Antithrombin inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by monocytes in vitro through inhibition of Egr-1 expression.

BACKGROUND: Antithrombin (AT) improves the outcome of septic patients with intravascular coagulation. However, the mechanisms underlying the therapeutic benefits of AT are not fully understood. Tumor necrosis factor-alpha (TNF-alpha) plays a critical role in the development of organ failure and intravascular coagulation in sepsis. AIM: This study aimed to elucidate a molecular mechanism by which AT inhibits TNF-alpha production. METHODS: Human peripheral monocyte was stimulated by lipopolysaccharide (LPS) and TNF-alpha concentration in media was measured. Levels of phosphorylation of extracellular signal-regulated protein kinases (ERK) 1/2 and early growth response factor-1 (Egr-1) were estimated by western blotting or by electrophoretic mobility shift assay. RESULTS: Antithrombin (3 U mL(-1)) inhibited TNF-alpha production by monocytes stimulated with LPS. Conversely, chemically modified AT that lacks affinity for heparin did not. AT inhibited the phosphorylation of ERK 1/2 and decreased the expression of Egr-1 in LPS-stimulated monocytes. However, it did not affect the activation of either nuclear factor-kappaB or activator protein-1. Pretreatment with KT5720, a protein kinase A inhibitor, reversed the inhibitory effect of AT on the LPS-induced phosphorylation of ERK1/2. Although 2 U mL(-1) AT slightly inhibited TNF-alpha production by LPS-stimulated monocytes, it significantly inhibited TNF-alpha production in the presence of a low concentration of beraprost, a stable derivative of prostacyclin. CONCLUSIONS: These observations suggest that AT might inhibit LPS-induced production of TNF-alpha by inhibiting the increase in Egr-1 expression in monocytes via interaction with heparin-like substances expressed on the cell surface.

Intra-operative monitoring of vagal nerve activity with wire electrodes.

A monitoring system for tracking the electromyogram (EMG) of the vocal cords with wire electrodes embedded in an endotracheal tube was designed to identify the recurrent laryngeal nerve during thyroidectomy. Our recent experience in two cases suggests that vagal nerve activity can be correctly detected by recording of the EMG of the vocal cords using a special endotracheal tube embedded with wire electrodes.

Differential regulation of aquaporin expression in astrocytes by protein kinase C.

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens, that increase plasma membrane water permeability in secretory and absorptive cells. In astrocytes of the central nervous system (CNS), using the reverse transcription-polymerase chain reaction (RT-PCR), we previously detected AQP3, 5 and 8 mRNAs in addition to the reported AQP4 and 9. However the mechanisms regulating the expression of these AQPs are not known. In this study, we investigated the effects of a protein kinase C (PKC) activator on the expression of AQP4, 5 and 9 in cultured rat astrocytes. Treatment of the cells with TPA caused decreases in AQP4 and 9 mRNAs and proteins in time- and concentration-dependent manners. The TPA-induced decreases in AQP4 and 9 mRNAs were inhibited by PKC inhibitors. Moreover, prolonged treatment of the cells with TPA eliminated the subsequent decreases in AQP4 and 9 mRNAs caused by TPA. Pretreatment of cells with an inhibitor of protein synthesis, cycloheximide, did not inhibit the decreases in AQP4 and 9 mRNAs induced by TPA. These results suggest that signal transduction via PKC may play important roles in regulating the expression of AQP4 and 9.

Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation.

Aquaporins (AQPs) are a family of water-selective transporting proteins with homology to the major intrinsic protein (MIP) of lens [Cell 39 (1984) 49], that increase plasma membrane water permeability in secretory and absorptive cells. In the central nervous system (CNS), we detected the transcripts of AQP3, 5 and 8 in addition to the previously reported transcripts of AQP4 and 9 in astrocytes, of AQP3, 5 and 8 in neurons, of AQP8 in oligodendrocytes, and none of them in microglia using RNase protection assay and the reverse transcription-polymerase chain reaction (RT-PCR). Hypoxia evoked a marked decrease in the expression levels of AQP4, 5 and 9, but not of AQP3 and 8 mRNAs, and in astrocytes in vitro subsequent reoxygenation elicited the restoration of the expression of AQP4 and 9 to their basal levels. Interestingly, AQP5 showed a transient up-regulation (about 3-fold) and subsequent down-regulation of its expression within 20 h of reoxygenation after hypoxia. The changes in the profiles of AQP expression during hypoxia and reoxygenation were also observed by Western blot analysis. These results suggest that AQP5 may be one of the candidates for inducing the intracranial edema in the CNS after ischemia injury.

