A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+]i of principal cells in rat cortical collecting duct

Cell Calcium 49 (2011) 35–42

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A nicardipine-sensitive Ca2+ entry contributes to the hypotonicity-induced increase in [Ca2+ ]i of principal cells in rat cortical collecting duct You Komagiri, Kazuyoshi Nakamura, Manabu Kubokawa ∗ Department of Physiology, Iwate Medical University School of Medicine, 19-1 Uchimaru, Morioka 020-8505, Japan

a r t i c l e

i n f o

Article history: Received 13 July 2010 Received in revised form 15 November 2010 Accepted 15 November 2010 Available online 10 December 2010 Keywords: Hypotonicity Nicardipine Renal cortical collecting duct Ca2+ channel Intracellular Ca2+

a b s t r a c t We examined the mechanisms involved in the [Ca2+ ]i response to the extracellular hypotonicity in the principal cells of freshly isolated rat cortical collecting duct (CCD), using Fura-2/AM fluorescence imaging. Reduction of extracellular osmolality from 305 (control) to 195 mosmol/kgH2 O (hypotonic) evoked transient increase in [Ca2+ ]i of principal cells of rat CCDs. The [Ca2+ ]i increase was markedly attenuated by the removal of extracellular Ca2+ . The application of a P2 purinoceptor antagonist, suramin failed to inhibit the hypotonicity-induced [Ca2+ ]i increase. The [Ca2+ ]i increase in response to extracellular hypotonicity was not influenced by application of Gd3+ and ruthenium red. On the other hand, a voltage-gated Ca2+ channel inhibitor, nicardipine, significantly reduced the peak amplitude of [Ca2+ ]i increase in the principal cells. In order to assess Ca2+ entry during the hypotonic stimulation, we measured the quenching of Fura2 fluorescence intensity by Mn2+ . The hypotonic stimulation enhanced quenching of Fura-2 fluorescence by Mn2+ , indicating that a Ca2+ -permeable pathway was activated by the hypotonicity. The hypotonicitymediated enhancement of Mn2+ quenching was significantly inhibited by nicardipine. These results strongly suggested that a nicardipine-sensitive Ca2+ entry pathway would contribute to the mechanisms underlying the hypotonicity-induced [Ca2+ ]i elevation of principal cells in rat CCD. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction The renal cortical collecting duct (CCD) is well known as an important nephron segment which operates the urinary concentrating mechanism. The tubular fluid osmolality passing into CCD changes continuously within the hypotonic range during the transition from diuresis to antidiuresis condition and vice versa [1]. In the presence of arginine vasopressin, which increases the water permeability of apical membrane of principal cells, hypotonic tubular fluid entering this segment is reabsorbed [2]. In general, hyperand hypotonic solutions are known to change the cell volume, and these changes recover via mechanisms called regulatory volume increase (RVI) and regulatory volume decrease (RVD), respectively [3]. Since the principal cells of CCD occasionally exposed to the hypotonic luminal fluid, RVD would be required to maintain the functional significance of CCD. RVD is mainly driven by the extrusion of inorganic solute (K+ and − Cl ) in many cells [4]. In addition, a variety of epithelial cell types including toad bladder cells, rabbit proximal tubule cells and cultured intestinal 407 cells show a rise in intracellular free Ca2+ during hyposmotic cell swelling [5–7], and intracellular Ca2+ has been

∗ Corresponding author. Tel.: +81 19 651 5111; fax: +81 19 654 8340. E-mail address: [email protected] (M. Kubokawa). 0143-4160/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.ceca.2010.11.006

