THAT changes in ionic gradients affect

Transcription

1 Electrolytes and Arterial Muscle Contractility By W. ALAN DODD, AND EDWIN E. DANIEL, PH.D. With the technical assistance of Kathleen Robinson THAT changes in ionic gradients affect contractility and excitability has been shown in skeletal muscle, 1 ' 4 cardiac muscle ' and nerve. 7 " 1 The reduction of external sodium causes reduction and eventual loss of excitability in nerve and skeletal muscle, supposedly because depolarization is due to the entry of sodium into the cell. The precise effect of alterations in external sodium on contractility of muscle is not known. Contraction of skeletal muscle and cardiac muscle is associated with potassium loss. Increasing the external potassium or decreasing internal potassium tends to cause contracture and a decrease in excitability in cardiac and skeletal muscle. 11 " 12 There are only a few studies of arterial muscle contractility and ionic gradients. Eecent studies by Leonard 13 have shown that potassium-free solutions enhance contractility induced by electrical stimulation and inhibit relaxation resulting eventually in contracture. This change in contractility was postulated to be the result of lowered intracelluar potassium. Bohr et al. 14 have obtained somewhat different results finding a marked and progressive reduction in contractility (to epinephrine) in potassium-free solution. These workers suggested the increase of the Ki/Ko ratio increased the threshold for response. The role of the sodium ion has been investigated by Bohr et al. 14 A decrease of sodium (to 8 meq./l.) produced an increased response to epinephrine; and an increase of sodium (from normal of 11 meq./l. to 1 meq./l), a decrease in response. The purpose of this series of experiments is first, to study arterial contractility (not excitability) in al- From the Department of Pharmacology, University of British Columbia, Vancouver, B. C. Supported by the Life Insurance Medical Research Fund. Beceivod for publication November 3, 199. Circulation Research, Volume VIII, March I96 41 tered ionic gradients (varying sodium and potassium) ; second, by tissue analysis to demonstrate any relation between tissue electrolytes and manifest response; and third, to attempt to further elucidate the electrolyte distribution in arterial muscle. Methods Tissue Preparation Male rabbits, 1. to 2 Kg., were used. The animals were killed by a blow at the foramen magnum and the entire thoracic aorta from the arch to the posterior attachment of the diaphragm rapidly removed. Control tissues were taken immediately, blotted free of blood and weighed. Spiral strips of thoracic aorta were prepared, 1 keeping the tissues in oxygenated Krebs-Binger medium at 3 C. Strips cm. were set up in a bath to record tension changes. Tension Recordings The arterial muscle strip was suspended in a muscle bath at a constant temperature under a tension of 1 Gm. An RCA No. 734 transducer was used to convert mechanical tension changes into electrical recording on the Sanborn Recorder Model No B. Tissue Equilibrium The strips were equilibrated for 2 hours at 3 C, in buffered Krebs-Ringer (ph 7.4) oxygenated with per cent dioxide in oxygen. This equilibration period allowed for stretching and increase in sensitivity as noted by Furehgott. 18 If the applied tension diminished during this period, it was restored by stretching the muscle strip. Drugs and Stimulation The drugs used for stimulation were 1-epinephrine bitartrate, histamine acid phosphate, acetylcholine bromide and pitressin. They were diluted with distilled water in such concentrations that a constant volume of.2 ml. was added to a bath of 3 ml. The concentrations, expressed in terms of the salt, were those which would produce maximal contractions (as determined by preliminary experiments) : epinephrine 1~ Gm./ml. bath solution, histamine 1" 4 Gm./ml. bath solution, acetylcholine 1" 3 Gm./ml. bath solution and pitressin 6 1~ 2 units/ml, bath solution.

