Published November 1, 1968
`
`Volume and Twitch Tension Changes in Single Muscle Fibers in Hypertonic Solutions CARLO CAPUTO From the Centro de Bioffsica, Instituto Venczolano de Investigaciones Cicnttiicas (IVIG), Caracas, Venezuela. Dr. Caputo's present address is Department of Physiology, Duke University Medical Center, Durham, North Carolina 27706 ABSTRACT Single muscle fibers were exposed to solutions made hypertonic (approximately 460 milliosmols/kg water) by addition of either NaC1, glycerol, urea, acetamide, ethylene glycol, or propylene glycol. The changes in either the fiber twitch tension or the volume were measured. In the case of NaC1 both fiber volume and twitch tension fall rapidly to 64 and 27 % of the respective initial value. These two values were maintained for the duration of the exposure. In the case of the other substances, the fiber volume and twitch tension also de- creased but in these cases the effect was transient and the fibers recovered their initial volume and twitch tension. The rate of recovery in the different hyper- tonic media increased in the order: glycerol < urea < ethylene glycol < pro- pylene glycol < acetamide. In the cases of the last three substances, the initial twitch value was recovered in less than 5 min and even surpassed. However, on returning to normal Ringer the fibers' ability to twitch or to develop potassium contractures was lost. The return of the fibers to normal Ringer after exposure to these hypertonic solutions causes a transient swelling of the fibers. However, when fibers were swelled by exposure to hypotonic media, they did not lose their ability to twitch on return to the normal Ringer. It is well-known (1-5) that hypertonic solutions, prepared by adding proper amounts of NaC1 or sucrose to the normal Ringer solution, selectively affect the twitches and potassium contractures of frog muscle fibers, without impair- ing the conduction of normal action potentials or development of contractures induced by caffeine. The effect of hypertonic solutions has been interpreted (3-5) in terms of the structural changes that occur at the level of the sarco- plasmic reticulum and the transverse tubule system (6-7), and in terms of the changes in ionic strength that occur when the fibers shrink (8-10, and per- sonal communication by Dr. Grundfest). In the course of previous ' work (3), a few unreported experiments were 793
`
`The Journal of General Physiology
`
`MYLAN INST. EXHIBIT 1087 PAGE 1
`
`MYLAN INST. EXHIBIT 1087 PAGE 1
`
`

`

`Published November 1, 1968
`
`794 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 52 • 1968 carried out using as osmotic agents some nonelectrolyte substances, with molecular radii smaller than that of sucrose. It was found that in this case the results obtained were at least qualitatively similar to those obtained by Yamaguchi et al. (I 1), who used glycerol hypertonic solutions. In fact, it was found that upon exposure to such hypertonic media, the fiber twitches dimin- ished transiently, and then recovered, in some cases even surpassing their original value. It was thought to be of interest to find out whether these changes in the twitching ability were accompanied by changes in the fiber volume. In fact, transient volume changes would be expected in the case of hypertonic solutions prepared with solutes to which the fiber membrane is permeable. However, Yamaguchi et al. (11) found that the weight loss which occurred when whole sartorius muscles were placed in glycerol hypertonic solutions, was not transient, at least during the exposure period in their experiment. Recently, Krolenko and Adamjan (12) have shown that the expected volume changes do in fact occur when single muscle fibers are ex- posed to solutions made hypertonic by addition of different penetrating substances. Howell and Jenden (13) have reported that frog toe muscles after having been exposed to a Ringer solution to which 400 rnM glycerol had been added, upon return to the normal Ringer solution, lose their ability to twitch, and that at this time drastic changes are observed in the structure of the transverse tubule system. This observation has been confirmed by Eisenberg and Eisen- berg (14). These interesting observations have been followed by that of Eisenberg and Gage (15) who found that the fiber membrane capacity diminished after the same treatment. In the course of the present work, the older observations have been repeated and extended in order to clarify whether fiber volume changes are associated with these effects. It was also thought of interest to study the effect of hypo- tonic solutions on the contractile properties of muscle fibers, to test whether there is any effect different from that of hypertonic solutions. Beside NaC1, the solutes used as osmotic agents in this work were glycerol, propylene glycol, ethylene glycol, urea, acetamide, and sucrose. Single muscle fibers were used throughout these experiments in order to better follow the time courses of these effects by diminishing the diffusion delays. A partial report of this work has been presented at the XXIV International Congress of Physiological Sciences, Washington, D. C., 1968. MATERIALS AND METHODS Single muscle fibers dissected from the semitendinosus muscle of the frog Rana pipiens were used. The dissection procedure, the experimental chamber, and the setup for registering tension, were the same as described previously (3). In the tension experi- ments, the fibers were stimulated at a frequency of 0.5 shock per see.
`
`MYLAN INST. EXHIBIT 1087 PAGE 2
`
`MYLAN INST. EXHIBIT 1087 PAGE 2
`
`

