Juice of Bryophyllum pinnatum (Lam.) inhibits oxytocin-induced increase of the intracellular calcium concentration in human myometrial cells.
The use of preparations from Bryophyllum pinnatum in tocolysis is supported by both clinical (retrospective comparative studies) and experimental (using uterus strips) evidence. We studied here the effect of B. pinnatum juice on the response of cultured human myometrial cells to stimulation by oxytocin, a hormone known to be involved in the control of uterine contractions by increasing the intracellular free calcium concentration ([[[Ca.sup.2+]].sub.i]).
In this work, [[[Ca.sup.2+]].sub.i] was measured online during stimulation of human myometrial cells (hTERT-C3 and M11) with oxytocin, which had been pre-incubated in the absence or in the presence of B. pinnatum juice. Since no functional voltage-gated [Ca.sup.2+] channels could be detected in these myometrial cells, the effect of B. pinnatum juice was as well studied in SH-SY5Y neuroblastoma cells, which are known to have such channels and can be depolarised with KCl.
B. pinnatum juice prevented the oxytocin-induced increase in [[[Ca.sup.2+]].sub.i] in hTERT-C3 human myometrial cells in a dose-dependent manner, achieving a ca. 80% inhibition at a 2% concentration. Comparable results were obtained with M11 human primary myometrial cells. In hTERT-C3 cells, prevention of the oxytocin-induced increase in [[[Ca.sup.2+]].sub.i] was independent of the extracellular [Ca.sup.2+] concentration and of voltage-dependent [Ca.sup.2+]-channels. B. pinnatum juice delayed, but did not prevent the depolarization-induced increase in [[[Ca.sup.2+]].sub.i] in SH-SY5Y cells.
Taken together, the data suggest a specific and concentration-dependent effect of B. pinnatum juice on the oxytocin signalling pathway, which seems to corroborate its use in tocolysis. Such a specific mechanism would explain the rare and minor side-effects in tocolysis with B. pinnatum as well as its high therapeutic index.
[C] 2010 Elsevier GmbH. All rights reserved.
Keywords:Bryophyllum pinnatum Crassulaceae Oxytocin Intracellular calcium concentration Tocolysis
Plants of the genus Bryophyllum (family Crassulaceae) occur in tropical Africa, America and Asia, Hawaii, India, China, Australia and Madagascar, and have been traditionally used in these regions in multiple pathological situations (Yadav and Dixit 2003; Lans 2006). In Europe the use of remedies prepared from the species Bryophyllum pinnatum [(Lam.), syn. Kalanchoe pinnata (Lam.), Bryophyllum calycinum (Salisb.)] is limited almost exclusively to anthroposophic medicine (Hamre et al. 2006; Simoes-Wust and Rist 2007), where it was introduced by Rudolf Steiner in 1921 (Daems 1982). Several components have been identified in B. pinnatum preparations, which are likely to have biological effects and might therefore have therapeutic potential, namely bufadienolides (McKenzie et al. 1987; Yamagishi et al. 1988; Yamagishi et al. 1989; Supratman et al. 2000; Supratman et al. 2001), flavonoids (Cao et al. 2005; Muzitano et al. 2006a), flavonoid glycosides (Gaind and Gupta 1971; Muzitano et al. 2006b), phenols (Gaind and Gupta 1973) and organic acids (Marriage and Wilson 1971). Our previous work on the pharmaceutical characterisation of an aqueous extract directly obtained from juice of B. pinnatum as the one used in the present study (Erni 2006), revealed the presence of flavonoids, cinnamic acid derivatives and bufadienolides (ca. 66[mu]g/100ml; Rist et al. 2007). The later were present in higher concentrations in similar extract from Bryophyllum daigremontiana (ca. 215 [mu]g/100 ml; Rist et al. 2007), in which several structural types have been previously identified (Wagner et al. 1986).
