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PubMed1991

Secondary active transport of water across ventricular cell membrane of choroid plexus epithelium of Necturus maculosus.

1. The interaction between Cl-, K+ and H2O fluxes were studied in the ventricular membrane of the choroid plexus epithelium from Necturus maculosus by means of ion-selective microelectrodes. The flux of H2O was measured by means of K+ electrodes as the dilution or concentration of intracellular choline ions, Ch+i. 2. In one series of experiments Cl- was readministered to the ventricular solution of tissues incubated in media with low Cl- concentrations. The resulting influx of Cl- was associated with an instantaneous influx of K+ and H2O. 3. Both the Cl- and the K+ influxes were reduced by the diuretic furosemide but were unaffected by inhibitors of Na+, K(+)-ATPase or changes in membrane potentials induced by Ba2+. Since the influx of K+ proceeds against its electrochemical gradient and is unaffected by changes in membrane potentials, the membrane exhibits secondary active, electroneutral transport of K+. 4. The influx of water, initiated simultaneously with the influx of K+ and Cl-, commenced before these ions had changed the osmolarity of the intracellular solution significantly. The influx of H2O could proceed against an osmotic gradient. The influx stopped when 100 mmol l-1 of mannitol was added to the ventricular solution at the same time as the Cl- ions. The influx of H2O was inhibited by K+ removal, furosemide or high external Ba2+ (10 mmol l-1), but not by strophanthidin, ouabain or low concentrations of Ba2+ (0.5 mmol l-1). The influx could not continue with other permeable anions, NO3-, acetate- or SCN-, replacing Cl-. 5. In another series of experiments Cl- was removed from the ventricular solution of tissues bathed in saline solutions with normal concentrations of Cl-. The resulting efflux of Cl- was associated with an instantaneous efflux of K+ and H2O. This efflux of H2O could proceed against an osmotic gradient of up to 70 mosmol l-1. This effect was inhibited by furosemide, in which case the water fluxes were entirely dependent on the osmotic gradients and the osmotic water permeability Lp of the ventricular membrane. 6. The data suggest that there is a coupling between the flux of KCl and of water in the ventricular membrane, which implies that the reflection coefficient sigma for KCl under the given circumstances is less than one. I suggest that the ability of leaky epithelia to transport against osmotic gradients depends on such a coupling, which derives from the properties of the proteins through which K+, Cl- and H2O leave the cell.

Animals↗

PubMedDate not supplied

Carbonic anhydrase and red blood cell anion exchange in the neotenic aquatic salamander, Necturus maculosus.

Carbonic anhydrase (CA) activity in blood and other tissues and red blood cell (rbc) anion exchange were measured in the mud puppy, Necturus maculosus, in order to gain insight into the strategy for CO2 transport used by these neotenic salamanders and to further explore evolutionary relationships between rbc CA activity and anion exchange in nonmammalian vertebrates. CA activity was detectable in all of the tissues examined, but CA activity in blood was much lower than that in most vertebrates. There was no indication, however, that additional CA had been incorporated into the membrane fraction of other tissues to compensate for this low blood CA activity. In further contrast to most other animals, low levels of CA activity were also detectable in mud puppy plasma. Preliminary characterization of the rbc CA indicated that the Type II, fast-turnover enzyme was indeed present, but that there are a very low number of active sites in mud puppy rbc's. Further experiments showed that the rbc's were highly permeable to anions and that the relative rate of anion flux could be inhibited by 4, 4-diisothiocyanostilbene-2,2-disulphonic acid. Thus, the process of CO2 transport in the blood of mud puppies probably involves components of the Jacobs-Stewart cycle, as in most other vertebrates.

Adaptation, Physiological↗

PubMed1975

Micropuncture study of the renal responses of the urodele amphibian Necturus maculosus to injections of arginine vasotocin and an anti-aldosterone compound.

