Chemorheological studies of the effect of heparin on the course of coagulation.

The influence of sodium heparin on viscoelastic change during coagulation was determined in vitro for whole blood samples from ten normal subjects at heparin concentrations ranging from 0 to 1.45 units/(ml whole blood). A four-parameter chemorheological model was used to describe the time course of coagulation as measured by the Weissenberg Rheogoniometer. One parameter compares closely with the whole blood activated partial thromboplastin time, while the other three may be related to the chemical kinetics of clotting. The chemorheological model and experimental techniques were then tested in a dog preparation. It was found that rheological measurements are more self-consistent than either thrombelastography or the activated partial thromboplastin time for the assay of in vivo heparin in two dogs.

Exercise training increases coronary transport reserve in miniature swine.

Female yucatan miniature swine were trained on a treadmill (ET) or were cage confined (C) for 16-22 wk. The ET pigs had increased exercise tolerance, heart weight-to-body weight ratio, and skeletal muscle oxidative capacity. After anesthesia the left anterior descending coronary artery was cannulated and pump perfused with blood while aortic, central venous, and coronary perfusion pressures, electrocardiogram, heart rate, and coronary blood flow were monitored. Capillary permeability-surface area product (PS) for EDTA was determined with the single-injection indicator-diffusion method by use of an organ model based on the Sangren-Sheppard equations for capillary transport. Coronary blood flow (CBF) and PS were compared before and during maximal adenosine vasodilation with coronary perfusion pressures at 120 mmHg. Results indicate that there were no differences in base-line CBF or PS between C and ET groups. alpha-Receptor blockade with phentolamine and/or prazosin, before adenosine vasodilation, produced increases in PS in C pigs but had little effect in ET pigs. During maximal vasodilation with adenosine, ET pigs had greater CBF (447 +/- 24 vs. 366 +/- 27 ml.min-1.100 g-1) and greater PS (83 +/- 9 vs. 55 +/- 7 ml.min-1.100 g-1) than the C group. It is concluded that ET induces an increased coronary transport capacity in miniature swine that includes a 22% increase in blood flow capacity and a 51% increase in capillary exchange capacity.

Modeling the effect of flow heterogeneity on coronary permeability-surface area.

In 11 anesthetized pigs, the left anterior descending coronary artery (LAD) was cannulated and pump perfused with blood before and during maximum adenosine vasodilation. For LAD plasma flows (F) ranging from 0.42 to 3.6 ml.min-1.g perfused tissue-1, we injected radiolabeled microspheres to measure heterogeneity and used the multiple indicator-dilution method to measure permeability-surface area product (PS) for EDTA. Heterogeneity of flow from the LAD was expressed as relative dispersion (RD) = standard deviation of flow/mean flow. Values of RD, corrected for tissue sample size using fractal theory, ranged from 13 to 87%, approaching 16-35% at high F. We developed a "variable-recruitment model" of regional heterogeneous capillary transport to correct PS for flow heterogeneity and capillary surface area recruitment. Values of PS ranged from 0.14 to 0.96 ml.min-1.g-1. Accounting for heterogeneity increased PS values by 0-18% compared with homogeneous values. Results revealed PS to be proportional to flow up to F = 1.5-2.1 ml.min-1.g-1 and then was constant at higher flows. The initial increase of PS with F may be due to capillary recruitment. When full recruitment is reached, PS becomes independent of F. We conclude that flow heterogeneity is significant but not readily predictable in the pig myocardium and that the use of microspheres to correct indicator-dilution data for flow heterogeneity improves the interpretation of multiple-tracer studies, particularly when tracers are used to study interventions that may alter flow distribution.

Effects of ligation and embolization on Kf and multiple tracer measurements in dog lungs.

