J. Phy8iol. (1982), 333, pp. 39-52 39With 9 text-figuremPrinted in Great Britain

THE INFLUENCE OF INJECTED CYCLIC AMP PROTEINKINASE CATALYTIC SUBUNIT ON THE SODIUM EFFLUX IN

BARNACLE MUSCLE FIBRES

BY E. EDWARD BITTAR, GEOFFREY CHAMBERSAND EDMOND H. FISCHER*

From the Department of Physiology, University of Wisconsin, Madison, WI 53706and the Department of Biochemistry*, University of Washington,

Seattle, WA 98195, U.S.A.

(Received 13 January 1982)

SUMMARY

1. A study has been made ofthe response of the Na efflux in single barnacle musclefibres to the injection of cyclic AMP protein kinase catalytic subunit (CSU).

2. (i) Injected CSU stimulates the Na efflux in unpoisoned and ouabain-poisonedfibres. (ii) Stimulation in both situations is always sustained, and (iii) stimulationof the ouabain-insensitive Na efflux is observed with as low a concentration as107 M-CSU.

3. (i) The response to injected CSU depends on [Ca]. but is unaffected by priorexternal application of verapamil. (ii) An effect fails to occur in fibres cooled to 0 'C.

4. (i) Injection of 5'-guanylylimidodiphosphate (Gpp(NH)p) following peakstimulation by CSU causes a further sustained stimulatory response. (ii) InjectionofCSU following peak stimulation byGpp(NH)p causes afurthersustained stimulatoryresponse. (iii) Injection of undialysed cholera toxin following CSU causes little or noeffect, whereas injection of dialysed cholera toxin causes a stimulatory effect.Injection ofCSU following undialysed cholera toxin causes little or no effect, whereasits injection after dialysed cholera toxin causes a fairly appreciable effect.

5. (i) The response to CSU is reduced by prior injection of protein kinase type 2regulatory subunit (RI,) but not when RI, is injected following peak response to CSU.(ii) Injection of protein kinase inhibitor partially reverses the response to CSU.

6. (i) Injection of Mg2+ fully reverses the response to CSU. (ii) Fe and Zn causeonly partial reversal.

7. (i) External imipramine but not trifluoperazine reduces the response to CSU.(ii) Diphenylhydantoin is ineffective.

8. These results support the view that CSU behaves as a modulator of theouabain-insensitive Na efflux and that Mg2+, Fe and Zn are inhibitors of the catalyticreaction.

INTRODUCTION

The mechanism whereby injected cyclic AMP stimulates the ouabain-insensitiveNa efflux in barnacle muscle fibres is still unknown (Bittar, Chambers & Schultz,1976). In recent studies, it has been found that the response to injected cyclic AMP

E. E. BITTAR, 0. CHAMBERS AND E. H. FISCHER

is markedly reduced by pre-injecting pure protein kinase inhibitor (PKI) (Bittar,Demaille, Fischer & Schultz, 1979). Such an observation is in keeping with the cyclicAMP-protein kinase theory put forward by Ashby & Walsh (1972, 1973), accordingto which cyclic AMP activates the holoenzyme by dissociating it into R and C(regulatory and catalytic subunits), and PKI acts by binding free catalytic subunit.On this theory, then, the transducer action of cyclic AMP is to dissociate cyclicAMP-protein kinase, and the freeing of catalytic subunit rather than the formationof an active ternary complex, viz. RC *cAMP, is regarded as the essential step leadingto stimulation of the Na efflux. Support for this line of thinking comes from workshowing that the RC 'cyclic AMP complex formed as an intermediate in the reaction(Builder, Beavo & Krebs, 1980; Chau, Huang, Romero, Biltonen & Huang, 1980)

R2C2+4 cAMP cAMP4R2C2 R2cAMP4+ 2C

is at most 10% as active as catalytic subunit (Builder, Beavo & Krebs, 1981).There is much interest at present in the problem of protein phosphorylation,

particularly since it promises to provide new insight into the mechanism of regulationof membrane structure and function. Thus, studies of catalytic subunit are importantin themselves, but they are also important from the standpoint of the role ofphosphorylation-dephosphorylation reactions in the operation and behaviour of theouabain-insensitive Na efflux e.g. in barnacle muscle fibres. The main object of theinvestigation now reported was twofold: fir8t, to obtain information about thekinetics of the Na efflux in unpoisoned and ouabain-poisoned fibres following theinjection of catalytic subunit, and secondly, to determine the behaviour of the effluxin ouabain-poisoned fibres towards injected catalytic subunit under a variety ofconditions, including the injection of Mg2+ and trace elements. As will be shown,experiments of this type provide a useful approach to studying the problem of howcyclic AMP is able to modulate the ouabain-insensitive Na efflux in barnacle musclefibres.

