Uptake of materials by the intact liver. The exchange of glucose across the cell membranes.

D-Glucose equilibrates within liver cells. We have studied its process of entry into and exit from these cells with the multiple indicator dilution technique. Labeled red cells (a vascular indicator), labeled sucrose (an extracellular reference), and labeled D-glucose were rapidly injected into the portal vein, and from serially sampled hepatic venous blood, normalized outflow-time patterns were obtained. The labeled red cell curve rises to an early high peak, and decays rapidly; and that for sucrose reaches a later and lower peak and decays less rapidly, but generates an equivalent area. The curve for labeled D-glucose begins with that for labeled sucrose, gradually rises to a peak which is later and substantially lower than that for sucrose, and then decreases slowly. At high glucose levels this curve assumes a squared-off shape, rises fairly quickly to its highest level, at the time of the sucrose peak, and then slowly decreases. Phlorizin and galactose infusion result in the emergence of a pronounced early peak, under the sucrose peak; and the curve for tracer L-glucose approaches that for sucrose. We resolve from the D-glucose curves, by model analysis, two components: throughout material, which has not entered the cells; and exchanging material, which has entered and later returned to the circulation. The analysis provides estimates of the kinetic entrance and exist coefficients; and from these, saturation of both the entrance and exit processes was evident. The characteristic transport parameters were determined. For both entrance and exit, a common Km, 2,170 mg/100 ml, and transport maximum, 5.13 mg s-1 (ml intracellular fluid)-1, were found. Both these values are exceedingly large. Several other phenomena were defined which additionally characterize the transport process: phlorizin and galacose produced competitive inhibition; the transport process was found to be relatively stereospecific; and sudden infusion of hypertonic glucose produced counter-transport of labeled D-glucose.

On the uptake of materials by the intact liver. The concentrative transport of rubidium-86.

In this study we use the multiple indicator dilution technique to outline the kinetic mechanisms underlying the uptake of rubidium, a cation which, in the steady state, is concentrated by hepatic parenchymal cells. We inject a mixture of (51)Cr-labeled red blood cells (a vascular reference substance), (22)Na (which is confined to the extracellular space, the expected extravascular distribution space for rubidium, in the absence of cellular uptake), and (86)Rb into the portal vein and obtain normalized outflow patterns, expressed as outflowing fractions of each injected mass per milliliter vs. time. The labeled red cell curve rises to the highest and earliest peak and decays rapidly. That for labeled sodium rises to a later and lower peak, and decays less rapidly. Its extrapolated recovery is equal to that for the red cells. The observed (86)Rb curve consists of two parts: an early clearly defined peak of reduced area, related to the (22)Na peak in timing; and a later tailing, obscured by recirculation, so that total outflow recovery cannot be defined (even though it would be expected to be the same). We model the concentrative uptake of (86)Rb and find two corresponding outflow fractions: throughput material, which sweeps past the cell surface as a wave delayed with respect to the vascular reference (tracer which has not entered cells); and exchanging material (tracer which has entered cells and later returns to the circulation). We find that the outflow form of the rubidium curve, the presence of both a relatively clearly defined throughput component and a relatively prolonged low-in-magnitude tailing, is consequent to the concentrative character of the transport mechanism, to the presence of an influx rate constant many times the efflux rate constant. The modeling which we develop is general, and has potential application in situations where transport is nonconcentrative.

On the uptake of materials by the intact liver. The transport and net removal of galactose.

D-galactose, a monosaccharide rapidly phosphorylated within liver cells, is irreversibly removed from the portal circulation. We have studied the kinetic relations between the hepatic cell entry process and the metabolic sequestration process, by means of the multiple indicator dilution technique. Labeled red blood cells (a vascular indicator), labeled sucrose (an extracellular reference), and labeled galactose were rapidly injected into the portal vein, and from rapidly sampled hepatic venous blood, normalized outflow-time patterns were secured. The labeled red cell curve rises to the highest and earliest peak, and decays rapidly; and that for labeled sucrose rises to a later and lower peak. Its extrapolated recovery is equivalent to that of the labeled red cells. At low blood galactose concentrations, the labeled galactose appears at the outflow with labeled sucrose, but is much reduced in magnitude, and exhibits a long tailing. Its outflow recovery is much reduced. At high blood galactose concentrations, the initial part of the profile increases towards that for labeled sucrose, the tailing becomes much larger in magnitude, and the outflow recovery becomes virtually complete. We have modeled the uptake of labeled galactose, and find two parts to the predicted outflow pattern, corresponding to our experimental observations; throughput material, which sweeps past the cell surface in the extracellular space; and returning material, which has entered the cells but escaped the sequestration process. Analysis of the data by use of this model provides estimates of both transmembrane fluxes and rates of sequestration. The capacity of the process subserving cell entry is found to be 40 times that for phosphorylation; and, whereas the K(m) value for sequestration is less than 15 mg/100 ml, that for entry is approximately 500 mg/100 ml. Both processes are relatively stereospecific; the entry of the L-stereoisomer is very slow and it undergoes no significant amount of metabolic sequestration. The sequestration process produces a lobular intracellular concentration gradient; and this gradient, in turn, produces some uncertainty in the estimate of the true K(m) value for the sequestration process.

