Takashi Kumazawa

Optical recordings of taste responses from fungiform papillae of mouse in situ.

1 Single taste buds in mouse fungiform papillae consist of ≈50 elongated cells (TBCs), where fewer than three TBCs have synaptic contacts with taste nerves. We investigated whether the non‐innervated TBCs were chemosensitive using a voltage‐sensitive dye, tetramethylrhodamine methyl ester (TMRM), under in situ optical recording conditions. 2 Prior to the optical recordings, we investigated the magnitude and polarity of receptor potentials under in situ whole‐cell clamp conditions. In response to 10 mM HCl, several TBCs were depolarized by ≈25 mV and elicited action potentials, while other TBCs were hyperpolarized by ≈12 mV. The TBCs eliciting hyperpolarizing receptor potentials also generated action potentials on electrical stimulation. 3 A mixture of 100 mM NaCl, 10 mM HCl and 500 mM sucrose depolarized six TBCs and hyperpolarized another three TBCs out of 13 identified TBCs in a taste bud viewed by optical section. In an optical section of another taste bud, 1 M NaCl depolarized five TBCs and hyperpolarized another two TBCs out of 11 identified TBCs. 4 The number of chemosensitive TBCs was much larger than the number of innervated TBCs in a taste bud, indicating the existence of chemosensitivity in non‐innervated TBCs. There was a tendency for TBCs eliciting the same polarity of receptor potential to occur together in taste buds. We discuss the role of non‐innervated TBCs in taste information processing.

Neuroblastoma cell as a model for a taste cell: mechanism of depolarization in response to various bitter substances.

The mouse neuroblastoma cell (N-18 clone) was used as a model for a taste cell. The N-18 cell was found to be reversibly depolarized by various bitter substances. The minimum concentrations of bitter substances which induced depolarization (threshold concentration) varied greatly with the type of the substance. There was a good correlation between the threshold concentrations for various bitter substances in the N-18 cell and those in the human taste responses. The input membrane resistance was little changed during the depolarization induced by the bitter substances. Replacement of Na+ and Cl- with impermeable ions had practically no effect on the depolarization response to the bitter substances and reduction of calcium concentration from 1.8 to 0.2 mM led to a slight increase in the responses. It was suggested that the depolarization of the N-18 cell by bitter substances mainly stems from changes in the phase-boundary potential at the outer surface of the cell.

Receptor mechanisms of bitter substances.

The receptor mechanism of bitter substances was discussed from the following points of views. (a) Both electrostatic and hydrophobic interactions of bitter substances with taste receptor membranes contribute to reception of bitter substances having a positive charge. (b) In the frog, the responses to bitter substances are easily adapted. The presence of Ca ion in the medium prolongs the responses. (c) Bitter substances elicit electrical responses in nongustatory cells such as neuroblastoma cells and olfactory cells, suggesting that bitter substances induce the response by nonreceptor-mediated mechanism. (d) There is also a possibility that receptors for some bitter substances are G-protein coupled. We cloned G-protein coupled receptors from bovine taste tissues. (e) A specific inhibitor of bitter taste has been desired in pharmaceutical and food sciences, but it has not been available. We found that a lipoprotein made of phosphatidic acid and beta-lactoglobulin selectively inhibits the responses to bitter substances in the frog and humans. Binding of the lipoprotein to the receptor sites for bitter substances leads to suppression of the response.

Drug Delivery System Using Nano-Magnetic Fluid

Nano-size magnetic magnetide is considered important for various medical applications. Dynamic motion of the magnetic particle is investigated in two essential models from a theoretical point of view. One is a drag model of the magnetic particle in an artery. The second is a pull model towards the surface of artery. Threshold conditions of external variables are obtained by dimensional analysis. On the basis of all these results, it is concluded that the movement of magnetic fluids can be controlled by external magnetic fields in blood vessels.

Cell-type-dependent action potentials and voltage-gated currents in mouse fungiform taste buds.

Taste receptor cells fire action potentials in response to taste substances to trigger non‐exocytotic neurotransmitter release in type II cells and exocytotic release in type III cells. We investigated possible differences between these action potentials fired by mouse taste receptor cells using in situ whole‐cell recordings, and subsequently we identified their cell types immunologically with cell‐type markers, an IP3 receptor (IP3R3) for type II cells and a SNARE protein (SNAP‐25) for type III cells. Cells not immunoreactive to these antibodies were examined as non‐IRCs. Here, we show that type II cells and type III cells fire action potentials using different ionic mechanisms, and that non‐IRCs also fire action potentials with either of the ionic mechanisms. The width of action potentials was significantly narrower and their afterhyperpolarization was deeper in type III cells than in type II cells. Na+ current density was similar in type II cells and type III cells, but it was significantly smaller in non‐IRCs than in the others. Although outwardly rectifying current density was similar between type II cells and type III cells, tetraethylammonium (TEA) preferentially suppressed the density in type III cells and the majority of non‐IRCs. Our mathematical model revealed that the shape of action potentials depended on the ratio of TEA‐sensitive current density and TEA‐insensitive current one. The action potentials of type II cells and type III cells under physiological conditions are discussed.

