Cell-free layer and wall shear stress variation in microvessels

In this study, we simulated multiple red blood cells flowing through straight microvessels with the immersed-boundary lattice-Boltzmann model to examine the shear stress variation on the microvessel surface and its relation to the properties of cell-free layer. Significant variation in shear stress has been observed due to the irregular configuration of blood cells flowing near the microvessel wall. A low shear stress is typically found at locations where there is a cell flowing close to the wall, and a large shear stress at locations with a relatively wide gap between cell and wall. This relationship between the shear stress magnitude and the distance between cell and wall has been attributed to the reverse pressure difference developed between the front and rear sides of a cell flowing near the vessel wall. We further studied the effects of several hemodynamic factors on the variation of shear stress, including the cell deformability, the flow rate, and the aggregation among red blood cells. These simulations show that the shear stress variation is less profound in situations with wider cell-free layers, since the reverse pressure difference around the edge cells is less evident, and the influence of this pressure difference on wall shear stress becomes weaker. This study also demonstrates the complexity of the flow field in the gap between cell and wall. More precise experimental techniques are required accurately measure such shear stress variation in microcirculation.     

Expression of P-glycoprotein in human cerebral cortex microvessels.

P-Glycoprotein (P-gp) is an ATP-dependent efflux transporter that extrudes non-polar molecules, including cytotoxic substances and drugs, from the cells. It was initially found in cancer cells and then was shown to be a normal component of complex transport systems working at the blood-brain barrier (BBB). Previous studies have demonstrated that, in the brain, P-gp is localized on the luminal plasmalemma of BBB endothelial cells and that it may interact with the caveolar compartment of these cells. The aim of this study was to identify the site of cellular expression of P-gp in human brain in situ and to morphologically determine whether an association may exist between P-gp and caveolin-1, a structural and functional protein of the caveolar frame. The study was carried out on human cerebral cortex by immunoconfocal microscopy with antibodies to both P-gp and caveolin-1. The results show that P-gp marks the microvessels of the cortex and that the transporter is localized in the luminal endothelial compartment, where it co-localizes with caveolin-1. The demonstration of this co-localization of P-gp with caveolin-1 contributes a morphological backing to biochemical studies on P-gp/caveolin-1 relationships and leads us to suggest that interactions between these molecules may occur at the BBB endothelia.     

Membrane-bound lipoxygenase of rat cerebral microvessels

The microvessels isolated from rat cerebral cortex has arachidonate lipoxygenase activity, which was not due to possible contamination of the platelets. The major product was identified to be 12-hydroxyeicosatetraenoic acid. After homogenization and sonication of the microvessel preparations, the lipoxygenase activity was recovered both in the membrane- and the cytosol-fractions, whereas that in the platelets was recovered in the cytosol fraction. Membrane-bound lipoxygenase of the microvessels has apparent Km value of 3.8 microM for arachidonic acid, which was corresponded to 1/5 of that in the platelet enzyme. Microvessel lipoxygenase was inhibited by nordihydroguaiaretic acid but not by indomethacin.     

Protection against radical peroxidations in cerebral aging in cerebral capillaries and microvessels]

Systemic injection of substances that generate free radicals into the rat induces deleterious alteration of various tissues (as demonstrated by the production of conjugated dienes and malondialdehyde) but only has a slight effect on the brain. This shows that the blood-brain barrier has important protective properties. In fact the cerebral capillaries and microvessels have superoxide dismutase, glutathione peroxidase, and catalase activities higher than those found in the rest of the cerebral tissue during aging. These activities vary little, except for catalase which decreases. But, curiously, the concentrations of Mn, Cu, and Zn are not related to enzymatic activity, although the micro-elements are necessary for the activity of superoxide dismutase. On the other hand, during aging, the capillaries and cerebral microvessels undergo extensive modifications at the level of the polyunsaturated fatty acids: for example, the concentration of arachidonic acid decreases by half.     