2 42 DODD, DANIEL Table 1 Relationship of Drugs to Tension and Maximum Bate of Contraction in Krebs-Binger Max.1 Gm Katet Gm./sec Epinephrine 3 rate Hiistamine % % t. max. rate * T-max. = maximum tension developed in grams, t rate = maximum rate of tension increase in Gm./sec. Acetylcholine % % t. max. rate Pitressin % % t. max. rate All drugs were left in contact with the tissues for minutes, then washed out thoroughly. One ml. of a 1 per cent sodium nitrate solution was used sometimes to facilitate relaxation of aorta strips following a contraction. A period of 1 minutes was allowed from the time the strip had reached basal tension to the next stimulation. Control responses were run on each tissue, using epinephrine, histamine, acetylcholine and. pitressin before altering the external medium. Extracellular Electrolyte Changes Changes in bathing media were accomplished by a complete washing of the chamber and a 1- minute period of equilibration to ensure complete extracellular exchange before stimulation was begun. The increase in potassium content was achieved by adding potassium chloride as a solid resulting in final concentrations of 1 meq./l. and 2 meq./l. Tissue Analysis Tissues for analysis, taken at various phases of altered response, were blotted dry and weighed. Since the test strip was an insufficient amount for analysis, a. similar strip was subjected to the same experimental procedure, with the exception of stretching. The analytical methods used are described by Daniel. 18 Fat extraction by the method of Lowry and Hastings was used. 17 Microelectrometric titration for chloride was employed. 18 Results Drug Stimulation in Controls Results showed that epinephrine produced the largest increase in tension (expressed as per cent of initial tension, ti_:1, where tm is the maximal tension developed and ti the initial tension). Histamine produced the next highest increase in tension, and acetylcholine the least increase in tension. All drugs produced a sigmoid shaped contraction and relaxation curve. Pitressin produced no response. The maximum rates of contraction and relaxation were.11 Gm./sec. and.4 Gm./sec, respectively. Occasionally, it was noted that the muscle would begin to relax in the presence of acetylcholine after 2 to 3 minutes of exposure. Relaxation after stimulation by epinephrine often did not begin for to 1 minutes after removal of the drug, and took up to 6 minutes for completion. The addition of 1 per cent sodium nitrite increased the rate of relaxation, but relaxation still followed a sigmoid curve (table 1). Variation in External Sodium Sodium Chloride Replaced by Sucrose There was often an initial increase in basal tension following immersion * of the strip in y * sucrose-krebs or in full sucrose-krebs media, up to 1. Gm. at a rate of.1 to.2 Gm./sec. This increment of tension disappeared in to 1 minutes. Responses to all drugs were decreased in y sucrose-krebs and still further in full sucrose-krebs media, but this decrease in response occurred gradually and contractions still could be produced up to 2 hours after complete removal of the external sodium (fig. 1). No potentiation of response was noted. The time taken to develop maximal tension and relaxation increased in the sodium-poor and sodium-free solutions. The rate of tension increase, as well as the maximum tension developed, were less than in the controls (table 2). Diminution of the response to drugs in media with reduced sodium was Circulation Research, Volume VIII, March 19GO

3 ARTERIAL MUSCLE CONTRACTILITY 43 _ 8 _ / / RESPONSE 6 _ : 4. A 2 _ ' A o ' 1 1 ^ ^ ' J 2 3 TIME ( Hou.cs) Figure 1 Response of aortic strips in sodium-free sucrose-krebs medium. % response tm ti in Na-free medium, tm - ti Control ti ivhere tm = maximum tension developed ti = initial tension Symbols = acetylcholine o = epinephrine /\ = histamine proportionately the same, irrespective of whether epinephrine, histamine or acetylcholine was used. Re-immersion in Krebs-Rmger restored responses to control levels in about 6 minutes. Sodium Replaced by Choline Chloride Equilibration (1 minutes) in sodium-free choline chloride Krebs medium produced a Circulation Research, Volume VIII, March 196 induced responses. variable change in basal tension; frequently a decrease in tension was noted. As with the sucrose medium, a decrease in the druginduced tension increment was the only change noted. The rates of contraction and relaxation were prolonged (table 2). Responses were obtained for 2 to 2y2 hours, slightly longer in the choline-krebs medium than in the

