Shigehiko Narumi

Characteristics of memory impairment following lesioning of the basal forebrain and medial septal nucleus in rats

Memory impairment in rats with lesions of the basal forebrain (BF) and medial septal nucleus (MS) including cell bodies of the cortical and septohippocampal cholinergic systems, respectively, were compared in order to evaluate the functional contribution of the two cholinergic systems to memory. Biochemical assay revealed that lesioning of the BF and MS resulted in marked and selective decreases in both choline acetyltransferase and acetylcholinesterase activities in the cerebral cortex and hippocampus, respectively. Rats with BF lesions exhibited a severe deficit in a passive avoidance task; acquisition of passive avoidance by repeated training was sluggish, and the acquired response was rapidly eliminated in a subsequent extinction test. However, only slight impairment of passive avoidance was observed in rats with MS lesions. Memory impairment in rats with BF or MS lesions was also investigated using two spatial localization tasks, the Morris water task and the 8-arm radial maze task. Both BF and MS lesions elicited a significant impairment in the Morris water task that required reference memory, as demonstrated by an apparent increase in the latency to escape onto a hidden platform in a large water tank. The impairment was much more obvious in the BF-lesioned rats. In contrast, in the radial maze task primarily requiring working memory, rats with lesions of the MS showed severe disruption, exhibiting a marked increase in total errors, a decrease in the number of initial correct responses, and an apparent change in the strategy pattern. However, corresponding changes in the rats with BF lesions were slight. These results suggest that BF lesions may lead to substantial long-term memory impairment while MS lesions may primarily produce short-term or working memory impairment, indicating a qualitatively different contribution of the two cholinergic systems to memory. It is also suggested that these two experimental animal models may be useful for evaluation of therapeutic drugs for senile dementia of the Alzheimer type.

STIMULATORY EFFECTS OF SUBSTANCE P ON NEURITE EXTENSION AND CYCLIC AMP LEVELS IN CULTURED NEUROBLASTOMA CELLS

Abstract— Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS.

γ-butyrolactone-γ-carbonyl-histidyl-prolinamide citrate (DN-1417) : A novel TRH analog with potent effects on the central nervous system

Abstract A novel TRH analog, γ-butyrolactone-γ-carbonyl-histidyl-prolinamide citrate (DN-1417) with very low TSH-releasing activity markedly increased motor activity in the mice, with and without reserpine administration. DN-1417 also promoted arousal from pentobarbital or ethanol sleep and from loss of consciousness induced by concussive head injury, and reversed hypothermia induced by pentobarbital and reserpine. Circling behavior was provoked by comparatively low doses of DN-1417 in mice with unilateral striatal lesion, and the effect was prevented by pimozide and α-methyl-p-tyrosine. The results indicate that DN-1417 is more potent and longer-acting than TRH in the CNS behavioral paradigms, and that some of the effects may be relevant to central monoaminergic systems.

TRH and its novel analog (DN-1417): antipentobarbital action and involvement of cholinergic mechanisms.

Possible neuroanatomical loci and the mode of action of thyrotropin-releasing hormone (TRH) or its analog, gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate (DN-1417), in reducing the pentobarbital-induced sleeping time were investigated by using an intracerebral microinjection technique in rats. Intravenous, intraperitoneal or intracerebroventricular (ICV) injection of TRH or DN-1417 produced a dose-related reduction of the sleeping time induced by pentobarbital. TRH or DN-1417 given into the posterior hypothalamic regions including the dorsal premammillary nucleus, lateral hypothalamic area and posterior nucleus of hypothalamus had a significant pentobarbital sleep shortening action in low doses. Injection of these peptides into the dorsomedial nucleus of thalamus, mesencephalic reticular formation, medial septal nucleus or hippocampus was also effective, in comparatively low doses. However, higher doses were required to elicit the effect when the injections were made into the nucleus accumbens, lateral preoptic area or caudate nucleus. In this respect, the parietal cortex was insensitive to TRH or DN-1417. The pentobarbital sleep shortening action of TRH or DN-1417 injected peripherally or into the hypothalamic regions was markedly antagonized by ICV or intrahypothalamic pretreatment with atropine methyl bromide. On the contrary, ICV injection of atropine methyl bromide had a weak or no antagonizing action on the effect of TRH injected ICV or into the reticular formation, medial septal nucleus or hippocampus. These results suggest that possible neuroanatomical sites mediating the pentobarbital sleep shortening action of TRH or DN-1417 may be posterior hypothalamic regions, dorsomedial nucleus of thalamus, reticular formation, medial septal nucleus or hippocampus. A cholinergic mechanism may also be involved in the effect of TRH on the hypothalamus.

