Andrew I. Spielman

Gγ13 colocalizes with gustducin in taste receptor cells and mediates IP3 responses to bitter denatonium

Gustducin is a transducin-like G protein selectively expressed in taste receptor cells. The α subunit of gustducin (α-gustducin) is critical for transduction of responses to bitter or sweet compounds. We identified a G-protein γ subunit (Gγ13) that colocalized with α-gustducin in taste receptor cells. Of 19 α-gustducin/Gγ13-positive taste receptor cells profiled, all expressed the G protein β3 subunit (Gβ3); ~80% also expressed Gβ1. Gustducin heterotrimers (α-gustducin/Gβ1/Gγ13) were activated by taste cell membranes plus bitter denatonium. Antibodies against Gγ13 blocked the denatonium-induced increase of inositol trisphosphate (IP3) in taste tissue. We conclude that gustducin heterotrimers transduce responses to bitter and sweet compounds via α-gustducins regulation of phosphodiesterase (PDE) and Gβγs activation of phospholipase C (PLC).

ANALYSIS OF CHARACTERISTIC HUMAN FEMALE AXILLARY ODORS: QUALITATIVE COMPARISON TO MALES

Odors produced in the human female axillae are of both biological and commercial importance. Several studies have suggested that extracts from female underarm secretions can alter the length and timing of the female menstrual cycle. In addition, more than 1.6 billion dollars are spent annually on products to eliminate or mask the axillary odors. Our recent studies have determined that the characteristic axillary odors in males consist of C6–C11, saturated, unsaturated and branched acids, with (E)-3-methyl-2-hexenoic acid (3M2H) being the major compound in this mixture. The 3M2H appears to be carried to the skin surface bound to two proteins in the axillary secretions. Data reported here show that the same mixture of odorous compounds is found in female axillary secretions, with several minor qualitative differences. Separation of the female apocrine secretions into aqueous and organic soluble fractions demonstrated that 3M2H, and several other members of the acids in the characteristic odor, are released by hydrolysis with base. Electrophoretic separation of the proteins found in the aqueous phase of female apocrine secretions revealed a pattern identical to that seen in males. The qualitative similarity of the acidic constituents making up the characteristic axillary odors of both females and males as well as the proteins present in the aqueous phase suggest a similar origin for axillary odors in both sexes.

Generation of inositol phosphates in bitter taste transduction

It is probable that there is a diversity of mechanisms involved in the transduction of bitter taste. One of these mechanisms uses the second messengers, inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Partial membrane preparations from circumvallate and foliate taste regions of mice tongues responded to the addition of known bitter taste stimuli by increasing the amount of inositol phosphates produced after 30 s incubation. Addition of both the bitter stimulus, sucrose octaacetate and the G-protein stimulant, GTP gamma S, led to an enhanced production of inositol phosphates compared with either alone. Pretreatment of the tissue samples with pertussis toxin eliminated all response to sucrose octaacetate plus GTP gamma S, whereas pretreatment with cholera toxin was without effect. Western blots of solubilized tissue from circumvallate and foliate regions probed with antibodies to the alpha-subunit of several types of G-proteins revealed bands reactive to antibodies against G alpha i1-2 and G alpha o, with no apparent activity to antibodies against G alpha i3. Given the results from the immunoblots and those of the toxin experiments, it is proposed that the transduction of the bitter taste of sucrose octaacetate in mice involves a receptor-mediated activation of a Gi-type protein which activates a phospholipase C to produce the two second messengers, IP3 and DAG.

Sour Ageusia in Two Individuals Implicates Ion Channels of the ASIC and PKD Families in Human Sour Taste Perception at the Anterior Tongue