4 44 DODD, DANIEL Table 2 Alterations in Epinephrine-Induced Contractions of Babbit Aorta by Variations in the External Medium Medium Basal tension Gm. Control 1. Per cent meq. K+/L. 1 meq. K+/L. 2 meq. K+/L. 2 meq. Na + /L. Sucrose meq. Na + /L- Choline Max. rate of tension increase Gm./sec..7 of control Tension increase Gm. 2. value* Max. tension Gm *Figures are an average for all responses up to Vi hour in the various media. sucrose-krebs medium. Epinephrine produced contraction after the tissue no longer contracted in response to the administration of acetylcholine and histamine (fig. 2). Variation in External Potassium Decreased External Potassium No significant variation in baseline tension was observed during equilibration. A slight decrease in rate of maximal tension increase in response to various drugs was observed. The relaxation phase was prolonged, specially during the early phase in which relaxation was more rapid. Sodium nitrite increased the rate of relaxation in potassium-free medium, but not to the same extent as in the controls. Contractions were initially enhanced and then progressively decreased; however, even after i y2 hours in potassium-free medium, substantial responses were obtained (fig. 3). When histamine was the test drug, the phase of enhancement was often absent. 72 Increased External Potassium Increase in the potassium concentration to 1 meq./l. caused no change in basal tension during equilibration. Contractions induced by epinephrine and histamine were slightly greater than the controls, and the maximum tension developed was about 1 per cent greater than the control (table 2). Relaxation was slowed and only 8 to 9 per cent completed after 1% hours (1 hour was the maximum time for relaxation of control responses). When the external potassium was increased to 2 meq./l., there was no initial increase in basal tension. Epinephrine and histamine produced responses in which the rate of maximal tension increase and tension developed was greater than the control (table 2). Relaxation was progressively less complete (even using sodium nitrite) until the resting tension was almost the same as the maximal tension at the height of contraction. Tissue Analysis Krebs-Singer Medium Strips were immersed in Krebs-Ringer for 4 to 6 hours before being subjected to altered electrolyte medium. A series was run to establish control values for electrolytes of tissues undisturbed in Krebs-Ringer medium. There was a significant gain in sodium (96.9 to meq./kg.) and chloride from (7.2 to 94.6 meq./kg. wet weight). Potassium appeared to decrease (27.1 to 21.1 meq./kg. wet weight) but this was not statistically significant. Tissue water was significantly increased from 663 to 714 ml./kg. (table 3). That this electrolyte gain was not in proportion to their extracellular concentrations (i.e., the medium) is shown by the fact that the increase in sodium space is almost times that of the chloride space. Calculations indicated that about 1 meq./kg. wet weight of the sodium taken up in Krebs' solution could not be accounted for by expansion of extracellular fluid. This probably was chiefly sodium which entered cells in exchange for potassium. At least 6 to 1 meq./kg. wet weight of potassium may have been exchanged in this way, but the variability of the potassium data make precise conclusions impossible. Sodium-Free Solutions (Sucrose-Krebs - Choline-Krebs) The importance of alterations in tissue electrolytes induced by lowering the external sodium concentration in bringing about the observed changes in contractility was the next question investigated. Circulation Research, Volume VIII, March 196

5 ARTERIAL MUSCLE CONTRACTILITY 4 IfeO _ 14 - ACH - Z EcyuaL Responses % 12. RESPONSE _ 8 _ A A A " * " 1 A»( n AY ^> A I 2 3 TIME ( Hours) Figure 2 Response of aortic strips in sodium-free choline-krebs medium. % response = tm ti in Na-free medium tm - ti Control ti where tm = maximum tension developed ti = initial tension Symbols = aeetylcholine o = epinephrine /\ = histamine In sodium-free medium, contractions gradually diminished, as previously described. An attempt was made to discover any correlation between alteration in contractility and in tissue electrolyte concentration. The data from tissues exposed to media were combined in table 4, arranged according to the contractility of the tissue at the time of analysis. While no definite relationship was found between sodium content and degree of depression of response, a correlation appeared between contractility and potassium content. Tissues with decreased contractility had decreased potas- Circulation Research, Volume VIII, March 196 induced responses. sium concentrations. In tissues with potassium concentrations of about 17. mbq./kg., or less, no contractions were produced. Table correlates tissue electrolytes and time of exposure to sodium-free media. Several things of interest were noted here. The chloride concentration decreased to about 17 meq./kg. wet weight within the equilibration period of 2 minutes and remained at about this value for 2% hours. This suggests that the diffusible chloride (presumably extracellular in location) equilibrates rapidly and that a substantial quantity (11 to 13

