Yasutaka Takagi

Regulation of acetylcholine release in vivo from rat hippocampus by monoamines as revealed by novel column-switching HPLC with electrochemical detection.

To clarify monoaminergic regulation of acetylcholine (ACh) release in the rat hippocampus, the effects of administration of monoamines through a dialysis probe on extracellular ACh levels were examined using in vivo brain microdialysis combined with a novel column-switching HPLC system. Infusion of dopamine or 5-hydroxytryptamine, but not noradrenaline, increased ACh levels. The ACh levels also increased following infusion of apomorphine and 5-methoxy-N,N-dimethyltryptamine. These results demonstrate that hippocampal ACh release is regulated by dopamine and 5-hydroxytryptamine.

Differential effects of hypoxia on ligand binding properties of nicotinic and muscarinic acetylcholine receptors on cultured bovine adrenal chromaffin cells.

Abstract: Hypoxia is known to disturb neuronal signal transmission at the synapse. Presynaptically, hypoxia is reported to suppress the release of neurotransmitters, but its postsynaptic effects, especially on the function of neurotransmitter receptors, have not yet been elucidated. To clarify the postsynaptic effects, we used cultured bovine adrenal chromaffin cells as a model of postsynaptic neurons and examined specific binding of l‐[3H]nicotine (an agonist for nicotinic acetylcholine receptors: nAChRs) and 22Na+ flux under control and hypoxic conditions. Experiments were performed in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). Scatchard analysis of the specific binding to the cells revealed that the KD under hypoxic conditions was twice as large as that under control conditions, whereas the Bmax was unchanged. When the specific [3H]nicotine binding was kinetically analyzed, the association constant (k1) but not the dissociation constant (k−1) was decreased to 40% of the control value by hypoxia. When the binding assay was performed using the membrane fraction, these changes were not observed. Nicotine‐evoked 22Na+ flux into the cells was suppressed by hypoxia. In contrast, specific [3H]quinuclidinyl benzilate binding to the intact cells was unaffected by hypoxia. These results demonstrate that hypoxia specifically suppresses the function of nAChRs (and hence, neuronal signal transmission through nAChRs), primarily by acting intracellularly.

Characterization of a Dopamine‐Releasing Action of 6R‐l‐erythro‐Tetrahydrobiopterin: Comparison with a 6S‐Form

Abstract: 6R‐l‐erythro‐Tetrahydrobiopterin (6R‐BH4) is a cofactor for aromatic l‐amino acid hydroxylases and nitric oxide synthase. Recently, we have reported that independently of its cofactor activities, 6R‐BH4 acts from the outside of neurons in the brain to enhance the release of monoamine neurotransmitters such as dopamine. To characterize the pharmacological properties of the action, we examined the effects of 6S‐BH4, a diastereoisomer of 6R‐BH4, on dopamine release in the rat striatum by using brain microdialysis and compared its effects with those of 6R‐BH4. Perfusion of 6S‐BH4 or 6R‐BH4 through the dialysis probe increased extracellular dopamine levels (an index of in vivo dopamine release) concentration dependently; the maximal increase by 6S‐BH4, was one‐sixth of that by 6R‐BH4. 6S‐BH4 increased extracellular DOPA levels in the presence of NSD 1015, an inhibitor of aromatic l‐amino acid decarboxylase (an index of in vivo tyrosine hydroxylase activity), to an extent similar to the increase induced by 6R‐BH4. The increase in the DOPA levels induced by either of the pteridines was abolished after pretreatment of rats with α‐methyl‐p‐tyrosine (an inhibitor of tyrosine hydroxylase). Under the same conditions, the 6S‐BH4‐induced dopamine release was abolished, but most of the 6R‐BH4‐induced increase persisted. Coadministration of 6S‐BH4 with 6R‐BH4 inhibited the increase in dopamine release induced by 6R‐BH4 alone. These results show that 6R‐BH4 stimulates dopamine release by acting at the specific recognition site on the neuronal membrane, and that 6S‐BH4 acts as an antagonist of 6R‐BH4 at this site, although it has cofactor activities.

Effects of bunazosin hydrochloride on ciliary muscle constriction and matrix metalloproteinase activities.

Purpose:To clarify the mechanism by which bunazosin hydrochloride (BZ), a selective &agr;1-adrenoceptor antagonist, increases uveoscleral outflow. Methods:The effects of BZ on matrix metalloproteinase (MMP) activities in cultured monkey ciliary muscle cells, and on phenylephrine hydrochloride-induced constriction in bovine ciliary muscles, were examined. Also, the possible additive ocular hypotensive effects of BZ and latanoprost (LP) were evaluated in ocular normotensive monkeys. Results:Although BZ at 10−7 to 10−5 M did not increase MMP-2, -3, and -9 activities in the culture medium of ciliary muscle cells, BZ at 10−7 to 10−5 M inhibited phenylephrine hydrochloride-induced constriction in ciliary muscles. The maximal reduction in intraocular pressure of concomitant administration of BZ and LP was greater than that of BZ alone and tended to be greater than that of LP alone. Conclusion: These findings, in normotensive monkeys, indicate that the mechanism whereby BZ increases uveoscleral outflow is independent of an effect on MMPs and is partly due to relaxation of the ciliary muscle. This effect is different from that of LP, which might help to explain the finding that topical concomitant administration of BZ and LP increased AUC(0–6h) value (IOP reduction) to 201% and 145% of BZ and LP given alone, respectively.

