L-3,4-dihydroxyphenylalanine-induced c-Fos expression in the CNS under inhibition of central aromatic L-amino acid decarboxylase.

L-3,4-dihydroxyphenylalanine (DOPA) is a neurotransmitter candidate. To map the DOPAergic system functionally, DOPA-induced c-Fos expression was detected under inhibition of central aromatic L-amino acid decarboxylase (AADC). In rats treated with a central AADC inhibitor, DOPA significantly increased the number of c-Fos-positive nuclei in the paraventricular nuclei (PVN) and the nucleus tractus solitarii (NTS), and showed a tendency to increase in the supraoptic nuclei (SON), but not in the striatum. On the other hand, DOPA with a peripheral AADC inhibitor elevated the level of c-Fos-positive nuclei in the four regions, suggesting that DOPA itself induces c-Fos expression in the SON, PVN and NTS. In rats treated with 6-hydroxydopamine (6-OHDA) to lesion the nigrostriatal dopamine (DA) pathway, DOPA significantly induced c-Fos expression in the four regions under the inhibition of peripheral AADC. However, under the inhibition of central AADC, DOPA did not significantly increase the number of c-Fos-positive nuclei in the four regions, suggesting that DOPA at least in part induces c-Fos expression through its conversion to DA. It was likely that the 6-OHDA lesion enhanced the response to DA, but attenuated that to DOPA itself. In conclusion, we proposed that the SON, PVN and NTS include target sites for DOPA itself.

Tonic function of nicotinic receptors in stress-induced release of L-DOPA from the nucleus accumbens in freely moving rats.

We investigated whether stress induces the release of L-3,4-dihydroxyphenylalanine (DOPA) and dopamine from the nucleus accumbens in conscious rats and characterized the stress-induced response. Electrical foot-shock stress induced both DOPA and dopamine release, measured by microdialysis, from the nucleus accumbens in freely moving rats. Pretreatment of rats with mecamylamine completely blocked stress-induced DOPA release, but only partially blocked dopamine release. Diazepam did not affect the foot-shock-induced release of DOPA, while the same dose of diazepam partially blocked the stress-induced release of dopamine. These findings suggest a tonic function of central nicotinic receptors in stress-induced DOPA release from the nucleus accumbens in conscious rats.

Autoradiographic studies using L-[(14)C]DOPA and L-DOPA reveal regional Na(+)-dependent uptake of the neurotransmitter candidate L-DOPA in the CNS.

We previously proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the CNS. Receptor and transporter molecules for L-DOPA, however, have not been determined. In the present study, in order to localize the uptake sites of L-DOPA in the CNS, we performed autoradiographic uptake studies using L-[14C]DOPA and L-[3H]DOPA in the uptake study on rat brain slice preparations, and further analyzed the properties of L-DOPA uptake. Image analysis of the L-[14C]DOPA autoradiogram showed a unique heterogeneous distribution of uptake sites in the brain. The intensity was relatively high in the cerebral cortex, the hypothalamus, the cerebellum and the hippocampus, while the density was moderate or even low in the striatum and the substantia nigra. L-DOPA and phenylalanine, but not dopamine (10mM) were able to almost completely inhibit the uptake of L-[14C]DOPA to basal levels. Microautoradiographic studies using L-[3H]DOPA revealed accumulation of dense grains in the median eminence, the supraoptic nucleus of the hypothalamus, the cerebral cortex (layer I) and the hippocampus. In the cerebellum, grains formed in clusters surrounding the Purkinje cells. This grain accumulation was concluded to be in Bergmann glial cells, since the morphological pattern of grain accumulation was similar to that of the immunoreactivity of the glutamate aspartate transporter, a marker protein for Bergmann glial cells. In the hippocampus, the grain density significantly decreased under Na(+)-free conditions. In addition, grain density also decreased in the absence of Cl(-). In contrast, grains in the choroid plexus and the ependymal cell layer, were not affected by the absence of Na(+). These findings indicated that the uptake of L-DOPA occurs via various types of large neutral amino acid transport mechanisms. It appears that neuronal and/or glial cells, which take up L-DOPA in a Na(+)-dependent manner, exist in the CNS. Our finding further supports the concept that L-DOPA itself may act as a neurotransmitter or neuromodulator.

