Electrogenic sodium pumping in Mytilus smooth muscle

ELECTROGENIC MYTILUS H.

SODIUM SMOOTH

YAMACUCHI

PUMPING MUSCLE

IN

and B. M. TWAROC

Department of Physiology. University of Massachusetts Medical School. Worcester Massachusetts. 01605 and the Department of Anatomical Sciences. School of Basic Health Sciences, Health Sciences Center, State University of New York at Stony Brook, Stony Brook, New York, I 1794, U.S.A.

(Rec&rd

6 Augusr

1979)

Abstract-l. The possible contribution of an electrogenic sodium pump to the resting membrane potential (E,) of smooth muscle of Myths has been investigated using glass microelectrodes. 2. E, hyperpolarized by I2 mV in K +-free artificial sea water (ASW), repolarized to the original level upon returning to control ASW. E, was unchanged in ASW containing 10m4 M ouabain or strophanthidin during the first 2 hr. 3. Slight membrane depolarization (5-6 mV) occurred after prolonged soaking in K’-free or cardiac glycoside-containing ASW. 4. After restoration to control ASW, the membrane transiently hyperpolarized. The hyperpolarization was blocked by IO-“ M ouabain. 5. The transient hyperpolarization was also demonstrated in the preparation exposed to isotonic NaCI-EGTA solution during soaking in K+-free ASW. 6. The evidence suggested the existence of an electrogenic sodium extrusion mechanism: however, its activity in normal resting membrane seemed restricted.

MATERIALS AND METHODS

INTRODUCTION

The response to physiological solutions containing reduced potassium (K’) concentrations or cardiac glycosides has been widely employed to determine whether an electrogenic sodium pump (Na+ pump) contributes to the resting membrane potential (Thomas, 1972a). Many excitable cells depolarize after one or both of these treatments, presumably because an electrogenic pump is inhibited, e.g. crayfish stretch receptor (Nakajima & Takahashi, 1966; Sokolove & Cooke, 197 I), barnacle photoreceptor (Koike et al., 1971); Lirnulus photoreceptor (Brown & Lisman, 1972); snail neuron (Kerkut & Thomas. 1965; Lambert rt al.. 1974); Anisodoris giant cell (Gorman & Marmor. 1970); Aplq’sia neuron (Russell & Brown, 1972). Rapid depolarization in reduced K+ and cardiac glycosides is particularly noticeable in many mammalian smooth muscles where the electrogenie Na’ pump has been reported to contribute significantly to the resting membrane potential: taenia cob (Casteels et al., 1971; Tomita & Yamamoto, 1971); uterine muscle (Taylor er al., 1970); portal vein, (Kuriyama et al., 1971); urinary bladder (Kurihara & Creed, 1972); ileum. (Bolton, 1973); jejunum (Connor & Prosser, 1974); duodenum (Connor et al., 1974). In the present study, attempts were made to investigate whether an electrogenic Nat pump functions in the anterior byssus retractor muscle (ABRM) of Mytilus eddis L. and whether it contributes significantly to the resting membrane potential. A preliminary account of this study has been published (Yamaguchi & Twarog. 1974). 265

A ganglion-free ABRM was mounted in a Lucite chamber and perfused with artificial seawater (ASW). The chamber contained about I ml of perfusion fluid after the muscle was mounted. The perfusion rate was about I.5 ml per min. The resting membrane potential (E,) was measured bv a elass microelectrode filled with 2.7 M KCI. 3&60 Ma cn resistance, connected to a capacity neutralized amplifier (Bioelectric NF-I) and a cathode ray oscilloscope (Tektronix 565). The resting membrane potential of the ABRM fell between -60 and -70 mV. inside negative. When the membrane was penetrated with a microelectrode, the E, initially was near -55 mV then it increased by 7-8 mV in the following few seconds. After E, reached a stable value, the microelectrode was withdrawn and the difference between stable value and zero was recorded. This method allowed frequent accurate readings of E,. The experiments were performed in a cold room, at 17’C. In experiments testing the effect of cold on E,, the muscle was mounted in a small Lucite chamber jacketed by a thermoelectric device which was capable of regulating temperature between 5 and 3O’C. In order to achieve temperatures down to - l.O’C, a solution of 500/ water. 50% methanol regulated at - l3’C by a Lauda (K-2R) refrigerated circulating bath was circulated through the thermoelectric device. Temperature was monitored by a thermistor placed on the muscle near the recording site. Warming and cooling required about 7 min.

