We have examined the cultured human bronchial epithelial cells (16HBE) to learn if changes in Cl⁻ concentration or osmolality stimulate the cells to release ATP and to determine whether its release is cyclic AMP (cAMP)- and/or Ca²⁺-dependent by using the luciferin-luciferase luminometric assay. In a control solution (290 mosmol kg H₂O⁻¹), the external ATP concentration and the rate of ATP release were 0.52 ± 0.20 nM and 0.036 ± 0.034 pmol min⁻¹, respectively. Upon hypotonicity (205 mosmol kg H₂O⁻¹), they increased to 7.0 ± 1.3 nM and 3.1 ± 0.6 pmol min⁻¹, respectively, at 6 min, then decreased. At the peak, the rate of ATP release is estimated to be 6.2×10⁴ ATP molecules s⁻¹ per cell. An accumulation of the released ATP for the initial 10 min increased significantly (p < 0.005) by 71.5% in the presence of forskolin (10 μM), adenylyl cyclase activator, however, it was abolished (p < 0.001) by pretreatment with BAPTA-AM (25 μM), a membrane permeable Ca²⁺ chelator. On the other hand, neither low Cl²⁻ (75 mM, isotonic) nor hypertonicity (+NaCl or +mannitol, 500 mosmol kg H₂O⁻¹) could significantly increase the ATP release. Further, forskolin or ionomycin (a Ca²⁺ ionophore) or, both, failed to stimulate ATP release under the isotonic condition. In conclusion, first, hypertonicity and changes in Cl⁻ concentrations are not effective signals for the ATP release; second, hypotonicity-induced ATP release is potentiated by the level of intracellular Ca²⁺ and cAMP; and third, a biphasic increase in ATP release and its low rate at the peak support the hypothesis that ATP is released through a non-conducting pathway model, such as exocytosis, or through a volume-dependent, ATP-conductive anion channel.

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We investigated the effect of muscle contraction velocity on cardiorespiratory responses during exercise. Eight males (23 ± 2 years, 175 ± 5 cm, 64 ± 6 kg, mean ± SD) performed 3-min repetitive one-leg extension exercises at various angular velocities (30, 60, 120, and 240 deg/s) with a controlled relaxation interval, relatively constant (duty cycle = 1:1, A trial) and absolutely constant (relaxation time = 0.75 s, B trial) at a total work of 2,100–2,400 J in an isokinetic mode, using a Cybex II dynamometer. We measured heart rate (HR), mean blood pressure (MAP), minute ventilation (V˙E), and oxygen uptake (V˙O₂) during the exercise. The angular velocity significantly affected the increase in HR, MAP, V˙E, and V˙O₂ at the end of exercise from resting in both A and B trials (e.g., MAP: 12 ± 2, 10 ± 2, 11 ± 2, and 18 ± 2 mmHg in the A trial). The result suggests that muscle contraction velocity affects cardiorespiratory responses during repetitive isokinetic exercise.

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The developmental changes in acetylcholine (ACh)-induced vasodilator response in isolated rabbit spinal arterioles were investigated with special reference to endogenous nitric oxide (NO) and vasodilator prostaglandins. Spinal arterioles from juvenile (1–3 months) and adult (12–14 months) Japanese white rabbits were dissected, cannulated, and perfused. In both age groups, ACh produced a comparable vasodilation of the arterioles preconstricted with KCl (20–40 mM). In juveniles, a major part of the ACh-induced vasodilation was eliminated by the cyclo-oxygenase inhibitor indomethacin (10 μM), whereas the effect of the NO synthase inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME, 30 μM) was smaller. In contrast, L-NAME completely eliminated the ACh-induced vasodilation in the adult group, which was partially restored with additional treatment with L-arginine (1 mM). Vasodilations induced by isocarbacyclin (a prostacyclin analogue) and cilostazol (a cAMP-phosphodiesterase inhibitor) was attenuated in the adult, whereas that induced by sodium nitroprusside was unaltered. These results suggest that the mechanisms of ACh-induced vasodilation of rabbit spinal arterioles change with the development: prostanoid-associated in juveniles and NO-associated in the adult. Such age-related modulation of the mediator roles of prostanoids and NO might represent an increase in NO production and a decreased sensitivity of the vascular smooth muscle to cAMP-mediated responses with age.

