INTRODUCTION

Osteoblasts, which are bone-forming cells, are a main target cell for calciotropic hormones including 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃).¹ 1,25(OH)₂D₃, the most biologically active metabolite of vitamin D₃, generates biological responses via genomic as well as rapid, nongenomic mechanisms (reviewed in Refs. 1-3). Genomic effects comprise the regulation of the transcription of different genes via interaction of the hormone with a nuclear receptor (nVDR) which in turn interacts with hormone response elements in the promoter region of those specific genes (4, 5). In contrast, nongenomic effects (6, 7) are likely initiated at the cell membrane level and seem to involve a putative membrane receptor for 1,25(OH)₂D₃ (8), a variety of second messengers (2), and the modulation of ion channel activity (9-11).

A wide variety of ion channels has been described in primary cultured osteoblasts and osteoblast-like cell lines (9-21). In particular, chloride channel activity in osteoblasts has been postulated to be related to rapid changes in the electrical state of the cell and a regulatory cellular volume response observed under the influence of different hormones acting on the bone (22-25).

To date, only a few published papers have shown the modulatory effects of 1,25(OH)₂D₃ on ion channel activity in target cells, which are believed to represent one of the first examples of a nongenomic response. It has been demonstrated in osteoblasts that the hormone facilitates the opening of L-type Ca²⁺ channels (9, 10); these responses occurred within seconds after the addition of 1,25(OH)₂D₃. Recently, we reported in a preliminary communication that 1,25(OH)₂D₃ also increases an outwardly rectifying anion conductance in ROS 17/2.8 cells (11). In this report we study via whole cell patch-clamp techniques the relative ability of the conformationally flexible 1,25(OH)₂D₃ and related analogs to modulate chloride channels in ROS 17/2.8 cells. Our results demonstrate that the rapid effects of 1,25(OH)₂D₃ on chloride channels are determined by specific structural components of the agonist ligand. Thus the 1-hydroxy epimer 1,25(OH)₂D₃ was found to be an antagonist of 1,25(OH)₂D₃, and the 6-s-cis locked 1,25(OH)₂-lumisterol was significantly more potent than the 6-s-trans locked 1,25(OH)₂-tachysterol in stimulating Cl currents in these bone cells. This is also the first extended report on the participation of Cl channels in nongenomic effects of 1,25(OH)₂D₃.

EXPERIMENTAL PROCEDURES

ROS 17/2.8 cells (obtained from M. C. Farach-Carson) were cultured in Ham's F-12 medium (Sigma) containing 5% fetal bovine serum (Sigma) and 5% Serum Plus (JRH Biosciences, Woodland, CA), as described previously (11). For patch-clamp experiments, cells were plated at very low density in 35-mm tissue culture dishes. Prior to recordings, the cells were washed at least three times with the electrophysiological external solution to remove the medium completely.

Initially we used recording solutions designed by others (9). The composition of the external (extracellular) solution was: 110 mM N-methyl-D-glucamine, 20 mM Ba²⁺, 130 mM glutamate, 10 mM Hepes, and 20 mM glucose (pH 7.4, adjusted with N-methyl-D-glucamine). The pipette (intracellular) solution contained: 100 mM N-methyl-D-glucamine, 150 mM Hepes, 2 mM Mg²⁺, 2 mM Ca²⁺, and 20 mM EGTA. For the recording of Cl currents, we used an external solution (modified from Ref. 23) consisting of 140 mM tetraethylammonium-Cl, 0.5 mM MgCl₂, 1.3 mM CaCl₂, 20 mM BaCl₂, and 10 mM Hepes (pH 7.4, adjusted with tetraethylammonium-OH). The corresponding pipette solution contained 140 mM CsCl, 1.2 mM MgCl₂, 1 mM EGTA, and 10 mM Hepes (pH 7.4, adjusted with CsOH). The osmolarity of the solutions was 290 mosmol/kg (adjusted with glucose). The presence of 20 mM external Ba²⁺ allowed the simultaneous recording of inward currents through Ca²⁺ channels. A high concentration of tetraethylammonium⁺ outside and Cs⁺ inside the cell prevented the recording of K⁺ channel activity.