B. pinnatum was first introduced in 1970 at the Community Hospital of Witten-Herdecke, Germany, as a tocolytic for preventing premature labour (Hassauer et al. 1985). A retrospective analysis from an obstetric clinical practice revealed better outcomes and less side-effects in patients treated with B. pinnatum (ethanolic tincture, 33%) compared to treatment with fenoterol (Daub 1989). More recently, the tolerability and the tocolytic outcome of patients treated with B. pinnatum (aqueous extract, 5%) was compared with those of patients treated with beta-agonists in a retrospective clinical study with closely matched pairs (n = 67) (Plangger et al. 2006). Treatment with B. pinnatum revealed similar effectiveness but less side-effects than treatment with beta-agonists currently used in clinic as standard therapy for prevention of pre-term delivery (Plangger et al. 2006). B. pinnatum (aqueous extract, 100 mg/ml) inhibited the ex vivo contractility of human term myometrium strips (biopsies from caesarean section, n = 14) under physiological conditions (Gwehenberger et al. 2004).
A tight regulation of the intracellular free calcium concentration ([[[Ca.sup.2+]].sub.i]) is known to play a crucial role in the myometrial contractant and relaxant signalling pathways (Sanborn et al. 1998). An oxytocin-induced sensitization of the cellular contractile apparatus to [Ca.sup.2+] is thought to play an important role in term and pre-term labour (reviewed in Shmygol et al. 2006; Arthur et al. 2007). In the absence of contractions, the [[[Ca.sup.2+]].sub.i] is rather low. Activation of surface receptors and/or depolarization of the plasma membrane increases significantly [[[Ca.sup.2+]].sub.i] due to influx of extracellular [Ca.sup.2+] and/or activation of signalling pathways which result in the release of [Ca.sup.2+] from intracellular stores such as the endoplasmatic reticulum (Sanborn et al. 1998; Berridge et al. 2003).
In the present work, the effect of B. pinnatum juice on [[[Ca.sup.2+]].sub.i] changes occurring in cultured human myometrial cells in response to oxytocin has been investigated.
Materials and methods
Human myometrial hTERT-C3 immortalized by transfection of a telomerase reverse transcriptase (hTERT-C3 cells) (Condon et al. 2002; Devost and Zingg 2007) were provided by Dr. H.H. Zingg (McGill University, Montreal, Quebec, Canada). Human primary human myometrial M11 cells (Devost and Zingg 2007) were obtained from John A. Copland (Mayo Clinic College of Medicine, Jacksonville, FL). SH-SY5Y human neuroblastoma cells were obtained from the European Collection of Cell Cultures - United Kingdom (ECACC, Salisbury, Wiltshire, UK).
hTERT-C3 human myometrial cells (Condon et al. 2002; Devost and Zingg 2007) were cultured in a 1:1 mixture of DMEM and F-12 supplemented with 10% heat-inactivated fetal bovine serum (all from Gibco-Invitrogen, Carlsbad, CA, USA) and antibiotics (100 U/ml penicillin and 100 [mu]g/ml streptomycin, Lonza-BioWhittaker, Basel, Switzerland). Primary human myometrial cells M11 (Devost and Zingg 2007) were maintained in DMEM with high glucose, supplemented with 10% fetal bovine serum (all from Gibco-Invitrogen) and antibiotics (100 U/ml penicillin and 100 [mu]g/ml streptomycin, Lonza-BioWhittaker). SH-SY5Y human neuroblastoma cells were cultured in a 1:1 mixture of DMEM and F-12 supplemented with 10% heat-inactivated fetal bovine serum (all from Gibco-Invitrogen), antibiotics (100 U/ml penicillin and 100 [mu]g/ml streptomycin, Lonza-BioWhittaker), 1% non-essential amino acids, 7.35 mg/l glutamate and 55 mg/l sodium-pyruvate (all from Sigma-Aldrich, St. Louis, MO, USA). All cell lines were cultured at 37[degrees]C, in an atmosphere of 5% [CO.sub.2]-95% air and 90% humidity. When cells reached near confluence, they were detached with trypsin (Gibco-Invitrogen) and plated at a 1:2, 1:3 or 1:6 dilutions. Medium was changed every 3 days.
