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Freezing point and melting point of barnacle muscle fibers
JEAN-PIERRE CAILLB' DCpnrf~menf dp Biophysique, Faculfk de MPdecine, L~niversifk de Sherbrooke, Sherbrooke, P.Q., Canada 9 8 H 5Wr$
Received September 24, 8982
CABLI.~, J.-P. 1983. Freezing point and melting point of barnacle muscle fibers. Can. J . H'hysiol. PharrnacoI. 61: I B 16- H 121. The freezing point and the melting point of myoplasm were measured with two experimental models. Hn all samples, a
supercooled stage was reached by lowering the temperature of the sample to approximately --T°C, and the freezing of the sample was nlechanically induced. The freezing process was associated with a phase transition in the interstices between the contractile filaments. In intact muscle fibers, the freezing point showed a structural component (0.43"Cj9 and the melting point indicated that the intracelIular and the cxtracelluIar cornpartrnents are isotonic. When the sample of myoplasm, previously inserted in a cylindrical cavity was incubated in an electrolyte solution, the freezing point showed a sta-idctural component similar to that of the intact rgluscle fiber, but the melting point was Iower than the freezing and the melting points of the embedding sol~ation. This was interpreted as evidence that the counterions arourad the contractile filarrlents occupied a nonnegljlgible fraction of the intracellular cornpartnnent.
G A I L L ~ , J.-P. 1983. Freezing p i n t and naelting point of barnacle ~nuscle fibers. Clan, J. PizysioI. Pharn-8acicol. 6%: 11 16- 1121. On a raaesw-6 les points de congelatio~n et de fusion du nagroplasrne B ['aide de deux modeles expCrimentaux. Dans tous %es
kchantilleaa~s, on a atteint un stade de surfusion en abaissant la tempkrature de I'6chantilIon a pres de -7°C et 1'6chantillon fut amen6 ii Iq6tat congel& par Lane action rr~6canique. Le processus de co~lgelation Ctait associC B une transiti(~n dc phase dans les interstices des filamer~ts contractiles. Dans les fibres musckalaires iratactes, le point de congklation rnontra uane compsante structurale (0,43"C), et le point de fusion indiqua que les compartiments intracellulaires et extracelllulaires etaient isotoniques. On a ins&-$ 1'Cshantillon de ~nyop%asme dans uaae caviti cylindrique, pour I'increber ensuire dans une so%ution Ciestrolytique; le point de congiiarion rnontra une cornpusante structurale similaire a celle de la fibre rankasculaire intacte alors que le point de fusion fut infkrieur auw points de congClation et de fusion de %a solution d'inclusion. On a inte~prdt6 ce fait conamze Ctant la preuve que les contre-ions entaurant les filaiments contractiles occupaient une fraction non negligeable du col-npartiment inti-acellulaire -e.
[Traduit par le journal]
The depression of the freezing paint in biological cells was first measured by Sahbatani (1901) in muscle and liver cells. Thereafter, n-aany investigators par- ticipated in the controversy iabout the values of the freezing point of the cytoplasm (Opie 2954; Pichotka 1952; Conway and McCc~rmack 1953; Brodsky et al. 11956). Blssh et al. (1963) confirmed that the freezing point of the cell was lower than the freezing point o f the embedding solution in gly cero1-extracted muscle. These investigators propc~sed that this difference was due to the network of' the contractile filaments.
In barnacle mkascBe fibers, laser Raman investigation of water indicated that 20% of the anycpplasrnic water remained in a liquid state at --5 and at - 10°C (Pezolet et al. 1978). More recently, the melting of water i ~ a
the melting points were measured in large cells isolated from the depressor muscle of the barnacle (Balanus rzubillrs).
The freezing point of the myoplasm was analyzed as the sum of a structural and an osmotic component. Successive cycles of freezing and melting did not mod- ify the freezing point or the melting point. In intact muscle cells the intracellular and extracellular cornpart- ments were isotc~nic. When the water content was con- trolled, the structural component of the freezing-point depression was similar in the intact muscle cell and in samples of rnycsplasrn incubated in sollaticsn c~f electro- lytes, but in the samples of ~r~yoplasm incubated, the n~elting point was lower than the meking point of the embedding solution.