1. Necturus maculosus kidney function has been examined using standard clearance techniques and renal tubular micropuncture methodology. 2. Throughout, cyanocobalamin (vitamin B12) has been used to monitor glomerular filtration rate (GFR) and tubular water movements. It was established that this substance was handled by the Necturus kidney in a similar manner to inulin. It can be readily analysed, together with renal electrolytes, by electron microprobe techniques. 3. Profiles of transtubular gradients (TF:P ratios) along the nephron were established for osmolarity, sodium, potassium, calcium and cobalt (of cyanocobalamin). 4. Ureteral urine is always hyposmotic with respect to plasma and the site of dilution of the plasma ultrafiltrate is within the distal segment. 5. Up to 30% of the filtrate is isosmotically reabsorbed along the proximal tubule; the tubular fluid:plasma ratio for osmolarity and sodium is around 1, and the TF:P for cobalt of cyanocobalamin is about 1.4 by the end of this segment. 6. The renal effects of the neurohypophysial hormone arginine vasotocin (AVT) and an aldosterone antagonist (SC14266; Soldactone) have been examined. 7. AVT was consistently antidiuretic causing both a decreased GFR and an enhanced distal tubular reabsorption of water. 8. SC14266 also increased distal tubular reabsorption of water. Such an effect differs from that found in higher vertebrates, and may indicate a "glucocorticoid-type" of renal action for aldosterone in amphibians.

Aldosterone↗

PubMed1992

Immunocytochemical survey of putative neurotransmitters in taste buds from Necturus maculosus.

To investigate synaptic mechanisms in taste buds and collect information about synaptic transmission in these sensory organs, we have examined taste buds of the mudpuppy, Necturus maculosus for the presence of neurotransmitters and neuromodulators. Immunocytochemical staining at the light microscopic level revealed the presence of serotonin-like and cholecystokinin-like (CCK) immunoreactivity in basal cells in the taste bud. Nerve fibers innervating taste buds were immunoreactive for vasoactive intestinal peptide-like (VIP), substance P-like, and calcitonin gene-related peptide-like (CGRP) or compounds closely related to these substances. Immunoreactivity for tyrosine hydroxylase (TH) and choline acetyltransferase (ChAT) in the taste cells and nerve fibers was absent. These data suggest that serotonin, CCK, VIP, substance P, and CGRP are involved in synaptic transmission or neuromodulation in the peripheral organs of taste. No evidence was found for cholinergic or adrenergic mechanisms on the basis of the absence of immunocytochemical staining for key enzymes involved in these two transmitter systems.

Animals↗

PubMed1989

Effects of parathyroid hormone on cyclic-AMP concentrations of in vitro Necturus maculosus gastric antrum.

The effects of bovine parathyroid hormone (bPTH1-84) on the stimulation of intracellular cyclic-AMP [cAMP] were investigated in an in vitro preparation of Necturus maculosus antral mucosa. When the antrum was exposed to 1, 5, 10, or 100 nM bPTH1-84, there was an approximately 2-fold nonlinear increase in tissue [cAMP] over basal values. The pretreatment of the antral mucosa with 1 mM isobutylmethylxanthine (IBMX, a phosphodiesterase inhibitor) increased with detectability of mucosal [cAMP]. The addition of 1, 5, 10, or 100 nM bPTH1-84 to tissues pretreated with IBMX resulted in an approximately 3.5-fold linear increase in mucosal [cAMP] over basal values. The time course of the generation of mucosal cAMP to 10 nM bPTH1-84 resulted in a small but significant transient increase at 2.5 min after the addition of bPTH1-84 but no change in the medium [cAMP]. In tissues pretreated with 1 mM IBMX the response to 10 nM bPTH1-84 was a large biphasic increase of [cAMP] at 2.5 min that progressively declined to near basal values by 15 min. There was also a significant sustained increase in the [cAMP] in the bathing medium at 2.5 min of tissues pretreated with IBMX followed by 10 nM bPTH1-84. These results suggest the presence of an adenylate cyclase that can be activated by a mammalian bPTH1-84 in elevating intracellular cAMP levels in the N. maculosus antral mucosa.

1-Methyl-3-isobutylxanthine↗

PubMed1982

A micro-electrode study of oligopeptide absorption by the small intestinal epithelium of Necturus maculosus.

