Bruce P. Bryant

Alkylamides that produce tingling paresthesia activate tactile and thermal trigeminal neurons.

Alkylamides isolated from the fruit of Xanthoxylum, Szechuan pepper, produce a strong tingling sensation in the mouth. In order to determine the peripheral basis of this sensation, extracellular nerve recordings were obtained from the lingual nerve of rats. The primary pungent compound, hydroxy-alpha-sanshool (HO-alpha-S), altered the levels of spontaneous activity in cool-sensitive fibers as well as inducing activity in tactile fibers, cold nociceptors and silent fibers that were insensitive to innocuous thermal or tactile stimuli. Moreover, tactile or thermal sensitivity was induced in fibers that were initially insensitive to touch or cooling. The neuronal distribution of sensitivities to capsaicin and to HO-alpha-S indicate that this compound affects neurons mediating innocuous sensations. HO-alpha-S may be useful as a model stimulus for studies of paresthesia.

Acetazolamide specifically inhibits lingual trigeminal nerve responses to carbon dioxide

The goal of this study was to examine the role of the enzyme, carbonic anhydrase, in oral trigeminal chemoreception with particular regard to the reception of CO2. Using both single and multiunit recordings of trigeminal neurons in the lingual nerve of rat, we measured responses to cool (24 degrees C), noxiously hot (55 degrees C) and cold (8 degrees C) H2O, NH4Cl and supersaturated solutions of CO2 (24 degrees C and 33 degrees C). The importance of peripheral carbonic anhydrase was tested by inhibiting enzyme activity with acetazolamide (15 mg/kg b.w.). Single unit responses to CO2 and HCl suggest that neural sensitivity to CO2 is not simply a function of extraepithelial pH. Responses to CO2 were significantly inhibited by acetazolamide while the responses to thermal stimuli and NH4Cl were not. The results support a role for carbonic anhydrase in trigeminal responses to CO2. Furthermore, the results suggest that intraepithelial acidification mediated by carbonic anhydrase may be the basis for sensitivity to CO2.

Specificl-arginine taste receptor sites in the catfish, Ictalurus punctatus: biochemical and neurophysiological characterization

We report here the characterization of the arginine binding site(s) and corroborative neurophysiological studies. Binding of L-[3H]arginine to Fraction P2 from taste epithelium was measured by a modification of the method of Krueger and Cagan. Parameters for measuring maximal binding activity were established for both duration of incubation and pH of medium. At pH 7.8, the apparent single rate constant for association (kobs) at 4 degrees C was 4.72 x 10(+5).M-1.min-1. Dissociation was more complex, yielding two rate constants of 1.77.min-1 and 8.34 x 10(-3).min-1. These data suggest the presence of two affinity states for L-arginine. The KD values as calculated from the ratio k-1/k+1 were 1.3 x 10(-6) M and 1.8 x 10(-8) M. Homologous inhibition studies of L-arginine binding were not fit by a simple mass action relationship (Hill Coefficient 0.79), but were best fit by a two-site model with IC50 values of 1.6 x 10(-6) M for the high affinity state and 9 x 10(-4) M for the low affinity state. Multiunit neural recordings examined the stimulatory effectiveness of a number of guanidinium-containing compounds. Compared with L-arginine, only L-arginine methyl ester and L-alpha-amino-beta-guanidino propionic acid (L-AGPA) were effective stimuli. Cross-adaptation experiments demonstrated that at 10(-4) M L-arginine methyl ester, L-AGPA and, to a lesser extent, D-arginine were effective cross-adapting stimuli to 10(-6) M L-arginine. In competition binding studies L-arginine methyl ester, L-AGPA and D-arginine also inhibited binding of L-[3H]arginine (10(-6) M), but each recognized only one affinity state. Inhibition by the poorly cross-adapting stimuli L-glutamate, glycine and L-alanine occurred only above 10(-3) M, indicating that the binding sites for L-arginine are selective. These studies suggest that there are at least two affinity states of L-arginine binding, that the binding sites are specific, and that effective agonists of L-arginine receptors must contain a guanidinium group and an unblocked L-alpha-amino group.

