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Action of 1,10-phenanthroline transition metal chelates and their constituents on the rat isolated diaphragm preparation
Chem.-Biol. Interactions, @Eisevier/North-Holland
289
18 (1977) 289-294 Scientific Publishers, Ltd.
ACTION OF 1,lOPHENANTHROLINE TRANSITION METAL CHELATES AND THEIR CONSTITUENTS ON THE RAT ISOLATED DIAPHRAGM PREPARATION
P.G. FARNWORTH*,
A. SHULMAN*,**
and A.T. CASEY*
Unit of Medical Chemistry and Department Melbourne, Parkville, 3052 (Australia)
of
Inorganic
Chemistry,
University
of
(Received March 21st, 1977) (Accepted June 14th, 1977)
SUMMARY
The acti0n.s of two related series of fully coordinated, divalent l,lOphenanthroline transition metal chelates have been investigated on the rat isolated diaphragm muscle-phrenic nerve preparation and, where possible, compared with those of their constituent metal ions and ligands. Each member of both series of chelates produced blockade of neuromuscular transmission, although mechanistically not of a uniform type, and several elicited varying degrees of muscle contracture. The kinetic reactivity of the metal chelate appeared to be an important factor determining the nature of the biological response and profound differences in response were observed between labile and inert chelates differing in some cases by only one electron in the 3d shell.
INTRODUCTION
The type of response produced by fully co-ordinated, divalent l,lOphenanthroline transition metal chelates on the guinea-pig isolated ileum [l] and atrium [2] depends on the nature of the constituent metal ion and ligand. Differences in response most likely reflect the differential formation or effect of an active species which may be the intact chelate cation, the liberated metal ion or ligand or a ternary complex formed with components of the biological tissue [3]. The present report extends earlier studies on the action of such chelates and their constituents on cholinergic nicotinic transmission in the rat isolated diaphragm muscle-phrenic nerve preparation [ 3,4] .
*Present address: School of Chemistry, University of Melbourne, Parkvihe, 3052, Australia. **To whom enquiries should be addressed. Abbreviations: P, l,lO-phenanthroline; TMP, 3,4,7,8-tetramethyl-1,lOphenanthroline
290 MATERIALS
AND METHODS
The test compounds were the bis Cu(II), Cd(II), Zn(I1) and Mn(I1) and the tris Co(II), Fe(II), Ni(I1) and Ru(I1) chelates of l,lO-phenanthroline (P) and 3,4,7,8-tetramethyl-l,lO-phenanthroline (TMP). The Fe(R) and Ni(I1) chelates were tested as sulphates, the Ru(I1) compounds as dichlorides and the remainder as diacetates; the Cd(I1) chelate of TMP was too insoluble in the biological suspending medium to study. The actions of the chelates and their corresponding metal ions (as sulphates) and free ligands (as hydrochlorides) were compared at equimolar concentrations (0.5 mM) on hemidiaphragm preparations from male Wistar rats. The force of muscle contraction and contracture were registered by transducer and electronic pen recorder. Experimental details are given in the legend to Fig. 1. RESULTi
AND DISCUSSION
The metal ions had little effect on the response of the diaphragm preparation with the exception of Cu’+ and Cd”, both of which transiently stimulated and then depressed muscle contraction and induced muscle contracture (Table I, Fig. 1). TMP initially produced rapid blockade of the response to indirect stimulation as well as muscle contracture, and subsequently progressive depression of contractions induced by direct muscle stimulation (Table I, Fig. 1). On the other hand, P produced transient stimulation of force followed by slow contracture and depression of the response to both indirect and direct stimulation; substantial recovery after drug washout occurred only in Ptreated