Wise, 1974), and this was shown to be associated with an increase in the rate of 45Ca. Denmark (Received 18 August 1978) by tetracaine (104 M).

Transcription

1 J. Physiol. (1979), 292, pp With 11 text-ftgurew Printed in Great Britain THE EFFECT OF HYPEROSMOLARITY AND INSULIN ON RESTING TENSION AND CALCIUM FLUXES IN RAT SOLEUS MUSCLE BY T. CLAUSEN, A. B. DAHL-HANSEN* AND J. ELBRINKt From the Institute of Physiology, University of Arhus, 8 Arhus C., Denmark (Reeived 18 August 1978) SUMMARY 1. The effet of hyperosmolarity on resting tension and on the fluxes of Na and Ca has been haraterized in isolated soleus musles of the rat. 2. When the osmolarity of the inubation medium was inreased by the addition of non-permeant solutes (1-4 m-osmole), the tension showed a rapid dosedependent rise whih ould be maintained for up to 6 min. 3. Tension development was unaffeted by tubourarine (2 x 14 M), onsiderably diminished by the omission of Na or Ca from the inubation medium, and inhibited by tetraaine (14 M). 4. The addition of mannitol or surose (2 mm) indued a prompt stimulation of the influx of 22Na and 45Ca. Both in the absene and the presene of extraellular Ca hyperosmolarity stimulated the washout of 45Ca from preloaded musles. Tetraaine (5 x 14 M) suppressed the effets of hyperosmolarity on both the influx and the efflux of 45Ca, but only gave a modest redution in the stimulation of 22Na influx. 5. Insulin (5-1 mu./ml.) indued a onsiderable further rise in the resting tension of musles exposed to mannitol or surose (2 mm). This effet was seen in a gluose-free medium and ould be abolished by the addition of insulin antibody. 6. It is onluded that hyperosmolarity leads to a rise in the onentration of free Ca2+ ions in the saroplasm, partly due to a mobilization of Ca from intraellular pools, but to a onsiderable extent supplemented from extraellular soures. Under these onditions, insulin further augments the Ca2+ ion level in the ytoplasm. INTRODUCTION Exposure of skeletal musle to hypertoni solutions has been used to dissoiate oupling of membrane exitation from mehanial ontration (Hodgkin & Horowiz, 1957; Tigyi & Shih-Fang, 1962). In onnexion with suh studies it was noted that in amphibian musles hypertoniity per se eliited a rise in resting tension (Hill, 1968; Isaason, 1969 Lannergren, 1973; Lannergren & Noth, 1973; Homsher, Briggs & Wise, 1974), and this was shown to be assoiated with an inrease in the rate of 45Ca * Present address: Med. Afd. A., Rigshospitalet, Blegdamsvej, 21 Kobenhavn, Denmark. t Present address: Department of Pharmaology, University of Alberta, Edmonton, Alberta, TG6 2H /79/ $ The Physiologial Soiety

2 56 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK release from reloaded musles (Isaason, 1969). Similar studies have not yet been arried out with mammalian musles; hene, the primary purpose of the present work was to assess the role of extraellular and intraellular alium and sodium ions in hyperosmolarity-indued tension development in intat rat skeletal musle. Hypertoniity stimulates heat prodution (Hill, 1968; Yamada, 197), glyogenolysis (Clausen, 1968) and gluose transport (Kuzuya, Samols & Williams, 1965; Clausen, Gliemann, Vinten & Kohn, 197; Clausen, Elbrink & Dahl-Hansen, 1975). Sine all of these effets might be due to an alteration in the distribution of Ca2+ ions, it was of interest to determine whether hypertoniity ould produe a rise in the ytoplasmi Ca2+ ion level and to haraterize this with respet to rate of onset, time ourse and dependene on extraellular ioni milieu. Attempts were made to obtain this information from reordings of resting tension and parallel measurements of 45Ca-washout, 5Ca uptake and 22Na uptake. The results indiate that in soleus and extensor digitorum longus musles of the rat, exposure to hypertoni media indues ontratures. In soleus musle the initiation of these ontratures appears to depend on a ellular soure of Ca2+ ions, whereas extraellular alium is required for the maintenane of the inrease in tension. When the intraellular free Ca2+ ion onentration had been inreased in a hypertoni medium, insulin ould indue a onsiderable further rise in tension whih may reflet an additional mobilization of Ca. This is in agreement with the earlier observation that the hormone an stimulate the release of 45Ca from preloaded soleus musles (Clausen & Martin, 1977). Part of the results have briefly been desribed in preliminary reports (Clausen, Elbrink & Martin, 1974; Clausen et al. 1975). METHODS All experiments were performed with fed or 48 hr fasted Wistar rats weighing 6-7 g. Soleus or extensor digitorum longus musles weighing 2-3 mg were isolated and inubated as desribed in detail elsewhere (Kohn & Clausen, 1971; Chinet, Clausen & Girardier, 1977). The standard inubation medium was Krebs-Ringer biarbonate buffer (Cohen, 1951) ontaining one half of the reommended onentration of Ca (1.27 mm). The ph of the buffer was maintained at 7-4 by ontinuous gassing with 95 %:5 % CO2. The experiments were arried out at 3 'C in order to prevent spontaneous ontratures (see Kohn & Clausen, 1971). Tension measurements For the reording of tension, the musles were mounted vertially in plasti holders whih were plaed in a small test tube in a onstant temperature bath. A stainless-steel ring was tied to the distal tendon and hooked to a platinum wire whih also ould serve as an eletrode for diret stimulation. In the experiments with soleus musles, a small piee of bone was left on the proximal tendon whih was attahed by a stainless-steel wire to an isometri fore transduer built at the Institute. Voltage was applied to the transduer from a 6 V DC power supply (Struers Sientifi Instruments, Copenhagen) and the ontratures were reorded on a Servograph pen reorder Model REC 51 with a REA 31 pendrive (Radiometer Co., Copenhagen). A Harvard isometri fore transduer (Model 373) with an eletroni reording module (Model 35) was used for the determination of single twith tension. In these experiments, the musles were stimulated with a Harvard Model 344 stimulator delivering square wave pulses of -3-5 mse duration with the voltage adjusted to times threshold. Before tension hanges were indued, the musles were allowed to equilibrate for 3 min. Preliminary experiments had shown that the tetani tension was maximal when the initial tension was between 1 and 2 g. The resting tension was routinely set at 1 g and ould be maintained for several hours. To indue hypertoni ontratures, the buffer was quikly aspirated from the hamber and replaed by the same medium