Indicator dilution measurements of extravascular water in the lungs.

Multiple indicator dilution studies of the pulmonary circulation were carried out in conscious, resting and exercising, and anesthetized dogs under conditions where there was no pulmonary edema. Labeled red cells, water, and albumin were injected together into the pulmonary artery, and effluent dilution patterns were obtained from the descending thoracic aorta. The product of the mean transit time differences between labeled water and red cells, and the pulmonary water flow was used to estimate extravascular parenchymatous water; and this was expressed as a proportion of the water content of the blood-drained lung at postmortem examination. These estimates of the proportional water content were found to increase with flow, and to approach an asymptotic value. Reconsideration of the flow patterns in capillaries, however, led to the postulate that extravascular water should be calculated, utilizing as the appropriate vascular reference a substance that uniformly labels the water in red cells and plasma, and which is confined to the circulation, rather than a tracer that only labels red cells. The mean transit time of this substance is approximated by the sum of the mean transit times of labeled red cells and albumin, each weighted according to the proportion of the water content of blood present in that phase. The values for lung water content so computed also increased with flow, and appeared to approach an asymptote that corresponded to approximately two-thirds of the wet lung weight. The estimated values for the water space after pentobarbital anesthesia corresponded to the lower values obtained in the resting conscious animals. When the anesthetized animals were also bled, the estimated water space was disproportionately large, in relation to the previous values. These experimental results support the hypothesis that dilutional estimates of the lung water space reflect pulmonary capillary filling; that this filling increases with exercise; and that a relative increase in filling also occurs as part of the response to hemorrhage.

Assessment of liver microcirculation in human cirrhosis.

Alterations in the liver microcirculation were characterized by use of the multiple-indicator dilution technique in 25 cirrhotic patients undergoing hemodynamic evaluation of portal hypertension. Hepatic vein outflow dilution curves were obtained after portal vein or hepatic artery injections of a vascular reference substance (labeled erythrocytes) and of diffusible substances (labeled albumin and sucrose). In 23 of these patients (19 with alcoholic cirrhosis and 4 with postnecrotic cirrhosis), unimodal erythrocytes and albumin curves were obtained; the immediately accessible albumin space ranged from normal values (that were substantially larger than the erythrocyte space) to low values (that were little larger than the erythrocyte space). In parallel with this, the hepatic extraction of indocyanine green decreased and was correlated with the albumin space (r = 0.821, P less than 0.001). The form of labeled sucrose curves showed progressive changes indicating limited diffusion into the interstitial space. In contrast, bimodal curves were found in two patients (with macronodular cirrhosis); a large proportion of all labels appeared simultaneously in the early part of the outflow curves. Model analysis of the unimodal data indicated that the spectrum of findings could best be explained by progressive development of a barrier to exchange by progressive capillarization of the microvascular bed, and the form of the bimodal data suggested that large vessel shunting was occurring. Both changes, in turn, will contribute to the reduced extraction of protein-bound materials in cirrhosis.

Plasma expansion effect on cardiac capillary and adrenergic exchange in intact dogs.

The effect of plasma volume expansion on transcapillary exchange and norepinephrine release in the heart was examined in pentobarbital sodium-anesthetized dogs by use of the multiple indicator-dilution technique. Animals were studied under basal conditions and following infusion of the plasma expander, dextran. Catheters were placed in coronary artery and coronary sinus in a closed-chest preparation. Labeled albumin, sucrose, and norepinephrine were injected into the coronary artery and outflow-dilution curves were secured. Analysis of these provided parameters reflecting coronary flow and permeability-surface product, and a norepinephrine tracer kinetic-bulk model provided simultaneous estimates of the rate of norepinephrine release into the myocardial interstitial space. The infusion of dextran resulted in a large increase in coronary flow without significant changes in myocardial norepinephrine release; at the same time the permeability-surface product values increased, amplifying the capacity of the higher flow to deliver substrates to sarcolemmal cells. The findings indicate that plasma volume expansion increases transcapillary exchange in the heart without activating the cardiac sympathetic system.

