Acid-activated cation currents in rat vallate taste receptor cells

Sour taste is mediated by acids with the degree of sourness a function of proton concentration. Recently, several members of the acid-sensing ion channel subfamily (ASICs) were cloned from taste cells and proposed to mediate sour taste. However, it is not known whether sour responses in taste cells resemble the responses mediated by ASICs. Using the whole cell patch-clamp technique and Na(+) imaging, we have characterized responses to acid stimuli in isolated rat vallate taste cells. Citric acid (pH 5) induced a large, rapidly activating inward current in most taste cells tested. The response showed various degrees of desensitization with prolonged stimulation. Current amplitudes were pH dependent, and adapting with acidic bath solutions reduced subsequent responses to acid stimulation. Amiloride (100-500 microM) partially and reversibly suppressed the acid-induced current. The current-voltage relationship showed reversal potential near the Na(+) equilibrium potential, suggesting that the current is carried predominantly by Na(+). These data were consistent with Na(+) imaging experiments showing that acid stimulation resulted in increases in intracellular Na(+). Taken together, these data indicate that acid-induced currents in vallate taste cells share general properties with ASICs expressed in heterologous cells and sensory neurons that express ASIC subunits. The large amplitude of the current and its existence in a high percentage of taste cells imply that ASICs or ASIC-like channels may play a prominent role in sour-taste transduction.     

Characterization of chloride currents and their noradrenergic modulation in rat taste receptor cells.

Taste receptor cells contain a heterogeneous array of voltage-dependent ion conductances that are essential components for the transduction of gustatory stimuli. Although mechanistic roles have been proposed for several cationic conductances, the understanding of anionic currents is rudimentary. This study characterizes biophysical and pharmacological properties of chloride currents in rat posterior taste cells using whole cell patch-clamp recording technique. Taste cells express a heterogeneous array of chloride currents that displayed strong outward rectification, contained both calcium-dependent and calcium-independent components, and achieved a maximal conductance of almost 1 nS. Reversal potentials altered predictably with changes in chloride concentration. Currents were sensitive to inhibition by the chloride channel pharmacological agents DIDS, SITS, and niflumic acid but were insensitive to 9-AC. Adrenergic enhancement of chloride currents, present in other cell types, was tested on taste cells with the beta-adrenergic agonist isoproterenol (ISP). ISP enhanced the outwardly rectifying portion of the chloride current. This enhancement was calcium dependent and was blocked by the beta-adrenergic antagonist propranolol. Collectively these observations suggest that chloride currents may participate not only in usually ascribed functions such as stabilization of the membrane potential and volume regulation but additionally play active modulatory roles in the transduction of gustatory stimuli.     

A transient receptor potential channel expressed in taste receptor cells

We used differential screening of cDNAs from individual taste receptor cells to identify candidate taste transduction elements in mice. Among the differentially expressed clones, one encoded Trpm5, a member of the mammalian family of transient receptor potential (TRP) channels. We found Trpm5 to be expressed in a restricted manner, with particularly high levels in taste tissue. In taste cells, Trpm5 was coexpressed with taste-signaling molecules such as alpha-gustducin, Ggamma13, phospholipase C-beta2 (PLC-beta2) and inositol 1,4,5-trisphosphate receptor type III (IP3R3). Our heterologous expression studies of Trpm5 indicate that it functions as a cationic channel that is gated when internal calcium stores are depleted. Trpm5 may be responsible for capacitative calcium entry in taste receptor cells that respond to bitter and/or sweet compounds.     

