EXTRACELLULAR SPACE MEASUREMENT IN CANINE FEMORAL ARTERY UNDER TENSION Thesis for the Degree of M. S. MiCHTGAN STATE UNIVERSTTY CARL R. BECK 1975 f‘W“ I m ‘32“ w W 3:65 T, {‘5 TH ABSTRACT u EXTRACELLULAR SPACE MEASUREMENT IN CANINE FEMORAL ARTERY UNDER TENSION BY Carl R. Beck The extracellular space (ECS) of canine femoral artery under tension was estimated using the tracer uptake technique. The vessels were placed under tension via a cannulated stainless steel rod. When sucrose 14C was used as the tracer, the tissue was so sensitive to tension that this tracer is believed to enter the smooth muscle cells. The ECS was 44.6 ml/lOO gm tissue for a "relaxed" artery, 54.4 ml/lOO gm tissue for an artery in "moderate" tension and 66.0 ml/lOO gm tissue for one in "tight" tension. However, when inulin 14C was used as a tracer, no statisti- cal difference could be detected between vessels under dif- ferent degrees of tension. The inulin space was 41.3 ml/lOO gm tissue. The total water volume (measured by the weight change after evaporation) was 76.0 ml/lOO gm tissue. This leaves an intracellular volume of 34.7 ml/lOO gm tissue. When the potassium concentration in the surrounding fluid was lowered to 25% of the normal concentration, the Carl R. Beck sucrose space did not change. The inulin space, however, dropped from 40.5 m1/100 gm tissue to 36.0 ml/lOO gm tissue when the potassium concentration was lowered. The water volume remained unchanged which indicates that the intra- cellular volume increased from 35.5 ml/lOO gm tissue to 40.0 ml/lOO gm tissue with a decrease in the potassium concentration. EXTRACELLULAR SPACE MEASUREMENT IN CANINE FEMORAL ARTERY UNDER TENSION BY Carl R. Beck A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Chemical Engineering 1975 To my parents ii ACKNOWLEDGMENTS The author would like to express his appreciation to his academic advisor, Dr. Donald K. Anderson, for his guidance, encouragement and assistance. Appreciation is also given to Dr. Jerry B. Scott and Dr. Won H. Lee for their advise and assistance during the course of this study. The author also wishes to thank Mrs. Josephine J. Johnston for the preparation of the solutions so necessary in this work. The financial support of the Michigan Heart Associa— tion is gratefully acknowledged. The understanding and encouragement of the author‘s wife, Susie, is sincerely appreciated. iii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . LIST OF TABLES . . . . . . . . LIST OF FIGURES . . . . . . . . INTRODUCTION . . . . . . . . . BACKGROUND AND THEORY . . . . . . Two Methods of Measurement of ECS Complications of Measurement of ECS A Comparison of Tracers . . The Intracellular and Water Space . EXPERIMENTAL PROCEDURES AND APPARATUS Artery Procurement . . . . Tissue Preparation . . . . Incubation and Washout . . Measurements and Calculations Apparatus and Solutions . . RESULTS 0 O O O O O O O O 0 The Total Water Space . . . Sucrose Space as a Function of Inulin Space . . . . . . The Effect of Low Potassium . DISCUSSION 0 O O O O O O O . CONCLUSION . . . . . . . . . MPENDIX . C O O O O O O O O BIBLIOGRAPHY I 0 O O O O O O 0 iv Tension Page iii vi 10 11 l3 13 13 14 15 21 22 22 22 24 26 31 35 36 40 LIST OF TABLES Table Page 1. Tracers used or tested for ECS measurement . . 7 2. ECS and Water Space measurements in vascular smooth muscle . . . . . . . . . . . 9 3. Summary of the results . . . . . . . . 33 Figure 1. LIST OF FIGURES Sketch of a helically cut artery segment, showing the circularly arranged cells . . Flow diagram of the experimental procedure . Uptake of sucrose 14C as a function of time in canine femoral artery . . . . . . Uptake of inulin 14C as a function of time in canine femoral artery . . . . . . Sucrose Space as a function of tension in canine femoral artery . . . . . . . Sucrose space as a function of rod diameter for samples of canine femoral artery . . Direct comparison of inulin and sucrose spaces in canine femoral artery . . . . Sucrose space as a function of the potassium concentration in canine femoral artery . Inulin space as a function of the potassium concentration in canine femoral artery . vi Page l6 19 20 23 25 27 29 30 INTRODUCTION Electrical potential differences are found across the membranes of all living cells. Changes in these potential differences are responsible for the electro- chemical impulses conducted by nerve cells and the contrac- tile characteristics of muscle cells. These potential differences are in part established by an energy consuming active transport mechanism which preferentially pumps certain