Trifluoromethyl ketone-based inhibitors of apoptosis in cerebellar granule neurons.

A variety of aromatic trifluoromethyl ketone derivatives has been studied as inhibitors of apoptosis in cerebellar granule neurons (CGNs). Among them, alpha-trifluoromethyl diketone (2) and benzyl trifluoromethyl ketone (11) were found to be apoptosis inhibitors which can prevent a neurodegenerative disease. Compounds 2 and 11 showed neuroprotection effect on low K+-induced apoptosis in CGNs. Furthermore, these compounds effectively suppressed DNA fragmentation accompanied with apoptosis. The neuroprotection mode of 2 and 11 was not related to inhibition of caspase-3.

Effect of glucocorticoid on expression of rat MUC5AC mRNA in rat gastric mucosa in vivo and in vitro.

The gastric mucus is an important factor in the gastric mucosal protection from acid, pepsin and various reagents (alcohol, aspirin, etc.). MUC5AC is the mucin secreted from surface mucous cells, and belongs to the gel-forming mucin. We examined the regulation of rat MUC5AC (rMUC5AC) mRNA by glucocorticoid in vivo and in vitro, comparing it with that of pepsinogen (Pg) mRNA. By adrenal gland resection, rMUC5AC and Pg mRNA levels and Pg content in rats significantly decreased to 70%, 46% and 42% of those in the sham operated controls, respectively. With the treatment of hydrocortisone (1, 5 and 50 mg/kg), Pg mRNA level and Pg content in adrenalectomized rats was restored. On the other hand, the rMUC5AC mRNA level exceeded the control with 1 or 5 mg/kg injections of hydrocortisone, but drastically decreased to 18% of sham operation levels with it (50 mg/kg). Similar results were obtained in normal rats with the treatment of hydrocortisone (50 mg/kg). Mucus and DNA content of cultured rat gastric epithelial cells were not affected by hydrocortisone, but rMUC5AC mRNA level was significantly decreased in a dose-dependent manner. From the in vivo and in vitro results, at least a physiological concentration of glucocorticoid was necessary in the expression of rMUC5AC mRNA. However, high dose of hydrocortisone directly suppressed the expression of rMUC5AC mRNA. These results suggested that hydrocortisone might directly cause the suppression and indirectly the enhancement of the mucin biosynthesis.

Dosing time-dependent pharmacological effects of anti-metabolites for rat cardiac graft.

Antimetabolites such as methotrexete and 6-mercaptopurine have been shown to have circadian variations in their toxicities. However, chronopharmacological profiles of mizoribine (Miz) that is newly synthesized as an anti-metabolic agent for immunosuppression, have not been evaluated. In this study, we examined the dosing time-dependent alterations in the pharmacokinetics and pharmacodynamics of Miz. In addition, chronopharmacology of azathiopurine (Aza) was also evaluated to compare with that of Miz. Initially, Miz (10 and 20 mg/kg) or Aza (20 mg/kg) was orally administered at 8:00 hr or 20:00 hr for 3 weeks to rats. To reveal the dosing time-dependent difference of pharmacokinetics, Miz (20 mg/kg) was orally given at 8:00 hr or 20:00 hr and blood was obtained for 12 hours. Finally, Miz (20 mg/kg) or Aza (20 mg/kg) was administered at 8:00 hr or 20:00 hr to rats with heterotopic allogeneic heart grafts. The Miz group treated at 8:00 hr and Aza group treated at 20:00 hr showed severe myelosuppression compared with their each opposite dosing time. AUC of Miz in the morning trial was twice as high as that in the evening trial. The graft survival durations of the Miz- and Aza-treated groups were significantly longer than those of the respective control groups, but were not affected by dosing time of each agent. These results suggest that the toxicity, but not efficacy of Miz is varied with the dosing time. The chronotoxicological phenomenon of Miz might be, at least in part, explained by the dosing time-dependent difference in serum drug concentrations and apparent clearance.

Antioxidative activity of Allium victorialis L. extracts.

