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Sodium in smooth T. S. MA AND muscle relaxation D. BOSE Department of Pharmacology and Therapeutics,, University Manitoba, Canada R3E OW3 Winnipeg, that this muscle, after undergoing depolarization and contraction in response to elevated external potassium, did not relax when the stimulus was removed, although the membrane potential recovered from depolarization. The present work was done to elucidate the mechanism whereby taenia coli fails to relax in the absence of sodium and also to utilize this phenomenon to elucidate the nature of movement of calcium in the tissue during relaxation. A preliminary account of this work has been presented to the Annual Meeting of the Canadian Federation of Biological Societies (17) l MATERIALS AND METHODS Guinea-pig taenia coli, about 20-25 mm in length, was dissected from adjoining circular muscle and suspended either in organ baths or in test tubes placed inside aluminum heating blocks. Resting tension was adjusted to obtain optimum length (L,) for contraction (usually 0.5 g). In a few experiments, uterine horns were obtained from albino rats (300 g) injected with diethylstilboesterol (1 mg/kg ip) 24 and 48 h before the experiment. The muscles were maintained at pH 7.3 taenia coli; uterus; D600; lanthanum; Na+-Ca++ exchange and a temperature of 37°C. One hundred percent oxygen was used for aerating the various bathing media shown in Table 1. Both for isometric tension as well as for CENTRAL TO THE UNDERSTANDING of relaxation in isotope flux studies, the tissues were preincubated in smooth muscle is the knowledge of how the elevated normal sodium-containing medium for 30 min followed cytoplasmic free calcium concentration is reduced. The by switching to the appropriate media indicated in Tamechanisms considered have ranged from active or pas- ble 2. Intracellular calcium was estimated by the lansive transcellular efflux of the ion to intracellular se- thanum method of van Breemen et al. (26). This estiquestration by sarcoplasmic reticulum and/or mitochonmate may be an approximation of the real value because dria. Attempts to demonstrate active extracellular it is not certain whether the disposition of lanthanum is transport of calcium in taenia coli or uterus have not wholly extracellular and also because lanthanum may been successful (11, l&22). Goodford (11) has proposed a or may not block calcium influx and efflux at the same two-stage calcium removal process in the membrane rate (10, 13). However, it is being assumed that such consisting first of an energy-requiring process followed error will exist in both the control as well as test cases. by a passive process, probably involving dissolution of After following the protocols outline in Table 2, lancalcium salts from the membrane. A passive efYlux of thanum (10 mM) was added to 45Ca-containing incubacalcium coupled to influx of sodium has been found in a tion medium for 5 min. This was followed by three lonumber of excitable tissues, e.g., squid axon and heart min-long isotope-free washes with the test medium in (1, 20). Reuter et al, (19) gave some evidence for the the presence of 10 mM lanthanum. The tissues were existence of such a mechanism in vascular smooth mus- then removed and lightly blotted dry. After weighing, cle. Lanthanum, which blocks transmembrane influx they were each dissolved in 300 ~1 of NCS (Amershaml and efflux of calcium, causes relaxation of a contracted Searle Corp.) after addition of 20 ~1 of water at 50” C and muscle. This has been considered, along with other later neutralized with 20 ~1 6 N acetic acid to reduce reasons, to indicate that intracellular sequestration of chemiluminescence and mixed with 10 ml scintillation calcium can mediate relaxation in the rabbit aorta (25). medium consisting of 6 g 2,5diphenyloxazole (PPO) per Katase and Tomita (15) studying the effect of sodium- liter of toluene. Radioactivity was measured in a Philfree environment on guinea-pig taenia coli observed lips scintillation counter. Fifty microliters of the radio- Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 MA, T. S., AND D. BOSE. Sodium in smooth muscle reluxation. Am. J. Physiol. 