Ionophoric properties of atropine: Complexation and transport of Na1, K1, Mg21 and Ca21 ions across a liquid membrane

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Ionophoric properties of atropine:

complexation and transport of Na ⁺ , K

+ , Mg ²⁺ and Ca ²⁺ ions across a liquid membrane

Latifa Rabi

, Adnane Moutaouakkil

& Mohamed Blaghen

a

Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology , Biotechnology and Environment, Faculty of Sciences Aïn Chock, University Hassan II–Aïn Chock , Km 8 route d’El Jadida, BP. 5366 Mâarif 20100 Casablanca, Morocco

Published online: 15 Apr 2008.

To cite this article: Latifa Rabi , Adnane Moutaouakkil & Mohamed Blaghen (2008) Ionophoric properties of atropine: complexation and transport of Na

, K

, Mg

and Ca

ions across a liquid membrane, Natural Product Research: Formerly Natural Product Letters, 22:6, 547-553, DOI:

10.1080/14786410701592620

To link to this article: http://dx.doi.org/10.1080/14786410701592620

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Natural Product Research, Vol. 22, No. 6, 15 April 2008, 547–553

Ionophoric properties of atropine: complexation and transport of Na

, K

, Mg

and Ca

ions across a liquid membrane

LATIFA RABI, ADNANE MOUTAOUAKKIL* and MOHAMED BLAGHEN Unit of Bio-industry and Molecular Toxicology, Laboratory of Microbiology, Biotechnology and Environment, Faculty of Sciences Aı¨n Chock, University Hassan II – Aı¨n

Chock, Km 8 route d’El Jadida, BP. 5366 Maˆarif 20100 Casablanca, Morocco

The activity of atropine on the complexation and transport of Na^þ, K^þ, Mg^2þand Ca^2þions across a liquid membrane was investigated using a spectrophotometric method. Atropine is a natural drug that blocks muscarinic receptors. It is a competitive antagonist of the action of acetylcholine and other muscarinic agonists. Atropine is shown to extract Na^þ, K^þ, Mg^2þand Ca^2þions from an aqueous phase into an organic one with a preference for Ca^2þions. According to a kinetic study, divalent cations (Mg^2þand Ca^2þ) are more rapidly transported than monovalent ones (Na^þand K^þ). In both complexation and transport, the flux of the ions increases with the increase of atropine concentration. Atropine might act on the membrane permeability; its complexation and ionophoric properties shed new lights on its therapeutic proprieties.

Keywords: Atropine; Complexation; Transport; Ionophore; Mono- and divalent cations

1. Introduction

The study of cation–ionophore binding is one of the fundamental subjects for understanding molecular recognition [1]. It is well known that some kinds of peptides and natural ionophores, such as monensin and valinomycin, play important roles as ion carriers in the typical metal ion transport system [2]. This later is one of the fundamental mechanisms for accumulation of energy, and the medium by which muscle is controlled and information passed on in living systems [2].

Atropine is an anti-cholinergic drug that is derived from the plant Atropa belladonna [3]. It is a widely used as a competitive antagonist against the activation by acetylcholine of the cholinergic muscarinic receptors [4] and also shown to have a parasympatholytic effect [5]. In vitro, for effective pharmacological blockade of muscarinic receptors, atropine is usually used within the range of 10 nM to 10 M. It is also used therapeutically, often by subcutaneous route, in doses of 0.5–10 mg [6]. Atropine can also be applied topically for ophthalmic purposes, intravenously in the treatment of food poisoning due to ingestion of anti-cholinergic plant alkaloids [7], and as an

*Corresponding author. Tel.:þ212-22-230680/84. Fax:þ212-22-230674. Email: moutaouakkil@hotmail.com

Natural Product Research

ISSN 1478-6419 print/ISSN 1029-2349 onlineß2008 Taylor & Francis http://www.tandf.co.uk/journals

DOI: 10.1080/14786410701592620

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antidote for organophosphate poisoning [8]. Other investigations have shown atropine to demonstrate a wide range of biological activities including vagolytic effects, relaxation action of vascular smooth muscle [4], suppression of gland and mucous secretions [9] and treatment of cyanide-induced neurotoxicity in the presence of Ca

and thiosulfate [10]. It has also been used to treat peptic ulcer by reducing the production of stomach acid. The biochemical mechanism through which atropine exerts its activity is not yet clearly established. Therefore, in the light of its wide therapeutic applications, if not properly administered, overdose of atropine can result in adverse effects associated with the suppression of cholinergic systems, manifested as dry mouth, blurred vision, respiratory failure, convulsions, urinary retention, mydriasis, tachycardia and flushing [7].

