Involvement of autophagy in the maintenance of rat intervertebral disc homeostasis: an in-vitro and in-vivo RNA interference study of Atg5.

OBJECTIVE: In the largest avascular low-nutrient intervertebral disc, resident cells would utilize autophagy, a stress-response survival mechanism by self-digestion and recycling wastes. Our goal was to elucidate the involvement of autophagy in disc homeostasis through RNA interference of autophagy-related gene 5 (Atg5). DESIGN: In vitro, small interfering RNAs (siRNAs) targeting autophagy-essential Atg5 were transfected into rat disc cells. Cell viability with levels of autophagy including Atg5 expression, apoptosis, and senescence was assessed under serum starvation and/or pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. In vivo, time-course autophagic flux was monitored following Alexa Fluor® 555-labeled Atg5-siRNA injection into rat tail discs. Furthermore, 24-h temporary static compression-induced disruption of Atg5 siRNA-injected discs was observed by radiography, histomorphology, and immunofluorescence. RESULTS: In disc cells, three different Atg5 siRNAs consistently suppressed autophagy with Atg5 protein knockdown (mean 44.4% [95% confidence interval: -51.7, -37.1], 51.5% [-80.5, -22.5], 62.3% [-96.6, -28.2]). Then, Atg5 knockdown reduced cell viability through apoptosis and senescence not in serum-supplemented medium (93.6% [-0.8, 21.4]) but in serum-deprived medium (66.4% [-29.8, -8.6]) further with IL-1ß (44.5% [-36.9, -23.5]). In disc tissues, immunofluorescence detected intradiscal signals for the labeled siRNA even at 56-d post-injection. Immunoblotting found 56-d autophagy suppression with prolonged Atg5 knockdown (33.2% [-52.8, -5.3]). With compression, Atg5 siRNA-injected discs presented radiographic height loss ([-43.9, -0.8]), histological damage ([-5.5, -0.2]), and immunofluorescent apoptosis ([2.2, 22.2]) and senescence ([4.1, 19.9]) induction compared to control siRNA-injected discs at 56 d. CONCLUSIONS: This loss-of-function study suggests Atg5-dependent autophagy-mediated anti-apoptosis and anti-senescence. Autophagy could be a molecular therapeutic target for degenerative disc disease.

Pharmacological inhibition of mTORC1 but not mTORC2 protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism through Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth. We hypothesized that mTOR is influential in the intervertebral disc-largest avascular, low-nutrient organ. Our objective was to identify the optimal mTOR inhibitor for treating human degenerative disc disease. DESIGN: mTOR complex 1 (mTORC1) regulates p70/ribosomal S6 kinase (p70/S6K), negatively regulates autophagy, and is controlled by Akt. Akt is controlled by phosphatidylinositol 3-kinase (PI3K) and mTOR complex 2 (mTORC2). mTORC1 inhibitors-rapamycin, temsirolimus, everolimus, and curcumin, mTORC1&mTORC2 inhibitor-INK-128, PI3K&mTOR inhibitor-NVP-BEZ235, and Akt inhibitor-MK-2206-were applied to human disc nucleus pulposus (NP) cells. mTOR signaling, autophagy, apoptosis, senescence, and matrix metabolism were evaluated. RESULTS: mTORC1 inhibitors decreased p70/S6K but increased Akt phosphorylation, promoted autophagy with light chain 3 (LC3)-II increases and p62/sequestosome 1 (p62/SQSTM1) decreases, and suppressed pro-inflammatory interleukin-1 beta (IL-1ß)-induced apoptotic terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity (versus rapamycin, 95% confidence interval (CI) -0.431 to -0.194; temsirolimus, 95% CI -0.529 to -0.292; everolimus, 95% CI -0.477 to -0.241; curcumin, 95% CI -0.248 to -0.011) and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage, senescent senescence-associated beta-galactosidase (SA-ß-gal) positivity (versus rapamycin, 95% CI -0.437 to -0.230; temsirolimus, 95% CI -0.534 to -0.327; everolimus, 95% CI -0.485 to -0.278; curcumin, 95% CI -0.210 to -0.003) and p16/INK4A expression, and catabolic matrix metalloproteinase (MMP) release and activation. Meanwhile, dual mTOR inhibitors decreased p70/S6K and Akt phosphorylation without enhanced autophagy and suppressed apoptosis, senescence, and matrix catabolism. MK-2206 counteracted protective effects of temsirolimus. Additional disc-tissue analysis found relevance of mTOR signaling to degeneration grades. CONCLUSION: mTORC1 inhibitors-notably temsirolimus with an improved water solubility-but not dual mTOR inhibitors protect against inflammation-induced apoptosis, senescence, and matrix catabolism in human disc cells, which depends on Akt and autophagy induction.