3 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION to whih surose, D-mannitol or other agents to the required osmolarity had been added. Alternately, an aliquot of the agent was added diretly to the buffer. Both methods gave idential results, but leaner reords were obtained by the addition of the aliquot. An inrease of 1 #uosmole/ml. brought the osmolarity to 1-32 x that of the standard medium (1-32 x K.R.). In the experiments where insulin was added the buffer ontained 41 g/1 ml. (w/v) dialysed bovine serum albumin. 'IMeasurements of 45Ca- and 22Na-fluxes The uptake of u5ca was measured by inubating musles with ontinuous gassing in 3 ml. buffer ontaining.5 4u/ml. of 45Ca. After inubation, the musles were blotted, the tendons removed to ensure homogeneity of the tissue, and the musles homogenized in 5 g/1 ml. (w/v) trihloroaeti aid using a rapid homogenizer (Elado, Type X12). Following entrifugation, aliquots of the lear supernatant were taken for ounting and the amount of 45Ca taken up expressed on the basis of the speifi ativity of the inubation medium. Control experiments showed that there was no preferential binding of 45Ca to the protein sediment. In some experiments the ellular uptake of '5Ca was alulated by deduting the amount of 45Ca present in the water spae available to [U-14C]surose. In order to obtain an alternative estimate of the amount of 45Ca taken up into the ytoplasm, the inubation with 45Ca was ombined with a wash in ieold Ca-free buffer to whih EGTA ( 5 mm) had been added so as to favour the removal of Ca from the interstitial spae. This proedure was based on the observation that in the old, the frational loss of ellular uca orresponds to 2 % of the total tissue ontent per minute (Clausen et al. 1975). From this it ould be expeted that following a wash of 3 min, the major part of the 45Ca onfined to the ells would be retained (see Table 2). With this tehnique, the unertainties involved in orreting for extraellular *Ca were avoided, and it was thus possible to assess the early effets of hyperosmolarity on 45Ca uptake. A similar ombination of inubation and old-wash had earlier proved satisfatory for the study of 22Na influx (Clausen & Kohn, 1977). In some of the experiments, 22Na (-5 1u/ml.) was added together with the 45Ca in order to measure the uptake of the two isotopes simultaneously. 45Ca efflux was determined in musles from whih the tendons had been arefully reseted. The tehniques for the measurement of the frational loss of 45Ca have been desribed earlier (Clausen et al. 1975). The radioativity of the samples was measured by liquid sintillation spetrometry using a toluene/triton-x 1 sintillation mixture. Quenhing was determined by the external standard hannels ratio method. The results are expressed as mean values ± S.E. and the statistial signifiane of differene was determined by two-tailed t tests. Where appliable, paired t tests were used. Chemials, isotopes and hormone All hemials were of analytial grade. Ouabain and tetraaine hydrohloride were purhased from Merk Co., bovine serum albumin (fration V) and aetylholine from Sigma Chemial Co. Tubourarine waas a produt of Burroughs Wellome Co. 45Ca (26 m/m-mole) was obtained from the Danish Atomi Energy Commission, Ris. [U-'4C]surose (35 m/m-mole) and 22Na (23 m/m-mole) from the Radio-hemial Centre, Amersham, U.K. Mono-omponent pork insulin, lot no. MC-S 82381, was a gift from the Novo Researh Laboratories, Copenhagen, and the anti-insulin rabbit serum was kindly provided by Dr Hans Orskov, Arhus Muniipal Hospital. 57 RESULTS The time ourse of the effet of hyperosmolarity on tension in rat soleus musle is shown in Fig. 1. Exposure of the musle to a medium ontaining 2 mm-surose led to a rapid rise in tension whih reahed a maximum in approximately 3 min. Thereafter tension gradually delined, at times taking 1 hr or more to return to the initial level. Sine the rate of deline was variable and ould not be related to the magnitude of the initial inrease, the ontratures were most onveniently desribed by the maximum tension obtained (Tp) and the time it took to reah this value

4 58 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK (tlp). With 2 mm-surose, Tp orresponded to about 6 % of the maximum tetani tension as measured in the standard inubation medium. In order to determine whether the reorded rise in tension was due to the inevitable shrinkage of the musle fibres, the effet of quikly releasing the tension was assessed. From Fig. 1B it an be seen that if this is done at a moment when the ontrature is well developed, there is again a rise in tension whih reahes the value expeted from the gradual deline of an uninterrupted ontrature. The same results were Surose (2 mm) 2 A, Cn C Surose (2 mm) so~~~~ee I l I II Time (min) Fig. 1. A, effet of hyperosmolarity on resting tension of rat soleus musle. Soleus musles were inubated in Krebs-Ringer biarbonate buffer at 3 OC. The ph ofthe buffer was maintained at 7*4 by ontinuous gassing with 95 % 2:5% CO2. The resting tension was 1 g. The hyperosmolar ontrature was indued by adding an aliquot of surose at a final onentration of 2 mm to the buffer. The hange in tension was reorded for 6 min. For further details see Methods. B, effet of quik-release on tension of rat soleus musle after indution of a hyperosmolar ontrature. A ontrature was indued as desribed above. At 'reset', tension was quikly dereased to resting tension and allowed to reover spontaneously. For further details see Methods. obtained with four other musles, whereas in three preparations tension was only partly reovered. Furthermore, as the rise in tension ould be bloked by agents whih did not prevent shrinkage of the ells (see below), it an be assumed to represent the result of an ativation of the ontratile filaments. Hyperosmolarity has been shown to stimulate seretion (Case & Clausen, 1973), and it was therefore possible that the release of aetylholine from nerve endings ould have ontributed to the rise in tension. This possibility was further supported by the reent observation that hyperosmolarity stimulates neuroseretion from motor nerve endings in frog musle (Shimoni, Alnaes & Rahamimoff, 1977). However, when 2 x 1-5 M-tubourarine was added 15 min before mannitol (2 mm) there was no signifiant hange in the response (Table 1). Aetylholine at onentrations up to 1-2 M indued only a small and transient rise in tension.