For the [[[Ca.sup.2+]].sub.i] measurements, hTERT-C3 cells were plated on solid black 96-well polystyrene microplates (Corning Inc., Schiphol-Rijk, The Netherlands) at a density of 31,250 cells/well (i.e. ca. 98,730 cells/[cm.sup.2]) 2 days before experiments were performed, to allow cells to fully recover from trypsinisation. M11 and SH-SY5Y cells were plated under similar conditions, except that the cell densities used were 25,000 cells/well (ca. 79,000 cells/[cm.sub.2]) and 62,000 cells/well (ca. 200,000 cells/[cm.sub.2]) respectively. In all cases, the cells were confluent on the day of the experiment, as assessed by phase-contrast microscopy, using cells seeded on a clear-bottom 96-well microplate.
B. pinnatum plants were provided by Weleda Brazil. A voucher specimen number 292.697 is deposited at the herbarium of Rio de Janeiro's Botanical Garden, Brazil. The plants were harvested in Brazil (19.03.07) by D. Magano, Weleda Brazil, before flowering, in the morning. After macroscopic confirmation of the identity, the fresh plant material was placed in a refrigerated box and immediately sent by airplane to Weleda Arlesheim, Switzerland. The plants were further kept refrigerated and processed within 3 days upon arrival by mechanical pressing in a roller mill to obtain B. pinnatum juice; 580 mg juice were obtained per g fresh plant. The procedure used corresponded to the production process for the active ingredient of Weleda Bryophyllum 50% (Weleda AG, Arlesheim). The juice was frozen and sent to the laboratory in Cantanhede, Portugal, where the cell biology experiments were performed. The suspension was thawed, homogenized, sonicated in an ultrasonic bath (Sonorex RK100) for 30s and then filtered, using a 595.5, 70 mm diameter filter (Whatman/Schleicher & Schuell, Kent, UK). Aliquots of the filtered juice were re-frozen and stored at -20[degrees]C. For each experiment, a freshly thawed aliquot was used.
HPLC profiling of B. pinnatum juice
Lyophilized B. pinnatum juice was reconstituted in methanol (corresponding to 2.15 ml B. pinnatum juice in 1 ml methanol), filtrated and submitted to HPLC using an Agilent 1100 HPLC-DAD (Agilent, Waldbronn, Germany) system equipped with a Waters SunFire C18 column (150 mm x 3 mm i.d., 3.5-[mu]m particle size, Waters, Baden-Dattwil, Switzerland) and a Waters SunFire C18 guard column (3 mm x 20 mm). The peaks separation was achieved under the following conditions: solvent A = 0.1% formic acid, solvent B = acetonitrile; linear gradient 0-30 min, 5-100% B in A; flow: 500 [mu]l/min; column temperature: 25[degrees]C; detection: 298 nm; DAD spectra: 190-400 nm; sample: 1 ml; injection volume: 10 [mu]l.
[[[Ca.sup.2+]].sub.i] measurements using the fluorescent [Ca.sup.2+] indicator Fura-2
hTERT-C3, M11 and SH-SY5Y cells were loaded with the [[[Ca.sup.2+]].sub.i] fluorescent indicator Fura-2 (Grynkiewicz et al. 1985) by incubation with 10 [mu]M of the membrane permeable acetoxymethylester derivative Fura-2/acetoxymethylester (Molecular Probes-Invitrogen, Carlsbad, CA, USA, reconstituted in DMSO as a 1 mM stock) and 0.06% (w/v) Pluronic F-127 (Molecular Probes-Invitrogen), in serum and antibiotics-free culture medium (40 [mu]/well). In all cases Fura-2/acetoxymethylester loading was performed for 1 h at 37[degrees]C in an atmosphere of 5% [CO.sub.2]-95% air and 90% humidity. The medium was then replaced by fresh culture medium without serum nor antibiotics and the cells - with exception of the SH-SY5Y which were directly submitted to the next step - were further incubated for 30 min to allow full hydrolysis of Fura-2/acetoxymethyl by cellular esterases, resulting in cytosolic capture of the membrane impermeant Fura-2. The cells were then washed twice with 100 [mu]l sodium salt solution (140 mM NaCl, 5 mM KC1, 1 mM [CaCl.sub.2],1 mM [MgCl.sub.2],10 mM glucose, 10 mM HEPES-[Na.sup.+], pH 7.4). For extracellular [Ca.sup.2+]-free experiments, a buffer with similar composition, but without [CaCl.sub.2], was used. The buffer was replaced again (100 [mu]/well) immediately prior to pre-treating the cells or not with B. pinnatum juice or nifedipine.
