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J Physiol. Feb 1, 2002; 538(Pt 3): 891–899.
PMCID: PMC2290104

Coupling of vasopressin-induced intracellular Ca2+ mobilization and apical exocytosis in perfused rat kidney collecting duct

Abstract

Arginine vasopressin (AVP) regulates the osmotic water permeability of the kidney collecting duct by inducing exocytotic insertion of aquaporin-2 into apical membrane. The coupling between AVP-induced intracellular Ca2+ mobilization and apical exocytosis was investigated in isolated perfused rat inner medullary collecting duct (IMCD) segments using confocal fluorescence microscopy. Changes of [Ca2+ ]i in IMCD cells were measured with fluo-4. A novel confocal imaging technique using a styryl dye, FM1-43, was developed to monitor real-time exocytosis induced by arginine vasopressin. AVP (0.1 nm) triggered a rapid increase of [Ca2+]i in IMCD cells, followed by sustained oscillations. Ratiometric measurement of [Ca2+]i confirmed that the observed [Ca2+]i oscillation was a primary event and was not secondary to changes in cell volume. The frequencies of [Ca2+]i oscillations in each IMCD cell were independent and time variant. 1-Deamino-8-d-arginine vasopressin (a V2 receptor agonist, 0.1 nm) simulated the effects of AVP by triggering [Ca2+]i oscillations. In the absence of extracellular Ca2+, ryanodine (0.1 mm) inhibited AVP-induced Ca2+ mobilization. AVP (0.1 nm) triggered accumulative apical exocytosis in IMCD cells within 20 s after application. Pre-incubating the IMCD with an intracellular Ca2+ chelator, BAPTA, prevented AVP-induced intracellular Ca2+ mobilization, apical exocytosis, and increase of osmotic water permeability. These results indicate that AVP, via the V2 receptor, triggers a calcium signalling cascade observed as [Ca2+]i oscillations in the IMCD and that intracellular Ca2+ mobilization is required for exocytotic insertion of aquaporin-2.

Arginine vasopressin (AVP) regulates the osmotic water permeability (Pf) of renal collecting duct, conferring the precise control of renal excretion of water. AVP increases Pf in collecting ducts by triggering translocation and insertion of aquaporin-2 (AQP2) to the apical membrane of principal cells in the collecting duct (Wade et al. 1981; Nielsen et al. 1995a). These responses depend on the binding of AVP to basolateral V2 vasopressin receptors in collecting duct cells, which leads to the activation of adenylyl cyclase and an increase of cyclic AMP level in the cells (Chabardes et al. 1996). Cyclic AMP-dependent protein kinase A is then activated, which subsequently phosphorylates AQP2 at Ser256 (Nishimoto et al. 1999). However, the steps that lead to the eventual insertion of AQP2 into apical membrane are not known. A number of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which are known to regulate exocytosis of synaptic vesicles, have been identified in renal collecting duct (Sollner et al. 1993). These include syaptobrevin-2 (Nielsen et al. 1995b), synaptotagmin VIII (Kishore et al. 1998), syntaxin-4 (Mandon et al. 1996) and SNAP23 (Inoue et al. 1998). It was hypothesized that the trafficking and targeting of AQP2 vesicles might resemble that of synaptic vesicles (Knepper & Inoue, 1997). Studies of a wide variety of vesicular-trafficking processes have pointed to a key role for localized increases in intracellular Ca2+ concentration ([Ca2+]i) in triggering the fusion of vesicles to their target membranes (Burgoyne & Morgan, 1998). This raises the possibility that the exocytotic insertion of AQP2 is also Ca2+ dependent. Studies from other investigators showed that 10 nm AVP was required to induce a detectable increase of [Ca2+]i in perfused inner medullary collecting duct (IMCD) (Star et al. 1988). However, 0.01–0.1 nm AVP is sufficient to increase cyclic AMP production and to induce a submaximal increase of Pf in IMCD (Star et al. 1988). Detection of Ca2+ mobilization from a perfused IMCD based on conventional fluorescence imaging might be compromised by out-of-focus fluorescence and asynchronous [Ca2+]i oscillations in individual cells. Conventional methods for measuring changes in Pf of a perfused IMCD also do not provide sufficient spatial and temporal resolution to capture the dynamics of AVP-induced exocytosis in individual IMCD cells.

