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THE ACTION OF POTASSIUM ON MUSCLE-PREPARATIONS FROM INVERTEBRATES

BY GEORGE P. WELLS.From the Laboratory of the Marine Biological Association, Plymouth, and the

Department of Physiology and Biochemistry, University College, London.

{Received 8th December 1927.)

(With Twelve Text-figures.)

C O N T E N T S .

IntroductionPart I . The crop of Aplysia . . . .

TechniqueThe action of potassiumThe potassium : calcium ratioThe action of rubidium and caesiumThe action of lithiumThe action of ammoniumDiscussion .

Part I I . The crop of HelixTechnique .The action of potassiumComparison with vertebrate plain muscle

Part I I I . The heart of Mdia .Technique .The action of calcium and sodiumThe action of potassiumComparison with plain muscle .

Summary . . . . . . .References . . . . . . .

INTRODUCTION.

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WHEREAS the action of electrolytes on vertebrate contractile tissues has been ex-tensively studied, observations on invertebrate preparations are few and far between.The outstanding literature in this field is reviewed in a recent paper by Hogben(1925), who has also contributed observations on a number of invertebrate muscles.

In the present communication the observations of Hogben are extended withregard to the action of potassium on molluscan and crustacean muscle. The workfalls into three parts. In the first part, dealing with the crop of the sea hare, theisotonic responses of the preparation to changes in the potassium concentrationare described, the relation between potassium and calcium and the possibility ofsubstituting other bases for potassium are explored, and the mode of action ofpotassium on muscles is discussed. In the second part, dealing with the crop ofthe snail, the responses of molluscan and vertebrate plain muscle to various

The Action of Potassium on Muscle-preparations from Invertebrates 259

potassium concentrations are compared. In the third part, dealing with the heartof the spider crab, these responses are compared with those of a striped musclepreparation under like conditions.

I. THE CROP OF APLYSIA.

TECHNIQUE.

The crop of the sea hare, Aplysia punctata, is admirably suited for an investi-gation of this nature. It is hardy, and will exhibit active movements for hoursin sea-water; further, owing to the peculiar nature of the molluscan circulationits muscle-fibres are readily accessible to bathing fluids. There is no closedcapillary system in Aplysia, the arteries discharging blood into irregular lacunae inthe tissues. These lacunae open freely into the large blood-space in which theoesophagus lies; moreover they have no lining endothelium, so that fluids used tobathe the excised and suspended preparation can rapidly reach the surfaces of themuscle cells without having to pass through any layers of tissue.

For an account of the minute anatomy of the crop see Bottazzi and Enriques(1901). The muscle fibres are plain, fusiform cells, with an axial cylinder ofsarcoplasm containing the nucleus1. Both longitudinal and circular layers arepresent. There is also a nerve-net, but it has not been established that thespontaneous contractions are neurogenic.

In these experiments the crop was removed, tied at both ends, and suspendedin a tube similar to that used by Hogben for the same purpose but of capacity35 c.c. The movements were recorded by means of a light isotonic lever. Sus-pended in this way the crop may show three distinct types of behaviour: (a) rela-tively rapid contractions of frequency about 5 to 10 per minute, (b) slower tone-waves similar to those of the tortoise auricle, and (c) as a result of variation inthe composition of the medium, gross changes in tone upon which the rhythmicphenomena are superposed. Regular tone-waves did not often appear in thesolutions used in this research, and the observations here recorded apply only to(a) and (cf.

As has already been said, the crop will contract actively for hours in sea-water.Hogben (1925) has shown that it is possible to use magnesium-free mixtures, theactual proportions chosen by him being in parts by volume of half molar chlorides:Na 100, K 5 to 10, Ca 10 to 12-5, pH 6-8. The aim of the work here describedwas to elucidate the action on the muscle of monovalent cations; nevertheless forreasons given below experiments in the presence of various proportions of divalentmetals have been performed. I used either calcium 0-012 molar and magnesium0-058 molar (i.e. approximately the concentrations found in sea-water) or mag-nesium-free mixtures whose calcium concentration varied in different experimentsfrom 0-014 t 0 o*O44 molar.

1 A valuable review of the different kinds of muscle-fibre found in gasteropods has beenPublished by Plenk (1924).

2 For a full account of the behaviour of the preparation in sea-water see Bottazzi (1898).17-2

z6o GEORGE P. WELLS

All the chlorides used were of Analytical Reagent standard. In order to excludevariations in osmotic pressure they were made up in solutions isotonic with thesea-water in the Plymouth tanks, i.e. 0-4 molar Ca and Mg chlorides, o-6 molarmonovalent metal chlorides, all mixtures being made from these solutions. Experi-ments were made at varying pH, from 6-8 to 7-8; during any one experiment thepH. was constant to within 0*2. The solutions were buffered by adding one dropof saturated Na2HPO4 per c.c. (i.e. about 8 x 10-4 molar phosphate) and adjustedwith NaOH or HC1. The temperature was recorded but not controlled; as mostof the experiments were performed in a cool cellar in which all solutions werekept, significant variations in temperature did not occur.

THE ACTION OF POTASSIUM.

In a few preliminary experiments the effect on the crop of adding isotonic KC1to sea-water was tried, but in most of the work the following procedure wasadopted. Two stock solutions were prepared by adding to o*6 molar NaCl andKC1 respectively the same amount of calcium and magnesium. By mixing thesestock solutions in various proportions, mixtures of any desired potassium concen-tration could be obtained without varying the divalent ion content; the potassium

B

I 1 1 I I I I II 1 1 I 1 I I I t 1 1 I I I I I I I IFig. 1. Crop of Aplysia: action of potassium. Mg absent. Ca 0-014 M. pHy-2. 16-5° C. A,C,E:o-oi 1 M K; B: K withdrawn; D: 0-053 M K. The time signal marks one minute intervals in allfigures of the Aplysia crop.

is increased at the expense of the sodium. The same method was employed inexperiments, to be described later, on the action of rubidium, caesium, andammonium. It is convenient to describe separately the action of potassium ontone and on the rhythmic movements.

To deal first with tone, if the crop is bathed by a solution containing o-oi MKC1 (i.e. a concentration about equal to that found in sea-water) it responds by


tonic contraction if this solution is changed for one containing no potassium, orcontaining potassium in excess (Fig. 1). In this respect, as will be shown later, thepreparation behaves like vertebrate plain muscle. Both effects are rapidly reversible.

The form of the response either to potassium withdrawal or to potassiumexcess is variable. The lever may rise directly to a level at which it remains con-stant, but often it rises swiftly to a height above its ultimate level, to which itslowly descends. In magnesium-free mixtures of calcium content 0*01.4 molar boththe contraction due to potassium lack and that due to potassium excess may besustained for at least an hour.

The contraction produced by potassium excess has a wider amplitude than thatproduced by potassium withdrawal. If the crop is contracted by potassium lack,addition of a small amount of potassium (e.g. o-oi M) produces rapid relaxation,but addition of large amounts (0-05 M or more) produces further contraction.