6 EPl. HIST. ACh % RESPONSE 1 - _ - * x> H * A A n TIME 1 ( flour's) Figure 3 Response of aortic strips in potassium-free Krebs medium. % response = tm in Na-free medium, ti tm - ti Control ti where tm = maximum tension developed ti = initial tension Symbols = acetylcholine "1 o = epinephrine ^ induced responses. A = histamine meq./kg. wet weight) is more firmly fixed in some manner, either intracellularly or in an extracellular solid phase. Such a large portion of noiidiffusible chloride will produce a significant error in calculation of extracellular space, using the chloride space, and also secondarily in the cellular electrotyte concentration and cell water. There appears to be a rapid loss of sodium within the first 2 minutes. Subsequently, the I loss of sodium continues slowly into both media. After a period of 2 minutes in sucrose-krebs medium, about 94 meq./l. of sodium and 78 meq./l. of chloride had been lost per Kg. wet weight, i.e., in a ratio of.83, which is not too different from the ratio of sodium to chloride (.91) in Krebs-Ringer, but there may have been some loss of sodium unaccompanied by chloride during this 2 minutes. Similar calculations could not be i 8 Circulation Research, Volume VIII, March I96

7 ARTERIAL MUSCLE CONTRACTILITY 47 Electrolyte Contents of Control Tissue s ' la 7 7 s * s * 96.9 ± ± 4. 3 a a + W. «g S 27.1* ± ±4. /Kg. 3 S i 7.2 ± ±3.3 bo 663 ± ±11. Table 3 Na+ meq. dry w ± ± ± ±12. Clsp 633 ± ±22 Nasp 6 ± ±32 *This value is slightly lower than that of other control groups (+3 meq./kg.). t Tissues taken directly from the animal. "in vivo"t control "Krebs" control made for loss of sodium into eholine-krebs solution, but the fact that more sodium was lost in this medium suggests that choline ion but not sucrose may exchange for bound or cellular sodium. Interpretations, however, must be made with caution owing to lack of large numbers of tissues and to the degree of individual variation. The mechanism of the apparently greater loss of sodium into choline-krebs medium than sucrose-krebs medium cannot be decided from these data, though the possibility has been suggested 19 that the choliue ion exchanges for bound or cellular sodium. At the end of 2y2 hours in sodium-free solution, about 19.8 meq./kg. wet weight sodium remained in the tissue (average sucrose- and choline-krebs). Obviously a substantial portion of aorta sodium diffuses only very slowly out of the tissue. This slow loss of sodium suggests that there is a form of sodium (cellular) which is not freely diffusible with the extracellular spaces. Whether or not there is a more firmly fixed fraction, as with chloride, could not be determined from these data. Potassium concentration diminished progressively (reaching 17.7 meq./kg. wet weight average sucrose and choline) after 14 minutes. No difference in the rate of potassium loss in sucrose and choline solution is evident. Tissue water did not change significantly. 'otaxxium-free Solutions Correlation potassium content and contractility. Analyses were made of electrolyte concentrations in aortal strips which had been Circulation Research, Volume VIII, March 1S6 exposed to potassium-free solutions for varying periods. Evidence from table 6 shows that changes in tissue electrolytes cannot be definitely correlated with the alterations in contractility found in this medium. It is interesting to note that substantial losses in potassium to concentrations of 3. meq./kg. wet weight are still compatible with contractility. Such findings raise doubts as to whether or not the loss of contractility in Na-free solutions can be attributed solely to loss of potassium, since the concentrations of potassium averaged about 17 meq./kg. wet weight in those instances. Electrolytes and Exposure. There was no apparent potassium loss in the 2-minute equilibration period (21.1 to 2.4 meq./kg. wet weight). A progressive loss continued, so that after 7% to 8% hours in K-free solutions, about 3. meq./kg. wet weight remained. Even at such concentrations, contractility remained. No significant changes were noted in the sodium or chloride content or in the total tissue water (table 7). Increased External Potassium Owing to the small numbers of tissues analyzed after exposure to increases in potassium concentrations of 1 meq./l. and 2 meq./l., no statistical data are included at this time. Preliminary results indicated however, that the electrolyte composition of aorta exposed to increased external potassium up to 2 hours is not essentially different from the composition of strips exposed to Krebs- Ringer for 4 to 6 hours. No differences were noted between strips exposed to 1 and 2 meq./l.

8 48 DODD, DANIEL Table 4 Correlation of Tissue Electrolytes and Response in Sodium-Free Medium'' Per cent Na+ K> of control No. of meq./kg. meq./kg. HsO response animals ml./kg. (No response) ± ± ± ± ± ± ±1 62 ±17 68 ±13 *Combination of data from tissues exposed to sucrose-krebs and choline-krebs. Discussion Distribution of Tissue Electrolytes The sodium content of aorta (96.9 to 19.2 meq./kg. wet weight) is considerably higher than that of skeletal muscle (1 to 3 meq./ Kg.). 2 The potassium content of aorta (3 to 4 meq./kg. wet weight) is less than skeletal muscle potassium content (11 to 114 meq./kg.) Aorta chloride (7 meq./kg.) is greater than that of skeletal muscle (1 to 2 meq./kg.). The total water of the aorta (64 to 67 meq./kg.) is less than that of skeletal muscle (7 to 77 meq./kg.). The sum of cations (Na + K) about 14 meq./kg. was about the same in vascular muscle as in skeletal muscle and less than that of cardiac muscle, taenia coli, and uterus. 