Selective interference of mTORC1/RAPTOR protects against human disc cellular apoptosis, senescence, and extracellular matrix catabolism with Akt and autophagy induction.

OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1ß) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1ß-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1ß-induced senescence-associated beta-galactosidase (SA-ß-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1ß-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

Expression of cyclooxygenase-2 and nitrotyrosine in human gastric mucosa before and after Helicobacter pylori eradication.

To determine whether cure of Helicobacter pylori infection influences the expression of COX-2 and nitrotyrosine in the distal stomach of humans, biopsy specimens were examined immunohistochemically. H. pylori infection was determined using a rapid urease test, culture and histology. Positive staining of COX-2/nitrotyrosine in the epithelium was expressed as the percentage of stained cells to the total epithelial cells. There was a significant increase in COX-2/nitrotyrosine staining in H. pylori -positive subjects compared with H. pylori -negative subjects. Cure of the infection resulted in a significant decrease in both COX-2/nitrotyrosine staining in all patients (52.1+/-12.1% vs 15. 4+/-7.2%, P<0.001; and 57.3+/-13.6% vs 36.1+/-18.0%, P<0.01, respectively). However, immunoreactivity of COX-2/nitrotyrosine was observed in all cases with intestinal metaplasia even after the cure of H. pylori infection.Thus, cure of H. pylori infection may decrease the risk of gastric carcinogenesis due to COX-2 and NO-related compounds in gastric mucosa but not in those patients with intestinal metaplasia.

Thrombotic thrombocytopenic purpura developed suddenly during interferon treatment for chronic hepatitis C.

A 57-year-old man had abnormal hepatic function identified in April 1994. In October 1994, chronic hepatitis C was diagnosed. Based on the findings of a liver biopsy, administration of recombinant interferon (rIFN)-alpha2b was begun. In the 16th week of treatment, the patient experienced headache and fever and developed a markedly decreased, platelet count and hemolytic anemia. He was admitted on May 19, 1995 and thrombotic thrombocytopenic purpura (TTP) was diagnosed. He died on the 3rd hospital day. The causes of TTP have yet to be elucidated, but in this patient the occurrence of TTP appeared to be related to the IFN treatment for chronic hepatitis C.

Action spectrum for sporulation and photoavoidance in the plasmodium of Physarum polycephalum, as modified differentially by temperature and starvation.

The plasmodium of the myxomycete Physarum polycephalum sporulates in bright natural environments, suggesting a relationship between photobehavior and sporulation. Thus, the action spectra for two light-dependent phenomena as well as the effects of other environmental conditions have been studied. Sporulation like photo-avoidance responded to UVC (near 270 nm) and near IR (near 750 nm) in addition to the well-documented UVA (near 350 nm) and blue (near 460 nm) regions. Sporulation and photoavoidance had similar sensitivities in the shorter wavelengths, while the former was about 100 times more sensitive in near IR. The plasmodium moved away from light in a wide spectral range. Starvation and high temperature at 31 degrees C (25 degrees C in standard conditions) reduced photoavoidance to UVA and to blue light, respectively. A high fluence rate of UVC suppressed the rhythmic contraction of the plasmodium, and the action spectrum peaked at 270 nm. These results indicate that the Physarum plasmodium may stay at brighter places not by positive phototaxis but by weakening the negative phototaxis to sunlight or by other possible taxes such as hydrotaxis. There may be at least four different photo-systems in the plasmodium.