Background The perception of sour taste in humans is incompletely understood at the receptor cell level. We report here on two patients with an acquired sour ageusia. Each patient was unresponsive to sour stimuli, but both showed normal responses to bitter, sweet, and salty stimuli. Methods and Findings Lingual fungiform papillae, containing taste cells, were obtained by biopsy from the two patients, and from three sour-normal individuals, and analyzed by RT-PCR. The following transcripts were undetectable in the patients, even after 50 cycles of amplification, but readily detectable in the sour-normal subjects: acid sensing ion channels (ASICs) 1a, 1β, 2a, 2b, and 3; and polycystic kidney disease (PKD) channels PKD1L3 and PKD2L1. Patients and sour-normals expressed the taste-related phospholipase C-β2, the δ-subunit of epithelial sodium channel (ENaC) and the bitter receptor T2R14, as well as β-actin. Genomic analysis of one patient, using buccal tissue, did not show absence of the genes for ASIC1a and PKD2L1. Immunohistochemistry of fungiform papillae from sour-normal subjects revealed labeling of taste bud cells by antibodies to ASICs 1a and 1β, PKD2L1, phospholipase C-β2, and δ-ENaC. An antibody to PKD1L3 labeled tissue outside taste bud cells. Conclusions These data suggest a role for ASICs and PKDs in human sour perception. This is the first report of sour ageusia in humans, and the very existence of such individuals (“natural knockouts”) suggests a cell lineage for sour that is independent of the other taste modalities.

Ceramide triggers intracellular calcium release via the IP 3 receptor in Xenopus laevis oocytes

Ceramide, a product of sphingomyelin turnover, is a lipid second messenger that mediates diverse signaling pathways, including those leading to cell cycle arrest and differentiation. The mechanism(s) by which ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopus laevis oocytes, ceramide-induced maturation is associated with the release of intracellular calcium stores. Ceramide caused a dose-dependent elevation in the second messenger inositol 1,4,5-trisphosphate (IP3) via activation of Gq/11α and phospholipase C-βX. Elevation of IP3, in turn, activated the IP3 receptor calcium release channel on the endoplasmic reticulum, resulting in a rise in cytoplasmic calcium. Thus our study demonstrates that cross talk between the ceramide and phosphoinositide signaling pathways modulates intracellular calcium homeostasis.Ceramide, a product of sphingomyelin turnover, is a lipid second messenger that mediates diverse signaling pathways, including those leading to cell cycle arrest and differentiation. The mechanism(s) by which ceramide signals downstream events have not been fully elucidated. Here we show that, in Xenopus laevis oocytes, ceramide-induced maturation is associated with the release of intracellular calcium stores. Ceramide caused a dose-dependent elevation in the second messenger inositol 1,4,5-trisphosphate (IP(3)) via activation of G(q/11)alpha and phospholipase C-betaX. Elevation of IP(3), in turn, activated the IP(3) receptor calcium release channel on the endoplasmic reticulum, resulting in a rise in cytoplasmic calcium. Thus our study demonstrates that cross talk between the ceramide and phosphoinositide signaling pathways modulates intracellular calcium homeostasis.

Comparison of the Effects of Phospholamban and Jasmone on the Calcium Pump of Cardiac Sarcoplasmic Reticulum EVIDENCE FOR MODULATION BY PHOSPHOLAMBAN OF BOTH Ca2+ AFFINITY AND Vmax (Ca) OF CALCIUM TRANSPORT

Regulation of the calcium pump of the cardiac sarcoplasmic reticulum by phosphorylation/dephosphorylation of phospholamban is central to the inotropic and lusitropic effects of β-adrenergic agonists on the heart. In order to study the mechanism of this regulation, we first obtained purified ruthenium red-insensitive microsomes enriched in sarcoplasmic reticulum membranes. The kinetics of microsomal Ca2+ uptake after phospholamban phosphorylation or trypsin treatment, which cleaves the inhibitory cytoplasmic domain of phospholamban, were then compared with those in the presence of jasmone, whose effects on the kinetics of fast skeletal muscle Ca2+-ATPase are largely known. All three treatments increased Vmax(Ca) at 25°C and millimolar ATP; phosphorylation and trypsin decreased the Km(Ca), while jasmone increased it. Trypsin and jasmone increased the rate of E2P decomposition 1.8- and 3.0-fold, respectively. The effects of phospholamban phosphorylation and jasmone on the Ca2+-ATPase activity paralleled their effects on Ca2+ uptake. Our data demonstrate that phospholamban regulates E2P decomposition in addition to the known increase in the rate of a conformational change in the Ca2+-ATPase upon binding the first of two Ca2+. These steps in the catalytic cycle of the Ca2+-ATPase may contribute to or account for phospholambans effects on both Vmax(Ca) and Km(Ca), whose relative magnitude may vary under different experimental and, presumably, physiological conditions.

