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Components of resting membrane electrogenesis in Lepidopteran skeletal muscle
J. Insect Physiol. Vol. 35, No. 9, pp. 659-666, 1989
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COMPONENTS ELECTROGENESIS
Copyright 0 1989Pergamon Press plc
OF RESTING MEMBRANE IN LEPIDOPTERAN SKELETAL MUSCLE
JILL DAWSON’, M. B. A. DJAMGOZ*‘, J. HARD& and S. N. IRVING’ ‘Department of Pure and Applied Biology, Neurobiology Group, Imperial College of Science, Technology and Medicine, London SW7 2BB and *Wellcome Research Laboratories, Berkhamstead, Herts HP4 2DY, England (Received 5 July 1988; revised 9 January 1989)
Abstract-The partial contributions of K+, Na+, Cl-, Mg2+ and an electrogenic, metabolic pump to resting membrane electrogenesis in skeletal muscles of Chile purrelk (Lepidoptera) have been quantified. The contributions of the ions were evaluated by ionic substitution experiments, whilst the role of the pump was investigated by cooling and application of ouabain. In response to changes in extracellular K+, Na+ and Cl- activities, membrane potentials changed in rapid, graded, sustained and reversible steps. For a IO-fold change in the extraccllular activity, the maximal changes in the membrane potentials were 31 mV (K+ ), 2 mV (Na+) and 17 mV (Cl- ). In constrast, changing the extracellular Mg2+ concentration had no immediate effect on resting potentials. Cooling preparations by 14°C from room temperature resulted in rapid membrane depolarizations of some 12 mV. However, application of 1 mM ouabain in Ringers containing varying concentrations of K+ had no effect on the membrane potential. It followed, therefore, that a metabolic mechanism was also involved in electrogenesis, but this was unlikely to be an ouabain-sensitive (Na+-K+) ATPase. The contributions of K+, Na+, Cl- and the metabolic pump were sufficient to account quantitatively for the prevailing resting membrane potential (mean, -46 mV) with an error margin of some 7%. Key Word Index:
Insect, muscle, ion, pump, ouabain
INTRODUCTION
Haemolymph ion levels in Lepidopteran insects are highly unusual, being relatively high in K+, Mg*+ and Ca2+ and low in Na+, compared with those of most other insects (Djamgoz, 1987). Lepidopteran skeletal muscles are physiologically interesting, therefore, since they are bathed directly in this medium (Piek and Njio, 1979). Data from intracellular ionic activity measurements, presented in an earlier paper (Djamgoz and Dawson, 1989), enabled us to make several predictions concerning possible mechanisms underlying resting membrane electrogenesis in bodywall (skeletal) muscles of larval Chile partellus (Lepidoptera). We show that the K+ equilibrium potential was significantly more positive than the resting potential (E,,,). We proposed, therefore, that the membrane must be permeable to an ion, in addition to K+, with an equilibrium potential more negative than the E,,,. The mean value of the intracellular Na+ activity was on average greater than extracellular, and a negative Na+ equilibrium potential prevailed, although this was still more positive than the E,,,. The intracellular Cl- activity was less than the value required for passive distribution, i.e. the Cl- equilibrium potential was more negative than the E,,,. Cl- could also, therefore, be contributing directly to the E,,, (Djamgoz and Dawson, 1989). Mechanisms of resting membrane electrogenesis in skeletal muscles of Lepidopteran insects were studied previously in Sphinx ligustri (Huddart, 1966b, c, 1967: Wareham, Duncan and Bowler, 1975); Actius *To whom all correspondence should be addressed.
selene, Bombyx mori (Huddart, Philosamia Cynthia (Yamaguchi,
1966a-c, 1967); Lockshin and Woodward, 1972; Piek, Njio and Mantel, 1973); Antheraea polyphemus (Rheuben, 1972); Ephestia kuhniella (Deitmer and Rathmayer, 1976; Dietmer, 1977); and reviewed extensively by Djamgoz (1987). In essence, these past studies suggested that the resting membrane potential had several components. K+ was found to have been involved consistently and to have had a dominant role. Inconsistent results were obtained with Na+, however, whereby reducing the amount of extracellular Na+ was found in one case to hyperpolarize, and in another, to depolarize the E,,, (Rheuben, 1972; Huddart, 1966a, respectively). As regards the role of Cl-, some reports suggested that this ion makes no contribution to the E,,, (Rheuben, 1972; Yamaguchi et al., 1972; Piek et al., 1973). On the other hand, others showed that sustained changes in the E,,, are obtained when the extracellular Cl- concentration is altered, implying that Cl- was involved directly in membrane electrogenesis (Huddart, 1967; Wareham et al., 1975). The causes of these apparently different results in the existing literature are not clear, and their evaluation has also been made somewhat difficult, since none of the previous studies attempted quantitatively to explain the generation of the whole E,,, in a given species. In the present study, we have attempted to achieve a unified view of the generation of the E,,, in skeletal muscles of a single species of Lepidoptera by investigating the relative contributions of a number of possibly electrogenic mechanisms, and determining whether collectively these would account completely for the prevailing E,,,.
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1 OF-
- to-
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-5o-
'L
-6O30
, -1
10
30 3
11
1
30
I
I
I
1 12
CK'I. (mM)
mln
Fig. 1. Chart recording of a K+ substitution experiment of a Chilo larval muscle cell. The bath K+ concentration ([K+ I,,) is indicated on the lowermost trace (Na+ was used as the substitute). E,,,, resting membrane potential. Before withdrawing the micro-electrode from the cell, the preparation was returned to normal Ringer ([K’]* = 30 mM) to ensure that the original value of E,,, was recorded. Impalement of the cell and the withdrawal of the micro-electrode are denoted by downward- and upward-going arrow-heads, respectively. MATERIALS AND METHODS
Experiments were carried out on segmental bodywall (skeletal) muscle of the final-instar larvae of the rice stem-borer, Ckilo partelius. Each insect was dissected dorsally to expose the ventral longitudinal muscles (Djamgoz and Dawson, 1989). Preparations were initially bathed in a saline modified from Weevers (1966), containing the following concentrations (in mM): 12 NaCl, 30 KCl, 18 MgCl,, 1.5 NaHCO,, 1.5 NaHPO,, 8 CaCl, , 117 glucose; pH 7 (Djamgoz and Dawson, 1989). Each preparation was allowed to equilibrate in this Ringer for at least 30 min prior to impalement of cells. Other details were as follows. intracellular micro-electrodes These were drawn from borosilicate glass capillaries (1 mm o.d./0.6mm i.d.) on a ventral puller. They were filled with 2.5 M KC1 and those with tip resistances in the range 10-20 MR (tip potentials <5 mV) were used. The micro-electrode was connected to a micro-probe system (WPI M701), the output of which was monitored on a chart recorder. Ionic substitution experiments K’, Na+, Cl- and Md+ substitution experiments were carried out. Modified salines were based on the Ringer solution. All solutions wece isoosmotic at 280 mOsm. Solution changes were performed manually using two disposable syringes. Each solution change was complete within some 30 s. Details of the individual sets of solutions used were as follows. K+ substitution solutions. Five different Ringerbased physiological salines with the following values of K+ concentration (in mM) were used: 1, 3, 10, 30 (normal) and 42. Na+ was used as the substitute. Na + substitution solutions. Only three substitution solutions were used, since preliminary tests indicated
that the contribution of Na+ to the E,, although consistent, was small. In order to cover a IO-fold change in extracellular Na+ concentration, the following values (in mM) were used: 15 (normal), 3.8 and 1.5. MgCI, and glucose were used simultaneously as substitutes for NaCi to maintain isoosmolarity and a constant extracellular Cl- activity. CI- substitution solutions. Five different Ringerbased physiological salines with the following Clconcentration values (in mM) were used: 0.94, 3.2, 9.4, 32 and 94 (normal). Gluconate salts were used as direct substitutes for Cl salts. lugz+ substitution solution. The role of Mg2+ in resting membrane electrogenesis was assessed by reducing the extracellular Mg2+ concentration IO-fold (18-1.8 mM) using choline chloride and glucose as substitutes to maintain iso-osmolarity and a constant extracellular Cl- activity. Since this procedure was found not to affect the E,,,, no further M8+ substitution was performed. Metabolic inhibition experiments Two types of metabolic inhibition were used. In order to test the effect of general metabolic inhibition on resting membrane electrogenesis, the temperature of the preparation was lowered from 20°C (normal room temperature) to 6”C, by introducing ice-cooled Ringer onto the preparation. In order to test inhibition specifically of the (Na+-K+) ATPase on the E,, 1 mM ouabain was applied to preparations either by adding to the normal Ringer or by including it in a series of K+ substitution solutions. RESULTS
The effect of K+ on membrane potential Figure 1 shows a chart recording of a typical experiment in which the effect on E, of varying the
Membrane potential of Lepidopteran muscle
-36.