Resynthesis of phosphatidylinositol 4,5-bisphosphate mediates adaptation of the caffeine response in rat taste receptor cells

Caffeine, a prototypic bitter stimulus, produces several physiological actions on taste receptor cells that include inhibition of K_IR and K_V potassium currents and elevations of intracellular calcium. These responses display adaptation, i.e. their magnitude diminishes in the sustained presence of the stimulus. Levels of the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP₂) are well known to modulate many potassium channels, activating the channel by stabilizing its open state. Here we investigate a putative relationship of K_IR and K_V with PIP₂ levels hypothesizing that inhibition of these currents by caffeine might be allayed by PIP₂ resynthesis. Using standard patch-clamp techniques, recordings of either potassium current from rat posterior taste receptor cells produced essentially parallel results when PIP₂ levels were manipulated pharmacologically. Increasing PIP₂ levels by blocking phosphoinositide-3 kinase with wortmannin or LY294002, or by blocking phospholipase C with U73122 all significantly increased the incidence of adaptation for both K_IR and K_V. Conversely, lowering PIP₂ synthesis by blocking PI4K or using the PIP₂ scavengers polylysine or bovine serum albumin reduced the incidence of adaptation. Adaptation could be modulated by activation of protein kinase C but not calcium calmodulin kinase. Collectively, these data support two highly novel conclusions: potassium currents in taste receptor cells are significantly modulated by PIP₂ levels and PIP₂ resynthesis may play a central role in the gustatory adaptation process at the primary receptor cell level.     

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

 Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840–G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 μM) and cholecystokinin-(26-33)-peptide amide (CCK₈, 0.1 μM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK₈-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-α and, to a lesser extent, PKC-ɛ. In addition, TPA-induced inhibition of the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-α. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca²⁺]_i. Taken together, the present findings are in agreement with the idea that PKC-α-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca²⁺]_i oscillations and eventually to inhibition of high-affinity Ca²⁺ signalling through the native CCK receptor in pancreatic acinar cells. Received: 15 September 1997 / Accepted: 2 October 1997     

Adrenergic nerve fibres and mast cells: correlation in rat thymus

The interactions between adrenergic nerve fibres and mast cells (MCs) were studied in the thymus of adult and old rats by morphological methods and by quantitative analysis of images (QAIs). The whole thymus was drawn in adult (12 months old) rats: normal, sympathectomized or electrostimulated. Thymuses from the above-mentioned animals were weighed, measured and dissected. Thymic slices were stained with eosin orange for detection of microanatomical details and with Bodian's method for identification of the whole nerve fibres. Thymic MCs were stained with Astrablau. Histofluorescence microscopy was used for staining of adrenergic nerve fibres. Finally, all morphological results were submitted to the QAIs and statistical analysis of data. Our results suggest that after surgical sympathectomy, the greater part of adrenergic nerve fibres disappear while related MCs appear to show less evident fluorescence and few granules. On the contrary, electrostimulation of the cervical superior ganglion induced an increase in the fluorescence of adrenergic nerve fibres and of related MCs.     

Responses to di-sodium guanosine 5'-monophosphate and monosodium L-glutamate in taste receptor cells of rat fungiform papillae

The 5'-ribonucleotide guanosine 5'-monophosphate (GMP) is used widely as an umami taste stimulus and a potent flavor enhancer as it synergistically increases the umami taste elicited by monosodium glutamate. Transduction mechanisms for GMP and its synergy with glutamate are largely unknown. Using whole-cell patch-clamp and Ca(2+) imaging, we examined responses to GMP, glutamate, and a mixture of GMP and glutamate in taste-receptor cells of rat fungiform papillae. Our electrophysiological results showed that GMP induces responses that are similar to those of glutamate, e.g., an outward current, an inward current, or a biphasic response. Our Ca(2+) imaging results showed that applications of GMP, glutamate, and the mixture increased intracellular Ca(2+) levels. Interestingly, both patch-clamp and Ca(2+) imaging showed that some taste cells can respond to GMP and glutamate independently, indicating that glutamate and GMP likely activate different receptors. Simultaneous application of GMP and glutamate resulted in synergistic responses in a subset of cells; both response intensity and number of responding cells were increased. Most responses to GMP, as well as the synergy between GMP and glutamate, were suppressed by 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) in patch-clamp recordings. Together, our results suggest that intracellular cAMP- and Ca(2+)-mediated pathways are involved in umami taste transduction for GMP and its synergistic responses with glutamate.     

