Phenytoin-Induced Elevation of the Intracellular Calcium Concentration by Stimulation of Calcium-Sensing Receptors in Gingival Fibroblasts *

Background: The mechanism concerning gingival overgrowth as a side effect of phenytoin, a therapeutic drug for epilepsy has been still unclear. As one of mechanisms, by measuring the intracellular calcium concentration ([Ca]i) of the gingival fibroblasts, it has been advocated that there is relationship between gingival overgrowth and phenytoin-induced alterations in the [Ca]i in gingival fibroblasts. To confirm that phenytoin elevates the [Ca]i, and if so, to find out its mode of action. Methods: The [Ca]i was measured with the Ca-sensitive fluorescent dye fura-2/AM. Cells were soaked in a flexiperm chamber and perfused by a saline. Drugs at appropriate concentrations were added to the perfusate. Results: Phenytoin concentration-dependently elevated the [Ca]i. NPS2390, a calcium-sensing receptor (CaSR) blocker, significantly suppressed the phenytoin-induced [Ca]i elevation. U73122, a phospholipase C (PLC) inhibitor, inihibited the phenytoin-induced [Ca]i elevation. TMB-8, a blocker of inositol triphophate (IP3) receptors in ER, significantly depressed the phenytoin-induced [Ca]i elevation. m-3M3FBS, a PLC activator, enhanced the phenytoin-induced [Ca]i elevation. From the findings obtained, it is discussed as follows: The Ca-free saline and NPS2390, a CaSR antagonist, inhibited the phenytoin-induced [Ca]i rise; These results indicate that CaSRs exist in gingival fibroblasts and that CaSRs are involved in the phenytoin-induced [Ca]i rise; U73122 and TMB-8 depressed the phenytoin-induced [Ca]i elevation and furthermore, m-3M3FBS enhanced the phenytoin-induced [Ca]i elevation, showing that the Ca release from the ER is involved in the phenytoin-induced [Ca]i elevation. Conclusion: We have concluded that phenytoin elevates the [Ca]i by activating CaSRs and enhancing the Ca release from the Ca stores in gingival fibroblasts.


Introduction
Gingival overgrowth as a side effect of phenytoin, a therapeutic drug for epilepsy has been still now observed [1].However, the mechanism concerning this symptom has been still unclear.Various mechanisms of gingival overgrowth induced by phenytoin have been advocated.It has been thought that there are mainly 2 causes, that is, collagen accumulation and fibroblast proliferation in gingiva.For example, concerning collagen accumulation, gingival overgrowth is due to only increase in collagen or inhibition of its degradation.Narayanan et al. [2] concluded that overproduction of collagen by cells from phenytoin-induced hyperplastic gingiva results from an increased steady state level of collagen mRNA and not decreased collagen degradation.However, Kanno et al. [3] proposed that collagen accumulation may be attributed to a decrease in its degradation rather than to an increase in collagen synthesis.Furthermore, Akhter et al. [4] also claimed that gingival overgrowth seems to be induced by the disruption of homeostasis of collagen synthesis and degradation in gingival connective tissue, predominantly through the inhibition of collagen phagocytosis of gingival fibroblasts.In addition, concerning fibroblast proliferation, Cockey et al. [5] speculated that drug-induced gingival overgrowth may occur due to direct or indirect stimulation of the proliferation of some populations of fibroblasts.González et al. [6] and Kato et al. [7] also proposed the increase in fibroblast proliferation.In contrast, Fujimori et al. [8] reported that a reduced rate of apoptosis contributes to the accumulation of gingival fibroblasts.On the hand, as the other mechanism, by measuring the intracellular calcium concentration ([Ca 2+ ]i) of the gingival fibroblasts, Modéer et al. [9] have advocated that there is relationship between gingival overgrowth and phenytoin-induced alterations in the [Ca 2+ ]i in gingival fibroblasts.
Nifedipine, an anti-hypertension drug, as well as phenytoin, develops gingival overgrowth.Regarding to nifedipine, we have claimed the involvement of the [Ca 2+ ]i in its action and that recently advocated the modified "Calcium trigger theory" [10].That is, we have inferred that nifedipine evokes gingival overgrowth by triggering the [Ca 2+ ]i elevation in gingival fibroblasts.
Thus, the present study aims to confirm that phenytoin elevates the [Ca 2+ ]i similarly to nifedipine, and identufy the mode of action.Above all, we investigated actions of modulators of calcium-sensing recaptors (CaSRs) and the transmission from CaSRs to endoplasmic reticula (ER), since it is reported that CaSRs are related to cell proliferation [11] and that signals from CaSRs are transmitted to ER [12,13].

