely understood (Urushidani & Forte, 1997; Okamoto & Forte, 2001). Although each stimulus can by itself elicit acid secretion, the physiological response is the result of synergistic interactions.
Studies of acid secretion at the cellular level have been based on isolated oxyntic cells or glands in vitro (Berglindh & Obrink, 1976; Michelangeli, 1978). The measurement of secretory activity in these preparations has relied upon indirect indices of activation. These include oxygen consumption, extracellular pH transients, intracellular pH and morphological changes (Berglindh et al. 1976; Michelangeli, 1978; Thibodeau et al. 1994). Acridine orange fluorescence has been used to localize the site of acid secretion in isolated gastric glands (Berglindh et al. 1980). However, this method is not suitable for HCl secretion measurements in live cells or glands due to a high degree of quenching upon accumulation and binding to cellular structures.
A breakthrough in the estimation of acid secretion in gastric glands came with the introduction of the aminopyrine (AP) technique (Berglindh et al. 1980). AP is an weak amine base that can be protonated at acid pH and accumulates in acid spaces as a function of the pH gradient (Berglindh, 1984). Stimulation of acid secretion leads to an enhanced accumulation ratio of radioactively labelled AP (AP ratio). This index has been invaluable in our understanding of the mechanisms of activation of the parietal cell by secretagogues (Berglindh, 1990).
However, for more detailed studies at the cellular level and for kinetic studies, the AP ratio method presents many disadvantages, the main one being the lack of spatio-temporal resolution. The site of AP accumulation within the gland cannot be assessed and the rate of accumulation is rather slow once a pH difference has been established by stimulation, making kinetic studies of H+ transport activation rather difficult or impossible. Long incubation times are also necessary, requiring sampling periods of at least 5 min. In addition, only population studies can be made.
To gain insight into the kinetics of activation of the secretory process we have used Lysosensor Yellow-Blue (LYB), a new acidotropic fluorescent pH indicator. Lysosensor dyes have been used to stain acidic spaces such as lysosomes in cultured cells and secretory vesicles in endocrine cells (Hurwitz et al. 1997; Diwu et al. 1999; Blackmore et al. 2001). Recently, LYB has been used to visualize the lumen and secretory canaliculi of resting and histamine-stimulated isolated gastric glands (Athmann et al. 2000). However, these dyes have not been used to measure changes in organelle pH or acid secretion in 'real time'. In this paper, we characterize the fluorescence of this optical reporter molecule in the hydrogen-concentrating spaces of live gastric glands, and report the first measurements of the kinetics of activation of H+ transport by secretagogues in the lumen and secretory canaliculi in gastric glands in real time at 1 s resolution, with simultaneous measurements of [Ca2+]i.
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METHODS |
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Gland preparation and dye loading
Isolated rabbit gastric glands are long tubular structures consisting of several cell types, including parietal, chief, ECL and mucous cells. Glands were prepared by collagenase digestion as described previously (Michelangeli et al. 1989; Berglindh, 1990). All experiments were carried out in accordance with the guiding principles for the care and use of laboratory animals of the Bioethics Committee at IVIC (Ministry of Science and Technology, Venezuela). Briefly, New Zealand White rabbits were anaesthetized and killed by I.V. injection of pentobarbital (50 mg kg-1). The stomach was perfused through the descending aorta under high pressure. The mucosa was scraped from the underlying muscularis, minced, and digested with collagenase Type IA at 37 °C for 30 min. Glands were separated by sedimentation and resuspended in Ringer medium (RM). For experiments involving simultaneous measurements of [Ca2+]i and H+ secretion, gland suspensions were incubated for 30 min at room temperature with 1 µM LYB and 5 µM Fluo-3 AM.