Pulmonary clearance of circulating endothelin-1 in dogs in vivo: exclusive role of ETB receptors.

The pulmonary circulation plays an important role in the removal of circulating endothelin-1 (ET-1). Plasma ET-1 levels are increased in pulmonary hypertensive states of various etiologies (e.g., idiopathic, heart failure, and congenital anomalies) in proportion to the severity of pulmonary hypertension. It is possible that reduced pulmonary clearance of this peptide contributes to the hyperendothelinemia of those pathologies. The ETA and ETB receptors are abundant in lung tissues: on the vascular endothelium, the ETB receptor is predominant and may contribute to ET-1 extraction through receptor-mediated endocytosis. We designed experiments to determine and quantify the importance of the ETA and ETB receptors in the pulmonary extraction of circulating ET-1 in anesthetized dogs. The single-pass cumulative tracer ET-1 extraction by the lung was measured with the indicator-dilution technique before and 5 min after intrapulmonary injection of the specific ETA antagonist BQ-123 (n = 5, 120-960 nmol) and the specific ETB antagonist BQ-788 (n = 6, 1,000 nmol). The inhibitors had no significant effect on pulmonary and systemic hemodynamics. Mean cumulative pulmonary ET-1 extraction was not modified by BQ-123 [control (C): 36 +/- 4%, antagonist (A): 34 +/- 6%] but was completely abolished by BQ-788 (C: 34 +/- 6%, A: 0 +/- 2%, P < 0.001). The pulmonary rate constant (K) for ET-1 removal was also unaffected by BQ-123 (C: 0.050 +/- 0.0085 s-1, A: 0.047 +/- 0.012 s-1) but significantly decreased and became close to zero after BQ-788 (C: 0.058 +/- 0.014 s-1, A: 0.009 +/- 0.007 s-1, P < 0.1). We conclude that the ETB receptor is completely and exclusively responsible for pulmonary ET-1 removal in vivo. Future studies are needed to show whether desensitization or downregulation of the ETB receptor may contribute to the increase in circulating ET-1 levels in conditions associated with pulmonary hypertension. This novel pulmonary endothelial cell function may play a protective role by modulating circulating ET-1 levels in the systemic circulation.

Pulmonary removal and production of endothelin in the anesthetized dog.

The single-bolus multiple-indicator-dilution technique was used to evaluate pulmonary removal of tracer 125I-labeled endothelin-1 in seven anesthetized dogs. Simultaneously, pulmonary arterial and aortic blood samples were obtained and assayed to determine the levels of immunoreactive endothelin-1. When 125I-endothelin-1 was compared with a plasma vascular reference (Evans blue dye), there was a single passage mean extraction of 31 +/- 8%. In contrast, there was no significant difference between immunoreactive endothelin-1 levels measured in blood samples from the pulmonary artery and the aorta (1.26 +/- 0.58 and 1.37 +/- 0.50 pg/ml, respectively; P = 0.47). The absence of an arteriovenous difference for bulk endothelin-1 across the lungs in the presence of tracer data indicating a substantial uptake implies that an amount of endothelin-1 quantitatively more or less equal to that removed is produced by the lung. The shapes of the dilution curves suggest that the tracer endothelin uptake by the lung is a one-way process without vascular reentry of tracer. We conclude that the dog lung is an important site for both uptake and release of endothelin-1.

Microsphere and dilution measurements of flow and interstitial space in dog heart.

The effects of coronary flow on cardiac capillary permeability-surface area products and interstitial spaces were examined at rest and after hemodilution in the canine heart. Multiple-indicator-dilution experiments and left atrial injections of microspheres were carried out in closed-chest anesthetized animals at rest and after plasma expansion with dextran. Plasma expansion was utilized to produce a large increase in coronary perfusion compared with control conditions. Values for plasma flow per unit interstitial space, derived from analysis of the indicator-dilution data, were found to correlate closely with average vascular plasma flow per gram, calculated from the cardiac microsphere data; the one reflects the other. With an increase in flow, cardiac capillary permeability-surface area product values were found to increase substantially, whereas the average sucrose extravascular or cardiac interstitial spaces remained stable. Consequently the dilution parameter, flow per unit interstitial space, which is independent of tracer loss, provided a good reflection of flow per weight of tissue in the heart, without the additional requirement for a flow probe.

