We tested the hypothesis that estrogen administration would retard immobilization-induced muscle atrophy in adult male rats. The rats were injected for 24 days with either estrogen (40 μg/kg⁻¹, β-estradiol 3-benzoate in olive oil vehicle), or vehicle alone. At day 14 of estrogen treatment, the hindlimb muscles of one leg were immobilized in plantar flexion position by the use of a plaster cast. Following 10 days of immobilzation, the atrophic and the contralateral soleus muscles were both removed and analyzed to determine the level of muscle atrophy along with the measurement of the protein levels of Cu-Zn-superoxide dismutase (Cu-Zn-SOD), heat shock protein 72 (HSP72), and selected proteases. Compared to placebo animals, estrogen treatment significantly reduced (–35%) muscle atrophy. Further, estrogen significantly abridged the expression of the calcium-activated protease, calpain, in the atrophied hindlimb muscle. In contrast, estrogen treatment did not alter the protein levels of HSP72 in the immobilized soleus muscle. These results support the postulate that estrogen attenuates the rate of disuse muscle atrophy, partly because of reductions in immobilization-induced calcium-activated protease levels.

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Furosemide, a blocker of Na⁺/K⁺/2Cl⁻ cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers. The aim of the present study was to investigate whether an NKCC blocker affects cancer cell growth. If so, we would clarify the mechanism of this action. We found that poorly differentiated gastric adenocarcinoma cells (MKN45) expressed the mRNA of NKCC1 three times higher than moderately differentiated ones (MKN28) and that the NKCC in MKN45 showed higher activity than that in MKN28. A cell proliferation assay indicates that furosemide significantly inhibited cell growth in MKN45 cells, but not in MKN28 cells. Using flow cytometrical analysis, we found that the exposure to furosemide brought MKN45 cells to spend more time at the G₀/G₁ phase, but not MKN28 cells. Based on these observations, we indicate that furosemide diminishes cell growth by delaying the G₁-S phase progression in poorly differentiated gastric adenocarcinoma cells, which show high expression and activity of NKCC, but not in moderately differentiated gastric adenocarcinoma cells with low expression and NKCC activity.

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Nitric oxide (NO) modulates the activity of an inwardly rectifying K⁺ channel in cultured human proximal tubule cells. In this study, we investigated which NO synthase (NOS) isoform(s) was involved in the endogenous production of NO and hence the regulation of channel activity. The patch-clamp experiments using the cell-attached mode showed that a nonselective NOS inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME; 100 μM), suppressed channel activity, whereas a NOS substrate, L-arginine (500 μM), stimulated it. A neuronal NOS (nNOS)/inducible NOS (iNOS)-selective inhibitor, 1-(α,α,α-trifluoro-o-tolyl)-imidazole (TRIM; 100 μM), suppressed channel activity to the same extent as L-NAME. TRIM also blocked the stimulatory effect of L-arginine. In contrast, an NO donor, sodium nitroprusside (10 μM) or 8-bromoguanosine 3′,5′-cyclic monophosphate (100 μM) stimulated channel activity even in the presence of TRIM. RT-PCR revealed that iNOS mRNA alone was expressed in most of the cultures, i.e., 34 out of 40. In the other 6 cases, endothelial NOS (eNOS) and iNOS mRNA were simultaneously expressed. This finding was confirmed at the protein level by Western blotting. Indeed, in the patch-clamp experiments TRIM sometimes failed to suppress the channel activity, but the following addition of L-NAME suppressed it. However, since the suppressive effect of TRIM was usually similar to that of L-NAME, the involvement of eNOS in K⁺ channel regulation would be relatively low. These results suggest that iNOS plays a pivotal role in the endogenous production of NO under the basal condition, which is involved in the activity of the inwardly rectifying K⁺ channel in cultured human proximal tubule cells.

