Journa/or

Neural T,.ansm~slon

J. Neural Transmission 43, 27--45 (1978)

by Springer-Verlag 1978

of Temperature

The Influence on Neuromuscular

Performance

F. F. F o l d e s , S. K u z e l, E . S. Vizir, and A.

Deery

Departments of Anesthesiology, Montefiore Hospital and Medical Center and Albert Einstein College of Medicine, Bronx, New York, N.Y., U.S.A. With 3 Figures Received December 30, 1977

Summary The influence of lowering the temperature, by 10 ~ increments, from 37 ~ to 17 ~ on the twitch (Pt) and tetanic (Po) tension during direct and indirect stimulation, on presynaptic acetylcholine (ACh) release and on muscle acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) activity were investigated in vitro on the rat's phrenic-nerve-hemidiaphragm preparation. Decreasing the temperature from 37 ~ to 17 ~ caused a progressive increase of the isometric Pt to 195.8 + 9.6 (S.E. of mean) and 169.6 + 2.9 ~ of control with direct and indirect stimulation respectively. This change in temperature also increased twitch duration and time to peak Pt by factors of about 4 and 6 respectively with both direct and indirect stimulation. The Po/Pt ratio did not change significantly between 37 ~ and 27 ~ but dropped sharply between 27 ~ and 17 ~ With direct stimulation tetanus was only maintained in 50 0/0 of the experiments at 37 ~ and in none at 27 ~ or 17 ~ With indirect stimulation tetanus was maintained in all experiments at 37 ~ and 27 ~ and in none at 17 ~ Post-tetanic facilitation was greater with indirect than direct stimulation and at higher than at lower temperatures. Post-tetanic exhaustion, with both direct and indirect stimulation, was only observed at 37 ~ 1 Present address: Department of Anesthesiology, Kanazawa University School of Medicine, Kanazawa, Japan. Present address: Department of Pharmacology, Semmelweis University School of Medicine, Budapest, Hungary.

0300-9564/78/0043/0027/$ 03.80

28

F. F. Foldes, S. Kuze, E. S. Vizi, and A. Deery:

Presynaptic ACh release (pmol. g-1 . min-1) at rest and with stimulation rates of 0.1 to 50 Hz decreased by more than 60 % as temperature was lowered from 37 ~ to 17 ~ Cooling from 37 ~ to 17 ~ caused a similar decrease in the volley output (pmol 9g-1. volley-i) of ACh. Muscle-AChE and BuChE activities decreased by 34 and 52 0/0 respectively when the temperature was lowered from 37 ~ to 17 ~ The findings presented indicate that the site of the facilitating effect of cooling on Pt is the muscle fiber. The facilitation is caused by the delay of the relaxation of the contracted muscle, causing prolongation of the active state and increased tension development. The decreased speed of nerve conduction and ACh release caused by cooling adversely affects neuromuscular transmission. This, however, is partially counteracted by decreased muscle-ChE activity and increased sensitivity of the postjunctional membrane to ACh caused by cooling. Key words: acetylcholine release, influence of temperature on; cholinesterase activity, influence of temperature on; temperature, influence of on neuromusucular performance; twitch and tetanic tension, influence of temperature on. Introduction Dating back to the end of the 19th Century (Gad and Heymans, 1890) numerous publications dealt with the influence of temperature on the isometric twitch (P0 and tetanic (Po) tension of muscles. Most of the reported experiments were carried out on isolated (Hartree and Hill, 1921; Hill, 1938, 1951; Kelley and Fry, 1958) or in situ (Walker, 1951) amphibian (frog or toad) muscles. In the in vitro experiments usually direct and in those with in situ muscles indirect supramaximal stimulation was used. In the mammalian experiments, effect of temperature on the in situ indirectly stimulated triceps surae (Walker, 1951), or the directly or indirectly stimulated gastrochemius (Doudournopoulos and Chatfield, 1959) or the in vitro directly stimulated soleus or extensor digitorum longus (Close and Hob, 1968) or the indirectly stimulated phrenic nerve-diaphragm preparation (Brown et aI., 1948; Harris and Leach, 1968; Segawa et al., 1967) were used. The effect of temperature on the directly and indirectly elicited Pt and Po on the same muscle (rat gastrochemius) were only compared in one in vivo study (Doudournopoulos and Chatfield, 1959). Search of the literature failed to reveal similar studies on in vitro mammalian muscle preparations. Although, depending on the experimental conditions (e.g., species, temperature range explored), there has been some variation in the reported findings (Walker, 1960), most investigators concluded that decreasing the temperature increased Pt and decreased Po (Hill, 1951), increasing the temperature had the opposite effect and