Allium victorialis L. (Liliaceae, "Hon-Gyoujya Nin-Niku" in Japanese) was successively extracted with hexane, acetone, methanol and 70% methanol and the extracts were further separated into a total of twenty-five fractions by silica gel and ODS column chromatographies. The biological activities of these four extracts and 25 column fractions were compared. The cytotoxic activity of all extracts and fractions against two oral tumor cell lines was significantly higher than that against normal human gingival fibroblasts, suggesting their tumor-specific action. Three methanol column fractions [M2, M3, M6] and a 70% methanol column fraction [70M6] most effectively reversed the multidrug resistance (MDR) against L5178 mouse T cell lymphoma. The electron spin resonance (ESR) spectroscopy showed that methanol column fractions and 70% methanol extracts produced the highest amount of radical(s) and most efficiently scavenging O2*-, generated by the hypoxanthine-xanthine reaction system, suggesting that the same substances in these fractions display both prooxidant and antioxidant properties. They showed no anti-human immunodeficiency virus (HIV) or anti-Helicobacterpylori activity. These data suggest the medicinal efficacy of Allium victorialis extract.

ONO-1603, a potential antidementia drug, delays age-induced apoptosis and suppresses overexpression of glyceraldehyde-3-phosphate dehydrogenase in cultured central nervous system neurons.

Primary cultures of rat cerebral cortical cells and cerebellar granule cells die by an apoptotic mechanism after more than 2 weeks in cultures in the absence of medium change and glucose supplement, a process termed age-induced apoptosis of cultured neurons. Our preliminary study has shown that age-induced apoptosis of cerebellar granule cells is protected by pretreatment with tetrahydroaminoacridine (THA), an antidementia drug. In this study, we systematically compared the neuroprotective effects of THA with those of (S)-1-[N-(4-chlorobenzyl)succinamoyl]pyrrolidine-2-carbaldehyde (ONO-1603), a novel prolyl endopeptidase inhibitor and potential antidementia drug. Both ONO-1603 and THA effectively delay age-induced apoptosis of cerebral and cerebellar neurons, as demonstrated morphologically with toluidine blue and fluorescein diacetate/propidium iodide staining or biochemically by DNA laddering analysis on agarose gels. ONO-1603 is about 300 times more potent than THA, with a maximal protective effect at 0.03 and 10 microM, respectively. ONO-1603 shows a wide protective range of 0.03 to 1 microM in contrast to a narrow effective range of 3 to 10 microM for THA. Moreover, ONO-1603 is nontoxic to neurons, even at the high concentration of 100 microM, whereas THA elicits severe neurotoxicity at a dose of >/=30 microM. Both ONO-1603 and THA robustly suppress overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) mRNA and accumulation of GAPDH protein in a particulate fraction of cultured neurons undergoing age-induced apoptosis. Because we documented that GAPDH overexpression participates in neuronal apoptosis induced by various insults, we conclude that the neuroprotective actions of ONO-1603 and THA appear to be mediated by suppression of this protein overexpression.

Nuclear localization of overexpressed glyceraldehyde-3-phosphate dehydrogenase in cultured cerebellar neurons undergoing apoptosis.

We recently reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is directly involved in cytosine arabinonucleoside (ara-C)- and low K+-induced neuronal death of cultured cerebellar granule cells. The former is entirely due to apoptosis, whereas the latter involves both apoptosis and necrosis. We examined the subcellular distribution of the overexpressed GAPDH occurring during apoptosis by using both subcellular fractionation and immunocytochemistry with a monoclonal antibody directed against this overexpressed protein. When immature cerebellar neurons were exposed to ara-C, an overexpression of GAPDH was observed, primarily in the nuclear fraction. In contrast, low K+ exposure of mature cerebellar neurons induced the overexpression of GAPDH not only in the nuclear fraction but also in the mitochondrial fraction. In both paradigms, no significant change of GAPDH levels occurred in the microsomal and cytosolic fractions. Moreover, pretreatment with GAPDH antisense oligonucleotide or classic apoptotic inhibitors clearly suppressed the accumulation of GAPDH protein in these subcellular loci. This discrete nuclear localization of GAPDH during apoptosis was supported further by immunoelectron microscopy. Quantitative assessment of GAPDH immunogold labeling revealed that a approximately 5-fold increase in the intensity of gold particles was observed within the nucleus of apoptotic cells. Thus, the current results raise the possibility that neuronal apoptosis may be triggered by GAPDH accumulation in the nucleus, resulting in perturbation of nuclear function and ultimate cell death.

Overexpression of glyceraldehyde-3-phosphate dehydrogenase is involved in low K+-induced apoptosis but not necrosis of cultured cerebellar granule cells.

