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Localization of sodium and potassium ions in a flight muscle of Pieris brassicae
J. Insect
Ph~siol..
1917, Vol. 23. pp. 919 to 929. Pergamon
Press. Pri,ued
ill Great Britain.
LOCALIZATION OF SODIUM AND POTASSIUM IONS IN A FLIGHT MUSCLE OF PIERIS BRASSICAE K. D. NJIO and T. PIEK Pharmacolcgical
Laboratory.
University
of Amsterdam.
(Recerwd
Polderwcg
21 Decmhrr
104. Amsterdam.
The Netherlands
19761
Abstract-T he ultrastructural localization of sodium and potassium ions in the longitudinal flight muscle of f’ieris hrassicar was carried out irr situ and after incubation in various media. The results indicate a rtzlatively high sodium and a low potassium content of the lumen of the transverse tubular system. Inca bation in a propionate saline results in an accumulation of potassium ions in the mitochondria and a decrease in concentration in the myoplasm.
INTRODUCTION
STUDIESin the last 3Qyr on the sarcotubular system of skeletal muscle fibres have led to the conclusion that the transverse !.ubular system (TTS) is continuous with the “surface” plasma membrane. Therefore, the lumen of the TTS is considered to be in continuity with the extracellular space. This has been demonstrated for vertebrates. crustaceans and insects (cf. ELDER, 1975). The continuity of the TX-lumen with the extracellular spice does. however, not necessarily mean that the ionic, composition in both spaces must be identical. PIF.K ( 974. 1975) suggested that a highly active sodium-potassium exchange pump might be operating in the extensive transverse tubular membrane. This could lead to a concentration of sodium chloride in the TTS-lumen. which is much higher than in the extracellular space and may produce a significant liquid junction potential at the opening of the TTS. In muscle fibrzs of P/lilo.sartriLccythiu the synaptic region is separated from the extracellular space by a glial cell. Only very long and narrow channels lead from the synaptic cleft to the outer environment (PIEK. 1974, 1975). Therefore, the postsynaptic membrane may be considered as a part of the invaginated plasma membrane and the fluid inside the invaginated parts of the membrane (mainly the TTS) is the outer medium for the postsynaptic membrane. The fact that these muscle fibres show hyperpolarizing inhibitory pos&ynaptic potentials. due to an activation for chloride ions, indicate that a high chloride equilibrium potential is present across the postsynaptic membrane and that therefore the fluid in the TTSlumen has a high concentration of chloride ions. No argument has beer presented for the presence of a high concentration of sodium ions in the TTS. The present paper deals with attempts to estimate the concentrations of sodium and potassium ions inside and outside the plasma membrane including the TTS-lumen. The techniques used are based on earlier
work on vertebrate muscle (ZADUNAIXY, 1966: SHIINA et al., 1968: SHIINA and MIZUHIRA, 1970).
MATERIALS
AND
METHODS
The longitudinal flight muscle of Pieris h~ssicc~ L. was dissected and put into the fixative either immediately or after incubation for 60min in various media. The composition of these media is summarized in Table 1. For the detection of sodium and potassium ions the method of SHIINA and MIZ~~~IRA (1970) was slightly modified. For sodium ion detection 1 vol of 49” osmium tetroxide + 3 vol of 3”” potassium hexahydroxoantimonate (V), K[Sb(OH),], (= potassium pyroantimonate) in distilled water or in 50 mmol per liter potassium phosphate buffer pH 7.4 was used as a fixative. For potassium ion detection I”;, glutaraldehyde + 3”, sodium hexanitrocobaltate (III). Na,[Co(NO,),]. in 30 mmol/l sodium phosphate buffer pH 7.4 was used. In later experiments the washing in yellow ammonium sulfide solution as described by SHIINA and MIZUHIRA (1970) was omitted in order to avoid the precipitation of a magnesiumammonium-phosphate complex. The cor~trols were fixed in the same fixatives but the reagents. without i.e. K[Sb(OH,]. resp. Na,[Co(N02),]. After fixation the pieces of muscle were washed in the buffer. subsequently dehydrated in ethanol and embedded in Epon according to LUFT (1961). The ultrathin sections were stained with uranyl acetate. In order to find an explanation for the low concentration of potassium in homogenized muscles after incubation in propionate (PINK uf ul., 1977). muscle preparations were tried for potassium ions after incubation in propionate saline containing per liter Napropionate 74 mmol, K-propionate 10 mmol CaCl, 6 mmol, and sucrose 170 mmol.
920
K. D. NJIO AND T. PIE-s mitochondria (Fig. 4). After incubation for 60min in medium V (74 mmol/l choline Cl) no precipitate was formed but some granules were present on the inside of the lamina basalis (Fig. 5). The localization of the sodium ions is summarized in Table 2. In the controls no precipitate was observed. In the preparations made for the localization of potassium ions it was often very difficult, if not impossible, to decide whether the precipitate was present in the sarcoplasmic reticulum (SR) or in the subsarcolemma1 cistemae (SSC). In the urltrraferl muscle fine precipitate was found in the region of the SR and of the SSC, on the myofibrils and in the mitochondria (Fig. 6). Occasionally a big granule was seen in the TTS, but this was never observed when the washing in yellow ammonium sulfide was omitted. After incubation for 60 min in medium I containing/l IO mmol KCl. 56 mmol NaCl and 9 mmol MgC12. the precipitate was found in the region of the SSC and the SR and in the mitochondria. As in the untreated muscle the big granules in the TTS were only seen after washing in yellow ammonium sulfide. After incubation for 60min in medium II containing no KCl, and 66 mmol NaCl and 9 mmol MgClJl only the big granules in the TTS were found, but only after washing with yellow ammonium sulfide. The localization of the precipitate after incubation for 60min in medium III containing no NaCl and per liter 10 mmol KC1 and 37 mmol MgCl,. was the same as after incubation in medium I. Again the big granules in the TTS were only seen after washing in yellow ammonium sulfide. After incubation for 60 min in medium IV containing/l 10 mmol KCl, 74 mmol NaCl and no MgC12, precipitate was found on the myofibrils, in the region of the SSC and the SR. Occasionally some precipitate was observed in the mitochondria. The same localization of the precipitate was found after incubation for 60 min in medium V containing/l 10 mmol KC1 and 74mmol choline Cl (Fig. 7). After incubation in propionute S&P precipitate was only found in the mitochondria (Fig. 8). The localization of the potassium ions are summarized in Table 3. In the controls some big granules were found in the TTS after washing in yellow ammonium sulfide. otherwise no precipitate was observed.