Temperature and Neuromuscular Performance

29

that the primary site of action of cooling is the muscle fiber itself. Cooling was also reported to decrease presynaptic acetylcholine (ACh) release at the neuromuscular (n.m.) junction (Fatt and Katz, 1952; Liley, 1956; Boyd and Martin, 1956) and increase the sensitivity of the postjunctional membrane to depolarization by ACh and succinylcholine (SCh) (Harris and Leach, 1968). The purpose of the present study was: a) To compare the effects of cooling on the direct and indirect Pt and Po in the same in vitro mammalian preparation; and b) to determine the effect of cooling not only on Pt and Po, but also on presynaptic ACh release and musclecholinesterase (ChE) activity and thereby obtain some information on the relative significance of temperature changes on the pre- and postsynaptic components of neuromuscular function.

Methods

Isolated Hemidiaphragm Preparation of the Rat Isolated phrenic nerve-hemidiaphragm preparations (B~lbring, 1946) of male Sprague-Dawley rats of 250 to 300 g body weight were used. This preparation was selected because its contractility and resting potential remain stable for several hours (Liillmann, 1958). Animals were stunned by a blow on the head and decapitated. The hemidiaphragms were dissected and mounted in organ baths in 70 ml mammalian Krebs' solution (NaC1 113.0; KC1 4.7; CaCI~ 2.5; KH.2PO4 1.2; MgSO4 0.6; NaHCOa 25.0 and glucose 11.5 mM). The Krebs' solution was equilibrated with 5 ~ CO.2-95 % 0.2 gas mixture. The resting tension of the hemidiaphragms was adjusted to 10 g. The temperature of the organ bath was kept at the desired level with a thermostatically controlled water bath. To facilitate cooling or heating, crushed ice or hot water were added to the water bath. Supramaximal, square wave stimuli (Grass $9 stimulator) of 0.2 msec and 2.0 msec duration were used for indirect and direct stimulation, respectively. The stimulation rate, except when stated otherwise was 0.1 Hz. Isometric Pt and Po were recorded with Grass FT03 force displacement transducers on a Grass Model 50 polygraph. The paper speed, except when stated otherwise, was 5 mm-min -1. To eliminate the "indirect" component or direct stimulation (Foldes, Brodman, Kranzler, Underwood, and Hemsworth, 1969) complete block of n.m. transmission was produced by the addition of 2 #g. ml-~ d-tubocurarine chloride (d-Tc) to the organ bath before measurement of the control Pt. In the tetanus experiments, stimuli of 50 Hz were applied for 10 sec. The time interval between successive tetani was at least 20 rain. In addition to the measurement of the Pt and Po the twitch duration and the time to peak tension were also recorded. The paper speed during these latter measurements was 100 mm 9 sec"~.

30

F.F. Foldes, S. Kuze, E. S. Vizi, and A. Deery:

Assay of Acetylcholine In the experiments designed to determine the influence of temperature on presynaptic ACh release the hemidiaphragms were suspended in 6 ml organ baths in eserinized (2 #g 9ml "1) Krebs' solution. Ai~ 90 min during which period the initially high rate of resting ACh release became stabilized (Boros, Foldes, and Torda, unpublished) the released ACh was collected for bio-assay. The successive collection periods at stimulation rates of 0, 0.1, 0.5 and 2 H z were 20 rain and at 50 H z was 1 min. ACh was measured with a modification (Vizi, Foldes, and Theunissen, 1975) of the guinea-pig ileum assay of Paton and Vizi (1969). The assay was carried out on a length of guinea-pig ileum suspended in 3.5 ml Krebs' solution at 33 ~ The aboral end was connected to an openended polyethylene tube which projected through the bottom of the organ bath to allow the luminal contents to be extruded from the ileum without contaminating the bath. Since prolonged isometric tension induces contractions of the ileum a sof~ spring of 1 g 9 cm -1 compliance was inserted between the proximal end of the ileum and the FT03 transducer to minimize this spontaneous activity. To reduce the release of endogenous ACh, morphine sulphate (10 #g 9 mD) was added to the assay bath. Samples of control ACh solutions freshly made up in 0.9 0/0 (w/v) NaC1 solution were added to the assay bath at intervals of 1 min in volumes of 0.1 to 0.4 ml. The esimate of the ACh content of samples was based on repeated control dose-responses curves. Changes of bath fluid were made by overflow. The activity of assay sample could always be abolished by pretreatment of the assay organ with atropine (1 # g . ml "1) or by the addition of purified plasma ChE (EC 3.1.1.7) to the samples; moreover, mepyramine (2 #g" m1-1) had no effect on the contractile responses. The ACh released and assayed was expressed in pmol. g-1. min-1 or p m o l ' g-1- volley -1 (for calculation see Vizi et al., 1975).

