J Pharmacol Sci 97, 000 – 000 (2005) Journal of Pharmacological Sciences ©2005 The Japanese Pharmacological Society Full Paper Lafutidine-Induced Increase in Intracellular Ca2+ Concentrations in PC12 and Endothelial Cells

Kana Kunieda1, Akiyoshi Someya1, Syunji Horie1, Hirofusa Ajioka2, and Toshihiko Murayama1,*

1Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba 260-8675, Japan 2Tokushima Research Center, Taiho Pharmaceutical Co., Ltd., Kawauchi-cho, Tokushima 771-0194, Japan

Received September 14, 2004; Accepted November 8, 2004

Abstract. Lafutidine, a H2 receptor antagonist, exerts gastroprotective effects in addition to gastric antisecretory activity. The gastrointestinal protective effects of lafutidine are mediated by capsaicin-sensitive neurons, where capsaicin excites neurons by opening a member of the transient receptor potential channel family (TRPV1). Since the effect of lafutidine on the 2+ 2+ intracellular Ca concentration ([Ca ]i) in cells has not been elucidated, we investigated the 2+ lafutidine response to [Ca ]i in rat pheochromocytoma PC12 and human endothelial cells. Lafutidine at pharmacological concentrations greater than 1 mM induced a sustained increase in 2+ [Ca ]i in the presence of extracellular CaCl2 in PC12 cells, while capsaicin showed dual effects 2+ 2+ on [Ca ]i in PC12 cells, where it activated TRPV1 and inhibited store-operated Ca entry. The 2+ 2+ thapsigargin (an activator of store-operated Ca entry)-induced increase in [Ca ]i in PC12 cells was inhibited by capsaicin and SKF96365, an inhibitor of store-operated Ca2+ entry, and the lafutidine response was inhibited by capsaicin but not by SKF96365. In endothelial cells, 2+ lafutidine induced an increase in [Ca ]i in a SKF96365-insensitive manner. These results suggest that lafutidine stimulates Ca2+ entry via the capsaicin-sensitive pathway but not the SKF96365- sensitive pathway. The possible role of store-operated Ca2+ entry induced by lafutidine on gastrointestinal function is also discussed.

Keywords: lafutidine, intracellular Ca2+ concentration, PC12 cell, human umbilical vein endothelial cell, store-operated Ca2+ entry

Introduction lafutidine at concentrations ranging from 0.1 to 1 M, which alone has no effect, enhances ileal contraction Lafutidine, ((M)-2-(furfurylsulfinyl)-N-[4-[4-(piperid- induced by capsaicin in the isolated guinea pig ileum inomethyl)-2-pyridyl]oxy-(Z)-2-butenyl] acetamide), is (11). Since the effects of capsaicin with and without a novel antiulcer drug that exhibits long-lasting gastric lafutidine are completely inhibited by tetrodotoxin, antisecretory effects due to blockade of the histamine H2 lafutidine appears to activate myenteric neurons in the receptor (1, 2). In addition, lafutidine exerts gastropro- ileum. However, the mechanism(s) responsible for tective effects independent of its antisecretory action (3, lafutidine activation by capsaicin-sensitive neurons has 4). These gastroprotective effects (3, 4, 5 – 7) and not been clarified. 2+ 2+ intestinal protective effects (8, 9) of lafutidine are due The intracellular free Ca concentration ([Ca ]i) is to the activation of capsaicin-sensitive neurons. strictly regulated, since Ca2+ is an important trigger of Nishihara et al. (10) reported that lafutidine, which alone numerous neuronal functions including neurotransmitter shows no effect, enhances the capsaicin-induced release release. In addition, capsaicin stimulates Ca2+ entry of calcitonin gene-related peptide (CGRP) from neurons through the vanilloid VR1 receptor/TRPV1 (a member in the rat stomach. Also, recently we showed that of transient receptor potential channel family), a receptor for capsaicin (12, 13), and then induces the release of *Corresponding author. FAX: +81-43-226-2875 neurotransmitters including CGRP. In the present study, E-mail: [email protected]