Table 1. Composition of the media, in which the longitudinal flight muscle of P. hrassicae was incubated for 60min Concn
KCI NsCl MU, CaCI, Choline Cl NaHCO, SUCrO%
mmal!l
I
II
III
IO 56 9 6 _
~~ 66 9 6
IO ~~ 37 6
I 180
I 180
IV
V
IO 71
IO
6
6 73 ~ 170
_ ~ 210
I 180
All media were used in potassium ion detection, the media I, III. IV, and V were also used in sodium ion detec-
tion.
RESULTS In the preparations made for the localization of the sodium ions the sarcoplasm and the mitochondria were badly swollen when the fixative was dissolved in distilled water. Because of this swelling the exact localization of the precipitate was extremely difficult. Therefore, phosphate buffer was used instead of distilled water. In the untreated muscle sodium ions were present in high concentration in the TTS (Figs. 1 and 2) and in the region of the sarcolemma. on the inside and outside of the lamina basalis (Fig. 2). We also observed some precipitate on the myofibrils, but we could not find any precipitate concentrated on the I-band as described by SHIINA and MIZUHIRA (1970). Some precipitate was also found in the mitochondria (Figs. 1 and 2). After incubation for 60 min in medium I containing/l 10 mm01 KCl, 56mmol NaCl and 9 mmol MgCl, , the localization of the precipitate was the same as in the untreated muscle, but to a lesser amount. After incubation for 60min in medium III containing no NaCl and 37 mmol MgCl, per liter, some big granules were found in the TTS and on the lamina basalis, and sporadically in the mitochondria (Fig. 3). After incubation for 60 min in medium IV containing 74 mmol NaCl/I and no MgCl,, the precipitate was heavily concentrated on the inside and outside of the lamina basalis and in the TTS. A fine precipitate was found on the myofibrils and in the
Table 2. Localization
NP, no precipitate:
ions
IO mm01 K 56 mmol Na 9 mmoi Mg medium I
IO mmol K 0 mmol Na 37 mmol Mg medium III
IO mm4 K 74 mmol Na 0 mmol Mg medium IV
IO mmol K 0 nmml Nn (1 mmal Mg medtum V
11 II II NP
(11 (1)
111 (11
II I I NP NP
NP
It NP
(1) (1) 1 NP
Untreated muscle Oufslde lamina basalis Inside lamma basalis TTS SR Myofibnls Mllochondria
of sodium
*
(1) some precipitate;
1 considerable
(1) I
(11
precipitate;
li heavy
precipitate.
Ilr NP NP NP NP
Control NP NP NP NP NP NP
Fig. 1. T’he distribution of antimonate precipitate in an untreated flight muscle of Pieris hrassicue. (b) and (I:) are details of (a) showing triadic and dyadic structures containing precipitate in the TTSlumen. Scale: 1 pm.
Fig. 2. The distribution of antimonate precipitate in the untreated flight Precipitate is found on the lamina basalis on both sides of an intercellular
muscle of Pirris hrussicue. space (arrow). Scale: 1 Arm.
Fig. 3. The distribution of antimonate precipitate after incubation for 60 min in medium 111 (no Na -. 37 mmol/l Mg”). Precipitate is found on the lamina basalis and some granules are seen in the TTSlumen. This precipitate is probably due to magnesium. Scale: 1 /rrn.
Fig. 4. After incubation for 60min in medium IV (74mmol/l Nat. no Mg*+) a heavy antimonate Some granules are seen in the prec ipitate is found on the lamina basalis and in the TTS-lumen. mitochondria and on the myofibrils. Scale: 1 pm. Fig. 5. Alter incubation
for 60 min in choline saline (medium V) only some antimonate found on the inside of the lamina basalis. Scale: 1 pm.
granules
are
Fig. 6. Distribution granules are found
of cobaltate precipitate in the untreated flight muscle of Pieris on the myofibrils, in the mitochondria and in the subsarcolemmal sarcoplasmic reticulum. Scale: 1pm.
hrc~~sicu~. The
cisternae
and
Fig. 7. After incubation for 60min in choline saline (medium V) cobaltate precipitate is found on the myofibrils. in the mitochondria and in the subsarcolemmal cisternae and sarcoplasmic reticulum. (b) is a detail of (a). Scale: 1 pm.
Fig. 8. After
incubation
for 60min in propionate saline cobaltate precipitate mitochondria. (b) is a detail of (a). Scale: 1 pm.
is only
found
in I.he
Sodium
and potassium
ions in insect muscle
Table
3. Localization
of potassium
927
ions
I@mmol K 56 mm4 Na 9 mmol Mg medium
0 mmol K 66 mmol Na 9 mmol Mg medwm II
IO mmol K 0 mmol Na 37 mmol Mg medium III
10 mm01 K 74 mmol Na 0 mmol Mg medium IV
IO mmol K 0 mmol Na 0 mmol Mg medium V
NP NP*
NP NP*
NP NP
NP NP
I
NP NP* NP
NP NP*
I i
i
(1) hP
f 1
I
Propionate Salme
Colltr01
I_amna
basahs TTS SR,SSC Myofibrlls Mltochondrn
NP. no precipitate; 1 precipitate * Precipitate 01114 after washing
in yellow ammonium
I i NP
cil
NP NP NP NP I
NP NP* NP NP NP
sulfide solution.