In the present article, we show atropine to be able to complex Na

, K

, Mg

and Ca

ions from aqueous picrate solution into an organic medium and to facilitate the transport of these metal cations through a chloroform liquid membrane model.

2. Results and discussion

The mobile carrier theory for ionophore-facilitated cation transport through membranes has been supported strongly by the finding of a correlation between the ability of the ionophores to induce transport from an aqueous medium into an organic phase via the mechanism of complexation [11].

Extensive research has been conducted to discover natural compounds that are able to complex and to transport mono- and divalent ions [12–15]. In general, two design features must be incorporated into the ionophore to achieve high selectivity for a particular guest; (i) the pocket of the ionophore must be an appropriate size to bind the guest, and (ii) a pre-organized structure is needed to reduce the entropic and enthalpic costs of complexation.

Structure examination of atropine, an anti-cholinergic drug considered as one of the most biologically active molecules, reveals that atropine is a simple piperidine alkaloid, because it consists of carbon ring into which a nitrogen atom is inserted. Moreover, atropine contains different oxygenated groups, able, in precise conformation, to complex mono-and/or divalent ions.

The ability of atropine to complex Na

, K

, Mg

and Ca

ions was studied using two solutions in equilibrium: a chloroform solution containing atropine and an aqueous picrate solution. The metal picrate salt, insoluble in chloroform, is extracted as an atropine complex. The decrease in absorption of picrate in the aqueous phase allows the evaluation of cations complexing efficiency of atropine.

Indeed, complexation experiments showed that atropine had remarkable activities with all of the metal ions tested, but with different affinities (figure 1). The reactivity was in the order Ca

> Mg

> K

> Na

. Thus, 10.14 10

M of Ca

was complexed by atropine within 20 min, whereas only 5.65 10

M of Mg

, 4.07 10

M of K

and 2.47 10

M of Na

were complexed by atropine within the same period of time (figure 1). The rate of complex formation depends on atropine concentration (figure 2).

In control experiments without atropine no complexation occurred.

In order to study ionophoric properties of atropine, we tested its ability to act as a carrier in the transport of Na

, K

, Mg

and Ca

ions through a liquid membrane.

In the system used, the metal picrate in aqueous phase I moved, as a complex, through

548

et al.

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the chloroform solution containing atropine and was released into aqueous phase II.

Ions tested were all transported by atropine but at different rates, in the order Mg

> Ca

> K

> Na

(figure 3). Divalent cations (Mg

and Ca

) were transported faster than monovalent ones (K

and Na

). Indeed, within 30 min at atropine concentration of 2.10 10

M, 29.94 10

M of Mg

, 25.47 10

M of Ca

, 9.15 10

M of K

and 5.19 10

M of Na

were transported (figure 3).

As for the complexation, the transport rate depends also on atropine concentration (figure 4(a) and (b)). For example after 15 min, the concentrations of Mg

and Ca

transported were, respectively, 17.69 10

M and 15.45 10

M at atropine concentration of 2.10 10

M. Whereas at the same time period, only 8.09 10

M of Mg

and 8.06 10

M of Ca

were transported at a lower atropine concentration

0 2 4 6 8 10 12

0 5 10 15 20 25 30

Time (min) Ions complexed (10−5 M)

Figure 1. Kinetics of complexation of Na^þ, K^þ, Mg^2þand Ca^2þions by atropine. Transfer of sodium picrate (-*-), potassium picrate (--), magnesium picrate (-g-) and calcium picrate (-

˙

0 2 4 6 8 10 12

0 5 10 15 20 25 30

Time (min) Ions complexed (10−5 M)

Figure 2. Effect of atropine concentration on complexation kinetics of Ca^2þions. Concentration of calcium picrate complexed as a function of time at different concentrations of atropine (-s-; 1.0510⁵M), (-

˙

Ionophoric properties of atropine

549

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(1.05 10

M) (figure 4(a) and (b)). In control experiments without atropine no transport occurred.

The essential conclusions which can be drawn is that all ions tested form complexes and are transported across chloroform by atropine, with a preference for divalent cations, Mg

and Ca

. This difference in the affinities is probably in part responsible of therapeutic effects of atropine.