barnacle muscle fibers was studied with differential scanning calorimetry (Aubain et al. 1980). This study Methods
confirmed the presence of water in a liquid state at - 10 M"5clefibers7
-=ye'. we then inrerest on water The experiinents were performed oaa isolated barnacle
undergoing a phase transition at temperatures between rnluscle fibers (Caill6 and H i k c 1972: Caililik 1975). The barnacles ( R ~ I I C J ~ U S nuhilus) were caught in the region of
-5 and OoC? which may with Vancouver (Canada) and kept in an aquarillm for a lyte solution filling the interstices between the con- of tractile filaments of the muscle cell. The freezing and ~~~~l~ were isolated from the depressor in a
sa~lution (barnacle Ringer) containing in miillirnolar: Na' , '~ecipient of a Cherchcur Boaarsier from the Fonds de la 450; K' , 8; @a2", 20; ?dg2+. 10: CI-, 518; Tris (hydroxy-
recherche en sant6 du QuCbec. methyl) aa~aiinomethane, 25. 'Phe pH was acf.iusted to 7.6 with
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HC1 (Winke and Gayton 197 1) . Two experimental models were used. In 1nodcB A the fibers were isolated in barnacle Ringer and then gei~tly pulled into a glass capillary, the di- ameter of which fitted the diameter of the fiber as previously described (Gail16 and Winke 1972). ' h e measurement c~f the freezing point was carried out irnrnediately without any other treatnnent sf the sarnple. In rnodel B, isolated muscle fibers were rinsed in a solution of sucrose and gently pulled into a cylindrical cavity 1 cm Bong. Ry rinsing the isolated rnaisclc: fibers in hypcrtonic sucrose before their introdrmcticm into the glass capillary, samples of cytoplasn~ with rctfuced water content could be prepared. Again the cavity's diameter was chosen to fit the diameter of the fiber. During this operation. the length of the fiber was kcpt at approximately the length of the fiber in vivo; the excess of rnyoplasrla at hot11 ends cjf the glass capillary was trirn~ned. This pcrrnitted the cytoplasm to be in contact with an elcctrolyte soitation at both open ends of the glass capillary (fiber-filled capillary). Becrnuse thc volume of the myoplasrn is restricted by the cavity. the hydration of the san~ple can be prevented even when the cytoplasm is immersed in a hypotonic solution. When the muscle fiber i s inserted without damage, the sample of myoplasm will not expand out of the cavity. As previously described (Caille and Hinke H972), those samples of myoplasm which remained translucent were selected fc9r nncasuremelats of the freezing and melting points.
Elecfrolybe solutions In rntjdel I% the samples were incubated ina an electrolyte
solution for at least 18 h at 4'C before the freezing and melting points were measured. The electrolyte solution contailred in rnillirnolar: K". 0 to 500; @I- , 0 $0 500; T r i ~ (hydroxyrnethyl) aminomethane, 25; EGTA, 0.5.
M~asurc~nenrs c$ the f r~ez i tm~ and melsita,q points The freezing and ~aeltrng points were inensured by n-son-
itoring the temperature daaring a cycle of wpercooling, freezing and melting of the sample. The temperature was measured with a thermocouple (copper-Cornstantan Type T) introduced inside the san~plc. The diameter of the thcrmo- couple was approxirrnatc1y 0.23 mrn and the diameter s f thc muscle fibers was between l .O and 1.2 mm. The electrical pstent~al at the terminail5 of the therrnocouplc was recorded with a rnicrovoItmeter and a XI' plotter. In preliminary experiments, bhc measurcmcnts were taken wath tuo thermo- couples measailring differential tea-r~peratures. Since this method did not Increase significantly the accuracy in the calibration of the thern~ocouples, it was not included in the protocol.
The freezing poant of NaC1 solutlon5 was used to calibrate the thermocoaaple An exanlple of the calibration 1s presented in Fig. IA with examples of the temperature changes d u n g cycles of freezing and melting of saanples of water (Fig. 1 R ) and cytoplasm (Fig. 1C). During these cycles the temperature sf the sample bas first lowered below the freezing tem- peraturc At approximately -4 to -6'6 (af lower tem- peratures were reached it mil% bc specified) a mechanical shock induced the freezing of the samplc and the following temperature changes were observed (Fag. 1B): the tem- perature remained constant h r a shore pcrit~d, then began to lower. When the temperature reached approain~ately --4 to
-6°C the sample was placed at room tcmperature (approxi- mately 20°C) and its tempcrattmre bcgan to rise. FV'itho~at the mechanical shock, samples of rnyoplasm remained in a super- cooled stage for many hours.
