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Recognition of sweet, bitter and umami tastes requires the nonvesicular release from taste bud cells of ATP, which acts as a neurotransmitter to activate afferent neural gustatory pathways (1). However, how ATP is released to fulfil this function is not fully understood. Here we show that calcium homeostasis modulator 1 (CALHM1), a voltage-gated ion channel (2,3), is indispensable for taste-stimuli-evoked ATP release from sweet-, bitter- and umami-sensing taste bud cells. Calhm1 knockout mice have severely impaired perceptions of sweet, bitter and umami compounds, whereas their recognition of sour and salty tastes remains mostly normal. Calhm1 deficiency affects taste perception without interfering with taste cell development or integrity. CALHM1 is expressed specifically in sweet/bitter/umami-sensing type II taste bud cells. Its heterologous expression induces a novel ATP permeability that releases ATP from cells in response to manipulations that activate the CALHM1 ion channel. Knockout of Calhm1 strongly reduces voltage-gated currents in type II cells and taste-evoked ATP release from taste buds without affecting the excitability of taste cells by taste stimuli. Thus, CALHM1 is a voltage-gated ATP-release channel required for sweet, bitter and umami taste perception., Tastes are sensed by dedicated receptor cells in taste buds, which are composed of three anatomically distinct types of cells: types I, II and III. Only type III cells, which [...]

cAMP is a second messenger implicated in sensory transduction for taste. The identity of adenylyl cyclase (AC) in taste cells has not been explored. We have employed RT-PCR to identify the AC isoforms present in taste cells and found that AC 4, 6, and 8 are expressed as mRNAs in taste tissue. These proteins are also expressed in a subset of taste cells as revealed by immunohistochemistry. Alterations of cAMP concentrations are associated with transduction of taste stimuli of several classes. The involvement of particular ACs in this modulation has not been investigated. We demonstrate that glutamate, which is a potent stimulus eliciting a taste quality termed umami, causes a decrease in cAMP in forskolin-treated taste cells. The potentiation of this response by inosine monophosphate, the lack of response to D-glutamate, and the lack of response to umami stimuli in nonsensory lingual epithelium all suggest that the cAMP modulation represents umami taste transduction. Because cAMP downregulation via ACs can be mediated through G[[alpha].sub.i] proteins, we examined the colocalization of the detected ACs with G[[alpha].sub.i] proteins and found that 66% of AC8 immunopositive taste cells are also positive for gustducin, a taste-specific G[[alpha].sub.i] protein. Whether AC8 is directly involved in signal transduction of umami taste remains to be established. immunohistochemistry; glutamate; umami; taste transduction; gustducin

In mammalian taste buds, Type I cells comprise half of all cells. These are termed "glial-like" based on morphologic and molecular features, but there are limited studies describing their function. We tested whether Type I cells sense chemosensory activation of adjacent chemosensory (i.e., Types II and III) taste bud cells, similar to synaptic glia. Using Gad2;;GCaMP3 mice of both sexes, we confirmed by immunostaining that, within taste buds, GCaMP expression is predominantly in Type I cells (with no Type II and ~28% Type III cells expressing weakly). In dissociated taste buds, GCaMP1 Type I cells responded to bath-applied ATP (10-100 μM) but not to 5-HT (transmitters released by Type II or III cells, respectively). Type I cells also did not respond to taste stimuli (5 μM cycloheximide, 1 mM denatonium). In lingual slice preparations also, Type I cells responded to bath-applied ATP (10-100 lM). However, when taste buds in the slice were stimulated with bitter tastants (cycloheximide, denatonium, quinine), Type I cells responded robustly. Taste-evoked responses of Type I cells in the slice preparation were significantly reduced by desensitizing purinoceptors or by purinoceptor antagonists (suramin, PPADS), and were essentially eliminated by blocking synaptic ATP release (carbenoxolone) or degrading extracellular ATP (apyrase). Thus, taste-evoked release of afferent ATP from type II chemosensory cells, in addition to exciting gustatory afferent fibers, also activates glial-like Type I taste cells. We speculate that Type I cells sense chemosensory activation and that they participate in synaptic signaling, similarly to glial cells at CNS tripartite synapses. [ABSTRACT FROM AUTHOR]

