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.

Expression of hypothalamic corticotropin-releasing hormone-like immunoreactivity in isolated ACTH deficiency: a report of an autopsied case.

A 77-yr-old man died of metastising adenocarcinoma of the lung. He had been treated with hydrocortisone for isolated ACTH deficiency for 9 yr. At autopsy, the pituitary and the adrenal glands were atrophic. Lymphocyte infiltration was not observed in the pituitary, the adrenal and the thyroid glands. Immunohistochemistry revealed selective loss of ACTH-like immunoreactivity in the pituitary gland whereas CRH-immunoreactive cells were found in the paraventricular nucleus of the hypothalamus. This is the first report demonstrating immunohistochemical examinations of the pituitary and the hypothalamus in isolated ACTH deficiency.

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.

Roles and mechanisms of action of pituitary adenylate cyclase-activating polypeptide (PACAP) in growth hormone and prolactin secretion.

It appears that PACAP has a direct action on somatotrophs to release GH. The intracellular signal transduction mechanisms for PACAP might be similar to but partly distinct from those for GRH. PACAP might play a role in GH secretion induced by serotoninergic mechanisms but not in ultradian rhythm of GH secretion in the rat. PACAP can stimulate PRL release from the pituitary in rats possibly through indirect paracrine effect. In addition, PACAP might participate in regulation of PRL secretion via hypothalamic VIP.

Involvement of cyclic guanosine 3',5'-monophosphate in nitric oxide-induced glucagon secretion from pancreatic alpha cells.

It has been reported that nitric oxide (NO) is a positive modulator of glucagon release. The involvement of cyclic guanosine 3',5'-monophosphate (cGMP) in NO-induced glucagon secretion and the possible role of NO in glucagon release induced by l-arginine were investigated in mouse clonal alpha-cell line clone 6 (alpha TC6) cells, which predominantly secrete glucagon. NOC12, an NO donor, elicited an increase in glucagon release from alpha Tc6 cells in perifusion and static incubation. An inhibitor of cGMP-dependent protein kinase inhibited NOC12-induced glucagon release. NOC12 (1 mmol/L) also increased the cellular level of cGMP. In addition, a permeable cGMP agonist increased glucagon release. l-arginine (15 mmol/L) increased perifusate concentrations of glucagon and nitrite in alpha Tc6 cells, which were inhibited by N(G)-nitro-L-arginine methyl ester. NO synthase (NOS) activity was shown in alpha Tc6 cells by l-citrulline formation assay. Our present findings suggest that NO plays a stimulating role in glucagon release from the alpha cells, and that a cGMP-dependent pathway is involved in NO action. These findings also provide further evidence that l-arginine might play a stimulating role in regulating glucagon secretion, at least partly, through generation of NO in the islets.

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.

Effects of dopamine and L-DOPA on survival of PC12 cells.

The effects of dopamine and L-DOPA on survival were examined in differentiated PC12 cells. Addition of dopamine to the culture medium at 3-30 microM prevented cell death induced by depletion of serum and nerve growth factor (NGF). At 100 microM, dopamine induced cell death. The cell-protective effect of dopamine was not affected by nomifensine, an inhibitor of dopamine uptake, or pargyline, an inhibitor of monoamine oxidase, suggesting that dopamine is working outside the cell. The cell-protective effect of dopamine was blunted by SCH-23390, a D(1) antagonist, but not sulpiride, a D(2) antagonist, indicating that the cell protective effect of dopamine is mediated by D(1) receptors in PC12 cells. L-DOPA also protected PC12 cells from cell death induced by depletion of serum and NGF at low concentrations and showed toxicity at high concentration. The effect of L-DOPA was unchanged after inhibition of conversion of L-DOPA to dopamine by m-hydroxybenzylhydrazine (NSD-1015), an inhibitor of DOPA decarboxylase, suggesting that L-DOPA itself is working for cell protection. Intracellular Ca(2+) concentration and mitogen-activated protein (MAP) kinase activity were increased by both dopamine and L-DOPA. The effects of dopamine and L-DOPA on cell survival were blunted by nicardipine, a Ca(2+) channel blocker, and PD-98059, an inhibitor of MAP kinase kinase (MEK). These results taken together raised the possibility that dopamine and L-DOPA protect PC12 cells from cell death at low concentrations by activating MAP kinase activity via elevation of intracellular Ca(2+) concentration.

