Erythropoietin modulates angiotensin II- or noradrenaline-induced Ca2+ mobilization in cultured rat vascular smooth-muscle cells
Tetsu Akimoto1,
Eiji Kusano1,,
Nobuya Fujita2,
Koji Okada2,
Osamu Saito1,
Shuichi Ono1,
Yasuhiro Ando1,
Sumiko Homma1,
Toshikazu Saito2 and
Yasushi Asano1
1 Departments of Nephrology and
2 Endocrinology and Metabolism, Jichi Medical School, Tochigi, Japan
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Abstract
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Background. It has been reported that human recombinant erythropoietin (rHuEpo) modulates the sensitivity of the cardiovascular system to angiotensin II (Ang II) or noradrenaline (NA). In the present study, we explored the effect of rHuEpo on the responsiveness of Ang II- or NA-induced cytosolic free calcium ([Ca2+]i) mobilization in cultured rat vascular smooth-muscle cells (VSMC).
Methods. [Ca2+]i concentrations in VSMC were measured by using the calcium-sensitive fluorescent dye fura-2.
Results. The addition of rHuEpo (250 U/ml) alone induced elevation in [Ca2+]i, which remained significantly elevated above basal level for at least 60 min in the presence of extracellular Ca2+. Pre-incubation with specific protein kinase C (PKC) inhibitor calphostin C (1 µmol/l) significantly reduced the peak and the sustained elevations of [Ca2+]i. Pre-treatment with rHuEpo for 60 min increased both basal [Ca2+]i and the changes in [Ca2+]i by Ang II or NA in a dose-dependent manner in the presence of extracellular Ca2+. The synergistic effects of rHuEpo with Ang II or NA were also retained when VSMC were bathed in the Ca2+-free medium after the pre-incubation of rHuEpo. Conversely, they were diminished in the presence of extracellular Ca2+ combined with intracellular Ca2+ release inhibitor 8-(NN-diethylamino)octyl-1,3,4,5-trimethoxybenzoate (TMB-8). The synergistic effects of rHuEpo were also diminished by PKC depletion or by PKC inhibitor.
Conclusions. These observations suggest that rHuEpo has synergistic effects on Ang II- or NA-induced [Ca2+]i mobilization, particularly on intracellular Ca2+ release, in VSMC. This may be a potential mechanism contributing to hypertension associated with rHuEpo therapy.
Keywords: angiotensin II; cytosolic free Ca2+ mobilization; erythropoietin; noradrenaline; protein kinase C; vascular smooth-muscle cells
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Introduction
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Erythropoietin (Epo) is a glycoprotein growth factor, synthesized mainly in the kidney, which regulates the survival, proliferation, and differentiation of the erythroid precursor cells [1].
Recombinant human erythropoietin (rHuEpo) is now widely used to treat anaemia in end-stage renal disease; it is effective and safe in clinical use [2]. However, since the introduction of rHuEpo in the treatment of renal anaemia, hypertension has emerged as one of its major side-effects [24]. Although the precise mechanism for this hypertension remains to be elucidated, several studies suggest that increased blood viscosity [3,5], loss of hypoxic vasodilatation [6], failure of adequate cardiovascular autoregulation [7], endothelin release [8,9], vascular endothelial dysfunction [10,11], genetic predisposition to hypertension [12,13], elevation of cytosolic free calcium ([Ca2+]i) in VSMC [14], and inhibition of nitric oxide synthesis [15,16] may be factors in rHuEpo-induced hypertension.
It has been reported that [Ca2+]i, particularly in VSMC, is one of the major determinants of vascular tone [17]. Also it is well known that several vasoconstrictors such as angiotensin II (Ang II) or noradrenaline (NA) have induced elevation of [Ca2+]i in several cell lines, including VSMC [1821].