Turning now to the automatic movements of the crop, if the preparation isbathed in sea-water the addition of a little KC1 excites both frequency andamplitude. Addition of large quantities (enough to raise the K content to 0-038 Mor higher) has a depressing action; the amplitude falls off, the rhythm becomesirregular, and is finally reversibly inhibited. Similar phenomena may be observedin the artificial media employed in this investigation. Movements of wide amplitudeare supported by mixtures containing o-oi M- K, but concentrations of 0-05 orhigher depress the rhythm very rapidly. (Movements may appear for some timein the presence of 0-05 M K, but in my experience they are never as regular orvigorous as those seen with o*oi M K.) Absence of potassium, like excess, pro-duces an irregular rhythm; the amplitude is very greatly diminished, and movementgenerally ceases altogether in ten minutes or less. These observations harmonisewith the results of Heymans (1923), who found that the heart of Aplysia beatsatisfactorily when perfused with a fluid containing 0*0114 molar potassium saltsand that either lack or excess of potassium arrested the beat.

The plain muscle of Aplysia therefore resembles the great majority of spon-taneously contracting tissues in being excited by potassium up to a certain optimumconcentration, above which the beats are depressed.

THE POTASSIUM: CALCIUM RATIO.

There are several different ways in which potassium can be imagined as actingon a living system. Before attempting to analyse the mechanism by which potassiumaffects tone, a distinction may be drawn between the following two types of action i

(1) Actions which depend on a balance between potassium and calcium. Manyproperties of protoplasm or of its inorganic analogues depend upon the ratio ofmonovalent to divalent metals in the medium; e.g. the degree of dispersion oflecithin sols in NaCI + CaCl2 mixtures (Neuschlosz, 1920) or the duration ofmovement of barnacle larvae in diluted van't Hoff solutions (Loeb, 1915) areunaffected by wide variations in the total electrolyte content, provided that a par-ticular balance between monovalent and divalent cations be maintained. In many

262 G E O R G E P . W E L L S

cases, potassium is a much more powerful calcium antagonist than sodium, sothat in experiments in which a roughly constant concentration of sodium is presentthese properties will be a function of the potassium : calcium ratio and inde-pendent of the absolute concentration of either metal. As an example of this classwe may cite the heart of the frog over a certain limited range of concentrations;either reduction of the potassium to one-quarter or fourfold increase in the calciumproduces 10 per cent, decrease in frequency in about five minutes, while potassiumand calcium may be simultaneously increased threefold without obvious disturbanceof the rhythm (Daly and Clark, 1921)—results which suggest an analogy with thephenomena of salt antagonism in artificial models. If, for example, the excitationprocess occurs at a surface analogous to the oil-soap emulsions of Clowes, potassiumand calcium may be imagined as affecting antagonistically the tendencies to ex-ternalisation of the two phases.

(2) Actions which depend only on the absolute concentration of potassium.Potassium has specific properties which are independent of other substances inthe medium; e.g. its radioactivity, its high ionic mobility, and the formation ofcomplex ions with certain proteins (Rona and Petow, 1923). Physiological effectsdue to these properties will be a function of the concentration of potassium andnot of the ratio between potassium and any other metal. As an example of thisclass we may consider tone in the mammalian uterus. Tate and Clark (1921)showed that calcium excess contracts the uteri of the rabbit and cat but it relaxesthose of the rat and guinea-pig; potassium excess contracts all four uteri. Appa-rently calcium excess affects nerve endings while potassium excess affects themuscle substance. When the two metals are applied simultaneously in excess theirindependent actions are simply added together: in species where each by itselfcontracts the uterus they augment each other's effects, but in species where theyact in opposed senses they tend to counteract each other. Here it is not a questionof K/Ca ratio at all, but simply the summation of two factors which act inde-pendently of each other.

It is of course possible that calcium and potassium could antagonise each othereven when acting independently on different parts of the cell. If, for example,potassium produced some effect by penetrating into the cell interior, calcium mightprevent or delay this action by diminishing the permeability of the cell membraneto potassium. Moreover, in the uteri of the rat and guinea-pig, as already pointedout, although calcium and potassium act on spatially distinct seats, their actionsare in opposite senses and tend to neutralise each other. It follows that an empiri-cally demonstrated antagonism between the two metals on an isolated organ doesnot necessarily imply an interpretation in terms of their antagonistic action ontest-tube colloidal systems. If, however, it be found that potassium and calciumare not antagonists in respect of any vital process but that they affect it inthe same sense, then it is at least clear that their mode of action is not of thistype: artificial models on which the two metals act antagonistically may be excluded.

The relation between the action of potassium and the calcium concentrationin the crop of Aplysia was investigated in two ways: by experimenting in the


presence of various calcium concentrations, and by attempting to reverse thesymptoms of potassium lack by increasing the Na/Ca ratio. Evidently if thephenomena are controlled by the ratio of K to Ca more potassium will be requiredto produce any given effect if the calcium concentration is high than if it is low,and the results of potassium deficiency will be reversible by decreasing the calciumto a corresponding extent.

To deal first with experiments in which the calcium concentration was constant,the relaxing action on tone of o-oi M K and the contracting action of 0-05 M Khave been observed in the presence of 0-012MCa + 0-058MMg and in mag-nesium-free mixtures containing from 0-014 t o 0-044 M Ca. Moreover in a fewexperiments they have been seen when divalent metals were absent altogetherfrom the bathing fluid. When the crop is immersed in isotonic NaCl it firstcontracts fully and then partially relaxes; rapid movements of small amplitudemay be seen, suggestive of the twitchings of a frog's sartorius in NaCl rather thanof the normal activity of the crop. It is now very excitable, responding by con-tractions of short duration to the mechanical stimulus of changing the medium.Under these conditions addition of 0-012 M Kproduces a perceptible loss of tone, observableafter the brief contraction due to the me-chanical disturbance has passed off, while0-055 M K or higher concentrations producethe usual tonic contraction (Fig. 2). Thesefacts show clearly that the action of potassiumon tone depends on its absolute concentrationand not on the ratio of potassium to calciumin the medium.

Confirmation of this conclusion is ob-tained from experiments in which the calciumconcentration was varied. Hogben, workingwith a mixture containing NaCl 0-42 M, pig. 2. CroP of Aplyda: action of potas-KC1 O-O2I My CaCl2 0-053 M, P^- "̂̂ » found sium in the absence of calcium or magne-'.1 . . . , j 1 r 1 • J j * : sium. *H 7*2. A: K absent, o-6oMNaCl;that withdrawal of calcium produced tonic B £>. O.OI2 M K C ^ O.^ M NaCl;contraction followed by gradual relaxation, C: o-17 MKC1,0-43 MNaCl. As the fallingwhiif " thp nrlmarv pflFprt of inrreasino- the line after £> was too faint to be photographedwnne tne primary ettect or increasing me i t h a s b e e n t o u c h e d in with Chinese White,amount of calcium is depressant." I haveobtained similar results in the absence of potassium. When the crop is contracted bypotassium lack, increasing the Na/Ca ratio produces further contraction and de-creasing the ratio produces loss of tone; the twitching movements often seen underthese conditions are accelerated by increase and retarded by decrease in the ratio.It may, however, be noted that although moderate increase in the calcium concen-tration (to O ' iMCa or more) depresses tone, very great excess (0-4 M CaClg byitself) calls forth a smooth tonic contraction.