Characterization of Human Fungiform Papillae Cells in Culture

The ability to maintain human fungiform papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Here we describe a method for enzymatically isolating human cells from fungiform papillae obtained by biopsy and maintaining them in culture for more than 7 passages (7 months) without loss of viability and while retaining many of the functional properties of acutely isolated taste cells. Cells in these cultures exhibited increases in intracellular calcium when stimulated with perceptually appropriate concentrations of several taste stimuli, indicating that at least some of the native signaling pathways were present. This system can provide a useful model for molecular studies of the proliferation, differentiation, and physiological function of human fungiform papillae cells.

Biochemical enrichment and biophysical characterization of a taste receptor for L-arginine from the catfish, Ictalurus puntatus

BackgroundThe channel catfish, Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers.ResultsBoth PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 kDa which may be interpreted as a component of a multimeric receptor/channel complex.ConclusionsThe data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata.

Isolation and culture of human fungiform taste papillae cells

Taste cells are highly specialized, with unique histological, molecular and physiological characteristics that permit detection of a wide range of simple stimuli and complex chemical molecules contained in foods. In human, individual fungiform papillae contain from zero to as many as 20 taste buds. There is no established protocol for culturing human taste cells, although the ability to maintain taste papillae cells in culture for multiple cell cycles would be of considerable utility for characterizing the molecular, regenerative, and functional properties of these unique sensory cells. Earlier studies of taste cells have been done using freshly isolated cells in primary culture, explant cultures from rodents, or semi-intact taste buds in tissue slices. Although each of these preparations has advantages, the development of long-term cultures would have provided significant benefits, particularly for studies of taste cell proliferation and differentiation. Several groups, including ours, have been interested in the development and establishment of taste cell culture models. Most attempts to culture taste cells have reported limited viability, with cells typically not lasting beyond 3-5 d. We recently reported on a successful method for the extended culture of rodent taste cells. We here report for the first time the establishment of an in vitro culture system for isolated human fungiform taste papillae cells. Cells from human fungiform papillae obtained by biopsy were successfully maintained in culture for more than eight passages (12 months) without loss of viability. Cells displayed many molecular and physiological features characteristic of mature taste cells. Gustducin and phospholipase C β2, (PLC-β2) mRNA were detected in many cells by reverse transcriptase-polymerase chain reaction and confirmed by sequencing. Immunocytochemistry analysis demonstrated the presence of gustducin and PLC-β2 expression in cultured taste cells. Cultured human fungiform cells also exhibited increases in intracellular calcium in response to appropriate concentrations of several taste stimuli indicating that taste receptors and at least some of the signalling pathways were present. These results sufficient indicate that taste cells from adult humans can be generated and maintained for at least eight passages. Many of the cells retain physiological and biochemical characteristics of acutely isolated cells from the adult taste epithelium to support their use as a model taste system. This system will enable further studies of the processes involved in proliferation, differentiation and function of mammalian taste receptor cells in an in vitro preparation. Human fungiform taste papillae used for establishing human fungiform cell culture were donated for research following proper informed consent under research protocols that were reviewed and approved by the IRB committee. The protocol (#0934) was approved by Schulman Associates Institutional Review Board Inc., Cincinnati, OH. Written protocol below is based on published parameters reported by Ozdener et al. 2011.

The Birth of the Most Important 18th Century Dental Text: Pierre Fauchard’s Le Chirurgien Dentist

Pierre Fauchard (1678–1761) is considered the father of modern dentistry. His seminal book, Le Chirurgien Dentiste, ou Traité des Dents (1728), is the discipline’s first complete work. During the five years preceding its publication (1723–1728), Pierre Fauchard sought the opinions, contributions, and “approbation” (approval) of 19 of his colleagues: six physicians, 12 surgeons, and one dentist. The first and most important contributor to the manuscript was Jean Devaux, surgeon and mentor to Fauchard. The next six reviewers were illustrious physicians and scientists of the time: Philippe Hecquet, Jean-Claude Adrien Helvetius, Jean Baptiste Silva, Antoine DeJussieu, Raymond Jacob Finot, and Antoine Benignus Winslow. The subsequent 12 reviewers were all sworn-surgeons (certified by St. Côme), including a lone dentist, Laudumiey, surgeon-dentist to His Majesty, Philip V, King of Spain. Fauchard knew that for dentistry to be regarded as a learned profession, and perhaps for Fauchard to be recognized as its leader, he needed the support and approval of the establishment before publishing his book. This is an account of how he attained this endorsement.