5 H YPEROSMOLARIT Y, Ca TRANSPORT AND TENSION 59 Both Tp and tp were quite variable in musles from different animals. In thirteen experiments where ontratures were indued by the addition of 2 mm-mannitol, Tp ranged from 1P28 to 2.49 g (mean +S.E.: g) and tp from 12 to 252 see (mean + S.E.: see). With surose (2 mm) the Tp was g and tp see (mean values + s.e. of thirty-three experiments). These variations may be related to the metaboli status of the rats. Thus, in musles from animals fasted for 48 hr, the maximum tension was somewhat smaller and later in onset than in those obtained from fed animals (Table 1; see also Table 5 and Fig. 11). The addition of D-gluose (1 mm) did not affet the response of the musles from fed animals (Table 1). TABLE 1. Effet of tubourarine, fasting and gluose on the response of rat soleus musle to hyperosmolarity. Contratures were indued by adding mannitol at a final onentration of 2 mm to the bathing medium. Further details are given in the legend to Fig. 1A and the Methods setion. Mean values ± s.e. for peak tension (T.) and the time interval between the onset of the ontrature and peak tension (tp) are given. The number of experiments is indiated in parentheses. Experimental Tp tp onditions (g) P (se) P Control (4) 191± 23 D-tubourarine (4) (2 x 1-5M) Control 1.8 ±.12 (13) 186 ± < X2 < *2 48 hr fasting (6) J J Control (5) MM-D-gouose 1* (5) When four pairs of musles were exposed to the same standard hyperosmolar stimulus, the variation in Tp amounted to 3% and that of tp to 16 %. Therefore, in most of the experiments with agents whih affeted the ontratures the ontralateral musle was used as ontrol. Both mannitol and surose were found to inrease the tension in a dose-dependent fashion. Fig. 2 shows the effet of various onentrations of surose. With 1 mm, the inrease in tension was hardly measurable and not signifiantly different from the resting tension (P >.5). At higher osmolarities, the initial rise in tension and the maximum tension obtained were dependent upon the inrease in osmolarity. However, the large rise in tension obtained with 4 mm-surose ould only be maintained for a few minutes and then dereased to approximately that found with 2 mm. These experiments indiated that the rise in tension was a funtion of the osmoti gradient reated aross the plasma membrane. When the osmolarity was augmented with agents whih penetrate the plasma membrane, only transient ontratures ould be obtained. As shown in Fig. 3 urea, whih permeates faster than erythritol, also gives a smaller response. On the other hand, surose and mannitol indued losely similar ontratures. In extensor digitorum longus musle 2 mm-surose did not inrease the resting

6 51 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK Surose 3 la C I- 5 1 Time (min) Fig. 2. Dose-response relationship for hyperosmolar ontratures of rat soleus musle. Contratures were indued by adding an aliquot of surose to the Krebs-Ringer biarbonate buffer. The onentrations of surose used were: A-A, 1 mm (n = 4); A ---A, 2 mm (n = 9) and A..A, 4 mm (n = 4). For further details see legend of Fig. 1 A and Methods. The vertial bars indiate one s.e. where this exeeds the size of symbols used. 1-8 j 1*4 IF 'a 1* 1 *8 Mannitol (13) Erythritol (7) Surose (33) Urea (4) 1*4 I I 1 2 Time (min) Fig. 3. Effet of mannitol, surose, erythritol, and ura on resting tension of rat soleus musle. Contratures were indued by adding these agents at a final onentration of 2 mm to the buffer. For further details see legend of Fig. 1IA and Methods. The number of observations is indiated in parentheses. The vertial bars indiate one s5.e where this exeeds the size of the symbols.

7 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION 511 tension. With 4 mm the ontratures developed appreiable tension and were of short duration. In four musles Tp was g and the duration se. The role of Ca The tension development indued by hyperosmolarity was found to depend on the extraellular onentration of alium (Fig. 4). When approximately 15 min after Mannitol CaCI mm 2 2~~~~~~~ B Mannitol I CaCI2, 1-27 mm C Time (min) Fig. 4. Effet of medium Ca onentration on hyperosmolar ontratures of rat soleus musle. Contratures were indued by adding mannitol at a final onentration of2 mm to the buffer. In panel A the medium Ca onentration was 1-27 mm, whih was inreased 15 min after the onset of the ontrature to 2-54 mm. In panel B the medium alium onentration was -1 mm, whih was inreased 15 min after the onset of the ontrature to 1*27 mm. In panel C the medium ontained 5 mm-egta, and Ca was omitted; 15 min after the onset of the ontrature Ca was added to a final onentration of 1-27 mm. Eah urve represents the mean of four or five experiments. For further details see legend of Fig. 1 A and Methods. The vertial bars represent 1 s.e. when this exeeds the size of the symbols used. the onset of a ontrature indued by mannitol (2 mm) the Ca onentration was inreased from 1*27 to 2-54 mm, a small but signifiant (P <.1) rise in tension was observed (Fig. 4A). Conversely, if the Ca onentration was lowered to 1 mm, TV was diminished and tp appreiably shortened (from se to se, P <.1). These ontratures were phasi and lasted about 5 min. If the normal Ca