232(l): C59-C66, 1977 or Am. J. Physiol.: Cell Physiol. l(1): C59-C66, 1977. -The contraction of guinea-pig taenia coli due to high K+ could not be reversed by washing when Na+ was absent from the medium. Reintroduction of Na+ (7 mM or more) caused relaxation. Similar results were obtained with rat uterus. The effect of sodium replacement was not due to change in ionic strength because equal or higher osmoles of choline, Ca++, K+, Mn++, or Mg++ had little or no effect. Persistence of contraction in the Na+-free medium was not due to a “catch state” of the contractile apparatus. Impairment of Ca+ removal from the cytoplasm rather than persistent increase in Ca+ influx seemed to sustain the mechanical response. This was because D600 (a calcium influx blocker) failed to completely relax K+-induced contraction in the absence of Na+ and also because the ability of EGTA to produce relaxation was reduced in the absence of Na+. Measurement of tissue calcium content using the lanthanum method revealed coincident, decrease in tissue calcium and tension to control level during Na+-mediated relaxation. The results suggest a role for transmembrane Na+-Ca++ exchange in causing the Na+-mediated relaxation of taenia undergoing Na+-free contracture. of Manitoba, C60 TABLE T. S. MA 1. Composition Medium NaCl of bathing Sum crose “pF* repeated 4 times or more unless specifically in RESULTS. media KC1 MgSO, CaC12 Glu- cow TrisHCl 4.5 4.5 5.9 5.9 1.2 1.2 1.2 1.2 2.5 2.5 2.5 2.5 11.5 11.5 11.5 11.5 10 10 10 10 4 TABLE 120 0 120 0 0 240 0 240 2. Time in various 0-Na i- K” + %a 1.4 1.4 0 0 media mentioned Normal Na + Wa 0-Na + 45Ca 0-Na + Lat 45Ca + Normal Na + 4sCa 1 58 0 0 0 0 2 3 4 5 30 30 30 30 10 10 10 IO 18 16 14 0 0 0 4 0 2 4 4 min 10 t 10 mM. active incubation medium were treated in the same manner as above before counting. 22Na+ uptake by the blotted whole tissue was measured by y-spectrometry. Uptake per gram of tissue was expressed as a percent of the counts per milliliter of the medium. Quick-release experiments were performed to assess the intensity of the active state by the method of Ritchie (21). The lower end of the muscle was fixed to a Grass FT-03C force displacement transducer while the upper end was connected by a jeweler’s chain to a rotary solenoid described more fully by Bose and Bose (4). This device was capable of making rapid length reductions of adjustable magnitude within 10 ms when activated by a gated DC power supply. Electron microscopic localization of lanthanum in taenia was done by exposing two normal as well as two taenia strips, depleted of sodium for 30 min, to LaCl, (20 mM) for 30 min. At the end of this period, glutaraldehyde was added to the bathing medium in a concentration of 3%. After 10 min the bathing temperature was reduced to 4”C, and after 50 min the muscles were washed with cacodylate buffer. The muscles were postfixed in osmic acid for 30 min and stained with uranyl acetate for 60 min. After dehydration in alcohol, muscle pieces were embedded in Epon, and ultrathin sections were examined in a Hitachi HS-8 electron microscope. At least 200 cells were examined in each muscle, Only cells with reasonably intact membrane boundary were studied. Drugs and chemicals used in this study were: ethylene glycol-bis-( P-aminoethylether)-N, N’-tetraacetic acid (EGTA; Eastman); D600 (Knoll); diethylstilbesterol (Hanovol; Homer); 45Ca and 22Na (New England Nuclear Corp); inorganic chemicals were Analar grade from Fisher Scientific Co., and sucrose was obtained from Sigma Chemical Co+ Statistical evaluation for multiple grouped data was done by analysis of variance in conjunction with Duncan’s new multiple range test. Experiments involving comparisons between two sets of data were done using a t test designed for unpaired observation with unequal variances or a paired-t test (19)* Every experiment was Guinea-pig taenia coli exhibited spontaneous activity in the normal sodium-containing medium buffered with Tris-HCl, The rate was more rapid than that observed in Krebs-Henseleit solution containing bicarbonate as a buffer. Addition of KC1 (70 mM) caused a rapid phasic contraction followed by a sustained tonic contraction, When the excess potassium was washed out, the muscle promptly relaxed to the base line (Fig+ 1). In muscle exposed to the