Determination of Muscle Cholinesterase Activity Muscle-ChE activity of the intact hemidiaphragms was measured with the radiometric assay of Ehrenpreis, Mittag, and Patric (1970) at 37 ~ 27 ~ and 17 ~ The phrenic nerve-hemidiaphragm preparations were suspended in 70 ml mammalian Krebs' solution containing 1 rag. m1-1 sodium acetate trihydrate (Krebs'-acetate) in twin organ baths. Either 1X 10-5 M ACh, butyrylcholine (BuCh) or acetyl-fi-methylcholine (MeCh) in which the acetyl or butyryl groups were labelled with 14C were used as substrates. The calculated specific activities of ACh, MeCh and BuCh were 3.84, 3.30 and 3.57/~c. #tool "1, respectively. The actual specific activities, however, were measured in each experiment and were used for the calculation of the results. Acetate was added to the Krebs' solution to minimize the uptake of 14C-acetate or -butyrate by the muscle. The preparations were washed 3 times, 5 rain apart with Krebs'-acetate

Temperature and Neuromuscular Performance

31

before the addition of the substrates to remove as much as possible of the solubilized ChE. After addition of the substrates 3 min were allowed for the equilibration of the enzymes with the substrates before the first (0 rain) 4 ml samples were removed. Additional 4 ml samples were taken at 5, 10 and 20 min with ACh and BuCh and at 15, 30 and 60 rain with MeCh substrate. A 2 ml aliquot of each sample was mixed immediately with 0.1 ml of 4 X 10-a M eserine sulfate. The other 2 ml aliquot was incubated at 37 ~ for 10 rain before the addition of eserine. This second aliquot was used to determine ChE activity due to solubilized enzymes remaining in the samples. Aflcer all samples necessary for the assay of enzymes activity at 37 ~ had been obtained, the bath temperature was decreased to 27 ~ The preparation was washed 3 times with Krebs' solution and was allowed to equilibrate at 27 ~ with fresh Krebs'-acetate. Labelled substrate was again added and samples collected according to the schedule used at 37 ~ Subsequently enzyme activity was also assayed in an identical manner at 17 ~ At the end of the experiment, the diaphraghm was dissected free of its tendon and costal attachments and its wet weight recorded. All samples were kept in an ice bath until further workup. Subsequently 1 ml aliquot of each eserinized sample were passed through a Bio-Rad 50W-X8, 200--400 mesh column, equilibrated with 0.1 M sodium phosphate buffer of p H 7.2. The effluents and washings obtained after the passage of a 1.0 and an 0.5 ml aliquot of phosphate buffer through the column.s were collected in scintillation vials containing 10 ml of Bray's solution and counted in a Packard Model 3375 liquid scintillation counter. Plotting the radioactivity (cpm. m1-1) of the immediately eserinized samples obtained at various intervals, at the same temperature, against time showed a linear relationship. Therefore with ACh and BuCh the 20 min and with MeCh the 60 rain data were used for the calculation of the hydrolysis rates of the substrates. The hydrolysis rates were expressed as nmol. g-l_ tissue, hour 1. The results with ACh and BuCh substrates were calculated with the formula: nmol. g-1. hour-1 = (cpm 9m1-1)2~t - [(cpm 9ml- 1)0t+(cpm - mlq)2~ X 3 X V W X cpm. nmol "1 In this formula (cpm. ml'l)~~ is total cpm at 20 min, (cpm. ml-1)~ is total cpm at 0 min, (cpm. ml'l)~~ is cpm at 20 min due to solubilized enzyme activity, V is the volume of the system before the 20 rain sampling, W is the wet weight (g) of the hemidiaphragm and cpm. nmo1-1 is the specific activity determined before the start of ea& experiment. A similar formula into which the 60 rain values were substituted was used for the calculation of the hydrolysis rates of MeCh. The statistical significance of the differences observed at various temperatures were evaluated with the "paired" t-test. Q~0 values were also calculated for the various parameters measured at different temperatures.