Characteristics of protective effects of NMDA antagonist and calcium channel antagonist on ischemic calcium accumulation in rat hippocampal CA1 region.

Effects of excitatory amino acid receptor antagonists and voltage-dependent Ca(2+) channel antagonists on ischemia-induced intracellular free Ca(2+) accumulation in rat hippocampal slices were examined. Ischemia caused a large Ca(2+) accumulation in CA1 region but a small Ca(2+) accumulation in CA3 and dentate gyrus regions. When applied during ischemia, the NMDA receptor antagonist MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate) inhibited the ischemic Ca(2+) accumulation only in the CA1, but the non-NMDA receptor antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) inhibited it in all the three regions. The L-type Ca(2+) channel antagonists nifedipine and verapamil inhibited the ischemic Ca(2+) accumulation only in the CA1 region, but omega-conotoxin, a N- and L-type Ca(2+) channel antagonist inhibited the Ca(2+) accumulation in all the three regions of the hippocampus. When applied after 5-min ischemia, nifedipine but not MK-801, inhibited sustained postiscehmic Ca(2+) elevation in the CA1 region but not in the CA3 and dentate gyrus regions. These findings suggest that the enhanced ischemia-induced Ca(2+) accumulation in the CA1 region is mediated via activation of both NMDA receptors and L-type-like Ca(2+) channels. It appears that sustained postischemic Ca(2+) elevation in the CA1 region is mediated via activation of L-type-like Ca(2+) channels, but not of NMDA receptors.

Inhibition by L-3,4-dihydroxyphenylalanine of hippocampal CA1 neurons with facilitation of noradrenaline and gamma-aminobutyric acid release.

Electrophysiological studies were performed to elucidate whether L-3,4-dihydroxyphenylalanine (L-DOPA) acted on hippocampal CA1 neurons, since this drug has been reported to act as a neurotransmitter in the hypothalamus and striatum. Hippocampal slices (450 microM thick) obtained from male Wistar rats (4-7 weeks of age) were placed in a bath (maintained at 30+/-1 degrees C) continuously perfused with artificial cerebrospinal fluid. The population spikes elicited by electrical stimuli applied to the Schaffer collateral/commissural fibers were recorded in the hippocampal CA1 region, using a glass micropipette filled with 3 M NaCl. Drugs were applied in the bath through a perfusion system. The population spikes were inhibited by L-DOPA (1 nM-10 microM) with a bell-shaped concentration-response curve (n=7-15). Maximum inhibitory effects were obtained at 100 nM. L-DOPA cyclohexyl ester, a putative L-DOPA recognition site antagonist, antagonized the L-DOPA-induced inhibition of population spike. However, the inhibition remained unaffected in the presence of 3-hydroxybenzylhydrazine, an aromatic amino acid decarboxylase inhibitor. Furthermore, bath application of either phentolamine, an alpha-adrenoceptor antagonist, or bicuculline, a GABA(A) receptor antagonist, antagonized the inhibitory effects of L-DOPA on population spikes. In addition, bicuculline (1 microM) antagonized the inhibition of population spike induced by 6-fluoronorepinephrine (10 microM), an alpha-adrenoceptor agonist, while phentolamine (10 microM) did not affect the muscimol (1 microM)-induced inhibition. These results suggested that L-DOPA itself acted on L-DOPA recognition sites to release noradrenaline, and that the latter facilitates gamma-aminobutyric acid (GABA) release via alpha-adrenoceptors located on the GABA-containing cells and/or their nerve terminals, thereby inhibiting the population spikes in the hippocampal CA1 field.

Endogenously released DOPA is a causal factor for glutamate release and resultant delayed neuronal cell death by transient ischemia in rat striata.