Sarcoanere-length muclsuremenf The measurements of the freezing ancf ~r~elting points
of ~nuscle fibers with experirnentaI rnodel A were performed as a failnctiom of sarcolnere length (L,). To obtain &, the fiber iri theocapi~Iary was placed in a laser beam (We-Ne, A - 6428 A ( 1 A = 0. I nm)). The sarcomerc length was calculated from thc dilfracted pattern recerdeai on a paper scrccn or ow film.
kt'atev corltrnt Finally. the sample of n~yoplasrn mas weighed and dried to
a constant weight at 95°C. AIB the valraes rep~brtb'd are rneans k SEM, unless men-
tioned otherwise. The data wcre tested for statistical signifi- cance wlth the Student's b-test. the significance level chosen was P < 0.85.
Results The freezing and melting points 04' myopkcssrn, e-plpleri-
mentnl m o d ~ l , ~ A and B Hn both experimental models (A arad IS), the freezing-
point depression for myoplasrn was consistently larger than the freezing-point depression for the equilibrating solution. The freezing and melting temperatures of the barnacle Ringer scp%ution were identical for both phase changes (-- 1-83 5 0.02, n - 6). Identical temperature prc~files were obtained when the precooling to melting sequences were repeated 2-3 times. Within the pre- cooling range, -5 to - 15'C, the freezing poima was found to be independent of the supercoc8led tan- perature. Also. the melting point was found to be inde- pendent of the Bkeezing - warming profile (Figs. I B and IC).
As indicated in Figs. H B and lC, the melting point was obtained fror~l the inilecfjon point in the warming curve. This method was at least validated for water and ionic soIutions since the melting points csf the latter are known. Freeakrag and melting poilkt,~ VCTSU,G sarcLp?nerc leragt!z,
e.xp~v-imk>ntal model A The freezing and melting points were naeasured on
samples of rnyoplasm with different sarcornere lengths. Thc reslalts are summarized in Fig. 2. The ~nelting point was not dependent on the sarcomerc length. The slope sf the mclting te~nperaturc versus sarcomere length re- lation (0.024"C/pna) was not different from zero. The freezing tcmperature increased with sarcornere length (0.054"C/p~-n) but P did not reach the Ievel0.85. The freezing and mclting temperature of the extracellular solutioa~ was - li .83"C. Freezing and melting poirlts hand the water content qf
yz~y~rpkusm, experimental model B Experimental model B was used for this study. The
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CAN. J . PHYSIOI>. PHAKMilCOL VOL. 61, 1983
Frc. 1 . Two examples of temperature monitc>ring during the freezing (F) and melting (M) of water (B) and of mycsplasrn (6). In A calibration curve oT the thermocouple, the x-axis is the temperature of freezing of the solutions used as standards. The thermocouple output was fixed to 800 for the solution with the larger freezing point depression.
fibers in the capillary were selected with sarcomere lengths between 8 1.5 and 13 pm which corresponds to the sarcomere lengths of the fibers in situ. The water content was varied from 79.3 to 41.6 g/100 wet-weight as described in Methods. Once in the capillary, all fiber samples were incubated for 18 h in a buffered solution with 0.072 g-moll%, osrnosity. The ficezing and melting points of these fibers are shown in Fig. 3 as a function of their water contents.
When the water content was reduced there was a significant increase in the freezing-point depression (upper curve). There was alw an increase in the melting-point depression (lower curve) although not as large as the increase in the freezing-point depression. The lower data (trianglesj indicate the constancy of the freezing point of the equilibrating solution.
SARCBMEWE LENGTH ( pfw 1
FIG. 2. The freezing (upper diagram) and melting [lower diagram) temperatures of single muscle fibers (experimental model A) as a function of sarcomere length. Linear regression coefficients were used to draw the lines.