In this study the confocal imaging technique developed in rabbit cortical collecting duct was adopted to rat IMCD to monitor the spatial and temporal variations of [Ca2+]i induced by AVP in individual cells (Yip & Kurtz, 1995). To investigate the Ca2+ dependence of the AVP signalling cascade and exocytosis in intact IMCD, a novel confocal imaging technique using a styryl dye (FM1-43) was implemented to detect the real-time apical exocytosis in IMCD cells. The results provide direct evidence that 0.1 nm AVP triggers intracellular Ca2+ mobilization and apical exocytosis in intact IMCD, and that the manifestation of apical exocytosis in IMCD is Ca2+ dependent.

METHODS

Isolation and perfusion of single IMCD segments

Experiments were conducted in IMCD isolated from rat kidney. All experiments were performed under protocols approved by the University of South Florida's Animal Care and Use Committee. Male Sprague-Dawley rats from Harlan Farms (80–120 g) were treated with furosemide intraperitoneally (5 mg) for 20–30 min, then killed by anaesthetic overdose (using 5 % halothane in a chamber through a Fluotec Mark-3 vaporizer). Furosemide treatment was used to wash out the medullary osmolarity gradient, in order to minimize osmotic shock when medulla was placed in isotonic dissection solution (Chou et al. 1995). The kidneys were rapidly removed through a midline abdominal incision, and placed in an ice-cold dissecting solution. The dissection solution consisted of (mm): NaCl, 120; NaHCO3, 25; K2HPO4, 2; MgSO4, 1.2; CaCl2, 2; glucose, 5.5; sodium acetate, 5. Terminal IMCD segments were dissected from the inner half of the inner medulla (Chou et al. 1995). The isolated piece of IMCD was then transferred to a temperature-controlled perfusion chamber (Vestavia, AL, USA) mounted on a Zeiss Axiovert 100TV inverted microscope, which was coupled to a laser scanning unit (Bio-Rad MRC100) equipped with a krypton-argon laser and three photomultiplier detectors. The IMCD was then cannulated and perfused with glass concentric pipettes using the method developed by Burg (1972). Luminal and bath perfusate were identical to the dissecting solution. All solutions were gassed with 95 % O2-5 % CO2 before use, and the pH was adjusted to 7.4. For Ca2+-free perfusate, CaCl2 was replaced by 2 mm EGTA.

Measurement of intracellular calcium

The changes of [Ca2+]i in IMCD cells induced by AVP or other pharmacological agents were determined from the confocal fluorescence images of perfused tubules as described previously (Yip & Marsh, 1996, 1997). In brief, changes in [Ca2+]i of IMCD were monitored with emission intensity of fluo-4, or the emission ratio of fluo-4/Fura-Red. When excited at 488 nm, fluo-4 exhibits an increase in green fluorescence (525 nm) on Ca2+ binding, whereas Fura-Red shows a decrease in red fluorescence (640 nm). Because the emission ratio of fluo-4/Fura-Red is a ratiometric index, it is independent of changes in cell volume (Lipp & Niggli, 1993; Yip & Marsh, 1996, 1997). Fluo-4 AM alone (5 μm) or a mixture of 5 μm fluo-4 AM and 25 μm Fura-Red AM (Molecular Probes, OR, USA) was loaded into the IMCD from peritubular solution at room temperature for 20 min. The tubule was then washed, and incubated at 37 °C for another 30 min to allow de-esterification prior to the measurements. Confocal fluorescence images were acquired at 0.5 Hz from the lower surface of the perfused IMCD with the 488 nm laser line. All images were collected with a Zeiss × 40 plan apochromat objective (NA 1.2, water immersion) at a zoom factor of around 3, which covered the field of six to eight IMCD cells. Residence time of the laser on the IMCD for each image was 0.4 s.

