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Cardiovascular Research 1998 37(1):263-270; doi:10.1016/S0008-6363(97)00207-1
© 1998 by European Society of Cardiology
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Copyright © 1998, European Society of Cardiology

In permeabilised endothelial cells IP3-induced Ca2+ release is dependent on the cytoplasmic concentration of monovalent cations

Peter G Wood and James I Gillespie*

The Department of Physiological Sciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, UK

* Corresponding author. Tel. +44 191 2226988; Fax +44 191 2226988; E-mail: J.I.Gillespie@newcastle.ac.uk

Received 24 April 1997; accepted 25 July 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: IP3-induced Ca2+ release from the intracellular stores plays a role in the production of vasoactive substances in the endothelium. In many cells, Ca2+ release is accompanied by an inward movement of K+ whose function may be to dissipate the potential difference created by the loss of positive charge from the internal stores. The existence of such a mechanism in endothelial cells was investigated. Methods: Using saponin-permeabilised bovine aortic endothelial (BAE) cells, the effects of K+ on the IP3-induced 45Ca2+ release were investigated. Results: Replacement of K+ with NMG inhibited IP3 (3 µM)-induced 45Ca2+ release by 55%. The ability of other ions to allow IP3-induced 45Ca2+ release was found to be K+=Na+>Cs+>Rb+>>Co2+. The K+ channel blockers TEA, 4AP and 3,4-DAP were found to significantly inhibit IP3-induced 45Ca2+ release by 16%, 36% and 27%, respectively. Conclusions: The data suggest that Ca2+ release from intracellular stores is partly dependent on a movement of K+ through K+ channels in the store membranes. In contrast, 9AA (400 µM) and substitution with Co2+ abolished the response. Therefore, K+ is important for IP3-induced 45Ca2+ release, but other ions are also likely to act as counter-ions. 9AA and Co2+ probably act on sites other than those involving ER monovalent cation channels. The possibility that a counter-ion system plays a role in the activation of endothelial cells is discussed.

KEYWORDS IP3, inositol 1,4,5-trisphosphate; BAE, bovine aortic endothelial; NMG, N-methyl-D-glucamine; TEA, tetraethyl ammonium; 4AP, 4-aminopyridine; 3,4-DAP, 3,4-diaminopyridine; 9AA, 9-aminoacridine; CICR, Ca2+-induced-Ca2+ release; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; DMEM, Dulbecco's modified Eagle's medium; SDS, sodium dodecyl sulfate; cpm, counts per minute; Tris, (hydroxymethyl)-methylamine; choline, [2-hydroxyethyl]trimethyl-ammonium; SERCA, sarco/endoplasmic reticulum ATPase; NO, nitric oxide


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
In endothelial cells stimulation with agonists, such as histamine or bradykinin, leads to an elevation of intracellular Ca2+. This rise is thought to underlie the production of vasoactive substances, which affect adjacent vascular smooth muscle cells and play a role in the regulation of vascular tone [1]. In general, the Ca2+ required for cell activation is derived from one or both of two sources: Ca2+ influx across the plasma membrane and Ca2+ release from the endoplasmic reticulum (ER) through the activation of IP3 receptors and Ca2+-induced-Ca2+ release (CICR) mechanisms [2–4]. In endothelial cells, agonist stimulation leads to the production of IP3 and the release of stored Ca2+ via the IP3/Ca2+ release channel [3]. However, ‘classical’ CICR has been difficult to demonstrate in endothelial cells, although a novel form of CICR has been reported [5].

Ca2+ signalling in endothelial cells is known to be complex involving oscillations in intracellular Ca2+ [6, 7]. Mathematical models have been developed for non-excitable cells, where counter-ion movements across the ER have been predicted to play a critical role in dampening Ca2+ oscillations [8]. However, in endothelial cells, the mechanisms underlying these complex events remain to be fully understood.