DISCUSSION To study basic processes underlying physiological phenomena electraphysiologists try to describe the electrochemical properties of membrane of cells and fibres. In axonal membranes. for instance, the membrane potential can be explained by passive permeation of ions. whit? have different concentrations on both sides of the membrane. and for which the membrane has different relative permeabilities. However, in other structures (e.g. some muscle fibres) this simple electrocherlical relation seems to be insufficient to describe the observed phenomena. The discrepancies found are often explained by assuming that one is not well informed about the ionic composition inside as well as outside the membrane. The electron microscopic techniques developed for the localization of sodium ions (KOMNICK. 1962) and potassium ions (SHIINA and MIZ~I-~IRA 1970) might throw some light on the presence of differences in ionic composition inside and outside the membranes. Unfortunately these techniques a-e far from quantitative. They can only discriminate between no precipitate. some precipitate, considerable precipitate and a heavy prccipitate. In this paper this evaluation of the intensity of the precipitation has been adapted. According to the intensity of the precipitation of the sodium hexah;:droxoantimonate (V) complex the conclusion may be drawn that the muscle fibres of P. hmMm~ fixed directly after the integument had been opened. probably contain more sodium in the T-B-lumen than outside the surface plasma membrane. An important question is, however. how reliable the data are. According to TORACE; and LAVALLE (1970) the use of an aqueous solution of hexahydroxoantimonate as a specific histochemical marker for sodium ions is not ‘gery reliable, since at physiological levels potassium as well as sodium are precipitated. According to SHIII\ A et nl. (I 970) precipitates of calcium and magnesium complexes may be formed too. In addition a pH of less than 7.2 appears to favour the production of non-specific precipitates (TORACX and LAVALLE. 1970; SHIINA or d. 1970). In the present experiments the pH of the fixative was 7.4. The use of the phosphate buffer did not affect the formation
of the precipitate of sodium ions as stated by TORACK and LAVALLE (1970) and SHIINA et al. (1970). The molarity of our buffer, however. was lower (50mmol/l) than used by TORACK and LAVALLE (200 mmol/l) and SHIINA et ~1.(100 mmol/l). Nevertheless it is likely that magnesium and possibly also calcium ions may contribute to the precipitates. After incubation of the muscle for one hour in a sodium and magnesium free medium. containing/l IO mmol potassium ions and 6 mmol calcium ions (med. V), no precipitate was found, except a few grains between the plasma membrane and the lamina basalis. It is unlikely that this is a potassium or calcium precipitate, since the highest concentration of these ions is considered to be present inside the plasma membrane. Therefore, it can be concluded that in our experiments hexahydroxoantimonate does not precipitate with either potassium or calcium. Moreover, the precipitation of calcium with the antimonate is performed in a solution at pH 7.8 (SATO et al., 1975; SAETERSDAL rt al.. 1974). After incubation in a medium without magnesium. containing 74 mmol/I sodium ions (med. IV) the precipitate was concentrated on both sides of the lamina basalis and in the TTS. Some precipitate was found in the myofibrils and in the mitochondria, somewhat more than in the untreated preparations. The concentration at the level of the lamina basalis was higher than in the untreated muscle, probably due to the fact that the sodium ion concentration in the saline is higher in the haemolymph. RAMSAY(1953) found 9.0mmolfl in the haemolymph of P. hrmsicac~ larvae. In four species adult Lepidoptera the sodium content of the haemolymph was not more than 16 mmol/l (cf. FLORKIN and JEUSIAUX, 1974). The fact that incubation with 37 mmol per liter magnesium ions (med. III) causes a precipitate with antimonate in the TTS that is less heavy than in the untreated preparations. indicates that the heavy precipitate in the TTS in the untreated preparations may mainly consist of sodium antimonate. It must be stated that the sodium detection method is very difficult. Occasionally a muscle showed a diffusely dispersed, electron-opaque precipitate, even after incubation in a medium without sodium or magnesium ions. We were not able to detect the cause
928
K. D. NJIO AND T. PIEK
of this precipitate. Presumably it is due to a change in pH in the muscle and/or the fixative which caused the antimonate to precipitate. This diffuse precipitate is, however, easily recognized as an artefact. In accordance with SHIINA and MIZUHIRA (1970) precipitates of potassium ions were found scattered on the myolibrils and in the mitochondria. In contrast to SHIINA and MIZUHIFU (1970), who also localized potassium in the lumen of the TTS. we did not conclude that the precipitate in the TTS was due to the presence of potassium. The reason for this view is that in the experiments with untreated muscles and after incubation in medium I (9 mmol/l MgCl, and III (37 mmol/l MgC1J made with and without the washing in yellow ammonium sulfide, the localization of the precipitate was the same in all cases, only the granules in the TTS had disappeared when the washing was omitted. Moreover, in the absence of magnesium-ions (media IV and V) no precipitate in the TTS was formed. Therefore, these granules probably represent the precipitation of the insoluble magnesiumammonium-phosphate complex. Outside the TTS the precipitate could only be found scattered on the SR, the SSC, and the myofibrils. It is uncertain whether the precipitate localized at the SR and SSC is situated inside or outside the membranes of these structures. It is also questionable whether calcium ions are involved in the precipitation. Precipitate near the SR and SSC. however, have only been found