Cells were pre-treated in the absence or in the presence of B. pinnatum juice or nifedipine (Sigma-Aldrich) for 5 min at 37[degrees]C (inside the microplate reader), except for experiments with SH-SY5Y cells, which were performed at 25[degrees]C. Cells were then stimulated with 100 nM oxytocin (Sigma-Aldrich) (Devost and Zingg 2007), by adding 10[mu]l of a 1.1 [mu]M stock solution (using a multichannel micropipette), or with 50 mM KCl (Merck, Darmstadt, Germany), by adding 10[mu]l of a 550mM stock solution. SH-SY5Y cells were stimulated with 10 mM KCl by adding 1 [mu]l of a 1 M stock solution. In [Ca.sup.2+]-free experiments, oxytocin was prepared in [Ca.sup.2+]-free sodium salt solution. At the end of the experiments, calibration of fluorescence was achieved by permeabilising cells with digitonin (Sigma-Aldrich), by adding 10 [mu]l of a 2.4 mM stock solution (200 [mu]M final concentration), to obtain maximum Fura-2 fluorescence, followed by addition of 10 [mu]l of Tris-EGTA pH 10.2 solution (Tris 1 M, EGTA 200mM, pH 10.2) to chelate [Ca.sup.2+] and obtain minimum fluorescence (Rosario et al. 1989). For extracellular [Ca.sup.2+]-free experiments, the [Ca.sup.2+]-free sodium salt solution was replaced by [Ca.sup.2+]-containing sodium salt solution prior to adding digitonin.
Data acquisition and analysis
Fluorescence was measured at emission 510 nm using two alternating excitation wavelengths (340 and 380 nm; Grynkiewicz et al. 1985) using a microplate fluorescence reader (Spectramax Gemini EM, Molecular Devices, with SoftMax[R] Pro software). The microplate reader was set at "top-read kinetics", PMT at "high", and temperature at 37[degrees]C for M11 and hTERT-C3 cells, or at 25[degrees]C in the case of SH-SY5Y cells. During basal conditions (in the absence and in the presence of B. pinnatum juice or nifedipine) and following oxytocin or KCI stimulation, Fura-2 fluorescence was read every 5 s in the experiments with hTERT-C3 or M11 cells and each fluorescence time point corresponded to an average of 18 reads. For SH-SY5Y cells, Fura-2 fluorescence was measured every 3 s and each fluorescence time point corresponded to an average of 13 reads. Intracellular (i.e. cytosolic) free [Ca.sup.2+] concentration ([[Ca.sup.2+].sub.i]) was calculated using the following formula, according to Grynkiewicz et al. (1985):
[[[Ca.sup.2+]].sub.i] = [K.sub.d] x Q[[R - [R.sub.min]]/[[R.sub.max] - R]],
where R = [F.sub.340nm]/[F.sub.380nm] (F: fluorescence intensity); [R.sub.max] and [R.sub.min] =[F.sub.340nm]/[F.sub.380nm] at maximum [Ca.sub.2+] concentration (after digitonin) and at minimum [Ca.sub.2+] concentration (after EGTA), respectively; Q = [[F.sub.min]/[F.sub.max] at 380nm ([F.sub.max:] after digitonin; [F.sub.min]: after EGTA); and [K.sub.d] is the dissociation constant of the [Ca.sup.2+]/Fura-2 complex and was taken as 224 nM for experiments performed at 37[degrees]C (Grynkiewicz et al. 1985) or as 263 nM for those performed at 25[degrees]C (Shuttleworth and Thompson 1991).
