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Biomedical subjects

M B Wolf

Publications and source records attributed to M B Wolf.

At least 19 recordsLinked to original sources
PubMed2001

Effect of advanced glycation end products on oxidative stress in endothelial cells in culture: a warning on the use of cells studied in serum-free media.

AIMS/HYPOTHESIS: Alterations in vascular permeability and oxidative stress are characteristics of endothelial dysfunction in diabetic vascular disease. Since AGE-proteins have been hypothesized to mediate these effects, we studied the effects of AGE-bovine serum albumin on endothelial monolayer permeability and intracellular glutathione. METHODS: AGE-BSA was prepared by incubating BSA for 30 days at 37 degrees C with 0.5 mol/l glucose and 0.2 mol/l phosphate buffer, pH 7.4. Permeability to fluorescently labelled BSA was assessed in a bovine pulmonary artery endothelial cell monolayer preparation. Glutathione was measured by an enzymatic assay. RESULTS: AGE-BSA concentrations greater than 3 to 4 micromol/l produced maximal increases in permeability (6-8 times basal) within 3 to 4 h of incubation with the cells. This effect persisted for at least 48 h. However, BSA incubated in the absence of glucose produced similar effects. Dialysis of the AGE-BSA showed that low molecular weight components contained the permeability-increasing activity. Phosphate buffer used to prepare the AGE-BSA, at concentrations equivalent to those present in phosphate-buffered saline and in the AGE preparation (approximately 5 mmol/l), produced similar permeability increases at equivalent incubation times. Metal chelators (0.5 mmol/l) or inclusion of fetal bovine serum (10-20 %) blocked these permeability increases. These increases in permeability were associated with a decrease in endothelial glutathione, both inhibited by 10 mmol/l N-acetylcysteine, and a loss of cell-to-cell and cell-to-matrix adhesion molecules. CONCLUSION/INTERPRETATION: Trace amounts of redox-active metal ions in biological buffers could induce oxidative stress and alterations in cellular functions attributed to AGE-proteins in vitro. It is important to use metal-free phosphate and bicarbonate buffers in studies on cell biology in vitro, especially in serum-free media.

Actins↗
PubMed2000

Interaction of endothelin-1 and nitric oxide in endothelial barrier failure in the cat hindlimb.

OBJECTIVE: To determine the interactions of endothelin-1 (ET-1) and nitric oxide (NO) in the regulation of endothelial barrier function in skeletal muscle. METHODS: The protein sieving coefficient (1 - sigma f) was measured as an index of microvascular permeability in the isolated, perfused cat hindlimb preparation. The measurement was made to determine 1) the effects of ET-1 and NO on basal permeability by blocking the ETA receptor with BQ123 and NO production with the NO-synthase inhibitors L-NAME or L-NMMA; 2) if elevated NO (SNAP) affects permeability; and 3) the interaction of ET-1 and NO by ascertaining if NO-synthase inhibition or elevated NO can block the ET-1-induced permeability increases. Additionally, vascular resistance was determined under these conditions to see if increased microvascular pressures or increased shear stress might play a role in the permeability changes. RESULTS: Blocking either the ETA receptor or basal NO production did not affect basal permeability. Likewise, raising NO levels did not affect this permeability. Blocking the ETA receptor blocked the ability of ET-1 to cause a profound barrier failure. Increased NO also could block this ET-1-induced effect. Blocking the ETA receptor or elevating NO blocked the 2.5-fold increase in vascular resistance induced by ET-1. CONCLUSIONS: Since the ETA receptor does not reside on skeletal muscle endothelium, it is not likely that ET-1 acts directly on the endothelium to produce its effects. It could act through 1) increases in shear stress secondary to an ET-1-induced vasoconstriction; 2) ET-1-induced increases in microvascular pressure sufficient to cause an inflammatory reaction; or 3) stimulation of other cell types, such as leukocytes, to release inflammatory mediators that could damage the endothelium.

Animals↗
PubMed1999

Endothelin-1 induces endothelial barrier failure in the cat hindlimb.

