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.

Involvement of tetrahydrobiopterin in trophic effect of erythropoietin on PC12 cells.

Tetrahydrobiopterin (BH(4)) synthesis is reported to be stimulated by nerve growth factor (NGF) in PC12 cells, suggesting involvement of BH(4) in the trophic effect of NGF. We have recently reported that erythropoietin (EPO) and BH(4) enhance survival of PC12 cells. In the present study, we investigated involvement of BH(4) in the trophic effect of EPO on PC12 cells. Cellular BH(4) content was increased by EPO (10(-10) to 10(-8) M) in a dose- and time-related manner. EPO (10(-10) to 10(-8) M) increased the viable cell number of PC12 cells. In addition to EPO, BH(4) (1, 3, and 10 microM) increased the viable cell number of PC12 cells. Administration of 0.3 mM 2,4-diamino-6-hydroxypyrimidine, an inhibitor of BH(4) synthesis, blunted EPO-induced increases in BH(4) content and the viable cell number of PC12 cells. These results taken together suggest that BH(4) is involved in the trophic effects of EPO on PC12 cells.

Effect of insulin on nitric oxide synthase-like immunostaining of arteries in various organs in Zucker diabetic fatty rats.

OBJECTIVE: Recently we reported that insulin treatment improved hypertension by inducing nitric oxide synthase (NOS) in Zucker diabetic fatty (ZDF) rats. In the present study, we investigated subtypes of NOS induced by insulin in arteries in various organs of ZDF rats using immunohistochemistry. DESIGN: Following treatment with insulin, localization of two subtypes of NOS in the arterial tissues of various organs was identified. METHODS: Following 4 weeks of s.c. injection of insulin, the aorta, cerebral cortex, pancreas and kidney were stained with polyclonal anti-endothelial NOS (eNOS) or anti-inducible NOS (iNOS) antibodies. RESULTS: In the aortic tissue, eNOS-like immunostaining was observed equally in the insulin-treated group and the control group, whereas iNOS-like immunostaining was present more densely in the insulin-treated group. In the cerebral artery, eNOS-like immunostaining was observed in the endothelium and was enhanced in the insulin-treated group. In the control group, iNOS-like immunostaining was absent in the cerebral artery, whereas immunostaining was densely observed in the insulin-treated group. In the interlobular artery of the pancreas, both eNOS-like and iNOS-like immunostaining was present in the control group and was enhanced in the insulin-treated group. In kidney, both eNOS-like and iNOS-like immunostaining was more densely present in the arterial tissue of the insulin-treated group. CONCLUSIONS: These results taken together suggest that insulin treatment induced NOS in arteries in various organs and that iNOS was more strongly induced than eNOS by insulin treatment in the ZDF rat.

Age-related structural changes in the human midbrain: an MR image study.

We measured midbrain structures of 194 subjects without neurological disorders, using T2-weighted MR imaging. Age was negatively correlated with the maximum anteroposterior distance of the midbrain through the substantia nigra (MD), and the average distance from the substantia nigra to the red nucleus (SNRND), while a positive correlation was found between aging and the maximum distance of the substantia nigra (SND). Significant left-right differences were revealed in MD, SND, SNRND and the area of the red nucleus (RNA), which was possibly responsible for cerebral hemispheric dominance or handedness. There were gender differences in MD and the maximum interpeduncular distance (IPD) in age-matched groups. Age-related structural changes of the midbrain may have a close relation to a decline in motor performance with aging. These findings provide essential information to evaluate the MR images of neurodegenerative disorders.

[Three elderly cases of hyperostosis cranii with various clinical symptoms].

We report three elderly patients with hyperostosis cranii (HC). Patient 1 had two episodes of unconsciousness; patient 2, headache; and patient 3, dementia. On the basis of the classification of Moore using skull films, patients 1 and 2 showed hyperostosis frontoparietalis and patient 3 had hyperostosis frontalis interna. Electroencephalography showed transient generalized spike and slow wave complexes over the frontal lobes in patient 1. Magnetic resonance (MR) images showed frontal lobes compressed by the thickness of the frontal bones in all patients and thickened parietal bones in patients 1 and 2. Because findings in our series and in the literature suggest that HC may show unexpected neuropsychiatric symptoms, HC should be checked in elderly patients whose presenting symptoms include epilepsy, dementia, psychiatric disease, headache and so on. MR images should reveal the relationship between clinical symptoms and the deformation of brains by the skull.