1 2 K Kunieda et al we focused on the effects of capsaicin and lafutidine on in Dulbecco’s modified Eagle’s medium (DMEM) 2+ [Ca ]i in cells. supplemented with 5% fetal bovine serum (FBS) and 5% Rat pheochromocytoma cell line PC12 is a well- horse serum at 37LC under 5% CO2 as previously established model used for the investigation of signaling described (16), while HUVECs were cultured on mechanisms in neurons. Previously, we reported that collagen-coated dishes in DMEM/Ham F12 (1:1) capsaicin stimulates arachidonic acid release and supplemented with 10% FBS and 10 ng/mL human formation in PC12 cells (14). The basic-fibroblast growth factor (PeproTech, Rocky Hill, 2+ existence of TRPV1 mRNA in PC12 cells was recently NJ, USA) as previously reported (21). [Ca ]i in PC12 observed (15), and we found that TRPV1 protein is cells and endothelial cells were measured as previously expressed and capsaicin induces Ca2+ entry via TRPV1 described (16) with minor modifications. In brief, cells in PC12 cells (16). In addition, Choi and Kim (17) on dishes were loaded with 3 M Fura 2-acetoxymethyl reported that capsaicin acts as an inhibitor of store- ester (Dojindo, Kumamoto) for 30 min at 37LC in operated Ca2+ entry in PC12 cells. Thus, PC12 cells are a normal Tyrode’s buffer (135 mM NaCl, 5.4 mM KCl, 2+ useful model for studying Ca regulation by capsaicin 1.8 mM CaCl2, 1.8 mM MgCl2, 1mM HEPES, 5mM and/or lafutidine. In the present study, we show that glucose, pH 7.4) containing 0.05% cremophor EL 2+ 2+ lafutidine induces an increase in [Ca ]i by Ca influx (Nacalai Tesque, Kyoto). The cells were then washed from extracellular spaces in PC12 cells. Since lafutidine and detached from the dishes under a gentle stream of causes an increase in gastric mucosal blood flow buffer. Detached cells were next washed by centrifuga- (GMBF) in the gastric microvasculature (4, 18), the tion (800 P g, 30 s) at 4LC, and resuspended. In some 2+ lafutidine response on [Ca ]i was also examined in experiments, extracellular CaCl2 was omitted from the human umbilical vein endothelial cells (HUVECs). assay mixture. Finally, fluorescence readings were taken Lafutidine, in addition to being an antagonist of with a Hitachi F-2500 spectrophotometer (Hitachi, To- 2+ histamine H2 receptors, may act on store-operated Ca kyo). 2+ entry in cells. The possible role of increase in [Ca ]i induced by lafutidine on lafutidine-induced pharmaco- Cell viability logical effects such as gastrointestinal protective effects Cell viability was estimated by the leakage of lactate and the modification of ileum contraction is also dehydrogenase as previously described (22), where discussed. leakage (%) was defined as the ratio of lactate dehydrogenase activity in the buffer to total activity Materials and Methods [%  (extracellular activity) / (extracellular activity and remaining cellular activity) P 100] per tube. Chemicals Capsaicin, capsazepine, and were pur- Statistics chased from Sigma (St. Louis, MO, USA). Adenosine- Data were presented as means M S.E.M. for three 5'-O-(3-thiotriphosphate) (ATPS) was purchased from independent experiments performed in duplicate. The Boehringer-Mannheim (Mannheim, Germany). Thapsi- experiments, in which a batch of cells were used, were gargin and 1-[-(3-(4-methoxyphenyl)propoxy)-4-meth- performed on different days, and the difference in oxyphenyl]-1H-imidazole hydrochloride (SKF96365) duplicate determinations for an experiment was within were purchased from Wako (Osaka). Lafutidine was 10%. The results were reproducible when different supplied from Tokushima Research Center of Taiho batches of cells were used. Statistical analyses were Pharmaceutical Co. Ltd. (Tokushima). These reagents performed using the two-tailed Student’s t-test for were diluted with deionized water or a minimum volume comparison of two groups and by one-way analysis of of dimethyl sulfoxide. The final concentrations of capsa- variance followed by Dunnett’s multiple comparison test icin, thapsigargin, lafutidine, ATPS, and SKF96365 in for comparison of more than three groups. Values of the assay mixture were selected based on previous P0.05 were regarded as being statistically significant. studies (16, 17, 19, 20). The final concentration of dimethyl sulfoxide in the assay mixtures was less than Results 1%, and the pHs of the assay mixtures containing 2+ reagents were 7.3 – 7.5. The vehicles did not show any Effect of lafutidine on [Ca ]i in PC12 cells 2+ 2+ significant effects on [Ca ]i. Lafutidine (1 – 10 mM) induced an increase in [Ca ]i in PC12 cells in a concentration-dependent manner 2+ Cells and measurement of [Ca ]i (Fig. 1, Panel A). The lafutidine (3 mM)-induced PC12 cells were cultured on collagen-coated dishes response reached a maximum within 30 s after addition Ca2+ Entry by Lafutidine 3