32

F.F. Foldes, S. Kuze, E. S. Vizi, and A. Deery:

Results

Effects o~ Temperature

on Twitch Tension

Lowering the temperature from 37 ~ to 17 ~ increased both the directly and indirectly elicited Pt (Fig. 1) prolonged twitch duration and delayed the development of maximal Pt (Fig. 2). Elevating the temperature from 17 ~ to 37 ~ caused a decrease of similar magnitude of the above parameters. Statistical analysis of the tabulated data (Table 1) indicates that with each 10 ~ decrease of the temperature from 37 ~ to 17 ~ the values of the various parameters became significantly (p < 0.001) higher, increasing the temperature from 17 ~ to 37 ~ had the opposite effect. The influence of temperature on Pt at corresponding temperatures was significantly (p < 0.002 to 0.001) greater with direct than with indirect stimuta-

TEMPERATURE 37

INDIRECT =-COOLING . . . . . . . . . . . . . . 35 32 29 27

TEMPERATURE 17

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Fig. 1. The influence of temperature on the twitch tension (Pt) of the directly and indirectly stimulated phrenic nerve-hemidiaphragm preparation of the rat. Experimental conditions: Mammalian Krebs' solution areated with 95 ~ 0'.,--5 ~ COx at temperatures indicated; supramaximal square wave stimuli of 2.0 and 0.2 ms duration with direct and indirect stimulation respectively; stimulation rate 0.1 Hz; isometric recording paper, speed 0.25 mm 9 in experiments with direct stimulation neuromuscular transmission blocked by tubocurarine. Note that decreasing the temperature increased and increasing the temperature decreased Pt during both direct and indirect stimulation

33

Temperature and Neuromuscular Performance

tion. There was no difference in the influence of temperature on twitch duration and time required for development of peak tension with direct or indirect stimulation.

Effects of Temperature on Tetanic Response Observations on the influence of temperature on tetanic response are summarized in Table 2. Decrease of temperature from 37 ~ to 27 ~ caused no significant change in Po or in the Po/Pt ratio with DIRECT

STIMULATION TEMPERATURE

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Fig. 2. The influence of temperature on the twitch duration and the time to peak tension of the directly and indirectly stimulated phrenic nerve-hemidiaphragm preparation of the rat. Experimental conditions as in Fig. 1 except that paper speed was 100 mm. sec-1. Note that decreasing the temperature increases twitch duration and delays development of peak tension. Increasing the temperature has the opposite effect 3 Journal of Neural Transmission43/1

F. F. Foldes, S. Kuze, E. S.Vizi, and A. Deery:

34

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F. F. Foldes, S. Kuze, E. S. Vizi, and A. Deery:

36

either direct or indirect stimulation. At 17 ~ however, both P,, and Po/Pt ratio was significantly tess (p < 0.05), than at 27 ~ or 37 ~ With direct stimulation tetanus was maintained at 37 ~ in 2 out of 4 experiments and in none at 27 ~ and 17 ~ With indirect stimulation tetanus was maintained in all experiments at 37 ~ and 27 ~ and in none at 17 ~ Post-tetanic facilitation with direct stimulation xx

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Temperature and Neuromuscular Performance

37

was moderate and not significantly different at various temperatures. With indirect stimulation post-tetanic facilitation was higher at 37 ~ and 27 ~ than at 17 ~ (p < 0.001). Post-tetanic exhaustion was observed only at 37 ~ both with direct and indirect stimulation.

Temperature Dependance of Acetylcholine Release ACh release was significantly higher at various rates of stimulation (p < 0.02 to p < 0.001; paired t-test) than at rest (Fig. 3 a). The volley output of ACh decreased progressively from 0.1 to 2 Hz (Fig. 3 b). Increasing the stimulation rate from 2 to 50 Hz caused no further decrease of the volley output. Decreasing the temperature from 3 7 ~ to 17~ caused a progressive decrease of both the resting and stimulated ACh release. The reduction of ACh release (pmol 9g-1. rain-i) was about 40 ~ of control between 37 ~ and 27 ~ and about 30 ~ between 27 ~ and 17 ~ The differences were significant (t-test) between the p < 0.02 and p < 0.001 levels. The influence of decreasing temperatures on the volley output of ACh was similar (Fig. 3 b).