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. PATIENTS AND DESIGN: rhGH was administrated in 8 patients with adult GH deficiency, three males and five females, aged from 24 to 69 years, mean (+/- SD) of 48.8 +/- 16.4 years, for 1 year by means of continuous subcutaneous infusion (CSI) at a flow rate of 0.036 U/kg/day using a portable syringe pump. Blood samples were obtained in the morning after an overnight fast every week for 1 month, followed by each month before and after the start of rhGH administration. RESULTS: Mean (+/- SE) plasma GH levels increased from 0.24 +/- 0.09 microg/l to 2.32 +/- 0.23 microg/l 1 week after the start of rhGH administration to maintain a steady state. Plasma IGF-I levels increased from 70.1 +/- 13.8 microg/l to 282.8 +/- 70.6 microg/l 1 week after the start of rhGH administration to maintain the steady state. Plasma EPO levels increased from 25.9 +/- 2.6 IU/l to 37.6 +/- 4.2 IU/l and 34.3 +/- 3.6 IU/l at 1 week and 2 weeks after the start of rhGH administration, respectively, and then decreased gradually to 14-9 +/- 2.1 IU/l at 10 months after the start of rhGH administration. Reticulocyte counts increased from 0.88 +/- 0.06% to 1.49 +/- 0.21% at 1 week. Hb concentrations increased from 103 +/- 5 g/l to 106 +/- 5 g/l at 2 weeks after the start of rhGH administration, and then increased gradually to reach the normal range. CONCLUSIONS: We conclude that EPO secretion was stimulated in the initial 2 weeks after the start of CSI of rhGH in anaemic patients with adult GH deficiency. Increased Hb concentrations after long-term administration of rhGH might be explained by direct stimulatory effects of rhGH and IGF-I on erythroid cells, which was accompanied by suppressed EPO secretion, in combination with a more generalized indirect impact of rhGH on physical activety. These findings suggest a beneficial effect of rhGH replacement in anaemic patients with adult GH deficiency.

A case of myotonic dystrophy (MD) associated with glucose-induced hyperinsulinemia followed by reactive hypoglycemia and increased number of cytosine-thymine-guanine (CTG) trinucleotide repeats in MD gene.

A 39-year-old man with myotonic dystrophy consulted our hospital for nausea, vomiting and dizziness that occurred after 75 g oral glucose tolerance test (OGTT). Reexamination of OGTT revealed remarkable hyperinsulinemia (622 microU/ml) followed by reactive hypoglycemia (50 mg/dl) and such hypoglycemic symptoms as nausea, vomiting, dizziness and palpitation. DNA analysis of the circulating lymphocytes revealed increased (1,500 times) number of cytosine-thymine-guanine (CTG) trinucleotide repeats in myotonic dystrophy protein kinase (DM kinase) gene. Gel chromatographic analysis of the plasma in combination with sensitive enzyme immunoassay of insulin revealed that the ratio of proinsulin to total immunoreactive insulin was elevated at fasting (12.9%), and was decreased to 8.9% at 60 min after glucose administration. These findings may indicate that biologically active authentic insulin was predominantly secreted after glucose administration in the present case. This is the first case report of myotonic dystrophy with hyperinsulinemia associated with reactive hypoglycemia induced by oral glucose administration.

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.

Molecular and electrical heterogeneity of circulating human erythropoietin measured by sensitive enzyme immunoassay.

