Kiyoshige Takayama

The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism.

Apelin is an endogenous ligand of the human orphan receptor APJ. We detected apelin-like immunoreactivity in the adipocytes, gastric mucosa, and Kupffer cells in the liver. We also detected apelin-like immunoreactivity localized within the endothelia of small arteries in various organs. Further, it was found that mean arterial pressure after the administration of apelin-12, apelin-13, and apelin-36 at a dose of 10 nmol/kg in anaesthetized rats was reduced by 26+/-5, 11+/-4, and 5+/-4 mm Hg, respectively. In the presence of a nitric oxide (NO) synthase inhibitor, the effect of apelin-12 on blood pressure was abolished. Furthermore, the administration of apelin-12 (10 nmol/kg) in rats produced a transitory elevation of the plasma nitrite/nitrate concentration from a basal level of 21.4+/-1.6 to 27.0+/-1.5 microM. Thus, apelin may lower blood pressure via a nitric oxide-dependent mechanism.

The comparison of effects of various anesthetics on expression of Fos protein in the rat brain

The purpose of this study was to compare effects of various anesthetic drugs on expression of Fos protein in the supraspinal neurons of rats. Optimum anesthesia was maintained under monitoring of EEG. Degree of Fos expression was evaluated by counts of Fos-immunoreactive (ir) neurons. Urethane induced the largest amount of Fos-ir neurons (n = 8439), while fentanyl/midazolam the smallest (n = 112). alpha-Chloralose, halothane, pentobarbital sodium and urethane/alpha-chloralose induced amounts in between. The results indicate that fentanyl/midazolam is the most recommendable anesthetic drug in the Fos expression study.

Neuronal expression of Fos protein in the rat brain after baroreceptor stimulation

The purpose of this study was to identify the CNS neurons that express Fos protein after repeated activation of the baroreceptor reflex. This was done in Wistar rats anesthetized with urethane and alpha-chloralose with careful physiological controls. The intact control rat showed few Fos-immunoreactive (ir) neurons, whereas the anesthetized control rat showed many Fos-ir neurons in the CNS from the medulla oblongata to the forebrain. After repeated stimulation of baroreceptors by pressor responses to phenylephrine (dose), we counted the amounts of Fos-ir neurons (response). The correlation coefficient of the dose-response relationship was high, and significant only in the medial part of the nucleus tractus solitarii (NTS) in the medulla and periaqueductal gray (PAG) in the midbrain, whereas it was comparatively high but insignificant in the commissure and lateral parts of the NTS, caudal and rostral ventrolateral medulla, periambiguus nucleus, dorsal and ventral medullary reticular nuclei, lateral parabrachial nucleus, paraventricular nucleus thalamus, and dorsomedial nucleus hypothalamus. No significant correlation was found in the humoral control nuclei in the preoptico-hypothalamic structure. Fos expression was never detected in the sensory neurons in the ganglia petrosum and nodosum, and in the sympathetic preganglionic neurons in the intermediolateral nucleus of the thoracic spinal cord. This study shows that Fos expression in the CNS neurons is induced not only by baroreceptor stimulation but also by anesthesia and/or sham-operation, and that Fos expression in the NTSm and PAG neurons faithfully responds to baroreceptor stimulation.

Circulatory and respiratory responses to glutamate stimulation of the lateral parabrachial nucleus of the cat

Electrical stimulation of the dorsal part of the lateral parabrachial nucleus (Pbl) induced a pressor response associated with a brief hyperpneic response, whereas glutamate stimulation induced no response, suggesting that the response to electrical stimulation may be due to excitation of passing fibers. Electrical stimulation of the lateral part of the Pbl induced a pressor and hyperpneic response, while glutamate stimulation induced either a pressor-depressor or single depressor response associated with a hyperpneic and polypneic response, suggesting that the response to electrical stimulation of the lateral part may be due to excitation of cell bodies. WGA-HRP study clarified that the cells in the region around the ventrolateral edge of the brachium conjunctivum project to the cells in the C1 area and subfacial cell group of the rostral ventrolateral medulla (RVLM), suggesting that the cells in this region may send a message to the sympathetic premotor and respiratory premotor cells in the RVLM. Inversely, the cells in the C1 area and the subfacial cell group project to the lateral part of the Pbl, suggesting mutual innervation between the Pbl and RVLM.

