Kiyoyuki Kitaichi

Sinoatrial Node Dysfunction and Early Unexpected Death of Mice With a Defect of klotho Gene Expression

Background—Homozygous mutant mice with a defect of klotho gene expression (kl/kl) show multiple age-related disorders and premature death from unknown causes. Methods and Results—The kl/kl mice subjected to 20-hour restraint stress showed a high rate (20/30) of sudden death, which was associated with sinoatrial node dysfunction (conduction block or arrest). Heart rate and plasma norepinephrine of kl/kl mice, unlike those of wild-type (WT) mice, failed to increase during the stress. Intrinsic heart rate after pharmacological blockade of autonomic nerves in kl/kl mice was significantly lower than that in WT mice (380±33 versus 470±44 bpm; n=7). The sinus node recovery time after an overdrive pacing (600 bpm, 30 seconds) in kl/kl mice was significantly longer than in WT mice (392±37 versus 233±24 ms; n=6). In isolated sinoatrial node preparations, the positive chronotropic effect of isoproterenol was significantly less, whereas the negative chronotropic effect of acetylcholine was significantly greater in kl/kl than in WT mice. There was no degenerative structural change in the sinoatrial node of kl/kl mice. The precise localization of klotho was analyzed in newly prepared klotho-null mice with a reporter gene system (kl−geo). Homozygous kl− geo mice showed characteristic age-associated phenotypes that were almost identical to those of kl/kl mice. In the kl− geo mice, klotho expression was recognized exclusively in the sinoatrial node region in the heart in addition to parathyroid, kidney, and choroid plexus. Conclusions—In the heart, klotho is expressed solely at the sinoatrial node. klotho gene expression is essential for the sinoatrial node to function as a dependable pacemaker under conditions of stress.

Role of Tumor Necrosis Factor-α in Methamphetamine-Induced Drug Dependence and Neurotoxicity

Tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, is now emerging as an important modulator of the function of the CNS. Methamphetamine (METH) is a widely abused psychostimulant that causes euphoria, hyperactivity, and drug dependence. High doses of METH cause long-term neurotoxicity in dopaminergic neurons. In this study, we investigated a role of TNF-α in METH-induced dependence and neurotoxicity. Repeated treatment with METH (2 mg/kg for 5 d) in rats induced a significant increase in TNF-α mRNA and protein expression in the brain. Exogenous TNF-α (1-4 μg) blocked locomotor-stimulating and rewarding effects of METH, as well as METH (4 mg/kg; four times at 2 hr intervals)-induced dopaminergic neurotoxicity in mice. To examine a role of endogenous TNF-α in behavioral and neurochemical effects of METH, we used mice with targeted deletions of the TNF-α gene. TNF-α-(-/-) mice showed enhanced responses to the locomotor-sensitizing, rewarding, and neurotoxic effects of METH compared with wild-type mice. We also examined the role of TNF-α in METH-induced dopamine (DA) release and uptake in vitro and in vivo in C57BL/6 mice. Exogenous TNF-α (4 μg) attenuated the METH-induced increase in extracellular striatal DA in vivo and potentiated striatal DA uptake into synaptosomes in vitro and in vivo. Furthermore, TNF-α activated vesicular DA uptake by itself and diminished the METH-induced decrease in vesicular DA uptake. Our findings suggest that TNF-α plays a neuroprotective role in METH-induced drug dependence and neurotoxicity by activating plasmalemmal and vesicular DA transporter as well as inhibiting METH-induced increase in extracellular DA levels.

Protective effects of minocycline on behavioral changes and neurotoxicity in mice after administration of methamphetamine

The effects of minocycline on behavioral changes and neurotoxicity in the dopaminergic neurons induced by the administration of methamphetamine (METH) were studied. Pretreatment with minocycline (40 mg/kg) was found to attenuate hyperlocomotion in mice after a single administration of METH (3 mg/kg). The development of behavioral sensitization after repeated administration of METH (3 mg/kg/day, once daily for 5 days) was significantly attenuated by pretreatment with minocycline (40 mg/kg). A reduction in the level of dopamine (DA) and its major metabolite, 3,4-dihydroxyphenyl acetic acid (DOPAC), in the striatum after the repeated administration of METH (3 mg/kg x 3, 3-h interval) was attenuated in a dose-dependent manner by pretreatment with and the subsequent administration of minocycline (10, 20, or 40 mg/kg). Furthermore, minocycline (40 mg/kg) significantly attenuated a reduction in DA transporter (DAT)-immunoreactivity in the striatum after repeated administration of METH. In vivo microdialysis study demonstrated that pretreatment with minocycline (40 mg/kg) significantly attenuated increased extracellular DA levels in the striatum after the administration of METH (3 mg/kg). In addition, minocycline was not found to alter the concentrations of METH in the plasma or the brain after three injections of METH (3 mg/kg), suggesting that minocycline does not alter the pharmacokinetics of METH in mice. Interestingly, METH-induced neurotoxicity in the striatum was significantly attenuated by the post-treatment and subsequent administration of minocycline (40 mg/kg). These findings suggest that minocycline may be able to ameliorate behavioral changes as well as neurotoxicity in dopaminergic terminals after the administration of METH. Therefore, minocycline could be considered as a useful drug for the treatment of several symptoms associated with METH abuse in humans.

