Studies on the metabolism and disposition of the new retinoid 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl] benzoic acid. 2nd communication: absorption, distribution and excretion after single and consecutive subcutaneous administration in rats.

4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)carbamoyl]benzoic acid (CAS 94497-51-5, Am-80) is a new synthetic retinoid which has been shown to have a potent topical antisporiatic activity. The accumulation characteristics of Am-80 were examined in rats after a single and consecutive subcutaneous administration of 14C-labeled Am-80 once a day for 24 days, at a daily dose of 0.2 mg/kg. As compared with the single administration, Tmax (1-2 h) and Cmax (about 50 ng eq./ml) of the blood radioactivity were not altered markedly after the consecutive administration. During the daily subcutaneous dosing, the blood level of radioactivity at 24 h after each dosing was also very low. These findings suggested that accumulation in the blood was low after long term consecutive administration of Am-80. The plasma levels of total radioactivity and the proportion of unchanged Am-80 to the total plasma radioactivity, being about 80% at 2 h after administration, and plasma elimination half-life of Am-80, being approximately 3 h, appeared to be hardly affected by the consecutive administration. The cumulative excretion of radioactivity at 168 h after the final dosing was 6.7% and 89.1% in the urine and feces, respectively. The radioactivity remaining in the carcass at this time was about 3% of the total dose. The excretion profile was not altered by the consecutive administration. In most tissues, the concentration of radioactivity at 24 h after each dose reached a steady-state within 24 doses. At 2 h after the consecutive administration for 24 days, the highest concentration of radioactivity was found in the liver followed by the adrenal gland. Accumulation and delayed elimination of radioactivity in most tissues, especially in the adrenal gland, fat, skin and epididymis, were evidently observed as predicted from the previous study where 14C-Am-80 was administered at a dose of 1 mg/kg. The profile of accumulation and retention of radioactivity after the consecutive administration may be considered as a common characteristic of retinoids, such as etretinate.

Differential expression of c-fos mRNA in rat prefrontal cortex, striatum, N. accumbens and lateral septum after typical and atypical antipsychotics: an in situ hybridization study.

The regional difference in the expression of c-fos mRNA induced by typical and atypical antipsychotics was determined in prefrontal cortex, striatum, N. accumbens and lateral septum in rats by in situ hybridization. Two typical antipsychotics, haloperidol (2 mg/kg) and fluphenazine (2 mg/kg), and three atypical antipsychotics, (-)sulpiride (100 mg/kg), clozapine (20 mg/kg) and OPC-14597 (40 mg/kg), were used. Brains were fixed with 4% paraformaldehyde 45 min after drug administration (i.p.). Brain sections of 30 microns-thickness were made in a cryostat and hybridized with 35S-labelled for c-fos oligonucleotide probe. These sections were apposed to X-ray films and the autoradiograms were semi-quantitatively analysed by computer-assisted densitometry. All antipsychotics used increased c-fos mRNA expression in N. accumbens shell, a region of the forebrain associated with limbic systems. On the other hand, two typical antipsychotics (haloperidol and fluphenazine) that cause a high incidence of acute motor side effects increased the expression of c-fos mRNA in the dorsolateral striatum, an extrapyramidal region primarily involved in motor control. Only clozapine induced c-fos mRNA in the medial prefrontal cortex and lateral septum. These results strongly suggest that the shell region of N. accumbens may be a common site of therapeutic action of antipsychotics.

Nicotine protects against the dexamethasone potentiation of kainic acid-induced neurotoxicity in cultured hippocampal neurons.

To elucidate the neuroprotective effect of nicotine, we investigated whether nicotine may attenuate dexamethasone potentiation of kainic acid-induced neurotoxicity. Primary hippocampal culture was pre-treated with nicotine for 24 h followed by dexamethasone (10(-4) M) for 24 h. Then, cultures were exposed with kainic acid (10(-4) M) and cellular viability was determined by LDH effluxmetry. Nicotine pre-treatment (10(-9)-10(-7) M) dose-dependently attenuated dexamethasone potentiation of kainic acid-induced neurotoxicity. These results may support the epidemiological data suggesting a neuroprotective effect of cigarette smoking on Alzheimer's disease or Parkinson's disease.

