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Capillary and cellular barriers to ouabain transport in the heart
MICROVASCULAR RESEARCH
7, 84-88 (1974)
Capillary and Cellular Barriers to Ouabain Transport
in the Heart*
WALTERN. DURAN~ AND DAVID L. YUDILEVICH Labaratorio G, Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Clasificador 198, Santiago 1, Chile Received June 12.1973 Serial barriers to blood-tissue transport of [3H] ouabain were studied by means of the indicator dilution method in the isolated, spontaneously beating dog heart perfused at constant blood flow. While [i4C] sucrose served as an indicator of the accessibleinterstitialspace(EVD), [51Cr] hemoglobinwastheintravascularreference. The data were analyzed according to the method of Martin de Julian and Yudilevich (1964). Capillary extraction of ouabain and sucrose were similar and in agreement with their molecular weights. In all the experiments EVD-ouabain was larger (range : 3.0-5.5) than EVD-sucrose. The similarity of the capillary extraction of both molecules indicates that the transport of ouabain across capillary walls occurs by passive diffusion while the difference in EVD is interpreted as the reflection of an uptake mechanism for ouabain operating at thelevel of a second barrier, presumably the myocardial cell membrane.
INTRODUCTION
Much information has been gathered about the mechanisms of action of cardiac glycosides on cardiac muscle as well as on other tissues; however, little is known about blood-tissue transport of these compounds. We report experiments here showing that, in the heart, transcapillary exchange of ouabain is a passive diffusion process whose rate agrees with its molecular size. It is also shown that an,uptake mechanism operates at the level of a second barrier. It is suggested that the myocardial cell membrane is the second barrier observed. MATERIALS
AND METHODS
Four experiments were performed on isolated, spontaneously beating dog hearts, perfused with blood at constant flow (Alvarez and Yudilevich, 1969; Duran, Alvarez and Yudilevich, 1973). Blood of reduced hematocrit (20 per cent) was pumped from a reservoir via the aorta to the coronary bed. Venous outflow, collected by inserting tubing into the right ventricle through the right auricle, was measured with a graduated cylinder and stopwatch. Plasma flow was determined from blood flow and hematocrit. 1 Supported by USPHS, NIH Grant No. HE-05929 and Comision National de Investigation Cientllica y Technologica de Chile, CONICYT, Grant No. 77/68. ’ On leave from Departamento de Fisiologla, Instituto de Ciencias Biologicas, Universidad Catolica de Chile. Present address: Department of Physiology & Pharmacology, Duke University Medical Center,Durham,NorthCarolina27710. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
84
85
OUABAIN TRANSPORT BARRIERS
Heart rate and perfusion pressurewere measuredand recorded. Under the experimental conditions the heart performed no external work. Capillary and cellular transport of ouabain were investigated by the indicator diffusion method (Martin de Julian and Yudilevich, 1964). For this purpose, a mixture of radioactive tracers was injected into the aorta. Collection of the venous effluent (60 samples) was started immediately and continued for a period of about 3 min, depending on the blood flow. Only one injection of tracers was given to each heart preparation. The tracer mixture contained [3H]ouabain, [‘4C]sucrose with [Wrlhemoglobin as nondiffusible reference (Alvarez and Yudilevich, 1969).The concentration of ouabain in the injectate was in the range of 10v7M. Sucrosewas used in this study as an
z -2 -g
loo-
.* .
rxl '; ,! z
*
.
.*
50.
-5
E(O).O.LO . 0
.5
1.0
1.5
t (II/C
211
2.5
3.0
(1)
FIG. 1. Graphical analysis of Martin de Julian and Yudilevich (1964). C(r) and c(t) are the venous concentrations of the intravascular and diffusible tracers relative to the amount injected, respectively, as a function of time. At is the interval between two successive samples. k is the reciprocal of the slope. Plasmaflow:26ml/minlOOg.
