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Previously, we reported that specific lower dose of sodium 2,3-dimercapto-1-propanesulfonic acid (DMPS) which is an antidote to heavy metal intoxication, inversely enhanced cisplatin (CDDP)-induced antitumor activity to S-180 cell-bearing mouse. This activity was only weak with meso-2,3-dimercaptosuccinic acid (DMSA), however. This study investigated the effects of lower doses of DMPS or DMSA on the nephrotoxicity and kinetics of CDDP. Kidney and blood isolated from female mice which received CDDP with or without DMPS or DMSA once daily for 4 days were provided for measuring levels of blood urea nitrogen (BUN) and transporter proteins (OCT2: organic cation transporter; MATE1: multidrug and toxin extrusion) mRNA, and CDDP-originated platinum, and TUNEL staining of renal tubular cells. DMPS or DMSA reduced effectively CDDP-induced BUN, and caused a moderate reduction of platinum in kidney. Additionally, both dimercapto-compounds restored the CDDP-reduced mRNA levels of transporter proteins (OCT2 and MATE1), and apparently suppressed the CDDP-induced apoptosis. These results suggest that DMPS, as well as DMSA, at approximate 17-fold dose (μmol/kg) of CDDP, has an enough potential to reverse the CDDP nephrotoxicity, and concomitant use of DMPS considering both dose and timing for administration is potentially useful for preventing nephrotoxicity and enhancing antitumor activity during CDDP chemotherapy.

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase–activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

We investigated the enhancing effect of two metal-chelating compounds, 2,3-dimercapto-1-propanesulfonic acid (DMPS) and meso-2,3-dimercaptosuccinic acid (DMSA), on the antitumor activity of cisplatin (CDDP). In the in vivo experiments, DMPS showed a clear synergistic effect and significantly enhanced the antitumor activity of CDDP in terms of survival and life span in mice transplanted with ascites sarcoma 180 cells (S180 cells) at a dose of <100 μmol/ kg, s.c., but not at a dose of >500 μmol/kg. On the other hand, DMSA did not enhance the antitumor activity of CDDP. DMPS (50 μmol/kg, s.c.) combined with CDDP also potently suppressed [3H]thymidine uptake in S180 cells implanted in mice, whereas DMSA did not. In the in vitro experiments, DMPS (10−6to 10−5M) produced a time- and dose-dependent decrease in intracellular Ca2+concentrations ([Ca2+] i) in S180 cells and, in combination with CDDP, yielded a significant increase in intracellular platinum accumulation compared to that in cells treated with CDDP alone. These results indicate that DMPS used in combination with CDDP may be of considerable benefit in enhancing the cytotoxicity of CDDP in tumor cells, especially at a low dose. The results also suggest that the enhancing effect of DMPS is closely related to a decrease in [Ca2+] iand that the suitable dose and adequate administrational time of DMPS are important for its effective action.

We investigated the inhibitory role of γ-aminobutyric acid A (GABA A) receptors on amylase release and the evidence for functional coupling with central-type benzodiazepine receptors in rat parotid acinar cells. Muscimol and GABA decreased isoprenaline-induced amylase release. This effect was blocked by bicuculline, a GABA A-receptor antagonist, and enhanced by clonazepam, a central-type benzodiazepine-receptor agonist, and diazepam, a central- and peripheral-type benzodiazepine-receptor agonist. Although bicuculline completely blocked the combination effect of GABA A-receptor agonist and clonazepam, it did not completely block the combination effect with diazepam. These results suggest that protein secretion is suppressively regulated by GABA Areceptors coupled with central-type benzodiazepine receptors.

Recent studies indicate that an endogenous co-agonist for an N-methyl-D-aspartate (NMDA) receptor–related glycine site, D-serine, is synthesized by serine racemase and is metabolized by D-amino acid oxidase (DAO) and that acute treatment with morphine augments the gene expression of serine racemase and DAO in rat brain. To further elucidate the mechanism underlying the activation of NMDA receptors following chronic opioid administration, we have evaluated the effects of the chronic administration of morphine on the mRNA and protein expressions of serine racemase and DAO and on the contents of D-serine in several areas of the rat brain. Repeated administration of morphine for 30 days produced a significant augmentation of both the mRNA and protein expressions of serine racemase in all the brain regions, whereas no significant change in the protein expression of DAO was observed in all the brain regions. Furthermore, the chronic administration caused a slight but significant elevation in the concentration of D-serine in the cortex, striatum, and hippocampus. These results indicate the elevated D-serine level following the chronic morphine treatment could at least in part be involved in the activation of NMDA receptors via the glycine site.