Parathyroid hormone stimulates adenylate cyclase in rat cerebral microvessels

We have studied the effect of parathyroid hormone (PTH) on adenylate cyclase of microvessels isolated from rat cerebral cortex. Native bovine (b) PTH-(1–84), the synthetic amino-terminal fragment bPTH-(1–34) and the synthetic analog [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH- (1–34) amide stimulated adenylate cyclase in a dose-dependent manner with apparent ED₅₀ values of 16 nM, 6.3 nM and 15 nM respectively. The stimulation by bPTH was greatly enhanced by guanosine triphosphate. The PTH antagonist, [Nle⁸, Nle¹⁸, Tyr³⁴]-bPTH-(3–34) amide inhibited the action of bPTH-(1–84) and bPTH-(1–34). In summary, PTH stimulated adenylate cyclase in rat cerebral microvessels in a very similar manner to its stimulation in the renal cortex.     

Oxygen tension in cerebral microvessels in acute anaemia in rats

Using polarographic oxygen microelectrodes, distribution of oxygen tension (pO2) in the rat cerebral arterioles (with a lumen diameter of 8-80 microm) and venules (with a lumen diameter of 8-120 microm) has been studied in acute reduction of haemoglobin concentration in the blood. Isovolumic haemodilution with 5 % albumin solution has been performed stepwise from 14 g/dl (control) to 10 g/dl (step 1), 7 g/dl (step 2) and to 4.6 g/dl (step 3). It was shown that step 1 of haemodilution led to no impairment of oxygen supply to the brain cortex. Step 2 resulted in moderate increase of pO2 in arterioles, whereas in venules oxygen tension fell down substantially (on the average, to 32 mm Hg). Step 3 resulted insignificant increase of pO2 in arterioles. A further fall of pO2 (to 27 mm Hg) in studied venules was recorded. The portion of venules with low pO2 grew to 31% (only 3 % in control). Microregions with a near-to-zero pO2 were recorded in some capillaries. This indicates presence of hypoxic zones in brain tissue. Hypoxic and anoxic microregions originate at this stage of anemia in locations with relatively low and/or impaired blood supply.     

Prostaglandin D synthase in microvessels from the cat cerebral cortex

Microvessels, a mixture composed predominantly of small arterioles and capillaries (7–80μ diameter), were isolated from the rat cerebral cortex by selective nylon sieving and glass bead elutriation. The morphology and purity of the microvessel and cerebral cortex filtrate (virtually free of vascular contamination) were monitored by light microscopy and by the activity of several enzymes: γ-glutamyl transpeptidase, GSH-transferase, prostacyclin synthase and PGD synthase. Prostacyclin and PGD synthesizing activities as well as γ-glutamyl transpeptidase activity were localized to the microvessels of the rat cerebral cortex whereas GSH-S-transferase was restricted to the non-vascular filtrate function. The characteristics of the PGD synthase were similar to those of the purified enzyme previously described for the rat brain. The microvessel (MV) PGD synthase was localized to the cytosol fraction of the microvessels and did not require reduced glutathione for activity. The enzyme was inhibitd by pre-incubation with p-hydroxymercuribenzoate (lmM) or N-ethylmaleimide (lmM). The MV PGD synthase saturated at 15–20μM PGH₂, exhibited an apparent K_M of 9.6μM, and a pH optimum of 8.0–8.1. These findings suggest roles for both prostacyclin and PGD synthesis by the rat cerebral vasculature in the autoregulation of cerebral blood flow and/or function. These studies also indicate that the major source of PGI₂ and PGD₂ synthesis by rat brain homogenates is the microvasculature.     

Diabetes-related changes in the protein composition of rat cerebral microvessels

To determine the effect of diabetes mellitus on cerebral microvessel protein composition, post translational modification of proteins with glucose and malondialdehyde (MDA) was determined and the abundant protein species found in cerebral microvessels isolated from control and streptozotocin-induced diabetic rats were studied. Two dimensional gel electrophoresis and computer assisted densitometry revealed that only one out of 25 quantitated proteins was significantly altered in diabetic rats after 5 weeks of uncontrolled hyperglycemia. The level of glycosylation of cerebral microvessel protein mixture was significantly increased in diabetic rats compared to control rats (168.8±25 vs 109.5±4.8 nmol/mg) (p<0.05). Western blot analysis of cerebral microvessel proteins from diabetic rats using a specific antibody against MDA-modified proteins revealed three protein spots with molecular weights of approximately 60,000 Kd. These were shown not to be contaminants from cerebral tissue or plasma proteins modified with MDA. It is concluded that short duration of streptozotocin-induced diabetes mellitus in rats is associated with some qualitative changes in protein composition of cerebral microvessels. These changes may contribute to the diabetes-related alterations in the blood-brain barrier.     

