PARTVIII. PERMEATION MECHANISM ACROSS BIOLOGICAL MEMBRANES: I1

BIOLOGICAL APPLICATIONS OF IONOPHORES: THEORY AND PRACTICE* Berton C. Pressman and Norbert0 T. deGuzman

Department of Pharmacology University of Miami School of Medicine Miami, Florida 33152

The discovery of the ion~phores'-~ has provided the means for studying a wide range of ion-selective processes. These compounds of moderate molecular weight (ca. 500-1500) form lipid-soluble complexes which provide a vehicle for a wide variety of cations to transverse lipid barriers, hence the term ionophore signifying i ~ n - b e a r e r .A~ series of representative ionophores is shown in FIGURE 1. Ionophore-mediated transport has not only been employed to perturb biological systems for study, but has also provided clues to the molecular mechanisms by which the biological membranes themselves transport and

VAllWbYYClW

CYCLOHEXYL fTHER

EWWA l TW l

B

MONlWS W l

MACROLIDE AClWS

WG I ERC IW I

Pa?

FIGURE1. Structures of representative neutral (valinomycin, enniatin B, macrolide actins and dicyclohexyl-18-crown-6)and carboxylic (monensin, nigericin) ionophores. *This work was supported by gifts from Hoffman La-Roche and Eli Lilly and by Grant No. M73A64 from the Heart Association of Greater Miami and Grants No. HL-14434 and HL-16,117 from the National Institutes of Health.

373

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Annals New York Academy of Sciences

discriminate between ions. Accordingly, ionophore transport and complexation have been intensively studied in a multitude of bulk phase and lipid bilayers systems. A practical offshoot of this work has been the development of ionophore-based liquid-exchange ion-selective electrodes, which, in their standard and micro variations, have further extended the impact of ionophores in biology. General reviews of this subject are contained in References 4 and 5. The first clue to the unique properties of ionophores was the observation that valinomycin induces the energy-dependent uptake of K , but not Na+, by mitochondria.’ Those ionophores whose existence was earlier revealed by the mitochondria1 test system interact primarily with alkali ions:. although they exhibit a diversity of cationic discrimination More recently it was recognized that the ionophore A-23187 is highly selective for divalent cations’ and that X-537A and several of its derivatives are broad-spectrum ionophores, capable of complexing and transporting not only alkali ions but polyvalent inorganic ions and complicated organic amines as Its ability to transport such key biological control agents as Ca” and the catecholamines led us to explore the effects of X-53lA on the isolated heart and ultimately on the intact organism. Initial results were encouraging and indicated that ionophores have unprecedented physiological effects with pharmacological implications which, we are encouraged to believe, will develop into yet another major area of ionophore-related studies. This paper will focus on the pharmacological properties of X-537A and related ionophores and examine how these correlate with their properties in other systems. 2) belongs to the class of carboxylic ionophores that X-537A (FIGURE contain a single carboxyl group which is usually deprotonated in its cationic complexes. In most cases the resulting negative charge of the ionophore offsets that of the cation complexed so that for 1 : l complexes with monovalent ions the complex as a whole is electrically neutral. In parallel fashion X-537A forms a 2 : l neutral complex with Ba“, which has been characterized by x-ray crystallography. The cation is sandwiched unequally between two deprotonated ionophore molecules termed “unprimed” (six ligands; see FIGURE2) and primed (three ligands) ,I3 X-537A contains an aromatic group that provides several interesting molecular properties. It gives rise to three absorption bands at 315 nm (aromatic hydroxyl), 245 nm (aromatic carboxyl), and 210 nm (aromatic ring proper). Although the 3 15-nm band shows a small shift on protonation-deprotonation, no useful shift occurs during the transition between the free anionic and complexed forms. The 315-nm and 245-nrn bands, along with the weakly absorbing but highly asymmetric ketonic carbonyl peak at 290 nm, give rise to C D bands that can be used as indices of molecular conformation and

FIGURE2. Structure of the “unprimed” moiety of X-537A as it occurs in the barium salt. It is assumed that the liganding pattern for monovalent cations is similar.