`

`Published November 1, 1968
`
`CARLO CAPIJTO Volume and Tension Changes 795 For measuring the volume changes caused by the experimental solutions, a special chamber was employed, which allowed rapid change of the solutions, and which could be mounted on the mechanical stage of a Nikon microscope. The fiber was fixed at both extremities on steel hooks, one of which was fixed and the other of which could be moved in order to stretch the fiber beyond slack length. The fiber was laid in a groove running longitudinally in the bottom of the chamber, and was covered by a glass coverslide over its whole length. A small segment of the fiber lay on a small plastic pedestal which was smeared with vaseline to prevent movement during the solution change. This portion of the fiber was photographed before, during, and at different times after the solution change. A Zeiss water immersion objective was used. The fiber diameters were determined from the photographic enlargements. Special care was taken to measure the diameter in the same fiber region in each of the photographs of the experimental series. Blinks (16) has shown that the calcula- tion of the fiber volume, assuming a circular cross-section, is a cause of error since muscle fibers have an elliptical rather than a circular cross-section. However, since in the present experiments it was necessary to make measurements immediately after the solution changes, the method described by Blinks (16) could not be used. Moreover since I was interested in measuring only the rate of change of the fiber volume and not the volume itself, it has been assumed that the value of the radius squared is pro- portional to the fiber volume. Action potentials were recorded intracellularly by means of glass microelectrodes filled with KCI 3 W. A Bioelectric NF1 amplifier (Bioelectric Instruments, Yonkers, N. Y.) with neutralized input capacity connected to an oscilloscope was used. The normal Ringer solution had the following composition in rnM: NaCI 115; KC1 2.5; CaC12 1.8; Na2HPO4, 2.15, and NaH2PO4 0.85. The osmolality of this solution was measured with a freezing point osmometer and was found to be 230 milliosmols/ kg of water. The hypotonic solution contained 40 m_M of NaC1 instead of 115 and its os- molality was 80 milliosmols/kg of water. The hypertonic solutions were prepared by adding 230 rnM of the nonelectrolyte substances, or 115 n~ of NaCI to the normal Ringer solution. In these cases the osmolalities of these solutions were approximately double those of normal Ringer solution. All the experiments were carried out be- tween 20 ° and 22°C. RESULTS Fig. 1 shows the effect on the volume and the twitch tension of two different single fibers of a solution made hypertonic by adding NaC1. The upper graph shows the shrinking of the fiber caused by this solution. In this and the next experiments the volume change during the fiber shrinking or swelling is considered to be proportional to the change in the value of the radius squared; these values are expressed in per cent of the initial value. In this case it may be observed that the value of r 2 falls to 61 a~o of the initial value in less than 30 sec. This new value is maintained for the entire time of exposure. The equilibrium value for three fibers was found' to be 64 4- 2~ of the normal. It is interesting to notice that this value is quite similar to that obtained by Blinks (16) who
`
`MYLAN INST. EXHIBIT 1087 PAGE 3
`
`MYLAN INST. EXHIBIT 1087 PAGE 3
`
`

