Effect of bradykinin on intracellular signalling systems in a rat clonal dental pulp-cell line

Archs oral Bid. Vol. 38, No. I, pp. 4348, 1993 Printedin Great Britain.All rightsreserved

Copyright0

0003-9969/93 $6.00 + 0.00 1993 PergamonPressLtd

EFFECT OF BRADYKININ ON INTRACELLULAR SIGNALLING SYSTEMS IN A RAT CLONAL DENTAL PULP-CELL LINE TOMOYUKI KAWASE, MICHIAKI ORIKASA

Department

of Pharmacology,

and

Niigata University, School Niigata 951, Japan

AKITOSHI SUZUKI

of Dentistry,

Gakkocho-dori,

(Accepted 18 August 1992) Summary-The cloned pulp-cell line RDP4-1 increases CAMP production, hydrolyses phosphoinositide (PI) and mobilizes calcium in response to prostaglandin E, (PGE,) and PGF,-a. The effect of bradykinin (BK) on intracellular signalling systems and DNA synthesis was studied in these cells. BK (10pM) transiently increased cytoplasmic free calcium ([Car’li) both in the presence and absence of external calcium. After stimulation with BK (IOpM), cells did not respond significantly to PGE, (O.Spg/ml). Pretreatment with indomethacin (30 PM) inhibited the [Ca2+]i increment by BK (10 PM), but not by the

subsequent addition of PGE, (0.5 pg/ml). Also, pretreatment with F’GE, (0.5 pg/ml) blocked the action of BK (10 PM). BK (0.1-100 pM) stimulated PI hydrolysis and CAMP production in a dose-dependent manner. Both the PI and the CAMP responses were inhibited by indomethacin (30 PM), as was the calcium response. BK (0.01-10 PM) also stimulated release of arachidonic acid and its metabolites dose-dependently. However, prolonged exposure to BK in serum-deficient medium did not exert any effect on DNA synthesis. RDP 4-l cells, therefore, appear to respond to BK with increased CAMP production, PI hydrolysis and calcium mobilization. The inhibition of these effects of BK by indomethacin raises the possibility that cycle-oxygenase product(s), especially PGE, or PGE,-like compounds, may be responsible for evoking these effects. These results indicate that BK may stimulate or modulate cell metabolism in the dental pulp. Key words: proliferation

bradykinin,

dental

pulp

cells, inositol

INTRODUCTION

phosphate,

cytoplasmic

free calcium,

CAMP,

cell

olism. A series of reports by Inoki and his co-workers (Kudo, Kuroi and Inoki, 1986a; Kudo et al., 1986b; Inoki and Kudo, 1986; Inoki, Kudo and Wei, 1987) has shown that this peptide activates a trypsin-like enzyme and stimulates secretion of an opioid peptide. An established cell line (RDP 4-1) from rat incisor pulp (Kawase, Orikasa and Suzuki, 1990) would serve as a useful system to study the effect of bradykinin and other factors on dental pulp metabolism. We have shown (Kawase et al., 1990) that PGE, and PGF_+ stimulate intracellular signalling systems through receptor-mediated pathways in RDP 4-l cells. To explore the regulation of dental pulp metabolism in inflammatory processes we have now examined the effects of bradykinin on the intracellular signalling systems [inositol phosphate, cytoplasmic free calcium ([Ca2+]i and CAMP] and the release of arachidonic acid in RDP 4-l cells. The effect of prolonged exposure to bradykinin on DNA synthesis was also examined.

Bradykinin, a nanopeptide (Arg-Pro-Pro-Gly-PheSer-Pro-Phe-Arg), is generated in inflammation in various tissues; it is thought to bind to specific receptors on cells, resulting in many biological effects such as vasodilation and hypotension (Regoli and Barabe, 1980), pain (Berkowitz and Way, 1971), increased vascular permeability (Barabe et al., 1979) and increased intestinal motility and chloride secretion (Manning et al., 1982; Cuthbert and Margolius, 1982). In the intracellular signalling systems, bradykinin increases CAMP and cGMP, mobilizes intracellular calcium, and hydrolyses phosphoinositide in various tissues (Zensen et al., 1984) and cells (Bareis et al., 1983; Pidikiti et al., 1985; Portilla and Morrison, 1986; Fu, Okano and Nozawa, 1988). Recently, Moolenaar and his co-workers (Tilly et al., 1987) reported that bradykinin stimulated DNA synthesis and cell division through an inositol phospholipid-mediated pathway in A431 cells, and that this mechanism was distinguishable from that of epidermal growth factor. Bradykinin is also generated in dental pulp tissue; however, little is known of its effect on pulp metab-