Glutamate is implicated in neuronal cell death. Exogenously applied DOPA by itself releases neuronal glutamate and causes neuronal cell death in in vitro striatal systems. Herein, we attempt to clarify whether endogenous DOPA is released by 10 min transient ischemia due to four-vessel occlusion during rat striatal microdialysis and, further, whether DOPA, when released, functions to cause glutamate release and resultant delayed neuronal cell death. Ischemia increased extracellular DOPA, dopamine, and glutamate, and elicited neuronal cell death 96 h after ischemic insult. Inhibition of striatal L-aromatic amino acid decarboxylase 10 min before ischemia increased markedly basal DOPA, tripled glutamate release with a tendency of decrease in dopamine release by ischemia, and exaggerated neuronal cell death. Intrastriatal perfusion of 10-30 nM DOPA cyclohexyl ester, a competitive DOPA antagonist, 10 min before ischemia, concentration-dependently decreased glutamate release without modification of dopamine release by ischemia. At 100 nM, the antagonist elicited a slight ceiling effect on decreases in glutamate release by ischemia and protected neurons from cell death. Glutamate was released concentration-dependently by intrastriatal perfusion of 0.3-1 mM DOPA and stereoselectively by 0.6 mM DOPA. The antagonist elicited no hypothermia during and after ischemia. Endogenously released DOPA is an upstream causal factor for glutamate release and resultant delayed neuronal cell death by brain ischemia in rat striata. DOPA antagonist has a neuroprotective action.

DOPA cyclohexyl ester, a competitive DOPA antagonist, protects glutamate release and resultant delayed neuron death by transient ischemia in hippocampus CA1 of conscious rats.

In rat striata, DOPA released is a causal factor for glutamate release and resultant delayed neuron death by four-vessel occlusion. Nanomolar DOPA cyclohexyl ester (CHE), a potent and relatively stable competitive DOPA antagonist, protects these events. We tried to clarify whether DOPA CHE protects these events in hippocampal CA1 pyramidal cell layers most vulnerable against ischemia. Five to 10 min ischemia caused slight to mild glutamate release in 10 min samples during microdialysis and mild to severe neuron death 96 h after reperfusion. DOPA and dopamine were under assay limit in this design, but were basally detected by 20 min sampling and released by 20 min ischemia. In 10 min samples, intrahippocampal perfusion of 100 nM DOPA CHE 10 min before ischemia for 70 min did not inhibit glutamate release by 10 min ischemia, while it abolished glutamate release and protected delayed neuron death by 5 min ischemia. DOPA CHE is neuroprotective under a mild ischemic condition in rat hippocampus CA1.

L-DOPA cyclohexyl ester is a novel potent and relatively stable competitive antagonist against L-DOPA among several L-DOPA ester compounds.

We explored L-DOPA esters with chemically bulky structures to find a potent stable competitive antagonist against L-DOPA, compared to DOPA methyl ester (DOPA ME). In anesthetized rats, DOPA cyclohexyl ester (DOPA CHE), DOPA cyclopentyl ester (DOPA CPE) and DOPA cyclopentyldimethyl ester (DOPA CPDME) at 1 microgram microinjected into depressor sites of the nucleus tractus solitarii elicited or tended to elicit more marked antagonism against depressor responses to 60 ng L-DOPA, compared to DOPA ME. At 100 ng, DOPA CHE elicited the most potent antagonism. At 1 microgram, duration of the antagonistic activity of DOPA CHE was approximately three times longer than that of DOPA ME. During microdialysis of the nucleus accumbens, conversion from DOPA CHE at 1 microM perfused via probes to extracellular L-DOPA was the lowest among these compounds and less than one half of that from DOPA ME. Binding studies showed that the recognition site for L-DOPA differs from ionotropic glutamatergic, dopaminergic D1 and D2 receptors. We recently found that L-DOPA evoked by transient ischemia may act as a DOPA CHE-sensitive causal factor for glutamate release and resultant neuronal cell death. DOPA CHE is the most potent, relatively stable competitive antagonist against L-DOPA and is a useful mother compound to develop neuroprotective drugs.

Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA.

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.

Successful management of severe tricuspid regurgitation associated with hydrops fetalis in a case of dysplastic tricuspid valve.

We report a case with hydrops fetalis and severe tricuspid regurgitation due to dysplastic tricuspid valve, diagnosed in utero and followed after birth. The patient was successfully managed on the basis of useful echocardiographic informations.

Role of cdk5 and tau phosphorylation in heterotrimeric G protein-mediated retinal growth cone collapse.