Recently several studies reported that rHuEpo changes the sensitivity to Ang II or NA in the cardiovascular system [13,2224]. In addition, Neusser et al. [14] observed that rHuEpo increases [Ca2+]i and enhances the Ang II-induced [Ca2+]i mobilization in VSMC, although the mechanism is unknown. Therefore in the present study we explore the effect of rHuEpo on the responsiveness of Ang II- or NA-induced [Ca2+]i mobilization in VSMC. We observe that rHuEpo enhances either Ang II- or NA-induced [Ca2+]i mobilization in VSMC. Also, we suggest the possible involvement of the protein kinase C pathway in the synergistic effects of rHuEpo on [Ca2+]i mobilization induced by each agent.
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Subjects and methods
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Materials
Staurosporine, calphostin C, Ang II, NA, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma Chemical Co. (St. Louis, MO). Prazosin, propranolol, and 8-(NN-diethylamino)octyl-1,3,4,5-trimethoxybenzoate (TMB-8) were also purchased from Sigma Chemical Co. The acetoxymethyl ester of fura-2 (fura-2/AM) was obtained from Dojin chemicals (Kumamoto, Japan). rHuEpo (epoetin beta) was donated by Chugai Pharmaceutical Company (Tokyo, Japan). 2-ethoxy-1-[[2 1-(1H-tetrazol-5yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylic acid (CV11974), which is one of the potent non-peptide Ang II type-1 receptor (AT1R) antagonists, was donated by Takeda Pharmaceutical Company (Osaka, Japan).
Cell culture
VSMC were prepared from rat aorta of male SpragueDawley (SD) rats (150200 g) as previously described [25]. In brief, cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (ICN Biomedicals, Osaka, Japan), 100 U/ml penicillin, and 100 mg/ml streptomycin (Life Technology Inc., Rockville, MD). Cells grown to confluence were detached by a treatment with 0.125% trypsin and 0.02% EDTA and reseeded in secondary cultures. Cells were used between passages 3 and 10.
Measurement of cytosolic free calcium concentration ([Ca2+]i)
[Ca2+]i of VSMC was estimated by the fura-2 fluorescence as described previously [26]. The cells cultured on glass slides were rinsed with physiological salt solution (PSS) containing 135 mmol/l NaCl, 5 mmol/l KCl, 1 mmol/l CaCl2, 1 mmol/l MgCl2, 5.5 mmol/l D glucose, and 10 mmol/l N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid (HEPES), pH 57.2. Then they were incubated with 5 µmol/l fura-2/AM for 60 min at 37C. After aspiration of the fura-2/AM solution, the glass slides were rinsed and placed in quartz cuvettes at 37°C in a fluorescence spectrometer RF 5000 (Shimazu, Tokyo, Japan). The fluorescence was collected at 510 nm (bandwidth 5 nm) with excitation wavelengths of 340 nm and 380 nm in the ratio mode. The effectors were added after a stable fluorescence signal (R) was achieved. [Ca2+]i was determined as described by Grynkiewicz et al. [27], using the following equation: [Ca2+]i (nM)=Kdx [(R-Rmin)/(RmaxR)]xb, where R is the ratio of fluorescence of sample at 340 and 380 nm, and Rmax and Rmin were determined by treating the cells with 500 µmol/l digitonin and 10 mmol/l MnCl2 respectively. The term b is the ratio of fluorescence of fura-2 at 380 nm in Ca2+-free conditions to 1 mmol/l external Ca2+ plus digitonin. Kd is the dissociation constant of fura-2 for Ca2+, reported to be 224 nm at 37°C [27].
Statistics
The results are expressed as mean±SEM. Data were analysed by analysis of variance combined with Fisher's protected least significant difference (Fisher's PLSD). Differences with P<0.05 were considered to be significant. Each figure is representative one of at least four separate experiments.
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Results
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Effect of rHuEpo on [Ca2+]i
First we explored the effect of rHuEpo per se on [Ca2+]i in VSMC to investigate the mechanism by which pre-treatment with rHuEpo increases the basal [Ca2+]i. As shown in Figure 1
, the addition of rHuEpo (250 U/ml) induced an elevation in [Ca2+]i, which reached a peak within 0.1 min and remained significantly elevated above basal level for at least 60 min in the presence of extracellular Ca2+. On the other hand, although the rHuEpo also induced a significant increase in [Ca2+]i in the absence of extracellular Ca2+, the sustained elevation of [Ca2+]i that was observed in the presence of extracellular Ca2+ was diminished. In addition the peak level of increase in [Ca2+]i was significantly reduced.