Plasma type II phospholipase A2 levels in patients with acute pancreatitis.

Changes in the blood levels of type II phospholipase A2 (PLA2) were investigated over time in patients with acute pancreatitis from an early stage after manifestation of the disease. The serum level of type II PLA2 at the first examination and the maximum level during the course of illness were both correlated with the severity of the disease. Serum levels of type II PLA2 were significantly higher in patients with acute pancreatitis complicated by multiple organ failure (349.1 +/- 146.6 ng/ml) than in those with acute pancreatitis not complicated by multiple organ failure (66.9 +/- 50.1 ng/ml). The serum levels of type II PLA2 were also significantly higher in patients who eventually died (316.8 +/- 150.5 ng/ml) than in those who survived (148.9 +/- 167.9 ng/ml). There was a significant correlation between the serum levels of type II PLA2 and those of TNF-alpha during the course of illness (r = 0.8037, p < 0.0001). The serum levels of type II PLA2 reliably reflected the severity of acute pancreatitis even in the early stages of the disease. These results suggest that type II PLA2 may be closely involved in the pathophysiology of acute pancreatitis.

[Tracheal tube/laryngeal mask exchange to prevent coughing in lung volume reduction surgery].

We report the use of laryngeal mask airway (LMA) to facilitate smooth emergence from anesthesia in 3 patients who underwent lung volume reduction surgery (LVRS) for severely symptomatic emphysema. To prevent coughing during emergence we replaced the endotracheal tube with LMA while patient was still in deep anesthesia. Emergence was smooth without coughing in all three patients, and ventilation with facemask was not needed after removal of LMA. We believe this technique is safe and beneficial for anesthetic management of LVRS.

[One-stage surgery of minimally invasive direct coronary artery bypass, abdominal aortic aneurysm repair and carotid endarterectomy].

A 74 year-old man with bladder transitional carcinoma had severe multivascular disease; coronary artery stenosis, abdominal aortic aneurysm and right internal carotid artery stenosis. First, transurethral bladder tumor resection (TUR-Bt) was performed twice but in the second TUR-Bt, no carcinoma cell was found. One stage surgery of minimally invasive direct coronary artery bypass (MIDCAB), abdominal aortic aneurysm (AAA) repair and carotid endarterectomy (CEA) was proposed. MIDCAB was performed first. Inspite of the bradycardia, heart oppression by stabilizer and coronary artery clamping, blood pressure and ST segments were stable. With heparinization and the chest left open, AAA repair was carried out. On aorta clamping and declamping, blood pressure and heart rate were stable. After completion of AAA repair, heparinization was reversed with protamine. Chest and abdominal wounds were closed simultaneously. CEA was performed lastly, because the patient had no cerebral ischemic symptom and no risk of cardiopulmonary bypass. After the operation, no neurologic deficit appeared. This experience of one stage surgery was reported with review of literatures. One stage surgery is a possible approach to the patients with severe multivascular disease.

Nuclear matrix protein (NMP) levels in patients with acute pancreatitis.

Nuclear matrix protein (NMP) is an index of apoptosis. We measured NMP in 22 patients with acute pancreatitis and investigated the relationship between severity and NMP. We also measured tumor necrosis factor-alpha (TNF-alpha) and investigated the relationship between NMP and TNF-alpha. The NMP value increased significantly as the pancreatitis became more increasingly severe, and the NMP values were significantly higher in the group with multiple organ dysfunction syndrome (MODS) than in the group without MODS. A comparison of the NMP values in the group that survived and the group that died revealed higher NMP values in the former. A significant correlation was found between the NMP values and the TNF-alpha values, suggesting that apoptosis may contribute to the pathophysiology of acute pancreatitis.

