24R,25-(OH) 2 Vitamin D 3 Inhibits 1,25-(OH) 2 Vitamin D 3 and Testosterone Potentiation of Calcium Channels in Osteosarcoma Cells*

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1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 52, Issue of December 27, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. 24R,25-(OH) 2 Vitamin D 3 Inhibits 1,25-(OH) 2 Vitamin D 3 and Testosterone Potentiation of Calcium Channels in Osteosarcoma Cells* (Received for publication, June 19, 1996, and in revised form, September 5, 1996) Kazuhiro Takeuchi and Sandra E. Guggino From the Department of Medicine, The Johns Hopkins School of Medicine, Baltimore, Maryland Calcium influx via L-type calcium channels in osteoblast cells causes a rapid (in seconds) elevation in intracellular calcium initiated by plasma membrane receptors for 1,25-dihydroxyvitamin D 3 (1,25-D 3 ). 24R,25- Dihydroxyvitamin D 3 (24,25-D 3 ) alone, in concentrations up to 200 nm, does not cause potentiation of calcium currents in osteoblasts, but it does inhibit the current potentiation by 1,25-D 3. To determine how various steroids interact in their potentiation of calcium channels, the action of vitamin D 3 analogues and testosterone with calcium channels in the rat osteoblast-like cell line ROS 17/2.8 was investigated. Bath additions of both 1,25-D 3 and testosterone at doses below K1 2 (the dose causing 50% left shift in the current-voltage relationship) are additive in their ability to potentiate calcium channels. When 1,25-D 3 and testosterone are added together at concentrations that would cause a maximal shift in the current-voltage relationship by each agent alone (V max ), the effect of these steroids is not additive. Taken together these data suggest one population of calcium channels is activated by 1,25-D 3 or testosterone. The shift in the current-voltage relationship caused by 1,25-D 3 is reduced by 1,25-dihydroxyvitamin D 3 (1,25-D 3 ), an agent which is thought to act specifically on the plasma membrane receptor for 1,25-D 3, but the potentiation caused by testosterone is not blocked by 1,25-D 3. However, 24,25-D 3 inhibits the left shift in the peak current-voltage relationship mediated by either 1,25-D 3 and testosterone. This result implies that 1) 1,25-D 3 directly displaces 1,25-D 3 but not testosterone from its plasma membrane receptor, and 2) the rapid (in seconds) stimulatory effects of 1,25-D 3 and testosterone on calcium channels are mediated by separate plasma membrane receptors for testosterone and 1,25-D 3, which are blocked by another receptor for 24,25-D 3. The interaction of these three receptors with L-type calcium channels is pertussis toxin-sensitive. A newly described function of 1,25-D 3 1 and its analogues is the rapid (in seconds) regulation of intracellular calcium concentration, first reported in chicken duodenum (1) and later in bone osteoblast cells (2 12). It has been documented by patch * This work was funded by National Institutes of Health Grant DK43423 (to S. E. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: 929 Ross Bldg., 720 Rutland Ave., Baltimore, MD Tel.: ; Fax: The abbreviations used are: 1,25-D 3,1,25-dihydroxyvitamin D 3 ; 1,25-D 3,1,25-dihydroxyvitamin D 3 ; 24,25-D 3,24R,25-dihydroxyvitamin D 3 ; VDR, vitamin D 3 receptor; PTX, pertussis toxin. This paper is available on line at clamp recordings of whole-cell barium currents (2, 3), by 45 Ca 2 uptake (5, 7), and by fluorescence measurements of intracellular calcium, using the calcium indicator acetoxymethyl Quin2 (10), that 1,25-D 3 (2) and other vitamin D 3 metabolites (3, 5, 7, 8, 12 14) stimulate calcium influx into rat osteosarcoma cells (2, 3, 5, 7, 8, 12, 14) and human and rat osteoblasts (4, 6) in primary culture. Guggino et al. (15) and Caffrey and Farach- Carson (2) have shown the presence of L-type calcium channels in ROS 17/2.8 osteoblast-like osteosarcoma cells. These channels, first described in nerve, brain, cardiac and skeletal muscle, are voltage-dependent, conduct barium and calcium, and are blocked by the dihydropyridine antagonist nitrendipine, blocked by the phenylalkylamine antagonist verapamil, and stimulated by the dihydropyridine agonist BayK 8644 (16 19). Using the patch clamp recording technique, Caffrey and Farach-Carson (2) reported that physiological concentrations of 1,25-D 3 potentiate L-type calcium channel openings in ROS 17/2.8 cells. The same vitamin D 3 analogues that cause potentiation of calcium channels by the patch clamp technique (3) also cause calcium influx into ROS 17/2.8 cells (5), thus the potentiation of L-type channel current is considered an initial part of the signal transduction process associated with the plasma membrane vitamin D 3 receptors. The rapid action of 1,25-D 3 is not associated with steroid binding to a nuclear vitamin D 3 receptor (VDR) binding site because Zhou et al. (13) found several 1,25-D 3 analogues stimulated 45 Ca 2 transport in duodenum despite poor binding affinity for the intestinal nuclear VDR. Farach-Carson et al. (5) likewise reported that these same analogues stimulated 45 Ca 2 influx into ROS 17/2.8 cells yet bound poorly to the nuclear VDR of ROS 17/2.8. Yukihiro et al. (3) reported that the potentiation of calcium channels by these analogues is not compatible with VDR being involved in signal transduction because the structural features like double bonds at carbon 16 and 23 (25-hydroxy-16,23E-diene-D 3 ) (Fig. 1, A C) or addition of a hydroxyethyl group at the 1-carbon position (1-(1 -hydroxyethyl)-25-hydroxy-d 3 ), which cause activation of rapid calcium signal transduction (5), virtually eliminate binding to VDR (3). Although it has been reported that 1,25-D 3 and several analogues potentiate calcium channel openings, some vitamin D 3 analogues with inhibitory effects have also been reported. Norman et al. (8) reported 1,25-D 3 decreased rapid transcaltachia stimulated by 1,25-D 3 in intestine. Yukihiro et al. (3) reported that 1,25-D 3 reduced potentiation of calcium current caused by 1,25-D 3 and that 24,25-D 3 alone, even in high concentration (200 nm), does not alter calcium currents in ROS 17/2.8 cells, but it reduced the potentiation of calcium channels by the 1,25-D 3 analogue 1-(1 -hydroxyethyl)-25-(oh)-d 3.On the other hand, Li et al. (21) reported that 10 M 24,25-D 3 reduced the amplitude of barium currents in UMR 106 cells, via a protein kinase C-dependent mechanism which takes several minutes to develop.