Pressman & deGuzman:

375

Ionophores

TABLE1 RELATIVE AFFINITIES OF IONOPHORES FOR DIVALENT CATIONS OBTAINED FROM TWO-PHASE DISTRIBUTION STUDIES* Ionophores Cation Mg++

ca++

sr++

Ba++

Acet yl-X-5 3 7A

X-537A

Br-XS 37A

Dhnemycin

.26 .29 1.8 72

.38 1 .o 8.5 2600

.54 2.8 18 5600

.01 .08 .20

* Complex formation was determined from the migration of the test cation from aqueous Tricine buffer (pH 9.0) into 70% toluene: 30% n-butanol. The [ionophore] was 0.5 mM and the two phase KA calculated from the following expression which assumes 2:l complex formation: KA = [M++ Ionophore-2 erg] /[MH,o] . [Ionophoreorg] 2. All values were normalized by dividing by the two phase KA for the Ca"-X-537A complex. that are unique for each complexation " The aromatic hydroxyl and carboxyl groups collectively constitute a fluorophore, exciting at 3 13 nm and emitting at 420 nm.I5 Each complexation species has its own characteristic fluorescent yield that is sufficiently different from that of the uncomplexed anion species to serve as a basis for affinity titrations. X-537A is readily susceptible to chemical modification. The aromatic ring may be substituted para to the phenolic hydroxyl (Br, C1, I, NOz), the phenolic hydroxyi may be acetylated or the ketonic carbonyl reduced," all of which produce functional ionophores with altered cationic selectivities. In order to ascertain whether the ability of X-537A to complex Bat+ extends to the biological trigger ion Ca++,complexation affinities were initially measured by two-phase distribution to establish ionophore affinities." The principle involved is the redistribution of the inherently hydrophilic test cation into a standard lipid phase upon the formation of a lipid-soluble complex with the test ionophore." ' TABLE1 shows that although complexation affinity falls off with decreasing ionic radius of the cation complexed, X-537A does form readily detectable complexes with Ca". For the sake of comparison, the divalent cation affinities of other carboxylic ionophores were measured. The next most effective one is dianemycin, which is lower in affinity for divalent ions by an order of magnitude; monensin and nigericin show still lower affinities for Ca". In the course of studying the effects of X-537A on biological systems, we observed that this ionophore forms a complex with the buffer Tris, which is a primary amine. In order to assay the ability of X-537A to complex amines in general by the two-phase partition procedure, ethanolamine was chosen as a model amine since it is highly hydrophilic, remains in the aqueous phase unless complexed, and is available isotopically labeled. As seen in TABLE 2, it readily forms lipid-soluble complexes with X-537A and to a considerably lesser extent with other carboxylic ionophores. The structural analogy between ethanolamine (H-CHOH-CH~NHZ) and the catecholamines such as norepinephrine [ (HO) G H 4 H O H - C H ? N H 2 ] is readily apparent and directed us to discover the important ability of X-S37A to complex catecholamines. The strong preference observed of X-537A for

376

Annals New York Academy of Sciences TABLE 2 Kn OF ORGANIC AMINE-IONOPHORE COMPLEXES*

Ionophore

Ethanolamine

Norepinephrine

Epinephrine

163

9.8

31

4.2

9 5

0.9 1.8

~

X-537A Dianemycin Monensin Nigericin

415

65 3 1.2

~-

’* The same system described in TABLE 1 was used. KA was calculated on the assumption of 1:l complexes with the monovalent cations. norepinephrine over epinephrine, presumably due to steric hindrance for complexation by the N-methyl group of the latter, parallels the selectivity of the biological a-adrenergic receptor for which it might be considered a model. The fluorescence properties of X-537A and its derivatives ultimately provided a nonisotopic method applicable for determining complexation affinities in a single phase. The effects of low concentrations of the test ions on the change in ionophore fluorescence were plotted as double reciprocal Langmuir saturation isotherms; the slopes of the resulting plots yielded the complexation affinities, while extrapolation of the linear isotherms to infinite cation concentration indicated the true fluorescent yield of the ionophore complexation species under saturation conditions. Often, for example, for Li’, Mg“, Ca++,and Sr”, these isotherms displayed abrupt changes in slope, indicative of the formation of different species of complexes, an interpretation also supported by CD spectra.” It should be noted that the two-phase distribution procedure for measuring complex formation selects for electrically neutral species; any tendency to form electrically charged complexes would set up interphasic potentials which would oppose further migration of cation to the nonpolar phase. The formation of electrically charged cation-ionophore complexes, the existence of which is implied by the ability of X-537A to carry current through a lipid bilayer,’” is under no such restriction in a homogeneous bulk phase. Preliminary titrations indicate that at low cation concentrations the first complex detectable in ethanol is 1:1, ionophore:ion, for ions of all valences. It should be noted that the 1:1 complexes of all polyvalent cations are charged and therefore could carry current. Thus, the complexes formed during singlephase fluorescence titrations may be different species from those governing cation migration in the two-phase test system described above, and those responsible for carrying current through the lipid bilayer. Haynes and Pressman have already reported that the fluorescence quantum efficiency of anionic uncomplexed X-357A in ethanol is about 0.75.” Ketodihydro-X-537A is about equally fluorescent while the decrease in fluorescence quantum efficiency of Br-X-537A by a factor of ten does not introduce undue difficulties into the titration procedure. From TABLE 3 we see that the alkali cation complexes of all three ionophores show qualitatively similar preferences for ions. Although by the partition method the most favored ion is Cs+ as opposed to Rb’,” this could reflect differences in cation desolvation energies between water and ethanol. The dihydro derivative shows considerably less affinity for alkali ions than the other two ionophores, a condition most marked for the nonspherical “pseudoalkali” ion Ag’.

