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Effect of insulin on the membrane resistance of frog skeletal muscle
LIFE SCIENCES Vol . 6, pp . 959-963, 1967 . Printed in Great Britain.
Pergamon Presa Ltd .
EFFECT OF INÔULIN ON THE MEMBRANE RESISTANCE OF FROG SKELETAL MUSCLE'
w, c .
de Metro
Department of Pharmacology, School of Medicine, San Juan, Puerto Rico (Received 13 January 1967 ; in final form 8 February 1967) Zierler3 (1959) and Willie and Manery2 (1960) have shown that the addition of insulin to Ringer's fluid prevents the loss of K+ ions from isolated frog and rat skeletal muscles while increasing the oxygen consumption . Zierler 3 (1959) described also an increase of membrane potential with insulin. However no precise infornsation is available concerning the influence of insulin on membrane cauhictance . In the present experiments this problen was investigated in the isolated sartorius muscle of the frog .
It was found that insulin increases both the
membrane potential and membrane resistance in a glucose free medium .
The
increase on membrane resistance was also observed in a C1-free isotonic, K2SOq solutions, indicating that insulin decreases the K conductance of the muscle fibres . N~aterial and Methods Isolated sartorius rustles from Rang pipiers were used .
The muscles were
dissected out and maintained in a glucose free Ringer's fluid containing (mM) : NaCl, 120 ; KC1, 2 .5 ; CaC1 2 , 1 .8 ; and H003, 2 .2 .
An isotonic K2SOq solution
free of C1 - ions was used in same experiments.
In all sulfate solutions, an
extra amount of Ca (8 mM) was added as CaSOq to provide enough ionized calciua in solution .
Trangmenbrane resting potentials were recorded with intracellular
microelectrodes filled with 3 M KC1 or ä~tpotassitarcitrate . ~ This work has been supported by grant HE 10897-01 (CVA) from National Heart Institute. 959
The membrane
960
INSULIN ACTION
Vol . 6, No . 9
resistance was measured by introducing two microelectrodes about 50 v apart, in the same fibre.
Pulse of imaard current (700 cosec in duration with an inten-
sity of up to about 10 -~ A) were injected through one microelectrode and the voltage changes were recorded with the other one.
All measurements of membrane
resistance were expressed in teams of "effective resistance" (Vo/Im) .
Its
average value in a grcrap of muscle fibres in Ringer's fluid was compared with the average value from another group of fibres of the same muscle after 1 hour of equilibration in Ringer's fluid containing 80 u ./1 . of insulin (crystalline zinc insulin-Squibb) .
The pH of this solution was kept the same as that of
Ringer's fluid (7 .2) . Results and discussion The addition of insulin to Ringer's fluid increased the average resting membrane potential by 4 to 6 mV .
This increase on membrane polarization (see
Table 1) was also observed with much smaller amounts of insulin (1 u./1 .) . TABLE 1 EFFECT OF INSULIN atd TEIE AVERAGE VALiIFS OF NIfTiBRANE POTENTIAL AND "EFFECTIVE RESISTANCE" OF SAR'PORIUS M[1SCLE FIBERS IN RINGER'S FLUID birscle
Effective Resistance (n X 105)
Resting membrane potential (mV)
$ D.E .R .*
Ringer's fluid
Insulin 80 u./1
Ringer's fluid
1
92 .5
96 .8 ~ 0.98 (22)
7 .4
t 0.06 (20)
9.3 * 0 .10 (22)
25
2
93 .2 = 1 .1 (20)
98 .4 = 1 .0 (18)
4 .9 = 0 .05 (20)
5.3 * 0 .13 (18)
8
3
90 .4 = 1 .2 (20)
96 .3
3 .2 = 0.02 (20)
4 .4 = 0 .085 (22)
37
4*
85 .6 ! 0.50
90
3 0 .78 (20)
t 1 .1 (22)
Insulin 80 u ./1
= 0.70
_ - Standard ermr of the mean . Figures in brackets ~ number of fibres used ; temperature, 25°C . * - In rotrscle no . 4 the amount of insulin added to Ringer's fluid was 1 u ./1 . ! - $ Difference in effective resistance .