Ischemia-induced changes in catecholamine release and their mechanisms: a study using cultured bovine adrenal chromaffin cells

Ischemia-induced changes in neurotransmitter release and their mechanisms were examined using cultured bovine adrenal chromaffin cells. When the cells were incubated in glucose-free media equilibrated with 0% O2/100% N2 (ischemia), ATP content decreased and reached the minimum level within 40 min. Control incubation was done in media equilibrated with 21% O2 in N2. After 10-min incubation under ischemic conditions, basal catecholamine (CA) release was elevated and the elevation persisted up to 90 min. High K(+)-evoked CA release was transiently enhanced at 10 min, but after that, it decreased to reach the minimum level at 60 min. At 10 min, cytosolic free Ca2+ concentration ([Ca2+]i) and 45Ca2+ uptake of the resting cells (basal values) and high K(+)-evoked increases in these two parameters were unchanged, but CA release from permeabilized cells in response to Ca2+ in media was augmented. After 60-min incubation under ischemic conditions, basal [Ca2+]i was elevated: the elevation was observed even in the absence of extracellular Ca2+. In contrast, high K(+)-evoked increases in [Ca2+]i and in 45Ca2+ uptake were suppressed, but basal 45Ca2+ uptake into intact cells and CA release from permeabilized cells were unchanged. These results suggest that in an early phase (10 min) of ischemia, both basal and stimulation-evoked CA release are augmented because of increased sensitivity of exocytotic machinery to Ca2+. In the late phase (60 min), basal CA release is augmented because of an increase in basal [Ca2+]i, which is due to accumulation of Ca2+ derived from intracellular Ca2+ pools: stimulation-evoked CA release is suppressed because of inhibition of stimulation-evoked increase in [Ca2+]i, which is due to functional disturbance of voltage-dependent Ca2+ channels.

Enhancement of acetylcholine release in the hippocampus by 6R-L-erythro-5,6,7,8-tetrahydrobiopterin is mediated by 5-hydroxytryptamine

Recently, we reported that 6R-L-erythro-tetrahydrobiopterin (6R-BH4), a natural cofactor for L-aromatic amino acid hydroxylases, enhances in vivo release of acetylcholine (ACh) in the rat hippocampus: the enhancement was abolished after depletion of brain catecholamines and 5-hydroxytryptamine (5-HT) by pretreatment with reserpine. In the present study, we have used in vivo brain microdialysis to clarify the neuronal mechanism involved in the enhancement of ACh release by 6R-BH4. After depletion of catecholamines by pretreatment of rats with alpha-methyl-p-tyrosine, 6R-BH4 added to the perfusion fluid still induced an increase in extracellular ACh levels monitored by microdialysis as an index of ACh release. In contrast, after depletion of 5-HT by pretreatment with p-chlorophenylalanine, most of the 6R-BH4-induced enhancement was eliminated. Exogenous 5-HT and dopamine (DA) but not noradrenaline added to the perfusion fluid stimulated ACh release with 5-HT being far more potent. Intraperitoneal administration of 5-hydroxytryptophan and L-DOPA also enhanced ACh release, presumably by their conversion to 5-HT and catecholamines, respectively. Administration of 6R-BH4 increased hippocampal 5-HT release, as indicated by increased extracellular levels of the major 5-HT metabolite, 5-hydroxyindoleacetic acid. These results suggest that 6R-BH4 stimulates ACh release in the hippocampus, mainly by augmenting release of 5-HT, a potent stimulator of ACh release, and partly by augmenting release of DA.

Distinct roles of second and third intracellular loops of human endothelin receptors in the selective activation of G alpha s/G alpha i.

Endothelin-1 (ET-1) stimulated cAMP formation in Chinese hamster ovary cells stably expressing human wild-type ET(A) (CHO/hET(A) cells) and inhibited the formation in cells expressing human wild-type ETB (CHO/hETB cells), suggesting a selective coupling of hET(A) and hETB with G alpha s and G alpha i, respectively. To find out the receptor domain(s) that determined the selective coupling, a series of chimeric receptors between hET(A) and hETB were expressed on CHO cells and the effect of ET-1 on cAMP formation in each cell line was tested. hET(A) with the replacement of the second and/or third intracellular loop (ICLII and/or III) to the corresponding region(s) of hETB failed to transmit the stimulatory effect of ET-1. hETB with the replacement of ICLIII to the corresponding region of hET(A) failed to transmit the inhibitory effect of ET-1. A chimeric receptor with ICLII of hETB and with ICLIII of hET(A) failed to transmit either effect. These results indicated the roles of ICLII and III of hETR as major determinants of the selective coupling of hET(A)/hETB with G alpha s/G alpha i, respectively.