Biochemical and Biophysical Research Communications 361 (2007) 522–527 www.elsevier.com/locate/ybbrc
Oestrogen directly inhibits the cardiovascular L-type Ca2+ channel Cav1.2 Nina D. Ullrich
a,b,*
, Alexandra Koschak c, Kenneth T. MacLeod
a
a Imperial College London, Cardiac Medicine, NHLI London, UK University of Bern, Department of Physiology, Bu¨hlplatz 5, CH-3012 Bern, Switzerland University of Innsbruck, Department of Pharmacology and Toxicology, Innsbruck, Austria
b c
Received 3 July 2007 Available online 23 July 2007
Abstract Oestrogen can modify the contractile function of vascular smooth muscle and cardiomyocytes. The negative inotropic actions of oestrogen on the heart and coronary vasculature appear to be mediated by L-type Ca2+ channel (Cav1.2) inhibition, but the underlying mechanisms remain elusive. We tested the hypothesis that oestrogen directly inhibits the cardiovascular L-type Ca2+ current, ICaL. The effect of oestrogen on ICaL was measured in Cav1.2-transfected HEK-293 cells using the whole-cell patch-clamp technique. The current revealed typical activation and inactivation profiles of nifedipine- and cadmium-sensitive ICaL. Oestrogen (50 lM) rapidly reduced ICaL by 50% and shifted voltage-dependent activation and availability to more negative potentials. Furthermore, oestrogen blocked the Ca2+ channel in a rate-dependent way, exhibiting higher efficiency of block at higher stimulation frequencies. Our data suggest that oestrogen inhibits ICaL through direct interaction of the steroid with the channel protein. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Oestrogen; Hormones; L-type Ca2+ channel; Heterologous expression; Whole-cell patch-clamp
Oestrogen (17b-oestradiol), the main female reproductive steroid hormone, is primarily produced in ovaries and adrenal glands, but in presence of the cytochrome P450 aromatase, it can also be locally synthesized from precursor steroids such as progesterone, testosterone or androstendione [1]. Apart from its indisputable function and importance in the reproductive system, where it regulates endometrial thickness in the uterus, oestrogen has great influence on a variety of other tissues and organs. In bone, oestrogen is implicated in the regulation of the bone density [2]; in the brain, it contributes significantly to the mechanisms involved in long-term potentiation and memory [3,4] and in breast tissue, oestrogen stimulates cell proliferation that can result in the development of breast cancer. Although it can have adverse effects on breast tissue, oestrogen may have some beneficial effects on the cardio* Corresponding author. Address: University of Bern, Department of Physiology, Bu¨hlplatz 5, CH-3012 Bern, Switzerland. Fax: +41 31 631 4611. E-mail address:
[email protected] (N.D. Ullrich).
0006-291X/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.07.054
vascular system [5–7] though the extent of these remains controversial. It has been shown that oestrogen significantly influences the balance of blood lipids in favour of HDL cholesterol [8]. In the heart, it has a negative inotropic effect and leads to a dilation of the coronary vasculature [9]. Together, these actions may partly explain why women, before the onset of menopause, are relatively protected from the development of cardiovascular disease compared with age-matched men [10]. The exact mechanism by which oestrogen exerts its effects on the cardiovascular system remains largely unknown. Classically, oestrogens cross the cell surface membrane easily and bind to the ligand binding domain of nuclear oestrogen receptors (ERs). Binding leads to phosphorylation of serine residues on the receptor which influences the recruitment of coactivators allowing the DNA binding domain of the receptor to interact with native DNA so enhancing gene transcription. However, recent studies revealed alternative pathways of oestrogen action, which do not involve alterations in gene transcription [11]. Discovery of the existence of extra-nuclear ERs at or near
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the plasma membrane has increased our awareness of rapid, non-genomic mechanisms of oestrogen-mediated intracellular signalling [12]. Apart from its receptor binding and subsequent activation of diverse signalling pathways, oestrogen might also have additional modes of action on certain effectors, like, for example, ion channels. Valverde and colleagues have shown that oestrogen directly activates large conductance Ca2+-activated K+ channels in smooth muscle cells [13,14]. In hippocampal neurons, Wu and colleagues reported that 17b-oestradiol induced a rapid response of the neuronal L-type Ca2+ channel, resulting in its activation and an increase in basal [Ca2+]i. This rise in intracellular Ca2+ activates Src and ERK signalling pathways that are involved in oestrogen-induced neurite outgrowth and neuroprotective effects by production of the antiapoptotic protein Bcl-2 [15]. We have shown that oestrogen rapidly reduces contraction of ventricular cardiomyocytes [16]. This effect is mediated by inhibition of Ca2+ influx via the cardiac L-type Ca2+ channel which is involved in the first steps of the excitation–contraction coupling mechanism. However, it is not known if this oestrogen-induced inhibition is mediated directly on the ion channel. Recent studies conducted in our laboratory showed that in cardiomyocytes derived from ERa and ERb knock-out mice [17], oestrogen produced the same inhibiting effect on the Ca2+ current, ICaL, and myocyte contraction as in wild-type tissue. Therefore, we tested the hypothesis that oestrogen may directly interact with the Ca2+ channel. Heterologous expression of Cav1.2 in an ER-deficient human cell line (HEK-293) allowed us to determine the direct regulatory effects of oestrogen on the functional properties of the L-type Ca2+ channel. We show that oestrogen significantly reduces ICaL and changes the conduction properties of the ion channel.