Our purpose was to see whether endothelin- (ET) 1 could produce a change in the endothelial membrane barrier to protein in skeletal muscle. Previous studies in other tissues have suggested that ET-1 affects this barrier, but the measurement methods used could not exclude vascular protein extravasation due to microvascular pressure changes or the effects of changes in perfused capillary surface area. We measured the protein sieving coefficient, a specific measure of the permeability of the membrane to protein, in the isolated, perfused cat hindlimb preparation. The integral-mass balance method determined this coefficient from the changes in hematocrit and plasma protein concentration induced by a period of transvascular fluid filtration. The data clearly indicate that ET-1 produces a dose (1-20 nM) dependent increase in permeability indicative of barrier dysfunction. Hence, elevated ET levels may contribute to the perivascular edema, hemoconcentration, and impaired tissue perfusion found in systemic inflammatory response syndromes and related diseases.

Animals↗
PubMed1998

Voltage-gated sodium channels in cardiac microvascular endothelial cells.

The goal of this study was to determine whether inward Na+ or Ca2+ currents could be measured in cardiac microvascular endothelial cells (CMEC). CMEC were isolated from rat ventricular muscle and studied during days 1-4 in culture. Differential uptake of fluorescently labeled acetylated low-density lipoproteins (LDL) indicated that the primary culture contained > 90% CMEC. Membrane currents were measured with the use of the whole cell arrangement of the patch-clamp technique with a Cs+ internal solution to prevent contamination by outward K+ currents. Voltage steps positive to -30 mV resulted in the activation of a fast, inward Na+ current (INa). In 20 cells examined, the peak inward current measured at 0 mV was 2.1 pA/pF. The half-maximal voltage required for inactivation of INa was -45 mV, and the current recovered from inactivation with a time constant of 10 ms. Inward currents were eliminated by replacement of external sodium with N-methylglucamine and were blocked by both tetrodotoxin (TTX) (dissociation constant = 5 nM) and saxitoxin (50 nM). Stimulation of protein kinase C, through application of phorbol 12,13-dibutyrate, resulted in an increase in the amplitude of INa without any change in the voltage dependence of current activation. Thus the endothelium of cardiac microvessels may be unique in expressing voltage gated, TTX-sensitive Na+ channels.

Animals↗
PubMed1998

Prediction of permeability-surface area product data by continuous-distribution pore models.

OBJECTIVE: To investigate the ability of continuous-distribution pore models to accurately predict permeability-surface area product (PS) experimental data in skeletal muscle. METHODS: Models having a water-only (WO) pathway and continuous distributions of microvascular transport-pathway sizes were fit to solute reflection-coefficient (sigma) experimental data (approximately 0.5-16 nm Stokes radii) obtained from skeletal muscle to determine optimal parameter values. Without further modification, these models were used to predict experimental PS values obtained from the literature for small solutes ranging in size from NaCl to inulin and for three proteins, alpha-lactalbumin, ovalbumin, and albumin (approximately 0.23-3.7 nm radii). The protein PSs were determined from fluorescent tracer-diffusion curves and a nonlinear model of tracer diffusion in the cat hindlimb preparation. The model's PS predictions were compared to those of a discrete-pore model previously developed and a fiber-matrix (FM) model. RESULTS: A log-normal (LN) continuous pore-size distribution plus WO-pathway model (three free parameters) fit the sigma data to within the 95% confidence intervals of each of eight solutes spanning a 32-fold size range and was nearly as close to the data as was the two discrete-pore plus WO-pathway model (four free parameters). Both models closely described the PS data for nine solutes spanning a 14-fold size range. The fit of a fiber-matrix plus WO-pathway model (three free parameters) to the sigma data was much poorer than for the other models. CONCLUSIONS: The LN and two-discrete-pore models accurately describe sigma and PS experimental data in cat and human skeletal muscle. Therefore, experimental data resulting from complex microvascular transport processes are well characterized by simple pore models.

Albumins↗
PubMed1996

Determination of the magnitude of the water-exclusive pathway in cat skeletal muscle microvasculature.

