The behavior of human neutrophils during flow through capillary pores

The passage times of individual human neutrophils through single capillary-sized pores in polycarbonate membranes were measured with the resistive pulse technique, and results were compared to those obtained from the micropipette aspiration of entire cells. Pore transit measurement serves as a useful means to screen populations of cells, and allows for protocols that measure time dependent changes to the population. Neutrophils exhibited a highly linear pressure/flow rate relationship at aspiration pressures from 200 Pa to 1,500 Pa in both the pore and pipette systems. Cellular viscosity, as determined by the method of Hochmuth and Needham, was 89.0 Pa.s for the pore systems and 134.9 Pa.s for the pipette systems. These results are in general agreement with recent values of neutrophil viscosity published in the literature. Extrapolation of the observed linear flow response revealed an apparent minimum pressure for whole cell aspiration significantly above the threshold pressure predicted by Evans' liquid drop model. However, whole cell aspiration was achieved in both the pore and pipette systems at pressures below this extrapolated minimum, although the calculated cellular viscosity was greatly increased. The implications of these two regimes of cell deformation is unclear. This behavior could be explained by shear thinning of the material in the cell body. However the origin of this phenomenon may be in the cortical region of the cell, which exhibits an elastic tension that may be deformation rate dependent.     

The variance of transit times of indicators through a vascular bed

A new method is developed of determining the variance of the times of transit of an indicator through a vascular bed (or through any perfused organ) from the time-course of the arterial-venous concentration difference, observed between two steady states. The method is illustrated using previously published data on arterial and hepatic venous radioactivities following a single injection of ¹³¹I-labelled serum albumin in man.     

Determination of erythrocyte transit times through micropores. I--Basic operational principles

To study the transit times of each red blood cell passing through cylindrical micropores and in order to evaluate sub-population of cells with regard to their deformability, we have developed a new system called the cell transit time analyser (CTTA). By using an AC voltage (100 KHz) across a special filter, we measure the electrical conductance change produced by the cells passing through the pores under a known driving pressure. This computer based device provides the distribution of transit times tau for 2000 cells in 1 minute and as a result the mean transit time [tau]. Experiments with red cells were designed to evaluate the flow behavior of both normal cells and cells whose mechanical properties were artificially altered. Cell volume was changed by use of non-isotonic media. Cell shape and cell volume were modified by varying the pH of the suspending buffer. Results of these experiments are: 1) a skew distribution of transit times towards high tau values for both control cells and artificially altered cells is observed: 2) [tau] is minimum for isotonic conditions and increases sharply for either hypotonic or hypertonic media: 3) [tau] is minimum at physiological pH and increases for either acid or alcaline changes of pH.     

Interleukin-6 induces demargination of intravascular neutrophils and shortens their transit in marrow

Recombinant human interleukin-6 (IL-6) causes both a thrombocytosis and leukocytosis. The thrombocytosis is caused by an accelerating thrombocytopoiesis, but the mechanism of the leukocytosis is unknown. This study was designed to determine the relative contributions of marrow stimulation and intravascular demargination to the IL-6 induced neutrophilia. IL-6 (2 microgram/kg), administered intravenously to rabbits, caused a biphasic neutrophilia with an initial peak at 3 h and a second peak at 9 h. Using the thymidine analog 5'-bromo-2'-deoxyuridine (BrdU) to label dividing polymorphonuclear leukocytes (PMNs) in the bone marrow, we showed that IL-6 treatment mobilizes PMNs from the marginated pool into the circulating pool at 2-6 h with a decrease in L-selectin expression on PMNs and also accelerates the release of PMNs from the postmitotic pool in the bone marrow at 12-24 h. We have concluded that IL-6 causes a biphasic neutrophilia wherein the first peak results from the mobilization of PMNs into the circulating pool from the marginated pool and the second peak results from an accelerated bone marrow release of PMNs.     