2+ Fig. 1. The effect of lafutidine on [Ca ]i in PC12 cells. PC12 cells were incubated with the indicated concentrations of lafutidine (LAF) in the presence of 1.8 mM CaCl2 (Panel A). Each individually performed recording is presented on the same 2+ panel. Panel B shows quantitative analyses of lafutidine on [Ca ]i in PC12 cells in the presence (open circle) or absence (closed a circle) of extracellular CaCl2. Each value represents the mean M S.E.M. of three independent experiments. P0.05, significantly different from the value without lafutidine. In Panel C, cells were stimulated with 3 mM lafutidine in the absence of extracellular CaCl2, and CaCl2 (1.8 mM at final concentration) was subsequently added to the assay mixture as indicated by the horizontal bar. PC12 cells were incubated with 300 M ATPS (Panel D) or 3 mM lafutidine (Panel E) and then further stimulated with 3 mM lafutidine (Panel D) or 300 M ATPS (Panel E) in the presence of CaCl2. Each Panel (A, C, D, and E) shows typical recordings made more than three times.

2+ and was sustained for at least 3 min. In some 207 M 37 nM (n  3), respectively. The [Ca ]i level experiments, the lafutidine response slowly and slightly induced by 1 mM lafutidine in the presence of 100 M decreased 1 or 2 min after addition. The concentration- capsazepine was 273 M 38 nM (n  3), and the net dependence of lafutidine concerning the maximal increase induced by lafutidine was almost the same as 2+ increase in [Ca ]i is shown in Panel B. Although the that in the control cells. The addition of 3 mM lafutidine 2+ lafutidine response was not saturated under the tested caused a sustained increase in [Ca ]i in the presence of conditions, its effects at concentrations above 10 mM 100 M capsazepine, where the net increases were could not be determined because of its poor solubility. 150 – 200 nM for two experiments. Also, the addition of 2+ Lafutidine (3 mM) did not induce an increase in [Ca ]i 100 M histamine and 3 mM famotidine, another when the extracellular CaCl2 was omitted (Panel C). antagonist of the , showed no 2+ However, subsequent addition of CaCl2 to the effect on [Ca ]i in PC12 cells (data not shown). extracellular solution evoked a sustained increase in The addition of 300 M ATPS evoked a transient 2+ 2+ [Ca ]i to a level above the control. Since lafutidine is increase in [Ca ]i and the peak value (approximately proposed to interact with capsaicin-sensitive neurons, 500 nM) was observed 5 – 10 s after addition in PC12 we investigated the effect of capsazepine, an antagonist cells (Panel D) as previously reported (19). Lafutidine at 2+ 2+ of TRPV1, on the lafutidine-induced increase in [Ca ]i. 3 mM still induced a sustained increase in [Ca ]i in the Treatment with capsazepine marginally but not ATPS-treated cells. Furthermore, the addition of 2+ 2+ significantly induced a sustained increased in [Ca ]i, 300 M ATPS still induced an increase in [Ca ]i in where the levels 2 min after vehicle and 100 M 3 mM (Panel E) and 10 mM (data not shown) lafutidine- capsazepine addition were 127 M 27 nM (n  3) and treated cells, although the peak value induced for the 4 K Kunieda et al