Thyrotropin-releasing hormone (TRH) and its analog (DN-1417): Interaction with pentobarbital in choline uptake and acetylcholine synthesis of rat brain slices

TRH or its analog DN-1417 (gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-proliamide) given 15 min after intravenous (i.v.) administration of pentobarbital (30 mg/kg) markedly shortened the pentobarbital-induced sleeping time in rats. This effect was almost completely abolished by intracerebroventricular pretreatment with atropine methylbromide (20 micrograms/rat), thereby suggesting the involvement of cholinergic mechanism. The action mechanism was investigated using rat brain slices. TRH (10(-6)-10(-4)M) or DN-1417 (10(-7)-10(-5)M) caused significant increases in the uptake of [3H]-choline into striatal slices. TRH(10(-4)M) or DN-1417(10(-5)M) also stimulated the conversion of [3H]-choline to [3H]-acetylcholine in striatal slices. A 30% reduction of acetylcholine synthesis from [3H]-choline in hippocampal slices and a 40% reduction of [3H]-choline uptake in slices of cerebral cortex, hippocampus and hypothalamus were observed in rats pretreated with pentobarbital (60 mg/kg, i.v.). TRH or DN-1417 (20 mg/kg, i.v.) given 15 min after the administration of pentobarbital markedly reversed both of the pentobarbital effects. Direct application of pentobarbital (5 X 10(-4)M) to slices in vitro also caused a 20-40% reduction of [3H]-choline uptake of cerebral cortex, hippocampus and diencephalon. A concomitant application of TRH(10(-4)M) or DN-1417(10(-5)M) and pentobarbital abolished the pentobarbital effect. These results provide neurochemical evidence that the antagonistic effects of TRH and DN-1417 on pentobarbital-induced narcosis are closely related to alterations in the rat brain choline uptake and acetylcholine synthesis, which are considered to be measures of the activity of cholinergic neurons.

Effects of gastric acid stimulants and inhibitors on the activities of HCO3−-stimulated, Mg2+-dependent ATPase and carbonic anhydrase in rat gastric mucosa

Abstract The effects of gastric acid stimulants or inhibitors, in vivo and in vitro , on the activities of HCO 3 − -stimulated , Mg 2+ -dependent ATPase ( (HCO 3 − −Mg 2+ )-ATPase , ATP phosphohydrolase, EC 3.6.1.3) and carbonic anhydrase in rat gastric mucosa were investigated in order to elucidate the significance of and the functional relationship between these enzymes. 1. 1. Subcutaneous treatment with carbachol (25–400 μg/kg) produced gastric juice secretion in 3-h pylorus-ligated rats. This drug also increased the Mg 2+ -dependent ATPase (Mg 2+ -ATPase) and carbonic anhydrase activities of the homogenate of rat gastric mucosa. 2. 2. Subcutaneous treatment with gastric acid stimulants, i.e. carbachol, tetragastrin and histamine, stimulated gastric juice secretion in 3-h pylorus-ligated rats. These reagents also increased the Mg 2+ -ATPase and carbonic anhydrase activities in the mitochondrial fraction of rat gastric mucosa. 3. 3. Pretreatment with atropine (5 mg/kg, subcutaneously) or acetazolamide (20 mg/kg, subcutaneously) prevented the carbachol-induced increase of Mg-ATPase and carbonic anhydrase activities in the mitochondrial fraction. 4. 4. In vitro effects of gastric acid stimulants and inhibitors: Incubation of Mg 2+ -ATPase or (HCO 3 − -Mg 2+ )-ATPase with histamine (10 −3 M), carbachol (10 −3 M) or tetragastrin (10 −5 M) had no effect on the activity of the enzyme. These reagents did not stimulate the enzyme activity, directly. Thiocyanate (10 −2 M) inhibited the activity of the enzyme by about 30–40%. Ouabain (10 −2 M) had no effect on the activity. From these results, it was obvious that Mg 2+ -ATPase and carbonic anhydrase activities in the mitochondrial fraction of the gastric mucosa correlated with gastric acid secretion. It is likely that carbonic anhydrase is functionally linked to Mg 2+ -ATPase in rat gastric mucosa.

Activation and phosphorylation of carbonic anhydrase by adenosine 3′,5′-monophosphate-dependent protein kinases

Abstract Two isozymes of carbonic anhydrase (carbonate dehydratase, EC 4.2.1.1) from bovine erythrocyte were obtained on DE 52 cellulose column chromatography, one of which was activated in the presence of ATP and Mg2+ by adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase from hog muscle, or bovine brain, and the other one was not. The activation of the enzyme by the protein kinase was dependent on preincubation time and concentration of protein kinase, cyclic AMP and ATP. The concentrations of cyclic AMP and ATP required to give half-maximal activation of carbonic anhydrase by protein kinase were about 1.6·10−6 and 1.5·10−5 M, respectively. The activation of carbonic anhydrase was also observed in the presence of the cyclic 3′,5′-monophosphate derivatives of inosine, guanosine, uridine and cytidine as well as with N 6 -2′-O- dibutyryladenosine 3′,5′-monophosphate by using a much higher concentration. Protein kinase modulator inhibited the activation of carbonic anhydrase by protein kinase. The protein kinase was no longer capable of activating carbonic anhydrase when it was boiled previously. Carbonic anhydrase was phosphorylated by the protein kinase, and its reaction was cyclic AMP dependent. Phosphorylation of carbonic anhydrase was dependent on incubation time and protein concentration. The results indicate that carbonic anhydrase is activated by cyclic AMP-dependent protein kinase, and that its activation is associated with phosphorylation of the enzyme protein.