Vol .
6,
No .
96 1
INSULIN ACTION
9
The action of insulin on the average "effective resistance" is also shave in Table 1 .
An increase of this resistance was observed .
Although quantitatively
variable the effect was always present after 1 hour of equilibration with the hormone. Since at resting condition the total membrane conductance is given by the s~ of potassium and chloride conductances, the effect of insulin could be interpreted as a decrease of K+ or C1
conductances or of both .
Qn the other
hand, it is known that Zn++ ions decrease the chloride conductance in muscle fibres (Mashima and Washiol , 1964) .
The presence of Zn~ ions in the insulin
employed could be the cause of the observed fall on membrane conductance . To remove the contribution of C1
ions to the total membrane conductance,
experiments were performed in C1-free SO4 solutions .
Isolated sartorius
muscles were equilibrated during 50 minutes in a solution containißg mainly Na2S04 .
During this period the muscle twitched spontaneously.
The muscle was
then immersed in isotonic KZSO4 solution during 30 minutes and measurements of membrane resistance were made before and after the addition of insulin. Microelectrodes filled with 2 M potassium citrate were used to pass current . In Table 2, the effect of insulin on the average "effective resistance" in three depolarized sartorius muscles is presented.
A marked increase in ~-
brane resistance was observed after the addition of the hormone .
This increase
which was more prono~aiced than that noted in Ringer's fluid (see Table 1) reached a value of 134+
in rustle X12
of Table 2 .
These results indicate that insulin decreases the potassiua caductance of the muscle cell membrane .
Ebwever, since insulin is a protein, the decrease
an membrane conductance could be a consequence of a general property of proteins .
Bcperimenta perfonoed with another protein (bovine sera albumin)
showed that its addition (3 .5 ug/ml)
to isotonic KZSO~ solution did not
increase appreciably the membrane resistance (see Table 3), as insulin did (see Table 2) .
962
INSULIN ACTION
Vol . 6, No . 9
TABLE 2 EFFECT OF INSULIN aV AVERAGE '~FFF~.TIVE RESISTANCE" OF FROG SAR'T0~2I11S MÜSCLE IN IS07~1IC K2S04 SOL[TI'IOtd Muscle
Effective resistance (n X 10 ) Isotonic K2~4
1
2 .3
2 3
~ Difference in Effective Resistance
Insulin 80 u./1
= 0 .095 (16)
4 .3 = 0 .17 (18)
79
1.04 * 0 .10 (20)
2 .4 ! 0 .16 (20)
134
2.51 = 0 .11 (20)
3.4 * 0 .19 (22)
36
* - Standard error of the neon . Figures in brackets ~ number of muscle fibres used ; temperature, 25°C . TABLE 3 EFFECT OF BOVINE SERIh1 ALBIMIIV ON TF~ AVERAGE EFFECTIVE RESISTANCE OF FROG SARTORIIIS MUSCLE IN I90r1nIdIC K2S04 Effective Resistance (n X 10 )
Muscle
Without protein
1
2 :24
t 0 .10 (14)
2
2 .34 = 0.11 (16)
With protein (3 .5 ug/ml") 2 .3
3 0.13 (22)
2 .39 r 0.15 (22)
~ - Standard error of the mean . Fi~rts in brackets, number of muscle fibres used ; temperature, 25°C . w - S.S rg/s1 cornspands approximately to the pmtein content of 80 u ./1 of insulin .
Vol . 6, No . 9
INSULIN ACTION
96 3
It seems reasonable to caulude Eras these experisents that the increase on mesbrane resistance observed in isotonic 82904 solution with insulin, is due to a specific action of the hoimoaie on potassüa conductance . Aclmawledgement-The author is üdebted to Professor J. dei Castilio for helpful discussion . References 1.
H. Meshima and H. Washio, Jap. J. Physiol . 14, 538 (1964) .
2.
L.B . 9nillie and J.F . Manery, Am . J. Physiol . 198, 67 (1960) .