ASW had the following ionic composition (mM); Na+ 428; K+ 10; Ca*’ 10; Mg2+ 20; Cl- 548. The pH was adjusted to 7.3 with 50mMTris+HCI. K’-deficient (OK+)ASW was prepared by replacing Na+ with K’ on an equimolar basis.

266

t-1. YAMACILXHI

iI

I

2

3

and 5. M. TWAKOG

3

HOURS

Fig. 1. Membrane potentials (E,) of the ABRM in normal and K ‘-free ASW measured with glass-microefectrodes. A- the muscle was in K +-free ASW for 2 hr. then normal ASW was admitted: E, ~iyperpo~~iriz~d in K”-free ASW and rcpolari7ed upon restoration of K’. B--E, of the muscle soaked in K+-free ASW for 3 hr; E, hyperpolarized in I<+-free ASW, but additional hyperpolarization of 12 mV was observed on restoration of K’. E, depolarized slightly after I hr in K+-free ASW in both A and B.

1

i

3 HOURS

of the transient hyperpolarization. Fig. 1. Blockade A--0uahain (10m4 M) was perfused during the soaking period in K’-free ASW for 45 min prior to the restoration of KC. Normal ASW containing ouabain (10 4 M) was then perfused to replace K”-free ASW. B--E, from another muscle in K+-free ASW. Normal ASW containing

10s4 ouabain was perfused at the end of soaking in K “-free ASW. A slight hyperpolari7ation was recorded.

at the time of K+ readmission. A slight hyperpolarizThe data were analyzed by a paired f-test and the membrane potentials were expressed as mean j SE. In one case. analysis of variance was made according to Duncan’s multiple range test (Duncan. 1955) in order to determine the significance

of the difference

between

treatments.

RESULTS Rrstiq

Iflef7lhYullP potrrrtiuls

irt 0 K ‘- nsw

Typical intracellular measurements of the resting membrane potentials (E,) in normal and K+-free ASW are displayed in Fig. I. Each point represents one reading of E,. The average resting membrane potential of this muscle in normal ASW was -64.3 mV in A and -60.1 mV in B. When 0 Kc ASW was introduced, E, hyperpolarized by 12.1 & 0.37 mV (a = 28) within 40min. The hyperpolarization frequently diminished by ‘1-3 mV after I hr of soaking in 0 K’ ASW, probably due to loss of int~ccllLllar K;. Upon readmission of K’, E, repolarized to the original level with a slightly faster time course than the initial ~lyperpolar~zation (Fig. IA). If the duration of soaking in 0 K+ ASW exceeded 3 hr. however, an additional hyperpolarization of ICI2 mV was observed following readmission of K + (Fig. I H). The hyperpolarization was a short, transient phenomenon. When this transient hyperpolarization occurred, rcpoiarization was delayed. Repolarization time was proportjonal to the duration of soaking in 0 K ’ ASW; it was 25-30 min in a muscle after a 3 hr exposure, and 40-50 min for a muscle after 4 hr treatment in 0 K+ ASW.

ation

seen, but it was less than the control. It be noted that adding ouabain to 0 K+ ASW

was

should

(Fig. IA) caused no change in the membrane potential. The changes in the resting membrane potential in normal ASW in the presence and absence of ouabain are shown in Fig. 3. IO-” M ouabain was added to normal ASW. Responses were similar in all four muscles tested; the resting membrane potential did

not change for the first 2 hr, but the membrane depolarized by 5.2 i 0.75 mV, ranging from 2.7 to 8.6 mV after 4 hr in ouabain. Washing off ouabain caused a transient hyperpolarization similar to the response observed in a muscle after treatment with 0 KC ASW. Similar responses were obtained with low4 M strophanthidin

The experiment

was repeated

with IO-’ M ouabain

(n = 5). Three muscles showed no change of E, in the presence of this agent and no hyperpolarization was observed on washing off ouabain following 4 hr treatment. In another two muscles, the membrane depolar-

,E -80 The transient hyperpolarization following readmission of K+ was blocked by 10d4 M ouabain (Fig. 2). In Fig. 2A. the muscle was exposed to K’ ASW for 3 hr. and 10-‘ouabain was perfused. This exposure to ouabain completeIy blocked transient hyperpolarization. In Fig. ZB, 10m4M ouabain was introduced

in three muscles.