8 512 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK onentration (1-27 mm) was restored 15 min after the addition of mannitol, a large inrease in tension was onsistently observed (Fig. 4B). The maximum tension thus obtained exeeded that reorded in the normal medium. When Ca was omitted and EGTA (.5 mm) added to obtain a further redution in the Ca2+ ion onentration, Tp was signifiantly diminished (P <.5) and tp learly shortened (Fig. 4C). These experiments indiated that although the presene of alium in the inubation medium was not essential for the initial effet of hyperosmolarity on tension, alium made available from the extraellular phase was of major signifiane in maintaining the ativation of the ontratile apparatus. Therefore, it was of interest to assess the effet of hyperosmolarity on the uptake of Ca. TABLE 2. Effet of hyperosmolarity on 5C'a uptake in rat soleus musle. Soleus musles were inubated for 6 min in Krebs-Ringer biarbonate buffer without or with mannitol and either 45Ca (-5 1/ml.) or 1 mm-[u-"4c]surose ( 5 /z/ml.) to determine the surose spae. The musles were then blotted, and following resetion of the tendons, homogenized in 5 % trihloroaeti aid. The amount of 45Ca- or "4C-ativity taken up was determined by ounting the lear supernatant obtained after entrifugation. On the basis of the speifi ativity of45ca in the inubation medium, the amount of "5Ca taken up was expressed as n-mole per g wet weight and orreted for the redution in tissue wet weight indued by the hyperosmolar medium. In another experiment, three groups of musles were inubated with 45Ca (-5,u/ml.) and then washed 3 x 1 min in ie-old Ca-free buffer ontaining -5 mm-egta. The amount of 45Ca retained was determined as desribed above. Eah value is given as the mean + S.E. with the number of observation in parentheses After orre- 45 Ca retained tion for "Ca in after wash in Total 45Ca surose spae the old Experimental uptake (n-mole/g (n-mole/g (n-mole/g onditions wet-wt.) P wet wt.) wet wt.) P Controls (16) (16) (11) 2 mm-mannitol (5) < (5) (5) < -1 3mM-mannitol (6) < (6) (6) < -1 From Table 2 it an be seen that the addition of mannitol (2-3 mm) inreases the uptake of 45Ca. Also when the results are orreted for the hanges in extraellular spae, it is evident that the amount of4ca taken up into the tissue ompartment not available to [14C]surose is augmented. It should be noted that the results are orreted for the hanges in wet weight following from the inevitable shrinkage in the hypertoni media. In order to avoid the unertainties involved in these orretions and at the same time determine whether the inreased uptake seen in hypertoni media was in part the result of more 45Ca being bound to the outer ell surfae, parallel experiments were performed in whih the exposure to 45Ca was ombined with a subsequent wash in ie-old Ca-free medium ontaining -5 mm-egta. WVith this proedure, it was found that the major part of the 45Ca taken up into the tissue ompartment not available to ['4C]surose was retained and that the musles whih had been inubated in the hyperosmolar medium retained appreiably more than the ontrols. A ertain loss of ellular 45Ca is likely to our during the washing proedure beause even in an ie-old washout medium, the slow phase of the washout orresponds to around -2 % of total tissue 45Ca per minute (Clausen et al. 1975). In view of the similarity between the results obtained with these two proedures, it seemed

9 HYPEROSMOLARlTY, Ca TRANSPORT AND TENSION 513 justified to use the ombination of inubation and old-wash to assess the time ourse of 45Ca uptake in normal and hyperosmolar media. From Fig. 5 it an be seen that the addition of mannitol (2 mm) to the inubation medium leads to a lear-ut inrease in the amount of 45Ca taken up and retained by the musles. This effet ould be deteted within the first 5 min of exposure to the hyperosmolar medium and showed a further inrease with time (see also below). 5 Mannitol (2 mm) 4 a, 3. +a i,, 3 - > Controls X Duration of inubation (min) Fig. 5. Effet of hyperosmolarity on 45Ca uptake in rat soleus musle. Soleus musles were inubated for the indiated interval of time in buffer ontaining 45Ca (-2,u/ml.) without or with mannitol (2 mm). They were then washed 3 x 1 min in ie-old Ca-free buffer ontaining -5 mm-egta, blotted, tendons rejeted and homogenized in 5% TCA. The amount of 45Ca retained after the wash was determined as desribed in the legend to Table 2. Eah point represents the mean of three to fourteen observations with bars denoting 2 x s.e. The observation that hyperosmolarity eliited small ontratures even at very low extraellular onentrations of Ca2+ indiated that alium was initially made available to the ontratile filaments from intraellular pools. The frational loss of 45Ca from preloaded tissues is dependent on the ytoplasmi onentration of free Ca2+ ions available for transport (Shatzman, 1973; Ashley, Caldwell & Lowe, 1972) and the release of Ca2+ from an intraellular pool into the ytoplasm may be deteted as an inrease in the rate of 45Ca washout. From Fig. 6A it an be seen that in a medium from whih Ca had been omitted and EGTA (-5 mm) had been added, the addition of mannitol (2 mm) promptly stimulates the washout of 45Ca from 17 PHY 292

10 514 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK pre-loaded soleus musles. In the standard inubation medium ontaining 1-27 mm-ca, mannitol (2 mm) produed roughly the same relative rise in the frational loss of 45Ca (Fig. 6B). This response was not affeted by the addition of D-tubourarine (1-5 M). Both surose and mannitol (1-4 mm) stimulated the release of "5Ca in (18 Mannitol (2 mm) -16 A -14 P+ + Mannitol (12 - (1-6Control o (>8 - Min'.2-1_ L (8 B -6 + Mannitol (4 -- Control -2 _L Duration of washout (min) Fig. 6. Effet of hyperosmolarity on 45Ca washout in rat soleus musle. Soleus musles were preloaded by inubation for 6 min in buffer ontaining 45Ca (2,s/ml.). Eah individual musle was then plaed in a polyethylene basket attahed to a tube (whih served at the same time as gas inlet) and transferred through a series of tubes ontaining 3 ml. of non-radioative buffer. In A, the washout medium ontained no alium and -5 mm-egta. In B, the standard inubation medium ontaining 1-27 mm-ca was used. Mannitol (2 mm) was inluded in the washout medium from 9 min onwards. The frational loss of45ca was alulated as desribed elsewhere (Clausen et al. 1975). Eah point represents the mean of five observations with bars denoting S.E. a dose-dependent fashion (results not shown). Urea, and erythritol, whih were found to indue a transient rise in tension (see Fig. 3) also stimulated the washout of 45Ca (Fig. 7A and B), but the inrease in the frational loss of 45Ca was smaller and of shorter duration than that eliited by mannitol or surose (Fig. 7C).