sodium-free medium containing an additional 70 mM potassium, there was an initial contraction which persisted over an observation period of 60 min when the extra potassium was washed out after ZO30 min. Changing over to normal sodium-containing medium rapidly relaxed the muscle. Relaxation at a slower rate was also seen by raising sodium concentration to as little as 7 mM. When a different experimental protocol was employed, taenia coli was transferred from normal sodium medium to Na+-free medium. A rapid contraction which lasted for 15-20 min ensued. When tension returned to base line, addition of extra KC1 (70 mM) resulted in a sustained contraction which, as in the previous case, persisted even though the high external potassium was washed out with the Na+-free medium, These persistent contractions in the Na+-free medium after KC1 was washed out will be referred to as Na+-free contractures These experiments confirm the work of Katase and Tomita (15). Experiments similar to the above were also done on six rat uterine horns, Contractions were elicited in response to KC1 (70 mM) in the presence or after 30-40 min absence of external Na+. The KCl-induced contractile response was reversed on washing when Na+ was present but not when Na+ was absent (Fig. 2). Further experiments were done in taenia strips to investigate whether the persistent contraction in the absence of sodium was due to: a) effect of low ionic strength; b) “catch state” analogous to molluscan smooth muscle; c) increased membrane permeability to calcium; d) decreased calcium removal from the cytoplasm either by intracellular sequestration or transcellular extrusion. Effect of low ionic strength. To examine the mechanism of sodium-mediated relaxation of Na+-free contrac- t -Ad t NK/K NK SK/K + SK -L NK FIG. 1. Effect of KC1 (70 mM) on guinea-pig taenia coli in presence of normal Na (NK/K) or Na-free medium (SK/K). KC1 washed out with sodium-containing (NK) or Na-free medium (SK) as indicated. Maintained contracture in presence of SK was rapidly reversed by introduction of normal sodium at NK. Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 Group * 70 mM. D. BOSE RESULTS mM Normal Na Na free PO, free, normal Na PO, free, Na free AND SODIUM AND SMOOTH MUSCLE C61 RELAXATION A t NK/K --- __t-----1 min FIG. 2. Effect of KC1 (70 mM) on rat uterus: A: in presence of normal Na (NK/K) or (B) in absence of (SK/K). KC1 washed out with NK or SK as indicated. NK I3 SK/K T-r-SK tures, it was considered necessary to examine whether it was an ionic strength effect or due to Na+ per se. Na+free contractures were obtained in eight preparations as described earlier. Various ions were added to the medium. NaCl, KCl, choline chloride, MgSOq, MnCl,, or CaCl, were added in concentrations of 10 mM which resulted in ionic strength increases of 0.02 for the first three salts, 0.04 for MgS04, and 0.03 for the last two salts. Relaxation was measured at the end of 5 min NaCl was the most potent relaxation of all agents tested. Choline chloride, KCl, and CaC12 had minimal relaxant effect. MgS04 and MnCl, were intermediate in effect (Fig. 3). It should be pointed out that these smaller effects were obtained with amounts of these salts that caused much larger increases in ionic strength than NaCl. These results suggest that the relaxant action of NaCl is a distinct effect of Na+ ions, although there may be a small effect due to a contribution to ionic strength. Catch state. In molluscan smooth muscle, the catch state tension can be maintained at very little energy cost and is associated with a markedly reduced “active state” (14). To test whether such a phenomenon maintained Na-free contracture in the taenia, active state was estimated by the “quick-release method.” Notwithstanding the criticism (5) that the method of testing can itself alter the active state, it was assumed that any error would have been present in both the control and test conditions. Four preparations were stimulated by the addition of KC1 (70 mM) to the normal Na-containing solution. After a steady contraction was obtained, the muscle length was rapidly reduced by 6%. The tension rapidly decreased followed by a secondary increase which was measured over the next 10 s, In the presence of Na, the muscle redeveloped 0.9 t 0.1 g tension, and the total active tension attained was 62% of the level before release. The strips were then transferred to Nafree medium for 30 min, and Na-free contractures were obtained. Quick release of these muscles caused similar initial decrease in tension followed by an increase of 0.7 2 0.2 g tension (Fig. 4). Active state as measured by this method was not significantly reduced by Na-free medium (P > 0.05; n = 4). Na-free contracture, therefore, does not fulfill one of the most important criteria for the catch state. 