METHODS

Material&. Specimens of the barnacle, Balanu8 nubilu8, were obtained from Mr David King, FridayHarbor, Washington and maintained at a temperature of - 12 0 in an Instant Ocean aquarium.The composition (mM) of the aquarium sea water as determined by flame emission and atomicabsorption spectroscopy was about: Na, 465; K, 10; Ca, 10, and Mg, 60. Its pH was- 8-0.

Di88edion and cannulation. Single muscle fibres were isolated by dissection from the depressormuscle bundles and cannulated, as previously described (Bittar, Chen, Danielson, Hartmann &Tong, 1972). The fibres used were 3-5 cm in length and 1-2 mm in width.The micro-injector. This was of the type described by Bittar & Tallitsch (1975) where the volume

of fluid discharged per injection was about 0-4 sl#. Since the average intrafibre fluid volume wasroughly 40 ,ul., dilution by the myoplasm ofthe injected test solution maybe taken as being 100-fold.

Solution&. The experiments were carried out with artificial sea water (ASW) having the followingcomposition (mM): NaCl, 465; KCI, 10; CaCI2, 10; MgCl2, 10; NaHCO3, 10; and pH 7'8. Solutionsnominally free of Ca2+ were prepared by simply omitting CaCI2 and raising NaCI in an osmoticallyequivalent amount.

Radioactivity mea8urement8. 22NaCl (0'2 mCi) in aqueous solution was supplied by Amersham-Searle Corporation, Arlington Heights, IL. The solution was dried down and then redissolved indistilled water (0-2 ml.) so that a volume of -04 ,sl. gave at least 700,000 counts/min. Theprocedures used for collecting the effluent following the injection of 22NaCl into cannulated fibres,and for counting its activity, as well as the activity remaining in the fibre at the end of theexperiment, were those described by Bittar, Caldwell & Lowe (1967). A Beckman 'Biogamma'

40

CATALYTIC SUBUNIT ON BARNACLE MUrSCLE

gamma counter was used for counting the samples. The data obtained was processed by means ofan Apple II computer programmed to calculate 22Na efflux (counts/min per min) and the fractionalrate constant for 22Na loss. Stimulation of Na efflux was computed by taking the difference betweenthe maximum rate constant and the rate constant immediately before the onset of stimulation.Where a second successive stimulatory response was elicited, its size was estimated by taking thedifference between the two combined responses and the first response. Inhibition was computedby taking the difference between the rate constant at maximal inhibition, or that found byextrapolating the last few points of the inhibitory phase, back to the time of application of theagent, and the rate constant before the onset of inhibition. The results given in this paper aremeans+S.E. of mean and significance levels were estimated by using Student's unpaired t test.

/ Except where stated otherwise, all experiments were carried out at room temperature, 23+ 1 'C.Cold experiments. The experiments done at 0 'C were as described by Chambers & Bittar (1981).

The cannulated fibres were bathed in a recessed Plexiglas cold block through which coolant at atemperature of -2 'C flowed. This maintained the ASW bathing the fibres at 0 'C.

Cyclic AMPprotein kinase catalytic and regulatory subunits. Both catalytic and regulatory subunitsof cyclic AMP-dependent protein kinase type 2 were prepared from bovine heart, as described byBechtel, Beavo & Krebs (1977) and Peters, Demaille & Fischer (1977). The sample of RI, subunitsfree of cyclic AMP was kept at -20 'C in 5000 glycerol containing 4 mM-MOPS, 100 mm-KC1.0-2 mM-ethylene diamine tetraacetic acid (EDTA) and 1 mm-mercaptoethanol at pH 7-0. Thesample of catalytic subunits was in 5000 glycerol containing 30 mM-KH2PO4. 150 mM-KCl.0 1 mM-dithiothreitol and 0-1 mM-EDTA, pH 6 8. Both samples were dialysed against 3 mM-HEPESin water, pH 72 in the cold room and subsequently concentrated by applying Sephadex G-10 tothe dialysis bag.

Protein kinase inhibitor. PKI was prepared from rabbit skeletal muscle and purified to homogeneit .as described by Demaille, Peters & Fischer (1977).

