Rie Fujiyama

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.

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.

Strain difference in amiloride-sensitivity of salt-induced responses in mouse non-dissociated taste cells.

The chorda tympani nerve responses to NaCl in a mouse strain, C57BL/6 are known to be much more sensitive than those in BALB/c. We compared the NaCl-induced responses obtained from taste cells of the fungiform papillae in these two strains of mice. Amiloride inhibited, in the same degree, the responses induced by a bath-application of normal extracellular solution (NES) containing 140 mM NaCl in either taste cells of C57BL/6 and BALB/c mice. In contrast, amiloride inhibited 62% of responses induced by an apically applied 0.5 M NaCl in the C57BL/6 strain, but only 33% of responses in the BALB/c strain. These results suggest that the difference in amiloride-sensitivity between taste cells in both strains mainly derives from the difference in density of functional amiloride sensitive Na+ channels at the apical receptive membrane but not at the basolateral membrane.

Effects of experimentally induced inflammation on temporomandibular joint nociceptors in rats

Response properties of nociceptors in the temporomandibular joint (TMJ) and surrounding area under experimental inflammation were investigated using an in vitro TMJ-nerve preparation in the rat. Nociceptive units (receptor and innervating nerve fiber) were classified into the following subtypes: Adelta-high-threshold mechanonociceptor (HTM), Adelta-polymodal nociceptor (POLY), C-HTM and C-POLY. In the inflamed joint, mechanical thresholds tended to be lower; however, the reaction to bradykinin was not identified as clearly as in control. Experimentally induced inflammation increased the proportion of heat-sensitive units and lowered heat threshold significantly. These results suggest that inflammation may sensitize nociceptors in the temporomandibular joint, and cause hyperalgesia and allodynia.

Non-selective cation channel in bullfrog taste cell membrane

USING single channel recordings of the patch clamp method, we found first that cation channels were present in the bullfrog taste cell membranes. These channels were widely distributed over the taste cell membrane. The conductance examined with inside-out patches was 30.7 ± 5.7 pS (mean ± S.D., n = 13). The permeability ratio of the cation channel to monovalent cations was PK: Pcs: PNa = 1.18: 1.00: 0.70, indicating that the channels are non-selective.

Inositol 1,4,5-trisphosphate activates non-selective cation conductance via intracellular Ca2+ increase in isolated frog taste cells

The effect of intracellular Ca2+ increase was analysed in isolated frog taste cells under the whole‐cell patch clamp. External application of a Ca2+‐ionophore, ionomycin (3 μm) induced the sustained inward current of −200 ± 17 pA (mean ± SE, n = 23) at – 50 mV in taste cells. The ionomycin‐induced response was observed in most of the cells exposed in the drug, but not when 10 mm BAPTA (1,2‐bis (o‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid) was included in the pipette (eight cells). Steady‐state I–V relationships of ionomycin‐induced currents were almost linear and reversed at – 8 ± 1 mV (n = 23). The simultaneous removal of Na+ and Ca2+ from the external solution eliminated the response completely (three cells). Intracellular dialysis with 1 mm Ca2+ or 50 μm inositol 1,4,5‐trisphosphate (IP3) in K+‐internal solution also induced an inward current in the taste cells. The Ca2+‐induced and IP3‐induced responses were observed in 82% and 36% of the cells dialysed with the drugs, respectively. The Ca2+‐induced and IP3‐induced currents were inhibited by external Cd2+ (1–2 mm). The reversal potentials of the inward currents were – 15 ± 3 mV (n = 9) in Ca2+ dialysis and – 11 ± 3 mV (n = 13) in IP3 dialysis. The half‐maximal Ca2+ concentration in the pipette to induce the inward current was ≈ 170 μm. The results suggest that IP3 can depolarize the taste cell with mediation by intracellular Ca2+.

Serotonin inhibits voltage-gated sodium current by cyclic adenosine monophosphate-dependent mechanism in bullfrog taste receptor cells

We have investigated the effect of 5-hydroxytryptamine (serotonin) (5-HT) on the membrane properties of bullfrog taste receptor cells (TRCs) using patch-clamp technique. External application of 5-HT reversibly suppressed the voltage-gated Na(+) current (I(Na)) in about half of the TRCs sampled. The magnitude of suppression of peak I(Na) was dependent on the holding potential of the cell. Forskolin and cyclic adenosine monophosphate (cAMP) mimicked the suppressive effect of 5-HT on I(Na), but an internal protein kinase A-inhibitor potentiated I(Na). These results suggest that 5-HT suppresses I(Na) of bullfrog TRCs via protein kinase A-dependent phosphorylation, resulting in suppression of the excitability of bullfrog TRCs.

Saccharin activates cation conductance via inositol 1,4,5‐trisphosphate production in a subset of isolated rod taste cells in the frog

The transduction mechanism of the conductance activated by saccharin was analysed in isolated bullfrog taste cells under whole‐cell voltage‐clamp. Bath application of 30 mm saccharin induced an inward current of −34 ± 12 pA (mean ± SEM, n = 10) at a membrane potential of −50 mV in 10 (23%) of 44 rod cells. The concentration–response relationship for the saccharin‐gated current was consistent with that of the gustatory neural response. The saccharin‐induced current was accompanied with a conductance increase under internal low Cl– condition (ECl = −56 mV), suggesting that saccharin activated a cation conductance. The reversal potential of the saccharin‐induced current was −17 ± 2 mV (n = 10). Intracellular dialysis of 0.5 mm guanosine 5′‐O‐(2‐thiodiphosphate) (GDP‐β‐S) completely blocked the saccharin‐induced response, suggesting the involvement of a G protein in the transduction. The dialysis of heparin (1 mg/mL) also inhibited the response almost completely, but the dialysis of 1 mm 8‐Br‐cAMP did not affect the response significantly. Intracellular 50 µm inositol 1,4,5‐trisphosphate (1,4,5 InsP3) also induced the inward current in five (38%) of 13 rod cells, but intracellular Pasteurella multocida toxin (5 µg/mL, Gαq‐coupled PLC activator) did not elicit any response in the cells. The results suggest that saccharin mainly activates a cation conductance in frog taste cells through the mediation of IP3 production.

Properties of Na+-dependent K+ conductance in the apical membrane of frog taste cells

Na(+)-dependent K+ conductance observed in apical membrane of isolated bullfrog taste cells was analyzed using various modifications of the patch clamp method. When the apical receptive membrane of a taste cell was exposed to 0.5 M NaCl using a pipette perfusion method, an inward current appeared in the cell-attached patch membrane. The permeability ratio of Na+ to K+ (P(Na)/P(K)) calculated from the reversal potential was 0.3. When an outside-out patch from the receptive membrane was exposed to 0.25 M NaCl solution, two types of K+ channel, whose conductance were 35.8 and 9.4 pS respectively, were activated. These channels were reversibly activated by increasing internal Na+ concentration. In the whole cell configuration, the outward current evoked by a voltage ramp from -80 to 80 mV was significantly suppressed by replacement of Na+ by N-methyl-D-glucamine+ (NMDG+). The Na(+)-dependent outward current was blocked by 10 mM BaCl2, with positive shift of the zero-current potential in the whole cell recordings, indicating that the outward current including Na(+)-dependent K+ component contributes to maintenance of the resting potential. These results suggest that small conductance Na(+)-dependent K+ channels, which are also permeable to Na+, are involved in salt signal transduction in frog taste cells.