In isolated blood-perfused dog lungs, the capillary filtration coefficient (Kf) and the permeability-surface area product of urea (PS) were measured to determine their responses to two different methods of altering filtration area: lobe ligation (LL, n = 5) and glass bead embolization (GBE, n = 4) during constant perfusion rates (700 +/- 45 ml/min). When two of three lobes were ligated, Kf decreased (1.36 +/- 0.13 to 0.58 +/- 0.23 g.min-1.cmH2O-1; P less than 0.05), but PS did not change (2.02 +/- 0.4 to 1.71 +/- 0.3 ml/s). Kf per gram of perfused blood-free dry lung weight was unchanged by LL (0.051 +/- 0.17 to 0.052 +/- 0.18 g.min-1.cmH2O-1), indicating that surface area per gram measured by Kf remained the same. However, PS per gram dry lung doubled (0.07 +/- 0.016 to 0.146 +/- 0.06 ml/s; P less than 0.05) after LL, suggesting that recruitment occurred in the remaining lobe. When three lobes were embolized with 200-microns glass beads (0.48 +/- 0.01 g beads/kg body wt), PS decreased (2.1 +/- 0.22 to 0.94 +/- 0.09 ml/s; P less than 0.05), but Kf was not altered (1.01 +/- 0.17 to 1.04 +/- 0.18 g.min-1.cmH2O-1). The constancy of Kf after GBE implies that the vascular pressure increase during the Kf measurement was transmitted to both blocked and flowing vessels and thereby measured the same filtration area before and after GBE. PS decreased significantly after GBE because of a loss of perfused surface area by the beads blocking flow in small arterial vessels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of flow heterogeneity on the measurement of capillary exchange in the lung.

The effects of flow heterogeneity on the measurement of transcapillary escape of small molecules for perfused in situ sheep lungs were evaluated. Lungs were studied at five flows (1.5-5.0 l/min) ranging from zone 1 to zone 3 conditions. At each flow, multiple indicator-dilution curves were collected using 14C-labeled urea (U) or butanediol (B) as the diffusing tracer, and radiolabeled 15-microns microspheres were injected. The lungs were removed, dried, sectioned, weighed, and counted for microsphere radioactivity. Flow heterogeneity expressed as relative dispersion, decreased with increasing flow, from 0.838 +/- 0.179 (mean +/- SD, n = 8) to 0.447 +/- 0.119 (n = 6). We applied homogeneous flow models of capillary exchange to compute permeability-surface area product (PS) and a related parameter, D1/2S, for diffusing tracers. (D is effective diffusivity of capillary exchange.) PS and D1/2S increased to a maximum with increasing flow, but the ratio of D1/2SU to D1/2SB remained constant. A new model incorporating flow heterogeneity and recruitment (the variable recruitment model) was used. The variable recruitment model described the effects of flow on capillary recruitment, but incorporating heterogeneity into the computation did not alter D1/2S values from those computed assuming homogeneous flow.

Pulmonary vascular resistance distribution and recruitment of microvascular surface area.

To test the hypothesis that the distribution of hemodynamic resistance is involved in the control of pulmonary capillary surface area, we measured permeability-surface area product (PS) and longitudinal resistance distribution (LRD) as functions of perfusion rate in isolated rabbit lungs under zone II conditions (n = 10) and through the zone II-III transition (n = 4). PS, considered to be indicative of functioning capillary surface area, was measured with the aid of the diffusion-limited tracer [14C]propanediol, whereas LRD was determined using a viscous bolus technique. LRD was seen to change character with increasing flow and increasing PS/surface area, becoming bimodal with low central resistance as full capillary recruitment was approached in zone III. Effects of hypoxic ventilation were studied in zone II in five lungs; it was found that hypoxia altered the LRD and eradicated the normoxic dependence of PS/surface area on perfusion rate. It was concluded that LRD is involved in the determination of functioning capillary surface area.

Exercise training-induced increase in coronary transport capacity.

The objective of this study was to measure effects of exercise training on coronary flow heterogeneity, microvascular transport, and hemodynamics. Five miniature swine were trained on a treadmill (ET) for 16 wk; five control pigs (C) were confined to cages for the same period. At the end of that period we used the multiple indicator dilution method to measure permeability-surface area product (PS) to EDTA over a range of flow (F) in an anesthetized, open-chest preparation. We found that the heterogeneity of flow as measured by microspheres decreased with increasing F, but that ET had no clear effect on heterogeneity. We evaluated PS from the indicator concentration curves, taking into account flow heterogeneity and variations in capillary recruitment throughout the bed. In both C and ET pigs we observed an increase in PS with F until a maximum value of PS was reached at full recruitment. This relationship between PS and F was unaltered by ET. However, hemodynamic resistance was significantly reduced by ET, and F was higher at a given perfusion pressure after training. Since PS increases with F, ET pigs had higher PS values at typical coronary artery pressures.

Effect of exogenous adenosine on resistance, capillary permeability-surface area and flow in ischemic canine myocardium.