Freezing point and osmosity of'thp embrdding snlrction, regression line the experimental points experimental model B gives a measure of the structural effect on the freezing-
Once again, experimental model B was used. The point depression at zero osmosity. fiber samples were equilibrated for 18 h in solutions which varied in osrnos-ity from 0.008 to 0.41 g-mol/L. Efl-ct o f p H on the ji-cczing and melting temperatures The freezing points of these fibers are shown in Flg. 4 of myopla.sm, e>,~perirnentcml model B as a linear function of osmosity. The y-intercept of the Just as the freezing-point depression increases with
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WATER CONTENT (g / 100 g wet weight
FIG. 3. Freczing and n-nclting temperatures of n-nyoplasrn for different water content of the sample experimental model B. 'The osrnosity s f the exterrial solution was 0.072 (g- n-mol/L). The freezing point is represented by filled circles and the melting point by filled squares. The freering temperature of the external rolution is represented by triangles. Values are means z SEILI. 'Fhe rnunnber of 1nea.surernents are between 7 and 13. 'Fhe curves were drawn by hand.
ostraosity so it should also increase with pH because the counterion concentration (hence osmosity) increases around the mysfilairlents with pH. To test this hypoth- esis. fibers were incubated for 18 h cither in one solu- tion at pH 7.0 or one at pH 10.0. The osmosity of the solution was 0.01 g-mol/& and contained 1 mM Tris- (hydroxymetky1)aminoethane and 0.5 mM ECTA. As shown in Table 1, the fibers equilibrated in the pH 10.0 solution had higher freezing-point and melting-point depressions.
Discussion Large depressions of the freezing point of the intra-
cellular medium have bcen interpreted as evidence of this nledium being hypertonic in relation to the extra- cellular fluid (Sabbatani 190 1 ; Opic 1954; arad Pichotka 1952) but large depressions of the freezing point have also been reported in systems containing networks of polyn~ers and rrnay be explained in terms of a reduction in the volume available h r the formation of crystals owing to the presencc of the network, so that only small crystals can be formed. Bloch et al. (1963) applied this hypothesis to the freezing point of ~nyoplasm.
The monitoring of tempemture (Fig. 1B) observed wlth water during freezing indicated that the rise time of the thermocouple was fast enough. As the melting of myoplasrn is stemmed over a few degrees (Aubain et al.
FIG. 4 . Freezing temperatures of my oplasrn (experimental m d c l B) as a function of the oslnosity of the cxternal solu- tion. The data were grouped according to the water content of the myoplasm. 'The filled squares correspond to water contents between 69 and 7 1.9 g/ 100 g wet weight and the filled triangles to water contents between 73 and 76 g/ 100 g wet weight. The freezlng temperature of the external solution is represented by the filled circlcs. 'Fhe straight lines are linear regressions calculated with the least \quare method.
1980) the melting point is difficult to determine. When the freezing and nrelting sequences were re-
peated on the same sample, ide~atical results were ob- tained. This differs frorrn the results of Bloch et al. (1963) who observed, on glycerinated muscle, tkat the freezing point depression was larger than that of the embedding solution in the first fr-reczing, but the freezing and melting temperatures were almost equal in the second cycle. This may be due to an additional condition impc~sed on our experime~atal model that pre- vented the hydration of myoplasm.
The freezing and ilmelting points of glycerinated muscle are similar to those observed on some gels (Bloch et al. 1963). The freezing-temperature depres- sion is partly due to a structural effect which may be obtained from the difference between the freezing temperature and the melting temperature. Our results will be analysed with the hypothesis tkat the freezing and melting processes in myoplasm are sitrlilar to the freezing and melting in gels and in glycerinated muscle.
Experimental model A was chosen to study the freezing and melting processes of freshly isolated fibers. The melting temperature of thc myoplasm of the barnacle muscle fiber was not dependent on the sarco- mere length and was not different from the freezing and melting tennperatures of the barnacle Ringer (- 1.83"G) in which the muscle fiber was immersed. So, the intra-
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CAN. J . PHYSBOE. PHAWMACUL. VOE. 61. 1983
TABLE 1. Effect of pH on the freezing and melting points of myoplasm"
pH of embedding Water content Freezing point Melting point solution" (g/ 100 g wet weight) i0C) ("C)
"The expe,rin~ental n~odei W was used in these measuren~ents. "'B'lle osrnosity of the cnlbeddirlg solution was 0.008 and 0.01 g-moL/L. "Values in parentheses are the number of Irleasurement:, taken.