2 Chloride Distribution and ECFV It is generally considered that so much of the tissue chloride is located extracellularly, and in a form which is free to equilibrate with plasma water, that the chloride space is not significantly different from the ECFV. Evidence from these experiments indicates that there is a fraction of freely diffusible chloride which is largely lost within 2 minutes and a fraction of more tightly bound chloride. Inclusion of this bound fraction of chloride in calculations of the chloride space would yield an erroneously high value for the ECFV, assuming that the ECFV has equilibrated in 2 minutes, as suggested by Daniel. 19 Calculations, using corrected values for diffusible chloride, yield an ECFV of 483 ml./kg. This is a more reasonable value than 633 ml./kg., since the total tissue water was found to be about 64 to 66 ml./kg. Intracellular potassium calculated, using the corrected ECFV of 483 ml./kg., is about 192 meq./kg. cell water, which is not an absurd value. Intracellular sodium calculated in the same manner is 18 meq./ml. cell water. The total cation (Na + K) is about 3 meq./ ml. cell water, which is similar to values of other smooth muscle. 2 It is likely that a substantial portion of tissue univalent cation is in bound form in view of such values and of the evidence that animal cells are in osmotic equilibrium. 2 Sodium Distribution It is not yet possible to describe the distribution of tissue sodium with certainty. However, some indication as to the location of aorta sodium has been obtained. One fraction, lost rapidly with chloride, is probably extracellular (and freely diffusible). The remaining sodium is lost more slowly, or not at all. Some sample calculations will help to elucidate its possible location. The control tissue sodium was 96.9 meq./kg., of which.483 ml./ Kg. 148 meq./l. (concentration of Na in rabbit plasma) or 71. meq./kg. was in the ECFV. If the amount of tissue sodium, (17.1 meq./kg.) which remains following 2*4> hours exposure to choline-krebs medium is assumed to be bound, then the 8.8 meq./kg. which is not bound, or accounted for in the ECFV, may be intracellular. Tissues exposed to Krebs- Ringer medium for 4 to 6 hours had an average total sodium content of meq./kg., of which 62 ml./kg meq./l., or 8.9 meq., was in the ECFV. Assuming no change in the amount of bound sodium, approximately 17.4 meq./kg. of sodium could not be accounted for in the ECFV, in exchange for potassium or in a bound form. This represents an increase of 9.1 meq./kg. of intracellular sodium which is taken up either in exchange for another cation (choline) or with an anion. Circulation Research, Volume VIII, March 196

9 ARTERIAL MUSCLE CONTRACTILITY 4 Table Correlation of Electrolytes and Exposure Time in Sodium-Free Media Time in sodium-free 2 minutes* 4-8 minutes 12-1 minutes Na* meq Sucrose 7t 32.9 ± ± ± 6.6./Kg. Choline 29.1 ± ± ± 6.2 K+ meq./kg. Sucrose 31.7 ± ± ± 2.9 Choline 2.3 ± ± ±.4 Cl" meq./kg. Sucrose Choline 17.1 ± ± ± ± ± 2.S 18.1 ± 3.8 'Tissues were equilibrated for 1 minutes, then stimulated; hence 2 minutes elapsed before tissues were removed from the medium, t Number of animals. H=O Sucrose 644 ±U 641 ±1 636 ± 3 ml./kg. Choline 686 ±12 67 ± ±1 The increase in ECFV (calculated by subtracting the bound chloride) may not be strictly accurate in view of the possibility that chloride enters intact or damaged cells. Even so, it seems probable that there is a considerable portion of tissue sodium existing intracelhilarly and in an exchangeable or an unbound form. Sodium Gradient and Contractility It has been postulated that the sodium gradient (Na o /Nai) of the vascular muscle cell is a basic determinant of vascular muscle tone.- 1 Evidence in support of this view is offered from the observations with intestinal muscle that acute reduction of sodium in the medium results immediately in an increase in tension followed by a relaxation to basal tension as the tissue equilibrates; and that reponse to drugs was increased following a 3- minute equilibration period in sodium-poor media, and decreased following exposure to high external sodium concentrations. The immediate increase in basal tension upon exposure to Na-free media was often observed in these experiments. To explain this phenomenon, using the sodium gradient, it has to be assumed that the extracellular phase equilibrates with the bathing medium within seconds something difficult to accept in view of the compact histologic structure of the aorta and data from other smooth muscle. 