Effect of osmolarity on potassium transport in isolated cerebral microvessels

Potassium transport in microvessels isolated from rat brain by a technique involving density gradient centrifugation was studied in HEPES buffer solutions of varying osmolarity from 200 to 420 mosmols, containing different concentration of sodium chloride, choline chloride, or sodium nitrate. The flux of 86Rb (as a tracer for K) into and out of the endothelial cells was estimated. Potassium influx was very sensitive to the osmolarity of the medium. Ouabain-insensitive K-component was reduced in hypotonic medium and was increased in medium made hypertonic with sodium chloride or mannitol. Choline chloride replacement caused a large reduction in K influx. Potassium influx was significant decrease when nitrate is substituted for chloride ion in isotonic and hypertonic media, whereas a slight decrease was found in hypotonic medium. The decrease of K influx in the ion-replacement medium is due to a decrement of the ouabain-insensitive component. Potassium efflux was unchanged in hypotonic medium but was somewhat reduced in hypertonic medium. The marked effect of medium osmolarity on K fluxes suggests that these fluxes may be responsible for the volume regulatory K movements. The possible mechanism of changes of K flux under anisotonic media is also discussed.     

Regulation of CO production in cerebral microvessels of newborn pigs

Carbon monoxide (CO) is produced from heme by heme oxygenase-2 (HO-2) in cerebral blood vessels. Gas chromatography-mass spectrometry was used on piglet cerebral microvessels to address the hypothesis that CO production is regulated by heme delivery and HO-2 catalytic activity. CO production appears to be substrate limited because heme and its precursor aminolevulinate increase CO production. Ionomycin also increases CO production. However, CO production from exogenous heme was the same in Ca-replete medium, Ca-free medium with ionomycin, and Ca-replete medium with ionomycin. Phorbol myristate acetate increases CO production but does not change the catalytic activity of HO-2. Also, the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine has no effect on the HO-2 catalytic activity. Protein tyrosine kinase inhibition reduces HO-2 catalytic activity. Inhibition of protein tyrosine phosphatases increased HO-2 catalytic activity. Therefore, regulation of CO production by cerebral microvessels can include changing heme availability and HO-2 catalytic activity. HO-2 catalytic activity is stimulated by tyrosine phosphorylation.     

Adrenergic receptors on cerebral microvessels in control and parkinsonian subjects

The binding of adrenergic ligands (3H-prazosin, 3H-clonidine, 3H-dihydroalprenolol) was studied on a preparation of cerebral microvessels in the prefrontal cortex and putamen of control and Parkinsonian subjects. The adrenergic receptor density in microvessels of control patients was less than 0.5% and 3.3% respectively of the total binding. A significant decrease in the number of alpha-1 binding sites was observed on microvessels in the putamen of patients with Parkinson's disease.     

Prostaglandin D synthase in microvessels from the rat cerebral cortex

Microvessels, a mixture composed predominantly of small arterioles and capillaries (7-80 micro diameter), were isolated from the rat cerebral cortex by selective nylon sieving and glass bead elutriation. The morphology and purity of the microvessel and cerebral cortex filtrate (virtually free of vascular contamination) were monitored by light microscopy and by the activity of several enzymes: gamma -glutamyl transpeptidase, GSH-S-transferase, prostacyclin synthase and PGD synthase. Prostacyclin and PGD synthesizing activities as well as gamma-glutamyl transpeptidase activity were localized to the microvessels of the rat cerebral cortex whereas GSH-S-Transferase was restricted to the non-vascular filtrate fraction. The characteristics of the PGD synthase were similar to those of the purified enzyme previously described for the rat brain. The microvessel (MV) PGD synthase was localized to the cytosol fraction of the microvessels and did not require reduced glutathione for activity. The enzyme was inhibited by pre-incubation with p-hydroxymercuribenzoate (ImM) or N-ethylmaleimide (ImM). The MV RGD synthase saturated at 15-20 microM PGH2, exhibited an apparent Km of 9.6 microM, and a pH optimum of 8.0-8.1. These findings suggest roles for both prostacyclin and PGD synthesis by the rat cerebral vasculature in the autoregulation of cerebral blood flow and/or neural function. These studies also indicate that the major source of PGI2 and PGD2 synthesis by rat brain homogenates is the microvasculature.     