Na+-H+ exchange activity in taste receptor cells

mRNA for two Na(+)-H(+)-exchanger isoforms 1 and 3 (NHE-1 and NHE-3) was detected by RT-PCR in fungiform and circumvallate taste receptor cells (TRCs). Anti-NHE-1 antibody binding was localized to the basolateral membranes, and the anti-NHE-3 antibody was localized in the apical membranes of fungiform and circumvallate TRCs. In a subset of TRCs, NHE-3 immunoreactivity was also detected in the intracellular compartment. For functional studies, an isolated lingual epithelium containing a single fungiform papilla was mounted with apical and basolateral sides isolated and perfused with nominally CO(2)/HCO(3)(-)-free physiological media (pH 7.4). The TRCs were monitored for changes in intracellular pH (pH(i)) and Na(+) ([Na(+)](i)) using fluorescence ratio imaging. At constant external pH, 1) removal of basolateral Na(+) reversibly decreased pH(i) and [Na(+)](i); 2) HOE642, a specific blocker, and amiloride, a nonspecific blocker of basolateral NHE-1, attenuated the decrease in pH(i) and [Na(+)](i); 3) exposure of TRCs to basolateral NH(4)Cl or sodium acetate pulses induced transient decreases in pH(i) that recovered spontaneously to baseline; 4) pH(i) recovery was inhibited by basolateral amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), HOE642, and by Na(+) removal; 5) HOE642, MIA, EIPA, and amiloride inhibited pH(i) recovery with K(i) values of 0.23, 0.46, 0.84, and 29 microM, respectively; and 6) a decrease in apical or basolateral pH acidified TRC pH(i) and inhibited spontaneous pH(i) recovery. The results indicate the presence of a functional NHE-1 in the basolateral membranes of TRCs. We hypothesize that NHE-1 is involved in sour taste transduction since its activity is modulated during acid stimulation.     

Sweet taste receptor mechanisms

Chemicals of diverse structures can elicit sweet response in humans, but marked species difference in response to sweet-tasting compounds exists among mammalinan species. In a number of species a distinct group of chorda tympani nerve fibers predominantly responds to sucrose and Na saccharin. Sweet response in certain mammals can be selectively blocked by a number of compounds, including gymnemic acid and ziziphin. Cu and Zn salts also selectively suppress chorda tympani nerve response to sweet stimuli in rodents. Probably Cu2+ and Zn2+ compete with sweet stimuli for binding to receptor molecules. A recent attempt to isolate a protein from monkey tongues revealed the presence of a thaumatin-binding protein of M.W. of approximately 50,000. This protein was predominantly found in taste papillae, with lesser amounts found in non-taste tissue.     

Analyses of taste nerve responses with special reference to possible receptor mechanisms of umami taste in the rat

Umami substances such as monopotassium L-glutamate (MPG) and 5'-inosine monophosphate (IMP) elicit a unique taste called 'umami' in humans. To elucidate the umami receptor mechanism in rats, we examined taste responses of the chorda tympani nerve by using three ionotropic glutamate receptor agonists, NMDA, KA and AMPA, a mGluR4 agonist, L-AP4, and a specific mGluR4 antagonist, MAP4, and an anti-sweet peptide, gurmarin. When IMP was added, synergistic responses were shown only for MPG and L-AP4, but not for NMDA, KA and AMPA. MAP4 enhanced the responses to MPG and L-AP4. Gurmarin suppressed the synergistic responses to mixtures of MPG and IMP or L-AP4 and IMP. These results suggest that glutamate and L-AP4 bind both the sweet-responsive macromolecule and mGluR4, but the synergism occurs only on the macromolecule.     