Fluorescence intensity values (in relative fluorescence units) measured at 340 and 380 nm were taken from the stabilized signals obtained at basal conditions (after incubation or not with the inhibitors) and after digitonin or EGTA treatment. Following stimulation with oxytocin, the fluorescence values were taken from the time point at the peak of the response. The relative fluorescence units readings were converted to [[[Ca.sup.2+]].sub.i] values (in nM) using the formula described above (on a Microsoft-Excel[TM] spreadsheet). The variation of intracellular [Ca.sup.2+] concentration ([DELTA][[[Ca.sup.2+]].sub.i]) in each well was calculated by subtracting the peak of [[[Ca.sup.2+]].sub.i] after stimulation with oxytocin or KCl from the respective value during basal readings. The average [DELTA][[[Ca.sup.2+]].sub.i] was measured in replicate wells containing control and B. pinnatum (in some cases replaced by nifedipine)-incubated cells; data obtained with B. pinnatum (or nifedipine)-incubated cells were then expressed as a percentage of the control (cells incubated with buffer only). Typically, each experiment consisted of three or four control wells plus three or four wells containing cells incubated with inhibitors, and all cells were stimulated and measured simultaneously.
The dose-response curve for the effect of B. pinnatum juice [log(% juice)] on the ([[Ca.sup.2+]].sub.i] response to oxytocin, the determination of the corresponding [IC.sub.50] by curve-fitting and a multigroup comparison (one-way ANOVA followed by Dunnett's test) were performed using the software GraphPad Prism[TM] Other graphics and statistical analyses (paired samples t-test) were done on Microsoft-Excel[TM]. Results are expressed either as data of a representative experiment (out of 3-5) or as mean[+ or -] S.E.M. of 3-5 independent experiments. When appropriated, P-values are shown.
HPLC profiling of B. pinnatum juice
A chromatographic fingerprinting of B. pinnatum juice is depicted in Fig. 1. This juice is composed of multiple compounds, such as cinnamic acid- (peaks 1 - 3), flavonoid- (peaks 4-9), and bufadienolide-type constituents (peaks 10-12). These were tentatively identified based on fractionation, thin-layer chromatography, DAD-UV spectra, and/or HPLC-MS data. It has to be noted that, compared to cinnamic acids and flavonoids, most bufadienolides show lower UV absorbances (low [epsilon] values) at the detection wavelength of 298 nm. The low intensities of peaks 10-12, therefore, should not be misinterpreted.
[FIGURE 1 OMITTED]
Characterisation of the oxytocin-induced increase of[[Ca.sup.2*]] in human myometrial cells in vitro
Stimulation of hTERT-C3 cells with 100nM oxytocin induced a transient increase of the [[[Ca.sup.2+]].sub.i] (Fig. 2). The peak [[[Ca.sup.2+]].sub.i] response was observed at about 30 s after stimulation, and was followed by a slower decrease in the Fura-2 fluorescence back to resting conditions. Similar responses were observed when the hTERT-C3 myometrial cells were stimulated with oxytocin in [Ca.sup.2+]-free medium (Fig. 2), with the maximally attained [[[Ca.sup.2+]].sub.i] corresponding to 85% of the value obtained in the presence of 1 mM [Ca.sup.2+].
