We studied the viability of high-performance liquid chromatography and mass spectrometry (LC/MS) as a selective and sensitive analytical method for measuring blood concentrations of the local anesthetic ropivacaine. Ropivacaine was effectively separated using a reverse-phase column and monitored at 275 m/z ion. The LC/MS method allowed measurement of concentrations of ropivacaine of lower than 75 ng/mL. The standard curve was linear and in the range of < 1.5 μg/mL. Recovery of ropivacaine in plasma samples was over 90% after precipitation of plasma protein with trichloroacetic acid. The method was tested on the pharmacokinetics of plasma ropivacaine after single intravenous or subcutaneous administration in rabbits. The pharmacokinetic parameters showed a one-compartment model and a mean elimination half-life of 0.54 ± 0.05 h and 2.83 ± 0.51 h after administration at doses of 0.4 mg/kg, i.v. and 5 mg/kg, s.c., respectively. These values were in approximate agreement with previously obtained results in dogs. The results of the present study demonstrated that the LC/MS method was highly selective and sensitive for the measurement of ropivacaine, indicating that it offers a useful tool for monitoring the therapeutic effects and determining the pharmacokinetic parameters of this drug in blood.
We analyzed the effect of low birth weight on pancreatic β cell mass. We used pregnant C57BL6J mice, and we reduced their food supply by 30% during the late gestational period and examined the changes in the metabolism and pancreatic β cell mass. Pancreatic β cell mass at birth was greatly decreased in the mice of the food restriction group (RG) as compared to the mice of the control group (CG). The body weight of RG mice exhibited a “catch-up growth” pattern and became equivalent to that of CG mice 7 days after birth, and thereafter exceeded that of CG mice; however, the pancreatic β cell mass in RG mice remained lower than that in CG mice at the age of 4 weeks. A high-fat diet significantly increased the pancreatic β cell mass in RG mice as compared to that in CG mice at 12 weeks of age. However, RG mice fed on high-fat diets tended to exhibit a decrease in the pancreatic β cell mass at approximately 20 weeks of age. The plasma insulin concentrations also tended to be decreased in RG mice after 24 weeks of age as compared to those of CG mice. These results thus indicate that the growth of pancreatic β cells is insufficient in RG mice, and pancreatic β cell failure can easily develop as a consequence of insulin resistance.
The spontaneously diabetic Torii (SDT) rat has recently been established as a model of type 2 diabetes mellitus (DM). The usefulness of this rat model for the study of diabetic voiding dysfunction was investigated. Male SDT rats and male Sprague-Dawley (SD) rats were used. Voiding function was evaluated by a metabolic cage study and cystometry. Total voided volume for 24 h, mean voided volume, and urinary frequency for 24 h were significantly greater in SDT rats at the age of 36 weeks. From cystometry mean inter-micturition interval (IMI) was significantly longer in SDT rats at the age of 22 and 36 weeks. In SDT rats mean IMI was significantly longer at the age of 36 weeks than at the age of 22 weeks. Mean voiding pressure was significantly higher in SDT rats at the age of 22 and 36 weeks. In the present study, SDT rats showed typical diabetic voiding dysfunction similar to other diabetic rat models. It was suggested that activity of the bladder afferent pathways is decreased and the urethral relaxation mechanism is impaired in SDT rats. In addition, SDT rats are suitable to study chronic diabetic voiding dysfunction because they survive without insulin treatment for as long as 60 weeks.
Age-related changes of physiological and biochemical properties were examined in the diaphragm muscle, which has particularly high activation compared to that of other skeletal muscles. The diaphragm from 10-week-, 50-week- and 100-week-old male Wistar rats were used to measure in vitro isometric contractile properties, sarcoplasmic reticulum (SR) Ca2+-ATPase activity, and myosin heavy chain (MHC) isoform composition. Although there were no significant differences in specific twitch tension of the diaphragm among the groups, there was significant reduction in specific tetanic tension in the 50-week to 100-week groups. The contraction time and 1/2 relaxation time of twitch contraction extended with aging, and significant differences were found between 10-week-old and 100-week-old diaphragms. Regarding the activity of SR Ca2+-ATPase, the pattern of age-related change was similar to that in the 1/2 relaxation time and there was a significant difference between 10-week-old and 100-week-old diaphragms. There was a significant increase in the relative composition of the MHC I isoform in 100-week-diaphragms compared to that in 10-week-old diaphragms and a concomitant decrease in the relative composition of fast myosin was noted. These findings demonstrated that older diaphragms have slower contraction and relaxation speeds, and these alterations were attributed to changes in SR Ca2+-ATPase activity and MHC isoform composition.