shown to play a crucial role in activation of membrane transport mechanisms to extrude solutes and water [7–10]. Many investigators have explored the mechanisms underlying the increase in [Ca2+ ]i induced by the hypotonic stimulation and proposed that various components regarding Ca2+ entry and/or Ca2+ release from intracellular stores are involved in the process of the [Ca2+ ]i increase [11–15]. In several types of epithelial cells, mechanosensitive cation channels have been suggested to be involved in the [Ca2+ ]i elevation induced by hypotonic stimulation [16–18]. On the other hand, it was reported that extracellular ATP released upon osmotic cell-swelling induced Ca2+ mobilization from internal Ca2+ store via purinergic receptors in human epithelial cells [15]. Recently, TRPV4 channel was suggested to be involved in Ca2+ influx during the hypotonicity in M-1 cell lines, derived from mouse CCD [19]. In rat CCD, Hirsch et al. [20] reported that enhancement of a large-conductance Ca2+ -activated K+ channel activity induced by hypotonic stimulation was dependent on extracellular Ca2+ . However, the precise mechanisms underlying hypotonicityinduced Ca2+ response in the principal cells of CCD are still unclear. The aim of the present study is to clarify the mechanisms responsible for the increase in [Ca2+ ]i induced by extracellular hypotonicity in principal cells of freshly isolated rat CCDs. We present evidence that a nicardipine-sensitive Ca2+ entry pathway plays a key role in the hypotonicity-induced [Ca2+ ]i increase in the principal cells of rat CCDs.

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Fig. 1. Effect of the exposure to hypotonic solution on the BK channel in principal cells of freshly isolated rat CCDs. (A) Representative trace of cell-attached recording before, during and after exposure to hypotonic solution. The numbered parts (1, 2 and 3) of the upper trace are displayed in the expanded time course in the lower part of the figure. Hypotonic stimulation (open square) was applied for 8 min. (B) Effect of paxilline on the hypotonicity-induced increase in single-channel activity in the cell-attached recording configuration. Paxilline (10 ␮M, filled square) was applied extracellularly for 2 min.(C) Effect of extracellular Ca2+ removal during the hypotonic stimulation on the large-conductance K+ channel activity. The hypotonicity-induced enhancement of BK channel activity was not observed when extracellular Ca2+ was removed from the bath solution. Extracellular Ca2+ was changed from 1 to 0 mM during the periods indicated by the horizontal bar.

2. Materials and methods 2.1. Preparation of rat CCD Animals were sacrificed according to the guidelines stipulated by the ethics committee for animal treatment of Iwate Medical University. Male Sprague–Dawley rats (5–10 weeks old) were fed with a standard rat diet. Rats were killed by cervical dislocation after inhalation anesthesia by halothane. The kidneys were immediately removed and cut into thin slices with razor blade. The kidney slices were stored in ice cold phosphate buffered saline (PBS) containing 2.5 mM MgCl2 . Isolation of CCD from the kidney slice and exposure of the luminal surface of principal cells were performed according to the methods which we employed previously [21]. In brief, CCDs were dissected with forceps under a stereoscopic microscope and attached to a cover glass coated with Cell-Tak (Becton Dickinson, Franklin Lakes, NJ). The cover glass was placed in a chamber mounted on an inverted microscope and CCD was split open with a sharpened micropipette to expose the luminal surface. 2.2. Solutions The control bath solution contained (mM): 140 NaCl; 5 KCl; 1 MgCl2 ; 1 CaCl2 ; 5 d-glucose; 10 HEPES. Osmolality of the control

solution was adjusted to 305 mosmol/kgH2 O using a freezing-point osmometer (Vogel OM-801, Giessen, Germany). The hyposmotic solution (195 mosmol/kgH2 O) was composed of (mM): 80 NaCl; 5 KCl; 1 MgCl2 ; 1 CaCl2 ; 5 d-glucose; 10 HEPES. To make Ca2+ free solution, CaCl2 was omitted and 2 mM EGTA was added. The pH of the pipette and bath solutions used in the present study was adjusted to 7.4, and the bath temperature during the experiments was kept at 35–37 ◦ C by a heater controller (model TC-324B, Warner Instruments, Hamden, CT).