Brain distribution of idebenone and its effect on local cerebral glucose utilization in rats

To investigate the possible action sites of a cerebral metabolism activator, idebenone, (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone), its distribution in the brain and effect on local cerebral glucose utilization (LCGU) were studied in normal (WKY) and stroke-prone spontaneously hypertensive rats (SHRSP) with cerebrovascular lesions. 14C-Idebenone distributed rapidly into the brain after intravenous administration (10 mg/kg), and the total 14C contents in the brain at peak time corresponded to 0.45-0.56% of the dosages. An autoradiographic study showed that the 14C levels were higher in the white than in the gray matter. When 14C-idebenone was administered orally (100 mg/kg) and intraperitoneally (30 mg/kg), the total 14C levels were not markedly different among the brain regions of the rats. The concentration of unchanged idebenone was higher in the cerebral cortex, thalamus, and cerebellum than that in the other brain regions. Studies on LCGU demonstrated that idebenone (30 mg/kg/day, i.p., for 3 days) improved the reduction of LCGU in SHRSP with stroke, especially in the temporal cortex, thalamus dorsomedial nucleus, subthalamic nucleus, mamillary body, hippocampus dentate gyrus, caudate-putamen, inferior colliculus, and cerebellar nucleus. Based on these results, possible action sites of idebenone for its main pharmacologic effects are discussed.

Possible role of cyclic AMP in gastric and secretion in rat. Activation of carbonic anhydrase

Abstract 1. 1. Administration of theophylline and gastric acid stimulants, such as histamine, tetragastrin and carbachol, increased the adenosine 3′:5′-cyclic monophosphate (cyclic AMP) level 2–3-fold as comparred with the control. 2. 2. N 6 -2′-O- Dibutyryl cyclic AMP (dibutyryl cyclic AMP) stimulated gastric acid secretion and activity of carbonic anhydrase in rat gastric mucosa. 3. 3. In vitro addition of cyclic AMP ( 10 −5 −10 −7 M ) to the supernatant fraction (cytosol) of rat gastric mucosa caused a concentration dependent potentiation of carbonic anhydrase activity. It is considered that this activation was mediated through protein kinase activation.

A TRH analog (DN-1417): Motor stimulation with rearing related to catecholaminergic mechanisms in rats

The effect of an analog of TRH, gamma-butyrolactone-gamma-carbonyl-histidyl-prolinamide citrate (DN-1417) on motor activity was studied in rats. Peripheral administration of DN-1417 (0.2-20 mg/kg, i.p.) caused a significant, dose-dependent increase in total spontaneous motor activity, with a definite increase in rearing behaviour. Both increases in spontaneous motor activity and rearing behaviour were markedly inhibited by pretreatment with chlorpromazine (1, 5 mg/kg, i.p.), haloperidol (0.1, 0.5 mg/kg, i.p.), pimozide (1 mg/kg, i.p.) or alpha-methyltyrosine (250 mg/kg, i.p.). Only stimulation of rearing behaviour was selectively attenuated by phenoxybenzamine (5 mg/kg, i.p.) or FLA-63 (25 mg/kg, i.p.) at doses producing no significant effect on spontaneous motor activity. Although propranolol (10 mg/kg, i.p.) and methysergide (10 mg/kg, i.p.) had no effect, atropine (10 mg/kg, i.p.) and mecamylamine (10 mg/kg, i.p.) respectively potentiated and counteracted the effects of DN-1417. Concerning the stimulation of spontaneous motor activity, the nucleus accumbens and lateral hypothalamic area were most sensitive to DN-1417, and the lateral hypothalamic area was the most sensitive site for the stimulation of rearing. Furthermore, DN-1417 (5 X 10(-5) M) significantly enhanced the spontaneous release of [3H]dopamine from the rat nucleus accumbens slices in vitro. These findings indicate that the motor stimulatory action of DN-1417 appears to be mediated primarily via a dopaminergic mechanism by enhancing the release of dopamine from nerve terminals, including the nucleus accumbens in the mesolimbic dopamine system, and, in turn, the rearing may be mediated via noradrenergic mechanism.