Fluorimetric measurements and imaging
The emission and excitation spectra of LYB in solution at different pH values, and LYB fluorescence of loaded gland suspensions were obtained using a spectrofluorimeter (Photon Technology International) equipped with a magnetic stirrer, temperature control and computer data acquisition. Fluorescence imaging and photometry of stained glands were carried out using a fluorescence microscope (Nikon TE-300) equipped with a cooled CCD camera (Newcastle Photometric Systems), shutters and a filter wheel for excitation wavelength changes. Glands stained with LYB and/or Fluo-3 were mounted on polylysine-coated coverslips and placed in a flow-through perfusion microscope chamber. Fluorescence was measured from up to 16 regions of interest at 16 bit resolution. Images were recorded at 8 bit resolution. All measurements were made at room temperature. Fluorescence data were normalized according to the equation: 
F/F0 = (F - Fb)/(Fb - background), where Fb is the fluorescence just before secretagogue addition.
Solutions and materials
Buffer solutions of different pH values were prepared by mixing different proportions of two solutions containing 50 mM citric acid + 150 mM NaCl, or 50 mM sodium citrate. Buffer pH was determined with an electrode. RM contained (mM): 145 NaCl, 5 KCl, 1 MgSO4, 1.8 CaCl2, 11 glucose, 20 Hepes; pH 7.45, 310 mosmol kg-1. LYB and Fluo-3 AM were purchased from Molecular Probes. All other reagents were from Sigma.
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RESULTS |
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Spectral characteristics of the proton-sensitive probe LYB
LYB is a pyridyl oxazole with an alkyl amino side chain (Diwu et al. 1999; Fig. 1A). The only groups capable of being protonated with a predicted pKa between 0 and 14 are the pyridyl and the alkyl amino groups, with values of 3.2 and 8.8, respectively (predictions were obtained using the ACD/I-Lab service). The protonation of either group would lead to an accumulation of the dye in glandular acid spaces in the presence of a pH gradient.
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Figure 1. Fluorescence of Lysosensor Yellow-Blue (LYB) as a function of pH A, structure of LYB showing the two groups capable of being protonated with predicted pKa values. B and C, LYB (1 µM) emission and excitation spectra, respectively, at different pH values. a.u., arbitrary units. D and E, normalized fluorescence at excitation (Exc) or emission (Ems) maxima, respectively, plotted against pH. Boltzmann fits gave an average pKa value of 4.2. F, fluorescence of 0.5 mM LYB as a function of pH. Since it was difficult to obtain this concentration in a cuvette, LYB solutions at pH 2.2, 4.3 and 7.7 were placed on a coverslip and the 530 nm fluorescence was measured with a microscope using excitation at 340 and 380 nm. | ||
The fluorescence of 1 µM LYB changed with pH, as shown in the excitation and emission spectra in Fig. 1B and C. Maximal fluorescence values were plotted against pH. Boltzmann fits obtained from the excitation and emission spectra gave a single pKa of around 4.2 (Fig. 1D and E). This value is close to that predicted for the pyridyl group, suggesting that this group is responsible for the pH-dependent change, and is in accordance with previously described effects of chemical modification on fluorescence (Diwu et al. 1999). Excitation or emission ratios could be used to calculate the pH in acid spaces, provided the spectral characteristics do not change upon accumulation. With a pH gradient between the extracellular space and the gland lumen of 3 pH units in the resting state (Athmann et al. 2000), the dye would reach an estimated concentration of 1 mM for 1 µM external dye. Therefore, we studied the pH dependence of fluorescence at high concentration. At a concentration of LYB of 0.5 mM there was a strong increase in fluorescence for both excitation wavelengths (340 and 380 nm) between pH 4.3 and 2.2 (Fig. 1F). The increase observed at 340 nm did not follow the behaviour predicted by the spectra obtained at low dye concentration (see Fig. 1B). In the pH range 4.3-7.7, the dye became rather insensitive to pH at high concentration. This was found to be due to concentration-dependent quenching at pH values between 7.7 and 4.3, but not at lower values (data not shown).