2+ treated cells was lower than that for the control cells. sustained increase in [Ca ]i induced by thapsigargin was Lafutidine at 3 and 10 mM did not stimulate lactate reduced by half and significantly inhibited by 30 M dehydrogenase leakage for at least 30 min. For this, the SKF96365 (Panel A), but the lafutidine response was values of lactate dehydrogenase leakage in the vehicle- not affected (Panel B). The effect of 10 M SKF96365 treated, lafutidine (10 mM)-treated, and control PC12 on the thapsigargin response was found to be limited cells were under 5% of the total activity. and/or marginal (data not shown). 2+ Capsaicin alone evoked a transient increase in [Ca ]i Effects of SKF96365 and capsaicin on the lafutidine- in PC12 cells; a peak increase was observed after 10 – 2+ 2+ induced increase in [Ca ]i 20 s and the [Ca ]i levels decreased within 30 s after Thapsigargin, an inhibitor of Ca2+-ATPase found in capsaicin addition (Panel C) as previously reported (16). intracellular Ca2+ stores, was used to activate store- The increase induced by capsaicin was completely operated Ca2+ entry by inducing Ca2+ store depletion inhibited by an antagonist of TRPV1 (16). In addition to independently of receptor activation. Addition of 5 M the effect on TRPV1, capsaicin inhibits store-operated 2+ 2+ thapsigargin induced a sustained increase in [Ca ]i in Ca entry in PC12 cells (17). We previously reported 2+ PC12 cells (Fig. 2, Panel A) as previously reported (17, that the sustained increase in [Ca ]i induced by 5 M 23). The magnitude and time course of the thapsigargin thapsigargin is inhibited by 300 M capsaicin and that response were similar to those of the 3 mM lafutidine pretreatment with capsaicin abolishes the thapsigargin response. Next, we investigated the effects of inhibitors response (16). Similar to the thapsigargin response 2+ 2+ of store-operated Ca entry on the lafutidine- and the (Panel D), the sustained increase in [Ca ]i induced by thapsigargin-induced responses. SKF96365 is an inhibi- 3 mM lafutidine was inhibited by 300 M capsaicin tor of store-operated Ca2+ channels (17, 20). The (Panel E). Quantitative analyses of the capsaicin

2+ Fig. 2. The effects of capsaicin and SKF96365 on the lafutidine- or thapsigargin-induced increase in [Ca ]i in PC12 cells. PC12 cells were stimulated with 5 M thapsigargin (TG, Panel A) or 3 mM lafutidine (LAF, Panel B) and then further incubated with 30 M SKF96365 (SKF) 2 min after the first stimulation in the presence of CaCl2. In Panel C, cells were stimulated with 300 M capsaicin (CAP) and then further stimulated with 3 mM lafutidine. PC12 cells were stimulated with 5 M thapsigargin (Panel D) or 3 mM lafutidine (Panel E) and then further incubated with 300 M capsaicin. Each Panel shows typical recordings made more than three times, and Panel F shows quantitative analyses of the inhibitory effect of capsaicin on the 3 mM lafutidine-induced 2+ increase in [Ca ]i. Each data point was measured 1 min after the addition of capsaicin and is shown as percentage of the lafutidine response measured just before the addition of capsaicin. The values represent the means M S.E.M. of three independent experiments. aP0.05, significantly different from the value without capsaicin. Ca2+ Entry by Lafutidine 5

2+ Table 1. The effects of capsaicin and SKF96365 on the lafutidine-induced increase in [Ca ]i in PC12 cells and HUVECs PC12 cells Thapsigargin PC12 cells Lafutidine HUVECs Lafutidine

2+ Net increase in [Ca ]i by stimuli (% of control) Experiment I (1 min after addition) Vehicle 95.2 M 6.3 83.2 M 8.9 90.1 M 7.5 Capsaicin 45.4 M 5.5a 40.2 M 4.2a 40.3 M 3.5a SKF96365 40.1 M 1.5a 80.3 M 3.3 80.4 M 4.8 Experiment II (Pretreatment) Capsaicin 10.1 M 1.5c 40.2 M 2.9b 15.3 M 3.6c