Neutrophil transit times through pulmonary capillaries: the effects of capillary geometry and fMLP-stimulation

The deformations of neutrophils as they pass through the pulmonary microcirculation affect their transit time, their tendency to contact and interact with the endothelial surface, and potentially their degree of activation. Here we model the cell as a viscoelastic Maxwell material bounded by constant surface tension and simulate indentation experiments to quantify the effects of (N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-stimulation on its mechanical properties (elastic shear modulus and viscosity). We then simulate neutrophil transit through individual pulmonary capillary segments to determine the relative effects of capillary geometry and fMLP-stimulation on transit time. Indentation results indicate that neutrophil viscosity and shear modulus increase by factors of 3.4, for 10(-9) M fMLP, and 7.3, for 10(-6) M fMLP, over nonstimulated cell values, determined to be 30.8 Pa.s and 185 Pa, respectively. Capillary flow results indicate that capillary entrance radius of curvature has a significant effect on cell transit time, in addition to minimum capillary radius and neutrophil stimulation level. The relative effects of capillary geometry and fMLP on neutrophil transit time are presented as a simple dimensionless expression and their physiological significance is discussed.     

Determination of erythrocyte transit times through micropores. II-- Influence of experimental and physicochemical factors

A new red blood cell filtration system, termed the Cell Transit Time Analyzer (CTTA), has been developed in order to measure the individual transit times of a large number of cells through cylindrical micropores in special "oligopore" filters: the system operates on the electrical conductometric principle and employs special computer software to provide several measures of the resulting transit time histogram. Using this system with filters having pore diameters of 4.5 or 5.0 cm and length to diameter ratios of 3.0 to 4.7, we have evaluated the effects of several experimental factors on the flow behavior of normal and modified human RBC. Our results indicate : 1) linear PBC pressure - flow behavior over a driving pressure range of 2 to 10.5 cm H2O with zero velocity intercepts at delta P = 0, thus suggesting the Poiseuille - like nature of the flow; 2) resistance to flow or "apparent viscosities" for normal RBC which are between 3.1 to 3.9 cPoise and are independent of driving pressure and pore geometry; 3) increased flow resistance (i.e., increased transit times) for old versus young RBC and for RBC made less deformable by DNP-induced crenation or by heat treatment at 48 degrees C; 4) increased mean transit time and poorer reproducibility when using EDTA rather than heparin as the anticoagulant agent. Further, using mixtures of heat-treated and normal RBC and various percentile values of the transit time histogram. We have been able to demonstrate the presence of sub-populations of rigid cells and thus the value of measurements which allow statistical analyses of RBC populations.     

Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.     

Determination of erythrocyte transit times through micropores. II. Influence of experimental and physicochemical factors

A new red blood cell filtration system, termed the Cell Transit Time Analyzer (CTTA), has been developed in order to measure the individual transit times of a large number of cells through cylindrical micropores in special "oligopore" filters; the system operates on the electrical conductometric principle and employs special computer software to provide several measures of the resulting transit time histogram. Using this system with filters having pore diameters of 4.5 or 5.0 microns and length to diameter ratios of 3.0 to 4.7, we have evaluated the effects of several experimental factors on the flow behavior of normal and modified human RBC. Our results indicate: 1) linear RBC pressure-flow behavior over a driving pressure range of 2 to 10.5 cm H2O with zero velocity intercepts at delta P = 0, thus suggesting the Poiseuille-like nature of the flow; 2) resistance to flow or "apparent viscosities" for normal RBC which are between 3.1 to 3.9 cPoise and are independent of driving pressure and pore geometry; 3) increased flow resistance (i.e., increased transit times) for old versus young RBC and for RBC made less deformable by DNP-induced crenation or by heat treatment at 48 degrees C; 4) increased mean transit time and poorer reproducibility when using EDTA rather than heparin as the anticoagulant agent. Further, using mixtures of heat-treated and normal RBC and various percentile values of the transit time histogram, we have been able to demonstrate the presence of sub-populations of rigid cells and thus the value of measurements which allow statistical analyses of RBC populations.     

Vertical gradient of pulmonary capillary transit times

The key determinants of alveolar capillary perfusion are transit times and the extent of recruitment. Capillaries are known to be heavily recruited in the dependent lung, but there are no direct data that bear on how capillary transit times might be affected by gravity. We directly determined mean capillary transit times on the surface of the upper, middle, and lower lung by measuring the passage of fluorescent dye through the capillaries using in vivo television microscopy. In anesthetized dogs, mean capillary transit times averaged 12.3 s in the upper lung, 3.1 s in the midlung, and 1.6 s in the lower lung. This near order of magnitude variation in speed of blood transit establishes that there is a vertical gradient of capillary transit times in the lung. As expected, dependent capillary networks were nearly fully recruited, whereas relatively few capillaries were perfused in the upper lung. The lengthy transit times and sparsely perfused capillary beds in the upper lung combine to provide a substantial part of pulmonary gas exchange reserve.     