We have reported that overexpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) is involved in age-induced apoptosis of the cultured cerebellar granule cells that grow in a depolarizing concentration (25 mM) of KCI. The present study was undertaken to investigate whether GAPDH overexpression also occurs and participates in apoptosis of the cerebellar granule cells that result from switching the culturing conditions from high (25 mM) to low (5 mM) concentrations of KCl. We found that exposure of granule cells to low potassium (K+) for 24 hr induces not only apoptosis but also necrotic damage. The latter is supported by the morphological observations that a subpopulation of neurons showed cell swelling, extensive cytoplasmic vacuolization, damaged mitochondria, and apparently intact nuclei. Treatments with two antisense but not sense oligodeoxyribonucleotides directed against GAPDH attenuated low K+-induced neuronal death by approximately 50%. Morphological inspection revealed that GAPDH antisense oligonucleotides preferentially blocked low K+-induced apoptosis with little or no effect on necrotic damage. Similar to antisense oligonucleotides, actinomycin-D partially inhibited low K+-induced death of granule cells with a predominant effect on apoptosis. In contrast, cycloheximide almost completely blocked low K+-induced neuronal death and seemed to prevent both apoptotic and necrotic damage. The levels of GAPDH mRNA and protein were markedly increased in a time-dependent manner after low K+ exposure. The overexpression of GAPDH mRNA and protein was completely blocked by cycloheximide, actinomycin-D, and its antisense but not sense oligonucleotides. Taken together, these results lend credence to the view that exposure of cerebellar granule cells to low K+ induces both apoptosis and necrosis and that only the apoptotic component involves overexpression of GAPDH.

ONO-1603, a potential antidementia drug, shows neuroprotective effects and increases m3-muscarinic receptor mRNA levels in differentiating rat cerebellar granule neurons.

We have reported that the antidementia drug tetrahydroaminoacridine (THA; 30 microM) is neuroprotective and neurotrophic and selectively increases m3-muscarinic acetylcholine receptor (mAChR) mRNA levels in differentiating cerebellar granule cells. Here, we examined whether novel prolyl endopeptidase inhibitor ONO-1603, a potential antidementia drug, induces similar effects in these cerebellar neurons. Supplement of ONO-1603 (0.03 microM) to cultures grown in 15 mM KCl-containing media was found to markedly promote neuronal survival and neurite outgrowth and enhance [3H]N-methylscopolamine binding to mAChRs. Moreover, ONO-1603 increased the level of m3-mAChR mRNA and stimulated mAChR-mediated phosphoinositide turnover. The common actions of ONO-1603 and THA suggest that these properties could be related to their putative antidementia activities and that this model system may be used to screen for drugs effective in the treatment for Alzheimer's disease.

An antisense oligodeoxynucleotide to glyceraldehyde-3-phosphate dehydrogenase blocks age-induced apoptosis of mature cerebrocortical neurons in culture.

We recently reported that the age-induced apoptotic death of cultured cerebellar neurons is correlated with an increased expression of a particulate-bound 38-kDa protein that we identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). To determine whether this phenomenon of GAPDH overexpression occurs in other cell types, we selected primary cultures of cerebrocortical cells for testing, because under normal culture conditions, cortical neurons die progressively after 15 days in vitro. As with cerebellar neurons, this age-induced neuronal death involves ultrastructural changes and internucleosomal DNA fragmentation characteristic of apoptosis and is effectively prevented by actinomycin-D and cycloheximide. Moreover, a GAPDH antisense oligodeoxyribonucleotide arrested this cortical neuronal death for about 4 to 5 days and thus was more effective than cycloheximide. By contrast, its corresponding sense oligonucleotide had no effect. Additionally, the age-induced apoptosis of cortical neuronal cultures is effectively protected by aurintricarboxylic acid and tetrahy-droaminoacridine (an antidementia drug). Before cell death, GAPDH mRNA levels increased by about 2-fold and the increase was blocked by the above-mentioned neuroprotective agents and the GAPDH antisense, but not sense, oligonucleotide. The effects of antisense oligonucleotide are more robust in the present case than those found with cerebellar neurons, and they indicate a significant, though at present not defined, role of GAPDH in the apoptotic process occurring in these two types of neurons.

Clinical pharmacology of multiple-dose losartan, an angiotensin II receptor antagonist, in patients with essential hypertension.