In preliminary experiments, we measured the time-dependent change in [Ca]i induced by the vehicle alone (exactly the same medium as that in which rHuEpo was dissolved). There was no transient or sustained elevation of [Ca]i by the vehicle alone. In addition, no potentiation of Ang II- or NA-induced [Ca2+]i was observed when cells were pre-treated with vehicle alone. Therefore these results suggest that rHuEpo per se potentiates [Ca2+]i induced by Ang II or NA.
We have previously demonstrated that rHuEpo induced the activation of protein kinase C (PKC) in VSMC [15]. Therefore we further explored the role of PKC in the rHuEpo-induced elevation of [Ca2+]i in VSMC. As shown in Figure 1
, pre-incubation with calphostin C (1 µmol/l), which is a specific PKC inhibitor, significantly reduced the peak level of the increase in [Ca2+]i induced by rHuEpo. Furthermore the sustained elevation of [Ca2+]i observed in the presence of extracellular Ca2+ was also diminished.
Effect of rHuEpo on Ang II- or NA-induced elevations of [Ca2+]i
We explored the effect of rHuEpo pre-treatment (250 U/ml, 60 min) on Ang II- or NA-induced elevations of [Ca2+]i in the presence of extracellular Ca2+. Figure 2
shows the typical tracings of [Ca2+]i after administration of either Ang II or NA both in the rHuEpo pre-treated (I) and untreated (II) cells. Both the basal level and the change of [Ca2+]i induced by either Ang II or NA were much higher in the former cells than in the latter. Also, the changes of [Ca2+]i induced by both agents were completely blocked by their specific receptor antagonists CV-11974 or prazosin, regardless of pre-treatment of rHuEpo respectively.

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Fig. 2. Typical tracing of [Ca2+]i after administration of Ang II (1 µmol/l) or NA (10 µmol/l) in rHuEpo-pre-treated (I) and untreated (II) VSMC. (A) Effect of Ang II (1 µmol/l) induced change of [Ca2+]i in the presence or absence of AT1R antagonist CV-11974 (1 µmol/l). (B) Effect of NA (10 µmol/l)-induced change of [Ca2+]i in the presence or absence of 1R antagonist prazosin (1 µmol/l). The traces are representative of more than six similar experiments.
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Furthermore, we examined whether rHuEpo pre-treatment had any effect on dose-dependent Ang II- or NA-induced [Ca2+]i increase. Basal [Ca2+]i in rHuEpo-untreated cells was 50.3±1.3 nmol/l (n=25), and in rHuEpo pre-treated cells (n=25) it increased significantly (P<0.01) to 79.8±4.3 nmol/l. Although both Ang II and NA elevated [Ca2+]i in a dose-dependent manner regardless of the rHuEpo pre-treatment, pre-incubation with rHuEpo significantly enhanced the elevation of [Ca2+]i (Figure 3A
,B
).

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Fig. 3. Effect of rHuEpo on Ang II- or NA-induced increase in [Ca2+]i in the presence of extracellular Ca2+. VSMC were pre-treated with fura2/AM (60 min) in the presence or absence of rHuEpo (250 U/ml) prior to stimulation. (A) Dose-dependent effect of Ang II-induced maximum increase in [Ca2+]i in either rHuEpo-pre-treated or untreated cells. (B) Dose-dependent effect of NE-induced maximum increase in [Ca2+]i in rHuEpo pre-treated or untreated cells. Data are means±SE of at least six separate experiments. *P<0.05, **P<0.01 vs rHuEpo-untreated.
