Keisuke Migita

Gain and Loss of Channel Function by Alanine Substitutions in the Transmembrane Segments of the Rat ATP-Gated P2X2 Receptor

ATP opens ionotropic P2X channels through a process that is poorly understood. We made an array of mutant rat P2X2 channels containing unique alanine substitutions in the transmembrane segments with the goal of identifying possible secondary structure and mapping gating domains in the pore. Alteration of channel function was measured as a change in ATP potency, 2′-3′-O-(4-benzoylbenzoyl)ATP (BzATP) efficacy, and deactivation kinetics. Four mutants (V45A, Y47A, V51A, and D349A) failed to respond to ATP. Seven (H33A, Q37A, I40A, L41A, Y43A, F44A, and I50A) of 22 mutations in the first transmembrane segment (TM1) produced channels with altered potencies, and two mutants (Y43A and F44A) were active in the absence of agonist. The pattern of hits was consistent with a helical secondary structure. In contrast, nine (I328A, P329A, N333A, L338A, T339A, S340A, G342A, G344A, and S345A) of 24 mutations in the second transmembrane segment (TM2) resulted in a change in potency, but no regular pattern of impact was apparent. Many of the same mutations that altered ATP potency also changed the relative efficacy of the partial agonist BzATP. Together, these data suggest that both TM1 and TM2 participate in the conformational changes that occur during receptor activation and help to define domains involved in conformational switching within or near the pore.

Morphologic changes of dendritic spines of striatal neurons in the levodopa-induced dyskinesia model.

Maladaptive plasticity at corticostriatal synapses plays an important role in the development of levodopa‐induced dyskinesia. Recently, it has been shown that synaptic plasticity is closely linked to morphologic changes of dendritic spines. To evaluate morphologic changes of dendritic spines of two types of striatal medium spiny neurons, which project to the internal segment of globus pallidus or the external segment of globus pallidus, in the levodopa‐induced dyskinesia model, we used 6‐hydroxydopamine‐lesioned rats chronically treated with levodopa. Dendritic spines were decreased and became enlarged in the direct pathway neurons of the model of levodopa‐induced dyskinesia. The same levodopa treatment to normal rats, in which no dyskinesia was observed, also induced enlargement of dendritic spines, but not a decrease in density of spines in the direct pathway neurons. These results suggest that a loss and enlargement of dendritic spines in the direct pathway neurons plays important roles in the development of levodopa‐induced dyskinesia.

On the role of the first transmembrane domain in cation permeability and flux of the ATP-gated P2X2 receptor.

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.

Modulation of P2X receptors in dorsal root ganglion neurons of streptozotocin-induced diabetic neuropathy

Painful diabetic neuropathy causes hyperalgesia and does not respond to commonly used analgesics such as non-steroidal anti-inflammatory drugs or opioids at doses below those producing disruptive side effects. In the present study, we examined the effect of P2X receptor antagonists, which are known to modulate the pain pathway, on mechanical hyperalgesia in streptozotocin (STZ)-induced diabetic mice. The paw withdrawal frequency measured by von Frey filaments, began to significantly increase 5 days after STZ injection and was maintained for more than 14 days. Intrathecal administration of P2X receptor antagonists (PPADS and TNP-ATP) inhibited the mechanical allodynia in diabetic mice. The levels of P2X(2) and P2X(3) receptors mRNA were significantly increased in diabetic mice at 14 days after the intravenous injection of STZ. These results suggest that the upregulation of P2X(2), P2X(3) and/or P2X(2/3) receptor in DRG neurons is associated with mechanical allodynia in STZ-induced diabetic mice.

Morphological and electrophysiological changes in intratelencephalic-type pyramidal neurons in the motor cortex of a rat model of levodopa-induced dyskinesia

Levodopa-induced dyskinesia (LID) is a major complication of long-term dopamine replacement therapy for Parkinsons disease, and becomes increasingly problematic in the advanced stage of the disease. Although the cause of LID still remains unclear, there is accumulating evidence from animal experiments that it results from maladaptive plasticity, resulting in supersensitive excitatory transmission at corticostriatal synapses. Recent work using transcranial magnetic stimulation suggests that the motor cortex displays the same supersensitivity in Parkinsons disease patients with LID. To date, the cellular mechanisms underlying the abnormal cortical plasticity have not been examined. The morphology of the dendritic spines has a strong relationship to synaptic plasticity. Therefore, we explored the spine morphology of pyramidal neurons in the motor cortex in a rat model of LID. We used control rats, 6-hydroxydopamine-lesioned rats (a model of Parkinsons disease), 6-hydroxydopamine-lesioned rats chronically treated with levodopa (a model of LID), and control rats chronically treated with levodopa. Because the direct pathway of the basal ganglia plays a central role in the development of LID, we quantified the density and size of dendritic spines in intratelencephalic (IT)-type pyramidal neurons in M1 cortex that project to the striatal medium spiny neurons in the direct pathway. The spine density was not different among the four groups. In contrast, spine size became enlarged in the Parkinsons disease and LID rat models. The enlargement was significantly greater in the LID model than in the Parkinsons disease model. This enlargement of the spines suggests that IT-type pyramidal neurons acquire supersensitivity to excitatory stimuli. To confirm this possibility, we monitored miniature excitatory postsynaptic currents (mEPSCs) in the IT-type pyramidal neurons in M1 cortex using whole-cell patch clamp. The amplitude of the mEPSCs was significantly increased in the LID model compared with the control. This indicates that the IT-type pyramidal neurons become hyperexcited in the LID model, paralleling the enlargement of spines. Thus, spine enlargement and the resultant hyperexcitability of IT-type pyramidal neurons in M1 cortex might contribute to the abnormal cortical neuronal plasticity in LID.

