Keiko Hoshi

Injectable porous hydroxyapatite microparticles as a new carrier for protein and lipophilic drugs.

Hydroxyapatite (Ca10 (PO4)6(OH)2) is a biodegradable material that forms a major component of bones and teeth. We prepared injectable spherical porous hydroxyapatite microparticles (SP-HAp) as a drug carrier by the spray-drying method. We then examined the usefulness of SP-HAp as a carrier for drugs such as interferon alpha (IFNalpha), testosterone enanthate (TE), and cyclosporin A (CyA). SP-HAp had an average diameter of 5 mum and a porosity of approximately 58%. It could be injected subcutaneously through a 27-gauge needle. SP-HAp was observed to be biodegradable. The speed of degradation of SP-HAp could be regulated by altering the calcination temperature. IFNalpha was adsorbed well to SP-HAp particles, but INFalpha was released faster from the particles, than the particles could degrade in both in vitro and in vivo experiments. Addition of human serum albumin and zinc (reinforcement) to IFNalpha-adsorbed SP-HAp caused marked prolongation of release in vivo. The in vivo release of testosterone enanthate and CyA from SP-HAp preparation, which was easily injectable, was similarly prolonged to that from the oil preparation. In conclusion, the SP-HAp seems to be useful as a biodegradable and subcutaneously injectable drug carrier. It is suggested that the reinforcement of the SP-HAp is very effective on the sustained release of drugs.

IgG anti-GalNAc-GD1a antibody inhibits the voltage-dependent calcium channel currents in PC12 pheochromocytoma cells.

We investigated the effects of IgG anti-GalNAc-GD1a antibodies, produced by immunizing rabbits with GalNAc-GD1a, on the voltage-dependent calcium channel (VDCCs) currents in nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells. VDCCs currents in NGF-differentiated PC12 cells were recorded using the whole-cell patch-clamp technique. Immunized rabbit serum that had a high titer of anti-GalNAc-GD1a antibodies inhibited the VDCCs currents in the NGF-differentiated PC12 cells (36.0+/-9.6% reduction). The inhibitory effect of this serum was reversed to some degree within 3-4 min by washing with bath solution. Similarly, application of purified IgG from rabbit serum immunized with GalNAc-GD1a significantly inhibited the VDCCs currents in PC12 cells (30.6+/-2.5% reduction), and this inhibition was recovered by washing with bath solution. Furthermore, the inhibitory effect was also observed in the GalNAc-GD1a affinity column binding fraction (reduction of 31.1+/-9.85%), while the GalNAc-GD1a affinity column pass-through fraction attenuated the inhibitory effect on VDCCs currents. Normal rabbit serum and normal rabbit IgG did not affect the VDCCs currents in the PC12 cells. In an immunocytochemical study using fluorescence staining, the PC12 cells were stained using GalNAc-GD1a binding fraction. These results indicate that anti-GalNAc-GD1a antibodies inhibit the VDCCs currents in NGF-differentiated PC12 cells.

Cav2.1 Voltage-dependent Ca2+ Channel Current is Inhibited by Serum from Select Patients with Guillain-Barré Syndrome

To investigate the pathophysiological mechanisms of immune-mediated peripheral neuropathies, we studied the effects of sera from patients with Guillain-Barré syndrome (GBS) on the Cav2.1 voltage-dependent calcium channel (VDCC) current in Purkinje cells. Using the whole-cell recording technique, Cav2.1 VDCC current was measured in cerebellar Purkinje cells in the presence of serum from GBS patients with acute motor axonal neuropathy (AMAN) or acute inflammatory demyelinating polyneuropathy (AIDP). The AMAN patient sera significantly inhibited the Cav2.1 VDCC current compared with healthy volunteer sera, and this inhibition was fully reversible by washing out the AMAN serum. Similarly, IgG purified from AMAN sera also inhibited the Cav2.1 VDCC current. However, the activation and inactivation kinetics of the Cav2.1 VDCC currents were not affected by serum from an AMAN patient. Moreover, the VDCC current of Purkinje cells was also inhibited by IgG anti-GM1 monoclonal antibody (anti-GM1 mAb). In an immunocytochemical study using double fluorescence staining, Purkinje cells were stained by monoclonal IgG anti-GM1 mAb. In contrast, AIDP patient and healthy volunteer sera did not affect the Cav2.1 VDCC current. These results suggest that in some case of GBS, particularly of AMAN patients with IgG anti-GM1 mAb, muscle weakness may be induced by dysfunction of Cav2.1 VDCC functioning at the motor nerve terminals.