ions across the membranes, and is influenced by the diffusion of ions through the membranes because of concentration differences. In studies aimed at attempting to better understand these processes, there are several parameters for which it would be useful to have numerical values. Two such parameters are the intracellular and extracellular volumes of the tissue, since the distribution of total volume is important in the interpretation of data relative to both active and passive transport across cell membranes. There are several methods for measuring the extra- cellular space (ECS) of a particular type of tissue. The "anatomical" ECS can be calculated by measuring the appropriate areas on electronmicrographs of sections pro- cured from the tissue. However, electronmicrographs are typically taken of an area which is densely populated with cells, excluding a relatively "cell free" fraction of the tissue. This cell free tissue fraction contains a variety of materials, such as connective tissue, fibro- blasts, glandular tissue and interstitial cells, of which an electronmicrograph would be of little value since the degree of ion permeability would be unknown. Since an ionic distribution study requires knowledge of the ECS which includes all the space accessible to the ions, this method is undesirable (7). The method most often used for this type of study is the tracer uptake technique. With this method a tracer molecule which cannot enter the cells, is allowed to diffuse into the ECS. The amount of this tracer taken up by the tissue is then measured to determine the ECS. This was the method chosen for this investigation. The ECS of different types of tissue vary consider- ably because of the different structures and the varying complexity of these structures. Even among different kinds of blood vessels there is considerable variation in structure. Therefore, the ECS must be determined for each particular tissue of interest. In this work the canine femoral artery was investigated. In actual physiological conditions, arteries are under tension resulting from blood pressure in the lumen. In metabolically active tissue, the arteries constrict in response to an increase in blood pressure and dilate in response to a decrease in blood pressure. This phenomenon is called the "Bayliss response" or myogenic response. It tends to maintain blood flow relatively constant deSpite changes in blood pressure. The arteries used in this work were cannulated with a stainless steel rod to mimic the tension produced by blood pressure. Traditionally, the ECS of blood vessels has been measured using helically cut strips, see Figure 1. By cutting the vessels in this way, excess moisture on the. surface of the tissue can be removed by blotting, and muscle cell damage will be reduced, since the cells are arranged in a circular fashion around the vessel. Reduction of cell damage is critical since a damaged cell may allow the tracer to enter, increasing the ECS (24). The helical cuts were not necessary in this study because the arteries were cannulated with a stainless steel rod which prevented moisture from being trapped in the lumen. Consequently, only the outer surface of the vessel required blotting and the vessel sustained less cell damage. Another, more recent observation in vascular smooth muscle, is vasoconstriction in a reduced potasSium environ— ment (14). If constriction significantly changes the dimensions of smooth muscle cells, these changes should be reflected in measurements of the ECS and the total water space. In this study the ECS and the total water space m uscle cells I"’ 1" l‘.' r 1 h r l r were measured in sections of artery subjected to a potassium concentration which was 25% of the normal physiological potassium environment. These volumes were then compared to those that were measured in the "normal" potassium solution. BACKGROUND AND THEORY Two Methods of Measurement of ECS There are two methods with which the accessible, or free extracellular volume may be calculated. One method is to use the Donnan equilibrium principle (9). This Inethod requires data on the amounts of circulatory space, (zonnective tissue, and nerve tissue in the ECS, the water aand chloride content of these tissues, and the total eelectrolyte content. These data can be determined and tJIiS method works well for skeletal muscle, however in snnooth muscle there is evidence of cation and anion binding Mfllich would invalidate the calculations (8, 25). The other technique is based on tracer uptake, Vflnere a tracer is chosen which will presumably not enter time muscle cells, or will do so at a very slow rate. This Ennasents the problem of choosing the prOper tracer. Table 1 lists some tracers which have been used or tested for ECS measurements in smooth muscle tissue. .Erom this large array’of'choices one must determine which.is mot effective f0r'measurement in a specific type of tissue. l.