Release of stored intracellular Ca2+ involves a movement of positive charge from the internal stores. In striated muscle, the amount of positively charged Ca2+ released from the internal stores is large [9], and this positive charge transfer from the sarcoplasmic reticulum (SR) results in a potential difference across the SR membrane, with the lumen becoming negative with respect to the cytoplasm. Unless such a potential is eliminated, the resulting electrochemical gradient would be expected to curtail the further release of stored Ca2+. In isolated skeletal and cardiac muscle, it has been shown that Ca2+ release from the SR is accompanied by a counter-movement of ions whose function may be to dissipate such a potential and thereby sustain Ca2+ release [10–13]. This charge movement appears to be predominantly a cation influx carried by K+ via a high conductance cation channel [14]. A counter-ion mechanism has been proposed in a variety of other excitable and non-excitable cells such as hepatocytes [15]and microsomes from brain [16], and may therefore be an important factor influencing Ca2+ release in endothelial cells. This paper describes experiments to identify the existence of a counter-ion movement accompanying IP3-induced Ca2+ release from internal Ca2+ stores in saponin-permeabilised endothelial cells. If a counter-ion movement exists, then replacement of K+ with a large impermeant cation should affect IP3-induced Ca2+ release. The permeabilised cell approach allows precise manipulation of the intracellular solution surrounding the ER without the possibility of altering ER/SR proteins during cell fractionation. It has long been recognised that ‘skinned’ preparations are particularly useful in the study of SR counter-currents as they form preparations which are physiologically relevant [17]. The data presented here shows that Ca2+ release is, in part, dependent on the cytoplasmic concentration of K+. The possibility that these ionic systems play a role in the activation of endothelial cells is discussed.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Tissue preparation and cell culture techniques
Endothelial cell preparation was carried out as previously described [5]and in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute for Health. From each isolation, cells were seeded on to 12-well culture plates and maintained in 95% air/5% CO2 at 37°C. Cells were plated at a density of 50 000–75 000 cells per well and fed with modified Dulbecco's modified Eagles medium (DMEM) every 48 h. Each well contained 1 ml culture medium and confluence was achieved in approximately 7 days. Studies have shown that quiescent and growing cells behave differently, therefore cells were fed 24 h before use to remove endothelial cell growth factors and promote quiescence [18]. Cells were used 5–6 days after seeding when the cultures were approximately 95% confluent.

2.2 45Ca2+ efflux from saponin-permeabilised cultured cells
Culture medium was removed and cells washed in balanced salt solutions (BSS) (mM); 135 NaCl, 5.9 KCl, 12 Hepes, 1.5 CaCl2, 1.2 MgCl2, and 10 D-glucose (pH 7.3), before being placed on a mechanical shaker. Cells were permeabilised at room temperature (22°C) with 40 µg/ml saponin for 10 min in ‘skinning solution’ (mM): 120 KCl, 10 Hepes, 2 MgCl2, 1 ATP and 1 EGTA (pH 7.0). The efficiency of permeabilisation was routinely checked using trypan blue and traces of saponin were removed by rinsing the cells with loading buffer (mM); 120 KCl, 10 Hepes, 5 MgCl2, 5 ATP, 0.44 EGTA, 5 NaN3 and 0.12 CaCl2 (pH 6.88). In basal efflux experiments, cell permeabilisation and viability were checked by applying a standard pulse of IP3 (3 µM) to control wells (data not shown).

Permeabilised cells were then loaded with 45Ca2+ (10 µCi/ml) in ‘loading buffer’ for 12 min at 22°C and subsequently washed three times in isotope-free ice cold ‘loading buffer’ to terminate loading. 45Ca2+ efflux was measured in a solution containing (mM); 120 KCl, 10 Hepes, 3 EGTA, 5 NaN3, 1 ATP (pH 6.88). NaN3 was used to inhibit the uptake of 45Ca2+ into mitochondrial stores. Concentrations of CaCl2 and MgCl2 required to give a free Ca2+ concentration of 100 nM were calculated using the REACT programme [19]. Solutions were exchanged every 2 min, and bi-directional 45Ca2+ efflux (i.e. in the absence of the Ca2+-ATPase inhibitor thapsigargin [20]) during that interval was measured by liquid scintillation counting. Radioactive Ca2+ remaining in the cells at the end of the experiment was determined by solubilising the cells with 2% (w/v) SDS in distilled water. 45Ca2+ remaining in the wells after this treatment was negligible. The loss of 45Ca2+ at each time point (i.e. fractional loss) was calculated from the cpm as a fraction of the total 45Ca2+ remaining (including the SDS fraction) within the cells at that time point. Changes in fractional loss ({Delta}) were calculated as the difference between the fractional loss at the peak of the stimulus response and the fractional loss at the point immediately preceding the stimulus. The standard efflux buffer was modified accordingly by substituting K+ with the large cation substitutes NMG, Tris or choline. IP3-dependent 45Ca2+ release was induced by addition of a 2-min pulse of IP3 after a steady baseline flux was obtained. Experiments were performed in the continuous presence of modified solutions or K+ channel blockers, unless stated otherwise. For each experiment the control response was taken to be that obtained in 120 mM KCl. Experiments were performed at 22°C as semi-intact cell preparations tend to deteriorate more rapidly at higher temperatures [21].