Projections of upper structure to the spinal cardioacceleratory center in cats: an HRP study using a new microinjection method

We studied, with the horseradish peroxidase (HRP) method, the supraspinal structure projections to the cardioacceleratory center in the intermediolateral nucleus (ILN) of T3-4 segments of the cat spinal cord. A fiber-filled double-barrel coaxial electrode was devised. The inner barrel electrode, filled with 3 M NaCl solution, was used for stimulating intraspinal structures to determine the cardioacceleratory center. The outer barrel electrode with an Agar stop, filled with a 20% HRP solution, was used for injecting HRP by pulses of positive electric current. In every experiment, HRP was injected into 3 points within the cardioacceleratory center at intervals of 1 mm. Out of 36 experiments, 10 showed that the HRP injection sites were centered in and almost confined to the ILN, and the population of the HRP-labeled cells was not less than 50. Some 1146 HRP-labeled cells were thus identified. They were distributed in the medulla oblongata (72.1%), pons (10.2%), midbrain (8.5%) and hypothalamus (9.2%). They were concentrated in the medullary reticular formation (37.8%), median raphe (26.9%) and pontine reticular formation (10.2%). Contrary to expectation, the HRP-labeled cells were few in the nucleus tractus solitarii (3.7%), paraventricular hypothalamic area (3.3%) and nucleus locus coeruleus (none).

Distribution of glutamate- and GABA-immunoreactive neurons projecting to the cardioacceleratory center of the intermediolateral nucleus of the thoracic cord of SHR and WKY rats : a double-labeling study

We aimed at (1) determining the distribution of glutamate (Glu)- and gamma-aminobutyric acid (GABA)-containing neurons in the brainstem with projections to the cardioacceleratory sympathetic preganglionic neurons in the intermediolateral nucleus (IML) of the upper thoracic cord and (2) determining whether such afferent projections in spontaneously hypertensive rats (SHR) differ from those of control Wistar-Kyoto (WKY) rats. We used a combination of electrophysiological methods to determine the site of HRP injection in the spinal cord and double-labeling methods for plotting the distribution of Glu- and GABA-immunoreactive neurons with projections to this site. HRP/Glu-labeled neurons (possibly glutamatergic) and HRP/GABA-labeled neurons (possibly GABAergic) were detected in 27% and 7% of the total HRP-labeled neurons of the central autonomic nuclei of 3 SHR rats and 3 WKY rats. HRP/Glu-labeled neurons were distributed predominantly ipsilaterally in 20 nuclei of the medulla oblongata, pons and hypothalamus, while HRP/GABA-labeled neurons were distributed in 7 nuclei of the medulla oblongata. No significant differences were found between the average percentages of HRP/Glu-labeled and HRP/GABA-labeled neurons in SHR and WKY rats. These findings indicate that: (1) the Glu-containing neurons represent a greater proportion than the GABA-containing neurons, (2) the proportions of these neurons appear to be similar in WKY and SHR rats and (3) generation of inbred tachycardia and hypertension in SHR rats can not be attributed to the topological and quantitative differences in the distribution of the glutamatergic and GABAergic neurons in the central autonomic nuclei.

Sites of origin and termination of gastric vagus preganglionic neurons: an HRP study in the rat

Using the horseradish peroxidase (HRP) method, we studied the relation between sites and forms of gastric vagus preganglionic neurons in the rat and gastric regions on which they project. Fifty microliters of a physiological saline solution containing 3 mg of HRP was injected into 6 different gastric regions: cardia, lesser curvature, pyloric antrum, greatger curvature, ventral corpus and dorsal corpus. After a survival period of 24h, the medulla oblongata was serially sectioned and processed for histochemical demonstration of HRP. In the medulla oblongata, HRP-labeled cells were distributed in 2 regions: (1) in the regions within the dorsal vagus nucleus (DVN); (2) in the regions within the ambiguous complex. The labeled cells in the DVN were observed in every experiment, while ones in the ambiguous complex were only seen in the cardia and lesser curvature experiments. In the ventral corpus experiment the labeled cells were observed dominantly on the left side and in the dorsal corpus experiment dominantly on the right side. The majority of the labeled cells in the ambiguous complex were medium-sized and multipolar in shape, while the majority of ones in the DVN were medium-sized and fusiform in shape.