Ion currents through pores. The roles of diffusion and external access steps in determining the currents through narrow pores.

External access steps, which may include restricted aqueous diffusion, are introduced into a kinetic model for ion transport through narrow pores. The conductance-concentration relation and the concentration dependence of the biionic permeability are calculated using two alternative assumptions: (a) access to the mouth of the pore is allowed only when no ion is within the lumen or at either mouth; (b) ions remain at the mouth only very transiently. With either assumption the concentration dependence of the fluxes is the same as in previous treatments in which all steps in access were lumped into a single process. Also as before, the biionic permeability ratio is independent of concentration so long as the lumen is never doubly occupied. For narrow pores, such as those formed by gramicidin A, the slowest external portion of the access process must occur close to the pore's mouth, and thus the region an ion must occupy to gain access is small. As a consequence, the probability of finding an ion within this region is also small. On this basis, it is argued that the second assumption is appropriate for these pores. The kinetic equations that result are identical to those used by Urban, B., S.B. Hladky, and D.A. Haydon (1980, Biochim. Biophys. Acta. 602:331-354).     

Gastrointestinal transit times in young and old cats

Ageing results in a decrease in apparent nutrient digestibility in the gastrointestinal (GI) tract. The aim of this study was to investigate whether the rate of gastric emptying or total GI transit times differed between young (3.0+/-0.9 years) and senior (11.6+/-1. 4 years) cats. Gastric emptying rates were measured using [1-(13)C]octanoic acid and total transit times with chromium oxide. No significant differences (P>0.05) were observed in either the rate of gastric emptying or total transit time between young and senior cats although senior cats exhibited a larger variability in total transit time compared to the younger cats (35.71+/-14.06 and 26. 46+/-5.80 h, respectively). The results of this study indicate that the observed reduction in nutrient digestibility in ageing cats is not due to alterations in the rate of passage of digesta through the GI tract.     

The effect of the solvent viscosity on the migration of small molecules through the structure of myoglobin.

The migration rate of small molecules through the structure of proteins can be monitored by quenching the light emitted from an excited optical probe located within the protein. In the present study we examined the influence of the solvent viscosity on the migration rate of the quencher anthraquinone sulfonate through myoglobin towards an excited Zn protoporphyrin molecule at the binding site of the protein. The solvent viscosity was increased by adding dextrans of different molecular weight but forming isoviscous solutions. The results demonstrate that the migration rate in the protein decreases with increasing solvent viscosity. This suggests that the fluctuations on the protein structure, which make the above migration possible, are affected by the solvent macroviscosity.     

Distribution of pulmonary capillary transit times in recruited networks

When pulmonary blood flow is elevated, hypoxemia can occur in the fastest-moving erythrocytes if their transit times through the capillaries fall below the minimum time for complete oxygenation. This desaturation is more likely to occur if the distribution of capillary transit times about the mean is large. Increasing cardiac output is known to decrease mean pulmonary capillary transit time, but the effect on the distribution of transit times has not been reported. We measured the mean and variance of transit times in single pulmonary capillary networks in the dependent lung of anesthetized dogs by in vivo videofluorescence microscopy of a fluorescein dye bolus passing from an arteriole to a venule. When cardiac output increased from 2.9 to 9.9 l/min, mean capillary transit time decreased from 2.0 to 0.8 s. Because transit time variance decreased proportionately (relative dispersion remained constant), increasing cardiac output did not alter the heterogeneity of local capillary transit times in the lower lung where the capillary bed was nearly fully recruited.     

Comparison of direct and indirect measurements of pulmonary capillary transit times

Using in vivo microscopy, we made direct measurements of pulmonary capillary transit time by determining the time required for fluorescent dye to pass from an arteriole to a venule on the dependent surface of the dog lung. Concurrently, in the same animals, pulmonary capillary transit time was measured indirectly in the entire lung using the diffusing capacity method (capillary blood volume divided by cardiac output). Transit times by each method were the same in a group of five dogs [direct: 1.75 +/- 0.27 (SE) s; indirect: 1.85 +/- 0.33 s; P = 0.7]. The similarity of these transit times is important, because the widely used indirect determinations based on diffusing capacity are now shown to coincide with direct measurements and also because it demonstrates that measurements of capillary transit times on the surface of the dependent lung bear a useful relationship to measurements on the capillaries in the rest of the lung.