Takenori Miyamoto

IP3- and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish.

SummaryOlfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP3 or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP3 were blocked by ruthenium red, a blocker of an IP3-gated Ca2+-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP3 in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP3 or cAMP when the buffering capacity for Ca2+ of the pipette solution was increased, when extracellular Ca2+ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Cai] that opened Ca2+-activated K+ channels. The results suggest that different conductances modulated by either IP3 or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca2+-activated K+ channels contribute to the termination of the IP3 and cAMP responses.

Ionic basis of receptor potential of frog taste cells induced by acid stimuli.

1. The ionic mechanism underlying the receptor potential in frog taste cells induced by acid stimuli was studied with single microelectrodes by replacing superficial and interstitial fluids of the tongue with modified saline solutions. 2. The removal of Na+, Ca2+ and Cl‐ from the normal interstitial fluid did not affect the receptor potential induced by acid stimuli. Interstitial 100 mM‐K+ saline did not affect the acid response. 3. The receptor potential was reduced greatly when Ca2+ was removed from the superficial saline, but was increased when the Ca2+ concentration was elevated. The removal of superficial Cl‐ did not affect the receptor potential. The receptor potential elicited by superficial Ca2+‐free saline was partly due to Na+. Li+, K+, NH4+ or choline + substituted for Na+ in producing the receptor potential. The amiloride‐sensitive Na+ channel on the receptor membrane did not contribute to the receptor potential. With pure water adaptation of the tongue surface, the mean magnitude of the acid response was 35% of the control. 4. The receptor potential was unaffected by superficial tetrodotoxin (TTX) but was blocked by superficial Ca2+ antagonists such as Co2+ and Cd2+. Sr2+ substituted for Ca2+ in generating the receptor potential. 5. The receptor potentials observed under various concentrations of superficial Ca2+ became smaller when Na+ was present in the superficial fluid, indicating a competition between Ca2+ and Na+. 6. It is concluded that a large portion of the receptor potential induced by acid stimuli is produced by cations passing through a tastant‐gated Ca2+ channel on the taste receptor membrane. Both divalent (Ca2+, Sr2+) and monovalent (Na+, Li+, K+, NH4+, choline+) cations can pass through the Ca2+ channel. The other mechanism responsible for the remaining part of the receptor potential is discussed.

Acid and salt responses in mouse taste cells

Acid and salt responses of taste cells induced by natural stimulation have not been investigated with exception of early studies with conventional microelectrode method, due to the toxicity of high concentration of salt or low pH of acid stimuli applied to isolated taste cells. This indicates that the application of rapid and localized stimulation to the apical membrane of taste cells is necessary for recording of natural responses to salt or acid stimuli using patch clamp technique. Recently we have developed a procedure to accomplish the quasi-natural condition including rapid, localized stimuli to the apical receptive membrane and the maintenance of taste bud polarity. In this review, we present our recent results obtained under quasi-natural condition using patch clamp techniques, comparing with the previously proposed hypothesis. One of our major finding is the fact that the acid-induced responses of taste cells in the mouse fungiform papillae are never suppressed by amiloride but an apical proton-gated conductance and a basolateral Cl(-) conductance possibly contribute to sour transduction. On the other hand, salt-induced responses are suppressed by amiloride, although the salt-induced responses recorded from a single cell involve both amiloride-sensitive and -insensitive components. Furthermore, the amiloride-insensitive component of salt responses possibly consists of multiple subcomponents including an apical sodium-gated nonselective cation conductance and a basolateral Cl(-) conductance. Recent reports also support the hypothesis that both acid and salt responses require specific receptor mechanisms of inorganic cations such as H(+) and Na(+) at the apical receptive membrane.

Cationic and anionic channels of apical receptive membrane in a taste cell contribute to generation of salt-induced receptor potential

Abstract 1. 1. After ionic composition of superficial fluid (ISF) and interstitial fluid (ISF) of the frog Rana catesbeiana) tongue had mostly been changed with a low Na+ saline solution, the relations between membrane potentials and receptor potentials in a frog taste cell evoked by various concentrations of NaCl and various types of salts were analyzed to examine permeability of the taste receptive membrane to cations and anions. 2. 2. The mean reversal potentials for depolarizing potentials of a taste cell in response to 0.05 M, 0.2 M and 0.5 M Nad were -40.0, 6.4 and 28.8 mV, respectively. 3. 3. When adding an anion channel blocker, SITS, to a NaCl solution the reversal potential for receptor potential with NaCl plus SITS became about twice as large than with NaCl alone. 4. 4. Reversal potentials for 0.2 M NaCl, LiCl, KCl and NaSCN were 6.4, 25.4, −1.0 and −7.8 mV, respectively, indicating that permeability of the apical taste receptive membrane to cations of Cl− salts is arranged in the order of Li+ > Na+ > K+ and that the permeability to anions of Na+ salts is arranged as SCN− > Cl− 5. 5. It is concluded that in the case of NaCl stimulation, Na+ and Cl− of NaCl stimulus permeate NaCl-gated cationic and anionic channels at the apical taste receptive membrane in generating receptor potentials.