During axonal growth, repulsive guidance cues cause growth cone collapse and retraction. In the chick embryo, membranes from the posterior part of the optic tectum containing ephrins are original collapsing factors for axons growing from the temporal retina. We investigated signal transduction pathways in retinal axons underlying this membrane-evoked collapse. Perturbation experiments using pertussis toxin (PTX) showed that membrane-induced collapse is mediated via G(o/i) proteins, as is the case for semaphorin/collapsin-1-induced collapse. Studies with Indo-1 revealed that growth cone collapse by direct activation of G(o/i) proteins with mastoparan did not cause elevation of the intracellular Ca(2+) level, and thus this signal transduction pathway is Ca(2+) independent. Application of the protein phosphatase inhibitor okadaic acid alone induced growth cone collapse in retinal culture, suggesting signals involving protein dephosphorylation. In addition, pretreatment of retinal axons with olomoucine, a specific inhibitor of cdk5 (tau kinase II), prevented mastoparan-evoked collapse. Olomoucine also blocks caudal tectal membrane-mediated collapse. These results suggest that rearrangement of the cytoskeleton is mediated by tau phosphorylation. Immunostaining visualized complementary distributions of tau phospho- and dephosphoisoforms within the growth cone, which also supports the involvement of tau. Taking these findings together, we conclude that cdk5 and tau phosphorylation probably lie downstream of growth cone collapse signaling mediated by PTX-sensitive G proteins.

An L-dopaergic relay from the posterior hypothalamic nucleus to the rostral ventrolateral medulla and its cardiovascular function in anesthetized rats.

We have proposed that L-3,4-dihydroxyphenylalanine (L-DOPA) is a neurotransmitter in the central nervous system [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. Herein, we attempt to clarify whether lesions in the posterior hypothalamic nucleus decrease the tissue content of L-DOPA in the rostral ventrolateral medulla. We also attempt to clarify whether or not endogenous L-DOPA is evoked by electrical stimulation of the posterior hypothalamic nucleus. It is possible that evoked L-DOPA functions as a transmitter candidate to activate pressor sites of the rostral ventrolateral medulla in anesthetized rats. Electrolytic lesions were made in the bilateral posterior hypothalamic nucleus by a monopolar direct current of 2 mA for 10 s, 10 days before measurements. The effect of the lesions was to selectively decrease the tissue content of L-DOPA by one-half in the right rostral ventrolateral medulla. Decreases in the amounts of dopamine, noradrenaline or adrenaline were not observed. Decreases were also not evident in the right caudal ventrolateral medulla. During microdialysis of the right rostral ventrolateral medulla, extracellular basal levels of L-DOPA and three types of catecholamine were consistently detectable by high-performance liquid chromatography with electrochemical detection. Tetrodotoxin (1 microM) perfused into the right rostral ventrolateral medulla gradually decreased basal levels of L-DOPA by 25%; it decreased basal levels of noradrenaline and adrenaline by 25-30% and dopamine levels by 40%. Intensive electrical stimulation of the ipsilateral posterior hypothalamic nucleus (50 Hz, 0.3 mA, 0.1 ms duration, twice for 5 min at an interval of 5 min) selectively caused the release of L-DOPA in a repetitive and constant manner. The stimulation was accompanied by hypertension and tachycardia. However, catecholamines were not released. Tetrodotoxin suppressed the release of L-DOPA, but partially inhibited hypertension with only a slight inhibition of tachycardia evoked by stimulation of the posterior hypothalamic nucleus. L-DOPA methyl ester, a competitive L-DOPA antagonist, was bilaterally microinjected into pressor sites of the rostral ventrolateral medulla at 1.5 microg x 2 and 3 microg x 2. The antagonist dose-dependently and consistently antagonized pressor and tachycardiac responses to mild transient stimulation of the unilateral posterior hypothalamic nucleus (33 Hz, 0.2 mA, 0.1 ms duration, for 10 s). In addition, the antagonist alone (3 microg x 2) elicited hypotension and bradycardia. These results show that an L-DOPAergic relay may project from the posterior hypothalamic nucleus directly to pressor sites of the rostral ventrolateral medulla and/or indirectly to certain neurons near pressor sites in microcircuits of the same region. When released, L-DOPA appears to function tonically to activate pressor sites; it also appears to be involved in the maintenance and regulation of blood pressure and heart rate.