2 ,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels FIG. 1. Structure of 1,25-dihydroxyvitamin D 3, 24,25-dihydroxyvitamin D 3, 25-hydroxy-16,23E-diene-D 3. The structure of 1,25-dihydroxyvitamin D 3 is shown in A, 24,25-dihydroxyvitamin D 3 in B, 25-hydroxy-16,23E-diene-D 3 in C. 1-(1 -Hydroxyethyl)-25-(OH)-D 3 (discussed in the Introduction) is 1,25-D 3 with a 1 -hydroxyethyl group substituted for the 1 -hydroxyl group. Androgen receptors have been shown in osteoblast-like cells (22, 23), where, besides the classical functions of sex steroids, rapid actions (in seconds) have also been examined. Lieberherr et al. (10, 11) using fura-2 fluorescence reported that both estrogen and testosterone stimulated calcium influx into osteoblasts which was blocked by calcium channel blockers nitrendipine and verapamil. She found that rapid actions were also produced by testosterone linked to bovine serum albumin, suggesting that this rapid action was induced by a membrane receptor and not by a nuclear receptor (11). She also found this signal transduction pathway was blocked by pertussis toxin (10, 11). In order to elucidate the role of testosterone and vitamin D 3 metabolites on potentiation of calcium channels, we asked several questions regarding the relationship between these pathways. 1) Do sex steroids and vitamin D 3 metabolites potentiate calcium channels in the same manner? 2) Do the steroids potentiate the same population of calcium channels? 3) Do steroids act on the same or separate receptors? 4) Are these steroid receptors coupled via G proteins to calcium channels? In this study we tested the interaction of 1,25-D 3, its analogues, testosterone, and pertussis toxin to evaluate how these steroids potentiate calcium channel function. MATERIALS AND METHODS Cell Culture ROS 17/2.8 cells were grown in 5% CO 2 at 37 C in Ham s F12 medium (Life Technologies, Inc.) containing 5% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 0.1 mg/ml kanamycin buffered with 25 mm HCO 3 and 14 mm Hepes using NaOH to adjust the ph to 7.4. For patch clamp experiments, cells were seeded at low density as single cells onto pieces of coverslips. Patch clamp experiments were performed within 24 h after plating, on single cells after overnight incubation in 1% charcoal-treated serum. The data in each table are compiled from one seeding of cells from which control and test experiments were alternated on the same day. Test Substances 1,25-Dihydroxyvitamin D 3 (1,25-D 3 ), 1,25-dihydroxyvitamin D 3 (1, 25-D 3 ), 24R,25-dihydroxyvitamin D 3 (24,25- D 3 ), 25-hydroxy-16,23E-diene D 3 (25(OH)-16,23E-diene-D 3 ) were kindly provided by Dr. Milan Uskokovic (Hoffmann-La Roche) (Fig. 1) Hydroxyandrost-4-en-3-one (testosterone) was from Sigma. These steroids were stored in the dark as stock solutions in absolute ethanol at 20 C until used. Solutions The perforated-patch recording technique was used for measuring inward barium currents under voltage clamp conditions. The composition of the pipette solution was 100 mm KOH, 150 mm Hepes, 20 mm EGTA, 2 mm CaCl 2,2mMMgCl 2,10mMK 2 HPO 4 buffered to ph 7.4 with KOH and the osmolarity adjusted to 290 mosm/kg using K-Hepes. Amphotericin B, an antibiotic that creates small nonselective pores in the membrane to allow ion flow, was added to this solution at a final concentration of 240 mg/ml, and then the mixture was added to the tip of the pipette. The pipette was then backfilled with pipette saline solution onto the antibiotic saline mixture. The composition of the initial external solution was 140 mm NaCl, 5 mm KCl, 20 mm Hepes buffered to ph 7.4 with NaOH, and the osmolarity adjusted to 290 mosm with Na-Hepes. After whole cell currents were established, a solution which consisted of 115 mm BaCl 2 and 20 mm Hepes buffered to FIG. 2.Whole cell recordings of barium currents in ROS 17/2.8 cells in the absence (control) and the presence of BayK. A, the cell membrane potential was held at 70 mv; 10-mV step pulses were applied for 300 ms between 60 and 50 mv with a 2-s interval between pulses. B-a and B-b, voltage-dependent current traces (control) and in the presence of 1 M BayK C, bath addition of 1 M BayK 8644 (Ç) caused the peak of the current-voltage relationship to shift negatively along the voltage axis from 23.4 to 7.6 mv (vertical lines). The amplitude of the current increased in the presence of BayK 8644 (B and C). BayK 8644 was added at the end of all experiments to verify that the maximal left shift was comparable in all experiments. Peak increase in current occurred 1 min after bath addition of BayK ph 7.4 with tetraethylammonium hydroxide, was added to the initial external solution so the final concentration of barium was 20 mm. Barium was used as a current carrier for two reasons. Barium current through L-type channels is known to be larger than calcium currents, and barium inhibits the activation of potassium channels. External tetraethylammonium was also used as a potassium channel blocker. Hepes, a nonpermeant anion, was used to eliminate inward currents via anion conductance. Using these conditions the inward barium current is stimulated by BayK 8644, completely blocked by nitrendipine, and displays the voltage-gating characteristics indicative of L-type calcium channels. Current Measurement A pipette was placed to the surface of a cell, and then gentle suction was applied until a tight seal of about 10 Gohm was formed. After about 3 10 min the amphotericin B diffused into