`

`Published November 1, 1968
`
`796 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 5 `-3 • 1968 measured the change in fiber cross-section. Therefore, in the range of os- molalities used here, the assumption that r 2 is proportional to fiber volume seems justified. The lower portion of the figure shows the change in twitch tension for another fiber after exposure to the same hypertonic solution. In this 120 I00 o ._ 80 "d 60 -ISOTONIC I HYPERTONIC (NaCI) [__ I I I I I I 0 I 2 3 4 5 TIME (minl I ,I ,, l 6 7 8 HYPERTONIC NaCI ISOTONIC i [ ISOTONIC tI!ililILIIINI!I!NIIL11LIII I I I I I I I I I I I I I I I I I I I_I I I I I I I I I 0 30 60 90 120 150 180 210 240 270 TIME (sec) FIGUR~ 1. The graph in the upper portion of this figure shows an experiment in which a single fiber was exposed to a hypertonic medium prepared by addition of NaC1. The shrinking of the fiber in this solution is expressed in terms of the value of the radius squared, in per cent of the value measured in the normal Ringer. The oscilloscope record in the lower portion of the figure shows the effect of the same hypertonic medium on the isometric twitch tension of a single fiber. The fiber was stimulated at a frequency of 0.5 per sec. Fiber diameter 75 #. Notice the difference between the time scales of the two experiments. case the tension fell to 36% of the initial tension. For seven fibers the mean was found to be 27 4- 2% of the original value. From these experiments it appears that after exposure to a hypertonic medium prepared by adding NaCI, both fiber volume and twitch tension values fall rapidly and the new values are maintained for the duration of the exposure. Recovery was observed only after removing the excess NaC1. In these experiments the maximum exposure time was 10 min.
`
`MYLAN INST. EXHIBIT 1087 PAGE 4
`
`MYLAN INST. EXHIBIT 1087 PAGE 4
`
`

`

`Published November 1, 1968
`
`CARLO CAPUTO ~rOlU~'/,~ and Tension Changes 797 Figs. 2-6 show the results obtained in experiments similar to those of Fig. 1, but in which the hypertonic solutions used were prepared by adding 230 rnM of glycerol, urea, ethylene glycol, propylene glycol, or acetamide to the normal medium. From the figures it appears that the fibers shrank rapidly after exposure to the respective hypertonic solution; however, this effect was transient, and the fibers returned slowly toward the original volume. The 140 120 ISOTONIC I "E IOO o ~ 80 60 HYPERTONIC (GLYCEROL) I o I 1 1 I I I I I I 2 3 4 5 6 7 8 9 TIME (rain) HYPERTONIC GLYCEROL ISOTONIC [ I Iso'roN~C I [ l ] I f I I f f f I l I I I f I I f r 1 | f I O 30 60 90 120 150 180 210 TIME (sec) FIOURE 2. Effect of the glycerol hypertonic medium on the volume (upper graph) and twitch tension (lower record) of two different single fibers. The diameter of the second fiber was 70 #. same phenomenon was observed in the case of the twitch tension. The rate of recovery in the different hypertonic media, both of the original volume and the original twitch tension, increased following the order glycerol < urea ethylene glycol < propylene glycol < acetamide. On return to the normal solution the fiber volume transiently increased by varying amounts, according to the solute and then returned slowly toward the original value. These transient volume changes, observed when the fibers were exposed to the respective hypertonic media and then returned to the
`
`MYLAN INST. EXHIBIT 1087 PAGE 5
`
`MYLAN INST. EXHIBIT 1087 PAGE 5
`
`