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Recent studies indicate a possible role of nitric oxide (NO) in regulating leukocyte-endothelial cell interactions, which plays a key role in the tumor immunity. The purpose of the present study is aimed to observe the tumor hemodynamics intravitally and to clarify the effect of NO on tumor microcirculation by means of a real-time confocal laser-scanning microscope using NO-reactive indicators. Visualization of localization of NO and the leukocyte behavior was made in the mesenteric microvessels of an experimental tumor model rat. Production of NO was clearly visualized along the endothelium of the tumor-free rats, but scarcely found in the newly formed tumor microvessels. A higher level of NO production was observed in a solid tumor region, where a more marked decrease in the leukocyte-endothelial cell interactions was observed. Local administration of a NO synthase (NOS) inhibitor increased leukocyte adhesion. This indicates that tumor-derived NOS creates the tumor specific microenvironment of the immature angiogenic tumor vessels, thereby modulating leukocyte behavior.

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The redox states of albumin in serum and synovial fluid (SF) were analyzed in 35 female patients with temporomandibular joint disorders and 9 asymptomatic female healthy volunteers by high-performance liquid chromatographic (HPLC) analysis. Human serum albumin (HSA) is divided by HPLC analysis into three fractions: mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA-1 and HNA-2, oxidized forms). It was found that the characterized values of albumin in SF of patients with the disorders were the fraction of HMA (f (HMA), 65.8 ± 9.1%), the fraction of HNA-1 (f (HNA-1), 31.3 ± 8.8%), and the fraction of HNA-2 (f (HNA-2), 2.9 ± 0.9%), respectively, and similarly the values of albumin in serum of same subjects were f (HMA), 79.4 ± 4.0%; f (HNA-1), 18.7 ± 3.8%; and f (HNA-2), 1.9 ± 0.5%. The HNA fractions in SF were significantly higher than those in serum (p < 0.0001). The SF of patients and even control showed more oxidative condition compared with sera of the same subjects. Each fraction of SF showed significant difference in control (p < 0.05), and greater significance in patients (p < 0.0001). Furthermore, the HNA-2 fraction (f (HNA-2), 2.9 ± 0.9%) in SF of the patients with temporomandibular joint disorders revealed a significantly higher value (p < 0.01) than that (f (HNA-2), 1.7 ± 0.3%) of controls. These findings suggested that SF of the temporomandibular joint is more locally oxidized than serum; thus oxidative factors are concerned in the pathogenesis of the temporomandibular joint disorders. It was also suggested that synovial albumin may play a scavenging role against the intraarticular oxidative stress.

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Pathophysiological functions of cardiac cystic fibrosis transmembrane conductance regulator (cCFTR) in ischemia are not well known. Using neonatal rat ventricular cardiomyocytes in primary culture in this study, we thus examined whether the CFTR protein is expressed and is functioning as a cAMP-activated anion channel on the plasma membrane under ischemic conditions. After the cells were subjected to simulated ischemia (O₂ and glucose deprivation), an up-regulation of the CFTR expression was transiently observed in the membrane fraction by Western blot. A peak expression of mature CFTR protein was found at 3 h of ischemia, and thereafter the signal diminished gradually. In contrast, the results of Northern blot indicated that the expression level of CFTR mRNA changed little until 3 h of ischemia, whereas the level slightly decreased after 8 h of ischemia. An immunohistochemical examination showed, in agreement with the results of Western blot analysis, that the expression of CFTR protein on the plasma membrane became most prominent at 3 h of ischemia, whereas the plasmalemmal CFTR signal was markedly reduced after 8 h of ischemia. Whole-cell recordings showed that the cardiomyocytes responded to cAMP with an activation of time- and voltage-independent currents that contained an anion-selective component sensitive to CFTR Cl^– channel blockers (NPPB and glibenclamide) but not to a stilbene-derivative conventional Cl⁻ channel blocker (SITS). This cAMP-activated Cl⁻ channel current was found to be enhanced after an application of ischemic stress for 3 to 4 h. These findings indicate that a plasmalemmal expression of CFTR is transiently enhanced under glucose-free hypoxic conditions presumably because of a posttranslational control.