Light irradiation induces fragmentation of the plasmodium, a novel photomorphogenesis in the true slime mold Physarum polycephalum: action spectra and evidence for involvement of the phytochrome.

A new photomorphogenesis was found in the plasmodium of the true slime mold Physarum polycephalum: the plasmodium broke temporarily into equal-sized spherical pieces, each containing about eight nuclei, about 5 h after irradiation with light. Action spectroscopic study showed that UVA, blue and far-red lights were effective, while red light inhibited the far-red-induced fragmentation. Difference absorption spectra of both the living plasmodium and the plasmodial homogenate after alternate irradiation with far-red and red light gave two extremes at 750 and 680 nm, which agreed with those for the induction and inhibition of the fragmentation, respectively. A kinetic model similar to that of phytochrome action explained quantitatively the fluence rate-response curves of the fragmentation. Our results indicate that one of the photoreceptors for the plasmodial fragmentation is a phytochrome.

The tumour-localizing properties of porphyrin derivatives.

The tumour-localizing abilities of various kinds of porphyrin derivatives in tumour-bearing hamsters were assessed by nitrogen-pulsed laser spectrofluorometry (N2-PLS). On examination of porphine derivatives (from haemoglobin), it was found that the dimer and acetylated and amidated compounds had a high affinity for tumour tissue; the dimer and hydroxylated compound of phorbine derivatives (from chlorophyll) also showed a high affinity. Furthermore, of the metalloporphines (gallium, zinc and indium complexes), those which contained hydrophilic groups showed a high affinity for tumour tissue; of the metallophorbines (gallium, zinc and indium complexes), those which contained hydrophobic groups showed a high affinity. A correlation was found between the side-chain structure of the porphyrins and metalloporphyrins and their affinity for tumour tissue.

Effect of plasma alpha1-acid glycoprotein concentration on the accumulation of lidocaine metabolites during continuous epidural anesthesia in infants and children.

OBJECTIVE: Alpha1-acid glycoprotein (AAG) is an acute-phase protein that is responsible for binding basic drugs such as lidocaine (LDC). The effect of AAG on the duration of LDC during continuous epidural anesthesia in infants and young children was investigated. PATIENTS, MATERIALS AND METHODS: Plasma levels of LDC and its active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), were monitored in 20 infants and children, 5 months to 6 years of age, who received continuous epidural infusion of 2.5 mg kg(-1) LDC hourly during abdominal or thoracic surgeries. RESULTS: Plasma LDC concentrations were constant after the first hour of injection. In contrast, the concentrations of MEGX and GX increased continuously during epidural infusion in all patients. The plasma AAG concentration correlated significantly (r = 0.814, p<0.001) with the steady-state LDC level. In addition, significant inverse correlation was observed between the plasma AAG concentration and the accumulation rate of MEGX (r = 0.742, p = 0.002). The plasma AAG concentration and the accumulation rate of GX correlated weakly (r = 0.474, p = 0.035). There was no correlation between the age of the patient and the plasma AAG concentrations (r = 0.295, p = 0.206). CONCLUSION: Our results suggest that the plasma AAG concentration is a valuable index in preventing the toxicity caused by accumulation of MEGX during continuous epidural anesthesia of LDC.

Chromatographic determination of free lidocaine and its active metabolites in plasma from patients under epidural anesthesia.

