Inorganic Chemistry in Biology and Medicine - American Chemical

1 Molecular and Biological Properties of Ionophores BERTON C. PRESSMAN, GEORGE PAINTER, and M O H A M M A D FAHIM

Downloaded by 117.253.205.125 on April 6, 2016 | http://pubs.acs.org Publication Date: December 22, 1980 | doi: 10.1021/bk-1980-0140.ch001

Department of Pharmacology, University of Miami, Miami, F L 33101

The ionophores are a group of natural and synthetic compounds which form lipid-soluble cation complexes which can transport cations across low polarity barriers such as organic solvents and l i p i d s (1). From a biological standpoint, the most important low polarity barrier is the l i p i d bilayer which l i e s within biological membranes; ionophores possess unique and potent biological properties which derive from their a b i l i t y to perturb transmembrane ion gradients and e l e c t r i c a l potentials. Each ionophore has i t s own characteristic ion selectivity pattern arising from the interaction between the conformational options of the host ionophore and the effective atomic radius and charge density of the guest cation. The a b i l i t y of ionophores to complex and transport cations has an ever growing l i s t of applications in experimental biology and technology and may ultimately provide the basis for novel cardiovascular drugs. Ionophores are also intriguing i n t e l lectually as objects for study of chemical and physical complexation processes at the molecular level and as challenges to the state of the art of chirally selective organic synthesis (2) . Several reviews are available for expanding the description of ionophores provided here (3,4,5). General Structural Features of Ionophores Several of the general structural features of ionophores are illustrated i n Figure 1. A l l ionophores deploy an array of liganding oxygen atoms about a cavity in space into which the complexed cation f i t s . X-ray crystallography reveals that the principal bonding energy is provided by induced dipolar interaction between the complexed cation and those specific oxygens which are filled in. Valinomycin consists of alternating residues of hydroxyacids and aminoacids constituting a cyclic dodecadepsipeptide. In space the ring undulates defining a bracelet 4 Å. high and 10 Åin diameter. The liganding oxygens, the ester carbonyls, form a three

0-8412-05 8 8-4/ 80/47-140-003$05.00/0 ©

1980 American Chemical Society

Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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INORGANIC


VANCOMYCIN

CYCLOHEXYL ETHER

CHEMISTRY IN BIOLOGY A N D MEDICINE

ENNIATIN B

MONENSIN

MACROLIDE ACTINS

NIGERICIN

Figure 1. Structures of representative ionophores. The oxygen atoms that x-ray crystallography indicates to be primarily involved in liganding to cations are filled in.


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dimensional cage which accommodates K ( r = 1.33 X) much more snugly than N a ( r = 0.95 ft) r e s u l t i n g i n a K : N a preference of 10,000:1 ( 4 ) . E n n i a t i n B i s a c y c l i c hexadepsipeptide; the smaller r i n g r e s u l t s i n a r e l a t i v e l y planer array of l i g a n d i n g oxygen atoms; the more open and more f l e x i b l e cage r e s u l t s i n a K : N a d i s c r i m i n a t i o n of only 3:1 ( 6 ) . A new f e a t u r e appears i n the c y c l i c t e t r a e s t e r s , the macrol i d e nactins. In a d d i t i o n to the e s t e r carbonyls, four heteroc y c l i c ether oxygens p a r t i c i p a t e i n complexation; the oxygens are arranged a t the apices o f a cubic cage. F i v e v a r i a n t n a c t i n s are known depending whether 0-4 o f the R groups a r e methyls (nonactin) or e t h y l s (monactin, d i n a c t i n , t r i n a c t i n , t e t r a n a c t i n ) ( 7 ) . While the aforementioned ionophores are Streptomyces metabol i t e s , the crown p o l y e t h e r s , the depicted prototype of which i s dicyclohexyl-18-crown-6, are s y n t h e t i c ( 8 ) . Although they l a c k the i n t r i c a t e conformations of the n a t u r a l ionophores a r i s i n g from m u l t i p l e asymmetric carbon atoms, t h e i r molecular l i g a n d i n g prope r t i e s are analogous. While they are l e s s e f f i c i e n t i o n c a r r i e r s , t h e i r l a c k of l a b i l e linkages confers increased chemical s t a b i l i t y ; they f i n d extensive use i n organic synthesis f o r s o l u b i l i z i n g e l e c t r o l y t e s , e.g. enolates, i n nonpolar solvents thereby prov i d i n g r e a c t i v e naked anions (9) . The ionophores thus f a r described l a c k i o n i z a b l e groups and are c o l l e c t i v e l y c l a s s i f i e d as n e u t r a l ionophores; t h e i r complexes acquire the net charge of whatever i o n i s complexed. We s h a l l now examine two r e p r e s e n t a t i v e s of the c a r b o x y l i c subclass of ionophores. Only the a n i o n i c form of these ionophores complex cations, hence they form e l e c t r i c a l l y n e u t r a l z w i t t e r i o n i c complexes. This d i s t i n c t i o n i s fundamental f o r e x p l a i n i n g the profound d i f f e r e n c e s i n b i o l o g i c a l behavior of the ionophore subclasses, hence we pref e r c a r b o x y l i c ionophore to the term polyether a n t i b i o t i c used by Westley ( 5 ) . The l a t t e r term, furthermore, leads to f u n c t i o n a l ambiguity with the e t h e r e a l macrolide n a c t i n s and crown polyethers which are n e u t r a l ionophores. The n a t u r a l l y o c c u r r i n g c a r b o x y l i c ionophores, t y p i f i e d by monensin, l a c k the s t r u c t u r a l redundancy of the n e u t r a l ionophores. Monensin c o n s i s t s of a f o r m a l l y l i n e a r array of heteroc y c l i c e t h e r - c o n t a i n i n g r i n g s , however the molecular c h i r a l i t y a r i s i n g from the r i n g s and asymmetric carbons favors the molecule assuming a q u a s i - c y c l i c c o n f i g u r a t i o n . A d d i t i o n a l s t a b i l i z a t i o n of the r i n g i s conferred by h e a d - t o - t a i l hydrogen bonding. In a d d i t i o n to i t s l i g a n d i n g ether oxygens, monensin has a p a i r of l i g a n d i n g hydroxyl oxygens (10). The t a i l p o r t i o n of n i g e r i c i n c l o s e l y resembles monensin, however, an a d d i t i o n a l tetrahydropyranol r i n g t h r u s t s the head carboxyl group i n t o the complexation sphere. Thus, i n a d d i t i o n to the induced d i p o l e i o n bonds p r e v i o u s l y described, n i g e r i c i n complexes feature a true i o n i c bond. Despite major s i m i l a r i t i e s i n s t r u c t u r e , n i g e r i c i n p r e f e r s K over N a by a f a c t o r of 100 while monensin p r e f e r s N a over K"" by a f a c t o r of 10 (11) . +