Accumulation and response of LYB in gastric glands
The kinetics of dye accumulation and response in isolated gland suspensions was studied with the aid of a spectrofluorimeter and in single glands with a microscope. Addition of 1 µM LYB to a cuvette containing a suspension of isolated gastric glands resulted in a step increase in fluorescence excited at 325 nm (Fig. 2A). This corresponds to the fluorescence of LYB at pH 7.4 in the extracellular medium. Immediately, the fluorescence at 325 nm started to decrease, with a concomitant increase in the fluorescence at 388 nm. It can be concluded from the spectra (Fig. 1B) that the dye was moving from the extracellular space into an acidic compartment within the gland. After 5 min, the fluorescence at 388 nm reached a maximum, whereas that at 325 nm continued to decrease, indicating that the dye continued to accumulate in acidic spaces.
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Figure 2. Uptake of LYB by gastric glands and response to forskolin stimulation A, time course of the change in fluorescence brought about by the addition of 1 µM LYB to a gland suspension, measured in a spectrofluorimeter. Excitation (Exc) alternated between 325 and 388 nm; emission was set at 530 nm. Forskolin (Forsk; 2.5 µM) and nigericin (Nig; 5 µM) were added as indicated. a.u., arbitrary units. B and C, emission and excitation spectra, respectively, of LYB accumulated in glands, taken before and after forskolin stimulation and after nigericin addition, as indicated by the asterisks in A. Fluorescence spectra of LYB in buffer are shown in the background as a reference. D, increase in LYB fluorescence after the addition of forskolin to a single gland, measured with the aid of a microscope. Glands were perfused continuously with Ringer medium (RM) containing 1 µM LYB. Fluorescence excitation was alternated between 340 and 380 nm; the emission filter selected for 510-530 nm. Traces are from selected areas representing the gland lumen and a parietal cell (PC), as indicated. E, fluorescence images (380 nm) taken at the times (min) shown in D (indicated by numbers) showing the accumulation of LYB in the intracellular canaliculi of parietal cells and in the gland lumen. An increase in fluorescence intensity and lumen volume was observed after forskolin stimulation, which disappeared after addition of nigericin. Scale bar, 15 µm. | ||
As forskolin is a potent stimulant of acid secretion (Chew, 1983), we used it to characterize the dye response after its accumulation. Forskolin induced an increase in fluorescence at 388 nm with a delay of about 2 min. This was accompanied by a small increase at 325 nm, as predicted for high dye concentrations. Dissipation of the pH gradient with the H+-K+ exchanger nigericin decreased the fluorescence at 388 nm with a concomitant increase at 325 nm, due to unprotonated dye release to the extracellular medium. Excitation and emission spectra of the LYB that had accumulated in the isolated gland suspension before and after stimulation with forskolin, and after addition of nigericin, during these experiments are shown in Fig. 2B and C, respectively. The fluorescence spectra of LYB in buffer are shown in the background as a reference. In resting or stimulated glands, both fluorescence spectra at the isosbestic points were lower than the values obtained in buffer solution. This may be attributable to quenching by concentration. Spectra obtained after exposure to nigericin closely followed the spectra of dye in buffer at pH 7.4, due to dilution of the dye in the bath solution. Comparison of resting vs. stimulated glands showed changes in the spectra only at wavelengths above the isosbestic points. This fact precludes ratiometric calibration of the accumulated dye as a function of actual pH. However, stimulation of acid secretion can be monitored as changes in fluorescence excited at 380 nm.
A similar experiment was performed using a fluorescence microscope (Fig. 2D). The trace shows LYB fluorescence of a luminal region and a parietal cell of a single gland at excitation wavelengths of 340 and 380 nm, under continuous dye perfusion. After dye equilibration, forskolin stimulation provoked an increase in the fluorescence excited at 380 nm, starting about 2 min following stimulation and reaching a maximum after 10-15 min. The change at 340 nm was small and similar to that observed in the gland suspension. Images obtained before and after stimulation with forskolin (at 380 nm) showed that LYB accumulated exclusively in the intracellular canalicular system of parietal cells and the gland lumen (Fig. 2E). After stimulation, fluorescence increased along the entire length of the gland lumen, and to a lesser extent, in some parietal cells. Addition of nigericin reduced the fluorescence to near zero at both wavelengths due to release of the dye from the gland and washout by the superfusion fluid (an animated sequence of all images of this experiment (video 1) is presented as supplementary material at The Journal of Physiology online; http://www.jphysiol.org/cgi/content/full/537/3/735). In other experiments, when the gland was preloaded with LYB (30 min) and then washed free of extracellular dye by continuous perfusion, the dye remained in the same spaces within the gland for up to 24 h. Subsequent stimulation with forskolin revealed a similar pattern of fluorescence change at the temporal and spatial level (see below).
