Motoi Sohmiya

Effects of erythropoietin on neuronal activity

Abstract: Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca2+ uptake, intracellular Ca2+ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10‐12‐10‐10M) increased 45Ca2+ uptake and intracellular Ca2+ concentration in PC12 cells in a dose‐related manner; these increases were inhibited by nicardipine (1 μM) or anti‐EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5‐day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10‐13‐10‐10M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti‐EPO antibody. Incubation with EPO (10‐13‐10‐10M) stimulated mitogen‐activated protein kinase activity in PC12 cells. EPO (10‐13‐10‐10M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti‐EPO antibody. Following a 4‐h incubation with EPO (10‐14‐10‐10M), NO production was increased, which was blunted by nicardipine and anti‐EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.

Neuronal protection from apoptosis by pituitary adenylate cyclase-activating polypeptide

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known to have trophic effects on neurons. Apoptosis of PC12 cells was induced by depletion of serum and nerve growth factor (NGF) from culture medium. Not only high potassium-induced Ca2+ channel activation but PACAP-38 at physiological concentrations (10[-10] to 10[-8] M) protected PC12 cells from apoptosis. PACAP-38 increased Ca2+ uptake and intracellular Ca2+ concentrations in PC12 cells. The effects of PACAP-38 on cell survival and Ca2+ channels were eliminated by inhibitors for Ca2+ channels and protein kinase A, and mimicked by 8-bromo-cAMP. Mitogen-activated protein (MAP) kinase activity was stimulated by PACAP-38. These findings implicate that PACAP protects PC12 cells from apoptosis by activating Ca2+ channels via the cAMP-protein kinase A pathway to stimulate MAP kinase cascade.

Stimulating effect of erythropoietin on the release of dopamine and acetylcholine from the rat brain slice

We investigated the effects of erythropoietin (EPO) in rat hippocampal and striatal slices, where EPO receptors have been known to exist. EPO stimulated dopamine release from rat striatal slices. Acetylcholine (ACh) release from rat hippocampal slices was not affected by EPO, but high K(+)-induced ACh release was considerably enhanced by EPO. Nitric oxide (NO) production from the hippocampus and the striatum was not affected by EPO. NO-synthase activity was not changed by EPO in the hippocampus or the striatum. These results suggest that EPO stimulates dopamine- and acetylcholine-release without affecting NO production.

Effect of long-term administration of recombinant human growth hormone (rhGH) on plasma erythropoietin (EPO) and haemoglobin levels in anaemic patients with adult GH deficiency

OBJECTIVE We investigated the effect of recombinant human GH (rhGH) on erythropoietin (EPO) and haemoglobin (Hb) concentrations in anaemic patients with adult GH deficiency.

Effect of erythropoietin on nitric oxide production in the rat hippocampus using in vivo brain microdialysis

In order to investigate the role of erythropoietin (EPO) in the hippocampus, we studied the effect of EPO on nitric oxide (NO) production in the rat hippocampus using brain microdialysis. The dialysis probe was stereotaxically inserted into the rat hippocampus 24 h before the dialysis experiment. The perfusion fluid (Krebs-HEPES buffer, pH 7.4) was collected at 15-min intervals under freely moving conditions and NO metabolites (NOx) in the perfusate were immediately measured using a NOx-analyzing high performance liquid chromatography (HPLC)system. Following the collection of four fractions, 1 microl of EPO (10(-10) M, 10(-8) M and 10(-6) M) or vehicle (saline) was gently injected into the hippocampal tissue. The perfusion fluid was collected for 3 h after the injection. The NOx levels were unchanged by the injection of vehicle alone. After the injection of EPO, NOx levels gradually increased. The EPO-induced increase in NOx levels was significant at 10(-6) M EPO. The EPO-induced increases in NOx levels were eliminated in the presence of anti-EPO antibody. The increase in NOx levels induced by EPO was blunted by nicardipine, a Ca2+ channel blocker, but not by MK-801, an antagonist of N-methyl-D-aspartate (NMDA) receptors. These findings, taken together, suggest that EPO increased NO production in the rat hippocampus by activating voltage-gated Ca2+ channels, but not through NMDA receptors.