The expression of mRNA encoding the cardiac dihydropyridine-sensitive calcium channel in skeletal muscle developing in vivo and primary cultures derived from skeletal muscle was investigated. Expression of the mRNA at high concentration was detected in the earliest stage in vivo but diminished rapidly as myofibers mature. Also, there was concomitant diminishing of cardiac calcium channel mRNA. In contrast, mRNA for the skeletal muscle-specific calcium channel accumulates gradually in developing skeletal muscle.

Abstract Background "Type II"/Receptor cells express G protein-coupled receptors (GPCRs) for sweet, umami (T1Rs and mGluRs) or bitter (T2Rs), as well as the proteins for downstream signalling cascades. Transduction downstream of T1Rs and T2Rs relies on G-protein and PLCβ2-mediated release of stored Ca2+. Whereas Gαgus (gustducin) couples to the T2R (bitter) receptors, which Gα-subunit couples to the sweet (T1R2 + T1R3) receptor is presently not known. We utilized RT-PCR, immunocytochemistry and single-cell gene expression profiling to examine the expression of the Gαq family (q, 11, 14) in mouse taste buds. Results By RT-PCR, Gα14 is expressed strongly and in a taste selective manner in posterior (vallate and foliate), but not anterior (fungiform and palate) taste fields. Gαq and Gα11, although detectable, are not expressed in a taste-selective fashion. Further, expression of Gα14 mRNA is limited to Type II/Receptor cells in taste buds. Immunocytochemistry on vallate papillae using a broad Gαq family antiserum reveals specific staining only in Type II taste cells (i.e. those expressing TrpM5 and PLCβ2). This staining persists in Gαq knockout mice and immunostaining with a Gα11-specific antiserum shows no immunoreactivity in taste buds. Taken together, these data show that Gα14 is the dominant Gαq family member detected. Immunoreactivity for Gα14 strongly correlates with expression of T1R3, the taste receptor subunit present in taste cells responsive to either umami or sweet. Single cell gene expression profiling confirms a tight correlation between the expression of Gα14 and both T1R2 and T1R3, the receptor combination that forms sweet taste receptors. Conclusion Gα14 is co-expressed with the sweet taste receptor in posterior tongue, although not in anterior tongue. Thus, sweet taste transduction may rely on different downstream transduction elements in posterior and anterior taste fields.

How taste buds detect NaCl remains poorly understood. Among other problems, applying taste-relevant concentrations of NaCl (50-500mM) onto isolated taste buds or cells exposes them to unphysiological (hypo/hypertonic) conditions. To overcome these limitations, we used the anterior tongue of male and female mice to implement a slice preparation in which fungiform taste buds are in a relatively intact tissue environment and stimuli are limited to the taste pore. Taste-evoked responses were monitored using confocal Ca2+ imaging via GCaMP3 expressed in Type 2 and Type 3 taste bud cells. NaCl evoked intracellular mobilization of Ca2+ in the apical tips of a subset of taste cells. The concentration dependence and rapid adaptation of NaCl-evoked cellular responses closely resembled behavioral and afferent nerve responses to NaCl. Importantly, taste cell responses were not inhibited by the diuretic, amiloride. Post hoc immunostaining revealed that >80% of NaCl-responsive taste bud cells were of Type 2. Many NaCl-responsive cells were also sensitive to stimuli that activate Type 2 cells but never to stimuli for Type 3 cells. Ion substitutions revealed that amiloride-insensitive NaCl responses depended on Cl- rather than Na+. Moreover, choline chloride, an established salt taste enhancer, was equally effective a stimulus as sodium chloride. Although the apical transducer for Cl- remains unknown, blocking known chloride channels and cotransporters had little effect on NaCl responses. Together, our data suggest that chloride, an essential nutrient, is a key determinant of taste transduction for amiloride-insensitive salt taste. [ABSTRACT FROM AUTHOR]