2.3. Electrophysiology Principal cells of the tubule were identified visually. Single channel currents were recorded under cell-attached patch-clamp configuration. For current recording, tubules were superfused with the control solution as described above. The patch-clamp pipettes, which were pulled from borosilicate glass capillaries (Harvard Apparatus Inc., Holliston, MA) with a vertical puller (PC-10, Narishige, Tokyo, Japan), had resistances of 3–5 M when filled with a standard K-rich pipette solution containing (mM): 145 KCl; 2 MgCl2 ; 1 EGTA; 10 HEPES. An Axopatch-200B patch-clamp amplifier (Molecular Devices, Sunnyvale, CA) was used to measure the membrane currents. The amplifier was driven by pClamp8 soft-

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ware. For analysis, the membrane currents were filtered through an eight-pole bessel filter (Multifunction Filter 3611, NF corporation, Yokohama, Japan) at 500 Hz and sampled at 2.5 kHz. Subsequent current analysis was performed using programs supplied with Clampfit9 software. Channel activity was determined by NPo , which was calculated from an amplitude histogram as NPo =

N 

n · tn ,

n=1

where N is the number of open channels observed during a given time period in the patch, Po is the open probability, n is the number of channels observed at the same time, and tn is the probability that n channels are simultaneously open. 2.4. Measurement of [Ca2+ ]i A fluorescent Ca2+ indicator, Fura-2 was used to measure [Ca2+ ]i using a dual-wavelength fluorescence imaging system (InCyt IM2TM fluorescence imaging system, Intracellular Imaging Inc., Cincinnati, OH).The ratio of fluorescence at excitation of 340 and 380 nm (F340 /F380 ) with emission at 510 nm was used to indicate the relative [Ca2+ ]i . To load Fura-2, CCDs attached to cover glasses were incubated for 1 h at room temperature with a 5 ␮M concentration of the acetoxymethyl ester form of Fura-2 (Fura-2 AM: Dojindo, Kumamoto, Japan) in PBS containing Mg2+ (2.5 mM). After loading, the tubules were split open as described above. The cover glasses were placed on a Petri dish on the stage of a Nikon inverted microscope. The CCDs were continuously perfused with experimental solutions through polyethylene tubes connected to a peristaltic pump at a flow rate of 3 ml/min. The bath solution could be changed in about 1 min using the present perfusion system. Cells were illuminated every 10 s with lights at 340 and 380 nm, and the respective images were captured by monochrome charge-coupled device camera. 2.5. Test substances Stock solutions of nifedipine (10 mM, Sigma, St Louis, MO) and nicardipine (10 mM, Sigma) were prepared in DMSO and ethanol, respectively. Stock solution of paxilline (20 mM, Sigma) was prepared in DMSO. Stock solutions of suramin (100 mM, Sigma) and ruthenium red (10 mM, Sigma) were prepared in distilled water. These stocks were stored at −20 ◦ C, thawed immediately prior to use, and diluted with bath solution to the individual final concentrations as described in the text. 2.6. Statistics Data are presented as the mean ± standard error of the mean (SEM) and n (number of observations). Statistical significance was determined by using Student’s t-test or ANOVA in conjunction with the Bonferroni t-test. A p values of less than 0.05 were considered significant. 3. Results 3.1. The effect of hypotonic stimulation on the BK channel activity in rat CCDs In many types of epithelial cells, it has been reported that Ca2+ -dependent K+ channel is involved in K+ efflux in the processes of RVD [22,23]. We examined the effect of hypotonic stimulation on the ionic conductance observed in the apical mem-