1. The effects of some amino acids and oligopeptides on the electrical properties of the brush-border membrane of the small intestine of the mudpuppy, Necturus maculosus were studied in vitro using micro-electrodes. 2. A number of amino acids (glycine, L-proline and L-leucine) and small peptides (carnosine, glycyl-L-proline, L-leucyl-L-leucine, glycylglycine and glycylglycylglycine) depolarized the brush-border membrane. This was associated with a reduction of input resistance. Tetraglycine did not appreciably reduce the membrane potential. 3. Evidence is presented that the electrical effects caused by application of the peptides are not solely due to the effects of the products of peptide hydrolysis. Furthermore there appears to be more than a single system available to the peptides. 4. Stereospecificity is found for both amino acids and peptides but appears to be more marked for the peptides. 5. Peptide-induced depolarizations are less markedly reduced in the absence of external Na+ than are the depolarizations caused by the amino acids. 6. These results are discussed with respect to the mechanism and significance of amino acid and peptide transport in the intestinal tract.

Amino Acids↗

PubMed1991

Water permeability of ventricular cell membrane in choroid plexus epithelium from Necturus maculosus.

1. The osmotic water permeability Lp and the relations between the flows of H2O, K+ and Cl- were studied in the ventricular membrane of the epithelium from the choroid plexus of Necturus maculosus. 2. The flows were induced by abrupt changes in external osmolarity of the ventricular solution. Solution changes were convective and no effects of unstirred layers could be detected on measured parameters. 3. The initial rate of change in intracellular concentrations of K+ and Cl- was monitored by double-barrelled ion-selective microelectrodes. 4. The initial rate of flux of H2O could be monitored as the changes in the concentration of intracellular choline ions (Ch+i). When 0.5 mmol l-1 of choline chloride was added to the external solutions, Ch+i attained values of 1-5 mmol l-1. The dilution or concentration of Ch+i could be recorded by K+ electrodes since the sensitivity of these to Ch+ is more than 50 times greater than to K+. 5. The Lp of the ventricular membrane of the epithelium was 1.4-2.1 x 10(-4) cm s-1 (osmol l-1)-1 and independent of the direction of the induced water flux. Lp was unchanged in tissues adapted to osmolarities of half the physiological value. 6. The efflux of H2O induced by mannitol was associated with an instantaneous efflux of K+ which was inhibited by furosemide. The fluxes had a ratio of 40 mmol l-1. The influx of H2O induced by the removal of NaCl from the ventricular solution was associated with an instantaneous influx of K+. The H2O influx had a ratio to the flux of K+ of 70 mmol l-1. 7. The efflux of H2O induced by mannitol was associated with an efflux of Cl- which was inhibited by furosemide. The ratio of the two fluxes was in the range 15-44 mmol l-1. 8. The conclusion is that the Ch+ method gives a reliable measure of the movement of H2O across the ventricular membrane. The magnitude of the Lp and its relevance to transepithelial transport are discussed. The osmotically induced H2O movement is accompanied by furosemide-sensitive fluxes of K+ and Cl- of the same magnitude. This suggests that co-transport between H2O and KCl can take place in the membrane.

Animals↗

PubMed1977

The permeability of the skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus.

1. The permeability of the isolated skin of a neotenous urodele amphibian, the mudpuppy Necturus maculosus, to Na, Cl, urea and water was measured. 2. Unidirectional transcutaneous flux measurements and the action of ouabain and amiloride, showed that there was normally no active Cl or Na transport, nor a Cl/Cl exchange diffusion process. 3. Amphotericin B initiated a transcutaneous potential difference and short-circuit current, which could be inhibited by ouabain. 4. The short-circuit current was nearly equivalent to the net Na Transport and this was also inhibited by ouabain. 5. A transcutaneous active Na transport mechanism thus appears to be incipient in the mudpuppy but is limited by a low permeability of the outer barrier of the cells. 6. Vasotocin increased the skin's diffusion permeability for water but had no effect on the influx of Na or urea. 7. The function of Necturus skin is in several respects unique compared to that of other amphibians.

Amiloride↗

PubMed1987

Ultrastructure of apical specializations of taste cells in the mudpuppy, Necturus maculosus.