11 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION Kd 6 ' L5_-- A Ure -8 m &7 LL U Cu 6-a Duration of washout (min) Fig. 7. Effet of hyperosmolarity on 45Ca washout from rat soleus musle. Experimental onditions as desribed in the legend to Fig. 6. From 9 min and onwards, the osmolarity of the washout medium was inreased by the addition of urea (A), erythritol (B) or surose (C). Eah point represents the mean of three observations with bars denoting S.E. The role of Na Sine an inreased influx of Na+ is a deisive step in the normal ativation of the ontratile apparatus, it was of interest to determine whether the ontratures eliited by hypertoniity were in some way dependent on extraellular Na+. When all NaCl in the medium was replaed by an iso-osmoti amount of surose, the ontratures eliited by mannitol (2 mm) were signifiantly smaller and had a slower onset (Table 3). When the NaCl was replaed by LiCl, only a minute rise in tension was deteted. In this medium eletrial stimulation produed virtually the same twith tension as in the standard medium indiating that the exitability of the musles was preserved. On the other hand, inhibition of ative Na-K transport with ouabain aused no signifiant hange in the effet of mannitol (Table 3) even though the ontratile response to eletrial stimulation showed a onsiderable and progressive derease under these onditions. Thus, following 3 min of exposure to ouabain (.2 mm), twith tension was diminished by more than 8 %. When K+ was omitted from the inubation medium, mannitol (2 mm) ould still indue ontratures, again indiating that ative Na-K transport was not essential for the ontratile response to hyperosmolarity. I7-2

12 516 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK Tetraaine, whih has previously been shown to inhibit Na influx in rat soleus musle (Dahl-Hansen & Clausen, 1973), was found to abolish the effet of mannitol (2 mm) on tension. When the loal anaestheti was added during the ontrature, a rapid drop in tension was produed (Fig. 8). TABLE 3. Effet of Na replaement and of ouabain on hyperosmolar ontratures of rat soleus musle. Contratures were indued by adding mannitol at a final onentration of 2 mm to the Krebs-Ringer biarbonate buffer. For further details see legend to Table 1 and Methods. Mean values + S.E. are given for the peak tension, Tp, and for the time interval between the onset of the ontrature and peak tension, tp. The number of observations is indiated in parentheses Experimental onditions Control NaCl replaed by surose Control NaCl replaed by LiCl Control x2 mm-ouabain TP (g) 1-58± -11 (4)) 1-28 ± -14 (4)) (4)X 1*4± *6 (4)) 1-89±-11(4) 2*18±-38 (4)J P <.2 <.1 tp (see) 198± ) 261± j 198 ± ± 35J p <.5 < '5 > -1 Surose X Tetraaine I-I 1-1 1! 5 Time (min) Fig. 8. Effet of tetraine on tension developed during hyperosmolar ontratures in rat soleus musle. Contratures were indued by adding surose at a final onentration of 2 mm to the buffer. Tetraaine, -1 m i, was added 18 min after the onset of the ontrature. For further detail see legend of Fig. 1A and Methods. The vertial bars represent 1 s.e. The urve represents the mean of four experiments. These experiments suggested that Na influx was required for eliiting the tension development in hyperosmolar media. Therefore, it was of interest to haraterize the hanges in Na influx and their possible relation to Ca flux. From Table 4 it an be seen that mannitol (2 mm) stimulates the uptake of 22Na by 89 % during the first 5 min of exposure. Simultaneously it was found that the uptake of 5Ca was also stimulated during this early phase. When the musles were pre-exposed to mannitol

13 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION 517 _ ~ e S v o v C v v v v CT _-= --,, _ QCC t _ 6 6 -_ t- - C-Il 4-,IC 6 in:,it: - OC _~. _ X -4 ~ v v C C~ 6 V 6 V - C) C 6 V >S I rw C-e m- '". +r _~ _ R 4;S -.,.- _ ~ Co -R C CD _l~ _l _ C- C mc C- C lt-c: C-C (M _ C; "I x,4 Ci 4.; = 3,_ ;z e v C 4-1-.e 4- E D ;. _- S:4 C) _: -C -- C- - o~ :ti - _ n C;.- _ E ; CẒ C- C.- :3. t. =.SL C-C- - -O q -C^ C e.; 4D~. _ L ;4

14 518 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK before the inubation with 22Na and 45Ca, the effets beame more pronouned, partiularly on the uptake of 45C. Sine it is more onvenient to measure 22Na influx when ative Na extrusion is bloked (Clausen & Kohn, 1977) the major part of these experiments was performed in the presene of ouabain (1- M), whih, as shown above, did not interfere with the effet of hyperosmolarity on tension. Other experiments (data not presented) showed that the effets of mannitol on 22Na and 45Ca uptake were dose-dependent and were also eliited by surose or erythritol (2 mm). As reported previously (Dahl-Hansen & Clausen, 1973) tetraaine was found to -1 _ Tetraaine (-5 mm) Mannitol (2 mm) / < / ~~~~~Mannitol alone W /,,, Mannitol + tetraaine -5 o--6control U_ n: 3 S.E.of meant - _ Duration of washout (min) Fig. 9. Effet of hyperosmolarity and tetraaine on '5Ca washout from rat soleus musle. Experimental onditions as desribed in the legend to Fig. 6. Tetraaine (.5 mm) or mannitol were added to the washout medium at the times indiated by the arrows. Eah point represents the mean of three observations with bars denoting S.E. inhibit the uptake of 22Na, but as shown in Table 4, the loal anaestheti does not prevent the stimulating effet of mannitol. Thus, in the presene of tetraaine the relative rise (195 %) was even larger than in its absene (134%). On the other hand, tetraaine abolished the effet of mannitol on 45Ca uptake, indiating that it is possible to dissoiate these two events. From these findings it seemed reasonable to onlude that the marked inhibitory effet of tetraaine on hyperosmolarity-indued tension was related to an effet on the flux of Ca rather than on sodium transport. It was of interest, therefore, to determine the effet of tetraaine on the response of45ca washout to mannitol. From Fig. 9 it an be seen that the stimulation indued by 2 mm-mannitol is suppressed by the loal anaestheti (.5 mm). This effet is statistially signifiant (P <.5) as evaluated over the first 2 min of exposure to mannitol.