Pressman & deGuzman:

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377

In the alkaline earth series increasing ionic radius from Ca" to Bat+ greatly increases cation affinity for X-537A. It is noteworthy that the affinity of dihydro-X-537A for Cat+ is 66% of that of the parent compound; for K it is 15%, while for ethanolamine it is less than 3%. The differential effects of modifications of a parent ionophore on its ability to translocate K', Ca", 2nd catecholamines could be optimized for a given pharmacological application 3y a systematic and comprehensive survey of derivatives. The wide variety of amines that complex X-537A provides a means for obtaining information about the ionophore binding site. The progressive gradual loss of affinity from methylamine to octylamine could be ascribed to increasing molecular "jiggling" of the hydrocarbon chain from thermal agitation. Introducing a second N-methyl group into either methylamine or ethanolamine is equivalently unfavorable for complexation, presumably due to steric hindrance (cf. norepinephrine vs. epinephrine, TABLE 2). For the sake of comparison K.4 values for some higher valent complexes of X-537A are also given. All inorganic cations tested gave definite complexes with X-537A including all available lanthanides, Cu", Zn", Fe", etc. Net transport velocities across membranes depend on a more complex reaction sequence than is involved in equilibrium affinity measurements, hence the latter predict only roughly what potential biological effects a given ionophore is capable of. An ion that forms a poor complex with an ionophore is not likely to TABLE3 KnOF IONOPHORECOMPLEXES IN ABSOLUTE ETHANOL*

X-537A Li+ Na+

K+ Rb+ cs+ NH,

+

T1+ &+

HO-CH,-CH,-NH:

Ma*

cia

+

SI'+ Ba'+

Mn" Law

Th* CH,NH: C,H,NH: C,H,NH: C,H,NH: C,H, 7 NH: (CH&,=NH,* (CH,),d

.1 6.7 67 77 59 5.3 50 11 11 7.4 7.6 25 1000 83 170 67 8.0 6.9 6.5 6.1 5.6 1.3 .3

Br-X537A

H,-537A

.2

.4

17 40 53 38 10 25 24 8 45

20 83 67 33

.5

10 11 10 .4

.7

.I .3 4 5 5.5 6.5 22

* 1 0 m M triethanolarnine partially neutralized with 5 m M HClO, present as buffer. AU K A expressed as mM-'.

Annals New York Academy of Sciences

378

FIGURE 3. Vertically stacked bulk phase transport system. Transport is followed by measuring the migration of isotope from the lower to the upper phase. Further details about this system are given in Reference 12.

be transported effectively by it; one that forms so tight a complex that it cannot be readily dissociated is an equally poor candidate for transport. Accordingly we devised the vertically stacked bulk transport test system shown in FIGURE3 which stimulates more closely the dynamic conditions that occur during ionophore-mediated membrane transport. The system has been described previously" and is dependably reproducible when replicate determinations are carried out, either simultaneously or in sequential runs. TABLE4 reveals that the alkali ion-transport rates follow the lyotropic Rb' > Na' (for convenience these experiments are restricted to series Cs' the readily available isotopically labeled cations). The poorer affinity of dihydro-X-537A shown in monovalent cation affinities in TABLE 3 is more apparent under the dynamic conditions of transport in TABLE4. On the other hand all three ionophores are about equally effective in transporting divalent ions, despite the extraordinarily high equilibrium affinity of X-537A for Bat+. This supports the earlier-stated inference that the complexation affinities seen in TABLE3 may not be those of the same complex species which most easily cross lipid barriers. Conversely Ca" appears to be transported unusually efficiently by comparison with its KAvalues.