The action of sodium and calcium is illustrated by the following experiment.At the beginning the crop was bathed by Na 0-58 M, K o-on M, Ca 0-014 M,

• 1 1 1 1 1 1 1 l_J_l I I I I I—t I I I I

264 GEORGE P. WELLS

pH 7-1, 170 C. Solutions lacking potassium and lacking both potassium andcalcium were applied in the following order, exposure to each solution being forabout 7 minutes:

(i) Na + Ca + K. Normal rhythmic activity.(ii) Na + Ca. Tonic contraction; rhythm inhibited.(iii) Na only. Further contraction; rapid beats of small amplitude appear

(about 7 per min.).(iv) Na + Ca. Partial relaxation; the beats are slowed (about 3 per min.).(v) Na + Ca + K. Complete relaxation; appearance of rhythmic contractions

of normal amplitude and frequency (about 4 per min.).

Evidently, since withdrawal of potassium and of calcium both augment tone, thetwo metals are acting in the same sense—a further proof that the tone depends onthe absolute concentrations of K and Ca and not on the K/Ca ratio.

A similar conclusion has been reached by Jendrassik and Annau (1925) formammalian plain muscle; " Fur den Darm ist nicht nur der K/Ca quotient wichtig,sondern vielmehr die absolute Konzentration der einzelnen Ionen."

It may be pointed out that potassium and calcium apparently act as antagonistson the rhythm, calcium slowing and potassium exciting the spontaneous move-ments. In this respect the movements resemble the twitchings of amphibianvoluntary muscle in calcium-free solutions (Mines, 1908) or the heart-beat of thefrog: but it should be remembered that even in the latter case potassium : calciumantagonism only occurs over a limited range of concentrations—for althoughcalcium can antagonise the depressing action of 0-008 M KCl it cannot antagoniseo-oi2 M KCl (Clark, 1926)—so that even here the absolute concentrations may bemore important than the K/Ca ratio.

THE ACTION OF RUBIDIUM AND CAESIUM.

The possibility of substituting other cations for potassium was investigated.Hogben (1925) has shown that if the crop is bathed by a fluid containing

already 0-049 M KCl, further addition of 0-0062 M KCl, 0-0062 M RbCl, or0-025 M CsCl all produce tonic contraction of about the same magnitude. Thatis to say, the contracting action of potassium excess is paralleled by rubidium andcaesium. I have carried out experiments to test whether the relaxing action ofsmall quantities of potassium is also exerted by these metals, and whether theycan restore the rhythm of a preparation arrested by potassium lack. These pointswere investigated in magnesium-free mixtures containing 0-014 molar calcium.

Rubidium acts excellently as a potassium substitute. When the crop is con-tracted by potassium lack, addition of 0-0058 molar rubidium relaxes the pre-paration and brings about a prompt return of the rhythmic movements (Fig. 3).Concentrations of 0-0029, o-on or 0-022 molar will also restore rhythmic activity,but the relaxation is best seen with 0-0058 molar rubidium. My impression is thatrubidium is between one and two times as active as potassium.


Owing to the small amount of caesium chloride at my disposal it was onlypossible to perform nine experiments on three crops. It was found that caesiumcan restore the rhythmic activity and bring about relaxation of the preparationshortened by potassium lack. Withdrawal of caesium produces a contraction asrapid and vigorous as that produced by withdrawing potassium, but it was observedin all the experiments performed that when caesium is added to the contractedcrop it does not relax as rapidly as it will on adding potassium. Addition of caesiumappears to have a stimulating effect which obscures its potassium-like action; thusin Fig. 4, on adding caesium there is a swift relaxation similar to that producedby potassium, followed at once by a contraction of equal extent which slowlypasses off. Occasionally a similar contraction was observed on adding smallquantities of rubidium. In respect of the action of caesium the crop of Aplysia

I > 1 / 1 1 1 / 1 ) 1 1 1 1 1 1 1 1 i 1 1 > i f .

Fig. 3. Crop of Aplysia: action of rubi-dium. Mg absent. Ca 0-014 M. i>H 7-2.16-7° C. A, E: o-on M K; B: K with-drawn; C: K absent, 0-0058 M Rb;

D: Rb withdrawn.

1 1 1 r 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i r 1 J 1 1 1 1

Fig. 4. Crop of Aplysia: action of caesium.Mg absent. Ca 0-014 Af. P& 7'2. i7'3° C.Same crop as Fig. 2. A, C: o-on M K;B, D: K withdrawn; 2?: K absent, 0-022 M Cs;

F: Cs withdrawn.

resembles the gut of the rabbit, for in the latter preparation caesium has apotassium-like action complicated by a tendency to produce contractures evenin low concentrations (Jendrassik and Annau, 1925).

I used concentrations of 0-0058, o-on, 0*022, 0-033, °'°43> a n d o*c>53 molarcaesium. That which most nearly resembled o-on molar potassium was 0-022molar. It may be inferred that caesium is about half as active as potassium.

We may say then that in the absence of potassium 0-0058 molar rubidium or0-022 molar caesium exert a relaxing action on tone and an exciting action on therhythm similar to those of o-on molar potassium. Now in Hogben's experimentquoted above, the potassium concentration was already high, so that furtheraddition of potassium produced not relaxation but contraction. Under thesecircumstances he found that addition of 0-0062 molar rubidium or 0-025 m o k rcaesium also produced contraction. The effect of adding rubidium or caesiumtherefore depends on the amount of potassium already present, and is always thesame as that of adding equivalent quantities of potassium.

266 GEORGE P. WELLS

THE ACTION OF LITHIUM.

In a small number of experiments the action of lithium on the preparationshortened by potassium lack was tried. Substitution of lithium for one-tenth orone-third of the sodium, without alteration in the calcium concentration (0*014 M),caused loss of tone, and reversing this substitution caused contraction; but lithiumdid not bring about any revival of the rhythmic activity of the preparation. Substi-tution of lithium for the whole of the sodium caused swift, complete relaxation,whereas substitution of potassium for the sodium would, of course, cause toniccontraction. It appears that lithium in any concentration exerts a depressingaction on tone, and is thereforedifferent from potassium, which de-presses in low concentrations andcontracts in high.

The response to lithium issomewhat different when potassiumis present. Under these conditions,if all the sodium is replaced bylithium the crop first relaxes ab-ruptly, then contracts, then slowlyrelaxes again; on making the reversechange an opposite cycle of pheno-mena appears (Fig. 5). These re-sponses are quite unlike anythingseen with potassium, rubidium or ' •" l ' LJ~^ r r , • 1 . . . i 1 , 1 1 t 1 • . 1caesium; from the point of view of Fig- 5 ' ,Cro,p °* Aply™: action,of

o !l th ium m t h e pre"' r sence of potassium. pH yz. 16-5 C.comparative physiology they are A, C:Nao-s7M+Ko-on M+Ca. 0-014M;interesting in that a precisely similar -B: Li 0-57 M substituted for the sodium.series of changes appears in the rabbit's intestine when lithium is substituted forone-fifth c** one-half of the sodium in a Tyrode solution (see Jendrassik andAnnau, 1925, Fig. 6).

THE ACTION OF AMMONIUM.

It is well known that K+, Rb+ and Cs+ form a group of cations, related bothin their physical and physiological properties. On the other hand, the ammoniumion, although related to these metals in its physical properties, has not receivedattention as a potassium substitute, at least in recent discussion. In its physio-logical action ammonium chloride varies between two modes of behaviour; it maybehave like potassium chloride, or it may exert specific actions of its own. Toillustrate this point, the changes in length of muscles produced by monovalentcations may be considered.