Materials and methods Transient expression of Cav1.2 in mammalian cells. Human embryonic kidney cells (HEK-293) were maintained at 37 °C in an atmosphere of 95% O2 and 5% CO2 in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich Company, UK) supplemented with 10% (v/v) foetal bovine serum (Gibco, UK), 2 mM L-glutamine, and 100 U/ml of penicillin–streptomycin. For transient Ca2+ channel expression, cells were plated onto 10 cm tissue culture dishes 12 h before transfection with Ca2+ phosphate precipitation using standard protocols. HEK-293 cells were transiently transfected with human Cav1.2a1 subunit cDNA (3 lg) together with the auxiliary subunits b2a (2 lg) and a2d1 (2.5 lg) as well as 1.5 lg of pUC18 carrier DNA. For identification of positively transfected cells, green fluorescence protein (GFP, 1 lg cDNA) was added to the transfection solution. Cav1.2transfected cells were incubated at 37 °C in 5% CO2 until used in experiments. 24 h after transfection, cells were transferred to 35 mm glass bottom microwell dishes (MatTek Corporation, Ashland, USA) which served as recording chambers. Currents were measured 2–5 days after transfection. Voltage–clamp studies. Membrane currents were measured using whole-cell procedures with an Axopatch (series 1D) amplifier (Axon Instruments Inc., Foster City, CA, USA) controlled by pClamp8 software
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(Axon Instruments, Union City, CA). Capacity current and series resistance compensation were carried out using analogue techniques. Cells were patch-clamped using low-resistance borosilicate glass micropipettes with resistances between 2 and 6 MX. Starting from a holding potential of 80 mV, the voltage protocol to determine the voltage dependence of current activation (I–V curve) consisted of depolarizing 100-ms steps to various test potentials. Effects of oestrogen were monitored continuously using 100-ms test pulses to the voltage of maximal current amplitude (Vmax) determined by the I–V protocol. Single test runs were repeated at a rate of 0.2 Hz, unless otherwise stated. Voltagedependent current inactivation was determined by applying 1-s pulses to various pre-potentials (Vpre) followed by a test step to Vmax. Data points were fitted with the Boltzmann equation, y = A2 + (A1 A2)/ (1 + exp((V V0)/k)), where A1, A2, V0, and k are the initial value, final value, mid-point voltage, and the slope factor, respectively. Solutions. Macroscopic whole-cell Ca2+ current was recorded using the following solutions (in mM). The pipette solution contained 135 CsCl, 10 Cs–ethylene glycol bis (2-aminoethyl ether)-N,N,N’N’-tetraacetic acid (EGTA), 10 N-2-hydroxyethylpiperazine-N’’-2-ethane sulphonic acid (Hepes), 4 MgATP, and 1 MgCl2, at pH 7.2 adjusted with CsOH. The external solution contained 150 NaCl, 15 CaCl2, 1 MgCl2, and 10 Hepes, at pH 7.4 adjusted with NaOH. The effect of 17b-oestradiol (50 lM) was tested on Cav1.2. Stock solution of the test substance was prepared in dimethyl sulfoxide (DMSO) to a concentration of 100 mM in order to minimize the final content of DMSO in solution. No DMSO-mediated effect was observed. Continuous superfusion of the cells and rapid solution exchange was assured by a gravity driven multi-solution exchange system. All measurements were performed at 37 °C. Data analysis and statistics. Data were analysed using Clampfit 8.1 (Axon Instruments) and Origin 6.1 (Microcal Software, Northampton, MA, USA). All data are presented as mean ± SEM, n being the number of cells patched. Statistical significance was determined by unpaired Student’s t test, P values below 0.05 were considered significant.