NG-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, increases extracellular GABA in the striatum of the freely moving rat.

We investigated the effect of an NO synthase inhibitor, NG-monomethyl-L-arginine (L-NMMA), on the levels of endogenous GABA in the rat striatum using in vivo microdialysis. Rats were perfused with the artificial CSF containing L-NMMA (0.1, 0.3 and 1.0 mM) or its inactive isomer D-NMMA (1.0 mM) for 1 h. Infusion of L-NMMA, but not its D-isomer, dose-dependently increased GABA concentration. Co-infusion with tetrodotoxin (1 microM) did not antagonize the increase of GABA induced by L-NMMA. These results show that decreased NO activity enhances GABA release even in the absence of depolarization of GABA neurones. We conclude that NO may be directly acting on GABA nerve terminals and tonically inhibiting GABA release or synthesis under basal conditions.

[A case of small cell lung cancer that developed during therapy for myasthenia gravis].

A 56-year-old man had been complaining of progressive proximal muscle weakness and bilateral ptosis before his first admission to our hospital. He received an injection of edrophonium chloride, which resulted in remarkable improvement of muscle strength. Electromyographic studies revealed a compound muscle action potential that decreased after repetitive stimulation at a high rate (15 Hz). He was regarded as having myasthenia gravis (MG) rather than Eaton-Lambert syndrome because of these findings. Eighteen months after successful treatment of MG with oral anticholinesterase medication, he complained of an abdominal mass. The mass was found to be a tumor that had metastasized from a primary small cell carcinoma of the lung. Cases of MG with small cell carcinoma of the lung seem to be very rare, and the details of the relationship between them remain unknown. In this patient, MG may have developed by paraneoplastic mechanisms. This hypothesis is interesting, since it has been demonstrated recently by molecular biological techniques that small cell carcinomas of the lung express nicotinic acetylcholine receptors.

Effect of systemic dexamethasone on extracellular amino acids, GABA and acetylcholine release in rat hippocampus using in vivo microdialysis.

In order to elucidate the mechanism of the cytotoxic effect of glucocorticoid on the rat hippocampus, we studied the effects of dexamethasone, a synthetic glucocorticoid, on neurotransmitter release from the hippocampus by in vivo microdialysis. Dexamethasone (10 mg/kg) was administered i.p. and the extracellular concentrations of the amino acids (glutamate, aspartate, glycine), GABA and acetylcholine were determined in the hippocampus of freely moving rats. Glutamate concentration increased by 20-25% after dexamethasone administration and this effect continued for 1 hour. The extracellular concentration of the other neurotransmitters, however, did not show any significant changes. From these results, we concluded that increased extracellular glutamate may be involved, at least in part, in the cytotoxic effect of glucocorticoid on hippocampal neurons.

Effect of caerulein on expression of the immediate-early genes c-fos and zif/268 in the rat brain.

The effect of caerulein, an analog of cholecystokinin-8, on expression of the immediate-early genes c-fos and zif/268 was studied in the rat brain using Northern blot analysis and an in situ hybridization technique. Intraperitoneal injection of caerulein did not change the basal c-fos and zif/268 expression. Administration of the convulsant, pentylenetetrazole (PTZ), caused a dramatic increase of c-fos and zif/268 mRNAs in the hippocampus and dentate gyrus. Pretreatment with caerulein suppressed the PTZ-induced c-fos and zif/268 expression. It is considered that systemically administrated caerulein modifies neuronal activities by exerting a suppressed effect on induction of the immediate-early genes.

[Secondary autonomic diseases--diabetes mellitus].