OBJECTIVE: We developed a simple and selective assay method for simultaneous determination of free lidocaine (LDC) and its active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX) in plasma, by using high-performance liquid chromatography (HPLC). The method was applied to the plasma concentration monitoring in continuous epidural anesthesia with LDC. MATERIALS AND METHODS: Free fraction was separated from plasma by using an ultrafiltration technique. Free and total LDC, MEGX and GX in plasma were analyzed by HPLC equipped with ordinary octadecylsilyl silica (ODS) column and ultraviolet (UV) detector. PATIENTS: Five male patients with cancer who received epidural injection of 1.5% LDC for 5 hours in elective thoracic surgery, were enrolled to determine the plasma levels of total and free LDC, MEGX and GX. RESULTS AND DISCUSSION: The calibration curve for free LDC, MEGX and GX were linear at the concentration of 25 to 1,000 ng ml(-1) (r = 0.9998 - 0.9999). The recoveries for LDC, MEGX and GX from plasma water were ranged 73.2-89.1%. The coefficient variations for intra- and inter-day assay for LDC, MEGX and GX were less than 4.1%. The detection limit ofeach drug was 20 ng ml(-1). Plasma-free MEGX after 180 min epidural injection was higher than free LDC, even though the total concentration of MEGX was 4 times lower than that of LDC. The percentages of free fraction for LDC, MEGX and GX were 11.7, 48.5 and 78.3% after 5-hour epidural administration of LDC. Since the free fraction of MEGX and GX increases and exceeds the concentration of free LDC during continuous epidural anesthesia, accumulation of these toxic metabolites should be carefully monitored as well as LDC. CONCLUSION: The present method is a reliable technique and can be applied to monitoring free LDC, MEGX and GX, which provide us beneficial information as to the LDC metabolism and toxicity.

Plasma lidocaine, monoethylglycinexylidide, and glycinexylidide concentrations after epidural administration in geriatric patients.

BACKGROUND AND OBJECTIVES: The purpose of this study was to evaluate the effect of age on the pharmacokinetics of lidocaine after epidural administration. METHODS: Two percent lidocaine with epinephrine (5 microg/mL) was administered in two different age groups: an adult group (age 42 +/- 6 years, n = 10) and an elderly group (age 77 +/- 4 years, n = 10). Concentrations of lidocaine and its active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), were measured in plasma samples obtained after 15, 30, 45, 60, 90, 120, 150, and 180 minutes of administration using high-performance liquid chromatography with ultraviolet detection. RESULTS: No significant differences in plasma concentrations of lidocaine and its metabolites were observed between the two groups during the 3 hours of study. However, the elderly group showed significantly longer mean residence times (MRTs) and lower plasma clearance of lidocaine during the period compared with the adult group (P < .05). Plasma concentration ratios of MEGX/lidocaine were significantly lower in the elderly group after 2 hours of lidocaine administration (P < .05). CONCLUSIONS: The increase in plasma lidocaine concentration after epidural anesthesia in elderly patients was not as high as anticipated. However, the elderly patients showed longer MRTs, lower clearance, and lower ratios of MEGX/lidocaine than did the adult (middle-age) patients.

Plasma concentrations of lidocaine and its principal metabolites during continuous epidural infusion of lidocaine with or without epinephrine.

BACKGROUND AND OBJECTIVES: The purpose of this study was to evaluate the effect of epinephrine on the absorption of lidocaine and the accumulation of active metabolites of lidocaine during continuous epidural anesthesia. METHODS: Lidocaine was administered as an initial bolus of 5 mg/kg of 2% lidocaine solution followed by continuous infusion at 2.5 mg/kg/h. Patients in group I (n = 10) received lidocaine alone and patients in group II (n = 10) received lidocaine + epinephrine (5 pg/mL). Concentrations of lidocaine and its active metabolites, monoethylglycinexylidide (MEGX) and glycinexylidide (GX), were measured in plasma samples obtained after 15 minutes, 30 minutes, and 1, 2, and 3 hours of infusion using high-performance liquid chromatography with ultraviolet detection. RESULTS: Plasma lidocaine concentrations were higher in group I for the first 30 minutes; however, after 1 hour the levels were the same. Plasma MEGX and GX increased continuously in both groups. MEGX levels the were significantly higher in group I, but there was no significant difference in the sum of lidocaine + MEGX after 2 hours. There was no significant difference in GX levels between the two groups. CONCLUSIONS: With respect to continuous epidural administration, addition of epinephrine to lidocaine solutions is ineffective after 2 hours for reducing the potential for systemic toxicity, because the sum of the plasma concentrations of lidocaine and its principal active metabolite, MEGX, are unaffected.