When only fluo-4 was loaded into the IMCD, emission was collected with a 522/35 nm bandpass filter at 0.5 Hz and stored digitally. Emissions were collected simultaneously with a 522/35 nm bandpass filter and a 580LP longpass filter on two separate photomultipliers at 0.5 Hz when both fluo-4 and Fura-Red were loaded into the IMCD. The spatial and temporal variations of [Ca2+]i after the introduction of AVP (Bachem, CA, USA) into the peritubular perfusate were monitored in individual IMCD cells during playback of the stored fluorescence images using the software (Time Course/Ratiometric Software Module) supplied by Bio-Rad. The fluo-4/Fura-Red emission ratio of the fluorescence image from each IMCD cell was calculated after subtraction of the dark current and background fluorescence at each emission wavelength. Calibration of fluorescence emission of fluo-4 in individual IMCD cells was performed by incubating the IMCD with a non-fluorescent Ca2+ ionophore, 4-bromo-A23187 (10−5m), in the presence of extracellular Ca2+ (10 mm) for 15 min to saturate the fluo-4 with Ca2+, and thereby obtain maximal fluorescence (Fmax). The minimal fluorescence (Fmin) was measured after incubating the IMCD with Ca2+-free perfusate (containing 2 mm EGTA) and 4-bromo-A23187 for another 15 min (Burnier et al. 1994; Chan et al. 2001). Fluorescence intensity (F) was converted to [Ca2+]i in individual IMCD cells using the equation:

equation image

where Kd is the dissociation constant (345 nm; provided by Molecular Probes, OR, USA). Since fluo-4 is a non-ratiometric dye, photobleaching and leakage during repeated image acquisitions would render calibrations unreliable. Consequently, calibrations were only performed in tubules that involved a single exposure of 0.1 nm vasopressin.

Spectral analysis of time series

Time series of [Ca2+]i variations in individual IMCD cells induced by AVP were sampled at 0.5 Hz for spectral analysis using Fast Fourier Transform. Each time series of about 400 points was distributed into four overlapping segments with 50 % overlap, to reduce the variance of the power spectrum. Each segment was subjected to linear trend removal, and was operated on by a cosine window function to minimize leakage. Power spectral density was calculated by averaging the power spectral density from all segments derived from a given time series (Yip et al. 1991).

Real-time monitoring of apical exocytosis in IMCD

Vasopressin-induced apical exocytosis was monitored by including 2 μm FM1-43 (Molecular Probes, OR, USA) in the luminal perfusate to label the apical membrane of IMCD cells. The changes in apical fluorescence of FM1-43 were used as an index of accumulative exocytosis (Smith & Betz, 1996; Cochilla et al. 1999). Confocal images were acquired at the mid-plane of the perfused IMCD (excited by 488 nm laser line, emission collected with a 580LP longpass filter) so that the apical membrane of IMCD cells was clearly discerned on both sides of the lumen. A similar optical sectioning approach to image endothelial cells of perfused arterioles has been reported previously (Yip & Marsh, 1996). Confocal images were collected at 20 s intervals before and after 0.1 nm AVP was added to the peritubular perfusate. The spatial and temporal variations of FM1-43 apical fluorescence induced by AVP were measured in IMCD cells from the stored fluorescence images.