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To identify which organelles contained inositol trisphosphate (InsP₃) receptor type 2 (InsP₃R2) in adrenal medullary (AM) cells, immunocytochemical and biochemical studies were performed on AM cells of several species. InsP₃R2-like immunoreactive materials produced by two different anti-InsP₃R2 antibodies (Abs) (Chemicon and Sigma) were distributed in rat AM cells in agreement with BODIPY-FL-InsP₃ binding sites. For two other Abs (KM1083 and Santa Cruz), some of the anti-InsP₃R2 immunoreactive materials were stained with an anti-dopamine-β-hydroxylase Ab, but not by BODIPY-FL-InsP₃. BODIPY-FL-thapsigargin binding sites were consistent with a distribution of the endoplasmic reticulum (ER) identified by an anti-calnexin Ab, and a prior application of thapsigargin significantly eliminated BODIPY-FL-thapsigargin bindings, suggesting that BODIPY-FL-thapsigargin bindings were mediated by thapsigargin, but not the fluorescence molecule. The anti-InsP₃R2 Ab that produced stainings consistent with BODIPY-FL-InsP₃ bindings recognized a protein with about 250 kDa. A fractional analysis of bovine adrenal medullae revealed that the 250 kDa InsP₃R2 was detected in a crude membrane fraction, but not in a secretory granule fraction. The results suggest that the InsP₃R2 was present in the ER, but not in secretory granules in AM cells.

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A large amount of evidence suggests that acupuncture stimulation enhances the experimental pain threshold in various animal models. Acupuncture analgesia is mediated by the endogenous opioid system, and the analgesic response to acupuncture shows individual variation. This study identified and characterized the genes that differ between high responders (HR) and low responders (LR) on acupuncture stimulation, using a cDNA microarray. Fifteen participants were stimulated at the LI 4 acupuncture point, and the finger withdrawal latency (FWL) test was performed to classify the HR and LR groups. Total RNA was then extracted from blood samples from each group and used as a template to synthesize cDNA. The cDNA was applied to Code Link^TM UniSet Human 20K microarray chips. The Symptom Checklist 90-Revised (SCL-90-R) was also analyzed as a measure of psychological variation. The FWL was significantly elevated in the HR group after acupuncture stimulation, whereas there was little increase in the LR group. The ratio of HR to LR subjects was 9:6. We found that 353 and 22 genes were up- and downregulated, respectively, in the HR group. However, the SCL-90-R profiles did not differ significantly between the two groups. These results suggest that the individual variation in acupuncture analgesia, verified by measuring the FWL in the HR and LR groups, resulted from genetic inheritance rather than differences in the psychological environment.

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Inspiratory muscle conditioning at a lower or higher lung volume based on the principles of muscle thixotropy causes acute changes in end-expiratory chest wall and lung volumes. The present study aimed to demonstrate the time course of effects of this conditioning on both end-expiratory chest wall volume and thoracoabdominal synchrony. We measured chest wall motion with respiratory induction plethysmography at 0.5, 1, 2, 3, and 6 min after conditioning at three different lung volumes in 15 healthy men. After conditioning at total lung capacity – 20% inspiratory capacity, increases in end-expiratory chest wall volume were significant at 0.5, 1, and 2 min (P < 0.05), being most obvious at 0.5 min (Δ0.24 ± 0.20 liter). After conditioning at residual volume, reductions in end-expiratory chest wall volume were significant at any time point (P < 0.05), being most obvious at 0.5 min (Δ0.16 ± 0.08 liter). Conditioning at functional residual capacity had little effect on the volume. Spirometric inspiratory capacity at 6 min after conditioning at residual volume (2.68 ± 0.35 liter) was higher than the baseline value (2.53 ± 0.31 liter, P < 0.05). Reductions in the phase angle, quantified by the Konno-Mead diagram, occurred after conditioning at residual volume at any time point (P < 0.05), being most obvious at 2 min (Δ3.47 ± 3.02 degrees). In conclusion, there is a 6-min time course of changes in end-expiratory chest wall volume after conditioning. More synchronous motion between the rib cage and abdomen occurs after conditioning at residual volume.