Pulmonary angiotensin-converting enzyme substrate hydrolysis during exercise.

We examined exercise-induced changes in indicator-dilution estimates of the angiotensin-converting enzyme first-order kinetic parameter, the ratio of a normalized maximal enzymatic conversion rate to the Michaelis constant (Amax/Km), which, under stable enzymatic conditions, will vary with the pulmonary vascular surface area accessible to vascular substrate, the extravascular lung water (an index of the proportion of lung tissue perfused), and the central blood volume (from pulmonary trunk to aorta). Experiments were performed in 10 mongrel dogs at rest and through two increasing levels of treadmill exercise, with the use of two vascular space tracers (labeled erythrocytes and albumin), a water space tracer ([1,8-14C]-octanediol), and a vascular endothelium surface area marker, benzoyl-Phe-Gly-Pro ([3H]BPGP), which is a pharmacologically inactive angiotensin-converting enzyme substrate. The exercise-induced increase in cardiac output was accompanied by a linear increase in central blood volume, and dilutional extravascular lung water rapidly increased to an asymptotic proportion close to 100% of postmortem vascular lung water. There was an average 55% [3H]BPGP hydrolysis, which did not vary with flow, and the computed Amax/Km increased linearly with exercise. We conclude that exercise results in complete lung tissue recruitment and increases the pulmonary vascular surface area available for BPGP hydrolysis linearly with flow, so that pulmonary vascular recruitment continues after full tissue recruitment.

Kinetics of pulmonary uptake of serotonin during exercise in dogs.

The multiple indicator-dilution technique was employed in the exercising dog to evaluate the effect of increasing activity on the pulmonary extraction and kinetics of removal of tracer 3H-labeled serotonin (5-HT) and on the measured central blood volume and tracer-accessible extravascular lung water. 51Cr-labeled red blood cells, 125I-labeled albumin, and 14C-labeled 1,8-octanediol were injected with labeled 5-HT at rest and at two increasing levels of exercise (lower and higher in 9 dogs). Blood flow approximately tripled at the highest level of exercise, and the central blood volume increased linearly with increasing blood flow. The tracer-accessible extravascular lung water increased in the transition from rest to low-level exercise and stabilized at an average proportion of 0.85 of the gravimetric extravascular lung water at the higher values of blood flow. The average labeled 5-HT extraction at rest was 42 +/- 11%, and this slowly decreased with increase in flow. The calculated permeability-surface area product for 5-HT increased approximately directly with increasing blood flow. We conclude that exercise results in an increase in the central blood volume that is accompanied by an increase in the tracer-accessible extravascular lung water (lung tissue recruitment) over low exercise levels, with no change at higher levels of exercise, and that the pulmonary capillary surface area subserving 5-HT uptake increases almost linearly with flow over the range of flows attained.

Effects of flow, perfusion pressure, and oxygen consumption on cardiac capillary exchange.

The roles of blood flow, local oxygen consumption, and perfusion pressure on cardiac transcapillary exchange were characterized in closed-chest anesthetized dogs by use of the multiple-indicator dilution technique. Occlusion of the carotid arteries or injection of dipyridamole increased coronary flow to significantly higher values compared with a group of animals in a basal state obtained in a previous study. Carotid occlusion resulted in a significant increase in perfusion pressure and myocardial oxygen consumption, whereas these two variables were significantly reduced after dipyridamole. For the whole group of animals, the capillary permeability-surface area product for sucrose increased with coronary flow, which appeared to be the important controller for this microcirculatory exchange parameter. Perfusion pressure and myocardial oxygen consumption also regulated permeability-surface area product values, although to a lesser extent than flow. The heterogeneity of transit times in the capillaries was reduced at high coronary flow values, despite large differences in the cardiac utilization of oxygen. The data suggest that cardiac capillary exchange responds mostly to hemodynamic changes originating at the precapillary level.

Use of norepinephrine uptake to measure lung capillary recruitment with exercise.