50 I NaCl CholineCI KCI Cdl, MnCI, MgSO, FIG. 3. Effect of various ions (10 mM) on Na-free contracture in taenia. Each bar represents mean * SE of 3-4 experiments. Asterisks (*P < 0.05; ** P < 0.01) denote significant difference (t test for unequal variance) from relaxation due to NaCl. normal Na Na-f ree 15 gr FIG. 4. Effect of quick induced tension in taenia deprivation of Na. 6% SEC qr release (qr) (6% of L,) on KC1 (70 mM) in presence of normal Na or after 30 min Effect on Ca++ permeability. Katase and Tomita (15) suggested that the Na-free contracture was maintained by a sustained elevation of Ca permeability. Experiments were done with D600, a methoxy derivative of verapamil known to decrease activated Ca influx (9). Eight strips of taenia were divided into two groups. One group was exposed to normal Na-containing medium while the other was exposed to Na-free medium for 20 min. Both groups of muscles were then exposed to KC1 (70 mM). After a steady contraction was obtained, D600 (0.4 PM) was added to the medium and the rate of relaxation was measured. Complete relaxation took place in the presence of Na in 19.3 -+ 1.5 min (Fig. 5). Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 <-t C62 T. S, MA 0600 0.4uM _ 1 191 5min D600 SK 1 SK/K _-_----I-_ Incomplete relaxation was obtained with D600 (51.0 t 8.8%; P > 0,OS) in the absence of Na, TI,2 for relaxation in the presence or absence of external Na were 4.7 t 0*3 and 25.5 -+ 0.2 min, respectively (P < O.Ol)* These results indicate that part of the Na-free contracture was dependent on continued Ca influx. An observation that dictated against increase in Ca permeability being the major factor was that EGTA (15 mM) relaxed a KC1 contraction in the presence of Na much more rapidly than a Na-free contracture (Tllz of 0.66 t 0.07 min vs. 4.81 -+ 0,96 min; P < 0.01, n = 4, Fig. 6). The incomplete relaxation by D600 in the Na-free medium is highly suggestive of impaired Ca removal from the cytoplasm being an important factor in Na-free contracture. Whether this effect of Na was on the plasma membrane or some intracellular site (s) was examined in the following experiments. Differentiation of transsarcolemmal Ca extrusion and intracellular Ca sequestration. Taenia strips, divided into five groups, were exposed to various media, some of which contained tracer amounts of 45Ca, as indicated in Table 2. 45Caexchange was terminated by adding La (10 mM) to the incubation medium as described in METHODS.As shown in Table 3, tissues in group 2 exhibited Na-free contracture of 3.0 t 0.3 g when the extra KC1 was washed out. This was associated with a significant increase in tissue 45Cacontent. Addition of normal Na to :4iiL t NK/K ___-------t NK 1 NK muscle in groups 3 and 4 for 2 or 4 min caused relaxation of 86.8 t 1.5 and 96 t 1.7%, respectively. Tissue calcium elevation was completely reversed under these conditions. Lanthanum (10 mM) was administered for 4 min to muscles of group 5 prior to reintroduction of normal sodium. Not only was the relaxation caused by a 4-min exposure to sodium reduced (44.4 t 2.7% compared to 96 t 1.7% with Na+), but also the 45Cacontent of the tissue did not decrease as much as in the absence of lanthanum pretreatment (Table 3). These results indicate that the cellular fraction of calcium elevated due to the absence of Na, and which to a certain extent was responsible for the maintained contracture, was completely eliminated from the tissue during sodium-mediated relaxation. Therefore, an outward transmembrane calcium movement rather than intracellular sequestration seems to be more important for relaxation in the taenia under the conditions of this study. The possible effect of