The construct with NCX₁ of dog was kindly provided by Dr. Luis Beauge (Laboratorio de Biophisica, Cordoba, Argentina) and linearised with XbaI, then transcribed into cRNA in vitro using mMESSAGEmMACHINESP6 kit (Ambion, USA). The final concentration of cRNA was adjusted to 0.2 ng/nL.

The plasmid pQE30-CyPA was kindly provided by Dr. Xu Sheng (Drug Discovery and Design Center and State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences). Expression and purification of the hCyPA protein was performed as described elsewhere [21] [22] . Gels and buffers used for native PAGE were made according to the standard Laemmli SDS protocol omitting the SDS. Native gels (12% polyacrylamide) were run at 12 mA and 45 min and stained with Coomassie Brilliant Blue R-250. The molecular weight of hCyPA was slightly less than 20 kDa (compare Figure 1) in line with the reported weight of 18 kDa. Unstained protein-molecular-weight marker was from Fermentas Life Science (USA).

Xenopus oocytes were used as expression for NCX₁ and as a model system to test the effects of herbal extracts and CyPA. This expression system is particularly suited because endogenous ion channels and transporters are functionally expressed only to a low extend, and hence, exogenous current components can easily be extracted. Females of the clawed toad Xenopus laevis (purchased from Maosheng Bio-Technology Com., Shanghai, China) were anaesthetised in a bath medium containing 1 g/L tricaine (Sandoz, Basel, Switzerland) and kept on ice. Parts of ovary were removed and treated with 0.5 or 0.25 mg/mL collagenase (Sigma) for 2 - 4 h, or overnight, respectively. Full-grown prophase-arrested

oocytes of Dumont stages V and VI [23] were selected for cRNA injection, and cultured with daily changed G-ORi solution or C-ORi solution (ORi plus 0.07 μg/mL gentamycin (Sigma) or ciprofloxacin hydrochloride (Sigma), respectively) at 20˚C. For expression of NCX₁ about 1.5 ng NCX₁-cRNA was microinjected into an ooctye at a flow-rate of 8 nL/s. Uninjected oocytes served as controls.

For investigating the effect of intracellular hCyPA, oocytes were first microinjected with NCX₁-cRNA and divided into 4 groups. The cells of each group were additionally microinjected with hCyPA (5, 10, 20 or 40 nl per oocytes, respectively, at a concentration of 4.27 µg/µL). For investigating extracellular effect of hCyPA, oocytes were first microinjected with NCX₁-cRNA and divided into 4 groups. The cells of each group were then incubated with different amounts of hCyPA; 200 µL of incubation medium contained 1, 2, 4 or 8 µL of hCyPA, respectively (4.27 µg/µL). Thereafter, oocytes were cultured in G-ORi or C-ORi at 20˚C for up to 2 days.

Since NCX is electrogenic, NCX-mediated current is a measure for transporter activities. To investigate the function of NCX₁, membrane currents were measured by conventional two-electrode voltage clamp (TEVC) using Turbo TEC-03 with Cell Works software (NPI electronic, Tamm, Germany). Glass microelectrodes were filled with 3 M KCl, and balanced in ORi solution for at least 30 min before recording. Before measurements, oocytes were loaded with Na⁺ by incubating the cells in Na⁺-loading solution for 30 min [24] . Thereafter, cells were kept for at least 30 min in post-loading solution (see solutions). Steady-state current-voltage dependencies were determined by averaging membrane currents during the last 20 ms of 200-ms rectangular voltage pulses from −150 to +30 mV in 10 mV increments that were applied from a holding potential of −60 mV. NCX-dependent current was determined as the difference of total membrane current in the absence and presence of 2 mM NiCl₂ as a specific inhibitor of NCX. The data were collected after analogue filtering at 300 kHz and analysed by Origin software (OriginLab Corp., USA). All experiments were performed at room temperature (about 25˚C).

The composition of ORi was (in mM): 90 NaCl, 2 KCl, 2 CaCl₂ and 5 MOPS (adjusted to pH 7.4 with Tris). To elevate intracellular Na⁺, cells were incubated for 30 min in “Na-loading solution” consisting of (in mM): 110 NaCl, 2.5 Na- citrate, 5 MOPS (adjusted to pH 7.4 with Tris) and stored thereafter for at least another 30 min in “Post-loading solution” consisting of (in mM): 100 NaCl, 5 BaCl₂, 20 TEA, 5 MOPS (adjusted to pH 7.4 with Tris). Standard test solutions contained (in mM): 100 Na-gluconate, 2 CaCl₂, 0.1 niflumic acid, 5 MOPS, and 0 or 2 mM NiCl₂ (adjusted to pH 7.4 with Tris).

Herbal extracts were kindly provided from Shanghai Institute Materia Medica (CAS) by Drs. CG Huang and CH Ma (supercritical fluid extraction of root of Acorus tatainowii Schott) and by Dr. LJ Xuan (dried ethanol extracts of Ilex pubescence and Gossampinus malabarica), and were dissolved in DMSO. Final concentration of the herbal extracts in test solution was 40 mg/L.