Agents. Ouabain, HEPES and GTP as the sodium salt were obtained from Sigma ChemicalCompany, St Louis, MO. 5'-Guanylylimidodiphosphate (Gpp(NH)p) sodium was applied by P-L.Biochemicals, Milwaukee, WI. Imipramine and trifluoperazine were gifts from Smith, Kline &French Laboratories, Philadelphia, PA'and diphenylhydantoin (sodium salt) was a gift from Parke.Davis & Company, Chicago, IL. Verapamil HCI was supplied by Knoll Pharmaceutical Company.Whippany, NJ. Cholera toxin was purchased from Schwartz/Mann. Orangeburg. NJ. Whenreconstituted to 0-1 ml. with water, the solution contained - 10-4 M-cholera toxin. 0-5 m-Tris.0-01 M-Na2EDTA, 003 M-NaN3 and 2 M-NaCl. pH 7-5.

RESULTS

Response of the Na efflux to injected cyclic AMP protein kinase catalytic subunitAn earlier paper of Bittar et al. (1979) had shown that injection of catalytic subunits

(from bovine heart muscle) into unpoisoned and ouabain-poisoned fibres causedsustained stimulation of the Na efflux. Because these experiments were carried outwith a sample of catalytic subunits which was - 8000 pure. they were repeated usingpure cAMP-protein kinase type 2 catalytic subunits (CSP) isolated from bovine heartmuscle. The results obtained show that injection of 10-4 M-CStU into unpoisonedfibres caused a sustained stimulatory response, the magnitude of which averaged1 6 + 160 (n = 9). They also show that injection of 10-4 M-CSU into fibres pre-treatedwith 10-4 M-ouabain caused a sustained stimulatory response, the magnitude of whichaveraged 279+ 330 (n = 22). Thus, there can be no doubt, that CSU is able to behaveeas a modulator of the ouabain-insensitive Na efflux and that the characteristic featureof the observed stimulatory response is its sustained nature.To exclude the possibility that the response of the ouabain-insensitice Na efflux

to injected CSTU might, involve activation and recruitment of latent Na+-K+-AT1'asesites, fibres treated with 10( 4M-ouabain were exposed to 103 NM-ouabain followingthe onset, of peak stimulation by injected 10-4 M-CSLt. The results of these experiments

41

E. E. BITTAR, G. CHAMBERS AND E. H. FISCHER

show that the 10-fold increase in ouabain concentration was without effect (n = 6).In additional experiments, however, fibres were treated with 10-4 M and 10-3 M-ouabain in succession following injection of CSU. As illustrated in Fig. 1, injectionofCSU caused sustained stimulation ofthe Na efflux (in the order of 103 + 12 %, n = 6),while 1o-4 and 10-3 M-ouabain had opposite effects. The step-down ofNa efflux causedby 10-4 M-ouabain (averaging 51+3 %, n = 6) resembles that seen when 10-4 M-ouabain was applied following peak stimulation by injected cyclic AMP (Schultz &Bittar, 1978). It implies that the effect of CSU does not involve inactivation ofmembrane Na+-K+-ATPase. As for the stimulatory response (averaging 69+ 11 %,n = 6) seen following external application of 10-3 M-ouabain, it is sustained rather

3 10-1 M-ouabain 10- M-ouabain

t 2 10~M-CSU ni.0

z

0C

UE 0 20 40 60 80 100 120 140 160 180

Time (min)Fig. 1. Injection of 10-4 M-CSU into a fibre, followed by external application of 10-4 M-

ouabain and then 10-3 M-ouabain.

than transitory. The cause of this sustained response on applying 10-3 M-ouabain isnot clear. Since this is also what happens when Gpp(NH)p is injected following theonset ofsustained stimulation ofthe ouabain-insensitive Na efflux by injected choleratoxin (Bittar & Nwoga, 1982a), it is tempting to suggest that ouabain acts byreducing myoplasmic pCa. This is in line with the accepted view that cardiotonicsteroids raise the intracellular level of free Ca. Failure of 10-3 M-ouabain to producea similar step-up in the Na efflux in fibres treated with 10-4 M-ouabain prior to theinjection of 10-4 M-CSU can be explained by supposing that injected CSU leads toa rise in myoplasmic pCa, possibly as the result of increased Ca2+ uptake by thesacroplasmic reticulum or Ca2+ outflux (or both).