-44
IO -
-20
w’ -46.
2
-
’
-48. -50.
-30
-
-40
-
-50
-
-60
-
w’
-52. 1
5
IO CK’I,
50 fmM)
Fig. 2. Graph showing the relationship between the resting membrane potential (,I&) and the extracellular K+ concentration ([K+],; mM; logarithmic scale). Data points denote means + SEs (n = 7; each cell from a different preparation). The maximal slope of the relationship is 30mV/lO-fold change in [K+b.
_,15
3.6
CNo’3,
extracellular K+ concentration ([K+],) in the range 1 mM to 42 mM was measured. E, responses to changes in [K+],, were fast, sustained, graded and reversible. Similar data were obtained from a total of eight cells, each from a different preparation. The average relationship between E, and the logarithm of [K+]o is shown in Fig. 2. The points follow a curve rather than a straight line. The maximal slope of the relationship is seen in the range 30 mM-42 mM and has a value of 30 mV/ IO-fold change in [K+ 1,. This is significantly different from the slope of 58 mV IO-fold change in [K+], predicted from a cell membrane permeable only to K+. In turn, this result agrees with our earlier conclusion in the K+ equilibrium potential is markedly different from the E,,, in these muscles (Djamgoz and Dawson, 1989). The effect of Na+ on membrane potential The response of the membrane to alterations in the extracellular Na+ concentration ([Na+],) was small but consistent. Figure 3 shows a representative chart recording of an experiment in which a cell was impaled with a micro-electrode in normal Ringer @la+], = 15 mM), and the effect of solutions containing [Na+],‘s of 3.8 mM and 1.5 mM on E,,, were measured. Clearly, changing pa+],, has sustained and reversible effects on the E,,,. Similar results were obtained from seven cells from seven different preparations, and data are plotted as a group in Fig. 4. In every cell, decreasing wa+10 caused the E, to hyperpolarize by some 2 mV/lO-fold change in ma+ Jo. It was concluded, therefore, Na+ makes a finite contribution to generation of the E,,,. The eflect of Cl- on membrane potential Figure 5 shows a chart recording of a typical experiment in which the effect of varying the extracellular Cl- concentration ([Cl-],) in the range 0.94 mM-94mM on E,,, was measured. Cells were impaled in the Ringer containing the lowest Clconcentration and subsequently exposed to Ringers in order of increasing Cl- concentration. This was necessary because decreasing the [Cl-], was found to cause membrane depolarization and frequent contraction of the muscle cell, leading to expulsion of the
15
( 1.5 1 (mhl)
-
6 min
Fig. 3. Chart recording of a Na+ substitution experiment on a Chile larval muscle cell. E,,,, resting membrane potential; [Na+ ]o, extracellular Na+ concentration (mM), as indicated on the lowermost trace. Before withdrawing the microelectrode out of the cell, the preparation was returned to normal Ringer @Ia+ ]a = 15 mM) to ensure that the original value of E,,, was obtained. Arrow-heads, as in Fig. 1 legend.
micro-electrode. Clearly, as [Cl-], was increased, E,,, rapidly hyperpolarized in sustained and graded steps (Fig. 5). Similar data were obtained from seven cells from seven different preparations. The average dependence of E,,, on the logarithm of [Cl-l, is shown in Fig. 6. The maximal average change in E,,, for a IO-fold change in [Cl-], was 17 mV. It was con-
-36-
w’
-40
-
-42
-
-44
-
-46
-
-46
-
-50
-
-52
-
I
1
1
IIII
5
10
20
CNo+l, ImM)
Fig. 4. Composite graph showing the relationship between the resting membrane potential (E,,,) and the extracellular Na+ concentration ([Na+b; mM; logarithmic scale) for seven different muscle cells from Chile larvae. It illustrat** .__ the small but consistent effect of changing pa+ b on E,,,. The results are presented as a family of curves due to the wide range of E,,,s encountered and the small size of the effect.
JILL DAWSQN
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et al.
-6OL 94 32 94 094
3.2
r
I
,
0.64
I tmM1
W-1. I 2 nil”
I
Fig. 5. Chart recording of Cl- substitution experiment on a Chile larval muscle cell, illustrating the sustained effects on the resting membrane potential (I$,,) of changing the extracellular Cl- concentration ([Cl-k; mM; lowermost trace). Before withdrawing the microelectrode from the cell, the preparation was returned to the original low-Cl_ Ringer ([Cl-], = 0.94 mM) to ensure that the E,,, changes were reversible. Arrow-heads, as in Fig. 1 legend. that Cl- makes a significant contribution to generation of the E,,,.
eluded, therefore, Lack
of eflect of
Mg2+ on membrane potential
Mgr+ concentration ([Mg2+10) by a factor of 10 was found to have no significant effect of the E,. A typical recording of such an experiment is shown in Fig. 7. Similar data obtained from five cells, each from a different preparation, showed clearly that any E,,, change that occurred following the application of the low-Mg’+ Ringer was small (< 1 mV), inconsistent in direction and irreversible. It was concluded, therefore, that M$+ is not involved in resting membrane electrogenesis in these cells. Reduction
of extracellular
Effect of metabolic inhibition of membrane potential A typical effect on the E,,, of reducing the temperature of a preparation from normal (~20°C) by exposing it to ice-cooled Ringer solution is shown in Fig. 8. Introduction of the cold Ringer led to a rapid and sustained depolarization of the E,,,, cooling
by 14°C resulting in an average E,,, depolarization of 12f2mV (mean+SE, n=7). In contrast, application of 1 mM ouabain to a preparation for several minutes had no discemable effect on the E,,, (Fig. 9a). We also investigated whether this lack of effect might be due to the high level of K+ in the Ringer, since Baker and Willis (1970) previously showed that ouabain competes with extracellular K+ in binding to the (Na+-KC ) ATPase. Thus, the effect of 1 mM ouabain on E,,, was tested also at the two extreme values of the [K+], range used (i.e. 1 mM and 42 mM). In both cases, inclusion of ouabain in the modified bathing solution had no effect of its own on the E, (Fig. 9b). Taken together, these results led to the conclusion that a temperature-sensitive (possibly metabolic) component makes a significant, hyperpolarizing contribution to resting membrane electrogenesis in’these
-3o-
-
-35.
h - -4ow'-45. -JO-
\__I
10 -,
1.0
CMg2’3, tmMl Fig. 6. Graph showing the relationship between the resting membrane potential (E,,,) and the extracellular Cl- concentration ([Cl-]& n&f; logarithmic scale). Data points denote means f SE’s (n = 7; each cell from a different preparation). The maximum slope of the relationship is 17 mV/lO-fold change in [Cl-],.
,
18
-2
min
Fig. 7. Chart recording of a Mg*+ substitution experiment on a Chifo larval muscle cell, showing that changing the extracellular Mg*+ concentration ([MS*+]o from 18 mM to 1.8 mM (lowermost trace) has no effect on the resting membrane potential (E,,,). Arrow-heads, as in Fig. I legend.
Membrane potential of Lepidopteran muscle
663
1
20
20
6
1
T (*C)
I 2 min
I
Fig. 8. Chart recording of an experiment in which the effect of cooling a C&lo larval preparation by 14°C on the resting membrane potential (E,,,) was measured. It shows the reversible, sustained depolarization in E,,,which resulted. The change in the temperature (T) is indicated on the lowermost trace. Arrow-heads, as in Fig. 1 legend.