preparations (Table I, Fig. 1). It would appear that P and TMP act at both the neuromuscular junction and directly on the diaphragm muscle. In addition, these lipophihc bases with strong chelating capacity appear to have local anaesthetic-like action and to interfere with Ca*+dependent processes [ 2, published observations] . With the exception of the labile Cu(I1) chelate, and to a minor extent the less labile Cd(I1) and Mn(I1) compounds, the P chelates blocked the response to indirect but not to direct stimulation without producing muscle contracture; hence, their actions were generally dissimilar to those of their constituent metal ions and ligand (Table I). In particular, the highly inert Ni(I1) chelate rapidly and reversibly blocked transmission while the corresponding highly labile Cu(I1) compound, although initially producing slower but typical blockade, subsequently showed what appeared to be the combined effects of the chelate cation and its constituents (Fig. 1). Thus, it seems likely that the blocking action of P chelates is mediated predominantly by the doublycharged chelate cation and that the more rapid blockade shown by the Ni(I1) compound reflects its more inert character [ 51. The TMP chelates all produced blockade of the responses to both indirect and direct stimulation as well as muscle contracture; moreover, their onset of action and speed of blockade were slower than those shown by the corre-
L--_
:
5 min
4P
M
M
M
M
M
M
__--
M
t&P
.M
M
M
M
C”?MP
Fig. 1. The effects on the rat isolated diaphragm muscle-phrenic nerve preparation of NiZ+ and Cu”+ (as sulphates), P and TMP (as hydrochlorides), and the chelates derived from them, all at 0.5 mM; the anion of the tris Ni(I1) chelates (Nip and NiTMP) was sulphate and of the bis Cu(I1) chelates (CUP and CuTMP) was diacetate. The diaphragm preparation was suspended in Krebs-Henseleit solution gassed with 95% O,-5% CO, at 37°C. The nerve was stimulated supramaximally (0.5-1.5 V) at a frequency of 0.2 c/set, each of 0.5 msec duration. Following blockade to indirect nerve stimulation by test drug, the diaphragm muscle was stimulated directly (100 V for 30 WC), through a muscle electrode (M); the frequency and duration of the stimulus was unchanged. The recording speed was 5 mm/min, A = drug added; v = drug washed out.
P chelates, and in most cases recovery was negligible 4 h after washout (Table I). Since generally the metal ions showed little activity, while TMP produced rapid and pronounced blockade and contracture, it seems likely that TMP chelate-induced muscle contracture is due substantially to liberated TMP while blockade of transmission represents the junctional action of a charged species (which may be the parent chelate and/or an intermediate or ternary metal-containing complex) and the junctional and/ or local anaesthetic-like action of TMP. The much more pronounced consponding
M
+
+++
*
8.8 * 0.6
+++
+++
*
*e
17.1 * 1.9
10.0 f 1.4
+++
++
7.8 f o.2d
7.5 f 1.7
_c
48.5 + 9.5b *
4.7 f 0.4
Cd
8.1 f 0.6
cu
23.7 f 2.4
Ligand only
++
61.0 f 6.5d
5.7 f 0.4
2.7 f 0.2
Zn
+++
3.7 f o.5d
f
9.9 + 0.7
3.6 * 0.2
Mn
**
+
12.1 f 0.8
10.2 + 1.0
2.1 f 0.1
Ru
*
+
9.3 * 0.5
6.7 f 0.8
1.6 * 0.1
Ni
**e
+++
3.4 f 0.1
7.6 * 0.7
2.1 f 0.1
co
*
+
7.4 * 0.8
6.9 * 0.7
2.1 + 0.1
Fe
aEach result represents the mean of at least five experiments * S.E. b50% recovery did not occur in all preparations. c- = 50% recovery did not occur within 240 min. dSome precipitation may appear with time in the bathing solution at this concentration which elicits a greater response than that obtained at lower concentrations showing no visible precipitate. The reason for the very slow action of the Zn(I1) chelate is unknown. eCould not be studied owing to insolubility (*) or unavailability (**). f- = Negligible blockade within 60 min.
