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Cardiovascular Research 1998 37(1):254-262; doi:10.1016/S0008-6363(97)00206-X
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Copyright © 1998, European Society of Cardiology

Regional variation in appearance of vascular contractile endothelin-B receptors following organ culture

Mikael Adnera,*, Erik Uddmana, Lars Olaf Cardellb and Lars Edvinssona

aDivision of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden
bDepartment of Otorhinolaryngology, Malmö University Hospital, Malmö, Sweden

* Corresponding author. Tel. +46 46175331; Fax +46 46137277; E-mail: mikael.adner@med.lu.se

Received 14 April 1997; accepted 30 July 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The aim of this study was to investigate the appearance of contractile endothelin (ET)-B receptors following organ culture in different vascular regions. Method: The contractile responses of vascular smooth muscle induced by ET-1 and the selective ETB receptor agonist sarafotoxin 6c (S6c) were investigated in circular segments representing eight vascular regions in the rat (aorta, femoral artery, mesenteric artery, branch of the mesenteric artery, proximal and distal parts of the caudal artery, femoral and mesenteric veins). To allow the ETB receptor to be expressed, the segments were placed in organ culture for 1 to 5 days. Pharmacological characterisation of the ET receptors was performed in mesenteric arterial segments. All contractile responses were measured in percentage of K+-induced contraction. Results: ET-1 induced strong concentration-dependent contractions of all fresh (not cultured) segments. S6c had negligible effects on all fresh vessels with the exception of the mesenteric vein, where a small contraction was seen. After 1 day of organ culture all tested segments, with the exception of aorta and the proximal part of the caudal artery, showed concentration-dependent contractile responses to S6c which were further augmented after 5 days of culture. The ET-1-induced responses were only slightly affected by organ culture. Contractions induced by S6c were more enhanced in small arteries and veins than in larger arteries. Furthermore, the S6c-induced response was more pronounced in the mesenteric region as compared to the hindlimb. In fresh mesenteric arterial segments FR139317 (ETA receptor antagonist) and bosentan (ETA/ETB receptor antagonist) but not IRL 2500 (ETB receptor antagonist) shifted the ET-1-induced concentration–response curve in parallel to the right. In contrast, after organ culture the S6c-induced concentration–response curves were shifted parallel to the right in the following potency order: IRL 2500>bosentan>FR139317. Conclusion: During normal conditions, the ETA receptor is the dominating mediator of endothelin-induced contraction in eight different vascular regions. Furthermore, this study indicates that most of the vessels have the ability to develop contractile ETB receptors and that this plasticity differs in vascular regions.

KEYWORDS Endothelin; ETA receptor; ETB receptor; Organ culture; Plasticity; Rat; Sarafotoxin


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelin (ET)-1, a potent vasoconstrictor produced by endothelial cells, is known to mediate its effects in mammals through two distinct G-protein coupled seven transmembrane receptor subtypes, the ETA and the ETB receptors [1–3]. Intravenous infusion of ET-1 elicits a transient decrease followed by a slowly developing long-lasting rise in arterial blood pressure [1]. The initial vasodilatation is believed to be mediated via endothelial ETB receptors through the formation of NO or prostacyclin [4, 5]. The subsequent contraction was initially believed to be mediated by smooth muscle ETA receptors but recent studies have shown that it can in part be mediated by smooth muscle ETB receptors in some vascular regions [6–8].

The precise localisation of the vascular level where the ETB contractions appear is in several in vivo studies undefined [6–8]. Yet, it has been shown in cat skeletal muscle that the ETB receptor-induced vasoconstriction is most pronounced in the resistance arteries, particularly in the small arterioles [5]. In vitro studies have revealed that ETB receptors seem to be more abundant in low pressure systems such as the venous circulation and the pulmonary circulation [9, 10]. In contrast, some studies of arteries with a luminal diameter greater than that of resistance arteries (100–300 µm [11]) also show ETB receptor-mediated contraction [12–14]. The physiological implication of the heterogeneous distribution of contractile ETB receptors is unclear and this is further complicated by a suggested local cross-talk between ETA and ETB receptors when both are present in the tissue [15, 16].