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.

Reverse correlation between urine nitric oxide metabolites and insulin resistance in patients with type 2 diabetes mellitus.

We studied the possible relationship between nitric oxide (NO) production and insulin resistance in patients with type 2 diabetes mellitus. Urine NO metabolites (NOx) were measured as an index for NO production by HPLC combined with a Cd column, Griess reaction and a spectrophotometer in 403 healthy control subjects and 102 hospitalized patients with type 2 diabetes. Glucose infusion rate (GIR) was measured as a reverse index for insulin resistance by euglycemic glucose clamp study using an artificial pancreas in 20 of 102 diabetic patients. Urine NOx was lower in the patients with type 2 diabetes than in healthy control subjects (mean+/-SE: 3.18 +/-0.02 versus 3.25 +/-0.01 log[-micromol/gCr], p<0.01). Urine NOx was correlated with body mass index (BMI) in 102 diabetic patients (r= -0.372, p<0.001), but not related to either age, sex, fasting plasma glucose, HbA1c or blood pressure. Urine NOx was correlated with GIR independent of BMI in 20 diabetic patients (r=0.774, P<0.0001). These findings suggest that NO production is closely related with insulin resistance in patients with type 2 diabetes.

The role of 6R-tetrahydrobiopterin in the nervous system.

In addition to its cofactor activities for aromatic L-amino acid hydroxylases and nitric oxide synthase (NOS), 6R-tetrahydrobiopterin (6R-BH(4)) shows diverse actions on neurons. Dopamine release from the rat striatum or PC12 cells was stimulated by 6R-BH(4). The action of 6R-BH(4) was independent of its cofactor activities and stereospecific. Ca(2+) channels in rat brain and PC12 cells were activated by 6R-BH(4) via cAMP-protein kinase A pathway. Membrane potential of PC12 cells was deplorized by 6R-BH(4). Thus, it is assumed that 6R-BH(4) acts on its specific action site (possibly outside of the cell membrane) to stimulate dopamine release by activating Ca(2+) channels. Apoptosis induced by depletion of serum and nerve growth factor in PC12 cells was prevented by 6R-BH(4). The cell surviving effect of 6R-BH(4) was also mediated by activation of Ca(2+) channels and cAMP-protein kinase A pathway. However, since 6R-BH(4) did not activate mitogen activated protein kinase, it did not support neuronal differentiation. Nitric oxide (NO)-induced cell death was prevented by 6R-BH(4) in PC12 cells. NOS activity was not changed by exogenous 6R-BH(4), but NO metabolites in culture medium were decreased by 6R-BH(4). When endogenous 6R-BH(4) was reduced by inhibition of biosynthesis, cell death was induced in PC12 cells. Superoxide is observed to be generated during autoxidation of 6R-BH(4). Superoxide producing system mimicked the cell protective action of 6R-BH(4) against NO toxicity. Thus, it is considered that 6R-BH(4) protects PC12 cells against NO toxicity by generating superoxide during its autoxidation. These results raised the possibility that 6R-BH(4) is a self-protective factor against NO toxicity in NO producing neurons. Our findings indicate that 6R-BH(4) regulates neuronal activities in the brain and that 6R-BH(4) can be a promising drug for neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease.

Twenty-kilodalton human growth hormone (20K hGH) secretion from growth hormone-secreting pituitary adenoma cells in vitro.

Circulating human growth hormone (GH) consists of several molecular isoforms. Increased proportion of circulating non-22K hGH and 20K hGH was reported in active acromegaly. In this study, we studied the release of 20K and 22K hGH from cultured GH-producing human pituitary adenoma cells in vitro. Pituitary adenoma cells obtained from 6 acromegalic patients were cultured and submitted to perifusion experiments. Concentrations of 20K and 22K hGH in the serum and the perifusion effluent were determined by specific enzyme-linked immunosorbent assays recently developed. The %20K value varied in a wide range from 3.58 to 8.72% in vitro and was lower than in the serum (mean+/-SD: 6.57+/-1.88% vs 9.08+/-2.12%, P<0.05). There was no correlation between the %20K values in vitro and in vivo (r=0.31, P>0.05). The in vitro secretions of 20K and 22K hGH were in parallel and strongly correlated (r=0.953, P<0.001). These findings suggest that different GH-producing pituitary adenoma cells secrete 20K hGH in variable amounts and that the proportion of 20K hGH in the serum might be affected by metabolic clearance of hGH isoforms. It was also suggested that 20K and 22K hGH might be secreted in toto from GH-producing human pituitary adenoma cells.