The average resting membrane potentials of seven preparations before treating with cardiac glycosides at 10m4 M, were -64.4 & 0.56 mV. These were depolarized to -59.2 mV at the end of a 4 hr treatment. The peak of the transient hyperpolarization after washing off cardiac glycosides ranged from 9. f to 16.7 mV (12.4 + 1.46mV).

10-4~ ouaboin 0

I

2

3

2

HOURS

Fig. 3. E, in the presence of lo-’ ouabain. Ouabain added to ASW caused no change in E, for the initial 2 hr. Depolarization of S-4 mV was observed after 3-hr soaking in ouabain. Washing off ouabain caused the transient hyperpolarization.

Sodium

pumping

in smooth

261

muscle

Table I, The resting membrane

potentials rewarming

0

I

2

0

I

Figures

’ HOURS

Fig. 4. Effect of “sodium loading” on transient hyperpolarization following K + restoration. A treatment supposed to increase the intracellular Na+ concentration was tested (see text). The ABRM was subjected to K+-free ASW soaking for 2 hr. In each control (first half) and test (second 3mMEGTA at open arrow, half) experiment,

3 mM EGTA isotonic NaCl at filled arrow, were applied for 5 min. On restoration

of K+. observed.

hyperpolarization

was

ized and hyperpolarization was observed when ouabain was washed off, the results were virtually identical with those of lo-4 M ouabain or strophanthidin. It appears that only muscles depolarized in ouabain demonstrate hyperpolarization on withdrawal of ouabain. EfSeectqf‘irxreased

ir~tracellular

;

.

qj:_~_;~:;::, 0

* 10

20

30

40

60 MIN

Fig. 5. Plot of the transient hyperpolarization following the treatment with EGTA solutions for various durations. Each point was the mean of 4-7 penetrations in every 5 min after restoration of K+ (time 0). Control (0). exposure

k 0.56

are expressed

-43.9 mean

* 0.96

-60.6

) 0.60

f SE. in mV.

sion of K+, as much as 15 mV hyperpolarization occurred. The significant experimental difference between these two trials was that before the first readmission of K+. the muscle was exposed to 0 K+ ASW-EGTA, while before the second, it was exposed to the isotonic NaCllEGTA. The experiment was repeated in three other muscles and the hyperpolarization ranged from 8 to 18 mV with an average of 12mV. Figure 5 is the plot of transient hyperpolarization following readmission of K’ obtained from the same preparation. The averaged value of E, demonstrated the effect of duration of treatment with the isotonic NaCl-EGTA solution. With the increase in exposure to test solution, the hyperpolarization was increased and became faster, yet repolarization was greatly delayed.

Na’

A novel method for increasing the intracellular Na+ concentration was employed, since the cells are too small to permit injection of Na+. Solutions containing EGTA have been used to increase membrane permeability (Twarog & Muneoka, 1973; Muneoka & Ichige. 1973). The experiment was performed as follows: The resting membrane potentials in normal ASW were measured, the muscle was equilibrated in 0 Kf ASW for I hr, then it was exposed to 0 K+ ASW containing 3 mM EGTA (control) for 5 min and then the muscle was restored to 0 K+ ASW for another hr. After this period, normal ASW was readmitted and the membrane potential was measured. The same muscle was again subjected to 0 K+ ASW, and after I hr, isotonic NaCl containing 3 mM EGTA (test) was introduced for 5 min. The muscle was restored to 0 K’ ASW for another hr and then normal ASW was readmitted. The results are shown in Fig. 4. Following the first readmission of K+, there may be some hyperpolarization of 4-5 mV. but this was not statistically significant. After the second readmis-

-651

-54.3

cold and

Rewarming (6’C)

Cold ( - I “C)

Control (6-C) EnI

during

for 5 min (0) and 15 min (+).