indicator of the interstitial spaceavailable to ouabain becauseit diffuses passively across heart capillaries (Alvarez and Yudilevich, 1969)and has a molecular weight close to that of ouabain (342 and 585, respectively). Sodium (Yudilevich and Alvarez, 1967; Ziegler and Goresky, 1971a) and sucrose (Ziegler and Goresky, 1971b) were previously reported to provide an estimate of the accessibleextravascular space during a single passagein the heart. Capillary fractional extraction (E(O)),fractional turnover rate (k), and extravascular distribution volume (EVD) for the diffusible substanceswere calculated from dilution curves (in which the venous concentrations at any time (t) of the diffusible (c(t)) and intravascular (C(t)) tracers were expressed as a fraction of the amount injected). E(O), defined as 1 - lim c(t)/C(t), t + 0, was computed directly from the average of the ratios of c(t) to C(t) in the early samples (1~6-11) since these ratios were relatively constant (Alvarez and Yudilevich, 1969; Duran et al., 1973). Fractional turnover rate was obtained by plotting i [C(t) - c(t)]At/C(t)
(where At is the interval
0
between two successivesamples) against c(t)/C(t) (Martin de Julian and Yudilevich, 1964). On such a plot (Fig. l), k is the reciprocal of the slope, and the intercept is
86
DURAN AND YUDILEVICH
1 - E(0). The extravascular distribution volumes of ouabain and sucrose were calculated from the expression EVD = E(0) F/k, where F is plasma flow. The methods for processing the radioactive samples have been previously published (Yudilevich and Martin de Julian, 1963; Yudilevich and Alvarez, 1967). RESULTS The experimental results obtained in one heart preparation are shown in Fig. 1. E(O)‘s for both molecules are similar indicating that the capillary barrier does not distinguish between the two molecular species. However, k-sucrose is higher than k-ouabain indicating that back-diffusion of ouabain into the blood stream occurs at a slower rate than that of sucrose. These findings were consistently observed in all four heart preparations (Table 1). TABLE 1 CAPILLARY EXTRACTION(E(O)), FRACTIONAL TURNOVER RATE (k, m&l), AND EXTRAVASCULAR DISTRIBUTION VOLUMJZ (EVD, ml/l00 g) STUDIEDIN THE HEART DIJRINGA SINGLEPASSAGE
k
E(O) Experiment no. 18 20 21 30
Heart rate’ 90 86 108 108
Plasma flow’ Ouabain 26 26 18 45
0.32 0.38 0.29 0.32
Sucrose Ouabain 0.40 0.42 0.37 0.25
EVD Sucrose Ouabain Sucrose
0.23 0.27 0.11 0.96
1.41 1.15 0.77 2.20
36.1 36.6 47.4 15.0
7.3 9.4 8.6 5.1
0 Heart rate is given as min-‘; plasma flow is expressed as ml/min 100 g.
From the work of Alvarez and Yudilevich (1969) flow rates that would yield E(O)sucrose in the range of 0.30-0.40 were chosen. E(O)-sucrose, close to prediction, varied from 0.25 to 0.42 while E(O)-ouabain varied from 0.29 to 0.38. In three out of four experiments E(O)-sucrose was slightly higher than E(O)-ouabain. These observations suggest that no special mechanisms for the transport of ouabain exist at the capillary wall. Table 1 lists, besides E(O), the calculated values of k and EVD for sucrose and ouabain. The accessible extravascular volume for sucrose ranged from 5.1 to 9.4 ml/ 100 g. The variation of the size of EVD-sucrose was not immediately related to flow rate. The values for EVD-ouabain varied from 15.0 to 47.4 ml/100 g. Within the limited number of observations, an inverse correlation of EVD-ouabain with flow was found in this series of experiments; however, more experimental data are certainly needed to evaluate more properly this relationship. Despite the fall with increasing flow rate EVD-ouabain was larger than EVD-sucrose in each heart preparation. DISCUSSION The possibility of studying serial barriers to blood-tissue transport in various organs, particularly in the heart, by means of the single-injection indicator dilution technique
OUABAIN TRANSPORT BARRIERS
87
