Ken Lee

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.

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.

Ca2+ entry channels involved in contractions of rat aorta induced by endothelin-1, noradrenaline, and vasopressin.

Endothelin-1 (ET-1) has been shown to activate three types of Ca2+ channel, namely two Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC), and that these channels can be discriminated by Ca2+ channel blockers such as LOE 908 (a blocker of NSCC-1 and NSCC-2) and SK&F 96365 (a blocker of NSCC-2 and SOCC). This study pharmacologically compared Ca2+ entry channels involved in contractions of rat thoracic aorta without endothelium induced by ET-1, noradrenaline (NA), or arginine-vasopressin (AVP). These agonists-induced contractions of aortic rings without endothelium and increases in the intracellular free Ca2+ concentration ([Ca2+]i) of cultured aortic smooth muscle cells were abolished by removal of extracellular Ca2+. A blocker of L-type voltage-operated Ca2+ channel (VOCC), nifedipine had no effect on the responses to ET-1, but it suppressed the responses to NA and AVP to 70% and 65% of control responses, respectively. LOE 908 partially suppressed the nifedipine-resistant responses to ET-1 and AVP, but not those to NA. SK&F 96365 also partially suppressed the nifedipine-resistant responses to ET-1 and AVP, whereas it abolished the responses to NA. LOE 908 in combination with SK&F 96365 abolished the nifedipine-resistant responses to either of the agonists. These results show that the contraction of rat aorta involves different Ca2+ entry channel depending on agonists: (a) NSCC-1, NSCC-2, and SOCC for ET-1; (b) VOCC and SOCC for NA; and (c) VOCC, NSCC-1, NSCC-2, and SOCC for AVP.

Enhancement of in vivo tyrosine hydroxylation in the rat adrenal gland under hypoxic conditions.

We examined the effects of hypoxia (8% O2) on in vivo tyrosine hydroxylation, a rate‐limiting step for catecholamine synthesis, in the rat adrenal gland. The hydroxylation rate was determined by measuring the rate of accumulation of 3,4‐dihydroxyphenylalanine (DOPA) after decarboxylase inhibition. One hour after hypoxic exposure. DOPA accumulation decreased to 60% of control values, but within 2 h it doubled. At 2 h, the apparent Km values for tyrosine and for biopterin cofactor of tyrosine hydroxylase (TH) in the soluble fraction were unchanged, whereas the Vmax value increased by 30%. The content of total or reduced biopterin was unchanged, but the content of tyrosine increased by 80%. Tyrosine administration had little effect on DOPA accumulation under room air conditions but enhanced DOPA accumulation under hypoxia. After denervation of the adrenal gland, the hypoxia‐induced increase in DOPA accumulation and in the Vmax value was abolished, whereas the hypoxia‐induced increase in tyrosine content was persistent. These results suggest that in vivo tyrosine hydroxylation is enhanced under hypoxia, although availability of oxygen is reduced. The enhancement is the result of both an increase in tyrosine content coupled with increased sensitivity of TH to changes in tyrosine tissue content and of an increase in dependence of TH on tyrosine levels. The increase in the sensitivity of TH and in the Vmax value is neurally induced, whereas the increase in tyrosine content is regulated by a different mechanism.

A novel action of 6R-l-erythro-5,6,7,8-tetrahydrobiopterin, a cofactor for hydroxylases of phenylalanine, tyrosine and tryptophan: Enhancement of acetylcholine release in vivo in the rat hippocampus

Recently, we have reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for hydroxylases of phenylalanine, tyrosine and tryptophan, has a dopamine releasing action in the rat striatum independent of its cofactor role. Here we studied the effects of 6R-BH4 on acetylcholine (ACh) release in the rat hippocampus using brain microdialysis. Intracerebroventricular injection of 6R-BH4 dose-dependently increased extracellular ACh levels monitored by brain microdialysis. Biopterin, an oxidized form of 6R-BH4, and 6S-BH4, an unnatural diastereoisomer of 6R-BH4, had little effect on the ACh levels. The 6R-BH4-induced increase in ACh levels was eliminated after inhibition of voltage-dependent Na+ channels by tetrodotoxin, but not after depletion of catecholamines by reserpine. These results show that 6R-BH4 has direct ACh releasing action in vivo.