—-Tracers used or tested for ECS measurement. Tracer Reference Arabinose 2 Mannitol 2 Sucrose l, 2, 24, 25 Raffinose 2 Inulin 2, 15, 19, 24, 25 Fructose 6 Sorbitol 12 Albumin 2, 25 Sulfate 3, 25 Ferrocyanide 25 Polyglucose 12 Ethanesulfonate 12, 13 EDTA 17 Thiosulfate 18, 19 Thiocyanate 15 Calcium 12 Lithium 12 Chloride 12 Sodium 12 Bromide 3 Complications of Measurement of ECS The extracellular volume of smooth.muscle tissue is filled with a complex heterogeneous mixture of materials, including collagen, blood vessels, nerve and Schwann cells, macrophages, fibroblasts, elastic tissue and a gel-like matrix of mucopolysaccharides. It is in this twisted maze that the tracer must diffuse. This labrinth is further complicated by "micropinocytic" or "plasmalemmal" vesicles which appear to be connected with an intracellular endo~ plasmic reticulum and could quite possibly increase the effective ECS (7). Thus the problem becomes one of accessibility of the tracer to the desired space. The ideal tracer would enter only those spaces which are available to the free (unbound) extracellular water and solutes. The differences in the tracers are reflected in the different values for ECS which have been reported. Some representative values of ECS for vascular smooth muscle are given in Table 2. Many investigators have used chloride as a tracer, since it is known to be excluded from most cells and is very small sterically. However, recent observations indicate a large fraction of chloride may be present inside the cells of smooth muscle and the possibility exists that chloride may be "bound" either extracellularly or intra- cellularly (25, 10). TABLE 2.--ECS and Water Space measurements in vascular smooth muscle. MuScle Type ECS Method Water Reference mouse femoral artery 30 electronmicro. -- 23 pig carotid artery 39 electronmicro. -- 22 cow carotid artery 39 thiosulfate -- 18 dog carotid artery 44 EDTA 72.2 17 dog carotid artery 25 inulin -- 16 dog carotid artery 36 inulin 73.6 11 dog carotid artery 37-39 inulin —- 20 dog carotid artery 40 sucrose 70.1 25 dog lingual artery 47 EDTA 75.5 17 rat aorta 35 inulin 68.3 15 rabbit aorta 62 inulin 72.7 4 dog aorta 44 inulin -- 20 rat portal vein 45 sucrose 77.9 1 ECS, ml/lOO gm tissue Water Space, gm/lOO gm tissue 10 A Comparison of Tracers A few investigators have used the uptake technique in smooth muscle to compare different tracers. Barr and Malvin (2) examined seven tracers in canine intestine and found sucrose, inulin and raffinose to measure approxi— mately the same space and not to approach the total tissue water with long incubation times. They suggested that these tracers may be a suitable means of estimating the ECS in this tissue. An analysis performed by Goodford and Leach (12) indicated that the inulin volume increased in the guinea-pig taenia coli when the tissue was treated with the enzyme hyaluronidase which metabolizes hyaluronic acid. Since hyaluronic acid has been found in the inter- spaces of many tissues, this data suggests that inulin is sterically inhibited by hyaluronic acid from entering portions of the ECS, an observation previously predicted in a partition study by Ogston and Phelps (21). As a result of this observation and a comparison of data from other papers, monosaccharides or disaccharides were recom- mended for ECS measurement. Villamil §t_al, (25) tested five tracers on canine carotid arteries. In that investi- gation hyaluronidase had no effect on the inulin space. However, they found that inulin could only partially pene- trate isolated dense adventitia suggesting that an obstruc— tion to inulin diffusion occurs in the adventitia, rather than in the mucopolysaccharide matrix of the ECS. Since 11 the sucrose space approached but never exceeded the total tissue water when the cellular barriers were metabolically abolished and because the values were near those calculated by combined light and electron microsc0py, sucrose was recommended as the best ECS indicator of the five tested. Although these studies all seem to ratify sucrose as the most reliable extracellular marker, other investigators advocate the use of larger molecules (8). In fact, most authors have chosen inulin for this function (5). Some evidence has been shown