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INORGANIC CHEMISTRY IN BIOLOGY A N D M E D I C I N E

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Dynamics of Ionophore-Mediated

Transport

N e u t r a l Ionophores. The r e l a t i o n s h i p between e q u i l i b r i u m ionophore a f f i n i t i e s and dynamic b i o l o g i c a l transmembrane t r a n s port i s d e t a i l e d i n Figure 2. The transport c y c l e c a t a l y z e d by n e u t r a l ionophores i s given on the l e f t . Ionophore added to a b i o l o g i c a l membrane p a r t i t i o n s predominately i n t o the membrane. A p o r t i o n of the ionophore d i f f u s e s to the membrane i n t e r f a c e where i t encounters a hydrated c a t i o n . A l o o s e encounter complex i s formed followed by replacement of the c a t i o n i c h y d r a t i o n sphere by engulfment of the c a t i o n by the ionophore. The dehydrated complex i s l i p i d - s o l u b l e and hence can d i f f u s e across the membrane. The c a t i o n i s then rehydrated, r e l e a s e d , and the uncomplexed ionophore f r e e d to r e t u r n to i t s i n i t i a l s t a t e w i t h i n the membrane. The net r e a c t i o n c a t a l y z e d i s the movement of an i o n with i t s charge across the membrane. Two independent f a c t o r s determine the thermodynamic gradient governing net transport by n e u t r a l ionophores: the membrane pot e n t i a l , i . e . A E ^ B , and the concentration gradient, [ M + ] ^ / [ M + ] B • At e q u i l i b r i u m , the e l e c t r o c h e m i c a l p o t e n t i a l (a combined f u n c t i o n of e l e c t r i c a l and concentration terms) of M*" on s i d e A becomes equal to the e l e c t r o c h e m i c a l p o t e n t i a l of on s i d e B, i . e . PMA PMB* of experimentally measurable parameters, the relationship =

I

N

T

E

R

M

S

A E ^

=

-59

mV

log

[ M ] +

A

/ [ M ] +

B

a p p l i e s . This s i g n i f i e s that i f the e l e c t r i c a l term, A E ^ B , exceeds the concentration term, 59 mV l o g [M^/Mj], the i o n w i l l flow down the p o t e n t i a l gradient and d i s s i p a t e i t ( e l e c t r o p h o r e t i c transport mode). I f the concentration term exceeds the p r e - e x i s t ing p o t e n t i a l term, the movement of down i t s concentration term w i l l increase AE^jg ( e l e c t r o g e n i c t r a n s p o r t ) . The r e l e v a n t s i g n i f i c a n c e of t h i s transport mode i s that n e u t r a l ionophores perturb not only the transmembrane i o n gradients of b i o l o g i c a l systems but a l s o t h e i r transmembrane e l e c t r i c a l p o t e n t i a l s . Since the l a t t e r are so important i n b i o l o g i c a l c o n t r o l , i t i s not s u r p r i s i n g that the n e u t r a l ionophores a r e exceedingly t o x i c towards i n t a c t animals. C a r b o x y l i c Ionophores. C a r b o x y l i c ionophore-mediated t r a n s port i s d e t a i l e d on the l e f t o f F i g u r e 2. The form assumed w i t h i n the membrane a t the s t a r t o f the transport c y c l e i s an e l e c t r i c a l l y n e u t r a l z w i t t e r i o n , M^-I"; a n i o n i c f r e e I " i s presumably too p o l a r to be s t a b l e a t that l o c u s . When t h i s species d i f f u s e s to the membrane i n t e r f a c e , i t i s subject to s o l v a t i o n ; the c a t i o n can be hydrated and removed from the complex. The r e s u l t a n t h i g h l y p o l a r I " i s o b l i g e d to remain at the i n t e r f a c e u n t i l a new charge partner, represented by N+'R^O, a r r i v e s . Once i n p o s i t i o n , N +


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PRESSMAN E T A L .

Properties

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exchanges i t s s o l v a t i o n H2O f o r the oxygen l i g a n d i n g system of I forming l i p i d compatible N+.I" which then d i f f u s e s across the membrane. There the process i s reversed and N i s exchanged f o r M+. The ionophore then reenters the membrane as M+I"" thereby completing the c a t a l y t i c c y c l e . The net r e a c t i o n i s the movement of N+ across the membrane i n exchange f o r M without an accompanying net charge t r a n s l o c a t i o n . T h i s i s presumably an e s s e n t i a l requirement f o r t o l e r a n c e of a p p r e c i a b l e concentrations of ionophores by animals, i . e . c a r b o x y l i c ionophores are r e l a t i v e l y nont o x i c compared to n e u t r a l ionophores. In other words, the a b i l i t y of c a r b o x y l i c ionophores to a l t e r p h y s i o l o g i c a l processes i n a pharmacologically u s e f u l manner stems from t h e i r c a p a b i l i t y to a l t e r transmembrane i o n gradients without d i r e c t l y s h o r t c i r c u i t ing the transmembrane p o t e n t i a l s of e l e c t r i c a l l y a c t i v e c e l l s . The formation and d i s s o c i a t i o n of ionophore-cation complexes i s e q u i v a l e n t to the displacement of the primary c a t i o n s o l v a t i o n sphere by the ionophore l i g a n d i n g atoms. The s o l v a t e d l i g a n d i n g groups approach the s o l v a t e d c a t i o n u n t i l they a t t a i n a p p o s i t i o n . They then i n t e r a c t v i a an a s s o c i a t i v e interchange mechanism analogous to an S 2 mechanism (12). Formation of the t r a n s i t i o n s t a t e i n v o l v e s extension of the c a t i o n to both the e n t e r i n g l i g a n d and the departing c a t i o n s o l v a t i o n sphere. In the process, the l e s s r i g o r o u s l y d e f i n e d s o l v a t i o n sphere of the l i g a n d i s a l s o d i s charged. The ionophore then engulfs the c a t i o n , i t s l i g a n d i n g groups p r o g r e s s i v e l y d i s p l a c i n g the molecules of the c a t i o n s o l v a t i o n s h e l l i n a concerted f a s h i o n . In the case of the c a r b o x y l i c ionophores, the i n i t i a l stage p r i o r to the formation of the t r a n s i t i o n complex i s a simple i o n p a i r . Although they vary widely i n s t r u c t u r e and conformation, the c a r b o x y l i c ionophores f e a t u r e a v a r i e t y of heteroatoms c o n s t i t u t ing a l i g a n d i n g system which operates by means of induced d i p o l e s . The magnitude of the d i p o l e s i n c r e a s e s p r o g r e s s i v e l y by i n d u c t i o n as approached by the c a t i o n and u l t i m a t e l y produces a s o l v a t i o n system stronger than that of the bulk phase s o l v e n t . Whereas the i n d i v i d u a l s o l v a t i o n molecules, w i t h i n the primary s o l v a t i o n sphere of a c a t i o n , exchange independently with the bulk s o l v e n t , the l i g a n d s of an ionophore, h e l d together by a common backbone, must behave i n a cooperative manner. Intramolecular hydrogen bonding and s u b s t i t u e n t s which favor c y c l i c conformations (e.g. spirane systems) promote the s t a b i l i t y of complexes. Consequently, the v a r i o u s c a t i o n a f f i n i t y and s e l e c t i v i t y p a t t e r n s which charact e r i z e each ionophore a r i s e from the p r e c i s e s p a c i a l depolyment of l i g a n d i n g heteroatoms as determined by molecular conformation (13,14). +