The gustatory system encodes information about chemical identity, nutritional value, and concentration of sensory stimuli before transmitting the signal from taste buds to central neurons that process and transform the signal. Deciphering the coding logic for taste quality requires examining responses at each level along the neural axis—from peripheral sensory organs to gustatory cortex. From the earliest single-fiber recordings, it was clear that some afferent neurons respond uniquely and others to stimuli of multiple qualities. There is frequently a "best stimulus" for a given neuron, leading to the suggestion that taste exhibits "labeled line coding." In the extreme, a strict "labeled line" requires neurons and pathways dedicated to single qualities (e.g. sweet, bitter, etc.). At the other end of the spectrum, "across-fiber," "combinatorial," or "ensemble" coding requires minimal specific information to be imparted by a single neuron. Instead, taste quality information is encoded by simultaneous activity in ensembles of afferent fibers. Further, "temporal coding" models have proposed that certain features of taste quality may be embedded in the cadence of impulse activity. Taste receptor proteins are often expressed in nonoverlapping sets of cells in taste buds apparently supporting "labeled lines." Yet, taste buds include both narrowly and broadly tuned cells. As gustatory signals proceed to the hindbrain and on to higher centers, coding becomes more distributed and temporal patterns of activity become important. Here, we present the conundrum of taste coding in the light of current electrophysiological and imaging techniques at several levels of the gustatory processing pathway. [ABSTRACT FROM AUTHOR]

Key points: Orosensory thermal trigeminal afferent neurons respond to cool, warm, and nociceptive hot temperatures with the majority activated in the cool range.Many of these thermosensitive trigeminal orosensory afferent neurons also respond to capsaicin, menthol, and/or mustard oil (allyl isothiocyanate) at concentrations found in foods and spices.There is significant but incomplete overlap between afferent trigeminal neurons that respond to oral thermal stimulation and to the above chemesthetic compounds.Capsaicin sensitizes warm trigeminal thermoreceptors and orosensory nociceptors; menthol attenuates cool thermoresponses. When consumed with foods, mint, mustard, and chili peppers generate pronounced oral thermosensations. Here we recorded responses in mouse trigeminal ganglion neurons to investigate interactions between thermal sensing and the active ingredients of these plants – menthol, allyl isothiocyanate (AITC), and capsaicin, respectively – at concentrations found in foods and commercial hygiene products. We carried out in vivo confocal calcium imaging of trigeminal ganglia in which neurons express GCaMP3 or GCAMP6s and recorded their responses to oral stimulation with thermal and the above chemesthetic stimuli. In the V3 (oral sensory) region of the ganglion, thermoreceptive neurons accounted for ∼10% of imaged neurons. We categorized them into three distinct classes: cool‐responsive and warm‐responsive thermosensors, and nociceptors (responsive only to temperatures ≥43–45 °C). Menthol, AITC, and capsaicin also elicited robust calcium responses that differed markedly in their latencies and durations. Most of the neurons that responded to these chemesthetic stimuli were also thermosensitive. Capsaicin and AITC increased the numbers of warm‐responding neurons and shifted the nociceptor threshold to lower temperatures. Menthol attenuated the responses in all classes of thermoreceptors. Our data show that while individual neurons may respond to a narrow temperature range (or even bimodally), taken collectively, the population is able to report on graded changes of temperature. Our findings also substantiate an explanation for the thermal sensations experienced when one consumes pungent spices or mint. Key points: Orosensory thermal trigeminal afferent neurons respond to cool, warm, and nociceptive hot temperatures with the majority activated in the cool range.Many of these thermosensitive trigeminal orosensory afferent neurons also respond to capsaicin, menthol, and/or mustard oil (allyl isothiocyanate) at concentrations found in foods and spices.There is significant but incomplete overlap between afferent trigeminal neurons that respond to oral thermal stimulation and to the above chemesthetic compounds.Capsaicin sensitizes warm trigeminal thermoreceptors and orosensory nociceptors; menthol attenuates cool thermoresponses. [ABSTRACT FROM AUTHOR]