Anti-emetic effects of a novel NK-1 receptor antagonist HSP-117 in ferrets

We have developed a non-peptide compound, HSP-117, antagonist of the tachykinin NK-1 receptor. Binding of 3H-substance P (SP) to the membranes of IM-9 cells was inhibited by the antagonists HSP-117 and CP-99,994, the inhibitory activity of HSP-117 being about 50-fold that of CP-99,994. The SP-induced firing responses of single neuron activity in slices of the nucleus tractus solitarius of ferrets were inhibited by 10 microM HSP-117. Intracerebroventricular injection of HSP-117 significantly inhibited retching and vomiting induced by copper sulphate and morphine and the inhibitory effect of HSP-117 on emesis was greater than that of CP-99,994. These results indicate that (1) HSP-117 is a potent anti-emetic agent, blocking NK-1 receptors in the nucleus tractus solitarius and (2) NK-1 receptors in the nucleus tractus solitarius play an important role in emesis induced by broad-spectrum emetic stimuli.

Modulation of the arterial baroreceptor reflex by the vasopressin receptor in the area postrema of the hypertensive rats

The role of arginine8-vasopressin (AVP) in regulation of the baroreceptor reflex in the area postrema was examined in anesthetized hypertensive rats. The sensitivity of the baroreceptor reflex in a one-kidney one clip (1K1C) hypertensive rats was increased in only the initial stage (2 weeks), in association with increase in blood pressure, and then returned to the normal level. This increase in the sensitivity of the baroreceptor reflex in the initial stage was reversed by microinjection of a V1 or V2 antagonist (1 microg) into the area postrema. AVP V2 receptor mRNA was expressed temporarily in the area postrema in this period. These results suggest that vasopressin receptors in the area postrema is important in regulating the sensitivity of the baroreceptor reflex.

Effects of arginine-vasopressin on neuronal interaction from the area postrema to the nucleus tractus solitarii in rat brain slices.

The effects of vasopressin (AVP) on area postrema (AP) neurons and the neuronal connection between the AP and nucleus tractus solitarii (NTS) were investigated electrophysiologically in slices preparation of the medulla oblongata of rats. In the AP, 27.9% of 129 neurons were excited by AVP and 20.5% were inhibited. The excitation was blocked by a V1 receptor antagonist. Synaptic transmission of the AP to the NTS was mainly mediated by non-NMDA receptors. Local application of AVP to the AP activated the NTS neurons. This activation was blocked by an NMDA antagonist. These results suggest that the excitation originating in the AP is conveyed to the NTS via non-NMDA receptors and modified by NMDA receptor activation secondly. These processes may be important in regulation of the arterial baroreceptor reflex.

Inhibition of glucose transporter 1 induces apoptosis and sensitizes multiple myeloma cells to conventional chemotherapeutic agents

Despite the recent development of anti-myeloma drugs, the prognosis of high-risk multiple myeloma remains poor. Therefore, new effective treatment strategies for this disease are needed. It has been reported that high intensity of 18-fluorodeoxyglucose positron emission tomography is high-risk factor in myeloma, suggesting that glucose uptake can be therapeutic target in high-risk myeloma. In this study, we addressed the utility of glucose transporter 1 (GLUT1) as a therapeutic target for myeloma with increased glucose uptake. We found myeloma cell lines with elevated glucose uptake activity via GLUT1 up-regulation. STF-31, a selective GLUT1 inhibitor, completely suppressed the glucose uptake activity and induced apoptosis in GLUT1 expressing myeloma cells. On the other hand, this agent little shows the cytotoxicity in normal peripheral blood mononuclear cells. Moreover, STF-31 synergistically enhanced the cell death induced by melphalan, doxorubicin, and bortezomib. GLUT1 may be promising therapeutic target in myeloma with elevated glucose uptake.

Drebrin immunoreactivity in the striatum of a rat model of levodopa-induced dyskinesia.

Levodopa‐induced dyskinesia has been suggested to result from maladaptive plasticity at corticostriatal synapses. Synaptic plasticity is based upon morphologic changes of dendritic spines. To elucidate whether the morphologic changes of spines occur in the striatum of rat models of levodopa‐induced dyskinesia, we examined immunoreactivity of drebrin, an actin‐binding protein localized in dendritic spines of excitatory synapses, using 6‐hydroxydopamine‐lesioned rats repeatedly treated with levodopa. The cross‐sectional area of drebrin‐immunoreactive organelles, putative spines, in the dopamine‐denervated striatum of the levodopa‐induced dyskinesia model was greater than that of the Parkinsons disease model. Immunoelectron microscopic examinations confirmed that drebrin‐immunoreactive spines became enlarged in the dopamine‐denervated striatum of the levodopa‐induced dyskinesia model, but not in the Parkinsons disease model. These results suggest that the development of levodopa‐induced dyskinesia is associated with enlargement of dendritic spines at corticostriatal excitatory synapses.