muscles. This mechanism does not, however, correspond to a ouabain-sensitive (Na+-K+) ATPase. DISCUSSION
The contributions of five different, possibly electrogenie mechanisms to generation of the resting membrane potential in skeletal muscles of a single Lepidopteran species (Chilo partellus) have been investigated for the first time. Four of these (K+, Na+, Cl- and a metabolic pump) were found to be involved in membrane electrogenesis, and the relative contribution of each was quantified. In contrast, Mg2+ had no direct role in the process. Each mechanism is first discussed separately and then all are considered collectively. Potassium
The contribution of K+ to resting membrane electrogenesis in skeletal muscles of a number of Lepidopteran species (noted in the Introduction) was studied earlier. The resting membranes were found consistently to ha;e an appreciable permeability to K+, such that the membrane potential changed by a maximum of 30-35 mV for a lo-fold change in the activity (Huddart, 1966c; extracellular K’ Yamaguchi et al., 1972; Rheuben, 1972; Wareham et al., 1975; Dietmer, 1977). In Chile, E,,, depolarized by a maximum of 30 mV for a IO-fold increase in [KC],. In the K+ substitution experiments, Na+ was used as a convenient substitute. However, since Na+ itself has a finite role in membrane electrogenesis, the contribution of K+ to the E, needs to be reevaluated. In the [K + lo range 30-42 mM, [Na+ IOwas reduced from 15 to 3 mM, and the latter change alone would be expected to result in about 1 mV hyperpolarization of the E,,, (see Fig. 4). Thus, the maximal slope of the relationship between the E,,, and [K+l, is probably nearer 31 mV/lO-fold change in the
latter. The involvement of K+ in resting membrane electrogenesis in skeletal muscles of Chile is therefore quantitatively very similar to other Lepidoptera. Importantly, K+ is clearly not the only ion involved in this process, however. A similar conclusion was reached earlier by demonstrating that the K+ equilibrium potential in these muscles is markedly different (more positive) than the prevailing E,,, (Djamgoz and Dawson, 1989). In all Lepidopteran species studies, including Chile, the membrane potential was found to be relatively insensitive to [K+l, changes in the range below the normal. Piek (1975) showed that if the skeletal muscles of Philosamia were “conditioned” for 5 h in a low-K+ Ringer, then the relationship between E,,, and [K+],, became log-linear over a much wider range of [K+ ],,‘s.In contrast, Huddart (1966b), using a similar experimental protocol in Sphinx and Actias muscles, found that the E,,, differed from the EK by about 20mV in the [K+k, range 20-2OOmM, and in the range l-20 mM, E,,, was still relatively insensitive to changes in [K+],,. Whilst the cause(s) of this disagreement is not certain, an overall limitation of experiments involving soaking muscles in artificial salines for up to 6 h would be that it is not clear if the physiological integrity of the cell membranes was maintained, i.e. it is not known whether the E,,, changes recorded were reversible and/or whether the preparations suffered irreversible deterioration. Nevertheless, both the intracellular K+ activity measurements (Djamgoz and Dawson, 1989) and K+ substitution data (this study) from Chile skeletal muscles consistently suggest that K+ is involved in resting membrane electrogenesis only to a limited extent. Chloride
We previously that the chloride
showed in Chilo skeletal muscles equilibrium
potential
is significantly
664
JILL DAWSON
3
et al. A
1
(a)
-2o-
E
w’
-3o-
-4o-
1
-5o'-
tb)
A
5 min
,
0
-10 T E
w’
-20
-
Ii
-30
t’
b
30
42 L
I
1 CK’I,
42 ,-,
30
(mM)
Fig. 9. Lack of effect of 1mM ouabain on the resting membrane potential (E,,,) of Chile larval muscle cells. (a) Chart recording of an experiment in which ouabain was simply introduced onto the preparation in normal Ringer ([K+l, = 30 mM). The duration of ouabain application is indicated by the horizontal bar above the E, trace. (b) Chart recording of an experiment showing that ouabain has no effect on the E,,, in Ringers containing lower or higher K+ concentrations (1 and 42 mM, respectively) than normal (30 mM). Extracellular K+ concentrations ([K+k,s) are indicated the lowermost trace; applications of ouabain-containing solutions one denoted by thick lines. Arrow-heads, as in Fig. I legend.
more negative than the E,,, (Djamgoz and Dawson, 1989). This result could not be explained by any artefact of measurement and raised the possibility that Cl- was not in passive equilibrium with the E,,,. This has been confirmed in the present study by demonstrating that changing [Cl-], has graded, sustained and reversible effects on the E,,,, in accordance with the hypothesis that Cl- makes a direct contribution to resting membrane electrogenesis in these muscles. Huddart (1967) and Wareham et al. (1975) working on Sphinx found that changing [Cl-], produced sustained changes in the E,,,of skeletal muscles, and therefore, made a similar proposal. In Sphinx, E,,, hyperpolarized by lOmV/lO-fold increase in [Cl-], (Huddart, 1967), compared with 17 mV in Chile (this study). Huddart (1967) also showed that the intracellular Cl- concentration decreased by only 6% when the extracellular value was reduced 200-fold, indicating a low permeability of the membranes to Cland/or the presence of a regulatory mechanism (e.g. a Cl- pump) for maintaining intracellular Cl- at a relatively steady level. It is not known if Cl- extra-
sion from cells is coupled to another ion (e.g. HCO; ) and also if this mechanism is electrogenic (Russell, 1980). Somewhat different effects of Cl- have been reported in some other Lepidopteran species, however, Yamaguchi et al. (1972) showed that increasing the extracellular Cl - concentration 6-fold hyperpolarized the E,,, of skeletal muscles of Philosamiu pupae only transiently, the E,,, gradually repolarizing over 160 min to become some 20 mV more positiue than the original level. Rheuben (1972) found in adult Antherueu that in a Cl--free medium, the Em initially depolarized and then returned to the original level within 10 min. In an extensive study on various skeletal muscles of Philosumiu, Piek et al. (1973) showed that the E,,, largely hyperpolarized if monocarboxylates were used as Cl- substitutes, whilst with citrate (a trivalent carboxylate) and sulphate, rapid and large depolarizations were seen, lasting for the whole duration (10 min) of the Cl--free saline application. In view of the rather limited work done on the
Membrane potential of Lepidopteran muscle involvement of Cl- in skeletal muscles of Lepidoptera, the cause(s) of the discrepancies in the existing literature is not clear at present. In the case of C&lo, however, both the intracellular Cl- data (Djamgoz and Dawson, 1989) and the result of the Cl- substitution experiments (this study) consistently suggest that Cl- has a significant, direct role in resting membrane electrogenesis in the skeletal muscles.
665
possibly by affecting levels of intracellular Mg’+ and the activity of M$+-dependent ATPase(s). Metabolic pump
The maintenance of the E,,, in Chile skeletal muscles was found to be extremely temperature-sensitive such that cooling by 14°C led to rapid membrane depolarization by some 12 mV. Comparable effects of metabolic inhibition have been described previously. Thus, application of 0.5 mM dinitrophenol depolarSodium ized the membrane potentials of skeletal muscles of Changing ma+], produced graded and reversible Philosamia and Sphinx, some 50% of E,,,s being lost effects on the E,,,, and thereby suggested that Na+ is over 2 h (Huddart and Wood 1966; Piek et al., 1973). involved to a limited extent in membrane electroAn even greater degree of depolarization was obgenesis. The change in E,,, was 2 mV/lO-fold change served resulting from anoxia in skeletal muscles of in [Na+ ]o. Rheuben (1972) also found that reducing Antheraea (Rheuben, 1972). In the latter study, it was [Na+10 hyperpolarized the E, of the skeletal muscles also established that during the anoxia-induced memof adult Antheruea, the maximal change being about brane depolarization, the intracellular K+ concentra5 mV per IO-fold change in ma+ I,,. However, a tion did not change, so that effect could not be due different result was obtained by Huddart (1966a) in to a shift in the K+ equilibrium potential; a small but skeletal muscles of Bombyx, whereby lowering [Na+], significant decrease in membrane input impedance was found to &polarize the E,,,. One possible cause was observed, but this was not related to K+ permeof this discrepancy is that under the conditions of ability (Rheuben, 1972). Generally similar results Huddart’s (1966a) experiments, again involving soakwere obtained in skeletal muscles of Philosamia by ing for long periods, lowering [Nat], produced a Piek et al. (1973). Taken together, the available data secondary, opposite effect which suppressed the direct clearly suggest, therefore, that a part of the E, in action of Na+. For example, lowering ma+],, was skeletal muscles of Lepidoptera is metabolic in origin. found to lead to a proportional decrease in intracelIn several other cell types also, direct contributions to lular Na+ concentration (Huddart, 1966a), which in resting membrane electrogenesis by electrogenic turn might have influenced the activity of a metabolic pumps have been found. In some systems (e.g. squid mechanism and therefore brought about a secondary giant axons), the contribution of the pump is very change in the E,,, in the ‘wrong’ direction. We have small (De Weer and Geduldig, 1973), whilst in others already suggested that a metabolic mechanism is (e.g. Lettre cells), the membrane potential appears to active in generating a part of the E,,, in Chile muscles be generated exclusively by metabolic pump activity (also, see later). (Bashford and Pastemak, 1984, 1986). It is interesting also to note that in skeletal muscles Interestingly, the metabolic mechanism in Chile of larval Calliphoru eryfhrocephafu, we previously muscle is not inhibited by ouabain, irrespective of the showed that lowering [Na+ 1, leads to a biphusic extracellular K+ concentration (see also Rheuben, change of the E,,,, an initial, transient hyperpolariza1972), and it may, therefore, be different from a depolarization tion and a delayed, sustained conventional (Na+-K+)ATPase. This may not be (Djamgoz and Dawson, 1988). The latter depolarizaso surprising, considering that the primary function tion of the E, was attributed to a reduction in the of a conventional (Na+-K+)ATPase is to maintain trans-membrane Na+ and K+ gradients, in inward partial K+ permeability of the membrane. Such an and outward directions, respectively. We have aleffect does not appear to operate in Chilo muscles, ready demonstrated in Chilo skeletal muscles that the since lowering [Nat], simply produced sustained Na+ gradient is in the outward direction whilst the hyperpolarizations of the E, (Fig. 3). K+ gradient, although outward, is much less than Magnesium usual (Djamgoz, 1987; Djamgoz and Dawson, 1989). Thus, the main functional characteristic of Removing the bulk of Mg*+ from the bathing medium had no immediate effect on the E,,, (Fig. 7), a conventional (Na+-K+)ATPase may not be suitthereby suggesting that M$+ does not play a signifi- able for maintenance of the unconventional transcant, direct role in resting membrane electrogenesis gradients membrane Na+ and K+ concentration in Chilo muscles. Similar results were reported in prevailing in skeletal muscles of Lepidoptera, and two other Lepidopteran insects: Antheraea poly- this situation may be consistent with the mechanism phemus (Rheuben, 1972), and Philosamia Cynthia in Lepidoptera being pharmacologically different, i.e. ouabain-insensitive. On the other hand, in the central (Yamaguchi et al., 1972). In skeletal muscles of final nervous system of Lepidoptera, where transinstar larvae and pupae of Antheraea pernyi, however, reducing (Mg2+ I,,resulted in depolarizations of the E,,, membrane Na+ and K+ activity gradients are conventional due to presence of a well-established blood(Weevers, 1966). The direction of the E,,, change brain-barrier (Lane, 1972), ouabain-specific binding implied that the role of Mg2+ in resting membrane (Na+-K+ )electrogenesis was not direct. McCann (1963) also sites indicative of a ouabain-sensitive found in the heart muscles of A. polyphemus that in ATPase have been observed (Rubin, Stirling and a Mg*+-free medium, E,,,s depolarized gradually such Stahl, 1983). Multiple molecular forms of (Na+-K+)ATPase with varying sensitivity to cardiac glycosides that some 77% of the E, was lost over 2 h. Taken together, these results imply that Mg2+ might have are known to exist in the vertebrate central nervous system (Sweadner, 1979; Specht and Sweadner, a metabolic effect on Lepidopteran skeletal muscles,
666
JILL
Table
I.