L-dopaergic components in the caudal ventrolateral medulla in baroreflex neurotransmission.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is probably a transmitter of the primary baroreceptor afferents terminating in the nucleus tractus solitarii; L-DOPA functions tonically to activate depressor sites of the caudal ventrolateral medulla, which receives input from the nucleus tractus solitarii [Misu Y. et al. (1996) Prog. Neurobiol. 49, 415-454]. We have attempted to clarify whether or not L-DOPAergic components within the caudal ventrolateral medulla are involved in baroreflex neurotransmission in anesthetized rats. Electrolytic lesions of the right nucleus tractus solitarii (1 mA d.c. for 10 s, 10 days before measurement) selectively decreased by 45% the tissue content of L-DOPA in the dissected ipsilateral caudal ventrolateral medulla. Electrolytic lesions did not decrease dopamine, norepinephrine and epinephrine levels. During microdialysis of the right caudal ventrolateral medulla, extracellular levels of L-DOPA, norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid were consistently detectable using high-performance liquid chromatography with electrochemical detection. However, extracellular dopamine levels were lower than the assay limit. Baroreceptor activation by i.v. phenylephrine selectively evoked L-DOPA without increasing the levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. This L-DOPA release was suppressed by acute lesion in the ipsilateral nucleus tractus solitarii. Intermittent stimulation of the right aortic depressor nerve (20 Hz, 3 V, 0.3 ms duration, for 30 min) repetitively and constantly caused L-DOPA release, hypotension and bradycardia, without increases in levels of norepinephrine, epinephrine and 3,4-dihydroxyphenylacetic acid. Local inhibition of L-DOPA synthesis with alpha-methyl-p-tyrosine (30 microM) infused into the ipsilateral caudal ventrolateral medulla gradually decreased basal levels of L-DOPA and 3,4-dihydroxyphenylacetic acid without decreasing norepinephrine and epinephrine. The inhibition of L-DOPA synthesis interrupted L-DOPA release and decreased by 65% depressor responses elicited by aortic nerve stimulation; however, it produced no effect on bradycardic responses. CoCl2 (119 ng), a mainly presynaptic inhibitory transmission marker, and L-DOPA methyl ester (1 microg), a competitive L-DOPA antagonist, when microinjected into depressor sites of the right caudal ventrolateral medulla, reduced by 60% depressor responses to transient ipsilateral stimulation of the aortic nerve (20 Hz, 3 V, 0.1 ms duration, for 10 s). No changes in bradycardic responses were observed. There may exist an L-DOPAergic relay from the nucleus tractus solitarii to the caudal ventrolateral medulla. L-DOPAergic components in the caudal ventrolateral medulla are involved in baroreflex neurotransmission via a baroreceptor-aortic depressor nerve-nucleus tractus solitarii-caudal ventrolateral medulla relay in the rat.

Growth cone neuropilin-1 mediates collapsin-1/Sema III facilitation of antero- and retrograde axoplasmic transport.

Collapsin-1/Sema III, a member of the semaphorin family, has been implicated in axonal pathfinding as a repulsive guidance cue. Cellular and molecular mechanisms by which collapsin-1 exerts its action are not fully understood. Collapsin-1 induces growth cone collapse via a pathway which may include neuropilin-1, a cellsurface collapsin-1 binding protein, as well as intracellular CRMP-62 and heterotrimeric G proteins. We previously identified a second action of collapsin-1, the facilitation of antero- and retrograde axoplasmic transport. This response occurs via a mechanism distinct from that causing growth cone collapse. To investigate the possible involvement of neuropilin-1 in the action of collapsin-1 on axoplasmic transport, we produced a soluble neuropilin-1 (sNP-1) lacking the transmembrane and intracellular region. sNP-1 progressively displaced the dose-response curve for collapsin-1 to induce growth cone collapse to higher concentrations. sNP-1 also inhibited collapsin-1-induced augmentation of both antero- and retrograde axoplasmic transport. Furthermore, an anti-neuropilin-1 antibody blocked the collapsin-induced axoplasmic transport. These results together indicate that neuropilin-1 mediates collapsin-1 action on axoplasmic transport. To visualize collapsin-1 binding to endogenous neuropilin-1, we used a truncated collapsin-1-alkaline phosphatase fusion protein (CAP-4). CAP-4 stains the growth cone, neurite, and cell body. However, local application of collapsin-1 to growth cone but to neither neurite nor cell body promotes axoplasmic transport. Thus, growth cone NP-1 mediates the facilitatory action of collapsin-1 on antero- and retrograde axoplasmic transport.