Taste buds are innervated by neurons whose cell bodies reside in cranial sensory ganglia. Studies on the functional properties and connectivity of these neurons are hindered by the lack of markers to define their molecular identities and classes. The mouse geniculate ganglion contains chemosensory neurons innervating lingual and palatal taste buds and somatosensory neurons innervating the pinna. Here, we report single cell RNA sequencing of geniculate ganglion neurons. Using unbiased transcriptome analyses, we show a pronounced separation between two major clusters which, by anterograde labeling, correspond to gustatory and somatosensory neurons. Among the gustatory neurons, three subclusters are present, each with its own complement of transcription factors and neurotransmitter response profiles. The smallest subcluster expresses both gustatory- and mechanosensoryrelated genes, suggesting a novel type of sensory neuron. We identify several markers to help dissect the functional distinctions among gustatory neurons and address questions regarding target interactions and taste coding. [ABSTRACT FROM AUTHOR]

Olfactory epithelium (OE) has a lifelong capacity for neurogenesis due to the presence of basal stemcells. Despite the ability to generate short-term cultures, the successful in vitro expansion of purified stem cells from adult OE has not been reported. We sought to establish expansion-competent OE stem cell cultures to facilitate further study of the mechanisms and cell populations important in OE renewal. Successful cultures were prepared using adult mouse basal cells selected for expression of c-KIT. We show that c-KIT signaling regulates self-renewal capacity and prevents neurodifferentiation in culture. Inhibition of TGFβ family signaling, a known negative regulator of embryonic basal cells, is also necessary for maintenance of the proliferative, undifferentiated state in vitro. Characterizing successful cultures, we identified expression of BMI1 and other Polycomb proteins not previously identified in olfactory basal cells but known to be essential for self-renewal in other stem cell populations. Inducible fate mapping demonstrates that BMI1 is expressed in vivo by multipotent OE progenitors, validating our culture model. These findings provide mechanistic insights into the renewal and potency of olfactory stem cells. [ABSTRACT FROM AUTHOR]

Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste. [ABSTRACT FROM AUTHOR]

A repeated 82 nucleotide sequence (copy frequency estimated at [is approx. =]1.5 X 10.sup.5 copies in the rat genome) has been described as a component of primary transcripts and assorted [...]

Taste buds are clusters of polarized sensory cells embedded in stratified oral epithelium. In adult mammals, taste buds turn over continuously and are replenished through the birth of new cells in the basal layer of the surrounding non-sensory epithelium. The half-life of cells in mammalian taste buds has been estimated as 8-12 days on average. Yet, earlier studies did not address whether the now well-defined functional taste bud cell types all exhibit the same lifetime. We employed a recently developed thymidine analog, 5-ethynil-2'-deoxyuridine (EdU) to re-evaluate the incorporation of newly born cells into circumvallate taste buds of adult mice. By combining EdU-labeling with immunostaining for selected markers, we tracked the differentiation and lifespan of the constituent cell types of taste buds. EdU was primarily incorporated into basal extragemmal cells, the principal source for replenishing taste bud cells. Undifferentiated EdU-labeled cells began migrating into circumvallate taste buds within 1 day of their birth. Type II (Receptor) taste cells began to differentiate from EdU-labeled precursors beginning 2 days after birth and then were eliminated with a half-life of 8 days. Type III (Presynaptic) taste cells began differentiating after a delay of 3 days after EdU-labeling, and they survived much longer, with a half-life of 22 days. We also scored taste bud cells that belong to neither Type II nor Type III, a heterogeneous group that includes mostly Type I cells, and also undifferentiated or immature cells. A non-linear decay fit described these cells as two sub-populations with half-lives of 8 and 24 days respectively. Our data suggest that many post-mitotic cells may remain quiescent within taste buds before differentiating into mature taste cells. A small number of slow-cycling cells may also exist within the perimeter of the taste bud. Based on their incidence, we hypothesize that these may be progenitors for Type III cells. [ABSTRACT FROM AUTHOR]

Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. ThisATPexcites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Hereweshow that extracellularATPis enzymatically degraded to adenosine withinmousevallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells,weshow thatmanyReceptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. [ABSTRACT FROM AUTHOR]

The article focuses on a study conducted on mice to examine the role of P2X receptors in the regulation of adenosine 5'-triphosphate (ATP) secretion in taste buds. The study found that presynaptic P2X receptors may generate autocrine positive feedback for stimulating ATP secretion. It informs that various methods were used during the study including functional magnetic resonance imaging and electron microscopy.

Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABAA and GABAB receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABAA receptor subunits β2, δ, and π, as well as GABAB receptors. In contrast, presynaptic cells express the GABAA β3 subunit and only occasionally GABAB receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds. [ABSTRACT FROM AUTHOR]

Background: The neuropeptide, oxytocin (OXT), acts on brain circuits to inhibit food intake. Mutant mice lacking OXT (OXT knockout) overconsume salty and sweet (i.e. sucrose, saccharin) solutions. We asked if OXT might also act on taste buds via its receptor, OXTR. Methodology/Principal Findings: Using RT-PCR, we detected the expression of OXTR in taste buds throughout the oral cavity, but not in adjacent non-taste lingual epithelium. By immunostaining tissues from OXTR-YFP knock-in mice, we found that OXTR is expressed in a subset of Glial-like (Type I) taste cells, and also in cells on the periphery of taste buds. Single-cell RT-PCR confirmed this cell-type assignment. Using Ca2+ imaging, we observed that physiologically appropriate concentrations of OXT evoked [Ca2+]i mobilization in a subset of taste cells (EC50 ∼33 nM). OXT-evoked responses were significantly inhibited by the OXTR antagonist, L-371,257. Isolated OXT-responsive taste cells were neither Receptor (Type II) nor Presynaptic (Type III) cells, consistent with our immunofluorescence observations. We also investigated the source of OXT peptide that may act on taste cells. Both RT-PCR and immunostaining suggest that the OXT peptide is not produced in taste buds or in their associated nerves. Finally, we also examined the morphology of taste buds from mice that lack OXTR. Taste buds and their constituent cell types appeared very similar in mice with two, one or no copies of the OXTR gene. Conclusions/Significance: We conclude that OXT elicits Ca2+ signals via OXTR in murine taste buds. OXT-responsive cells are most likely a subset of Glial-like (Type I) taste cells. OXT itself is not produced locally in taste tissue and is likely delivered through the circulation. Loss of OXTR does not grossly alter the morphology of any of the cell types contained in taste buds. Instead, we speculate that OXT-responsive Glial-like (Type I) taste bud cells modulate taste signaling and afferent sensory output. Such modulation would complement central pathways of appetite regulation that employ circulating homeostatic and satiety signals. [ABSTRACT FROM AUTHOR]