In earlier studies we have revealed that human omental arteries after 1 to 5 days of organ culture elicit an ETB receptor mediated contraction which appears together with a concomitant increase in ETB receptor mRNA [17, 18]. The mechanism behind this phenomenon is at present unknown, but may be relevant to the adaptation of some parts of the circulation in different physiological (e.g. increase of blood flow [19]) and pathophysiological conditions (e.g. congestive heart failure and arteriosclerosis [20–22]). The aim of the present study was to examine if there are variations between vascular regions in the spontaneous increase of ET receptor mediated contraction following organ culture.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Tissue preparation and organ culture procedure
Male Wistar-Kyoto rats (250 g; Møllegården, Denmark) were anaesthetised with CO2 and killed by a cut through the heart. The thoracic aorta, the superior mesenteric artery, the first branch of the mesenteric artery, the mesenteric vein, the femoral artery, the femoral vein and the ventral caudal artery were immediately taken out, immersed in cold (4°C) sterile Dulbecco's modified Eagle's medium (DMEM), dissected free from adherent tissue under cold sterile conditions and cut into 1–2 mm long circular segments. The caudal artery was further divided into a proximal part (20 mm closest to the body) and a distal part (the last 20 mm of the end of the tail). Segments from each vessel were divided into two groups; one which was analysed within 1 h (fresh) and the other which was incubated in culture medium for 1 or 5 days. Segments for culture were placed in a 48 well plate (one segment in each well) containing 1 ml DMEM (for composition see below) and incubated at 37°C in humidified 5% CO2 in air as described previously [18]. Presence of the endothelium was verified by staining with 5% silver nitrate followed by light microscopy [23]. The experiments were approved by the Animal Ethics Committee, Lund University, Lund, Sweden.

2.2 In vitro pharmacology
The vasomotor reactivity was analysed in temperature-controlled (37°C) tissue baths containing a buffer solution (see below). The solution was continuously equilibrated with 5% CO2 in O2 resulting in a pH of 7.4. The vessel segments were mounted on two L-shaped metal prongs. One prong was connected to a force displacement transducer (FTO3C, Grass Instr., Quincy, USA) attached to a MacLab unit (ADInstruments, Hastings, UK) for continuous registration of isometric tension by the Chart software (ADInstruments). The other prong was connected to a displacement device, allowing adjustment of the distance between the two parallel prongs. A passive tension of 2–4 mN and 0.5–1 mN was applied to the arterial segments and venous segments, respectively. The tension was chosen with regard to variation in outer diameter and length of individual segments. The specimens were subsequently allowed to stabilise at the selected level of tension for 60 min. The contractile capacity of each tissue segment was then examined through exposure to a potassium (K+)-rich buffer solution (60 mM) which had the same composition as the standard solution except that equimolar concentration of NaCl was exchanged for KCl. Concentration–response curves for the agonists were obtained by cumulative application of the peptides. In the antagonist experiments, FR139317, bosentan and IRL 2500 were tested at the same concentration (10 µM) to obtain maximal blocking effects and preceded agonist addition by 15–20 min.

2.3 Solutions and drugs
Buffer solution was of the following composition in mM: 119 NaCl, 15 NaHCO3, 4.6 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 1.5 CaCl2 and 5.5 glucose. Serum-free DMEM (1000 mg l–1 D-glucose) contained sodium pyruvate (100 mg l–1) (Gibco BRL, Paisley, UK) and was supplemented with penicillin (100 U ml–1) and streptomycin (100 µg ml–1) (Gibco BRL). The sources of the agonists and antagonists were: ET-1, S6c (Auspep, Parkville, Australia), FR139317; (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl]amino-4-methylpentanoyl]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl)propionic acid (Fujisawa Pharmaceuticals Co., Osaka, Japan), bosentan (Ro 47-0203); 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2'-bipyrimidin-4-yl]-benzenesulfonamide (F. Hoffman-La Roche, Basel, Switzerland) and IRL 2500 (N-(3,5-dimethylbenzoyl)-N-methyl-(D)-(4-phenylphenyl)-alanyl-L-tryptophan) (Ciba-Geigy, Takarazuka, Japan). The peptides were dissolved in 0.1% w/v bovine serum albumin (Sigma, St. Louis, USA) in double-distilled water and further diluted in buffer solution.