in preparations incubated in potassium containing media. and not in medium II, which contains 6 mmol CaClJ but no potassium. It is therefore probably that the precipitate scattered on the SR and SSC is actually potassium. Varying the potassium concentration in the incubation medium did not result in distinct differences in precipitation with nitrocoboltate. Therefore, the muscles were incubated for one hour in a medium in which most of the chloride was replaced by propionate. PIEK rt uI. (1977) had found that propionate saline causes reduction of the potassium content in extracts of muscle homogenates. though they could not explain this finding. The results of the present experiments suggest a reduction of the potassium content of the myoplasm and an enhanced concentration of potassium ions inside the mitochondria. This finding agrees with the low potassium concentration found in extracts of muscle homogenates bathed in a propionate saline (PIEK et al. 1977). In Exopterygota (cockroaches, locusts. and stick insects) the recorded muscle fibre resting potentials fit those calculated from the internal and external potassium concentrations (HOYLE, 1955; WOOD, 1957). However. in Endopterygota (mealworm and moth) the recorded resting membrane potentials differ considerably from the theoretical values (BELTON and GRUNDFEST, 1962a, b; HUDDART. 1966). It is generally accepted that the high concentration of potassium ions in the myoplasm at least contributes to the generation of the resting membrane poten-
tial. This view is strongly supported by the observations that increase of the potassium concentration in the bath reduced the resting membrane potential with a slope, approximately predicted by the Nernst equation for a potassium electrode. The propionate saline. however. did reduce the potassium content measured in the homogenate and caused also a distinct decrease in potassium localized in the myoplasm (PIK er trl. 1977). Nevertheless, the resting membrane potential is largely increased. This suggests that other ions might be involved in the generation of the membrane potential. The conclusion is that potassium ion detection with nitrocobaltate does not enable us to localize the potassium very well, but that it is highly probable that potassium ions are not abundant inside the lumen of the TTS. Sodium and magnesium ions, however, are present inside the TTS, sodium ions in a concentration that might be higher than in the haemolymph or bathing fluid of the muscle. Since arguments are presented for a high chloride concentration in the TTS-lumen (PIEK. 1974, 1975) the conclusion that the invaginated muscle fibre membrane of Lepidoptera, and possibly also of other insects. creates inside the TTS-lumen and inside the synaptic spaces its own external ionic environment, is supported by the present results, indicating a low potassium and high sodium content of the fluid in the TTS-lumen. Acknowledgenlents-The authors thank Professor Dr. C. VAN DER MEER for his professional advice and Mr. G. A. C. BELLING and Mr. P. MANTEL for their technical assistance.
REFERENCES BELTON P. and GRUNDFEST H. (1963a) The K-permeability of the muscle fibre membrane of the mealworm (Trnehrio molifor) larva. J. gen. Physiof. 54, S90A. BELTON P. and GRUNDFEST H. (1962b) Potassium activation and K-spikes in muscle fibres of the mealworm larva (Terwhrio rtloliforl. Avt. J. Physiol. 203. X8-594. ELIIER H. Y. (1975) Muscle structure. In Inset MUSCIC~. (Ed. by USHERWOOD P. N. R.). Academic Press. New York. FLORKIN, M. and JEUNIAUX Ch. (1974) Haemolymph: composition. In T/W Ph~sioiogy of fmecra (Ed. by RCCKSTEINM.) 5. Academic Press. New York. HOYLE G. (1955) The effect of some common cations on neuromuscular transmission in insects. J. Ph~siol.. .!,o~it/.
127. 9&103. HUDDART H. (1966) The effect of potassium ions on resting and action potentials in lepidopteran muscle. Cor~lp, Biocl1em. PIlV:siOl. 18. 13l-140. KOMNICK H. (1962) Elektronenmicroskopische Lokahsation von Na+ und Cl- in Zellen und Geweben. Proroplasma 55, 414-418. LUFT J. H. (1961) Improvements in epoxyresin embedding methods. .f. Biophys. hiochm. Cytol. 9. 409%414. PIEK T. (1974) Ion barriers in muscle fibres. .4rch. irlr. Physiol. Biochim. 82. 337-339. POX T. (1975) Ionic and electrical properties. In I,lsecr Muscle (Ed. by USHERWOOD P. N. R.). Academic Press, New York.
Sodium
and potassium
PIES: T.. MANTEL P . and WIJSMANJ. P. M. (I 977) Effects 01‘ monocarboxyl+tes and external pH on some parameters of insect muscle tibres. J. Irtsect Physiol., in press. RAMSAY J. A. (1953) Active transport of potassium by Malpighian tubules of insects. J. esp. Biol. 30, 358-369. SAETERSDALTHV. S., MYKLEBUST R., BERG JUSTESEN N. P. and CATO OLSEN W. (1974) Ultrastructural localization of calcium in the pigeon papillary muscle as demonstrated by cytochemical studies and X-ray microanalysis. Cell Tis.xre Res. 155. 57-74. SATO T.. HERMAN L., CHANDLER J. A.. STRACHER A., and DETWILLER C. (1975) Localization of a thrombin-sensitive calcium pool in platelets. J. Histochern. Cytochern. 23, 103-106. SHIINA S. J. and MIZUHIRA V. (1970) Subcellular locahzation of sodium and potassium ions in skeletal muscle. .4cta Histochc~rn. Cytochm. 3, 7479.
ions in insect muscle
929
SHIINA S. J., MIZUHIRA V.. AMAKAWA T., and FUTAESAKA Y. (1970) An analysis of the histochemical procedure for sodium ion detection. J. Histochrm. Cytochem. 18, 644649. SHIINA S. J., MIZUHIRA V., UCHIDA K.. AMA~WA T., and TS~JZI K. (1968) Electron microscopic study on the distribution of sodium and potassium in heart muscle cell. J. electron rnicrosc. 17. 267-268. TORACK R. M. and LAVALL~ M. (1970) The specificity of the pyroantimonate technique to demonstrate sodium. J. Histochrm Cytochem 18. 635-643. WOOD D. W. (1957) The effect of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. 138, 119-139. ZADUNAISKY J. A. (1966) The localization of sodium in the transverse tubules of skeletal muscle. J. Cell Biol. 131, cl I-~16.