Unique case of growth hormone (GH) deficiency accompanied by clinical anophthalmia, hypoplastic orbits, digital dysplasia, short stature, obesity, and diabetes mellitus.

A 43-year-old female was admitted to our hospital for polydipsia and hyperglycemia. She had total blindness and globes were not recognized by inspection, indicating clinical anophthalmia. Physical examination revealed short stature, obesity, prematurely gray hair, shortness of fingers and toes, syndactyly, and multiple dental caries. Laboratory examination showed hyperglycemia, increased glycosilated hemoglobin (HbA1c) and insulin resistance on euglycemic glucose clamp. Blunted growth hormone (GH) secretion was shown in response to insulin-induced hypoglycemia, arginine infusion, and GH-releasing hormone (GHRH) loading test, and in 24 h spontaneous GH profile. Magnetic resonance imaging (MRI) and computed tomography (CT) showed dysostosis of orbit, defect of optic nerve, enlarged suprasellar cistern, and prolonged pituitary stalk. This may be the first report of a unique case with GH deficiency accompanied by clinical anophthalmia, hypoplastic orbits, digital dysplasia, short stature, obesity, and diabetes mellitus.

A New Device to Introduce Self-Injection of Insulin by His Non-Dominant Hand in a Patient with Hemiplegia

Dominant hand dysfunction due to cerebrovascular accident or fracture makes it more difficult to self-inject insulin. This would likely lead to diminishing a patients quality of life. We made a new device to introduce self-injection of insulin by a patients non-dominant hand and tested it. This device was built into a 600-g block of wood 11.5 cm x 8 cm x 8 cm, to be used with the InnoLet insulin kit system (Novo Nordisk Pharmaceuticals Inc., Bagsvaerd, Denmark). It had an insulin injector clamp on the front and a needle holder on the top. The bottom and the back were covered with silicon rubber, which allows the devices own weight to affix it on a table. The insulin injector is placed upright in a holder and fastened with a bar. A needle is installed on the insulin injector with a needle cap. After this cap was removed, the patient could remove any air bubbles by pushing 2 units of insulin through the needle. After the insulin injector was unfastened from the device, the patient injected the insulin subcutaneously into his abdomen or thigh. Then, the insulin injector was removed from the device. We introduced this device in a 59-year-old man with type 2 diabetes mellitus who had suffered from ischemic cerebral infarction in the left middle cerebral artery distribution, resulting in complete right hemiparesis. Our patient mastered this procedure within a few days. At the time of discharge, he could self-inject regular human insulin in a dose of 16 units in the morning, 6 units at noon, and 8 units in the evening. Two weeks after he was admitted to our hospital, he continued independent insulin self-injection three times per day without any help. His hemoglobin A(1c) level gradually decreased until it reached 5.7%. The self-injection of insulin may be introduced with a new device by the non-dominant hand in a patient with diabetes having a disabled dominant hand.

Effects of rat galanin and galanin message associated peptide (GMAP) on rat growth hormone secretion and stimulating effect of γ-aminobutyric acid on galanin release from rat hypothalamus

Immunoreactive galanin and galanin message associated peptide (GMAP) were detectable in rat hypothalamus in the concentration of 563 +/- 23 and 14.3 +/- 3.1 fmol/hypothalamus, respectively. gamma-Aminobutyric acid (GABA) elicited a dose-related increase in galanin release from rat hypothalamic fragments, which was inhibited by picrotoxin, a GABA antagonist. Growth hormone (GH) secretion from rat anterior pituitary cells were stimulated by rat galanin, but not by GMAP. These findings suggest that hypothalamic galanin, but not GMAP, may play roles in GH secretion induced by GABAergic mechanisms in the rat.