Results and discussion Properties of ICaL in HEK293 cells HEK-293 cells do not express any endogenous cardiovascular L-type Ca2+ channels and also lack expression of the oestrogen receptors, ERa and ERb [18], both being essential requirements for this study. The channel forming Cav1.2a1 subunit was co-expressed with the cardiac-specific auxiliary subunits b2a and a2d1. In heart, several isoforms of the b2 subunit have been identified, of which b2a has been shown to enhance functional channel expression in the plasma membrane and the characteristic current inactivation properties of Cav1.2 [19]. Fig. 1 shows the basic characteristics of nifedipine-sensitive ICaL in Cav1.2transfected HEK-293 cells. Membrane current evoked by depolarization from 80 mV (holding potential) to +10 mV exhibited the typical features of the cardiovascular ICaL showing rapid activation upon membrane depolarization followed by slow inactivation. Application of 10 lM nifedipine completely inhibited ICaL (99 ± 3% at Imax). The current–voltage relationships of ICaL before and during maximal block are summarized in Fig. 1B showing typical peak activation at +10 mV and reversing at +65 mV under the respective conditions. Current block by nifedipine revealed some outwardly rectifying background conductance that might be due to Cl currents naturally
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Fig. 1. Nifedipine-sensitive ICaL in Cav1.2-transfected HEK-293 cells. (A) Representative current traces of ICaL during activation by depolarizing 100-ms pulses from the holding potential (VH = 80 mV) to +10 mV (black trace, see voltage step protocol in upper panel). Membrane currents were normalized to the cell capacitance. Application of 10 lM nifedipine completely blocked ICaL (grey trace). (B) The mean current–voltage relationship of ICaL measured during steady-state ICaL in control solution and during maximal inhibition by nifedipine (10 lM, n = 5). Currents were elicited by 100-ms voltage steps from VH to different potentials ranging from 40 to +70 mV (increment 10 mV) and normalized to Imax.
present in HEK-293. These results formed the basis for the following study, where the effect of oestrogen on ICaL was investigated. Oestrogen inhibited ICaL in HEK-293 cells Oestrogen rapidly decreased heterologously expressed ICaL in HEK-293 cells. Fig. 2 summarizes the effect of oestrogen on ICaL. Paired analysis of the current before and during maximal inhibition revealed that, at 50 lM, 17boestradiol reduced ICaL by 49 ± 6% (n = 6, Fig. 2A
and B). Residual ICaL could be completely blocked by 100 lM CdCl2 (Fig. 2A,C, and D). The oestrogen concentration at which current was reduced by 50% corresponds to previous findings in cardiomyocytes, where similar high concentrations were required to reduce ICaL [20–22]. The physiological relevance of such concentrations might be questioned, since plasma levels of circulating oestrogen are much lower, even taking into consideration oscillating variations [6,18]. However, it has been shown that oestrogen can be locally synthesized in the presence of the Cytochrome Cyp450 aromatase, which turns androgenic
Fig. 2. Inhibition of ICaL by 17b-oestradiol and CdCl2. (A) The time course of current inhibition during repetitive depolarizing 100-ms pulses from the holding potential ( 80 mV) to Vmax. 50 lM 17b-oestradiol significantly reduced ICaL by 50% in the representative experiment. Application of 100 lM CdCl2 completely inhibited the residual ICaL. During wash-out with control solution, ICaL only partially returned to initial current levels, reaching new steady-state after approximately 50% return from the initial oestrogen-induced inhibition. (B) The effect of oestrogen on ICaL. Currents were measured at Vmax and normalized to Imax. Paired analysis of the current before (Ctrl) and at the end of oestrogen application revealed significant inhibition of ICaL to 49 ± 6% by 50 lM 17b-oestradiol (n = 6). (C) Single, representative current traces elicited by depolarizing steps to Vmax (upper panel) before (black trace), during maximal block by 17b-oestradiol (dark grey trace), and by CdCl2 (light grey trace). (D) The current–voltage relationship of ICaL from the experiment shown in (C) during steady-state ICaL in control solution (circles), 17b-oestradiol (triangles), and CdCl2 (diamonds). Inhibition of ICaL by the steroid was similar at all voltages. CdCl2 blocked ICaL by 79% at Imax and revealed a non-specific outwardly rectifying background current.