OBJECTIVE: To measure the magnitude of the water-exclusive pathway in cat skeletal muscle. METHODS: The osmotic reflection coefficient (sigma d) was measured for sucrose, raffinose and cyancobalamine using osmotic transient techniques in the isolated, perfused cat hindlimb preparation at sufficiently high perfusate flows (60-80 ml/min-1 100 g-1) so that solute diffusion was not a factor. Microvascular filtration coefficient values required for the sigma d determination were measured using the capillary filtration coefficient (CFC) technique at these high flows. With these sigma d data and macromolecular reflection coefficient data from a previous study, discrete pore-modeling techniques were used to estimate the magnitude of the water movement through the water-exclusive pathway. RESULTS: CFC values increased significantly at very high flows ( > 80 ml/ min-1 100 g-1), but these values were unchanged from control at the lower flows used to measure sigma d. The sigma d values for sucrose and raffinose were 0.41 +/- 0.03 SE and 0.42 +/- 0.03 SE, respectively, in 12 limbs. In the same limbs, the sigma d for cyancobalamine was 0.52 +/- 0.03 SE, which was significantly (p < 0.05) larger, consistent with a larger Stokes-Einstein radius for this molecule. A 3-pathway model (small and large pores and a water-exclusive pathway) was fit to the data. The result was that 41 +/- 4% (95% confidence interval) of total water flow makes use of the water-exclusive pathway in this preparation. CONCLUSIONS: The very high fraction of water flow through the water-exclusive pathway in cat skeletal muscle suggests that this pathway is of major importance in microvascular water movement under normal conditions. Failure to take this finding into account can lead to inaccuracies in the estimation of parameters for pathways which carry solute.

Animals↗
PubMed1994

Effect of cold on ischemia--reperfusion-induced microvascular permeability increase in cat skeletal muscle.

The effects of reduced temperature during ischemia (I) upon microvascular permeability and resistance increases during reperfusion (R) were assessed in skeletal muscle. Protein solvent-drag reflection coefficients (sigma f) and changes in vascular resistance were measured during 37 degrees C reperfusion of isolated, whole-blood perfused cat hindlimbs after the limbs had been subjected to 3.5 h of ischemia at several temperatures. sigma f was determined from the disappearance rates of water and protein from the circulating perfusate during a period of induced microvascular fluid filtration. The I/R procedure at 37 degrees C caused sigma f to fall from approximately 0.85 to approximately 0.5, indicating a large increase in microvascular permeability. Hypothermic ischemia at 30, 22, 17, or 12 degrees C totally abolished this drop in sigma f. However, when the ischemia was at approximately 5 degrees C, there was a significant fall in sigma f to approximately 0.7, which was similar to the value we found previously with 5 h of continuous perfusion at this low temperature. The normothermic I/R procedure led to an increase in vascular resistance of approximately 250% above the value measured prior to I/R. Hypothermic ischemia totally abolished this resistance increase, except for the lowest temperature, for which the increase was 150%. Therefore, hypothermia can prevent the microvascular dysfunction caused by 3.5 h of ischemia at 37 degrees C in this preparation. However, when the temperature was reduced too far (approximately 5 degrees C), a cold injury to the microvascular resulted in permeability and resistance increases.

Animals↗
PubMed1994

Identification of microvascular transport pathways in skeletal muscle.

The solvent-drag reflection coefficient (sigma f) was measured from plasma disappearance (integral-mass balance method) for native albumin and four fluorescent solutes of radii from 2 to 16 nm in the isolated, plasma-perfused cat hindlimb preparation. The data for the smallest solutes were measured > 2 h after tracer addition and at high filtration rates to avoid underestimation of sigma f due to tracer diffusion. A two-pore model was fit (small-pore and large-pore radii, approximately 3.5 and 23 nm, respectively, 84% of hydraulic capacity in small pores) to these data using an objective computer-based estimation procedure. In the model, membrane sigma f was determined by flow weighting the sigma f values for the two pathways. Also, the phenomenon of volume circulation among the pathways was included. In different limbs, the permeability-surface area (PS) product was measured for the smallest solute, alpha-lactalbumin, from its perfusate-disappearance transient and a linear diffusion model. The PS value estimated was 0.11 +/- 0.026 (95% confidence limits) ml.min-1 times 100 g muscle-1. These PS values were found to be coincident with those predicted using parameter sets derived from the multiparameter 95% confidence space consistent with the two-pore model fits. The two-pore model also closely predicted PS data for small solutes from other studies in skeletal muscle; however, it failed to adequately describe small-molecule transport data from osmotic transient studies. It was necessary to add a water-exclusive pathway (40% of total hydraulic capacity) to account for these latter data; however, the predictions with this addition were still consistent with the data measured in the present study. We conclude that pore models can describe both macromolecular and small solute reflection coefficient and PS data in skeletal muscle.