>

TABLE 4 BULKPHASETRANSPORT RATESFOR CATION COMPLEXES OF IONOPHORES*

Na'

Rb' CS'

ca2+ Sr '+ Ba ?+

Tm" Ethanolamine Norepinephrine Epinephrine Isopropylnorepinephrine

X-537A

Br-X537A

39 45 118 119 54 89

46 132 128 57 88

3.1 104 52 65

47

5.6

17

55 29 9 6

Dihydro-XJ37A 1.4 2.1

1.2

0.5

* Solvent system employed is shown in FIGURE3. Rates are expressed as nmol/hr cation appearing in the upper phase. Turnover numbers may be calculated from the information that the 1-ml organic barrier layer (which was approximately 1 cm in height) contained 100 nmol ionophore.

Pressman & deGuzman:

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Transport in the amine series parallels the affinity constants seen in TABLE 2, except that discrimination between norepinephrine and epinephrine is less sharp. Determinations with other biogenic amines establish the rank order of transport capability of X-537A: dopamine > serotonin > ethanolamine norepinephrine epinephrine isopropylnorepinephrine. The collective in v i t r o studies of X-537A and its derivatives described indicate a diversity of complexation species and an amenability to examination by a wide variety of techniques. It seemed to us remarkable if these ionophores could not produce profound and interesting biological perturbations, particularly in view of the potentiality of X-537A for transporting the key biological control cations, Ca++and the biogenic amines, and for shifting the resting potentials of excitable membranes by altering alkali ion gradients. Because of the well-established effects of Ca” and catecholamines on the cardiovascular system we choje to test X-537A with a standard heart preparation. When the ionophore is perfused through spontaneously beating rabbit or guinea pig hearts (modified Langendorff preparation), increases in force of contraction with some increase in rate are observed in the dose range 10-aM - 10-5M.10-”These results were confirmed in the rat heart by the Williamson group.’’ Preparations from animals “reserpinized” to deplete catecholamines showed markedly diminished contractility responses to X-537A.“ At a constant perfusion pressure the perfusion rate was increased by the ionophore. Since in this preparation the perfusion fluid normally flows through the coronary arteries, which regulate flow by their hydraulic resistance, this indicated that the ionophore induces a dilation of the coronary vasculature that could even be observed in animals that had previously been heavily reserpinized. These preliminary observations encouraged us to extend our studies to the standard subject for pharmacological evaluation of cardiovascular drugs, the anesthetized The time course of the response of a dog to X-537A 4. The effect of X-537A upon the maximal rate of pressure is shown in FIGURE

>

>

FIGURE4. Continuous monitoring of the response of an anesthetized dog to X-537A (2 mg/kg). (Upper truce) left ventricular dP/dt is the rate of rise of pressure of the left ventricle as obtained from a pressure transducer tipped catheter. (Middle trace) aortic pressure. This tracing merges with the left ventricular dP/dt trace after 15 minutes. (Lower truce) mean blood flow in the left anterior descending coronary artery obtained from an electromagnetic flow probe. This trace becomes the uppermost one 10 minutes after the administration of X-537A.

380

Annals New York Academy of Sciences

development (dP/dt) in the left ventricle, which is conventionally regarded as an index of the contractile state of the heart, is shown in the upper trace of FIGURE 4. This function begins to rise almost immediately upon administration of a large dose of X-537A (2 mg/ kg) , attaining a value of almost threefold the control value within twenty minutes of the drug injection. The middle trace, aortic pressure, eventually undergoes a marked elevation after an initial transient decrease. This and other evidence (data from cardiac output) indicate that there is an initial drop in systemic vascular resistance paralleling the previously cited drop in coronary resistance that was noted in the isolated heart. As contractility rises, accompanied by an increased cardiac output, the drop in aortic pressure is gradually replaced by an increase. The lower tracing shows the blood flow through a major coronary artery (left anterior descending) as measured continuously by means of an electromagnetic flow probe. Coronary flow rises much more sharply and earlier than contractility or aortic pressure and in this experiment, after twenty minutes, was fully ten times higher than the control value, indicative of a striking drop in coronary vascular resistance. The hemodynamic responses to X-537A over an extended period of time in a series of six dogs are presented in FIGURE5. The upper trace shows a definite but minimal increase in heart rate. The rise in contractility, left ventricular dP/dt, is only slightly less than that shown for the representative dog in FIGURE 4 and attenuates slowly and gradually during the four-hour observation period. The initial rise in aortic (arterial) pressure is less sustained, returning to the control value after two hours. Cardiac output, a complicated function of contractility, systemic resistance and other factors that govern the venous return to the heart, shows an initial doubling which attenuates during the first hour and then persists at a mildly elevated level for four hours. Total peripheral resistance, calculated by dividing aortic pressure by flow, i.e., cardiac output, shows a decided initial drop during the first half hour, followed by a minimal decrease during the remainder of the observation period. These results collectively indicate a stimulation of the pumping characteristics of the heart and a decrease in vascular resistance which might be