Antigen receptor signalling in apoptosis-resistant mutants of WEHI 231 cells

Ligation of membrane immunoglobulin M (mIgM) induces cell cycle arrest and apoptosis in the WEHI 231 B-lymphoma cell line. The molecular mechanisms which link receptor ligation and the nuclear events that underlie this response, have yet to be fully elucidated. Here we have examined the signals induced following mIgM cross-linking in variants of WEHI 231 that no longer undergo apoptosis in response to this stimulus. Tyrosine phosphorylation of cellular substrates in two of the variants is identical to that seen in wild-type cells but in one of the mutants, VS2.12, a restricted set of substrates becomes tyrosine phosphorylated. In a second variant (E8), mIgM cross-linking does not induce elevation of intracellular Ca2+, although tyrosine phosphorylation of PLCgamma2 is induced to an equivalent extent to that seen in WEHI 231 cells. A third variant, 2E10.F9, is resistant to apoptosis despite the fact that all signals analysed appear to be similar to those induced in wild-type cells. Our findings show that resistance to apoptosis can arise as a result of mutations affecting discrete stages of the mIgM signalling pathway. The mutant lines reported here show defects that have not yet been identified in previous studies and are likely to be useful tools in dissecting the signalling of cell death in B lymphocytes.     

Adrenergic innervation of the rat hypothalamus

The adrenergic innervation of the hypothalamus was studied by measuring hypothalamic adrenaline levels following surgical transection of the lower brain stem or electrolytic lesion of the medullary adrenaline-containing cell groups. The adrenaline levels in some hypothalamic nuclei and in the median eminence showed a slight decrease after partial transection of the medulla oblongata, whilst there was a pronounced decrease (by 59-78%) 7-10 days following total hemisection or unilateral lesion of the C1-catecholaminergic cell group in the medulla oblongata.     

Adrenergic receptor hyperactivity--a cause for pseudopheochromocytoma?

Two patients with classical signs and symptoms of pheochromocytoma, are described. In these two patients the 24-hr urinary excretion of dopamine, norepinephrine, epinephrine and their metabolites were normal or low, both in basal states or after attacks (spontaneous or provoked). However, the 24-hr urinary excretion of cyclic AMP was exceedingly high in one patient (13000 nmol/gm creatinine), and elevated in the second (5920 nmol/gm creatinine). Both patients were benefited by treatment with a combination of alpha and beta adrenergic blocking drugs. The first patient, after stopping treatment, developed hypertensive crisis and died. Post-mortem examination did not detect a pheochromocytoma or any other abnormality which could explain the excessively elevated cyclic AMP. In the second patient, extensive X-ray and CT examinations were negative for pheochromocytoma. A hypothesis is developed linking the symptoms and biochemical findings of both patients to an abnormality of the adrenergic receptor.     

Intracellular pH and calcium signalling in rat pancreatic acinar cells

 Intracellular free Ca²⁺ signals, which occur in many secretory cell types after the binding of some secretagogues to their membrane receptors, are due to Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular space. There is also growing evidence for a modulatory role of intracellular pH in Ca²⁺ metabolism. In fact it has been proposed that Ca²⁺ stores in pancreatic acinar cells may be loaded by Ca²⁺/H⁺ exchange. The aim of this paper was to establish the effect of intracellular pH on Ca²⁺ signalling in pancreatic acinar cells. Application of the proton carrier nigericin impairs Ca²⁺ mobilization in response to cholecystokinin (CCK-8), and application of membrane-permeant bases or acids inhibits CCK-8-evoked intracellular Ca²⁺ oscillations. Both nigericin and a cell-permeant weak base release Ca²⁺ from internal stores. However, cytosolic acidification by removal of extracellular Na⁺ had no effect on the resting or stimulated cytosolic Ca²⁺ concentration. After depletion of Ca²⁺ stores by a maximal concentration of CCK-8, nigericin and ionomycin released a residual Ca²⁺ pool. Taken together, our results show that in pancreatic acinar cells Ca²⁺ signals require the existence of subcellular gradients of pH and indicate the presence of acidic pools of Ca²⁺. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

Angiotensin II receptor signalling

Angiotensin II plays a key role in the regulation of body fluid homeostasis. To correct body fluid deficits that occur during hypovolaemia, an animal needs to ingest both water and electrolytes. Thus, it is not surprising that angiotensin II, which is synthesized in response to hypovolaemia, acts centrally to increase both water and NaCl intake. Here, we review findings relating to the properties of angiotensin II receptors that give rise to changes in behaviour. Data are described to suggest that divergent signal transduction pathways are responsible for separable behavioural responses to angiotensin II, and a hypothesis is proposed to explain how this divergence may map onto neural circuits in the brain.