Fractional
membrane
contributions
electrogenesis
Maximal
change
change
Nat
to resting
Chile parfeflus
in &/IO-fold Fractional
of ion (mV)
K+
31
Nat
2
Cl
and Cl
muscles of larval
in extracellular,
concentration
Ion
of K+,
in skeletal
DA WS0li
contribution
of ion to f?, (%) 53 3
I7
29 .___
The
fractional obtaind
contribution
by expressing
in the extracellular fraction
of each
the maximal activity
of 58 (theoretical
permeable remain
(%)
of
change
the
maximum
to that ion), assuming
ion
ion
Total
85%
(third
column)
in &,/IO-fold (second
column)
for a membrane
that internal
was
change as a
selectively
ion concentraions
the same.
1984). Further electrophysiological aspects of metabolic pumps in insect muscles have been discussed more extensively by Djamgoz (1986).
et al.
Dietmer J. W. (1977) Electrical properties of skeletal ~~ of the flour moth larva, Ephestia kuhniella 2. fibres (Lepidoptera). J. Insect Physiol. 23, 33-38. Dietmer J. W. and Rathmayer W. (1976) Calcium action potentials in larval muscle fibres of the moth Ephestiu kuhnielia Z. (Lepidoptera). J. camp. Physiol. 112, 123-132. . - Djamgoz M. B. A. (1986) Electrophysiological aspects of metabolic pumping in insect muscle. Comp. Biochem. Physiol. 84A, 207-2 15. Djamgoz M. B. A. (1987) Insect muscle: intracellular ion concentrations and mechanisms of resting potential generation. J. Insect Physiol. 33, 287-314. Djamgoz M. B. A. and Dawson J. (1988) Quantitative analysis of resting membrane electrogenesis in insect (Diptera) skeletal muscles. II. Testing of a model involving contributions from potassium and sodium ions, and the anomalous effect of reducing extracellular sodium. J. exp. Biol. 136, 433449. Djamgoz M. B. A. and Dawson J. (1989) Ion-sensitive micro-electrode measurements of intracellular K+, Na+ and Cl- activities in lepidopteran skeletal muscle. J. Insect Physiol. 35, 165-l 73. Huddart H. (1966a) The effect of sodium ions on resting and action potentials in skeletal muscle fibres of Eombyx mori.
Summation of the fractional contributions of K +, Na +, Cl- and the presumed metabolic pump to resting membrane electrogenesis It would be worthwhile considering whether the Archs int. Physiol. Biochim. 74, 592602. four electrogenic mechanisms investigated here would Huddart H. (1966b) Ionic composition of haemolymph and quantitatively account for the whole E,,, prevailing in myoplasm in the Lepidoptera in relation to their memChile skeletal muscles. Using the results of the ionic brane potentials. Archs inr. Physiol. Biochim 74, 603613. substitution experiments, the fractional contributions Huddart H. (1966c) The effect of potassium ions on resting and action potentials in Lepidopteran muscle. Comp. of K+, Na+ and Cl- to E,,, can be calculated, as Biochem. Physiol. 18, 131-140. outlined and summarised in Table 1. Thus, K+, Na+ Huddart H. (1967) Effect of chloride ions on moth skeletal and Cl- collectively can account for 85% of resting muscle fibres. Comp. B&hem. Physiol. ZOA, 355-359. membrane electrogenesis. It would follow, therefore, Huddart H. and Wood D. W. (1966) The effect of DNP on that the remaining 15% i.e. -7 mV (of the mean E, the resting potential and ionic content of some insect of -46 mV) was due to at least one other electrogenic skeletal muscle fibres. Comp. Biochem. Physiol. 18, mechanism. Cooling the cells caused an average 681688. depolarization of 12 mV, 2 mV of which could be due Lane N. J. (1972) Fine structure of a Lepidopteran nervous system and its accessibility to peroxidase and lanthanum. to the effect of temperature on the equilibrium potenZ. Zellforsch. mikrosk. Anat. 131, 205-222. tials of the three ions. The average observed depolarization attributable to the metabolic pump was McCann F. V. (1963) Electrophysiology of an insect heart. J. gen Physiol. 46, 803-821. therefore 3 mV larger than predicted. It may be possible that a part of the depolarizing effect of Piek T. and Njio K. D. (1979) Morphology and electrochemistry of insect muscle fibre membrane. Ado. Insect lowering the temperature was mediated indirectly by Physiol. 15, 185-249. a rise in intracellular Ca2+ activity leading to opening Piek T.. Njio K. D. and Mantel P. (1973) Effects of anions of Caz+-dependent K+ channels (Deitmer and and dinitrophenol on resting membrane potentials of Rathmayer, 1976). In conclusion, within an error insect muscle libres. J. Insecf Physiof. 19, 2373-2392. limit of some 7%, the contributions of K+, Na+, ClRheuben M. (1972) The resting potential of moth muscle fibre. J. Physiol. 225, 529-554. and an electrogenic pump are sufficient to generate Rubin A. L., Stirling C. E. and Stahl W. L. (1983) ‘H-ouathe E,,, of Chifo skeletal muscles. We cannot, however. bain autoradiography in the abdominal nerve cord of the exclude minor involvement of other ions (e.g. Ca’+, hawk moth, Manduca sexta. J. exp. Biol. 104, 217-230. H+), and further work is required to elucidate the Russell J. M. (1980) Anion transport mechanisms in neunature of the proposed electrogenic pump. rones. Ann. N. Y. Acad. Sci. 341, 510-523. Acknowledgemeni-This study was supported by a SERC CASE studentship to Jill Dawson (ICI Agrochemicals as collaborating body). REFERENCES
Baker P. F. and Willis J. S. (1970) Potassium ions and the binding of cardiac glycosides to mammalian cells. Nature 226, 521-523. Bashford C. L. and Pasternak C. A. (1984) Plasma membrane potential of Lettre cells does not depend on cation gradients but on pumps. J. memb. Biol. 79, 275-284. Bashford C. L. and Pasternak C. A. (1986) Plasma membrane potential of some animal cells is generated by ion pumping, not by ion gradients. Trends Biochem. Sci. 11, 113-116. De Weer P. and Geduldig D. (1973) Electrogenic sodium pump in squid giant axon. Science, N. Y. 179, 13261328.