Diabetic autonomic neuropathy is an established disease entity. Nevertheless, it was not until 1945 that diabetic autonomic neuropathy became a target for extensive systemic research works. This is partly because of insidious progress of this disorder and lack of precise techniques of examinations. In this review article, signs and symptoms of diabetic autonomic disorders characteristic of each visceral organ are described. Among them, such symptoms as painless myocardial infarction and hypoglycemia without warning symptoms deserve special attention. It is essential for clinical doctors to realize that autonomic neuropathy is the most important part for understanding diabetic pathology.

Activin receptor mRNA is expressed early in Xenopus embryogenesis and the level of the expression affects the body axis formation.

Activin is a member of the transforming growth factor beta (TGF-beta) and possesses various activities in cellular control phenomena. During Xenopus embryonic development, activin is thought to act as a natural mesoderm-inducing factor. We isolated here the Xenopus activin receptor cDNA from Xenopus tadpole cDNA library and examined the expression of the Xenopus activin receptor gene during the course of early embryonic development. The Xenopus activin receptor has an 87% homology at the level of deduced amino acid sequence with the mouse activin receptor, and using the cDNA obtained, three bands of mRNA with different lengths were detected in Xenopus embryos throughout early embryogenesis. We synthesized activin receptor mRNA in vitro and tested the effect of the injection of the mRNA into Xenopus fertilized eggs on subsequent development. When the synthetic mRNA was injected into uncleaved fertilized eggs, embryos with reduced trunk structure were formed. However, when the mRNA was injected into the ventral blastomeres at the 16-cell stage, embryos with a secondary body axis were formed. These results indicate the importance of the function of activin receptor in the regulatory mechanism for body axis formation.

Effect of age on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites in the rat brain studied by in vitro autoradiography.

Receptors for excitatory amino acid, L-glutamate, have been classified into three subtypes named as N-methyl-D-aspartate (NMDA), quisqualate (QA) and kainate receptors. In the present study, an effect of age on binding sites of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA), a QA agonist, was studied in the rat brain through quantitative in vitro autoradiography. 3H-AMPA binding sites were most concentrated in the hippocampus and cerebral cortex where glutamate receptors have been demonstrated to play a role in synaptic transmission. In aged rats, 3H-AMPA binding sites in the hippocampus and cerebral cortex were not significantly changed. In our previous studies, it was noticed that strychnine-insensitive glycine receptors, which functionally coupled with NMDA receptors, showed marked age-dependent decreases in telencephalic regions. It has been shown that the glutamatergic neuronal system is involved in learning and memory. Nevertheless, it is considered that AMPA binding sites are not involved in the decline of neuronal functions, especially impairment of learning and memory, accompanying with aging process.

Influence of age on N-methyl-D-aspartate antagonist binding sites in the rat brain studied by in vitro autoradiography.

The N-methyl-D-aspartate (NMDA) receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In this study, an effect of age on NMDA antagonist binding sites was investigated through quantitative in vitro autoradiography with 3H-3-((+)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). 3H-CPP binding sites were most concentrated in the hippocampus and cerebral cortex where NMDA receptors have been demonstrated to be involved in synaptic transmission. In aged rats, 3H-CPP binding sites in the hippocampus and cerebral cortex were not significantly changed. As for other brain regions, there was an age-dependent decline of binding sites only in the caudate-putamen and nucleus accumbens. Our previous study revealed that strychnine-insensitive glycine receptors were markedly reduced in telencephalic regions in the aged rat brain. Taking these findings into consideration, it is concluded that glycine receptors but not NMDA antagonist binding sites are severely altered in telencephalic regions of aged animals. It is considered that within the NMDA receptor complex, glycine receptors may be selectively affected in the aging process.

Cholecystokinin increases intracellular Ca2+ concentration in cultured striatal neurons.