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Cholecystokinin receptors (CCK-Rs) have been classified into two subtypes: CCK-AR (1R) and -BR (2R). We generated CCK-AR(–/–), CCK-BR(–/–), and CCK-AR(–/–)BR(–/–) mice and found that the gastric emptying of a liquid meal was increased in CCK-BR(–/–) and AR(–/–)BR(–/–) mice, compared with wild-type and CCK-AR(–/–) mice. Given that enhanced gastric emptying leads to eating, food intake after overnight fasting was examined, as was the effect of CCK-8S on food intake. Male mice 6–8 months of age were deprived of food for 16 h with free access to water, after which they were injected intraperitoneally (0.1 ml/mouse) with either vehicle or CCK-8 (0.3, 1.0, or 3.0 nmol/mouse), and their food intake was monitored for 4 h. CCK-8S inhibited food intake in wild-type and CCK-BR(–/–) mice, but not in CCK-AR(–/–) or AR(–/–)BR(–/–) mice. Unexpectedly, we observed a lower food intake in CCK-AR(–/–)BR (–/–) mice treated with vehicle than in mice of the other genotypes. To examine the mechanism of decrease in food intake in CCK-AR(–/–)BR(–/–) mice, the involvement of ghrelin was determined in wild-type and CCK-AR(–/–)BR(–/–) mice. Fasting plasma ghrelin levels were significantly lower in CCK-AR(–/–)BR(–/–) mice than in wild-type mice, and no increase in response to fasting was observed in CCK-AR(–/–)BR(–/–) mice. An administration of acyl-ghrelin produced a small increase in food intake in CCK-AR(–/–)BR(–/–) mice, but not to the levels of wild-type mice. In conclusion, CCK-AR(–/–)BR(–/–) mice showed lower food intake as well as lower response to exogenous ghrelin, and a lower plasma ghrelin level after fasting, though which receptor is more important is unknown.

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The functional residual capacity (FRC) and airway resistance (R_aw) of the rat were measured, using a newly designed body plethysmograph (BPG), the inner environment of which was maintained at body temperature and was water-vapor saturated. The subjects were anesthetized and tracheally intubated male Wistar rats (n = 15). After measuring the FRC and R_aw, we analyzed the effects of inhaled methacholine (Mch, 0–8 mg/ml) on R_aw.The determined FRC was 5.37 ± 0.22 ml (mean ± SE). An almost linear relationship between box pressure and respiratory flow was obtained when the difference between box-gas temperature and the rectal temperature of the rat was less than 1.0°C. The R_aw at FRC was 0.230 ± 0.017 cmH₂O/ml/s. It increased proportionally with increases in the Mch concentration. When the dynamic changes in R_aw were analyzed, the R_aw was found to progressively increase during expiration; this increase continued throughout inspiration. Thus in the rat, R_aw is not simply a function of changes in lung volume. In conclusion, the humidity- and temperature-adjusted BPG provided an absolute and possibly dynamic value of R_aw.

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The aim of this study was to determine the daily rhythms in the blood serum of homocysteine in horses. Ten thoroughbred horses, five athletic (trained for 1 h, 6 days a week) and five sedentary, were used. Blood samples were collected on each subject every 4 h for two days by means of the jugular vein. On each individual sample, the serum concentration of homocysteine was assessed. The results obtained during the experimental period indicated the existence of a daily rhythm of serum homocysteine in sedentary and athletic horses. They also demonstrated that in horses, physical exercise influences the daily rhythm of serum homocysteine.

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We report a new system for monitoring sensations of many body parts as well as comprehensively showing the distribution of overall skin temperature (T_sk) and temperature-related sensations. The system consists of a console with 52 levers to report temperature-related sensations and software that facilitates the visualization of the distribution of T_sk and temperature-related sensations by displaying them on a model of the human body. The system’s utility was demonstrated with a physiological experiment involving three males and three females. They were exposed to step changes of ambient temperature from 23°C to 33°C. We measured T_sk at 50 points, and the subjects concurrently provided estimates of local temperature sensation and thermal comfort/discomfort at 25 loci. This system greatly facilitates the perception and analysis of spatial relationships and differences in temperature and sensation in various areas of the body.

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