The effects of adenosine on capillary-tissue exchange have not been evaluated. Although adenosine is a known vasodilator, its effects on nutritive flow are unknown. We therefore measured the influence of adenosine on resistance and capillary exchange in normal and mildly ischemic myocardium in 11 anesthetized, heparinized dogs. Flow to the left anterior descending artery was measured and controlled through an extracorporeal shunt from the carotid artery. Capillary permeability-surface area for sucrose (PS) was determined using the multiple-tracer technique in which mixtures of isotopes were injected into the coronary arterial shunt. We found that in the normal myocardium, intracoronary infusion of adenosine at 8 +/- 2 (S.E.M.) micrograms/ml of plasma significantly lowered resistance and increased PS. In the ischemic myocardium, however, large doses of adenosine (200 +/- micrograms/ml of plasma) lowered resistance but failed to increase PS. We interpret these results to mean that in mild, flow-reduction ischemia, adenosine did not lead to a recovery of capillary surface area for exchange, but simply increased flow through capillaries which had already been functioning. Nutritional flow was not enhanced.

Effects of hyaluronidase and protamine on resistance and transport after coronary flow reduction in heparinized dogs.

We have measured the effects of hyaluronidase, protamine and a mixture of the two drugs on coronary flow resistance and transcapillary exchange in 20 heparinized, anesthetized dogs in which flow to the left anterior descending coronary artery was supplied through an extracorporeal shunt from the carotid artery. Multiple-tracer measurements were made by injecting a mixture of isotopes into the shunt and sampling from the coronary sinus. These were carried out under base-line conditions, after 1 hr of reduced flow to the left anterior descending coronary artery and after a 2nd hr during which the drug under study was infused into the ischemic zone. The results of multiple-tracer measurements were expressed as extravascular water volume (VT), extravascular sucrose volume (VS) and microvascular permeability surface area for sucrose. It was found that flow reduction significantly reduced VT, VS and permeability surface area. Resistance increased during the period of reduced flow after an initial decrease immediately upon flow reduction. In five dogs, a mixture of hyaluronidase and protamine significantly lowered coronary flow resistance and increased permeability surface are, VT, VS and VS/VT compared to preinfusion, reduced flow values. Infusion of hyaluronidase alone had significantly less effect in five other dogs, whereas infusion of protamine alone (N = 5) and saline (N = 5) did not affect resistance or transport. It is concluded that protamine enhances the effects of hyaluronidase when heparin is present, probably by preventing its blockade of hyaluronidase action.

Tracer exchange in the normal and ischemic coronary circulation.

Proper function of the coronary blood-tissue exchange system may be important in the preservation of myocardium threatened by ischemia. We have undertaken studies aimed at elucidating the functions of this system under baseline and ischemic conditions. The exchange of [14C]sucrose between the coronary capillaries and extravascular space has been studied with the multiple-tracer method. Protein transport has been examined by measuring the deposition of labeled albumin and by collecting cardiac lymph. Results indicate that reduced-flow ischemia decreases functioning capillary surface area but increases permeability to small molecules and protein. Hyaluronidase and adenosine can restore flow after partial occlusion of the coronary artery. However, only hyaluronidase restores capillary surface to its baseline value. Thus, ischemic effects on exchange are not controlled merely by hemodynamic factors. Reduced-flow ischemia in the heart can induce a vascular permeability change in the lung circulation. We conclude that capillary and interstitial transport are altered significantly by ischemia. Preservation of the proper function of these processes may be important in protecting the ischemic myocardium.

Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis.

We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.

Concurrent increases in resistance and transport after coronary obstruction in dogs.

We have extended earlier studies on coronary vascular permeability-surface area (PS) for [14C]sucrose to include observations of the transcoronary flow resistance before and after partial occlusion of the left anterior descending coronary artery. Multiple-tracer (MT) studies were conducted on 13 anesthetized dogs by inserting an isotope mixture (125I-albumin, 51Cr-red blood cells, [14C]sucrose, 3HOH) into a cannula connecting the carotid and the left anterior descending coronary artery. Analysis of blood sampled from the coronary sinus allowed calculation of PS, extravascular 3HOH volume (VT), and extravascular [14C]sucrose volume (VS). Flow reduction significantly reduced VT and VS and increased PS/VT relative to base line. Coronary resistance distal to the obstruction (R) decreased significantly immediately after flow reduction but then, over the low-flow period, increased significantly above this initial low-flow value. The mean R during reduced flow normalized to R immediately after flow reduction (Ri) correlated significantly with PS/VT during ischemia. Because R/Ri is not correlated with VT alone, we speculate that capillary permeability alterations may play a role in the deterioration of myocardial perfusion during ischemia.