+

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Conformational Studies of a Representative C a r b o x y l i c Ionophore, Salinomycin

(15),

Salinomycin, a r e p r e s e n t a t i v e c a r b o x y l i c ionophore ( F i g u r e 3) i s a p a r t i c u l a r l y s u i t a b l e model f o r studying the dynamic


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NCITMl sMi I

CHEMISTRY IN BIOLOGY A N D MEDICINE

Sill

cmixYiid

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\r r-r-M r-i*M M HI TtMUKMIN

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Figure 2. Different modes of ionophore-mediated transmembrane transport. Neutral ionophore-mediated transport is depicted on the left and carboxylic ionophoremediated transport, on the right. The individual transport steps are detailed in the text.


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conformational aspects of complexation. The c i r c u l a r dichroism (CD) a r i s i n g from the n -> TT* t r a n s i t i o n of the C - l l carbonyl i s s e n s i t i v e to molecular environment and serves as a probe to report the c h i r a l i t y i n i t s v i c i n i t y . CD enables us to evaluate the conformational p e r t u r b a t i o n s produced by a l t e r i n g the p o l a r and p r o t i c p r o p e r t i e s of the s o l v e n t system. Systematic p e r t u r b a t i o n of the s o l u t i o n conformation of salinomycin by an appropriate choice of s o l v e n t s r e v e a l s that i o n a f f i n i t y and s e l e c t i v i t y are v a r i a b l e , conformationally determined, p r o p e r t i e s . Representative CD s p e c t r a of protonated salinomycin, i t s K complex and i t s uncomplexed anion are presented i n F i g u r e 4 . No s i g n i f i c a n t s h i f t of the negative 2 9 0 nm peak occurs w i t h solvent change or l i g a n d i n g s t a t e ; Beer's law i s obeyed from 1 0 " ^ to 1 0 " " 6 M . The f u n c t i o n most s u i t a b l e f o r r e l a t i n g CD s p e c t r a to the conformation of a molecule i s the r o t a t i o n a l strength (R£) of the observed e l e c t r o n i c t r a n s i t i o n ( 1 6 ) . Since the Gaussian a p p r o x i mation appears to h o l d f o r the salinomycin CD curves, Rj was c a l c u l a t e d from [8] and wavelength by a standard equation ( 1 7 ) . Figure 5 i l l u s t r a t e s the e f f e c t of solvent changes on the R£ of the ionophore f r e e a c i d and i t s anion. Kosower's Z values proved e m p i r i c a l l y an e f f e c t i v e f u n c t i o n f o r ranking s o l v e n t s according to t h e i r i n t e g r a t e d p o l a r and p r o t i c p r o p e r t i e s ( 1 8 ) . The | R J | of the f r e e a c i d decreases l i n e a r l y with a small p o s i t i v e slope as the Z values r i s e . In c o n t r a s t , the | R Q | of the uncomplexed anion, the species p a r t i c i p a t i n g i n complexation, drops sharply between Z values of 8 0 and 8 3 , v a r y i n g l i t t l e above and below these v a l u e s . Thus, the conformation of the anion tends toward one of two metastable s t a t e s depending upon s o l v e n t Z value. The r o l e of the s o l v e n t i n determining e q u i l i b r i u m s o l u t i o n conformation can best be understood i n terms of f u n c t i o n a l group stabilization. In p o l a r p r o t i c media the e q u i l i b r i u m conformation of the uncomplexed a n i o n i c ionophore i s determined by the s o l v a t i o n of the carboxylate anion and the p o l a r l i g a n d i n g groups. Thus, two d i s t i n c t s o l v e n t e f f e c t s are o p e r a t i v e , s o l v a t i o n of the p o l a r l i g a n d i n g groups r e s u l t i n g i n conformational s t a b i l i z a t i o n due to decreased d i p o l e - d i p o l e r e p u l s i o n and maximization of the s o l v a t i o n energy of the anion. The protonated ionophore responds only to the s o l v a t i o n of p o l a r l i g a n d i n g groups. Thus, Figure 5 provides i n s i g h t i n t o the r e l a t i v e importance of each of these f a c t o r s i n determining e q u i l i b r i u m s o l u t i o n conformation. The p e r t u r b a t i o n of conformation due to s o l v a t i o n of p o l a r l i g a n d i n g groups alone, as i n the protonated ionophore, causes only a s l i g h t change i n conformation, i . e . a small change i n |Rol> over a l a r g e range of Z v a l u e s . However, i o n i z a t i o n of the protonated form of the ionophore profoundly changes i t s response to s o l v e n t s . A t Z values > 8 3 , the carboxylate i s s t a b i l i z e d by i t s p r o t i c , p o l a r environment. The r e s u l t i n g s o l v a t i o n sphere i n f l u e n c e s the conformation s t r o n g l y as evidenced by the very low | R Q | values (Figure 3 ) . As the Z values f a l l , and the s o l v e n t becomes l e s s


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INORGANIC CHEMISTRY IN BIOLOGY A N D MEDICINE

M Figure 4. CD spectra of the carboxylic acid free anion and K complex forms of salinomycin. The free anionic form was generated by the addition of excess tri-nbutylamine and the K complex by the addition of excess KSCN.