Perceiving Nasal Patency through Mucosal Cooling Rather than Air Temperature or Nasal Resistance

Adequate perception of nasal airflow (i.e., nasal patency) is an important consideration for patients with nasal sinus diseases. The perception of a lack of nasal patency becomes the primary symptom that drives these patients to seek medical treatment. However, clinical assessment of nasal patency remains a challenge because we lack objective measurements that correlate well with what patients perceive.The current study examined factors that may influence perceived patency, including air temperature, humidity, mucosal cooling, nasal resistance, and trigeminal sensitivity. Forty-four healthy subjects rated nasal patency while sampling air from three facial exposure boxes that were ventilated with untreated room air, cold air, and dry air, respectively. In all conditions, air temperature and relative humidity inside each box were recorded with sensors connected to a computer. Nasal resistance and minimum airway cross-sectional area (MCA) were measured using rhinomanometry and acoustic rhinometry, respectively. General trigeminal sensitivity was assessed through lateralization thresholds to butanol. No significant correlation was found between perceived patency and nasal resistance or MCA. In contrast, air temperature, humidity, and butanol threshold combined significantly contributed to the ratings of patency, with mucosal cooling (heat loss) being the most heavily weighted predictor. Air humidity significantly influences perceived patency, suggesting that mucosal cooling rather than air temperature alone provides the trigeminal sensation that results in perception of patency. The dynamic cooling between the airstream and the mucosal wall may be quantified experimentally or computationally and could potentially lead to a new clinical evaluation tool.

Biochemical studies of taste sensation. XIII. Enantiomeric specificity of alamine taste receptor sites in catfish,Ictalurus punctatus

Specific binding of amino acid taste stimuli is known to occur to a sedimentable fraction (P2) from catfish (Ictalurus punctatus) taste epithelium or to purified plasma membranes from that fraction. L-Alanine, a potent taste stimulus for the catfish, binds in a reversible and saturable manner to these preparations. The extent to which the enantiomeric stimuli, L- and D-alanine, interact with the same or different receptor/transduction processes is investigated here both electrophysiologically and biochemically. With an electrophysiological assay, L-alanine was the more potent stimulus across a concentration range of 10(-9)-10(-3) M, yet both enantiomers displayed approximately the same threshold. The concentration-electrophysiological response functions for each enantiomer were different. That of L-alanine was approximately linear across the (log) concentration range while that of D-alanine was non-linear, with small but definitely observable responses being noted from 10(-9)-10(-5) M D-alanine, and larger incremental responses thereafter. With most of the nerve bundle preparations studies, L- and D-alanine cross-adapted one another, but this cross-adaptation was not always complete. Experiments in which both L- and D-alanine were present in a 1:1 mixture of equally stimulatory concentrations suggested the existence of receptor or transduction processes unique to each enantiomer. Biochemically binding studies demonstrated high affinity binding sites for both enantiomers with values of Kd-app for L-alanine of 1.5 microM and for D-alanine of 25 microM. For both enantiomers, additional lower-affinity binding sites were observable. The capacity of the lower-affinity sites was particularly great for D-alanine. The enantiomers competed one with the other for binding, with L-alanine showing greater competitive ability than D-alanine at low concentrations. For the high affinity sites, double-reciprocal plots of the data suggested a competitive mechanism. The lower affinity sites for D-alanine were less accessible to L-alanine compared with the high affinity sites of D-alanine. Both the biochemical and electrophysiological results indicate that while a portion of the responses to L- and D-alanine occurs through a common receptor/transduction process, there exist independent receptor/transduction processes for the enantiomers, L- and D-alanine.

Multiple types of sensory neurons respond to irritating volatile organic compounds (VOCs): Calcium fluorimetry of trigeminal ganglion neurons

&NA; Many volatile organic compounds (VOCs) are significant environmental irritants that stimulate somatosensory nerve endings to produce pain and irritation. We measured intracellular calcium in cultured trigeminal ganglion neurons to characterize the cellular mechanisms and chemical structural determinants underlying sensitivity to VOCs. Trigeminal neurons responded to homologous series of alcohols (C4–C7) as well as saturated and unsaturated aldehydes in a concentration dependent manner. Ranked in terms of threshold to recruit neurons by compounds of the same carbon chain length, enaldehyde<aldehyde<alcohol. Unlike aldehydes and alcohols that displayed ascending concentration curves, recruitment of neurons by enaldehydes (C4–C7) appeared to saturate, consistent with a mechanism that is restricted in its neural distribution. Using pentanol, pentanal and pentenal as model compounds, we found that many but not all cool/cold‐sensitive and capsaicin‐sensitive neurons responded with increases in intracellular calcium. These VOCs also stimulated other neurons that were insensitive to cooling and capsaicin. Because not all cooling‐ and all capsaicin‐sensitive neurons responded to the model VOCs, it is highly unlikely that known nociceptive ion channels such as TRPV1 or TRPA1 mediate sensitivity to these compounds. For pentanol, pentanal and pentenal, induced calcium influx was dependent on the presence of extracellular calcium. Responses of all neurons to pentanal and pentenal were also dependent upon extracellular sodium. Responses to pentanol were variably dependent on sodium. The distribution of sensitivity suggests that VOC irritation may be mediated by an as yet unidentified mechanism(s) that is/are distributed across different modalities of neurons.