Characterization of nicotinic acetylcholine receptors on cultured bovine adrenal chromaffin cells using modified l-[3H]nicotine binding assay

SummaryTo characterize the properties of nicotinic acetylcholine receptors (nAChRs) in autonomic ganglia, we examined l-[3H]nicotine binding to membrane fraction prepared from cultured bovine adrenal chromaffin cells, using a modified filtration method. Binding of l-[3H]nicotine to non-treated glass fiber filters interfered with the detection of specific binding to the membrane fraction. Presoaking glass fiber filters in 3% or higher concentrations of polyethyleneimine (PEI) solution (sixty times higher than earlier used concentration) for at least 5 h could reduce the binding of l-[3H]nicotine to the filters to the background level. Specific l-[3H]nicotine binding to the membrane fraction was detected only when the membrane fraction was prepared in Ca2+- and Mg2+ (EDTA, EGTA and protease inhibitors were added)-free buffer. Specific binding of l-[3H]nicotine was saturable and reversible. Both computer program and Scatchard analysis revealed a single class of high affinity binding sites with an average Kd of 8.9 nM and a Bmax of 42.5 fmol/mg protein. The Hill coefficient was 0.98. In inhibition studies, both cholinergic agonists (carbachol and l-nicotine) and ganglionic agonists (lobeline and 1,1-dimethyl-4-phenylpiperazinium iodide) were much effective in inhibiting l-[3H]nicotine binding, whereas both neuromuscular blocking (α-bungarotoxin and d-tubocurarine) and ganglionic blocking agents were less effective. These results suggest that high affinity nicotinic binding sites on adrenal chromaffin cells are nAChRs of the ganglion-type, which have properties different from nAChRs on the neuromuscular junction but similar to nAChRs in the brain.

Effects of hypoxia on the catecholamine release, Ca2+ uptake, and cytosolic free Ca2+ concentration in cultured bovine adrenal chromaffin cells.

Abstract: The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K+‐induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media pre‐equilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K+‐induced catecholamine release was inhibited by hypoxia to ∼40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura‐2) gradually increased and reached a plateau of ∼1.0 μM at 30 min, whereas the level was constant in the control medium (∼200 nM). High K+‐induced increases in [Ca2+]I were inhibited by hypoxia to ∼30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K+‐induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K+‐induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K+‐induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage‐dependent Ca2+ channels.

A nonisotopic method for determination of the in vivo activities of tyrosine hydroxylase in the rat adrenal gland.

A rapid and reliable method for determination of in vivo activities of tyrosine hydroxylase in the rat adrenal gland is presented. This method involves determining the rate of accumulation of 3,4-dihydroxyphenylalanine (Dopa) in the adrenal gland after decarboxylase inhibition by NSD 1015, using HPLC with electrochemical detection after purification of the acid-deproteinized tissue extract with Bio-Rex 70 columns followed by alumina batch method. Purification of the sample with alumina adsorption alone, a method usually used for purification of catecholamines and Dopa, was ineffective: epinephrine and norepinephrine, which are present in high concentrations, interfered with an accurate determination of Dopa, and dopamine, which is retained strongly on the reverse-phase column, interfered with a rapid analysis. Purification with Sephadex G-10 columns followed by alumina adsorption was also ineffective. After purification with columns of weak cation-exchange resins such as Bio-Rex 70 or Amberlite CG-50 followed by alumina adsorption, most of the epinephrine and norepinephrine was removed and dopamine was eliminated. Thus a rapid and accurate determination of Dopa could be made. Of the two cation exchangers, Bio-Rex 70 was more effective. Accumulation of Dopa in the adrenal gland was linear up to 30 min after administration of NSD 1015 and a plateau was reached with doses over 10 mg/kg. Using this method, we investigated the effects of immobilization stress, reserpine, and hypoxia on in vivo activities of tyrosine hydroxylase in the adrenal gland.

Determination of Serotonin Turnover in the Rat Brain Using 6-Fluorotryptophan

Abstract: After intraperitoneal injection of rats with 6‐fluorotryptophan (6‐FT), brain 5‐hydroxytryptamine (5‐HT) levels decreased exponentially over 1 h. Depletion was dose‐dependent and maximum depletion was observed at 200 mg/kg. 6‐FT (200 mg/kg) did not significantly alter the content of 5‐hydroxyindoleacetic acid. Turnover rates of 5‐HT obtained by the 6‐FT and other methods were fairly consistent. 6‐FT had little effect on the content of noradrenaline and dopamine. These data suggest that 6‐FT completely inhibits tryptophan hydroxylase, in vivo, without affecting the release of 5‐HT from 5‐HT neurons and with little effect on the activities of tyrosine hydroxylase. Therefore, 6‐FT is a good pharmacological tool for studying the turnover rate of 5‐HT in the brain.