Animals↗
PubMed1993

Platelet activating factor-induced microvascular permeability increases in the cat hindlimb.

Changes in microvascular permeability induced by platelet activating factor (PAF) were measured in the isolated, perfused cat hindlimb preparation, and compared to the effect produced by another inflammatory mediator, histamine. Permeability was assessed from changes in the protein reflection coefficient, as measured from changes in hematocrit and protein concentration resulting from microvascular fluid filtration. The findings were 1) PAF produces transient increases in permeability similar to histamine, but PAF is approximately 30 times as potent; 2) the permeability changes induced by 76 nM PAF can be totally inhibited by the specific PAF receptor blocker WEB-2086, but the blocker can only partially inhibit 380 nM PAF, a dose that produces a maximal increase in permeability; 3) Diphenhydramine (2 microM), an H1-receptor blocker, totally inhibits the transient permeability increase produced by 2 microM histamine; 4) Cimetidine (2 or 20 microM), an H2 blocker, could not inhibit this latter increase; 5) Isoproterenol (1 microM), a beta-agonist, totally inhibited the permeability increase produced by 1 microM histamine, but 10 microM isoproterenol only partially inhibited the maximal permeability increase produced by 10 microM histamine; 6) Isoproterenol could not inhibit PAF's permeability effect; and 7) PAF's effects were unchanged by depletion of white blood cells in the perfusate. These results suggest that PAF and histamine work through different pathways to increase permeability, but the final step of endothelial contraction, which opens large inter-endothelial gaps, occurs in response to both mediators. In addition, when concentrations of these inflammatory agents are sufficient to produce maximal permeability increases, as can occur in shock situations, then the permeability increases are more sustained and resistant to receptor inhibition.

Animals↗
PubMed1992

Transport parameter estimation from lymph measurements and the Patlak equation.

Two methods of estimating protein transport parameters for plasma-to-lymph transport data are presented. Both use IBM-compatible computers to obtain least-squares parameters for the solvent drag reflection coefficient and the permeability-surface area product using the Patlak equation. A matrix search approach is described, and the speed and convenience of this are compared with a commercially available gradient method. The results from both of these methods were different from those of a method reported by Reed, Townsley, and Taylor [Am. J. Physiol. 257 (Heart Circ. Physiol. 26): H1037-H1041, 1989]. It is shown that the Reed et al. method contains a systematic error. It is also shown that diffusion always plays an important role for transmembrane transport at the exit end of a membrane channel under all conditions of lymph flow rate and that the statement that diffusion becomes zero at high lymph flow rate depends on a mathematical definition of diffusion.

Animals↗
PubMed1992

Effects of cold on vascular permeability and edema formation in the isolated cat limb.

We investigated the effects of cold temperatures on microvascular protein permeability in the isolated constant-flow perfused cat hindlimb. The perfusates were 20% cat plasma-80% albumin-electrolyte solution (low-viscosity perfusate, approximately 1 cP) or whole blood (high-viscosity perfusate, approximately 4 cP). The time at low temperature (less than 10 degrees C) was less than 3 h (short term) or greater than 5 h (long term). Decreases in the solvent drag reflection coefficient (sigma f) indicated increases in permeability. The sigma f's were determined with the integral-mass balance method from measurement of changes in protein concentration and hematocrit induced by fluid filtration into the tissues. Short-term cold exposure did not increase permeability with either a low- or a high-viscosity perfusate, whereas long-term exposure with limb temperatures of approximately 5 degrees C significantly increased permeability when the perfusate was whole blood. In addition, we verified our previous prediction that flow had to be reduced to 6-8 ml.min-1.100 g-1 to avoid the hydrostatic edema caused by short-term perfusion with whole blood at approximately 5 degrees C. Also, we found that at approximately 3 degrees C histamine's permeability-increasing effect was totally abolished, whereas at approximately 20 degrees C this effect was partially inhibited. Hence, constant-flow perfusion at low temperature with whole blood can cause edema by a pressure-dependent mechanism, whereas long-term perfusion with this perfusate at low temperatures can cause a permeability increase that further compounds edema formation. Histamine is not responsible for this permeability increase.