In some muscles excess of ammonium chloride, like potassium chloride, causescontracture; thus Zoethout (1903) recorded contracture of frog's sartorii immersed

The Action of Potassium on Muscle-preparations from Invertebrates 2.67

in isotonic KC1, RbCl, CsCl or NH4C1 but not in NaCl or LiCl, and similarphenomena were found by Crozier (1916) in the cloacal musculature of holo-thurians. Gellhorn (1926), working on plain muscle from calves' arteries, foundthat addition of KC1, RbCl, CsCl or NH4C1 to Tyrode's solution caused con-tracture, while NaCl had no effect and LiCl caused loss of tone. The possibilitythat these mechanical effects are causally connected with electrical changes isindicated by the results of Hober (1905), who showed, by immersing one-half ofa frog's sartorius in pure alkali metal chloride and the other half in Ringer, thatKC1, RbCl, CsCl or NH4C1 make the immersed half negative to the half in Ringer,while NaCl has no effect and LiCl makes it positive. Contrasting with the fore-going examples of ammonium-potassium parallelism are the results of Fienga(1910), who found, on the oesophagus of the fowl, that potassium excess causedthe usual tonic contraction, while ammonium chloride added to Ringer had ahighly characteristic effect; it caused loss of tone with occasional isolated con-tractions of wide amplitude and brief duration. The writer has observed a verysimilar response to ammonium excess in the intestine of the frog. There areapparently two effects which ammonium excess can produce; on some preparationsit causes contracture like potassium excess, while in others it has a characteristicdepressing action.

The fact that ammonium salts may be potassium-like or may show specificactions was noted by Mathews (1907), who pointed out that an ammonium chloridesolution contains several potential effective agents. In the first place, the NH4C1molecule may dissociate into NH4

+ + Cl~, and it is reasonable to suppose that thepotassium-like actions of ammonium chloride are due to the potassium-likeammonium ion. But in addition the solution will contain a certain number ofuncharged molecules of NH4OH and NH3. Mathews suggests that the specificactions of ammonium salts are due to these uncharged groupings; and recentlythe work of Jacobs (1923) and others has shown that ammonia can penetrate intocells in an uncharged form and raise the alkalinity of the cell interior. It is probabletherefore that in an ammonium chloride solution the potassium-like action of theammonium ion is complicated by a simultaneous penetrating action of neutralammonia. The oesophagus of the fowl or the intestine of the frog may be regardedas being particularly sensitive to the latter agent, so that the former is masked;on the other hand, in preparations resistant to ammonia it should be possible todemonstrate ammonium-potassium parallelism.

In view of the theoretical interest of this question, the action of ammoniumchloride on the Aplysia crop was investigated in a large number of experiments.It was found that the crop is not depressed by ammonium—regular rhythmicmovements may be shown in the presence of 0-05 M NH4 for at least 20 minutes—and in exposures of this duration the action of ammonium closely parallels thatof potassium. It may be pointed out that the cases of ammonium-potassiumparallelism quoted above are confined to excesses of the two bases; so far as thewriter is aware there has hitherto been no demonstration that the effect of potassiumlack in plain muscle can be compensated by addition of an ammonium salt.

268 GEORGE P. WELLS

The action of low ammonium concentrations on the Aplysia crop is illustratedin Fig. 6; when the crop has been contracted by potassium withdrawal, additionof 0-041 M ammonium produces both prompt relaxationand return of the rhythmic movements. High concentra-tions of the two cations are also similar, for if theammonium concentration is increased to an excessivevalue tonic contraction and depression of the rhythm re-sult. On comparing Figs. 1 and 6 it will be seen that asolution containing 0-041 M ammonium corresponds toone containing o-on Mpotassium; withdrawal or excessof ammonium produces effects like those of withdrawalor excess of potassium.

Potassium is about five times as active as ammonium,but this figure is only approximate; owing to individual .1 I I 1 f 1 I 1 1 1 f .1variations in appearance of the responses, the method Fis- «• Crop of Aplysia: ao

r . r 1 1 r tion of ammonium. Me ab-of comparmg experiments performed on a number of s e n t C a O.OI4 M ^ H T2

different crops cannot give precise quantitative results. 16-5° C.As with rubidium and caesium, the actions of am- ^ : ? r O I ? ^ l K ;

. . . . . . c B: K withdrawn;monium and potassium are additive. Addition or am- c. K absent, 0-041MNH4;monium to a solution containing already at least o-on M D: K absent> 0-16 M NH4.potassium produces effects exactly like those produced by increasing the potassium—i.e. tonic contraction and, in sufficient quantity, inhibition of the rhythm. More-over the speeds with which ammonium and potassium exert their effects on thetissue are about equal. These facts show that the mechanism by which ammoniumchloride acts is identical with that of potassium chloride.

The potassium-like action of ammonium has been observed in the presenceand absence of magnesium, and with calcium concentrations from 0-014 to 0-044molar.

DISCUSSION.The results recorded above may be summarised as follows: in a mixture of

NaCl 0-58 Mand CaCl2 0-014 Mthe crop is contracted and the rhythm paralysed;substitution of potassium, rubidium, caesium or ammonium for a small fractionof the sodium causes relaxation and rhythmic activity, but substitution of any ofthese bases for a large fraction of the sodium causes further contraction andincreases the paralysis (Fig. 7). Substitution of lithium for part or all of thesodium does not produce similar effects. The amount of potassium required toproduce any given effect on tone is independent of the calcium concentration.

In the light of ammonium-potassium parallelism two rival hypotheses of themode of action of potassium may be discussed: (1) that it acts in virtue of itsradioactivity, and (2) that it acts in virtue of its high ionic mobility. Since theaction of potassium, at least on tone, is independent of alkali: calcium antagonisms,we may leave aside hypotheses based on analogy with protein or lipoid suspensoids,or with emulsions of oil and soap solution.


To deal first with Zwaardemaker's hypothesis: Clark (1922) has already pointedout that the quantitative relationship between the actions of Rb, Cs and K oncontractile tissues makes it improbable that the action of K is due to the beta rayswhich it emits. Whereas Rb is at least seven times as radioactive as K and Cs hasno detectable radioactivity, Rb is only about twice as active and Cs is half as activeas K on the kitten's uterus—quantitative relationships which have been confirmedin the present investigation of molluscan muscle. Further evidence againstZwaardemaker's view is afforded by the potassium-like action of ammonium.Loeb (1921) has already pointed out that on the eggs of Fundulus ammonium andpotassium chlorides act similarly and that since the NH4 group does not emitbeta rays, the action of K cannot in this case be due to its radioactivity. So far asthe writer is aware, a detailed parallelism involving both rhythm and tone has not

A B

* 1 1 1 1 1 ! ( ( 1 1 III 111 I H I L 1 1 1 1 1 1 I l l l l f l l l l l !Fig. 7. Crop of Aplysia: action of ammonium on a preparation contracted by potassium lack.Mg absent. Ca 0-014 M. pH 7-4. K absent.

I. A, C: NH4 absent; B: 0-053 M N H 4 .II. A, C: NH4 absent; B: 027 M NH4.

hitherto been worked out for a muscular tissue; the observations recorded in thepresent paper show however that the same argument applies to plain muscle. Itwould appear from these considerations that the hypothesis is untenable.