the cell membrane under the patch pipette causing the capacitance to increase, at which time the experiment was initiated. The cell membrane potential was held at 70 mv; 10 mv step pulses were applied for 300 ms between 60 and 50 mv with a 2-s interval between pulses (Fig. 2). Currents were monitored on an EPC-7 patch clamp amplifier (List Electronics, Darmstadt, Germany) and visualized on a Nicolet digital oscilloscope (Nicolet Instrument, Madison, WI) after filtering at 1 khz through a Bessel filter (Frequency Devices, Haverhill, MA). Test substances (vitamin D 3 metabolites and testosterone) were dissolved in a bath solution with 10% ethanol then added to the bath solution by pipette. The test substances were diluted into the bath in a 1 to 100 ratio giving a final dilution of ethanol of 1/1000. Controls for addition of ethanol did not cause left shift or changes in the amplitude of calcium channel currents. It took about 30 s to add test substances. After the initiation of drug addition, the first currents were recorded within 30 s, and the data were sampled once per min afterward. The data which

3 24,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels TABLE I Effect of 1,25-dihydroxyvitamin D 3 on the peak of the current-voltage relationship of inward barium currents in ROS 17/2.8 cells The cumulative data for 10 experiments involving sequential addition of three increasing concentrations of 1,25-D 3 is shown as average S.E. of the shift in the peak of the current-voltage relationship compared with control. The average left shift is increased by 1,25-D 3 in a dose-dependent manner. The effect of BayK 8644, measured at the end of each experiment, is to assess the maximum of the shift in current, which was always constant and much greater than that of the steroids (n 10). 1,25-D nm 5nM 50 nm BayK 8644, 1 M No. of experiments Peak shift (mv) TABLE II Effect of increasing concentrations of testosterone on the peak of the current voltage relationship of inward barium currents in ROS 17/2.8 cells The cumulative data for 12 experiments involving sequential addition of three increasing concentrations of testosterone is shown as average S.E. of the shift in the peak of the current-voltage relationship compared with control. The average left shift is increased by testosterone in a dose-dependent manner. BayK 8644 is added at the end of all experiments to verify that the maximal left shift is comparable in all experiments (n 12). Testosterone 0.1 nm 1nM 10 nm BayK 8644, 1 M No. of experiments Peak shift (mv) were recorded just after adding samples already showed about 80% left shift, and the data at 1.5 min showed a maximal steady state value (Fig. 4). Data were stored and analyzed on a computer (IBM-PC compatible 386, AST) using the P-clamp software version 5.5 (Axon Instruments, Foster City, CA). At the end of each experiment BayK 8644 (1 M) was added to determine the maximal current through the L-type calcium channels and to standardize the data between experiments (Fig. 2). This amount of BayK 8644 was previously shown in control cells (15) to cause a maximal increase in barium current and a maximal left shift in barium current. This concentration of BayK 8644 usually caused large currents in the range of 20 mv; thus, as a result of the rather high resistance of the amphotericin patch, the clamp was not totally effective in this voltage range. However, the smaller currents caused by steroids, which were evaluated in this work, were adequately clamped. BayK 8644 was used at the end of each experiment to ensure that after three sequential steroid additions the maximal barium currents could be stimulated. The peak of the current-voltage relationship was estimated by drawing a line joining the three largest current amplitudes. Using an equation that weights the slope of the line on either side of the maximal measured negative current, the peak current was estimated. Thus, the peak current was estimated by the equation {(a b)/(a b a c)} 10 mv e, where a is the maximal current measured by the depolarization protocol; b is the current measured 10 mv more positive and c is the current measured 10 mv more negative than maximal current. e is the difference between the voltage depolarization causing the maximal measured current and 0 mv. All data are represented as mean S.E. For statistical analyses, the Student s t test was used, and p 0.05 was recognized as statistically significant. RESULTS Effects of Vitamin D 3 and Testosterone The activation of L-type calcium channels by steroids was investigated using the perforated patch clamp technique. Using 20 mm barium in the external medium to carry inward currents, the maximal potentiation of L-type calcium channels was confirmed at the end of each experiment by the stimulation of currents in response to the dihydropyridine agonist BayK 8644 (Fig. 2, B-b and C, Tables I and II). A concentration of 1 M BayK 8644 monitored the maximal peak inward current via L-type calcium channels, as well as determining the maximal left shift in the peak of the current-voltage relationship (Fig. 2). Bath addition of 1,25-D 3 caused the peak of the current-voltage relationship to shift negatively along the voltage axis but did not increase the amplitude of currents (Fig. 3B) as shown previously (3). This left shift in the current-voltage relationship occurred within 30 s of drug application (Fig. 4) (a function of diffusion time of the drug to the cell surface) and remained constant over a period of 30 min, the usual length of the recording time for stable patches. The maximal left shift for 1,25-D 3 was constant for all the experiments recorded in this paper, throughout several different stock tissue culture preparations, and for various serum lots, although the magnitude decreased to 10 mv from values of 20 mv measured earlier by our laboratory (3). 