Toshihiro Dohi

Phorbol esters alter functions of the expressed dopamine transporter.

Recent elucidation of the amino acid sequences of the neurotransmitter transporters reveals several consensus sequences for phosphorylation by kinases including protein kinase C. Protein kinase C activation did modulate the function of the rat dopamine transporter expressed in COS cells. Cell treatment with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) reduced the affinity of binding of the radiolabeled cocaine analog [3H](-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (WIN 35,428) without affecting its Bmax. The uptake of [3H]dopamine was reduced by treatment with PMA in a staurosporine-sensitive manner. Kinetic analysis revealed that the inhibitory effect of PMA on [3H]dopamine uptake was due to reduced uptake velocity and a small reduction of affinity for Na+, without changed affinity for dopamine. 1-Oleoyl-2-acetyl-sn-glycerol (OAG) mimicked these actions of PMA. These results demonstrate that activation of protein kinase C alters dopamine transporter functions in both ligand recognition and substrate translocation. These phosphorylation phenomena in vitro suggest the possibility that phosphorylation could modulate the activity of this important dopaminergic synaptic regulator under physiological conditions.

Stimulation of Cyclic ADP-ribose Synthesis by Acetylcholine and Its Role in Catecholamine Release in Bovine Adrenal Chromaffin Cells

Cyclic ADP-ribose (cADPR) is suggested to be a novel messenger of ryanodine receptors in various cellular systems. However, the regulation of its synthesis in response to cell stimulation and its functional roles are still unclear. We examined the physiological relevance of cADPR to the messenger role in stimulation-secretion coupling in cultured bovine adrenal chromaffin cells. Sensitization of Ca2+-induced Ca2+release (CICR) and stimulation of catecholamine release by cADPR in permeabilized cells were demonstrated along with the contribution of CICR to intracellular Ca2+ dynamics and secretory response during stimulation of intact chromaffin cells. ADP-ribosyl cyclase was activated in the membrane preparation from chromaffin cells stimulated with acetylcholine (ACh), excess KCl depolarization, and 8-bromo-cyclic-AMP. ACh-induced activation of ADP-ribosyl cyclase was dependent on the influx of Ca2+ into cells and on the activation of cyclic AMP-dependent protein kinase. These and previous findings that ACh activates adenylate cyclase by Ca2+ influx in chromaffin cells suggested that ACh induces activation of ADP-ribosyl cyclase through Ca2+ influx and cyclic AMP-mediated pathways. These results provide evidence that the synthesis of cADPR is regulated by cell stimulation, and the cADPR/CICR pathway forms a significant signal transduction for secretion.

Spinal Antiallodynia Action of Glycine Transporter Inhibitors in Neuropathic Pain Models in Mice

Neuropathic pain is refractory against conventional analgesics, and thus novel medicaments are desired for the treatment. Glycinergic neurons are localized in specific brain regions, including the spinal cord, where they play an important role in the regulation of pain signal transduction. Glycine transporter (GlyT)1, present in glial cells, and GlyT2, located in neurons, play roles in modulating glycinergic neurotransmission by clearing synaptically released glycine or supplying glycine to the neurons and thus could modify pain signal transmission in the spinal cord. In this study, we demonstrated that i.v. or intrathecal administration of GlyT1 inhibitors, cis-N-methyl-N-(6-methoxy-1-phenyl-1,2,3,4-tetrahydronaphthalen-2-yl methyl)amino methylcarboxylic acid (ORG25935) or sarcosine, and GlyT2 inhibitors, 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)-methyl]benzamide (ORG25543) and (O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-L-serine) (ALX1393), or knockdown of spinal GlyTs by small interfering RNA of GlyTs mRNA produced a profound antiallodynia effect in a partial peripheral nerve ligation model and other neuropathic pain models in mice. The antiallodynia effect is mediated through spinal glycine receptor α3. These results established GlyTs as the target molecules for the development of medicaments for neuropathic pain. However, these manipulations to stimulate glycinergic neuronal activity were without effect during the 4 days after nerve injury, whereas manipulations to inhibit glycinergic neuronal activity protected against the development of allodynia in this phase. The results implied that the timing of medication with their inhibitors should be considered, because glycinergic control of pain was reversed in the critical period of 3 to 4 days after surgery. This may also provide important information for understanding the underlying molecular mechanisms of the development of neuropathic pain.