110,

, *I11 11111111 f1111111

1

FIGURE 5. Hemodynamic effects of X-537A ( 2 mg/ kg) on a series of six dogs. Measurements were made on records such as represented in FIGURE 4. Values are the series means with

Pressman & deGuzman:

-c 58 0-

-

r"

MIAI

' I CORONA)

AII~RIAL'O~

20-

* . Y

5

100.

38 1

un c o i o i ~ i vIIIW

li-i-i '

Ionophores

'

MllUTtS

conid

FIGURE 6. Effects of X-537A (2 mg/ kg) on the consumption of O2by the left ventricle. PO, measurements were made on blood entering (aorta) and leaving (coronary sinus) the left ventricle. These were converted to actual 0,content of the basis of the hemoglobin content and pH of the blood. Left ventricular 0,consumption was obtained as the product of the atrioventricular AO, across the ventricle and the flow of blood through the coronary artery, and extrapolated to the whole ventricle from the measured ratio of myocardium supplied by this vessel to the total mass of the left ventricle.

useful during pathological cardiovascular conditions such as those that occur in acute pump failure or shock. FIGURE 5 depicts some of the effects of X-537A on the circulatory parameters of the heart itself. The upper trace shows the series average of the increase in coronary blood flow, which is somewhat more modest than the example chosen in FIGURE 4. The 0,content of the coronary sinus blood draining the left ventricle rises markedly as a consequence of the increased flow rate 6). The stippled area represents the extracthrough the myocardium (FIGURE tion of O,, i.e., the atrioventricular difference, from which the left ventricular OZ consumption is calculated by multiplying it by the coronary blood flow through the ventricular muscle. The 0,consumption shows a slight, but significant rise, which returns to the control values within thirty minutes. The implications of these data on cardiac work efficiency are shown in FIGURE 7. The small increases in ventricular 0 2 consumption have been contrasted with the marked increase in the hydraulic work calculated as the product of cardiac output and effective pressure (mean aortic pressure). This I

1"

,

irri iimicuiii 0, toxswriiov

FIGURE 7. Effects of X-537A on cardiac work efficiency.

LA 0

30

90 YlNUltS

60

120

150

382

Annals New York Academy of Sciences

derived index of external cardiac work shows an approximately 100% increase, which was sustained over a period of two hours. If we divide cardiac work by 0 2 consumption we see that X-537A induces a considerable increase in cardiac work output per unit 0:consumed, i.e., a marked improvement in cardiac efficiency. This indicates that it is possible to improve cardiac performance under conditions of limited Oxsupply, such as occur in coronary vessel disease (often accompanied by angina), or in selected regions of the heart following an infarction. This is in sharp contrast to the effect of catecholamines, the administration of which increases cardiac work, but only at the expense of a considerable augmentation of tissue O2consumption, which would be contraindicated under conditions with accompanying ischemia. The doubling of the work efficiency of the heart by ionophores might appear implausible. However, reasonable values for the thermodynamic cardiac efficiency of a barbiturate-anesthetized dog would be about 15-20%, and a doubling of this value would not call into question any thermodynamic principles. The heart is known to increase its 0 2 consumption in response to increased heart rate and pressure; since the major component of the ionphore-induced work increase is pow (a consequence of decreased peripheral resistance) rather than pressure, the minimal increase in myocardial respiration is understandable. Our original rationalization for administration of X-537A to dogs was the hope that the mobilization of calcium and catecholamines would prove advantageous for cardiac performance.10-12We have recently surveyed a series of other carboxylic ionophores and were startled to observe that they duplicated most of the cardiovascular properties of X-537A; even those ionophores are virtually incapable of transporting calcium and catecholamines in vitro (especially 8 five of these have been compared true of monensin and X-206).2' In FIGURE with X-537A in terms of the dose necessary to produce a doubling of left ventricular dP/dt. They are all more potent than X-537A; e.g., only one seventh the dose of A-204, is required to produce the same effect as 1 mg/kg X-537A. Their potency to increase cardiac output roughly parallels their effects on contractility. The carboxylic ionophores show qualitative differences in response patterns as well. Thus, the dose of monensin that doubles left ventricular dP/dt appears to be more effective than an equivalent inotropic dose of X-537A in increasing coronary blood flow. The maximal responses