1,25-D 3 caused a left shift in the current-voltage relationship in a dose-dependent manner (Fig. 3B). The averages of the left shifts of the peak currents are shown in Table I. A maximal left shift for 1,25-D 3 (V max ) was obtained at a concentration of 50 nm ( mv, n 10) and the V1 2, the concentration causing 50% of the maximal left shift of the current-voltage relationship, was 0.37 nm (Fig. 5). Bath addition of testosterone also caused the peak of the current-voltage relationship to shift negatively in a dose-dependent manner (Fig. 6, Table II). The V max of testosterone was slightly higher than 1 nm. The V1 2 was nm (Fig. 7). The Interaction of 1,25-D 3, Vitamin D 3 Analogues, and Testosterone We investigated the interaction of these steroids to determine whether their effects on calcium channels were additive. A dose of 0.5 nm 1,25-D 3 (near the V1 2 for 1,25-D 3 ) and 0.1 nm testosterone (near the V1 2 for testosterone) were additive in their effect on the left shift of the peak of the current-voltage relationship of inward barium current. The total left shift in the current-voltage relationship was mv (n 5) when 0.1 nm testosterone was added after 0.5 nm 1,25-D 3, which by itself caused an initial left shift of mv (n 5) (Table III, Experiment A). 25(OH)-16,23E-Diene-D 3, a more potent analog of 1,25-D 3, which is specific for activating calcium channels, but has less than 1% binding affinity to VDR (24), has a V max of 0.1 nm (data not shown). A concentration of testosterone near the V1 2 (0.1 nm) added after 0.05 nm 25(OH)- 16,23E-diene-D 3 (also near the V1 2) (Fig. 8) is additive in its ability to shift the peak of the current-voltage relationship of the inward barium current (Table III, Experiment B). However, when 10 nm 1,25-D 3 (a V max dose) and 1 nm testosterone (a V max dose) were added together, the left shift in the peak of the current-voltage relationship was no larger than that present when each agent was added alone (Table III, Experiment C). For example, 10 nm 1,25-D 3 followed by 1 nm testosterone caused a left shift of mv (n 3) compared with a left shift of mv (n 3) when 1,25-D 3 was added alone (Table III, Experiment C). 100 nm 1,25-D 3 has been shown previously to reduce the potentiation of calcium channels by 1,25-D 3 (3). 1,25-D 3 by itself does not cause a left shift of the peak of inward barium currents at a concentration of up to 100 nm (Table IV, Experiments A and B, line 1) (3), but the left shift in the currentvoltage relationship was reduced by 1,25-D 3 to 32% of control for 1,25-D 3 and 47% of control for 25(OH)-16,23E-diene-D 3 (Table IV, Experiments A and B). On the other hand, the left shift in the current-voltage relationship caused by testosterone was maintained constant even when 100 nm 1,25-D 3 was

4 ,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels FIG. 3.Whole cell recordings of barium currents in the absence (control) and the presence of 1,25-dihydroxyvitamin D 3. A, the pulse protocol was the same as in Fig. 2A, but only the step pulses between 0 and 40 mv are shown for clarity. B-a are representative control current traces (top) and the resulting current-voltage relationship (below). B-b d are the current traces and the current-voltage relationships of the same cell as B-a given three increasing, sequentially added concentrations of 1,25-D 3. Bath addition of 1,25-D 3 caused, in a dose-dependent manner, the peak current of the current-voltage relationship to shift from 25 mv in control to more negative values along the voltage axis. The vertical line shows the peak of the current-voltage relationship for each concentration of 1,25-D 3 ( 25.0 mv for control; 19.1 mv for 0.5 nm 1,25-D 3, a left shift of 5.9 mv; 15.0 mv for 5 nm 1,25-D 3, a left shift of 10.0 mv; and 14.6 mv for 50 nm 1,25-D 3, a left shift of 10.4 mv). The magnitude of the current did not increase with addition of 1,25-D 3. The symbols for each voltage are as follows:, 0 mv;, 10mV;Ç,20mV;,30mV;,40mV(B-b d). Current traces shown were measured 1.5 min after addition of steroids, when the maximal left shift in current occurs (Fig. 4). added before testosterone. The left shifts of mv for 0.1 nm, mv for 1 nm, and mv for 10 nm testosterone (Table IV, Experiment D) were not significantly different (p 0.05) when compared with the left shifts seen in the absence of 1,25-D 3 (Table IV, Experiment C). Thus, regardless of whether testosterone was present in concentrations below the V1 2 or severalfold above the V1 2, 1,25-D 3 had no effect. 