The second messenger, 3′,5′-cyclic adenosine monophosphate (cAMP), is known to be modulated in taste buds following exposure to gustatory and other stimuli. Which taste cell type(s) (Type I/glial-like cells, Type II/receptor cells, or Type III/presynaptic cells) undergo taste-evoked changes of cAMP and what the functional consequences of such changes are remain unknown. Using Fura-2 imaging of isolated mouse vallate taste cells, we explored how elevating cAMP alters Ca2+ levels in identified taste cells. Stimulating taste buds with forskolin (Fsk; 1 μm) + isobutylmethylxanthine (IBMX; 100 μm), which elevates cellular cAMP, triggered Ca2+ transients in 38% of presynaptic cells ( n= 128). We used transgenic GAD-GFP mice to show that cAMP-triggered Ca2+ responses occur only in the subset of presynaptic cells that lack glutamic acid decarboxylase 67 (GAD). We never observed cAMP-stimulated responses in receptor cells, glial-like cells or GAD-expressing presynaptic cells. The response to cAMP was blocked by the protein kinase A inhibitor H89 and by removing extracellular Ca2+. Thus, the response to elevated cAMP is a PKA-dependent influx of Ca2+. This Ca2+ influx was blocked by nifedipine (an inhibitor of L-type voltage-gated Ca2+ channels) but was unperturbed by ω-agatoxin IVA and ω-conotoxin GVIA (P/Q-type and N-type channel inhibitors, respectively). Single-cell RT-PCR on functionally identified presynaptic cells from GAD-GFP mice confirmed the pharmacological analyses: Cav1.2 (an L-type subunit) is expressed in cells that display cAMP-triggered Ca2+ influx, while Cav2.1 (a P/Q subunit) is expressed in all presynaptic cells, and underlies depolarization-triggered Ca2+ influx. Collectively, these data demonstrate cross-talk between cAMP and Ca2+ signalling in a subclass of taste cells that form synapses with gustatory fibres and may integrate tastant-evoked signals. [ABSTRACT FROM AUTHOR]

Abstract: The taste-selective G protein, α-gustducin (α-gus) is homologous to α-transducin and activates phosphodiesterase (PDE) in vitro. α-Gus-knockout mice are compromized to bitter, sweet and umami taste stimuli, suggesting a central role in taste transduction. Here, we suggest a different role for Gα-gus. In taste buds of α-gus-knockout mice, basal (unstimulated) cAMP levels are high compared to those of wild-type mice. Further, H-89, a cAMP-dependent protein kinase inhibitor, dramatically unmasks responses to the bitter tastant denatonium in gus-lineage cells of knockout mice. We propose that an important role of α-gus is to maintain cAMP levels tonically low to ensure adequate Ca2+ signaling. [Copyright &y& Elsevier]

Cyclic AMP (cAMP) and Ca2+ are two ubiquitous second messengers in transduction pathways downstream of receptors for hormones, neurotransmitters and local signals. The availability of fluorescent Ca2+ reporter dyes that are easily introduced into cells and tissues has facilitated analysis of the dynamics and spatial patterns for Ca2+ signaling pathways. A similar dissection of the role of cAMP has lagged because indicator dyes do not exist. Genetically encoded reporters for cAMP are available but they must be introduced by transient transfection in cell culture, which limits their utility. We report here that we have produced a strain of transgenic mice in which an enhanced cAMP reporter is integrated in the genome and can be expressed in any targeted tissue and with tetracycline induction. We have expressed the cAMP reporter in b-cells of pancreatic islets and conducted an analysis of intracellular cAMP levels in relation to glucose stimulation, Ca2+ levels, and membrane depolarization. Pancreatic function in transgenic mice was normal. In induced transgenic islets, glucose evoked an increase in cAMP in b-cells in a dose-dependent manner. The cAMP response is independent of (in fact, precedes) the Ca2+ influx that results from glucose stimulation of islets. Glucose-evoked cAMP responses are synchronous in cells throughout the islet and occur in 2 phases suggestive of the time course of insulin secretion. Insofar as cAMP in islets is known to potentiate insulin secretion, the novel transgenic mouse model will for the first time permit detailed analyses of cAMP signals in β-cells within islets, i.e. in their native physiological context. Reporter expression in other tissues (such as the heart) where cAMP plays a critical regulatory role, will permit novel biomedical approaches. [ABSTRACT FROM AUTHOR]