The pharmacokinetic and pharmacodynamic alterations of multiple doses of losartan, an angiotensin II receptor antagonist, were examined in nine patients with essential hypertension. Participants were given placebo once daily for the first 7 days (from day -7 to day -1), and then 50 mg of losartan for the next 9 days (from day 1 to day 9). The 24-hour blood pressure was measured on days -1, 1, and 7 and blood samples for measurement of losartan and its active metabolite, E-3174, were obtained on days 1 and 7. Plasma concentrations of uric acid and plasma clearance were determined before and during treatment with losartan, and at the end of the study. Pharmacokinetic parameters after the seventh dose, including maximum plasma concentration (Cmax) and time to Cmax (tmax) of losartan and E-3174, did not differ significantly from those after the first dose. The blood pressure lowering effect of losartan, however, was significantly greater after the seventh dose than after the first dose. Plasma uric acid decreased and its plasma clearance (ClUA) increased significantly during repeated administration with losartan. These values returned to pretreatment levels after the end of treatment. These results suggest that although the pharmacokinetic profiles of losartan and E-3174 do not change during repeated administration, the blood pressure lowering effect in hypertensive patients is greater after multiple doses than after a single dose.

Evidence that glyceraldehyde-3-phosphate dehydrogenase is involved in age-induced apoptosis in mature cerebellar neurons in culture.

Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.

Lack of diurnal variation in glomerular filtration rates in the elderly.

The present study was undertaken to examine the effect of age on diurnal variation in glomerular filtration rate (GFR). The GFR, as estimated by creatinine clearance (Clcr), was determined during a 24-hour period in 10 younger (mean +/- SD age 42 +/- 9 years) and 10 older (mean age 75 +/- 4 years) patients with hypertension. Significant diurnal variations in Clcr were observed in the younger patients, with a peak during the day and trough during the night. Such were not observed in the older patients, however. These results suggest that diurnal variation in GFR is affected by age. Chronopharmacologic profiles of drugs, which are mainly excreted in urine by glomerular filtration, might be altered in these patients.

Effect of ranitidine on renal clearance of lomefloxacin.

OBJECTIVE: To examine the effect of ranitidine on the renal clearance of lomefloxacin. SETTING: Department of Clinical Pharmacology, Jichi Medical School. METHODS: Lomefloxacin 200 mg and ranitidine 300 mg or its placebo were given orally in a randomised, double-blind, crossover design. Blood and urine samples were obtained during a 24-h period after dosing. RESULTS: The area under the plasma concentration-time curve and the elimination half-life of lomefloxacin were significantly increased following coadministration with ranitidine. These effects were caused by significant decreases in total (7.8%) and renal (22%) clearance of lomefloxacin. In contrast, creatinine clearance and urinary excretion of electrolytes were not influenced by ranitidine. CONCLUSION: As lomefloxacin and ranitidine are excreted in urine by renal tubular secretion, the present results suggest that the renal tubular secretion of lomefloxacin is diminished by ranitidine. As the reduction in lomefloxacin clearance is only marginal, it is probable that the drug interaction observed in this study is not of clinical significance.

Glyceraldehyde-3-phosphate dehydrogenase is over-expressed during apoptotic death of neuronal cultures and is recognized by a monoclonal antibody against amyloid plaques from Alzheimer's brain.

The age-induced apoptotic death of cerebellar neurons in culture is associated with over-expression of a 38-kDa particulate protein identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Both the age-induced apoptosis and the 38-kDa protein overexpression were effectively suppressed by the presence of tetrahydroaminoacridine, an antidementia drug, or aurintricarboxylic acid. This over-expressed 38-kDa protein and purified GAPDH were found to react with a monoclonal antibody (mAb), Am-3, which was raised against amyloid plaques from Alzheimer's brain, but not with mAb, AmT-1, which was produced using synthetic amyloid beta peptide. These results raise the possibility that GAPDH is also involved in the neurodegeneration during the development of Alzheimer's disease.

Effect of losartan, an angiotensin II receptor antagonist, on response of cortisol and aldosterone to adrenocorticotrophic hormone.

Many imidazole derivatives are shown to inhibit adrenal steroid biosynthesis. The present study was undertaken to examine an effect of another imidazole derivative, losartan (an angiotensin II receptor antagonist), on responses of cortisol and aldosterone to adrenocorticotrophic hormone (ACTH). Nine patients with essential hypertension were given placebo orally for 7 days and 50 mg of losartan for the next 9 days. Response of serum cortisol and plasma aldosterone to intramuscular ACTH injection were determined before and at the end of the treatment with losartan. Serum cortisol and plasma aldosterone significantly increased after ACTH injection in both periods of treatment (placebo and losartan). The increments in these parameters during treatment with losartan were not significantly different from those during treatment with placebo. These results suggest that the inhibitory effect of losartan on adrenal steroid biosynthesis is negligible.

Recombinant human growth hormone and protein metabolism of burned rats and esophagectomized patients.