Methamphetamine-induced dopaminergic neurotoxicity is regulated by quinone formation-related molecules

Recently, the neurotoxicity of dopamine (DA) quinone formation by auto‐oxidation of DA has focused on dopaminergic neuron‐specific oxidative stress. In the present study, we examined DA quinone formation in methamphetamine (METH)‐induced dopaminergic neuronal cell death using METH‐treated dopaminergic cultured CATH.a cells and METH‐injected mouse brain. In CATH.a cells, METH treatment dose‐dependently increased the levels of quinoprotein (protein‐bound quinone) and the expression of quinone reductase in parallel with neurotoxicity. A similar increase in quinoprotein levels was seen in the striatum of METH (4 mg/kg X4, i.p., 2 h interval)‐injected BALB/c mice, coinciding with reduction of DA transporters. Furthermore, pretreatment of CATH.a cells with quinone reductase inducer, butylated hydroxyanisole, significantly and dose‐dependently blocked METH‐induced elevation of quinoprotein, and ameliorated METH‐induced cell death. We also showed the protective effect of tyrosinase, which rapidly oxidizes DA and DA quinone to form stable melanin, against METH‐induced dopaminergic neurotoxicity in vitro and in vivo using tyrosinase null mice. Our results indicate that DA quinone formation plays an important role, as a dopaminergic neuron‐specific neurotoxic factor, in METH‐induced neurotoxicity, which is regulated by quinone formation‐related molecules.

Continuous positive airway pressure improves daytime baroreflex sensitivity and nitric oxide production in patients with moderate to severe obstructive sleep apnea syndrome.

Individuals with obstructive sleep apnea syndrome (OSAS) are at high risk for cardiovascular morbidity and mortality. The effects of OSAS severity and nocturnal continuous positive airway pressure (CPAP) on daytime baroreflex sensitivity (BRS) and nitric oxide (NO) production were investigated in OSAS patients. Fifty-one consecutive males with OSAS and 29 age-matched healthy men underwent the Valsalva test and standard polysomnography. Patients with an apnea-hypopnea index (AHI) of ≥20 episodes per hour were randomized to receive CPAP treatment for 3 months (n=14) or no such treatment (n=19). The BRS index measured from the overshoot phase (phase IV) of the Valsalva maneuver and plasma NO concentration were significantly lower, whereas the AHI, oxygen desaturation time, arousal index, percentage of sleep stage 1, and systolic blood pressure were significantly greater, in patients with an AHI of ≥20/h than in those with an AHI of <20/h or in controls. The 24-h urinary excretion of norepinephrine was significantly reduced and the plasma NO concentration was significantly increased after one night of CPAP. The BRS index for phase IV and the Valsalva ratio were significantly increased in the CPAP group after the 3-month treatment period but remained unchanged in the non-CPAP group of OSAS patients. The daytime BRS index and NO production were thus inversely related to the severity of OSAS, and successful CPAP treatment improved these parameters in patients with moderate to severe OSAS. CPAP may therefore reduce the risk of cardiovascular complications due to endothelial dysfunction or increased sympathetic activity.

Reversal of anticancer drug resistance by macrolide antibiotics in vitro and in vivo.

1. The combined effects of the macrolide antibiotics erythromycin, josamycin, clarithromycin and YM17K (3,4′‐dideoxy mycaminosyl tylonolide hydrochloride) on in vitro intracellular accumulation of vinblastine or cyclosporine (Cs)A and on the in vivo antitumour activity of vinblastine were investigated using mouse leukaemia P388 cells (P388/S) and anticancer drug‐resistant (P388/ADR) cells. These effects were compared with those of a calcium antagonist (verapamil) or immunosuppressants (FK506 and CsA).