Transmural gradients of oxygen tension on cerebral microvessels in rats

Using modified oxygen needle microelectrodes, vital microscopy with video-recording facilities, measurements of tissue oxygen tension (PO2) profiles near the cortical arterioles and transmural PO2 gradients on pial arterioles of the rat were performed. At control transmural PO2 gradient averaged 1.17 +/- 0.06 mm Hg/microm (mean +/- SEM, n = 40). Local dilatation of the arteriolar wall (microapplication of sodium nitroprusside approximately 2 x 10(-7) M) resulted in marked drop of the transmural PO2 gradient to 0.68 +/- 0.04 mm Hg/microm (p < 0.001, n = 38). The important finding of the study is the dependence of the transmural PO2 gradient on the vascular tone of pial arterioles. The data presented allow to conclude that O2 consumption of the arteriolar wall lies within the range for surrounding tissue and O2 consumption of the endothelial layer and, apparently, has no substantial impact on transmural PO2 gradient.     

Mechanism of glutamate stimulation of CO production in cerebral microvessels

Dilation of piglet pial arterioles to glutamate involves carbon monoxide (CO) produced from heme by heme oxygenase-2 (HO-2). Piglet cerebral microvessels and endothelial and smooth muscle cells grown on microcarrier beads were used to address the hypothesis that glutamate increases endothelial CO production by increasing HO-2 catalytic activity. CO was measured by gas chromatography/mass spectrometry. Glutamate increased CO production from endogenous heme by cerebral microvessels, endothelial cells, and smooth muscle cells. Glutamate increased the conversion of exogenous heme to CO. Protein tyrosine kinase inhibition blocked glutamate stimulation of CO production. Inhibition of protein tyrosine phosphatases stimulated CO production. Conversely, neither phorbol myristate acetate nor H-7 changed glutamate stimulation of CO production. The mechanism of HO-2 stimulation by glutamate appears to be independent of cytosolic Ca, because stimulation of CO production by glutamate was the same in Careplete medium, Ca-free medium with ionomycin, and Careplete medium with ionomycin. Therefore, glutamate appears to increase HO-2 catalytic activity in cerebral microvessels via a tyrosine kinase mediated pathway.     

Red blood cell velocity and oxygen tension measurement in cerebral microvessels by double-wavelength photoexcitation.

Because the regulation of microcirculation in the cerebral cortex cannot be analyzed without measuring the blood flow dynamics and oxygen concentration in cerebral microvessels, we developed a fluorescence and phosphorescence system for estimating red blood cell velocity and oxygen tension in cerebral microcirculation noninvasively and continuously with high spatial resolution. Using red blood cells labeled with fluorescent isothiocyanate to visualize red cell distribution and using the oxygen quenching of Pd-meso-tetra-(4-carboxyphenyl)-porphyrin phosphorescence to measure oxygen tension enabled simultaneous measurement of blood velocity and oxygen tension. We examined how the measurement accuracy was affected by the spatial resolution and by the excitation laser light passing through the targeted microvessel and exciting the oxygen probe dye in the tissue beneath it. Focusing the excitation light into the microvessel stabilized the phosphorescence lifetime at each spatial resolution; moreover, it greatly reduced phosphorescence from the brain tissue. Animal experiments involving acute hemorrhagic shock demonstrated the feasibility of our system by showing that the changes in venular velocity and oxygen tension are synchronized to the change in mean arterial pressure. Our system measures the red cell velocity and oxygen concentration in the cerebral microcirculation by using the differences in luminescence and wavelength between fluorescence and phosphorescence, making it possible to easily acquire information about cerebral microcirculatory distribution and oxygen tension simultaneously.     