lanthanum on sodium entry into the tissue and thereby on Ca efflux was tested with 22Na. Figure 7 depicts the tissue 22Nacontent (expressed as a % of medium counts) in the presence or absence of lanthanum. In all experiments (n = 3 in each group) the tissues were rinsed in isotope-free medium for 30 s before residual radioactivity was measured. In tissues preexposed to lanthanum (10 mM) for 5 min followed by exposure to lanthanum and 22Nafor 30 min, there was a small decrease in sodium exchange which was statistically significant (P < 0.05). In other experiments, the tissues were additionally exposed for 30 or 60 min to ouabain (30 PM) in order to decrease sodium pumping and thereby increase sodium content of the tissue. Both 3. Effect on intracellular isometric tension Ca and TABLE Group Treatment 1 2 3 4 5 NK Control 0-Na contracture NK 2’ NK 4’ La pretreated * Data n = 4-15 in each group. as 0% and in group II as 100%. Ca Content, pmolkg 162.2 329.3 151.5 164.7 249.6 i + 2 + f 11.4 16.8 22.2 16.8 16.8 % Ca* 0 100 -6.4 2 11.6 1.5 k 8.8 52.3 2 8.8 % Tension+ 0 100 13.2 2 1.5 4.0 2 1.7 44.1 2 2.7 Data normalized by considering Ca content t Relative to 0-Na contracture tension. in group I FIG. 6. Effect of EGTA (15 mM) on tension response of taenia due to KC1 (70 mM) in presence of normal Na (A) or in absence of Na (B). 8 SK/K D. BOSE Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 FIG. 5. Effect of D600 (0.4 PM) on tension response of taenia due to KC1 (70 mM) in presence of normal Na (NK/K) or in absence of Na (SK/W. AND SODIUM AND SMOOTH MUSCLE RELAXATION C63 Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 well as removal of the inhibitory effect of troponin on actin-mediated increase in nucleotide-bound myosin ATPase activity. This explanation is not applicable to our results because the contracture can partly be reversed by EGTA or by a calcium antagonist D600. Since D600 does not affect smooth muscle actomyosin ATPase (personal communication), the Na-free contracture is mediated by Ca. Our findings with small concentrations of these cations differ from those of Gordon et al. (12) who found K and Na to be equally effective as relaxants of skeletal muscle and Katase and Tomita (15) who, using very high concentrations of various ions, found them all to relax taenia undergoing Na-free contracture. We feel that the ionic strength effects are best analyzed with submaximal amounts of ions such as we Na-22 60 90 have done. Uwabain 30um 30 60 Molluscan smooth muscle exhibits a catch state under Time (min) certain circumstances (24). This condition is characterFIG . 7 . ““Na content of taenia expressed as tissue countslmin g-l ized by sustained contraction during which active state as a percent of medium countslmin ml-*. Open bar denotes content in is markedly reduced. This possibility was ruled out in absence and hatched bar denotes content in presence of LaC&(lO the taenia during Na-free contracture because intensity mM). A is after 30 min exposure to 22Na; B and C, additional 30 and 60 min after exposure to 9Ja and ouabain (3 x IO-” M). Results of active state was well maintained. shown are mean + SE for 3 experiments. In the absence of the above two possible mechanisms, attention was focused on the possibility of Na-free contracture being due to either an increase in CA permeacontrol and lanthanum&eated muscles showed significant increase in tissue 22Nacontent over the correspond- bility and/or inhibition of Ca removal from the contractile system. It is not possible to completely rule out the ing nonouabain-treated ones. However, a small inhibition was seen in the lanthanum-treated muscle com- first alternative. The inability of D600 to fully relax Nafree contracture contrasts sharply with the effect of this pared to control muscles. This inhibition was significantly different (P < 0.01) only in the tissues that had drug on a potassium-contracted muscle (in presence of normal sodium) and dictates against increased permeabeen exposed to ouabain for 60 min. bility being the sole mechanism for the contracture. ELectron microscopy. Electron microscopic examinaAdditionally, EGTA is less effective in relaxing a Nation revealed that most of the lanthanum was localized free contracture than a