All chemicals used were obtained from Sigma, UK. Tissue culture materials were from Gibco, UK. 45Ca2+ was purchased from ICN Biomedical, CA, USA.

Data are expressed as mean±standard error of the mean (s.e.m.). Where appropriate, data were analysed using paired t-tests and a P- value of <0.05 was considered significant. Asterisks indicate the level of significance as follows: **P<0.005, ***P<0.0005.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 K+ sensitivity of IP3-induced 45Ca2+ release in permeabilised cells
Fig. 1A shows typical data from 45Ca2+ efflux experiments in which cells were bathed in a standard intracellular solution containing 120 mM K+ and where indicated, the cells were exposed to a 2-min pulse of 3 µM IP3. If a movement of net positive charge into the ER is required to counter-balance the loss of Ca2+ during stimulation, then removal of K+ from the bathing solution should affect 45Ca2+ release. Replacement of K+ with NMG significantly reduced mean IP3 evoked 45Ca2+ release by 55% compared with the control response (P<0.005, n = 18). The sensitivity of the release process to K+ was determined by varying the K+ concentration in the efflux buffer from 0 to 120 mM and measuring the 45Ca2+ released by a standard dose of IP3. Fig. 1B shows that as the concentration of K+ is increased there is also a progressive increase in IP3-induced 45Ca2+ loss. This increase is unlikely to be due to K+ affecting the rate of 45Ca2+ re-uptake by the Ca2+-ATPase, as thapsigargin (2 µM) did not alter the response (data not shown). These observations suggest that IP3-induced 45Ca2+ release is partly dependent on cation movement into the internal stores. However, maximum inhibition of the release was at a concentration of around 3 mM K+ and not 0 mM. If the amount of free Ca2+ released is in the 1–100 µM range then it might be expected that 3 mM K+ would provide sufficient monovalent charge to counter-balance the movement of Ca2+. Therefore, other factors must be involved in the movement of counter-ions and the possibility of alternative ion pathways must be taken into account.


Figure 1
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  		Fig. 1 The effect of K+ on IP3-induced 45Ca2+ release from saponin-permeabilised BAE cells. (A) Mean data showing the fractional loss of 45Ca2+ in a solution containing 120 mM K+ ({circ}) and 0 mM K+ (bullet). Where indicated (by the horizontal bar), a 2-min pulse of IP3 (3 µM) was applied. (B) Analysis of data following the protocol described in A where {Delta} fractional loss was determined for solutions containing the K+ concentration indicated (n = 18).

 
3.2 Effects of other K+ substitutes on IP3-induced 45Ca2+ release
The nature of the process linking monovalent cations to Ca2+ release was investigated using a range of K+ substitutes. Fig. 2A shows mean data from a series of experiments where cells were bathed continuously in solutions containing the large organic cations Tris or choline. The mean IP3-induced Ca2+ release was reduced by either Tris or choline, by 36% and 25%, respectively (n = 12). These reductions were not statistically different to that seen with NMG (Fig. 2B), indicating that each may simply be acting as a cation substitute and not via a pharmacological interaction with the Ca2+ release mechanism.