`

`Published November 1, 1968
`
`798 THE JOURNAL OF GENERAL PHYSIOLOGY - VOLUME 52 " I968 isotonic solution, are expected when the solutes used are able to penetrate the fiber membrane. In the cases of glycerol and urea the exposure time was not sufficiently long to allow the fiber to reach the original volume and twitch values. For seven fibers in the case of glycerol and three fibers in the case of urea, the 140 120 .o~ "~ I00 'S ,-4 -- 80 ¢,1 60 HYPERTONIC (UREA} lSOTONICl [ 0 I 2 I I I I l __ TIME (min) ,ISOTONIC ,_ [ HYPERTONIC UREA I ISOTONIC 1 ? T I I T 1 f T I I ~ T ] 1 1 [ I ? t T I T I f 1 i ~ [ 0 30 60 90 120 150 180 210 240 ~70 TIME (sec) FIGURE 3. Effect of the urea hypertonic medium on the fiber volume (upper graph) and twitch tension (lower record) of two different single fibers. In the lower tension record, a small contracture developed following the change to the hypertonic medium. The diameter of the second fiber was 80 ~. minimum values of r 2 expressed in per cent of the initial value were found to be respectively: 65 4- 1 and 60 4. 3 (mean 4- SEM). After 5 min exposure to the respective hypertonic medium these values were found to have increased to 72 4- 2 and 73 4- 4o~ of the initial value. On return to the isotonic medium r 2 was found transiently to have increased to 119 4- 4 and 122 4- 5%. In the cases of both glycerol and urea the volume changes were accompanied by twitch tension changes; immediately after exposure to the hypertonic media the twitch tension fell to 25 4- 3~o (seven fibers) and 25 4- 5% (five fibers) for the two solutes, and after 2-5 min these values had increased to 40 4- 4 and
`
`MYLAN INST. EXHIBIT 1087 PAGE 6
`
`MYLAN INST. EXHIBIT 1087 PAGE 6
`
`

`

`Published November 1, 1968
`
`CARLO CAPUTO Volume and Tension Changes 799 63 4- 10%. On return to the normal solution the twitches recovered their normal value; occasionally a small potentiation was observed. In the case of the experiments carried out with hypertonic solutions pre- pared with ethylene glycol, propylene glycol, and acetamide, after rapidly 70 -ISOTONICI 6O 50 40 30 ~ ~o 3 I00 ~ 90 SO 70 6O 50 HYPERTONIC (ET. GLYCOL) I I l I I I 0 I ;~ 3 4 5 TIME (rnin) = t = I ill i 6 7 8 g I0 I ~' HYPERTONIC ETHYLENE "GLYCOL ISOTONIC ] I ISOTONIC N![HIII I]I II[lllllli [1 ; l f f .f l l f I I 0 ! 1 f I l f f f I f I t I I I t I t I 1 f I I f I l I 0 50 60 9 120 150 180 210 240 270 300 330 360 TIME (see,) FIOURE ft. Effect of the ethylene glycol hypertonic medium on the volume and twitch tension of two different fibers. In the tension experiment the fiber recovered the initial twitch tension value while still immersed in the hypertonic medium. In other fibers the initial value was even surpassed. In this and the next two figures, notice the disappear- ance of the twitch response after reimmersion in the normal medium. The diameter of the fiber used in the tension experiment was 90 #. reaching a minimum value of 66 ± 4 (four fibers), 69 -4- 2 (five fibers), and 69 -4- 2 (five fibers) respectively, the normal value of r * was recovered within the 5 rain exposure period. At this time the equilibrium values were found to be 97 -4- 3, 101 -4- 1, and 101 -4- 1%. In these cases the fibers swell considerably on being returned to the normal medium. During this transient swelling the
`
`MYLAN INST. EXHIBIT 1087 PAGE 7
`
`MYLAN INST. EXHIBIT 1087 PAGE 7
`
`