Patch-clamp recordings (26) were performed with an Axopatch 1C patch-clamp amplifier (Axon Instruments, Foster City, CA). Patch pipettes were fabricated from Drummond capillaries (Drummond Scientific Co., Broomall, PA), coated with Sylgard elastomer (Dow Corning Corp., Midland, MI) to reduce capacitative transients, and fire-polished. Experiments were carried out at room temperature. Currents were low-pass-filtered at 1 kHz and digitized every 100 µs. Cell membrane capacitance and series resistance were electronically compensated prior to the recording of currents. Leak current compensation was done by subtracting the leak current elicited by an hyperpolarizing pulse from 70 mV to 80 mV. In all experiments, depolarizing pulses were applied at intervals of 2-4 s.

1,25(OH)₂D₃, 25-OH-D₃, and 1,25(OH)₂D₃ (analog HL) were obtained from M. Uskokovic (Hoffmann-La Roche); 1,25(OH)₂-lumisterol₃ (analog JN) and 1,25(OH)₂-tachysterol (analog JB) from W. H. Okamura (University of California, Riverside, CA) (27). Vitamin D analogs were stored in the dark as stock solutions in absolute ethanol at 20 °C and added to the bath solution containing 1 mg/ml cytochrome c, which was used as a nonspecific protein carrier. Appropriate controls for cytochrome c and a final ethanol concentration of 0.01% or less were carried out before the addition of the analogs to the bath. CdCl₂ (Sigma) and nifedipine (Sigma) were used as specific Ca²⁺ channel blockers and were added to the bath from aqueous and ethanolic stock solutions, respectively. 4,4-Diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, Sigma) was used as a specific Cl channel blocker and was added to the bath from a stock solution made in the recording medium. Cholesterol (Calbiochem) and -estradiol (Sigma) were added to the bath from a stock solution in ethanol.

RESULTS

Effects of 1,25(OH)₂D₃ on Ion Currents in ROS 17/2.8 Cells

The structures of the conformationally flexible 1,25(OH)₂D₃ and other analogs employed in this study are shown in Fig. 1.

Structures of 1,25(OH)₂D₃ and its analogs.

We first recorded the activity of previously described voltage-dependent L-type Ca²⁺ channels in the ROS 17/2.8 cells (9, 10, 15). Fig. 2 shows the effect mediated by 1,25(OH)₂D₃ on Ba²⁺ currents when it was added to the bath at a final concentration of 0.5 nM. This effect, which have been described before (9, 10, 15), was characterized by a drastic shift of I/V relations of about 25 mV to more negative potentials in the case of this particular cell and developed over the course of the first few minutes after the addition of the hormone. In this experiment, an increase in the concentration of 1,25(OH)₂D₃ to 5 nM did not cause any additional effect, although this result varied among different cells. We recorded a mean shift of 10.4 ± 2.6 mV in 8 out of 22 cells (36%) studied under the same conditions. These 1,25(OH)₂D₃-sensitive inward Ba²⁺ currents were almost completely blocked by 100 µM Cd²⁺, a Ca²⁺ channel blocker, subsequently added to the bath. As postulated previously (9), this modification of the voltage sensitivity of Ca²⁺ channels in osteoblasts by 1,25(OH)₂D₃ may result in the facilitation of Ca²⁺ channel opening by the hormone for Ca²⁺ uptake at membrane potentials close to the resting value, which has been reported to be in the range of 10 to 40 mV in osteoblasts (25, 28, 29).

Effect of 1,25(OH)₂D₃ on inward barium currents in a ROS 17/2.8 cell.

In a preliminary communication we reported that 1,25(OH)₂D₃ also affects Cl channel activity in the ROS 17/2.8 cells (11). In the presence of 100 µM Cd²⁺ in the bath which effectively blocks Ca²⁺ channel activity, an outward current was recorded in the range of 30 to 80 mV in the glutamate-containing external solution (see "Experimental Procedures") in approximately 80% of the cells, as shown in Fig. 3. The addition of a final concentration of 0.05 nM 1,25(OH)₂D₃ to the bath caused, in the case of this particular cell, a 1.2-fold increase of the outward current measured at 80 mV over the course of 2.5 min. The time course of this effect is shown in Fig. 3B. After a lag period of about 45 s, which could be attributed to the time required for diffusion of the steroid to the cell and/or the stimulation of cellular signal transduction pathways, the rapid increase of the current took place over the course of the next 30 s. 1,25(OH)₂D₃ promoted the increase of outward currents in 14 out of 15 cells (93%) studied under the same recording conditions. This enhancing effect was dependent on the concentration of the hormone in the range of 0.05-50 nM (see also Fig. 5).