Ph~siol..
1917, Vol. 23. pp. 919 to 929. Pergamon
Press. Pri,ued
ill Great Britain.
LOCALIZATION OF SODIUM AND POTASSIUM IONS IN A FLIGHT MUSCLE OF PIERIS BRASSICAE K. D. NJIO and T. PIEK Pharmacolcgical
Laboratory.
University
of Amsterdam.
(Recerwd
Polderwcg
21 Decmhrr
104. Amsterdam.
The Netherlands
19761
Abstract-T he ultrastructural localization of sodium and potassium ions in the longitudinal flight muscle of f’ieris hrassicar was carried out irr situ and after incubation in various media. The results indicate a rtzlatively high sodium and a low potassium content of the lumen of the transverse tubular system. Inca bation in a propionate saline results in an accumulation of potassium ions in the mitochondria and a decrease in concentration in the myoplasm.
INTRODUCTION
STUDIESin the last 3Qyr on the sarcotubular system of skeletal muscle fibres have led to the conclusion that the transverse !.ubular system (TTS) is continuous with the “surface” plasma membrane. Therefore, the lumen of the TTS is considered to be in continuity with the extracellular space. This has been demonstrated for vertebrates. crustaceans and insects (cf. ELDER, 1975). The continuity of the TX-lumen with the extracellular spice does. however, not necessarily mean that the ionic, composition in both spaces must be identical. PIF.K ( 974. 1975) suggested that a highly active sodium-potassium exchange pump might be operating in the extensive transverse tubular membrane. This could lead to a concentration of sodium chloride in the TTS-lumen. which is much higher than in the extracellular space and may produce a significant liquid junction potential at the opening of the TTS. In muscle fibrzs of P/lilo.sartriLccythiu the synaptic region is separated from the extracellular space by a glial cell. Only very long and narrow channels lead from the synaptic cleft to the outer environment (PIEK. 1974, 1975). Therefore, the postsynaptic membrane may be considered as a part of the invaginated plasma membrane and the fluid inside the invaginated parts of the membrane (mainly the TTS) is the outer medium for the postsynaptic membrane. The fact that these muscle fibres show hyperpolarizing inhibitory pos&ynaptic potentials. due to an activation for chloride ions, indicate that a high chloride equilibrium potential is present across the postsynaptic membrane and that therefore the fluid in the TTSlumen has a high concentration of chloride ions. No argument has beer presented for the presence of a high concentration of sodium ions in the TTS. The present paper deals with attempts to estimate the concentrations of sodium and potassium ions inside and outside the plasma membrane including the TTS-lumen. The techniques used are based on earlier
work on vertebrate muscle (ZADUNAIXY, 1966: SHIINA et al., 1968: SHIINA and MIZUHIRA, 1970).
MATERIALS
AND
METHODS
The longitudinal flight muscle of Pieris h~ssicc~ L. was dissected and put into the fixative either immediately or after incubation for 60min in various media. The composition of these media is summarized in Table 1. For the detection of sodium and potassium ions the method of SHIINA and MIZ~~~IRA (1970) was slightly modified. For sodium ion detection 1 vol of 49” osmium tetroxide + 3 vol of 3”” potassium hexahydroxoantimonate (V), K[Sb(OH),], (= potassium pyroantimonate) in distilled water or in 50 mmol per liter potassium phosphate buffer pH 7.4 was used as a fixative. For potassium ion detection I”;, glutaraldehyde + 3”, sodium hexanitrocobaltate (III). Na,[Co(NO,),]. in 30 mmol/l sodium phosphate buffer pH 7.4 was used. In later experiments the washing in yellow ammonium sulfide solution as described by SHIINA and MIZUHIRA (1970) was omitted in order to avoid the precipitation of a magnesiumammonium-phosphate complex. The cor~trols were fixed in the same fixatives but the reagents. without i.e. K[Sb(OH,]. resp. Na,[Co(N02),]. After fixation the pieces of muscle were washed in the buffer. subsequently dehydrated in ethanol and embedded in Epon according to LUFT (1961). The ultrathin sections were stained with uranyl acetate. In order to find an explanation for the low concentration of potassium in homogenized muscles after incubation in propionate (PINK uf ul., 1977). muscle preparations were tried for potassium ions after incubation in propionate saline containing per liter Napropionate 74 mmol, K-propionate 10 mmol CaCl, 6 mmol, and sucrose 170 mmol.
920
K. D. NJIO AND T. PIE-s mitochondria (Fig. 4). After incubation for 60min in medium V (74 mmol/l choline Cl) no precipitate was formed but some granules were present on the inside of the lamina basalis (Fig. 5). The localization of the sodium ions is summarized in Table 2. In the controls no precipitate was observed. In the preparations made for the localization of potassium ions it was often very difficult, if not impossible, to decide whether the precipitate was present in the sarcoplasmic reticulum (SR) or in the subsarcolemma1 cistemae (SSC). In the urltrraferl muscle fine precipitate was found in the region of the SR and of the SSC, on the myofibrils and in the mitochondria (Fig. 6). Occasionally a big granule was seen in the TTS, but this was never observed when the washing in yellow ammonium sulfide was omitted. After incubation for 60 min in medium I containing/l IO mmol KCl. 56 mmol NaCl and 9 mmol MgC12. the precipitate was found in the region of the SSC and the SR and in the mitochondria. As in the untreated muscle the big granules in the TTS were only seen after washing in yellow ammonium sulfide. After incubation for 60min in medium II containing no KCl, and 66 mmol NaCl and 9 mmol MgClJl only the big granules in the TTS were found, but only after washing with yellow ammonium sulfide. The localization of the precipitate after incubation for 60min in medium III containing no NaCl and per liter 10 mmol KC1 and 37 mmol MgCl,. was the same as after incubation in medium I. Again the big granules in the TTS were only seen after washing in yellow ammonium sulfide. After incubation for 60 min in medium IV containing/l 10 mmol KCl, 74 mmol NaCl and no MgC12, precipitate was found on the myofibrils, in the region of the SSC and the SR. Occasionally some precipitate was observed in the mitochondria. The same localization of the precipitate was found after incubation for 60 min in medium V containing/l 10 mmol KC1 and 74mmol choline Cl (Fig. 7). After incubation in propionute S&P precipitate was only found in the mitochondria (Fig. 8). The localization of the potassium ions are summarized in Table 3. In the controls some big granules were found in the TTS after washing in yellow ammonium sulfide. otherwise no precipitate was observed.