[FIGURE 2 OMITTED]
The L-type [Ca.sup.2+]-channel blocker nifedipine did not affect significantly the [[[Ca.sup.2+]].sub.i] response to oxytocin in hTERT-C3 cells at 10 [mu]M, a concentration known to effectively and specifically block this type of channels (McCleskey et al. 1986; data not shown), suggesting that L-type [Ca.sup.2+]-channels are not involved (at least primarily) in oxytocin-induced [[[Ca.sup.2+].sub.i] rise in these cells. This was corroborated by results showing that depolarization of hTERT-C3 cells with 50 mM KCl did not induce a detectable increase of the [[Ca.sup.2+]].sub.i], in the presence of an extracellular [[Ca.sup.2+]] of 1 mM (Fig. 2). Prevention of oxytocin-induced increase in [[Ca.sup.2+].sub.i] by B. pinnatum juice in human myometrial cells
Using the same experimental set-up we performed dose-response experiments to study the effect of B. pinnatum juice [0.1-2% (v/v)] on the [[[Ca.sup.2+]].sub.i] responses induced with oxytocin. B. pinnatum juice inhibited oxytocin-induced (100 nM) increase in cytosolic [[Ca.sup.2+].sub.i] in hTERT-C3 cells in a concentration-dependent manner (Fig. 3A). The calculated [IC.sub.50] value was 0.94% (v/v). A comparable though somewhat weaker inhibitory effect on oxytocin-induced increase in [[[Ca.sup.2+].sub.i] was observed in M11 cells treated with B. pinnatum juice (Fig. 3B). In further experiments, hTERT-C3 cells were pre-treated under similar experimental conditions, in the absence or in the presence of 2% B. pinnatum juice, but in [Ca.sup.2+]-free conditions. B. pinnatum significantly inhibited the observed oxytocin-induced increase of [[[Ca.sup.2+]].sub.i] in extracellular [Ca.sup.2+]-free conditions, indicating that the effect is independent of extracellular [[Ca. sup.2+]] (Fig. 4).
[FIGURE 3 OMITTED]
[FIGURE 4 OMITTED]
Effect of B, pinnatum juice on voltage-dependent increase of [[[Ca.sup.2+]].sub.i] in a neuroblastoma model
The neuroblastoma SH-SY5Y cells, which are known to express L-type [Ca.sup.2+]-channels (Hirota and Lambert 1997), responded to stimulation with 10 mM KCl with a typical transient increase of the [[[Ca.sup.2+]].sub.i], returning to near basal levels shortly (about 5 min) thereafter, as observed with the fluorescent indicator Fura-2 (Fig. 5). As expected, when the SH-SY5Y cells were pre-incubated for 5 min with 10 [mu]M of the L-type [Ca.sup.2+]-channel inhibitor nifedipine, the maximal [[[Ca.sup.2+]].sub.i] response to KCl was inhibited by about 60% (Fig. 6). When SH-SY5Y cells were pre-incubated with 2% B. pinnatum juice for 5 min, the variation of [DELTA][[[Ca.sup.2+]].sub.i] in response to KCl was similar to that observed in control cells (Fig. 6). However, SH-SY5Y cells pre-incubated with B. pinnatum juice showed a slower [[[Ca.sup.2+]].sub.i] increase than control cells pre-incubated with buffer only (Fig. 5).