On the other hand, potassium-ammonium parallelism is distinctly favourableto the view put forward by Mines (1912) that Na and K act "by passing fromone region to another, carrying their charges and so setting up differences ofpotential between the various parts." As Mines has pointed out, the ability of anion so to do will depend primarily on its mobility, and since the mobility of NH4

+

differs from that of K+ by only 1 per cent., a similarity between the physiologicalactions of the two ions is to be expected. The following figures of migrationvelocities are taken from Michaelis (1926, p. 176):

Li 33-44 R b 67*5Na 43-55 Cs 68K 64-67 NH4 64

It will be seen that K+, Rb+, Cs+ and NH4+, which are related in their physio-logical action,- also form a group of rapidly moving cations. It is not necessary to


assume that the ions penetrate at all deeply into the cell; they could act by alteringpotential conditions at the surface membrane, and the recent work of Clark (1926)has made it probable that this is their seat of action.

The great drawback to this view is that the difference in mobility betweenNa+ and K+ is not very considerable; it could be imagined is underlying a quanti-tative difference between the two metals, but it is hardly sufficient to account forthe very striking contrast in physiological behaviour that is found between them.However, this difficulty has been removed by the recent work of Michaelis andhis co-workers. Michaelis (1925) has explored the properties of the dried collodionmembrane, which resembles biological membranes in many respects. He hasshown that this membrane is permeable to monovalent cations, which penetrateit at speeds depending on their mobilities in free aqueous solution, but the differencebetween any two cations is vastly greater in the membrane than in free solution.This effect is due to the pores in the membrane being so small that the zone oforiented water molecules surrounding the cations includes molecules adhering tothe walls of the passages; thus the normal retarding effect of hydration is greatlyincreased. The magnitude of this retardation is such that whereas in free solutionK+ is 1-5 times as mobile as Na+, in the pores of the membrane it is 6-9 timesas mobile. Relative to the permeability of the membrane for potassium its per-meability for sodium is insignificant. Michaelis and Fujita (1925) found that thepotential developed between various mixtures of NaCl + KCl depended almostentirely on the K concentration, the effect of the Na being negligible; that is tosay, in the small pores differences in mobility are so exaggerated that in NaCl + KClmixtures K has an apparently specific action in determining the potential.

It is therefore clear that if Na+ and K+ can be imagined as moving in verysmall canals, the difference in mobility would be sufficiently increased to accountfor an apparent specificity in the action of the latter metal. Recent work (Pantin,1926) has indicated that the surface membrane is a network of protein moleculesheld together by their aliphatic groups, on to which a layer of lipoid molecules isadsorbed. It is reasonable to suppose that in such a network the passages wouldbe small enough for the walls to exert a considerable attraction on the waterenvelopes of the ions, in which case, as in the dried collodion membrane, theaction of the heavily hydrated sodium ion would be insignificant compared withthat of potassium. If K+ acts by penetrating into the cell membrane and settingup an electrical double layer, its action would be paralleled by the rapidly movingRb+, Cs+ and NH4+, while the slower Na+ and Li+ would be ineffective. Moreovercalcium could only influence the process indirectly, e.g. by affecting the propertiesof the walls of the canals.

These suggestions are intended only as a tentative basis for further work. Ifpotassium acts by setting up potential differences at the surfaces of cells, it wouldbe premature to discuss further its polarising action in the absence of potentialmeasurements in various mixtures of NaCl + KCl + CaCl2. It may however benoted in confirmation: (1) that Jendrassik and Annau (1925) working on the mam-malian intestine, found large alterations of the external sodium concentration to have


effects not unlike those of small alterations in the same sense of the potassium con-centration, i.e. the difference between the two metals is not qualitative but a verygreat quantitative one; and (2) that the dried collodion membrane resembles thecell membrane in its permeability to NH3 and NH4+; the ammonium ion pene-trates the former at a speed nearly equal to that of potassium, but ammoniamolecules differ from other non-electrolytes in having a method of rapid pene-tration, at present unexplained, which is out of all proportion to their mobility infree aqueous solution (Fujita, 1925).

II. THE CROP OF HELIX.

It is difficult to compare quantitatively the action of potassium on the Aplysiacrop with its action on vertebrate preparations because of the great difference inabsolute concentrations of electrolytes in the bloods. The following experimentson snails were undertaken in order to compare mammalian and amphibian plainmuscle with that of molluscs whose blood has the same osmotic pressure. Twospecies of snails were used: Helix pomatia, the edible snail, and Helix aspersa, thecommon garden snail. The facts to be described are true of both species.

TECHNIQUE.

The histology of the crop, which is in all essentials similar to that of Aplysia,has been described by von Haffner (1924). The procedure of isolating the pre-paration and recording its changes in length was the same as that adopted forAplysia, except that the salivary glands, which clothe a large part of the Helixcrop, were dissected away in order to make its whole surface immediately accessibleto bathing fluids. The movements shown are on the whole similar in both crops,but the rapid rhythmical contractions are better seen in Aplysia', on the other hand,the crop of Helix shows spontaneous changes in tone of considerable amplitudewhich may assume a slow rhythmic character.

The blood of Helix is roughly isotonic with frog Ringer. I used one-eighthmolar chlorides of sodium, potassium, and calcium, all mixtures being bufferedwith sodium phosphate and adjusted to pH 7-6 ± 0-2. Lovatt Evans (1912) andHogben (1925) have shown that the presence of magnesium is unnecessary for theheart of Helix pomatia, which resembles that of air-breathing vertebrates in thisrespect. It may be inferred that the crop also is suited by magnesium-free solu-tions. As regards calcium concentration, Hogben used for the heart, in parts byvolume of one-eighth molar chlorides, Na 100, K 2 to 8, Ca 12; on the otherhand, Lovatt Evans used Ringer, which may be approximated tobyNa 100, K 1-7,Ca i-o. Evidently since the heart will beat in either of these solutions it is tolerantof a wide range of calcium concentrations. In the present investigation concen-trations were used varying in different experiments from i-o to 12 c.c. CaClg per100 c.c. monovalent chlorides—i.e. from 0*0012 to 0*013 molar.

272 GEORGE P. WELLS


It was invariably found that if the crop is bathed with a solution containingNa and K in the ratio ioo : 2 it will shorten if the potassium is either withdrawnor greatly increased. The contractions are rapidly reversible. The responses arewell seen in the presence of calcium concentrations comparable to those generallyemployed for vertebrate muscle, but they also occur with the highest calciumconcentrations used in this research.

As with Aplysia, if the crop is contracted by a potassium-free solution, theaddition of a little K causes relaxation, but the addition of high concentrationscauses further contraction (Fig. 8).

Although the primary object of these experiments was to investigate changesin tone, the fact, clearly illustrated in Fig. 8 II, that the rhythmic contractionsare reversibly depressed by potassium lack is of interest. In this respect the crop

n

Fig. 8. Crop of Helix pomatia: action of potassium. Two different crops. pH: I, 7-8; II, 7-4.Solutions in parts by volume of one-eighth molar chlorides.

I. A, C: Na 100, Ca 3, K o; B : Na 100, Ca 3, K 2 (= 0-0024 M K ) .II . A, C: Na 100, Ca 3, K o; B: Na 100, Ca 3, K 10 (=o-on M K).

Base-line interrupted once every five minutes. Compare Fig. 7.

differs from the heart, which was found by Lovatt Evans and by Hogben to beatfor a considerable time in the absence of potassium. It may be pointed out thatthere is also a histological distinction; the heart muscle of Helix shows simplestriation.