15 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION 519 The ytoplasmi Na+ is likely to inrease during the ell shrinkage indued by hyperosmolarity. Therefore, Ca2+ influx might be favoured via a Na+-Ca+ exhange mehanism. In view of the following, however, this seems to be of minor signifiane: ouabain, whih inreases the ytoplasmi Na+ onentration, auses little hange in tension, and no signifiant rise in "6Ca influx (see Table 4). In the presene of tetraaine or in the Na+-substituted media, the ontratures are onsiderably smaller in spite of the fat that hypertoniity still indues ell shrinkage. Finally, both the ontratures and the aelerated 45Ca entry were detetable for a long time after the initial ell shrinkage had taken plae. Measurements of the Na ontent of the tissue showed that 6 min after the onset of exposure to hyperosmolarity, the intraellular Na onentration was not signifiantly higher than in the ontrols. The effet of insulin Earlier studies had shown that insulin indued a release of 45Ca from preloaded epididymal fat pads (Clausen, 1969), fat ells (Clausen et al. 1974), and soleus musles Mannitol 4 - {Insulin 1 m-u./ml 3 4o / A I- Mannitol + ~~~Anti-inulninsmm Time (min) Fig. 1. Effet of insulin and anti-insulin serum on tension developed during a hyperosmolar ontrature in rat soleus musle. The ontrature was indued by adding mannitol at a final onentration of 2 mm to the buffer whih ontained -1 g/ 1 ml. (w/v) albumin. Insulin, 1 m-u./ml., was added 15 min after the onset of the ontrature (panel A), while in the ontralateral musle anti-insulin serum was added a few minutes before the hormone (panel B). For further details see legend of Fig. 1 A and Methods. of the rat (Clausen & Martin, 1977). These observations indiated that the hormone ould indue a mobilization of Ca2+ ions from a ellular pool and preliminary experiments showed that in a hyperosmolar medium, insulin indued a further rise in tension (Clausen et al. 1975). The present desription of the hyperosmolarity-indued ontratures onstituted a basis for a more detailed study of this otherwise poorly haraterized effet of insulin.

16 52 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK I C: t- +l o~ v1 v ~ C+ +l t- v 6 CS +l 'd, +l - t- +ll 1-22<Q r i- C)+l +l m 6 - r- - +l,-4-4 +l r- _ E- X- C5._ C -f M-."._ ;_._

17 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION 521 When added at a onentration of 1 m-u./ml., insulin did not affet resting tension or single twith tension. Also, when the same onentration of insulin was added before hyperosmolar ontratures were indued, neither the Tp nor the tp values were hanged. On the other hand, when the hormone was introdued 15 min after the onset of a ontrature indued by mannitol (2 mm), a pronouned further rise in tension was produed (Fig. 1A). This response was abolished if guinea-pig anti-insulin serum was added a few minutes before the insulin (Fig. 1B). This agent Mannitol Insulin. 1 m-u./mi /- C 1-5/ 1- D Time (min) Fig. 11. Effet of insulin on tension developed during a hyperosmolar ontrature in soleus musles from fed and fasted rats. Contratures were indued by adding mannitol at a final onentration of 2 mm to the buffer whih ontained.1 g/1 ml. (w/v) albumin. Insulin, 1 m-u./ml., was added 15 min after the onset of the ontrature. For further details see legend of Fig. 1 A and Methods. - musles from fed rats (the urve represents the mean of eight experiments); O - -- O, musles from rats fasted for 48 hr (the urve represents the mean of four experiments). The vertial bars represent 1 S.E. of the mean. See also Table 5. in itself had no effet on tension, and as neither bovine serum albumin (5,g/ml.) nor ZnCl2 (16/tM) produed any detetable hange in tension, it is reasonable to assume that the ontratures observed in the presene of insulin are speifi for this hormone. Quik-release experiments, arried out as desribed above, indiated that the tension generated in the presene of insulin was due to an ative proess. In three experiments, resetting of the tension to 1 g was followed by an inrease to a level omparable to that reorded in the ontralateral insulin-treated musles; in one pair of musles, however, the reovery of tension was inomplete. Table 5 summarizes the results of experiments with various onentrations of insulin. With 5 m-u./ml. a rise in tension ould onsistently be demonstrated, whereas at lower onentrations only a small and variable effet was seen. The magnitude of the inrease in tension and the time required to reah this maximum showed very little hange in the onentration range from 5 to 1 m-u./ml. The latent period was between 3 and 5 min. Even though musles from 48 hr fasted rats showed a

18 522 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK smaller response to mannitol (2 mm), insulin produed virtually the same maximum rise in tension as in the musles from fed ontrols, albeit with a longer time lag (Fig. 11 and Table 5). DISCUSSION The present work demonstrates that like amphibian musles, mammalian musles respond to hypertoniity by a rise in tension whih is dose-dependent and in the ase of rat soleus, of suffiient magnitude and duration to be analysed in relation to ion transport phenomena. The quik release experiments indiated that this ontrature is aused by an ative proess and, in agreement with the onlusion of LIannergren & Noth (1973), is most likely to be aused by a normal interation of the ontratile filaments. An alternative interpretation was suggested by the observation that hypertoniity stimulates reatine phosphate hydrolysis (Homsher et al. 1974) and that an ensuing ATP depletion might give rise to an inrease in tension similar to that produed by treatment of soleus musles with 2,4-dinitrophenol (Clausen et al. 1975). However, unlike 2,4-dinitrophenol, the addition of mannitol (2 mm) produed no signifiant derease in the ATP ontent, even after 6 min of exposure (T. Clausen, unpublished observations). Even though hypertoniity may ause a release of aetylholine from the motor nerve endings (Hubbard, Jones & Landau, 1968; Shimoni et al. 1977) it seems unlikely that this an have eliited the ontratures. In agreement with studies on amphibian musles (Homsher et al. 1974) tubourarine did not prevent the rise in tension. There is also abundant evidene that the resting membrane potential is little affeted by hypertoniity unless the toniity is greatly inreased or the time of exposure prolonged (Hodgkin & Horowiz, 1957; Tigyi & Shih-Fang, 1962; Andersson, 1972). Furthermore, hypertoni ontratures ould be indued even when the onentration gradients for Na and K were onsiderably redued and the membrane potential diminished (see Table 3 and Clausen & Flatman, 1977). Finally, it ould be ruled out that the reorded rise in tension was the simple outome of passive mehanial phenomena in relation to the hange in ell volume. In ontrast to the ontratures obtained with mannitol and surose, an inrease in the osmolarity of the buffer by the addition of permeant solutes (urea and erythritol) only led to a small and transient tension development. Therefore, the establishment of an osmoti gradient aross the plasma membrane is suffiient to eliit a ontrature, but the rise in tension will only persist if this gradient is maintained long enough to produe a lasting redution in ell volume. Hene, a derease in ell volume seems essential, and this would suggest that the Ca2+ ion level might beome high enough to initiate ontratures beause of the inevitable inrease in the onentration of all ytoplasmi solutes. However, aording to the results of Weber, Herz & Reiss (1964), the rise due to the volume redution (alulated as 65 % for an experiment with 2 mm-mannitol) is insuffiient to ativate the ontratile apparatus. An inrease in the ioni strength of the inubation medium has been shown to derease the uptake of Ca in isolated vesiles of the saroplasmi retiulum (Martinosi & Feretos, 1964; Carvalho & Leo, 1967; de Meis, 1971; Gattass & De Meis, 1975) and diminish the binding of alium to isolated sarolemma (Sulakhe, Drummond