Time for 50% blockade (min)f Muscle contracture
Metal ions
Time for 50% blockade (min) Time for 50% recovery (min)c Muscle contracture
TMP Series
Time for 50% blockade (min) Time for 50% recovery (min) Muscle contracture
P Series
Effect
Chelates of
COMPARATIVE ACTIONS OF DIVALENT TRANSITION METAL CHELATES OF l,lO-PHENANTHROLINE (P) AND 3,4,7,8TETRAMETHYL-l,lO-PHENANTHROLINE (TMP) AND THEIR CONSTITUENT METAL IONS AND LIGANDS (0.5 mM) ON THE RAT ISOLATED DIAPHRAGM MUSCLE-PHRENIC NERVE PREPARATION SUSPENDED IN PHYSIOLOGICAL KREBS-HENSELEIT SOLUTIONa
293 tracture produced by the labile Cu(II), Mn(II), Co(I1) and Zn(I1) chelates than by the more inert Ni(II), Fe(I1) and Ru(I1) compounds (Table I) is consistent with these views. Labile chelates would undergo rapid metal ion or ligand exchange with components of the diaphram preparation whereas such exchange would be slower with more inert compounds [ 51; the responses shown by the Cu(I1) and Ni(I1) chelates of P and TMP (Fig. 1) support this suggestion. Similar correlations between the biological response and the kinetic reactivity of the present series of chelates have been observed in other systems [ 2,6-101 including the guinea-pig isolated ileum where, of the TMP chelates, only highly inert compounds appear to release acetylcholine [l] . Preliminary mechanistic studies on the ability of added Ca*+ (2.5 mM), K+ (6.7 mM) or neostigmine (0.17-0.67 r.lM) to modify the actions of the metal chelates and their constituent ligands and active metal ions (Table I), each at 0.1-0.5 mM, have been carried out on the rat isolated diaphragm preparation. The inert P chelates produced both presynaptic Mg*+-likeand postsynaptic (+)-tubocurarine-like actions to varying degrees (see also refs. 3 and 4); similar (+)-tubocurariform blockade has been reported for related chelates [ll] . The inert TMP chelates had no demonstrable Mg*?like action and showed little non-depolarizing blockade while blockade produced by Cd*‘, and to a lesser extent Cu*‘, was substantially presynaptic in nature (see also ref. 12). On the other hand, the mechanisms whereby the labile P and TMP chelates block neuromuscular transmission are complex and still under investigation. The time course and magnitude of muscle contracture produced by Cu*‘, P and TMP, and their active Cu(I1) and Ni(I1) chelates were influenced in each case, in a graded manner, by varying the Ca*+ and Mg*+ concentrations of the bathing fluid from 0.01 to 5.0 mM and from 8 to 30 mM respectively. This suggests that such contracture may reflect metal ion-, ligand- or chelateinduced, surface-mediated modification of the intracellular Ca*+ concentration or more direct involvement by the active species of the intracellular, Ca*+-dependent contractile mechanism as suggested for chinoform [13] . Thus, the present series of metal chelates show multiple actions on the rat isolated diaphram preparation that vary with the nature of the metal ion and ligand. It is important that a variety of complex biological tissues [2,6-101 including the rat diaphragm preparation, can distinguish readily between labile and inert chelates that differ by only one electron in the 3d shell [5] , for example, labile 3d9 Cu(I1) and inert 3d* Ni(I1) chelates. REFERENCES 1
2
A. Shulman, G.M. Laycock, E.J. Arii;ns and A.R.H. Wigmans, The action of selected metal complexes on receptor systems of isolated organs. Part I. The action of selected ruthenium(I1) phenanthroline chelates on the cholinergic mechanism of the rat intestine and guinea-pig ileum, Eur. J. Pharmacol., 9 (1970) 347. H. Grossman, A. Shulman and C. Bell, Action of fully co-ordinated l,lO-phenanthroline transition metal chelates on the guinea-pig isolated atrium, Experientia, 29 (1973) 1522.