MATERIALS AND METHODS

Cells and cell culture The RDP 4-1 cell line, derived from rat dental pulp, was established and cloned as described by Kawase et al. (1990). The cells produced abundant type I collagen and showed alkaline phosphatase activity. In the cells, PGE, stimulated CAMP production, phos-

Abbreuiafions: BSA, bovine serum albumin;

HIFCS, heatinactivated fetal calf serum; c(-MEM; a-minimum essential medium; PG, prostaglandin, PI, phospholipase; TCA, trichloracetic acid. 43

44

TOMOYUKI KAWASE et al

phoinositide hydrolysis and calcium mobilization, while PGE+ evoked the last two of these events. The cells were routinely maintained in !x-MEM supplemented with 10% (v/v) HIFCS (Hazleton, St Lenexa, KS, U.S.A.) under a humidified atmosphere of 95% air and 5% CO, at 37°C. For passaging, cells were treated with 0.25% trypsin plus 0.02% EDTA for 3 min, centrifuged at 1000 rev/min for 5 min after addition of serum, and resuspended with the culture medium for seeding. The medium was changed every 48 h unless otherwise specified. The cells were seeded into multiwell plates at a density of approx. 5 x IO3cells/cm* for each experiment. Determination of intracellular CAMP After 4 days of culture, the cell monolayers in 24-multiwell plates (3.6-4.0 x IO5 cells/well) were washed with serum-free medium and preincubated with 0.1 mM isobutylmethyl xanthine for 30 min in 20 mM Hepes-buffered g -MEM containing 0.1% (w/v) BSA (buffer A, pH 7.2). Cells were then stimulated with bradykinin (Peninsula Laboratories Inc., Belmont, CA, U.S.A.) at the specified concentrations for 5 min. The reaction was terminated by immediate aspiration of the medium and addition of ice-cold 6% (w/v) TCA. The cell extraction and the radioimmunoassay of CAMP were as described previously (Kawase et a/., 1989; Kawase, Orikasa and Suzuki, 1991 b; Kawase and Suzuki, 1990). Measurement qf’ [Ca Z-1]i The cell monolayers in Bionique chambers (1.6-2.0 x lo6 cells/chamber) were loaded with Fura 2/AM (Dojin Chemical Laboratories, Kumamoto, Japan) in 20 mM Hepes-buffered Hanks balanced salt solution containing 0.1% (w/v) BSA (buffer B, pH 7.2). After washing and incubation for an appropriate period, cells were treated with bradykinin in buffer B. [Ca*+]i was measured using a Nikon microspectrofluorometric system (Tokyo, Japan) at 37”C, as described elsewhere (Kawase and Suzuki, 1988, 1990; Kawase, Ishikawa and Suzuki, 1988; Kawase et al., 1990). Each optical field held three to five cells, and Fura 2 was excited at 340 and 380 nm, with the emitted fluorescence monitored at 510 nm for both excitation wavelengths. [Ca’+]i was expressed using the 340/380 nm emission fluorescence ratios. Determination of water-soluble inositol phosphates The cells in subconfluent cultures (3 days in culture) in 6-multiwell plates were labelled with 3 pCi/ml [‘HI-myo-inositol (80.0 Ci/mmol, Amersham) in c(MEM containing 10% HIFCS for 24 h. The medium was removed and the unincorporated [‘HI-myoinositol was washed away with serum-free medium. Cells (1.6-l .8 x lo6 cells/well) were preincubated for I5 min in buffer A in the presence of 10 mM LiCl and then treated with bradykinin for 2 min. In a preliminary study, we found that accumulation of inositol trisphosphate reached steady-state within 2 min of stimulation and did not rapidly return to the resting levels in the presence of 10mM LiCl. The reaction was ended and water-soluble inositol phosphates were extracted, as described previously (Kawase et al., 1990; Kawase, Orikasa and Suzuki, 1991a). [-‘HIinositol phosphate, inositol bisphosphate and inositol