Measurement of the [Ca 2+ ]i
The [Ca 2+ ]i was measured with the Ca 2+ -sensitive fluorescent dye fura-2/AM (Dojindo Laboratories, Kumamoto, Japan).Cells were kept in a buffer comprising 135 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 20 mM HEPES-NaOH (pH 7.4).They were loaded with the dye by incubation in 5 μM fura-2/AM for 45 min at 37˚C.Cells were then washed to remove excess fura-2/AM and then incubated in fresh buffer (without fura-2/AM) for 15 min after incubation to allow intracellular cleavage of the acetoxymethyl ester conjugate (and thus activation) of fura-2.Excitation light from a xenon lamp was passed through a filter (340 nm or 360 nm).The emission wavelength for analyses was 500 nm.Changes in the fluorescence intensity of fura-2 in cells were recorded with a video-imaging analysis system (FC-400, Furusawa Laboratory Appliance, Kawagoe, Japan).The [Ca 2+ ]i was determined as the ratio of the fluorescence stimulated by excitation at 340 nm or 360 nm compared with a standard calibration curve obtained using a Calcium Calibration Buffer Kit I (Molecular Probes, Eugene, OR, USA).
To minimize leakage of fura-2, cells were kept at 32˚C during fluorescence measurements using a bath temperature controller (DTC-100A; DIA Medical Systems, Kunitachi, Japan).Cells were soaked in a flexiperm chamber containing 0.5 ml of saline and perfused at 8.0 ml/min with a tubing pump system (Master flex 7524-10; Cole-Parmer Instrument Company, Barrington, IL, USA).Drugs at appropriate concentrations were added to the perfusate.The drug-containing perfusates were switched by 6-Way Cock (Daiwa Co. Ltd., Matsumoto, Japan).To ensure that fura-2 fluorescence was maintained within the linear range (i.e., did not become saturated), we selected for analyses cells with a basal [Ca 2+ ]i in the range 50 -200 nM.

Statistical Analyses
Data are the mean ± standard error of the mean (SEM) and the number of observations (N).Statistical analyses of the data were undertaken by the Student's two-sided paired t-test.Differences between mean values were considered significant if the probability of error (p) was less than 0.05.

Results
In order to determine whether or not phenytoin elevates the [Ca 2+ ]i similarly to nifedipine, effects of phenytoin on the [Ca 2+ ]i were examined.Phenytoin (10 -