294 3
4 5 6
7
8
9 10
11 12 13
A. Shulman and F.P. Dwyer, Metal chelates in biological systems, in F.P. Dwyer and D.P. Mellor (Eds.), Chelating Agents and Metal Chelates, Academic Press, New York, 1964, pp. 383-439. F.P. Dwyer, E.C. Gyarfas, R.D. Wright and A. Shulman, Effect of inorganic complex ions on transmission at a neuromuscular junction, Nature (Lond.), 179 (1957) 425. F. Basolo and R.G. Pearson, Mechanisms of Inorganic Reactions. A Study of Metal Complexes in Solution, Wiley, New York, 1967, pp. 149-151. H.M. Butler, A. Hurse, E. Thurksy and A. Shulman, Bactericidal action of selected phenanthroline chelates and related compounds, Aust. J. Exp. Biol. Med. Sci., 47 (1969) 541. G. Cade, M. Cohen and A. Shulman, The action of phenanthroline metal chelates and related substances on Erysipelothrix rhusiopathiae and Fusiformis nodosus, Aust. Vet. J., 46 (1970) 387. A. Shulman, G. Cade, L. Dumble and G.M. Laycock, The lethal action of l,lOphenanthroline transition metal chelates and related compounds on dermatophytes and Candida albicans, Arzneim-Forsch., 22 (1972) 154. A. Shulman and D.O. White, Virostatic activity of l,lO-phenanthroline transition metal chelates: a structure-activity analysis, Chem.-Biol. Interact., 6 (1973) 407. A. Shulman, G.M. Laycock and T.R. Bradley, Action of l,lO-phenanthroline transition metal chelates on P388 mouse lymphocytic leukaemic cells, Chem.-Biol Interact., 16 (1977) 89. D.B. Taylor, K.P. Callahan and I. Shaikh, Synthesis of a bifunctional coordination complex of osmium with curariform activity, J. Med. Chem., 18 (1975) 1088. N. Toda, Neuromuscular blocking action of cadmium and manganese in isolated frog striated muscles, Eur. J. Pharmacol, 40 (1976) 67. K. Hiratsuka, T. Mayahara, S. Yamada and T. Kinoshita, Actions of chinoform and oxine on the isolated phrenic nerve-diaphragm preparation of the rat: the accumulation of chinoform by rat diaphragm strip, Jpn. J. Pharmacol., 24 (1974) 23.
289
18 (1977) 289-294 Scientific Publishers, Ltd.
ACTION OF 1,lOPHENANTHROLINE TRANSITION METAL CHELATES AND THEIR CONSTITUENTS ON THE RAT ISOLATED DIAPHRAGM PREPARATION
P.G. FARNWORTH*,
A. SHULMAN*,**
and A.T. CASEY*
Unit of Medical Chemistry and Department Melbourne, Parkville, 3052 (Australia)
of
Inorganic
Chemistry,
University
of
(Received March 21st, 1977) (Accepted June 14th, 1977)
SUMMARY
The acti0n.s of two related series of fully coordinated, divalent l,lOphenanthroline transition metal chelates have been investigated on the rat isolated diaphragm muscle-phrenic nerve preparation and, where possible, compared with those of their constituent metal ions and ligands. Each member of both series of chelates produced blockade of neuromuscular transmission, although mechanistically not of a uniform type, and several elicited varying degrees of muscle contracture. The kinetic reactivity of the metal chelate appeared to be an important factor determining the nature of the biological response and profound differences in response were observed between labile and inert chelates differing in some cases by only one electron in the 3d shell.
INTRODUCTION
The type of response produced by fully co-ordinated, divalent l,lOphenanthroline transition metal chelates on the guinea-pig isolated ileum [l] and atrium [2] depends on the nature of the constituent metal ion and ligand. Differences in response most likely reflect the differential formation or effect of an active species which may be the intact chelate cation, the liberated metal ion or ligand or a ternary complex formed with components of the biological tissue [3]. The present report extends earlier studies on the action of such chelates and their constituents on cholinergic nicotinic transmission in the rat isolated diaphragm muscle-phrenic nerve preparation [ 3,4] .
