Kunio Koshimura

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.

Enhancement of Dopamine Release In Vivo from the Rat Striatum by Dialytic Perfusion of 6R‐l‐erythro‐5,6,7,8‐Tetrahydrobiopterin

We have previously reported that intracerebroventricular administration of 6R‐L‐erythro‐5,6,7,8‐tetrahydrobiopterin (6R‐BH4), a cofactor for tyrosine hydroxylase, enhances biosynthesis of 3,4‐dihydroxyphenylethylamine (dopamine) in the rat brain. In the present study, we have more precisely examined the effects of 6R‐BH4 on dopamine release in vivo from the rat striatum using brain microdialysis. The amount of dopamine collected in striatal dialysates was determined using HPLC with electrochemical detection after purification with an alumina batch method. When the striatum was dialyzed with Ringer solution containing various concentrations of 6R‐BH4 (0.25,0.5, and 1.0 mM), dopamine levels in striatal dialysates increased in a concentration‐dependent manner. Biopterin had little effect on dopamine levels in dialysates. The 6R‐BH4‐induced increase in dopamine levels in dialysates was abolished after pretreatment with tetrodotoxin (50 μM) added to the perfusion fluid, but after pretreatment with nomifensine (100 mg/kg, intraperitoneal injection), an inhibitor of dopamine uptake mechanism, a larger increase was observed. After inhibition of tyrosine hydroxylase by pretreatment with α‐methyl‐p‐tyrosine (250 mg/kg, intraperitoneal injection), most of the increase persisted. These results suggest that 6R‐BH4 has a dopamine‐releasing action, which is not dependent on biosynthesis of dopamine.

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.

Effects of Hypoxia on the Activity of the Dopaminergic Neuron System in the Rat Striatum as Studied by In Vivo Brain Microdialysis

The purpose of the present study is to clarify the effects of hypoxia on the activity of the dopaminergic neurons in the brain and its mechanism of action. For this purpose, the effects of hypoxia on the extracellular levels of 3,4‐dihy‐droxyphenylethylamine (dopamine) were examined in the rat Striatum using in vivo brain microdialysis in the presence or absence of pretreatment with either tetrodotoxin (a blocker of voltage‐dependent sodium channels) or nomifensine (a blocker of dopamine reuptake). Exposure to various degrees of hypoxia (15, 10, and 8% O2 in N2) increased dopamine levels in striatal dialysates to 200, 400, and 1,100%, respectively, of the control value. On reoxygenation, dopamine levels in the dialysates rapidly returned to the control level. Reexposure to hypoxia increased the dopamine levels to the same extent as during the first exposure. After addition of tetrodotoxin (40 mUM) to the perfusion fluid or pretreatment with nomifensine (100 mg/kg, i.p.), exposure to hypoxia no longer increased the dopamine levels. These results suggest that although hypoxia induces an increase in the extracellular dopamine levels (hence, an apparent increase in the activity of the dopaminergic neurons), this increase is not the result of an increase in dopamine release itself, but rather the result of inhibition of the dopamine reuptake mechanism.

Effects of choline administration on in vivo release and biosynthesis of acetylcholine in the rat striatum as studied by in vivo brain microdialysis.

The purpose of the present study is to clarify the effects of the administration of choline on the in vivo release and biosynthesis of acetylcholine (ACh) in the brain. For this purpose, the changes in the extracellular concentration of choline and ACh in the rat striatum following intracerebroventricular administration of choline were determined using brain microdialysis. We also determined changes in the tissue content of choline and ACh. When the striatum was dialyzed with Ringer solution containing 10 μM physostigmine, ACh levels in dialysates rapidly and dose dependently increased following administration of various doses of choline and reached a maximum within 20 min. In contrast, choline levels in dialysates increased after a lag period of 20 min following the administration. When the striatum was dialyzed with physostigmine‐free Ringer solution, ACh could not be detected in dialysates both before and even after choline administration. After addition of hemicholinium‐3 to the perfusion fluid, the choline‐induced increase in ACh levels in dialysates was abolished. Following administration of choline, the tissue content of choline and ACh increased within 20 min. These results suggest that administered choline is rapidly taken up into the intracellular compartment of the cholinergic neurons, where it enhances both the release and the biosynthesis of ACh.

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.

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 μM prevented cell death induced by depletion of serum and nerve growth factor (NGF). At 100 μM, 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 D1 antagonist, but not sulpiride, a D2 antagonist, indicating that the cell protective effect of dopamine is mediated by D1 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 Ca2+ 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 Ca2+ 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 Ca2+ concentration. J. Neurosci. Res. 62:112–119, 2000.

Effects of transient forebrain ischemia and reperfusion on function of dopaminergic neurons and dopamine reuptake in vivo in rat striatum.

To clarify functional changes of dopaminergic neurons and dopamine (DA) reuptake during and after ischemia, extracellular DA levels in striatum were determined using in vivo brain microdialysis in a 4-vessel occlusion model of male Wistar rats with and without pharmacological interventions. Without interventions, the extracellular DA levels markedly increased during ischemia, but upon reperfusion, rapidly returned to control level. Infusion of tetrodotoxin, a blocker of voltage-dependent Na+ channels, was without effect on the DA surge during ischemia, but decreased the DA levels after reperfusion to the same extent as in control rats. Pretreatment with nomifensine, an inhibitor of DA reuptake, was also without effect on the surge, but reduced the rate of DA decline after reperfusion to one-fifth of the rate without the pretreatment. When nomifensine was administered 40 min after reperfusion, extracellular DA levels increased to the same extent as in control rats. Infusion of high K+ 1 h after reperfusion induced a smaller increase in extracellular DA levels than that in control rats. It took 96 h for this reduced response to high K+ stimulation to recover after reperfusion. These results suggest that the DA surge during ischemia is mainly derived from action potential-independent DA release (means dysfunction of dopaminergic neurons), although activity of DA reuptake is completely inhibited. After reperfusion, the basal function of dopaminergic neurons and activity of DA reuptake rapidly recover, but the neurons are functionally disturbed to release less DA in response to a given stimulus for several days.

Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates growth hormone release from GH3 cells through type II PACAP receptor

Effect of pituitary adenylate cyclase activating polypeptide (PACAP) on growth hormone (GH) release from GH3 cells was studied in a dynamic superfusion system. PACAP-38 and PACAP-27 stimulated GH release from superfused GH3 cells. The stimulatory effect of PACAP-38 was comparable to those of vasoactive intestinal polypeptide (VIP) and PACAP-27 at a concentration of 1 nM, but the duration of action was more prolonged in PACAP-38 than in the other two peptides. PACAP(6-38), a selective antagonist of PACAP, as well as a VIP antagonist blunted the GH release induced by PACAP-38 and VIP. An antagonist of GH-releasing factor (GRF) at a concentration of 1 microM, however, did not affect the GH release induced by PACAP-38. These findings suggest that PACAP and VIP stimulate GH release from GH3 cells through type II PACAP receptor but not through the GRF receptors.

L-dopa administration enhances exocytotic dopamine release in vivo in the rat striatum.

Peripheral administration of L-3,4-dihydroxyphenylalanine (L-DOPA) methylester increased extracellular levels of DOPA and dopamine (DA) in the rat striatum monitored by in vivo brain microdialysis. The increase in DA levels persisted after inhibition of DA reuptake by nomifensine. Administration of blockers of voltage-dependent Na+ (tetrodotoxin) or Ca2+ (NKY-722) channels through the dialysis membrane completely eliminated the increase in DA levels. These results demonstrate that the L-DOPA-induced DA release is exocytotic in nature and hence, derived from neurons in the striatum.