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We further analysed the dose-dependent effect of rHuEpo pre-treatment on Ang II (1 µmol/l)- or NA (10 µmol/l)-induced elevations of [Ca2+]i. Pre-treatment with rHuEpo increased basal level of [Ca2+]i in a dose-dependent manner (Table 1
). In addition, pre-treatment with rHuEpo also enhanced Ang II- or NA-induced elevation of [Ca2+]i in a dose-dependent manner (Table 1
).
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Table 1. Dose-dependent effect of rHuEpo pre-treatment on Ang II (1 µmol/l)- or NA (10 µmol/l)-induced changes of [Ca2+]i in the presence of extracellular Ca2+. Data are means±SE of four separate experiments. *P<0.05, **P<0.01 vs controls
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Effect of PKC activator on Ang II- or NA-induced increase in [Ca2+]i
We next examined the effect of PMA pre-treatment, which is a potent activator of PKC, on Ang II (1 µmol/l)- or NA (10 µmol/l)-induced elevation of [Ca2+]i in the presence of extracellular Ca2+. Pre-treatment of PMA for 60 min prior to stimulation with these agents increased the basal level of [Ca2+]i in a dose-dependent manner (Table 2
). Moreover, pre-treatment of PMA also enhanced Ang II- or NA-induced elevation of [Ca2+]i in a dose-dependent manner (Table 2
).
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Table 2. Dose-dependent effect of PMA pretreatment on Ang II (1 µmol/l)- or NA (10 µmol/l)-induced changes of [Ca2+]i in the presence of extracellular Ca2+. Both Ang II and NA rapidly induced the elevation of [Ca2+]i, which reached a peak level within 0.1 min. Data are means±SE of four separate experiments. **P<0.01 vs controls
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Effect of ionomycin on Ang II- or NA-induced increase in [Ca2+]i
To investigate the contribution of the elevated basal [Ca2+]i level to the enhancement of Ang II- or NA-induced [Ca2+]i elevation, we further analysed the effect of ionomycin, a calcium ionophore, pre-treatment in the presence of extracellular Ca2+; as well as the effect of PMA, pre-treatment of ionomycin increased basal level of [Ca2+]i in a dose-dependent manner (Table 3
). In addition, pre-treatment of ionomycin also enhanced Ang II (1 µmol/l)- or NA (10 µmol/l)-induced elevation of [Ca2+]i in a dose-dependent manner (Table 3
).
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Table 3. Dose-dependent effect of ionomycin pretreatment on Ang II (1 µmol/l) or NA (10 µmol/l)-induced changes of [Ca2+]i in the presence of extracellular Ca2+. Both Ang II and NA rapidly induced the elevation of [Ca2+]i, which reached a peak level within 0.1 min. Data are means±SE of four separate experiments. *P<0.05, **P<0.01 vs controls
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Effect of rHuEpo, PMA, and ionomycin pre-treatment on Ang II- or NA-induced intracellular Ca2+ release and extracellular Ca2+ influx
It has been reported that both Ang II and NA induce [Ca2+]i mobilization by intracellular Ca2+ release and by extracellular Ca2+ in several cell lines, including VSMC [1821,40]. Therefore we further explored their contribution to the synergistic effects of rHuEpo, PMA, and ionomycin pre-treatment.
After pre-treatment with rHuEpo (250 U/ml), PMA (10 nmol/l), or ionomycin (10 nmol/l) for 10 min in the presence of extracellular Ca2+, VSMC were bathed in the extracellular Ca2+-free medium prior to simulation with Ang II or NA to investigate the effect of rHuEpo, PMA, and ionomycin pre-treatment on Ang II (1 µmol/l)- or NA (10 µmol/l)-induced intracellular Ca2+release. As shown in Table 4
, basal [Ca2+]i was significantly elevated in each pre-treated group. In addition, the elevations of [Ca2+]i induced by Ang II or NA were also enhanced in these groups.