Effect of 1,25(OH)₂D₃ on outward currents in a ROS 17/2.8 cell.

Fold increase of outward currents in ROS 17/2.8 cells mediated by 1,25(OH)₂D₃ in the absence and presence of 1 nM 1,25(OH)₂D₃.

Permeability studies carried out by replacing the main anion in the recording solution are summarized in Table I. We found that the 1,25(OH)₂D₃-sensitive outward current was permeable to glutamate and Cl, suggesting a poor anion discrimination of this channel. On the other hand, gluconate in the external solution significantly decreased outward currents, as expected for a less permeable anion. The addition of 200 µM DIDS, a specific Cl channel blocker, to the bath blocked the 1,25(OH)₂D₃-enhanced outward currents, as shown in Fig. 4. This blockade by DIDS was time- and voltage-dependent. It developed over the course of 1-2 min after the addition of the agent to the bath. As described before for the blockade by DIDS of the cAMP-activated Cl currents in primary cultured rat osteoblasts (22) and of the mechanosensitive Cl channels in ROS 17/2.8 cells (23), these 1,25(OH)₂D₃-sensitive Cl currents were strongly reduced by the agent but were not completely blocked. DIDS was shown to be effective at a concentration of 200 µM when any of the anions shown in Table I were used.

Table I. Permeability of the 1,25(OH)2D3-sensitive outward conductance in ROS 17/2.8 cells to different anions The outward current amplitude was measured at 80 mV. Concentrations of ions are in mM. NMDG, N-methyl-D-glucamine; TEA, tetraethylammonium. External solution Internal solution Current amplitude Main anion Main cation Main anion Main cation pA 130 glutamate 110 NMDG+ 150 Hepes 100 NMDG+ 178  ± 19 (n = 9) 140 Cl 140 TEA+ 140 Cl 140 Cs+ 486  ± 106 (n = 14) 140 gluconate 140 Na+ 140 Cl 140 Cs+ 5  ± 3 (n = 5)

Blockade of 1,25(OH)₂D₃-promoted outward currents in ROS 17/2.8 cells by the specific Cl channel blocker DIDS.

Fig. 4 also shows the outward rectification of these Cl currents. No inward currents were recorded at membrane potentials below the reversal potential (E_rev) for Cl in the Cl-containing solutions (see "Experimental Procedures"). The E_rev for Cl calculated from the Nernst equation gave a value of 6 mV, which is very close to the value found from the recordings.

Antagonist Effects of the 1-Hydroxy Epimer 1,25(OH)₂D₃

The synthetic analog 1,25(OH)₂D₃ (analog HL), which only differs from the natural metabolite in the orientation of the hydroxy group on carbon 1 (see Fig. 1), has been shown by this laboratory to inhibit the rapid activation of transepithelial calcium transport by 1,25(OH)₂D₃ in chick intestine (30) and ⁴⁵Ca²⁺ uptake in ROS 17/2.8 cells (31). Analog HL has also been shown to reduce the agonist potentiation by 1,25(OH)₂D₃ of Ca²⁺ channels in ROS 17/2.8 cells (32). In this work, we investigated the effects of 1,25(OH)₂D₃ on outward Cl currents in the osteoblastic cells. No enhancement of the Cl currents by 1 nM 1,25(OH)₂D₃ was found in 44% of the studied cells, while we recorded a modest 1.1 ± 0.3-fold increase of the outward currents at 80 mV in the remaining 56% of the cases (9 out of 16 cells) (data not shown). The addition of 0.05-5 nM 1,25(OH)₂D₃ to the bath in the presence of 1 nM HL promoted a significantly lower increase of the outward anion current if compared with the increments recorded for the hormone alone (see Fig. 5). On the contrary, when the concentration of 1,25(OH)₂D₃ was raised to 50 nM, the increase of the outward currents in the presence of the -stereoisomer was similar to that measured for the hormone alone, suggesting that both ligands may be competing for the same receptor. These results with HL suggest that the 1-hydroxy epimer 1,25(OH)₂D₃ may act as an antagonist of the 1,25(OH)₂D₃ effects on Cl channels in osteoblasts as it blocked the effects of the hormone on these cells.