The first interaction of taste stimuli with lingual chemoreceptors occurs on the apical membrane of taste cells, since only that portion is exposed to the oral cavity. To gain better insight into this interaction, we examined the pore region of taste buds in Necturus maculosus with scanning electron microscopy (SEM), transmission electron microscopy, and high-voltage electron microscopy. SEM of the pore reveals a patchwork distribution of three morphologically distinct types of apical specializations: long and branched (LB) microvilli, short and unbranched (SU) microvilli, and bundles of stereocilia. As demonstrated in thin and thick sections, LB microvilli are specializations of dark cells, SU microvilli are the apical specializations of light cells, and stereocilia arise from a cell that has the cytoplasmic markers characteristic of light cells. When left in place, the pore mucus completely covers the SU microvilli and partially covers the LB microvilli. However, stereocilia project above the surface and thus are highly exposed to taste stimuli in the oral cavity. These three morphologically distinct types of apical specializations may reveal functional differences among taste cells. The initial interaction between chemical stimulus and taste cell, and possibly chemoreceptor specificity itself, may be influenced by the morphology of the apical ending.

Animals↗

PubMed1992

Influence of temperature and photoperiod on plasma melatonin in the mudpuppy, Necturus maculosus.

A melatonin (MEL) radioimmunoassay employed previously only in mammals was used to estimate plasma MEL in a salamander, Necturus maculosus. Validation procedures included thin-layer chromatography of plasma extracts, parallel inhibition curves of authentic MEL and serially diluted plasma and plasma extracts and quantitative recovery of authentic MEL added to pooled Necturus plasma. A diel cycle of plasma MEL was demonstrated in mudpuppies acclimatized for a minimum of 3 weeks under a 12L:12D photoperiod and 15 +/- 1 degrees and sampled in late March. The MEL cycle persisted under a reversed photoperiod (lights on, 1800 hr), but the amplitude of the MEL peak was diminished, and the peak was more than 180 degrees out of phase with the corresponding peak under a normal photoperiod. In animals acclimated to 5 degrees in mid-June under a 12L:12D photoperiod, the diel cycle of plasma MEL continued, but both midphotophase and midscotophase concentrations were reduced compared with 15 degrees controls. The diel cycle was also present in animals acclimated to 25 degrees, but the decrease in MEL concentrations was less marked than that in 5 degrees animals. Photoperiod is apparently the primary cue for cycles in plasma MEL in Necturus, but the cycle can be influenced by temperature.

Animals↗

PubMed1992

Autocrine and paracrine role of steroids during spermatogenesis: studies in Squalus acanthias and Necturus maculosus.

Due to a cystic mode of spermatogenesis and zonal testes, the spiny dogfish shark (Squalus acanthias) and salamander (Necturus maculosus) are advantageous for studying the relationship between steroidogenesis and germ cell development. Direct analysis of steroidogenic enzymes and steroid receptors in staged tissue samples, together with light and electron microscopic observation, suggests that androgens and estrogens produced by Sertoli cells or Leydig cells immediately before or after spermiation serve as intratesticular regulators of events specific to premeiotic stages of spermatogenesis. These correlative data are consistent with limited observations in mammalian testis and provide a conceptual framework for direct testing of functional interactions between spermatogenesis and steroidogenesis in future studies.

Animals↗

PubMed1993

Modulation of Cl-, K+, and nonselective cation conductances by taurine in olfactory receptor neurons of the mudpuppy Necturus maculosus.

Odors are transduced by processes that modulate the membrane conductance of olfactory receptor neurons. Olfactory neurons from the aquatic salamander, Necturus maculosus, were acutely isolated without enzymes and studied with a resistive whole-cell method to minimize loss of soluble intracellular constituents. 55 of 224 neurons responded to the test compound taurine at concentrations between 10 nM and 100 microM. Four different conductance changes were elicited by taurine: an increased Cl- conductance (33%), an increased nonselective cation conductance (15%), a decreased Cl- conductance (15%), and a decreased K+ conductance (15%); in addition, responses too small to be characterized were elicited in some neurons. In most cases, taurine appeared to modulate only a single conductance in any particular cell. Modulation of each conductance was dose dependent, and each response ran down quickly in the normal whole-cell mode, presumably due to washout of a diffusible component in the transduction pathway. Modulation of taurine-sensitive conductances caused either inhibitory or excitatory responses. A similar diversity of responses in vivo would produce a complex pattern of electrical activity that could encode the identity and characteristics of an odor.