3.
K. L. .Zierler, Am. J. Physiol. 197, 515 (1959) .
Pergamon Presa Ltd .
EFFECT OF INÔULIN ON THE MEMBRANE RESISTANCE OF FROG SKELETAL MUSCLE'
w, c .
de Metro
Department of Pharmacology, School of Medicine, San Juan, Puerto Rico (Received 13 January 1967 ; in final form 8 February 1967) Zierler3 (1959) and Willie and Manery2 (1960) have shown that the addition of insulin to Ringer's fluid prevents the loss of K+ ions from isolated frog and rat skeletal muscles while increasing the oxygen consumption . Zierler 3 (1959) described also an increase of membrane potential with insulin. However no precise infornsation is available concerning the influence of insulin on membrane cauhictance . In the present experiments this problen was investigated in the isolated sartorius muscle of the frog .
It was found that insulin increases both the
membrane potential and membrane resistance in a glucose free medium .
The
increase on membrane resistance was also observed in a C1-free isotonic, K2SOq solutions, indicating that insulin decreases the K conductance of the muscle fibres . N~aterial and Methods Isolated sartorius rustles from Rang pipiers were used .
The muscles were
dissected out and maintained in a glucose free Ringer's fluid containing (mM) : NaCl, 120 ; KC1, 2 .5 ; CaC1 2 , 1 .8 ; and H003, 2 .2 .
An isotonic K2SOq solution
free of C1 - ions was used in same experiments.
In all sulfate solutions, an
extra amount of Ca (8 mM) was added as CaSOq to provide enough ionized calciua in solution .
Trangmenbrane resting potentials were recorded with intracellular
microelectrodes filled with 3 M KC1 or ä~tpotassitarcitrate . ~ This work has been supported by grant HE 10897-01 (CVA) from National Heart Institute. 959
The membrane
960
INSULIN ACTION
Vol . 6, No . 9
resistance was measured by introducing two microelectrodes about 50 v apart, in the same fibre.
Pulse of imaard current (700 cosec in duration with an inten-
sity of up to about 10 -~ A) were injected through one microelectrode and the voltage changes were recorded with the other one.
All measurements of membrane
resistance were expressed in teams of "effective resistance" (Vo/Im) .
Its
average value in a grcrap of muscle fibres in Ringer's fluid was compared with the average value from another group of fibres of the same muscle after 1 hour of equilibration in Ringer's fluid containing 80 u ./1 . of insulin (crystalline zinc insulin-Squibb) .
The pH of this solution was kept the same as that of
Ringer's fluid (7 .2) . Results and discussion The addition of insulin to Ringer's fluid increased the average resting membrane potential by 4 to 6 mV .
This increase on membrane polarization (see
Table 1) was also observed with much smaller amounts of insulin (1 u./1 .) . TABLE 1 EFFECT OF INSULIN atd TEIE AVERAGE VALiIFS OF NIfTiBRANE POTENTIAL AND "EFFECTIVE RESISTANCE" OF SAR'PORIUS M[1SCLE FIBERS IN RINGER'S FLUID birscle
Effective Resistance (n X 105)
Resting membrane potential (mV)
$ D.E .R .*
Ringer's fluid
Insulin 80 u./1
Ringer's fluid
1
92 .5
96 .8 ~ 0.98 (22)
7 .4
t 0.06 (20)
9.3 * 0 .10 (22)
25
2
93 .2 = 1 .1 (20)
98 .4 = 1 .0 (18)
4 .9 = 0 .05 (20)
5.3 * 0 .13 (18)
8
3
90 .4 = 1 .2 (20)
96 .3
3 .2 = 0.02 (20)
4 .4 = 0 .085 (22)
37
4*
85 .6 ! 0.50
90
3 0 .78 (20)
t 1 .1 (22)
Insulin 80 u ./1
= 0.70
_ - Standard ermr of the mean . Figures in brackets ~ number of fibres used ; temperature, 25°C . * - In rotrscle no . 4 the amount of insulin added to Ringer's fluid was 1 u ./1 . ! - $ Difference in effective resistance .