The effects of recombinant human growth hormone (rhGH) on protein metabolism were investigated. In the experimental study, 31 male Sprague-Dawley rats were divided into two groups. One group received 200 mU/day of rhGH for 3 consecutive days, before and after being burned (GH group). The other group received no rhGH as a control. Cumulative nitrogen balance after being burned was significantly higher in the GH group, and the rates of whole-body protein turnover, synthesis, and breakdown were significantly higher in the GH group. An increase of synthesis greater than that of breakdown resulted in an improved nitrogen balance in the GH group. Protein content of the liver and the gastrocnemius muscle were also significantly greater in the GH group. In the clinical study, 13 patients receiving esophagectomy for esophageal cancer were studied. Six of them received 24 U/day of rhGH for 5 consecutive postoperative days (GH group). Cumulative nitrogen balance on postoperative days was significantly higher in the GH group than in the control group. On the 3rd postoperative day, the rate of whole-body protein synthesis was significantly greater in the GH group; those of turnover and breakdown also increased in the GH group. The arteriovenous difference of amino acid composition revealed that uptake of branched-chain amino acids into the leg muscles was significantly elevated and that release of phenylalanine and tyrosine from the muscles was significantly reduced in the GH group. Hepatic function was not affected by the administration of rhGH, and rhGH inhibited the rise of blood urea nitrogen and total bilirubin after esophagectomy.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronopharmacology of enalapril in hypertensive patients.

The pharmacokinetics and pharmacodynamics of enalapril, an angiotensin converting enzyme inhibitor, are reported to vary with the time of administration. The present study was undertaken to examine whether the effect of enalapril on plasma bradykinin (BK), substance P and prostaglandin E2 (PGE2), which are likely to be involved in the mechanism of enalapril-induced cough, might also be affected by its time of administration. Enalapril 5 mg or placebo was given orally at 10:00 h (day trial) or 22:00 h (night trial) to 12 patients with essential hypertension. Serum concentrations of total drug (enalapril + enalaprilat, its active metabolite) during the day and night trials did not differ significantly at any time. However, serum enalaprilat tended to be higher and its maximum concentration greater in the day trial than in the night trial. Blood pressure 24 h after administration of enalapril was reduced at 22:00 h, but not at 10:00 h. Plasma BK tended to increase following enalapril administration at 10:00 h, but not at 22:00 h. Remarkable increases in plasma BK were observed in two patients in the day trial and one of them also complained of cough. However, no such increase in plasma BK or subsequent adverse effect were recorded in the night trial. Plasma substance P and PGE2 did not change significantly following enalapril administration either in the day or night trial. The results suggest that the response of BK to enalapril is affected by the time of administration. In patients who complain of cough during treatment with enalapril during the daytime, this adverse effect might be diminished or eliminated by a switch to night-time administration.

Administration time-dependent change in the effect of spironolactone in aged rats.

We have previously demonstrated that the natriuretic effect of spironolactone, a competitive antagonist of mineralocorticoid, varies with its administration time in young rats. The present study was undertaken to examine the influence of aging on chronopharmacological profiles of the agent. Spironolactone (10 and 50 mg/kg) was given orally at 12 am or 12 pm in young (12 weeks old) and aged (28 months old) Wistar rats. The 8-hour urine was collected after each administration, and the urinary sodium excretion was determined. The urinary sodium excretion increased dose-dependently following spironolactone in the young and aged groups of rats. The increments in this parameter in the 12 pm trial were significantly greater than those of the 12 am trial in the young rats. However, such an administration time-dependent difference in the effect of spironolactone was diminished and did not reach statistical significance in the aged animals. These results suggest that the mode of the administration time-dependent change in the effect of spironolactone is altered with age.

Tetrahydroaminoacridine increases m3-, but not m2-, muscarinic acetylcholine receptor mRNA levels in differentiating cerebellar granule cells.

We used Northern blot hybridization to determine whether 9-amino-1,2,3,4-tetrahydroacridine (THA), a potential antidementia drug, selectively altered the levels of muscarinic acetylcholine receptor (mAChR) mRNA in differentiating cerebellar granule cells. Granule cells were cultured for 8 days in media containing 15 mM K+, 25 mM K+ or 15 mM K+ plus 30 microM THA. High K+ markedly increased the levels of m2- and m3-mAChR mRNA in the surviving cells. In contrast, THA increased the levels of m3-mAChR mRNA, but had little or no effect on m2-mAChR mRNA levels. These results suggest that THA selectively up-regulates the synthesis of m3-mAChR mRNA.