RESULTS

AVP-induced [Ca2+]i oscillations in IMCD

A confocal fluorescence image of a fluo-4-loaded IMCD acquired from the lower surface is shown in Fig. 1. Individual IMCD cells were clearly discerned. There was fluorescence heterogeneity in different regions of the perfused tubule. This was due to the fact that cells were on different focal planes and were loaded with the different amount of dye. The simultaneous time courses of fluo-4 emission intensity in four adjacent cells from an IMCD are shown in Fig. 2. When 0.1 nm AVP was introduced into the peritubular perfusate, there were immediate increases in the emission intensity in all cells. The emissions reached maxima within 10–20 s, and then started to oscillate. These observations indicated that AVP triggered an initial increase in [Ca2+]i in IMCD, which was followed by periodic oscillations in [Ca2+]i. The oscillations of [Ca2+]i in adjacent cells were not synchronous and had different oscillatory frequencies. These observations suggest that the [Ca2+]i oscillations in each cell are independent and that there is no intracellular Ca2+ coupling between IMCD cells. A typical record of AVP-induced oscillations in fluo-4 emission and the corresponding power spectrum are shown in Fig. 3. Despite the prominent periodic oscillations in the time series, there was no single dominant oscillatory frequency in the power spectrum. Most of the power spectral density was found between 0.02 and 0.08 Hz (2–5 cycles min−1). Similar distributions of power spectral density were found in all analysed records, which was reflected in the mean normalized power spectrum (Fig. 3).

Figure 1
Confocal fluorescence image of a perfused IMCD loaded with fluo-4
Figure 2
Oscillations of fluo-4 emission in adjacent IMCD cells
Figure 3
Spectral analysis of oscillations in fluo-4 emission

Since fluo-4 is not a ratiometric calcium dye, oscillations in the emission intensity could be due to rhythmic variations of cell volume rather than oscillations in [Ca2+]i. This possibility was tested by monitoring the variations of emission ratio in fluo-4/Fura-Red when the perfused IMCD was exposed to 0.1 nm AVP. Figure 4 shows the emission ratio and the emission intensities from fluo-4 and Fura-Red from a single IMCD cell. There was an immediate increase in the emission ratio induced by AVP, which was accompanied by 180 deg out-of-phase oscillations in the emission intensity of fluo-4 and Fura-Red. These observations confirm that the oscillations observed in the emission intensity from fluo-4 are the result of [Ca2+]i oscillations and are not secondary to changes in cell volume.

Figure 4
Oscillations in fluo-4/Fura-Red emission ratio in an IMCD cell

It is known that AVP regulates IMCD water permeability via a basolateral V2 vasopressin receptor (Ecelbarger et al. 1996). To test whether AVP-induced intracellular Ca2+ mobilization is also mediated by the V2 vasopressin receptor, changes in fluo-4/Fura-Red emission ratio following exposure of IMCD to either 0.1 nm AVP or 0.1 nm dDAVP (1-deamino-8-d-arginine vasopressin, a specific V2 receptor agonist) were compared. Figure 5 shows the time courses of mean normalized emission ratios. AVP and dDAVP induced nearly identical profiles of changes in [Ca2+]i. Together, these observations indicate that the same subtype of vasopressin receptor mediates the regulation of water permeability and the mobilization of intracellular Ca2+ in IMCD. Using fluo-4 alone as a calcium-reporting dye, the mean baseline and peak [Ca2+]i induced by 0.1 nm AVP are 77 ± 11 and 218 ± 21 nm, respectively (52 cells/six tubules). Pre-incubation of IMCD with 50 μm BAPTA AM for 30 min prevented AVP-induced intracellular Ca2+ mobilization (Fig. 5).

Figure 5
dDAVP simulates and BAPTA abolishes AVP-induced calcium mobilization

To determine whether the AVP-mobilized Ca2+ is derived from endogenous Ca2+ stores or extracellular Ca2+, changes in fluo-4 emission intensity were monitored while IMCD was perfused with Ca2+-free solution. Figure 6A is the mean normalized time course of changes in emission intensity in response to 0.1 nm AVP. AVP was capable of increasing [Ca2+]i in the absence of extracellular Ca2+, but sustained oscillations were not observed. Pre-incubation with 0.1 mm ryanodine in the absence of extracellular Ca2+ totally inhibited AVP-induced Ca2+ mobilization (Fig. 6B). These observations show that AVP induces Ca2+ release from ryanodine-sensitive Ca2+ stores to produce the initial increase in [Ca2+]i, but extracellular Ca2+ is required to sustain the [Ca2+]i oscillations.