2.4 Calculation and statistics
All data are expressed as mean values±s.e.m., and n refers to the number of rats which the vessel segments were obtained from. Contractile responses in each vessel segment are expressed as a percentage of the contraction induced by 60 mM K+ and Emax values refer to the maximum contractile effect of an agonist (mN values for Emax are included in Table 1). pEC50, the negative logarithm of the molar concentration that produced a half-maximum contraction was calculated from the straight-line equation between the concentration above and below the midpoint of the concentration–response curve. pKB (negative logarithm of the molar concentration of an antagonist which reduces the effect of agonist to half the concentration) was approximated using the equation pKB=–log([B]/(r–1)) in which [B] denotes the concentration of the antagonist and r denotes the ratio of the EC50 value with the antagonist and the EC50 value of the control experiment [24]. To arbitrarily compare differences of the effect of the ETB receptor, ETB ratio was calculated from the of Emax for S6c and the Emax for ET-1 in paired segments. A ratio of the K+ contraction after organ culture and the mean value for the K+ contraction in fresh segments was calculated in order to estimate the change in contractile capacity as a result of organ culture.


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  		Table 1 Contractile responses for ET-1 and sarafotoxin 6c (S6c) in fresh and cultured segments of different vascular regions in rat

 
Mann–Whitney U-test and Kruskal–Wallis test for multiple groups together with Bonferroni correction were used for unpaired analyses. Wilcoxon signed-rank and Friedman test for multiple groups were used for paired analyses. Linear regression analysis was performed to elucidate the correlation for the changed contractile capacity and the increased ETB receptor-induced contraction due to organ culture. Differences were considered significant at P-values <0.05.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Regional contractile effects of ET-1 and S6c in fresh segments
In fresh segments, ET-1 induced a strong concentration-dependent contraction of all vessels tested with the following potency order; femoral vein>mesenteric vein>aorta>branch of the mesenteric artery>distal part of the caudal artery>mesenteric artery>proximal part of the caudal artery>femoral artery (Table 1, Fig. 1). With the exception of the mesenteric vein, S6c induced no significant contraction in any of the vessel segments tested. The mesenteric vein responded with a weak but nevertheless potent contraction (Table 1, Fig. 1).


Figure 1
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  		Fig. 1 Contraction induced by cumulative concentrations of endothelin-1 and sarafotoxin 6c in fresh vessels (bullet) and vessels cultured for 1 ({triangleup}) or 5 days ({square}). The contraction of each segment tested was calculated as a percentage of the potassium (K+)-induced contraction in the same segment, and each point represents the mean of all segments tested with error bars representing s.e.m. n = 5–9.

 
3.2 Regional contractile effects of ET-1 and S6c in cultured segments
Vessels from five regions of interest, the femoral artery, the mesenteric artery, a branch of the mesenteric artery, the distal part of the caudal artery and the femoral vein displayed concentration-dependent contraction to S6c as the result of organ culture (Table 1, Fig. 1). The mesenteric vein showed a significantly enhanced contraction following organ culture. The S6c-induced vasoconstriction was increased with time of culture and, after 5 days of culture, the branch of the mesenteric artery, the mesenteric and the femoral veins reached Emax values that did not significantly differ from the responses induced by ET-1. S6c failed to induce significant contraction of the aorta and the proximal caudal artery, even though small contractions occasionally were seen in some of the cultured segments.

The response to ET-1 was not markedly changed following organ culture in relation to the K+-induced contraction. However, concomitant with the trend of decreased K+-induced contraction there was a general decrease of contraction for ET-1 as measured in absolute values (mN) at day 5 (Table 1). Yet, ET-1 elicited strong and potent concentration-dependent contraction in all the vessel segments tested (Fig. 1). After organ culture all vessels with the exception of aorta displayed a tendency towards increased Emax values but these were only significant in the caudal arteries. A significant decrease in Emax was seen in aorta after 1 day but this may be due to an increased sensitivity to K+ contraction. The pEC50 values for ET-1 were significantly higher in the mesenteric artery branch and the distal caudal artery after 5 days of organ culture. In the femoral artery, the mesenteric artery and the mesenteric vein the potency tended to increase with time of organ culture but was this not significant.