Lactoferrin, a member of the transferrin family, is iron-binding and a strongly cationic 76 kDa glycoprotein. In breast milk it is secreted in high concentrations from glandular epithelia and is also present in other exocrine fluids including saliva. In the present study, we examined the biological mechanisms of apoptosis induced by pepsin-digested-lactoferrin peptide (Lfn-p) in the human oral squamous cell carcinoma cell line SAS. We found that treatment with Lfn-p induced cell death with apoptotic nuclear changes, preceded by the cleavage of caspase-3 and poly (ADP-ribose) polymerase (PARP) in the apoptotic cells. Treatment with Lfn-p induced phosphorylation of extracellular signal-regulated kinase (ERK1/2), a member of the MAP kinase family, at early stages of apoptosis. Another MAP kinase, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), was also phosphorylated by treatment with Lfn-p. Pretreatment of SAS cells with SP600125, a JNK/SAPK inhibitor, diminished Lfn-induced apoptosis, as assessed by determining released lactate dehydrogenase activity. On the other hand, the MEK1 inhibitors PD98059 or U0126 showed no effect on repression of cell death, but rather an increase. These results suggest that JNK/SAPK activation may play an important role in Lfn-p-induced apoptotic cell death of human oral squamous cell carcinoma cells.

The effects of various anticonvulsants on local anaesthetics procaine- and lidocaine-induced convulsions were investigated in rats. Pretreatment with diazepam (2.5–5 mg/kg, intraperitoneally) and clonazepam (5–10 mg/kg, intraperitoneally) completely protected the rats against both local anaesthetic-induced convulsions. Phenobarbital (12.5–50 mg/kg, subcutaneously) also significantly decreased the incidence of both convulsions and prolonged their latencies. Carbabazepine (10–40 mg/kg, intraperitoneally) did not completely repress both convulsions, but it prolonged their latencies. Phenytoin (5–20 mg/kg, intraperitoneally) and primidone (30–60 mg/kg, intraperitoneally) markedly enhanced both local anaesthetic-induced convulsions, as shown by shortening of latency and increase in mortality. Valproate (100–200 mg/kg, intraperitoneally) produced a protective effect against procaine-induced convulsions, while it strongly enhanced lidocaine-induced convulsions. These results suggest that the benzodiazepines are effective drugs to prevent neurotoxicity induced by local anaesthetics, while phenytoin and primidone potentiate them.

This study examined the influence of benzodiazepine receptors on amylase release from rat parotid acinar cells. Diazepam (10−8–10−6M), which is a potent agonist of both central- and peripheral-type benzodiazepine receptors, dose dependently decreased amylase release induced by isoprenaline and carbachol, which are β-adrenoceptor and muscarinic receptor agonists, respectively. The maximum inhibitory response was obtained with 10−6M diazepam: amylase release was decreased to 57% (isoprenaline) and 39% (carbachol) of maximal levels, while these responses were completely inhibited by propranolol and atropine, respectively. Clonazepam and 7-chloro-1,3-dihydro-1-methyl-5-(p-chlorophenyl)-2H-1,4-benzodiazepine-2-one (Ro 5-4864), which are selective agonists of central- and peripheral-type benzodiazepine receptors, respectively, also produced a significant and dose-dependent decrease in isoprenaline-induced amylase release. The inhibitory potency was diazepam>clonazepam>Ro 5-4864. Flumazenil and 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK 11195), which are selective antagonists of central- and peripheral-type benzodiazepine receptors, respectively, dose dependently blocked the inhibition of isoprenaline-induced amylase release by diazepam. At a concentration of 10−5M, flumazenil and PK 11195 restored amylase release to ∼75% of that in the presence of isoprenaline alone. The combination of both antagonists completely prevented the inhibition by diazepam. Similarly, the inhibitory responses of clonazepam and Ro 5-4864 were completely blocked by flumazenil and PK 11195, respectively. These results suggest that, in rat parotid acinar cells, benzodiazepines inhibit β-adrenoceptor and muscarinic receptor-stimulated amylase release and that both central- and peripheral-type benzodiazepine receptors contribute to this inhibitory regulation.