Expression and localization of Kv1 potassium channels in rat dorsal and ventral spinal roots

We investigated the expression and localization of Kv1 channels in dorsal spinal roots (DRs) and ventral spinal roots (VRs) in rats. Among Kv1.1-1.6 tested by RT-PCR, mRNAs of Kv1.1, 1.2, and 1.5 were moderately expressed, those of Kv1.3 and Kv1.6 were weakly expressed, and that of Kv1.4 was hardly expressed at all in both DRs and VRs, whereas all six mRNAs were detected in spinal cord. Western blotting revealed that the major immunoreactive proteins were Kv1.1 and Kv1.2 in both DRs and VRs. Quantitative analysis indicated that levels of Kv1.1 and Kv1.2 protein were significantly higher in DRs than VRs. Immunohistochemical examination showed that Kv1.1 and Kv1.2 were colocalized in juxtaparanodal regions of axons in both DRs and VRs. Finally, immunoprecipitation experiments revealed that Kv1.1 and Kv1.2 were coassembled. These findings indicate that Kv1 subtypes in DRs and VRs are somewhat different from those in spinal cord, and that the numbers of Kv1.1 and Kv1.2 channels are higher in DRs than VRs.

Preventative effects of 1,3-dimethyl- and 1,3-dimethyl-N-propargyl-1,2,3,4-tetrahydroisoquinoline on MPTP-induced Parkinson's disease-like symptoms in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is well known as an exogenous dopaminergic neurotoxin that induces Parkinsons disease-like symptoms. In addition, 1,2,3,4-tetrahydroisoquinoline (TIQ) derivatives have been investigated as endogenous MPTP mimetic compounds that structurally resemble selegiline, a commercially available drug for treating Parkinsons disease. In the present study, we examined the ability of 1,3-dimethyl-TIQ (1,3-diMeTIQ) and 1,3-dimethyl-N-propargyl-TIQ (1,3-diMe-N-proTIQ) to prevent MPTP-induced Parkinsons disease-like symptoms in mice and to prevent 1-methyl-4-phenylpyridinium ion (MPP+, an active metabolite of MPTP)-induced cytotoxicity in vitro, including its structural stereoselectivity. Repeated administration of MPTP induced bradykinesia, a symptom of behavioral abnormality; this was prevented by both 1,3-diMeTIQ and 1,3-diMe-N-proTIQ pretreatments. Pretreatment with 1,3-diMeTIQ did not prevent the MPTP-induced decrease in dopamine content in the striatum or the decrease in the number of tyrosine hydroxylase-positive cells in the substantia nigra. On the other hand, 1,3-diMe-N-proTIQ prevented these Parkinsons disease-like symptoms; in particular, the trans-isomer of this agent showed potent protective effects. However, the ability of the trans-1,3-diMe-N-proTIQ isomer to prevent MPP+-induced PC12 cell death was weaker than that of its cis-isomer. Thus, stereoisomers of 1,3-diMe-N-proTIQ exhibit different effects; cis-1,3-diMe-N-proTIQ inhibits MPP+-induced cytotoxicity while trans-1,3-diMe-N-proTIQ exhibits neuroprotective effects primarily through MPTP-related biological events in mice. These results also indicate the possibility of utilizing, at least in part, the stereoselective efficacy of 1,3-diMe-N-proTIQ against MPTP and/or MPP+-induced adverse states.

The importance of brain PGE2 inhibition versus paw PGE2 inhibition as a mechanism for the separation of analgesic and antipyretic effects of lornoxicam in rats with paw inflammation.

Objectives Lornoxicam is a non‐selective cyclooxygenase inhibitor that exhibits strong analgesic and anti‐inflammatory effects but a weak antipyretic effect in rat models. Our aim was to investigate the mechanism of separation of potencies or analgesic and antipyretic effecls of lornoxicam in relatioin to its effect on prostaglandin E2 (PGE2) production in the inflammatory paw and the brain.

Involvement of the protein kinase Cγ isoform in development of tolerance to nitrous oxide–induced antinociception in mice