[FIGURE 5 OMITTED]
[FIGURE 6 OMITTED]
To address the main goal of this study--characterisation of the effect of B. pinnatum juice on oxytocin-induced increase of intra-cellular [Ca.sup.2+] in myometrium cells--an experimental set-up was established to monitor [[[Ca.sup.2+]].sub.i] in myometrial cells. The first experiments showed that treatment of the cells with oxytocin induced an increase of [[[Ca.sup.2+]].sub.i] in the presence ofa physiological extracellular [Ca.sup.2+] concentration (1 mM). The cellular response to oxytocin in [Ca.sup.2+]-free conditions was comparable to that obtained in the presence of 1 mM extracellular [Ca.sup.2+]] (Fig. 2), indicating that the effects of oxytocin in these cells are mainly mediated by mobilization of [Ca.sup.2+] from intracellular stores. Nevertheless, duration of the response was shorter in [Ca.sup.2+]-free conditions ([[[Ca.sup.2+]].sub.i] returned to basal levels faster), suggesting that extracellular [[Ca.sup.2+]] influx is necessary to refill the intracellular [Ca.sup.2+] stores (Putney 2007). Treatment of hTERT-C3 cells with 50 mM KCl, which should be sufficient to open voltagegated [Ca.sup.2+] channels (if expressed in these cells; Rosario et al. 1989) did not affect significantly the [[[Ca.sup.2+]].sup.i] (Fig. 2), suggesting that voltage-dependent [Ca.sup.2+]-channels are absent or inactive in hTERT-C3 cells. These results are in agreement with previous studies using human myometrial cell populations (Molnar and Hertelendy 1990) and single cells (Thornton et al. 1992). The same applies to our in vitro observations with myometrial cells showing that [[[Ca.sup.2+]].sub.i] increase in response to oxytocin is mostly insensitive to nifedipine (a potent inhibitor of the voltage-dependent [Ca.sup.2+] channels; Holda etal. 1996).
Our results clearly show that B. pinnatum juice prevents oxytocin-induced increase in [[[Ca.sup.2+]].sub.1] in hTERT-C3 human myometrial cells in a dose-dependent manner. This effect was observed at concentrations that are comparable with those of an aqueous extract from B. pinnatum that inhibit the contractility of human term myometrium strips (5 x [10.sup.3] - 1 x [10.sup.4] mg/l; Gwehenberger et al. 2004), conferring physiological relevance to our data. The inhibitory effect observed in hTERT-C3 cells was also seen with M11 primary myometrial cells, even though it was somewhat weaker. This may be associated with the lower effect of oxytocin stimulation in M11 cells, which is possibly due to a lowerexpression of oxytocin receptors as compared to hTERT-C3 cells (Devost and Zingg 2007). Since M11 cells are primary, non-transformed and non-tumoural cells with finite dividing capacity, only a limited number of experiments could be carried out with these cells.
At least in hTERT-C3 myometrial cells, the inhibitory effect of B. pinnatum juice on the oxytocin-induced [[[Ca.sup.2+]].sub.i] increase does not seem to be mediated by an inhibition of L-type [Ca.sup.2+]-channels, since the oxytocin-induced [[[Ca.sup.2+]].sub.i] increase was essentially independent of the extracellular [Ca.sup.2+]-concentration and insensitive to the L-type [Ca.sup.2+]-channel blocker nifedipine. Conversely, B. pinnatum might inhibit the oxytocin-induced signal transduction pathway (for a review see Arthur et al. 2007), either by preventing the binding of oxytocin to the corresponding receptor and/or inhibiting other intracellular proteins involved in the signalling cascade leading to [Ca.sup.2+] release from intracellular compartments. The elucidation of the exact mechanism of action of B. pinnatum juice on myometrial cells and of which juice components are relevant for its effect on [[[Ca.sup.2+]].sub.i] warrants further investigations. Several of the numerous already detected components of B. pinnatum extract's components can conceivably affect [[[Ca.sup.2+]].sub.i] homeostasis. For instance, quercetin--a flavonoid known to be present in the leaves of B. pinnatum (Cao et al. 2005)--can promote a fast [Ca.sup.2+]-release from mitochondria (Ortega and Garcia 2009). Also bufadienolides (Van der Walt et al. 1997; Poindexter et al. 2007), among which bufalin (Bick et al. 2002) might increase [[[Ca.sup.2+]].sub.i] in cardiomyocytes. Whereas these compounds seem to lead to an increase in [[[Ca.sup.2+]].sub.i] xanthomicrol, a flavone present in Brickellia paniculata, seems to inhibit voltage-dependent [Ca.sup.2+]-channels in rat uterus (Ponce-Monter et al. 2006).