Table 1. Composition of the media, in which the longitudinal flight muscle of P. hrassicae was incubated for 60min Concn
KCI NsCl MU, CaCI, Choline Cl NaHCO, SUCrO%
mmal!l
I
II
III
IO 56 9 6 _
~~ 66 9 6
IO ~~ 37 6
I 180
I 180
IV
V
IO 71
IO
6
6 73 ~ 170
_ ~ 210
I 180
All media were used in potassium ion detection, the media I, III. IV, and V were also used in sodium ion detec-
tion.
RESULTS In the preparations made for the localization of the sodium ions the sarcoplasm and the mitochondria were badly swollen when the fixative was dissolved in distilled water. Because of this swelling the exact localization of the precipitate was extremely difficult. Therefore, phosphate buffer was used instead of distilled water. In the untreated muscle sodium ions were present in high concentration in the TTS (Figs. 1 and 2) and in the region of the sarcolemma. on the inside and outside of the lamina basalis (Fig. 2). We also observed some precipitate on the myofibrils, but we could not find any precipitate concentrated on the I-band as described by SHIINA and MIZUHIRA (1970). Some precipitate was also found in the mitochondria (Figs. 1 and 2). After incubation for 60 min in medium I containing/l 10 mm01 KCl, 56mmol NaCl and 9 mmol MgCl, , the localization of the precipitate was the same as in the untreated muscle, but to a lesser amount. After incubation for 60min in medium III containing no NaCl and 37 mmol MgCl, per liter, some big granules were found in the TTS and on the lamina basalis, and sporadically in the mitochondria (Fig. 3). After incubation for 60 min in medium IV containing 74 mmol NaCl/I and no MgCl,, the precipitate was heavily concentrated on the inside and outside of the lamina basalis and in the TTS. A fine precipitate was found on the myofibrils and in the
Table 2. Localization
NP, no precipitate:
ions
IO mm01 K 56 mmol Na 9 mmoi Mg medium I
IO mmol K 0 mmol Na 37 mmol Mg medium III
IO mm4 K 74 mmol Na 0 mmol Mg medium IV
IO mmol K 0 nmml Nn (1 mmal Mg medtum V
11 II II NP
(11 (1)
111 (11
II I I NP NP
NP
It NP
(1) (1) 1 NP
Untreated muscle Oufslde lamina basalis Inside lamma basalis TTS SR Myofibnls Mllochondria
of sodium
*
(1) some precipitate;
1 considerable
(1) I
(11
precipitate;
li heavy
precipitate.
Ilr NP NP NP NP
Control NP NP NP NP NP NP
Fig. 1. T’he distribution of antimonate precipitate in an untreated flight muscle of Pieris hrassicue. (b) and (I:) are details of (a) showing triadic and dyadic structures containing precipitate in the TTSlumen. Scale: 1 pm.
Fig. 2. The distribution of antimonate precipitate in the untreated flight Precipitate is found on the lamina basalis on both sides of an intercellular
muscle of Pirris hrussicue. space (arrow). Scale: 1 Arm.
Fig. 3. The distribution of antimonate precipitate after incubation for 60 min in medium 111 (no Na -. 37 mmol/l Mg”). Precipitate is found on the lamina basalis and some granules are seen in the TTSlumen. This precipitate is probably due to magnesium. Scale: 1 /rrn.
Fig. 4. After incubation for 60min in medium IV (74mmol/l Nat. no Mg*+) a heavy antimonate Some granules are seen in the prec ipitate is found on the lamina basalis and in the TTS-lumen. mitochondria and on the myofibrils. Scale: 1 pm. Fig. 5. Alter incubation
for 60 min in choline saline (medium V) only some antimonate found on the inside of the lamina basalis. Scale: 1 pm.
granules
are
Fig. 6. Distribution granules are found
of cobaltate precipitate in the untreated flight muscle of Pieris on the myofibrils, in the mitochondria and in the subsarcolemmal sarcoplasmic reticulum. Scale: 1pm.
hrc~~sicu~. The
cisternae
and
Fig. 7. After incubation for 60min in choline saline (medium V) cobaltate precipitate is found on the myofibrils. in the mitochondria and in the subsarcolemmal cisternae and sarcoplasmic reticulum. (b) is a detail of (a). Scale: 1 pm.
Fig. 8. After
incubation
for 60min in propionate saline cobaltate precipitate mitochondria. (b) is a detail of (a). Scale: 1 pm.
is only
found
in I.he
Sodium
and potassium
ions in insect muscle
Table
3. Localization
of potassium
927
ions
I@mmol K 56 mm4 Na 9 mmol Mg medium
0 mmol K 66 mmol Na 9 mmol Mg medwm II
IO mmol K 0 mmol Na 37 mmol Mg medium III
10 mm01 K 74 mmol Na 0 mmol Mg medium IV
IO mmol K 0 mmol Na 0 mmol Mg medium V
NP NP*
NP NP*
NP NP
NP NP
I
NP NP* NP
NP NP*
I i
i
(1) hP
f 1
I
Propionate Salme
Colltr01
I_amna
basahs TTS SR,SSC Myofibrlls Mltochondrn
NP. no precipitate; 1 precipitate * Precipitate 01114 after washing
in yellow ammonium
I i NP
cil
NP NP NP NP I
NP NP* NP NP NP
sulfide solution.