3.3 Regional differences in ETB receptor-induced contraction
In order to evaluate the plasticity of the ETB receptors, the S6c response was expressed as a ratio of ET-1-induced contraction in paired segments (x-axes in Fig. 2). The ETB ratio was higher in arteries with smaller diameter than in corresponding proximal arteries (i.e. the mesenteric branch vs. the superior mesenteric artery and the distal caudal artery vs. the proximal caudal artery; P<0.05 day 1 and P<0.01 day 5). Furthermore, veins showed a higher ETB ratio than corresponding arteries (i.e. the mesenteric vein vs. the superior mesenteric artery and the femoral vein vs. the femoral artery; P<0.05 day 5). The ETB ratio for the mesenteric vessels (superior artery and vein) showed a higher ETB ratio than the hindlimb vessels (femoral artery and vein) (P<0.05, day 1).


Figure 2
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  		Fig. 2 Linear regression analysis of the ratio for the contraction induced by K+ day 0 and day 1 (a) or day 5 (b) with the ETB ratio. The ETB ratio was calculated from the Emax for S6c and ET-1 in paired segments. The regression analyses were calculated from each experiment for each vessel. Every point was thereafter grouped in the type of vessels, each point representing the mean value with error bars representing s.e.m. of both the K+ ratio and the ETB ratio. Linear regression analyses showed no significant correlation between the reduction of the K+ contraction after organ culture and the increase of ETB receptor induced contraction after 1 day (P = 0.48, r2=0.01, n = 53) or after 5 days (P = 0.06, r2=0.07, n = 52) of organ culture.

 
3.4 Linear regression analysis of potassium-induced contraction vs. the ETB receptor-evoked contraction
The K+-induced contraction, used as a reference of the contractile capacity was not different in segments exposed to ET-1 or S6c. However, the K+-induced contraction showed a trend towards a decrease after 5 days of organ culture and was significantly attenuated in the mesenteric artery branch and the mesenteric vein (Fig. 2). Linear regression analysis was performed to determine if the reduction of the K+-induced contraction correlated with the simultaneous increase of the ETB receptor mediated contraction. A ratio of the contraction induced by K+ day 0 and day 1 or day 5 was compared with the ETB ratio. The resulting analysis indicated no statistically significant relation between the reduction of the K+ contraction and the increase in ETB receptor reactivity after 1 day or 5 days of organ culture (P = 0.48; r2=0.01 and P = 0.06; r2=0.07, respectively) (Fig. 2).

3.5 Pharmacological characterisation of the contractile responses
For pharmacological characterisation of the ET-receptors the superior mesenteric artery and its branch (fresh and cultured 5 days) were exposed to different specific ET-receptor antagonists. The selective ETA receptor antagonist FR139317, the most potent antagonist of the ET-1 contraction in fresh segments, caused a 141-fold rightward shift of the concentration–response curve in a parallel fashion (Fig. 3a). Bosentan, a mixed ETA/ETB receptor antagonist, caused a potent parallel rightward shift (67-fold), whereas the selective ETB receptor antagonist, IRL 2500, did not affect the contraction induced by ET-1 in fresh segments. The calculated pKB values from the single concentration of the antagonist used (10 µM) were 7.14 for FR139317 and 6.82 for bosentan. None of the Emax values were significantly altered by the antagonists.


Figure 3
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  		Fig. 3 Contraction induced by cumulative concentrations of endothelin-1 of fresh mesenteric arteries (a) and sarafotoxin 6c of mesenteric arteries cultured for 5 days (b) without ({circ}) and with either the ETA receptor antagonist FR139317 (bullet), the mixed ETA and ETB receptor antagonist bosentan ({blacktriangleup}) or the ETB receptor antagonist IRL 2500 ({blacksquare}). Each antagonist was added to the bath 15–20 min before the agonists for a final concentration of 10 µM. The contraction of each segment tested was calculated as a percentage of the potassium (K+)-induced contraction in the same segment, and each point represents the mean of all segments tested with error bars representing s.e.m. n = 6–9 in each group.