Glycine transporter inhibitors as a novel drug discovery strategy for neuropathic pain

Injury to peripheral or spinal nerves following either trauma or disease has several consequences including the development of neuropathic pain. This syndrome is often refractory against conventional analgesics; and thus, novel medicaments are desired for its treatment. Recent studies have emphasized that dysfunction of inhibitory neuronal regulation of pain signal transduction may be relevant to the development of neuropathic pain. Glycinergic neurons are localized in specific brain regions and the spinal cord, where they play an important role in the prevention of pathological pain symptoms. Thus, an enhancement of glycinergic control in the spinal cord is a promising strategy for pain relief from neuropathic pain. Glycine transporter (GlyT) 1 and GlyT2, which are located in glial cells and neurons, respectively play important roles by clearing synaptically released glycine or supplying glycine to glycinergic neurons to regulate glycinergic neurotransmission. Thus, an inhibition of GlyTs could be used to modify pain signal transmission in the spinal cord. Recently developed specific inhibitors of GlyTs have made this possibility a reality. Both GlyT1 and GlyT2 inhibitors produced potential anti-nociceptive effect in various neuropathic pain models, chronic and acute inflammatory models in animals. Their anti-allodynia effects are mediated by the inhibition of GlyTs following activation of spinal glycine receptor alpha3. These results established GlyTs as target molecules for medicaments for neuropathic pain. Moreover, the phase-dependent anti-allodynia effects of GlyT inhibitors have provided important information on effective therapeutic strategies and also understanding the underlying molecular mechanisms of the development of neuropathic pain.

Evidence for GABAA receptor agonistic properties of ketamine: Convulsive and anesthetic behavioral models in mice

We examined the potentiation by ketamine of the &ggr;-aminobutyric acidA (GABAA) receptor function using convulsive and anesthetic behavioral models in adult male ddY mice. General anesthetic potencies were evaluated by a rating scale, which provided the data for anesthetic scores, loss of righting reflex, duration, and recovery time. All drugs were administered intraperitoneally. Small subanesthetic doses of ketamine did inhibit tonic seizures induced by a large dose of the GABAA receptor antagonist bicuculline (8 mg/kg). The 50% effective dose value was 15 (95% confidence limits 10–22) mg/kg. Even large anesthetic doses (100–150 mg/kg) did not suppress clonic seizures in 50% of the animals. The GABAA receptor agonist, muscimol (0.32–1.12 mg/kg), potentiated ketamine-induced anesthesia in a dose-dependent fashion (P < 0.05). Similarly, the benzodiazepine receptor agonist, diazepam (1–3 mg/kg), augmented ketamine anesthesia in a dose-dependent manner (P < 0.05). Bicuculline (2–5 mg/kg) dose-dependently antagonized ketamine-induced anesthesia (P < 0.05). Neither the benzodiazepine receptor antagonist, flumazenil (2–20 mg/kg), nor the GABA synthesis inhibitor, l-allylglycine (200 mg/kg), affected the anesthetic action of ketamine. These results suggest that ketamine has GABAA receptor agonistic properties and that ketamine-induced anesthesia is mediated, at least in part, by GABAA receptors. Implications We examined the potentiation by ketamine of the &ggr;-aminobutyric acidA receptor function using convulsive and anesthetic behavioral models in mice. Subanesthetic doses of ketamine-inhibited tonic convulsions induced by the &ggr;-aminobutyric acidA receptor antagonist bicuculline. The &ggr;-aminobutyric acidA receptor agonist, muscimol, potentiated ketamine-induced anesthesia. Bicuculline antagonized ketamine anesthesia, but the benzodiazepine receptor antagonist, flumazenil, and the &ggr;-aminobutyric acid synthesis inhibitor, l-allyglycine, did not. The effects of ketamine on the &ggr;-aminobutyric acidA receptors appear to correlate with its anesthetic actions.

Enhancement of Stimulation‐Evoked Catecholamine Release from Cultured Bovine Adrenal Chromaffin Cells by Forskolin

Abstract: Acetylcholine (ACh) increased cyclic AMP levels in cultured bovine chromaffin cells with a peak effect at 1 min after the addition. Pretreatment with forskolin (0.3 μM) enhanced the ACh‐evoked cyclic AMP increase. The catecholamine (CA) release induced by ACh was enhanced by forskolin, but forskolin alone did not enhance the CA release. The effect of forskolin increased dose‐dependently up to 1 μM, but decreased at higher concentrations. Dibutyryl cyclic AMP (DBcAMP) also enhanced ACh‐evoked CA release, but the effect was less potent than that of forskolin. Forskolin enhanced both [3H]norepinephrine ([3H]NE) and endogenous CA release evoked by 30 mMK+ from cells that were preloaded with [3H]NE. The effects of forskolin were substantial when CA release was evoked with low concentrations of ACh or excess K+, but decreased with higher concentrations of the stimulants. Forskolin also enhanced the CA release induced by ionomycin and veratrine, or by caffeine in Ca2+‐free medium. The potentiation by forskolin of the ACh‐evoked CA release was manifest in low Ca2+ concentrations in the medium, but decreased when Ca2+ concentration was increased. These results suggest that cyclic AMP may play a role in the modulation of CA release from chromaffin cells.