Figure 2
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  		Fig. 2 (A) Mean data showing fractional loss in response to a pulse of IP3 (5 µM) at the time indicated. Cells were bathed in solutions where K+ was replaced with Tris ({diamondsuit}), choline ({blacksquare}) or NMG ({blacktriangleup}). (B) Analysis obtained from experiments in A plotted as mean {Delta} fractional loss, showing the inhibitory effects of K+ substitution on the IP3 response with Tris, choline or NMG (n = 12).

 
The ability of other cations (Na+, Rb+, Cs+ or Co2+) to substitute for K+ was also examined. Fig. 3A shows mean data from such experiments and Fig. 3B shows the relative magnitude of the peak responses compared to the K+ control value. Replacement with Na+ or Rb+ did not significantly reduce mean 45Ca2+ release compared to that determined with K+. However, using Cs+ as a cation substitute, the mean release was significantly reduced by 36% (n = 12) and was abolished by the divalent cation Co2+ (P<0.0005, n = 12) (Fig. 3B).


Figure 3
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  		Fig. 3 The effect of cation substitutions on IP3-induced 45Ca2+ release. (A) Mean data showing the fractional loss of 45Ca2+ in Cl–-based solutions containing K+ ({circ}), Na+ ({diamondsuit}), Rb+ ({blacksquare}) or Cs+ ({blacktriangleup}). Where indicated (by the horizontal bar), a 2-min pulse of IP3 (3 µM) was applied. (B) Analysis obtained from experiments in A, plotted as mean {Delta} fractional loss showing how K+, Na+, Rb+, Cs+ or Co2+ affect fractional loss in response to a standard pulse of IP3 (3 µM) (n = 12).

 
3.3 Effects of K+ channel blockers on IP3-induced and basal 45Ca2+ release
The data obtained suggest the operation of a monovalent cation-dependent mechanism involved in Ca2+ release from the ER. This mechanism may involve the K+ channels described in other cell types [10–13]. In order to explore this possibility, the actions of several K+ channel blockers (TEA, 4AP, 3,4-DAP and 9AA) were examined. Fig. 4 shows that high doses of TEA (5 mM), 4AP (2 mM) and 3,4-DAP (2 mM) significantly reduced the mean IP3-induced 45Ca2+ response by 16%, (P<0.05), 37% (P<0.005) and 27% (P<0.005, n = 8), respectively. This reduction is similar to that seen with the substitution experiments using Tris, choline and Cs+. Taken together these data support the idea that there is a functional K+ channel on the ER of endothelial cells.


Figure 4
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  		Fig. 4 The effects of the K+ channel blockers TEA, 4AP, 3,4 DAP and 9AA on IP3-induced 45Ca2+ release in BAE cells. The histogram shows mean {Delta} fractional loss in response to a 2-min pulse of IP3 (3 µM) as previously described (n = 8).

 
In contrast, 9AA (1 mM) was found to abolish IP3-induced 45Ca2+ release (P<0.0005, n = 16) (Fig. 5A). Fig. 5B illustrates the dose response relationship of the 9AA effect and shows that the half maximal concentration is approximately 0.01 mM.


Figure 5
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  		Fig. 5 (A) Mean data showing the effect of 9AA on IP3-induced fractional loss of 45Ca2+. 9AA was applied where indicated (by the lower horizontal bar) and was present throughout the remainder of the experiment. Where indicated (by the upper horizontal bar), a 2-min pulse of IP3 (3 µM) was applied. KCl control ({circ}) and control plus 400 µM 9AA (bullet). (B) Mean data following the protocol described in A and used to construct a histogram showing the dose–response relationship between 9AA concentration and {Delta} fractional loss of 45Ca2+ (n = 16).

 
9AA was also found to significantly decrease the basal efflux of 45Ca2+ in a dose-dependent manner (P<0.05, n = 4) (Fig. 6A). The highly significant effects of 9AA on IP3-induced and basal efflux of 45Ca2+ could not be mimicked by other K+ channel blockers. TEA, 4AP and 3,4-DAP have no discernible effect on basal efflux (data not shown). Fig. 6B shows how substituting K+ with NMG caused a small, transient rise in the basal release. However, overall, the removal of K+ from the bathing medium did not significantly alter the basal efflux of 45Ca2+.