CIRDIAC OUlPUl

FIGURE 8. Comparison of the cardiovascular effects of various carboxylic ionophores. Dosages were adjusted to produce comparable effects on cardiac contractility, left ventricular dP/dt for X-537A this represented 1 mg/kg. Total cardiac output (less the coronary flow) was measured by the thermal dilution technique. Coronary flow was monitored by means of an electromagnetic probe placed on the left anterior descending coronary artery.

Pressman & deGuzman:

Ionophores

383

in all three parameters induced by subtoxic doses of monensin seem to be somewhat greater than those produced by equivalent doses of X-537A. It thus emerges that despite our original rationalization, the ability to transport calcium and catecholamines well is not essential for a carboxylic ionophore to exert pharmacologically useful cardiovascular effects. A multitude of physiological effects have been ascribed to the calcium-mobilizing effects of X-537A (and A23 187), e.g., coagulation of platelets?-" release of histamine from mast cells,2s. fertilization of eggs,*'. and a variety of calcium-mediated exocytotic processes." The direct mobilization of catecholamines has been found to release catecholamines from chromaffin granules3oand peripheral adrenergic neurons.31 Accordingly, there might be a distinct advantage in avoiding unwanted side effects by selecting the ionophore with the narrowest cation-selectivity spectrum compatible with cardiovascular activity for further therapeutic development. Earlier reports of at least partial inhibition of the cardiovascular effects of ionophores by catecholamine-depleting or p-blocking agents implicate catecholamines in their action.''' '* The discovery that carboxylic ionophores virtually devoid of in vitro catecholamine transport capacity can have potent cardiovascular activity suggests that whatever the involvement of catecholamines is, it is indirect. These effects are not shared by all types of ionophores since the neutral subclass, e.g., valinomycin and the macrotetrolide actins, are cardiovascular depressants and exceedingly toxic. At this juncture we surmise that the essential property of an ionophore required for useful cardiovascular applications is the ability to induce some reequilibration of alkali ions, i.e., Na' for K exchange, across certain membranes in critical control centers without causing undue changes in membrane potentials (as would the neutral ionophores). Their mechanism of action now seems much more complicated than was previously assumed. We thus see how the concept of ionophores, conceived in the $mitochondrion and matured in the test tube, has reemerged in a new biological context, that of providing a means of controlling biological processes for therapeutic purposes. The stimulation of cardiovascular function by carboxylic ionophores might be expected to find applications in chronic heart failure, reversal of shock of various etiologies, limiting the damage from myocardial ischemia and coronary insufficiency (encompassing angina). Finally it is hoped that these studies will serve as the prototype for a chemotherapeutic tactic of wide applicability: the alteration of membrane permeability by appropriately designed ionophores. Acknowledgments

The technical assistance of Frank Lattanzio, Rick Barrios, and Valeria Gomez is gratefully acknowledged. We are also indebted to Dr. Peter Somani for many stimulating and helpful discussions. References 1. MOORE,C. & B. C. PRESSMAN. 1964. Mechanism of action of valinomycin on mitochondria. Biochem. Biophys. Res. Commun. 15: 562-567.

3 84 2. 3.

8.

9. 10.