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precursor steroids into oestrogen by aromatization [1]. Expression of Cyp450 and sufficient local biosynthesis of oestrogen in cardiomyocytes have been confirmed by Grohe and colleagues [1], providing good evidence that oestrogen levels might locally rise to concentrations above serum levels and thus to levels high enough to modulate effectors such as the cardiac L-type Ca2+ channel. During wash-out of the steroid with control solution, ICaL only partially recovered from block by 17b-oestradiol (see time course of current inhibition, Fig. 2A). The incomplete reversibility of ICaL after steroid application may be due to its hydrophobic nature and possible incorporation into the plasma membrane. Fig. 2C shows sample traces of ICaL, elicited by 100-ms depolarizations to Vmax, before and at the end of steroid application as well as during complete inhibition by CdCl2. The respective current–voltage relationships are illustrated in Fig. 2D showing that oestrogen significantly decreased ICaL at all voltages and shifted the reversal potential to hyperpolarized potentials. Constant intracellular perfusion with the patch pipette, the rapid time course of the effect (less than 1 min, Fig. 2A), and the lack of ERs in this preparation make it highly unlikely that genomic effects of oestrogen are involved in this inhibition. Therefore direct channel blockade is assumed. It has previously been shown that testosterone directly inhibited the L-type Ca2+ channel through direct interaction with the pore forming a1c subunit [21,23]. In order to investigate this issue, we looked at the activation and inactivation properties of the channel in the absence and presence of the steroid oestrogen. 17b-oestradiol influenced current gating of ICaL Comparison of the voltage-dependent activation properties of ICaL in control solution and during steady-state inhibition by 50 lM 17b-oestradiol revealed a significant oestrogen-induced leftward shift of the activation curve (Fig. 3, triangles). Tail currents (Itail) of ICaL, obtained after stepping back from various pre-potentials (Fig. 3), were normalized to the maximum Itail (Itail/max). The resulting data were averaged and then fitted with the Boltzmann equation. Under our experimental conditions, the midvoltage points V0.5,act for current activation in control and 17b-oestradiol were +21.3 ± 0.8 mV (n = 12) and +13.7 ± 0.6 mV (n = 8), respectively (P < 0.0001). The slope factors kact were 13.7 ± 0.7 and 10.8 ± 0.5 for each fit (P = 0.0071). Furthermore, comparison of the voltage-dependent inactivation properties of ICaL in control solution and during steady-state inhibition by 50 lM 17b-oestradiol also showed significant oestrogen-induced shift of the inactivation curve to more negative potentials. Data points were collected by calculating the fraction of open channels (Fopen) at Vmax after conditioning pulses of 1-s duration to various pre-potentials (Vpre, Fig. 3). V0.5,inact shifted from 7.6 ± 0.7 mV in control solution (n = 7) to
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Fig. 3. Effect of 17b-oestradiol on voltage-dependent current properties of ICaL. The graph shows the activation (triangles) and inactivation (circles) curve of ICaL in control solution (black) and during administration of 50 lM 17b-oestradiol (grey). Currents were normalized to Imax and averaged. Data points were fitted with the Boltzmann equation. Values for half-maximal current activation obtained from tail currents before and during steroid application were V0.5,act(Ctrl) = +21 ± 1 mV, n = 12, V0.5,act(Oestrogen) = +14 ± 1 mV, n = 8 (P = 0.0001). 17b-oestradiol significantly shifted the fraction of open channels (Fopen) in dependence of the pre-potentials to more negative potentials compared with control (V0.5,inact(Ctrl) = 8 ± 1 mV, n = 7, V0.5,inact(Oestrogen) = 21 ± 2 mV, n = 5, P < 0.0001).