Specht S. C. and Sweadner K. J. (1984) Two different Na, K-ATPases in the optic nerve: cells of origin and axonal transport. Proc. natn. Acad. Sci. U.S.A. 81, 12361238. Sweadner K. J. (1979) Two molecular forms of (Na+ + K+ )-stimulated ATPase in brain: separation and difference in affinity for strophanthidin. J. biol. Chem. 254, 606&6067.
Wareham A. C., Duncan C. J. and Bowler K. (1975) The resting potential of the muscle membrane of moths. Como. Biochem. Phvsiol. SZA, 295-298. Weevers R. de G. (1966) a Lepidopteran saline; effects of inorganic cation concentration on sensory, reflex and motor responses in a herbivorous insect. J. exp. Biol. 44, 163-175. Yamaguchi H., Lockshin R. A. and Woodward D. J. (1972) The intersegmental muscle of silkmoths. Ionic components of activity. J. Insecr Physiol. 18, 243-258.
0022-1910/89 $3.00 + 0.00
Prmted in Great Britain. All rights reserved
COMPONENTS ELECTROGENESIS
Copyright 0 1989Pergamon Press plc
OF RESTING MEMBRANE IN LEPIDOPTERAN SKELETAL MUSCLE
JILL DAWSON’, M. B. A. DJAMGOZ*‘, J. HARD& and S. N. IRVING’ ‘Department of Pure and Applied Biology, Neurobiology Group, Imperial College of Science, Technology and Medicine, London SW7 2BB and *Wellcome Research Laboratories, Berkhamstead, Herts HP4 2DY, England (Received 5 July 1988; revised 9 January 1989)
Abstract-The partial contributions of K+, Na+, Cl-, Mg2+ and an electrogenic, metabolic pump to resting membrane electrogenesis in skeletal muscles of Chile purrelk (Lepidoptera) have been quantified. The contributions of the ions were evaluated by ionic substitution experiments, whilst the role of the pump was investigated by cooling and application of ouabain. In response to changes in extracellular K+, Na+ and Cl- activities, membrane potentials changed in rapid, graded, sustained and reversible steps. For a IO-fold change in the extraccllular activity, the maximal changes in the membrane potentials were 31 mV (K+ ), 2 mV (Na+) and 17 mV (Cl- ). In constrast, changing the extracellular Mg2+ concentration had no immediate effect on resting potentials. Cooling preparations by 14°C from room temperature resulted in rapid membrane depolarizations of some 12 mV. However, application of 1 mM ouabain in Ringers containing varying concentrations of K+ had no effect on the membrane potential. It followed, therefore, that a metabolic mechanism was also involved in electrogenesis, but this was unlikely to be an ouabain-sensitive (Na+-K+) ATPase. The contributions of K+, Na+, Cl- and the metabolic pump were sufficient to account quantitatively for the prevailing resting membrane potential (mean, -46 mV) with an error margin of some 7%. Key Word Index:
Insect, muscle, ion, pump, ouabain
INTRODUCTION
Haemolymph ion levels in Lepidopteran insects are highly unusual, being relatively high in K+, Mg*+ and Ca2+ and low in Na+, compared with those of most other insects (Djamgoz, 1987). Lepidopteran skeletal muscles are physiologically interesting, therefore, since they are bathed directly in this medium (Piek and Njio, 1979). Data from intracellular ionic activity measurements, presented in an earlier paper (Djamgoz and Dawson, 1989), enabled us to make several predictions concerning possible mechanisms underlying resting membrane electrogenesis in bodywall (skeletal) muscles of larval Chile partellus (Lepidoptera). We show that the K+ equilibrium potential was significantly more positive than the resting potential (E,,,). We proposed, therefore, that the membrane must be permeable to an ion, in addition to K+, with an equilibrium potential more negative than the E,,,. The mean value of the intracellular Na+ activity was on average greater than extracellular, and a negative Na+ equilibrium potential prevailed, although this was still more positive than the E,,,. The intracellular Cl- activity was less than the value required for passive distribution, i.e. the Cl- equilibrium potential was more negative than the E,,,. Cl- could also, therefore, be contributing directly to the E,,, (Djamgoz and Dawson, 1989). Mechanisms of resting membrane electrogenesis in skeletal muscles of Lepidopteran insects were studied previously in Sphinx ligustri (Huddart, 1966b, c, 1967: Wareham, Duncan and Bowler, 1975); Actius *To whom all correspondence should be addressed.
selene, Bombyx mori (Huddart, Philosamia Cynthia (Yamaguchi,
1966a-c, 1967); Lockshin and Woodward, 1972; Piek, Njio and Mantel, 1973); Antheraea polyphemus (Rheuben, 1972); Ephestia kuhniella (Deitmer and Rathmayer, 1976; Dietmer, 1977); and reviewed extensively by Djamgoz (1987). In essence, these past studies suggested that the resting membrane potential had several components. K+ was found to have been involved consistently and to have had a dominant role. Inconsistent results were obtained with Na+, however, whereby reducing the amount of extracellular Na+ was found in one case to hyperpolarize, and in another, to depolarize the E,,, (Rheuben, 1972; Huddart, 1966a, respectively). As regards the role of Cl-, some reports suggested that this ion makes no contribution to the E,,, (Rheuben, 1972; Yamaguchi et al., 1972; Piek et al., 1973). On the other hand, others showed that sustained changes in the E,,, are obtained when the extracellular Cl- concentration is altered, implying that Cl- was involved directly in membrane electrogenesis (Huddart, 1967; Wareham et al., 1975). The causes of these apparently different results in the existing literature are not clear, and their evaluation has also been made somewhat difficult, since none of the previous studies attempted quantitatively to explain the generation of the whole E,,, in a given species. In the present study, we have attempted to achieve a unified view of the generation of the E,,, in skeletal muscles of a single species of Lepidoptera by investigating the relative contributions of a number of possibly electrogenic mechanisms, and determining whether collectively these would account completely for the prevailing E,,,.
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1 OF-
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-5o-
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30 3
11
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I
I
1 12
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Fig. 1. Chart recording of a K+ substitution experiment of a Chilo larval muscle cell. The bath K+ concentration ([K+ I,,) is indicated on the lowermost trace (Na+ was used as the substitute). E,,,, resting membrane potential. Before withdrawing the micro-electrode from the cell, the preparation was returned to normal Ringer ([K’]* = 30 mM) to ensure that the original value of E,,, was recorded. Impalement of the cell and the withdrawal of the micro-electrode are denoted by downward- and upward-going arrow-heads, respectively. MATERIALS AND METHODS
Experiments were carried out on segmental bodywall (skeletal) muscle of the final-instar larvae of the rice stem-borer, Ckilo partelius. Each insect was dissected dorsally to expose the ventral longitudinal muscles (Djamgoz and Dawson, 1989). Preparations were initially bathed in a saline modified from Weevers (1966), containing the following concentrations (in mM): 12 NaCl, 30 KCl, 18 MgCl,, 1.5 NaHCO,, 1.5 NaHPO,, 8 CaCl, , 117 glucose; pH 7 (Djamgoz and Dawson, 1989). Each preparation was allowed to equilibrate in this Ringer for at least 30 min prior to impalement of cells. Other details were as follows. intracellular micro-electrodes These were drawn from borosilicate glass capillaries (1 mm o.d./0.6mm i.d.) on a ventral puller. They were filled with 2.5 M KC1 and those with tip resistances in the range 10-20 MR (tip potentials <5 mV) were used. The micro-electrode was connected to a micro-probe system (WPI M701), the output of which was monitored on a chart recorder. Ionic substitution experiments K’, Na+, Cl- and Md+ substitution experiments were carried out. Modified salines were based on the Ringer solution. All solutions wece isoosmotic at 280 mOsm. Solution changes were performed manually using two disposable syringes. Each solution change was complete within some 30 s. Details of the individual sets of solutions used were as follows. K+ substitution solutions. Five different Ringerbased physiological salines with the following values of K+ concentration (in mM) were used: 1, 3, 10, 30 (normal) and 42. Na+ was used as the substitute. Na + substitution solutions. Only three substitution solutions were used, since preliminary tests indicated
that the contribution of Na+ to the E,, although consistent, was small. In order to cover a IO-fold change in extracellular Na+ concentration, the following values (in mM) were used: 15 (normal), 3.8 and 1.5. MgCI, and glucose were used simultaneously as substitutes for NaCi to maintain isoosmolarity and a constant extracellular Cl- activity. CI- substitution solutions. Five different Ringerbased physiological salines with the following Clconcentration values (in mM) were used: 0.94, 3.2, 9.4, 32 and 94 (normal). Gluconate salts were used as direct substitutes for Cl salts. lugz+ substitution solution. The role of Mg2+ in resting membrane electrogenesis was assessed by reducing the extracellular Mg2+ concentration IO-fold (18-1.8 mM) using choline chloride and glucose as substitutes to maintain iso-osmolarity and a constant extracellular Cl- activity. Since this procedure was found not to affect the E,,,, no further M8+ substitution was performed. Metabolic inhibition experiments Two types of metabolic inhibition were used. In order to test the effect of general metabolic inhibition on resting membrane electrogenesis, the temperature of the preparation was lowered from 20°C (normal room temperature) to 6”C, by introducing ice-cooled Ringer onto the preparation. In order to test inhibition specifically of the (Na+-K+) ATPase on the E,, 1 mM ouabain was applied to preparations either by adding to the normal Ringer or by including it in a series of K+ substitution solutions. RESULTS
The effect of K+ on membrane potential Figure 1 shows a chart recording of a typical experiment in which the effect on E, of varying the
Membrane potential of Lepidopteran muscle
-36.