Effects of sympathetic nerve blockades on low-frequency oscillations of human earlobe skin blood flow.

Earlobe skin blood flow measured by laser Doppler flowmetry often shows low-frequency oscillations with a frequency around 0.1 Hz. We evaluated the effects of different sympathetic blocking techniques on the oscillations. Power spectrum of 5-min time series of beat-to-beat peak earlobe skin blood flow showed a distinct spectral peak at a frequency around 0.1 Hz (mean +/- SD, 0.107+/-0.016 Hz). The power of the spectral peak measured as coefficient of component variance (CCV) was diminished with total spinal anesthesia (TSA, n = 4) and cervicothoracic epidural anesthesia (CTEA, n = 4; P<0.05 for both). The CCV was unchanged significantly with bilateral thoracic sympathetic ganglionic excision (TSGE, n = 5). Right stellate ganglion block (RSGB, n = 6) caused a trend towards an increase in the CCV on the right (blocked)-side (P = 0.072) but no change on the left (intact)-side. Cross-spectrum analysis revealed that the low-frequency oscillations were not coherent between the right- and left-side earlobes or with low-frequency oscillations in systolic blood pressure either before or after RSGB. TSA and CTEA are known to cause extensive sympathetic nerve blockade in a broad area, while TSGE and RSGB are known to cause local and partial sympathetic blockade. Our results suggest that although the low-frequency oscillations in earlobe skin blood flow may be mediated non-neurally, magnitude of the oscillations may be modified by sympathetic vasomotor tone.

Induction of blood-brain barrier properties in immortalized bovine brain endothelial cells by astrocytic factors.

The blood-brain barrier (B-BB) protects the free passage of substances into the brain and maintains the homeostasis of the central nervous system. It is commonly accepted that astrocytes surrounding brain endothelial cells influence the B-BB formation and the exhibition of B-BB function of capillaries. To begin the in vitro study on the B-BB, it is essential to obtain a homogenous and sufficient supply of brain endothelial cells as well as astrocytes. We thus immortalized the bovine brain endothelial cell (BBEC) by transfection of the SV40 large T antigen and obtained a single clone, t-BBEC-117, which retained the brain endothelial cell phenotype. Astrocyte in co-culture was found to tighten the intercellular contacts of the immortal cells resulting in a reduced L-glucose permeability, and its conditioned medium (CM) augmented a B-BB phenotype, alkaline phosphatase (ALP) activity. Among known astrocytic factors, only fibroblast growth factor-basic (bFGF) could mimic the actions of astrocytes as measured by L-glucose permeability and ALP activity. Moreover, anti-bFGF antibody canceled 90% of ALP activation by astrocyte CM. Basic FGF, however, failed to induce other B-BB phenotypes such as the expressions of multidrug resistance (mdr) and glucose transporter (GLUT-1) genes. These data suggest that bFGF is one of the most plausible astrocytic factors to induce the B-BB properties of immortal brain endothelial cells together with some unknown factors in the astrocyte CM.

Molecular cloning of two bovine aquaporin-4 cDNA isoforms and their expression in brain endothelial cells.

Two cDNA isoforms of bovine aquaporin-4 (bAQP4-A and bAQP4-B) were newly isolated. Sequence analysis of both cDNAs revealed open reading frames of 972 (bAQP4-A) and 906 nucleotides (bAQP4-B) with deduced proteins of 323 (bAQP4-A) and 301 amino acid residues (bAQP4-B). Partial 5'-genomic sequence analysis showed that the 5'-noncoding sequences specific to bAQP4-A and -B transcripts were contained in distinct exons, exon 0 for bAQP4-A and new exon X for bAQP4-B. RNase protection assay demonstrated the definite expression of both isoforms in bovine brain. The deduced amino acid sequence of bAQP4-A was highly homologous to the human (97%), rat (95%), and mouse (93%) AQP4. Reverse transcription-PCR detected the expression of AQP4 mRNAs in bovine brain endothelial cells as well as in a variety of bovine organs such as brain, lung, spleen, and kidney. Northern blot analysis indicated that a 6.0 kb message is predominantly expressed in bovine brain and lung.