trisphosphate were separated by anion-exchange chromatography over AG l-X8 (Bio-Rad, formate form) by the method of Brown et al. (1987). Samples of elution fractions were diluted into liquid scintillation fluid (Atomlight, NEN Research Products) and the radioactivity was measured in a fl-counter. Measurement of release of arachidonic acid and its metabolites Release of [3H]-arachidonic acid and its metabolites was assessed essentially as described by Kawase, Orikasa and Suzuki (1991~). The cells cultured for 3 days in 24-multiwell plates were labelled with 0.42 pCi/ml [‘HI-arachidonic acid (209.4 Cij mmol, Amersham) for 24 h in a-MEM containing 10% HIFCS. After labelling, cells were washed three times with serum-free medium and then treated with bradykinin in buffer A. Incubations were ended by transferring the medium to centrifuge tubes on ice. Tubes were immediately centrifuged (10 min, 27,000g) to remove cell debris. A sample of each supernatant was diluted into Atomlight and the radioactivity was measured in a /j’-counter. Measurement of DNA synthesis [3H]-Thymidine incorporation was assessed in active growing cells, as described previously (Kawase et al., 1990, 1991a). After being cultured in a-MEM containing 10% (v/v) HIFCS for the specified periods (24-72 h), cells were washed with serum-free a-MEM and exposed to bradykinin or epidermal growth factor for 24-48 h in a-MEM containing 1% (v/v) HIFCS and 0. I % (w/v) BSA. The growth factor, a well-known mitogen, was used as the positive control. [‘HI-Thymidine (47 Ci/mmol, Amersham) was added to cells at final concentration 0.1 pCi/ml and cells were further incubated for 2 h. At the end of the period, cells were washed three times with ice-cold, phosphate-buffered saline and ice-cold 10% (w/v) TCA and subsequently with ethanol+ther (3 : 1, v/v) to remove residual TCA. Cells were dissolved into 0.3 ml of 0.5N NaOH-0.5% (v/v) Triton X-100 solution. Each well was washed with distilled water and these washes were combined with the original cell extracts. The radioactivity was measured in a /l-counter. Statistical analysis The results in the figures are expressed as the mean + SEM. The Student’s t-test was used for statistical analysis of the data. RESULTS

Ejkct of bradykinin on [Ca’+]i, and accumulation of inositol phosphates and CAMP Bradykinin ( 3 0.5 g M) rapidly increased [Ca*+ ]i in the single cells in confluent cultures (Fig. I); it returned to basal levels within 1.5 min. Even after treatment with EGTA (2mM) to remove external calcium, cells responded to bradykinin (10 PM) with increased [Ca*+ Ii. This increase in [Ca*+ ]i was reduced to 60-80% of that in the normal condition ([Ca’+]out = 1.26 mM). On the other hand, pretreatment with bradykinin (10 PM) markedly reduced the increase by PGE, (0.5 pg/ml) in [Ca*+]i. In cells

Bradykinin

/-

g 1.3

Indo.

p. -z*

LL b z 1.3

BtK

B+K + Indo.

J ‘x

and intracellular

-s

-

PdE2

[ -k1.1 P&p

min

B’K

Fig. I. Effects of bradykinin (BK) and PGE, on [Ca’+]i in single RDP 4-l cells. The Fura 2-loaded cells in conguent cultures were stimulated with BK (10 PM) in the presence or absence of EGTA (2 mM) (upper). Cells were successively stimulated with BK (10 PM) and PGE, (0.5 pg/ml) in the presence or absence of indomethacin (30 PM) (middle), or with PGE, (0.5 pg/ml) and BK (10 PM) (lower). [Ca*+]i was measured fluorometrically. The traces are representative of at least three separate experiments.