Figure 6
Ryanodine blocks AVP-induced calcium mobilization

AVP-induced apical exocytosis in IMCD cells

Apical exocytotic activity was monitored optically by measuring the changes in apical FM1-43 fluorescence of IMCD cells. FM1-43 is a styryl dye that is non-fluorescent in aqueous solution, is impermeable to the cell membrane, and is fluorescent when bound to membranes (Cochilla et al. 1999). By measuring the membrane capacitance in chromaffin cells, Smith & Betz (1996) demonstrated that the increase in membrane surface area during induced exocytosis was associated with a proportional increase in FM1-43 membrane fluorescence. Figure 7 is a series of time-lapsed images showing the increase in apical FM1-43 fluorescence intensity induced by AVP. There was a gradual increase in apical fluorescence after the exposure to AVP, which indicated that there was an accumulative increase in apical membrane surface area as a result of AVP-induced AQP2 exocytosis. There was no such increase in apical fluorescence in the timed control (image not shown). When apical FM1-43 fluorescence was monitored in individual IMCD cells, increase in FM1-43 fluorescence could be detected within 20 s. The apical fluorescence intensity then increased continuously for about 30 min before reaching a plateau. Some IMCD cells showed signs of saturation as early as 25 min. The apical fluorescence intensity increased by 100 % within the first 10 min, and increased by 200 % after 30 min (Fig. 8). Pre-incubating IMCD with 50 μm BAPTA AM for 30 min completely inhibited the AVP-induced increase in apical FM1-43 fluorescence (Fig. 8) and Ca2+ mobilization (Fig. 5). These observations provide the first direct real-time evidence that AVP induces apical exocytosis in intact IMCD cells, and that intracellular Ca2+ mobilization is required for the AVP-induced exocytosis.

Figure 7
Time-lapsed confocal fluorescence image of a perfused IMCD with 2 μm FM1-43 in the lumen (a-h)
Figure 8
BAPTA inhibits AVP-induced apical exocytosis

DISCUSSION

By utilizing confocal fluorescence microscopy in perfused IMCD, subnanomolar AVP induces a detectable increase of [Ca2+]i. Two elements in the imaging system contribute to the successful visualization of [Ca2+]i changes in IMCD induced by subnanomolar AVP. Fluorescence images were collected from the lower surface of the perfused tubule confocally so that the variations of fluorescence signal could be determined from individual cells without the complication of out-of-focus fluorescence. The sensitivity to detect Ca2+ mobilization was enhanced when emission intensity was estimated from individual cells as compared with the whole tubule (Fig. 2E). Unexpectedly, AVP induced asynchronous oscillations in [Ca2+]i in IMCD cells. These asynchronous oscillations confounded the detection of Ca2+ mobilization when a photomultiplier was used as the detector (Star et al. 1988). The epifluorescence signal registered by a photomultiplier from a perfused IMCD is a measure of total fluorescence collected from a multiple of cells located in different focal planes and cannot discern the variations of [Ca2+]i in individual cells. Fluo-4, which is a derivative from the more commonly used fluo-3, was used as a Ca2+-reporting dye. The absorption peak of fluo-4 is closer to the excitation line of the argon laser (488 nm) than fluo-3, which improves the signal-to-noise ratio during image acquisition.