COMPARISON WITH VERTEBRATE PLAIN MUSCLE.

A mixture of N :K ratio 100 : 2, contains about 0-018 per cent. KC1 (theprecise value depends of course on the volume of CaCl2 added). As stated above,if the Helix crop is bathed by such a solution, it responds by tonic contractioneither to potassium withdrawal or to potassium excess. Similar behaviour hasbeen recorded in the following vertebrate preparations: blood-vessels of the frog,perfusion fluid containing 0-015 per cent. KC1 (Clark, 1922), uterus and gut ofthe rabbit, bathing fluid containing o-oi per cent. KC1 (Clark, 1922), uteri ofthe rabbit, rat, guinea-pig and kitten, bathing fluid containing 0*042 per cent. KC1(Tate and Clark, 1921), intestines of the rabbit, cat and guinea-pig, bathing fluidcontaining 0-02 per cent. KC1 (Jendrassik and Annau, 1925). The action ofpotassium on tone in the plain muscle of Helix and that of vertebrates is thereforequantitatively identical.


The following considerations enable this comparison to be extended to Aplysia.As with land vertebrates, during the evolution of land molluscs from their marineancestors the blood has been diluted without any great alteration in the relativeproportions of sodium, potassium and calcium. Since Helix blood is roughlyequivalent to Aplysia blood diluted five times with water, the cells of Aplysia arepresumably in equilibrium with a solution containing five times as much potassiumas that which bathes the tissues of Helix. Now for Helix a solution of Na : K,ratio 100 :2, contains 0-002 molar KC1, and we have seen that in Aplysia a solutioncontaining five times this concentration has the same physiqlogical properties; itsupports active rhythmical movement, and tonic contraction and inhibition of therhythm result if potassium is either withdrawn or increased. If therefore wedisregard the absolute concentration and consider potassium as a fraction of thetotal electrolyte content of the blood, the identity between its actions on the cropsof Helix and of Aplysia becomes manifest.

Hogben (1925) found that withdrawal of potassium from the media employedby him produced relaxation of the crops of Aplysia and Helix, and inferred thatthese preparations differ from vertebrate plain muscle, which contracts whenpotassium is withdrawn. The reason for this result is that he used abnormallyhigh potassium concentrations. For Helix he used, in parts by volume of one-eighth molar chlorides, Na 100, K 10, Ca 2 to 10 (i.e. about o-oi molar KC1); andfor Aplysia he gives a figure showing relaxation on withdrawing 0-041 molarpotassium. Now it has already been pointed out that the contraction producedby great potassium excess is more vigorous than that produced by potassium lack,and that addition of an excessive amount of potassium to the crop in a potassium-free solution causes, not relaxation, but further contraction; evidently whenpotassium is withdrawn again from such a solution the result will be partialrelaxation. But if the potassium content is comparable to that usually employedfor vertebrate muscle, withdrawing potassium causes contraction (Fig. 8).

III. THE HEART OF MAIA.

An extensive account of the action of electrolytes on the hearts of Homarusand Maia has been published by Hogben (1925), attention being chiefly focussedon the rhythmic contractions. Now in response to alteration in composition ofthe perfusion fluid, the striped muscle of the crustacean heart may show changesin tone; although small in extent relatively to the amplitude of the rhythmic con-tractions, these tone-changes are quite as definite as those of the Aplysia crop.As these tone-changes have not yet been thoroughly worked out in relation to thepotassium concentration, it was thought worth while to do so, in order to comparethe behaviour of crustacean striped muscle with plain muscle in this respect. Thework was done on the heart of the spider crab, Maia squinado.

TECHNIQUE.

The decapod heart consists of striped muscle-fibres forming a syncytium, anddiffering from vertebrate heart muscle in the relatively large amount of sarcoplasm

BjEB-viii x


they contain. In their physiological properties they resemble the voluntary ratherthan the cardiac fibres of vertebrates—e.g. they are readily tetanised. The hemhas no endothelial layer, the perfusion fluid coming into direct contact with thesurfaces of the muscle-fibres (see von Wettstein, 1915, for the anatomy of theheart and pericardial sinus, and Gadzikiewicz, 1904, for the histological structure).The question whether the rhythm is neurogenic or myogenic has been reviewe'dby Clark (1927) and left undecided.

An excellent technique for perfusing the heart under constant pressure is tobe found in the paper by Hogben. In the work here described the same methodswere adopted with one minor modification: I found it more convenient to insertthe cannula into the sternal artery of Maia than into the superior abdominal.

As the results of these experiments are intended for comparison with thoseobtained on Aplysia, the chemical environment was made as similar as possiblein the two cases. The perfusion fluids for Maia were made by mixing o*6 M NaClor KC1 and 0-4 M CaCl2, buffered with Na2HPO4, and adjusted to a pR between7-0 and 7-4. As with Aplysia, in order to regularise the osmotic pressure increaseof potassium was compensated by equal decrease of the sodium concentration,the calcium concentration being strictly constant in any one experiment (e.g. inFig. 10 II, addition of 0*053 M K is accompanied by decrease of the Na from 0*58to 0*527 M). The great majority of the experiments were performed with a calciumconcentration of 0*014 or 0*015 M.

Hogben has shown that the heart will beat normally for two hours or morein a mixture containing only sodium and calcium chlorides. In the work hererecorded the effects of adding various amounts of potassium to such a potassium-free perfusion fluid are described. It may be pointed out that Hogben exploredthe action of potassium on the crustacean heart from a rather different angle; hestarted with a mixture containing already 0*0037 M K and described the resultsof increasing potassium to higher concentrations. Consequently he did not giveany account of the immediate effect of changing from a potassium-free perfusionfluid to one containing a small amount of potassium. Moreover Hogben usedHomarusy and, as will be shown later, there is a difference in the way the hearts ofthe lobster and spider crab respond to potassium excess.

THE ACTION OF CALCIUM AND SODIUM.

As the heart of Maia will beat regularly for some time with a perfusion fluidcontaining only Na and Ca, it is possible, as a preliminary to investigation of theaction of potassium, to study the responses of the heart to simple variation in theNa : Ca ratio. The effects of calcium lack and excess (up to 0*097 M) have alreadybeen described by Hogben, but it will be convenient to summarise them here.My own experiments are fully confirmatory of Hogben*s results on this point.

The heart generally beats satisfactorily in a mixture of NaCl + CaCl2, pK 7*0,containing 0*015 M Ca—but, as Hogben has pointed out, the optimum calciumconcentration varies from heart to heart. As might be expected, the concentration

The Action of Potassium on Muscle-preparations from Invertebrates

required to call forth the symptoms of any given degree of calcium excess or lackvaries correspondingly, and the figures quoted in the following paragraph are onlyto be regarded as rough averages.

Changing from a balanced Na + Ca mixture to NaCl produces abrupt toniccontraction and cessation of the rhythm; effects which are both rapidly reversible(Fig. 9). Slight calcium deficiency (e.g. 0-007 M Ca) produces acceleration of therhythm, irregularity, and a slow, steady rise of tone. The irregularity becomesmore and more pronounced as perfusion continues, and, as the tone gradually rises,the heart approaches the condition seen in NaCl. Moderate excess of calcium has

B

Fig. 9. Heart of Maia: perfused with various mixtures of o-6 M NaCl and 0-4M CaCl2. pH 7*0.12° C.