Animals↗

PubMed1989

Cardiovascular regulation in the mudpuppy Necturus maculosus at rest and during short term exercise.

Prebranchial blood pressure (Pva), heart rate (fH) and plasma catecholamine concentration were measured in the mudpuppy Necturus maculosus at rest and during exercise. During exercise, both fH and Pva increased, as did the plasma noradrenaline concentration. There was no significant effect on Pva after injection of the adrenergic neuron blocker, bretylium, in resting animals, neither did bretylium affect the exercise-induced increase in Pva. This suggests that there is no adrenergic nervous tone on the vasculature at rest or during exercise. The alpha-adrenoceptor antagonist yohimbine had no effect on the resting Pva in the bretylium-treated animals, but it abolished the increase in Pva during exercise. This is compatible with the view of no influence on Pva by the plasma catecholamines at rest, while during exercise the increase in plasma noradrenaline concentration is responsible for the increase in Pva. Injection of atropine elevated resting fH, and reduced or abolished cardiac arrythmia, indicating an inhibitory cholinergic tone on the heart at rest, and that variations in this tone are responsible for the intrinsic variation seen in untreated animals. After the atropine treatment, there was still an increase in fH during exercise. The beta-adrenoceptor antagonist sotalol decreased fH in resting atropinized animals, and inhibited the exercise-induced tachycardia, implying that there is an additional beta-adrenoceptor-mediated adrenergic tone, affecting the heart both at rest and during exercise. The cholinergic tone decreased during exercise with a concomitant increase in adrenergic tone. Falck-Hillarp fluorescent histochemistry was used to study the presence of adrenergic nerve fibres and other catecholamine-storing cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Arachidonic acid may mediate the galanin-induced hyperpolarization in parasympathetic neurons from Necturus maculosus.

The effects of arachidonic acid (AA) and compounds that inhibit intracellular signalling pathways on membrane potential and galanin-induced hyperpolarizations were investigated in parasympathetic neurons from Necturus maculosus. Treatment for 10-90 min with 10-20 microM 4-bromophenacylbromide or 10 microM cyclosporin A caused a progressive decrease in the amplitude of galanin-induced hyperpolarizations without any change in resting membrane potential. The galanin-induced hyperpolarization was not altered following a 10-120 min treatment with the protein kinase inhibitor H-7. These results indicated that phospholipase A2 activation, but not protein kinase activation, may be required for the galanin-induced hyperpolarization. Arachidonic acid (20-100 microM) caused a concentration-dependent membrane hyperpolarization of the parasympathetic neurons and a decrease in the amplitude of the galanin-induced hyperpolarization. These data indicate that phospholipase A2-catalyzed liberation of AA may be involved in the galanin-induced membrane hyperpolarization observed in mudpuppy parasympathetic neurons.

Acetophenones↗

PubMed1982

A fine-structural study of interstitial cell changes in the testes of Necturus maculosus during a portion of the annual cycle, and possible evidence for local feedback control by seminiferous epithelium.

The developmental stages of the interstitial cells of Leydig in Necturus maculosus were studied in testes of animals obtained in November and December. As a consequence of the caudal to cranial progression of the wave of spermatogenesis, samples obtained from cranial, central, and caudal portions of these testes exhibit regional differences in the morphology of their Leydig cells. In these samples, fibroblast-like stromal cells with large, dense, elongated nuclei and thin sheets of cytoplasm surround the cranial seminiferous lobules that usually contain spermatozoa. Such stromal cells possess a small juxtanuclear Golgi apparatus, profiles of smooth and rough endoplasmic reticulum, and mitochondria with tubular and shelf-like cristae. In the central area of the testis, interstitial cells with oval nuclear profiles and a finer chromatin pattern surround degenerating seminiferous lobules. The abundant cytoplasm of these cells contains predominantly smooth endoplasmic reticulum interspersed with a few lipid droplets. These cells contain mitochondria that are packed with tubular cristae. Inthe caudal part of the testis, the Leydig cells have a round nucleus with finely dispersed chromatin. Numerous lipid droplets accumulate in the ample cytoplasm, which is filled with smooth endoplasmic reticulum. The juxtanuclear Golgi apparatus becomes enlarged; associated with it are vesicles with an electron-dense content. The cristae of the mitochondria are more numerous and have tubules of 50-nm diameter mixed with arrays of small tubules 25-30 nm in diameter. The appearance of morphologically mature interstitial cells of Leydig only adjacent to degenerated seminiferous lobules inthe caudal portion of the testis suggests the possibility of local feedback control of Leydig-cell development.