Discussion
Modéer et al. [9] reported that phenytoin elevates the [Ca 2+ ]i under the condition of the normal extracellular calcium concentration.They claimed that there is relationship between phenytoin-induced alterations in [Ca  Cell proliferation and progression through the cell cycle are Ca 2+ -dependent [14].Munaron [15] concluded that most peptidic growth factors that bind to tyrosine kinase receptors and trigger complex intracellular signal transduction pathways finally leading to cell proliferation.Among the early events induced by growth factors, cytosolic calcium increase plays a key role.There are various mechanisms of [Ca 2+ ]i elevation such as the Ca 2+ release from ER and influx through VOCs (voltage-operated Ca 2+ channels), ROCs (receptor-operated Ca 2+ channels), and TRP (transient receptor potential) channels [16].Kwak et al. [11] have insisted that the extracellular CaSR is expressed in mouse mesangial cells and modulates cell proliferation.Thus, we aimed at roles of CaSRs, since we had already pharmacologically confirmed that CaSRs exist in gingival fibroblasts by observing that CaSR agonists (gentamicin, neomycin, spermine, LaCl 3 , and verapamil) elevated the [Ca 2+ ]i [10].Thus, effects of the Ca 2+ -free saline and NPS2390, a CaSR antagonist, were examined.The Ca 2+ -free saline and NPS2390 inhibited the phenytoin-induced [Ca 2+ ]i rise (Figure 2).These results indicate that CaSRs exist in gingival fibroblasts and that CaSRs are involved in the phenytoin-induced [Ca 2+ ]i rise.
By finding out the stimulatory effect of the CaSR on H + /K + -ATPase activity in gastric parietal cells, Remy et al. [12] have reported that signals from CaSRs are transmitted to ER, which is prevented by TMB-8, an IP 3 receptor blocker in ER.Thus, we examined effects of modulators of signal transmission from CaSRs to ER.Since stimulation of CaSRs induces PLC activation [12], effects of U73122, a phospholipase C inhibitor [17], were examined.U73122 depressed the phenytoin-induced [Ca 2+ ]i elevation and furthermore, TMB-8, also inhibited the phenytoin-induced [Ca 2+ ]i elevation (Figure 3).On the other hand, m-3M3FBS, a phospholipase C activator [11], enhanced the phenytoin-induced [Ca 2+ ]i elevation (Figure 4).PLC hydrolyzes PIP 2 and yields IP 3 , which diffuses to ER [18] and releases Ca 2+ from them [13].From these facts, our observations show that the Ca 2+ release from the ER is involved in the phenytoininduced [Ca 2+ ]i elevation.
From the findings obtained, we have concluded that phenytoin elevates the [Ca 2+ ]i by activating CaSRs and enhancing the Ca 2+ release from the Ca 2+ stores in gingival fibroblasts.

Figure 4 .
Figure 4. Enhancement of the phenytoin-induced [Ca 2+ ]i elevation by m-3M3FBS.m-3M3FBS (20 μM), a phospholipase C activator, enhanced the phenytoin-induced [Ca 2+ ]i elevation.The upper trace shows a representative time course of the [Ca 2+ ]i in the case of m-3M3FBS application.N = 24, **** p < 0.001.val fibroblasts and its action was concentration (10 -200 μM) dependent (Figures 1(a) and (b)).Cell proliferation and progression through the cell cycle are Ca 2+ -dependent[14].Munaron[15] concluded that most peptidic growth factors that bind to tyrosine kinase receptors and trigger complex intracellular signal transduction pathways finally leading to cell proliferation.Among the early events induced by growth factors, cytosolic calcium increase plays a key role.There are various mechanisms of [Ca 2+ ]i elevation such as the Ca 2+ release from ER and influx through VOCs (voltage-operated Ca 2+ channels), ROCs (receptor-operated Ca 2+ channels), and TRP (transient receptor potential) channels[16].Kwak et al.[11] have insisted that the extracellular CaSR is expressed in mouse mesangial cells and modulates cell proliferation.Thus, we aimed at roles of CaSRs, since we had already pharmacologically confirmed that CaSRs exist in gingival fibroblasts by observing that CaSR agonists (gentamicin, neomycin, spermine, LaCl 3 , and verapamil) elevated the [Ca 2+ ]i[10].Thus, effects of the Ca 2+ -free saline and NPS2390, a CaSR antagonist, were examined.The Ca 2+ -free saline and NPS2390 inhibited the phenytoin-induced [Ca 2+ ]i rise (Figure2).These results indicate that CaSRs exist in gingival fibroblasts and that CaSRs are involved in the