2 If the increase in response to drug stimula- Circulation Research, Volume VIII. March J9S tion observed in rat colon, 21 and if a similar increase in response seen in rabbit aorta following a 1-minute equilibration in sodiumpoor medium, 14 are due to an altered Na o /Nat gradient, the same effect was to be expected when tissues are exposed to sodium-free media for 1 minutes. This was not the case in these experiments; instead, a progressive decrease in response was obtained on exposure to Na- free media. Previous reference has been made to the fact that a portion of the chloride is bound and a re-calculation of the chloride space yielded a value of 483 ml./kg. The amount of sodium in the ECFV after 4 to hours exposure to Krebs-Ringer medium (allowing for the increase in chloride space) M r as approximately 8.9 meq./kg. The total measured tissue sodium after exposure to Krebs-Ringer medium for the same period of time was meq./kg. Therefore, when all the extracellular diffusible sodium is lost i.e., when the ECFV has equilibrated, the tissue sodium should be about 4. meq./kg., which is only slightly higher than the values seen after 2 minutes of exposure to both of the sodum-free media. This would indicate that little intracellular sodium has been lost in 2 minutes. If contractility depended upon the presence of extracellular sodium to maintain the proper sodium gradient, then removal of external sodium might be expected to produce loss of

10 46 DODD, DANIEL Table 6 Correlation of Tissue Electrolytes and Response in Potassium-Free Medium Per oent response No. of animals Na+ meq./ke ± ± ± ± ±13.8 K+ meq./ke ± ± ± ± ± 13.8 cimeq./ke. 9.6 ± ± ,± ± ± 9. HO ml./ke. 74 ± ±1 721 ± ±1 71 ± 9 Cl space 742 ± ±8 74 ± ± ±73 Na space 988 ±2 741 ±3 776 ± ±32 9 ±88 response or paralysis. This was not observed for a considerable period of time. A decrease of the sodium gradient in the light of the present study results in depression, but not immediate abolition, of contractility. Potassium Gradient and Contractility The relationship between the potassium gradient and threshold for response has been studied in nerve, 8 skeletal muscle, 3 ' 4 uterus 22 and taenia coli. 23 Basically at equilibrium, log K /K o C= resting potential = threshold for response, where Kj is intracellular potassium, K o is extracellular potassium and C is a constant. Bohr 14 suggests that a similar relationship exists between the potassium gradient and aortic smooth muscle responsiveness to physiologic stimuli such that as external potassium increases relative to internal potassium, the response increases. Strictly speaking, these experiments were not designed to test the threshold response. However, an increase in rate of contraction and in tension developed was observed in media containing potassium at concentrations of 1 meq./l and 2 meq./ L, which, may indirectly tend to support this hypothesis. For the relation between potassium gradient and contractility to be valid, an opposite effect should be observed when extracellular potassium is lowered. This is only partly confirmed by the recent study. In fact, instead of a decrease in contractility, potentiation of response was usually seen after 1 minutes in potassium-free media. Since histamine did not often produce such potentiation, but eqinephrine and acetylcholine invariably did, it is possible that response to different drugs may be affected in different ways by altering potassium concentrations and/or gradients. That the loss of arterial muscle fiber potassium causes an increased response, a slower rate of relaxation and eventually contracture has been suggested. 13 Tissue exposed to potassium-free Krebs for 7y2 to 8% hours contained about 3. meq./kg. potassium (or 37.6 meq./l cell water) and averaged 8 per cent of control response. On exposure to sodiumfree medium, when contractility was no longer present, tissue potassium averaged 17. meq./ Kg. wet weight (121.3 meq./l cell water). This latter value may not be entriely accurate when all possible sources of error are considered (loss of cell water, damaged cells, etc.), but if the corrections could be applied, the intracellular potassium would probably be even greater. Thus the loss of fiber potassium does not necessarily cause an increase in response ; in fact, the level of intracellular potassium, per se, does not seem greatly to influence arterial contractility. The actual external potassium in potassiumfree medium bathing the tissue is probably.1-.2 meq./l., or less, because of constant washings. The gradient Ki/K o is therefore greater than 18. In sodium-free solutions, Circulation Research, Volume VIII, March 196

11 ARTERIAL MUSCLE CONTRACTILITY 461 Table 7 Correlation of Electrolytes and Time of Exposure in Potassium-Free Medium Time in K-free med. 2 minutes 2 minutes 2 hours 2% hours 6V& hours 7% hours 8% hours No. of animals Na+ meq./kg ± it ± ± 7.6 K+ med./kg. 2.4 ± ± ± ± 7.6 ClmEd./Kg ± ± ± ± 7.6 HJO ml./kg. 716 ± ± 8 71 ± ± 6. Cl space 734 ±2 71 ± ± ±42 Na space 92 ± ± ±8 72 ± 4.3 Average per cent of control response K,/K o (using 121.3/.79) is about 21. Even if the K in this latter case were actually per cent larger, an unlikely possibility, the gradient would not be larger than that in potassium-free media. If so, then the eventual decrease in contractility in potassium-free media must be caused by other means. In view of these figures, it seems possible that the potassium gradient may be correlated with contractility in this case. Because of the lack of a complete correlation between contractility and the potassium gradient, the roles