has already been pointed out (Duran and Yudilevich, 1969; Yudilevich, 1970). The characteristics of the capillary barrier are mainly reflected in the early part of the dilution curves whereas those of deeper diffusion barriers affect the latter part. The use of substancesrestricted to the interstitial space,in addition to the intravascular tracer and other test molecules, increasesthe potentiality of this methodological approach. The close correlation between E(O)-ouabain and E(O)-sucroseshows that the passage of ouabain across capillary walls occurs by passive diffusion. In fact, little distinction between ouabain (MW 585) and sucrose (MW 342) is made at the capillary wall. This finding is in agreement with the correlation between E(0) and free-diffusion in water coefficients shown for several molecules in the heart (Alvarez and Yudilevich, 1969). E(0) also shows that ouabain was not bound to plasma proteins in our experimental conditions. Our results demonstrate that a distinction between ouabain and sucrose is made beyond the capillary wall. Inasmuch as the sameaccessibleinterstitial volume, measured by sucrose, is available to both molecular species the EVD-ouabain/EVD-sucrose ratio (average 4.3) indicates that a cellular uptake mechanism is available to ouabain but not to sucrose. Our methods do not distinguish whether ouabain binds to specific receptors on the myocardial cell membrane (Kuschinsky, Ltillmann, and van Zwieten, 1968) or is transported to intracellular binding sites (Conrad and Baxter, 1964). The average accessibleextravascular volume of distribution calculated for sucrose in these experiments (7.6 ml/100 g) is in accordance with the value of 8.4 ml/100 g reported by Ziegler and Goresky (1971b). Theseauthors also found values for EVD-Na and EVD-sulfate comparable to those of sucrose (Ziegler and Goresky, 1971a, b). Yudilevich and Alvarez (1967) reported larger extravascular volumes for sodium (average 17.2ml/ 100g). Yudilevich and Alvarez (1967), in experiments done on separate hearts, and Ziegler and Goresky (1971a), performing several runs in the same heart, noted that EVD was relatively independent of flow. This finding is confirmed, over a narrow range and in different hearts, for sucrose in the present observations. The agreement of the values for EVD-sucrose reported in the literature by use of the same technique and the relative flow independence of the calculated accessibleextravascular space support the adequacy of using the single-injection indicator dilution technique for the study of diffusion barriers located beyond the capillary wall. The identification of the accessible extravascular space for sucrose (or sodium) and the true interstitial spaceas well as other possible limitations of the method, however, still need further investigation. ACKNOWLEDGMENTS The authors are grateful to Dr. E. M. Renkin for his helpful criticisms and suggestions. The excellent technical assistance of Eddie Gonzalez, Carlos Heldt, Humberto Letelier, Carlos Lizana, Jorge Mendez, and Fernando Rodriguez is acknowledged. REFERENCES ALVAREZ, 0. A., AND YUDILEVICH,D. L. (1969). Heart capillary permeability to lipid-insoluble molecules. J. Physiol. London202,45-58. CONRAD,L. L., AND BAXTER,D. J. (1964). Intracellular distribution-of digoxin-H3 in the hearts of rats and dogs demonstrated by autoradiography and its relationship to changes in myocardial contractile force. J. Pharmacol. Exp. Ther. 145210-214.
88
DURkN AND YUDILEVICH
DURAN, W. N., AND YUDILEVICH,D. L. (1969). Capillary and cellular transport of rubidium, glucose, sucrose, and ouabain in the dog heart. XII Annual Meeting, Sociedad de Biologla de Chile, Panimavida, Chile. (Abstract). DURAN, W. N., ALVAREZ,0. A., AND YUDILEVICH,D. L. (1973). Influence of maximal vasodilation on glucose and sodium blood-tissue transport in canine heart. Microvasc. Res. 6,347-359. KUSCHINSKY,K., L~~LLMANN, H., ANDVANZWIETEN,P. A. (1968). A comparison of the accumulation and release of JH-ouabain and 3H-digoxin by guinea pig heart muscle. Brit. J. Pharmacol. 32,598608. MARTINDEJULIAN,P., AND YUDILEVICH,D. L. (1964). A theory for the quantification of transcapillary exchange by tracer dilutioncurves. Amer. J. Physiol. 207,162-168. YUDILEVICH,D. L. (1970). Serial barriers to blood-tissue transport studied by single injection indicator diffusion technique. In “Capillary Permeability” (C. Crone and N. A. Lassen, eds.), pp. 115-128. Munksgaard, Copenhagen. YUDILEVICH,D. L., ANDALVAREZ,0. A. (1967). Water, sodium, and thiourca transcapillary diffusion in the dog heart. Amer. J. Physiol. 213,308-314. YUDILEVICH,D. L., AND MARTINDE JULIAN, P. (1963). A method of processing data when several mixtures of elements are resolved in their components. Applications to mixtures of Fesg, Na2* and I131. Znt. J. Appl. Radiat. Zsotop. 14,563-570. ZIEGLER,W. H., AND GORESKY,C. A. (1971a). Transcapillary exchange in the working left ventricle of the dog. Circ. Res. 29,181-207. ZIEGLER,W. H., AND GORESKY,C. A. (1971b). Kinetics of rubidium uptake in the working dog heart. Circ. Res. 29,208-220.