We used the multiple indicator-dilution technique with norepinephrine, a vascular endothelium surface marker, to study the pulmonary vascular changes in awake exercising dogs. The vascular space tracers, labeled erythrocytes and albumin, and a water space tracer, 1,8-octanediol, were injected with the norepinephrine, and right atrium-aortic root dilution curves were obtained in nine dogs, at rest and at two increasing levels of exercise. Extravascular lung water multiple tracer dilutional estimates increased with flow and rapidly approached a maximal asymptotic value representing 75% of the postmortem lung weight. The ratio of the extravascular lung water measured in this way to that measured gravimetrically also increased, to reach an asymptotic proportion of close to 100%. The transit time-defined central vascular space increased linearly with flow; the ratio of lung tissue space to lung vascular space, therefore, decreased with increasing flow. The mean tracer upslope norepinephrine extractions at rest and at the two levels of exercise were 17 +/- 1.2, 14 +/- 0.8, and 15 +/- 0.8% (SE). With the use of the Crone approximation, we computed permeability-surface area products for norepinephrine; these increased linearly with flow. If permeability does not change, the increase in the permeability-surface area product with flow can be attributed to capillary recruitment. We conclude that when all lung tissue has become accessible to 1,8-octanediol delivered via the perfused vascular space, there is nevertheless further recruitment, with increase in flow, of vascular surface that can extract norepinephrine.

Transport, binding, and metabolism of sulfate conjugates in the liver.

Sulfate conjugates are a heterogeneous class of polar, anionic metabolites that result from the conjugation of endogenous and exogenous compounds. Sulfate conjugates exhibit a high degree of binding to albumin, the extent of which usually exceeds those of their parent compounds. Preponderant direct and indirect evidence suggests that sulfation activity is slightly higher in the periportal than in the perivenous (centrilobular) region of the liver, but recent immunohistochemical studies imply that specific isoforms of the sulfotransferases may also be preferentially localized in the perivenous region. Entry of sulfate conjugates into the liver cell is poor unless discrete carriers are present. Although known transport carriers exist for the sulfated bile acids, the specificity of the carriers for drug sulfate conjugates is presently unknown. The removal of sulfates is usually by way of biliary excretion while, on occasion, sulfates can be desulfated and participate in futile cycling with their parent compounds. The binding, transport, and hepatic elimination of various drug sulfate conjugates are examined. Non-recirculating studies carried out in the perfused rat liver with the multiple indicator dilution technique under varying input sulfate conjugate concentrations have provided essential information on the effects of vascular (red blood cells and plasma protein) binding on transport and removal of the conjugates. These studies clearly demonstrate the need to study protein binding, transmembrane transfer characteristics across the liver basolateral (sinusoidal) and canalicular membranes, and enzyme zonation in a distributed-in-space fashion in order to properly define the handling of sulfate conjugates in the liver.

The 1994 G. Malcolm Brown Lecture. Biological barriers and medicine.

Career support provides the essential element needed for the detailed development of thematic areas of inquiry. Dr Goresky outlines how career support provided for him the substratum for the investigation and definition of tracer exchange processes in the liver. Classical interstitial substances (labeled albumin, inulin, sucrose, and sodium) and labeled water were found to undergo flow limited distribution in the liver. An excluded volume phenomenon created systematic variation in accessible interstitial space (that is, Disse space) values, smaller for larger molecular weight substances. Labeled rubidium entered liver cells in a concentrative fashion (return from cells was very small, early in time). Labeled glucose liver cell entry was found to exhibit the characteristics of a carrier-mediated membrane transport process. Labeled galactose showed saturation of intracellular sequestration, as well as of the cell entry process, the former at much lower galactose concentrations. Straight-chain labeled monohydric alcohols were found to enter liver cells in a flow-limited fashion. Labeled ethanol consumption, when related to underlying steady ethanol values, exhibited Michaelis-Menten kinetics. A substantial extra intracellular enzymic space of distribution was found for the C3- and C4- straight-chain alcohols, predicted by the kinetics. Development of the tracer approach allowed the differentiation and characterization of the processes by which substances penetrate the cell membrane barrier and those resulting in intracellular sequestration.

Kinetic interpretation of hepatic multiple-indicator dilution studies.

The single-injection, multiple-indicator dilution approach, together with an analysis based on a model of microcirculatory events, provides a way of determining the composition of and the rates of processes in the liver. By comparison with a vascular reference, labeled red cells, a set of interstitial materials are found to enter the Disse space in a delayed-wave, flow-limited fashion, larger materials being excluded from a proportion of the space, and labeled water is found to undergo flow-limited distribution into the liver cells. In contrast, many other substances exhibit barrier-limited exchange with the liver cells; their tracer outflow profiles consist of throughput material (which has entered the interstitium but not the liver cells) and of returning material (which has entered the cells, later to return to the vascular space). Intracellular metabolic sequestration or biliary secretion reduces the outflow recovery of returning tracer; it also results in decreasing steady-state concentrations along sinusoids and cells and, where the cell membrane is limiting, in a step-down in concentration across it.