that sucrose enters smooth muscle cells (6, 9) and therefore a larger molecule should be used. The strongest indication of this is given by Bozler and Lavine (6) who noticed that the smooth muscle cells of frog stomach swell in isosmotic sucrose solution and the sucrose Space was larger than inulin space. In this investigation inulin and sucrose were used as ECS markers because they appeared to be the best repre- sentatives of both large and small molecules. The Intracellular and Water Space The intracellular volume is determined by subtract- ing the ECS from the total volume available to the solutes. One way total volume is determined is through the use of a tracer, such as urea, which is assumed to enter all the available space, both intracellular and extracellular (1). It has been shown however, that urea Space exceeds the 12 total water content of certain smooth muscle tissue (2) and therefore binding of urea may be occurring. Another popular method for determining total avail— able space is to weigh the tissue to obtain a "wet weight." Then the tissue is heated in an oven to remove all the moisture, and a water-free "dry-weight" is obtained. The difference is taken to be the total water volume available to the tissue. This method requires the removal of extraneous solution from the outside of the tissue. The most reproducible method of achieving this according to Hagemeijer et_al. (15) is with.absorbant paper. A method very similar to this was adopted here. Table 2 lists some values of water space. EXPERIMENTAL PROCEDURES AND APPARATUS Artery Procurement Mongrel dogs ranging in weight from 20 to 40 kg were anesthetized by intravenous injection of sodium pento- barbitol (30 mg/kg) and ventilated with a mechanical posi- tive pressure respirator via an intratracheal tube. The femoral artery was surgically exposed, ligated, and cannulated with a stainless steel rod. The artery and rod were then removed from the hindlimb and placed in a vial containing oxygenated Ringers solution at 37°C for trans- portation to another laboratory. The arteries were approximately 1.5 cm in length, 1.5 mm in diameter, and weighed about 18 mg. Tissue Preparation The artery, attached to the stainless steel rod on which it was originally cannulated, was placed in a petri dish (filled with oxygenated Ringers solution) while the loose adventitia was removed. At this time the artery was moved along the rod with a pair of forceps to establish the degree of tension. 13 14 Three criteria of tension were distinguishable: (l) a relaxed tissue was defined as one which could be easily slipped off the rod, i.e., held on the rod only by the surface tension of the solution. A tissue sample of this size was very difficult to obtain, as a perfect com- promise had to be made between "too loose" and some degree of tension. A loose artery presumably, would trap water between the steel rod and the inner wall of the vessel, which would add to the wet weight of the vessel. (2) An artery was defined as being under moderate tension if it exhibited some degree of resistance to being dis- placed on the rod. (3) A tight artery was one which was difficult to move along the rod. After the mounted tissue had been examined in the petri dish, it was taken from the Ringers solution and the residual moisture was removed by blotting the tissue and rod on Kimwipe disposable wipers. The artery and rod com- bination were then weighed on a Torbal balance and the "wet weight" obtained from the difference of this weight and the weight of the rod. The time lapse between artery procurement and the wet weight determination was approxi- mately 30 minutes. Incubation and Washout Immediately after weighing, the mounted artery was placed in a centrifuge tube containing the incubation medium, in which a mixture of 95% O2 and 5% CO2 was 15 continuously bubbling, see Figure 2. The incubation medium consisted of 3 ml of Ringers solution, plus 0.05 ml of labelled sucrose or 0.10 ml of labelled inulin solution and was kept near 37°C by a water bath. After the tissue was incubated in the labelled Ringers solution for 30 minutes it was blotted as des— cribed previously. The artery was slipped off the rod and placed in another centrifuge tube containing distilled water, 5 ml for a sucrose experiment or 3 ml for an inulin experiment. After one hour the tracer was assumed to have equilibrated throughout the tissue and the solution. Measurements and Calculations Calculation of the Water Volume. After the washout step, the tissue was removed from the washing medium and placed on a watchglass in a Cenco constant temperature oven at 105°F to evaporate the water. A 24 hour period in the oven was sufficient for the tissue to achieve a constant weight. This dry weight was then subtracted from the wet weight previously calculated to give the total water weight (or water volume if a density of 1.0 is assumed). The Number of Counts in the Tissue. A 1.0 ml aliquot of the washing medium was placed in a scintillation vial containing 10 ml of Insta-Gel liquid scintillation fluid, and counted on a Packard Tri-Carb liquid scintillation spectrometer. Since the volume of the washing medium is 16 0.05 ml of incubation medium Dilution 10 ml water (sucrose) or 20 ml water (inulin) .. 