`

`Published November 1, 1968
`
`800 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 5 2 • 1968 mean maximum values oft ~ were found to be 142 4- 10, 141 4- 4, and 151 4- 3~o of the isotonic value. It was also found that the diminution in the twitch size caused by these hypertonic media was transient, the mean minimum values being respectively 26 4- 2, 61 4- 6, and 24 4- 2%. Immediately after the minimum values had been reached, the twitches started to recover their original values. The rates of recovery of the twitches varied according to the NYPERTONIC (PROR GLYCOL) 140 ISOTONIC [ [ 120 "G 'F_ o "~ lO0 's 80 60 2__ f ,, I ? I f 1 i I | I 1 0 I 2: 3 4 5 6 7 8 9 tO TIME (rain) HYPERTONIC PROPYLENE GLYCOL ISOTONIC l-- ] ISOTONIC fir ell Ill l ll[tt t 1 ] I ] i I I I I I I I ~ I I ? ] 0 50 60 90 120 150 180 TIME (see) FIGURE 5. Effect of the propylene glycol hypertonic medium on the volume and twitch tension of two different fibers. It may be observed how the initial twitch tension was re- covered more rapidly than the initial volume. The diameter of the fiber used in the tension experiment was 70 #. solute. The times necessary for the fibers to recover 750/0 of their original twitch values were 81 4- 10 sec for eight fibers exposed to ethylene glycol, 49 4- 4 sec for five fibers exposed to acetamide, and 12 4- 3 sec for seven fibers ex- posed to propylene glycol. In the case of propylene glycol the twitch recovery rate was faster than that expected from the rate of volume recovery. After ex- posures varying from 3-5 min, the twitch tension reached equilibrium values of 128 4- 10, 153 4- 23, and 197 4- 18. For the three solutes in these cases, it was found that on returning the fibers to the isotonic medium the twitch was
`
`MYLAN INST. EXHIBIT 1087 PAGE 8
`
`MYLAN INST. EXHIBIT 1087 PAGE 8
`
`

`

`Published November 1, 1968
`
`CARLO CAPUTO Volume and Tension Changes 8oz rapidly abolished, as is clearly shown in Figs. 4-6. In the case of the fibers exposed to the propylene glycol solution, the twitch was recovered relatively soon after their return to the normal medium, as is shown in Fig. 7. A slower recovery was obtained in the case of some of the fibers exposed to the aceta- mide and ethylene glycol solutions. HYPERTONIC (ACETAMIDE) 160 - ISOTONIC I 140 120 -- I00 "6 ~" 80 60 I I I I I 1 t I I f / 0 I 2 5 4 5 6 7 8 9 I o TIME (rain) HYPERTONIC ACETAMID'~" ISOTON]CJ ' ] ISOTONIC II tml l lllllllllll1111 lllllilltlllfll I IIt[ l_L l ' ' ~'o ' ' Lo' ' ~S ' ' ,~'o' ' ,;o' ' ,~o' ' 2io' ' ~;,o' '~-~o' '33o' ' ~o' ' TIME (sec) Fmtm.E 6. Effect of the acetamide hypertonic medium on the volume and twitch tension of two different fibers. The second fiber diameter was 70/~. The loss of the twitch under these conditions is similar to that described by Howell and Jenden (13) and Gage and Eisenberg (17), when whole muscles were returned to a normal isotonic solution after a prolonged exposure to a 430 rn~ glycerol medium. The work of these authors shows that the loss of the contractile response is accompanied by and most probably caused by disruption of the transverse tubule membranes. Gage and Eisenberg (17) also showed that the loss of the contractile response was not due to inexcita- bility of the fiber membrane. This has been confirmed during the present work. Fig. 8 shows an experi-
`
`MYLAN INST. EXHIBIT 1087 PAGE 9
`
`MYLAN INST. EXHIBIT 1087 PAGE 9
`
`