DISCUSSION To study basic processes underlying physiological phenomena electraphysiologists try to describe the electrochemical properties of membrane of cells and fibres. In axonal membranes. for instance, the membrane potential can be explained by passive permeation of ions. whit? have different concentrations on both sides of the membrane. and for which the membrane has different relative permeabilities. However, in other structures (e.g. some muscle fibres) this simple electrocherlical relation seems to be insufficient to describe the observed phenomena. The discrepancies found are often explained by assuming that one is not well informed about the ionic composition inside as well as outside the membrane. The electron microscopic techniques developed for the localization of sodium ions (KOMNICK. 1962) and potassium ions (SHIINA and MIZ~I-~IRA 1970) might throw some light on the presence of differences in ionic composition inside and outside the membranes. Unfortunately these techniques a-e far from quantitative. They can only discriminate between no precipitate. some precipitate, considerable precipitate and a heavy prccipitate. In this paper this evaluation of the intensity of the precipitation has been adapted. According to the intensity of the precipitation of the sodium hexah;:droxoantimonate (V) complex the conclusion may be drawn that the muscle fibres of P. hmMm~ fixed directly after the integument had been opened. probably contain more sodium in the T-B-lumen than outside the surface plasma membrane. An important question is, however. how reliable the data are. According to TORACE; and LAVALLE (1970) the use of an aqueous solution of hexahydroxoantimonate as a specific histochemical marker for sodium ions is not ‘gery reliable, since at physiological levels potassium as well as sodium are precipitated. According to SHIII\ A et nl. (I 970) precipitates of calcium and magnesium complexes may be formed too. In addition a pH of less than 7.2 appears to favour the production of non-specific precipitates (TORACX and LAVALLE. 1970; SHIINA or d. 1970). In the present experiments the pH of the fixative was 7.4. The use of the phosphate buffer did not affect the formation
of the precipitate of sodium ions as stated by TORACK and LAVALLE (1970) and SHIINA et al. (1970). The molarity of our buffer, however. was lower (50mmol/l) than used by TORACK and LAVALLE (200 mmol/l) and SHIINA et ~1.(100 mmol/l). Nevertheless it is likely that magnesium and possibly also calcium ions may contribute to the precipitates. After incubation of the muscle for one hour in a sodium and magnesium free medium. containing/l IO mmol potassium ions and 6 mmol calcium ions (med. V), no precipitate was found, except a few grains between the plasma membrane and the lamina basalis. It is unlikely that this is a potassium or calcium precipitate, since the highest concentration of these ions is considered to be present inside the plasma membrane. Therefore, it can be concluded that in our experiments hexahydroxoantimonate does not precipitate with either potassium or calcium. Moreover, the precipitation of calcium with the antimonate is performed in a solution at pH 7.8 (SATO et al., 1975; SAETERSDAL rt al.. 1974). After incubation in a medium without magnesium. containing 74 mmol/I sodium ions (med. IV) the precipitate was concentrated on both sides of the lamina basalis and in the TTS. Some precipitate was found in the myofibrils and in the mitochondria, somewhat more than in the untreated preparations. The concentration at the level of the lamina basalis was higher than in the untreated muscle, probably due to the fact that the sodium ion concentration in the saline is higher in the haemolymph. RAMSAY(1953) found 9.0mmolfl in the haemolymph of P. hrmsicac~ larvae. In four species adult Lepidoptera the sodium content of the haemolymph was not more than 16 mmol/l (cf. FLORKIN and JEUSIAUX, 1974). The fact that incubation with 37 mmol per liter magnesium ions (med. III) causes a precipitate with antimonate in the TTS that is less heavy than in the untreated preparations. indicates that the heavy precipitate in the TTS in the untreated preparations may mainly consist of sodium antimonate. It must be stated that the sodium detection method is very difficult. Occasionally a muscle showed a diffusely dispersed, electron-opaque precipitate, even after incubation in a medium without sodium or magnesium ions. We were not able to detect the cause
928
K. D. NJIO AND T. PIEK
of this precipitate. Presumably it is due to a change in pH in the muscle and/or the fixative which caused the antimonate to precipitate. This diffuse precipitate is, however, easily recognized as an artefact. In accordance with SHIINA and MIZUHIRA (1970) precipitates of potassium ions were found scattered on the myolibrils and in the mitochondria. In contrast to SHIINA and MIZUHIFU (1970), who also localized potassium in the lumen of the TTS. we did not conclude that the precipitate in the TTS was due to the presence of potassium. The reason for this view is that in the experiments with untreated muscles and after incubation in medium I (9 mmol/l MgCl, and III (37 mmol/l MgC1J made with and without the washing in yellow ammonium sulfide, the localization of the precipitate was the same in all cases, only the granules in the TTS had disappeared when the washing was omitted. Moreover, in the absence of magnesium-ions (media IV and V) no precipitate in the TTS was formed. Therefore, these granules probably represent the precipitation of the insoluble magnesiumammonium-phosphate complex. Outside the TTS the precipitate could only be found scattered on the SR, the SSC, and the myofibrils. It is uncertain whether the precipitate localized at the SR and SSC is situated inside or outside the membranes of these structures. It is also questionable whether calcium ions are involved in the precipitation. Precipitate near the SR and SSC. however, have only been found in preparations