(30 PM) for 15 min, bradykinin, but not PGE, failed to increase [Ca2+]i. In addition, pretreatment with PGE, blocked the bradykinin-induced increase in [Ca2+ Ii. In the presence of LiCl(l0 mM), [‘HI-myo -inositol labelled cells were treated with bradykinin pretreated

with

indomethacin

signalling

45

systems

(O.l-100pM) for 2min (Fig. 2). Bradykinin (10-100 PM) significantly increased the accumulation of inositol di- and triphosphate in a dose-dependent manner. However, pretreatment with indomethacin (30 ,LIM) inhibited the bradykinin-induced hydrolysis of phosphoinositide (Fig. 3). In cells pretreated with indomethacin, bradykinin (100 PM) did not increase the accumulation of the three inositol phosphates. In the presence of 0.1 mM isobutylmethyl xanthine, cells were treated with bradykinin (O.Ol100 p M) for 5 min (Fig. 4). Bradykinin (0.1-100 p M) increased cAMP accumulation in a dose-dependent manner, but this increase was relatively small. Pretreatment with indomethacin (30 ,uM) inhibited the bradykinin-stimulated production of CAMP (data not shown). Effects of bradykinin on release of arachidonic acid and its metabolites

Bradykinin (1 PM) stimulated release of arachidonic acid and its metabolites in a time-dependent manner (Fig. 5). The release increased rapidly and reached steady-state within 10min both in untreated and bradykinin-treated cells. Bradykinin

1

i

2.0

* 1.5

*

1.0

1

0

0

0.1

1

10

100

BK (JIM)

Fig. 2. Dose-response curve for bradykinin (BK)-stimulated phosphoinositide hydrolysis in RDP 4-1 cells. Each value is the mean If: SEM of five incubations from a single experiment representative of three separate experiments. *p < 0.05, **p < 0.02, ***p co.01 compared with each control level (Student’s t-test). IPl, -2, -3: inositol phosphate, diphosphate, triphosphate.

L

dh_Jl IPl

ok..,,

I

IP2

IP3

Fig. 3. Inhibitory effect of indomethacin on bradykinin (BK)-stimulated hydrolysis of phosphoinositides in RDP 4-1 ceils. [‘HI--inositol-labelled cell monolayers preincubated for I5 min in buffer A containing LiCl(10 mM) in the presence (solid column) or absence (open column) of indomethacin (30pM), and then stimulated with BK [(A) 1 PM, (B) 10 PM] for 2 min. Each value is the mean & SEM of three incubations from a single experiment representative of three separate experiments. *p < 0.05, **p < 0.02 compared with each control level (Student’s t-test). Other abbreviations as in Fig. 2.

46

TOMOYUKI KAWASE et

al.

(0.01-10 PM) increased the release of arachidonic acid at 10min in a dose-dependent manner (Fig. 6). Eflects of bradykinin and epidermal growth factor on DNA synthesis Assay for DNA synthesis was done under three different conditions that possibly affect regulation of bradykinin receptors. First, cells precultured for 24 h in a-MEM containing 10% HIFCS were exposed to bradykinin or epidermal growth factor at the specified concentrations for 50 h in u-MEM containing 1% HIFCS and 0.1% BSA [Fig. 7(A)]. The growth factor (1.6 nM-1.6 p M) markedly stimulated DNA synthesis in a dose-dependent manner, while bradykinin failed to do so. Second, cells precultured for 48 h in c(-MEM containing 10% HIFCS were exposed to these peptides for 26 h in the experimental medium [Fig. 7(B)]. The growth factor (1.6nM1.6p M) also stimulated DNA synthesis in a dose-dependent manner, while bradykinin did not. Third, cells precultured for 72 h in the serum-sufficient medium were exposed to these peptides for 26 h in the experiment medium [Fig. 7(C)]. The growth factor (16 nM-1.6 PM) dose-dependently stimulated DNA synthesis, but the increases were relatively smaller than those in the former two cases. Bradykinin also had no mitogenic effect in this case.