In Experiment I, cells were incubated with 5 M thapsigargin or 3 mM lafutidine for 2 min as a first stimulus; and then they were further incubated with vehicle, 300 M capsaicin, or 30 M SKF96365 as a second stimulus. Each 2+ value was measured 1 min after the second stimuli and is shown as the percentage of a net increase in [Ca ]i measured just before the second stimuli. In Experiment II, cells were incubated with 300 M capsaicin for 2 min and then 2+ stimulated with 5 M thapsigargin or 3 mM lafutidine. The data is shown as percentages of the net increase in [Ca ]i 2+ in the control cells. The absolute increases in [Ca ]i induced by thapsigargin and lafutidine are shown in Figs. 1 – 3. Each data point indicates the mean M S.E.M. of three independent experiments. aP0.01, significantly different from the vehicle-treated group. bP0.01 and cP0.001, significantly different from the control group. responses are shown in Panel F and Table 1. The 3 mM lafutidine and famotidine (data not shown). 2+ increase in [Ca ]i induced by lafutidine tended to Addition of 300 M capsaicin alone caused a 2+ decrease slightly faster compared with the thapsigargin transient increase in [Ca ]i in HUVECs (data not response in PC12 cells. Also, pretreatment with 300 M shown), like in PC12 cells. However, we could not capsaicin for 2 min markedly inhibited the 3 mM detect TRPV1 protein in endothelial cells by immunob- lafutidine response (Panel C) and almost completely lotting using anti-TRPV1 antibody (sc-12498, Santa abolished the thapsigargin response (Table 1). Pretreat- Cruz Biotech) against human TRPV1 under our ment with 30 M SKF96365 for 2 min markedly conditions. It should be determined whether TRPV1 is inhibited the thapsigargin response (70 – 80% inhibi- expressed in HUVECs or not. tion), but only slightly affected the lafutidine response (20 – 30% inhibition). Discussion

2+ Effect of lafutidine on [Ca ]i in endothelial cells The present study showed that lafutidine and not Next, we examined whether lafutidine induces an famotidine at pharmacological concentrations above 2+ 2+ increase in [Ca ]i in endothelial cells (HUVECs). 1 mM induces a sustained increase in [Ca ]i in PC12 Similar to PC12 cells, lafutidine induced a sustained cells, a neuronal model cell line, and HUVECs, an endo- 2+ increase in [Ca ]i in the endothelial cells (Fig. 3, thelial model cell line. The stimulatory effects of 3 and Panel A). The lafutidine response was concentration- 10 mM lafutidine were not due to cell toxicity because dependent and the 10 mM lafutidine response in endo- (i) receptor stimulation using ATPS still induced an in- 2+ thelial cells was greater than that in PC12 cells. How- crease in [Ca ]i in lafutidine-treated PC12 cells, and (ii) ever, famotidine at 3 and 10 mM had no effect (data not lafutidine showed little and/or marginal effects on lac- shown). The lafutidine response in endothelial cells was tate dehydrogenase leakage from PC12 cells for at least significantly inhibited by 300 M capsaicin, but not by 30 min. These results suggest that lafutidine increases 2+ 30 M SKF96365 (Table 1), and pretreatment of endo- [Ca ]i in cells without inducing cell toxicity. thelial cells with 300 M capsaicin for 2 min also inhib- 2+ ited the increase in [Ca ]i induced by following chal- Non-involvement of TRPV1 and histamine receptors on 2+ lenge with 3 mM lafutidine. The sustained increase in the lafutidine-induced increase in [Ca ]i 2+ [Ca ]i induced by 5 M thapsigargin in endothelial cells As previously mentioned, PC12 cells express TRPV1 was inhibited by capsaicin and SKF96365 (data not mRNA and its protein (15, 16). Thus, lafutidine may act shown). Although 100 M histamine caused a rapid on TRPV1 and therefore stimulate Ca2+ influx in PC12 2+ 2+ increase in [Ca ]i in HUVECs as previously reported cells. Capsaicin elicited a transient increase in [Ca ]i in (24), the histamine response was not inhibited by 1 and PC12 cells, but the lafutidine response was long-lasting 6 K Kunieda et al