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Table 4. Effect of rHuEpo (250 U/ml), ionomycin (10 nmol/l), and PMA (10 nmol/l) pretreatment in the presence of extracellular Ca2+ on Ang II- or NA-induced intracellular Ca2+ release. After pretreatment of rHuEpo, ionomycin, and PMA in the presence of extracellular Ca2+, VSMC were then bathed in the extracellular Ca2+-free medium containing 5 mmol/l EGTA prior to simulation with Ang II or NA. Data are means±SE of four separate experiments. **P<0.01 vs controls
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Next, VSMC were treated with TMB-8, a potent inhibitor of intracellular Ca2+release, in the presence of extracellular Ca2+, for 10 min prior to stimulation with Ang II or NA. Although basal [Ca2+]i was significantly elevated in each of the pre-treated groups, there was no significant enhancement in the elevation of [Ca2+]i induced by Ang II or NA in either of the pre-treated groups (Table 5
).
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Table 5. Effect of rHuEpo (250 U/ml), ionomycin (10 nmol/l), and PMA (10 nmol/l) pretreatment in the presence of extracellular Ca2+ on Ang II- or NA-induced extracellular Ca2+ influx. After pretreatment of rHuEpo, ionomycin, and PMA in the presence of extracellular Ca2+, VSMC were then treated with TMB-8 (10 µmol/l) for 10 min prior to simulation with Ang II or NA. Data are means±SE of four separate experiments. **P<0.01 vs controls
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After VSMC were pre-treated with rHuEpo (250 U/ml), PMA (10 nmol/l), or ionomycin (10 nmol/l) in the absence of extracellular Ca2+, VSMC were bathed in the Ca2+-containing PSS prior to simulation with Ang II or NA. Basal [Ca2+]i was not significantly elevated in either of the pre-treated groups (data not shown). In addition, there was also no significant enhancement in Ang II- or NA-induced elevation of [Ca2+]i in either of the pre-treated groups (Table 6
).
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Table 6. Effect of rHuEpo (250 U/ml), ionomycin (10 nmol/l), and PMA (10 nmol/l) pretreatment in the absence of extracellular Ca2+ on Ang II- or NA-induced extracellular Ca2+ influx. After pretreatment with indicated agents, cells were then bathed in the Ca2+-containing PSS for 15 s prior to simulation with Ang II or NA. Data are means±SE of four separate experiments
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Effect of rHuEpo or PMA pre-treatment on Ang II- or NA-induced [Ca2+]i mobilization in PKC depletion or PKC inhibitor
We further analysed the effect of rHuEpo and PMA pre-treatment on Ang II or NA induced elevation of [Ca2+]i in PKC-depleted VSMC. Since it is known that pre-treatment with phorbol ester for 24 h causes the downregulation of PKC in VSMC [28], VSMC were pre-treated with PMA (1 µmol/l) for 24 h. Then VSMC were further incubated with rHuEpo (250 U/ml) or PMA (10 nmol/l) for 60 min prior to stimulation with Ang II (1 µmol/l) or NA (10 µmol/l). There was neither significant elevation in basal [Ca2+]i nor enhancement of Ang II- or NA-induced [Ca2+]i elevation by either rHuEpo or PMA pre-treatment (data not shown).
In addition, when cells were loaded with fura-2/AM combined with 1 µmol/l calphostin C, which is a specific inhibitor of PKC, neither significant elevation in basal [Ca2+]i nor enhancement in Ang II- or NA-induced elevation of [Ca2+]i were observed in the presence of 250 U/ml rHuEpo. Basal level of [Ca2+]i in the presence or absence of rHuEpo prior to stimulation with Ang II were 46±1.8 and 45±1.9 nmol/l respectively; the maximal changes in [Ca2+]i induced by Ang II were 129±16.0 and 149±14.0 nmol/l respectively. In addition, basal levels of [Ca2+]i in the presence or absence of rHuEpo prior to stimulation with NA were 45±2.1 and 43±2.3 nmol/l respectively; the maximal changes in [Ca2+]i induced by NA were 153±16.0 and 146±17.0 nmol/l respectively.
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Discussion
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The present study clearly demonstrates that rHuEpo has synergistic effects on Ang II- or NA-induced [Ca2+]i mobilization, particularly on intracellular Ca2+ release, in VSMC. Furthermore, results from our study suggest that PKC pathway might be involved in these effects, since PMA mimics the effects of rHuEpo and those effects were diminished in the PKC-depleted VSMC.