`

`Published November 1, 1968
`
`80,-, THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 52 • 1968 ment in which the action potential and twitch tension of a single fiber were registered simultaneously in normal Ringer, after 5 min of exposure to an ethylene glycol medium, and finally after 5 rain of reimmersion in the normal Ringer. It may be observed that in the hypertonic medium the action potential is rather similar to that obtained initially. At this time the twitch has recovered its initial value. It may be observed, however, that the tension development is slightly delayed. After reimmersion in the normal solution the twitch is lost, while the action potential is not abolished. The change in the shape of the action potential; i.e., the abolition of the afterpotential is similar to that described by Gage and Eisenberg (17). The same results were obtained with three other fibers. HYPERTONIC PROPYLENE GLYCOL ISOTONIC l l ISOTONIC H !I! , ,,,H,,HlII!t111111111 IIIII l/illlllim 111 ~ -I10 rng _ ...................................... ,,,,,,,,,u,,i n, nll,lllllllllltilllllllllllllUilllllllllllllUlllUlll[llllll[![! I I I I [ [ I I [ I r [ T I I I l I I [ I ~ I T I [ I I I I t 0 $0 60 90 120 150 180 210 240 2'70 300 TIME (see) FIouP~ 7. Oscilloscope records showing the disappearance and the slow recovery of the twitch response in the normal Ringers' following exposure to the propylene glycol hypertonic medium. The first trace shows what happened when the fiber was exposed to the hypertonic medium and then returned to the normal solution; the second trace is continuous with the first and shows recovery of the twitch. The entire run lasted ap- proximately 600 sec. Fiber diameter 70 #. Unpublished experiments also showed that when the fiber was returned to the normal solution after exposure to these hypertonic solutions, the potassium contractures were also diminished or abolished; however, caffeine contractures could still be obtained under these conditions. The Effect of Hypotonic Solutions In this work the abolition of the twitch response after treatment with hyper- tonic solutions was observed in the case of those solutes which rapidly pene- trate the fibers. On the other hand other authors have shown that a similar phenomenon occurs after a longer exposure to a glycerol hypertonic medium (13-17). When the fibers loaded with these penetrating substances are reimmersed in the normal isotonic medium, a transient swelling occurs as shown by the
`
`MYLAN INST. EXHIBIT 1087 PAGE 10
`
`MYLAN INST. EXHIBIT 1087 PAGE 10
`
`

`

`Published November 1, 1968
`
`CARLO CAPUTO Volume and Tension Changes 803 experiments of Krolenko and Adamjan (12) and by the experiments reported here. It was thought to be of interest to see whether the fiber swelling per se might be responsible for the disruption of the transverse tubule membranes and for the impairment of the ability of the fiber to twitch. Therefore, the effect of swelling induced by hypotonic solutions was studied. The top part of Fig. 9 shows the effect on the volume of a single fiber of a hypotonic solution prepared with only 40 mu of NaCI. It may be observed my 0 -40 -80 my 0 -40 -80 -40 -80 ,, lltilll 0 4 6 8 ! msec i45 mg 45 mg 45mg FIOURE 8. Oscilloscope records showing the action potential and the twitch tension of a single fiber registered simul- taneously. The upper record was obtained with the fiber in normal Ringer. The middle record was obtained after 5 rain exposure to the ethylene glycol hypertonic medium; after this time the twitch had re- covered its isotonic value. The lowest record was obtained 5 rain after returning the fiber to the normal Ringer; the action potential, although affected, was not lost while the twitch had disappeared. The dotted horizontal bars indicate the twitch peak tension values. Fiber diameter 60 #. that upon exposure to this solution the fiber swells rapidly to a value of r* equal to 160% of the initial value. This value is in agreement with the fiber volume increase caused by a similar solution, reported by Blinks (16). It may also be observed that this value is maintained during the entire period of exposure and that the effect is reversible. In the bottom part of Fig. 9 the effect of this solution on the twitch tension is shown. In this case the twitch disappeared rapidly after the solution change. However, the most conspicu- ous fact in this experiment is that the twitch is rapidly recovered when the fiber is returned to the normal medium. These results suggest that the fiber swelling, that occurs when the fibers are returned to the isotonic medium
`
`MYLAN INST. EXHIBIT 1087 PAGE 11
`
`MYLAN INST. EXHIBIT 1087 PAGE 11
`
`