 
In cultured arteries, S6c induced a concentration–response curve that was shifted significantly to the right in a parallel fashion by all three antagonists without significant alteration of the maximum contractile effect. IRL 2500 was the most potent antagonist and demonstrated a 1086-fold shift, bosentan caused a 124-fold shift and FR139317 a 5-fold shift. The pKB value for the single antagonist concentration used (10 µM) were 8.04, 7.09 and 5.66, respectively (Fig. 3b).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The potential to develop ETB receptor mediated contraction in eight different vascular regions following organ culture was studied. During control conditions the ETA receptor showed to be the dominating contractile endothelin receptor in all investigated vascular beds. After organ culture, six out of the eight vessels showed marked increase in ETB receptor contraction. This plasticity was more apparent in small as compared to large arteries, more marked in veins than in arteries, and was most pronounced in the mesenteric region.

Fresh vessels from all eight regions responded to ET-1. In addition, the mesenteric vein responded to S6c by a significant contraction. This indicates the presence of a dominating ETA receptor in all vessels and a mixed population of ETA and ETB receptors in the mesenteric vein. However, since the S6c-induced response in the mesenteric vein was weak relative to the ET-1-induced response, it is likely that ETA is the dominating contractile receptor also in the mesenteric vein. The hypothesis of a dominating contractile ETA receptor in vessels with a simultaneous presence of contractile ETB receptors, like the mesenteric vein, is in agreement with previous data [9, 25, 26]. The finding that ET-1 generally appears to be more potent in veins than in arteries is in accordance with earlier studies [27]. The potency of ET-1 differed among the fresh vessels but showed similar order as described earlier (aorta>superior mesenteric artery>rat caudal artery) [28].

All vessel segments tested, with the exception of the aorta and the proximal caudal artery, showed a concentration-dependent contractile response to S6c after 1 day of organ culture. These responses were further augmented after 5 days, similar as that described in human omental arteries [17]. The enhancement of both potency and maximum effect for S6c is probably related to an increased number of ETB receptors as described by Black and Leff [29]. This suggestion is in agreement with a recent study which has shown that the increased effect and the concomitant increase of mRNA for the ETB receptor after organ culture are both abolished after treatment with the transcriptional inhibitor actinomycin D [30].

As reflected by the ETB ratio, large differences of the magnitude for S6c-induced contraction between the vessels were apparent. Smaller arteries showed a more pronounced ability to develop ETB receptor responses than the corresponding larger arteries. In vivo studies of the human forearm and the cat skeletal muscle indicate that ETB receptors are more prominent in resistance arteries [5, 8]. Similar observations have been demonstrated in the isolated third branch of mesenteric artery of the rat [31]. The preference for ETB receptors in small arteries may explain why the increase in contractile ETB receptors after organ culture was abolished or lower in larger arteries. The veins demonstrated a higher ETB ratio than the corresponding arteries, which may reflect that ETB receptors are favoured in the low pressure systems [10]. Regional differences in response to organ culture with a proportionally higher ETB ratio in the mesenteric as compared to the femoral vessels is in line with in vivo studies showing strong ETB receptor responses in the mesenteric vascular bed as compared to a weak or an abolished ETB receptor mediated contraction in the hindlimb circulation [6, 7].

In six of the eight vessels studied, the maximum contractile effect of ET-1 was not significantly altered even after 5 days of culture (in relation to K+-induced contraction). However, similar to the pattern of the concentration–response curve evoked by S6c, the potency of ET-1 tended to increase in the vessels that developed ETB receptor mediated contractions. Thus, in vessels where S6c had no effect after organ culture, the pEC50 of ET-1 did not change, which suggests that the ETA receptor-mediated contraction was not affected by the organ culture procedure. This is in agreement with earlier study on human omental arteries, showing that ETA receptors have the capability to elicit at least 70% of the ET-1-induced contraction after 5 days of culture [18]. Furthermore, since ET-1 is known to bind with similar affinity to the ETA and ETB receptors [32, 33], the enhanced potency for ET-1 may reflect ETB receptor development, rather than a sign of a parallel increase in ETA receptors. However, the caudal arteries showed a significant increase in the ET-1 response after organ culture. The enhanced ET-1 response in the distal caudal artery can at least in part be explained by the increased ETB receptor effect, whereas it cannot be ruled out that an increase of ETA receptors may have occurred in the proximal caudal artery.