showed that capsaicin but not lafutidine inhibits the specific binding of [3H]resiniferatoxin in neuronal cells. Mimaki et al. (18) showed that lafutidine alone at 2+ 0.1 mM does not increase [Ca ]i in human embryo kidney cells that express TRPV1. These findings and reports suggest that lafutidine activates Ca2+ entry in PC12 and endothelial cells in a TRPV1-independent manner. Lafutidine is an antagonist of histamine H2 receptors (1, 2). In some cases, a receptor antagonist may act as a partial agonist. The activation of histamine H2 receptors stimulates cyclic AMP accumulation, but the change in 2+ [Ca ]i induced by histamine is small or negative in PC12 cells (26). In the present study, lafutidine caused 2+ an increase in [Ca ]i in PC12 cells, but histamine and famotidine had no effect. In HUVECs, histamine 2+ induces a rapid increase in [Ca ]i and regulates the expression of various molecules via histamine H1 but not H2 receptors (21, 24). However, lafutidine did not change the Ca2+ response induced by histamine in endo- 2+ thelial cells. Thus, a sustained increase in [Ca ]i induced by lafutidine in cells does not appear to be mediated by histamine H1 and H2 receptors.

Role of store-operated Ca2+ entry on the lafutidine- 2+ induced increase in [Ca ]i Ca2+ channels that are activated by intracellular store- 2+ Fig. 3. The effect of lafutidine on [Ca ]i in HUVECs. In Panel A, depletion and/or by thapsigargin were recently classi- HUVECs were stimulated with the indicated concentrations of fied as belonging to the TRP classic (TRPC) subfamily lafutidine (LAF) in the presence of extracellular CaCl2. Each individually performed recording is presented on the same panel. and as members of TRP ion channel family (27, 28). Panel B shows quantitative analyses of the effects of lafutidine on mRNAs encoding TRPC1-6 have also been detected in 2+ [Ca ]i in HUVECs, and the values represent the means M S.E.M. of a PC12 cells (20). As with thapsigargin, lafutidine elicited three independent experiments. P0.05, significantly different from 2+ a long-lasting increase in [Ca ]i in PC12 cells. Similar to the value without lafutidine. 2+ this, lafutidine causes a sustained increase in [Ca ]i in endothelial cells, where mRNA and/or the proteins of similar to that of the thapsigargin response (Figs. 1 and some TRPC are expressed (29, 30). In addition to its 2). Capsazepine is known to be an effective antagonist of action on TRPV1, capsaicin acts as an inhibitor of store- TRPV1, although capsazepine is somewhat nonspecific. operated Ca2+ entry induced by thapsigargin in PC12 Recently, Phillips et al. (25) reported that mutation of rat cells (16, 17) and inhibits store-operated Ca2+ entry in TRPV1 at several positions decreased the IC50 values human leukemia HL-60 cells (31) and Jurkat T cells 2+ for capsazepine inhibition of the TRPV1-mediated (32). The sustained increase in [Ca ]i induced by response. Although capsazepine almost completely lafutidine was significantly inhibited by capsaicin in inhibited the capsaicin response (16), the inhibitory PC12 and endothelial cells. Furthermore, pretreatment effect of capsazepine on the lafutidine-induced increase with capsaicin inhibited the thapsigargin- and lafutidine- 2+ in [Ca ]i was limited in PC12 cells. Resiniferatoxin (a induced responses in PC12 cells and the lafutidine potent agonist of TRPV1, Ref. 13) at 10 M did not response in endothelial cells. These results suggest the inhibit the thapsigargin response and capsaicin inhibited possible involvement of store-operated Ca2+ entry in the 2+ the response in the presence of 10 M ruthenium red (an lafutidine-induced increase in [Ca ]i in cells. antagonist of TRPV1, Ref. 13) in PC12 cells (data not Treatment with SKF96365 significantly inhibited the shown), as previously reported (17). Lafutidine caused a thapsigargin responses, but barely affected the lafutidine 2+ sustained increase in [Ca ]i in HUVECs. The pharmaco- responses in PC12 and endothelial cells. In PC12 cells, logical characteristic of its response in HUVECs was diacylglycerol-activated Ca2+ channels probably consist- similar to that in PC12 cells. Nishihara et al. (10) ing of TRPC3/6 proteins are different from the Ca2+ Ca2+ Entry by Lafutidine 7 channels activated by acetylcholine or