These results indicate that LYB senses pH changes in the intracellular canaliculi of parietal cells and in the gland lumen of the isolated gastric gland. The strong and fast changes in fluorescence at 380 nm due to acidification can be used to measure the kinetics of pH changes in the acidic compartments of living cells, which would be impossible to obtain using methods based on AP accumulation.
Simultaneous measurement of [Ca2+]i and carbachol-stimulated gastric acid secretion
The spectral properties of LYB, with a useful excitation at 380 nm and emission at 530 nm, enable the measurement of acid changes together with changes in Ca2+ using Fluo-3 excited at 490 nm. In all subsequent experiments, isolated glands were preloaded with both Fluo-3 and LYB and then washed by perfusion in the microscope chamber. Figure 3 shows the time course of changes in [Ca2+]i and H+ secretion in a single gastric gland, during stimulation with carbachol. Traces corresponding to Fluo-3 and LYB fluorescence from a single representative parietal cell and LYB fluorescence in the adjacent lumen are presented in Fig. 3A. Images of double-stained glands show that Fluo-3 stained predominantly parietal cells, whereas LYB stained the intracellular canaliculi of parietal cells and the gland lumen (Fig. 3B). Carbachol stimulation produced a transient increase in [Ca2+]i that peaked and descended to a plateau that was higher than the resting level of fluorescence, as long as the stimulant was present. Peak [Ca2+]i was reached about 11 s after the initiation of the signal rise (10.8 ± 0.5 s, mean ± S.E.M.; n = 48 cells; 8 glands from 8 animals). Photometry showed similar and synchronous Ca2+ responses in all parietal cells. As shown widely in parietal cells, these fluorescence changes correspond to an initial Ca2+ release from intracellular stores and subsequent Ca2+ influx through the basolateral membrane (Chew, 1986; Negulescu & Machen, 1988; Michelangeli et al. 1989).
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Figure 3. Carbachol stimulation rapidly increases [Ca2+]i and H+ secretion in single gastric glands Glands were double-stained with LYB and Fluo-3 and then washed by perfusion with RM. Fluorescence was excited at 387 and 500 nm for LYB and Fluo-3, respectively, and recorded at 530 nm. The H2 receptor antagonist cimetidine (100 µM) was present throughout the experiment. A, normalized Fluo-3 and LYB fluorescence of a single representative parietal cell and LYB fluorescence in the adjacent luminal region during carbachol (CCh; 10 µM) stimulation, as indicated. B, LYB (top) and Fluo-3 (bottom) pseudocoloured images taken at the time points (s) indicated by the numbers in A. Scale bar, 10 µm. C, amplification of time scale during the rise phase indicated in A. Acid in the lumen was detected 3 s after the initiation of the [Ca2+]i rise recorded in an adjacent parietal cell. D, derivative of the LYB signal as a function of time superimposed on the Ca2+ signal. Representative experiment (see Table 1). | ||
The fluorescence of LYB in the gland lumen increased promptly after stimulation by carbachol; the lumen also appeared to increase in volume (Fig. 3B, arrow). The effect of carbachol on LYB fluorescence in parietal cells was barely detectable, although significant. The averaged results of acid accumulation in cell and luminal regions from eight glands isolated from eight animals are presented in Table 1. Acid in the lumen was detected as early as 3 s after the initiation of the [Ca2+]i rise recorded in an adjacent parietal cell (Table 1), much earlier than the time at which peak [Ca2+]i was attained (Fig. 3C). Under the time resolution (1 point s-1) conditions of this study, the activation of acid secretion appeared to be synchronous in all luminal regions and in parietal cells. The maximal LYB fluorescence signal in the lumen was reached about 40 s after stimulation (Table 1), when [Ca2+]i had already peaked and decreased to the plateau level. After removal of carbachol from the perfusion fluid, Fluo-3 fluorescence returned to near-basal levels, whereas LYB fluorescence remained relatively stable. The first derivative of the LYB signal is a function of the rate of net acid accumulation in a given space (Fig. 3D). This derivative was transient, even under conditions of continuous stimulation. This may be the result of a short transient pulse of acid induced by carbachol, concomitant with the Ca2+ release phase of the spike. Alternatively, the sustained level of LYB fluorescence could be the result of a new steady state achieved with continuous secretion and bulk flow of acid or diffusion from the measured space. In fact, in some experiments the LYB signal declined with time, suggesting that these phenomena contribute to the overall signal in specific areas. It is worth noting that the carbachol response (Ca2+ and H+) was similar in the presence or absence of cimetidine, supporting the notion that endogenous histamine does not play a significant role in the carbachol-induced response under these conditions (Lindstrom et al. 1997; Athmann et al. 2000).