Animals↗

PubMed1985

Neuropeptides in the gastrointestinal canal of Necturus maculosus. Distribution and effects on motility.

The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut. Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum. Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa. The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like-bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity. It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.

Animals↗

PubMed1998

Uptake and release of neurotransmitter candidates, [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid, in taste buds of the mudpuppy, Necturus maculosus.

Neurotransmitters in vertebrate taste buds have not yet been identified with confidence. Serotonin, glutamate, and gamma-aminobutyric acid (GABA) have been postulated, but the evidence is incomplete. We undertook an autoradiographic study of [3H]serotonin, [3H]glutamate, and [3H]GABA uptake in lingual epithelium from the amphibian, Necturus maculosus, to determine whether taste bud cells would accumulate and release these substances. Lingual epithelium containing taste buds was incubated in low concentrations (0.4-6 microM) of these tritiated transmitter candidates and the tissue was processed for light microscopic autoradiography. Merkel-like basal taste cells accumulated [3H]serotonin. When the tissue was treated with 40 mM K+ after incubating the tissue in [3H]serotonin, cells released the radiolabelled transmitter. Furthermore, depolarization (KCl)-induced release of [3H]serotonin was Ca-dependent: if Ca2+ was reduced to 0.4 mM and 20 mM Mg2+ added to the high K+ bathing solution, Merkel-like basal cells did not release [3H]serotonin. In contrast, [3H]glutamate was taken up by several cell types, including non-sensory epithelial cells, Schwann cells, and some taste bud cells. [3H]glutamate was not released by depolarizing the tissue with 40 mM K+. [3H]GABA uptake was also widespread, but did not occur in taste bud cells. [3H]GABA accumulated in non-sensory epithelial cells and Schwann cells. These data support the hypothesis that serotonin is a neurotransmitter or neuromodulator released by Merkel-like basal cells in Necturus taste buds. The data do not support (nor rule out) a neurotransmitter role for glutamate or GABA in taste buds.

Animals↗

PubMed1997

Bitter taste transduction of denatonium in the mudpuppy Necturus maculosus.

Bitter substances are a structurally diverse group of compounds that appear to act via several transduction mechanisms. The bitter-tasting denatonium ion has been proposed to act via two different G-protein-regulated pathways, one involving inositol 1,4, 5-trisphosphate and raised intracellular calcium levels, the other involving phosphodiesterase and membrane depolarization via a cyclic nucleotide-suppressible cation channel. The aim of the present study was to examine these transduction mechanisms in taste cells of the mudpuppy Necturus maculosus by calcium-imaging and whole-cell recording. Denatonium benzoate increased intracellular calcium levels and induced an outward current independently of extracellular calcium. The denatonium-induced increase in intracellular calcium was inhibited by U73122, an inhibitor of phospholipase C, and by thapsigargin, an inhibitor of calcium transport into intracellular stores. The denatonium-induced outward current was blocked by GDP-beta-S, a blocker of G-protein activation. Neither resting nor denatonium-induced intracellular calcium levels were affected by inhibition of phosphodiesterase (with IBMX) or adenylate cyclase (with SQ22536) or by raising intracellular cyclic nucleotides directly (with cell permeant analogs). Our results support the hypothesis that denatonium is transduced via a G-protein cascade involving phospholipase C, inositol 1,4,5-trisphosphate, and raised intracellular calcium levels. Our results do not support the hypothesis that denatonium is transduced via phosphodiesterase and cAMP.

1-Methyl-3-isobutylxanthine↗