Animals↗
PubMed1991

Microvascular permeability transients due to histamine in cat limb.

We measured the protein solvent drag reflection coefficient (sigma f) and the capillary filtration coefficient (CFC) before and after adding 1 or 10 microM histamine to the recirculating fluid (20% plasma, remainder albumin and electrolytes, hematocrit of 1-2%) perfusing the isolated cat hindlimb preparation. Transient sigma f measurements were made at 3- to 15-min intervals after histamine using a modification of the steady-state integral-mass balance method. CFC measurements were made at approximately 10-min intervals after histamine in separate experiments. A 1 microM dose of histamine caused sigma f to fall from approximately 0.8 to approximately 0.3 in 2-3 min; sigma f then returned to control in approximately 20 min. CFC response to the 1 microM histamine was a peak increase approximately 2 times control and a return to control in approximately 40 min. A 10 microM dose caused sigma f to fall rapidly to near zero. In general, recovery was much slower than for the 1 microM dose, most of the limbs not returning to control by 40 min after histamine. CFC measurements after 10 microM histamine increased only approximately 5 times control even though sigma f was near zero at the same time. CFC remained above control for approximately 60 min. The combined sigma f and CFC data could be described quantitatively if histamine simultaneously opened both short-lived large gaps (approximately 1,000 A) and a longer-lived pathway that sieved protein like the normal pathway and if the numbers of channels of each pathway closed exponentially with 4- and 15-min time constants, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Effects of cold on microvascular fluid movement in the cat limb.

We investigated the effects of low temperatures down to approximately 5 degrees C on postcapillary resistance (Rv) and isogravimetric capillary pressure (Pci) in the isolated constant-flow-perfused cat hindlimb to see if a low-temperature-induced increase in Rv and decrease in Pci could lead to an increase in filtration pressure and edema formation. A low-viscosity perfusate (20% cat plasma, 80% albumin-electrolyte solution; viscosity approximately 1 cP) was used. Isoproterenol (10(-7) M) was added to vasodilate the limb and achieve normal microvascular permeability. Rv and Pci were estimated from the slope and zero-flow intercept, respectively, of the straight-line fit to the isogravimetric venous pressure vs. flow data. Rv and Pci were determined in each experiment at an initial 37 degrees C control, at a lowered temperature (30, 23, 15, or 5-10 degrees C), and then at 37 degrees C again. The ratio of Rv at the low temperatures relative to the initial 37 degrees C control increased almost linearly as temperature was reduced. The increase was 3.4 times control at the lowest temperature. Pci decreased significantly from control only in the lowest temperature group where the change was -5.4 mmHg. Analysis of our data with the low-viscosity perfusate shows that the limb can become edematous if temperature is lowered to approximately 5 degrees C unless venous pressure (Pv) is lowered to venous collapse and flow reduced to less than approximately 20 ml.min-1.100g-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Filtration coefficient in cat hindlimb using protein concentration changes.

The maximum value of capillary filtration coefficient (CFC) in maximally vasodilated cat skeletal muscle is disputed. It was hypothesized that the wide range of reported values was caused by the inability of gravimetric and volumetric measurements of tissue volume to separate transcapillary filtration from vascular volume changes. Consequently, we developed a method of measuring filtration rates from changes in venous protein concentration using Evan's blue-labeled albumin in the isolated hindlimb (pentobarbital sodium anesthesia). The filtration coefficient (PFFC) calculated from these filtration rates after a step in venous pressure should not be influenced by vascular volume changes. When the perfusate flow rate through the hindlimb was greater than 15 ml.min-1.100 g muscle-1, PFFC was 0.0085 +/- 0.0015 (SD, n = 8) ml.min-1.mmHg-1.100 g muscle-1. PFFC was observed to be unvarying from 1 to 12 min after the venous pressure elevation, in contrast to CFC values, which fall during the same period. It is argued that the difference between CFC and PFFC values is caused by vascular volume changes.

Algorithms↗
PubMed1989

Measurement of osmotic reflection coefficient for small molecules in cat hindlimbs.