Stimulation with short pulses of carbachol elicits repetitive [Ca2+]i and H+ secretion responses
To investigate whether carbachol-stimulated secretion was transient or reflected a new steady state, we stimulated gastric glands with a sequence of short-duration carbachol pulses and simultaneously recorded [Ca2+]i in parietal cells and H+ accumulation in the lumen. Figure 4 (top panel) shows representative traces of Fluo-3 fluorescence in a parietal cell and LYB fluorescence in an adjacent luminal area of a single gastric gland. Repetitive and short (20 s) stimulations with carbachol produced repetitive responses in Fluo-3 and LYB fluorescence. Each carbachol pulse induced a transient increase in [Ca2+]i. This type of response has been described before, and was explained by the release of Ca2+ from intracellular stores during stimulation and their recharge during the interstimulus period (Negulescu & Machen, 1988). LYB fluorescence increased in steps with each stimulation, each time attaining a new level that was higher than that obtained with the previous stimulation. The LYB response following the first stimulation was characterized by a rapid increase, attaining a peak, which was followed by a decrease to a sustained level that was higher than basal. This biphasic response was observed in about half of the luminal areas in this series of experiments. In subsequent stimulations, the decrease during the second phase was largely attenuated. The first derivative of the LYB fluorescence change (Fig. 4, bottom panel) revealed repetitive transients associated with each stimulation. These transients were temporally related to the release phase of the Ca2+ spike, as shown above for a single and sustained stimulation (Fig. 3D). In all luminal areas, the derivative of the LYB signal corresponding to the first stimulation was higher than those that followed, as was the [Ca2+]i response.
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Figure 4. Repetitive stimulation with carbachol elicits repetitive responses in [Ca2+]i and H+ secretion General experimental details are as given in Fig. 3. Gastric glands were continuously perfused with RM and stimulated at the indicated times with 10 µM carbachol (CCh). Top, representative traces of Fluo-3 fluorescence recorded in a parietal cell and LYB fluorescence in an adjacent luminal region. Bottom, first derivative of the LYB signal as a function of time. The traces are representative of 30 pairs of luminal and parietal cell areas obtained from 3 glands, isolated from 3 animals. | ||
These experiments indicate that each stimulation with carbachol produced a transient accumulation of acid within the lumen of the gland that was closely associated with Ca2+ release from stores. The decrease in LYB fluorescence, observed especially during the first stimulation, may have diverse explanations. Bulk flow out of the gland due to a rise in hydrostatic pressure following HCl and water secretion may lead to a decrease in fluorescence. However, this would drag the fluorophore out of the gland, precluding any further LYB response. The intraluminal bulk movement of fluid and/or diffusion from the selected area into the contiguous one may also account for some of this decrease during the first pulse. In fact, this can be observed in some cases when all areas are jointly analysed. Some back-flux of H+ may occur, which would decrease luminal acidity. If this were the case, the rate of back-flux should increase with the gradient, which was not observed during the experiment involving repetitive stimulations. Another possible explanation for the decrease in fluorescence may be the secretion of alkali and/or proteins, such as pepsinogen and mucins, from chief and other non-parietal cells that would buffer H+ in the lumen (Koelz et al. 1982; Allen et al. 1993).