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brane of principal cells of rat CCDs. In the apical membrane of principal cells of mammalian CCDs, BK channel is functionally expressed [24,25]. As shown in Fig. 1A, about 5 min exposure to the hypotonic solution induced prominent increase in single-channel activity with mean unitary amplitudes of 5.6 ± 0.5 pA (n = 5) in a cell-attached patch at the pipette potential of 0 mV. The increased channel activity was sustained during hypotonic stimulation (8 min). The NPo value of the single-channel events increased from 0.022 ± 0.013 to 1.12 ± 0.44 (n = 5) after 8 min exposure to hypotonic stimulation. Then, returning the solution to the control gradually reduced the channel activity in a few minutes. To confirm whether the channel activated by the extracellular hypotonicity is BK channel, a membrane permeant BK channel blocker, paxilline (10 ␮M, [26–28]) was added to the bath solution when the channel was activated by the hypotonicity. As shown in Fig. 1B, the hypotonicity-induced activation of single-channel events was completely abolished by paxilline, indicating that the channel activated by hypotonicity is BK channel in the principal cell of rat CCDs. In cell-attached patches on the apical membrane of principal cells of CCDs, small-conductance inward rectifier K+ channels (SK channel, [29,30]) current was also observed. It has been demonstrated that SK channel plays a key role in K+ secretion in the CCD [31]. However, SK channel activity was not influenced by the hypotonic condition in this study (data not shown). In the next, we examined whether the hypotonicity-induced activation of BK channel was observed in the absence of extracellular Ca2+ . As shown in Fig. 1C, Ca2+ free hypotonic solution failed to activate the BK channel, but the channel was activated after adding 1 mM Ca2+ to the hypotonic solution. These results suggest that extracellular Ca2+ is indispensable for the hypotonicity-induced BK channel activation. 3.2. The [Ca2+ ]i response to hypotonic stimulation in principal cells of CCDs To determine whether the potentiation of BK channel activity by the exposure to the hypotonic solution was the result of increase in the cytosolic Ca2 , we examined the effect of hypotonic stimulation on [Ca2+ ]i of principal cells in rat CCDs. As shown in Fig. 2A, the transient increase in the cytosolic Ca2+ was observed after cells were exposed to the hypotonic solution. Hypotonic stimulation increased the Fura-2 fluorescence ratio from 1.02 ± 0.04 (basal) to 1.49 ± 0.12 (peak, n = 8). To clarify the source of the rise in [Ca2+ ]i , we first investigated the change in [Ca2+ ]i when extracellular Ca2+ was removed before and during exposure of the cell to hypotonic solution. Five minutes after the removal of extracellular Ca2+ , cells were stimulated with hypotonic solution in the absence of external Ca2+ . As shown in Fig. 2B, removal of extracellular Ca2+ reduced the basal [Ca2+ ]i , and [Ca2+ ]i elevation was largely reduced during the hypotonic stimulation. The hypotonicity-induced [Ca2+ ]i elevation was restored when Ca2+ was reintroduced to the hypotonic solution (Fig. 2B). The magnitude of hypotonicity-induced [Ca2+ ]i increase in the absence of extracellular Ca2+ was 16.6 ± 1.3% (n = 8) of that observed in the presence of extracellular Ca2+ . The transient elevation in [Ca2+ ]i in response to hypotonic solution was strongly dependent upon the presence of extracellular Ca2+ . As mentioned in Section 2, hypotonic solution was prepared simply by reducing NaCl concentration. Thus, we examined the possibility that lowering Na+ and Cl− might affect the cytosolic Ca2+ concentration through Na+ /Ca2+ exchanger or changes in membrane potential. Isotonic replacement of 60 mM NaCl with 120 mM mannitol (low NaCl solution) did not influence the cytosolic Ca2+ , but hypotonicity after removal of mannitol elevated [Ca2+ ]i

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Fig. 2. [Ca2+ ]i responses to hypotonic stimulation in principal cells of freshly isolated rat CCDs. (A) A typical trace of [Ca2+ ]i changes induced by hypotonic stimulation in principal cells of rat CCDs. Hypotonic stimulation (open square) was applied for 8 min. (B) Effect of extracellular Ca2+ removal on the hypotonicity-induced [Ca2+ ]i increase. Extracellular Ca2+ was changed from 1 to 0 mM during the periods indicated by the horizontal bar. (C) Effect of lowering extracellular NaCl concentration on [Ca2+ ]i in rat CCD. A typical trace showing effect of replacement of extracellular 60 mM NaCl by 120 mM mannitol on [Ca2+ ]i in rat CCD. Extracellular NaCl was decreased from 140 to 80 mM during the periods indicated by the filled square.

(Fig. 2C). These results indicate that the increase in [Ca2+ ]i was not due to the low NaCl but to the hypotonicity. When cells were stimulated twice by hypotonic solution with an interval at least 10 min, the mean magnitude of [Ca2+ ]i increase in 2nd responses normalized to that in the 1st responses was 1.00 ± 0.14 (= Ratio2nd /Ratio1st , n = 8). Although there is no statistical significance between 1st and 2nd responses, [Ca2+ ]i increase in the 2nd response was various compared to the 1st response (maximally changed by ±40%). Thus, in the following some experiments, to evaluate the effect of blockers on the hypotonicity-induced [Ca2+ ]i increase, the 2nd responses in the presence of blockers were compared to the 2nd responses in the absence of blockers. The 2nd response was normalized to the each 1st response in the absence of blocker.