It became of interest next to construct a concentration-response curve for thestimulatory action of injected CSU on the ouabain-insensitive Na efflux. In view ofthe fact that the sensitivity of these fibres to CSU tended to vary quite widely amongbarnacle specimens, the experiments were deliberately carried out by using fibres fromthe same barnacle specimen. From the results summarized in Fig. 2, it is apparentthat the fibres in this case were very sensitive to injected CSU. This is particularlytrue when 100-fold dilution by the myoplasm is taken into account. Sensitivity ofthis degree is reminiscent of that seen in fibres from some barnacles when exposedto high [K]0 (Bittar & Nwoga, 1982b), or usually, when injected with cyclic AMP(Bittar, Chambers & Schultz, 1976). If one now regards the size of the responseobtained by injecting 10-5 M-CSU (or more precisely by adding 10-7 M-CSU to themyoplasm) as being comparable to that seen following the injection of 10-2 M-cyclicAMP, the question then arises: what is the total internal concentration of cyclic

42

CATAL YTIC SUBUNIT ON BARNACLE MUSCLE

AMP-protein kinase in these fibres? An answer cannot yet be provided, but in rabbitskeletal muscle, for example, the concentration of CSU is 0 4 /SM, assuming uniformdistribution of the enzyme (Hoffman, Bechtel & Krebs, 1977). Such considerationssuggest that dissociation of the total available cyclic AMP-protein kinase in barnaclefibres would require the injection of cyclic AMP in a concentration exceeding 10-2 M,and hence justify the past use of 3 x 10-2 M-cyclic AMP (Bittar et al. 1976).

3

n = 5

106 10-S 10-4[CSUJ (M)

Fig. 2. Concentration-response curve for the stimulator effect on the ouabain-insensitiveNa efflux of injected CSU. Vertical bars indicate + S.E. of mean. Numbers given are

the measurements done.

10-1 M-ouabain '1

_~~~~~A~mz bq4C44- X

c0 N.- -'.r4-0 4Q Wm 0I-U-

20 Ca-ASW

1 10-4 M-CSU inj.

lr . .~~~~~~~~~',20 40 60 80 100 120 140 160 180 200

Time (min)

Fig. 3. Response of the ouabain-insensitive Na efflux into nominally Ca2+-free ASW tothe injection of 1O-4 M-CSU and subsequently, to the restoration of external Ca21.

Response to CSU before and after omitting external Ca2+

The transitory response of the Na efflux to injected cyclic AMP is known to dependon external Ca2+ (Bittar et al. 1976). As shown in Fig. 3, the same is true of theresponse to CSU, since the injection of 10-4 M-CSU into an ouabain-poisoned fibresuspended in Ca2+-free ASW caused a rather small stimulatory response (averaging82 + 17 %, n = 3), and when external Ca2+ was restored there was a marked step-upinthe efflux averaging233 + 27 % , n = 3), whichwas sustained. In parallel experiments,where 10-4 M-CSU was injected into fibres suspended in 10 mM-Ca2+-ASW, the

15

g 10

x-

c .-

0

a

I-

e~~~~~~~~~ a -f l .__

43

-1)"W

E. E. BITTAR, G. CHAMBERS AND E. H. FISCHER

sustained stimulatory response (averaging 171 + 51 %, n = 3, P > 0-2) was reducedby 48 + 9 % (n = 3) on sudden omission of external Ca2 . Subsequent addition of10 mM-Ca2+ to the bathing medium led to complete restoration of the sustainedstimulatory response.

Response to CSUfollowing reduction in external pHBittar et al. (1976) reported that the size of the response to injected cyclic AMP

is augmented by prior protonation of the bathing medium. The results show thatinjection of 10-4 M-CSU stimulates the ouabain insensitive Na efflux into 3 mm-HEPES-ASW (pH 6-3) but the value obtained, that of 228+ 33% (n = 6), is notsignificantly different from 356 + 103% obtained with control fibres, n= 6 (Pbeing > 0-3).

NoK0 If

+J

° - g 0 m-CSU inj./UL

0 20 40 60 80 100 120 140 160 180 200 220Time (min)

Fig. 4. Lack of effect following injection of 10-4 M-CSU into a fibre cooled to 0 0C andappearance of the effect following restoration of the environmental temperature to 24 'C.

Lack of effect of verapamil on the response to CSUTo explore the possibility that part of the response to injected CSU might be

due to activation ofCa2+ channels, experiments were done in which ouabain-poisonedfibres were exposed to 10-4 M-verapamil prior to the injection of 10-4 M-CSU. Theresults show a sustained stimulatory response averaging 113 + 29% (n = 5), comparedwith 132 + 32% in companion controls (P > 0-5). This result agrees with unpublisheddata showing that verapamil fails to influence the response to the injection of 10-3 M-cyclic AMP (Bittar, E. E. & Nwoga, J.).