Prolonged exposure to nitrous oxide (N2O) results in development of acute tolerance to its antinociceptive effect. Cross-tolerance to N2O-induced antinociception is also observed in morphine-tolerant animals. Despite increasing evidence of tolerance development to N2O-induced antinociception, the details of the mechanisms that underlie this tolerance remain unknown. The present study was conducted to investigate the involvement of brain protein kinase C (PKC) isoform in these two types of tolerance to N2O-induced antinociception in mice. Prolonged exposure (41 min in total, including 30 min pre-exposure and 11 min of antinociceptive testing) to 70% N2O produced a reduction in N2O-induced antinociception, indicating development of acute tolerance. The prolonged exposure to 70% N2O caused an activation of PKCgamma isoform in the brain, but not the PKCepsilon isoform. Pretreatment with a PKCgamma-antisense oligonucleotide but not the corresponding mismatch oligonucleotide (i.c.v.) prevented the development of acute tolerance to N2O-induced antinociception. Chronic morphine treatment (10 mg/kg, s.c., b.i.d. for 5 days) resulted in development of tolerance to morphine-induced antinociception and cross-tolerance to N2O-induced antinociception. The development of tolerance to morphine and cross-tolerance to N2O were both inhibited by pretreatment with PKC inhibitor, chelerythrine (1 nmol, i.c.v.). Morphine-tolerant mice showed an activation of PKC within the brain, which was suppressed by pretreatment with chelerythrine (1 nmol, i.c.v.). Thus, activation of brain PKC, in particular, the PKCgamma isoform, appears to play an important role in the development of both acute tolerance and cross-tolerance to N2O-induced antinociception in mice.

IgM anti-GQ1b monoclonal antibody inhibits voltage-dependent calcium current in cerebellar granule cells.

Miller-Fisher syndrome (MFS), which is known to be associated with anti-GQ1b antibodies and to cause ataxia, is a variant of an acute inflammatory neuropathy. However, the pathogenic role of anti-GQ1b antibodies remains unclear. In this study, we investigated the effects of mouse IgM anti-GQ1b monoclonal antibody (IgM anti-GQ1b mAb) on the spontaneous muscle action potential of a rat spinal cord-muscle co-culture system and on the voltage-dependent calcium channel (VDCC) current in cerebellar granule cells and Purkinje cells using the whole-cell patch clamp technique. The frequency of spontaneous muscle action potential of the innervated muscle cells was transiently increased by IgM anti-GQ1b mAb and then was blocked completely, which was the same finding as reported previously. Moreover, the cerebellar granule cell VDCC current was decreased by 30.76+/-7.60% by 5 microg/mL IgM anti-GQ1b mAb, whereas IgM anti-GQ1b mAb did not affect the VDCC current in cerebellar Purkinje cells. In immunocytochemistry, IgM anti-GQ1b mAb stained the whole cell surface of cerebellar granule cells, but not that of Purkinje cells. Therefore, the clinical symptoms of Miller-Fisher syndrome, such as cerebellar-like ataxia, may be explained by the inhibitory effects of anti-GQ1b antibodies on VDCC current in cerebellar granule cells.

Ca2+ Channel Currents Inhibited by Serum from Select Patients with Guillain-Barré Syndrome

We performed an electrophysiological study demonstrating inhibition of spontaneous muscle action potentials within a coculture of rat muscle and spinal cord by exposure to serum, as well as purified IgG, from patients with the acute motor axonal neuropathy (AMAN) variant of Guillain-Barré syndrome (GBS). However, exposure to serum from two patients with the acute inflammatory demyelinating polyneuropathy (AIDP) form of GBS had no effect. Using a whole-cell recording technique, we then investigated the effects of serum and purified IgG from patients with GBS on voltage-dependent calcium channel (VDCC) currents in nerve growth factor-differentiated PC12 cells. Serum from patients with GBS (AMAN) inhibited VDCC currents in PC12 cells, which was fully reversible by washing with the bath solution. Similarly, purified IgG from the serum of two patients with GBS (AMAN) also inhibited VDCC currents in PC12 cells. In contrast, sera from patients with AIDP and healthy volunteers did not affect VDCC currents in PC12 cells. These results suggest that muscle weakness in some patients with GBS might be induced by inhibition of Ca2+ channel currents within motor nerve terminals.

Glycosylation and Cell Surface Expression of Kv1.2 Potassium Channel are Regulated by Determinants in the Pore Region

Voltage-gated K+ channels contain six membrane spanning segments and a pore-forming domain. We used site-directed mutation to examine the role of specific amino acids in the extracellular region of the pore in Kv1.2. When expressed in CHO cells, a K+ current was not observed for mutants S356A, S360A, T383A and T384A. However, coexpression of the Kvβ2 subunit and the S360A mutant resulted in a robust peak current. Immunocytochemistry for Kv1.2 showed staining throughout the cytoplasm in cells coexpressing the β2 and S360A, whereas only the perinuclear region was stained in cells expressing the S360A mutant. Western blotting revealed that the major immunoreactive protein in wild-type- and mutant-expressing cells is 60-kDa, but 87-kDa bands were also detected in cells expressing wild-type Kv1.2 and cells coexpressing β2and S360A. These results suggest that amino acids in the pore region help regulate ion permeability or cellular trafficking by affecting glycosylation of Kv1.2.