GA (CAS 1405-86-3, purity ≥95%) and α-asarone (CAS 2883-98-9, purity 98%) were purchased from SIGMA. Stock solutions of 1 or 100 mM were prepared in DMSO and diluted to the final concentration in the test solution. DMSO concentrations in all test solutions were below 1%, which was without effect on the membrane currents.

Analysed data were represented as means (±SEM) from N experiments. Means were considered as significantly different by Student’s t test on the basis of p < 0.05.

In control oocytes not injected with cRNA of NCX₁ Ni²⁺-sensitive could never be detected (Figure 2(a)), confirming that the cells do not express significant endogenous Na⁺/Ca²⁺ exchanger on the plasma membrane [26] . On the contrary,

oocytes being injected with cRNA of NCX₁ exhibited huge currents, in particular at positive potentials in outward direction. Interestingly, the NCX-dependent current could even exceed 10 µA, which can hardly be mediated by a carrier protein even at high density in the cell membrane. Nevertheless, such large currents had been considered to be mediated by NCX (see e.g. [27] ). On the other hand, Xenopus oocytes exhibit Ca²⁺-activated Cl⁻ current (see [28] ). Therefore, an alternative interpretation would be that the transporter operates at these potentials in reversed mode accumulating Ca²⁺ at the intracellular membrane surface. This accumulated Ca²⁺ would activate the Cl⁻ channels. After blocking NCX₁ by Ni²⁺ also the Cl⁻ channels will no longer be activated.

To reduce this Ca²⁺-dependent background current, we used as an inhibitor of the Cl⁻ channels 100 nM niflumic acid [29] in the test solutions, which indeed led to a considerable reduction of the Ni²⁺-sensitive outward-directed current (Figure 2(b)); in addition the inward-directed current was enhanced. Figure 2(c) illustrates that reduction of external Cl⁻ from 100 mM to 6 mM , also led to strong inhibition of the outward-directed current. Therefore, in most experiments, if not state otherwise, we used niflumic acid and the Cl⁻-reduced test solution as given in the Materials & Methods Section. The still remaining Ni²⁺- sensitive current in this standard test solution was considered to be mediated by NCX₁. In the following we will differentiate between “NCX₁-dependent” (or total Ni²⁺-sensitive) and “NCX₁-mediated” current.

For a first rough screening we looked for drug effects on total Ni²⁺-sensitive current. To determine this current, we used standard external oocyte-Ringer’s-like solution (ORi) in the absence of niflumic acid without and with 2 mM NiCl₂. The extracts of Ilex and Gossampinus showed slight, but statistically significant inhibition at 40 mg/L by about 15% or 25%, respectively; the Acorus extract showed slight stimulation by about 15% of the Ni²⁺-sensitive outwardly directed current at +10 mV (Table 1). We also tested several pure compounds in addition to the Acorus extract α-asarone, which showed slight inhibition by 10% (Table 1). Out of several tested drugs only the GA from Glycyrrhiza galabra exhibited significant and clear inhibition by about 60% at 40 mg/L (Table 1). In the following, therefore, our focus was on the effect of GA. Since the scatter of current measurements under voltage clamp to positive potentials in general is pretty large, we will concentrate on the analysis at negative potentials.

In the standard experiment, membrane currents were measured in different solutions with low Cl⁻ and 100 nM niflumic acid that were applied usually in the sequence:

The currents measured in the respective 0Ni²⁺ solution before and after the

application of 2 mM Ni²⁺ were averaged to partially compensate for small drift with time. The currents in 2 mM Ni²⁺ were then subtracted to obtain the NCX₁- mediated current. In stable experiments, the sequence could be repeated with another drug concentration. In some experiments an abbreviated protocol was applied with the solution sequence

The result of the effect of 20 μM GA on the current-voltage dependence of NCX-mediated current is illustrated in Figure 3(a) showing a significant inhibition of the activity of the exchanger over the entire potential range. A more detailed analysis of the concentration dependency yielded an IC₅₀ value of about 40 μM at −100 mV (Figure 3(b)). The inhibition did not significantly depend on membrane potential.

Microinjection of hCyPA up to 86 ng/µL (corresponding to 4.8 µM within the cytoplasm, calculated by assuming an oocyte volume of 1 µL) hardly affected the NCX₁-mediated current (Figure 4(a)). Interestingly, extremely high concentration (9.6 µM) obviously stimulated the current by about 30%. Despite the large error bars, the current increase is statistically significant.

Incubation of NCX₁-expressing oocytes in 1.2 µM hCyTA resulted in significant inhibition of NCX-mediated current (p < 0.05 compared to untreated cells); higher concentration 4.8 µM produced only insignificantly more inhibition (Figure 4(b)). Despite the considerable scatter of data an IC₅₀ value for 50% inhibition of less than 1 µM could be estimated (see inset of Figure 4(b)). For the experiments described above, the oocytes were incubated in the respective hCyPA solution for 2 days. One hour of incubation already showed some tendency of inhibition (about 10%), but only after one day maximum inhibition could be detected. From 2 batches of oocytes we found that the current at −100 mV was after 1 h of incubation reduced to 0.87 ± 0.16 and after one day to 0.34 ±

0.06 compared to untreated cells.