Lack of effect of injected CSU at 0 'CInjected cyclic AMP is able to stimulate the Na efflux in fibres cooled down to 0 'C

(Bittar et al. 1976). By contrast, injected CSU is ineffective, as shown in Fig. 4. Suddenrewarming to 24 'C caused an 'overshoot' which exceeds the original base line (at24 'C) by 109+36% (n = 4). As reported by Chambers & Bittar (1981), rewarmingofthese fibres is followed by almost complete restoration ofthe Na efflux to its originalcontrol level. Such results imply that the response to injected CSU is metabolicallycoupled, or that the development of a response to CSU requires a certain membraneconformation or both. It also implies that cyclic AMP is able to exert an effect at0 'C by a mechanism which is unrelated to cyclic AMP-protein kinase dissociation.

44

CATAL YTIC SUBUNIT ON BARNA(JLE MUSC(LE

Response to CSU before and after injecting Gpp(NH)pEvidence is available that GTP and its non-hydrolysable analogue, Gpp(NH)p,

when injected into ouabain-poisoned fibres, stimulate the Na efflux to about the sameextent by a mechanism largely involving activation of Ca2+-channels. There also isevidence that the injection of cholera toxin into ouabain-poisoned fibres causes asustained stimulation of the Na efflux and that subsequent injection of GTP orGpp(NH)p leads to a further sustained response (Bittar & Nwoga, 1982a; Bittar,E. E. & Nwoga, J., unpublished data). It thus seemed of interest to find out whethersimilar kinetic results are obtained by injecting GTP and Gpp(NH)p before or afterCSU. Shown in Fig. 5 is that injection of0-25 M-Gpp(NH)p, following peak stimulation

3 - 10-4 M-ouabainf0-25 M-Gpp(NH)p inj.

_2_l

0

Gpp(NHop, followed by 10-M-CSU(lower inj.

5 MGpN pcueafrhrspuia0-25m-Gpp(NH inj. ( o

0 20 40 60 80 100 120 140 160 180 200Time (min)

Fig. 5. Effects on the ouabain-insensitive Na efflux of internal application of 3 S,followed by 025 m-Gpp(NH)p (upper panel), and of internal application of 0-25±-Gpp(NH)p, followed by 1l-4 MeCSU (lower panel).

by injected 1s-4 m-CSU, resulted in a further separae ftimulatory response. Alsoshowncess in poisoinjection of 1t-4 MiCSU following peak stimulation by injected0t25 m-Gpp(NH)p caused a further step-up in the Na efflux. (Estimates of theresponse to CSU prior to injecting Gpp(NH)p gave a value of 329+ 36 %/, n = 6, whichis not significantly different from 372k+n81 a found when CSe was injected afterGpp(NH)p, n = 6. Nor is 372 + 81a significantly greater than the value of 256 + 59 oobtained for the size of the response to injected Gpp(NH )p). Collectively, these resultssupport the suggestion that it is possible to separate functionally two transportprocesses in poisoned fibres: one that is mediated by cyclic AMP-protein kinase andthe other mediated by internal free Ca2+ presumably involving the calmodulin/Ca-dependent protein kinase.

Response to CSU before and after cholera toxinIllustrated in Fig. 6A is that injection of 10-4 M-cholera toxin (CT) caused a marked

sustained stimulation of the ouabain-insensitive Na efflux (averaging 509+79°/%,n = 3), and that subsequent injection of 10-4 m-CSU had little or no effect (25 + 25 %/ *

45

46 E. E. BITTAR, G. CHAMBERS AND E. H. FISCHERA

4 10-4 M-oUabain

x

0

M C10-4 MCSU ij

z 2 5 X 1010 m-CT inj MlCSUi.

0C0

(U

20 40 60 80 100 120 140 160 180Time (min)

C

so 3 -10 M-OUabainlX 10-4 M-CSU min.

0

XO 5 X .- -DTi

z

0C0

L 0 20 40 60 80 100 120 140 160 180Time (min)

C1 3 1-0 m-ouabainf

X 5 X f0o b xM-DCT i.