24,25-D 3, in concentrations up to 200 nm, did not cause a left shift in the current-voltage relationship when added alone (Table V, Experiments A and B, line 1) (3). We tested the effect of 24,25-D 3 on calcium channel potentiation produced by other steroids. When 100 nm 24,25-D 3 was applied before 5 nm 1,25- D 3, the resulting left shift of the peak current-voltage relationship was only mv (n 4) (Table V, Experiment A, line 2) as compared with mv (n 5) when 1,25-D 3 was applied alone (Table V, Experiment A, line 5). Likewise, the left shift produced by 0.1 nm 25(OH)-16,23E-diene-D 3 was reduced to 48% of control when 24,25-D 3 was added to the bath before 25(OH)-16,23E-diene-D 3 (Table V, Experiment B, line 2). Therefore, the presence of 24,25-D 3 blunted the left shift of the current-voltage relationship caused by either 1,25-D 3 or 25(OH)-16,23E-diene-D 3. Similarly, 24,25-D 3 reduced the left shift in the current-voltage relationship caused by testosterone (Table V, Experiment D). When 100 nm 24,25-D 3 was added before 0.1, 1.0, or 10 nm testosterone, the left shift at each concentration was significantly decreased in the presence of 24,25-D 3 (p 0.05). The final left shift, when 100 nm 24,25-D 3 was added in combination with testosterone, was mv for 0.1 nm testosterone, mv (n 4) for 1 nm testosterone, and mv (n 4) for 10 nm testosterone, respectively (Table V, Experiment D), as compared with mv (n 4) for 0.1 nm, mv (n 4) for 1 nm, and mv (n 4) for 10 nm when testosterone was added alone (Table V, Experiment C). Thus, regardless of whether testosterone was added below the V1 2 or above the V1 2, 24,25-D 3 effectively decreased the left shift in barium current. Effects of Pertussis Toxin (PTX) on the Potentiation Caused

5 24,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels FIG. 4.Time course of effect of 1,25-dihydroxyvitamin D 3. The first currents were recorded within 30 s of drug application (a function of diffusion time for addition of drug to the bath), and the data were sampled once per min afterward. The left shift in the current-voltage relationship occurred within 30 s and reached constant value by 1.5 min (n 6). All data points shown in the tables and other figures were obtained at the 1.5-min time point after steady state left shift was reached. FIG. 5. Dose-response curve for the effect of 1,25-dihydroxyvitamin D 3 on the left shift of inward barium currents. The average left shift was increased by 1,25-D 3 in a dose-dependent manner. V1 2 (the concentration causing a 50% of the maximal left shift) was 0.37 nm (n 10). by 1,25-D 3, 24,25-D 3, Testosterone We investigated the effects of PTX to determine whether the potentiation of calcium channels caused by steroids was mediated via G-proteins. Cells were preincubated for min with 500 ng/ml PTX, and then 5nM1,25-D 3 (V max dose) or 1 nm testosterone (V max dose) was added to the cell as described previously. The cells preincubated with PTX showed a decreased left shift in the currentvoltage relationship caused by 1,25-D 3 or testosterone. For 1,25-D 3, PTX reduced the left shift to under 30% of that caused by 1,25-D 3 alone in 7/14 cells. The average left shift of mv (n 14) caused by 1,25-D 3 in cells preincubated with PTX was significantly different (p 0.01) when compared with the left shifts seen in the cells without preincubation (Table VI, Experiment A). For testosterone, PTX reduced the left shift to under 50% of that caused by testosterone alone (4/15 cells). The average left shift of mv (n 15) caused by testosterone in cells preincubated in PTX was significantly different (p 0.05) compared with control group (Table VI, Experiment B). Under normal circumstances 24,25-D 3 does not cause a left shift in the current-voltage relationship ( mv) (n 6), but cells preincubated in PTX showed a left shift of mv in 8/9 cells (Table VI, Experiment C). The left shift was significantly different (p 0.01) compared with the control group. All PTX-treated cells had normal responses to BayK DISCUSSION Intracellular calcium concentration in most cells is regulated by the release of calcium from intracellular calcium storage compartments (25, 26) and calcium influx through calcium channels (2, 15, 27, 28). The rapid increase of intracellular calcium concentration caused by vitamin D 3 analogues and other steroid hormones is an area of increasing investigation (2 13, 29, 30). 1,25-D 3 and its analogues rapidly activate inward calcium movement in osteoblasts or osteoblast-like cells (2 7, 12). Using acetoxymethyl Quin2 fluorescent calcium indicator, Lieberherr (6) concluded, based on inhibition by dihydropyridine drugs like nitrendipine, that 70% of the increase in intracellular calcium concentration caused by 1,25-D 3 was from calcium influx via L-type calcium channels, suggesting this is a major pathway for calcium influx. Furthermore, she found that testosterone and estrogen also caused increases in intracellular calcium (10, 11). We have verified by the patch clamp technique that 1,25-D 3 and testosterone both act by the same mechanism (a left shift in the current-voltage relationship) to potentiate the opening of L-type calcium channels. FIG. 6.Whole cell recordings of barium currents in the absence (control) and the presence of testosterone. The current-voltage relationship of one representative cell given three sequentially added, increasing concentrations of testosterone. Bath addition of testosterone caused the peak of the current-voltage relationship to shift negatively along the voltage axis in a dose-dependent manner. Testosterone did not increase the amplitude of barium currents. The vertical line shows the estimated peak for the currentvoltage relationship for each concentration of testosterone versus control. The peak for control is 26.6 mv; a peak for 0.1 nm testosterone is 23.0 mv, a left shift of 3.6 mv; a peak for 1 nm testosterone is 15.1 mv, a left shift of 11.5 mv; a peak for 10 nm testosterone is 13.5 mv, a left shift of 13.1 mv. The symbols for the voltages are as follows:, 0 mv;, 10mV;Ç,20mV;,30mV.