DISCUSSION

Dental pulp is easily irritated and readily inflamed. Thus, the production and action of chemical mediators involved in the pulp have been studied mainly at tissue level. Prostaglandins, the mediators most studied, are produced in pulp tissue (Hirafuji, Satoh and Oguro, 1980), but their action therein is poorly understood. Using an established cell line (RDP 4-l), we showed that PGE, and PGF,-LY stimulated some of the intracellular signalling systems, and we suggested that these PGs have some role in regulating dental

** T

I

0

10

20

30

Time (mid

Fig. 5. Time course of bradykinin-stimulated release of arachidonic acid (AA) and its metabolites in RDP 4-1 cells. The [‘HI-AA-labelled cell monolayers were treated with bradykinin (I PM) for the indicated periods before radioassay of the medium. Each value is the mean f SEM of three incubations from a single experiment representative of two separate experiments. pulp metabolism (Kawase et al., 1990). Bradykinin, a well-known chemical mediator of cell activity, is thought to be converted from kininogens released from the blood vessels into the interstitial space (Inoki et al., 1987). Inoki and his co-workers (Kudo et al., 1986a, b; Inoki and Kudo, 1986; Inoki et al., 1987) proposed a negative feedback action of bradykinin in pulp cells, whereby it stimulates release of enkephalins, which consequently inhibit its production by inhibiting the release of kininogens from the blood vessels. It is possible that the B, type of bradykinin receptor mediates this action independently of the generation of PGs (Kudo T., personal communication). However, there is less information about the effects of bradykinin on intracellular signalling systems in pulp cells.

I

i

C

2 ;

***

150-

,j+G

tzloo2 2

50 -

3l-

O+iz%TE! . . BK (JIM)

Fig. 4. Dose-response curve for bradykinin (BK)-stimulated CAMP accumulation in RDP 4-1 cells. Each value is the mean f SEM of three incubations from a single experiment representative of three separate experiments. *p < 0.02, **p < 0.005 compared with the control level (Student’s f-test).

0 0

10 0.01

0.1

1

BK (JJM)

Fig. 6. Dose-response curve for bradykinin (BK)-stimulated release of arachidonic acid (AA) and its metabolites in RDP 4-1 cells. [3H]-AA-labelled cell monolayers were treated with BK at the indicated concentrations before radioassay of the medium. Each value is the mean k SEM of three incubations from a single experiment representative of three separate experiments. *p < 0.02, **p < 0.01, ***p < 0.005 compared with the control level (Student’s I-test).

41

Bradykinin and intracellular signalling systems

Peptides t-log M 1

Fig. 7. Effects of bradykinin and epidermal growth factor on DNA synthesis in RDP 4-l cells. Cells preincubated for 24 h (A), 48 h (B) or 72 h (C), and then exposed to bradykinin (0) or growth factor (0) for 48 h (A) or 24 h (B, C). Incorporation of [‘HI-thymidine into the acid-insoluble precipitate was then measured. Each value is the mean It SEM of three incubations of a single experiment representative of two to three separate experiments. *p < 0.005, **p < 0.001 compared with each control level (Student’s t-test).

To our knowledge, we here provide the first evidence that bradykinin can stimulate CAMP production, calcium mobilization and PI hydrolysis of phosphoinositide in a cell line derived from rat dental pulp (RDP 4-l). Based on Berridge’s review (1987), the small (2@40%) inhibition of bradykinin-induced calcium mobilization by EGTA indicates that bradykinin increases [Ca’+]i by stimulting calcium influx and mainly by releasing calcium from intracellular stores through the production of inositol trisphosphate. The action of bradykinin on these signalling systems, which was accompanied by release of arachidonic acid, was blocked by inhibition of cyclooxygenase by indomethacin. This indicates that the generation of cycle-oxygenase product(s) derived from arachidonic acid, i.e. PG(s), is responsible for evoking the bradykinin response. Concerning the CAMP system, our findings are compatible with most other reports (Bareis et al., 1983; Jelsema, Moss and Manganiello, 1985; Dixon et al., 1990). In contrast to these Zensen et al. (1984) showed that bradykinin increased CAMP production by a mechanism inde-