0M=13).Inparalle0lm-CSU in.i

0

0

U- 0 20 40 60 80 100 120 140 160 180Time (min)

Fig. 6. A, effects on the ouabain-insensitive Na efflux of internal application of 5 x 10- M-undialysed cholera toxin (CT), followed by 10= m-CSU. B, effects on the ouabain-insensitiveNa efflux of internal application of 5 X 1O- M-dialysed cholera toxin (DCT), followed by10s4MfCSU. C, effects on the ouabain-insensitive Na efflux of internal application of10-' m-CSU, followed by 5 x 10-6m-DCT.

n = 3). In parallel experiments, injection of 10O4 M-CSU caused 442±+68 % stimulationof the ouabain-insensitive Na efflux (n = 3), and subsequent injection of -10O4 m-CTcaused 7+7 % stimulation, n = 3. This type ofexperiment was repeated, but this timea sample of CT was dialysed against 3 mm-HEPES and this dialysed cholera toxinused for micro-injection. Shown in Fig. 6B is that injection of -5 X 1O-5 m-dialysed

CATAL YTIC SUBUNIT ON BARNACLE MUSCLE

cholera toxin (DCT) caused a sustained stimulation of the ouabain-insensitive Naefflux (averaging 243 +59 %, n = 3) and that subsequent injection of 10-4 M-CSUcaused a stimulation in the order of 112 +44 % (n = 3). In parallel experiments,injection of 10-4 M-CSU caused sustained stimulation of the efflux (averaging442+ 68 %, n = 3), and subsequent injection of - 5 x 10-5 M-DCT caused 95 + 9%stimulation (n = 3) (Fig. 6C). Taken together, these results suggest that one (ok more)of the constituents, e.g. Na, of the aqueous vehicle used for cholera toxin suppressesthe catalytic reaction or interferes with the process of protein phosphorylation, andthat dialysis removes the inhibitor.

10, M-Ouabainf

X 2 1o-4 M-PKI inj.

0 20 40 60 80 100 120 140 160 180 200Time (min)

Fig. 7. Reversal by internal application of 1O-4 M-PKI of the response to injected104 M-CSU.

Effect of PKI before and after CSUAs shown in Fig. 7, injection of 10-4 M-PKL following peak stimulation by injected

10-4 M-CSU caused almost complete reversal of the response. However, such apronounced effect proved to be the exception rather than the rule (viz. 30 ±5°/%reversal of the step-up, n = 11). In further experiments, 10-4 M-CSU was injected20 m4 after injecting 10- M-PKI. The results show 30 ± 2 % stimulation (n = 3),compared with 120+36% stimulation in companion controls, n = 3 (P < 0-1). Withdepletion ofour supply ofPKI, more experiments along these lines could not be done.

Response to CSU before and after protein iinase type 2 regulatory subunit (RCS)The cyclic AMP-dependent protein kinase theory states that activation by cyclic

AMP leads to dissociation of the holoenzyme into regulatory subunits (R2 cAMP4) andcatalytic subunits (2C), the latter acting as an active kinase (Beavor Bechtel &Krebs31974). On this theory, one would expect that injection of regulatory subunits (fromprotein kinase type 1 or 2) would reduce the response to CSU. The results obtainedby injecting 5x 10-6 M-CSU into ouabain-poisoned fibres 30 mmn after injecting2 x 10-5 M-R11 showed 135+19% rsstimulation (n = 13), compared with 212 ± 28%stimulation found in controls, n = 13 (P < 0 05). When, however, the effect Of Rh1was examined by injecting a2px 10-5 M-soution following peak stimulation byinjected 5 x10k 6 M-CSU, two kinds of results were obtained: one where Rob wascompletely ineffective (n = 4) and the other where injected RI, caused a step-up inthe stimulated efflux (averaging 176 ± 20%, n = 9). An explanation of this effect isunavailable.

47

E. E. BITTAR, G. CHAMBERS AND E. H. FISCHER

Effect of RI, on the response to Ca2+ injectionOne possible explanation of the failure of injected RI, to reverse the response to

CSU is that activation of Ca2+-dependent proteases is sufficient to render RI,ineffective. Since it is known that injection of CaCl2 causes a stimulatory responseof the ouabain-insensitive Na efflux which is reduced by pre-injecting PKI (Mason-Sharp & Bittar, 1981), experiments were done in which 1 M-CaCl2 was injected 30 minafter injecting 2 x 10-5 M-RII. The results showed 349 +69% stimulation (n = 8),compared with 727 + 67 % stimulation in companion controls, n = 9 (P < 0-01). Thisis strong evidence that the lack of effect of RI, in the preceding experiments was notlikely to be due to protease activation by increased internal free Ca2+.