Capillary osmotic reflection coefficients (sigma) for NaCl, urea, sucrose, and raffinose were measured in the isolated, perfused cat hindlimb using the osmotic transient technique. sigma were determined from the ratio of the maximum rate of transcapillary absorption [delta Jv(max)] to the increase in the osmotic pressure (25-35 mosmol/kg H2O) in the arterial inflow (delta pi a) produced by adding one of the molecules to an albumin-electrolyte perfusate containing isoproterenol (greater than 10(-7) M). delta Jv (max) was determined from organ weight and delta pi a from perfusate osmolalities. For each molecule, the delta Jv(max)/delta pi a ratio increased monotonically with perfusate flow rates (Q) to Q greater than 100 ml.min-1.100 g-1. This ratio was independent of the size of the delta pi a. Apparent sigma values were calculated by dividing these ratios by the capillary hydraulic capacity determined in other studies. At low Q, apparent sigma was comparable to the approximately 0.1 values found by others in skeletal muscle. At the highest Q, apparent sigma for these molecules were at least 0.5. These data are consistent with at least 50% of transcapillary water flow moving through a water-exclusive pathway.

Algorithms↗
PubMed1989

Effects of elevated venous pressure on capillary permeability in cat hindlimbs.

We investigated the effects of elevated venous pressure, Pv, (up to 140 mmHg) on the solvent drag reflection coefficient, sigma f, for protein and on the capillary filtration coefficient, CFC, in the isolated cat hindlimb perfused at constant flow. The perfusate contained 30% cat plasma and the remainder was a dialyzed albumin-electrolyte mixture. Cat red cells were added to a hematocrit of approximately 2%. sigma f was measured from the changes in hematocrit and plasma protein concentration (Integral-Mass Balance method) resulting from the fluid filtration caused by the Pv elevation. CFC was measured from the slope of the limb weight recording 2-4 min after the Pv elevation. sigma f decreased linearly from 0.807 (Pv less than 50 mmHg) to approximately 0.2 at 140 mmHg. CFC increased linearly from 0.0086 ml.min-1.mmHg-1.100 g-1 to about 0.04 over the same pressure range. A weight-independent filtration coefficient calculated from the change in hematocrit and a measurement of the initial perfusate volume gave comparable results, except at the very highest of pressures, where this coefficient was sometimes 20-40% less than CFC. Successive sigma f determinations at Pv at about 40 mmHg did not return to control after an initial measurement in which Pv was approximately 110 mmHg. Pore-theory analysis of the data suggests that the elevated Pv causes large pores to open as opposed to the stretching of small pores. Also, these large pores may remain open for a period of hours.

Animals↗
PubMed1987

Blood and isoproterenol reduce capillary permeability in cat hindlimb.

In an earlier study, plasma was observed to counteract the permeability-increasing effects of blood-free perfusion when papaverine was present in the perfusate. To determine if this plasma effect was still present in the absence of papaverine, cat hindlimbs were perfused with a blood-free albumin-electrolyte solution, and capillary filtration coefficient (CFC) was measured. These data were compared with CFC values obtained when blood, plasma, or isoproterenol were present in the albumin perfusate. In separate groups of animals, CFC was observed to be 0.017 +/- 0.006 (SD, n = 26) ml X min-1 X mmHg-1 X 100 g muscle-1 during blood-free albumin perfusion, 0.012 +/- 0.002 (n = 24) when blood was present, 0.014 +/- 0.003 when isoproterenol was present, and 0.010 +/- 0.002 (n = 33) when both blood and isoproterenol were present. In a separate study, it was observed that 1) isoproterenol could reduce CFC by 11% when added to blood-free perfusate, 2) plasma had a similar but smaller and more variable effect than isoproterenol, 3) plasma had no consistent effect on CFC when added to a blood-free albumin perfusate containing isoproterenol, and 4) blood could reduce CFC significantly (P less than 0.005), from 0.014 +/- 0.003 to 0.0094 +/- 0.002 (n = 7), when added to a blood-free albumin perfusate containing isoproterenol. It was concluded that, in the absence of papaverine, the principal CFC-reducing effect of blood lay in the cell fraction and that isoproterenol had a small CFC-reducing effect under blood-free conditions. This result contrasts with the plasma effect reported earlier.

Animals↗