Quick increase of pulmonary blood flow in response to an acute alveolar hypoxia in human subjects.

The effects of acute alveolar hypoxia on pulmonary blood flow were studied in upright standing human subjects by means of a constant expiration technique combined with continuous analysis of the C2H2 fraction in the expired gas. The subject rapidly and deeply inhaled a gas mixture containing 4.87% C2H2, 0.99% O2, N2 for balance and exhaled the alveolar gas via a constant flow valve. The Po2, of the alveolar gas fell to 37.6+/-5.6 mmHg. The C2H2 curve showed a clear downward inflection 7 to 10 sec after the completion of washout of the dead space gas. This inflection indicates an increase of the C2H2 uptake rate produced by an increase in pulmonary blood flow. The pulmonary blood flow calculated for the time range of 10.0 to 12.5 sec after the establishment of acute alveolar hypoxia (6.5+/-1.11/min) was significantly larger than the blood flow under normoxia (5.5+/-0.91/min). This increase associated with a quick rise in systemic arterial blood pressure which was probably induced by a chemical reflex.

An analysis of water movement between myocardial tissue and capillary blood during reactive hyperemia.

The colloid osmotic pressure (COP) of blood from the great cardiac vein was continuously measured by means of a membrane colloid osmometer during the reactive hyperemia following temporary occlusion of the anterior descending branch of the left coronary artery in anesthetized open-chest dogs. The COP increased sharply after releasing the occlusion, then decreased below the preocclusion level before gradually returning to it. These findings indicate that a measurable amount of water moved from the capillary blood into the myocardial tissues and then flowed back slowly into the capillary blood. To analyse the factors affecting this water movement, a method is proposed in which the Starling mechanism is combined with the interstitial volume elasticity and a steady-state solution of a Navier-Stokes equation. The pressure observed with a catheter wedged into a branch of the great cardiac vein was used as a measure of capillary perfusion pressure. During the coronary arterial occlusion, the filtration constant increased while the volume elasticity of the myocardial interstitial spaces decreased rapidly. The filtration constant and volume elasticity of the interstitial space under normal conditions were approximately estimated to be 2.4 X 10(-11) cm/(sec.dyn.cm-2) and 1.1 X 10(7) dyn.cm-2, respectively.

Non-uniform oxygen supply to the left ventricular myocardium by systolic perfusion of coronary artery.

The left anterior descending branch of the coronary artery (LAD) of anesthetized, open-chest dogs was perfused by intraventricular pressure (systolic perfusion) and aortic blood pressure (aortic perfusion) alternately. When the LAD perfusion was switched from aortic perfusion to the systolic one, the subendocardial PO2 decreased to 9.8 mmHg, on an average, in 1 to 2 min from the initial level of 18.9 mmHg obtained during the aortic perfusion. On total occlusion of LAD, the subendocardial PO2 fell to 4.5 mmHg, while the subepicardial PO2 showed no significant change. These results suggest that a small amount of oxygen is transported to the subendocardium during the systolic phase despite the impaired blood flow to the subendocardium and that the oxygen supply to subepicardium can be supported even though the LAD perfusion is restricted to the systolic phase.

Effects of temperature and transfer from seawater to freshwater on blood microrheology in Pacific salmon.