of other cations (Ca + Mg) and/or anions which are imdoubtedly concerned with arterial contractility, are becoming increasingly more important to investigate. Many studies have been undertaken seeking a relation between intracellular and extracellular electrolyte abnormalities and hypertension. In chronic hypertension, 24 " 28 the chemical composition of the rat aorta is altered in such a way that the sodium, potassium and water content increases. These increases are thought to be intracellular. In transient hypertension, 21* - *1 the electrolyte shifts are not consistent. Pitressin produced an increase in blood pressure Avith an accompanying increase in aortal sodium. No changes were found in potassium content. Hypertensive drugs, such as norepinephrine, failed to produce any significant alterations in arterial wall sodium, but a produced decrease in potassium content. Thus it seems that chronic or "fixed" hypertension is accompanied by an increase in tissue sodium, Circulation Research, Volume VIII, March 196 potassium and water, while acute or "transient" hypertension is not accompanied by any consistent electrolyte shift. It might not be unreasonable, then, to suggest that the transition from early hypertension to fixed hypertension is correlated with a more or less permanent increase in tissue cation. The relation of increased tissue cations (Na + K) to arterial responsiveness in "fixed" hypertension is still being investigated. In vivo studies have demonstrated vascular hyper-responsiveness in hypertensive subjects. 32 ' 3S In vitro studies using arterial strips from hypertensive rats have failed to confirm this finding ' The relation of increased arterial cation content to the mechanism of hypertension in terms of altered vascular contractility is still unknown. Summary Arterial contractility in response to various drugs was studied in media designed to alter ionic gradients of sodium and potassium across the cell membrane. Tissues were analyzed to determine the effects of these procedures on tissue electrolytes and to demonstrate any correlation between tissue electrolytes and response. It was found that the contractile responses progressively decreased in sodium-free media, disappearing in 2 to 2y2 hours. A decrease in external potassium, initially caused a potentiation of response. Thereafter, a decreased response was manifested, yet contractility remained even after 7 to 8 hours in potassium-

12 462 DODD, DANIEL free medium. Increased external potassium caused an increase in response. There was no correlation between the content of sodium and potassium and response in potassium-free solutions, but in sodium-free solutions a positive correlation between contractility and potassium content existed. Chloride appeared to exist in 2 fractions, a diffusible fraction, and a more tightly bound fraction. Calculation of the ECFV based on the bound chloride yielded values which otherwise would have been absurd. Sodium appeared to be distributed in 3 fractions; diffusible and extracellular, not diffusible over the duration of our experiments and slowly diffusible and presumably intracellular, the latter fraction being possibly capable of cation exchange. Evidence obtained does not suggest that the sodium gradient, per se, is responsible for contractility. The concentration of intracellular potassium does not influence contractility directly. However, the potassium gradient may in part determine vascular muscle contractility. Summario in Interlingua Le coutructilitiitc lie nuisculo arterial in responsa a viirie drogas esseva studiate in medios preparate con le objectivo de alterar le gradientes ionic de natrium e kaliuin ab mi latere al altere del membrana cellular. Specimens de tissu esseva nnulysate pro determinar le efeectos del mentionate manovras super le electrolytos tissutal e pro demonstrar le existentia possibile de un correlation inter le electrolytos tissutal e le responsa. Esseva constatate que le responsas contractile clecresceva progressivemente in medios libcrc de natrium. Illos dispareva coinpletemente intra 2 a 2M; boras. Un reduction del kaliuin externe causava initialmente un potentiation del responsa. Postea un redueite responsa esseva manifeste, sed tracias de contnictilitate remaneva presente iticsmo post 7 a 8 lioras in medios libere de kalium. Augmeiito del kaliuin externe causava un augmento del responsa. Esseva trovate uulle correlation inter le contento de natrium e le responsa in solutiones libere de kalium, sed in solutiones libere de natrium le contractilitate esseva positivemente correlationate eon le contento de kaliuin. Chloruro pareva exister in 2 frnctionos, un fraction diffusibile e un plus firmemente ligate fraction. Le calculation del volumine de tluido extracellular super le base del ligate chloruro producova valores plausibile. Super le base do ambe fractiones le valores haberea essite absurde. Natrium pareva essor dist.ribuite in tres fractiones: (1) "Diffusibile e extracellular, (2) non diffusibile in le spatio de tempore de nostre experiinentos, e (3) lentemente diffusibile e presumitemente intracellular. Iste ultimo fraction es possibileiuente capace de excanibio cationic. Le observationes facito in le curso de iste studios non suggere que le gradiente de natrium es per se responsabile pro le pbenoineno del contractilitatc. Le concentration de kalium intracellular non exerce un influentia directe super le contractilitate. Tamen, le gradient e de kalium pote detorminar in parte lo contractilitate de musculo vascular. References 1. CON WAY, E. J.: Nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. Phj'siol. Rev. 37: 84, BOYLE, P. J., AND CONWAY, E. J.: Potassium accumulation in muscle and associated changes..7. Physiol. : 1, JENEKICK, H. P., AND GERARD, R. \V.: Membrane potential and threshold of single muscle fibers. J. Cell. & Comp. Physiol. 