10* -a


+

+

WmLiKT* (urn)

Figure 5. Rotational strengths of the carboxylic acid and free anion forms of salinomycin as a function of solvent Z values

Figure 6. K :Na* selectivity (l/K +: 1/K +) of salinomycin as a function of solvent Z value +

DNa

Dk


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able to s t a b i l i z e the charge, s t a b i l i z a t i o n i s achieved by a t i g h t h e a d - t o - t a i l (C^-O-LyH) hydrogen bond. The formation of t h i s bond r e s u l t s i n a compression of the l i g a n d i n g c a v i t y , the l i m i t of which i s determined by d i p o l e - d i p o l e r e p u l s i o n . A p p l i c a t i o n of the Octant Rule (16) to computer models of the anion c o r r o b o r a t e s that t i g h t e n i n g of the h e a d - t o - t a i l bond should be accompanied by a concomitant i n c r e a s e i n | R Q | . F i g u r e 4 i n d i c a t e s that CD can be employed to determine comp l e x a t i o n K s (see Table I ) . The r a t i o of the Na :K+ K s , i . e . K : N a s e l e c t i v i t y , a l s o shows a sharp s h i f t between Z values of 80 and 83 ( c f . F i g u r e 6). Thus, the a b i l i t y of the complexing form of the ionophore to d i s c r i m i n a t e between ions depends s t r o n g l y upon environmental i n f l u e n c e s on conformation. Changes i n i n t e r - l i g a n d d i s t a n c e s and l i g a n d o r i e n t a t i o n s e f f e c t e d by changes i n ionophore conformation manifest themselves by a d e t e r minative a l t e r a t i o n of the f r e e energy of complexation. CD was u t i l i z e d to o b t a i n the s o l v e n t dependency of the conformation of the cation-ionophore complex as w e l l as Kp's. S a t u r a t i o n isotherms were p l o t t e d from l i n e a r computer f i t s of 1/[cation] versus 1 / A R £ ; the slopes y i e l d e d Kpj's w h i l e e x t r a p o l a t i o n of R J to i n f i n i t e c a t i o n c o n c e n t r a t i o n provided the R^'s of the c a t i o n - s a t u r a t e d ionophore. I t i s important to note that the c a t i o n i t s e l f i s a s i g n i f i c a n t v i n c i n a l moiety, which by v i r t u e of i t s charge, p o l a r i z a b i l i t y and l o c a t i o n with respect to the chromophore of concern, can modify the r o t a t i o n a l s t r e n g t h of the chromophore. Comparison of the | R £ | values f o r the N a and K complexes of salinomycin i n Table I with the | R J | values f o r salinomycin anion i n F i g u r e 5 shows an i n c r e a s e i n the magnitude of | R £ | upon comp l e x a t i o n i n a l l s o l v e n t s . T h i s corresponds to a change i n conformation upon complexation, i . e . r e o r i e n t a t i o n of the ionophore about the c a t i o n . A p p l i c a t i o n of the Octant Rule to computer generated models of salinomycin i n d i c a t e s that t h i s r e o r i e n t a t i o n i s a c o n s t r i c t i o n of the l i g a n d i n g oxygens which surround the cation. The extent of t h i s c o n s t r i c t i o n c o r r e l a t e s with the s t a b i l i t y of the complex i n d i c a t e d by i t s ( c f . Table I ) . T

+

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+

X-ray c r y s t a l l o g r a p h i c s t u d i e s confirm that a l l c a t i o n i c complexes of c a r b o x y l i c ionophores have t h e i r l i g a n d i n g atoms o r i ented toward a c e n t r a l c a v i t y . The extent to which t h i s conformat i o n would be a l t e r e d i n the absence of a bound c a t i o n due to the mutual e l e c t r o s t a t i c r e p u l s i o n of the d i p o l a r oxygen atoms would, i n turn, be modulated by the m o b i l i t y of the backbone supporting the l i g a n d s . We conclude that the dynamics of molecular conformation assoc i a t e d w i t h s a l i n o m y c i n complexation i n a l l l i k e l i h o o d extend at l e a s t to the other n a t u r a l l y o c c u r r i n g c a r b o x y l i c ionophores. The i n f l u e n c e of ionophore environment, e.g. s o l v e n t , on ionophore conformation i s p a r t i c u l a r l y s i g n i f i c a n t when c o n s i d e r i n g the environmental continuum encountered by an ionophore when t r a n s v e r s i n g a b i o l o g i c a l membrane.


Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980. 2

2

D

T

1.69 1.71

•5 1.17x10" •5 5.52x10" •5 5.48x10"

•5 3.12x10" •5 5.69x10" •5 5.45x10"

1.77

1.47

1.17

+

4

1

|R |Na xl0 o 1

1.03x10"

+

•4 4.89x10"

K K

•4 3. 87x10"

+

•3 1.84x10"

D

K Na

Q

3 8 1

+

1.70

1.73

1.80

1.75

1.21

1

|R |K xl0 o

T

T + + and R o f Na and K Complexes

3 8

* A p r i o r i we would expect a p r o g r e s s i v e drop i n K^'s as the s o l v e n t Z values decrease s i n c e the energies r e q u i r e d to desolvate the c a t i o n s (12) and ionophore (38) p r i o r to complexation decrease p r o g r e s s i v e l y . The r i s e i n apparent values i n s o l v e n t s of low Z values can be accounted f o r by p r o g r e s s i v e i n c r e a s e s i n i o n p a i r i n g which reduce the a c t u a l c a t i o n c o n c e n t r a t i o n , i . e . c a t i o n a c t i v i t y , a v a i l a b l e f o r complexation. P r e l i m i n a r y c o r r e c t i o n s f o r i o n p a i r i n g by means of Bjerrum's equation, however, do not s i g n i f i c a n t l y a l t e r the c a t i o n s e l e c t i v i t y patterns reported here.