1.0 ml of the dilution 10 ml Insta-Gel number of counts 2 C dil number of counts 95% Oz Incubation Medium 3 ml of Ringers soln. +0.05 m1 sucrose soln. or 0.10 ml inulin soln. artery removed from the rod Wash 5 ml water (sucrose) or 3 ml water (inulin) 1.0 ml of the wash 10 ml Insta-Gel C “7 0 Figure 2.——Flow diagram of the experimental procedure. 17 either 3 or 5 m1 (depending on whether inulin or sucrose was used) the number of counts in the tissue is approximated by equation 3.1. Ct = CWO(I) 3.1 Ct = the number of counts in the tissue and CWO = the number of counts in the washout aliquot. I = 5 if sucrose was used or I = 3 if inulin was used. This equation assumes that the amount of tracer left in the tissue is negligible (for an artery 1.5 cm in length and 1.5 mm in diameter mounted on a 0.9 mm diameter rod, the total tissue volume is 0.34% of the washout medium in a sucrose solution or 0.57% of the washout medium in an inulin solution). The Number of Counts in the Incubation Medium. A 0.05 ml aliquot of the incubation medium was diluted with 10 m1 of water if sucrose was used, or 20 ml of water if inulin was used. A 1.0 m1 aliquot of this dilution was then mixed with 10 ml of Insta—Gel and gave approximately the same total counts as the washout sample. The number of counts in the incubation medium is a function of the number of counts obtained from this dilution. Since there was 3.05 ml in the incubation medium and a .05 ml aliquot was removed this relationship is given by equation 3.2. C. = C im dil(D) (3.05/0.05) 3.2 18 Cim = the number of counts in the incubation medium and Cdil = the number of counts in the dilution aliquot. D = the dilution, and is 20.05 ml for an inulin experiment or 10.05 ml for a sucrose experiment. Calculation of ECS. If it is assumed that at the end of the 30 minute incubation period, the incubation medium is in equilibrium with the ECS, then the concentra- tion of tracer in both volumes must be equal as in equation 3.3. Ct/Vo = Cim/3.05 ml 3.3 V0 = ECS and 3.05 ml = the volume of the incubation medium. Once V0 is known, Vi (the intracellular volume) can be calculated by equation 3.4. Vt = the total water volume, obtained by weighing. The Time Studies, Time studies were performed with both tracers to determine how long incubation must proceed in order to equilibrate the tracer between the incubation medium and the ECS. These studies indicated that 30 minutes was sufficient for both sucrose and inulin, see Figures 3 and 4. In these experiments a tissue was incubated for 5, 10, 15, 30 and 60 minutes with a 60 minute washout in Ringers solution in between each incubation. l9 .mnmuum Hmuoamm maficmo ca mEHu mo coauocdm m mm 0 vH mmonosm mo mxmumsll.m musmwm 355:. 5 ms: a pm .1 a r 0 ii s .2 m m TON a k. ,8 w. '8 .m. . SW... 18 w m in o [E w. -8 m 20 s p. a i .. .wumuum Hmuoamm mcwgmo cw oEHu mo GOHHUGSM N mm 0 GHHDCH mo mxmumslt.v mHsmam «H 85:2: 5 me: anssp, )0 Mi 001nm ‘aoeds uunu| 21 These results are similar to those obtained by Arvill (l) for sucrose. Apparatus and Solutions Stainless steel rods were fashioned from syringe needles ranging from 15 to 24 guage. The tips were cut off and both ends were filled with solder. The tips of the rods were then polished smooth to prevent damage to the tissue during cannulation. The composition of the normal potassium Ringers solution used was as follows: 7.66 gm NaCl, 0.316 gm KCl, 0.141 gm MgCl 1.9 gm NaHCO 1.0 gm glucose, 8.33 cc 10% 2’ 3’ calcium gluconate, brought to one liter with water. For low potassium Ringers the solution was made isosmolar with NaCl. These solutions were oxygenated by bubbling with a mixture of 95% O2 and 5% CO2 for 30 to 60 minutes. Stock sucrose solution was made up of 0.00714 millimoles of a 14 mCi/millimole sample of 14c labelled sucrose purchased from ICN Pharmaceuticals. This gave a concentration of 0.00143 millimoles/ml when diluted with 5 m1 of