Time-dependent expression of hypertonic effects on bullfrog taste nerve responses to salts and bitter substances.

We previously showed that the hypertonicity of taste stimulating solutions modified tonic responses, the quasi-steady state component following the transient (phasic) component of each integrated taste nerve response. Here we show that the hypertonicity opens tight junctions surrounding taste receptor cells in a time-dependent manner and modifies whole taste nerve responses in bullfrogs. We increased the tonicity of stimulating solutions with non-taste substances such as urea or ethylene glycol. The hypertonicity enhanced phasic responses to NaCl>0.2M, and suppressed those to NaCl<0.1M, 1mM CaCl2, and 1mM bitter substances (quinine, denatonium and strychnine). The hypertonicity also enhanced the phasic responses to a variety of 0.5M salts such as LiCl and KCl. The enhancing effect was increased by increasing the difference between the ionic mobilities of the cations and anions in the salt. A preincubation time >20s in the presence of 1M non-taste substances was needed to elicit both the enhancing and suppressing effects. Lucifer Yellow CH, a paracellular marker dye, diffused into bullfrog taste receptor organs in 30s in the presence of hypertonicity. These results agreed with our proposed mechanism of hypertonic effects that considered the diffusion potential across open tight junctions.

Reconstituted Ion Channels of Frog Fungiform Papilla Cell Membrane

Abstract We identified a Cl− channel, two K+ channels and a cAMP-gated channel which were isolated from bullfrog fungiform papilla cell membranes and incorporated into phospholipid bilayers using the tip-dip method. The 156 pS channels were inhibited by 100 μM 4, 4′-diisothiocyanostilbene-2, 2′-disulfonic acid (DIDS) and displayed the reversal potential identical to the equilibrium potential of Cl−, it was identified as a Cl− channel. Two types of K+ channel had unitary conductances of 79 and 43 pS, which may correspond to those of Ca2+-activated and cAMP-blockable K+ channels observed in isolated intact frog taste cell membranes, respectively. These results suggest that the tip-dip method is useful for stable investigation of the properties of ion channels already identified in the taste cell. Furthermore, the 23 pS channels were newly found and were activated directly by internal cAMP as cyclic nucleotide-gated (CNG) nonselective cation channels established in olfactory receptor cells. Thus, our results suggest the possibility that besides Cl− and K+ channels, the cAMP-gated channels contribute to taste transduction.

Evaluation of the Influence on the Living Body as a New Transdermal Therapeutic System Magnetic Nanoparticles

As a delivery carrier, the nanoparticles are broadly applied in various fields for excellent infiltration characteristics. In recent years, there has been new rearch was investigated in the medical area, but the research of influence on a living body is seldom studied. In this study, the influence on the living body as new transdermal carrier system using magnetic nanoparticles was investigated. The application of magnetic nanoparticles coated medication on the mice model investigated that the effect of treatment on the atopic dermatitis. The skin tissues were stained, and the section of skin tissues was observed by the optical Microscope. In vivo efficacy studies in mice model revealed highly efficient improvement of AD-like skin lesions. Furthermore the magnetic nanoparticles are useful as novel delivery drug carrier via human dermal papilla cell experiment. The results indicated that the magnetic nanoparticles are useful as new the transdermal carrier , and it proved that it is a highly efficient system useful as novel medicine delivery system.

Study on the Application of Magnetic Nanoparticles as Drug Delivery Particles on the Atopic Dermatitis

It is well known that the advances in the biomedicinal applications of magnetite nanoparticles were studied recently. As a drug delivery tools, it only affects on the target region of illness. We investigated the application of magnetic nanoparticles as drug delivery tools by the mice model of atopic dermatitis. The results involve that after magnetic nanoparticles are applied to the skin, the condition of atopic dermatitis becomes much better than the former one of atopic mice model. It only affects on the illness region and healthy regions almost have no influence. It is suitable for the medical treatment technique usage on the atopic dermatitis.

Bio-medicine Coating on Surface of Magnetic Nanoparticles and Its Safety Evaluation

The composite magnetic nanoparticles of coated SiO nano film with about 8 nm size can be dispersed in various liquid media, widely known as magnetic fluids or ferrofluids with both magnetic and liquid properties. In this paper, a bio-medicine coating technology on surface of magnetic nanoparticles and the optimum fabrication condition and the magnetism of composed bio-nanoparticles are investigated. Through observation of micro-structure of the bio-nanopaticles on coating surface, and evaluation of magnetic property and safety to apply to biomaterials, we know the bio-medicine coated on surface of magnetic nanoparticles is suitable of bio-solutions into surface of biomaterials.