76.7

2

90% DI0XANE/H 0*

80.2

83.6

87.6

79.6

DI0XANE/H 0

MeOH

DI0XANE/H 0

Z

E f f e c t of Solvent Z Value on of Salinomycin

EtOH*

80%

50%

SOLVENT

Table I


w 2 o 2 w

a

>

i

1

GO H 2 a 5 r

§

m

X

o o

2 o *> o >

1.

PRESSMAN E T A L .

Properties

of

Ionophores

13

The extension to ionophore s e l e c t i v i t y of a hypothesis based on analogy with the r i g i d matrices of i o n s e l e c t i v e g l a s s e s (19) i s i n c o n s i s t e n t with the dynamic conformational aspect of i o n s e l e c t i v i t y developed i n the present paper. Furthermore, the conformational options of ionophores are not n e c e s s a r i l y a graded f u n c t i o n of environmental p o l a r i t y but may d i s p l a y sudden s h i f t s between metastable s t a t e s over narrow p o l a r i t y ranges. Electros t a t i c i n t e r a c t i o n s between ions and induced d i p o l e s undoubtedly play a determinative r o l e i n c a t i o n complexation by ionophores, but the a b i l i t y of the ionophore to a l t e r i t s conformation cannot be ignored as i t i s i n the assumption of i s o s t e r i s m (19).


Pharmacological P r o p e r t i e s of C a r b o x y l i c Ionophores Pharmacological E f f e c t s . Although both n e u t r a l and carboxyl i c ionophores have been e x t e n s i v e l y employed as t o o l s f o r i n v i t r o s t u d i e s of b i o l o g i c a l systems f o r the reasons d e t a i l e d prev i o u s l y , only the c a r b o x y l i c ionophores are s u f f i c i e n t l y t o l e r a t e d by i n t a c t animals to produce w e l l defined pharmacological responses. We i n i t i a l l y examined the c a r d i o v a s c u l a r e f f e c t s of l a s a l o c i d because of i t s a b i l i t y to transport the key b i o l o g i c a l c o n t r o l agents, C a ^ and catecholamines (20,21). However, we l a t e r discovered that c a r b o x y l i c ionophores s e l e c t i v e f o r a l k a l i ions were even more potent i n evoking the same responses (22). F i g u r e 7 i l l u s t r a t e s the two d i s t i n c t primary c a r d i o v a s c u l a r e f f e c t s produced by monensin. At low c o n c e n t r a t i o n s , 50 yg/kg, i t produces a d i r e c t d i l i t a t i o n , i . e . r e l a x a t i o n of the smooth muscle of the coronary a r t e r i e s , manifested by a m u l t i f o l d i n crease i n coronary blood flow. At t h i s l e v e l or below, no other e f f e c t s occur. I f the dose i s i n c r e a s e d to 0.2 mg/kg, an i n o t r o p i c response f o l l o w s the i n i t i a l coronary d i l i t a t i o n . T h i s response, an i n c r e a s e i n c a r d i a c c o n t r a c t i l i t y , can be monitored as the maximum r a t e of r i s e of pressure i n the l e f t v e n t r i c l e , LV dP/dt max. Other parameters p a r a l l e l the i n o t r o p i c e f f e c t . Following an i n i t i a l drop caused by d i l i t a t i o n of the systemic a r t e r i e s , mean blood pressure r i s e s as does pulse pressure, the i n t e r v a l between lowest ( d i a s t o l i c ) and h i g h e s t ( s y s t o l i c ) t r a n s i e n t pressures; the r a t e of blood pumped by the h e a r t ( c a r d i a c output) a l s o r i s e s . The two d i s t i n c t e f f e c t s are thus an i n c r e a s e i n coronary flow, which r a p i d l y f o l l o w s i n j e c t i o n of the ionophore, followed by an i n o t r o p i c response, which only appears at h i g h e r doses. The r e s o l u t i o n by dosage of the two ionophore responses i s c l e a r l y apparent i n the dose-response p l o t of F i g u r e 8. Coronary flow r i s e s p r o g r e s s i v e l y u n t i l i t plateaus at 10-50 yg/kg monensin. Higher doses cause a secondary i n c r e a s e i n flow r e f l e c t i n g the r i s e i n a t r i a l pressure which d r i v e s blood through the c o r o n a r i e s . Only 2.5 yg/kg ( i . e . 2.5 ppb) are s u f f u c i e n t to double the b a s a l flow r a t e . I t i s p o s s i b l e to detect the i n creased flow of 1 yg/kg (1 ppb) with s t a t i s t i c a l confidence. +


14



1.15 Ml/Kg

M Bt/Kl

Figure 7. Cardiovascular response of a typical anesthetized dog to monensin. A low dose (0.05 mg/kg) was first introduced iv (dissolved in ethanol), and after an interval of an hour to permit the animal to return to basal conditions, a higher dose (0.2 mg/kg) was administered. The lowest tracing (mean LAD CF.) is the timeaveraged flow measured by a magnetic flow probe encircling the left anterior descending coronary artery. The AP trace gives the diastolic-systolic pressure range recorded from a catheter in the aorta. LV dP/dt max, the index of cardiac contractility, was obtained from a manometer-tipped catheter inserted in the left ventricle. The measured pressure was converted to its derivative to record dP/dt directly.

0

10

20

30

40

SO

60

70

80

90. lAo

MONENSIN INJECTED Figure 8. Dose-response curve of coronary flow vs. monensin in the dog. Data replotted from Ref. 37, as a function of dose at a fixed time interval of 5 min after injection.


1.

PRESSMAN

E T AL.