-44
IO -
-20
w’ -46.
2
-
’
-48. -50.
-30
-
-40
-
-50
-
-60
-
w’
-52. 1
5
IO CK’I,
50 fmM)
Fig. 2. Graph showing the relationship between the resting membrane potential (,I&) and the extracellular K+ concentration ([K+],; mM; logarithmic scale). Data points denote means + SEs (n = 7; each cell from a different preparation). The maximal slope of the relationship is 30mV/lO-fold change in [K+b.
_,15
3.6
CNo’3,
extracellular K+ concentration ([K+],) in the range 1 mM to 42 mM was measured. E, responses to changes in [K+],, were fast, sustained, graded and reversible. Similar data were obtained from a total of eight cells, each from a different preparation. The average relationship between E, and the logarithm of [K+]o is shown in Fig. 2. The points follow a curve rather than a straight line. The maximal slope of the relationship is seen in the range 30 mM-42 mM and has a value of 30 mV/ IO-fold change in [K+ 1,. This is significantly different from the slope of 58 mV IO-fold change in [K+], predicted from a cell membrane permeable only to K+. In turn, this result agrees with our earlier conclusion in the K+ equilibrium potential is markedly different from the E,,, in these muscles (Djamgoz and Dawson, 1989). The effect of Na+ on membrane potential The response of the membrane to alterations in the extracellular Na+ concentration ([Na+],) was small but consistent. Figure 3 shows a representative chart recording of an experiment in which a cell was impaled with a micro-electrode in normal Ringer @la+], = 15 mM), and the effect of solutions containing [Na+],‘s of 3.8 mM and 1.5 mM on E,,, were measured. Clearly, changing pa+],, has sustained and reversible effects on the E,,,. Similar results were obtained from seven cells from seven different preparations, and data are plotted as a group in Fig. 4. In every cell, decreasing wa+10 caused the E, to hyperpolarize by some 2 mV/lO-fold change in ma+ Jo. It was concluded, therefore, Na+ makes a finite contribution to generation of the E,,,. The eflect of Cl- on membrane potential Figure 5 shows a chart recording of a typical experiment in which the effect of varying the extracellular Cl- concentration ([Cl-],) in the range 0.94 mM-94mM on E,,, was measured. Cells were impaled in the Ringer containing the lowest Clconcentration and subsequently exposed to Ringers in order of increasing Cl- concentration. This was necessary because decreasing the [Cl-], was found to cause membrane depolarization and frequent contraction of the muscle cell, leading to expulsion of the
15
( 1.5 1 (mhl)
-
6 min
Fig. 3. Chart recording of a Na+ substitution experiment on a Chile larval muscle cell. E,,,, resting membrane potential; [Na+ ]o, extracellular Na+ concentration (mM), as indicated on the lowermost trace. Before withdrawing the microelectrode out of the cell, the preparation was returned to normal Ringer @Ia+ ]a = 15 mM) to ensure that the original value of E,,, was obtained. Arrow-heads, as in Fig. 1 legend.
micro-electrode. Clearly, as [Cl-], was increased, E,,, rapidly hyperpolarized in sustained and graded steps (Fig. 5). Similar data were obtained from seven cells from seven different preparations. The average dependence of E,,, on the logarithm of [Cl-l, is shown in Fig. 6. The maximal average change in E,,, for a IO-fold change in [Cl-], was 17 mV. It was con-
-36-
w’
-40
-
-42
-
-44
-
-46
-
-46
-
-50
-
-52
-
I
1
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IIII
5
10
20
CNo+l, ImM)
Fig. 4. Composite graph showing the relationship between the resting membrane potential (E,,,) and the extracellular Na+ concentration ([Na+b; mM; logarithmic scale) for seven different muscle cells from Chile larvae. It illustrat** .__ the small but consistent effect of changing pa+ b on E,,,. The results are presented as a family of curves due to the wide range of E,,,s encountered and the small size of the effect.
JILL DAWSQN
662
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,
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I tmM1
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I
Fig. 5. Chart recording of Cl- substitution experiment on a Chile larval muscle cell, illustrating the sustained effects on the resting membrane potential (I$,,) of changing the extracellular Cl- concentration ([Cl-k; mM; lowermost trace). Before withdrawing the microelectrode from the cell, the preparation was returned to the original low-Cl_ Ringer ([Cl-], = 0.94 mM) to ensure that the E,,, changes were reversible. Arrow-heads, as in Fig. 1 legend. that Cl- makes a significant contribution to generation of the E,,,.
eluded, therefore, Lack
of eflect of
Mg2+ on membrane potential
Mgr+ concentration ([Mg2+10) by a factor of 10 was found to have no significant effect of the E,. A typical recording of such an experiment is shown in Fig. 7. Similar data obtained from five cells, each from a different preparation, showed clearly that any E,,, change that occurred following the application of the low-Mg’+ Ringer was small (< 1 mV), inconsistent in direction and irreversible. It was concluded, therefore, that M$+ is not involved in resting membrane electrogenesis in these cells. Reduction
of extracellular
Effect of metabolic inhibition of membrane potential A typical effect on the E,,, of reducing the temperature of a preparation from normal (~20°C) by exposing it to ice-cooled Ringer solution is shown in Fig. 8. Introduction of the cold Ringer led to a rapid and sustained depolarization of the E,,,, cooling
by 14°C resulting in an average E,,, depolarization of 12f2mV (mean+SE, n=7). In contrast, application of 1 mM ouabain to a preparation for several minutes had no discemable effect on the E,,, (Fig. 9a). We also investigated whether this lack of effect might be due to the high level of K+ in the Ringer, since Baker and Willis (1970) previously showed that ouabain competes with extracellular K+ in binding to the (Na+-KC ) ATPase. Thus, the effect of 1 mM ouabain on E,,, was tested also at the two extreme values of the [K+], range used (i.e. 1 mM and 42 mM). In both cases, inclusion of ouabain in the modified bathing solution had no effect of its own on the E, (Fig. 9b). Taken together, these results led to the conclusion that a temperature-sensitive (possibly metabolic) component makes a significant, hyperpolarizing contribution to resting membrane electrogenesis in’these
-3o-
-
-35.
h - -4ow'-45. -JO-
\__I
10 -,
1.0
CMg2’3, tmMl Fig. 6. Graph showing the relationship between the resting membrane potential (E,,,) and the extracellular Cl- concentration ([Cl-]& n&f; logarithmic scale). Data points denote means f SE’s (n = 7; each cell from a different preparation). The maximum slope of the relationship is 17 mV/lO-fold change in [Cl-],.
,
18
-2
min
Fig. 7. Chart recording of a Mg*+ substitution experiment on a Chifo larval muscle cell, showing that changing the extracellular Mg*+ concentration ([MS*+]o from 18 mM to 1.8 mM (lowermost trace) has no effect on the resting membrane potential (E,,,). Arrow-heads, as in Fig. I legend.