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Lipopolysaccharide (LPS) stimulates peripheral mononuclear cells (PBMC) to synthesize or release pyrogenic cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha (TNF-α). Nuclear factor-kappa B (NF-κB) influences inflammatory responses through the regulation of genes encoding cytokines. In the present study, experiments were carried out to determine whether an inhibition of NF-κB mechanisms causes an inhibition of pyrogenic cytokine synthesis or release from PBMC and results in antipyresis. Intravenous administration of the supernatant fluids obtained from the human PBMC incubated with LPS caused feverlike hyperthermia in rabbits. The febrile responses were in parallel with the levels of IL-1β, IL-6, and TNF-α in supernatant fluids. Both the fever and the increased levels of these cytokines in supernatant fluids were decreased by incubating LPS-PBMC with NF-κB inhibitors, including pyrrolidine dithiocarbamate, sodium pyrithione, N-acetyl-cysteine, and curcumin. Moreover, an intravenous administration of LPS (0.5–2 μg/kg) produced dose-dependent fever in the rabbits. The fevers were in parallel with the levels of IL-1β, IL-6, and TNF-α in rabbit serum. A pretreatment of rabbits with an intravenous injection of pyrrolidine dithiocarbamate, sodium pryithione, N-acetyl-cysteine, or curcumin 1 h before the intravenous administration of LPS significantly attenuated the LPS-induced fever and/or increased levels of these cytokines in the serum of rabbits. Furthermore, pretreatment with an intravenous dose of anti–IL-1β, anti–IL-6, or anti–TNF-α monoclonal antibody significantly attenuated the fever induced by the intravenous injection of LPS in rabbits. The antipyretic effects exerted by anti–L-1β monoclonal antibody were greater than those exerted by anti–L-6 or anti–NF-α monoclonal antibody. The data indicate that NF-κB activation correlates with an LPS-induced synthesis or a release of cytokines (in particular, IL-1β) from PBMC and triggers fever. Blocking NF-κB mechanisms in the PBMC with NF-κB inhibitors may be an effective strategy in the fever therapy.

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The blue-sheep, pika, and yak live in the Tibetan highlands at an altitude of 6,100 m and are typical mammals adapted to high-altitudes. These animals have a long history of habitation at high-altitudes and are considered to be "animals completely adapted to high-altitudes" because of their physiological and morphological traits that are well adapted to high-altitude environments. To evaluate the physiological characteristics of high-altitude adaptation in the blue-sheep, changes in the pulmonary hemodynamics during exposure to simulated-altitudes at 0, 2,300, and 4,500 m were examined by means of a climatic chamber in Qinghai Province, China (altitude 2,300 m). Seven blue-sheep inhabiting the mountains (3,000 m) of Qinghai Province, China, were compared with 5 pigs raised in the same area as controls. The primary items of measurement were the body weight (BW), systemic arterial pressure (Psa), pulmonary artery pressure (Ppa), hematocrit (Ht), left ventricular weight (LVW), right ventricular weight (RVW), and blood gas profile. The principal findings of this study are: (1) Ht, an index of right ventricular hypertrophy (RVW/LVW), and oxygen consumption (V˙O₂) were significantly lower in the blue sheep compared with the pigs; (2) When the animals were exposed to simulated-altitudes at 0, 2,300, and 4,500 m, Ppa increased significantly in tandem with altitude elevation in both species, but the increases were significantly smaller in the blue-sheep; and (3) Ppa/Psa, an index of the right ventricular load, increased with the altitude in both species, but the increases were smaller in the blue sheep. From these observations, low Ht and RVW/LVW and significant attenuation of hypoxic pulmonary vasoconstriction (HPV) in the blue-sheep is considered to be characteristics of animals completely adapted to high-altitudes, such as the pika.

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The blood flow dynamics in the early chick embryo were visualized by using a video-imaging method without invasion to the circulatory system. The movement of juvenile blood cells in the dorsal aorta was tracked and the flow velocity of blood cells calculated by using an image processor and a computer.

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