Figure 6
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  		Fig. 6 (A) Mean data illustrating how 9AA effects basal fractional loss of 45Ca2+ in a dose-depenent manner. The histogram shows {Delta} fractional loss between the time point when 9AA (400 µM) was applied and after 4 min of efflux had occurred (n = 4). (B) Mean data showing how a step change into K+-free solution (•) (at the time indicated by the horizontal bar) does not significantly affect the basal efflux of 45Ca2+ compared to control ({circ}) (n = 4).

 
In an attempt to provide an alternative pathway for cation movement across the Ca2+-containing internal membranes, the K+ ionophore valinomycin was included in the bathing solutions. Valinomycin (2 mM) had no significant effect on the control response or the block induced by 9AA (Fig. 7). This suggests that 9AA exerts its effects by acting on sites other than those of the monovalent cation channels situated on the ER internal membrane, or alternatively 9AA blocks the channels formed by valinomycin.


Figure 7
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  		Fig. 7 The combined affects of valinomycin (2 mM) and 9AA (400 µM) on IP3-induced 45Ca2+ release. The histogram shows mean {Delta} fractional loss in response to a 2-min pulse of IP3 (5 µM) and was calculated as previously described (n = 6).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The data presented suggest that the release of Ca2+ by IP3-activated mechanisms in BAE cells may be limited if the ER membrane potential is not dissipated. This may be particularly important in cells such as BAEs, where intracellular Ca2+ has been reported to oscillate [6]. Oscillations are thought to be controlled by complex positive and negative feedback mechanisms on the IP3/Ca2+ release channel involving cytoplasmic and intra luminal Ca2+ [2, 22, 23]. The development of a negative potential across the ER membrane could also curtail further Ca2+ release. The potential difference across the ER membrane would rapidly dissipate and allow the further release of Ca2+ in a cyclical manner that could lead to the generation of oscillations.

The presence of a counter-current system to dissipate trans-sarcoplasmic reticular potential differences is well described in skeletal [10–13]and cardiac muscle SR [24, 25]. SR K+ channels appear to be the most abundant and have been isolated, purified and reconstituted into lipid bilayers [26, 27]. SR K+ channels have also been detected in preparations of SR from aortic smooth muscle cells [28]. Although the specific function of these channels has not been described in smooth muscle, it is likely that their role is also to dissipate potential differences across the SR. The experiments described in this paper are consistent with an inward movement of K+, facilitating the sustained release of 45Ca2+ during IP3-induced mobilisation from the internal stores in BAE cells. A similar K+ dependence of IP3-induced Ca2+ release has also been suggested to occur in hepatocytes [15], brain microsomes [16]and platelet membrane vesicles [29]. Collectively, these data indicate a widespread association between cytoplasmic K+ and the Ca2+ contained in IP3-sensitive stores. It is now well established that Ca2+ release from IP3-sensitive stores terminates, even in the continued presence of IP3; however the exact mechanisms responsible remain controversial. Most proposals have focused on the rapid hydrolysis of IP3 causing receptor deactivation, effects of cytoplasmic and/or luminal Ca2+ concentration on specific Ca2+ binding sites on the IP3 receptor/channel complex, and intrinsic deactivation properties of the IP3 receptor [30]. The present study raises the possibility that in endothelial cells, the K+ concentration in the cytoplasm and/or ER lumen may also be able to influence the release of stored Ca2+ and thus indirectly affect vascular tone and blood flow.

The putative monovalent cation channel in BAE cells appears to be relatively non-selective, therefore Na+, Rb+ and K+ are equally effective in facilitating the release of stored 45Ca2+ in response to IP3. Complete substitution of K+ with Cs+ allowed a reduced response, suggesting these ions are also capable of carrying charge across the internal store membranes, but less effectively than either K+, Na+ or Rb+. This observation is in agreement with a recent study using cerebellar microsomes which has postulated a role for K+ as a ‘co-factor’ in Ca2+ channel opening [31].

The possibility should be considered that NMG, the largest cation substitute used in these experiments, may itself block the IP3/Ca2+ release channel or have other pharmacological properties. However, this is unlikely to account for the K+ sensitivity observed, as no statistical differences were found between NMG, Tris and choline despite their differing structures and molecular weights (Mr 195, 121 and 140, respectively).