Furthermore, atropine has an important potential for complexing metal ions, particularly Ca

and this may suggest additional uses as a tool for studying cellular functions mediated by changes in Ca

. In the other hand, Ca

shift is still the primary factor mediating cellular responses, although other factors may also play significant roles in atropine pharmacology. Moreover, the higher affinity of atropine for Ca

ions reinforce the investigations reported by Choi et al. [16], that may provide an indication of mechanism through which atropine exerts its effect, especially a relaxation effect, on isolated rabbit corpus carvernosal smooth muscle. This relaxation may be mediated by decreasing intracellular calcium sequestration, and probably by direct change of calcium transport via voltage dependent calcium channel or sacroplasmic reticulum. This observation may be a tie between a relaxation effect and ionophoretic activity of atropine.

Potential pharmacological application of drugs is in the area of nuclear medicine.

Ionophores may favourably modify the systemic distribution of the radionucleotide

201

Ti, which is used for the radioimagining of myocardial infarcts [17]. The same principle should be extended to improve the resolution of organ imaging by radionucleotides for the diagnosis of tumours and pathological conditions. Increasing membrane permeability by ionophore administration may also be effective in treating heavy-metal toxicity.

Since the balance between Na

, K

, Mg

and Ca

ions plays a crucial role in biology, including the involvement of potassium and sodium in nerve impulse transmission which depends upon the efficient transport of cations across cellular membranes. It would be of interest to study the rate of transport of these ions through an intact cell membrane and the effect of atropine on cellular functions. Studies in

0 5 10 15 20 25 30

0 5 10 15 20 25 30

Time (min) Ions transported (10−6 M)

Figure 3. Kinetics of transport of Na^þ, K^þ, Mg^2þand Ca^2þions by atropine. The concentration of Na^þ (-*-), K^þ(--), Mg^2þ(-g-) and Ca^2þ(-

˙

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et al.

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biological systems in the presence of these ions may provide a better model for atropine mediated selective complex-formation and transport. It may also help to explain its biological activity.

3. Experiment

3.1. Kinetics of complex formation

The interaction between atropine and Na

, K

, Mg

and Ca

ions using the extraction procedure described by Frensdorff [18] was studied: the aqueous solutions

0 5 10 15 20 25 30

0 5 10 15 20 25 30

Time (min) Ions transported (10−6 M)

0 5 10 15 20 25 30 (a)

(b)

0 5 10 15 20 25 30

Time (min) Ions transported (10−6 M)

Mg²⁺ Ca²⁺

Figure 4. Effect of atropine concentration on transport kinetics of Ca^2þ (A) and Mg^2þ (B) ions.

Concentration of Ca^2þand Mg^2þions as a function of time at different concentrations of atropine (-*-;

1.0510⁵M), (-f-; 2.1010⁵M).

Ionophoric properties of atropine

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were prepared from standard stock solutions of NaOH, KOH, Mg(OH)

, Ca(OH)

and picric acid. Atropine was dissolved in the organic phase. Equal volumes of the two solutions in screw-cap beaker flasks were thoroughly stirred on a vortex junior mixer.

All extractions were conducted in a constant room temperature at 25 0.5

C.

Five millilitres of an aqueous solution, consisting of metal picrate (10

M) and metal nitrate (5 10

M), and 5 mL of a chloroform solution of atropine at the concentration of 1.05 10

M or 2.10 10

M were placed in a beaker, and the organic layer was stirred at constant speed. The rate of complex-formation was monitored by measuring the decrease of the adsorption at 355 nm of the metal picrate in the aqueous solution. Each experiment was carried out in triplicate.

3.2. Transport kinetics

Experiments on the transport of Na

, K

, Mg

and Ca

ions were carried out in a cylindrical glass cell (3.2 cm i.d.) containing a cylindrical glass-walled tube (2.0 cm i.d.), separating two aqueous phases (phase I and phase II) [19]. The aqueous phase I contained metal picrate (10

M), metal nitrate (0.05 M) and metal hydroxide (5 10

M) in 5 mL of double-distilled water, while the aqueous phase II consisted of 5 mL of double-distilled water. The two phases were separated by 5 mL of chloroform containing atropine at the concentration of 1.05 10

M or 2.10 10

M.

The absorption at 355 nm of the metal picrate transported into aqueous phase II was measured at regular time intervals.

To confirm the results obtained by the spectrophotometric procedure, we have used the atomic absorption technique. At the end of each experiment, the aqueous phase II was recuperated for the purpose to dose the ion tested using a flame photometer (Jenway) type PFP7 with specific filters for Na

, K

, Mg

and Ca

.

Acknowledgements

This work was supported by the Moroccan CNRST and the urban community of Casablanca. The authors would like to thank Dr Nourrddine Chafik (Sothema Laboratories) for helpful corrections of the text.

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