19 HYPEROSMOLARITY, Ca TRANSPORT AND TENSION 523 & Ng, 1973). Both these effets ould ontribute to a net rise in the ytoplasmi Ca2+ ion level. The rate of Ca efflux is a funtion of the onentration of free Ca2+ ions available for transport in the ytoplasm (Ashley et al. 1972; Shatzman, 1973), and sine hypertoniity also stimulated 4Ca washout in a Ca-free medium, it seems reasonable to assume that Ca2+ ions are released from ellular pools. Studies on amphibian musles have led to a similar onlusion (Lannergen & Noth, 1973; Homsher et al. 1974). The results obtained with tetraaine support the idea that in hypertoni media Ca2+ ions are released from the saroplasmi retiulum. Loal anaesthetis affet the flux of Ca2+ ions aross the plasma membrane as well as their release from ertain ellular pools. In addition to bloking affeine ontratures (Feinstein & Paimre, 1969) loal anaesthetis also inhibit ontratures indued in hypertoni media (see above and Saito, 1971; LUnnergren & Noth, 1973). Hertz & Weber (1968) have shown that affeine an release Ca ions from the saroplasmi retiulum and studies with isolated fragments of saroplasmi retiulum indiate that loal anaesthetis an prevent the leakage of Ca ions (Feinstein & Paimre, 1969). It is therefore likely that the soure of Ca ions whih initiate the affeine and hypertoni ontratures is the same. It should be noted that a affeine ontrature an be eliited in Ca-free media (Frank, 196) whereas Ca ions from the extraellular environment seem to be required for the full development of hypertoni ontratures. MGrath & Shepherd (1976) also onluded that in their smooth musle preparation the effet of hypertoniity on tension was mediated by a mehanism whih did not involve a diret ation on the ell membrane. The experiments performed with Ca-depleted -media indiate that extraellular Ca is required for the maintenane of the ontratures indued by hypertoniity. Homsher et al. (1974) have demonstrated that hypertoniity stimulates the uptake of XCa, and in the present experiments a similar effet ould be deteted within the first 5 min of exposure to a medium ontaining mannitol (2 mm). In view of the low ytoplasmi onentration of free Ca2+ ions, even a modest inrease in alium entry ould lead to a marked relative rise in the ytoplasmi Ca2+ ion level, in partiular under onditions where the Ca learing apaity of ellular organelles is redued. The observation that tetraaine abolished the stimulating effet of hypertoniity on 45Ca influx suggests that the inhibitory effet of the loal anaestheti on tension development is not solely due to interferene with the release of Ca2+ ions from the saroplasmi retiulum. The role of extraellular Na+ is more diffiult to assess. In ontrast to the observations of Saito (1971) and Homsher et al. (1974), the maximum tension of hypertoni ontratures in rat soleus musles is dereased when NaCl is replaed by surose. Thus, Na+ ions seem to be required and, in aordane with this ontention, hypertoniity was found to produe a marked stimulation of 22Na influx. However, this effet was not appreiably altered by tetraaine at a onentration ( 5 mm) whih ompletely abolished the rise in tension. It is generally assumed that during a normal ation potential an inrease in Na influx is of importane in eliiting a release of Ca2+ ions from the saroplasmi retiulum into the ytoplasm. The experiments with tetraaine indiate that when the Ca release mehanism is inhibited, even a large rise in Na influx annot trigger a ontrature. It should be noted that tetraaine prevented the stimulation of 45Ca uptake.