The steady level attained after the rapid rise during carbachol stimulation may reflect the contribution of a steady state where some or all of the above-mentioned factors may operate. However, the step rise in fluorescence following repetitive carbachol stimulation strongly suggests the activation (and deactivation) of a transient process.
Activation of the gastric secretory process by histamine and forskolin
Histamine is a powerful cAMP-mediated stimulant of acid secretion by the isolated gastric gland (Chew et al. 1980; Berglindh, 1990). It has also been shown to increase [Ca2+]i in gastric glands and parietal cells (Chew, 1986; Michelangeli et al. 1989; Ljungstrom & Chew, 1991). Figure 5A shows recordings of [Ca2+]i and acid secretion in two representative cells and adjacent gland lumina (a and b) under stimulation by 100 µM histamine. A trace showing LYB fluorescence obtained from the entire gland area was also included. Parietal cells within the gland responded to histamine stimulation with non-synchronous [Ca2+]i oscillations (Fig. 5A), as has been reported previously (Ljungstrom & Chew, 1991). The oscillations were rather complex, but in general had a frequency of about 1 cycle min-1, with an irregular period, and were attenuated during the experiment under the continuous presence of histamine. In some parietal cells, the response was somewhat different, exhibiting a large peak of [Ca2+]i increase followed by attenuated oscillations.
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Figure 5. Simultaneous measurements of [Ca2+]i and H+ secretion during stimulation by histamine General experimental details are as given in Fig. 3. Gastric glands were continuously perfused with RM and stimulated with 100 µM histamine (Hist). A, recordings of Fluo-3 and LYB fluorescence in two representative parietal cells and adjacent gland lumina (a and b) as indicated in the LYB images in B. A trace (black) of LYB fluorescence from the entire gland is included. B, LYB (top) and Fluo-3 (bottom) images taken during stimulation with histamine at the indicated times (min). The initiation of acid secretion in different cells is indicated by arrows. Representative experiment (see Table 1). Scale bar, 10 µm. | ||
Acid secretion started in parietal cells between 30 and 300 s after the initiation of the [Ca2+]i rise (Table 1). In contrast to the situation with carbachol, the activation of H+ secretion by histamine in parietal cells was not synchronous in all cells within a gland. This is shown in the selected traces and in the image sequence (Fig. 5B, arrows; see also video 2 published as supplementary material at The Journal of Physiology online; http:/www.jphysiol.org/cgi/content/full/537/3/735). Increases in LYB fluorescence intensity and the volume of the intracellular canaliculi in parietal cells and gland lumen were large and progressive. As secretion in all cells did not start synchronously, there may be luminal regions in which fluorescence increases before that in adjacent parietal cells, due to bulk fluid movement along the gland lumen (Fig. 5Ab). Conversely, the rate of fluorescence increase stimulated by histamine was sustained both in the lumen and in the parietal cell. Maximal LYB fluorescence in the lumen was reached between 240 and 600 s after stimulation (Table 1). These changes were significantly larger and slower that those stimulated by carbachol. Moreover, there were no increases in LYB fluorescence associated with the spikes during [Ca2+]i oscillations, as was observed with [Ca2+]i transients obtained with repetitive carbachol stimulations.
The responses observed following direct stimulation of adenyl cyclase by forskolin were reminiscent of the effects of histamine (Fig. 6). Parietal cells responded to forskolin stimulation (5 µM) with increases in [Ca2+]i following a non-synchronous oscillatory pattern (Fig. 6A). In this particular cell, the response consisted of a large peak [Ca2+]i increase followed by attenuated oscillations. Acid secretion was detected at about the same time in parietal cells and the gland lumen, with a delay of about 80 s after the initiation of the [Ca2+]i rise (Table 1). As observed with histamine, the activation of H+ secretion by forskolin in parietal cells was not synchronous in all cells within a gland. Increases in LYB fluorescence intensity and the volume of the intracellular canaliculi and gland lumen were large and progressive, with a sustained rate (Fig. 6). Maximal LYB fluorescence in the lumen was reached about 500 s after stimulation (Table 1). These changes were significantly larger than those stimulated by carbachol, but were not different from values obtained with histamine stimulation. As observed with histamine, there were no increases in LYB fluorescence associated with the spikes observed during [Ca2+]i oscillations elicited by forskolin.