The natural metabolite 25-OH-D₃ promoted an increase in Cl outward currents in 12 out of 14 (86%) ROS 17/2.8 cells within the first 5 min when added to the external solution at a concentration of 0.05-5 nM. In the case of the cell shown in Fig. 6A, 5 nM 25-OH-D₃ caused a 2.7-fold increase of the current at 80 mV. On the other hand, 25-OH-D₃ did not cause any significant modification of I/V relations for inward Ba²⁺ currents in the range of 0.05-50 nM (see Fig. 6B), which agrees with previously published results (9, 10). The specificity of this effect of 25-OH-D₃ on Cl currents, which was not found on Ba²⁺ currents in the same cellular system, may be indicative of different modulatory mechanisms underlying the mode of action of the vitamin D metabolites.

Effects of 25-OH-D₃ on inward Ba²⁺ and outward Cl currents in ROS 17/2.8 cells.

Enhancement of Cl Currents by a 6-s-cis Locked and a 6-s-trans Locked Analog

It has been recently shown that synthetic vitamin D analogs locked in the 6-s-cis position (steroid-like molecules, see Fig. 1) are potent agonists for the rapid effects of the hormone, while 6-s-trans locked conformers (extended forms) are inactive for the same responses in target cells (27, 33). Noticeable transcaltachia-promoting effects in chick intestinal epithelium and ⁴⁵Ca²⁺ uptake by ROS 17/2.8 cells have been described specifically for the synthetic conformer 1,25(OH)₂-lumisterol₃ (analog JN), which is locked in the 6-s-cis position (27). We found that 1-10 nM analog JN caused a 2.2 ± 0.7-fold increase of the outward anion conductance in 7 out of 10 ROS 17/2.8 cells (70%). This response did not differ significantly from the effects promoted by 0.5 nM 1,25(OH)₂D₃ (p < 0.05, see Fig. 7). On the other hand, 1,25(OH)₂-tachysterol (analog JB), a synthetic 6-s-trans conformer which has been shown to be inactive in both transchaltachia and ⁴⁵Ca²⁺ influx in osteoblasts (27) promoted only a modest 0.8 ± 0.3-fold increase of Cl currents at 80 mV when applied at 1-10 nM in 80% of the cells. In this case, the response differed significantly from the enhancing effect exerted by 0.5 nM 1,25(OH)₂D₃ (p < 0.05, Fig. 7).

Comparative effects of analogs of 1,25(OH)₂D₃ and other steroids on outward currents in ROS 17/2.8 cells.

The magnitude of the effects on Cl currents promoted by the different analogs of 1,25(OH)₂D₃ used in this work is shown comparatively in Fig. 7. Note that the effects promoted by 1-10 nM JN and 1-10 nM JB on these Cl currents differed significantly from each other (p < 0.01).

Specificity of the Response by 1,25(OH)₂D₃ Analogs

The specificity of ion channel responses to 1,25(OH)₂D₃ analogs was investigated by means of the evaluation of possible effects exerted by other steroids on the Cl outward currents in ROS 17/2.8 cells. Steroid hormones have been demonstrated to modify ion channel activity in different cells (34-36). As also shown in Fig. 7, 50 nM cholesterol and 0.01-10 nM -estradiol did not have any significant effect on the Cl currents studied in this work.

DISCUSSION

It has been demonstrated in osteoblasts that different hormones taking part in the process of bone remodeling affect ion channel activity in the plasma membrane. More specifically, electrophysiological measurements carried out on the bone-forming cells have shown that the secosteroid 1,25(OH)₂D₃ increases Ca²⁺ influx through voltage-activated Ca²⁺ channels by facilitating the opening of the channels at membrane potentials close to the resting value (9, 10) and also enhances an outward K⁺ current activated by depolarization (37). However, the precise mechanisms for ion channel modulation by the hormone and their physiological role remain unknown.