Possible Involvement of the Drug Transporters P Glycoprotein and Multidrug Resistance-Associated Protein Mrp2 in Disposition of Azithromycin

ABSTRACT P glycoprotein and multidrug resistance-associated protein 2 (Mrp2), ATP-dependent membrane transporters, exist in a variety of normal tissues and play important roles in the disposition of various drugs. The present study seeks to clarify the contribution of P glycoprotein and/or Mrp2 to the disposition of azithromycin in rats. The disappearance of azithromycin from plasma after intravenous administration was significantly delayed in rats treated with intravenous injection of cyclosporine, a P-glycoprotein inhibitor, but was normal in rats pretreated with intraperitoneal injection erythromycin, a CYP3A4 inhibitor. When rats received an infusion of azithromycin, cyclosporine and probenecid, a validated Mrp2 inhibitor, significantly decreased the steady-state biliary clearance of azithromycin to 5 and 40% of the corresponding control values, respectively. However, both inhibitors did not alter the renal clearance of azithromycin, suggesting the lack of renal tubular secretion of azithromycin. Tissue distribution experiments showed that azithromycin is distributed largely into the liver, kidney, and lung, whereas both inhibitors did not alter the tissue-to-plasma concentration ratio of azithromycin. Significant reduction in the biliary excretion of azithromycin was observed in Eisai hyperbilirubinemic rats, which have a hereditary deficiency in Mrp2. An in situ closed-loop experiment showed that azithromycin was excreted from the blood into the gut lumen, and the intestinal clearance of azithromycin was significantly decreased by the presence of cyclosporine in the loop. These results suggest that azithromycin is a substrate for both P glycoprotein and Mrp2 and that the biliary and intestinal excretion of azithromycin is mediated via these two drug transporters.

Effect of Endotoxin on P-Glycoprotein-Mediated Biliary and Renal Excretion of Rhodamine-123 in Rats

ABSTRACT The effects of Klebsiella pneumoniae endotoxin on the biliary excretion and renal handling of rhodamine-123 were investigated in rats at different times after intraperitoneal injection (1 mg/kg of body weight). The typical substrates for P glycoprotein, i.e., cyclosporine, colchicine, and erythromycin, inhibited the biliary clearance of rhodamine-123, whereas a substrate for organic cation transporter, cimetidine, did not inhibit clearance, suggesting that rhodamine-123 is transported mainly by P glycoprotein. The biliary, renal, and tubular secretory clearances of rhodamine-123 and the glomerular filtration rate significantly decreased 6 h after injection of endotoxin but returned to control levels by 24 h. These results suggest that endotoxin-induced decreases in P-glycoprotein-mediated biliary excretion and renal handling of rhodamine-123 were probably due to impairment of P-glycoprotein-mediated transport ability. Pretreatment with pentoxifylline (50 mg/kg) significantly inhibited endotoxin-induced increases in tumor necrosis factor alpha (TNF-α) levels in plasma, which ameliorated the endotoxin-induced reduction of the biliary excretion of rhodamine-123. It is likely that endotoxin-induced impairment of the transport of rhodamine-123 is caused, in part, by overproduction of TNF-α. The effect of endotoxin on the expression of P-glycoprotein mRNA in liver and kidneys of rats was investigated by using a reverse transcriptase PCR. The expression of Mdr1a mRNA in both liver and kidney decreased 6 h after endotoxin injection and returned to control levels after 24 h, whereas the expression of Mdr1b mRNA in liver increased at both times and that in kidney decreased at 24 h. These findings suggest that K. pneumoniae endotoxin dramatically decreases P-glycoprotein-mediated biliary and renal excretion of rhodamine-123 probably by decreasing the expression of Mdr1a, which is likely due to increased plasma TNF-α levels.

Outcome measurement of extensive implementation of antimicrobial stewardship in patients receiving intravenous antibiotics in a Japanese university hospital

Background:  Antimicrobial stewardship has not always prevailed in a wide variety of medical institutions in Japan.

Mice lacking the schizophrenia-associated protein FEZ1 manifest hyperactivity and enhanced responsiveness to psychostimulants

FEZ1 (fasciculation and elongation protein zeta 1), a mammalian ortholog of Caenorhabditis elegans UNC-76, interacts with DISC1 (disrupted in schizophrenia 1), a schizophrenia susceptibility gene product, and polymorphisms of human FEZ1 have been associated with schizophrenia. We have now investigated the role of FEZ1 in brain development and the pathogenesis of schizophrenia by generating mice that lack Fez1. Immunofluorescence staining revealed FEZ1 to be located predominantly in gamma-aminobutyric acid-containing interneurons. The Fez1(-/-) mice showed marked hyperactivity in a variety of behavioral tests as well as enhanced behavioral responses to the psychostimulants MK-801 and methamphetamine. In vivo microdialysis revealed that the methamphetamine-induced release of dopamine in the nucleus accumbens was exaggerated in the mutant mice, suggesting that enhanced mesolimbic dopaminergic transmission contributes to their hyperactivity phenotype. These observations implicate impairment of FEZ1 function in the pathogenesis of schizophrenia.