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Figure 6. Simultaneous measurements of [Ca2+]i and H+ secretion during stimulation by forskolin General experimental details are as given in Fig. 3. Gastric glands were continuously perfused with RM and stimulated with 5 µM forskolin (Forsk). A, normalized Fluo-3 and LYB fluorescence of a single representative parietal cell and LYB fluorescence in the adjacent luminal region during forskolin stimulation, as indicated. A trace (black) of LYB fluorescence from the entire gland is included. B, pseudocoloured LYB fluorescence images taken at the indicated times (min) during the experiment. Representative experiment (see Table 1). Scale bar, 10 µm. | ||
In some cases, stimulation with forskolin or histamine resulted in an increase in gland volume to a point at which the lumen suddenly opened at one end and the secreted fluid containing the fluorophore was released. In this case, fluorescence of LYB, but not Fluo-3, dropped rapidly to a level that was much lower than basal (data not shown).
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DISCUSSION |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our experiments reveal for the first time the kinetics of activation, at 1 s resolution, of H+ secretion by the parietal cell in relation to changes in [Ca2+]i elicited by carbachol and histamine. We characterized the dynamic responses of LYB that were used to sense pH changes in the intracellular canaliculi and lumen of gastric glands. LYB accumulates exclusively in acid spaces, with good retention and no binding to cellular structures. Once accumulated, the dye can be washed out of the extracellular space and its pH-dependent fluorescence followed in specific areas of the gland in real time. In addition, LYB could possibly be used with other fluorescein-like indicators to study the coupling of H+ transport with other intracellular events such as membrane fusion, cytoskeletal dynamics, changes in Ca2+ in intracellular stores and cytoplasmic pH, for example, in gastric glands and in a variety of animal and plant cells.
The signal transduction cascade in the parietal cell involves second-messenger changes, protein phosphorylation, cytoskeletal dynamics, ion channel activation, membrane fusion and translocation. Activation of the gastric H+ pump by these processes results ultimately from the availability of substrate, K+ on the luminal side, and an increase in the number of pumps exposed by membrane translocation (Urushidani & Forte, 1997; Agnew et al. 1999; Okamoto & Forte, 2001). This series of phenomena occur on a yet unknown time scale. With our simultaneous measurements of LYB and Fluo-3 fluorescence, we found striking differences in the kinetics of activation of H+ transport induced by cholinergic and histaminergic agonists and their relationship to the changes in [Ca2+]i.
The stimulation of secretion by carbachol reported in this study occurs much faster and is shorter in duration than that reported using the AP ratio method. Maximal AP accumulation was observed after 15 min of stimulation and declined with time (Berglindh et al. 1976). Accumulation of a weak base such as AP (or LYB) in acid spaces is rather slow once a pH gradient has been established. The time course of LYB accumulation shown in Fig. 2 indicates that it may take as long as 15 min for the weak base to accumulate in an acid space. The kinetics of AP accumulation in gastric glands may reflect the rate of permeation of AP rather than the rate of secretion, which is shown here to take place in a few seconds.
Sustained stimulation by carbachol showed a fast increase in LYB fluorescence that reached a plateau within the 1st minute. This stable level of fluorescence may be due to a transient secretion or may reflect a steady state, with continuous secretion and output occurring by diffusion or bulk flow. The experiment shown in Fig. 4 indicates that each stimulation with carbachol produced a transient accumulation of acid within the lumen of the gland. The step pattern of LYB fluorescence (acid accumulation) appears to correspond to the transient pulses of acid secreted.