BACKGROUND: We set up a sensitive sandwich enzyme immunoassay (EIA) of human erythropoietin (EPO) and further investigated molecular weight and structural heterogeneity, and electrical heterogeneity of the circulating EPO in normal human plasma without any concentrating procedure. MATERIALS AND METHODS: We set up a sensitive sandwich EIA of EPO using anti-EPO rabbit Fab'-peroxidase conjugate and anti-EPO IgG coated polystyrene balls. Standard EPO and plasma samples obtained from three normal subjects were applied to gel chromatographic analysis. Plasma samples obtained from three normal subjects were subjected to preparative isoelectric focusing with a column (pH 3.5-10). RESULTS: The minimal detectable quantity was 0. 15 mIU mL-1 using 100 microL sample. There was a good parallelism between diluted human plasma samples and the standard EPO in the assay. Rat plasma EPO was cross-reacted in the assay. There was a good correlation between EPO levels determined by the EIA and those of radioimmunoassay (y = 0.897x + 0.564, r = 0.957). Gel chromatographic analysis of standard EPO revealed a single peak. On the other hand, gel chromatographic analysis of unconcentrated plasma samples obtained from three normal subjects revealed at least four components of immunoreactive EPO. Preparative isoelectrical focusing revealed at least four major components and some other small peaks in normal human plasma samples. CONCLUSION: These findings indicate the first evidence for molecular and electrical heterogeneity of circulating EPO in normal human plasma without any concentrating procedure.

Effect of long-term treatment with recombinant human growth hormone on erythropoietin secretion in an anemic patient with panhypopituitarism.

We studied the effect of treatment with recombinant human GH in an anemic patient with panhypopituitarism in which hemoglobin (Hb) concentration remained as low as 11.0 g/dl in spite of appropriate replacement with thyroid and adrenocortical hormones. Recombinant human GH was subcutaneously and constantly infused for 12 months using a portable syringe pump at a rate of 0.25 U/kg/week. After the treatment with human GH plasma erythropoietin (EPO) levels increased from 12.2 to 25.1 mIU/ml, with a concomitant increase of Hb concentration to 13.6 g/dl. When the administration of human GH was interrupted, both plasma EPO levels and Hb concentrations decreased. There was a close correlation between plasma GH and EPO levels before and during the human GH administration (y=2.444x+1 3.423, r=0.641, p<0.05). Plasma GH levels were well correlated with Hb concentrations before and during human GH administration (y=0.529x+11.313, r=0.690, p<0.01). Plasma IGF4 levels were also correlated with Hb concentrations (y=0.007x+10.874, r=0.832, p<0.001), but not with plasma EPO levels. These findings suggest that GH treatment may be useful in anemic patients with panhypopituitarism.

Effects of erythropoietin on neuronal activity.

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(-10) M) increased 45Ca2+ uptake and intracellular Ca2+ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 microM) 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(-10) M) 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(-10) M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10(-13)-10(-10) M) 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(-10) M), 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.

Therapeutic use of continuous subcutaneous infusion of recombinant human erythropoietin in malnourished predialysis anemic patients with diabetic nephropathy.

We compared the effect of recombinant human erythropoietin (rhEPO) administration by continuous s.c. infusion (CSI) with that of a weekly bolus s.c. injection (SBI) in five malnourished predialysis anemic patients with diabetic nephropathy. rhEPO was either continuously infused at a flow rate of 6000 IU per week (36 IU/h) (CSI group) or injected s.c. at a dose of 6000 IU once a week (SBI group) for 4 weeks, in a cross-over comparative study with a washout period of 4 weeks. Mean+/-S.D. plasma EPO levels increased from a basal value of 18.0+/-4.9 mIU/ml to a steady state value of 70.5+/-38.9 mIU/ml 2 weeks after the start of CSI of rhEPO (P < 0.05). Increases in reticulocyte count above the basal value were greater in the CSI group than the SBI group at 3 weeks after the start of treatment (0.94+/-0.35% vs -0.03+/-0.46%, P < 0.05). Increases in Hb concentration above the basal value were much greater in the CSI group than the SBI group at 4 weeks after the start of treatment (2.56+/-0.77 g/dl vs 0.28+/-0.62 g/dl, P < 0.05). These findings suggest that rhEPO administration by CSI is more effective than by SBI for improving anemia in malnourished predialysis patients with diabetic nephropathy.

Stimulation of erythropoietin secretion by continuous subcutaneous infusion of recombinant human GH in anemic patients with chronic renal failure.