Properties

of

15

Ionophores

Mechanism of the Pharmacological E f f e c t s . Table I I compares the jln v i t r o ion c a r r y i n g c a p a c i t y of a s e r i e s of ionophores with t h e i r i n o t r o p i c potency. Appreciable r a t e s of C a or c a t e c h o l amine (norepinephrine) transport are observed only f o r l a s a l o c i d , the ionophore of the group with the poorest i n o t r o p i c potency. Extremely wide ranges of Ca + and norepinephrine t r a n s p o r t capacity are seen with no c o r r e l a t i o n with i n o t r o p i c potency. The Ca2+-selective A-23187 gives only a s p o r a t i c i n o t r o p i c response with the i n t a c t dog. The c o r r e l a t i o n between i n o t r o p i c potency and N a transport c a p a c i t y i s l e s s negative and i s w i t h i n the realm of l i k e l y d i f f e r e n c e s between the p r o p e r t i e s of the e x p e r i mental solvent b a r r i e r system and those of a c t u a l b i o l o g i c a l membranes. When the a c t i v i t i e s of ionophores are compared on the b a s i s of the quantity required to r e l e a s e a standard amount of K from e r y t h r o c y t e s , c h i e f l y i n exchange f o r Na , the c o r r e l a t i o n with i n o t r o p i c potency i s even b e t t e r . C e l l s i n general c o n t a i n high K : N a l e v e l s and are bathed i n e l e c t r o l y t e s containing low K+:Na r a t i o s . Inducing ionophoremediated exchange-diffusion t r a n s p o r t thermodynamically favors l o s s of i n t r a c e l l u l a r K f o r a roughly equivalent amount of Na . Since the r e l a t i v e increase i n c e l l u l a r N a induced by ionophores i s considerably greater than the r e l a t i v e l o s s of K , we i n f e r that the gain i n i n t r a c e l l u l a r Na , r e f l e c t e d by the more conveni e n t l y measured r e l e a s e of K+, i s more s i g n i f i c a n t than the l o s s of K per se. An a d d i t i o n a l f a c t o r i s that d i f f e r e n t b i o l o g i c a l membranes, e.g. erythrocytes and mitochondria, respond d i f f e r e n t l y to ionophores (23). A l l things taken i n t o c o n s i d e r a t i o n , the data of Table I I are reasonably supportive of a mechanism of a c t i o n of ionophores i n v o l v i n g i n i t i a t i o n of an increase i n i n t r a c e l l u l a r Na . 2 +

2

+

+


+

+

+

+

+

+

+

+

+

+

+

Many of the e f f e c t s of ionophores appear to i n v o l v e an i n crease i n i n t r a c e l l u l a r Ca +. Increased c o n t r a c t i l i t y implies an increased a v a i l a b i l i t y of i n t r a c e l l u l a r Ca^+ to t r i g g e r the i n t e r a c t i o n of a c t i n and myosin. At higher concentrations, monensin p r o g r e s s i v e l y induces c o n t r a c t i o n of the r e s t i n g heart ( c o n t r a c ture) i n d i c a t i n g that C a a c t i v i t y becomes too elevated to allow normal r e l a x a t i o n (24). Increased i n t r a c e l l u l a r C a a c t i v i t y also activates secretory c e l l s (25). I n h i b i t i o n s t u d i e s i n d i c a t e that the i n o t r o p i c e f f e c t of monensin i s mediated i n p a r t by the r e l e a s e of c a t e c h o l amines from the adrenals and/or the h e a r t i t s e l f (22). Monensin a l s o discharges catecholamines from disaggregated bovine chromaff i n c e l l s i n c u l t u r e (26,27), and induces the r e l e a s e of a c e t y l c h o l i n e a t the neuromuscular j u n c t i o n (28). Thus, the s e c r e t i o n s t i m u l a t o r y a c t i v i t y of monensin a l s o supports the concept that increased i n t r a c e l l u l a r N a a c t i v i t y produces a r i s e i n i n t r a cellular C a a c t i v i t y s u f f i c i e n t to s t i m u l a t e C a - a c t i v a b l e cells. Two hypotheses f o r the conversion of a primary i n c r e a s e i n i n t r a c e l l u l a r N a a c t i v i t y to a subsequent increase i n i n t r a c e l l u 2

2 +

2 +

+

2 +

2 +

+


Martell; Inorganic Chemistry in Biology and Medicine ACS Symposium Series; American Chemical Society: Washington, DC, 1980. .37

7.7

12.1

13.1

+

X-206

Salinomycin

A-204

+

.000025

6.1

Monensin .000009

.00015

low

.01

.003

.1

.002

20

-

2

31

27

+

+

+

-

41

10.0

10.9

7.2

18.0

16.4

-

4.1

(1.0)

Erythrocyte K Release

I n o t r o p i c potencies were compared as the i n v e r s e of the ionophore dose r e q u i r e d to double LV max dP/dt. C a , norepinephrine and N a t r a n s p o r t r a t e s were obtained i n the v e r t i c a l l y stacked three phase system described i n r e f . (39). E r y t h r o c y t e K r e l e a s e potency was measured as the i n v e r s e o f the c o n c e n t r a t i o n r e q u i r e d to r e l e a s e 10 mM K from washed human e r y t h r o c y t e s suspended i n mock plasma c o n t a i n i n g 5 mM KC1, 145 mM NaCl and 10 mM TRIS c h l o r i d e , pH 7.4.

2 +

-

-

4.3

Dianemycin

A-23187

.002

.000025

2.8

Nigericin

1.4

-

-

-

2.0

Septamycin 0.001

-

-

-

1.5

Lysocellin

.000009

(1.0)

(1.0)

(1.0)

(1.0)

Na Transport

Lasalocid

Norepinephrine Transport

Ca Transport

Inotropic Potency 2 +

Comparison of I n o t r o p i c Potency of Ionophores with jln v i t r o Transport P r o p e r t i e s

Ionophore

Table I I


a

o 3 w

w

a

>

8

3 w 3 r

g H

=

>

2 o

ON

1.

PRESSMAN E T A L .

Properties

of

17

Ionophores

2 +

lar C a are p l a u s i b l e . One would be an exchange-diffusion c a r r i e r i n the plasma membrane p e r m i t t i n g the l a r g e C a activity gradient (a 10"3 M e x t r a c e l l u l a r , a 10" M i n t e r i o r ) to permit entry of Ca + i n t o the c e l l i n exchange f o r Na . (On thermodynamic grounds one would expect the exchange r a t i o to be 3-4 N a expelled f o r each C a taken up). Thus, making more i n t r a c e l l u l a r N a a v a i l a b l e f o r exchange, or i n thermodynamic terms reducing the gradient against which N a must move (a 10~ M i n t r a c e l l u l a r , a 10-1 M e x t r a c e l l u l a r ) , would favor the entry of C a . A c r i t i c a l e v a l u a t i o n of t h i s hypothesis has appeared i n a recent review (29). An a l t e r n a t e mechanism would be the r e l e a s e of i n t r a c e l l u l a r ^ bound C a by displacement by Na+. This i s f e a s i b l e s i n c e the gross chemical C a i n t r a c e l l u l a r concentration i s ca. 10"" 3 M while i t r e q u i r e s only 10"~6 - 10~ M C a a c t i v i t y to a c t i v a t e c o n t r a c t i o n or s e c r e t i o n . There might w e l l e x i s t purposeful C a N a ion-exchange s i t e s w i t h i n c e l l s so designed that only a small r e l a t i v e N a a c t i v i t y change i n the mM range would t r i g g e r a l a r g e relative C a a c t i v i t y change i n the yM range which would be s u f f i c i e n t to a c t i v a t e Ca -dependent i n t r a c e l l u l a r processes. 2 +