Membrane potential of Lepidopteran muscle
663
1
20
20
6
1
T (*C)
I 2 min
I
Fig. 8. Chart recording of an experiment in which the effect of cooling a C&lo larval preparation by 14°C on the resting membrane potential (E,,,) was measured. It shows the reversible, sustained depolarization in E,,,which resulted. The change in the temperature (T) is indicated on the lowermost trace. Arrow-heads, as in Fig. 1 legend.
muscles. This mechanism does not, however, correspond to a ouabain-sensitive (Na+-K+) ATPase. DISCUSSION
The contributions of five different, possibly electrogenie mechanisms to generation of the resting membrane potential in skeletal muscles of a single Lepidopteran species (Chilo partellus) have been investigated for the first time. Four of these (K+, Na+, Cl- and a metabolic pump) were found to be involved in membrane electrogenesis, and the relative contribution of each was quantified. In contrast, Mg2+ had no direct role in the process. Each mechanism is first discussed separately and then all are considered collectively. Potassium
The contribution of K+ to resting membrane electrogenesis in skeletal muscles of a number of Lepidopteran species (noted in the Introduction) was studied earlier. The resting membranes were found consistently to ha;e an appreciable permeability to K+, such that the membrane potential changed by a maximum of 30-35 mV for a lo-fold change in the activity (Huddart, 1966c; extracellular K’ Yamaguchi et al., 1972; Rheuben, 1972; Wareham et al., 1975; Dietmer, 1977). In Chile, E,,, depolarized by a maximum of 30 mV for a IO-fold increase in [KC],. In the K+ substitution experiments, Na+ was used as a convenient substitute. However, since Na+ itself has a finite role in membrane electrogenesis, the contribution of K+ to the E, needs to be reevaluated. In the [K + lo range 30-42 mM, [Na+ IOwas reduced from 15 to 3 mM, and the latter change alone would be expected to result in about 1 mV hyperpolarization of the E,,, (see Fig. 4). Thus, the maximal slope of the relationship between the E,,, and [K+l, is probably nearer 31 mV/lO-fold change in the
latter. The involvement of K+ in resting membrane electrogenesis in skeletal muscles of Chile is therefore quantitatively very similar to other Lepidoptera. Importantly, K+ is clearly not the only ion involved in this process, however. A similar conclusion was reached earlier by demonstrating that the K+ equilibrium potential in these muscles is markedly different (more positive) than the prevailing E,,, (Djamgoz and Dawson, 1989). In all Lepidopteran species studies, including Chile, the membrane potential was found to be relatively insensitive to [K+l, changes in the range below the normal. Piek (1975) showed that if the skeletal muscles of Philosamia were “conditioned” for 5 h in a low-K+ Ringer, then the relationship between E,,, and [K+],, became log-linear over a much wider range of [K+ ],,‘s.In contrast, Huddart (1966b), using a similar experimental protocol in Sphinx and Actias muscles, found that the E,,, differed from the EK by about 20mV in the [K+k, range 20-2OOmM, and in the range l-20 mM, E,,, was still relatively insensitive to changes in [K+],,. Whilst the cause(s) of this disagreement is not certain, an overall limitation of experiments involving soaking muscles in artificial salines for up to 6 h would be that it is not clear if the physiological integrity of the cell membranes was maintained, i.e. it is not known whether the E,,, changes recorded were reversible and/or whether the preparations suffered irreversible deterioration. Nevertheless, both the intracellular K+ activity measurements (Djamgoz and Dawson, 1989) and K+ substitution data (this study) from Chile skeletal muscles consistently suggest that K+ is involved in resting membrane electrogenesis only to a limited extent. Chloride
We previously that the chloride
showed in Chilo skeletal muscles equilibrium
potential
is significantly
664
JILL DAWSON
3
et al. A
1
(a)
-2o-
E
w’
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-4o-
1
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tb)
A
5 min
,
0
-10 T E
w’
-20
-
Ii
-30
t’
b
30
42 L
I
1 CK’I,
42 ,-,
30
(mM)
Fig. 9. Lack of effect of 1mM ouabain on the resting membrane potential (E,,,) of Chile larval muscle cells. (a) Chart recording of an experiment in which ouabain was simply introduced onto the preparation in normal Ringer ([K+l, = 30 mM). The duration of ouabain application is indicated by the horizontal bar above the E, trace. (b) Chart recording of an experiment showing that ouabain has no effect on the E,,, in Ringers containing lower or higher K+ concentrations (1 and 42 mM, respectively) than normal (30 mM). Extracellular K+ concentrations ([K+k,s) are indicated the lowermost trace; applications of ouabain-containing solutions one denoted by thick lines. Arrow-heads, as in Fig. I legend.
more negative than the E,,, (Djamgoz and Dawson, 1989). This result could not be explained by any artefact of measurement and raised the possibility that Cl- was not in passive equilibrium with the E,,,. This has been confirmed in the present study by demonstrating that changing [Cl-], has graded, sustained and reversible effects on the E,,,, in accordance with the hypothesis that Cl- makes a direct contribution to resting membrane electrogenesis in these muscles. Huddart (1967) and Wareham et al. (1975) working on Sphinx found that changing [Cl-], produced sustained changes in the E,,,of skeletal muscles, and therefore, made a similar proposal. In Sphinx, E,,, hyperpolarized by lOmV/lO-fold increase in [Cl-], (Huddart, 1967), compared with 17 mV in Chile (this study). Huddart (1967) also showed that the intracellular Cl- concentration decreased by only 6% when the extracellular value was reduced 200-fold, indicating a low permeability of the membranes to Cland/or the presence of a regulatory mechanism (e.g. a Cl- pump) for maintaining intracellular Cl- at a relatively steady level. It is not known if Cl- extra-
sion from cells is coupled to another ion (e.g. HCO; ) and also if this mechanism is electrogenic (Russell, 1980). Somewhat different effects of Cl- have been reported in some other Lepidopteran species, however, Yamaguchi et al. (1972) showed that increasing the extracellular Cl - concentration 6-fold hyperpolarized the E,,, of skeletal muscles of Philosamiu pupae only transiently, the E,,, gradually repolarizing over 160 min to become some 20 mV more positiue than the original level. Rheuben (1972) found in adult Antherueu that in a Cl--free medium, the Em initially depolarized and then returned to the original level within 10 min. In an extensive study on various skeletal muscles of Philosumiu, Piek et al. (1973) showed that the E,,, largely hyperpolarized if monocarboxylates were used as Cl- substitutes, whilst with citrate (a trivalent carboxylate) and sulphate, rapid and large depolarizations were seen, lasting for the whole duration (10 min) of the Cl--free saline application. In view of the rather limited work done on the
Membrane potential of Lepidopteran muscle involvement of Cl- in skeletal muscles of Lepidoptera, the cause(s) of the discrepancies in the existing literature is not clear at present. In the case of C&lo, however, both the intracellular Cl- data (Djamgoz and Dawson, 1989) and the result of the Cl- substitution experiments (this study) consistently suggest that Cl- has a significant, direct role in resting membrane electrogenesis in the skeletal muscles.