`

`Published November 1, 1968
`
`804 THE JOURNAL OF GENERAL PHYSIOLOGY ° VOLUME 52 • ~968 following the exposure to the hypertonic media prepared with nonelectrolytes, is not the factor responsible for the twitch loss. Although in this case the twitch was completely abolished upon exposure of the fiber to the hypotonic solution in other experiments only a considerable twitch diminution was observed. This effect is almost certainly due to the diminution of the external Na +. This 160 140 o 120 al ISOTONIC ISOTONIC HYPOTONIC (40 mM NoCl) I I f I f I I TIME (rain) HYPOTONIC I~OTONIC I I ISOTONIC IllilL J 1 ! 0 ,lllllllllllll }oomg ! I I I I I I ! I I 20 40 60 80 I00 TIME (sec) FIotr~ 9. Effect of a hypotonic solution on the volume and twitch tension of two different fibers. In the lower record the fiber was stimulated with a frequency of 0.5 stimulus per sec. The loss of the twitch is attributed to the reduction of external sodium ions. Notice that after reimmersion in the isotonic medium the twitch is rapidly recov- ered. Fiber diameter 70 #. seems to be confirmed by the fact that when isosmotic amounts of sucrose were substituted for the NaC1 withdrawn, the same effect was observed. DISCUSSION Hypertonic solutions prepared with penetrating solutes induce transient changes in the volume and in the twitch tension of single muscle fibers. The fiber volume changes described in this work confirm the results obtained by Krolenko and Adamjan (12).
`
`MYLAN INST. EXHIBIT 1087 PAGE 12
`
`MYLAN INST. EXHIBIT 1087 PAGE 12
`
`

`

`Published November 1, 1968
`
`CARLO CAPUTO Volume and Tension Changes 805 Yamaguchi et al. (11) reported transient changes in the twitch tension of single fibers and whole muscle exposed to 430 rnM glycerol hypertonic solu- tions. However, they did not observe transient changes in the volume of whole muscles (measured by weighing the muscles). They considered that the mechanical response of the fiber, abolished by the glycerol hypertonic me- dium, recovered even though the fibers remained in the dehydrated state. Similar results were obtained using urea, but not when mannitol, glucose, monoacetin, or sucrose were used. From the results obtained in the present and in previous work (18) it may be considered that the fiber membrane is sparingly permeable to glycerol and urea; from considerations of molecular radii it may be supposed that it is almost impermeable to mannitol, glucose, monoacetin, and sucrose. Therefore, the failure of Yamaguchi et al. (11) to observe a weight recovery after the weight loss caused by immersing the muscle in the glycerol and urea hypertonic solutions, might be attributed to a too short exposure time (less than 1 hr) for these substances, and also to the use of whole muscle. On the other hand, the partial recovery of the twitch of whole muscles could be explained by assuming that only the few outermost fibers recovered the twitch. These considerations are based on the results obtained in the experiments reported here, and on the work of Krolenko and Adamjan (12). These authors showed that prolonged immersion of single fibers in hypertonic solutions, prepared by the addition of many different substances including those used here, causes volume changes of the same type as those reported in the present work. From these experiments it appears that the membrane permeability to the solutes used in these experiments follows the order: glycerol < urea < ethylene < glycol < propylene glycol < acetamide. This order with the exception of glycerol and urea is the same as that found by Krolenko and Adamjan. This is in agreement with previous work (18) in which it was found that the relationship A/AX, representing the ratio between the total area for diffusion and the length of diffusion pathways for glycerol, urea, and ethylene glycol, was respectively 0.09, 0.14, and 0.26. These results are similar to those obtained by Collander and Barlund studying the penetration of nonelectro- lytes in Chara (19). Table I shows a comparison between the dimensions of the molecular radii and of the off-water partition coefficients of the nonelec- trolyte substances used in this work. The test compounds are listed according to the above-mentioned permeability sequence. The values of the molecular radii and partition coefficients were taken from Goldstein and Solomon (20) and Collander and Barlund (19), respectively. This table shows that no clear-cut relationship is evident between the membrane permeability to these substances and their molecular dimensions or their olive oil-water partition coefficient. The fiber volume changes that occur in hypertonic solutions appear to be,
`
`MYLAN INST. EXHIBIT 1087 PAGE 13
`
`MYLAN INST. EXHIBIT 1087 PAGE 13
`
`