Propofol-induced Anesthesia in Mice Is Mediated by γ-aminobutyric Acid-a and Excitatory Amino Acid Receptors

To elucidate the role of &ggr;-aminobutyric acid (GABA)A receptor complex and excitatory amino acid receptors (N-methyl-d-aspartate [NMDA] and non-NMDA receptors) in propofol-induced anesthesia, we examined behaviorally the effects of GABAergic and glutamatergic drugs on propofol anesthesia in mice. All drugs were administered intraperitoneally. General anesthetic potencies were evaluated using a righting reflex assay. The GABAA receptor agonist muscimol potentiated propofol (140 mg/kg; 50% effective dose for loss of righting reflex) induced anesthesia. Similarly, the benzodiazepine receptor agonist diazepam and the NMDA receptor antagonist MK-801 augmented propofol anesthesia, but the non-NMDA receptor antagonist CNQX did not. In contrast, the GABAA receptor antagonist bicuculline antagonized propofol (200 mg/kg; 95% effective dose for loss of righting reflex) induced anesthesia. However, neither the benzodiazepine receptor antagonist flumazenil, the GABA synthesis inhibitor l-allylglycine, nor the NMDA receptor agonist NMDA reversed propofol anesthesia. Conversely, the non-NMDA receptor agonist kainate enhanced propofol anesthesia. These results suggest that propofol-induced anesthesia is mediated, at least in part, by both GABAA and excitatory amino acid receptors.

Molecular cloning and characterization of rat trp homologues from brain

Identification of trp (transient receptor potential) gene from Drosophila photoreceptor and subsequent molecular cloning of the human cDNA homologues suggest its participation in capacitative calcium entry (CCE) or so called store-operated Ca2+ channel (SOC). We identified five different trp-related amplifications of reverse-transcription-polymerase chain reaction (RT-PCR) from rat brain; these corresponded to mouse trp homologues, mtrp1,3,4,5,6 and were distributed in various tissues with multiple expression levels. Two cDNAs, homologous to Drosophila trp from rat brain, designated rtrp3 and rtrp6, were isolated and characterized. By RT-PCR analysis, mRNAs of rtrp3 and rtrp6 were found to be expressed differently in brain and other tissues. In situ hybridization analysis revealed that rtrp6 mRNA was preferentially expressed in hippocampal dentate gyrus and cortical layers II and III. Expression of rat TRP3 and TRP6 in COS cells revealed an increase in CCE, as compared to that in the mock-transfected COS cells of the control. Isolation of cDNAs of rat trp gene family provides a useful model for studying mechanism of CCE.

Dominant Negative Isoform of Rat Norepinephrine Transporter Produced by Alternative RNA Splicing

We have cloned from rat brain a family of alternatively spliced cDNAs from a single gene, which encodes a norepinephrine transporter (NET) having variations at the 3′-region including both coding and noncoding regions. This produces two transporter isoforms, rNETa and rNETb, which differ at their COOH termini. The rNETa isoform reveals a COOH terminus homologous to human NET and transports norepinephrine. In contrast, rNETb revealed no detectable transport function but reduced functional expression of rNETa when both isoforms were expressed in the same cell. Thus, rNETb potentially functions as a dominant negative inhibitor of rNETa activity. Co-expression of rNETb with a γ-aminobutyric acid transporter (rGAT1), a serotonin transporter (rSERT), and a dopamine transporter (rDAT) reduced their transport activity. No reduction was found with the glutamate/aspartate transporter (rGLAST). Alternative RNA splicing of NET suggests a novel mechanism for the regulation of synaptic transmission.

Tyrosine-533 of rat dopamine transporter : involvement in interactions with 1-methyl-4-phenylpyridinium and cocaine

To improve our understanding of structure-function relationships for neurotransmitter transporters, we performed site-directed mutagenesis of the rat dopamine transporter (DAT) and assessed the functions of the mutants in transiently-expressing COS cells. Tyrosine-533 of rat DAT lies in the 11th transmembrane region, where the corresponding amino acid of human DAT is phenylalanine. Alanine substitution of tyrosine-533 (Y533A) conferred an increased affinity for 1-methyl-4-phenylpyridinium (MPP+). Phenylalanine substitution of tyrosine-533 (Y533F) increased the velocity of MPP+ uptake but decreased DATs affinity for MPP+. Cocaines potency in inhibiting dopamine uptake was unchanged with Y533A, but increased with Y533F. Differences in the uptake kinetics and inhibitory potency of cocaine between rat and human DATs were similar to the differences observed between the wild-type and Y533F mutants DATs. Tyrosine-533 may be important for the DAT function and for species differences in transporter functions, including differential sensitivities to cocaine and 1-methyl-1,2,3,6-tetrahydropyridine (MPTP) in humans and rats.