7, 84-88 (1974)
Capillary and Cellular Barriers to Ouabain Transport
in the Heart*
WALTERN. DURAN~ AND DAVID L. YUDILEVICH Labaratorio G, Departamento de Biologia, Facultad de Ciencias, Universidad de Chile, Clasificador 198, Santiago 1, Chile Received June 12.1973 Serial barriers to blood-tissue transport of [3H] ouabain were studied by means of the indicator dilution method in the isolated, spontaneously beating dog heart perfused at constant blood flow. While [i4C] sucrose served as an indicator of the accessibleinterstitialspace(EVD), [51Cr] hemoglobinwastheintravascularreference. The data were analyzed according to the method of Martin de Julian and Yudilevich (1964). Capillary extraction of ouabain and sucrose were similar and in agreement with their molecular weights. In all the experiments EVD-ouabain was larger (range : 3.0-5.5) than EVD-sucrose. The similarity of the capillary extraction of both molecules indicates that the transport of ouabain across capillary walls occurs by passive diffusion while the difference in EVD is interpreted as the reflection of an uptake mechanism for ouabain operating at thelevel of a second barrier, presumably the myocardial cell membrane.
INTRODUCTION
Much information has been gathered about the mechanisms of action of cardiac glycosides on cardiac muscle as well as on other tissues; however, little is known about blood-tissue transport of these compounds. We report experiments here showing that, in the heart, transcapillary exchange of ouabain is a passive diffusion process whose rate agrees with its molecular size. It is also shown that an,uptake mechanism operates at the level of a second barrier. It is suggested that the myocardial cell membrane is the second barrier observed. MATERIALS
AND METHODS
Four experiments were performed on isolated, spontaneously beating dog hearts, perfused with blood at constant flow (Alvarez and Yudilevich, 1969; Duran, Alvarez and Yudilevich, 1973). Blood of reduced hematocrit (20 per cent) was pumped from a reservoir via the aorta to the coronary bed. Venous outflow, collected by inserting tubing into the right ventricle through the right auricle, was measured with a graduated cylinder and stopwatch. Plasma flow was determined from blood flow and hematocrit. 1 Supported by USPHS, NIH Grant No. HE-05929 and Comision National de Investigation Cientllica y Technologica de Chile, CONICYT, Grant No. 77/68. ’ On leave from Departamento de Fisiologla, Instituto de Ciencias Biologicas, Universidad Catolica de Chile. Present address: Department of Physiology & Pharmacology, Duke University Medical Center,Durham,NorthCarolina27710. Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
84
85
OUABAIN TRANSPORT BARRIERS
Heart rate and perfusion pressurewere measuredand recorded. Under the experimental conditions the heart performed no external work. Capillary and cellular transport of ouabain were investigated by the indicator diffusion method (Martin de Julian and Yudilevich, 1964). For this purpose, a mixture of radioactive tracers was injected into the aorta. Collection of the venous effluent (60 samples) was started immediately and continued for a period of about 3 min, depending on the blood flow. Only one injection of tracers was given to each heart preparation. The tracer mixture contained [3H]ouabain, [‘4C]sucrose with [Wrlhemoglobin as nondiffusible reference (Alvarez and Yudilevich, 1969).The concentration of ouabain in the injectate was in the range of 10v7M. Sucrosewas used in this study as an
z -2 -g
loo-
.* .
rxl '; ,! z
*
.
.*
50.
-5
E(O).O.LO . 0
.5
1.0
1.5
t (II/C
211
2.5
3.0
(1)
FIG. 1. Graphical analysis of Martin de Julian and Yudilevich (1964). C(r) and c(t) are the venous concentrations of the intravascular and diffusible tracers relative to the amount injected, respectively, as a function of time. At is the interval between two successive samples. k is the reciprocal of the slope. Plasmaflow:26ml/minlOOg.