`

`Published November 1, 1968
`
`806 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 52 • 1968 up to a certain extent, the cause of the changes in the twitch tension. The interpretation of this phenomenon appears to be open to discussion. There is some evidence showing that the increase in the ionic strength in the interior of the fibers caused by water loss may affect the contractile mechanism itself (8-10). On the other hand the changes in the structure of the sarcoplasmic retic- ulum, that occur when the muscle fibers are exposed to hypertonic solutions (6-7), might affect the excitation-contraction coupling mechanism (5). In fact it has been shown (2-4) that when single fibers are exposed to hypertonic solutions, with double the normal tonicity and prepared with a nonpenetrating solute, caffeine contractures are not depressed but potentiated. Furthermore, the rate of tension development of the caffeine contractures is not affected (3). This effect is opposite to that occurring when the contractile responses are triggered by depolarization of the membrane. TABLE I Compound Olive oil-water partition Mean molecular radius~; coefficient* (XIO*) A Glycerol 0.07 2.74 Urea 0.15 2.03 Ethylene glycol 0.49 2.24 Propylene glycol 5.7 2.61 Acetamide 0.83 2.27 * Values taken from reference 19. Values taken from reference 20. A combination of these two factors seems worth considering in order to explain the effect of hypertonic solutions. Podolsky and Sugi (9) have found that solutions of three times normal tonicity diminish by a factor of 10 the shortening velocity of fibers activated by caffeine. Unpublished experiments from this laboratory have shown that the isometric tension developed during caffeine contractures is impaired by the same tonicity. It could be assumed that up to a certain tonicity, the morphological changes in the elements of the sarcoplasmic reticulum affect the excitation-contraction coupling mecha- nism. In this case the responses induced by the depolarization of the mem- brane are affected, while the responses induced by caffeine, which activates the contractile material without appreciable depolarization of the fiber mem- brane (21 ), are not affected. When the fiber shrinkage reaches a certain value, the ionic strength inside the fibers may affect directly the contractile proteins. This assumption, however, is not in total agreement with the recent work of April et al. (10 a). These authors did not find evidence of any threshold value
`
`MYLAN INST. EXHIBIT 1087 PAGE 14
`
`MYLAN INST. EXHIBIT 1087 PAGE 14
`
`

`

`Published November 1, 1968
`
`CARLO CAPUTO Volume and Tension Changes 807 for the effect of ionic strength on the contractile ability of crayfish muscle fibers, activated by iontophoretic injections of calcium ions. In the case of ethylene glycol, acetamide, and propylene glycol, a potentia- tion of the twtich, of different magnitude for each solute, was observed in the fibers that had recovered their original volumes. This potentiation cannot be accounted for at the present time. However, if it is assumed that the permea- bilities of the transverse tubules and sarcoplasmic reticulum membranes to the substances used here differ from that of the fiber membrane, it would be possible to think that structural changes occur at the level of these structures without being accompanied by fiber volume changes. On the other hand a direct specific interaction of the solutes with some structure or reaction in- volved in the excitation-contraction coupling cannot be ruled out. It would be of interest, therefore, to study by electron microscopy the internal fiber structure under the conditions in which the potentiation occurs. Another interesting phenomenon is the dissociation of the excitation-con- traction coupling mechanism, which occurs when the fibers are returned to the isotonic medium after they have regained their initial volume in the nonelectrolyte hypertonic media. Under these conditions the action potential, although altered in shape, is not abolished. These