21.1 ± 1.6 mV in 50 lM 17b-oestradiol (n = 5; Fig. 3, circles, P < 0.0001). The values for the slope factor kinact were not significantly different (Ctrl = 12.6 ± 0.64, Oestrogen = 10.5 ± 1.4, P = 0.1619). Thus, in addition to the general blocking effect of oestrogen on ICaL, our results also show that oestrogen changes the activation and availability characteristics of ICaL. The leftward shift in both V0.5,act and V0.5,inact during oestrogen administration provides clear evidence for a direct interaction of the steroid with ion channel conduction in a way that significantly reduces the area of the window current compared with ICaL under control conditions. In a native environment like the heart, oestrogen-mediated decrease in the area of window current and a shift to hyperpolarized voltages would render the cell less sensitive to spontaneous activity. This is especially important in situations of increased excitability through pathological action potential prolongation or in the case of early afterdepolarizations (EADs), when the plateau phase of the action potential would be long enough to re-activate L-type Ca2+ channels at these depolarized potentials. However, although oestrogen reduced ICaL in cardiomyocytes [16,20,22,24], its effect on activation and availability of the Ca2+ channel was not unequivocally confirmed [24]. Such difference in the data might be due to the distinct cellular environment of HEK-293 cells compared with freshly isolated cardiac cells. Use-dependence of oestrogen-mediated inhibition of ICaL In order to investigate the nature of current block by oestrogen in more detail, we tested whether the effect of oestrogen on ICaL depends on the functional state of the
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Fig. 4. Activity and frequency-dependence of oestrogen-induced current inhibition. (A) Oestrogen-mediated block of ICaL was tested using repetitive stimulation of current at 0.2 Hz by depolarization from VH ( 100 mV) to Vmax (indicated by open bars). After reaching steady-state in control solution, stimulation was halted and 17b-oestradiol (50 lM, black bar) was administered for one minute before current stimulation restarted. Current decrease by oestrogen was measured at Vmax until new steady-state levels were reached (n = 3). Currents were normalized to Imax, and data points during the decreasing phase of the currents were fitted with a mono-exponential function, y = y0 + x*exp( x/s), where x and s are the amplitude and the decay constant, y0 is the offset current. Please note the decrease in ICaL at restart of the stimulation protocol in 17b-oestradiol (resting channel block). (B) Comparison of the effect of use dependence on the inhibitory effect of 17b-oestradiol on ICaL. Stimulation of ICaL by depolarizing voltage steps from VH ( 80 mV) to Vmax was repeated at varying frequency. After reaching steady-state current levels in control solution, 17b-oestradiol was applied. Normalized current is plotted as a function of time at different stimulation rates. The results of current stimulation at a rate of 0.5 Hz and 0.2 Hz are shown. Data points were fitted with a mono-exponential function. Higher stimulation rate induced faster 17b-oestradiol-mediated inhibition of ICaL (s0.2Hz = 41.7 ± 12.1 s, n = 7, s0.5Hz = 22.7 ± 4.7 s, n = 3).
Ca2+ channel, i.e., if the channel needs to be activated in order for oestrogen to inhibit current flow [25]. Fig. 4A summarizes our data on the channel’s state-dependence. Application of oestrogen was performed at highly negative membrane potential (VH = 100 mV) to ensure that most channels would be in a closed state. Current stimulation after an exposure time long enough to ensure full solution exchange of the recording chamber and current inhibition revealed that a fraction of Ca2+ channels (27 ± 5%, n = 3) were already inhibited by 17b-oestradiol. These results show that oestrogen does not only require the channels to be open in order to induce effective current inhibition, but significantly inhibits the channels in the resting state. Furthermore, we tested whether the frequency of current activation had any influence on the oestrogen-mediated inhibition of ICaL. Repetitive stimulation of ICaL by depolarizing pulses to Vmax was given at two different frequencies (0.2 and 0.5 Hz). 17b-oestradiol (50 lM) was applied after a steady-state ICaL was reached and the rate of current inhibition at different frequencies was compared. Fig. 4B shows averaged traces for oestrogen-mediated decrease in Imax at 0.2 Hz (n = 7) and 0.5 Hz (n = 3). Data points were fitted with a mono-exponential function to calculate the time constant (s) for current decrease at the respective conditions. ICaL was inhibited by oestrogen more rapidly at the higher stimulation frequencies indicating an accumulation of channel block. Taken together, this study provides a broad characterization of the effects of oestrogen on the cardiac L-type Ca2+ channel offering a number of facts to support the hypothesis that oestrogen directly inhibits ICaL. Significant current inhibition was obtained by direct interaction with the channel protein in an activity- and rate-dependent
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