Fig. 3. Effect of purinergic receptor antagonist on the hypotonicity-induced [Ca2+ ]i increase. (A) Typical traces showing the effect of extracellular ATP on [Ca2+ ]i in the absence (upper trace) or presence (lower trace) of purinergic receptor antagonist, suramin (100 ␮M). ATP (10 ␮M, open squares) was applied for 90 s. Suramin (100 ␮M, filled square) was applied for 5 min. (B) Typical traces of [Ca2+ ]i responses to the hypotonic stimulation in the absence (left panel) or presence (right panel) of suramin (100 ␮M, filled square) obtained from the same cell. Open square indicates the period of hypotonic stimulation. (C) Summarized data of the effect of suramin on hypotonicity-induced increase in [Ca2+ ]i . The difference in ratio between the basal and peak level of 2nd responses in the absence (control) and presence of suramin (n = 6) was normalized to the 1st response. Data were expressed as mean ± SEM.

3.3. Effect of purinergic antagonist on the hypotonicity-induced [Ca2+ ]i increase In some tissues, including epithelial cells, it has been reported that extracellular ATP, released by hypotonic cell swelling contributed to the hypotonicity-induced [Ca2+ ]i increase through the purinergic receptors[15,32]. Thus, we investigated the contribution of ATP to the hypotonicity-induced [Ca2+ ]i elevation. In rat CCDs, extracellular ATP was reported to elicit the [Ca2+ ]i increase via P2 Y purinoceptor [33]. Indeed, application of 10 ␮M ATP to the bath solution produced [Ca2+ ]i enhancement in principal cells of freshly isolated rat CCDs. In the presence of suramin, a purinergic recep-

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tor antagonist, [Ca2+ ]i elevation induced by ATP was completely abolished (Fig. 3A). On the other hand, the hypotonicity-induced [Ca2+ ]i elevation was not inhibited by suramin (Fig. 3B and C, Ratio2nd /Ratio1st = 0.90 ± 0.15, n = 6). 3.4. Effect of cation channel blockers on the hypotonicity-induced [Ca2+ ]i increase We next investigated the possibility that Ca2+ influx through the Ca2+ permeable cation channels contributes to the hypotonicityinduced [Ca2+ ]i increase by using several cation channel blockers. It has been reported that trivalent cations, Gd3+ and La3+ strongly inhibited the stretch-activated non-selective cation channels [18,34]. In some experiments of this study, application of GdCl3 produced a rapid decrease in the basal Ca2+ level of principal cells in rat CCDs (Fig. 4A right panel). However, regardless of whether the decrease of basal [Ca2+ ]i was observed, the presence of Gd3+ (30 ␮M) in the bath solution during the 2nd stimulation had no effect on hypotonicity-induced [Ca2+ ]i elevation (Ratio2nd /Ratio1st = 0.96 ± 0.058, n = 5, Fig. 4C). Similar results were obtained with CCDs tested with La3+ (30 ␮M, Ratio2nd /Ratio1st = 0.89 ± 0.019, n = 5). Even when the concentration of La3+ was raised to 5 mM, no significant effect was observed (Ratio2nd /Ratio1st = 0.89 ± 0.13, n = 4). To investigate the contribution of TRPV4 channels, which was reported to play an important role in response to the osmotic change in the CCD cell line, M-1cells [19], ruthenium red was applied during the second hypotonic stimulations (Fig. 4B). Ruthenium red (100 ␮M) did not have any significant effect on the hypotonicity-induced [Ca2+ ]i increase (Ratio2nd /Ratio1st = 0.94 ± 0.082, n = 5, Fig. 4C). Moreover, we studied the effects of several inhibitors of voltagegated Ca2+ channel on the hypotonicity-induced increase in [Ca2+ ]i in principal cells of rat CCDs. Cd2+ has been widely used to block voltage-gated Ca2+ channels [35]. In rat CCDs, application of 100 ␮M Cd2+ , the concentration of which was high enough to inhibit voltage-gated Ca2+ channels induced progressive increase in basal [Ca2+ ]i of principal cells. Thus, we could not assess the effect of Cd2+ (100 ␮M) on the hypotonicity-induced [Ca2+ ]i increase (data not shown). The effect of dihydropyridine L-type Ca2+ channel antagonists on the hypotonicity-induced [Ca2+ ]i increase was examined. 10 ␮M nifedipine was applied during the second stimulation of hypotonicity. There was no significant difference in the 2nd response to the hypotonicity in the presence (Ratio2nd /Ratio1st = 1.04 ± 0.06, n = 5) and absence (Ratio2nd /Ratio1st = 1.00 ± 0.14, n = 8) of nifedipine (Fig. 5B). In contrast, 10 ␮M nicardipine significantly reduced the hypotonicity-induced [Ca2+ ]i increase by about 70% (Ratio2nd /Ratio1st = 0.34 ± 0.08, n = 5, p < 0.05, Fig. 5A and B).