7

2

+

+

2 +

+

+

2

2 +

2 +

2 +

5

2 +


2 +

+

+

2 +

2+

Impact of Ionophores on Man

and Animals

C a r b o x y l i c Ionophores and E f f i c i e n c y of Feed Conversion by L i v e s t o c k . A strong note of relevance to s t u d i e s of the chemical and pharmacological p r o p e r t i e s of c a r b o x y l i c ionophores d e r i v e s from the l a r g e s c a l e use of monensin as a l i v e s t o c k feed a d d i t i v e . The r a t i o n a l e i s that c a r b o x y l i c ionophores c o n t r o l endemic c o c c i d i o s i s i n the p o u l t r y gut (30) and promote a more f a v o r a b l e fermentation of c e l l u l o s e i n the bovine rumen (31). In e i t h e r case, the net r e s u l t i s the economically important increased e f f i c i e n c y of conversion of feed i n t o meat. Pharmacokinetics of Ionophore Absorption. We have developed a s e n s i t i v e chemical assay f o r c a r b o x y l i c ionophores (which w i l l be published elsewhere) based on t h e i r a b i l i t y to form l i p i d s o l u ble complexes with c a t i o n s . We can detect as l i t t l e as 1 part per b i l l i o n (ppb) monensin i n 2 ml of blood plasma or t i s s u e . For a comparison y a r d s t i c k , c u r r e n t feeding regimens c a l l f o r ca. 30 parts per m i l l i o n (ppm) i n c a t t l e feed (32) and as much as 100 ppm i n p o u l t r y feed (33). T y p i c a l l y , a cow i n g e s t s about 0.3 g (^ 1 ppm) monensin/day. As p r e v i o u s l y observed i n F i g u r e 7, as l i t t l e as 1 ppl> (based on body weight) produces a detectable p h y s i o l o g i c a l e f f e c t on the dog. In order to e s t a b l i s h the pharmacokinetic r e l a t i o n s h i p s between o r a l l y ingested and i n t r a v e n o u s l y i n j e c t e d monensin, we c a r r i e d out p r e l i m i n a r y s t u d i e s of monensin blood l e v e l s i n the dog. In F i g u r e 9 we see that i n j e c t e d monensin c l e a r s from the plasma with a t ^ of ^ 2.5 minutes which we presume i s too r a p i d for the operation of normal e l i m i n a t i o n mechanisms. Hence, i t i s


18



2SII 20N ISM

it!i\

sec

1000 MIREHSIR

f 1IMI/K( I.I. IKE

2**1/11 HAL USE

60

90 120 ISO MINUTES AFTER 00SE

Figure 9. Pharmacokinetics of monensin in the dog. In the upper trace, 100 fig/kg monensin was injected into a barbiturate-anesthetized dog with a manometer-tipped catheter in the left ventricle to measure dP/dt. Blood samples were taken at various periods and 2 mL samples of plasma obtained by centrifugation for ionophore assay. Note that the monensin cleared the blood rapidly and that the cardiac responses persisted. Subsequent assays revealed the monensin entered the dog tissues, particularly the lungs. The lower trace compares the pharmacokinetics of the injected dose with those obtained from a nonanesthetized dog that received the monensin orally (2 mg/kg) as a concentrate applied to a small quantity of feed. The plasma levels obtained by administration of an oral dose approached those obtained by injection, indicating that the major portion of the oral dose passed through the plasma and into the tissues before being eliminated.



1.

PRESSMAN E T A L .

Properties

of

Ionophores

19

reasonable to assume that the ionophore l e a v i n g the plasma i s taken up by the t i s s u e s . T h i s would not at a l l be unexpected cons i d e r i n g the high l i p i d : w a t e r p a r t i t i o n c o e f f i c i e n t of ionophores. I t i s supported by the delayed and p e r s i s t e n t e l e v a t i o n of the i o n o p h o r e - s e n s i t i v e c a r d i a c f u n c t i o n parameter, LV dP/dt. Prel i m i n a r y t r i a l s of a v a r i a t i o n of our assay adapted f o r whole t i s s u e s i n d i c a t e that i n the r a b b i t the major p o r t i o n of monensin appears i n the t i s s u e s w i t h i n 10 minutes f o l l o w i n g i . v . i n j e c t i o n , a t concentrations roughly p a r a l l e l i n g the degree of blood p e r f u sion: lung > h e a r t > kidney > l i v e r , muscle, f a t . The lower graph of F i g u r e 8 compares the time course of appearance i n the plasma of i n j e c t e d and o r a l l y administered monensin doses i n the dog. The o r a l dose appears i n the blood more slowly but produces more sustained ionophore blood l e v e l s . The time concentration i n t e g r a l gives an index of the q u a n t i t y of the drug which passes through the plasma; r a t e of entry and c l e a r ance from the blood a f f e c t only the shape of the curve, not the net i n t e g r a l . The i n t e g r a l can be c a l i b r a t e d by comparison with the i n t e g r a l of a known dose administered d i r e c t l y i n t o the b l o o d Although d i f f e r e n t animals and d i f f e r e n t dose l e v e l s were used, the r a t i o of the i . v . i o r a l dose i n t e g r a l s are approximately prop o r t i o n a l to the 1:20 r a t i o s of the net doses administered. This s i g n i f i e s that a major p o r t i o n , i f not a l l of the o r a l l y ingested monensin dose, passes through the blood stream of the dog before being e l i m i n a t e d . In the r a b b i t , a h e r b i v o r e , one might p r e d i c t absorption of o r a l doses would be slower. We can detect o r a l l y administered monensin doses i n r a b b i t plasma, but only a f t e r a couple of hours f o l l o w i n g i n g e s t i o n . We have not yet completed the more prolonged plasma l e v e l - t i m e p r o f i l e s i n t h i s s p e c i e s . The Need f o r Increased S u r v e i l l a n c e of the Exposure of Man to Ionophores. From the l i p i d s o l u b i l i t y of monensin and other ionophores, we would p r e d i c t they should have no trouble e q u i l i b r a t i n g across b i o l o g i c a l membrane systems i n c l u d i n g the gut. This i s c e r t a i n l y the case f o r the two d i v e r s e species observed, the dog, a c a r n i v o r e , and the r a b b i t , a h e r b i v o r e . A c c o r d i n g l y , we i n f e r that there i s ample opportunity f o r monensin and other c a r b o x y l i c ionophores administered o r a l l y to l i v e s t o c k to d i s t r i b ute s y s t e m i c a l l y and exert a pharmacological e f f e c t on the r e c i p i ent animal. Furthermore, the r e s u l t a n t p h y s i o l o g i c a l e f f e c t s may be p a r t of the mechanism by which ionophores produce t h e i r improved feed conversion e f f i c i e n c y . There are f u r t h e r i n f e r e n c e s which d i r e c t l y a f f e c t man. If the ionophores do pervade the t i s s u e s , i t i s p o s s i b l e that man may become exposed to pharmacologically competent and p o t e n t i a l l y d e t r i m e n t a l l e v e l s of ionophores through h i s meat supply. Based on l i m i t e d pharmacokinetic and t o x i c o l o g i c a l data, the F.D.A. has s e t upper p e r m i s s i b l e l e v e l s of 0.05 ppm i n meat f o r human consumption (34). The isotope r e s i d u e s t u d i e s of Herberg et a l . r e p o r t that under c u r r e n t feeding procedures c a t t l e l i v e r



