Kunio Iwata

Effects of a novel thyrotropin-releasing hormone analogue, JTP-2942, on extracellular acetylcholine and choline levels in the rat frontal cortex and hippocampus.

The effects of a novel thyrotropin-releasing hormone (TRH) analogue, N alpha-[(1S,2R)-2-methyl-4-oxocyclopentanecarbonyl]-L-histidyl-L-pr olinamide (JTP-2942) on acetylcholine (ACh) release and on the extracellular choline level were investigated in rat frontal cortex and hippocampus by microdialysis, and were compared with effects of TRH. JTP-2942 (0.3 mg/kg i.p.) produced a marked (> 300%) and persistent increase of ACh release in both the frontal cortex and hippocampus, while TRH (3 mg/kg i.p.) caused a significant but transient increase of ACh to about 200% in the frontal cortex. Both drugs significantly decreased the choline levels in both brain regions. Investigation of the effects of JTP-2942 (0.001-1 mM) and TRH (1 and 10 mM) on ACh release and choline levels when perfused through the dialysis probe revealed that JTP-2942 had a greater effect than TRH in both the frontal cortex and the hippocampus. The action of JTP-2942 was about 1000-fold more potent than that of TRH in both brain regions. Oral administration of JTP-2942 at a dose of 10 mg/kg markedly and persistently increased the release of ACh and at doses of 1-10 mg/kg decreased the extracellular choline level in the frontal cortex and hippocampus. These results also suggest that JTP-2942 has some selectivity for the hippocampus compared to the frontal cortex after both systemic administration and local injection. The increase of ACh release caused by JTP-2942 was completely antagonized by perfusion with tetrodotoxin (TTX, 1 microM), suggesting that the action of JTP-2942 on cholinergic neurons was mediated via neuronal activity.

B16-G4F mouse melanoma cells: An MSH receptor-deficient cell clone

The two mouse melanoma cell lines B16‐F1 and B16‐G4F retain their melanogenic capacity when cultured in vitro. Melanotropic peptides such as α‐melanocyte‐stimulating hormone (α‐MSH) induce formation and release of melanin pigment in B16‐F1 cells. In contrast, B16‐G4F cells do not respond to α‐MSH. Using receptor‐binding analysis and photoaffinity crosslinking we demonstrate that the lack of response of B16‐G4F cells to α‐MSH is due to the absence of functional MSH receptors from the cell surface. Northern blot analysis of receptor mRNA revealed that MSH receptor mRNA is not expressed in B16‐G4F cells. These cells represent a new tool for the study of signal pathways related to the control of melanogenesis in melanoma cells.

A rapid simple screening method for skin tumor promoters using mouse peritoneal macrophages in vitro

The enhancing effects in vitro of a potent skin-tumor promoter, phorbol myristate acetate (PMA), and its derivatives on nitroblue tetrazolium (NBT) reduction, phagocytosis and cell spreading of mouse peritoneal macrophages were compared. The enhancing effects on these markers of macrophage function, especially NBT reduction, were found to correlate well with the skin-tumor promoting activities of the phorbol esters. Another potent promoter, teleocidin, strongly enhanced NBT reduction, although teleocidin is structurally different from phorbol esters. Other tumor promoters, namely saccharin, phenobarbital, cantharidin and Tween 60 did not enhance NBT reduction by macrophages. These data indicate that this test system is appropriate for screening of PMA-type tumor promoters such as phorbol esters and teleocidin.

Effect of JTP-2942, a novel thyrotropin-releasing hormone analogue, on pentobarbital-induced anesthesia in rats

The effects of a novel thyrotropin-releasing hormone (TRH) analogue, N alpha-((1S,2R)-2-methyl-4-oxocyclopentylcarbonyl)-L-histidyl-L-pro linamide monohydrate (JTP-2942), on pentobarbital-induced anesthesia in rats were investigated and compared with those of TRH. Intravenous administration of both JTP-2942 and TRH caused a dose-dependent decrease in the recovery time from pentobarbital-induced anesthesia. The minimum effective doses of JTP-2942 and TRH were respectively 0.03 and 1 mg/kg. The effect of JTP-2942 was antagonized by intraperitoneal scopolamine (0.5 mg/kg). Intraperitoneal JTP-2942 (1 mg/kg) caused an increase of acetylcholine release and a decrease of choline release in the frontal cortex and hippocampus of pentobarbital-treated rats. In addition, JTP-2942 ameliorated the decrease of hemicholinium-3-sensitive high-affinity choline uptake and the increase of acetylcholine in these brain regions. However, JTP-2942 had no effect on choline acetyltransferase activity or the choline content, which were also not changed by pentobarbital. Our results indicate that the effect of JTP-2942 on pentobarbital-induced anesthesia was about 30 times more potent than that of TRH, and suggest that JTP-2942 may act by accelerating acetylcholine turnover.