Effect oj’cold

on E,

Our previous observations indicated that the membrane of the ABRM depolarizes when the temperature is lowered (Hidaka et al., 1977). The depolarization was larger than that predicted from the Nernst equation, suggesting that E, is in part regulated by a metabolic process. Experiments were performed here to see the effects of cooling and warming on E,. The temperature employed was between f6.0 and - I.O”C. The results are illustrated in Table I. E, at 6’C was - 54.3 _t 0.56 mV (n = 9). a decrease of about IO mV from the membrane potentials at 17°C. When the temperature was lowered to - l.O”C within 7 min. the and reached membrane depolarized quickly -43.9 k 0.96 mV (11= 5). The difference was highly significant (P < 0.01). Three muscles were rewarmed to 6°C. Exposure to cold (- 1°C) never exceeded IOmin. E, hyperpolarized significantly to -60.6 k 0.60 mV following rewarming. DISCUSSION In the present study, the membrane of the ABRM invariably hyperpolarized in 0 K + ASW. This hyperpolarization probably reflects the increased concentration gradient for K+. An ABRM equilibrated in 0 K’ ASW for less than 2 hr repolarized to the original level upon readmission of K+. However, in muscles soaked in 0 K+ ASW for longer periods, the membrane further hyperpolarized when K+ was readmitted. This hyperpolarization was a transient phenomenon. Transient hyperpolarization following readmission of Kf has been observed in many tissues (Adrian & Slayman. 1966; Rang & Ritchie, 1968; Livengood & Kusano, 1972; Gorman & Marmor. 1970; Taylor et al., 1970) and has been attributed to activation of an

26X

H. YAMAGLICHIand B. M. TWAROG

Na+ pump. The observations on the ABRM are consistent with the suggestion that an clectrogenic Na+ pump is activated. The transient hyperpolarization made the membrane potential more negative than the potential previously attained in 0 K + ASW. and it was completely blocked by IO-” ouabain. In the case of the transient hyperpolarization after long soaking in ouabain or strophanthidin, an electrogenic Na+ extrusion was probably the underlying mechanism. Na+ loading would occur in the presence of cardiac glycosides and the increased Na’ ; would activate the electrogenic Na+ pump when the inhibitor was removed. It should be noted that only muscles which depolarized in the presence of cardiac glycosides displayed hyperpolarization as the inhibitory agent was washed off. An unusual observation was that cardiac glycosides appeared to be readily washed off even after hours of treatment. It is well known that cold temperatures reduce metabolic activity and can be an effective tool to depress metabolic pumps. Hyperpolarization which overshoots the control membrane potential has been observed in rewarmed muscles (Magaribuchi et al., 1973) and also in neurons (Carpenter & Alving, 1968). This hyperpolarization was considered to be due to activation of an electrogenic Na+ pump. Na’ loading was achieved by inhibition of this pump. However. there is considerable evidence that cold increased sodium influx in squid axon (Cohen & Landowne. 1974). An Increase m PNa would facilitate Na+ loading and cause stimulation of the pump upon rewarming. The evidence indicates that activation of an electrogenie Na+ pump can be demonstrated under certain circumstances. These circumstances include long exposure to 0 K + ASW or ASW containing cardiac glycosides, and also exposure of the muscle to isotonic NaCl containing EGTA. As much as 18 mV of hyperpolarization was observed after the ABRM was exposed to the NaCl-EGTA. It appears that intracellular Nat accumulation is greatest after this last type of treatment. Although evidence for the presence of an electrogenic Na+ pump in the ABRM is strong, inhibitors have no immediate effect on the resting membrane. Na+ loading apparently requires at least 3 hr with 0 K’ ASW or cardiac glycosides, suggesting that the resting Nat pump is operating extremely slowly or that the Na+-K+ exchange pump is neutral in the resting muscle. Results of the present study did not discriminate between these alternatives. The effect of cardiac glycosides were not observed continuously in the same cell, rather E, was obtained by averaging values from multiple penetrations. Thus, the method lacks the resolution of continuous observation. In squid axon (DeWeer & Geduldig, 1973) and snail neuron (Thomas, 1972b), membrane depolarization was observed immediately following perfusion of cardiac glycosides. There is growing evidence that the Na+ pump of the resting membrane is electrogenic (Brinley & Mullins. 1974; Thomas, 1969), which might suggest that a slow time course of the depolarization in the ABRM by Na pump inhibitors was due to the fact that the pump operated at a slow rate. A number of tissues in the presence of Na+ pump inhibitors (cold, cardiac glycosides) behave like the ABRM. Instead of electrogenic