6 ,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels FIG. 7. Dose-response curve for the left shift of the currentvoltage relationship caused by testosterone. The average left shift is increased by testosterone in a dose-dependent manner. V1 2 was calculated to be nm (n 12). TABLE III Interaction of 1,25-dihydroxyvitamin D 3, and a vitamin D 3 analog with testosterone on barium currents Experiments A and B, In these experiments 0.5 nm 1,25-D 3 (near V 1/2 dose) or 0.05 nm 25(OH)-16,23E-diene-D 3 (near V 1/2 dose) was added to the bath first; the left shift in the peak of the current-voltage relationship was measured, and then 0.1 nm testosterone (near V 1/2 dose) was added to the bath, and the effect of the analogues added together was measured. The data show additive effects. Experiment C, 10 nm 1,25-D 3 (at V max dose) was added to the bath first and a current-voltage relationship measured, and then 1 nm testosterone (at V max dose) was added to the bath, followed by a second measurement of the current-voltage relationship. The effect of these agents was not additive when both steroids were added at a V max dose. Average peak shift compared to control (mv) Experiment A 1,25-D nm a Testosterone 0.1 nm with 1,25-D nm a BayK M No. of experiments (n x) 5 Experiment B 25(OH)-16,23E-diene-D nm b Testosterone 0.1 nm with 25(OH)-16,23E-diene-D nm b BayK M No. of experiments (n x) 6 Experiment C 1,25-D 3 10 nm c Testosterone 1 nm with 1,25-D 3 10 nm c BayK M No. of experiments (n x) 3 a p b p c Not significant. FIG. 8. Dose-response curve for the left shift of the currentvoltage relationship caused by 25-hydroxy-16,23E-diene-D 3. The average left shift is increased by testosterone in a dose-dependent manner. V1 2 was calculated to be nm (n 5). To determine whether calcium movement is initiated by the well characterized nuclear steroid receptor, VDR, or a totally separate plasma membrane receptor, 1,25-D 3 and its analogues have been tested on transepithelial movements of 45 Ca 2 in intestine (transcaltachia) (1, 5, 7, 20) or 45 Ca 2 influx (2, 5, 7, 12) or whole cell currents (2 4) in osteosarcoma cells. The OH group at the 1 position of 1,25-D 3 is well known to confer potency on VDR (29 31) (Fig. 1A). However, other parts of the vitamin D 3 structure like a double bond in the C/D side chain (Fig. 1, A C) are permissive for the rapid movements of calcium associated with L-type calcium channels. Zhou et al. (13), Farach-Carson et al. (5), and Yukihiro et al. (3) reported stimulatory effects of analogues with double bonds at the 23- carbon position. These data indicate that synthetic analogues that stimulate calcium influx have different structural features that are not compatible with an affinity at the nuclear VDR binding site. Current thinking suggests that regulation of calcium channel openings may be initiated by a different ligand binding receptor from that which initiates the genomic action. Thus, in order to eliminate the possibility of a VDR-type binding site, we verified all experiments involving 1,25-D 3 with the analogue 25(OH)-16,23E-diene-D 3 which acts only to stimulate calcium channels and has minimal binding to VDR (24). Unlike BayK 8644, these steroids do not increase the amplitude of currents but rather shift the current-voltage relationship (Figs. 3B and 6 and Tables I and II), suggesting a mechanism of action on calcium channels which involves alteration in the voltage dependence of the channel. In addition, 1,25-D 3, 25(OH)-16,23E-diene-D 3, and testosterone all had the same V max for the left shift, suggesting they alter the voltage dependence by the same amount. The fact that these steroids all have about 1 3 the efficacy of BayK 8644 implies that they are less potent than dihydropyridine agonists in potentiation of total calcium channel current or that they act only on a portion of the population of all calcium channels. We investigated the effect of combinations of 1,25-D 3, its analogues, and testosterone on calcium channel currents in rat osteoblast-like cells in order to better understand the interaction between the steroid receptors and the calcium channels they modulate. 1,25-D 3, 25(OH)-16,23E-diene-D 3, and testosterone, when supplied at doses near the V1 2, add together to shift the peak of the currentvoltage relationship (Table III, Experiments A and B), but when given at their V max concentration their effects are not additive (Table III, Experiment C), implying that these agents converge on the same population of calcium channels. Norman et al. (8, 9) reported the inhibitory effect of 1,25-D 3 on the transcaltachia stimulated by 1,25-D 3. Yukihiro et al. (3) demonstrated the inhibitory effect of 1,25-D 3 on the peak shift of the current-voltage relationship of currents potentiated by 1,25-D 3. Therefore, we evaluated effects of 1,25-D 3 on the potentiation caused by 1,25-D 3, 25(OH)-16,23E-diene-D 3, and testosterone. 1,25-D 3 reduced the left shift in the calcium channel current-voltage relationship potentiated by 1,25-D 3 or 25(OH)-16,23E-diene-D 3 but did not alter the potentiation caused by testosterone (Table IV). Because 1,25-D 3 is very similar to 1,25-D 3 in structure, it may block directly at the plasma membrane 1,25-D 3 receptor but does not compete at the plasma membrane testosterone receptor which probably has a very different binding site. Interestingly, 24,25-D 3, by itself, stimulates transcaltachia in the duodenum (30). In ROS 17/2.8 cells, 24,25-D 3 inhibits 45 Ca 2 influx and reduces whole-cell barium currents at very positive membrane potentials when concentrations greater than 1.0 nm are added to the bath (5). In patch clamp experiments measuring barium currents of ROS 17/2.8 cells, 24,25-D 3 caused no left shift of the peak of the current-voltage relationship when added in concentrations between 0.5 and 200 nm, although it does decrease calcium currents at very positive membrane potentials (3). In UMR 106 cells, 10 nm 24,25-D 3 increased the amplitude of barium current, whereas a high concentration (10 M) of 24,25-D 3 reduced the amplitude of the