11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

Annals New York Academy of Sciences PRESSMAN, B. C. 1965. Induced transport of ions in mitochondria. Proc. Nat. Acad. Sci. U.S.A. 53: 1076-1083. PRESSMAN, B. C., E. J. HARRIS,W. S. JACGER & J. H. JOHNSON.1967. Antibiotic-mediated transport of alkali ions across lipid barriers. Proc. Nat. Acad. Sci. U S A . S8: 1949-1956. PRESSMAN,B. C. 1973. Alkali metal chelators-the ionophores. In Inorganic Biochemistry. G. L. Eichhorn, Ed.: 203-226. Elsevier. New York, N.Y. OVCHINNIKOV, Yu. A., V. T. IVANOV, & A. M. SHKROB.1974. Membrane-active complexones. B.B.A. Library, Vol. 12. Elsevier. New York, N.Y. PRESSMAN, B. C. 1968. Lonorphorous antibiotics as models for biological transport. Fed. Proc. 27: 1283-1288. PRESSMAN,B. C. & D. H. HAYNES. 1969. Ionophorous agents as mobile ion carriers. In the Molecular Basis of Membrane Function. D. C. Tosteson, Ed.: 221-246. Prentic Hall, Inc. Englewood Cliffs, N.J. PRESSMAN, B. C. & M. J. HEEB.1972. Permeability studies on erythrocyte ghosts with ionphorous antibiotics. In Symposium on Molecular Mechanisms of Action of Antibiotics which Affect Membrane Permeability. E. Munoz, F. Garcia-Ferrandiz and D. Vazquez, Eds.: 603-614. Elsevier. New York, N.Y. REED, P. W. & H. A. LARDY.1972. A23187: A divalent cation ionphore. J. Biol. Chem. 247: 6970-6977. PRESSMAN,B. C. 1973. Properties of ionphores with broad range cation selectivity. Fed. Proc. 32: 1698-1703. PRESSMAN,B. C. 1972. Carboxylic ionphores as mobile carriers for divalent ions. In The Role of Membranes in Metabolic Regulation. M. A. Mehlman and R. W. Hanson, Eds.: 149-164. Academic Press. New York, N.Y. PRESSMAN,B. C. & N. T. DE GUZMAN.1974. New ionophores for old organelles. Ann. N.Y. Acad. Sci. 227: 380-391. JOHNSON,S. M., J. HERRIN,S. J. LIU & I. C. PAUL.1970. The crystal and molecular structure of the barium salt of an antibiotic containing a high proportion of oxygen. J. Am. Chem. SOC.92: 4428-4430. DEGANI,H., H. L. FRIEDMAN, G. NAVON& E. M. KOSSOWER.1973. Fluorometric complexing constants and circular dichroism measurements for antibiotic X-537A with univalent and bivalent cations. Chem. Commun.: 431-432. CASWELL,A. H. & B. C. PRESSMAN.1972. Kinetics of transport of divelent cations across sarcoplasmic reticulum vesicles induced by ionophores. Biochem. Biphys. Res. Commun. 49: 292-298. WESTLEY,J. W., E. P. OLIVETO,J. BERGER,R. H. EVANS,JR., R. GLASS,A. & T. WILLIAMS.1973. Chemical transformations of antiSTEMPEL,V. TOOME biotic X-537A. J. Med. Chem. 16: 397-403. HAYNES,D. H. & B. C. PRESSMAN.1974. X-537A: A CaZ+ionophore with a polarity-dependent and complexation-dependent fluorescent signal. J. Membr. Biol. 16: 195-205. SCHAFFER,S. W., B. SAFER,A. SCARPA& J. R. WILLIAMSON. 1974. Mode of action of the calcium ionophores X-537A and A23187 on cardiac contractility. Biochem. Pharmacol. 23: 1609-1617. DEGUZMAN, N. T. & B. C. PRESSMAN.1974. The inotropic effects of the calcium ionophore X-537A in the anesthetized dog. Circulation 4 9 1972-1977. DEGUZMAN, N. T. & B. C. PRESSMAN.1974. Cardiovascular effects of the inotropic ionophore X-537A. Circulation SO Suppl. In: 36. DEGUZMAN, N. T. & B. C. PRESSMAN.1975. The carboxylic ionophores-a new group of inotropic agents. Am. J. Cardiol. 35: 131. MASSINI,P. & E. F. LUSCHER.1974. Some effects of ionophores for divalent cations on blood platelets: Comparison with the effects of thrombin. Biochem. Biophys. Acta 372: 109-1 17.