There was a possibility that the modulation of water permeability by AVP induced changes in the cell volume of IMCD and, since fluo-4 is not a ratiometric dye, rhythmic variations of cell volume could be manifested as oscillations in fluo-4 emission. The oscillations of fluo-4 emission were accompanied by 180 deg out-of-phase oscillations in emission from Fura-Red when both dyes were loaded into IMCD cells. Rhythmic cell volume variations should result in synchronous oscillations in both fluo-4 and Fura-Red emission. These observations confirm that AVP triggers oscillations in [Ca2+]i but not in cell volume. This is the first report that a physiological hormonal signal triggers [Ca2+]i oscillations in an intact epithelium. This preparation provides a unique opportunity to study the mechanism of [Ca2+]i oscillations under physiological conditions. Simultaneous monitoring of [Ca2+]i in adjacent IMCD cells indicated that each cell oscillated at a different frequency and amplitude. No intercellular Ca2+ coupling in the form of synchronous oscillations was observed. Intercellular Ca2+ coupling is usually mediated through gap junctions (Christ et al. 1992). An ultra-structural study has concluded that there are no gap junctions between IMCD cells (Pricam et al. 1974), which is consistent with the current observations.

FM1-43 has been used to study exocytosis and vesicle recycling in isolated cells in different tissues (Terasaki, 1995; Smith & Betz, 1996; Cochilla et al. 1999). FM1-43 membrane fluorescence is an index of accumulative exocytosis because the endosomes formed by endocytosis still stay close to the apical membrane and contribute to the fluorescence signal. By taking advantage of the ability to label the apical membrane of IMCD cells by FM1-43 independently of the basolateral membrane through the luminal perfusate, and the ability to monitor apical FM1-43 fluorescence of IMCD in real time using the optical sectioning capability of confocal fluorescence microscopy (Yip & Marsh, 1996), it was demonstrated for the first time that AVP induces rapid and continuous exocytosis in the apical membrane of intact IMCD cells. Wall et al. (1992) reported that the first detectable increase of Pf in perfused IMCD induced by AVP was after 40 s, and 50 % of the increase in Pf occurred within 10 min. The same time profile of increase in apical FM1-43 fluorescence was found in IMCD cells, in that 50% of the increase was observed in the first 10 min (Fig. 8). The apical FM1-43 fluorescence after 30 min of 0.1 nm AVP stimulation was about three times the pre-stimulation baseline. A similar increase in Pf was also found after 30 min of 0.1 nm AVP stimulation (Chou et al. 2000). Pf is a variable measured from the whole perfused IMCD and is limited to 2–3 samples min−1, while FM1-43 fluorescence is monitored in individual cells and can be sampled up to 2 Hz. Therefore, compared with Pf measurement, the measurement of FM1-43 offers improved temporal and spatial resolution to study the antidiuretic action of AVP.

In LLC-PK1 cells transfected with an AQP-2-c-myc construct, AQP2 is recycled multiple times between intracellular vesicles and plasma membrane following AVP simulation (Katsura et al. 1996; Gustafson et al. 2000). It is also known that endocytosed membrane containing water channels is rapidly recycled back to the surface in response to vasopressin stimulation in toad bladder (Coleman & Wade, 1994). These observations indicate that the trafficking and recycling of AQP2 vesicles can be monitored by tracking the endocytosed membrane instead of AQP2 protein. Since accumulative exocytosis in IMCD was detected by monitoring apical FM1-43 fluorescence intensity, it demonstrated the flexibility of pre-labelling endocytosed vesicles carrying AQP2 using FM1-43 by sequential addition and removal of AVP. This approach might provide the opportunity to study the regulation of fusion, retrieval and directional trafficking of AQP2 vesicles in intact IMCD cells (Marples et al. 1998). These protocols have been applied successfully to study endocytosis and membrane recycling of synaptic vesicles (Cousin & Robinson, 1999).