Distribution and projection of the medullary cardiovascular control neurons containing glutamate, glutamic acid decarboxylase, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rats

This study was aimed at showing the distribution and projection of the medullary cardiovascular control neurons that contain a standard neurotransmitter or a related enzyme in the rat. A small amount of HRP was injected into either the depressor area of the caudal ventrolateral medulla (D-CVLM) or the pressor area of the rostral ventrolateral medulla (P-RVLM). Using an immunohistochemical method, we identified HRP-labelled neurons which were stained with antiserum to glutamate (Glu), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT). Our findings are summarized as follows. (1) The Glu-containing neurons in the nucleus tractus solitararii (NTS) project to the D-CVLM (n = 279, 100% assumed as a standard value) and P-RVLM (n = 225, 81% against the standard), indicating divergent excitatory projection. (2) The GAD-containing neurons in the NTS (n = 74, 27% against the standard) project to the P-RVLM, indicating the convergent inhibitory projection. (3) The projections of the TH-containing neurons from the NTS (n = 19, 7% against the standard) and CVLM (n = 4, 1% against the standard) to the P-RVLM are weaker than those of the GAD-containing neurons, suggesting that the catecholaminergic neurons play a minor role in inhibition of the sympathetic activity of the P-RVLM neurons. These results suggest that the glutamatergic NTS neurons excite both the P-RVLM and D-CVLM neurons, and the gamma-aminobutyric acid (GABA)ergic NTS and CVLM neurons inhibit the sympathetic activity of the P-RVLM neurons.

Increase in nitric oxide and cyclic GMP of rat cerebellum by radio frequency burst‐type electromagnetic field radiation.

1. Using rat cerebellum supernatant, the effects of radio frequency (RF) burst‐type electromagnetic (EM) field radiation on the production of cyclic GMP were examined under various conditions. The radiation was generated by a generator coil, and set at a 10 MHz radiation frequency, a 50% burst time, a 10 kHz burst rate and a 5 V peak‐to‐peak generator voltage. 2. When the cerebellum supernatant was incubated with both exogenous L‐arginine (nitric oxide (NO) donor) and NADPH, and irradiated by an RF burst‐type EM field, the production of cyclic GMP was increased significantly from a level of 21‐22 nmol min‐1 (g tissue)‐1 to 25‐26 nmol min‐1 (g tissue)‐1. By contrast, such an effect was not found when the cerebellum supernatant was irradiated by an RF volley‐type EM field. 3. When neither L‐arginine nor NADPH were added to the cerebellum supernatant, the production of cyclic GMP was lowered to a level of 6 nmol min‐1 (g tissue)‐1 and the radiation effect was not found. When the cerebellum supernatant was chelated with EDTA, the production of cyclic GMP was lowered to a level of 7 nmol min‐1 (g tissue)‐1 and the radiation effect was not found. 4. Incubation with Methylene Blue, a guanylate cyclase inhibitor, lowered the production of cyclic GMP to a level of 10‐12 nmol min‐1 (g tissue)‐1, and the radiation effect did not occur. On incubation with a NO synthase inhibitor, either NG‐methyl‐L‐arginine or N omega‐nitro‐L‐arginine methyl ester, the production of cyclic GMP was lowered to a level of 10‐12 nmol min‐1 (g tissue)‐1 or 5‐9 nmol min‐1 (g tissue)‐1 respectively, and the radiation effect was not observed. 5. Using electrochemical NO probes, the production of NO in the cerebellum supernatant was detected. The concentration of NO increased gradually after the onset of the EM field radiation. The radiation effect persisted, and reached a maximum after the cessation of the radiation. 6. In an in vivo study, the arterioles of the frog web were dilated by the radiation, and this radiation effect was almost completely abolished by the addition of a NO synthase inhibitor. This indicates that radiation activates NO synthase and ultimately induces vasodilatation.

Urocortin 1 reduces food intake and ghrelin secretion via CRF2 receptors

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.