depolarizing, E, hyperpolarizes following reduction of KJ (Carpenter & Alving, 1968; Gorman & Marmor, 1970; Tomita & Yamamoto, 1972; Lambert e’t al., 1974). In view of the fact that M~tilus ABRM is a marine molluscan smooth muscle. it is of interest to knou whether high external Na’ has an inhibitory effect on the electrogenic Nat pump. Baker & Connelly (1966) using oxygen consumption as an index for the activity of the Nat pump have reported that pump activity in crab nerve is inhibited by high Na,:. In squid axon. the rate of ouabain-sensitive Nat efflux is inhibited by an increased concentration of external Na + (Baker et a/., 1969). Similar inhibitory effects of Na,’ on the ouabain-sensitive Na’-K+ pump has been reported in hisodoris (Gorman & Marmor. 1970) as well as other cells (Sjodin & Medici. 1975; Garrahan & Glynn, 1967). There is no definite evidence available to explain the extremely low activity of the electrogenic Na+ pump in the ABRM. Inhibition of the Na+--K+ pump by high external Na+ could play a role in this. A~krlo~/cn(/rnlc,,rls -These studies were supported by Grants from the United States Public Health Service. NS 10554 and NS 12857.

REFERENCES ADRIAN R. H. & SLAYMA?. C. L. (lY66) Membrane potential and conductance during transport of sodium, potassium and rubidium in frog muscle. J. Ph~.siol. 184, 970. BAUK P. F. & CWN~LLY C. M. (1966) Some properties of the external activation site of the sodium pump in crab nerve. J. Pk!siol. 185, 770. BAKER P. F., BLAI!STI:I\ M. P.. KEYNES R. D.. MANIL J.. SHAW T. I. & ST~IUHARIX R. A. (1969) The ouabainsensitive Ruxes of sodium and potassium in squid giant axons. J. Phpsiol. 200. 459. BOLTON T. B. (1973) Effects of electrogenic sodium pumping on the membrane potential of longitudinal smooth muscle from terminal ileum of guinea-pig. J. PI~~s/ol. 228, 693. BRIXLICYF. J. & ML.LLINS L. J. (1974) Effects of membrane potential on sodium and potassium effluxes in squid axons. ,411~ 3’. I! Acud. SC;. 242, 406. BKOWS J. E. & LISMAU J. E. (1972) An electrogenic sodium pump in Limulm ventral photoreceptor cells. J. qoz. Phq‘siol. 59, 720. CARPENTER D. 0. & ALVINC; B. 0. (1968) A contribution ol an electrogenic Na’ pump to membrane potential in Aplysiu neurons. J. qerl. P/I@/. 52, I. CASTEELSR.. DROOGMANSG. & HEUDRIWX H. (1971) Electrogenic sodium pump in smooth muscle cells of the guinea-pig’s taenia coli. J. Ph~srol. 217, 297. COHEN L. B. & LANWWNE D. (1974) The temperature dependence of the movement of sodium ions associated with nerve impulses. J. Phwol. 236. 95. CONNOR C. & PROSSER C. L. (1974) Comparison of ionic effects on longitudinal and circular muscle of cat jeJunum. Am. J. Physiol. 226, 1212. CONNOR J. A.. PROSSER C. L. & WkEMS W. A. (1974) A study of pace-maker activity in intestinal smooth muscle. .I. Physiof. 240, 67 I. DEWkkR P. & GI:utILIxG P. (1973) Electrogenic sodium pump in squid giant axon. Scienw 179, 1326. DLWTAIL D. B. (1955) Multiple range and multiple F tests. Bi0nwtric.s 1 I. I

Sodium

pumping

GARRAHAN P. .I. & GLYNN I. M. (1967) The sensitivity

the sodium

pump

to external

sodium.