Effect of propofol on norepinephrine-induced increases in [Ca2+]i and force in smooth muscle of the rabbit mesenteric resistance artery.

BACKGROUND: Propofol (2,6-diisopropylphenol) possesses vasodilating activity in vivo and in vitro. The propofol-induced relaxation of agonist-induced contractions in small resistance arteries has not been clarified. METHODS: The effect of propofol was examined on the contractions induced by norepinephrine and high K+ in endothelium-denuded rabbit mesenteric resistance artery in vitro. The effects of propofol on the [Ca2+]i mobilization induced by norepinephrine and high K+ were studied by simultaneous measurement of [Ca2+]i using Fura 2 and isometric force in ryanodine-treated strips. RESULTS: Propofol attenuated the contractions induced by high K+ and norepinephrine, the effect being greater on the high K+-induced contraction than on the norepinephrine-induced contraction. In Ca2+-free solution, norepinephrine produced a transient contraction resulting from the release of Ca2+ from storage sites that propofol attenuated. In ryanodine-treated strips, propofol increased the resting [Ca2+]i but attenuated the increases in [Ca2+]i and force induced by both high K+ and norepinephrine. In the presence of nicardipine, propofol had no inhibitory action on the residual norepinephrine-induced [Ca2+]i increase, whereas it still modestly increased resting [Ca2+]i, as in the absence of nicardipine. CONCLUSIONS: In smooth muscle of the rabbit mesenteric resistance artery, propofol attenuates norepinephrine-induced contractions due to an inhibition both of Ca2+ release and of Ca2+ influx through L-type Ca2+ channels. Propofol also increased resting [Ca2+]i, possibly as a result of an inhibition of [Ca2+]i removal mechanisms. These results may explain in part the variety of actions seen with propofol in various types of vascular smooth muscle.

[Anesthetic management for microlaryngeal surgery with high frequency jet ventilation and intravenous application of pentazocine and propofol].

Microlaryngeal surgery was performed with total intravenous anesthesia using pentazocine and propofol in 20 patients. The patients were paralyzed by suxamethonium infusion and ventilated by high frequency jet ventilation via the laryngoscope. In place of the opioid analgesics commonly used in TIVA for microlaryngeal surgery, pentazocine was given. All but one of the patients received pentazocine 15 m.g. intramuscularly as a premedication and then another 15 m.g. intravenously for induction of anesthesia. Average time of surgeries was 33.6 minutes. Average doses of pentazocine and suxamethonium given during surgeries were 369 m.g. and 286 m.g., respectively. This technique allowed stable anesthesia to be achieved with rapid postoperative recovery, without serious complications like intraoperative return of awareness.

[Effects of ulinastatin on PMNL and vascular endothelial injury in patients undergoing open heart surgery with CPB].

Activated leukocytes are thought to contribute to respiratory dysfunction, alterations in microvascular permeability, disseminated intravascular coagulation, and thrombosis, all of which can complicate cardiopulmonary bypass (CPB). We have measured the levels of circulating proinflammatory cytokines (IL-6, 8), polymorphonuclear leukocytes elastase (PMNL-E), and vascular endthelial factors (ET-1, TM, sICAM-1) in patients undergoing open heart surgery with CPB. Patients were divided into a control group and a ulinastatin group. We have examined the effects of ulinastatin on these humoral mediators and postoperative pulmonary function. Every factor except IL-8 increased after CBP in control group. IL-6 and PMNL-E declined sharply to normal level in a few hours, but it took several days after surgery for ET-1, TM, and sICAM-1 to return to preoperative levels. Ulinastatin significantly suppressed the elevation of PMNL-E after CPB, indirectly suppressing the increase of other factors. There was no significant relationship between levels of humoral mediators and postoperative pulmonary function between the two groups. Our results suggest that ulinastatin alleviates the damage of vascular endothelium due to CPB (first attack), and this may be beneficial to reduce excessive inflammatory reaction against secondary insults.