Effect of injection of Mg2+ before and after CSUEvidence is available that the stimulatory response to injected GTP or Gpp(NH)p

(Bittar & Nwoga, 1982 a), high [K]o (Bittar & Nwoga, 1982b) and injected cyclic AMP(Bittar, E. E. & Nwoga, J., unpublished) is abolished by injecting Mg2+. The resultsof experiments show that injection of 10-4 M-CSU into ouabain-poisoned fibrespre-injected with 0-5 M-MgCl caused 70+ 38% stimulation (n = 5), compared with267 + 46% stimulation found in companion controls, n = 5, (P < 0-02). In additionalexperiments, 05 M-MgCl2 was injected following peak stimulation by CSU. Mg2+caused almost complete reversal of the response in each of the four fibres tested.

Response to Zn and Fe after CSUPrevious work with trace elements led to evidence that injected Zn and Fe partially

reverses the sustained stimulatory response of the ouabain-insensitive Na efflux tothe injection of GTP (Bittar & Nwoga, 1981). Shown in Fig. 8 is that injection of0 5 M-ZnCl2 (upper panel) and 0 5 m-FeCl2 (lower panel) caused partial reversal of thesustained stimulatory response to injected CSU (Zn:48+6 % reversal, n = 5 andFe :50+ 11 % reversal, n = 6). Such results are consistent with a mechanism in whichFe and Zn inhibit the catalytic reaction (Miyamoto, Kuo & Greengard, 1969; Rosen,Rangel-Aldao & Erlichman, 1977) or interfere with phosphorylation of membranesubstrate proteins.

Response to CSU before and after calmodulin inhibitorsThe antidepressant, imipramine, and the antipsychotic drug, trifluoperazine

(TFP), are able to reduce the size of the response to injected GTP (Bittar & Nwoga,1982 a) and high [K]o (Bittar & Nwoga, 1982b). The results of experiments show thatinjection of 10-4 M-CSU 20 min after external application of 5 x 10-5 M-imipraminecaused 32 + 23% stimulation (n = 6), compared with 227 + 59% stimulation obtainedwith control fibres, n = 6 (P < 0 02). In parallel experiments, external applicationof 5 x 10-5 M-imipramine following peak stimulation by CSU was found to lead to aprompt step-down in the efflux (averaging 32 +4 %, n = 6), as shown in Fig. 9.

In the next experiments the effect of TFP was examined. Injection of 10-4 M-CSU20 min following external application of 1O-- M-TFP led to 148+ 32% stimulation(n = 6), compared with 171 + 60% stimulation obtained with control fibres, n = 6(P > 0 5). In parallel experiments, application of 10-5 M-TFP following peak stimu-

48

CATALYTIC SUBUNIT ON BARNACLE MUSCLE 49

lation by CSU was without effect in the five fibres tested. Such results make it difficultto interpret those obtained with imipramine. Although an explanation of thediscrepancy is unavailable, experience with these drugs indicates that while imipramineis consistently effective, e.g. in reducing the response to injected GTP, the same isnot true of TFP.

3 10-4 M-ouabain2 2 l ~~~~~~~~~0-5M-ZnCI2 inj.

2

x ~~~~10-4 MCSU ni ,

0

z 00-5 M-FeCI2 inj.

0

0' m-CSU'i/.U-

00 20 40 60 80 100 120 140 160 180

Time (min)Fig. 8. Partial reversal by internal application of0 5 M-ZnCl2 (upper panel) and 0(5 M-FeCl2,

(lower panel) of the response to injected 10-4 M-CStl.

x 10-4 M-ouabain

o 50 Mm-imipramineZ 10-4 M-CSU i. 4'

0C0

01

U. 20 40 60 80 100 120 140 160 180

Time (min)Fig. 9. Partial reversal by external application of 5 X 10- Mjilflipr~ailine Of' thle response

to injected 10Y4 MCSU.

Lack of effect of diphenylhydafitoin (DPH) on the response to CiSUTo strengthen the argument that the sustained stimulator, response to injected

CSU does not involve calmodulin/Ca-dependent protein kinase. experiments were

designed in which 10 M-CSU was injected 20 mi2 after external application of10-4 M-DPH. This resulted in 258 ± 70 00 stimulation (ti = 3). compared with327 ± 550o stimulation in companion controls. n = 3 (11 < 0-5). In parallel experiments.external application oft yM-DPH following peak stimulatiiplonh v CSU wasco et-elyineffe fftive in the three fibres tested