cellular and extracellular compartments in these ~a~uscle fibers were isotonic. In intact nluscle fibers the distance between the filaments of myosin decreases wlnera the sarcolnere length increases (Gayton and Elliott 1980), SCP the stnactura! component of the freeking point de- pression should be more important at long sarcomere lengths. However. the dispersion of the results did not permit us to conclude that the freezing temperature sig- nificantly increased at long sarcomere lengths. The dis- persion of the rcsuHts cannot be attributed to the thermo- couple used because the values obtained ial NaCH solutions were very reproducible (Fig. 1'4). Tht: lack sf a significant increase in the freezing-point depression with the sarcomere length may be explained by thc very small decrease in interfilament distance that occurs when the sarcomere length is increased from 9 to 14 prn (Gayton and Elli~pet 1980). At a sarcomere length of 82 p,m the difference between the freezing and the melting temperatures obtained with the regression coef- ficients indicates a structural cornponent of 0.43OC in the freezing temperature. This value was lower than the one observed (0.8OC) on glycerinated fibers by BHoch et al. (1963).
The depression of the freezing point allows the testing of a hypothesis concerning the electrolyte con- tent of the interstice between the contractile filaments in the fiber-filled capillary (CaillC and Hinke 1972) be- cause the freezing-point depression of the mycbplasm can be attributed to a phase transition in this interstice. This hypothesis proposes that the clcctrolyte content of the external solution and that of the lraterstice arc the same. With experimental model B a linear relation was observed between the freezing point of the nayoplasm and the osrnosity of the embedding solution, When the osmosity of the external soltation reaches zero, the de- pression of the Breezing point should only be structural for the hypothesis to be valid. 'Fhe values obtained for the structural component of freezing temperature de- pression 0.95 and 1 . 1 O°C for water contents of 73.1 and 70 g/100 g wet weight, respectively, were larger than the ones reported by Bloch et al. (1963) for glycerol- extracted ridductor muscle of Mytklt~s etlulis, and for isolated fibers reported above. This conflicting result
casts doubts on the validity of thc hypothesis stated above.
The smaller volume of thc interstice caused an im- portant increase in the depression of the freezing point when time water content was reduced (experimental naodel B). Furthermore, the melting pomt was signifi- cantly lower than the freezi~mg and melting poirats of the embedding solution (Fig. 3). This observation suggests that the ionic strength in the interstice is higher than that of the embedding solution when the cell membrane is removed and the water content of the cell is kept as in the intact fiber.
Following the interpretation of the Donnan potential nleasured between the cytc~plasnn of these nrbescle cells and the embedding solution, presented by I-Hinke f 1988) and by Elliott and Bai-tels (1982) thc lowering of the melting temperature army result from an excess of coun- terions in the intermyofi'ilanleiats phase. To test this in- terpretation, the effect of pH on the freezing and n-nelting temperature was measured. The melting de- pression should be larger, especially at low osanosity when the pH of the embedding solution is increased, as was observed (see Table 1 ) .
The structural connponcnt of the freezing point in experimental model B, Fig. 3. was 6).4(BoC with a water content of 76 g/100 g wet weight. This value is close to that obtained in intact muscle cells with experimental model A (0.43"C). The strlacttnral component of the f'reezing point was similar in both experimental nlodels.
The freezing point of nnyopiasm measured either in intact m~lscle cells or Hn fiber-filled capillaries contains a structural component which is comparable for similar water contents (0.30°C for 76 g/100 g wet weight). In intact 1nusc1e cells the melting point indicated that the intrricellular cornpanmen1 is isotonic with the extra- cellular scplution, but if the surfice rne~a~brane is re- moved and an increase in the water content of the sample is prevented, the melting point depression of the myoplasm becomes larger than that of the embedding solution. This can be explained as excess s f counterions in the interstice between the filaments, due to the net
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negative charge of the myofilaments (Caille 1979 and Hinke 1980). These results emphasize the importance sf the cell membrane in the raaaintenarace of the osmotic equilibrium between the intracellular content and the external medium.
The author thanks Dr. E. Ruiz-Ceretti for her critical reading of the manuscript, Mrs. Michelins Delorme- Fournier for her assistance in this research and R4rs. G. Josephfowich for typing the manuscript. This research was generously supported by the Medical Research Council of Canada.
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BWCI-I, Q7.. D. H. WALTERS, and W. KL~HN. 1963. Struc- turally caused freezing point depression of biological tis- sues. J . Gen. Physiol. 46: 605-615.
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G a ~ r o ~ , D. C. , and G. F. El,l,!rr'n'. 1980. Structural and osmotic studies of single giant fibres of barnacle muscle. J . Muscle Res. Cell. Motil. 1: 391 -407.
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