water. Stock inulin solution was made up of 50 mg of a 1.0 uCi/mg sample of 14 C inulin (also purchased from ICN) diluted with 10 ml of water to give a concentration of 5 mg/ml. RESULTS The Total Water Space The total water space remained the same regardless of which tracer was used, the degree of tension, or the concentration of potassium. The total water volume deter- mined from all the experiments of this study (n = 75) was 76.0 gm/lOO gm tissue (i 3.1 s.d.). This value agrees with the water space reported for canine lingual artery (75.7 gm/lOO gm tissue (17)). Sucrose Space as a Function of Tension It became apparent after several experiments that, not only did the rod in the lumen of the artery change the measured sucrose volume, but the tissue was extremely sensitive to the degree of tension under which it was placed. When tension was removed, i.e. the artery was judged to be "relaxed," the sucrose space averaged 44.6 ml/lOO gm tissue (i 0.08 s.d.), see Figure 5., This value closely approximates the sucrose space measured by Arvill et al. (45.4 ml/100 gm tissue (1)). Assuming a water 22 23 701 n =13* n=17* 10- Sucrose Space, ml/lOO gm of tissue Relaxed Moderate Tight Figure 5.--Sucrose space as a function of tension in canine femoral artery. 0, extracellular; A, intra- cellular (calculated by difference). *, statis— tically different from control at P < .002. 24 space of 76.0 gm/lOO gm tissue, this would leave an intra— cellular volume of 31.4 m1/100 gm tissue. Arteries which were defined to be under "moderate" tension had an average sucrose space of 54.4 ml/lOO gm tissue (: 5.0 s.d.), which gives an intracellular volume of 21.6 ml/lOO gm tissue. Tissues which were judged to be "tight" gave even larger sucrose volumes. The average was 66.0 ml/lOO gm tissue (i 4.6 s.d.). The corresponding intracellular volume was 10.0 ml/lOO gm tissue. In three experiments, two pieces of tissue were taken from a single animal and the arteries were cannulated with rods having different outside diameters. This was an attempt to find pairs of arteries which were as close in size as possible. In all three cases, the artery cannulated with the larger rod (and thus under a greater degree of tension) gave a larger sucrose space. There was no apparent effect of varying the placement of the larger rod (i.e. in the distal or proximal piece of tissue), see Figure 6. Inulin Space The inulin space measured under "moderate" tension was 40.5 ml/lOO gm tissue (i 4.8 s.d.). This volume could not be differentiated to a 95% confidence level from the inulin space measured under the "tight" condition, which was 44.9 ml/lOO gm tissue (i 4.1 s.d.). If the "tight“ and "moderate" data is combined, the inulin space becomes 25 60- % 40 £4. 55- 0” e E5 01 § 2 50* 0/0 EE 8.. s 0/0 U) 3, 45~ e U 3 (I) 40 .67 .d9 rod diameter, cm Figure 6.—-Sucrose space as a function of rod diameter for samples of canine femoral artery. 'Points connected by a line are measurements taken from two segments of the same artery mounted on different rods. 26 41.3 m1/100 gm tissue (i 4.9 s.d.) which leaves an intra- cellular space of 34.7 ml/lOO gm tissue. In eight experiments a direct comparison of inulin and sucrose volume was attempted (see Figure 7). In seven of these, two adjacent sections of an artery were cannulated with a pair of rods which had the same diameter. One of these tissues was then incubated in a sucrose solution, and the other in an inulin solution. The respective volumes measured with different tracers could then be compared. In the eighth experiment, four sections of a single artery were cannulated by two pair of rods of two different diameters. The results showed the sucrose space to be greater than the inulin space in each of the nine pairs of tissue. The "tight" arteries, as expected, had greater sucrose volumes than the "moderate" tension vessels. This trend was not as well defined with the inulin space however. The Effect of Low Potassium Sucrose Space. The sucrose space, measured for "relaxed" tissue in a low potassium environment (1.0 mEq/l) was 45.9 ml/lOO gm tissue (i 6.2 s.d.), which would give an intracellular volume of 30.1 ml/lOO mg tissue. The sucrose space measured in this way could not be differenti- ated from the 44.6 ml/lOO gm tissue which was determined with normal potassium. When the tissue was placed under tension from the rod on which it was cannulated (no distinction was 27 75‘ 70-+ / / ”Op — / 60 / / . 55- 4/ / CK \\\\ \ \ o 40- : Inulin and Sucrose, ml/100 gm of tissue U1 0 1 \:°\ \ 35 l , l Inulin Sucrose Figure 7.