20 524 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK The inhibitory effet of redued extraellular Na+ on the response to hypertoniity might also be taken as an indiation that the ontrature was an outome of ation potentials being generated by the hypertoni media. The experiments with tubourarine exlude that ation potentials are eliited at the end-plate. From the slow time ourse of the ontratures, it seems that the ativation of the ontratile apparatus is muh slower than what is normally seen following an ation potential. Furthermore, in the presene of ouabain, the ontrations indued by ation potentials due to eletrial stimulation dereased muh more with time than those indued by hypertoniity. Therefore, it seems unlikely that typial ation potentials are involved in the mehanism of hypertoniity ontratures. The above-mentioned data lend support to the following interpretation: hypertoniity leads to an inrease in the intraellular ioni strength, whih favour the release of Ca2+ ions from the inner surfae of the plasma membrane and in partiular from the saroplasmi retiulum. This seems to be assoiated with an inreased permeability of the plasma membrane to Na and Ca, whih auses a further rise in the ytoplasmi Ca2+ ion level with ensuing ativation of the ontratile apparatus. Under these onditions, where the Ca2+ onentration is maintained at a higher level than normal, insulin indues a further rise in tension. Although this effet is only seen with relatively large onentrations of the hormone, it seems to be speifi and unrelated to hanges in gluose transport and metabolism. The insulin-indued rise in tension is due to normal interation of the ontratile filaments and presumably reflets a further rise in the ytoplasmi Ca2+ ion level. In media of normal toniity insulin was shown to stimulate the washout of 45Ca from soleus musles (Clausen & Martin, 1977) and isolated ultured myotubes (Shudt, Gaertner & Pette, 1976) whereas in ardia musle an inotropi effet ould be demonstrated (Lee & Downing, 1975; Nudel, Lee & Downing, 1977; Snow 1976). These findings may be taken to indiate that the hormone eliits a mobilization of Ca into the ytoplasm. In a medium of normal toniity this did not lead to any detetable rise in tension of soleus musles, presumably beause the Ca2+ ions were aumulated into ell organelles or transported out of the ell. In the hypertoni medium, where the Ca2+ ion learing apaity of the saroplasmi retiulum may be impaired (Gattass & De Meis, 1975), a further release of Ca2+ ions into the ytoplasm is likely to augment the onentration to a level where the ontratile apparatus is ativated. At the present time it is not known from whih ellular pool the Ca2+ ions are mobilized by insulin. Shudt et al. (1976) have proposed that insulin an stimulate the release of mitohondrial alium and for fat ells there is evidene that a labile fration of the mitohondrial Ca pool is inreased (MDonald, Bruns & Jarett, 1976). In plasma membranes isolated from liver, insulin was shown to diminish the binding apaity for alium (Shlatz & Marinetti, 1972), and in adipoyte ghosts, a similar effet was reported (Kissebah, Clarke, Vydelingum, Hope-Gill, Tulloh & Fraser, 1975). Although suh effets ould all ontribute to an elevation of the ytoplasmi Ca2+ ion level, it is diffiult to determine whether they are diret, and their signifiane for the many other ations of insulin is as yet hypothetial (for reviews see Czeh, 1977; Clausen, 1978).

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22 T. CLAUSEN, A. B. DAHL-HANSEN AND J. ELBRINK 526 HOMSEmR, E., BRIGGS, F. N. & WISE, R. M. (1974). Effets of hypertoniity on resting and ontrating frog skeletal musles. Am. J. Phy8iol. 226, HUBBARD, J. I., JONES, S. F. & LANDAU, E. M. (1968). An examination of the effets of osmoti pressure hanges upon transmitter release from mammalian motor nerve terminals J. Phy8iol. 197, lsaacson, A. (1969). Caffeine-indued ontratures and related alium movements of musle in hypertoni media. Experientia 25, KISsEBAH, A. H., CLARKE, P., VYDELINGUM, N., HOPE-GIL, H., TULLoE, B. & FRASER, T. R. (1975). The role of alium in insulin ation, III. Calium distribution in fat ells; its kinetis and the effets of adrenaline, insulin and proaine-hcl. Eur. J. din. Inve8t. 5, KOHN, P. G. & CLAUSEN, T. (1971). The relationship between the transport of gluose and ations aross ell membranes in isolated tissues. VI. The effet of insulin, ouabain, and metaboli inhibitors on the transport of 3--methylgluose and gluose in rat soleus musles. Biohim. biophy8. Ata 225, KUZUYA, T., SAMOLS, E. & WmLirAms, R. H. (1965). Stimulation by hyperosmolarity of gluose metabolism in rat adipose tissue and diaphragm in vitro. J. bil. Chem. 24, LXNERGREN, J. (1971). The effet of low-level ativation of the mehanial properties of isolated frog musle fibres. J. gen. Phyaiol. 59, LANNERGREN, J. & NoTH, J. (1973). Tension in isolated frog musle fibres indued by hypertoni solutions. J. gen. Phyaiol. 61, LEE, J. C. & DOWNING, S. E. (1976). Effets of insulin on ardia musle ontration and responsiveness to norepinephrine. Am. J. Phy8iol. 23, MARTONOSI, A. & FERETOS, R. (1964). Saroplasmi retiulum. Correlation between adenosine triphosphatase ativity and Ca2+ uptake. J. biol. Chem. 239, MCDONALD, J. M., BRuwS, D. E. & JARETT, L. (1976). The ability of insulin to alter the stable alium pools of isolated adipoyte subellular frations. Biohem. biophy8. Rem. Common. 71, MGRATH, M. A. & SEMPHERD, J. T. (1976). Hyperosmolarity: Effets on nerves and smooth musle of utaneous veins. Am. J. Physiod. 231, NUDEL, D. B., LEE, J. C. & DOWNING, S. E. (1977). Reiproal inhibition of ardia responses to norepinephrine and insulin. Am. J. Phy-iol. 233, SAITO, K. (1971). The first major omponent of tension of the skeletal musle in hypertoni solution. Jikei. med. J. 18, SCHATZMANN, H. T. (1973). Dependene on alium onentration and stohiometry of the alium pump in human red ells. J. Physiol. 235, SCHUDT, C., GAERTNER, U. & PErwE, D. (1976). Insulin ation on gluose transport and alium fluxes in developing musle ells in vitro. Eur. J. Biohem. 68, SHIMONI, Y., ALNAES, E. & RAHAmaoFr, R. (1977). Is hyperosmoti neuroseretion from motor nerve endings a alium-dependent proess? Nature, Lond. 267, SCMLATZ, L. & MARINE=, G. V. (1972). Hormone-alium interations with the plasma membrane of ratliver ells. Siene, N.Y. 176, SNOW, T. R. (1976). Study of the harateristis of the inotropi effet of insulin in rabbit papillary musle. Experientia 32, SULAm, P. V., DRummoND, G.I. & NG, D. C. (1973). Calium binding by skeletal musle sarolemma. J. biol.chem. 248, TIaYI, J. & SHI -FANG, F. (1962). The effet of hypertoni solution on the ontration, resting and ation potential of the musle fibre. Ata phy8iol. hung. 22, WEBER, A., HERz, R. & REISS, I. (1964). The regulation of myofibrillar ativity by alium. Pro.R. So. B 16, YAXADA, K. (197). The inrease in the rate of heat prodution of frog's skeletal musle aused by hypertoni solutions. J. Physiol. 28,