The actions of the K+ channel blockers, TEA, 4AP and 3,4-DAP suggest that BAE cells possess a cation channel of the K+ family. However, other workers have noted variable activities of these blockers depending on the exact preparation used and in bilayer studies, from which side of the membrane this compound is applied [32]. Garcia and Miller tested a number of K+ channel blockers on isolated SR vesicles and found most to be significantly more effective when applied from the luminal, as opposed to cytosolic side. The observation that 4AP and 3,4-DAP reduce the IP3 response to a level not significantly different to that of complete K+ replacement suggest that they completely block the putative cation channel in BAE cells.

Preventing the movement of K+, either by complete removal of this ion, or applying effective K+ channel blockers, still enabled a reduced IP3 response (45–70% of control). The charge movement associated with this residual K+-insensitive component may be balanced by the redistribution of other ions such as Mg2+, H+ or Cl– as has been suggested to occur in skeletal muscle [17]. Recently SR Cl– channels have been identified in a number of investigations [17, 33]. Ongoing work on BAE cells is showing that removal of Cl– from the intracellular bathing solution can also affect the magnitude of the IP3 response [34]. Alternatively, IP3-induced Ca2+ release in endothelial cells may have a component which is independent of counter-ion movements and can therefore proceed despite the possible build-up of a potential difference across the ER.

Data using the larger K+ channel blocker, 9AA has yielded some particularly interesting results. The differences between 4AP, 3,4-DAP and the extremely potent 9AA, could be accounted for if 9AA abolishes IP3-induced 45Ca2+ release by interfering with the IP3/receptor channel complex, either at the level of IP3 binding, or by channel modulation. This finding is in agreement with other studies on brain microsomes [35]and reinforced by the finding that in BAE cells, the addition of the K+ ionophore, valinomycin, did not prevent the abolition of the IP3 response by 9AA (Fig. 7). This effect may alternatively be due to 9AA blocking the channels formed by valinomycin.

When K+ was substituted with the divalent cation Co2+, IP3-induced 45Ca2+ release was not possible. If 9AA and Co2+ were to block the putative channel in addition to the IP3 receptor/channel complex, 9AA and Co2+ would be expected to abolish IP3-induced 45Ca2+ release as demonstrated. This finding is consistent with a report using nitric oxide (NO) sensitive electrodes where it was demonstrated that Co2+, but not the voltage-dependent Ca2+ channel blocker verapamil, completely inhibited NO production in aortic endothelial cells [36].

It is known that a thapsigargin-sensitive Ca2+ leak pathway operates at physiological resting internal Ca2+ concentrations (100–300 nM) in some cells [37]. The data suggest that 9AA might prevent this basal 45Ca2+ leak occurring and lead to a significant reduction in this basal 45Ca2+ efflux (Fig. 6A). Replacement of K+ with NMG did not produce a similar effect on basal 45Ca2+ efflux (Fig. 6B), suggesting that basal 45Ca2+ release is not dependent on cytoplasmic K+ or relies on an alternative counter-ion. Certainly the effects of Cl– substitution on IP3-induced 45Ca2+ release in BAE cells, suggest the involvement of ionic currents being carried by a variety of different ions to maintain electroneutrality across the internal store membranes. Clearly, in endothelial cells, the roles of H+, Mg2+ and Cl– in IP3-induced Ca2+ release require more detailed examination.

In summary, our results indicate that in BAE cells, a K+ counter-ion system operates to facilitate the release of Ca2+ from intracellular stores. However, the mechanism of ion movement is likely to be more complex than a simple Ca2+-out–K+-in counter-current system, given that an IP3-induced 45Ca2+ release remains in the complete absence of K+. In investigating these complex phenomena, caution should exercised regarding the use of 9AA and Co2+ which appear to have direct interactions with the internal Ca2+ release mechanisms in BAE cells.

Time for primary review 33 days.


   Acknowledgements
 
We gratefully acknowledge support from the British Heart Foundation. Parts of the results have appeared in abstract form [38]. The authors would like to thank Dr. H. Otun and Dr. J. Morgan for critically reading the manuscript and for helpful discussions.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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