Reperfusion of the ischemic canine myocardium: effect on vascular exchange and resistance.

In order to see if changes in hemodynamic resistance following reperfusion of ischemic myocardium could be related to alterations in microvascular exchange, we measured resistance (R), permeability surface-area for sucrose (PS), and distribution volumes for tritiated water (V) and for sucrose (VS) in nine anesthetized dogs in which blood to the left anterior descending coronary artery was supplied via a shunt from the carotid artery. Measurements were made during four periods: baseline, reduced coronary artery flow, reperfusion, and a second period of reduced flow. Increase in resistance following reperfusion (R2 = 1.8 +/- 3, R4 = 2.5 +/- .5 mmHg/min/ml, mean +/- s.e.m.) was significantly greater than in nine control dogs in which reperfusion was omitted. Also, the series of interventions including reperfusion lowered PS and V (PS4/PS1 = .54 +/- .07, V4/V1 = .58 +/- .08). Our results suggest that increases in resistance due to reperfusion may be accompanied by a loss in functioning capillary surface area.

Analysis of the influences of the E5 transforming protein on kinetic parameters of epidermal growth factor binding and metabolism.

The E5 protein of the bovine papillomavirus induces cellular transformation when transfected into NIH 3T3 cells, and the extent of focal transformation is enhanced by cotransfection with the epidermal growth factor (EGF) receptor (Martin et al., Cell 59:21-32, 1989). To determine whether E5 affects EGF:receptor interactions we analyzed the kinetics of 125I-EGF processing using a mathematical model that enabled us to evaluate rate constants for ligand association (ka), dissociation (kd), internalization (ke), recycling (kr), and degradation (kh). These rate constants were measured in NIH 3T3 cells transfected with the human EGF receptor (ER cells) and in cells transfected with both the EGF receptor and E5 (E5/ER cells). We found that the rate constant for 125I-EGF association ka was significantly decreased in E5/ER cells, but was apparently occupancy-independent in both cell lines. The 125I-EGF dissociation rate constant kd was significantly lower in E5 transformed cells, and increased with occupancy in both cell lines. This suggests that E5 alters the receptor before or during EGF binding so that ligand association is slower; however, once complexes are formed, EGF is bound more tightly to the receptor. Rate constants for internalization ke were also found to be occupancy-dependent, although at a given level of occupancy ke was similar for both cell lines. Also, there was no apparent effect of E5 on the recycling rate constant kr. The 125I-EGF degradation rate constant kh was 30% lower in E5 transformed cells, and was occupancy-independent. The overall effect of E5 is to stabilize intact EGF:receptor complexes which may alter mitogenic signaling of the receptor.

Rate constants for binding, dissociation, and internalization of EGF: effect of receptor occupancy and ligand concentration.

We measured the kinetic parameters for interaction of epidermal growth factor (EGF) with fetal rat lung (FRL) cells under two sets of experimental conditions and applied sensitivity analysis to see which parameters were well-defined. In the first set of experiments (method 1), the kinetics of internalization and dissociation of radiolabeled EGF were measured with a temperature-shift protocol in medium initially devoid of free ligand. The initial concentration of radiolabeled EGF bound to the cell surface corresponded to levels of receptor occupancy ranging from approximately 200 receptors per cell to approximately 18,000 receptors per cell, a level at which EGF binding approaches saturation. In the second set of experiments (method 2), carried out at a constant temperature, we began with no surface-bound or internalized ligand. The initial free ligand concentration was varied from 0.2 to 50 ng/mL. In both sets of experiments, we measured surface-bound, internalized, and free 125I-EGF as functions of time and evaluated the parameters of a mathematical model of endocytosis. Sensitivity analysis showed that three rate constants were well-defined in this combination of two experimental approaches: ke, the endocytic rate constant; ka, the association rate constant; and kd, the dissociation rate constant. The endocytic parameter ke was found to be independent of initial surface receptor occupancy (method 1); there was some indication that it increased with initial free ligand concentration in method 2. Neither kd nor ka was found to change with extent of initial surface receptor occupancy or initial free ligand concentration, respectively, a finding of significance, since diffusion theory predicts these parameters will vary with surface receptor occupancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Parameter identification in coronary pressure flow models: a graphical approach.