indicator of the interstitial spaceavailable to ouabain becauseit diffuses passively across heart capillaries (Alvarez and Yudilevich, 1969)and has a molecular weight close to that of ouabain (342 and 585, respectively). Sodium (Yudilevich and Alvarez, 1967; Ziegler and Goresky, 1971a) and sucrose (Ziegler and Goresky, 1971b) were previously reported to provide an estimate of the accessibleextravascular space during a single passagein the heart. Capillary fractional extraction (E(O)),fractional turnover rate (k), and extravascular distribution volume (EVD) for the diffusible substanceswere calculated from dilution curves (in which the venous concentrations at any time (t) of the diffusible (c(t)) and intravascular (C(t)) tracers were expressed as a fraction of the amount injected). E(O), defined as 1 - lim c(t)/C(t), t + 0, was computed directly from the average of the ratios of c(t) to C(t) in the early samples (1~6-11) since these ratios were relatively constant (Alvarez and Yudilevich, 1969; Duran et al., 1973). Fractional turnover rate was obtained by plotting i [C(t) - c(t)]At/C(t)
(where At is the interval
0
between two successivesamples) against c(t)/C(t) (Martin de Julian and Yudilevich, 1964). On such a plot (Fig. l), k is the reciprocal of the slope, and the intercept is
86
DURAN AND YUDILEVICH
1 - E(0). The extravascular distribution volumes of ouabain and sucrose were calculated from the expression EVD = E(0) F/k, where F is plasma flow. The methods for processing the radioactive samples have been previously published (Yudilevich and Martin de Julian, 1963; Yudilevich and Alvarez, 1967). RESULTS The experimental results obtained in one heart preparation are shown in Fig. 1. E(O)‘s for both molecules are similar indicating that the capillary barrier does not distinguish between the two molecular species. However, k-sucrose is higher than k-ouabain indicating that back-diffusion of ouabain into the blood stream occurs at a slower rate than that of sucrose. These findings were consistently observed in all four heart preparations (Table 1). TABLE 1 CAPILLARY EXTRACTION(E(O)), FRACTIONAL TURNOVER RATE (k, m&l), AND EXTRAVASCULAR DISTRIBUTION VOLUMJZ (EVD, ml/l00 g) STUDIEDIN THE HEART DIJRINGA SINGLEPASSAGE
k
E(O) Experiment no. 18 20 21 30
Heart rate’ 90 86 108 108
Plasma flow’ Ouabain 26 26 18 45
0.32 0.38 0.29 0.32
Sucrose Ouabain 0.40 0.42 0.37 0.25
EVD Sucrose Ouabain Sucrose
0.23 0.27 0.11 0.96
1.41 1.15 0.77 2.20
36.1 36.6 47.4 15.0
7.3 9.4 8.6 5.1
0 Heart rate is given as min-‘; plasma flow is expressed as ml/min 100 g.
From the work of Alvarez and Yudilevich (1969) flow rates that would yield E(O)sucrose in the range of 0.30-0.40 were chosen. E(O)-sucrose, close to prediction, varied from 0.25 to 0.42 while E(O)-ouabain varied from 0.29 to 0.38. In three out of four experiments E(O)-sucrose was slightly higher than E(O)-ouabain. These observations suggest that no special mechanisms for the transport of ouabain exist at the capillary wall. Table 1 lists, besides E(O), the calculated values of k and EVD for sucrose and ouabain. The accessible extravascular volume for sucrose ranged from 5.1 to 9.4 ml/ 100 g. The variation of the size of EVD-sucrose was not immediately related to flow rate. The values for EVD-ouabain varied from 15.0 to 47.4 ml/100 g. Within the limited number of observations, an inverse correlation of EVD-ouabain with flow was found in this series of experiments; however, more experimental data are certainly needed to evaluate more properly this relationship. Despite the fall with increasing flow rate EVD-ouabain was larger than EVD-sucrose in each heart preparation. DISCUSSION The possibility of studying serial barriers to blood-tissue transport in various organs, particularly in the heart, by means of the single-injection indicator dilution technique
OUABAIN TRANSPORT BARRIERS
87