Early postmenopausal bone loss is prevented by estrogen and partially by 1alpha-OH-vitamin D3: therapeutic effects of estrogen and/or 1alpha-OH-vitamin D3.

A total of 79 Japanese women who were within 5 years of menopause were randomly assigned 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] 1.0 microg/day, conjugated estrogens 0.625 mg/day, a combination of both, or control (no treatment). Lumbar spine and proximal femur bone mineral density (BMD) and biochemical indices were monitored over 2 years. In the 1alpha(OH)D3-treated group, there was a nonsignificant decrease in lumbar spine BMD compared with controls, and no significant loss in the femoral neck compared with controls. In the estrogen-treated group, there was a nonsignificant increase in spine BMD (+2.17% in the first year and +1.71% in the second year), and no loss in femoral neck BMD. The combination of conjugated estrogens +1alpha(OH)D3 was more effective in increasing BMD in the spine (+3. 68% in the first year and +3.63% in the second year) and femur (+2. 56% in the first year and +4.44% in the second year) BMD. There was a significant difference in lumbar spine BMD in both the first and second years between the combination-treated group and the 1alpha(OH)D3-treated and control groups (P < 0.01). Serum osteocalcin (OC) significantly decreased in the combination-treated group (-23.8% in the first year) and the estrogen-treated group (-37. 6% and -41.2% at 6 and 18 months, respectively), and serum alkaline phosphatase (Alp) decreased significantly in the first year in the combination-treated (-31.5%), estrogen-treated (-27.3%), and 1alpha(OH)D3-treated (-7.9%) groups, whereas serum OC increased (+45. 4% in the first year) in women without treatment. The results of this study indicate that early postmenopausal bone loss in the femoral neck is prevented by conjugated estrogens, 1alpha(OH)D3, or both, whereas bone loss in the spine is not prevented by 1alpha(OH)D3. Estrogen proves effective in preventing early postmenopausal bone loss by markedly inhibiting bone turnover. Moreover, a synergistic bone-sparing effect can be expected when estrogen is administered concomitantly with 1alpha(OH)D3 rather than when used alone.

Some interactions of L-DOPA and its related compounds with glutamate receptors.

L-DOPA is probably a transmitter and/or modulator in the central nervous system (1). L-DOPA methyl ester (DOPA ME) is a competitive L-DOPA antagonist. However, it remains to be clarified whether there exist L-DOPAergic receptors. In Xenopus laevis oocytes injected with rat brain poly(A)+ RNA, L-DOPA induced small inward currents with ED50 of 2.2 mM at a holding potential of -70 mV. The currents were abolished by kynurenic acid or CNQX. Similar L-DOPA-currents were seen in oocytes co-injected with AMPA receptors, GluRs1,2,3 and 4. In brain membrane preparations, L-DOPA inhibited specific binding of [3H]-AMPA with IC50 of 260 microM. This inhibition was not modified by 200 microM ascorbic acid, an antioxidant. L-DOPA did not inhibit binding of [3H]-ligands of MK-801, kainate, DCKA and CGP39653. DOPA ME and L-DOPA cyclohexyl ester, a novel, potent and competitive antagonist (2), inhibited specific binding of [3H]-MK-801 with respective IC50 of 1 and 0.68 mM, but elicited no effect on that of the other [3H]-ligands. With low affinities, L-DOPA acts on AMPA receptors, while competitive antagonists act on NMDA ion channel domain. L-DOPAergic agonist and antagonist may not interact on ionotropic glutamate receptors. DOPA ME-sensitive L-DOPA recognition sites (1) seem to differ from glutamate receptors.

GABA may function tonically via GABA(A) receptors to inhibit hypotension and bradycardia by L-DOPA microinjected into depressor sites of the nucleus tractus solitarii in anesthetized rats.