normal contraction due to eleeither in the extracellular or micropinocytic vesicles vated potassium which argues against’an increased Ca (Fig. 8). In several instances, mitochondria were seen permeability. approaching these surface vesicles, but no electron One has now to consider the effect of Na on Ca reopaque lanthanum aggregates were found to associate moval mechanism in the taenia. Smooth muscle cells with these. Occasionally what looked like lanthanum are characterized by three distinct features: large extraaggregates were seen within cytoplasmic regions. However, the clearly rounded outline of these deposits so cellular space, small cell size, and numerous pinocytic closely resembled those in the pinocytic vesicles that it vesicles in the plasma membrane (21). All these characwas felt that perhaps the former represented deposits of teristics will tend to favor easy exchange between the cells and the external medium. Considering these, it is surface vesicles that were overlying the cytoplasmic attractive to consider the cell membrane as an imporregion. Relative to the pinocytic vesicles, the extracellutant site for calcium extrusion during relaxation of the lar region had much less lanthanum. This suggests that smooth muscle cells. However, controversy still exists the lanthanum trapped in these vesicles is not easily with regard to the presence of Na-Ca exchange mechawashed out by the fixatives and buffer. nism across the smooth muscle sarcolemmal membrane, although there is greater acceptance of this mechanism DISCUSSION in the squid axon and cardiac muscle (1, 20). Reuter et Persistence of smooth muscle contracture due to high al. (19) found that in rabbit aortic smooth muscle 45Ca K+ in Na-free medium even after the stimulant was efflux decreased in a Na-free medium by approximately removed has been systematically analyzed in the 35% and promptly increased when the Na-containing taenia, and the observation has also been extended to solution was reintroduced, and they suggested a Na+the rat uterus. sensitive efYlux mechanism for Ca. Van Breemen et al. Replacement of Na by sucrose caused a decrease in (26), on the other hand, showed that in rabbit aorta the external ionic strength. Gordon et al. (12) showed that Ca gradient does not depend on the Na gradient, espeskinned frog sartorius muscle developed tension in a low cially under conditions of elevated intracellular calionic strength medium. Weber and Murray (27) have cium. Furthermore, they showed that the relaxation referred to the fact that tropomyosin can dissociate from brought about by reintroduction of normal sodium in a the contractile apparatus when the ionic strength is high-K contracted aortic strip was not accompanied by reduced. This can lead to loss of calcium sensitivity as Ca extrusion, indicating that transmembrane calcium C64 T. S. MA AND D. BOSE movement was not an important mechanism for relaxa- tioil. Intracellular Ca concentration as measured by the La method can be used reliably as an index for the testing of transmembrane Na-Ca coupled exchange only if other intracellular Ca sequestering sites, e.g., mitochondria or sarcoplasmic reticulum, do not complicate the picture. This error may be different in various tissues. It is possible that Na-CA exchange exists in aortic tissue as has been shown by Reuter et al. (19). Nevertheless, it may not be a mandatory process for reducing cellular Ca during relaxation. The fact that large elastic arteries contain a particularly large volume of sarcoplasmic reticulum, comprising approximately 5% of the cell volume (8), suggests a large intracellular Ca-binding capacity. It is likely that relaxation in aorta involves two processes: the faster one involving intracellular Ca binding and a slower one involving transmembrane Na-Ca exchange. The more extreme argument by van Breemen et al. (26) and Deth and van Breeman (7) that Ca++ extrusion is not a necessary process for aortic relaxation based on the finding that La, a Ca efflux blocker, at a concentration of 2 mM relaxed tissues contracted by K+, Li+, norepinephrine, high pH, or ouabain, requires further evaluation because the time course of the Ca-efflux blocking effect of La is not known (i.e., it may require a longer time to block