665
possibly by affecting levels of intracellular Mg’+ and the activity of M$+-dependent ATPase(s). Metabolic pump
The maintenance of the E,,, in Chile skeletal muscles was found to be extremely temperature-sensitive such that cooling by 14°C led to rapid membrane depolarization by some 12 mV. Comparable effects of metabolic inhibition have been described previously. Thus, application of 0.5 mM dinitrophenol depolarSodium ized the membrane potentials of skeletal muscles of Changing ma+], produced graded and reversible Philosamia and Sphinx, some 50% of E,,,s being lost effects on the E,,,, and thereby suggested that Na+ is over 2 h (Huddart and Wood 1966; Piek et al., 1973). involved to a limited extent in membrane electroAn even greater degree of depolarization was obgenesis. The change in E,,, was 2 mV/lO-fold change served resulting from anoxia in skeletal muscles of in [Na+ ]o. Rheuben (1972) also found that reducing Antheraea (Rheuben, 1972). In the latter study, it was [Na+10 hyperpolarized the E, of the skeletal muscles also established that during the anoxia-induced memof adult Antheruea, the maximal change being about brane depolarization, the intracellular K+ concentra5 mV per IO-fold change in ma+ I,,. However, a tion did not change, so that effect could not be due different result was obtained by Huddart (1966a) in to a shift in the K+ equilibrium potential; a small but skeletal muscles of Bombyx, whereby lowering [Na+], significant decrease in membrane input impedance was found to &polarize the E,,,. One possible cause was observed, but this was not related to K+ permeof this discrepancy is that under the conditions of ability (Rheuben, 1972). Generally similar results Huddart’s (1966a) experiments, again involving soakwere obtained in skeletal muscles of Philosamia by ing for long periods, lowering [Nat], produced a Piek et al. (1973). Taken together, the available data secondary, opposite effect which suppressed the direct clearly suggest, therefore, that a part of the E, in action of Na+. For example, lowering ma+],, was skeletal muscles of Lepidoptera is metabolic in origin. found to lead to a proportional decrease in intracelIn several other cell types also, direct contributions to lular Na+ concentration (Huddart, 1966a), which in resting membrane electrogenesis by electrogenic turn might have influenced the activity of a metabolic pumps have been found. In some systems (e.g. squid mechanism and therefore brought about a secondary giant axons), the contribution of the pump is very change in the E,,, in the ‘wrong’ direction. We have small (De Weer and Geduldig, 1973), whilst in others already suggested that a metabolic mechanism is (e.g. Lettre cells), the membrane potential appears to active in generating a part of the E,,, in Chile muscles be generated exclusively by metabolic pump activity (also, see later). (Bashford and Pastemak, 1984, 1986). It is interesting also to note that in skeletal muscles Interestingly, the metabolic mechanism in Chile of larval Calliphoru eryfhrocephafu, we previously muscle is not inhibited by ouabain, irrespective of the showed that lowering [Na+ 1, leads to a biphusic extracellular K+ concentration (see also Rheuben, change of the E,,,, an initial, transient hyperpolariza1972), and it may, therefore, be different from a depolarization tion and a delayed, sustained conventional (Na+-K+)ATPase. This may not be (Djamgoz and Dawson, 1988). The latter depolarizaso surprising, considering that the primary function tion of the E, was attributed to a reduction in the of a conventional (Na+-K+)ATPase is to maintain trans-membrane Na+ and K+ gradients, in inward partial K+ permeability of the membrane. Such an and outward directions, respectively. We have aleffect does not appear to operate in Chilo muscles, ready demonstrated in Chilo skeletal muscles that the since lowering [Nat], simply produced sustained Na+ gradient is in the outward direction whilst the hyperpolarizations of the E, (Fig. 3). K+ gradient, although outward, is much less than Magnesium usual (Djamgoz, 1987; Djamgoz and Dawson, 1989). Thus, the main functional characteristic of Removing the bulk of Mg*+ from the bathing medium had no immediate effect on the E,,, (Fig. 7), a conventional (Na+-K+)ATPase may not be suitthereby suggesting that M$+ does not play a signifi- able for maintenance of the unconventional transcant, direct role in resting membrane electrogenesis gradients membrane Na+ and K+ concentration in Chilo muscles. Similar results were reported in prevailing in skeletal muscles of Lepidoptera, and two other Lepidopteran insects: Antheraea poly- this situation may be consistent with the mechanism phemus (Rheuben, 1972), and Philosamia Cynthia in Lepidoptera being pharmacologically different, i.e. ouabain-insensitive. On the other hand, in the central (Yamaguchi et al., 1972). In skeletal muscles of final nervous system of Lepidoptera, where transinstar larvae and pupae of Antheraea pernyi, however, reducing (Mg2+ I,,resulted in depolarizations of the E,,, membrane Na+ and K+ activity gradients are conventional due to presence of a well-established blood(Weevers, 1966). The direction of the E,,, change brain-barrier (Lane, 1972), ouabain-specific binding implied that the role of Mg2+ in resting membrane (Na+-K+ )electrogenesis was not direct. McCann (1963) also sites indicative of a ouabain-sensitive found in the heart muscles of A. polyphemus that in ATPase have been observed (Rubin, Stirling and a Mg*+-free medium, E,,,s depolarized gradually such Stahl, 1983). Multiple molecular forms of (Na+-K+)ATPase with varying sensitivity to cardiac glycosides that some 77% of the E, was lost over 2 h. Taken together, these results imply that Mg2+ might have are known to exist in the vertebrate central nervous system (Sweadner, 1979; Specht and Sweadner, a metabolic effect on Lepidopteran skeletal muscles,
666
JILL
Table
I.
Fractional
membrane
contributions
electrogenesis
Maximal
change
change
Nat
to resting
Chile parfeflus
in &/IO-fold Fractional
of ion (mV)
K+
31
Nat
2
Cl
and Cl
muscles of larval
in extracellular,
concentration
Ion
of K+,
in skeletal
DA WS0li
contribution
of ion to f?, (%) 53 3
I7
29 .___
The
fractional obtaind
contribution
by expressing
in the extracellular fraction
of each
the maximal activity
of 58 (theoretical
permeable remain
(%)
of
change
the
maximum
to that ion), assuming
ion
ion
Total
85%
(third
column)
in &,/IO-fold (second
column)
for a membrane
that internal
was
change as a
selectively
ion concentraions
the same.
1984). Further electrophysiological aspects of metabolic pumps in insect muscles have been discussed more extensively by Djamgoz (1986).
et al.
Dietmer J. W. (1977) Electrical properties of skeletal ~~ of the flour moth larva, Ephestia kuhniella 2. fibres (Lepidoptera). J. Insect Physiol. 23, 33-38. Dietmer J. W. and Rathmayer W. (1976) Calcium action potentials in larval muscle fibres of the moth Ephestiu kuhnielia Z. (Lepidoptera). J. camp. Physiol. 112, 123-132. . - Djamgoz M. B. A. (1986) Electrophysiological aspects of metabolic pumping in insect muscle. Comp. Biochem. Physiol. 84A, 207-2 15. Djamgoz M. B. A. (1987) Insect muscle: intracellular ion concentrations and mechanisms of resting potential generation. J. Insect Physiol. 33, 287-314. Djamgoz M. B. A. and Dawson J. (1988) Quantitative analysis of resting membrane electrogenesis in insect (Diptera) skeletal muscles. II. Testing of a model involving contributions from potassium and sodium ions, and the anomalous effect of reducing extracellular sodium. J. exp. Biol. 136, 433449. Djamgoz M. B. A. and Dawson J. (1989) Ion-sensitive micro-electrode measurements of intracellular K+, Na+ and Cl- activities in lepidopteran skeletal muscle. J. Insect Physiol. 35, 165-l 73. Huddart H. (1966a) The effect of sodium ions on resting and action potentials in skeletal muscle fibres of Eombyx mori.
Summation of the fractional contributions of K +, Na +, Cl- and the presumed metabolic pump to resting membrane electrogenesis It would be worthwhile considering whether the Archs int. Physiol. Biochim. 74, 592602. four electrogenic mechanisms investigated here would Huddart H. (1966b) Ionic composition of haemolymph and quantitatively account for the whole E,,, prevailing in myoplasm in the Lepidoptera in relation to their memChile skeletal muscles. Using the results of the ionic brane potentials. Archs inr. Physiol. Biochim 74, 603613. substitution experiments, the fractional contributions Huddart H. (1966c) The effect of potassium ions on resting and action potentials in Lepidopteran muscle. Comp. of K+, Na+ and Cl- to E,,, can be calculated, as Biochem. Physiol. 18, 131-140. outlined and summarised in Table 1. Thus, K+, Na+ Huddart H. (1967) Effect of chloride ions on moth skeletal and Cl- collectively can account for 85% of resting muscle fibres. Comp. B&hem. Physiol. ZOA, 355-359. membrane electrogenesis. It would follow, therefore, Huddart H. and Wood D. W. (1966) The effect of DNP on that the remaining 15% i.e. -7 mV (of the mean E, the resting potential and ionic content of some insect of -46 mV) was due to at least one other electrogenic skeletal muscle fibres. Comp. Biochem. Physiol. 18, mechanism. Cooling the cells caused an average 681688. depolarization of 12 mV, 2 mV of which could be due Lane N. J. (1972) Fine structure of a Lepidopteran nervous system and its accessibility to peroxidase and lanthanum. to the effect of temperature on the equilibrium potenZ. Zellforsch. mikrosk. Anat. 131, 205-222. tials of the three ions. The average observed depolarization attributable to the metabolic pump was McCann F. V. (1963) Electrophysiology of an insect heart. J. gen Physiol. 46, 803-821. therefore 3 mV larger than predicted. It may be possible that a part of the depolarizing effect of Piek T. and Njio K. D. (1979) Morphology and electrochemistry of insect muscle fibre membrane. Ado. Insect lowering the temperature was mediated indirectly by Physiol. 15, 185-249. a rise in intracellular Ca2+ activity leading to opening Piek T.. Njio K. D. and Mantel P. (1973) Effects of anions of Caz+-dependent K+ channels (Deitmer and and dinitrophenol on resting membrane potentials of Rathmayer, 1976). In conclusion, within an error insect muscle libres. J. Insecf Physiof. 19, 2373-2392. limit of some 7%, the contributions of K+, Na+, ClRheuben M. (1972) The resting potential of moth muscle fibre. J. Physiol. 225, 529-554. and an electrogenic pump are sufficient to generate Rubin A. L., Stirling C. E. and Stahl W. L. (1983) ‘H-ouathe E,,, of Chifo skeletal muscles. We cannot, however. bain autoradiography in the abdominal nerve cord of the exclude minor involvement of other ions (e.g. Ca’+, hawk moth, Manduca sexta. J. exp. Biol. 104, 217-230. H+), and further work is required to elucidate the Russell J. M. (1980) Anion transport mechanisms in neunature of the proposed electrogenic pump. rones. Ann. N. Y. Acad. Sci. 341, 510-523. Acknowledgemeni-This study was supported by a SERC CASE studentship to Jill Dawson (ICI Agrochemicals as collaborating body). REFERENCES
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