E. E. BITTAR, G. CHAMBERS AND E. H. FISCHER

DISCUSSION

The results described in this paper clearly show that the ouabain-insensitive Naefflux is sensitive to the injection of pure catalytic subunits (CSU) and that thecharacteristic response is one of sustained stimulation. Kinetics of this type are notunexpected, since they are also seen following the injection of cholera toxin (Bittar& Nwoga, 1982a) which is known to produce persistent activation of membraneadenylate cyclase, and hence raised cyclic AMP levels (Gill, 1977). However, incontrast to CSU and cholera toxin, injected cyclic AMP is found to consistently causeonly transitory stimulation ofthe Na efflux (Bittar et al., 1976). Transitoriness is simplyexplained by supposing that a large fraction of the injected cyclic AMP is rapidlydegraded by the phosphodiesterase system. That the observed effect of CSU is ofphysiological significance is indicated by the fact that the ouabain-insensitive Naefflux is markedly sensitive to CSU, even when as low a concentration as a10-7 M-solution is injected. Such a result would be expected only if CSU behaves asan active kinase, paralleling the situation where injected cyclic AMP assumablycauses dissociation of cyclic AMP-protein kinase into regulatory and catalyticsubunits. The extent to which the response to CSU is altered by PKI or RI, wasexamined but while the results obtained endorse the cyclic AMP-protein kinasetheory, there are circumstances (e.g. injection of RI, following peak stimulation byinjected CSU) in which further stimulation, rather than reversal is seen. Whether thisis so because of proteolysis of RI, following its injection is not yet clear. Proteolysisof both PKI and RI, during storage and following injection could account for theinability of these proteins to abolish the response to CSU completely. A similar stateof affairs has been reported in experiments with Aplysia neurones (Levitan & Adams,1981).

It is a fact of some interest that trace elements, e.g. Fe and Zn, are able to reversethe response to CSU partially. The only reasonable interpretation of this is that theFe and Zn in millimolar concentrations are inhibitory to the catalytic reaction, asreported by Rosen et al. (1977). In retrospect, this finding provides a satisfactoryexplanation of the ability of trace elements to partially reverse the sustainedstimulation of the ouabain-insensitive Na efflux by injected GTP fibres pre-exposedto aldosterone (Bittar & Nwoga, 1981). By the same token, the effect of injected Mg2+in practically reversing the response to CSU can be ascribed to inhibition of thecatalytic reaction. This interpretation is based on evidence that the Km for Mg2+ ofthe phosphotransferase reaction lies in the region of 1-7 mm, and that excess Mg2+is inhibitory (Rosen et al., 1977; Chiu & Tao, 1978). A second but less likelyexplanation is that excess Mg2+ activates a putative Mg2+-dependent phosphoproteinphosphatase, thus leading to dephosphorylation ofany involved membrane substrateproteins. The existence of this enzyme in barnacle muscle has not yet been establishedbut it is known, for example, that rabbit skeletal muscle possesses such an enzyme(Yang, Vandenheede, Goris & Merlevede, 1980).The effect of omission of external Ca2+ is to reduce the magnitude of the response

to CSU. This is in keeping with the concept that the catalytic reaction controls theNa/Ca carrier which is responsible for the transport ofNa outwards and Ca2+ inwards,especially under conditions where a raised internal Na pre-exists. The possibility that

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CATAL YTIC SUBUNIT ON BARNACLE MUSCLE

injected CSU may lead to activation of Ca2+ channels was considered but on the basisof the results obtained with verapamil no evidence could be found that this is the casehere. The lack of an effect with trifluoperazine but not with imipramine is not easyto explain. A similar unresponsiveness, however, was seen with DPH. Thus, theinference may be drawn that calmodulin-sensitive proteins, e.g. calmodulin/Ca2+-protein kinase is not involved in the response to CSU.As yet there is no evidence suggesting that one of the rate-limiting factors is the

number of vacant phosphate acceptor sites. A glance at the concentration-responsecurve indicates that the response obtained by injecting 10-4 M-CSU may not evenapproach Vmax. An exactly similar relationship between concentrations and responseis seen following the injection of cyclic AMP (Bittar et al. 1976; Schultz & Bittar,1978). One likely explanation of this phenomenon is an augmented activity of theputative Mg2+-dependent phosphoprotein phosphatase. Whether this is true andwhether variation in sensitivity of the Na efflux to injected CSU (and cyclic AMP)also depends on the extent to which activity of this enzyme is governed by the proteininhibitor 1 and inhibitor 2 (Stewart, Hemmings, Cohen, Goris & Merlevede, 1981)must remain the subject of future investigation.

Financial support from the Graduate School Research Committee, the Wisconsin HeartAssociation and National Science Foundation is gratefully acknowledged.

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