I. A, C, E: 0-015M Ca; B: 0-030M Ca; D: 00077M Ca.II. A, C: 0-015 M Ca; B: Ca absent, o-6 M Na; D: 0-4 M Ca, Na absent.

In all figures of the Maia heart the base-line is interrupted once a minute.

effects exactly opposite to those of calcium lack—slowing of the rhythm, and lossof tone. With concentrations of about 0-07 M Ca the heart stops at once indiastole. Although moderate calcium excess causes loss of tone, I find that verygreat excess (e.g. 0*4 M Ca, Fig. 9 II) produces complete relaxation followed bya slow tonic contraction1. This contraction is reversible, but only very slowly;after about half an hour's perfusion with a balanced salt solution the heart fromwhich Fig. 9 was traced had relaxed fully and was beginning to give irregular beats.

A point noticed during this work is that in general the heart responds morequickly to increase in calcium concentration than it does to decrease. The effects

1 This effect is more clearly seen in the adductor muscle of the claw, which is contracted byisotonic NaCl or CaCl2, but relaxes in NaCl 100 c.c. +CaCl2 4 c.c. The NaCl contraction is accom-panied by spontaneous twitches and is rapidly reversible; the CaCl2 contraction is smooth and onlyslowly reversible.

18-2

276 GEORGE P. WELLS

of calcium lack appear relatively slowly and are rapidly reversible, while those ofcalcium excess appear rapidly but are more slowly reversible. This observationtallies with the view that calcium forms an insoluble calcium-lipoid compound atthe surface of the cell.


The action of potassium after a potassium-free fluid is illustrated in Fig. 10.As with Aplysia, the effects on tone and on the rhythmic beats may be describedseparately.

A B CFig. 10. Heart of Maia: action of potassium. Ca 0-014 M. pH T"2-

I. A,C:K absent; B: 00058 M K.II. A, C: K absent; B: 0-053 M K.

At X the drum was stopped for three minutes. II was taken one minute after I.

To deal first with the rhythm; low concentrations of potassium have two effects.The immediate effect of changing from a potassium-free solution to one containinga little potassium is a temporary depression; the rhythm may be greatly slowed,or the heart may even stop altogether for about a minute as in Fig. 10 I. In twoor three minutes, when the temporary depression has passed off, the permanentaction of the potassium, which is to accelerate the rhythm, can be seen. The wholephenomenon is evidently analogous to the "Paradoxe de Potassium" found byLibbrecht (1921) in the frog's heart—indeed, it is difficult to believe, in view ofthe very close resemblance between Fig. 10 I and Libbrecht's Fig. 1, that there isany essential physiological difference in the relation of the two hearts to potassium.At first sight the fact that the Maia heart will beat for two hours or more in the


absence of potassium, suggests that the ionic requirements of crustacean muscleare simpler, but it should be remembered that the rapid disturbance of functionproduced in the frog's heart by perfusion with potassium-free Ringer is due tofailure of the special structures at the auriculo-ventricular and sino-auricularjunctions, and not to any general loss of rhythmic contractility. The sinus beatsand the ventricle remains excitable for over an hour in the absence of potassium(Clark, 1920). Since the decapod heart is not subdivided, corresponding to thesinus rather than to the whole amphibian heart, the long survival of activity inthe absence of potassium is not surprising.

High concentrations of potassium invariably depress the rhythm, but myrecords show considerable variation in the way this result is brought about: e.g.with 0-028 M K both amplitude and frequency are diminished, but either theformer or the latter may be the more rapidly affected; in some cases this amountof potassium evokes a very irregular rhythm reminiscent of that seen when calciumis deficient. The heart is a complex system, the normal cycle depending on theintegration of various processes—excitation, conduction of the impulse, and actualcontraction—and by supposing that the relative sensitivity to potassium excess ofthe mechanisms underlying these processes is not constant in different individuals,the variation in visible response can be accounted for. My results therefore confirmthe conclusion reached by Hogben; small amounts of potassium excite in the longrun, while large amounts depress the rhythm.

Turning now to tone: low concentrations (0-0029 or 0-0057 M K) produce adrop of tone which persists until the potassium is withdrawn again. The amplitudeof this effect is small, but it is nevertheless perfectly definite; it appears on therecord as a simultaneous shift of diastolic and systolic level (see Fig. 10 I at C).In one experiment it was shown very clearly by a heart which, for reasons unknown,would not beat; the lever traced a simple horizontal line with an abrupt drop onchanging from a potassium-free solution to one containing 0-0057 or o-on M K,and a rise on withdrawing the potassium. A similar tone-drop to that shown inFig. 10 appears in published figures of the frog's heart (e.g. Libbrecht, 1921,Fig. 1), but owing to the presence in the frog of a layer of plain muscle underthe endothelium, which would be expected to give a similar reaction, it is unsafeto attribute it to the cardiac muscle fibres. In the crab's heart, on the other hand,there is no plain muscle, so there can be no doubt that these changes in lengthoccur in the striated myocardium.

High concentrations (0-028 or more) produce tonic contraction, but this issometimes complicated by an effect in the opposite sense. On adding a largequantity of potassium there may be simple contraction, persisting as in Fig. 10 IIuntil the potassium is withdrawn again, but with different individuals underidentical experimental conditions another type of response may be seen: the con-traction is followed by a falling of the lever as in Fig. 11 I. This depression oftone appears in a minority of my records; e.g. with 0-053 M K four out of elevenhearts showed the depression in varying degrees. I have never seen depressionin the presence of potassium concentrations exceeding o-i M, which produce full

278 GEORGE P. WELLS

tonic contraction of the heart. These facts show that the characteristic actionof potassium excess is to contract the preparation, and that the depression is asecondary effect which appears in some cases only and partly obscures the first.

The ultimate accelerating action on the rhythm of low potassium concen-trations may be due to a K/Ca antagonism, since in potassium-free mixturesdecreasing the calcium accelerates the beat. Hogben found that the slowing actionof calcium excess was antagonised by potassium. It should be noted, however,that, as with Aplysia, addition of a little K and increase in the Ca act similarly indepressing tone, so that in this respect there is no antagonism between the twometals. The depressing action on tone of 0-0057 M K and the contracting actionof 0-053 M K have been observed in the presence of calcium concentrationsranging from 0-0078 to 0-029

\ B

Fig. 11 (see text).I. Heart oiMaia. Cao-oisM. p H y o . n ° C . A, C: K absent; B: 0-053 MK.

II . Heart oZ Homarus. Ca 0-014 M. pH 7'2. 180 C. A, C: K absent; B: 0-053 M K.