Fig. 4. Effects of non-selective cation inhibitors on the hypotonicity-induced [Ca2+ ]i increase. (A) Typical [Ca2+ ]i responses to hypotonic stimulation in the absence (left panel) or presence (right panel) of 30 ␮M Gd3+ . (B) Typical [Ca2+ ]i responses to hypotonic stimulation in the absence (left panel) or presence (right panel) of 100 ␮M ruthenium red. Open squares indicate the period of hypotonic stimulation. Gd3+ or ruthenium red was applied during the periods indicated by filled squares. (C) Summarized data of the effect of cation channel blockers on hypotonicity-induced increase in [Ca2+ ]i . The difference in ratio between the basal and peak level of 2nd responses in the absence and presence of Gd3+ and ruthenium red was normalized to the 1st response. Data were expressed as mean ± SEM (n = 5 for Gd3+ , n = 5 for ruthenium red).

3.5. The Mn2+ -quenching of Fura-2 fluorescence intensity Mn2+ is known to pass through many Ca2+ permeable channels and quench Fura-2 fluorescence [36,37]. To obtain the direct evidence that Ca2+ entry was triggered by hypotonic stimulation, we measured the Mn2+ -induced quenching of Fura-2 fluorescence intensity excited at 360 nm (the isosbestic point for Fura-2) as an indicator of Ca2+ influx. In the absence of Mn2+ , hypotonic stimulation induced rapid reduction in the Fura-2 fluorescence of 10.8 ± 1.2% (n = 10), which was followed by a gradual recovery to the initial fluorescence level during the hypotonic stimulation (Fig. 6A, left panel). The rapid decrease of Fura-2 fluorescence might be due to the dilution of intracellular Fura-2 by cell swelling. The application of MnCl2 (200 ␮M) to the iso-

tonic extracellular solution caused a slow progressive quenching of fluorescence intensity in Fura-2/AM-loaded cells (Fig. 6A, right panel). The reduction of Fura-2 fluorescence intensity was largely enhanced after hypotonic stimulation, indicating that Mn2+ entry was increased by hypotonicity (Fig. 6A, right panel). At the end of the hypotonic stimulation, the Fura-2 fluorescence intensity was reduced by 28.4 ± 5.3% (Fig. 6C, n = 5) in the presence of MnCl2 compared with that immediately before hypotonic stimulation. In contrast, the hypotonicity-induced enhancement of Fura-2 fluorescence quenching was significantly attenuated in the presence of 10 ␮M nicardipine (Fig. 6B and C, 9.8 ± 1.4%, n = 5, p < 0.05).

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Fig. 5. Effects of voltage-gated Ca2+ channel blockers on the hypotonicity-induced [Ca2+ ]i increase. (A) Typical [Ca2+ ]i responses to hypotonic stimulation in the absence (left panel) or presence (right panel) of 10 ␮M nicardipine. Open squares indicate the period of hypotonic stimulation. Nicardipine was applied during the periods indicated by filled square. (B) Summarized data of the effect of nifedipine and nicardipine on hypotonicity-induced increase in [Ca2+ ]i . The difference in ratio between the basal and peak level of 2nd responses in the absence and presence of nifedipine and nicardipine was normalized to the 1st response. Data were expressed as mean ± SEM (n = 5 for nifedipine, n = 5 for nicardipine). *p < 0.05 vs. control.