The Effect of Temperature on Muscle Cholinesterase Activity Lowering the temperature from 37 ~ to 27 ~ decreased the hydrolysis rate of ACh, MeCh and BuCh to about 83, 96, and 70 % of control rates respectively (Table 3). These differences are significant (paired t-test) at the p < 0.01, 0.05 and 0.025 levels respectively. Further decrease of the temperature to 17 ~ decreased hydrolysis Fig. 3. The influence of stimulation rate and temperature on acetylcholine release in the rat's phrenic nerve-hemidiaphragm preparation. Experimental conditions as in Fig. 1 except that the volume of the organ baths was 6 ml, Hemidiaphragms incubated in Krebs' solution containing 2/zg/ml eserine for 90 rain before the first collection period. The successive collection periods with stimulation rates of 0, 0.1, 0.5 and 2 Hz were 20 rain, with 50 Hz 1 rain. The ACh content of the samples was assayed on a segment of guinea-pig ileum at 33 ~ (Paton and Vizi, 1969; Vizi et al., 1975). Values represents means of 4 to 5 experiments, Horizontal bars indicate SE of the mean. The differences between the resting and stimulated ACh release at 3 7 ~ were significant (paired t-test) between the p ~ 0.02 (x) and p ~ 0.001 (• levels (Fig. 3 a). Both at rest and at various stimulation rates ACh release decreased progressively with decreasing temperatures. The significance of the differences (t-test) between 37 ~ and 27 ~ were between p < 0.01 (+) and p ( 0.001 (++) and between 27 ~ and 17 ~ between p ( 0.02 (e) and p ~ 0.01 (o e) levels. Volley output [(stimulated releaseresting release)/number of stimuli] decreased with increasing stimulation rates and with decreasing temperatures at the same stimulation rate (Fig. 3 b)

38

F. F. Foldes, S. Kuze, E. S. Vizi, and A. Deery:

rates of ACh, MeCh and BuCh to 61, 66 and 48 ~ of control respectively. These values are significant between the p < 0 . 0 1 and p < 0.001 levels. Table 3. Influence of temperature on tissue-bound acetylcholinesterase

(ACHE) and butyrylcholinesterase (BuChE) activity in the rat's hemidiaphragm preparation Hydrolysis rates (nmol 9g-1. hour-l) with various substrates

Temperatures (~

ACh

MeCh

BuCh

37 27 17

290 + 10.1 239 +_ 8.1"** 179_+ 6.1 ***'~

50 + 1.4 48 + 0.8* 33+_0.7*":"

169 +_19.3 119 + 24.7** 81_+ 9.1"**

AChE (EC 3.1.1.7), BuChE (EC 3.1.1.8) and total-ChE activities was determined with '4C labelled acetyl-fi-methylcholine (MeCh), butyrylcholine (BuCh) and ACh substrates. Substrate concentrations 1 X 10-5 M. For details of experimental procedure see Methods. Means + SE of means of 4 experiments. *, "**, *** and ':'*** indicate significance (paired t-test) between hydrolysis rates at 37 ~ and 27 ~ or 37 ~ and 17 ~ at the p < 0.05, 0.03, 0.01 and 0.001 levels respectively.

Discussion The Qi0 values (Table 4) of the various parameters of neuromuscular activity indicate that between 37 ~ and 17 ~ lowering of temperature increased Pt, twitch duration and the time required for the development of maximal twitch tension with both direct and indirect stimulation. Po increased moderately during direct and remained unchanged during indirect stimulation between 37 ~ and 27 ~ In contrast between 27 ~ and 17 ~ Po decreased with both direct and indirect stimulation. Presynaptic ACh release at rest and at stimulation rates of 0.1 to 50 H z decreased at about the same rate between 37 ~ and 27 ~ and 27 ~ and 17 ~ AChE activity decreased insignificantly between 37 ~ and 27 ~ and at a more rapid rate between 27 ~ and 17 ~ The rate of decrease of BuChE activity between 37 ~ and 27 ~ and 27 ~ and 17 ~ was about the same and between 37 ~ and 17 ~ the overall decrease of BuChE activity (about 52 ~ was greater than that of AChE activity (about 34 ~

Temperature and Neuromuscular Performance

39

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