We have proposed that DOPA is a transmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. GABA may inhibit DOPAergic transmission. Drugs were microinjected into depressor sites of the NTS in anesthetized rats. DOPA (10-60 ng) elicited dose-dependent depressor responses. GABA (3-300 ng) elicited dose-dependent pressor responses. Nipecotic acid (100 ng) elicited pressor responses. Bicuculline (10 ng) elicited depressor responses. Responses to DOPA (30 ng) were inhibited by pretreatment with GABA and nipecotic acid, but potentiated by bicuculline, when vascular responses to pretreated drugs returned to basal levels. DOPA ME, a competitive DOPA antagonist, did not displace specific [3H]GABA binding. Prior DOPA ME (1 microg) inhibited by one-half pressor responses to 300 ng GABA. GABA seems to inhibit tonically via GABA(A) receptors depressor responses to DOPA and to elicit pressor responses partially by inhibition of tonic function of endogenous DOPA to activate depressor sites in the NTS. These findings further support the above proposal.

The evidence for tonic GABAergic regulation of basal L-DOPA release via activation of inhibitory GABA(A) receptors in the nucleus tractus solitarii of anesthetized rats.

We have proposed that DOPA is a neurotransmitter of the primary baroreceptor afferents terminating in the rat nucleus tractus solitarii (NTS). GABA is a putative inhibitory neuromodulator for baroreflex inputs in the NTS. Thus, GABA may inhibit DOPAergic transmission in the NTS. We tried to clarify whether basal DOPA release is inhibited by muscimol, a GABA(A) agonist, and facilitated by bicuculline, a GABA(A) antagonist, during microdialysis of the NTS in anesthetized rats. DOPA release was consistently detectable. Muscimol 10-100 microM perfused via probes gradually inhibited concentration-dependently DOPA release. Peak 30% inhibition occurred 2 h after perfusion. Muscimol (30 microM)-induced inhibition was antagonized by non-effective 10 microM bicuculline. Bicuculline (30 microM) elicited peak 30% facilitation of DOPA release 2 h after perfusion. Endogenous GABA seems to regulate tonically basal DOPA release via activation of inhibitory GABA(A) receptors in the rat NTS. These findings further support the above proposal.

[A DOPA antagonist, DOPA cyclohexyl ester inhibits transient brain ischemia-induced release of glutamate and delayed neuronal cell death in striatal and hippocampal region of in vivo rats].

We have previously obtained evidence that DOPA is probably involved in an upstream process of mechanisms for in vivo neuronal cell death in striatum. We attempted to clarify whether or not this is also the case in hippocampal region of conscious Wistar rats. Four vessels were occluded for 5 min during microdialysis of hippocampus. DOPA, dopamine and glutamate (Glu) in perfusates collected every 10 min were measured by HPLC-ECD and spectrophotometer. Delayed neuronal cell death in hippocampus was evaluated 96 hr after ischemia. Five-min transient brain ischemia induced Glu release, with the peak being 2.5-fold of a basal release at the fraction immediately after ischemia. The release of DOPA and dopamine was not consistently detectable, but an increase was sometimes observed during and after ischemia. Delayed neuronal cell death was slight to moderate with 5-min ischemia. Intrastriatal perfusion of DOPA cyclohexyl ester (DOPA CHE) at 100 nM, a novel stable potent competitive DOPA antagonist, almost completely inhibited the ischemia-induced glutamate release, and protected hippocampal neurons from delayed cell death. Endogenously released DOPA itself seems to act on its recognition site and to behave as a causal and/or deteriorating factor on glutamate release and resultant delayed neuronal cell death by transient ischemia in rats.

Melittin, a metabostatic peptide inhibiting Gs activity.

Some basic amphiphilic peptides are known to directly stimulate heterotrimeric GTP-binding proteins (G proteins). Mastoparan and melittin are known to stimulate Gi activities. Here, we found melittin inhibited guanine nucleotide-dependent adenylyl cyclase activity in synaptic membranes of the rat cerebral cortex. However, in insect cell membranes overexpressing specific heterotrimeric G proteins using baculovirus expression system, melittin showed unique effects different from those by mastoparan on G protein activities. This peptide markedly stimulated Gi1 and G11 activities, whereas it did inhibit Gs activities. Kinetic studies revealed that the inhibition of Gs activity by melittin is attributed to the inhibition of GDP release in exchange for added guanine nucleotides (or the association of guanine nucleotides). Thus, melittin may be the first metabostatic peptide inhibiting G protein (Gs) activity, and both mechanisms through the stimulation of Gi and inhibition of Gs might be involved in the melittin-induced inhibition of adenylyl cyclase.