4. Discussion Using Ca2+ imaging, we demonstrated that a nicardipinesensitive Ca2+ influx pathway was activated by extracellular hypotonicity and played a crucial role in the hypotonicity-induced [Ca2+ ]i increase in principal cells of fleshly isolated rat CCDs. Removal of extracellular Ca2+ markedly reduced the hypotonicity-induced [Ca2+ ]i increase, suggesting that extracellular Ca2+ was indispensable for the [Ca2+ ]i elevation. In the human epithelial cell (Intestine 407), it was reported that extracellular ATP released upon cell swelling enhanced [Ca2+ ]i rise via the stimulation of purinergic receptors [15]. In addition, it was demonstrated that extracellular ATP produced the rise in [Ca2+ ]i through the P2 Y receptors in rat CCDs [33]. Indeed, rapid increase in [Ca2+ ]i of principal cells was observed after application of ATP in the preparation of this study (Fig. 3A). However, a purinergic receptor antagonist suramin (100 ␮M), the concentration of which was high enough to inhibit the ATP-induced [Ca2+ ]i increase failed to influence the [Ca2+ ]i response to the hypotonic stimulation in the principal cell of rat CCDs (Fig. 3B). It was indicated that purinergic receptor mediated signaling pathway did not contribute to the mechanisms underlying the hypotonicity-induced [Ca2+ ]i increase.

Recently, some members of TRP cation channel family have been reported to possess Ca2+ permeability and be activated by various mechanical stimuli [11,19,38]. Particularly, it was reported that Ca2+ influx through TRPV4 channel in response to cell swelling and/or shear stress produced a rise in [Ca2+ ]i in renal epithelial cells [19,39]. In the nephron, TRPV4 channel proteins were expressed in the constitutively or conditionally water-impermeant segments, including CCD [40]. In addition, a recent report demonstrated that the expression of TRPV4 was critical for Ca2+ influx induced by hypotonicity and fluid flow in the M-1 cell line, derived from the mouse CCD [19]. In our study, however, application of ruthenium red, which has been known to block TRPV4 channel did not influence the hypotonicity-induced [Ca2+ ]i responses (Fig. 4B). In the epithelial cells of CCD, expression of other TRP channel isoforms, TRPC3 and TRPC6, was also reported [41]. It has been demonstrated that TRPC6 was activated by hypotonic cell swelling in heterologous expression system [42]. TRPC family has been reported to be blocked by La3+ and Gd3+ at micromolar concentration. In our study, the extent of the rise in [Ca2+ ]i elicited by hypotonic stimulation was not changed in the presence of extracellular La3+ or Gd3+ (30 ␮M). Jung et al. [43] reported that micromolar concentration of La3+ or Gd3+ potentiated TRPC5 channel, which was also activated by mechanical stimuli. In contrast, milli-molar concentrations had an inhibitory effect on TRPC5 currents. In the rat CCDs, even at 5 mM, La3+ did not inhibited the hypotonicityinduced [Ca2+ ]i increase. Taken together, these results suggest that TRP channels, which are activated by mechanical stimuli, did not contribute to the [Ca2+ ]i elevation observed after hypotonic stimulation in rat CCDs. On the other hand, the decrease in basal [Ca2+ ]i observed after application of Gd3+ might be due to the inhibition of Gd3+ sensitive cation channels activated by the fluid flow [19,39]. It was reported that nifedipine-sensitive Ca2+ conductance was observed in the rabbit proximal tubules and connecting tubules [44,45]. In addition, in the rabbit thick ascending limb, it was demonstrated that a high concentration (100 ␮M) nifedipine inhibited the rise in [Ca2+ ]i induced by reduction of extracellular osmolarity [46]. In this study, hypotonicity-induced [Ca2+ ]i enhancement was not inhibited by nifedipine but was largely inhibited by nicardipine. Moreover, the treatment of nicardipine significantly attenuated Mn2+ influx during the hypotonic stimulation. These results indicated that a nicardipine-sensitive Ca2+ entry pathway was activated by the reduction of extracellular osmolality and contributed to the hypotonicity-induced [Ca2+ ]i increase in the principal cells of rat CCDs. However, it is still uncertain to what extent the Ca2+ influx through this pathway contributes to the hypotonicity-induced [Ca2+ ]i increase in rat CCDs. In rabbit CCDs, it was reported that not only IP3 -sensitive Ca2+ store but also caffeinesensitive store was functionally expressed [47]. In rabbit TALH cells, it was suggested that Ca2+ -induced Ca2+ release contributed to the Ca2+ signaling, which underlies the RVD mechanisms [46]. In this study, we cannot exclude the possibility of the involvement of the Ca2+ mobility from intracellular store in the hypotonicity-induced [Ca2+ ]i increase of principal cells in rat CCDs. Although the Ca2+ release from the store might play an important role in hypotonicityinduced [Ca2+ ]i increase, it would be possible that the reduction of Ca2+ entry by nicardipine inhibited the Ca2+ -induced Ca2+ release in rat CCDs. In this study, it remains unclear what factors trigger the activation of nicardipine-sensitive Ca2+ entry pathway. Membrane stretch or changes in membrane potential induced by hypotonicity might activate the nicardipine-sensitive Ca2+ influx. Further electrophysiological studies will be necessary to address these issues. In the epithelial cells of CCD, immunohistochemical studies have shown that ␣1c , a subunit of cardiac L-type Ca2+ channel was expressed at the basolateral and apical membrane [48]. However, in general, cardiac L-type Ca2+ channel is completely inhibited by 10 ␮M nifedipine [49], suggesting that cardiac L-type Ca2+ chan-