Hogben, working with Homarns, found two opposed actions of K excess. For0-031 M K he gives a figure showing tonic contraction and depression of the beatscoupled with a curious periodic irregularity (Hogben, 1925, Fig. 5), but he statesthat "when the amount of potassium is greatly increased, the heart is brought toa standstill in a more or less relaxed condition." In this respect the heart ofHomarus is peculiar; the great majority of muscle preparations (including the frog'sheart) are contracted by sufficient potassium excess. As Hogben has not publisheda figure of diastolic stoppage produced in the lobster heart by excess of potassium,I made experiments on four Homarus hearts to compare this effect with the de-pression occasionally seen in Maia. Although the number of experiments is small,my records suggest very strongly that the difference between the two species isone of degree rather than of kind. In Fig. 11 II, it is shown that the heart ofHomarus stops in a nearly relaxed condition in the presence of 0-053 M K, but


when the record is carefully compared with the upper figure an essential similarityis revealed; in both species potassium excess tends to produce two effects inopposite senses, but whereas in Maia the depression is relatively slight and oftendoes not appear at all, in Hotnarus it is so pronounced that the heart stops in arelaxed condition. The contracting action of great potassium excess in Homarusis clearly shown by the fact that the heart stops in full systole if potassium besubstituted for all the sodium in a perfusion fluid containmg only NaCl 0-58 Mand CaCl2 0-014 M (Fig. 12). Whatever may be the cause of this depression, it iscertainly not such a general effect as the contraction.

The two hearts also resemble each other in their response to low potassiumconcentrations. I have records of the Homarus heart (Ca 0-014 M) which arevery similar to Fig. 10 I, both as regards the drop in tone and the temporaryinhibition of the beat.

Fig. 12. Heart of Homarus'. great potassium excess. pH 7*2. 170 C.A, C: 0-58 MNa , 0-014 M Ca; B: 0-58 M K, 0-014 MCa.

We may say then that in the hearts of Maia and Homarus, addition of lowconcentrations of potassium depresses tone and their withdrawal produces a slightrise, while addition of high concentrations produces contraction; the latter effect,particularly in Homarus, may be complicated by a secondary depression.

COMPARISON WITH PLAIN MUSCLE.

So far as tone is concerned, it is clear that potassium acts very similarly onthe plain muscle of the Helix and Aplysia crops and on the striped muscle of theMaia and Homarus hearts. If Figs. 8 and 10 be compared, it will be seen that inboth kinds of muscle a small amount of potassium causes a drop of tone and alarge amount causes contraction.

There is, however, a quantitative difference between the heart of Maia and thecrop of Aplysia. In Maia, 0*0029 or 0-0057 M K produces a drop of tone whichpersists until potassium is withdrawn again, but concentrations of about o-on M Kproduce an effect intermediate between that seen with low concentrations and thatseen with high. The immediate effect of changing from a potassium-free perfusionfluid to one containing o-on M K i s a drop of tone and temporary inhibition,just as in Fig. 10 I, but this is followed by a slow rise of tone so that in two minutesthe tone is at a higher level than that assumed in the absence of potassium. (At figureof this effect has been published by Hogben, 1925, Fig. 3.) At this stage withdrawalof potassium produces a loss of tone. Now with Aplysia, o-on M K exerts a


relaxing action like that of 0-0057 M K on the heart of Maia, and concentrationsof o-O2 to 0-05 M K have to be applied before the tone rises to a higher levelthan that seen in the absence of potassium. Apparently although the responsesto various potassium concentrations are qualitatively identical in the two cases,less than half as much potassium is required to produce any given effect in theMaia heart than in the Aplysia crop.

As regards the rhythm, the distinction is here encountered that whereaspotassium lack immediately arrests the rhythm of the crop or heart of Aplysia,the heart of Maia, like that of Helix, will beat for hours when potassium is absentfrom the external medium. This may mean only a quantitative difference, e.g.in the speed with which potassium is washed out. With this exception, the re-lations of the two preparations to potassium concentration are very similar; aslight addition of isotonic KC1 to the Aplysia crop suspended in sea-water or tothe Maia heart perfused with a solution containing 0-0057 M K produces accelera-tion and increased amplitude, while high concentrations of potassium (0-053 M K)depress the movements in both cases.

We may note in this connection that the "paradox" seen in the heart of Maiawhen a potassium-free perfusion fluid is replaced by one containing a little Kmay also be paralleled in the Aplysia crop. Generally when the latter preparationis contracted by potassium lack, addition of o-on M K produces a prompt re-laxation and return of the rhythmic movements, as in Fig. 1, but sometimes inthe presence of magnesium or with high calcium concentrations (0*044 M Ca)a different type of recovery was seen: on adding o-oi 1 M K the crop relaxes atonce, but the rhythmic movements are not immediately revived—indeed, suchirregular movements as were shown in the potassium-free fluid are inhibited—andfor about five minutes the preparation remains quiescent in the relaxed condition.After this pause rhythmic contractions abruptly start, a revival which may beaccompanied by slight rise of tone. This phenomenon is evidently related to the"paradoxe Lahmung" seen in the mammalian intestine (Jendrassik, 1924). Whenthe fact that all the responses of the Aplysia crop are slower than those of cardiacmuscle is taken into account, the phenomenon just described is suggestively similarto the temporary inhibition seen in the hearts of Maia and Rana—a resemblancewhich lends strong support to the view that the mechanism of spontaneouscontraction is fundamentally the same in striped and plain muscle.

In further confirmation of this view, the two preparations react in like mannerto variation in the Na/Ca ratio of potassium-free mixtures. As has been shownabove, in both kinds of muscle the effect of moderate calcium excess is to depresstone while calcium lack causes contraction; moreover in both kinds of musclepure isotonic CaCl2 calls forth a smooth, steady tonic contraction which is onlyslowly reversible. Lastly, decrease in the ratio Na/Ca slows both the heart-beatof Maia and the twitching movements of the Aplysia crop, while increase ac-celerates them.

To sum up: during the present investigation no evidence was obtained thatthe mechanism of response to electrolytes differs fundamentally in striped and

The Action qf Potassium on Muscle-preparations from Invertebrates 281

plain muscle. So far as essentials are concerned the responses of the Maia heartare exactly like those of the Aplysia crop, except that in the former preparationthe rhythmical movements are more regular and frequent, and the amplitude oftone changes is relatively smaller, than in the latter.

SUMMARY.

(1) When the crop of Aplysia is bathed by a solution containing c o n M KC1,tonic contraction and inhibition of the rhythmic movements result if potassium iseither withdrawn or added in excess.

(2) The action of potassium on tone in the Aplysia crop is independent of thecalcium concentration.

(3) Rb, Cs and NH4 act like potassium on the Aplysia crop, but Li does not.Rb is about twice as active, Cs about one-half as active, and NH4 about one-fifthas active as potassium.

(4) The changes of tone seen in the Helix crop when the potassium concen-tration is varied are identical with those seen in vertebrate plain muscle underlike conditions.

(5) Addition of a little potassium to the heart of Maia perfused with apotassium-free fluid causes a drop of tone. High concentrations of potassium havetwo opposed effects, a contracting action and a secondary depressing action.

(6) The action of potassium on the striped muscle of the Maia heart is essen-tially like its action on molluscan or vertebrate plain muscle.

The work on Aplysia and Maia was carried out at the laboratory of the MarineBiological Association at Plymouth during the autumn of 1926 and the summerof 1927; that on Helix at the department of Physiology and Biochemistry, Uni-versity College, London, during February and June 1927. I am glad of thisopportunity to express my gratitude to Dr Allen and his staff for many courtesiesextended to me during my stay at Plymouth. I wish also to thank Professor LovattEvans for his kindness while I was working in his laboratory, Mr A. IX Hobsonfor allowing me to examine his tracings and sections of the Aplysia crop, andMr C. F. A. Pantin for his never-failing interest and advice during the progressof the work.

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282 GEORGE P. WELLS

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