Analysis of urinary nitric oxide metabolites in healthy subjects.

Nitric oxide (NO) has divergent actions under physiological and pathological conditions. NO is rapidly decomposed to nitrite (NO2-) and nitrate (NO3-). Since these metabolites are stable, they are good indices of NO production under various conditions. In the present study, we measured NO2- and NO3- concentrations in the urine collected from 62 hospital controls and 504 healthy subjects by means of a new HPLC system combined with Griess reaction. NOx was the sum of NO2- and NO3-. There was no considerable inter-day variation in urinary NO metabolite levels, and there was close correlation between NO2-, NO3- and NOx values in spot urine obtained in the early morning and those in 24-h stored urine in hospital controls. Urinary NO metabolite levels, which were corrected by creatinine (Cr) excretion and expressed on a logarithmic scale, showed normal distribution and were independent of sex and age in healthy subjects. The normal ranges of urinary NO2-, NO3- and NOx levels were estimated as 17-72 micromol/g Cr, 1,023-2,818 pmol/g Cr, and 1,071-2,951 micromol/g Cr, respectively. We also found that urinary NO metabolite levels were lower than normal range in patients with various diseases.

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.

Thyrotropin-releasing hormone stimulates nitric oxide release from GH3 cells.

Constitutive nitric oxide synthase (NOS) is expressed in the rat adenohypophysis but the mechanisms regulating its activity at the cellular level remain to be elucidated. The effect of TRH on nitric oxide release from GH3 cells was studied by means of reverse-phase HPLC to measure NO-2 and NO-3 concentrations in the incubation medium, and by polarography using electrodes specific for NO. Medium NO-2 concentrations in the incubation medium were dependent on the incubation time, and were further increased by sodium nitroprusside (SNP) or high potassium. NO-3 was detectable only in the presence of 100 microM SNP. Addition of L-arginine increased medium NO-2 concentrations. Diamino-hydroxypyrimidine decreased medium NO-2 concentrations, which were restored by the addition of (6R)-5, 6, 7, 8-tetrahydro-L-biopterin (THB). TRH elicited dose-related increases in medium NO-2 concentrations and in nitric oxide-specific currents, which were abolished by Nomega-nitro-L-arginine methyl ester. TRH failed to increase medium NO-2 concentrations in cells loaded with an intracellular Ca2+-chelating agent. The findings suggest that mobilization of intracellular Ca2+ by TRH stimulation activates Ca2+-dependent NOS in GH3 cells.

Enhancement of neuronal survival by 6R-tetrahydrobiopterin.

Recently, we have reported that 6R-tetrahydrobiopterin activates Ca2+ channels in neuronal cells independently of its cofactor activities. Several reports indicate that depolarization-induced activation of Ca2+ channels enhances neuronal survival. Here, we investigated the effects of 6R-tetrahydrobiopterin on survival of differentiated PC12 cells. Depletion of serum and nerve growth factor caused cell death, which was prevented by high potassium. 6R-Tetrahydrobiopterin also prevented death of PC12 cells cultured without serum and nerve growth factor in a dose-related manner at physiological concentrations (1-100 microM). However, surviving cells cultured with 6R-tetrahydrobiopterin showed undifferentiated form. 6S-Tetrahydrobiopterin, a diastereoisomer of 6R-tetrahydrobiopterin, also had a cell-surviving effect, but it was less potent as compared with that of 6R-tetrahydrobiopterin. The cell-surviving effect of 6R-tetrahydrobiopterin was eliminated by a Ca2+ channel blocker, but persisted in the presence of an inhibitor for tyrosine hydroxylase, dopamine, L-DOPA, an inhibitor for nitric oxide synthase and a nitric oxide generator. The effect of 6R-tetrahydrobiopterin was mimicked by a cyclic-AMP analogue and inhibited by an inhibitor for protein kinase A. Ca2+ channel activity was preserved but dopamine-releasing activity was disturbed in surviving cells cultured with 6R-tetrahydrobiopterin. 6R-Tetrahydrobiopterin had no effect on mitogen-activated protein kinase. These findings suggest that, independently of its cofactor activities and mitogen-activated protein kinase cascade, 6R-tetrahydrobiopterin enhances survival of PC12 cells by activating Ca2+ channels via the cyclic-AMP-protein kinase A pathway, but that 6R-tetrahydrobiopterin does not preserve neuronal character induced by nerve growth factor.