Blood of Pacific salmon was studied with particular interest in red blood cell (RBC) deformability in relation to migration. Blood samples were taken via cardiac puncture or chronic cannula placed in the dorsal aorta and heparinized. As an index of RBC deformability the mean passage time of single RBCs through micropores of 8 micron in diameter and 10 micron in length was determined under a pressure difference of 10 cmH2O. Despite about 100 mOsmol/l difference in plasma osmolality, there was no marked difference in RBC passage time between fish in seawater and those well acclimatized to freshwater. However, it seemed probable that a transient decrease in RBC passage time, i.e., an increase in RBC deformability, occurred immediately following transfer from seawater to freshwater. Plasma osmolality decreased to about 300 mOsmol/l within 1 hr after the transfer and showed no fluctuations thereafter. The temperature dependence of RBC deformability was much smaller in comparison with those previously observed in yellowtail and carp; salmon RBCs were still highly deformable even at 5 degrees C, a possible temperature of cold river water.

Transvascular osmotic flow reflected by changes in plasma oncotic pressure of anesthetized dog.

To assess overall bodily transvascular fluid flow due to osmotic imbalance between blood and interstitial fluid during or after addition of hypertonic saline, sugar-, albumin- and dextran solutions or CO2 mixture to blood, arterial blood colloid osmotic pressure (COP) of anesthetized dogs was continuously measured with a needle-type colloid osmometer. Plasma volume change (delta Vp) was estimated from the changes in either the equivalent albumin concentration (C) or the albumin concentration equivalent to plasma COP, which was well confirmed in inanimate model experiments. Carbon dioxide inhalation caused RBC swelling (1.5% volume increase/10 mmHg of PCO2). Intravenous injection of hypertonic solutions resulted in transient osmotic flow (8.5 +/- 2.2 ml/g of solute per kg of animal weight by NaCl, and 1.7 +/- 0.4 by glucose), and albumin and dextran also induced fluid flow (1.3 +/- 0.4 by albumin and 2.2 +/- 0.7 by dextran), which depended on van't Hoff's law and reflection coefficient of solutes.

Hypoxic reduction in blood flow velocity in pulmonary arterioles and capillaries.

A small ring chamber (I.D. = 6 mm) was placed on the exposed lung of anaesthetized bullfrogs. A localized hypoxia was induced in the ring chamber by introducing nitrogen in it. Blood flow velocity in pulmonary microvessels was measured by means of a laser Doppler microscope. The mean blood flow velocity was 1.98 +/- 0.45 and 1.52 +/- 0.10 mm/sec during the control condition in arterioles and capillaries, respectively. It was then reduced by the localized hypoxia to 1.63 +/- 0.32 and 1.33 +/- 0.08 mm/sec in arterioles and capillaries, respectively. The reduction, when expressed in the percentage ratio to the control flow velocity in each blood vessel group, was significantly larger in arterioles than in capillaries. A phase delay in the pulsation of the flow velocity contour was detected only in arterioles. These differences between pulmonary arterioles and capillaries in response to the localized hypoxia may be attributed to the dense interconnection of capillary network extending beyond the localized hypoxic area to the normoxic area.

White blood cell adhesion to endothelium and rheological behavior in microvessels of overinflated frog's lung.

The flow behaviors of white blood cells (WBCs) in frog's pulmonary microvessels were recorded and analyzed by means of a microscope-TV camera system. When the flow velocity in arterioles was reduced to a level lower than 1 mm/sec by a moderate overinflation of the exposed lung, WBCs rolled on the endothelial surface, frequently came in contact with the capillary orifice and passed it quickly without deformation. The time length which was required for WBCs to pass through the capillary orifice was shorter than the time length for red blood cells. This observation suggested that WBCs were no hinderance to blood delivery from arterioles to the capillary network in the normal and moderate overinflation of the lung. However, when the lung was strongly overinflated and the center line flow velocity was reduced to 0.1 mm/sec, WBCs adhered to the endothelium in ten minutes. The adhering WBCs could not be detached by the recovery of the blood flow. It seemed probable that a large shear stress up to 100 to 200 dynes/cm2 was necessary to pull down the interaction between the adhering WBCs and the endothelium.