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Fig. 6. Effect of nicardipine on the Mn2+ influx in rat CCDs. (A) The time course of change in Fura-2 fluorescence excited at 360 nm was monitored in the absence (left panel) and presence (right panel) of Mn2+ . The relative fluorescence intensity was normalized to the control condition before adding Mn2+ . The slow decrease of Fura-2 fluorescence was observed after application of MnCl2 (200 ␮M, arrow). Hypotonic stimulations (open squares) were applied for 8 min. (B) Effect of nicardipine (10 ␮M) on the hypotonicity-induced enhancement of Fura-2 fluorescence quenching. Nicardipine was applied during the periods indicated by filled square. Hypotonic stimulations (open squares) were applied for 8 min. (C) The percentage fluorescence reduction during the hypotonic stimulation is summarized. Data indicate as mean ± SEM (n = 10 for hypotonic, n = 5 for hypotonic + Mn2+ , n = 5 for hypotonic + Mn2+ + nicardipine). *p < 0.05 vs. hypotonic + Mn2+ .

nel was not involved in the hypotonicity-induced [Ca2+ ]i increase in this study. It was also reported that an ␣-subunit of T-type Ca2+ channel (Cav 3.1) was also expressed in CCDs [50]. Recently, it was demonstrated that all three subtypes of T-type Ca2+ channels (Cav 3.1, Cav 3.2 and Cav 3.3) were potently blocked by nicardipine, but not by nifedipine [51]. Therefore, it would be possible that a Ca2+ channel composed of Cav 3.1 subunit might involve in the nicardipine-sensitive Ca2+ entry pathway in rat CCDs. Determination of the molecular basis of this Ca2+ influx pathway will be required to clarify the mechanisms underlying the hypotonicityinduced [Ca2+ ]i increase in rat CCDs. Since CCD is often exposed to hypotonic luminal fluid, cell volume regulation in response to osmotic stress would be important to maintain the cellular function. Although precise role of the nicardipine-sensitive Ca2+ entry pathway in the function of CCDs is still unknown, it is highly likely that the Ca2+ entry during the hypotonicity plays a key role in volume regulation mechanisms.

Acknowledgements This study was partly supported by Grants-in-Aid for Young Scientists (B) from the Japan Society for the Promotion of Science (to Y.K., 21790212) and grant from the Corporation for Private School of Japan (to M.K., 2008–2009).

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