A longstanding question in taste research concerns taste coding and, in particular, how broadly are individual taste bud cells tuned to taste qualities (sweet, bitter, umami, salty, and sour). Taste bud cells express G-protein-coupled receptors for sweet, bitter, or umami tastes but not in combination. However, responses to multiple taste qualities have been recorded in individual taste cells. We and others have shown previously there are two classes of taste bud cells directly involved in gustatory signaling: "receptor" (type II) cells that detect and transduce sweet, bitter, and umami compounds, and "presynaptic" (type III) cells. We hypothesize that receptor cells transmit their signals to presynaptic cells. This communication between taste cells could represent a potential convergence of taste information in the taste bud, resulting in taste cells that would respond broadly to multiple taste stimuli. We tested this hypothesis using calcium imaging in a lingual slice preparation. Here, we show that receptor cells are indeed narrowly tuned: 82% responded to only one taste stimulus. In contrast, presynaptic cells are broadly tuned: 83% responded to two or more different taste qualities. Receptor cells responded to bitter, sweet, or umami stimuli but rarely to sour or salty stimuli. Presynaptic cells responded to all taste qualities, including sour and salty. These data further elaborate functional differences between receptor cells and presynaptic cells, provide strong evidence for communication within the taste bud, and resolve the paradox of broad taste cell tuning despite mutually exclusive receptor expression. [ABSTRACT FROM AUTHOR]

ATP has been shown to be a taste bud afferent transmitter, but the cells responsible for, and the mechanism of, its release have not been identified. Using CHO cells expressing high-affinity neurotransmitter receptors as biosensors, we show that gustatory stimuli cause receptor cells to secrete ATP through pannexin 1 hemichannels in mouse taste buds. ATP further stimulates other taste cells to release a second transmitter, serotonin. These results provide a mechanism to link intracellular Ca2+ release during taste transduction to secretion of afferent transmitter, ATP, from receptor cells. They also indicate a route for cell-cell communication and signal processing within the taste bud. [ABSTRACT FROM AUTHOR]

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca2+. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca2+. Specifically, 5 μM thapsigargin or 3 μM A-23187 (calcium ionophore), both of which increase intracellular Ca2+ concentration ([Ca2+]i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca2+, suggesting that it is dependent on Ca2+ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase Cβ2 (i.e., cells that elevate [Ca2+]i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca2+ may represent independent second messenger signals downstream of taste receptors. [ABSTRACT FROM AUTHOR]

Taste buds are aggregates of 50 -100 cells, only a fraction of which express genes for taste receptors and intracellular signaling proteins. We combined functional calcium imaging with single-cell molecular profiling to demonstrate the existence of two distinct cell types in mouse taste buds. Calcium imaging revealed that isolated taste cells responded with a transient elevation of cytoplasmic Ca2+ to either tastants or depolarization with KCl, but never both. Using single-cell reverse transcription (RT)-PCR, we show that individual taste cells express either phospholipase C β2 (PLCβ2) (an essential taste transduction effector) or synaptosomal-associated protein 25 (SNAP25) (a key component of calcium-triggered transmitter exocytosis). The two functional classes revealed by calcium imaging mapped onto the two gene expression classes determined by single-cell RT-PCR. Specifically, cells responding to tastants expressed PLCβ2, whereas cells responding to KCl depolarization expressed SNAP25. We demonstrate this by two methods: first, through sequential calcium imaging and single-cell RT-PCR; second, by performing calcium imaging on taste buds in slices from transgenic mice in which PLCβ2-expressing taste cells are labeled with green fluorescent protein. To evaluate the significance of the SNAP25-expressing cells, we used RNA amplification from single cells, followed by RT-PCR. We show that SNAP25-positive cells also express typical presynaptic proteins, including a voltage-gated calcium channel (α1A), neural cell adhesion molecule, synapsin-II, and the neurotransmitter-synthesizing enzymes glutamic acid decarboxylase and aromatic amino acid decarboxylase. No synaptic markers were detected in PLCβ2 cells by either amplified RNA profiling or by immunocytochemistry. These data demonstrate the existence of at least two molecularly distinct functional classes of taste cells: receptor cells and synapse-forming cells. [ABSTRACT FROM AUTHOR]