--Direct comparison of inulin and sucrose spaces in canine femoral artery. Connected points are from adjacent sections of the same artery. Dashed lines correspond to arteries under "tight" tension and continuous lines corres- pond to arteries under "moderate" tension. 28 attempted between "moderate" and "tight" in this study) and in a low potassium environment, the sucrose space became 59.3 ml/lOO gm tissue (i 8.2 s.d.). This would leave an intracellular space of 16.7 ml/lOO gm tissue. This sucrose volume was also indistinguishable from that obtained from the normal potassium tension data (59.4 ml/lGO gm tissue i 7.5 s.d.). These results are given in Figure 8. Inulin Space. Unlike the sucrose space, the inulin space in a low potassium environment differed significantly from the normal potassium data. With just "moderate" tension considered, the low potassium inulin space was 36.0 ml/lOO gm tissue (i 4.1 s.d.) which yields an intra- cellular Space of 40.0 ml/lOO gm tissue. The inulin Space under "moderate" tension in a normal potassium environment ras 40.5 ml/lOO gm tissue which differs from the low potassium value with a level a Significance of 0.013. These results are shown in Figure 9. 29 70- T T 60“ _l_ 50- I i (D 3 I :g 40- .l- E D1 8 S 30‘ E 8‘ A 20- “3’ e m 10- relaxed tension relaxed tension normal K+ low K+ Figure 8.--Sucrose Space as a function of the potassium concentration in canine femoral artery. 30 70. 60~ 50— 40~ i—el l———l Inulin Space, ml/100 gm of tissue normal K+ low K” Figure 9.--Inulin Space as a function of the potassium concentration in canine femoral artery. DISCUSSION In one experiment, the sucrose Space measured for a "tight" vessel was 73.0 ml/lOO gm tissue in contrast to a water space of 76.0 ml/lOO gm tissue. If sucrose does not enter the muscle cells and is not bound, then the intra- cellular space was 3 ml/100 gm tissue or approximately 3% of this particular piece of tissue. These proportions are hard to believe. In an investigation of canine carotid artery, Villamil e£_al. (25) have Shown that binding of sucrose is unlikely, since the sucrose space never exceeded the total tissue water when the cellular barriers were abolished by metabolic inhibition. Sucrose has, however, shown evidence of entering smooth muscle cells (1, 6, 9). Bozler and Lavine (6) for example, measured sucrose volumes as high as 67-85% of the muscle weight in frog stomach (the water Space averaged 81%). These large volumes were measured in tissue that had previously been incubated in a 2mM calcium chloride solution which caused the muscle cells to swell. It is known that vascular smooth muscle contracts in low potassium (14). This study 31 32 indicates a decrease in inulin Space in a low potassium environment, with the water Space remaining constant imply- ing an increase in the cell volume. If the muscle cells swell with contraction they could be exhibiting the same effect achieved by Bozler and Lavine with calcium chloride. Certainly the increase they observed in the sucrose Space is similar to the increase seen in this study with tissue under tension. Cell swelling with low potassium contraction can be explained by considering the action of the Na-K pump. This pump is believed to transport more sodium ions out of a cell than it transports potassium ions into the cell. Since this active transport mechanism is Slowed down by lowering the external potassium concentration, it seems likely that an excess of ions would accumulate internally. This would increase the osmolarity inside the cell and consequently water would be drawn in and effect cell swelling. In contrast to sucrose, inulin Space remained relatively constant with respect to tension. A summary of the data is given in Table 3. It seems likely that if sucrose is entering the muscle cells under tension while the inulin space remains unchanged under the same condi- tions, that the inulin molecule is sterically inhibited from entering the cells. The molecular weight of inulin is 7000 compared to 342 for sucrose. The inulin space (41.3 m1/100 gm tissue) corresponds quite well to other values reported for comparable tissues, see Table 2. 33 TABLE 3.