Vol .
6,
No .
96 1
INSULIN ACTION
9
The action of insulin on the average "effective resistance" is also shave in Table 1 .
An increase of this resistance was observed .
Although quantitatively
variable the effect was always present after 1 hour of equilibration with the hormone. Since at resting condition the total membrane conductance is given by the s~ of potassium and chloride conductances, the effect of insulin could be interpreted as a decrease of K+ or C1
conductances or of both .
Qn the other
hand, it is known that Zn++ ions decrease the chloride conductance in muscle fibres (Mashima and Washiol , 1964) .
The presence of Zn~ ions in the insulin
employed could be the cause of the observed fall on membrane conductance . To remove the contribution of C1
ions to the total membrane conductance,
experiments were performed in C1-free SO4 solutions .
Isolated sartorius
muscles were equilibrated during 50 minutes in a solution containißg mainly Na2S04 .
During this period the muscle twitched spontaneously.
The muscle was
then immersed in isotonic KZSO4 solution during 30 minutes and measurements of membrane resistance were made before and after the addition of insulin. Microelectrodes filled with 2 M potassium citrate were used to pass current . In Table 2, the effect of insulin on the average "effective resistance" in three depolarized sartorius muscles is presented.
A marked increase in ~-
brane resistance was observed after the addition of the hormone .
This increase
which was more prono~aiced than that noted in Ringer's fluid (see Table 1) reached a value of 134+
in rustle X12
of Table 2 .
These results indicate that insulin decreases the potassiua caductance of the muscle cell membrane .
Ebwever, since insulin is a protein, the decrease
an membrane conductance could be a consequence of a general property of proteins .
Bcperimenta perfonoed with another protein (bovine sera albumin)
showed that its addition (3 .5 ug/ml)
to isotonic KZSO~ solution did not
increase appreciably the membrane resistance (see Table 3), as insulin did (see Table 2) .
962
INSULIN ACTION
Vol . 6, No . 9
TABLE 2 EFFECT OF INSULIN aV AVERAGE '~FFF~.TIVE RESISTANCE" OF FROG SAR'T0~2I11S MÜSCLE IN IS07~1IC K2S04 SOL[TI'IOtd Muscle
Effective resistance (n X 10 ) Isotonic K2~4
1
2 .3
2 3
~ Difference in Effective Resistance
Insulin 80 u./1
= 0 .095 (16)
4 .3 = 0 .17 (18)
79
1.04 * 0 .10 (20)
2 .4 ! 0 .16 (20)
134
2.51 = 0 .11 (20)
3.4 * 0 .19 (22)
36
* - Standard error of the neon . Figures in brackets ~ number of muscle fibres used ; temperature, 25°C . TABLE 3 EFFECT OF BOVINE SERIh1 ALBIMIIV ON TF~ AVERAGE EFFECTIVE RESISTANCE OF FROG SARTORIIIS MUSCLE IN I90r1nIdIC K2S04 Effective Resistance (n X 10 )
Muscle
Without protein
1
2 :24
t 0 .10 (14)
2
2 .34 = 0.11 (16)
With protein (3 .5 ug/ml") 2 .3
3 0.13 (22)
2 .39 r 0.15 (22)
~ - Standard error of the mean . Fi~rts in brackets, number of muscle fibres used ; temperature, 25°C . w - S.S rg/s1 cornspands approximately to the pmtein content of 80 u ./1 of insulin .
Vol . 6, No . 9
INSULIN ACTION
96 3
It seems reasonable to caulude Eras these experisents that the increase on mesbrane resistance observed in isotonic 82904 solution with insulin, is due to a specific action of the hoimoaie on potassüa conductance . Aclmawledgement-The author is üdebted to Professor J. dei Castilio for helpful discussion . References 1.
H. Meshima and H. Washio, Jap. J. Physiol . 14, 538 (1964) .
2.
L.B . 9nillie and J.F . Manery, Am . J. Physiol . 198, 67 (1960) .
3.
K. L. .Zierler, Am. J. Physiol. 197, 515 (1959) .