Osmotic reflection coefficient for total plasma protein in lung microvessels

The osmotic reflection coefficient (sigma) for total plasma proteins was estimated in 11 isolated blood-perfused canine lungs. Sigma's were determined by first measuring the capillary filtration coefficient (Kf,C in ml X min-1 X 100g-1 X cmH2O-1) using increased hydrostatic pressures and time 0 extrapolation of the slope of the weight gain curve. Kf,C averaged 0.19 +/- 0.05 (mean +/- SD) for 14 separate determinations in the 11 lungs. Following a Kf,C determination, the isogravimetric capillary pressure (Pc,i) was determined and averaged 9.9 +/- 0.5 cmH2O for all controls reported in this study. Then the blood colloids in the perfusate were either diluted or concentrated. The lung either gained or lost weight, respectively, and an initial slope of the weight gain curve (delta W/delta t)0 was estimated. The change in plasma protein colloid osmotic pressure (delta IIP) was measured using a membrane osmometer. The measured delta IIP was related to the effective colloid osmotic pressure (delta IIM) by delta IIM = (delta W/delta t)0/Kf,C = sigma delta IIP. Using this relationship, sigma averaged 0.65 +/- 0.06, and the least-squares linear regression equation relating Pc,i and the measured IIP was Pc,i = -3.1 + 0.67 IIP. The mean estimate of sigma (0.65) for total plasma proteins is similar to that reported for dog lung using lymphatic protein flux analyses, although lower than estimates made in skeletal muscle using the present methods (approximately 0.95).     

Cell-free translation in reversed micelles.

Cell-free translation in reversed micelles (RM) of surfactants in organic solvents is demonstrated using as an example the synthesis of human interleukin-2 by the wheat germ translation system solubilized in Brij 96 (oleyl-poly(10)oxyethylene ether) RM in cyclohexane. The translation system components and the product were recovered from the RM system by acetone precipitation. The recovery and translation reaction yields depended on the degree of surfactant hydration. The translation yields in Brij 96 RM were close to that observed in regular aqueous solution. The Brij 96 RM system is regarded as a promising media for the cell-free synthesis of hydrophobic proteins. Meanwhile, no translation reaction was observed in Aerosol OT (sodium bis(2-ethylhexyl) sulfosuccinate) RM in octane, which presumably is due to the ability of Aerosol OT to bind Mg2+ ions necessary for the functioning of the translation apparatus.     

Role of microtubules in the regulation of metabolism in isolated cerebral microvessels

We used¹³C-labeled substrates and nuclearmagnetic resonance spectroscopy to examine carbohydrate metabolism invascular smooth muscle of freshly isolated pig cerebral microvessels(PCMV). PCMV utilized[2-¹³C]glucose mainlyfor glycolysis, producing[2-¹³C]lactate.Simultaneously, PCMV utilized the glycolytic intermediate [1-¹³C]fructose1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing[1-¹³C]glucose withonly minor[3-¹³C]lactateproduction. The dissimilarity in metabolism of[2-¹³C]FBP derivedfrom [2-¹³C]glucosebreakdown and metabolism of exogenous[1-¹³C]FBPdemonstrates that carbohydrate metabolism is compartmented in PCMV.Because glycolytic enzymes interact with microtubules, we disruptedmicrotubules with vinblastine. Vinblastine treatment significantlydecreased[2-¹³C]lactate peakintensity (87.8 ± 3.7% of control). The microtubule-stabilizing agent taxol also reduced[2-¹³C]lactate peakintensity (90.0 ± 2.4% of control). Treatment with both agentsfurther decreased[2-¹³C]lactateproduction (73.3 ± 4.0% of control). Neither vinblastine, taxol,or the combined drugs affected[1-¹³C]glucose peakintensity (gluconeogenesis) or disrupted the compartmentation ofcarbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest thatthey produce their effects on glycolytic rate by competing withglycolytic enzymes for binding, not by affecting the overall assemblystate of the microtubule network. Glycolysis, but not gluconeogenesis,may be regulated in part by glycolytic enzyme-microtubule interactions.