20

INORGANIC

CHEMISTRY IN

BIOLOGY A N D

MEDICINE

may accumulate over ten times t h i s l e v e l of monensin as a combinat i o n of parent compounds and m e t a b o l i t e s of unknown pharmacologic a l e f f e c t s (35). This data was obtained 12 hours a f t e r administ r a t i o n of tagged monensin. One might surmise that r e s i d u e s would be a p p r e c i a b l y higher f o r an animal butchered a s h o r t e r p e r i o d of time f o l l o w i n g i t s l a s t exposure to monensin. T h i s i s p a r t i c u l a r l y s i g n i f i c a n t i n that l i t e r a t u r e s u p p l i e d to farmers advises that no withdrawal p e r i o d i s necessary. C u r r e n t l y a v a i l a b l e methods f o r assaying monensin i n v o l v e cumbersome e x t r a c t i o n procedures, t h i n l a y e r chromatography and d e t e c t i o n by means of bioautographs w i t h microorganisms whose s e n s i t i v i t y to ionophores and t h e i r m e t a b o l i t e s (36) may or may not p a r a l l e l mammalian s e n s i t i v i t y . The simple chemical assay method we have developed can provide a more r a t i o n a l b a s i s f o r a s s i g n i n g p e r m i s s i b l e r e s i d u e l e v e l s , f o r r o u t i n e l y monitoring products a r r i v i n g a t the market, and a s c e r t a i n i n g whether s t i p u l a t e d ionophore withdrawal periods are being complied w i t h . A d d i t i o n a l complications y e t to be evaluated d e r i v e from the notably poor b i o d e g r a d a b i l i t y of monensin. Reports i n d i c a t e that c a t t l e f e c a l l y e l i m i n a t e 75% of ingested monensin without degrad a t i o n . Furthermore, 60-70% of the monensin survives 10 weeks i n cubation a t 37° (34). Current manuring p r a c t i c e s render i t prudent to determine whether crops o r garden produce take up s i g n i f i cant q u a n t i t i e s of c a r b o x y l i c ionophores or whether the o b v i o u s l y l a r g e s o i l burdens of such compounds f i n d t h e i r way i n t o water supplies. We have long been i n t e r e s t e d i n the p o s s i b i l i t y that the c a r d i o v a s c u l a r e f f e c t s of c a r b o x y l i c ionophores could be harnessed to provide new drugs f o r the treatment of disease s t a t e s such as heart f a i l u r e and shock. There may, however, be subpopulations of man f o r whom ionophores may be p a r t i c u l a r l y t o x i c . For example, a t o x i c i n t e r a c t i o n between monensin and d i g i t a l i s on the dog h e a r t has been r e p o r t e d (37). Our o r a l a b s o r p t i o n data do i n d i cate that i f a u s e f u l human therapeutic a p p l i c a t i o n can be est a b l i s h e d , ionophores could be administered as drugs o r a l l y . Summary We have described how the unique p h y s i c a l p r o p e r t i e s of i o n o phore molecules l e a d to b e t t e r understanding of t h e i r unique b i o logical effects. Ionophores have been a p p l i e d as t o o l s f o r b i o l o g i c a l r e s e a r c h , as commercially important l i v e s t o c k feed a d d i t i v e s f o r i n c r e a s i n g the e f f i c i e n c y of meat production, and i n v e s t i g a t e d as p o t e n t i a l l y u s e f u l drugs i n man. E x p e r t i s e derived from s t u d i e s of the molecular p r o p e r t i e s of ionophores has been u t i l i z e d to design a simple assay procedure which g i v e s promise f o r p r o v i d i n g more r a t i o n a l safeguards f o r man i n the widespread use of ionophores i n food p r o d u c t i o n . L a s t l y , i n view of the burgeoning i n c r e a s e s i n the s c a l e of commercial ionophore usage,


1.

PRESSMAN E T A L .

Properties

of

Ionophores

21

i t appears urgent that we i n c r e a s e our understanding i n depth of the p h y s i o l o g i c a l and metabolic e f f e c t s o f ionophores and t h e i r pharmacological and t o x i c o l o g i c a l r a m i f i c a t i o n s . Acknowledgements


We wish to acknowledge the a s s i s t a n c e o f Ms. Georgina Del V a l l e and Mr. Frank L a t t a n z i o i n the development of the i o n o phore assay and Drs. L. A l l e n and M. K o l b e r i n h e l p i n g program the computer s t u d i e s . We are indebted to E l i L i l l y f o r samples o f monensin and A.H. Robbins and Kaken Chemical Co. (Japan) f o r s a l i n o m y c i n . These s t u d i e s were supported i n p a r t by NIH grant HL-23932 and a grant from the F l o r i d a A f f i l i a t e of the American Heart A s s o c i a t i o n .

Literature 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

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RECEIVED July 17, 1980.


Inorganic Chemistry in Biology and Medicine - American Chemical

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