Although it has been established that pancreatic cholecystokinin (CCK) receptors are coupled to phosphatidylinositol turnover, the events which follow activation of CCK receptors in the central nervous system have not received much attention. In this paper, changes in intracellular Ca2+ concentration ([Ca2+]1) in response to CCK peptides were measured in single cultured rat striatal neuron by fura-2 fluorometry. CCK peptides dose-dependently increased [Ca2+]i in a monophasic manner. The order of the potencies of CCK peptides to increase [Ca2+]i was as follows: caerulein greater than CCK-8 greater than desulfated CCK-8 greater than CCK-4. The effect of caerulein was completely blocked in a Ca2(+)-depleted medium. In addition, omega-conotoxin GVIA completely inhibited the effect of caerulein, while neither nifedipine nor verapamil affected it. Our results indicate that CCK receptors couple to N-type voltage-sensitive Ca2+ channels in cultured rats striatal neurons.

Influence of age on NMDA receptor complex in rat brain studied by in vitro autoradiography.

N-methyl-D-aspartate (NMDA) receptors are known to play an important role in learning and memory and to be involved in neuron cell death accompanying cerebral ischemia, seizures, and Alzheimer's disease. The NMDA receptor complex has been considered to consist of an L-glutamate recognition site, a strychnine-insensitive glycine modulatory site, and a voltage-dependent cation channel. In the present study, effects of age on an L-glutamate recognition site and a glycine site were examined in rat brain by quantitative in vitro autoradiography with [3H]-CPP and [3H]-glycine. Both [3H]-glycine and [3H]-CPP binding sites were most abundant in the hippocampus and cerebral cortex, and they showed a similar distribution pattern throughout the brain. [3H]-glycine binding sites were severely decreased in the telencephalic regions, including the hippocampus and cerebral cortex, in aged brain. Conversely, [3H]-CPP binding sites were well preserved in these brain areas. In the mid-brain regions and cerebellum, neither [3H]-glycine nor [3H]-CPP binding sites changed in the aged brain. Our results indicate that within the NMDA receptor complex, glycine receptors are primarily affected in the aging process.

Mapping of somatostatin receptor localization in rat brain: forebrain and diencephalon.

Using quantitative in vitro receptor autoradiography, minute distributions of 125I-Tyr11-somatostatin (SS)-14 binding sites were investigated in the rat forebrain and diencephalon. In the cerebral cortex, there was a high density of receptors observed in layers V-VI and a low density in layers I-IV. The entorhinal cortex displayed the highest receptor density of the cerebral cortices. The olfactory system had a high SS receptor density. The anterior olfactory nucleus, nucleus of the lateral olfactory tract, medial habenular nucleus and the basolateral amygdaloid nucleus showed moderate densities. In the limbic system, the CA1 and subiculum regions had high receptor densities. More detailed observations revealed high receptor densities in the oriens, radiatum and lacunosum layers and a much lower density in the pyramidal cell layer. The caudate putamen and substantia nigra showed low receptor densities, while the claustrum displayed the highest density of receptors in the rat brain. These data were not consistent with those of previous studies using 125I-SS-28 and 125I-201-995, which had shown that the high receptor density area in the basolateral amygdaloid group was identified as the lateral amygdaloid nucleus, and that the pyramidal cell layer in the hippocampus showed high receptor densities.

Ontogeny of phorbol ester receptors in rat brain studied by in vitro autoradiography.

The ontogeny of phorbol ester receptors, which have been considered to correspond to protein kinase C, in the rat brain was studied through in vitro autoradiography with 3H-phorbol 12,13-dibutyrate (3H-PDBu). The distribution of 3H-PDBu binding sites in the adult rat brain was similar to the previous reports by other researchers. The developmental pattern of 3H-PDBu binding sites varied with brain region. 3H-PDBu binding sites in the amygdala, thalamus, stratum pyramidale of CA 1 of the hippocampus, dentate gyrus, superior colliculus, substantia nigra, interpeduncular nucleus and cerebellar molecular layer were postnatally increased to adult levels and after that they remained constant. On the other hand, in the stratum oriens and stratum radiatum of CA 1 of the hippocampus, and in the lateral and medial geniculate bodies, 3H-PDBu binding sites reached peaks at 21 or 28 days of postnatal age and after that they declined to adult levels. The cerebellar granular layer showed a low level of 3H-PDBu binding sites throughout all the ontogenetic stages. A distinct ontogenetic pattern of phorbol ester receptors in various regions of the brain may reflect a role of protein kinase C in the neural development of each discrete area.