Ionic mechanism of generation of receptor potential in response to quinine in frog taste cell

The ionic mechanism of generation of the receptor potential in a frog taste cell elicited by quinine-HCl (Q-HCl) was studied with an intracellular recording technique by replacing the superficial and interstitial fluids of the tongue with various saline solutions. The taste cells whose receptor membranes were adapted to normal saline and deionized water generated depolarizing receptor potentials at Q-HCl concentrations higher than 2 and 0.01 mM, respectively. The input resistance of taste cell during Q-HCl stimulation scarcely changed. The receptor potential did not change even when the membrane potential level was broadly changed. The magnitude of the receptor potential was increased by reducing the concentration of superficial Cl- on the taste receptor membrane, but was independent to the concentration of superficial Na+. Injection of Cl- into a taste cell increased the receptor potential to 170%. The magnitude of receptor potential was decreased to 20-30% by removing interstitial Na+ or Cl- or both surrounding the basolateral membrane of taste cell. Furosemide (1 mM) added to the interstitial fluid decreased the receptor potential to 15%, while interstitial ouabain (0.1 mM) and superficial SITS (0.1 mM) did not influence it. From these results, we conclude: (1) an electroneutral Na+/Cl- cotransport occurs through the basolateral membrane of a taste cell in the resting state, so that Cl- accumulates inside the cell. (2) Q-HCl stimulation induces the active secretion of Cl- across the taste receptor membrane, resulting in a depolarizing receptor potential.

Membrane properties of isolated frog taste cells: three types of responsivity to electrical stimulation.

The whole cell clamp technique was applied to an isolated frog taste cell to examine the detailed membrane properties. The electrical responses could be classified into 3 types. The taste cells of the active type generated a large spike potential under current clamp and a large early inward current followed by a small outward current. The taste cells of the inactive type did not generate a spike but showed a small inward current followed by a large outward current. The distribution of each type was 15, 70 and 15%, respectively.

Potent excitatory effect of maitotoxin on Ca channels in the insect skeletal muscle

The effect of maitotoxin (MTX), the most potent marine toxin as yet known, was studied using the skeletal muscle of the larval meal worm,Tenebrio molitor. In normal saline,Tenebrio muscles responded with the spike to direct stimulation. In the saline containing tetraethylammonium (TEA) the all-or-none action potential which had characteristic plateau was elicited by membrane depolarization. When MTX (5×10−9 to 10−8 g/ml) in the TEA saline was added, the plateau of action potential was prolonged more than in the saline containing TEA alone. Furthermore, MTX lowered the threshold, so that action potentials were readily evoked in the saline containing MTX. In either case, effects, of MTX were antagonized by Co2+. These results suggest that MTX activates the voltage-dependent Ca2+ channels in the insect muscle.

Whole-cell recording from non-dissociated taste cells in mouse taste bud

A method for the whole-cell recording from non-dissociated taste cells within mouse taste bud is described. The lingual epithelial sheet containing the taste buds was peeled free from the tongue by injecting a proteolytic enzyme, elastase, under the lingual epithelium and by incubating it in normal Tyrode solution at 30 degrees C. The preparation consisting of a taste bud and a small piece of the lingual epithelium was obtained by further the incubation in divalent cation-free Tyrode solution. After holding the small piece of the epithelium by a holding pipette loaded with continuous negative pressure for keeping the orientation of the taste bud, whole-cell configuration was established in a non-dissociated taste cell within the taste bud with a patch pipette containing Lucifer Yellow. Taste stimuli or blockers were applied from the third pipette placed near the taste pore under the continuous flow of bathing solution. Under this condition, we could simultaneously accomplish patch-clamping, visualization of taste cell morphology, localized taste stimulation and maintenance of microenvironment around the taste organ. Rapid responses to a relatively high concentration of salt stimuli were also obtained.

DIFFERENTIAL DISTRIBUTION OF TWO CA2+-DEPENDENT AND -INDEPENDENT K+ CHANNELS THROUGHOUT RECEPTIVE AND BASOLATERAL MEMBRANES OF BULLFROG TASTE CELLS

We could identify two types of K+ channels, of 80 and 40 pS conductance, respectively, in the bullfrog taste cell membrane using excised and cell-attached configurations of the patch-clamp technique. The taste cell membrane could be divided into four membrane parts — receptive area, apical process, cell body and proximal process. The 80-pS K+ channels were dependent on voltage and Ca2+ and were located exclusively on the receptive membrane and the apical process membrane. The 40-pS K+ channels were independent of voltage and Ca2+. The open probability of 40-pS K+ channels was decreased by the simultaneous presence of cyclic adenosine monophosphate (cAMP) and adenosine triphosphate (ATP), and the suppressive effect was antagonized by protein kinase inhibitor (PKI). Although 40-pS K+ channels were found in a high density on the receptive and apical process membranes, the channels also were present in the other two parts of the taste cell membrane. These results suggest that the two different types of K+ channel in the bullfrog taste cells may play different roles in gustatory transduction.

Distributions of sensory spots in the hand and two-point discrimination thresholds in the hand, face and mouth in dental students

Densities of pressure, pain and temperature spots in the back of the hand in 551 students and two-point discrimination thresholds in the hand, the face and the mouth in 684 students were measured. The mean numbers of pressure, pain, warm and cold spots in the back of the hand were 24.7/cm2, 130.5/cm2, 3.4/cm2 and 9.1/cm2, respectively. The mean thresholds of two-point discrimination were 1.7 mm in the tip of the tongue, 2.4 mm in the upper lip, 5.5 mm in the lower jaw, 7.5 mm in the palm, 8.8 mm in the forehead, and 11.8 mm in the back of the hand. There were mostly no differences between males and females in the values of sensory spots and two-point discrimination thresholds.