The use of ET-1 and S6c as tools to characterise the responses reside in their affinity to the ETA and ETB receptors [32, 33]. In order to confirm our conclusions regarding the ET receptors studied, a more extensive receptor characterisation was performed on vessels from one of the regions, the mesenteric arteries, by use of different ET receptor antagonists. The selective ETA receptor antagonist FR139317, induced a 141-fold parallel shift of the ET-1-induced concentration–response curve in fresh vessels. The combined ETA/ETB receptor antagonist bosentan caused a 67-fold rightward displacement of the ET-1 curve whereas the selective ETB receptor antagonist IRL 2500 was without effect. These data support the idea of a dominating contractile ETA receptor in fresh vessels. The approximated pKB values obtained are in accordance with previous obtained pA2 values for the antagonists [34, 35]. After 5 days of organ culture the three antagonists all displaced the S6c curve to the right with the following order of the approximated pKB values: IRL 2500>bosentan>FR139317. This clearly indicates the development of an ETB receptor-related response.

There was no change in K+-induced contraction after 1 day of organ culture reflecting no differentiation of the smooth muscle cells. However, there was a trend to attenuation of the contractile capacity, in most of the vessels studied after 5 days of organ culture, which was illustrated both by the reduced K+-induced contraction and the Emax induced by ET-1. This is in accordance with earlier studies demonstrating decreased K+-induced contraction at different times in different vessel types after organ culture [17, 36, 37]. However, there was no correlation between the K+-induced contraction and the apparent ETB receptor alteration after organ culture, which suggests that these are two separate events.

The different regional appearance of the ETB receptor mediated contraction demonstrated that there is a heterogeneity between different vessel types which may reflect that vessels are composed of variable smooth muscle populations as suggested by Daemen and De Mey [38]. The reason for the appearance of ETB receptors in the vascular smooth muscle cells following organ culture [17, 18, 30]is still unknown, but may reflect the presence of an intrinsic mechanism for plasticity of smooth muscle cells expressing ETB receptors. The switch from expressing ETA receptors in early passages of cultured rat vascular smooth muscle cells to expressing ETB receptors in late passages [39]show change of phenotype of the vascular smooth muscle cells during certain conditions. During organ culture, the vessels are not exposed to intraluminal pressure or blood flow, or exposed to humoral and neuronal signals. The role of the endothelium may be a key point. However, denudation of the endothelium in different vessel types do not affect the responses to ETB receptor agonists in fresh or cultured vessel segments [40]. The increased amount of smooth muscle ETB receptors during increases in blood flow [19], in congestive heart failure [20]and in coronary artery disease [21, 22]suggest that an altered phenotype may contribute to vascular remodelling, in such circumstances. Thus, the marked differences in the ability to develop the contractile ETB receptors in various vascular regions may reflect a very complex role for endothelins in different pathophysiological conditions.

Time for primary review 28 days.


   Acknowledgements
 
This study was supported by the Swedish Society of Medicine, the Swedish Medical Research Council (Grant nos. 05958, 11294, 11432 and 11717), the Medical Faculty, Lund University, Sweden, Anna Lisa and Sven-Eric Lundgrens Foundation for Medical Research, The Swedish Heart Lung Foundation and Astra Draco Research Foundation. The authors wish to thank Dr. Jo Mori, Fujisawa Pharmaceutical Co., Dr. Martine Clozel, F. Hoffman-La Roche AG, and Dr. Okada, Ciba-Geigy Japan Ltd., for supplying us with FR139317, bosentan and IRL 2500, respectively.


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

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