In previous studies [15,16], we reported that rHuEpo inhibited cytokine-stimulated nitric oxide production in cultured rat VSMC, which was the same origin of the cells used in the present study. In one of the former studies [16] we demonstrated that EpoR mRNA was expressed in VSMC by using RT-PCR analysis, and that the addition of anti-EpoR antibody, which recognizes the extracellular domain of EpoR, reversed the inhibitory effect of rHuEpo. In addition, using western blotting analysis, we recently documented that rHuEpo could induce phosphorylation of EpoR in cultured rat VSMC [unpublished data]. Furthermore, we have already confirmed that rHuEpo (250 U/ml) induced an elevation of [Ca]i, which reached a peak within 1 min and remained significantly elevated above basal level for at least 60 min. The degree of the elevation of [Ca]i at 60 min after the administration of rHuEpo was similar to that of elevation of basal [Ca]i observed when cells were loaded with fura-2/AM and rHuEpo for 60 min. In the presence of anti-EpoR antibody, however, we could not observe the transient and sustained elevation of [Ca]i [unpublished data]. Taking these findings into consideration, results of the present study strongly suggest that an Epo receptor-mediated mechanism may be involved in the potentiation of Ang II and NA on [Ca]i in VSMC.
Interestingly, in addition to the synergistic effects of rHuEpo pre-incubation on Ang II- or NA-induced [Ca2+]i mobilization, rHuEpo also induced the elevation of basal [Ca2+]i prior to stimulation with each agent. It is reported that rHuEpo could induce the elevation of [Ca2+]i in several cell lines, including VSMC [24,2933]. Our present data suggest that rHuEpo per se elevates [Ca2+]i in VSMC by at least two mechanisms, namely stimulation of intracellular Ca2+ release and extracellular Ca2+ influx, since elevations of [Ca2+]i were observed in both the presence and the absence of extracellular Ca2+. Furthermore, although elevation of [Ca2+]i remained significantly above basal level for at least 60 min in the presence of extracellular Ca2+, the sustained elevations of [Ca2+]i were diminished in the absence of extracellular Ca2+.
Previous studies demonstrated that phorbol esters, which are a strong activator of PKC, have been shown to stimulate 45Ca2+ entry in VSMC and they lead to elevations of [Ca2+]i in VSMC [3436]. In addition, we have previously demonstrated that rHuEpo induced the activation of PKC in VSMC [16]. Therefore we further explored the role of PKC in rHuEpo-induced elevation of [Ca2+]i. Consistent with these previous findings, it is suggested that the PKC pathway may be involved in the sustained extracellular Ca2+ influx, since pre-incubation with a specific PKC inhibitor, calphostin C, diminished the sustained elevations of [Ca2+]i. Also, similar results were observed when the VSMC were pre-incubated with a broad type of serinethreonine kinase inhibitor staurosporine (data not shown). These observations suggested that the elevations of basal [Ca2+]i after pre-incubation with rHuEpo prior to stimulation with Ang II or NA in the presence of extracellular Ca2+ could be explained by reference to this sustained elevation of [Ca2+]i, namely, sustained extracellular Ca2+ influx induced by activation of PKC.
Recent observations have indicated that there are several receptor subtypes for Ang II, i.e. AT1R and AT2 receptor [37]. Also, there is evidence proving that the
-adrenergic receptor, which could be a receptor of NA, comprises a heterogeneous family [38]. Consistent with previous studies [19,39], our present results demonstrate that Ang II and NA induce mobilization of [Ca2+]i via AT1R or
1R in VSMC respectively. When the VSMC were pre-treated with rHuEpo, although both the basal level and the changes of [Ca2+]i induced by Ang II or NA were much higher than in rHuEpo-untreated cells, the change in [Ca2+]i induced by both of them was also blocked by the specific AT1R antagonist CV-11974 and the
1R antagonist prazosin respectively. Therefore it is suggested that both agents induce the mobilization of [Ca2+]i via AT1R or
1R, regardless of pre-treatment of rHuEpo.