We have investigated the effect of human GH on erythropoietin (EPO) secretion in eight anemic patients with chronic renal failure (CRF) (three males and five females, aged from 46 to 83 years). Recombinant human GH was infused subcutaneously at a flow rate of 2 microg/kg body weight per 0.1 ml/h for 72 h using a portable infusion pump. Blood samples were obtained immediately before and 2, 4, 6, 12, 24, 36, 48, 60, 72, 84, 96, 108, 120 and 168 h after the start of GH infusion. Storage urine samples were obtained before and 24, 48 and 72 h after the start of the infusion. The mean (+/- S.E.M.) basal plasma GH levels increased from 1.9 +/- 0.3 to 18.8 +/- 0.7 microg/l during the GH infusion. Plasma IGF-I levels increased 12 h after the start of GH treatment and the mean peak values of 403.6 +/- 38.5 microg/l were obtained at 72 h. Plasma EPO levels increased 6 h after the start of GH infusion, and the peak values of 38.4 +/- 11.6 IU/l were obtained at 96 h (P < 0.05 vs basal values 24.5 +/- 7.2 IU/l). Reticulocyte counts increased from 28.7 +/- 5.2 x 10(3)/microl to 40.3 +/- 8.0 x 10(3)/l at 108 h, 43.6 +/- 9.2 x 10(3)/microl at 120 h and 41.7 +/- 7.7 x 10(3)/microl at 160 h (P < 0.05). Serum urea nitrogen decreased at 72 h (P < 0.05), whereas there was no significant change in urinary excretion of nitrogen. Hemoglobin levels were not significantly changed throughout the experimental period. These findings indicate that human GH has a stimulating effect on EPO secretion in anemic patients with CRF.

[Fluctuations of physical function affected by sleep-awake rhythm--endocrine system].

Pituitary hormone secretion is changed by sleep-awake rhythm, which also regulates by the hypothalamo-pituitary axis. The endocrine rhythm is also affected by such factors as aging and environment conditions. Nocturnal secretion of growth hormone is known to be induced by slow-wave sleep. Plasma prolactin levels seem to increase during REM sleep. Serum thyroid stimulating hormone levels increase during the night. Plasma adrenocorticotropin and cortisol levels increase in the early morning and decreased in the night, which are not related to the sleep stage. The sleep-related hormone secretion is not shown in the patients with disordered hypothalamo-pituitary axis. The evaluation of sleep-related changes in pituitary hormone is important to assess the hypothalamo-pituitary function.

An acromegalic patient with pulsatile secretion of growth hormone (GH) coincident with the slow-wave sleep.

A 43-year-old woman was admitted to our hospital for further treatment of acromegaly with high plasma GH and IGF-I levels after transsphenoidal adenomectomy and subsequent treatment with bromocriptine. Physical examination and magnetic resonance imaging (MRI) showed an active acromegalic appearance with residual pituitary macroadenoma. Laboratory findings revealed an increase in basal levels of plasma GH (21.3 microg/L) and plasma IGF-I (470 ng/ml). Plasma GH levels were suppressed from 21.3 microg/L to 9.9 microg/L following oral administration of 75 glucose and did not respond to either TRH or LHRH injection. When plasma GH levels were measured after repeated blood sampling every 20 min for 24 h and sleep stages were analyzed, there were three GH peaks during the night which corresponded to the slow-wave sleep. Mean plasma GH levels which corresponded to the slow-wave sleep stages were much greater than those of other sleep stages during the night. After the patient was treated with intermittent sc injections of octreotide (40 microg/every 2 h, 480 microg/day) in combination with oral administration of bromocriptine (15 mg/day, t.i.d.), episodic GH release was somewhat suppressed but plasma GH levels were slightly increased, corresponding to the slow-wave sleep stage during the night. Mean plasma GH levels were much higher during the sleeping period than the waking period for 24 h before and after the treatment. These findings suggest that GH secretion is correlated to the slow-wave sleep in this particular patient with pituitary GH producing adenoma.

A case of autoimmune hypophysitis associated with asymptomatic primary biliary cirrhosis.

We report a 61-year old male patient with panhypopituitarism complicated with asymptomatic primary biliary cirrhosis (PBC). T1-weighted magnetic resonance imaging demonstrated high intensity of the anterior pituitary gland. There was no mass lesion or enlargement of the pituitary gland or the stalk. Immunoblot analysis of the patient's sera with rat pituitary antigens revealed a band with a molecular size of 22 kD. Anti-M2 mitochondrial antibody has been consistently positive for five years. Liver biopsy revealed portal hepatitis with periportal infiltration of the inflammatory cells. This is the first case report of autoimmune hypophysitis complicated with asymptomatic PBC.