We recently described in a preliminary report the enhancing effect of 1,25(OH)₂D₃ on Cl currents in the osteoblastic cell line ROS 17/2.8 cells (11). In the present work, we describe in more detail the effects of 1,25(OH)₂D₃ and related structural analogs on these currents, this being the first extended report on a Cl conductive membrane pathway involved in the rapid responses of this secosteroid.

We found an outwardly rectifying voltage-dependent Cl current activated upon depolarization in ROS 17/2.8 cells that can be increased by external application of physiological concentrations of the hormonally active 1,25(OH)₂D₃. This effect was found in 93% of the studied cells. Since patch-clamp currents are defined on the basis of the direction of the movement of positive charges, an outward current in the case of anions represents an influx of the negative charges into the cell. Therefore, according to our results, the depolarization of the osteoblast membrane from the resting value (40 to 10 mV, according to Refs. 28 and 29) activates an influx of anions, Cl being the most abundant one under physiological conditions.

The potentiation of the anion currents by 1,25(OH)₂D₃ was dependent upon the concentration of the hormone and had a maximal increase at 0.5-5 nM followed by an attenuation at 50 nM. A biphasic effect of 1,25(OH)₂D₃ was also previously described for other nongenomic effects of the hormone including the promotion of Ca²⁺ uptake in ROS 17/2.8 cells and the rapid Ca²⁺ transport process in the chick intestinal epithelium (transcaltachia) (9, 33).

The enhancing effect of 1,25(OH)₂D₃ on the anion currents in the osteoblastic cell line developed rapidly, in the course of only a few seconds to minutes. The rapidity of these effects suggests that they are different from the classical steroid receptor-mediated nuclear effects and are part of an increasing list of 1,25(OH)₂D₃-mediated nongenomic effects described in different target cells (2, 3, 30, 33, 38-41).

Structure-function studies carried out with different structurally related 1,25(OH)₂D₃ analogs on different target cellular systems have proved to be very useful in defining the molecular steps involved in the mechanisms of action of the hormone (reviewed in Ref. 1). In the case of rapid nongenomic effects postulated to be initiated at the plasma membrane level, the use of synthetic analogs has led to the discovery that some structural forms are "preferred" over others which have been shown to be more effective in genomic processes. The natural secosteroid 1,25(OH)₂D₃ exists as a continuum of potential shapes extended from the 6-s-cis (steroid-like) to the 6-s-trans (extended, see Fig. 1) which may interact with the receptors. It has been shown recently that synthetic 6-s-trans locked analogs are inactive in both rapid and some genomic responses, while 6-s-cis locked analogs are potent agonists of membrane-initiated nongenomic effects (27). In the present work, we demonstrate for the first time that the 6-s-cis locked analog 1,25(OH)₂-lumisterol₃ significantly increased outwardly rectifying voltage-activated Cl currents in ROS 17/2.8 cells, whereas the enhancing effects by 1,25(OH)₂-tachysterol, the corresponding 6-s-trans conformer, were significantly lower (see Fig. 7).

In a related study the 1 epimer of 1,25(OH)₂D₃, 1,25(OH)₂D₃, which has been shown to block the rapid effects of the hormone (30-32) and to bind to the cellular membrane in osteoblasts (42), remarkably decreased the potentiation of Cl currents by 1,25(OH)₂D₃ in ROS 17/2.8 cells (see Fig. 5), suggesting that it may also act as an antagonist of ion channel responses by the hormone. We conclude that inversion of the orientation of the hydroxyl on carbon 1 of the hormone (1 for 1) may be enough to block the response of cell ion channels by competitively binding to the same surface receptor as a first step in the process.

Finally, the specific effects found for the natural metabolite 25-OH-D₃ on Cl currents but not on Ba²⁺ currents in ROS 17/2.8 cells (see Fig. 6) open the possibility to the existence of different cellular modulatory mechanisms underlying the control of membrane ionic pathways and the electrical state of the cell by active 1,25(OH)₂D₃.

Although there is growing evidence that 1,25(OH)₂D₃ may exert its rapid, nongenomic effects by interacting with a putative mVDR and by initiating a series of molecular pathways involving second messengers, future experiments need to be carried out to elucidate the molecular steps linking the hormonal signal and the specific enhancement of voltage-dependent outward Cl currents in osteoblasts.