pendent of its effect on PGE, production in renal medulla. As for other signalling systems, Burch and Axelrod (1987) clearly demonstrated that bradykinininduced hydrolysis of phosphoinositide was dissociated from PG production in Swiss 3T3 fibroblasts. As far as we know, PG production has been rarely reported to mediate the action of bradykinin on the calcium and the inositol phosphate signalling systems. However, our results do not provide a clear answer to this controversy. In fact, inhibition of the action of bradykinin by indomethacin indicates involvement of cycle-oxygenase products, whereas lack of time-lag expected in the bradykinin-stimulated increase in [Ca2+]i indicates mediation of a PGindependent mechanism. However, both PGE, and bradykinin failed to induce significant changes in [Ca’+]i in the bradykinin- and the PGE,-stimulated cells, respectively. This phenomenon is explained by exogenous or (bradykinin-generated) endogenous PGE, desensitizing PGE, receptors and consequently inhibiting the action of bradykinin or PGE,. The PG-independent mechanism cannot satisfactorily explain all these phenomena. We therefore suggest that a cycle-oxygenase product(s), probably PGE, , is responsible for the action of bradykinin, at least in the pulp-derived cells. We failed to obtain satisfactory data on the effect of bradykinin on DNA synthesis. Because of downregulation of the bradykinin receptor in trypsinized endothelial cells (Sung et al., 1989) and cells cultured in bradykinin-containing medium, we tried to vary the duration of preculture in serum-sufficient medium. Epidermal growth factor stimulated DNA synthesis under all conditions tested. However, bradykinin had no mitogenic effect at any duration of preculture. Was this due to the CAMP signalling system? In differentiated cells, including osteoblastic cells (Yamaguchi et al., 1988; Kawase et al., 199la), the CAMP system is generally thought to be associated with induction of growth arrest and cellular differentiation. In RDP 4-l cells, however, PGE, at high concentrations (0.5-5 pg/ml) stimulated DNA synthesis and CAMP production. Thus, it is plausible that the amount of PGEp produced by bradykinin is sufficient for stimulating these signalling systems but not for enhancing DNA synthesis in these cells. In conclusion, bradykinin appears to stimulate CAMP, inositol phosphate and the calcium signalling systems in the dental pulp-derived RDP 4-l cell line via generation of cycle-oxygenase product(s), especially PGE, or PGE,-like compound. However, these initial responses do not result in enhancement of cell proliferation. These results indicate that bradykinin, like PGE,, may modulate cell metabolism in the dental pulp.

REFERENCES

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48

TOMOYUK~KAWASE et al.

Berkowitz B. A. and Way E. L. (1971) Analgesic activity and central nervous system distribution of the optical isomers of pentazocine in the rat. J. Pharmac. exp. Ther. 177, 500-508. Berridge M. J. (1987) Inositol trisphosphate and diacylglycerol: two interacting second messengers. A. Ret>. Biochem. 56, 159-193. Brown K. D., Blakeley D. M., Hamon M. H., Laurie M. S. and Corps A. N. (1987) Protein kinase C-mediated negative feedback inhibition of unstimulated and bombesinestimulated phosphoinositide hydrolysis in Swiss-mouse 3T3 cells. Biochem. J. 245, 631439. Burch R. M. and Axelrod J. (1987) Dissociation of bradykinin-induced prostaglandin formation from phosphatidyl-inositol turnover in Swiss 3T3 tibroblasts: evidence for G protein regulation of phospholipase A,. Proc. natn. Acad. Sci., U.S.A. &4, 63746378. Cuthbert A. W. and Margolius H. S. (1982) Kinins stimulate net chloride secretion by the rat colon. Br. J. Pharmac. 75, 587-588. Dixon B. S., Breckon R., Fortune J., Vavrek R. J., Stewart J. M.. Marzec-Calvert R. and Linas S. L. (1990) Effects of kihins on cultured arterial smooth muscle.’ Am. J. Physiol. 258 C, 299-308. Fu T., Okano Y. and Nozawa Y. (1988) Bradykinin-induced generation of inositol 1,4,5_trisphosphate in fibroblasts and neuroblastoma cells: effect of pertussis toxin, extracellular calcium, and down-regulation of protein kinase C. Biochem. biophys. Res. Commun. 157, 142991435. Hirafuji M., Satoh S. and Ogura Y. (1980) Prostaglandins in rat dental pulp tissue. J. dent. Res. 59, 1535-1540. Inoki R. and Kudo T. (1986) Enkephalins and bradykinin in dental pulp. Trend Pharmac. Sci. 7, 275-277. Inoki R., Kudo T. and Wei E.-Q. (1987) Significance of the production of bradykinin in inflamed dental pulp. Microcirculation-an Update (Eds M. Tsuchiya et al.) Vol. 2, p. 111. Elsevier Science, Amsterdam. Jelsema C. L., Moss J. and Manganiello V. C. (1985) Effect of bradykinin on prostaglandin production by human skin fibroblasts in culture. Meth. Enzym. 109, 48&503. Kawase T. and Suzuki A. (1988) Phosphatidic acid-induced calcium mobilization in osteoblasts. J. Biochem. 103, 581-582. Kawase T. and Suzuki A. (1990) Initial responses of a clonal osteoblast-like cell line, MOB 3-4, to phosphatidic acid in vitro. Bone Miner. 10, 61-70. Kawase T., Ishikawa I. and Suzuki A. (1988) NaF-induced CaZ+ mobilization is dependent upon the culture density in a parathyroid hormone-responsive osteoblast-like cell line. Lz$ Sci. 43, 2241-2247. Kawase T., Ishikawa I., Orikasa M. and Suzuki A. (1989) Aluminium enhances the stimulatory effect of NaF on