thapsigargin, in lafutidine (9, 10). In the present study, we found that 2+ 2+ that diacylglycerol-activated Ca channels have a lower lafutidine causes a sustained increase in [Ca ]i in endo- sensitivity to inhibition by SKF96365 than do acetylcho- thelial cells. Store-operated Ca2+ entry probably via line-activated channels, and maximal Ca2+ entry due to TRPC and not a transient Ca2+ influx is required for NO diacylglycerol is greater than that observed in the release in endothelial cells and vasodilation (35). Thus, 2+ presence of thapsigargin (20). The sensitivity of the lafutidine-induced increase in [Ca ]i in endothelial lafutidine response to pretreatment with capsaicin in cells may contribute to the gastroprotective effect of PC12 cells was lower than that in endothelial cells. Our lafutidine via NO production. However, lafutidine alone findings and these reports suggest the existence of a does not affect NO production in the rat gastric mucosa diversity of store-operated Ca2+ channels in cells. Lafuti- (10). Lafutidine given intraperitoneally hardly increases dine may activate capsaicin-sensitive and SKF96365- GMBF under basal conditions, although lafutidine insensitive Ca2+ channels in cells. Thus, the identifica- augmented the increase in GMBF induced by mucosal tion of target ion channels of lafutidine should be acidification (18). The intragastric administration of determined in the future. lafutidine causes a sustained increase in GMBF, but this effect is totally attenuated by ablation of capsaicin- Possible role of Ca2+ entry on gastrointestinal protec- sensitive neurons (4). Thus, the lafutidine effect on 2+ tion induced by lafutidine [Ca ]i in endothelial cells does not appear to be involved We found that lafutidine at concentrations greater directly in the gastroprotective effect of lafutidine. 2+ than 1 mM induces a sustained increase in [Ca ]i Lafutidine enhances CGRP release from afferent probably via store-operated Ca2+ entry in PC12 cells, neurons in response to capsaicin in the rat stomach (10). although the maximally induced increase was limited as CGRP stimulates NO production in endothelial cells, compared with that brought about by receptor stimula- and this NO then participates in the regulation of GMBF tion. In PC12 cells, Ca2+ entry activated by store through vasodilation in the gastric microvasculature (36, 2+ 2+ 2+ depletion rather than Ca release from intracellular Ca 37). An increase in [Ca ]i induced by lafutidine in endo- pools is an important source of Ca2+ required to trigger thelial cells may enhance NO production induced by exocytosis (33), and receptor stimulation of PC12 cells stimuli such as CGRP and resulting vasodilation in 2+ expressing TRPC4 and not control PC12 cells provides endothelial cells. The effects of lafutidine on [Ca ]i sufficient Ca2+ to trigger neurotransmitter release (34). levels in neuronal and endothelial cells in gastrointesti- Lafutidine at concentrations less than 10 M, which nal tissues should be examined in the future. alone exerts no effect, enhances the capsaicin-induced release of CGRP from afferent neurons in the rat Acknowledgments stomach (10) and capsaicin-induced contraction via acetylcholine release in isolated guinea pig ileum (11). This study was supported in part by Grants-in-Aid for Although we could not detect a significant increase in Scientific Research from the Ministry of Education, 2+ [Ca ]i induced by lafutidine at concentrations less than Culture, Sports, Science, and Technology, Japan. 1 mM in a model cell line (PC12 cells), lafutidine at M 2+ order may cause an increase in [Ca ]i in the neurons in References gastrointestinal tissues. A possible explanation for the lafutidine responses in gastrointestinal tissues is that it 1 Shibata M, Yamaura T, Inaba N, Onodera S, Chiba Y, Ohnishi enhances neurotransmitter release by maintaining a H. Gastric antisecretory effect of FRG-8813, a new histamine H2 2+ receptor antagonist, in rats and dogs. Eur J Pharmacol. [Ca ]i level high for a sustained period in cells in 1993;235:245–253. cooperation with other stimuli such as capsaicin. It 2+ 2 Onodera S, Shibata M, Tanaka M, Inaba N, Yamaura T, Ohnishi i should be determined whether the [Ca ] responses H. 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