Not only oxytocin-elicited release of [Ca.sup.2+] from intracellular stores, but also opening of voltage-gated [Ca.sup.2+]-channels is likely to be involved in the physiological regulation of uterus contractions (Shmygol et al. 2006). Therefore, we also addressed the possible effect of B. pinnatum juice on these channels. Since the myometrium cells used in the above experiments appeared not to express functional voltage-gated [Ca.sup.2+] channels, the effect of B. pinnatum juice on voltage-dependent increase of [[[Ca.sup.2+]].sub.i] was investigated in undifferentiated SH-SY5Y neuroblastoma cells. These cells are known to express L-type voltage-sensitive calcium channels and to respond to KCl stimulation with a fast and transient increase of [[[Ca.sup.2+]].sub.i] (Hirota and Lambert 1997), which could be corroborated by our data. B. pinnatum juice delayed, but did not prevent, the increase in [[[Ca.sup.2+]].sub.i] induced by KCl in SH-SY5Y cells, suggesting that it does not block voltage-gated [Ca.sup.2+] channels. As to be expected nifedipine, a potent inhibitor of the voltage-dependent [Ca.sup.2+] channels, significantly reduced the KCl-evoked increase of [[[Ca.sup.2+]].sub.i] in the SH-SY5Y cell-model. Given that [[[Ca.sup.2+]].sub.i] is tightly controlled and reacts to alterations in the membrane potential in numerous tissues, the effect of B. pinnatum juice of delaying (but not blocking) the activation of voltage-dependent [Ca.sup.2+] channels might be clinically advantageous and explain why the patients treated with B. pinnatum experience only few, dose-independent and minor side-effects (Plangger et al. 2006).
In summary, our in vitro results show that B. pinnatum juice inhibits the oxytocin-induced increase of [[[Ca.sup.2+]].sub.i] in human myometrial cells in a dose-dependent, but extracellular [Ca.sup.2+]-independent manner. Such an inhibition is compatible with the tocolytic properties of B. pinnatum and can be explained through a specific effect on the oxytocin signalling pathway.
We acknowledge Dr. H. Zingg, Dr. D. Devost and J.A. Copland for kindly providing hTERT-C3 and M11 cells. We are indebted to Dr. K. Ndjoko for helping with the HPLC-analysis of B. pinnatum juice. We also thank Dr. C. Zimmermann for technical tips regarding the juice manipulation. This work was financed by Weleda AG (Arlesheim, Switzerland).
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A.P. Simoes-Wust (a), *, (1) M. Graos (b), (1), C.B. Duarte (b), (c), R. Brenneisen (d), M. Hamburger (e), M. Mennet (f), M.H. Ramos (f), (2), M. Schnelle (f), R. Wachter (g), A.M. Worel (h), U. von Mandach (g)
(a) Research Department, Paracelsus Hospital, Richterswil, Switzerland
(b) Biocant - Center for innovation in Biotechnology, Cantanhede, Portugal
(c) Center for Neuroscience and Cell Biology and Department of Zoology, University of Coimbra, Coimbra, Portugal
(d) Department of Clinical Research, University of Bern, Bern, Switzerland
(e) Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
(f) Clinical Research, Weleda AG, Arlesheim, Switzerland
(g) Department of Obstetrics, University Hospital Zurich, Zurich, Switzerland
(h) Medical Department, Weleda AG, Arlesheim, Switzerland
* Corresponding author at: Research Department, Paracelsus Hospital, Bergstrasse 16, CH-8805 Richterswil, Switzerland. Tel.: +41 44 787 24 93: fax: +41 44 787 2351.
E-mail address: email@example.com (A.P. Simoes-Wust).
(1) These authors contributed equally to the work and should be considered as joint first authors.
(2) Left Weleda in the meantime.
0944-7113/$ - see front matter [C] 2010 Elsevier GmbH. All rights reserved.
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|Author:||Simoes-Wust, A.P.; Graos, M.; Duarte, C.B.; Brenneisen, R.; Hamburger, M.; Mennet, M.; Ramos, M.H.;|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Article Type:||Clinical report|
|Date:||Oct 1, 2010|
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