The confident identification of parameters is important in the practical application of physiological models. However, the task of parameter identification is often complicated by interactions among parameters and by the fact that the sensitivity of the model to changes in a given parameter is generally a function of all the other parameters. Here we illustrate a graphical approach to parameter identification that allows the modeler to visualize the behavior of the model, the sensitivity functions, and certain functions characteristic of parameter interdependence. The visual display can be generated over any desired portion of parameter space. The technique is applied to a simple, four-parameter, myocardial pump model of the coronary circulation. The results indicate that over specified ranges of parameters, it is possible to distinguish among the four parameters of the model: the ratio of proximal-to-distal resistance, alpha; the overall resistance of the vascular bed, R; the compliance of the vascular bed, C; and a parameter, kappa, relating tissue pressure to left ventricular pressure. It was found that in order to identify all parameters uniquely, it was necessary to regress upon both coronary inflow and outflow.

Deduction of pulmonary microvascular hematocrit from indicator dilution curves.

We have developed a new model describing the relationship between plasma and red cell tracers flowing through the lung. The model is the result of an analysis of the transport of radiolabeled plasma albumin between two flowing phases and shows that differences between red cell and plasma tracer curves are related to microvascular hematocrit. The model was tested in an isolated, blood-perfused dog lung preparation in which we injected 51Cr-labeled red cells and 125I-labeled plasma albumin into the pulmonary artery. From the tracer concentration-time curves at the venous outflow, we calculated hr, the ratio of microvascular hematocrit to large-vessel hematocrit. In 18 baseline experiments, hr = 0.92 +/- 0.01 (mn +/- sem) at a blood flow rate of 10.7 +/- 0.3 ml s-1. We determined the effects of (a) glass bead embolization, (b) alloxan, and (c) lobe ligation on hr. Embolization attenuated the separation between plasma and red cells (increased hr), probably as a consequence of passive vasodilation. Alloxan enhanced separation of plasma and red cells (decreased hr), possibly as a result of arteriolar vasoconstriction. Ligation of a fraction of the perfused tissue at constant flow did not cause significant change in hr in the remaining perfused tissue. The model assumes that large-vessel transit times are uniform and that all dispersion occurs in the microvasculature. A theoretical analysis apportioning dispersion between large and small vessels disclosed that the error associated with these assumptions is likely to be less than 15% of the measured hr. We conclude from this study that the microvascular hematocrit model describes experimental plasma and red cell curves. The results imply that hr can be readily deduced from tagged red cells and plasma and can be accounted for in calculating permeability-surface area in diffusing tracer experiments.

The use of microsoft excel as a user interface for biological simulations.

We used Microsoft Excel 4.0 for Windows running on a PC-486 to develop a user interface for two biological simulation models: a lung fluid balance model and a fractal model of the pulmonary circulation. The simulation programs were written in the C programming language, while the user interface was written in the macro language of Excel. The interface builds input data files for the simulation programs and provides a mechanism for displaying relevant information from output files produced from the simulations. Input fields are partially protected so that the user cannot modify certain portions of the spreadsheet. The Excel interface is used to build models from different available components and to select appropriate parameters for these models. The developed interface was also useful for running models in the batch mode. After selecting changes in lung fluid balance parameters, the interface allows users to find new steady state values by automatically running the model and adjusting initial conditions. Several different graphical options allow users to easily investigate the effects of selecting particular models and parameters. Techniques used in developing our user interface can be extended to most biological simulation programs which manipulate input and output data files.

Regional measurement of the Gd-DTPA tissue partition coefficient in canine myocardium.

Quantifiable MRI perfusion studies using the contrast agent Gd-DTPA require measurement or estimation of the tissue partition coefficient (lambda) for tracer kinetic modeling. Radiotracer techniques were used to obtain regional lambda measurements from the left ventricles of five dogs. Measurements were analyzed to determine whether spatial heterogeneity was a major component of lambda variability. No systematic variations were identified in terms of radial position, short-axis slice location, or wall position. The high lambda variability seen in this study and in cited data of others may be due in part to tissue heterogeneity in interstitial volume, plasma volume, and perfusate hematocrit.