Induction of nitroblue tetrazolium reduction in mouse peritoneal macrophages by tumor promoters and inhibition of the induced nitroblue tetrazolium reduction by some inhibitors

Two polyacetates, aplysiatoxin and debromoaplysiatoxin, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA), mezerein and teleocidin enhance nitroblue tetrazolium (NBT) reduction in mouse peritoneal macrophages in vitro. The ED50 values for NBT reduction of these 5 TPA-type tumor promoters were 4.2 ng/ml for TPA, 36 ng/ml for mezerein, 0.53 ng/ml for teleocidin, 1.5 ng/ml for aplysiatoxin and 108 ng/ml for debromoaplysiatoxin. The NBT reduction induced by the 5 tumor promoters is inhibited by 2 inhibitors of tumor promotion, retinoic acid and dibromoacetophenone. The possibility that tumor promotion by TPA-type tumor promoters involves similar mechanisms such as superoxide anion radicals release in cell membranes is discussed.

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced nitroblue tetrazolium reduction in mouse peritoneal macrophages by various tumor promotion inhibitors

The effects of various inhibitors on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced reduction of nitroblue tetrazolium (NBT) in mouse peritoneal macrophages were investigated. The reduction was inhibited by phospholipase A2 inhibitors, such as dibromoacetophenone, the lipoxygenase inhibitor nordihydroguaiaretic acid, an NADH-dehydrogenase inhibitor, the microfilament inhibitor cytochalasin B, oxygen radical scavengers such as superoxide dismutase, antioxidants such as butyl hydroxyanisole and non-specific inhibitors such as retinoic acid. The reduction was not affected by the cyclooxygenase inhibitor indomethacin or the H2O2 scavenger catalase.

Effect of JTP-2942, a Novel TRH Analogue, on Cognitive Function and Learning in Rodents

Thyrotropin-releasing hormone (TRH) is a hypothalamic hormone that releases thyrotropin and prolactin from the anterior pituitary, and is widely distributed throughout the central nervous system (CNS).1–5 It has been shown to have various central actions, such as promoting locomotor activity, increasing body temperature in animals, attenuating pentobarbital induced sleep, and promoting recovery from traumatic loss of consciousness in mice.6–10 TRH also increases cholinergic neuronal activity11–14 and improves memory in amnesia models.15 Currently TRH and its analogues are under clinical investigation for treating spinal cord injury, traumatic brain injury, and dementia including Alzheimer’s disease.16–18 However, TRH has some disadvantages for therapeutic use, including a short duration of action and unsuitability for oral administration. Accordingly, we synthesized a novel TRH analogue, Nα-WS, 2R)-2-methyl-4-oxocyclopentanecarbonyl]-L-histidyl-L-prolineamide monohydrate (JTP2942), with a longer duration of action on the CNS and fewer hormonal effects than TRH.19–21 JTP-2942 has been shown to markedly increase CNS cholinergic neuronal activity22 and improve memory in amnesia models and aged animals.23,24 The callosal-neocortical system is implicated in interhemispheric transfer of the engram and the lateralization of information.25–27 The present study investigated whether JTP-2942 could improve cognitive function and learning in rats subjected to a passive avoidance test after transection of the corpus callosum, and investigated the mechanism of action of this drug using the transcallosal response.

Effects of a Novel TRH Analog, JTP-2942, on Cholinergic and Monoaminergic Neurons in the Brain

Thyrotropin-releasing hormone (TRH) is a hypothalamic hormone that releases thyrotropin and prolactin from the anterior pituitary gland. TRH and its receptor are widely distributed throughout the brain, including the amygdala, medulla, cerebral cortex, septal region, and hippocampus,1–4 suggesting that TRH may play an important role as a neurotransmitter as well as a neuromodulator regulating the functions of cholinergic5–7 and monoaminergic neurons.8,9 At present, several TRH analogues designed to reduce the hormonal actions and to increase the potency and duration of the central actions of TRH are under clinical investigation for the treatment of spinal cord injury, traumatic brain injury, and Alzheimer’s disease.10,11 A novel TRH analogue, Nα-[(1S, 2R)-2-methyl-4-oxocyclopentanecarbonyl]-L-histidyl-L-prolineamide (JTP-2942), with a cyclopentanone structure substituted for the pyroglutamyl moiety of TRH, has recently been demonstrated to have an increased therapeutic potency compared with the parent compound.12 JTP-2942 has been shown to cause reversal of experimental amnesia13 and to enhance both learning and memory in rodents in a Morris water maze study. The present study was performed to clarify the effects of JTP-2942 on cholinergic and monoaminergic neurons.