We conducted two studies to examine the hypothesis that lower urinary tract obstruction induces excessive production of prostaglandin E2 (PGE2) in the bladder in young boys, with consequent overactive bladder (OAB) symptoms. The subjects were boys aged less than 15 years old who were scheduled to undergo surgery in our department from October 2006 to March 2008. In study 1 (n = 25), the patients were divided into two groups based on the presence or absence of lower urinary tract obstruction. In study 2 (n = 38), the patients were classified by age. The PGE2 level in the bladder was determined by washing with saline before the operation and urinary symptoms were evaluated using the Pediatric Lower Urinary Tract Scoring System. In study 1, the PGE2 level in the bladder of patients with lower urinary tract obstruction was higher than in those without obstruction (49.1 ± 37.4 vs. 21.5 ± 10.1 pg/mL, P = 0.0475). In study 2, the PGE2 level in the bladder was negatively correlated with age (r = -0.379, P = 0.0207). A higher level of PGE2 is found in boys with bladder outlet obstruction due to urethral stricture and in younger boys, and this elevated level of PGE2 may induce OAB symptoms.
We recently demonstrated that endothelin-1 (ET-1) was strongly expressed in inflamed gingival tissues, but the biological role of ET-1 in gingival tissue remains unknown. This study focused on the relationship between ET-1 and interleukin-1β (IL-1β), an important cytokine during the periodontal inflammatory process. We determined the protein levels of ET-1 and IL-1β in gingival tissues from patients and examined whether ET-1 could regulate the expression of the IL-1β gene and protein in oral epithelial cells and fibroblasts in vitro. There was a significant correlation between the levels of ET-1 and IL-1β in 26 gingival tissues, as determined by ELISA. Following the confirmation of two specific ET-1 receptors (ETA and ETB receptors) on the cultured cells, the effects of ET-1 stimulation on IL-1β mRNA and protein expression were evaluated by RT-PCR and ELISA, respectively. The IL-1β mRNA and protein levels were enhanced by ET-1 stimulation in a dose-dependent manner, and the enhancement of IL-1β was inhibited by ETA or ETB receptor antagonists. These findings indicate that ET-1 is involved in the regulation of IL-1β expression in gingival tissues and suggest that ET-1 signaling to the cells may be a therapeutic target for treating IL-1β-dependent inflammatory responses.
GPR7, now known as a receptor of neuropeptide B and neuropeptide W, is expressed in neurons of the suprachiasmatic nucleus (SCN), the mammalian circadian center. By the quantitative in situ hybridization, we demonstrated that GPR7 mRNA showed a significant circadian rhythm in the SCN showing a peak at early subjective night in both light-dark and constant dark. We characterized the circadian feature of GPR7-knockout mice, but the period length and the phase-dependent phase shift to light exposure were not disordered in GPR7-knockout mice. Moreover, the food-anticipatory behavior in restricted feeding schedule was observed in this gene-deleted mouse similar to wild-type. These results indicate that the role of GPR7 may be subtle or limited in relation to the circadian clock despite its robust expression in the SCN.
In this study, we demonstrate that a cyanine dye, lumin, significantly suppressed cytopathic effect by herpes simplex virus (HSV)-1 toward human amnionic FL cell and also it reduced replication of HSV-1 in a dose-dependent manner. In addition, lumin additively augmented the antiviral effect of interferon (IFN)-α. Furthermore, fluorescence microscopic study showed that lumin (not IFN-α) itself remarkably induced alkalinization of intracellular organelle, suggesting the inhibition of virus invasion into the cells. These results suggest that lumin exerts an antiviral action against HSV-1 with the independent pathways of IFN-α and also it would become a therapeutically effective drug in clinical practice.