*Present address: School of Chemistry, University of Melbourne, Parkvihe, 3052, Australia. **To whom enquiries should be addressed. Abbreviations: P, l,lO-phenanthroline; TMP, 3,4,7,8-tetramethyl-1,lOphenanthroline
290 MATERIALS
AND METHODS
The test compounds were the bis Cu(II), Cd(II), Zn(I1) and Mn(I1) and the tris Co(II), Fe(II), Ni(I1) and Ru(I1) chelates of l,lO-phenanthroline (P) and 3,4,7,8-tetramethyl-l,lO-phenanthroline (TMP). The Fe(R) and Ni(I1) chelates were tested as sulphates, the Ru(I1) compounds as dichlorides and the remainder as diacetates; the Cd(I1) chelate of TMP was too insoluble in the biological suspending medium to study. The actions of the chelates and their corresponding metal ions (as sulphates) and free ligands (as hydrochlorides) were compared at equimolar concentrations (0.5 mM) on hemidiaphragm preparations from male Wistar rats. The force of muscle contraction and contracture were registered by transducer and electronic pen recorder. Experimental details are given in the legend to Fig. 1. RESULTi
AND DISCUSSION
The metal ions had little effect on the response of the diaphragm preparation with the exception of Cu’+ and Cd”, both of which transiently stimulated and then depressed muscle contraction and induced muscle contracture (Table I, Fig. 1). TMP initially produced rapid blockade of the response to indirect stimulation as well as muscle contracture, and subsequently progressive depression of contractions induced by direct muscle stimulation (Table I, Fig. 1). On the other hand, P produced transient stimulation of force followed by slow contracture and depression of the response to both indirect and direct stimulation; substantial recovery after drug washout occurred only in Ptreated preparations (Table I, Fig. 1). It would appear that P and TMP act at both the neuromuscular junction and directly on the diaphragm muscle. In addition, these lipophihc bases with strong chelating capacity appear to have local anaesthetic-like action and to interfere with Ca*+dependent processes [ 2, published observations] . With the exception of the labile Cu(I1) chelate, and to a minor extent the less labile Cd(I1) and Mn(I1) compounds, the P chelates blocked the response to indirect but not to direct stimulation without producing muscle contracture; hence, their actions were generally dissimilar to those of their constituent metal ions and ligand (Table I). In particular, the highly inert Ni(I1) chelate rapidly and reversibly blocked transmission while the corresponding highly labile Cu(I1) compound, although initially producing slower but typical blockade, subsequently showed what appeared to be the combined effects of the chelate cation and its constituents (Fig. 1). Thus, it seems likely that the blocking action of P chelates is mediated predominantly by the doublycharged chelate cation and that the more rapid blockade shown by the Ni(I1) compound reflects its more inert character [ 51. The TMP chelates all produced blockade of the responses to both indirect and direct stimulation as well as muscle contracture; moreover, their onset of action and speed of blockade were slower than those shown by the corre-
L--_
:
5 min
4P
M
M
M
M
M
M
__--
M
t&P
.M
M
M
M
C”?MP
Fig. 1. The effects on the rat isolated diaphragm muscle-phrenic nerve preparation of NiZ+ and Cu”+ (as sulphates), P and TMP (as hydrochlorides), and the chelates derived from them, all at 0.5 mM; the anion of the tris Ni(I1) chelates (Nip and NiTMP) was sulphate and of the bis Cu(I1) chelates (CUP and CuTMP) was diacetate. The diaphragm preparation was suspended in Krebs-Henseleit solution gassed with 95% O,-5% CO, at 37°C. The nerve was stimulated supramaximally (0.5-1.5 V) at a frequency of 0.2 c/set, each of 0.5 msec duration. Following blockade to indirect nerve stimulation by test drug, the diaphragm muscle was stimulated directly (100 V for 30 WC), through a muscle electrode (M); the frequency and duration of the stimulus was unchanged. The recording speed was 5 mm/min, A = drug added; v = drug washed out.