Capsaicin and its analogs induce ion channels in planar lipid bilayers

The irritating, pungent compound, capsaicin (10-20 microM), induces the formation of non-selective ion channels with a wide variety of conductances in protein-free lipid bilayers form from a mixture of zwitterionic phospholipids. The channel-forming activity of capsaicin and four of its analogs followed the sequence: resiniferatoxin > capsaicin = pelargonic acid vanillylamide > methylcapsaicin >> veratrylamine. The ability to form channels correlated with the biological activity of these compounds observed in other studies that measured 45Ca uptake into rat dorsal root ganglion cells. The correlation obtained suggests that an interaction with the lipid bilayer may be an important component of the biological activity of capsaicin.

The Zebrafish as a Model for Nociception Studies

Nociception is the sensory mechanism used to detect cues that can harm an organism. The understanding of the neural networks and molecular controls of the reception of pain remains an ongoing challenge for biologists. While we have made significant progress in identifying a number of molecules and pathways that are involved in transduction of noxious stimuli, from the skin through the sensory receptor cell and from this to the spinal cord on into the central nervous system, we still lack a clear understanding of the perceptual processes, the responses to pain and the regulation of pain perception. Mice and rat animal models have been extensively used for nociception studies. However, the study of pain and noiception in these organisms can be rather laborious, costly and time consuming. Conversely, the use of Drosophila and Caenorhabditis elegans may be affected by the large evolutionary distance between these animals and humans. We outline here the reasons why zebrafish presents a new and attractive model for studying pain reception and responses and the most interesting findings in the study of nociception that have been obtained using the zebrafish model. J. Cell. Physiol. 228: 1956–1966, 2013.

Reduction in carbonic anhydrase activity in the tongue epithelium and submandibular gland in zinc-deficient rats.

We investigated the effects of zinc deficiency on carbonic anhydrase (CA) activity in the tongue epithelium and submandibular gland in rats. Male 4-week-old SD rats were given free access to a diet containing 2.2 (zinc-deficient), 4.1 (low-zinc), or 33.7 (zinc-sufficient) mg zinc/kg diet for 6 weeks. Rats in the fourth group (receiving 33.7 mg zinc/kg) were pair-fed against the zinc-deficient rats. Biochemical analysis at the end of the experimental period indicated that zinc deficiency significantly reduced CA activity in the tongue epithelium and submandibular gland, and the CA activity levels in these tissues seemed to parallel the dietary zinc levels. By enzyme histochemistry, an intensely positive reaction for CA was observed in the middle and basal regions of the taste buds in the circumvallate papilla in the zinc-sufficient and pair-fed (control) rats. The cells in von Ebners glands also showed a strong positive reaction in control rats, although only a weak reaction product was found in zinc-deficient rats. These results suggest that CA activity is affected by the dietary content of zinc, which is considered to be an indispensable factor for the maintenance of normal taste sensation.

Calcium responses of chicken trigeminal ganglion neurons to methyl anthranilate and capsaicin

SUMMARY Using digital fluorescence imaging, we determined the effects of methyl anthranilate (MA), an avian irritant, and capsaicin (CAP), a mammalian irritant, on intracellular calcium ([Ca2+]i) in chicken trigeminal neurons. Concentration–response functions indicated that the threshold for inducing increases in [Ca2+]i was higher for CAP (30 μmol l–1) than for MA (10 μmol l–1). The maximum magnitudes of [Ca2+]i in response to MA and CAP were compared after normalization to 40 mmol l–1 KCl. At equal concentrations (300 μmol l–1), trigeminal neurons responded with a greater change in [Ca2+]i to MA (78% of KCl) than to CAP (43% of KCl). Furthermore, at 300 μmol l–1, 48% of neurons responded to MA whereas only 16% responded to CAP. The increases in [Ca2+]i induced by both MA and CAP were dependent upon extracellular calcium. While the calcium responses to MA were also dependent on extracellular sodium, responses to CAP were not. There were separate but overlapping populations of neurons sensitive to MA and CAP. Taken together, the higher threshold concentration of CAP, the higher response magnitude to MA than CAP and the greater number of neurons sensitive to MA than CAP provide a rationale for the observed behavioral differences of birds to these two compounds. Finally, the findings that the calcium responses to MA and CAP have different ion dependencies and that there are separate populations sensitive to these compounds suggest different transduction mechanisms mediating chicken trigeminal responses to MA and CAP.