Antihypertensive effect of insulin via nitric oxide production in the Zucker diabetic fatty rat, an animal model for non-insulin-dependent diabetes mellitus.

It has been reported that insulin treatment improves hypertension in patients with diabetes mellitus. The mechanisms of the antihypertensive effect of insulin, however, remain to be fully elucidated. In the present study, we investigated a possible involvement of nitric oxide (NO) in insulin-induced reduction of blood pressure using the Zucker diabetic fatty (ZDF) rat, an animal model of non-insulin-dependent diabetes mellitus. The animals were divided into three groups and treated for 4 weeks with daily subcutaneous injections of insulin (25U/kg body weight) with or without oral administration of l-nitro-arginine methyl ester (L-NAME, 50mg/kg/day body weight as drinking water), an inhibitor of NO synthase (NOS). Saline solution was injected subcutaneously in the control groups. During the experimental period, body weight gain was greater in the insulin-treated groups than in the control groups whereas water intake was considerably decreased in the insulin-treated groups. Insulin treatment resulted in a decrease in plasma glucose and blood pressure, and an increase in both NO metabolites (NOx) in the plasma and NOS activity in the aorta tissue. L-NAME treatment blunted not only the antihypertensive effect of insulin but also the changes in NOx and NOS activity. These findings suggest that insulin reduces blood pressure in the ZDF rat by stimulating NOS activation and NO production.

Endogenous nitric oxide inhibits growth hormone secretion through cyclic guanosine monophosphate-dependent mechanisms in GH3 cells.

Constitutive nitric oxide synthase (NOS) is expressed in rat adenohypophysis and clonal GH3 cells. The mechanisms of action of nitric oxide (NO) to inhibit hormone secretion and the possible role of (6R)-5, 6, 7, 8-tetrahydro-L-biopterin (THB) in the action of endogenous NO were studied in GH3 cells. Inhibiting NOS with N(G)-nitro-L-arginine or trapping NO with oxyhemoglobin enhanced both the basal and TRH-stimulated rat GH release. Sodium nitroprusside did not further decrease either the basal or the TRH-stimulated GH secretion, suggesting that endogenous NO exerted the maximal inhibitory effect. Inhibition of de novo synthesis of THB increased GH secretion. A cyclic guanosine-monophosphate (cGMP) antagonist did not increase the basal GH secretion but enhanced TRH-induced GH release. These findings suggest that endogenous NO plays an inhibitory role on basal GH release and TRH-stimulated hormone release from GH3 cells in an autocrine or paracrine fashion, at least partly, through a cGMP-dependent pathway. It is also suggested that endogenous THB plays a role in NO production and subsequent inhibition of hormone secretion in GH3 cells.

Self-protection of PC12 cells by 6R-tetrahydrobiopterin from nitric oxide toxicity.

Nitric oxide (NO) has cytotoxic effects but NO producing neurons are resistant to NO toxicity. These results suggest the presence of self-protecting factors for NO toxicity. Recently, 6R-tetrahydrobiopterin (6R-BH4), a cofactor for NO synthase (NOS), has been reported to degrade NO raising the possibility that 6R-BH4 acts as a self-protecting factor for NO toxicity. In PC12 cells which have NOS, three-day culture with sodium nitroprusside (SNP) or NOC-12, NO generators, at 10-100 microM increased nitrite and nitrate concentrations in the culture medium and induced death of PC12 cells. Coadministration of 6R-BH4 (10 or 30 microM) with SNP or NOC-12 prevented cell death with reduction of nitrite and nitrate in the medium. Inhibition of 6R-BH4 synthesis by 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor for GTP cyclohydrolase I, decreased cellular 6R-BH4 content and viable cell number. The inhibiting effects of DAHP were restored by exogenous 6R-BH4. NOS activity, as estimated by nitrite concentrations in the medium, was unchanged by DAHP. Hypoxanthine and xanthine oxidase, which produce superoxide, mimicked the cell-protecting effect of 6R-BH4 which is reported to generate superoxide during its autoxidation. These results suggest that 6R-BH4 acts as a self-protecting factor for NO toxicity with generation of superoxide in NO-producing neurons.