P chelates, and in most cases recovery was negligible 4 h after washout (Table I). Since generally the metal ions showed little activity, while TMP produced rapid and pronounced blockade and contracture, it seems likely that TMP chelate-induced muscle contracture is due substantially to liberated TMP while blockade of transmission represents the junctional action of a charged species (which may be the parent chelate and/or an intermediate or ternary metal-containing complex) and the junctional and/ or local anaesthetic-like action of TMP. The much more pronounced consponding
M
+
+++
*
8.8 * 0.6
+++
+++
*
*e
17.1 * 1.9
10.0 f 1.4
+++
++
7.8 f o.2d
7.5 f 1.7
_c
48.5 + 9.5b *
4.7 f 0.4
Cd
8.1 f 0.6
cu
23.7 f 2.4
Ligand only
++
61.0 f 6.5d
5.7 f 0.4
2.7 f 0.2
Zn
+++
3.7 f o.5d
f
9.9 + 0.7
3.6 * 0.2
Mn
**
+
12.1 f 0.8
10.2 + 1.0
2.1 f 0.1
Ru
*
+
9.3 * 0.5
6.7 f 0.8
1.6 * 0.1
Ni
**e
+++
3.4 f 0.1
7.6 * 0.7
2.1 f 0.1
co
*
+
7.4 * 0.8
6.9 * 0.7
2.1 + 0.1
Fe
aEach result represents the mean of at least five experiments * S.E. b50% recovery did not occur in all preparations. c- = 50% recovery did not occur within 240 min. dSome precipitation may appear with time in the bathing solution at this concentration which elicits a greater response than that obtained at lower concentrations showing no visible precipitate. The reason for the very slow action of the Zn(I1) chelate is unknown. eCould not be studied owing to insolubility (*) or unavailability (**). f- = Negligible blockade within 60 min.
Time for 50% blockade (min)f Muscle contracture
Metal ions
Time for 50% blockade (min) Time for 50% recovery (min)c Muscle contracture
TMP Series
Time for 50% blockade (min) Time for 50% recovery (min) Muscle contracture
P Series
Effect
Chelates of
COMPARATIVE ACTIONS OF DIVALENT TRANSITION METAL CHELATES OF l,lO-PHENANTHROLINE (P) AND 3,4,7,8TETRAMETHYL-l,lO-PHENANTHROLINE (TMP) AND THEIR CONSTITUENT METAL IONS AND LIGANDS (0.5 mM) ON THE RAT ISOLATED DIAPHRAGM MUSCLE-PHRENIC NERVE PREPARATION SUSPENDED IN PHYSIOLOGICAL KREBS-HENSELEIT SOLUTIONa
293 tracture produced by the labile Cu(II), Mn(II), Co(I1) and Zn(I1) chelates than by the more inert Ni(II), Fe(I1) and Ru(I1) compounds (Table I) is consistent with these views. Labile chelates would undergo rapid metal ion or ligand exchange with components of the diaphram preparation whereas such exchange would be slower with more inert compounds [ 51; the responses shown by the Cu(I1) and Ni(I1) chelates of P and TMP (Fig. 1) support this suggestion. Similar correlations between the biological response and the kinetic reactivity of the present series of chelates have been observed in other systems [ 2,6-101 including the guinea-pig isolated ileum where, of the TMP chelates, only highly inert compounds appear to release acetylcholine [l] . Preliminary mechanistic studies on the ability of added Ca*+ (2.5 mM), K+ (6.7 mM) or neostigmine (0.17-0.67 r.lM) to modify the actions of the metal chelates and their constituent ligands and active metal ions (Table I), each at 0.1-0.5 mM, have been carried out on the rat isolated diaphragm preparation. The inert P chelates produced both presynaptic Mg*+-likeand postsynaptic (+)-tubocurarine-like actions to varying degrees (see also refs. 3 and 4); similar (+)-tubocurariform blockade has been reported for related chelates [ll] . The inert TMP chelates had no demonstrable Mg*?like action and showed little non-depolarizing blockade while blockade produced by Cd*‘, and to a lesser extent Cu*‘, was substantially presynaptic in nature (see also ref. 12). On the other hand, the mechanisms