The parallelism between the Ca2+ release component of the spikes and the transient increases in H+ transport into the lumen strongly suggests that secretion is triggered by Ca2+ release. It has been demonstrated that cholinergic agonists elicit inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ mobilization from intracellular stores, with a secondary increase in Ca2+ permeability (Chew & Brown, 1986; Berridge, 1997). The Ca2+ response appears to be essential for H+ secretion as both are blunted by [Ca2+]i buffering with BAPTA (Michelangeli et al. 1989; Negulescu et al. 1989). Our experiments show clearly a relationship between the increase in [Ca2+]i and the onset of H+ secretion during single or repetitive cholinergic stimulation, with very fast activation kinetics. It is worth pointing out that acid secretion under carbachol stimulation was observed clearly in the lumen, whereas a barely detectable increase was measured in the cell. This may have been due to the fact that the signal in the lumen is the result of contributions from many individual parietal cells, in which secretion may have not been detectable.
Histamine stimulation induced an increase in [Ca2+]i that followed a complex oscillatory pattern, confirming earlier reports (Ljungstrom & Chew, 1991). This response may not be related to increases in IP3 concentration (Chew & Brown, 1986). Oscillations may be coupled to changes in cAMP and protein kinase A phosphorylation of ion channels in the plasma membrane or the endoplasmic reticulum (Joseph & Ryan, 1993; Bugrim, 1999). An activator of adenyl cyclase, forskolin, also induced oscillations similar to those elicited by histamine, suggesting that the effect of histamine on [Ca2+]i is mediated by cAMP.
On the other hand, histamine and forskolin do not trigger a fast acid response, as observed with carbachol, even though they both induce elevations of [Ca2+]i and cAMP (Chew et al. 1980; Chew, 1986; Michelangeli et al. 1989). In contrast, H+ secretion stimulated by histamine and forskolin had a delayed onset, a larger and more sustained response, and no temporal relationship with the [Ca2+]i oscillations. Although in many experiments a large [Ca2+]i transient was observed following stimulation with histamine or forskolin, we never observed an acid secretion response that was as fast as that with carbachol. Therefore [Ca2+]i and acid secretion may be temporally coupled during cholinergic stimulation, but uncoupled during histamine stimulation. However, Ca2+ may play a role in histamine-stimulated secretion on another time scale, since BAPTA also reduces the AP response in isolated gastric gland suspensions (Michelangeli et al. 1989; Negulescu et al. 1989).
The [Ca2+]i changes evoked by carbachol and histamine may have a different spatial distribution and may be regulated locally in relation to the H+-K+ pumps. It has been shown recently that, after histamine stimulation, the Ca2+ signal was first evident at the basolateral region of parietal cells, followed by a faint wave spreading to the apical pole (Athmann et al. 2000). On the other hand, cholinergic stimulation of pancreatic acinar cells caused Ca2+ release mostly in the apical region (Thorn et al. 1993; Xu et al. 1996). This might also occur in the parietal cell.
The striking differences in the kinetics of activation of H+ secretion by the two classes of secretagogues indicate that two distinct mechanisms are operating during the final stimulation of the pump, in spite of both producing a [Ca2+]i response. Those changes occurring in a differential spatio-temporal domain may trigger secretion through distinct activation cascades and/or physical structures. We hypothesize that by increasing apical [Ca2+]i, cholinergic agonists activate pumps that are already present in the apical membrane, while histamine activates secretion by a slower mechanism involving the cAMP-dependent translocation of pumps to the intracellular canaliculus.
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Acknowledgements
This research was supported by CONICIT (Venezuela), grant S1-95000-519. The helpful and encouraging comments of two anonymous reviewers are gratefully acknowledged.
Supplementary material
The online version of this paper (http://www.jphysiol.org/cgi/content/full/537/3/735) contains supplementary material entitled: 'Animated sequence of images of H+ secretion in a gastric gland stimulated by forskolin' (video 1) and 'Animated sequence of images of [Ca2+]i and H+ secretion in a gastric gland stimulated by histamine' (video 2).
Corresponding author
F. Michelangeli: CBB-IVIC, PO Box 21827, Caracas 1020 A, Venezuela.
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