incubated in potassium containing media. and not in medium II, which contains 6 mmol CaClJ but no potassium. It is therefore probably that the precipitate scattered on the SR and SSC is actually potassium. Varying the potassium concentration in the incubation medium did not result in distinct differences in precipitation with nitrocoboltate. Therefore, the muscles were incubated for one hour in a medium in which most of the chloride was replaced by propionate. PIEK rt uI. (1977) had found that propionate saline causes reduction of the potassium content in extracts of muscle homogenates. though they could not explain this finding. The results of the present experiments suggest a reduction of the potassium content of the myoplasm and an enhanced concentration of potassium ions inside the mitochondria. This finding agrees with the low potassium concentration found in extracts of muscle homogenates bathed in a propionate saline (PIEK et al. 1977). In Exopterygota (cockroaches, locusts. and stick insects) the recorded muscle fibre resting potentials fit those calculated from the internal and external potassium concentrations (HOYLE, 1955; WOOD, 1957). However. in Endopterygota (mealworm and moth) the recorded resting membrane potentials differ considerably from the theoretical values (BELTON and GRUNDFEST, 1962a, b; HUDDART. 1966). It is generally accepted that the high concentration of potassium ions in the myoplasm at least contributes to the generation of the resting membrane poten-
tial. This view is strongly supported by the observations that increase of the potassium concentration in the bath reduced the resting membrane potential with a slope, approximately predicted by the Nernst equation for a potassium electrode. The propionate saline. however. did reduce the potassium content measured in the homogenate and caused also a distinct decrease in potassium localized in the myoplasm (PIK er trl. 1977). Nevertheless, the resting membrane potential is largely increased. This suggests that other ions might be involved in the generation of the membrane potential. The conclusion is that potassium ion detection with nitrocobaltate does not enable us to localize the potassium very well, but that it is highly probable that potassium ions are not abundant inside the lumen of the TTS. Sodium and magnesium ions, however, are present inside the TTS, sodium ions in a concentration that might be higher than in the haemolymph or bathing fluid of the muscle. Since arguments are presented for a high chloride concentration in the TTS-lumen (PIEK. 1974, 1975) the conclusion that the invaginated muscle fibre membrane of Lepidoptera, and possibly also of other insects. creates inside the TTS-lumen and inside the synaptic spaces its own external ionic environment, is supported by the present results, indicating a low potassium and high sodium content of the fluid in the TTS-lumen. Acknowledgenlents-The authors thank Professor Dr. C. VAN DER MEER for his professional advice and Mr. G. A. C. BELLING and Mr. P. MANTEL for their technical assistance.
REFERENCES BELTON P. and GRUNDFEST H. (1963a) The K-permeability of the muscle fibre membrane of the mealworm (Trnehrio molifor) larva. J. gen. Physiof. 54, S90A. BELTON P. and GRUNDFEST H. (1962b) Potassium activation and K-spikes in muscle fibres of the mealworm larva (Terwhrio rtloliforl. Avt. J. Physiol. 203. X8-594. ELIIER H. Y. (1975) Muscle structure. In Inset MUSCIC~. (Ed. by USHERWOOD P. N. R.). Academic Press. New York. FLORKIN, M. and JEUNIAUX Ch. (1974) Haemolymph: composition. In T/W Ph~sioiogy of fmecra (Ed. by RCCKSTEINM.) 5. Academic Press. New York. HOYLE G. (1955) The effect of some common cations on neuromuscular transmission in insects. J. Ph~siol.. .!,o~it/.
127. 9&103. HUDDART H. (1966) The effect of potassium ions on resting and action potentials in lepidopteran muscle. Cor~lp, Biocl1em. PIlV:siOl. 18. 13l-140. KOMNICK H. (1962) Elektronenmicroskopische Lokahsation von Na+ und Cl- in Zellen und Geweben. Proroplasma 55, 414-418. LUFT J. H. (1961) Improvements in epoxyresin embedding methods. .f. Biophys. hiochm. Cytol. 9. 409%414. PIEK T. (1974) Ion barriers in muscle fibres. .4rch. irlr. Physiol. Biochim. 82. 337-339. POX T. (1975) Ionic and electrical properties. In I,lsecr Muscle (Ed. by USHERWOOD P. N. R.). Academic Press, New York.
Sodium
and potassium
PIES: T.. MANTEL P . and WIJSMANJ. P. M. (I 977) Effects 01‘ monocarboxyl+tes and external pH on some parameters of insect muscle tibres. J. Irtsect Physiol., in press. RAMSAY J. A. (1953) Active transport of potassium by Malpighian tubules of insects. J. esp. Biol. 30, 358-369. SAETERSDALTHV. S., MYKLEBUST R., BERG JUSTESEN N. P. and CATO OLSEN W. (1974) Ultrastructural localization of calcium in the pigeon papillary muscle as demonstrated by cytochemical studies and X-ray microanalysis. Cell Tis.xre Res. 155. 57-74. SATO T.. HERMAN L., CHANDLER J. A.. STRACHER A., and DETWILLER C. (1975) Localization of a thrombin-sensitive calcium pool in platelets. J. Histochern. Cytochern. 23, 103-106. SHIINA S. J. and MIZUHIRA V. (1970) Subcellular locahzation of sodium and potassium ions in skeletal muscle. .4cta Histochc~rn. Cytochm. 3, 7479.
ions in insect muscle
929
SHIINA S. J., MIZUHIRA V.. AMAKAWA T., and FUTAESAKA Y. (1970) An analysis of the histochemical procedure for sodium ion detection. J. Histochrm. Cytochem. 18, 644649. SHIINA S. J., MIZUHIRA V., UCHIDA K.. AMA~WA T., and TS~JZI K. (1968) Electron microscopic study on the distribution of sodium and potassium in heart muscle cell. J. electron rnicrosc. 17. 267-268. TORACK R. M. and LAVALL~ M. (1970) The specificity of the pyroantimonate technique to demonstrate sodium. J. Histochrm Cytochem 18. 635-643. WOOD D. W. (1957) The effect of ions upon neuromuscular transmission in a herbivorous insect. J. Physiol. 138, 119-139. ZADUNAISKY J. A. (1966) The localization of sodium in the transverse tubules of skeletal muscle. J. Cell Biol. 131, cl I-~16.