whereby the labile P and TMP chelates block neuromuscular transmission are complex and still under investigation. The time course and magnitude of muscle contracture produced by Cu*‘, P and TMP, and their active Cu(I1) and Ni(I1) chelates were influenced in each case, in a graded manner, by varying the Ca*+ and Mg*+ concentrations of the bathing fluid from 0.01 to 5.0 mM and from 8 to 30 mM respectively. This suggests that such contracture may reflect metal ion-, ligand- or chelateinduced, surface-mediated modification of the intracellular Ca*+ concentration or more direct involvement by the active species of the intracellular, Ca*+-dependent contractile mechanism as suggested for chinoform [13] . Thus, the present series of metal chelates show multiple actions on the rat isolated diaphram preparation that vary with the nature of the metal ion and ligand. It is important that a variety of complex biological tissues [2,6-101 including the rat diaphragm preparation, can distinguish readily between labile and inert chelates that differ by only one electron in the 3d shell [5] , for example, labile 3d9 Cu(I1) and inert 3d* Ni(I1) chelates. REFERENCES 1
2
A. Shulman, G.M. Laycock, E.J. Arii;ns and A.R.H. Wigmans, The action of selected metal complexes on receptor systems of isolated organs. Part I. The action of selected ruthenium(I1) phenanthroline chelates on the cholinergic mechanism of the rat intestine and guinea-pig ileum, Eur. J. Pharmacol., 9 (1970) 347. H. Grossman, A. Shulman and C. Bell, Action of fully co-ordinated l,lO-phenanthroline transition metal chelates on the guinea-pig isolated atrium, Experientia, 29 (1973) 1522.
294 3
4 5 6
7
8
9 10
11 12 13
A. Shulman and F.P. Dwyer, Metal chelates in biological systems, in F.P. Dwyer and D.P. Mellor (Eds.), Chelating Agents and Metal Chelates, Academic Press, New York, 1964, pp. 383-439. F.P. Dwyer, E.C. Gyarfas, R.D. Wright and A. Shulman, Effect of inorganic complex ions on transmission at a neuromuscular junction, Nature (Lond.), 179 (1957) 425. F. Basolo and R.G. Pearson, Mechanisms of Inorganic Reactions. A Study of Metal Complexes in Solution, Wiley, New York, 1967, pp. 149-151. H.M. Butler, A. Hurse, E. Thurksy and A. Shulman, Bactericidal action of selected phenanthroline chelates and related compounds, Aust. J. Exp. Biol. Med. Sci., 47 (1969) 541. G. Cade, M. Cohen and A. Shulman, The action of phenanthroline metal chelates and related substances on Erysipelothrix rhusiopathiae and Fusiformis nodosus, Aust. Vet. J., 46 (1970) 387. A. Shulman, G. Cade, L. Dumble and G.M. Laycock, The lethal action of l,lOphenanthroline transition metal chelates and related compounds on dermatophytes and Candida albicans, Arzneim-Forsch., 22 (1972) 154. A. Shulman and D.O. White, Virostatic activity of l,lO-phenanthroline transition metal chelates: a structure-activity analysis, Chem.-Biol. Interact., 6 (1973) 407. A. Shulman, G.M. Laycock and T.R. Bradley, Action of l,lO-phenanthroline transition metal chelates on P388 mouse lymphocytic leukaemic cells, Chem.-Biol Interact., 16 (1977) 89. D.B. Taylor, K.P. Callahan and I. Shaikh, Synthesis of a bifunctional coordination complex of osmium with curariform activity, J. Med. Chem., 18 (1975) 1088. N. Toda, Neuromuscular blocking action of cadmium and manganese in isolated frog striated muscles, Eur. J. Pharmacol, 40 (1976) 67. K. Hiratsuka, T. Mayahara, S. Yamada and T. Kinoshita, Actions of chinoform and oxine on the isolated phrenic nerve-diaphragm preparation of the rat: the accumulation of chinoform by rat diaphragm strip, Jpn. J. Pharmacol., 24 (1974) 23.