Age-related changes of strychnine-insensitive glycine receptors in rat brain as studied by in vitro autoradiography.

Age-related changes of strychnine-insensitive glycine receptors in the rat brain were studied through quantitative in vitro autoradiography with 3H-glycine. 3H-glycine binding sites were most concentrated in the hippocampus, cerebral cortex, and olfactory tubercle, and moderate densities of binding sites were located in the striatum, nucleus accumbens, amygdala, and certain thalamic nuclei. Low densities of 3H-glycine binding sites were observed in the lateral septal nucleus, midbrain nuclei such as the superior colliculus and central gray matter, and granule cell layer of the cerebellum. In aged animals, severe decline of 3H-glycine binding sites was observed in the telencephalic regions including the hippocampus and cerebral cortex. On the other hand, decrease of binding sites in the midbrain nuclei was of lesser degree, and there were no changes in the cerebellum. These results suggest that the decrease of glycine receptors in particular brain regions has some relation with changes of neuronal functions associated with aging process in these areas. The glutamatergic neuronal system, particularly the N-methyl-D-aspartate (NMDA) subtype, has been considered to play an important role in learning and memory. Taking into consideration that strychnine-insensitive glycine receptors are contained in the NMDA receptor complex, the present study implies that the decrease of glycine receptors may be involved in impairments of learning and memory occurring in aged brains.

Quantitative autoradiographic localization of the M1 and M2 subtypes of muscarinic acetylcholine receptors in the monkey brain.

The distribution of muscarinic acetylcholine receptors (mAChR) was investigated in the monkey brain by means of quantitative in vitro autoradiography. 3H-QNB, 3H-pirenzepine (PZ) and 3H-AF-DX 116 were used for labelling total mAChR, M1 and M2 receptors, respectively. 3H-PZ and 3H-AF-DX 116 showed specificity to each receptor subtype in the monkey brain. On sections containing the putamen and globus pallidus, the sum of Bmax values of 3H-PZ and 3H-AF-DX 116 binding sites was almost close to that of 3H-QNB binding sites. Autoradiographic distributions of muscarinic subtype receptors in the monkey brain were similar to those reported in the rat brain; that is, M1 receptors were dominant in most areas of the telencephalon, while M2 receptors were richly distributed in the brainstem and cerebellum. However, some nuclei of the brainstem such as the central gray matter, superior colliculus, substantia nigra, nucleus of the oculomotor nerve, pontine nucleus and inferior olivary nucleus, had relatively high ratios of M1 receptors in the monkey brain. In addition, the cortical lamminar distribution of M2 receptors noticed in the rat was not observed in the monkey brain. Knowledge about the localizations of M1 and M2 receptors in various brain regions in the monkey brain will increase our understanding of the functions of the brain cholinergic system in the primate.

Somatostatin increases intracellular Ca2+ concentration in cultured rat hippocampal neurons.

Changes of intracellular Ca2+ concentration [( Ca2+]i) in response to somatostatin were measured in cultured rat hippocampal neurons on a single cell basis by fura-2 fluorometry. Somatostatin increased [Ca2+]i dose-dependently and this effect was completely blocked in either Ca2+-depleted medium or LaCl3-containing medium. In addition, omega-conotoxin GVIA completely inhibited the effect of somatostatin. Our results indicate that somatostatin receptors couple with N-type voltage-sensitive Ca2+ channels in cultured rat hippocampal neurons.