J. Physiol.

of 192,

17s. GORMAS. A. L. F. & MARMOR M. F. (1970) Temperature

dependence of the sodium-potassium permeability ratio of a molluscan neurone. J. Physiol. 210, 919. HIDAKA T.. TWAROG B. M. YAMAC;UCHIH. & MUNEOKA Y. Neurotransmitter action on the membrane of Mytilus smooth muscle: 11. Dopamine. Gen. Pharmac. 8, 87. K~,.KKUT G. A. & THOMAS R. C. (1965) An electrogenic sodium pump in snail nerve cells. Camp. Biochem. Pkysiol. 14. 617. KOIKE H., BROWS H. M. & HAG~WARA S. (1971) Hyperpolarization of a barnacle photoreceptor membrane following illumination. J. qrn. Physiol. 57. 723. KUKIYAMA H.. OSHIMA K. & SAKAMOTO Y. (1971) The membrane properties of the smooth muscle of the guinea-pig portal vein in isotonic and hypertonic solutions. J. Pllysiol. 217, 179. KUKIHAKA S. & CK~:I.D K. E. (1972) Changes in the membrane potential of the smooth muscle cells of the guinea pig urinary bladder in various environments. Jap. J. Physiol. 22, 667. LAMHIKT J. D. C.. KI.RKIIT G. A. & WALKER R. J. (1974) The elcctrogenic sodium pump and membrane potential of identified neurones in Hr,li\- aspersa. Camp. Biochrm. Physiol. 47A. X97. LIV~N~;OOD D. R. & KUSANO K. (1972) Evidence for an electrogenic sodium pump in follower cells of the lobster cardiac ganglion. J. !Veuropltysro/. 25, 170. MAGAKIRCIcHIT.. ITO Y. & KCRIYAMA H. (1973) Activation of an electrogenic sodium pump in the smooth muscle cell membrane of guinea pig taenia coli during recovery after cold treatment. Jap. J. Physiol. 23. 25. MUNEOKA Y. & ICHIGE Y. (1973) Effect of serotonin in calcium contraction in depolarized anterior byssus MUXEOKA Y. & ICHIGE Y. (1973) Effect of sorotonin in calcium contraction in depolarized anterior byssus

in smooth

muscle

269

retractor muscle of Mytilus (In Japanese). Jap. J. Smooth Muscle Res. 9, 248. NAKAJIMA S. & TAKAHASHI K. (1966) Post-tetanic hyperpolarization and electrogenic Na pump in stretch receptor neurone of crayfish. J. Physiol. 187. 105. RANG H. P. & RITCHIE J. M. (1968) On the electrogenic sodium pump in mammalian non-myelinated nerve fibres and its activation by various external cations. J. Physiol. 1%. 183. RUSSELLJ. M. & BROWN A. M. (1972) Active transport of potassium by the giant neuron of the Aplysia abdominal ganglion. J. yen. Physiol. 60, 519. SJODINR. A. & MEDICI A. (1975) Inhibitory effect of external sodium ions on the potassium pump in striated muscle. Yaturr 255, 632. SOKOLOVEP. G. & CCIOKE I. M. (1971) Inhibition of impulse activity in a sensory neuron by an electrogenic pump. J. gen. Physiol. 57, 125. TAYLOR G. S., PATON D. M. & DANIEL E. E. (1970) Characteristics of electrogenic sodium pumping in rat myometrium. J. gen. Physiol. 56, 360. THOMAS R. C. (1969) Membrane current and intracellular sodium changes in a snail neurone during extrusion of injected sodium. J. Physiol. 201, 495. THOMAS R. C. (1972a) Electrogenic sodium pump in nerve and muscle. Physiol. Rev. 52, 563. THOMAS R. C. (1972b) Intracellular sodium activity and the sodium pump in snail neurones. J. Physiol. 220, 55. TOMITAT. & YAMAMOTOY. (1971) Effects of removing the external potassium on the smooth muscle of guinea-pig taenia coli. J. Physiol. 212, 851. TWAROG B. M. & MUNEDKA Y. (1973) Calcium and the control of contraction and relaxation in a molluscan catch muscle. Cold Spring Harh. Symp. Quant. Biol. 37. 489. YAMAGUCHI H. & TWAROG B. M. (1974) Electrogenic sodium pumping in Mytilus smooth muscle. Physiologist 17, 363.