7 24,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels TABLE IV Effect of 1,25-dihydroxyvitamin D 3 on agonist potentiation of barium currents Experiments A and B, in these experiments 100 nm 1,25-D 3 was added to the bath first and then 5 nm 1,25-D 3 (at V max dose) or 0.1 nm 25(OH)-16,23E-diene-D 3 (at V max dose) was added to the bath afterward. 100 nm 1,25-D 3 did not cause a left shift in the current-voltage relationship when added by itself (line 1). 1,25-D 3 decreased the left shift caused by 1,25-D 3 or 25(OH)-16,23E-diene-D 3 (line 2) compared with the current-voltage relationship measured in cells to which the agent was added alone (line 5). Experiment C, in these experiments testosterone was added alone. The peak left shift was increased by testosterone in a dose-dependent manner. Experiment D, 100 nm 1,25-D 3 was added to the bath first, then testosterone was added to the bath afterward. The shift in current-voltage relationship was compared for each concentration of testosterone. The peak-shift induced by testosterone was not decreased by 1,25-D 3 (Experiment D) when compared with cells which received testosterone alone even when testosterone was added at doses in 10-fold excess of saturation concentration needed to cause a left shift in the current-voltage relationship (Experiment C). 1 M BayK 8644 added at the end of these experiments produced consistent effects ( , n 11). When the results were compared using a Student s test, c and d were significantly different at p 0.01 and p 0.05, respectively, but e, f, and g were not statistically different. Average peak shift compared to control (mv) Experiment A 1,25-D nm (alone) a 1,25-D 3 5nMwith 1,25-D nm b BayK 8644, 1 M No. of experiments (n x) 4 1,25-D 3 5nM(alone) a,b Experiment B 1,25-D nm (alone) c 25(OH)-16,23E-diene-D nm with 1,25-D nm d BayK 8644, 1 M No. of experiments (n x) 4 25(OH)-16,23E-diene-D nm (alone) c,d Testosterone 0.1 nm 1 nm 10 nm Experiment C Peak shift (mv) e f g No. of experiments Testosterone with 100 nm 1,25-D nm 1nM 10 nm Experiment D Peak shift (mv) e f g No. of experiments a c p d p e g Not significant. TABLE V Inhibitory effect of 24,25-dihydroxyvitamin D 3 on agonist-stimulated barium currents Experiments A and B, in these experiments 100 nm 24,25-D 3 was added to the bath first and then 5 nm 1,25-D 3 (at V max dose) or 0.1 nm 25(OH)-16,23E-diene-D 3 (at V max dose) was added to the bath afterward. 100 nm 24,25-D 3 did not cause a left shift in the current-voltage relationship when added alone, but the presence of 24,25-D 3 decreased the left shift caused by the subsequent addition of 1,25-D 3 or 25(OH)- 16,23E-diene-D 3 when compared with that value found when each agent was added alone. Experiment C, in these experiments where testosterone was added alone, the peak left shift was increased in a dose-dependent manner. Experiment D, 100 nm 24,25-D 3 was added to the bath first; the current-voltage relationship was measured (100 nm 24,25-D 3 did not cause a left shift in the current-voltage relationship; mv) (n 4), and then a concentration of testosterone was added to the bath, and the current-voltage relationship was measured again. 24,25-D 3 decreased the calcium channel potentiation stimulated by each concentration of testosterone whether testosterone was added at dose below or above saturation of the testosterone receptor. The shift in the peak of the current-voltage relationship (Experiment D) was compared with cells of the same pass exposed to three increasing concentrations of testosterone (Experiment C). The left shift caused by 1 M BayK 8644 at the end of these experiments was constant ( , n 12). When Student s test was used to compare control and experimental conditions the differences were significant. Average peak shift compared to control (mv) Experiment A 24,25-D nm (alone) a 1,25-D 3 5nMwith 24,25-D nm b BayK M No. of experiments (n x) 4 1,25-D 3 5nM(alone) a,b Experiment B 24,25-D nm (alone) c 25(OH)-16,23E-diene-D nm with 24,25-D nm d BayK M No. of experiments (n x) 4 25(OH)-16,23E-diene-D nm (alone) c,d Testosterone (alone) 0.1 nm 1 nm 10 nm Experiment C Peak shift (mv) e f g No. of experiments Testosterone with 100 nm 24,25-D nm 1nM 10 nm Experiment D Peak shift (mv) e f g No. of experiments a e p f, g p current (21). The left shift in current-voltage relationship caused by 24,25-D 3 occurs within 1.5 min, suggesting a direct effect on the gating mechanism of the channel. Changes in the amplitude of barium currents occur over min, probably to involve phosphorylation of calcium channel by protein kinase A and C pathway (21). Thus, 24,25-D 3, by itself, can mediate effects on calcium channels via multiple mechanisms. 24,25-D 3 also inhibits the left shift in the current-voltage relationship caused by 1,25-D 3, 25(OH)-16,23E-diene-D 3,or testosterone (Table V). 24,25-D 3 blocks potentiation of calcium channels caused by two separate steroid signaling pathways, that of 1,25-D 3 and testosterone. Nemere et al. (32) reported separate binding proteins for 1,25-D 3 and 24,25-D 3 in chick intestinal epithelium basolateral membranes. Our data are consistent with the idea that 24,25-D 3 may act via an independent receptor which is separate from the receptors for 1,25-D 3 or testosterone and which can decrease potentiation of calcium currents caused by 1,25-D 3 or testosterone. Classically, 24,25-D 3 has been thought to be a less active form of 1,25-D 3 because the genomic action is the same as 1,25-D 3, although the affinity at the VDR binding site is 2000 less than that for 1,25-D 3 (33). Although 24,25-D 3 and 1,25-D 3 both cause the rapid movement of calcium in transcaltachia, 24,25-D 3 also has rapid physiological effects which are independent of 1,25-D 3. Langston et al. (34) showed, using 45 Ca 2 uptake, that different patterns of calcium transport in cultured chondrocyte cells were modulated by 1,25-D 3 and 24,25-D 3. For example, growth zone chondrocytes responded primarily to 1,25-D 3 by stimulating both transmembrane 45 Ca 2 influx and efflux, whereas resting zone chondrocytes respond to 24,25-D 3 by an inhibition of influx and efflux of 45 Ca 2. We find 24,25-D 3 inhibits the action of 1,25-D 3 on L-type calcium channels. Based on previous work showing that L-type calcium channel agonists stimulate bone matrix production and bone resorption (15), the regulation of calcium influx via L-type calcium channels may be very important for bone metabolism. Thus, a new role for 24,25-D 3 may be inhibition of that component of bone remodeling mediated via the nongenomic 1,25-D 3 receptor through L-type calcium channels. Two modes of calcium channel modulation via G-proteins