Both Ang II and NA mediate their effects on [Ca2+]i mobilization by inositol 1,4,5-triphosphate (IP3)-induced intracellular Ca2+ release, resulting in a rapid and transient increase in [Ca2+]i, and by influx of extracellular Ca2+ through Ca2+ channels, resulting in a sustained elevation of [Ca2+]i in several cell lines, including VSMC [1821,40]. It is reported that IP3-dependent intracellular Ca2+ release is controlled by changes in the [Ca2+]i in a biphasic manner. Ca2+ at submicromolar concentrations enhances IP3-dependent Ca2+ release from intracellular Ca2+ stores, while Ca2+ at higher concentrations inhibits it [4143]. We have demonstrated that pre-treatment of rHuEpo induced the elevation of basal [Ca2+]i by the extracellular Ca2+ influx mediated by PKC activation. In addition, when the basal [Ca2+]i was elevated, the synergistic effects of rHuEpo were observed on the Ang II- or NA-induced intracellular Ca2+ release (Table 4
) but not on the extracellular Ca2+ influx (Table 5
).
Importantly, neither of the synergistic effects of rHuEpo on the Ang II- or NA-induced [Ca2+]i mobilizations nor the elevations of the basal [Ca2+]i were observed when the VSMC were pre-treated with rHuEpo in the absence of extracellular Ca2+ (Table 6
). These results suggest that the mechanism of the synergistic effects of rHuEpo on Ang II- or NA-induced [Ca2+]i mobilizations may be explained by reference to the elevation of basal [Ca2+]i induced by PKC, which may be activated by an rHuEpo-mediated extracellular Ca2+ influx. This mechanism is further supported by the findings that pre-treatment of PMA (Table 2
) and calcium ionophore ionomycin (Table 3
) demonstrated effects similar to those of rHuEpo. In contrast, the synergistic effects of rHuEpo or PMA on Ang II or NA induced mobilization of [Ca2+]i were diminished in the PKC-depleted cells. Therefore these observations suggest that the elevations of basal [Ca2+]i may contribute to the synergistic effects of rHuEpo on Ang II or NA by enhancing IP3-dependent Ca release from intracellular stores.
There are several studies suggesting that the pre-treatment of phorbol esters, including PMA, inhibited not only receptor-coupled inositol phospholipid metabolism but also [Ca2+]i mobilization induced by several vasoconstrictors [4446]. However, PMA concentrations used in these studies were relatively high doses compared with those used in the present study. Indeed, PMA pre-treatment with the concentrations above 10 nmol/l did not have the synergistic effects on Ang II- or NA-induced mobilizations of [Ca2+]i, but rather diminished them (data not shown). Therefore, it could be assumed that PMA might control the mobilizations of [Ca2+]i as well as the changes in the [Ca2+]i in a biphasic manner. Further studies, however, are required in order to clarify the effects of phorbol esters on the [Ca2+]i mobilizations.
In summary, our results demonstrate that rHuEpo has synergistic effects by a PKC-dependent pathway on Ang II- or NA-induced [Ca2+]i mobilization, which is involved in the regulation of vascular tone, particularly on intracellular Ca2+ release, in VSMC. These synergistic effects of rHuEpo may be one of the potential mechanisms that contribute to hypertension associated with the treatment of rHuEpo.
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Acknowledgments
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We would like to thank Mrs Yuko Watanabe for her technical assistance. Part of the work in the manuscript was presented in the abstract form in the 31st Annual Meeting of the American Society of Nephrology (Am J Soc Nephrol 1998; 9: 413A (Abstr)). This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (10671003), and a grant from Jinseihinketsu-kenkyukai and Jin-kenkyukai.
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Notes
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Correspondence and offprint requests to: Eiji Kusano MD, Department of Nephrology, Jichi Medical School, Yakushiji 3311-1, Minamikawachi, Tochigi 329-0498, Japan. 
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Received for publication: 21.12.99
Revision received 31. 8.00.