calcium efflux to the same degree as compared to influx). Freeman and Daniel (10) found that 2 mM La do not block efflux completely in rabbit aorta. Furthermore, we have observed that the Nainduced relaxation of taenia undergoing the Na-free contracture, which was blocked by prior incubation with 10 mM La, was not blocked by 2.5 mM La. This suggests that lower concentrations of La do not block Ca efflux fast enough. In the taenia, addition of La (10 mM) completely relaxed a potassium-stimulated muscle probably because influx of Ca was blocked more rapidly than efflux. Relaxation is not seen if Ca efflux mechanism is inhibited by Na removal. The data on 22Na uptake indicate that La probably inhibits Ca efflux directly rather than by inhibition of Na influx. Katase and Tomita (15) studied the Na-induced relaxation of Na-free contracture in guinea-pig taenia coli. They observed that the relaxation produced by Na at 15°C was only slightly slower than at 35°C. An average Q10 of about 1.4 was obtained between 35 and 15°C. The low Q1,, suggests that the removal of intracellular free Ca is Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 FIG. 8. Electron microscopic wpearance of taenia exposed to LaCl, (20 mM) in presence of normal Na. Most of electron dense area due to La deposits ( t ) is located within micropinocytic vesicles. SODIUM AND SMOOTH MUSCLE C65 RELAXATION These experiments themselves do not argue against the presence of transmembrane Na-Ca exchange in living tissue. The La method for measuring intracellular Ca (25) has been criticized, and ca uti on against using the Larest .stant intracellul .ar pool as a measure of Ca invol ved in EC coupling is probably justified (13). However, it is gratifying to note that the concentration of lanthanum employed by us not only attenuated sodium-induced 45Caloss from the taenia, but also reduced relaxation. The results of electron microscopy in the taenia also differ from those in the stripped uterine preparation (13). Although we could not demonstrate deposits of lanthanum (applied in the ionic form) in intracellular structures such as mitochondria, we cannot be absolutely certain that some lanthanum did not go inside the cell, although one would have expected some to be deposited on the inside of the cell membrane or on the mitochondrial membrane. The discrepancy between our results and those of Hodgson et al. (13) merely indicates gross tissue differences in lanthanum permeability. We intend our results to qualitatively depict the role of Na in controlling Ca movement and relaxation and do not wish to make any quantitative claims about the contractile Ca-pool in this tissue. It is interesting to note sodium ions are also involved in promoting relaxation in the rat portal vein (2). Both taenia and portal vein have a sparse sarcoplasmic reticulum (8). Whether the effect of sodium relaxation is prominent in muscles where sarcolemma plays a greater role in relaxation than sarcoplasmic reticulum remains to be established. Kroeger et al. (16) have proposed that P-adrenergic relaxation of myometrium by isoprenaline is due to calcium extrusion, Our own unpublished observation that isoprenaline fails to relax taenia as well as diethylstilboesterol-treated uterine muscle in the absence of Na can be taken to support the role of Na in drugmediated calcium extrusion mechanism(s). In conclusion, our results are best explained by postulating a role for transmembrane Na+-Ca++ exchange in causing relaxation of taenia undergoing Na+-free contracture. This work was supported by grants from the Medical Research Council of Canada and the University of Manitoba. The expert assistance of Paul Hazelton and Maria Prasher in doing the electron microscopy is gratefully acknowledged. T. S. Ma is the recipient of a University of Manitoba graduate scholarship. Present address of T. S. Ma: Dept. of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada. Send requests for reprints to: D. Bose, Dept. of Pharmacology and Therapeutics, University of Manitoba, 770 Bannatyne Ave., Winnipeg, Manitoba, Canada R3E OW3. Received for publication 24 November 1975. REFERENCES 1. BAKER, P. F., M. P. BLAUSTEIN, A. L. HODGKIN, AND R. A. STEINHARDT. The influence of Ca on Na efflux in squid axons. J. Physid., London 200: 431-458, 1969. 