8 ,25-(OH) 2 Vitamin D 3 Regulates Calcium Channels TABLE VI Pertussis toxin (PTX) inhibits potentiation of barium currents by 1,25-dihydroxyvitamin D 3, testosterone and 24,25-dihydroxyvitamin D 3 In the control group (Experiment A), 5 nm 1,25-D 3 (at V max dose) was added to the bath without preincubation of PTX. 5 nm 1,25-D 3 caused 9.4 mv left shift in the current-voltage relationship (line 1). In the PTX-treated group, cells from the same pass and cell split were preincubated with 500 ng/ml PTX for min, and then 5 nm 1,25-D 3 (at V max dose) was added to the bath afterward. Some cells preincubated with PTX showed a decrease in the left shift caused by the addition of 1,25-D 3 (line 2) when compared with control (line 1). PTX reduced the magnitude of the left shift to under 30% of control in 7/14 cases. The left shift caused by 1 M BayK 8644 at the end of these experiments was mv when 5 nm 1,25-D 3 alone was added; mv when 5 nm 1,25-D 3 PTX where added together. When Student s test was used to compare 1,25-D 3 alone and 1,25-D 3 with PTX, the differences were significant. Experiment B, in the control group, 1 nm testosterone (at V max dose) was added to the bath without preincubation of PTX. 1 nm testosterone caused 9.1 mv left shift in the current-voltage relationship (line 1). In the PTX-treated group, cells were preincubated with 500 ng/ml PTX for min, and then 1 nm testosterone (at V max dose) was added to the bath afterward. Some cells preincubated with PTX showed a decrease in the left shift caused by the addition of testosterone when compared with control which was measured in other cells from the same pass and split. PTX reduced the magnitude of the left shift to under 50% in 4/15 cases. The left shift caused by 1 M BayK 8644 at the end of these experiments was constant and not significantly different (1 nm testosterone alone, mv; 1 nm testosterone PTX, mv). When Student s test was used to compare testosterone alone and testosterone with PTX, the differences were significant. Experiment C, in the control group, 100 nm 24,25-D 3 was added to the bath without preincubation of PTX. 100 nm 24,25-D 3 does not cause left shift in the current-voltage relationship ( mv, n 6) (line 1). In the PTX-treated group, cells were preincubated with 500 ng/ml PTX for min, and then 100 nm 24,25-D 3 was added to the bath and barium currents were measured as previously described. Cells preincubated with PTX showed a significant left shift caused by the addition of 24,25-D 3 of mv, 8/9 cells showed left shift greater than control (line 2), when compared with control which was measured in other cells from the same pass and split (line 1). The left shift caused by 1 M BayK 8644 at the end of these experiments was constant and not significantly different (100 nm 24,25-D 3 alone, mv; 100 nm 24,25-D 3 PTX, mv). When Student s test was used to compare the left shift of 24,25-D 3 alone compared to 24,25-D 3 with PTX, the differences were significant. Peak shift compared to control (mv) Number of experiments (n x) Experiment A 1,25-D 3 (5 nm) a 7 1,25-D 3 (5 nm) PTX (500 ng/ml) a 14 Experiment B Testosterone (1 nm) b 7 Testosterone (1 nm) PTX (500 ng/ml) b 15 Experiment C 24,25-D 3 (100 nm) c 6 24,25-D 3 (100 nm) PTX (500 ng/ml) c 9 a p b p c p have been reported. Schultz et al. (35), Imoto et al. (36), and Yatani and Brown (37) reported that G-proteins directly modulate channel activity in neurons and cardiac tissue, respectively. However, Trautwein and Heschler (38) and Sculptoreanu et al. (39) found that G-protein-mediated phosphorylation by campdependent protein kinase was also important for regulation of calcium channels. The potentiation of calcium channels by 1,25-D 3 and testosterone which was reduced in PTX-treated cells suggests that the PTX-sensitive G-protein (G i ) couples these steroid receptors to calcium channels. We do not know whether the effect of PTX is direct or via phosphorylation because the cells were incubated for min in PTX. However, an increase in the current amplitude of the barium current mediated by phorbol ester occurs over 10 min (data not shown) suggesting that in ROS cells phosphorylation events can occur over the time frame of the PTX experiments. A PTX-insensitive G-protein may cause potentiation of calcium channels through the 24,25-D 3 receptor, because under normal conditions 24,25-D 3 does not cause a left shift in the currentvoltage relationship, but when incubated with PTX, 24,25-D 3 does potentiate calcium channels. Smrcka et al. (40) and Taylor et al. (41) reported a G q protein (a PTX-insensitive G-protein) which activates phospholipase C. This enzyme induces phosphoinositol turnover, generation of inositol trisphosphate, and diacylglycerol which cause calcium release from endoplasmic reticulum and protein kinase C activation. Grosse et al. (42) reported increased production of inositol phosphate by 24,25- D 3. Because in the whole-cell patch clamp system PTX needs at least min to act, signal transduction pathways beyond G-proteins may also alter calcium channel function. The late induced potentiation by 24,25-D 3 may also be a result of phosphorylation coupled to a PTX-insensitive G-protein like G q in accord with the results found by Li et al. (21) that 24,25-D 3 affects calcium channel current by phosphorylation. Thus, the steroid receptors for 1,25-D 3, 24,25-D 3, and testosterone initiate signal transduction pathways via G-proteins, which may activate multiple signaling pathways to feed back regulating calcium channel function. In summary, we have evaluated the interaction between vitamin D 3 metabolites and testosterone on calcium channels in rat osteoblast-like osteosarcoma ROS 17/2.8 cells. 1,25-D 3, 25(OH)-16,23E-diene-D 3 (a potent 1,25-D 3 analogue specific for calcium channel potentiation), and testosterone each potentiate the openings of L-type calcium channels in a dose-dependent manner. 1,25-D 3 and testosterone open calcium channels cooperatively. In the presence of PTX, 24,25-D 3 also causes calcium channel potentiation. On the other hand, 24,25-D 3 reduces the potentiation caused by 1,25-D 3 or testosterone. These findings suggest 24,25-D 3 functions separately from 1,25-D 3, perhaps by a separate nongenomic receptor, to alter calcium channel potentiation. Data presented here offer important insights into the order of the signaling events in this cascade. Acknowledgment We thank Dr. Shigefumi Yukihiro for helpful discussions regarding data analysis. REFERENCES 1. 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