To determine whether the AVP-induced Ca2+ mobilization is linked with apical exocytosis, the [Ca2+]i of IMCD was clamped by incubating the IMCD with 50 μm BAPTA AM for 30 min. This protocol of loading BAPTA not only nullified the Ca2+ mobilization induced by 0.1 nm AVP (Fig. 5), but also prevented the apical exocytosis (Fig. 8). The same protocol also inhibited the increase of Pf induced by 0.1 nm AVP (Chou et al. 2000). These results indicate that manifestation of AVP-induced exocytotic insertion of AQP2 requires intracellular Ca2+ mobilization. These observations are consistent with the notion that exocytotic fusion of AQP2 vesicles resembles that of synaptic vesicles, both involving SNARE proteins and an increase in [Ca2+]i (Knepper & Inoue, 1997). Vesicle fusion might not be the only Ca2+-dependent step in regulating AQP2 exocytosis. Ryanodine (in the absence of extracellular Ca2+) and calmodulin inhibitors blocked AVP-induced trafficking of AQP2 vesicles in cultured IMCD cells (Chou et al. 2000). These observations indicate that there are Ca2+-sensitive steps in the trafficking process of AQP2 vesicles.

The transduction of many cellular stimuli results in [Ca2+]i oscillations in both excitable and non-excitable cell types. Signalling information is suggested to be encoded in the frequency of such oscillations (Berridge & Galione, 1988). The present study is the first to report that a physiological hormonal signal (0.1 nm AVP) triggers [Ca2+]i oscillations in intact renal epithelium, and to show that Ca2+ mobilization is linked to the regulation of a physiological variable, the Pf. AVP is known to induce [Ca2+]i oscillations in isolated or cultured cells such as vascular smooth muscle cells (Wu et al. 1995), insulin-secreting cells (Schofl et al. 1995), and glomerular mesangial cells (Hajjar & Bonventre, 1991). The spectral analysis of [Ca2+]i oscillations in IMCD indicated that most of the oscillations were found between 0.02 and 0.08 Hz. Detailed spectral properties of [Ca2+]i oscillations have been rarely reported in the available literature. It is not known whether this pattern of power spectrum is unique to intact renal epithelium or is common to other preparations. In general, oscillatory frequencies reported from primary cultures derived from kidney are one to two orders of magnitude less than those of our observations, and are beyond the resolution limit of the IMCD power spectra. Specifically, vasopressin induces [Ca2+]i to oscillate at the range of 0.001 Hz to 0.01 Hz in cultured mesangial cells (Hajjar & Bonventre, 1991). Removal of extracellular Na+ triggers [Ca2+]i oscillations in primary culture of rabbit collecting duct at 0.01 Hz (Koster et al. 1993).

Oscillation of [Ca2+]i has been suggested as a more efficient and robust signal than the global sustained increase of cytoplasmic Ca2+ in regulating the physiological process (Berridge & Galione, 1988). Removal of extracellular Ca2+ in perfused IMCD did not prevent the initial rise of [Ca2+]i induced by AVP, but inhibited the sustained oscillations (Fig. 6). Type 1 ryanodine receptors were colocalized with AQP2 in the apical membrane of IMCD (Chou et al. 2000). Ryanodine in the absence of extracellular Ca2+ completely eliminated the AVP-induced intracellular Ca2+ mobilization (Fig. 6). These observations suggest that the AVP-induced [Ca2+]i oscillations involve intracellular Ca2+ released from ryanodine-sensitive stores and the influx of extracellular Ca2+. The maximal increase of Pf induced by 0.1 nm AVP in the absence of extracellular Ca2+ is only half of that in the presence of extracellular Ca2+ (Chou et al. 2000). These observations suggest that some signalling information for AQP2 vesicle translocation and/or exocytotic fusion is encoded in the oscillations of [Ca2+]i. It has been shown that oscillations in [Ca2+]i evoke oscillatory bursts of exocytosis in pituitary gonadotropes (Tse et al. 1993). It is possible that [Ca2+]i oscillations are required to prolong the exocytotic activity induced by AVP in IMCD.

Acknowledgments

The author acknowledges Drs E. Bennett, C.-L. Chou, M. A. Knepper and D. J. Marsh for their helpful suggestions. This study was support by NIH Grant DK-15968, HL-59156 and a Grant-In-Aid from the American Heart Association, Florida/Puerto Rico Affiliate.

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