has already been pointed out (Duran and Yudilevich, 1969; Yudilevich, 1970). The characteristics of the capillary barrier are mainly reflected in the early part of the dilution curves whereas those of deeper diffusion barriers affect the latter part. The use of substancesrestricted to the interstitial space,in addition to the intravascular tracer and other test molecules, increasesthe potentiality of this methodological approach. The close correlation between E(O)-ouabain and E(O)-sucroseshows that the passage of ouabain across capillary walls occurs by passive diffusion. In fact, little distinction between ouabain (MW 585) and sucrose (MW 342) is made at the capillary wall. This finding is in agreement with the correlation between E(0) and free-diffusion in water coefficients shown for several molecules in the heart (Alvarez and Yudilevich, 1969). E(0) also shows that ouabain was not bound to plasma proteins in our experimental conditions. Our results demonstrate that a distinction between ouabain and sucrose is made beyond the capillary wall. Inasmuch as the sameaccessibleinterstitial volume, measured by sucrose, is available to both molecular species the EVD-ouabain/EVD-sucrose ratio (average 4.3) indicates that a cellular uptake mechanism is available to ouabain but not to sucrose. Our methods do not distinguish whether ouabain binds to specific receptors on the myocardial cell membrane (Kuschinsky, Ltillmann, and van Zwieten, 1968) or is transported to intracellular binding sites (Conrad and Baxter, 1964). The average accessibleextravascular volume of distribution calculated for sucrose in these experiments (7.6 ml/100 g) is in accordance with the value of 8.4 ml/100 g reported by Ziegler and Goresky (1971b). Theseauthors also found values for EVD-Na and EVD-sulfate comparable to those of sucrose (Ziegler and Goresky, 1971a, b). Yudilevich and Alvarez (1967) reported larger extravascular volumes for sodium (average 17.2ml/ 100g). Yudilevich and Alvarez (1967), in experiments done on separate hearts, and Ziegler and Goresky (1971a), performing several runs in the same heart, noted that EVD was relatively independent of flow. This finding is confirmed, over a narrow range and in different hearts, for sucrose in the present observations. The agreement of the values for EVD-sucrose reported in the literature by use of the same technique and the relative flow independence of the calculated accessibleextravascular space support the adequacy of using the single-injection indicator dilution technique for the study of diffusion barriers located beyond the capillary wall. The identification of the accessible extravascular space for sucrose (or sodium) and the true interstitial spaceas well as other possible limitations of the method, however, still need further investigation. ACKNOWLEDGMENTS The authors are grateful to Dr. E. M. Renkin for his helpful criticisms and suggestions. The excellent technical assistance of Eddie Gonzalez, Carlos Heldt, Humberto Letelier, Carlos Lizana, Jorge Mendez, and Fernando Rodriguez is acknowledged. REFERENCES ALVAREZ, 0. A., AND YUDILEVICH,D. L. (1969). Heart capillary permeability to lipid-insoluble molecules. J. Physiol. London202,45-58. CONRAD,L. L., AND BAXTER,D. J. (1964). Intracellular distribution-of digoxin-H3 in the hearts of rats and dogs demonstrated by autoradiography and its relationship to changes in myocardial contractile force. J. Pharmacol. Exp. Ther. 145210-214.
88
DURkN AND YUDILEVICH
DURAN, W. N., AND YUDILEVICH,D. L. (1969). Capillary and cellular transport of rubidium, glucose, sucrose, and ouabain in the dog heart. XII Annual Meeting, Sociedad de Biologla de Chile, Panimavida, Chile. (Abstract). DURAN, W. N., ALVAREZ,0. A., AND YUDILEVICH,D. L. (1973). Influence of maximal vasodilation on glucose and sodium blood-tissue transport in canine heart. Microvasc. Res. 6,347-359. KUSCHINSKY,K., L~~LLMANN, H., ANDVANZWIETEN,P. A. (1968). A comparison of the accumulation and release of JH-ouabain and 3H-digoxin by guinea pig heart muscle. Brit. J. Pharmacol. 32,598608. MARTINDEJULIAN,P., AND YUDILEVICH,D. L. (1964). A theory for the quantification of transcapillary exchange by tracer dilutioncurves. Amer. J. Physiol. 207,162-168. YUDILEVICH,D. L. (1970). Serial barriers to blood-tissue transport studied by single injection indicator diffusion technique. In “Capillary Permeability” (C. Crone and N. A. Lassen, eds.), pp. 115-128. Munksgaard, Copenhagen. YUDILEVICH,D. L., ANDALVAREZ,0. A. (1967). Water, sodium, and thiourca transcapillary diffusion in the dog heart. Amer. J. Physiol. 213,308-314. YUDILEVICH,D. L., AND MARTINDE JULIAN, P. (1963). A method of processing data when several mixtures of elements are resolved in their components. Applications to mixtures of Fesg, Na2* and I131. Znt. J. Appl. Radiat. Zsotop. 14,563-570. ZIEGLER,W. H., AND GORESKY,C. A. (1971a). Transcapillary exchange in the working left ventricle of the dog. Circ. Res. 29,181-207. ZIEGLER,W. H., AND GORESKY,C. A. (1971b). Kinetics of rubidium uptake in the working dog heart. Circ. Res. 29,208-220.