42: 197, : Muscle membrane potential, resistance, and external potassium chloride..1. Cell. & Comp. Physiol. 42: 427, GREEN, J. P., GIARMAN, N..T., AND SALTEK, AV. T.: Combined effects of calcium and potassium on contractility and excitability of the mammalian myocardium. Am. J. Physiol. 171: 174, "WEIDIIANN, S.: Resting and action potentials of cardiac muscle. Ann. New York Acad. Se. Cambridge Phil. Soc. 26: 339, : 663, HODGKIN, A. L.: Tonic basis of electrical activity in nerve and muscle. Biol. Rev. Cambridge Phil. Soc. 26: 339, 19]. 8., AND KATZ, B.: Effect of sodium ions on the electrical activity of the giant axon of the squid. J. Physiol. 18: 37, KEYNES, R. D.: Ionic movements during nervous activity. J. Physiol. 114: 119, HODGKIN, A. L.: Ionic exchange and electrical activity in nerve and muscle. Arch. Sc. Physiol. 31: 11, HADJU, S.: Bioassay for cardiac active principles based on the staircase phenomenon of the frog heart. J. Pharm. & Exper. Therap. 12: 9, VICK, R. L., AND KAHN,,T. B., JR.: Effects of ouabain and veratridine on potassium movement in the isolated and guinea pig heart. J, Pharm. & Exper. Therap. 121: 389, 197. Circulation Research. Voluvir. VIII. March 19G

13 ARTERIAL MUSCLE CONTRACTILITY LEONARD, E.: Alteration of contractile response of artsry strips by a potassium-free solution, cardiac glycosides and changes in stimulation frequency. Am. J. Physiol. 189: 18, BOHR, D. F., BRODIE, D. C, AND CHEU, D. H.: Effect of electrolytes on arterial muscle contraction. Circulation 17: 746, FURCHGOTT, R. F., AND BHADRAKOM, S.: Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs. J. Pharm. & Exper. Therap. 18: 129, DANIEL, E. E., AND BASS, P.: Besponses of smooth and striute muscle to alterations in extracellular electrolytes, Am. J. Physiol. 187: 247, LOWRY, O. H., AND HASTINGS, A. B.: Histochemical changes associated with aging. I. Methods and calculations. J. Biol. Chein. 143: 27, WHITTAM, R. A.: Convenient micromethod for the estimation of tissue chloride. J. Physiol. 128: 6, DANIEL, B. K, AND DANIEL, B. N.: Effects of ovarian hormones on the content and distribution of cation in intact and extracted rabbit and cat uterus. Canad. J. Biochem. & Physiol. 3: 12, : Smooth muscle electrolytes. Canad. J. Biochem. & Physiol. 36: 8, FRIEDMAN, S. M., JAMIESON, J. D., AND FRIED- MAN, C. L. : Sodium gradient, smooth muscle tone and blood pressure regulation. Circulation Res. 7: 44, CSAPO, A.: Relation of threshold to the potassium gradient in the myometrium. J. Physiol. 133: 14, BORN, G. V. R., AND BULBRING, E.: Movement of potassium between smooth muscle and the surrounding fluid. J. Physiol. 131: 69, BRAUN-MENENDEZ, E.: Wator and electrolytes in Experimental Hypertension. Ciba Foundation Symposium on Hypertension. Boston, Little, Brown and Company, 194, p. 23S. 2. TOBIAN, L., JR., AND BINION, J. T.: Tissue cations and water in arterial hypertension. Circulation : 74, FREED, S. C, ST. GEORGE, S., AND ROSEMAN, R. H.: Arterial wall potassium in renal hypertensive rats. Circulation Res. 7: 219, TOBIAN, L., AND REDLEAF, P. D.: Ionic composition of aorta in renal and adrenal hypertension. Am. J. Physiol. 192: 32, , AND BINION, J.: Artery wall electrolytes in renal and desoxycorticosterone hypertension. J. Clin. Invest. 33: 147, DANIEL, E. E., DODD, A., AND HUNT, J.: Effects of pitressin and isoprotereuol on aorta electrolytes. Arch, intern.it. pharmacodyn. 119: 43, , DAWKINS, O., AND HUNT, J.: Selective depletion of rat aorta, potassium by small pressor doses of norepinephrine. Am. J. Physiol. 19: 67, , AND : Aorta and.smooth muscle electrolytes during early and late hypertension. Am. J. Physiol. 19: 71, LEE, R. E., AND HOLZE, E. A.: Peripheral vascular heniodynamics in the bulbar conjunctive of subjects with hypertensive vascular disease. J. Clin. Invest. 3: 39, GOLDENBERG, M., PlNES, K. L., BALDWIN, E. I)E F., GREENE, G. D., AND ROH, C. E.: Hemodynuinic response of mail to norepinephrine and opinephrine and its relation to the problem of hypertension. Am. J. Med. : 792, REDLEAF, P. D., AND TOBIAN, L.: Question of vascular hyper-responsiveness in hypertension Circulation Res. 6: ]8, 19S. 3. MATLOV, S.: Comparative reactivities of aortic strips from hypertensive and normotensive rats to epinephrine and levarterenol. Circulation Res. 7: 196, 199. Circulation Research, Volume VIII, March 196