prostaglandin E, synthesis in a clonal osteoblast-like cell line, MOB 3-4, in vitro. J. Biochem. 106, 8-10. Kawase T., Orikasa M. and Suzuki A. (1990) A clonal prostaglandin-responsive cell line (RDP 4-l) derived from rat dental pulp. Bone Miner. 11, 163-175. Kawase T., Orikasa M. and Suzuki A. (199la) Effects of prostaglandin E, and F,a on cytoplasmic pH in a clonal osteoblast-like cell line, MOB 3-4. J. CeN. Physiol. 146, 141-147. Kawase T., Orikasa M. and Suzuki A. (1991b) Phorbol ester (TPA) reduces prostaglandin E,-stimulated CAMP production by desensitization of prostaglandin E, receptors in a clonal osteoblast-like cell line, MOB 3-4. Calc. Tiss. Int. 48, 1677175. Kawase T., Orikasa M. and Suzuki A. (1991~) Aluminumfluoride- and epidermal growth factor-stimulated DNA synthesis in MOB 3-4-F2 cells, Pharmac. Toxic. 69, 330-337. Kudo T., Kuroi M. and Inoki R. (1986a) In uifro production and release of opioid peptides in the tooth pulp induced by bradykinin. ieuropeptide 7, 391-397. Kudo T., Chang H.-L., Kuroi S., Wakisaka S., Akai M. and Inoki R. (1986b) Influence of bradykinin and substance P on the met-enkephalin-like peptide content in the rat incisor pulp. Neuropeptide 7, 399405. Manning D. C., Snyder S. H., Kanchur J. F., Miller R. J. and Field M. (1982) Bradykinin receptor-mediated chloride secretion in intestinal function. Nature 199, 25&259. Pidikiti N., Gamer0 D., Gamer0 J. and Hassid A. (1985) Bradykinin-evoked modulation of cytosolic Ca2+ concentrations in cultured renal epithelial (MDCK) cells. Biothem. hiophys. Res. Commun. 130, 807-813. Portilla D. and Morrison A. R. (1986) Bradykinin-induced changes in inositol trisphosphate mass in MDCK cells. Biochem. biophys. Res. Commun. 140, 644649. Regoli D. and Barabe J. (1980) Pharmacology of bradykinin and related kinin. Pharmac. Rev. 32, 146. Sung C.-P., Arleth A. J., Shikano K., Zabko-Potapovich B. and Berkowitz B. A. (1989) Effect of trypsinization in cell culture on bradykinin receptors in vascular endothelial cells. Biochem. Pharmac. 38, 69-99. Tilly B. C., van Paridon P. A., Verlaan I., Wirtz K. W. A., de Laat S. W. and Moolenaar W. H. (1987) Inositol phosphate metabolism in bradykinin-stimulated human A431 carcinoma cells. Biochem. J. 244, 129-135. Yamaguchi D. T., Hahn T. J., Beeker T. G., Kleeman C. R. and Muallem S. (1988) Relationship of CAMP and calcium messenger systems in prostaglandin-stimulated UMR-106 cells. J. Biol. Chem. 263, 10,745-10,753. Zensen T. V., Rapp N. S., Spry L. A. and Davis B. B. (1984) Independent effects of bradykinin on adenosine 3’,5’monophosphate and prostaglandin E, metabolism by rabbit renal medulla. Endocrinology 114, 541-544.