-—Summary of the results. Potassium Tension ECS SD SEM n Sucrose normal relaxed 44.6 0.8 0.5 3 normal moderate 54.4 5.0 1.2 17 normal tight 66.0 4.6 1.3 13 normal moderate 59.4 7.5 1.4 2 & tight low relaxed 45.9 6.2 4.4 2 low moderate 59.3 8.2 2.6 10 & tight Inulin normal tight 44.9 4.1 2.1 4 normal moderate 40.5 4.8 1.1 17 low moderate 36.0 4.1 1.4 9 normal moderate 41.3 4.9 1.1 21 & tight Water Space--76.0 ml/lOO gm tissue, SD 3.1, SEM 0.4 ECS, ml/lOO gm tissue SD, standard deviation SEM, standard error of the mean n, number of points 34 Villamil et_al, (25) combined light and electron microscopy of canine carotid artery to calculate an extra- cellular volume (including solids) of 55.7 ml/lOO gm tissue which they called "anatomical space." These investigators measured the water fraction of collagen and elastin fibers at 70% and concluded that if the ECS contained only elastin and collagen then the accessible volume would be 39 ml/100 gm tissue. They then called this number the "lowest possi- ble estimate" of the ECS. Extrapolating this to the water space measured in this study (76.0 gm/lOO gm tissue) and using the same anatomical Space, a value of 42.3 ml/lOO gm tissue is obtained. This could be considered an "upper estimate" if the true extracellular water fraction was between 70 and 76%. CONCLUS ION It is apparent that sucrose is an unreliable measure of ECS in canine femoral artery when the tissue is placed under tension with a cannulating rod. Of the two tracers tested, inulin appears to be the best choice when measuring ECS with the artery under tension. The inulin Space of 41.3 ml/lOO gm tissue corresponds quite well to the "lower estimate" of 39 ml/100 gm tissue suggested by Villamil et_al. (25), and the "upper estimate" of 42.3 ml/lOO gm tissue determined in this work. The inulin tracer also exhibited sufficient sensitivity to changes in the potassium environment. The cell volume increased from 35.5 ml/lOO gm tissue to 40.5 ml/lOO gm tissue when subjected to a potassium concentration in the surrounding medium which was 25% of the normal concentration. 35 APPENDIX TABULATED EXPERIMENTAL DATA Exp. water vol Vi/100g Vi/Vo No. VO/100g wet wt. calculated calculated Sucrose, Normal Potassium, Relaxed Tension 26 45.20 0.8031 35.11 0.7768 29 43.70 0.7799 34.70 0.7847 32A 44.94 0.8152 36.58 0.8139 Sucrose, Normal Potassium, Moderate Tension 27 57.04 0.8088 23.85 0.4181 28 57.17 0.7884 21.67 0.3791 32B 47.31 0.7876 31.44 0.6646 33 63.88 0.7825 14.36 0.2248 34 61.94 0.7815 16.21 0.2616 35A 55.42 0.7736 21.94 0.3958 358 56.53 0.7574 19.21 0.3399 36A 50.33 0.7654 26.21 0.5208 37A 58.93 0.7650 17.57 0.2982 41A 56.23 0.7554 19.31 0.3434 43A 51.23 0.7448 23.25 0.4538 438 49.39 0.7536 25.96 0.5256 46B 56.75 0.7622 19.46 0.3430 47B 47.80 0.7227 24.47 0.5118 48B 54.18 0.7583 21.66 0.3997 36 37 Exp. water vol Vi/lOOg Vi/Vo No. VO/100g wet wt. calculated calculated 50B 54.28 0.7375 19.47 0.3587 51B 46.68 0.7734 30.66 0.6568 Sucrose, Normal Potassium, Tight Tension 3 61.67 0.7302 11.35 0.1841 5 67.26 0.7532 8.05 0.1197 7 63.00 0.7870 15.70 0.2492 22 65.84 0.8158 15.74 0.2390 23 65.33 0.7865 13.32 0.2039 24 59.78 0.7811 18.32 0.3065 30 67.30 0.8024 12.94 0.1923 31 70.76 0.7834 7.58 0.1071 38A 72.43 0.7432 1.90 0.0262 39A 60.23 0.7447 14.24 0.2364 40A 73.00 0.7361 0.61 0.0084 42A 61.35 0.7414 12.78 0.2084 45A 69.37 0.7381 4.44 0.0640 Sucrose, Low Potassium, Relaxed Tension 11 41.56 0.8255 40.99 0.9861 14 50.30 0.8447 34.17 0.6794 38 Exp. water vol Vi/100g Vi/Vo No. VO/lOOg wet wt. calculated calculated Sucrose, Low Potassium, Moderate and Tight Tension 8 49.55 0.7940 29.85 0.6024 9 57.49 0.7978 22.22 0.3878 10 48.48 0.7874 30.25 0.6240 12 52.90 0.7871 25.81 0.4879 16 75.56 0.7863 3.07 0.4060 17 56.11 0.7500 18.89 0.3367 18 60.23 0.7600 15.77 0.2618 19 65.07 0.8026 15.20 0.2335 20 63.99 0.7888 14.89 0.2327 21 63.15 0.7727 14.13 0.2237 Inulin, Normal Potassium, Moderate Tension 368 38.38 0.7761 39.23 1.0221 373 39.87 0.7769 37.81 0.9483 413 41.08 0.7681 35.73 0.8697 43C 48.44 0.7588 27.44 0.5663 43D 40.41 0.7419 33.78 0.8359 44A 37.15 0.7473 37.58 1.0117 443 45.07 0.7621 31.14 0.6910 44C 49.70 0.7651 26.81 0.5395 53B 38.28 0.7619 37.92 0.9906 54B 33.31 0.7350 40.19 1.2065 39 Exp. water vol Vi/loog vi/Vo No. vO/loog wet wt. calculated calculated 55B 45.06 0.7333 28.27 0.6275 56B 45.05 0.7125 26.20 0.5815 57B 37.97 0.7222 34.25 0.9022 58B 33.22 0.7059 37.33 1.1251 59B 36.02 0.7260 36.58 1.0156 60B 38.96 0.7238 33.42 0.8577 61B 40.01 0.7050 30.49 0.7620 Inulin, Normal Potassium, Tight Tension 38B 43.27 0.7451 31.24 0.7220 39B 44.79 0.7285 28.07 0.6267 40B 40.98 0.7510 34.12 0.8327 42B 50.62 0.7425 23.63 0.4668 Inulin, Low Potassium, Moderate Tension 53A 32.72 0.7692 44.20 1.3506 54A 41.57 0.7179 30.23 0.7272 55A 34.05 0.7050 36.45 1.0706 56A 37.85 0.7240 34.55 0.9127 57A 36.28 0.7194 35.66 0.9830 58A 31.12 0.7220 41.08 1.3202 59A 30.85 0.7358 42.73 1.3851 60A 41.46 0.7286 31.40 0.7572 61A 38.36 0.7036 32.00 0.8341 10. 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