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1974. Effects of the 23. WHITE, J. G., H. R. RAO GUNDU& J. N. GERRARD. inonophore A23187 on blood platelets. Am. J. Pathol. 77: 135-144. M. B. & C. FRASER.1975. Secretion and aggregation of human 24. FEINSTEIN,

platelets induced by calcium ionophores. Inhibition by PGE, and dibutyryl cyclic AMP. J. Gen. Physiol. In press. 25. FORMAN, J. C., J. L. MONGAR & B. D. GOMPERTS. 1973. Calcium ionophores and movement of calcium ions following the physiological stimulus to the secretory process. Nature (London) 245: 249-251. 26. COCHRANE, D. E. & W. W. DOUGLAS. 1974. Calcium-induced extrusion of secretory granules (exocytosis) in mast cells exposed to 48/80 or the ionophores A23187 and X-537A. Proc. Nat. Acad. Sci. U.S.A. 71: 408-412. 1974. R. A., D. EPEL, E. J. CARROLL, JR. & R. YANAGIMACHI. 27. STEINHARDT, Is calcium ionophore a universal activator for unfertilized eggs? Nature 252: 4143. 28. CHAMBERS, E. L., B.

c. PRESSMAN,& BIRGIT ROSE. 1-974. The activation of sea urchine eggs by the divalent ionophores A23187 and X-537A. Biochem. Biophys. Res. Commun. 6 0 226-132. 29. DOUGLAS, W. W. Exocytosis and the exocytosis-vesiculation sequence: with special reference to neurohypophysis, chromaffin and mast cells, calcium and calcium ionophores. I n Secretory Mechanisms of Exocrine Glands. N. A. Thorn and 0. H. Petersen, Ed.: 116-136. Munksgaard. Copenhagen, Denmark. R. G. & A. SCARPA.1974. Catecholamine equilibration gradients 30. JOHNSON, of isolated chromaffin vesicles induced by the ionophore X-537A. FEBS Lett. 47: 117-121.

THOA,N. B., J. L. COSTA,J. Moss & I. J. KOPIN.1974. Mechanism of release of norepinephrine from peripheral adrenergic neurones by the calcium ionophores X-537A and A23187. Life Sci. 14: 1705-1719. 32. SCHWARTZ, A., R. M. LEWIS,H. G. HANLEY, R. G. MUNSON,F. D. DIAL& M. V. RAY. 1974. Hemodynamic and biochemical effects of a new positive inotropic agent antibiotic R02-2985. Circulation Res. 34: 102-1 11.

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Discussion DR. GREEN: A very intriguing question is posed by these remarkable studies, and it has to do with the mechanism of entry of calcium into the cardiac cell. Presumably there are active processes physiologically involved in the movements of calcium; that is, calcium just doesn’t flow passively across the cell membrane. And yet, these remarkable effects are presumably achieved by a passive mechanism. It is hard to believe that these ionophores are acting in any way other than in a passive capacity. How can you combine both active and passive processes in the regulation of cardiac function? DR. PRESSMAN: Cardiology is a rather arcane field, although not quite as much as cardiologists would have us believe. There is a well-known property of cardiac cells: They seem to have inherently a calcium for sodium exchange system. So if we bring sodium into a cell, the increased sodium is now capable of driving an uptake of calcium. Now, since the contractility of the heart is improving, ultimately somewhere at the bottom there has got to be an increase in the amount of calcium that can bs delivered to the myofibril during contraction. But it now appears not to be getting there directly, as we

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originally thought, by the ionophore carrying the calcium in. However, it may turn out that the movement of calcium is secondary to the movement of sodium. The curious thing is that we got the right results, but for the wrong reason: that is, the calcium is getting in indirectly and not directly. All of these ionophores move sodium. There is a gradient of sodium across the cell that is greater on the outside than on the inside. So if we induce a passive leak of sodium into the cell, we will raise the intracellular sodium level, which can then cause an increase passively in the amount of calcium within the cell. DR.YUMAN:It is interesting that X-537A, either in your studies or in Schwartz’s, seems to have rather prolonged effects. If X-537A were continually releasing catecholamines, the effects certainly wouldn’t be that long-lasting. It is intriguing that X-537A causes some effects that are similar to those of dopamine; at least these effects are seen on the renal blood flow and the cardiovascular system, aren’t they? DR.PRESSMAN: Yes, but dopamine would give rise to a response like that of the other catecholamine-like substances. It would stimulate the myocardial oxygen consumption. Ionophores have some effects that are similar to those of dopamine, and resemble dopamine more than they do the other catecholamines. Some differences still remain, however.

Biological applications of ionophores: theory and practice.

PARTVIII. PERMEATION MECHANISM ACROSS BIOLOGICAL MEMBRANES: I1 BIOLOGICAL APPLICATIONS OF IONOPHORES: THEORY AND PRACTICE* Berton C. Pressman and Nor...
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