2. BIAMINO, cf., AND B. JOHANSSON. Effects of Ca and Na on con- tractile Pfluegers tension Arch. in the smooth muscle 321: 143-158, 1970. of the rat portal vein. 3. BOSE, D. Mechanism of mechanical inhibition of smooth muscle by ouabain. Brit. J. Pharmacol. 55: 111-116, 1975. 4. BOSE, D,, AND R. BOSE. Mechanics of guinea-pig taenia coli smooth muscle during anoxia and rigor. Am. J. Physiol. 229: 324-328, 1975. 5. BRADY, A. J. Active state in cardiac muscle. PhysioZ. Rev. 48: Downloaded from http://ajpcell.physiology.org/ by 10.220.33.3 on April 29, 2017 mainly controlled by a physical process, which may be Na-Ca exchange. The clear-cut evidence for transmembrane Ca movement during Na-mediated relaxation of Na-free contracture in guinea-pig taenia coli in contrast to the conflicting finding in rabbit aorta m ay be due to tissu .e difference. The spontaneously active spike generating taenia coli contains a relatively smaller amount of sarcoplasmic reticulum, comprising approximately only 2% of the cell volume (8). It might be that under the conditions studied, transmembrane Na-Ca exchange rather than intracellular binding is the important process for relaxation of taenia. Recently, Casteels and van Breeman (6) and Raeymaekers et al. (18) have presented evidence against the transmembrane Na-Ca exchange process in guinea-pig taenia coli. Some of their most important evidence against such a process was that exposing the muscle to a potassium-free solution caused a progressive decrement of Na gradient across the cell membrane but failed to augment intracellular Ca content. One aspect of the problem not considered in this argument is that elevation of intracellular Na in taenia coli has been shown by us (2) to cause a lesser calcium accumulation due to high K stimulus. Whether this was due to activation of a sodium-dependent Ca punp or due to a decrease in Ca permeability is not known. Casteels and van Breeman (6) found that substitution of all extracellular Na+ with choline but not lithium reduced 45Ca++ efflux, and as little as 7 mM Na+ reversed this effect completely. They considered the choline effect to be due to a change in extracellular monovalent cation composition. It is possible that in smooth muscle, unlike squid axon, lithium can mimic sodium in causing coupled calcium movement. Our own results indicate that lithium can substitue for Na, although to a lesser extent, in relaxing Na-free contracture (unpublished observation). Therefore, the possibility of a Na+mediated Ca++ exchange in taenia, at least in the range of Na+ concentrations around 10 mM, cannot be dismissed. One can invoke, alternatively, the possibility of a Na-dependent Ca++ pump. The low Q10of Na+-induced relaxation (15) argues against the effect of Na+ on a energy-dependent Ca++ pump. Raeymaekers et al. (18) suggested that the Na-Ca exchange which they observed represented only extracellular Na-Ca exchange. This is probably true considering the 0-Na effect which they studied was 100 min after efYlux started (in the presence of Na), whereas our data showed that the cellular Ca and tension can be lowered to control level within 4 min of exposure of the muscle to Na after a 0-Na contraction. Experiments involving denatured taenia coli (18) show that extracellular Na-Ca exchange still occurred in dead tissue. C66 T, S. MA 16 17 18 19 20. 21. . - - - - 22. 23. 24. 25. 26. 27. D. BOSE coli. J. PhysioZ. London, 224: 489-500, 1972. KROEGER, E. A., J. M. MARSHALL, AND C. P. BIANCHI. Effect of isoproterenol and D600 on calcium movements in rat myometrium. J. Pharmacol. Exptl. Therap. 193: 309-316, 1975. MA, T. S., AND D. BOSE. Impairment of smooth muscle relaxation in the absence of sodium. Proc. Can. Fed. BioZ. Sot. 18: 567,1975. RAEYMAEKERS, L., I?. Wuytack, and R. CASTEELS. Na-Ca exchange in taenia coli of the guinea-pig. Pfluegers Arch. 347: 329340, 1974. REUTER, H., M. I? BLAUSTEIN, AND G. HAEUSLER. Na-Ca exchange and tension development in arterial smooth muscle. PhiZ. Trans. Roy. S&c. London, Ser. B. 265: 87-94, 1974. REUTER, H., AND N. SEITZ. The dependence of Ca-efflux from cardiac muscle on temperature and external ion composition. J. 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